Detection of Chlamydia psittaci DNA in avian clinical samples by polymerase chain reaction

A polymerase chain reaction (PCR) assay was developed to detect Chlamydia psittaci DNA in faeces and tissue samples from avian species. Primers were designed to amplify a 264 bp product derived from part of the 5' non-translated region and part of the coding region of the ompA gene which encodes the major outer membrane protein. Amplified sequences were confirmed by Southern hybridization using an internal probe. The sensitivity of the combined assay was found to be between 60 to 600 fg of chlamydial DNA (approximately 6 to 60 genome copies). The specificity of the assay was confirmed since PCR product was not obtained from samples containing several serotypes of C. trachomatis, strains of C. pneumoniae, the type strain of C. pecorum, nor from samples containing microorganisms commonly found in the avian gut flora. In this study, 404 avian faeces and 141 avian tissue samples received by the Central Veterinary Laboratory over a 6 month period were analysed by PCR, antigen detection ELISA and where possible, cell culture isolation. PCR performed favourably compared with ELISA and cell culture, or with ELISA alone. The PCR assay was especially suited to the detection of C. psittaci DNA in avian faeces samples. The test was also useful when applied to tissue samples from small contact birds associated with a case of human psittacosis where ELISA results were negative and chlamydial isolation was a less favourable method due to the need for rapid diagnosis.

Partial identification of spirochaetes from two dairy cows with digital dermatitis by polymerase chain reaction analysis of the 16S ribosomal RNA gene

Specimens taken postmortem from typical lesions of digital dermatitis in two dairy cows were tested by the polymerase chain reaction (PCR) for the presence of a spirochaetal 16S rRNA gene. Seven different assays detected the gene in the samples from both cows. Two of the PCR products were sequenced and a comparison of the nucleotide sequences revealed that the spirochaete belonged to the genus Treponema and was closely related to Treponema denticola. A PCR specific for the detection of the digital dermatitis-associated treponeme was developed.

Sequence heterogeneity of an mpb70 gene analogue in Mycobacterium kansasii

The protein antigen MPB70 is a major component of culture supernatants of Mycobacterium bovis and Is an active ingredient of bovine PPD used for skin-testing cattle for tuberculosis. we have shown that Mycobacterium kansasii possesses a similar gene that cross-reacts in a PCR test for M. bovis. Single strand conformational polymorphism analysis, and the DNA sequence of the PCR product, shows differences between M. kansasii strains, supporting the suggestion that M. kansasii is not a homogeneous species.

The variant rpoS allele of S-enteritidis strain 27655R does not affect virulence in a chick model nor constitutive curliation but does generate a cold-sensitive phenotype

Adherence of pathogenic Escherichia coli and Salmonella spp. to host cells is in part mediated by curli fimbriae which, along with other virulence determinants, are positively regulated by RpoS. Interested in the role and regulation of curli (SEF17) fimbriae of Salmonella enteritidis in poultry infection, we tested the virulence of naturally occurring S. enteritidis PT4 strains 27655R and 27655S which displayed constitutive and null expression of curli (SEF17) fimbriae, respectively, in a chick invasion assay and analysed their rpoS alleles. Both strains were shown to be equally invasive and as invasive as a wild-type phage type 4 strain and an isogenic derivative defective for the elaboration of curli. We showed that the rpoS allele of 27655S was intact even though this strain was non-curliated and we confirmed that a S. enteritidis rpoS::str(r) null mutant was unable to express curli, as anticipated. Strain 27655R, constitutively curliated, possessed a frameshift mutation at position 697 of the rpoS coding sequence which resulted in a truncated product and remained curliated even when transduced to rpoS::str(r). Additionally, rpoS mutants are known to be cold-sensitive, a phenotype confirmed for strain 27655R. Collectively, these data indicated that curliation was not a significant factor for pathogenesis of S. enteritidis in this model and that curliation of strains 27655R and 27655S was independent of RpoS. Significantly, strain 27655R possessed a defective rpoS allele and remained virulent. Here was evidence that supported the concept that different naturally occurring rpoS alleles may generate varying virulence phenotypic traits. (C) 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

The frequent detection of a treponeme in bovine digital dermatitis by immunocytochemistry and polymerase chain reaction

A study was carried out to determine whether spirochaetes are frequently associated with digital dermatitis in United Kingdom (UK) dairy cattle. Histopathological examination of lesions using a silver stain showed a large number of unidentified spirochaete-like organisms present in digital dermatitis hoof skin tissue in all examined biopsies. Immunocytochemical staining demonstrated that spirochaetes in skin lesions were identified by polyclonal antisera to Borrelia burgdorferi, Treponema denticola and Treponema vincentii (again all biopsies were positively stained), whereas monoclonal antibodies to B. burgdorferi and any Treponema pallidum did not stain any organisms in all biopsies. A PCR of 16S rRNA, previously shown to be specific for a new treponeme, was employed and produced positive results from 82.4% of digital dermatitis tissues. It is concluded that this spirochaete (or related spirochaetes), which is similar to human oral treponemes, is frequently associated with, and may be responsible for, pathological changes in digital dermatitis. (C) 1998 Elsevier Science B.V.

Colonisation of the chicken caecum by afimbriate and aflagellate derivatives of Salmonella enterica serotype Enteritidis

A semi-quantitative cloacal-swab method was used as an indirect measure of caecal colonisation of one-day old and five-day old chicks after oral dosing with wild-type Salmonella enterica serovar Enteritidis PT4 and,genetically defined isogenic derivatives lacking the ability to elaborate flagella or fimbriae. Birds of both ages were readily and persistently colonised by all strains although there war a decline in shedding by the older birds after about 21 days. There were no significant differences in shedding of wild-type or mutants in single-dose experiments. In competition experiments, in which five-day old birds were dosed orally with wild-type and mutants together, shedding of non-motile derivatives was significantly lower than wild-type, At 35 days post infection, birds were sacrificed and direct counts of mutants and wild-type from each caecum were determined. Whilst there appeared to be poor correlation between direct counts and the indirect swab method, the overall trends shown by these methods of assessment indicated that flagella and not fimbriae were important in caecal colonisation in these models. Crown Copyright (C) 1999 Published by Elsevier Science B.V.

The role of flagella, but not fimbriae, in the adherence of Salmonella enterica serotype Enteritidis to chick gut explant

To gain an understanding of the role of fimbriae and flagella in the adherence and colonisation of Salmonella enterica serotype Enteritidis in chickens, an in-vitro gut adherence assay was developed and used to assess the adherence of a wild-type Enteritidis strain and isogenic non-fimbriate and non-flagellate mutant strains. Enteritidis strain S1400/94, a clinical isolate virulent in chickens, was shown to possess genes which encoded type 1, SEF14, SEF17, plasmid-encoded and long polar fimbriae. Mutant strains unable to elaborate these fimbriae were created by allelic exchange. Each fimbrial operon was inactivated by the insertion of an antibiotic resistance gene cassette. In addition, fliC, motAB and cheA loci, which encode the major subunit of the flagellum, the energy-translation system for motility and one of the chemotaxis signalling proteins, respectively, were similarly inactivated. Non-flagellate mutant strains were significantly less adherent than the wild-type strain, whereas mutant strains defective for the elaboration of any of the types of fimbriae adhered as well as the wild-type strain. A flagellate but non-motile (paralysed) mutant strain and a smooth-swimming chemotaxis-deficient mutant strain were shown to be less adherent than the wild-type strain, but that observation depended on the assay conditions used.

Fimbriae- and flagella-mediated association with and invasion of cultured epithelial cells by Salmonella enteritidis

Salmonella enteritidis expresses flagella and several finely regulated fimbriae, including SEF14, SEF17 and SEF21 (type 1). A panel of mutants was prepared in three strains of S. enteritidis to elucidate the role of these surface appendages in the association with and invasion of cultured epithelial cells. In all assays, the naturally occurring regulatory-defective strain 27655R associated with tissue culture cells significantly more than wild-type progenitor strains LA5 and S1400/94. Compared with wild-type strains, SEF14 mutants had no effect on association and invasion, whereas SEF17, SEF21 and aflagellate mutants showed significant reductions in both processes. Histological examination suggested a role for SEF17 in localized, aggregative adherence, which could be specifically blocked by anti-SEF17 sera and purified SEF17 fimbriae. SEF21-mediated association was neutralized by mannose and a specific monoclonal antibody, although to observe enhanced association it was necessary for the bacteria to be in fimbriate phase prior to infection. Additionally, aflagellate mutants associated and invaded less than motile bacteria. This study demonstrated the potential for multifactorial association and invasion of epithelial cells which involved SEF17 and SEF21 fimbriae, and flagella-mediated motility.

Non-curliation of Escherichia coli O78 : K80 isolates associated with IS1 insertion in csgB and reduced persistence in poultry infection

The elaboration of curli fimbriae by Escherichia coli is associated with the development of a lacy colony morphology when groan on colonisation factor antigen agar at 25 degrees C. Avian colisepticaemia E. coli isolates screened for curliation by this culture technique showed lacy and smooth colonial morphologies and the genetic basis of the non-curliated smooth colonial phenotype was analysed. Two smooth E, coli O78:K80 isolates possessed about 40 copies of the IS1 element within their respective genomes of which one copy insertionally inactivated the csgB gene, the nucleator gene for curli fibril formation. One of these two isolates also possessed a defective rpoS gene which is a known regulator of curli expression. In the day-old chick model, both smooth isolates were as invasive as a known virulent O78:K80 isolate as determined by extent of liver and spleen colonisation post oral inoculation but were less persistent in terms of caecal colonisation. (C) 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Attaching and effacing lesions in the large intestine of an eight-month-old heifer associated with Escherichia coli O26 infection in a group of animals with dysentery

Escherichia coli O26:K60, with genetic attributes consistent with a potentially human enterohaemorrhagic E coli was isolated from the faeces of an eight-month-old heifer with dysentery. Attaching and effacing lesions were identified in the colon of a similarly affected heifer examined postmortem, and shown to be associated with E coli O26 by specific immunolabelling.

Brucella melitensis 16M: characterisation of the galE gene and mouse immunisation studies with a galE deficient mutant

The galE gene of Streptomyces lividans was used to probe a cosmid library harbouring Brucella melitensis 16M DNA and the nucleotide sequence of a 2.5 kb ClaI fragment which hybridised was determined. An open reading frame encoding a predicted polypeptide with significant homology to UDP-galactose-4-epimerases of Brucella arbortus strain 2308 and other bacterial species was identified. DNA sequences flanking the B. melitensis galE gene shared no identity with other gal genes and, as for B. abortus, were located adjacent to a mazG homologue. A plasmid which encoded the B. melitensis galE open reading frame complemented a galE mutation in Salmonella typhimurium LB5010, as shown by the restoration of smooth lipopolysaccharide (LPS) biosynthesis, sensitivity to phage P22 infection and restoration of UDP-galactose-4-epimerase activity. The galE gene on the B. melitensis 16M chromosome was disrupted by insertional inactivation and these mutants lacked UDP-galactose-4-epimerase activity but no discernible differences in LPS structure between parent and the mutants were observed. One B. melitensis 16M galE mutant, Bm92, was assessed for virulence in CD-1 and BALB/c mice and displayed similar kinetics of invasion and persistence in tissues compared with the parent bacterial strain. CD-1 mice immunised with B. melitensis 16M galE were protected against B. melitensis 16M challenge. Crown Copyright (C) 1999 Published by Elsevier Science B.V.

Effect of pH, temperature and surface contact on the elaboration of fimbriae and flagella by Salmonella serotype Enteritidis

Survival of enteric pathogens exposed to various environmental stresses depends upon a number of protective responses, some of which are associated with induction of virulence determinants. Flagella and fimbriae are putative virulence determinants of Salmonella spp, and ELISAs specific for the detection of flagella and SEF21, SEF14 and SEF17 fimbriae were used to assess the effect of temperature and pH upon their elaboration by isolates of Salmonella serotype Enteritidis in planktonic growth and on the surface of two-dimensional gradient agar plates, For three phage type 4 isolates of Enteritidis of comparative clinical provenance, similar phenotypes for the elaboration of these surface antigens were observed. SEF14 fimbriae were elaborated in planktonic growth at 37 degrees C, but not 20 degrees C, at pH 4.77 and above but not at pH 4.04; whereas on agar gradient plates SEF14 fimbriae were elaborated poorly but with best yields at pH 4.04, SEF17 fimbriae were elaborated in planktonic growth at 20 degrees C, but not at 37 degrees C, at pH 6.18 and above but not at pH 5.09 or below; whereas on agar gradient plates SEF17 fimbriae were elaborated well even at pH 4.65, SEF21 fimbriae were expressed very poorly under all conditions tested, Planktonic growth at 37 degrees C induced least flagella whereas growth at 20 degrees C, and particularly surface growth at lower pH values, induced a 'hyper-flagellate' phenotype, Single colonies allowed to form on gradient agar plates were shown to generate different colonial morphologies which were dependent on initial pH. These results demonstrate that the physicochemical environment is an important determinant of bacterial response, especially the induction of putative virulence factors.

Infection of gnotobiotic calves with Escherichia coli O157 : H7 strain A84

Six colostrum-deprived, hysterotomy-derived calves were maintained under sterile conditions and fed a milk replacer diet. At five days of age, five of the calves were dosed orally with 10(9)cfu of Escherichia coli O157: H7 strain A84. They were killed after, one, two, six, 12 and 24 days, and samples were taken for bacteriological and pathological examination. The sixth uninfected control calf was killed at seven days of age and matched samples were taken for pathological comparison. The animals remained normal throughout the observation period. Bacteriological data indicated a heavy bacterial load of strain A84 throughout the gastrointestinal tract but the bacterium was not found in liver, kidney or muscle. No evidence of attaching and effacing' lesions in the small or large intestine was found although there was a mild inflammatory response in the intestinal tract, consisting mainly of infiltrating eosinophils.