Article Detection of Leptospira spp. in wildlife reservoir hosts in Ontario through comparison of immunohistochemical and polymerase chain reaction genotyping methods Karen E. Shearer, Michael J. Harte, Davor Ojkic, Josepha DeLay, Douglas Campbell Abstract — A total of 460 kidney samples from wildlife (beavers, coyotes, deer, foxes, opossums, otters, raccoons, skunks) were obtained from road-kill and hunter/trapper donations in Ontario between January 2010 and November 2012. The objectives of the study were to detect Leptospira spp. by immunohistochemistry and polymerase chain reaction (PCR), to map presence of leptospires in wildlife relative to livestock and human populations, and to characterize positive samples by sequencing and comparison to leptospires known to affect domestic animals and humans. The proportion of samples that tested positive ranged from 0% to 42%, with the highest rates in skunks and raccoons. Leptospira spp. were present in kidneys of wildlife across Ontario, particularly in areas of high human density, and areas in which livestock populations are abundant. The PCR was too weak in most samples to permit genotyping and examination of the relationship between the leptospires found in this study and those affecting domestic animals and humans. Résumé — Détection de Leptospira spp. chez des hôtes du réservoir faunique en Ontario par la comparaison des méthodes de génotypage de réaction immunohistochimique et d’amplification en chaîne par la polymérase. Un total de 460 échantillons de reins provenant de la faune (castors, coyotes, cerfs de Virginie, renards, opossums, loutres, ratons-laveurs, moufettes) ont été obtenus d’animaux tués sur la route et de dons de chasseurs et de trappeurs en Ontario entre janvier 2010 et novembre 2012. Les objectifs de l’étude étaient de détecter Leptospira ssp. par immunohistochimie et amplification en chaîne par la polymérase afin de cartographier la présence des leptospires dans la faune en rapport avec les populations de bétail et d’humains et de caractériser les échantillons positifs par le séquençage et la comparaison avec des leptospires reconnus comme affectant les animaux domestiques et les humains. La proportion des échantillons qui ont montré un résultat positif s’échelonnait de 0 à 42 %, et les taux les plus élevés se retrouvaient chez les moufettes et les ratons-laveurs. Leptospira spp. était présent dans les reins de la faune partout en Ontario, particulièrement dans les régions à forte densité humaine et dans les régions où les populations de bétail sont abondantes. L’amplification en chaîne par la polymérase était trop faible dans la plupart des échantillons pour permettre le génotypage et l’examen de la relation entre les leptospires trouvés dans cette étude et ceux touchant les animaux domestiques et les humains. (Traduit par Isabelle Vallières) Can Vet J 2014;55:240–248

Fleming College School of Environmental & Natural and Resource Sciences, Fish & Wildlife Program, 200 Albert St. South, Lindsay, Ontario K9V 5E6 (Shearer, Harte); Animal Health Laboratory (Ojkic, DeLay), Canadian Cooperative Wildlife Health Centre, Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1 (Campbell). Address all correspondence to Dr. Karen Shearer; e-mail: [email protected] Reprints will not be available from the authors. Sources of support: UG Agreement through the Animal Health Strategic Investment fund (AHSI) managed by the Animal Health Laboratory of the University of Guelph. This project was supported by the Ontario Ministry of Agriculture, Food, and Rural Affairs (OMAFRA). Additional funding provided by Wyeth Animal Health. Disclaimer: Karen Shearer is now employed by Zoetis. Use of this article is limited to a single copy for personal study. Anyone interested in obtaining reprints should contact the CVMA office ([email protected]) for additional copies or permission to use this material elsewhere. 240

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L

Introduction

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Materials and methods Samples An effort was made to obtain samples from a broad geographical area of Ontario and to include species in which leptospiral infection had previously been documented. Samples were obtained based on availability through established networks of hunters, trappers, government agencies, and road-kills. Information regarding the location of each sample was recorded as either lot and concession or geographic co-ordinates, depending on the source of the information. Carcasses were either sampled on-site, or brought to Fleming College, Lindsay, Ontario, and frozen at 220°C, for processing at a later date. Animals from which samples were collected were categorized as adult or immature based on the size of the animal, and male or female whenever possible. Gloves were changed between processing each sample to avoid cross contamination. Instruments used were either single use, or disinfected by soaking in 10% hypochlorite solution for a minimum of 5 min. The right kidney was sectioned such that a mid-sagittal section of 0.5 cm was halved. Of the resulting 2 kidney samples, one was preserved in 10% buffered formalin, and the other was frozen at 220°C in a plastic bag. Frozen samples were maintained at 220°C until sent to the Animal Health Laboratory (AHL), Guelph, Ontario for IHC and PCR.

Immunohistochemistry Formalin-fixed kidney sections from each animal were processed for light microscopy and embedded in paraffin, and 4-mm sections were mounted on charged slides. Immunostaining was carried out using an automated stainer (Dako Autostainer; Dako, Mississauga, Ontario). Following deparaffinization and rehydration, sections were treated sequentially with 3% hydrogen peroxide and Proteinase K (Dako; 12 min). Tissue sections were incubated with rabbit polyclonal anti-Leptospira spp. antiserum (National Veterinary Services Laboratory, Ames, Iowa, USA) for 30 min at room temperature. A labeled streptavidinbiotin detection system (LSAB2, Dako) was used with NovaRed (Vector Laboratories, Burlington, Ontario) as chromogen, and slides were counterstained with Harris hematoxylin. Sections of known Leptospira spp.-positive skunk kidney were used as a positive tissue (run) control. For negative reagent controls, duplicate sections of each control and test tissue were subjected to the same immunohistochemical procedure with substitution of non-immune rabbit serum at similar protein concentration for the primary antiserum. All IHC slides were interpreted by a veterinary pathologist and were scored as positive or negative for presence of leptospiral antigen.

Polymerase chain reaction (PCR) Total nucleic acids were extracted from homogenized samples using the MagMAXTM-96 Viral RNA Isolation Kit (Life 241

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eptospirosis affects many species of livestock, wild animals, and humans (1–6). Clinical signs of leptospirosis in both humans and domestic animals are not specific and can be confused with other diseases; therefore, the presence of the disease is not always recognized (7,8). Economic losses due to leptospirosis in livestock include the effects of acute and chronic disease and associated reduced reproductive parameters, abortion, mastitis, uveitis in horses, and death in any species (1,2,6). Human disease has mainly been diagnosed in those working with livestock, but has been known to affect other risk groups including outdoor enthusiasts (7–13). Feral and wild animals are reservoir hosts for leptospires and often do not exhibit clinical signs of disease (14–16). It is thought that these reservoirs act as a source of infection for humans and domestic animals, which then may become a source of infection for other animals and humans (3,7,14). Lending support to this, seroconversion in wildlife has been documented in animals living near livestock that have been affected by the disease (17,18). However, it is uncertain what role wildlife play in maintaining the disease outside target populations of domestic animals or humans. The distribution of leptospires in wildlife in Ontario relative to domestic animals and humans is not well understood and further information is needed about the relative importance of leptospires affecting various wild animals (16,19,20). In reservoir hosts leptospires reside in the kidney and are shed in the urine (3). Detection of infection in wildlife has often focused on serology (16,21), but interpretation of serological testing can be difficult due to low responses to infection in adapted hosts, cross-reactivity between serovars, and the need for samples to be collected in series to confirm acute infection (19,22). Also, antibodies may persist while the organism is no longer present in the kidneys (16,23). Culture, the historical gold standard of testing, can take weeks or months to provide results and may result in false negatives (22,24). Immunohistochemistry (IHC) can be used to detect Leptospira spp. antigen in tissue but cannot provide information regarding either the serovar of leptospires present or the relevance of infection of the wildlife host to the humans or domestic animals in the vicinity (22,25). Polymerase chain reaction (PCR) techniques hold the best promise for timely results, and could be used on samples from live animals (26,27). On its own PCR does not give information about the serovar, but recent studies have related genetic identification of leptospires to serovar group (28–32). Therefore, PCR coupled with genotyping could confirm a current infection and can also be related to serovar, which may be helpful in determining the role of wildlife in the maintenance of this disease in humans and domestic animals. Previous studies of leptospirosis in Ontario wildlife have focused on small geographical areas, and often on particular wildlife species (16–19). There may be additional species of wildlife harboring leptospires in Ontario that have not been identified, as well as geographic areas that have not been surveyed, including areas where humans and domestic animals reside. The purpose of this study was to provide a prevalence

estimate for the presence of leptospires in wildlife, to map Leptospira spp. infection of wildlife relative to livestock and human populations, and to genotype leptospires identified for comparison to those of known pathogenicity in humans and domestic animals.

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Figure 1.  Leptospirosis positive wildlife in relation to cattle population per census consolidated subdivision.

Technologies) on a MagMAXTM Express-96 Magnetic Particle Processor (Life Technologies) and subjected to PCR (28). Nucleotide sequences of PCR positive samples were determined and compared to reference sequences (29).

human density and livestock data used are representative of the 2011 Census of Agriculture and Canadian Census of Population and Dwellings.

Mapping

Results for IHC by species, age, and gender are listed in Table 1. A total of 460 kidney samples were collected from 2010 and 2012, of which 104 (23%) were positive for Leptospira spp. antigen; 2 additional samples had suspicious staining. The highest prevalences of Leptospira spp. infection were in adult skunks (42%) and raccoons (33%), followed by beavers (9%), foxes (8%), and opossums (6%). There was no association between IHC-positive samples and age or gender, in the animals where age and gender were determined. Sampling locations are depicted relative to human and livestock populations (Figures 1–4), and in terms of number of positive samples per location (Figure 5). Samples were taken from areas that included relatively high densities of humans, cattle, swine, and horses. The majority of positive samples appear to be in areas with the greatest human population density (Figure 4), secondly in areas of high cattle density (Figure 1), and thirdly in areas of high swine density (Figure 2). Samples were not obtained from some of the areas of relatively high horse density.

ArcMap10 (Esri, Redlands, California) was used to project sample location data of IHC positive and suspicious samples as well as negative samples. All sample-specific data were linked to the projected points to allow for queries by attributes. Shape files including Province/territory boundary files, and river boundary files (polygons), Consolidated Census Subdivision (CCS) boundary files, and human population center layers were obtained from Statistics Canada (33). A population center is defined as having a minimum population of 1000 persons and a population density of at least 400 persons/km2, based on the 2011 census population count. Human population files contain the boundaries of all population centers defined for the 2011 census. Agricultural data were obtained in tabular form, and were linked to the consolidated census subdivision boundary layers using a common attribute (CCS boundaries). Total numbers of livestock per census area were divided by number of animals per census area, to give comparison of densities. All 242

Results

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Figure 2.  Leptospirosis positive wildlife in relation to pig population per census consolidated subdivision.

Table 2 shows a comparison of IHC and PCR results. Of 437 samples, 341 of the positive PCR samples were in agreement with IHC, and 17 of the negative PCR samples were in agreement with IHC. The remaining 23 samples of 460 tested had discordant results. Samples were screened by real-time PCR (rRT-PCR); however, genotyping could not be performed on rRT-PCR product, but was attempted on 3 samples that were amplified by conventional PCR. Two fox samples showed the closest match to Leptospira interrogans serovar Pomona type kennewicki. The third sample was from skunk, but genotyping data were inconclusive. All other rRT-PCR-positive samples could not be further characterized.

Discussion Skunks have been described as possible maintenance hosts for leptospires, and as such they present a risk for transmission to other species (18,19). Infected animals are host reservoirs if they permanently maintain the pathogen and if transmission of the infection has been documented from the reservoir host to the target population. In a previous study in Ontario, out of 9 skunks that were examined 3 were culture-positive for L. pomona. The current study sampled a larger number of CVJ / VOL 55 / MARCH 2014

skunks, and found a higher percentage of skunks containing leptospires. Since the type of Leptospira spp. was not determined, it cannot be determined if these skunks would have been capable of transmitting to humans or domestic livestock. The majority of wildlife samples in this study were obtained from raccoons, which comprised 53% of animals tested, and also were the species most commonly sampled from areas of high human density. The percentage of positive raccoon samples is consistent with another study in urban Ontario, which found 31% of raccoons to be seropositive for Leptospira serovars Grippotyphosa and Pomona (16). The role of raccoons in epidemiology of infection has been debated (15,16,18,21). Jardine et al (16) argued that there was no evidence that raccoons were persistently infected, suggesting that they may not be maintaining the pathogen in the environment. McGowan et al (18) suggested that raccoons may be an amplifier host, rather than a long-term maintenance host because, in spite of natural infections having been previously documented, the authors found raccoons very difficult to infect experimentally with Leptospira serovar Pomona. The presence of Leptospira spp. antigen by IHC in the present study suggests that the raccoons were infected; however, since neither the serovar nor the genotype of the organism could be 243

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Figure 3.  Leptospirosis positive wildlife in relation to horse population per census consolidated subdivision.

determined, the likelihood of transmission to a target population of humans or domestic animals is unknown. Of the fox samples, 8% were positive and all of the positive samples were from males. The percentage of positive samples is consistent with a previous study of foxes in Ontario which found 6% to be seropositive for L. Pomona and L. Autumnalis (19). Kidney samples from 3 of those foxes were classified as positive for L. interrogans serovar Pomona type kennewicki, which is consistent with this study in which the best match for the fox samples where genotyping was attempted was also serovar Pomona type kennewicki. Severe nephropathy in 8 of the foxes was also found in that study, which was interpreted to indicate that the fox is more likely to function as an amplifier and indicator host, and less likely to be a maintenance host due to the poorly balanced host-parasite relationship (19). The current study also found interstitial nephritis in 3 of the male juvenile foxes. This study found 1 beaver positive for Leptospira spp. antigen. Beavers have not previously been documented to harbor Leptospira spp. in Ontario; however, in a survey of 25 beavers in Louisiana 1 was positive for L. australis by culture and a second beaver was serologically positive for L. autumnalis and L. pomona (34). 244

This is the first report of positive opossum in Ontario. One previous study found the opossum to be positive for various Leptospira spp. serovars (35); whereas, another study found no opossums to be positive for the serovars tested (36). Infection with leptospires was not identified in deer, coyote, or otter samples in this study using either IHC or PCR. This may be due to the small number of samples tested from these species, a low prevalence of leptospiral infection in those populations sampled, or geographic variation in infection rate and the location of the sample collection. A previous study of leptospirosis in deer in Ontario in January of 1962 examined deer for seroconversion and by tissue culture (17). The study aimed to compare deer located in close proximity to outbreaks of leptospirosis in swine and cattle with those in areas of reduced contact with livestock. The geographic area studied where deer had reduced contact to livestock in 1962 is the same area of low human and livestock density from which the deer samples were obtained in the current study. Abdulla et al (17) found infection in deer was higher in those deer with increasing proximity to the cattle where the outbreaks occurred. They also detected Leptospira serovar Pomona in deer from the area of reduced contact with livestock, albeit to a lesser degree. In contrast, the CVJ / VOL 55 / MARCH 2014

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Figure 4  Leptospirosis positive versus negative wildlife samples relative to human populations.

current study did not detect Leptospira spp., using IHC, in any of the deer sampled from this area. The status of surrounding livestock herds with respect to infection with Leptospira spp. at the time of sample collection in the current study is unknown. Therefore, it is possible that these deer had no opportunity for exposure to the organism from domestic animals. Since these deer are located in the same area, and positive samples were found during the time of an outbreak in livestock, this may question the hypothesis of deer maintaining the infection outside of a livestock population. The results from coyote are consistent with a study in Yellowstone Park where 0% to 17% of the samples were positive, with the infection occurring mostly in juveniles (37). The ages of the coyotes in this study were not documented; however, they were considered adults, and as such, the absence of positive samples is not surprising. Otters were included in the current study because of their aquatic habitat; the numbers were low, all but 1 of the otter samples were from rural areas and none were positive. In a recent study, seropositivity for L. interrogans in this species was 50% in areas of high human density, and 0% in areas of low human density, which raised the question of the effect of human CVJ / VOL 55 / MARCH 2014

density and population growth on prevalence of this infection in wildlife (38). Maps of human, cattle, swine, and horse density with the overlay of positive and negative samples appear to show an association of Leptospira infection in wildlife with increased human density. This is consistent with findings in other studies (36,38). It is unlikely that the wildlife are being exposed to leptospires from humans; however, areas of increased human density also provide food sources which facilitate an increased density of urban wildlife, feral rodents, as well as exposure to domestic animals, such as dogs, which may be a source of infection for wildlife. In examination of the livestock density maps (Figures 1 to 4), association of wildlife positive samples with increasing livestock density is less apparent than that with humans. It is possible that vaccination programs instituted in response to outbreaks in the past have reduced the disease in livestock, and therefore reduced the source of infection for wildlife in rural agricultural areas. Haydon et al (39) note that control of a disease, versus eradication, can be achieved by directing control at the target population or at blocking transmission between reservoir and target. It is possible that an overall reduction of leptospirosis in livestock has resulted in a lower incidence in 245

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Figure 5.  Distribution of locations that had more than 1 positive sample, 1 positive sample, or no positive sample.

wildlife in these rural areas. If this were the case, then the ability of wildlife to serve as reservoirs of infection for domestic animals would be questionable, as it would indicate that they may not be independently maintaining the disease within their own populations, but rather wildlife infection may be the result of re-introduction of leptospires into wildlife populations from domestic animals. Nonetheless, since Leptospira spp. were identified in wildlife in areas of concentrated human and agricultural livestock density, these are areas in which the organism may be present in untreated water and damp soils, serving as an environmental source of infection for domestic animals, humans, and wildlife. This geographical information is important to veterinarians, as it has been noted that clustering of cases of canine leptospirosis by clinic within geographic areas suggested differences in awareness or in diagnosis by veterinarians (40). Detection of leptospires in wildlife in these areas, therefore, may increase the awareness of veterinarians to consider a differential diagnosis of leptospirosis in their patients, or to vaccinate domestic species at risk. Culture is considered the gold standard for detection of Leptospira spp., but it was not attempted in this study due to anticipated low quality of samples; most of the samples had 246

some degree of autolysis due to unavoidable delays in collection and freezing, and all samples were frozen and thawed prior to sectioning of the kidney. Therefore, culture results could not be used to relate PCR results to IHC. It was expected that the PCR results would have been more sensitive in the detection of leptospires than IHC; however, the majority of discordant results were IHC-positive and PCR-negative. It is possible that autolysis or freeze-thaw cycles of some samples may have negatively impacted the performance of PCR assays. Another possibility is that genetic variation of field strains may have reduced the effectiveness of the PCR test. The discordant results may also reflect variation in Leptospira spp. distribution within individual kidneys and difference in pathogen load between the 2 areas sampled for the IHC and PCR tests. Cross-contamination of samples during processing is unlikely, as precautions were taken to avoid this. Since PCR results were mostly too weak to genotype, conclusions regarding whether or not the type of Leptospira spp. that was detected is pathogenic to humans and domestic livestock cannot be confirmed. However, the presence of Leptospira spp. in the kidneys of a variety of wildlife species across Ontario was confirmed by IHC, including in areas of relatively high human and agricultural livestock density. More CVJ / VOL 55 / MARCH 2014

Table 1.  Results for immunohistochemistry (IHC) by species, age, and gender Species

Total tested

IHC positive or suspicious

Positive males

Positive females by agea,d

Beaver (Castor canadensis)  11  1 (9%) NDb

Juvb ND Adult ND

Juvb ND Adult ND

Coyote (Canis latrans)   5  0 (0%)  0/3 (0%)

Juvc ND   0/2 (0%) Adult ND

Juvc ND Adult ND

Deer (Odocoileus viginianus)   12   0 (0%)   0/5 (0%)

Juv 0 (0%)   0/7 (0%) Adult 0 (0%)

Juv 0/1 (0%) Adult 0/6 (0%)

Fox (Vulpes vulpes)   73   6 (8%)   6/45 (13%)

Juv 5/29 (17%)   0/27 (0%) Adult 1/15 (6%)

Juv 0/18 (0%) Adult 0/8 (0%)

Opossum (Didelphis virginiana)   53   3 (6%)   3/29 (10%)

Juv 0 (0%)   0/23 (0%) Adult 1/17 (6%)

Juv 0/8 (0%) Adult 0/10 (0%)

Otter (Lutra canadensis)   28   0 (0%)   0/20 (0%)

Juv 0 (0%)   0/8 (0%) Adult 0 (0%)

Juv 0 (0%) Adult 0 (0%)

Raccoon (Procyon lotor) 245 82 (33%) 57/176 (32%)

Juv 4/19 (21%) 24/66 (36%) Adult 39/126 (31%)

Juv 7/18 (39%) Adult 11/33 (33%)

Skunk (Mephitis mephitis)   33 14 (42%) 11/22 (50%)

Juv 5/11 (45%)   3/10 (30%) Adult 6/11 (55%)

Juv 3/9 (33%) Adult 0 (0%)

ND ND

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Positive males Positive by agea,d femalesd

ND — Not determined. a Age estimates were based on relative size. b Gender of beavers was not recorded. c Age estimates were not recorded. d Gender and age status was presented as available and does not equal the total number of animals tested due to unknowns. 95% confidence intervals for proportions of animals testing positive for leptospirosis by species: beaver 0.23% to 41.2%, coyote 0% to 52.1%, deer 0% to 26.4%, fox 3.1% to 17.0%, opossum 1.2% to 15.7%, otter 0% to 12.3%, raccoon 27.2% to 39.3%, skunk 20.5% to 49.9%.

Table 2.  Results for immunohistochemistry (IHC) compared to results for polymerase chain reaction (PCR) (n = 437)

IHC Positive

IHC Negative

PCR Positive PCR Negative

17   4 75 341

IHC — 2 samples were neither positive nor negative, but “suspicious,” both were PCR negative; 21 samples were PCR inconclusive, 9 were IHC negative, 12 were IHC positive.

work is needed to determine the relationship between leptospiral organisms in wildlife and those in domestic animals and humans, as well as the role of various wildlife species in the maintenance of leptospires in their respective populations.

Acknowledgments Thanks to the Ontario Ministry of Natural Resources, Canadian Wildlife Service, and Darcy Alkerton of Nuisance Wildlife Control Inc. for assistance in obtaining samples for the study. Thanks to Fleming students Bronwen Hennigar, Paul Merritt, and Lindsay Robbins for their help in processing samples. CVJ

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20. Prescott JF, McEwen B, Taylor J, Woods PJ, Abrams-Ogg A, Wilcock B. Resurgence of leptospirosis in dogs in Ontario: Recent findings. Can Vet J 2002;43:955. 21. Randall JE, Bauman K, King M, Gompper ME. A serologic assessment of exposure to viral pathogens and leptospira in an urban raccoon (Procyon lotor) population inhabiting a large zoological park. J Zoo Wildl Med 2007;38:18–26. 22. Wild CJ, Greenlee JJ, Bolin CA, Barnett JK, Haake DA, Cheville NF. An improved immunohistochemical diagnostic technique for canine leptospirosis using antileptospiral antibodies on renal tissue. J Vet Diagn Invest 2002;14:20–24. 23. Harkin KR, Roshto YM, Sullivan JT, Purvis TJ, Chengappa MM. Comparison of polymerase chain reaction assay, bacteriologc culture, and serologic testing in assessment of prevalence of urinary shedding of leptospires in dogs. J Am Vet Med Assoc 2003;222:1230–1233. 24. Surujballi O, Mallory M. Competitive enzyme-linked immunosorbent assay for detection of Leptospira interrogans serovar Pomona antibodies in bovine sera. Clin Diagn Lab Immunol 2001;8:40–43. 25. Szeredi, L, Haake DA. Immunohistochemical identification and pathologic findings in natural cases of equine abortion caused by leptospira infection. Vet Pathol 2006;43:755–761. 26. Harkin KR, Roshto YM, Sullivan JT. Clinical application of a polymerase chain reaction assay for diagnosis of leptospirosis in dogs. J Am Vet Med Assoc 2003;222:1224–1229. 27. Smythe LD, Smith IL, Smith GA, et al. A quantitative PCR (TaqMan) assay for pathogenic Leptospira spp. BMC Infect Dis 2002;2:13. 28. Palaniappan RU, Chang YF, Chang CF, et al. Evaluation of lig-based conventional and real time PCR for the detection of pathogenic leptospiras. Mol Cell Probes 2005;2:111–117. 29. Ahmed A, Engelberts MFM, Boer KR, Ahmed N, Hartskeerl RA. Development and validation of a real-time PCR for detection of pathogenic Leptospira species in clinical materials. PLoS ONE 4(9): e7093. doi:10.1371/journal.pone.0007093. 30. Ramadass P, Jarvis BDW, Corner RJ, Penny D, Marshall RB. Genetic characterization of pathogenic Leptospira species by DNA hybridization. Int J Syst Bacteriol 1992;42:215–219.

31. Yasuda PH, Steigerwalt AG, Sulzer KR, Kaugmann AF, Rogers F, Brenner DJ. Deoxyribonucleic acid relatedness between serogroups and serovars in the family Leptospiraceae with proposals for seven new Leptospira species. Int J Syst Bacteriol 1987;37:407–415. 32. Brenner DJ, Kaufmann AF, Sulzer KR, Steigerwalt AG, Rogers FC, Weyant RS. Further determination of DNA relatedness between serogroups and serovars in the family Leptospiraceae with a proposal for Leptospira alexanderi sp. nov. and four new Leptospira genomospecies. Int J Syst Bacteriol 1999;49:839–858. 33. Boundary Files. Statistics Canada. Available from: www.12.statcan.gc.ca/ census-recensement/2011/geo/bound-limit/bound-limit-eng.cfm Last accessed November 20, 2013. 34. Stuart BP, Crowell WA, Adams WV, Morrow DT. Spontaneous renal disease in beaver in Louisiana. J Wildl Dis 1978;14:250–253. 35. Barr TRB. Infectious disease in the opossum: A review. J Wildl Manage 1963;27:53–71. 36. Richardson DJ, Gauthier JL. A serosurvey of leptospirosis in Connecticut peridomestic wildlife. Vector Borne Zoonotic Dis 2003; 4:187–193. 37. Gese EM, Schultz RD, Johnson MR, Williams ES, Crabtree RL, Ruff RL. Serological survey for diseases in free-ranging coyotes (Canis latrans) in Yellowstone National Park, Wyoming. J Wildl Dis 1997;33:47–56. 38. Gaydos JK, Conrad PA, Gilardi VK, Blundell GM, Ben-David M. Does human proximity affect antibody prevalence in marine foraging river otters (Lontra Canadensis)? J Wildl Dis 2007;43:116–123. 39. Haydon DT, Cleaveland S, Taylor LH, Laurenson MK. Identifying reservoirs of infection: A conceptual and practical challenge. Emerg Infect Dis 2002;8:1468–1473. 40. Gillian DA, Berke O, Reid-Smith R, Ojkic D, Prescott JF. Increase in seroprevalence of canine leptospirosis and its risk factors, Ontario 1998–2006. Can J Vet Res 2009;73:167–175.

Book Review Compte rendu de livre The Cat, Clinical Medicine and Management Little SE. Elsevier Saunders, St. Louis, Missouri, USA, 2012. 1398 pp. ISBN 9781-4377-0660-4, Hardcover and electronic versions, $215 CDN (text).

O

ur knowledge and understanding of felines have come a long way! Never again will they be considered just “small dogs.” Nor will reference material for cats be limited to a few chapters in a small animal text. On the other hand, Dr. Little has achieved the unimaginable task of compiling the current state of knowledge about felines into one volume. I found this book to be very user friendly and pleasant to read. It is very complete in its breadth of detail. Feline behavior and internal medicine topics are covered well, and the chapters on feline reproduction and pediatrics may well be very handy as such cases appear in clinical practice from time to time. I was most interested to see details on the feline genome although this mostly makes for background reading. Chapters on the fundamentals of a feline only practice and population medicine

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address changing attitudes towards care of cats in different settings. And finally, the most unique area of the text covered the management of concurrent and chronic diseases. So often we are treating multiple conditions together, and getting the balance just right can be a challenge. The text is littered with photographs, figures, algorithms, tables, and boxes of key information. Material is organized by body system, using a logical stepwise plan through diagnosis and treatment. Common procedures are outlined in detail, using plenty of photographs. The text is also available electronically. Dr. Little has drawn knowledge and expertise from over 60 well-respected veterinarians. Together they have produced a practical and helpful “go to” text. Dr. Little aims to raise the bar on the quality of feline medicine, and as she says, “cats do not give up their secrets easily”. It is her hope that this text will guide and encourage, and in my opinion, she has thoroughly succeeded.

Reviewed by Janeen Junaid, DVM, Small Animal Veterinarian, Hamilton, Ontario. CVJ / VOL 55 / MARCH 2014

Detection of Leptospira spp. in wildlife reservoir hosts in Ontario through comparison of immunohistochemical and polymerase chain reaction genotyping methods.

Détection deLeptospiraspp. chez des hôtes du réservoir faunique en Ontario par la comparaison des méthodes de génotypage de réaction immunohistochimiq...
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