Учесталост налаза Salmonella spp. на труповима бројлера и њихов значај за безбедност хране / Frequency of Salmonella spp. findings on broiler carcassess at slaughterline and it‟s significance for the food safety

Based on the literature data, it is assumed that in the human salmonellosis, the important role have poultry meat and poultry products, as the increasing resistance to antibiotics complicates the treatment of salmonellosis. Therefore, in this doctoral dissertation the framework is set to review the sources of salmonella infection in broiler farms and to determine the frequency of Salmonella spp. presence on the carcasses of broilers at slaughter line. Besides, the goal of this research is to compare isolates from broiler carcasses with salmonella strains isolated in cases of illness in humans as well as to assess the sensitivity of salmonella isolates to antibiotics. In order to determine the extent of the presence of Salmonella spp., оn the slaughter line of broilers, during four year period (2008-2011), 520 samples of broiler neck skin, formed from 1560 broiler carcasses on slaughter line, were examined. In order to determine the frequency of Salmonella spp. presence on broiler carcasses, and to detect contamination sources, 200 samples of broiler neck skin, formed by 600 carcasses, from three slaughterhouses of different capacities, were examined. For detection of Salmonella spp. in broiler skin samples, automated qualitative test miniVidas method (bioMerieux, France) is used as screening method. Samples that were screened tested positive were streaked on selective media - xylose lysine deoxycholate agar (XLD agar) and differential medium - Rambach agar (Merck, Germany) in order to isolate Salmonella spp. Phenotypic identification of isolates of Salmonella spp. was performed using conventional and commercial biochemical tests (with automatic identification system API 20E bioMérieux, France) and serological typing by using polyvalent, or monovalent antisera, according to Kauffman-White scheme. Genotypic identification of Salmonella spp. isolated from poultry carcasses, were performed with molecular-biological method (gel electrophoresis in pulsed field - PFGE). Phylogenetic comparison of Salmonella spp. isolated from poultry carcasses with strains of Salmonella spp. isolated cases of the disease in humans, is carried out by the genome analysis - CHEF-DR III (Bio-Rad, California). Sensitivity of isolates of Salmonella spp. the antimicrobials tested by the disk diffusion method on Mueller-Hinton agar according to CLSI guide disk usage (ampicillin 10μg, cefotaxime 30 mg, ciprofloxacin 5 mg, gentamicin 10 mg, nalidixic acid 30 mg, tetracycline 30 mg, trimethoprim sulfamethoxazole 30 mg) manufacturer Oxoid, Bashingstoke ,UK. Tests for the presence of Salmonella spp. in broiler neck skin samples were performed using SRPS ISO 6579. Detection of Salmonella spp. on the carcasses of slaughtered broilers ranged from 5,46 ± 0,95 to 6,04 ± 0,87 positive samples of 50 tested samples. In 2011. hygiene conditions of production processes were improved and the average findings of Salmonella spp. on the carcasses of slaughtered broilers decreased to 2,27 ± 1,19 positive samples, on 50 tested samples. In order to determine the source of contamination of broiler carcasses on the slaughter line the origin of broilers was monitored. The largest number of broilers with finding Salmonella spp. originated from farm I - 11 (55%). During the new production cycle, more tests were performed on farm I. Examining the swabs from drinkers, feeders and walls of the object on the site and at the junction with the floors, as well as samples of feed and water supply, salmonella was not detected. Next test was performed in the middle of the next broiler breeding cycle, when salmonella was detected in 100% samples of shoe covers, rugs and culled carcasses and in 20% of samples of insects. Further investigation was performed on the day old chicken carcasses from the transportation truck. Presence of salmonella was not detected. The gained results excluded the hatchery as the source of Salmonella spp. infection. The results of this study showed that Salmonella spp. infection of broilers comes from the atmosphere and from farm living vectors. After the application of strict biosecurity measures on farms, the salmonella finding was reduced from 55% to 10% of the samples of neck skin of broilers in the slaugherhouse. Salmonella isolated from broiler neck skin samples were identified with serological typing as serovars of S. Infantis (6,7: r: 1,5). Based on the results of PFGE, Salmonella isolated from broiler neck skin are classified into seven genotypes or genetic profiles. Salmonella isolates within the same genetic profile showed the degree of genetic similarity of 100%. The name of each genetic profile was formed according to marking in which the first letter refers to the type of microorganism, the following three letters to the serovars identified, then the next two letters refer to the used enzyme, then the four numbers that begin with 0001. SINFXB0001 profile was found in 11 strains of which 8 originated from the neck skin of broiler chickens (4, 5, 7, 8, 10, 11, 12, 15), while the three strains were isolated in the case of human disease (18,19, 20) . Profiles SINFXB0002 (16, 17) and SINFXB0003 (2, 3) were found in isolates from broiler neck skin, while the profile SINFXB0004 (21, 22) was found in strains isolated in the case of human diseases. Profile SINFXB0005 (9), SINFXB0006 (14) and SINFXB0007 (1) was determined with only one isolate. The similarity between the genetic profile was 92%. Antimicrobial activity of the microorganism of the present profile greatly depends on the drug used in broiler farms, higher than that of the drug of choice used in the treatment of humans. One isolate, originating from the neck skin of broilers, SINFXB0002 profile, was resistant to ampicillin, cefotaxime / clavulanic acid and nalidixic acid, and other isolates of the same profile was resistant to ampicillin, tetracycline and nalidixic acid. Profiles SINFXB0003, SINFXB0005, and SINFXB0006 SINFXB0007 which showed identical antimicrobial resistance to ampicillin, cefotaxime / clavulanic acid, nalidixic acid and tetracycline, originated from the neck skin of broilers. SINFXB0004 profile showed identical antimicrobial resistance, and as a predominant profile SINFXB0001 ampicillin, nalidixic acid and tetracycline. Resistance profile of salmonella to antibiotics originating from broiler skin and from affected humans, did differ. Resistance was determined to nalidixic acid and ampicillin (95,5%), tetracycline (91%) and cefotaxime (68,25%). Salmonella isolates were susceptible and intermediately susceptible to trimethoprim / sulfamethoxazole, ciprofloxacin and gentamicin. Comparing the additional antimicrobial profiles it was found that the isolates which are derived from poultry were resistant to ampicillin, cefotaxime / clavulanic acid, nalidixic acid and tetracycline, while isolates originating from human disease showed resistance to ampicillin, tetracycline and nalidixic acid.