Emerging and well-characterized chlamydial infections detected in a wide range of wild Australian birds.

Birds can act as successful long-distance vectors and reservoirs for numerous zoonotic bacterial, parasitic and viral pathogens, which can be a concern given the interconnectedness of animal, human and environmental health. Examples of such avian pathogens are members of the genus Chlamydia. Presently, there is a lack of research investigating chlamydial infections in Australian wild and captive birds and the subsequent risks to humans and other animals. In our current study, we investigated the prevalence and genetic diversity of chlamydial organisms infecting wild birds from Queensland and the rate of co-infections with beak and feather disease virus (BFDV). We screened 1114 samples collected from 564 different birds from 16 orders admitted to the Australia Zoo Wildlife Hospital from May 2019 to February 2021 for Chlamydia and BFDV. Utilizing species-specific quantitative polymerase chain reaction (qPCR) assays, we revealed an overall Chlamydiaceae prevalence of 29.26% (165/564; 95% confidence interval (CI) 25.65-33.14), including 3.19% (18/564; 95% CI 2.03-4.99%) prevalence of the zoonotic Chlamydia psittaci. Chlamydiaceae co-infection with BFDV was detected in 9.75% (55/564; 95% CI 7.57-12.48%) of the birds. Molecular characterization of the chlamydial 16S rRNA and ompA genes identified C. psittaci, in addition to novel and other genetically diverse Chlamydia species: avian Chlamydia abortus, Ca. Chlamydia ibidis and Chlamydia pneumoniae, all detected for the first time in Australia within a novel avian host range (crows, figbirds, herons, kookaburras, lapwings and shearwaters). This study shows that C. psittaci and other emerging Chlamydia species are prevalent in a wider range of avian hosts than previously anticipated, potentially increasing the risk of spill-over to Australian wildlife, livestock and humans. Going forward, we need to further characterize C. psittaci and other emerging Chlamydia species to determine their exact genetic identity, potential reservoirs, and factors influencing infection spill-over.

Real-Time Fluorometric Isothermal LAMP Assay for Detection of Chlamydia pecorum in Rapidly Processed Ovine Abortion Samples: A Veterinary Practitioner's Perspective.

Traditional methods of detecting Chlamydia pecorum in tissue samples such as polymerase chain reaction or cell culture are laborious and costly. We evaluated the use of a previously developed C. pecorum LAMP assay using minimally processed ovine samples. Cotyledon (n = 16), foetal liver (n = 22), foetal lung (n = 2), and vaginal (n = 6) swabs, in addition to cotyledon (n = 6) and foetal liver (n = 8) tissue samples, were rapidly processed and used for LAMP testing without DNA extraction. Overall, LAMP test results were highly congruent with the in-house reference qPCR, with 80.43% (37/46; 72.73% positive agreement (PA); 84.75% negative agreement (NA)) overall agreeance for swab samples, and 85.71% (12/14; 80% PA; 88.89% NA) overall agreeance for tissue samples. Out of the 11 total discrepant results, discrepancy was mainly observed in samples (n = 10) with less than 100 copies/µL C. pecorum DNA. While sensitivity could be improved, the simplicity, low cost, and accuracy of detection makes this test amenable for use at point-of-care for detecting C. pecorum in sheep.

Chlamydia Psittaci ST24: Clonal Strains of One Health Importance Dominate in Australian Horse, Bird and Human Infections.

Chlamydia psittaci is traditionally regarded as a globally distributed avian pathogen that can cause zoonotic spill-over. Molecular research has identified an extended global host range and significant genetic diversity. However, Australia has reported a reduced host range (avian, horse, and human) with a dominance of clonal strains, denoted ST24. To better understand the widespread of this strain type in Australia, multilocus sequence typing (MLST) and ompA genotyping were applied on samples from a range of hosts (avian, equine, marsupial, and bovine) from Australia. MLST confirms that clonal ST24 strains dominate infections of Australian psittacine and equine hosts (82/88; 93.18%). However, this study also found novel hosts (Australian white ibis, King parrots, racing pigeon, bovine, and a wallaby) and demonstrated that strain diversity does exist in Australia. The discovery of a C. psittaci novel strain (ST306) in a novel host, the Western brush wallaby, is the first detection in a marsupial. Analysis of the results of this study applied a multidisciplinary approach regarding Chlamydia infections, equine infectious disease, ecology, and One Health. Recommendations include an update for the descriptive framework of C. psittaci disease and cell biology work to inform pathogenicity and complement molecular epidemiology.

Real-time fluorometric and end-point colorimetric isothermal assays for detection of equine pathogens C. psittaci and equine herpes virus 1: validation, comparison and application at the point of care.

BACKGROUND: C. psittaci has recently emerged as an equine abortigenic pathogen causing significant losses to the Australian Thoroughbred industry, while Equine herpesvirus-1 (EHV-1) is a well-recognized abortigenic agent. Diagnosis of these agents is based on molecular assays in diagnostic laboratories. In this study, we validated C. psittaci and newly developed EHV-1 Loop Mediated Isothermal Amplification (LAMP) assays performed in a real-time fluorometer (rtLAMP) against the reference diagnostic assays. We also evaluated isothermal amplification using commercially available colorimetric mix (cLAMP), and SYBR Green DNA binding dye (sgLAMP) for "naked eye" end-point detection when testing 'real-world' clinical samples. Finally, we applied the C. psittaci LAMP assays in two pilot Point-of-Care (POC) studies in an equine hospital. RESULTS: The analytical sensitivity of C. psittaci and EHV-1 rt-, and colorimetric LAMPs was determined as one and 10 genome equivalents per reaction, respectively. Compared to reference diagnostic qPCR assays, the C. psittaci rtLAMP showed sensitivity of 100%, specificity of 97.5, and 98.86% agreement, while EHV-1 rtLAMP showed 86.96% sensitivity, 100% specificity, and 91.43% agreement. When testing rapidly processed clinical samples, all three C. psittaci rt-, c-, sg-LAMP assays were highly congruent with each other, with Kappa values of 0. 906 for sgLAMP and 0. 821 for cLAMP when compared to rtLAMP. EHV-1 testing also revealed high congruence between the assays, with Kappa values of 0.784 for cLAMP and 0.638 for sgLAMP when compared to rtLAMP. The congruence between LAMP assays and the C. psittaci or EHV-1 qPCR assays was high, with agreements ranging from 94.12 to 100% for C. psittaci, and 88.24 to 94.12% for EHV-1, respectively. At the POC, the C. psittaci rt- and c-LAMP assays using rapidly processed swabs were performed by technicians with no prior molecular experience, and the overall congruence between the POC C. psittaci LAMPs and the qPCR assays ranged between 90.91-100%. CONCLUSIONS: This study describes reliable POC options for the detection of the equine pathogens: C. psittaci and EHV-1. Testing 'real-world' samples in equine clinical setting, represents a proof-of-concept that POC isothermal diagnostics can be applied to rapid disease screening in the equine industry.

In vitro analysis of genetically distinct Chlamydia pecorum isolates reveals key growth differences in mammalian epithelial and immune cells.

Chlamydia (C.) pecorum is an obligate intracellular bacterium that infects and causes disease in a broad range of animal hosts. Molecular studies have revealed that this pathogen is genetically diverse with certain isolates linked to different disease outcomes. Limited in vitro or in vivo data exist to support these observations, further hampering efforts to improve our understanding of C. pecorum pathogenesis. In this study, we evaluated whether genetically distinct C. pecorum isolates (IPA, E58, 1710S, W73, JP-1-751) display different in vitro growth phenotypes in different mammalian epithelial and immune cells. In McCoy cells, shorter lag phases were observed for W73 and JP-1-751 isolates. Significantly smaller inclusions were observed for the naturally plasmid-free E58 isolate. C. pecorum isolates of bovine (E58) and ovine origin (IPA, W73, JP-1-751) grew faster in bovine cells compared to a porcine isolate (1710S). C. pecorum isolates could infect but appear not able to complete their developmental cycle in bovine peripheral neutrophil granulocytes. All isolates, except 1710S, could multiply in bovine monocyte-derived macrophages. These results reveal potentially important phenotypic differences that will help to understand the pathogenesis of C. pecorum in vivo and to identify C. pecorum virulence factors.

Detection of a range of genetically diverse chlamydiae in Australian domesticated and wild ungulates.

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.