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Bovine rotavirus (BRV) is the main cause of acute gastroenteritis in calves, resulting in significant economic losses to the cattle industry worldwide. Additionally, BRV has multiple genotypes, which could enable cross-species transmission, thereby posing a significant risk to public health. However, there is a problem of multiple genotypes coexisting in BRV, and the cross-protection effect between different genotypes of rotavirus strains is not effective enough. Therefore, mastering clinical epidemic genotypes and using epidemic genotype strains for vaccine preparation is an effective means of preventing and controlling BRV. In this study, BRV strain DQ2020 in MA104 cells was identified by transmission electron microscopy (TEM), reverse transcription polymerase chain reaction (RT-PCR), and colloidal gold immunochromatographic test strips. The whole genome of BRV strain DQ2020 was sequenced and pathogenicity in suckling mice was assessed. The results showed that after 10 passages in MA104 cells, BRV strain DQ2020 induced cytopathic effects. Wheel-shaped virus particles (diameter, ~80 nm) were observed by TEM. A target band of 382 bp was detected by RT-PCR, a positive band was detected with the colloidal gold immunochromatographic test strips, and significant green fluorescence was observed by indirect immunofluorescence (IFA). The highest median tissue culture infectious dose of strain DQ2020 after 9 passages in MA104 cells was 10−4.81 viral particles/0.1 mL. Based on phylogenetic analysis of 11 gene fragments, the genotype of BRV strain DQ2020 was G6-P[1]-I2-R2-C2-M2-A11-N2-T6-E2-H3, confirming transmission of the G6-P[1] genotype in Chinese cattle herds. Further analysis showed that the isolated strain was a reassortant of bovine (VP7, VP6, NSP3, and NSP5), human (VP4, VP1, VP2, VP3, NSP2, and NSP4), and ovine (NSP1) rotaviruses. BRV strain DQ2020 caused damage to the intestinal villi of suckling mice and diarrhea, confirming pathogenicity. In summary, this study identified a reassortant strain of bovine, human, and ovine rotavirus that is pathogenic to lactating mice, and conducted whole genome sequence analysis, providing valuable insights for the genetic evolution of the virus and the development of vaccines. [ABSTRACT FROM AUTHOR]

Bovine rotavirus (BRV) is the main cause of acute gastroenteritis in calves, resulting in significant economic losses to the cattle industry worldwide. Additionally, BRV has multiple genotypes, which could enable cross-species transmission, thereby posing a significant risk to public health. However, there is a problem of multiple genotypes coexisting in BRV, and the cross-protection effect between different genotypes of rotavirus strains is not effective enough. Therefore, mastering clinical epidemic genotypes and using epidemic genotype strains for vaccine preparation is an effective means of preventing and controlling BRV. In this study, BRV strain DQ2020 in MA104 cells was identified by transmission electron microscopy (TEM), reverse transcription polymerase chain reaction (RT-PCR), and colloidal gold immunochromatographic test strips. The whole genome of BRV strain DQ2020 was sequenced and pathogenicity in suckling mice was assessed. The results showed that after 10 passages in MA104 cells, BRV strain DQ2020 induced cytopathic effects. Wheel-shaped virus particles (diameter, *80 nm) were observed by TEM. A target band of 382 bp was detected by RT-PCR, a positive band was detected with the colloidal gold immunochromatographic test strips, and significant green fluorescence was observed by indirect immunofluorescence (IFA). The highest median tissue culture infectious dose of strain DQ2020 after 9 passages in MA104 cells was 10-4.81 viral particles/0.1 mL. Based on phylogenetic analysis of 11 gene fragments, the genotype of BRV strain DQ2020 was G6-P[1]-I2-R2-C2-M2-A11-N2-T6-E2-H3, confirming transmission of the G6-P[1] genotype in Chinese cattle herds. Further analysis showed that the isolated strain was a reassortant of bovine (VP7, VP6, NSP3, and NSP5), human (VP4, VP1, VP2, VP3, NSP2, and NSP4), and ovine (NSP1) rotaviruses. BRV strain DQ2020 caused damage to the intestinal villi of suckling mice and diarrhea, confirming pathogenicity. In summary, this study identified a reassortant strain of bovine, human, and ovine rotavirus that is pathogenic to lactating mice, and conducted whole genome sequence analysis, providing valuable insights for the genetic evolution of the virus and the development of vaccines. [ABSTRACT FROM AUTHOR]

Abstrak Diare merupakan penyakit yang menyebabkan angka kesakitan yang tinggi pada pedet dan kematian neonatal. Penyakit ini dapat disebabkan oleh beberapa agen penyakit yang berbeda. Rotavirus Grup A (RVA) atau Bovine Rotavirus merupakan salah satu agen infeksi penyebab diare pada pedet. Selanjutnya, diare neonatal pada pedet dapat berdampak pada kerugian ekonomi bagi ternak sapi perah dan sapi potong di seluruh dunia karena menyebabkan gangguan pertumbuhan, meningkatnya biaya perawatan, dan/atau kematian pada hewan sakit. Prevalensi Bovine Rotavirus dapat berbeda antar negara di seluruh dunia. Sirkulasi Bovine Rotavirus pada sapi telah dilaporkan di beberapa negara tetapi sirkulasi Bovine Rotavirus pada sapi di Indonesia belum diketahui. Untuk mengetahui prevalensi Rotavirus grup A atau Bovine Rotavirus (BRV), 100 sampel feses dikoleksi dari pedet dengan gejala klinis diare atau tidak diare di Kabupaten Bogor, Jawa Barat pada tahun 2021. Sampel dianalisis terhadap urutan yang mengkode protein kapsid bagian dalam VP6 (subkelompok) menggunakan Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). Lima dari 100 sampel feses sapi (5%) terdeteksi positif BRV. Pada penelitian ini menunjukkan bahwa kelompok Rotavirus atau Bovine Rotavirus (BRV) telah bersirkulasi di antara ternak sapi di Indonesia, khususnya Kabupaten Bogor. Sampel positif Rotavirus grup A atau Bovine Rotavirus (BRV) dapat diidentifikasi dengan metode diagnosis dini (RT-PCR). Kata kunci : Bovine Rotavirus; RT PCR; Bogor Abstract Diarrhea is the most disease that cause high morbidity in calves and neonatal mortality. This disease can be caused by several different infectious agents. Group A rotaviruses (RVA) or Bovine Rotavirus are one of the infectious agents causing diarrhea in calves. Then, Neonatal calf diarrhea can impact to economic losses to dairy and beef cattle herds worldwide, in consequence of growth disorders, value of treatment, and/or death of sick animals. The prevalence of Bovine Rotavirus can become different in the worldwide. The circulation of these bovine rotavirus in calves from the some region has already been demonstrated but the circulation of bovine rotavirus in Indonesia is not known. To investigate the prevalence of A group Rotaviruses or Bovine Rotavirus (BRV), 100 fecal samples were collected from calves with diarrhea or no diarrhea in Bogor district, West Java at 2021. The samples were analyzed for sequences encoding the inner capsid protein VP6 (subgroup) using RT-PCR. Five of 100 specimens of bovine fecal (5%) were detected positive as BRV positive. In this study, A group Rotaviruses or Bovine Rotavirus (BRV) have been circulated among cattle herds in Indonesia, particularly Bogor District. The positive samples of A group Rotaviruses or Bovine Rotavirus (BRV) can be identified using the early diagnosis method (RT-PCR). Keywords : Bovine Rotavirus; RT PCR; Bogor District

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. [ABSTRACT FROM AUTHOR]

Abstract Numerous infectious diseases in cattle lead to reductions in body weight, milk production, and reproductive performance. Cattle are primarily vaccinated using inactivated vaccines due to their increased safety. However, inactivated vaccines generally result in weaker immunity compared with live attenuated vaccines, which may be insufficient in certain cases. Over the last few decades, there has been extensive research on the use of the Newcastle disease virus (NDV) as a live vaccine vector for economically significant livestock diseases. A single vaccination dose of NDV can sufficiently induce immunity; therefore, a booster vaccination dose is expected to yield limited induction of further immune response. We previously developed recombinant chimeric NDV (rNDV-2F2HN), in which its hemagglutinin-neuraminidase (HN) and fusion (F) proteins were replaced with those of avian paramyxovirus 2 (APMV-2). In vitro analysis revealed that rNDV-2F2HN expressing human interferon-gamma had potential as a cancer therapeutic tool, particularly for immunized individuals. In the present study, we constructed rNDV-2F2HN expressing the bovine rotavirus antigen VP6 (rNDV-2F2HN-VP6) and evaluated its immune response in mice previously immunized with NDV. Mice primarily inoculated with recombinant wild-type NDV expressing VP6 (rNDV-WT-VP6), followed by a booster inoculation of rNDV-2F2HN-VP6, showed a significantly stronger immune response than that in mice that received rNDV-WT-VP6 as both primary and booster inoculations. Therefore, our findings suggest that robust immunity could be obtained from the effects of chimeric rNDV-2F2HN expressing the same or a different antigen of a particular pathogen as a live attenuated vaccine vector.

Introduction: Calf diarrhea is a significant condition that has a strong effect on the cattle industry, resulting in huge economic losses annually. Bovine torovirus (BToV), bovine enterovirus (BEV), bovine norovirus (BNoV), bovine coronavirus (BCoV), bovine rotavirus (BRV), and bovine viral diarrhea virus (BVDV) are key pathogens that have been implicated in calf diarrhea. Among these viruses, there remains limited research on BToV, BEV, and BNoV, with no available vaccines or drugs for their prevention and control. Although commercial vaccines exist for BCoV, BRV, and BVDV, the prevalence of these diseases remains high. Methods: To address this issue, we developed a multiplex real-time fluorescence quantitative PCR method for detecting BToV, BEV, BNoV, BCoV, BRV, and BVDV. This method can be used to effectively monitor the prevalence of these six viruses and serve as a reference for future prevention and control strategies. In this study, we specifically designed primers and probes for the BNoV Rdrp, BEV 5'UTR, BToV M, BCoV N, BRV NSP5, and BVDV 5'UTR genes. Results: This method was determined to be efficient, stable, and sensitive. The lowest detectable levels of plasmids for BNoV, BEV, BToV, BRV, BCoV, and BVDV were 1.91 copies/μL, 96.0 copies/μL, 12.8 copies/μL, 16.4 copies/μL, 18.2 copies/μL, and 65.3 copies/μL, respectively. Moreover, the coefficients of variation for all six detection methods were < 3%; they also exhibited a strong linear relationship (R² ≥ 0.98), and an amplification efficiency of 90%-110%. A total of 295 fecal and anal swabs were collected from calves with diarrhea in Guangdong, China. The positive rates for BToV, BEV, BNoV, BCoV, BR, and BVDV were determined to be 0.34% (1/295), 6.10% (18/295), 0.68% (2/295), 1.36% (4/295), 10.85% (32/295), and 2.03% (6/295), respectively. Notably, BEV and BRV exhibited the highest prevalence. Discussion: Additionally, this study identified the occurrence of BToV and BNoV in Guangdong for the first time. In summary, this study successfully established an effective method for detecting several important bovine viruses; ultimately, this holds strong implications for the future development of the cattle industry. [ABSTRACT FROM AUTHOR]

Simple Summary: Rotavirus is one of the pathogens that cause diarrhea in calves. Bovine rotavirus poses difficulties in diagnosis and prevention due to its complex serology and large differences between strains, so the establishment of a rapid and effective detection kit has a wide range of applications for the early diagnosis and timely treatment of viral infections. In this study, we expressed the bovine rotavirus VP6 protein by a eukaryotic expression system, immunized BALB/c mice to collect serum, and established an indirect ELISA for VP6 protein. Our results showed that the prepared indirect ELISA has the advantages of good specificity and high sensitivity, which provides technical support for the effective detection and epidemiological investigation of bovine rotavirus at a later stage. The objective of this study was to develop an indirect ELISA utilizing a polyclonal antibody against bovine rotavirus (BRV) VP6 protein. To achieve this, pcDNA3.1-VP6, a recombinant eukaryotic expression plasmid, was constructed based on the sequence of the conserved BRV gene VP6 and was transfected into CHO-K1 cells using the transient transfection method. The VP6 protein was purified as the coating antigen using nickel ion affinity chromatography, and an indirect ELISA was subsequently established. The study found that the optimal concentration of coating for the VP6 protein was 1 μg/mL. The optimal blocking solution was 3% skim milk, and the blocking time was 120 min. The secondary antibody was diluted to 1:4000, and the incubation time for the secondary antibody was 30 min. A positive result was indicated when the serum OD450 was greater than or equal to 0.357. The coefficients of variation were less than 10% both within and between batches, indicating the good reproducibility of the method. The study found that the test result was positive when the serum dilution was 217, indicating the high sensitivity of the method. A total of 24 positive sera and 40 negative sera were tested using the well-established ELISA. The study also established an indirect ELISA assay with good specificity and sensitivity for the detection of antibodies to bovine rotavirus. Overall, the results suggest that the indirect ELISA method developed in this study is an effective test for detecting such antibodies. [ABSTRACT FROM AUTHOR]

Bovine rotaviruses (BRVs) are significant causative agents of severe diarrhea in newborn calves, resulting in substantial economic losses in the livestock industry. Inhibition of bovine rotavirus using extracts prepared from a Cucurbitaceae plant, which contains trypsin protease inhibitors, might offer a potential anti-rotaviral effect in vitro. Ecballium elaterium (E. elaterium) belongs to the Cucurbitaceae family, indigenous to the Mediterranean, contains E. elaterium trypsin isoinhibitors (EETIso), and has been used in traditional medicine. This study aimed to evaluate the in vitro efficacy of E. elaterium extract against bovine rotavirus infections. Ethanol extracts were prepared from E. elaterium seeds and fruit juice, and their non-toxic concentrations were determined using MA-104 cells. The cells were infected with bovine rotavirus in the presence of E. elaterium extract. The results demonstrated a significant decrease in the rotavirus titer in vitro upon treatment with the E. elaterium extract, suggesting its potential as a therapeutic agent against bovine rotavirus-induced diarrhea in calves. The utilization of E. elaterium extract may contribute to reduced calf mortality, lower medication costs, and improved economic value in cattle farming.

Bovine rotaviruses (BRVs) are significant causative agents of severe diarrhea in newborn calves, resulting in substantial economic losses in the livestock industry. Inhibition of bovine rotavirus using extracts prepared from a Cucurbitaceae plant, which contains trypsin protease inhibitors, might offer a potential anti-rotaviral effect in vitro. Ecballium elaterium (E. elaterium) belongs to the Cucurbitaceae family, indigenous to the Mediterranean, contains E. elaterium trypsin isoinhibitors (EETIso), and has been used in traditional medicine. This study aimed to evaluate the in vitro efficacy of E. elaterium extract against bovine rotavirus infections. Ethanol extracts were prepared from E. elaterium seeds and fruit juice, and their non-toxic concentrations were determined using MA-104 cells. The cells were infected with bovine rotavirus in the presence of E. elaterium extract. The results demonstrated a significant decrease in the rotavirus titer in vitro upon treatment with the E. elaterium extract, suggesting its potential as a therapeutic agent against bovine rotavirus-induced diarrhea in calves. The utilization of E. elaterium extract may contribute to reduced calf mortality, lower medication costs, and improved economic value in cattle farming. [ABSTRACT FROM AUTHOR]

Neonatal calf diarrhea remains one of the most important problems faced by livestock, causing great economic losses. Fecal samples were collected from 100 diarrheic calves in Al-Fayoum governorate, Egypt during 2021, and 2022, to investigate the prevalence of Enterotoxigenic Escherichia coli (ETEC), Salmonella Typhimurium, Klebsiella pneumoniae, Clostridium perfringens, bovine rotavirus, bovine coronavirus, and Cryptosporidium parvum which are the major enteropathogens associated with neonatal calf diarrhea, the prevalence of enteropathogens were 58%, 29%, 34%, 14%, 35%, 8%, and 65% respectively. Molecular characterization was performed to confirm the E. coli, Klebsiella pneumoniae, Salmonella Typhimurium, Clostridium perfringens, and Cryptosporidium isolates and to detect some virulent genes associated with their pathogenicity. All the bacterial isolates gave a clear band with 16S rRNA. In E. coli, virulent genes (K99, F41, phoA) were detected, also; Salmonella strains were found positive for the invA and sopB gene, while all Clostridium perfringens strains were tested positive for Alpha and Beta toxin but negative for Epsilon toxin. On the other hand, all Klebsiella pneumoniae isolates were tested positive with iutA and fimH genes. Also, the in-vitro antibiotic sensitivity testing of bacterial isolates was applied. Statistical analysis was carried out to determine the potential influence of age factor on the reported prevalence of concurrent infections, which revealed that the animals age significantly affected the infection prevalence in all pathogens inversely excepts those infected by Klebsiella was affected by age directly, and those infected by E. coli, or Cryptosporidium, were not affected by age at all. Good hygienic management and good vaccination program are very important to overcome acute diarrhea in neonate calves and the misuse of antibiotic revealed the presence of multidrug resistance isolates of some enteropathogenic bacteria. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Northwest A & F University - Natural Science Edition is the property of Editorial Department of Journal of Northwest A&F University (Natural Science Edition) and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The changes in the composition of intestinal microbiota and metabolites have been linked to digestive disorders in calves, especially neonatal calf diarrhea. Bovine rotavirus (BRV) and bovine coronavirus (BCoV) are known to be the primary culprits behind neonatal calf diarrhea. In this study, we analyzed changes in the fecal microbiota and metabolites of calves with neonatal diarrhea associated with BRV and BCoV infection using high-throughput 16S rRNA sequencing and metabolomics technology. The microbial diversity in the feces of calves infected with BRV and BCoV with diarrhea decreased significantly, and the composition changed significantly. The significant increase of Fusobacterium and the reductions of some bacteria genera, including Faecalibacterium, Bifidobacterium, Ruminococcus, Subdoligranulum, Parabacteroides, Collinsella, and Olsenella, etc., were closely related to diarrhea associated with BRV and BCoV infection. Metabolites in the feces of BRV and BCoV-infected calves with diarrhea were significantly changed. Phosphatidylcholine [PC; 16:1(9 Z)/16:1(9 Z)], lysophosphatidylethanolamine (LysoPE; 0:0/22:0), lysophosphatidylcholine (LysoPC; P-16:0) and LysoPE (0:0/18:0) were significantly higher in the feces of BRV-infected calves with diarrhea. In contrast, some others, such as desthiobiotin, were significantly lower. BRV infection affects glycerophospholipid metabolism and biotin metabolism in calves. Two differential metabolites were significantly increased, and 67 differential metabolites were significantly reduced in the feces of BCoV-infected calves with diarrhea. Seven significantly reduced metabolites, including deoxythymidylic acid (DTMP), dihydrobiopterin, dihydroneopterin triphosphate, cortexolone, cortisol, pantetheine, and pregnenolone sulfate, were enriched in the folate biosynthesis, pantothenate and CoA biosynthesis, pyrimidine metabolism, and steroid hormone biosynthesis pathway. The decrease in these metabolites was closely associated with increased harmful bacteria and reduced commensal bacteria. The content of short-chain fatty acids (SCFAs) such as acetic acid and propionic acid in the feces of BRV and BCoV-infected calves with diarrhea was lower than that of healthy calves, which was associated with the depletion of SCFAs-producing bacteria such as Parabacteroides, Fournierella, and Collinsella. The present study showed that BRV and BCoV infections changed the composition of the calf fecal microbiota and were associated with changes in fecal metabolites. This study lays the foundation for further revealing the roles of intestinal microbiota in neonatal calf diarrhea associated with BRV and BCoV infection. [ABSTRACT FROM AUTHOR]

Simple Summary: We conducted a metataxonomic analysis to compare the taxonomic composition and functional profile of the fecal microbiota in healthy and diarrheic calves across three clinical periods, aiming to gain a better understanding of the changes. The findings here might provide information about pathogenic microbiota in diarrheic calves, which were found to be more enriched than in healthy calves. The objective of the present study was to identify changes in fecal microbiota and predict the functional features of healthy calves and those infected with rotavirus over time. Six Holstein calves (average body weight 43.63 ± 1.19 kg, age-matched within 5–7 d) were randomly selected and distributed into two groups which contained three calves each. Fecal samples were taken 3 days before inoculation and on days 1 and 7 post-inoculation. The 16S rRNA gene amplicon sequencing was performed. Bacterial diversity tended to decrease in the rota group, as indicated by the alpha (evenness, p = 0.074 and Shannon, p = 0.055) and beta (Bray–Curtis dissimilarity, p = 0.099) diversity at 1 day post-inoculation. Differences in the bacterial taxa between healthy and rota-infected calves were detected using a linear discriminant analysis effect size (LDA > 2.0, p < 0.05). Rota calves had a higher abundance of certain bacterial taxa, such as Enterococcus, Streptococcus, and Escherichia-Shigella, and a lower abundance of bacteria that contribute to the production of short-chain fatty acids, such as Alistipes, Faecalibacterium, Pseudoflavonifractor, Subdoligranulum, Alloprevotella, Butyricicoccus, and Ruminococcus, compared to the healthy calves. The observed changes in the fecal microbiota of the rota-infected group compared to the healthy group indicated potential dysbiosis. This was further supported by significant differences in the predicted functional metagenomic profiles of these microbial communities. We suggest that calves infected with bovine rotavirus had bacterial dysbiosis, which was characterized by lower diversity and fewer observed genera than the fecal microbiota of healthy calves. [ABSTRACT FROM AUTHOR]

A bovine rotavirus (BRV) isolate from Kirsehir was isolated from feces of a neonatal calf with diarrhea, identified, and sequenced by shotgun sequencing. Its genotype constellation is G10-P[5]-I2-R2-C2-M2-A3-N2-T6-E2-H3. The structural genes and the non-structural genes NSP1, NSP3, and NSP4 of the Kirsehir isolate were similar in sequence to those of BRVs identified in Turkey. However, VP2, NSP2, NSP4, and NSP5/6 showed similarity to those of rotaviruses from different animal hosts. These findings not only expand our current understanding of the diversity of rotaviruses but also contribute to our understanding of the evolution of rotaviruses at both the national and global levels and reinforce the significance of conducting further research on rotaviruses in Turkey. [ABSTRACT FROM AUTHOR]

Copyright of Firat Universitesi Saglik Bilimleri Veteriner Dergisi is the property of Firat Universitesiu, Saglik Bilimleri Enstitusu and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Background: Neonatal calf diarrhea, which is the most common cause in calf deaths, leads to significant economic losses in dairy farming around the world. Diarrhea develops due to infectious and non-infectious reasons. Group A Rotaviruses (RVA) are the leading and predisposing factor for acute neonatal gastroenteritis. Methods and results: In this study, 20 diarrheic fecal samples were collected from one farm in Balıkesir province of Turkey. During virus isolation, a total of 2 stool samples were detected to produce cytopathogenic effects in MA-104 cell line. The two samples (RV-36, RV-38) were tested positive with antigen ELISA kits detecting RVA antigens. In order to detect the presence of rotavirus viral nucleic acid in cell supernatants, VP6 gene region-specific RT-PCR test was performed and the samples RV-36 and RV-38 were positive for RVA viral nucleic acid. By RT-PCR using genotype specific primers, both the isolates RV-36 and RV-38 formed amplicons compatible with G10 and P[11] genotypes of RVA. RVA nucleic acids segments were also visualized by poliacrilamide gel electrophoresis (PAGE) method. The phylogenetic tree constructed according to the VP6 gene region showed that these isolates were in the Rotavirus A group and in the I2 cluster same as other bovine and some human RVA isolates. Conclusion: Succesful isolation of RVA G10P[11] was echieved in the cattle farm. As rotaviruses play the most important role in the etiology of diarrhea in newborn calves respected genotype G10P[11] should be considered in selection of the vaccines applied to the dams. Those isolates can be further evaluated as vaccine candidate. [ABSTRACT FROM AUTHOR]

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.

The objective of this study was to develop an indirect ELISA utilizing a polyclonal antibody against bovine rotavirus (BRV) VP6 protein. To achieve this, pcDNA3.1-VP6, a recombinant eukaryotic expression plasmid, was constructed based on the sequence of the conserved BRV gene VP6 and was transfected into CHO-K1 cells using the transient transfection method. The VP6 protein was purified as the coating antigen using nickel ion affinity chromatography, and an indirect ELISA was subsequently established. The study found that the optimal concentration of coating for the VP6 protein was 1 μg/mL. The optimal blocking solution was 3% skim milk, and the blocking time was 120 min. The secondary antibody was diluted to 1:4000, and the incubation time for the secondary antibody was 30 min. A positive result was indicated when the serum OD450 was greater than or equal to 0.357. The coefficients of variation were less than 10% both within and between batches, indicating the good reproducibility of the method. The study found that the test result was positive when the serum dilution was 217, indicating the high sensitivity of the method. A total of 24 positive sera and 40 negative sera were tested using the well-established ELISA. The study also established an indirect ELISA assay with good specificity and sensitivity for the detection of antibodies to bovine rotavirus. Overall, the results suggest that the indirect ELISA method developed in this study is an effective test for detecting such antibodies.

Abstract Background Neonatal calf diarrhea (NCD) is the leading cause of calf morbidity and mortality in beef cattle. Cow’s vaccination in last stage of pregnancy is one of the most important measures to mitigate the risk of NCD outbreaks. The aim of this study was to evaluate the efficacy of prepartum single dose vaccination against NCD, especially Bovine Rotavirus type A (BoRVA) and Bovine Coronavirus (BCoV), in Nelore dams and offspring. A total of 117 pregnant cows (n = 81) and heifers (n = 36) were distributed in two groups, vaccinated (VAC: cows = 40; heifers = 19) and non-vaccinated (NVAC: cows = 41; heifers = 17). Vaccination occurred between 60 to 50 days before the expected calving date with a single dose of a water-in-oil (W/O) vaccine, and NVAC group received a dose of saline solution 0.9%. Blood samples were collected before vaccination and 30 days after to evaluate the antibody (Ab) response. Specific IgG1 Abs against BoRVA and BCoV were measured by using an Enzyme Linked Immuno Sorbent Assay (ELISA). Calves’ births were monitored, and the transference of passive immunity was evaluated. Diarrhea was monitored in the first 30 days of age, and fecal samples were collected for identification of the etiological agent. Results Higher titers of IgG1 Ab against BoRVA and BCoV was observed in the VAC group than NVAC group in the cow (P

Rotavirus ribonucleic acid was extracted from 16 fecal samples of the serologically positive diarrheic calves using Latex agglutination test (LAT) and Immunochrmatographic assay (ICA). The extracted RNA was submitted to Reverse transcriptase polymerase chain reaction (RT-PCR) to detect VP7 and VP4 genes and the positive samples were 100% (16/16) and 81.25% (13/16), respectively. The amplified products were subjected to G and P-genotyping by semi-nested multiplex PCR using of G6, G8 and G10 genotyping and P1, P5 and P11 genotyping primers, respectively. G6 was detected in 10 (62.50%) of 16 samples and G10 was diagnosed in 5 (31.25%) of 16 samples and one (6.25%) sample did not react with any G primer used. P5 was detected in 9 (56.25%) of 16 samples, P11 was diagnosed in 3 (18.75%) of 16 samples, mixed infection with P5+P11 was observed in 1 (6.25%) of 16 samples and 3 (18.75%) samples did not react with any P primer used. G and P genotypes combination revealed that G6P5 was in 50% (8/16), G10P11 in 12.50% (2/16), G10P5 in 6.25% (1/16), G6P11 in 6.25% (1/16), G10 (P5+P11) in 6.25% (1/16), G6P? in 6.25% (1/16), G10P? in 6.25% (1/16), and G?P? in 6.25% (1/16). These results suggest that the detected genotypes can used as dominant strains for the formulation of an appropriate vaccine against BRV in Assiut Governorate. In conclusion, RTPCR and Semi-nested multiplex PCR can used as rapid and confirmatory test for detection of nucleic acid and genotypes of Rotavirus, G and P genotypes combination in the present study revealed that G6P5, G6P11, G10P5 and G10P11 were circulating genotypes in bovine population in Assiut governorate. G6P5 strain was the most common of all strain diagnosed in other fecal samples. The presence of various combinations of G and P genotypes among field isolates of BRV suggests that genetic reassortment frequently occurred between viral strains with genes encoding different G and P genotypes. Finally, presence of different genotypes of Rotaviruses emphasizes their simultaneous monitoring in animals for the development and optimization of Rotavirus vaccines. [ABSTRACT FROM AUTHOR]

The objective of the present study was to identify changes in fecal microbiota and predict the functional features of healthy calves and those infected with rotavirus over time. Six Holstein calves (average body weight 43.63 ± 1.19 kg, age-matched within 5–7 d) were randomly selected and distributed into two groups which contained three calves each. Fecal samples were taken 3 days before inoculation and on days 1 and 7 post-inoculation. The 16S rRNA gene amplicon sequencing was performed. Bacterial diversity tended to decrease in the rota group, as indicated by the alpha (evenness, p = 0.074 and Shannon, p = 0.055) and beta (Bray–Curtis dissimilarity, p = 0.099) diversity at 1 day post-inoculation. Differences in the bacterial taxa between healthy and rota-infected calves were detected using a linear discriminant analysis effect size (LDA > 2.0, p < 0.05). Rota calves had a higher abundance of certain bacterial taxa, such as Enterococcus, Streptococcus, and Escherichia-Shigella, and a lower abundance of bacteria that contribute to the production of short-chain fatty acids, such as Alistipes, Faecalibacterium, Pseudoflavonifractor, Subdoligranulum, Alloprevotella, Butyricicoccus, and Ruminococcus, compared to the healthy calves. The observed changes in the fecal microbiota of the rota-infected group compared to the healthy group indicated potential dysbiosis. This was further supported by significant differences in the predicted functional metagenomic profiles of these microbial communities. We suggest that calves infected with bovine rotavirus had bacterial dysbiosis, which was characterized by lower diversity and fewer observed genera than the fecal microbiota of healthy calves.

Background: Neonatal calf diarrhea (NCD) is the leading cause of calf morbidity and mortality in beef cattle. Cow's vaccination in last stage of pregnancy is one of the most important measures to mitigate the risk of NCD outbreaks. The aim of this study was to evaluate the efficacy of prepartum single dose vaccination against NCD, especially Bovine Rotavirus type A (BoRVA) and Bovine Coronavirus (BCoV), in Nelore dams and offspring. A total of 117 pregnant cows (n = 81) and heifers (n = 36) were distributed in two groups, vaccinated (VAC: cows = 40; heifers = 19) and non-vaccinated (NVAC: cows = 41; heifers = 17). Vaccination occurred between 60 to 50 days before the expected calving date with a single dose of a water-in-oil (W/O) vaccine, and NVAC group received a dose of saline solution 0.9%. Blood samples were collected before vaccination and 30 days after to evaluate the antibody (Ab) response. Specific IgG1 Abs against BoRVA and BCoV were measured by using an Enzyme Linked Immuno Sorbent Assay (ELISA). Calves' births were monitored, and the transference of passive immunity was evaluated. Diarrhea was monitored in the first 30 days of age, and fecal samples were collected for identification of the etiological agent. Results: Higher titers of IgG1 Ab against BoRVA and BCoV was observed in the VAC group than NVAC group in the cow (P < 0.0001) and total dams categories (P < 0.0001). The titer of specific IgG1 Abs in the calves' serum reflected the dams response, observing higher IgG1 Ab titers for BoRVA (P < 0.0016) and BCoV (P < 0.0095) in the offspring born to VAC cows and higher IgG1 Ab titers for BoRVA(P < 0.0171) and BCoV (P < 0.0200) in the offspring born to VAC total dams. The general incidence of diarrhea observed was 18.6% (11/59) and 29.3% (17/58) in the calves born to the VAC and NVAC group, respectively. Conclusions: Prepartum vaccination with a single dose of the vaccine tested increased the titers of IgG1 Ab against BCoV and BoRVA, and it could be used as a preventive strategy to decrease the NCD occurrence in Nelore calves. [ABSTRACT FROM AUTHOR]

Bovine diarrhea is a major concern in the global bovine industry because it can cause significant financial damage. Of the many potential infectious agents that can lead to bovine diarrhea, bovine rotavirus (BRV) is a particular problem due to its high transmissibility and infectivity. Therefore, it is important to prevent the proliferation of BRV using an early detection system. This study developed an affinity peptide-based electrochemical method for use as a rapid detection system for BRV. A BRV-specific peptide was identified via the phage display technique and chemically synthesized. The synthetic peptide was immobilized on a gold electrode through thiol-gold interactions. The performance of the BRV specific binding peptides was evaluated using square wave voltammetry. The developed detection system exhibited a low detection limit (5 copies/mL) and limit of quantitation (2.14 × 102 copies/mL), indicating that it is a promising sensor platform for the monitoring of BRV. [ABSTRACT FROM AUTHOR]

Bovine rotavirus A (BRVA) is a frequent causative agent of diarrhea in neonatal calves. Accurate and rapid diagnosis is crucial to prevent calf mortality from BRVA induced diarrhea. Currently, variety of diagnostic methods are being used to detect BRVA from calves’ feces: antibody-based rapid test and ELISA, and molecular-based RT-PCR and RT-qPCR. The aim of the study was to evaluate the accuracy (sensitivity and specificity) of the rapid test (Immunochromatography), ELISA, and RT-PCR assays, using RT-qPCR as the gold standard, in detection of BRVA in diarrheic calves’ fecal samples. One hundred (n=100) clinically diarrheic fecal samples were tested with four different diagnostic tools. The percent of samples positive by rapid test, ELISA, RT-PCR and RT-qPCR was 10%, 16%, 17%, and 33%, respectively. The agreement between different assays was 75% to 99%. The highest agreement was observed between ELISA and RT-PCR assay (99%). The lowest agreement was recorded (75%) between rapid test and RT-qPCR. The sensitivity of the rapid test, ELISA, and RT-PCR were 30%, 49%, and 52%, respectively when compared to the reference test (RT-qPCR), whereas specificity was 100% for all assays. In conclusion, none of the frequently used diagnostic tests showed a satisfactory level of sensitivity to identify BRVA in calves’ feces. Therefore, the use of a more sensitive rapid test should be used to identify infected calves in field conditions in order to prevent calf mortality from rotaviral diarrhea.

Aim: Coronaviruses and Rotaviruses are important virologic factors for both animal and human health in Turkey and the world. Bovine Rotavirus (BRV) and Bovine Coronavirus (BCoV) in cattle cause significant economic losses. The aim of this study was to determine the presence of BRV and BCoV in Anatolian buffaloes which were on the same farms with cattle. For this purpose, presence of these two viruses were investigated by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and BRV-BCoV Rapid tests and sensitivity and specificity ratios of these two tests were compared. Materials and Methods: In this study, 230 Anatolian buffaloes were clinically evaluated in cattle farms in Afyonkarahisar region. Fecal samples were collected from 27 buffaloes which had clinical signs (weakness, dehydration, vomiting, watery consistency and yellow stool). The fecal samples were evaluated by Rapid Test and RT-PCR for Bovine Rotavirus and Bovine Coronavirus. The analyzes were performed according to the procedure of the commercial RT-PCR and rapid kits. Results: The RT-PCR results were positive as 22.2% (6/27) for BRV and 3.7% (1/27 27/1) for BcoV while Rota-Corona Rapid test results were negative in all samples. When compared with RT-PCR results for both viruses, the rapid test sensitivity and specificity was determined as 0% and 100%, respectively. In addition, positive rates of BRV was statistically important as BCoV rate in analyzed samples (p Conclusion: In conclusion, low sensitivity of rapid test may be due to the change in the amount of virus scattered throughout the course of enteric infections.

Copyright of Chinese Journal of Preventive Veterinary Medicine / Zhongguo Yufang Shouyi Xuebao is the property of Chinese Journal of Preventive Veterinary Medicine Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Bovine rotavirus A (BRVA) is a frequent causative agent of diarrhea in neonatal calves. Accurate and rapid diagnosis is crucial to prevent calf mortality from BRVA induced diarrhea. Currently, variety of diagnostic methods are being used to detect BRVA from calves' feces: antibody-based rapid test and ELISA, and molecular-based RT-PCR and RT-qPCR. The aim of the study was to evaluate the accuracy (sensitivity and specificity) of the rapid test (Immunochromatography), ELISA, and RT-PCR assays, using RT-qPCR as the gold standard, in detection of BRVA in diarrheic calves' fecal samples. One hundred (n=100) clinically diarrheic fecal samples were tested with four different diagnostic tools. The percent of samples positive by rapid test, ELISA, RT-PCR and RT-qPCR was 10%, 16%, 17%, and 33%, respectively. The agreement between different assays was 75% to 99%. The highest agreement was observed between ELISA and RT-PCR assay (99%). The lowest agreement was recorded (75%) between rapid test and RT-qPCR. The sensitivity of the rapid test, ELISA, and RT-PCR were 30%, 49%, and 52%, respectively when compared to the reference test (RT-qPCR), whereas specificity was 100% for all assays. In conclusion, none of the frequently used diagnostic tests showed a satisfactory level of sensitivity to identify BRVA in calves' feces. Therefore, the use of a more sensitive rapid test should be used to identify infected calves in field conditions in order to prevent calf mortality from rotaviral diarrhea. [ABSTRACT FROM AUTHOR]

Bovine rotavirus A (RVA) and bovine coronavirus (CoV) are the two main viral enteropathogens associated with neonatal calf diarrhea. The aim of the present work was to study the impact of group and individual housing systems in the epidemiology of RVA and CoV infection. Eleven calves reared in individual housing (FA) and nine calves in group housing (FB) were monitored during the first 7 weeks of life. Stool and serum samples were screened for RVA and CoV antigens by ELISA. IgG1 antibodies (Ab) to both antigens were also measured. From the 160 fecal samples collected, the proportion of positive samples to RVA and CoV was significantly higher in FB (23.6%) than in FA (9%) (p = 0.03). The geometric mean of colostral IgG1 Ab titers to CoV and RVA in FA (IgG1 anti-CoV 1024 and anti-RVA 1782.9) was lower than in FB (IgG1 anti-CoV 10,321.2 and anti-RVA 4096) at birth. Calves less than 2 weeks of life from FB had a higher risk of being infected by RVA (OR = 4.9; p = 0.01) and CoV (OR = 17.15; p = 0.01) than calves from FA. The obtained results showed that there was higher RVA and CoV shedding in group-housed calves than in individual-housed animals. [ABSTRACT FROM AUTHOR]

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. ​., Competing Interests: The authors declare that there is a patent related to this work (application no. 2024102313502). The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Wang, Wang, Chang, Jiang, Han, Wang, Jing, Zhao and Yin.)

Copyright of Chinese Journal of Preventive Veterinary Medicine / Zhongguo Yufang Shouyi Xuebao is the property of Chinese Journal of Preventive Veterinary Medicine Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Eurasian Journal of Veterinary Sciences is the property of Eurasian Journal of Veterinary Sciences and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Rotavirus can be detected in stool samples by using serological and molecular methods. An easy and rapid dual gene RTPCR assay for the simultaneous amplification of the capsid protein VP4 (structural) and rotavirus enter toxin NSP4 (nonstructural protein) has been developed. We validated the assay by comparing the results of single target PCR with dual-gene PCR products. [ABSTRACT FROM AUTHOR]

Group A rotaviruses (RVAs) are major enteric pathogens causing infections in calves. To investigate the epidemiological characteristics and genetic diversity of bovine rotavirus (BRV), 233 fecal samples were collected from calves with diarrhea in northeast China. The samples were analyzed for sequences encoding the inner capsid protein VP6 (subgroup) and the outer capsid proteins VP7 and VP4 (G and P type, respectively) using RT-PCR. Ten of the 233 samples (4.3%) were identified as BRV positive and were used for virus isolation and sequence analysis, revealing that all strains analyzed were of the G6P[1] genotype. The isolates exhibited high VP6 sequence identity to the USA cow RVA NCDV strain (>99% amino acid identity) and were further shown to be closely related to Japanese cow RVA BRV101 and Israelian human RVA G6P[1] strains, with >99% amino acid identity to VP7 and VP4 proteins, respectively. Comparative analyses of genome-predicted amino acid sequences between the isolates and the NCDV strains indicated that the antigenicity and infectivity of the strains isolated had changed. In this study, BRV genotypes and the genetic diversity among vaccinated cattle herds were monitored to provide epidemiological data and references for early diagnosis, allowing for early detection of new, potentially pathogenic RVA strains.

This study presents whole genomes of seven bovine rotavirus strains from South Africa and Mozambique. Double-stranded RNA, extracted from stool samples without prior adaptation to cell culture, was used to synthesise cDNA using a self-annealing anchor primer ligated to dsRNA and random hexamers. The cDNA was subsequently sequenced using an Illumina MiSeq platform without prior genome amplification. All strains exhibited bovine-like artiodactyl genome constellations (G10/G6-P[11]/P[5]-I2-R2-C2-M2-A3/A11/A13-N2-T6-E2-H3). Phylogenetic analysis revealed relatively homogenous strains, which were mostly related to other South African animal strains or to each other. It appears that these study strains represent a specific bovine rotavirus population endemic to Southern Africa that was derived through multiple reassortment events. While one Mozambican strain, MPT307, was similar to the South African strains, the second strain, MPT93, was divergent from the other study strains, exhibiting evidence of interspecies transmission of the VP1 and NSP2 genes. The data presented in this study not only contribute to the knowledge of circulating African bovine rotavirus strains, but also emphasise the need for expanded surveillance of animal rotaviruses in African countries in order to improve our understanding of rotavirus strain diversity.

Bovine rotavirus (BRV), bovine parvovirus (BPV), and bovine viral diarrhea virus (BVDV) are the pathogens that cause diarrhea primarily in newborn calves. A mixed infection of BRV, BPV, and BVDV makes clinical diagnosis difficult. In this study, we designed dual-priming oligonucleotide (DPO) primers the VP6 gene of BRV, VP2 gene of BPV, and 5′UTR gene of BVDV and synthesized gold nanoparticles (GNPs) with an average diameter of 10 nm. We combined the DPOs with the GNPs to develop a DPO-nanoPCR assay for detecting BRV, BPV, and BVDV. The annealing temperature, primer concentration, and GNP concentration were optimized for this assay. Compared to a conventional PCR assay, the DPO-nanoPCR assay allowed the use of a wider range of annealing temperatures (41–65°C) to effectively amplify target genes. PCR amplification was the most efficient at 56.2°C using conventional primers. The optimal volume of all the primers (10 μM) was 1.0 μL. The optimal volume of GNPs (10 nM) for all the reactions was 0.5 μL. The detection limits of DPO-nanoPCR for pMD19-T-VP6, pMD19-T-VP2, and pMD19-T-5′UTR were 9.40 × 102 copies/μL, 5.14 × 103 copies/μL, and 4.09 × 101 copies/μL, respectively; and those using conventional PCR were 9.40 × 104 copies/μL, 5.14 × 105 copies/μL, and 4.09 × 104 copies/μL, respectively. The sensitivity of DPO-nanoPCR was at least 100-fold higher than that of conventional PCR. The specificity detection showed that the DPO-nanoPCR was able to specifically detect BRV, BPV, and BVDV. Use of clinical samples indicated that target viruses can be detected accurately. Thus, DPO-nanoPCR is a new powerful, simple, specific, and sensitive tool for detecting mixed infections of BRV, BPV, and BVDV.

Introduction: Calf diarrhea is a significant condition that has a strong effect on the cattle industry, resulting in huge economic losses annually. Bovine torovirus (BToV), bovine enterovirus (BEV), bovine norovirus (BNoV), bovine coronavirus (BCoV), bovine rotavirus (BRV), and bovine viral diarrhea virus (BVDV) are key pathogens that have been implicated in calf diarrhea. Among these viruses, there remains limited research on BToV, BEV, and BNoV, with no available vaccines or drugs for their prevention and control. Although commercial vaccines exist for BCoV, BRV, and BVDV, the prevalence of these diseases remains high., Methods: To address this issue, we developed a multiplex real-time fluorescence quantitative PCR method for detecting BToV, BEV, BNoV, BCoV, BRV, and BVDV. This method can be used to effectively monitor the prevalence of these six viruses and serve as a reference for future prevention and control strategies. In this study, we specifically designed primers and probes for the BNoV Rdrp, BEV 5'UTR, BToV M, BCoV N, BRV NSP5, and BVDV 5'UTR genes., Results: This method was determined to be efficient, stable, and sensitive. The lowest detectable levels of plasmids for BNoV, BEV, BToV, BRV, BCoV, and BVDV were 1.91 copies/μL, 96.0 copies/μL, 12.8 copies/μL, 16.4 copies/μL, 18.2 copies/μL, and 65.3 copies/μL, respectively. Moreover, the coefficients of variation for all six detection methods were < 3%; they also exhibited a strong linear relationship (R 2 ≥ 0.98), and an amplification efficiency of 90%-110%. A total of 295 fecal and anal swabs were collected from calves with diarrhea in Guangdong, China. The positive rates for BToV, BEV, BNoV, BCoV, BR, and BVDV were determined to be 0.34% (1/295), 6.10% (18/295), 0.68% (2/295), 1.36% (4/295), 10.85% (32/295), and 2.03% (6/295), respectively. Notably, BEV and BRV exhibited the highest prevalence., Discussion: Additionally, this study identified the occurrence of BToV and BNoV in Guangdong for the first time. In summary, this study successfully established an effective method for detecting several important bovine viruses; ultimately, this holds strong implications for the future development of the cattle industry., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Meng, Chen, Jiang, Chen, Guo, Ma, Jia and Li.)

The most common worldwide diarrhoea-causing agents in neonatal calves are Cryptosporidium spp. (Crypto), bovine rotavirus (BRV), bovine coronavirus (BCoV), and enterotoxigenic Escherichia coli F5 (K99) (ETEC). Crypto is a zoonotic pathogen of diarrhoea in humans, particularly for children and immunocompromised adults. Four weighted-stratified random-effect meta-analyses including meta-regression analyses were performed to calculate the worldwide mean prevalence of Crypto and associated concurrent infections with BRV, BCoV and ETEC, as well as their potential influencing factors. The meta-analysis incorporated 28 studies (56 substudies) in 17 countries that determined the presence or absence of concurrent infections with Crypto in the global calf population. Approximately half of all considered studies presented here were conducted in Europe independently of the type of infections with Crypto. Within Europe, the highest estimated mean Crypto-BRV prevalence was identified in Ireland (16.7%), the highest estimated mean Crypto-BCoV prevalence was detected in the United Kingdom (4.3%), and the highest estimated mean Crypto-ETEC prevalence across the literature was determined in Turkey (4.7%). The chance of detecting BRV, BCoV, and ETEC in calves with diarrhoea was 0.8 (confidence interval (CI): 0.6–1.0), 0.7 (CI: 0.5–1.0) and 0.6 (CI: 0.4–0.9) lower in the presence of Crypto compared to calves without Crypto. This may indicate an inhibitory effect between BRV, BCoV, ETEC, and Crypto in calves. The variance in the published prevalence across the literature can mainly be explained by the “diagnostic” factor (R2 min–max: 0.0–40.3%), followed by the “health status of the sampled animals” (R2 min–max: 1.4–27.3%) and “geographical region” (R2 min–max: 5.9–23.6%).

Multiple enteropathogens such as bovine rotavirus (BRV), bovine coronavirus (BCoV), Escherichia coli K99 (ETEC) and Cryptosporidium spp. (Crypto) are the most common causes of calf diarrhoea during the first 30 days of animal age. Three weighted-stratified random-effects meta-analyses were performed to calculate the worldwide prevalence of mixed infections of the causative agents (i.e., BRV-BCoV, BRV-ETEC, BRV-Crypto) and their potential influencing factors. The meta-analysis covered 41 studies (94 sub-studies) in 21 countries that determined the presence or absence of mixed infections in global calf populations. The highest worldwide estimated pooled prevalence was identified for BRV-Crypto (6.69%), followed by BRV-BCoV (2.84%), and BRV-ETEC (1.64%). The chance of detecting BCoV in calves with diarrhoea was 1.83 higher in the presence of BRV compared to calves without BRV, whereby an inhibition effect (odds ratio: 0.77) was determined between BRV and Crypto infections. The diagnostic methods were identified as a significant influencing factor in the detection of all considered mixed infections, while the other analysed factors differed in relation to their effect on prevalence. In contrast to BRV-BCoV, the prevalence of BRV-ETEC and BRV-Crypto mixed infections followed the course of individual ETEC and Crypto prevalence related to the age class of the sampled animals.

Bovine rotavirus (BRV), bovine parvovirus (BPV), and bovine viral diarrhea virus (BVDV) are the pathogens that cause diarrhea primarily in newborn calves. A mixed infection of BRV, BPV, and BVDV makes clinical diagnosis difficult. In this study, we designed dual-priming oligonucleotide (DPO) primers the VP6 gene of BRV, VP2 gene of BPV, and 5′UTR gene of BVDV and synthesized gold nanoparticles (GNPs) with an average diameter of 10 nm. We combined the DPOs with the GNPs to develop a DPO-nanoPCR assay for detecting BRV, BPV, and BVDV. The annealing temperature, primer concentration, and GNP concentration were optimized for this assay. Compared to a conventional PCR assay, the DPO-nanoPCR assay allowed the use of a wider range of annealing temperatures (41–65°C) to effectively amplify target genes. PCR amplification was the most efficient at 56.2°C using conventional primers. The optimal volume of all the primers (10 μM) was 1.0 μL. The optimal volume of GNPs (10 nM) for all the reactions was 0.5 μL. The detection limits of DPO-nanoPCR for pMD19-T-VP6, pMD19-T-VP2, and pMD19-T-5′UTR were 9.40 × 102 copies/μL, 5.14 × 103 copies/μL, and 4.09 × 101 copies/μL, respectively; and those using conventional PCR were 9.40 × 104 copies/μL, 5.14 × 105 copies/μL, and 4.09 × 104 copies/μL, respectively. The sensitivity of DPO-nanoPCR was at least 100-fold higher than that of conventional PCR. The specificity detection showed that the DPO-nanoPCR was able to specifically detect BRV, BPV, and BVDV. Use of clinical samples indicated that target viruses can be detected accurately. Thus, DPO-nanoPCR is a new powerful, simple, specific, and sensitive tool for detecting mixed infections of BRV, BPV, and BVDV. [ABSTRACT FROM AUTHOR]

Bovine rotavirus (BRV) is one of main pathogens responsible for diarrhea, fever, and vomiting. In this study, we developed a colloidal gold immunochromatographic test strip for detecting BRV according to the principle of double-antibody sandwich. The monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) were prepared and purified. On the strip, the purified mAbs labeled with the colloidal gold were used as the detector, and the goat anti-mouse antibodies and purified pAbs were coated on the nitrocellulose membranes as the control line and the test line, respectively. We optimized different reaction conditions, including the amount of mAbs, the pH of colloidal gold solution, coating solution, blocking solution, sample pad treatment solution, antibody concentration in control line, and antibody concentration in detection line. In specificity assay, the strip had high specificity in detecting BRV. No cross-reaction was observed in detecting other viruses. The detection sensitivity of the strip was found to be 1 × 103 TCID50/0.1 mL. Two hundred twenty clinical samples were detected with the strip compared to reverse transcription-polymerase chain reaction. No false-negative or false-positive results were found, and the results obtained by the two methods were similar. In conclusion, we developed a novel immunochromatographic strip to rapidly detect BRV. The strip developed exhibited high sensitivity and specificity for BRV detection. It could be a rapid, convenient, and effective method for the rapid diagnosis of BRV infection in the fields. [ABSTRACT FROM AUTHOR]

• We developed a 1-step TaqMan real-time PCR method for detection of the Bovine Group A Rotavirus (BRVA). • The detection limits of the method for BRVA was 3.47 copies/μL. • This method has the advantages of rapid detection and avoidance of cross-contamination. • The method was applied in the detection of BRVA in 96 clinical samples. • The method provides reliable technological support for the detection, prevention, and control of BRVA infection. The purpose of this study was to develop a 1-step real-time quantitative fluorescence polymerase chain reaction (QF-PCR) method for detecting Bovine Group A Rotavirus (BRVA). The primers and probe were designed targeting the VP6 gene of BRVA. The standard substance was obtained through in vitro transcription. The primers, probe concentration, and annealing temperatures were optimized to determine the optimal system and conditions for the reaction. The specificity, sensitivity, and repeatability of the method were assessed and compared with a reported real-time QF-PCR method for clinical samples. The results indicated that the detection method can achieve a sensitivity of 3.47 copies/μL and exhibit good specificity by exclusively detecting BRVA without cross-reactivity to other common pathogens in cattle and sheep. The standard curve exhibited a robust linear correlation, and the amplification efficiency was calculated to be 105%. The intra-group and inter-group coefficients of variation were less than 2%. A total of 96 clinical samples were tested and compared with the real-time QF-PCR method that was reported. The coincidence rate was 90.63% (87/96). Furthermore, the clinical samples revealed that the prevalence of BRV in cattle from Fujian Province was 85.42% (82/96). This study has successfully developed a 1-step real-time QF-PCR method for BRVA, which offers an efficient and sensitive technical support for the rapid diagnosis and epidemiological investigation of BRVA. [ABSTRACT FROM AUTHOR]

Purpose: Bovine Rotavirus and Bovine Coronavirus are the most important causes of diarrhea in newborn calves and in some other species such as pigs and sheep. Rotavirus VP8 subunit is the major determinant of the viral infectivity and neutralization. Spike glycoprotein of coronavirus is responsible for induction of neutralizing antibody response. Methods: In the present study, several prediction programs were used to predict B and T-cells epitopes, secondary and tertiary structures, antigenicity ability and enzymatic degradation sites. Finally, a chimeric antigen was designed using computational techniques. The chimeric VP8-S2 antigen was constructed. It was cloned and sub-cloned into pGH and pET32a(+) expression vector. The recombinant pET32a(+)-VP8-S2 vector was transferred into E.oli BL21CodonPlus (DE3) as expression host. The recombinant VP8-S2 protein was purified by Ni-NTA chromatography column. Results: The results of colony PCR, enzyme digestion and sequencing showed that the VP8-S2 chimeric antigen has been successfully cloned and sub-cloned into pGH and pET32a(+).The results showed that E.coli was able to express VP8-S2 protein appropriately. This protein was expressed by induction of IPTG at concentration of 1mM and it was confirmed by Ni–NTA column, dot-blotting analysis and SDS-PAGE electrophoresis. Conclusion: The results of this study showed that E.coli can be used as an appropriate host to produce the recombinant VP8-S2 protein. This recombinant protein may be suitable to investigate to produce immunoglobulin, recombinant vaccine and diagnostic kit in future studies after it passes biological activity tests in vivo in animal model and or other suitable procedure.

The scientific community has reported several cases of microbes that exhibit elevated rates of antibiotic resistance in different regions of the planet. Due to this emergence of antimicrobial resistant microorganisms, the use of antibiotics as promoters of livestock animals’ growth is being banned in most countries around the world. One of the challenges of agricultural immunology therefore is to find alternatives by modulating the immune system of animals in drug-independent safe food production systems. In this regard, in an effort to supplant antibiotics from bovine feeds, several alternatives were proposed including the use of immunomodulatory probiotics (immunobiotics). The purpose of this review is to provide an update of the status of the modulation of intestinal antiviral innate immunity of the bovine host by immunobiotics, and the beneficial impact of immunobiotics on viral infections, focused on intestinal epithelial cells (IECs). The results of our group, which demonstrate the capacity of immunobiotic strains to beneficially modulate Toll-like receptor 3-triggered immune responses in bovine IECs and improve the resistance to viral infections, are highlighted. This review provides comprehensive information on the innate immune response of bovine IECs against virus, which can be further investigated for the development of strategies aimed to improve defenses in the bovine host.

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., Competing Interests: DL was employed by Tecon Biology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Chen, Li, Xu, Li, Ma, Liu, Yuan, Zhang, Fu and Shi.)