Characterization of rotaviruses isolated from pigs with diarrhoea in Venezuela

The prevalence of porcine rotavirus infection was studied in 15 different herds located in the north-western region of Venezuela. The presence of rotavirus was studied by direct electron microscopy (EM) and by an enzyme-linked immunosorbent assay (ELISA). From 136 samples analyzed during the six months of the study (September 1983-February 1984), 38 (27.9%) were found to be positive for rotaviruses, with infection more common in animals that were 4-6 weeks old. Atypical rotaviruses were not detected in any of the samples examined. Most rotavirus positive specimens were subgrouped using specific monoclonal antibodies in an ELISA test. The majority of the samples (26 out of 38) were found to exhibit Subgroup I antigenicity. Only two specimens, collected from the same herd in two consecutive months, were found to belong to Subgroup II. To characterize further the circulating rotaviruses, electrophoretic analysis of the RNA genome was performed on samples selected from nine different herds. Great variability in the RNA electropherotypes was observed. No correlation was found between subgroup specificity and the migration of the two smaller segments (Genes 10 and 11), as has been described for human rotaviruses.     

Use of electron microscopy and an enzyme-linked immunosorbent assay for the detection of rotaviruses in neonatal calf diarrhea.

Electron microscopy and an enzyme-linked immunosorbent assay were compared for the diagnosis of rotaviruses associated with neonatal calf diarrhea. One hundred percent correlation was observed when 125 samples were tested by both techniques. Both techniques were equally efficient in detecting rotaviruses. The enzyme-linked immunosorbent assay was more sensitive but being specific could not detect other viruses.     

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.     

Use of a fast test to detect rotavirus in feces

The commercially available immunoassay "OnSite Rotavirus" was used for the detection of animal rotaviruses in 113 faecal samples. The sensitivity of the test was 88% and the specificity 96% compared with reference methods (EIA, EM). This test would detect approximately 4.4 x 10(6) to 1.8 x 10(7) virus particles per ml. The presence of virus could be demonstrated in fresh faecal samples from cattle, horses and pigs within a few minutes. The rotaviruses of group A were identified independently of the virus serotype. Further results and additional problems of using this test kit are described.     

Rapid coagglutination test for detection of rotaviruses in turkeys

Avian rotaviruses present in fecal samples were readily detected using a staphylococcal protein-A coagglutination test on a white porcelain plate. Staphylococci, which produced large amounts of protein-A, were coated with rabbit anti-avian rotavirus serum. The antibody-coated staphylococci were agglutinated specifically by rotavirus present in the fecal sample. The macroscopic agglutination reaction occurred within a few minutes. A total of 40 fecal samples were tested by the coagglutination test. The sensitivity and specificity of the coagglutination test were compared with those of electron microscopy, enzyme-linked immunosorbent assay, and tissue-culture virus-isolation methods. Of the 31 fecal samples positive for rotavirus on electron microscopy, 27 (87%) were positive on coagglutination test. Of the nine electron-microscopy-negative samples, seven (78%) were also negative on coagglutination test. It was concluded that the staphylococcal protein-A coagglutination test can be used as a simple, rapid screening test for avian rotavirus.     

Antiviral activity of Alpinia katsumadai extracts against rotaviruses

In vitro anti-rotavirus activity of Alpinia katsumadai (AK) extracts were evaluated against bovine G8P[7] and porcine G5P[7] rotaviruses in two different assay strategies, a mixed treatment assay and a post treatment assay. In the mixed treatment assay, six AK extracts [AK-1 (EtOH extract), AK-3 (H(2)O layer), AK-5 (40% methanol fraction), and AK-9-11 (H(2)O extract, polysaccharide fraction, supernatant fraction)] exhibited inhibitory activities against G5P[7] rotavirus with the EC(50) values ranging from 0.7±0.4 to 33.7±6.5 μg/mL. Extracts AK-1, AK-3, and AK-5 inhibited rotavirus infection against G8P[7] rotavirus, the with EC(50) values of 8.4±2.2 μg/mL, 6.5±0.8 μg/mL and 8.4±5.0 μg/mL, respectively. By hemagglutination inhibition (HI) assay, six AK extracts completely inhibited viral adsorption onto human RBCs in both strains of rotaviruses at less than 11 μg/mL. However, in the post treatment assay, there was no anti activity shown against both strains of rotaviruses. As a result, six AK extracts were attributed mainly to having a strong interaction with hemagglutinin protein on the outer surface of rotavirus, resulting to blockage of viral adsorption.     

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.     

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.     

Production and characterization of monoclonal antibodies against an avian group A rotavirus.

Fifteen monoclonal antibodies (MAbs) against an avian group A rotavirus were cloned and characterized. Eight of the 15 MAbs had neutralizing activity (N-MAbs). Five of the N-MAbs (1G1, 5B8, 4E2, 3G1, 2E3) were VP4-specific by radioimmunoprecipitation assay (RIPA), and two N-MAbs (2D11, 6E8) were possibly VP7-specific (faint bands by RIPA). One N-MAb (4H12) of undefined protein specificity cross-reacted with serotype 3 simian rotaviruses. The other seven N-MAbs did not cross-react with any of the eight distinct serotypes of human and mammalian rotaviruses tested. Of the seven non-neutralizing MAbs, three were VP6-specific (3H10, 4B12, 5F6), two were VP8-specific (6C9, 1D1), one was VP4-specific (4E9), and one was of undefined protein specificity (1B11). Four non-neutralizing MAbs recognized only avian group A rotavirus in cell-culture immunofluorescence tests (6C9, 1D1, 4E9 and 5F6), whereas two MAbs (3H10 and 4B12) cross-reacted with all human and animal rotaviruses tested. The MAb 1B11 did not recognize any human rotavirus serotypes but cross-reacted with all nonhuman animal rotavirus serotypes. The MAbs produced in this study should be useful for the detection and further characterization of avian group A rotaviruses.     

Comparison of polyacrylamide gel electrophoresis, an enzyme-linked-immunosorbent assay, and an agglutination test for the direct identification of bovine rotavirus from feces and coelectrophoresis of viral RNAs

The dsRNA concentrated polyacrylamide gel electrophoresis (CPAGE) detected rotavirus directly from 19% of 77 stool specimens from diarrheic calves. A commercial enzyme-linked immunosorbent assay (ELISA) detected 25%, latex agglutination test, 23%, and polyacrylamide gel electrophoresis (PAGE), 19%. Establishing CPAGE as the "standard," the commercial ELISA and the latex agglutination test both had higher sensitivity (84%) than PAGE (79%). However, PAGE produced the highest specificity (100%), followed by agglutination (88%) and ELISA (84%). The commercial ELISA had a slightly higher sensitivity than agglutination, PAGE, and CPAGE, but the ELISA specificity was generally lower. The latex agglutination test had a lower sensitivity than ELISA, but specificity was higher. Agglutination had similar negative predictive values (94%), compared with agglutination and PAGe, but had the lowest positive predictive value (a measure of accuracy) (70%). Agreement with CPAGE was highest for PAGE (94.8%), followed by agglutination (87%) and ELISA (84.4%). The calculated percentages of total disagreement with all other tests indicated that ELISA differed from the other rotavirus detection assays in 10.4% of the cases, agglutination in 7.8%, PAGE in 2.6%, and CPAGE in 1.3%. The 2 PAGE assays allowed the detection of atypical rotaviruses from feces based on the characteristic "super-short" migration pattern of the 11 genomic segments of rotaviruses and of other members of the Reoviridae.     

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.     

Serotyping and antigenic comparison of some animal rotaviruses isolated in China.

Eight strains of rotaviruses isolated from diarrheal animals (4 from calves and 4 from piglets) in China were compared by serotyping with reference animal rotavirus strains (bovine NCDV, porcine OSU and simian SA-11 and human rotavirus Wa strain). Two-way cross neutralization test showed no antigenic difference between all 4 local strains of bovine rotavirus (BRV007, BRV014, HN-7 and BRV6555) and reference NCDV, so they belonged to rotavirus serotype 6 (bovine rotavirus serotype 1 or NCDV-serotype). Meanwhile, the four strains of Chinese porcine rotavirus could be determined into 2 different serotypes. One (Li99) was neutralised to a high titer with the antiserum against reference OSU virus and probably related to OSU (serotype 5 or porcine serotype 1). The other three strains (Lin71, Nan86 and Jiang150) were antigenically obviously different from Li99 and did not react with the antiserum against OSU. They were tentatively considered as porcine rotavirus serotype 2. All the strains of bovine and porcine rotavirus did not cross-neutralise with simian SA-11 and human Wa strain. There was also no antigenic relationship between bovine rotaviruses and porcine rotaviruses.     

Routine isolation and cultivation of bovine rotaviruses in cell culture

Using the bovine embryonic kidney cell line, Aubek, bovine rotaviruses were routinely isolated from fecal samples of calves with diarrhea. Of 125 fecal samples positive for rotavirus by immune electron microscopy and the enzyme-linked immunosorbent assay, 61 isolates were recovered and cultivated continuously.     

A case-control study of selected pathogens including verocytotoxigenic Escherichia coli in calf diarrhea on an Ontario veal farm.

A case-control study of diarrheal disease in veal calves was conducted over a three month period on a single large veal farm in southern Ontario. One hundred diarrheic calves (cases) were identified by visual examination of their feces. Each case was matched to two nondiarrhetic controls from the same room on the same day, and a fecal sample was obtained from each animal. Fecal consistency of cases and controls was observed daily for one week following sample collection. Control calves which developed diarrhea during that period were excluded from the study. Breed, sex and the date and nature of antimicrobial drugs administered to each calf were recorded. Moisture content of fecal samples was measured by weighing samples before and after oven drying. Samples were screened for verocytotoxigenic Escherichia coli (VTEC) using a Vero cell assay, for enterotoxigenic E. coli (ETEC) using an immunoblot procedure with anti-K99 monoclonal antibodies, and for Salmonella species using modified semi-solid Rappaport-Vassiliadis medium. A latex agglutination test was used to detect rotaviruses, and samples were examined for cryptosporidia using sucrose wet mounts. No VTEC were identified in cases or controls. One calf was positive for Salmonella and three were positive for ETEC. Rotaviruses were detected in four cases and four controls. A significant positive association was found between diarrhea and infection with Cryptosporidium. This study thus provided no evidence of an association between diarrhea and infection with either VTEC, ETEC, Salmonella spp. or rotaviruses in the population examined. On the other hand our results do suggest that Cryptosporidium infection may promote transient diarrheal disease in veal calves in Ontario.     

Typing by Polymerase Chain Reaction of Buffalo Rotaviruses Isolated in Italy

Six group A rotaviruses were isolated in Italy from buffaloes during different outbreaks of calf diarrhoea occurring in the same dairy herd over a 5-year period of observation. The isolates were characterized by polymerase chain reaction assay for G- and P-type antigens. G8 and P1 were the types most frequently isolated.     

Typing by polymerase chain reaction of buffalo rotaviruses isolated in Italy.

Six group A rotaviruses were isolated in Italy from buffaloes during different outbreaks of calf diarrhoea occurring in the same dairy herd over a 5-year period of observation. The isolates were characterized by polymerase chain reaction assay for G- and P-type antigens. G8 and P1 were the types most frequently isolated.     

Primary isolation and identification of avian rotaviruses from turkeys exhibiting signs of clinical enteritis in a continuous MA 104 cell line

Avian rotaviruses were isolated from turkeys with enteritis using MA 104 cell line. MA 104 cells were suitable for primary isolation and propagation of avian rotaviruses. Trypsin appeared essential for the enhancement of infectivity and the occurrence of cytopathic effect (CPE). Serum neutralization (SN), electron microscopy (EM), and analysis of genomic RNA were done to identify and confirm the identity of rotaviruses. Electrophoretic migration patterns of genomic RNA from avian rotaviruses were examined, and they were compared with those from mammalian rotaviruses. The migration patterns differed between these groups.