Defining immune correlates during latent and active chlamydial infection in sheep

Abstract

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.

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

“…Assessment of these cytokines revealed virtually no IL-4 production but some clear and variable antigen-specific IL-10 production in all vaccine groups. This data is consistent with previous studies assessing cellular immunity to sheep experimentally infected with C. abortus where elevated IFN-γ and IL-10 was observed [43,49]. Although the focus of this study was evaluation of vaccine efficacy rather than detailed dissection of any protective immune response generated by the candidate vaccines, the analyses conducted here with these signature cytokines highlight the difficulties and complexities in identifying immunological correlates of protection.…”

“…PBMC were isolated from venous blood, counted, adjusted to 2 × 10 6 cells/mL in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% heat-inactivated Foetal Bovine Serum (FBS), 2 mM L-glutamine, gentamicin (50 µg/mL), 100 IU/mL penicillin, 50 µg/mL streptomycin and 50 µM β-mercaptoethanol (Sigma-Aldrich, Dorset, UK) and cultured, according to previously described protocols [49]. To the cells, 100 µL of purified C. abortus EB antigen (5 µg/mL) as prepared in Section 2.2, 100 µL of ConA (5 µg/mL; extract from Concanavalia ensiformis, ICN Biochemicals, Cleveland, OH, USA) or 100 µL of medium alone were added in quadruplicate wells for each treatment.…”

“…To the cells, 100 µL of purified C. abortus EB antigen (5 µg/mL) as prepared in Section 2.2, 100 µL of ConA (5 µg/mL; extract from Concanavalia ensiformis, ICN Biochemicals, Cleveland, OH, USA) or 100 µL of medium alone were added in quadruplicate wells for each treatment. Antigenspecific recall responses were assessed by analysis of the culture supernatants collected after 96 h for cytokines Interferon-gamma (IFN-γ), Interleukin (IL)-4 and IL-10, as described previously [49]. Supernatants were stored at −70 • C until further analysis.…”

“…Quantification was performed by generating a standard curve using known concentrations of recombinant bovine IFN-γ (Endogen-Pierce, Waltham, MA, USA). IL-4 and IL-10 were measured in an indirect ELISA using antibody pairs (mAb clones CC313 and CC314 for IL-4; CC318 and CC320 for IL-10, BioRad Antibody Laboratories, Oxford UK) and quantified using recombinant bovine (rbov) IL-4 of known concentration and recombinant ovine IL-10 of known biological activity [49].…”

Presence of Chlamydia abortus in colostrum, milk and vaginal discharge samples of sheep

“…The main route of transmission of C. abortus is the ingestion of the microorganism that has been eliminated in vaginal secretions, placental membranes or abortions that contaminate the environment, mainly the water, food and bedding of animals (Nietfeld, 2001;Wattegedra et al, 2020). Both sheep and goats can excrete C. abortus vaginally within two weeks after delivery or abortion; being able to remain infectious for several weeks in the environment in which they are found, a situation that increases the spread and permanence of the microorganism in the herd (Papp et al, 1994;Longbottom and Coulter, 2003;Rodolakis and Laroucau, 2015).…”