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.

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.

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.

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.

An immunoinformatics study reveals a new BoLA-DR-restricted CD4+ T cell epitopes on the Gag protein of bovine leukemia virus.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leucosis (EBL), which has been reported worldwide. The expression of viral structural proteins: surface glycoprotein (gp51) and three core proteins - p15 (matrix), p24 (capsid), and p12 (nucleocapsid) induce a strong humoral and cellular immune response at first step of infection. CD4+ T-cell activation is generally induced by bovine leukocyte antigen (BoLA) region- positive antigen-presenting cells (APC) after processing of an exogenous viral antigen. Limited data are available on the BLV epitopes from the core proteins recognized by CD4+ T-cells. Thus, immunoinformatic analysis of Gag sequences obtained from 125 BLV isolates from Poland, Canada, Pakistan, Kazakhstan, Moldova and United States was performed to identify the presence of BoLA-DRB3 restricted CD4+ T-cell epitopes. The 379 15-mer overlapping peptides spanning the entire Gag sequence were run in BoLA-DRB3 allele-binding regions using a BoLA-DRB- peptide binding affinity prediction algorithm. The analysis identified 22 CD4+ T-cell peptide epitopes of variable length ranging from 17 to 22 amino acids. The predicted epitopes interacted with 73 different BoLA-DRB3 alleles found in BLV-infected cattle. Importantly, two epitopes were found to be linked with high proviral load in PBMC. A majority of dominant and subdominant epitopes showed high conservation across different viral strains, and therefore could be attractive targets for vaccine development.

Impacts of commingling preconditioned and auction-derived beef calves on bovine respiratory disease related morbidity, mortality, and weight gain.

Introduction: Stressors predisposing to bovine respiratory disease (BRD) upon arrival in the feedlot, include the ranch to feedlot transition and mixing cattle from multiple sources. Preconditioning (PC) reduces multiple stressors, but commingling PC and auction-derived (AD) calves in a feedlot may increase BRD risk. Our objective was to evaluate PC calf performance over the first 40 days in the feedlot and determine impacts of commingling with varying proportions of AD calves (25, 50, and 75%). Methods: Calves were either preconditioned at one ranch (n = 250) or mixed-source and bought from a local auction (n = 250). At arrival, calves were assigned into 1 of 5 pens: 100 PC, 75 PC, 50 PC, 25 PC, and 0 PC, reflecting the percentage of PC calves in a 100-head pen. Results: Over 40 days, morbidity in pen 100 PC was lower compared to 0 PC (24 vs. 50%, P < 0.001) and varied in commingled pens, being highest (63%) in 25 PC and least (21%) in 50 PC (P < 0.05). There were 3 AD deaths in 0 PC and 2 deaths in 25 PC. The AD calves in 0 PC were 3 times more likely to get BRD than PC calves in 100 PC; however, AD calves gained 0.49 kg/d more than PC calves (P < 0.0001). Ignoring pen placement, AD calves were 2.76 times more likely to get BRD but gained 0.27 kg/d more than PC calves (P < 0.0001). Commingling did not affect morbidity of PC (P = 0.5) or AD calves (P = 0.96), implying commingling did not affect health. Calves in 25 PC were 3.39 times more likely to get BRD than those in the 100 PC (P < 0.001). Furthermore, 25 PC calves gained the most (1.08 kg/d), followed by 50 PC (0.62 kg/d) and 75 PC (0.61 kg/d), compared to 100 PC (P < 0.05). Calf weight at arrival modified ADG (P < 0.05). Discussion: In conclusion, PC calves had lower morbidity over the first 40 days, irrespective of commingling. With larger variations in arrival weight, there was no advantage of PC for ADG in the first 40 days. The unknown weaning strategies and comparable arrival weights of AD calves may have contributed to greater ADG in AD calves.

A Single Intranasal Dose of Bacterial Therapeutics to Calves Confers Longitudinal Modulation of the Nasopharyngeal Microbiota: a Pilot Study.

To address the emergence of antimicrobial-resistant pathogens in livestock, microbiome-based strategies are increasingly being sought to reduce antimicrobial use. Here, we describe the effects of intranasal application of bacterial therapeutics (BTs) on the bovine respiratory microbiota and used structural equation modeling to investigate the causal networks after BT application. Beef cattle received (i) an intranasal cocktail of previously characterized BT strains, (ii) an injection of metaphylactic antimicrobial (tulathromycin), or (iii) intranasal saline. Despite being transient colonizers, inoculated BT strains induced longitudinal modulation of the nasopharyngeal bacterial microbiota while showing no adverse effect on animal health. The BT-mediated changes in bacteria included reduced diversity and richness and strengthened cooperative and competitive interactions. In contrast, tulathromycin increased bacterial diversity and antibiotic resistance and disrupted bacterial interactions. Overall, a single intranasal dose of BTs can modulate the bovine respiratory microbiota, highlighting that microbiome-based strategies have potential in being utilized to mitigate bovine respiratory disease in feedlot cattle. IMPORTANCE Bovine respiratory disease (BRD) remains the most significant health challenge affecting the North American beef cattle industry and results in $3 billion in economic losses yearly. Current BRD control strategies mainly rely on antibiotics, with metaphylaxis commonly employed to mitigate BRD incidence in commercial feedlots. However, the emergence of multidrug-resistant BRD pathogens threatens to reduce the efficacy of antimicrobials. Here, we investigated the potential use of novel bacterial therapeutics (BTs) to modulate the nasopharyngeal microbiota in beef calves, which are commonly administered metaphylactic antibiotics to mitigate BRD when sourced from auction markets. By direct comparison of the BTs with an antibiotic commonly used for BRD metaphylaxis in feedlots, this study conveyed the potential use of the BTs to modulate respiratory microbiome and thereby improve resistance against BRD in feedlot cattle.

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.

Efficacy of Commercial Infectious Bronchitis Vaccines against Canadian Delmarva (DMV/1639) Infectious Bronchitis Virus Infection in Layers.

Vaccination is the most important way to control infectious bronchitis (IB) in chickens. Since the end of 2015, the Delmarva (DMV)/1639 strain of infectious bronchitis virus (IBV) has caused significant damage to the layer flocks in Eastern Canada. The efficacy of a combination of existing IB vaccines licensed in Canada was assessed against experimental challenge with this IBV strain. The layer pullets were vaccinated during the rearing phase with live attenuated IB vaccines of Massachusetts (Mass) + Connecticut (Conn) types followed by an inactivated IB vaccine of Mass + Arkansas (Ark) types and then challenged with the Canadian IBV DMV/1639 strain at 30 weeks of age. Protection was evaluated based on the egg laying performance, immune responses, viral shedding, and viral genome loads and lesions in IBV target organs. The vaccinated challenged hens were protected from the drop in egg production observed in the non-vaccinated challenged hens. Early (5 dpi) anamnestic serum antibody response was measured in the vaccinated challenged hens as well as a significant level of antibodies was detected in the oviduct washes (14 dpi). In contrast, hens in the non-vaccinated challenged group showed delayed (12 dpi) and significantly lower serum antibody response. Viral RNA loads were reduced in the respiratory, alimentary, and reproductive tissues of the vaccinated challenged hens compared to the non-vaccinated challenged hens. Compared to the control groups, the vaccinated challenged hens had less marked microscopic lesions in the trachea, kidney, magnum, and uterus. Our experimental model demonstrated inconclusive results for cell-mediated immune responses and viral shedding. Overall, the vaccination program used in this study minimized viral replication and histopathological changes in most IBV target organs and protected challenged hens against drop in egg production.

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.

Alberta dairy farmers' and veterinarians' opinion about bovine leukemia virus control measures.

A high herd and within-herd prevalence of Bovine Leukemia Virus (BLV) infections in the dairy herds of North America and the negative effects thereof caused the Alberta dairy industry to initiate the development of an on farm BLV control program. Because BLV control is dependent on the commitment of the farmer, potential barriers were identified and farmers' and veterinarians' points of view toward different control options were investigated to inform how the control program might be adjusted. Conversations with these stakeholders were sought and four focus groups with farmers and eleven interviews with veterinarians were conducted. Testing for BLV, the most common BLV control strategies (testing/culling/segregation/management), as well as on farm best management practices (BMP) to prevent the transmission of BLV, were discussed. The thematic analysis of these conversations resulted in the following findings: Testing of animals was considered important for BLV control, but the financial investment was prohibitive for farmers. Test and cull as well as test and segregation approaches of test positive animals were considered efficient BLV control measures, but impractical and not feasible due to the supply managed Alberta dairy industry (i.e. milk is produced based on demand), with a high prevalence. The management of test positive animals with BMP to prevent new infections and thereby decreasing the within-herd prevalence was considered the only realistic BLV control strategy. The most important barriers for suggested BMP were the cost for some BMP, the inconvenience of performing other BMP, as well as difficulties in performing some BMP consistently and well. Additionally, a lack of knowledge about BLV and its control were identified as an important barrier. On the contrary, farmers indicated being inclined to implement BMP they considered feasible or that were considered a standard within the industry. Further, if BMP increased convenience on farm, they were considered easy to implement. Farmers and veterinarians agreed in many, but not all cases. For example, the single use of examination sleeves was met with differing opinions (i.e. considered doable by farmers while veterinarians assumed it to be too costly). In conclusion, stakeholders' awareness and communication amongst each other (e.g. veterinarians and farmers) about BLV and its control has to be highlighted in order to manage BLV infection successfully. In addition, by communicating and understanding barriers and motivators for specific BMP, important barriers could be identified (e.g. difficulties while changing needles), and solutions found (e.g. tool belt for needles), thereby improving BLV control efforts on farm.

Train driver experience: A big data analysis of learning and retaining the new ERTMS system.

Now and in the coming years a new European Rail Traffic Management System (ERTMS) is being deployed on a larger scale. In the system level chosen in the Netherlands (Level 2) lineside signals will disappear and the train driver will receive all information needed for safe and efficient train operation through an interface inside the train cab. Furthermore, procedures, driving skills and communication will alter. Many train drivers will thus need to be trained in driving with ERTMS. Train operating companies (TOC) have to decide on efficient and effective strategies to organise this extensive training volume and to reassure that drivers become and remain proficient after training when roll-out of ERTMS may not have been fully completed. In this paper the central questions to be answered are: How many hours/duties does a driver have to do during a certain period of time on an ERTMS track section to become proficient with ERTMS (learning period), and to stay proficient (retaining period)? The approach taken is unique for rail industry (to the authors' knowledge) and is based on big data analyses. In this study, we were able to study the collective performance of train drivers, by big data analysis while at the same time maintaining privacy through pseudonymisation of the train driver database and their individual performances, characteristics and other potential privacy sensitive data. Databases containing detailed information on all planned and accomplished train service times/positions of the last three years (2015-2017) of a large TOC were combined with personal data from HR databases (age, experience, ERTMS training history, job assessment results, etcetera). From the data, delays caused by rolling stock and train operating related issues were identified. The amount and duration of these delays were correlated with the personal data to test research hypotheses. Again, at the same time we maintained privacy of the individuals' personal data. Although the research has some limitations due to the small amount of existing ERTMS lines, and associated rolling stock, the results show some clear correlations. Thus, quantifiable directions to the minimum amount of ERTMS driving per 3 months during the first year and in the years after are presented. The results are now being translated into models for training and rostering related to the phase of ERTMS roll-out.

Contagious Ecthyma Dermatitis as a Portal of Entry for Erysipelothrix rhusiopathiae in Muskoxen (Ovibos moschatus) of the Canadian Arctic.

Erysipelothrix rhusiopathiae was detected immunohistochemically in contagious ecthyma (orf virus) dermatitis in two muskoxen (Ovibos moschatus), harvested and found dead in 2014 and 2015, respectively, on Victoria Island, Canada. This may help target further research on E. rhusiopathiae epidemiology and mechanisms of infection in muskoxen, recently associated with widespread mortalities in Canada's Arctic.

Pathogenesis and host responses in lungs and kidneys following Canadian 4/91 infectious bronchitis virus (IBV) infection in chickens.

The infectious bronchitis virus (IBV) 4/91 was one of the common IBV variants isolated in Eastern Canada between 2013 and 2017 from chicken flocks showing severe respiratory and production problems. We designed an in vivo experiment, using specific pathogen free (SPF) chickens, to study the pathogenesis of, and host response to, Canadian (CAN) 4/91 IBV infection. At one week of age, the chickens were infected with 4/91 IBV/Ck/Can/17-038913 isolate. Swab samples were collected at predetermined time points. Five birds from the infected and the control groups were euthanized at 3, 7- and 10-days post-infection (dpi) to collect lung and kidney tissues. The results indicate IBV replication in these tissues at all three time points with prominent histological lesions, significant immune cell recruitment and up regulation of proinflammatory mediators. Overall, our findings add to the understanding of the pathogenesis of 4/91 infection and the subsequent host responses in the lungs and kidneys following experimental infection.

Evaluation of Recombinant Herpesvirus of Turkey Laryngotracheitis (rHVT-LT) Vaccine against Genotype VI Canadian Wild-Type Infectious Laryngotracheitis Virus (ILTV) Infection.

In Alberta, infectious laryngotracheitis virus (ILTV) infection is endemic in backyard poultry flocks; however, outbreaks are only sporadically observed in commercial flocks. In addition to ILTV vaccine revertant strains, wild-type strains are among the most common causes of infectious laryngotracheitis (ILT). Given the surge in live attenuated vaccine-related outbreaks, the goal of this study was to assess the efficacy of a recombinant herpesvirus of turkey (rHVT-LT) vaccine against a genotype VI Canadian wild-type ILTV infection. One-day-old specific pathogen-free (SPF) White Leghorn chickens were vaccinated with the rHVT-LT vaccine or mock vaccinated. At three weeks of age, half of the vaccinated and the mock-vaccinated animals were challenged. Throughout the experiment, weights were recorded, and feather tips, cloacal and oropharyngeal swabs were collected for ILTV genome quantification. Blood was collected to isolate peripheral blood mononuclear cells (PBMC) and quantify CD4+ and CD8+ T cells. At 14 dpi, the chickens were euthanized, and respiratory tissues were collected to quantify genome loads and histological examination. Results showed that the vaccine failed to decrease the clinical signs at 6 days post-infection. However, it was able to significantly reduce ILTV shedding through the oropharyngeal route. Overall, rHVT-LT produced a partial protection against genotype VI ILTV infection.

Pathogenicity of the Canadian Delmarva (DMV/1639) Infectious Bronchitis Virus (IBV) on Female Reproductive Tract of Chickens.

Infectious bronchitis virus (IBV) infection causes significant economic losses to various sectors of the poultry industry worldwide. Over the past few years, the incidence of false layer syndrome in Eastern Canadian layer flocks has been associated with the increased prevalence of the IBV Delmarva (DMV)/1639 strain. In this study, 1-day-old specific-pathogen-free (SPF) hens were infected with the Canadian DMV/1639 strain and observed until 16 weeks of age in order to determine if the IBV DMV/1639 strain is causing false layer syndrome. Early after infection, the virus showed a wide tissue distribution with characteristic gross and histopathological lesions in the respiratory tract and kidney. Around 60-70% of the infected hens demonstrated continuous cloacal viral shedding until the end of the experiment (at 16 weeks) which was associated with high IBV genome loads detected in the cecal tonsils. The experiment confirmed the field observations that the Canadian DMV/1639 strain is highly pathogenic to the female reproductive tract causing marked cystic lesions in the oviduct. Moreover, significant histopathological damage was observed in the ovary. Our study provides a detailed description of the pathological consequences of the IBV DMV/1639 strain circulating in an important poultry production sector.

Vulvar squamous cell carcinoma associated with Equus caballus papillomavirus type 2 infection in a Japanese mare.

Equus caballus papillomavirus type 2 (EcPV2) infection has been associated with genital squamous cell carcinoma (SCC) development in horses. However, very few reports on EcPV2-associated disease in Asia exist. Our study characterizes pathological and virological features of an EcPV2-associated vulvar SCC from a Japanese mare. Conventional PCR, in situ hybridization, reverse-transcriptase PCR and immunohistochemistry confirmed the presence and distribution of EcPV2 within the lesion and suggested that p53 degradation may not be the mechanism by which this virus induces neoplastic transformation. The complete viral sequence in this Japanese case shows near perfect sequence homology with European reference strains of EcPV2, which may be useful when considering the target for future EcPV2 vaccine development. This report also serves to highlight the importance of EcPV2 in female (vulvar) neoplasia, which is less commonly recognized than EcPV2-induced male (penile or preputial) neoplasia. Finally, the SCC described in this mare was an unusual acantholytic variant that has not been reported previously in horses. It is the first report of EcPV2 identified from genital SCC in Asia and underscores the likely worldwide distribution of this virus and its consistent association with equine genital neoplasia.

11 years of regular access to subsidized veterinary services is associated with improved dog health and welfare in remote northern communities.

Access to veterinary services can have positive impacts on animal health and welfare, and on human mental and physical health and well-being; however, many communities worldwide lack access to such services. At their request, the 5 communities of the Sahtu Settlement Area, Northwest Territories, Canada, have received annual access to preventive veterinary services through the University of Calgary's Northern Community Health Rotation since 2008. To determine the reach of the program, we conducted a dog census in 2017. We then conducted a chart review of 11 years of dog medical records from 2008 to 2018 to evaluate how the reach of the program, the uptake of veterinary services, and dog population demographics, health and welfare measures changed over the duration of the program. In the chart review, we used either multi-level logistic regression or generalized linear models, to determine how seven variables, including age, sex, breed, body condition, deworming, vaccination, and sterilization status upon clinic entry, changed over the course of program delivery. Our results suggest that program reach, veterinary service uptake, and dog demographic, health and welfare measures improved over time. We observed high rabies vaccination coverage in some communities (48 %-83 % of the dog population) and moderate overall sterilization status (25 %-56 % of the dog population) with female dog sterilization more common than male (75 % of dogs leaving the 2017 clinics, compared to 43 %). Several dog demographic, health, and welfare measures, including age, body condition, and vaccination, deworming, and sterilization status, were significantly better in later years of the program (all p < 0.001). Differences among communities, both in dog population numbers from the 2017 census (40-89 dogs) and in the uptake of veterinary services in 2017 (48 %-83 % of the dog population), were notable. Vaccination uptake was directly related to clinic attendance, but sterilization was impacted by additional factors, including community members' acceptance of the procedure. Some unintended consequences were noted, however, including the potential effect of sterilization on the availability of traditional dog breeds in the communities. Overall, our study findings demonstrate that subsidized veterinary services provided over a regular and extended period of time benefit animal population demographics, health and welfare, and could have positive impacts on human well-being. The framework of community collaboration and long-term commitment developed through this program serves as a model for achieving common health goals among communities in need and veterinary service providers.

PCIP-seq: simultaneous sequencing of integrated viral genomes and their insertion sites with long reads.

The integration of a viral genome into the host genome has a major impact on the trajectory of the infected cell. Integration location and variation within the associated viral genome can influence both clonal expansion and persistence of infected cells. Methods based on short-read sequencing can identify viral insertion sites, but the sequence of the viral genomes within remains unobserved. We develop PCIP-seq, a method that leverages long reads to identify insertion sites and sequence their associated viral genome. We apply the technique to exogenous retroviruses HTLV-1, BLV, and HIV-1, endogenous retroviruses, and human papillomavirus.

Whole-Blood Transcriptome Analysis of Feedlot Cattle With and Without Bovine Respiratory Disease.

Bovine respiratory disease (BRD) is one of the main factors leading to morbidity and mortality in feedlot operations in North America. A complex of viral and bacterial pathogens can individually or collectively establish BRD in cattle, and to date, most disease characterization studies using transcriptomic techniques examine bronchoalveolar and transtracheal fluids, lymph node, and lung tissue as well as nasopharyngeal swabs, with limited studies investigating the whole-blood transcriptome. Here, we aimed to identify differentially expressed (DE) genes involved in the host immune response to BRD using whole blood and RNA sequencing. Samples were collected from heifers (average arrival weight = 215.0 ± 5.3 kg) with (n = 25) and without (n = 18) BRD at a commercial feedlot in Western Canada. RNAseq analysis showed a distinct whole-blood transcriptome profile between BRD and non-BRD heifers. Further examination of the DE genes revealed that those involved in the host inflammatory response and infectious disease pathways were enriched in the BRD animals, while gene networks associated with metabolism and cell growth and maintenance were downregulated. Overall, the transcriptome profile derived from whole blood provided evidence that a distinct antimicrobial peptide-driven host immune response was occurring in the animals with BRD. The blood transcriptome of the BRD animals shows similarities to the transcriptome profiles obtained from lung and bronchial lymph nodes in other studies. This suggests that the blood transcriptome is a potential diagnostic tool for the identification of biomarkers of BRD infection and can be measured in live animals and used to further understand infection and disease in cattle. It may also provide a useful tool to increase the understanding of the genes involved in establishing BRD in beef cattle and be used to investigate potential therapeutic applications.