Removing bovine leukemia virus infected animals with high proviral load leads to lowering within-herd prevalence and new case reduction.

Bovine leukosis is prevalent in the North American dairy industry and its effect on animal health and production is widely documented. However, not all bovine leukemia virus (BLV) infected animals transmit the virus equally. Animals with high BLV proviral loads (HPL) are associated with higher transmission risks and therefore, their removal may reduce transmission and eventually within-herd prevalence. We aimed to evaluate the impact of selectively removing HPL cows on the within-herd BLV prevalence and incidence rate of BLV infection in 10 dairy herds. Annual blood and/or milk samples were collected from adult cows over 3 years. Positivity with BLV were determined by ELISA tests and proviral loads in blood of BLV-positive animals were estimated with BLV SS1 quantitative PCR assays. Herd managers were encouraged to consider the proviral load when making culling decisions and implement BLV control practices. High proviral load cows had the highest relative risk of removal indicating the farmers prioritized HPL cows for culling. The within-herd BLV prevalence decreased significantly in 4 herds whereas BLV incidence rate decreased in 9 herds. Over the 3 years, the proviral load demonstrated a relatively stable level, suggesting a single proviral load test in an adult cow may suffice to make culling decisions.

Effects of bovine leukemia virus seropositivity and proviral load on milk, fat, and protein production of dairy cows.

The objective was to evaluate the effects of bovine leukemia virus (BLV) infection, as determined by BLV seropositivity and proviral load, on 305-d milk, fat, and protein production of dairy cows. A cross-sectional study was conducted among 1,712 cows from 9 dairy herds in Alberta, Canada. The BLV status was assessed using an antibody ELISA, whereas BLV proviral load in BLV-seropositive cattle was determined with quantitative PCR. Dairy Herd Improvement 305-d milk, fat, and protein production data were obtained for all enrolled cattle. Differences in these milk end points were assessed in 2 ways: first, by categorizing cows based on BLV serostatus (i.e., BLV positive or negative), and second, by categorizing based on BLV proviral load (i.e., BLV negative, low proviral load [LPL] BLV positive, and high proviral load [HPL] BLV positive). A mixed-effect multivariable linear regression model was used to assess differences in milk parameters. We found that BLV positivity, adjusted for parity and natural log-transformed somatic cell count (SCC), was not associated with reduction in 305-d milk, fat, or protein production. However, significant reductions in 305-d milk, fat, and protein yield occurred in HPL cows, but not in LPL cows, compared with BLV-negative cows, when adjusted for parity number and natural log-transformed SCC. In summary, BLV proviral load may predict effects of BLV infection on milk, fat, and protein production.

Herd-level prevalence of bovine leukemia virus, Salmonella Dublin and Neospora caninum in Alberta, Canada, dairy herds using ELISA on bulk tank milk samples.

Endemic infectious diseases remain a major challenge for dairy producers worldwide. For effective disease control programs, up-to-date prevalence estimates are of utmost importance. The objective of this study was to estimate the herd-level prevalence of bovine leukemia virus (BLV), Salmonella Dublin, and Neospora caninum in dairy herds in Alberta, Canada using a serial cross-sectional study design. Bulk tank milk samples from all Alberta dairy farms were collected 4 times, in December 2021 (n = 489), April 2022 (n = 487), July 2022 (n = 487), and October 2022 (n = 480), and tested for antibodies against BLV, S. Dublin, and N. caninum using ELISAs. Herd-level apparent prevalence was calculated as positive samples divided by total tested samples at each time point. A mixed effect modified Poisson regression model was employed to assess the association of prevalence with region, herd size, herd type, and type of milking system. Apparent prevalence of BLV was 89.4, 88.7, 86.9 and 86.9% in December, April, July, and October, respectively, whereas for S. Dublin apparent prevalence was 11.2, 6.6, 8.6, and 8.5%, and for N. caninum apparent prevalence was 18.2, 7.4, 7.8, and 15.0%. For BLV, S. Dublin and N. caninum, a total of 91.7, 15.6, and 28.1% of herds, respectively, were positive at least once, whereas 82.5, 3.6, and 3.0% of herds were ELISA-positive at all 4 times. Compared with the north region, central Alberta had a high prevalence (prevalence ratio (PR) = 1.13) of BLV-antibody positive herds, whereas south Alberta had a high prevalence (PR = 2.56) of herds positive for S. Dublin antibodies. Furthermore, central (PR = 0.52) and south regions (PR = 0.46) had low prevalence of N. caninum-positive herds compared with the north. Hutterite colony herds were more frequently BLV-positive (PR = 1.13) but less frequently N. caninum-positive (PR = 0.47). Large herds (>7,200 L/day milk delivered ∼ > 250 cows) were 1.1 times more often BLV-positive, whereas small herds (≤3,600 L/day milk delivered ∼ ≤ 125 cows) were 3.2 times more often N. caninum-positive. For S. Dublin, Hutterite-colony herds were less frequently (PR = 0.07) positive than non-colony herds only in medium and large stratum but not in small stratum. Moreover, larger herds were more frequently (PR = 2.20) S. Dublin-positive than smaller herds only in non-colony stratum but not in colony stratum. Moreover, N. caninum prevalence was 1.6 times higher on farms with conventional milking systems compared with farms with an automated milking system. These results provide up-to-date information of the prevalence of these infections that will inform investigations of within-herd prevalence of these infections and help in devising evidence-based disease control strategies.

Immunopathogenesis of the Canadian Delmarva (DMV/1639) infectious bronchitis virus (IBV): Impact on the reproductive tract in layers.

IBV infection may lead to reduced egg production and poor egg quality in layer flocks. The DMV/1639 strain was recently identified as one of the most dominant IBV variants isolated from Canadian layer flocks with egg production problems. The current study aimed to investigate the immunopathogenesis of the Canadian DMV/1639 strain in laying chickens. Specific-pathogen-free (SPF) layers were infected at the peak of lay (29 weeks; n = 10) with an uninfected control group (n = 10). Egg production in the infected group dropped to 40% by the fifth day post-infection (dpi). Five birds from the infected and the control groups were euthanized at 5 and 10 dpi. Ovarian regression and shortened oviduct with marked histopathological changes were observed in the infected group at 10 dpi. An increase in the IBV viral load in reproductive tissues was accompanied by a significant recruitment (p < 0.05) of KUL01+ macrophages and CD4+ and CD8+ T cell subsets at 10 dpi. Additionally, anti-IBV antibody response was detected in serum and locally in the reproductive tract washes of the infected group. Overall, our findings contribute to the understanding of the pathogenicity of the Canadian DMV/1639 strain and the subsequent host responses in the reproductive tract of chickens.

Pilot implementation of a newly developed bovine leukemia virus control program on 11 Alberta dairy farms.

We developed a custom bovine leukemia virus (BLV) control program for the Alberta dairy industry, consisting of a risk assessment and a comprehensive list of best management practices (BMP) aimed at prevention of BLV transmission between cattle. This control program was implemented on 11 farms for approximately 1 yr. Blood samples were collected from all cattle ≥12 mo old, and serum was tested with a commercial ELISA. Risk assessments were performed on each farm, risk-connected on-farm management was identified, and management changes expected to prevent transmission of BLV between cattle were suggested by the first author and agreed upon with each farmer. Throughout the following year, all participating farmers were visited multiple times to identify and overcome barriers to implementation and to monitor progress. After approximately 1 yr of implementing BLV control, all cattle ≥12 mo old on farm with a negative or no previous test result were sampled, and the within-herd prevalence was determined. The median number of cattle on farm that were ≥12 mo was 195 (range 110-524). The initial prevalence averaged 39% (13-66%). On average, 5 BMP (3-7) were suggested to each farmer. On average, 4 BMP (1-7) were implemented. At the second sampling, the average within-herd prevalence of all animals that tested positive (including the previous sampling) was 36% (12-62%). Eight farms reduced their within-herd BLV prevalence, within-herd prevalence stayed constant on 1 farm, and it increased on 1 farm. The remaining farm terminated their participation before the second sampling. The number of seroconversions per farm ranged from 3 to 109, highlighting the success of some producers to minimize new infections. The risk assessment was proven to be a valuable tool to identify flaws in on-farm management, although risk assessment score was unrelated to the within-herd BLV prevalence. Finally, it appeared that implementation of BMP aimed at prevention of BLV transmission between cattle could reduce within-herd BLV prevalence when farmers committed to their implementation.

Invited review: Bovine leukemia virus-Transmission, control, and eradication.

Bovine leukemia virus (BLV) infection, endemic in North American dairy herds, has production-limiting effects. A literature review of available papers published since 1995 concerning BLV transmission and its control was conducted. Although confirmed transmission routes were reviewed (blood, natural breeding, in utero, colostrum, and milk), there is still a lack of detailed information on other specific risks for transmission (e.g., contact transmission and hoof-trimming knives). Eradication of BLV has been achieved by combined management, segregation, and culling approaches. In contrast, although sole implementation of best management practices aimed at prevention of BLV transmission has decreased within-herd BLV prevalence, it has not eradicated BLV from a herd. Therefore, control and eradication of BLV by best management practices only should be further investigated. Additionally, the role of proviral load in infected cattle was investigated. Cattle with a high proviral load seem to be more likely to infect others, whereas those with a very low proviral load seem to have low risks of transmitting BLV. Information on proviral load could be taken into account when controlling BLV in high-prevalence herds. In conclusion, there is a need for detailed, large-scale studies investigating roles of specific transmission routes, knowing proviral load of infected individuals.

Economic evaluation of 4 bovine leukemia virus control strategies for Alberta dairy farms.

Bovine leukemia virus (BLV) is a production-limiting disease common in North American dairy herds. To make evidence-based recommendations to Canadian dairy producers and their consultants regarding cost and financial benefits of BLV on-farm control, an economic model that takes the supply-managed milk quota system into account is necessary. Alberta-specific input variables were used for the presented analysis. A decision tree model program was used to evaluate economic aspects of decreasing a 40% BLV within-herd prevalence on dairy farms by implementing various control strategies over 10 yr. Investigated strategies were (1) all management strategies, including 3 options for colostrum management; (2) some management strategies; (3) test and cull; and (4) test and segregate. Each of these strategies was compared with a no control on-farm approach. The prevalence for this no-control approach was assumed to stay constant over time. Each control strategy incurred specific yearly cost and yielded yearly decreases in prevalence, thereby affecting yearly partial net revenue. Infection with BLV was assumed to decrease milk production, decrease cow longevity, and increase condemnation of carcasses at slaughter from cattle with enzootic bovine leukosis, thereby decreasing net revenue. Cows infected with BLV generated a yearly mean partial net revenue of Can$7,641, whereas noninfected cows generated Can$8,276. Mean cost for the control strategies ranged from Can$193 to Can$847 per animal over 10 yr in a 146-animal herd. Net benefits of controlling BLV on farm, as compared with not controlling BLV, per cow in a 146-animal herd over a 10-yr period for each strategy was: Can$1,315 for all management strategies (freezer); Can$1,243 for all management strategies (pasteurizer); Can$785 for all management strategies (powdered colostrum); Can$1,028 for some management strategies; Can$1,592 for test and cull; and Can$1,594 for test and segregate. Consequently, on-farm BLV control was financially beneficial. Even though negative net benefits were possible and expected for some iterations, our sensitivity analysis highlighted the overall robustness of our model. In summary, this model provided evidence that Canadian dairy farmers should be encouraged to control BLV on their farm.

Comparative features of infections of two Massachusetts (Mass) infectious bronchitis virus (IBV) variants isolated from Western Canadian layer flocks.

BACKGROUND: Infectious bronchitis virus (IBV) is one of the leading causes of mortality and morbidity in chickens. There are numerous serotypes and variants, which do not confer cross protection resulting in failure of currently used IBV vaccines. Although variant IBV isolates with major genetic differences have been subjected to comparative studies, it is unknown whether minor genetic differences in IBV variants within a serotype are different in terms of pathogenesis and eliciting host responses. Two Massachusetts (Mass) variant IBV isolates recovered from commercial layer flocks in the Western Canadian provinces of Alberta (AB) and Saskatchewan (SK) were compared genetically and evaluated for their pathogenicity, tissue distribution and ability to recruit and replicate in macrophages. RESULTS: Although whole genome sequencing of these two Mass IBV isolates showed low similarity with the M41 vaccinal strain, they had an identical nucleotide sequence at open reading frames (ORFs) 3a, 3b, envelop (E), matrix (M), 5a and 5b. The rest of the ORFs of these 2 IBV isolates showed 99.9% nucleotide similarity. However, upon experimental infection, we found that the IBV isolate originating from AB was different to the one that originated in SK due to higher tracheal lesion scores and lower lung viral replication and lower genome loads in cecal tonsils. Nevertheless, both IBV isolates elicited host responses characterized by significant macrophage recruitment to the respiratory tract and there was evidence that both IBV isolates replicated within tracheal and lung macrophages. CONCLUSIONS: Overall, this study shows that Mass variant IBV isolates, although possessing minor genetic variations, can lead to significant differences in pathogenicity in young chickens. Further studies are required to investigate the pathogenicity of these two Mass variant IBV isolates in laying hens.

Short communication: Evaluation of 5 different ELISA for the detection of bovine leukemia virus antibodies.

Although Canadian dairy herds have been infected with bovine leukemia virus (BLV) for years, recent research has put new emphasis on the potential negative effects of this infection. Consequently, BLV control is becoming more favorable; however, BLV control cannot be successful without identifying infected animals. Bovicheck BLV (Biovet, Saint-Hyacinthe, QC, Canada) is currently the only assay licensed by the Canadian Centre for Veterinary Biologics. The first goal of this study was, therefore, to determine the reproducibility of the Bovicheck BLV assay for serum samples derived from Canadian cattle. The second goal was to evaluate and compare 5 different ELISA and determine their test characteristics using serum samples from Canadian herds. The considered ELISA were Bovicheck BLV, ID Screen BLV Competition (IDvet, Grabels, France), Idexx Leukosis Serum X2 Ab Test (Idexx Europe B.V., Hoofddorp, the Netherlands), Svanovir BLV gp51-Ab (Svanova, Uppsala, Sweden), and the Serelisa BLV Ab Mono Indirect (Synbiotics, Lyon, France). Eighty serum samples from Canadian cattle provided by Prairie Diagnostic Services (PDS; Saskatoon, SK, Canada) and an additional 80 serum samples from Canadian dairy and beef herds were used for the study. The Bovicheck BLV assay yielded the same results for all PDS-derived samples, implying a high level of reproducibility and robustness of this assay. Additionally, the comparison of the assays' results showed high agreement between assays, with Cohen's kappa values between κ = 0.91 and κ = 1. Furthermore, using original test results of the field samples as true status, relative diagnostic sensitivity and specificity were calculated. Relative diagnostic sensitivity of all tests was 100%. False-positive results were probable; therefore, the following relative diagnostic specificities were determined: 100% for Bovicheck BLV, Idexx Leukosis Serum X2, and Svanovir BLV; 95% for ID Screen BLV; and 97% for Serelisa BLV. When considering other test characteristics, ID Screen BLV is exceptional due to considerable practical advantages.

Discovery of herpesviruses in Canadian wildlife.

Herpesviruses (HVs) have a wide range of hosts in the animal kingdom. The result of infection with HVs can vary from asymptomatic to fatal diseases depending on subtype, strain, and host. To date, little is known about HVs naturally circulating in wildlife species and the impact of these viruses on other species. In our study, we used genetic and comparative approaches to increase our understanding of circulating HVs in Canadian wildlife. Using nested polymerase chain reaction targeting a conserved region of the HV DNA polymerase gene, we analyzed material derived from wildlife of western and northern Canada collected between February 2009 and Sept 2014. For classification of new virus sequences, we compared our viral sequences with published sequences in GenBank to identify conserved residues and motifs that are unique to each subfamily, alongside phylogenetic analysis. All alphaherpesviruses shared a conserved tryptophan (W856) and tyrosine (Y880), betaherpesviruses all shared a serine (S836), and gammaherpesviruses had a conserved glutamic acid (E835). Most of our wildlife HV sequences grouped together with HVs from taxonomically related host species. From Martes americana, we detected previously uncharacterized alpha- and beta-herpesviruses.

Genomic analysis of the multi-host pathogen Erysipelothrix rhusiopathiae reveals extensive recombination as well as the existence of three generalist clades with wide geographic distribution.

BACKGROUND: Knowledge about how bacterial populations are structured is an important prerequisite for studying their ecology and evolutionary history and facilitates inquiry into host specificity, pathogenicity, geographic dispersal and molecular epidemiology. Erysipelothrix rhusiopathiae is an opportunistic pathogen that is currently reemerging in both the swine and poultry industries globally. This bacterium sporadically causes mortalities in captive marine mammals, and has recently been implicated in large-scale wildlife die-offs. However, despite its economic relevance and broad geographic and host distribution, including zoonotic potential, the global diversity, recombination rates, and population structure of this bacterium remain poorly characterized. In this study, we conducted a broad-scale genomic comparison of E. rhusiopathiae based on a diverse collection of isolates in order to address these knowledge gaps. RESULTS: Eighty-three E. rhusiopathiae isolates from a range of host species and geographic origins, isolated between 1958 and 2014, were sequenced and assembled using both reference-based mapping and de novo assembly. We found that a high proportion of the core genome (58 %) had undergone recombination. Therefore, we used three independent methods robust to the presence of recombination to define the population structure of this species: a phylogenetic tree based on a set of conserved protein sequences, in silico chromosome painting, and network analysis. All three methods were broadly concordant and supported the existence of three distinct clades within the species E. rhusiopathiae. Although we found some evidence of host and geographical clustering, each clade included isolates from diverse host species and from multiple continents. CONCLUSIONS: Using whole genome sequence data, we confirm recent suggestions that E. rhusiopathiae is a weakly clonal species that has been shaped extensively by homologous recombination. Despite frequent recombination, we can reliably identify three distinct clades that do not clearly segregate by host species or geographic origin. Our results provide an essential baseline for future molecular epidemiological, ecological and evolutionary studies of E. rhusiopathiae and facilitate comparisons to other recombinogenic, multi-host bacteria.

'WE CAN'T GET WORMS FROM COW DUNG': REPORTED KNOWLEDGE OF PARASITISM AMONG PASTORALIST YOUTH ATTENDING SECONDARY SCHOOL IN THE NGORONGORO CONSERVATION AREA, TANZANIA.

Records at the Endulen Hospital in the Ngorongoro Conservation Area (NCA), Tanzania, reveal that soil-transmitted helminth infections and protozoa are consistently in the top ten diagnoses for Maasai pastoralists, indicating a significant public health concern. Nevertheless, Maasai pastoralist adaptations to life in close proximity to livestock and to unreliable access to water raise important questions about experiences of, and resiliency to, parasitic infections. Though these infections are particularly prevalent among youth in low- and middle-income countries (LMIC), a focus on resiliency highlights local capacity to recover from and prevent illness. For instance, how is human parasitism perceived and experienced among communities displaying behaviours that studies have associated with transmission of diarrhoeal diseases, such as open defecation? Among these communities, how is parasitism seen to impact the health and development of children? And, what resources are available to endure or mitigate this heavy disease burden among affected communities? This study draws on formative research carried out in May 2014 in anticipation of an innovative school-based and youth-driven water, sanitation and hygiene education intervention rolled out in two boarding schools in the NCA in subsequent months. The initiative is grounded in a One Health approach to health promotion, drawing on partnerships in medicine, public health and veterinary medicine to appreciate the unique interactions between humans, animals and the environment that shape well-being among pastoralist communities. Qualitative data generated through group discussions with secondary school youth (n=60), Maasai teachers (n=6) and a women's group (n=8) in the NCA convey existing knowledge of the prevalence, prevention and treatment of human parasitism. An underlying principle of the larger initiative is to engage youth as creative agents of change in developing and sustaining locally relevant health promotion strategies. Findings highlight practical knowledge around certain 'neglected tropical diseases', namely helminths, among pastoralist communities in the NCA, in turn feeding into the development of the science fair and related interventions.

Acute BVDV-2 infection in beef calves delays humoral responses to a non-infectious antigen challenge.

Immunosuppressive effects of an intranasal challenge with non-cytopathic bovine viral diarrhea virus (BVDV) 2a (strain 1373) were assessed through acquired and innate immune system responses to ovalbumin (OVA). Concurrent BVDV infection was hypothesized to delay and reduce the humoral response to ovalbumin (administered on days 3 and 15 post-inoculation). Infected animals followed the expected clinical course. BVDV titers, and anti-BVDV antibodies confirmed the course of infection and were not affected by the administration of OVA. Both the T-helper (CD4(+)) and B-cell (CD20(+)) compartments were significantly (P < 0.05) reduced in infected animals, while the gamma-delta T-cell population (Workshop cluster 1+, WC1(+)) decreased slightly in numbers. Infection with BVDV delayed the increase in OVA IgG by approximately 3 d from day 12 through day 21 post-inoculation. Between days 25 and 37 post-inoculation following BVDV infection the IgM concentration in the BVDV- group decreased while the OVA IgM titer still was rising in the BVDV+ animals. Thus, active BVDV infection delays IgM and IgG responses to a novel, non-infectious antigen.

Une infection aiguë par le BVDV-2 chez les veaux retarde les réponses humorales face à un test à l'aide d'un antigène non infectieux. Les effets immunosuppressifs d'une inoculation défin intranasale à l'aide du virus non cytopathogène de la diarrhée virale bovine (VBVD) 2a (souche 1373) ont été évalués par les réactions acquises et innées du système immunitaire à l'ovalbumine (OVA). On a émis l'hypothèse que l'infection concomitante par le VBVD retardait et réduisait la réaction humorale à l'ovalbumine (administrée aux jours 3 et 15 après l'inoculation). Les animaux infectés ont suivi le cheminement clinique prévu. Les titres de BVDV et les anticorps anti-BVDV ont confirmé le déroulement de l'infection et ils n'ont pas été affectés par l'administration d'OVA. Les compartiments des lymphocytes T auxiliaires (CD4+) et des cellules B (CD20+) étaient significativement réduits (P < 0,05) chez les animaux infectés, tandis que la numération de la population de cellules T gamma-delta (WC1+) a diminué légèrement. L'infection par le VBVD a retardé l'augmentation de l'OVA IgG d'environ 3 jours, à compter du jour 12 jusqu'au jour 21 après l'inoculation. Entre les jours 25 et 37 après l'inoculation suivant l'infection par le BVDV, la concentration d'IgM dans le groupe VBVD a diminué tandis que le titre d'OVA IgM augmentait toujours chez les animaux positifs pour le VBVD. Par conséquent, l'infection active par le VBVD retarde les réactions IgM et IgG face à un antigène non infectieux nouveau.(Traduit par Isabelle Vallières).

Pathogens at the livestock-wildlife interface in Western Alberta: does transmission route matter?

In southwestern Alberta, interactions between beef cattle and free-ranging elk (Cervus elaphus) may provide opportunities for pathogen transmission. To assess the importance of the transmission route on the potential for interspecies transmission, we conducted a cross-sectional study on four endemic livestock pathogens with three different transmission routes: Bovine Viral Diarrhea Virus and Bovine Herpesvirus 1 (predominantly direct transmission), Mycobacterium avium subsp. paratuberculosis (MAP) (indirect fecal-oral transmission), Neospora caninum (indirect transmission with definitive host). We assessed the occurrence of these pathogens in 28 cow-calf operations exposed or non-exposed to elk, and in 10 elk herds exposed or not to cattle. We characterized the effect of species commingling as a risk factor of pathogen exposure and documented the perceived risk of pathogen transmission at this wildlife-livestock interface in the rural community. Herpesviruses found in elk were elk-specific gamma-herpesviruses unrelated to cattle viruses. Pestivirus exposure in elk could not be ascertained to be of livestock origin. Evidence of MAP circulation was found in both elk and cattle, but there was no statistical effect of the species commingling. Finally, N. caninum was more frequently detected in elk exposed to cattle and this association was still significant after adjustment for herd and sampling year clustering, and individual elk age and sex. Only indirectly transmitted pathogens co-occurred in cattle and elk, indicating the potential importance of the transmission route in assessing the risk of pathogen transmission in multi-species grazing systems.

Detection and Phylogenetic Analysis of Orf Virus and Muskox Rhadinovirus 1 from Muskoxen (Ovibos moschatus) in the Canadian Arctic.

Orf virus (genus Parapoxvirus) has been associated with gross skin lesions on muskoxen (Ovibos moschatus) from Victoria Island, Nunavut, Canada, where muskox populations are experiencing population declines. Orf virus causes painful proliferative and necrotizing dermatitis upon viral replication and shedding, which may lead to animal morbidity or mortality through secondary infections and starvation. Herpesvirus, known to cause gross lesions on skin and mucosa during active viral replication, has also been documented in muskoxen but to date has not been associated with clinical disease. Our objective was to characterize the variation of orf virus and herpesvirus in wild muskoxen of the Canadian Arctic. Tissue samples including gross skin lesions from the nose, lips, and/or legs were opportunistically collected from muskoxen on Victoria Island, Nunavut and Northwest Territories, and mainland Nunavut, Canada, from 2015 to 2017. Sampled muskoxen varied in age, sex, location, hunt type, and body condition. Tissues from 60 muskoxen were tested for genetic evidence of orf virus and herpesvirus infection using PCR targeting key viral genes. Tissues from 38 muskoxen, including 15 with gross lesions, were also examined for histological evidence of orf virus and herpesvirus infection. Eleven muskoxen (10 from Victoria Island and one from mainland Nunavut) with gross lesions had microscopic lesions consistent with orf virus infection. Muskox rhadinovirus 1, a gammaherpesvirus endemic to muskoxen, was detected in 33 (55%) muskoxen including 17 with gross lesions. In all tissues examined, there was no histological evidence of herpesvirus-specific disease. Sequencing and characterization of amplified PCR products using phylogenetic analysis indicated that a strain of orf virus, which appears to be unique, is likely to be endemic in muskoxen from Victoria Island and mainland Nunavut. Many of the muskoxen are also subclinically infected with a known muskox-endemic strain of herpesvirus.

Evolutionary analysis of inter-farm transmission dynamics in a highly pathogenic avian influenza epidemic.

Phylogenetic studies have largely contributed to better understand the emergence, spread and evolution of highly pathogenic avian influenza during epidemics, but sampling of genetic data has never been detailed enough to allow mapping of the spatiotemporal spread of avian influenza viruses during a single epidemic. Here, we present genetic data of H7N7 viruses produced from 72% of the poultry farms infected during the 2003 epidemic in the Netherlands. We use phylogenetic analyses to unravel the pathways of virus transmission between farms and between infected areas. In addition, we investigated the evolutionary processes shaping viral genetic diversity, and assess how they could have affected our phylogenetic analyses. Our results show that the H7N7 virus was characterized by a high level of genetic diversity driven mainly by a high neutral substitution rate, purifying selection and limited positive selection. We also identified potential reassortment in the three genes that we have tested, but they had only a limited effect on the resolution of the inter-farm transmission network. Clonal sequencing analyses performed on six farm samples showed that at least one farm sample presented very complex virus diversity and was probably at the origin of chronological anomalies in the transmission network. However, most virus sequences could be grouped within clearly defined and chronologically sound clusters of infection and some likely transmission events between farms located 0.8-13 Km apart were identified. In addition, three farms were found as most likely source of virus introduction in distantly located new areas. These long distance transmission events were likely facilitated by human-mediated transport, underlining the need for strict enforcement of biosafety measures during outbreaks. This study shows that in-depth genetic analysis of virus outbreaks at multiple scales can provide critical information on virus transmission dynamics and can be used to increase our capacity to efficiently control epidemics.

Contrasting results of culture-dependent and molecular analyses of Mycobacterium avium subsp. paratuberculosis from wood bison.

Reduced to near extinction in the late 1800s, a number of wood bison populations (Bison bison athabascae) have been re-established through reintroduction initiatives. Although an invaluable tool for conservation, translocation of animals can spread infectious agents to new areas or expose animals to pathogens in their new environment. Mycobacterium avium subsp. paratuberculosis, a bacterium that causes chronic enteritis in ruminants, is among the pathogens of potential concern for wood bison management and conservation. In order to inform translocation decisions, our objectives were to determine the M. avium subsp. paratuberculosis infection status of wood bison herds in Canada and to culture and genetically characterize the infective strain(s). We tested fecal samples from bison (n = 267) in nine herds using direct PCR for three M. avium subsp. paratuberculosis-specific genetic targets with different copy numbers within the M. avium subsp. paratuberculosis genome. Restriction enzyme analysis (REA) and sequencing of IS1311 were performed on seven samples from five different herds. We also evaluated a panel of different culture conditions for their ability to support M. avium subsp. paratuberculosis growth from feces and tissues of direct-PCR-positive animals. Eighty-one fecal samples (30%) tested positive using direct IS900 PCR, with positive samples from all nine herds; of these, 75% and 21% were also positive using ISMAP02 and F57, respectively. None of the culture conditions supported the growth of M. avium subsp. paratuberculosis from PCR-positive samples. IS1311 REA and sequencing indicate that at least two different M. avium subsp. paratuberculosis strain types exist in Canadian wood bison. The presence of different M. avium subsp. paratuberculosis strains among wood bison herds should be considered in the planning of translocations.

The modification and evaluation of an ELISA test for the surveillance of Mycobacterium avium subsp. paratuberculosis infection in wild ruminants.

BACKGROUND: Enzyme-linked immunosorbent assay (ELISA) is often used to test wildlife samples for Mycobacterium avium subsp. paratuberculosis (MAP) infection. However, commercially available kits are only validated for use with domestic ruminant species. A literature review was performed to document the current use of MAP serum ELISA in wild and semi-domestic ruminants. We then modified and evaluated a commercial ELISA kit (IDEXX Mycobacterium paratuberculosis Antibody Test Kit) for use with species for which it was not originally developed: elk (Cervus elaphus), bison (Bison bison) and caribou (Rangifer tarandus). We tested the affinity of different conjugates for immunoglobulin G (IgG) isolated from these species, performed checkerboard tests to determine the optimal dilutions of samples and conjugates, and established cut-off values using two different methods: a Receiver Operational Curve on a panel of known samples for elk, and an alternate method involving a panel of unknown serum samples for the three species. RESULTS: We found that the anti-bovine conjugate included in the IDEXX ELISA kit has limited affinity for elk, bison, and caribou IgG. Protein G showed good affinity for IgG of all three species, while anti-deer conjugate also bound elk and caribou IgG. Using Protein G with elk serum, a cut-off sample-to-positive (S/P) value of 0.22 was selected, resulting in a sensitivity and specificity of 73% and 90%, respectively, whereas, using an anti-deer conjugate with elk serum, an S/P cut-off value of 0.29 gave a sensitivity of 68%, with 100% specificity. Cut-off values for bison and caribou using the Protein G conjugate were 0.17 and 0.25 respectively. CONCLUSIONS: Due to incomplete reporting and a lack of test validation, it is difficult to critically appraise results of many sero-surveys that have previously been done for MAP in wildlife. Commercial ELISA kits may have limited or no capacity to detect antibodies from species other than for which they were developed. In order to generate reliable test results, it is essential to evaluate the test and perform modifications if deemed necessary. Despite the challenges inherent to wildlife diagnostics, we have shown that several methods can be used to improve confidence in test results.

Next-generation sequencing approach to investigate genome variability of Parapoxvirus in Canadian muskoxen (Ovibos moschatus).

In 2016, the first orf virus, a double-stranded DNA (dsDNA) virus of the genus parapoxvirus, from a muskox was isolated on Victoria Island, Nunavut (NU), Canada. We used deep sequencing on DNA extracted from orf virus-positive tissues from wild muskoxen from locations on Victoria Island and the adjacent mainland. Orf virus sequence reads derived from four samples were nearly identical. The consensus sequences generated from pooled reads of MxOV comprises of a large contiguous sequence (contig) of 131,759 bp and a smaller right terminal contig of 3552 bp, containing all coding sequences identified as Parapoxvirus. Individual gene comparisons reveal that MxOV shares genetic characteristics with reference strains from both sheep and goat origin. Recombination analysis using Bootscan, MAXCHI, GENECONV, CHIMAERA, SISCAN, and RDP algorithms within the RDP4 software predicted recombination events in two virulence factors, and a large 3000 bp segment of the MxOV genome. Partial B2L nucleotide sequences from strains around the world and other North American isolates were compared to MxOV using MUSCLE alignments and RAxML phylogenetic trees. MxOV was identical to our previously characterized isolate, and shared similarity with orf virus isolated from sheep and goats. The phylogenetic grouping of partial B2L nucleotide sequences did not follow the sample geographic distribution. More full genomes of orf virus, or at least full B2L gene squences, in wildlife are needed especially in North America to better understand the epidemiology of the disease in muskoxen.

Effectiveness of TRIzol in Inactivating Animal Pathogens.

Introduction: Safe handling of biological samples sourced from wild ecosystems is a pressing concern for scientists in disparate fields, including ecology and evolution, OneHealth initiatives, bioresources, geography, veterinary medicine, conservation, and many others. This is especially relevant given the growing global research community and collaborative networks that often span international borders. Treatments to inactivate potential pathogens of concern during transportation and analysis of biospecimens while preserving molecular structures of interest are necessary. Objective: We provide a detailed resource on the effectiveness and limitations of TRIzol™ Reagent, a product commonly used in molecular biology to inactivate bacterial and viral pathogens found in wild animals. Methods: By literature review, we evaluate the mode of action of TRIzol Reagent and its main components on bacterial and viral structures. We also synthesize peer-reviewed literature on the effectiveness of TRIzol in inactivating a broad range of infectious bacteria and viruses. Key Findings: TRIzol Reagent inactivation is based on phenol, chaotropic salts, and sodium acetate. We find evidence of widespread efficacy in deactivating bacteria and a broad range of enveloped viruses. The efficacy against a subset of potential pathogens, including some nonenveloped viruses, remains uncertain. Conclusion: Available evidence suggests that TRIzol Reagent is effective in inactivating a broad spectrum of bacteria and viruses from cells, tissues, and liquids in biological samples when the matrices are exposed to at least 10 min at room temperature to the reagent. We highlight areas that require additional research and discuss implications for laboratory protocols.