Waddlia chondrophila is a strict intracellular bacterium belonging to the Chlamydiales order, which also encompasses important human and animal pathogens, including Chlamydia trachomatis and Chlamydia psittaci. Like all other members of the order, W. chondrophila displays a biphasic developmental cycle, alternating between infectious extracellular and replicative intracellular forms.First isolated from a bovine aborted foetus, W. chondrophila was later demonstrated to be associated, in women, with adverse pregnancy outcomes, such as miscarriage. Furthermore, recent studies have demonstrated a high prevalence of anti-W. chondrophila antibodies in asymptomatic women who consulted doctors regarding infertility problems, and in the male patients of infertile couples. Additionally, a role for this bacterium in respiratory tract infections has been suggested, as it has also been recovered from respiratory samples.Although the cause of miscarriage is not identified in ∼50% of cases, nowadays, no systematic diagnostic testing for the presence of W. chondrophila is routinely performed. Implementing large-scale testing in women with reproductive health issues would undoubtedly increase our knowledge of W. chondrophila prevalence and its implications for disease outcomes.

Background/Objective: Recently, we published three studies describing the development and optimization of a new, safe, and efficacious vaccine to protect sheep from ovine enzootic abortion, which is caused by the zoonotic pathogen Chlamydia abortus. The vaccine, which can be delivered through a single inoculation, is based on a detergent-extracted outer membrane protein (chlamydial outer membrane complex or COMC) preparation of the pathogen. This study aimed to optimize the vaccine further by comparing the effects of different adjuvants on protective efficacy. Methods: We evaluated the effectiveness of three different vaccines (2.5 µg COMC) formulated with one of three adjuvants (Montanide ISA 70VG, Montanide ISA 61VG, and QuilA) to reduce the rate of abortion, placental load and pathology, and post-partum vaginal shedding of organisms in comparison to our benchmark 20 µg COMC/Montanide ISA 70 VG vaccine and a challenge control group of animals. The humoral and cellular immunological responses to vaccination and to challenge were also assessed. Results: The two low-dose Montanide formulated vaccines resulted in low abortion rates of 3.2 and 8.1% for ISA 70 VG and ISA 61 VG, respectively, which were comparable to the benchmark vaccine group (2.7%) and considerably lower than the QuilA (23.7%) and challenge control (36.8%) groups. Similarly, the Montanide-adjuvanted groups had much lower bacterial loads (range: 136–431 genome copies) on vaginal swabs post-parturition than the QuilA (8.9 × 104 copies) and challenge control (2.4 × 105 copies) groups. Conclusions: The results showed that both Montanide adjuvants are more effective for maximizing COMC vaccine efficacy than the QuilA adjuvant and result in much lower bacterial shedding of the pathogen post-parturition, which is important for minimizing potential transmission to naïve animals.

Chlamydia abortus is an obligate intracellular bacterium primarily affecting ruminants and causing reproductive failures in sheep and goats. As a recognised zoonotic pathogen, C abortus can cause obstetric and respiratory infections in humans, with severe consequences in pregnant women and immunocompromised individuals. Previous studies have identified novel C abortus strains in avian species, closely related to the zoonotic Chlamydia psittaci, with some strains associated with cases of human pneumonia. In this Personal View, we review the evolving taxonomy of C abortus in light of these novel avian strains, propose subspecies distinctions, and describe the different zoonotic risks associated with both avian and ruminant strains. Challenges in diagnostics, host preference, and case detection are discussed, emphasising the need for a One Health approach to fully understand and mitigate these risks. With increasing reports of human C abortus infections, precise strain classification and differentiation between avian and ruminant subspecies, along with targeted surveillance, are essential to limit their zoonotic transmission.

Background/Objective: Virus-based vaccine vectors have been widely utilised in commercial vaccines, predominantly for virus infections. They also offer promise for bacterial diseases, for which many vaccines are sub-optimal or ineffective. It is well-established for chlamydial infections, including ovine enzootic abortion, that the major outer membrane protein (MOMP) antigen is protective. Immune responses strongly associated with controlling Chlamydiae include cellular interferon-gamma (IFN-γ) production. Methods: A study was conducted to compare the ability of a modified Orf virus vector directly with a modified sheep maedi visna virus vector to deliver the C. abortus antigen ompA and stimulate vaccine-induced responses in sheep. The Orf virus-based vaccine (mORFV-ompA) was found to be more effective in stimulating MOMP-specific antibodies and cellular antigen-driven IFN-γ in immunised sheep. This mORFV-ompA vaccine was assessed in a follow-up immunogenicity investigation in sheep, where the cellular and humoral immune responses elicited following immunisation with the live or inactivated vaccine were determined. Sheep were immunised intramuscularly with a live mORFV-ompA (n = 10) or an inactivated mORFV-ompA (n = 10). An additional group of 10 sheep served as unvaccinated controls. Results: Serological anti-MOMP antibodies and cellular recall responses of peripheral blood mononuclear cells to the native C. abortus antigen were assessed. Immunisation with either the live or inactivated mORFV-ompA-induced anti-MOMP immunoglobulin-G. Antigen-specific cellular responses, characterised by the secretion of IFN-γ and interleukin (IL)-17A, with negligible IL-10 and no IL-4, were detected in lymphocyte stimulation assays from both mORFV groups. No antibody responses to the mORFV platform were detected following immunisations. Conclusions: Both live and inactivated vaccines have the potential to be a platform technology for deployment in sheep. This addresses a notable gap in veterinary vaccine development where the induction of both humoral responses and cellular responses is required without using an adjuvant. The successful use of the MOMP candidate antigen suggests potential utility for bacterial disease deployment.

Pathogens frequently produce proteins to evade or inhibit host immune responses. One such protein is lymphostatin from attaching and effacing Escherichia coli (also known as lymphocyte inhibitory factor A; LifA), which influences intestinal colonization and inhibits mitogen- and antigen-activated proliferation of T lymphocytes and pro-inflammatory cytokine synthesis. Here, we report the cloning, purification and characterization of a LifA homologue from Chlamydia pecorum. The predicted 382 KDa protein (CPE2_0552) exhibited 36% identity and 55% similarity over 3171 amino acids to lymphostatin from enteropathogenic E. coli strain E2348/69. CPE2_0552 shares glycosyltransferase and cysteine protease motifs required for lymphostatin activity, including similarity in the tertiary structure of these domains predicted by AlphaFold 3. Purified CPE2_0552 did not share the L-shaped globular structure of lymphostatin when analyzed by transmission electron microscopy. CPE2_0552 inhibited concanavalin A-stimulated proliferation of bovine T cells in a concentration-dependent manner, with an inhibitory dose 50 (ID50) of 812 pg/mL. This was 38-fold higher than the ID50 of E. coli E2348/69 lymphostatin tested in parallel on T cells from the same donors (21 pg/mL), but was similar to another LifA homologue from E. coli O157:H7 (ToxB). Moreover, CPE2_0552 inhibited the secretion of interferon gamma (IFNγ), a key cytokine that influences the outcome of Chlamydia infections. At the concentrations at which CPE2_0552 inhibited T lymphocyte proliferation and IFNγ secretion, negligible cytotoxicity was observed after 72 h of stimulation. Our study indicates that E. coli lymphostatin belongs to a wider family of lymphocyte-inhibitory molecules that exist in distantly related bacterial pathogens.

Background/Objective: Chlamydia abortus, the cause of ovine enzootic abortion, is a zoonotic bacterial pathogen and one of the most infectious causes of foetal death in sheep worldwide. Although the disease can be controlled using commercial inactivated and live whole-organism vaccines, there are issues with both, particularly concerning efficacy and safety. Recently, we have described the development of a new COMC (chlamydial outer membrane complex) vaccine based on a detergent-extracted outer membrane protein preparation of the pathogen, which can be delivered in a single inoculation and is both efficacious and safe. Methods: In this study, we have evaluated the COMC vaccine further in a dose–response titration of the chlamydial antigen content of the vaccine (from 20 to 2.5 µg in seven experimental groups) using an established pregnant sheep challenge model. Results: No obvious dose–response relationship was observed across the groups, with a single abortion event occurring in four of the groups and three in the lowest dose group (2.5 µg). No abortions occurred in the 15 and 10 µg groups. The abortion rates (0–14%) were significantly below that of the challenge control group (33%). A similar reduction in bacterial shedding of infectious organisms following parturition was observed in the vaccinated groups compared to the challenge control group, which is important in terms of reducing potential transmission to naive animals. Conclusions: The results show that a dose of 10 µg antigen in the vaccine will be optimal in terms of maximising efficacy, reducing shedding at parturition, and ensuring it is cost-effective to produce for commercial manufacture.

Chlamydia abortus is a bacterial pathogen that causes enzootic abortion of ewes (EAE), a disease of sheep and goats with potential risks to human health. Accurate detection is essential for disease management and prevention. While serological tests have been widely used for herd screening, they are lacking in terms of sensitivity and specificity. PCR methods, particularly real-time PCR, are now preferred due to their improved sensitivity and accuracy for confirmation of clinical cases and for use in routine surveillance. This study aimed to compare and evaluate the analytical parameters and diagnostic performance of two commercial PCR tests and four in-house PCR methods for their suitability for diagnosing EAE. Two of the in-house methods are based on the ompA gene, which is specifically used to detect C. abortus in ruminants. The other two tests target different genetic markers, that also allow the detection of avian C. abortus strains. The study found that all PCR tests had a similar analytical sensitivity and were effective in identifying C. abortus in clinical samples, such as placentas, vaginal swabs and fetal organs especially in animals showing symptoms. However, detection in asymptomatic carriers was more difficult. One of the ompA-based assays showed superior diagnostic accuracy, particularly compared to the commercial kits, with a diagnostic sensitivity of 98.0 % and a diagnostic specificity of 96.6 % and is thus the one recommended for routine testing. The study also concluded that the interpretation criteria of the commercial kits should be reviewed to improve their reliability.

Chlamydia abortus causes the disease ovine enzootic abortion, which is one of the most infectious causes of foetal death in small ruminants worldwide. While the disease can be controlled using live and inactivated commercial vaccines, there is scope for improvements in safety for both sheep and human handlers of the vaccines. We have previously reported the development of a new prototype vaccine based on a detergent-extracted outer membrane protein preparation of C. abortus that was determined to be more efficacious and safer than the commercial vaccines when administered in two inoculations three weeks apart. In this new study, we have developed this vaccine further by comparing its efficacy when delivered in one or two (1 × 20 µg and 2 × 10 µg) doses, as well as also comparing the effect of reducing the antigen content of the vaccine by 50% (2 × 5 µg and 1 × 10 µg). All vaccine formulations performed well in comparison to the unvaccinated challenge control group, with no significant differences observed between vaccine groups, demonstrating that the vaccine can be administered as a single inoculation and at a lower dose without compromising efficacy. Future studies should focus on further defining the optimal antigen dose to increase the commercial viability of the vaccine.

Coxiella burnetii is an important zoonotic bacterial pathogen of global importance, causing the disease Q fever in a wide range of animal hosts. Ruminant livestock, in particular sheep and goats, are considered the main reservoir of human infection. Vaccination is a key control measure, and two commercial vaccines based on formalin-inactivated C. burnetii bacterins are currently available for use in livestock and humans. However, their deployment is limited due to significant reactogenicity in individuals previously sensitized to C. burnetii antigens. Furthermore, these vaccines interfere with available serodiagnostic tests which are also based on C. burnetii bacterin antigens. Defined subunit antigen vaccines offer significant advantages, as they can be engineered to reduce reactogenicity and co-designed with serodiagnostic tests to allow discrimination between vaccinated and infected individuals. This study aimed to investigate the diversity of antibody responses to C. burnetii vaccination and/or infection in cattle, goats, humans, and sheep through genome-wide linear epitope mapping to identify candidate vaccine and diagnostic antigens within the predicted bacterial proteome. Using high-density peptide microarrays, we analyzed the seroreactivity in 156 serum samples from vaccinated and infected individuals to peptides derived from 2,092 open-reading frames in the C. burnetii genome. We found significant diversity in the antibody responses within and between species and across different types of C. burnetii exposure. Through the implementation of three different vaccine candidate selection methods, we identified 493 candidate protein antigens for protein subunit vaccine design or serodiagnostic evaluation, of which 65 have been previously described. This is the first study to investigate multi-species seroreactivity against the entire C. burnetii proteome presented as overlapping linear peptides and provides the basis for the selection of antigen targets for next-generation Q fever vaccines and diagnostic tests.

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.

Here, we report the complete genome sequences of Pasteurella multocida strains P504190 and P504188/1, which were isolated from the diseased lungs of a sow and her piglet, respectively. Despite the unusual clinical presentation, whole-genome sequence typing revealed both strains to be capsular type D and lipopolysaccharide (LPS) group 6, commonly found in pigs.

The placenta is the organ that allows the exchange of oxygen and nutrients between maternal and foetal blood, supplying the requirements of the growing foetus. Consequently, any factor that alters placental integrity may affect foetal nutrition, viability and lamb birth weight. Reproductive diseases, such as ovine enzootic abortion (OEA), impact on foetal viability due to the reduction in the functional area for maternofoetal exchange. This study aimed to consider the impact of pathological features of OEA placental lesions on lamb birth weight and indirectly on foetal viability. To investigate the relationship between birth weight and various OEA-related parameters, data from 562 lambs/foetuses from animals experimentally challenged with Chlamydia abortus strain S26/3 and from uninfected animals were analysed. The parameters investigated included the number of foetuses/lambs delivered (single/multiple), foetus/lamb sex, length of gestation, the proportion of placentas affected by lesions (percentage of gross placental pathology), foetal viability (live/aborted) and the number of C. abortus organisms shed in post-parturition vaginal excretions. The results suggest that the length of gestation and the proportion of placentas affected by lesions are the main contributors to birth weight variability, whereas the other factors, including foetal viability (live or aborted outcomes), were found to be less relevant co-variables. The study determined the strongest positive and negative correlations between birth weight were with the length of gestation and the extent of placental pathology, respectively. These results may indicate that economic losses associated with OEA infections result not only from aborted foetuses but also from the surviving lambs that are born weaker and consequently are more susceptible to diseases.

The bacterium Coxiella burnetii can cause the disease Q-fever in a wide range of animal hosts. Ruminants, including sheep, are thought to play a pivotal role in the transmission of C. burnetii to humans; however, the only existing livestock vaccine, namely, Coxevac® (Ceva Animal Health Ltd., Libourne, France), a killed bacterin vaccine based on phase I C. burnetii strain Nine-Mile, is only approved for use in goats and cattle. In this study, a pregnant ewe challenge model was used to determine the protective effects of Coxevac® and an experimental bacterin vaccine based on phase II C. burnetii against C. burnetii challenge. Prior to mating, ewes (n = 20 per group) were vaccinated subcutaneously with either Coxevac®, the phase II vaccine, or were unvaccinated. A subset of pregnant ewes (n = 6) from each group was then challenged 151 days later (~100 days of gestation) with 106 infectious mouse doses of C. burnetii, Nine-Mile strain RSA493. Both vaccines provided protection against C. burnetii challenge as measured by reductions in bacterial shedding in faeces, milk and vaginal mucus, and reduced abnormal pregnancies, compared to unvaccinated controls. This work highlights that the phase I vaccine Coxevac® can protect ewes against C. burnetii infection. Furthermore, the phase II vaccine provided comparable levels of protection and may offer a safer and cost-effective alternative to the currently licensed vaccine.

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 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, including C. psittaci strain 84/2334 that was isolated from a parrot. Methods Strain 84/2334 was analysed by multi-locus sequence typing of seven housekeeping genes and by analysis of five species-discriminant proteins. The strain was de novo sequenced for comparison with representative strains from chlamydial species. 16S rRNA and whole genome phylogenetic analyses, network and recombination analyses, and analyses of key virulence-associated genes were conducted to explore its evolutionary relationship with both C. psittaci and C. abortus. Results Typing, 16S rRNA and whole genome phylogenetic analyses, as well as network and recombination analyses showed that strain 84/2334 clusters closely with C. abortus. Analyses also suggested a closer evolutionary relationship with classical C. abortus strains, than to two other avian C. abortus or C. psittaci strains. Analyses of virulence-associated genes that exhibit greatest diversity within chlamydial species, reveal greater diversity than present in sequenced C. abortus genomes. The strain also possesses an extrachromosomal plasmid, as found in C. psittaci species but absent from all sequenced classical C. abortus strains. Conclusion The results are consistent with 84/2334 being a C. abortus ancestral strain, suggesting that it should be reclassified as C. abortus. The identification of a C. abortus strain bearing an extra-chromosomal plasmid has implications for plasmid-based transformation studies providing a potential route for future vaccine development.

Aims The order Chlamydiales comprises a broad range of bacterial pathogens and endosymbionts, which infect a wide variety of host species. Within this order, members of the family Parachlamydiaceae, which includes Parachlamydia and Neochlamydia species, have been particularly associated with infections in both humans and cattle, including having a potential pathogenic role in cases of bovine abortion. While the route of transmission has yet to be defined, it has been hypothesised that asymptomatic carriage and contamination of the immediate environment may be a route of inter-animal transmission. We investigated the asymptomatic carriage of Chlamydia-related organisms in healthy cattle. Methods & Results DNA was isolated from nasal and rectal swabs obtained from 38 healthy dairy heifers. A Chlamydiales sp. 16S rRNA qPCR was performed on each sample. A total of 18/38 nasal samples and all 38/38 rectal samples were identified as positive for Chlamydiales sp. Each positive sample was sequenced confirming the presence of DNA belonging to the Parachlamydiaceae. Conclusions The presence of Parachlamydiaceae DNA in nasal and rectal swab samples of healthy cattle provides evidence for the asymptomatic carriage of parachlamydial organisms within cattle. Significance & Impact of the Study The study provides evidence of potential routes of environmental contamination that could provide a route for inter-animal and animal transmission of Parachlamydiaceae.

The presence and zoonotic transfer of four different avian Chlamydia spp. was assessed in an epidemiological study in a psittacine bird population and its owners. Fecal swabs from 84 pet birds and pharyngeal swabs from 22 bird owners were collected from 21 locations in Flanders. Samples were examined using established and novel PCR platforms combined with culture on PCR-positive samples. Chlamydiaceae DNA was detected in 33 of 84 (39.3%) birds. The predominant part of the avian infections could be attributed to C. psittaci (22 of 84; 26.2%), followed by C. avium (11 of 84; 13.1%). C. gallinacea and C. abortus were not detected in birds or humans. C. psittaci was the only species detected in pet bird owners (4 of 22; 18.2%), stressing its zoonotic importance. This study showed that C. psittaci and the more recently discovered novel avian species C. avium are undoubtedly present in the Flemish psittacine bird population. Our results justify additional research in a larger psittacine bird population and its owners, focusing on C. psittaci and C. avium. In the meantime, increased awareness among pet bird owners and the implementation of preventive measures in the pet bird industry is advised to limit the circulation of established and novel emerging avian chlamydial species.

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.

We report the complete genome sequence of Chlamydia abortus MRI-10/19, recovered from the infected placenta of a sheep that had been vaccinated with the commercial live attenuated C. abortus 1B vaccine strain. Comparative analysis revealed 1 single nucleotide polymorphism (SNP) difference and 4 indels compared to the vaccine strain.

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.