DOI: 10.7589/2013-08-215

Journal of Wildlife Diseases, 50(3), 2014, pp. 699–702 # Wildlife Disease Association 2014

Sylvatic Plague in a Canadian Black-Tailed Prairie Dog (Cynomys ludovicianus) Kym S. Antonation,1,6 Todd K. Shury,2 Trent K. Bollinger,3 Adam Olson,1 Philip Mabon,1 Gary Van Domselaar,4 and Cindi R. Corbett1,5 1Bioforensics Assay Development and Diagnostics, National Microbiology Laboratory, Public Health Agency of Canada, 1015 Arlington St., Winnipeg, Manitoba, R3E 3R2, Canada; 2Parks Canada Agency, 52 Campus Dr., Saskatoon, Saskatchewan, S7N 5B4, Canada; 3Canadian Cooperative Wildlife Health Centre, 52 Campus Dr., Saskatoon, Saskatchewan, S7N 5B4, Canada; 4 Bioinformatics Section, National Microbiology Laboratory, Public Health Agency of Canada, 1015 Arlington St., Winnipeg, Manitoba, R3E 3R2, Canada; 5Department of Medical Microbiology, Room 543, 745 Bannatyne Ave., University of Manitoba, Winnipeg, Manitoba R3E 0J9, Canada; 6Corresponding author (email: [email protected])

vessels of the brain, liver, kidney, heart, and adrenal gland. Pulmonary alveoli were filled with protein-rich edema fluid and the spleen contained a marked neutrophil and fibrin exudate with a myriad of small bacteria. Based on gross and histologic findings, a preliminary diagnosis of bacterial septicemia, likely due to Yersinia pestis, was made. Identification of this isolate, named ‘‘EBD10-058’’ was confirmed using an National Microbiology Laboratory inhouse (unpublished) real-time PCR assay targeting three regions of the plasmid and chromosome in addition to conventional bacteriologic tests (identification of the capsular F1 antigen and CDC A1122 bacteriophage). In addition, two singlenucleotide polymorphism (SNP) assays were undertaken to rapidly determine the origin of the strain (Vogler et al. 2008). These assays demonstrated the presence of a biovar Orientalis, North America–specific SNP, and lack of the Y. pestis CO92–specific SNP. Whole genomic sequencing was performed using the genome analyzer IIx (Illumina Inc, San Diego, California, USA) following the manufacturer’s instructions, generating single end reads (150-bp length). De novo sequence assemblies were completed using Edena v3 (Hernandez et al. 2008), followed by reference mapping of the EBD10-058 de novo contigs to another characterized North American Y. pestis (CO92) using SMALT map version 0.6.4 (Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire, UK). Unmapped

ABSTRACT: In 2010, a black-tailed prairie dog (Cynomys ludovicianus) was found dead in Grasslands National Park, Saskatchewan, Canada. Postmortem gross and histologic findings indicated bacterial septicemia, likely due to Yersinia pestis, which was confirmed by molecular analysis. This is the first report of Y. pestis in the prairie dog population within Canada.

In July 2010, a black-tailed prairie dog (Cynomys ludovicianus) was found deceased on the Larson Prairie Dog Colony in Grasslands National Park, Saskatchewan, Canada (49u6900, 107u24900). Concurrently, researchers from Canadian Wildlife Service observed that prairie dogs at a separate colony in the park had disappeared, and anecdotal evidence indicated that active prairie dogs were last observed on the 4.6-ha colony in October 2009. Although prairie dog population abundance is highly variable among years, the population during summer 2010 was 50–70% below the long-term average (Parks Canada unpubl. data). Hypothesized causes for the population decline included climate (drought) or predation; it was also hypothesized that plague may have been acting in a supporting role. The prairie dog was submitted for necropsy with suspected sylvatic plague. On gross necropsy, the prairie dog was in excellent condition. The spleen was moderately enlarged and there was generalized mild to moderate congestion and edema of all tissues. Histologic examination of tissues revealed thrombi containing small gram-negative bacilli and moderate numbers of neutrophils in numerous blood 699

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TABLE 1. Thirty-three high-quality single-nucleotide polymorphisms (SNPs) identified between Yersinia pestis EBD10-058 and CO92 genome sequences. High quality is defined as .53 coverage, .85% consensus among reads, and minimum of 250 bp of consecutively mapped sequence flanking on either side. The SNP names indicated are as they appear in original publications (as denoted by footnotes). ‘‘Bx’’ SNPs are those found in this study.

SNP name

CO92 genome position

CO92/ EBD10-058 base change

Coverage in EBD10-058 (3)

% of reads as SNP

Substitution

16a 2a B1 B2 4a Nb B3 17a 5a B4 B5 6a M, 7b P, 8a Q, 9b B6 B7 Ob B8 10a 11a 12a R, 19c 20a B9 B10 B11 B12 B13 B14 S, 14b 15a

150946 351821 432785 904485 917155 1178178 1821543 1939828 1939841 1971663 2122883 2273616 2278317 2300659 2619611 2905077 2966696 2968425 3096234 3608932 3647867 3655609 3739401 3886839 4039597 4102752 4159046 4166467 4413466 4430475 4579183 4624135

C/A T/G C/T T/C A/G T/C C/T T/G A/G A/G C/T G/C A/G T/G T/G C/A C/G A/G G/C T/C C/T T/C C/A T/C A/C C/T G/A A/T G/A G/A A/G C/G

47 40 31 39 55 48 30 40 43 44 46 51 45 42 48 33 32 36 17 47 44 43 42 36 58 46 33 40 48 47 41 43

97.87 100.00 100.00 97.44 100.00 100.00 96.67 100.00 100.00 100.00 97.83 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 98.28 100.00 100.00 100.00 96.00 97.87 97.56 100.00

Synonymous Nonsynonymous Nonsynonymous Nonsynonymous Nonsynonymous Noncoding Synonymous Synonymous Nonsynonymous Noncoding Noncoding Nonsynonymous Nonsynonymous Nonsynonymous Nonsynonymous Synonymous Synonymous Noncoding Noncoding Nonsynonymous Nonsynonymous Nonsynonymous Synonymous Synonymous Noncoding Synonymous Nonsynonymous Nonsynonymous Nonsynonymous Synonymous Nonsynonymous Nonsynonymous

a

CO92 gene ID (gene name)

YPO0138 (pckA) YPO0342 YPO0415 YPO0824 YPO0837 YPO1597 (fabH) YPO1701 YPO1701

YPO2000 YPO2005 YPO2029 YPO2328 YPO2583 YPO2638

YPO3243 YPO3273 YPO3275 YPO3352 YPO3481

(gmhA) (pssA) (clpB) (ydjJ) (ibeB)

YPO3674 YPO3721 YPO3722 YPO3928 YPO3942 YPO4060 YPO4103

(metH) (argE) (glgB) (fdhD) (trmE)

Auerbach et al. 2007.

b

Touchman et al. 2007.

c

Vogler et al. 2008.

regions of CO92 (,4 mapped reads) were utilized to identify regions missing within EBD10-058. The reference mapping analyses revealed that the genetic material for EBD10-058 was encoded on one chromosome and three plasmids of similar size to that of Y. pestis CO92 (Parkhill et al. 2001). There did not appear to be any large regions of divergence. We identified SNPs from referencemapped pileups using FreeBayes version

0.9.6 (Garrison and Marth 2012). Regions were visualized using Tablet version 1.12.09.03 (Milne et al. 2010). For SNP analysis only high-quality SNPs that had .53 coverage, .85% consensus among reads, and minimum of 250 bp of consecutively mapped sequence flanking on either side were used. Once these SNPs were identified in EBD10-058, alignments to 12 publically available North American Y. pestis strains were conducted using

0 0 4 0 3 5 0 9 8 8 0 0 9 8 8 0 11 11 10 9 9 0 0 11 11 10 9 9 0 12 12 1 1 10 9 9 0 15 15 15 14 14 11 10 10 0 16 15 15 15 14 14 11 10 10 0 18 18 17 17 17 16 16 13 12 12

CA88-4125 CO92 AS200901539 AS200901509 BA200901990 BA200902009 AS200901434 AS200902147 BA200901703 AS200901156 BA200901799 EBD10-058

0 23 21 21 20 20 20 19 19 16 15 15 EBD10-058 CA88-4125 CO92 AS200901539 AS200901509 BA200901990 BA200902009 AS200901434 AS200902147 BA200901703 AS200901156 BA200901799

TABLE 2.

Single-nucleotide polymorphism matrix using core genome pair-wise comparisons of publicly available North American Yersinia pestis isolates.

LETTERS

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progressive Mauve aligner (Darling et al. 2010). Thirty-three SNPs between EBD10058 and CO92 (Table 1) were found. This included 14 unique SNPs (10 coding and four noncoding) not previously reported. The remaining 19 SNPs were previously reported between CO92 and other sequenced North American strains (Auerbach et al. 2007; Touchman et al. 2007). Comparison of gene content within EBD10-058 to other North American Y. pestis strains revealed that there were few to no differences, confirming the genetic monomorphic nature of 1.ORI North American Y. pestis strains (Morelli et al. 2010). A SNP matrix table (Table 2) illustrates relatedness and provides context among publicly available isolates that would otherwise yield a linear tree structure. The sequence has been deposited at DDBJ/EMBL/GenBank under accession number ATWT00000000. In Canada, there have been very few documented outbreaks of sylvatic plague. Sporadic outbreaks were reported in ground squirrels (species not reported) in Alberta and Saskatchewan in the 1930s and a 1939 report describes an outbreak in farmed mink (Mustela vison), presumably the result of using ground squirrels as a food source; the mink rancher also died (Wobeser et al. 2009). A more recent report described two cases of plague among bushy-tailed woodrats (Neotoma cinerea) in southern British Columbia (Lewis 1989). Although confirmed animal cases of plague are rare, the disease is present in the western provinces as evidenced by detection of antibodies to Y. pestis in domestic dogs (Canis lupus familiaris) and cats (Felis catus) in southern Saskatchewan and Alberta (Leighton et al. 2001). In contrast, plague occurs and is reported frequently in US Gunnison’s prairie dog (Cynomys gunnisoni) populations throughout the midwestern states (Friggens et al. 2010; Busch et al. 2011). Wobeser et al (2009) theorized that plague in Canada is less visible than in the US because colonial rodents that are highly

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susceptible to the disease, such as prairie dogs, are confined to a small area of southern Saskatchewan, in Grasslands National Park. This is the first report of sylvatic plague in Canadian black-tailed prairie dogs and the first Canadian isolate to be subject to molecular analysis. As expected, this isolate belongs to the biovar Orientalis group (1.ORI branch, specific to North America), further confirming that all Y. pestis in North America seem to have arisen from one introduction event (Morelli et al. 2010). In addition, 14 unique SNPs were found. This parallels previous studies that have shown a range of two to 18 unique SNPs for each North American Y. pestis strain compared to CO92 (Auerbach et al. 2007; Touchman et al. 2007). Hopefully, with enhanced surveillance and molecular characterization of future isolates we can define Canadian-specific SNPs and contribute further to the understanding of the radiation of plague in Canada. We thank B. Balcewich and K. Hayden for technical expertise. This work was supported by the Public Health Agency of Canada, the Canadian Cooperative Wildlife Health Centre, and Parks Canada. The views and opinions expressed herein are those of the authors only and do not necessarily represent the views and opinions of the Public Health Agency of Canada, the Canadian Food Inspection Agency, or the Government of Canada. LITERATURE CITED Auerbach RK, Tuanyok A, Probert WS, Kenefic L, Vogler AJ, Bruce DC, Munk C, Brettin TS, Eppinger M, Ravel J, Wagner DM, Keim P. 2007. Yersinia pestis evolution on a small timescale: Comparison of whole genome sequences from North America. PLoS One 2:e770. Busch JD, Van AR, Cordova J, Colman RE, Keim P, Rocke TE, Leid JG, Van Pelt WE, Wagner DM. 2011. Population differences in host immune factors may influence survival of Gunnison’s prairie dogs (Cynomys gunnisoni) during plague outbreaks. J Wildl Dis 47:968–973. Darling AE, Mau B, Perna NT. 2010. progressiveMauve: Multiple genome alignment with gene

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