Comparative study of rotavirus strains of bovine and rabbit origin

Newborn calves were susceptible to infection with a rotavirus strain isolated from rabbits and, conversely, rabbits 4–5 weeks of age became ill and some died when inoculated with the rotavirus strain of bovine origin. However, the latter isolate appeared to be more virulent than the rabbit isolate in either calves or rabbits. Moreover, in reciprocal cross protection tests, carried out in calves and in rabbits, the rabbit rotavirus antiserum afforded only weak protection to challenge infection with either the homologous or the heterologous virus. By contrast, the protective level of the bovine rotavirus antiserum was relatively high.     

The efficacy of colostrum from cows vaccinated with rotavirus in protecting calves to experimentally induced rotavirus infection

Calves which were continuously fed colostrum from vaccinated cows for the first ten days of life, were fully refractory to experimental infection with strain 81/36 F of bovine rotavirus. By contrast, the response to virus exposure of calves which were treated with normal colostrum was identical to that of the control calves, in that they underwent severe diarrhea and a significant slowing of the growth rate. The antibody titer in the milk of vaccinated cows tends to decline rapidly so that it no longer provides any protective effect. Two alternatives were considered feasible in improving prophylaxis for rotavirus infections: (a) the continuous feeding of calves with 1st day colostrum as part of the ration throughout the period of greatest risk (first week of life), or (b) enhancing the efficacy of the vaccine in pregnant cows to the point where antibody concentration in the milk would remain at a protective level.     

牛A群轮状病毒G10型XJ-2022株分离鉴定及基因型分析

【目的】 试验旨在对新疆某规模牛场患腹泻疾病的犊牛进行病原学鉴定及基因型分析。【方法】 采用抗原诊断试剂盒方法对在新疆某牛场随机采集的15份腹泻犊牛粪便样品进行检测,对抗原检测结果为阳性的样品进行反复冻融和过滤处理,然后将样品接种于Marc-145细胞进行病毒的分离和传代。对分离毒株进行间接免疫荧光试验（IFA）和负染电镜观察进一步确定病原。对分离株的VP6和VP7基因进行PCR扩增测序,并对其进行基因相似性比对和进化树分析。【结果】 抗原诊断试剂盒结果显示,3份粪便样品呈牛轮状病毒（Bovine rotavirus,BRV）抗原阳性。将阳性粪便样品分别接种于Marc-145细胞,仅有1份样品连续盲传至第9代出现明显的细胞病变效应（CPE）。IFA结果显示,接种该分离株的Marc-145细胞有亮绿色荧光,对照组未见荧光。电镜观察可见约65 nm的圆形病毒粒子,并命名为XJ-2022株。经PCR扩增获得VP6和VP7基因的目的条带,长度分别为1 356和342 bp。基因相似性比对和遗传进化分析表明,VP6基因与人源A群轮状病毒参考株DB2015-066（LC367318.1）相似性最高且遗传进化亲缘关系最近;VP7基因与牛源G10轮状病毒参考株XJX2（MN937506.1）相似性最高,遗传进化亲缘关系最近,确定该分离株为A群G10型轮状病毒。【结论】 试验成功分离到基因型为A群G10型BRV XJ-2022株,该毒株为新疆地区首次发现的多宿主来源的基因重配病毒。     

Studies on cross protection induced in calves by rotaviruses of calves, children and foals.

Inoculation at birth with a live attenuated strain of a bovine rotavirus isolated in the USA (scourvax-reo) induced protection in five gnotobiotic calves seven to 21 days later against a UK isolate of pathogenic bovine rotavirus. However, no protection was induced in three calves challenged three to five days after vaccination. There was a close antigenic relationship demonstrated between the two bovine rotavirus isolates. In contrast only one of three gnotobiotic calves inoculated with foal rotavirus, and one of three with human rotavirus, were protected against bovine rotavirus challenge. Protection in these two calves correlated with high heterologous immunofluorescent antibody titre (320 or greater), although the neutralising antibody titres was less than 20.     

犊牛轮状病毒和大肠杆菌混合感染检测

黑龙江省某规模化奶牛场部分新生犊牛发生了严重腹泻且犊牛病死率较高。为了确诊,无菌采集病料,通过病毒检测、细菌分离、生化试验、细菌和病毒特异性基因PCR扩增、动物致病性试验及药敏试验等方法进行了病毒抗原和微生物检测。结果显示,病毒检测中牛轮状病毒（BRV）呈阳性,PCR扩增得到400 bp的特异性片段;从病料中分离到1株含K99和F41兼性菌毛抗原的高致病性产肠毒素大肠杆菌（ETEC）,该分离菌株对恩诺沙星、头孢唑啉、氯霉素敏感,对其他抗生素有一定的耐药性。流行情况调查结合病原学检查确诊该牛场新生犊牛严重腹泻是由BRV和大肠杆菌混合感染造成的,根据诊断结果进行了针对性地防制,取得了良好的防控效果。     

Detection and Genetic Evolution of Diarrhea-related Viruses in Chongqing Beef Cattle

In order to investigate the pathogen prevalence of bovine viral diarrhea in Chongqing, this study carried out an investigation of bovine viral diarrhea virus (BVDV), bovine coronavirus (BCV), bovine rotavirus (BRV) and bovine astrovirus (BAstV) on a total of 81 diarrhea samples of beef cattle which were collected from Chongqing by RT-PCR. After the PCR products were sequenced, phylogenetic analysis was performed with Mega 6.0 software. From 81 samples, the positive rate of BRV,BAstV and BVDV were 66.7%,8.6% and 7.4%, respectively,BCV was not detected.Phylogenetic analysis showed that 5 strains of BRV sequences were clustered into a small branch, which had significant genetic distance with other VP6 sequences in GenBank; 5 strains of BVDV, Chinese and Denmark strains were clustered into one branch,genetic relationship were close; 5 strains of BAstV clustered into a branch with Hongkong strain, but there was still an obvious genetic distance. The result showed that calves under the age of half year were the main group of beef cattle with diarrhea in Chongqing. BRV was an important cause of diarrhea in Chongqing, the genetic diversity of BRV,BAstV and BVDV could be reference to further concern.     

重庆肉牛腹泻相关病毒的检测及遗传进化分析

为了解重庆市肉牛病毒性腹泻的病原流行情况,本研究对重庆市8个肉牛养殖场的81份腹泻粪便样本中牛病毒性腹泻-黏膜病病毒（bovine viral diarrhea virus,BVDV）、牛冠状病毒（bovine coronavirus,BCV）、牛轮状病毒（bovine rotavirus,BRV）和牛星状病毒（bovine astrovirus,BAstV）4种致腹泻病毒进行了RT-PCR检测,对PCR产物进行测序,用Mega 6.0软件进行系统发育分析。结果显示,BRV、BAstV和BVDV检出率分别为66.7%、8.6%和7.4%,BCV未检出。遗传进化分析结果表明,测序的5个BRV单独聚为一小支,与GenBank中其他VP6序列有明显的遗传距离;BVDV与中国株和丹麦株聚为一支,遗传关系最近;5个BAstV单独聚为一支,与中国香港株遗传关系最近,但仍有明显的遗传距离。本试验结果表明,重庆地区肉牛腹泻主要发生在6月龄以下犊牛,BRV是该地区肉牛腹泻的重要原因,BRV、BAstV和BVDV 3种病毒的遗传多样性值得进一步关注。     

Detection of rotavirus in faecal specimens with a monoclonal antibody enzyme-linked immunosorbent assay: Comparison with polyclonal antibody enzyme immuno-assays and a latex agglutination test

Monoclonal antibodies have been produced against the 81/36F strain of rotavirus. One of them, was chosen as diagnostic reagent: it showed high ELISA reactivity with all the bovine, human and porcine rotavirus strains tested and reacted with VP6, structural protein product known to support the common rotavirus antigen.

A sandwich ELISA procedure using the chosen monoclonal as “capture and detecting” antibody was performed to detect rotavirus in faecal samples from experimentally inoculated newborn calves: it always gave a negative response with meconium and a positive response for the stool specimens which rotavirus have been isolated. This assay was compared with Enzygnost and Slidex Rota Kit tests and with a non-commercial sandwich ELISA test using polyclonal antibodies: it showed more sensitivity than the agglutination test and was as sensitive as the other two tests to detect rotavirus in routine diagnostic material. The test evaluated showed no equivocal results.     

The Test of Polyinfection of Bovine Rotavirus and Escherichia coli

Newborn calves in a dairy farms suffered from severe diarrhea disease with high mortality in Heilongjiang province.In order to make a definite diagnosis,the pathological samples were collected aseptically and the viral antigens and microorganism were detected by virus detection,bacteria separation,biochemical test,PCR for the specific gene of virus and bacteria,animal pathogenicity test and antimicrobial susceptibility test. The results showed that the virus detection of bovine rotavirus was positive and specific gene fragment of bovine rotavirus was amplified. One E. coli strain with K99 and F41 adhesin antigens which belonged to highly pathogenic enterotoxigenic E. coli was isolated. This strain was sensitive to enrofloxacin,cefamedin and chloramphenicol,however,it showed resistance for other antibiotics. A polyinfection of bovine rotavirus and E. coli were eventually confirmed through epidemic investigation and pathological diagnosis. It had significant preventive effect by taking effective measures according to the diagnosis results.     

一例冠状病毒、轮状病毒混合感染引起犊牛腹泻的诊治

2022年3月,河南省某规模化奶牛养殖场发生一起新生犊牛腹泻病例,在流行病学调查、临床症状观察、病理剖检的基础上,采集6头腹泻犊牛的新鲜粪便进行了寄生虫卵检查、小球隐孢子虫、牛轮状病毒、冠状病毒、大肠杆菌K99抗体检测,血液样品进行了牛病毒性腹泻病毒抗原检测,组织样品进行了细菌分离。结果表明牛轮状病毒抗原6头阳性,阳性率100%;冠状病毒抗原4头阳性、2头阴性,阳性率66.7%;其他病原均为阴性。根据临床症状、病理变化和实验室检测结果,确诊该病例为牛冠状病毒和轮状病毒混合感染引起的犊牛腹泻。根据诊断结果,采取了改善饲养管理、补液、收敛、止泻等综合性防治措施,疫情得到了有效的控制,为临床防治犊牛腹泻提供了参考。     

猪轮状病毒弱毒苗免疫研究

用猪源轮状病毒弱毒株－Ｎａ８６Ｆ９０制成的疫苗先后免疫母猪２５０头，乳中抗ＲＶ的ＩｇＡ最高滴度为８１９２０，对照组最高滴度仅有１６０；产后１个月，免疫组滴度仍维持在５１２０以上，而对照组仅４０；免疫组仔猪哺乳期轮状病毒检出率下降３７．２％，断奶仔猪育成率由７５％提高到９０％以上，断奶后窝重由平均１３０～１５０ｋｇ，提高到２００～２５０ｋｇ     

Longitudinal study of bovine rotavirus group A in newborn calves from vaccinated and unvaccinated dairy herds

Reports of rotavirus excretion in calves usually result from cross-sectional studies, and in face of the conflicting results regarding protection of calves born to vaccinated dams against diarrhea, the aim of the present study was to evaluate rotavirus excretion in dairy calves born to vaccinated or unvaccinated dams, to identify the genotypes of bovine rotavirus group A (RVA) strains isolated from these animals as well as to investigate characteristics of the disease in naturally occurring circumstances throughout the first month of life. Five hundred fifty-two fecal samples were taken from 56 calves, 28 from each farm and, in the vaccinated herd, 11/281 samples (3.91%) taken from six different calves tested positive for RVA while in the unvaccinated herd, 3/271 samples (1.11%) taken from 3 different calves tested positive. The genotyping of the VP7 genes showed 91.2% nucleotide sequence identity to G6 genotype (NCDV strain), and for the VP4 gene, strains from the vaccinated herd were 96.6% related to B223 strain, while strains from the unvaccinated herd were 88% related to P[5] genotype (UK strain). Genotypes found in this study were G6P[11] in the vaccinated herd and G6P[5] in the unvaccinated herd. All calves infected with rotavirus presented an episode of diarrhea in the first month of life, and the discrepancy between the genotypes found in the commercial vaccine (G6P[1] and G10P[11]) and the rotavirus strains circulating in both vaccinated and unvaccinated herds show the importance of keeping constant surveillance in order to avoid potential causes of vaccination failure.     

Detection of avian-like rotavirus A VP4 from a calf in Japan

A total of 568 normal feces from calves on a beef farm in Fukui Prefecture, Japan, in 2011–2012 were examined by RT-semi-nested PCR for rotavirus A (RVA) VP4 genes. Through partial sequencing and BLAST analyses of 84 VP4-positive specimens, we identified an avian-like RVA strain, N2342, which shares highest nucleotide identity (80.0%) with known avian-like bovine strain 993/83, in one specimen. Phylogenetic analysis also revealed a close genetic relationship between N2342 and avian RVAs, suggesting bird-to-cattle transmission. We observed frequent contact of wild birds with calves in the farm, suggesting that these birds were the source of the virus.     

Prevalence of enteric pathogens in the feces of healthy beef calves

Fecal specimens from 136 healthy beef calves (1 day to 12 weeks of age) were examined for the presence of infectious agents known to cause enteric disease in calves. The calves were selected from 22 herds in which all calves were free of clinically apparent enteric disease. Salmonella sp, enterotoxigenic Escherichia coli, Cryptosporidium, and coronavirus were not detected in any of the calves. Three calves were infected with rotavirus and 1 calf was infected with Yersinia enterocolitica. Campylobacter-like bacteria were isolated from 50 of 130 calves, with 36 of the calves positive for C jejuni. Seemingly, clinically normal calves may be infected more often with enterotoxigenic E coli, Cryptosporidium, coronavirus, or rotavirus in herds in which some calves have enteric disease than in herds free of major enteric disease. Campylobacter jejuni was well adapted to the bovine host and was of similar prevalence in diarrheal and nondiarrheal calves. The K99 positive, nonenterotoxigenic E coli was isolated from the feces of 16 healthy calves. Information in addition to the presence of K99 antigen is useful when diagnosing enterotoxic colibacillosis in calves.     

Identification of a bovine enteric syncytial virus as a nongroup A rotavirus

An atypical or nongroup A rotavirus was identified in feces obtained from gnotobiotic calves in which fecal preparations originally derived during an epizootic of neonatal calf diarrhea had been serially passaged. The epizootic was previously reported to be caused by a noncharacterized viral agent that induced the formation of epithelial syncytia on small intestinal villi of experimentally infected calves. This bovine, nongroup A rotavirus was found to be antigenically related to a described atypical rotavirus of rats by immunofluorescence and by enzyme immunoassay. Complementary DNA derived from the atypical rat rotavirus cross hybridized with RNA obtained from the bovine virus, but not with RNA extracted from group A rotaviruses. Complementary DNA derived from SA-11 group A rotavirus cross hybridized with other group A rotavirus RNA, but not with RNA obtained from either the rat or bovine nongroup A isolates. Additionally, another similar, if not identical, bovine atypical rotavirus was identified in a second epizootic of neonatal calf diarrhea that occurred several hundred kilometers and 8 months apart from the original epizootic.     

牛冠状病毒、牛轮状病毒和致病性大肠杆菌混合感染致犊牛腹泻的研究

为了明确新疆石河子某规模化奶牛场犊牛腹泻病死率较高的原因。本研究对腹泻犊牛的粪样及病死犊牛肠道内容物进行病原检测,并对分离菌株进行毒力基因检测、耐药基因检测、动物致病性试验及药敏试验。最终明确病因主要由牛冠状病毒(BCoV)和牛轮状病毒(BRV)和大肠杆菌混合感染引起。药敏试验表明,分离到的大肠杆菌对28种抗生素产生了严重的耐药,仅对多粘菌素和呋喃妥因敏感,并且发现该菌同时携带2种毒力基因和10种耐药基因。本研究分离的一株大肠杆菌携带有丰富的耐药基因,具有多重耐药性,为该奶牛场上临床治疗加大了难度。而本试验结果为该养殖场提供科学的临床药物指导,为该牛场治疗此次犊牛腹泻提供了依据,同时为新疆犊牛腹泻混合感染的防控提供了病例参考和技术资料。     

A study on neonatal calf diarrhea induced by rotavirus

This review summarizes the results of a study on rotaviruses isolated from calves affected by neonatal diarrhea.

The results indicated that rotavirus infection is widespread and supported the evidence for an etiologic role of these viruses in neonatal diarrhea. Differences in virulence among bovine rotaviruses appeared also to be confirmed.

Conventionally reared calves were fully susceptible to the experimental infection induced by rotaviruses originating from heterologous hosts, i.e. monkeys, pigs and rabbits.

When rotavirus strains of bovine, simian and rabbit origin were compared by cross neutralization tests, it was found the simian and porcine strains were indistinguishable and both appeared to relate antigenically to the bovine strain.

Finally, it was proven that feeding newborn calves with colostrum and first milk of their dams, previously vaccinated with an inactivated adjuvanted rotavirus vaccine, could prevent the neonatal diarrhea from occurring.     

Detection and differentiation of bovine group A rotavirus serotypes using polymerase chain reaction-generated probes to the VP7 gene.

Dot and Northern blot hybridization assays were developed to detect and differentiate group A bovine rotavirus serotypes using radiolabeled serotype 6 (Nebraska calf diarrhea virus [NCDV] and United Kingdom [UK] strains) or serotype 10 (Crocker [Cr] strain) VP7 gene probes. Partial length VP7-specific cDNA encompassing areas of major sequence diversity were generated by the polymerase chain reaction (PCR) using either cloned VP7 genes (NCDV and UK strains) or reverse transcribed mRNA (Cr strain) as templates. Radiolabeled probes prepared from the PCR-generated cDNA were tested at various stringency conditions to optimize the hybridization assays. At high stringency conditions (52 C, 50% formamide, 5 x standard saline citrate), the NCDV, UK, and Cr probes serotypically differentiated bovine rotavirus isolates in RNA samples prepared from cell culture propagated viruses or in fecal specimens from infected gnotobiotic calves. The sensitivity and specificity of NCDV and Cr VP7 probes were characterized in dot blot hybridization assays, and the probes were estimated to detect at least 1 ng of viral RNA. The serotyping results obtained using VP7 probes were similar to those obtained using serologic assays. Further development of these assays may provide a useful means for the rapid detection and differentiation of bovine rotavirus serotypes in fecal samples from calves in the field.     

Effect of combined rotavirus and Escherichia coli in neonatal gnotobiotic calves

Gnotobiotic calves (24 hours old) were monoinfected with calf rotavirus (CRV) strain NCDV, an enterotoxigenic Escherichia coli (ETEC) strain B44 (K99+), or a nonenterotoxigenic E coli (NETEC) strain 123 (K99-). Calves also were dually infected with CRV and either ETEC or NETEC. Eighteen calves equally allotted between 6 treatment groups were used in these studies: Noninfected controls--group A; CRV--group B; ETEC--group C; NETEC--group D; CRV + ETEC--group E; and CRV + NETEC--group F. Severe diarrhea and villous atrophy were observed in calves of treatment groups B, C, E, and F. Mortality was present only in treatment groups C and E as result of ETEC infection. There were no significant differences in the clinical responses or enteric lesions between treatments B and F, although a significant increase in the concentrations of NETEC was demonstrated in calves dually infected with CRV + NETEC (group F) as compared with calves monoinfected with NETEC (group D). Calves inoculated with ETEC (group C) had severe villous atrophy, neutrophilic infiltration of intestinal lumen, and moderate enterocyte necrosis. Calves dually inoculated with CRV + ETEC (group E) had the most extensive and severe lesions, similar to those in group C, plus a pronounced necrotic fibrino-hemorrhagic enteritis. Infection of enterocytes by CRV did not affect in any way the adherence of ETEC to the intestinal mucosa. Dual viral and bacterial infections of the same enterocytes were evident.