Herein, we present novel quantitative loop-mediated isothermal amplification (qLAMP) and reverse-transcription qLAMP (RT-qLAMP) assays for the detection of five viruses commonly implicated with the onset and progression of bovine respiratory disease (BRD): Bovine Alphaherpesvirus Type 1 (BHV-1), Bovine Adenovirus Type 3 (BAV-3), Bovine Respiratory Syncytial Virus (BRSV), Bovine Viral Diarrhea Virus Type 1 (BVDV-1), and Bovine Parainfluenza Virus Type 3 (BPIV-3). Using contrived samples spiked with whole viruses, we found that our extraction-free assays have limits of detection between 30 and 1,057 copies per reaction (1.8% final sample concentration) with minimal sample processing. Using dual-tipped swabs and 1.4 mL resuspension volumes, these limits of detection are on the order of 2 × 10 5 copies per swab for BAV-3 and BHV-1 and between 6.31 × 10 6 to 8.22 × 10 6 copies per swab in the case of BPIV-3, BRSV, and BVDV-1. Analytical sensitivities ranged from 73 - 100% and analytical specificities ranged from 90 - 100%. Additionally, we introduced a streamlined pipeline to minimize the experimental workload to design, screen, select, and characterize LAMP performance for developing assays. The assays targeting these BRD viruses can be utilized to develop colorimetric LAMP assays that enable the sensitive and specific detection of these viruses' chute side to aid in diagnosing and treating BRD. The associated development pipeline enables more rapid development of LAMP-based diagnostic tools targeting emerging pathogens.

The dairy industry in India is emerging as one of the key agribusiness sectors, significantly contributing to the national economy. However, despite its high significance, the dairy business is vulnerable to various diseases. The bovine respiratory syncytial virus (BRSV) is the most common viral infection affecting the respiratory system, especially in calves. The association of BRSV with the development of pneumonia is well-established globally; however, only limited research has been conducted on its prevalence and associated pathology in India. This study aims to investigate the pathology and prevalence of BRSV among bovine calves under one year of age. Thirty calves necropsied from June 1st, 2021, to June 30th, 2022, were included in the research to investigate BRSV pathology using cytological, gross, histopathological, and immunohistochemical examinations. A total of 228 samples were collected for BRSV detection, comprising 30 tissue samples from necropsy, 130 samples from clinically ill calves (121 nasal and 9 ocular swabs), and 68 nasal swabs from apparently healthy calves. In cases where BRSV was detected, the main gross findings during necropsy were cranioventral consolidation, rubbery and non-collapsing lungs having rib impressions. Histopathologically, necrotising bronchiolitis and bronchitis, thickening of the interalveolar septa, and multiple numbers of syncytial cells in the alveolar lumen, along with the infiltration of inflammatory cells, mainly lymphocytes, were evident. The cytological examination revealed multi-nucleated cells along with the infiltration of inflammatory cells, mainly lymphocytes. Immunohistochemical examination revealed the presence of viral antigen in syncytial cells along with cells from the bronchial and bronchiolar epithelium, including inflammatory cells, predominantly macrophages. The prevalence of BRSV was found to be 50% (15/30; 95% CI: 33.2-66.8) in necropsied samples and 23.73% (47/198; 95% CI: 18.0-30.3) in clinical samples tested by nested RT-PCR targeting the nucleoprotein (N) gene having an amplicon size of 435bp. This study concludes that BRSV causes significant morbidity and mortality in bovine calves and requires timely diagnosis and management to prevent heavy economic losses.

Development of multiplex reverse-transcription digital PCR assay for co-detection of bovine leukemia virus and bovine viral diarrhea virus in cattle.

Polyanhydride particle-based vaccines overcome several limitations of current vaccines owing to their ability to encapsulate different types of antigenic payloads, provide tunable release kinetics of payloads, and induce protective immunity against multiple respiratory infections. In this work, two particle synthesis methods were compared by analyzing the structure and antigenicity of released proteins from particles made by these methods. Flash nanoprecipitation is a lab-scale method to synthesize protein-loaded particles. Spray drying is a scalable method that allows for the production of protein-loaded particles. The polyanhydride copolymer used for both synthesis methods was composed of a 20:80 M ratio of 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane and 1,6-bis(p-carboxyphenoxy)hexane. The three proteins used in this work are bovine serum albumin (a model, globular protein), and SARS-CoV-2 spike and bovine RSV post-F protein (both clinically relevant proteins). The release kinetics of the encapsulated proteins were studied, and the structure of the released proteins was analyzed using gel electrophoresis and fluorescence spectroscopy. The antigenicity of the released spike and post-F was estimated based on the binding of positive mouse sera from previously inoculated mice. Our results indicate that both flash nanoprecipitation and spray drying resulted in particle formulations that provided a burst release of protein followed by a sustained period of release. The primary and tertiary structures and the antigenicity of the released proteins were maintained consistently across both methods. Altogether, these studies indicate that spray drying can be used to generate particles that stabilize encapsulated antigens and for at-scale particle synthesis in the future.

Abstract Our objective was to evaluate the effects of late-gestation chromium (CR) supplementation of beef cows on the physiology of the offspring. Twenty-four multiparous Brangus cows [6.3 ± 2.45 yr of age; body condition score (BCS) = 5.5 ± 0.88; body weight (BW) = 603 ± 65.8 kg] were assigned to 1 of 2 treatments (3 cows/pasture; 4 pasture/treatment): 1) control (no CR) or 2) CR. The CR propionate (KemTRACE Chromium 0.4%, Kemin Industries) was added to the free-choice mineral supplement (mineral target intake of 113 g/head/d) at a rate of 1.6% (CR target intake of 6.3 mg/cow/d) and was offered from d -94 to calving (d 0). After calving, all animals were allocated to a single pasture and received the control mineral until weaning (d 214). On d 0, before treatment application, calves were vaccinated against infectious bovine rhinotracheitis (IBR), parainfluenza-3 (PI3) virus, bovine viral diarrhea virus type 1 (BVDV-1) and 2 (BVDV-1), bovine respiratory syncytial virus (BRSV), and Mannheimia haemolytica. Blood samples were collected from cows on d -94, -29, 81, and 214 and calves on d 0, 81 and 214. The free-choice mineral intake was similar between control and CR treatments (60.6 and 55.8 ± 8.49 g/cow/d; P = 0.71), and the CR intake was approximately 3.11 mg/cow/day. On cows, the CR supplementation tended to decrease (P = 0.09) the concentration of serum aspartate aminotransferase (AST) and reduced (P = 0.02) the concentration of alanine aminotransferase (ALT) on d 81. No other effects were detected (P ³ 0.15) for other serum biochemistry variables evaluated on cows. Maternal CR supplementation reduced (P = 0.03) the concentration of ALT and increased the serum titer concentration of BVDV-I in calves. No other effects were detected (P ³ 0.17) for other serum biochemistry variables and serum titer concentration against other viruses on offspring. Our findings suggest maternal chromium supplementation during late gestation reduces serum AST and ALT in cows and reduces serum ALT, and increases the response of vaccination of offspring.

Background/Objectives: A new multivalent vaccine (DIVENCE® PENTA), containing Bovine viral diarrhoea virus (BVDV) types 1 and 2 recombinant proteins, live gE/tk double gene deleted Bovine Herpesvirus type 1 (BoHV-1 or IBR), live attenuated Bovine respiratory syncytial virus (BRSV) and inactivated parainfluenza-3 virus (PI-3) has been designed to protect cattle against the main viral pathogens associated with Bovine respiratory disease (BRD). The aim of this study was to demonstrate the efficacy of DIVENCE® PENTA against experimental infections with BVDV-1, BVDV-2, IBR, BRSV and PI-3 in young calves. Methods: Ten-week-old calves were given two intramuscular doses three weeks apart. The efficacy was evaluated by means of an experimental challenge three weeks after vaccination. Serology, clinical signs, rectal temperature, white blood cell count, viral shedding and lung lesions were monitored after the challenge. Results/Conclusions: The results demonstrated a significant sparing of BRD in calves vaccinated with DIVENCE® PENTA, as evidenced by fewer clinical signs, lower rectal temperatures, reduced viral shedding and less severe pulmonary lesions compared to control animals. A significant reduction in hyperthermia, leukopenia and viraemia post-challenge was also observed, highlighting the efficacy of the multivalent vaccine against BVDV types 1 and 2, IBR, BRSV and PI-3 in young calves.

This study investigated the possible occurrence of pulmonary disease in beef cattle from 13 municipalities within the State of Mato Grosso (MT), Brazil. The state of MT is a leading player in beef cattle production in Brazil, but with comparatively few data relative to the occurrence of pulmonary disease or ovine gammaherpesvirus 2 (OvGHV2)-related infections in cattle. Pulmonary samples from 44 beef cattle, with ages ranging between 18 and 28 months, were collected during slaughter and processed to determine the patterns of pulmonary lesions. Additionally, duplicate fragments were used in immunohistochemical (IHC) assays designed to detect malignant catarrhal fever (MCFV) antigens and in molecular assays to amplify 10 agents associated with the development of bovine respiratory disease (BRD). Interstitial pneumonia (IP) was diagnosed in most of the lungs (98%; 43/44) evaluated from all municipalities. MCFV antigens were detected in 37% (16/43) of the animals with IP. Only four pathogens were amplified by molecular assays within the lungs of cattle with IP: OvGHV2 (23%; 10/43), bovine viral diarrhea virus (12%; 5/43), bovine coronavirus (7%; 3/43), and Mannheimia haemolytica (2%; 1/43). The nucleic acids of bovine respiratory syncytial virus, bovine alphaherpesvirus 1, bovine parainfluenza virus 3, Pasteurella multocida, Histophilus somni, and Mycoplasmopsis (Mycoplasma) bovis were not amplified. Singular infections in cattle from municipalities were associated with MCFV (n = 3) and OvGHV2 (n = 2), while OvGHV2 occurred in all dual and triple infections. However, in four animals with IP, none of the disease pathogens identified were detected. Statistically, MCFV antigens were more frequently (p = 0.048) detected in the lungs of female (75%; 12/16) cattle with IP relative to males (25%; 4/16). Additionally, there was a positive correlation (p < 0.001) between the IHC detection of MCFV antigens within the bronchial epithelium and the epithelium of peribronchial glands of cattle with IP. This is the first study to statistically demonstrate that female cattle are at greater risk of developing MCFV-related infections as compared to male animals. The detection of OvGHV2 in singular and multiple infections during this investigation supports earlier studies that associate this pathogen with the development of pulmonary disease in cattle, indicating that OvGHV2 can contribute to the etiology of IP. Additionally, the detection of OvGHV2-induced infections in asymptomatic cattle suggests that all animals were subclinically infected, confirming that subclinically OvGHV2-induced infections may be widespread in ruminants from Brazil. Furthermore, the occurrence of atypical interstitial pneumonia cannot be discarded, particularly in animals with IP but without any associated pathogen. These initial findings suggest the need for a more elaborate investigation to understand the dynamics of pulmonary disease within this state.

ABSTRACTBovine respiratory disease complex (BRDC), a multifactorial syndrome driven by viral and bacterial co‐infections, poses significant challenges to cattle health in northern China. We performed a large‐scale epidemiological and phylogenetic investigation (2022–2024) to identify BRDC pathogens in the region. A total of 5052 samples from symptomatic Holstein calves (nasal swabs, sera, tissues) were analysed using virological, bacteriological and molecular methods. Viral pathogens—bovine viral diarrhoea virus (BVDV), bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus‐3 (BPIV‐3) and bovine herpesvirus‐1 (BHV‐1)—dominated infections, with pronounced seasonal peaks in colder months. The prevalence of non‐cytopathic BVDV highlighted clinical difficulties. Bacterial infections involved Mycoplasma bovis (M. bovis), Mannheimia haemolytica and Pasteurella multocida, with M. bovis prevalent in mixed infections. Phylogenetic analysis revealed biological connections between Chinese isolates and international strains (e.g., BVDV subgenotypes 1a/1d and BPIV‐3c), showing global pathogen flow. Transmission electron microscopy revealed the presence of viral popular structure. Meanwhile, seasonality strongly influenced viral dynamics, while bacterial detection remained stable, involving environmental and management factors. Pathogen co‐infections demonstrate the complexity of BRDC. This study provides the first comprehensive BRDC pathogen profile for northern China, emphasising the need for region‐specific control strategies, including multipathogen vaccines and improved biosecurity.

Human and bovine respiratory syncytial virus (RSV) are significant causes of morbidity and mortality in human and cattle populations worldwide, respectively. RSV disease is characterized by deleterious inflammatory immune responses as well as generation of radical oxygen species in the airways. Recent reports have shown antiviral and anti-inflammatory activity of NRF2 agonists and immunometabolite derivatives 4-octyl-itaconate (4-OI) and dimethyl fumarate (DMF), suggesting their potential to protect against viral-induced inflammation. Here, we evaluated whether 4-OI or DMF impact human and bovine RSV replication and its associated inflammatory response in vitro and the efficacy of these NRF2 agonists in preventing RSV disease in a murine model. We observed that 4-OI and DMF inhibited the early inflammatory response to RSV as well as reduced infectious titers in epithelial cells. Moreover, mice treated with 4-OI or DMF were partially protected against RSV-induced weight loss and airway inflammation and showed reduced viral loads and interleukin-6 levels in the lung. Overall, these results support the use of NRF2 agonists 4-OI and DMF in the prevention of RSV disease in target populations.

During disease, there may be increased local demands for zinc (Zn) and vitamin A to support pathogen response. This study evaluates the effects of intranasal Zn and vitamin A treatments on steers experimentally infected with bovine respiratory disease (BRD) pathogens, bovine respiratory syncytial virus (BRSV) and Mannheimia haemolytica, hypothesizing that steers treated with Zn and vitamin A (VA) will have improved recovery to BRD challenge. Forty-eight Angus crossbred steers (333 ± 4.2 kg) were utilized in two groups with identical challenge timelines. The day prior to challenge (d −1), steers were shipped for 6 hours. On d 0, steers were administered an aerosol inoculation with ~104 TCID50/mL BRSV strain 375 followed by an intratracheal inoculation with M. haemolytica (1.42 × 108 CFU strain D153, serotype A1) on d 5. On d 4, steers received intranasal treatments: zinc (IN ZN; 50 mg Zn oxide nanoparticles), vitamin A (IN VA; 200,000 IU as retinyl palmitate), a combination of zinc (50 mg) and vitamin A (200,000 IU; IN VA + ZN) or no treatment (CON). Statistics were analyzed using the Mixed procedure of SAS 9.4 (Cary, NC) and contrast statements were utilized to determine the effects of Zn, VA and intranasal treatment. Disease challenge resulted in mostly mild, subclinical signs of disease. There was an interaction for plasma VA (TRT × Day P < 0.01) where VA treated steers (IN VA and IN VA + ZN) had sustained plasma VA concentrations on d 5, when ZN and CON had decreased plasma VA. After challenge (d 19), liver VA concentrations were increased in IN VA (IN VA P = 0.03) and IN ZN (IN ZN P = 0.05) treated steers. Zn treated steers (ZN and ZN + VA) tended to have increased gene expression of matrix metalloproteinase 9 (P = 0.06) on d 5 and cellular retinol binding protein 1 (P = 0.08) on d 7 in cells collected from nasopharyngeal swabs. Additionally, immune cell populations from bronchoalveolar lavage were altered with increased CD11b expression on neutrophils (IN VA P = 0.01) and CD11c on macrophages (IN ZN P = 0.08) on d 7. During a mild disease challenge, intranasal Zn and VA treatments impacted lung inflammatory environment and nutritional immunity, suggesting potential benefits in mild or deficient nutritional statuses.

Bovine respiratory disease complex (BRDC) is one of the primary causes of morbidity, mortality, and economic loss in cattle worldwide. Accurate and rapid identification of causative pathogenic agents is essential for effective disease management and control. In this study, a novel multiplex fluorescence-based quantitative polymerase chain reaction (qPCR) assay was developed for the simultaneous detection of eight major pathogens associated with BRDC. The targeted pathogens included the following: bovine viral diarrhea virus (BVDV), bovine parainfluenza virus type 3 (BPIV3), bovine respiratory syncytial virus (BRSV), bovine coronavirus (BcoV), Mycoplasma bovis (M.bovis), Pasteurella multocida (PM), Mannheimia haemolytica (MH), and infectious bovine rhinotracheitis virus (IBRV). The assay was rigorously optimized to ensure high specificity with no cross-reactivity among targets. The limit of detection (LOD) was determined to be as low as 5 copies per reaction for all target pathogens. The coefficient of variation (CVs) for both intra-assay and inter-assay measurements were consistently below 2%, demonstrating excellent reproducibility. To validate the clinical utility of the assay, a total of 1012 field samples were tested, including 504 nasal swabs from Farm A and 508 from Farm B in Jiangsu Province. BVDV, BcoV, PM, and MH were detected from Farm A, with a BVDV-positive rate of 21.63% (109/504), BcoV-positive rate of 26.79% (135/504), PM-positive rate of 28.77% (145/504), and MH-positive rate of 15.08% (76/504). Also, BcoV, PM, MH, and IBRV were detected from Farm B, with a BcoV-positive rate of 2.36% (12/508), PM-positive rate of 1.38% (7/508), MH-positive rate of 14.76% (75/508), and IBRV-positive rate of 5.51% (28/508). Notably, a significant proportion of samples showed evidence of mixed infections, underscoring the complexity of BRDC etiology and the importance of a multiplex diagnostic approach. In conclusion, the developed multiplex qPCR assay provides a reliable, rapid, and cost-effective tool for simultaneous detection of multiple BRDC-associated pathogens, which will hold great promise for enhancing disease surveillance, early diagnosis, and targeted intervention strategies, ultimately contributing to improved BRDC management and cattle health outcomes.

Background/Objectives: This study aimed to determine the changes in BVDV (bovine viral diarrhea virus), BoHV-1 (bovine herpesvirus-1), and BRSV (bovine respiratory syncytial virus) antibody levels until weaning in calves who ingested colostrum from vaccinated dairy cattle. Additionally, it aimed to measure the antibody levels induced by the vaccine administered before and after socialization after weaning. Methods: Exposure to respiratory viral and bacterial agents was monitored by PCR analysis using nasal swabs at regular intervals from birth to weaning (pre-colostral and after the 2nd, 7th, 15th, 25th, 35th, 45th, 55th, and 65th days). The levels of colostral BVDV, BoHV-1, and BRSV antibodies were monitored using an enzyme-linked immunosorbent assay (ELISA) at the same intervals from birth to weaning (pre-colostral and after the 2nd, 7th, 15th, 25th, 35th, 45th, 55th, and 65th days). Results: The highest level of maternal antibodies in the blood was detected on day 7. BoHV-1, BVDV, and BRSV antibody levels decreased steadily until weaning by 69.14%, 38%, and 53%, respectively. Conclusions: Vaccination strategies should be planned by considering the presence of maternally derived antibodies and minimizing stress that may negatively affect vaccine titers, thus maximizing vaccine efficacy in calves.

Little is known regarding the timing of initial establishment of bovine respiratory disease complex (BRDC)-associated bacterial and viral pathogens. The objective was to evaluate the presence of BRDC-associated bacteria and viruses in the upper respiratory tract of newborn calves within the first 24h of life. Commercial beef cows (n = 26) were allocated to indi­vidual pens before calving. A left nasal swab (LNS), right na­sal swab (RNS) and vaginal swab were collected from each cow immediately following parturition (0h). Calf (n = 26) LNS and RNS sampling occurred at 0h, 6h, 12h and 24h post-calv­ing. Samples were submitted to a diagnostic lab for bacterial culture (n = 26 pairs) and real-time PCR (rtPCR; n = 10 pairs) to isolate and identify bacteria and to detect bacterial and viral nucleic acid. While we were unable to isolate bacteria of interest using culture, rtPCR yielded some success. At birth, Histophilus somni (Hs) was detected in 4/30 dam swabs and 1/20 calf swabs, Mannheimia haemolytica (Mh) in 1/30 dam swabs and 1/20 calf swabs, Mycoplasma bovis (Mb) in 4/30 dam swabs and 1/20 calf swabs, Pasteurella multocida (Pm) in 2/30 dam swabs, bovine herpes virus type-1 (BHV1) in 1/30 dam swabs, and bovine respiratory syncytial virus (BRSV) in 1/30 dam swabs. In 6h calf swabs, Hs was detected in 3/20, Mb in 1/20, and BRSV in 1/20. Associations of bacteria and viruses be­tween dam and offspring remain unclear as establishment of the neonatal microbiome appears complex and more sensitive methods may be needed.

ABSTRACTBackgroundBovine Respiratory Disease Complex (BRDC) represents a multifactorial infection that poses a significant threat to animal health, leading to severe and fatal pneumonia outbreaks in large herds.ObjectiveIn this study, the molecular diagnosis of Bovine Parainfluenza Virus Type 3 (BPIV3) and Bovine Respiratory Syncytial Virus (BRSV) was targeted in nasal swab samples collected from cattle with clinical signs of respiratory infections. Furthermore, this study aimed to identify possible environmental and herd management‐related risk factors associated with the occurrence of these infections in herds/farms where BPIV3 and BRSV were detected, using statistical methods.MethodsTherefore, a total of 200 nasal swab samples were randomly collected from 24 different cattle herds with respiratory infection outbreaks, representing 10% of each herd population.ResultsIn the study, which utilized the RT‐PCR technique, viral nucleic acid of BPIV3 was detected in 8% (16/200) of the samples, and BRSV was detected in 11.5% (23/200) of the samples. In addition, logistic regression models incorporating both fixed and multiple variables identified the following risk factors for BRSV infection: in the univariate regression analysis, quarantine status, air quality, the period of disease occurrence and previous occurrence of BRDC in the farm; and in the multivariate analysis, the disease period, herd size, bedding type and qualitative air quality were significant risk factors for the presence of infection in the sampled herds. For BPIV3, univariate regression analysis indicated that animal transport, housing type, ventilation and the duration from infection onset to sampling were risk factors, while in multivariate analysis, age, duration from infection onset to sampling, and air quality were identified as risk factors for infection in the sampled herds.ConclusionA statistical relationship was demonstrated between BRSV and BPIV3 infections in cattle and certain herd, environmental and infection‐related factors, identifying these factors as risk factors contributing to the occurrence of these infections.

The purpose of this study was to determine the feasibility of facility-level wastewater surveillance in the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in skilled nursing facility (SNF) wastewater using three concentration methods, as well as a proof-of-concept for antimicrobial resistance (AR) genes/organisms detection. Wastewater effluent samples were collected from an SNF over an 8-week period. Wastewater was concentrated using electronegative membrane filtration (enMF), polyethylene glycol precipitation, and Nanotrap® magnetic virus particles (NP). Quantification of the genome copy concentration from SARS-CoV-2 and bovine respiratory syncytial virus (BRSV), a SARS-CoV-2 surrogate spiked into all samples, was performed with droplet digital polymerase chain reaction (ddPCR). Wastewater sample aliquots were also enriched in microbiological culture media and screened for organisms with AR phenotypes on selective and differential agars. Multiplex real-time PCR was used to detect a broad array of carbapenem resistance genes. SARS-CoV-2 was detected and quantified from a single enMF-concentrated wastewater sample. The highest concentration of BRSV came from enMF-concentrated samples. Klebsiella, Enterobacter, Citrobacter, and Escherichia coli exhibiting AR phenotypes were successfully detected using culture-dependent approaches. Culture-independent, multiplex PCR indicated that blaKPC was the main carbapenemase gene detected in wastewater samples. Facility-level wastewater surveillance could be a useful strategy for SNFs.

Bovine respiratory syncytial virus (BRSV) is an important pathogen affecting the respiratory health of ruminants and causes pulmonary lesions with occasional systemic involvement. This study aimed to detect and characterize BRSV in susceptible hosts exhibiting clinical signs of respiratory distress and pulmonary lesions in Maharashtra, India. A total of 391 samples (295 lung tissues and 96 nasal swabs), were collected from cattle and buffaloes showing respiratory signs and pulmonary lesions. Nested PCR was employed for the molecular detection of BRSV. Gross and histopathological changes were further analyzed in positive cases. The overall incidence of BRSV was 13.55%, with a higher detection rate in buffaloes (27.73%) compared to cattle (9.09%). Clinically, BRSV-positive cases exhibited nasal discharge, cough, fever, wheezing, and conjunctivitis, consistent with respiratory distress. Gross pathological findings included focal to multifocal consolidation in the cranioventral lung regions and emphysema and edema in the caudal lobes. Histopathological examination revealed bronchiolar epithelial necrosis, hyperplasia, neutrophilic and mononuclear infiltration, and the presence of syncytial cells. Molecular characterization demonstrated high genetic similarity among isolates, indicating a common viral strain circulating in the region. However, nucleotide variations, leading to nonsynonymous amino acid changes, suggested genetic diversity among circulating strains. This study highlights BRSV as a critical respiratory pathogen in cattle and buffaloes, particularly affecting buffaloes with a higher incidence. The findings underscore the need for sustained surveillance, targeted control measures, and vaccine development to mitigate the disease's impact on animal health and productivity. This is the first report of BRSV detection and characterization in Maharashtra, India.

Bovine respiratory syncytial virus (BRSV) infection is a part of the bovine respiratory disease complex. This is one of the most significant problems in both the dairy and beef production sector, inflicting severe economic damage to the industry. BRSV manifests clinically as a respiratory syndrome, affecting both upper and lower respiratory tract, including bronchiolitis with dyspnea and wheezing. It has been shown previously that these symptoms caused by IL-4/IL-13 domination in the immune response are associated with an antibody isotype switch to IgE. Prostaglandin production, such as PGE2 is another factor contributing to the pathogenesis of the disease. In this work we demonstrated the effects of ibuprofen and antiviral fusion protein inhibitor (FPI) separately and combined. We showed the synergistic effect of ibuprofen in combination with FPI on antiviral effects and suppression of PGE2, resulting in improved cytoplasmic toll-like receptor recognition and humoral immune responses mediated by antimicrobial peptide in lungs. We also demonstrated a Th1/Th2 balance shift towards a Th2 response in lungs and mediastinal lymph nodes, favorable to IL-4/IL-13 responses. This shift may explain the factors contributing to higher viral loads and the lack of histopathological improvement with ibuprofen administered without FPI. Additionally, we demonstrated that endocannabinoids may play a crucial role as natural regulators of the inflammation, adaptive immune response, and resolution of the inflammatory process.

The bovine respiratory disease complex poses a significant threat to the cattle industry, necessitating a multifaceted approach to address its occurrence. The syndrome is caused by various pathogens such as bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus type 3 (BPIV3), bovine viral diarrhea virus (BVDV), bovine adenovirus type 3 (BAV3), Mycoplasma bovis (Mb), and infectious bovine rhinotracheitis virus (IBRV). The confluence of these pathogens causes substantial economic losses to the cattle industry. Although preventive and control measures have been implemented, containment of bovine respiratory diseases continues to present a formidable challenge, highlighting the need for innovative diagnostic and intervention strategies. In this study, we designed specific primers targeting six conserved pathogen genes (N of BRSV, M of BPIV3, 5'UTR of BVDV, Hexon of BAV3, oppF of Mb, and gB of IBRV). Subsequently, we established a multiplexed fluorescent real-time quantitative PCR (qPCR) assay for simultaneous detection of these pathogens. The developed method exhibited high specificity and sensitivity, with the lowest detection limits for plasmid DNA standards of BRSV, BPIV3, BVDV, BAV3, Mb, and IBRV being 70.1, 40.4, 15.1, 74.4, 69.6, and 4.99 copies/μL, respectively. The coefficients of variation determined by the assay established in this study were <4%, and the amplification efficiency was 93.84%-111.60%, which showed the reliability and stability of the method. The detection rates for BRSV, BPIV3, BVDV, BAV3, Mb, and IBRV were 7.59% (17/224), 11.61% (26/224), 8.04% (18/224), 22.32% (50/224), 27.23% (61/224), and 8.04% (18/224), respectively. All 224 cows were cases of natural disease. Fifty-six diseased cattle were infected with a mixture of two or more of the six pathogens at a mixed infection rate of 25% (56/224). Therefore, this study successfully developed a highly efficient, rapid, specific, and sensitive multiplex qPCR method to detect major pathogens associated with bovine respiratory diseases. This advancement is expected to significantly influence the future of the cattle industry and serve as a valuable reference for subsequent research in this field.

Background: Bovine respiratory syncytial virus (BRSV) is a major pathogen of the bovine respiratory disease complex and causes regular severe winter outbreaks of respiratory disease in cattle. It is therefore responsible for important economic losses for the farming industry. In this study, the genetic diversity of the circulating BRSV strains in Belgium, which has not been assessed since the end of the 1990s, was investigated. Methods: We analyzed 51 BRSV-positive samples collected from 2015 to 2023. This study is the first report on the circulation of BRSV subgroup VIII in Belgium. Furthermore, co-circulation of subgroups II and III was recorded in the same period. Four commercially available vaccine strains marketed in Belgium were also included in the analysis and they clustered with subgroup II or III. Results: Our findings indicate that different strains of BRSV are circulating in Belgium, including those from subgroups II and VIII, with the subgroup VIII strains being particularly distant from the commercially available vaccine strains. Conclusions: These results highlight the importance of ensuring that the available vaccines efficiently protect against strains from circulating subgroups and assessing the potential circulation of attenuated vaccine strains.

Efficacy of intranasal vaccines containing bovine corona virus or bovine respiratory syncytial virus and parainfluenza virus type 3 in young calves with or without maternally derived antibodies