IntroductionThis study employs bulk RNA sequencing, PCR, and ELISA assays to analyze the pathological factors affecting the outcomes of C. pecorum ocular infections in koalas. It investigates the immune responses and gene expression profiles associated with various stages of koala ocular chlamydiosis.MethodsA cohort of 114 koalas from Queensland, Australia were assessed, with 47% displaying clinical signs of ocular disease. Animals were classified into three cohorts: acute active disease (G1), chronic active disease (G2), and chronic inactive disease (G3), along with subclinical Chlamydia pecorum positive (H2) and healthy (H1) cohorts.ResultsAnalysis of clinical, microbiological, humoral immune and cellular immune biomarkers revealed varying chlamydial loads and anti-chlamydial IgG levels across disease grades, with a negative correlation observed between ocular chlamydial load and anti-chlamydial IgG. Koala ocular mucosa gene expression analysis from 27 koalas identified shared expression pathways across disease cohorts, with a significant upregulation of IFNγ expression and tryptophan metabolism in all disease stages.DiscussionThese findings help elucidate immune response dynamics and molecular pathways underlying koala ocular chlamydiosis, providing insights crucial for disease management strategies.

IntroductionChlamydia trachomatis, a gram-negative obligate intracellular bacterium, commonly causes sexually transmitted infections (STIs). Little is known about C. trachomatis transmission within the host, which is important for understanding disease epidemiology and progression.MethodsWe used RNA-bait enrichment and whole-genome sequencing to compare rectal, vaginal and endocervical samples collected at the same time from 26 study participants who attended Fijian Ministry of Health and Medical Services clinics and tested positive for C. trachomatis at each anatomic site.ResultsThe 78 C. trachomatis genomes from participants resolved into two major clades of the C. trachomatis phylogeny (the “prevalent urogenital and anorectal” clade and “non-prevalent urogenital and anorectal” clade). For 21 participants, genome sequences were almost identical in each anatomic site. For the other five participants, two distinct C. trachomatis strains were present in different sites; in two cases, the vaginal sample was a mixture of strains.DiscussionThe absence of large numbers of fixed SNPs between C. trachomatis genomes within many of the participants could indicate recent acquisition of infection prior to the clinic visit without sufficient time to accumulate significant genetic variation in different body sites. This model suggests that many C. trachomatis infections may be resolved relatively quickly in the Fijian population, possibly reflecting common prescription or over-the-counter antibiotics usage.

Background The obligate intracellular bacterial family Chlamydiaceae comprises a number of different species that cause disease in various vertebrate hosts including humans. Chlamydia suis, primarily found in the gastrointestinal tract of pigs, is the only species of the Chlamydiaceae family to have naturally gained tetracycline resistance (TetR), through a genomic island (Tet-island), integrated into the middle of chromosomal invasin-like gene inv. Previous studies have hypothesised that the uptake of the Tet-island from a host outside the Chlamydiaceae family was a unique event, followed by spread among C. suis through homologous recombination. In vitro recombination studies have shown that Tet-island exchange between C. suis strains is possible. Our aim in this study was to gain a deeper understanding of the interclade recombination of the Tet-island, among currently circulating C. suis field strains compared to in vitro-generated recombinants, using published whole genome sequences of C. suis field strains (n = 35) and in vitro-generated recombinants (n = 63). Results We found that the phylogeny of inv better reflected the phylogeny of the Tet-island than that of the whole genome, supporting recombination rather than site-specific insertion as the means of transfer. There were considerable differences between the distribution of recombinations within in vitro-generated strains compared to that within the field strains. These differences are likely because in vitro-generated recombinants were selected for a tetracycline and rifamycin/rifampicin resistant background, leading to the largest peak of recombination across the Tet-island. Finally, we found that interclade recombinations across the Tet-island were more variable in length downstream of the Tet-island than upstream. Conclusions Our study supports the hypothesis that the occurrence of TetR strains in both clades of C. suis came about through interclade recombination after a single ancestral horizontal gene transfer event.

ABSTRACTPacific Islanders (PI) are a vulnerable population with a high prevalence of Chlamydia trachomatis (Ct) sexually transmitted infections and remain underrepresented in research. Here, 258 vaginal and 92 paired endocervical samples from women of diverse ethnicities in Fiji were analyzed using metagenomic shotgun sequencing. Using VALENCIA, a sub-community state type (subCST) classifier, 93 (36.04%) vaginal and 38 (41.30%) endocervical microbiomes did not match reference subCSTs. We, therefore, designed four unique subCSTs, IV-D0, IV-D1, IV-D2, and IV-E—differentially dominated by Gardnerella vaginalis, Prevotella spp., and Lactobacillus iners—to improve microbiome classifications. Ct-infected paired endocervical and vaginal microbiomes were significantly more likely to have differential species relative abundance (81.58%) than uninfected pairs (35.84%; adjusted odds ratio, 7.93; 95% confidence interval, 2.93–21.93; P < 0.0001). Among paired microbiomes, the endocervix had a significantly higher alpha diversity and independently a higher alpha diversity for high-risk human papilloma virus (HPV) types compared to low-risk and no HPV. The Ct-infected endocervical network compared to all others had smaller clusters—indicating lower species abundance and decreased weight of interactions among bacterial groups within clusters—and no interactions with potentially beneficial Lactobacillus spp. These data suggest a distinct influence on this network from Ct-infected endocervical cells. Furthermore, the high relative abundance of G. vaginalis and Prevotella spp. may generate polymicrobial biofilms that predispose to and/or promote Ct and possibly HPV persistence and pathogenicity. While these data expand on the existing repertoire of endocervical and vaginal microbiomes and fill in knowledge gaps regarding PIs, prospective studies are needed to define causal associations to develop successful interventions.IMPORTANCEChlamydia trachomatis (Ct) is the most common sexually transmitted bacterium globally. Endocervical and vaginal microbiome interactions are rarely examined within the context of Ct or among vulnerable populations. We evaluated 258 vaginal and 92 paired endocervical samples from Fijian women using metagenomic shotgun sequencing. Over 37% of the microbiomes could not be classified into sub-community state types (subCSTs). We, therefore, developed subCSTs IV-D0, IV-D1, IV-D2, and IV-E—dominated primarily by Gardnerella vaginalis—to improve classification. Among paired microbiomes, the endocervix had a significantly higher alpha diversity and, independently, higher diversity for high-risk human papilloma virus (HPV) genotypes compared to low-risk and no HPV. Ct-infected endocervical networks had smaller clusters without interactions with potentially beneficial Lactobacillus spp. Overall, these data suggest that G. vaginalis may generate polymicrobial biofilms that predispose to and/or promote Ct and possibly HPV persistence and pathogenicity. Our findings expand on the existing repertoire of endocervical and vaginal microbiomes and fill in knowledge gaps regarding Pacific Islanders.

Background Pacific Islanders are a vulnerable population with a high prevalence of Chlamydia trachomatis ( Ct ) sexually transmitted infections (STIs) and remain underrepresented in research. Here, 258 vaginal and 92 paired endocervical samples from women of diverse ethnicities in Fiji were evaluated using metagenomics to characterize microbial relative abundance, composition and networks including associations with Neisseria gonorrhoeae , human papilloma virus (HPV), Mycoplasma genitalium , Candida and bacterial vaginosis (BV). Results Pacific Islander ethnicities and age <25 years were significantly associated with Ct infection. Using VALENCIA, a sub-community state type (subCST) classifier, 93 (36.04%) vaginal and 38 (41.30%) endocervical microbiomes did not match reference subCSTs. Four unique subCSTs were developed to better classify Pacific Islander microbiomes: IV-D0, dominated by Gardnerella vaginalis ; IV-D1, high/moderate G. vaginalis relative abundance with Prevotella spp.; IV-D2, high/moderate G. vaginalis with Lactobacillus iners ; and IV-E, moderate Prevotella spp . Ct positive endocervical and paired vaginal microbiomes were significantly more likely to have differential species relative abundance (81.58%) than Ct negative pairs (35.84%; AOR: 7.93; 95% CI: 2.93-21.93; P <0.0001). A significantly higher alpha diversity was found for iTaukei ethnicity, all subCST-IV types, BV, and Ct for vaginal microbiomes. For endocervical microbiomes, higher diversity was significant for subCST-IV-A, subCST-IV-D1, and subCST-IV-E, and high-risk HPV types. Overall, there was a significantly higher diversity for the endocervix in paired microbiomes. Vaginal microbiomes showed significant divergence in community composition as above and for Candida. Endocervical composition varied significantly by subCST type and Ct status. Gut and BV-associated bacterial clusters were present in Ct positive and negative paired endocervical and vaginal microbiome networks but were smaller with fewer bacterial and no Lactobacillus spp. interactions in Ct -infected endocervical networks where G. vaginalis generated polymicrobial biofilms along with Ct likely influence pathogenicity. Conclusions Fijian endocervical and vaginal microbiomes represent divergent microbial abundance and compositions, especially for Pacific Islander ethnicities, with distinct subCSTs compared to other global populations. The higher microbial diversity of the endocervix with prevalent G. vaginalis , L. iners and Prevotella spp. suggest that these microbiomes/networks may predispose to and/or promote chlamydial and HPV pathogenesis. Prospective studies are needed to further define causal associations to develop successful interventions.

The Chlamydiaceae are a family of obligate intracellular, gram-negative bacteria known to readily exchange DNA by homologous recombination upon co-culture in vitro , allowing the transfer of antibiotic resistance residing on the chlamydial chromosome. Among all the obligate intracellular bacteria, only Chlamydia ( C .) suis naturally integrated a tetracycline resistance gene into its chromosome. Therefore, in order to further investigate the readiness of Chlamydia to exchange DNA and especially antibiotic resistance, C. suis is an excellent model to advance existing co-culture protocols allowing the identification of factors crucial to promote homologous recombination in vitro . With this strategy, we co-cultured tetracycline-resistant with rifamycin group-resistant C. suis , which resulted in an allover recombination efficiency of 28%. We found that simultaneous selection is crucial to increase the number of recombinants, that sub-inhibitory concentrations of tetracycline inhibit rather than promote the selection of double-resistant recombinants, and identified a recombination-deficient C. suis field isolate, strain SWA-110 (1-28b). While tetracycline resistance was detected in field isolates, rifampicin/rifamycin resistance (RifR) had to be induced in vitro . Here, we describe the protocol with which RifR C. suis strains were generated and confirmed. Subsequent whole-genome sequencing then revealed that G530E and D461A mutations in rpoB , a gene encoding for the β-subunit of the bacterial RNA polymerase (RNAP), was likely responsible for rifampicin and rifamycin resistance, respectively. Finally, whole-genome sequencing of recombinants obtained by co-culture revealed that recombinants picked from the same plate may be sibling clones and confirmed C. suis genome plasticity by revealing variable, apparently non-specific areas of recombination.

Chlamydia trachomatis ( Ct ) is a major global public health concern causing sexually transmitted and ocular infections affecting over 130 million and 260 million people, respectively. Sequelae include infertility, preterm birth, ectopic pregnancy, and blindness. Ct relies on available host tryptophan pools and/or substrates to synthesize tryptophan to survive.

Chlamydia are a genus of successful obligate intracellular pathogens spread across humans, wildlife, and domesticated animals. The most common species reported in livestock in this genus are Chlamydia abortus, Chlamydia psittaci, Chlamydia suis, and Chlamydia pecorum. Chlamydial infections trigger a series of inflammatory disease-related sequelae including arthritis, conjunctivitis, pneumonia, and abortion. Other bacteria in the phylum Chlamydiae have also been reported in livestock and wildlife but their impact on animal health is less clear. Control of chlamydial infections relies on the use of macrolides, fluoroquinolones, and tetracyclines. Tetracycline resistance (TETR) reported for porcine C. suis strains in association with the use of tetracycline feed is a potentially significant concern given experimental evidence highlighting that the genetic elements inferring TETR may be horizontally transferred to other chlamydial species. As documented in human Chlamydia trachomatis infections, relapse of infections, bacterial shedding post-antibiotic treatment, and disease progression despite chlamydial clearance in animals have also been reported. The identification of novel chlamydiae as well as new animal hosts for previously described chlamydial pathogens should place a renewed emphasis on basic in vivo studies to demonstrate the efficacy of existing and new antimicrobial treatment regimes. Building on recent reviews of antimicrobials limited to C. trachomatis and C. suis, this review will explore the use of antimicrobials, the evidence and factors that influence the treatment failure of chlamydial infections in animals and the future directions in the control of these important veterinary pathogens.

Chlamydiae are globally widespread obligate intracellular bacteria, which several species are a well-recognized threat to human and animal health. In Australia, the most successful chlamydial species are the infamous koala pathogen C. pecorum, and C. psittaci, an emerging pathogen associated with zoonotic events. Little is known about infections caused by other chlamydial organisms in Australian livestock or wildlife. Considering that these hosts can be encountered by humans at the animal/human interface, in this study, we investigated genetic diversity of chlamydial organisms infecting Australian domesticated and wild ungulates. A total of 185 samples from 129 domesticated (cattle, horses, sheep, and pigs) and 29 wild (deer) ungulate hosts were screened with C. pecorum and C. psittaci species-specific assays, followed by a screen with pan-Chlamydiales assay. Overall, chlamydial DNA was detected in 120/185 (65%) samples, including all ungulate hosts. Species-specific assays further revealed that C. pecorum and C. psittaci DNA were detected in 27% (50/185) and 6% (11/185) of the samples, respectively, however from domesticated hosts only. A total of 46 "signature" 16S rRNA sequences were successfully resolved by sequencing and were used for phylogenetic analyses. Sequence analyses revealed that genetically diverse novel as well as traditional chlamydial organisms infect an expanded range of ungulate hosts in Australia. Detection of the C. psittaci and C. pecorum in livestock, and novel taxa infecting horses and deer raises questions about the genetic make-up and pathogenic potential of these organisms, but also concerns about risks of spill-over between livestock, humans, and native wildlife.

Chlamydia pecorum and Chlamydia abortus are related ruminant pathogens endemic to different global regions. Potential co-infections combined with the lack of species-specific serological assays challenge accurate diagnosis. Serological screening revealed low C. abortus seropositivity with the peptide-based ELISA (1/84; 1.2%) in Australian sheep yet moderate seropositivity in a Swiss flock with history of C. abortus-associated abortions (17/63; 26.9%). By whole cell antigen complement fixation tests (CFT) and ELISA, chlamydial seropositivity was significantly higher in all groups, suggesting cross-reactivity between these two chlamydial species and non-specificity of the tests. However, only C. pecorum DNA could be detected by qPCR in Chlamydia seropositive Australian animals screened, suggesting chlamydial seropositivity was due to cross-reactivity with endemic C. pecorum infections. These results suggest ascribing Chlamydia seropositivity to chlamydial species in livestock using whole-cell antigen CFT or ELISA should be treated with caution; and that peptide-based ELISA and qPCR provide greater chlamydial species-specificity.

Background Chlamydia pecorum is a globally significant livestock pathogen causing pathology and production losses. The on-farm infection and serological dynamics and the relevance of existing diagnostic tools for diagnosing C. pecorum in livestock remains poorly characterized. In this study, we characterized the antigen and antibody dynamics of this pathogen in a longitudinal study of prime lamb production, utilizing the infection focused C. pecorum-specific 16S rRNA qPCR assay and serology based chlamydial Complement fixation Test (CFT). Methods The study consisted of 76 Border Leicester mixed sex lambs (39 females and 37 males) that were sampled bimonthly from 2–10 months of age in a commercial farm operating in Central NSW, Australia. Blood/plasma was analysed for CFT antibodies, and swabs from conjunctival, rectal and vaginal sites were analysed for C. pecorum shedding using qPCR. We assessed the temporal and overall dynamics of C. pecorum in lambs, including detailed description and comparison of qPCR and CFT, the timing of first detection by either diagnostic method, the lag between infection and antibody response; and the distribution of qPCR load and CFT antibody titre over time. Results Over the study period, C. pecorum was highly prevalent (71.0% by qPCR, 92.1% by CFT, 96.0% by both), with 21.1% (16/76) lambs shedding ≥1, 000 qPCR copies/µl (denoted as high shedders). C. pecorum shedding (as evidence of infection) were first observed at two months of age (14.4%) with a significant peak of infection occurring at six months of age (34.2%), whereas seroconversions peaked at eight months of age (81.5%). 52.6% of C. pecorum qPCR and CFT positive lambs became qPCR negative by 10 months of age, indicating clearance of chlamydial infection. Although CFT is utilised for on-farm detection of active infection, we confirm that it lagged behind qPCR detection (average lag 1.7 ± 2.1 months) and that the proportion of qPCR positives simultaneously identified by CFT was low with 2/11 (18.1%), 0/13, 17/25 (68.0%), 5/7 (71.4%) and 1/10 (10.0%) concurrent seroconversions occurring at two, four, six, eight and 10 months of age, respectively. Discussion This work reveals rapid rates of C. pecorum infection and widespread exposure during lamb production. The comparison of molecular and serological diagnostic agreement longitudinally, supports the use of qPCR as an important ancillary tool for the detection of active infections in conjunction with chlamydial CFT for routine veterinary diagnostics. Development of rapid Point-of-Care (POC) tools for diagnosing active infection would be valuable for producers and veterinarians.

Lamydia pecorum is a globally recognised livestock pathogen that is capable of causing severe and economically significant diseases such as arthritis in sheep and cattle. Relatively little information is available on the clinical progression of disease and the long-term effects of asymptomatic and symptomatic chlamydiosis in sheep. Recent studies in calves indicate that endemic C. pecorum infections may reduce growth rates. To investigate the clinical health parameters and production impacts of endemic C. pecorum infection in an Australian commercial lamb flock, we performed bimonthly sampling and clinical health assessments on 105 Border Leicester lambs from two to ten months of age. Chlamydial status was investigated via serology and species-specific quantitative PCR. Throughout the study period, conjunctivitis remained a persistent clinical feature while signs of arthritis (e.g. palpable synovial joint effusions) resolved in a subset of lambs while persisting in others. Clinical disease and C. pecorum infection were highest at six months of age (weaning). As previously reported, peak seroconversion tends to occur two months after the onset of clinical symptoms (6 months of age), with lambs clearing chlamydial infection by 10 months of age, despite ongoing disease still being present at this time. Notably, the presence of chlamydial infection did not affect lamb mass or growth rates throughout the study. At necropsy, C. pecorum was not detected within the joints of lambs with chronic arthritis. Molecular analysis of the strains in this flock suggest that the infecting strains circulating in this flock are clonal C. pecorum pathotypes, denoted ST 23, commonly associated with conjunctivitis and polyarthritis in Australian sheep. This study provides a platform for further research in the epidemiology and disease transmission dynamics of C. pecorum infections in sheep.

BackgroundChlamydia pecorumis a globally significant livestock pathogen causing pathology and production losses. The on-farm infection and serological dynamics and the relevance of existing diagnostic tools for diagnosingC. pecorumin livestock remains poorly characterized. In this study, we characterized the antigen and antibody dynamics of this pathogen in a longitudinal study of prime lamb production, utilizing the infection focusedC. pecorum-specific 16S rRNA qPCR assay and serology based chlamydial Complement fixation Test (CFT).MethodsThe study consisted of 76 Border Leicester mixed sex lambs (39 females and 37 males) that were sampled bimonthly from 2–10 months of age in a commercial farm operating in Central NSW, Australia. Blood/plasma was analysed for CFT antibodies, and swabs from conjunctival, rectal and vaginal sites were analysed forC. pecorumshedding using qPCR. We assessed the temporal and overall dynamics ofC. pecorumin lambs, including detailed description and comparison of qPCR and CFT, the timing of first detection by either diagnostic method, the lag between infection and antibody response; and the distribution of qPCR load and CFT antibody titre over time.ResultsOver the study period,C. pecorumwas highly prevalent (71.0% by qPCR, 92.1% by CFT, 96.0% by both), with 21.1% (16/76) lambs shedding ≥1, 000 qPCR copies/µl (denoted as high shedders).C. pecorumshedding (as evidence of infection) were first observed at two months of age (14.4%) with a significant peak of infection occurring at six months of age (34.2%), whereas seroconversions peaked at eight months of age (81.5%). 52.6% ofC. pecorumqPCR and CFT positive lambs became qPCR negative by 10 months of age, indicating clearance of chlamydial infection. Although CFT is utilised for on-farm detection of active infection, we confirm that it lagged behind qPCR detection (average lag 1.7 ± 2.1 months) and that the proportion of qPCR positives simultaneously identified by CFT was low with 2/11 (18.1%), 0/13, 17/25 (68.0%), 5/7 (71.4%) and 1/10 (10.0%) concurrent seroconversions occurring at two, four, six, eight and 10 months of age, respectively.DiscussionThis work reveals rapid rates ofC. pecoruminfection and widespread exposure during lamb production. The comparison of molecular and serological diagnostic agreement longitudinally, supports the use of qPCR as an important ancillary tool for the detection of active infections in conjunction with chlamydial CFT for routine veterinary diagnostics. Development of rapid Point-of-Care (POC) tools for diagnosing active infection would be valuable for producers and veterinarians.

Arthritis and kerato-conjunctivitis caused by Chlamydia pecorum in lambs are difficult to diagnose and treat. We tested the ability of a prototype C. pecorum vaccine (SC-vaccine), comprised of C. pecorum major outer membrane protein (MOMP-G) and polymorphic membrane protein G (PmpG), to trigger a Chlamydia-specific humoral and cell-mediated immune response in lambs and pregnant ewes. Vaccinations with the SC-vaccine (one and two injections) were very well tolerated by all ewes and lambs. Although the overall immune responses of ewes to SC-vaccination was poor, their lambs showed stronger antigen-specific immune response than lambs from control vaccine ewes. SC-vaccination in lambs triggered production of systemic anti-MOMP-G and anti-PmpG IgG antibodies and secretory IgA in the ocular mucosa. Double vaccination caused statistically significant increases in the height and duration of the humoral response. Antigen-specific IFN-γ was produced in the peripheral blood mononuclear cells of vaccinated lambs.

Chlamydia pecorum is a globally recognised livestock pathogen due to the significant clinical and economic impact it poses to livestock producers. Routine serological diagnosis is through a complement fixation test (CFT), which is often criticised for cross-reactivity, poor sensitivity and specificity. Although serology remains the preferred method in veterinary diagnostic laboratories, serological assays based on surface antigens of C. pecorum have not been established until now. In this study, we evaluated the use of two chlamydial recombinant protein antigens (PmpG and MOMP-G) by a direct IgG ELISA method for detection of ovine anti-chlamydial antibodies. Using the Pepscan method we then identified B cell epitopes across PmpG and MOMP-G proteins, in lambs with (a) naturally occurring asymptomatic C. pecorum infections (b) C. pecorum-associated polyarthritis and (c) recombinant PmpG and MOMP-G vaccine. Plasma IgG antibodies to PmpG in natural infection of lambs were detected earlier in infection than CFT and served as an acute phase marker. Antibodies to MOMP-G IgG were significantly heightened in lambs with C. pecorum-associated polyarthritis. PmpG and MOMP-G specific B-cell epitope mapping revealed epitope responses in immunised lambs cluster with some of the epitope responses in naturally infected lambs. B-cell epitope mapping further revealed that lambs with polyarthritis recognised several unique PmpG (50% frequency, peptide 8, 25, 40, 41 and 50) and MOMP (50% frequency, peptide 50) epitopes in comparison to asymptomatic infections. The findings of this study will have implications towards improved serodiagnosis of C. pecorum infections in livestock and inform the downstream development of alternative peptide-based antigens for future C. pecorum vaccine studies.

BACKGROUND:Arthritis is an economically significant disease in lambs and is usually the result of a bacterial infection. One of the known agents of this disease is Chlamydia pecorum, a globally recognised livestock pathogen associated with several diseases in sheep, cattle and other hosts. Relatively little published information is available on the clinical, diagnostic and pathologic features of C. pecorum arthritis in sheep, hindering efforts to enhance our understanding of this economically significant disease. In this case series, a combination of standard diagnostic testing used routinely by veterinarians, such as the Chlamydia complement fixation text (CFT), veterinary clinical examinations, and additional screening via C. pecorum specific qPCR was used to describe putative chlamydial infections in five sheep flocks with suspected ovine arthritis.CASE PRESENTATION:Five separate cases involving multiple lambs (aged six to ten months) of different breeds with suspected C. pecorum arthritis are presented. In two of the five cases, arthritic lambs exhibited marked depression and lethargy. Arthritis with concurrent conjunctivitis was present in four out of five lamb flocks examined. Chlamydia CFT demonstrated medium to high positive antibody titres in all flocks examined. C. pecorum shedding was evident at multiple sites including the conjunctiva, rectum and vagina, as determined via qPCR. Two of the five flocks received antimicrobials and all flocks recovered uneventfully regardless of treatment.CONCLUSION:This case series highlights the features a field veterinarian may encounter in cases of suspected ovine chlamydial arthritis. Our analysis suggests a presumptive diagnosis of chlamydial arthritis in lambs can be made when there is evidence of joint stiffness with or without synovial effusion and elevated chlamydia antibody titres. C. pecorum-specific qPCR was found to be a useful ancillary diagnostic tool, detecting Chlamydia positivity in low or negative CFT titre animals. Variables such as symptom duration relative to sampling, sheep breed and farm management practices were all factors recorded that paint a complex epidemiological and diagnostic picture for this disease. These case studies serve to provide a platform for further research to improve diagnostic testing and new treatment and control strategies for C. pecorum infections in sheep.

Human immunodeficiency virus type 1 (HIV-1) viral genes nef and tat play an important role in disease progression. In this study we characterized the Nef and Tat proteins from a group of HLA-B57 typed pediatric perinatally infected long-term survivors (LTS) with ≥10 years of infection. We identified 19 therapy-naive LTS after screening 250 children from an Indian pediatric cohort. Nef and tat amplified from plasma virus showed that all the LTS harbored HIV-1 subtype C. The two B57(+) children showed mutations, deletions, and insertions in experimentally defined B57 epitopes in the virus that are likely to be escape mutants. Only GW12 (GPGVRYPLTFGW) and YY9 (YTPGPGIRY) were conserved, while the remaining 90% (18/20) of the epitopes showed some degree of mutations. The most variable epitopes were RR15, SE15, QP15, KF9, HW9, YT9, and GF15. To our knowledge this is the first study from India in which characterization of Nef and Tat from LTS has led to information on genetic alterations in these genes that are associated with slow disease progression, and can provide an important lead in future studies.