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 psittaci by using SYBR green real-time PCR

Chlamydophila psittaci is the causative agent of human psittacosis and avian chlamydiosis. This zoonotic pathogen is frequently transmitted from infected birds to humans. Therefore proper and rapid detection of C. psittaci in birds is important to control this disease. We developed a method for detecting C. psittaci by using SYBR Green Real-time PCR based on targeting the cysteine-rich protein gene (envB) of C. psittaci. This one step procedure was highly sensitive and rapid for detection and quantification of C. psittaci from fecal samples. This assay was also able to detect other zoonotic Chlamydophila species such as C. abortus and C. felis. The assay is well suited for use as a routine detection method in veterinary medicine.     

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.     

Chlamydophila psittaci DNA detection in the faeces of cage birds

In this study, we investigated the shedding of Chlamydophila psittaci in faecal samples from cage birds using PCR testing. A total of 47 faeces samples were collected from four different aviaries. Main symptoms determined after clinical investigation and owner histories of the birds showed that the birds had respiratory system problems changing from mild to severe. They also showed conjunctivitis, diarrhoea or no symptoms at all. DNA extractions from faeces were performed with the QIAamp DNA Stool Mini Kit. Following PCR with Cp. psittaci specific primers, 43 (91.5%) samples were determined to harbour-specific DNA. Only one bird from each aviary was found to be negative by PCR. As all the samples from birds showing clinical signs were PCR positive, these signs could be correlated to psittacosis in these birds. Cp. psittaci shedding in faeces was detected in all the aviaries. After restriction analysis of PCR amplicons with AluI enzyme, all the isolates showed the same RFLP (Restriction Fragment Length Polymorphism) patterns with the control Cp. psittaci DNA. PCR following QIAamp DNA stool mini kit extraction of faecal samples was found to be a rapid, specific, sensitive, reproducible test, which did not need additional nested PCR of samples.     

Chlamydial diseases of domestic animals--zoonotic potential of the agents and diagnostic issues

The role of chlamydiae as agents of a number of important animal and human diseases is still the subject of intensive research. Recently, a proposal for taxonomic reclassification of this group of obligate intracellular bacteria was published, which was based on a large amount of new data on genetic relatedness. According to this proposal, the family Chlamydiaceae now comprises two genera (Chlamydia and Chlamydophila) with 9 largely host-related species. The previously accepted classification scheme had distinguished 4 species within the genus Chlamydia. The most important animal chlamydiosis with zoonotic character is psittacosis, a systemic disease in psittacine birds of acute, protracted, chronic or subclinical manifestation. The analogous infection in domestic and wild fowl is known as ornithosis. Avian strains of C. psittaci (new classification: Chlamydophila psittaci) can also infect humans, the symptoms being mainly unspecific and influenza-like, but severe pneumonia, endocarditis and encephalitis are also known. The main group of persons facing an elevated risk of infection includes those having frequent contact with domestic and companion birds at work or in their spare time. In Germany, the annual average of notified cases is approximately 100. Cases of transmission to humans were repeatedly reported in connection with enzootic abortion in sheep (causative agent: C. psittaci or Chlamydophila abortus, respectively). Various chlamydial species occur as pathogens and commensals as well in cattle, pigs, horses, and cats. The assessment of the actual epidemiological importance is, however, often difficult because of their almost ubiquitous spread. Likewise, those strains of C. pneumoniae (new classification: Chlamydophila pneumoniae) found in several animal species can not yet be assessed for pathogenic properties. The possibilities for diagnostic detection of chlamydiae have considerably improved following the introduction of molecular methods, particularly the polymerase chain reaction (PCR), which permits direct identification from clinical specimens and differentiation of species.     

Development of a Chlamydophila psittaci species-specific and genotype-specific real-time PCR

A Chlamydophila psittaci species-specific real-time PCR targeting the rDNA ribosomal spacer was developed as well as a genotype-specific real-time PCR targeting the Cp. psittaci outer membrane protein A (ompA) gene. The SYBR Green-based species-specific real-time PCR detected Cp. psittaci genotypes A to F, and the recently discovered E/B genotype. The genotype-specific real-time PCR could easily distinguish genotypes C, D, F by use of TaqMan probes. Genotypes A, B and E could not be distinguished from each other by simply using TaqMan probes. For this purpose, non-fluorescent competitor oligonucleotides, had to be used next to the TaqMan probes. Genotype E/B could only be detected by use of a minor groove binder (MGB) probe. Both real-time PCR assays allowed reproducible, sensitive (10 rDNA or ompA copies/microL DNA extract) and specific detection of Cp. psittaci DNA. The genotype-specific real-time PCR was compared to ompA sequencing and ompA restriction fragment length polymorphism (RFLP) analysis using five Cp. psittaci field isolates (99, 61/8, 7344/2, 8615/1 and 7778B15) each consisting of two different genotypes. The currently developed real-time PCR assays were used in a case study on a veterinary school and a turkey farm. In the veterinary school, Cp. psittaci genotypes D, E/B and F infection were detected in all five groups of turkeys, and one veterinarian who was taking care of all these turkeys. On the turkey farm, the presence of two Cp. psittaci genotype B infection waves was demonstrated in one randomly selected turkey, the first wave at the age of 6 weeks, and the second at the age of 12 weeks.     

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.     

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.     

Chlamydia-related abortions in cattle from Graubunden, Switzerland

In 2001, the first case of bovine chlamydial abortion was reported in canton Graubunden, Switzerland. In this region, Chlamydophila (Cp.) abortus is endemic in small ruminants. Hence, we aimed to investigate the incidence of chlamydia-related abortions in cattle from Graubunden. During breeding seasons of 2003-2004, formalin-fixed and paraffin-embedded placenta specimens (n = 235) from late-term abortions in cattle were analyzed by histopathology, immunohistochemistry with a Chlamydiaceae-specific monoclonal antibody against chlamydial lipopolysaccharide (LPS), and 2 different polymerase chain reaction (PCR) methods (16 S ribosomal ribonucleic acid [rRNA] PCR, intergenic spacer [IGS-S] PCR), followed by PCR product sequencing. In 149 of 235 cases (63.4%), histopathologic lesions such as purulent and/or necrotizing placentitis were observed. Chlamydial antigen was clearly demonstrated in immunohistochemistry in only 1 of 235 cases (0.4%). Cp. abortus or Cp. psittaci was found in 12 of 235 (5.1%) and 10 of 235 cases (4.2%) by 16 S rRNA PCR and IGS-S PCR, respectively. However, we detected, by 16 S rRNA PCR, 43 of 235 cases (18.3%) to be positive for chlamydia-like organisms. In contrast to the situation in small ruminants in the canton Graubunden, bovine abortion from Cp. abortus seems not to play an important role. Nevertheless, zoonotic potential should be taken into account when handling abortion material from cattle. The significance of chlamydia-like isolates other than Waddlia chondrophila remains an open question in abortion and needs further investigation.     

Ovine enzootic abortion: seroprevalence in Switzerland using a competitive enzyme-linked immunosorbent assay (cELISA)

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.     

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.     

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.     

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.     

Molecular characterisation and ovine live vaccine 1B evaluation toward a Chlamydophila abortus strain isolated from springbok antelope abortion

Chlamydiosis is a zoonosis with a worldwide distribution. The reservoir of susceptible hosts is large and includes birds and both domestic and wild mammals. Chlamydial infection, determined serologically, seems to be widespread among wild ruminants in the Paris zoo (France). In February 2003, an abortion case was reported within the springbok (Antidorcas marsupialis) herd of the zoo. PCR assay using primers targeting the polymorph membrane protein gene (pmp) family was performed on both vaginal swab and placenta samples revealing the presence of Chlamydophila. The inoculation into chicken embryos of an infected placenta extract led to the successful isolation of a C. abortus strain referred to as ASb1. The omp1 gene coding the major outer membrane protein (momp) and the 16S-23S rRNA spacer region of ASb1 were compared to those of various strains by restriction fragment length polymorphism (RFLP). The RFLP analysis showed that this isolate belonged to Chlamydophila abortus species and is highly related to known domestic ruminant's strains causing abortion. The efficacy of a live vaccine 1B, based on a temperature-sensitive mutant of the ovine abortion reference strain AB7, was tested. Protection-challenge experiments in a mouse model show that the ASb1 strain led to mice abortions and that vaccination with 1B vaccine provided them with effective protection.     

Improved sensitivity of PCR for Chlamydophila using pmp genes.

Primers targeting the conserved pmp gene family of Chlamydophila abortus were evaluated for their ability to improve the polymerase chain reaction (PCR) sensitivity. In purified DNA, specific pmp primers (named CpsiA and CpsiB) allowed at least a 10-fold increase of the PCR sensitivity compared to the specific ompA primers for C. abortus, but also for C. psittaci and C. caviae strains. No amplification was observed on C. felis, C. pecorum, C. pneumoniae and Chlamydia trachomatis strains. Tested on contaminated specimens such as genital swabs, the PCR sensitivity observed with CpsiA/CpsiB was also better than with the ompA primers. This study demonstrated that these specific pmp primers could serve as valuable, sensitive and common tools for a specific Chlamydophila diagnosis in ruminant, avian and human diseases. Digestion by AluI of the CpsiA/CpsiB fragments allowed a specific discrimination of the strains in function of their hosts and/or their serotypes.     

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.     

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.     

An investigation into the role of Chlamydophila spp. in bovine upper respiratory tract disease

An outbreak of upper respiratory tract disease was investigated in a group of 17 housed home-bred calves on a mixed dairy, beef and sheep farm in Devon. Conjunctival swabs were collected and tested for Chlamydophila spp. DNA using a PCR test that detects Chlamydophila abortus and Chlamydophila psittaci. Six of the calves tested gave a positive result. Further epidemiological observations and laboratory testing indicated that the adult dairy cows, from which the affected calves originated, were the most likely source of infection.     

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%).     

Evaluation of bioaerosol sampling techniques for the detection of Chlamydophila psittaci in contaminated air

Chlamydophila (C.) psittaci, a category B bioterrorism agent, causes respiratory disease in birds and psittacosis or parrot fever in man. The disease spreads aerogenically and no vaccines are available for either birds or man. Highly sensitive C. psittaci bioaerosol monitoring methods are unavailable. We evaluated: (1) dry filtration for collecting C. psittaci from contaminated air using different samplers and membrane filters, (2) impingement into different liquid collection media by use of the AGI-30 impinger and the BioSampler and (3) impaction into newly designed C. psittaci media utilizing the MAS-100 aerosol impactor. For personal bioaerosol sampling, we recommend the use of a gelatin filter in combination with the IOM inhalable dust sampler at an airflow rate of 2L/min. This allowed the detection of 10 organisms of C. psittaci by both PCR and culture. For stationary bioaerosol monitoring, sampling 1000L of air in 10min with the MAS-100 impactor and ChlamyTrap 1 impaction medium was most efficient and made it possible to detect 1 and 10 C. psittaci organisms by PCR and culture, respectively. ChlamyTrap 1 in combination with the MAS-100 impactor might also be applicable for bioaerosol monitoring of viruses.