Limitations of bacterial culture, viral PCR, and tulathromycin susceptibility from upper respiratory tract samples in predicting clinical outcome of tulathromycin control or treatment of bovine respiratory disease in high-risk feeder heifers.

A cross-sectional prospective cohort study including 1026 heifers administered tulathromycin due to high risk of clinical signs of bovine respiratory disease (BRD), measured poor association between BRD clinical outcomes and results of bacterial culture and tulathromycin susceptibility from BRD isolates of deep nasopharyngeal swabs (DNS) and adequate association with viral polymerase chain reaction (PCR) results from nasal swabs. Isolation rates from DNS collected on day-0 and at 1st BRD-treatment respectively were: Mannheimia haemolytica (10.9% & 34.1%); Pasteurella multocida (10.4% & 7.4%); Mycoplasma bovis (1.0% & 36.6%); and Histophilus somni (0.7% & 6.3%). Prevalence of BRD viral nucleic acid on nasal swabs collected exclusively at 1st BRD-treatment were: bovine parainfluenza virus type-3 (bPIV-3) 34.1%; bovine viral diarrhea virus (BVDV) 26.3%; bovine herpes virus type-1 (BHV-1) 10.8%; and bovine respiratory syncytial virus (BRSV) 54.1%. Increased relative risk, at 95% confidence intervals, of 1st BRD-treatment failure was associated with positive viral PCR results: BVDV 1.39 (1.17-1.66), bPIV-3 1.26 (1.06-1.51), BHV-1 1.52 (1.25-1.83), and BRSV 1.35 (1.11-1.63) from nasal swabs collected at 1st BRD-treatment and culture of M. haemolytica 1.23 (1.00-1.51) from DNS collected at day-0. However, in this population of high-risk feeder heifers, the predictive values of susceptible and resistant isolates had inadequate association with BRD clinical outcome. These results indicate, that using tulathromycin susceptibility testing of isolates of M. haemolytica or P. multocida from DNS collected on arrival or at 1st BRD-treatment to evaluate tulathromycin clinical efficacy, is unreliable.

Bovine Respiratory Syncytial Virus (BRSV) Infection Detected in Exhaled Breath Condensate of Dairy Calves by Near-Infrared Aquaphotomics.

Bovine respiratory syncytial virus (BRSV) is a major contributor to respiratory disease in cattle worldwide. Traditionally, BRSV infection is detected based on non-specific clinical signs, followed by reverse transcriptase-polymerase chain reaction (RT-PCR), the results of which can take days to obtain. Near-infrared aquaphotomics evaluation based on biochemical information from biofluids has the potential to support the rapid identification of BRSV infection in the field. This study evaluated NIR spectra (n = 240) of exhaled breath condensate (EBC) from dairy calves (n = 5) undergoing a controlled infection with BRSV. Changes in the organization of the aqueous phase of EBC during the baseline (pre-infection) and infected (post-infection and clinically abnormal) stages were found in the WAMACS (water matrix coordinates) C1, C5, C9, and C11, likely associated with volatile and non-volatile compounds in EBC. The discrimination of these chemical profiles by PCA-LDA models differentiated samples collected during the baseline and infected stages with an accuracy, sensitivity, and specificity >93% in both the calibration and validation. Thus, biochemical changes occurring during BRSV infection can be detected and evaluated with NIR-aquaphotomics in EBC. These findings form the foundation for developing an innovative, non-invasive, and in-field diagnostic tool to identify BRSV infection in cattle.

Isolation, identification, and phylogenetic analysis of subgroup III strain of bovine respiratory syncytial virus contributed to outbreak of acute respiratory disease among cattle in Northeast China.

Bovine respiratory syncytial virus (BRSV) is a clinically important causative agent of acute respiratory diseases in postweaning calves and feedlot cattle and causes numerous economic losses to the cattle industry. In June 2018, an outbreak of an acute respiratory disease occurred among 4- to 10-month-old calves on three intensive beef cattle farms in Heilongjiang Province, Northeast China, with a 27.42% morbidity rate (329/1200) and a > 25% mortality rate (85/329). Using next-generation sequencing, we comprehensively analyzed microbial diversity in the lung samples of the diseased cattle and found that the causative agent of this epidemic outbreak is mainly a bovine orthopneumovirus named BRSV strain DQ. We then isolated and confirmed the virus by RT-PCR and an indirect immunofluorescence assay. Phylogenetic analysis of genes G, F, N, NS1, NS2, and SH of BRSV strain DQ showed that this strain shares the highest genetic similarity with strains USII/S1, 15489, V41, and NY487834 belonging to subgroup III of BRSV. This is the first report of subgroup III strain of BRSV presence in China. Heilongjiang Province is a major cattle-breeding province in China; therefore, it is necessary to test for BRSV in the cattle trade and to conduct region-extended epidemiological surveillance for BRSV in China.

Molecular characterization and comparison of diagnostic methods for bovine respiratory viruses (BPIV-3, BRSV, BVDV, and BoHV-1) in field samples in northwestern Turkey.

The aim of this study was to evaluate the compatibility among virus isolation (VI), ELISA, and PCR for diagnosis of the major viral agents (BPIV-3, BRSV, BVDV, and BoHV-1) responsible for BRD in the field samples. For that purpose, a total of 193 samples (133 nasal swabs and 60 lung tissue samples) from cattle with respiratory signs in northwestern Turkey were examined. For VI, all the samples were inoculated at least 3 blind passages onto MDBK cell culture. In addition, the samples were tested by hemadsorption assay and RT-PCR for BPIV-3; nested RT-PCR for BRSV; immunoperoxidase monolayer assay, antigen-ELISA, and RT-PCR for BVDV; and antigen-ELISA and PCR for BoHV-1. The detected 1 (0.52%) BPIV-3 isolate was found to be in the genotype BPIV-3c. No BRSV isolate could be obtained, while 5 (2.59%) samples were evaluated positive in nested-RT PCR. The presence of BVDV antigen in 10 (5.18%) samples and the BVDV genome in 5 (2.59%) samples were detected, while non-cytopathogenic BVDV isolates were obtained only in 2 (1.04%) samples. The detected BVDV strains fell into the genetic clusters of BVDV-1a, -1f, and -1l. For detection of BoHV-1, although viral isolation and Ag-ELISA results were negative, presence of BoHV-1.1 genome was detected in 2 (1.04%) samples. By the results of VI, ELISA, and PCRs, 10.88% (21/193) of samples were found positive for the evaluated viruses. Depending on the obtained data, combined uses of the diagnostic methods were evaluated to be more reliable for routine diagnosis of bovine respiratory viruses.

Bovine respiratory disease in beef calves supported long transport stress: An epidemiological study and strategies for control and prevention.

BRD is associated with infectious agents, but management and transport-stress are trigger factors. Metaphylactic administration of antimicrobial reduces colonization of respiratory tract by pathogens, but the development of antibiotic-resistance raises public health concerns leading to propose new control strategies. The study analyzed nasopharyngeal swabs of 231 imported cattle, 10% of 49 trucks, transported from France to southern Italy and, through Real-time PCR identified the prevalence of the involved pathogens speculating on strategies to reduce the impact of BRD. The samples were tested by Real-time PCR, for the detection of bovine coronavirus (BCoV), bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus (BPiV), bovine adenovirus (BAdV), Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis. Yates-corrected chi squared, or Fisher's exact test were used to compare both animal-health status and positivity/negativity to pathogens, and the relationship between presence/absence of clinical signs and Real-time PCR-positivity. H. somni and BCoV were the most frequently identified pathogens. In BRD-diagnosed cattle, BAdV was detected in 13.8% (19/138), BRSV in 14.5% (20/138) and BPiV in 4.3% (6/138). Healthy cattle were mostly positive for H. somni (89.2%, 83/93). A statistically significant association was observed between clinical signs and positivity to M. haemolytica (p value = 0.016). Although mass-medication and vaccination are used for BRD control, it still remains a primary health problem. Our results highlight that the nasopharyngeal microbiota could be affected by transport and that strategies to enhance calf immunity for reducing BRD-risk development would be more effective if applied at farm of origin prior to loading.

Sequence and unique phylogeny of G genes of bovine respiratory syncytial viruses circulating in Japan.

Bovine respiratory syncytial virus (BRSV) is an etiologic agent of bovine respiratory disease. The rapid evolutionary rate of BRSV contributes to genetic and antigenic heterogeneity of field strains and causes occasional vaccine failure. We conducted molecular epidemiologic characterization of BRSV circulating in Japan to obtain genetic information for vaccine-based disease control. Phylogenetic analysis of G and F gene sequences revealed that all of the isolated Japanese BRSV strains clustered in the same genetic subgroup, which was distinct from the 9 known groups. We assigned the Japanese group to subgenotype X. The Japanese isolates formed 2 temporal clusters: isolates from 2003 to 2005 clustered in lineage A; isolates from 2017 to 2019 formed lineage B. The alignment of the deduced amino acid sequences of the G gene revealed that the central hydrophobic region responsible for viral antigenicity is conserved in all of the isolates; unique amino acid mutations were found mainly in mucin-like regions. Our results suggest that BRSV has evolved uniquely in Japan to form the new subgenotype X; the antigenic homogeneity of the viruses within this group is inferred.

Microbial diversity involved in the etiology of a bovine respiratory disease outbreak in a dairy calf rearing unit.

The etiological agents involved in a bovine respiratory disease (BRD) outbreak were investigated in a dairy heifer calf rearing unit from southern Brazil. A battery of PCR assays was performed to detect the most common viruses and bacteria associated with BRD, such as bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), bovine alphaherpesvirus 1 (BoHV-1), bovine coronavirus (BCoV), bovine parainfluenza virus 3 (BPIV-3), Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis. Bronchoalveolar lavage fluid (BALF) samples were taken from 21 heifer calves (symptomatic n = 15; asymptomatic n = 6) that, during the occurrence of the BDR outbreak, were aged between 6 and 90 days. At least one microorganism was detected in 85.7 % (18/21) of the BALF samples. Mixed infections were more frequent (72.2 %) than single infections (27.7 %). The interactions between viruses and bacteria were the most common in coinfections (55.5 %). The frequencies of BRD agents were 38.1 % for BRSV, 28.6 % for BVDV, 33.3 % for BCoV, 42.85 % for P. multocida, 33.3 % for M. bovis, and 19 % for H. somni. BoHV-1, BPIV-3, and M. haemolytica were not identified in any of the 21 BALF samples. Considering that BALF and not nasal swabs were analyzed, these results demonstrate the etiological multiplicity that may be involved in BRD outbreaks in dairy calves.

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.

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.

Co-infection of epithelial cells established from the upper and lower bovine respiratory tract with bovine respiratory syncytial virus and bacteria.

Bovine respiratory disease complex is a major disease affecting the global cattle industry. Multiple infections by viruses and bacteria increase disease severity. Previously, we reported that bovine respiratory syncytial virus (BRSV) infection increases adherence of Pasteurella multocida to human respiratory and bovine kidney epithelial cells. To examine the interaction between the virus and bacteria in bovine respiratory cells, we generated respiratory epithelial cell lines from bovine trachea (bTEC), bronchus (bBEC), and lung (bLEC). Although all established cell lines were infected by BRSV and P. multocida susceptibility differed according to site of origin. The cells derived from the lower respiratory tract (bBEC and bLEC) were significantly more susceptible to BRSV than those derived from the upper respiratory tract (bTEC). Pre-infection of bBEC and bLEC with BRSV increased adherence of P. multocida; this was not the case for bTEC. These results indicate that BRSV may reproduce better in the lower respiratory tract and encourage adherence of bacteria. Thus, we identify one possible mechanism underlying severe pneumonia.

Bovine respiratory syncytial virus in experimentally exposed and rechallenged calves; viral shedding related to clinical signs and the potential for transmission.

BACKGROUND: Bovine respiratory syncytial virus (BRSV) is an important respiratory pathogen worldwide, detrimentally affecting the economy and animal welfare. To prevent and control BRSV infection, further knowledge on virus shedding and transmission potential in individual animals is required. This study aimed to detect viral RNA and infective virions during BRSV infection to evaluate duration of the transmission period and correlation with clinical signs of disease. The outcome of BRSV re-exposure on calves, their housing environment and effect of introduction of sentinel calves was also investigated. A live animal experiment including 10 calves was conducted over 61 days. Initially, two calves were inoculated with a non-passaged BRSV field isolate. Two days later, six naïve calves (EG: Exposed group) were introduced for commingling and four weeks later, another two naïve calves (SG: Sentinel group) were introduced. Seven weeks after commingling, EG animals were re-inoculated. Clinical examination was performed daily. Nasal swabs were collected regularly and analysed for viral RNA by RT-ddPCR, while virus isolation was performed in cell culture. BRSV serology was performed with ELISA. RESULTS: All the EG calves seroconverted and showed clinical signs of respiratory disease. Viral RNA was detected from days 1-27 after exposure, while the infective virus was isolated on day 6 and 13. On day 19, all animals were seropositive and virus could not be isolated. Total clinical score for respiratory signs corresponded well with the shedding of viral RNA. The SG animals, introduced 27 days after exposure, remained negative for BRSV RNA and stayed seronegative throughout the study. Inoculation of the EG calves seven weeks after primary infection did not lead to new shedding of viral RNA or clinical signs of disease. CONCLUSION: Viral RNA was detected in nasal swabs from the calves up to four weeks after exposure. The detection and amount of viral RNA corresponded well with the degree of respiratory signs. The calves were shedding infective virions for a considerable shorter period, and naïve calves introduced after four weeks were not infected. Infected calves were protected from reinfection for at least seven weeks. This knowledge is useful to prevent spread of BRSV.

Genetic analysis of bovine respiratory syncytial virus in Croatia.

Bovine respiratory syncytial virus (BRSV) represents an important causative agent of respiratory tract disease in cattle. This study describes the genetic diversity of BRSV strains detected in beef cattle herds in Croatia during four consecutive years, from the end of 2011 to April 2016. Genetic diversity of circulating Croatian strains is reflected in their clustering within three different genetic subgroups. Analysis of representative BRSV G gene sequences revealed that infections in Croatia were caused by BRSV strains belonging to two new subgroups (VII and VIII identified herein for the first time). In 2014-2016, the subgroup VII strains were replaced with BRSV strains clustered in the previously unidentified subgroup VIII. Furthermore, co-circulation of subgroup II and new subgroup VIII strains in Croatia was recorded in the same time period. Sequences of Croatian BRSV strains within subgroups II and VII revealed unique mutations within an essential immunodominant region, demonstrating continuous evolution of viral mechanisms for immune escape.

Bovine respiratory syncytial virus infection enhances Pasteurella multocida adherence on respiratory epithelial cells.

Primary infection with bovine respiratory syncytial virus (BRSV) predisposes cattle to secondary infection with bacteria that cause bovine respiratory disease complex (BRDC). However, the interaction between BRSV and bacteria is unclear. This in vitro study examined the adherence of Pasteurella multocida (PM) to BRSV-infected cells was assessed in colony forming unit assays, by flow cytometry analysis, and by indirect immunofluorescence analysis (IFA) of epithelial cells (A549, HEp-2, and MDBK). An in vitro model based on infection of BRSV-infected epithelial cells revealed that PM adherence to BRSV-infected cells was 2- to 8-fold higher than uninfected cells. This was confirmed by flow cytometry analysis and IFA. Epithelial cell expression of mRNA encoding cytokines and chemokines increased after exposure to PM, but increased further after co-infection with BRSV and PM. BRSV-mediated adherence of PM to epithelial cells may underlie the serious symptoms of BRDC.

Temporary carriage of bovine coronavirus and bovine respiratory syncytial virus by fomites and human nasal mucosa after exposure to infected calves.

BACKGROUND: In order to prevent spread of the endemic pathogens bovine coronavirus (BCoV) and bovine respiratory syncytial virus (BRSV) between herds, knowledge of indirect transmission by personnel and fomites is fundamental. The aims of the study were to determine the duration of viral RNA carriage and the infectivity of viral particles on fomites and human nasal mucosa after exposure to BCoV and BRSV. During two animal infection experiments, swabs were collected from personnel (nasal mucosa) and their clothes, boots and equipment after contact with calves shedding either virus. Viral RNA was quantified by RT-qPCR or droplet digital RT-PCR (RT-ddPCR), and selected samples with high levels of viral RNA were tested by cell culture for infectivity. RESULTS: For BCoV, 46% (n = 80) of the swabs from human nasal mucosa collected 30 min after exposure were positive by RT-qPCR. After two, four and six hours, 15%, 5% and 0% of the swabs were positive, respectively. Infective virions were not detected in mucosal swabs (n = 2). A high viral RNA load was detected on 97% (n = 44) of the fomites 24 h after exposure, and infective virions were detected in two of three swabs. For BRSV, 35% (n = 26) of the human nasal mucosa swabs collected 30 min after exposure, were positive by RT-ddPCR, but none were positive for infective virions. Of the fomites, 89% (n = 38) were positive for BRSV RNA 24 h after exposure, but all were negative for infective viruses. CONCLUSIONS: The results indicate that human nasal mucosa can carry both BCoV and BRSV RNA after exposure to virus shedding calves, but the carriage seems short-lived and the transmission potential is likely limited. High viral loads on contaminates fomites 24 h after exposure to infected animals, and detection of infective BCoV, indicate that contaminated fomites represent a significant risk for indirect transmission between herds.

Genetic characterization of bovine respiratory syncytial virus strains isolated in Italy: evidence for the circulation of new divergent clades.

Bovine respiratory syncytial virus (BRSV) is circulating in cattle in Europe. Although vaccination helps control the disease, its prevalence within and among herds remains high. Previous genetic characterization studies revealed a strict geographic correlation between viral variants; on the other hand, they showed the emergence of new variants in northern Europe. Few studies have described BRSV distribution, and little is known about the genetic features of BRSV strains circulating in Italy. We studied sample-positive tests for BRSV, and sequenced the coding regions of the G and N proteins to determine the presence of divergent variants. Two different sets of sequences were found, including in samples from animals from vaccinated herds. The 2 groups of sequences correspond to 2 time periods and suggest an active role of herd immunity in preventing the spread of infection. Our findings that different strains of BRSV are circulating in Italy and that the virus is evolving rapidly highlight the importance of updating vaccination strategies.

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.

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.

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.

Bovine respiratory syncytial virus and bovine coronavirus antibodies in bulk tank milk - risk factors and spatial analysis.

Bovine respiratory syncytial virus (BRSV) and bovine coronavirus (BCoV) are considered widespread among cattle in Norway and worldwide. This cross-sectional study was conducted based on antibody-ELISA of bulk tank milk (BTM) from 1347 herds in two neighboring counties in western Norway. The study aims were to determine the seroprevalence at herd level, to evaluate risk factors for BRSV and BCoV seropositivity, and to assess how these factors were associated with the spatial distribution of positive herds. The overall prevalence of BRSV and BCoV positive herds in the region was 46.2% and 72.2%, respectively. Isopleth maps of the prevalence risk distribution showed large differences in prevalence risk across the study area, with the highest prevalence in the northern region. Common risk factors of importance for both viruses were herd size, geographic location, and proximity to neighbors. Seropositivity for one virus was associated with increased odds of seropositivity for the other virus. Purchase of livestock was an additional risk factor for BCoV seropositivity, included in the model as in-degree, which was defined as the number of incoming movements from individual herds, through animal purchase, over a period of five years. Local dependence and the contribution of risk factors to this effect were assessed using the residuals from two logistic regression models for each virus. One model contained only the x- and y- coordinates as predictors, the other had all significant predictors included. Spatial clusters of high values of residuals were detected using the normal model of the spatial scan statistic and visualized on maps. Adjusting for the risk factors in the final models had different impact on the spatial clusters for the two viruses: For BRSV the number of clusters was reduced from six to four, for BCoV the number of clusters remained the same, however the log-likelihood ratios changed notably. This indicates that geographical differences in proximity to neighbors, herd size and animal movements explain some of the spatial clusters of BRSV- and BCoV seropositivity, but far from all. The remaining local dependence in the residuals show that the antibody status of one herd is influenced by the antibody status of its neighbors, indicating the importance of indirect transmission and that increased biosecurity routines might be an important preventive strategy.

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.