Outbreak of Chlamydia psittaci Infection in a Commercial Psittacine Breeding Aviary in Argentina.

Chlamydiosis, caused by Chlamydia psittaci is a bacterial infection found in at least 465 species of birds worldwide. It is highly contagious among birds and can spread to humans. In birds, the disease can manifest itself in acute, subacute, and chronic forms with signs including anorexia, diarrhea, lethargy, weight loss, or, occasionally, mucopurulent or serous oculonasal discharge. This article describes an outbreak of chlamydiosis that occurred in a commercial psittacine breeding aviary in 2021 in Buenos Aires province, Argentina. In total, 16 juvenile blue-fronted parrots, more than 60 blue-fronted parrot chicks, and 2 adult macaws died during the outbreak. In all cases, clinical signs were weight loss, diarrhea, yellowish green excrement, and respiratory distress. The necropsy of four juvenile blue-fronted parrots, two blue-fronted parrot chicks, and two adult macaws revealed cachexia, hepatomegaly, splenomegaly, splenic petechial hemorrhages, ascites, pulmonary edema, and hydropericardium. Histologically, multifocal lymphoplasmacytic and heterophilic airsaculitis, multifocal lymphoplasmacytic and necrotizing hepatitis with intracytoplasmic elementary bodies, multifocal necro-heterophilic hepatitis, multifocal lymphoplasmacytic nephritis, and diffuse heterophilic pneumonia were found. A presumptive diagnosis was established based on gross and microscopic lesions, and it was confirmed using immunohistochemistry and polymerase chain reactions. The sequencing and phylogenetic analysis of the ompA gene revealed genotype A and B of Chlamydia psittaci.

Chlamydia gallinacea in Brazilian backyard chicken farms.

Avian chlamydiosis is a bacterial infectious disease of birds, considered until recently caused only by Chlamydia psittaci, that now includes the newly described species C. buteonis, C. avium, and C. gallinacea, associated with several avian hosts. Since its recognition as a species in 2014 and having chickens as one of its main hosts, C. gallinacea has already been described in backyard poultry on all continents. The present study aimed to survey by molecular techniques the presence and species of Chlamydia spp. in backyard chickens from three states of the southern region of Brazil (Paraná-PR, Santa Catarina-SC, and Rio Grande do Sul-RS). DNA extracted from cloacal swab samples were tested by polymerase chain reaction (PCR) for different species of Chlamydia, namely Chlamydiaceae (23 S rRNA gene), C. psittaci (ompA gene), C. avium (enoA gene) and C. gallinacea (gidA and enoA genes). The 16 S rRNA gene was used for sequencing and phylogenetic analysis. A total of 582 backyard chicken samples were collected and grouped in 238 pools, from 134 properties in 59 municipalities. Chlamydiaceae was detected in 25.2% (60/238) of the samples, in 38.8% (52/134) of the properties and in 66.1% (39/59) of the municipalities. None of the samples yielded positive PCR results for C. psittaci or C. avium. For C. gallinacea, the overall percentage was 16.3% (39/238) according to the results of gidA and enoA genes. Sequence analysis confirmed that the samples corresponded to C. gallinacea. This is the first report of C. gallinacea in Brazil.

Diversity and genetic characterization of Chlamydia isolated from Siamese crocodiles (Crocodylus siamensis).

Chlamydiosis, an infection caused by several Chlamydia species, has been reported in Nile, saltwater, and Siamese crocodiles. Despite its widespread reports in various countries, including Thailand, genetic information on Chlamydia species remains limited. This study presents a whole-genome-based characterization of Siamese crocodile-isolated Chlamydia. The results showed that Siamese crocodile Chlamydia contained a single circular chromosome with a size of 1.22-1.23 Mbp and a plasmid with a size of 7.7-8.0 kbp. A plasmid containing eight coding sequences (CDSs) was grouped in a ß lineage. A chromosome sequence had approximately 1,018-1,031 CDSs. Chlamydial factors involving virulence were documented in terms of the presence of cytotoxins and several virulence factors in the chromosomes of Siamese crocodile Chlamydia. The analysis of antimicrobial resistance genes in the Chlamydia genome revealed that the most common resistance genes were associated with aminoglycosides, fluoroquinolones, macrolides, tetracyclines, and cephalosporins, with loose matching (identities between 21.12 % and 74.65 %). Phylogenetic analyses, encompassing the assessments of both whole proteome and nine taxonomic markers, revealed that Siamese crocodile Chlamydia was separated into three lineages (lineages I-III) with high bootstrapping statistic support. Interestingly, isolate 12-01 differed genetically from the others, suggesting that it is a new member of Chlamydia. The study findings indicate that Siamese crocodiles are susceptible hosts to Chlamydia, involving more than one species. This study is the first employing the highest number of whole-genome data on Siamese crocodile Chlamydia and provides better insights into pathogen genetics.

Immunohistochemical characterization of the immune cell response during chlamydial infection in the male and female koala (Phascolarctos cinereus) reproductive tract.

Chlamydiosis is one of the main causes of the progressive decline of koala populations in eastern Australia. While histologic, immunologic, and molecular studies have provided insights into the basic function of the koala immune system, the in situ immune cell signatures during chlamydial infection of the reproductive tract in koalas have not been investigated. Thirty-two female koalas and 47 males presented to wildlife hospitals with clinical signs suggestive of Chlamydia infection were euthanized with the entire reproductive tract collected for histology; immunohistochemistry (IHC) for T-cell (CD3ε, CD4, and CD8α), B-cell (CD79b), and human leukocyte antigen (HLA)-DR markers; and quantitative real-time polymerase chain reaction (rtPCR) for Chlamydia pecorum. T-cells, B-cells, and HLA-DR-positive cells were observed in both the lower and upper reproductive tracts of male and female koalas with a statistically significant associations between the degree of the inflammatory reaction; the number of CD3, CD4, CD79b, and HLA-DR positive cells; and the PCR load. CD4-positive cells were negatively associated with the severity of the gross lesions. The distribution of immune cells was also variable according to the location within the genital tract in both male and female koalas. These preliminary results represent a step forward towards further exploring mechanisms behind chlamydial infection immunopathogenesis, thus providing valuable information about the immune response and infectious diseases in free-ranging koalas.

Serological and molecular survey of brucellosis and chlamydiosis in dromedary camels from Tunisia.

The present sero-epidemiological survey was designed and conducted to scrutinize the current status of camel-related brucellosis and chlamydiosis in Tunisia. Whole blood and serum samples were collected from 470 dromedaries (Camelus dromedarius) from eight different Tunisian governorates. Serum samples were subjected to indirect enzyme-linked immunosorbent assay (iELISA). The detection of Brucella and Chlamydia DNA was performed using conventional PCR targeting the bcsp-31 and 16 S rRNA gene, respectively. Overall, 10/470(2.12%) and 27/470 (5.75%) camels were revealed seropositive to Brucella and Chlamydia, respectively. Multivariate logistic regression analysis showed different risk factors associated with these infections. Meaningful high rates of seropositivity of brucellosis (9.5%; p = 0.000; OR=64.193) and chlamydiosis (22.6%; p = 0.000; OR=42.860) were noted among camels showing previous abortions in particular for aged females. Besides, Chlamydia seropositivity is significantly important during winter (12.5%; p = 0.009; OR= 27.533), and in camels raised in small farms (11.4%, p = 0.000, OR=86.052). Molecular analysis revealed no positivity from all analyzed blood samples. These findings indicate the involvement of camels in the epidemiology of these abortive infectious diseases. This raises awareness and serious public health concern for infectious camel diseases in order to develop further diagnostic improvements and effective control strategies.

New insight on chlamydiae.

This article provides an overview of the current knowledge on chlamydiae, which are intracellular bacteria belonging to the Chlamydiaceae family. Whole-genome sequencing leads to great increases in the available data about Chlamydia spp. Recently, novel chlamydial taxons in various hosts living in different environments have been recognised. New species and taxons with Candidatus status have been recorded mainly in birds and reptiles. Chlamydia gallinacea is an emerging infectious agent in poultry with indirectly confirmed zoonotic potential. Recently, a new group of avian C. abortus strains with worldwide distribution in various wild bird families has been described. The definition of C. abortus species became outdated with the discovery of these strains and has been amended. It now includes two subgroups, mammalian and avian, the latter including all isolates hitherto referred to as atypical C. psittaci or C. psittaci/C. abortus intermediates.

Epidemiology of Chlamydia sp. infection in farmed Siamese crocodiles (Crocodylus siamensis) in Thailand.

BACKGROUND: Although Chlamydia sp. causes widespread disease outbreaks in juvenile crocodiles in Thailand, data regarding the epidemiology, and risk factors of such infections are limited. The aim of this study was to investigate the prevalence and possible risk factors associated with Chlamydia sp. infections on Siamese crocodile (Crocodylus siamensis) farms in Thailand. A cross-sectional study was conducted from July to December 2019. Samples were collected from 40 farms across six regions in Thailand. Conjunctival, pharyngeal, and cloacal swab samples were analyzed for Chlamydiaceae nucleic acids using semi-nested PCR followed by phylogenetic analysis based on the ompA gene fragment. Risk factors of infection were analyzed using chi-square and univariate regression to calculate odds ratios. RESULTS: The prevalence of Chlamydia sp. infection across all regions was 65%. The ompA phylogenetic analysis showed that Chlamydia sp. detected in this study was genetically closely related to Chlamydia crocodili and Chlamydia caviae. The risk factors for infection were water source, reusing treated wastewater from the treatment pond, not disposing of leftover food, low frequency of water replacement in the enclosure of juvenile crocodiles, and lack of water replacement after the death of a crocodile. CONCLUSION: The prevalence of Chlamydia sp. infection in farmed crocodiles in Thailand was 65% during the study period. Cloacal swabs were superior to conjunctival and pharyngeal swabs due to their higher sensitivity in detecting Chlamydia sp., as well as their lower invasiveness. Good management and biosecurity in crocodile farming can reduce the risk of Chlamydia sp.

Carriage of antibiotic resistance genes to treatments for chlamydial disease in koalas (Phascolarctos cinereus): A comparison of occurrence before and during catastrophic wildfires.

Growing reports of diverse antibiotic resistance genes in wildlife species around the world symbolises the extent of this global One Health issue. The health of wildlife is threatened by antimicrobial resistance in situations where wildlife species develop disease and require antibiotics. Chlamydial disease is a key threat for koalas in Australia, with infected koalas frequently entering wildlife hospitals and requiring antibiotic therapy, typically with chloramphenicol or doxycycline. This study investigated the occurrence and diversity of target chloramphenicol and doxycycline resistance genes (cat and tet respectively) in koala urogenital and faecal microbiomes. DNA was extracted from 394 urogenital swabs and 91 faecal swabs collected from koalas in mainland Australia and on Kangaroo Island (KI) located 14 km off the mainland, before (n = 145) and during (n = 340) the 2019-2020 wildfires. PCR screening and DNA sequencing determined 9.9% of samples (95%CI: 7.5% to 12.9%) carried cat and/or tet genes, with the highest frequency in fire-affected KI koalas (16.8%) and the lowest in wild KI koalas sampled prior to fires (6.5%). The diversity of cat and tet was greater in fire-affected koalas (seven variants detected), compared to pre-fire koalas (two variants detected). Fire-affected koalas in care that received antibiotics had a significantly higher proportion (p < 0.05) of cat and/or tet genes (37.5%) compared to koalas that did not receive antibiotics (9.8%). Of the cat and/or tet positive mainland koalas, 50.0% were Chlamydia-positive by qPCR test. Chloramphenicol and doxycycline resistance genes in koala microbiomes may contribute to negative treatment outcomes for koalas receiving anti-chlamydial antibiotics. Thus a secondary outcome of wildfires is increased risk of acquisition of cat and tet genes in fire-affected koalas that enter care, potentially exacerbating the already significant threat of chlamydial disease on Australia's koalas. This study highlights the importance of considering impacts to wildlife health within the One Health approach to AMR and identifies a need for greater understanding of AMR ecology in wildlife.

Design and characterization of a multi-epitope vaccine targeting Chlamydia abortus using immunoinformatics approach.

Chlamydiosis is a widespread ailment affecting humans, livestock, and wildlife, caused by C. abortus, a member of the Chlamydia genus. This disease leads to reproductive disorders in bovines and poses a zoonotic risk, resulting in adverse outcomes such as abortion, stillbirths, weak offspring, endometritis, repeat breeding, and perinatal mortality. However, current chlamydiosis vaccines have limitations in terms of safety, efficacy, and stability, necessitating the development of effective and safe alternatives. In this study, our objective was to design a multi-epitope vaccine (MEV) targeting all strains of C. abortus using bioinformatics and immunoinformatics approaches. We identified highly antigenic and non-allergic proteins (yidC, yajC, secY, CAB503, and CAB746) using VaxiJen and AlgPred tools. Physicochemical analyses and secondary structure predictions confirmed protein stability through ProtParam and SOPMA methods. Furthermore, we employed IEDB-AR, NETMHCpan, and ToxinPred2 tools to predict cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), and B-cell epitopes, resulting in the identification of conserved epitopes for further analysis. The MEV construct, consisting of 545 amino acids, incorporated the adjuvant Beta defensin-3, along with 9 CTL epitopes and 21 HTL epitopes linked by EAAAK, KK, and AAY linkers. We assessed the safety and immunogenicity of the vaccine through comprehensive evaluations of antigenicity, toxicity, allergenicity, and physicochemical properties. Structural stability and quality were examined using 3D modeling via the ab initio approach with the Robetta platform. Molecular docking analysis explored the compatibility of the MEV with Toll-like receptor 9 (TLR9) using ClusPro, while molecular dynamics simulation with the DESMOND Maestro software predicted the stability and flexibility of the docked complex. Despite promising in silico findings, further wet lab investigations are crucial to validate the safety and efficacy of the MEV. Successful development and validation of this MEV hold significant potential in combatting chlamydiosis in both animal and human populations.Communicated by Ramaswamy H. Sarma.

Determining the Prevalence of Avian Chlamydiosis in Wild Amazona Species From Brazil Using Molecular Testing and Clinical Signs.

Avian chlamydiosis is a disease that occurs in birds, especially parrots, and is caused by the Gram-negative bacterium Chlamydia psittaci. Wild Animal Screening Centers in Brazil receive, maintain, treat, and place (preferably to nature) wild animals recovered from illegal trafficking. We performed molecular testing for avian chlamydiosis in parrots from the genus Amazona that were presented to these centers. Cloacal swab samples were collected from 59 parrots (Amazona species) and transported in aqueous or culture medium. The samples were subsequently submitted for DNA extraction by the boiling method, polymerase chain reaction (PCR) amplification using CPF/CPR primers, and agarose gel electrophoresis. Conjunctivitis, nasal discharge, and poor body condition were the clinical signs associated with a differential disease diagnosis of avian chlamydiosis. Transport medium did not have an effect on the test results. The prevalence of C psittaci in the samples was 37% (22/59, 95% confidence interval: 25-49). There was a significant (P = 0.009) association between the PCR test results and clinical signs. Follow-up testing was conducted on a subgroup of 14 individuals that initially tested negative on PCR; 50% (7/14) of these birds were found to be positive within 24 days of the first test. The results of this study confirm the feasibility of using the CPF/CFP primer-based PCR to detect C psittaci in Amazona species, describe a less costly method of transporting biological material for DNA extraction, and evaluate the temporal aspect for obtaining positive results through molecular testing for C psittaci in Amazona species.

Prevalence of asymptomatic infections of Chlamydia psittaci in psittacine birds in Korea.

Avian chlamydiosis is an acute or chronic bacterial disease of birds. Chlamydia psittaci is the primary agent of the disease. It is also an important zoonotic pathogen. Chlamydia avium and Chlamydia gallinacea have also been recognized as potential causative agents of the disease. Clinical signs of this disease can vary in severity. Asymptomatic infections of Chlamydia have commonly been reported in various birds worldwide. In this study, we investigated the distribution of Chlamydia species in healthy psittacine birds in Korea. A total of 263 samples (pharyngeal/cloacal swabs and faeces) were collected from psittacine birds of 26 species in five zoos, five parrot farms and seven parrot cafes between 2020 and 2021. Ages of these birds had a wide range (1 month to 30 years). During sample collection, no bird showed any clinical signs indicating diseases such as chlamydiosis. Samples were tested for the presence of Chlamydia spp. using real-time PCR assays. Chlamydia spp. were detected in 168 (63.9%) samples and C. psittaci was detected in 96 (36.5%) samples. However, C. avium and C. gallinacea were not detected. There were no significant differences in the prevalence of asymptomatic infections in birds among three types of housing environments. Regarding ompA genotypes, 87 C. psittaci-positive samples had genotype A based on sequence analysis (n = 28) and genotype-specific real-time PCR (n = 59). Other positive samples were untyped (n = 9). Overall findings showed high prevalence of asymptomatic infections of C. psittaci in psittacine birds in Korea, posing a significant hazard to public health.

Prevalence and Risk Factors of Avian Chlamydiosis Detected by Polymerase Chain Reaction in Psittacine Birds in Thailand.

This study surveyed avian chlamydiosis, with the aim to estimate the prevalence and potential risk factors associated with Chlamydia psittaci infection in psittacine birds kept as domestic pets in Thailand. Oropharyngeal swabs were collected from 120 psittacine birds that were randomly selected from hospitals in the central (Bangkok) and northeastern regions (Khon Kaen) of Thailand between 2019 and 2021. The oropharyngeal swabs were subject to polymerase chain reaction testing to detect the C psittaci ompA gene. The prevalence of C psittaci was 2.5% (3/ 120, 95% confidence interval = 0.3-5.3). Of the 3 positive birds, 1 was a Forpus parrot (Forpus species)(CP43TH) and 1 was an African grey parrot (Psittacus erithacus)(CP49TH) from Bangkok; both were juvenile birds with clinical signs of disease. The third positive bird (CP12TH) was a subclinical adult sun conure (Aratinga solstitialis) from Khon Kaen. Two sequences of samples that were previously identified in human psittacosis cases (accession numbers MK032053.1 and HM450409.1) were also examined. Since there was a low number of infected birds, potential associations between C psittaci infection and various environmental variables (eg, cage cleaning, synanthropic birds, quarantine of new birds, and overcrowding) were assessed by Fisher exact tests. This study provides estimates of the prevalence and potential risk factors associated with C psittaci infection in psittacine birds from central (Bangkok) and the northeastern regions (Khon Kaen) of Thailand. The detection of C psittaci in captive psittacine birds demonstrates that there is a possibility for bird-to-bird transmission as well as some zoonotic potential for the human caretakers of these birds. Furthermore, larger-scale studies should be conducted to confirm these findings.

Unusual cases of chlamydiosis in psittacine birds.

Avian chlamydiosis is a common disease found in domesticated and nondomesticated avian species caused by several species of chlamydiae including but not limited to Chlamydia psittaci, Chlamydia avium, Chlamydia gallinacea, Chlamydia buteonis, and Chlamydia ibidis. Generally, early in the disease course, birds present with mild nonspecific clinical signs associated with gastrointestinal and respiratory tract disease. During end-stage disease, birds may present in a severe state of emaciation, dehydration, and/or acute death with no known history of prior illness. Between 2000 and 2009, 14 unusual cases of avian chlamydiosis were submitted to the California Animal Health and Food Safety Laboratory System. Histologic lesions noted in the 14 birds included meningoencephalomyelitis (3 of 13, 23%), otitis media (3 of 8), bursitis (9 of 11, 81%), nephritis (8 of 13, 61%), and orchitis (1 of 8). Corresponding immunopositive chlamydiae intracytoplasmic inclusions were detected in all tissues. Positive immunolabeling was detected in optic nerves (5 of 10, 50%), meninges (5 of 13, 38%), and endothelial cells (14 of 14, 100%) in the absence of significant microscopic lesions. This study highlights unusual gross, histological, and immunohistochemical findings of chlamydiosis in psittacines and highlights the importance of a thorough diagnostic approach when confirming or excluding chlamydiosis in psittacine birds.

Prevalence of New and Established Avian Chlamydial Species in Humans and Their Psittacine Pet Birds in Belgium.

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.

Chlamydiosis and cystic dilatation of the ovarian bursa in the female koala (Phascolarctos cinereus): Novel insights into the pathogenesis and mechanisms of formation.

Infection with Chlamydia pecorum is one of the main causes of progressive decline of koala (Phascolarctos cinereus) populations in Eastern Australia. Pathological changes associated with the chlamydial infection in the genital tract of female and male koalas have been widely described with reports of acute and chronic lymphoplasmacytic inflammation and the description of the cystic dilatation of the ovarian bursa. Although these disease manifestations can result in severe chronic inflammation, structural changes and even sterility, only limited data is currently available on the organism's distribution and associated histopathological and ultrastructural changes within the upper genital tract of affected females. This study examined the pathogenesis of the most common pathological lesion associated with chlamydiosis in female koalas, the cystic dilation of the ovarian bursa starting from the evidence that Chlamydia spp. induces disruption of the intercellular junctions in the epithelium of the reproductive organs in humans. Histology, immunohistochemistry (IHC) and transmission electron microscopy (TEM) were performed to evaluate the structural features and the expression of epithelial cell and cellular junctions' markers in affected bursae from 39 Chlamydia-infected female koalas. Epithelial cells from the ovarian bursae of one affected animal examined by transmission electron microscopy showed severe widening of the intercellular space, as morphologic evidence of disrupted permeability of the epithelial barrier. The epithelial cell-cell junctions markers E-cadherin, ß-catenin and ZO-1 expressions were significantly reduced in samples from cystic bursae when compared to normal tissue samples (P < 0.0001). On the other end, a significantly higher expression of the proliferation marker Ki67 was observed in cystic bursae compared to control samples (P < 0.0001). As these proteins are required to maintain epithelial functional integrity and cell-cell adhesive interactions, their loss may permanently impair and affect female koala fertility and suggest the molecular basis of the pathogenesis of the cystic accumulation of bursal fluid within this tissue.

An outbreak of systemic chlamydiosis in farmed American alligators (Alligator mississippiensis).

Chlamydia spp are reported to causes systemic disease in a variety of hosts worldwide including few reports in crocodilians. Disease presentations vary from asymptomatic to fulminant disease, some of which are zoonotic. The aim of this study was to describe the pathological, immunohistochemical, and molecular findings associated with the occurrence of a previously unreported Chlamydia sp infection causing a major mortality event in farmed American alligators (Alligator mississippiensis). The outbreak presented with sudden death in juvenile alligators mainly associated with necrotizing hepatitis and myocarditis, followed by the occurrence of conjunctivitis after the initial high mortality event. The widespread inflammatory lesions in multiple organs correlated with intralesional chlamydial organisms identified via immunohistochemistry and confirmed by 23S rRNA-specific real-time quantitative polymerase chain reaction (qPCR) for Chlamydiaceae bacteria. By sequencing and phylogenetic analysis of the OmpA gene, this uncultured Chlamydia sp grouped closely with Chlamydia poikilothermis recently described in snakes. This study highlights the significance of such outbreaks in farmed populations. Enhanced epidemiological monitoring is needed to gain further insight into the biology of Chlamydia sp in alligators, disease dynamics, risk factors, and role of carrier animals.

Pharmacokinetic Profile of Doxycycline in Koala Plasma after Weekly Subcutaneous Injections for the Treatment of Chlamydiosis.

Six mature, male koalas (Phascolarctos cinereus), with clinical signs of chlamydiosis, were administered doxycycline as a 5 mg/kg subcutaneous injection, once a week for four weeks. Blood was collected at standardised time points (T = 0 to 672 h) to quantify the plasma doxycycline concentrations through high-pressure liquid chromatography (HPLC). In five koalas, the doxycycline plasma concentration over the first 48 h appeared to have two distinct elimination gradients; therefore, a two-compartmental analysis was undertaken to describe the pharmacokinetic (PK) profile. The average ± SD maximum plasma concentration (Cmax) was 312.30 ± 107.74 ng/mL, while the average time ± SD taken to reach the maximum plasma concentration (Tmax) was 1.68 ± 1.49 h. The mean ± SD half-life of the distribution phase (T1/2 α) and the elimination phase (T1/2 ß) were 10.51 ± 7.15 h and 82.93 ± 37.76 h, respectively. The average ± SD percentage of doxycycline binding to koala plasma protein was 83.65 ± 4.03% at three different concentrations, with a mean unbound fraction (fu) of 0.16. Using probability of target attainment modelling, doxycycline plasma concentrations were likely to inhibit 90% of pathogens with the doxycycline minimum inhibitory concentration (MIC) of 8.0-31.0 ng/mL, and the reported doxycycline MIC to inhibit Chlamydia pecorum isolates at the area under the curve/minimum inhibitory concentration (AUC/MIC) target of ≥24. All koalas were confirmed to be negative for Chlamydia pecorum using loop-mediated isothermal amplification (LAMP), from ocular and penile urethra swabs, three weeks after the last doxycycline injection.

A multiplex real-time PCR assay for differential identification of avian Chlamydia.

Avian chlamydiosis is an acute or chronic disease of birds after infection by Chlamydia. Although Chlamydia psittaci is the primary agent of the disease, two additional species, Chlamydia avium and Chlamydia gallinacea, have also been recognized as potential disease agents. Therefore, the diagnosis of avian chlamydiosis requires differential identification of these avian Chlamydia species. The objective of the present study was to develop a multiplex real-time polymerase chain reaction (PCR) assay to rapidly differentiate between these three species of avian Chlamydia (C. psittaci, C. avium, and C. gallinacea) as well as to detect the genus Chlamydia. Specific genetic regions of the three species were identified by comparative analysis of their genome sequences. Also, the genus-specific region was selected based on 23S rRNA sequences. PCR primers and probes specific to the genus and each species were designed and integrated in the multiplex real-time PCR assay. The assay was highly efficient (94.8-100.7%). It could detect fewer than 10 copies of each target sequence of the genus and each species. Twenty-five Chlamydia control and field DNA samples were differentially identified while 20 other bacterial strains comprising 10 bacterial genera were negative in the assay. This assay allows rapid, sensitive, and specific detection of the genus and the three species of avian Chlamydia in a single protocol that is suitable for routine diagnostic purposes in avian diagnostic laboratories.

Identification of Campylobacter jejuni and Chlamydia psittaci from cockatiel (Nymphicus hollandicus) using metagenomics.

BACKGROUND: In July 2015, the carcasses of 11 cockatiels were submitted for disease diagnosis to the Avian Disease Division of the Animal and Plant Quarantine Agency of Korea. The cockatiels, which appeared dehydrated and underweight, had exhibited severe diarrhea and 22 % mortality over 2 weeks. Traditional diagnosis did not reveal the causes of these symptoms. METHODS: We conducted metagenomics analysis on intestines and livers from the dead cockatiels using Illumina high-throughput sequencing. To obtain more accurate and longer contigs, which are required for further genetic characterization, we compared the results of three de novo assembly tools (metaSPAdes, MEGAHIT, and IDBA-UD). RESULTS: Sequence reads of Campylobacter jejuni (C. jejuni) and Chlamydia psittaci (C. psittaci) were present in most of the cockatiel samples. Either of these bacteria could cause the reported symptoms in psittaciformes. metaSPAdes (ver.3.14.1) identified the 1152 bp flaA gene of C. jejuni and the 1096 bp ompA gene of C. psittaci. Genetic analysis revealed that flaA of C. jejuni was recombinant between C. jejuni and Campylobacter coli, and that ompA of C. psittaci isolated from cockatiel was closely related to strains isolated from humans. CONCLUSIONS: C. jejuni and C. psittaci were detected in cockatiels in the Republic of Korea using metagenomic analysis. This approach is useful for understanding pathogens of pet birds. Three de novo assemblers were compared to obtain accurate contigs from large quantities of reads, and sequences of C. jejuni and C. psittaci generated by metaSPAdes were analyzed.

A Review of Chlamydial Infections in Wild Birds.

The Chlamydia are a globally distributed genus of bacteria that can infect and cause disease in a range of hosts. Birds are the primary host for multiple chlamydial species. The most well-known of these is Chlamydia psittaci, a zoonotic bacterium that has been identified in a range of wild and domesticated birds. Wild birds are often proposed as a reservoir of Chlamydia psittaci and potentially other chlamydial species. The aim of this review is to present the current knowledge of chlamydial infections in wild avian populations. We focus on C. psittaci but also consider other Chlamydiaceae and Chlamydia-related bacteria that have been identified in wild birds. We summarise the diversity, host range, and clinical signs of infection in wild birds and consider the potential implications of these infections for zoonotic transmission and avian conservation. Chlamydial bacteria have been found in more than 70 species of wild birds, with the greatest chlamydial diversity identified in Europe. The Corvidae and Accipitridae families are emerging as significant chlamydial hosts, in addition to established wild hosts such as the Columbidae. Clarifying the effects of these bacteria on avian host fitness and the zoonotic potential of emerging Chlamydiales will help us to understand the implications of these infections for avian and human health.