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.

Natural Mycoplasma Infection Reduces Expression of Pro-Inflammatory Cytokines in Response to Ovine Footrot Pathogens.

Ovine footrot is a complex multifactorial infectious disease, causing lameness in sheep with major welfare and economic consequences. Dichelobacter nodosus is the main causative bacterium; however, footrot is a polymicrobial disease with Fusobacterium necrophorum, Mycoplasma fermentans and Porphyromonas asaccharolytica also associated. There is limited understanding of the host response involved. The proinflammatory mediators, interleukin (IL)-1ß and C-X-C Motif Chemokine Ligand 8 (CXCL8), have been shown to play a role in the early response to D. nodosus in dermal fibroblasts and interdigital skin explant models. To further understand the response of ovine skin to bacterial stimulation, and to build an understanding of the role of the cytokines and chemokines identified, primary ovine interdigital fibroblasts and keratinocytes were isolated, cultured and stimulated. The expression of mRNA and protein release of CXCL8 and IL-1ß were measured after stimulation with LPS, D. nodosus or F. necrophorum, which resulted in increased transcript levels of IL-1ß and CXCL8 in the M. fermentans-free cells. However, only an increase in the CXCL8 protein release was observed. No IL-1ß protein release was detected, despite increases in IL-1ß mRNA, suggesting the signal for intracellular pre-IL-1ß processing may be lacking when culturing primary cells in isolation. The keratinocytes and fibroblasts naturally infected with M. fermentans showed little response to the LPS, a range of D. nodosus preparations or heat-inactivated F. necrophorum. Primary single cell culture models complement ex vivo organ culture models to study different aspects of the host response to D. nodosus. The ovine keratinocytes and fibroblasts infected with M. fermentans had a reduced response to the experimental bacterial stimulation. However, in the case of footrot where Mycoplasma spp. are associated with diseased feet, this natural infection gives important insights into the impact of multiple pathogens on the host response.

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.

A Trifecta of New Insights into Ovine Footrot for Infection Drivers, Immune Response, and Host-Pathogen Interactions.

Footrot is a polymicrobial infectious disease in sheep causing severe lameness, leading to one of the industry's largest welfare problems. The complex etiology of footrot makes in situ or in vitro investigations difficult. Computational methods offer a solution to understanding the bacteria involved and how they may interact with the host, ultimately providing a way to identify targets for future hypothesis-driven investigative work. Here, we present the first combined global analysis of bacterial community transcripts together with the host immune response in healthy and diseased ovine feet during a natural polymicrobial infection state using metatranscriptomics. The intratissue and surface bacterial populations and the most abundant bacterial transcriptomes were analyzed, demonstrating that footrot-affected skin has reduced diversity and increased abundances of not only the causative bacterium Dichelobacter nodosus but also other species such as Mycoplasma fermentans and Porphyromonas asaccharolytica. Host transcriptomics reveals the suppression of biological processes related to skin barrier function, vascular functions, and immunosurveillance in unhealthy interdigital skin, supported by histological findings that type I collagen (associated with scar tissue formation) is significantly increased in footrot-affected interdigital skin compared to outwardly healthy skin. Finally, we provide some interesting indications of host and pathogen interactions associated with virulence genes and the host spliceosome, which could lead to the identification of future therapeutic targets.

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.

Hemagglutinin protein of Peste des Petits Ruminants virus (PPRV) activates the innate immune response via Toll-like receptor 2 signaling.

The toll-like receptor (TLR) family comprises both cell-surface and intracellular receptors that recognize different types of pathogen-associated molecular patterns (PAMPs) leading to the production of pro-inflammatory cytokines and subsequent development of adaptive immunity. TLR2 is a cell-surface receptor initially thought to act as a bacterial sentinel but also shown to recognize a number of viral glycoproteins. In this study, we sought to characterize the role of TLR2 in the activation of the immune response by peste des petits ruminants virus (PPRV), a morbillivirus of the Paramixoviridae family that causes an acute, highly contagious disease in goats and sheep. Using human embryonic kidney (HEK) 293 cells stably expressing human (h)TLR2 but lacking any other TLR, we found that PPRV induces IL-8 production in a dose-dependent manner. That activation is only observed in cells expressing hTLR2 and is greatly reduced when the receptor is blocked by pretreatment with specific antibody. We identified hemagglutinin (H) as the viral protein responsible of TLR2 activation by performing the same assays with purified recombinant mammalian-expressed H protein. Exogenous addition of recombinant H protein to cell culture induces high levels of interleukin (IL)-8 only in TLR2-expressing cells. Moreover, H engagement on TLR2 in the monocytic cell line THP-1 activates extracellular-signal-regulated kinase (ERK) signaling. Stimulation of primary ovine dendritic cells with either inactivated PPRV or purified recombinant H protein results in transcription of pro-inflammatory cytokines and the secretion of the Th1-polarizing cytokine IL-12. The role of these host immune mechanisms in the control of PPR is discussed.

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.

The Veterinary Immunological Toolbox: Past, Present, and Future.

It is well-recognized that research capability in veterinary species is restricted by a lack of immunological reagents relative to the extensive toolboxes for small rodent biomedical model species and humans. This creates a barrier to the strategic development of disease control solutions for livestock, companion animals and wildlife that not only affects animal health but can affect human health by increasing the risk of transmission of zoonotic pathogens. There have been a number of projects aimed at reducing the capability gaps in the veterinary immunological toolbox, the majority of these focusing on livestock species. Various approaches have been taken to veterinary immunological reagent development across the globe and technological advances in molecular biology and protein biochemistry have accelerated toolbox development. While short-term funding initiatives can address specific gaps in capability, they do not account for long-term sustainability of reagents and databases that requires a different funding model. We review the past, present and future of the veterinary immunological toolbox with specific reference to recent developments discussed at the International Union of Immunological Societies (IUIS) Veterinary Immunology Committee (VIC) Immune Toolkit Workshop at the 12th International Veterinary Immunology Symposium (IVIS) in Seattle, USA, 16-19 August 2019. The future availability of these reagents is critical to research for improving animal health, responses to infectious pathogens and vaccine design as well as for important analyses of zoonotic pathogens and the animal /human interface for One Health initiatives.

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.

Immunological Homeostasis at the Ovine Placenta May Reflect the Degree of Maternal Fetal Interaction.

Successful mammalian pregnancies are a result of complex physiological, endocrinological, and immunological processes that combine to create an environment where the mother is tolerant to the semi-allogeneic fetus. Our knowledge of the mechanisms that contribute to maternal tolerance is derived mainly from human and murine studies of haemochorial placentation. However, as this is the most invasive type of placentation it cannot be assumed that identical mechanisms apply to the less invasive epitheliochorial placentation found in other species such as ruminants. Here, we examine three features associated with reproductive immune regulation in a transformed ovine trophoblast cell line and ex-vivo ovine reproductive tissues collected at term, namely: major histocompatibility complex (MHC) expression, Indoleamine 2,3 dioxygenase-1 (IDO-1) expression, and Natural Killer (NK) cell infiltration. High levels of MHC class I protein expression were detected at the surface of the trophoblast cell line using a pan-MHC class I specific monoclonal antibody. The majority of MHC class I transcripts isolated from the cell line clustered with classical MHC alleles. Transcriptional analysis of placental tissues identified only classical MHC class I transcripts. We found no evidence of constitutive transcription of IDO-1 in either the trophoblast cell line or placental tissues. Ex-vivo tissues collected from the materno-fetal interface were negative for cells expressing NKp46/NCR1. Collectively, these observations suggest that the relatively non-invasive synepitheliochorial placentation found in sheep has a more limited requirement for local immunoregulation compared to the more invasive haemochorial placentation of primates and rodents.

A Novel 3D Skin Explant Model to Study Anaerobic Bacterial Infection.

Skin infection studies are often limited by financial and ethical constraints, and alternatives, such as monolayer cell culture, do not reflect many cellular processes limiting their application. For a more functional replacement, 3D skin culture models offer many advantages such as the maintenance of the tissue structure and the cell types present in the host environment. A 3D skin culture model can be set up using tissues acquired from surgical procedures or post slaughter, making it a cost effective and attractive alternative to animal experimentation. The majority of 3D culture models have been established for aerobic pathogens, but currently there are no models for anaerobic skin infections. Footrot is an anaerobic bacterial infection which affects the ovine interdigital skin causing a substantial animal welfare and financial impact worldwide. Dichelobacter nodosus is a Gram-negative anaerobic bacterium and the causative agent of footrot. The mechanism of infection and host immune response to D. nodosus is poorly understood. Here we present a novel 3D skin ex vivo model to study anaerobic bacterial infections using ovine skin explants infected with D. nodosus. Our results demonstrate that D. nodosus can invade the skin explant, and that altered expression of key inflammatory markers could be quantified in the culture media. The viability of explants was assessed by tissue integrity (histopathological features) and cell death (DNA fragmentation) over 76 h showing the model was stable for 28 h. D. nodosus was quantified in all infected skin explants by qPCR and the bacterium was visualized invading the epidermis by Fluorescent in situ Hybridization. Measurement of pro-inflammatory cytokines/chemokines in the culture media revealed that the explants released IL1ß in response to bacteria. In contrast, levels of CXCL8 production were no different to mock-infected explants. The 3D skin model realistically simulates the interdigital skin and has demonstrated that D. nodosus invades the skin and triggered an early cellular inflammatory response to this bacterium. This novel model is the first of its kind for investigating an anaerobic bacterial infection.

Pathogenic outcome following experimental infection of sheep with Chlamydia abortus variant strains LLG and POS.

This study investigated the pathogenesis of two variant strains (LLG and POS) of Chlamydia abortus, in comparison to a typical wild-type strain (S26/3) which is known to be responsible for late term abortion in small ruminants. Challenge with the three strains at mid-gestation resulted in similar pregnancy outcomes, with abortion occurring in approximately 50-60% of ewes with the mean gestational lengths also being similar. However, differences were observed in the severity of placental pathology, with infection appearing milder for strain LLG, which was reflected in the lower number of organisms shed in vaginal swabs post-partum and less gross pathology and organisms present in placental smears. Results for strain POS were somewhat different than LLG with a more focal restriction of infection observed. Post-abortion antibody responses revealed prominent differences in seropositivity to the major outer membrane protein (MOMP) present in elementary body (EB) preparations under denaturing conditions, most notably with anti-LLG and anti-POS convalescent sera where there was no or reduced detection of MOMP present in EBs derived from the three strains. These results and additional analysis of whole EB and chlamydial outer membrane complex preparations suggest that there are conformational differences in MOMP for the three strains. Overall, the results suggest that gross placental pathology and clinical outcome is not indicative of bacterial colonization and the severity of infection. The results also highlight potential conformational differences in MOMP epitopes that perhaps impact on disease diagnosis and the development of new vaccines.

Enhancing the toolbox to study IL-17A in cattle and sheep.

The development of methods to detect cytokine expression by T cell subsets in ruminants is fundamental to strategic development of new livestock vaccines for prevention of infectious diseases. It has been possible to detect T cell expression of IFN-γ, IL-4 and IL-10 in ruminants for many years but methods to detect expression of IL-17A are relatively limited. To address this gap in capability we have cloned bovine and ovine IL-17A cDNAs and expressed biologically-active recombinant proteins in Chinese Hamster Ovary (CHO) cells. We used the transfected CHO cells to screen commercially-available antibodies for their ability to detect IL-17A expression intracellularly and in culture supernates. We demonstrate that an ELISA for bovine IL-17A detects native ovine IL-17A. Moreover, the constituent polyclonal antibodies (pabs) in the ELISA were used to enumerate peripheral blood mononuclear cells (PBMC) expressing IL-17A from cattle and sheep by ELISpot. We identified two monoclonal antibodies (mabs) that detect recombinant intracellular IL-17A in CHO cells by flow cytometry. One of these mabs was used to detect native intracellular IL-17A expression in PBMC in conjunction with cell surface phenotyping mabs [CD4+ve, CD8+ve and Workshop Cluster 1 (WC-1)+ve gamma-delta (γδ)] we show that distinct T cell subsets in cattle (defined as CD4+ve, CD8+ve or WC-1+ve) and sheep (defined as CD4+ve or WC-1+ve) can express IL-17A following activation. These novel techniques provide a solid basis to investigate IL-17A expression and define specific CD4+ve T cell subset activation in ruminants.

Phenotypic and functional analysis of monocyte populations in cattle peripheral blood identifies a subset with high endocytic and allogeneic T-cell stimulatory capacity.

Circulating monocytes in several mammalian species can be subdivided into functionally distinct subpopulations based on differential expression of surface molecules. We confirm that bovine monocytes express CD172a and MHC class II with two distinct populations of CD14(+)CD16(low/-)CD163(+) and CD14(-)CD16(++)CD163(low-) cells, and a more diffuse population of CD14(+)CD16(+)CD163(+) cells. In contrast, ovine monocytes consisted of only a major CD14(+)CD16(+) subset and a very low percentage of CD14(-)CD16(++)cells. The bovine subsets expressed similar levels of CD80, CD40 and CD11c molecules and mRNA encoding CD115. However, further mRNA analyses revealed that the CD14(-)CD16(++) monocytes were CX3CR1(high)CCR2(low) whereas the major CD14(+) subset was CX3CR1(low)CCR2(high). The former were positive for CD1b and had lower levels of CD11b and CD86 than the CD14(+) monocytes. The more diffuse CD14(+)CD16(+) population generally expressed intermediate levels of these molecules. All three populations responded to stimulation with phenol-extracted lipopolysaccharide (LPS) by producing interleukin (IL)-1ß, with the CD16(++) subset expressing higher levels of IL-12 and lower levels of IL-10. The CD14(-)CD16(++) cells were more endocytic and induced greater allogeneic T cell responses compared to the other monocyte populations. Taken together the data show both similarities and differences between the classical, intermediate and non-classical definitions of monocytes as described for other mammalian species, with additional potential subpopulations. Further functional analyses of these monocyte populations may help explain inter-animal and inter-species variations to infection, inflammation and vaccination in ruminant livestock.

Exploiting ovine immunology to improve the relevance of biomedical models.

Animal models of human disease are important tools in many areas of biomedicine; for example, in infectious disease research and in the development of novel drugs and medical devices. Most studies involving animals use rodents, in particular congenic mice, due to the availability of a wide number of strains and the ease with which they can be genetically manipulated. The use of mouse models has led to major advances in many fields of research, in particular in immunology but despite these advances, no animal model can exactly reproduce all the features of human disease. It is increasingly becoming recognised that in many circumstances mice do not provide the best model and that alternative species may be more appropriate. Here, we describe the relative merits of sheep as biomedical models for human physiology and disease in comparison to mice, with a particular focus on reproductive and respiratory pathogens.

Suppression of ovine lymphocyte activation by Teladorsagia circumcincta larval excretory-secretory products.

Teladorsagia circumcincta is an important pathogenic nematode of sheep. It has been demonstrated previously that stimulation of murine T lymphocytes with excretory-secretory (ES) products derived from fourth stage larvae of T. circumcincta (Tci-L4-ES) results in de novo expression of Foxp3, a transcription factor intimately involved in regulatory T cell function. In the current study, Foxp3⁺ T cell responses in the abomasum and the effects of Tci-L4-ES on ovine peripheral blood mononuclear cells (PBMC) following T. circumcincta infection were investigated. T. circumcincta infection resulted in a significant increase in numbers of abomasal Foxp3⁺ T cells, but not an increase in the proportion of T cells expressing Foxp3. Unlike in mice, Tci-L4-ES was incapable of inducing T cell Foxp3 expression but instead suppressed mitogen-induced and antigen-specific activation and proliferation of ovine PBMC in vitro. This effect was heat labile, suggesting that it is mediated by protein(s). Suppression was associated with up-regulation of interleukin-10 (IL-10) mRNA, and specific monoclonal antibody neutralisation of IL-10 resulted in a 50% reduction in suppression, indicating involvement of the IL-10 signaling pathway. Suppression was significantly reduced in PBMC isolated from T. circumcincta infected vs. helminth-naïve lambs, and this reduction in suppression was associated with an increase in Tci-L4-ES antigen-specific T cells within the PBMC. In conclusion, we have identified a mechanism by which T. circumcincta may modulate the host adaptive immune response, potentially assisting survival of the parasite within the host. However, the impact of Tci-L4-ES-mediated lymphocyte suppression during T. circumcincta infection remains to be determined.

Intranasal infection with Chlamydia abortus induces dose-dependent latency and abortion in sheep.

BACKGROUND: Latency is a key feature of the animal pathogen Chlamydia abortus, where infection remains inapparent in the non-pregnant animal and only becomes evident during a subsequent pregnancy. Often the first sign that an animal is infected is abortion occurring late in gestation. Despite this, little is understood of the underlying mechanisms that control latency or the recrudescence of infection that occurs during subsequent pregnancy. The aim of this study was to develop an experimental model of latency by mimicking the natural route of infection through the intranasal inoculation of non-pregnant sheep with C. abortus. METHODOLOGY/PRINCIPAL FINDINGS: Three groups of sheep (groups 1, 2 and 3) were experimentally infected with different doses of C. abortus (5×10(3), 5×10(5) and 5×10(7) inclusion forming units (IFU), respectively) prior to mating and monitored over 2 breeding cycles for clinical, microbiological, pathological, immunological and serological outcomes. Two further groups received either negative control inoculum (group 4a,b) or were inoculated subcutaneously on day 70 of gestation with 2×10(6) IFU C. abortus (group 5). Animals in groups 1, 2 and 5 experienced an abortion rate of 50-67%, while only one animal aborted in group 3 and none in group 4a,b. Pathological, microbiological, immunological and serological analyses support the view that the maternal protective immune response is influenced by initial exposure to the bacterium. CONCLUSIONS/SIGNIFICANCE: The results show that intranasal administration of non-pregnant sheep with a low/medium dose of C. abortus results in a latent infection that leads in a subsequent pregnancy to infection of the placenta and abortion. In contrast a high dose stimulates protective immunity, resulting in a much lower abortion rate. This model will be useful in understanding the mechanisms of infection underlying latency and onset of disease, as well as in the development of novel therapeutics and vaccines for controlling infection.

New challenges for vaccination to prevent chlamydial abortion in sheep.

Ovine enzootic abortion (OEA) is caused by the obligate intracellular Gram-negative bacterium Chlamydia abortus. OEA remains a common cause of infectious abortion in many sheep-rearing countries despite the existence of commercially available vaccines that protect against the disease. There are a number of confounding factors that influence the uptake and use of these vaccines, which includes an inability to discriminate between infected and vaccinated animals (DIVA) using conventional serological diagnostic techniques. This suggests that the immunity elicited by current vaccines is similar to that observed in convalescent, immune sheep that have experienced OEA. The existence of these vaccines provides an opportunity to understand how protection against OEA is elicited and also to understand why vaccines can occasionally appear to fail, as has been reported recently for OEA. Interferon-gamma (IFN-γ), the cytokine that classically defines Th1-type adaptive immunity, is a strong correlate of protection against OEA in sheep and has been shown to inhibit the growth of C. abortus in vitro. Humoral immunity to C. abortus is observed in both vaccinated and naturally infected sheep, but antibody responses tend to be used more as diagnostic markers than targets for strategic vaccine design. A future successful DIVA vaccine against OEA should aim to elicit the immunological correlate of protection (IFN-γ) concomitantly with an antibody profile that is distinct from that of the natural infection. Such an approach requires careful selection of protective components of C. abortus combined with an effective delivery system that elicits IFN-γ-producing CD4+ve memory T cells.

Identification of CD4+CD25 high Foxp3+ T cells in ovine peripheral blood.

Regulatory T cells (Treg) are an important subset of T lymphocytes which play a key role in maintaining peripheral immunological tolerance. The most studied subpopulation of Treg in mice and humans are natural Treg, which differentiate in the thymus and are identified by expression of CD4, high levels of IL-2Rα (CD25), and forkhead box P3 (Foxp3), a transcription factor intimately associated with Treg function. We and others have previously identified Foxp3(+) T cells in ovine tissue, suggesting that Treg exist in this species. However, the existence of putative natural Treg in sheep, as identified by co-expression of CD4, CD25 and Foxp3, has yet to be determined. In this study we demonstrate that the anti-rat/mouse Foxp3 monoclonal antibody FJK-16s cross-reacts with ovine Foxp3. Using a transfected Chinese hamster ovary cell line that constitutively expresses recombinant ovine Foxp3 as a positive control, we have developed a sensitive triple-labelling flow cytometry protocol to simultaneously label CD4, CD25 and Foxp3. We demonstrate that Foxp3(+) T lymphocytes exist in ovine peripheral blood, and that the majority of Foxp3 expression occurs within the CD4(+)CD25(hi) population. These results are consistent with those seen in other mammalian species and indicate that putative natural Treg exist in sheep.

Immunological paradigms and the pathogenesis of ovine chlamydial abortion.

Successful mammalian pregnancy involves complex immunological interactions between the mother and foetus that are not yet fully understood. A number of immunological paradigms have been established to explain the failure of the maternal immune system to reject the semi-allogeneic foetus, mainly based on studies in mice and humans. However, as placental structure, gestation periods and number of concepti per pregnancy can vary greatly between mammals, it is not always clear how applicable these immunological paradigms are to reproduction in other species. Here, we discuss the predictions of three important immunological paradigms in relation to the pathogenesis of ovine enzootic abortion (OEA), a common cause of infectious abortion in sheep and other ruminants. OEA is caused by the intracellular Gram-negative bacterium Chlamydophila abortus that exhibits a tropism for placental trophoblast. The paradigms of particular relevance to the pathogenesis of OEA are as follows: (i) intracellular bacterial infections are controlled by T(H)1-type CD4(+ve) T cells; (ii) indoleamine 2,3-dioxygenase is expressed in the placenta to prevent immunological rejection of the semi-allogeneic foetus; and (iii) pregnancy is a maternal T(H)2-type phenomenon. We discuss the relevance and validity of these paradigms for chlamydial abortion and reproductive immunology in sheep.