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1.
Two commercial enzyme-linked immunosorbent assays (ELISA), the CHEKIT-CHLAMYDIA which uses inactivated Chlamydophila psittaci antigen, and the Chlamydophila abortus ELISA produced by the Institut Pourquier which uses a recombinant fragment of the 80-90 kDa protein, were evaluated with the objective to determine whether the new ELISAs would perform as improved alternatives to the complement fixation test (CFT) for the serological diagnosis of ovine enzootic abortion (OEA). The results were compared to those obtained by the CFT and the competitive ELISA (cELISA). The tests were assessed with a panel of 17 serum samples from specific pathogen-free (SPF) lambs experimentally infected with various subtypes of Chlamydophila pecorum, with sera from 45 C. abortus-infected pregnant sheep and from 54 sheep free of OEA. The C. abortus ELISA was identified as being more specific and sensitive than the other tests. The 4 assays were evaluated further with 254 sera from flocks with documented OEA, from flocks with no history of abortion and from animals after abortion of unknown cause. The C. abortus ELISA by the Institut Pourquier identified less OEA-positive sera than the other assays though it identified correctly 9 of 10 OEA-positive flocks. The basis of the discordant results is discussed.  相似文献   

2.
A new commercially available ELISA (ELISAr-Chlamydia) for detecting antibodies against Chlamydophila abortus has been evaluated using sheep field serum samples. The ELISA is based on a recombinant antigen which expresses part of a protein from the 80-90kDa family that is specific to C. abortus. Sera (105) from six flocks with confirmed ovine chlamydial abortion (OEA) outbreaks were used in this study, as well as sera (258) from 18 flocks which had suffered no OEA in the last lambing. The ELISAr-Chlamydia was compared with the complement fixation test (CFT) and with an ELISA using purified C. abortus elementary bodies (ELISA-EB), employing as reference technique a comparative microimmunofluorescence test that differentiates C. abortus infection from Chlamydophila pecorum infection. The results showed that the sensitivity of ELISAr-Chlamydia was 90.9% with a specificity of 85.9%, the sensitivity of CFT was 71.0% with a specificity of 83.6%, while the sensitivity of ELISA-EB was 95.2% and the specificity was 54.2%. Furthermore, ELISAr-Chlamydia was the test with fewer false positives resulting from positive reactivity to C. pecorum, although 15% of the sera positive for C. pecorum but negative for C. abortus antibodies reacted positively. This study demonstrated with field material that ELISAr-Chlamydia provides the most balanced results between sensitivity and specificity, especially in flocks with no clinical OEA but reactivity to C. abortus.  相似文献   

3.
Chlamydial infections in small ruminants.   总被引：1，自引：0，他引：1  
Chlamydophila abortus (formerly Chlamydia psittaci) is one of the most important causes of reproductive failure in sheep and goats, especially in intensively managed flocks. The disease is usually manifested as abortion in the last 2 to 3 weeks of gestation, regardless of when the animal was infected. Ewes that abort are resistant to future reproductive failure due to C. abortus, but they become inapparent carriers and persistently shed the organism from their reproductive tracts during estrus. Chlamydophila pecorum is the other member of the genus that affects small ruminants, and it is recognized as a primary cause of keratoconjunctivitis in sheep and goats and of polyarthritis in sheep.  相似文献   

4.
Recombinant major outer membrane proteins (rMOMP) of Chlamydophila (Ch.) abortus, Ch. pecorum, and Chlamydia (C.) suis were used as antigens to distinguish chlamydial species-specific antibodies in (i) immune sera from six rabbits and three pigs raised against native purified elementary bodies, (ii) serum samples from 25 sows vaccinated with Ch. abortus, and (iii) 40 serum samples from four heifers experimentally infected with Ch. abortus. All post-exposition sera contained chlamydial antibodies as confirmed by strong ELISA seroreactivities against the chlamydial LPS. For the rMOMP ELISA mean IgG antibody levels were at least 5.8-fold higher with the particular rMOMP homologous to the chlamydial species used for immunisation or infection than with heterologous rMOMPs (P <0.001). Preferential rMOMP ELISA reactivities of sera were confirmed by Western blotting. The results suggest that the entire chlamydial rMOMP could provide a species-specific serodiagnostic antigen.  相似文献   

5.
Bacteria of the family Chlamydiaceae are obligate intracellular pathogens of human and animals. Chlamydophila pecorum is associated with different pathological conditions in ruminants, swine and koala. To characterize a coding tandem repeat (CTR) identified at the 3' end of incA gene of C. pecorum, 51 strains of different chlamydial species were examined. The CTR were observed in 18 of 18 tested C. pecorum isolates including symptomatic and asymptomatic animals from diverse geographical origins. The CTR were also found in two strains of C. abortus respectively isolated from faeces from a healthy ewe and from a goat belonging to asymptomatic herds, but were absent in C. abortus strains isolated from clinical disease specimens, and in tested strains of C. psittaci, C. caviae, C. felis and C. trachomatis. The number of CTR repeats is variable and encode several motifs that are rich in alanine and proline. The CTR-derived variable structure of incA, which encode the Chlamydiaceae-specific type III secreted inclusion membrane protein, IncA, may be involved in the adaptation of C. pecorum to its environment by allowing it to persist in the host cell.  相似文献   

6.
OBJECTIVE: To compare the sensitivity and specificity of Chlamydophila abortus antibody assays, to find a suitable serological assay for testing sheep for export. DESIGN: Comparison of results from known positive and negative sheep populations. PROCEDURE: Fifty-five positive and fifty negative sera were analysed by four enzyme linked immunosorbent assays (ELISA), three using recombinant antigens based on the chlamydial polymorphic outer membrane proteins (POMP90-3, POMP90-4, POMP80-90) and one using a synthetic peptide based on chlamydial major outer membrane proteins (MOMP-P). They were also analysed by complement fixation tests (CFT) using crude antigens from chlamydia isolated from an Australian sheep, a Californian parakeet and a Texan turkey. Assay sensitivity and specificity were expressed as point estimates and 95% confidence intervals. Results were compared using McNemar's test for paired samples. RESULTS: ELISA sensitivity ranged from 70 to 98% and complement fixation test sensitivity from 60 to 96%; with POMP90-3 > POMP90-4 > CFT (parakeet) > CFT (turkey) > POMP80-90 > MOMP-P > CFT (sheep). There was no significant difference from POMP90-3 to POMP80-90 (P > 0.05). ELISA specificity ranged from 88 to 100% and CFT specificity was 100% for all three antigens; with CFT and POMP90-4 > MOMP-P > POMP80-90 > POMP90-3. There was no significant difference from CFT to POMP80-90 (P > 0.05). Changing the CFT cut-off from 1:32 to 1:4 substantially reduced the specificity with little improvement in sensitivity. CONCLUSION: Assays using POMP90-4, POMP80-90, CFT (parakeet) and CFT (turkey) had equivalent sensitivity and specificity; none of the ELISAs were more specific than any CFT. The POMP80-90 ELISA is recommended as an alternative to CFT (parakeet) but as its specificity is not ideal the search for a more specific assay should continue.  相似文献   

7.
The present study gives an overview over the seroprevalence of ovine enzootic abortion in Switzerland. 639 sheep flocks out of eight cantons in Switzerland were examined by a competitive enzyme linked immunosorbent assay (cELISA) for antibodies against Chlamydophila abortus (Chlamydia psittaci serotype 1), the agent causing ovine enzootic abortion. The eight cantons included Aargau, Bern, Zürich, Appenzell-Ausserrhoden, Appenzell-Innerrhoden and Fribourg, the Vallais and the Graubünden. They were representative for 57% of the Swiss sheep flocks and for 60% of Swiss sheep population. In total, almost 19% (118) of the examined flocks were seropositive. Seroprevalence was the highest in Graubünden with 41%; this requires further examination and the evaluation of the need for a monitoring and controlling program. The examination of pooled sera made it possible to test a large number of samples with a reasonable amount of work. Higher sensitivity (92.9%) and specificity (97.6%) than the complement fixation test (CFT) in combination with testing of pooled sera makes the cELISA to be an usuable tool for serological screening on flock level.  相似文献   

8.
Objective   To examine the incidence of positive results in a complement fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA) for Chlamydophila abortus in Australian sheep and how this incidence differs with state of origin, age, sex, breed and property. To examine the consequences in relation to rejection of breeder sheep for export.
Design   Collection of blood samples from 891 sheep on 109 properties in southern Australia. All samples had a unique, coded property identification.
Procedure   The samples were tested using the Institut Pourquier Chlamydophila abortus antibody ELISA (rELISA) and a CFT. Residual maximum likelihood analyses of the sample to positive ratio of the corrected optical density for the rELISA and generalised linear mixed model analyses of the CFT outcomes were carried out.
Results   The sample to positive ratio of the corrected optical density values of the rELISA did not differ between sex, age, breed or state of origin, but differed greatly between properties. The CFT outcome did not differ between age, breed or state of origin, but differed greatly between properties and was more often positive with rams than with ewes.
Conclusion   Positive outcomes to C. abortus antibody tests are very common in Australia. Rams have a particularly high incidence of positive results with the CFT. Rejection of sheep and property consignments is likely to be very common with all tests and situations examined except for the CFT (at 1:32 dilution) in ewes.  相似文献   

9.
Chlamydophila (C.) abortus, responsible for chlamydial abortion (commonly known as Enzootic Abortion of Ewes [EAE]), causes major financial losses to the sheep industry worldwide. There remain many uncertainties surrounding the epidemiology of EAE. The aim of this study was to construct an epidemiological model to simulate EAE based on current knowledge of the disease, and in doing so, identify knowledge gaps that need to be addressed through further research. Key parameters that impact upon the development of the disease, such as the rate of contact between na?ve ewes and infected material, are defined. Sensitivity analysis was undertaken for parameter values that are unknown to explore their impact upon the pattern of disease. The simulated results show the importance of the transmission rate (i.e. contact) and the number of infected replacements introduced at the start of an outbreak. Depending upon the rate of transmission, the year in which the peak number of affected ewes occurs and the number of years over which a high number of animals are affected varies. This suggests that a better understanding of the underlying processes that drive transmission of C. abortus is needed. Furthermore, if infected ewes could be identified prior to parturition, when they shed the organism in large numbers, the impact of EAE on sheep flocks could be greatly reduced.  相似文献   

10.
The efficacy of Chlamydophila abortus vaccine strain 1B in protecting against two selected Chlamydophila pecorum strains, isolated from an aborted goat (M14) in Morocco and a ewe (AB10) in France, was investigated in a mouse model, by comparing the reduction in number of bacteria in the placentas of vaccinated mice challenged intraperitoneally at 11 days of pregnancy with the reference C. abortus (AB7) and C. pecorum (M14, or AB10) strains, to those of unvaccinated mice. Vaccine 1B was shown to provide effective protection against the field strains of C. pecorum, since it significantly reduced the placental Chlamydophila colonisation. The two C. pecorum strains were not sufficiently abortifacient in mice to use reduction in abortion as a criterion of protection.  相似文献   

11.
The Dutch national Brucella abortus eradication programme for cattle started in 1959. Sporadic cases occurred yearly until 1995; the last infected herd was culled in 1996. In August 1999 the Netherlands was declared officially free of bovine brucellosis by the European Union. Before 1999, the programme to monitor the official Brucella-free status of bovine herds was primarily based on periodical testing of dairy herds with the milk ring test (MRT) and serological testing of all animals older than 1 year of age from non-dairy herds, using the micro-agglutination test (MAT) as screening test. In addition, serum samples of cattle that aborted were tested with the MAT. The high number of false positive reactions in both tests and the serum agglutination test (SAT) and complement fixation test (CFT) used for confirmation seemed to result in unnecessary blockade of herds, subsequent testing and slaughter of animals. For this reason, a validation study was performed in which three indirect enzyme-linked immunosorbent assays (ELISAs), the CFT and the SAT were compared using a panel of sera from brucellosis-free cattle, sera from experimentally infected cattle, and sera from cattle experimentally infected with bacteria which are known to induce cross-reactive antibodies (Pasteurella, Salmonella, Yersinia, and Escherichia). Moreover, four ELISAs and the MRT were compared using a panel of 1000 bulk milk samples from Brucella-free herds and 12 milk samples from Brucella abortus- infected cattle. It is concluded that the ELISA obtained from ID-Lelystad is the most suitable test to monitor the brucelosis free status of herds because it gives rise to fewer false-positive reactions than the SAT.  相似文献   

12.
AIM: To determine the performance characteristics of two enzyme-linked immunosorbent assays (ELISAs) manufactured by Institut Pourquier (IP) for the detection of antibodies against Neospora caninum in bovine sera. METHODS: Sera from 526 cattle were assayed in two ELISAs (IP) for the detection of anti-N. caninum antibodies. Results from a further ELISA (IDEXX) were used to provide the "gold standard"N. caninum infection status of the cattle and the ELISA results assessed by two-graph receiver operating characteristic (TG-ROC) analysis. RESULTS: TG-ROC analysis suggested changes to one of the IP ELISA protocols, arriving at a cut-off threshold that was different to the one recommended by the manufacturer. With that change, both of the ELISAs performed with high sensitivity and specificity (in excess of 98%) for bovine sera. CONCLUSIONS: The analysis of the two IP ELISAs when used on individual bovine sera demonstrated high sensitivity and specificity. TG-ROC analyses optimised the cut-off point suggested by the manufacturer for one of these commercial diagnostic assays and found agreement with the manufacturer's cut-off regarding the other assay. This will help with the accurate identification of infected animals and thereby contributing to the control of neosporosis.  相似文献   

13.

Background

Reports worldwide indicate high prevalence of Chlamydophila spp. infection in cattle. To assess the prevalence in Sweden, 525 cows in 70 dairy herds with reproductive disorders was investigated.

Methods

To detect antibodies two commercially available kits were used. Moreover, 107 specimens, including vaginal swabs, organ tissues and milk were analysed by Polymerase Chain Reaction (PCR).

Results

Two (0.4%) cows were seropositive in the Pourquier Cp. abortus ELISA. The seroprevalence with the Chekit ELISA was 28% with no difference between cases and controls. Five specimens were positive in real-time PCR and further analysed by nested PCR. Cp. pecorum was confirmed by partial omp1 DNA sequencing of the nested PCR product of vaginal swabs from control cows.

Conclusion

The results suggest that Cp. abortus infection is absent or rare in Swedish cows whereas Cp. pecorum is probably more spread. They also suggest that Chlamydophila spp. are not related to reproduction disorders in Swedish cattle.  相似文献   

14.
An automated indirect enzyme-linked immunosorbent assay (I-ELISA) for the serological diagnosis of bovine brucellosis was developed and validated in-house. A total of 4,803 cattle sera from South Africa (n = 3,643), Canada (n = 652), Germany (n = 240), France (n = 73) and the USA (n = 195) was used. The South African panel of sera represented 834 sera known to be positive by the Rose Bengal test (RBT), serum agglutination test (SAT) and complement fixation test (CFT), 2709 sera that were negative by CFT, and 100 sera from animals vaccinated with a standard dose of Brucella abortus strain 19. Overseas sera were obtained from reference non-vaccinated brucella-free cattle (n = 834), naturally infected (n = 72), experimentally infected (n = 71), and vaccinated animals (n = 83). Also 100 sera collected from cattle in Canada and known to be positive by competitive ELISA (C-ELISA) were used. The intermediate ranges ("borderline" range for the interpretation of test results) were derived from two-graph receiver operating characteristics analysis. The lowest values of the misclassification cost-term analysis obtained from testing overseas panels, covered lower I-ELISA cut-off PP values (0.02-3.0) than those from local panels (1.5-5.0). The relatively low cut-off PP values selected for I-ELISA were due to the fact that the positive control used represents a very strong standard compared to other reference positive sera. The greater overlap found between negative and positive cattle sera from South Africa than that between reference overseas panels was probably due to the different criteria used in classifying these panels as negative (sera from true non-diseased/non-infected animals) or positive (sera from true diseased/infected animals). The diagnostic sensitivity of the I-ELISA (at the optimum cut-off value) was 100% and of the CFT 83.3%. The diagnostic specificity of I-ELISA was 99.8% and of the CFT 100%. Estimate of Youden's index was higher for the I-ELISA (0.998) than that for the CFT (0.833). Analysis of distribution of PP values in sera from vaccinated and naturally infected cattle shows that in vaccinated animals all readings were below 31 PP where in infected ones these values represented 43%. Therefore, it appears that I-ELISA could be of use in identifying some naturally infected animals (with values > 31 PP), but more sera from reference vaccinated and infected animals need to be tested to further substantiate this statistically. Of 834 sera positive by RBT, SAT and CFT, 825 (98.9%) were positive in the I-ELISA. Compared to C-ELISA the relative diagnostic sensitivity of the I-ELISA was 94% and of the CFT 88% when testing 100 Canadian cattle sera. Of 258 South African cattle sera, of which 183 (70.9 %) were positive by the I-ELISA and 148 (57.4 %) by the CFT, 197 (76.4%) were positive by C-ELISA when re-tested in Canada. One has to stress, however, that Canadian C-ELISA has not been optimised locally. Thus, the C-ELISA was probably not used at the best diagnostic threshold for testing South African cattle sera. This study shows that the I-ELISA performed on an automated ELISA workstation provides a rapid, simple, highly sensitive and specific diagnostic system for large-scale detection of antibodies against B. abortus. Based on the diagnostic accuracy of this assay reported here, the authors suggest that it could replace not only the currently used confirmatory CFT test, but also the two in-use screening tests, namely the RBT and SAT.  相似文献   

15.
AIM: To determine the aetiology of a recurring and severe form of infectious keratoconjunctivitis (IKC) in sheep. METHODS: Five sheep flocks that had experienced a severe form of IKC were examined. Clinical history, conjunctival swabs and blood samples were collected from affected animals. Culture for bacteria, and also specifically for Mycoplasma and Chlamydophila spp, and detection of Mycoplasma conjunctivae DNA by polymerase chain reaction (PCR) were attempted. Serum samples were tested for antibodies to M. agalactiae, M. capricolum, M. conjunctivae and Chlamydophila spp. RESULTS: Mycoplasma conjunctivae DNA was detected using PCR in 3/5 flocks, and in all flocks antibodies to M. conjunctivae were detected in sera. A pure growth of Branhamella ovis was cultured from conjunctival swabs from a small proportion of sheep in two flocks. No other pathogens were detected. CONCLUSIONS: This investigation demonstrated that M. conjunctivae was a primary pathogen causing severe IKC in sheep, and is the first report of detection of this organism in sheep in New Zealand. Introduction of clinically normal carrier sheep appeared to have caused the outbreaks. KEYWORDS: Infectious keratoconjunctivitis, Mycoplasma conjunctivae, Chlamydophila pecorum, Branhamella ovis, polymerase chain reaction, ELISA, complement fixation test.  相似文献   

16.
A dot-immunobinding assay (DIA), using as antigen a sonic extract of Brucella abortus dotted on nitrocellulose bound to a plastic strip, was employed for the detection of Brucella antibodies in 666 sheep sera. The results were compared with the complement fixation test (CFT). All the 242 sera belonging to two flocks were found to be negative by DIA. CFT was negative in 239 cases, whereas three samples showed anti-complementary activity. Of the 424 sera from the remaining three flocks, 98 were positive by both tests and six were positive in DIA, but negative in CFT. In addition, 14 of the 19 anti-complementary sera were also positive by DIA.  相似文献   

17.
Two monoclonal antibodies (MAbs) conjugated with horseradish peroxidase were used independently in a competitive enzyme immunoassay (cEIA) to detect Brucella specific antibodies in 1120 sera from Brucella-free cattle, 61 from cattle known to be infected with B. abortus, and 207 sera from vaccinated calves. The results were compared to those obtained in the complement fixation test (CFT). The cEIA with both MAbs proved to be more sensitive than the CFT because no false-negative results were obtained. In addition, discrimination between sera from infected and vaccinated animals was more evident.  相似文献   

18.
Chlamydophila abortus-DNA was detected using a touchdown enzyme time-release (TETR)-polymerase chain reaction (PCR) assay as an improved test for sensitive and rapid diagnosis of abortion in small ruminants. Two hundred and fifty two placentae, liver or spleen tissue samples from aborting ewes and goats or aborted lambs and kids in which C. abortus infection was suspected were examined by TETR-PCR and the results were compared with cell culture. Sixty-five tissue samples were found to be TETR-PCR positive while only 56 samples were cell culture-positive. After resolution of discrepant samples with a confirmatory nested PCR assay, TETR-PCR had a sensitivity of 97% and a specificity of 99.5% while culture had a sensitivity of 84.8% and a specificity of 100%. The analytical sensitivity of the TETR-PCR assay was determined with DNA extracted from 4-fold serial dilution of C. abortus B577 culture and found to be 0.25 inclusion-forming unit per PCR. No reduction in the analytical sensitivity was noted when the assay was tested with mouse liver samples spiked with 4-fold serial dilution of C. abortus B577 culture. No target product was amplified when DNA from Chlamydophila pecorum was tested. TETR-PCR used in this study is a practical, rapid, sensitive and specific assay that could be used for the detection of C. abortus in infected tissue samples. We recommend the use of this assay as a supplemental diagnostic tool for detection of C. abortus in infected tissue samples.  相似文献   

19.
In a chronological study of sera collected from eight adult cattle vaccinated with 3 X 10(-10) cfu of Brucella abortus S19, antibody of each of the four major isotypes was measured by indirect enzyme immunoassay (ELISA) and by direct and modified complement fixation tests (CFT). Six of the cattle gave antibody responses to the vaccine strain that commenced between days 5 and 8 for all the isotypes in the ELISA, peaked by 1 to 4 months and then declined to low levels by 10 months. Direct CFT and modified CFT titers were measurable by 7 or 8 days post-vaccination, and peaked by 1 month; direct CFT titers disappeared by 5 months while the modified CFT titers lingered for 10 months. Two animals gave cyclical direct CFT and modified CFT antibody responses, a cyclical IgG1 response, a low IgG2 and an elevated IgA response. The amplitude of the cycles was uniform over three cycles while the wavelength increased with time. A year post-vaccination, B. abortus S19 was isolated twice from milk from one of the animals (no attempt was made to culture B. abortus from the other). Sera from B. abortus naturally infected cattle were analysed for comparison.  相似文献   

20.
The aim of this study was to estimate the diagnostic value of different commercially available ELISA kits for the detection of bluetongue virus (BTV) antibodies in infected and vaccinated animals. The relative specificity of ELISA kits was evaluated using a panel of sera originating from healthy cattle, never vaccinated nor exposed to BTV. All ELISA kits applied had a high relative specificity (99.3 - 100%). The relative sensitivity of ELISA kits assessed using a panel of sera collected from BTV infected cattle was also high and similar for all the kits (97.3 - 100%). However, the relative sensitivity evaluated on the basis of testing vaccinated animals was different: the highest sensitivity was found for Ingenasa, PrioCHECK and ID VET ELISAs (96.5 - 98.3%). Slightly lower sensitivity was calculated for Pourquier and LSI kits (82.8% and 85.4%, respectively) and much lower sensitivity was found for VMRD ELISA kit (69.5%). The repeatability of BTV ELISA kits was expressed as a coefficient of variation (CV) of results of sera tested 5 times in the same day and in different days by the period of 2 months, by the same person, in the same conditions, and by using the same equipment. The CVs of sera tested in all ELISA kits ranged from 6.1 to 9.8% and were below 10% threshold adopted as a maximum for the acceptable repeatability of the method. In conclusion, it can be stated that the applied ELISA kits can be a valuable diagnostic tool for the serological monitoring studies in the BTV contaminated premises. All the methods are very specific and sensitive when testing BTV infected animals. Nevertheless, the Ingenasa and PrioCHECK can be the most useful in sero-surveillance of livestock following vaccination.  相似文献   

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