European Chlamydia abortus livestock isolate genomes reveal unusual stability and limited diversity, reflected in geographical signatures.

BACKGROUND: Chlamydia abortus (formerly Chlamydophila abortus) is an economically important livestock pathogen, causing ovine enzootic abortion (OEA), and can also cause zoonotic infections in humans affecting pregnancy outcome. Large-scale genomic studies on other chlamydial species are giving insights into the biology of these organisms but have not yet been performed on C. abortus. Our aim was to investigate a broad collection of European isolates of C. abortus, using next generation sequencing methods, looking at diversity, geographic distribution and genome dynamics. RESULTS: Whole genome sequencing was performed on our collection of 57 C. abortus isolates originating primarily from the UK, Germany, France and Greece, but also from Tunisia, Namibia and the USA. Phylogenetic analysis of a total of 64 genomes shows a deep structural division within the C. abortus species with a major clade displaying limited diversity, in addition to a branch carrying two more distantly related Greek isolates, LLG and POS. Within the major clade, seven further phylogenetic groups can be identified, demonstrating geographical associations. The number of variable nucleotide positions across the sampled isolates is significantly lower than those published for C. trachomatis and C. psittaci. No recombination was identified within C. abortus, and no plasmid was found. Analysis of pseudogenes showed lineage specific loss of some functions, notably with several Pmp and TMH/Inc proteins predicted to be inactivated in many of the isolates studied. CONCLUSIONS: The diversity within C. abortus appears to be much lower compared to other species within the genus. There are strong geographical signatures within the phylogeny, indicating clonal expansion within areas of limited livestock transport. No recombination has been identified within this species, showing that different species of Chlamydia may demonstrate different evolutionary dynamics, and that the genome of C. abortus is highly stable.

Exposure to viral and bacterial pathogens among Soay sheep (Ovis aries) of the St Kilda archipelago.

We assessed evidence of exposure to viruses and bacteria in an unmanaged and long-isolated population of Soay sheep (Ovis aries) inhabiting Hirta, in the St Kilda archipelago, 65 km west of Benbecula in the Outer Hebrides of Scotland. The sheep harbour many metazoan and protozoan parasites but their exposure to viral and bacterial pathogens is unknown. We tested for herpes viral DNA in leucocytes and found that 21 of 42 tested sheep were infected with ovine herpesvirus 2 (OHV-2). We also tested 750 plasma samples collected between 1997 and 2010 for evidence of exposure to seven other viral and bacterial agents common in domestic Scottish sheep. We found evidence of exposure to Leptospira spp., with overall seroprevalence of 6·5%. However, serological evidence indicated that the population had not been exposed to border disease, parainfluenza, maedi-visna, or orf viruses, nor to Chlamydia abortus. Some sheep tested positive for antibodies against Mycobacterium avium subsp. paratuberculosis (MAP) but, in the absence of retrospective faecal samples, the presence of this infection could not be confirmed. The roles of importation, the pathogen-host interaction, nematode co-infection and local transmission warrant future investigation, to elucidate the transmission ecology and fitness effects of the few viral and bacterial pathogens on Hirta.

Ectopic lymphoid tissue formation in the lungs of mice infected with Chlamydia pneumoniae is associated with epithelial macrophage inflammatory protein-2/CXCL2 expression.

Infection with Chlamydia pneumoniae (Cp) accounts for around 10% of community acquired bacterial pneumonia and has been associated with other chronic inflammatory conditions. We describe a C57/Bl6 murine model of Cp lung infection characterized by a dose-dependent, resolving neutrophilia followed by lymphocytic infiltration of the lungs. By 21 days post-infection, mice exhibit a T helper type 1 (Th1) polarized serum antibody response with local mucosal antibody secretion and organization of ectopic lymphoid tissue which persisted in the absence of detectable Cp DNA. Macrophage inflammatory protein (MIP)-2/CXCL2, which recruits neutrophils and lymphocytes and is associated with ectopic lymphoid tissue formation, was secreted in the lungs post-infection. In vitro, lung epithelial cells up-regulated MIP-2/CXCL2 in response to both rough lipopolysaccharide (reLPS) and Cp infection. We conclude that Cp infection can have long-term inflammatory effects on tissue that persist after clearance of active infection.

Identification of Chlamydophila abortus and the development of lesions in placental tissues of experimentally infected sheep.

Chlamydophila (C.) abortus is a major cause of infectious abortion in sheep in many countries. Twenty-one pregnant sheep were experimentally infected intranasally with C. abortus at 70 days of gestation (dg). Thereafter, a number of animals were killed at weekly intervals and a post-mortem examination was carried out. Evidence of chlamydial infection in the placenta was determined by isolation of the bacterium by tissue culture and detection of C. abortus DNA by real-time polymerase chain reaction (real-time PCR). In addition, histopathological changes in the placenta were assessed, as was the detection of chlamydial antigen by immunohistochemistry (IHC). Evidence of placental infection was observed as early as 2 weeks after inoculation, and while only relatively low numbers of bacteria were isolated by culture and/or detected by real-time PCR prior to 113-114dg, at 119-121dg, it was more numerous. This study, using the four criteria for assessment of infection, showed that while C. abortus gained access to the placenta as early as 85dg, characteristic histopathological changes were not apparent until 119/121dg. While the chronology of when the bacterium arrived in the placenta and subsequent lesion development is remarkable for its consistency this paper provides more reliable data on the former which in turn now allows study of the factors that permit its access to this tissue and govern its multiplication and the ensuing triggering of damage.

Abortion storm induced by the live C. abortus vaccine 1B strain in a vaccinated sheep flock, mimicking a natural wild-type infection.

Chlamydia abortus is responsible for enzootic abortion (known as ovine enzootic abortion (OEA) and enzootic abortion of ewes (EAE)) in both sheep and goats and has major economic implications for the farming industry worldwide. A virulence-attenuated mutant strain of C. abortus (strain 1B) is currently commercially available as a live attenuated vaccine for immunization of sheep and goats in several European countries. Following an abortion storm in a French flock of 200 ewes that occurred two years after vaccination of 36 replacement ewes with the commercial 1B vaccine strain, the vaginal swabs of 3 vaccinated and 7 unvaccinated aborted ewes and 12 of the 13 dead fetuses were found to be positive for C. abortus by real-time PCR. Genotyping of the samples, using vaccine-specific SNP markers, identified all as positive for the vaccine-type strain. The recent vaccination of this flock with the attenuated commercial vaccine strain, the large number of abortion cases observed in ewes irrespective of vaccination status, the high C. abortus load detected in vaginal swabs or abortion tissues and the identification of specific vaccine-type markers in these samples strongly suggest that the 1B strain has been transmitted from vaccinated to naïve animals, thus mimicking a natural wild-type infection.

Vaccination against chlamydial infections of man and animals.

Vaccination is the best approach for controlling the spread of chlamydial infections, in animal and human populations. This review summarises the progress that has been made towards the development of effective vaccines over the last 50 years, and discusses current vaccine strategies. The ultimate goal of vaccine research is to develop efficacious vaccines that induce sterile, long-lasting, heterotypic protective immune responses. To date, the greatest success has been in developing whole organism based killed or live attenuated vaccines against the animal pathogens Chlamydophila abortus and Chlamydophila felis. However, similar approaches have proved unsuccessful in combating human chlamydial infections. More recently, emphasis has been placed on the development of subunit or multicomponent vaccines, as cheaper, safer and more stable alternatives. Central to this is a need to identify candidate vaccine antigens, which is being aided by the sequencing of representative genomes of all of the chlamydial species. In addition, it is necessary to identify suitable adjuvants and develop methods for antigen delivery that are capable of eliciting mucosal and systemic cellular and humoral immune responses. DNA vaccination in particular holds much promise, particularly in terms of safety and stability, although it has so far been less effective in humans and large animals than in mice. Thus, much research still needs to be done to improve the delivery of plasmid DNA, as well as the expression and presentation of antigens to ensure that effective immune responses are induced.

The activation of rabbit intestinal adenylate cyclase by cholera toxin.

Brush-border and basal-lateral membranes were prepared from rabbit intestinal epithelial cells by differential centrifugation and MgCl2 precipitation. The ADP-ribosylation of proteins in these fractions when incubated with [adenylate-32P]NAD+ and cholera toxin was investigated. Three proteins of molecular mass 45, 40 and 37 kDa were labelled in a toxin-dependent manner in each membrane fraction. The incorporation of 32P-labelled ADP-ribose was 18-fold greater in brush-border membranes than in basal-lateral membranes, comparable to the enrichment of sucrase (marker enzyme for the brush border) in these membranes. There was a 20% release of the 40 and 45 kDa proteins from the brush-border membrane following this ADP-ribosylation. Activation of adenylate cyclase by both cholera toxin and sodium fluoride was 2.7- and 2.3-fold greater, respectively, in basal-lateral membranes than in brush-border membranes, comparable to the enrichment of Na+/K+-ATPase (marker enzyme for the basal-lateral membrane) in these membranes. The effect of sodium fluoride on membranes pretreated with cholera toxin revealed no increase in adenylate cyclase activity above that due to the toxin. This presumably means that both toxin and fluoride activate adenylate cyclase by the same regulatory protein. The results show that cholera toxin catalyzes the ADP-ribosylation of regulatory proteins in the brush-border membrane, and these proteins then migrate to the basal-lateral membrane where they activate the catalytic component of adenylate cyclase.

Subunit structure of calgranulins A and B obtained from sputum, plasma, granulocytes and cultured epithelial cells.

The calcium-binding proteins calgranulins A and B co-purified with an elastase-specific inhibitor after the affinity and cation-exchange chromatography of the perchloric acid-soluble fraction of pooled sputum collected from patients with chronic obstructive pulmonary disease (Sallenave, J.-M. and Ryle, A.P. (1991) Biol. Chem. Hoppe-Seyler 372, 13-21). The calgranulins were separated from the inhibitor by reverse-phase FPLC. Protein blot analysis of the calgranulin fraction in the absence of reducing agent revealed a band of 25 kDa corresponding to the disulphide-bonded heterodimerization of the two monomer components. Similar results were obtained from the immunoprecipitation and protein blot analysis of plasma, granulocytes and cultured epithelial cells. This implies that the calgranulins exist in the heterodimeric form in secretions in vivo. Their association with pancreatic elastase during the affinity chromatography stage of purification implicates them in the tissue destruction elicited by the inflammatory response in chronic obstructive pulmonary diseases.

Immunopathology of Chlamydophila abortus infection in sheep and mice.

Chlamydophila abortus targets the placenta, causing tissue damage, inflammation and abortion (enzootic abortion of ewes). It is one of the main infectious causes of abortion in ewes, resulting in major economic losses to agricultural industries worldwide. Although ruminants and pigs are the principal hosts, humans are also susceptible to infection. Control of disease requires a host inflammatory response, which is likely to contribute to pathology and abortion. Mouse models have been widely used to provide insight into the role of specific immune cells in controlling infection and disease. The use of such model systems for investigating the mechanisms of abortion, latency, persistence, and immunity to reinfection will result in the identification of novel vaccine control strategies for sheep.

Late production of CXCL8 in ruminant oro-nasal turbinate cells in response to Chlamydia abortus infection.

Chlamydia abortus is an obligate intracellular bacterium that is an important cause of ovine abortion worldwide. There are reports of abortions in cattle, but these are very rare compared to the reported incidence in sheep. The bacterium is transmitted oro-nasally and can establish a sub-clinical infection until pregnancy, when it can invade the placenta and induce an inflammatory cascade leading to placentitis and abortion. Early host-pathogen interactions could explain differential pathogenesis and subsequent disease outcome in ruminant species. In this study, we assessed the ability of sheep and cattle oro-nasal turbinate cells to sense and respond to C. abortus infection. The cells expressed toll like receptor (TLR) 2, TLR4, nucleotide oligomerization domain (NOD) 1 and NOD-like receptor pyrin domain containing 3 (NLRP3) mRNA. In response to C. abortus infection, both ovine and bovine turbinate cells produce CXCL8 mRNA and protein late in the bacterial developmental cycle, but do not produce IL-1ß or TNF-α. The UV-inactivated bacteria did not elicit a CXCL8 response, suggesting that intracellular multiplication of the bacteria is important for activating the signalling pathways. The production of innate immune cytokines from cattle and sheep turbinate cells in response to C. abortus infection was found to be largely similar.

Identification of the 1B vaccine strain of Chlamydia abortus in aborted placentas during the investigation of toxaemic and systemic disease in sheep.

CASE HISTORY: One hundred and forty Cheviot and 100 Suffolk cross Mule primiparous 1-2-year-old ewes, from a flock of about 700 ewes, were vaccinated with an attenuated live 1B strain Chlamydia abortus vaccine about 4 weeks before ram introduction (September 2011). Between 08 March and 01 April 2012, 50 2-year-old ewes aborted and 29 of these died, despite antimicrobial and anti-inflammatory treatment and supportive care. PATHOLOGICAL FINDINGS: Seven fetuses and three placentae from five 2-year-old ewes were submitted for pathological investigation. The aborted fetuses showed stages of autolysis ranging from being moderately fresh to putrefaction. Unusual, large multifocal regions of thickened membranes, with a dull red granular surface and moderate amounts of grey-white surface exudate were seen on each of the placentae. Intracellular, magenta-staining, acid fast inclusions were identified in Ziehl Neelsen-stained placental smears. Immunohistochemistry for Chlamydia-specific lipopolysaccharide showed extensive positive labelling of the placental epithelia. LABORATORY FINDINGS: Molecular analyses of the aborted placentae demonstrated the presence of the 1B vaccine-type strain of C. abortus and absence of any wild-type field strain. The vaccine strain bacterial load of the placental tissue samples was consistent with there being an association between vaccination and abortion. DIAGNOSIS: Initial laboratory investigations resulted in a diagnosis of chlamydial abortion. Further investigations led to the identification of the 1B vaccine strain of C. abortus in material from all three of the submitted aborted placentae. CLINICAL RELEVANCE: Timely knowledge and understanding of any potential problems caused by vaccination against C. abortus are prerequisites for sustainable control of chlamydial abortion. This report describes the investigation of an atypical abortion storm in sheep, and describes the identification of the 1B vaccine strain of C. abortus in products of abortion. The significance of this novel putative association between the vaccine strain of C. abortus and severe clinical disease is unknown. Aspects of the approach that is described are relevant to the investigation of all outbreaks of ovine abortion, irrespective of the diagnosis. Awareness of the changing role of C. abortus as a major global cause of abortion ought to reinforce the importance of monitoring of adequate biosecurity in those countries which are currently free from chlamydial abortion.

Effect of antipsychotic drugs on the molecular action of cholera toxin in rabbit intestinal epithelial cells.

Antipsychotic drugs of known antidiarrhoeal and anticalmodulin activity inhibited the cholera-toxin-catalysed ADP-ribosylation of proteins of Mr 37,000, 40,000 and 45,000 (thought to be regulatory components of the adenylate cyclase complex) that was previously shown to occur in plasma membranes from rabbit intestinal epithelial cells [(1989) Biochim. Biophys. Acta 1014, 289-297]. There was no obvious correlation between the different activities of the drugs. The drugs also inhibited adenylate cyclase activity, but in this case the inhibition correlated well with the known IC50 values of the drugs for anticalmodulin activity and with their antidiarrhoeal activities.

Single channel analysis of recombinant major outer membrane protein porins from Chlamydia psittaci and Chlamydia pneumoniae.

We recently demonstrated that the major outer membrane protein of Chlamydia psittaci, the primary vaccine candidate for combating chlamydial infections, functions as a porin-like ion channel. In this study, we have cloned, expressed and functionally reconstituted recombinant major outer membrane proteins from C. psittaci and Chlamydia pneumoniae and analysed them at the single channel level. Both form porin-like ion channels that are functionally similar to those formed by native C. psittaci major outer membrane protein. Also, like the native channels, recombinant C. psittaci channels are modified by a native major outer membrane protein-specific monoclonal antibody. This is the first time that native function has been demonstrated for recombinant chlamydial major outer membrane proteins. Future bilayer reconstitution will provide a strategy for detailed structure/function studies of this new subclass of bacterial porins and the work also has important implications for successful protein refolding and the development of improved subunit vaccines.

Molecular cloning and characterisation of a putative aspartate proteinase associated with a gut membrane protein complex from adult Haemonchus contortus.

A cDNA was isolated from an adult Haemonchus contortus cDNA expression library the deduced amino acid sequence of which showed significant homology to mammalian pepsinogen sequences. The library was screened with antisera raised against Haemonchus galactose-containing glycoprotein complex, a gut membrane protein complex with aspartyl proteinase activity which has shown considerable potential as a protective antigen. The amino acid sequence obtained corresponded very closely in part to the N-terminal amino acid sequences of two polypetides within the complex. The enzyme was shown to be almost exclusively expressed by the blood-feeding parasite stages. The cDNA was expressed in E. coli, and antibody produced to the recombinant protein bound to the luminal surface of the gut in the adult parasite. The proteinase may play a central role in digesting the blood meal and is considered a potential sub-unit vaccine candidate.

Diagnosis of ovine enzootic abortion using an indirect ELISA (rOMP91B iELISA) based on a recombinant protein fragment of the polymorphic outer membrane protein POMP91B of Chlamydophila abortus.

Chlamydophila abortus is of major economic importance worldwide as one of the principal causes of abortion in sheep. Serological diagnosis of infection by the complement fixation test (CFT) is complicated by false positive reactions resulting from cross-reactive antibodies to Chlamydophila pecorum. To improve diagnosis an indirect enzyme-linked immunosorbent assay (iELISA) based on a recombinant protein fragment of the C. abortus polymorphic outer membrane protein POMP91B (rOMP91B iELISA) was assessed using a panel of 281 sera from experimentally and naturally infected sheep. The iELISA performed well, being more sensitive (84.2%) and specific (98.5%) than the CFT. Furthermore, the iELISA was better at differentiating C. abortus- from C. pecorum-infected animals. The new rOMP91B iELISA test will prove a valuable tool for the routine serodiagnosis of C. abortus infection.

Immunoelectron microscopic localisation of the OMP90 family on the outer membrane surface of Chlamydia psittaci.

The putative outer membrane location of the OMP90 (formerly POMP) family from the ovine abortion strain of Chlamydia psittaci was investigated by immunoelectron microscopy. Using a non-embedding technique, antigens were shown to be localised on the outer membrane surface of both elementary and reticulate bodies, the infectious and non-infectious forms of Chlamydiae respectively. Antibodies affinity-purified against the expressed amino- and carboxy-terminal halves of one of the family members. OMP90A, demonstrated that the amino half is surface-exposed while the carboxyl half is most probably localised internally. Surface localisation on elementary bodies indicates the importance of these proteins as protective antigen candidates.

Identification of a multigene family coding for the 90 kDa proteins of the ovine abortion subtype of Chlamydia psittaci.

While attempting to identify genes and their corresponding antigens that could be used to improve the current methods of diagnosing Chlamydia psittaci infection which causes enzootic abortion in ewes, two candidate clones were isolated from a lambda gt 11 genomic DNA expression library of ovine abortion subtype (strain S26/3) C. psittaci. These clones contained fragments of a gene coding for a group of three chlamydial proteins of approximately 90 kDa which appeared as major immunogens by immunoblotting experiments, indicating their potential as diagnostic or possibly protective antigens. Southern blotting of S26/3 genomic DNA using the two clones as probes identified a family of three or four genes. These represent the first example of protein gene duplication reported in Chlamydia.

Ovine chlamydial abortion: characterization of the inflammatory immune response in placental tissues.

Ovine chlamydial abortion is a serious cause of fetal mortality in several sheep-rearing countries. The causal agent, Chlamydophila abortus (Chlamydia psittaci), does not generally induce clinical signs in the ewe other than abortion; this is associated with macroscopically visible damage in the placenta, which may be inflamed and thickened. To investigate the nature of the placental inflammation, seven pregnant sheep were inoculated subcutaneously at 70 days' gestation with C. abortus (strain S 26/3). A further five pregnant sheep received control inoculum by the same route at the same stage of pregnancy. Three of the infected ewes produced stillborn lambs and four produced live lambs. Lesions characteristic of chlamydial infection were present in all placentas except for two from one ewe that gave birth to twins. Histopathological examination of placental tissues from aborted fetuses showed a mixed inflammatory cell infiltrate with vasculitis and thrombosis in the mesenchyme of the intercotyledonary membranes. Cells expressing the macrophage-associated molecule CD 14 were found to be numerous, as were cells expressing major histocompatibility complex class II (MHC II) molecules. Many cells expressing messenger RNA (mRNA) encoding for tumour necrosis factor-alpha (TNF-alpha) were demonstrated, but few cells expressing interferon gamma mRNA and none expressing interleukin-4 mRNA were detected. The fetal immune response included small numbers of CD4+ and CD8+ cells, gamma delta T cells and B cells. It is concluded that abortion is the result of several factors, including destruction of tissue by C. abortus, vascular thrombosis, and an inflammatory response by the fetus. Production of TNF-alpha by fetal macrophages expressing MHC II molecules may be of considerable significance in the pathogenesis of abortion.

Risk factors associated with Lawsonia intracellularis in English pig farms.

Porcine proliferative enteropathy (PPE) caused by the bacterium Lawsonia intracellularis causes considerable economic loss to the pig industry. The objective of this study was to evaluate the seroprevalence of L. intracellularis exposure in different age groups of pigs (growers to finishers) within English farms and to identify potential risk factors. Samples were obtained in a cross-sectional study of 147 farms between 2008 and 2009. Twelve samples (six growers and six finishers) from each farm were tested for L. intracellularis by antibody ELISA. At animal level there was a significant positive linear trend between seroprevalence and age in weeks (r(2)=2.65, P<0.001), with seroprevalence lowest (24.73%) at 11 weeks and highest (93.33%) at 24 weeks. At farm level, seroprevalence was significantly lower in growers than finishers (56.80% vs. 94.26%, P<0.001). Farms reporting minor Salmonella problems and those that brought boars onto the farm had higher odds of testing positive in growers (OR 5.69 and 4.31, respectively. On the other hand, farms where producers considered temperature as an important stress factor (OR=0.3) and which had more than two sites on which pigs are kept (OR=0.16) were less likely to test positive in growers. The current study confirmed the high prevalence of L. intracellularis in English pig farms. The potential risk factors and further information of the disease impact on the farm productivity will aid the development of appropriate control strategies through better understanding of the disease.

Endemic and emerging chlamydial infections of animals and their zoonotic implications.

The Chlamydiae are a diverse group of obligate intracellular Gram-negative bacteria that are known to infect a wide variety of host species and are responsible for a wide range of diseases in animals and man. Many of these organisms have been extensively characterized and their zoonotic implications recognized. Studies of human disease first provided evidence for the disease-causing potential of Chlamydia-related bacteria; however, there is now increasing evidence that a number of these organisms may also be the causative agents for a number of pathogenic conditions of livestock that had previously remained undiagnosed. The aim of this review is to draw together the evidence for the role of the newly emerging chlamydial infections in livestock disease, the current understanding of their roles in human disease and highlight the potential for zoonotic transmission.