Bovine Respiratory Syncytial Virus Research Articles

Highly pathogenic Bovine Respiratory Syncytial virus variant in a dairy herd in Italy.

Bovine respiratory syncytial virus (BRSV) is an economically significant pathogen in cattle production worldwide. Usually, it is detected in outbreaks of respiratory disease, most often during the winter period. During the middle of October 2018, a serious outbreak of respiratory disease occurred in a cattle farm comprising about 300 heads located in Central Italy. The herd was affected by a severe flu‐like syndrome unresponsive to any antibiotic treatment. Within 3 weeks, 39 adult animals died, and 12 abortions occurred. Direct and indirect laboratory tests were performed to detect the main pathogens causing the respiratory disease of the affected cattle. The results of laboratory investigations provided evidence of an acute and severe BRSV syndrome characterized by unusual mortality. In order to investigate the molecular underpinnings of this syndrome, phylogenetic analysis of the BRSV strain detected from the outbreak was carried out. The sequence analysis showed that the strain was genetically divergent from BRSV strains previously identified in Italy, as it showed high sequence similarity of more than 97% with strains isolated during a major BRSV epizootic that occurred in Sweden, Norway and Denmark during 2010–2011. The infection of the herd in Italy with this BRSV strain was likely due to the introduction of animals imported into Italy from abroad.

Bovine Respiratory Syncytial Virus Decreased Pasteurella multocida Adherence by Downregulating the Expression of Intercellular Adhesion Molecule-1 on the Surface of Upper Respiratory Epithelial Cells

The synergistic infection of bovine respiratory syncytial virus (BRSV) and Pasteurella multocida (PM) may predispose cattle to develop severe pneumonia. Previously, we reported that BRSV infection significantly decreased PM adherence to the upper respiratory epithelial cells. It may allow bacteria to invade into the lower respiratory tract and lead to severe pneumonia. To investigate whether BRSV infection regulates the cell surface adherence receptor on bovine trachea epithelial cells (bTECs), we performed proteomic and functional analyses. BRSV infection decreased the expression of intercellular adhesion molecule-1 (ICAM1) on bTECs. Inhibition and knockdown experiments using anti-ICAM1 antibody and siRNAs targeting ICAM1 indicated that PM adherence to bTECs was dependent on ICAM1 expression. These data suggest that under normal conditions bTECs may capture PM in the upper respiratory tract, while BRSV infection reverses this mechanism. The proposed gateway function of bTECs is disrupted by BRSV infection that may facilitate bacterial invasion into the lower respiratory tract and lead to secondary or more severe respiratory infection.

The Effect of Co-Administration of Inactivated Vaccines Against Bovine Respiratory Disease and Neonatal Calf Diarrhoea

Abstract A pilot study was performed to evaluate the safety and serological responses after co-administration of two multivalent inactivated vaccines to pregnant cattle. One vaccine was directed against bovine respiratory disease (BRD) and contained antigens of bovine respiratory syncytial virus (BRSV), parainfluenza 3 virus (PI3) and Mannheimia haemolytica (Mh). The second vaccine targeted neonatal calf diarrhoea (NCD) and was composed of inactivated antigens of bovine rotavirus (BRV), bovine coronavirus (BCV) and E. coli. The use of these combinations have been used more and more by veterinary practitioners as there exist some clear evidence that both vaccines improves the passive protection via the colostrum for the relevant pathogens. However, up until now, no safety or efficacy data has been available concerning such co-administrations. The safety of both vaccines and the serological responses to the BRD vaccine has been evaluated when used at the same time, but without mixing and compared to the responses to the administration of each vaccine independently. There was no evidence of any negative effect on calving or calf health in any of the vaccinated animals. The antibody levels against BRSV and Mh in the sera of the calves from cows vaccinated with both vaccines were not significantly different from the levels in the sera of calves vaccinated with the BRD vaccine alone. The results from this pilot study demonstrated that the co-administration of the two multivalent inactivated vaccines had no detrimental effect on the safety or serological responses to the BRD vaccine compared to the independent use of the vaccines.

A Single Shot Pre-fusion-Stabilized Bovine RSV F Vaccine is Safe and Effective in Newborn Calves with Maternally Derived Antibodies.

Achieving safe and protective vaccination against respiratory syncytial virus (RSV) in infants and in calves has proven a challenging task. The design of recombinant antigens with a conformation close to their native form in virus particles is a major breakthrough. We compared two subunit vaccines, the bovine RSV (BRSV) pre-fusion F (preF) alone or with nanorings formed by the RSV nucleoprotein (preF+N). PreF and N proteins are potent antigenic targets for neutralizing antibodies and T cell responses, respectively. To tackle the challenges of neonatal immunization, three groups of six one-month-old calves with maternally derived serum antibodies (MDA) to BRSV received a single intramuscular injection of PreF, preF+N with MontanideTM ISA61 VG (ISA61) as adjuvant or only ISA61 (control). One month later, all calves were challenged with BRSV and monitored for virus replication in the upper respiratory tract and for clinical signs of disease over one week, and then post-mortem examinations of their lungs were performed. Both preF and preF+N vaccines afforded safe, clinical, and virological protection against BRSV, with little difference between the two subunit vaccines. Analysis of immune parameters pointed to neutralizing antibodies and antibodies to preF as being significant correlates of protection. Thus, a single shot vaccination with preF appears sufficient to reduce the burden of BRSV disease in calves with MDA.

Dynamics of neutralizing antibodies against Bovine respiratory syncytial virus in a dairy herd from Santa Fe Province, Argentina

Bovine respiratory syncytial virus (BRSV) is one of the most relevant agents responsible for respiratory disease in cattle from both dairy and beef farms. BRSV is spread by horizontal contact causing a constant presence of seropositive animals that favors viral circulation throughout the year. Moreover, reinfections with BRSV are frequent between animals regardless of their age as BRSV does not confer long-lasting protective immunity. Several studies have demonstrated the circulation of BRSV in cattle from different regions of the world; however, little is known about the dynamics of BRSV infection in cows before and after they begin lactation. The aim of this work was to study the dynamics of BRSV neutralizing antibodies from birth up to 36 months of age in a closed dairy herd of Argentina specifically around the lactation period. Passive maternal antibodies against BRSV started to decrease monthly and became almost undetectable at 8 months of age. We detected two potential infection points at months 11 and 27 after birth, in which 30% and 45% of the animals showed seroconversion, respectively. Specifically, an increase in the proportion of seropositive cows after the start of lactation suggests that they became reinfected around the time they began lactating. We demonstrate the importance of understanding BRSV dynamics in a closed dairy herd to review the vaccination schedule of the animals to achieve protection against BRSV infection.

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.

Effects of Ibuprofen and Fusion Protein Inhibitor (FPI) on neutrophil extracellular traps (NETs) in the lung tissue of calves experimentally infected with Bovine Respiratory Syncytial Virus (BRSV)

Abstract Neutrophil extracellular traps is a mode of neutrophil death targeting extracellular pathogens. They are composed of a complex of processed chromatin bound to granular and cytoplasmic proteins expelled from the cell onto pathogens. Although NETs play a role in trapping and killing extracellular pathogens they may be injurious and are considered to play a role in a wide array of inflammatory diseases. The exact contribution of NETs to clearance of pathogens versus inflammatory injury is poorly understood. Bovine respiratory syncytial virus (BRSV), like RSV, causes severe respiratory disease. In this study, we quantified NETs in lung tissue of calves experimentally infected with BRSV after treating with the anti-inflammatory drug Ibuprofen and/or an antiviral drug (FPI). Twenty four calves were infected with BRSV, placed into 6 treatment groups and treated with ibuprofen, FPI or both. Calves were euthanized on day 10 post infection and the lungs evaluated for pathological changes that were correlated with the IHC data. Cryosections of lung tissue were stained with primary antibodies [Rabbit anti citrinated histones(citH3), Rabbit anti neutrophil elastase (NE) and counter stained with secondary antibodies conjugated to Alexa 594 or 488. Slides were examined using an EVOS fluorescence microscope with at least 10 fields from each lung section at ×400 magnification evaluated. NETs were analyzed and quantitated using Materials Image Processing and Automated Reconstruction (MIPAR™) with the total NETs score calculated as the average of NETs in the 10 fields. Results show that a combination of Ibuprofen and FPI treatment from day 3 following experimental infection had a significant reduction in clinical score and average NETs in lung tissue.

Safety, immunogenicity, and protective efficacy of a recombinant vaccine against RSV: Evidence from murine and bovine models

Abstract The human Respiratory Syncytial virus (RSV) is a leading cause of lower respiratory tract infection in infants worldwide. Despite its health burden, there are no licensed vaccines to prevent human RSV-caused disease. In mice, immunization with a single subcutaneous dose of 3 × 105 CFUs of a recombinant BCG expressing hRSV N protein (rBCG-N-hRSV) is safe, and induces robust, long-lived humoral and cellular Th1/Th17 immunity, which prevents lung damage after an intranasal hRSV challenge. Vaccinated mice display reduced weight loss, less neutrophil airway infiltration, and reduced lung viral loads, when compared to unimmunized controls. To further explore the safety, immunogenicity and efficacy of this formulation, we carried out a vaccination and challenge study on a bovine RSV - neonatal calf infection model. Five day old calves received a subcutaneous dose of the rBCG-N-hRSV vaccine and a booster vaccination after 14 days. On day 28, calves were inoculated via aerosol challenge with ~104 TCID50/mL of BRSV strain 375. Clinical data was registered daily until euthanasia on day 7 post infection. Virus-specific IgA and neutralization activity increased in vaccinated calves at 7dpi, and the animals mounted a robust virus-specific IFN-γ/IL-17 response observed after PBMC antigen recall assays. Following bRSV challenge, we observed reduced clinical disease compared to unvaccinated control calves, without signs of vaccine-enhanced disease. Reduction trends in viral shedding and lung viral load were observed in vaccinated animals, as well as a significantly reduced neutrophil infiltration in BAL. These results suggest that the rBCG-N-hRSV vaccine would be safe for neonatal use and support further investigation in clinical studies.

Immunogenic potency of engineered exosomes for prevention of respiratory syncytial virus

Abstract Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract disease in infants and young children. Bovine RSV is a common cause of respiratory disease in young cattle and is a useful model to study RSV infection. There is currently no vaccine for RSV treatment in humans, and the vaccines available for BRSV have limited efficacy in the field. Exosomes (30–150nm) are cell-derived extracellular vesicles that play critical roles in cellular communication by carrying various molecules. Since exosomes secreted by virus or tumor infected cells can deliver cell-specific antigens and proteins to immune cells, immunogenic exosomes have emerged as a new vaccine platform for prevention of viral infections or therapy for cancer treatment. Here, dendritic-cell derived exosomes were engineered to present peptides from the RSV M, NS and L proteins, purified from PDMS microfluidic systems and were evaluated as an efficacious surface engineered antigenic exosome vaccine against RSV and BRSV in mice. The immunogenicity of the exosomes was confirmed in vitro by measuring their capacity to induce antigen-specific T cell clonal proliferation and IFNγ production. The in vivo immunogenicity and efficacy of the engineered exosomes was investigated by immunizing animals subcutaneously with escalating doses of engineered exosomes and measuring antigen-specific T cell responses in the spleen and lungs, and assessing morbidity and mortality. Collectively, dendritic-cell derived surface engineered antigenic exosomes have the capacity to elicit antigen-specific CD8 T cell activation in vitro and in vivo, and the potential to be developed as a vaccine platform to prevent RSV and other viral infections in humans and animals.

Ursolic acid moderates Th17-mediated immunity in respiratory syncytial virus infections of young cattle

Abstract Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract disease in infants and young children. Bovine RSV is a common cause of respiratory disease in young cattle with high genetic and immunological similarity to human RSV, making it a relevant clinical model. While RSV is highly prevalent in both humans and cattle, the disease pathogenesis remains poorly understood and therapeutic options are limited. Th17-mediated immunity is intrinsically linked to RSV infections and characterized by the production of the inflammatory cytokine IL-17A; however, excessive IL-17 production may contribute to immunopathology by exacerbating neutrophil recruitment and activation in the lungs. Herein, we sought to elucidate the role of Th17-mediated immune responses by inhibiting Th17 cell differentiation with ursolic acid (UA), a small molecule agonist of the RORγt transcription factor. Calves were prophylactically treated with UA before being challenged with aerosolized bovine RSV. Challenged calves were monitored for clinical disease, gross and microscopic lung pathology, and immunologic responses in the periphery and respiratory tract. Local and systemic neutrophils and monocytes were analyzed for phagocytic and oxidative burst functions. Cellular adaptive immune responses were measured in the peripheral blood and airways by antigen recall assays. Our results suggest that UA-treatment may result in reduced RSV-associated immunopathology by shifting the immune response from Th17-mediated immunity to a more productive Th1-mediated immune response.

Detection of bovine viral diarrhea virus genotype 1 in aerosol by a real time RT-PCR assay

BackgroundAs a pestivirus of the Flaviviridae family, bovine viral diarrhea virus (BVDV), has imposed a large burden on animal husbandry worldwide, and such virus can be transmitted mainly through direct contact with other infected animals and probably via aerosols. In the present study, we aimed to develop a real-time RT-PCR method for detection of BVDV-1 in aerosol samples.MethodsA pair of primers specific for highly conserved regions of the BVDV-1 5′-UTR was designed. The standard curve and sensitivity of the developed assay were assessed based on 10-fold serial dilutions of RNA molecular standard. The specificity of the assay was evaluated with other pestiviruses and infectious bovine viruses. The clinical performance was examined by testing 169 aerosol samples.ResultsThe results showed that a good linear relationship existed between the standard curve and the concentration of template. The lowest detection limit was 5.2 RNA molecules per reaction. This assay was specific for detection of BVDV-1, and no amplification was found for other pestiviruses such as classical swine fever virus (CSFV), border disease virus (BDV), and common infectious bovine viruses, including BVDV-2, infectious bovine rhinotracheitis virus (IBRV), bovine parainfluenza virus type 3 (BPIV-3), bovine respiratory syncytial virus (BRSV), bovine ephemeral fever virus (BEFV) and bovine coronavirus (BcoV). The assay was highly reproducible with low variation coefficient values (CVs) for intra-assay and inter-assay. A total of 169 aerosol samples collected from six dairy herds were tested using this method. The results showed that the positive detection rate of BVDV-1 was 17.2% (29/169), which was significantly higher compared with the conventional RT-PCR. Additionally, the positive samples (n = 29) detected by real-time RT-PCR were verified by BVDV RPA-LFD, and a concordance rate of 100% was obtained between them.ConclusionsTaken together, we developed a real-time RT-PCR assay for quantitative analysis of BVDV-1 in aerosol samples, and our finding provided valuable insights into the risk on aerosol transmission of BVDV-1.

Using Biosecurity Measures to Combat Respiratory Disease in Cattle: The Norwegian Control Program for Bovine Respiratory Syncytial Virus and Bovine Coronavirus.

Bovine respiratory disease (BRD) cause important health problems in all cattle husbandry systems. It contributes substantially to the use of antimicrobial substances and compromises animal welfare and the sustainability of the cattle industry. The existing preventive measures of BRD focus at the individual animal or herd level and include vaccination, mass treatment with antimicrobials and improvement of the animal's environment and general health status. Despite progress in our understanding of disease mechanism and technological development, the current preventive measures are not sufficiently effective. Thus, there is a need for alternative, sustainable strategies to combat the disease. Some of the primary infectious agents in the BRD complex are viruses that are easily transmitted between herds such as bovine respiratory syncytial virus (BRSV) and bovine coronavirus (BCoV). This conceptual analysis presents arguments for combatting BRD through improved external biosecurity in the cattle herds. As an example of a population-based approach to the control of BRD, the Norwegian BRSV/BCoV control-program is presented. The program is voluntary and launched by the national cattle industry. The core principle is classification of herds based on antibody testing and subsequent prevention of virus-introduction through improved biosecurity measures. Measures include external herd biosecurity barriers and regulations in the organization of animal trade to reduce direct and indirect transmission of virus. Improved biosecurity in a large proportion of herds will lead to a considerable effect at the population level. Positive herds are believed to gain freedom by time if new introduction is avoided. Vaccination is not used as part of the program. Dissemination of information to producers and veterinarians is essential. We believe that reducing the incidence of BRD in cattle is essential and will lead to reduced antimicrobial usage while at the same time improving animal health, welfare and production. Alternative approaches to the traditional control measures are needed.

Immunofluorescence and molecular diagnosis of bovine respiratory syncytial virus and bovine parainfluenza virus in the naturally infected young cattle and buffaloes from India

Pneumonia in bovines is a multifactorial disease manifestation leading to heavy economic losses. Infections of bovine respiratory syncytial virus (BRSV) and bovine parainfluenza virus-3 (BPI-3) are among the important contributing factors for the development of pneumonia in young animals. These viral agents either primarily cause pneumonia or predispose animals to the development of pneumonia. Although, the role of BRSV and BPI-3 in the pathogenesis of pneumonia is well established, there are no reports of involvement of BRSV and BPI-3 from Indian cattle and buffaloes suffering from pneumonia. In the present investigation, we performed postmortem examinations of 406 cattle and buffaloes, which were below twelve months of age. Out of 406 cases, twelve (2.95%) cases were positive for BRSV and fifteen (3.69%) cases were positive for BPI-3, screened by reverse transcriptase polymerase chain reaction (RT-PCR). Further, positive cases were confirmed by sequence analysis of RT-PCR amplicons and direct immunofluorescence antibody test (d-FAT) in paraffin-embedded lung tissue sections. BRSV positive cases revealed characteristic findings of bronchiolar epithelial necrosis, thickened alveolar septa by mononuclear cells infiltration and edema; alveolar lumens were filled with mononuclear cells and numerous syncytial cells were seen having intracytoplasmic inclusions. The BRSV antigen distribution was found to be in bronchiolar and alveolar epithelium and syncytial cells in the lung sections. In fifteen cases, where BPI-3 was detected, bronchointerstitial pneumonia in the majority of cases with thickened alveolar septa by mild macrophage infiltration, hyperplasia of type-II pneumocytes and bronchiolar necrosis along with syncytial cells having intracytoplasmic inclusions in the majority of cases were observed. The BPI-3 antigen distribution was found to be in bronchiolar and alveolar epithelium and syncytial cells in the lung sections. RT-PCR amplicons of BRSV and BPI-3 obtained were sequenced and their analysis showed homology with already available sequences in the NCBI database. It is the first report of detection of BRSV and BPI-3 from pneumonic cases by RT-PCR and d-FAT from cattle and buffaloes of India, indicating the need for more epidemiological studies.

Demonstration of Protection Provided by a Single Dose Modified Live BRSV Vaccine against Virulent BRSV Challenge

Bovine respiratory syncytial virus (BRSV) is an economically important cause of respiratory disease in cattle worldwide. The virus is an important respiratory pathogen in naïve cattle of all ages, but as with other respiratory syncytial viruses, it primarily affects young calves. The virus has also been implicated as one of the viral agents involved in the inducing phase of bovine respiratory disease complex. Efficacy of vaccines developed to protect against BRSV related respiratory disease must be demonstrated in models that exhibit clinically relevant respiratory disease in susceptible cattle. The model used in this trial involves experimental infection with a virulent field isolate of BRSV, resulting in induction of severe clinical disease and associated pneumonic lesions.

A randomized controlled trial of a combination of antiviral and nonsteroidal anti-inflammatory treatment in a bovine model of respiratory syncytial virus infection.

Bovine respiratory syncytial virus (RSV) is a valid model for human RSV and an important bovine pathogen. Very early administration of ibuprofen and GS-561937, a fusion protein inhibitor (FPI), have separately been shown to decrease the severity of bovine RSV. Our aims were to determine how long after RSV inoculation ibuprofen and GS-561937 can be administered with clinical benefit and whether using both was better than monotherapy. We conducted a blinded randomized placebo controlled trial of ibuprofen, GS-561937 (FPI), or combinations of the two initiated at 3 or 5 days after artificial infection with bovine RSV in 36 five to six-week-old Holstein calves (Bos taurus). We measured clinical scores, respiratory rate, and viral shedding daily for 10 days following inoculation. We estimated the average effect for each drug and compared treatment arms using mixed effects models. We found a significant decrease in clinical scores only in the combined treatment arms. This benefit was greater when treatment was initiated at 3 days rather than 5 days post infection with decreased clinical scores and lower respiratory rates at both time points. Ibuprofen alone started on day 3 increased, and FPI with ibuprofen started on day 3 decreased, viral shedding. Dual therapy with Ibuprofen and FPI, on average, decrease clinical severity of illness in a bovine model of RSV when started at 3 and 5 days after infection.

Determination of serum amyloid A, haptoglobin and hepcidin levels in calves with endemic viral pneumonia

The aim of this study was to investigate serum amyloid A, haptoglobin and hepcidin levels in respiratory infections caused by various viral agents in calves. Pneumonia is an infectious or non-infectious disease affecting the lungs of all animals, and during the inflammation, blood levels of acute phase proteins increase rapidly. Hepcidin is a hormone that has many functions and is known as type II acute phase reactant. The blood samples were collected from a total of 20 calves to form the study group. The control group consisted of 10 healthy animals. Sera were analyzed with ELISA for antigen detection. According to the results, Bovine herpesvirus type-1 (BHV-1) was positive in 3 calves, Bovine viral diarrhea virus (BVDV) was positive in 3 calves, Bovine respiratory syncytial virus (BRSV) was positive in 1 calf, and Bovine parainfluenza virus type 3 (BPI3) was positive in 1 calf. Adenovirus infection was detected in 5 animals together with other viruses. Mixed infections were observed in 12 calves. Haptoglobin, serum amyloid A and hepcidin values were measured in sera. According to the results, statistically significant differences were observed between the two groups for the haptoglobin (P<0.01), serum amyloid A (P<0.05) and hepcidin (P<0.05). It was concluded that these parameters can be used to support the diagnosis of respiratory infections of the calves.

Pathology and immunohistochemical evaluation of lungs of cattle slaughtered at metropolitan abattoirs in Nigeria and Ghana

ABSTRACTThere was a dearth of information on pathology and causal agents of bovine pneumonia in West Africa. This cross-sectional study conducted at four major metropolitan abattoirs in Nigeria and Ghana was to evaluate the pathology and to immunohistochemically demonstrates viral and bacterial pathogens of bovine pneumonia in West Africa. Out of the 20,605 cattle lungs examined at post-mortem using standard inspection procedures, 136 samples grossly showed pneumonic lesions and 99 randomly selected lung samples were fixed in 10% neutral buffered formalin for histopathological and immunohistochemical examination. The overall prevalence of pneumonia was 0.66%, with 0.72% prevalence in Ibadan, Nigeria and 9.68% prevalence in Ghana. Age and breed were observed to be among the predisposing factors to pneumonia in cattle. Histologically, bronchopneumonia (0.65%), broncho-interstitial pneumonia (0.13%), and interstitial pneumonia (0.08%) were the prominent type of pneumonias observed. Immunohistochemically, 0.8% was positive for bovine PI-3, 0.9% for bovine RSV, 1.0% for Mannheimia haemolytica (MH), and 0.6% for Pasteurella multocida (PM). There were a few interactions of pathogens: PI3 and MH (0.01%), RSV and MH (0.01%), PM and MH (0.02%). This was the first study that immunohistochemically demonstrated bacterial and viral antigens in naturally occurring pneumonia in cattle in Nigeria and Ghana.

Systemic and local immune responses of weaned beef calves vaccinated post-transportation and at the time of a mild respiratory tract infection

To examine the effects of transport stress and concur-rent respiratory infection on bovine vaccine responses, 75 previously weaned beef calves were randomly assigned to 1 of 3 treatments (n=25/group). Group 1 calves were not transported, but were vaccinated (NTV). Both Groups 2 (vaccinated TV) and 3 (not vaccinated TUV) were transported for 12 h. Twelve h after transport, calves in NTV and TV groups were vaccinated intranasally with modified-live bovine respiratory syncytial virus (BRSV), bovine herpesvirus −1 (BHV-1), and parainfluenza virus type 3 (PI3V), and subcutaneously with modified-live bovine viral diarrhea virus (BVDV) types 1 and 2 vaccine with Mannheimia hemolytica (Mh) leukotoxoid vaccine. Nasal secretions and serum were collected pre- and post-vaccination for measurement of nasal interferon alpha, beta, and gamma, IgA to BHV-1 and BRSV, and serum neutralizing (SN) titers to BHV-1, BRSV, and BVDV types 1 and 2.
 At vaccination some calves had nasal discharge and fever. Pre-vaccination nasal swabs, tested for respiratory viruses, were negative. During the 21-d study, 6 calves developed BRD and eventually recovered. BHV-1 and BVDV 1 and 2 SN titers were significantly higher in vaccinated than nonvaccinated calves on d 14 and 21. BVDV2 titers were significantly higher in TV than NTV. Vaccination stimulated systemic, but not mucosal, antibody responses. Cattle can mount a humoral response to vaccination in spite of transport and mild respiratory disease.

Evaluation of novel multiplex qPCR assays for diagnosis of pathogens associated with the bovine respiratory disease complex

Bovine respiratory disease complex is the most common disease requiring the use of antimicrobials in industrial calf production worldwide. Pathogenic bacteria (Mannheimia haemolytica (Mh), Pasteurella multocida (Pm), Histophilus somni (Hs), and Mycoplasma bovis) and a range of viruses (bovine respiratory syncytial virus, bovine coronavirus, bovine parainfluenza virus type 3, bovine viral diarrhea virus and bovine herpesvirus type 1) are associated with this complex. As most of these pathogens can be present in healthy and diseased calves, simple detection of their presence in diseased calves carries low predictive value. In other multi-agent diseases of livestock, quantification of pathogens has added substantially to the predictive value of microbiological diagnosis. The aim of this study was to evaluate the ability of two recently developed quantitative PCR (qPCR) kits (Pneumo4B and Pneumo4V) to detect and quantify these bacterial and viral pathogens, respectively.Test efficiencies of the qPCR assays, based on nucleic acid dilution series of target bacteria and viruses, were 93–106% and 91–104%, respectively, with assay detection limits of 10–50 copies of nucleic acids. All 44 strains of target bacteria were correctly identified, with no false positive reactions in 135strains of non-target bacterial species. Based on standard curves of log10 CFU versus cycle threshold (Ct) values, quantification was possible over a 5-log range of bacteria. In 92 tracheal aspirate samples, the kappa values for agreement between Pneumo4B and bacterial culture were 0.64-0.84 for Mh, Pm and Hs. In an additional 84 tracheal aspirates, agreement between Pneumo4B or Pneumo 4V and certified diagnostic qPCR assays was moderate (0.57) for M. bovis and high (0.71–0.90) for viral pathogens. Thus Pneumo4 kits specifically detected and quantified the relevant pathogens.

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.