Ellagic acid inhibits bovine rotavirus in vitro and in vivo

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.

BackgroundBovine rotavirus (BRV) infection is common in young calves. This viral infection causes acute diarrhea leading to death. Rapid identification of infected calves is essential to control BRV successfully. Therefore development of simple, highly specific, and sensitive detection method for BRV is needed.ResultsA reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and optimized for rapid detection of BRV. Specific primer sets were designed to target the sequences of the VP6 gene of the neonatal calf diarrhea virus (NCDV) strain of BRV. The RT-LAMP assay was performed in a water bath for 60 minutes at 63°C, and the amplification products were visualized either directly or under ultraviolet light. This BRV specific RT-LAMP assay could detect 3.32 copies of subtype A BRV. No cross-reactions were detected with other bovine pathogens. The ability of RT-LAMP to detect bovine rotavirus was further evaluated with 88 bovine rectal swab samples. Twenty-nine of these samples were found to be positive for BRV using RT-LAMP. The BRV-specific-RT-LAMP results were also confirmed by real-time RT-PCR assay.ConclusionsThe bovine rotavirus-specific RT-LAMP assay was highly sensitive and holds promise as a prompt and simple diagnostic method for the detection of group A bovine rotavirus infection in young calves.

Background: Neonatal calf diarrhea is one of the most important problems of calf breeding in the world. It causes serious economic losses by causing deaths in calves in the 1st 10 days of their lives. Although many factors cause calf diarrhea, Escherichia coli (E. coli), Salmonella spp., Campylobacter spp., viral agents such as Bovine Rotavirus (BRV) and Bovine Coronavirus (BCoV) and parasitic agents such as Cryptosporidium spp., Eimeria spp. and Toxocara spp. frequently plays a role in calf diarrhea. In addition to these infectious factors, the formation of diarrhea has been linked to a number of other factors, including unfavorable barn conditions, mass rearing, inadequate cleaning and disinfection of barn tools, delaying the feeding of colostrum to newborn calves, failing to do so, and failing to disinfect the umbilical cord after delivery. The identification of the microorganisms and virulence factors responsible for diarrhea in newborn calves will direct the development of preventative measures and control strategies. The present study aimed to determine various virulence factors (Stx1, Stx2, STa, eaeA, K99 and F41) of Escherichia coli isolated from fecal samples and to investigate the prevalence of BRV, BCoV and E. coli K99 agents which play a role in the etiology of neonatal calf diarrhea in Burdur province. Materials, Methods & Results: This study was carried out in 75 different cattle farms with diarrhea problem in Burdur province and its districts. Rectal swab samples were taken from 90 calves with diarrhea between 0-4 weeks of age, using 2 sterile swabs from each animal. The swabs are used for the detection of BRV, BCoV and E. coli K99 by direct ELISA, and the isolation of E. coli. The swabs were cultured on blood agar including 7% sheep blood and MacConkey agar. The isolated bacteria were identified by conventional bacteriological culture methods such as Gram staining, triple tube method, oxidase. The bacteria isolated and identified as E. coli were stored at -20 ºC by using. Enzyme immunobinding assay (ELISA) was positive in 45 of the calf fecal samples. BRV was detected in 28 (31.11%) of samples, BCoV in 15 (16.67%) and E. coli K99 in 11 (12.22%) samples by ELISA. While BRV and BCoV were detected together in 5.56% of the samples, BRV and E. coli K99 were detected in 1.11% of samples, BCoV, E. coli K99 were detected in 1.11% of samples and all three infections were detected together in 1.11% of samples. The virulence factors of E. coli was investigated by polymerase chain reaction (PCR) and Stx1, K99, F41 and eaeA virulence genes were determined in 2, 5, 3, 4 of samples, respectively. K99 and F41 antigens were detected together only in 2 of the E. coli isolates. The according to ages, it was determined that the highest BRV was detected between 1-7 days of age and 8-15 days of age. While E. coli was detected in 5 of 1-day-old calves, BCoV was also detected in 1 of these calves. Discussion: In this study, it has been determined that neonatal calf diarrhea is mostly caused by BRV in Burdur province, followed by BCoV and E. coli. Even though E. coli was recovered from the samples, the inability to extract virulence factors suggests that additional virulence factors might potentially be involved in the infection. It was concluded that the determination of virulence factors of E. coli isolates can be a guide in the preparation of protection and control strategies in calf diarrhea. Keywords: calf diarrhea, bovine coronavirus, E. coli, bovine rotavirus, ELISA, PCR.

The correlation between maternal serum antibodies in beef calves at 2 days old and protection against diarrhea induced by natural bovine rotavirus (BRV) infection was examined. Virus neutralizing (VN) antibody titers against BRV in sera from calves that developed diarrhea by BRV infection within 14 days of age (BRV-diarrheal calves) were significantly lower than those from calves that had no diarrhea. In the BRV-diarrheal calves, a positive correlation was found between the VN antibody titers and age of the onset of diarrhea. There were negative correlations between the VN antibody titers and duration of the diarrhea, VN antibody titers and cumulative diarrhea scores, and the VN antibody titers and duration of virus shedding. These results suggest that the VN antibody titers against BRV in newborn calf serum could be an indicator of protection against BRV-induced diarrhea.

BackgroundBovine rotavirus (BRV) is a non-enveloped dsRNA virus that cause neonatal calf diarrhea. Lipid rafts are cholesterol-enrich membrane mircodomains that play a vital role in many cellular processes. In this study, the effect of cellular cholesterol depletion on infection of MA-104 cells with bovine rotavirus was investigated.ResultsWe demonstrated that cholesterol depletion of the plasma membrane by MβCD had no effect on BRV binding to cells but significantly impaired BRV entry in a dose-dependent manner and the effect was partially reversed by addition of exogenous cholesterol, suggesting the reduction of BRV infection by MβCD was specifically due to cholesterol depletion. Cholesterol depletion after virus entry did not reduce BRV replication, whereas affected virus assembly.ConclusionsTaken together, our results demonstrate that cell membrane cholesterol is essential to BRV infectivity.

Bovine rotavirus (BRV) causes massive economic losses in the livestock industry worldwide. Elucidating the pathogenesis of BRV would help in the development of more effective measures to control BRV infection. The MA-104 cell line is sensitive to BRV and is thereby a convenient tool for determining BRV–host interactions. Thus far, the role of the microRNAs (miRNAs) of MA-104 cells during BRV infection is still ambiguous. We performed Illumina RNA sequencing analysis of the miRNA libraries of BRV-infected and mock-infected MA-104 cells at different time points: at 0 h post-infection (hpi) (just after 90 min of adsorption) and at 6, 12, 24, 36, and 48 hpi. The total clean reads obtained from BRV-infected and uninfected cells were 74,701,041 and 74,184,124, respectively. Based on these, 579 were categorized as known miRNAs and 144 as novel miRNAs. One hundred and sixty differentially expressed (DE) miRNAs in BRV-infected cells in comparison with uninfected MA-104 cells were successfully investigated, 95 of which were upregulated and 65 were downregulated. The target messenger RNAs (mRNAs) of the DE miRNAs were examined by bioinformatics analysis. Functional annotation of the target genes with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) suggested that these genes mainly contributed to biological pathways, endocytosis, apoptotic process, trans-Golgi membrane, and lysosome. Pathways such as the mammalian target of rapamycin (mTOR) (mml-miR-486-3p and mml-miR-197-3p), nuclear factor kappa B (NF-κB) (mml-miR-204-3p and novel_366), Rap1 (mml-miR-127-3p), cAMP (mml-miR-106b-3p), mitogen-activated protein kinase (MAPK) (mml-miR-342-5p), T-cell receptor signaling (mml-miR-369-5p), RIG-I-like receptor signaling (mml-miR-504-5p), AMP-activated protein kinase (AMPK) (mml-miR-365-1-5p), and phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) signaling (mml-miR-299-3p) were enriched. Moreover, real-time quantitative PCR (qPCR) verified the expression profiles of 23 selected DE miRNAs, which were consistent with the results of deep sequencing, and the 28 corresponding target mRNAs were mainly of regulatory pathways of the cellular machinery and immune importance, according to the bioinformatics analysis. Our study is the first to report a novel approach that uncovers the impact of BRV infection on the miRNA expressions of MA-104 cells, and it offers clues for identifying potential candidates for antiviral or vaccine strategies.

Egg yolk IgY: Protection against rotavirus induced diarrhea and modulatory effect on the systemic and mucosal antibody responses in newborn calves

Background Bovine rotavirus (BRV) and bovine coronavirus (BCoV) are the most common viral agents in neonatal calf diarrhea and result in serious economic consequences. The aim of the study was to determine the epidemiology of those viruses in randomly selected dairy farms of Addis Ababa. Methods A cross-sectional study was conducted from November 2018 to April 2019 using a probability proportional to size (PPS) sampling technique. A total of 110 calves, less than 30 days of age, from 57 dairy herds were involved in the study. Associated factors of herds and calves were collected using semistructured interviews from farm owners and through physical observation of selected calves. Fecal samples were collected and analyzed using the sandwich ELISA method. Data generated from both semistructured interviews and laboratory investigation were analyzed using STATA_MP version 15. Results From the total 110 calves, 42 (38.18%) had diarrhea during the survey. The prevalence of bovine rotavirus and coronavirus was 3.64% (4/110) and 0.91% (1/110), respectively. Diarrhea, feeding colostrum timing, and sex of the neonatal calves had statistically significant association with bovine rotavirus infection (p < 0.05). All rotavirus-positive neonatal calves were identified in small scale dairy farms and in dairy farms that reported mortality though they lack statistically significant association. Only one coronavirus case was detected among the neonatal calves. The case was identified among small scale herds and in a herd with diarrheal cases. The sex of the coronavirus calf was female, diarrheic, and among 11-20 days old. Conclusion The prevalence of rotavirus and coronavirus infections in neonatal calves was seldom in dairy farms of the study area. Rotavirus was more common than coronavirus, and further studies should be initiated on other (infectious and noninfectious) causes of neonatal calf diarrhea in the area.

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

VP8* antigen produced in tobacco transplastomic plants confers protection against bovine rotavirus infection in a suckling mouse model

Bovine rotavirus (BRV) is one of the main pathogens that cause acute diarrhea in calves under one month of age. Passive immunization has been recognized as an effective way to prevent and treat BRV infection. Recent studies have shown that 10% of bovine antibodies possess an ultra-long CDR H3 domain, which has been shown to be the smallest antigen-binding domain. Due to the extremely small size of ultra-long CDR H3 antibodies, the phage display method was utilized to obtain ultra-long CDR H3 antibodies targeting BRV, providing a new approach for the prevention and/or treatment of BRV. Here, we report the preparation of BRV-specific bovine ultra-long CDR H3 antibodies obtained by constructing and screening a phage display library containing approximately 8.55 × 109 individual clones. Through three rounds of bio-panning, we identified 92 candidate clones, of which 79 exhibited specific binding activity in phage ELISAs. The recombinant bovine ultra-long CDR H3 antibodies could specifically bind to BRV in ELISAs and cell immunofluorescence assays. The neutralizing activity was further confirmed through virus neutralization tests. In the calf model experiment, the recombinant bovine ultra-long CDR H3 antibodies could relieve the symptoms of diarrhea, reduce both the amount and duration of virus release, and increase the survival in calves experimentally infected with BRV. Therefore, BRV-specific bovine ultra-long CDR H3 antibodies could serve as an effective agent for the prevention and treatment of BRV infection. At the same time, the development of ultra-long CDR H3 antibodies using phage display screening technology provides a new approach for developing biological agents for the prevention and control of infectious diseases in bovines.

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. 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+. 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. 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. ​.

Ellagic acid mitigates sodium arsenite-induced renal and hepatic toxicity in male Wistar rats.

The aim of this study was to investigate whether ellagic acid (EA) treatment can prevent changes in contractile function and Ca2+ regulation of cardiomyocytes in pathologic cardiac hypertrophy. Groups were assigned as Con group; an ISO group in which the rats received isoproterenol alone (5mg/kg/day); and an ISO + EA group in which the rats received isoproterenol and EA (20mg/kg/day) for 4weeks. Subsequently, fractional shortening, intracellular Ca2+ signals, and L-type Ca2+ currents of isolated ventricular myocytes were recorded. Protein expression levels were also determined by the Western blotting method. The survival rate was increased, and the upregulated cardiac hypertrophy markers were significantly attenuated with the EA treatment. The fractional shortening and relaxation rate of myocytes was decreased in the ISO group, whereas EA significantly improved these changes. Ventricular myocytes of the ISO + EA rats displayed lower diastolic Ca2+ levels, higher Ca2+ transients, shorter Ca2+ decay, and higher L-type Ca2+ currents than those of ISO rats. Protein expression analyses indicated that the upregulated p-PLB and p-CaMKII expressions were restored by EA treatment, suggesting improved calcium handling in the ISO + EA rat heart. Moreover, ISO rats displayed significantly increased expression of p-22phox and p47phox subunits of NOX2 protein. Expression of the p22phox subunit was reduced with EA administration, while the decrease in p47phox did not reach a significant level. The increased ROS impairs Ca2+ homeostasis and contractile activity of cardiac myocytes, whereas chronic EA administration prevents Ca2+ dysregulation and functional abnormalities associated with pathological cardiac hypertrophy via the diminution of oxidative stress.

Calf diarrhea continues to be the major problem of calves in the neonatal period. The effect of zeolites has been increasingly studied in ruminant health in recent years. In the present study, the efficacy of cristobalite, a zeolite, in neonatal calf diarrhea was studied first time. For this purpose, twenty-five neonatal calves with diarrheas were divided into two groups, and Group 1 (n=12) received conventional treatment and Group 2 (n=13) received cristobalite (Zoosorb 10 mg/kg) orally 3 times a day in addition to conventional treatment. Escherichia coli k99 and CS31a, bovine rotavirus and bovine coronavirus were isolated from fecal samples at the beginning of the treatment, on the third day and before discharge. It was determined that the recovery period in Group 2 was 0.95 (20.6%) days shorter than in Group 1 (p⟨0.05) while no viral agents were found on the fifth day in Group 2, viral shedding continued in 4 of 5 calves in Group 1. In conclusion, the study revealed that cristobalite speeds the recovery time and possibly decreases viral shedding in neonatal calf diarrhea, demonstrating a remarkable efficiency in the treatment.