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Canadian Journal of Veterinary Research Revue Canadienne de Recherche Vétérinaire

Canadian Journal of Veterinary Research Revue Canadienne de Recherche Vétérinaire

ArticlesFarm-level prevalence and risk factors for detection of hepatitis E virus, porcine enteric calicivirus, and rotavirus in Canadian finisher pigsBarbara Wilhelm, Danielle Leblanc, David Leger, Sheryl Gow, Anne Deckert, David L. Pearl, Robert Friendship, Andrijana Rajic, Alain Houde, Scott McEwen. . . . . . . . . . . . . . . . . . . . . . . . . . 95

Genetic diversity of Streptococcus suis serotype 2 isolated from pigs in BrazilDaniela Sabatini Doto, Luisa Zanolli Moreno, Franco Ferraro Calderaro, Carlos Emilio Cabrera Matajira, Vasco Tulio de Moura Gomes, Thais Sebastiana Porfida Ferreira, Renan Elias Mesquita, Jorge Timenetsky, Marcelo Gottschalk, Andrea Micke Moreno. . . . . . . . . . . . . . . . . . 106

Comparison of 2 commercial single-dose Mycoplasma hyopneumoniae vaccines and porcine reproductive and respiratory syndrome virus (PRRSV) vaccines on pigs dually infected with M. hyopneumoniae and PRRSVChanghoon Park, Ikjae Kang, Hwi Won Seo, Jiwoon Jeong, Kyuhyung Choi, Chanhee Chae . . . . . . . . . . . . 112

Safety and early onset of immunity with a novel European porcine reproductive and respiratory syndrome virus vaccine in young pigletsMichael Piontkowski, Jeremy Kroll, Christian Kraft, Teresa Coll . . . . . . . . . . . . . . 124

Development of a double-monoclonal antibody sandwich ELISA: Tool for chicken interferon-g detection ex vivoHua Dai, Zheng-zhong Xu, Meiling Wang, Jun-hua Chen; Xiang Chen, Zhi-ming Pan, Xin-an Jiao . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

Effects of ketamine and lidocaine in combination on the sevoflurane minimum alveolar concentration in alpacasPatricia Queiroz-Williams, Thomas J. Doherty, Anderson F. da Cunha, Claudia Leonardi . . . . . . . . . . . . . . . . . . . . . . .141

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Article

2016;80:95–105 The Canadian Journal of Veterinary Research 95

Farm-level prevalence and risk factors for detection of hepatitis E virus, porcine enteric calicivirus, and rotavirus in Canadian finisher pigsBarbara Wilhelm, Danielle Leblanc, David Leger, Sheryl Gow, Anne Deckert, David L. Pearl,

Robert Friendship, Andrijana Rajic, Alain Houde, Scott McEwen

A b s t r a c tHepatitis E virus (HEV), norovirus (NoV), and rotavirus (RV) are all hypothesized to infect humans zoonotically via exposure through swine and pork. Our study objectives were to estimate Canadian farm-level prevalence of HEV, NoV [specifically porcine enteric calicivirus (PEC)], and RV in finisher pigs, and to study risk factors for farm level viral detection. Farms were recruited using the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) and FoodNet Canada on-farm sampling platforms. Six pooled groups of fecal samples were collected from participating farms, and a questionnaire capturing farm management and biosecurity practices was completed. Samples were assayed using validated real-time polymerase chain reaction (RT-PCR). We modeled predictors for farm level viral RNA detection using logistic and exact logistic regression. Seventy-two herds were sampled: 51 CIPARS herds (15 sampled twice) and 21 FoodNet Canada herds (one sampled twice). Hepatitis E virus was detected in 30/88 farms [34.1% (95% CI 25.0%, 44.5%)]; PEC in 18 [20.5% (95% CI: 13.4%, 30.0%)], and RV in 6 farms [6.8% (95% CI: 3.2%, 14.1%)]. Farm-level prevalence of viruses varied with province and sampling platform. Requiring shower-in and providing boots for visitors were significant predictors (P , 0.05) in single fixed effect mixed logistic regression analysis for detection of HEV and PEC, respectively. In contrast, all RV positive farms provided boots and coveralls, and 5 of 6 farms required shower-in. We hypothesized that these biosecurity measures delayed the mean age of RV infection, resulting in an association with RV detection in finishers. Obtaining feeder pigs from multiple sources was consistently associated with greater odds of detecting each virus.

R é s u m éLe virus de l’hépatite E (VHE), le norovirus (NoV), et le rotavirus (RV) sont tous suspectés être des agents zoonotiques associés à une exposition aux porcs ou à la viande de porc. Les objectifs de la présente étude étaient d’estimer, dans des fermes canadiennes, la prévalence de VHE, NoV [spécifiquement le calicivirus entérique porcin (CEP)], et le RV chez des porcs en finition, et d’étudier les facteurs de risque pour la détection virale à la ferme. Les fermes ont été recrutées à l’aide des plateformes d’échantillonnage à la ferme du Programme intégré canadien de surveillance de la résistance aux antimicrobiens (PICRA) et de FoodNet Canada. Six groupes d’échantillons amalgamés de matières fécales ont été récoltés dans les fermes participantes, et un questionnaire relevant les pratiques de gestion à la ferme et les mesures de biosécurité a été complété. Les échantillons ont été analysés au moyen d’une méthode validée de réaction d’amplification en chaîne par la polymérase en temps réel (RT-PCR). Des prédicteurs de détection de l’ARN viral sur la ferme ont été modélisés à l’aide de régressions logistiques et de régressions logistiques exactes. Soixante-douze troupeaux ont été échantillonnés : 51 troupeaux du programme CRIPA (15 troupeaux échantillonnés deux fois) et 21 troupeau du programme FoodNet Canada (un troupeau échantillonné deux fois). Le VHE a été détecté dans 30/88 fermes [34,1 % (IC 95 % : 25,0 %, 44,5 %)], CEP dans 18 [20,5 % (IC 95 % : 13,4 %, 30,0 %)], et RV dans 6 fermes [6,8 % (IC 95 % : 3,2 %, 14,1 %)]. La prévalence des virus dans les fermes variait selon la province et la plate-forme d’échantillonnage. Une douche obligatoire avant l’entrée dans la porcherie et le fait de fournir des bottes aux visiteurs s’avéraient des prédicteurs significatifs (P < 0,05) pour la détection du VHE et du CEP, respectivement, dans une analyse par régression logistique mixte à effet fixe unique. Ceci contrastait avec le fait que toutes les fermes positives pour RV fournissaient des bottes et des couvre-tout, et 5 des 6 fermes exigeaient une douche à l’entrée. Nous émettons l’hypothèse que ces mesures de biosécurité ont retardé l’âge moyen d’une infection par le RV, ce qui résultait en une association entre la détection de RV et les animaux en finition. L’acquisition de porcs en croissance de sources multiples était constamment associée avec une probabilité plus grande de détecter chaque virus.

(Traduit par Docteur Serge Messier)

Department of Population Medicine, University of Guelph, Guelph, Ontario N1G 2W1 (Wilhelm, Pearl, Friendship, Rajic, McEwen); Agriculture and Agri-Food Canada, Food Research and Development Centre, 3600 Casavant Blvd. West, St-Hyacinthe, Quebec J2S 8E3 (Leblanc, Houde); Laboratory for Foodborne Zoonoses, Public Health Agency of Canada, 160 Research Lane, Suite 103, Guelph, Ontario N1G 5B2 (Leger, Deckert); Laboratory for Foodborne Zoonoses, Public Health Agency of Canada, Western College of Veterinary Medicine, 52 Campus Drive, Saskatoon, Saskatchewan S7K 3J8 (Gow).

Address all correspondence to Dr. Barbara Wilhelm; telephone: 780-853-7820; fax: 780-853-6682; e-mail: [email protected]

Received July 9, 2015. Accepted October 5, 2015.


96 The Canadian Journal of Veterinary Research 2000;64:0–00

I n t r o d u c t i o nThe RNA viruses hepatitis E virus (HEV), norovirus (NoV), and

rotavirus (RV), have all been hypothesized over the past decade to infect humans zoonotically (1,2,3). A recent scoping review identified a small number of zoonotic cases [HEV n = 3; NoV n = 0; RV n = 40 (zoonoses n = 3; human-animal re-assortants n = 37)] categorized as “likely” zoonoses (4). Several researchers have assayed Canadian pigs for detection of HEV: a survey of 70 Quebec farms reported 34% categorized as HEV-infected (5); the prevalence of HEV RNA shedding in 51 close-to-market pigs on one infected farm in Québec was estimated at 42.2% (6). Hepatitis E virus isolates obtained from 2 locally acquired Canadian Hepatitis E cases in the province of Quebec demonstrated close (. 97% nt) sequence identity between case isolates and those collected from pigs in the same province over part of the ORF2 gene (7). Canadian pig farms have been sampled for NoV, which was detected in 30 of 120 samples from 10 Ontario farms (8), and in Quebec, in which NoV RNA was detected in samples from 4 of 20 farms (9). Rotavirus RNA was detected on 9 of 10 Ontario pig farms (10) and more recently in 9 of 10 Quebec finisher farms (11). A recent Canadian study reported detection of HEV on retail pork livers (12). However, a Canadian national survey of finisher pigs to estimate the prevalence of these viruses has not been conducted to date. Our study objectives, therefore, were to estimate farm-level prevalence of HEV, porcine enteric calicivirus (PEC), and RV in Canadian finisher pigs, and to investigate potential associations between farm level detection and farm management practices.

M a t e r i a l s a n d m e t h o d s

Recruitment and sample collectionExpected farm-level and individual-level prevalence of HEV in

finisher pigs were estimated by a meta-analysis (MA) of 12 pub-lished North American investigations of HEV detection in fecal samples in pigs aged 4 to 6 mo using the Comprehensive Meta-analysis 2 software (Biostat; Englewood, New Jersey, USA) and the method of moments (13). Hepatitis E virus was used in preference over PEC and RV for sample size calculations, as this was the virus for which we were able to obtain the most comprehensive dataset. Heterogeneity, or across study variation in viral prevalence, was investigated by calculation of Higgins’ I2 (14) and T2, an estimate of 2 or true variance in prevalence across studies (15). Sample size calculations to estimate the number of pooled fecal samples to detect HEV RNA on-farm, using the MA summary estimates of within-farm and farm-level prevalence (desired precision 1/2 10%), were conducted in EpiTools (16).

Two sampling platforms were used to recruit pig farms: the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) sampling platform for finisher pigs (17), and the FoodNet Canada (FC) sampling platform (8,10,18). The CIPARS maintains a national network of participating veterinarians, each of whom invite client farms to participate in the CIPARS program by completing annual questionnaires regarding management prac-tices and herd performance, and allowing collection of pooled fecal

samples from finisher pigs (17). For this project, each CIPARS veteri-narian was sent an electronic invitation to participate. Those agreeing received a package containing invitation letters for the clients they recruited, and a consent form for the participating farmers. During the farm visit, the CIPARS farm program questionnaire capturing herd-level data on demographics, current management practices, biosecurity status, pig inventory, and herd performance, was com-pleted by the producer with the help of the herd veterinarian. Data volunteered by the farmer were then validated by the veterinarian and entered on the questionnaire. Missing or incomplete data, or those entries falling outside pre-set logical limits, were further investigated by a telephone call or e-mail to the veterinarian when completed questionnaires were received by CIPARS.

Similarly, during a routine visit for FC sample collection, each pig farm participating in the FC sentinel pig farm program was invited to participate. The FC farms were a subset of 100 farms located in a single province, originally selected as a random sample of 5000 pig farms, stratified by size (18). During the farm visit, a question-naire identical to the CIPARS farm program questionnaire with the exception of the CIPARS antimicrobial use questions, which were not administered to the FC farmers was completed by the producer with the help of the FC sample collection technician.

All fecal samples and questionnaires used farm herd codes as identifiers; the identities of the participating farms were known only to the CIPARS herd veterinarians or the FC program technician. This project received approval from the Research Ethics Board of the University of Guelph.

All sample collection, including those samples collected from FC farms, followed the CIPARS sampling protocol. Briefly, 5 fresh fecal samples were collected into one specimen jar and mixed with a stir stick. One jar was collected from each of 6 close-to-market pens, and shipped by courier to the Public Health Agency of Canada (PHAC) diagnostic laboratory in St. Hyacinthe, Quebec. Samples were then divided, and part of each pooled fecal sample originating from a farm participating in this project was forwarded to the Agriculture and Agri-Food Canada laboratory (AAFC) in St. Hyacinthe, where they were held frozen at 280°C prior to batch processing and test-ing. Samples from FoodNet Canada farms were stored in a freezer at 280°C at the University of Guelph until completion of sampling, when they were forwarded by courier to AAFC St. Hyacinthe.

Viral RNA extraction from fecesFecal samples were thawed and a 1 g portion of sample was fur-

ther diluted 1:5 (p/v) in PBS pH 7.4 (Life Technologies, Burlington, Ontario). After vigorous shaking, mixtures were centrifuged at 4000 3 g for 20 min and supernatants were considered as viral suspensions. A volume of 13 mL of SDS 10%, 0.69 mL of protein-ase K (20 mg/mL) and 4.8 3 103 PFU of feline calicivirus used as sample process control were added to 130 mL of viral suspension. The mixture was incubated at 37°C for 1 h. The RNA was extracted from 140 mL of the treated viral suspension with QIAampViral RNA mini (Qiagen, Toronto, Ontario) and the robotic workstation system QIAcube for automated sample purification of RNA; the QIAcube manufacturer’s protocol was used. The extracted RNA was recov-ered in 60 mL volume of AVE solution and frozen at 280°C until further use.



Table I. Description of variables and participating farms

CIPARS FCVariable Categories Number of samples (%) Number of samples (%)General and Farm Descriptors Herd veterinarian Unique identifier n = 17 veterinarians N = 1 technician for each veterinarian

Herd identifier n = 51 herds N = 21 herds

Number of days sampled 1 = 1 day 1 = 36 herds 1 = 20 herds 2 = 2 days 2 = 15 herds 2 = 1 herd

Province Province A 108 samples, 18 herds Province B 72 samples, 12 herds Province C 114 samples, 19 herds 132 samples, 22 herds Province D 102 samples, 17 herds

Production system used Continuous 192 (48.5%) 96 (72.8%) All-in-all-out 204 (51.5%) 18 (13.6%) Missing observations 18 (13.6%)

Month collected January 18 0 February 6 0 March 48 0 April 12 0 May 12 0 June 12 0 July 18 0 August 48 0 September 42 36 October 36 30 November 96 54 December 48 12

Season Winter 72 (18.2%) 0 (0%) Spring 36 (9.1%) 0 (0%) Summer 108 (27.3%) 36 (27.3%) Fall 180 (45.5%) 96 (72.7%)

Finishers onsite 0–2000 240 (60.6%) 120 (90.9%) 2001–4000 150 (37.9%) 12 (9.1%) $ 4000 6 (1.5%) 0 (0%) Mean (SD) Mean (SD)

Finishers onsite, 1706 (SD 1172) 810 (SD 1175) numerical value

Biosecurity predictors Multiple source No 342 (86.4%) 114 (86.4%) Yes 54 (13.6%) 6 (54.5%) Not captured 0 (0%) 12 (9.1%)

Boots provided No 66 (16.7%) 6 (4.5%) Yes 330 (72.3%) 114 (86.4%) Not captured 0 (0%) 12 (9.1%)

Coveralls provided No 72 (18.2%) 48 (36.4%) Yes 324 (81.8%) 66 (16.7%) Not captured 0 (0%) 18 (13.6%)



Viral RNA detectionDetection of HEV, PEC, and RV was done using the quantitative

real-time polymerase chain reaction (qRT-PCR) detection systems previously described (12) adapted in Brilliant II RT-PCR Core reagent kit, 1-Step (Agilent Technologies Canada, Mississauga, Ontario). Briefly, a sample of 2.5 mL of RNA template was used in a final volume of 25 mL of core RT-PCR buffer containing 2.5 mL of MgCl2, 1.0 mL of dNTP, 1.25 mL of bovine serum albumin at 20 mg/mL, each of the forward and reverse primers and the TaqMan probe, 1 mL of the reference dye diluted 1:500, 1 mL of reverse transcriptase diluted 1:10, and 1.25U of SureStart TaqDNA polymerase (Qiagen). Porcine enteric calicivirus, NoV G II, and RV were detected in a monoplex assay and HEV/FCV in duplex assay.

Viral quantificationExtracted nucleic acids showing amplification inhibition in

the first detection step were treated with OneStep PCR Inhibitor Removal kit (Zymo Research, Irvine, California, USA) before retest-ing. Addition of 2 mL of Hepatitis A virus RNA (3.98 3 104 Tissue cul-ture infectious dose) to each assay was used as amplification control. For each detection system, a calibration curve was generated using 10-fold serial dilutions of the plasmid constructs to the correspond-ing virus in a 5 ng/mL salmon sperm solution. Arithmetic mean load was determined using the MX-pro V4.0 software (Stratagene, La Jolla, California, USA).

Data storage, cleaning, and analysisData pertaining to detection of viral RNA in samples, and ques-

tionnaire responses from each participating farm were captured in Excel spreadsheets (Microsoft, Mississauga, Ontario) and forwarded to a central repository. Data were cleaned in Excel and exported to Stata IC 13 (StataCorp, College Station, Texas, USA) for descriptive statistics and modeling. Missing data received their own unique code. However, we found that a small group (n = 3) of FC respon-dents had not answered the entire group of biosecurity questions (Table I). Therefore, we deleted this subset of farms from the “bios-ecurity” variable analyses. We checked the correlation among pre-

dictor variables to avoid issues associated with collinearity. Where collinear variables were eligible for inclusion in the full model, the variable with the most complete set of observations was selected.

In assessing the effect of a predictor variable in univariable analy-sis, we included random intercepts for herd and sampling event using the “xtmelogit” command (19) in Stata 13 to account for the hierarchical data structure. We estimated the proportion of variance occurring at each level of the model, assuming that the lowest level variance on the logit scale was π2/3, based on use of the latent vari-able technique (20). All variables for which P , 0.20 in univariable analysis were considered for inclusion in the multivariable model. However, where full multivariable models failed to converge, pre-cluding multivariable analysis, only single fixed effect mixed models are presented.

For sparse datasets exact logistic regression was employed, with farm-level detection of viral RNA as the outcome of interest, and a farm was categorized “positive” if viral RNA were detected in one or more of 6 pooled samples collected on-farm.

Re s u l t sMeta-analysis of 12 published North American surveys of finisher

swine yielded a summary estimate of within-farm HEV prevalence in finisher pigs of 23.0% [95% confidence interval (95% CI): 13.2%, 35.8%] with Higgins’ I2 value of 89.31% and T2 of 1.18, suggesting marked heterogeneity across studies. Calculation of the number of pooled samples (consisting of 5 samples from individual animals) required to demonstrate freedom from viral presence, using the meta-analysis estimate of 23.0% prevalence on-farm, confirmed that 3 pooled samples would be sufficient to identify freedom from disease. The standard operating procedure of the CIPARS on-farm swine surveillance system (i.e., collection of 6 pooled samples per farm), was adopted for the purposes of this study, on all participat-ing farms including the FC farms, given the heterogeneity in the dataset supporting the MA estimate of prevalence. Meta-analysis of 5 published North American surveys of finisher pigs yielded a summary estimate of farm-level HEV prevalence in finisher pigs of 30.0% (95% CI: 14.7%, 51.6%) with Higgins’ I2 value of 71.9% and T2

Table I. (continued)

Boot dip provided No 288 (72.7%) 96 (72.7%) Yes 108 (27.3%) 12 (9.1%) Not captured 0 (0%) 24 (18.2%)

Biosecurity sign No 78 (19.7%) 0 (0%) Yes 318 (80.3%) 114 (86.4%) Not captured 0 (0%) 18 (13.6%)

Shower-in No 216 (54.5%) 90 (68.2%) Yes 180 (45.5%) 24 (18.2%) Not captured 0 (0%) 18 (13.6%)

Downtime required No 120 (30.3%) 96 (72.7%) Yes 276 (69.7%) 12 (9.1%) Not captured 0 (0%) 24 (18.2%)CIPARS — Canadian Integrated Program for Antimicrobial Resistance Surveillance; FC — FoodNet Canada.



of 0.92, suggesting marked heterogeneity across studies. Calculation of the number of farms required to estimate a prevalence of 30% with desired precision yielded a target of 75 farms participating.

Farm recruitment and sample collectionAll 26 CIPARS veterinarians were approached to participate

in this study in the summer of 2011. Nine veterinarians enrolled and they recruited 17 finisher herds in total. In the fall of 2011 the FC farms were invited by the FC program technician to participate, and 20 of 28 enrolled with one farm being tested twice. At the end of 2011, CIPARS veterinarians were again invited to participate. In 2012, 15 veterinarians participated, enrolling 34 new farms, and re-testing 15 farms.

A total of 528 samples from 72 herds from 4 provinces (A, B, C, and D) with 6 pooled samples per herd were collected over a 17-month period and stored at 280°C until further use. A summary of the characteristics of participating farms in both sampling plat-forms is presented in Table I. Overall, relative to CIPARS farms, FC farms fed fewer finisher pigs; all were located in province C, and most practiced continuous pig flow (72.7%) as opposed to all-in-all-out flow. Compared with CIPARS farms, more FC farms provided

boots (86.4% versus 72.3%), but fewer provided coveralls prior to entry (16.7% versus 81.8%); fewer required shower-in (18.2% versus 45.5%), and fewer required downtime after visiting another pig farm, prior to entry (9.1% versus 69.7%). Sampling was only conducted September to December, for FC farms. “Retest” was included as a categorical variable in the dataset as 16 farms were sampled twice.

Data were missing from a sub-group of FC farms for the follow-ing biosecurity variables: provision of boots or coveralls, use of a boot dip, posting a biosecurity sign, requirement for shower-in, or downtime, and obtaining pigs from multiple sources.

Hepatitis E virusHepatitis E virus RNA was detected from one or more samples

collected from 19 of 66 CIPARS farms (estimated herd-level preva-lence 28.8%; 95% CI: 19.3%, 40.6%), and 11 of 22 FC farms (estimated herd-level prevalence 50.0%, 95% CI: 30.7%, 69.3%). Hepatitis E virus RNA was detected from 35/396 individual pooled samples col-lected from CIPARS farms (estimated sample-level prevalence 8.8%; 95% CI: 6.4%, 12.2%), and 38/132 pooled samples from FC farms (estimated sample-level prevalence 28.8%; 95% CI: 21.8%, 37.0%). Hepatitis E virus prevalence by province is presented in Table II.

Table II. Prevalence and mean load of hepatitis E virus, porcine enteric calicivirus, and rotavirus in Canadian finisher pigs

Hepatitis E virus Porcine enteric calicivirus Rotavirus Farms Samples Farms Samples Farms Samples Number Number Number Number Number Number positive positive positive positive positive positive /number /number /number /number /number /number sampled (%) sampled (%) sampled (%) sampled (%) sampled (%) sampled (%)National 30/88 (34.1%) 73/528 (13.8%) 18/88 (20.5%) 38/528 (7.2%) 6/88 (6.8%) 8/528 (1.5%) Province A 3/18 (16.7%) 3/108 (2.8%) 2/18 (11.1%) 3/108 (2.8%) 2/18 (11.1%) 2/108 (1.9%) Province B 6/12 (50.0%) 14/72 (19.4%) 3/12 (25.0%) 7/72 (9.7%) 1/12 (8.3%) 1/72 (1.4%) Province C 14/41 (34.1%) 42/246 (17.1%) 6/41 (14.6%) 13/246 (5.3%) 3/41 (14.3%) 5/246 (2.0%) Province D 7/17 (41.2%) 14/102 (13.7%) 7/17 (41.2%) 15/102 (14.7%) 0/17 0/102

Month January 1/3 (33.3%) 1/18 (5.6%) 1/3 (33.3%) 1/18 (5.6%) 0/3 (0%) 0/18 (0%) February 0/1 (0%) 0/6 0/1 (0%) 0/6 (0%) 0/1 (0%) 0/6 (0%) March 2/8 (25.0%) 7/48 (14.6%) 1/8 (12.5%) 1/48 (2.1%) 0/8 (0%) 0/48 (0%) April 1/2 (50.0%) 2/12 (16.7%) 0/2 (0%) 0/12 (0%) 0/2 (0%) 0/12 (0%) May 0/2 (0%) 0/12 0/2 (0%) 0/12 (0%) 0/25 (0%) 0/12 (0%) June 1/2 (50.0%) 2/12 (16.7%) 1/2 (50.0%) 3/12 (25.0%) 0/2 (0%) 0/12 (0%) July 0/3 (0%) 0/18 (0%) 1/3 (33.3%) 6/18 (33.3%) 1/3 (33%) 1/18 (6%) August 1/8 (12.5%) 1/48 (2.1%) 1/8 (12.5%) 1/48 (2.1%) 1/8 (12.5%) 1/48 (2.1%) September 3/13 (23.1%) 3/78 (3.8%) 1/13 (7.7%) 2/78 (2.6%) 1/13 (7.7%) 2/78 (2.6%) October 8/11 (72.7%) 20/66 (30.3%) 2/11 (18.2%) 3/66 (4.5%) 1/11 (9.1%) 1/66 (1.5%) November 8/25 (32.0%) 26/150 (17.3%) 5/25 (20.0%) 10/150 (6.7%) 2/25 (8.0%) 3/150 (2.0%) December 5/10 (50.0%) 11/60 (18.3%) 5/10 (50.0%) 11/60 (18.3%) 0/10 (0%) 0/60 (0%)

GM/g (all samples) 619 401 gc/ga 31 644 gc/g 275 277 gc/gSD 5 819 952 gc/g 442 402 gc/g 6 051 227 gc/g

GM/g (‘positive’ samples) 4 360 583 gc/g 388 556 gc/g 18 200 000 gc/gSD 15 000 000 gc/g 1 521 130 gc/g 48 800 000 gc/ga gc/g = genomic copies per gram of sample.GM — geometric mean; HEV — hepatitis E virus; PEC — porcine enteric calicivirus; RV — rotavirus; SD — standard deviation.



Table III. Single fixed effect logistic regression modeling predictors of viral detection in Canadian finisher pigs: general and farm descriptors

Hepatitis E virus HEV Porcine enteric calicivirus PEC Rotavirus RV ORa OR Model OR OR Model ORb OR ModelVariable (95% CI) P-value P-value (95% CI) P-value P-value (95% CI) P-value P-valueSampling platform 0.01 0.29 0.0007 CIPARS Referent Referent Referent FC 14.58 (1.82, 0.01 0.12 (0.003, 0.29 0.05c (0, 0.24) 0.0001 116.93) 5.89)

Province 0.20 0.56 0.01 A Referent Referent Referent B 16.65 (0.62, 0.09 3.50 (0.06, 0.54 0.73 (0.31, 0.74 447.63) 188.94) 1.34) C 8.92 (0.73, 0.09 1.58 (0.06, 0.79 0.63 (0.28, 0.33 109.04) 44.15) 1.50) D 10.82 (0.50, 0.26 10.82 (0.24, 0.22 0.06c (0.35) 0.0005 232.98) 485.40)

Year of sampling 0.08 0.58 , 0.0001 Year 1 Referent Referent Referent Year 2 0.19 (0.03, 0.08 1.95 (0.18, 0.58 48.35c (8.26, , 0.0001 1.19) 21.05) Inf)

Re-test 0.67 0.15 0.002 No Referent Referent Referent Yes 2.06 (0.07, 0.67 8.45 (0.45, 0.15 0.08 (0, 0.46) 0.001 58.72) 158.54)

Season 0.07 0.93 0.003 Winter Referent Referent Referent Spring 1.58 (0.03, 0.82 1.42 (0.005, 0.90 1.0 (0, Inf) 74.37) 402.51) Summer 0.31 (0.02, 0.42 1.05 (0.02, 0.98 14.34 (2.41, 0.0009 5.47) 70.60) Inf) Fall 5.45 (0.42, 0.20 2.37 (0.05, 0.65 7.04 (1.19, 0.03 72.20) 102.61) Inf)

Production type 0.34 No convergenced 0.01 Referent Referent Referent

Continuous All-in-all-out 2.29 (.31, 0.42 2.87 (1.30, 0.007 16.80) 6.26) Not captured 184.26 (0.09, 0.18 1.41 , 0.0001 3.87e 1 5) (0, 9.48)

Finishers on-farm 0.99 (0.99, 0.07 0.07 1.00 (0.99, 0.80 0.78 No convergence 1.00) 1.00)

Farm size No convergence No convergence 0.0003

0 to 2000 finishers Referent Referent Referent

2 to 4000 finishers 0.58 (0.19, 0.34 1.51)

. 4000 finishers 6.93 (1.96, 0.003 22.18) a Mixed logistic regression with random intercepts for “farm” and “sampling event” with “sample positive” as the outcome measure.b Exact logistic regression with “farm positive” as the outcome measure.c Mean unbiased estimate. dMaximum memory allocated in Stata = 2 Megabytes.CIPARS — Canadian Integrated Program for Antimicrobial Resistance Surveillance; FC — FoodNet Canada; HEV — hepatitis E virus; Inf — infinity; OR — odds ratio; PEC — porcine enteric calicivirus; RV — rotavirus; 95% CI — 95% confidence interval.



Mean viral load in positive samples was 4 360 583 genomic copies (gc)/g (SD = 15 000 000 gc/g).

We performed mixed logistic regression modeling HEV RNA detection in a sample as the outcome measure, with a single fixed effect and random intercepts for farm and sampling event (Tables III, IV). The following predictors were eligible for inclusion in the full multivariable model (i.e., P , 0.20): sampling platform — with FC farms having significantly (P , 0.05) greater odds of HEV RNA detection on-farm relative to CIPARS farms; province — with province A farms having reduced odds of HEV detection relative to the other provinces; year of sampling, with farms sampled in year 2 having reduced odds of HEV detection relative to year 1 sampling; season — with samples collected in spring or fall having greater odds of HEV detection relative to winter; and the number of finisher pigs on-farm — with larger farms having reduced odds of HEV detection. Requiring shower-in was associated with significantly (P , 0.05) reduced odds of HEV detection.

In an intercept-only model, with random intercepts for herd and sampling event, 45.1% of the variance occurred at the individual sample level (i.e., the lowest hierarchical level of the data), with 32.6% of the variance occurring at the herd level, and 22.3% at the sampling event. Similar proportions were observed for each single fixed effect model.

The full multivariable model failed to converge, as did most models incorporating a subset of the eligible variables, precluding investigation of interaction or confounding.

Porcine enteric calicivirusPorcine enteric calicivirus RNA was detected from one or more

samples collected from 17 of 66 CIPARS farms (estimated farm-level prevalence 25.8%; 95% CI: 16.8%, 37.4%), and 1 of 22 FC farms (farm-level prevalence 4.6%; 95% CI: 0.80%, 21.8%). Porcine enteric calici-virus RNA was detected from 36 of 396 individual pooled samples collected from CIPARS farms (estimated sample-level prevalence

Table IV. Single fixed effect logistic regression modeling predictors of viral detection in Canadian finisher pigs: biosecurity practicesa

Hepatitis E virus Porcine enteric calicivirus Rotavirus ORb OR Model ORb OR Model ORc OR ModelVariable (95% CI) P-value P-value (95% CI) P-value P-value (95% CI) P-value P-valueFinishers from 0.57 0.77 0.0003 multiple sources No Referent 0.57 Referent 0.77 Referent 0.0006 Yes 2.14 1.48 4.42 (0.16, 29.34) (0.10, 21.63) (1.89, 9.89)

Boots provided 0.63 0.01 No Referent 0.63 Referent 0.04 Referent 0.01 Yes 1.82 0.08 0.04 9.14d (0.16, 21.02) (0.007, 0.83) (1.64, Inf)

Coveralls provided 0.62 0.60 0.003 No Referent Referent Referent 0.0001 Yes 0.38 0.44 0.26 0.31 17.32d (0.03, 4.51) (0.02, 3.51) (3.07, Inf)

Shower-in 0.05 0.67 , 0.0001 No Referent Referent Referent Yes 0.16 0.05 0.68 0.70 8.59d , 0.0001 (0.03, 0.96) (0.10, 4.26) (3.43, 25.74)

Biosecurity sign 0.74 0.20 0.006 No Referent 0.74 Referent 0.20 Referent 0.004 Yes 1.51 0.20 10.06d (0.13, 17.44) (0.02, 2.22) (1.77, Inf)

Downtime required 0.57 0.44 0.16 No Referent 0.57 Referent 0.44 Referent 0.22 Yes 1.83 0.43 0.59d (0.23, 14.22) (0.05, 3.56) (0.27, 1.34) a The three FoodNet Canada farms for which data for all biosecurity predictors were missing, were dropped from the dataset for these analyses.b Mixed logistic regression with random intercepts for “farm” and “sampling event” with “sample positive” as the outcome measure.c Exact logistic regression with “farm positive” as the outcome measure. d Mean biased estimate.HEV — hepatitis E virus; Inf — infinity; PEC — porcine enteric calicivirus; RV — rotavirus; 95% CI — 95% confidence interval.



9.1%; 95% CI: 6.6%, 12.3%), and 2 of 132 pooled samples from FC farms (estimated sample level prevalence 1.5%; 95% CI: 0.4%, 5.9%). Porcine enteric calicivirus prevalence by province is presented in Table II. Mean viral load in positive samples was 388 556 gc/g (SD = 1 521 130 gc/g). In mixed logistic regression with single fixed effect and random intercepts for farm and sampling event, providing boots on entry to the unit was associated with significantly (P , 0.05) reduced odds of PEC detection. Samples collected during re-test of a farm were associated with increased (P = 0.15) odds of PEC detection. However, a multivariable model including both of these predictors failed to converge.

In an intercept-only model, with random intercepts for herd and sampling event, 53.9% of the variance occurred at the individual sample level (i.e., the lowest hierarchical level of the data), with negligible variance occurring at the herd level, and 46.1% at the sampling event. Similar proportions were observed for each single fixed effect model.

RotavirusRotavirus RNA was detected from one or more samples collected

from 6/66 CIPARS farms (estimated farm-level prevalence 9.1%, 95% CI: 4.2%, 18.5%), and 0 of 22 FC farms. Rotavirus RNA was detected from 8 of 396 individual pooled samples collected from CIPARS farms (estimated sample level prevalence 2.0%, 95% CI: 1.0%, 3.9%), and 0 of 132 pooled samples from FC farms. Rotavirus prevalence by province is presented in Table II. Mean viral load in positive samples was 18 200 000 gc/g (SD = 48 800 000 gc/g). In univariable exact logistic regression with “farm positive” as the outcome measure

(Table III), both sampling platform and province were significant (P , 0.05) predictors for RV detection, with farms from province A having greatest odds of RV detection, and FC farms reduced odds of RV detection as compared with CIPARS farms (OR = 0.05, 95% CI: 0, 0.24). Production type was a significant predictor for RV detection, with farms practicing all-in-all-out pig flow having significantly increased odds relative to farms using continuous flow (OR = 2.87, 95% CI: 1.30, 6.26). Farms obtaining grow-finish pigs from multiple sources had greater odds of RV detection (OR = 4.42, 95% CI: 1.89, 9.89) as compared with those only using one source. Provision of boots and coveralls for visitors, requiring a shower prior to enter-ing the unit, and posting a biosecurity sign at the entry to the site, all were significantly (P , 0.05) associated with increased odds of RV detection in finisher pigs.

Overall, FC farms had greater odds of HEV RNA relative to CIPARS farms, and reduced odds of RV RNA detection. Province A had lower odds of HEV relative to the other provinces, and province D had lower odds of RV RNA detection relative to province A.

D i s c u s s i o nThe overall farm-level prevalence of HEV in finishers in our

survey (34.1%, 95% CI: 25.0%, 44.5%) is comparable with published North American estimates ranging from 25% to 68% (6,21). The FC farms tend to be smaller multi-species farms as compared with CIPARS farms (Deckert & Friendship, 2015, University of Guelph, personal communication). Other species, including cats, dogs, cattle, sheep, goats, and rabbits have been reported to be capable

CIPARS 2011Referring veterinarians N = 9Farms invited N = 93Farms enrolled n = 17

AAFC St-HyacintheRT-PCR assays

CIPARSHEV Farms positive n = 19/66 (28.8%) Samples positive n = 35/396 (9%)

PEC Farms n = 17/66 (25.8%) Samples n = 36/396 (9.1%)

RV Farms n = 6/66 (9.1%) Samples n = 8/396 (2.0%)

FCHEV Farms n = 11/22 (50%) Samples n = 38/132 (28.8%)

PEC Farms n = 1/22 (5.4%) Samples n = 2/132 (21.5%)

RV Farms n = 0/22 (0%) Samples n = 0/132 (0%)

CIPARS 2012Referring veterinarians N = 15Farms invited N = 87Farms enrolled n = 34Re-tests n = 15

FC 2011Program technician: N = 1Farms invited N = 28Farms enrolled n = 21Re-tests n = 1

Figure 1. Study recruitment and sample collection process.AAFC — Agriculture and Agri-Food Canada laboratory; CIPARS — Canadian Integrate Program for Antimicrobial Resistance Surveillance; FC — FoodNet Canada; HEV — Hepatitis E virus; PEC — Porcine enteric calicivirus; RT-PCR — Real time reverse transcriptase polymerase chain reaction; RV — Rotavirus.



of HEV sero-conversion and/or shedding (3), and it is possible that HEV shedding of other species on-farm may have contributed to the introduction of HEV to the pig herd. The significant association between requiring shower-in and reduced odds of HEV RNA detec-tion on-farm (OR = 0.16, 95% CI: 0.03, 0.96) is consistent with the possibility that asymptomatic human shedders could be a potential exposure source of HEV for pigs, as has been hypothesized for pork (7). Conditional estimates of effect would be required to fur-ther investigate this finding, which was not possible in this dataset.

Overall farm-level prevalence of PEC was 20.5% (95% CI: 13.4%, 30.0%). In comparison, a Dutch study reported PEC RNA detected in 3- to 9-month-old pigs on only 2 of 100 farms sampled (22) and a US investigation detected PEC RNA in 9 of 9 farms sampled (23). The significant association estimated between provision of boots upon entry to the unit, and reduced odds of PEC RNA detection on-farm (OR = 0.08, 95% CI: 0.007, 0.83), is consistent with an agent transmitted via the fecal-oral route.

Rotavirus had the lowest farm-level prevalence of the 3 viruses of interest in this survey. A recent Vietnamese survey reported a farm-level RV prevalence of 74% (77/104) (24). However, with a smaller sample size, an Italian survey reported a numerically lower prevalence (3 of 8 herds) surveyed (25). In comparison with HEV and PEC, RV had a different geographic distribution, with province “A” having greater odds of RV detection on-farm, and no RV detected in province “D” farms.

While few of the predictor variables from this study, with the exception of geographic region, have been investigated in other published research, we generally expected that the “biosecurity” variables (providing boots/boot dip/coveralls, requiring shower-in/downtime) would be associated with reduced odds of detection, and sampling platform and province could be significant predictors, for viral RNA detection on-farm. For example, investigators in France have reported a significant association between lack of hygiene mea-sures such as failure to provide boots, and detection of HEV in the livers of slaughter pigs (26). In our study, obtaining finishers from multiple sources was the one predictor variable consistently associ-ated with increased odds of viral RNA detection. This is similar to other research, which reported that obtaining pigs from more than one source was a significant (P , 0.05) predictor for the presence of Salmonella spp. on-farm (27,28) in cross-sectional studies of 109 and 359 farms, respectively.

However, the measures of association for some predictor vari-ables differed in magnitude and, in some cases, direction, across the 3 viruses studied, although the primary transmission route for all 3 is the fecal-oral route. The varying relationships between predictor variables and outcome across the 3 viruses may reflect differences in viral biology and relative importance of various pos-sible methods of introduction/transmission. On an infected farm, incident HEV infection occurs most frequently in the nursery (21), RV infection occurs most frequently in suckling pigs, and mean age of PEC infection has varied across studies (29,30). In our study, RV detection in finishers was significantly associated with adoption of widely accepted biosecurity measures such as provision of boots and coveralls (Table III). Possibly, given the tendency for RV infection to occur early in the pig’s life, the application of these biosecurity measures may functionally delay the mean age of RV infection. This

could result in biosecurity measures such as provision of boots being significantly associated with RV detection in finishers, particularly in farrow-to-finish herds.

The fact that sampling platform was a significant predictor for HEV and RV RNA was noteworthy. However, it highlights the potential importance of stratified sampling in surveillance programs; some farms (e.g., small scale/mixed species), although perhaps not producing a large proportion of market animals, may be important biologically in pathogen transmission and control.

Our study has several limitations. The farmers who participated in this survey are volunteers, suggesting the potential for selection bias, by functionally using an incomplete definition to define the eligible population (31). There is no national sampling frame of finisher farms with which our study group might be compared with regards to important demographic or management param-eters. However, CIPARS also operates an abattoir-level sampling platform that includes cecal sampling of pigs. The close similarity between CIPARS farm level antimicrobial resistance estimates, and those obtained from the national abattoir component of CIPARS (which randomly samples slaughter pigs and is therefore unaffected by producer or veterinarian willingness to participate), indicates that the convenience sample of farms obtained by the CIPARS on-farm sampling platform is likely representative of Canadian pig production (17). Unfortunately, however, no similar mechanism exists for estimating the similarity of the FC farms to the national herd.

Additionally, participants self-reported farm management and biosecurity practices. A minority (n = 20) of respondents did not complete some survey questions, including those pertaining to farm biosecurity practices, with 3 FC respondents not answering any of these questions. We hypothesize that the missing biosecurity data could be missing not at random, perhaps reflecting respondents’ reluctance to report that fairly widely endorsed biosecurity mea-sures (32) were not practiced. If the 3 farms for which data were missing were, in fact, not using these measures, their inclusion in the analysis would have increased the observed protective effect of these biosecurity measures.

While the diagnostic sensitivity and specificity of the RT-PCR assays used in this study have been investigated in fecal samples collected from other populations (e.g., humans, younger pigs), the diagnostic sensitivity and specificity of these assays in fecal samples from finisher pigs are not known. Additionally, the RV assay targets the NSP3 gene of group A RVs, and its performance in detecting RVs of other serotypes such as group C RVs, which are also reported in finisher pigs (33) is also unknown. A Brazilian survey of diarrheic pigs 1 to 4 weeks old identified 144 fecal samples inconclusively categorized using polyacrylamide gel assay. Of these, 19 and 5 were identified as group B and C RVs, respectively, using group-specific RT-PCR, highlighting both the potential for their detection on-farm, and the requirement for appropriate assays (34).

Biosecurity practices in pig farms, and farmers’ attitudes towards them, have been increasingly investigated in North America and Europe, with sow replacement policy, and protocols around trucking of livestock being consistently recognized as important components of a farm biosecurity protocol (35). These practices were not captured by our questionnaire, which was originally designed to capture data



pertinent to development of antimicrobial resistance. The extent to which missing predictors/confounders may have affected our estimates is therefore unknown.

Our recruitment and ultimately our sample size was limited by both the lack of a complete sampling frame of pig farms in Canada, and the relatively recent swine flu pandemic of 2009, which par-ticipating veterinarians reported decreased farmer willingness to participate in this project. It is possible that a larger sample size, and therefore increased study power, would have resulted in more of the predictor variables being significantly associated with viral detec-tion. Additionally, we sampled the finisher pigs at only one point in their lives. It is possible that some of the predictors investigated (e.g., all-in-all-out pig flow) might be significantly associated with delaying, but not preventing, viral infection. Longitudinal studies would be helpful in understanding viral kinetics, and the effects of predictors (i.e., biosecurity/management practices) on mean age of infection. We identified knowledge gaps in addition to the lack of a Canadian sampling frame. Detection of HEV, PEC, and RV on Canadian finisher farms suggests the potential for occupational exposure to these viruses amongst swine workers. Field surveys and research synthesis have supported the hypothesis that swine workers and veterinarians have increased odds of exposure to HEV (36,37). However, the extent of Canadian illness attributable to occupational exposure (if any) remains unknown. This reflects both the challenges in data collection (HEV infection is not federally notifiable in Canada) and possibly also incomplete understanding of the causal complement, or combination of risk factors, required to produce clinical disease after viral exposure.

In conclusion, all 3 viruses were detected in finishing pigs, sug-gesting occupational exposure to feeder pigs is a potential source of human exposure to these viruses. Overall, HEV had the greatest farm-level prevalence of the 3 viruses studied; sampling platform was a significant predictor of HEV and RV viral RNA detection in finishers in this dataset. The significance, effectiveness, and direction of effect of the biosecurity practices captured (provision of boots, or coveralls, on entry; requiring shower-in prior to entry; use of boot dip; posting biosecurity sign; requiring downtime prior to entry) varied across the 3 viruses studied. The management procedure consistently associated with greater odds of viral RNA detection for all 3 viruses, was obtaining feeder pigs from multiple sources.

A c k n o w l e d g m e n t sWe gratefully acknowledge the assistance of Bryan Bloomfield,

Danielle Daignault, Louise Bellai, Karen Richardson, the CIPARS vet-erinarians, and the farmers participating in this study, as well as the funding support of OMAFRA — University of Guelph Partnership Research Program (Grant # 027118).

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Article


I n t r o d u c t i o nStreptococcus suis infection is one of most important diseases in

the swine industry worldwide. Streptococcus suis is also considered an emerging zoonotic pathogen that causes septicemia and menin-gitis in humans (1). From the 35 serotypes described thus far, S. suis serotype 2 is most commonly associated with disease in both pigs and humans in most countries (2).

The most common virulence markers associated with serotype 2 strains are suilysin (SLY; sly), muramidase-released protein (MRP; mrp) and extracellular factor (EF; epf) (3,4). A correlation between the mrp1/epf1/sly1 genotype and clinical human and porcine isolates has already been suggested (5,6). Although the mrp1/epf1/sly1 genotype is the most prevalent worldwide, the mrp2/epf2 genotype appears to be increasing among human and porcine S. suis isolates in Asia, Europe, and North America (2).

Genetic diversity of Streptococcus suis serotype 2 isolated from pigs in Brazil

Daniela Sabatini Doto, Luisa Zanolli Moreno, Franco Ferraro Calderaro, Carlos Emilio Cabrera Matajira, Vasco Tulio de Moura Gomes, Thais Sebastiana Porfida Ferreira, Renan Elias Mesquita,

Jorge Timenetsky, Marcelo Gottschalk, Andrea Micke Moreno

A b s t r a c tStreptococcus suis is an emerging zoonotic pathogen that causes septicemia, meningitis, arthritis, and pneumonia in swine and humans. The present study aimed to characterize the genetic diversity of S. suis serotype 2 isolated from pigs showing signs of illness in Brazil using pulsed-field gel electrophoresis (PFGE), single-enzyme amplified fragment length polymorphism (SE-AFLP), and profiling of virulence-associated markers. A total of 110 isolates were studied, 62.7% of which were isolated from the central nervous system and 19.1% from the respiratory tract. Eight genotypes were obtained from the combination of virulence genes, with 43.6% and 5.5% frequencies for the mrp1/epf1/sly1 and mrp2/epf2/sly2 genotypes, respectively. The presence of isolates with epf gene variation with higher molecular weight also appears to be a characteristic of Brazilian S. suis serotype 2. The PFGE and SE-AFLP were able to type all isolates and, although they presented a slight tendency to cluster according to state and year of isolation, it was also evident the grouping of different herds in the same PFGE subtype and the existence of isolates originated from the same herd classified into distinct subtypes. No further correlation between the isolation sites and mrp/epf/sly genotypes was observed.

R é s u m éStreptococcus suis est un agent pathogène zoonotique en émergence responsable de septicémies, des méningites, d’arthrites, et de pneumonies chez les porcs et les humains. La présente étude visait à caractériser la diversité génétique de souches de S. suis sérotype 2 isolées au Brésil de porcs montrant des signes de maladie à l’aide des techniques suivantes : électrophorèse en champs pulsés (PFGE), polymorphisme des fragments amplifiés par un enzyme unique (SE-AFLP), et profilage des marqueurs de virulence. Un total de 110 isolats a été étudié, 62,7 % isolats provenant du système nerveux central et 19,1 % du tractus respiratoire. Huit génotypes furent obtenus de la combinaison de gènes de virulence, avec des fréquences de 43,6 % et 5,5 % pour les génotypes mrp1/epf1/sly1 et mrp2/epf2/sly2, respectivement. La présence d’isolats avec la variation du gène epf et un poids moléculaire plus élevé semble être également une caractéristique de S. suis sérotype 2 d’origine brésilienne. Les méthodes PFGE et SE-AFLP ont été en mesure de permettre le typage de tous les isolats et, bien qu’il y avait une légère tendance à se regrouper selon l’état et l’année d’isolement, il était également évident qu’il y avait du regroupement d’isolats provenant de différents troupeaux dans le même sous-type de PFGE et de l’existence d’isolats provenant du même troupeau classifiés dans des sous-types différents. Aucune autre corrélation entre le site d’isolement et les génotypes de mrp/epf/sly ne fut observée.


Laboratório de Sanidade Suína e Virologia, Faculdade de Medicina Veterinária e Zootecnia (Sabatini Doto, Zanolli Moreno, Ferraro Calderaro, Cabrera Matajira, de Moura Gomes, Porfida Ferreira, Mesquita, Micke Moreno); Instituto de Ciências Biomédicas, Departamento de Microbiologia, Universidade de São Paulo, São Paulo, Brazil (Timenetsky); Faculté de Médecine Vétérinaire, Université de Montréal, Québec (Gottschalk).

Address all correspondence to Dr. Moreno; telephone: 155 02 111 3091 1377; fax: 155 02 111 3091 7928; e-mail: [email protected]

Dr. Moreno’s current address is FMVZ/USP, Av. Prof. Dr. Orlando Marques de Paiva, 87, Cidade Universitária, CEP 05508 270, São Paulo/SP, Brazil.

Received July 8, 2015. Accepted October 1, 2015.



Few data are available regarding the epidemiology and molecular characteristics of S. suis serotype 2 isolated from pigs showing signs of illness in South America (7). Therefore, the aim of this study was to characterize the genetic diversity of S. suis serotype 2 isolated from diseased pigs in Brazil by pulsed-field gel electrophoresis (PFGE), single-enzyme amplified fragment length polymorphism (SE-AFLP) and profiling of virulence-associated genes.


Bacterial strains and phenotypic identificationA total of 133 S. suis strains were isolated from 103 pigs with

clinical signs of meningitis, septicemia, arthritis, and/or pneu-monia that originated from 88 herds in different Brazilian states (São Paulo, Santa Catarina, Paraná, Pernambuco Bahia, Minas Gerais, and Rio Grande do Sul) during the period of 2001 to 2003. Animal samples were plated onto Columbia blood agar base containing 5% sheep blood supplemented with SR-126 (Oxoid, Cambridge, United Kingdom). The presumptive bacteriological identification was based on the presence of a-hemolysis on blood agar; the pro-duction of amylase; the absence of growth in brain-heart-infusion (BHI) medium supplemented with 6.5% NaCl, inulin, and trehalose fermentation; and a lack of mannitol and sorbitol fermentation, as well as a negative Voges-Proskauer test. From each animal examined, 1 to 6 different S. suis colonies were selected isolated from different body sites (CNS, lungs, thoracic cavity, and peritoneum).

Species identification and profiling of virulence-associated markers

Genomic DNA was purified according to Boom et al (8) proto-col, and S. suis identification was confirmed through gdh ampli-fication according to the primers described by Okwumabua et al (9). The epf and sly virulence genes were amplified through con-ventional PCR as previously described by Wisselink et al (10) and King et al (11), respectively. For partial amplification of the mrp gene, primers mrp-Fw (TGCTTCATCAGAACCAAC) and mrp-Rv (GAGAATTTCAATGCTCCAG) (binding sites 311-1189 bp; accession no. X64450.1) were applied (M. Gottschalk, University of Montreal, personal communication). Amplicons were stained using BlueGreen (LGC Biotecnologia, Sao Paulo, Brazil) and separated by electrophoresis using 1.5% agarose gels and 100 bp DNA lad-der (New England BioLabs, Ipswich, Massachusetts, USA) for the amplicons size estimation.

SerotypingSerotype 2 was identified by using the coagglutination test as

described by Gottschalk, et al (12).

Molecular typing by SE-AFLP and PFGEIsolates were also genotyped through SE-AFLP and PFGE. The

SE-AFLP was done according to McLauchlin et al (13) protocol. DNA fragments were detected by electrophoresis at 24 V for 26 h using a 2% agarose gel stained using BlueGreen (LGC Biotecnologia). The PFGE culture conditions, plug preparation, and DNA extrac-tion followed the protocol described previously by Vela et al (14).

The restriction enzyme SmaI (New England BioLabs) was used for DNA digestion at 30°C for 24 h. Electrophoresis was done using 1% SeaKem Gold agarose (Cambrex Bio Science Rockland, New Jersey, USA) and a CHEF-DR III System (Bio-Rad Laboratories, California, USA) with 0.53 TBE at 14°C. DNA fragments were separated at 6 V/cm at a 120° fixed angle, with pulse times from 0.5 to 40 s for 18 h. Gels were stained with 13 SYBR Safe; (Invitrogen Corporation, Carlsbad, California, USA) for 30 min and imaged under UV trans-illumination. Lambda DNA-PFGE marker (New England BioLabs) was used for fragment size determination.

The SE-AFLP and PFGE fingerprint patterns were analyzed by a comprehensive pairwise comparison of restriction fragment sizes using the Dice coefficient. The mean values obtained from the Dice coefficient were applied in UPGMA (BioNumerics 7.5; Applied Maths NV, Sint-Martens-Latem, Belgium) to generate dendrograms. A similarity cut-off value of 90% was used to analyze SE-AFLP clusters. For the PFGE analysis, the isolates were consid-ered in different pulsotypes when they differed by $ 4 bands and assigned into subtypes when they differed by 1 to 4 bands (15). Discriminatory indexes were calculated according to Hunter and Gaston (16).

Re s u l t s

Virulence-associated gene profilingOne hundred and ten of the 133 S. suis strains isolated in this

study were characterized as serotype 2. Specifically, 62.7% were isolated from the central nervous system; 19.1% from the respira-tory tract; and 18.2% from joint, peritoneum, pericardium/heart, and blood samples. From the 21 S. suis serotype 2 strains isolated from the respiratory tract, only 2 originated from nasal swabs and, therefore, were considered as non-invasive isolates. The remaining strains were obtained from lungs and thoracic cavity samples of animals with pneumonia.

The presence of mrp, epf, and sly genes was characterized by the visualization of fragments of 879, 626, and 1818 bp, respectively. The epf gene can present fragment size variations (EF variation — epfv) with possible amplicons of 1278, 1505, 2313, 2537, and 2993 bp. Of

Table I. Classification of Streptococcus suis serotype 2 isolates among virulence genotypes and isolation sites

Isolation site CNS RT OthersGenotype N (%) N (%) N (%) N (%)mrp1 epf2 sly2 1 (0.9) 0 1 (4.8) 0mrp1 epf2 sly1 1 (0.9) 0 1 (4.8) 0mrp2 epf1 sly1 4 (3.6) 3 (4.4) 0 1 (5.0)mrp2 epf2 sly2 6 (5.5) 4 (5.8) 1 (4.8) 1 (5.0)mrp2 epf1 sly2 14 (12.7) 10 (14.5) 3 (14.2) 1 (5.0)mrp1 epfv sly1 17 (15.5) 12 (17.4) 0 5 (25.0)mrp1 epf1 sly2 19 (17.3) 7 (10.1) 6 (28.6) 6 (30.0)mrp1 epf1 sly1 48 (43.6) 33 (47.8) 9 (42.8) 6 (30.0)CNS — central nervous system; RT — respiratory tract; Others — joints, blood, peritoneum, heart or pericardium; epfv — EF variation.



Figure 1. Pulsed-field gel electrophoresis (PFGE) dendrogram showing the relationship among Streptococcus suis serotype 2 pulsotypes.



Figure 2. Dendrogram showing the relationship among the Streptococcus suis serotype 2 isolates single-enzyme amplified fragment length polymor-phism (SE-AFLP) patterns.



the isolates, 15.5% presented EF variation (higher molecular weight). Eight genotypes were obtained from the combined presence of mrp, epf, and sly genes (Table I). Only 5.5% of serotype 2 isolates were negative for all of the studied genes.

Molecular typingThe PFGE analysis resulted in 14 pulsotypes (A — N), which were

divided into 2 main clusters with over 55% of genetic similarity (Figure 1). One of these clusters comprised exclusively pulsotype A, which consists of 86 isolates that were further classified into sub-types A1 to A31. The other main cluster comprised the remaining 23 S. suis isolates divided among 13 pulsotypes and respective subtypes. Among the A1 to A31 subtypes, there is a tendency to cluster accord-ing to isolates state and year of origin, although it is also evident that some isolates from different herds were grouped into the same subtype (e.g., A3 and A12) and that some isolates from the same herd were classified into distinct subtypes (e.g., H103 and H108). However, no correlation between the isolation sites and mrp/epf/sly genotypes was observed.

The SE-AFLP analysis resulted in 20 patterns (P1 to P20) distrib-uted among 3 primary clusters with 65% similarity (Figure 2). It exposes pattern P11, which grouped 61 S. suis isolates from different origin, isolation site, and virulence genotypes. With the exception of this pattern, the isolates tended to group according to state and year of origin, but no further correlation with mrp/epf/sly genotypes was observed. Interestingly, mrp1/epfv/sly1 isolates presented high genetic similarity and appeared to cluster closely in both techniques.

The discriminatory indexes for PFGE using SmaI and SE-AFLP using HindIII were 0.97 and 0.67, respectively. Even though both techniques enabled S. suis serotype 2 molecular typing and the PFGE appears to be more discriminative, SE-AFLP presents the advantage of being faster, more economically viable and less troublesome than PFGE.

D i s c u s s i o nThe high frequency of S. suis serotype 2 isolation from the central

nervous system corroborates previous studies of swine infection (14,17), as well as the characterization of this pathogen as one of the most common causes of meningitis in Asia (2). The high frequencies of mrp1/epf1/sly1 and mrp1/epfv/sly1 genotypes among serotype 2 isolates from diseased pigs also corroborate previous reports of S. suis infection in Europe and China (5,6).

The only 2 S. suis non-invasive isolates studied (SS166 and SS167) presented the genotypes mrp2/epf1/sly2 and mrp1/epf1/sly2, respec-tively; nevertheless, they grouped together with mrp1/epf1/sly1 isolates in both PFGE and SE-AFLP. The presence of isolates that presented epf gene variation with higher molecular weight appears to be a characteristic of Brazilian S. suis serotype 2 as previously described by Martinez et al (7). Even though we detected a lower incidence than Martinez et al (7), the mrp1/epfv/sly1 genotype was also obtained from diseased animals contradicting the statement that EF variant strains are weakly virulent (4).

We also observed the presence of mrp2/epf2/sly2 genotype in invasive strains, which is unprecedented in Brazil; however, they also grouped together with mrp1/epf1/sly1 isolates in both PFGE

and SE-AFLP. Differing from the European and Asian scenario, the MRP2/EF2/SLY2 and MRP1/EF2/SLY2 phenotypes appear to be common in virulent North American S. suis serotype 2 strains (2,18,19). Therefore, further studies are necessary to understand the distribution and pathogenicity of mrp2/epf2/sly2 isolates in the continent.

Although both PFGE and SE-AFLP had been previously applied for S. suis typing, their clustering patterns were highly associated with mrp/epf/sly genotypes and isolate origins (14,20–22). The greater genetic heterogeneity observed in this study could be related to the distinct origin and year of isolation, and it stands out in the existence of isolates originating from the same herd classified into distinct PFGE subtypes and even different patterns in both techniques, such as the isolates from herd 108.

The greater genetic heterogeneity observed for S. suis serotype 2 originated from the same herd and could be related to the introduc-tion of infected animals and the herd’s high turnover. Therefore, the high variation of S. suis virulence genotypes and genotypic patterns in the Brazil demand attention to quality of the sanitary status of the traded animals and the implications for their treatment and the pathogen control among respective herds.

Nevertheless, serotype 2 isolated from diseased pigs also appears to be more variable than human strains (23). Therefore, although the molecular epidemiology of S. suis infection continues to improve worldwide, further studies are necessary to enhance the knowledge regarding virulence genotype relevance and distribution in diseased animals and carriers, and its geographical variability.

A c k n o w l e d g m e n t sThis study was supported by FAPESP (grant 2015/01532-1),

CAPES and CNPq (grant 309062/2014-4).

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humans: The Chinese experience and the situation in North America. Ani Health Res Rev 2007;8:29–45.

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3. Jacobs AAC, Loeffen PLW, Van De Berg AJG, Storm P. Identifica-tion, purification and characterization of thiol-activated hemo-lysin (suilysin) of Streptococcus suis. Infect Immun 1994;62: 1742–1748.

4. Vecht U, Wisselink HJ, Van Dijk JE, Smith HE. Virulence of Streptococcus suis type 2 strains in newborn germfree pigs depends on phenotype. Infect Immun 1992;60:550–556.

5. Silva L, Baums CG, Rehm T, Wisselink H, Goethe R, Valentin-Weigand P. Virulence-associated gene profiling of Streptococcus suis isolates by PCR. Vet Microbiol 2006;115:117–127.

6. Wei Z, Li R, Zhang A, et al. Characterization of Streptococcus suis isolates from the diseased pigs in China between 2003 and 2007. Vet Microbiol 2009;137:196–201.



7. Martinez G, Pestana de Castro AF, Ribeiro Pagnani KJ, Nakazato G, Dias da Silveira W, Gottschalk M. Clonal distribution of an atypical MRP1, EF*, and suilysin1 phenotype of virulent Streptococcus suis serotype 2 strains in Brazil. Can J Vet Res 2003; 67:52–55.

8. Boom R, Sol CJA, Salimans MMM, Jansen CL, Wertheim-Van Dillen PM, Van Der Noorda J. Rapid and simple method for purification of nucleic acids. J Clin Microbiol 1990;28:495–503.

9. Okwumabua O, O’Connor M, Shull E. A polymerase chain reaction (PCR) assay specific for Streptococcus suis based on the gene encoding the glutamate dehydrogenase. FEMS Microbiol Lett 2003;218:79–84.

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12. Gottschalk M, Higgins R, Jaques M, Mittal KR, Henrichsen J. Description of 14 new capsular types of Streptococcus suis. J Clin Microbiol 1989;27:2633–2636.

13. McLauchlin J, Ripabelli G, Brett MM, Threlfall EJ. Amplified fragment length polymorphism (AFLP) analysis of Clostridium perfringens for epidemiological typing. Int J Food Microbiol 2000;56:21–28.

14. Vela AI, Goyache J, Tarradas C, et al. Analysis of genetic diver-sity of Streptococcus suis clinical isolates from pigs in Spain by pulsed-field gel electrophoresis. J Clin Microbiol 2003;41: 2498–2502.

15. Van Belkum A, Tassios PT, Dijkshoorn L, et al. Guidelines for the validation and application of typing methods for use in bacterial epidemiology. Clin Microbiol Infect 2007;13:1–46.

16. Hunter PR, Gaston MA. Numerical index of the discriminatory ability of typing systems: An application of Simpson’s index of diversity. J Clin Microbiol 1988;26:2465–2466.

17. Del’Arco AE, Santos JL, Bevilacqua PD, Faria JE, Guimarães WV. Swine infection by Streptococcus suis: A retrospective study. Arq Bras Med Vet Zootec 2008;60:878–883.

18. Fittipaldi N, Fuller TE, Teel JF, et al. Serotype distribution and production of muramidase-released protein, extracellular factor and suilysin by field strains of Streptococcus suis isolated in the United States. Vet Microbiol 2009;139:310–317.

19. Gottschalk M, Lebrun A, Wisselink H, Dubreuil JD, Smith H, Vecht U. Production of virulence-related proteins by Canadian strains of Streptococcus suis capsular type 2. Can J Vet Res 1998; 62:75–79.

20. Baums CG, Verkuhlen GJ, Rehm T, et al. Prevalence of Streptococcus suis genotypes in wild boars of northwestern Germany. App Environ Microbiol 2007;73:711–717.

21. Rehm T, Baums CG, Strommenger B, Beyerbach M, Valentin-Weigand P, Goethe R. Amplified fragment length polymor-phism of Streptococcus suis strains correlates with their profiles of virulence-associated genes and clinical background. J Med Microbiol 2007;56:102–109.

22. Tharavichitkul P, Wongsawan K, Takenami N, et al. Correlation between PFGE Groups and mrp/epf/sly genotypes of human Streptococcus suis Serotype 2 in northern Thailand. J Pathog 2014. doi: /10.1155/2014/350416 Available from: http://www.hindawi.com/journals/jpath/2014/350416 Last accessed January 6, 2016.

23. Berthelot-Herault F, Marois C, Gottschalk M, Kobisch M. Genetic diversity of Streptococcus suis strains isolated from pigs and humans as revealed by pulsed-field gel electrophoresis. J Clin Microbiol 2002;40:615–619.


Article


I n t r o d u c t i o nMycoplasma hyopneumoniae is the etiological pathogen of enzootic

pneumonia, which is characterized by a chronic, nonproductive cough (1). Infection of M. hyopneumoniae causes considerable eco-nomic losses, due to decreased growth rates, high feed conversion ratios, increased medication costs, and the susceptibility of sick pigs to infection by other organisms (1,2). Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) is an enveloped, single-stranded, positive-sense RNA virus belonging to the Arteriviridae family in the order Nidovirales (3) that can cause reproductive prob-lems in sows and respiratory problems in growing pigs (4).

In pigs, respiratory disease is multifactorial and complex and is caused by sequential or concurrent infections with several viral or

bacterial pathogens; therefore, the name porcine respiratory disease complex (PRDC) is used to describe this disease (5,6). The economic impact of PRDC is tremendous, mainly due to decreased fattening performance and the cost of medication (7,8). Currently, the use of antibiotics for controlling PRDC is limited due to increased risk of antimicrobial resistance and residue in carcasses (9). Therefore, vaccinations are of prime importance and are routinely applied worldwide.

Since coinfection with M. hyopneumoniae and PRRSV is one of the most economically important situations in PRDC (10), vaccina-tion of pigs with both M. hyopneumoniae and PRRSV is necessary to control PRDC efficiently. The commercial modified live PRRSV vaccine (Ingelvac PRRS MLV; Boehringer Ingelheim Vetmedica, St. Joseph, Missouri, USA) was first licensed for worldwide use in

Comparison of 2 commercial single-dose Mycoplasma hyopneumoniae vaccines and porcine reproductive and respiratory syndrome virus (PRRSV)

vaccines on pigs dually infected with M. hyopneumoniae and PRRSVChanghoon Park, Ikjae Kang, Hwi Won Seo, Jiwoon Jeong, Kyuhyung Choi, Chanhee Chae

A b s t r a c tThe objective of this study was to compare the efficacy of 2 different commercial Mycoplasma hyopneumoniae vaccines and porcine reproductive and respiratory syndrome virus (PRRSV) vaccines in regard to growth performance, microbiological and immunological analyses, and pathological observation from wean to finish (175 d of age). Pigs were administered M. hyopneumoniae and PRRSV vaccines at 7 and 21 d of age, respectively, or both at 21 d old and then challenged with both M. hyopneumoniae and PRRSV at 49 d old. Significant (P , 0.05) differences were observed between the 2 vaccinated challenged groups in average daily weight gain, nasal shedding of M. hyopneumoniae, M. hyopneumoniae-specific interferon-g secreting cells, and macroscopic and microscopic lung lesions. Induction of interleukin-10 following PRRSV vaccination does not interfere with the immune responses induced by M. hyopneumoniae vaccine. The present study demonstrated that the single-dose vaccination regimen for M. hyopneumoniae and PRRSV vaccine is efficacious for controlling coinfection with M. hyopneumoniae and PRRSV based on clinical, microbiological, immunological, and pathological evaluation.

R é s u m éL’objectif de la présente étude était de comparer l’efficacité de deux vaccins commerciaux différents contre Mycoplasma hyopneumoniae et le virus du syndrome reproducteur et respiratoire porcin (VSRRP) quant aux performances de croissance, aux analyses microbiologiques et immunologiques, et les observations pathologiques chez des porcs du sevrage à la finition (175 j d’âge). Les porcs ont reçu les vaccins M. hyopneumoniae et VSRRP à 7 et 21 j d’âge, respectivement, ou les deux à 21 j et par la suite soumis à une infection défi avec M. hyopneumoniae et VSRRP à l’âge de 49 j. Des différences significatives (P < 0,05) ont été observées entre les deux groupes vaccinés et challengés pour les paramètres suivants : le gain quotidien moyen, l’excrétion nasale de M. hyopneumoniae, le nombre de cellules secrétant de l’interféron-g spécifique à M. hyopneumoniae, et les lésions pulmonaires macroscopiques et microscopiques. L’induction d’interleukine-10 suivant la vaccination pour VSRRP n’a pas interférée avec les réponses immunitaires induites par le vaccin M. hyopneumoniae. Cette étude a démontré qu’une vaccination avec une dose unique de vaccin contre M. hyopneumoniae et le VSRRP est efficace pour limiter une co-infection par ces deux agents si on se base sur les évaluations clinique, microbiologique, immunologique et pathologique.


Seoul National University, College of Veterinary Medicine, Department of Veterinary Pathology, 1 Gwanak-ro, Gwanak-gu, 151-742, Seoul, Republic of Korea.

Drs. Changhoon Park and Ikjae Kang contributed equally to this work.

Address all correspondence to Dr. Chanhee Chae; telephone: 182-2-880-1277; fax: 182-2-871-5821; e-mail:[email protected]

Received April 15, 2015. Accepted November 20, 2015.



1994. In 2012, another new commercial modified live PRRSV vaccine (Fostera PRRS; Zoetis, Florham Park, New Jersey, USA) was intro-duced to the international market to control respiratory disease in growing pigs. A comparison of both single-dose M. hyopneumoniae and PRRSV vaccines together, therefore, is more practical and mir-rors field conditions, rather than a comparison of each single dose M. hyopneumoniae and PRRSV vaccines by themselves. The objective of the present study was to compare the efficacy of 2 commercial single-dose M. hyopneumoniae vaccines and PRRSV vaccines in regard to virological and immunological analysis, pathological observation, and growth performance from wean to finish using a challenge model.


Commercial vaccinesTwo types of commercial M. hyopneumoniae vaccines were used in

this study: A — the inactivated M. hyopneumoniae bacterin (RespiSure-One; Zoetis) given as one 2.0-mL dose at 7 d of age and B — the inac-tivated M. hyopneumoniae bacterin (Ingelvac MycoFLEX; Boehringer Ingelheim Vetmedica) given as one 1.0-mL dose at 21 d of age. Two types of commercial PRRSV vaccines were used in this study: A — the modified live PRRSV vaccine (Fostera PRRS; Zoetis) given as one 2.0-mL dose at 21 d of age, and B — the modified live PRRSV vac-cine (Ingelvac PRRS MLV; Boehringer Ingelheim Vetmedica) given as one 2.0-mL dose at 21 d of age. All vaccines used in this study were administered according to the manufacturer’s label claims with regards to timing and route of injection (intramuscularly).

InoculaMycoplasma hyopneumoniae strain SNU98703, used as inocu-

lum, was isolated from lung samples from postweaned pigs with severe enzootic pneumonia (11). Mycoplasma hyopneumoniae strain SNU98703 caused typical lesions consisting of peribronchial, peri-bronchiolar, and perivascular lymphoid hyperplasia in experimen-tally infected pigs (11). The PRRSV strain SNUVR090851 (type 2 genotype, lineage 1, GenBank no. JN315685), used as inoculum, was isolated from lung samples from different newly weaned pigs in Chungcheung Providence in 2010 (12). This virus strain caused interstitial pneumonia characterized by thickened alveolar septa with increased numbers of interstitial macrophages and lymphocytes and by type II pneumocyte hyperplasia in experimentally infected pigs (12).

AnimalsA total of 60 colostrum-fed, cross-bred, conventional piglets were

purchased at 5 d of age from an M. hyopneumoniae- and PRRSV-free commercial farm, based on serological testing of breeding herd and long-term clinical and slaughter history. All piglets were negative for M. hyopneumoniae, PRRSV, and porcine circovirus type 2 (PCV2) according to routine serological testing. Mycoplasma hyopneumoniae was not detected in the nasal samples by the real-time polymerase chain reaction (PCR) (13). The PRRSV and PCV2 were not detected in the serum and nasal samples by the real-time polymerase chain reaction (PCR) (14,15).

Experimental designA total of 60 pigs were randomly divided into 4 groups: Zoetis-

Vac (n = 20 pigs), BI-Vac (n = 20 pigs), positive control (n = 10), and negative control (n = 10) groups using the random number genera-tion function (Excel; Microsoft Corporation, Redmond, Washington, USA). In the Zoetis-Vac group, pigs were immunized with the M. hyopneumoniae A bacterin and the PRRSV A vaccines at 7 and 21 d of age, respectively. In BI-Vac group, pigs were immunized with both the M. hyopneumoniae B bacterin (left side of the neck) and PRRSV B vaccine (right side of the neck) at 21 d of age. At 49 d of age [0 days post challenge (dpc)], pigs in the Zoetis-Vac, BI-Vac, and positive control groups were intratracheally administered a 10 mL dose of a lung homogenate of M. hyopneumoniae strain SNU98703 (1:100 dilution in Friis medium) at a final concentra-tion of 104 to 105 color-changing units (CCU)/mL in the morning, as previously described (16). In the afternoon of the same day, same pigs were intranasally administered 3 mL of type 2 PRRSV (strain SNUVR090851; 2nd passage in MARC-145 cells) containing 1.2 3 105 TCID50/mL. Blood samples from each pig were collected by jugular venipuncture at 242, 228, 0, 14, 28, 63, 91, and 126 dpc. Sterile polyester swabs (Fisher Scientific, Pittsburgh, Pennsylvania, USA) were used to swab the nasal mucosa of both nostrils, reaching deeply into the turbinates at 242, 228, 0, 14, 28, 63, 91, and 126 dpc. Swabs were stored in 5 mL plastic tubes (Fisher Scientific) containing 1 mL of sterile saline solution. Pigs were sedated with an intrave-nous injection of azaperon (Stresnil; Janssen Pharmaceutica, Beerse, Belgium) and then euthanized by electrocution for necropsy at 175 d of age (126 dpc). Tissues were collected from each pig at necropsy. All of the methods were approved by the Seoul National University Institutional Animal Care and Use Committee.

Clinical evaluationFollowing M. hyopneumoniae and PRRSV inoculation, the pigs were

monitored daily for physical condition and scored weekly for clinical respiratory disease severity using scores ranging from 0 (normal) to 6 (severe dyspnea and abdominal breathing) (17). Observers were blinded to vaccination and challenge status. Pigs were observed daily at the same time of day. Rectal body temperature was recorded daily from 0 to 21 dpc.

Assessment of growth performanceThe live weight of each pig was measured at 3 (228 dpc),

7 (0 dpc), 10 (21 dpc), 16 (63 dpc), and 25 (126 dpc) weeks of age. The average daily weight gain (ADWG, grams/pig per day) was analyzed over 4 time periods: between 3 and 7; 7 and 10; 10 and 16; and 16 and 25 weeks of age. The ADWG during these various pro-duction stages was calculated as the difference between the starting and final weights divided by the duration of the stage. Data from dead pigs were included in the calculation.

SerologyThe serum samples were tested using the commercially avail-

able M. hyopneumoniae and PRRSV enzyme-linked immunosorbent assay (ELISA; IDEXX M.hyo Ab Test and PRRS X3 Ab Test, IDEXX Laboratories, Westbrook, Maine, USA). Serum virus neutralization



Figure 1. Mean values of the clinical sign scores (A) and the rectal body temperature (B) in the different groups: Zoetis-Vac (), BI-Vac (), positive control (), and negative control () groups. a,bSignificant (P , 0.05) difference between groups.

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tests for PRRSV were also done using the challenge strain, as pre-viously described (18,19). Serum samples were considered to be positive for neutralizing antibodies (NA) if the titer was greater than 2.0 (log2) (20).

Quantification of M. hyopneumoniae DNADNA was extracted from the nasal swabs using a kit (QIAamp

DNA Mini Kit; QIAGEN, Crawley, United Kingdom). The DNA

extracts were then used to quantify the M. hyopneumoniae genomic DNA copy numbers by real-time PCR, as previously described (13).

Quantification of PRRSV RNARNA was extracted from serum samples and nasal swabs using

a kit (QIAamp Viral RNA Mini Kit; QIAGEN) to quantify type 2 PRRSV genomic cDNA copy numbers by real-time PCR, as previ-ously described (14).

Table I. Average daily weight gain (ADWG, g/pig per day), proportion of viremic pig and nasal shedder, pathology, in-situ hybridization of Mycoplasma hyopneumoniae (Mhp) and immunohistochemistry of porcine reproductive and respiratory syndrome virus (PRRSV) among 4 groups at different days post challenge (dpc)

Positive Negative Zoetis-Vac* BI-Vac† control controlNumber of pigs 20 20 10 10

Number of dead pigs 0 1 2 0

ADWG (wk old) 3–7 328 6 28 324 6 24 327 6 19 330 6 18 7–10 613 6 26 601 6 30 603 6 28 615 6 26 10–16 806 6 34a 786 6 33a 729 6 31b 802 6 35a

16–25 746 6 30a 698 6 25b 623 6 27c 750 6 31a

3–25 669 6 25a 640 6 23b 595 6 20c 670 6 24a

Mhp nasal shedders (dpc) 14 11/20a 13/20a 8/10a 0/10 28 11/20a 12/19a 9/9b 0/10 63 9/20a 11/19a 9/9b 0/10 91 7/20a 8/19a 6/8a 0/10 126 6/20a 7/19a 5/8a 0/10

PRRSV viremic pigs (dpc) 14 11/20a 12/20a 10/10b 0/10 28 6/20a 6/19a 9/9b 0/10 63 2/20a 1/19a 3/9a 0/10 91 1/20a 1/19a 1/8a 0/10 126 0/20a 0/19a 1/8a 0/10

PRRSV nasal shedders (dpc) 14 10/20a 12/20a 10/10b 0/10 28 6/20a 7/19a 8/9b 0/10 63 1/20a 1/19a 3/9a 0/10 91 1/20a 1/19a 1/8a 0/10 126 0/20a 0/19a 0/8a 0/10

Macroscopic lung lesion score 2.17 6 0.63a 3.48 6 0.65b 10.75 6 5.83c 0.53 6 0.38d

Microscopic lung lesion score 2.21 6 0.34a 3.10 6 0.36b 5.43 6 0.45c 0.87 6 0.35d

Mhp DNA score 0.40 6 0.51a 0.55 6 0.51a 1.50 6 0.52b 0

PRRSV antigen score 1.95 6 3.14 2.05 6 3.25 2.70 6 3.02 0* Pigs that had received RespiSure-One bacterin at 7 days old and Fostera PRRS vaccine at 21 days old followed by a dual challenge at 49 days old.† Pigs that had received Ingelvac MycoFLEX bacterin and Ingelvac PRRS vaccine at 21 days old followed by a dual challenge at 49 days old.Different letters (a, b, and c) indicate significant (P , 0.05) difference among groups.



Enzyme-linked immunospot assayMycoplasma hyopneumoniae and PRRSV antigens were prepared as

previously described (21,22). The numbers of M. hyopneumoniae- and PRRSV-specific interferon-g secreting cells (IFN-g-SC) stimulated by challenging M. hyopneumoniae and PRRSV were determined in peripheral blood mononuclear cells (PBMC) as previously described (23–25). The results were expressed as the numbers of IFN-g-SC per million PBMC.

Quantification of interleukin-10 secretionThe interleukin-10 (IL-10) protein levels were quantified in the

supernatants of PBMC cultures (2 3 106 cells per well; 250 mL) in vitro for 20 h with challenging type 2 PRRSV (0.01 of MOI) or PHA (10 mg/mL) by using a commercial ELISA (Pig Interleukin-10 ELISA kit; Cusabio Biotech, Wuhan, China) according to the manufacturer’s instructions. Detection limits for IL-10 were 1.5 pg/mL.

Macroscopic lung lesion scoresThe total extent of macroscopic lung lesions was estimated and

calculated as previously described (26). The frequency distribution of the lung lesion scores for each lung lobe was calculated by treat-ment (16,26).

Morphometric analysisFor the morphometric analysis of pulmonary histopathological

changes, 3 lung sections were taken from ventromedial part of the right and left caudal lobe, and right middle lobe. Lung sections were examined blindly and given an estimated severity score for the mea-sures of pulmonary pathology (i.e., atelectasia, epithelial necrosis, hemorrhage, airway plugging, epithelial hyperplasia, interstitial change, and leukocyte infiltration) (26). Each individual measure was given a score ranging from 0 (no lesion visible) to 3 (severe lesions). These scores were added to obtain an overall score for each section.

In-situ hybridization and immunohistochemistryIn-situ hybridization (ISH) for M. hyopneumoniae was done as

previously described (27). Immunohistochemistry (IHC) for PRRSV was done as previously described (28). The results of ISH and IHC were analyzed morphometrically as previously described (29,30).

Statistical analysisContinuous data (rectal body temperatures, ADWG, quantified

M. hyopneumoniae DNA and PRRSV RNA, M. hyopneumoniae and PRRSV serology, M. hyopneumoniae- and PRRSV-specific IFN-g-SC, PRRSV-specific IL-10, macroscopic lung lesion scores, and PRRSV

Figure 2. Mean values of the genomic copy numbers of Mycoplasma hyopneumoniae (Mhp) DNA in the nasal swabs in the different groups: Zoetis-Vac (), BI-Vac (), and positive control () groups. a,b,cSignificant (P , 0.05) difference between groups.



antigen scores) were analyzed using a repeated measures analysis of variance (ANOVA). If the repeated measures ANOVA showed a significant effect, Tukey’s multiple comparison test was done at each time point. Discrete data (clinical respiratory scores, mortality

rate, microscopic lung lesion scores, and M. hyopneumoniae DNA scores) were analyzed using the Mann-Whitney test. The correlation between the ADWG and lung lesions was assessed by Spearman’s correlation. A value of P , 0.05 was considered significant.

Figure 3. Mean values of the genomic copy numbers of PRRSV RNA in the serum samples (A) and nasal swabs (B) in the different groups: Zoetis-Vac (), BI-Vac (), and positive control () groups. a,b,cSignificant (P , 0.05) difference between groups.



Figure 4. Immunological responses against Mycoplasma hyopneumoniae (Mhp). Mean values of the anti- M. hyopneumoniae antibody levels. Mean number of M. hyopneumoniae-specific interferon-g secreting cells (IFN-g-SC) in the different groups: Zoetis-Vac (), BI-Vac (), and positive control () groups. a,b,cSignificant (P , 0.05) difference between groups.

Figure 5. Immunological responses against Mycoplasma hyopneumoniae (Mhp). Mean number of M. hyopneumoniae-specific interferon-g secreting cells (IFN-g-SC) in the different groups: Zoetis-Vac ( ), BI-Vac ( ), and positive control ( ) groups. a,b,cSignificant (P , 0.05) difference between groups.



Figure 6. Immunological responses against porcine reproductive and respiratory syndrome virus (PRRSV). A — Mean val-ues of the anti-PRRSV antibody levels. B — Mean values of PRRSV-specific neutralizing antibodies (NA). Zoetis-Vac (), BI-Vac (), and positive control () groups. a,b,cSignificant (P , 0.05) difference between groups.



Figure 7. Immunological responses against porcine reproductive and respiratory syndrome virus (PRRSV). Mean number of PRRSV-specific interferon-g secreting cells (IFN-g-SC) in the different groups: Zoetis-Vac ( ), BI-Vac ( ), and positive control ( ) groups. a,b,cSignificant (P , 0.05) difference between groups.

Figure 8. Level of challenging type 2 porcine reproductive and respiratory syndrome virus (PRRSV)-specific interleukin (IL)-10 in the different groups; Zoetis-Vac (), BI-Vac (), and positive control () groups. a,b,cSignificant (P , 0.05) difference between groups.



Re s u l t s

Clinical evaluationPigs in the Zoetis-Vac and BI-Vac groups remained normal

throughout the study, as measured by their respiratory scores and rectal temperatures, whereas moderate to severe respiratory signs were observed in the positive controls (Figures 1A and B). The mean rectal temperature (ranging from 39.8°C to 40.2°C) was significantly higher (P , 0.05) in the positive control groups than in the Zoetis-Vac and BI-Vac groups at 3, 4, 5, 6, and 7 dpc (Figure 1B). The mean respiratory scores were significantly higher (P , 0.05) in the posi-tive control group than in the Zoetis-Vac and BI-Vac groups from 14 to 56 dpc, and at 70, 91, and 98 dpc. The overall mortality rate was significantly lower (P , 0.05) for the vaccinated challenged pigs (0%, 0/20 in the Zoetis-Vac group; 5%, 1/20 in the BI-Vac group) than the positive control group (20%; 2/10) (Table I). Diagnostic results indicated that mortality was primarily related to severe pneumonia.

Growth performanceNo significant difference in the ADWG was observed between

Zoetis-Vac and BI-Vac group during weeks 3 to 10. However, during weeks 10 ot 16, the ADWG of Zoetis-Vac group was significantly higher (P , 0.05) than that of BI-Vac group. The overall growth performance (from 3 to 25 wk of age) in pigs from the Zoetis-Vac group was significantly higher (P , 0.05) than that of pigs from the BI-Vac group (Table I). The other significant results are summarized in Table I.

Quantification of M. hyopneumoniae DNA in nasal swabs

Prevalence rates of M. hyopneumoniae positive pigs are summa-rized in Table I. At the time of the challenges, no genomic copies of M. hyopneumoniae were detected in any of the nasal samples from any of the 4 groups. Pigs in the Zoetis-Vac and BI-Vac groups had a significantly lower (P , 0.05) number of genomic copies of M. hyo-pneumoniae in their nasal swabs than the positive controls throughout the experimental period (Figure 2). Pigs in the Zoetis-Vac group had a significantly lower (P , 0.05) number of genomic copies of M. hyo-pneumoniae in their nasal swabs than pigs in the BI-Vac group from 14 to 63 dpc. No genomic copies of M. hyopneumoniae were detected in any of the nasal samples from negative controls throughout the experimental period.

Quantification of PRRSV RNA in sera and nasal swabs

Prevalence rates of PRRSV positive pigs are summarized in Table I. At the time of the challenges, no genomic copies of PRRSV were detected in any of the serum samples or nasal swabs from any of the 4 groups. Pigs in the Zoetis-Vac and BI-Vac groups had a significantly lower (P , 0.05) number of genomic copies of PRRSV in their sera and nasal swabs than pigs in positive controls at 14 and 28 dpc (Figure 3). No genomic copies of PRRSV were detected in any of the serum samples or nasal swabs from the negative controls throughout the experimental period.

Immunological responses of M. hyopneumoniaePigs in the Zoetis-Vac group had significantly higher (P , 0.05)

anti-M. hyopneumoniae antibody levels (Figure 4) and numbers of M. hyopneumoniae-specific IFN-g-SC (Figure 5) at various dpc compared to pigs in the BI-Vac and negative control groups. The other significant results are summarized in Figure 4. No anti-M. hyo-pneumoniae antibodies or M. hyopneumoniae-specific IFN-g-SC were detected in the negative controls.

Immunological responses of PRRSVPigs in the Zoetis-Vac and BI-Vac groups had significantly higher

(P , 0.05) anti-PRRSV antibody levels from 0 to 28 dpc compared to positive control group (Figure 6A). The PRRSV-specific NA was detected in pigs in the Zoetis-Vac and BI-Vac groups at 91 and 126 dpc only (Figure 6B). Pigs in the Zoetis-Vac and BI-Vac groups had significantly higher (P , 0.05) numbers of PRRSV-specific IFN-g-SC from 0 to 28 dpc compared to positive control group (Figure 7). No anti-PRRSV antibodies, PRRSV-specific NA, or PRRSV-specific IFN-g-SC were detected in the negative controls.

The PRRSV-specific IL-10After stimulation with challenging type 2 PRRSV, IL-10 gradually

increased until 0 dpc and thereafter it decreased until 63 dpc in pigs in the Zoetis-Vac and BI-Vac groups (Figure 8). In positive controls, IL-10 had gradually increased at 28 dpc, after which it decreased until 63 dpc. Pigs in the Zoetis-Vac group had significantly lower (P , 0.05) levels of IL-10 than pigs in the BI-Vac group at 0 dpc. Pigs in the Zoetis-Vac and BI-Vac groups had significantly lower (P , 0.05) levels of IL-10 than pigs in the positive group at 28 dpc. Interleukin-10 was not detected in negative controls.

Lung lesion scoresPigs in the Zoetis-Vac group had significantly lower (P , 0.05)

scores for macroscopic and microscopic pulmonary lesions than pigs in the BI-Vac group at 126 dpc. The other significant results of scores for pulmonary lesions are summarized in Table I. Additionally, a negative correlation was found between ADWG and macroscopic lung lesion (r = 20.578, P , 0.01), and between ADWG and micro-scopic lung lesion (r = 20.538, P , 0.01).

In-situ hybridization of M. hyopneumoniaePigs in the Zoetis-Vac and BI-Vac groups had significantly

(P , 0.05) lower amounts of M. hyopneumoniae DNA in their lungs than positive controls (Table I).

Immunohistochemistry for PRRSVThere was no significantly different number of PRRSV-positive

cells per unit in the lungs from pigs in the Zoetis-Vac, BI-Vac, or positive control groups (Table I).

D i s c u s s i o nThe present study demonstrated that the single-dose vaccination

regimen for M. hyopneumoniae bacterin and PRRS modified live vac-cine is efficacious for controlling coinfection with M. hyopneumoniae



and PRRSV from wean to finish based on clinical, microbiological, immunological, and pathological evaluation. Vaccination of pigs with M. hyopneumoniae and PRRSV is able to reduce the levels of mycoplasmal nasal shedding, PRRSV viremia, and severity of lung lesions compared to unvaccinated challenged pigs. In contrast with earlier findings that vaccination with the PRRSV vaccine reduced the efficacy of the M. hyopneumoniae bacterin (31), sequential or con-current single-dose vaccination with M. hyopneumoniae and PRRSV could not reduce the efficacy of the M. hyopneumoniae bacterin or the PRRSV vaccine in this study. Our results agree with previous findings in which no negative influence of the PRRSV vaccination on M. hyopneumoniae vaccine efficacy was observed (32,33). Interestingly, high levels of IL-10 were detected in pigs during the first 4 to 5 wk following PRRSV vaccination. Because IL-10 is a known potent immunosuppressive cytokine (34), induction of IL-10 following PRRSV vaccination may cause interference of the mycoplasmal vaccine’s efficacy. However, M. hyopneumoniae-specific IFN-g-SCs gradually increased at the same time as the gradual increase of IL-10, indicating that levels of IL-10 following PRRSV vaccination are not enough to interfere with immune responses induced by the mycoplasmal vaccine.

Selection of an appropriate challenge virus is critical when com-paring 2 PRRSV vaccines, which are only 91.7% homologous to each other. The Ingelvac PRRS MLV vaccine virus is from lineage 5 and the Fostera PRRS vaccine virus is from lineage 8. A challenge strain was chosen that was not closely related to either vaccine virus and had a similar level of homology to the 2 vaccine viruses, based on open reading frame 5 (ORF5) nucleotide sequences. Strain SNUVR090851 is from lineage 1 with homologies of 87.2% and 85.9% to Fostera PRRS and Ingelvac PRRS MLV, respectively. However, our results should be interpreted carefully because only ORF5 of the genome is used for the comparison. Therefore, additional studies are needed to confirm heterogenicity using full genome of challenge and vaccine viruses.

Pathological observation is also important parameter to evalu-ate the mycoplasmal and PRRSV vaccine because a reduction in M. hyopneumoniae- and PRRSV-induced lesions is correlated with improved weight gain (35–38). In the present study, coinfection with M. hyopneumoniae and PRRSV induces severe lung lesions, which are similar to typical PRDC. The most striking and consistent microscopic lesions were severe interstitial pneumonia with some degree of peribronchial and peribronchiolar fibrosis and lymphoid tissue hyperplasia. A reduction in lung lesions was correlated with improved weight gain, as previous studies have shown (35–38).

Comparison of 2 vaccines is more practical and may be reflected field conditions because M. hyopneumoniae and PRRSV are 2 major contributors to PRDC (10). The 2 commercial vaccines used in this study were shown to be efficacious in controlling coinfection with M. hyopneumoniae and PRRSV based on clinical, microbiological, immu-nological, and pathological evaluation under experimental conditions.

A c k n o w l e d g m e n t sThis research was supported by Zoetis, contract research funds

of the Research Institute for Veterinary Science (RIVS) from the College of Veterinary Medicine, and by Brain Korea Plus Program for Creative Veterinary Science Research in the Republic of Korea.

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Article


I n t r o d u c t i o nPorcine reproductive and respiratory syndrome virus (PRRSV) is

a major cause of economic concern in the swine industry owing to losses resulting from decreased growth performance and increased mortality rates in weaned pigs (1–4). Vaccination with a modi-fied live virus (MLV) vaccine is considered to be of value for decreasing these losses; however, the diversity of the virus tends to interfere with the protection provided by vaccines. Currently

the virus has 2 main genotypes, type I (European) and type II (North American), which are genetically and antigenically dif-ferent (1,3,5–11). Furthermore, 4 subtypes of type I PRRSV based on sequence analyses of open reading frame 5 (ORF5) have been identified (8). Although similarities exist between and within the type I strains, there is enough diversity that vaccines may not induce sufficient cross-protection against heterologous strains. Pileri et al (12) stated that the genetic diversity of PRRSV is such that all chal-lenge situations in the field could be considered heterologous. Some

Safety and early onset of immunity with a novel European porcine reproductive and respiratory syndrome virus vaccine in young piglets

Michael Piontkowski, Jeremy Kroll, Christian Kraft, Teresa Coll

A b s t r a c tPorcine reproductive and respiratory syndrome virus (PRRSV) can be difficult to manage in commercial settings. A novel type I PRRSV vaccinal strain (94881) was evaluated for safety and efficacy/onset of immunity (OOI) in piglets. In 2 experiments, groups of piglets were vaccinated intramuscularly (IM) at approximately 14 d of age with a maximum-range commercial dose, an overdose, or a placebo in experiment 1 and either a minimum-range commercial dose or a placebo in experiment 2. The piglets in experiment 1 were evaluated for local and systemic reactions from days −2 through 14 after vaccination. The piglets in experiment 2 were challenged with a virulent heterologous type I PRRSV isolate 14 d after vaccination and observed once daily for general health from days −1 through 12 after vaccination and once daily for clinical signs associated with challenge from days 13 through 24 after vaccination. The average daily weight gain (ADWG) and the results of serologic and viremia testing were evaluated in experiments 1 and 2. Lung lesion scores and results of testing for PRRSV in lung tissue were evaluated in experiment 2. In experiment 1 the vaccine was shown to be safe, as there were no relevant differences between the vaccinated piglets and the piglets given a placebo. In experiment 2 the vaccine’s efficacy, with an OOI of 14 d after vaccination, was established, as the vaccinated and challenged piglets exhibited significantly lower lung lesion scores, viremia, viral load in lung tissue, and total clinical sign scores, along with a significantly greater ADWG, compared with the placebo-vaccinated and challenged piglets.

R é s u m éLa gestion du virus du syndrome reproducteur et respiratoire porcin (VSRRP) peut être difficile dans un environnement de production commerciale. Une nouvelle souche vaccinale du VSRRP de type 1 (94881) a été évaluée d’un point de vue sécurité et efficacité/début de l’immunité (DDI) chez des porcelets. Dans deux expériences, des groupes de porcelets ont été vaccinés par voie intramusculaire (IM) à l’âge d’environ 14 j avec une dose commerciale maximale, une surdose, ou un placebo dans l’expérience 1 et une dose commerciale minimale ou un placebo dans l’expérience 2. Les porcelets dans l’expérience 1 furent évalués pour des réactions locale et systémique à compter du jour 22 jusqu’au jour 14 post-vaccination. Les porcelets dans l’expérience 2 furent soumis à une infection défi avec un isolat virulent hétérologue de VSRRP de type 1 14 j après la vaccination et observés une fois par jour pour leur état de santé général du jour 21 jusqu’au jour 12 après la vaccination et une fois par jour pour des signes cliniques associés avec l’infection du jour 13 au jour 24 après l’infection. Le gain moyen quotidien (GMQ) et les résultats des analyses sérologiques et de virémie ont été évalués dans les expériences 1 et 2. Les pointages de lésions pulmonaires et les résultats de détection du VSRRP dans le tissu pulmonaire ont été évalués dans l’expérience 2. Dans l’expérience 1, le vaccin s’est montré sécuritaire étant donné qu’il n’y avait aucune différence significative entre les porcelets vaccinés et les porcelets ayant reçu un placebo. Dans l’expérience 2, l’efficacité du vaccin, avec un DDI de 14 j après la vaccination, a été établie, étant donné que les porcelets vaccinés, et soumis à une infection défi avaient des valeurs significativement moins élevées de pointage de lésions pulmonaires, de virémie, de charge virale dans le tissu pulmonaire, et des pointages de signes cliniques totaux, avec un GMQ significativement plus élevé, comparativement au porcs vaccinés avec un placebo et soumis à une infection défi.


Boehringer Ingelheim Vetmedica, 2621 North Belt Highway, St. Joseph, Missouri 64506, USA.

Address all correspondence to Dr. Jeremy Kroll; e-mail: [email protected]

Received May 28, 2015. Accepted November 20, 2015.



studies have determined that the level of genetic similarity between the vaccine strain and the challenge strain was not necessarily an accurate predictor of vaccine efficacy (4,13), whereas others have concluded that the level of protection a vaccine provides against PRRSV infection may depend on the degree of relatedness between the vaccine and challenge strains (7,10).

It has been reported that MLV vaccines against PRRSV are at least partially effective at reducing symptoms and viremia, as well as virus shedding (14). Furthermore, MLV vaccines based on a single virus strain offer consistently good protection against homologous challenge; however, protection against heterologous challenge is neither as consistent nor as effective (2,6,14). In an attempt to broaden vaccine protection, a vaccine containing 5 type II strains of attenuated PRRSV was developed; this vaccine, however, was found to offer no more effective protection against heterologous challenge than a single-strain vaccine (15). Geldhof et al (5) summarized the difficulties facing development of an effective PRRSV vaccine by reporting that infection with different strains leads to diverse viro-logic and immunologic effects (i.e., various degrees of homologous and heterologous protection) and that the susceptibility of PRRSV strains to antibody neutralization likewise differs. Since new strains, including highly pathogenic ones (3,16), continue to be found, it is important to continue to search for a single-strain vaccine that is effective in the face of heterologous challenge. One important fac-tor to consider in this search is the virulence of the challenge strain: it should reflect what is found currently in the field. In the past, clinical signs of PRRSV infection have been difficult to reproduce with a single-strain type I PRRSV challenge, through either virus administration or contact with infected pigs (1,4,7,12,17,18). In addi-tion to efficacy concerns, the safety of PRRS MLV vaccines has been questioned. It has been reported that these vaccines may cause clini-cal signs of respiratory disease and decreased growth performance, as well as viremia, and that the virus may subsequently spread to other naïve animals when administered to piglets (2,5,9,14,19). Additionally, PRRS MLV vaccines have been found to initiate pro-tection in a relatively delayed manner, at around 3 to 4 wk after administration (2,17,18).

The present study was conducted to evaluate the safety and efficacy of a new European-derived PRRS MLV vaccine based on a novel type I strain (1) [European Collection of Cell Cultures accession numbers 11012501 (parent strain) and 11012502 (attenuated strain)] when administered to young piglets in the face of a disease-inducing heterologous PRRSV challenge.


AnimalsThe protocols were reviewed and approved by the Contract

Research Organization’s Institutional Animal Care and Use Committee before the study was started. Two experiments were conducted to establish safety (experiment 1) and efficacy/onset of immunity (OOI) (experiment 2) with 32 and 50, respectively, com-mercial mixed-breed castrated male and female piglets. The piglets in experiment 1 were blocked by weight and randomly assigned to group A (1A, n = 11), B (1B, n = 11), or C (1C, n = 10), except for

the last block, which was assigned to either 1A or 1B. The piglets in experiment 2 were assigned to 1 of 3 treatment groups in the follow-ing manner: the piglets were blocked by weight, assigned a random number by means of the random number function in Microsoft Excel (Microsoft Corporation, Redmond, Washington, USA), and ranked in numerically ascending order by block; then the 2 lowest numbers of each block were assigned to group A (2A, n = 20), the next 2 numbers were assigned to group B (2B, n = 20), and the highest number was assigned to group C (2C, n = 10).

The piglets in experiment 2 were administered ceftiofur crystalline- free acid (Excede; Pharmacia & Upjohn Company, Division of Pfizer, New York, New York, USA) intramuscularly (IM) in the right ham, according to label directions, at the time of arrival. The piglets in groups 1A, 1B, and 2A received the test vac-cine (Ingelvac PRRSFLEX EU; Boehringer Ingelheim Vetmedica, St. Joseph, Missouri, USA) (1) at the maximum-range commercial dose, at an overdose, and at the minimum-range commercial dose, respectively, whereas the piglets in groups 1C, 2B, and 2C were administered a placebo. All the piglets were 14 6 3 d of age at the time of treatment and were clinically healthy. The piglets in groups 1A, 1B, and 1C were monitored for local and systemic reac-tions to determine vaccine safety. The piglets in groups 2A and 2B were challenged with a heterologous strain of type I PRRSV, and those in group 2C were not challenged, serving as negative controls, to evaluate vaccine efficacy and OOI.

All the piglets were housed in multiple raised pens (5 or 6 pigs per pen) equipped with a nipple waterer, a feeder, and plastic slat-ted flooring. Each group was housed in separate but similar rooms for the duration of the experiments. The rooms were biosafety level 2-compliant, thermostat-controlled, and mechanically ventilated, with high-efficiency particulate air filtering, to ensure biosecurity. Additionally, appropriate measures were taken to prevent accidental cross-contamination from vaccinates and/or challenged animals. All the piglets were fed an age-appropriate commercially available ration medicated with tiamulin, 35 g/tonne, and chlortetracycline, 400 g/tonne (Lean Metrics Infant; Purina Mills, St. Louis, Missouri, USA). Feed and water were available ad libitum.

Serologic and viremia testing before vaccinationBlood was collected from all the piglets before vaccination.

Briefly, venous blood was collected into serum separator tubes and allowed to clot at room temperature. Aliquots of serum were dispensed into appropriate tubes and held at either 2°C to 8°C or −70°C 6 10°C before testing. The samples held at 2°C to 8°C were tested for PRRSV antibodies at the Boehringer Ingelheim Vetmedica Health Management Center, Ames, Iowa, USA, with a commercially available enzyme-linked immunosorbent assay (ELISA) kit (IDEXX HerdChek PRRS X3 ELISA; IDEXX Laboratories, Westbrook, Maine, USA). Results were reported as negative [sample to positive (S/P) ratio of , 0.4] or posi-tive (S/P ratio $ 0.4). The samples held at −70°C 6 10°C were tested for PRRSV RNA by quantitative polymerase chain reaction (qPCR; bioScreen GmbH, Münster, Germany) as described by Revilla-Fernandez et al (20) with use of the 23 TaqMan Universal PCR Kit (Applied Biosystems, Foster City, California, USA) with AmpErase uracil N-glycosylase (Applied Biosystems), the EU6-MGB



(CTGTGAGAAAGCCCGGAC) probe, and the primers EU6-343f-plus (GTRGAAAGTGCTGCAGGYCTCCA; sense) and EU6-462r-plus (CACGAGGCTCCGAAGYCCW; antisense). A PCR run was consid-ered valid when the plasmid standard curve was linear, its R2 value was greater than or equal to 0.95, and the nontemplate control did not cross the threshold line. Test samples were evaluated for the presence or absence of a signal crossing the threshold, shown as the cycle threshold (Ct) value. For reporting purposes the samples were designated as positive or negative according to the point at which the PCR signal crossed the threshold in all replicates. Genomic equiva-lence (GE) was determined with use of the plasmid standard. The standard curve was generated and then used to calculate the initial concentration of an unknown sample by comparing its Ct value with the standard curve. The resulting initial concentration of the reaction (GE per reaction) was extrapolated to the amount of GE per milliliter.

VaccinationAll piglets in both experiments were administered the test vac-

cine or placebo at 14 6 3 d of age. Those in groups 1A and 1B were vaccinated IM once with 1 mL of the vaccine (according to the manufacturer ’s label instructions), at the maximum-range commercial dose (the maximum release dose of the vaccine at the time of production) and at an overdose (10 times the release dose), respectively. The piglets in group 1C served as negative controls and were administered 1 mL of sterile phosphate-buffered saline IM. The piglets in group 2A were vaccinated IM with 1 mL of the vaccine at the minimum-range commercial dose (minimum dose that provides protection). The piglets in groups 2B and 2C were injected IM with 1 mL of a lyophilized placebo product containing the inert vaccinal material without the PRRS MLV fraction.

Observations after vaccinationThe piglets in experiment 1 were observed once daily for clini-

cal signs of disease in terms of behavior (recumbency, shivering, lethargy, unconsciousness, or death), respiration (mild to severe coughing, sneezing, abdominal breathing, or rapid respiration), and digestion (vomiting, diarrhea, or change in appetite), as well as for other relevant clinical signs (e.g., hernia, thinness, lameness, and edema around the eyes) on days −2 and −1 and from days 1 to 14 after vaccination, as well as twice on day 0 (i.e., just before and at 4 h after vaccination). The piglets in experiment 2 were observed once daily for general health on days −1 to 12 after vaccination.

After vaccination, all injection sites in the piglets in experiment 1 were clearly identified with a circle by means of a black water-resistant marker and were examined for redness, swelling, heat, and pain on day 0 at 4 h after vaccination and once daily on days 1 to 14 after vaccination.

Challenge inoculationOn day 14 after vaccination the piglets in groups 2A and 2B were

challenged with the virulent, low-passage type I PRRSV isolate 205817, at a median tissue culture infective dose of 1 3 104.71 per 3 mL. The challenge inoculum was derived from isolate 190136, originally obtained from the lung tissue of a newborn piglet on a farm showing typical reproductive signs of PRRSV (abortions in sows and weakness in newborn piglets) during an outbreak in Lower

Saxony, Germany, in April 2004. The challenge isolate (205817) and the vaccine master seed isolate (94881) are heterologous members of type I PRRSV, subtype 1, isolated from geographically distinct regions in Germany and exhibiting less than 87% genetic identity within the complete genome (or 88% with the ORF5/ORF7 data) (21).

Observations after challengeClinical observations were conducted on the piglets in experiment 2

once daily from days 13 to 24 after vaccination (days −1 to 10 after challenge) by personnel that were blinded to the treatment-group assignment. Each piglet was visually examined in the pen before handling and was scored for clinical signs including coughing (none, soft or intermittent, harsh or severe and repetitive, or dead) and abnor-malities of respiration (normal, panting/rapid respiration, dyspnea, or dead) and behavior (normal, mild to moderate lethargy, severely lethargic/recumbent, or dead).

Rectal temperature was determined with a self-calibrating digital thermometer in the piglets in experiment 1 on days −2 and −1 and from days 1 to 14 after vaccination, as well as twice on day 0 (just before and at 4 h after vaccination), and in the piglets in experiment 2 from days 13 to 24 after vaccination (days −1 to 10 after challenge). A normal physiological range was considered to be 38.7°C to 39.9°C, and fever was defined as an increase in temperature to 40.0°C or beyond.

In experiment 1 the individual body weight was measured in all the piglets on days −2, 0, and 14 after vaccination, and the aver-age daily weight gain (ADWG) was determined from days 0 to 14 after vaccination. In experiment 2 the individual body weight was measured in all the piglets on days 0, 14, and 24 after vaccination, and the ADWG was calculated from days 0 to 14 and days 14 to 24 after vaccination.

Serologic and viremia testing for PRRSBlood was collected via jugular venipuncture from the piglets in

experiment 1 on days 7 and 14 after vaccination and from the piglets in experiment 2 on days 7, 14, 17, 21, and 24 after vaccination. Blood was processed for serum and used to determine the S/P ratio of anti-bodies against PRRSV by ELISA and PRRSV viremia (log10 GE/mL) by qPCR as described.

Postmortem examination and lung studiesAll the piglets in experiment 1 were euthanized by sedation and

then electrocution and underwent necropsy on day 14 after vacci-nation. Each injection site was palpated, incised, and evaluated for gross lesions. Additionally, the thoracic and abdominal cavities were exposed and examined for gross lesions. Two lung tissue samples containing lesions were collected from 1 piglet (in group 1A). One sample was placed in a Whirlpak (United States Plastic Corporation, Lima, Ohio, USA) and the other in a container with 10% formalin and submitted to the Iowa State University Veterinary Diagnostic Laboratory, Ames, Iowa, USA. The fresh sample was cultured for bacteria and the formalin-fixed sample examined histopatho-logically and by immunohistochemistry testing for PRRSV, porcine circovirus 2 (PCV-2), and Mycoplasma hyopneumoniae antigens.

All the piglets in experiment 2 were similarly euthanized and underwent necropsy on day 24 after vaccination (day 10 after



challenge). The thoracic cavity was exposed, and the heart and lungs were removed. The lungs were examined for gross lesions, and the percentage of each lobe that was abnormal was recorded. Total lung lesion scores were determined as a percentage of lung involvement, calculated according to a weighting formula that accounts for the relative weight of each of the 7 lobes. The assessed percentage of lung lobe area with typical lesions was multiplied by the lobe factor (i.e., left apical = 0.05, left cardiac = 0.06, left diaphragmatic = 0.29, right apical = 0.11, right cardiac = 0.10, right diaphragmatic = 0.34, and intermediate = 0.05), and the total weighted lung lesion score was determined.

Lung samples were collected from all the piglets in experiment 2 for quantitation of PRRSV in the lung tissue. Samples in Whirlpaks were stored at 270°C 6 10°C until thawed and liquefied into a homogenate, subjected to RNA extraction procedures, and then tested for PRRSV RNA by qPCR as described. The results were reported as log10 GE/mL for left and right/intermediate lung samples.

Statistical analysesThe statistical analyses were conducted and data summaries

prepared by Dr. Martin Vanselow (Biometrie & Statistik, Hannover, Germany) using SAS software release 8.2 or later (SAS Institute, Cary, North Carolina, USA).

For experiment 1, all data listings and summary statistics by treat-ment group, including mean, median, standard deviation, and/or frequency distribution, were generated for all primary variables, including local (injection-site) reactions, systemic (behavioral, respiratory, digestive, and other, as well as fever) reactions, and ADWG. Specifically, the proportions of piglets in each group with any abnormal clinical observation, with a specific clinical observation (behavioral, respiratory, digestive, or other score greater than 0), with an increase in rectal temperature greater than 1.5°C when compared with baseline (day 0), with any injection-site reaction score greater than 0, or with a specific injection-site reaction score greater than 0 for at least 1 d from 0 1 4 h to 14 d after vaccination were evalu-ated by Fisher’s exact test; the numbers of days per animal with any abnormal clinical observation and with any injection-site reac-tion from 0 1 4 h to 14 d after vaccination were evaluated by the Wilcoxon Mann–Whitney test; the mean daily rectal temperature for each group from 0 1 4 h to 14 d after vaccination and the initial mean body weight for each group on day 0 were evaluated by analysis of variance (ANOVA); and the mean ADWG for each group from 0 to 14 d after vaccination was evaluated by the t-test.

For experiment 2, data were analyzed with the assumption of a randomized block design, and all tests on differences were designed as 2-sided at an a-value of 5%. Frequency tables of animals with at least 1 positive clinical finding between days 1 and 12 after vaccina-tion, animals with at least 1 positive clinical finding from days 15 to 24 after vaccination (days 1 to 10 after challenge), and animals with positive ELISA results were generated, and differences between groups were compared by Fisher’s exact test. Differences in mean daily rectal temperature between treatment groups, as compared with baseline, were tested by ANOVA and t-tests, and least-squares (LS) means of groups and differences between LS means with 95% confidence intervals were calculated from the ANOVA. Comparisons

between treatment groups for total lung lesion scores, maximum and mean clinical scores (for coughing and abnormalities of respi-ration and behavior, as well as for all 3 added together) per animal for days 15 to 24 after vaccination, the PRRSV qPCR (viremia) data (evaluated separately for each day), as well as the area under the curve (AUC) for individual responses between days 14 and 24 and between days 17 and 24 after vaccination were analyzed with the Wilcoxon Mann–Whitney test.

Re s u l t sAfter vaccination no piglets in groups 1A and 1B (single-dose and

overdose vaccination, respectively) exhibited abnormal behavior, whereas 1 piglet in group 1C (placebo) was lethargic on days 8 and 9. There was no significant difference in behavior (P = 0.4762) between the vaccinated groups and the nonvaccinated group. Conversely, at least 1 piglet in each group exhibited abnormal respiration for a minimum of 1 d after vaccination: 1 piglet in group 1A, 2 piglets in group 1B, and 3 piglets in group 1C exhibited mild coughing; sneez-ing was noted for 3 piglets in group 1C; and severe coughing was noted for 1 piglet. There was no significant difference in respiration (P $ 0.1486) between the vaccinated groups and the nonvaccinated group. No piglets exhibited any abnormal digestive findings after vaccination. Several abnormal clinical findings not related to PRRSV vaccination (designated as “other”) were also noted: 1 piglet in group 1A was noted as thin on days 7 to 10 after vaccination (likely a result of decreased feed intake due to competition at the feeder) but was otherwise normal; 1 piglet in group 1B was found to have rectal prolapse on days 6 to 12; and 1 piglet in group 1B was noted as having a scrotal hernia on days 10 to 14. There was no significant difference in “other” clinical signs (P $ 0.4762) between the vacci-nated groups and the nonvaccinated group. Likewise, no significant differences in clinical observations were noted between either of the vaccinated groups and the nonvaccinated group when all categories of clinical observation were combined (P $ 0.3615) or in the num-ber of days piglets exhibited any clinical abnormalities (P = 0.2662 and 0.7726 for groups 1A and 1B, respectively) (Table I). No clinical abnormalities related to PRRSV were noted in any piglets in experi-ment 2 after vaccination and before challenge (i.e., on days −1 to 12 after vaccination); however, 1 piglet in group 2B had a lesion anterior to the right front leg beginning 9 d after vaccination.

At the injection site, redness, heat, and pain were not noted in any piglet in experiment 1, but 1 piglet each in groups 1A (single dose) and 1B (overdose) exhibited minimal swelling (palpable only) for at

Table I. Number of days on which piglets exhibited any abnormal clinical signs after vaccination against porcine reproductive and respiratory syndrome virus (PRRSV)

Number Number Group of piglets of days P-valuea

1A (maximum dose) 11 4 0.26621B (overdose) 11 7 0.77261C (placebo) 10 8 NAa In comparison with group 1C.NA — not applicable (no analysis conducted).



least 1 d after vaccination; no piglets in group 1C (placebo) exhibited swelling after vaccination. These differences were not significant (P = 1.0000) (Table II).

After challenge, abnormal respiration was observed in 2 (10%) of the 20 vaccinates (group 2A) and 6 (30%) of the 20 challenge con-trols (group 2B); however, these proportions were not significantly different (P = 0.2351). Groups 2A and 2B had maximum abnormal respiration scores of 1 (panting/rapid respiration) and 2 (dyspnea), respectively, a difference that was not significant (P = 0.1872); both groups had a median maximum respiration score of 0. The mean respiration score for group 2A was lower than that for group 2B (0.02 versus 0.07; P = 0.1394). Similarly, although coughing was observed in more of the group 2B piglets than in the group 2A piglets [11 of 20 (55%) versus 6 of 20 (30%)], the difference was not significant (P = 0.2003). Groups 2A and 2B had maximum scores for coughing of 1 (soft or intermittent; median score of 0) and 2 (harsh or severe and repetitive; median score of 1), respectively; these differences were not significant (P = 0.1129). The mean coughing score was lower for group 2A than for group 2B (0.07 versus 0.17; P = 0.0535). Abnormal behavior was observed in significantly fewer piglets (P = 0.0012) in group 2A than in group 2B: 0 of 20 versus 9 of 20 (45%). Group 2A had a significantly lower (P = 0.0012) maximum behavior score than group 2B: 0 (normal) versus 1 (mild to moderate lethargy). The mean behavior score for group 2A was significantly lower (P = 0.0012) than that for group 2B: 0.00 versus 0.12. When combined, the percentages of piglets with total clinical scores greater than 0 were 30% (6 of 20)

in group 2A and 65% (13 of 20) in group 2B and were not signifi-cantly different (P = 0.0562). Group 2A had a significantly lower maximum total score than group 2B: 1 versus 4 (P = 0.0072). The mean total scores for group 2A were significantly lower (P = 0.0103) than those for group 2B: 0.08 versus 0.35. No clinical signs were observed in the negative controls (group 2C) at any time after challenge, and the group had a score of 0 for each parameter.

For the piglets in experiment 1, all the mean rectal temperatures were within the normal physiological range, and no piglets exhibited an increase in rectal temperature of 1.5°C or more above baseline on any day after vaccination. In experiment 2, the mean and LS mean rectal temperatures for the piglets in group 2A (vaccinated and challenged) were 39.77°C on the day before challenge (day 13 after vaccination) and ranged from 39.69°C to 40.68°C after challenge (on days 1 and 2 after challenge, respectively), for the piglets in group 2B (nonvaccinated and challenged) they were 39.39°C on the day before challenge and ranged from 39.77°C to 40.61°C after chal-lenge (on days 2 and 6, respectively), and for the piglets in group 2C (nonvaccinated and not challenged) they remained at 39.68°C or less throughout the study. The LS means were significantly lower (P , 0.0001) for the piglets in group 2B than for those in group 2A the day before challenge (39.39°C versus 39.77°C), the day of chal-lenge (39.37°C versus 39.76°C), and 2 d after challenge (39.77°C versus 40.68°C). There were no other significant differences (P $ 0.0528) between the 2 groups on any day, as the rectal temperatures for both groups were at or above 40°C from days 4 (group 2B) and 5 (group 2A) through day 10 after challenge (Figure 1).

In experiment 1 there were no significant differences in mean body weight between the groups on days 0 and 14 after vaccina-tion (P = 0.7582 and P $ 0.2273, respectively) and no significant differences (P = 0.1562 and 0.1628, respectively) in ADWG from 0 to 14 d after vaccination (Table III). In experiment 2 there were no significant differences in LS mean body weight between the piglets in groups 2A and 2B on days 0 (P = 0.8743) and 14 (P = 0.4297) after vaccination. However, on day 24 after vaccination (day 10 after chal-lenge) the LS mean body weight of the piglets in group 2A (10.26 kg)

Table II. Number of days after vaccination on which any injection-site reaction was noted

Number Number of daysGroup of piglets Minimum Maximum Median 95% CI Mean P-valuea

1A (maximum dose) 11 0 4 0 0 0.4 1.00001B (overdose) 11 0 3 0 0 0.3 1.00001C 10 0 0 0 0 0.0 NAa In comparison with group 1C.CI — confidence interval; NA — not available.

Table III. Mean body weight and average daily weight gain (ADWG) on days 0 to 14 after vaccination in experiment 1

Weight (kg) ADWGGroup Day 0 Day 14 P-valuea (kg/d) P-valuea

1A 4.5 8.1 0.7582 0.3 0.15621B 4.3 8.0 $ 0.2273 0.3 0.16281C 4.4 8.6 NA 0.3 NAa In comparison with group 1C.NA — not available.

Figure 1. Mean rectal temperatures of piglets after challenge with a heterologous strain of type I porcine reproductive and respiratory syn-drome virus (PRRSV) (groups 2A and 2B) 14 d after administration of the test vaccine (Ingelvac PRRSFLEX EU; Boehringer Ingelheim Vetmedica, St. Joseph, Missouri, USA) at the minimum-range commercial dose (group 2A) or a placebo (groups 2B and 2C). The asterisks indicate a significant difference (P , 0.0001) in the temperatures for group 2B compared with group 2A on days −1, 0, and 2 after challenge.

Days after challenge

Deg

rees

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was significantly higher (P = 0.0063) than that of group 2B (8.87 kg). Likewise, the difference in LS mean ADWG between groups 2A and 2B was not significant (P = 0.1889) from days 0 to 14 after vac-cination; however, from days 14 to 24 (days 0 to 10 after challenge) the LS mean ADWG was significantly higher (P = 0.0003) for the piglets in group 2A compared with those in group 2B (Table IV). Therefore, the piglets vaccinated 14 d before challenge had an ADWG significantly higher than the piglets that were not vaccinated before challenge.

All the piglets in experiment 1 were PRRSV-seronegative on day 0 after vaccination. On day 14 all 11 piglets in group 1A and 10 (91%) of the 11 piglets in group 1B were seropositive, whereas the 10 pig-lets in group 1C remained seronegative. These results indicate that the single dose and overdose of vaccine were effective at causing an immune response. All the piglets in experiment 2 were PRRSV-seronegative on days 0 and 7 after vaccination. On day 14 the propor-tion of seropositive piglets was significantly higher (P , 0.0001) in group 2A than in group 2B: 17 of 20 (85%) versus 0 of 20. Three days after challenge (17 d after vaccination) the proportion of seropositive piglets in group 2A had increased to 19 of 20 (95%); the piglets in group 2B remained seronegative. On day 7 after challenge (day 21 after vaccination) 11 of 20 (55%) of the piglets in group 2B were seropositive, a significant difference (P # 0.0012) from the 20 of 20 in group 2A. On day 10 after challenge (day 24 after vaccination), however, 20 (100%) and 19 (95%) of the piglets in groups 2A and 2B, respectively, were PRRSV-seropositive. All the piglets in group 2C remained seronegative throughout the experiment.

No PRRSV RNA was detected in the serum of any piglets in experiment 2 on day 0. On days 7 and 14 after vaccination the pig-lets in group 2A had median values of 3.00 and 3.32 log10 GE/mL, respectively, whereas group 2B (nonvaccinated) remained free of PRRSV RNA; however, on day 17 (day 3 after challenge) the piglets in group 2B had significantly higher values (P , 0.0001) than those in group 2A: 8.18 versus 6.72 log10 GE/mL. There were no significant differences (P $ 0.0565) between the 2 groups on days 21 and 24 after vaccination (days 7 and 10 after challenge), the values for group 2A being 7.38 and 7.15 log10 GE/mL, respec-tively, and those for group 2B being 7.87 and 7.27 log10 GE/mL, respectively. However, on days 17 to 24 after vaccination (days 3 to 10 after challenge) the daily AUC for group 2A was significantly lower (P = 0.0039) than the AUC for group 2B: 49.52 GE/mL versus

54.35 GE/mL. No PRRSV RNA was detected in the serum of any piglet in group 2C (Figure 2).

No injection-site abnormalities or gross lesions in the abdominal and thoracic cavities were noted at necropsy for any of the piglets in experiment 1, except for 1 piglet in group 1A that had exhibited mild coughing for 1 d and was found to have minor lung lesions. After sampling and testing, the lesions were determined to be a result of infection with Bordetella bronchiseptica. The piglet was negative for PRRSV, PCV-2, and M. hyopneumoniae in IHC testing. Interestingly, the 4 group 1C piglets that exhibited sneezing, mild coughing, or severe coughing did not have gross lesions at necropsy. Additionally, at the time of necropsy the weight of the group 1A piglet that had been observed as thin for 4 d was found to be within the range of the other piglets in the group, and no gross lesions were noted. Thus, the sneezing, coughing, and temporary thinness noted for the piglets were not associated with vaccination. In experiment 2, the mean total lung lesion score was significantly lower (P = 0.0002) for group 2A than for group 2B: 27.368% versus 54.841% (Table V).

Although PRRSV RNA was detected in the lung tissue of all pig-lets in groups 2A and 2B, the mean qPCR values were significantly

Table IV. Least-squares mean weight and ADWG on days 0 to 14 and 14 to 24 after vaccination and days 0 to 10 after challenge with a virulent heterologous type I PRRSV isolate 14 d after vaccination in experiment 2

ADWG (kg/d) Weight (kg) On days 0 to 14 On days 0 to 10Group Day 0 Day 14a Day 24b P-valuec after vaccination P-valued after challenge P-valued

2A (20 vaccinates) 4.14 7.64 10.26 0.0063

0.25 0.1889

0.26 0.0003

2B (20 controls) 4.17 7.39 8.87 0.23 0.15a Day 0 after challenge.b Day 10 after challenge.c For the difference between groups 2A and 2B on day 24 after vaccination. On days 0 and 14 after vaccination there were no significant differences between the 2 groups (P = 0.8743 and 0.4297, respectively).d For differences between groups 2A and 2B.

Figure 2. Amount of PRRSV RNA in the serum as tested by quantitative polymerase chain reaction. The genomic equivalence (GE) of the initial concentration of the reaction (GE per reaction) was extrapolated to the amount of GE per milliliter. The asterisks indicate significantly greater amounts (P , 0.0001) for the piglets in group 2A compared with those in groups 2B and 2C on days 7 and 14 after vaccination and signifi-cantly lower amounts (P , 0.0001) for the piglets in group 2A 2 d after challenge (day 17 after vaccination) compared with the nonvaccinated piglets in group 2B.

Days after vaccination

log 10

GE

/mL



lower (P = 0.0101) in group 2A than in group 2B at the time of nec-ropsy (day 24 after vaccination; day 10 after challenge): 7.47 log10

GE/mL versus 7.88 log10 GE/mL.

D i s c u s s i o nVaccinating weaned piglets with a PRRS MLV vaccine is a benefi-

cial practice for a variety of reasons, such as increased ADWG and decreased frequency of clinical signs in piglets exposed to PRRSV (1,5,21). The effectiveness of a PRRS MLV vaccine in protecting pig-lets from clinical signs is important because clinical disease generally leads to decreased health and growth. In addition to being effective, a PRRS MLV vaccine must be safe to administer (i.e., not cause local or systemic reactions and result in minimal viremia and virus shed-ding). Safety was evaluated in this study by monitoring for local and systemic reactions (including through clinical assessment) and calculating the ADWG; effectiveness was determined primarily from the total lung lesion scores, with supportive parameters including the results of clinical assessment, serologic study, viremia testing after vaccination, ADWG, and viral load in lung tissues. According to these criteria the data clearly demonstrated that vaccination with the novel type I PRRSV 94881-based MLV vaccine (Ingelvac PRRSFLEX EU) is a safe option for reducing the incidence of disease associated with PRRSV. Specifically, in experiment 1 young piglets (16 to 17 d of age) that had been vaccinated once with either the maximum-range commercial dose or an overdose of the test vac-cine did not exhibit any relevant differences in local or systemic reactions when compared with nonvaccinated placebo recipients, and in experiment 2 no vaccinated piglets exhibited clinical abnor-malities associated with vaccination. Likewise, in both experiments there were no significant differences between the vaccinated groups when compared with the nonvaccinated controls in regard to indi-vidual clinical signs, including behavior, respiration, digestion, and others, as well as in regard to total clinical signs after vaccination. However, the piglets in group 1B (those receiving a vaccine over-dose) had significantly higher rectal temperatures than the piglets in group 1C (those receiving a placebo) on days 3, 5, 7, 9, and 11 after vaccination, and the piglets in group 2A (challenged vaccinates) had significantly higher rectal temperatures than those in group 2B (challenged piglets that had not been vaccinated) on days −1 and 0 after challenge; however, these differences were not biologically relevant. Thus, the significance of elevated rectal temperatures is ambiguous. Nevertheless, the serologic results from experiment 2 show that a single dose of the vaccine at the minimum commercial

range induces immunity 14 d after vaccination when administered to very young piglets (14 6 3 d of age). Moreover, when compared with the nonvaccinated piglets the vaccinated piglets in experiment 2 had significantly lower lung lesion scores, viremia, viremia over time (AUC), viral load in lung tissue, behavior scores, and total clinical scores, and they had a significantly higher ADWG after challenge (i.e., 14 to 24 d after vaccination).

Several previously reported studies examined the clinical effec-tiveness of MLV PRRS vaccines. Martelli et al (4) found that a single IM or intradermal (ID) dose of an MLV vaccine (based on the type I PRRSV vaccinal strain DV) administered to 5-week-old piglets offered approximately 70% protection against clinical signs of disease when the piglets were challenged with a heterologous type I Italian wild-type strain (05R1421) through natural exposure 45 d after vac-cination. The challenge strain was shown to be virulent, as 100% of the controls had increased rectal temperatures, increased lethargy, and decreased appetite throughout the study compared with 90% of the vaccinated animals, which showed consistently less severe signs of disease. However, the frequency and severity of clinical signs in all the groups may have been complicated by the presence of other pathogens within the herd (i.e., PCV-2, M. hyopneumoniae, Pasteurella multocida, and Streptococcus sp.). Various reports have indicated that PRRSV may have an immunosuppressive effect, thus making pigs more susceptible to secondary infections (5,12,14). Additionally, the exposure levels of the field challenge will vary among herds and between individual pigs, such that it is difficult to determine the occurrence of a vaccinal effect or if some animals were less exposed to the challenge virus. Another study evaluated a single-strain MLV vaccine (based on the EU vaccinal strain ALL-183) (19) administered on 2 occasions, 3 wk apart, to 5-month-old gilts that were challenged with a heterologous Lelystad strain 28 d after initial vaccination (18). In this trial it was determined that the vaccine had no significant effect on clinical signs, rectal temperatures, and ADWG; however, the challenge strain was not considered virulent. On the other hand, there were significant differences between the vaccinated and nonvaccinated pigs in level of viremia and viral load in lungs and tonsils after heterologous challenge, as in the present study. In contrast, the vaccine did not induce a serologic response to PRRSV until 21 d after initial vaccination; the ELISA S/P ratio was negative (, 0.4) on day 14 after vaccination, whereas in the present study there was a positive strong serologic response, an S/P ratio $ 0.4, in 17 out of 20 piglets on day 14. Likewise, another study found that pigs vaccinated once at 7 wk of age with a commercially available attenuated vaccine based on the type I strain DV experienced a

Table V. Total lung lesion scores (percentages of lung involvement) for the piglets in experiment 2 at necropsy on day 24 (day 10 after challenge)

Groupa Minimum Maximum Median 95% CI IQR Mean P-value2A 0.06 59.30 27.550 12.270 40.600 29.515 27.368

0.00022B 13.86 91.60 55.200 47.300 66.500 21.850 54.8412C 0.00 0.06 0.000 0.000 0.000 0.000 0.006 NIa The 20 group 2A piglets were vaccinated with the minimum-range commercial dose of the test vaccine, whereas the other 2 groups received a placebo. The 20 group 2B piglets were challenged with the same strain as group 2A, whereas the 10 group 2C piglets were not challenged.IQR — interquartile range; NI — not included in the statistical analysis; CI — confidence interval.



shortened duration of viremia compared with pigs vaccinated twice, at 5 and 9 wk of age, with a commercially available inactivated vac-cine based on the type I strain P120 or with either of 2 experimental inactivated vaccines based on the type I field isolates 07 V063 and 08 V194 in the face of fever-inducing heterologous challenge with either 07 V063 or 08 V194 (resulting in temperatures . 39.5°C and # 40.6°C, respectively) at 13 wk of age (5). Trus et al (11) reported a significant reduction in severity and duration of fever as well as in the AUC for viremia in 4- and 7-week-old pigs vaccinated with an MLV PRRS vaccine (type I strain DV) and challenged with a highly pathogenic PRRSV strain (Lena, isolated from a farm in the Republic of Belarus) 8 and 6 wk, respectively, after vaccination when compared with nonvaccinated controls. However, differences in gross lung lesions between the groups were not significant. This trial used older pigs that were challenged much later after vac-cination, compared with the present study, in which the younger piglets were challenged 14 d after vaccination. Another study using an MLV PRRS vaccine based on the European DV strain found that vaccinated 4-week-old pigs exhibited significantly reduced viremia levels and shorter duration of viremia, compared with nonvaccinated controls, when challenged with a heterologous strain (3267, isolated from a Portuguese farm) by contact with inoculated pigs 37 d after vaccination (12). The challenge strain was not known to induce noteworthy clinical signs outside of mild respiratory symptoms, which is characteristic of type I PRRSV strains; however, PRRSV was detected by means of qPCR in the lung tissue of all vaccinated and nonvaccinated pigs that were subsequently euthanized (n = 5 and 4, respectively). In contrast, in the present study the vaccinated piglets had significantly lower scores for total clinical signs, reduced PRRS viral load in the lung tissue, and, most importantly, significant reductions in scores for PRRSV-specific lung lesions compared with the nonvaccinated piglets after challenge. Roca et al (22) found that a commercially available MLV vaccine based on type I PRRSV strain VP046 BIS administered to 4-week-old pigs offered partial protection against challenge with a highly virulent type II strain (HP-PRRS21) 6 wk after vaccination. In that trial the vaccinated piglets showed a significant reduction in fever on 2 d (days 11 and 12 after challenge), significantly increased weight gain, decreased lethargy and/or anorexia, and decreased viremia (which was detected only on day 7 after challenge); however, the vaccine did not significantly reduce the score for lung lesions. In comparison, in the present study the vaccinated piglets had significantly lower LS mean rectal temperatures 1 d after challenge, significantly increased ADWG in the postchallenge period, a significantly lower incidence of abnormal behavior (lethargy), and significantly reduced viremia (on day 3 after challenge and AUC on days 3 to 10 after challenge) and lung abnormalities compared with the nonvaccinates after challenge. Prieto et al (13) evaluated the efficacy of multiple doses of vaccine before challenge: 4-week-old pigs were vaccinated on 3 occasions, 21 d apart, with the type I strain DV, beginning at 28 d of age, and were challenged with the type I PRRSV strain 5710 (isolated in Spain) 4 wk after the final vaccination. There were no significant differences between the vaccinated and nonvaccinated pigs in clini-cal signs or virus titer in all tissues collected at necropsy. However, the clinical signs were less severe in the vaccinated pigs, and the virus was found less frequently in their tissues, compared with the

nonvaccinated pigs. Another study using the MLV PRRS European DV strain determined (by virus titration in bronchoalveolar lavage fluids and serum by immunoperoxidase monolayer assay) that pigs vaccinated at 5 wk of age either IM or ID were fully protected from homologous wild-type challenge but only partially protected from heterologous challenge (with an Italian strain) at 49 d after vaccina-tion, as evidenced by significantly lower mean virus titers in serum samples from the vaccinated pigs as compared with the nonvacci-nated controls (4). Antibodies to PRRSV began developing around 7 d after vaccination; however, 100% seroconversion did not occur until 35 d after vaccination, whereas in the present study the pigs in groups 1A, 1B, and 2A were 100%, 91%, and 85% seropositive, respectively, on day 14 after vaccination. Likewise, the data from the present study show that the novel PRRSV vaccinal strain 94881 was effective against viremia after challenge, the vaccinates having significantly lower mean log10 GE/mL values on day 3 after chal-lenge and a significantly lower AUC for 3 to 10 d after challenge compared with the controls.

Effectiveness of vaccination was shown in the above instances in a manner similar to that in the present study (i.e., through decreased clinical signs, viremia, and viral load in lung tissue, as well as increased ADWG). However, no other vaccine was shown to positively affect all these parameters in a single trial involving challenge with a heterologous European-derived, type I PRRSV that induced clinical signs and lung lesions. In contrast to the previously reported studies, the present study showed significant reductions in lung abnormalities in young pigs vaccinated with the PRRS 94881 MLV vaccine. Moreover, to our knowledge this is the first time a 14-d OOI has been reported in piglets vaccinated before 3 wk of age.

Most PRRS MLV vaccines are considered safe if there are no significant local and/or systemic reactions, as well as differences in clinical signs, after vaccination (5,7,9,13,17); however, little is known about the likelihood of European-derived PRRS MLV vaccinal strains to cause viremia as well as the shedding of vaccine virus and subse-quent transmission to naïve pigs. One study aimed to alleviate this knowledge gap by evaluating the safety of 3 vaccines based on type I strains that are commercially available in Europe (9): 4-week-old pigs were vaccinated with 3 PRRS MLV vaccines based on the strains VP046 BIS, ALL-183, and DV, and unvaccinated sentinel pigs were introduced at day 3 after vaccination. As with the present study, the vaccines were all deemed clinically safe, as no significant differences were noted in clinical signs between vaccinated and nonvaccinated pigs, and vaccination did not negatively affect ADWG. In contrast, the authors found that the vaccinated groups had varied mild lung lesion scores on 1 or more necropsy days (i.e., 7, 14, and 21 d after vaccination) that decreased over time. Additionally, all the vaccine strains induced viremia (in 57.3% to 88% of the pigs), and PRRSV was found in lung tissues in all the groups (in 20% to 66.7%). These results are contrary to the data from the present study, as no pigs in experiment 1 had lung lesions associated with vaccination, and PRRSV was not isolated from lung tissues at the time of necropsy even after the pig had received an overdose of the vaccine. Although the vaccinated piglets in experiment 2 also exhibited viremia, the level was significantly less than in the nonvaccinated group after challenge. Moreover, the vaccinated groups in the safety trial (9) shed virus in oropharyngeal and nasal secretions as well as feces (4.89%



to 6.67% of all samples tested positive); thus, the sentinel pigs for each group became viremic and subsequently shed virus (2.22% to 5.71% of all samples tested positive). Although the vaccinated pigs in the present study did exhibit viremia, there was no evidence that the vaccine virus infected their lungs even after an overdose. Additionally, good biosecurity practices ensured that all negative control animals remained negative for all parameters tested. Other studies have also reported the presence of vaccine virus in the blood after vaccination (4,9,13,17).

These studies, as well as the present data, support the knowledge that the variability of PRRSV continues to confound the development of a single-strain vaccine that may be effective against the virus in the field. Although it is difficult to compare these data in a side-by-side manner, similarities and trends do exist. Generally, vaccination with an MLV vaccine has been found to offer significant protection against certain aspects of PRRSV infection (viremia, clinical signs, decreased productivity, etc.). The present study offers evidence that a vaccine based on the novel type I PRRSV strain 94881 administered to very young piglets may offer similar, if not improved, protec-tion against the negative clinical and economic effects of PRRSV infection.

In conclusion, a vaccine that is safe and effective in preventing the detrimental effects of type I PRRSV in a timely manner is an essential part of swine production practices. The results from this study support the clinical safety and efficacy of Ingelvac PRRSFLEX EU when administered to piglets approximately 14 d of age. When the vaccine was administered once at the maximum-range com-mercial dose or as an overdose, there were no significant differences in clinical signs attributable to the vaccine; only transient, minimal swelling was noted at the injection site of 1 piglet, and ADWG was not affected. Through the significant reduction in lung lesions, viremia, viral load in lung tissue, and clinical signs, as well as the improvement in ADWG, it has been shown that administration of a single IM dose of the novel PRRS 94881 MLV vaccine to piglets induced the response required to build protective immunity by 14 d after vaccination. Consequently, a vaccine formulated from the type I PRRS viral strain 94881 has been proven to be a safe and effective method of protection against the detrimental effects of virulent PRRSV infection in young piglets.

A c k n o w l e d g m e n t sThe authors thank Dr. Ryan Saltzman, Dr. Lyle Kesl, Dr. Stephan

Pesch, Mr. Rex Smiley, Dr. Alicia Zimmerman, and Ms. Sarah Layton for technical assistance.

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10. Mengeling WL, Lager KM, Vorwald AC, Koehler KJ. Strain specificity of the immune response of pigs after vaccination with various strains of porcine reproductive and respiratory syndrome virus. Vet Microbiol 2003;93:13–24.

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12. Pileri E, Gibert E, Soldevila F, et al. Vaccination with a geno-type 1 modified live vaccine against porcine reproductive and respiratory syndrome virus significantly reduces viremia, viral shedding and transmission of the virus in a quasi-natural experi-mental model. Vet Microbiol 2015;175:7–16.

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Article


I n t r o d u c t i o nInterferon-g (IFN-g; also called type II interferon), a cytokine

produced predominantly by T-helper type 1 (TH1) cells and Natural Killer cells in response to antigenic or mitogenic stimulation (1,2), plays a critical role in initiating and regulating cell mediated immu-nity, which is a central player in initiating the TH1 response against intracellular pathogens (3,4).

Chicken provides an important animal model of a number of intracellular infections. Like its mammalian counterpart, chicken IFN-g (ChIFN-g) strongly upregulates the expression of class II major histocompatibility complex (MHC) proteins (5–7) so that anti-microbial and antiviral activities of chickens are improved (5,7–10). The ChIFN-g also enhances immunity against tumors and parasites (11–15). Previous studies showed that the level of IFN-g following antigenic/mitogenic stimulation allows for accurate evaluation of cell-mediated immunity (CMI) (16,17). Unfortunately, methods of detecting ChIFN-g are limited. So far, ChIFN-g is commonly detected based on its ability to inhibit viral replication in vitro or activate the HD11 macrophages. Both of these assays are labor-intensive, time-consuming, and nonspecific methods that exhibit low sensitivity

and are difficult to standardize. Although real-time PCR (RT-PCR) or Northern blot can detect very low levels of ChIFN-g, these meth-ods can be used to analyze ChIFN-g only at the mRNA, but not the protein level.

Therefore, a qualitative and quantitative assay to accurately and efficiently determine ChIFN-g levels in biological samples is extremely urgent, especially to study response to infections induced by intracellular bacteria, parasites, and viruses. Until now, there were 2 kinds of assays reported that could successfully evaluate ChIFN-g in the protein level (18,19). One is a monoclonal antibody (mAb)-based direct binding enzyme-linked immunosorbent assay (ELISA), the other is a quantitative ELISA based on the combination of a rabbit anti-ChIFN-g serum with a mAb. Both of them could measure ChIFN-g in a variety of formats and the latter is more sensitive than the former. But these assays are still limited and need to be improved in detecting trace amounts of ChIFN-g. To address this problem, this study was designed to develop a ChIFN-g-specific ELISA. We have used recombinant ChIFN-g, which was generated before (20) to develop mAbs against ChIFN-g. Using these antibod-ies we have developed a capture ELISA system for the detection of both recombinant and native ChIFN-g.

Development of a double-monoclonal antibody sandwich ELISA: Tool for chicken interferon-g detection ex vivo

Hua Dai, Zheng-zhong Xu, Meiling Wang, Jun-hua Chen, Xiang Chen, Zhi-ming Pan, Xin-an Jiao

A b s t r a c tThe aim of the present work was to develop reagents to set up a chicken interferon-g (ChIFN-g) assay. Four monoclonal antibodies (mAbs) specific for ChIFN-g were generated to establish sandwich ELISA based on 2 different mAbs. To improve the detection sensitivity of ChIFN-g, a double-monoclonal antibody sandwich ELISA was developed using mAb 3E5 as capture antibody and biotinylated mAb 3E3 as a detection reagent. The results revealed that this ELISA has high sensitivity, allowing for the detection of 125 to 500 pg/mL of recombinant ChIFN-g, and also has an excellent capacity for detecting native ChIFN-g. This ELISA was then used to detect ChIFN-g level in chickens immunized with a Newcastle disease virus (NDV) vaccine, the immunized chicken splenocytes were stimulated by NDV F protein as recall antigen. From our results, it appears that the sensitivity range of this sandwich ELISA test is adequate to measure the ex vivo release of ChIFN-g.

R é s u m éL’objectif de la présente étude était de développer des réactifs afin de mettre au point une épreuve de détection de l’interféron-g de poulet (ChIFN-g). Quatre anticorps monoclonaux (AcMo) spécifiques pour ChIFN-g ont été produits afin d’avoir un ELISA sandwich reposant sur deux AcMo différents. Afin d’améliorer la sensibilité de détection de ChIFN-g, un test ELISA sandwich a deux anticorps monoclonaux a été développé utilisant l’AcMo 3E5 comme anticorps de capture et l’AcMo 3E3 biotinylé comme réactif de détection. Les résultats ont démontré que ce test ELISA possède une sensibilité élevée, permettant la détection de 125 à 500 pg/mL de ChIFN-g recombinant, et ayant également une excellente capacité à détecter le ChIFN-g original. Ce test ELISA a par la suite été utilisé pour détecter les quantités de ChIFN-g chez des poulets immunisés avec un vaccin contre la maladie de Newcastle (NDV), les cellules de la rate des poulets immunisés ont été stimulées par la protéine F du NDV comme antigène de rappel. À partir de nos résultats, il semble que la plage de sensibilité de ce test ELISA sandwich est adéquate pour mesurer la libération ex vivo de ChIFN-g.


Jiangsu Key Lab of Zoonosis, Yangzhou University, China (Dai, Xu, Chen, Chen X, Pan, Jiao); College of Medicine, Yangzhou University, China (Wang).

Address all correspondence to Dr. Xin-an Jiao; e-mail: [email protected]

Received April 21, 2015. Accepted July 20, 2015.




ChickensFour-week-old, white Laihang, specific pathogen free (SPF)

chickens and 8-week-old BALB/C mice used in this study were pro-vided by the Comparative Medical Center of Yangzhou University (Yangzhou, China), the animals were housed and handled at the Animal Biosafety Facilities and all procedures were approved by the Institutional Animal Experimental Committee.

Vaccines, plasmids, and recombinant proteinsThe Newcastle disease mild, living (La Sota strain) vaccine

(100992007, Wuhan Chopper Biology, Wuhan, Hubei, China) was used to vaccinate chickens. Chickens were immunized via the oculo-nasal route according to the manufacturer’s instructions. Recombinant plasmid pVAX1-ChIFN-g was provided by Jiangsu Key Lab of Zoonosis (Jiangsu, China). Newcastle disease virus recall antigen (recombinant protein of NDV F protein) (21), bovine IFN-g (BovIFN-g), cervine IFN-g (CerIFN-g), chicken IFN-a (ChIFN-a), and chicken interleukin 4 (ChIL-4), all provided by Jiangsu Key Lab of Zoonosis.

Production of recombinant ChIFN-g proteins and native ChIFN-g

The production and purification of Escherichia coli-derived recom-binants, histidine (His)-tagged ChIFN-g (His-ChIFN-g, 330 mg/mL), glutathione S-transferase(GST)-tagged ChIFN-g (GST-ChIFN-g, 1500 mg/mL) and the baculovirus-derived recombinant Bac-ChIFN-g (the supernatant of recombinant virus infected Sf9) were described previously (20,21).

Natural ChIFN-g was produced by concanavalin A (Con) A-stimulation of spleen cells from SPF chickens. Splenocyte suspen-sions were prepared as described (22) and adjusted to 107 cells/mL in the growth medium RPMI 1640 (Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA) containing 10% inac-tivated fetal bovine serum (Hyclone; Thermo Fisher Scientific), 100 U penicillin/mL, 100 mg streptomycin/mL. Then, 2.5 3 106 cells (250 mL) per well were transferred into flat-bottomed 24-well plates. Equal volumes (250 mL) of medium containing 6, 12, or 24 mg/mL of final concentration Con A were added in triplicate, and cultures were incubated for 4 d. Negative controls received 250 mL RPMI 1640 medium only. After 4 d of incubation at 41°C, 5% CO2, supernatant was harvested from each well for the measurement of ChIFN-g production.

Production of monoclonal antibodiesThe 8-week-old BALB/c mice were immunized at an interval

of 2 wk by subcutaneous injections with 100 mg of recombinant His-ChIFN-g emulsified in Freund’s adjuvant (Sigma-Aldrich, St. Louis, Missouri, USA), and boosted intravenously with 100 mg of His-ChIFN-g without adjuvant 3 d prior to fusion. Hybridomas were tested for the presence of antibodies against GST-ChIFN-g by using an ELISA (see below). Positive cells were cloned 3 times by limiting dilution. The immunoglobulin (Ig) sub-class of mAbs was determined using a mouse mAb isotyping kit (Sigma-Aldrich,)

according to the manufacturer’s instructions. BALB/c mice were injected intraperitoneally using positive hybridoma cell line secreting anti-ChIFN-g mAb. Ascites fluids containing abundant anti-ChIFN-g mAb were produced by these immunized mice and purified by protein A chromatography (GE Healthcare, Wauwatosa, Wisconsin, USA). Purified mAbs were biotinylated using standard methods (GenScript Biotechnique Company, Nanjing, Jiangsu, China).

The ChIFN-g ELISA for screening of ChIFN-g antibodies

The 96-well plates (XiaMen YunPeng Biotechnique Company, Xiamen, Fujian, China) were coated with purified GST-ChIFN-g diluted at 1.5 mg/mL in 0.05 M/L carbonate-bicarbonate buffer (pH 9.6) for 24 h at 4°C, 50 mL each well. Subsequently, the plates were washed 3 times using phosphate-buffered saline (PBS) con-taining 0.05% Tween-20 (PBS-T). The plates were then blocked with PBS-T containing 10% (v/v) newborn bovine serum (Lanzhou National Hyclone Bio-engineering Corp., Lanzhou, Gansu, China) for 4 h at 37°C. After washing, 50 mL of hybridoma supernatants were added for 1 h at 37°C and then washed as above. Binding of antibod-ies was revealed using horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Sigma-Aldrich), incubated for 30 min at 37°C. The HRP activity was revealed by addition of o-phenylenediamine (OPD) for 15 min at 37°C. The reaction was stopped by addition of 2 M H2SO4, 490 nm wavelength was used to detect the color devel-opment due to HRP reacting with OPD, OD490 value indicates the presence of mAbs against ChIFN-g.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western-blotting

Proteins were separated by SDS-PAGE on a 12% polyacrylamide gel (pH 8.8) with a 5% stacking gel (pH 6.8), in Tris-glycine run-ning buffer (pH 8.7), and transferred onto Polyvinylidene Fluoride (PVDF) membrane. The membranes were blocked with 5% milk protein in PBST, then incubated with anti-ChIFN-g mAbs in blocking buffer for 1 h. After washing, the membranes were incubated with HRP-conjugated goat anti-mouse IgG (Sigma-Aldrich) in blocking buffer for 0.5 h. Washing 5 times, the membranes were developed using western blotting detection reagent [PBS containing 25% 3, 39-diaminobenzidine (DAB) and 0.03% H2O2]. This DAB substrate solution deposits a brown specific stain in the presence of HRP, then the reaction was quickly stopped by distilled water. After air drying the chromogenic membrane was scanned by the scanner (2580, Epson China Co., Beijing, China).

(COS)-7 cells, Cercopithecus aethiops kidney cells transformed by SV40, were seeded in 24-well-plates and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen, Thermo Fisher Scientific) containing 10% fetal bovine serum (Hyclone) and incu-bated overnight to 50% cell confluent at 37°C, 5% CO2. Recombinant plasmid pVAX1-ChIFN-g and control plasmid pVAX1 were trans-duced into COS-7 cells, respectively, using lipofectin reagent (Invitrogen; Thermo Fisher Scientific) following the manufacturer’s instruction. After an additional 48 h incubation at 37°C, 5% CO2, the supernatant of each well was discarded, plates were washed 3 times using ice cold PBS (pH 7.2), and directly fixed by ice cold methanol for 5 min at room temperature. Then methanol was removed and



allow to air dry. After PBS washing, an optimal concentration of mAb was added and incubated for 1 h at room temperature in a shaker, 50 times/min (TS-2, Kylin-Bell Lab Instrument Co., Haimen, Jiangsu, China). After washing, the second antibody fluoresceine isothiocya-nate (FITC)-conjugated goat anti-mouse IgG (H 1 L) (R&D systems, Minneapolis, Minnesota, USA) was added into each well for 30 min incubation at room temperature in dark. After the washing steps, fluorescence of each well was observed and photos were taken by fluorescence microscope (TS 100, Nikon, Japan).

Antigen-capture ELISATo determine the ability of mAbs to capture ChIFN-g in various

biological test samples, 96-well ELISA plates were precoated with serial dilutions of purified mAbs in 0.01 M PBS at 4°C for 24 h, 100 mL each well. Precoated wells were washed 3 times in PBS-T, then blocked with 2% BSA (in PBS-T, Sangon Bio-engineering Corporation, Shanghai, China) overnight at 4°C. After blocking, 100 mL of each test sample diluted in 1% BSA (in PBS) was added to individual wells and incubated for 2 h at 37°C, followed by a washing step, 100 mL of an optimized dilution of the biotin-labeled mAbs were added and incubation was continued for 1 h at 37°C. The plates were washed 5 times in PBS-T, then incubated with 100 mL of avidin-HRP (diluted in 1: 2500 in PBS-T containing 1% BSA, 20% inactivated newborn bovine serum) for 30 min at 37°C, then exposed to 3,39,5,59-tetramethylbenzidine (TMB) peroxidase substrate solution (eBioscience, San Diego, California, USA). The reaction was stopped by addition of 2 M H2SO4, OD450 of each sample was measured.

Establishment of a double-mAb sandwich ELISA for the detection of ChIFN-g

In a checker-board analysis, all 4 mAbs were used as capturing or revealing antibodies, and His-ChIFN-g was used to identify com-patible mAb sandwich partners as well as to determine the optimal concentration of coating antibody. The specificity of this sandwich ELISA was verified using Bac-ChIFN-g and native ChIFN-g secreted from splenocytes activated by Con A.

The ChIFN-g stimulated by specific NDV F protein recall antigen

Four-week-old SPF chickens were vaccinated with the Newcastle disease mild vaccine. Three weeks after vaccination, splenocytes from each chicken were isolated for stimulation ex vivo. The NDV F protein was used as a recall antigen to test the response of spleno-

cytes as measured by ChIFN-g production in the supernatant. Spleen cell suspensions were prepared as described previously. Splenocytes were adjusted to 107 cells/mL in RPMI 1640 and 100 mL cells per well were transferred into flat-bottomed 96-well plates. Equal vol-umes of medium containing NDV F protein recall antigen (2, 10, 20, 40 mg/mL) were added in triplicate and cultures were incubated for 4 d at 37°C, 5% CO2. Negative controls received 100 mL RPMI 1640 medium only. After 24 h, 48 h, 72 h, and 96 h of incubation, 100 mL cell supernatant was removed from each well and assayed for ChIFN-g production.

Re s u l t s

Generation and characterization of mAbs to ChIFN-g

From 2 independent fusions, 4 hybridomas (1G10, 2C3, 3E3, 3E5) were cloned and selected for further study based upon their strong ChIFN-g ELISA reactivity. The IgG isotypes of 1G10, 2C3, 3E3, and 3E5 were IgG1, IgG1, IgG2a, and IgG1, respectively. The specificity of the 4 mAbs to ChIFN-g was confirmed by ELISA, Western blot, and

Table I. Characterization of monoclonal antibodies (mAbs) specificity to chicken interferon-g (ChIFN-g)

Reactivity of chicken cytokines OD490 nma

mAb ChIFN-g BovIFN-g CerIFN-g ChIFN-a ChIL-41G10 2.049 6 0.093 0.071 6 0.017 0.090 6 0.004 0.067 6 0.001 0.090 6 0.0062C3 2.097 6 0.092 0.076 6 0.002 0.085 6 0.005 0.080 6 0.005 0.080 6 0.0073E3 2.352 6 0.062 0.079 6 0.001 0.097 6 0.003 0.063 6 0.039 0.070 6 0.0023E5 1.845 6 0.075 0.085 6 0.002 0.087 6 0.003 0.065 6 0.001 0.072 6 0.005a Recombinant ChIFN-g, BovIFN-g, CerIFN-g, ChIFN-a, and ChIL-4 obtained by E. coli were used as coating antigen (1.5 mg/mL) in the ELISA. The indicated mAbs (1 mg/mL) were incubated for binding to cytokine-coated wells and their binding was detected by reacting with HRP-labeled goat anti-mouse IgG.Values are means 6 SD.

Figure 1. Western blotting analysis of anti-chicken interferon-g (ChIFN-g) monoclonal antibody. Lane 1 and Lane 3 — control proteins from isopropyl-beta-pyranoside-D-1-thio-Galactose (IPTG) induced recombinant bacteria BL21-(pGEX-6P-1) and BL21(DE)3-(pET) were stained with anti-ChIFN-g mAb 3E3; Lanes 2 and 4: purified GST-ChIFN-g and His-ChIFN-g, derived from E. coli, were stained with anti-ChIFN-g mAb 3E3.

GST-ChIFN-g

His-ChIFN-g

1 2 3 4



immunofluorescence analysis. All the results showed that 4 mAbs provided positive identification of recombinant ChIFN-g without cross-reaction with other control proteins (Table I, Figures 1 and 2).

The establishment of a double-mAb sandwich ELISA for recombinant ChIFN-g

In order to detect native ChIFN-g, an antigen-capture ELISA sys-tem was set up using purified and biotinylated mAbs as capture and detection antibodies, respectively. Checker-board analysis revealed that even very low levels of recombinant ChIFN-g could be detected when the biotin-labeled 3E3 was used as the detection antibody in combination with any of the other 4 mAbs used as capturing antibody (Figure 3). A double-mAb sandwich ELISA allowing the detection of 125–500 pg/mL of His-ChIFN-g with low background was achieved using mAb 3E5 as the capture antibody (85 mg/mL) and biotinylated mAb 3E3 as the detection antibody (0.67 mg/mL) (Figures 4a, 4b, 5b).

Sandwich ELISA for detection of native ChIFN-gAfter establishment of the double-mAb sandwich ELISA shown

in Figure 3, the specificity of this ELISA was evaluated by detect-ing recombinant ChIFN-g (Bac-ChIFN-g, His-ChIFN-g) as well as native ChIFN-g which is in the supernatant secreted from Con A activated chicken splenocytes. The ELISA could detect not only the recombinant ChIFN-g but also the native ChIFN-g (Figure 5a), and is not affected by recombinant ChIL-4 protein (Figure 5b). When the concentration of Con A in the cultured medium was increased from 0 to 12 mg/mL, the level of ChIFN-g detected also increased. Concentrations of Con A greater than 12 mg/mL did not further increase the level of ChIFN-g detected (Figure 6). Furthermore, extending the time over which splenocytes were incubated with Con A also led to an increase in the levels of ChIFN-g detected, although the degree of increase was less pronounced after 48 h.

In addition, we found that increasing levels of ChIFN-g were detected when the number of splenocytes was increased from 1 3 106 cell/mL to 5 3 106 cell/mL (Figure 7). These results indi-cate that this double-mAb sandwich ELISA is suitable to measure ChIFN-g in a variety of settings.

Sandwich ELISA for detection of native ChIFN-g stimulated by NDV recall antigen

Splenocytes from 4-week-old NDV immunized chickens were examined 3 wk after live vaccination for their ability to produce ChIFN-g upon ex vivo stimulation. We found that splenocytes from vaccinated chickens produced ChIFN-g after NDV-specific antigen recall stimulation with NDV F proteins. The ELISA could detect the highest production of ChIFN-g after 96 h of exposure to 20–40 mg/mL of NDV F proteins (Figure 8). There was no significant difference in ChIFN-g production from the antigen-stimulated cultures of unim-munized birds (data not show). These results suggest that this capture ELISA is able to detect native ChIFN-g released ex vivo.

D i s c u s s i o nInterferon-g is a significant regulatory cytokine in animals’ and

birds’ immune systems, and is therefore an important indicator of immune function. Due to the lack of efficient methods, the study of this important cytokine was hindered. Therefore, it is urgent to establish a convenient assay to detect this important cytokine espe-cially on the protein level.

Previous studies have reported that mAbs with high biological activity could be developed using recombinant antigens derived from E. coli, and could be used to establish a potent sandwich ELISA (24–26). In this paper, 4 specific mAbs against recombinant ChIFN-g were generated, and the specificity of the 4 mAbs was confirmed by ELISA, Western blot, and immunofluorescence analysis. In order to

Figure 3. Antibody combinations of different monoclonal antibodies (mAbs). Biotinylated 3E3 as detecting antibody and any of the 4 mAbs as capture reagent to establish the double-mAb sandwich ELISA.

His-ChIFN-g (ng/mL)

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2C3 6 mg/mL

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500 250 125 62.5 32 16 8 4 2 1 BSA

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0Figure 2. Immunofluorescent staining analysis of anti-chicken interferon-g (ChIFN-g) monoclonal antibody (mAb). Recombinant plasmid pVAX1-ChIFN-g was transduced into COS-7 cells, after fixing step, cells were stained with anti-ChIFN-g mAb 3E3.

100 mm



find the optimal mAb combinations, the 4 mAbs were purified and used in different combinations, with the most sensitive mAb pair (3E5, 3E3) subsequently selected as capture and detecting reagents to develop a capture ELISA with high-sensitivity and low-background.

Our results demonstrated that this capture ELISA based on mAbs could detect both recombinant and native forms of ChIFN-g. Increasing culture time, mitogen concentration, and numbers of sple-nocytes activated led to increased detection of ChIFN-g, indicating

that this assay is adequate to measure in vitro release of ChIFN-g. Furthermore, this capture ELISA was successfully used to detect limited native ChIFN-g production stimulated ex vivo by NDV recall antigen, suggesting that this ELISA is likely to be useful for measur-ing native IFN-g in biological samples and for studying the function of native IFN-g in vivo.

This sandwich ELISA will be particularly useful in examining the role of ChIFN-g in the induction of the immune response to several

Figure 5. Specificity of chicken interferon-g (ChIFN-g) sandwich ELISA. A — Serial 2-fold dilutions of His-ChIFN-g (20 ng/mL) or Bac-ChIFN-g (the supernatant of recombinant virus infected Sf9) and native ChIFN-g produced by Con A-activated chicken splenocytes were tested in the sandwich ELISA. B — Serial 2-fold dilutions of His-ChIFN-g, His-ChIL-4, and His-ChIFN-g plus His-ChIL-4 were tested in the sandwich ELISA. In this assay, mAb 3E5 was used as coating antigen and the detection reagent was biotinylated 3E3. There is no significant difference between His-ChIFN-g, His-ChIL-4, plus His-ChIFN-g groups (P = 0.1884).

A B

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64.00 32.00 16.00 8.00 4.00 2.00 1.00 0.50 0.25 0.12 0.06 0.03 0 1 2 3 4 5 6 7 8 9 10

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Figure 4. Establishment of double-monoclonal antibody sandwich ELISA. A — Optimal coating concentration of mAb 3E5. Using mAb 3E5 as capture anti-body at the concentration range from 5.4 to 85 mg/mL, and biotinylated mAb 3E3 as detecting antibody, when the coating concentration of 3E5 reached 85 mg/mL, it is the most sensitive combination for the detection of limiting amounts of recombinant chicken interferon-g (ChIFN-g). B — Sensitivity of double-monoclonal antibody sandwich ELISA. Detection limit of His-ChIFN-g (64 ng/mL) was tested in this double-monoclonal antibody sandwich ELISA when 3E5 coated as 85 mg/mL, biotinylated mAb 3E3 used as 0.67 mg/mL.

A B

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3E5 42.5 mg/mL

3E5 21.3 mg/mL

3E5 10.7 mg/mL

3E5 5.4 mg/mL

His-ChIFN-g

64 32 16 8 4 2 1 0.5 0.25 1% BSA 64.00 32.00 16.00 8.00 4.00 2.00 1.00 0.50 0.25 0.12 0.06 0.03

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infectious agents and in the development of protective responses in the chicken. Additionally, this assay could be used to detect ChIFN-g as a surrogate for a robust Th1 response in several chicken models of infectious diseases. We are currently investigating whether the mAbs reported here can be applied to intracellular cytokine staining by flow cytometry or to ELISPOT assays to enumerate IFN-g-secreting cells. At the very least, these antibodies can serve as useful reagents to develop additional specific IFN-g immunoassays.

A c k n o w l e d g m e n t sThe authors thank Dr. Vidya C. Sinha for critically reading the

manuscript. This work was supported by grants from National Natural Science Foundation of China under agreement number 30972171, 81472815, National Key Technology R&D Program (2011BAK10B01-05), and from a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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Figure 6. Detection of mitogenic response of chicken splenocytes stimu-lated with different concentration of Con A at 0, 6, 12, and 24 mg/mL using the sandwich ELISA (P , 0.0001).

Time (h)

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24 h 48 h 72 h 96 h

4.0

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Figure 7. Mitogenic response of different numbers of chicken splenocytes stimulated with Con A (12 mg/mL) using the sandwich ELISA (P , 0.0001).

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1 3 106 cell/mL

2.5 3 106 cell/mL

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F 2 mg/ml

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Figure 8. Interferon-g (IFN-g) responses induced by Newcastle disease virus (NDV) La Sota vaccine. Optimal concentration of recall antigen (at 40 mg/mL) and the time of the harvest for antigen-specific chicken interferon-g (ChIFN-g) production of splenocytes from immune chickens. Supernatants of stimulated splenocytes were harvested after 24, 48, 72, or 96 h of activation. The ChIFN-g production was determined by the ChIFN-g sandwich ELISA (P , 0.005).

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Article


I n t r o d u c t i o nThe use of multidrug regimens to maintain general anesthesia

allows a decrease in the minimum alveolar concentration (MAC) of volatile anesthetics, and is generally associated with a lesser decrease in arterial blood pressure than when inhalational anesthesia alone is used (1). Ketamine and lidocaine infusions are commonly used in combination with inhalational anesthetics, such as sevoflurane (SEVO), for the maintenance of anesthesia.

Ketamine infusions decreased the MAC of isoflurane in alpacas (2), cats (3), dogs (4,5), and goats (6,7); the MAC of enflurane in dogs (8); the MACSEVO in dogs (9) and rats (10), and the MAC of halothane in horses (11). Intravenous (IV) infusions of lidocaine decreased the MAC of isoflurane in a variety of species, including dogs (4,12), cats (13), goats (7), and horses (14), and the MACSEVO in dogs (9,15) and horses (16). The hypnotic effects of lidocaine combined with ketamine were demonstrated to be synergistic in mice (17), and the combination had an additive effect in decreasing the MAC of isoflurane in goats

Effects of ketamine and lidocaine in combination on the sevoflurane minimum alveolar concentration in alpacas

Patricia Queiroz-Williams, Thomas J. Doherty, Anderson F. da Cunha, Claudia Leonardi

A b s t r a c tThis study investigated the effects of ketamine and lidocaine in combination on the minimum alveolar concentration of sevoflurane (MACSEVO) in alpacas. Eight healthy, intact male, adult alpacas were studied on 2 separate occasions. Anesthesia was induced with SEVO, and baseline MAC (MACB) determination began 45 min after induction. After MACB determination, alpacas were randomly given either an intravenous (IV) loading dose (LD) and infusion of saline or a loading dose [ketamine = 0.5 mg/kg body weight (BW); lidocaine = 2 mg/kg BW] and an infusion of ketamine (25 mg/kg BW per minute) in combination with lidocaine (50 mg/kg BW per minute), and MACSEVO was re-determined (MACT). Quality of recovery, time-to-extubation, and time-to-standing, were also evaluated. Mean MACB was 1.88% 6 0.13% and 1.89% 6 0.14% for the saline and ketamine 1 lidocaine groups, respectively. Ketamine and lidocaine administration decreased (P , 0.05) MACB by 57% and mean MACT was 0.83% 6 0.10%. Saline administration did not change MACB. Time to determine MACB and MACT was not significantly different between the treatments. The quality of recovery, time-to-extubation, and time-to-standing, were not different between groups. The infusion of ketamine combined with lidocaine significantly decreased MACSEVO by 57% and did not adversely affect time-to-standing or quality of recovery.

R é s u m éLa présente étude visait à examiner les effets d’une combinaison de kétamine et de lidocaïne sur la concentration alvéolaire minimale de sevoflurane (CAMSEVO) chez des alpagas. Huit alpagas mâles entiers et en santé ont été étudiés en deux occasions distinctes. L’anesthésie a été induite avec du SEVO, et la détermination de la CAM de base (CAMB) débutée 45 min après l’induction. Après détermination de la CAMB, les alpagas ont reçu par voie intraveineuse (IV), sur une base aléatoire, une dose de charge (DC) et une infusion de saline ou une dose de [kétamine = 0,5 mg/kg de poids corporel (PC); lidocaïne = 2 mg/kg PC] et une infusion de kétamine (25 mg/kg PC par minute) en combinaison avec de la lidocaïne (50 mg/kg PC par minute), et la CAMSEVO re-déterminée (CAMT). La qualité de la récupération, le temps pour extuber, et le temps pour se tenir debout ont également été évalués. La CAMB moyenne était de 1,88 % 6 0,13 % et de 1,89 % 6 0,14 % pour les groupes saline et kétamine 1 lidocaïne, respectivement. L’administration de kétamine et de lidocaïne entraîna une diminution (P < 0,05) de 57 % de CAMB et la CAMT moyenne était de 0,83 % 6 0,10 %. L’administration de saline n’a pas changé la CAMB. Le temps pour déterminer la CAMB et la CAMT n’était pas significativement différent entre les groupes de traitement. La qualité de la récupération, le temps pour extuber, et le temps pour se tenir debout n’étaient pas significativement différents entre les groupes. L’infusion de kétamine combinée à la lidocaïne a diminué significativement la CAMSEVO de 57 % et n’affecta pas négativement le temps pour se tenir debout ou la qualité de la récupération.


Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana 70803, USA (Queiroz-Williams, da Cunha); Department of Large Animal Clinical Sciences, Veterinary Medicine School, University of Tennessee, Knoxville, Tennessee 37996, USA (Doherty); and the Department of Biostatistics, School of Public Health, Louisiana State University, New Orleans, Louisiana 70112, USA (Leonardi).

Address all correspondence to Dr. Patricia Queiroz-Williams; telephone: 225-578-9600; fax: 225-578-9559; e-mail: [email protected]




(7), and the MACSEVO in dogs (9). Although ketamine decreased the MAC of isoflurane in alpacas when administered at 40 mg/kg body weight (BW) per minute (2), there is no information on the effect of either lidocaine alone or lidocaine in combination with ketamine on the MAC of volatile anesthetics in camelids.

The objective of this study was to investigate the effects of a constant rate infusion of lidocaine and ketamine in combination on the MACSEVO in alpacas. It was hypothesized that the combination would significantly decrease the MACSEVO.


AnimalsEight healthy, intact male, adult (2.5- to 10-year-old) alpacas,

weighing between 42.3 kg and 60.5 kg, were used in the study. Alpacas were determined to be healthy on the basis of history, physi-cal examination, complete blood cell count, and serum biochemical profile. Food was withheld for 18 h prior to anesthesia but water was permitted. The study was approved by the Institutional Animal Care and Use Committee of the Louisiana State University School of Veterinary Medicine.

Experimental designAlpacas were used in a randomized 8 3 2 crossover design

(Wichman-Hill procedure for pseudo-random number generation). Briefly, each alpaca was studied on 2 different occasions, getting each treatment once, with a washout period of at least 10 d between treatments. Baseline MAC (MACB) and post-treatment MAC (MACT) for SEVO were measured for each anesthetic occasion. Investigators were aware of the selected treatments.

AnesthesiaAnesthesia was induced with SEVO at 6% (SevoFlo; Abbott

Laboratories, Illinois, USA) in oxygen (5 L/min) delivered by face mask from a circle system attached to a small animal anesthetic machine (SurgiVet Small Animal Veterinary Anesthesia Machine; Smiths Medical, Ohio, USA). After endotracheal intubation, alpacas were placed in left lateral recumbency and anesthesia was main-tained with SEVO in oxygen (3 L/min). Monitoring (Passport 2 Datascope Gas Module SE; Datascope, New Jersey, USA) consisted of electrocardiogram (ECG), indirect blood pressure, pulse oximetry, and continuous gas analysis for capnography (PE9CO2), end-tidal oxygen (PE9O2), and end-tidal sevoflurane (FE9SEVO), and body temperature. Gas sample was drawn from the proximal portion of the endotracheal tube at a rate of 150 mL/min. The gas analyzer was calibrated, according to the manufacturer’s instructions at the start of each experiment. Indirect blood pressure was monitored using an oscillometric technique, and a suitably sized cuff (width approximately 40% of limb circumference) placed between the elbow and carpal joint. Ventilation was controlled (SAV 2500 Anesthesia Ventilator; SurgiVet, Wisconsin, USA) to achieve PE9CO2 between 30 and 45 mmHg. Body temperature was monitored continuously via an esophageal probe and maintained within normal limits (37.5°C to 38.6°C) using a forced warm air device (Bair Hugger; 3M, Minnesota, USA). Hemoglobin oxygen saturation was estimated using a probe

placed on the tongue. A 16-gauge (51 mm) IV catheter (Abbocath-T; Venisystems, Illinois, USA) was placed into the right jugular vein for administration of a Normosol-R solution (Hospira, Illinois, USA) at 3 mL/kg BW per hour, and ketamine (Ketathesia; Butler Health Supply, Ohio, USA) and lidocaine (Lidocaine Injectable; Sparhawk Laboratories, Kansas, USA) administration. An 18-gauge (51 mm) IV catheter (Abbocath-T; Venisystems, Illinois, USA) was placed into the saphenous vein at the medial aspect of the left hind limb for blood sampling for drug analysis. Catheters were placed percutaneously and aseptically after animals were anesthetized and recumbent on the surgical table.

Determination of baseline minimum alveolar concentration

Approximately 45 min after induction of anesthesia, and with FE9SEVO held constant at 2.3 vol% for at least 20 min, determination of the MACB for SEVO was initiated. A noxious stimulus, which con-sisted of clamping a claw between the jaws of a 25.4 cm Vulsellum forceps, was delivered. The forceps were closed tightly to the first or second ratchet, depending on the claw size, just below the coro-nary band, and the claw was moved continuously for 1 min, or until purposeful movement occurred. Purposeful movement was defined as gross movement of the head or extremities. Coughing, stiffening of the neck or limbs, or chewing was not considered purposeful movement. If purposeful movement occurred, the FE9SEVO was increased by 0.1 vol%, otherwise it was decreased by 0.1 vol%, and the stimulus was re-applied after a 20-minute equilibration period. Claws were clamped in a rotating manner to prevent overuse of individual claws. The MACB for SEVO was defined as the mean of the highest concentration that resulted in gross purposeful move-ment and the lowest concentration that prevented that movement. The MACB was determined in duplicate for each anesthetic occasion and the mean of the mean values was used for statistical analysis. Time-to-MACB was recorded as the time from intubation to the completion of MACB determination, in duplicate.

Drug administrationAfter MACB determination, alpacas were randomly given one of

the following 2 IV treatments as a loading dose (LD) and constant rate infusion (CRI), as follows:• Saline: 0.9% NaCl solution (LD of 10 mL followed by a CRI at

the volume of ketamine 1 lidocaine calculated for that specific animal)

• Ketamine 1 lidocaine: ketamine (LD of 0.5 mg/kg BW followed by CRI of 25 mg/kg BW per minute) combined with lidocaine (LD of 2 mg/kg BW followed by CRI of 50 mg/kg BW per minute)Loading doses were made up to a final volume of 10 mL in nor-

mal saline (0.9% NaCl) and given over 3 min. The CRI was started immediately after the loading dose was administered. For each anes-thetic occasion, the LD and CRI of ketamine 1 lidocaine were gently shaken several times into the syringes for homogeneous mixture of both drugs, ketamine and lidocaine. The LD and CRI were delivered using a syringe pump (Medfusion; Medex, Duluth, Georgia, USA).

Post-treatment MAC (MACT) determination began 30 min after the start of the LD with the FE9SEVO held constant for at least 20 min at each alpaca’s MACB value. MACT was determined in duplicate



(MACT1 and MACT2) using the same methodology as described for MACB. After MACT determination, SEVO and ketamine 1 lidocaine infusion were discontinued and alpacas were allowed to recover. The time-to-MACT determination was defined as the time from starting the CRI of ketamine 1 lidocaine to the completion of MACT in duplicate. Approximately 6 mL of blood was collected from a saphenous catheter for analysis of ketamine and lidocaine plasma concentrations at the time of each MACT determination (MACT1 and MACT2). The blood was placed into lithium heparin tubes and the plasma was harvested and stored at 280°C until analysis.

Recovery evaluationAlpacas were placed in sternal recumbency for recovery with their

heads elevated on a pad. A quality recovery score system was made and used to subjectively evaluate (by the same investigator, PQW) alpacas’ recovery using a 3-point scale as follows:• Score 1 = no drooping of eyelids; alert and able to hold head up

and move head around while in sternal recumbency; no struggling or paddling; and standing on first attempt.

• Score 2 = mild drooping of eyelids; partially able to hold head up and to move head around while in sternal recumbency; mild struggling and paddling; more than 2 attempts to stand.

• Score 3 = moderate drooping of eyelids; unable to hold head up or to move head around while in sternal recumbency; moderate paddling movements and unwillingness to remain in sternal recumbency; more than 2 attempts to stand.Extubation was performed after chewing motions were present.

Time-to-extubation was defined as time (min), from discontinuing ketamine 1 lidocaine and SEVO to extubation. Time-to-standing (min), was defined as the time from discontinuing ketamine 1 lidocaine and SEVO to standing. Although animals were allowed to recovery freely, they were encouraged to stand up with noise stimulation (hand clapping and calling alpacas’ names).

Drug analysisSamples were prepared for analysis by a protein precipitation

method. A standard curve of ketamine, lidocaine, and lidocaine metabolites, monoethylglycinexylidide (MEGX), and glycinexylidide (GX) was prepared from 0, 50, 100, 500, 1000, 5000, and 10 000 ng/mL.

A TSQ Vantage triple quadrupole mass spectrometer with a Transend turboflow LC system (Thermo Scientific) was used for the positive ion electrospray (ESP1) analysis. An Eclipse Plus C18 column,

3.0 3 100 mm; 3.5 mm particle size (Agilent) was used for the analysis with an injection size of 10 mL. The mobile phases used were: A — 0.1% formic acid in H2O, and B — 0.1% formic acid in acetonitrile with a flow rate of 300 mL/min. The gradient was as follows: 0 to 1.5 min — 90% A: 10% B; 1.5 to 5 min — 2% A: 98% B; 5.5 to 10 min — 90% A: 10% B.

Multiple reaction monitoring analysis was performed for all com-pounds. Ion transitions used for quantization were 292 . 152 m/z for the internal standard (Morphine-d6); 235.16 . 86.15 m/z for lidocaine; 238.08 . 125.05 m/z for ketamine; 207.12 . 58.16 m/z for MEGX, and 179.13 . 122.15 m/z for GX. Dwell for each transition was 0.10 s and collision energy was optimized according to each com-pound. Mass spectrometric conditions were optimized for lidocaine by infusion of a pure standard. Although, the tube lens optimization was according to each compound.

Statistical analysisTreatment effect on MACB, MACT, percent change in MAC, time

to MACB, time to MACT, time-to-extubation, and time-to-standing were determined using the MIXED procedure of SAS (SAS system; SAS Institute, Cary, North Carolina, USA). Treatment (saline or ketamine 1 lidocaine), period (1 or 2) and sequence (1: saline first and ketamine 1 lidocaine second or 2: ketamine 1 lidocaine first and saline second) were included in the model as fixed effects. Plasma concentration of ketamine, lidocaine, and lidocaine metabolites (MEGX and GX) were analyzed including sequence and time of MACT determination (1 or 2) in the model as fixed effect. Alpaca within sequence was included as a random effect in all the above models. Recovery scores were analyzed using the GLIMMIX pro-cedure of SAS using the model previously described for the non-plasma variables. The multinomial distribution with a cumulative logit link was implemented. Results are reported as least squares means 6 SEM, unless stated otherwise. Significance was declared at P , 0.05.

Re s u l t sThe least-squares mean of SEVO MACB was 1.88% 6 0.13% and

1.89% 6 0.14% for the saline and K 1 L groups, respectively, and did not significantly differ between treatments (Table I). Ketamine combined with lidocaine significantly (P = 0.0001) decreased MACB by 56.9% 6 4.6%. Saline did not significantly (P = 0.898) change MACB. Time to determine MACB and MACT was not significantly

Table I. Effect of IV saline (0.9% NaCl) solution (control group) and ketamine (LD: 0.5 mg/kg BW IV; CRI: 25 mg/kg BW per minute) combined with lidocaine (LD: 2 mg/kg BW, IV; CRI: 50 mg/kg BW per minute) on sevoflurane MAC in alpacas (mean 6 SEM)

Treatment MACB MACT Change (%)a Time MACBb Time MACT

b

Saline 1.88 6 0.13c 1.84 6 0.10a 20.61 6 4.6c 276 6 31c 145 6 25c

K 1 L 1.89 6 0.13c 0.83 6 0.10b 256.9 6 4.6d,e 230 6 31c 169 6 25c

a Percentage change from baseline MAC = [(MACT erMACB)/MACB] 3 100.b Time (min) to complete MACB and MACT determination in duplicate.c,d Values in the same columns with different letters are significantly different (P , 0.05).e Value significantly different from 0.LD — loading dose; MAC — minimum alveolar concentration; MACB — baseline MAC; MACT — post-treatment MAC



different between the treatments (Table I). Plasma concentrations (ng/mL) of lidocaine and its main metabolites, GX and MEGX, and ketamine are reported in the Table II. The mean plasma concentration was 1797 ng/mL for lidocaine, 748 ng/mL for ketamine, 217 ng/mL for GX, and 927 ng/mL for MEGX. A significant (P = 0.005) increase in GX plasma concentration was observed from MACT1 to MACT2 determination. Although no other significant difference was observed in plasma concentration of ketamine (P = 0.860), lidocaine (P = 0.905), and the MEGX metabolite (P = 0.093) between MACT1 and MACT2. The mean induction time was 11.7 min and mean arterial blood pressure in all alpacas was greater than 70 mmHg at all times. The time-to-extubation (P = 0.522) and time-to-standing (P = 0.737) did not significantly differ between treatment groups. The mean time-to-extubation was 18.13 6 3.22 min for saline and 21.13 6 3.22 min for ketamine 1 lidocaine. The mean time-to-standing was 45.25 6 4.90 min for saline and 42.88 6 4.90 min for ketamine 1 lidocaine. The median recovery score was 1 for saline treatment and 1.5 for K 1 L treatment. There was no significant difference (P = 0.404) between treatments for quality of recovery, although one alpaca in the K 1 L group received a score of 3.

D i s c u s s i o nThe MAC method used in this study consisted of bracketing

SEVO concentrations up and down by 0.1%. In the present study, the mean baseline MACSEVO was 1.89%, which is less than the MACSEVO (2.33%) reported in another study in alpacas (18). A variation in MAC values of up to 20% within species is well-accepted in MAC studies (19). Larger variations in MAC values due to different experimental designs (differences in type and intensity of noxious stimulus and the subjectivity of purposeful movement assessment), have been reported (20). Interestingly, in a study on dogs (21), it was speculated that a low number of subjects (low study power) could contribute to MAC variation among different species. The difference in MACSEVO values for alpacas between the present study and the study done by Grubb et al (18), could be explained by the different of type of noxious stimuli used (clamping a claw versus electrical stimulus, respectively) and the subjective assessment of the purposeful move-ment (different investigators). One could also speculate if the power of these studies contributed to the MAC variation (8 alpacas versus 6 alpacas, respectively).

The administration of ketamine 1 lidocaine was associated with a 57% decrease in the MACSEVO, and this is consistent with the reported effects of the combination on MACSEVO in dogs (9).

In a previous study in alpacas (2), an infusion of ketamine at 40 mg/kg BW per minute decreased the MACSEVO by 37%, but plasma concentrations of ketamine were not reported. In goats (6), a similar infusion rate of ketamine decreased the MAC of isoflurane by 28.7% at a plasma concentration of 592 ng/mL, which is less than the mean value of 782 ng/mL achieved herein. Thus, it is likely that in the present study, the contribution of ketamine to MAC reduction was greater than 28.7%. In dogs, ketamine infusion of 40 mg/kg BW per minute resulted in a plasma concentration of 824 6 196 ng/mL with an isoflurane MAC decrease of 33%. Yet, in cats (3), a ketamine infu-sion of 23 mg/kg BW per minute resulted in mean plasma ketamine concentrations of 1750 ng/mL and was associated with an isoflurane MAC decrease of 45% 6 17%. A study in dogs (22) reported how ketamine pharmacokinetics can be greatly variable even within same group of species. Therefore, the discrepancies in the plasma ketamine concentrations among studies cited here are probably due to differences in the ketamine’s pharmacokinetics among species.

Lidocaine infusions of 50 mg/kg BW per minute are associated with decreases in MACSEVO between 22% in dogs (9) and 27% in horses (16) at plasma concentrations of approximately 1500 ng/mL and 2200 ng/mL, respectively. In goats, an infusion of 100 mg/kg BW per minute resulted in a plasma concentration of lidocaine compa-rable (1900 ng/mL) to the present study, and was associated with a decrease in the isoflurane MAC of 18% (7). Based on these studies, it is unlikely that lidocaine was associated with more than a 25% decrease in the MACSEVO herein. Lidocaine metabolism occurs mainly by oxidative dealkylation via cytochrome P-450 enzymes. Dealkylation produces MEGX and GX, the 2 main metabolites of lidocaine, with the MEGX being further metabolized to GX (23). After an IV administration of lidocaine, plasma concentrations of MEGX and, especially, GX increase with time while lidocaine rapidly decreases (24). Therefore, the difference in the GX plasma concentra-tions between MACT1 and MACT2 could be expected according to the time elapsing for the post-treatment MAC determinations.

A weakness in our study was that each drug’s effect on MAC was not assessed separately and therefore, the individual contribution of each drug to the decrease in MAC is unknown.

The interaction between ketamine and lidocaine on volatile anesthetic-induced immobility in other species was considered to be additive (7,9). However, it is difficult to compare studies because of differences in species and drug infusion rates; and, in addition, plasma concentrations of the drugs differed even when similar infusion rates were used (4,7,9,12,16). For example, higher infu-sion rates (100 mg/kg BW per minute) of ketamine and lidocaine in dogs decreased the MACSEVO by approximately 63% (9), which is comparable to the decrease of 57% in the MACSEVO in the pres-ent study. In goats (7), an infusion of ketamine at 50 mg/kg BW per minute and lidocaine at 100 mg/kg BW per minute decreased the MAC of isoflurane by 69%. In a clinical study in horses (1), ketamine at 50 mg/kg BW per minute combined with lidocaine at 50 mg/kg BW per minute reduced the MAC of isoflurane by 40%. On the con-trary, lower infusion rates of ketamine at 10 mg/kg BW per minute combined with lidocaine at 20 mg/kg BW per minute in a clinical

Table II. Plasma concentration of ketamine, lidocaine, and lidocaine metabolites, monoethylglycinexylidide (MEGX), and glycinexylidide (GX) at the time of post-treatment MAC determination (MACT1 and MACT2) in 8 alpacas given ketamine (LD: 0.5 mg/kg BW IV; CRI: 25 mg/kg BW per minute) combined with lidocaine (LD: 2 mg/kg BW IV; CRI: 50 mg/kg BW per minute), expressed as mean 6 SEM

MACT1 MACT2

GX 204 6 69b 229 6 69a

MEGX 884 6 213a 971 6 213a

Lidocaine 1803 6 239a 1791 6 241a

Ketamine 788 6 303a 708 6 325a

a,b Values in the same row with different letters are significantly different (P , 0.05).



study in sheep (25) reduced the requirement for isoflurane by 23%. The doses of ketamine and lidocaine used in the present study were based on studies in alpacas (2) and goats (6,7).

In conclusion, the infusion of ketamine combined with lidocaine, at the doses used in this study, decreased the MACSEVO by 57% in alpacas, and did not affect the quality of recovery from anesthesia. Based on these results, IV administration of ketamine combined with lidocaine, during SEVO anesthesia, may be a safe source to achieve a balanced anesthesia in alpacas.

A c k n o w l e d g m e n tThe authors thank John Ladner (RVT) for technical assistance.

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Breed- and age-related differences in canine mammary tumorsHyun-Woo Kim, Ha-Young Lim, Jong-Il Shin, Byung-Joon Seung, Jung-Hyung Ju, Jung-Hyang Sur

A b s t r a c tTriple-negative breast cancer is a type of breast cancer that does not express the genes for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER-2). It is an important and clinically relevant condition as it has a poor prognosis and is difficult to treat. Basal-like triple-negative cancer is highly prevalent in both African-Americans and adolescents. We therefore examined whether such a cancer likewise occurs in specific breeds and age groups in dogs, focusing on basal-like triple-negative cancer in particular. In this study, 181 samples from dogs with malignant mammary carcinoma from the 5 most common breeds and 2 age groups in Korea were analyzed. Histological classification and molecular subtyping, including assessment of immunohistochemical findings, were carried out. Twenty-five of 28 (89.3%) triple-negative carcinomas were identified as basal-like triple-negative carcinomas. Analysis of associations of classified factors revealed that the shih tzu breed (9/25, 36.0%) and advanced-age (19/25, 76.0%) groups were characterized by higher prevalence of basal-like triple-negative tumors with diverse histological types and of a higher grade. These results suggest that breed- and age-related differences can be identified in canine mammary carcinoma and, notably, in the shih tzu breed and at older ages. Further investigation of these distinguishing characteristics of the shih tzu breed is warranted.

R é s u m éLe cancer mammaire triple-négatif est un type de cancer mammaire qui n’exprime pas les gènes pour les récepteurs de l’œstrogène (RO), les récepteurs de la progestérone (RP), et les récepteurs pour le facteur de croissance épidermique humain-2 (REH-2). Il s’agit d’une condition importante et cliniquement significative étant donné son mauvais pronostic et la difficulté à le traiter. Le cancer triple-négatif de type basal est très prévalent chez les afro-américains et les adolescents. Nous avons donc voulu savoir si chez les chiens ce type de cancer est rencontré également dans des races spécifiques et dans des groupes d’âge précis, en se concentrant sur des cancers triple-négatifs de type basal en particulier. Dans la présente étude réalisée en Corée, 181 échantillons provenant de chiens avec des carcinomes mammaires malins des cinq races les plus communes et de deux groupes d’âge ont été analysés. Une classification histologique et un sous-typage moléculaire, incluant une évaluation des trouvailles immunohistochimiques, ont été réalisés. Vingt-cinq des 28 (89,3 %) des carcinomes triple-négatifs ont été identifiés comme étant des carcinomes triple-négatifs de type basal. Une analyse des associations des facteurs classés a révélé que les groupes de chiens de la race Shih tsu (9/25, 36,0 %) et d’un âge avancé (19/25, 76,0 %) étaient caractérisés par une prévalence plus élevée de tumeurs triple-négatives de type basal avec des types histologiques variés et de grade plus élevé. Ces résultats suggèrent que des différences reliées à la race et à l’âge peuvent être identifiés dans les carcinomes mammaires canins et, notamment, chez la race Shih tsu et à un âge plus avancé. Des études supplémentaires de ces caractéristiques distinctives de la race Shih tsu sont justifiées.


Department of Veterinary Pathology, Small Animal Tumor Diagnostic Center, College of Veterinary Medicine, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Korea.

Address all correspondence to Dr. Jung-Hyang Sur; telephone: 82-2-450-4153; fax: 82-2-455-8124; e-mail: [email protected]

Received June 8, 2015. Accepted October 26, 2015.



such as CK5/6, CK14, CK17, and EGFR (11). Because of a particularly poor prognosis, basal-like triple-negative tumors warrant thorough investigation (10,11,13).

In humans, the occurrence of breast cancer shows race- and age-related differences (14,15). The discovery that African-American female patients with breast cancer tended to have a lower survival rate than Caucasian female patients triggered studies of racial differences in breast cancer (14,15). Different aspects of breast cancer related to race and age have since been reported (16). The total number of breast cancer patients was not remarkable in African-American women or younger subjects, but breast cancers with worse prognosis, particularly the triple-negative cancers, tended to occur more often in African-American female patients and younger patients (17–21). These find-ings are presumed to be related to genetic differences among races.

Recently, basal-like triple-negative tumors have also been identi-fied in dogs (22,23). The dog may be a reliable animal model for human breast cancer (23,24). Therefore, establishing breed-related and age-related differences in mammary tumors in dogs would be meaningful for both human and canine studies. This study aimed to determine the differences in expressions of histological types, molecular types, and other applicable factors in canine mammary cancer and to determine the characteristics of canine mammary carcinoma, with particular reference to the race- and age-related differences in expressions of basal-like triple-negative cancer.


Sample selectionEntire samples used to diagnose canine mammary tumors were

obtained from the Department of Veterinary Pathology, Konkuk University Animal Teaching Hospital, Seoul, Korea. Primary selec-tion was limited to canine malignant mammary carcinoma diagnosed from 2003 to 2013 and consisted of 672 samples. All 672 samples were analyzed according to breed and the 5 most frequent breeds (Maltese, 148; Yorkshire terrier, 99; shih tzu, 72; poodle, 63; and Cocker spaniel, 53) were divided by age into subjects below and above 10 y of age, with 20 samples extracted randomly for each subgroup (, 10 and $ 10 y). The final study population consisted of 20 dogs in each age subgroup for each of the 5 breed groups, i.e., 40 dogs in each breed group for a total of 200 samples. As samples excluded after initial screening were not artificially replaced, 181 samples were analyzed.

ImmunohistochemistryFour-micrometer sections of formalin-fixed paraffin-embedded

tissues were fixed on slides and deparaffinated by xylene, followed by serial rehydration by graded ethanol. Slides were washed 3 times with phosphate-buffered saline (PBS). A 3% hydrogen peroxide solution diluted with PBS was used to block endogenous peroxidase activity. Antigen retrieval was then carried out by the microwave retrieval method or enzyme retrieval method, selected according to the primary antibody.

Microwave retrieval (750W, 60Hz, 15 min) in pH 9.0 Tris-ethylenediamine tetra-acetic acid (EDTA) buffer was applied to the sample for anti-ER (clone: ER88; BioGenex, Fremont, California, USA), PR (PR10A9; Immunotech SAS, Marseille, France), HER-2/

neu (CB1L; BioGenex), CK5/6 immunostaining (D5/16/B4; Dako, Glostrup, Denmark), and CK14 (LL002; Abcam, Cambridge, UK); 0.05% proteinase K (37°C, 30 min) enzyme digestion was applied for staining with anti-EGFR antibody (31G7; Abcam).

After PBS washing, slides were covered with 5% normal goat serum for 30 min for anti-ER staining to block nonspecific binding. Primary antibody staining was conducted. Incubation for 3 h at room temperature was applied for anti-ER (1:60), HER-2/neu (1:100), and CK14 (1:300) and overnight at 4°C for anti-PR (1:500), CK5/6 (1:100), and EGFR (1:50) antibody staining. PBS washing was carried out, followed by 2-step immunolabeling with secondary antibody-HRP conjugation for 40 min. Visualization was achieved using DAB1 chromogen (REAL EnVision Kit; Dako). Finally, slides were washed with distilled water and counterstained with Gill’s hematoxylin and coverslips were applied.

Tumor classificationHistological subtyping was carried out using HE-stained slides

according to previously proposed criteria (25). Histological subtypes included carcinoma arising in a mixed tumor, carcinoma-complex type, simple carcinoma, intraductal papillary carcinoma, solid carcinoma, ductal carcinoma, carcinoma-anaplastic, carcinoma- micropapillary invasive, comedocarcinoma, and squamous cell carcinoma.

In the molecular classification of tumors, types of tumors that are positive for hormone receptor expression are called luminal types. Tumors with positive expression for ER or PR and negative expression for HER-2 were classified as luminal A type and tumors with positive expression for ER or PR and negative expression for HER-2 were clas-sified as luminal B type. Types of tumors that were hormone-receptor negative, but were positive for HER-2 receptor expression, were clas-sified as HER-2-overexpressing type and tumors that were negative for the expression of all 3 receptors were classified as triple-negative type. After identifying triple-negative cancer samples, basal-marker expression was sequentially evaluated for subtyping basal-like triple negative cancers. Triple-negative cancers expressing at least 1 basal marker were considered basal-like triple-negative cancers.

Grading was done by evaluating the sum of the scores of tubule formation, mitoses, and nuclear pleomorphism (26). Grade 1 indi-cated well differentiated tumors, Grade 2 indicated moderately differentiated tumors, and Grade 3 indicated poorly differentiated tumors.

Immunohistochemical analysisAnti-ER or anti-PR staining was considered positive if there was

more than 10% expression of clear nuclear staining. Evaluation of anti-HER-2 staining was based on the Hercep test and more than 10% of complete plasma membrane expression was considered positive. CK5/6, CK14, and EGFR were used as basal markers and areas of interest with more than 5% of membrane expression were considered basal-positive.

Statistical analysisThe Statistical Package for Social Science 17.0 software (SPSS,

Chicago, Illinois, USA) was used for statistical analysis. Analysis of fre-quency, Pearson’s Chi-square test, and Fisher’s exact test were applied and P-values less than 0.05 (P , 0.05) were considered significant.



Figure 1. A — Carcinoma arising in a mixed tumor, mammary gland, canine. Hematoxylin and eosin (H&E). Bar = 35 mm. B — Simple carcinoma, mammary gland, canine. H&E. Bar = 35 mm. C — Squamous cell carcinoma, mammary gland, canine. H&E. Bar = 35 mm. D — Comedocarcinoma, mammary gland, canine. H&E. Bar = 35 mm. E — Carcinoma-complex type, canine. H&E. Bar = 35 mm. F — Ductal carcinoma, mammary gland, canine. H&E. Bar = 35 mm. G — Carcinoma-micropapillary invasive, mammary gland, canine. H&E. Bar = 35 mm. H — Carcinoma-anaplastic, mammary gland, canine. H&E. Bar = 35 mm.

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Figure 2. A — Positive expression of estrogen receptor in simple carcinoma. Immunohistochemistry (IHC), mammary gland, canine. Bar = 35 mm. B — Negative expression of estrogen receptor in solid carcinoma. Immunohistochemistry (IHC), mammary gland, canine. Bar = 35 mm. C — Positive expression of progesterone receptor in ductal carcinoma. IHC, mammary gland, canine. Bar = 35 mm. D — Negative expression of progesterone receptor in complex carcinoma. IHC, mammary gland, canine. Bar = 35 mm. E — Positive expression of human epithelial growth factor receptor-2 in ductal carcinoma. IHC, mammary gland, canine. Bar = 35 mm. F — Negative expression of human epithelial growth factor receptor-2 in ductal carcinoma. IHC, mammary gland, canine. Bar = 35 mm.

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Re s u l t s

Clinical dataThe 181 dogs with malignant mammary carcinoma ranged in

age from 3 to 17 y (mean: 9.4 6 2.72 y). Ninety-two dogs (50.8%) were , 10 y and 89 dogs (49.2%) were $ 10 y. The 181 dogs were divided into 5 groups based on breeds with the highest prevalence for canine malignant mammary carcinoma as follows: 38 Cocker spaniels (20 , 10 y, 18 $ 10 y), 36 Maltese (18 , 10 y, 17 $ 10 y), 36 poodles (18 , 10 y, 18 $ 10 y), 34 shih tzus (18 , 10 y, 16 $ 10 y), and 35 Yorkshire terriers (18 , 10 y, 17 $ 10 y).

Histological classification and molecular phenotype

The malignant canine mammary carcinomas were categorized into histological types according to the classification proposed by Goldschmidt et al (25) based on hematoxylin and eosin (HE) stain-ing (Figure 1). The 181 samples were divided into the following histological types: 71 (39.2%) carcinoma arising in a mixed tumor;

47 (26.0%) carcinoma-complex types; 36 (19.9%) simple carcinomas; 8 (4.4%) intraductal papillary carcinomas; 7 (3.09%) solid carcino-mas; 5 (2.8%) ductal carcinomas; 4 (2.2%) squamous cell carcinomas; 1 (0.6%) carcinoma-micropapillary invasive; 1 (0.6%) comedocarci-noma; and 1 (0.6%) carcinoma-anaplastic.

Two major types of tumor, carcinoma arising in a mixed tumor and complex type carcinoma, accounted for more than half (65%) of the samples. According to molecular phenotype, the luminal A type was the most prevalent at 42.5%, followed by the luminal B and triple-negative types (15.5%), with the HER-2-overexpressing type being the least common (6.1%). Overall, hormonal receptor positive type (ER positive or PR positive) mammary tumors accounted for 78% of the samples and the triple-negative type, indicating negative expres-sion for anti-ER, anti-PR, and anti-HER-2 antibodies, accounted for 15.5% (Figure 2).

Characteristics of tumor according to breedCorrelations between breeds and characteristics of malignant

canine mammary carcinoma are listed in Table I. Statistically sig-nificant associations were not found between the breed and the

Table I. Breed-based statistical analysis of malignant mammary carcinoma of canines

Breed Cocker Yorkshire spaniel Maltese Poodle Shih tzu terrier (n = 38) (n = 36) (n = 36) (n = 34) (n = 35) PHistological type Carcinoma — anaplastic (n = 1) 1 (100.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0.115 Carcinoma — complex type (n = 47) 12 (25.5) 13 (27.7) 8 (17.0) 5 (10.6) 9 (19.1) Carcinoma — micropapillary invasive (n = 1) 0 (0.0) 1 (100.0) 0 (0.0) 0 (0.0) 0 (0.0) Simple carcinoma (n = 36) 11 (30.6) 7 (19.4) 7 (19.4) 5 (13.9) 6 (16.7) Solid carcinoma (n = 7) 1 (14.3) 1 (14.3) 1 (14.3) 3 (42.9) 1 (14.3) Carcinoma arising in a mixed tumor (n = 71) 9 (12.7) 15 (21.1) 18 (25.4) 13 (18.3) 16 (22.5) Comedocarcinoma (n = 1) 0 (0.0) 0 (0.0) 0 (0.0) 1 (100.0) 0 (0.0) Ductal carcinoma (n = 5) 1 (20.0) 1 (20.0) 0 (0.0) 1 (20.0) 2 (40.0) Intraductal papillary carcinoma (n = 8) 3 (37.5) 0 (0.0) 2 (25.0) 2 (25.0) 1 (12.5) Squamous cell carcinoma (n = 4) 0 (0.0) 0 (0.0) 0 (0.0) 4 (100) 0 (0)

Molecular type HER-2 overexpressinga (n = 11) 5 (45.5) 1 (9.1) 0 (0.0) 1 (9.1) 4 (36.4) 0.096 Luminal Ab (n = 77) 18 (23.4) 14 (18.2) 16 (20.8) 13 (16.9) 16 (20.8) Luminal Bc (n = 65) 12 (18.5) 19 (29.2) 14 (21.5) 10 (15.4) 10 (15.4) Triple negatived (n = 28) 3 (10.7) 4 (14.3) 6 (21.4) 10 (35.7) 5 (17.9)

Triple negative Non-triple negative (n = 153) 35 (22.9) 34 (22.2) 30 (19.6) 24 (15.7) 30 (19.6) 0.111 Triple negative (n = 28) 3 (10.7) 4 (14.3) 6 (21.4) 10 (35.7) 5 (17.9)

Grade 1 (n = 118) 29 (24.6) 25 (21.2) 23 (19.5) 19 (16.1) 22 (18.6) 0.017 2 (n = 52) 8 (15.4) 13 (25.0) 11 (21.2) 8 (15.4) 12 (23.1) 3 (n = 11) 1 (9.1) 0 (0.0) 2 (18.2) 7 (63.6) 1 (9.1)a ER2/PR2/HER-21.b ER1 or PR1/HER-22.c ER1 or PR1/HER-21.d ER2/PR2/HER-22.P , 0.05.



histological classification and molecular phenotype. Histological grade revealed decreasing prevalence from Grade 1 to Grade 3 for all the breeds (P , 0.05). Grade 1 was most common (65.2%), fol-lowed by Grade 2 (28.7%), and Grade 3 (6.0%). The prevalence of Grade 3 was higher in the shih tzu breed, however, than in the other breeds (7/34, 63.6%).

Characteristics of tumor according to ageData from the 2 age groups are summarized in Table II. Luminal A

(44/77, 57.1%) and luminal B type (38/65, 58.5%) tumors were associ-ated with the group of younger dogs (under 10 y), whereas HER-2-overexpressing (8/11, 72.7%) and triple-negative (21/28, 75.0%) types were related to the older group (10 y and over) (P , 0.05). Grade 1 accounted for 65.2% of all subjects, followed by Grade 2 at 28.7%, and Grade 3 at 6.1%. Of note, Grade 3 was seen more in the older group ($ 10 y) (10/11, 90.9%) than in the younger group (, 10 y) (1/11, 9.1%).

Analysis of tumor characteristics according to grade

There were significant correlations (P , 0.05) among grade and dog breed, age group, histological type, and molecular phenotype. The results of the Chi-square test based on grade are given in Table III. Complex-type carcinoma (27/47, 57.4%), simple carcinoma (28/36, 77.8%), carcinoma arising in a mixed tumor (54/71, 76.1%), ductal carcinoma (4/5, 80.0%), and intraductal papillary carcinoma (5/8, 62.5%) were more predominant in the lower grades. There was a tendency for higher grade expression to be in the solid carcinoma (4/7, 57.1%) and squamous cell carcinoma (3/4, 75.0%). Although there was only 1 case of each, the anaplastic carcinoma (1/1, 100%) and comedocarcinoma (1/1, 100%) were both Grade 3. Grade 1 tended to dominate in all molecular phenotypes, but the triple-negative subtype accounted for 45.4% (5/11) of Grade 3.

Table II. Age-based statistical analysis of malignant mammary carcinoma of canines

Age groups , 10 y $ 10 y (n = 92) (n = 89) PHistological type Carcinoma — anaplastic (n = 1) 0 (0.0) 1 (100.0) 0.061* Carcinoma — complex type (n = 47) 32 (68.1) 15 (31.9) Carcinoma — micropapillary invasive (n = 1) 1 (100.0) 0 (0.0) Simple carcinoma (n = 36) 18 (50.0) 18 (50.0) Solid carcinoma (n = 7) 3 (42.9) 4 (57.1) Carcinoma arising in a mixed tumor (n = 71) 32 (45.1) 39 (54.9) Comedocarcinoma (n = 1) 0 (0.0) 1 (100.0) Ductal carcinoma (n = 5) 3 (60.0) 2 (40.0) Intraductal papillary carcinoma (n = 8) 3 (37.5) 5 (62.5) Squamous cell carcinoma (n = 4) 0 (0.0) 4 (100.0)

Molecular type HER-2 overexpressinga (n = 11) 3 (27.3) 8 (72.7) 0.005 Luminal Ab (n = 77) 44 (57.1) 33 (42.9) Luminal Bc (n = 75) 38 (58.5) 27 (41.5) Triple negatived (n = 28) 7 (25.0) 21 (75.0)

Triple negative Non-triple negative (n = 153) 85 (55.6) 68 (44.4) 0.003 Triple negative (n = 28) 7 (25.0) 21 (75.0)

Grade 1 (n = 118) 61 (51.7) 57 (48.3) 0.013 2 (n = 52) 30 (57.7) 22 (42.3) 3 (n = 11) 1 (9.1) 10 (90.9)a ER2/PR2/HER-21.b ER1 or PR1/HER-22.c ER1 or PR1/HER-21.d ER2/PR2/HER-22.P , 0.05.* Fisher’s exact test.



Characteristics of basal-like triple-negative mammary cancer

Twenty-five of the tumors were basal-like triple-negative tumors (Figure 3), which represented 89.3% of the triple-negative type can-cers. The most frequent histological type was carcinoma arising in a mixed tumor (12/25, 48%) and the most common breed was the shih tzu (9/25, 36%) (Table IV). Grade 1 (16/25, 64%) was the predomi-nant grade within the basal-like triple-negative tumors and Grade 3 was solely expressed by the shih tzu breed, except for 1 Poodle. The shih tzu breed had the broadest spectrum of histological types. By age, 76% (19/25) of the total cases were $ 10 y of age and 36.8% (7/19) of these dogs were shih tzus.

D i s c u s s i o nDetermining and categorizing the characteristics of breast cancer

may help to treat this disease and to establish the causative factors of the cancer (27). A molecular-based classification scheme has been proposed (28), in which triple-negative breast cancer and basal-like breast cancer are problematic in terms of having comparatively poor prognoses and therapeutic challenges (19).

Many factors have been implicated in the pathogenesis of breast cancer, including hormonal influences, environmental and heredi-

tary factors, and age (18,29). Investigations into the characteristics of tumor development yielded higher incidences of triple-negative breast cancer in young patients and African-American women (12,14). These predilections of race and age appear to be related to the hereditary factors causing breast cancer.

The dog is one of the most common species that develops mam-mary tumors and can therefore serve as an acceptable model for human breast cancer. Basal-like triple-negative tumors have also been identified in dogs (22,23). In this study, we investigated asso-ciations between types and characteristics of tumors. We especially focused on breed and age group by using previously reported criteria. Among the tumor types, particular emphasis was placed on the basal-like triple-negative tumor. Characteristics of basal-like triple-negative cancers were analyzed by frequency and relations between certain clinicopathological parameters were evaluated.

While the types of tumor expression did not clearly differ accord-ing to breed, the shih tzu breed exhibited some distinguishing char-acteristics. In analyzing histological type, types of tumors known to have better prognoses were not commonly expressed in shih tzus, but higher expression rates of types of tumors known to have worse prognoses were the most outstanding characteristics of the shih tzu breed compared with the other breeds. Tumors with better prognoses are carcinoma arising in a mixed tumor, carcinoma-complex type, and simple carcinoma, while tumors with worse prognoses include

Table III. Statistical analysis of malignant canine mammary carcinoma based on grade

Grade 1 2 3 (n = 118) (n = 42) (n = 11) PHistological type Carcinoma — anaplastic (n = 1) 0 (0.0) 0 (0.0) 1 (100.0) 0.000 Carcinoma — complex type (n = 47) 27 (57.4) 20 (42.6) 0 (0.0) Carcinoma — micropapillary invasive (n = 1) 0 (0.0) 1 (100.0) 0 (0.0) Simple carcinoma (n = 36) 28 (77.8) 8 (22.2) 0 (0.0) Solid carcinoma (n = 7) 0 (0.0) 3 (42.9) 4 (57.1) Carcinoma arising in a mixed tumor (n = 71) 54 (76.1) 15 (21.1) 2 (2.8) Comedocarcinoma (n = 1) 0 (0.0) 0 (0.0) 1 (100.0) Ductal carcinoma (n = 5) 4 (80.0) 1 (20.0) 0 (0.0) Intraductal papillary carcinoma (n = 8) 5 (62.5) 3 (37.5) 0 (0.0) Squamous cell carcinoma (n = 4) 0 (0.0) 1 (25.0) 3 (75.0)

Molecular type HER-2 overexpressinga (n = 11) 10 (90.9) 0 (0.0) 1 (9.1) 0.010* Luminal Ab (n = 77) 45 (58.4) 28 (36.4) 4 (5.2) Luminal Bc (n = 65) 46 (70.8) 18 (27.7) 1 (1.5) Triple negatived (n = 28) 17 (60.7) 6 (21.4) 5 (17.9)

Triple negative Non-triple negative (n = 153) 101 (66.0) 46 (30.1) 6 (3.9) 0.016 Triple negative (n = 28) 17 (60.7) 6 (21.4) 5 (17.9)a ER2/PR2/HER-21.b ER1 or PR1/HER-22.c ER1 or PR1/HER-21.d ER2/PR2/HER-22.P , 0.05.* Fisher’s exact test.



all of the other types of tumors. In molecular phenotype analysis, triple-negative type tumors were more common in the shih tzu than in other breeds. Overall, the number of subjects decreased from Grade 1 to Grade 3, but the shih tzu group had the highest percent-age of Grade 3 subjects. Of the 25 basal-like triple-negative tumors in

28 triple-negative mammary tumors, 36% (9/25) were from shih tzu dogs. The shih tzu group had a broader distribution of histological types, whereas histological types of tumors in other breeds tended towards better prognostic types, such as carcinoma-complex type and carcinoma arising in a mixed tumor (Figure 3). Although expres-sions of grade in other groups were primarily Grade 1 or Grade 2, a higher proportion of Grade 3 was a distinguishing characteristic of the shih tzu breed.

When analyzing the characteristics of tumors according to age groups, we ascertained that histological types of mammary tumors with the worse prognosis tended to develop in older dogs ($ 10 y), particularly in the case of basal-like triple-negative tumors. Regarding molecular phenotype, both luminal A and luminal B types of tumors were more likely to be expressed in the younger age group, while HER-2-overexpressing types and triple-negative types of tumors tended to be expressed in the older age group. Analysis by Fisher’s exact test revealed that the histological types with better prognoses, such as complex carcinoma, carcinoma arising in a mixed tumor, and simple-type carcinoma, were significantly associated with younger age.

In contrast, triple-negative tumors, which are known to have a worse prognosis in human studies and poor response to treatment, were more common in the older group of dogs. Basal-like triple-negative tumors were expressed more frequently in the older group [19 of 25 cases (76.0%)], with the shih tzu being the most common breed among these 19 subjects [36.8% (7/19)].

Additional analysis was carried out according to tumor grade. An association was identified between tumor types with worse prognosis and higher grades and relations between triple-negative tumors and Grade 3. Some histological types of tumors known to have worse prognoses showed higher grades (P , 0.05). Molecular phenotype analysis that showed grade 3 tumors had the highest percentage of triple-negative type tumors.

To summarize the results, breeds did not show statistical signifi-cance by tumor molecular phenotypes, but the shih tzu breed most frequently developed basal-like triple-negative tumors and was sig-nificantly associated with Grade 3. In age, the older group of $ 10 y was associated with basal-like triple-negative and Grade 3 tumors.

Breed-related differences in the development of basal-like triple-negative tumors in dogs are similar to the race-related characteristics of African-Americans in the development of human breast cancer. The shih tzu breed was not the most common breed to develop mammary carcinoma, but it showed a higher prevalence of basal-like triple-negative mammary tumors than all the other breeds, just as African-Americans have the highest prevalence of human triple-negative breast cancer.

Basal-like triple-negative carcinomas were associated with both older non-shih tzu subjects and older shih tzu breed subjects. Differences were revealed between humans and dogs in develop-ing basal-like triple-negative tumors because there was a higher prevalence of these tumors in the older dogs (. 10 y). In humans, younger patients are usually more likely to develop these tumors.

Menopause is one of the important standards in classifying when breast cancer develops in humans. When breast cancer develops dur-ing the postmenopausal period, it does not develop under conditions of typical cyclic hormonal influence. As there is no physiological

Figure 3. A — Positive expression of epidermal growth factor recep-tor in simple carcinoma. IHC, mammary gland, canine. Bar = 35 mm. B — Positive expression of cytokeratin 14 in carcinoma arising in a mixed tumor. IHC, mammary gland, canine. Bar = 35 mm. C — Positive expres-sion of cytokeratin 5/6 in squamous cell carcinoma, metastasized. IHC, mammary gland, canine. Bar = 35 mm.

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menopausal period in dogs, they do not experience cessation of ovar-ian hormone release and the accompanying lobular involution of the mammary tissue. To correctly determine the age-related differences between dogs and humans, further studies will be needed to clarify and exclude the effects of a change in hormone status, accompanied by the effects of aging on tumor development or to provide corre-spondent factors in the dog, such as an ovariohysterectomy.

Although the results were not identical to those in human cases, there were considerable breed- and age-related differences in canine tumor characteristics. There were enough disparities in the expres-sion of tumor types by breed to consider that certain hereditary factors may influence cancer development. We can therefore infer that this prevalence is based on genetic inequality.

Associations between triple-negative tumors and the BRCA1 mutation have recently been investigated in humans (30–33). The BRCA1 mutation is a hereditary factor and representative mutation of genes that can cause breast cancers. In the present study, associa-tions of canine breed and age with basal-like triple-negative tumors have been investigated. In addition, associations of the BRCA muta-tion with canine mammary tumors have been investigated in many other studies (34–35). One study found noticeable characteristics of the shih tzu breed in overexpression of BRCA1 (36). Because the antibody used in this study was limited to exon 11 mutations of the human BRCA1 gene, these results cannot fully reflect the various BRCA1 mutations in dogs. Considering that 84% of the human and canine BRCA1 genes are homologous (37), however, these common characteristics of the shih tzu breed are noteworthy.

Previous studies suggest that there are racial differences in triple-negative breast cancer (29), that BRCA mutation locus varies among races (38–40), and that there is an association between the occurrence of triple-negative tumors and BRCA mutation (6,33). Further stud-ies must be conducted on canine breeds, however, to better under-stand breed-related hereditary factors that can influence the type of tumor that develops, basal-like triple-negative cancers of specific breeds, and the specific locus of BRCA1 gene mutation, as well as to establish associations between certain breeds and basal-like triple-negative cancers with BRCA1 mutations. When considering

those common characteristics in the shih tzu breed, triple-negative tumors and BRCA1 mutation seem to be affected by certain heredi-tary influences in tumor development. There may be an association between basal-like triple-negative cancers and BRCA1 mutation as well as other genetic factors in tumor development. Further study to investigate and understand canine mammary cancers may be useful for canine mammary cancer to serve as an effective model in human studies.

A c k n o w l e d g m e n t sThe authors thank Rae-Hwa Jang for her excellent technical assis-

tance. This article is part of the PhD thesis of Hyun-Woo Kim. This research was supported by the Basic Science Research Program of the National Research Foundation of Korea (NRF) and funded by the Ministry of Science, Information/Communication Technology (ICT) and Future Planning (2014R1A2A2A01003470).

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Histological type Carcinoma — complex type (n = 4) 1 (25.0) 1 (25.0) 0 (0.0) 1 (25.0) 1 (25.0) Simple carcinoma (n = 1) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 100 (100.0) Solid carcinoma (n = 4) 0 (0.0) 1 (25.0) 1 (25.0) 2 (50.0) 0 (0.0) Carcinoma arising in a mixed tumor (n = 12) 1 (8.3) 1 (8.3) 4 (33.3) 2 (16.7) 4 (33.3) Intraductal papillary carcinoma (n = 2) 0 (0.0) 0 (0.0) 1 (50.0) 1 (50.0) 0 (0.0) Squamous cell carcinoma (n = 2) 0 (0.0) 0 (0.0) 0 (0.0) 2 (0.0) 0 (0)

Grade 1 (n = 16) 2 (12.5) 2 (12.5) 4 (25.0) 4 (25.0) 4 (25.0) 2 (n = 4) 0 (0.0) 1 (25.0) 1 (25.0) 1 (25.0) 1 (25.0) 3 (n = 5) 0 (0.0) 0 (0.0) 1 (20.0) 4 (80.0) 0 (0.0)



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Article


I n t r o d u c t i o nIntraocular pressure (IOP) is maintained by a balance between

the production of aqueous humor and its outflow (1). Anesthetic agents can alter intraocular pressure by changing the rate of aqueous production or outflow or by increasing extraocular muscle tone (2). When choosing an anesthetic protocol for intraocular surgeries, the effect of the protocol on intraocular pressure must be taken into account (2,3). Abrupt increases in IOP throughout anesthesia of patients undergoing ophthalmic surgery can have significant effects. For example, minimal increases in IOP (30 to 35 mmHg) in glau-comatous animals can significantly lower axoplasmic flow within the optic nerve, resulting in further nerve injury (4). Inadvertent perforation of a deep corneal ulcer before or during surgery due to IOP elevations can complicate the surgical procedure and worsen the postoperative prognosis (5,6).

Alfaxalone is a synthetic neuroactive steroidal anesthetic that is injectable and acts as an agonist at gamma-aminobutyric acid A (GABAA) receptors within the central nervous system (CNS) (7). Alfaxalone was first introduced into veterinary medicine in 1971 (8) and is currently licensed as Alfaxan (Jurox, Kansas City, Missouri, USA) for use in several countries, including Canada, Australia, and most of Europe. Anesthesia can be induced and maintained with alfaxalone, producing rapid and smooth induction with excellent muscle relaxation (9–11). The cardiopulmonary effects of alfaxalone have been well-investigated in dogs (9–12). The reported effects of alfaxalone on IOP in dogs are varied (13,14). In 1 study without pre-anesthetic medication, a single bolus of alfaxalone induced a transient nonsignificant increase in IOP, followed by a significant reduction in IOP (14). In another study, a significant increase in IOP was observed when alfaxalone was administered after premedication with acepromazine and hydromorphone (13).

The effects of intravenous alfaxalone with and without premedication on intraocular pressure in healthy dogs

Bianca S. Bauer, Barbara Ambros

A b s t r a c tThe objective of this study was to investigate the effects of intravenous alfaxalone with and without premedication on intraocular pressure (IOP) in healthy dogs. Thirty-three dogs were randomized to receive 1 of 3 treatments: acepromazine [0.03 mg/kg body weight (BW)] with butorphanol (0.2 mg/kg BW) intramuscularly (IM), followed by intravenous (IV) alfaxalone (1.5 mg/kg BW); dexmedetomidine (0.002 mg/kg BW) with hydromorphone (0.1 mg/kg BW) IM, followed by alfaxalone (1 mg/kg BW) IV; and saline 0.9% (0.02 mL/kg BW) IM, followed by alfaxalone (3 mg/kg BW) IV. Intraocular pressure (IOP) was measured at baseline, 15 min, and 30 min after premedication, after pre-oxygenation, after administration of alfaxalone, and after intubation. After induction and after intubation, the IOP was significantly increased in all groups compared to baseline. While premedication with acepromazine/butorphanol or dexmedetomidine/hydromorphone did not cause a significant increase in IOP, the risk of vomiting and the associated peak in IOP after dexmedetomidine/hydromorphone should be considered when selecting an anesthetic protocol for dogs with poor tolerance for transient increases in IOP.

R é s u m éL’objectif de la présente étude était d’examiner les effets de l’administration intraveineuse (IV) d’alfaxalone avec et sans prémédication sur la pression intraoculaire (PIO) chez des chiens en santé. Trente-trois chiens ont été randomisés pour recevoir un des trois traitements : acépromazine [0,03 mg/kg de poids corporel (PC)] avec du butorphanol (0,2 mg PC) par voie intramusculaire (IM), suivi d’alfaxalone (1,5 mg/kg PC) IV; dexmedetomidine (0,002 mg/kg PC) avec de l’hydromorphone (0,1 mg/kg PC) IM, suivi d’alfaxalone (1 mg/kg PC) IV; et saline 0,9 % (0,02 mL/kg PC) IM, suivi d’alfaxalone (3 mg/kg PC) IV. La PIO a été mesurée au départ, 15 min et 30 min après la prémédication, après pré-oxygénation, après administration d’alfaxalone, et après intubation. Suite à l’induction et après intubation, la PIO était augmentée de manière significative dans tous les groupes comparativement à la valeur de base. Bien que la prémédication avec les combinaisons acépromazine/butorphanol ou dexmedetomidine/hydromorphone n’a pas causée d’augmentation significative de la PIO, le risque de vomissements et le pic associé de la PIO suite à l’administration de dexmedetomidine/hydropmorphone devraient être pris en considération lors de la sélection d’un protocole d’anesthésie pour des chiens avec une faible tolérance à une augmentation transitoire de la PIO.


Department of Small Animal Clinical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Drive, Saskatoon, Saskatchewan S7N 5B4.

Address all correspondence to Dr. Bianca Bauer; telephone: (306) 966-7083; fax: (306) 966-7174; e-mail: [email protected]

Received June 30, 2015. Accepted November 4, 2015.



Pre-anesthetic medication is commonly used to calm the patient, reduce anesthetic requirements, promote smooth induction and recovery from anesthesia, and provide analgesia (15). Combinations of acepromazine and butorphanol or dexmedetomidine and hydro-morphone are commonly used as pre-anesthetic medications in dogs (15,16). Although the effect of combined acepromazine and butorphanol on IOP has been evaluated in 1 study (17), the com-bination of dexmedetomidine and hydromorphone on IOP has not yet been studied. The objective of this study was to deter-mine the effects on IOP in healthy dogs after the administration of alfaxalone alone compared with alfaxalone administered after pre-anesthetic medication with acepromazine-butorphanol and dexmedetomidine-hydromorphone.

M a t e r i a l s a n d m e t h o d sThirty-three healthy shelter and client-owned dogs undergoing

elective surgery procedures were used for this study. Informed owner consent was obtained and the protocol was approved by the University of Saskatchewan’s Animal Research Ethics Board and fol-lowed the guidelines provided by the Canadian Council on Animal Care. Breed, gender, and body weight were recorded. Age was not recorded as definitive age could not be established for most patients, but all dogs were considered adults on the basis of physical and dental examination. Pre-anesthetic physical examination and blood work consisting of packed cell volume (PCV), total protein (TP), blood glucose, and blood urea nitrogen (BUN) were conducted in all cases and dogs were classified according to the American Society of Anesthesiologists (ASA) classification.

Before entering the study, all dogs underwent a complete ophthal-mic examination (Schirmer tear tests, intraocular pressure measure-ment with rebound tonometry, slit lamp biomicroscopy, and indirect ophthalmoscopy) by a veterinary ophthalmologist (BB) who was blinded to the treatment group. Dogs that were deemed unhealthy on physical examination or with abnormal PCV, TP, BUN, or oph-thalmic examination were excluded from the study. Brachycephalic breeds and aggressive dogs that were difficult to restrain were also excluded from the study.

All IOP measurements were taken by a veterinary ophthalmologist (BB) using rebound tonometry (Tonovet; Icare Finland Oy, Helsinki, Finland) and both eyes [oculus uterque (OU)] were measured in all subjects at all time points. Each IOP obtained was an average of 6 readings and only measurements with , 2.5% variance were used. All animals were restrained in sternal position with the head raised, avoiding pressure against the globe, jugular veins, or eyelids.

Dogs were allocated into 1 of 3 treatment groups by random drawing, using an envelope containing the assignment to a treat-ment. The main investigators (BA and BB) were unaware of which premedication combination had been administered. The 3 treatment groups were: Group ABA — acepromazine [0.03 mg/kg body weight (BW)] (Atravet; Ayerst Laboratories, Montreal, Quebec) with butor-phanol (0.2 mg/kg BW) (Torbugesic; Wyeth Animal Health, Guelph, Ontario) intramuscularly (IM), followed by intravenous (IV) alfaxa-lone (1.5 mg/kg BW) (Alfaxan; Abbott Laboratories, Saint Laurent, Quebec); Group DHA — dexmedetomidine (0.002 mg/kg BW) (Dexdomitor; Pfizer Canada, Mississauga, Ontario) with hydro-

morphone (0.1 mg/kg BW) (Hydromorphone HCl; Sandoz Canada, Quebec City, Quebec) IM, followed by alfaxalone (1 mg/kg BW) IV; and Group SA — saline 0.9% (0.02 mL/kg BW) (0.9% Sodium Chloride; Hospira, Montreal, Quebec) IM, followed by alfaxalone (3 mg/kg BW) IV.

After pre-medication, an intravenous catheter was placed in a cephalic vein and dogs were pre-oxygenated with a fitting face mask for 3 min. If dogs did not tolerate the application of the face mask, i.e., became agitated and struggled, pre-oxygenation was discontinued and procedures were paused for 3 min. Anesthesia was then induced with intravenous alfaxalone. The prepared dose of alfaxalone was administered by hand injection over 60 s, after which jaw tone was assessed by a board-certified anesthesiologist (BA). If jaw tone was not lost after 20 s, further boluses of 0.5 mg/kg BW of alfaxalone were given over 20 s until jaw tone was absent such that intubation could be achieved. After induction was complete, post-induction intraocular pressures were obtained right away, followed immediately by intubation by a board-certified anesthesiologist (BA). The number of attempts and any difficulty in orotracheal intubation were recorded. Intraocular pressures were measured at the follow-ing 6 time points: baseline at initial ophthalmic examination (BL); 15 min after premedication (15); 30 min after premedication (30); after pre-oxygenation and before induction (Post O2); immediately after administration of alfaxalone (Post A); and after intubation, before connection to the anesthetic breathing system (Post INT).

Quality of sedation at time point 30, anesthesia induction, and intubation was scored by a board-certified anesthesiologist (BA) blinded to the treatment (Appendix 1). Behavior after premedica-tion was observed and any incidences of vomiting or retching were recorded. After the final IOP measurement, dogs continued on to the planned surgical procedure. All procedures were carried out by the same 2 people (BA and BB) between 0800 and 1400 h.

Statistical analysisA commercially available software package (Graph Pad Prism 6

for MAC OS X; Graph Pad Software, San Diego, California, USA) was used for statistical analyses. Normality was tested by the Kolmogorov-Smirnov test. A paired t-test was done to compare IOP between right and left eye at each time point. A 1-way analysis of variance (ANOVA) with Tukey’s post-hoc test was used to evalu-ate within-group changes in IOP. Between-treatment effects in IOP were analyzed with 2-way repeated measures ANOVA with Tukey’s post-hoc test. Data are reported as mean 6 standard deviation (SD). Sedation, induction, and intubation scores were compared with a Mann-Whitney test and data are presented as a median. Population and drug data were compared among groups with independent t-tests. A value of P , 0.05 was considered significant.

Re s u l t sThirty-three dogs were initially included in the study, but 2 dogs

in the saline group were excluded from data processing due to signif-icant differences between the IOP in the right and left eye. As there were no significant differences between the right and left eye in the remaining 31 dogs at any time point, the data from both eyes were pooled for subsequent analysis. Of the 31 dogs included in the data



analysis, there were 17 mixed breed, 3 border collies, 3 Weimaraners, 2 Siberian huskies, 2 Australian shepherds, 1 Labrador retriever, 1 Staffordshire terrier, 1 Rottweiler, and 1 Portuguese water dog.

Body weight, sedation, induction and intubation scores, base-line IOP, and the number of dogs requiring alfaxalone top-ups, and alfaxalone top-up dose are provided in Table I. There were no significant differences in body weight and baseline IOP (P . 0.05) among groups. Sedation scores were significantly different between the ABA [median 3 (2 to 4)] and SA groups (median 1) (P , 0.0001) and between the DHA [median 2 (1 to 3)] and SA groups (median 1) (P = 0.0002), but not between the ABA and DHA groups (P = 0.09). Two dogs in the DHA group vomited, but no other side effects were observed. All anesthetic regimes were successful in eliminating

laryngeal reflex and preventing cough; 4 dogs required alfaxalone top ups (Table I). There were no difficulties in carrying out orotra-cheal intubation. All dogs were successfully intubated on the first attempt and all subjects were intubated within 2 min of the begin-ning of the alfaxalone injection.

When comparing IOP among groups, a significant differ-ence was observed between the SA (15.4 6 3.7 mmHg) and ABA (19.0 6 2.6 mmHg) groups (P , 0.01) at 15 min (Table II). No other significant differences were observed among treatment groups (P . 0.05, Table II). In the ABA group, IOP was significantly differ-ent among BL (15.9 6 2.1 mmHg) and post A (20.3 6 3.9 mmHg, P , 0.05) and post INT (22.3 6 4.0 mmHg, P , 0.001); among 15 (19.0 6 2.6 mmHg) and 30 (15.3 6 4.6 mmHg, P , 0.01) and post O2

Table I. Baseline characteristics for dogs administered ABA (acepromazine, butorphanol, alfaxalone) (n = 11), DHA (dexmedetomidine, hydromorphone, alfaxalone) (n = 11), and SA (saline, alfaxalone) (n = 9)

Variable ABA group DHA group SA groupBody weight (kg) 20.1 6 10.4 18.4 6 6.0 17.8 6 7.3Baseline IOP (mmHg) 15.9 6 2.1 16.0 6 2.9 16.9 6 3.4Sedation score (1 to 5) 3 (2 to 4)* 2 (1 to 3)* 1*Induction score (1 to 3) 1 1 (1 to 2) 1 (1 to 2)Intubation score (1 to 4) 1 (1 to 2) 1 (1 to 2) 1 (1 to 2)Number of dogs requiring alfaxalone top up 2 1 1Alfaxalone top-up dose (mg/kg) 1 6 0.5 1 0.5Data are expressed as mean 6 SD or median (range). Treatment groups were compared with unpaired t-test (body weight), 2-way analysis of variance (ANOVA) for repeated measures [intraocular pressure (IOP)], and Mann-Whitney test (sedation scores, induction, and intubation scores).No significant differences were observed among treatments, except for sedation scores. * Sedation scores between SA and ABA and between SA and DHA were significantly different.P-values , 0.05 were considered significant.

Table II. Mean 6 SD values for intraocular pressure (mmHg) recorded at different time points in healthy dogs receiving either ABA (acepromazine, butorphanol, alfaxalone) (n = 11), DHA (dexmedetomidine, hydromorphone, alfaxalone) (n = 11), or SA (saline, alfaxalone) (n = 9)

TreatmentsTime points SA ABA DHABL 16.9 6 3.4 15.9 6 2.1 16.0 6 2.915 15.4 6 3.7* 19.0 6 2.6 16.3 6 3.430 15.4 6 3.5 15.3 6 4.6b 16.2 6 4.3Post O2 15.9 6 3.1 14.5 6 3.4b 15.3 6 4.4Post A 21.8 6 3.9aaa,bbbb,cccc,dddd 20.3 6 3.9a,ccc,dddd 19.6 6 3.5aa,dd

Post INT 22.4 6 3.9aaa,bbbb,ccccc,dddd 22.3 6 4.0aaa,cccc,ddd 22.2 6 3.5aaa,b,cccc,dddd

P-values , 0.05 were considered significant.BL — Baseline; 15 to 15 min after premedication; 30 to 30 min after premedication; Post O2 — Post pre-oxygenation; Post A — Post alfaxalone administration; and Post INT — Post intubation.* Values are significantly different between group ABA and SA (P , 0.01).a Significantly different from baseline value for the respective treatment.b Significantly different from (15) value for the respective treatment.c Significantly different from (30) value for the respective treatment.d Significantly different from post O2 value for the respective treatment.(a) P , 0.05; (aa) P , 0.01;(aaa) P , 0.001; (aaaa) P , 0.0001.



(14.5 6 3.4 mmHg, P , 0.001); among 30 and post A (P , 0.0001) and post INT (P , 0.0001), among post O2 and post A (P , 0.0001) and post INT (P , 0.0001). In the DHA group, IOP did not dif-fer among BL (16.0 6 2.9 mmHg) and 15 min (16.3 6 3.4 mmHg) or 30 min (16.2 6 4.3 mmHg) after premedication (P . 0.05). There was, however, a significant difference among BL and post A (19.6 6 3.5 mmHg, P , 0.01) and post INT (22.2 6 3.5 mmHg, P , 0.0001); among post O2 (15.3 6 4.4 mmHg) and post A (P , 0.01) and post INT (P , 0.0001); between 15 and post INT (P , 0.0001); and between 30 and post INT (P , 0.0001). For the SA group, IOP was significantly different between BL (16.9 6 3.4 mmHg) and post A (21.8 6 3.9 mmHg, P , 0.001) and post INT (22.4 6 3.9 mmHg, P , 0.0001); between 15 (15.4 6 3.7 mmHg) and post A and post INT (P , 0.0001); between 30 (15.4 6 3.5 mmHg) and post A and post INT (P , 0.0001); between post O2 (15.9 6 3.1 mmHg) and post A and post INT (P , 0.0001).

D i s c u s s i o nThis was a clinical study that investigated the effects of 2 common

pre-anesthetic medication protocols, followed by induction with alfaxalone, on IOP in healthy dogs scheduled for routine surgery. The drug combinations and doses used in this study were those routinely used to achieve a level of sedation and induction necessary for intubation. The baseline IOP values obtained in this study before treatment are in agreement with the generally accepted normal range for IOP in dogs (15 to 18 mmHg) (18).

It has previously been reported that IOP does not significantly increase after IM administration of combined acepromazine/ butorphanol (17) or acepromazine/hydromorphone (13,19). Our results using combined acepromazine/butorphanol show a signifi-cant difference in IOP at 15 min. Although statistically significant, we do not believe that this is clinically significant. Our results also demonstrate that no significant elevation in IOP occurs with the IM administration of dexmedetomidine in combination with hydromorphone.

To the authors’ knowledge, there are no previous reports evaluat-ing the effects on IOP of this particular drug protocol. One study, however, demonstrated an elevated IOP in dogs in lateral recum-bency after IV administration of dexmedetomidine and butorpha-nol (20). This study compared IV medetomidine (0.3 mg/m2) with butorphanol (6 mg/m2) to IV dexmedetomidine (0.3 mg/m2) with butorphanol (6 mg/m2) and, although both groups demonstrated sig-nificant increases in IOP relative to baseline, the dexmedetomidine/ butorphanol group was significantly higher than the medetomidine/butorphanol group (20). It was proposed that the significant differ-ences between the groups was due to the potency of the dexme-detomidine dose chosen for the dexmedetomidine/butorphanol group. The IOP elevation noted by Rauser et al (20) in both groups was deemed to be due to elevations in systemic vascular resistance and blood pressure indirectly influencing IOP. These IOP increases were not determined to be clinically significant, however, as the IOPs did not exceed 20 mmHg. In contrast, Artigas et al (21) determined that IV dexmedetomidine (0.005 mg/kg BW) given alone did not significantly increase IOP of healthy dogs in sternal recumbency (21). While low doses of dexmedetomidine should not increase systemic

vascular resistance enough to increase IOP, the dose used by Rauser et al (20) (approximately 0.01 mg/kg BW, IV) was sufficient to cause an increase in IOP in dogs.

Our study demonstrated that there is no increase in IOP at a 0.002 mg/kg BW dose of dexmedetomidine. The overall effect of dexmedetomidine combined with hydromorphone on IOP is mini-mal at the time points at which IOP was measured, which suggests that IM sedation with dexmedetomidine/hydromorphone at these dosages is a satisfactory option for surgical premedication when an increase in IOP is undesirable. However, the adverse effects of this combination need to be considered. Two out of 11 dogs in our study vomited shortly after sedation with dexmedetomidine and hydro-morphone. Coughing, retching, and vomiting and any maneuver that increases central venous pressure may induce a dramatic increase in IOP (2). This elevation in central venous pressure results in a steep increase in choroidal blood volume and IOP. Emesis and the associated increase in IOP is a possible side-effect of administering systemic m-opioid receptor agonist (22) and alpha-2 adrenergic recep-tor agonist (23). The risk of vomiting and the associated elevation in IOP after administration of dexmedetomidine and hydromorphone should be considered when selecting an anesthetic protocol for a patient at risk of globe rupture.

When comparing IOP among all 3 treatment groups, a statistically significant difference was observed between SA (15.4 6 3.7 mmHg) and ABA (19.0 6 2.6 mmHg) at 15 min after premedication. Given that IOP changes within the respective groups were not statistically significant compared to baseline values and remained within the normal range at the measured time points, this finding was deemed not clinically relevant. A significant increase in IOP has been demon-strated after induction of anesthesia in healthy dogs with propofol (13,24,25), alfaxalone (13), and a combination of ketamine, diazepam, and ketamine/diazepam (26). Our study findings are similar as IV induction with alfaxalone resulted in a statistically significant increase in IOP relative to baseline and 30 min post-premedication in all study groups. These results are consistent with the findings of Hasiuk et al (13), in which 1.5 mg/kg BW of IV alfaxalone was used for induction following premedication with IV acepromazine and hydromorphone in dogs and IOPs were measured in sitting or sternal recumbency.

In contrast to these findings, a more recent study observed a transient nonsignificant increase in IOP, followed by a significant reduction in IOP after a single bolus of 3 mg/kg BW of alfaxalone was administered to healthy non-premedicated dogs (14). There are several possible explanations for this difference in IOP after alfaxa-lone. Dogs in our study and in the study by Hasiuk et al (13) were sedated with acepromazine/butorphanol or dexmedetomidine/hydromorphone and acepromazine-hydromorphone, respectively. The simultaneous use of pre-anesthetic medication could have induced pharmacologic or pharmacokinetic interactions, which promoted an increase in IOP. Another more likely explanation is the different duration of the 3 studies. Costa et al (14) monitored IOP for 30 min after alfaxalone administration, whereas measurements of IOP pressure were stopped after orotracheal intubation in our dogs as well as in the study by Hasiuk et al (13). It is possible that a reduction in IOP at a later time point was missed due to the shorter monitoring window after alfaxalone administration.



Another difference between studies is orotracheal intubation following alfaxalone administration. Dogs in the study by Costa et al (14) were not intubated, while dogs in our study and in the study by Hasiuk et al (13) were intubated. Although intubation criteria were met for all the dogs herein and no obvious response to orotracheal intubation, such as gagging or coughing, was observed, IOP increased further in all 3 treatment groups post-intubation. This observation is consistent with the findings of Hasiuk et al (13). An increase in IOP of 5 mmHg secondary to laryngoscopy and intubation has been described in humans (27). Hofmeister et al (25) reported a significant increase in IOP in dogs after intubation. This increase in IOP is most likely related to the cardiovascular response to intubation. Laryngoscopy and tracheal intubation can cause tachy-cardia and hypertension due to sympathetic discharge caused by stimulation of the upper respiratory tract (28). While brief elevation of IOP during intubation is normally of little consequence, it can be harmful to patients with penetrating globe injuries.

Intraocular pressure during anesthesia can be affected in several ways. Anesthetic agents can alter intraocular pressures by changing the rate of aqueous production or outflow or by increasing extraocu-lar muscle tone and scleral rigidity (2). It has been suggested that extraocular muscle tone has a minimal effect on IOP during induc-tion of anesthesia in healthy dogs when propofol or alfaxalone are used (13). Aqueous humor production and outflow can be affected by changes in central nervous system (CNS) output, blood pressure (BP), central venous pressure (CVP), partial pressure of oxygen in arterial blood (PaO2), and partial pressure of carbon dioxide in arte-rial blood (PaCO2) (2). Hofmeister et al (24,29) demonstrated that in unpremedicated dogs, hypercapnea did not play a role in IOP changes with propofol. Although BP, CVP, PaO2, and PaCO2 were not specifically evaluated in this study, Ambros et al (10) determined that, in dogs premedicated with acepromazine and hydromorphone, PaCO2 increases significantly post-induction with alfaxalone or pro-pofol and that PaO2 is unaltered. A limitation of the present study is that PaO2 and PaCO2 were not measured and it is therefore possible that hypoventilation and hypercapnea may have played a role in the IOP alterations noted in our dogs.

Changes in CNS control of the production and outflow of aqueous humor may also play a role in alfaxalone induction in dogs similar to propofol induction in humans (30). As alfaxalone induces sig-nificant respiratory depression (10), it is possible that hypoxemia or elevated CVP during induction could also contribute to the increase in IOP observed in our study. All dogs were given oxygen for at least 3 min before induction, however, and were intubated within 2 min of the beginning of the alfaxalone injection. In our study, the pre-oxygenation had no significant effects on IOP as expected and hypoxemia would not be expected to develop within 2 min of the onset of apnea after pre-oxygenation (31). With regard to CVP, to avoid affecting ocular venous drainage, no pressure was placed on either external jugular vein during handling of the patients.

In conclusion, the results of this study show a statistically signifi-cant increase in IOP after induction with alfaxalone, with or with out pre-anesthetic medication. Premedication with acepromazine/butorphanol or dexmedetomidine/hydromorphone did not cause a significant increase in IOP and are satisfactory pre-anesthetic combinations to use alone or before anesthesia induction in dogs.

The risk of vomiting and the associated elevation in IOP after dex-medetomdine and hydromorphone are administered should be considered when selecting an anesthetic protocol for a patient with limited tolerance for short-lived increases in IOP. Additional studies are needed to evaluate the effect of alfaxalone on the eyes of patients with ocular diseases, such as glaucoma.

A c k n o w l e d g m e n tThis study was supported by the Companion Animal Health Fund,

Western College of Veterinary Medicine.

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Appendix

Scores for Sedation, Induction, and Intubation[from Maddern et al (32)]

Sedation ScoreI. No discernable effectII. Mild sedation: appears sleepyIII. Moderate sedation: very sleepy may be recumbent, but could be

arousedIV. Heavy sedation: recumbent, difficulty rousingV. Profound sedation: lateral recumbency, not arousable

Anesthesia Induction ScoreI. Smooth uneventful inductionII. Some mild paddling, twitching, excitementIII. Poor induction, pronounced paddling, twitching, excitement

Tracheal Intubation ScoreI. Smooth intubationII. Some mild coughingIII. Pronounced coughingIV. Swallowing, coughing, gagging — failed attempt



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12. Keates H, Whittem T. Effect of intravenous dose escalation with alfaxalone and propofol on occurrence of apnoea in the dog. Res Vet Sci 2012;93:904–906.

13. Hasiuk MM. A comparison of alfaxalone and propofol on intra-ocular pressure in healthy dogs. Vet Ophthalmol 2014;17:411–416.

14. Costa D, Leiva M, Moll X, Aguilar A, Peña T, Andaluz A. Alfaxalone versus propofol in dogs: A randomised trial to assess effects on peri-induction tear production, intraocular pressure and globe position. Vet Rec 2015;176:73.

15. Rankin DC. Sedatives and tranquilizers. In: Grimm KA, Lamont LA, Tranquilli WJ, Green SA, eds. Veterinary Anesthesia and Analgesia. 5th ed. Ames, Iowa:Wiley-Blackwell, 2015: 196–206.

16. Cornick JL, Hartsfield SM. Cardiopulmonary and behavioral effects of combinations of acepromazine/butorphanol and acepromazine/oxymorphone in dogs. J Am Vet Med Assoc 1992; 200:1952–1956.

17. Tamura EY, Barros PS, Cortopassi SR, Ambrosio AM, Fantoni DT. Effects of two preanesthetic regimens for ophthalmic surgery on intraocular pressure and cardiovascular measurements in dogs. Vet Ther 2002;3:81–87.

18. Featherstone HJ, Heinrich CL. Ophthalmic examination and diagnostics, Part 1: The eye examination and diagnostic pro-cedures. In: Gelatt KN, Gilger BC, Kern TJ, eds. Veterinary Ophthalmology. 5th ed. Vol. 2. Ames: John Wiley & Sons, 2013: 533–613.

19. Stephan DD,Vestre WA, Stiles J, Krohne S. Changes in intraocular pressure and pupil size following intramuscular administration of hydromorphone hydrochloride and acepromazine in clinically normal dogs. Vet Ophthalmol 2003;6:73–76.

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21. Artigas C, Redondo JI, Lopez-Murcia MM. Effects of intrave-nous administration of dexmedetomidine on intraocular pres-sure and pupil size in clinically normal dogs. Vet Ophthalmol 2012;15:79–82.

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25. Hofmeister EH, Williams CO, Braun C, Moore PA. Influence of lidocaine and diazepam on peri-induction intraocular pressures in dogs anesthetized with propofol-atracurium. Can J Vet Res 2006;70:251–256.

26. Hofmeister EH, Mosunic CB, Torres BT, Ralph AG, Moore PA, Read MR. Effects of ketamine, diazepam, and their combination on intraocular pressures in clinically normal dogs. Am J Vet Res 2006;67:1136–1139.

27. Ismail SA, Bisher NA, Kandil HW, Mowafi HA, Atawia HA. Intraocular pressure and haemodynamic responses to insertion of the i-gel, laryngeal mask airway or endotracheal tube. Eur J Anaesthesiol 2011;28:443–448.

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Article


I n t r o d u c t i o nSurgery is an essential part of veterinary medicine, regardless of

the area of practice. Sutures play a crucial role in surgery. The knot is the weakest part of a suture and is generally the site of failure (1–3). Of the many types of knots, the following 4 are commonly

used in small animal practice: square knot, surgeon’s knot, granny knot (technical error), and the sliding half-hitch.

The ideal knot should be quick and easy to make, while remaining safe and secure (4). A “safe knot” has been defined as a knot that breaks rather than unties due to slippage when tested to failure (5). Additionally, the knot should have as little affect as possible on the

Evaluation of the effect of 4 types of knots on the mechanical properties of 4 types of suture material used in small animal practice

Xytilis Avoine, Bertrand Lussier, Vladimir Brailovski, Karine Inaekyan, Guy Beauchamp

A b s t r a c tThe influence of the type of material used, knot configuration, and use of an additional throw on the tensile force at failure, the elongation, and the mode of failure of different configurations of linear sutures and knotted suture loops was evaluated in this in-vitro mechanical study. We hypothesized that all types of knots would significantly influence the initial force and elongation of suture materials and would influence the force and elongation at which the knotted loops break, but not their mode of failure. A total of 432 samples of 4 types of size 3-0 suture material (polydioxanone, polyglecaprone 25, polyglactin 910, and nylon), representing 9 configurations, were tested in a tensiometer. The configurations were 1 linear suture without a knot and the following loops: square (SQ) knot; surgeon’s (SU) knot; granny (GR) knot; and sliding half-hitch (SHH) knot using either 4 and 5 or 3 and 4 throws, depending on the material. For polydioxanone, SQ and SU knots did not decrease the initial force at failure of the suture. Granny (GR) and SHH knots decreased the tensile force at failure and elongation by premature failure of the loop. For polyglecaprone 25, all knots decreased the initial force at failure of the suture, with SHH being weaker than the other knots. For coated polyglactin 910, all knots decreased the initial force at failure of the suture and slippage increased significantly compared with the other 3 sutures. The use of SQ knots with 3 throws did not result in a safe knot. For nylon, knots did not alter the original mechanics of the suture. In conclusion, all knots and types of suture material do not necessarily have the same effect on the initial tensile force at failure of suture materials.

R é s u m éL’objectif de la présente étude était de déterminer, dans des conditions expérimentales contrôlées, l’influence de quatre types de nœud, du nombre de croisements et du type de matériel de suture utilisés sur le mode de rupture, l’élongation et la résistance de différentes configurations de boucles de suture. Un total de 432 échantillons de quatre types de fil de suture 3-0 (polydioxanone, polyglecaprone 25, polyglactin 910, nylon), représentant neuf configurations, ont été évalués à l’aide d’un tensiomètre. Plusieurs paramètres ont été analysés: la force atteinte lors de la rupture, l’élongation maximale de la boucle et son mode de rupture. Les différentes configurations sont: suture linéaire, nœud plat (NP), de chirurgien (NC), de vache (NV) et le nœud coulissant barré (NCB) en réalisant 4 et 5 croisements pour les monofilaments et 3 et 4 croisements pour le multifilament. Les différents matériaux et nœuds utilisés n’affectent pas tous de la même manière la force initiale du matériel de suture. Pour le polydioxanone, le NP et NC ne diminuent pas la force initiale du fil de suture. Le NV et le NCB diminuent la force initiale du fil de suture et diminuent son élongation par un bris prématuré du fil de suture. Pour le polyglecaprone 25, tous les nœuds réduisent la force initiale de la suture; le NCB est celui qui affecte le plus le matériel de suture. Pour le polyglactin 910 enrobé, tous les nœuds réduisent la force initiale de la suture; le glissement est augmenté de manière significative comparé aux trois autres types de matériels de sutures, une élongation significative de la boucle de suture a été observée avec la présence d’un NP avec trois croisements. Pour le nylon, les nœuds n’ont pas modifié les propriétés mécaniques d’origine du fil de suture. En conclusion, un NP et NC avec un polydioxanone 3-0 ne diminue pas la force initiale du fil de suture. Le polyglactin 910 enrobé est significativement associé à un glissement accru. Il n’y a pas de différence statistiquement significative à l’ajout d’un croisement à une boucle de suture 3-0.

(Traduit par les auteurs)

Department of Clinical Sciences (Avoine, Lussier) and Department of Pathology and Microbiology (Beauchamp), Faculty of Veterinary Medicine, Université de Montréal, 3200, rue Sicotte, St. Hyacinthe, Québec J2S 7C6; Department of Mechanical Engineering, École de technologie supérieure, 1100, rue Notre-Dame Ouest, Montréal, Québec H3C 1K3 (Brailovski, Inaekyan).

Address all correspondence to Dr. Xytilis Avoine; telephone: 1033 7 83 63 57 93; e-mail: [email protected]

The authors declare that there are no financial or other conflicts of interest related to this study.




suture material. Many factors influence the safety of a suture, includ-ing the type of material, the type of knot, the number of throws, and the surgeon’s experience. Various studies have been conducted on the configuration of suture loops, evaluating the knot configuration (4,6), the number of throws (7,8), and the type of material (1,4,9,10). To our knowledge, there are no reports in the literature that evaluate, under the same experimental conditions, the behavior of 3-0 knotted loops of suture material frequently used in small animal surgery.

The aim of this study was to evaluate, under controlled experi-mental conditions, the influence of the type of material used, knot configuration, and use of an additional throw on the tensile force at failure, the elongation, and the mode of failure of different configura-tions of linear sutures and knotted suture loops. We hypothesized that all types of knots would significantly influence the initial force and elongation of each of the linear sutures. Our second hypothesis was that all types of knots would significantly influence the force and elongation at which the knotted loops break, but not their mode of failure.


Suture materialFour types of size 3-0 suture materials were evaluated: polydioxa-

none (PDSII), polyglecaprone 25 (Monocryl), coated polyglactin 910 (Vicryl), and nylon (Ethilon) [Ethicon (Johnson & Johnson), Somerville, New Jersey, USA].

Suture configurationEach type of suture material was evaluated in 9 configurations:

1 linear strand of suture without a knot (control group) and 8 loop configurations. All knots were individually tied by the same operator (BL) using 2-handed hand ties wrapped around the combination of a 20-mL syringe (Terumo, Somerset, New Jersey, USA) and an adjacent metallic pin (3-mm diameter) so that the suture loops could easily be removed from the syringe after the metallic pin is withdrawn, thus minimizing friction-related microtrauma to the suture loops. The knot ears were cut to a standardized length of 5 mm (1,3). The syringe was fixed in a vice on a bench top to ensure consistent ten-sion when tightening the knots and all the knots were inspected for slippage before cutting the ears.

The 8 loop configurations were: square (SQ) knot; surgeon’s (SU) knot; granny (GR) knot; and the sliding half-hitch (SHH) knot, with a standard number of throws (4 for monofilament or 3 for multifilament) and another series was tied with a standard number of throws plus 1 (5 for monofilament or 4 for multifilament). For all configurations, the supplemental throws made over the initial throw consisted of square knots. The number of throws for monofilament (4) versus multifilament suture (3) was based on recommendations published earlier (11). For each configuration, 12 samples were tied (based on a pilot study). All knots were tied in an alternate sequen-tial order according to the type of knot; square, surgeon’s, granny, sliding half-hitch: square 11, surgeon’s 11, granny 11, sliding half-hitch 11. The work was divided into 4 sessions to prevent surgeon fatigue and creating a potential bias. A total of 432 samples was obtained.

Mechanical testingAll samples were tested using a materials-testing machine (Electro

Force 3200; Bose, Eden Prairie, Minnesota, USA). All samples were tested in the following sequential order for each type of suture: polydioxanone, polyglecaprone 25, coated polyglactin 910, and nylon. When placed in the materials-testing machine, each loop of tied suture was wrapped around 2 rods that were able to rotate, thus reducing constraints due to friction (Figure 1). The linear sutures were placed between 2 clamps using a previously described method (12).

Samples were subjected to an initial tension of 5 Newtons (N) for 3 min and an elongation rate of 5 mm per min (to avoid the influence of the strain rate on the behavior of the suture material) until failure occurred. Tensile force and elongation were recorded continuously with dedicated software (WinTest 4.0; Bose). The following param-eters were analyzed: (a) tensile force (N) measured at the failure of the loop; (b) elongation of the loop (mm) measured at the failure of the loop; and (c) mode of failure, defined as failure at the knot, failure of the suture excluding the knot, and slippage of the knot.

The mode of failure was determined by analyzing the “load- displacement” curves and visually inspecting the knotted loops. Video recordings were made to document all trials and were reviewed when needed to validate the mode of failure. For data analysis, failure forces and elongations at failure were considered for all the samples. Samples were excluded from the force data analysis only if the displacement limit of the testing machine was reached, but no failure occurred or if knot slippage resulted in the loop opening during testing (Table I). For elongation, however, loops that did not rupture at the end of the displacement were considered

Figure 1. Testing jig of the materials-testing system with a knotted loop of suture positioned between the rods.



for statistical analyses. For the mode of failure, all samples were considered for descriptive analyses.

Statistical analysesA linear model was used to compare the tensile force and elonga-

tion. Given the large number of comparisons, the alpha level was adjusted to declare a significant difference to be more conservative. The sequential Bonferroni method was used to make this adjust-ment. A descriptive analysis was used for the failure mode. In each

dataset, P , 0.05 was considered statistically significant. SAS version 9.3 software (SAS Institute, Cary, North Carolina, USA) was used to conduct the statistical analysis. A posteriori statistical analyses using logistic regression were used, with the occurrence of slippage as the dependent variable and the type of knot and number of throws as independent variables. For each type of knot, an exact Chi-square test was carried out to determine whether suture type was associ-ated with the prevalence of slippage. This was followed by pairwise comparisons among suture types.

Table 1. Results of mechanical sample testing for tensile force and elongation at failure

Tensile force Elongation at at failure (N) failure (mm)Suture material Knot configuration n Throws Mean SD Mean SDPolydioxanone Square knot 12 4 58.21 1.60 11.18 0.64 Surgeon’s knot 12 4 54.37 5.07 10.61 0.96 Granny knot 12 4 43.81★ 8.06 8.91 1.13 Sliding half-hitch knot 11* 4 44.53 4.99 8.48★ 1.80 Square knot 8* 5 60.28★ 3.85 11.71 0.47 Surgeon’s knot 10** 5 54.11 2.84 10.58 0.88 Granny knot 12 5 49.67★ 3.55 9.28 0.94 Sliding half-hitch knot 12 5 44.59★ 6.94 8.38★ 1.56Linear suture (without knot) 12 N/A 55.14 1.62 10.30 0.58

Polyglecaprone 25 Square knot 11** 4 63.46★ 3.64 10.69 0.4 Surgeon’s knot 12 4 57.65★ 8.25 10.39 0.88 Granny knot 12 4 55.19★ 6.10 9.97 0.52 Sliding half-hitch knot 12 4 52.18★ 3.52 9.46 0.35 Square knot 12 5 62.24★ 6.46 10.78 0.82 Surgeon’s knot 11* 5 58.34★ 5.45 10.82 0.58 Granny knot 12 5 53.24★ 7.72 9.59 0.79 Sliding half-hitch knot 12 5 54.06★ 4.68 9.77 0.66Linear suture (without knot) 12 N/A 74.27 6.71 10.92 1.10

Polyglactin 910 Square knot 10* 3 34.38★ 13.3 9.91★ 1.66 Surgeon’s knot 12 3 41.05★ 4.72 7.46 1.89 Granny knot 12 3 21.72★ 9.07 9.86★ 1.68 Sliding half-hitch knot 12 3 22.26★ 11.7 9.20★ 2.17 Square knot 12 4 41.56★ 7.79 6.98 1.01 Surgeon’s knot 12 4 39.92★ 10.8 6.62 1.37 Granny knot 12 4 28.63★ 11.3 9.47★ 2.38 Sliding half-hitch knot 12 4 33.99★ 10.2 9.02★ 2.54Linear suture (without knot)) 12 N/A 66.47 5.51 5.89 1.95

Nylon Square knot 12 4 40.41 2.16 9.86 0.32 Surgeon’s knot 12 4 37.84 3.76 9.46 1.20 Granny knot 11* 4 35.08 8.50 8.78 2.29 Sliding half-hitch knot 10* 4 34.38 4.68 8.42 1.43 Square knot 12 5 40.07 3.12 9.68 1.08 Surgeon’s knot 12 5 34.01 4.92 8.30 1.31 Granny knot 9* 5 37.24★ 3.02 9.41 2.04 Sliding half-hitch knot 12 5 35.71 2.46 7.83 0.60Linear suture (without knot) 12 N/A 40.74 2.68 9.00 1.57* Machine displacement limit was reached.** Erroneous data acquisition.★ Significant result (P , 0.05).N/A — Not applicable.



Re s u l t sA total of 432 samples were submitted for evaluation in the mate-

rials-testing machine. Seventeen specimens were excluded from data analysis of tensile force, 14 because the machine displacement limit was reached, but no failure occurred and 3 because of an erroneous data acquisition. The results of mechanical testing of the samples for tensile force and elongation are summarized in Table I.

Tensile force at failureInfluence of knot versus linear suture on force at failure — As

shown in Figure 2, for polydioxanone, the presence of a knot signifi-cantly decreased the tensile force at failure of the loops for granny and sliding half-hitch knots, but not for square and surgeon’s knots. For polyglecaprone 25 and coated polyglactin 910, all knots signifi-

cantly decreased the force at failure of the loops and for nylon, only the surgeon’s knot with an additional throw significantly decreased the force at failure of the loops.

Influence of types of knots on force at failure — As shown in Figure 3, for polydioxanone, the presence of a granny knot and a sliding half-hitch significantly decreased tensile force at failure of loops compared to square or surgeon’s knots. For polyglecaprone 25, the presence of a sliding half-hitch significantly decreased tensile force at failure compared to a square knot. For coated polyglactin 910, the presence of a granny knot or a sliding half-hitch significantly decreased tensile force at failure compared to a surgeon’s knot and for the nylon, there was no significant difference among the 4 types of knots.

Addition of supplemental throw — There was no statistically significant difference in the tensile force at failure of suture loops

Figure 2. Effect of 8 loop configurations on the tensile force Newton (N) at failure of 4 suture materials. All loop configurations are compared to linear suture for each type of suture material. Significant values are indicated by symbol:



associated with the addition of a supplemental throw for each type of the 4 suture materials.

Elongation at failureInfluence of knot versus linear suture on elongation at failure —

As shown in Figure 4, for polydioxanone, the presence of a sliding half-hitch significantly decreased the elongation of the loops at failure, which means the loop breaks earlier. For polyglecaprone 25 and nylon, there was no significant difference in loop elongation at failure. For coated polyglactin 910, significant loop elongation was observed with the presence of a square knot with 3 throws and with the granny and the sliding half-hitch knots.

Influence of types of knots on elongation at failure — As shown in Figure 5, for polydioxanone, the presence of a granny and a slid-ing half-hitch knot significantly decreased elongation at failure of loops compared to square or surgeon’s knots. For polyglecaprone 25, the presence of a sliding half-hitch significantly decreased elongation at failure compared to a square knot. For coated polyglactin 910 and nylon, the presence of the knot did not significantly influence the elongation at failure of the loop.

Addition of a supplemental throw — There was no statistically significant difference in the loop elongation associated with the addition of a supplemental throw for each type of suture material.

Mode of failurePolydioxanone — Of the 96 samples, 2 were discarded due to

erroneous data acquisition. Of the 94 samples, 89 (94, 6%) broke at the knot and 5 did not break (reached displacement limit).

Polyglecaprone 25 — Of the 96 samples, 1 was discarded due to erroneous data acquisition. Of the 95 samples 92 (96, 8%) broke at the knot, 2 broke outside the knot, none slipped, and 1 did not break (reached displacement limit).

Coated polyglactin 910 — Of the 96 samples, 48 (50%) broke at the knot, 45 (46, 8%) slipped, 1 broke outside the knot, and 2 did not break (reached displacement limit). Our a posteriori analyses showed that the prevalence of slippage is significantly increased when using coated polyglactin 910 compared to the other types of suture mate-rial. Furthermore, the type of knot (granny and sliding half-hitch) significantly increased slippage, but the number of throws did not.

Nylon — Of 96 samples, 89 (92, 7%) broke at the knot, 1 broke outside the knot, none slipped, and 6 did not break (reached dis-placement limit).

D i s c u s s i o nSuture failure can lead to both minor complications (delayed

healing, inappropriate scarring, or superficial infection) and major complications (wound separation, wound dehiscence, or deep infec-tion) that subsequently lead to catastrophic events, such as intestinal leakage, evisceration, and peritonitis (1–4,7). It is therefore important to objectively select the type of suture material used based on its intrinsic properties (strength, elongation, and failure mode) and the safety of the knot used. Moreover, depending on the type of suture material used, each type of knot will influence the tensile force and elongation at failure.

Our first hypothesis was that all types of knots would significantly influence the force and elongation at failure of each of the linear sutures. Our hypothesis with regard to the tensile force (Figure 2) was partially correct for polydioxanone, correct for polyglecaprone 25 and coated polyglactin 910, and incorrect for nylon, with the exception of the surgeon’s knot with 5 throws, which could be the result of a type-I error. Our hypothesis with regard to elongation (Figure 4) was partially correct for polydioxanone and coated poly-glactin 910 and incorrect for polyglecaprone 25 and nylon.

Figure 3. Effect of 4 loop configurations on the tensile force (N) at failure of 4 suture materials. Each knot configuration is compared to the other 3 configurations for each type of suture material. The number of throws is 4 for polydioxanone, polyglecaprone 25, and nylon and 3 for coated polyglactin 910.



Interestingly, in disagreement with several textbooks (13–15), we found that not all suture materials and knots affect the initial force at failure of linear suture materials and not to the same extent. For polydioxanone, polyglecaprone 25, coated polyglactin 910, and nylon, we documented a variation of the force at failure of the linear suture when using a knotted loop with a square knot of, respectively 5.6%, 214.6%, 248.3%, and 20.9%. When using a surgeon’s knot, the variations were: 21.4%, 222.4%, 238.2%, and 27.1%, respectively.

It has been reported that the knot is the weakest point in a suture line or a ligature, as tying a knot results in friction between 2 strands, which weakens the suture (13,15). Naleway et al (16) have shown that a single knot decreased the force at failure of nylon by 62%. It has also been previously reported that shear stresses at the point between the loop and the first throw of the loop weaken the

suture material (4). In our study, we observed this behavior for polydioxanone when using a sliding half-hitch knot; loop elongation decreased significantly at failure due to premature breakage of the loop compared to the linear suture. Furthermore, when comparing the knotted loops, the use of a granny knot or sliding half-hitch knot significantly decreased loop elongation at failure compared to the square or surgeon’s knots. This finding is also associated with premature breakage of the loops.

Our second hypothesis was that all types of knots would signifi-cantly influence the force and elongation at which the knotted loop breaks, but not their mode of failure. Our hypothesis with regard to tensile force (Figure 3) was correct for polydioxanone, partially cor-rect for polyglecaprone 25 and coated polyglactin 910, and incorrect for nylon. Our hypothesis with regard to elongation (Figure 5) was

Figure 4. Effect of 8 loop configurations on the elongation (mm) at failure of 4 suture materials. All knot configurations are compared to linear suture for each type of suture material. Significant values are indicated by symbol:



correct for polydioxanone, partially correct for polyglecaprone 25, and incorrect for coated polyglactin 910 and nylon. Furthermore, our a posteriori statistical analyses have shown that, for all knots, the use of coated polyglactin 910 significantly increased slippage versus breakage compared to polyglecaprone 25, polydioxanone, and nylon.

Regarding elongation in our study, each loop measured 2 cm in diameter, which corresponds to 62.8 mm. As the maximal elonga-tion observed in our study was 12 mm, this correlates to 19% of elongation. In the healing process, this value is within physiologi-cal tolerance to deformation (100% for granulation tissue) (14). We propose that elongation of suture loops as evaluated in our study has no clinical relevance.

The square knot and the surgeon’s knot are the most common knots used in surgical procedures (8). It is not uncommon for the novice surgeon to create granny knots than square knots (8,17,18). The impact of this technical error has been outlined in the present study (premature failure, influence on elongation at failure). A square knot can easily become a sliding half-hitch when tied by a novice or even an experienced surgeon. It has been reported that 80% of square knots can slip into a sliding half-hitch depending on the suture material, the configuration of the knot, and the surgeon’s tying skills (1,6,7,17,18).

Marturello et al (1) showed that the surgeon’s experience and training were significant factors affecting knot security. We empha-sized that a modification in the knot due to suboptimal technique could significantly affect the tensile force of a suture. Furthermore, it is important to recognize the consequences of tying a granny or sliding half-hitch knot by mistake, as these knots significantly decreased the tensile force and elongation at failure of the knotted loops because of the premature failure. We also noted that adding a supplemental throw did not significantly change our results. Marturello et al (1) also reported that 3 throws were as secure as 6 throws and, although the tensile failure load was greater with 6 throws, there was no significant difference in knot security with more than 4 throws for each suture material tested. It has also been reported by Van Rijssel et al (19) that the size of the suture material is more important than the number of throws for knot security.

When selecting polyglactin 910 for our study, we considered it as a true multifilament because there was no mention on the packaging (box and suture envelopes) that it was coated. After analyzing our data, it was difficult to explain the results obtained. When looking closer at the monograph inside the box, we realized that polyglactin 910 was coated, even though this was not specified on the packaging. This may explain why this suture did not exhibit the behavior of a multifilament but behaved instead as a coated suture. Interestingly, as shown in Figure 6, the square knot with 4 throws and the sur-geon’s knot exhibited the most constant behavior.

We have found that the mechanical properties of knotted loops using polyglactin 910 are similar to those reported in the literature (3,7,17,20,21). Polyglactin 910 seems to exhibit more slippage and its safety is uncertain, as the use of additional throws does not seem to be correlated with the safety of the knotted loop. In a study by Ching et al (8), when tensile strengths were evaluated for knots that failed by breakage and those that failed by slippage, the number of throws did not significantly affect the tensile strength of the polyglactin 910 (size 2-0) (8). Behm et al (20) reported that the failure rates for sizes 0 and 2-0 of polyglactin 910 were still fairly high (40%) even with 5 throws. As reported by Rodeheaver et al (21), when using polyglactin 910, knots were made secure by using 1 more throw than with coated polyglycolic acid sutures. Our findings are also consis-tent with the observations of Rosin et al (5) who evaluated the knot security of size 2-0 knotted sutures and observed that a surgeon’s knot was more secure than a square knot when using polyglactin 910. Finally, it has also been determined that, for size 0 polyglactin 910, knot security continued to improve until 5 throws were used (17).

Taking into account all the data evaluating the mechanical prop-erties of 4 types of size 3-0 suture materials from this study, we can conclude that for polydioxanone, the use of a square or a surgeon’s knot did not decrease the tensile force at failure of the linear suture and the use of a granny or sliding half-hitch knot decreased the tensile force at failure and significantly reduced the elongation at failure of the knotted loop because of the premature failure. These 2 knots should therefore be avoided. For polyglecaprone 25, all the

Figure 5. Effect of 4 loop configurations on the elongation (mm) at failure of 4 suture materials. All knot configurations are com-pared to each other for each type of suture material. The number of throws is 4 for polydioxanone, polyglecaprone 25, and nylon and 3 for coated polyglactin 910.

More elongation

Less elongation

Square

Surgeon

Granny

Sliding half-hitch



knots decreased the tensile force at failure of the linear suture and the sliding half-hitch knot was significantly less resistant than the other knots and should be avoided. For coated polyglactin 910, all knots decreased the tensile force at failure of the linear suture and this material was associated with a significantly high rate of slip-

page. Using a square knot with 3 throws with coated polyglactin 910 did not seem to result in a secure knot. For nylon, the tensile force and the elongation at failure were not influenced by the pres-ence of a knot. Overall, however, nylon seemed to be the weakest suture material; the initial tensile force was 73.9%, 54.9%, and 61.3%,

Figure 6. Load-displacement curves of coated polyglactin 910. Each line represents a tested suture loop: 12 loops were evaluated per knot configuration. Tensile force (N) versus displacement (mm).



respectively, of the force at failure of linear suture with polydioxa-none, polyglecaprone 25, and coated polyglactin 910.

This study had many limitations, including the difficulty of extrapolating the findings of an in-vitro experimental study to an in-vivo clinical situation. In addition, the knotted loops were not incubated in saline or plasma before mechanical testing, which might have mimicked a more physiological condition. Other suture materials, knot configurations, and numbers of throws could have been evaluated, thus evaluating fatigue, and cyclic loading was not tested. Further studies are warranted in order to evaluate the proper-ties of knotted loops in more physiological conditions.

The observations made in this study indicate that, when using dif-ferent types of size 3-0 suture material, the results from mechanical testing pertaining to tensile force, elongation, and mode of failure cannot be extrapolated among various types of sutures and knots.

A c k n o w l e d g m e n t sThe authors thank Le Fonds en santé des animaux de compagnie

(FSAC) and Mike and Valeria Rosenbloom for financial support.

Re f e r e n c e s1. Marturello DM, McFadden MS, Bennett RA, Ragetly GR, Horn G.

Knot security and tensile strength of suture materials. Vet Surg 2014;43:73–79.

2. Good MM, Good LB, Mclntire DD, Brown SA, Wai CY. Surgical knot integrity: Effect of suture type and caliber, and level of residency training. J Surg Educ 2013;70:156–160.

3. Muffly TM, Cook C, Distasio J, Bonham AJ, Blandon RE. Suture end length as a function of knot integrity. J Surg Educ 2009;66:276–280.

4. Muffly TM, Boyce J, Kieweg SL, Bonham AJ. Tensile strength of a surgeon’s or a square knot. J Surg Educ 2010;67:222–226.

5. Rosin E, Robinson G. Knot security of suture materials. Vet Surg 1989;18:269–273.

6. Zhao C, Hsu CC, Moriya T, et al. Beyond the square knot: A novel knotting technique for surgical use. J Bone Joint Surg Am 2013;95:1020–1027.

7. Trimbos JB. Security of various knots commonly used in surgical practice. Obstet Gynecol 1984;64:274–280.

8. Ching SS, Mok CW, Koh YX, Tan SM, Tan YK. Assessment of sur-gical trainees’ quality of knot-tying. J Surg Educ 2013;70:48–54.

9. Muffly T, McCormick TC, Dean J, Bonham A, Hill RF. An evalu-ation of knot integrity when tied robotically and conventionally. Am J Obstet Gynecol 2009;200:e18–20.

10. Davis DA, Pellowski DM, Rawdon EJ. All monofilament knots assume sliding conformation in vivo. Dermatol Surg 2013;39: 729–733.

11. Aanning HL, Hass T, Jorgensen DR, Wulf CA. Square not a run-ning knot. J Am Coll S Surg 2007;204:422–425.

12. Tensile Testing of Coir Fibres and Yarns. Instron (homepage on the Internet). Available from: http://www.instron.us/en-us/testing-solutions Last accessed January 3, 2016.

13. Fossum TW. Biomaterials, suturing and hemostasis. In: Fossum TW, ed. Small Animal Surgery. 3rd ed. St. Louis, Missouri: Mosby, 2007:57–78.

14. Hulse D, Hyman B. Fracture biology and biomechanics. In: Slatter D, ed. Textbook of Small Animal Surgery. 3rd ed. vol 2. Philadelphia, Pennsylvania: Saunders, 2003:1785–1792.

15. Tobias K, Johnston S. Instrument and tissue handling techniques. In: Veterinary Surgery: Small Animal. 1st ed. Vol 1. St. Louis, Missouri: Elsevier, 2012:211.

16. Naleway SE, Lear W, Kruzic JJ, Maughan C. Mechanical prop-erties of suture materials in general and cutaneous surgery. J Biomed Mater Res B Appl Biomater 2015;103:735–742.

17. Muffly TM, Kow N, Iqbal I, Barber MD. Minimum number of throws needed for knot security. J Surg Educ 2011;68:130–133.

18. Alzacko SM, Majid OW. “Security loop” tie: A new technique to overcome loosening of surgical knots. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;104:1–4.

19. Van Rijssel EJC, Trimbos JB, Booster MH. Mechanical perfor-mance of square knots and sliding knots in surgery: A compara-tive study. Am J Obstet Gynecol 1990;162:93–96.

20. Behm T, Unger JB, Ivy JJ, Mukherjee D. Flat square knots: Are 3 throws enough? Am J Obstet Gynecol 2007;197:172.e1–3.

21. Rodeheaver GT, Thacker JG, Owen J, Strauss M, Masteron T, Edlich RF. Knotting and handling characteristics of coated syn-thetic absorbable sutures. J Surg Res 1983;35:525–530.


Short Communication Communication brève


The Gram-negative bacterium, Anaplasma marginale, is the caus-ative agent of bovine anaplasmosis, a disease that affects ruminants worldwide (1). Anaplasma marginale is an obligate intra-erythrocytic bacterium that chronically infects wildlife, including: bison (Bison bison), some cervid species including elk (Cervus canadensis), white-tailed deer (Odocoileus virginianus), and mule deer (O. hemionus). Infections in wildlife may not cause any measurable level of mor-bidity or reach parasitemia levels that allow them to serve as reservoirs for the pathogen (1). Clinical signs can develop in cattle including anemia, splenomegaly, enlargement of the gallbladder, icterus, reduced weight gain, abortion, and in severe cases, death (2). Animals that survive acute infections of A. marginale may become chronically infected (1).

All species of Anaplasma typically use a tick as a biological vector, undergoing a migration and maturation in the tick before becom-ing infectious to the definitive vertebrate host (1). Ticks of the genus Dermacentor are the main biological vectors of A. marginale in North America (3). In Canada, the 2 key tick vectors are the Rocky Mountain wood tick, D. andersoni, which ranges from British Columbia to south-central Saskatchewan, and the American dog tick,

D. variabilis (3). The range of D. variabilis extends from Saskatchewan east to Nova Scotia and is found throughout the southern portion of Manitoba, often in high densities (4). Adult male D. variabilis and D. andersoni are known to take multiple blood meals and even feed on multiple hosts (5).

Anaplasma marginale may also be transmitted by other means. Vertical transmission can occur between an infected cow and naïve calf through trans-placental infection or through blood contact dur-ing the birthing process (6). Additionally, the infection can be spread mechanically by contaminated veterinary tools, an important mode of transmission in some regions (7). Under some circumstances, bit-ing flies, specifically deer flies and horse flies (Diptera: Tabanidae), may serve as mechanical vectors, transferring infected blood cells on their mouthparts between animals with varying degrees of suc-cess (8,9).

Anaplasma marginale is of economic importance in Canada, and has resulted in trade restrictions being imposed on Canadian cattle for export (1,10). Bovine anaplasmosis was a reportable disease in Canada until 2014 and, under the Health of Animals Act, required pro-ducers with infected herds to undertake a quarantine, test, and cull

Active surveillance of Anaplasma marginale in populations of arthropod vectors (Acari: Ixodidae; Diptera: Tabanidae) during and after an outbreak

of bovine anaplasmosis in southern Manitoba, CanadaMatthew E.M. Yunik, Terry D. Galloway, L. Robbin Lindsay

A b s t r a c tBovine anaplasmosis is the disease caused by the bacterium Anaplasma marginale. It can cause production loss and death in cattle and bison. This was a reportable disease in Canada until April 2014. Before then, infected herds were quarantined and culled, removing infected animals. In North America, A. marginale is biologically vectored by hard ticks (Acari: Ixodidae), Dermacentor variabilis and D. andersoni. Biting flies, particularly horse flies (Diptera: Tabanidae), can also act as mechanical vectors. An outbreak of bovine anaplasmosis, consisting of 14 herds, was detected in southern Manitoba in 2008. This outbreak lasted multiple rounds of testing and culling before eradication in 2011, suggesting local maintenance of the pathogen was occurring. We applied novel approaches to examine the vector ecology of this disease in this region. We did not detect A. marginale by screening of 2056 D. variabilis (2011 and 2012) and 520 horse flies (2011) using polymerase chain reaction (PCR).

R é s u m éL’anaplasmose bovine est une maladie causée par la bactérie Anaplasma marginale. Elle peut être responsable pour la perte de production et entrainer la mort du bétail et des bisons. La maladie devait être obligatoirement déclarée jusqu’en avril 2014. Avant cette date, les troupeaux infectés étaient mis en quarantaine et abattus pour éliminer les animaux contaminés. En Amérique du Nord, A. marginale est transmise par des vecteurs biologiques; les tiques dures (Acari: Ixodidae), Dermacentor variabilis et D. andersoni. Les mouches piquantes, en particulier les mouches à cheval (Diptera: Tabanidae), peuvent aussi agir comme vecteurs mécaniques. Une épidémie d’anaplasmose bovine touchant 14 troupeaux, a été détectée au Sud du Manitoba en 2008. Suite à une série de tests et d’abattages, cette épidémie fut éradiquée en 2011, suggérant qu’il se produisait localement un entretien du pathogène. Nous avons appliqué de nouvelles approches pour examiner l’écologie du vecteur de cette maladie dans cette région. Lors des tests de dépistage par réaction en chaîne pas polymérase (PCR), nous n’avons pas détecté A. marginale sur 2056 D. variabilis (2011 et 2012) et 520 mouches du cheval (2011).

(Traduit par Dre Florence Huby-Chilton)

Department of Entomology, University of Manitoba, Winnipeg, Manitoba R3T 2N2 (Yunik, Galloway); Public Health Agency of Canada, National Microbiology Laboratory, Winnipeg, Manitoba R3E 3R2 (Lindsay).

Address all correspondence to Matthew E.M. Yunik; telephone: 204-474-9257; fax: 204-474-7628; e-mail: [email protected]



program until there were no longer infected animals in the region (10,11). Vaccines are not currently available in North America and no antibiotics are currently licensed for use in Canada. The Canadian Food Inspection Agency (CFIA) conducted a Bovine Serological Survey (BSS) every 3 to 5 y to affirm that the national herd remained free of A. marginale as well as other pathogens. In 2007–2008, the BSS revealed that the prevalence of infection in the Canadian herd was higher than the acceptable 0.02% for A. marginale (11). A portion of the positive samples was traced to Manitoba. Enhanced surveillance efforts led to the detection of animals infected with A. marginale from herds belonging to 13 beef producers and 1 bison producer. A quarantine, test, and cull program was initiated in 2009 and con-cluded in the summer of 2011 (11). From a Canadian perspective, this outbreak was unique because infected animals were detected during multiple summers, requiring multiple rounds of culling to end local transmission. At the initiation of the program, producers who participated in the study had a cumulative beef cattle herd size of approximately 1278 animals (range of head per farm: 40 to 373), 15% of which were culled after the first round of testing by the CFIA. Prevalence of A. marginale infection ranged from 2.7% to 32.0% per herd. In the spring of 2011, 7 additional animals were culled from a herd of 115, while one additional animal was culled in the summer from a herd of approximately 30. These data are based on results from a questionnaire distributed by the CFIA to participating pro-ducers in 2011.

The exact role that arthropods, especially ticks, play in the epi-demiology of bovine anaplasmosis is understudied (1). Certain strains of A. marginale have varying ability to infect vertebrate and arthropod hosts (12–14). Additionally, the behavior, physiology, and life history of the tick vectors can vary throughout their geographic range (15). Only recently have we had the molecular tools to conduct large-scale, active surveillance of arthropod vectors in a pastoral set-ting, which has been suggested as a key element to understanding the ecology of bovine anaplasmosis. The objective of this study was to assess what role, if any, ticks and horse flies had in the potential maintenance A. marginale in the outbreak region in Manitoba.

In April 2011, we contacted CFIA personnel who were the pri-mary responding veterinarians responsible for the control of the anaplasmosis outbreak in southeastern Manitoba. Staff from the CFIA provided essential historical background on the presence of A. marginale in the region over previous years and initiated contact with producers who owned the pastures in which infected animals had been detected. Of those contacted, 9 producers participated in the study. Ticks were collected by drag sampling using a piece of white flannel (1.3 3 0.70 m), spread by a plastic spar, dragged by researchers at a leisurely pace through the pastures. Tick dragging was conducted on non-rainy days when the air temperature was above 10°C; after snow had melted in April until August when questing ticks were no longer present. The flannel, along with researchers’ clothes, was examined for ticks approximately every 10 m. Collected ticks were then placed in a 56-mL plastic vial with a perforated snap-top lid. Upon leaving the pasture, the date and location of the collection were recorded, and the vials were placed in a plastic bag with a moistened paper towel, and deposited in an insulated container for transport. The time allotted for drag sampling the over 18 km2 of pasture, distributed among the 9 par-

ticipating producers, was based on suitability of tick habitat, recent herd infection history, including number of animals culled per herd and pasture utilization, and producer interest. At least 100 ticks were collected from land owned by each producer involved in the study.

Once returned to the lab, ticks were identified based on mor-phological characteristics to be D. variabilis before being surface- sterilized in 4 different solutions, and grouped according to collec-tion site. The first solution consisted of one drop of Tween 80 per 10 mL of 0.5% bleach. The second solution was 0.5% benzalkonium chloride. The third solution was 70% ethanol. The final solution was filtered water. All ticks collected from the same pasture on the same day were placed in 50 mL centrifuge tubes and approximately 45 mL of the first solution was added. The tubes were then sealed and slowly and repeatedly inverted for 3 min. The solution was decanted and the next solution added. This process was repeated for all solu-tions. After decanting the filtered water, the ticks and centrifuge tube were dried with a paper towel; the ticks were replaced into the tube and frozen at 280°C for storage.

Horse flies were collected using a Manitoba Horse Fly Trap (16). In 2011, a trap was operated for 4 days from 9 a.m. to 8 p.m. in the first week of June when fly populations appeared to be at their highest. The trap was placed for the first 2 d on a pasture where fly intensity appeared to be the highest and where previously infected animals had been maintained in 2009–2010. On subsequent days, the trap was placed in close proximity to the herd that contained a cow which had an active A. marginale infection approximately one month prior. Once removed from the field, flies were killed by being placed in a freezer at 25°C for approximately 20 min before being transferred to a sterile plastic bag and placed in a freezer at 280°C.

Ticks were removed from storage at 280°C and sorted by loca-tion and date collected, as well as gender, and placed into pools of no more than 5. Each tick was then cut in half sagittally using a scalpel on a sterile Petri dish in a biosafety cabinet. The scalpel was disinfected between ticks by washing in 10% chlorine bleach solution followed by a rinse in 90% ethanol. Half of each tick was frozen indi-vidually in a 2 mL microtube (Sarstedt AG and CO, Newton, North Carolina, USA), while the other half remained in a designated pool. This ensured that individual positive ticks found in pools which tested positive for the presence of A. marginale could be identified. The ticks in each pool were then cut further into smaller pieces using a sterile scalpel to enhance DNA extraction.

Extraction of DNA from the pools was conducted using DNAeasy Blood and Tissue Kits (Qiagen, Austin, Texas, USA) following the blood and tissue protocol. All extracts were then stored in a freezer at 280°C. Extractions were screened for A. marginale DNA using real-time polymerase chain reaction (RT-PCR) with primers and a probe that had been successfully used in previous studies targeting the 16S rRNA gene (17). The RT-PCR was conducted using 96-well fast plates on a VIIATM 7 RT-PCR system (Applied Biosystems, Foster City, California, USA). The thermocycling regime consisted of an activation stage lasting for 2 min at 50°C, an initial denatur-ation lasting 10 min at 95°C, and 40 cycles of 95°C, and 60°C lasting for 15 s and 1 min, respectively. Each reaction contained 12.5 mL of TaqMan Universal Master Mix (2X) (Applied Biosystems), forward and reverse primers at a concentration of 0.667 mM each, a probe at a



concentration of 0.125 mM, and 5 mL of DNA extract. The remaining volume consisted of nuclease-free water. An isolate of A. marginale from an infected cow from the Manitoba outbreak in 2008 was used as a positive control. No template controls were also used.

Horse flies are potential mechanical vectors; therefore, only the mouthparts were tested for A. marginale as bacteria in the remaining part of the fly body should not be transmittable. Flies were sorted by the date collected and to the lowest taxonomic level possible. Mouthparts were removed from the flies and pooled in groups of no more than 5, keeping each trap date and taxonomic group separate. The pools of mouthparts were subjected to the same DNA extraction and PCR protocols that were used to screen the ticks for A. marginale.

In total, 1013 (487 males; 526 females forming 217 pools) and 1043 (493 males; 550 females forming 224 pools) D. variabilis were collected in 2011 and 2012, respectively. A total of 520 horse flies collected in 2011 were placed into 110 pools for testing. The total collection consisted of 2 genera: Tabanus (n = 116) and Hybomitra (n = 404). Tabanus similis was the most abundant species of the genus Tabanus; however, 3 T. vivax were also collected. There were 6 species of Hybomitra identified, H. illota (n = 61) and H. pechumani (n = 10). In order to minimize the amount of time between collection and testing for A. marginale, the remaining 4 Hybomitra species (n = 333) were not sorted to species level. However, H. nuda, H. lasiophthalma, H. frontalis, and H. epistates were all present in the samples. Because of the success of the CFIA’s quarantine, test, and cull program, the probability of collecting infectious horse flies in the summer of 2012 was seen as negligible; therefore, no flies were collected. The poten-tial for A. marginale to be harbored in overwintering adult ticks from one vector season to the next (18) or in potential wild reservoir hosts justified collecting ticks in 2012.

All ticks and flies tested were negative for A. marginale; positive controls always tested positive while no template controls always tested negative. As part of another study, a proportion of tick extracts were positive for the presence of a related tick-associated bacterium, Rickettsia montanensis, confirming that our DNA extractions were successful (19). The producers involved were informed of the results as they became available.

There are numerous factors that may have been responsible for our failure to detect A. marginale. The most likely factor was the success of the quarantine, test, and cull program that the CFIA enacted in 2009 and concluded in 2011, when all herds that had been infected, and neighboring herds, were deemed free of anaplasmosis. This success by the CFIA dramatically reduced the probability that A. marginale would be detected in the arthropod populations, par-ticularly the biting flies. Vector competency might have also been an issue. Although Dermacentor ticks collected in Canada have been experimentally shown to transmit A. marginale (13) some strains are not vectored by ticks (12,15). Finally, the lack of detection could also be caused by the absence of infected wild reservoirs that were previously in the area. Although the exact role wild reservoirs play in the maintenance of bovine anaplasmosis is not well-understood, O. virginianus and C. canadensis are present in southeastern Manitoba and are known to come in close proximity to livestock, providing a potential spatial link for pathogen transmission. These animals may also frequently cross the international border into Minnesota where anaplasmosis is classified as an endemic, non-regulated pathogen

(11). However, the herd of C. canadensis that routinely migrates between Minnesota and the outbreak region in Manitoba has been monitored for A. marginale in the past. On the basis of serological tests, the herd has been considered free of anaplasmosis since at least 2004 (20).

Although the results of this study were negative there are many aspects of this work that are novel and noteworthy. This was the first large-scale study to use field-collected ticks and flies from pastoral settings to examine their potential role in the transmission of A. marginale in North America. This was accomplished through the use of molecular techniques not previously applied to such specimens. The use of fly mouthparts as opposed to the whole fly, demonstrating the potential role of the fly as a mechanical vector, is also very important. In a preliminary study, whole horse fly heads were used for DNA extraction. A component of the fly’s large compound eyes inhibited conventional and real time PCR. This is also the first time the Manitoba Horse Fly Trap has been used for surveillance of A. marginale. This trap has been used to evaluate fly population composition and density, and to reduce fly pressure (16). Using this trap to study the epidemiology of a mechanically vectored disease agent spread by tabanids may provide data that are more reflective of what is occurring in nature rather than other methods, such as sweep netting near the cattle, because only unfed or partially fed host seeking flies are caught in the trap. Finally this work demonstrates the effectiveness of the CFIA’s former control measure for managing outbreaks of bovine anaplasmosis in areas where potential vectors are present.

A c k n o w l e d g m e n t sAntonia Dibernardo of the Public Health Agency of Canada at the

National Microbiology Laboratory, Winnipeg, provided training on laboratory techniques. Diana Dunlop assisted with fieldwork in 2012. Manitoba Conservation provided research permits for collecting ticks from crown land. Dr. Alvin Gajadhar of the CFIA, Saskatoon, Saskatchewan, provided the A. marginale isolate that served as the positive control. Staff from the CFIA in Winnipeg, Dr. Lynn Bates, Dr. Krista Howden, and those at the Steinbach district office, including Drs. Max Popp and Jolene Byers, offered insight and support. Dr. Wayne Tomlinson, Dr. Glen Dizer, and Peter Veldhuis, Manitoba Agriculture, Food and Rural Initiatives, provided advice and support. Drs. Kateryn Rochon and Kim Ominsky, University of Manitoba, offered their expertise. Dr. Neil Chilton, University of Saskatchewan provided feedback on the manuscript. This research was funded by Growing Forward, a program jointly funded by Manitoba Agriculture Food and Rural Initiatives, and Agriculture and Agri-Food Canada.

Additional and special thanks go to the producers who allowed access to their pastures and offered firsthand experience of the Anaplasma problem in southeastern Manitoba.

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Short Communication/ Communication brèveActive surveillance of Anaplasma marginale in populations of arthropod vectors (Acari: Ixodidae; Diptera: Tabanidae) during and after an outbreak of bovine anaplasmosis in southern Manitoba, CanadaMatthew E.M. Yunik, Terry D. Galloway, L. Robbin Lindsay . . . . . . . . . . . . . . . . . . . . . . . 171


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