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1.
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   

2.
Sera from cattle naturally infected with Brucella abortus (n = 160), vaccinated with B. abortus S19 (n = 88) or immunized with Yersinia enterocolitica O:9 (n = 25) or Escherichia coli O157:H7 (n = 80) were collected. The sera were compared for antibody content to the same bacteria by indirect enzyme immunoassay (IELISA), fluorescence polarization assay (FPA) and competitive enzyme immunoassay (CELISA). Cattle sera (n = 523) collected randomly from across Canada were tested in the same tests. Sera from the B. abortus infected group reacted positively in the brucellosis IELISA (IELISA(Br)), CELISA and FPA (FPA(Br)) and the Y. enterocolitica IELISA (IELISA(Ye)) while the Y. enterocolitica FPA (FPA(Ye)) detected antibody in 93.8% and the E. coli IELISA (IELISA(Ec)) 86.9% and the E. coli FPA (FPA(Ec)) 48.1%. About 70% of the sera from B. abortus S19 vaccinated animals reacted in the three IELISAs, 45% in the CELISA, and 37.7% in the FPA(Ec), 21.6% in the FPA(Br) and 5.7% in the FPA(Ye). Sera from E. coli O:157 exposed cattle reacted mainly in the IELISA(Ec) and FPA(Ec) although surprisingly 87.5% reacted in the IELISA(Ye) and only 3.8% in the IELISA(Br). No reactions were observed with these sera in the FPA(Br) and FPA(Ye) but one serum gave a low positive reaction in the CELISA. All sera from Y. enterocolitica O:9 exposed cattle reacted in the IELISA(Br) and IELISA(Ye) and 80% in the IELISA(Ec). In the CELISA, 44% gave a positive reaction and 64% were positive in the FPA(Br), 28% in the FPA(Ye) and 12% in the FPA(Ec). Of the 523 Canadian sera, about 50% reacted in the E. coli tests with only minor reactions in the Y. enterocolitica O:9 and B. abortus assays. From the data, the cross reaction between E. coli O157:H7, Y. enterocilitica O:9 and B. abortus is dependent on the test used. Thus, extensive cross reaction was observed with the IELISA with much less reactivity in the FPA and the CELISA.  相似文献   

3.
A competitive enzyme immunoassay (EIA) for the detection of circulating bovine antibodies to Brucella abortus has been developed using horseradish peroxidase conjugated monoclonal antibodies (MAb) raised against B. abortus cell surface antigens. Antibodies present in the serum of either vaccinated or infected cattle can apparently displace the conjugated MAb from the lipopolysaccharide antigen (LPS) in a quantitatively different manner allowing an assessment of immune status of the animal. The results from a panel of sera from animals with a known status of vaccination or infection indicated that the test was more selective in the detection and discrimination of infected from uninfected or immunized animals, than conventional complement fixation, agglutination or indirect enzyme immunoassay procedures.  相似文献   

4.
A second generation competitive enzyme immunoassay (CELISA) for detection of bovine antibody to Brucella abortus was developed to eliminate reagent variables in the assay. This assay was different from earlier CELISA formats in that it used recombinant protein A and protein G immunoglobulin receptors (PAG), labelled with horseradish peroxidase, thus eliminating the requirement for polyclonal anti-mouse-enzyme conjugate for detection. This allowed standardization of the assay. The CELISA uses a monoclonal antibody specific for a common epitope of the O-polysaccharide (OPS) of smooth lipopolysaccharide (SLPS) derived from B. abortus S1119.3. This antibody did not react with PAG. This monoclonal antibody was used to compete with antibody in the bovine test serum to the smooth lipopolysaccharide (SLPS) antigen. Reaction of bovine antibody was then measured directly with the PAG enzyme conjugate. In this case, development of colour in the reaction indicated a positive reaction. The performance characteristics of the new CELISA, sensitivity, specificity and exclusion of antibody of B. abortus S19 vaccinated animals, were very similar to those of the classical CELISA and to the indirect enzyme immunoassay (IELISA) when using sera deemed positive by isolation of the bacterium, either from individual animals or from some animals on the premises. All sera were tested by the buffered antigen plate agglutination test (BPAT) and the complement fixation test (CFT). Only samples positive on both BPAT and CFT were considered as positive and only samples negative on both tests were used considered negative. Sufficient samples from cattle, swine, sheep and goats to validate the test were included based on OIE guidelines suggesting inclusion of a minimum of 300 positive and 1000 negative samples.  相似文献   

5.
OBJECTIVE: To determine whether feeding tylosin, an antimicrobial growth promoter, to pigs was associated with increased risk of infection with and excretion of Salmonella enterica serotype Typhimurium. ANIMALS: 17 healthy pigs. PROCEDURE: A commercial pelleted dry feed was given in 2 feeding trials. In trial A, 11 pigs were given feed with tylosin, 11 pigs were given feed without tylosin, and 11 pigs were given feed with tylosin before and feed without tylosin after inoculation with S Typhimurium. In trial B, 44 pigs were given feed that contained tylosin, and 44 pigs were given feed without tylosin. Three weeks after the start of each trial, pigs were orally inoculated with approximately 5 x 10(6) colony-forming units of S Typhimurium. Feces were examined for S Typhimurium, using semiquantitative microbiologic techniques before and for 5 or 6 weeks after inoculation. Serum antibody titers against S enterica were measured by use of ELISA. RESULTS: None of the pigs developed clinical signs of salmonellosis. However, after inoculation, S Typhimurium was isolated from feces of most pigs, and all but 2 pigs developed serum antibodies against S enterica. Significant differences were not detected between experimental and control groups in either trial. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that tylosin fed as an antimicrobial growth promoter to pigs may not be an important factor in promoting infection with or excretion of S enterica serotype Typhimurium.  相似文献   

6.
Canine strongyloidiasis is a parasitic infection caused by the nematode Strongyloides stercoralis and presents a great zoonotic potential. Its confirmation, using coproparasitological methods, is difficult. The detection of serum specific antibodies, however, may facilitate the diagnosis. The aims of this study were to determine the presence of S. stercoralis through the use of parasitological methods and to detect specific antibodies to the parasite in serum samples from domestic dogs by using the indirect fluorescent antibody test (IFAT) on slides and the enzyme-linked immunosorbent assay (ELISA). A total of 215 dogs of various breeds, from the cities of Uberlandia, Araxá and Campo Belo in the State of Minas Gerais, were examined and distributed according to age into the following groups: (I) 19 males and 20 females of 1-2 months old; (II) 11 males and 20 females of 2-month- to 1-year-old and (III) 41 males and 104 females, from 1 to 7 years old. Coproparasitological results showed that 63/215 (29.3%) of the dogs presented some kind of parasite, with two (0.9%) dogs (one from Araxá and the other from Uberlandia) passing S. stercoralis larvae in the feces. Serological results revealed antibodies to S. stercoralis in 45/215 (20.9%) of the dogs, with seropositivity rates of 0% (0/39) in Group I, 22.6% (7/31) in Group II, and 26.2% (38/145) in Group III. No serological cross-reactivity between S. stercoralis and hookworms or Ascaridae was found. Hookworm infections were seen in 31 dogs, but only one of these dogs (infected with both hookworm and Cystoisospora spp.) was S. stercoralis seropositive by IFAT. The present study demonstrated, for the first time, natural S. stercoralis infections in dogs diagnosed by coproparasitological and serological methods. It was concluded that the detection of specific antibodies to S. stercoralis by IFAT and ELISA may contribute to the diagnosis of canine strongyloidiasis.  相似文献   

7.
The Danish government and cattle industry instituted a Salmonella surveillance program in October 2002 to help reduce Salmonella enterica subsp. enterica serotype Dublin (S. Dublin) infections. All dairy herds are tested by measuring antibodies in bulk tank milk at 3-month intervals. The program is based on a well-established ELISA, but the overall test program accuracy and misclassification was not previously investigated. We developed a model to simulate repeated bulk tank milk antibody measurements for dairy herds conditional on true infection status. The distributions of bulk tank milk antibody measurements for infected and noninfected herds were determined from field study data. Herd infection was defined as having either >or=1 Salmonella culture-positive fecal sample or >or=5% within-herd prevalence based on antibody measurements in serum or milk from individual animals. No distinction was made between Dublin and other Salmonella serotypes which cross-react in the ELISA. The simulation model was used to estimate the accuracy of herd classification for true herd-level prevalence values ranging from 0.02 to 0.5. Test program sensitivity was 0.95 across the range of prevalence values evaluated. Specificity was inversely related to prevalence and ranged from 0.83 to 0.98. For a true herd-level infection prevalence of 15%, the estimate for specificity (Sp) was 0.96. Also at the 15% herd-level prevalence, approximately 99% of herds classified as negative in the program would be truly noninfected and 80% of herds classified as positive would be infected. The predictive values were consistent with the primary goal of the surveillance program which was to have confidence that herds classified negative would be free of Salmonella infection.  相似文献   

8.
Brucella, a causative agent of brucellosis and facultative intracellular pathogen, has been isolated recently from a variety of wild mammals. In this study, serum samples from 115 Japanese wild boar (Sus Scrofa leucomystax) killed by hunters in the 4 Prefectures of Shikoku, Japan were tested for antibodies to Brucella spp. by means of the tube agglutination test (TAT) and enzyme-linked immunosorbent assay (ELISA) using antigens extracted with n-lauroylsarcosine. In 9 of the 115 samples (7.8%) antibodies to Brucella spp. were detected by TAT and ELISA. These results suggest that wild boar in Shikoku may be exposed to Brucella spp. or other cross-reactive pathogen infection.  相似文献   

9.
利用抗兔出血症病毒(RHDV)的特异性单克隆抗体(McAb)建立了ELISA阻断试验用以检测血清抗体,并与血凝抑制(HI)和琼脂扩散试验(ID)进行了比较。结果ELISA阻断试验敏感性超过HI,更远胜于ID,具有灵敏、特异、准确等优点,适于大批血清样品的快速检测。  相似文献   

10.
An ELISA was developed for the detection of Fasciola hepatica antibody in serum of cattle. The assay was applied to sera from 258 naturally infected cattle, 256 non-infected cattle and six calves experimentally infected with F. hepatica. The diagnostic sensitivity and specificity of the ELISA test was 98% (95% confidence intervals, 96-100%) and 96% (95% confidence intervals, 93-98%) respectively at a cut-off value of 15% positivity. The results using sera from the experimentally infected calves showed that antibodies were first detected 2-4 weeks after infection. The ELISA test was also compared to the commercially available Bio-X bovine F. hepatica ELISA kit. A subset of 39 positive sera and 47 negative sera were selected from the samples used to evaluate the in-house test. The results indicated that the agreement between the two tests was almost perfect (k statistic=0.82).  相似文献   

11.
In the chicken, maternal antibodies are transferred into the egg and subsequently transported into the developing embryo. IgG (called IgY) is the primary immunoglobulin isotype of the egg yolk. Their level in serum depends on the correct function of immunological system in laying hens. Many factors have a direct or indirect influence on antibody level in fowl. One of them is a commonly used antibiotic, but its influence on avian immune system is still unknown. The objective of the study was to determine the effect of enrofloxacin and chloramphenicol on the level of IgY antibody in serum and egg yolk after immunostimulation of hens with living cells of Salmonella enterica subsp. enterica serovar Enteritidis and lipopolisaccharide. Forty adult egg-laying Arbor Acres and Isa 215 hens (32 and 50 weeks old) from the reproductive flocks and 1640 of their eggs were used for the investigation. No clinical symptoms of any diseases were observed in birds during the entire breeding period. Additionally the birds were checked as free from Salmonella spp. in the beginning of the experiment. The birds were divided into 6 experimental and 2 control groups (5 birds in one group). The hens in the experimental groups were immunized with S. Enteritidis antigens: living bacteria and lipopolisaccharide and treated with enrofloxacin or chloramphenicol. Antibiotics were administered in drinking water for 10 days (from 3rd to 13th day of experiment). To indicate anti-S. Enteritidis, antibodies in sera and egg yolk were used indirectly on ELISA based on lipopolisaccharide from S. Enteritidis. As conjugate these were applied anti-chicken IgY with horseradish peroxidase and ABTS with H2O2 as obtained. Additionally, to detect antibody in serum, a rapid slide test was used with Pullognost and Enterognost standard antigens made in the laboratory. The study revealed that both antibiotics tested decreased the level of specific IgY in laying hens immunized with living bacteria and lipopolisaccharide. It seems that antibiotics have a suppressive effect on the immunological system. The strongest immunosuppressive effect was exerted by chloramphenicol.  相似文献   

12.
建立了ＡＧＩＤ用于猪伪狂犬病（Ｐｓｅｕｄｏｒａｂｉｅｓ,Ｐｒ）的诊断。对９８份被俭血清的ＡＧＩＤ结果与ＳＮ结果比较,当ＳＮ滴度大于１：８时两者的阳性检出符合率为１００％,ＳＮ滴度小于或等于１：８时,两者的阳性检出符合率为８７．５％,ＡＧＩＤ和ＳＮ总的阳性符合率为９４．４％,结果表明,ＡＧＩＤ对Ｐｒ可进行特异性诊断,流行病学调查和免疫动态监测。  相似文献   

13.
The purpose of this study was to evaluate the performance of the indirect enzyme immunoassay (IELISA) and the competitive enzyme immunoassay (CELISA) for the diagnosis of bovine brucellosis in comparison to conventional serological tests routinely used in Argentina. Serum samples (n = 3500), from Brucella-free herds, from vaccinated cattle and from naturally infected cattle, were tested by the following tests: buffered antigen agglutination test (BPAT), rose bengal test (RBT), 2-mercaptoethanol test (2-ME), complement fixation test (CFT), IELISA and CELISA. Sensitivity and specificity of the BPAT, RBT, IELISA and CELISA were determined relative to the 2-ME and the CFT. The CELISA was considered suitable for eliminating most serological reactions of vaccinated animals and was more specific than the other tests. The results indicate the potential use of the CELISA as a complementary assay in the brucellosis control and eradication program in Argentina and other countries, where Brucella abortusstrain 19 vaccination is mandatory.  相似文献   

14.
Escherichia coli (E. coli) strains were collected from young diarrheic calves in farms and field. Strains that expressed the K99 (F5) antigen were identified by agglutination tests using reference antibodies to K99 antigen and electron microscopy. The K99 antigen from a selected field strain (SAR-14) was heat-extracted and fractionated on a Sepharose CL-4B column. Further purification was carried out by sodium deoxycholate treatment and/or ion-exchange chromatography. Monoclonal antibodies to purified K99 antigen were produced by the hybridoma technique, and a specific clone, NEK99-5.6.12, was selected for propagation in tissue culture. The antibodies, thus obtained, were affinity-purified, characterized and coated onto Giemsa-stained Cowan-I strain of Staphylococcus aureus (S. aureus). The antibody-coated S. aureus were used in a co-agglutination test to detect K99+ E. coli isolated from feces of diarrheic calves. The specificity of the test was validated against reference monoclonal antibodies used in co-agglutination tests, as well as in ELISA. Specificity of the monoclonal antibodies was also tested against various Gram negative bacteria. The developed antibodies specifically detected purified K99 antigen in immunoblots, as well as K99+ E. coli in ELISA and co-agglutination tests. The co-agglutination test was specific and convenient for large-scale screening of K99+ E. coli isolates.  相似文献   

15.
Canine hepatozoonosis is caused by the tick-borne protozoon Hepatozoon spp. The prevalence of the infection in the Aegean coast of Turkey was investigated by examination of blood smear parasitology and polymerase chain reaction (PCR) using blood samples from 349 dogs collected from Central Aydin, Kusadasi, Selcuk, Central Manisa, Bodrum and Marmaris within the Aegean coast of Turkey. The indirect fluorescent antibody test (IFAT) for the detection of Hepatozoon canis antibodies was also used to detect the exposure rate to H. canis. PCR amplifying a 666bp fragment of 18S rRNA gene of Hepatozoon spp. was used in the epidemiological survey. The prevalence of Hepatozoon spp. infection was 10.6% by blood smear parasitology and 25.8% by PCR. IFAT revealed that 36.8% of serum samples were positive for antibodies reactive with Hepatozoon spp. The PCR products of 18S rRNA gene of Hepatozoon spp. isolated from six infected dogs, one isolate originating from each of the six different locations, were sequenced. The results of sequence analysis indicate that they are closely related to Indian and Japanese isolates of H. canis. This is the first epidemiological study on the prevalence of H. canis infection in the dog, in Turkey.  相似文献   

16.
A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed for measuring Aujeszky's disease virus (ADV) antigen concentration and an inhibition technique based on the former was developed for detection of antibodies to ADV. The results were checked by determining the cytopathic and serum neutralization titres. The correlation was satisfactory in both cases, with correlation coefficients above 0.8. When measuring ADV antigen concentration, the lower limit of detection was 10(3) TCID 50/0.2 ml. The sensitivity of ELISA in detecting antibodies to ADV was found to be superior to that of the serum neutralization test and, thus, enabled the testing of rabbit and guinea-pig sera.  相似文献   

17.
本研究以牛传染性鼻气管炎gE基因的原核表达产物为抗原建立检测IBRV抗体间接ELISA检测方法.通过DANStar比对分析牛传染性鼻气管炎病毒gE蛋白序列,通过亲和层析对重组表达产物进行纯化,经Western blot检测证明重组表达产物能被IBRV标准性鼻气管炎gE蛋白长度为500 bp的特异性肽序列,随后构建了pC...  相似文献   

18.
Mycoplasma gallisepticum is a poultry pathogen that causes respiratory disease and loss of egg production worldwide. A live attenuated vaccine, ts-11, has been used for control of M. gallisepticum in several countries. The rapid serum agglutination test is usually used as an indicator of flock response to vaccination; however, in some flocks, the detected response may be weak or absent. With the use of specific monoclonal antibodies against M. gallisepticum strain S6 pMGA in immunoaffinity purification, the major membrane antigen of ts-11 was purified. An indirect enzyme-linked immunosorbent assay (ELISA) was developed with the purified antigen, and its potential for detection of antibodies induced after ts-11 vaccination was compared with an indirect ELISA with M. gallisepticum strain S6 pMGA. In the presence of high levels of ts-11-induced antibodies, both antigens detected similar numbers of positive sera. However, when lower levels of antibodies were present, ts-11 pMGA showed a higher sensitivity than S6 pMGA. Further examination of ts-11 pMGA with Mycoplasma synoviae-infected chicken sera revealed that ts-11 pMGA is specific for M. gallisepticum antibodies. With a panel of sera from ts-11-vaccinated or non-ts-11-vaccinated field chickens, the ts-11 pMGA ELISA was found to be more sensitive than the commercial rapid serum agglutination test in detecting antibodies to ts-11 vaccine. The results from this study suggest that the major membrane antigen of M. gallisepticum may have slightly different antigenic profiles in different strains, thereby necessitating the use of autologous antigens in serodiagnostic assays to increase sensitivity of the tests for mycoplasma antibodies. Thus, the low level of antibody response after ts-11 vaccination is, at least partially, due to the low ability of the current diagnostic antigens to bind ts-11 antibodies.  相似文献   

19.
In this study, to evaluate the influence of strangles vaccination on serological test results, we investigated the changes in strangles serum antibody levels in horses after vaccination and subsequent intranasal challenge with S. equi. The horses were vaccinated for strangles with either a component vaccine (Group C) or a live vaccine (Group L). We measured changes in strangles serum antibody levels weekly for 20 weeks after vaccinating horses twice for strangles over a 3-week interval, and for 7 weeks after intranasal challenge with S. equi in the same horses. Serum antibody responses to the proline-glutamic acid-proline-lysine (PEPK) antigen with five repetitions (PEPK-5R) were higher at all times (up to 2.4-fold) following vaccination in Group C than in Group L, and the value peaked at 2.9-fold above the initial value after the second vaccination in Group C horses. However, the value was lower than that in horses infected with S. equi, and it gradually decreased, reaching the initial (week 0) value by the 15th week. Serum antibody responses to PEPK-5R after challenge with S. equi increased in both groups of horses, but the value tended to be lower than that reported for unvaccinated horses. In addition, the average value in Group C was 2.6-fold higher than that of Group L. These results suggest the serum antibody responses of horses infected with S. equi varies according to the type of vaccine with which they have been vaccinated. Although the serological diagnostic test for strangles in which PEPK-5R is used as an antigen is effective for the investigation of serum antibodies to strangles in vaccinated horses, the present data suggest it is necessary to consider the vaccination history when interpreting the results.  相似文献   

20.
An ELISA was compared with the plaque-reduction serum neutralization (PRSN) test, for detection of vesicular stomatitis virus (VSV) antibodies in cattle in a vesicular stomatitis enzootic region of Mexico. A total of 325 bovine serum samples were screened for VSV antibodies. The PRSN test was performed, using Vero cells. The ELISA contained gradient-purified VSV Indiana (Lab strain) and VSV New Jersey (Hazelhurst) as the antigens. Regression analysis and weighted kappa statistic were used to estimate measures of agreement between the 2 assays for detection of VSV antibodies. The ELISA method proved useful for serodiagnosis of vesicular stomatitis. The ELISA and PRSN test results were highly correlated for detection of VSV antibodies.  相似文献   

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