This report describes a rapid detection procedure for salmonellae from chicken feces by the combination of tetrathionate primary enrichment (preenrichment [PE])-bacterial lysis-capillary PCR and capillary gel electrophoresis. Pure Salmonella enterica serovar Enteritidis 64K was reisolated and detected by capillary PCR after buffered peptone water and nutrient broth, tetrathionate broth base Hajna (TTBH), and tetrathionate broth (TTB) preenrichments. When the same culture was mixed with intestinal homogenate, bacteriological reisolation and capillary PCR detection was achieved only by TTBH and TTB preenrichments. Capillary gel electrophoresis revealed that a Salmonella genus-specific 281-bp PCR product was detected when Salmonella strains but not non-Salmonella strains were tested. The detection limit of capillary PCR with whole-cell DNA extracted from pure Salmonella enterica serovars Enteritidis 64K, Typhimurium LT2-CIP60-62, and Gallinarum 64K was 3, 3, and 9 CFU ml ؊1 , respectively. The detection limit of capillary PCR from whole-cell DNA extracted from intestinal homogenate artificially contaminated with the same three strains was 3, 3, and 7 CFU ml ؊1 , respectively. We compared the results of the capillary PCR and bacteriological examination from the natural samples. Thirty-five of 53 naturally contaminated samples produced a specific PCR product. In 9 of the 35 PCR-positive samples, Salmonella could not be detected bacteriologically either by PE or a primary and delayed secondary enrichment (DSE) combination. In the 18 PCR-negative samples, 4 samples were found to harbor Salmonella by both PE and DSE and 14 samples were positive after DSE. Fifty-three additional intestinal homogenate samples, which were negative by their PE and DSE in bacteriological examination, were found to be also negative by their PCRs. The total time required to detect Salmonella with the capillary PCR method we used was approximately 20 h. If samples are from clinically diseased birds, the total time for PCR and detection is reduced to 2 h since the 18-h PE is not required. These results indicate that TTB enrichment, bacterial lysis, and genus-specific capillary PCR combined with capillary gel electrophoresis constitute a sensitive and selective procedure which has the potential to rapidly identify Salmonella-infected flocks.Salmonellae are among the major bacterial pathogens of poultry in Turkey and in the world (3,8). Prevention of Salmonella infection is important for poultry health and for the food industry, and prevention can be achieved only by good monitoring and screening programs (10,17,27). Salmonella detection by bacteriological methods usually requires 5 to 11 days (10, 27), and samples with low numbers of Salmonella cells, usually seen in subclinically infected chickens, may give false-negative results (7). Efforts have been made to reduce the time required and to increase the sensitivity of methods to detect Salmonella serovars in poultry samples (16,25). PCR with preincubation in an enrichment broth has been perform...

Aims: The present study describes the implementation of real‐time PCR to tetrathionate broth enrichment step of Salmonella detection in poultry. Methods and Results: Real‐time PCR with Salmonella invA‐specific primers and a standard bacteriological method was applied to detect Salmonella in tetrathionate enrichment cultures of 492 intestinal homogenates and 27 drag swabs from 47 poultry flocks. The number of positive individual samples by real‐time PCR and culture method was 65 (12·5%) and 35 (6·8%), respectively. The number of Salmonella‐positive flocks was 13 (27·7%) by both methods. PCR detection required 25 min for up to 32 samples. Melting curve analysis revealed the Tm for Salmonella‐specific PCR product as 87 ± 1°C. Conclusions: Implementation of real‐time PCR to tetrathionate broth enrichment step reduces the Salmonella detection time to 18 h and 25 min. Isolation of Salmonella should be carried out with PCR to determine the serovar. Significance and Impact of the Study: Real‐time PCR is a powerful tool in rapid and accurate Salmonella monitoring in poultry companies, together with standard bacteriology.

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.

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.The bacterial toxin cytolethal distending toxin (CDT) was initially described as having the ability to cause certain cultured cell lines to slowly distend and then die (18). More recently, several groups have shown that CDT causes sensitive cells to become apparently irreversibly blocked in either the G 1 (6,14) or G 2 phase of the cell cycle (4, 32, 36). CDT apparently invokes this block by causing direct DNA damage through the action of the CdtB protein, which is a member of a class of phosphodiesterases that includes nucleases (8,13,19). The CdtA and CdtC proteins appear to be required as accessory proteins, mediating binding to the CDT receptor on sensitive cells (20,21). CdtA and CdtC may also possess additional properties important for intoxication, but such roles have not yet been defined (12).CDT is produced by several different bacterial species, including Escherichia coli, Shigella spp., Campylobacter jejuni and related thermophilic campylobacters, Haemophilus ducreyi, Actinobacillus actinomycetemcomitans, and enterohepatic Helicobacter spp. (5,18,31,35,37). In all of these species, CDT activity is encoded by three adjacent genes, cdtA, cdtB, and cdtC, and the expression of all three genes is required for the production of active CDT (31, 34). CDT production by E. coli strains was first documented by Johnson and Lior (18), and the cdt genes from three different E. coli strains were subsequently cloned and sequenced (29,30,34). These sequence data indicated that there is heterogeneity present in the E. coli cdt genes. For example, the predicted amino acid sequences of CdtA, -B, and -C from E. coli strains 9142-88 and 6468/62 have 48, 61, and 42% identical and conserved amino acids, respectively (30, 34). The third strain sequenced, S5, is most closely related to strain 9142-88, with CdtA, C...

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.

In this study, 151 (18.6%) of 814 ceca obtained during in-line processing of 28 broiler (Hybro G, Avian, Arbor acres, and Cobb breeds) and 5 layer (Ross, Tetra SL, Isa Brown, and Brown Nick breeds) flocks in Turkey were found to be contaminated with four different Salmonella serovars. Only Salmonella enterica subsp. enterica Serovar Enteritidis (Salmonella Enteritidis) was recovered from layer birds, whereas Salmonella Enteritidis (81.5%). Salmonella Agona (7.6%), Salmonella Thompson (10.1%), and Salmonella Sarajane (0.8%) were isolated from broiler birds. Isolations of Salmonella Agona and Salmonella Thompson from poultry are reported for the first time in Turkey. The isolation of Salmonella Sarajane from chickens is the first report in the world. The standard method of National Poultry Improvement Plan, U.S. Department of Agriculture, was used to detect Salmonella from chicken cecal samples. Primary and delayed secondary enrichments (PE and DSE) were done in tetrathionate-Hajna broth (TTHB). Two different agar media, xylose lysine tergitol 4 (XLT4) and brilliant green with novobiocin (BGN) were used to observe, and compared for their isolation and selective differentiation of, Salmonella-suspected colonies. Isolated salmonellae were then biotyped and serotyped. Ninety-one and 151 salmonellae were isolated with XLT4 agar after PE and DSE, respectively. From the same samples, BGN agar was able to detect only 50 and 131 Salmonella after PE and DSE, respectively. The isolation rate with XLT4 was 11.2% (P < 0.01) with PE, and this rate increased to 18.6% after DSE. Also, the PE isolation rate (11.2%) with XLT4 agar was significantly higher (P < 0.01) than PE with BGN agar (6.1%). Salmonella was isolated from 39.3% (11 of 28) of the broiler flocks and from 60.0% (3 of 5) of the layers. The detection sensitivity of the isolation method was determined as 1 CFU g(-1) experimentally. These data demonstrate the presence of Salmonella Enteritidis, Salmonella Thompson, Salmonella Agona, and Salmonella Sarajane in chicken flocks in Turkey.

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Campylobacter jejuni produces a toxin called cytolethal distending toxin (CDT). Knowledge of the prevalence and homogeneity ofCampylobacter sp. cdt genes is incomplete. In this work, we identified four PCR primer pairs that collectively amplified cdt genes in all of the C. jejuni andCampylobacter coli strains tested. Restriction analyses of the cdt PCR products showed clear differences between thecdt genes of these two species, yet there were few heterogeneities noted between members of the same species. Consequently, it may be possible to speciate C. jejuni andC. coli isolates on the basis of restriction patterns within their cdt genes.