Comparative evaluation of eight serological assays for diagnosing Chlamydophila abortus infection in sheep

Chlamydophila abortus is one of the principal causes of late-term abortion (enzootic abortion of ewes or EAE) in sheep across Europe. Serological diagnosis of EAE is routinely carried out by the complement fixation test, although the interpretation of results can often be difficult because of cross reaction with Chlamydophila pecorum, which also commonly infects sheep. The purpose of this study was to evaluate and compare four ELISAs developed at Moredun Research Institute and based on whole C. abortus elementary bodies (EBs), an outer membrane preparation of the whole organism (SolPr) and two recombinant polymorphic outer membrane protein fragments (rOMP90-3 and rOMP90-4), with 3 commercial tests, the CHEKIT Chlamydophila Abortus, Pourquier ELISA Chlamydophila abortus and ImmunoComb Ovine Chlamydophila Antibody tests. The tests were evaluated using a panel of 202 sera from experimentally and naturally infected animals, as well as from EAE-free flocks. The EB, SolPr and CHEKIT ELISAs performed similarly to the CFT, all lacking in specificity by cross reacting with sera from C. pecorum infected animals. The ImmunoComb also lacked specificity with C. pecorum sera, but also badly cross reacted with sera from EAE-free flocks. The rOMP90-3, rOMP90-4 and Pourquier ELISAs were the most specific, although the Pourquier test appeared less sensitive with sera from naturally infected animals. Overall, the rOMP90-3 ELISA performed the best, with high sensitivity (96.8%) and no cross reaction with sera from C. pecorum infected animals or from EAE-free flocks (100% specificity) and so would be a suitable alternative to the CFT for the serological diagnosis of EAE.     

Experimental infection of pregnant ewes with enteric and abortion-source Chlamydophila abortus

Two groups of pregnant ewes were experimentally infected oronasally in midpregnancy. A faecal and an abortion-source isolate of Chlamydophila abortus were used. They were derived from a healthy ewe from a flock with no history of abortion, and from an aborted foetus in a farm with enzootic abortion. As assessed by modified Ziehl-Neelsen (MZN) staining, egg culture, antigen ELISA, the Clearview test and immunohistochemistry, inoculation resulted in placental and/or foetal infection in all ewes. Histopathology revealed placentitis in two and four ewes inoculated with the enteric or abortion-source isolate, respectively, in addition, these samples were immunohistochemically positive for chlamydial antigen. All six ewes infected with the enteric isolate and five of seven ewes infected with the abortion-source isolate showed evidence for a serological response by an indirect ELISA or CFT. Neither chlamydiae nor lesions were detected in the placentae and lambs of the uninfected control ewes, which remained seronegative. Our results suggest that enteric C. abortus can be associated with placental and foetal lesions in sheep.     

Evaluation of two commercial assays for the detection of Chlamydophila abortus antibodies

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.     

Field evaluation of a new commercially available ELISA based on a recombinant antigen for diagnosing Chlamydophila abortus (Chlamydia psittaci serotype 1) infection

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.     

Naturally occurring lesions of the uterine tube in sheep and serologic evidence of exposure to Chlamydophila abortus.

The uterine tubes from 405 ewes, collected at an abattoir, were assessed grossly and microscopically for abnormalities that correlated with serological evidence of exposure to Chlamydophila abortus. Gross lesions were found in 41 ewes and 86 had microscopic lesions. Enzyme immunoassay (EIA) of serum was used as an indication of exposure of individual ewes to C. abortus; 52 were found to be positive. Chi-squared analysis indicated no association between EIA-positive animals and lesions of the uterine tube.     

Longitudinal studies of naturally acquired Brucella abortus infection in sheep.

Naturally acquired Brucella abortus infections were studied during consecutive pregnancies in eight sheep and in their lambs over a period of 40 months to evaluate epizootiologic aspects of natural infection in sheep. Brucella abortus was isolated from the ewes following 16 of 26 natural terminations of pregnancy: from 5 of 6 ewes in the first year, from six of eight ewes in the second year, from two of six ewes in the third year, and from three of six ewes in the fourth year. Vaginal swab samples and milk samples were the most consistent source of the brucella organisms. Brucella abortus was isolated from three ewes when standard tube test seroagglutination titers were less than 1:100. In contrast, results of supplemental tests (card, 2-mercaptoethanol, complement-fixation, and Rivanol) remained positive during the study. During the 40 months, B abortus was isolated from 4 of 4 aborted fetuses, 2 of 5 stillborn lambs, 10 of 37 living lambs, and as an indicator of continuing infection, from 6 of 12 lambs born during the fourth year. Although B abortus has a definite host preference for cattle, this study demonstrated that under appropriate management conditions, sheep may be naturally infected and may remain infected for more than 40 months. Epizootiologic evaluation of all factors, including husbandry practices and exposure potential, should be utilized in determining the need to test other species that may have been exposed to cattle infected with B abortus.     

Amniotic and allantoic fluids from experimentally infected sheep contain immunoglobulin specific for Chlamydophila abortus

Chlamydophila abortus, the aetiological agent of enzootic abortion of ewes (EAE), replicates in trophoblast cells leading to their destruction and dissemination of the bacterium to foetal organs. To further understand the pathogenesis of EAE, amniotic and allantoic fluids were collected from experimentally infected pregnant ewes at 30 (7 samples from each fluid), 35 (8 samples from each fluid), 40 (10 samples from each fluid) and 43 (6 amniotic fluids and 7 allantoic fluids) days post-infection to determine pathogen numbers and other markers of infection. Whilst experimentally infected ewes had characteristic placental lesions, only two amniotic and seven allantoic fluid samples were positive for C. abortus by real-time PCR. In contrast, all amniotic and allantoic fluids were positive for immunoglobulin. Immunoglobulins were generally detected earlier in allantoic fluid than in amniotic fluid and the numbers of samples containing immunoglobulins increased as infection progressed. IgG in amniotic and allantoic fluids was shown to be specific for C. abortus, and reacted with the major outer membrane proteins, polymorphic outer membrane protein and macrophage infectivity potentiator protein. A comparison of two-dimensional immunoblots using purified IgG from the allantoic fluid, amniotic fluid, ewe serum and foetal serum of a C. abortus infected animal at 40 days post infection indicated a pattern of reactivity intermediate between that of the ewe serum and the foetal serum. Results suggest that a maternal source of immunoglobulin is predominant at 30 days post-infection but that foetal derived antibodies may be contributed at a later stage.     

Comparison of the dot-immunobinding assay with the complement fixation test for the detection of Brucella antibodies in sheep

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.     

Immunopathology of Chlamydophila abortus infection in sheep and mice

Chlamydophila abortus targets the placenta, causing tissue damage, inflammation and abortion (enzootic abortion of ewes). It is one of the main infectious causes of abortion in ewes, resulting in major economic losses to agricultural industries worldwide. Although ruminants and pigs are the principal hosts, humans are also susceptible to infection. Control of disease requires a host inflammatory response, which is likely to contribute to pathology and abortion. Mouse models have been widely used to provide insight into the role of specific immune cells in controlling infection and disease. The use of such model systems for investigating the mechanisms of abortion, latency, persistence, and immunity to reinfection will result in the identification of novel vaccine control strategies for sheep.     

Epidemiological modelling of chlamydial abortion in sheep flocks

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.     

Comparison of ELISA and CFT assays for Chlamydophila abortus antibodies in ovine sera

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.     

Molecular detection of Chlamydophila abortus in post-abortion sheep at oestrus and subsequent lambing

Enzootic abortion of ewes (EAE), resulting from infection with the bacterium Chlamydophila abortus (C. abortus), is a major cause of lamb loss in Europe. The purpose of this study was to assess the potential impact of the shedding of organisms in post-abortion ewes at oestrus and subsequent lambing on the epidemiology of EAE. Using a newly developed C. abortus specific real-time PCR assay, few chlamydial genomes could be detected in vaginal swabs taken from post-abortion ewes at oestrus. At subsequent parturition, all ewes lambed normally with no macroscopic or microbiological evidence of infection. Real-time PCR analysis of placental samples identified very few or no chlamydial genomes, which contrasted significantly with samples taken at the time of abortion, where an average of 2.7x10(7) chlamydial genomes per microgram of total tissue DNA was detected. Few genomes could also be detected from vaginal and cervical tissue samples and lymph nodes taken post-mortem. The results, although not discounting the possibility of a chronic low level persistent infection in post-abortion ewes, suggest that the low levels of chlamydial DNA detected during the periovulation period and at lambing do not significantly impact on the epidemiology of EAE. In terms of flock management, the products of abortion should be considered the major and principal source of infection for transmission to na?ve ewes.     

布鲁氏菌弱毒疫苗粘膜免疫及检测方法的研究

为研究布鲁氏菌弱毒疫苗粘膜免疫及其检测方法,本实验采用粘膜点眼途径对健康母羊接种布鲁氏杆菌猪2号疫苗(S2)、牛19号疫苗(A19)和羊强毒株(M16),筛选布鲁氏杆菌病鉴别检测方法。将12月龄～14月龄母羊60只随机分为3组,以常规疫苗推荐剂量进行半量粘膜点眼接种。采集血液、淋巴、脏器进行布鲁氏菌病血清学检测和细菌学分离以及PCR检测。结果表明:布鲁氏菌弱毒疫苗抗体水平持续6个月,其中血清学的试管凝集试验、半胱氨酸凝集试验与补体结合试验的阳性符合率达到100%。细菌分离期为6个月,乳腺、乳腺淋巴、髂淋巴分离率较高;而强毒株M16的抗体水平和细菌分离持续12个月以上。结果显示以常规血清学和细菌学检测方法在点眼免疫布鲁氏菌S2、A19苗6个月后可以进行野毒感染和疫苗免疫畜的鉴别诊断。     

Serologic survey of prevalence of ovine progressive pneumonia in Idaho range sheep.

Blood samples from 2,310 mature sheep in 3 Idaho range flocks were examined by agar gel immunodiffusion to determine the prevalence of ovine progressive pneumonia. The prevalence ranged from 58% for all ages combined in one flock to 90% of cull ewes in another flock. Age-specific prevalence rates increased from 16% in yearlings to 83% in ewes greater than or equal to 7 years old. Rambouillet sheep had a significantly (P less than 0.01) lower prevalence than sheep of 5 other breeds, whereas one-half Finnsheep crosses had a significantly (P less than 0.01) higher prevalence than sheep of other breeds. Within breed and age, there was no significant difference in reproductive performance between seropositive and seronegative ewes.     

Sarcocystosis in slaughterhouse sheep

The occurrence of sarcocystosis was studied in the sheep produced in the vicinity of Brno and killed in the slaughterhouse. Adspection, the homogenization method and the digestion method were used for the detection of muscular cysts. Antibodies to sarcocystosis were determined in the blood serum by the indirect fluorescence method. The total number of sheep examined was 157; sarcocysts were detected in the muscle of 102 sheep (64.9%). The highest incidence was recorded in ewes: 60 ewes (80%) out of 75 were positive. Out of the 157 blood serum samples examined by the indirect fluorescence method, antibodies were found in 105 samples (66.9%). The antibody response was 77.3% in ewes, 63.4% in lambs and 46.6% in rams. The highest titres (640-10 240) were obtained in 16 lambs and 26 ewes.     

Molecular evidence for chlamydial infections in the eyes of sheep

Ocular infections by chlamydiae are associated with ocular disease manifestations such as conjunctivitis and keratitis in humans and animals. Limited evidence exists that members of the order Chlamydiales can also cause ocular disease in sheep. In the current study, the prevalence of chlamydiae in the eyes of sheep was investigated by using PCR methods. Data obtained in sheep by broad-range 16S rRNA order Chlamydiales-specific PCR were compared to the prevalence of antibodies against chlamydiae detected by a competitive enzyme-linked immunosorbent assay (cELISA). Flocks tested included a clinically healthy flock and two flocks suffering from ocular disease and with histories of Ovine Enzootic Abortion (OEA). PCR detected DNA of Chlamydophila (Cp.) abortus and Cp. pecorum in the eyes of both healthy and sick animals but also identified Chlamydia (C.) suis and a variety of uncultured chlamydia-like organisms. Good correlation was found between the presence of Cp. abortus DNA in sheep conjunctival samples and seropositivity detected by cELISA. Despite these findings, no association was found between the presence of chlamydial DNA in the sheep conjunctival samples and the onset of clinical disease. These results suggest that the biodiversity of chlamydiae in the eyes of sheep is greater than that previously thought. Further investigations are needed to determine whether a causal relationship between infection by chlamydiae and ocular disease exists in these animals.     

Detection of the genome of Chlamydophila abortus in samples taken from the uteri of 304 sheep at an abattoir

A PCR was used to detect the genome of Chlamydophila abortus in samples of uterine tissue collected from 304 sheep by a sterile technique at an abattoir. The stage of pregnancy of the sheep was determined by measuring the dimensions of the embryo/fetus, and its morphology was recorded. Only samples from non-pregnant sheep and sheep up to 100 days of gestation were retained; the clinical history of the animals was unknown. The total prevalence of the chlamydial genome was 30.9 per cent, with a significantly higher prevalence in the pregnant animals (46.9 per cent). Higher detection rates were recorded during early gestation than during mid-gestation.     

Rechallenge of previously-infected pregnant ewes with Chlamydophila abortus

In an attempt to ascertain the means whereby previous exposure to Chlamydophila (C.) abortus can protect against the re-occurrence of enzootic abortion of ewes (EAE), ten previously-exposed ewes were intravenously rechallenged with a large infective dose of C. abortus during pregnancy. The patterns of development of chlamydial placentitis and its sequelae closely resembled that observed following first-time challenge of previously-na?ve ewes, although placentitis appeared to develop more slowly following rechallenge infection and none of the rechallenged ewes aborted. Chorioallantoic and foetal pathology and foetal immune responses were qualitatively similar whilst the local maternal response to C. abortus infection of the endometrium did not appear to differ in rechallenged and first-time challenged sheep. This demonstrates that if C. abortus reaches the foetal side of the placenta, a stereotypical response is elicited, regardless of the status of maternal immunity. Therefore it appears that in natural circumstances, acquired immunity of the dam protects against the re-occurrence of EAE by preventing the causative agent from reaching the susceptible foetal trophoblast.     

Application of touchdown enzyme time release (TETR)-PCR for diagnosis of Chlamydophila abortus infection

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.     

Texel sheep are more resistant to natural nematode challenge than Suffolk sheep based on faecal egg count and nematode burden

Limited information is available on differences between sheep breeds with respect to helminth resistance under temperate conditions. The present study was designed to confirm and extend preliminary findings on observed breed differences in resistance to naturally acquired gastrointestinal nematodes in Suffolk and Texel sheep. Three trials were carried out. In trial 1 (1999-2003) lambs co-grazed from birth were faecal sampled at various time points up to 17 weeks of age. Worm burden was assessed at 17 weeks of age from a minimum of six lambs per breed in each of the 3 years. In trial 2, faecal egg count (FEC) was determined on six farms with co-grazed Suffolk and Texel purebred lambs. In trial 3 (2001-2003), ewes were faecal sampled at winter housing. In all three trials, an influence of breed on resistance to naturally acquired trichostrongyle infection was demonstrated. In trial 1, significantly higher FEC and worm burden was observed in Suffolk compared with Texel lambs following natural challenge. In trial 2, FEC recorded in lambs from six farms confirmed the breed differences previously observed. A breed difference in resistance to GI parasites was also observed in older ewes. In both breeds, an age effect on the FEC was observed with younger ewes having greater FEC than older ewes.