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.     

Efficacy of live Chlamydophila abortus vaccine 1B in protecting mice placentas and foetuses against strains of Chlamydophila pecorum isolated from cases of abortion

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

Protection of pregnant mice against placental and splenic infection by three strains of Chlamydophila abortus with a live 1B vaccine

The efficacy of a live 1B vaccine against three strains of Chlamydophila abortus, AB16, LLG and POS, was assessed in pregnant mice in terms of the reduction in the levels of infection recorded in their placentas, fetuses and spleens. The vaccine was more effective against the AB16 strain than against the LLG and POS strains, suggesting that there are antigenic differences between the three strains.     

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.     

Induction of a protective immune response against swine Chlamydophila abortus infection in mice following co-vaccination of omp-1 DNA with recombinant MOMP

Chlamydophila abortus is the causative agent of abortion in pigs and pregnant women. Seroconversion rates were arranged from 11% to 80% in piglets and sows in China. These very high rates illustrate the scale of the problem in China and highlight the urgent need for the development of a C. abortus vaccine. An efficacious anti-chlamydial vaccine should induce not only strong mucosal and systemic T-helper type 1 (Th1) immune response but also give a humoral response that enhances Th1 activation following infection. In order to evaluate an active immune response of a combination of the major outer membrane protein (MOMP) DNA- and protein-based vaccines, 54 BALB/c mice were randomly assigned to six groups and inoculated intramuscularly with: (i) 100 microg pcDNA::MOMP, (ii) 10 microg r-MOMP, (iii) primed with 100 microg pcDNA::MOMP and boosted with 10 microg r-MOMP, (iv) primed-boosted with a combination of pcDNA::MOMP and r-MOMP simultaneously, (v) live-attenuated 1B vaccine, (vi) 100 microg pcDNA3.1 vector. All animals were vaccinated two times at 14 days intervals. Results showed that mice given DNA and r-MOMP induced higher antibody levels, higher T cells proliferation and an elevated level of chlamydial clearance in spleen, which was equivalent to the clearance of 1B vaccine. Mice administrated the DNA-primed/MOMP-boosted approach elicited moderate antibody levels, less T-lymphocyte proliferation and lower chlamydial clearance as compared with 1B vaccine. Co-immunization with DNA- and r-MOMP vaccine may provide novel ways for active immunization strategy against swine C. abortus.     

Efficacy of different commercial and new inactivated vaccines against ovine enzootic abortion

The protective efficacy of two inactivated commercial (A, B) and two new inactivated vaccines (M7, QS) against ovine enzootic abortion was determined in two separate experiments in sheep. Vaccine A contained chlamydiae propagated in chicken embryos, adjuvated with Marcol 82, and vaccine B contained chlamydiae cultured in cell monolayers, adjuvated with aluminium hydroxide. For the preparation of the experimental vaccines, Chlamydophila abortus AB7 strain was cultured in McCoy cells and adjuvated with QS-21 (QS) or Montanide ISA 773 (M7). The ewes were vaccinated twice subcutaneously and challenged at 90 days of gestation. Protection was evaluated by clinical, bacteriological and serological examinations, and compared to two control groups: one of infected but not vaccinated ewes, and another of vaccinated but not infected ewes. The experimental vaccines induced considerably better protection than the two commercial ones. The new vaccine M7 especially showed no abortions, a good antibody response, the highest newborn lamb weights and the lowest level of C. abortus shedding at lambing.     

Protection of mice against Chlamydophila abortus infection with a bacteriophage-mediated DNA vaccine expressing the major outer membrane protein

A bacteriophage-delivered DNA vaccine against Chlamydophila abortus was constructed by cloning a eukaryotic cassette containing the ompA gene (which expresses the Major Outer Membrane Protein) into a bacteriophage lambda vector. Four groups, each of 20 BALB/c mice were inoculated separately with the phage vaccine, a conventional DNA vaccine based on the same ompA expression cassette, a live attenuated vaccine (strain 1B) or the empty phage vector. The phage and DNA vaccines and empty phage vector were administered intramuscularly on days 0, 14 and 28; the attenuated vaccine was given once on day 0. Half the animals in each group were challenged on day 42 by intraperitoneal injection of live C. abortus and sacrificed on day 49. Phage-vaccinated mice developed moderate antibody levels against C. abortus and yielded higher levels of IFN-γ and IL-2 compared with the attenuated live vaccine group. Clearance of chlamydiae from spleens was significantly better in the attenuated vaccine group compared with the phage vaccine group, while both groups were significantly superior to the DNA vaccine and control groups (p<0.01). Although levels of protection in the mouse model were lower in phage-vaccinated animals, than in 1B vaccinated animals, phage vaccines offer several other advantages, such as easier handling and safety, potentially cheaper production and no chance of reversion to virulence. Although these are preliminary results in a model system, it is possible that with further optimisation immunization with phage vaccines may provide a novel way to improve protection against C. abortus infection and trials in large animals are currently being initiated.     

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.     

中国牛种布鲁菌弱毒疫苗株A19 SNP位点的研究

为鉴别我国牛种布鲁菌弱毒疫苗株A19与野生菌株,运用生物信息学方法结合基因测序,对疫苗株A19基因组SNP位点分析筛选,选取其中部分SNP位点,通过与布鲁菌常见种、生物型标准参考菌株和弱毒疫苗株基因组SNP位置核苷酸测序比较,验证SNP位点的A19特异性。结果表明,共筛选获得A19基因组29个SNP位点,验证ClpX G825-C825、LysR A605-C605、Omp2b G503-A503这3个SNP位点为A19（或S19）特异,揭示了A19基因组SNP位点分布情况,为疫苗株 A19与野生菌株鉴别提供了分子依据。     

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.     

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.     

Identification of Brucella abortus strain 19 by decreased ability to utilize erythritol as determined by gas liquid chromatography

A method to identify Brucella abortus strain 19 by erythritol utilization using gas liquid chromatography (GLC) was developed. A total of 69 strains of B. abortus (41 virulent field strain isolates and 28 strain 19 isolates) were tested. Following incubation of the isolate with a standard amount of erythritol, the erythritol present in the cell suspension was acetylated and measured by GLC. Field strains of B. abortus utilized an average of 90.9% of the erythritol, whereas vaccine strains utilized an average of 42.4%. This difference in erythritol utilization will allow a more rapid identification of B. abortus strain 19.     

New real-time PCR tests for species-specific detection of Chlamydophila psittaci and Chlamydophila abortus from tissue samples

Chlamydophila psittaci and Chlamydophila abortus are the causative agents of avian chlamydiosis (psittacosis) and ovine enzootic abortion, respectively. Both pathogens are known to possess zoonotic potential. Due to their close genetic relatedness, direct and rapid species identification is difficult. In the present study, new real-time PCR assays are reported for both species. The tests are based on highly specific probes targeting the ompA gene region and were conducted as duplex PCRs including an internal amplification control. The Cp. psittaci assay successfully passed a proficiency test at national level. Examination of field samples revealed Cp. psittaci as the dominating species in birds, but also Cp. abortus in a few psittacines. Real-time PCR assays for species-specific detection of Cp. psittaci and Cp. abortus are suited for routine diagnosis, which renders them important tools for the recognition of outbreaks of psittacosis and ovine enzootic abortion.     

Characterization of a murine model of intranasal infection suitable for testing vaccines against C. abortus

Mouse models have been widely used to test candidate vaccines against Chlamydophila abortus infection in mice. Although the induction of a systemic infection by endogenous or intraperitoneal inoculation is a useful tool for understanding the immune mechanism involved in the protection conferred by the vaccination, a different approach is necessary to understand other factors of the infection, such as mucosal immunity or the colonization of target organs. To test whether C. abortus intranasal model of infection in mice is a useful tool for testing vaccines in a first group of experiments mice, were infected intranasally with C. abortus to characterize the model of infection. When this model was used to test vaccines, two inactivated experimental vaccines, one of them adjuvated with QS-21 and another with aluminium hydroxide, and a live attenuated vaccine (strain 1B) were used. Non-vaccinated control mice died within the first 8 days, after displaying substantial loss of weight. Histologically, the mice showed lobar fibrinopurulent bronchointerstitial pneumonia. Prior immunization with QS-21 adjuvated vaccine or 1B vaccine presented mortality and the recipients showed a greater number of T cells in the lesions, especially CD8(+) T cells, than the control mice and mice immunized with vaccine adjuvated with aluminium hydroxide. The results confirm that the C. abortus intranasal model of infection in mice is a useful tool for testing vaccines.     

Brucella abortus INTA2, a novel strain 19 (Delta)bp26::luc (Delta)bmp18 double mutant lacking drug resistance markers

Brucella abortus INTA2, a novel mutant strain, was constructed by inactivation of two B. abortus S19 genes: bp26 and bmp18, with the objective of obtaining a mutant strain that could be compatible with a diagnostic test and have less residual virulence than strain 19. The double mutant was constructed by replacing a large section of the bp26 coding region with the luciferase (luc) coding gene, resulting in mutant strain B. abortus M1luc, followed by partial deletion of bmp18 coding sequence. Both genes were inactivated by allelic replacement assisted by sacB counter-selection. Luciferase expression was evaluated and confirmed that it is a valid marker in the construction of mutant strains. When B. abortus INTA2 was inoculated in BALB/c mice, significantly fewer colony forming units (CFUs) were recovered from mice spleens during initial phase of infection. No splenomegaly was observed in strain INTA2-immunized mice at any time suggesting that strain INTA2 has lost some residual virulence of the parental strain. Nevertheless, similar protection levels against virulent challenge were observed in mice immunized with strains INTA2 or S19. Although strain INTA2 would still induce O-side antibodies, it does not express BP26. This would allow differentiation of INTA2-vaccinated animals from animals infected with field strains by measuring anti-BP26 antibodies, either by an agglutination test or ELISA using BP26 as antigen. Altogether these results indicate that B. abortus INTA2 might be a promising vaccine strain against brucellosis.     

Seroprevalence of selected viral and bacterial pathogens in free-ranging European bison from the Białowieza Primeval Forest (Poland)

Blood was collected from bison that were selected between 1991 and 2001 for poor body condition, cachexia, lameness and balanoposthitis. Sera were tested for antibodies to bovine herpes virus type 1 (BHV-1), bovine viral diarrhoea virus (BVDV), foot-and-mouth disease virus (FMDV), parainfluenza virus type 3 (PI-3), Brucella abortus, Chlamydophila abortus, Coxiella burnetti, and Leptospira interrogans. The results of serological tests showed a prevalence of low titers of a serological reaction to Chlamydophila abortus (45.1%), bovine viral diarrhoea virus (29.5%), Leptospira interrogans (21.3%) and parainfluenza virus type 3 (13.9%). There were differences in the prevalence of antibodies to Ch. abortus between female and male bison. Futhermore, a relationship between age and antibodies to BVDV was observed in older bison. These results suggest that there is some transmission of bacterial and viral pathogens occurring between domestic and wild ruminants grazing in the same pastures in the peripherial areas of Bialowieza Primeval Forest.     

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.     

Enhanced efficacy of recombinant Brucella abortus RB51 vaccines against B. melitensis infection in mice

Brucella abortus strain RB51 is an attenuated rough strain, currently being used as the official live vaccine for bovine brucellosis in the USA and several other countries. In strain RB51, the wboA gene, encoding a glycosyltransferase required for the O-side chain synthesis, is disrupted by an IS711 element. Recently, we have demonstrated that strain RB51WboA, RB51 complemented with a functional wboA gene, remains rough but expresses low quantities of O-side chain in the cytoplasm. Mice vaccinated with strain RB51WboA develop greatly enhanced resistance against challenge with B. abortus virulent strain 2308. We have also demonstrated that overexpression of Cu/Zn superoxide dismutase (SOD) in strain RB51 (RB51SOD) significantly increases its vaccine efficacy against strain 2308 challenge. In this study, we constructed a new recombinant strain, RB51SOD/WboA, that over expresses SOD with simultaneous expression of O-side chain in the cytoplasm. We tested the vaccine potential of strains RB51SOD, RB51WboA, RB51SOD/WboA against challenge with virulent Brucella melitensis 16M and B. abortus 2308 in mice. In comparison with strain RB51, strain RB51SOD induced better protection against strain 2308, but not strain 16M, challenge. Similar to strain RB51WboA, vaccination with strain RB51SOD/WboA resulted in complete protection of the mice from infection with strain 2308. When challenged with strain 16M, mice vaccinated with either strain RB51WboA or strain RB51SOD/WboA were significantly better protected than those vaccinated with strain RB51 or RB51SOD. These results suggest that strains RB51WboA and RB51SOD/WboA are good vaccine candidates for inducing enhanced protection against B. melitensis infection.     

The virB-encoded type IV secretion system is critical for establishment of infection and persistence of Brucella ovis infection in mice

Brucella spp. are gram-negative intracellular bacterial pathogens that cause chronic infections. Brucella virulence factors include a type IV secretion system (T4SS) and its lipopolysaccharide (LPS), which are essential for persistence. However, the role of the virB-encoded T4SS has not been investigated in naturally rough Brucella species such as Brucella ovis. In this study, male 6-week old BALBc mice were infected with B. ovis, Brucella abortus, and their respective ΔvirB2 mutant strains. During early infection, B. ovis and B. abortus wild type strains were similarly recovered from spleen. Interestingly, in contrast to ΔvirB2 B. abortus that was recovered at similar levels when compared to the wild type strain, the ΔvirB2 B. ovis was markedly attenuated as early as 24h post infection (hpi). The ΔvirB2 B. ovis was unable to survive and multiply in murine peritoneal macrophages and extracellularly within the peritoneal cavity at 12 and 24 hpi with lower splenic colonization than the parental strain at 6, 12 and 24 hpi. In contrast, wild type B. abortus and ΔvirB2 B. abortus had a similar kinetics of infection in this model. As expected, the T4SS was essential for intracellular replication of smooth and rough strains in RAW macrophages at 48 hpi. These results suggest that T4SS is important for survival of B. ovis in murine model, and that a T4SS deficient B. ovis strain is cleared at earlier stages of infection when compared to a similar B. abortus mutant.     

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.