BackgroundA fast and simple two-step multiplex real-time PCR assay has been developed to replace the traditional, laborious Salmonella serotyping procedure. Molecular beacons were incorporated into the assay as probes for target DNA. Target sequences were regions of the invA, prot6E and fliC genes specific for Salmonella spp. Salmonella Enteritidis and Salmonella Typhimurium, respectively, the two most clinically relevant serotypes. An internal amplification positive control was included in the experiment to ensure the optimal functioning of the PCR and detect possible PCR inhibition. Three sets of primers were used for the amplification of the target sequences. The results were compared to those of the Kauffmann-White antigenic classification scheme.ResultsThe assay was 100% sensitive and specific, correctly identifying all 44 Salmonella strains, all 21 samples of S. Enteritidis and all 17 samples of S. Typhimurium tested in this work. Therefore, the entire experiment had specificity and sensitivity of 100%. The detection limit was down to 10 copies of DNA target per 25 μl reaction.ConclusionThe assay can amplify and analyse a large number of samples in approximately 8 hours, compared to the 4 to 5 days conventional identification takes, and is thus considered a very promising method for detecting the two major serotypes of Salmonella quickly and accurately from clinical and environmental samples.

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In 2007, serological evidence for foot-and-mouth disease (FMD) infection was found as a result of differential diagnostic testing of Cypriot sheep suspected to be infected with bluetongue or contagious ecthyma. Seropositive sheep and goats were subsequently uncovered on ten geographically clustered flocks, while cattle and pigs in neighbouring herds were all seronegative. These antibodies were specific for serotype-O FMD virus, reacting with both structural and non-structural (NS) FMD viral proteins. However, no FMD virus could be recovered from the seropositive flocks. FMD had not been recorded in Cyprus since 1964 and there has been no vaccination programme since 1984. Since all the seropositive animals were at least 3 years old and home-bred, it was concluded that infection had occurred approximately 3 years previously had passed un-noticed and died out spontaneously. It therefore appears that antibodies to FMD virus NS proteins can still be detected around 3 years after infection of small ruminants, but that virus carriers cannot be detected at this time. This unusual situation of finding evidence of historical infection in a FMD-free country caused considerable disruption and alarm and posed questions about the definition of what constitutes a FMD outbreak.

SUMMARYIntestinal thermophilic Campylobacter species produce stable patterns when subjected to bacterial restriction endonuclease DNA analysis (BRENDA); this technique is therefore of considerable value in epidemiological studies. BRENDA was used to examine thermophilic Campylobacter species from humans, wild and domestic animals. One hundred and ninety-four (61 %) of 316 isolates of Campylobacter jejuni from humans had BRENDA patterns which could be matched to those of animal isolates. Poultry appear to be a major source of infection for C. jejuni in humans with nearly half (49 7 %) of the human isolates giving patterns which were indistinguishable from those isolated from poultry. A total of 60 BRENDA types were identified from 316 human isolates and 11 of these had the same pattern as those isolated from poultry. One of the three Campylobacter coli BRENDA types recovered from poultry was indistinguishable from a human isolate type. Pigs appear to be only a minor source of C. coli infection for humans in New Zealand. Rats were found to be infected with strains of C. jejuni with BRENDA patterns indistinguishable from those infecting humans, poultry and a horse. None of the 102 isolates of Campylobacter species from wild birds gave BRENDA patterns similar to those of isolates from humans.

Ninety-nine Campylobacter coli isolates were examined by bacterial restriction endonuclease DNA analysis (BRENDA) with HindIII. Isolates from poultry from the same environment had identical patterns, patterns of isolates carried by suckling piglets were generally the same as those of isolates recovered from their dams, and one human patient yielded the same BRENDA type when sampled 6 weeks later. The 14 human isolates examined produced 11 distinct BRENDA types. Forty-three C. coli isolates from pigs were represented by 20 BRENDA types. Ten C. coli isolates from the feces of gulls yielded five different BRENDA types. Thirty-two C. coli isolates from live chickens and processed chicken yielded five different BRENDA types. Three human isolates had identical DNA patterns; two were from brothers living in the same house, and the third was from a human with no apparent relationship to the brothers. Another human isolate was identical to a poultry isolate. None of the pig strains had DNA patterns resembling those of human strains, nor were the DNA patterns like those of any strains recovered from poultry or gulls. Four C. coli isolates were subcultured onto agar 23 times over a period of 45 days, and their BRENDA patterns were preserved. BRENDA shows great promise for use in epidemiological studies of C. coli. Campvlobacter (oli was first described as Vibrio coli (7) and was thought to be the etiological agent of swine dysentry (6). Veron and Chatelain (36) classified the genus Cainpylobacteer and renamed V. coli as C. coli. This renaming has recently been accepted by the International Committee on Systematic Bacteriology (27). The feature which distinguishes C. coli from C. jejiuni is its inability to hydrolyze hippurate in the test described by Harvey (9) and Skirrow and Benjamin (30). Although C. (oli is less frequently isolated from cases of human diarrhea than is C. jejiunii (3, 4. 11, 14, 22, 31), there appears to be no obvious difference in disease severity for humans with either of these two species (29). Each year in New Zealand a number of acute enteritis cases in humans are found to be caused by C. (oli (D. M. Norris, personal communication), but their source is un-* Corresponding author.