Biomedical subjects
Publications and source records attributed to R F Bishop.
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Fecal flora of 12 children undergoing bone-marrow transplantation was monitored prospectively using comprehensive microbiological techniques. Diarrhea developed at least once in ten of the 12 children (83%), and a total of 24 episodes were recorded. Recognised gut pathogens were isolated from 11/21 (52%) diarrheal episodes where fecal specimens were obtained. Enteric pathogens identified included viral pathogens in 19% (rotaviruses, 'enteric' adenoviruses), parasites in 19% (cryptosporidium, Giardia lamblia) and cytotoxic C. difficile (14%). Excretion of clostridial species (including cytotoxin negative C. difficile, C. innocuum) occurred in 90% of diarrheal episodes when no enteric pathogen was identified. These results suggest that infection is often responsible for diarrhea associated with bone-marrow transplantation. Prophylaxis against enteric infection might reduce the morbidity and mortality associated with severe diarrhea in bone-marrow transplanted children.
An enzyme immunoassay utilizing neutralizing monoclonal antibodies to VP7 of four human group A rotavirus serotypes successfully typed rotaviruses in 71.4% (568 of 796) of fecal specimens. Sensitivity was enhanced by using homologous capture and detector antibodies. Serotyping was most successful with specimens stored for less than 3 years and containing 10(4) or more particles per ml.
Faeces obtained from 1,163 children (including 66 newborn babies) were analysed in parallel for the presence of rotavirus particles using two enzyme-linked immunosorbent assay kits. The kits had been formulated by the WHO Collaborating Centre for Reference and Research on Rotavirus (WHO-ELISA kit) and by DAKOPATTS (DAKO-ELISA kit) to be suitable for use in laboratories in developing countries. The kits were evaluated in laboratories in Burma, Chile, India, Mexico, Pakistan, Sri Lanka and the United Kingdom. Comparison of the results obtained with the two kits indicated that the DAKO-ELISA had an overall sensitivity of 97% and a specificity of 97% relative to the WHO-ELISA. In individual laboratories the DAKO-ELISA (K349) kit had a sensitivity in the range 90-100%, and a specificity of 85-100%. The kit showed a sensitivity of 100% and a specificity of 98% in assays on faeces obtained from newborn babies. We conclude that the DAKO-ELISA is as sensitive and specific as the WHO-ELISA for the detection of rotavirus antigen in faeces.
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The development of mucosal immunity is presumed to be the most important marker of rotavirus infection. The practical difficulties of obtaining small-bowel secretions stimulated this study of the antibody response to acute rotavirus infection at other sites. Forty-four infants admitted to the hospital with rotavirus gastroenteritis had serum, saliva, and feces collected at the acute phase (median, 5.5 days), during convalescence (median, 33.5 days), and 4 months later (median, 12.2 weeks). A subgroup of 19 children also had duodenal juice collected in parallel. Rotavirus-specific immunoglobulin G (IgG), IgA, secretory immunoglobulin, and IgM were measured and compared in all samples. The results showed that the estimation of antirotavirus serum IgM, serum IgG, duodenal juice IgA, and duodenal juice IgM by an enzyme immunoassay indicated an immune response to severe primary rotavirus infection in all children. Four months later, the levels of serum IgG and IgA served as the most sensitive markers of the preceding rotavirus infection. The predictive accuracies of immune responses at different sites in relation to a positive IgA immune response in the duodenum were calculated. Fecal IgA predicted duodenal IgA rotavirus antibodies with accuracies of 86% at 1 month and 92% at 4 months. The high sensitivity of serum IgM and IgG in detecting rotavirus infection and the high predictive accuracy of fecal IgA as an indicator of duodenal IgA abrogates the need for duodenal intubation to detect (or monitor) an immune response to rotavirus infection. This finding has important practical implications for epidemiological studies of acute diarrhea in children and in rotavirus vaccine trials.
Enzyme-linked immunosorbent assays were developed for the quantitation of anti-reovirus immunoglobulin G (IgG), IgA, and IgM in serum. The assays were specific, sensitive, and reproducible and measured antibodies to the reovirus group antigen. They should permit diagnosis of recent reovirus infection and longitudinal study of the development of reovirus group antibodies.
Rotaviruses were isolated from Australian farm animals (calf, pig and foal) with diarrhoea. Reciprocal cross neutralisation studies showed antigenic similarities with overseas animal strains and with human strains including a newly defined fifth human serotype.
Two group A, subgroup I, rotavirus strains possessing "super short" RNA patterns were adapted to growth in MA-104 cells. Both produced marked cytopathic effect in primary culture. Reciprocal cross-neutralization titers, polypeptide analysis, and the serum neutralizing antibody response of an infected child suggested that super short viruses are serotypically distinct from the four recognized human serotypes.
A neutralizing monoclonal antibody, B37:1, against a human rotavirus (B37) with a "super-short" RNA pattern was derived. The antibody gave serotype-specific reactions with a panel of standard rotaviruses and was reactive in an enzyme immunoassay. Epidemiological studies of this new human serotype should now be possible.
An enzyme immunoassay for serotyping human rotaviruses in stools and in cell culture was developed. Hyperimmune rabbit antisera to rotaviruses were used as capture antibodies, and rotavirus-neutralizing mouse monoclonal antibodies specific for serotypes 1, 2, 3, and 4 were used as detection reagents. Partial purification of monoclonal antibodies and inclusion of skim milk powder in antibody diluents contributed to assay specificity. The sensitivity of this assay was greater than that of a direct enzyme immunoassay in which rotaviruses of the appropriate serotype were adsorbed directly to the solid phase. When fecal extracts were concentrated threefold, this serotyping enzyme immunoassay was of equal specificity and approached the sensitivity of electron microscopy for rotavirus detection. This assay is simple and rapid and is suitable for serotyping the large numbers of isolates obtained from epidemiological studies and vaccine trials.
Twenty-three rotavirus strains obtained from the stools of 71 newborn babies were adapted to growth in MA-104 cells. Babies were housed in newborn nurseries of eight different obstetric hospitals in Melbourne between 1975 and 1979. All strains belonged to serotype 3 when reacted with serotype-specific neutralizing monoclonal antibodies in an enzyme immunoassay. Genome RNA of these 23 strains and of one stool virus not adapted to cell culture were compared by coelectrophoresis of mixtures of RNA. When strains were compared by coelectrophoresis of RNA for 4 h at 40 mA current, the majority appeared to be identical. Coelectrophoresis at 4 degrees C for 17 h at 10 mA current with 0.75-mm-thick polyacrylamide gels resulted in increased resolution of segments, revealing more genetic diversity than previously observed. Seventeen different electropherotypes showing slight variations in migration of one to seven segments were identified. Segments 5 and 7, 8, 9, 10, and 11 varied more frequently than segments 1, 2, 3, 4, and 6. Strains endemic in one hospital from 1975 to 1983 showed increased numbers of segmental changes over time. Differing patterns of reaction with two neutralizing monoclonal antibodies reacting with VP3 and VP7 were observed. Comparison of electropherotypes of three neonatal strains with a serotype 3 community strain showed marked differences in segment migration. The serotypic similarity, electropherotypic dissimilarity from community strains, and asymptomatic nature of most infections are additional evidence that these viruses infecting newborn babies form a unique group of rotaviruses.
Neutralizing monoclonal antibodies were derived to human rotaviruses RV-4 (serotype 1), RV-5 (serotype 2), and ST-3 (serotype 4). By enzyme immunoassay and fluorescent focus neutralization, eight of the antibodies appeared to be specific for the immunizing serotype, and so have potential as reagents for rotavirus serotyping by enzyme immunoassay. Seven of these were shown by Western blotting, enzyme immunoassay for antibody additivity, and reaction with rotavirus reassortants, to be directed against the major outer capsid glycoprotein. The remaining serotype-specific antibody immunoprecipitated the 84-kD outer capsid protein. One antibody reacted with all serotype 1 and 3 rotaviruses but not with serotypes 2 or 4. When tested with virus mutants, this antibody recognized an immunodominant determinant of neutralization shared between serotypes 1 and 3 on the major outer shell glycoprotein. Our results suggest that two outer capsid proteins possess determinants of neutralization, and that viruses of different serotypes may share immunodominant neutralization sites.
The etiology of acute diarrhea in children less than 42 months of age attending one pediatric hospital in Melbourne, Australia, was studied during a 7-month period encompassing the winter of 1984. Pathogens identified in 157 children treated as outpatients with mild disease were compared with those in 232 children hospitalized with severe disease. The pathogens (and frequencies among outpatients and inpatients, respectively) detected were rotaviruses (32.5 and 50.9%), enteric adenoviruses (8.9 and 7.4%), Campylobacter jejuni (7.2 and 1.3%), and Salmonella sp. (4.0 and 1.7%). Electropherotypes of rotavirus strains from outpatients and inpatients were compared. Two strains predominated during the 7 months of this study and were observed with equal frequency from outpatients and inpatients. Rotaviruses of the same electropherotype caused a wide spectrum of disease, with symptoms ranging from mild to severe, life-threatening diarrhea. The similarity of etiological agents identified from children with mild and severe forms of acute diarrhea suggests that the etiology of community enteric illness can be reasonably inferred from the etiology of inpatient disease in children in the same geographic area. During the winter epidemic period, the severity of symptoms associated with rotavirus infection in young children is likely to be determined by the inherent susceptibility of the host rather than by genetic differences in the strains of infecting rotaviruses.
Administration per os of 2 X 10(6) fluorescent cell-forming units of a human serotype 3 rotavirus (RV-3) protected all of nine gnotobiotic piglets against severe diarrheal disease when they were challenged 10 to 14 days later with 8 X 10(3) fluorescent cell-forming units of virulent wild-type porcine rotavirus (AT/76). The porcine virus was similar antigenically to porcine prototype strain OSU, previously described as antigenically distinct from all four recognized human serotypes. Administration of RV-3 was associated with the development of serum-neutralizing antibody to both RV-3 and AT/76 in piglets that excreted RV-3. Neutralizing antibody levels to RV-3 and AT/76 increased rapidly postchallenge. Vaccinated piglets were not immune to infection with AT/76 but showed no or minimal gastrointestinal symptoms after challenge. Control nonvaccinated piglets that were fed AT/76 developed severe dehydrating diarrhea and low levels of neutralizing antibody to AT/76 alone. The apparent heterologous clinical protection observed in this study could have been predicted from results of in vitro assays. Neutralization tests with reduction of fluorescence focus indicated a one-way cross-reaction between RV-3 and AT/76 such that hyperimmune antiserum to RV-3 neutralized porcine virus to moderate titer, but not vice versa. The results emphasize the importance of neutralizing antibody in protection against disease and the need to determine reciprocal cross-neutralization titers, rather than serotype alone, in order to predict the ability of rotavirus strains to cross protect.
Microflora of the gut was studied close to the onset of sporadic necrotizing enterocolitis (NEC) in neonates. Enteric flora in 25 babies with NEC differed from that in 23 matched controls. Bacteroides spp and lactobacilli were less common in babies with NEC compared with controls: 32 versus 61% (p = 0.03) and 12 versus 48% (p = 0.006), respectively. Clostridium perfringens was isolated from 40% of babies with NEC compared with 13% of controls (p = 0.03). It was present in 33% of babies with NEC aged less than 14 days but was not detected in control babies of the same age. The incidences of C. butyricum and C. difficile were similar in patients and controls. Colonization with C. perfringens in the absence of a protective barrier flora may be crucial in the pathogenesis of severe sudden onset NEC. Two potentially valuable marker factors to predict onset of this form of sporadic NEC are the use of fecal smears and estimation of fecal tryptic activity.
Cells producing neutralizing monoclonal antibodies to a serotype 3 human neonatal rotavirus strain RV-3 were derived by fusion of hyperimmunized mouse spleen cells with mouse myeloma cells. As ascites fluid, three rotavirus-neutralizing monoclonal antibodies were characterized by hemagglutination inhibition and reacted with 17 cultivable mammalian rotaviruses representing five virus serotypes, by fluorescent focus neutralization and enzyme immunoassay. Two antibodies, Mab RV-3:1 and Mab RV-3:2, reacted with the seven serotype 3 rotaviruses only. Mab RV-3:1 was shown to bind to the outer capsid glycoprotein gp34 of rotavirus when variants of SA 11 rotavirus were used, and it therefore appears to react with the major neutralization epitope of serotype 3 rotaviruses. The antibody Mab RV-3:3 was specific for an epitope of RV-3 rotavirus not present on any other rotavirus of any serotype tested, including another neonatal isolate of identical RNA electropherotype isolated from the same ward of the same hospital as RV-3 3 months earlier. These two viruses were also distinguishable by fluorescent focus neutralization, using antiserum to RV-3 virus. Western blot analysis showed binding of Mab RV-3:3 to the trypsin cleavage product of the outer capsid protein p86 of RV-3. This suggests that antigenic drift may have occurred among neonatal rotaviruses in Melbourne. These monoclonal antibodies will be useful in serotyping assays of rotaviruses directly in stool samples, and in further analysis of antigenic variation within the serotype.