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G F Browning

Publications and source records attributed to G F Browning.

At least 37 records · Page 2Linked to original sources
PubMed2001

Molecular epidemiology of Streptococcus uberis isolates from dairy cows with mastitis.

Pulsed-field gel electrophoresis and antimicrobial sensitivity testing were used as tools to investigate the epidemiology of Streptococcus uberis mastitis in dairy cows. A total of 62 different strains were found among 138 isolates from the four herds investigated, and between 10 and 26 different strains were found in each herd. There was no strain common to all four herds. Identical strains of S. uberis were detected from different quarters of individual cows and from cows within the same herd, suggesting that transmission from quarter to quarter and cow to cow had occurred. Despite the great variation in S. uberis strains, persistent infection with the same strain within a lactation was observed in most cows. Predominant strains were present in two herds. Preliminary investigations could not clarify why these particular strains might predominate, but in one herd there was a significant difference between the prevalence of clinical mastitis in quarters infected with the predominant strain and that in quarters infected with other strains, suggesting the greater virulence of the predominant strain. The wide variety of S. uberis strains found is consistent with an environmental source of S. uberis. However, evidence of direct transmission, the persistence of infection, and the predominance of particular strains in some herds indicate that S. uberis infections are epidemiologically complex and that the relative importance of these factors in the occurrence of mastitis may differ between herds.

Animals↗
PubMed2001

Multiplex polymerase chain reaction assay for simultaneous detection of Staphylococcus aureus and streptococcal causes of bovine mastitis.

To improve diagnosis of mastitis in dairy cattle, a multiplex polymerase chain reaction (PCR) assay was developed for the simultaneous detection of the four major bacterial causes of bovine mastitis, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis. The target sequence was the 16S to 23S rRNA spacer regions. The performance of the assay was examined with 117 milk samples collected from a subclinically infected herd, and the diagnostic specificities and sensitivities of the multiplex PCR were compared with conventional culture. PCR was significantly more sensitive than culture for detection of S. aureus and S. uberis, but there were no significant differences in sensitivities between PCR and culture for the detection of S. agalactiae and S. dysgalactiae. The results suggest that this multiplex PCR assay could be used as an alternative method in routine diagnosis for rapid, sensitive, and specific simultaneous detection of S. aureus, S. agalactiae, S. dysgalactiae, and S. uberis in milk samples.

Animals↗
PubMed2000

Transcription in Mycoplasma pneumoniae.

Very little is understood of the structure of mycoplasma promoters, and this limits interpretation of genomic sequence data in these species. In this study the transcriptional start points of 22 genes of Mycoplasma pneumoniae were identified and the regions 5' to the start point compared. Although a strong consensus -10 region could be seen, there was only a weak consensus in the -35 region. A high proportion of transcripts had heterogeneous 5'-ends and characterisation of the sequence of the 5'-ends of two transcripts established that the heterogeneity was derived from initiation of transcription at reduced levels between 1 and 4 bases 5' to the major starting point. In addition to this apparently unique feature, a high proportion of transcripts lacked a 5' untranslated leader region that could contain a ribosomal binding site. Such leaderless transcripts are seen rarely in other bacterial species. Although the promoter regions for a number of members of lipoprotein multigene families were examined, no obvious explanation for regulation of expression was apparent. Using the data from this study an improved matrix for prediction of M.pneumoniae promoters was derived. Application of this matrix to the sequences immediately 3' and 5' to each predicted start codon in the genome suggested that most M. pneumoniae transcriptional start points were likely to occur between 5 and 30 bases 5' to the start codon.

Base Composition↗
PubMed2000

A novel mechanism for control of antigenic variation in the haemagglutinin gene family of mycoplasma synoviae.

High-frequency phase and antigenic variation of homologous lipoprotein haemagglutinins has been seen in both the major avian mycoplasma pathogens, Mycoplasma synoviae and Mycoplasma gallisepticum. The expression and, hence, antigenic variation of the pMGA gene family (encoding these lipoproteins in M. gallisepticum) is controlled by variation in the length of a trinucleotide repeat motif 5' to the promoter of each gene. However, such a mechanism was not detected in preliminary observations on M. synoviae. Thus, the basis for control of variation in the vlhA gene family (which encodes the homologous haemagglutinin in M. synoviae) was investigated to enable comparison with its homologue in M. gallisepticum and with other lipoprotein gene families in mycoplasmas. The start point of transcription was identified 119 bp upstream of the initiation codon, but features associated with control of transcription in other mycoplasma lipoprotein genes were not seen. Comparison of three copies of vlhA revealed considerable sequence divergence at the 3' end of the gene, but conservation of the 5' end. Southern blot analysis of M. synoviae genomic DNA revealed that the promoter region and part of the conserved 5' coding sequence occurred as a single copy, whereas the remainder of the coding sequence occurred as multiple copies. A 9.7 kb fragment of the genome was found to contain eight tandemly repeated regions partially homologous to vlhA, all lacking the putative promoter region and the single-copy 5' end of vlhA, but extending over one of four distinct overlapping regions of the 3' coding sequence. Examination of sequential clones of M. synoviae established that unidirectional recombination occurs between the pseudogenes and the expressed vlhA, with duplication of pseudogene sequence and loss of the corresponding region previously seen in the expressed gene. Expression of the 5' end of two variants of the vlhA gene showed that they differed in their reaction with monoclonal antibodies specific for this region. These data suggest that the control of vlhA antigenic variation in M. synoviae is achieved by multiple gene conversion events using a repertoire of coding sequences to generate a chimeric expressed gene, with the greatest potential for variation generated in the region encoding the haemagglutinin. Thus, completely distinct mechanisms have been adopted to control antigenic variation in homologous gene families.

Amino Acid Sequence↗
PubMed2000

Lymphocytic infiltration in the chicken trachea in response to Mycoplasma gallisepticum infection.

A prominent feature of disease induced by Mycoplasma gallisepticum is a lymphoproliferative response in the respiratory tract. Although this is also seen in other mycoplasma infections, including Mycoplasma pneumoniae, the phenotype of the lymphocytes infiltrating the respiratory tract has not been determined. In this study, the numbers and distribution of lymphocytes in the tracheas of chickens infected with a virulent strain of M. gallisepticum were examined. Three groups of chickens were experimentally infected with M. gallisepticum and three unchallenged groups were used as controls. One infected and one control group were culled at 1, 2 and 3 weeks post infection. Tracheas were removed and examined for the presence and number of T cells carrying CD4, CD8, TCRgamma7, TCRalphabeta1 or TCRalphabeta2 markers. There was no significant difference in the number of CD8+ cells in the upper, middle and lower trachea. High numbers of both CD4+ and CD8+ cells were found with variable numbers of TCRalphabeta1+ and TCRalphabeta2+, but no TCRgammadelta+, cells throughout the time course. The distribution of CD4 cells was dispersed, while the CD8+ cells were clustered in follicular-like arrangements. No difference was detected in the distribution of TCRalphabeta1+ and TCRalphabeta2+ cells. The titre of mycoplasma genomes in the trachea decreased significantly from 1 to 2 weeks, while the mucosal thickness of the trachea increased significantly from 1 to 2 weeks then decreased from 2 to 3 weeks, indicating resolution of the lesions following control of infection. This study is the first to examine the phenotypes of T lymphocytes infiltrating the respiratory tract during mycoplasma infections. The findings suggest involvement of specific stimulation of CD8+ cells, particularly in the acute phase of disease.

Animals↗
PubMed2000

pMGA phenotypic variation in Mycoplasma gallisepticum occurs in vivo and is mediated by trinucleotide repeat length variation.

Chickens were infected with a pathogenic strain of Mycoplasma gallisepticum, and the expression of pMGA, the major surface protein, was inferred by examination of colonies from ex vivo cells. Within 2 days postinfection, 40% of cells had ceased the expression of the original pMGA surface protein (pMGA1.1), and by day 6, the majority of recovered cells were in this category. The switch in pMGA phenotype which had occurred in vivo was reversible, since most colonies produced from ex vivo progenitors exhibited frequent pMGA1. 1(+) sectors. After prolonged in vivo habitation, increasing proportions of recovered cells gave rise to variant pMGA colonies which had switched from the expression of pMGA1.1 to another gene, pMGA1.2, concomitant with the acquisition of a (GAA)(12) motif 5' to its promoter. Collectively, the results suggest that changes in M. gallisepticum pMGA gene expression in vivo are normal, common, and possibly obligate events for successful colonization of the host. Surprisingly, the initial cessation of pMGA1.1 expression occurred in the absence of detectable pMGA antibodies and seemed to precede the adaptive immune response.

Animals↗
PubMed2000

Colonization of the respiratory tract by a virulent strain of avian Escherichia coli requires carriage of a conjugative plasmid.

The E3 strain of E. coli was isolated in an outbreak of respiratory disease in broiler chickens, and experimental aerosol exposure of chickens to this strain induced disease similar to that seen in the field. In order to establish whether the virulent phenotype of this strain was associated with carriage of particular plasmids, four plasmid-cured derivatives, each lacking two or more of the plasmids carried by the wild-type strain, were assessed for virulence. Virulence was found to be associated with one large plasmid, pVM01. Plasmid pVM01 was marked by introduction of the transposon TnphoA, carrying kanamycin resistance, and was then cloned by transformation of E. coli strain DH5alpha. The cloned plasmid was then reintroduced by conjugation into an avirulent plasmid-cured derivative of strain E3 which lacked pVM01. The conjugant was shown to be as virulent as the wild-type strain E3, establishing that this plasmid is required for virulence following aerosol exposure. This virulence plasmid conferred expression of a hydroxamate siderophore, but not colicins, on both strain E3 and strain DH5alpha. Carriage of this plasmid was required for strain E3 to colonize the respiratory tracts of chickens but was not necessary for colonization of the gastrointestinal tract. However, the virulence plasmid did not confer virulence, or the capacity to colonize the respiratory tract, on strain DH5alpha. Thus, these studies have established that infection of chickens with E. coli strain E3 by the respiratory route is dependent on carriage of a conjugative virulence plasmid, which confers the capacity to colonize specifically the respiratory tract and which also carries genes for expression of a hydroxymate siderophore. These findings will facilitate identification of the specific genes required for virulence in these pathogens.

Animals↗
PubMed1999

Indirect enzyme-linked immunosorbent assay for detection of immunoglobulin G reactive with a recombinant protein expressed from the gene encoding the 116-kilodalton protein of Mycoplasma pneumoniae.

Serology remains the method of choice for laboratory diagnosis of Mycoplasma pneumoniae infection. Currently available serological tests employ complex cellular fractions of M. pneumoniae as antigen. To improve the specificity of M. pneumoniae diagnosis, a recombinant protein was assessed as a serodiagnostic reagent. A panel of recombinant proteins were expressed from a cloned M. pneumoniae gene that encodes a 116-kDa surface protein antigen. The recombinant proteins were assessed for reactivity with patient sera and the most antigenic was further assessed for its serodiagnostic potential by indirect enzyme-linked immunosorbent assay (ELISA). The ELISA based on the recombinant protein was equivalent in sensitivity to the commercial test (Serodia Myco II; Fujirebio Inc.) to which it was compared. Southern and Western blotting data suggested that the recombinant protein derived from the 116-kDa protein of M. pneumoniae could provide a species-specific diagnostic tool, although further assessment is required.

Animals↗
PubMed1998

Detection and strain differentiation of feline calicivirus in conjunctival swabs by RT-PCR of the hypervariable region of the capsid protein gene.

Reverse transcription-polymerase chain reaction (RT-PCR) was used to direct the amplification of a 670 to 680 base pair segment that included the hypervariable regions of the capsid protein gene of feline calicivirus (FCV). The segment was amplified from 13/13 cultivated FCV strains including 12 field isolates collected over 18 years. The sensitivities of culture and this RT-PCR for the detection of FCV in conjunctival swabs over the course of infection were then compared and correlated with clinical signs in 5 vaccinated and 3 unvaccinated experimentally-infected cats. Conjunctival swabs were taken daily from days 0 to 14 and on days 16, 19, 21 and 24 after challenge. FCV was detected in 19/144 swabs by RT-PCR and 16/144 swabs by culture. Virus detection correlated poorly with clinical signs regardless of the assay used. The Sau3AI restriction profiles of the RT-PCR products amplified from both cultivated strains and clinical samples were compared. All 13 cultivated isolates, including the 12 field isolates, exhibited different profiles, whereas all profiles from the experimentally-infected cats were identical, and matched the profile of the challenge/vaccine strain. This study has established that the RT-PCR assay described is as sensitive as culture for detection of FCV in conjunctival swabs, that a broad range of field isolates can be detected and rapidly differentiated by restriction endonuclease digestion, and that the assay thus meets the requirements for large scale epidemiological studies of FCV infections in cats. However, it has also shown that even the use of RT-PCR on conjunctival swabs alone is insufficient for accurate diagnosis of FCV infection in cats with conjunctivitis.

Animals↗
PubMed1998

Expression of the pMGA genes of Mycoplasma gallisepticum is controlled by variation in the GAA trinucleotide repeat lengths within the 5' noncoding regions.

We analyzed the segment of DNA which contains the expressed pMGA gene from one strain of Mycoplasma gallisepticum in normal (strain S6) cells and in cells in which pMGA1.1 gene expression had ceased as a consequence of in vitro culture in the presence of pMGA1. 1-specific antibodies. Sequence analysis of isolates lacking pMGA1.1 expression revealed that this gene, which is typically expressed, exhibited sequence changes within a region 5' to its promoter. Specifically, pMGA1.1(+) cells contained a (GAA)12 motif upstream of the promoter, whereas in pMGA1.1(-) cells the corresponding region contained a (GAA)10 motif; when such cells were grown in medium no longer containing pMGA-specific antibodies, pMGA1.1 was reexpressed and the 5' (GAA)12 motif was restored. Two other genes, pMGA1.9 and pMGA1.2, were also shown to acquire a (GAA)12 motif in clones which expressed these genes. The results imply the evolution by the pMGA genes of M. gallisepticum of a novel transcriptional requirement which facilitates rapid and reversible switches in the pMGA expression pattern.

Bacterial Proteins↗
PubMed1998

Expression of two members of the pMGA gene family of Mycoplasma gallisepticum oscillates and is influenced by pMGA-specific antibodies.

Certain monoclonal antibodies and polyclonal antisera directed to pMGA, the major protein of Mycoplasma gallisepticum, were tested for the ability to influence the surface phenotype of the cell population which resulted from their inclusion in growth medium. The polyclonal antiserum and one monoclonal antibody (MAb 66) resulted in an alteration of surface phenotype; specifically, populations of cells grown either on plates or in broth cultures which contained these reagents ceased the expression of pMGA and instead expressed an antigenically unrelated new polypeptide (p82). Upon the removal of antibody, the progeny of these cells regained pMGA expression and produced antigenically sectored colonies. The basis of this switch between pMGA+ and pMGA- states was shown to be transcriptional. The p82 polypeptide, the expression of which resulted from growth of cells in antibodies, was another member of the pMGA gene family and was located just downstream from the pMGA gene normally expressed by the M. gallisepticum cells used. Collectively the results of this work suggest that this organism has evolved an unusual means of altering the antigenic composition of its surface in response to antibodies or to other environmental cues.

Amino Acid Sequence↗
PubMed1998

Multigene families encoding the major hemagglutinins in phylogenetically distinct mycoplasmas.

Mycoplasma synoviae has two major membrane antigens, MSPA and MSPB, both of which are phase variable and which may be coordinately involved in adhesion of the organism to erythrocytes. A single gene (vlhA) from M. synoviae was characterized, and polypeptides were expressed from nonoverlapping 5' and 3' regions in Escherichia coli. The expression product of the vlhA 5' region reacted with specific reagents against MSPB, while that of the 3' region reacted with specific reagents against MSPA. Analysis of the predicted amino acid sequence showed a characteristic signal peptidase II cleavage site, and the presence of the acylation site was confirmed by identification of a lipid-associated membrane protein, similar in molecular mass to MSPB, in [3H]palmitate-labelled membrane proteins. Further sequence analysis of the vlhA gene revealed a high identity with the Mycoplasma gallisepticum pMGA1.7 gene, a member of a large translated family. The vlhA gene was shown to hybridize to multiple restriction fragments of the M. synoviae genome, suggesting that it was also a member of a multigene family. These findings indicate that coordinate phase variation of the two major surface antigens of M. synoviae WVU may be due to their expression from the same gene and that homologous gene families encode the major hemagglutinins of two phylogenetically distinct mycoplasmas. The presence of homologous multigene families in such phylogenetically distinct species, but not in the genomes of more closely related species, suggests that the families may have been transferred horizontally.

Amino Acid Sequence↗
PubMed1997

Comparison of Chlamydia psittaci from cats with upper respiratory tract disease by polymerase chain reaction analysis of the ompA gene.

Conjunctival swabs were taken from 168 cats with clinical signs of acute or chronic upper respiratory tract disease and tested for Chlamydia psittaci by the polymerase chain reaction (PCR) to amplify the ompA gene coding region. Twenty-two (13 per cent) were positive for C psittaci. The PCR products from positive samples were subjected to restriction endonuclease analysis with the restriction enzymes Alu I and Mse I. The fragments of DNA were detected on silver-stained polyacrylamide gels and the results were compared with the results obtained from chlamydial isolates from cats in Japan, France, the USA and the UK. All the strains had identical restriction patterns. When PCR is used as an epidemiological tool, feline chlamydial strains worldwide appear very similar.

Animals↗
PubMed1997

Strain differentiation of isolates of streptococci from bovine mastitis by pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) was examined as a tool to differentiate strains of streptococci isolated from clinical and sub-clinical cases of bovine mastitis. Analysis of SmaI chromosomal digests of Streptococcus agalactiae, S. dysgalactiae subsp. dysgalactiae and S. uberis isolates revealed intraherd and interherd strain relationships within each species. Comparison of S. agalactiae isolates from the same herd revealed little variability in their SmaI restriction patterns indicating a single strain originating from a common source of infection. However, comparison of S. agalactiae isolates between herds showed that each herd was infected with a distinct strain. The restriction profiles from S. dysgalactiae subsp. dysgalactiae and S. uberis were more diverse and often multiple strains of both species were present within an individual herd, with some herds containing more than one representative of a particular strain. Interestingly, it was only observed with S. dysgalactiae subsp. dysgalactiae that some strains were present in more than one herd. PFGE was found to be a useful and reproducible method for the discrimination of different strains of the three most important species of streptococci responsible for bovine mastitis and to offer a means to identify environmental sources of infection.

Animals↗
PubMed1997

Differential sensitivity of culture and the polymerase chain reaction for detection of feline herpesvirus 1 in vaccinated and unvaccinated cats.

The diagnostic sensitivities of the polymerase chain reaction (PCR) and culture were compared and correlated with clinical signs in 5 vaccinated cats and 3 unvaccinated cats that were experimentally infected with feline herpesvirus 1. Conjunctival swabs were taken each day from 0 to 14 days and on 21, 28 and 30 days after challenge. PCR (49.3%) was significantly more sensitive than culture (30.1%) as assessed by an adjusted McNemar's test to account for non-independence of results between days within each cat (P = 0.02). PCR was considerably more sensitive (34.1%) than culture (8.2%) in vaccinated cats (P = 0.001), whereas there was no significant difference in sensitivities in the unvaccinated cats, where the sensitivity of PCR was 74.5% and that of culture was 66.7% (P = 0.17). In vaccinated cats showing clinical signs, the sensitivities of culture and PCR were 14.8% and 55.6% respectively (P = 0.03), whereas in unvaccinated cats the sensitivities were 80.6% and 96.8% respectively (P = 0.07). This study suggests that disease due to feline herpesvirus 1 has been significantly underdiagnosed, particularly in vaccinated cats.

Animals↗