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.     

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.     

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.     

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.     

Serological analysis of Chlamydophila abortus in Australian sheep and implications for the rejection of breeder sheep for export

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.     

Prevalence of Chlamydophila abortus infection in domesticated ruminants in Taiwan.

This study is to (1) investigate the prevalence of Chlamydophila abortus infection in cows and goats in Taiwan, and (2) compare the genetic properties of Taiwanese isolates with abortion strains from other sources. Approximately 71% of aborted cows and 58% of aborted does had IgG against C. abortus in their sera. The seroprevalence rate in cows may be overestimated, because a certain degree of cross-reactivity with C. pecorum cannot be ruled out. Only 22.7% (from aborted cows) and 33.3% (from aborted dogs) of vaginal swabs that tested positive by polymerase chain reaction led to successful isolation of C. abortus by inoculation into chicken embryos, equivalent to 7.1% and 7.9% of isolation rates, respectively. The major outer membrane protein gene of 15 Taiwanese abortion isolates was compared with that of various strains by restriction fragment length polymorphism (RFLP) and nucleotide sequencing. Restriction enzyme CfoI was able to distinguish Taiwanese ruminant isolates, which have identical RFLP patterns, from C. felis (feline) and C. psittaci (avian) strains. Taiwanese isolates had 98.8-100% homology with known ruminant abortion strains and were phylogenetically closest to bovine LW508 strain.     

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.     

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.     

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.     

Experimental infection of sheep with Brucella abortus

A case of brodifacoum poisoning is described in a six-year-old male Kelpie cross working dog. The clinical features were severe exercise intolerance, haemorrhage from the oral and nasal cavities, dyspnoea and pale mucous membranes. Diagnosis was confirmed by demonstrating an abnormally long whole blood clotting time. The dog was treated successfully by administering 1 litre of whole blood intravenously, intramuscular vitamin K₁ and a three week course of oral vitamin K₃.

Experience at the Massey University Small Animal Clinic and Hospital has indicated that poisoning of dogs with the newer long acting anticoagulant rodenticides is becoming more common.     

一起山羊流产衣原体感染的诊断

陕西省某山羊养殖场发生了一起山羊流产病,经流行病学调查,临床症状和病理剖检观察进行初步诊断;利用布鲁菌虎红平板凝集试验、衣原体间接血凝试剂盒对10份流产母羊血清检测;对病料进行细菌学检测;依据GenBank收录的山羊流产性衣原体基因组设计1对特异性引物,通过PCR方法从病料中扩增衣原体特异性片段。结果表明,根据临床症状和病理变化初步怀疑为布鲁菌和鹦鹉热亲衣原体感染;10份血清检测结果为布鲁菌病血清全部阴性,衣原体感染血清全部为阳性;细菌学检测结果为阴性;PCR结果获得523bp基因片段,测序结果与鹦鹉热亲衣原体100%相似。依据流行病学调查,临床症状和病理剖检观察,病原学和血清学诊断,最终确诊该病为鹦鹉热亲衣原体引起的山羊地方流行性流产。     

流产嗜衣原体TaqMan-MGB荧光定量PCR检测方法的建立

为建立一种快速、准确检测流产嗜衣原体(C.abortus)Taq Man-MGB荧光定量PCR方法,本研究根据C.abortus主要外膜蛋白基因的特异保守序列设计引物及探针,并优化反应条件,建立了检测C.abortus的荧光定量PCR方法。结果表明,以重组质粒为标准品建立的标准曲线在1.6×103拷贝/μL~1.6×107拷贝/μL内具有良好的线性关系,相关系数为0.9999。该方法仅对C.abortus的靶基因扩增呈阳性,而对鹦鹉热嗜衣原体、家畜嗜衣原体、鼠衣原体、沙眼衣原体、肺炎嗜衣原体、猪源衣原体核酸扩增结果均为阴性,特异性强;其最低检出限为1.6拷贝/μL;组内和组间重复性试验变异系数均小于3%,具有良好的重复性。利用建立的方法和普通PCR方法同时对225份临床样品进行检测,结果显示荧光定量PCR检出率比普通PCR高4.5%,表现较高的灵敏度和准确性。本研究建立的方法对C.abortus的临床鉴别检测和疾病诊断具有重要意义。     

Identification of Chlamydophila abortus and the development of lesions in placental tissues of experimentally infected sheep

Chlamydophila (C.) abortus is a major cause of infectious abortion in sheep in many countries. Twenty-one pregnant sheep were experimentally infected intranasally with C. abortus at 70 days of gestation (dg). Thereafter, a number of animals were killed at weekly intervals and a post-mortem examination was carried out. Evidence of chlamydial infection in the placenta was determined by isolation of the bacterium by tissue culture and detection of C. abortus DNA by real-time polymerase chain reaction (real-time PCR). In addition, histopathological changes in the placenta were assessed, as was the detection of chlamydial antigen by immunohistochemistry (IHC). Evidence of placental infection was observed as early as 2 weeks after inoculation, and while only relatively low numbers of bacteria were isolated by culture and/or detected by real-time PCR prior to 113-114dg, at 119-121dg, it was more numerous. This study, using the four criteria for assessment of infection, showed that while C. abortus gained access to the placenta as early as 85dg, characteristic histopathological changes were not apparent until 119/121dg. While the chronology of when the bacterium arrived in the placenta and subsequent lesion development is remarkable for its consistency this paper provides more reliable data on the former which in turn now allows study of the factors that permit its access to this tissue and govern its multiplication and the ensuing triggering of damage.     

Chlamydophila abortus infection in the mouse: a useful model of the ovine disease

Chlamydophila (C.) abortus is an obligate intracellular bacterium able to colonize the placenta of several species of mammals, which may induce abortion in the last third of pregnancy. The infection affects mainly small ruminants resulting in major economic losses in farming industries worldwide. Furthermore, its zoonotic risk has been reported in pregnant farmers or abattoir workers. Mouse models have been widely used to study both the pathology of the disease and the role of immune cells in controlling infection. Moreover, this animal experimental model has been considered a useful tool to evaluate new vaccine candidates and adjuvants that could prevent abortion and reduce fetal death. Future studies using these models will provide and reveal information about the precise mechanisms in the immune response against C. abortus and will increase the knowledge about poorly understood issues such as chlamydial persistence.     

Prevalence and risk factors associated with Chlamydophila abortus infection in dairy herds in Jordan

A cross-sectional study was carried out to determine seroprevalence and to identify risk factors associated with Chlamydophila abortus infection in 62 nonvaccinated dairy herds (671 cows) in Jordan between January and June 2007. Information regarding herd management was recorded through a personal interview with farmers. Antibodies against C. abortus were detected using an ELISA test kit. Chi-square analysis and multivariable logistic regression model were used to identify risk factors associated with C. abortus seropositivity. The true prevalence of antibodies against C. abortus in individual cows and cattle herds were 19.9?% and 66.3?%, respectively. Univariable Chi-square analysis revealed three variables with P????0.25 that were further offered to multivariable logistic regression analysis. Small-sized herds were identified as a risk factor for seropositivity to C. abortus, while sweeping followed by water hosing and using disinfectants were identified as protective factors. Cows in the age groups of >8 and ??10?years old and >2 and ??6?years old had the highest and lowest significant seroprevalence to C. abortus, respectively. Results of this study indicated that C. abortus is highly prevalent in Jordan's dairy herds and Chlamydophila infection could be controlled by applying strict biosecurity measures in the dairy farms.     

Seroprevalence of Chlamydophila abortus infection in yaks (Bos grunniens) in Qinghai,China

Chlamydophila abortus is an important amphixenosis which in a wide range of animals, associated with reproductive disorders in yaks. In order to assess the prevalence of this infection in yaks in Qinghai, China, a cross-sectional study was carried out, and a total of 674 serum samples were collected from June to October 2012 in six counties, and antibodies to C. abortus were examined by indirect hemagglutination (IHA) test. The overall seroprevalence of C. abortus in yaks was 17.66 % (119/674), and the seroprevalence of antibodies to C. abortus in yaks ranged from 11.82 to 28.43 % among the six different areas, and the difference was statistically significant (P?<?0.05). The seropositivity of C. abortus infection in different age groups varied from 16.33 to 18.49 %, and prevalence in yaks of ≥3 year (18.49 %) was slightly higher than that in yaks of <3 year, but the differences among the age groups were not statistically significant (P?>?0.05). The seroprevalence of C. abortus infection in male yak (16.8 %) was slightly lower than that in females (17.85 %), and the difference was not statistically significant (P?>?0.05). So far, this is the first systematic and comprehensive investigation of C. abortus infectionin in yaks in this area.     

Subspecies variation in Greek strains of Chlamydophila abortus

The Greek chlamydial strains FAS, FAG, VPG and LLG, isolated from aborted sheep or goat foetuses, had been previously characterized as divergent on the basis of mouse cross-protection experiments, with LLG and its homologous POS significantly different from the rest in inclusion morphology, polypeptide profiles and reactivity with monoclonal antibodies. To determine the genetic basis of their divergence the 16S-23S ribosomal intergenic spacer was analysed by RFLP analysis of PCR 16SF2/23R amplicons. Using the restriction enzymes BfaI, SfcI, HpaI, BclI, DdeI and AclI, the strains were classified as Chlamydophila abortus. However, digestion with RsaI made it possible to differentiate strains FAS, FAG and VPG from strains LLG and POS, generating DNA fragments of 530/55 and 585bp, respectively. By subsequent sequence analysis of the 23S domain I rRNA gene only strain FAS was identical to reference strain A22 of C. abortus. Strains FAG and VPG presented an identical nucleotide deviation at position 593 of signature sequences. Strains LLG and POS presented three identical nucleotide deviations at positions 156, 186 and 307. Variation within the domain I signature sequences for the examined abortion strains was < or =0.69%. In conclusion, substantial genetic and biological diversity among strains of C. abortus was demonstrated, suggesting that subspecies variation status for certain strains may be applicable. Our findings suggest that differentiation may be possible at a subspecies level by RFLP analysis.     

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.     

Comparison of serological tests forBrucella melitensis infection in sheep

Summary A comparative study of the standard tube agglutination test (SAT), Rose Bengal plate agglutination test and counter immuno-electrophoresis (CIEP) was made on 647 sera from naturally aborting ewes, orchitic, in-contact and apparently healthy sheep with no history of vaccination against brucellosis. No individual test could detect all the 13 known positive reactors (the foetuses of which yieldedBrucella melitensis) but by combination of two tests all 13 were positive. The SAT detected more reactors during the early stage of infection while CIEP performed better in later stages of infection. All these tests may be carried out in a field laboratory at very low cost.

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Resumen Se llevó a cabo un estudio donde se comparó la efectividad de la aglutinación en tubo, la prueba Rosa de Bengala y la contrainmunoelectroforesis en 647 sueros de ovejas que habían abortado naturalmente, machos con orquitis, ovejas en contacto y ovejas aparentemente sanas, sin previa historia de vacunación contra brucelosis. Ninguna de las pruebas utilizadas pudo detectar individualmente todos los 13 reactores positivos (de los fetos de los cuales se aislóBrucella melitensis), pero la combinación de dos pruebas detectó todos los reactores positivos. La prueba de la aglutinación en tubo detectó la mayoría de reactores en los estadíos iniciales de la infección, mientras que la contrainmunoelectroforesis se comportó bien en estadíos iniciales de la infección, mientras que la contrainmunoelectroforesis se comportó bien en estadíos tartíos de la infección. Todas estas pruebas pueden realizarse, en laboratorios de campo a un costo bajo.

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Résumé Les auteurs ont réalisé une étude comparative entre l'agglutination en tube standard (SAT), le test d'agglutination sur lame au Rose Bengale (RBPT) et la contre immunoélectrophorèse (CIEP), à partir de 647 sérums prélevés sur des brebis ayant avorté naturellement ou sur des moutons atteints d'orchite ou ayant été en contact mais restés apparement sains et sans passé vaccinal contre la brucellose. Aucun test individuel n'a pu reconna?tre comme positif l'un des 13 animaux reconnus réagissant (dont les foetus hébergeaientBrucella melitensis), mais par combinaison des deux tests, tous les 13 se sont révélés positifs. Le SAT a décelé plus de réagissants au cours des premiers stades de l'infection. Par contre, le CIEP a été plus sensible dans les derniers stades. Tous ces tests peuvent être réalisés dans un laboratoire de terrain à, un co?t trés bas.