Infection of calves with in-vivo passaged bovine parainfluenza-3 virus, alone or in combination with bovine respiratory syncytial virus and bovine coronavirus.

Bovine respiratory disease complex is etiologically complex and usually involves co-infection by several agents, including bovine parainfluenza virus-3 (BPIV-3), bovine respiratory syncytial virus (BRSV), and bovine coronavirus (BCoV). Traditionally, vaccines have been tested in seronegative calves infected with a single in vitro-passaged agent, often with little disease, resulting in unvaccinated subjects. To overcome the potential problem of attenuation coincident with in vitro culture of the viruses, cocktails of field isolates of BPIV-3s and BCoVs were passaged in the lungs of neonatal colostrum-deprived calves. Lung lavage fluids were used as inocula, alone and in combination with in-vivo passaged BRSV, and aerosolized into a trailer containing conventionally reared 9-week-old weaned Holstein calves with decayed, but still measurable, maternal antibodies. Calves developed acute respiratory disease of variable severity. Upon necropsy, there were characteristic gross and histologic lesions in the respiratory tract, associated immunohistochemically with BPIV-3, BRSV, and BCoV. In-vivo passage of viruses is an alternative to in vitro culture to produce inocula to better study the pathogenesis of infection and more rigorously and relevantly assess vaccine efficacy.

Le complexe des maladies respiratoires bovines possède une étiologie complexe et implique habituellement une co-infection par plusieurs agents, incluant le virus parainfluenza bovin 3 (BPIV-3), le virus respiratoire syncitial bovin (BRSV) et le coronavirus bovin (BCoV). Traditionnellement, les vaccins ont été testés chez des veaux séronégatifs infectés avec un seul agent cultivé in vitro, présentant souvent peu de maladie, résultant en des sujets non-vaccinés. Afin de contrecarrer le problème potentiel d'atténuation associé à la culture in vitro des virus, des cocktails d'isolats de champs de BPIV-3 et de BCoV furent passés dans des poumons de veaux nouveau-nés privés de colostrum. Les liquides de lavage pulmonaire furent utilisés comme inoculum, seul et en combinaison avec des BRSV passés in vivo, et aérosolisés dans une remorque contenant des veaux Holstein sevrés élevés de manière conventionnelle âgés de 9 semaines ayant des anticorps maternels en déclin mais toujours mesurables. Les veaux ont développé une maladie respiratoire aiguë de sévérité variable. Lors de la nécropsie, il y avait des lésions macroscopiques et histologiques caractéristiques dans le tractus respiratoire, associées immuno-histochimiquement avec BPIV-3, BRSV et BCoV. Le passage in vivo de virus est une alternative à la culture in vitro afin de produire un inoculum permettant de mieux étudier la pathogénie de l'infection et d'évaluer plus rigoureusement et plus pertinemment l'efficacité de vaccins.(Traduit par Docteur Serge Messier).

Pathogen-specific risk factors in acute outbreaks of respiratory disease in calves.

Respiratory tract infections (bovine respiratory disease) are a major concern in calf rearing. The objective of this study was to identify pathogen-specific risk factors associated with epidemic respiratory disease in calves. A cross-sectional study was conducted, involving 128 outbreaks (29 dairy, 58 dairy-mixed, and 41 beef) in Belgium (2016-2018). A semiquantitative PCR for 7 respiratory pathogens was done on a pooled nonendoscopic bronchoalveolar lavage sample for each herd. Potential risk factors were collected by questionnaire and derived from the national cattle registration databank. Most outbreaks occurred between October and March, and single and multiple viral infections were detected in 58.6% (75/128) and 13.3% (17/128), respectively. Bovine coronavirus (BCV) was the most frequently isolated virus (38.4%), followed by bovine respiratory syncytial virus (bRSV; 29.4%) and parainfluenzavirus type 3 (PI-3; 8.1%). Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni were detected in 33.3, 41.2, 89.1, and 36.4% of the herds, respectively. Specific risk factors for BCV detection were detection of M. haemolytica [odds ratio (OR) = 2.8 (95% confidence interval = 1.1-7.5)], increasing herd size [OR = 1.3 (1.0-1.8) for each increase with 100 animals] and detection of BCV by antigen ELISA on feces in calves in the last year [OR = 3.6 (1.2-11.1)]. A seasonal effect was shown for bRSV only {more in winter compared with autumn [OR = 10.3 (2.8-37.5)]}. Other factors associated with bRSV were PI-3 detection [OR = 13.4 (2.1-86.0)], prevalence of calves with respiratory disease [OR = 1.02 (1.00-1.04) per 1% increase], and number of days with respiratory signs before sampling [OR = 0.99 (0.98-0.99) per day increase]. Next to its association with BCV, M. haemolytica was more frequently detected in herds with 5 to 10 animals per pen [OR = 8.0 (1.4-46.9)] compared with <5 animals, and in herds with sawdust as bedding [OR = 18.3 (1.8-191.6)]. Also, for H. somni, housing on sawdust was a risk factor [OR = 5.2 (1.2-23.0)]. Purchase of cattle [OR = 2.9 (1.0-8.0)] and housing of recently purchased animals in the same airspace [OR = 5.0 (1.5-16.5)] were risk factors for M. bovis. This study identified pathogen-specific risk factors that might be useful for the development of customized control and prevention and for the design of decision support tools to justify antimicrobial use by predicting the most likely pathogen before sampling results are available.

Triplex qRT-PCR with specific probe for synchronously detecting Bovine parvovirus, bovine coronavirus, bovine parainfluenza virus and its applications.

Bovine parvovirus (BPV), bovine coronavirus (BCoV) and bovine parainfluenza virus (BPIV) are common etiologies causing gastrointestinal and respiratory diseases in dairy herds. However, there are few reports on the synchronous detection of BPV, BCoV and BPIV. The present article aimed to develop a quick and accurate RT-PCR assay to synchronously detect BPV, BCoV and BPIV based on their specific probes. One pair universal primers, one pair specific primers and one specific probe was designed and synthesized. After the concentrations of primer and probe and annealing temperature were strictly optimized, the specificity, sensitivity and repeatability of the established triplex probe qRT-PCR were evaluated, respectively. The results showed the recombinant plasmids of pMD18-T-BPV, pMD18-T-BCoV and pMD18-T-BPIV were 554bp, 699bp and 704bp, respectively. The optimal annealing temperature was set at 45.0°C for triplex qRT-PCR. The triplex probe qRT-PCR can only synchronously detect BPV, BCoV and BPIV. Detection sensitivities were 2.0×102, 2.0×102 and 2.0×101 copies/µL for BPV, BCoV and BPIV, being 1000-fold greater than that in the conventional PCR. Detection of clinical samples demonstrated that triplex probe qRT-PCR had a higher sensitivity and specificity. The intra-assay and inter-assay coefficient of variation were lower than 2.0%. Clinical specimens verified that the triplex qRT-PCR had a higher sensitivity and specificity than universal PCR. In conclusion, this triplex probe qRT-PCR could detect only BPV, BCoV and BPIV. Minimum detection limits were 2.0×102 copies/µL for BPV and BCoV, and 2.0×101 copies/µL for BPIV. The sensitivity of this triplex probe qRT-PCR was 1000-fold greater than that in the conventional PCR. The newly qRT-PCR could be used to monitor or differentially diagnose virus infection.

Mycoplasma bovis and viral agents associated with the development of bovine respiratory disease in adult dairy cows.

The etiology and pathologic findings of bovine respiratory disease (BRD) in adult dairy cows (n = 35) from a commercial dairy herd in Southern Brazil were investigated. Pulmonary samples were examined for histopathologic patterns and specific features within these patterns, while immunohistochemical (IHC) assays were designed to detect the intralesional antigens of viral infectious disease agents and Mycoplasma bovis. Pneumonia was diagnosed in 91.4% (32/35) of these cases; neither pneumonia nor any of the infectious disease pathogens evaluated occurred in three cows. The presence of multiple respiratory pathogens in 75% (24/32) of these cases indicated the complex origin of pneumonia in cattle. Interstitial pneumonia, necrosuppurative bronchopneumonia and suppurative bronchopneumonia were the principal patterns of pulmonary disease identified by histopathology. The most frequent pathogens identified by IHC were bovine viral diarrhea virus (BVDV; n = 18), M. bovis (n = 16) and bovine alphaherpesvirus type 1 (BoHV-1; n = 14), followed by bovine respiratory syncytial virus (BRSV; n = 11) and bovine parainfluenza virus type 3 (BPIV-3; n = 5). Obliterative bronchiolitis and peribronchial lymphocytic cuffings were the characteristic histopathologic features associated with M. bovis. Necrohemorrhagic bronchitis with bronchial angiogenesis was associated with BoHV-1. Necrotizing bronchitis and bronchiolitis were associated with BVDV, BoHV-1 and BRSV. Ballooning degeneration of the bronchial and bronchiolar epithelia was associated with BRSV and BoHV-1. This is the first report from Brazil that correlated the histopathologic findings of BRD with the associated infectious disease agents by immunohistochemistry. M. bovis was frequently detected in the tissues of cows with fatal pulmonary disease during this study and may be a possible primary disease pathogen associated with the development of BRD in dairy cows. Additionally, the histopathologic features identified within patterns of pulmonary disease during this investigation may be an efficient diagnostic tool to associate histopathologic findings with specific agents of BRD in dairy cows.

Isolation of a Divergent Strain of Bovine Parainfluenza Virus Type 3 (BPIV3) Infecting Cattle in China.

Bovine parainfluenza virus type 3 (BPIV3) is one of the most important known viral respiratory pathogens of both young and adult cattle. It is also named "heat stress in transport", causing morbidity and mass death. New variants of BPIV3 have been detected or isolated in China since 2008. Here, we isolate one BPIV3 strain (named BPIV3 BJ) in Madin-Darby bovine kidney (MDBK) cells from nasal samples collected in China. Phylogenetic analysis showed that our isolate is related to BPIV3 of the genotype A. The comparison of BPIV3-BJ and the reference Chinese isolate NM09 showed that these strains are highly divergent. We found many differences in the amino acid composition in the nucleocapsid (NP) protein among these genotype A strains. Since the NP protein has been implicated in immunization studies, our BPIV3 isolate will be useful for the development of immune assays and vaccine studies. The diversity of BPIV3 lineages that we found in China indicated ongoing evolution for immune escape. Our study highlights the importance of genetic surveillance for determining the effect of BPIV3 variability on pathogen evolution and population-scale immunity.

The first serological report for genotype C bovine parainfluenza 3 virus in ruminant species of mid-northen Turkey: Traces from the past.

Bovine parainfluenza 3 virus (BPI3V)is one of the most important respiratory pathogens and a leading cause of serious respiratory illnesses in cattle, both independent of and in connection with other pathogens involved in the bovine respiratory disease complex (BRDC). In this study, we aimed to identify the historical circulation of genotype C bovine BPI3V (BPI3Vc) in Turkey using the archival serum samples of domestic ruminants that had been collected from six provinces of northern Anatolia in Turkey between 2009-2010. A total of 896 sera from cattle (n=442), sheep (n=330), and goats (n=124) were randomly selected and screened with a virus neutralization test in order to detect antibodies for BPI3Vc. The overall seropositivity rate was 21.09%, with seropositivity rates for cattle, sheep, and goats of 21.04%, 20.00%, and 24.19%, respectively. Neutralizing antibody titers for selected samples ranged between 1/4 to 1/512. This study represents the first serological study conducted using the first BPI3V isolate of Turkey.

Molecular survey of infectious agents associated with bovine respiratory disease in a beef cattle feedlot in southern Brazil.

We investigated the occurrence of infectious pathogens during an outbreak of bovine respiratory disease (BRD) in a beef cattle feedlot in southern Brazil that has a high risk of developing BRD. Nasopharyngeal swabs were randomly collected from steers ( n = 23) and assessed for the presence of infectious agents of BRD by PCR and/or RT-PCR assays. These included: Histophilus somni, Mannheimia haemolytica, Pasteurella multocida, Mycoplasma bovis, bovine respiratory syncytial virus (BRSV), bovine coronavirus (BCoV), bovine viral diarrhea virus (BVDV), bovine alphaherpesvirus 1 (BoHV-1), and bovine parainfluenza virus 3 (BPIV-3). Pulmonary sections of one steer that died with clinical BRD were submitted for pathology and molecular testing. The frequencies of the pathogens identified from the nasopharyngeal swabs were: H. somni 39% (9 of 23), BRSV 35% (8 of 23), BCoV 22% (5 of 23), and M. haemolytica 13% (3 of 23). PCR or RT-PCR assays did not identify P. multocida, M. bovis, BoHV-1, BVDV, or BPIV-3 from the nasopharyngeal swabs. Single and concomitant associations of infectious agents of BRD were identified. Fibrinous bronchopneumonia was diagnosed in one steer that died; samples were positive for H. somni and M. haemolytica by PCR. H. somni, BRSV, and BCoV are important disease pathogens of BRD in feedlot cattle in Brazil, but H. somni and BCoV are probably under-reported.

Identification of candidate protein markers of Bovine Parainfluenza Virus Type 3 infection using an in vitro model.

Bovine Parainfluenza Virus Type 3 (BPI3V) infections are often asymptomatic, causing respiratory tissue damage and immunosuppression, predisposing animals to severe bacterial pneumonia, the leading cause of Bovine Respiratory Disease (BRD) mortality. As with many pathogens, routine BPI3V serology does not indicate the presence of damaged respiratory tissue or active infection. In vitro proteomic marker screening using disease relevant cell models could help identify markers of infection and tissue damage that are also detectable during in vivo infections. This study utilised a proteomic approach to investigate in vitro cellular responses during BPI3V infection to enhance the current understanding of intracellular host-virus interactions and identify putative markers of in vivo infection. Through 2D gel electrophoresis proteomic analysis, BPI3V Phosphoprotein P and host T-complex Protein 1 subunit theta were found to be accumulated at the latter stages of infection within bovine fibroblasts. These proteins were subsequently detected using targeted multiple reaction monitoring (MRM) mass spectrometry in the plasma of animals challenged with BPI3V, with differential protein level profiles observed dependant on animal vaccination status. Potential mechanisms by which BPI3V overcomes host cellular immune response mechanisms allowing for replication and production of viral proteins were also revealed. Assessment of circulating protein marker levels identified through an in vitro approach as described may enable more effective diagnosis of active viral infection and diseased or damaged respiratory tissue in animals and allow for more effective utilisation of preventative therapeutic interventions prior to bacterial disease onset and significantly aid the management and control of BRD.

Genomic and antigenic characterization of bovine parainfluenza-3 viruses in the United States including modified live virus vaccine (MLV) strains and field strains from cattle.

This study investigated the genetic and antigenic characterization of parainfluenza-3 virus (PI3V) of cattle. Using molecular tests including real time PCR and viral genome sequencing, PI3V strains could be separated into PI3V types, including PI3V A, PI3V B, and PI3V C. Isolates from cattle with bovine respiratory disease clinical signs and commercial vaccines in the U.S. with MLV PI3V were typed using these molecular tests. All the MLV vaccine strains tested were PI3V A. In most cases PI3V field strains from calves receiving MLV vaccines were types heterologous to the vaccine type A. Also antigenic differences were noted as PI3V C strains had lower antibody levels than PI3V A in serums from cattle receiving MLV PI3V A vaccines. This study further demonstrates there is genetic variability of U.S. PI3V strains and also antigenic variability. In addition, isolates from cattle with BRD signs and receiving MLV vaccines may have heterologous types to the vaccines, and molecular tests should be performed to differentiate field from vaccine strains. Potentially the efficacy of current PI3V A vaccines should be evaluated with other types such a PI3V B and PI3V C.

The bovine paranasal sinuses: Bacterial flora, epithelial expression of nitric oxide and potential role in the in-herd persistence of respiratory disease pathogens.

The bovine paranasal sinuses are a group of complex cavernous air-filled spaces, lined by respiratory epithelium, the exact function of which is unclear. While lesions affecting these sinuses are occasionally reported in cattle, their microbial flora has not been defined. Furthermore, given that the various bacterial and viral pathogens causing bovine respiratory disease (BRD) persist within herds, we speculated that the paranasal sinuses may serve as a refuge for such infectious agents. The paranasal sinuses of clinically normal cattle (n = 99) and of cattle submitted for post-mortem examination (PME: n = 34) were examined by microbial culture, PCR and serology to include bacterial and viral pathogens typically associated with BRD: Mycoplasma bovis, Histophilus somni, Mannheimia haemolytica and Pasteurella multocida, bovine respiratory syncytial virus (BRSV) and bovine parainfluenza-3 virus (BPIV-3). Overall, the paranasal sinuses were either predominantly sterile or did not contain detectable microbes (83.5%: 94.9% of clinically normal and 50.0% of cattle submitted for PME). Bacteria, including BRD causing pathogens, were identified in relatively small numbers of cattle (<10%). While serology indicated widespread exposure of both clinically normal and cattle submitted for PME to BPIV-3 and BRSV (seroprevalences of 91.6% and 84.7%, respectively), PCR identified BPIV-3 in only one animal. To further explore these findings we investigated the potential role of the antimicrobial molecule nitric oxide (NO) within paranasal sinus epithelium using immunohistochemistry. Expression of the enzyme responsible for NO synthesis, inducible nitric oxide synthase (iNOS), was detected to varying degrees in 76.5% of a sub-sample of animals suggesting production of this compound plays a similar protective role in the bovine sinus as it does in humans.

Serologic survey for antibodies against three genotypes of bovine parainfluenza 3 virus in unvaccinated ungulates in Alabama.

OBJECTIVE To determine titers of serum antibodies against 3 genotypes of bovine parainfluenza 3 virus (BPI3V) in unvaccinated ungulates in Alabama. ANIMALS 62 cattle, goats, and New World camelids from 5 distinct herds and 21 captured white-tailed deer. PROCEDURES Serum samples were obtained from all animals for determination of anti-BPI3V antibody titers, which were measured by virus neutralization assays that used indicator (reference) viruses from each of the 3 BPI3V genotypes (BPI3V-A, BPI3V-B, and BPI3V-C). The reference strains were recent clinical isolates from US cattle. Each sample was assayed in triplicate for each genotype. Animals with a mean antibody titer ≤ 2 for a particular genotype were considered seronegative for that genotype. RESULTS Animals seropositive for antibodies against BPI3V were identified in 2 of 3 groups of cattle and the group of New World camelids. The geometric mean antibody titer against BPI3V-B was significantly greater than that for BPI3V-A and BPI3V-C in all 3 groups. All goats, captive white-tailed deer, and cattle in the third cattle group were seronegative for all 3 genotypes of the virus. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that BPI3V-A may no longer be the predominant genotype circulating among ungulates in Alabama. This may be clinically relevant because BPI3V is frequently involved in the pathogenesis of bovine respiratory disease complex, current vaccines contain antigens against BPI3V-A only, and the extent of cross-protection among antibodies against the various BPI3V genotypes is unknown.

Pathogens, patterns of pneumonia, and epidemiologic risk factors associated with respiratory disease in recently weaned cattle in Ireland.

We examined the pathogens, morphologic patterns, and risk factors associated with bovine respiratory disease (BRD) in 136 recently weaned cattle ("weanlings"), 6-12 mo of age, that were submitted for postmortem examination to regional veterinary laboratories in Ireland. A standardized sampling protocol included routine microbiologic investigations as well as polymerase chain reaction and immunohistochemistry. Lungs with histologic lesions were categorized into 1 of 5 morphologic patterns of pneumonia. Fibrinosuppurative bronchopneumonia (49%) and interstitial pneumonia (48%) were the morphologic patterns recorded most frequently. The various morphologic patterns of pulmonary lesions suggest the involvement of variable combinations of initiating and compounding infectious agents that hindered any simple classification of the etiopathogenesis of the pneumonias. Dual infections were detected in 58% of lungs, with Mannheimia haemolytica and Histophilus somni most frequently recorded in concert. M. haemolytica (43%) was the most frequently detected respiratory pathogen; H. somni was also shown to be frequently implicated in pneumonia in this age group of cattle. Bovine parainfluenza virus 3 (BPIV-3) and Bovine respiratory syncytial virus (16% each) were the viral agents detected most frequently. Potential respiratory pathogens (particularly Pasteurella multocida, BPIV-3, and H. somni) were frequently detected (64%) in lungs that had neither gross nor histologic pulmonary lesions, raising questions regarding their role in the pathogenesis of BRD. The breadth of respiratory pathogens detected in bovine lungs by various detection methods highlights the diagnostic value of parallel analyses in respiratory disease postmortem investigation.

DISEASE COMPLEXITY IN A DECLINING ALASKAN MUSKOX (OVIBOS MOSCHATUS) POPULATION.

The muskox ( Ovibos moschatus ) population inhabiting the eastern North Slope (ENS) of Alaska, US declined dramatically during 1999-2006, whereas populations in western Alaska (WA) were stable or increasing. To understand morbidity and mortality factors contributing to the decline, Alaska Department of Fish and Game conducted pathologic investigations of carcasses from 2005 until 2008. Additionally, archived sera from both ENS and WA muskoxen collected during 1984-92, before the documented beginning of the ENS decline; sera collected during 2000, near the beginning of the decline; and contemporary sera (from live capture-release, adult females) collected during 2006, 2007, and 2008 were analyzed to determine whether prevalence of antibody to potential pathogens differed in the two areas or changed over time. The pathogens investigated were those that were believed could cause lameness or poor reproduction or adversely affect general health. Furthermore, trace mineral levels, hemograms, and gastrointestinal parasites were evaluated in live adult females captured 2006-08. Pathologic investigations identified several comorbid conditions, including predation, polyarthritis caused by or consistent with Chlamydophila spp. infection, hoof lesions, copper deficiency, contagious ecthyma, verminous pneumonia, hepatic lipidosis suggestive of negative energy balance, and bacterial bronchopneumonia due to Trueperella pyogenes and Bibersteinia trehalosi . Pathogens suspected to be newly introduced in the ENS muskox population on the basis of serologic detection include bovine viral diarrhea, respiratory syncytial virus, Chlamydophila spp., Brucella spp., Coxiella burnetii , and Leptospira spp., whereas parainfluenza virus-3 antibody prevalence has increased in the WA population. Although multiple disease syndromes were identified that contributed to mortality and, in combination, likely limited the ENS muskox population, further holistic investigations of disease agents, trace mineral status, and nutritional factors in conjunction with intensive demographic and environmental analyses would provide a better understanding of factors that influence Alaskan muskox populations.

Seroprevalence of respiratory viral pathogens of indigenous calves in Western Kenya.

Most studies of infectious diseases in East African cattle have concentrated on gastro-intestinal parasites and vector-borne diseases. As a result, relatively little is known about viral diseases, except for those that are clinically symptomatic or which affect international trade such as foot and mouth disease, bluetongue and epizootic haemorrhagic disease. Here, we investigate the seroprevalence, distribution and relationship between the viruses involved in respiratory disease, infectious bovine rhinotracheitis virus (IBR), bovine parainfluenza virus Type 3 (PIV3) and bovine viral diarrhoea virus (BVDV) in East African Shorthorn Zebu calves. These viruses contribute to the bovine respiratory disease complex (BRD) which is responsible for major economic losses in cattle from intensive farming systems as a result of pneumonia. We found that calves experience similar risks of infection for IBR, PIV3, and BVDV with a seroprevalence of 20.9%, 20.1% and 19.8% respectively. We confirm that positive associations exist between IBR, PIV3 and BVDV; being seropositive for any one of these three viruses means that an individual is more likely to be seropositive for the other two viruses than expected by chance.

Detection and characterization of viruses as field and vaccine strains in feedlot cattle with bovine respiratory disease.

This study investigated viruses in bovine respiratory disease (BRD) cases in feedlots, including bovine herpesvirus-1 (BoHV-1), bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), bovine coronaviruses (BoCV) and parainfluenza-3 virus (PI3V). Nasal swabs were collected from 114 cattle on initial BRD treatment. Processing included modified live virus (MLV) vaccination. Seven BRD necropsy cases were included for 121 total cases. Mean number of days on feed before first sample was 14.9 days. Swabs and tissue homogenates were tested by gel based PCR (G-PCR), quantitative-PCR (qPCR) and quantitative real time reverse transcriptase PCR (qRT-PCR) and viral culture. There were 87/114 (76.3%) swabs positive for at least one virus by at least one test. All necropsy cases were positive for at least one virus. Of 121 cases, positives included 18/121 (14.9%) BoHV-1; 19/121 (15.7%) BVDV; 76/121 (62.8%) BoCV; 11/121 (9.1%) BRSV; and 10/121 (8.3%) PI3V. For nasal swabs, G-PCR (5 viruses) detected 44/114 (38.6%); q-PCR and qRT-PCR (4 viruses) detected 81/114 (71.6%); and virus isolation detected 40/114 (35.1%). Most were positive for only one or two tests, but not all three tests. Necropsy cases had positives: 5/7 G-PCR, 5/7 q-PCR and qRT-PCR, and all were positive by cell culture. In some cases, G-PCR and both real time PCR were negative for BoHV-1, BVDV, and PI3V in samples positive by culture. PCR did not differentiate field from vaccines strains of BoHV-1, BVDV, and PI3V. However based on sequencing and analysis, field and vaccine strains of culture positive BoHV-1, BoCV, BVDV, and PI3V, 11/18 (61.1%) of BoHV-1 isolates, 6/17 (35.3%) BVDV isolates, and 1/10 (10.0%) PI3V identified as vaccine. BRSV was only identified by PCR testing. Interpretation of laboratory tests is appropriate as molecular based tests and virus isolation cannot separate field from vaccine strains. Additional testing using sequencing appears appropriate for identifying vaccine strains.

Identification and genome characterization of genotype B and genotype C bovine parainfluenza type 3 viruses isolated in the United States.

BACKGROUND: Bovine parainfluenza 3 viruses (BPI3V) are respiratory pathogens of cattle that cause disease singly but are often associated with bovine respiratory disease complex (BRDC) in conjunction with other viral and bacterial agents. Bovine vaccines currently contain BPI3V to provide protection against the virus, but there is no current information regarding the BPI3V strains that are circulating in the U.S. RESULTS: A project was initiated to sequence archival BPI3V isolates to study viral evolution over time. This was done with a deep sequencing protocol that generated sequences of multiple RNA virus genomes simultaneously. Analysis of the BPI3V sequences revealed that, in addition to the genotype A (BPI3Va) viruses previously described in the United States, there were two additional genotypes of BPI3V circulating that had been described only in Australia (BPI3Vb) and Asia (BPI3Vc). The U.S. BPI3Vb and BPI3Vc isolates showed some divergence from the Australian and Asian strains; the BPI3Vb were 93 % similar to the Australian Q5592 strain and the BPI3Vc viruses were 98 % similar to the 12Q061 strain that was described in South Korea. Overall, the three genotypes were 82 to 84 % identical to each other and 80 % identical to the most similar human PI3V. Cross-neutralization studies using an APHIS/NVSL BPI3V reference serum showed that neutralization titers against the genotype B and C viruses were 4- to ≥16-fold less then the titer against the APHIS BPI3Va reference strain, SF-4. CONCLUSIONS: This study clearly demonstrated that BPI3Vb and BPI3Vc strains, previously thought to be foreign to the U.S., are indeed circulating in domestic livestock herds. Based on virus neutralization using polyclonal antisera, there were antigenic differences between viruses from these genotypes and the BPI3Va viruses that are included in currently marketed bovine vaccines. Further study of these viruses is warranted to determine pathogenic potential and cross-protection afforded by vaccination.

Patterns of detection of respiratory viruses in nasal swabs from calves in Ireland: a retrospective study.

A retrospective analysis was conducted to investigate the prevalence and seasonality of bovine viral diarrhoea virus (BVDV), bovine coronavirus (BoCV), bovine herpesvirus-1 (BoHV-1), bovine respiratory syncytical virus (BRSV) and parainfluenza virus-3 (PI3V) in calves (aged three months and below) in Ireland. Results from real-time PCR testing, including cycle threshold values, conducted on nasal swabs (single or pooled) submitted from 1364 respiratory disease outbreaks between January 1, 2008 and December 31, 2012 were included in this study. One or more viruses were detected in 34.6 per cent of submissions, with BoCV detected most frequently (22.9 per cent), followed by BRSV (11.6 per cent), PI3 V (7.0 per cent), BoHV-1 (6.1 per cent) and BVDV (5.0 per cent). The detection rate of all viruses was higher when pooled multiple swabs were submitted from outbreaks rather than single swabs, with these differences being significant for all except BVDV. Two or more viruses were detected in 39.4 per cent of positive submissions, with BoCV and BRSV most commonly present as one of the two partners in detection. With the exception of BVDV, which was detected all year round, the others showed a clear seasonal pattern, being most commonly detected in winter and spring.

Relevamiento serológico de anticuerpos contra enfermedades virales de interés sanitario en llamas (Lama glama) de la provincia de Jujuy, Argentina / Serological survey of antibodies aainst vital diseases of public health interest in llamas (Lama glama) from Jujuy rpovince, Argentina

Las poblaciones de llamas de Argentina se concentran principalmente en la provincia de Jujuy; su explotación representa un importante recurso económico de las comunidades altoandinas. El objetivo de este trabajo fue evaluar la seroprevalencia de anticuerpos contra algunos agentes virales asociados a enfermedades de impacto productivo en rodeos de llamas de Jujuy. Se analizaron 349 sueros de llamas adultas de 6 departamentos de la puna jujeña ubicados por encima de los 3300 msnm. Se obtuvo una prevalencia del 100 % para rotavirus grupo A y del 70 % para el virus parainfluenza-3 bovino, mientras que no se detectaron reactores para herpesvirus bovino 1, virus de la diarrea viral bovina, influenza A humana (H1N1) e influenza equina (H3N8). Los resultados obtenidos confirman la amplia distribución de rotavirus y virus parainfluenza y la baja susceptibilidad a herpesvirus y pestivirus en las tropas de llamas de la puna jujeña

Llama population from Argentina is mainly concentrated in the Andean Puna, Jujuy. Llamas represent an important economic resource for the Andean communities. The aim of this study was to investigate the prevalence of antibodies against viral antigens associated to viral diseases of economic impact (neonatal diarrhea, reproductive and respiratory syndromes). A total of 349 serum samples from adult llamas were analyzed. The obtained antibody prevalence was 100 % for Rotavirus A and 70 % for Bovine parainfluenza virus 3. In contrast, no reactors were detected to Bovine herpesvirus 1, Bovine viral diarrhea virus 1, Human influenza A virus (H1N1) and Equine influenza virus (H3N8). These results confirm the wide circulation of rotavirus and parainfluenza virus in Argentinean llamas and suggest that susceptibility to infection with bovine herpesvirus, pestivirus and influenza A viruses is low. This serologic survey provides novel information regarding the epidemiology of viral diseases affecting llamas from the Argentinean Andean Puna

Molecular characterization of a Korean bovine parainfluenza virus type 3 isolate.

Bovine parainfluenza virus type 3 (BPIV-3) was isolated from Korean native cattle that presented clinical signs of mild pneumonia. The complete genome of a representative isolate (12Q061) was sequenced. The newly identified strain, which was found to be distinct from the previously reported genotypes A (BPIV-3a) and B (BPIV-3b) and closely related to the Chinese strain SD0835, was tentatively classified as genotype C (BPIV-3c). Our results suggest a relationship between BPIV-3 genetic variation and the geographic location of its isolation. Identification of these new BPIV-3 genotypes may facilitate the development of improved diagnostic methods and vaccines. This is to our knowledge the first report of the identification and molecular characterization of BPIV-3 in Korea.