Animal Chlamydiae: A Concern for Human and Veterinary Medicine

The Chlamydiae are a phylum of obligate intracellular, Gram-negative bacteria with a biphasic lifecycle [...

Prevalence and molecular characterization of C. pecorum detected in Swiss fattening pigs

Chlamydia (C.) pecorum, an obligate intracellular bacterial species commonly found in ruminants, can also occur in pigs. However, its significance as a potential porcine pathogen, or commensal, is still unclear. In a previous study (Hoffmann et al. 2015), mixed infections of C. suis and C. pecorum were detected in 14 Swiss fattening pig farms. Using these samples, we aimed to investigate the infection dynamics of C. suis and C. pecorum mixed infections in these farms. In addition, we analyzed the genetic diversity of Swiss porcine C. pecorum strains in relation to globally circulating strains. In total, 1284 conjunctival and rectal swabs from 391 pigs, collected at the beginning and end of the fattening period, were tested during the course of this study. We determined the bacterial loads of C. suis and C. pecorum using species-specific real-time PCR (qPCR) and compared these results to already existing DNA-microarray and Chlamydiaceae qPCR data. Overall, C. suis and Chlamydiaceae copy numbers decreased in the course of the fattening period, whereas C. pecorum copy numbers increased. No association was found between clinical signs (conjunctivitis, lameness and diarrhea) and the bacterial loads. Preventive antibiotic treatment at the beginning of the fattening period significantly lowered the chlamydial load and outdoor access was associated with higher loads. Proximity to the nearest ruminants correlated with increased C. pecorum loads, indicating that C. pecorum could be transmitted from ruminants to pigs. Multi-locus sequence typing (MLST) and major outer membrane protein (ompA) genotyping revealed two novel sequence types (STs) (301, 302) and seven unique ompA genotypes (1-7) that appear to form a specific clade separate from other European C. pecorum strain

A Sarcoptes scabiei specific isothermal amplification assay for detection of this important ectoparasite of wombats and other animals

Background The globally distributed epidermal ectoparasite, Sarcoptes scabiei, is a serious health and welfare burden to at-risk human and animal populations. Rapid and sensitive detection of S. scabiei infestation is critical for intervention strategies. While direct microscopy of skin scrapings is a widely utilised diagnostic method, it has low sensitivity. PCR, alternatively, has been shown to readily detect mite DNA even in microscopy-negative skin scrapings. However, a limitation to the latter method is the requirements for specialised equipment and reagents. Such resources may not be readily available in regional or remote clinical settings and are an important consideration in diagnosis of this parasitic disease. Methodology A Loop Mediated Isothermal Amplification (LAMP) assay targeting the ITS-2 gene for S. scabiei was developed and evaluated on clinical samples from various hosts, previously screened with conventional S. scabies-specific PCR. Species specificity of the newly developed LAMP assay was tested against a range of DNA samples from other arthropods. The LAMP assays were performed on a real-time fluorometer as well as thermal cycler to evaluate an end-point of detection. Using skin scrapings, a rapid sample processing method was assessed to eliminate extensive processing times involved with DNA extractions prior to diagnostic assays, including LAMP. Results The S. scabiei LAMP assay was demonstrated to be species-specific and able to detect DNA extracted from a single mite within a skin scraping in under 30 minutes. Application of this assay to DNA extracts from skin scrapings taken from a range of hosts revealed 92.3% congruence (with 92.50% specificity and 100% sensitivity) to the conventional PCR detection of S. scabiei. Preliminary results have indicated that diagnostic outcome from rapidly processed dry skin scrapings using our newly developed LAMP is possible in approximately 40 minutes. Discussion We have developed a novel, rapid and robust molecular assay for detecting S. scabiei infesting humans and animals. Based on these findings, we anticipate that this assay will serve an important role as an ancillary diagnostic tool at the point-of-care, complementing existing diagnostic protocols for S. scabiei

Molecular and pathological insights into Chlamydia pecorum-associated sporadic bovine encephalomyelitis (SBE) in Western Australia

Background Despite its global recognition as a ruminant pathogen, cases of Chlamydia pecorum infection in Australian livestock are poorly documented. In this report, a C. pecorum specific Multi Locus Sequence Analysis scheme was used to characterise the C. pecorum strains implicated in two cases of sporadic bovine encephalomyelitis confirmed by necropsy, histopathology and immunohistochemistry. This report provides the first molecular evidence for the presence of mixed infections of C. pecorum strains in Australian cattle. Case presentation Affected animals were two markedly depressed, dehydrated and blind calves, 12 and 16 weeks old. The calves were euthanized and necropsied. In one calf, a severe fibrinous polyserositis was noted with excess joint fluid in all joints whereas in the other, no significant lesions were seen. No gross abnormalities were noted in the brain of either calf. Histopathological lesions seen in both calves included: multifocal, severe, subacute meningoencephalitis with vasculitis, fibrinocellular thrombosis and malacia; diffuse, mild, acute interstitial pneumonia; and diffuse, subacute epicarditis, severe in the calf with gross serositis. Immunohistochemical labelling of chlamydial antigen in brain, spleen and lung from the two affected calves and brain from two archived cases, localised the antigen to the cytoplasm of endothelium, mesothelium and macrophages. C. pecorum specific qPCR, showed dissemination of the pathogen to multiple organs. Phylogenetic comparisons with other C. pecorum bovine strains from Australia, Europe and the USA revealed the presence of two genetically distinct sequence types (ST). The predominant ST detected in the brain, heart, lung and liver of both calves was identical to the C. pecorum ST previously described in cases of SBE. A second ST detected in an ileal tissue sample from one of the calves, clustered with previously typed faecal bovine isolates. Conclusion This report provides the first data to suggest that identical C. pecorum STs may be associated with SBE in geographically separated countries and that these may be distinct from those found in the gastrointestinal tract. This report provides a platform for further investigations into SBE and for understanding the genetic relationships that exist between C. pecorum strains detected in association with other infectious diseases in livestock

Chlamydiaceae in wild, feral and domestic pigeons in Switzerland and insight into population dynamics by Chlamydia psittaci multilocus sequence typing

Feral pigeons, common wood pigeons and Eurasian collared doves are the most common representatives of the Columbidae family in Switzerland and are mostly present in highly populated, urban areas. Pigeons may carry various members of the obligate intracellular Chlamydiaceae family, particularly Chlamydia (C.) psittaci, a known zoonotic agent, and C. avium. The objective of the study was to identify the infection rates of common free-roaming pigeons for different Chlamydia species with the overall aim to assess the risk pigeons pose to public health. In this study, 431 pigeons (323 feral pigeons, 34 domestic pigeons, 39 Eurasian collared doves, 35 common wood pigeons) from several geographic locations in Switzerland were investigated for the presence of Chlamydiaceae. Samples consisted of pooled choanal-cloacal swabs (n = 174), liver samples (n = 52), and paired swab and liver samples from 205 pigeons (n = 410). All 636 samples were screened using a Chlamydiaceae family-specific 23S rRNA real-time PCR (qPCR). Subsequent species identification was performed by DNA-microarray assay, sequencing of a 16S rRNA gene fragment and a C. psittaci specific qPCR. In total, 73 of the 431 pigeons tested positive for Chlamydiaceae, of which 68 were positive for C. psittaci, four were C. avium-positive and one pigeon was co-infected with C. avium and C. psittaci. The highest infection rates were detected in feral (64/323) and domestic pigeons (5/34). Common wood pigeons (2/35) and Eurasian collared doves (2/39) revealed lower infection rates. Additionally, multilocus sequence typing of twelve selected C. psittaci-positive samples revealed closely related sequence types (ST) between and within different Swiss cities. Furthermore, liver and corresponding swab samples from the same bird were colonized by the same ST. Considering the high infection rates of C. psittaci in domestic and feral pigeons, close or frequent contact to these birds poses a human health risk

Multilocus sequence analysis provides insights into molecular epidemiology of Chlamydia pecorum infections in Australian sheep, cattle and koalas

Chlamydia pecorum is a significant pathogen of domestic livestock and wildlife. We have developed a C. pecorum-specific multilocus sequence analysis (MLSA) scheme to examine the genetic diversity of and relationships between Australian sheep, cattle, and koala isolates. An MLSA of seven concatenated housekeeping gene fragments was performed using 35 isolates, including 18 livestock isolates (11 Australian sheep, one Australian cow, and six U.S. livestock isolates) and 17 Australian koala isolates. Phylogenetic analyses showed that the koala isolates formed a distinct clade, with limited clustering with C. pecorum isolates from Australian sheep. We identified 11 MLSA sequence types (STs) among Australian C. pecorum isolates, 10 of them novel, with koala and sheep sharing at least one identical ST (designated ST2013Aa). ST23, previously identified in global C. pecorum livestock isolates, was observed here in a subset of Australian bovine and sheep isolates. Most notably, ST23 was found in association with multiple disease states and hosts, providing insights into the transmission of this pathogen between livestock hosts. The complexity of the epidemiology of this disease was further highlighted by the observation that at least two examples of sheep were infected with different C. pecorum STs in the eyes and gastrointestinal tract. We have demonstrated the feasibility of our MLSA scheme for understanding the host relationship that exists between Australian C. pecorum strains and provide the first molecular epidemiological data on infections in Australian livestock hosts

Isolation of tetracycline-resistant Chlamydia suis from a pig herd affected by reproductive disorders and conjunctivitis

Due to various challenges in diagnosing chlamydiosis in pigs, antibiotic treatment is usually performed before any molecular or antibiotic susceptibility testing. This could increase the occurrence of tetracycline-resistant Chlamydia (C.) suis isolates in the affected pig population and potentiate the reoccurrence of clinical signs. Here, we present a case of an Austrian pig farm, where tetracycline resistant and sensitive C. suis isolates were isolated from four finishers with conjunctivitis. On herd-level, 10% of the finishers suffered from severe conjunctivitis and sows showed a high percentage of irregular return to estrus. Subsequent treatment of whole-herd using oxytetracycline led to a significant reduction of clinical signs. Retrospective antibiotic susceptibility testing revealed tetracycline resistance and decreased susceptibility to doxycycline in half of the ocular C. suis isolates, and all isolates were able to partially recover following a single-dose tetracycline treatment in vitro. These findings were later confirmed in vivo, when all former clinical signs recurred three months later. This case report raises awareness of tetracycline resistance in C. suis and emphasizes the importance of preventative selection of tetracycline resistant C. suis isolates

The limitations of commercial serological assays for detection of chlamydial infections in Australian livestock

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

Evaluation of the relationship between Chlamydia pecorum sequence types and disease using a species-specific multi-locus sequence typing scheme (MLST)

Chlamydia pecorum is globally associated with several ovine diseases including keratoconjunctivitis and polyarthritis. The exact relationship between the variety of C. pecorum strains reported and the diseases described in sheep remains unclear, challenging efforts to accurately diagnose and manage infected flocks. In the present study, we applied C. pecorum multi-locus sequence typing (MLST) to C. pecorum positive samples collected from sympatric flocks of Australian sheep presenting with conjunctivitis, conjunctivitis with polyarthritis, or polyarthritis only and with no clinical disease (NCD) in order to elucidate the exact relationships between the infecting strains and the range of diseases. Using Bayesian phylogenetic and cluster analyses on 62 C. pecorum positive ocular, vaginal and rectal swab samples from sheep presenting with a range of diseases and in a comparison to C. pecorum sequence types (STs) from other hosts, one ST (ST 23) was recognised as a globally distributed strain associated with ovine and bovine diseases such as polyarthritis and encephalomyelitis. A second ST (ST 69) presently only described in Australian animals, was detected in association with ovine as well as koala chlamydial infections. The majority of vaginal and rectal C. pecorum STs from animals with NCD and/or anatomical sites with no clinical signs of disease in diseased animals, clustered together in a separate group, by both analyses. Furthermore, 8/13 detected STs were novel. This study provides a platform for strain selection for further research into the pathogenic potential of C. pecorum in animals and highlights targets for potential strain-specific diagnostic test development

The first genomic insight into Chlamydia psittaci sequence type (ST)24 from a healthy captive psittacine host in Australia demonstrates evolutionary proximity with strains from psittacine, human, and equine hosts

Chlamydia psittaci is a zoonotic pathogen that infects birds, humans, and other mammals. Notably, recent studies suggested the human-to-human transmission of C. psittaci, and this pathogen also causes equine reproductive loss in Australia. Molecular studies in Australia to date have focused on and described clonal sequence type (ST)24 strains infecting horses, wild psittacine, and humans. In contrast, the genetic identity of C. psittaci strains from captive psittacine hosts is scarce. In 2022, C. psittaci was detected in the faeces of a healthy captive blue-fronted parrot (Amazona aestiva). Genomic DNA was extracted and underwent whole-genome sequencing. Here we report the 1,160,701 bp circular chromosome of C. psittaci strain BF_amazon_parrot13 and the 7,553 bp circular plasmid pCpsBF_amazon_parrot13. Initial in silico multi-locus sequence typing and ompA genotyping revealed that BF_amazon_parrot13 belongs to the clonal ST24 lineage and has an ompA genotype A. Further context involved the genomes of 31 published ST24 strains, utilising a single-nucleotide variant (SNV) based clustering approach. Despite temporal, host, and biogeographical separation, a core-genome SNV-based phylogeny revealed that BF_amazon_parrot13 clustered in a distinct subcluster with seven C. psittaci strains from equines in Australia (maximum pairwise distance of 13 SNVs). BF_amazon_parrot13 represents the first complete C. psittaci ST24 genome from a captive psittacine in Australia. Furthermore, by using whole-genome sequencing to coordinate surveillance, we can also learn more about the possible health risks and routes of chlamydia transmission among people, livestock, wild animals, and domesticated animals