Diversifying selection identified in immune epitopes of bovine coronavirus isolates from Irish cattle.

Bovine betacoronavirus (BoCoV) is a pneumoenteric pathogen of cattle that is closely related to human coronavirus OC43. Vaccines are administered to protect against diseases caused by BoCoV, but knowledge gaps exist with regard to correlates of protection and the effect of immune evasion on driving evolution. In this study, immune epitopes were mapped onto BoCoV structural proteins, including spike and haemagglutinin esterase (HE), and then supported with targeted gene sequencing of Irish clinical isolates and selective pressure analysis. Increased prevalence of diversifying selection and amino acid changes in some mapped immune epitopes suggests that immune escape is selecting for non-synonymous mutations arising in these regions. Selection analysis and sequencing provided increased support for neutralising antibody (nAb) epitopes compared to others, suggesting that nAbs are an important arm of the immune response to BoCoV. Phylogenetic analysis of spike and HE sequences showed that Irish isolates from this study were in the European clade, except for one HE sequence that sat in the Asian/American clade, while the spike gene of this sample was in the European clade. Recombination between a European and an Asian/American isolate would give rise to such a sequence. This study has gathered evidence suggesting that pressure to evade the nAb response is contributing to BoCoV evolution.

Humoral Immune Response in Immunized Sheep with Bovine Coronavirus Glycoproteins Delivered via an Adenoviral Vector.

Bovine coronavirus (BCoV) is distributed globally and mainly causes different clinical manifestations: enteric diarrhea in calves, winter dysentery in adults, and respiratory symptoms in cattle of all ages. Low mortality and high morbidity are the hallmarks of BCoV infection, usually associated with substantial economic losses for the livestock industry. Vaccination, combined with the implementation of biosecurity measures, is the key strategy for the prevention of infections. This pilot study evaluates the immunogenicity of a recombinant vaccine containing two BCoV antigens (S and M) in sheep, compared to vaccines containing only the M or S protein. Three groups of sheep were inoculated intramuscularly at day 0 and day 21 with recombinant adenoviruses expressing BCoV S protein (AdV-BCoV-S), BCoV M protein (AdV-BCoV-M), or both proteins (AdV-BCoV-S + M). Serum antibodies were evaluated using immunofluorescence (IF) and serum neutralization (SN) tests. Moderate seroconversion was observed by day 21, but serum antibodies detected via SN increased from 1:27.5 (day 21) to 1:90 (day 28) in sheep inoculated with the recombinant AdV expressing both the S- and M-BCoV proteins. Based on the SN results, a repeated-measures ANOVA test indicated a more significant difference in immune response between the three groups (F = 20.47; p < 0.001). The experimental investigation produced satisfactory results, highlighting that the S + M recombinant vaccine was immunogenic, stimulating a valid immune response. Despite some inherent limitations, including a small sample size and the absence of challenge tests, the study demonstrated the efficacy of the immune response induced via the recombinant vaccine containing both S and M proteins compared to that induced via the individual proteins S or M.

Novel CRISPR/Cas13-based assay for detection of bovine coronavirus associated with severe diarrhea in calves.

Bovine coronavirus (BCoV) is one of the important causes of diarrhoea in cattle. The virus is responsible for the high fatality rate associated with acute diarrhoea in calves. Rapid and accurate tests need to be conducted to detect the virus and minimise economic losses associated with the disease. Nucleic acid-based detection assays including PCR is an accurate test for detecting pathogens. However, these tests need skilled personnel, time and expensive devices. In this study, we developed a novel assay for the detection of BCoV in clinical cases. This novel assay combined reverse transcription-recombinase polymerase amplification with CRISPR/Cas13 and conducted a rapid visualisation of cleavage activity using a Lateral Flow Device. A conserved sequence of the BCV M gene was used as a target gene and the assays were tested in terms of specificity, sensitivity and time consumption. The result showed the specificity of the assay as 100% with no false positives being detected. Ten copies of the input RNA were enough to detect the virus and perform the assay. It took up to forty minutes for reading the results. Conducted together, the assay should be used as a rapid test to clinically diagnose infectious pathogens including bovine coronavirus. However, the assay needed the RNA to be extracted from the clinical sample in order to detect the virus. Therefore, more studies are needed to optimise the assay to be able to detect the virus in the clinical sample without extracting the RNA.

Evaluation of bovine coronavirus in Korean native calves challenged through different inoculation routes.

Bovine coronavirus (BCoV) is a pneumoenteric virus that can infect the digestive and respiratory tracts of cattle, resulting in economic losses. Despite its significance, information regarding BCoV pathogenesis is limited. Hence, we investigated clinical signs, patterns of viral shedding, changes in antibody abundance, and cytokine/chemokine production in calves inoculated with BCoV via intranasal and oral. Six clinically healthy Korean native calves (< 30 days old), initially negative for BCoV, were divided into intranasal and oral groups and monitored for 15 days post-infection (dpi). BCoV-infected calves exhibited clinical signs such as nasal discharge and diarrhea, starting at 3 dpi and recovering by 12 dpi, with nasal discharge being the most common symptoms. Viral RNA was detected in nasal and fecal samples from all infected calves. Nasal shedding occurred before fecal shedding regardless of the inoculation route; however, fecal shedding persisted longer. Although the number of partitions was very few, viral RNA was identified in the blood of two calves in the oral group at 7 dpi and 9 dpi using digital RT-PCR analysis. The effectiveness of maternal antibodies in preventing viral replication and shedding appeared limited. Our results showed interleukin (IL)-8 as the most common and highly induced chemokine. During BCoV infection, the levels of IL-8, monocyte chemoattractant protein-1, and macrophage inflammatory protein-1ß were significantly affected, suggesting that these emerge as potential and reliable biomarkers for predicting BCoV infection. This study underscores the importance of BCoV as a major pathogen causing diarrhea and respiratory disease.

Brain invasion of bovine coronavirus: molecular analysis of bovine coronavirus infection in calves with severe pneumonia and neurological signs.

IMPORTANCE: Although the role of bovine coronavirus (BCoV) in calf diarrhea and respiratory disorders is well documented, its contribution to neurological diseases is unclear. OBJECTIVE: This study conducted virological investigations of calves showing diarrhea and respiratory and neurological signs. METHODS: An outbreak of diarrhea, respiratory, and neurological disorders occurred among the 12 calves in July 2022 in Istanbul, Türkiye. Two of these calves exhibited neurological signs and died a few days after the appearance of symptoms. One of these calves was necropsied and analyzed using molecular and histopathological tests. RESULTS: BCoV RNA was detected in the brain, lung, spleen, liver, and intestine of the calf that had neurological signs by real-time reverse transcription polymerase chain reaction. Immunostaining was also observed in the intestine and brain. A 622 bp S1 gene product was noted on gel electrophoresis only in the brain. Phylogenetic analysis indicated that the BCoV detected in this study had a high proximity to the BCoV strain GIb with 99.19% nucleotide sequence homology to the strains detected in Poland, Israel, Türkiye, and France. No distinct genetic lineages were observed when the brain isolate was compared with the respiratory and enteric strains reported to GenBank. In addition, the highest identity (98,72%) was obtained with the HECV 4408 and L07748 strains of human coronaviruses. CONCLUSIONS AND RELEVANCE: The strain detected in a calf brain belongs to the GIb-European lineage and shares high sequence homology with BCoV strains detected in Europe and Israel. In addition, the similarity between the human coronaviruses (4408 and L07748) raises questions about the zoonotic potential of the strains detected in this study.

Efficacy of Three Antimicrobials Against two SARS-COV-2 Surrogates, Bovine Coronavirus and Human Coronavirus OC43, on Hard or Soft Nonporous Materials.

The efficacy of three antimicrobials was evaluated against two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surrogates - bovine coronavirus (BCoV) and human coronavirus (HCoV) OC43 - on hard and soft nonporous materials. Three antimicrobials with three different active ingredients (chlorine, hydrogen peroxide, and quaternary ammonium compound + alcohol) were studied. Initially, a neutralization method was optimized for each antimicrobial. Then, we determined their efficacy against BCoV and HCoV OC43 in both suspension and on surfaces made with polyethylene terephthalate (PET) plastic and vinyl upholstery fabric. All tests were conducted under ambient environmental conditions with a soil load of 5% fetal bovine serum. After a 2-min exposure, all three antimicrobials achieved a >3.0 log10 reduction in viral titers in suspension. All three also reduced virus infectivity on both surface materials below the detection limit (0.6 log10 TCID50/carrier). Treatments in which the reduction in virus titer was <3.0 log10 were attributed to a decreased dynamic range on the carrier during drying prior to disinfection. The carrier data revealed that both surrogates were inactivated more rapidly (p <0.05) on vinyl or under conditions of high relative humidity. Three classes of antimicrobials were efficacious against both SARS-CoV-2 surrogate viruses, with BCoV demonstrating slightly less sensitivity compared to HCoV OC43. These findings also illustrate the importance of (1) optimizing the neutralization method and (2) considering relative humidity as a key factor for efficacy testing.

Isolation and Characterization of Contemporary Bovine Coronavirus Strains.

Bovine coronavirus (BCoV) poses a threat to cattle health worldwide, contributing to both respiratory and enteric diseases. However, few contemporary strains have been isolated. In this study, 71 samples (10 nasal and 61 fecal) were collected from one farm in Ohio in 2021 and three farms in Georgia in 2023. They were screened by BCoV-specific real-time reverse transcription-PCR, and 15 BCoV-positive samples were identified. Among them, five BCoV strains from fecal samples were isolated using human rectal tumor-18 (HRT-18) cells. The genomic sequences of five strains were obtained. The phylogenetic analysis illustrated that these new strains clustered with US BCoVs that have been detected since the 1990s. Sequence analyses of the spike proteins of four pairs of BCoVs, with each pair originally collected from the respiratory and enteric sites of one animal, revealed the potential amino acid residue patterns, such as D1180 for all four enteric BCoVs and G1180 for three of four respiratory BCoVs. This project provides new BCoV isolates and sequences and underscores the genetic diversity of BcoVs, the unknown mechanisms of disease types, and the necessity of sustained surveillance and research for BCoVs.

Prevalence and Evolutionary Characteristics of Bovine Coronavirus in China.

Bovine coronavirus (BCoV), bovine rotavirus, bovine viral diarrhea virus, and bovine astrovirus are the most common intestinal pathogenic viruses causing diarrhea in cattle. We collected 1646 bovine fecal samples from January 2020 to August 2023. BCoV was the major pathogen detected, with a positive rate of 34.02% (560/1646). Of the 670 diarrheal samples and 976 asymptomatic samples, 209 and 351 were BCoV-positive, respectively. Studying the relevance of diarrhea associated with BCoV has shown that the onset of diarrheal symptoms post-infection is strongly correlated with the cattle's age and may also be related to the breed. We amplified and sequenced the hemagglutinin esterase (HE), spike protein, and whole genomes of the partially positive samples and obtained six complete HE sequences, seven complete spike sequences, and six whole genomes. Molecular characterization revealed that six strains were branched Chinese strains, Japanese strains, and partial American strains from the GⅡb subgroup. Strains HBSJZ2202 and JSYZ2209 had four amino acid insertions on HE. We also analyzed ORF1a and found disparities across various regions within GIIb, which were positioned on separate branches within the phylogenetic tree. This work provides data for further investigating the epidemiology of BCoV and for understanding and analyzing BCoV distribution and dynamics.

Study on the susceptibility of bovine coronavirus to BALB/c mice.

There are no other bovine coronavirus (BCoV) infection models except calves, which makes efficacy evaluation of vaccines and pathogenic mechanism research of BCoV inconvenient owing to their high value and inconvenient operation. This study aimed to establish a mouse model of BCoV infection. BCoV was used to infect 4-week-old male BALB/c mice and the optimal infection conditions were screened, including the following infection routes: gavage, intraperitoneal injection, and tail vein injection at doses of 1 × 108 TCID50, 2 × 108 TCID50 and 4 × 108 TCID50. Using the optimal infection conditions, BALB/c mice were infected with BCoV, and their body weight, blood routine, inflammatory factors, autopsy, virus distribution, and viral load were measured at 1, 3, 5, and 7 days after infection. The results showed that the optimal conditions for infecting BALB/c mice with BCoV HLJ-325 strain were continuous oral gavage for 3 days with a dose of 4 × 108 TCID50. On the 7th day after infection, there was significant extensive consolidation of the lungs and thinning of the colon wall. Significant inflammation was observed in various organs, especially in the colon and alveoli, where a large number of inflammatory cells infiltrate. Both BCoV Ag and nucleic acid are positive in visceral organs. The viral load in the colon and lungs was significantly higher than that in the other organs (p < 0.001). BCoV-infected mice showed a decreasing trend in body weight starting from day 5, and there was a significant difference compared to the control group on days 6 and 7 (p < 0.001). The total number of white blood cells and lymphocytes began to decrease and was significantly lower than that in the control group 24 h after infection (p < 0.001), and gradually returned to the control level. The cytokine TNF-α, IL-1ß, and IL-6 showed an increasing trend, significantly higher than the control group on day 5 and 7 (p < 0.001). These results indicate that the BCoV HLJ-325 strain can infect BALB/c mice and cause inflammatory reactions and tissue lesions. The most significant effect was observed on the seventh day after infection with a dose of 4 × 108 TCID50 and three consecutive gavages. This study established, for the first time, a BALB/c mouse model of BCoV infection, providing a technical means for evaluating the immune efficacy of BCoV vaccines and studying their pathogenic mechanisms.

The Potential Roles of Host Cell miRNAs in Fine-Tuning Bovine Coronavirus (BCoV) Molecular Pathogenesis, Tissue Tropism, and Immune Regulation.

Bovine coronavirus (BCoV) infection causes significant economic loss to the dairy and beef industries worldwide. BCoV exhibits dual tropism, infecting the respiratory and enteric tracts of cattle. The enteric BCoV isolates could also induce respiratory manifestations under certain circumstances. However, the mechanism of this dual tropism of BCoV infection has not yet been studied well. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and play a dual role in virus infection, mediating virus or modulating host immune regulatory genes through complex virus-host cell interactions. However, their role in BCoV infection remains unclear. This study aims to identify bovine miRNAs crucial for regulating virus-host interaction, influencing tissue tropism, and explore their potential as biomarkers and therapeutic agents against BCoV. We downloaded 18 full-length BCoV genomes (10 enteric and eight respiratory) from GenBank. We applied several bioinformatic tools to study the host miRNAs targeting various regions in the viral genome. We used the criteria of differential targeting between the enteric/respiratory isolates to identify some critical miRNAs as biological markers for BCoV infection. Using various online bioinformatic tools, we also searched for host miRNA target genes involved in BCoV infection, immune evasion, and regulation. Our results show that four bovine miRNAs (miR-2375, miR-193a-3p, miR-12059, and miR-494) potentially target the BCoV spike protein at multiple sites. These miRNAs also regulate the host immune suppressor pathways, which negatively impacts BCoV replication. Furthermore, we found that bta-(miR-2338, miR-6535, miR-2392, and miR-12054) also target the BCoV genome at certain regions but are involved in regulating host immune signal transduction pathways, i.e., type I interferon (IFN) and retinoic acid-inducible gene I (RIG-I) pathways. Moreover, both miR-2338 and miR-2392 also target host transcriptional factors RORA, YY1, and HLF, which are potential diagnostic markers for BCoV infection. Therefore, miR-2338, miR-6535, miR-2392, and miR-12054 have the potential to fine-tune BCoV tropism and immune evasion and enhance viral pathogenesis. Our results indicate that host miRNAs play essential roles in the BCoV tissue tropism, pathogenesis, and immune regulation. Four bovine miRNAs (miR-2375, bta-miR-193a-3p, bta-miR-12059, and bta-miR-494) target BCoV-S glycoprotein and are potentially involved in several immune suppression pathways during the viral infection. These miRNA candidates could serve as good genetic markers for BCoV infection. However, further studies are urgently needed to validate these identified miRNAs and their target genes in the context of BCoV infection and dual tropism and as genetic markers.

Genetic characterization of bovine coronavirus strain isolated in Inner Mongolia of China.

BACKGROUND: Bovine coronavirus (BCoV) is implicated in severe diarrhea in calves and contributes to the bovine respiratory disease complex; it shares a close relationship with human coronavirus. Similar to other coronaviruses, remarkable variability was found in the genome and biology of the BCoV. In 2022, samples of feces were collected from a cattle farm. A virus was isolated from 7-day-old newborn calves. In this study, we present the genetic characteristics of a new BCoV isolate. The complete genomic, spike protein, and nucleocapsid protein gene sequences of the BCoV strain, along with those of other coronaviruses, were obtained from the GenBank database. Genetic analysis was conducted using MEGA7.0 and the Neighbor-Joining (NJ) method. The reference strains' related genes were retrieved from GenBank for comparison and analysis using DNAMAN. RESULTS: The phylogenetic tree and whole genome consistency analysis showed that it belonged to the GIIb subgroup, which is epidemic in Asia and America, and was quite similar to the Chinese strains in the same cluster. Significantly, the S gene was highly consistent with QH1 (MH810151.1) isolated from yak. This suggests that the strain may have originated from interspecies transmission involving mutations of wild strains. The N gene was conserved and showed high sequence identity with the epidemic strains in China and the USA. CONCLUSIONS: Genetic characterization suggests that the isolated strain could be a new mutant from a wild-type lineage, which is in the same cluster as most Chinese epidemic strains but on a new branch.

Molecular and Serological Detection of Bovine Coronaviruses in Marmots (Marmota marmota) in the Alpine Region.

In this study, virological surveillance focused on coronaviruses in marmots in the Alpine region in 2022, captured as part of a population control reduction program in the Livigno area. Seventy-six faecal samples were randomly collected from marmots at the time of capture and release and tested for genome detection of pan-coronavirus, pan-pestivirus, canine distemper virus, and influenza A and D virus. Nine faecal samples were positive in the Pan-CoV RT-PCR, while all were negative for the other viruses. Pan-coronavirus positives were further identified using Illumina's complete genome sequencing, which showed the highest homology with Bovine Coronavirus previously detected in roe deer in the Alps. Blood samples (n.35) were collected randomly from animals at release and tested for bovine coronavirus (BCoV) antibodies using competitive ELISA and VNT. Serological analyses revealed that 8/35 sera were positive for BCoV antibodies in both serological tests. This study provides molecular and serological evidence of the presence of BCoV in an alpine marmot population due to a likely spillover event. Marmots share areas and pastures with roe deer and other wild ruminants, and environmental transmission is a concrete possibility.

Development and application of one-step multiplex Real-Time PCR for detection of three main pathogens associated with bovine neonatal diarrhea.

Introduction: Neonatal calf diarrhea (NCD) is one of the most common diseases in calves, causing huge economic and productivity losses to the bovine industry worldwide. The main pathogens include bovine rotavirus (BRV), bovine coronavirus (BCoV), and Enterotoxigenic Escherichia coli (ETEC) K99. Since multiple infectious agents can be involved in calf diarrhea, detecting each causative agent by traditional methods is laborious and expensive. Methods: In this study, we developed a one-step multiplex Real-Time PCR assay to simultaneously detect BRV, BCoV, and E. coli K99+. The assay performance on field samples was evaluated on 1100 rectal swabs of diseased cattle with diarrhea symptoms and compared with the conventional gel-based RT-PCR assay detect BRV, BCoV, and E. coli K99+. Results: The established assay could specifically detect the target pathogens without cross-reactivity with other pathogens. A single real-time PCR can detect ~1 copy/µL for each pathogen, and multiplex real-time PCR has a detection limit of 10 copies/µL. Reproducibility as measured by standard deviation and coefficient of variation were desirable. The triple real-time PCR method established in this study was compared with gel-based PT-PCR. Both methods are reasonably consistent, while the real-time PCR assay was more sensitive and could rapidly distinguish these three pathogens in one tube. Analysis of surveillance data showed that BRV and BCoV are major enteric viral pathogens accounting for calves' diarrhea in China. Discussion: The established assay has excellent specificity and sensitivity and was suitable for clinical application. The robustness and high-throughput performance of the developed assay make it a powerful tool in diagnostic applications and calf diarrhea research. ​.

Development of a multiplexed Luminex assay for simultaneous detection of enteric viruses in cattle.

Viral and bacterial gastroenteritis and diarrhea have long been a problem in livestock with devastating effects on animal health and production causing a heavy financial burden on producers. Therefore, the bead-based multiplex detection assay was created for simultaneous detection of three livestock viral diarrheic agents viz. bovine rotavirus (BRV), bovine coronavirus (BCoV) and bluetongue virus (BTV). The primers and probes for triplex MAGPIX assay for simultaneous detection of three enteric viruses were designed and the assay was optimized for hybridization temperature, primer-probe and bead concentrations. The newly developed MAGPIX assay was used to determine the prevalence of these diarrhea-associated viruses by testing 200 fecal samples collected from Haryana state of India during 2018-2019. The limit of detection of the developed triplex assay was 1 × 105, 1 × 104, and 1 × 105 RNA copies for BRV, BCoV, and BTV, respectively, being lower than the reverse transcription-quantitative polymerase chain reaction (RT-qPCR). However, it was higher than the conventional RT-PCR, showing it to be more sensitive. The newly developed MAGPIX assay was a rapid, cost-effective and high throughput diagnostic tool for identification of three major entero-pathogenic diarrhea associated viruses, either alone or in tandem, with the aim to prevent and control viral diarrhea in animals.

No vaccine interference between bovine coronavirus and bovine herpesvirus-1 in a randomized trial when coadministrating two intranasal modified-live viral vaccines to neonatal calves.

OBJECTIVE: Compare immune responses induced by 2 commercial intranasal (IN) modified-live viral (MLV) vaccines given individually or coadministered and evaluate prevention of infection and lung pathology following bovine herpesvirus-1 (BHV-1) challenge. ANIMALS: 36 male Holstein calves (ages, 5 to 12 days). METHODS: In a randomized complete block design, each calf received an IN injection of either vaccine diluent (Placebo), an MLV vaccine containing bovine herpesvirus-1 (BHV-1; N3), bovine coronavirus vaccine (BC), or both N3 and BC (BC + N3) with a booster 4 weeks later. Nasal secretions and blood were collected weekly. Three weeks after the booster, the calves were challenged with BHV-1, sampled for virus shedding, and euthanized 10 days later to quantify lung pathology. The study period was September 7, 2020, to April 6, 2021. RESULTS: Calves were seropositive for BHV-1 and BC before vaccination. No significant difference in BC-specific serum immunoglobin G and nasal immunoglobin A antibody responses in the BC versus BC + N3 group or BHV-1-specific serum immunoglobin G and nasal immunoglobin A antibody responses in the N3 versus BC + N3 group. Cytokine responses to BHV-1 and BC did not differ among groups. BHV-1 shedding after challenge was significantly reduced in N3 groups versus Placebo and BC. There was a significant reduction in lung pathology in the N3 + BC group versus Placebo. CLINICAL RELEVANCE: This study provides evidence an MLV vaccine containing BHV-1 and an MLV BC vaccine can be coadministered to neonatal calves without significantly altering immune responses to the 2 viruses or compromising the prevention of BHV-1 respiratory disease. Calves receiving the BC + N3 vaccine had a significant reduction in lung pathology after BHV-1 aerosol challenge.

The Role of Mycoplasma bovirhinis in the Development of Singular and Concomitant Respiratory Infections in Dairy Calves from Southern Brazil.

The role of Mycoplasma bovirhinis in the development of pulmonary disease in cattle is controversial and was never evaluated in cattle from Latin America. This study investigated the respiratory infection dynamics associated with M. bovirhinis in suckling calves from 15 dairy cattle herds in Southern Brazil. Nasal swabs were obtained from asymptomatic (n = 102) and calves with clinical manifestations (n = 103) of bovine respiratory disease (BRD) and used in molecular assays to identify the specific genes of viral and bacterial disease pathogens of BRD. Only M. bovirhinis, bovine coronavirus (BCoV), ovine gammaherpesvirus 2 (OvGHV2), Histophilus somni, Pasteurella multocida, and Mannheimia haemolytica were detected. M. bovirhinis was the most frequently diagnosed pathogen in diseased (57.8%; 59/102) and asymptomatic (55.3%; 57/103) calves at all farms. BCoV-related infections were diagnosed in diseased (52%; 53/102) and asymptomatic (51.4%; 53/103) calves and occurred in 93.3% (14/15) of all farms. Similarly, infectious due to OvGHV2 occurred in diseased (37.2%; 38/102) and asymptomatic (27.2%; /28/103) calves and were diagnosed in 80% (12/15) of all farms investigated. Significant statistical differences were not identified when the two groups of calves were compared at most farms, except for infections due to OvGHV2 that affected five calves at one farm. These results demonstrated that the respiratory infection dynamics of M. bovirhinis identified in Southern Brazil are similar to those observed worldwide, suggesting that there is not enough sufficient collected data to consider M. bovirhinis as a pathogen of respiratory infections in cattle. Additionally, the possible roles of BCoV and OvGHV2 in the development of BRD are discussed.

Serological and Molecular Investigation of SARS-CoV-2 in Horses and Cattle in Switzerland from 2020 to 2022.

Horses and cattle have shown low susceptibility to SARS-CoV-2, and there is no evidence of experimental intraspecies transmission. Nonetheless, seropositive horses in the US and seropositive cattle in Germany and Italy have been reported. The current study investigated the prevalence of antibodies against SARS-CoV-2 in horses and cattle in Switzerland. In total, 1940 serum and plasma samples from 1110 horses and 830 cattle were screened with a species-specific ELISA based on the SARS-CoV-2 receptor-binding domain (RBD) and, in the case of suspect positive results, a surrogate virus neutralization test (sVNT) was used to demonstrate the neutralizing activity of the antibodies. Further confirmation of suspect positive samples was performed using either a pseudotype-based virus neutralization assay (PVNA; horses) or an indirect immunofluorescence test (IFA; cattle). The animals were sampled between February 2020 and December 2022. Additionally, in total, 486 bronchoalveolar lavage (BAL), oropharyngeal, nasal and rectal swab samples from horses and cattle were analyzed for the presence of SARS-CoV-2 RNA via reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Six horses (0.5%; 95% CI: 0.2-1.2%) were suspect positive via RBD-ELISA, and neutralizing antibodies were detected in two of them via confirmatory sVNT and PVNA tests. In the PVNA, the highest titers were measured against the Alpha and Delta SARS-CoV-2 variants. Fifteen cattle (1.8%; 95% CI: 1.0-3.0%) were suspect positive in RBD-ELISA; 3 of them had SARS-CoV-2-specific neutralizing antibodies in sVNT and 4 of the 15 were confirmed to be positive via IFA. All tested samples were RT-qPCR-negative. The results support the hypotheses that the prevalence of SARS-CoV-2 infections in horses and cattle in Switzerland was low up to the end of 2022.

A Rapid and Novel Multiplex PCR Assay for Simultaneous Detection of Multiple Viruses Associated with Bovine Gastroenteritis.

Bovine viral diarrheal virus (BVDV) and bovine coronavirus (BCoV) are prevalent viral infections in buffalo calves that result in significant economic losses globally. However, Bovine picobirnavirus (BPBV) Group I and II has been an emerging causes of gastrointestinal infection as has been detected with mixed of BVDV as well as BCV. To combat economic losses and viral infection, a rapid and innovative multiplex-PCR assay (M-PCR) was developed to simultaneously identify BVDV, BCV, and BPBV. The assay employed three primer pairs, each specific to a particular virus. Notably, the primers for BCV and BVDV, targeting the transmembrane (M) Mebus gene and 5'UTR genes, respectively, were self-designed. To validate the assay, 300 samples of buffalo calf feces were subjected to the standardized multiplex PCR. The results demonstrated that 54 (18%) samples tested positive for multiple viruses, with 16.67% samples infected by BVDV, 0.9% by BCoV, and 0.13% by BPBV, as detected by the M-PCR assay. In summary, this developed assay is characterized by high specificity, sensitivity, throughput, and speed, enabling the simultaneous detection of the three viruses in a single reaction tube. Consequently, it holds potential for epidemiological investigations. It is worth noting that, to the best of our knowledge, this is the first reported multiplex assay for the worldwide detection of BVDV, BCoV, and BPBV. This novel assay promises to aid in the detection of mixed infections in the gastrointestinal tract.

Establishment and application of multiplex droplet digital polymerase chain reaction assay for bovine enterovirus, bovine coronavirus, and bovine rotavirus.

Bovine enterovirus (BEV), bovine coronavirus (BCoV), and bovine rotavirus (BRV) are still the major worldwide concerns in the health care of cattle, causing serious economic losses in the livestock industry. It is urgent to establish specific and sensitive methods to detect viruses for the early control of diseases. Droplet digital PCR (ddPCR) has been proposed to effectively detect viral particles, and it does not involve Ct values or standard curves. In this study, we designed specific primers and probes, based on conserved regions of viral genomes, to optimize protocols for a dual ddPCR assay for detecting BCoV and BRV and a multiplex ddPCR assay for BEV, BCoV, and BRV. Sensitivity assays revealed that the lower limit of detection for qPCR was 1,000 copies/µL and for ddPCR for BEV, BCoV, and BRV, 2.7 copies/µL, 1 copy/µL and 2.4 copies/µL, respectively. Studying 82 samples collected from diarrheal calves on a farm, our dual ddPCR method detected BCoV, BRV, and co-infection at rates of 18.29%, 14.63%, and 6.1%, respectively. In contrast, conventional qPCR methods detected BCoV, BRV, and co-infection at rates of 10.98%, 12.2%, and 3.66%, respectively. On the other hand, studying 68 samples from another farm, qPCR detected BCoV, BRV, BEV, and co-infection of BCoV and BEV at rates of 14.49%, 1.45%, 5.80%, and 1.45%, respectively. Our multiplex ddPCR method detected BCoV, BRV, BEV, co-infection of BCoV and BEV, and co-infection of BRV and BEV. at rates of 14.49%, 2.9%, 8.7%, 2.9%, and 1.45%, respectively. Studying 93 samples from another farm, qPCR detected BCoV, BRV, BEV, and co-infection of BCoV and BEV was detected at rates of 5.38%, 1.08%, 18.28%, and 1.08%, respectively. Co-infection of BCoV, BRV, BEV, BCoV, and BEV, and co-infection of BRV and BEV, were detected by multiplex ddPCR methods at rates of 5.38%, 2.15%, 20.45%, 1.08%, and 1.08%, respectively. These results indicated that our optimized dual and multiplex ddPCR methods were more effective than conventional qPCR assays to detect these viral infections.

Presence of Equine and Bovine Coronaviruses, Endoparasites, and Bacteria in Fecal Samples of Horses with Colic.

Acute abdominal pain (colic) is one of the major equine health threats worldwide and often necessitates intensive veterinary medical care and surgical intervention. Equine coronavirus (ECoV) infections can cause colic in horses but are rarely considered as a differential diagnosis. To determine the frequency of otherwise undetected ECoV infections in horses with acute colic, fresh fecal samples of 105 horses with acute colic and 36 healthy control horses were screened for viruses belonging to the Betacoronavirus 1 species by RT-PCR as well as for gastrointestinal helminths and bacteria commonly associated with colic. Horses with colic excreted significantly fewer strongyle eggs than horses without colic. The prevalence of anaerobic, spore-forming, gram-positive bacteria (Clostridium perfringens and Clostridioides difficile) was significantly higher in the feces of horses with colic. Six horses with colic (5.7%) and one horse from the control group (2.8%) tested positive for Betacoronaviruses. Coronavirus-positive samples were sequenced to classify the virus by molecular phylogeny (N gene). Interestingly, in three out of six coronavirus-positive horses with colic, sequences closely related to bovine coronaviruses (BCoV) were found. The pathogenic potential of BCoV in horses remains unclear and warrants further investigation.