Isolation and characterisation of Brucella melitensis by bacteriological and molecular methods from livestock in North Cyprus.

In this study, the isolation, biotyping and molecular characterisation of Brucella melitensis from cattle, sheep and goats in North Cyprus are reported on. A total of 319 raw milk samples obtained from seropositive dairy livestock (190 cattle, 74 sheep and 55 goats) and tissue samples including the liver, spleen and abomasal contents obtained from 32 aborted foetal samples (5 cattle, 18 sheep and 9 goats) were analysed for the presence and characterisation of the agent. B. melitensis was isolated and identified from 90 out of 319 (28.2%) milk and 19 out of 32 (59.4%) foetal samples by conventional bacteriological methods. Identification of all 109 isolates was confirmed by using real-time PCR with genus and species-specific primers. Following the preliminary identification, 27 selected isolates representing various counties and herds were further analysed by conventional methods. Twenty (74.1%) isolates were identified as B. melitensis biovar 1 and seven (25.9%) were identified as B. melitensis biovar 3. The Bruce-ladder multiplex PCR assay revealed that all the isolates were field strains. The results of the present study confirmed the presence of B. melitensis in livestock including the cattle population in North Cyprus. Even though the majority of the samples came from seropositive cattle, Brucella abortus was not isolated in the study. The results also revealed the potential public health risk of brucellosis in livestock emphasising the need of implementing strict control and eradication strategies against the disease in animal populations in order to protect human health.

Direct diagnosis of Brucella species through multiplex PCR formed by a new method.

This study aimed to develop direct PCR methods, which enable the diagnosis of brucellosis agents from ruminant aborted fetus samples at species and genus levels, and determine the applicability of the newly developed methods. For this purpose, 137 lung, 137 liver, and 52 fetal stomach fluid samples belonging to 166 ruminant aborted fetuses (326 samples in total) were examined. Firstly, agent isolation and identification were performed and species-specific multiplex PCR (m-PCR) from the culture was applied to the samples. In addition, the Mayer-Scholl m-PCR method was modified and termed 'modified Mayer-Scholl', and genus specific Bcsp31 PCR was also modified with minor changes. Four different methods were applied to direct examination samples and the obtained results were compared. The conventional culture method was set as the standard method to which sensitivities and specificities of the molecular methods were calculated. According to the assessments on the basis of fetus (n = 166), sensitivity and specificity values for modified Mayer-Scholl m-PCR method were 94.11% and 98.76%, and the same indicators for the modified Bcsp31 PCR were 95.29% and 98.76%, respectively. When all organ samples were taken into account (n = 326), sensitivity and specificity values for the modified Mayer-Scholl m-PCR method were 85.38% and 98.06%, and for the modified Bcsp31 PCR, they were 83.62% and 98.06%, respectively. As a result, it was found that the diagnostic power of the tests were 'high' when results were evaluated at fetus level. On the other hand, it was found to be 'clinically useful' when evaluated at organ level. We concluded that species level identifications can be made through the modified Mayer-Scholl method, which is a direct m-PCR method, with a high diagnostic power by specifying DNAs belonging to Brucella species directly from clinical samples.