Diversity, impact and control of chlamydial infections in ruminants. David Longbottom Moredun Research Institute, Edinburgh, UK

Diversity, impact and control of chlamydial infections in ruminants David Longbottom Moredun Research Institute, Edinburgh, UK

Chlamydial diversity Chlamydiae Obligate intracellular bacteria Variety of diseases with a wide host range Biphasic developmental cycle: EB & RB

Chlamydial Developmental Cycle RB

Chlamydial diversity Chlamydiae Obligate intracellular bacteria Variety of diseases with a wide host range Biphasic developmental cycle: EB & RB Superkingdom: Bacteria (Eubacteria) Superphylum: Chlamydiae/Verrucomicrobia Group Phylum: Chlamydiae Class: Chlamydiae Order: Chlamydiales Family: Chlamydiaceae Taxonomic re-classification in 1999 [Everett et al.] Chlamydiaceae and Chlamydia-like organisms 2 genera: Chlamydia & Chlamydophila 9 species

Chlamydial diversity Since 1999 Chlamydia-like organisms reported from huge range of sources/hosts Waste water plants Rivers Arctic/Antarctic sediment Arthropods Isopod crustaceae Insects Spiders Scorpions Bats Reptiles Fish 2011 Single genus: Chlamydia

Chlamydial Taxonomy (2011) Phylum. Chlamydiae Class I. Chlamydiia class. nov. Order I. Chlamydiales Family I. Chlamydiaceae Genus I. Chlamydia Family II. Candidatus Clavichlamydiaceae Genus I. Candidatus clavichlamydia Family III. Criblamydiaceae Genus I. Criblamydia Family IV. Parachlamydiaceae Genus I. Parachlamydia Genus II. Neochlamydia Genus III. Protochlamydia gen. nov. Family V. Candidatus Piscichlamydiaceae Genus I. Candidatus Piscichlamydia Family VI. Rhabdochlamydiaceae fam. nov. Genus I. Rhabdochlamydia gen. nov. Family VII. Simkaniaceae Genus I. Simkania Genus II. Candidatus Fritschea Family VIII. Waddliaceae Genus I. Waddlia

Chlamydial Disease Species Chlamydia trachomatis Chlamydia pneumoniae Chlamydia muridarum Chlamydia suis Chlamydia caviae Chlamydia felis Chlamydia psittaci Chlamydia abortus Chlamydia pecorum Typical Host Humans Humans, Koala, Horse Mice, hamsters Swine Guinea pig Cats Birds, poultry Ruminants, swine Ruminants, swine Infection/Disease STI, PID, salpingitis, trachoma Pneumonia, Bronchitis Respiratory, Genital, (Model System) Enteric, Respiratory, Reproductive Ocular, Genital, (Model System) Conjunctivitis Respiratory Abortion Enteritis, Pneumonia, Conjunctivitis, Metritis, Polyarthritis, Mastitis, Encephalomyelitis

Chlamydia abortus: The most common diagnosed cause of infectious ovine abortion in the UK Veterinary Investigation Disease Analysis (VIDA) Ovine Fetopathies 1995-2009 4,5 2,9 1,2 1,2 7,4 24,1 12,3 43,5 44,7 14,9 26,9 4,1 6,8 0,7 0,7 1,6 2,5 Chlamydophila abortus Campylobacter spp Listeria monocytogenes Arcanobacterium pyogenes Toxoplasma gondii Salmonella spp Bacillus licheniformis Other Chlamydophila abortus Campylobacter spp Listeria monocytogenes Arcanobacterium pyogenes Diagnosis not reached Toxoplasma gondii Salmonella spp Bacillus licheniformis Other

Sheep, goats Chlamydophila abortus Enzootic Abortion of Ewes (EAE) / Ovine Enzootic Abortion (OEA) Cattle, pigs, deer, horses Humans

Enzootic Abortion of Ewes Pathogenesis and control of C. abortus infection Immunopathogenesis/molecular pathogenesis: Define mechanisms that trigger immunopathology - Maternal and foetal immune response Mechanisms underlying latency & persistence Identify and characterise the protective immune response - Innate and adaptive immunity Characterisation of the pathogen - membrane + exported proteins - genomic, transcriptomic & proteomic analyses - structure/function Control strategies: diagnosis (serological & molecular) prophylaxis flock management

EAE Pathogenesis at 6-8 days post-infection organism becomes undetectable in non-pregnant ewe latency involves suppression of the organism by interferon γ infection in female only becomes apparent during pregnancy organism detectable at 90-95 dg infection establishes in foetal chorionic epithelium (trophoblast) necrosis develops in placenta abortion occurs at 125-140 dg after abortion maternal antibody levels rise which coincides with the ewe developing protective immunity protective immunity involves both humoral and cell-mediated responses

McGeady et al 2006 Blackwell Publishing

Cotyledons associated with rounded elevations of the uterine endometrium called caruncles. Placental chorionic villi extend into the crypts of the uterine caruncle and interdigitate with the maternal septa At around 60 days gestation, haematomas develop at the bases of the chorionic villi, likely results from leakage of blood from peripheral capillaries in the maternal septal tips

Ovine Enzootic Abortion (OEA)

Ovine placentome

120 days gestation (50 dpi) Inclusions visible in chorionic epithelium

mab 13/5 (chlamydial LPS) Anti-LPS mab: chlamydial antigen & inclusions Vasculitis with luminal platelet adhesion Partial or complete thrombosis Thrombosis Pathological processes may be triggered by locally produced LPS and by interaction of chlamydial effectors and surface antigens LPS shown to cause thrombus formation and inducing TNFalpha production in mice

mab 49.1 (MHC II) Few CD4+ and CD8+ T cells in inflammatory infiltrate Few B cells Numerous cells expressing MHCII mononuclear Most cells consisted of monocytes/macrophages mab VPM 65 [CD14] (macro/mono) Macrophages (CD14) in deeper zones of inflammation in mesenchyme, plus some also in arterioles and arteries

mab VPM 65 (macro/mono) Few cells expressing IFN-gamma mrna Numerous mononuclear cell types expressing mrna encoding for TNF-alpha within infected arterioles and arteries as well as inflammatory exudate Results in inflammation and destruction of placental tissue TNF-alpha (mrna in situ)

Ovine chlamydial abortion placental pathology Summary C. abortus infects chorionic epithelial cells (trophoblast) Destruction of chorionic epithelium Release of chlamydial LPS Triggers foetal cell mediated immune response Monocyte/Macrophages/MHC II/TNF-α Inflammation/oedema placental membrane Arteritis Platelet adhesion/thrombosis Hormonal balance affected: progesterone ( ), oestradiol ( ), prostoglandin E2 ( ) affected (Leaver et al., 1989) precipitates abortion

EAE transmission Contaminated environment: vaginal discharges, infected placenta & foetus Venereal / mechanical Vertical transmission ABORTION 2-3 weeks pre-term WEAKLY LAMBS

Zoonoses

Human foetopathy and Chlamydia abortus 1956 C. psittaci identified placenta Giroud et al 1967 serology +ve Roberts et al 1977 - - Terskikh et al 1982 - - Beer et al 1985 C. abortus isolated / IHC Johnson et al 1985 serol + IHC McKinlay et al 2000 PCR +ve Kampinga et al

Human foetopathy and Chlamydia abortus Clinical signs 1 fever, headache, nausea, malaise 2 spontaneous contractions 3-8 days after onset delivery stillborn infant 3 disseminated intravascular coagulation (DIC) profound thrombocytopenia renal/hepatic dysfunction myocardial symptoms

Human foetopathy and Chlamydia abortus

Farmer / Scotland (28 weeks) Vet / Scotland (25 weeks) Farmer / Netherlands (26 weeks)

Pathogenesis: Chlamydia abortus Ovine infection Subacute/chronic Human infection Peracute/acute necrosis + inflammation (macrophages and TNF-α) grave risk to fetus minimal risk to ewe destruction trophoblast cells DIC grave risk for fetus and mother

Chlamydia abortus Vaccines: Vaccine development (DIVA vaccine) Commercial vaccines: live, attenuated ts mutant Enzovax (Intervet) Cevac Chlamydophila (Ceva Animal Health)

Vaccine strain discrimination CAB153 (SfcI) M S26/3 AB7 1B 1 2 3 4 5 CAB636 (HaeIII) M S26/3 AB7 1B 1 2 3 4 5 319 bp 177bp 142 bp 118bp 74 bp 44 bp CAB648 (Sau3AI) M S26/3 AB7 1B 1 2 3 4 5 Placental samples from 35 cases submitted: 88/89 bp 67bp 21 from non-vaccinated flocks: all wildtype genotype 14 from vaccinated flocks: 5 (8 samples) had 1B vaccine genotype 3 samples from an Enzovax trial (1994) also had 1B genotype

Vaccine strain discrimination CDS Genomic SNP Mutation Nucleotide identified in position of SNP class e S26/3, AB7 1B Field cases 1994 samples #1-2, 6-8 #15-17 CAB220 241667 G A Non G A A A CAB283 328034 C T Non C T T T 328010 T C Syn T C C C CAB469 542479 A G Syn A A A A Sequencing of 3 additional genes confirmed the presence of 1B in the 5 cases and 3 1994 samples Identical mutations were present both in 2008 and 1994 which suggests the vaccine has not changed Wheelhouse et al., 2010. Evidence of Chlamydophila abortus vaccine strain 1B as a possible cause of Ovine Enzootic Abortion. Vaccine 28, 5657-5663.

Ovine/Bovine fetopathies: VIDA Ovine Fetopathies 1995-2009 Bovine Fetopathies 1995-2009 2,9 1,2 1,2 4,5 7,4 12,3 43,5 5,2 23,2 0,2 5,4 2,3 0,2 9,2 11,9 2,4 26,9 1,7 5,3 22,6 10,3 0,2 Chlamydophila abortus Campylobacter spp Listeria monocytogenes Arcanobacterium pyogenes Toxoplasma gondii Salmonella spp Bacillus licheniformis Other Brucella abortus Arcanobacterium pyogenes Campylobacter Chlamydophila abortus Leptospira Listeria monocytogenes Salmonella spp Bacillus licheniformis Coxiella Neospora Fungi of any species IBR BVD Other 0,0 1,3 0,60,1 2,30,62,4 2,5 0,0 5,6 44,7 24,1 6,8 14,9 77,5 1,3 0,4 1,3 4,0 4,1 0,70,7 Chlamydophila abortus Campylobacter spp Listeria monocytogenes Arcanobacterium pyogenes Diagnosis not reached 1,6 2,5 Toxoplasma gondii Salmonella spp Bacillus licheniformis Other Brucella abortus Arcanobacterium pyogenes Campylobacter Chlamydophila abortus Leptospira Listeria monocytogenes Salmonella spp Bacillus licheniformis Coxiella Neospora Fungi of any species IBR BVD Other Fetopathy diagnosis not reached

Chlamydial diversity Isolation of chlamydia-like organism from aborted bovine fetus Dilbeck et al. 1990. Isolation of a previously undescribed rickettsia from an aborted bovine fetus. Kocan et al. 1990. Development of a rickettsia isolated from an aborted bovine fetus. Henning et al. 2002. Neospora caninum and Waddlia chondrophila strain 2032/99 in a septic stillborn calf. Discovery and ultra-structural analysis of several endosymbionts of free-living amoebae Lewis et al. 1990. Microbiological and serological studies of an outbreak of humidifier fever in a print shop [Hall s coccus]. Michel et al. 1994. Acanthamoeba from human nasal mucosa infected with an obligate intracellular parasite. Parachlamydia acanthamoeba Collingro et al. 2005. Candidatus Protochlamydia amoebophila', an endosymbiont of Acanthamoeba spp. Description of a conspicuous bacterium growing as a contaminant in a cell culture within cytoplasmic phagosomes Kahane et al. 1993. Description and partial characterization of a new Chlamydia-like microorganism. Simkania micro-organism Z Associated with community acquired pneumonia in adults, bronchiolitis in infants and COPD.

Emerging bovine chlamydial infections Growing body of evidence suggesting an association between bovine abortion, human respiratory infections and human miscarriage with infections due to chlamydia-like organisms Borel et al. 2006. Chlamydia-related abortions in cattle from Graubunden, Switzerland. [Parachlamydia] Borel et al. 2007. Parachlamydia spp. and related Chlamydia-like organisms and bovine abortion. Baud et al. 2007. Waddlia chondrophila, a potential agent of human fetal death. Baud et al. 2008. Emerging role of Chlamydia and Chlamydia-like organisms in adverse pregnancy outcomes. [Parachlamydia] Haider et al. 2008. Chlamydia-like bacteria in respiratory samples of community-acquired pneumonia patients. [Protochlamydia amoebophila, Waddlia chondrophila, and 'Candidatus Rhabdochlamydia porcellionis ] Ruhl et al. 2009. Evidence for Parachlamydia in bovine abortion. Baud et al. 2009. Evidence of maternal-fetal transmission of Parachlamydia acanthamoebae Greub et al. 2009. Parachlamydia acanthamoebae, an emerging agent of pneumonia. Lamoth et al. 2009. Parachlamydia and Rhabdochlamydia in premature neonates.

Initial investigation Pooled fetal tissue samples (brain, heart and/or placenta) from cases of bovine abortion submitted by SAC for Neospora testing Chlamydiales specific 16S rrna PCR [Ossewaarde & Meijer, 1999] 22 (26.5%) of 83 samples tested were Chlamydiales positive All Chlamydiaceae negative (23S rrna Chlamydiaceae qpcr) Serological, bacteriological and histopatological examination of samples identified no other infectious abortifacient agents Sequence data obtained for 15 of the 22 samples Sequences aligned with representative set of 22 Chlamydiales 16S rdna sequences plus an outgroup of 7 non-chlamydiales sequences Bayesian phylogenetic analysis 3 clusters Chlamydiaceae Rhabdochlamydiaceae / Simkaniaceae Parachlamydiaceae / Waddliaceae / Criblamydiaceae

Bayesian phylogenetic tree 5 / 15 10 / 15 Wheelhouse et al. 2010. Novel Chlamydia-like organisms as cause of bovine abortions, UK. Emerging Infectious Diseases 16, 1323-24.

Further investigations Pooled fetal tissue samples (brain, heart and/or placenta) from cases of undiagnosed bovine abortion submitted by SAC Chlamydiales specific 16S rrna PCR [Ossewaarde & Meijer, 1999] 71 (37.9%) of 187 samples tested were Chlamydiales positive 1 of 71 samples Chlamydiaceae positive (23S rrna Chlamydiaceae qpcr) C. abortus (Alere Technologies Array Tube microarray) 24 samples sequenced so far 106 sequences aligned using MAFFT multiple alignment program. Maximum Likelihood phylogenetic tree estimated using the RaxML program, with a GTR+I+G model and 100 bootstrap replicates Reconcile samples with pathological results

Case Study Undiagnosed bovine abortion placental samples (2) submitted by SAC Spring-calving beef herd with annual abortion rate of 4% Previous investigations failed to reveal cause Severe placentitis with intercotyledonary thickening & necrosis Histopath revealed necrosuppurative placentitis with vasculitis Granular cytoplasmic inclusions within trophoblasts Parachlamydia confirmed by IHC and 16S rrna PCR [Gilbert Greub/Nicole Borel]

Case Study Other samples subsequently submitted Mixed infections with B. licheniformis IHC positive for Parachlamydia Water samples from troughs in sheds tested by 16S rdna PCR 16S rrna gene clone library constructed 31 clones sequenced Organisms belonging to Parachlamydia & Neochlamydia genera Deuchande et al. 2010. Parachlamydia involvement in bovine abortions in a beef herd in Scotland. Veterinary Record 598-9. Wheelhouse et al. 2011. Detection of Chlamydia-like organisms in cattle drinking water. Veterinary Microbiology

Summary Chlamydia-like organism DNA identified in cases of bovine abortion Phylogenetic analysis has identified DNA as originating from Parachlamydia and Rhabdochlamydia Parachlamydia confirmed in trophoblast inclusions by IHC Parachlamydial DNA identified in water samples from drinking troughs DNA sequences match some of those obtained from fetal samples Environmental route of transmission Association causal role?? Isolation & pathogenesis studies Prevalence studies in UK

Acknowledgements Morag Livingstone Kevin Aitchison Kim Wilson Nick Wheelhouse Michelle Sait David Buxton Gary Entrican Mark Dagleish Kim Willoughby Frank Katzer Frank Wright (BioSS) Rita Deuchande (VLA) Jo Gidlow + VIOs (SAC) Gilbert Greub (Lausanne) Nicole Borel (Zurich) Karine Laroucau (Paris)