Field testing of a vaccine against eel diseases caused by Vibrio vulnificus

The field results of a vaccination programme against Vibrio vulnificus serovar E (biotype 2) in a Spanish eel farm are reported. A total of 9.5 million glass eels were vaccinated from January 1998 to March 2000 by prolonged immersion followed by 2 subsequent reimmunisations after 12 to 14 and 24 to 28 d, respectively. The acquired protection and the immune response against serovar E were estimated over a period of 6 mo after vaccination. A similar vaccination schedule was conducted with elvers in a Danish eel farm. In this case, the acquired protection and the immune response against serovar E and the new eel-pathogenic serovars, recently described in Denmark, were evaluated over a short term. The overall results show that the vaccine against V. vulnificus serovar E induces a satisfactory protective immunity during the main growth period of eels (around 6 mo) with a relative percentage survival of 62 to 86% and protects them against the new eel-pathogenic serovars. Vaccination of eels by immersion seems to be the best strategy to prevent diseases caused by V. vulnificus.     

Molecular characterization of Leptospira sp. strains isolated from human subjects in São Paulo, Brazil using a polymerase chain reaction-based assay: a public health tool

A polymerase chain reaction (PCR)-based assay which amplifies repetitive DNA elements present within bacterial genomes was used to characterize and differentiate Leptospira sp. Thirty-five strains from a reference culture collection and 18 clinical isolates which had been previously analyzed by cross agglutinin absorption test (CAAT) were evaluated by this technique. PCR results from analysis of the reference culture collection showed no bands corresponding to serogroups Australis, Autumnalis, Bataviae, Celledoni, Cynopteri, Djasiman, Panama, Pomona, Pyrogenes, and Tarassovi. However, the PCR method was able to clearly discriminate the serogroups Andamana, Ballum, Canicola, Grippotyphosa, Hebdomadis, Icterohaemorrhagiae, Javanica, Sejroe, Semaranga, and Shermani. Clinical isolates previously characterized by CAAT as serovar Copenhageni, serovar Castellonis, and as serovar Canicola were in agreement with PCR results. The clinical isolate previously characterized as serovar Pomona was not differentiated by PCR. Forty additional clinical isolates from patients with leptospirosis obtained in S?o Paulo, Brazil were also evaluated by this PCR method. Thirty-nine of these were determined to belong to serogroup Icterohaemorrhagiae (97.5%) and one to serogroup Sejroe (2.5%). These results demonstrate that the PCR method described in this study has utility for rapid typing of Leptospira sp. at the serogroup level and can be used in epidemiological survey.     

A comparison of a blocking ELISA and a haemagglutination inhibition assay for the detection of antibodies to Avibacterium (Haemophilus) paragallinarum in sera from artificially infected chickens

The ability of blocking ELISAs and haemagglutination-inhibition (HI) tests to detect antibodies in sera from chickens challenged with either Avibacterium (Haemophilus) paragallinarum isolate Hp8 (serovar A) or H668 (serovar C) was compared. Serum samples were examined weekly over the 9 weeks following infection. The results showed that the positive rate of serovar A specific antibody in the B-ELISA remained at 100% from the second week to the ninth week. In chickens given the serovar C challenge, the highest positive rate of serovar C specific antibody in the B-ELISA appeared at the seventh week (60% positive) and was then followed by a rapid decrease. The B-ELISA gave significantly more positives at weeks 2, 3, 7, 8 and 9 post-infection for serovar A and at week 7 post-infection for serovar C. In qualitative terms, for both serovar A and serovar C infections, the HI tests gave a lower percentage of positive sera at all time points except at 9 weeks post-infection with serovar C. The highest positive rate for serovar A HI antibodies was 70% of sera at the fourth and fifth weeks post-infection. The highest rate of serovar C HI antibodies was 20% at the fifth and sixth weeks post-infection. The results have provided further evidence of the suitability of the serovar A and C B-ELISAs for the diagnosis of infectious coryza.     

Screening method for Salmonella enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones by PCR-restriction fragment length polymorphism

Salmonella enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones (MICs of ciprofloxacin, 0.25 to 2 microg/ml) have a mutation at codon either Ser-83 or Asp-87 of gyrA gene. A screening method by PCR-restriction fragment length polymorphism (PCR-RFLP) was designed to screen the mutations at codon Ser-83 and Asp-87 of the gyrA gene of S. enterica serovar Typhi and serovar Paratyphi A clinical isolates. This method successfully screened the gyrA mutations of S. enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones.     

Multidrug-resistant Salmonella enterica serovar paratyphi A harbors IncHI1 plasmids similar to those found in serovar typhi

Salmonella enterica serovars Typhi and Paratyphi A cause systemic infections in humans which are referred to as enteric fever. Multidrug-resistant (MDR) serovar Typhi isolates emerged in the 1980s, and in recent years MDR serovar Paratyphi A infections have become established as a significant problem across Asia. MDR in serovar Typhi is almost invariably associated with IncHI1 plasmids, but the genetic basis of MDR in serovar Paratyphi A has remained predominantly undefined. The DNA sequence of an IncHI1 plasmid, pAKU_1, encoding MDR in a serovar Paratyphi A strain has been determined. Significantly, this plasmid shares a common IncHI1-associated DNA backbone with the serovar Typhi plasmid pHCM1 and an S. enterica serovar Typhimurium plasmid pR27. Plasmids pAKU_1 and pHCM1 share 14 antibiotic resistance genes encoded within similar mobile elements, which appear to form a 24-kb composite transposon that has transferred as a single unit into different positions into their IncHI1 backbones. Thus, these plasmids have acquired similar antibiotic resistance genes independently via the horizontal transfer of mobile DNA elements. Furthermore, two IncHI1 plasmids from a Vietnamese isolate of serovar Typhi were found to contain features of the backbone sequence of pAKU_1 rather than pHCM1, with the composite transposon inserted in the same location as in the pAKU_1 sequence. Our data show that these serovar Typhi and Paratyphi A IncHI1 plasmids share highly conserved core DNA and have acquired similar mobile elements encoding antibiotic resistance genes in past decades.     

Application and Validation of PFGE for Serovar Identification of Leptospira Clinical Isolates

Serovar identification of clinical isolates of Leptospira is generally not performed on a routine basis, yet the identity of an infecting serovar is valuable from both epidemiologic and public health standpoints. Only a small number of reference laboratories worldwide have the capability to perform the cross agglutinin absorption test (CAAT), the reference method for serovar identification. Pulsed-field gel electrophoresis (PFGE) is an alternative method to CAAT that facilitates rapid identification of leptospires to the serovar level. We employed PFGE to evaluate 175 isolates obtained from humans and animals submitted to the Centers for Disease Control and Prevention (CDC) between 1993 and 2007. PFGE patterns for each isolate were generated using the NotI restriction enzyme and compared to a reference database consisting of more than 200 reference strains. Of the 175 clinical isolates evaluated, 136 (78%) were identified to the serovar level by the database, and an additional 27 isolates (15%) have been identified as probable new serovars. The remaining isolates yet to be identified are either not represented in the database or require further study to determine whether or not they also represent new serovars. PFGE proved to be a useful tool for serovar identification of clinical isolates of known serovars from different geographic regions and a variety of different hosts and for recognizing potential new serovars.     

Characterization of flagellar antigens and insecticidal activities of Bacillus thuringiensis populations in animal feces

In total, 287 Bacillus thuringiensis isolates, recovered from feces of 28 zoo-maintained animal species, were examined for flagellar (H) antigenicity and insecticidal activity. Serologically, 209 isolates (72.8%) were allocated to the 8 H serogroups, 4 were untypable, and 74 were untestable. Among the 8 H serotypes detected, H3abc (serovar kurstaki) predominated at a high frequency of 88.0%, followed by H6 (serovar entomocidus) with a frequency of 7.7%. Insecticidal activity was associated with 67.2% of the fecal populations: 188 isolates were toxic to both Bombyx mori (Lepidoptera: Bombycidae) and Aedes aegypti (Diptera: Culicidae), 2 isolates were specific for B. mori, and 3 isolates were toxic to A. aegypti only. Of the isolates with dual toxicity, 97.9% belonged to the serovar kurstaki, producing bipyramidal parasporal inclusions. All of the H7 (serovar aizawai) isolates were toxic to both insects.     

The major outer-membrane proteins of Chlamydia trachomatis serovars A and B: intra-serovar amino acid changes do not alter specificities of serovar- and C subspecies-reactive antibody-binding domains.

The major outer-membrane protein (MOMP) of Chlamydia trachomatis is a promising candidate antigen for chlamydial vaccine development. We have sequenced the MOMP genes for a serovar A and a serovar B isolate and have compared these new sequences with those already reported. Intra-serovar changes in the inferred amino acid sequences of the surface-exposed variable segments known to be responsible for binding of neutralizing antibody were observed. Nevertheless, epitope mapping with solid-phase peptides showed that these intra-serovar changes did not affect the binding of serovar- and subspecies-specific, potentially protective antibodies. Variable segment 1 of C. trachomatis serovar A contained two adjacent antibody-binding sites, one of which was C-subspecies specific while the other was serovar A specific. Therefore the subspecies binding site for C-complex organisms is in variable segment 1, whilst that for B-complex organisms is in variable segment 4. This work shows that MOMP sequences are relatively stable within the serovar categorization for isolates taken decades apart from different continents. Within a given serovar, however, limited interchange of functionally related amino acids may occur without impairing the binding of serovar-specific antibody.     

Interserovar antitoxic immunity to Vibrio cholerae toxins

The immunochemical affinity of V. cholerae enterotoxins, serovars Inaba and Ogawa, has been shown in animal experiments on cross antitoxic immunity in the small intestine, the passive hemagglutination test and the toxin neutralization test. However, antitoxic interaction with both enterotoxins is characteristic only for antibodies to V. cholerae of serovar Inaba, while in animals immune to Ogawa toxin the choleragenic effect of enterotoxins produced by V. cholerae of both serovars in retained. The possible mechanisms of one-sided cross interserovar antitoxic immunity in cholera are discussed.     

赖型钩端螺旋体外膜蛋白基因结构比较性研究

用ＰＣＲ方法扩增不同毒力赖型钩体ＯｍｐＬ１基因片段,进行序列测定,用相关软件比较分析核苷酸序列、蛋白质二级结构以及限制性内切酶谱,不同毒力赖型钩体能扩增出９６０ｂｐ的片段,非致病Ｐａｔｏｃ株未能扩出相应片段,中国赖型参考株ＯｍｐＬ１序列（ＧｅｎｅＢａｎｋ Ｎｏ．ＡＦ２５０３１８）与流感伤寒型相应序列比较有９８个核苷酸差异,同源性为８９．８％,二级结构预测和氨基酸疏水图显示变异主要发生在跨膜蛋白的膜     

Monoclonal antibodies for a comparative serological study of strains of Vibrio anguillarum

Murine monoclonal antibodies against characteristic determinants of heat stable somatic antigens of typical serovar I (820/15/8 Kop) and serovar II (134/82/1 Kiel) of European Vibrio anguillarum strains were produced. Three stable hybridoma cell lines, called aVaI/1F4, aVaI/6F4 and aVaI/2H5, produced monoclonal antibodies each against a typical serovar I determinant of strain 820/15/8 Kop. One hybridoma cell line (aVaII/5A4) producing monoclonal antibodies against Vibrio anguillarum strain 134/82/1 Kiel (serovar. II) was established. Fourteen Vibrio strains and Aeromonas salmoniáda strains were serologically compared by Enzyme-Linked Immunosorbent Assay (Elisa ).     

胸膜肺炎放线杆菌apxIC基因插入失活突变株构建及免疫原性分析

胸膜肺炎放线杆菌是引起猪传染性胸膜肺炎(APP)的呼吸道病原菌,其分泌的Apx毒素是最重要的毒力因子之一。为构建APP突变弱毒菌株,在apxIC基因下游XhoI酶切位点处插入氯霉素抗性基因(Chlr)制备转移载体,通过电转化导入APP血清10型参考菌株(D13039)进行同源重组,筛选获得apxIC基因插入突变菌株D13039C-Chlr。该突变菌株特性鉴定结果表明其溶血活性完全丧失,可正常增殖和分泌ApxI毒素,连续10次传代后基因组中插入的Chlr基因可稳定遗传,利用5个剂量(2×108CFU~2×106CFU)对每组3只小鼠腹腔攻毒结果显示突变菌株毒力较母源菌株降低至少100倍以上,将突变菌株作为弱毒活疫苗经滴鼻途径免疫仔猪后利用APP血清1型(4074)和血清10型(D13039)菌株攻毒进行免疫原性鉴定,结果显示血清1型攻毒后非免疫组4头仔猪全部死亡而免疫组4头中死亡2头,非免疫组肺损伤指数(34.4)显著高于免疫组(17.5),血清10型攻毒后非免疫组肺损伤指数(17.5)也高于免疫组(10.5),同时鼻拭子和肺组织样品的细菌重分离数及PCR检测阳性数非免疫组也明显高于免疫组,表明突变菌株作为弱毒活疫苗对仔猪具有一定的交叉免疫保护力。该突变菌株的构建为鉴定ApxI毒素活性及研制具有交叉保护活性的APP弱毒活疫苗奠定了基础。     

An oral Salmonella vaccine promotes the down-regulation of cell surface Toll-like receptor 4 (TLR4) and TLR2 expression in mice

A single oral immunization with the Lon-protease-deficient Salmonella enterica serovar Typhimurium (strain CS2022) induced protective immunity in mice against a subcutaneous challenge with virulent Listeria monocytogenes as well as virulent Salmonella serovar Typhimurium. The populations of cell surface Toll-like receptor 4 (TLR4) and TLR2 on peritoneal macrophages decreased at week 6 after immunization. This population decrease was not reversed after a challenge with either Salmonella or Listeria. These results suggest that oral immunization with CS2022 induced immune tolerance correlated with the down-regulation of cell surface TLR expression. This down-regulation may in part account for the development of cross-protection against a Listeria challenge by immunization with CS2022.     

Naturally occurring isolates of Neisseria gonorrhoea, which display anomalous serovar properties, express PIA/PIB hybrid porins, deletions in PIB or novel PIA molecules

The por gene of Neisseria gonorrhoeae encodes the Protein I porin responsible for serovar specificity. In this study the por genes have been sequenced from clinical isolates which exhibited anomalous serovar reactivity. One group of `intermediate' strains differed significantly from both Protein IA and IB strains, were more closely related to IA but appeared to represent a distinct class of Protein I. Another strain was closely related to Protein IB of serovar IB-6 but contained a deletion of six amino acids in surface exposed loop 6 which removed epitopes recognised by IB specific monoclonal antibodies. The third group of strains, which reacted with both IA and IB specific monoclonal antibodies, expressed hybrid Protein I molecules containing both IA and IB epitopes. These strains appeared to originate from a double crossover between Proteins IA and IB with the amino and carboxy terminal residues homologous to IB while the surface exposed loop 6 demonstrated close homology to IA. This is the first demonstration of naturally occurring gonococci expressing a hybrid Protein IA/IB.     

Poultry-Associated Salmonella enterica subsp. enterica Serovar 4,12:d:− Reveals High Clonality and a Distinct Pathogenicity Gene Repertoire

A European baseline survey during the years 2005 and 2006 has revealed that the monophasic Salmonella enterica subsp. enterica serovar 4,12:d:− was, with a prevalence of 23.6%, the most frequently isolated serovar in German broiler flocks. In Denmark and the United Kingdom, its serovar prevalences were 15.15% and 2.8%, respectively. Although poultry is a major source of human salmonellosis, serovar 4,12:d:− is rarely isolated in humans (approximately 0.09% per year). Molecular typing studies using pulsed-field gel electrophoresis and DNA microarray analysis show that the serovar is highly clonal and lacks genes with known contributions to pathogenicity. In contrast to other poultry-associated serovars, all strains were susceptible to 17 antimicrobial agents tested and did not encode any resistance determinant. Furthermore, serovar 4,12:d:− lacked the genes involved in galactonate metabolism and in the glycolysis and glyconeogenesis important for energy production in the cells. The conclusion of the study is that serovar 4,12:d:− seems to be primarily adapted to broilers and therefore causes only rare infections in humans.     

Cloning and characterization of the gene encoding the z66 antigen of Salmonella enterica serovar Typhi

Z66 antigen-positive strains of Salmonella enterica serovar Typhi change flagellin expression in only one direction from the z66 antigen to the d or j antigen, which is different from the phase variation of S. enterica serovar Typhimurium. In the present study, we identified a new flagellin gene in z66 antigen-positive strains of S. enterica serovar Typhi. The genomic structure of the region containing this new flagellin gene was similar to that of fljBA operon of biphasic S. enterica serovars. A fljA-like gene was present downstream of the new flagellin gene. A rho-independent terminator was located between the new flagellin gene and the fljA-like gene. Hin-like gene was not present upstream of the new flagellin gene. We generated a mutant strain of S. enterica serovar Typhi, which carries a deletion of the new flagellin gene. Western blotting revealed that the 51-kDa z66 antigen protein was absent from the population of proteins secreted by the mutant strain. Southern hybridization demonstrated that the z66 antigen-positive strains of S. enterica serovar Typhi carried the new flagellin gene and fliC on two different genomic EcoRI fragments. When z66 antigen-positive strains were incubated with anti-z66 antiserum, the flagellin expression by S. enterica serovar Typhi changed from z66 antigen to j antigen. The new flagellin gene and the fljA-like gene were absent in the strain with altered flagellin expression. These results suggested that the new flagellin gene is a fljB-like gene, which encodes the z66 antigen of S. enterica serovar Typhi, and that deletion of fljBA-like operon may explain why S. enterica serovar Typhi alters the flagellin expression in only one direction from the z66 antigen to the d or j antigen.     

Serological analysis of serogroup icterohaemorrhagiae using monoclonal antibodies

Eighteen serovars (19 strains) of serogroup Icterohaemorrhagiae were serologically analyzed using 18 monoclonal antibodies against serovar copenhageni Shiromizu, M20 and serovar icterohaemorrhagiae RGA strains. The reaction patterns of the serovars against these monoclonal antibodies were different. According to these results, we divided the serovars, except for serovar tonkini, into the following three subgroups: Subgroup 1 reacted to many monoclonal antibodies including serovars icterohaemorrhagiae, copenhageni, hualien, monymusk, mankarso, and budapest. Subgroup 2 fell between subgroups 1 and 3 including serovars dakota, naam, bogvere, birkini, smithi, ndambari, gem, ndahambukuje and mwogolo. Subgroup 3 reacted to only a few monoclonal antibodies: serovars weaveri and sarmin. Serovar tonkini did not react to any of the monoclonal antibodies used. There is a possibility that serovar tonkini does not belong to serogroup Icterohaemorrhagiae. Further studies on the serological reactions of each strain revealed that it was impossible to distinguish the RGA strain from the serovar hualien LT11-31 strain, indicating that they may be identical. It was also observed that serovar copenhageni and monymusk seemed to be closely related. Serovars birkini and smithi, and serovars ndambari and gem were alike in their serological reactivities. Among the 18 monoclonal antibodies, RGAMA-1 was a unique antibody which reacted only to serovar icterohaemorrhagiae and serovar hualien, indicating that it must be the serovar icterohaemorrhagiae specific antibody. On the other hand, SHIRMA-2, 5, 6 reacted to all the serovars except for serovars weaveri, sarmin, and tonkini. These antibodies exhibited a broad reaction spectrum.     

The gene identification of bacterial species and serovariants by the polymerase chain reaction with universal oligonucleotides: the reidentification of earlier isolated strains of Yersinia pseudotuberculosis]

Genetic analysis of 19 standard strains belonging to 6 Yersinia species (Y. pestis, Y. pseudotuberculosis, Y. enterocolitica, Y. kirstensenii, Y. frederiksenii, Y. intermedia) revealed that gene typing by the method of polymerase chain reaction (PCR) with the use of universal primers permitted the identification of species in bacterial cultures by PCR patterns and the determination of Y. pseudotuberculosis serovars within 4 hours. By this method 23 Y. pseudotuberculosis strains (serovar 1), earlier isolated in different regions of the USSR from humans and rodents, were studied. The study showed that out of 14 strains of human origin only two strains could actually be classified with serovar 1, while the remaining strains were reidentified as belonging to serovar 5. Among 9 strains isolated from rodents those of serovar 1 prevailed (8 strains). The authors suppose that strains of serovar 5 cause outbreaks and sporadic cases of pseudotuberculosis, occurring considerably more often than it is commonly believed in the USSR.     

Decarboxylase activity involved in methyl ketone production by Staphylococcus carnosus 833, a strain used in sausage fermentation

Outbreaks of Vibrio parahaemolyticus gastroenteritis in the United States (Texas, New York and Pacific Northwest) in 1997-98 emphasized the need to develop molecular methods for identification and differentiation of these organisms. When outbreak isolates were analyzed for the enterobacterial repetitive intergenic consensus sequences, the Texas and New York outbreak isolates had a specific 850-bp DNA fragment that was absent in Pacific Northwest isolates. The 850-bp polymerase chain reaction (PCR) product was found in isolates of serovar O3:K6, which have an unusual potential to spread and cause infections. To develop a specific molecular detection method for serovar O3:K6, the nucleotide sequence of the 850-bp product was determined. The GenBank blast analysis did not show homology with any known Vibrio spp. gene sequences. Two PCR primers were designed to specifically amplify the unique sequences from serovar O3:K6 isolates. Genomic DNA from 10 Texas, eight New York, and seven Pacific Northwest outbreak isolates of V. parahaemolyticus was assayed by PCR. Texas and New York isolates were positive in the PCR assay, giving a 327-bp PCR product as predicted; however, Pacific Northwest isolates were negative, indicating the absence of the target gene. Texas and New York isolates were all serovar O3:K6; the Pacific Northwest isolates were not. The primers were tested with other Vibrio spp. and other closely related species and no amplification of the 327-bp PCR product was found. The PCR method can be used to specifically identify O3:K6 V. parahaemolyticus isolates in less than 6 h.