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Background Chlamydia abortus and Chlamydia psittaci are important pathogens of livestock and avian species, respectively. While C. abortus is recognized as descended from C. psittaci species, there is emerging evidence of strains that are intermediary between the two species, suggesting they are recent evolutionary ancestors of C. abortus . Such strains include C. psittaci strain 84/2334 that was isolated from a parrot. Our aim was to classify this strain by sequencing its genome and explore its evolutionary relationship to both C. abortus and C. psittaci . Results In this study, methods based on multi-locus sequence typing (MLST) of seven housekeeping genes and on typing of five species discriminant proteins showed that strain 84/2334 clustered with C. abortus species. Furthermore, whole genome de novo sequencing of the strain revealed greater similarity to C. abortus in terms of GC content, while 16S rRNA and whole genome phylogenetic analysis, as well as network and recombination analysis showed that the strain clusters more closely with C. abortus strains. The analysis also suggested a closer evolutionary relationship between this strain and the major C. abortus clade, than to two other intermediary avian C. abortus strains or C. psittaci strains. Molecular analyses of genes (polymorphic membrane protein and transmembrane head protein genes) and loci (plasticity zone), found in key virulence-associated regions that exhibit greatest diversity within and between chlamydial species, reveal greater diversity than present in sequenced C. abortus genomes as well as similar features to both C. abortus and C. psittaci species. The strain also possesses an extrachromosomal plasmid, as found in most C. psittaci species but absent from all sequenced classical C. abortus strains. Conclusion Overall, the results show that C. psittaci strain 84/2334 clusters very closely with C. abortus strains, and are consistent with the strain being a recent C. abortus ancestral species. This suggests that the strain should be reclassified as C. abortus . Furthermore, the identification of a C. abortus strain bearing an extra-chromosomal plasmid has implications for plasmid-based transformation studies to investigate gene function as well as providing a potential route for the development of a next generation vaccine to protect livestock from C. abortus infection.

Chlamydia abortus is one of the most commonly diagnosed causes of infectious abortion in small ruminants worldwide. Control of the disease (Enzootic Abortion of Ewes or EAE) is achieved using the commercial live, attenuated C . abortus 1B vaccine strain, which can be distinguished from virulent wild-type (wt) strains by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Published studies applying this typing method and whole-genome sequence analyses to cases of EAE in vaccinated and non-vaccinated animals have provided strong evidence that the 1B strain is not attenuated and can infect the placenta causing disease in some ewes. Therefore, the objective of this study was to characterise the lesions found in the placentas of ewes vaccinated with the 1B strain and to compare these to those resulting from a wt infection. A C . abortus -free flock of multiparous adult ewes was vaccinated twice, over three breeding seasons, each before mating, with the commercial C . abortus 1B vaccine strain (Cevac® Chlamydia, Ceva Animal Health Ltd.). In the second lambing season following vaccination, placentas (n = 117) were collected at parturition and analysed by C . abortus -specific real-time quantitative PCR (qPCR). Two placentas, from a single ewe, which gave birth to live twin lambs, were found to be positive by qPCR and viable organisms were recovered and identified as vaccine type (vt) by PCR-RFLP, with no evidence of any wt strain being present. All cotyledons from the vt-infected placentas were analysed by histopathology and immunohistochemistry and compared to those from wt-infected placentas. Both vt-infected placentas showed lesions typical of those found in a wt infection in terms of their severity, distribution, and associated intensity of antigen labelling. These results conclusively demonstrate that the 1B strain can infect the placenta, producing typical EAE placental lesions that are indistinguishable from those found in wt infected animals.

Livestock abortion is an important cause of productivity losses worldwide and many infectious causes of abortion are zoonotic pathogens that impact on human health. Little is known about the relative importance of infectious causes of livestock abortion in Africa, including in subsistence farming communities that are critically dependent on livestock for food, income, and wellbeing. We conducted a prospective cohort study of livestock abortion, supported by cross-sectional serosurveillance, to determine aetiologies of livestock abortions in livestock in Tanzania. This approach generated several important findings including detection of a Rift Valley fever virus outbreak in cattle; high prevalence of C. burnetii infection in livestock; and the first report of Neospora caninum , Toxoplasma gondii, and pestiviruses associated with livestock abortion in Tanzania. Our approach provides a model for abortion surveillance in resource-limited settings. Our findings add substantially to current knowledge in sub-Saharan Africa, providing important evidence from which to prioritise disease interventions.

Background To date, whole genome sequencing has been performed mainly for isolates of Chlamydia trachomatis , C. pneumoniae , C. psittaci and C. abortus , but only a few isolates of C. pecorum have been entirely sequenced and this makes it difficult to understand its diversity and population structure. In this study the genome of two C. pecorum strains isolated from the lung of an Alpine chamois affected with pneumonia (isolate PV7855) and the brain of a water buffalo affected with meningoencephalomyelitis (isolate PV6959), were completely sequenced with MiSeq system (Illumina) and analyzed in their most polymorphic regions. Results The genome length and GC content of the two isolates were found to be consistent with other C. pecorum isolates and the gene content of polymorphic membrane proteins and plasticity zone was found to be very similar. Some differences were observed in the phospholipase genes for both isolates and in the number of genes in the plasticity zone, such as the presence of some hypothetical proteins in PV6959, not present in any other genomes analyzed in this study. Interestingly, PV6959 possesses an extra pmp and has an incomplete tryptophan biosynthesis operon. Plasmids were detected in both isolates. Conclusions Genome sequencing of the two C. pecorum strains did not reveal differences in length and GC content despite the origin from different animal species with different clinical disease. In the plasticity zone, the differences in the genes pattern might be related to the onset of specific symptoms or infection of specific hosts. The absence of a tryptophan biosynthesis pathway in PV6959 may suggest a strict relationship between C. pecorum and its host.

Latency is a key feature of the animal pathogen Chlamydia abortus, where infection remains inapparent in the non-pregnant animal and only becomes evident during a subsequent pregnancy. Often the first sign that an animal is infected is abortion occurring late in gestation. Despite this, little is understood of the underlying mechanisms that control latency or the recrudescence of infection that occurs during subsequent pregnancy. The aim of this study was to develop an experimental model of latency by mimicking the natural route of infection through the intranasal inoculation of non-pregnant sheep with C. abortus. Three groups of sheep (groups 1, 2 and 3) were experimentally infected with different doses of C. abortus (5×10(3), 5×10(5) and 5×10(7) inclusion forming units (IFU), respectively) prior to mating and monitored over 2 breeding cycles for clinical, microbiological, pathological, immunological and serological outcomes. Two further groups received either negative control inoculum (group 4a,b) or were inoculated subcutaneously on day 70 of gestation with 2×10(6) IFU C. abortus (group 5). Animals in groups 1, 2 and 5 experienced an abortion rate of 50-67%, while only one animal aborted in group 3 and none in group 4a,b. Pathological, microbiological, immunological and serological analyses support the view that the maternal protective immune response is influenced by initial exposure to the bacterium. The results show that intranasal administration of non-pregnant sheep with a low/medium dose of C. abortus results in a latent infection that leads in a subsequent pregnancy to infection of the placenta and abortion. In contrast a high dose stimulates protective immunity, resulting in a much lower abortion rate. This model will be useful in understanding the mechanisms of infection underlying latency and onset of disease, as well as in the development of novel therapeutics and vaccines for controlling infection.

Ovine enzootic abortion (OEA) caused by the obligate intracellular bacterial pathogen Chlamydia abortus ( C. abortus ), is an endemic disease in most sheep-rearing countries worldwide. Following infection, C. abortus establishes a complex host–pathogen interaction with a latent phase in non-pregnant sheep followed by an active disease phase in the placenta during pregnancy leading to OEA. Improved knowledge of the host–pathogen interactions at these different phases of disease will accelerate the development of new diagnostic tests and vaccines to control OEA. Current evidence indicates that cellular immunity is essential for controlling C. abortus infection. We have previously described a model of mucosal (intranasal) infection of non-pregnant sheep with C. abortus that replicates the latent and active phases of OEA. We have investigated antigen-specific recall responses of peripheral blood mononuclear cells (PBMC) in sheep infected with C. abortus via the intranasal route to determine how these change during the latent and active phases of disease. By analysing cytokines associated with the major CD4 +ve T helper (T h ) cell subsets (Interferon-gamma (IFN-γ)/T h 1; Interleukin (IL)-4/T h 2; IL-17A/T h 17; IL-10/T regulatory ), we show that there is selective activation of PBMC producing IFN-γ and/or IL-10 during the latent phase following infection. These cytokines are also elevated during the active disease phase and while they are produced by sheep that are protected from OEA, they are also produced by sheep that abort, highlighting the difficulties in finding specific cellular immunological correlates of protection for complex intracellular pathogens.

Data are presented on the identification and partial characterisation of proteins comprising the chlamydial outer membrane complex (COMC) fraction of Chlamydia abortus (C. abortus)-the aetiological agent of ovine enzootic abortion. Inoculation with the COMC fraction is known to be highly effective in protecting sheep against experimental challenge and its constituent proteins are therefore of interest as potential vaccine candidates. Sodium N-lauroylsarcosine (sarkosyl) insoluble COMC proteins resolved by SDS-PAGE were interrogated by mass spectrometry using combined rapid monolithic column liquid chromatography and fast MS/MS scanning. Downstream database mining of processed tandem MS data revealed the presence of 67 proteins in total, including putative membrane associated proteins (n = 36), such as porins, polymorphic membrane proteins (Pmps), chaperonins and hypothetical membrane proteins, in addition to others (n = 22) that appear more likely to have originated from other subcellular compartments. Electrophoretic mobility data combined with detailed amino acid sequence information derived from secondary fragmentation spectra for 8 Pmps enabled peptides originating from protein cleavage fragments to be mapped to corresponding regions of parent precursor molecules yielding preliminary evidence in support of endogenous post-translational processing of outer membrane proteins in C. abortus. The data presented here will facilitate a deeper understanding of the pathogenesis of C. abortus infection and represent an important step towards the elucidation of the mechanisms of immunoprotection against C. abortus infection and the identification of potential target vaccine candidate antigens.

This study investigated the pathogenesis of two variant strains (LLG and POS) of Chlamydia abortus, in comparison to a typical wild-type strain (S26/3) which is known to be responsible for late term abortion in small ruminants. Challenge with the three strains at mid-gestation resulted in similar pregnancy outcomes, with abortion occurring in approximately 50-60% of ewes with the mean gestational lengths also being similar. However, differences were observed in the severity of placental pathology, with infection appearing milder for strain LLG, which was reflected in the lower number of organisms shed in vaginal swabs post-partum and less gross pathology and organisms present in placental smears. Results for strain POS were somewhat different than LLG with a more focal restriction of infection observed. Post-abortion antibody responses revealed prominent differences in seropositivity to the major outer membrane protein (MOMP) present in elementary body (EB) preparations under denaturing conditions, most notably with anti-LLG and anti-POS convalescent sera where there was no or reduced detection of MOMP present in EBs derived from the three strains. These results and additional analysis of whole EB and chlamydial outer membrane complex preparations suggest that there are conformational differences in MOMP for the three strains. Overall, the results suggest that gross placental pathology and clinical outcome is not indicative of bacterial colonization and the severity of infection. The results also highlight potential conformational differences in MOMP epitopes that perhaps impact on disease diagnosis and the development of new vaccines.

A variety of Chlamydia species belonging to the Chlamydiaceae family have been reported in birds. Until recently, C. psittaci was considered to be the most common avian species, although found in both birds and mammals, while C. abortus has only been found in mammals. Recently, a new group of avian C. abortus strains with worldwide distribution in various wild bird families has been described. In this study, whole genome sequencing (WGS) of three of these strains (15-70d24, 15-49d3 and 15-58d44, representing genotypes G1, G2 and 1V, respectively) that were isolated from wild birds were analysed. Genome assemblies based on both short-read Illumina and long-read Nanopore data indicate that these avian C. abortus strains show features characteristic of both C. abortus and C. psittaci species, although phylogenetic analyses demonstrate a closer relationship with classical C. abortus strains. Currently, species classification established by the ICSP Subcommittee on the taxonomy of Chlamydiae, determines that these avian C. abortus strains 15-70d24, 15-49d3 and 15-58d44 should be classified as C. abortus. However, the authors of this study conclude that the current taxonomic definition of C. abortus is outdated and should be amended to include two subgroups, mammalian and avian, the latter of which would include all isolates so far referred to as atypical C. psittaci or C. psittaci/C. abortus intermediates.

The enzootic abortion of ewes, caused by the bacterium Chlamydia abortus (C. abortus), is one of the main causes of abortion in sheep. There are multiple contributory factors, including chlamydial growth, host immune response, and hormonal balance, that result in different pregnancy outcomes, such as abortion, the birth of weak lambs that may die, or healthy lambs. This study aimed to determine the relationship between phenotypical patterns of immune cell infiltration and different pregnancy outcomes in twin-bearing sheep (both lambs born dead; one alive and one dead; both alive) when experimentally infected with C. abortus. Both the sheep uteri and placentae were collected after parturition. All samples were analysed for specific immune cell features, including cell surface antigens and the T-regulatory (Treg) cell-associated transcription factor and cytokines, by immunohistochemistry and in situ hybridisation. Some of these immunological antigens were evaluated in ovine reproductive tissues for the first time. Differential patterns of T helper/Treg cells revealed significant group effects in the placentae. It suggests the potential role that the balance of lymphocyte subsets may play in affecting different pregnancy outcomes in C. abortus-infected sheep. The present study provides novel detailed information about the immune responses observed at the maternofoetal interface in sheep at the time of pre-term abortion or lambing.