Chlamydia (C.) psittaci is the causative agent of avian chlamydiosis and human psittacosis. In this study, we extracted single-nucleotide polymorphisms (SNPs) from the whole genome sequences of 55 C. psittaci strains and identified eight major lineages, most of which are host-related. A combined PCR/high-resolution melting (HRM) assay was developed to screen for eight phylogenetically informative SNPs related to the identified C. psittaci lineages. The PCR-HRM method was validated on 11 available reference strains and with a set of 118 field isolates. Overall, PCR-HRM clustering was consistent with previous genotyping data obtained by ompA and/or MLST analysis. The method was then applied to 28 C. psittaci-positive samples from animal or human cases. As expected, PCR-HRM typing results from human samples identified genotypes linked to ducks and pigeons, a common source of human exposure, but also to the poorly described Mat116-like genotype. The new genotyping method does not require time-consuming sequencing and allows a quick identification of the source of infection.

Chlamydiaceae are obligatory intracellular bacteria causing acute and chronic diseases in animals and humans worldwide, with recently discovered species with a still unclear pathogenic potential (i.e., C. gallinacea). In Italy, Chlamydiaceae infections are underestimated both in animals and humans. To estimate the prevalence of Chlamydiaceae species in poultry and occupationally exposed workers on farm, a cross-sectional study was carried out in north-western Italy. A total of 2063 samples from 83 commercial and 31 backyard poultry farms were analysed using real-time PCRs for Chlamydiaceae screening and species typing. Chlamydiaceae were detected in 23 farms, with a herd prevalence of 20.2% (95%CI: 13.2–28.7), higher in backyard farms (38.7%; 95%CI: 21.8–57.8) compared to commercial ones (13.3%; 95%CI: 6.8–22.5). C. gallinacea was found in 18 chicken farms, both commercial and backyard, and C. psittaci only in 3 backyard farms. Exposure to wild birds and factors related to biosecurity resulted the main risk factors associated with Chlamydia positivity. Out of the 113 sputum samples collected from farmers, 16 tested positive to Chlamydiaceae, with a prevalence of 14.2% (95%CI: 8, 3–22). To the best of our knowledge, for the first time at international level, C. gallinacea was detected in humans with farmer positivity associated with farm infectious status, suggesting a bird-to-human transmission.

Chlamydiaceae occurrence has been largely evaluated in wildlife, showing that wild birds are efficient reservoirs for avian chlamydiosis. In this study, DNA extracted from cloacal swabs of 108 corvids from Northeast Italy was screened for Chlamydiaceae by 23S real-time (rt)PCR. The positive samples were characterised by specific rtPCRs for Chlamydia psittaci, Chlamydia abortus, Chlamydia gallinacea, Chlamydia avium, Chlamydia pecorum and Chlamydia suis. Cloacal shedding of Chlamydiaceae was detected in 12 out of 108 (11.1%, 5.9%–18.6% 95% CI) corvids sampled. Molecular characterisation at the species level was possible in 8/12 samples, showing C. psittaci positivity in only one sample from a hooded crow and C. abortus positivity in seven samples, two from Eurasian magpies and five from hooded crows. Genotyping of the C. psittaci-positive sample was undertaken via PCR/high-resolution melting, clustering it in group III_pigeon, corresponding to the B genotype based on former ompA analysis. For C. abortus genotyping, multilocus sequence typing was successfully performed on the two samples with high DNA load from Eurasian magpies, highlighting 100% identity with the recently reported Polish avian C. abortus genotype 1V strain 15-58d44. To confirm the intermediate characteristics between C. psittaci and C. abortus, both samples, as well as two samples from hooded crows, showed the chlamydial plasmid inherent in most C. psittaci and avian C. abortus, but not in ruminant C. abortus strains. The plasmid sequences were highly similar (≥99%) to those of the Polish avian C. abortus genotype 1V strain 15-58d44. To our knowledge, this is the first report of avian C. abortus strains in Italy, specifically genotype 1V, confirming that they are actively circulating in corvids in the Italian region tested.

The presence and zoonotic transfer of four different avian Chlamydia spp. was assessed in an epidemiological study in a psittacine bird population and its owners. Fecal swabs from 84 pet birds and pharyngeal swabs from 22 bird owners were collected from 21 locations in Flanders. Samples were examined using established and novel PCR platforms combined with culture on PCR-positive samples. Chlamydiaceae DNA was detected in 33 of 84 (39.3%) birds. The predominant part of the avian infections could be attributed to C. psittaci (22 of 84; 26.2%), followed by C. avium (11 of 84; 13.1%). C. gallinacea and C. abortus were not detected in birds or humans. C. psittaci was the only species detected in pet bird owners (4 of 22; 18.2%), stressing its zoonotic importance. This study showed that C. psittaci and the more recently discovered novel avian species C. avium are undoubtedly present in the Flemish psittacine bird population. Our results justify additional research in a larger psittacine bird population and its owners, focusing on C. psittaci and C. avium. In the meantime, increased awareness among pet bird owners and the implementation of preventive measures in the pet bird industry is advised to limit the circulation of established and novel emerging avian chlamydial species.

The cultivation of Chlamydia gallinacea, a recently identified species, is challenging due to the lack of an optimized protocol. In this study, several infection protocols were tested, including different cell lines, incubation temperatures, centrifugation methods and culture media. However, none were successful in field samples. The only exception was a chance co-culture with Trichosporon asahii, a microorganism commonly found in the chicken gut. This suggests that current in vitro methods may not be optimized for this species and that host-associated microorganisms may influence the in vivo growth of C. gallinacea, which is typically found in the chicken gut. These findings raise new questions and highlight the need for further investigation of microbial interactions within the host, particularly to understand their role in the proliferation of chlamydial species.

Members of the Chlamydiaceae family infect a wide range of vertebrate hosts, yet their diversity and ecology in remote insular ecosystems remain poorly understood. Two previous studies screened 2274 samples from seabirds and mammals collected across five sub-Antarctic islands in the Southern Indian and Atlantic Oceans between 2017 and 2022. Here, we further characterise 424 Chlamydiaceae positive samples, including members of the Chlamydia and Chlamydiifrater genera. Chlamydiifrater spp. were detected in almost all seabird species, particularly king penguins (Aptenodytes patagonicus) and lesser sheathbills (Chionis minor), with local prevalence reaching up to 57% and 78%, respectively. In this study, phylogenetic analyses of 16S and 23S rRNA genes revealed a distinct sub-Antarctic lineage structured into three host-associated clusters, likely structured by ecological interactions and spatial segregation. Chlamydia psittaci was detected in a limited number of penguin and sheathbill samples, with ompA- and MLST-based typing revealed at least two novel genotypes. In mammals from Amsterdam Island, C. muridarum-related strains were identified in brown rats (Rattus norvegicus), while C. felis-related strains were detected in sub-Antarctic fur seals (Arctocephalus tropicalis). This is the first evidence of C. felis in a wild non-feline host, raising the issue of spillover from introduced species (here, cats (Felis catus)), to native species. These findings demonstrate that Chlamydiaceae are diverse and widespread in sub-Antarctic wildlife, encompassing both endemic and potentially introduced lineages. This emphasizes the need for ongoing surveillance, including whole-genome sequencing, to enhance our understanding of the interactions between hosts, infectious agents and the environment, and to assess potential zoonotic risks to fieldworkers in these sensitive ecosystems.