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A total of 61 honey bees from different regions of the Republic of Serbia were analyzed for Clostridium botulinum(C. botulinum) spores. The microbiological methods and molecular methods (multiplex PCR/mPCR and PCR method) were utilized to examine multiple subunits of each honey bees samples. The C. botulinumspores in PCR-positive samples were estimated by the most probable number method (MPN). The presence of C. botulinumspores, by applying mPCR and PCR methods, was detected in 1 of the 61 honey bees (1.64%). Using MPN method, the number of spores in positive sample was 110/kg. Detection of C. botulinumspores directly from untreated honey bees, without prior enrichment, is impossible by applying PCR. Using conventional microbiological methods, detection of C. botulinumspores in dead honey bees is not possible without preenrichment. Therefore, conventional, microbiological methods are not suitable for the detection of C. botulinumspores in honey bees. In order to detect C. botulinumspores in honey bees using PCR methods, due to the small and/or unequal distribution of spores in the samples, it is desirable to use multiple subunits/replicates for each sample examined.

Q fever is a zoonotic disease caused by Coxiella burnetii, a gram-negative coccobacillus, which has been detected in a wide range of animal species, mostly domestic ruminants, but also in wild mammals, pets, birds, reptiles, arthropods (especially ticks), as well as in humans. Although the exposure to domestic animals in rural areas is regarded as the most common cause of the disease in humans, recent studies have shown that the role of pets in the epidemiology of Q fever has been increasingly growing. Although the primary route of infection is inhalation, it is presumed that among animals the infection circulates through ticks and that they are responsible for heterospecifi c transmission, as well as spatial dispersion among vertebrates. The aim of this study was to determine the presence and prevalence of C. burnetii in ticks removed from stray dogs, as well as to examine the distribution of tick species parasitizing dogs on the territory of Belgrade city. A PCR protocol targeting IS1111 repetitive transposon-like region of C. burnetii was used for the detection of C. burnetii DNA in ticks and the results were confi rmed by sequence analysis. In total, 316 ticks were collected from 51 stray dogs - 40 females (78.43%) and 11 males (21.57%). Three species of ticks were identifi ed: Rhipicephalus s anguineus (72.15%), Ixodes ricinus (27.53%) and Dermacentor reticulatus (0.32%). Out of 316 examined ticks, C. burnetii DNA was detected only in the brown dog tick R. sanguineus, with a total prevalence of 10.53% (24/228) . The high prevalence of C. burnetii in R. sanguineus, which is primarily a dog tick, indicates the importance of dogs in the epidemiology of Q fever in the territory of Belgrade.

The goal of this study was to compare the results of serological and conventional bacteriological methods with the results obtained using multiplex PCR Bruce-ladder assay. Based on the obtained results, the usability of the assay was assessed in regard to rapid diagnosis of canine brucellosis directly from the samples of reproductive organs of infected dogs. Out of 225 blood samples, 33 (14.67%) had a positive agglutination reaction. In this study, out of the 225 assayed reproductive organs of dogs, B. canis was isolated from 3 samples (1.33%), while the PCR Bruce-ladder assay detected two positive samples (0.88%). Two dogs from which B. canis was isolated, an antibody titer of 1/200 was established in blood serums, and third dog from which B. canis was isolated was negative using the tube agglutination test. From a total of 225 assayed organ samples, a positive PCR reaction was obtained from two samples. The obtained results show that the tube agglutination method remains the first choice for the detection of dogs infected with B. canis. In addition, whenever possible, it is necessary to try isolation. It is desirable to attempt the detection of B. canis in tissues using PCR, but the results may not be treated as definitive and reliable.

The aim of the study was to evaluate and adapt the PCR-based protocol that utilizes the developed serotype-specific primers to identify Salmonella entericaspecies and its serotypes that are most frequently isolated from poultry samples in Vojvodina. Using the slide agglutination test, 64 and 33 out of 107 Salmonellaisolates were identified as S. Infantis and S. Enteritidis, respectively, while ten isolates were identified as eight different Salmonellaserovars. Using the same isolates, presence of 993-bp (bcfCgene), 636-bp (steBgene) and 293-bp (sdf locus) amplicons in multiplex PCR unambiguously identified 31 isolates as S. Enteritidis. Two isolates identified as Enteritidis in slide agglutination test were not identified as such in PCR-based approach since they both were missing 293-bp long PCR product. Thirty-nine isolates produced a 727-bp amplicon in the specific simplex PCR, and thus were identified as S. Infantis. The greatest discrepancy in comparison to the results of conventional serotyping has been observed in the case of S. Infantis, since 25 more isolates were noted as S. Infantis by conventional serotyping. Seven isolates, with unexpected PCR profiles stayed unidentified by molecular typing, although they were serotyped as S. Typhimurium (1) and S. Infantis (6). S. Gallinarum serovar has to be additionally confirmed, since it shares the same PCR profile with S. Livingstone. Clearly, PCR-based identification has to be thoroughly checked, verified and adapted if it is to be applied as the routine identification protocol.

For the purpose of precise antibiotic susceptibility testing it is necessary to clearly distinguish Pseudomonasand Stenotrophomonasgenera, considering acquired resistance of Pseudomonasspecies, as well as the intrinsic resistance of Stenotrophomonasspecies. This is why in the identification of the 51 isolates originated from fish, the following methods were used: standard PCR, 16S rRNA gene sequencing, and MALDI-TOF.