Preliminary results of the determination of plasmid profiles of veterinary Salmonella isolates

A plasmid of 60 Md magnitude was recorded from 40 in 41 Salmonella (S.) typhimurium strains, including the Copenhagen minus variant. A plasmid of that kind had been described in the international literature as serovar-specific of S. typhimurium. One S. typhimurium strain was without plasmid. Five contained the 60-Md and other plasmids. No relationship was found to exist between the 60-Md plasmid and biovar as well as chemotherapeutic resistance. Further studies will be necessary for consistent information on virulence association of this plasmid and its serovar specificity. Plasmid profiles were also checked in four S. enteritidis strain and additional serovars.     

Use of a Salmonella typhimurium-derived virulence probe in the detection of Salmonella sp. and in the characterization of S. cholerae-suis virulence plasmids

A genetic probe encoding a virulence gene from Salmonella typhimurium was useful in the detection of Salmonella from feces during an outbreak of salmonellosis at a local dairy. A 3.2-kb BamHI restriction endonuclease fragment of the S. typhimurium virulence plasmid, pStSR100, has been useful as a DNA probe for both detection of Salmonella sp. and characterization of virulence plasmids from numerous field isolates. This virA probe hybridizes to a highly conserved gene carried on the large virulence plasmids of invasive Salmonella isolates. Colony blots prepared from feces directly plated onto MaConkey's agar failed to detect low numbers of Salmonella sp. However, hybridization of the VirA probe to vacuum blots or colony blots prepared from feces in tetrathionate enrichment broth incubated for 16 hours at 37 C was effective for detecting Salmonella sp. and resulted in an 85.9% correlation with culture results. The probe also demonstrated the highly conserved nature (96%) of the virulence gene among S. cholerae-suis isolate plasmids detected using Southern blot analysis.     

Relation of plasmids to virulence and other properties of salmonellae from avian sources

A collection of 185 isolates of 34 serovars of Salmonella from avian sources was examined for plasmids, drug resistance, biochemical properties, serum resistance, and virulence. No serovars other than S. enteritidis, S. typhimurium, and S. heidelberg showed evidence of serovar-associated plasmids. All S. enteritidis isolates carried a single plasmid of 36 Mdal and were resistant to guinea pig serum; one strain that was tested was virulent. Of 27 isolates of S. typhimurium, 11 possessed a 60-Mdal plasmid and 17 harbored a 2.3-Mdal plasmid. Among isolates of S. heidelberg, 21 of 24 carried a 2.2-Mdal plasmid. The only biochemical property that varied was fermentation of inositol, which tended to be related to serovar. Of 172 isolates, 54 were resistant to at least one drug. Multiple drug resistance was usually associated with R plasmids, and transmissible plasmids that encoded resistance to chloramphenicol and gentamicin were demonstrated. Of 117 isolates tested, 43 were resistant to guinea pig serum. Resistance appeared to be a characteristic of isolates rather than serovar and could not be related to plasmids. Twenty-five isolates highly resistant to guinea pig serum were all susceptible to the bactericidal action of chicken serum. In tests for virulence using intraperitoneally (i.p.) and orally inoculated Balb/c mice and day-old chicks, only i.p.-inoculated chicks proved useful in demonstrating large differences among isolates: LD50's ranged from 10(0) to 10(8).     

Plasmids of Salmonella muenster and their relation to virulence

The plasmid content of 104 strains of Salmonella muenster collected from bovine and human sources in Ontario from 1982 to 1984 was determined. The strains were classified into 17 different groups on the basis of the sizes of the plasmids they contained. Further division was made on the basis of the fragments obtained following digestion of the plasmids with restriction endonucleases BglI and BglII. Representative strains from each plasmid profile group were compared for resistance to serum and for virulence in mice. No differences in serum resistance or in LD50 by the oral or intraperitoneal route in mice could be associated with the presence of plasmids. Although the strains of S. muenster were recovered from clinical cases in cattle and humans, they were of low virulence for mice: the LD50 values were approximately 10,000-fold greater than that of Salmonella typhimurium.     

Temporal changes in the population genetics of Salmonella pullorum

Salmonella pullorum is the cause of pullorum disease, which is characterized by white diarrhea and a high mortality rate in poultry. During the 1990s, the serologic "pullorum" test has occasionally failed to detect infected birds during the early stage of disease. To determine if any recent genetic changes have taken place in S. pullorum to account for poor seroconversion sometimes observed in infected flocks, S. pullorum from 1990s outbreaks and strains isolated prior to the 1980s were typed by random amplified polymorphic DNA (RAPD). Of 40 S. pullorum isolates typed by this method, eight distinct DNA patterns were identified with one of three RAPD polymerase chain reaction primers. Sixty-two percent of S. pullorum isolates shared the same RAPD DNA pattern, and a major proportion of these strains were from recent flock infections. The RAPD patterns for S. pullorum were clearly distinct from the avian Salmonella group B isolates included in this analysis. The distribution of Salmonella virulence genes among avian Salmonella isolates was also examined. Eighty-five percent of the S. pullorum isolates had both the virulence plasmid gene, spvB, and the invasion gene, invA, with the same percentage positive for the Salmonella enteriditis fimbrial gene, sef. However, significant variability was observed among S. pullorum in their ability to invade avian epithelial cells, despite the presence of the Salmonella invasion gene in these isolates.     

PCR扩增invA基因特异性检测沙门氏菌

建立了扩增invA基因检测沙门氏菌的PCR方法。对收集的50个血清型123株沙门氏菌及7种27株非沙门氏菌进行PCR,2％琼脂糖电泳检查,结果所有沙门氏菌都扩增出了300bp的特异性产物,非沙门氏菌都未扩增出此目的条带。产物的特异性由slot blot杂交进一步证实。通过电泳判定结果,该法可检出扩增体系中10pg染色体DNA及10~2cfu的纽波特沙门氏菌50029。为下步克隆而设计的两个酶切点（Bam HI,Eco RI）对引物的特异性没有影响。本研究为沙门氏菌的检测提供了简洁、敏感、特异的新方法,同时为克隆invA基因做属特异性探针打下了基础。     

Virulence of Salmonella enteritidis phagetypes 4, 8 and 13 and other Salmonella spp. for day-old chicks, hens and mice.

Virulence of three Canadian poultry strains of Salmonella enteritidis, namely phagetypes (PT) 4, 8 and 13, and one Salmonella heidelberg strain was assessed in orally and intraperitoneally inoculated one-day old chickens and compared to the virulence of a human S. enteritidis PT 4 strain from the United Kingdom (UK). The two PT 4 strains were also compared in orally inoculated adult laying hens. In addition, orally inoculated Balb/c mice were used to evaluate virulence of the above strains and two strains of Salmonella typhimurium containing different plasmids. In orally inoculated one-day old chickens, the UK S. enteritidis PT 4 strain was more virulent than the Canadian PT 4 strain. The UK PT 4 strain was also more virulent and invasive in adult laying hens than the Canadian PT 4 strain. The S. enteritidis PT 8 strain and one S. typhimurium strain isolated from a chicken hatchery were the most virulent for orally inoculated Balb/c mice. This strain of S. typhimurium contained the 60 megadalton plasmid associated with virulence for Balb/c mice which was not present in the S. typhimurium strain isolated from a pig with septicemic disease.     

沙门菌肠毒素基因克隆及序列分析

研究常见的不同血清型沙门菌肠毒素(stn)基因核苷酸序列之间的差异及其分布情况。根据沙门菌的stn核苷酸序列设计一对引物,应用PCR技术,分别对肠炎沙门菌、鼠伤寒沙门菌和鸡白痢沙门菌进行PCR扩增,对扩增产物进行克隆及序列分析,并用所设计的引物检测7种血清型沙门菌(42株)。结果显示,3种沙门菌经PCR均扩增出749 bp的特异条带,DNA序列分析证实,沙门菌的stn核苷酸序列比较保守,42株沙门菌stn的检出率为100%。本试验成功克隆出沙门菌的stn,调查其在不同血清型沙门菌中的分布及序列分析,为进一步研究stn致病机理及研制减毒沙门菌活菌疫苗奠定了基础。     

鸡白痢沙门菌和鸡伤寒沙门菌PCR-RFLP鉴别

根据鸡白痢沙门菌和鸡伤寒沙门菌flic基因可变区两端的保守序列设计1对引物,PCR扩增出约866 bp的产物,用HinplI对PCR产物进行酶切,经RFLP分析区分鸡白痢沙门菌和鸡伤寒沙门菌.利用该技术对1株鸡白痢沙门菌标准株及2株鸡伤寒沙门菌标准株进行分子鉴别,结果与预计的RFLP模式相符,证明该方法可行.在此基础上...     

Serologic survey of slaughter-age ostriches (Struthio camelus) for antibodies to selected avian pathogens

Serum samples from 163 slaughter-age ostriches (Struthio camelus) in Ohio and Indiana were tested for antibodies to avian influenza virus (AIV), Newcastle disease virus (NDV), paramyxovirus (PMV) 2, PMV3, PMV7, infectious bursal disease virus (IBDV), Bordetella avium, Mycoplasma synoviae, Mycoplasma gallisepticum, Ornithobacterium rhinotracheale, Salmonella pullorum, Salmonella gallinarum, and Salmonella typhimurium. One ostrich had antibodies to AIV H5N9, 57% of the ostriches had antibodies to NDV, four ostriches had antibodies to both NDV and PMV2, and one ostrich had antibodies to NDV, PMV2, PMV3, and PMV7. None of the ostriches had antibodies to IBDV, B. avium, M. synoviae, M. gallisepticum, O. rhinotracheale, S. pullorum, S. gallinarum, and S. typhimurium. This is the first report of antibodies to avian influenza and PMV7 in ostriches in the United States.     

Virulence plasmids of Salmonella enterica--incidence and properties

Salmonella virulence plasmids (SVPs) are large and closely related low-copy plasmids harbored by certain serovars of Salmonella enterica subspecies enterica. These serovars not only comprise those of veterinary significance like Abortusequi, Abortusovis, Choleraesuis, Dublin and Gallinarum/Pullorum, but also Typhimurium and Enteritidis which currently are the most prevalent serotypes in humans and food animals. Experiments with several animal species gave evidence that SVPs increase Salmonella strains' capabilities to replicate in extraintestinal organs of infected hosts thus leading to death of those hosts more frequently and rapidly. The common feature of all SVPs is the "Salmonella plasmid virulence" locus (spv-locus), a highly conserved 7.8 kbp region that is most responsible for the SVP-encoded virulence phenotype of Salmonella. Although functional characterisation of spv gene products has made some progress the molecular mechanism of spv-mediated virulence has not been fully elucidated yet. Some SVPs carry additional gene loci causatively related to Salmonella virulence like the pef-operon of Typhimurium and Enteritidis strains which encodes an adhesive type of fimbria, or genes traT, rsk and rck which are involved in serum resistance. The frequent occurrence of SVPs in host-adapted serovars suggests that SVP-encoded factors represented selective advantages to some Salmonella variants in their effort to colonize certain new niches during Salmonella evolution. This study provides an overview over current knowledge about the virulence plasmids of Salmonella enterica.     

Characterization of Salmonella enteritidis strains.

A study was conducted to characterize 318 Salmonella enteritidis strains that were mainly isolated from poultry and their environment in Canada. Biotype, phagetype (PT), plasmid profile (PP), hybridization with a plasmid-derived virulence sequence probe, antibiotic resistance, outer membrane proteins (OMPs), and lipopolysaccharide (LPS) profiles were determined. Relationships of these properties to one another, and their diagnostic and pathogenic significance were assessed. Biotyping indicated that failure to ferment rhamnose was sometimes useful as a marker for epidemiologically related strains. Phagetyping was the most effective method for subdividing S. enteritidis; it distinguished 12 PTs. Phagetype 13 was occasionally associated with septicemia and mortality in chickens. The strains belonged to 15 PPs. A 36 megadalton (MDa) plasmid was found in 97% of the strains. Only the 36 MDa plasmid hybridized with the probe. Seventeen percent of the strains were drug resistant; all strains were sensitive to ciprofloxacin. Thirty-five of 36 strains possessed the same OMP profile, and 36 of 41 strains contained smooth LPS.     

Prevalence of the Salmonella plasmid virulence gene "spvD" in Salmonella strains from animals]

Strains of Salmonella isolated from animals in Germany (n = 878) were analysed for the presence of the spvD gene ("Salmonella plasmid virulence gene D") by DNA-DNA hybridization. The spvD gene was only detected in strains of serovars Typhimurium (93.3%), Enteritidis (97.1%), and Dublin (100%) as well as in two rough strains of Salmonella enterica. Salmonella isolates from mammals carried the gene more frequently (cattle 94.0%, horses 92.6%, pigs 73.7%) than those from birds (33.3%) or reptiles (4.5%). Due to its high prevalence in epidemiologically relevant salmonellae, the virulence factor spvD may represent a sensitive and specific target in various serovars for diagnostic or immunization strategies.     

A molecular epidemiologic investigation of Salmonella from a meat source to the feces of captive cheetah (Acinonyx jubatus).

Low cheetah (Acinonyx jubatus) birth rates were observed for a long time in a captive breeding facility in which Salmonella, which was possibly present in contaminated beef, was isolated from still-born lion (Panthera leo) cubs. Salmonella, including 14 isolates of Salmonella serovar typhimurium and 19 isolates of Salmonella serovar muenchen, was subsequently isolated 47 times from 378 meat samples at the facility during a 13-mo period. Salmonella, including 26 isolates of S. serovar typhimurium, 10 of S. serovar muenchen, and 11 other serovars, also was isolated 54 times from 119 fecal samples. Only three plasmid profiles were identified in 59 S. typhimurium isolates from both meat and fecal samples. Although random-amplified polymorphic DNA fingerprinting using different primers in the polymerase chain reaction was able to distinguish between S. typhimurium and S. muenchen and to demonstrate similar chromosomal DNA fingerprints in some of the isolates from meat and feces, the results were not consistent enough to prove that the Salmonella in the feces originated from contaminated meat. However, the predominance of only two serovars in the meat fed to carnivores and in the feces of these animals suggests that the meat was the source of the Salmonella organisms in the feces.     

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.     

基于等位基因特异性PCR原理建立鸡白痢沙门氏菌PCR方法

根据鸡白痢沙门氏菌与鸡伤寒沙门氏菌的rfbS基因在第237和598位碱基的不同,设计和合成等位基因特异性PCR引物,建立快速检测鸡白痢沙门氏菌的PCR方法,并应用该法对鸡白痢沙门氏菌临床分离样品进行了PCR鉴定。结果显示,该PCR方法能特异性地鉴定鸡白痢沙门氏菌,检测灵敏度达18 pg/μL DNA,4.7×10⁴ CFU/mL菌液,表明建立的等位基因特异性PCR方法能准确而快速地鉴定鸡白痢沙门氏菌。     

Use of molecular fingerprinting to assist the understanding of the epidemiology of Salmonella contamination within broiler production

1. We analysed Salmonella isolates by conventional sero- and phage-typing, as well as by molecular techniques within the broiler production chain in two integrated companies. The most prevalent serovars were selected for genetic fingerprinting. 2. Isolates were first screened by plasmid profiling; subsequently, the most common plasmid types within the prevalent zoonotic serovars (enteritidis and typhimurium) and S. agama were further characterised by PstI-SphI ribotyping, and XbaI pulsed field gel electrophoresis (PFGE). 3. Salmonella binza, S. kedougou, and S. 4,12:d:- were endemic in the feed mills over long periods of time, and a variety of plasmid types for each of the serovars were found in the premises. 4. A similar situation was found with S. binza and S. senftenberg within the hatchery in company B. The Salmonella serovars which were resident in those locations were also the ones most widely distributed throughout the broiler flocks. 5. Plasmid profiling was useful to subdivide clusters of isolates within serovars, but for each serovar a high percentage (36 to 79%) of the isolates tested fall within a prevalent plasmid type. 6. A more detailed genetic analysis of the isolates by a multiple typing approach allowed for further strain differentiation, and allowed some epidemiological conclusions to be drawn.     

Virulence determinants of Salmonella typhimurium from animal sources

Two hundred seventy-eight strains of Salmonella typhimurium isolated from 1973 to 1981 from animal sources in New York State were studied for possible virulence determinants and for a serotype-specific plasmid possibly linked with virulence. Of the strains, 98% possessed type-1 fimbriae. All strains possessed flagella and were motile. One hundred twenty-three strains (44%) treated with mitomycin C tested positive for the cholera-Escherichia coli heat labile family of toxins by a kinetics-based ELISA; when treated with mitomycin C and extracted with polymyxin B, 249 (90%) were positive in the kinetics-based ELISA. All strains were negative in the Biken test. A smooth cell wall was found in 99% of the strains. Sixty-one percent (169) of the strains had a 62-Md plasmid. Seventy-six (27%) of the strains had detectable plasmids ranging in size from 1 to 124 Md.     

HRP直接标记属特异性基因探针检测沙门氏菌的研究

以活化辣根过氧化物酶复合物（ＨＲＰ—ＰＢＱ—ＰＥＩ＋—ＮＨ３＋）直接标记沙门氏菌属特异性ＤＮＡ探针ｐＬＳ２和ｐＬＳ３，探针与靶ＤＮＡ杂交后催化发光底物，经增强型化学发光反应（ＥＣＬ），用普通Ｘ光胶片自显影（ＣＰＤ）检测沙门氏菌。经狭缝杂交（Ｓｌｏｔｂｌｏｔ），该法标记探针均可检测到０．１ｐｇ的纯质粒ＤＮＡ及１０３个未经培养的鼠伤寒沙门氏菌。Ｄｏｔ—ｂｌｏｔ杂交结果证明，ＨＲＰ标记的探针仅与沙门氏菌属细菌杂交，而与试验的其他肠道非沙门氏菌不杂交。本研究表明，ＨＲＰ直接标记基因探针化学发光自显影法检测沙门氏菌，安全、快速、简便，且有高度的敏感性和特异性，是一种有较大应用前景的非放射性标记探针杂交检测方法。     

Plasmid mediated antimicrobial resistance in Ontario isolates of Actinobacillus (Haemophilus) pleuropneumoniae.

The genetic basis of antimicrobial resistance in Ontario isolates of Actinobacillus (Haemophilus) pleuropneumoniae was studied. Two Ontario isolates of A. pleuropneumoniae were found to be resistant to sulfonamides (Su), streptomycin (Sm) and ampicillin (Amp). Resistance to Su and Sm was specified by a 2.3 megadalton (Mdal) plasmid which appeared to be identical to pVM104, which has been described in isolates of A. pleuropneumoniae from South Dakota. Southern hybridization showed that the 2.3 Mdal Su Sm plasmid was highly related to those Hinc II fragments of RSF1010 known to carry the Su Sm genes, but was unrelated to the remainder of this Salmonella resistance plasmid. Resistance to Su and Amp was specified by a 3.5 Mdal plasmid and appeared identical to pVM105 previously reported. The beta-lactamase enzyme had an isoelectric point of approximately 9.0. Southern hybridization showed no relationship to the TEM beta-lactamase. A third isolate of A. pleuropneumoniae was found to be resistant to chloramphenicol (Cm), Su and Sm by virtue of a 3.0 Mdal plasmid which specified a chloramphenicol acetyl transferase. We conclude that resistance to Su, Sm, Amp and Cm is mediated by small plasmids in A. pleuropneumoniae. Although the Su and Sm resistance determinants are highly related to those found in Enterobacteriaceae, the plasmids themselves and the beta-lactamase determinant are different.