What Is Your Diagnosis? Blood Smear From a North American Beaver ( <scp> <i>Castor canadensis</i> </scp> )

A wild juvenile North American beaver (Castor canadensis) of unknown sex was presented to the Zoo, Exotics and Wildlife Service at the Western College of Veterinary Medicine Veterinary Medical Centre for a whole body computed tomography (CT) scan under general anesthesia to exclude aspiration pneumonia prior to release. The beaver was found stuck in a human-made dam in mid-July for 3–4 days. In rehabilitation, sneezing was observed, but the patient was otherwise doing well. On assessment under general anesthesia, there were no respiratory signs, and the lungs were clear on auscultation. There were no overt abnormalities in the pulmonary parenchyma or pleural space on CT. An EDTA blood sample collected from the ventral tail vein was submitted to the Clinical Pathology laboratory of Prairie Diagnostic Services Inc., Saskatoon, for a complete blood count (CBC). A CBC was performed using the ADVIA 2120i Hematology Analyzer (Siemens Healthcare Limited) and interpreted according to ISIS physiological reference intervals for the North American Beaver, all ages combined, with no gender selection [1]. The erythrogram revealed a mild normocytic (MCV 86.2 fL [RI: 77.5–108.9 fL]), normochromic (MCHC 314 g/L [RI: 297–406 g/L]) anemia (HGB 88 g/L [RI: 100–155 g/L] and HCT 0.279 L/L [RI: 0.282–0.469 L/L]) with a reticulocyte count of 32 × 109/L. Due to the absence of published species-specific reference intervals for reticulocytes, it was unable to comment on regenerative versus non-regenerative response in this case. The leukogram revealed a mild left shift (Neutrophil bands 0.34 × 109/L [RI: 0.02–0.12 × 109/L]). Segmented neutrophil count was within normal limits (12.9 × 109 cells/L; RI 1.09–15.76 × 109 cells/L). The platelet count was elevated (588 × 109/L [RI: 4–388 × 109/L]). A blood smear prepared from the sample drawn is shown in Figure 1. The significance of the mild anemia is unknown. There was no evidence of significant ongoing hemolysis according to RBC morphology on blood smear review; thus, anemia may be secondary to hemorrhage, which is difficult to confirm or differentiate between external or internal hemorrhage with the available history. The leukogram revealed a mild left shift that can be attributed to acute inflammation. The thrombocytosis could be a reactive response secondary to inflammation and anemia. Microscopic evaluation of peripheral blood revealed occasional extracellular parasites, of ~40–50 μm long and ~5 μm wide with a deeply basophilic, elongated, curvy body, a flagellum running alongside the body of the parasite, which was enveloped in a thin transparent membrane (undulating membrane), and a deep blue, round, central nucleus. This morphology is most compatible with the trypomastigote stage of Trypanosoma (Figure 1A,B). Total genomic DNA was extracted from the submitted blood sample using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. The following amplicons were obtained using two different sets of primers (for 18S rRNA gene [2], and for the Trypanosoma-specific SSU rRNAgene [3]—Table 1). Primer set TryForD/TryRevH amplified a 567 bp fragment, whereas primer set BabAF/BabBR yielded a 1447 bp product. The samples were loaded onto a 1% agarose gel. The amplicons of interest were purified using a kit, GFX PCR DNA and Gel Ban Purification kit. (GE Healthcare UK Limited, Chalfont, Buckinghamshire). Thermocycling conditions and reaction-mix composition were identical to those reported by Pattullo et al. 2013 [4]. The samples were sequenced using Sanger low-throughput sequencing (National Research Council of Canada). A megaBLAST search yielded both Trypanosoma carassii and Trypanosoma cobitis producing 96.83% sequence identity to the 18S rRNA gene, and 95.42% sequence identity using the Trypanosoma-specific SSU rRNA gene. GenBank accession numbers OL963926.1 and AJ009143.1 showed the highest significant alignment. They were observed in both BLAST searches. There was significant sequence alignment to at least one hundred other Trypanosoma spp, such as T. rajae (MG878996.1-96.83%), T. remakii (OQ130041.1-95.42%), and Trypanosoma spp. Cobitis taenia, (KJ601720.1-95.42%); however, their sequence identity was less than the percentages that we had reported for Trypanosoma carassii (OL963926.1-96.83%) and Trypanosoma cobitis (AJ009143.1-96.83%), respectively. Trypanosomes are protozoan parasites, with representative species infecting nearly all vertebrate hosts. However, reports of Trypanosoma infections from Canada are very limited. Notably, there is one report of Trypanosoma (Megatrypanum) species identified in the blood culture of woodland caribou in north Alberta [5]. Numerous wild mammals in the southern United States are naturally infected with T. cruzi, enabling enzootic transmission of the parasite through continuous interactions with infected vectors. To the authors' knowledge, this is the first report of Trypanosoma species found in beavers of any genus. In this case, it is highly possible that the beaver was infected with either Trypanosoma carassii or Trypanosoma cobitis. Historically, these Trypanosome species have been identified as fish parasites [6]. We speculate that the beaver involved in this case may have acquired the Trypanosome infection, possibly by living in the same habitat with infected fish species. However, transmission via other vectors such as kissing bugs or aquatic leeches is also a possibility, which cannot be confirmed or excluded in this case. Although the clinical significance and pathogenesis of trypanosome infection in beavers are not discussed in the literature, this is likely a significant finding, especially for Canadian and North American One Health professionals, as well as the public, given the zoonotic potential of trypanosomiasis. Due to the lack of understanding of the pathogenesis of Trypanosoma infection in beavers, it is challenging to determine the contribution of Trypanosoma infection to the reported anemia in this patient. Unfortunately, there was no follow-up information available for this patient after transferring to a rehabilitation facility. We thank the staff of the clinical pathology laboratory, Prairie Diagnostic Services Inc., Saskatoon, for their technical support. Special thanks to LaRhonda Sobchishin, Research Technician, Department of Veterinary Pathology, Western College of Veterinary Medicine at the University of Saskatchewan, Saskatoon, Canada, for her support in capturing images. The authors declare no conflicts of interest.

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