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1.
It was observed that mild acidification (pH less than 4.0) together with solvent extraction of the soluble sonicate of a crude preparation of Babesia bigemina infected cattle erythrocytes caused a quantitative loss of B. bigemina-specific antigen. Cross-reacting antigen activities with Babesia bovis remained intact. These properties were utilized in an assay system wherein antibody response to the specifically depleted antigen preparation was subtracted from the response to the initial crude preparation leaving the net B. bigemina response. The radioimmunoassay based on this antigen system was verified using sera from known negative cattle and from cattle previously infected with B. bigemina, B. bovis or Anaplasma marginale. The following discrimination values were obtained: B. bigemina-positive sera less than 2% false negatives; negative sera, 2% false positives; B. bovis-positive sera, 4% false positives; A. marginale-positive sera, 0% false positives. Levels of cross-reactivity in the false positive results were in the "suspect" rather than positive class and in the case of B. bovis-positive sera, may have been due to non-specific antibodies induced by blood inoculation. In animals naturally infected with B. bovis only, there were no false positive reactions. B. bigemina antibodies were readily detectable in field sera for at least 10 months post-infection following infection by the cattle tick Boophilus microplus. This assay overcomes the problems of currently used tests for B. bigemina infection as it is both sensitive and specific and is able to discriminate between both field and laboratory infections of B. bigemina and B. bovis.  相似文献   

2.
An ammonium sulfate-soluble fraction of Taenia hydatigena cyst fluid (ThFAS) was further evaluated for use in the immunodiagnosis of cysticercosis. Analysis of ThFAS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein immunoblot analysis confirmed earlier reports of a highly specific, low molecular weight antigen in this preparation; in contrast, other components of ThFAS were shown to react nonspecifically. Antibodies against the less than 12-kD diagnostic antigen were detected in sera from 10 cattle and 4 swine inoculated with metacestodes of T saginata and T solium, respectively, but not in animals inoculated with Fasciola hepatica, Trichinella spiralis, Brucella abortus, or Toxoplasma gondii, or in noninoculated controls. Isolation and immobilization of the less than 12-kD antigen on a hydrophobic transfer membrane resulted in development of an unambiguous dipstick assay capable of correctly identifying fully developed (10-week) experimentally induced infections in cattle and swine. In addition, the dipstick assay was highly specific for diagnosis of the disease in human beings, and offers the potential of distinguishing between human clinical cases of cysticercosis and taeniasis. A similar reactive antigen of diagnostic potential was also identified and isolated from T crassiceps and T taeniaeformis cyst fluids.  相似文献   

3.
A reliable bovine viral diarrhea (BVD) viral antigen was prepared from BVD virus grown on Madin Darby bovine kidney (MDBK) cells by solubilizing the virus with detergent MEGA-10 (decanoyl-N-methylglucamide) followed by removal of hydrophobic proteins with Triton X-100 treatment. By these treatments, problems of high background associated with BVD viral antigen in the enzyme-linked immunosorbent assay (ELISA) were eliminated. With this new antigen, an ELISA was adapted to detect bovine serum antibody against BVD virus. The diagnostic specificity of the assay in 403 bovine sera collected from a BVD virus-free herd was 100%; in 296 bovine sera with serum neutralizing antibody titers of greater than or equal to 1:2, 289 sera were ELISA positive (relative sensitivity of 97.6%), two sera gave false negative reactions (0.7%) and five sera gave suspicious reactions (1.7%). These interpretations were based on positive/negative (P/N) ratio readings, i.e. a P/N ratio of less than 1.50, 1.50-1.99 and greater than or equal to 2.00 were interpreted as negative, suspicious and positive reactions, respectively. The ELISA results gave excellent agreement with serum neutralization in detecting both seropositive and seronegative animals (Kappa = 0.994). The ELISA assay was considered to be technically superior to the serum neutralization test for the routine detection of BVD viral antibody in bovine sera.  相似文献   

4.
An antigenic fraction (ThFAS) isolated from Taenia hydatigena metacestode cyst fluid was used in an ELISA to detect antibodies to T saginata in experimentally and naturally infected cattle. In 10 calves given 1,000 to 100,000 T saginata eggs (20% to 60% viability), IgG and IgM antibodies were detected in all the calves by post-inoculation week 3. Immunoglobulin G antibody values remained increased until calves were slaughtered at post-inoculation weeks 13 to 26. Six naturally infected calves (determined by postmortem examination) were considered positive, using the ELISA. Shared antigens were demonstrated between ThFAS and T saginata and T crassiceps; there were no shared antigens between ThFAS and Haemonchus contortus or Fasciola hepatica. Specific lectin binding to ThFAS indicated the presence of glycoconjugates. Immunoblot analysis indicated that a low molecular weight polypeptide (10,000 Mr) bears the immunodiagnostic antigen.  相似文献   

5.
The serological responses of dogs to cell wall and internal antigens of B. canis were studied in experimentally infected specific-pathogen-free (SPF) Beagles. Sera from infected and false positive field dogs also were examined. Cell wall antigens were extracted from B. canis by two procedures that employed either hot phosphate buffered saline (PBS) or sodium desoxycholate (SDC). Agar gel immunodiffusion (AGID) tests employing sera from experimentally infected SPF dogs were used to evaluate antigenic extracts. Extraction with PBS yielded two antigens; SDC extracted an antigen complex and sonication of PBS extracted cells liberated four internal antigens.Sera from field dogs that were negative for B. canis infection in repeated tests often had heterospecific antibodies. Such cross-reactive sere commonly gave “spur” (partial fusion) reactions with a positive reference serum when tested against the SDC cell wall antigen. In addition, false positive dogs did not have antibody to one of the cell wall antigens or to the internal antigens. In contrast, sera from infected field dogs commonly gave “identity” (fusion) reactions in the AGID test with two antigens in the SDC extract, and produced precipitin lines to one to four internal antigens.Examination of a library of sera obtained from experimentally infected SPF dogs over a period spanning 412 years revealed that none of the serodiagnostic tests employed (tube agglutination, slide agglutination, AGID) was accurate during the inital 12 weeks of infection; hemocultures were the most sensitive during this period. Tube and slide agglutination tests were initially sensitive, but they showed a lack of sensitivity and specificity after the bacteremic period ceased, as well as in their failure to exclude false positive reactions in field animals. Immunodiffusion tests that employed SDC or PBS extracts of B. canis cell walls were sensitive and accurate in identifying most infected dogs. After the bacteremia had ceased, however, AGID tests that employed cell wall antigens gave equivocal results. Immunodiffusion tests that employed sonicated (internal) antigens were sensitive shortly after the onset of bacteremia, and they had the advantage of detecting infected animals for at least 6 months following the cessation of bacteremia, a time when other serological tests gave equivocal results.  相似文献   

6.
Enzyme linked immunosorbent assay (ELISA) using a fraction of larval Taenia hydatigena cyst fluid antigen was carried out on 469 bovine sera collected at slaughter from feedlot cattle for the presence of anticysticercosis antibodies. Cysticerci, in low numbers, were found in the heart, tongue and/or masseter muscles of 84 of the 469 cattle at postmortem inspection. Only nine sera gave positive ELISA reactions and in only one of these nine animals were cysticerci found. Within the limitations of this study, the high rate of false negative and false positive reactions suggests that the ELISA with the antigen used is not a satisfactory procedure to diagnose cysticercosis in cattle, at least in animals with light infections.  相似文献   

7.
An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies in bovine sera against Parafilaria bovicola nematodes was developed and its sensitivity was compared with the immunodiffusion (ID) method. An exoantigen of P. bovicola which was shown to contain four major polypeptides was used in these procedures. The serological reactivity of the antigen polypeptides was defined by using the enzyme-linked immunoelectrotransfer blot technique (EITB) and whole-worm extract proteins. It identified only four serologically reactive polypeptides with sera from one experimentally infected calf and a verified field case. These two positive sera reacted mainly with four major antigens which coincided in molecular weights of the polypeptides of the exoantigenic preparation, namely, 43, 39, 28 and 25 KDa. Calves experimentally infected with P. bovicola showed a positive reaction with ELISA at 4 months after inoculation, and after this period a rapid increase in serum antibody response occurred. In these cases the ID reaction was observed for the first time at 7 months after inoculation. The specificity of an ELISA method using crude exoantigen preparation of P. bovicola was tested for the diagnosis of bovine parafilariasis. No cross-reactivity was detected when the P. bovicola exoantigen preparation was tested against sera from calves experimentally infected with Onchocerca lienalis, as well as against the sera from cattle naturally infected with Dictyocaulus viviparus or from cattle chronically infected with Ostertagia ostertagi. In addition, testing of 740 field sera from cattle in areas non-endemic and endemic for P. bovicola indicated a specificity of the antigen preparation used. Forty sera from laboratory-confirmed field cases of P. bovicola infection were tested by ELISA and immunodiffusion. All of these sera were ELISA positive, whereas only 70% of these were positive in the ID test. Seven (2.1%) of 328 sera from 21 herds from non-endemic P. bovicola areas were ELISA positive, as opposed to none in the ID test. Of the 94 sera from six herds in areas endemic for P. bovicola infection, 51 (54%) were ELISA positive whereas only 24 (26%) were positive in the ID test. When 56 slaughtered cattle, with varying degrees of meat condemnations due to parafilariasis, were tested for P. bovicola specific antibody, 91% of the serum samples were positive by ELISA. These results suggest that the exoantigen of P. bovicola can be used in a sensitive and reliable serological detection of parafilariasis by ELISA.  相似文献   

8.
An agglutination test with the use of formalin-killed antigen of the cell carrying the capsule was developed and used for the detection of antibody in swine naturally infected with Bordetella bronchiseptica. Under optimum antigen concentration and reaction temperature 210 or 60% of 342 serum samples tested from 42 conventional swine herds were positive for Bordetella infection. In contrast, only 34 or 10% of 342 nasal swabs from the same animals were positive for Bordetella by culture technique. The test was relatively free of cross-reactivity to related organism. However, 2.7 and 13.0% of sera from growing pigs and mature hogs, respectively, reacted with antigen of Pasteurella multocida. Because of this, only agglutinin reactions in 1:20 dilutions or higher to Bordetella were considered positive. The bulk of the antibody activity of selected sera tested from various age ranges of swine was mercaptoethanol sensitive, suggesting that serum antibody in Bordetella infection may be associated with immunoglobulin IgM. Because of the high agglutinability and stability of formalin-killed antigen the test may be useful as an auxiliary aid for the diagnosis of Bordetella infection where the organism cannot be identified by culture means.  相似文献   

9.
Serodiagnosis of bovine besnoitiosis by ELISA and immunofluorescence tests   总被引：1，自引：0，他引：1  
Sera from non-infected cattle and cattle infected with Anaplasma, Babesia, Theileria and Sarcocystis were tested for antibodies to Besnoitia in ELISA and immunofluorescence tests (IFT) with Besnoitia besnoiti of blue wildebeest origin as antigen. Only 2 out of 86 sera gave false positive reactions in ELISA and none in the IFT, indicating a high specificity for the tests. Three-hundred-and-three bovine sera from 3 farms in an area endemic for besnoitiosis were similarly tested and the results were correlated with clinical findings based on visual inspection for typical symptoms and the presence of cysts in the scleral conjunctiva. Most of the positive tests were observed in cattle older than 1 year. Of the cases with scleral cysts, 68,7% were positive in the ELISA and 81,74% in the IFT. However, 45,74% (ELISA) and 49,47% (IFT) of the clinically negative cattle were clinically positive, indicating a high incidence of clinically inapparent infection. These results indicate a relatively low sensitivity for these serological tests. An unexpected finding was that the ELISA remained negative for at least 60 days after experimental infection of the cattle, the maximum period for which tests were done, whereas the IFT became positive. No antibodies against B. besnoiti could be found in human sera. Besnoitia jellisoni antigen gave positive results with B. besnoiti antibodies in ELISA, but not in the IFT.  相似文献   

10.
The development of an enzyme-linked immunosorbent assay (ELISA) for the detection of Babesia ovis antibodies is described. In an initial study, a crude Babesia bovis antigen and a synthetic B. bovis-derived antigen (designated 11C5) were used to screen 46 B. ovis-positive and 55 negative sheep sera. A 95% correlation between the two antigenic preparations was found with the positive sera; no negative sera gave positive reactions. The synthetic antigen was then used in the screening of 1466 sera collected from sheep from 18 regions of Turkey. A high incidence of B. ovis-positive reactions was found from all regions (60-80%) in sheep over 1 year old, while from two smaller samples the incidence in young sheep was much less (28 and 52%). This test is superior to existing ones because the synthetic antigen can be produced in a highly reproducible state, is specific and is stable over extended periods of time.  相似文献   

11.
A reliable method was developed to produce a viral antigen preparation from porcine reproductive and respiratory syndrome virus (PRRSV) infected MARC-145 cells by solubilizing the virus with Triton X-100. This method eliminated problems previously encountered with high background reactions associated with PRRSV antigen or cell control antigen. With this new antigen, an indirect enzyme-linked immunosorbent assay (ELISA) was adapted to detect swine serum anti-body against PRRSV. In the ELISA, non-specific reactions associated with test serum samples have been eliminated by utilizing an effective blocking serum diluent. The ELISA is more sensitive than an indirect immunofluorescent assay (IFA), particularly with late-infection sera, while maintaining a high diagnostic specificity. In a comparison of IFA and ELISA using sera collected from 250 pigs of various ages from 5 herds that had PRRS histories, IFA revealed 178 positive samples and 72 negative samples. All of the IFA-positive sera were proven to be ELISA reactors. However, nearly one-half (34/72) of the IFA-negative samples were also ELISA reactors. The diagnostic specificity and sensitivity of the ELISA were 100% and 96.6% with 257 serum samples collected from known healthy PRRS-negative swine herds and 57 sera collected from infected swine at 6 to 56 days after infection, respectively. The ELISA is technically superior to IFA, time-efficient and cost-effective, and suitable for testing of a large number of samples over a short period of time.  相似文献   

12.
Serodiagnosis of Taenia solium cysticercosis in pigs was conducted by an enzyme-linked immunosorbent assay with an excretory-secretory antigen. The antigen was obtained by in vitro cultivation of the cysticerci in a synthetic medium RPMI 1640. The sensitivity and specificity of the ELISA in detecting infection in pigs reared on free range was 92% and 100%, respectively. In addition, 33.33% of pigs in which infection could not be detected at meat inspection were found positive by ELISA. However, none of the sera from a group of farm-reared pigs were positive. No cross reactions were observed in pigs that contained either the cysticerci of Taenia hydatigena or hydatid cysts.  相似文献   

13.
In this study, improvements were made in the technique and the preparation of the antigen. It was possible to perform three extractions and elutions resulting in a soluble reactive preparation from each batch of infected mouse brain. This led to an appreciable increase in the yield of highly reactive antigen. The presence of bluetongue antibodies was not detected in 13,210 sheep sera. Of the 13,486 bovine sera tested, only three questionable reactions were obtained. It was possible to determine that two of these animals were imported. Various isolation methods, including transmission trials to susceptible sheep followed by serological tests on the sheep sera, failed to confirm the infection in the three reactors.  相似文献   

14.
Groups of hog sera from endemic and non-endemic areas for swine trichinellosis in Yugoslavia were tested by ELISA using excretory-secretory (ES) antigens collected from T. spiralis muscle larvae maintained in vitro for 24, 48 or 72 h. The 24-h ES had the highest level of specificity for T. spiralis infection. Antigen preparations recovered after 48 or 72 h yielded an increasing rate of false-positive reactions. Additional antigens occurred in the 48- and 72-h ES preparations as determined by gel electrophoresis and monoclonal antibody binding. The occurrence of false-negative reactions was directly correlated with T. spiralis worm burdens. Hogs with muscle larvae densities greater than 10 larvae per gram were all positive by ELISA. Among 17 hogs with less than 10 larvae per gram, only one hog was negative by ELISA with 24-h ES antigen; the false-negative rate was higher with 48- and 72-h ES. These results show that ES antigen produced during the first 24 h of in vitro cultivation is highly specific for the immunodiagnosis of swine trichinellosis.  相似文献   

15.
An ELISA using a Trichinella spiralis spiralis excretory-secretory antigen was evaluated as a procedure for the diagnosis of trichinosis in swine in Canada. Field and experimental trials were carried out using both indirect serological (ELISA) and direct parasitological (pepsin-digestion) methods concurrently on serum and musculature, respectively, from each animal. The ELISA is a sensitive and specific test for the detection of Trichinella antibodies in porcine sera when present. The development of Trichinella antibodies appears to be dependent on the magnitude of the infection established, age of the infection when the animal is tested and the immunocompetence or response to infection of individual animals. False negative reactions were recorded in both field and experimental trials. In the field study, five of the 1009 swine examined were parasitologically positive with light infections ranging from 0.01 to 0.046 larvae per gram (la/g) of musculature yet all were serologically negative. Experimentally it was shown that Trichinella antibodies develop slowly, at least two to three months postinfection, in pigs with very light infections. Even in pigs which developed infections of 33 to 55 la/g of musculature, seroconversion occurred greater than 23 and less than 30 days postinfection. The immunocompetence or response to infection of individual pigs was variable as illustrated by one pig inoculated with 3000 infective larvae which had consistently lower titers compared to others in the same group despite the establishment of a muscle infection of 8.5 la/g of musculature. One false positive reaction was recorded in the experimental trial in an animal which had received 100 larvae and seroconverted at about three months postinfection.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

16.
Fifty six dogs of mixed age and sex were acquired from farms in the Otago/Southland region, and maintained at the Hydatid Research Unit, Taieri, where 43 were each fed two Taenia ovis cysts. All were bled fortnightly for six or 12 weeks. Coded sera were sent to Wallaceville Animal Research Centre for testing using ELISA, with antigen from T. ovis scoleces. Dog treatments were identified after all tests were complete. A discriminant level was derived from the mean absorbance value plus three standard deviations of 56 sera taken at time zero and 78 sera from serially bled uninfected dogs. None of these 134 sera registered as a false positive using this discriminant level. The data showed no significant deviation from normality, and the expected frequency of the occurrence of false positives is therefore less than 0.14%. Four weeks after infection 63% of dogs proved to be infected were serologically positive, rising to 78% after 6 weeks. When worms were removed by anthelmintic treatment, ELISA absorbance levels decreased. Four weeks after removal 70% of previously infected dogs remained positive, decreasing to 30% after 6 weeks.

Six weeks after infection the sensitivity of the test was 78%, and the specificity 63%. However, if dogs with positive ELISA absorbance levels, but which did not purge worms, were regarded as having had worms, the respective figures would be 82% and 100%. The latter figures are similar to our previously published laboratory results. The test is of comparable efficiency to arecoline purgation for surveillance, and has the additional advantage of detecting infection in the majority of those dogs that have been infected for three weeks or more but fail to pass worms on purgation, and a substantial proportion of those infected dogs that were treated by their owners prior to presenting them for purgation in order to avoid detection of infection.  相似文献   

17.
Due to almost identical lipopolysaccharide (LPS) O-antigens, infections with Yersinia enterocolitica serotype O:9 (YeO:9) cause false positive serological reactions (FPSR) in tests for Brucella and thus cause problems in National Brucella surveillance programs. As LPS are strong inducers of antibody responses it was hypothesized that cell-mediated immune responses to non-LPS antigens of the two bacteria can be used to separate immune responses to these two biologically very different infections. Following subclinical experimental infections with Brucella suis biovar 2, high interferon-gamma (IFN-gamma) assay responses with a commercial Brucella melitensis antigen preparation (Brucellergene OCB) preceded the development of antibodies. High IFN-gamma responses in the seven B. suis inoculated pigs with serological evidence of infection were consistent throughout a 20-week post-inoculation observation period. In contrast, IFN-gamma responses in two B. suis inoculated pigs without bacteriological or serological evidence of infection were below a cut-point of 25pg/ml at all samplings. IFN-gamma responses in repeated samplings from 5 uninfected control pigs and 18 pigs experimentally infected with YeO:9 were all negative, except for solitary false positives in 3.7% of the samples from both the experimentally YeO:9 infected pigs and control pigs. Skin tests using the same commercial Brucella antigen confirmed the ability of cell-mediated immune responses to differentiate between the two infections. In addition, a field evaluation of the diagnostic use of cell-mediated immune responses by IFN-gamma assay and skin test to resolve serological suspicions of Brucella was conducted in an YeO:9 infected pig herd. Following a screening of 200 pigs 39 pigs were identified with false positive serological Brucellosis reactions. While 36 of the 39 FPSR pigs were also FPSR in a second test, none of the pigs were test positive in whole blood IFN-gamma assay or Brucellergene OCB skin test. In conclusion, use of IFN-gamma assay and skin test as measurements of cell-mediated immune responses to non-LPS Brucella antigens were specific and sensitive in discriminating subclinical experimental infections with B. suis from both natural and experimental infections with YeO:9.  相似文献   

18.
An antigen was prepared from purified sheep hydatid-cyst fluid by gelfiltration on Sephadex G-200 followed by chromatography on DEAE-cellulose. In each case the first-peak material was used. This antigen, which migrated cathodically, was concentrated and used in immunoelectrophoretic analyses of 4X concentrated sera from sheep experimentally and naturally infected with Echinococcus granulosus and from uninfected sheep. Of 34 sheep with E. granulosus infection 31 were positive with the cathodic antigen while of 85 sheep without E. granulosus 8 were (falsely) positive. Many false positives appeared to be associated with heavy infections of Taenia ovis or T. hydatigena larvae. The “arc 5” immunoelectrophoresis test, which is the most specific immuno-diagnostic test for echinococcus infection in humans, was not able to specifically identify E. granulosus infections in sheep.  相似文献   

19.
A rapid, inexpensive and rugged serological test that distinguishes cattle and swine infected with Brucella sp. or Yersinia enterocolitica O:9 is described. The test protocol, which is an indirect enzyme immunoassay uses a high concentration of divalent cation chelating agents to minimize binding of Y. enterocolitica O:9 antibody to rough lipopolysaccharide antigen derived from B. abortus RB51. No false positive reactions were observed when testing 100 Canadian cattle and swine without any evidence of brucellosis. The assay detected 91.6% of cattle (n=155) and 93.5% (n=31) of swine infected with Brucella sp. Sera from 58 cattle and 38 swine exposed to Y. enterocolitica O:9 were negative while only 20 sera from 121 'false positive' reactors of unspecified origin gave low level positive reactions, eliminating 84% of the false positive reactions.  相似文献   

20.
When preparations containing smooth Brucella abortus lipopolysaccharide (LPS) were used as antigens in an ELISA, strong positive reactions were obtained with sera from sheep infected with Brucella melitensis or with Brucella ovis. Oxidation of the LPS with sodium metaperiodate greatly reduced the extent of the cross-reactions with antisera to B. ovis, with little effect on the reactions with antisera to smooth B. melitensis. Periodate oxidation of hot saline extract (HSX) antigen of B. ovis markedly reduced its reactivity in ELISA with anti-B. ovis sera and eliminated cross-reactivity with anti-B. melitensis sera. The reactivity of HSX was maintained after treatment with proteinase K.

A simple ELISA system, in which replicate samples from a single serum dilution were tested in parallel against both B. ovis HSX antigen and periodate-oxidised smooth phase B. abortus LPS, was evaluated. It was found to discriminate well between antibodies induced by vaccination or virulent infection with B. melitensis strains and those induced by infection with B. ovis.  相似文献   


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