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A specific PCR assay based on unique sequences of the rrs genes (16S rRNA) of Mycoplasma conjunctivae was developed for direct detection and identification of this pathogen from clinical material. DNA from eye swabs was amplified after a simple lysis step by either a single PCR with the M. conjunctivae specific primer pair McoR1 and McoF1, or by a nested PCR with the Mycoplasma genus specific primer pair MOLIGEN1-L and 16UNI-R in the first step and McoR1 and McoF1 in the second step. The specificity of the primer pair McoR1 and McoF1 was verified with purified DNA from the type strain, from 17 field isolates of M. conjunctivae and from several Mollicutes which are phylogenetically related to M. conjunctivae or which can be isolated from the same host animals. This method identified mycoplasma isolates from goat, sheep, ibex and chamois originating from different countries as M. conjunctivae. No cross amplifications with other mycoplasmas which are related to M. conjunctivae were observed. Eye swab samples containing known numbers of M. conjunctivae cells were analysed after direct lysis of the material. The detection level was estimated to be 20 cells per swab when the nested PCR procedure was used and 2 × 105 by the single PCR method. In an experimental infection model of sheep, the nested PCR method for detection of M. conjunctivae gave results which were comparable to mycoplasmal culture. These are the implications for diagnostic purposes: M. conjunctivae isolates can be identified by the one-step PCR method, whereas for detection and identification of M. conjunctivae in clinical material the two-step method should be used (higher sensitivity). [ABSTRACT FROM AUTHOR]

Mycoplasma conjunctivae was isolated four times from the eyes of nine Alpine ibex ( Capra ibex ibex) suffering from keratoconjunctivitis. The animals examined were affected in two different outbreaks in the Swiss Alps. Parasitological and bacteriological studies, including investigations for chlamydia and mycoplasmas, were performed. The results indicate that M. conjunctivae is the primary pathogenic agent causing infectious keratoconjunctivitis in this species. [ABSTRACT FROM AUTHOR]

Clostridium perfringens type C, which produces α- and β-toxin, causes severe haemorrhagic and necrotic enteritis in animals and humans. A polymerase-chain-reaction (PCR) assay was developed for the specific detection of the genes encoding α-, β-, ε- and enterotoxin of C. perfringens for rapid typing of C. perfringens strains, and especially for the identification of type C strains. Both the α- and β-toxin genes were detected directly in porcine C. perfringens type C cultures and also in type B and type C collection strains to a sensitivity of 103 cells without purification of the DNA. The α-toxin gene was detected in all types of C. perfringens. The ε-toxin gene was found in type B and type D, and the enterotoxin gene in some type A strains. Nine other species of Clostridium and a variety of intestinal pathogenic bacteria showed no signal for these toxin genes in this PCR assay. The α- and β-toxin genes PCR assay were used to identify C. perfringens strains isolated from intestinal contents of 36 necropsied piglets that had suddenly died or died after premonitory signs of diarrhoea. At necropsy, 20 piglets showed necrotizing enteritis (15 acute and 5 chronic cases) and were suspected to have suffered from a C. perfringens type C infection. All of them had C. perfringens which gave a positive PCR signal for α- and β-toxin genes, and, hence, were identified as type C strains. From the 16 other piglets with lesions other than necrotizing enteritis, C. perfringens strains with the α-toxin gene, but no β-toxin gene, were isolated. The necropsy findings and the anamnesis showed a very good correlation with the PCR identification of toxin genes. It was therefore concluded that the PCR-based toxin gene examination is a good alternative to the time-consuming, less specific, and more expensive mouse neutralization test in the routine diagnostic laboratory. [ABSTRACT FROM AUTHOR]

To determine the clinical significance and prevalence of Vero cytotoxin (VT)-producing E. coli (VTEC) in Swiss cattle, faecal swabs and milk samples of 93 cattle from two farms with calf diarrhoea, and of 54 cattle from two similar farms without clinical problems, were screened for VTEC by PCR and colony-blot hybridization. On average, 21% of all cows were positive for VTEC by PCR, without differences between farms with and without diarrhoea problems. By contrast, VTEC were detected by PCR in 60% of animals younger than 2 years from farms with diarrhoea problems, whereas only 32% of comparable control animals from farms without clinical problems had VTEC. No VTEC were detected in the milk samples. A variety of toxin types, antimicrobial-susceptibility patterns and serotypes were found by colony-blot hybridization, among the VTEC in individual herds. However, there was a significant relationship between the VT1-only toxin type and the presence of antimicrobial resistance and clinical disease in calves. No association could be found between levels of VTEC excretion and the presence of diarrhoea. This is the first study to report systematic quantitative data on numbers of VTEC in faecal samples of cattle positive for VT genes by PCR. [ABSTRACT FROM AUTHOR]

A total of 70 puppies and their dams, distributed in 14 litters, were submitted to weekly fecal examinations for C. difficile during the first 10 weeks after birth. During the study, 94.3% of the puppies and 42.9% of the dams harboured C. difficile at least once in their feces. We calculated that 58% of the puppies carried toxigenic C. difficile at least once during the survey. In the puppies, C. difficile carriage rates ranging from 3.1% to 67.1% were observed at different moments of the observation period. In comparison, C. difficile carriage rate was 1.4% in a control group of healthy dogs more than 3 months old. Discrepancies in the toxigenic phenotype of the C. difficile strains isolated in the same litter showed that the neonate dogs were transiently infected with different strains, and that the dam is often not the source of infection with C. difficile. We could not demonstrate any pathogenicity of C. difficile for neonate dogs. [ABSTRACT FROM AUTHOR]

A sterile plastic tube, a boric acid-glycerol-sodium formate tube and a dip-slide tube were compared for transport of dog urine for bacteriological examination under practice conditions, at ambient temperature. In a first part, 50 dog urine samples were cultured on agar plates and on a dip-slide kit within 2 h after collection and after storage at 20°C in a sterile tube and in a boric acid tube for 24 h and 48 h. Culture of the samples stored in the boric acid tube and culture on the dip-slide yielded results which correlated very well with those of the culture of fresh urine. However, culture of the samples stored in the sterile tube yielded up to 65 % false positive results. In a second part, a comparison was drawn with culture results of 100 dog urine specimens collected by different practitioners and simultaneously mailed to our laboratory in a sterile tube, in a boric acid tube and in a dip-slide kit. Samples sent in the boric acid and in the dip-slide tube showed comparable culture results. Culture of the samples sent in the plastic tube yielded 53 % false positive results in comparison with those of the samples preserved in boric acid. [ABSTRACT FROM AUTHOR]