Assessment of Metagenomic Sequencing and qPCR for Detection of Influenza D Virus in Bovine Respiratory Tract Samples.

High throughput sequencing is currently revolutionizing the genomics field and providing new approaches to the detection and characterization of microorganisms. The objective of this study was to assess the detection of influenza D virus (IDV) in bovine respiratory tract samples using two sequencing platforms (MiSeq and Nanopore (GridION)), and species-specific qPCR. An IDV-specific qPCR was performed on 232 samples (116 nasal swabs and 116 tracheal washes) that had been previously subject to virome sequencing using MiSeq. Nanopore sequencing was performed on 19 samples positive for IDV by either MiSeq or qPCR. Nanopore sequence data was analyzed by two bioinformatics methods: What's In My Pot (WIMP, on the EPI2ME platform), and an in-house developed analysis pipeline. The agreement of IDV detection between qPCR and MiSeq was 82.3%, between qPCR and Nanopore was 57.9% (in-house) and 84.2% (WIMP), and between MiSeq and Nanopore was 89.5% (in-house) and 73.7% (WIMP). IDV was detected by MiSeq in 14 of 17 IDV qPCR-positive samples with Cq (cycle quantification) values below 31, despite multiplexing 50 samples for sequencing. When qPCR was regarded as the gold standard, the sensitivity and specificity of MiSeq sequence detection were 28.3% and 98.9%, respectively. We conclude that both MiSeq and Nanopore sequencing are capable of detecting IDV in clinical specimens with a range of Cq values. Sensitivity may be further improved by optimizing sequence data analysis, improving virus enrichment, or reducing the degree of multiplexing.

The pulmonary virome, bacteriological and histopathological findings in bovine respiratory disease from western Canada.

The aetiology and pathogenesis of bovine respiratory disease (BRD) are complex and involve the interplay of infectious agents, management and environmental factors. Previous studies of BRD focused on ante-mortem samples from the upper respiratory tract and identified several unconventional viruses. The lung, however, is the primary location where significant BRD lesions are usually found and is a common post-mortem diagnostic specimen. In this study, results of high-throughput virome sequencing, bacterial culture, targeted real-time PCR and histological examination of 130 bovine pneumonic lungs from western Canadian cattle were combined to explore associations of microorganisms with different types of pneumonia. Fibrinous bronchopneumonia (FBP) was the predominant type of pneumonia (46.2%, 60/130) and was associated with the detection of Mannheimia haemolytica. Detection of Histophilus somni and Pasteurella multocida was associated with suppurative bronchopneumonia (SBP) and concurrent bronchopneumonia and bronchointerstitial pneumonia (BP&BIP), respectively. Sixteen viruses were identified, of which bovine parvovirus 2 (BPV2) was the most prevalent (11.5%, 15/130) followed by ungulate tetraparvovirus 1 (UTPV1, 8.5%, 11/130) and bovine respiratory syncytial virus (BRSV, 8.5%, 11/130). None of these viruses, however, were significantly associated with a particular type of pneumonia. Unconventional viruses such as influenza D virus (IDV) and bovine rhinitis B virus (BRBV) were detected, although sparsely, consistent with our previous findings in upper respiratory tract samples. Taken together, our results show that while virus detection in post-mortem lung samples is of relatively little diagnostic value, the strong associations of H. somni and M. haemolytica with SBP and FBP, respectively, indicate that histopathology can be useful in differentiating bacterial aetiologies.

Canine adenovirus type 1 causing neurological signs in a 5-week-old puppy.

BACKGROUND: Infectious canine hepatitis is a rarely encountered disease, that is caused by Canine Adenovirus-1. Clinical signs can vary dramatically, and neurological signs are rarely seen. Neurological manifestation of this disease is rarely reported in the veterinary literature. CASE PRESENTATION: A 5-week-old, male entire Husky cross puppy presented for a one-day history of abnormal neurological behaviour (circling, ataxia, vocalization and obtund mentation). The puppy was euthanized shortly after presentation due to rapid deterioration. Histopathology raised concerns for Canine Adenovirus 1 (CAdV-1) based on vasculitis in the brain and intranuclear inclusion bodies in endothelial cell and hepatocytes; immunohistochemistry on brain tissue confirmed CAdV-1 infection. CONCLUSIONS: This report discusses possible routes of infection and manifestations of adenovirus infections causing neurologic signs. It also provides a timely reminder that CAdV-1 should be considered a differential in unvaccinated dogs that present with neurological signs. Further studies are required to better understand the neurotrophic tendencies of this virus.

Detection and targeting insulin growth factor receptor type 2 (IGF2R) in osteosarcoma PDX in mouse models and in canine osteosarcoma tumors.

Osteosarcoma (OS) represents 3.4% of all childhood cancers with overall survival of 70% not improving in 30 years. The consistent surface overexpression of insulin-like growth factor-2 receptor (IGF2R) has been reported in commercial and patient-derived xenograft (PDX) OS cell lines. We aimed to assess efficacy and safety of treating PDX and commercial OS tumors in mice with radiolabeled antibody to IGF2R and to investigate IGF2R expression on canine OS tumors. IGF2R expression on human commercial lines 143B and SaOS2 and PDX lines OS-17, OS-33 and OS-31 was evaluated by FACS. The biodistribution and microSPECT/CT imaging with 111Indium-2G11 mAb was performed in 143B and OS-17 tumor-bearing SCID mice and followed by radioimmunotherapy (RIT) with 177Lutetium-2G11 and safety evaluation. IGF2R expression in randomly selected canine OS tumors was measured by immunohistochemistry. All OS cell lines expressed IGF2R. Biodistribution and microSPECT/CT revealed selective uptake of 2G11 mAb in 143B and OS-17 xenografts. RIT significantly slowed down the growth of OS-17 and 143B tumors without local and systemic toxicity. Canine OS tumors expressed IGF2R. This study demonstrates the feasibility of targeting IGF2R on OS in PDX and spontaneous canine tumors and sets the stage for further development of RIT of OS using comparative oncology.

Risk factors and prevalence of antibodies for Toxoplasma gondii in diaphragmatic fluid in wolverines (Gulo gulo) from the Northwest Territories, Canada.

Toxoplasma gondii, a zoonotic food borne parasite that can infect almost all warm-blooded animals including people, and ranks 4th among 24 most significant global foodborne parasites listed by the World Health Organization/United Nations Food and Agriculture Organization (FAO/WHO, 2014). Exposure to T. gondii has been reported in wildlife and people in the Canadian North, despite low densities of feline definitive hosts. The ecology of this host-parasite system could be affected by changing climate and landscape in boreal and sub-Arctic regions, and surveillance data are critically needed. Wolverines are an economically and culturally important species in northern Canada due to their valuable fur. Fluid obtained from diaphragmatic muscle of 127 wolverines (Gulo gulo) were tested for antibodies to T. gondii using an enzyme linked immunosorbent assay (ELISA). A seroprevalence of 62% (Confidence Interval (CI): 53-71%) was observed. This result indicates high levels of exposure, likely either through environmental contamination with T. gondii oocysts shed by infected wild felids, or consumption of carcasses/offal of other intermediate hosts containing tissue cysts with bradyzoites in tissues. We examined factors associated with seropositivity, including age, sex, harvest location, harvest location with respect to treeline, and body condition index. Adult (≥2 years) wolverines had 5.2 times higher odds of being sero-positive than juvenile (<1 years) wolverines. The highest seroprevalence was observed in wolverines from Sahtu and South Slave regions. Proportion of sero-positive wolverines harvested above and below the tree line was not significantly different (60% vs 65%). Age was the only significant predictor of T. gondii exposure in wolverines (using logistic regression analysis); further studies should target larger sample sizes. This study is an example of how fluid from diaphragmatic muscle can be used for screening for T. gondii antibodies in wolverines. The diaphragm, commonly collected for screening for another food borne parasite, Trichinella, in wildlife harvested for human consumption, can be used for screening of T. gondii exposure in wildlife. Due to their predatory and scavenging lifestyle and high trophic level, wolverines could serve as a sentinel species for T. gondii.

A novel multiplex PCR-electronic microarray assay for rapid and simultaneous detection of bovine respiratory and enteric pathogens.

Respiratory and enteric diseases continue to be two major causes of economic losses to the cattle industry worldwide. Despite their multifactorial etiology, the currently available diagnostic tests for bovine respiratory disease complex (BRDC) and bovine enteric disease (BED) are single-pathogen-tests. DNA microarray when combined with multiplex polymerase chain reaction (PCR) is a powerful tool in detection and differentiation of multiple pathogens in a single sample. This study reports development and initial validation of two independent highly sensitive and specific multiplex PCR-electronic microarray assays, one for the detection and differentiation of pathogens of the BRDC and the other for detection and differentiation of pathogens of the BED. The BRDC multiplex PCR-microarray assay was able to detect and differentiate four bacteria (Mannheimia haemolytica, Histophilus somni, Pasteurella multocida, and Mycoplasma bovis) and five viruses [bovine parainfluenza virus-3, bovine respiratory syncytial virus, bovine herpesvirus-1, bovine coronavirus (BCoV), and bovine viral diarrhea virus (BVDV)] associated with BRDC. The BED multiplex PCR- microarray- assay was able to detect and differentiate four bacteria (Clostridium perfringens, Escherichia coli, Salmonella enterica Dublin, and Salmonella enterica Typhimurium), three protozoa (Eimeria zuernii, Eimeria bovis, and Cryptosporidium parvum), and four viruses (bovine torovirus, bovine rotavirus, BCoV, and BVDV) associated with the BED. Both assays detected their respective targets individually or in combination when present. The limit-of-detection of each assay at the PCR amplification and DNA microarray levels was determined using previously titrated laboratory amplified target pathogens or using quantified synthetic nucleotides. Both assays showed very high analytical sensitivity and specificity, and were validated using a limited number of clinical samples. The BRDC and BED multiplex PCR- microarray-assays developed in this study, with further clinical validation, could be used in veterinary diagnostic laboratories for the rapid and simultaneous identification of pathogens to facilitate quick and accurate decision making for the control and treatment of these two economically important disease complexes. Furthermore, these assays could be very effective tools in epidemiological studies as well as for screening of healthy animals to identify carriers that may potentially develop BRDC or BED.

Seroprevalence of Cache Valley virus and related viruses in sheep and other livestock from Saskatchewan, Canada.

Cache Valley virus, an orthobunyavirus, is an important cause of ovine neonatal malformations. Information on the seroprevalence of this virus in Saskatchewan livestock populations is lacking. The objectives of this study were to determine the seroprevalence of Cache Valley virus and closely related viruses in sheep, cattle, goats, horses, and mule deer in Saskatchewan by performing a plaque-reduction neutralization test using Cache Valley virus. In total, sera from 130 sheep from 50 flocks were tested. Seroprevalence in sheep was 64.6% (84/130) and 94.0% (47/50) of flocks had 1 or more seropositive sheep. Antibodies to Cache Valley virus or closely related viruses were also detected in serum samples collected from cattle, goats, horses, and mule deer with seroprevalences of 20.0% (5/25), 33.3% (8/24), 69.0% (40/58), and 50.8% (33/65), respectively. These results suggest widespread exposure to Cache Valley virus or closely related viruses in domestic animals and mule deer in Saskatchewan.

Séroprevalence du virus de la Vallée Cache ou de virus connexes chez les moutons et d'autres animaux de cheptel en Saskatchewan, Canada. Le virus de la Vallée Cache, un orthobunyavirus, est une cause importante de malformations néonatales ovines. Il manque des renseignements sur la séroprévalence de ce virus dans les populations des cheptels de la Saskatchewan. Les objectifs de cette étude consistaient à déterminer la séroprévalence du virus de la Vallée Cache et des virus étroitement apparentés chez les moutons, les bovins, les chèvres, les chevaux et les cerfs mulets en Saskatchewan en réalisant un test de séro-neutralisation par réduction des plages en utilisant le virus de la Vallée Cache. Au total, le sérum provenant de 130 moutons dans 50 troupeaux a été testé. Chez les moutons, la séroprévalence était de 64,6 % (84/130) et 94,0 % (47/50) des troupeaux avaient un mouton ou plusieurs moutons séropositifs. Les anticorps pour le virus de la Vallée Cache ou les virus étroitement apparentés ont aussi été détectés dans les échantillons de sérum prélevés auprès des bovins, des chèvres, des chevaux et des cerfs mulets avec une séroprévalence de 20,0 % (5/25), de 33,3 % (8/24), de 69,0 % (40/58) et de 50,8 % (33/65), respectivement. Ces résultats suggèrent une vaste exposition au virus de la Vallée Cache ou à des virus étroitement apparentés chez les animaux domestiques et les cerfs mulets en Saskatchewan.(Traduit par Isabelle Vallières).

Primary bilateral corneal nerve sheath neoplasm in a dog.

A 15-year-old, neutered male, Shih Tzu cross developed progressive corneal stromal thickening and vascularization of the right eye, and 5 months later, of the left eye. Both eyes became blind due to extensive corneal opacification and were enucleated. Light microscopic examination revealed a diffuse corneal infiltrate of neoplastic mesenchymal cells, and immunohistochemistry revealed diffuse cytoplasmic vimentin immunoreactivity and variable cytoplasmic and nuclear immunoreactivity for S100 in the neoplastic cells. Transmission electron microscopy revealed desmosomes between contiguous cells, thread-like cytoplasmic processes coated with basement membrane, extracellular bundles of collagen, and axonal degeneration consistent with features of a nerve sheath neoplasm. This is the first report of primary, bilateral corneal nerve sheath sarcoma in a canine.

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).

Mucosal immunization of calves with recombinant bovine adenovirus-3 coexpressing truncated form of bovine herpesvirus-1 gD and bovine IL-6.

Previous studies have suggested an important role of the cytokine adjuvant IL-6 in the induction of mucosal immune responses in animals, including mice. Here, we report the in vivo ability of bovine adenovirus (BAdV)-3 expressing bovine (Bo) IL-6, to influence the systemic and mucosal immune responses against bovine herpesvirus (BHV)-1 gDt in calves. To co-express both antigen and cytokine, we first constructed a recombinant BAdV-3 expressing chimeric gDt.BoIL-6 protein (BAV326). Secondly, we constructed another recombinant BAdV-3 simultaneously expressing gDt and BoIL-6 using IRES containing a bicistronic cassette gDt-IRES.IL-6, (BAV327). Recombinant proteins expressed by BAV326 and BAV327 retained antigenicity (gDt) and biological activity (BoIL-6). Intranasal immunization of calves with recombinant BAV326, BAV327 or BAV308 (gDt alone) resulted in demonstrable levels of gDt-specific IgG responses in sera and IgA response in nasal secretions, in all animals. In addition, all calves developed complement-independent neutralizing antibody responses against BHV-1. However, no significant difference could be observed in the induction of systemic or mucosal immune response in animals immunized with recombinant BAV326 or BAV327 co-expressing BoIL-6. Moreover, there was no difference in the protection against BHV-1 challenge particularly in the amount of virus excretion in the nasal cavity in calves immunized with BAV326, BAV327 or BAV308. These data suggest that the BoIL-6 had no modulating effect on the induction of gDt specific mucosal and systemic immune responses in calves.