Serum antibodies to surface proteins of Chlamydia trachomatis as candidate biomarkers of disease: results from the Baltimore Chlamydia Adolescent/Young Adult Reproductive Management (CHARM) cohort.

We previously observed that the nine-member family of autotransported polymorphic membrane proteins (Pmps) of Chlamydia trachomatis is variably expressed in cell culture. Additionally, C. trachomatis-infected patients display variable Pmp-specific serum antibody profiles indirectly suggesting expression of unique Pmp profiles is an adaptive response to host-specific stimuli during infection. Here, we propose that the host response to Pmps and other outer surface proteins may correlate with disease severity. This study tests this hypothesis using an ELISA that measures serum IgG antibodies specific for the nine C. trachomatis Pmp subtypes and four immunodominant antigens (MOMP, OmcB, Hsp60, ClpP) in 265 participants of the Chlamydia Adolescent/Young Adult Reproductive Management (CHARM) cohort. More C. trachomatis-infected females displayed high Pmp-specific antibody levels (cut-off Indexes) than males (35.9%-40.7% of females vs. 24.2%-30.0% of males), with statistical significance for PmpC, F and H (P < 0.05). Differences in Pmp-specific antibody profiles were not observed between C. trachomatis-infected females with a clinical diagnosis of pelvic inflammatory disease (PID) and those without. However, a statistically significant association between high levels of OmcB-specific antibody and a PID diagnosis (P< 0.05) was observed. Using antibody levels as an indirect measure of antigen expression, our results suggest that gender- and/or site-specific (cervix in females vs. urethra in males) stimuli may control pmp expression in infected patients. They also support the possible existence of immune biomarkers of chlamydial infection associated with disease and underline the need for high resolution screening in human serum.

Does Active Oral Sex Contribute to Female Infertility?

Based on recent, historical, and circumstantial evidence, we present a multifactorial hypothesis that has potential direct implications on the epidemiology and management of chlamydial infection and disease in humans. We propose that (1) like its veterinary relatives, the oculogenital pathogen Chlamydia trachomatis evolved as a commensal organism of the human gastrointestinal (GI) tract primarily transmissible via the fecal-oral route; (2) in the modern era, C. trachomatis causes "opportunistic" infection at non-GI sites under conditions driven by improved sanitation/hygiene and reduced fecal-oral transmission; and (3) the rise in the practice of oral sex is contributing to the increased prevalence of C. trachomatis in the human GI tract. Infectious organisms produced in the GI tract and reaching the rectum may then chronically contaminate and infect the female urogenital tract, thereby potentially contributing to the most serious sequelae of chlamydial infection in women: pelvic inflammatory disease, ectopic pregnancy, and tubal factor infertility.

Analysis of Polymorphic Membrane Protein Expression in Cultured Cells Identifies PmpA and PmpH of Chlamydia psittaci as Candidate Factors in Pathogenesis and Immunity to Infection.

The polymorphic membrane protein (Pmp) paralogous families of Chlamydia trachomatis, Chlamydia pneumoniae and Chlamydia abortus are putative targets for Chlamydia vaccine development. To determine whether this is also the case for Pmp family members of C. psittaci, we analyzed transcription levels, protein production and localization of several Pmps of C. psittaci. Pmp expression profiles were characterized using quantitative real-time PCR (RT-qPCR), immunofluorescence (IF) and immuno-electron microscopy (IEM) under normal and stress conditions. We found that PmpA was highly produced in all inclusions as early as 12 hpi in all biological replicates. In addition, PmpA and PmpH appeared to be unusually accessible to antibody as determined by both immunofluorescence and immuno-electron microscopy. Our results suggest an important role for these Pmps in the pathogenesis of C. psittaci, and make them promising candidates in vaccine development.

Chlamydia caviae infection alters abundance but not composition of the guinea pig vaginal microbiota.

In humans, the vaginal microbiota is thought to be the first line of defense again pathogens including Chlamydia trachomatis. The guinea pig has been extensively used as a model to study chlamydial infection because it shares anatomical and physiological similarities with humans, such as a squamous vaginal epithelium as well as some of the long-term outcomes caused by chlamydial infection. In this study, we aimed to evaluate the guinea pig-C. caviae model of genital infection as a surrogate for studying the role of the vaginal microbiota in the early steps of C. trachomatis infection in humans. We used culture-independent molecular methods to characterize the relative and absolute abundance of bacterial phylotypes in the guinea pig vaginal microbiota in animals non-infected, mock-infected or infected by C. caviae. We showed that the guinea pig and human vaginal microbiotas are of different bacterial composition and abundance. Chlamydia caviae infection had a profound effect on the absolute abundance of bacterial phylotypes but not on the composition of the guinea pig vaginal microbiota. Our findings compromise the validity of the guinea pig-C. caviae model to study the role of the vaginal microbiota during the early steps of sexually transmitted infection.

Application of quantitative real-time polymerase chain reaction for the diagnosis of toxoplasmosis and enzootic abortion of ewes.

Toxoplasma gondii and Chlamydophila abortus are the 2 most common infectious causes of ovine abortion worldwide. These obligate intracellular pathogens are associated with severe placentitis leading to abortion or stillbirth in pregnant ewes, and resulting in significant economic losses. The objectives of the current study were the development, validation, and application of a duplex real-time polymerase chain reaction (PCR) assay capable of quantifying the burden of infection by T. gondii and C. abortus in material submitted for diagnostic purposes. The validation was carried out using samples from ewes experimentally infected with these organisms. Based on the numbers of genome copies detected, an arbitrary cutoff level was established to correlate with significant pathological changes sufficient to give rise to abortion. When the PCR assay was applied to samples from 66 Irish farms with naturally occurring outbreaks of ovine abortion, toxoplasmosis and enzootic abortion of ewes (EAE) accounted for 14% and 20% of the farms, respectively, while on 6% of the farms, there was evidence of dual infection. When standard diagnostic techniques including histopathological examination, serological analysis, chlamydial antigen detection, and bacteriological culture, were used on samples from the same farms, toxoplasmosis was diagnosed in 17% of farms, and EAE in 12%; dual infection was diagnosed on 3% of the farms. In general, good agreement was found between the PCR and the standard methods. The duplex real-time PCR assay developed in this study has proved to be a very sensitive and rapid tool that might provide a valuable addition to the methods currently available for routine diagnosis of ovine abortions.

Amniotic and allantoic fluids from experimentally infected sheep contain immunoglobulin specific for Chlamydophila abortus.

Chlamydophila abortus, the aetiological agent of enzootic abortion of ewes (EAE), replicates in trophoblast cells leading to their destruction and dissemination of the bacterium to foetal organs. To further understand the pathogenesis of EAE, amniotic and allantoic fluids were collected from experimentally infected pregnant ewes at 30 (7 samples from each fluid), 35 (8 samples from each fluid), 40 (10 samples from each fluid) and 43 (6 amniotic fluids and 7 allantoic fluids) days post-infection to determine pathogen numbers and other markers of infection. Whilst experimentally infected ewes had characteristic placental lesions, only two amniotic and seven allantoic fluid samples were positive for C. abortus by real-time PCR. In contrast, all amniotic and allantoic fluids were positive for immunoglobulin. Immunoglobulins were generally detected earlier in allantoic fluid than in amniotic fluid and the numbers of samples containing immunoglobulins increased as infection progressed. IgG in amniotic and allantoic fluids was shown to be specific for C. abortus, and reacted with the major outer membrane proteins, polymorphic outer membrane protein and macrophage infectivity potentiator protein. A comparison of two-dimensional immunoblots using purified IgG from the allantoic fluid, amniotic fluid, ewe serum and foetal serum of a C. abortus infected animal at 40 days post infection indicated a pattern of reactivity intermediate between that of the ewe serum and the foetal serum. Results suggest that a maternal source of immunoglobulin is predominant at 30 days post-infection but that foetal derived antibodies may be contributed at a later stage.

Monitoring clinical outcomes, pathological changes and shedding of Chlamydophila abortus following experimental challenge of periparturient ewes utilizing the natural route of infection.

Enzootic abortion of ewes (EAE) caused by Chlamydophila abortus is an important disease resulting in significant lamb loss in most sheep producing countries. Ewes are considered to be naturally infected with C. abortus via the oral-nasal route and may become persistent carriers, shedding during subsequent oestrous cycles and at lambing. The aim of this study was to monitor the clinical outcomes, pathological changes and shedding of C. abortus in 18 periparturient orally infected sheep for two breeding seasons. In the first season, C. abortus was detected by real-time PCR (rt-PCR) in 13/18 conjunctival swabs at oestrus. Three out of the 15 pregnant ewes gave birth to 1 live and 1 dead lamb, and 2 of them aborted. Following parturition/abortion, C. abortus was detected in 12/15 vaginal swabs and in all the collected foetal membranes. However, only those membranes containing high copy numbers of the bacterium displayed the EAE typical lesions. In the second season, none of the 13 pregnant ewes aborted, and 5 of them gave birth to dead or weak lambs. C. abortus was not detected in conjunctival or vaginal swabs at oestrus or parturition. The bacterium was detected at low levels in 36% of the foetal membranes, but with no evidence of histopathological lesions. These results indicate that C. abortus can be detected in a large proportion of animals during the first pregnancy after oral infection. However, this proportion is reduced at the subsequent breeding season, confirming the occurrence of a chronic low level persistent infection in post-abortion/lambing ewes.

First survey for Babesia bovis and Babesia bigemina infection in cattle from Central and Southern regions of Portugal using serological and DNA detection methods.

Incidence of bovine babesiosis in Portugal is currently unknown. In this study, a first survey of Babesia bovis and Babesia bigemina infection in cattle was carried out using blood samples from 406 clinically healthy individuals from different districts from Central and Southern regions of Portugal and analyzed by indirect enzyme linked immunosorbent assay (iELISA) and nested polymerase chain reaction (nPCR). Overall, serological testing revealed that 79% and 52% of cattle were positive for B. bovis and B. bigemina antibodies, respectively, whereas nPCR testing detected 71% and 34% cattle infected with B. bovis and B. bigemina protozoan, respectively. This is the first report of the prevalence of B. bovis and B. bigemina in cattle obtained by serological and DNA analysis studies in Central and Southern regions of Portugal. These data suggests high incidence of Babesia sp. infection in Portugal and can be used for designing adequate control programs.