Tissue and cellular tropism of Eptesicus fuscus gammaherpesvirus in big brown bats, potential role of pulmonary intravascular macrophages.

Gammaherpesviruses (γHVs) are recognized as important pathogens in humans but their relationship with other animal hosts, especially wildlife species, is less well characterized. Our objectives were to examine natural Eptesicus fuscus gammaherpesvirus (EfHV) infections in their host, the big brown bat (Eptesicus fuscus), and determine whether infection is associated with disease. In tissue samples from 132 individual big brown bats, EfHV DNA was detected by polymerase chain reaction in 41 bats. Tissues from 59 of these cases, including 17 from bats with detectable EfHV genomes, were analyzed. An EfHV isolate was obtained from one of the cases, and electron micrographs and whole genome sequencing were used to confirm that this was a unique isolate of EfHV. Although several bats exhibited various lesions, we did not establish EfHV infection as a cause. Latent infection, defined as RNAScope probe binding to viral latency-associated nuclear antigen in the absence of viral envelope glycoprotein probe binding, was found within cells of the lymphoid tissues. These cells also had colocalization of the B-cell probe targeting CD20 mRNA. Probe binding for both latency-associated nuclear antigen and a viral glycoprotein was observed in individual cells dispersed throughout the alveolar capillaries of the lung, which had characteristics of pulmonary intravascular macrophages. Cells with a similar distribution in bat lungs expressed major histocompatibility class II, a marker for antigen presenting cells, and the existence of pulmonary intravascular macrophages in bats was confirmed with transmission electron microscopy. The importance of this cell type in γHVs infections warrants further investigation.

Eptesipox virus-associated lesions in naturally infected big brown bats.

Bats have many unique qualities amongst mammals; one of particular importance is their reported tolerance to viruses without developing disease. Here, the authors present evidence to the contrary by describing and demonstrating viral nucleic acids within lesions from eptesipox virus (EfPV) infection in big brown bats. One hundred and thirty bats submitted for necropsy from Saskatchewan, Canada, between 2017 and 2021 were screened for EfPV by polymerase chain reaction (PCR); 2 had amplifiable poxvirus DNA. The lesions associated with infection were oral and pharyngeal ulcerations and joint swelling in 2/2 and 1/2 cases, respectively. These changes were nonspecific for poxvirus infection, although intracytoplasmic viral inclusion bodies within the epithelium, as observed in 2/2 bats, are diagnostic when present. Viral nucleic acids, detected by in situ hybridization (ISH), were observed in the epithelium adjacent to ulcerative lesions from both cases and within the joint proliferation of 1 case. A new isolate of EfPV was obtained from 1 case and its identity was confirmed with electron microscopy and whole genome sequencing. Juxtanuclear replication factories were observed in most cells; however, rare intranuclear virus particles were also observed. The significance of the presence of virus particles within the nucleus is uncertain. Whole genome assembly indicated that the nucleotide sequence of the genome of this EfPV isolate was 99.7% identical to a previous isolate from big brown bats in Washington, USA between 2009 and 2011. This work demonstrates that bats are not resistant to the development of disease with viral infections and raises questions about the dogma of poxvirus intracytoplasmic replication.

DNA sequencing confirms meningeal worm (Parelaphostrongylus tenuis) and muscle worm (Parelaphostrongylus andersoni) in white-tailed deer (Odocoileus virginianus): Implications for moose (Alces alces) management.

In North America, some moose populations are declining, and meningeal worm (Parelaphostrongylus tenuis) infections may be contributing. Moose are aberrant hosts for meningeal worm and develop severe pathology whereas white-tailed deer (WTD) are definitive hosts that experience minimal pathology and spread parasite larvae into the environment. Analyses of harvested WTD heads confirmed meningeal worm in Western Manitoba, Canada including in areas where moose have experienced population declines and are currently of management concern. The prevalence of larval meningeal worm from WTD feces in these areas are unknown, particularly because the dorsal-spined larvae (DSL) are morphologically indistinguishable from muscle worm (Parelaphostrongylus andersoni). To assess transmission risk of DSL, we investigated the spatial and temporal variation of prevalence in WTD feces from four areas (two with historical moose population declines and two without) sampled across two summers. We predicted higher prevalence of DSL in areas where moose are of management concern and surveys have shown higher meningeal worm prevalence in WTD heads. Further, we expected to only recover meningeal worm, as muscle worm has only been reported from caribou in more northern areas of Manitoba. We collected WTD feces by transect sampling, used the Baermann technique to obtain larvae, and sequenced partial cytochrome oxidase 1 and internal transcribed spacer 2 genes to confirm species identity. Zero-inflated models revealed that DSL prevalence did not differ temporally but was higher in areas where moose are of management concern. Genetic analyses revealed that meningeal worm and muscle worm were both present in Western Manitoba and co-occurred in three areas. Our results reveal novel insights into the geographic distribution of muscle worm and emphasize the importance of DNA sequencing for DSL identification. We suggest that concern for moose populations is warranted given the increased risk of parasite infection in some management areas.

Characterization of neurotropic HPAI H5N1 viruses with novel genome constellations and mammalian adaptive mutations in free-living mesocarnivores in Canada.

The GsGd lineage (A/goose/Guangdong/1/1996) H5N1 virus was introduced to Canada in 2021/2022 through the Atlantic and East Asia-Australasia/Pacific flyways by migratory birds. This was followed by unprecedented outbreaks affecting domestic and wild birds, with spillover into other animals. Here, we report sporadic cases of H5N1 in 40 free-living mesocarnivore species such as red foxes, striped skunks, and mink in Canada. The clinical presentations of the disease in mesocarnivores were consistent with central nervous system infection. This was supported by the presence of microscopic lesions and the presence of abundant IAV antigen by immunohistochemistry. Some red foxes that survived clinical infection developed anti-H5N1 antibodies. Phylogenetically, the H5N1 viruses from the mesocarnivore species belonged to clade 2.3.4.4b and had four different genome constellation patterns. The first group of viruses had wholly Eurasian (EA) genome segments. The other three groups were reassortant viruses containing genome segments derived from both North American (NAm) and EA influenza A viruses. Almost 17 percent of the H5N1 viruses had mammalian adaptive mutations (E627 K, E627V and D701N) in the polymerase basic protein 2 (PB2) subunit of the RNA polymerase complex. Other mutations that may favour adaptation to mammalian hosts were also present in other internal gene segments. The detection of these critical mutations in a large number of mammals within short duration after virus introduction inevitably highlights the need for continually monitoring and assessing mammalian-origin H5N1 clade 2.3.4.4b viruses for adaptive mutations, which potentially can facilitate virus replication, horizontal transmission and posing pandemic risks for humans.

Surveillance of Babesia odocoilei in wild and farmed cervid populations of Saskatchewan, Canada.

Objective: To investigate whether Babesia odocoilei could be detected from farmed and wild cervid diagnostic submissions prior to its first reported occurrence in Saskatchewan. Procedure: Polymerase chain reaction (PCR) for B. odocoilei was used to survey 85 fresh-frozen samples and 112 formalin-fixed, paraffin-embedded samples from Saskatchewan cervids submitted for necropsy between 2000 and 2014. Results: The PCR was positive for B. odocoilei in 1/84 white-tailed deer, 1/41 moose, 0/37 mule deer, and 1/35 elk. The positive elk was from a farmed herd, but the remaining 2 positive samples were from wild cervids. The positive moose sample was the earliest confirmed infection, dating back to 2008. Therefore, 1.5% of the study population tested positive over the 14-year period. Conclusion: There were low numbers of cervids infected with B. odocoilei in the study population. Clinical relevance: Babesiosis should be included as a differential diagnosis for disease in susceptible cervids when clinical signs are compatible; however, a lack of suggestive clinical signs or necropsy findings does not preclude infection. Thus, monitoring prevalence of the disease within Saskatchewan (and Canada) will likely require targeted surveillance.

Objectif: Déterminer si Babesia odocoilei pouvait être détectée dans les soumissions de diagnostic de cervidés d'élevage et sauvages avant sa première occurrence signalée en Saskatchewan. Procédure: La réaction d'amplification en chaîne par la polymérase (PCR) pour B. odocoilei a été utilisée pour étudier 85 échantillons fraîchement congelés et 112 échantillons fixés au formol et inclus en paraffine de cervidés de la Saskatchewan soumis à l'autopsie entre 2000 et 2014. Résultats: La PCR était positive pour B. odocoilei chez 1/84 cerf de Virginie, 1/41 orignal, 0/37 cerf mulet et 1/35 wapiti. Le wapiti positif provenait d'un troupeau d'élevage, mais les deux autres échantillons positifs provenaient de cervidés sauvages. L'échantillon d'orignal positif était la première infection confirmée, remontant à 2008. Par conséquent, 1,5 % de la population étudiée a été testée positive au cours de la période de 14 ans. Conclusion: Il y avait un faible nombre de cervidés infectés par B. odocoilei dans la population étudiée. Pertinence clinique: La babésiose devrait être incluse comme diagnostic différentiel de maladie chez les cervidés sensibles lorsque les signes cliniques sont compatibles; cependant, l'absence de signes cliniques évocateurs ou de résultats d'autopsie n'exclut pas l'infection. Ainsi, la surveillance de la prévalence de la maladie en Saskatchewan (et au Canada) nécessitera probablement une surveillance ciblée.(Traduit par Dr Serge Messier).

Species-specific PCR assay for the detection of Babesia odocoilei.

We developed a PCR assay for the detection of Babesia odocoilei based on the 18S rRNA gene. Multiple specimens of B. odocoilei were examined, and the assay consistently produced a small specific PCR product of 306 bp. The PCR assay was also challenged with DNA from 13 other Babesia species and 2 Theileria species, originating from 10 different host species; however, nonspecific DNA amplification and multiple banding patterns were observed, and the amplicon banding patterns varied between different isolates of the same species. Sensitivity was determined to be 6.4 pg of DNA, and an estimated 0.0001% parasitism. This assay can be utilized for species-specific differential detection of B. odocoilei.

White-nose syndrome is associated with increased replication of a naturally persisting coronaviruses in bats.

Spillover of viruses from bats to other animals may be associated with increased contact between them, as well as increased shedding of viruses by bats. Here, we tested the prediction that little brown bats (Myotis lucifugus) co-infected with the M. lucifugus coronavirus (Myl-CoV) and with Pseudogymnoascus destructans (Pd), the fungus that causes bat white-nose syndrome (WNS), exhibit different disease severity, viral shedding and molecular responses than bats infected with only Myl-CoV or only P. destructans. We took advantage of the natural persistence of Myl-CoV in bats that were experimentally inoculated with P. destructans in a previous study. Here, we show that the intestines of virus-infected bats that were also infected with fungus contained on average 60-fold more viral RNA than bats with virus alone. Increased viral RNA in the intestines correlated with the severity of fungus-related pathology. Additionally, the intestines of bats infected with fungus exhibited different expression of mitogen-activated protein kinase pathway and cytokine related transcripts, irrespective of viral presence. Levels of coronavirus antibodies were also higher in fungal-infected bats. Our results suggest that the systemic effects of WNS may down-regulate anti-viral responses in bats persistently infected with M. lucifugus coronavirus and increase the potential of virus shedding.

Isolation, characterization and prevalence of a novel Gammaherpesvirus in Eptesicus fuscus, the North American big brown bat.

Little is known about the relationship of Gammaherpesviruses with their bat hosts. Gammaherpesviruses are of interest because of their long-term infection of lymphoid cells and their potential to cause cancer. Here, we report the characterization of a novel bat herpesvirus isolated from a big brown bat (Eptesicus fuscus) in Canada. The genome of the virus, tentatively named Eptesicus fuscus herpesvirus (EfHV), is 166,748 base pairs. Phylogenetically EfHV is a member of Gammaherpesvirinae, in which it belongs to the Genus Rhadinovirus and is closely related to other bat Gammaherpesviruses. In contrast to other known Gammaherpesviruses, the EfHV genome contains coding sequences similar to those of class I and II host major histocompatibility antigens. The virus is capable of infecting and replicating in human, monkey, cat and pig cell lines. Although we detected EfHV in 20 of 28 big brown bats tested, these bats lacked neutralizing antibodies against the virus.

A persistently infecting coronavirus in hibernating Myotis lucifugus, the North American little brown bat.

Bats are important reservoir hosts for emerging viruses, including coronaviruses that cause diseases in people. Although there have been several studies on the pathogenesis of coronaviruses in humans and surrogate animals, there is little information on the interactions of these viruses with their natural bat hosts. We detected a coronavirus in the intestines of 53/174 hibernating little brown bats (Myotis lucifugus), as well as in the lungs of some of these individuals. Interestingly, the presence of the virus was not accompanied by overt inflammation. Viral RNA amplified from little brown bats in this study appeared to be from two distinct clades. The sequences in clade 1 were very similar to the archived sequence derived from little brown bats and the sequences from clade 2 were more closely related to the archived sequence from big brown bats. This suggests that two closely related coronaviruses may circulate in little brown bats. Sequence variation among coronavirus detected from individual bats suggested that infection occurred prior to hibernation, and that the virus persisted for up to 4 months of hibernation in the laboratory. Based on the sequence of its genome, the coronavirus was placed in the Alphacoronavirus genus, along with some human coronaviruses, bat viruses and the porcine epidemic diarrhoea virus. The detection and identification of an apparently persistent coronavirus in a local bat species creates opportunities to understand the dynamics of coronavirus circulation in bat populations.

Mule deer spatial association patterns and potential implications for transmission of an epizootic disease.

Animal social behaviour can have important effects on the long-term dynamics of diseases. In particular, preferential spatial relationships between individuals can lead to differences in the rates of disease spread within a population. We examined the concurrent influence of genetic relatedness, sex, age, home range overlap, time of year, and prion disease status on proximal associations of adult Rocky Mountain mule deer (Odocoileus hemionus hemionus) in a chronic wasting disease endemic area. We also quantified the temporal stability of these associations across different sex, age, and disease status classes. We used three years of high frequency telemetry data from 74 individuals to record encounters within 25 m of each other, and to calculate seasonal home range overlap measured by volume of intersection (VI). The strength of pairwise spatial association between adult mule deer was independent of genetic relatedness, age and disease status. Seasonal variation in association strength was not consistent across years, perhaps due to annual changes in weather conditions. The influence of home range overlap on association strength varied seasonally, whereby associations were stronger in pre-rut and fawning than in the rest of the seasons. The sexes of individuals also interacted with both VI and season. At increasing levels of VI, associations were stronger between females than between males and between females and males. The strongest associations in pre-rut were between males, while the strongest in rut were between females and males. The temporal stability of associations was markedly dependant on the sex and the diagnosis of the associating pair. Our findings highlight the importance of considering concurrent effects of biological and environmental factors when seeking to understand the role of social preference in behavioural ecology and disease spread. Applying this knowledge in epidemiological modelling will shed light on the dynamics of disease transmission among mule deer.

Toxocara pteropodis in Free-Ranging Indian Flying Foxes (Pteropus medius) in Sri Lanka.

Toxocara pteropodis, an intestinal nematode, occurs in several captive and free-ranging pteropid bat species. We report infection in free-ranging Indian flying foxes (Pteropus medius) in Sri Lanka and contribute to our understanding of parasites in free-ranging P. medius .

Calodium hepaticum in Jungle Cats ( Felis chaus ) in Sri Lanka.

Calodium hepaticum infection is rarely reported in carnivores. We describe two cases of C. hepaticum infection, causing liver lesions, in wild jungle cats ( Felis chaus ) in Sri Lanka.

White-Nose Syndrome Disease Severity and a Comparison of Diagnostic Methods.

White-nose syndrome is caused by the fungus Pseudogymnoascus destructans and has killed millions of hibernating bats in North America but the pathophysiology of the disease remains poorly understood. Our objectives were to (1) assess non-destructive diagnostic methods for P. destructans infection compared to histopathology, the current gold-standard, and (2) to evaluate potential metrics of disease severity. We used data from three captive inoculation experiments involving 181 little brown bats (Myotis lucifugus) to compare histopathology, quantitative PCR (qPCR), and ultraviolet fluorescence as diagnostic methods of P. destructans infection. To assess disease severity, we considered two histology metrics (wing area with fungal hyphae, area of dermal necrosis), P. destructans fungal load (qPCR), ultraviolet fluorescence, and blood chemistry (hematocrit, sodium, glucose, pCO2, and bicarbonate). Quantitative PCR was most effective for early detection of P. destructans, while all three methods were comparable in severe infections. Correlations among hyphae and necrosis scores, qPCR, ultraviolet fluorescence, blood chemistry, and hibernation duration indicate a multi-stage pattern of disease. Disruptions of homeostasis occurred rapidly in late hibernation. Our results provide valuable information about the use of non-destructive techniques for monitoring, and provide novel insight into the pathophysiology of white-nose syndrome, with implications for developing and implementing potential mitigation strategies.

Infectious Disease and Grouping Patterns in Mule Deer.

Infectious disease dynamics are determined, to a great extent, by the social structure of the host. We evaluated sociality, or the tendency to form groups, in Rocky Mountain mule deer (Odocoileus hemionus hemionus) from a chronic wasting disease (CWD) endemic area in Saskatchewan, Canada, to better understand factors that may affect disease transmission. Using group size data collected on 365 radio-collared mule deer (2008-2013), we built a generalized linear mixed model (GLMM) to evaluate whether factors such as CWD status, season, habitat and time of day, predicted group occurrence. Then, we built another GLMM to determine factors associated with group size. Finally, we used 3 measures of group size (typical, mean and median group sizes) to quantify levels of sociality. We found that mule deer showing clinical signs of CWD were less likely to be reported in groups than clinically healthy deer after accounting for time of day, habitat, and month of observation. Mule deer groups were much more likely to occur in February and March than in July. Mixed-sex groups in early gestation were larger than any other group type in any season. Groups were largest and most likely to occur at dawn and dusk, and in open habitats, such as cropland. We discuss the implication of these results with respect to sociobiology and CWD transmission dynamics.

Disease risks associated with free-ranging wild boar in Saskatchewan.

This study investigated the disease status of Saskatchewan's feral wild boar population. Whole carcasses, tissue samples, and/or serum from 81 hunter-killed boars from Saskatchewan were submitted to the Canadian Wildlife Health Cooperative (CWHC) between 2009 and 2014. Serological tests were negative for PRRS, H1N1, and H3N2 swine influenza, PCV-2, and TGE/PRCV in 22/22 boars and for Toxoplasma gondii and Mycoplasma hyopneumoniae in 20/20 boars. Of 20 boars whose sera were tested 20 were positive for Actinobacillus pleuropneumoniae, with 7 positive for, among other strains, serotype 14; 16 were positive for Lawsonia intracellularis, 1 was positive and 6 were suspicious for Salmonella spp. Polymerase chain reaction tests were negative for PRRS and PCV2 in 58/58 boars and positive for Torque teno virus in 1/8 boars. Digestion assays were negative for Trichinella spp. in 22/22 boars. The high seroprevalence of A. pleuropneumoniae serotype 14 is noteworthy as this serotype has not been previously reported in North America.

Risques de maladie associés au sanglier en liberté en Saskatchewan. Cette étude a examiné l'état des maladies de la population de sangliers féraux de la Saskatchewan. Des carcasses entières, des échantillons de tissus et/ou du sérum provenant de 81 sangliers tués par des chasseurs de la Saskatchewan ont été soumis à la Canadian Wildlife Health Cooperative (CWHC) entre 2009 et 2014. Les tests sérologiques étaient négatifs pour SRRP, l'influenza porcine H1N1 et H3N2, CVP-2 et GET/CVRP chez 22/22 sangliers et pour Toxoplasma gondii et Mycoplasma hyopneumoniae chez 20/20 sangliers. Parmi les 20 sangliers dont le sérum a été analysé, 20 présentaient des résultats positifs pour Actinobacillus pleuropneumoniae, et sept étaient positifs pour le sérotype 14, entre autres souches; 16 étaient positifs pour Lawsonia intracellularis, un était positif et six étaient suspectés pour Salmonella spp. Des tests d'amplification en chaîne par la polymérase ont été négatifs pour SRRP et CVP2 chez 58/58 sangliers et positifs pour le virus torque teno chez 1/8 des sangliers. Des épreuves de digestion ont été négatives pour Trichinella spp. chez 22/22 sangliers. La séroprévalence élevée du sérotype A. pleuropneumoniae 14 mérite d'être signalée car ce sérotype n'a pas été signalé antérieurement en Amérique du Nord.(Traduit par Isabelle Vallières).

Septicemic listeriosis in wild hares from Saskatchewan, Canada.

The bacterium Listeria monocytogenes causes disease in a wide variety of mammals including rabbits and hares. We describe naturally acquired metritis and septicemic listeriosis in wild female hares from Saskatchewan, Canada. Between April 2012 and July 2013, two white-tailed jackrabbits (Lepus townsendii) and a snowshoe hare (Lepus americanus) were presented to the Veterinary Medical Centre at the Western College of Veterinary Medicine, Saskatoon, Saskatchewan, Canada with nonspecific neurologic signs. The hares were euthanized and autopsied. Necrotizing fibrinosuppurative metritis was present in all. Additional findings in individual hares included fetal maceration, multifocal necrotizing myocarditis, multifocal hepatic necrosis, and nonsuppurative encephalitis. Listeria monocytogenes was cultured from multiple tissues in each hare. Although listeriosis in pregnant domestic rabbits has been studied, this is the first detailed description in wild North American hares. The epidemiology of listeriosis, including prevalence and the role of environmental sources and coprophagy in transmission among hares, requires further investigation.

Conspecific disturbance contributes to altered hibernation patterns in bats with white-nose syndrome.

The emerging wildlife disease white-nose syndrome (WNS) affects both physiology and behaviour of hibernating bats. Infection with the fungal pathogen Pseudogymnoascus destructans (Pd), the first pathogen known to target torpid animals, causes an increase in arousal frequency during hibernation, and therefore premature depletion of energy stores. Infected bats also show a dramatic decrease in clustering behaviour over the winter. To investigate the interaction between disease progression and torpor expression we quantified physiological (i.e., timing of arousal, rewarming rate) and behavioural (i.e., arousal synchronisation, clustering) aspects of rewarming events over four months in little brown bats (Myotis lucifugus) experimentally inoculated with Pd. We tested two competing hypotheses: 1) Bats adjust arousal physiology adaptively to help compensate for an increase in energetically expensive arousals. This hypothesis predicts that infected bats should increase synchronisation of arousals with colony mates to benefit from social thermoregulation and/or that solitary bats will exhibit faster rewarming rates than clustered individuals because rewarming costs fall as rewarming rate increases. 2) As for the increase in arousal frequency, changes in arousal physiology and clustering behaviour are maladaptive consequences of infection. This hypothesis predicts no effect of infection or clustering behaviour on rewarming rate and that disturbance by normothermic bats contributes to the overall increase in arousal frequency. We found that arousals of infected bats became more synchronised than those of controls as hibernation progressed but the pattern was not consistent with social thermoregulation. When a bat rewarmed from torpor, it was often followed in sequence by up to seven other bats in an arousal "cascade". Moreover, rewarming rate did not differ between infected and uninfected bats, was not affected by clustering and did not change over time. Our results support our second hypothesis and suggest that disturbance, not social thermoregulation, explains the increased synchronisation of arousals. Negative pathophysiological effects of WNS on energy conservation may therefore be compounded by maladaptive changes in behaviour of the bats, accelerating fat depletion and starvation.

Activation of innate immune-response genes in little brown bats (Myotis lucifugus) infected with the fungus Pseudogymnoascus destructans.

Recently bats have been associated with the emergence of diseases, both as reservoirs for several new viral diseases in humans and other animals and, in the northern Americas, as hosts for a devastating fungal disease that threatens to drive several bat species to regional extinction. However, despite these catastrophic events little Information is available on bat defences or how they interact with their pathogens. Even less is known about the response of bats to infection during torpor or long-term hibernation. Using tissue samples collected at the termination of an experiment to explore the pathogenesis of White Nose Syndrome in Little Brown Bats, we determined if hibernating bats infected with the fungus Pseudogymnoascus destructans could respond to infection by activating genes responsible for innate immune and stress responses. Lesions due to fungal infection and, in some cases, secondary bacterial infections, were restricted to the skin. However, we were unable to obtain sufficient amounts of RNA from these sites. We therefore examined lungs for response at an epithelial surface not linked to the primary site of infection. We found that bats responded to infection with a significant increase in lungs of transcripts for Cathelicidin (an anti-microbial peptide) as well as the immune modulators tumor necrosis factor alpha and interleukins 10 and 23. In conclusion, hibernating bats can respond to experimental P. destructans infection by activating expression of innate immune response genes.

Flavobacterium columnare: an important contributing factor to fish die-offs in southern lakes of Saskatchewan, Canada.

During June and July 2012, Buffalo Pound Lake and Blackstrap Lake in Saskatchewan, Canada were visited biweekly and surveyed for sick and dying fish. During this investigation, 2 fish kills were identified. Buffalo Pound experienced a large die-off of yellow perch (Perca flavascens) in June, while Blackstrap experienced a die-off of lake whitefish (Coregonus clupeaformis) in July. In excess of 50 fish were examined for gross lesions at each lake, and dead and moribund fish consistently had 1 or more of the following lesions: multifocal petechial cutaneous hemorrhage, skin ulceration, or branchial necrosis. Of these, 17 fish were collected for necropsy, and major tissues were submitted for histology. Aerobic bacterial culture was performed on 16 out of 17 fish. In 7 out of 8 (88%) yellow perch, the body wall had multiple areas of pale discoloration that corresponded to erosion and ulceration of the skin. Seven out of 8 (88%) whitefish had severe necrotizing branchiitis, and 8 out of 8 (100%) had severe epicardial parasitism, consistent with Ichthyocotylurus erraticus. Wet mounts of skin and gill lesions demonstrated filamentous bacteria with gliding motility, which often formed haystack-like arrangements. Flavobacterium columnare and Aeromonas hydrophila were cultured from skin and gill lesions of all fish. Based on the characteristic appearance and distribution of lesions, mortality was attributed to columnaris disease with secondary infection with A. hydrophila. The current study demonstrates that columnaris disease is an important contributor to fish kills in southern Saskatchewan lakes. However, further research is needed to determine what role environmental factors play in outbreaks of columnaris disease in prairie lakes.

Use of the polymerase chain reaction assay for the detection of Babesia odocoilei 18S ribosomal RNA in formalin-fixed tissues.

The effect of fixation and storage conditions on the performance of polymerase chain reaction (PCR) assays for Babesia odocoilei were examined using 3 different primer sets targeting the eukaryotic 18S ribosomal RNA gene, with variably sized products of 1,723 base pairs (bp), 483 bp, and 306 bp. All primer sets performed well on fresh-frozen tissue, and storage for 1 year at -20°C did not affect PCR performance. Formalin fixation markedly affected the amplicon length that could be amplified. However, DNA was successfully amplified after storage in formalin for 2 months using the primer set with a 483-bp product, and up to 6 months using the primer set with a 306-bp product. The latter primer set successfully differentiated B. odocoilei and Babesia microti DNA; however, further evaluation is required to confirm its specificity. Treatment of tissues with formic acid, at concentrations typically used to denature prions, degraded the DNA and made it unsuitable for PCR testing.