Nontyphoid Salmonella Prevalence, Serovar Distribution and Antimicrobial Resistance in Slaughter Sheep.

This study aimed to determine the current prevalence, serovar distribution and antimicrobial resistance rate and patterns of nontyphoid Salmonella (NTS) in slaughter sheep and their edible offal. While filling the gap of up to date related information in Turkey, data presented is also of significance since contamination of ovine meat, its products and offal with this pathogen is threat to public health due to their considerably high consumption rates in our country. Current NTS carriage in 200 apparently healthy slaughter sheep by ISO 6579:2002, 6579:2002/A1:2007 standard bacteriology (ISO) was 5% (10/200) (4 fecal content - 2%, 3 mesenterial lymph node - 1.5%, 3 kidney - 1.5%) out of 1,400 samples (0.7%), with no isolation from carcass, liver, gallbladder, spleen. Real-time PCR was in substantial agreement to ISO in confirming Salmonella-suspect isolates (Relative Trueness: 93.6%). S. Newport (40%) was the predominant serovar, followed by the second prevalent serovars as S. Typhimurium and S. Kentucky (20%), and by S. Umbilo and S. Corvallis (10%). Four and 6 out of 10 NTS isolates were susceptible (40%) and resistant (60%) to 18 antimicrobials, respectively. S. Typhimurium isolates were multidrug resistant (MDR) to tigecycline and sulphamethoxazole/trimethoprim, with one also resistant to cefepime. S. Corvallis was MDR to ampicillin, ciprofloxacin, norfloxacin and pefloxacin. The predominance of S. Newport and first isolation of S. Corvallis in sheep in the world; first time isolations of Newport, Kentucky, Corvallis, Umbilo serovars from sheep in Turkey; and high antimicrobial resistance rates obtained in majority of the isolates highlights study findings.

Salmonella prevalence and serovar distribution in healthy slaughter sheep and cattle determined by ISO 6579 and VIDAS UP Salmonella methods.

Prevalence of Salmonella in slaughter sheep and cattle was determined by International Organization for Standardization Method 6579 (ISO) and Vitek Immunodiagnostic Assay System UP Salmonella Phage Technology (VIDAS UP Salmonella SPT-VIDAS UP). A total of 400 healthy slaughter sheep (n = 200) and cattle (n = 200) carcass (C), fecal content (FC), mesenteric lymph node (MLN), liver (L), kidney (K), spleen (S) and gall bladder (GB) were randomly sampled and analysed. ISO and VIDAS UP results indicated 13 (3.25%) and 17 (4.25%) of 400 animals carried Salmonella, respectively, regardless of sample type. There was no isolation from L, S, GB, while 2 C (0.5%), 6 FC (1.5%), 7 MLN (1.75%), 3 K (0.75%) were contaminated with Salmonella. S. Typhimurium (27.8%), S. Enteritidis (22.2%), S. Newport (22.2%) were the three dominant serovars, followed by S. Kentucky (11.1%), S. Umbilo (5.6%), S. Corvallis (5.6%), and S. Albany (5.6%). Overall prevalence in 2800 samples was 0.46% by ISO and 0.61% by VIDAS UP. High relative trueness (RT: 99.79%) of VIDAS UP with a substantial agreement to ISO (κ value: 0.80) indicated its efficiency to accompany ISO to monitor Salmonella in slaughter animals. As the first report to evaluate ISO and VIDAS UP in detecting Salmonella from slaughter sheep and cattle, this current prevalence signifies a risk for public health in red-meat and related products in Turkey.

Salmonella serogroup detection in poultry meat samples by examining multiple colonies from selective plates of two standard culture methods.

This study aims to determine the serogroup profiles of randomly collected 46 chicken meat and 15 turkey meat samples, following the U.S. Food and Drug Administration's (FDA) Bacteriological Analytical Manual Chapter 5: Salmonella and International Organization for Standardization (ISO) Method 6579 culture methods. The total number of poultry meat samples with more than one serogroup isolated by the FDA and ISO culture methods were 10 (37.0%) and 21 (77.8%) of 27, respectively. Presence of multiple serogroups per sample was more frequently observed in chicken meat samples than in turkey meat samples. The profile of Salmonella serogroup isolates of chicken meat samples in descending order were serogroups D and E4 (15.8%), B and C2 (8.8%), C1 (5.3%), G (3.5%), and E1 and F (1.7%). The serogroup distribution of turkey meat sample isolates were serogroups B (27.2%), E4 (18.2%), and C2 (9.1%). On the basis of our findings that a selective plate in Salmonella culture method can harbor more than one serogroup, and that the FDA and ISO methods could detect different serogroups from chicken and turkey meats, we suggest screening multiple suspect colonies from each plate, if possible, and considering the collective and comparative use of the FDA and ISO culture methods and/or including several selective and differential media to ensure the detection of Salmonella and the possible detection of multiple serogroups from samples.

Evaluation of ISO 6579 and FDA-BAM methods to complement real-time polymerase chain reaction for the detection of Salmonella in naturally contaminated poultry meat and red meat.

In this study, we evaluated the Salmonella detection capability and compatibility of a LightCycler polymerase chain reaction (LC PCR) system with two bacteriological methods, United States Food and Drug Administration's Bacteriological Analytical Manual Chapter 5: Salmonella (FDA) and International Organization for Standardization Method 6579 (ISO). The aim was to determine which bacteriological method would support LC PCR for testing naturally contaminated poultry and red meat samples with Salmonella. Twenty three (50.0%) and 24 (52.2%) out of 46 chicken meat samples were positive for Salmonella by the FDA and ISO methods, respectively. Five of the 15 (33.3%) turkey meat samples were found to harbor Salmonella by both bacteriological methods. None of the red meat samples were positive for Salmonella using the FDA method. There was one red meat sample (3.3%) positive for Salmonella using ISO method. LC PCR results indicated that 23 (50.0%) and 31 (67.4%) of the DNA templates obtained from the 46 preenriched chicken meat FDA and ISO samples were positive for Salmonella. Salmonella detection rate from turkey meat samples by ISO LC PCR was 6.7%, whereas no detection was observed by FDA LC PCR. FDA LC PCR detection rate in red meat samples was 23.3%, whereas the ISO LC PCR was 43.3%. Relative accuracy rates of ISO LC PCR and FDA LC PCR were 67.4%, 60.0%, 53.3% and 56.5%, 66.7%, 76.7% for chicken, turkey, and red meats, respectively. We presume that the low relative accuracy problem, which can be related to the use of FDA and ISO preenrichments for template preparations in the PCRs, can be overcome by the use of primary enrichments of both FDA and ISO bacteriologies.

Real-time PCR culture and serology for the diagnosis of Mycoplasma gallisepticum in chicken breeder flocks.

This study aimed to compare a real-time PCR (rPCR) test with improved detection limit to serology and culture for the detection of Mycoplasma gallisepticum (MG) infection in chicken breeder flocks. Six hundred and forty-six blood and tracheal swab samples belonging to 31 grandparent chicken breeder flocks were tested by rPCR. The detection limit of rPCR was 0.9 pg microl(-1), with pure MG S6 strain DNA and 100 colony forming units (CFUs) ml(-1), where both pure culture and tracheal swabs were artificially spiked with the same strain. The seropositive flock rate based on both MG RPA and HI were calculated as 48.4% (15/31) and 32.3% (10/31), respectively, while culture- and rPCR-positive flock rates were 16.1% (5/31) and 29.0% (9/31), respectively. On flock-based analysis, culture method detected 5 out of 10 MG seropositive flocks (sensitivity 50%, specificity 100%), whereas rPCR detected 8 out of 10 flocks (sensitivity 80%, specificity 95%). Agreements between serology and culture, and serology and rPCR were 83.9% and 90.3%, respectively. On individual sample-based analysis, culture method detected 26 out of 78 MG seropositive chicken (sensitivity 33%, specificity 100%), whereas rPCR detected 51 out of 78 MG seropositive chickens (sensitivity 65%, specificity 96%). There was 91.9% and 91.4% agreement between serology and culture, and serology and rPCR, respectively. Results of this study indicate that the rPCR with improved in vitro detection limit could detect MG in seropositive chicken flocks. Therefore, we advise the use of rPCR and/or culture for confirmation of serology results obtained from screening MG infection in chicken flocks.

Inhibition of hepatocarcinogenesis by the deletion of the p50 subunit of NF-kappaB in mice administered the peroxisome proliferator Wy-14,643.

Wy-14,643 (WY) is a hypolipidemic drug that induces hepatic peroxisome proliferation and tumors in rodents. We previously showed that peroxisome proliferators increase NF-kappaB DNA binding activity in rats, mice, and hepatoma cell lines, and that mice deficient in the p50 subunit of NF-kappaB had much lower cell proliferation in response to the peroxisome proliferator ciprofibrate. In this study we examined the promotion of hepatocarcinogenesis by WY in the p50 knockout (-/-) mice. The p50 -/- and wild type mice were first administered diethylnitrosamine (DEN) as an initiating agent. Mice were then fed a control diet or a diet containing 0.05% WY for 38 weeks. Wild-type mice receiving DEN only developed a low incidence of tumors, and the majority of wild-type mice receiving both DEN and WY developed tumors. However, no tumors were seen in any of the p50 -/- mice. Cell proliferation and apoptosis were measured in hepatocytes by BrdU labeling and the TUNEL assay, respectively. Treatment with DEN + WY increased both cell proliferation and apoptosis in both the wild-type and p50 -/- mice; DEN treatment alone has no effect. In the DEN/WY-treated mice, cell proliferation and apoptosis were slightly lower in the p50 -/- mice than in the wild-type mice. These data demonstrate that NF-kappaB is involved in the promotion of hepatic tumors by the peroxisome proliferator WY; however, the difference in tumor incidence could not be attributed to alterations in either cell proliferation or apoptosis.

Salmonella profile in chickens determined by real-time polymerase chain reaction and bacteriology from years 2000 to 2003 in Turkey.

From years 2000 to 2003, Salmonella was investigated from a total of 1785 samples comprised of chicken intestinal samples, cloacal swabs, drag swabs, litter samples and chick dust samples collected from 191 poultry breeding flocks belonging to 15 different chicken breeding stock companies in the Marmara region, Turkey by a SYBR green-based real-time polymerase chain reaction (SGBRT-PCR), by a probe-specific real-time polymerase chain reaction (PSRT-PCR) and by standardized bacteriology as described in the manual of National Poultry Improvement Plan and Auxillary Provisions, United States Department of Agriculture. Between January 2000 and July 2001, Salmonella was detected at the rates of 5.87% and 4.10% out of a total of 1242 samples by SGBRT-PCR and bacteriology, respectively. From July 2001 until December 2003, Salmonella was found at rates of 11.42% and 5.52% from a total of 543 samples by PSRT-PCR and bacteriology, respectively. The dominant Salmonella serovar was determined as Salmonella enterica subsp. enterica Serovar Enteritidis (S. Enteritidis), while serogroup C1 and C2 in 2001 and serogroup E1 in 2002 were isolated as additional serovars. As a conclusion, S. Enteritidis seems to be the major problem in poultry breeding flocks in Turkey, and both of the real-time polymerase chain reaction methods were found more sensitive than standard bacteriology for the detection of Salmonella from poultry samples.

Patterns of variations in Escherichia coli strains that produce cytolethal distending toxin.

A collection of 20 Escherichia coli strains that produce cytolethal distending toxin (CDT) were analyzed for their virulence-associated genes. All of these strains were serotyped, and multiplex PCR analysis was used to ascertain the presence of genes encoding other virulence factors, including Shiga toxin, intimin, enterohemolysin, cytotoxic necrotizing factor type 1 (CNF1) and CNF2, heat-stable toxin, and heat-labile toxin. These CDT-producing strains possessed various combinations of known virulence genes, some of which have not been noted before. Partial cdtB sequences were obtained from 10 of these strains, and their predicted CdtB sequences were compared to known E. coli CdtB sequences; some of the sequences were identical to known CdtB sequences, but two were not. PCR primers based on sequence differences between the known cdt sequences were tested for their ability to detect CDT producers and to determine CDT type. Correlations between the type of CDT produced, the presence of other virulence properties, and overall strain relatedness revealed that the CDT producers studied here can be divided into three general groups, with distinct differences in CDT type and in their complement of virulence-associated genes.

Real-time polymerase chain reaction for Mycoplasma gallisepticum in chicken trachea.

In this work, we describe a rapid detection procedure for Mycoplasma gallisepticum from chicken tracheal swabs by real-time polymerase chain reaction (PCR) by LightCycler system, where we were able to monitor the amplification of the newly synthesized M. gallisepticum-specific PCR product as a proportionally increasing fluorescent signal by using the double-stranded DNA binding dye SYBR Green I and have identified M. gallisepticum-specific PCR products by DNA melting curve analysis by plotting the first negative derivative (-d[F1]dT) of fluorescence over temperature. Detection limits of the PCR were found to be 3 and 3000 colony-forming units ml(-1) with pure culture of M. gallisepticum and artificially spiked samples, respectively. Out of 96 tracheal swabs, 68 were taken from live chickens and 28 were taken by scraping the mucosal surface of the trachea (SMST) of necropsied chickens. All of the 18 PCR-positive results were from the swabs taken by the SMST method, whereas all of the samples taken from live chickens were negative. Thus, the PCR with the SMST method had a sensitivity and a specificity of 64.2% (18 of 28 chickens) and 100%, respectively. The total time required for template preparation from tracheal swab samples and real-time PCR was approximately 65 min. These results indicate that real-time PCR with the LightCycler technology is a rapid and sensitive test to identify M. gallisepticum-infected flocks if a proper sampling is applied.

Rapid detection of Salmonella from poultry by real-time polymerase chain reaction with fluorescent hybridization probes.

Detection of Salmonella by bacteriologic methods is known to be time consuming. Therefore, we have developed a real-time probe-specific polymerase chain reaction (PCR) to rapidly detect Salmonella invA gene-based PCR products from chicken feces and carcasses by a fluorescence resonance energy transfer assay. The sensitivity and the specificity of this system were determined as 3 colony-forming units ml(-1) and 100%, respectively. Overnight tetrathionate broth enrichment cultures of chicken feces and carcass samples were used in template preparation for PCR. Also, a standard bacteriology was performed (National Poultry Improvement Plan-U.S. Department of Agriculture, Bacteriological Analytical Manual-Food and Drug Administration Center for Food Safety and Applied Nutrition) for confirmation. Seventy-two cloacal swab, 147 intestine, and 50 carcass (neck) samples were examined. Thirteen (8.8%) and 25 (17%) of the intestinal samples were found to harbor Salmonella by bacteriology and PCR, respectively. Forty-five of 50 (90%) carcass samples were Salmonella positive by both methods. Salmonella was not detected from cloacal swab samples. Results indicate that this assay has the potential for use in routine monitoring and detection of Salmonella in infected flocks and carcasses.