Incidence of group A and atypical rotaviruses in Brazilian pig herds

The incidence of rotaviruses as a gastroenteritis causal agent in piglets was studied in 19 pig herds of Sao Paulo State, Brazil, during 1985. From 302 diarrhoea samples collected during January (summer), 65 were positive for rotavirus when analysed by enzyme-linked immunosorbent assay (ELISA) and polyacrylamide gel electrophoresis (PAGE). Sixty-two of these samples belonged to the classical group A rotavirus, three to atypical rotaviruses (ELISA negative and probably group B) and one elicited a mixed electropherotype of group A and atypical rotavirus and was ELISA positive. Atypical viruses appear to be very fragile and were rapidly degraded upon storage of samples at -20 degrees C. Three herds where atypical rotaviruses were present in January were sampled again in August (winter). Nine atypical isolates out of a total 21 positive samples (assayed by electron microscopy and PAGE) were detected again in two of them.     

Primary isolation of cytopathic bovine rotaviruses on fetal rhesus monkey kidney cells

Primary isolation of bovine rotaviruses was successfully performed on rolling cultures of MA104 cells following trypsin treatment of fecal samples and cells. Fifty-one fecal samples were obtained from 22 herds affected with naturally-occurring acute diarrhea in calves during a period of over two years. Rotavirus particles were demonstrated in only 10 fecal samples by electron microscopy. Fourteen cytopathic bovine rotaviruses were isolated from positive samples and could be serially cultivated on MA104 cells. The presence of virus was identified by specific immunofluorescence in infected cells. These data indicated that approximately 30% of the herds affected with acute diarrhea in their calves were associated with rotavirus infection.     

Relative prevalence of typical and atypical strains among rotaviruses from diarrheic pigs in conventional swine herds

Polyacrylamide gel electrophoresis was conducted on genomic RNA extracted from rotaviruses detected in diarrheic pigs from conventional swine herds. Ninety samples contained sufficient virus for RNA band visualization and genome classification. Genome profiles were characteristic of typical group A rotaviruses in 67.8% of the 90 samples, of group B rotaviruses in 10.0%, and of group C rotaviruses in 11.1%. In 11.1% of the samples, the presence of more than 11 bands suggested concurrent infection with more than 1 strain of rotavirus. In infections among nursing pigs, 76.4% were group A rotaviruses, 7.4% were group B, 7.4% were group C, and 8.8% were coinfections. In infections among weaned pigs, 40.9% were group A, 18.2% were group B, 22.7% were group C, and 18.2% were coinfections. Coelectrophoresis with prototype OSU and Gottfried strains revealed a great diversity in electropherotype among field strains of rotavirus.     

Epidemiology of typical and atypical rotavirus infections in New Zealand pigs

Polyacrylamide gel electrophoresis (PAGE) and enzyme-linked immunosorbent assay (ELISA) were employed to investigate the epidemiology of typical and atypical rotavirus infections in five piggeries. Of 152 faecal samples examined, 46 (30 per cent) were positive by ELISA for group A rotavirus. Rotaviruses with electrophoretic patterns resembling groups A, B and C were detected. At least two and up to five different rotavirus electrophoretypes (typical and/or atypical) were detected in each of the five piggeries. Out of 152 faecal samples examined, 28 (18 per cent) contained rotaviruses with group A electrophoretypes, 9 (6 per cent) with group C but only 1 with Group B. Six samples contained both group A and group C rotaviruses. No common electrophoretypes of group A or C rotaviruses were detected in these five piggeries. The PAGE technique was also used to analyze group A rotavirus isolated sequentially from another piggery over a three year period. A single electrophoretype was found during the first two years, but in the third year a different electrophoretype was detected.     

Rotavirus excretion by village pigs in Papua New Guinea

Cohort studies were conducted on 29 pigs from 3 villages in the Highlands of Papua New Guinea. Animals ranged in age from 9 d to 5 m old. Three hundred and twenty nine faecal samples were collected from individual pigs followed over 3 to 6 w periods, and were examined for group A rotavirus antigen by ELISA, and rotaviral genomic RNA by polyacrylamide gel electrophoresis (PAGE). Electron microscopy was also conducted on selected samples. Group A rotavirus was detected in the faeces of 16 pigs with infected individuals coming from all villages. Non-group A rotavirus resembling group C was found in faeces from pigs from 2 villages. All of the group A rotaviruses examined had the same electrophoretype and this was distinct from that of the common type infecting humans in the area at the time of the study. None of the group A positive samples reacted with monoclonal antisera specific for human group A rotaviruses of serotypes 1, 2, 3, 4, or 8. The non-group A rotaviruses also all had identical electrophoretypes. In contrast to previous findings in intensive piggeries, rotavirus infection did not occur in all young pigs and was not limited to young animals under 2 m of age. Infected pigs varied in age from 12 days to 20 weeks of age. This pattern of infection was attributed to the non-intensive husbandry situations in the villages, with less opportunity for transmission to occur than in intensive piggeries.     

Epidemiology of typical and atypical rotavirus infections in New Zealand pigs

Polyacrylamide gel electrophoresis (PAGE) and enzyme-linked immunosorbent assay (ELISA) were employed to investigate the epidemiology of typical and atypical rotavirus infections in five piggeries. Of 152 faecal samples examined, 46 (30 per cent) were positive by ELISA for group A rotavirus. Rotaviruses with electrophoretic patterns resembling groups A, B and C were detected. At least two and up to five different rotavirus electrophoretypes (typical and/or atypical) were detected in each of the five piggeries. Out of 152 faecal samples examined, 28 (18 per cent) contained rotaviruses with group A electrophor etypes, 9 (6 per cent) with group C but only 1 with Group B. Six samples contained both group A and group C rotaviruses. No common electrophoretypes of group A or C rotaviruses were detected in these five piggeries. The PAGE technique was also used to analyze group A rotavirus isolated sequentially from another piggery over a three year period. A singIe electrophoretype was found during the first two years, but in the third year a different electrophoretype was detected.     

Detection of a mammalian-like group A rotavirus in diarrhoeic chicken

The present investigation describes detection of a mammalian-like electropherogroup A rotavirus in chicken with diarrhoea. This also records the first detection of a rotavirus in an avian species from India. During the investigation 75 diarrhoeic faecal samples collected from adult chicken were screened for the presence of group A rotavirus antigen by sandwich ELISA. All three samples positive for rotavirus antigen revealed 11 bands of RNA in polyacrylamide gel electrophoresis (PAGE). In contrast to avian group A rotavirus, segment 5 was found to migrate closer to 6 as is the case with mammalian group A rotaviruses. Segments 7, 8 and 9 were found to migrate as a tight triplet, which is characteristic of group A rotavirus.     

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)     

Epizootiology of bovine rotavirus infection.

Published information on rotaviruses as pathogens, the source of virus infection and the method of transmission of infection under normal conditions are reviewed. The antigenic differences between rotavirus isolates from children, calves, pigs, foals and mice are discussed. Bovine rotaviruses isolated in the USA and the UK were shown to be closely related antigenically and the US vaccine strain protected calves from challenge with the UK rotavirus. Nineteen normally reared calves, with 20 or more ZnSO4 units of serum delta globulin, were susceptible to rotavirus inoculation at two days of age. They developed diarrhoea, showed body weight loss but recovered. Three calves with less than 10 ZnSO4 units of serum delta globulin developed diarrhoea and died. In a serological survey of 654 adult cows and calves from three herds, between 2 per cent and 37 per cent of individuals in a group had low rotavirus antibody titres and were probably susceptible to rotavirus infection. These were found in all age groups of animals studied, whether or not the group had suffered a recent rotavirus epizootic. It was not possible to predict whether an epizootic would develop on the basis of a serological survey.     

An ELISA optimized for porcine epidemic diarrhoea virus detection in faeces

Monoclonal antibodies to porcine epidemic diarrhoea virus (PEDV) membrane protein M were prepared and used for the comparative assessment of three blocking ELISA variants to detect PEDV. The competitive blocking ELISA (CB-ELISA) format showed the highest sensitivity, allowing detection of 10(2.5) plaque-forming units of PEDV/ml in culture medium. Its specificity was verified by inclusion of control samples containing transmissible gastroenteritis virus (TGEV) and rotavirus A in each analysis. Eighty porcine field samples of faeces obtained from 38 herds affected with diarrhoea were examined, and PEDV was found in 15 (19%) samples from 6 (16%) herds. The suitability of the CB-ELISA for the screening herds in epizootiologic situations is discussed.     

Diversity and zoonotic potential of rotaviruses in swine and cattle across Europe

Group A rotaviruses can infect both humans and animals. Individual rotavirus strains can occasionally cross species barriers and might hereby contribute to the emergence of new genotypes in heterologous hosts. The incidence and impact of zoonotic rotavirus are not well defined, and one reason for this is a lack of data about strains circulating in suspected reservoir animal hosts. In this study we report the incidence, genetic diversity, and molecular epidemiology of rotaviruses detected in domestic cattle and swine in 6 European countries. From 2003 to 2007, 1101 and more than 2000 faecal specimens were collected from swine and cattle, both healthy and diarrhoeic, and tested for rotaviruses. Viruses from positive stools were genotyped and a subset of strains was characterized by nucleotide sequencing and phylogenetic analysis of the VP7 (G) and VP4 (P) genes. Rotaviruses were detected in 43% of bovine samples and in 14% of porcine samples. In cattle, 10 different combinations of G and P types were identified and the most common strains were G6P[11] and G6P[5]. In swine, the number of identified G-P combinations was higher (n=21), however, no single combination was predominant across Europe. Newly described genotype specificities, P[27] and P[32], were identified in swine. When compared at the nucleotide sequence level, the identified porcine rotavirus strains and contemporary human strains grouped together phylogenetically, whereas bovine rotavirus strains formed separate clades. These data demonstrate large genetic diversity of porcine and bovine rotavirus strains across Europe, and suggest that livestock herds may serve as potential reservoirs for human infections.     

The significance of bredavirus as a diarrhea agent in calf herds in Lower Saxony]

The objective of this investigation was to determine the distribution of Bredavirus in cattle herds in Lower Saxony and to evaluate its significance as potential cause of diarrhea in calves. Fecal samples and paired blood samples of 119 diarrheic and 46 healthy calves up to two months of age were collected from herds where diarrhea of calves was a problem. Fecal samples were examined for Breda-, rota- and coronavirus by solid phase immune electron microscopy and by ELISA, for K99-positive E. coli and salmonella by microbiological methods, and for cryptosporidia in smears. Antibody titers against Bredavirus, total serum protein and serum gamma globulin content were evaluated in the blood samples. Bredavirus was found in fecal samples from 5% (n = 6) of diarrheic calves which came from four different herds, but not in healthy calves. Rotavirus (31.9%), coronavirus (18.5%) and cryptosporidia (29.9%) were detected more frequently in fecal samples than Bredavirus. In this investigation rotavirus, coronavirus and cryptosporidia were present in addition in all herds where Bredavirus was found. In contrast to the low percentage of fecal samples containing Bredavirus, antibody titers in 75% of calves confirmed the high prevalence of Bredavirus infection in the cattle population of Lower Saxony.     

Use of an enzyme-linked immunosorbent assay for detection of infection with pseudorabies virus on a herd basis.

The use of an ELISA that can differentiate between swine infected with pseudorabies virus (PRV) and swine vaccinated with a specific PRV vaccine was evaluated on an individual and herd basis, and a system for interpreting ELISA results on a herd basis was developed. In 17 herds, recently introduced replacement gilts, seronegative for PRV, were vaccinated with a thymidine kinase- and glycoprotein X (gpX)-deleted vaccine. After vaccination, blood samples were collected from these gilts approximately every 1 to 2 months for up to 19 months. Serum samples were analyzed for antibodies to gpX antigen, using a commercially available ELISA kit according to the manufacturer's protocol. Herd status was determined as positive, suspect, or negative, according to the serum sample:negative control (S:N) values of the samples collected from the herd. From the 17 herds, 130 evaluations were performed. On 49 (38%) of the 130 herd evaluations, 1 or more gilts had suspect test results. Additional testing was required in 19 (39%) of these 49 herd evaluations to determine the PRV infection status of the herd. Status of herds having gilts with suspect results and no positive results was usually negative after retesting. Herds having gilts with positive results were unlikely to have negative status after retesting.     

Atypical rotavirus in chickens in Argentina

In Argentina the presence of rotavirus was investigated in a chicken flock experiencing periodic episodes of diarrhoea during the winter of 1986. All the samples analysed were negative by the enzyme-linked immunosorbent assay (ELISA). However, when samples were observed by electron microscopy, particles which were indistinguishable from standard rotaviruses were detected in some samples. Ten of the 36 samples were positive after polyacrylamide gel electrophoresis (PAGE) analysis, all of them showing the same electropherotype. Based on these results these viruses were classified as rotavirus-like or atypical rotaviruses.     

The stability of porcine rotavirus in feces

Rotaviruses are known as major causal agents of diarrhea in humans and animals. They affect young animals in intensive rearing and cause great economic losses. This study evaluated the infectivity of porcine rotavirus maintained for 32 months at approximately 10 degrees C in the original stool specimens. Thirty stool specimens of 1-4-week-old piglets from breeding farms located in the southwest of the State of Parana were selected for this study. They were randomly chosen from stool samples positive for rotavirus RNA by polyacrylamide gel electrophoresis (PAGE) at the time of collection. The thirty stool samples maintained for 32 months were re-tested by PAGE and 11 out of 30 were still positive showing physical integrity of the eleven segments of viral RNA. In order to demonstrate the maintenance of viral infectivity processed fecal homogenates were inoculated in MA-104 cell cultures. After an average of three blind passages 5 out of 11 samples demonstrated cytopathic effect similar to that of a simian rotavirus (SA-11) used as positive control. To confirm these findings an immunofluorescence test was performed and typical cytoplasmatic granular fluorescence was observed. Electron microscopy of stool samples showed that most of the virus particles were single-shelled and some were found to be in advanced state of degradation. The viral nucleic acid extracted from six fecal specimens out of those that showed physical integrity of rotavirus RNA by PAGE were also amplified when submitted to RT-PCR demonstrating stability of viral RNA. We therefore concluded that porcine rotavirus infectivity is maintained for a long period of time in stool specimens at low temperature.     

Isolation and molecular characterisation of equine rotaviruses from Germany

A total of 26 rotavirus positive faecal samples of diarrhoeal foals, and 8 equine rotavirus isolates were examined. Viral RNA patterns were generated, G typing was performed by PCR, and a P[12]-specific DNA probe was developed for P typing. Furthermore, five equine rotavirus isolates were sequenced in the genomic regions coding for VP7 and part of VP4. Rotaviruses of genotype G3 P[12] were found in 22 faecal samples and G14 P[12] type could be found in 4 faecal samples. These findings confirm that in Germany G3 P[12] is the predominating type of equine rotaviruses.     

The isolation, propagation and characterization of tissue-cultured equine rotaviruses

From 105 field cases of diarrhea in neonatal or young foals, rotavirus was detected by electron microscopy (EM) and/or by enzyme-linked immunosorbent assay (ELISA) in the feces of 65 foals on 16 different premises. ELISA was performed with Rotazyme test kits developed by Abbot and Company for the detection of rotaviruses. Twenty-four field isolates from the feces of diarrheic foals with equine rotavirus infection as ascertained by EM were placed in MA-104 cell cultures after pretreatment of the viral suspension with 10 micrograms ml-1 of trypsin and incorporation of 0.5 micrograms ml-1 or 1 microgram ml-1 of trypsin in Earle's minimal essential medium (MEM), 2% lactalbumen hydrolysate, and antibiotics. The isolates that replicated in cell culture produced varying degrees of cytopathic effect. After the 24 isolates had been transferred 5 or 7 times in cell culture, viral particles were observed in 17 by EM, and 22 had positive ELISA tests as determined by visual color chart and spectrophotometric readings. Concentrated tissue-cultured viral antigen of 9 isolates fixed complement using Nebraska calf diarrhea rotavirus calf antiserum while four isolates gave negative results. The same 13 tissue-cultured viral suspensions failed to fix complement using reovirus antiserum. The 9th passages of two isolates (EID1 and EID2) yielded titers of 10(4.45) ml-1 TCID50 and of 10(4.95) ml-1 TCID50, respectively, as measured by cytopathic effect. After 13 tissue-cultured passages, 2 other isolates, EID3 and EID4, each had titers of 10(6.2) ml-1 TCID50 and of 10(5.95) ml-1 TCID, respectively. Cytoplasmic or intranuclear inclusions were not seen in any cells of the MA-104 infected cell cultures. Small, but distinct, plaques in MA-104 cell cultures were produced by the EID1 isolate. Polyacrylamide gel electrophoresis tests of EID1 and EID2 isolates at the 9th cell passage and EID3 and EID4 isolates at the 13th cell passage each showed that the RNA genome had 11 segments with a migrating pattern that was identical for each isolate and characteristic of rotaviruses. These 4 equine tissue-cultured isolates when tested by ELISA, utilizing a monoclonal antibody serum pool that cross-reacted with many rotavirus isolates, each gave positive values comparable to rotavirus antigen controls.