Avian Salmonella-Stained Microtest Antigens Produced on Solid Media

Procedures are described for the preparation of Salmonella pullorum and Salmonella typhimurium tetrazolium-stained microagglutination and microantiglobulin antigens with solid media for culture propagation. Cell suspensions were found to be more easily harvested from solid medium than from previously used liquid medium, and greater yields of antigen were obtained. Liquid medium microagglutination and microantiglobulin test antigens were found to be more sensitive than those prepared on solid medium. The microtest antigens produced on solid medium were easier to prepare and in serological tests gave titers more comparable to those demonstrated with the standard macroscopic tube agglutination test.     

Differentiation of Salmonella enterica serotype gallinarum biotype pullorum from biotype gallinarum by analysis of phase 1 flagellin C gene (fliC)

Salmonella enterica serotype gallinarum biotype gallinarum and biotype pullorum are non-motile and pathogenic avian strains. Biotype gallinarum causes fowl typhoid and biotype pullorum is the cause of pullorum disease in chickens. The two biotypes could be differentiated based on biochemical characteristics. However, conventional culture and biochemical assays are time-consuming, laborious and need sterile laboratory practices. Although the two biotypes, gallinarum and pullorum are non-motile, they possess the phase 1 flagellin C gene. The variable regions of the flagellin C gene from 41 biotype pullorum and 52 biotype gallinarum were amplified by colony-PCR and analyzed by single strand conformational polymorphism (SSCP) method. Differences in SSCP electrophoretic patterns were confirmed by nucleotide sequencing. In addition, PCR-RFLP with Hinp1I was also successfully applied to differentiate the two biotypes. These results suggested that the variable regions of fliC could be used as a genetic marker to differentiate biotype gallinarum from biotype pullorum.     

鸡白痢沙门菌ipaJ基因的克隆与鉴定

摘要：【目的】从鸡白痢沙门菌C79-13中克隆ipaJ基因,体外表达该蛋白后进行免疫原性分析。【方法】鸡白痢沙门菌C79-13与肠炎沙门菌50041进行抑制差减杂交后获得的片段PEA2、PE31和PE44与猪霍乱沙门菌C500疫苗株pSFD10质粒上ipaJ基因高度同源,拼接后获得了鸡白痢沙门菌完整的ipaJ基因序列。从鸡白痢沙门菌中克隆出ipaJ基因并将其构建到原核表达载体pET-30a(+)上,Western-blot检测体外表达蛋白的免疫原性,同时检测了该基因在鸡白痢沙门菌分离株中的分布。【结果】从鸡白痢沙门菌中克隆了大小为840 bp的ipaJ基因序列,并获得了体外原核表达的大小为37 kDa融合蛋白。该蛋白可与鸡白痢沙门菌阳性血清反应。PCR结果显示该基因广泛存在于鸡白痢沙门菌菌株中。 【结论】本文首次报道和克隆了鸡白痢沙门菌ipaJ基因,并证明了IpaJ蛋白具有免疫原性。     

Microagglutination Test for Detecting and Measuring Serum Agglutinins of Francisella tularensis

A microagglutination method utilizing stained antigen for detecting and measuring serum agglutinins against Francisella tularensis is described. The microagglutination and standard tube agglutination techniques were demonstrated to be comparable in sensitivity and specificity. Advantages of the micro method are rapidity and ease of performance, economy of reagents and, in particular, ease of interpreting specific reactivity.     

Methodological aspects of serological diagnosis in leptospirosis

The results of serological study for leptospirosis of 515 blood serum specimens from patients coming to different clinics of the Republic of Bangladesh, are presented. The sera were tested in microagglutination test. To exclude intergroup reactions and to enhance reliability of results, immunoglobulin classes were determined with the use of cysteine as a reducing agent and immunoabsorption test. In 51 patients (9.9%) antileptospiral antibodies were detected in titers from 1:20 to 1:1600 against pathogenic leptospires of different serological groups.     

A microagglutination test for blood typing rhesus monkeys, Macaca mulatta

We have developed a microagglutination test for typing rhesus monkey erythrocytes that is sensitive, accurate and easy to perform. The technique requires only microliter quantities of antiserum and cells, and agglutination is easily detected using an inverted microscope. An advantage of this technique is that the typing plates can be stored at -70 degrees C without loss of activity. The results of typing over 400 rhesus blood samples with this technique were 95% concordant with results using the standard microtitre agglutination technique. Preliminary results indicate that this test is also adaptable to typing human blood.     

Identification of campylobacteria isolated from Danish broilers by phenotypic tests and species-specific PCR assays

AIMS: To validate a phenotypic Campylobacter species identification method employed to identify campylobacters in broilers by comparison with campylobacterial species identification using various species-specific PCR analyses. METHODS AND RESULTS: From a collection of 2733 phenotypically identified campylobacterial cultures, 108 Campylobacter jejuni cultures and 351 campylobacterial cultures other than Camp. jejuni were subjected to various species-specific PCR assays. On the basis of the genotypic tests, it was demonstrated that Camp. jejuni and Camp. coli constituted approx. 99% of all cultures, while other species identified were Helicobacter pullorum, Camp. lari and Camp. upsaliensis. However, 29% of the 309 Camp. coli cultures identified by phenotypic tests were hippurate-variable or negative Camp. jejuni cultures, whereas some Camp. lari cultures and unspeciated campylobacter cultures belonged to H. pullorum. It was also notable that 2-6% of the cultures were, in fact, mixed cultures. CONCLUSIONS: The phenotypic identification scheme employed failed to appropriately differentiate Campylobacter species and particularly to identify the closely related species, H. pullorum. SIGNIFICANCE AND IMPACT OF THE STUDY: Future phenotypic test schemes should be designed to allow a more accurate differentiation of Campylobacter and related species. Preferably, the phenotypic tests should be supplemented with a genotypic strategy to disclose the true campylobacterial species diversity in broilers.     

A microagglutination test for blood typing rhesus monkeys, Macaca mulatta

Summary. We have developed a microagglutination test for typing rhesus monkey erythrocytes that is sensitive, accurate and easy to perform. The technique requires only μ1 quantities of antiserum and cells, and agglutination is easily detected using an inverted microscope. An advantage of this technique is that the typing plates can be stored at -70°C without loss of activity. The results of typing over 400 rhesus blood samples with this technique were 95% concordant with results using the standard microtitre agglutination technique. Preliminary results indicate that this test is also adaptable to typing human blood.     

Prevalence of Helicobacter pullorum in conventional, organic, and free-range broilers and typing of isolates

Helicobacter pullorum represents a potential food-borne pathogen, and avian species appear to be a relevant reservoir of this organism. In this study, the prevalence of H. pullorum was investigated at 30 conventional farms where 169 ceca from 34 flocks were tested, at eight organic farms where 39 ceca from eight flocks were tested, and at seven free-range farms where 40 ceca from eight flocks were tested. All of the ceca were obtained from healthy broiler chickens. Moreover, amplified fragment length polymorphism, pulsed-field gel electrophoresis, and automated ribotyping were employed to estimate the levels of genetic variability of H. pullorum broiler isolates within and between flocks. Overall, Gram-negative, slender, curved rods, identified as H. pullorum by PCR, were isolated at 93.3% of the farms tested. The percentage of positive free-range farms (54.2%) was significantly lower than that of conventional (100%) or organic (100%) farms (P < 0.001). The level of within-flock genetic variability, calculated as the number of flocks colonized by isolates genetically different by all of the typing methods, was 34.9%. Isolates showing identical profiles by each typing method were observed in 11.6% of the flocks, but they were never detected between flocks. However, groups of isolates clustered together with an overall similarity level of ≥85%. Our results suggest that even though a high level of genetic variability is attributable to H. pullorum broiler isolates, their hierarchical genotyping produces data useful for epidemiological investigations.     

Identification of unusual Campylobacter-like isolates from poultry products as Helicobacter pullorum

Twenty-six unclassified Campylobacter -like strains previously isolated from 15 chicken carcasses and caecal contents, together with two more strains isolated from chicken faeces on a different occasion, were identified as Helicobacter pullorum using various phenotypic identification methods. API Campy identification kits and a 16-test identification scheme developed for campylobacters failed to identify these bacteria, or identified them as Campylobacter spp. Eighteen strains (including the two isolated on a different occasion) were chosen for examination using a more comprehensive probabilistic identification scheme. Using this method, 14 of the 18 strains were identified as H. pullorum with ID scores >95% ; two strains were also identified as H. pullorum with lower ID scores. Of the remaining two strains, one was not identified with this scheme and the other was misidentified to the H. acinonyx pylori complex. Whole cell protein profiling by SDS-PAGE confirmed the identity of these isolates as H. pullorum , affirming the value of a polyphasic approach for accurately identifying campylobacteria. The comparatively high prevalence of H. pullorum in poultry determined in this study (60%) suggests that routine isolation and identification methods should be amended to enable a thorough evaluation of its role in human gastroenteritis and avian hepatitis. Some phenotypic characters useful in identifying poultry campylobacteria are presented which could be utilized, along with other technique(s), for improved differentiation of the campylobacteria that are found in poultry.     

Microantiglobulin Test for Detecting Salmonella typhimurium Agglutinins

A sensitive antiglobulin (AG) test procedure for the demonstration and experimental study of the agglutinin response of chickens infected orally with Salmonella typhimurium is described. A tetrazolium-stained S. typhimurium antigen was employed with microagglutination techniques and equipment for the first time in conducting the AG test. Results with the conventional macroscopy tube agglutination test for S. typhimurium and the 24-hr microtest were comparable; however, the AG test enhanced titers as much as 16 times, and these persisted at a significant level for as long as 4 months. This study is being extended to other Salmonella serotypes and possible field applications of the AG test procedure.     

禽大肠杆菌、肠炎、鼠伤寒、鸡白痢及鸡伤寒沙门菌多重PCR方法的建立及应用

【背景】大肠杆菌病和沙门菌病是最常见的家禽细菌性疾病,给养禽业造成严重经济损失。另外,禽大肠杆菌和沙门菌也是重要的人畜共患病原菌,可通过禽类及其产品传播给人类,对人类健康造成严重威胁。加强禽大肠杆菌和沙门菌的快速鉴别检测,对养禽业和公共卫生都具有重要意义。【目的】建立禽大肠杆菌、肠炎沙门菌、鼠伤寒沙门菌、鸡白痢沙门菌和鸡伤寒沙门菌的多重PCR检测方法。【方法】通过比较分析确定禽致病性大肠杆菌、肠炎沙门菌、鼠伤寒沙门菌、鸡白痢沙门菌和鸡伤寒沙门菌的特异靶标基因,设计5对特异性引物,通过条件优化建立多重PCR方法,分析该多重PCR方法的特异性、敏感性及可靠性。【结果】该方法能特异性地鉴定禽致病性大肠杆菌、肠炎沙门菌、鼠伤寒沙门菌、鸡白痢沙门菌和鸡伤寒沙门菌,每个PCR反应的最低检出限分别为103 CFU细菌和100 pg基因组DNA。临床分离菌株检测显示,多重PCR与传统血清学方法结果一致。【结论】建立的多重PCR方法能够快速鉴别禽致病性大肠杆菌和不同血清型沙门菌,对禽大肠杆菌病和沙门菌病的流行病学调查及临床检测具有重要意义。     

SEF14菌毛基因操纵子在肠炎沙门氏菌和D-群沙门氏菌的变异分析

【目的】本文旨在探索SEF14菌毛特异性表达于D-群沙门氏菌,特别是肠炎沙门氏菌以及都柏林沙门氏菌的原因。【方法】应用PCR扩增以及序列测定检测了18株鸡白痢沙门氏菌,11株肠炎沙门氏菌以及1株都柏林沙门氏菌标准株中sefA,sefD和sefR基因序列,并分析比对其序列变异。【结果】以11株肠炎沙门氏菌以及1株都柏林沙门氏菌染色体DNA为模板能成功扩增sefA,sefD以及sefR基因;从18株鸡白痢沙门氏菌中均能成功扩增sefA基因,但只有分离于1980年之前的7株分离菌能成功扩增sefD和sefR基因,而另11株1980年后分离菌PCR扩增sefD和sefR基因却无任何产物。比对PCR扩增产物测序结果发现,11株肠炎沙门氏菌以及1株都柏林沙门氏菌株中sefA,sefD以及sefR基因序列和已发表的序列(GenBank登录号为L11008,U07129和AF233854)100%同源;7株鸡白痢沙门氏菌sefD基因测序结果表明,在196位点处发生碱基缺失,造成移码突变,提前于氨基酸残基71位点处产生终止密码子。优化菌毛表达条件,体外抽提和纯化菌毛并进一步试验证明:肠炎沙门氏菌以及都柏林沙门氏菌体外能很好表达SEF14菌毛,但鸡白痢沙门氏菌在相同培养条件下却无任何表达迹象。【结论】SEF14菌毛操纵子亚单位基因sefA,sefD以及调节基因sefR在不同沙门氏菌中的变异情况可能是SEF14菌毛局限性表达的原因之一。     

Comparison of tests for the diagnosis of spontaneous encephalitozoonosis in rabbits

The effectiveness of immunofluorescence, complement fixation, microagglutination serologic tests, intradermal skin test, and detection of histologic lesions were compared for use in diagnosis of spontaneous encephalitozoonosis in rabbits. The India ink and microbead agglutination reactions were compared with immunofluorescence and complement fixation by testing 11 single or pooled sera. Serologic tests correlated best with each other and less well with intradermal tests or presence of lesions. Immunofluorescence, India ink reaction and microbead agglutination were equally useful in detecting antibodies to Encephalitozoon cuniculi. The intradermal test correlated best with the presence of detectable lesions.     

Genetic diversity in Helicobacter pullorum from human and poultry sources identified by an amplified fragment length polymorphism technique and pulsed-field gel electrophoresis

Helicobacter pullorum was first isolated from the faeces and carcasses of poultry and has been associated with human gastroenteritis. The aim of this study was to examine interstrain genetic diversity within H. pullorum. Two fingerprinting techniques were used: amplified fragment length polymorphism (AFLP) and pulsed field gel electrophoretic (PFGE) analysis. The 20 strains examined were from four countries and comprised 13 human isolates and seven poultry isolates. Their identity was confirmed by a species-specific PCR assay. The human and poultry isolates had distinct genotypes and most strains showed a high degree of genetic diversity. Genotyping also indicated a clonal origin for two strains from the same poultry flock, and established a close relatedness between three chicken carcass isolates from a processing plant. It is concluded that these two genotyping techniques will provide a useful basis for future epidemiological investigations of H. pullorum in poultry, and may provide a link with its possible causal role in human gastrointestinal infections.     

A simple method for the detection of antibodies to Encephalitozoon cuniculi in rabbits.

Rabbit antibodies against Encephalitozoon cuniculi were detected in an indirect microagglutination test using a bead substrate to which anti-rabbit immunoglobin G light and heavy chain antibodies were coupled. The test was positive using immune whole serum or F(ab)' and F(ab)'2 fragments of immunoglobin G but negative using the F(c) fragment. The reaction was blocked by saturating the beads with rabbit serum or by absorbing positive sera with excess Encephalitozoon cuniculi. The test provided a simple method to detect antibodies to Encephalitozoon cuniculi, did not require elaborate equipment and could be performed using frozen antigen.     

The diagnosis of antileptospiral antibodies in human subjects by solid-phase immunoenzyme analysis

The possibility of using the enzyme-linked immunosorbent assay (ELISA) for the diagnosis of leptospirosis has been shown. This method has proved to be more simple and sensitive than the leptospiral microagglutination and lysis test. The data on obtaining genus-specific leptospiral antigens are presented. As revealed in this study, the antigens obtained by the complex treatment of microbial cells with ultrasound and detergents show the maximum activity in ELISA. The optimum parameters of the ELISA system for the diagnosis of leptospirosis have been established.     

Serological investigation of the fish pathogen Edwardsiella ictaluri, cause of enteric septicemia of catfish.

The serological relationships among 32 isolates of Edwardsiella ictaluri obtained from fish were studied. The strains were extremely homogeneous in protein and lipopolysaccharide preparations as observed by sodium-dodecyl-sulfate polyacrylamide gel electrophoresis. Only minor variations were observed in the structural O-side chain subunits in three isolates; however, such variation did not preclude antigenic recognition by two E. ictaluri antisera in either microagglutination or Western blot immunoassays. The antigenic homogeneity of E. ictaluri was further demonstrated by microagglutination assays with both formalin-killed and heat inactivated cellular antigens. The minimal degree of antigenic variability observed suggested that most isolates of E. ictaluri compose a single antigenic serotype.     

Use of the immunofluorescent microagglutination reaction for the intrageneric differentiation of the immune response in chlamydiosis

The possibility of using, on principle, the immunofluorescent microagglutination test with Chlamydia fluorescent corpuscular antigen for the strain-specific differentiation of immune response induced by the causative agents of ornithosis (strain B), enzootic abortus ovis (strain EAE) and lymphogranuloma venereum (strain LV) has been experimentally demonstrated. The calculation of indices, characterizing the specificity of differences between the systems under comparison, by the method of S. Frasser and D. Berman (1965) has confirmed the significance of such differentiation.     

Use of microcapsular leptospiral antigens for detecting specific antibodies

The sensitivity of microcapsular leptospiral antigens, produced by Japan Lyophilization Laboratory and intended for use in tests for the detection of antibodies to leptospires in the sera of experimentally immunized laboratory animals, were studied. The comparative study of the microcapsular agglutination (MCA) test and other serological tests, such as the microagglutination (MA) test and the indirect enzyme immunoassay (EIA), was made. The leptospiral antigens under study were found to actively react with serospecific and group-specific antibodies. In infected guinea pigs and rabbits specific antibodies could be detected from days 3-4 in the MCA test and only from days 5-7 in the MA test. The average antibody level determined by titration in the MCA test was 3.3 times higher and in indirect EIA, 4.3 times higher than that determined by titration in the MA test. These data make it possible to recommend the use of microcapsular leptospiral antigens for the early diagnosis of leptospirosis.