Effects of sample storage and delayed secondary enrichment on detection of Salmonella spp in swine feces

OBJECTIVE: To determine effects of fecal sample storage and delayed secondary enrichment (DSE) on detection of Salmonella spp in swine feces. Sample Population-Fecal samples obtained from 84 pigs in a commercial herd. PROCEDURE: Each fecal sample underwent 3 storage treatments: no storage (ie, processed on the day of collection), storage at 4 C for 6 days, and storage at -15 C for 14 days. After assigned storage treatments, all samples were enriched in Rappaport-Vassiladias (RV) broth (single enrichment) and plated on XLT4 agar. Delayed secondary enrichment was performed, using single enrichment broths that were stored for 4 days at room temperature. RESULTS: Of 504 cultures, 186 (36.9%) were Salmonella positive. A difference in proportions of samples with positive results was not found between same-day processing and storage at 4 C for 6 days. Compared with use of single enrichment for 24 hours (34% positive), use of DSE resulted in a greater proportion (40%; P < 0.001) of samples with positive results. Estimated relative sensitivities for the storage methods were 0.90, 0.85, and 0.71 for same-day processing, storage at 4 C for 6 days, and storage at -15 C for 14 days, respectively. CONCLUSIONS: Where practical, processing of fecal samples on the day of collection is recommended, although storage at 4 C for several days does not result in marked loss of sensitivity. Improved detection associated with DSE warrants further investigation and optimization.     

Molecular characterization of Salmonella enteritidis isolates from Maine poultry and poultry farm environments.

Eighty-six Salmonella enteritidis isolates obtained during a surveillance program of poultry farms in Maine were subjected to phage-typing, plasmid profiling and fingerprinting, outer-membrane polypeptide analysis, and antimicrobial sensitivity testing. Isolates were obtained from a variety of sources, including poultry-farm environmental samples, chicken organ samples, human stool samples, cat feces, and live-trapped rats and mice. These isolates were compared with 21 S. enteritidis isolates originating outside of Maine. Phage types isolated in Maine included 13a (60%); 14b (29%); 23 (5%); 8 (2%); and 2 (2%). All S. enteritidis isolates from Maine carried plasmid DNA, and 97% of these isolates carried a 40.3-megadalton plasmid alone (6%) or in conjunction with several smaller plasmids (91%). All 52 phage-type 13a isolates harbored 40.3- and 3.0-megadalton plasmids. All 25 phage-type 14b isolates carried 3.3- and 1.3-megadalton plasmids, and 22 isolates also carried the 40.3-megadalton plasmid. All isolates displayed highly similar outer-membrane polypeptide profiles and were sensitive to a variety of antimicrobials commonly used against gram-negative organisms. The above data suggest that phage type and plasmid content may be related in the cases of phage-type 13a and 14b isolates, and that traditional plasmid-borne antimicrobial resistance determinants were not present in Maine isolates. Results also indicate that phage-typing can be a valuable epizootiological tool for monitoring the potential spread of these strains throughout the Northeast.     

Comparison of a Salmonella enteritidis-specific polymerase chain reaction assay to delayed secondary enrichment culture for the detection of Salmonella enteritidis in environmental drag swab samples

The California Egg Quality Assurance Program uses the delayed secondary enrichment culture method for detecting Salmonella Enteritidis in environmental drag swabs obtained from commercial layer complexes. The turnaround time for this method is variable and is dependent on the prevalence of Salmonella, level of the Salmonella identification, and capabilities of the performing laboratory. On a sample basis, a range of 4 to 8 days is required to identify a Salmonella sp. to the serogroup level. Additional time is required to serotype group D Salmonella isolates. A Salmonella Enteritidis-specific polymerase chain reaction (PCR) assay was developed for use on drag swabs and chick box papers, and had a turnaround time of 3-4 days. The delayed secondary enrichment culture method and the Salmonella Enteritidis-specific PCR assay were compared on 942 drag swab and 85 chick box paper samples submitted from 217 and 22 premises, respectively, as part of the California Egg Quality Assurance Program. The PCR assay identified 43 positive Salmonella Enteritidis samples from 22 premises, whereas the culture method identified 24 group D Salmonella-positive samples from 16 premises. There was a significant difference (P = 0.001) in the proportion of positive samples as determined by the two assays. Complete serotyping of the group D Salmonella-positive cultures confirmed Salmonella Enteritidis in all but one sample that was identified as Salmonella Jamaica and was negative by the PCR assay.     

The effect of environmental poultry samples on the pH of typical Salmonella pre-enrichment and enrichment media following incubation

The first step to detect Salmonella in feed and other dry contaminated samples is a pre-enrichment broth that can assist in the recovery of small numbers of stressed Salmonella cells. A previous study demonstrated that incubation of feed and feed ingredients in commonly used pre-enrichment media resulted in a low pH that injured or killed the Salmonella. The objective of this study was to determine which environmental samples and pre-enrichment and selective broths could interfere with the accuracy of Salmonella detection by allowing pH to drop. Samples were collected from commercial poultry operations. Triplicate 10-g subunits were dispensed into sterile 18-oz Whirl Pak bags and 90 mL of each of the media [lactose broth (LB), buffered peptone water (BPW), Universal Pre-enrichment (UP), minimal salts (M-9), tetrathionate broth (TT) and Rappaport-Vassiliadis broth (RV)] were added to the bags and incubated 24 h at 37°C (or 42°C for RV and TT). The pH was measured after incubation. With turkey litter, fluff, and eggshells, regardless of media, the pH never went below 5.7. Regardless of sample type, the lowest pH was 6.1, 6.2, and 6.4 for UP, M-9, and BPW, respectively. For the pre-enrichment medium commonly used in the US, (LB) the pH dropped to 4.7 to 4.9 with broiler litter and 4.2 for boot covers used in turkey or broiler houses. Many researchers testing these sample types are unaware of this potential for pH change during pre-enrichment and may be underestimating the presence of Salmonella.     

A comparison of two enrichment and two plating media for the isolation of Salmonella sp. from broilers.

Twenty duplicate cloacal swabs and the intestines of 98 broilers were cultured using Rappaport-Vassiliadis and tetrathionate as enrichment broths. These were plated to brilliant green and modified dulcitol brilliant green agars at one, two and seven days. Salmonellae were recovered with greater frequency from tetrathionate plated to modified dulcitol brilliant green than the other combinations.     

Ring-trial results for the cultural detection of Salmonella in poultry faeces

The Directive 2003/99/EG of the European Parliament and of the Council on the monitoring of zoonoses and zoonotic agents demands a quality management (QM) system for the execution of its monitoring programmes. Consequently the National Salmonella Reference Laboratory of Germany performed two ring-trials in 2005 and 2006 on the microbiological detection of Salmonella from poultry feces among all participating laboratories in the Federal States. Salmonella detection was performed according to the EN ISO 6579:2002 standard method which was modified according to the recommendations of the Community Reference Laboratory for Salmonella in Bilthoven, The Netherlands. This method uses modified-semisolid Rappaport-Vassiliadis Agar as the only selective enrichment. In 2005 twenty-four and in 2006 twenty-two laboratories participated.They received eight identical samples of the contamination levels L0 (no Salmonella), L1 (11 and 16 cfu per 10 g faeces respectively) and L2 (292 and 418 cfu per 10 g faeces respectively). For both years the data of 20 laboratories could statistically be evaluated. The relative accuracy of the respected results increased from 88.8% in 2005 to 98% in 2006. This is as well reflected in the improved COR- and Kappa-Indices. Taken all together the data show, that the modified-semisolid Rappaport-Vassiliadis protocol is a sensitive, established method for the detection of Salmonella from poultry faeces.     

如何降低家禽沙门氏菌感染的风险(续完)

3预防沙门氏菌传播的措施3.1卫生和消毒在引进新一批鸡群前,所有鸡舍和器具必须进行清洁消毒,特别是在前一批鸡群为沙门氏菌阳性的情况下,清洁消毒就更有必要。卫生措施的有效性可通过严格的细菌学定量检测验证,要求肠道细     

"养鸡生产中沙门氏菌病的预防与控制策略"专栏(四)沙门氏杆菌和弯曲杆菌在家禽群体中的动态变化特征

难道我们不想掌控自身命运吗?虽然这是-个很广博的问题,但如果你在考虑幼龄肉鸡群沙门氏菌感染这个特定话题以及考虑它与人食源性疾病可能的关系时,这个问题就具有特殊的意义.出席美国国家家禽改良计划(the National Poultry Improvement Plan,NPIP)第40届年会(两年一次)的大多数代表,在投票是否采用美国肉鸡扩繁群肠炎沙门氏菌监测方案时,对该特殊问题给出了肯定回答.     

Reducing risks of Salmonella infections in poultry

3预防沙门氏菌传播的措施 3.1卫生和消毒 在引进新一批鸡群前,所有鸡舍和器具必须进行清洁消毒,特别是在前一批鸡群为沙门氏菌阳性的情况下,清洁消毒就更有必要.卫生措施的有效性可通过严格的细菌学定量检测验证,要求肠道细菌计数小于103个/25 m2.     

饲料添加剂可降低家禽沙门氏菌的感染率

对家禽生产者而言,沙门氏菌出现在家禽中绝对是一件高成本的事故。饲料添加剂能通过多种方式来降低沙门氏菌感染的风险。精油化合物的抗菌特性会使家禽具有良好的健康状况,并可确保消费者的食品安全。     

Influence of enrichment media and application of a PCR based method to detect Salmonella in poultry industry products and clinical samples.

To attempt the rapid detection of Salmonella enterica, we have coupled a culture procedure with PCR amplification of the genus-specific invE/invA genes. The method was applied to different kinds of samples from the poultry industry and evaluated by using hydrolyzed feather meal, meat meal, litter and viscera, all experimentally inoculated with a known number of Salmonella followed by cultivation in selenite--cystine broth prior to the PCR reaction. The expected 457bp specific DNA fragment could be amplified from dilutions containing as few as 5.7CFU, indicating that the PCR technique can be successfully coupled with culture in an enrichment broth to distinguish Salmonella species from other enteric bacteria present in samples from the poultry industry. Tetrathionate broth proved to be a much better enrichment media compared to selenite-cystine when the presence of Salmonella was evaluated by PCR in 1-day-old chicks experimentally infected with known numbers of Salmonella. Samples included cecal tonsils and viscera, collected at 48h and 7 days postinfection. The PCR technique was more sensitive in detecting infected animals than the standard microbiological procedure, which detected only 47% of all PCR positive samples.     

Serotype-specific and serotype-independent strategies for preharvest control of food-borne Salmonella in poultry

Of more than 2500 identified Salmonella serotypes, only a small proportion are common in poultry flocks. However, there is an epidemiologically important connection between poultry products and human infections because many of the serotypes that are most prevalent in humans (such as Salmonella Typhimurium and Salmonella Enteritidis) are similarly common in poultry. The scope of food safety efforts for poultry products has been broadened in recent years to include more attention to animal production (or preharvest) issues. The goal of preharvest poultry food safety is to minimize opportunities for the introduction, persistence, and transmission of flock infections with Salmonella and other human pathogens. This objective can be pursued either by general strategies directed against all Salmonella serotypes (and in some instances against other pathogenic microorganisms as well) or by more specific strategies that are designed to act with precision against particular Salmonella serotypes with distinctive public health or economic significance. Risk assessment studies have recommended intervention at multiple steps in the farm-to-table continuum as the most productive overall approach. A comprehensive quality assurance strategy, encompassing both broadly based risk-reduction practices and targeted testing to detect pathogens of concern, has been associated with a lower incidence of Salmonella Enteritidis infections in both egg-laying flocks and humans in a number of countries. Although the emphasis in these types of programs is primarily on risk reduction, testing provides essential verification of the efficacy and cost-effectiveness of risk-reduction practices (and identifies flocks infected with uniquely problematic serotypes). Vaccination can enhance the short-term responsiveness of control programs to address problems involving specific serotypes of elevated significance.     

Results of salmonella isolation from poultry products, poultry, poultry environment, and other characteristics

Five hundred sixty-nine Salmonella were isolated out of 4745 samples from poultry products, poultry, and poultry environment in 1999 and 2000 from the Pacific northwest. These Salmonella were identified to their exact source, and some were serogrouped, serotyped, phage typed, and tested for antibiotic sensitivity. Food product samples tested included rinse water of spent hens and broilers and chicken ground meat. Poultry environment samples were hatchery fluff from the hatcheries where eggs of grandparent broiler breeders or parent broiler breeder eggs were hatched and drag swabs from poultry houses. Diagnostic samples were of liver or yolk sac contents collected at necropsy from the young chicks received in the laboratory. Of these samples tested, 569 were Salmonella positive (11.99%). Ninety-two Salmonella were serogrouped with polyvalent somatic antisera A-I and the polymerase chain reaction. Somatic serogroups B and C comprised 95.25% of all the Salmonella. Out of a total of 569 positive samples, 97 isolates of Salmonella were serotyped. A total of 16 serotypes and an unnamed Salmonella belonging to serogroup C1 were identified. The Salmonella serotypes were heidelberg (25.77%); kentucky (21.64%); montevideo (11.34%); hadar and enteritidis (5.15% each); infantis, typhimurium, ohio, and thompson (4.12% each); mbandaka and cerro (3.09% each); senftenberg (2.06%); berta, istanbul, indiana, and saintpaul (1.03% each); and an unnamed monomorphic Salmonella (2.06%). Ninety-two Salmonella were tested for drug sensitivity with nine different antimicrobials. All of the 92 Salmonella were resistant to erythromycin, lincomycin, and penicillin except one sample (S. berta), which was moderately sensitive to penicillin. All of the tested Salmonella were susceptible to sarafloxacin and ceftiofur. The percentages of Salmonella susceptible to sulfamethoxazole-trimethoprim, gentamicin, triple sulfa, and tetracycline were 97.83%, 92.39%, 86.96%, and 82.61%, respectively.     

E-nose identification of Salmonella enterica in poultry manure

1. A DiagNose II electronic nose (e-nose) system was tested to evaluate the performance of such systems in the detection of the Salmonella enterica pathogen in poultry manure.

2. To build a database, poultry manure samples were collected from 7 broiler houses, samples were homogenised, and subdivided into 4 portions. One portion was left as is; the other three portions were artificially infected with S. enterica.

3. An artificial neural network (ANN) model was developed and validated using the developed database.

4. In order to test the performance of DiagNose II and the ANN model, 16 manure samples were collected from 6 different broiler houses and tested using these two systems.

5. The results showed that DiagNose II was able to classify manure samples correctly as infected or non-infected based on the ANN model developed with a 94% level of accuracy.