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Veterinary Parasitology 188 (2012) 382–385 Contents lists available at SciVerse ScienceDirect Veterinary Parasitology jou rn al h om epa ge: www.elsevier.com/locate/vetpar Short communication Epidemiological survey of Neospora caninum infection in dogs from Romania R. Gavrea, V. Mircean, A. Pastiu, V. Cozma University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Faculty of Veterinary Medicine, Department of Parasitology, Calea an˘ an˘ atur 3-5, 400372 Cluj-Napoca, Romania a r t i c l e i n f o Article history: Received 8 February 2012 Received in revised form 21 March 2012 Accepted 23 March 2012 Keywords: Dogs Neosporosis Romania Epidemiology IFAT a b s t r a c t The aim of this study was to evaluate the seroprevalence of Neospora caninum infection in dogs from Romania at a national level and to detect possible correlations between the value of seroprevalence and a series of factors like sex, age, breed category and area. The prevalence of anti-N. caninum antibodies was determined in sera samples from 1114 dogs located in different regions of Romania using the IFAT method. Antibodies to N. caninum were found in 364 dogs (32.7%). The titers of anti-N. caninum antibodies varied from 1:50 to 1:800. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Neospora caninum is a protozoan parasite that causes abortions in cattle worldwide. In dogs, infection with this parasite can cause a severe disease characterized by vari- ous clinical signs as ataxia, ascending paralysis and other general nervous symptoms (Dubey and Lindsay, 1996). The infection can also induce myocardial, pulmonary and der- matological symptoms (Lindsay et al., 1999). In cattle neosporosis can persist in raising farms for many generations (Pare et al., 1996) that can serve as a reservoirs for N. caninum infection in dogs (Wouda et al., 1999). Dogs (Canis familiaris) have an important role in the epi- demiology of N. caninum infection as they are, together with coyotes (Canis latrans) and wolves (Canis lupus) the definitive hosts that shed oocysts in the environment (Gondim et al., 2004; Dubey et al., 2011). Some epidemiological studies presented a positive cor- relation between presence of dogs and cattle abortions Corresponding author. Tel.: +40 264 596 384x165; fax: +40 264 593 792. E-mail address: [email protected] (V. Cozma). induced by N. caninum infection (Pare et al., 1998). Some other studies showed that dogs raised near farms that pre- sented or not episodes of abortion in their history, had a higher seroprevalence than those raised in urban areas (Ferroglio et al., 2004). N. caninum has a wide distribution and the presence of specific antibodies is mentioned both in healthy dogs and in cattle (Dubey et al., 2007). For their detection various types of serological tests have been used: ELISA (Ghalmi et al., 2007), DAT (Packham et al., 1998) and IFAT (Dubey et al., 1988) tests. A large number of studies showed that IFAT expressed the fewest cross-reactivity reactions with other coccid- ian or non-coccidian parasites like T. gondii or H. heydorni (Dubey et al., 2007) and because of that is considered the golden-standard test used for detection of anti-N. caninum antibodies (Capelli et al., 2006). 2. Materials and methods A number of 1114 sera samples were collected from dogs located in different regions of Romania. Most of the samples were collected from Transylvania (511), Oltenia (173) and Dobrogea (132) (Table 1). 0304-4017/$ see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2012.03.044

Epidemiological survey of Neospora caninum infection in dogs from Romania

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Page 1: Epidemiological survey of Neospora caninum infection in dogs from Romania

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Veterinary Parasitology 188 (2012) 382– 385

Contents lists available at SciVerse ScienceDirect

Veterinary Parasitology

jou rn al h om epa ge: www.elsev ier .com/ locate /vetpar

hort communication

pidemiological survey of Neospora caninum infection in dogs fromomania

. Gavrea, V. Mircean, A. Pastiu, V. Cozma ∗

niversity of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Faculty of Veterinary Medicine, Department of Parasitology, Calea Mananatur 3-5,00372 Cluj-Napoca, Romania

r t i c l e i n f o

rticle history:eceived 8 February 2012eceived in revised form 21 March 2012ccepted 23 March 2012

a b s t r a c t

The aim of this study was to evaluate the seroprevalence of Neospora caninum infectionin dogs from Romania at a national level and to detect possible correlations between thevalue of seroprevalence and a series of factors like sex, age, breed category and area. Theprevalence of anti-N. caninum antibodies was determined in sera samples from 1114 dogs

eywords:ogseosporosisomania

located in different regions of Romania using the IFAT method. Antibodies to N. caninumwere found in 364 dogs (32.7%). The titers of anti-N. caninum antibodies varied from 1:50to 1:800.

© 2012 Elsevier B.V. All rights reserved.

pidemiologyFAT

. Introduction

Neospora caninum is a protozoan parasite that causesbortions in cattle worldwide. In dogs, infection with thisarasite can cause a severe disease characterized by vari-us clinical signs as ataxia, ascending paralysis and othereneral nervous symptoms (Dubey and Lindsay, 1996). Thenfection can also induce myocardial, pulmonary and der-

atological symptoms (Lindsay et al., 1999).In cattle neosporosis can persist in raising farms for

any generations (Pare et al., 1996) that can serve as aeservoirs for N. caninum infection in dogs (Wouda et al.,999).

Dogs (Canis familiaris) have an important role in the epi-emiology of N. caninum infection as they are, togetherith coyotes (Canis latrans) and wolves (Canis lupus) theefinitive hosts that shed oocysts in the environment

Gondim et al., 2004; Dubey et al., 2011).

Some epidemiological studies presented a positive cor-elation between presence of dogs and cattle abortions

∗ Corresponding author. Tel.: +40 264 596 384x165;ax: +40 264 593 792.

E-mail address: [email protected] (V. Cozma).

304-4017/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.vetpar.2012.03.044

induced by N. caninum infection (Pare et al., 1998). Someother studies showed that dogs raised near farms that pre-sented or not episodes of abortion in their history, hada higher seroprevalence than those raised in urban areas(Ferroglio et al., 2004).

N. caninum has a wide distribution and the presence ofspecific antibodies is mentioned both in healthy dogs andin cattle (Dubey et al., 2007). For their detection varioustypes of serological tests have been used: ELISA (Ghalmiet al., 2007), DAT (Packham et al., 1998) and IFAT (Dubeyet al., 1988) tests.

A large number of studies showed that IFAT expressedthe fewest cross-reactivity reactions with other coccid-ian or non-coccidian parasites like T. gondii or H. heydorni(Dubey et al., 2007) and because of that is considered thegolden-standard test used for detection of anti-N. caninumantibodies (Capelli et al., 2006).

2. Materials and methods

A number of 1114 sera samples were collected fromdogs located in different regions of Romania. Most of thesamples were collected from Transylvania (511), Oltenia(173) and Dobrogea (132) (Table 1).

Page 2: Epidemiological survey of Neospora caninum infection in dogs from Romania

R. Gavrea et al. / Veterinary Parasit

Table 1Number of samples collected from each region.

Region Number of samples

Banat 32Cris ana 92Maramures 37Transilvania 511Oltenia 255Dobrogea 132

for the parasite are eviscerated in the field and dogs have

Bucovina 37Moldova 18

The majority of samples were collected from males(645) and 469 samples were collected from females. 932pure breed dogs and 182 cross-breed dogs were sampled.Depending on their category the dogs were classified in:dogs raised in kennels, pets, farm dogs and hunting dogs.According to age the dogs were divided in three categories:under one year old (257), between 1 and 5 years old (572)and over 5 years old (285 samples). 641 samples were col-lected from dogs originated from urban areas and 473 fromrural areas.

All these sera samples were analyzed by IFAT methodusing MegaScreen FLUONEOSPORA commercial kit (Mega-cor Diagnostik, Austria). Briefly all sera samples werediluted at 1:50 dilution and 10 �l from each one were dis-tributed in slides in different wells. Positive and negativecontrol were used undiluted and in volume of 20 �l. Afterdistribution in wells the slides were incubated for 30 minat 37 ◦C in humid chamber. Rinsing with PBS and adding ofone drop of FITC anti-dog conjugate followed. Next, incu-bation at 37 ◦C for 30 min, adding two drops of mountingmedium and reading the results at UV light at 400×. Forpositive samples obtained at the cut-off dilution of 1:50,serial dilutions were made until negativity was reached.

All the obtained results were statistically analyzed usingEpi Info Program, Version 3.5.3.

All activities have been performed according to Euro-pean Animal Welfare Legislation (Directive 2010/63/EU).

3. Results

The interpretation of results was made at a cut-off dilu-tion of 1:50. For assessing the intensity of N. caninuminfection for positive samples serial solutions were madeuntil they reached negativity. In some cases the positivityfor N. caninum infection persisted until a dilution of 1:800.

After reading the results, a number of 364 sampleswere positive indicating a seroprevalence for neosporosisof 32.7%. Doubtful samples were considered as being neg-ative. At a dilution of 1:100 the seroprevalence reached avalue of 14.5% (95% CI 12.6–16.8%), at 1:200 it dropped at8.9% (95 CI 7.3–10.8%), at 1:400 was 4.1% (95% CI 3.1–5.5%).At the dilution of 1:800 the positivity reached a valueof 1.9%. All these differences were statistically significant,p < 0.0001.

At 1:50 dilution out of 932 samples collected from

mixed breed dogs, 299 were positive, indicating a sero-prevalence of neosporosis in this category of 32.1%.Regarding pure breed dogs, a similar value of 35.7%

ology 188 (2012) 382– 385 383

(65/182) was obtained. The difference was not statisticallysignificant (p > 0.05).

Males were affected in 29.6% (95% CI 26.1–33.3%) ofcases (dilution 1:50), value that decreases until 1.7% (95%CI 0.9–3.1%) at 1:800 dilution. Females were more affectedthan males, reaching a value of infection of 36.9% (95% CI32.5–41.5%) that dropped to 2.1% (95% CI 1.1–4%) at thedilution of 1:800.

At the initial dilution of 1:50, the highest seropreva-lence of N. caninum infection was detected in hunting dogs(54.5%) and none of the dogs kept in kennels were positivefor N. caninum infection (Table 2).

Most of the infected dogs (36.0%) were included in thesecond age group (1–5 years old), compared to the oneswith age under 1 year old (23.0%) and the third group (olderthan 5 years old, 34.7%). These differences were statisticallysignificant (p < 0.001), and they remained significant evenat next dilutions (1:100, 1:200). These aspects that can beobserved in Table 3.

34.5% (95% CI 30.2-39%) of the dogs that originated fromrural areas and 31.4% (95% CI 27.8-35.1%) from urban areaspresented anti-N. caninum specific antibodies at a dilutionof 1:50.

4. Discussions

This study aimed for detection of anti-N. caninum spe-cific antibodies in dogs from different regions of Romania. Anumber of 1114 sera samples were collected from 26 coun-ties. After testing of these sera samples by IFAT method,32.7% of them were positive for neosporosis. This per-centage indicates a higher rate of infection than the onesobtained in other European countries like Sweden 0.5%(Bjorkman et al., 1994); Austria 3.6% (Wanha et al., 2005),Germany 4% (Klein and Muller, 2001) and Czech Republic5.9% (Vaclavek et al., 2002).

Pure breed animals were more affected (35.7%) thanmixed breed dogs (32.1%) aspect that can be explained bythe fact that the number of samples collected from pure-breed animals was much lower than that of mixed breedanimals. The similar aspect was noticed by Vaclavek et al.(2007), Wanha et al. (2005), Fernandes et al. (2004) andGhalmi et al. (2009).

36.9% of females were positive for neosporosis, a higherpercentage than the one obtained in males (29.6). Thesame differences between males and females was obtainedby Wouda et al. (1999), unlike Ghalmi et al. (2009) andVaclavek et al. (2007) who have not noticed any significantdifferences between infection in both sexes.

The highest percentage of infection was obtained inhunting dogs (54.5%) compared to pets (34.8%). This dif-ference can be explained by dog feeding habits: the onesthat have outdoor access have more chances to come incontact with bovine infected tissues or with other interme-diate hosts of N. caninum. Hunting dogs can also get infectedwith N. caninum especially given the fact that most oftenhunted animals (like deers) that can be intermediate hosts

access to them.Increased seroprevalence of anti-N. caninum antibod-

ies in dogs from farms (50%) (Table 2) can be caused by

Page 3: Epidemiological survey of Neospora caninum infection in dogs from Romania

384 R. Gavrea et al. / Veterinary Parasitology 188 (2012) 382– 385

Table 2Percentage of positive animals from each service.

Category of animals 1:50 Serologicaltiters % a

1:100 Serologicaltiters % a

1:200 Serologicaltiters % a

1:400 Serologicaltiters % a

1:800 Serologicaltiters % a

Kennel 0. (0/4) 0. (0/4) 0. (0/4) 0. (0/4) 0. (0/4)Pet 34.8 (174/500) 16.2 (81/500) 8.8 (44/500) 4.2 (21/500) 1.8 (9/500)Shelter 31.2 (119/381) 10.8 (41/381) 7.6 (29/381) 2.6 (10/381) 1.0 (4/381)Guard 29.6 (64/216) 17.1 (37/216) 11.6 (25/216) 6.9 (15/216) 3.7 (8/216)Farms 50. (1/2) 0. (0/2) 0. (0/2) 0. (0/2) 0. (0/2)Hunting 54.5 (6/11) 27.3 (3/11) 9.1 (1/11) 0. (0/11) 0. (0/11)p 0.22 0.10 0.65 0.20 0.34

a Positive samples/total of collected samples from each category.

Table 3The results obtained at successive dilutions 1:100–1:800.

Age category 1:100 Serologicaltiters (95% CI)

1:200 Serologicaltiters (95% CI)

1:400 Serologicaltiters (95% CI)

1:800 Serologicaltiters (95% CI)

Less than 1 year old 7.4 (4.5–11.3) 4.7 (2.4–8) 2.3 (0.9–5) 1.6 (0.4–3.9)1–5 years old 15.9 (13.1–19.2) 9.6 (7.4–12.4) 3.8 (2.5–5.9) 1.7 (0.9–3.3)

(7.8–1518

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Over 5 years old 18.2 (13.9–23.2) 11.2p 0.0007 0.0

higher chance of them being fed with bovine fetusesr placentas infected with tachyzoites or parasitic cysts.nother possible explanation for this high seroprevalence

s the close contact between them and other wild animalsfoxes, wolves) or other domestic animals (horses, sheep oroats).

Dogs from rural areas (34.5%) were more affected thanogs from urban areas (31.4%), but there was no signifi-ant statistical difference (p > 0.05). A possible explanationf this phenomenon is the fact that animals from urbanreas come in contact with infected tissues or other pos-ible intermediate hosts much less than those from ruralreas. The latter may also present a higher percentage ofnfection due to their feeding habits: their owners do notook the meat offered as food.

The highest seroprevalence was obtained for dogs withge between 1 and 5 years old (36.0%) followed by dogsver 5 years old (34.7%). In similar studies (Barber andrees, 1996; Wouda et al., 1999; De Souza et al., 2002;anón-Franco et al., 2003), authors mentioned a signifi-antly higher seroprevalence in older dogs.

. Conclusions

Even if other studies have been performed in Roma-ia using IFAT in order to detect the seroprevalence ofeosporosis in dogs (S uteu et al., 2005, 2008) they wereerformed on a small number of animals (56 dogs in 2005nd 57 in 2008) and were limited to a small region of oneounty. This is the first large scale study on this diseasen involved a large number of animals (1114) that origi-ated from 26 counties of the country. In first studies theeroprevalence was 12.5% in 2005 (S uteu et al.) and 12.3 in008 (S uteu et al.), but in our study the value increased to

2.7%. This new obtained value shows us that in Romaniaeosporosis is present in a larger number of animals thane expected and prevention and control measures must be

mplemented.

.5) 6.3 (3.8–9.8) 2.5 (1–5)0.059 0.70

Acknowledgment

This study was supported by the Ministry of Educa-tion, Research and Innovation and National Center forProgramme Management from Romania, Grant PNII PC 52-177/2008.

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