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1.
In our studies on FimH adhesins expressed by different Salmonella serovars, we cloned and sequenced the fimH genes from Salmonella enterica ssp. Enterica ser. Gallinarum biovar Gallinarum and S. enterica ssp. Enterica ser. Gallinarum biovar Pullorum. Comparison of the nucleotide sequences revealed the presence of a single-nucleotide polymorphism (SNP) at position 544 bp from the A of the start codon of the fimH open reading frame (ORF). Further analysis of the restriction enzyme sites in fimH gene showed that the SNP at this position is responsible for a sequence specifically recognized by SacI in S. Gallinarum biovar Gallinarum only, making it possible to differentiate both biovars with the use of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Digestion of PCR amplicons of the fimH gene from S. Gallinarum biovar Gallinarum strains with SacI gave two DNA fragments of 554 and 472 bp and only one fragment of 1026 bp for S. Gallinarum biovar Pullorum. This allows a clear differentiation between these two biovars.  相似文献   

2.
Salmonella enterica subsp. enterica serovar Gallinarum biovars Gallinarum and Pullorum cause fowl typhoid and pullorum disease in avian species, respectively, and have been of considerable economic importance to the poultry industry in parts of the world. The definitive diagnosis of these diseases can be made only by isolation and identification of the causative agent. However, rapid identification of biovars Gallinarum and Pullorum is not easily feasible due to their common antigenic structure and genomic sequence similarity. We developed a duplex polymerase chain reaction (PCR) assay to identify and discriminate between strains of biovars Gallinarum and Pullorum. Duplex PCR primers were designed to target polymorphic regions of glgC and speC genes showing multiple mutations in the sequenced S. enterica subsp. enterica serovar Gallinarum 287/91 genome and were applied to the specific identification of biovars Gallinarum and Pullorum. Boiled lysates of 131 reference and field strains of Salmonella and other related Gram-negative bacteria were tested to validate the duplex PCR assay. All strains of biovars Gallinarum (n=53) and Pullorum (n=21) tested were correctly identified based on this assay (100% sensitivity) while the other strains (n=57) were PCR negative (100% specificity). These results demonstrate that a highly accurate biovar-specific duplex PCR assay can be performed for the rapid identification and discrimination of biovars Gallinarum and Pullorum from field isolates.  相似文献   

3.
Detection of the specific Salmonella serovar Gallinarum, which is divided into the biovars Pullorum and Gallinarum, is compulsory under the national hygienic and sanitary control regulations of France for breeding flocks whose offspring are exported. Our aim was to examine the suitability of bacteriologic and serologic methods routinely used in France to screen serum samples and organs for S. Gallinarum. Since bacteriologic reference techniques are designed to isolate the commonly occurring non-typhoid serovars, such as S. Typhimurium, S. Enteritidis, and others that cause outbreaks of foodborne illness, they may not be particularly suitable for detecting S. Pullorum and S. Gallinarum. This hypothesis was confirmed by the inoculation of 10-wk-old chickens and 1-d-old chicks with various strains of S. Pullorum and S. Gallinarum. The most reliable enrichment media were selenite cystine and Rappaport-Vassiliadis broths. Moreover, on the usual plating media, colonies were small, grew more slowly than the common serovars (in 48 h instead of 24 h), and had an unusual appearance. Since the rapid slide agglutination (RSA) test is based only on antigens from standard and variant strains of S. Pullorum, it may not readily detect S. Gallinarum. In our study, it detected infection in all 10-wk-old chickens inoculated with S. Pullorum strains but did not detect any antibodies against S. Gallinarum. Therefore, S. Gallinarum antigens must be added to the S. Pullorum antigens used in the RSA test in order to detect antibodies produced by birds infected with either biovar.  相似文献   

4.
Salmonella Gallinarum biovar Pullorum (S. Gallinarum biovar Pullorum) is the causative agent of pullorum disease (PD) in chickens which results in considerable economic losses to the poultry industries in developing countries. PCR-Signature Tagged Mutagenesis was used to identify virulence determinants of S. Gallinarum biovar Pullorum and novel attenuated live vaccine candidates for use against this disease. A library of 1800 signature-tagged S. Gallinarum biovar Pullorum mutants was constructed and screened for virulence-associated genes in chickens. The attenuation of 10 mutants was confirmed by in vivo and in vitro competitive index (CI) studies. The transposons were found to be located in SPI-1 (2/10 mutants), SPI-2 (3/10), the virulence plasmid (1/10) and non-SPI genes (4/10). One highly attenuated spiC mutant persisted in spleen and liver for less than 10 days and induced high levels of circulating antibody and protective immunity against oral challenge in young broiler chickens. The spiC mutant is a potential new vaccine candidate for use with chickens against this disease.  相似文献   

5.
Salmonella enterica serovar Gallinarum biovar Gallinarum (S. Gallinarum) is a host-specific pathogen causing systemic infection in poultry, which leads to significant economic losses due to high mortality. However, little is known about the dynamic process of systemic infection and pathogenic characteristics of S. Gallinarum in chickens. In the present study, we developed an oral infection model that reproduces the pathology of S. Gallinarum and clarified the host immune response of the infected chickens. Chickens at 20 days of age orally inoculated at a dose of 108 colony forming unit (CFU) showed typical clinical signs of fowl typhoid and died between 6 and 10 days post infection. The inoculated S. Gallinarum rapidly disseminated to multple organs and the bacterial counts increased in the liver and spleen at 3 days post infection. Pathological changes associated wirh inflammation in the liver and spleen became apparent at 4 days post infection, and increased expression of interferon (IFN)-γ and interleuikin (IL)-12 in the liver and spleen did not observed until 3 days post infection. These results indicate that S. Gallinarum rapidly spread to entire body through intestine, and the low-level of inflammatory responses in the liver during the early stage of infection may contribute to rapid, systemic dissemination of the bacteria. Our infection model and findings will contribute to the better understanding of the pathogenic mechanism of S. Gallinarum, and provide new insights into the prevention and control of fowl typhoid.  相似文献   

6.
为验证鸡白痢、鸡伤寒沙门菌分子分型方法的准确性,本研究以3株鸡白痢、鸡伤寒沙门菌国际参考菌株和306株国内分离株为研究对象,采用5种已知的分子分型方法开展验证性实验.结果发现,基于特定基因可变区的2种分型方法具有良好的特异性,其检测结果与生化分型结果一致,而3种基于特定基因单核苷酸多态性的分型方法则出现假阳性或假阴性的...  相似文献   

7.
1. The aim of the present study was to determine if the 9R-strain of the Salmonella Gallinarum live vaccine was responsible for having fowl typhoid outbreaks in chicken flocks from both chicken and turkey breeders as well as to verify the antimicrobial resistance of the isolates from the outbreaks.

2. The triplex polymerase chain reaction, standard antimicrobial test, beta-lactamase genes identification and Ion Torrent PMG whole-genome sequence were used in the field isolates and in the vaccine strain of S. Gallinarum.

3. The 60 tested isolates were not from vaccine origin and manifested high resistance to drugs from macrolide and quinolone groups. Whole-genome sequencing (WGS) and single nucleotide polymorphism analysis on selected isolates for core genes from Salmonella enterica confirmed the wild origin of these isolates and showed two possible sources of S. Gallinarum in the studied outbreaks.

4. S. Gallinarum isolated from fowl typhoid outbreaks in the studied period were not caused by the use of the SG9R live vaccine. The source of strains sequenced was diverse.  相似文献   

8.
An attempt was made to develop a vaccine against salmonellosis in poultry by formalizing the Salmonella toxins (enterotoxin plus cytotoxins) that have been found to be the main virulent products of the organisms. Formalized (FT) and carbonated (CT) toxoids were prepared from partially purified toxins of Salmonella enterica subspecies enterica ser. Weltevreden (BM-1643) and S. enterica ser. Gallinarum (L-19/a). There was no mortality in birds vaccinated with formalized toxoid of serovar Weltevreden plus Freund's complete adjuvant (FCA) following homologous or heterologous (S. enterica ser. Gallinarum and S. enterica ser. Typhimurium) challenges. Protection ranged from 50% to 83.3% in the groups immunized with other preparations of S. enterica ser. Weltevreden, i.e. with FT without FCA or with CT with or without FCA. Formalized toxoid prepared from S. enterica ser. Gallinarum (FTSG) toxins given with FCA afforded 100% protection against homologous challenge, but not against heterologous serovars. In the control group, only 16.7% of the birds survived in a subgroup challenged with S. enterica ser. Gallinarum, and none withstood challenge with S. enterica ser. Weltevreden or S. enterica ser. Typhimurium. No untoward reactions were observed in any of the immunized groups. Thus, the vaccine was considered to be potent and safe.  相似文献   

9.
Fowl typhoid and pullorum disease, caused bySalmonella enterica serovars Gallinarum biovarsSalmonella Gallinarum (S. Gallinarum) andSalmonella Pullorum (S. Pullorum), remain large threat to the organic poultry industry. These infections are serious threats to poultry health and overall flock viability especially at their early age. These avian pathogens cause a significant production loss and price increase of poultry products especially organic poultry products due to lack of antibiotics use. As a result, alternative intervention strategies have become an urgent demand of the farmers. Natural antimicrobials from plant products, such as bioactive compounds from berry juice byproducts can play important role in such a situation. In this study, we showed that blackberry and blueberry pomace extracts inhibitedS. Gallinarum andS. Pullorum growth in vitro. Two mg gallic acid equivalent (GAE)/mL blackberry or blueberry pomace extract reduced the growth of both pathogens by more than 5 logs at 24 h in broth. In semisolid poultry fecal medium, which contains poultry gut nutrients available to the gut bacteria, 1.0 mg GAE/mL of both extracts reduced the growth of these pathogens by more than 2 logs at 24, 48, and 72-h time points.S. Gallinarum andS. Pullorum were completely inhibited by 1.0 mg GAE/mL both extracts in water. The growth of probioticLactobacillus plantarum (L. plantarum) was unaffected in the presence of berry pomace extracts in broth but growth stimulation was observed in fecal medium when grown in presence of these extracts at 72 h. Moreover,L. plantarum completely inhibitedS. Gallinarum andS. Pullorum when co-cultured in fecal medium in the presence of 1.0 mg GAE/mL of both pomace extracts. In this study, we conclude that bioactive extracts from berry pomace are potential antimicrobials for organic producers which will modulate poultry gut microflora and improve productivity, product safety, and quality.  相似文献   

10.
Fast and accurate identification of Brucella suis at the biovar level is an important issue for public health laboratories because some of the biovars that infect suidae (boars and pigs) are pathogenic for humans while others are not. Since classical biovar typing methods are often time-consuming, hard to standardize and require high-level biosafety containment, methodological improvements are desirable. This article describes new single nucleotide polymorphism (SNP) signatures for the rapid identification and biovar characterization of B. suis. These SNPs were included together with previously described ones in real-time PCR assays applicable to low-biosafety conditions. Allelic profiles unique for each B. suis biovar were defined and the most relevant signatures were determined on a collection of 137 field strains of worldwide origin characterized previously. Biovars assigned with both present and classical methods were globally consistent except for some biovar 3 field strains which matched the allelic profile of biovar 1.  相似文献   

11.
The structure and serological specificities of the lipopolysaccharides (LPSs) from Salmonella enterica serovar Gallinarum biovar Pullorum were studied to provide an improved basis for the distinction between antigenic types and the development of improved diagnostic tests. The structure of the LPS O-polysaccharide (O-PS) from S. Pullorum standard, intermediate and variant antigenic type strains was determined by mass spectrometry, nuclear magnetic resonance spectroscopy and chemical analysis. The LPS of the three strains shared a common structural repeating oligosaccharide unit containing d-mannose, l-rhamnose, d-galactose and d-tyvelose (1:1:1:1). The O-PS of the variant type LPS contained an additional d-glucose residue linked to the O-4 position of the d-galactose residue. The O-PS of the intermediate type LPS was partially the same as that of the variant LPS, however, the molar ratio of the d-glucose component was lower with respect to the other glycose components. Serological specificities of the three antigenic type LPSs were examined with anti-S. Pullorum LPS monoclonal antibodies (Mabs). On immunoblots, Mabs to the standard type O-PS reacted with high molecular mass (HMM) and low molecular mass (LMM) LPS from the standard strain, and with LMM but not HMM LPS from the variant strain. Monoclonal antibodies to the variant type O-PS reacted with HMM but not LMM LPS from the variant strain, and did not react with HMM or LMM LPS from the standard strain. On ELISA, the standard, intermediate and variant antigenic type strains were differentiated by the relative reactivity with the anti-LPS O-PS Mabs. Several of the anti-LPS O-PS Mabs were specific for S. Pullorum and other serogroup D1 Salmonella, and are potentially useful for the development of improved diagnostic tests for these organisms.  相似文献   

12.
Salmonella enterica subspecies enterica serovar Gallinarum biovar Gallinarum is the causative agent of fowl typhoid in chickens, outbreaks of which have devastated poultry populations in Korea since 1992. In order to identify genetic differences among S. Gallinarum isolates, bacteria were examined using the random amplified polymorphic DNA (RAPD) method. Of 13 arbitrary primers screened initially, the primer designated as universal rice primer-6 (URP-6) was selected for subsequent typing assays because it produced a distinctive and reproducible DNA fingerprint for a S. Gallinarum reference strain. URP-6-based RAPD analysis assigned 30 S. Gallinarum isolates into 6 types, with 26 isolates (86.6%) belonging to 2 major RAPD types. The distribution of virulence genes in S. Gallinarum isolates was examined by Southern hybridization. All tested isolates had the invasion gene, invA, the virulence plasmid gene, spvB, and the S. Enteritidis fimbrial gene, sefC. The distribution of virulence genes among S. Gallinarum isolates did not correlate with any specific RAPD type.  相似文献   

13.
In 40 cases salmonellae of the serovar Salmonella (S.) gallinarum were culturally isolated from domesticated gallinaceous birds submitted for diagnostic purposes in the period of 1979-1989. On the basis of the cultural and biochemical features found 35 of them could be assigned to the biovar Pullorum and 5 to the biovar Gallinarum. Of 35 isolates of the biovar Pullorum, 29 were isolated from pure bred chickens of small fancy-exhibition type flocks, 4 from floor-housed adult brown hybrid laying hens and one each from broiler chicks and pheasant chicks (Phasianus colchicus). Acute to subacute courses of the pullorum disease were observed in the 4 flocks of brown hybrid hens. Of 5 isolates of the biovar Gallinarum, 4 were isolated from adult brown hybrid laying hens kept in battery cages and one from floor-housed brown hybrid pullets of the laying type. First cases of fowl typhoid occurred early in the summer of 1988. The disease was characterized by a peracute course in the 4 flocks of brown laying hens and by a more acute course in the pullet flock. The primary source of the fowl typhoid producing organisms was not elucidated.  相似文献   

14.
Salmonella enterica subspecies enterica infection remains a serious problem in a wide range of animals and in man. Poultry-derived food is the main source of human infection with the non-host-adapted serovars while fowl typhoid and pullorum disease are important diseases of poultry. We have assessed cecal colonization and immune responses of newly hatched and older chickens to Salmonella serotypes Enteritidis, Infantis, Gallinarum and Pullorum. S. Enteritidis and S. Infantis colonized the ceca more efficiently than S. Gallinarum and S. Pullorum. Salmonella infection was also associated with increased staining for B-lymphocytes and macrophages in the cecal tonsils of infected birds. S. Enteritidis infection in newly hatched birds stimulated the expression of CXCLi1 and CXCLi2 chemokines in the cecal tonsils, while S. Gallinarum up-regulated the expression of LITAF. In older chickens, S. Enteritidis infection resulted in a significantly higher expression of CXCLi2, iNOS, LITAF and IL-10 while S. Pullorum appeared to down-regulate CXCLi1 expression in the cecal tonsils. Data from spleens showed either no expression or down-regulation of the tested genes.  相似文献   

15.
The purpose of this investigation was to study the host specific infection of Salmonella Gallinarum in chickens and to determine the contribution of intestinal invasion and macrophage survival in relation to systemic infection in the host. This was carried out by comparing the kinetics of infection of S. Gallinarum to that of other Salmonella host-adapted (S. Cholerae-suis, S. Dublin and S. Typhimurium) and host-specific (S. Pullorum and S. Abortus-ovis) serovars. Establishment of the rate of colonisation in intestinal tissue, bursa and systemic sites was carried out by oral infection in day-old and week-old birds. Salmonella Gallinarum was the only serovar capable of causing systemic infection in chickens, however, general colonising ability in the intestine and bursa demonstrated no apparent selective advantage for S. Gallinarum. Further quantification of gastrointestinal invasion was carried out using ligated loops in the small intestine. Invasion in the jejunum of the chicken intestine over 3h demonstrated that Salmonella Typhimurium invasion was statistically higher (P<0.01) when compared with S. Gallinarum. Specific sites of high lymphoid tissue concentration in the chicken, including the bursa of Fabricius and caecal tonsils, were also targeted in invasion assays to investigate possible areas of tissue tropism. S. Typhimurium demonstrated significantly higher (P<0.01) invasion at these sites when compared with S. Gallinarum. Infection of chicken macrophages with S. Gallinarum did not demonstrate increased multiplication and survival intracellularly when compared with other Salmonella serotypes. The only difference seen was with S. Abortus-ovis, which demonstrated a significantly lower (P<0.05 to 0.001) intracellular survival. Together these data suggest that although S. Gallinarum host specificity in the chicken correlates with systemic infection, intestinal and lymphoid tissue invasion in the bursa and caeca, and macrophage survival does not influence this outcome.  相似文献   

16.
To establish a molecular differentiation method for Salmonella enterica subsp. enterica, a hyper-variable region of RNA polymerase beta-subunit (rpoB) of S. enterica subsp. enterica (I), serotype Typhimurium, and Escherichia coli were investigated through comparison of nucleotide sequence of the region. The hyper-variable region was identified at 612-937 of the gene. After PCR amplification of the region in the 17 serotypes and two biotypes of serotype Gallinarum of S. enterica subsp. enterica (I), the nucleotide sequences of the region were determined and compared. All serotypes were distantly related to E. coli with 82.8-84.7% identities in nucleotide sequence while showing 96.6-100% identities with each other. According to the phylogenetic analysis based on the sequenced region with the neighbor-joining method, relatedness of biotype Gallinarum to serotype Enteritidis and biotype Pullorum was determined. Biotype Gallinarum was more closely related to serotype Enteritidis than biotype Pullorum. These results suggested that the 612-937 variable region of rpoB might be useful for molecular evolutionary analysis of serotypes of S. enterica subsp. enterica (I).  相似文献   

17.
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18.
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19.
One hundred twenty-nine Brucella field strains isolated from cattle in Cantabria, Spain, from March 1999 to February 2003, were analysed by using the AMOS-ERY PCR assay and by Southern blot hybridisation with a probe from insertion sequence IS711. Most of the field isolates produced only the ery band in the AMOS-ERY assay and showed a hybridisation pattern identical to that exhibited by reference strains of biovars 5, 6 and 9 of Brucella abortus, but different from strain Tulya, belonging to biovar 3 of B. abortus. However, typing of these strains by standard methods demonstrated that they belonged to biovar 3 of B. abortus. These results indicated that B. abortus biovar 3 was not genetically homogeneous and at least could be divided in two. In one class, that we called biovar 3a, would be the Tulya strain, while the local field strains would belong to biovar 3b. Cloning and nucleotide sequencing of a DNA fragment containing an IS711 copy exclusive of the B. abortus field strains from biovar 3b and reference strains from biovars 5, 6 and 9, revealed the existence of a 5.4 kb deletion close to an IS711 copy. Based on these data, we designed a new primer, which together with the IS711 AMOS primer produced a PCR fragment of 1.7 kb only from the isolates of biovars 3b, 5, 6 and 9 of B. abortus. No amplification products were produced with these primers from strains of the rest of species and biovars of Brucella and from bacteria phylogenetically close to Brucella analysed in this work. Addition of this primer to the AMOS-ERY PCR primer cocktail allows the positive distinction of B. abortus biovars 3b, 5, 6 and 9 from the rest of Brucella species and biovars.  相似文献   

20.
Salmonella serovar Pullorum is a causative agent of pullorum disease (PD) in poultry and is responsible for severe economic losses to the poultry industry in many parts of the world. A definitive detection of Pullorum requires culture followed by serotyping and biochemical identification, a process that is tedious and takes several weeks to accomplish. We have developed a rapid allele-specific polymerase chain reaction (PCR) method based on the nucleotide polymorphism in rfbS gene sequence for the serotype-specific detection of Pullorum and its differentiation from the closely related Gallinarum. The specificity of this PCR assay was tested using DNA samples from Pullorum (n = 13), Salmonella serotypes other than Pullorum (n = 19), and closely related non-Salmonella organisms (n = 5). The PCR assay was highly serotype-specific as the PCR amplicon of 147 base pairs was observed only in the case of Pullorum, while all the other DNA samples tested PCR negative. A definitive identification of Pullorum cultures was possible in less than 3 hr. As little as 100 pg of SP DNA was detected. This allele-specific PCR method is highly specific as well as sensitive and may be an effective molecular tool in the rapid and serotype-specific detection of Pullorum and differentiation from other Salmonella species.  相似文献   

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