Biomedical subjects

G F Browning

Publications and source records attributed to G F Browning.

At least 55 records · Page 3Linked to original sources

PubMed1997

Mycoplasma synoviae has two distinct phase-variable major membrane antigens, one of which is a putative hemagglutinin.

Mycoplasma synoviae is a major pathogen of poultry, causing synovitis and respiratory infection. A cluster of 45- to 50-kDa membrane proteins is immunodominant in strain WVU-1853. Four distinct proteins were identified in this cluster by high-pressure liquid chromatography. Monoclonal antibodies and monospecific antisera against each established that they fell into two groups, MSPA and MSPB, each containing two members distinguishable by a difference in hydrophobicity. A 25- to 30-kDa membrane protein (MSPC) was shown to be antigenically related to the MSPB proteins. Considerable variation in the size and expression of MSPA and MSPB was observed among different strains of M. synoviae. Examination of expression in colonies of strain WVU-1853 established that both MSPA and MSPB (and MSPC) were phase variable. Immunostaining of MSPB (and MSPC) with monoclonal antibodies exhibited quantal variation, with three distinct levels observed between and within colonies. Hemadsorption by M. synoviae colonies was also found to be phase variable, with some colonies exhibiting sectorial expression of hemadsorption. Monospecific antisera against MSPA inhibited hemagglutination, but neither monoclonal antibodies nor monospecific antisera against MSPB could inhibit hemagglutination. However, loss of the capacity to hemadsorb by individual clones was associated with loss of expression of both MSPA and MSPB. These findings have elucidated the complexity of structure, function, and expression of the 45- to 50-kDa membrane protein cluster of M. synoviae, and they suggest that all members of the cluster may be involved in adhesion.

Antibodies, Monoclonal↗

PubMed1997

Prevalence of the virulence-associated gene of Rhodococcus equi in isolates from infected foals.

The prevalence of the plasmid-encoded virulence-associated gene (vapA) of Rhodococcus equi, as determined by PCR, was found to be 98% in isolates from 154 foals with pneumonia, confirming the strong association of vapA with virulence. The vapA genes from 60 representative isolates were compared by digestion with the restriction endonuclease HinfI, and no evidence of sequence variation was detected.

Actinomycetales Infections↗

PubMed1996

Equine rhinovirus 1 is more closely related to foot-and-mouth disease virus than to other picornaviruses.

Equine rhinovirus 1 (ERhV1) is a respiratory pathogen of horses which has an uncertain taxonomic status. We have determined the nucleotide sequence of the ERhV1 genome except for a small region at the 5' end. The predicted polyprotein was encoded by 6741 nucleotides and possessed a typical picornavirus proteolytic cleavage pattern, including a leader polypeptide. The genomic structure and predicted amino acid sequence of ERhV1 were more similar to those of foot-and-mouth disease viruses (FMDVs), the only members of the aphthovirus genus, than to those of other picornaviruses. Features which were most similar to FMDV included a 16-amino acid 2A protein which was 87.5% identical in sequence of FMDV 2A, a leader (L) protein similar in size to FMDV Lab and the possibility of a truncated L protein similar in size to FMDV Lb, and a 3C protease which recognizes different cleavage sites. However, unlike FMDV, ERhV1 had only one copy of the 3B (VPg) polypeptide. The phylogenetic relationships of the ERhV1 sequence and nucleotide sequences of representative species of the five genera of the family Picornaviridae were examined. Nucleotide sequences coding for the complete polyprotein, the RNA polymerase, and VP1 were analyzed separately. The phylogenetic trees confirmed that ERhV1 was more closely related to FMDV than to other picornaviruses and suggested that ERhV1 may be a member, albeit very distant, of the aphthovirus genus.

Amino Acid Sequence↗

PubMed1996

Prevalence of G and P serotypes among equine rotaviruses in the faeces of diarrhoeic foals.

Variant types of VP4 and VP7 gene segments of faecal rotaviruses from diarrhoeic foals were identified by restriction endonuclease digestion of reverse transcription/polymerase chain reaction (RT/PCR) products. The variants observed were correlated with serotypes by determination of the sequence of representative RT/PCR products (entire coding sequence for VP7 and the VP8 region of VP4) and comparison to published sequences of equine G and P serotype genes. Both G and P serotypes could be predicted for 95/116 (82%) strains, P serotype only for a further 8 (7%) strains and G serotype only for 1 (1%) strain. All characterised strains belonged to the same P serotype, P12, although minor sequence variations were observed. Of those strains able to be assigned to G serotypes, 84/96 (87.5%) belonged to serotype G3A, and 12/96 (12.5%) belonged to serotype G14. Comparison of G serotyping by ELISA to the RT/PCR method showed that serotyping equine rotaviruses by currently available ELISA methods was prone to error. This study establishes the restricted serotypic diversity of equine rotaviruses, and the significance of serotype G14.

Amino Acid Sequence↗

PubMed1996

Characterisation of haemolytic RTX toxins produced by Australian isolates of Actinobacillus pleuropneumoniae.

The haemolytic RTX toxins of 27 isolates of Actinobacillus pleuropneumoniae, representing all serovars that have been isolated in Australia, were characterised. The quantity of protein secreted by these isolates into the media was not significantly different between serovars, but haemolytic activity was detected only in the unconcentrated supernatants from cultures of serovar 1 and 5 isolates. Haemolytic activity in supernatants of serovar 2, 3 and 7 isolates was detected only after the supernatants were concentrated. On Southern hybridisation blots, genomic DNA of serovar 1 and 5 isolates contained regions that were similar to the cloned structural genes for ApxI (apxIA) and for ApxII (apxIIA). In contrast, genomic DNA of serovar 2, 3 and 7 isolates only contained regions similar to, if not identical with, the cloned apxIIA gene. The haemolytic activity of the culture supernatant depends on the type or composition of media and adaptability of the bacteria to in-vitro cultivation. Low passage cultures of A pleuropneumoniae, which were characterised by waxy colonies, produced significantly weaker haemolytic activity than A pleuropneumoniae after several passages in vitro.

Actinobacillus Infections↗

PubMed1995

A type-specific serological test to distinguish antibodies to equine herpesviruses 4 and 1.

We describe a type-specific ELISA, which distinguishes antibody to equine herpesvirus 4 (EHV4; equine rhinopneumonitis) and EHV1 (equine abortion virus) thereby identifying horses that have been infected with either or both of these antigenically related viruses. The antigens used are parts of the EHV4 and EHV1 glycoprotein G (gG) homologues expressed in E. coli as fusion proteins [Crabb and Studdert, 1993: J Virol 67: 6332-6338). The expressed proteins comprise corresponding regions of the gG molecules that are highly divergent and encompass strong, typespecific epitopes. Plasma samples from 97 Thoroughbred and 174 Standardbred horses were tested, all of which were unvaccinated. All horses were strongly EHV4 ELISA positive while 30% were EHV1 ELISA positive. The type-specificity of the EHV1 gG antigen was tested in cross-absorption experiments and it was found that 96% (66 of 69) of EHV1 ELISA positive horses were true EHV1 antibody positives. It was also shown that 100% (26 of 26) horses known to have been exposed to EHV1, either by infection or immunisation with EHV1, had significant levels of antibody against the EHV1 gG antigen (i.e., all horses recognised the EHV1 epitope(s) contained within this molecule). Maintenance of EHV1 gG antibody was examined by testing sera obtained from mares four years after confirmed EHV1 abortion. Seven out of 10 of these mares remained EHV1 ELISA positive. In summary, the ELISA is highly specific and is sufficiently sensitive to detect all horses previously infected with EHV4 and most previously infected with EHV1.

Animals↗

PubMed1995

Expression studies on four members of the pMGA multigene family in Mycoplasma gallisepticum S6.

A large family of related genes known as pMGA exists in the avian pathogen Mycoplasma gallisepticum but only a single member of this family was previously found to be expressed in one strain of this bacterium. In this work two unrelated strains of M. gallisepticum were also shown by amino-terminal sequencing to express a unique pMGA polypeptide in both cases. To investigate pMGA gene selection in M. gallisepticum, mRNA expression was analysed in M. gallisepticum strain 56 using reverse transcription-PCR (RT-PCR) and Northern blot techniques with probes for several members of the pMGA multigene family. It was shown that the pMGA message is 2.2 kb in size and is monocistronic. RT-PCR detected four different pMGA mRNA molecules but their relative yields were significantly affected by magnesium concentration. By quantitative Northern analysis, the relative abundances of the four pMGA mRNAs in M. gallisepticum S6 total RNA was determined: the pMGA1.1 mRNA predominated [1.88 ng (micrograms total RNA)-1] but at least three other pMGA genes were found to be transcribed but at much lower levels (20 to 40-fold lower). The pMGA1.1 mRNA is expressed at a level five times higher than the tuf gene, known to be one of the most abundantly expressed proteins in the prokaryotic cell. The start point of transcription for pMGA1.1 was determined and probable promoter assigned. From these data it appears likely that transcriptional control plays a major role in the selection of pMGA gene expression in the M. gallisepticum cell.

Amino Acid Sequence↗

PubMed1994

The organisation of the multigene family which encodes the major cell surface protein, pMGA, of Mycoplasma gallisepticum.

The genome of the avian pathogen Mycoplasma gallisepticum contains a number of related genes for putative adhesion molecules (pMGA). Cloning and sequence analysis of several pMGA genes suggested that all of them might be transcriptionally and translationally functional. Analysis of the gene sequence encoding the sole pMGA variant expressed in vitro in the S6 strain (pMGA1.1) revealed no unambiguous feature that could account for its unique expression. It is estimated that the pMGA gene family may contain up to 50 members, and its possible role is discussed herein.

Amino Acid Sequence↗

PubMed1994

Veterinary education in the era of information technology.

A major innovation in the delivery of the veterinary curriculum is being implemented at The University of Melbourne using the subject of systematic bacteriology and mycology as a pilot project. Students receive course information as interactive, multimedia databases. These consist of text and an associated library of catalogued digital images, movies and sounds. The databases employ a hypermedia information system to achieve efficient integration within and between subjects. The new delivery method encourages greater autonomy and more active learning roles for students than occurs in traditionally taught courses. Students will use their databases as the principal resource of information for undergraduate studies. A unique feature of this system for delivering the curriculum is that students will modify and expand their databases during the course. The ultimate aim is for students at graduation to receive, on disc, a copy of their own databases, adapted by themselves to their particular future professional needs. As graduate veterinarians they will continue to use their databases as a major resource for information and learning, thus providing continuity from undergraduate to continuing postgraduate education.

Animals↗

PubMed1994

Functional and structural changes of porcine alveolar macrophages induced by sublytic doses of a heat-labile, hemolytic, cytotoxic substance produced by Actinobacillus pleuropneumoniae.

Alterations in the size and functions of porcine alveolar macrophages exposed to sublytic amounts of heat-labile, hemolytic cytotoxin produced by Actinobacillus pleuropneumoniae (App) serotype 1, strain HS54 into the culture medium were studied in vitro. Alveolar macrophages were sensitive to the cytotoxin; treatment of the macrophages with low concentrations of cytotoxin (0.016 hemolytic unit) resulted in severe, irreversible cell swelling. However, high doses of cytotoxin (2.0 hemolytic units) were required to cause substantial cell death, as indicated by the influx of propidium iodide into and release of lactate dehydrogenase from cells. Macrophages exposed to low, sublytic doses of cytotoxin failed to migrate toward chemoattractant, were unable to attach to glass, and failed to phagocytize optimally opsonized erythrocytes. Macrophages already attached to glass surfaces detached when exposed to sublytic doses of cytotoxin. The swelling and impairment of functions of alveolar macrophages observed in this study could not be attributed to endotoxic effects, because heat treatment of the cytotoxin preparation for 60 minutes at 60 C resulted in complete loss of cytotoxicity. We conclude that sublytic doses of heat-labile, hemolytic cytotoxic substances produced by App depress alveolar macrophage function at concentrations likely to develop in association with acute pulmonary infection with App. The Apx (A pleuropneumoniae Rtx toxins) exotoxins secreted by the bacteria into culture medium were considered responsible for the toxic activity of the cytotoxin preparation. The Apx of the App field strain used in this study were likely to be similar to those of serotype-1 reference strain (S4707). Analysis by use of DNA-DNA hybridization indicated that genomic DNA of the field strain contained sequences similar to those encoding structural protein of ApxI (apxIA) and ApxII (apxIIA) of the serotype-1 reference strain. Therefore, Apx produced by the field strain of App used in this study are likely to be of similar pathogenic importance worldwide.

Actinobacillus pleuropneumoniae↗

PubMed1992

Human and bovine serotype G8 rotaviruses may be derived by reassortment.

The origin of, and relationship between human and bovine serotype G8 rotaviruses were investigated by genomic hybridisation. Radiolabelled mRNAs of human G8 rotaviruses 69M (isolated in Indonesia) and HAL1271 (isolated in Finland), and bovine rotaviruses KK3 (G10) and NCDV (G6), were used as probes. The products of liquid hybridisation between the probes and the genomic RNA of human and bovine rotaviruses, including bovine G8 rotavirus 678 (isolated in Scotland) and two other Finnish human G8 rotaviruses HAL1166 and HAL8590, were examined by separation in polyacrylamide gels. The genomes of Finnish human G8 rotaviruses were similar to those of bovine G6 and G10 rotaviruses. Neither Indonesian human G8 nor bovine G8 viruses had high levels of similarity to each other or to other bovine and human rotaviruses. Thus these three epidemiologically distinct G8 rotaviruses have different origins and may be derived by reassortment with rotaviruses of a third, as yet unknown, host species. The similarity between the Finnish isolates and the bovine isolate NCDV suggests that they have diverged recently and that these human G8 rotaviruses may be derived from a zoonotic infection, or alternatively, from the live rotavirus vaccine of bovine origin which has been used to vaccinate Finnish children.

Animals↗

PubMed1992

Identification of four VP4 serological types (P serotypes) of bovine rotavirus using viral reassortants.

A series of five reassortant viruses each containing the VP4 gene of a distinct bovine rotavirus and the VP7 gene of human rotavirus strain ST3 was prepared, and antisera to these were produced in rabbits. In neutralization tests, these antisera allowed the differentiation of the five original strains (from three different VP7 or G serotypes) into three or possibly four VP4 or P serotypes. All of a further seven bovine rotavirus strains adapted to cell culture were successfully typed by these antisera. There was a degree of cross-reaction between antiserum to the fourth bovine rotavirus P serotype and the predominant human rotavirus serotype. However, antisera raised in guinea-pigs to recombinant VP4 from this serotype showed the bovine serotype to be distinct. There was no significant serological relationship between these four bovine rotavirus P serotypes and previously described P serotypes from rotaviruses isolated from man and non-bovine animals. The predominant bovine rotavirus VP7 serotypes G6 and G10 tended to have distinct P serotypes also.

Animals↗

PubMed1992

Rotavirus serotype G3 predominates in horses.

Foal fecal group A rotavirus strains were characterized by electropherotype, serotype, and subgroup and shown to be distinctly different from rotaviruses of other mammals. Of 86 strains that were electropherotyped, 98% had similar profiles, with gene segments 3 and 4 close together and segments 7, 8, and 9 widely spaced. Of 70 strains that had sufficient detectable VP7 antigen to be serotyped by enzyme-linked immunosorbent assays (ELISAs), 63% were serotype G3 (39% were subtype G3A and 24% were subtype G3B), 4% were serotype G13, and 33% were untypeable. Serotypes G1, G2, G4, G5, G6, G9, G10, and G14 were not detected, although G5 and G14 strains have been identified among cultivable equine strains. Of 50 strains that had sufficient detectable VP6 antigen to be subgrouped by ELISAs, only 12% were able to be assigned to either subgroup I or II, with the remaining 88% belonging to neither subgroup.

Animals↗

PubMed1992

Evidence for two serotype G3 subtypes among equine rotaviruses.

Ten cultivable equine rotavirus isolates, two of North American, six of British, and two of Irish origin, were compared with standard rotavirus strains and with each other by cross neutralization, neutralization with a panel of monoclonal antibodies (MAbs), hybridization to a simian rotavirus (SA-11) VP7 gene probe, and reaction with rotavirus subgrouping and serotyping MAbs in enzyme-linked immunosorbent assays. Six isolates, two of which had previously been serotyped as G3 by other workers, were found to be serotype G3; one was confirmed to be G5, and three were not related to serotypes G1 to G10. The serotype G3 strains were divisible into two subtypes, G3A and G3B, on the basis of cross neutralization. This division was also apparent in reactions with neutralizing VP7-specific MAbs and in the liquid hybridization assay. Two of the isolates were not bound by either subgroup MAb, six were bound by both subgroup I and II MAbs, and two were bound by only the subgroup I MAb. The assays used in this characterization provide a range of epidemiological information for use in future field investigations.

Animals↗

PubMed1991

Increased sensitivity of a rotavirus serotyping enzyme-linked immunosorbent assay by the incorporation of CaCl2.

The sensitivity of a rotavirus serotyping enzyme-linked immunosorbent assay (ELISA) was improved by the addition of 0.5 mM CaCl2 to the washing buffer and reagent diluent. Twenty-nine of 63 (46%) previously untyped bovine and equine faecal rotavirus samples were serotyped in the modified assay. A differential response to Ca2+ ions was noted for different G-serotypes suggesting that serotyping assays performed without the inclusion of CaCl2 in the assay buffers may produce biased results.

Animals↗

PubMed1991

Homotypic and heterotypic serum and milk antibody to rotavirus in normal, infected and vaccinated horses.

The homotypic and heterotypic antibody response to rotavirus was determined in three pony mares and their foals. The normal concentrations of anti-rotavirus antibodies in mares' milk and mares' and foals' serum over the first 10 weeks post-partum were measured using IgA, IgG and rotavirus serotype-specific enzyme linked immunosorbent assays. Experimental infection of the foals with serotype 3 equine rotavirus produced a rapid, serotype-specific response which peaked 10 days after infection and a slower heterotypic response which peaked 32 days later. In contrast, vaccination of the mares with an inactivated, adjuvanted serotype 6 bovine rotavirus produced a heterotypic response similar to that of the homotypic response in both serum and milk, although the predominant response in serum was IgG, while in milk it was IgA. These results suggest that non serotype-restricted passive protection of foals against rotavirus may be achieved by parenteral vaccination of mares.

Animals↗

PubMed1991

Serological and genomic characterization of L338, a novel equine group A rotavirus G serotype.

A group A rotavirus designated L338 was isolated from the faeces of a diarrhoeic foal and was compared to 11 standard G serotype strains of group A rotaviruses by cross-neutralization. It was clearly distinct from serotypes G1 to G11 and thus representative of a novel rotavirus G serotype tentatively designated G13. The nucleic acid sequence of the virion protein 7 (VP7) coding region was determined and the deduced amino acid sequence compared to published sequences. Within VP7 regions A and B, L338 was clearly distinct from serotypes G1 to G12 (excluding G7 which has not been sequenced), but region C was very similar to those of G3 and G8. This further questions the significance of region C in determining serotype specificity of at least three distinct rotavirus G serotypes.

Amino Acid Sequence↗

PubMed1991

The prevalence of enteric pathogens in diarrhoeic thoroughbred foals in Britain and Ireland.

A survey of 77 normal and 326 diarrhoeic foals in Britain and Ireland from 1987 to 1989 revealed a significantly higher prevalence of Group A rotaviruses and Aeromonas hydrophila in diarrhoeic foals. The prevalence of cryptosporidia, potentially pathogenic Escherichia coli, Yersinia enterocolitica and Clostridium perfringens was similar in normal or diarrhoeic foals. Rotaviruses had a similar prevalence in all age groups of scouring foals up to three months of age, with an overall prevalence of 37 per cent among diarrhoeic foals. The number of cases of diarrhoea varied considerably from year to year, but in all three years of the survey rotavirus was a significant pathogen. A comparison of diagnostic tests for rotavirus in the faeces showed electron microscopy (EM) and polyacrylamide gel electrophoresis (PAGE) to have similar sensitivity. The Rotazyme ELISA test kit was found to have the same sensitivity as a combination of EM and PAGE. A. hydrophila had an overall prevalence of 9 per cent among diarrhoeic foals, although its prevalence was higher in some age groups. A. hydrophila has not been established previously as a significant enteric pathogen in foals. Other putative pathogens found at very low prevalence were coronavirus, the putative picobirnavirus, Campylobacter spp. and Salmonella spp. No evidence was found of synergistic effects between rotavirus, cryptosporidia and potentially pathogenic E. coli. Neither coccidia nor non-Group A rotaviruses were found in any of the samples examined.

Aeromonas↗