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.     

用质粒DNA指纹图谱法分析猪水肿病大肠杆菌的流行病学特征

从广西各地病死猪器官、组织中分离到17株细菌,经细菌形态、培养特性及生化反应鉴定为大肠杆菌。作本动物回归试验、小白鼠致病性试验和溶血试验结果表明,全部为致病性大肠杆菌。血清定型鉴定结果发现,菌株血清型分布不规律。17株致病性大肠杆菌的质粒指纹图谱显示:来源于不同猪群的菌株GP20-3和GP25-1以及GP18-9和GP8-3分别具有相同的质粒图谱,说明发病猪群传染源的首发菌相同。所有菌株都含有1条大小相同的质粒带,广西流行的猪水肿病主要是由在遗传学上具有高度同源相关性的大肠杆菌的广泛流行传播所引起。菌株GP18-9和GP8-3的质粒指纹图谱相同,但它们的血清型却不一致的现象,可作为自然条件下,同一质粒在不同血清型大肠杆菌中扩散转化的一个生物学依据。     

Transmission of Pasteurella multocida on California turkey premises in 1988-89.

Restriction endonuclease analysis (REA) of whole-cell DNA was used to determine possible sources of Pasteurella multocida for each outbreak of fowl cholera occurring in turkey flocks in eight commercial poultry companies in California from October 1988 to September 1989. Over this period, 179 isolates of P. multocida were obtained from dead turkeys in 80 meat and breeder flocks on 43 premises. P. multocida was isolated from wildlife on five premises. Isolates were characterized by subspecies, serotype, presence of plasmid DNA, and REA type. In 52 (65%) flocks, all isolates of P. multocida had the same REA pattern as the M9 live vaccine strain following digestion of DNA with the restriction enzyme SmaI. Field strains of P. multocida were obtained from 27 (34%) flocks, and one flock (1%) yielded both M9 and a field strain of the organism. REA of field strains of P. multocida revealed 17 different SmaI REA types. Based on matching SmaI REA types, potential sources of P. multocida were identified for 15 of the 28 flocks infected with field strains of the organism, and transmission between turkey premises was a possibility in only seven flocks.     

DNA fingerprinting of plasmid-containing serotype A: 3,4 Pasteurella multocida isolated from cases of fowl cholera in chickens and turkeys

The live, attenuated vaccine strains of Pasteurella multocida have been hypothesized to be responsible for homologous serotype outbreaks of fowl cholera on farms that use the commercial vaccines. We have further hypothesized that the naturally occurring Clemson University (CU) vaccine strain may be transformed to virulence by the acquisition of plasmid DNA. To test this hypothesis, we obtained seven homologous serotype (A:3,4) P. multocida isolates, all plasmid bearing, that were cultured from fowl cholera cases in vaccinated flocks and compared the isolates with the CU reference vaccine by molecular methods. Restriction fragment length polymorphisms (RFLPs) were detected by DNA/DNA hybridization with labeled probes specific for the cya, aroA, and rrn genes of P. multocida. The RFLPs obtained from BglII-digested genomic DNA probed with cya demonstrated no differences among the isolates. Although three isolates probed with aroA showed a RFLP identical to the vaccine strain, five isolates were distinctly different. Isolates probed with rrn grouped into three different restriction patterns that were dissimilar from that of the vaccine strain. Therefore, we have shown that these fowl cholera isolates are different from the CU vaccine strain and that these outbreaks were not vaccine related.     

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.     

Salmonella enteritidis and other Salmonella in laying hens and eggs from flocks with Salmonella in their environment.

Seven Canadian layer flocks with Salmonella enteritidis in their environment were investigated to determine the numbers of hens infected with S. enteritidis, the localization of S. enteritidis in organs of infected hens and the numbers of S. enteritidis-infected eggs produced by two affected flocks. By a microagglutination test (MAT) using S. pullorum antigens, these flocks had more seropositive hens (mean 51.9 +/- 16.9%) than two Salmonella-free flocks (mean 13.0 +/- 4.2%). Culture of tissues of 580 hens (433 seropositive) from the seven flocks detected 26 (4.5%) S. enteritidis-infected hens from two flocks. In one flock, 2/150 hens were infected with S. enteritidis phage type (PT) 8, which was confined to the ceca, and no Salmonella spp. were isolated from 2520 eggs (one day's lay). In the second flock, where 24/150 hens were infected with S. enteritidis PT13, extraintestinal infection was found in nine hens and involved the ovaries and/or oviduct in two hens. Salmonella enteritidis PT13 was isolated from one sample of egg contents and from one sample of cracked shells from among 14,040 eggs (one day's lay) from this flock. The overall prevalence of S. enteritidis-contaminated eggs from the two flocks with infected hens was less than 0.06%. Other Salmonella spp. isolated were S. heidelberg from 58 hens (10%), and S. hadar, S. mbandaka and S. typhimurium from one hen (0.2%) each. The MAT with antigens of S. pullorum had a sensitivity of 81% and a specificity of 24% for detecting S. enteritidis-infected hens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics of Salmonella spp. and Escherichia coli isolated from broiler flocks classified as "good" or "poor" producers

Cecal samples from 100 broiler flocks were cultured for Escherichia coli and Salmonella. Samples were selected from flocks classified as either "good" or "poor" producers by a production formula. In an attempt to identify predictors of flock productivity, isolates were studied for differences in antibiotic resistances, hemagglutination of erythrocytes, production of colicins, production of siderophores, type of hemolysis, resistance to host complement, and presence of plasmids. S. typhimurium (copenhagen) was isolated from one poor producing flock and three good producers. Salmonella isolates showed no significant differences in the parameters studied. The E. coli isolates showed significant differences only for the presence of plasmids. These data indicate that differences in host intestinal E. coli from good and poor producing flocks do not predict flock productivity.     

Field observations with Salmonella enteritidis bacterins

A study involving 11 commercial layer flocks was conducted to determine the efficacy of Salmonella enteritidis bacterins (autogenous or federally licensed). The criterion for evaluation of vaccine efficacy was the presence or absence of S. enteritidis in the environment, the organs of the bird (including ovary and oviduct), and eggs. Environmental, rodent, and organ specimens from dead birds as well as eggs were cultured throughout the life of the flock. All layers were obtained from pullet sources that were negative for S. enteritidis, as determined by organ and environmental cultures. Despite the use of S. enteritidis vaccination, 63.6% of the houses had S. enteritidis-positive environmental cultures and 100% of the flocks had S. enteritidis organ-culture-positive birds. The range of positive cultures for S. enteritidis in the environment in vaccinated flocks was between 0 and 45.5%. Birds in vaccinated flocks were organ-culture positive for S. enteritidis between 10% and 40% of the time. The unvaccinated portion of flocks in the same house and the unvaccinated flock in a complex had similar results compared with the vaccinated portion of the flocks.     

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.     

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).     

Salmonella contamination of poultry flocks in The Netherlands

The contamination of poultry in the Netherlands with Salmonella enteritidis was tested. For this, different methods (detection of S. enteritidis in faecal samples of 25 g; detection of S. enteritidis in cloacal swabs; detection of S. enteritidis by serological testing of antibodies in serum) were compared for their efficiency to detect S. enteritidis in flocks of poultry. Testing of faecal samples clearly yielded the best results. This method was used in a transmission study, in which 14 flocks descending from a contaminated primary mother flock were screened for the presence of S. enteritidis. The method was also used for screening 49 flocks of laying hens and 52 flocks of broiler chickens throughout the Netherlands. From the transmission study it became clear that S. enteritidis, phage type 2 (Dutch phage set) was isolated both from the mother flock and from five of the descendent flocks. Screening of poultry flocks for the presence of salmonella revealed that salmonella was present in 47% of the layer flocks and in 94% of the broiler flocks. S. enteritidis was isolated from 15% of the flocks screened.     

Detection of antibody to Salmonella enteritidis and S typhimurium in the yolk of hens' eggs

Enzyme-linked immunosorbent assays (ELISAs) have been developed to detect IgG antibodies to Salmonella enteritidis and S typhimurium in the yolk of hens' eggs. Better discrimination and more consistent results were obtained between eggs from experimentally infected and uninfected hens by using saline-dilution of yolk rather than chloroform extraction. Threshold absorbance values were determined in three salmonella-free flocks, and on the basis of these results ELISA optical density values greater than 0.25 were considered to be positive for antibodies to salmonella. Four flocks with a history of salmonella infection were examined; three contained birds which were seropositive for S enteritidis by ELISA and from which S enteritidis was isolated, and a large proportion of eggs from these birds contained antibody to S enteritidis. Eggs from the fourth flock had no detectable antibody, although serum antibody was detected in some birds. No salmonellae were isolated from the yolks of the eggs from any of the four flocks.     

Molecular biologic methods for epidemiological precision characterization of disease agents using Salmonella enteritidis from poultry as an example

The principles of molecular biological methods for epidemiological investigation of infectious bacteria are described. They were applied for differentiating S. enteritidis isolates originating from poultry. Among the methods described are the determination of the chromosomal DNA fingerprints (BRENDA), the methods of multilocus enzyme electrophoresis, plasmid profiles, and the outer membrane protein as well as lipopolysaccharide pattern. It turned out, that S. enteritidis strains isolated before 1987, in 1988 and the ones originating from Great Britain did not differ in anyone of these criteria. Consequently one has to assume, that the strains recently causing problems originate from the population of S. enteritidis which had existed in poultry before.     

Molecular and phenotypical characterization of Clostridium perfringens isolates from poultry flocks with different disease status

Due to the diminished use of growth-promoting antibiotics in the European Union, Clostridium perfringens induced necrotic enteritis and subclinical disease have become important threats to poultry health. A study was set up to genotypically and phenotypically characterise C. perfringens isolates from poultry flocks with different health status. Animals from healthy flocks were sampled by cloacal swabs, while intestinal and liver samples of animals suffering from necrotic enteritis were analysed. A total of 27 isolates was obtained from 23 broiler flocks without clinical problems and 36 isolates were obtained from 8 flocks with clinical problems. Using PFGE typing, high genetic diversity was detected between isolates from different flocks. Isolates derived from flocks where disease outbreaks occurred were clonal within each flock, but each flock harboured a different clone. All isolates were of toxin type A. Isolates from 5 out of 35 PFGE types carried the cpb2 gene, encoding the beta2 toxin, and isolates from 2 out of 35 PFGE types harboured the cpe gene, encoding the enterotoxin. In vitro alpha toxin production for all isolates was quantified by enzyme-linked immunosorbent assay. It was shown that in vitro alpha toxin production of C. perfringens isolates from diseased flocks was not higher than in vitro alpha toxin production from isolates derived from healthy flocks.     

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.     

鸡源大肠杆菌的耐药性监测及生物学特性研究

从山东省诸城地区6个集约化养鸡场采集50只病死鸡病料,从中分离鉴定得到32株大肠杆菌,对其进行了12种常规药敏试验,发现分离的菌株有不同程度的耐药性。对14株优势血清型菌株进行了质粒DNA提取分析,然后用Hind Ⅲ限制性内切酶对质粒进行了酶切。结果表明,质粒的得率为100%,来源相同的菌株具有相同或相似的质粒图谱,也就是说它们有共同的起源;来源不同的菌株的质粒图谱一般不同,即有不同的起源。将血清型和质粒图谱比较,结果发现,同一血清型可以有不同的质粒图谱,不同血清型可以有相似的质粒图谱,血清型与质粒图谱没有直接的联系。     

Genotyping Campylobacter jejuni strains isolated from the gut and oviduct of laying hens

Campylobacter jejuni frequently colonizes the avian intestine. Recent evidence suggests that this organism can also colonize the oviduct of laying hens. However, the source and role of this colonization are unknown. Isolates from the ceca, cloacae, and oviducts of 11 laying hens in three intensive egg-producing flocks were genotyped by Fla typing with the restriction fragment length polymorphism of the polymerase chain reaction product of the flaA and flaB genes (fla typing) and pulsed-field gel electrophoresis (PFGE). A diversity in fla types and PFGE types was observed within and between flocks. Individual birds could be colonized by different genotypes at various intestinal and oviduct sites. However, the oviduct of individual birds appeared to be colonized by only one genotype at the time of sampling. In two birds, matching isolates investigated from the intestinal and reproductive tracts were genotypically identical but different from those oviduct isolates found in other birds in the same flock. Interestingly, not all cecal isolates appeared to be equally able to colonize the oviduct. These results suggest that oviduct colonization may result from ascending infection via the cloaca and that some strains of C. jejuni may be better adapted than others to oviduct colonization.     

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.     

Characterization of Enterococcus hirae outbreaks in broiler flocks demonstrating increased mortality because of septicemia and endocarditis and/or altered production parameters

In Denmark, increased problems associated with streptococci and enterococci have been observed in broilers and broiler parent flocks, resulting in increased mortalities, uneven flocks, and subsequent downgrading and increased condemnations. Postmortem lesions associated with recent outbreaks due to Enterococcus hirae have been accompanied or dominated by septicemia and endocarditis. As a result of infection at an early age and relatively low mortality rates, outbreaks are not always clearly defined and may go unnoticed or may be attributed to poor chick quality. For the same reasons, the pathogenesis and epidemiology of observed outbreaks has only remained speculative. Four separate outbreaks associated with E. hirae infections in broiler flocks occurring between 1998 and 2002 have been investigated. Two of the outbreaks indicated evidence of two separate clones, with 89% and 79% of isolates involved in the individual outbreaks belonging to a single pulsed-field gel electrophoresis (PFGE) profile, respectively. Another outbreak (outbreak 4) demonstrated clear clonality, with all isolates demonstrating affinity to one of two PFGE profiles that differed by only two bands. However, all three outbreaks demonstrated a different clone. The remaining outbreak was nondonal, with isolates distributed between six separate PFGE profiles. One of the outbreaks (outbreak 4) was descended from a parent broiler flock previously associated with an outbreak of Streptococcus gallinaceus; the flock also exhibited septicemia and endocarditis. Initial indications suggested the possibility of vertical transmission of S. gallinaceus to the current broiler flock, causing infection. By extended phenotypic characterization and subsequent genetic characterization, including 16S rRNA sequencing, all strains from the four outbreaks were confirmed as E. hirae. This investigation highlights the problems associated with characterizing enterococci infections in broiler flocks.