The indirect fluorescent antibody test for bovine anaplasmosis

Summary

An indirect fluorescent antibody test was used succesfully for the serodiagnosis of experimental Anaplasma infections in cattle. Specific antibodies were detected three to ten days after anaplasma bodies werd found in the blood, and persistedat least 15 weeks post‐infection.

An American and an African stock of A. marginale were used to prepare antigens, and gave comparable results when tested on sera positive to either of these stocks, as well as to an A. centrale‐like stock from Korea.

There were no cross‐reactions with several Theileria, Babesia, Trypanosoma and Eperythrozoon species.     

Prevalence of antibodies to porcine adenovirus in swine by indirect fluorescent antibody test

An indirect fluorescent antibody test was developed for routine identification of a porcine adenovirus and its specific antibody. Two specific-pathogen-free young pigs were inoculated with the viral antigen prepared in continuous porcine kidney cell cultures, and their sera were used as an antibody reagent to standardize the test. Sera of adult pigs with respiratory problems, obtained from pig farms in Quebec, were tested for antibodies to this virus; 83 of 540 sera tested (15.2%) were found to be positive.     

An indirect fluorescent antibody test for antibodies to transmissible gastroenteritis of swine.

The indirect fluorescent antibody test was modified to provide a rapid technique for the detection, screening and titration of antibodies to transmissible gastroenteritis of pigs. Large numbers of slides containing transmissible gastroenteritis antigen were prepared by planting mixtures of infected and uninfected swine testicular cells onto multiwelled teflon-coated slides. After overnight incubation, about one-half of the cells in each well were infected which provided contrast to aid in detecting specific fluorescence in the presence of varying degrees of background staining. Following fixation, antigen slides were stored at -20 degrees C until used. The indirect fluorescent antibody test was compared to the virus neutralization test in both the screening and titration of swine sera containing transmissible gastroenteritis antibodies. The test was found to be sensitive and reliable and to offer certain advantages over the virus neutralization test.     

Identification and differentiation of Taylorella equigenitalis and Taylorella asinigenitalis by lipopolysaccharide O-antigen serology using monoclonal antibodies

Lipopolysaccharides (LPSs) from Taylorella equigenitalis, the causative agent of contagious equine metritis, and T. asinigenitalis were compared by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Lipopolysaccharide profiles of 11 T. equigenitalis strains were similar, but different from the profiles of 3 T. asinigenitalis strains, and the profiles of 2 T. asinigenitalis strains were similar to each other. The serological specificities of the LPSs from these 14 strains were examined by immunoblotting and enzyme-linked immunosorbent assay with monoclonal antibodies (MAbs) to the LPSs of the T. equigenitalis and T. asinigenitalis type strains and T. asinigenitalis strain 2329–98. A MAb to T. equigenitalis LPS O-polysaccharide (O-PS) (M2560) reacted with LPSs from all T. equigenitalis strains but did not react with LPSs from the 3 T. asinigenitalis strains or with 43 non-Taylorella bacteria. Three MAbs to the T. asinigenitalis type strain LPS O-PS or core epitopes (M2974, M2982, M3000) reacted with the homologous strain and T. asinigenitalis strain Bd 3751/05, but not with any of the other bacteria. Five MAbs to T. asinigenitalis 2329–98 LPS O-PS or core epitopes (M2904, M2907, M2910, M2923, M2929) reacted only with this strain. Proton nuclear magnetic resonance spectra of the O-PSs of the type strains of T. equigenitalis and T. asinigenitalis provided fingerprint identification and differentiation of these 2 organisms. The serological results were consistent with our previous finding that the O-antigen of the type strain of T. equigenitalis, being a linear polymer of disaccharide repeating [→4)-α-L-GulpNAc3NAcA-(1→4)-β-D-ManpNAc3NAcA-(1→] units, differs from that of the T. asinigenitalis O-antigen polymer that is composed of repeating [→3)-β-D-QuipNAc4NAc-(1→3)-β-D-GlcpNAmA-(1→] units. Lipopolysaccharide O-PS could be a specific marker for identification and differentiation of T. equigenitalis and T. asinigenitalis, and provide the basis for the development of specific detection assays for T. equigenitalis.     

The application of the indirect fluorescent antibody test in research on heartwater

The preparation of the antigen, details of the reagents, the titration of the antispecies conjugates and the execution of the indirect fluorescent antibody test are described. The sensitivity and specificity of the test and its applicability to the detection of antibodies to Cowdria ruminantium are recorded. The test is both highly specific and sensitive and can be applied to a wide range of studies on heartwater, including epidemiology, determination of the C. ruminantium infection rate of Amblyomma ticks and the evaluation of immunization against heartwater. The test can also be used to detect antibodies to the heartwater agent in the sera of game.     

间接荧光抗体试验诊断犬吉氏巴贝斯虫病

利用冷藏抗原玻片 ,用间接荧光抗体试验检测感染吉氏巴贝斯虫犬血清 ,具有特异性强、敏感性高、快速、准确、操作简单等特点。对疫区 82份犬血清的检查 ,阳性率为 37 8% ;对非疫区 50份犬血清的检查 ,假阳性率为 2 %。与血液涂片染色检查比较 ,该法检出率高 ,结果可靠 ,可用于犬吉氏巴贝斯虫病的诊断和流行病学调查     

Passive hemagglutination test for detection of antibodies against Taylorella (Haemophilus) equigenitalis in sera of mares

The passive hemagglutination (PHA) test was improved to enable the detection of antibodies to Taylorella (Haemophilus) equigenitalis in the sera of mares. Horse red blood cells (RBC) fixed with glutaraldehyde were compared with similarly treated RBC of a cow, pig and sheep for the PHA test. The horse RBC were superior to those of the other animals tested in detecting mares affected with contagious equine metritis (CEM). A PHA test using these cells as indicator and an antigen prepared from T. equigenitalis by sonication following treatment with hyaluronidase was the most satisfactory in terms of sensitivity and specificity. None of the 156 serum samples from clinically healthy mares without a history of contact with T. equigenitalis-infected stallions or mares showed PHA titers greater than 1:32 and only a few samples (7.1%) showed PHA titers of 1:32. Four of the 50 serum samples from mares affected with CEM showed PHA titers of 1:32, while most of the samples (92.0%) showed PHA titers greater than 1:32. The glutaraldehyde-fixed horse RBC sensitized with the antigen had the advantage of being reproducible for at least 7 months when preserved at 4 degrees C.     

An indirect fluorescent antibody test for the detection of antibody to swine infertility and respiratory syndrome virus in swine sera.

An indirect fluorescent antibody (IFA) test was developed and standardized to detect and quantitate antibody for swine infertility and respiratory syndrome (SIRS) virus in swine sera. Test results were evaluated using sera of pigs infected both experimentally and naturally with SIRS virus. The IFA test used swine alveolar macrophage (SAM) monolayers prepared in 96-well microplates and infected with SIRS virus. The monolayers were incubated with test sera, washed, and stained with fluorescein isothiocyanate-labeled rabbit anti-swine IgG. After another wash step, the monolayers were examined under a fluorescent microscope. A noninfected SAM control well was included for each sample. The antibody titers for each serum sample were recorded as the highest serum dilutions with specific cytoplasmic fluorescence but no fluorescence in the control wells. To evaluate the test, sera of 4 6-week-old pigs that had been infected with SIRS virus, 2 contact pigs, and 13 experimentally infected sows were used. In the experimentally infected pigs, antibody was first detected at 7 days postexposure (PE) and peaked (1:256-1,024) between 11 and 21 days PE. All 13 sow sera were negative at time of infection but were positive (1:64- greater than or equal to 1:1,024) at 14-26 days PE. Seven hundred twenty sera collected from 25 different swine farms with or without a history of SIRS were also tested. Of 344 sera from 15 swine farms with a clinical history of SIRS, 257 (74.7%) sera had IFA titers greater than or equal to 1:4, whereas 371 (98.7%) of 376 sera from herds with no history of SIRS were negative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modified indirect fluorescent antibody test for the serodiagnosis of Anaplasma marginale infections in cattle

A modified indirect fluorescence antibody technique was used for the serodiagnosis of Anaplasma marginale infections in cattle. Nonspecific antibodies adherent to infected erythrocytes were removed, using acidic glycine buffer. Evans blue was used as a counterstain.     

Development of a real time PCR for the detection of Taylorella equigenitalis directly from genital swabs and discrimination from Taylorella asinigenitalis

A discriminatory real time PCR for the detection of Taylorella equigenitalis, the causative agent of contagious equine metritis (CEM), and the related species T. asinigenitalis was developed for the direct examination of genital swabs. The 112bp amplicons produced from the two species were discriminated from each other using TaqMan probes labelled with different fluorophores. The TaqMan PCR was shown to be specific for the 16S ribosomal DNA of the two species of taylorella and did not cross-hybridise with the 16S ribosomal DNA of other bacteria tested. Direct amplification from genital swabs was shown to be equally sensitive to that of culture methods. Prevalence in a sample set from The Netherlands was shown to be equivalent to that demonstrated by culture. A companion real time PCR that amplified a fragment of the 16S rDNA gene of equine commensal bacteria was developed to ensure bacterial DNA was extracted from swab material supplied for testing. The use of a rapid and reliable real time PCR for the organism causing CEM should aid the control of this disease.     

The fluorescent antibody and indirect fluorescent antibody assays for diagnosing stunting syndrome of turkeys

The fluorescent antibody (FA) assay was developed for detecting the stunting syndrome agent (SSA) from intestinal tissue. Similarly, the indirect fluorescent antibody (IFA) assay was developed for detecting serum antibodies to SSA. Convalescent antiserum from turkeys orally immunized with SSA was found to be the primary antibody of choice for the FA assay. Intestinal jejunal samples from poults inoculated 3-4 days postinoculation (DPI) was found to be the best antigen source for the IFA assay. SSA was detected from the intestinal tracts of experimentally inoculated birds at 2 DPI with highest level of reactivity at 3 DPI by the FA assay. After 4 DPI the level of SSA infectivity of the intestines waned to low levels. Serum antibody was detected from experimentally inoculated birds as early as 7 DPI with all birds tested seroconverting by 12 DPI. These assays should prove useful for future studies concerning stunting syndrome.     

Evaluation of the indirect fluorescent antibody test for Babesia major and Theileria mutans in Britain.

Use of the indirect fluorescent antibody (IFA) tests is described to detect antibodies to Theileria mutans and Babesia major in the sera of infected cattle. When antisera against T mutans and B major were tested against homologous antigens high antibody titres were recorded: when they were tested against each other or against Babesia divergens antigen insignificant titres (1/40 or less) were recorded. Thus the test was found to be species specific. Animals recovered from T mutans and B major infections retained significant levels of IFA titres for 22 and 11 months respectively. It is suggested that the IFA test could be used for field survey of the piroplasms of cattle in Britain.     

Demonstration of the absence of intervening sequences (IVSs) within 16S rRNA genes of Taylorella equigenitalis and Taylorella asinigenitalis isolates

A total of 57 Taylorella equigenitalis (n=22) and Taylorella asinigenitalis (n=35) isolates was shown not to carry any intervening sequences (IVSs) within 16S rRNA gene sequences. By contrast, we have already shown the genus Taylorella group to carry several kinds of IVSs within the 23S rRNA gene sequences.