Pregnancy, indoleamine 2,3-dioxygenase (IDO) and chlamydial abortion: an unresolved paradox

Chlamydophila abortus infects the placental trophoblast in sheep, humans and mice, causing cell damage and inflammation that culminates in abortion. Host control of C. abortus appears to be heavily dependant on interferon (IFN)-gamma production. IFN-gamma induces expression of the enzyme indoleamine 2,3-dioxygenase (IDO), resulting in the degradation of intracellular pools of tryptophan, thereby depriving the organism of this essential growth nutrient. The anti-chlamydial effects of IFN-gamma can be reversed by the addition of exogenous tryptophan. This finding is consistent with studies of the C. abortus genome sequence that have revealed that the organism lacks the capability to synthesise tryptophan from host cell substrates and is therefore dependant on host tryptophan. This raises an interesting paradox since the placental trophoblast in humans and mice is known to constitutively express IDO and degrade tryptophan, a phenomenon that has been linked to maternal immunological tolerance of the semi-allogeneic fetus. This paradox is discussed in the context of immune modulation during pregnancy, tryptophan biosynthesis by Chlamydiaceae and differences in placental structures between sheep, humans and mice.     

Protective adaptive immunity to Chlamydophila abortus infection and control of ovine enzootic abortion (OEA)

Ovine enzootic abortion (OEA) remains a major problem in sheep-rearing countries despite the availability of protective vaccines. The causative agent, Chlamydophila abortus, is a Gram-negative bacterium that can induce a persistent, subclinical infection in non-pregnant sheep. The development of a new safe, effective and practical vaccine requires a detailed understanding of host-pathogen interactions and the identification of clear correlates of protection. Since disease (abortion) is only observed during pregnancy, the nature of host immunity to C. abortus and the specialised immunological features that permit maternal acceptance of the semi-allogeneic fetus are central to the pathogenesis of OEA. We review the current literature on persistence of C. abortus, host immunity to infection and mechanisms of abortion. We identify the key outstanding questions surrounding OEA and discuss the current knowledge gaps with a view to developing improved control strategies.     

Abortion in humans caused by Chlamydophila abortus (Chlamydia psittaci serovar 1)

On a farm housing cattle and goats an abortion storm occurred affecting 50% of the goats during the lambing season 2000/2001. In one of three investigated caprine abortions Chlamydophila abortus could be identified as etiology. During this time a pregnant woman (pregnancy week 19/20) had contact with aborting goats. She developed a severe generalized infection and aborted. The placenta contained Chlamydophila abortus shown by immunohistochemistry and PCR. Aim of the present case report is to alert veterinarians about the potential zoonotic risk of ovine/caprine abortions.     

Abortion in small ruminants in Switzerland: investigations during two lambing seasons (1996-1998) with special regard to chlamydial abortions

Abortion cases of 144 goats und 86 sheep were investigated etiologically during 2 lambing seasons (1996/1997, 1997/1998). Macroscopic inspection of fetus and placenta was completed by histopathology and bacteriological isolation of agents. In addition, immunohistologically the following antigens were labeled in formalin-fixed and paraffin-embedded tissue sections: Toxoplasma gondii, Neospora caninum, Chlamydophila abortus (formerly Chlamydia psittaci serovar 1) and Border Disease Virus. From farms with abortions caused by Chlamydophila abortus specific data were recorded. In 75% of abortion cases in sheep and in 59% of cases in goats an etiologic diagnosis could be substantiated. Chlamydophila abortus is the most commonly involved agent in the etiology of caprine and ovine abortion (sheep 39%, goats 23%), followed by Toxoplasma gondii (sheep 19%, goats 15%) and Coxiella burnetti (sheep 1%, goats 10%). All other agents are of minor importance. An infectious cause of abortion based on histopathologic findings without isolation of agents was observed in sheep (10%) and goats (21%). Malformation occurred in sheep (2%) and goats (3%) and lesions suggestive for Vitamin E/Selenium deficiency were seen in goats only (2%).     

Chlamydial infections in small ruminants.

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.     

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.     

Role of Chlamydophila abortus in ovine and caprine abortion in Sardinia, Italy

Between 1999-2003, 14321 sera and 646 abortion samples (498 foetuses and 148 placentae) were analysed from 807 sheep and goat farms distributed all over the island of Sardinia. After notification of abortion in a flock, sera collected at random from adult animals were examined to detect antibodies specific to Chlamydophila (C.) abortus by ELISA, whereas foetuses and placenta were analysed by PCR assay. Specific IgG antibodies were detected in 611 (4.8%) sheep and 106 (5.8%) goats. From a total of 2050 ovine and 151 caprine fetal samples including muscle, liver, abomasum, spleen, brain and placenta, 29 (1.4%) ovine and 1 (0.6%) caprine samples were C. abortus PCR-positive. Placenta was the tissue with the highest detection rate. These results indicate that the seroprevalence of C. abortus infection in sheep and goats is very low in Sardinia, and PCR results demonstrate that C. abortus has no significant role in abortion, especially in goats.     

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.     

Diagnosis of ovine chlamydial abortions by PCR-RFLP performed on vaginal swabs

Ovine enzootic abortion is an infectious and contagious disease clinically characterized by abortion and weak neonates, affecting sheep and goats. The etiological agent is Chlamydophila (C.) abortus, which is considered one of the most common animal pathogens of small ruminants; it has important economic implications and represents a significant zoonotic risk. Clinical diagnosis is often difficult because the clinical signs and the pathological lesions are not specific for C. abortus infection, in fact they can also be observed as a result of infections with other abortifacient agents. Moreover, the involvement of the laboratory is necessary to perform the definitive diagnosis. One hundred and seventeen vaginal swabs from sheep with clinical signs related to chlamydial infection were examined by a PCR-RFLP assay that demonstrated high specifity and sensitivity. Six samples were positive for C. abortus. Vaginal swabs are easy to handle and allow to deal with biohazardous material in safety conditions.     

Influence of the Th2 immune response established by Nippostrongylus brasiliensis infection on the protection offered by different vaccines against Chlamydophila abortus infection

Chlamydophila abortus is the aetiological agent of enzootic abortion in small ruminants in which it infects the placenta to cause abortion during the last trimester of gestation. In a mouse model, a Th1 immune response involving IFN-gamma production and CD8+ T cells is necessary for the infection to be resolved. The authors previously demonstrated that infection with Nippostrongylus brasiliensis, a rodent gastrointestinal nematode extensively used in experimental models to induce Th2 responses, alters the specific immune response against C. abortus infection, increasing bacterial multiplication in liver and reducing specific IFN-gamma production. The aim of the present work was to clarify whether a Th2 immune response has any influence on the success of vaccination using both inactivated and attenuated vaccines. The results showed that the Th2 response established prior to vaccination did not influence the induction of protection offered by the vaccines. However, the effectiveness of this protective response can be altered, depending on the adjuvant employed in the inactivated vaccines, when the Th2 response is established after vaccination, just before challenge with C. abortus.     

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.     

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.     

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.     

Detection of Chlamydophila abortus in ovine milk by immunomagnetic separation-polymerase chain reaction

The present study was carried out to investigate the presence of Chlamydophila abortus, the causative agent of ovine enzootic abortion, in milk samples collected from sheep flocks with and without the history of abortion in Eastern Turkey by means of immunomagnetic separation (IMS) in conjunction with the polymerase chain reaction (PCR). A total number of 201 milk samples collected from 10 flocks with abortion and four flocks without abortion were tested. In the analysis of the milk samples by IMS-PCR, correct amplification was obtained with three (1.5%) samples originating from one flock with abortion. In the digestion of PCR positive products by AluI, restriction profiles observed in all three samples were determined to be the same as C. abortus S26/3.     

贵州省山羊流产的病原学鉴定

为了解贵州省山羊流产与山羊痘的相关性,采用琼脂扩散试验和PCR法对本省10个市(县)流产羊群的血清和病料样本进行山羊痘抗原抗体及病原核酸检测,同时血清进行布氏杆菌抗体检测,流产胎儿病料进行羊流产亲衣原体病原核酸检测。结果发现山羊痘羊群流产率达37.1%(4329/11660),山羊痘血清抗体阳性率为38.2%(34/89),抗原阳性率为72.7%(32/44),流产胎儿山羊痘病毒核酸检出率为83.3%(10/12),发病羊群未检出布氏杆菌和羊流产亲衣原体感染。结果表明,山羊流产与山羊痘感染有一定关系,提示在山羊养殖中应加强饲养管理,防止山羊痘感染引起孕羊流产。     

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.     

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.     

Detection of Chlamydophila abortus in sheep and goat flocks in southern Italy by PCR using four different primer sets

An epidemiological survey was performed to detect the presence of Chlamydophila (C.) abortus and other members of the order Chlamydiales in ovine and caprine flocks with a history of abortion in southern Italy. Four pairs of primers were compared to evaluate their ability to detect Chlamydiales using purified DNA preparations and tissue samples from aborted foetuses with suspected chlamydial infections. As expected, amplification of DNA of the reference strain C. abortus using primer pairs U23F/23Sigr, 16SF2/23R, CTU/CTL and CpsiA/CpsiB produced fragments of about 600 bp, 585 bp, 1000 bp and 300 bp, respectively. The detection limits of the four PCR tests performed on serial DNA dilutions of the C. abortus reference strain were of 10 pg, 0.1 pg, 0.1 pg and 1 fg of DNA, respectively. The most sensitive amplification of DNA extracted from the organ tissues was obtained with primer pairs CpsiA/CpsiB, which detected Chlamydophila spp. DNA in all infected tissue samples. Only C. abortus was identified during the survey. The presence of this agent was confirmed in 3 out of 27 ovine and caprine flocks included in the survey suggesting that abortion due to C. abortus is uncommon in southern Italy.     

羊流产嗜衣原体病和弓形虫病IHA与ELISA诊断方法的比较

通过比较研究羊流产嗜衣原体与弓形虫抗体的间接血凝试验(IHA)与酶联免疫吸附试验(ELISA)检测结果,选择适宜贵州省山羊流产血清学调查的检测方法。通过IHA和ELISA两种方法对贵州省195份山羊血清进行羊流产嗜衣原体与弓形虫抗体的检测,统计并分析两种疫病的阳性符合率、阴性符合率和总符合率。结果表明,羊流产嗜衣原体与弓形虫IHA与ELISA的总符合率分别为77.95%和78.97%,阳性符合率均仅为50%,阴性符合率为80.45%和79.27%。说明在检测两种疫病血清抗体方面,IHA比ELISA更适合在贵州省基层推广。     

Regulated upon activation normal T-cell expressed and secreted (RANTES) contributes to abortion caused by Brucella abortus infection in pregnant mice

Brucella abortus (B. abortus) is a facultative intracellular pathogen that can survive inside macrophages and trophoblast giant cells, and the causative agent of brucellosis. In the present study, we found that production of regulated upon activation normal T-cell expressed and secreted (RANTES) due to B. abortus infection contributes to abortion in pregnant mice. B. abortus infected pregnant interferon-gamma (IFN-gamma) knockout mice died within 15 days of infection, but non-pregnant IFN-gamma knockout mice were still alive. With infection by wild type B. abortus, a large amount of RANTES production was observed in pregnant IFN-gamma knockout mice, and induction of RANTES was also observed in normal pregnant mice infected with the wild type, but not in those infected with the intracellular replication-defective mutant. Production of RANTES and IFN-gamma were inhibited in mice inoculated with the respective RANTES or IFN-gamma antibody. Neutralization of RANTES, induced by B. abortus infection, served to prevent abortion. These results indicate that the production and function of RANTES are correlated with IFN-gamma in pregnant mice infected with B. abortus.