Detection of rotavirus antigen in diarrhoeic and non-diarrhoeic piglets in Nigeria

Faecal samples from 96 piglets with acute gastroenteritis and 41 samples from non-diarrhoeic piglets were investigated for porcine rotavirus (PRV) antigens by enzyme-linked immunosorbent assay (ELISA). PRV was detected in 43 of 96 (44.8%) in the diarrhoeic group whilst none of the samples in the non-diarrhoeic group was positive for PRV (P less than 0.01). Age distribution of infection showed a higher infection rate of 30.2% in piglets aged 1-3 weeks, which was significantly higher (P less than 0.05) than in piglets aged 4-6 weeks, where the infection rate was 14.6 per cent. No PRV antigen was detected in diarrhoeic piglets above 6 weeks of age. Titration of 10 randomly selected ELISA-positive samples showed titres ranging from 512 to greater than or equal to 4.096.     

Detection and survival of group A rotavirus in a piggery

Samples of dust, faeces and effluent were collected from a piggery and examined for group A rotavirus, using a commercial ELISA test, electron microscopy and inoculation of MA-104 cells. Rotavirus antigen was demonstrated in samples collected from farrowing and weaner rooms but not from fattener and sow houses. Rotavirus antigen was also detected in samples collected from a weaner room which had been free of piglets for three months. A cytopathic porcine rotavirus (British isolate SW20/21) was kept at room temperature for four months; it survived with titres reduced by 2 log10. These observations suggest that the environment of commercial piggeries is an important source of rotaviral infection for young piglets.     

Detection of group a rotavirus strains circulating in calves in Tunisia

Faecal samples were collected from 89 dairy calves to determine the prevalence of rotavirus infection in Tunisia and the genomic diversity of bovine rotavirus strains. After screening of all faecal samples by enzyme-linked immunosorbent assay, rotavirus strains were analysed by RNA polyacrylamide gel electrophoresis and characterized antigenically by monoclonal antibodies to the VP6 subgroup. The VP7 genotype was determined by nested RT-PCR. Of the 89 calves tested, 27 (30%) were positive for rotavirus antigen. Four different long electrophoretypes were identified. All VP6 typeable strains carried the subgroup I specificity. G8 genotype was the most prevalent, but G6 and mixed strains G(6 + 8) were also detected.     

Molecular characterization of a novel bovine group A rotavirus

In this study, by partial sequence analysis of the genome segments encoding VP5* and VP7, we characterized a novel bovine group A rotavirus, namely, Tak2, that was detected from adult cattle diarrhea in Tochigi Prefecture, Japan. The nucleotide (nt) and deduced amino acid (aa) sequences of the genome segments encoding VP5* and half of the amino terminal portion of VP7 of Tak2 revealed a low identity with those of group A rotaviruses carrying previously published P and G type specificities (VP5*: nt identity, 61.6%-67.6% and aa identity, 58.0%-71.4%; half of the amino terminal portion of VP7: nt identity, 57.8%-73.5% and aa identity, 61.2%-70.9%). Additionally, phylogenetic analysis of the nt sequences of the genome segments encoding VP5* and half of the amino terminal portion of VP7 revealed that Tak2 formed a branch separate from the established P and G types. These results suggested that Tak2 could possess novel P and G types yet not reported among group A rotaviruses.     

Natural transmission of group A rotavirus within a pig population

Five litters of piglets born within two days of each other, together with their dams, were investigated for faecal excretion of group A rotavirus antigen from birth to two months old. All the 50 piglets in these litters became infected with the virus between 19 and 35 days old. Rotavirus excretion was first seen in one litter which was housed with other litters not included in this study. Two days later, piglets of the second litter in another farrowing room began to excrete rotavirus, and then infection spread to the other three litters in the same room. Within each of these litters, one or two piglets were infected early and thereafter infection spread to other piglets. It took four to 10 days for rotavirus to infect every piglet within a litter, and 16 days in total before all piglets in the five litters were infected. No rotavirus antigen was demonstrated in faeces from sows during the investigation period.     

An unusual case of concomitant infection with chicken astrovirus and group A avian rotavirus in broilers with a history of severe clinical signs

A molecular study of intestinal samples from 21 broiler flocks with a history of enteritis revealed that 23.8% and 14.3% were positive for chicken astrovirus (CAstV) and avian rotavirus (ARV), respectively. CAstV and group A ARV were simultaneously detected in only one broiler flock. Birds in this group developed the significant intestinal lesions characterized by frothy contents, paleness, and thin intestinal walls. In this report we present an unusual case of runting stunting syndrome (RSS) with a history of high mortality and growth retardation in broiler chickens. We also make the first identification of CAstV and group A ARV in broiler chickens in Korea.     

Isolation, identification and characterization of group A rotavirus from a chicken: the inner capsid protein sequence shows only a distant phylogenetic relationship to most other avian group A rotaviruses

Rotavirus particles were identified in the intestinal content of a 35-day-old stunted chicken. The virus was isolated, RNA pattern was analysed and the viral genome segment 6 was sequenced. In particular, the sequence data showed a very close similarity to the chicken rotavirus isolate Ch-1 (99.2% amino acid homology), this is distantly related to all known avian rotaviruses and supports the existence of different VP6 types amongst avian group A rotaviruses.     

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.     

Detection of a bovine group C rotavirus from adult cows with diarrhea and reduced milk production

Only two strains (Shintoku and porcine-like WD534tc) of group C rotavirus (GCR) from cattle have been reported to date. A GCR designated the Yamagata strain was the only pathogen detected in an outbreak of adult cow diarrhea accompanied by a decrease in milk production. The nucleotide sequences of the VP6 and VP7 genes from strain Yamagata were determined. Comparative sequence analysis showed that the sequence identities between strains Yamagata and Shintoku were markedly high in both VP6 gene (98.1%) and VP7 gene (93.5%), and that these strains belonged to the same clusters which were distinguished from GCRs from different host species in phylogenetic trees of these genes. These results suggested strongly that cattle species is one of the natural hosts of GCR infection, and that GCRs are a cause of adult cow diarrhea.     

我国A群牛轮状病毒感染的血清流行病学调查

为调查A群牛轮状病毒(BRV)在我国不同地区牛群中的感染和流行情况,本研究采用间接ELISA检测2005年～2006年期间在我国12个不同地区收集的1760份牛血清中抗A群BRV抗体。结果显示,其强阳性血清225份(12.8%);中等阳性血清1240份(70.4%);弱阳性血清279份(15.9%);阴性血清16份(1%)。总抗体阳性率高达99%。不同地区强阳性、中等阳性及弱阳性血清所占比例有所不同。结果表明,A群BRV在我国牛群中的感染和流行不但非常广泛,而且极为严重。本研究对我国BRV感染进行了较大规模的血清流行病学调查,其结果可为我国犊BRV腹泻疾病的防制提供重要的依据。     

猪A组轮状病毒VP4全基因在大肠杆菌中的表达与检测

以猪轮状病毒mRNA为模板,应用反转录聚合酶链式反应(RT-PCR)技术,扩增了2331bp的VP4全基因,通过T-A克隆技术,将PCR产物克隆至pGEM-T Vector中,成功地构建出克隆质粒pGEM-T-VP4.以BamHⅠ和SalⅠ双酶切pGEM-T-VP4和pGEX-6P-1,并将纯化的VP4基因亚克隆至融合型表达载体pGEX-6P-1中,构建出原核表达质粒pGEX-6P-VP4.将pGEX-6P-VP4转化至感受态E.coli BL21(DE3)中,经IPTG诱导和SDS-PAGE分析,可见约111.47 ku融合蛋白带.Western blot分析发现,该蛋白具有轮状病毒抗原性,从而为进一步研究轮状病毒的亚单位疫苗及DNA疫苗奠定基础.     

Prevalence of bovine group A rotavirus shedding among dairy calves in Ohio.

Fecal samples were collected from 450 neonatal calves, ranging from 1 to 30 days old, between May, 1988 and May, 1989 to estimate the prevalence of bovine group A rotavirus in a stratified random sample of Ohio dairy herds. Calves were from 47 dairy herds chosen to be representative of Ohio herds. Bovine group A rotavirus was detected in fecal samples by a cell culture immunofluorescence test (CCIF) and ELISA. Of 450 samples tested, 46 (10%) were positive by CCIF and 67 (15%) were positive by ELISA. The agreement beyond chance between the 2 assays was good (kappa = 0.65). The overall prevalence rate of rotavirus shedding was 16.4% (74/450). Forty-three percent (29/67) of the samples positive by ELISA were subgroup 1, none were subgroup 2, and the remaining 57% (38/67) could not be assigned to either subgroups 1 or 2. Thirty herds (62.5%) had at least 1 group A rotavirus-positive calf (mean number of samples per positive herd = 12.4), and 17 herds (37.5%) had no rotavirus-positive calves (mean number of samples per negative herd = 6.0). A live oral rotacoronavirus vaccine was used in neonatal calves of only 1 herd and 3 of 17 (17.6%) calves from this herd were positive for group A rotavirus. The percentage of the rotavirus-positive fecal samples from all calves (n = 450) when stratified by fecal consistency was as follows: 28.3% (13/46) had liquid feces; 25.6% (10/39) had semiliquid feces; 23.4% (22/94) had pasty feces; and 10.7% (29/271) had firm feces.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The shedding of group A rotavirus antigen in a newly established closed specific pathogen-free swine herd.

A longitudinal study was undertaken in a newly established specific pathogen-free (SPF) swine herd to determine the dynamics of rotavirus antigen shedding in a closed swine facility. Pregnant SPF gilts which populated the herd, and their offspring, were monitored weekly for three consecutive lactations. Fecal samples were assayed for the presence of group-specific viral antigen by a solid phase immunoassay (ELISA). Results indicate that in the week prior to farrow, 35% of samples from gilts/sows contained rotavirus antigen. During nursing, 37% of the gilts'/sows' fecal samples also contained virus antigen. Over the course of three farrowings, every gilt/sow in the herd excreted virus antigen. Virus antigen was present in 25% of the samples tested from nursing pigs and in 70% of the samples tested from pigs in the postnursing period; 95% of the litters excreted virus antigen either while nursing or postweaning. Seasonal incidence in virus antigen excretion was noted with proportionally more suckling pigs virus antigen-positive in summer and proportionally more sows/gilts positive during winter. Diarrhea occurred only rarely in the sampled population. Although piglets shed rotavirus subclinically, ELISA positive feces from piglets of each lactation caused severe disease when fed to neonatal gnotobiotic pigs. Electropherotyping of these passaged viruses indicated minor variation in RNA banding patterns over time.     

Detection of rotavirus infection by immunodiffusion

Three precipitin reactions associated with bovine rotavirus infection were demonstrable by immunodiffusion. One of the reactions has been utilized in a diagnostic test for the detection of rotavirus in faeces, or specific antibody to rotavirus group antigen in serum or faeces. The test, based on bovine materials, appeared to be group-specific and effective in demonstrating rotaviral antigen or antibody in other species of animals, including human beings. The procedure was as efficient as electron microscopy in detecting evidence of rotavirus in faeces of calves and a range of other species.