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.

Metadata beyond the sequence enables the phylodynamic inference of bovine viral diarrhea virus type 1a isolates from Western Canada.

Bovine viral diarrhea virus (BVDV) has been recognized as an important pathogen of livestock in Canada. The high mutation rate of this virus leads to a great degree of diversity between isolates resulting in the ability to infer precise evolutionary relationships. Many studies have attempted to elucidate the regional and global evolution of BVDV, but so far few have applied Bayesian methods to this end. We aimed to describe the molecular epidemiology and phylodynamics of BVDV 1a isolates in Western Canada using 5'UTR and E1-E2 sequence data, collection dates and locations. Sequences were obtained from isolates submitted to a diagnostic laboratory in Saskatoon, Canada. Path sampling and stepping stone model testing were employed to identify the model that best fit the data. We found that these Western Canadian isolates share a most recent common ancestor dated near 1909. Furthermore, the E1-E2 region shows a median substitution rate about ten times greater than the 5'UTR. It was also noted that caution should be exercised when inferring phylogenetic relationships using the 5'UTR alone, as it becomes difficult to resolve relationships within major clades. Phylogeographic and population size fluctuation estimates require more thorough sampling than was performed here to be reliable. We have found that there are significant gains to be made by utilizing a Bayesian analysis and by incorporating additional types of data beyond the sequence. These include the estimation of most common recent ancestor dates and the precise inference of transmission routes. Future work will expand upon these findings by more thoroughly sampling BVDV isolates spatially and temporally and further refining the Bayesian model employed here.

Equine protozoal myeloencephalitis caused by Neospora hughesi in an adult horse in Saskatchewan.

A protozoal parasite identified as Neospora hughesi was found in inflammatory lesions in the central nervous system of a Canadian-born adult horse presented with neurological signs. This is believed to be the first case of equine protozoal myeloencephalitis (EPM) caused by Neospora hughesi in a horse outside of the United States.

Canine distemper virus-associated encephalitis in free-living lynx (Lynx canadensis) and bobcats (Lynx rufus) of eastern Canada.

Between 1993 and 1999, encephalitis caused by morbillivirus was diagnosed by immunohistochemistry and histology in six lynx (Lynx canadensis) and one bobcat (Lynx rufus) in the eastern Canadian provinces of New Brunswick and Nova Scotia. Five of the six cases in lynx occurred within an 11-mo period in 1996-97. A second bobcat with encephalitis caused by unidentified protozoa and a nematode larva also had immunohistochemical evidence of neurologic infection by morbillivirus. The virus was identified as canine distemper virus (CDV) by reverse transcriptase polymerase chain reaction and nucleotide sequencing in four of five animals from which frozen tissue samples were available, and it was isolated in cell culture from one of them. To our knowledge, this is the first report of disease caused by CDV in free-living felids in North America.

Characterization of a Canadian mink H3N2 influenza A virus isolate genetically related to triple reassortant swine influenza virus.

In 2007, an H3N2 influenza A virus was isolated from Canadian mink. This virus was found to be phylogenetically related to a triple reassortant influenza virus which emerged in Canadian swine in 2005, but it is antigenically distinct. The transmission of the virus from swine to mink seems to have occurred following the feeding of animals with a ration composed of uncooked meat by-products of swine obtained from slaughterhouse facilities. Serological analyses suggest that the mink influenza virus does not circulate in the swine population. Presently, the prevalence of influenza virus in Canadian farmed and wild mink populations is unknown. The natural occurrence of influenza virus infection in mink with the presence of clinical signs is a rare event that deserves to be reported.

Innate immune responses induced by CpG oligodeoxyribonucleotide stimulation of ovine blood mononuclear cells.

Examples exist in the literature that demonstrate that treatment with immunostimulatory cytosine-phosphate-guanosine (CpG)-DNA can protect mice against infection by intracellular pathogens. There are, however, few studies reporting that CpG-DNA offers similar disease protection in other species. In this study, we assessed the potential of a class A and class B CpG oligodeoxynucleotide (ODN) to induce innate immune responses in sheep, an outbred species. Using peripheral blood mononuclear cells, we have for the first time demonstrated CpG-ODN-induced innate immune responses, including natural-killer-like activity [non-major histocompatibility complex (MHC)-restricted cytotoxicity], interferon-alpha secretion and 2'-5'A oligoadenylate synthetase activity, that could contribute to immune protection in sheep. The type and magnitude of these responses were dependent on ODN class and non-MHC-restricted killing was not associated with interferon-gamma production. The latter observation is in contrast with observations reported for mice and humans. These observations support the conclusion that differences in CpG-ODN-induced responses exist among species and that specific ODN sequences can significantly influence innate immune responses.

The effectiveness and limitations of immune memory: understanding protective immune responses.

Immune memory is the foundation of the practise of vaccination. Research on the molecular and cellular events leading to generation and development of memory T and B lymphocytes explain why there are heightened secondary immune responses after an initial encounter with antigen. In this review, we discuss how clonal expansion, targeted tissue localisation, more efficient antigen recognition and more proficient effector functions contribute to the improved effectiveness of memory cells. Despite the enhanced efficacy of memory cells and the recall immune response, there are numerous experimental and empirical examples in which protection provided by vaccines are short-lived, particularly against pathogens that replicate and cause pathology at their site of entry. In the absence of active immune effector activities, the ability of memory cells to respond quickly enough to control this type of infection is limited. The protective efficacy of bovine herpes virus-1 vaccines in experimental and field challenge conditions are used to illustrate the concept that full protection from disease conferred by vaccination requires the presence of active immune effector mechanisms. Thus, regardless of the many successful technological advances in vaccine design and better understanding of mechanisms underlining induction of memory responses by vaccination, we should recognise that vaccine immunoprophylaxis has limitations. Expectations for vaccines should be realistic and linked to the understanding of host immune responses and knowledge regarding the pathogen and disease pathogenesis.

Protection of chickens against Escherichia coli infections by DNA containing CpG motifs.

Synthetic oligodeoxynucleotides (ODN) containing CpG motifs (CpG-ODN) have been shown to be effective immunoprotective agents in murine models for a variety of viral, intracellular bacterial, and protozoan infections. Until now, the use of CpG-ODN to protect against extracellular bacterial infections has not been reported. The objective of this study was to investigate the effect of CpG-ODN against cellulitis and colibacillosis in broiler chickens, using a well-established model. At 22 days of age, birds received CpG-ODN by either the subcutaneous or intramuscular route. Three days later, a virulent isolate of Escherichia coli was applied to a scratch site on the caudal abdominal skin. Birds were examined for 10 days after the E. coli challenge, and pathological and bacteriological assessments were conducted on all birds. The control group of birds receiving no CpG-ODN((2007)) had a survival rate of 15%. In contrast, groups that received CpG-ODN((2007)), by either subcutaneous or intramuscular injection, had significantly higher survival rates (P < 0.0001). Furthermore, the size of the cellulitis lesion was significantly smaller in groups that received CpG-ODN((2007)) by the subcutaneous route (P < 0.01). A dose of as little as 3.16 micro g of CpG-ODN((2007)), delivered 3 days prior to challenge by either the subcutaneous or intramuscular route, significantly protected birds against E. coli infection (P < 0.01). This study demonstrates that CpG-ODN((2007)) has both local and systemic protective effects in broiler chickens. This is the first time that CpG-ODN((2007)) has been demonstrated to have an immunoprotective effect against an extracellular bacterial infection in any food animal species.

Bovine and ovine blood mononuclear leukocytes differ markedly in innate immune responses induced by Class A and Class B CpG-oligodeoxynucleotide.

Cytosine-phosphate-guanosine (CpG)-DNA can induce an impressive array of innate immune responses that may directly or indirectly contribute to the clearance of infectious agents. Assays, such as lymphocyte proliferative responses, have been used to demonstrate that the immunostimulatory activity of CpG-DNA is conserved among a broad range of vertebrate species, but no studies have been completed to determine if qualitative differences exist among species for CpG-oligodeoxynucleotide (ODN)-induced innate immune responses. In this study, we assessed the capacity of a Class A (ODN 2216) and a Class B (ODN 2007) CpG-ODN to induce innate immune responses in two closely related species, ovine (n = 28) and bovine (n = 29). The secretion of interferon (IFN)-alpha and IFN-gamma and non-major histocompatability complex (MHC)-restricted cytotoxic activity were assayed with CpG-ODN-stimulated peripheral blood mononuclear cells (PBMC). These investigations revealed significant interspecies and intraspecies variation in the responses. As expected, ODN 2216 was a potent inducer of IFN-alpha secretion by both bovine and ovine PBMC, but ODN 2007 also induced dose-dependent, CpG-specific IFN-alpha secretion by ovine PBMC. In contrast, a significant dose-dependent, CpG-specific IFN-gamma secretion response was only observed following ODN 2216 stimulation of bovine PBMC. Furthermore, both ODN 2216 and ODN 2007 induced CpG-specific non-MHC-restricted cytotoxicity with ovine but not bovine PBMC. Finally, there was not a single assay in which PBMC from all sheep or cattle responded at a detectable level. A striking aspect of these results is that such marked differences in CpG-ODN induced innate responses existed both between and within two closely related species.

Th1/Th2 biasing effects of vaccination in cattle as determined by real-time PCR.

Real-time polymerase chain reaction (PCR) is now becoming an accepted tool for measuring gene expression at the transcriptional level. In this study, a direct comparison between real-time PCR, enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunosorbent spot (ELISPOT) assay was performed. When interferon-gamma (IFN-gamma) gene expression was assessed, both ELISA and ELISPOT data strongly correlated to results obtained by real-time PCR. Real-time PCR was subsequently used to measure bovine IFN-gamma (bIFN-gamma) and bovine interleukin-4 (bIL-4) gene expression by antigen stimulated peripheral blood mononuclear cells (PBMC), isolated from bovine herpesvirus-1 (BHV-1) infected animals. BHV-1-infected animals were either non-vaccinated or vaccinated using one of two adjuvants prior to infection. With non-vaccinated infected animals, a Th1 bias occurred, based on IFN-gamma expression exceeding IL-4 expression. The level of cytokine expression, and the IFN-gamma/IL-4 ratio could be significantly affected, depending on the manner in which animals were vaccinated.