Comparative analysis of two genomes of Chlamydia pecorum isolates from an Alpine chamois and a water buffalo.

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.

Identification of Parachlamydiaceae DNA in nasal and rectal passages of healthy dairy cattle.

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.

Whole genome de novo sequencing and comparative genomic analyses suggests that Chlamydia psittaci strain 84/2334 should be reclassified as Chlamydia abortus species.

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.

Defining immune correlates during latent and active chlamydial infection in sheep.

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 Thelper (Th) cell subsets (Interferon-gamma (IFN-γ)/Th1; Interleukin (IL)-4/Th2; IL-17A/Th17; IL-10/Tregulatory), 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.

Analysis of Humoral Immune Responses to Surface and Virulence-Associated Chlamydia abortus Proteins in Ovine and Human Abortions by Use of a Newly Developed Line Immunoassay.

The obligate intracellular bacterium Chlamydia abortus is the causative agent of enzootic abortion of ewes and poses a significant zoonotic risk for pregnant women. Using proteomic analysis and gene expression library screening in a previous project, we identified potential virulence factors and candidates for serodiagnosis, of which nine were scrutinized here with a strip immunoassay. We have shown that aborting sheep exhibited a strong antibody response to surface (MOMP, MIP, Pmp13G) and virulence-associated (CPAF, TARP, SINC) antigens. While the latter disappeared within 18 weeks following abortion in a majority of the animals, antibodies to surface proteins persisted beyond the duration of the study. In contrast, nonaborting experimentally infected sheep developed mainly antibodies to surface antigens (MOMP, MIP, Pmp13G), all of which did not persist. We were also able to detect antibodies to these surface antigens in C abortus-infected women who had undergone septic abortion, whereas a group of shepherds and veterinarians with occupational exposure to C abortus-infected sheep revealed only sporadic immune responses to the antigens selected. The most specific antigen for the serodiagnosis of human C abortus infections was Pmp13G, which showed no cross-reactivity with other chlamydiae infecting humans. We suggest that Pmp13G-based serodiagnosis accomplished by the detection of antibodies to virulence-associated antigens such as CPAF, TARP, and SINC may improve the laboratory diagnosis of human and animal C abortus infections.

Genome sequencing and comparative analysis of three Chlamydia pecorum strains associated with different pathogenic outcomes.

BACKGROUND: Chlamydia pecorum is the causative agent of a number of acute diseases, but most often causes persistent, subclinical infection in ruminants, swine and birds. In this study, the genome sequences of three C. pecorum strains isolated from the faeces of a sheep with inapparent enteric infection (strain W73), from the synovial fluid of a sheep with polyarthritis (strain P787) and from a cervical swab taken from a cow with metritis (strain PV3056/3) were determined using Illumina/Solexa and Roche 454 genome sequencing. RESULTS: Gene order and synteny was almost identical between C. pecorum strains and C. psittaci. Differences between C. pecorum and other chlamydiae occurred at a number of loci, including the plasticity zone, which contained a MAC/perforin domain protein, two copies of a >3400 amino acid putative cytotoxin gene and four (PV3056/3) or five (P787 and W73) genes encoding phospholipase D. Chlamydia pecorum contains an almost intact tryptophan biosynthesis operon encoding trpABCDFR and has the ability to sequester kynurenine from its host, however it lacks the genes folA, folKP and folB required for folate metabolism found in other chlamydiae. A total of 15 polymorphic membrane proteins were identified, belonging to six pmp families. Strains possess an intact type III secretion system composed of 18 structural genes and accessory proteins, however a number of putative inc effector proteins widely distributed in chlamydiae are absent from C. pecorum. Two genes encoding the hypothetical protein ORF663 and IncA contain variable numbers of repeat sequences that could be associated with persistence of infection. CONCLUSIONS: Genome sequencing of three C. pecorum strains, originating from animals with different disease manifestations, has identified differences in ORF663 and pseudogene content between strains and has identified genes and metabolic traits that may influence intracellular survival, pathogenicity and evasion of the host immune system.

De Novo Whole-Genome Sequencing of Two Pathogenic Pasteurella multocida Type D:6 Strains Isolated from Pigs.

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 Immune Response in the Uteri and Placentae of Chlamydia abortus-Infected Ewes and Its Association with Pregnancy Outcomes.

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.

Efficacy of Phase I and Phase II Coxiella burnetii Bacterin Vaccines in a Pregnant Ewe Challenge Model.

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.

Prevalence of New and Established Avian Chlamydial Species in Humans and Their Psittacine Pet Birds in Belgium.

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.

Prospective cohort study reveals unexpected aetiologies of livestock abortion in northern Tanzania.

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.

Genome sequence of the Chlamydophila abortus variant strain LLG.

Chlamydophila abortus is a common cause of ruminant abortion. Here we report the genome sequence of strain LLG, which differs genotypically and phenotypically from the wild-type strain S26/3. Genome sequencing revealed differences between LLG and S26/3 to occur in pseudogene content, in transmembrane head/inc family proteins, and in biotin biosynthesis genes.

Identification, sequencing and molecular analysis of Chp4, a novel chlamydiaphage of Chlamydophila abortus belonging to the family Microviridae.

Members of the family Microviridae have been identified in a number of chlamydial species infecting humans (phage CPAR39 in Chlamydophila pneumoniae), other mammals (φCPG1 in Chlamydophila caviae, Chp2 in Chlamydophila abortus and Chp3 in Chlamydophila pecorum) and birds (Chp1 in Chlamydophila psittaci). This study describes the identification and genome sequencing of Chp4, an icosahedral, 4530 bp, ssDNA phage in C. abortus. Chp4 is predicted to contain eight ORFs, six of which could be assigned putative functions based on sequence similarity to characterized bacteriophage. Gene order and content were highly conserved amongst chlamydiaphage, with the highest sequence variability occurring in the IN5 and INS variable regions of the VP1 major coat protein, which has been associated with host cell recognition and binding. Phylogenetic analysis of VP1 indicated that Chp4 is a member of the Chlamydiamicrovirus, and is most closely related to phage φCPG1 and CPAR39.

Complete Genome Sequence of Chlamydia abortus MRI-10/19, Isolated from a Sheep Vaccinated with the Commercial Live C. abortus 1B Vaccine Strain.

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.

Whole Genome Sequencing and Comparative Genome Analyses of Chlamydia abortus Strains of Avian Origin Suggests That Chlamydia abortus Species Should Be Expanded to Include Avian and Mammalian Subgroups.

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.

Distribution and Severity of Placental Lesions Caused by the Chlamydia abortus 1B Vaccine Strain in Vaccinated Ewes.

Chlamydia abortus infects livestock species worldwide and is the cause of enzootic abortion of ewes (EAE). In Europe, control of the disease is achieved using a live vaccine based on C. abortus 1B strain. Although the vaccine has been useful for controlling disease outbreaks, abortion events due to the vaccine have been reported. Recently, placental pathology resulting from a vaccine type strain (vt) infection has been reported and shown to be similar to that resulting from a natural wild-type (wt) infection. The aim of this study was to extend these observations by comparing the distribution and severity of the lesions, the composition of the predominating cell infiltrate, the amount of bacteria present and the role of the blood supply in infection. A novel system for grading the histological and pathological features present was developed and the resulting multi-parameter data were statistically transformed for exploration and visualisation through a tailored principal component analysis (PCA) to evaluate the difference between them. The analysis provided no evidence of meaningful differences between vt and wt strains in terms of the measured pathological parameters. The study also contributes a novel methodology for analysing the progression of infection in the placenta for other abortifacient pathogens.

Efficacy of Two Chlamydia abortus Subcellular Vaccines in a Pregnant Ewe Challenge Model for Ovine Enzootic Abortion.

Chlamydia abortus, the aetiological agent of enzootic abortion of ewes, is a major cause of reproductive loss in small ruminants worldwide, accounting for significant economic losses to the farming industry. Disease can be managed through the use of commercial inactivated or live whole organism-based vaccines, although both have limitations particularly in terms of efficacy, safety and disease-associated outbreaks. Here we report a comparison of two experimental vaccines (chlamydial outer membrane complex (COMC) and octyl glucoside (OG)-COMC) based on detergent extracted outer membrane preparations of C. abortus and delivered as prime-boost immunisations, with the commercial live vaccine Cevac® Chlamydia in a pregnant sheep challenge model. No abortions occurred in either experimental vaccine group, while a single abortion occurred in the commercial vaccine group. Bacterial shedding, as a measure of potential risk of transmission of infection to naïve animals, was lowest in the COMC vaccinated group, with reductions of 87.5%, 86.4% and 74% observed for the COMC, OG-COMC and live commercial vaccine groups, respectively, compared to the unvaccinated challenge control group. The results show that the COMC vaccine performed the best and is a safer efficacious alternative to the commercial vaccines. However, to improve commercial viability, future studies should optimise the antigen dose and number of inoculations required.

De Novo Whole-Genome Sequencing and Annotation of Pathogenic Bovine Pasteurella multocida Type A:3 Strains.

Pneumonic pasteurellosis, caused by Pasteurella multocida, is a common respiratory infection of ruminants that has major economic and welfare implications throughout the world. Here, we report the annotated genome sequences of seven pathogenic strains of P. multocida that were isolated from cattle in the United Kingdom.

The 1B vaccine strain of Chlamydia abortus produces placental pathology indistinguishable from a wild type 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.

Complete Genome Sequence of Chlamydia avium PV 4360/2, Isolated from a Feral Pigeon in Italy.

We report here the whole-genome sequence of a Chlamydia avium isolate recovered from a feral pigeon in 1999 in Italy. Only one complete genome of a C. avium strain has been published so far. Future comparative analyses could provide valuable insights on the genomic evolution of the pathogen.