ORIGINAL ARTICLE

David Grant Æ Peter A. Todd Æ Tom Pennycott

Monitoring wild greenfinch (Carduelis chloris)for Salmonella enterica typhimurium

Received: 7 April 2006 / Accepted: 4 September 2006 / Published online: 24 October 2006� The Ecological Society of Japan 2006

Abstract The identification and monitoring of emerginginfectious diseases in free living wild birds is a challengeto wildlife biologists. In this study, a non-invasivemethodology for identifying salmonellosis in wildgarden birds was developed. We focussed on greenfinch,Carduelis chloris, which were found to have a seasonalpattern in the occurrence of Salmonella TyphimuriumDT 56(v). Principal components analysis of biometricdata indicated that low fat and low weight could beuseful indicators of Salmonella positive greenfinch. Acombination of biometrics taken from live birds, faecalanalysis, and behavioural observations provide aneffective and efficient system for identifying the presenceof salmonellosis within greenfinch.

Keywords Greenfinch Æ Salmonellosis Æ Diseasemonitoring Æ Wild birds Æ Principal components analysis

Introduction

During the past half century, emerging infectious dis-eases (EIDs) of free-living wild animals have beenincreasingly investigated as causes of declining birdnumbers and species (e.g. MacDonald 1963; Cizek et al.1994). Only recently, however, have health and diseaseissues been recognised as playing a pivotal role in both

conservation and wildlife biology (Osofsky and Hirsch2000).

The methods for collecting samples for examinationfor the presence of Salmonella have developed over theyears. Early research (e.g. Wilson and McDonald 1967)focused on carcasses of wild birds found dead or mori-bund by members of the public, whereas later studiessampled faeces or faecally contaminated material fromareas where wild birds congregated (Pennycott et al.2002). Species-level research required the use of invasivetechniques such as cloacal swabs from bird carcasses or(apparently) healthy wild birds captured alive (Refsumet al. 2003) or from culled birds (Monaghan et al. 1985).

The present study uses non-invasive techniques onindividual, living, resident species, with a focus on thegreenfinch (Carduelis chloris). Records of greenfincheswith Salmonella date back to the 1960s (Wilson andMcDonald 1967) and they have been identified as apotential endemic source of S. typhimurium, particularlyat sites that provide food for birds all year (Pennycottet al. 2002, 2005). Our objectives were to (1) develop anon-invasive methodology for the screening for Salmo-nella from living wild birds, (2) identify any seasonalpatterns in occurrence of S. typhimurium among com-mon garden species, and (3) investigate relationshipsbetween biometric measurements taken from wildgreenfinch and the recovery of S. typhimurium.

Methodology

Site description and sampling strategy

Live birds were caught and dead birds collected at a160 m2 garden site (55�26.48¢N, 004�31.10¢W) in Ayr,near Glasgow, Scotland, UK, from January to Decem-ber 2004. Bird food, predominantly black sunflowerseeds, was provided in four free-standing feeders and onthe ground bi-weekly for the duration of the study.

Live passerines were caught using potter traps and agarden trap. The traps were set for 6–8 h and once

D. Grant (&)Social Sciences Teaching Group,The Scottish Agricultural College, Auchincruive,Ayr KA6 5HW, UKE-mail: david.grant@sac.ac.ukTel.: +44-1292-525026

P. A. ToddDepartment of Biological Sciences,National University of Singapore,Singapore 117543, Singapore

T. PennycottThe Scottish Agricultural College Veterinary Services,Auchincruive, Ayr KA6 5HW, UK

Ecol Res (2007) 22: 571–574DOI 10.1007/s11284-006-0056-2

a bird was observed in a trap it was removed andprocessed. Mist nets were also used, but less frequently(approximately once a month). Birds caught alive hadBritish Trust for Ornithology (BTO) rings placed upontheir right tarsus for future identification. Faecalsamples were collected from bags, or from greaseproofpaper placed on the floor of small holding cages(30·15·25 cm3) that the birds were kept in after beingcaught.

A pilot study to test the efficacy of collecting faecalsamples during the average processing time of ringingand collecting biometric data from a bird (approxiamt-ely 15 min from point of capture to release) revealedthat the success rate was low (34%, n=102). By keepingbirds for up to a maximum of 45 min, in either cage orbag, the success rate for acquiring samples increasedsignificantly to 93% (n=172, G=110.07, v20.05 (1)=3.84,P<0.001); therefore, this approach was adopted for therest of the study. To avoid cross-contamination withother samples, bags were sterilised after each use bywashing at a minimum of 80�C (McCulloch 1998).

After ringing, the birds were sexed and aged (Svensson1992; Jenni and Winkler 1994). Each bird was weighedon digital scales (±0.01 g) before being scored for pec-toral muscle using the system described by Bairlien(1995) and fat using the BTO Ringing Committee Bio-metrics Working Group (BWG) system described byGosler (1996). Calliper measurements (±1.0 mm) weretaken of the flattened maximum wing chord, total headand bill length and bill depth from the distal edge of thenostril. All birds were released unharmed.

Bacteriological methodology

The analysis of faecal samples followed Pennycott et al.(2002). Samples were placed within sterile containersand mixed with a sterile swab before being cultured onMacConkey’s agar (Difco) and in selenite F broth. Bothwere incubated at 37�C for 18–24 h. The MacConkey’sagar plate was examined for non-lactose fermentingcolonies, which were typed using the API 20E system toobtain a biochemical profile of the isolates. The seleniteF broth culture was subcultured onto deoxycholate cit-rate lactose sucrose agar and further incubated at 37�Cfor 18–24 h. Using salmonella polyvalent O and poly-valent H antisera, slide agglutination tests were carriedout on any non-lactose fermenting colonies on DCLSagar. Isolates that were found to be positive to either oneor both antisera were typed by the API 10S system.Using the API 20E system further biochemical profilesof negative isolates were obtained. Isolates identified bythe API 10S system as Salmonella species were submittedto the Scottish Salmonella Reference Laboratory(SSRL) where they were identified as Salmonella enter-ica. Additional microtitre serotyping of their somaticand flagellar antigens was used to confirm the strains asS. typhimurium. The strains were subsequently phagetyped (Anderson et al. 1977).

Statistical analysis

To determine whether physical measurements couldhelp identify salmonella-positive birds, biometric datawere used as input variables in a principal componentsanalysis (PCA) performed on Minitab v14. Associa-tions among age, sex and likelihood of infection wereexamined with G tests. Correlations between traits andPCA axes were also tested for.

Results

Salmonella occurrence

A total of 336 live birds were captured and six carcassesfound; altogether 13 species were represented. The spe-cies caught most often were greenfinch (40.5%) andchaffinch (Fringilla coelebs, 27.4%). Of the 336 capturedbirds and 6 recovered carcasses, 18 individuals from fivespecies tested positive for Salmonella (Fig. 1). S. ty-phimurium DT 56(v) occurred in all five species, whereasS. typhimurium DT 40 was found only in two housesparrows (Passer domesticus). S. typhimurium DT 56(v)in the greenfinch shows a peak within the year, possiblya seasonal pattern, with all cases occurring duringJanuary and February (Fig. 1). While, on the whole,infected birds accounted for only a small percentage ofthose caught or found dead, in January and February28.6% of greenfinches tested positive. The two cases ofS. typhimurium DT 40 occurred in September andNovember.

Greenfinch biometrics

Biometric data were collected from all captured green-finch. G tests indicated that there was no significantassociation between the age or sex of a greenfinch and itslikelihood of being Salmonella positive or negative

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

9

8

7

6

5

4

3

2

1

0

Month

Salm

onel

la p

osi

tive

Starling (Sturnus vulgaris)

10

House sparrow (Passer domesticus)

Greenfinch (Carduelis chloris)

Blackbird (Turdus merula)

Chaffinch (Fringilla coelebs)

Fig. 1 The distribution of Salmonella-positive species over a12-month period. The greenfinch number includes four carcasses,two found in January and two in February. A chaffinch carcass,found in February, is also included

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(Gadj=0.378451, v20.05 (1)=3.84, P=0.936 andGadj=0.883772, v2 0.05 (1)=3.84, P=0.813771, respec-tively). Negative birds had a mean weight of 27.42 gcompared to 24.02 g for positive birds (Table 1). Thisdifference did not appear to be explained by the size ofthe birds, as the mean wing length was similar for po-sitive birds and negative birds, whereas the minimum fortotal head and bill length for positive birds was largerthan for negative birds (Table 1). Both fat and pectoralmuscle scores for positive birds had lower means andreduced ranges compared to negative birds.

Initial attempts to use PCA to identify infected birdsusing the full year data were confounded by post-breeding birds exhibiting depleted fat and body weight,traits similar to those of birds with Salmonella. A rep-resentative subset of data, i.e. the first quarter of 2004,was used instead as it excluded post-breeding birds whileincluding the months when Salmonella-positive green-finches were found. The first three axes of the PCA ac-counted for 73.6% of the total variance (Table 2).Significant negative Spearman rank correlations werefound between weight and PC1 (rs=�0.392, P<0.005,n=43) and fat score and PC1 (rs=�0.396, P<0.005,n=43). This suggested that as fat score and weight de-creased, the likelihood of a Salmonella-positive resultincreased. No significant correlation was found for billdepth and PC1 (rs=�0.335, P=0.28, n=43). The plotshowed a separation of the infected individuals fromhealthy birds with a grouping towards the negative ofPC1 (Fig. 2).

Discussion

A primary objective of this current study was to developan effective methodology to sample live, wild birds

utilising non-invasive techniques. Other samplingmethods of screening for salmonellosis, such as cloacalswabs and culling of live birds, are invasive, expensive,and not viable for use on projects such as nationwideringing schemes. By keeping birds for an extended per-iod, we increased the effectiveness of faecal sample col-lection from 34% (for 15-min holding times) to over90% (for 45-min holding times). This method of faecalsample acquisition is both time and cost efficient, highlyeffective, and could be utilised for large-scale screeningof avian diseases.

Since all cases of Salmonella-positive greenfincheswere found during January and February, there appearsto be a season for salmonellosis within this species at thisfeeding site. Pennycott et al. (2005) suggest a stress-re-lated trigger could activate the Salmonella, such asenvironmental conditions, e.g. cold weather, orbehavioural changes such as pre-breeding cycle. It is alsopossible that, at certain times of the year, some birdshave more vigorous immune systems as an adaptation tothe increased risk of exposure to bacterial and otherinfections (Moreno et al. 2003). Compared to the otherspecies in this study, the greenfinch appears to havesome trait that makes them more susceptible to Salmo-nella—although it is unclear how this may be linked withthe apparent seasonality in infection rates.

The biometric analysis shows that low fat and lowweight are important indicators for the field identifica-tion of late-stage Salmonella-positive birds. The resultssuggest that a greenfinch with a lower than normal bodyweight caught at this site during the months of January–March is likely to be infected with S. typhimuriumDT56(v). The correlation between fat (rs=�0.396) andpectoral muscle (rs=�0.371) and a positive Salmonellaresult suggests both measurements may be important indistinguishing infected birds. This finding, however,could be an artefact caused by the simplicity of thesystems used for recording fat scores and pectoral

Table 1 Summary of mean (and SE) biometric data for positive(n=6, Fig. 2) and negative (n=37) greenfinches caught (i.e.excluding moribund) in the first quarter of 2004

Measurement Positive birds Negative birds

Wing length (mm) 85.61 (1.83) 85.91 (2.02)Head and bill length (mm) 30.98 (0.43) 31.71 (0.80)Bill depth (mm) 8.83 (0.45) 9.93 (0.99)Fat (score) 0.00 (0.00) 0.95 (0.98)Pectoral muscle (score) 0.00 (0.00) 0.86 (0.44)Weight (g) 24.02 (1.31) 27.42 (1.81)

Table 2 PCA scores and Eigenvalues for biometrics from sixgreenfinches

Measurement PC1 PC2 PC3

Maximum wing length 0.033 0.405 0.814Total head and bill length 0.165 0.630 �0.473Bill depth 0.529 0.295 0.214Fat score 0.485 �0.288 �0.051Pectoral muscle score 0.393 �0.505 0.172Weight 0.550 0.118 �0.190

210-1-2-3-4

3

2

1

0

-1

-2

-3

principle component 1

prin

cipl

e co

mpo

nent

2

Fig. 2 Principal component analysis of six biometrics in green-finches. Open circle Salmonella-positive birds; closed circlesSalmonella-negative birds

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muscle scores (Bairlien 1995; Gosler 1996). For instance,none of the greenfinches captured were identified ashaving a full rounded pectoral muscle, with the scoresonly ranging between 0 and 2 (out of a possible range of0–4). The lack of sensitivity of fat and muscle scoringhas been discussed by Gosler et al. (1998), but, for fatscores at least, the current visual system remains the onlypracticable field technique (Rogers 2003).

Behavioural and visual identificationof infected greenfinch in the field

One of the clinical signs of salmonellosis is developmentof lesions in the upper digestive tract that results in thebird being unable to swallow (Refsum et al. 2003). Thisleads to a decrease in weight as the bird uses its storedenergy reserves: first fat followed by muscle. As thepectoral muscle is depleted the keel becomes more pro-nounced and sharp. The emaciated state of a bird in theadvanced stages of salmonellosis leaves it weak andunable to respond to threats in the same way a healthybird would. During the present study, four Salmonella-positive carcasses were recovered from cats and onenewly ringed bird, which was subsequently found to beSalmonella positive, was observed being caught by asparrowhawk (Accipiter nisus). Predators are apparentlyalso able to identify these weakened birds and prey uponthem. Predators could thus play an important role inmaintaining healthy populations of birds, as long as theydo not become susceptible to the disease themselves.

Kirkwood (1998) provided anecdotal evidence sug-gesting that Salmonella-positive birds were oftenlethargic, puffed up, feeding in isolation, and easilyapproachable. During this study greenfinches were seenshowing similar behaviour; however, unlike Kirkwood(1998), we were able to confirm infection by catching,processing, and taking faecal samples from these birds.Such behavioural observations could form the first stageof monitoring the health of greenfinch populations.Once diseased birds are suspected, then the second stageof catching birds and taking faecal samples using themethodology described here can be employed.

Whilst the present study does not identify the causesof salmonellosis in wild birds, and in fact raises moreissues about the complexity of factors and events thatlead to peaks in disease occurrence, it does provide aneffective two-stage non-invasive methodology for mon-itoring the health of greenfinch populations. There aresubstantial opportunities for conservation biologists toconduct research in collaboration with epidemiologistsand government bodies. Whilst S. typhimurium does notrepresent the same zoonotic danger as some other aviandiseases, it does pose a considerable threat as an epizo-otic disease, particularly amongst certain songbirds.

Research into salmonellosis and passerines is in an earlystage; however, with continued momentum, futurestudies adopting a multidisciplinary approach shouldeventually reveal the complex relationship between Sal-monella, bird populations, and the environment.

Acknowledgments This research would not have been possiblewithout financial assistance from the SAC Trust Fund and CJWildbird Foods, for which we are very grateful.

References

Anderson SE, Ward LR, De Saxe MJ, De Sa JDH (1977) Bacte-riophage-typing designations of Salmonella typhimurium. J Hyg78:297–300

Bairlien F (1995) European–African songbird migration networkmanual of field methods. Vogelwarte Helgoland, Wilhelm-shaven

Cizek A, Literak I, Heljicek K, Treml F, Smola J (1994) Salmonellacontamination of the environment and its incidence in wildbirds. J Vet Med 41:320–327

Gosler AG (1996) Environmental and social determinants of winterfat storage in the Great Tit Parus major. J Anim Ecol 65:1–17

Gosler AG, Greenwood JJD, Baker JK, Davidson NC (1998) Thefield determination of body size and condition in passerines: areport to the British Ringing Committee Bird Study 45:92.103

Jenni L, Winkler R (1994) Moult and ageing of European passe-rines. Academic, London

Kirkwood JK (1998) Population density and infectious disease atbird tables. Vet Rec 142:468

MacDonald JW (1963) Mortality of wild birds. Bird Study 10:91–108

McCulloch J (1998) Infection control: principles for practice. NurStd 13(1):49–53

Monaghan P, Shedden CB, Ensor K, Fricker CR, Girdwood RWA(1985) Salmonella carriage by herring gulls in the Clyde area ofScotland in relation to their feeding ecology. J Appl Ecol22:669–680

Moreno J, Briones V, Merino S, Ballesteros C, Sanz JJ, Tomas G(2003) Beneficial effects of cloacal bacteria on growth andfledging size in nestling Pied Flycatchers (Ficedula hypoleuca) inSpain. Auk 120:784–790

Osofsky SA, Hirsch KJ (2000) Conservation medicine: a veterinaryperspective. Conserv Biol 14:336–337

Pennycott TW, Cinderey RN, Park A, Mather HA, Foster G(2002) Salmonella enterica subspecies enterica serotype ty-phimurium and Escherichia coli O86 in wild birds at two gardensites in south-west Scotland. Vet Rec 151:563–567

Pennycott TW, Cinderey RN, Park A, Mather HA, Foster G,Grant D (2005) Further monitoring for Salmonella species andEscherichia coli O86 at a bird table in south-west Scotland. VetRec 157:477–480

Refsum T, Vikoren T, Handeland K, Kapperud G, Holstad G(2003) Epidemiologic and pathologic aspects of Salmonella ty-phimurium infection in passerine birds in Norway. J Wildl Dis39:64–72

Rogers CM (2003) New and continuing issues with using visible fatclasses to estimate fat stores of birds. J Avian Biol 34(2):129–133

Svensson L (1992) Identification Guide to European Passerines,BTO

Wilson JE, McDonald JW (1967) Salmonella infection in wildbirds. Br Vet J 123:212–219

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