Poxvirus infection in an American red squirrel (Tamiasciurus hudsonicus) from northwestern Canada.

There are two recognized poxviruses that are associated with disease in tree squirrels: squirrel fibroma virus (SQFV), Leporipoxvirus, which affects eastern grey squirrels (Sciurus carolinensis) in eastern North America, and squirrelpox virus (SQPV), a member of a newly identified poxvirus genus, which affects European red squirrels (Sciurus vulgaris) in the United Kingdom. In August 2008, a cutaneous poxvirus-associated disease was identified in a North American red squirrel (Tamiasciurus hudsonicus) from the Yukon Territory, Canada. The gross and microscopic appearance of the skin lesions was more consistent with SQPV than SQFV, and electron microscopy revealed poxvirions only within epithelial cells. Polymerase chain reaction (PCR) was used to identify poxvirus core protein encoding DNA in skin samples, and phylogenetic analysis showed that the inferred amino acid sequence was distinct from all other poxvirus species for which the core protein gene has been sequenced, including those of the genus Leporipoxvirus. Although the core protein sequence of SQPV was not available, comparison of the constructed phylogenetic tree to other published trees, based on major outer envelope proteins, revealed that the identified sequence occupies a position similar to SQPV in terms of its relationship to other poxviruses. However, PCR primers designed to amplify gene sequences encoding the SQPV major envelope protein and RNA polymerase did not amplify any sequences from infected tissues. These findings suggest that the virus present in this squirrel is a novel poxvirus of North American red squirrels. To our knowledge, this is the first case of poxvirus infection in Canadian squirrels outside of Ontario.

Development of cpn60-based real-time quantitative PCR assays for the detection of 14 Campylobacter species and application to screening of canine fecal samples.

Campylobacter species are important organisms in both human and animal health. The identification of Campylobacter currently requires the growth of organisms from complex samples and biochemical identification. In many cases, the condition of the sample being tested and/or the fastidious nature of many Campylobacter species has limited the detection of campylobacters in a laboratory setting. To address this, we have designed a set of real-time quantitative PCR (qPCR) assays to detect and quantify 14 Campylobacter species, C. coli, C. concisus, C. curvus, C. fetus, C. gracilis, C. helveticus, C. hyointestinalis, C. jejuni, C. lari, C. mucosalis, C. rectus, C. showae, C. sputorum, and C. upsaliensis, directly from DNA extracted from feces. By use of a region of the cpn60 (also known as hsp60 or groEL) gene, which encodes the universally conserved 60-kDa chaperonin, species-specific assays were designed and validated. These assays were then employed to determine the prevalence of Campylobacter species in fecal samples from dogs. Fecal samples were found to contain detectable and quantifiable levels of C. fetus, C. gracilis, C. helveticus, C. jejuni, C. showae, and C. upsaliensis, with the majority of samples containing multiple Campylobacter species. This study represents the first report of C. fetus, C. gracilis, C. mucosalis, and C. showae detection in dogs and implicates dogs as a reservoir for these species. The qPCR assays described offer investigators a new tool to study many Campylobacter species in a culture-independent manner.

Characterization and quantification of feline fecal microbiota using cpn60 sequence-based methods and investigation of animal-to-animal variation in microbial population structure.

The complex microbial community of the intestine plays a major role in animal health and diseases. Despite its significance to feline health and the significance of intestinal and fecal populations to the public health, little is known about the actual composition of the normal microbiota of the cat. To create a sequence-based inventory of feline fecal microbiota, we applied established methods exploiting the gene encoding the universal 60kDa chaperonin (cpn60) to create libraries of cloned cpn60 sequences from pooled fecal samples from five exclusively indoor and four outdoor, known predatory cats. Sequencing of 1248 clones from each library revealed diverse populations dominated by Actinobacteria (particularly bifidobacteria) and Firmicutes (particularly lactobacilli). To investigate the degree of animal-to-animal variation in species abundance, ten targets were selected from the libraries for analysis by quantitative real-time PCR. Quantitative PCR results showed substantial animal-to-animal variation in target abundance although most targets were detected in all cats. This study lays the foundation for future work aimed at understanding the dynamics of intestinal microbial communities and their role in feline health.