Prevalence of Campylobacter spp. in a subset of intensive poultry flocks in Ireland

Aim:  The aim of this study was to investigate the prevalence of Campylobacter species in a subset of intensive poultry flocks by examining samples collected in geographically disparate areas on the island of Ireland.
Methods and Results:  Faecal, water and environmental samples were collected from the interior of poultry houses on nine farms. Three cultural methods were used for Campylobacter isolation: direct plating, enrichment culture and a recovery method for emerging Campylobacter spp. Presumptive Campylobacter isolates were confirmed using biochemical tests and further identified to species level by multiplex PCR. All flocks sampled in this study were found to be contaminated with Campylobacter at the time of sampling. Structural and air samples taken from the interior of broiler houses were also found to be Campylobacter positive. All water samples were found to be Campylobacter negative. The Campycheck method was used for the isolation of emerging Campylobacter spp.
Conclusions:  Campylobacter spp. were recovered (as contaminants) from the poultry house interior, air and environmental samples in all intensive poultry flocks surveyed.
Significance and Impact of the Study:  This study highlights the need for improved biosecurity on selected poultry farms.     

Rapid detection of Campylobacter coli,C. jejuni,and Salmonella enterica on poultry carcasses by using PCR-enzyme-linked immunosorbent assay

Contamination of retail poultry by Campylobacter spp. and Salmonella enterica is a significant source of human diarrheal disease. Isolation and identification of these microorganisms require a series of biochemical and serological tests. In this study, Campylobacter ceuE and Salmonella invA genes were used to design probes in PCR-enzyme-linked immunosorbent assay (ELISA), as an alternative to conventional bacteriological methodology, for the rapid detection of Campylobacter jejuni, Campylobacter coli, and S. enterica from poultry samples. With PCR-ELISA (40 cycles), the detection limits for Salmonella and Campylobacter were 2 x 10(2) and 4 x 10(1) CFU/ml, respectively. ELISA increased the sensitivity of the conventional PCR method by 100- to 1,000-fold. DNA was extracted from carcass rinses and tetrathionate enrichments and used in PCR-ELISA for the detection of Campylobacter and S. enterica, respectively. With PCR-ELISA, Salmonella was detected in 20 of 120 (17%) chicken carcass rinses examined, without the inclusion of an enrichment step. Significant correlation was observed between PCR-ELISA and cultural methods (kappa = 0.83; chi-square test, P < 0.001) with only one false negative (1.67%) and four false positives (6.67%) when PCR-ELISA was used to screen 60 tetrathionate enrichment cultures for SALMONELLA: With PCR-ELISA, we observed a positive correlation between the ELISA absorbance (optical density at 405 nm) and the campylobacter cell number in carcass rinse, as determined by standard culture methods. Overall, PCR-ELISA is a rapid and cost-effective approach for the detection and enumeration of Salmonella and Campylobacter bacteria on poultry.     

Evaluation of an automated immunomagnetic separation method for the rapid detection of Salmonella species in poultry environmental samples

Automated immunomagnetic separation (AIMS), using Dynabeads anti-Salmonella (Dynal, Oslo), was evaluated for its ability to detect Salmonella spp. in poultry environmental samples in comparison with standard, culture-based method (Health Canada, Health Protection Branch, MFHPB-20). AIMS was found to be more reliable in detecting Salmonella from artificially inoculated enrichment broths at low levels and exhibited a 15.5% higher sensitivity value than the culture method.     

A comparison of conventional culture and three rapid methods for the detection of Salmonella in poultry feeds and environmental samples

Three rapid methods, an impedance method (Malthus 2000 Analyzer), a colorimetric DNA hybridization method (Gene-Trak) and a post-enrichment enzyme-linked immunosorbent assay (Salmonella-Tek) were compared with conventional culture for the detection of Salmonella in poultry feeds, and in fluff and dust samples from poultry housing. The percentage positive samples for Salmonella by each of the methods were 25.5% for conventional culture, 38.4% for the Malthus, 28.9% for the Gene-Trak and 28.5% for the Salmonella-Tek. By any method 60/153 (39.2%) of the samples tested were positive on confirmed culture.     

Direct detection of Salmonella spp. in estuaries by using a DNA probe.

A method for direct detection of Salmonella spp. in water was developed by using a commercially available DNA probe. Particulate DNA was extracted from 500- to 1,500-ml water samples collected from New York Harbor and Chesapeake Bay and used as a substrate for a salmonella-specific DNA probe in dot blot assays. The method detected salmonellae in water samples from 12 of 16 sites, including 6 sites where salmonellae could not be cultured. The specificity of the probe was evaluated, and cross-hybridization, although negligible, was used to set detection limits for the assay. Salmonella DNA bound the probe quantitatively, and from these results Salmonella DNA in the total particulate DNA in environmental samples could be estimated. The data obtained in this study indicate that Salmonella spp. often are not detected in water samples by culture methods, even when they are present in significant numbers.     

Direct detection of Salmonella spp. in estuaries by using a DNA probe

A method for direct detection of Salmonella spp. in water was developed by using a commercially available DNA probe. Particulate DNA was extracted from 500- to 1,500-ml water samples collected from New York Harbor and Chesapeake Bay and used as a substrate for a salmonella-specific DNA probe in dot blot assays. The method detected salmonellae in water samples from 12 of 16 sites, including 6 sites where salmonellae could not be cultured. The specificity of the probe was evaluated, and cross-hybridization, although negligible, was used to set detection limits for the assay. Salmonella DNA bound the probe quantitatively, and from these results Salmonella DNA in the total particulate DNA in environmental samples could be estimated. The data obtained in this study indicate that Salmonella spp. often are not detected in water samples by culture methods, even when they are present in significant numbers.     

Comparison of three rapid methods for identification of Salmonella spp

A study was carried out to compare three rapid methods for detection of Salmonella spp. The fluorogenic MUCAP test (Biolife, Italy), the SM-ID agar test (bioMérieux, France) and the Rambach agar test (Merck, Germany) were used in this study to examine 103 strains (69 Salmonella strains and 34 non- Salmonella strains). Two conventional culture media, Hektoen and Leifson agars, were also included. The sensitivities of the MUCAP, SM-ID, Rambach and Hektoen agar tests for pure strains were 100, 93, 88 and 99%, respectively, and their specificities were 74, 97, 76 and 59%, respectively. A total of 100 stool samples from patients with acute diarrhoea was also tested and showed great discrepancy between the different methods. In agreement with other investigators, it was found that the discriminating capacity of Rambach and SM-ID as primary plating media was very restricted. The MUCAP test was very sensitive, rapid and easy to perform but not very specific. In view of these results, it is essential to combine different methods for the accurate and reliable detection of Salmonella strains.     

The incidence of antimicrobial-resistant Salmonella spp on freshly processed poultry from US Midwestern processing plants

AIMS: To determine the incidence of antimicrobial-resistant Salmonella spp. on processed poultry (turkey) at Midwestern poultry plants. METHODS AND RESULTS: Two participating plants were visited at monthly intervals for a period of 1 year. Surface swabs were obtained from carcasses at two selected points on the production line, pre- and post-chill. In addition, samples of the chill water from chill tanks were also examined. Isolation and detection of Salmonella spp. from carcass swabs and chill water was carried out using standard enrichment techniques. Immunomagnetic separation was used to enhance the recovery of the pathogen. Salmonella isolates recovered were identified, serotyped and their antimicrobial resistance profiles determined using the National Antimicrobial Resistance Monitoring System. Results from the study indicated that the overall incidence of Salmonella was approx. 16.7%, with a greater incidence of the pathogen observed on pre-chill than post-chill carcasses. Salmonella isolates recovered displayed resistance to an average of four different antimicrobials. Approximately 15 different serotypes of Salmonella spp. were recovered, with Salmonella serotype Agona, Salmonella serotype Hadar, Salmonella serotype Heidelberg and Salmonella serotype Senftenberg being the most common. CONCLUSIONS: The incidence of Salmonella spp. was relatively low and isolates recovered showed significant degrees of antimicrobial resistance. Factors such as the processing plant examined, the season and farms that were presenting animals for processing influenced the incidence of the pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: Differences were observed in the serotypes of Salmonella recovered and the types of antimicrobial resistance found at the two plants. The study suggests that the use of antimicrobials at the farm level influences the creation of an environment that promotes the selection of antimicrobial-resistant Salmonella spp. The incidence, isolation and detection of Salmonella spp. on processed poultry are discussed.     

Evaluation of broiler litter with reference to the microbial composition as assessed by using 16S rRNA and functional gene markers

Very little is known about the microbial composition of animal bedding wastes, including poultry litter, and what is known has been deduced from standard culture methods, by which some fastidious organisms that exist in the environment may not be detected. We evaluated the bacterial composition of poultry litter by using a combination of culture and molecular detection. Total aerobic bacteria in poultry litter were detected by culture at 10(9) CFU/g of material. Enteric bacteria such as Enterococcus spp. and coliforms composed 0.1 and 0.01%, respectively, of the total aerobic cultivatable bacteria in poultry litter; no Salmonella strains were detected by culture. In order to characterize the most abundant bacterial groups, we sequenced 16S ribosomal DNA (rDNA) genes amplified by PCR with microbial community DNA isolated from poultry litter as the template. From the 16S rDNA library, 31 genera were identified. Twelve families or groups were identified with lactobacilli and Salinococcus spp. forming the most abundant groups. In fact, 82% of the total sequences were identified as gram-positive bacteria with 62% of total belonging to low G+C gram-positive groups. In addition to detection of 16S rDNA sequences associated with the expected fecal bacteria present in manure, we detected many bacterial sequences for organisms, such as Globicatella sulfidofaciens, Corynebacterium ammoniagenes, Corynebacterium urealyticum, Clostridium aminovalericum, Arthrobacter sp., and Denitrobacter permanens, that may be involved in the degradation of wood and cycling of nitrogen and sulfur. Several sequences were identified in the library for bacteria associated with disease in humans and poultry such as clostridia, staphylococci, and Bordetella spp. However, specific PCR targeting other human and veterinary pathogens did not detect the presence of Salmonella, pathogenic Escherichia coli, Campylobacter spp., Yersinia spp., Listeria spp., or toxigenic staphylococci. PCR and DNA hybridization revealed the presence of class 1 integrons with gene cassettes that specify resistance to aminoglycosides and chloramphenicol. Only from understanding the microbial community of animal wastes such as poultry litter can we manage animal disease and limit the impact of animal waste on the environment and human and animal health.     

APPLICATION OF POLYMERASE CHAIN REACTION-OLIGONUCLEOTIDE LIGATION ASSAY FOR THE DETECTION OF SALMONELLAE IN PROCESSED MEAT, POULTRY, FISH, AND PET FOODS

We have tested a rapid and sensitive DNA-based assay for the detection of Salmonella serovars in a number of different processed meat, fish, poultry, and pet food samples. This technique uses an enrichment broth cultivation followed by a Salmonella-specific polymerase chain reaction (PCR) and oligonucleotide ligation assay (OLA) to specifically detect amplified PCR products in an ELISA-based microtiter plate format. The combined cultivation and PCR-OLA techniques were compared with a conventional culture method and with DNA hybridizations of PCR products for the detection of Salmonella bacteria. Eighty-one different processed meat, poultry, and pet food samples were screened for the presence of Salmonella serovars after 24 h and 48 h of enrichment broth cultivation. After 24 h of incubation, one ground turkey sample was positive by both culture and PCR-OLA (100% sensitivity and 100% specificity). After 48 h of incubation, two additional samples (ground beef and a dog food sample) were positive by both culture and PCR-OLA (100% sensitivity and 100% specificity), and three other samples (two ground beef samples and one ground turkey) were positive only by PCR-OLA (96.1% specificity). All positive PCR-OLA results were confirmed in DNA hybridizations with an oligonucleotide specific for the amplified PCR product. When compared to conventional culture, the combined 48 h enrichment and PCR-OLA had a positive predictive value of 50% and a negative predictive value of 100%. We concluded that a combined cultivation and PCR-OLA could be used as a sensitive and specific presumptive screening method for detecting Salmonella serovars in processed meat, fish, poultry, and pet foods.     

Climate patterns governing the presence and permanence of salmonellae in coastal areas of Bahia de Todos Santos, Mexico

Despite the importance of salmonellae as one of the major causes of food-borne infections worldwide, data regarding the presence of these organisms in the environment are limited. We investigated the presence of Salmonella spp. in Bahia de Todos Santos (Baja California, Mexico) and evaluated the environmental factors that affect the occurrence of Salmonella spp. in this arid region. A total of 1,331 samples collected from 21 sites along the coast during a period of 3 years were analyzed for Salmonella spp. Geographical and seasonal distribution of Salmonella spp. was evaluated in association with environmental parameters and with human infections in the area. The incidence of Salmonella bacteria throughout the study was 4.8%, with the highest incidence detected in wastewater (16.2%), followed by stream water (10.6%), mollusks (7.4%), and seawater (2.3%). Twenty different serotypes were identified among the 64 Salmonella isolates. The dominant serotype was Typhimurium (23.4%), followed by Vejle (6.2%). The presence of Salmonella spp. in coastal areas was mostly confined to rainy periods and areas of stream discharges, and runoff was identified as the predominant factor influencing the transport of Salmonella bacteria from source points to the sea via streams. Isolation of Salmonella spp. was negatively and significantly associated with temperature, probably because of the effect of solar radiation in the decline of permanence of Salmonella bacteria. Conversely, human infections prevailed during the warmest months and were negatively correlated with the presence of Salmonella spp. in the marine environment.     

Evaluation of the MicroFoss system for the detection of Listeria species in environmental samples

The MicroFoss system was evaluated for its ability to detect Listeria species in environmental samples. The sensitivity and specificity of the MicroFoss were determined in relation to a standard culture method for Listeria detection. The sensitivities of both the MicroFoss and standard culture methods were similar (88.4%-MicroFoss, 90.7%-Culture) based on the total number of positive results obtained by both methods. The MicroFoss system detected Listeria spp. in 12 samples, which were not detected by culture, and the culture method detected Listeria spp. in 15 samples, which were not detected by the MicroFoss method. This was likely due to uneven distribution of low levels of Listeria organisms in the split sponge samples used to assess the performance of these test methods. The specificity value determined for the MicroFoss system was 92.7%. The majority of microbes causing false positive results in the MicroFoss system were Bacillus species, which were readily distinguishable from Listeria species by a simple Gram stain and morphological features. Listeria monocytogenes (89.4%-MicroFoss, 88.0%-Culture) and Listeria innocua (8.8%-MicroFoss, 7.7%-Culture) were the most common isolates of Listeria detected by the two test methods, with L. monocytogenes being the most predominant isolate detected. The highly comparable results and rapid nature of the MicroFoss system demonstrate its effectiveness as a detection system for species of Listeria in environmental samples. The fact that the sensitivity of the MicroFoss system was similar to that of the culture method and the Listeria results were obtained within 48 h of testing, support the use of the MicroFoss as an alternative rapid method for screening large numbers of environmental samples for Listeria spp.     

Fluorescent-antibody method useful for detecting viable but nonculturable Salmonella spp. in chlorinated wastewater.

An indirect fluorescent-antibody (IFA) technique, which employed adsorbed Behring polyvalent I O antiserum, was used to detect Salmonella spp. in environmental water systems. The IFA method used in this study detected 95% of Salmonella serotypes encountered in human infections in France, with a sensitivity threshold of 7.5 x 10(3) bacteria per ml of wastewater. Specificity was assessed by testing IFA against Salmonella-free seawater and a variety of bacteria other than Salmonella spp. When used to examine raw and chlorinated wastewater over a 2-month period, the IFA method was successful in detecting Salmonella spp. in all 12 of the samples examined, with total numbers determined to be 4.5 x 10(5) to 3.3 x 10(7) salmonellae per 100 ml. In comparison, for the same samples, enumeration by culture, using the most-probable-number technique, was effective in detecting Salmonella spp. in only four of eight raw-water samples and one of four chlorinated water samples tested. Three samples were further tested by using the direct viable count procedure combined with IFA and results showed that 5 to 31.5% of the Salmonella spp. enumerated by this method in chlorinated water were substrate responsive.     

Fluorescent-antibody method useful for detecting viable but nonculturable Salmonella spp. in chlorinated wastewater

An indirect fluorescent-antibody (IFA) technique, which employed adsorbed Behring polyvalent I O antiserum, was used to detect Salmonella spp. in environmental water systems. The IFA method used in this study detected 95% of Salmonella serotypes encountered in human infections in France, with a sensitivity threshold of 7.5 x 10(3) bacteria per ml of wastewater. Specificity was assessed by testing IFA against Salmonella-free seawater and a variety of bacteria other than Salmonella spp. When used to examine raw and chlorinated wastewater over a 2-month period, the IFA method was successful in detecting Salmonella spp. in all 12 of the samples examined, with total numbers determined to be 4.5 x 10(5) to 3.3 x 10(7) salmonellae per 100 ml. In comparison, for the same samples, enumeration by culture, using the most-probable-number technique, was effective in detecting Salmonella spp. in only four of eight raw-water samples and one of four chlorinated water samples tested. Three samples were further tested by using the direct viable count procedure combined with IFA and results showed that 5 to 31.5% of the Salmonella spp. enumerated by this method in chlorinated water were substrate responsive.     

Presence of Listeria and Salmonella spp. in retail chicken in Northern Ireland

AIMS: Retail packs of fresh chicken in Northern Ireland were sampled to determine the frequency with which they were contaminated with Salmonella and Listeria spp. METHODS: Packs of chicken were chosen from supermarkets ensuring a diverse range of EU producer codes were sampled. Salmonellas were isolated using BS EN 12824: 1998 methodology, biotyped and serotyped whilst Listeria spp. were isolated based on EN ISO 11290-1: 1996 procedures and identified using a multiplex PCR system utilizing genus and species specific primers. SIGNIFICANCE AND IMPACT OF THE STUDY: Only three of 205 samples yielded Salmonella spp. indicating that measures undertaken by the poultry industry to control this pathogen have apparently been successful. However, Listeria spp. were present in 38 of 80 samples tested (48%) and 14 (18%) yielded Listeria monocytogenes. Thus Salmonella controls do not markedly affect this pathogen and retail packs of raw chicken must be considered a potential source of L. monocytogenes, and appropriate precautions taken to prevent infection.     

Detection of Salmonella enterica serovar Typhi (S. Typhi) by selective amplification of invA, viaB, fliC-d and prt genes by polymerase chain reaction in mutiplex format

AIMS: Development of a PCR assay that can target multiple genes for rapid detection of Salmonella enterica serovar Typhi (S. Typhi) from water and food samples. METHODS AND RESULTS: PCR primers for invasion, O, H and Vi antigen genes, invA, prt, fliC-d and viaB were designed and used for the rapid detection of S. Typhi by multiplex PCR. Internal amplification control, which co-amplified with prt primers, was also included in the assay. The results showed that all cultures of Salmonella were accurately identified by the assay with no nonspecific amplification in other cultures. The assay had 100% detection probability when a cell suspension of 10(4) CFU ml(-1) (500 CFU per reaction) was used. Salmonella Typhi bacteria were artificially inoculated in the water and food (milk and meat rinse) samples and detected by mPCR after overnight pre-enrichment in buffered peptone water. No Salmonella bacteria could be detected from water samples collected from the field by mPCR or standard culture method. CONCLUSIONS: The developed mPCR assay provides specific detection of S. Typhi. SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid methods for detection of S. Typhi from complex environmental matrices are almost nonexistent. The mPCR assay reported in this study can be useful to identify S. Typhi bacteria in field environmental samples.     

Impact of concentration, temperature, and pH on inactivation of Salmonella spp. by volatile fatty acids in anaerobic digestion

It is known that the presence of volatile fatty acids may play a role in the inactivation of pathogens for systems that employ an acid phase reactor. This study was conducted to investigate the influence of volatile fatty acids on the inactivation of Salmonella spp. over a range of digestion temperatures. In this study, digesters that were treating municipal wastewater treatment plant sludges were operated at temperatures that ranged from 35 to 49 degrees C and had a solids residence time of 15 days. Samples collected from the effluent of the digesters were dosed with solutions containing acetic, propionic, and butyric acids alone and in mixtures, and the dosed effluents were analyzed for Salmonella spp. over time. In the first round of testing, the digester effluents were dosed with individual organic acids and also a mixture containing all three volatile fatty acids over a range of concentrations from 750 to 6000 mg/L, and the pH of the samples was fixed at a value of 5.5. In the second round of testing, the sample sludges were spiked with a fixed amount of organic acid mixture, and the pH was varied from 4.5 to 7.5. The reduction of Salmonella spp. in digester effluents, when dosed with volatile organic acids, was found to depend on pH, temperature, the chain length of the acids, and the concentration and composition of the acids present. Increases in temperature appeared to increase the inhibitory effects of the volatile organic acids. At mesophilic temperatures, acidic pHs resulted in a greater inhibition of Salmonella spp.; whereas at higher temperatures neutral pHs were found to be more inhibitory. The results suggest that acid phase digesters that operate at elevated temperatures and low pH can achieve substantial reduction of Salmonella spp.     

Efficacy of a commercial polymerase chain reaction-based assay for detection of Salmonella spp. in animal feeds

Salmonellosis is a cyclic problem in the food industry, to which animal feed has been contributory. Current conventional methods of Salmonella spp. detection require 96 h for detection and confirmation. With modern and just-in-time production schedules, a 96-h hold represents a significant expense in storage and decontamination. The commercially available assay, 'BAX for Screening/Salmonella' (BAX), is based on the principle of the polymerase chain reaction and may represent a significant decrease in assay time. Seven fresh feed formulations, two fresh feed ingredients, seven stored feeds and two stored feed ingredients were artificially contaminated with a primary poultry isolate of Salmonella typhimurium and analysed by conventional and BAX methodology. The results of BAX agreed with conventional plating results for 16 of 18 samples spiked with 1200 cfu 10 g(-1) of feed and 13 of 18 samples spiked with 40 cfu 10 g(-1) of feed. Indigenous Salmonella spp. were detected in five of eight samples of poultry diets by conventional methods. With BAX, Salmonella spp. could not be detected in any of the samples after only 7 h of enrichment but could be detected in two dietary samples after 13 h of enrichment and four dietary samples after 24 h of enrichment. Specific sequences of salmonella DNA that were extracted from poultry diets could be detected with BAX.     

Detection of Salmonella enterica serovar typhimurium by using a rapid, array-based immunosensor

The multianalyte array biosensor (MAAB) is a rapid analysis instrument capable of detecting multiple analytes simultaneously. Rapid (15-min), single-analyte sandwich immunoassays were developed for the detection of Salmonella enterica serovar Typhimurium, with a detection limit of 8 x 10(4) CFU/ml; the limit of detection was improved 10-fold by lengthening the assay protocol to 1 h. S. enterica serovar Typhimurium was also detected in the following spiked foodstuffs, with minimal sample preparation: sausage, cantaloupe, whole liquid egg, alfalfa sprouts, and chicken carcass rinse. Cross-reactivity tests were performed with Escherichia coli and Campylobacter jejuni. To determine whether the MAAB has potential as a screening tool for the diagnosis of asymptomatic Salmonella infection of poultry, chicken excretal samples from a private, noncommercial farm and from university poultry facilities were tested. While the private farm excreta gave rise to signals significantly above the buffer blanks, none of the university samples tested positive for S. enterica serovar Typhimurium without spiking; dose-response curves of spiked excretal samples from university-raised poultry gave limits of detection of 8 x 10(3) CFU/g.     

Influence of octanoic acid addition to medium on some volatile compounds and PR-toxin biosynthesis by Penicillium roqueforti

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 degrees 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.