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.EEA MercedesFil: Livingstone, Morag. Moredun Research Institute; Reino UnidoFil: Caspe, Sergio Gaston. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mercedes; ArgentinaFil: Caspe, Sergio Gaston. Moredun Research Institute; Reino UnidoFil: Longbottom, David. Moredun Research Institute; Reino Unid

Transcriptional analysis of in vitro expression patterns of chlamydophila abortus polymorphic outer membrane proteins during the chlamydial developmental cycle.

Chlamydophila abortus is the aetiological agent of ovine enzootic abortion. Sequencing, annotation and comparative analysis of the genome of C. abortus strain S26/3 has revealed variation in theloci encoding the polymorphic membrane proteins (Pmps). These Pmps resemble autotransporter proteins of the type V secretion system, suggesting an important role in chlamydial pathogenesis. The purpose of this study was to characterise the transcriptional expression patterns of this family during the developmental cycle of C. abortus. McCoy cells were infected with C. abortus and analysed for pmp mRNA expression over a 72 h period. Few pmp transcripts were detected in the early stages of the developmental cycle. Peakexpression occurred at 48 h post-infection (p.i.) other than for pmp5E, where it was observed at 24 h p.i. Overall, expression of pmps 5E, 18D and 10G were found to be 40 to 100-fold higher than the lowest expressing pmps (6H, 13G and 15G) at 24 h p.i., while pmps 18D and 17G were 14 to 16-fold higher than the lowest (11G, 14G and 15G) at 48 h. Levels of expression for all the other pmp genes were below one copy per genome at any time point. The expression of all the pmps reduced to near base-line levels by 60 hp.i. These results demonstrate that pmp expression in C. abortus is mid to late cycle, consistent with conversion of the reticulate body to the elementary body. The low level of pmp transcription may beindicative of heterogeneity in expression, suggesting a possible role for some of the Pmps in antigenic variation and chlamydial pathogenesis

The extent of placental pathology is negatively correlated to birth weight in ewes infected with the wild-type strain of Chlamydia abortus

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.EEA MercedesFil: Caspe, Sergio Gaston. Moredun Research Institute; Reino UnidoFil: Caspe, Sergio Gaston. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mercedes; ArgentinaFil: Caspe, Sergio Gaston. University of Edinburgh. Royal (Dick) School of Veterinary Studies; Reino UnidoFil: Palarea-Albaladejo, Javier. Biomathematics & Statistics Scotland; Reino UnidoFil: Palarea-Albaladejo, Javier. University of Girona. Deparment of Computer Sciences, Applied Mathematics and Statistics; EspañaFil: Livingstone, Morag. Moredun Research Institute; Reino UnidoFil: Wattegedera, Sean Ranjan. Moredun Research Institute; Reino UnidoFil: Milne, Elspeth. University of Edinburgh. Royal (Dick) School of Veterinary Studies; Reino UnidoFil: Sargison, Neil Donald. University of Edinburgh. Royal (Dick) School of Veterinary Studies; Reino UnidoFil: Longbottom, David. Moredun Research Institute; Reino Unid

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.EEA MercedesFil: Caspe, Sergio Gaston. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mercedes; ArgentinaFil: Caspe, Sergio Gaston. Moredun Research Institute; Reino UnidoFil: Caspe, Sergio Gaston. University of Edinburgh. Royal (Dick) School of Veterinary Studies; Reino UnidoFil: Palarea-Albaladejo, Javier. Biomathematics & Statistics Scotland; Reino UnidoFil: Underwood, Clare. Moredun Research Institute; Reino UnidoFil: Livingstone, Morag. Moredun Research Institute; Reino UnidoFil: Wattegedera, Sean Ranjan. Moredun Research Institute; Reino UnidoFil: Milne, Elspeth. University of Edinburgh. Royal (Dick) School of Veterinary Studies; Reino UnidoFil: Sargison, Neil Donald. University of Edinburgh. Royal (Dick) School of Veterinary Studies; Reino UnidoFil: Chianini, Francesca. Moredun Research Institute; Reino UnidoFil: Longbottom, David. Moredun Research Institute; Reino Unid

Antibody responses to recombinant protein fragments of the major outer membrane protein and polymorphic outer membrane protein POMP90 in Chlamydophila abortus-infected pregnant sheep

Chlamydophila abortus is one of the major causes of infectious abortion in pregnant sheep (enzootic abortion of ewes or EAE) worldwide. Organisms shed in infected placentas and uterine discharges at lambing time are the main sources of environmental contamination, responsible for transmission to susceptible animals and possible human contacts. In the present study, a recently developed test, based on a recombinant fragment of the polymorphic outer membrane protein POMP90 (rOMP90-4 indirect enzyme-linked immunosorbent assay [iELISA]) and one based on the variable segment 2 (VS2) region of the major outer membrane protein (MOMP) (MOMP VS2 iELISA) were compared using sera from C. abortus-infected ewes at different stages throughout pregnancy. The rOMP90 iELISA detected antibody much earlier in pregnancy than the MOMP iELISA, which, like the complement fixation test, detected antibody only at the time of abortion or lambing. No anti-MOMP antibody response could be detected in three of seven experimentally infected ewes. Furthermore, the rOMP90 iELISA detected antibody in an animal that seroconverted during the course of the study, which the MOMP iELISA failed to detect. Overall, the results show that the rOMP90-4 iELISA is considerably more sensitive than the MOMP VS2 iELISA for identifying animals infected with C. abortus. Earlier detection of infection will allow appropriate control measures to be taken to reduce environmental contamination, thus limiting the spread of infection, financial losses, and the possible risks of zoonotic transmission to humans

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

Experimental challenge of pregnant cattle with the putative abortifacient Waddlia chondrophila

Waddlia chondrophila is a Gram-negative intracellular bacterial organism that is related to classical chlamydial species and has been implicated as a cause of abortion in cattle. Despite an increasing number of observational studies linking W. chondrophila infection to cattle abortion, little direct experimental evidence exists. Given this paucity of direct evidence the current study was carried out to investigate whether experimental challenge of pregnant cattle with W. chondrophila would result in infection and abortion. Nine pregnant Friesian-Holstein heifers received 2 × 108 inclusion forming units (IFU) W. chondrophila intravenously on day 105–110 of pregnancy, while four negative-control animals underwent mock challenge. Only one of the challenged animals showed pathogen-associated lesions, with the organism being detected in the diseased placenta. Importantly, the organism was re-isolated and its identity confirmed by whole genome sequencing, confirming Koch’s third and fourth postulates. However, while infection of the placenta was observed, the experimental challenge in this study did not confirm the abortifacient potential of the organism

Genomic evidence that the live Chlamydia abortus vaccine strain 1B is not attenuated and has the potential to cause disease.

BACKGROUND: The live, temperature-attenuated vaccine strain 1B of Chlamydia abortus, the aetiological agent of ovine enzootic abortion (OEA), has been implicated in cases of vaccine breakdown. The aim of this study was to understand the nature of this attenuation through sequencing of the vaccine parent strain (AB7) and the derived mutant strains 1B and 1H, as well as to clarify the role of the vaccine strain in causing disease through comparative whole genome analysis. METHODS: Whole genome sequencing was performed on: vaccine parent strain AB7; N-methyl-N'-nitro-N-nitrosoguanidine (NTG)-induced temperature attenuated mutant strain 1B grown from the commercial live vaccines Cevac Chlamydia and Enzovax; strain 1H a reverted NTG mutant; and 5 strains isolated from cases of OEA originating from animals from the original vaccine safety trial (2 strains) or from vaccinated ewes or ewes exposed to vaccinated animals (3 strains). RESULTS: We confirmed that AB7 is in a different lineage from the reference strain S26/3. The genome of vaccine strain 1B contains ten single nucleotide polymorphisms (SNPs) created by the NTG treatment, which are identical to those found in strain 1H. The strains from OEA cases also cluster phylogenetically very tightly with these vaccine strains. CONCLUSIONS: The results show that C. abortus vaccine strain 1B has an identical genome sequence to the non-attenuated "reverted mutant" strain 1H. Thus, the protection of the 1B vaccine is unlikely to be due to the NTG induced SNPs and is more likely caused by the administration of high doses of C. abortus elementary bodies that stimulate protective immunity. Vaccine-identical strains were also isolated from cases of disease, as well as strains which had acquired 1-3 SNPs, including an animal that had not been vaccinated with either of the commercial live OEA vaccines, indicating that the 1B vaccine strain may be circulating and causing disease

Processing of Chlamydia abortus polymorphic membrane protein 18D during the chlamydial developmental cycle

BACKGROUND: Chlamydia possess a unique family of autotransporter proteins known as the Polymorphic membrane proteins (Pmps). While the total number of pmp genes varies between Chlamydia species, all encode a single pmpD gene. In both Chlamydia trachomatis (C. trachomatis) and C. pneumoniae, the PmpD protein is proteolytically cleaved on the cell surface. The current study was carried out to determine the cleavage patterns of the PmpD protein in the animal pathogen C. abortus (termed Pmp18D). METHODOLOGY/PRINCIPAL FINDINGS: Using antibodies directed against different regions of Pmp18D, proteomic techniques revealed that the mature protein was cleaved on the cell surface, resulting in a100 kDa N-terminal product and a 60 kDa carboxy-terminal protein. The N-terminal protein was further processed into 84, 76 and 73 kDa products. Clustering analysis resolved PmpD proteins into three distinct clades with C. abortus Pmp18D, being most similar to those originating from C. psittaci, C. felis and C. caviae. CONCLUSIONS/SIGNIFICANCE: This study indicates that C. abortus Pmp18D is proteolytically processed at the cell surface similar to the proteins of C. trachomatis and C. pneumoniae. However, patterns of cleavage are species-specific, with low sequence conservation of PmpD across the genus. The absence of conserved domains indicates that the function of the PmpD molecule in chlamydia remains to be elucidated