Villous atrophy in pigs orally infected with Salmonella cholerae-suis.

The macroscopic and microscopic appearance of villous atrophy in weaned pigs orally infected with Salmonella cholerae-suis is described. The other intestinal diseases of pigs in which villous atrophy occurs are discussed.     

Iron and transferrin in acute experimental Salmonella cholerae-suis infection in pigs

The effects of experimental Salmonella cholerae-suis inoculation with a virulent and an avirulent strain on serum iron (SI), total iron-binding capacity (TIBC), and transferrin (TF) were evaluated. Inoculation of virulent strain 38 was followed by significant (P less than 0.05) decreases of SI, TIBC, and TF. Exposure to avirulent strain 33 was followed by moderate decreases of SI, TIBC, and TF. When exposure to avirulent strain 33 was followed by challenge exposure with virulent strain 38, the SI, TIBC, and TF values remained at initial values or were higher. Negative correlation was observed between rectal temperature and SI and TIBC values, but was significant (P less than 0.0001) only 7 days after inoculation of the virulent strain 38.     

Hybridization studies with a DNA probe derived from the virulence region of the 60 Mdal plasmid of Salmonella typhimurium.

Plasmid DNA of 68 strains of Salmonella that belonged to 18 serovars and exhibited 48 different plasmid profiles was examined for hybridization with a 32P-labelled DNA probe which consisted of a 3750 base pairs (bp) HindIII-HindIII fragment derived from the virulence region of the 60 megadalton (Mdal) plasmid of Salmonella typhimurium. The 32 Mdal plasmid of S. cholerae-suis, the 50 Mdal plasmid of S. dublin, the 36 Mdal plasmid of S. enteritidis, the 60 Mdal plasmid of S. gallinarum, the 60 Mdal plasmid of S. pullorum, and the 60 Mdal plasmid of S. typhimurium, plasmids that have been associated with virulence, all hybridized with the probe. Digestion of plasmid DNA of these strains with PvuII and hybridization with the probe revealed that the plasmids of strains of all six serovars contained fragments of approximately 2520 and 1520 bp that hybridized with the probe. Similarly, hybridization with BglI digests of DNA of the virulence-associated plasmids of strains of these six serovars showed that all six plasmids contained a fragment of approximately 3690 bp that hybridized with the probe. No other plasmids of these strains nor any plasmids of 12 other Salmonella serovars hybridized with the probe. Chromosomal DNA did not hybridize with the probe. The 60 Mdal plasmids of S. gallinarum and S. pullorum showed similar digestion patterns with restriction endonucleases BglI, BglII and PvuII.     

Conjunctival and intramuscular vaccination of pigs with a live avirulent strain of Salmonella cholerae-suis

An avirulent mutant strain of Salmonella cholerae-suis was cloned for resistance to streptomycin and nalidixic acid. The mutant strain 33-13 also was used because of its avirulence and immunogenicity in mice. Weaned pigs were vaccinated with live strain 33-13; 5 pigs were vaccinated by conjunctivally administered 5.5 X 10(7) organisms (low dose), 5 were conjunctivally administered 5.5 X 10(9) organisms (high dose), and 5 pigs were administered 5.5 X 10(9) organisms (high dose) IM. Transient fever and transient fecal shedding of the vaccine strain developed in pigs vaccinated IM, but not in 2 groups of pigs vaccinated conjunctivally. After intratracheal administration of virulent strain 38-9, nonvaccinated control pigs (n = 9) developed persistent high fever, anorexia, bacteremia, diarrhea, and fecal shedding of strain 38-9, whereas vaccinated pigs remained afebrile and clinically normal. Nonvaccinated and uninfected sentinel pigs (n = 8) were kept in units of 2 pigs with each group of experimental pigs, and remained healthy throughout the experiment. Thirteen vaccinated and 7 nonvaccinated control pigs were killed 42 days after vaccination, and 2 vaccinated, 2 nonvaccinated, and 8 sentinel control pigs were killed 58 days after vaccination. Ten organs were evaluated by quantitative bacteriology on necropsy of all pigs for the presence of vaccine strain 33-13, and for virulent strain 38-9. Strain 33-13 was not found. Lung and liver, lesions were found in most of the nonvaccinated control pigs, with a high frequency of recovery of large numbers of strain 38-9 from the mesenteric lymph nodes, lungs, liver, and ileum.(ABSTRACT TRUNCATED AT 250 WORDS)     

The detection and determination of potential virulence of Salmonella spp. using PCR method

The aim of this study was to prove that PCR is a very useful method to identify Salmonella strains and to determine their virulence factors by amplification of characteristic genetic markers. Investigations included 5 strains of Salmonella which were obtained from pure cultures and 1 Salmonella strain from the mixed culture. Genotypic analysis of 6 examined strains revealed the 163-bp fragment of chromosomal DNA, which is the DNA rep. ori. gene, encoding the particular genus. In all of these strains 215-bp, 203-bp and 204-bp chromosomal DNA fragments were identified as representing the stn, stpA and spaO genes that confirmed their virulence. These amplification products were identified in both pure and mixed culture from pork. Sensitive and rapid PCR method may be used not only for identification of Salmonella strains and for determination of their virulence factors but also for routine microbiological diagnostic of food pathogens.     

Evaluation of an immunochromatography strip assay for the detection of Salmonella sp. from poultry.

The present study was conducted to evaluate the sensitivity and specificity of an immunochromatography-based diagnostic kit for Salmonella. The analytical sensitivity of the test when using pure colonies of different Salmonella species was in the range of 1 X 10(4) to 1 x 10(5) colony-forming units per milliliter. The strip detected 19 of 22 strains of Salmonella spp. but failed to detect S. worthington, S. choleraesuis var. kunzendorf and S. johannesburg. The strip did not detect 27 different enteric bacteria, including Escherichia coli O157:H7, Campylobacter jejuni, Shigella sonnei, and Vibrio parahaemolyticus. In direct testing of feces (n = 66) from chickens infected with Salmonella typhimurium, the strip had a sensitivity of 12.3% and a specificity of 100%. Evaluation of the strip assay (n = 510) after sample pre-enrichment in 2% buffered peptone water (BPW) yielded a sensitivity of 93.8% and specificity of 89% when compared to isolation and identification with xylose-lysine-tergitol 4 (XLT4) selective plating media. Subsequent enrichment in Hajna tetrathionate (TT) broth yielded a higher sensitivity (94.7%) and specificity (96.8%). The agreement (kappa) between the strip test and isolation was 0.004 in direct fecal testing, 0.82 in BPW, and 0.89 in TT broth. The assay could detect Salmonella sp. as early as 18-48 hours during pre-enrichment and enrichment compared to isolation on XLT4, which required an overnight incubation step for the presumptive isolation and identification of Salmonella.     

牛肉中沙门氏菌的检测

对95份牛肉样品进行沙门氏菌实验室检测,通过增菌、选择性分离培养及生化试验、血清学鉴定等检验,结果3份样品检出沙门氏菌,检出阳性率为3.15%。     

Distribution and function of plasmids in Salmonella enterica

Plasmids of Salmonella enterica vary in size from 2 to more than 200 kb. The best described group of plasmids are the virulence plasmids (50-100 kb in size) present in serovars Enteritidis, Typhimurium, Dublin, Cholerae-suis, Gallinarum, Pullorum and Abortus-ovis. They all encode spvRABCD genes involved in intra-macrophage survival of Salmonella. Another group of high molecular weight plasmids are plasmids responsible for antibiotic resistance. Since most of these plasmids are conjugative, besides storage of genetic information, they contribute to the spread of genes in bacterial populations. The low molecular weight plasmids are the last group of plasmids found in S. enterica. Some of them have been shown to increase resistance to phage infection due to the presence of restriction modification systems. Despite limited knowledge on their function, their presence or absence is frequently used for strain differentiation in epidemiological studies.     

贝类中副溶血弧菌和沙门氏菌荧光定量PCR快速检测方法的建立

为适应进口鲜活水产品的快速检验,有必要建立快速检测多种病原生物的方法。本文针对鲜活水产品中常见的副溶血弧菌和沙门氏菌,设计了特异的荧光引物,建立了检测贝类中这两种细菌的荧光定量PCR体系,并进行了特异性与重复性试验。结果表明,在相同的反应条件下,副溶血弧菌和沙门氏菌均得到了特异性扩增,而阴性对照菌株均未见扩增。副溶血弧菌标准曲线在1.53×105~1.53×109拷贝/μL之间、沙门氏菌标准曲线在4.89×106~4.89×1010拷贝/μL之间有较好的线性关系。与传统的检测方法相比较,该荧光定量PCR方法检测贝类中的副溶血弧菌和沙门氏菌更为快速准确,结果直观,可以满足口岸进口水产品快速检测的需要。     

Loss of virulence by heterophil-adapted Salmonella pullorum.

We report that reduction of virulence for day-old chicks was achieved after eight-times-repeated heterophil passage of Salmonella pullorum (SP). The virulent source strain SP-V caused 64% mortality and 89% internal organ as well as 89% cecal colonization 10 days after administration of 10(7) colony-forming units (CFU) to day-old chicks. Eight-times-repeated passage of SP in heterophils resulted in attenuated strain SP-A that was nonlethal and reduced colonization of internal organs from 89% for SP-V strain to 4.3% for SP-A strain 10 days after administration of 10(7) or 10(8) CFU to day-old chicks. Cecal colonization was reduced from 89% for SP-V strain to 0 for SP-A strain 10 days after administration of 10(7) or 10(8) CFU to day-old chicks.     

Use of pooled samples for the detection of Salmonella in feces by polymerase chain reaction.

Many epidemiological studies of Salmonella rely on conventional bacteriological culture methods to detect Salmonella in fecal samples. These culture-based methods are inefficient for epidemiological studies in populations with a low prevalence of Salmonella. The objective of this study was to optimize a protocol that uses pooled Salmonella enrichment broth cultures of bovine feces and polymerase chain reaction (PCR) for the detection of the invA gene of Salmonella in feces. In one field trial, 196 animals were sampled, and all samples were tested by culture, invA PCR on individual samples, invA PCR on pools of 5 samples, and BAX PCR on individual samples. All assays showed a high agreement on individual samples (kappa > or = 0.75). The invA PCR was run on each of 40 pools and detected 19 of 22 culture-positive pools. In another field trial, 152 samples were taken from 4 dairies, and the invA PCR was performed on pools of 5 samples in addition to bacteriological culture of individual samples. Salmonella was detected in 5 of the 32 pools (7 total positive samples) by both PCR and culture. One pool was PCR-positive but culture-negative. Pooling did not dramatically affect the performance of the invA PCR; most of the culture-positive samples were detected, including all of the samples when there were 4 or more Salmonella colonies on the agar plate. Based on these field trials, invA PCR on pooled samples appears to be an efficient method of Salmonella detection as long as Salmonella loads are not extremely low.     

Tagging and elimination of plasmids in Salmonella of avian origin

This study compared the effectiveness of a number of procedures designed to label and eliminate plasmids that may play a role in virulence in Salmonella. Twenty strains of Salmonella of 9 serovars were subjected to 3 methods for labelling plasmids with transposons. Strains containing labelled and unlabelled plasmids were exposed to physical and chemical curing agents. Plasmids in 9 of 20 strains of Salmonella were tagged by conjugation with a donor Escherichia coli containing a temperature-sensitive RP4 plasmid that carried the Tn1 transposon. Plasmids in 2 of 5 strains of Salmonella were labelled by conjugation with a donor E. coli that contained a F' tslac::Tn5 plasmid. Transduction of Salmonella with a P22 bacteriophage that carried a temperature-sensitive Tn10 transposon resulted in chromosomal insertion of Tn10 in 2 of 10 strains. Use of chemical curing agents resulted in curing of plasmids in only 6 of 17 strains. Two strains were cured by ethidium bromide, two by a combination of ethidium bromide and novobiocin, two by a combination of imipramine and methylene blue, and none by acridine orange, novobiocin, sodium dodecyl sulfate or rifampicin. In contrast, plasmids in 14 of 17 Salmonella strains were eliminated by incubation at 45.5 degrees C.     

Use of an alkaline phosphatase-labelled probe for the detection of Mycoplasma synoviae in chickens

Short nucleotides directly labelled to alkaline phosphatase (SNAP probes) are an interesting alternative to digoxigenin-labelled probes (DIG probes), because they reduce the number of steps necessary in dot blots for the detection of DNA or amplificate. This study examined the questions whether a SNAP probe might not only save time, but also increase the sensitivity of another PCR-based DNA probe test using a digoxigenin probe. Amplificates obtained by multispecies polymerase chain reaction (PCR), with either purified genomic DNA or DNA extracted from tracheal swabs taken in chicken flocks, were detected by both methods. The results for the clinical specimens were compared to culture. Under stringent conditions, the specificity and sensitivity obtained with the SNAP probe were comparable to the results obtained with the DIG probe. The quantities 10 fg (SNAP probe) and 100 fg (DIG probe) of purified Mycoplasma synoviae DNA were detected after amplification, but more positive clinical specimens were detected with the DIG probe. Under non-stringent conditions sensitivity with purified DNA did no change, but the coloration of the dots improved markedly, and more positive specimens could be detected with the SNAP probe than with the DIG probe, truly positives as confirmed by culture. Because cross-reaction with Mycoplasma gallisepticum and Mycoplasma imitans, two species with DNA that was also recognized by the multispecies primers, occurred under non-stringent conditions, it was concluded that, to take the full advantage of SNAP probes, their use in combination with species-specific primer pairs is recommended. PCR as a method for mycoplasma detection is however, always accompanied with serological and cultural methods. When a M. synoviae mono-infection is likely by serological results, non-stringent dot blot conditions and use of the SNAP probe will ease and improve the detection of mycoplasma.     

鸡粪中沙门氏菌和大肠杆菌O78多重PCR检测方法的建立

为建立同时快速检测沙门氏菌和大肠杆菌O78的方法,本研究根据沙门氏菌inv A基因和大肠杆菌O78 O-抗原特异基因序列设计引物,并在细菌16S r DNA区设计内参引物,通过各项参数调整优化,建立了能够直接从样品中同时检测沙门氏菌和大肠杆菌O78的多重PCR检测方法。结果显示:该方法可以同时扩增出O781 113 bp和沙门氏菌821 bp的特异片段以及细菌16S r DNA 527 bp的通用片段,而对于其他13种肠道致病菌只能扩增出527 bp的细菌同源片段,具有较强的特异性。该体系检测沙门氏菌和大肠杆菌O78的最低检出限分别为10~3cfu/m L和10~2 cfu/m L。增菌5 h,鸡粪模拟污染菌样品中沙门氏菌和大肠杆菌O78的检测灵敏度分别为10~3 cfu/m L和10~2 cfu/m L。本研究建立的检测方法能够在8 h内完成鸡粪样品中两种靶标菌的快速检测,对于沙门氏菌和大肠杆菌O78的鉴别诊断和快速检测具有重大意义。     

沙门氏菌分子生物学诊断技术的研究进展

本文主要介绍了沙门氏菌分子生物学方面的研究进展,将当前国内外的一些研究成果进行了汇总并分门别类。对该菌的分子生物学诊断技术的灵敏度和特异性进行了介绍,对此病原菌的实验室研究提供了多种方法,最后展望了其分子生物学方法应用于实践的前景和存在的问题为进一步开展基础研究提供了思路。     

Management and nutritional factors associated with the detection of Salmonella sp. from cattle fecal specimens from feedlot operations in the United States

In a convenience sample of 100 feedlot operations (included in the United States Department of Agriculture: Animal and Plant Health Inspection Service 1994 Cattle on Feed Evaluation), up to 25 cattle fecal samples were collected and tested for the presence of Salmonella from each of two pens (the pen which contained the most-recent arrivals, and the pen with cattle that had been on feed the longest). One or more Salmonella spp. were recovered from 38 (38.0%) of the 100 feedlots, 52 (26.0%) of the 200 pens and 273 (5.5%) of the 4977 fecal samples collected. Multivariable logistic regression indicated that feeding tallow and feeding whole cottonseed or cottonseed hulls within seven days prior to fecal sample collection was associated with an increased risk of finding Salmonella in a pen. Variables not found to be significantly associated with the detection of Salmonella in a pen included region, operation size, use of sprinklers, time on feed, type of cattle in the pen, number and concentration of cattle in a pen, feeding probiotics, and various other feeds.     

Estimating the prevalence of Salmonella spp. in swine herds--influence of sensitivity and specificity of Salmonella detection

Information about the proportion of truly Salmonella-free herds is required for an evaluation of the epidemiological situation, the development of control strategies and their implementation. Findings regarding the presence of salmonellas in faeces and intestinal lymph nodes as well as the presence of Salmonella antibodies in meat juice from slaughtered pigs were obtained in the context of a study conducted by a number of institutes. These data were used for an analysis of the validity of data on the prevalence of infected animals within herds and on the prevalence of infected herds. The proportion of batches or herds with exclusively negative individual findings was found to depend not only on the true proportion of truly Salmonella-free animals within herds but quite essentially also on the distribution of the proportion of infected animals within herds, the sensitivity of the methods of examination and sample sizes. When taking into account the existing dependencies, it was found that among the swine, the real numbers of Salmonella carriers were much higher than shown by bacteriological and serological examination. Regarding salmonellosis in swine, also a number of contaminated herds must be expected which is far higher than that shown by the number of herds with positive findings in at least one animal. Even a low contamination of all or almost all herds would result in the numbers of 'negative' batches observed, i.e. batches with exclusively negative individual findings. A rating of the salmonella exposure of herds as high, low, or very low is possible and may, and should be, used for measures of consumer protection, irrespective of the proportion of truly Salmonella-free herds.     

Efficacy of a novel virulence gene-deleted Salmonella Typhimurium vaccine for protection against Salmonella infections in growing piglets

We have previously developed a novel attenuated Salmonella Typhimurium (S. Typhimurium) ΔcpxR Δlon vaccine. This study was carried out to examine whether this vaccine could effectively protect growing piglets against Salmonella infection. Attenuated S. Typhimurium secreting the B subunit of Escherichia coli heat-labile enterotoxin was also used as a mucosal adjuvant. Pregnant sows in groups A and B were primed and boosted with the vaccine and mucosal adjuvant, whereas sows in groups C, D and E received PBS. Piglets in groups A and C were intramuscularly primed with formalin-inactivated vaccine and orally boosted with live vaccine, while piglets in groups B, D and E received PBS. Piglets in groups A, B, C, and D were challenged with a wild type virulent S. Typhimurium at the 11th weeks of age. Colostrum sIgA and IgG titers in vaccinated groups A and B sows were approximately 50 and 40 times higher than those of non-vaccinated groups C, D and E sows (P < 0.001). Serum IgG titers of group A piglets were also significantly higher than those of groups D and E piglets during the study (P < 0.001). Furthermore, no clinical signs were observed in group A piglets during the entire experimental period after the challenge, while diarrhea was observed in many of the piglets in groups B, C, and D. No Salmonella was isolated from fecal samples of the groups A and C piglets on day 14 after challenge, whereas the challenge strain was isolated from several piglets in groups B and D. These results indicate that vaccination of the piglets with the vaccine and mucosal adjuvant in addition to vaccination of their sows induced effective protection against Salmonella infections in the growing piglets.