Recent history of psittacosis in Australia: expanding our understanding of the epidemiology of this important globally distributed zoonotic disease.

Psittacosis is a human systemic disease caused by infection with Chlamydia psittaci. Shortly after reports emerged of a global pandemic associated with contact with imported parrots, Australian researchers including Macfarlane Burnet and others demonstrated that C. psittaci was widespread in Australian parrots. Australian cases over the last two decades have revealed that environmental exposure and contact with infected horses are also risk factors in an increasingly complicated epidemiological picture for this zoonotic disease.

Reclassification of Bisgaard taxon 37 and taxon 44 as Psittacicella melopsittaci gen. nov., sp. nov., Psittacicella hinzii sp. nov. and Psittacicella gerlachiana sp. nov. within Psittacicellaceae fam. nov. of the order Pasteurellales.

Bacteria isolated from lesions as well as apparently normal tissues of psittacine birds have previously been reported as taxon 37 and taxon 44 of Bisgaard. 16S rRNA gene sequence comparisons revealed a distant relationship to members of Pasteurellaceae at the species, genus and family levels. The polar lipid profile consisted of the major components phosphatidylethanolamine and phosphatidylglycerol. A new family Psittacicellaceae fam. nov. is proposed with the type genus Psittacicella gen. nov. The new genus Psittacicella includes the type species Psittacicella melopsittaci sp. nov. with type strain B96/4T (=CCUG 70858T=DSM 105476T), Psittacicella hinzii sp. nov. with type strain 111T (=CCUG 52861T=CCM 8842T) and Psittacicella gerlachiana sp. nov. with type strain EEAB3T1T (=CCUG 70857T=DSM 105477T). In addition to the major polar lipids, strain 111T possessed the non-identified aminophospholipids APL1 and APL2 and trace amounts of four lipids (L1-L4) whereas strain B94/4T showed the minor unidentified aminophospholipids APL3 and APL2 and trace amounts of unidentified lipid L3. These results demonstrate that strain B96/4T can be distinguished from 111T based on presence/absence of the unidentified lipids APL1 and APL3. The total polar lipid profile of strain EEAB3T1T differed from B96/4Tonly in one minor lipid. Strain B96/4T can further be distinguished from 111T by acid formation from trehalose and raffinose and the α-glucosidase test. Strains 111T and EEAB3T1T can be separated based on acid formation from trehalose and the α-glucosidase test. Strains B96/4T and EEAB3T1T can be separated by acid formation from raffinose and eight signature indels in the RpoB protein.

First case report of fatal Nocardia nova infection in yellow-bibbed lory (Lorius chlorocercus) identified by multilocus sequencing.

BACKGROUND: Nocardiosis is often a multi-systemic disease in humans and other mammals. Nocardiosis in birds is uncommon. Laboratory identification of Nocardia to the species level is difficult by traditional phenotypic methods based on biochemical reactions and hydrolysis tests, and is most accurately performed by sequencing multiple gene targets. CASE PRESENTATION: We report the first case of fatal Nocardia nova infection in a yellow-bibbed lory nestling in an oceanarium diagnosed by multilocus sequencing. Necropsy examination showed effacement of normal sternal musculature with yellowish, firm aberrant material, and diffuse infiltration of the lungs with nodular, tan to yellow foci. Histologically, severe granulomatous inflammation with marked necrosis was observed in the lung, spleen and sternal musculature. Fine, sometimes Gram-positive, 0.5-1 µm wide, branching and beaded filamentous organisms were visible within the lesions. They were acid-fast on Fite-Faraco stain. Tissue samples obtained from the sternum, liver, right lung and right kidney recovered Nocardia species. Sequencing of four gene loci and phylogenetic analysis of concatenated (gyrB-16S-secA1-hsp65) sequences revealed that the isolate was N. nova. CONCLUSIONS: We report the first case of N. nova infection in yellow-bibbed lorry (Lorius chlorocercus). The present case is the first one of which the species identity of the isolate was determined by multilocus sequencing. Molecular diagnosis is important for identifying the Nocardia to species level and understanding the epidemiology of nocardiosis in birds.

Influence of hand rearing and bird age on the fecal microbiota of the critically endangered kakapo.

The critically endangered New Zealand parrot, the kakapo, is subject to an intensive management regime aiming to maintain bird health and boost population size. Newly hatched kakapo chicks are subjected to human intervention and are frequently placed in captivity throughout their formative months. Hand rearing greatly reduces mortality among juveniles, but the potential long-term impact on the kakapo gut microbiota is uncertain. To track development of the kakapo gut microbiota, fecal samples from healthy, prefledged juvenile kakapos, as well as from unrelated adults, were analyzed by using 16S rRNA gene amplicon pyrosequencing. Following the original sampling, juvenile kakapos underwent a period of captivity, so further sampling during and after captivity aimed to elucidate the impact of captivity on the juvenile gut microbiota. Variation in the fecal microbiota over a year was also investigated, with resampling of the original juvenile population. Amplicon pyrosequencing revealed a juvenile fecal microbiota enriched with particular lactic acid bacteria compared to the microbiota of adults, although the overall community structure did not differ significantly among kakapos of different ages. The abundance of key operational taxonomic units (OTUs) was correlated with antibiotic treatment and captivity, although the importance of these factors could not be proven unequivocally within the bounds of this study. Finally, the microbial community structure of juvenile and adult kakapos changed over time, reinforcing the need for continual monitoring of the microbiota as part of regular health screening.

Prevalence of IgY antibodies against Erysipelothrix rhusiopathiae in a critically endangered parrot (kakapo, Strigops habroptilus) and associated responses to vaccination.

An enzyme-linked immunosorbent assay (ELISA) was developed to estimate levels of IgY antibody against the bacterium Erysipelothrix rhusiopathiae in serum samples collected from the critically endangered kakapo (Strigops habroptilus, Psittaciformes, Aves) before and after vaccination against this bacterium. Relative IgY antibody titres in pre-vaccination serum samples (n = 71 individual kakapo) were normally distributed with the exception of four outliers which displayed low IgY levels. Notably all four low IgY samples were collected from fledglings 3 - 6 months old. Pre-vaccination serum samples from nine nestlings <3 months old, seven of which were hatched in incubators and had no contact with either adult kakapo or their natural environment (e.g. soil), were found to have relatively high IgY levels, suggesting transfer of maternal IgY molecules to fledglings via the yolk. IgY levels in pre-vaccination serum samples from seven kakapo aged 25 - 30 months were also relatively high, suggesting that most kakapo naturally acquire anti- E.rhusiopathiae IgYs within their first 2 years. There was no evidence that vaccination increased the kakapo population's mean anti-E.rhusiopathiae IgY levels. However, there was a significant negative relationship between an individual bird's pre-vaccination IgY level and any subsequent increase following vaccination, suggesting that vaccination may only raise the IgY levels of birds with relatively low pre-vaccination IgY levels. A statistical model of the relationship between 'death from erysipelas' and sex, age and transfer from one to island sanctuary to another found that only transfer was significantly associated with death from erysipelas.

Genome sequences of the zoonotic pathogens Chlamydia psittaci 6BC and Cal10.

Chlamydia psittaci is a highly prevalent avian pathogen and the cause of a potentially lethal zoonosis, causing life-threatening pneumonia in humans. We report the genome sequences of C. psittaci 6BC, the prototype strain of the species, and C. psittaci Cal10, a widely used laboratory strain.

DNA detection and genotypic identification of potentially human-pathogenic microsporidia from asymptomatic pet parrots in South Korea as a risk factor for zoonotic emergence.

We detected and identified genotypes of human-pathogenic microsporidia in fecal samples from 51 asymptomatic captive-bred pet parrots in South Korea. Microsporidia were identified in 8 samples (15.7%); 7 parrots tested positive for Encephalitozoon hellem, and 1 parrot tested positive for both E. hellem and Encephalitozoon cuniculi. In genotypic identifications, E. hellem was present in genotypes 1A and 2B and E. cuniculi was present in genotype II. Pet parrots might be a source of human microsporidian infection.

Colourful parrot feathers resist bacterial degradation.

The brilliant red, orange and yellow colours of parrot feathers are the product of psittacofulvins, which are synthetic pigments known only from parrots. Recent evidence suggests that some pigments in bird feathers function not just as colour generators, but also preserve plumage integrity by increasing the resistance of feather keratin to bacterial degradation. We exposed a variety of colourful parrot feathers to feather-degrading Bacillus licheniformis and found that feathers with red psittacofulvins degraded at about the same rate as those with melanin and more slowly than white feathers, which lack pigments. Blue feathers, in which colour is based on the microstructural arrangement of keratin, air and melanin granules, and green feathers, which combine structural blue with yellow psittacofulvins, degraded at a rate similar to that of red and black feathers. These differences in resistance to bacterial degradation of differently coloured feathers suggest that colour patterns within the Psittaciformes may have evolved to resist bacterial degradation, in addition to their role in communication and camouflage.

Disseminated histoplasmosis with concurrent oral candidiasis in an Eclectus parrot (Eclectus roratus).

Disseminated histoplasmosis caused by Histoplasma capsulatum, a zoonotic fungal organism, is an important disease in animals and humans, particularly those with compromised immune systems. Reports of disseminated histoplasmosis in an avian species are not available within the current literature. Candida albicans, another fungal agent with zoonotic importance, is a commensal of the avian digestive tract that is often associated with opportunistic infections particularly in young or immunocompromised birds. This report describes a case of concomitant histoplasmosis and candidiasis in an Eclectus parrot (Eclectus roratus) characterized by severe granulomatous glossitis, blepharitis and osteomyelitis with numerous intrahistiocytic and extracellular yeasts (H. capsulatum) as well as intralesional hyphae, pseudohyphae and conidia (C. albicans). To our knowledge, co-infection with H. capsulatum and C. albicans has not been reported in an avian species.

A 25-year retrospective study of Chlamydia psittaci in association with equine reproductive loss in Australia.

Introduction. Chlamydia psittaci is primarily a pathogen of birds but can also cause disease in other species. Equine reproductive loss caused by C. psittaci has recently been identified in Australia where cases of human disease were also reported in individuals exposed to foetal membranes from an ill neonatal foal in New South Wales.Hypothesis/Gap Statement. The prevalence of C. psittaci in association with equine reproductive over time and in different regions of Australia is not known.Aim. This study was conducted to detect C. psittaci in equine abortion cases in Australia using archived samples spanning 25 years.Methodology. We tested for C. psittaci in 600 equine abortion cases reported in Australia between 1994 to 2019 using a Chlamydiaceae real-time quantitative PCR assay targeting the 16S rRNA gene followed by high-resolution melt curve analysis. Genotyping and phylogenetic analysis was performed on positive samples.Results. The overall prevalence of C. psittaci in material from equine abortion cases was 6.5 %. C. psittaci-positive cases were detected in most years that were represented in this study and occurred in Victoria (prevalence of 7.6 %), New South Wales (prevalence of 3.9 %) and South Australia (prevalence of 15.4 %). Genotyping and phylogenetic analysis showed that the C. psittaci detected in the equine abortion cases clustered with the parrot-associated 6BC clade (genotype A/ST24), indicating that infection of horses may be due to spillover from native Australian parrots.Conclusion. This work suggests that C. psittaci has been a significant agent of equine abortion in Australia for several decades and underscores the importance of taking appropriate protective measures to avoid infection when handling equine aborted material.

Metagenomic next-generation sequencing in the family outbreak of psittacosis: the first reported family outbreak of psittacosis in China under COVID-19.

Chlamydia psittaci infection in humans, also known as psittacosis, is usually believed to be an uncommon disease which mainly presents as community-acquired pneumonia (CAP). It is usually sporadic, but outbreaks of infection may occasionally occur. In outbreaks, diagnosis and investigations were usually hampered by the non-specificity of laboratory testing methods to identify C. psittaci. In this study, we use metagenomic next-generation sequencing (mNGS) in the diagnosis of a family outbreak of psittacosis under COVID-19. Three members of an extended family of 6 persons developed psittacosis with pneumonia and hepatic involvement with common symptoms of fever and weakness. Two newly purchased pet parrots, which had died successively, were probably the primary source of infection. Imagings show lung consolidations and infiltrates, which are difficult to be differentiated from CAP caused by other common pathogens. mNGS rapidly identified the infecting agent as C. psittaci within 48 h. The results of this work suggest that there are not characteristic clinical manifestations and imagings of psittacosis pneumonia which can differentiate from CAP caused by other pathogens. The use of mNGS can improve accuracy and reduce the delay in the diagnosis of psittacosis especially during the outbreak, which can shorten the course of the disease control. Family outbreak under COVID-19 may be related to the familial aggregation due to the epidemic. To our knowledge, this is the first reported family outbreak of psittacosis in China, and the first reported psittacosis outbreak identified by the method of mNGS in the world.

Genomic characterization of enteropathogenic Escherichia coli (EPEC) of avian origin and rabbit ileal loop response; a pet macaw (Ara chloropterus) as a possible zoonotic reservoir.

Enteropathogenic Escherichia coli (EPEC) constitutes one of the main causes of mortality in children in low- to medium-income countries. Diverse animal species have been linked as reservoirs, including birds. The aim of this study was to describe the genomic and phylogenetic features of an EPEC recovered from a pet macaw and further characterizing the macro and microscopic lesion in a rabbit ileal loop experimental model. The isolate was whole-genome sequenced (WGS) obtaining its genotypic and phenotypic in silico characteristics and inoculated in a rabbit experimental model with subsequently evaluating the strain's pathogenicity by scanning electron microscopy (SEM) and histopathology. The isolate was characterized as O109:H21-B1-ST40 typical EPEC, harboring several virulence factors of diarrheagenic E. coli. The macaw EPEC genome was located in a monophyletic clade of human and animal ST40 EPEC sequences. In vivo inoculation demonstrated severe hemorrhage with SEM and histopathological analysis confirming these lesions to be associated with intra-epithelial lymphocytes. Therefore, the isolate not only shared several genotypic and phylogenetic similarities with EPEC that affects humans and animals, but was able to induce severe tissue injury in a mammal model. These findings highlight the underrated role of pet birds as zoonotic reservoirs and the diversity in virulence factors being unraveled by new WGS studies.

Species, sex and geographic variation in chlamydial prevalence in abundant wild Australian parrots.

Chlamydia psittaci (order: Chlamydiales) is a globally distributed zoonotic bacterium that can cause potentially fatal disease in birds and humans. Parrots are a major host, yet prevalence and risk factors for infection in wild parrots are largely unknown. Additionally, recent research suggests there is a diverse range of novel Chlamydiales circulating in wildlife. We therefore sampled seven abundant parrot species in south-eastern Australia, taking cloacal swabs and serum from n = 132 wild adults. We determined C. psittaci and Chlamydiales prevalence and seroprevalence, and tested for host species, sex, geographical and seasonal differences, and temporal changes in individual infection status. Across all species, Chlamydiales prevalence was 39.8% (95% CI 31.6, 48.7), C. psittaci prevalence was 9.8% (95% CI 5.7, 16.3) and C. gallinacea prevalence was 0.8% (95% CI 0.1, 4.5). Other Chlamydiales species were not identified to species level. We identified two C. psittaci strains within the 6BC clade, which is highly virulent in humans. Seroprevalence was 37.0% (95% CI 28.5, 46.4). Host species (including crimson rosellas, galahs, sulphur-crested cockatoos and blue-winged parrots) differed in seroprevalence and Chlamydiales prevalence. Galahs had both highest Chlamydiales prevalence (54.8%) and seroprevalence (74.1%). Seroprevalence differed between sites, with a larger difference in males (range 20-63%) than females (29-44%). We reveal a higher chlamydial prevalence than previously reported in many wild parrots, with implications for potential reservoirs, and transmission risks to humans and other avian hosts.

Chlamydia psittaci in fulmars on the Faroe Islands: a causative link to South American psittacines eight decades after a severe epidemic.

A psittacosis epidemic linked to fulmar hunting occurred on the Faroe Islands in the 1930s. This study investigates a plausible explanation to the 20% human mortality in this outbreak. Phylogenetic analysis showed that Chlamydia psittaci isolated from fulmars were closely related to the highly virulent 6BC strains from psittacines and are compatible with an acquisition by fulmars of an ancestor of the 6BC clade in the 1930s. This supports the hypothesis that the outbreak on the Faroe Islands started after naïve fulmars acquired C. psittaci from infected dead parrots thrown overboard when shipped to Europe in the 1930s.

Isolation of Salmonella typhimurium from dead blue and gold macaws (Ara ararauna).

Two blue and gold macaw (Ara ararauna) chicks died of fatal salmonellosis in Buenos Aires Province, Argentina. The birds were histopathologically and microbiologically examined. Salmonella enterica subspecies enterica serovar Typhimurium was isolated from the liver, spleen, heart, lung, kidney, and intestine of both birds. All strains were susceptible to ampicillin, cephalothin, cefotaxime, enrofloxacin, nalidixic acid, gentamicin, streptomycin, chloramphenicol, fosfomycin, tetracycline, nitrofurantoin, and trimethoprim-sulfamethoxazole. The XbaI-PFGE profile of the Salmonella Typhimurium isolated from the two animals, which shared the same cage, was identical and showed a unique pattern compared with 301 isolates included in the PulseNet national database of Salmonella pulsed-field gel electrophoresis patterns. This is the first report that describes fatal cases of salmonellosis from blue and gold macaws.

Unusual multifocal granulomatous disease caused by actinomycetous bacteria in a nestling Derbyan parrot (Psittacula derbiana).

A nestling Derbyan parrot (Psittacula derbiana) was presented with unusual subcutaneous swellings of the thigh regions, and poor growth. Histological examination revealed actinomycetous bacteria associated with multifocal systemic granulomas. The clinical and pathological findings of the case are presented, and some relevant aspects of actinomycetous bacterial infections in mammals and birds are discussed. Although granulomatous disease is encountered at times in avian species, the actinomycetous bacteria (Nocardia and Actinomyces spp.) have rarely been reported in association with multifocal granulomatous disease in birds.

Yeasts and filamentous fungi in psittacidae and birds of prey droppings in midwest region of Brazil: a potential hazard to human health.

Birds of prey and from Psittacidae family are host to fungal microbiota and play an important role in the epidemiology of zoonoses. Few studies in the literature have characterized mycelial and yeast fungi in the droppings of these birds and correlated the isolates with the zoonotic potential of the microorganisms. Droppings from 149 birds were evaluated and divided into two groups: captive: Rhea americana araneipes, Primolius maracana, Ara ararauna, Ara chloropterus, Anodorhynchus hyacinthinus, Amazona aestiva, Ara macao macao, Ramphastos toco, Sarcoramphus papa, Busarellus nigricollis, Bubo virginianus nacurutu, Buteogallus coronatus, Buteogallus urubitinga urubitinga, Spizaetus melanoleucus, Spizaetus ornatus ornatus, Buteo albonotatus, Geranoaetus albicaudatus albicaudatus, Rupornis magnirostris magnirostris and Harpia harpyja, and quarantined birds: Amazona aestiva and Eupsitulla aurea. The fungal isolates were identified according to macroscopic (gross colony appearance), micromorphological and biochemical characteristics. Among birds displayed in enclosures, Aspergillus niger (41.1%) and Candida kefyr (63.8%) were the fungi most frequently isolated in Harpia harpyja and Ramphastos toco, respectively. For quarantined birds, the following percentages were observed in Eupsittula aurea , (76.6%) C. krusei, (84.4%) C. kefyr and (15.2%) C. famata, while in Amazona aestiva, (76.2%) C. krusei was observed. These findings indicate potentially pathogenic species in the bird droppings assessed, which constitute a risk of exposure for keepers and individuals who visit the zoo. Birds of the Cerrado and Pantanal of Mato Grosso (Central Western region of Brazil) could act in the epidemiological chain of important zoonoses.

Disease surveillance in wild Victorian cacatuids reveals co-infection with multiple agents and detection of novel avian viruses.

Wild birds are known reservoirs of bacterial and viral pathogens, some of which have zoonotic potential. This poses a risk to both avian and human health, since spillover into domestic bird populations may occur. In Victoria, wild-caught cockatoos trapped under licence routinely enter commercial trade. The circovirus Beak and Feather Disease Virus (BFDV), herpesviruses, adenoviruses and Chlamydia psittaci have been identified as significant pathogens of parrots globally, with impacts on both aviculture and the conservation efforts of endangered species. In this study, we describe the results of surveillance for psittacid herpesviruses (PsHVs), psittacine adenovirus (PsAdV), BFDV and C. psittaci in wild cacatuids in Victoria, Australia. Samples were collected from 55 birds of four species, and tested using genus or family-wide polymerase chain reaction methods coupled with sequencing and phylogenetic analyses for detection and identification of known and novel pathogens. There were no clinically observed signs of illness in most of the live birds in this study (96.3%; n = 53). Beak and Feather Disease Virus was detected with a prevalence of 69.6% (95% CI 55.2-80.9). Low prevalences of PsHV (1.81%; 95% CI 0.3-9.6), PsAdV (1.81%; 95% CI 0.3-9.6), and C. psittaci (1.81%; 95% CI 0.3-9.6) was detected. Importantly, a novel avian alphaherpesvirus and a novel avian adenovirus were detected in a little corella (Cacatua sanguinea) co-infected with BFDV and C. psittaci. The presence of multiple potential pathogens detected in a single bird presents an example of the ease with which such infectious agents may enter the pet trade and how novel viruses circulating in wild populations have the potential for transmission into captive birds. Genomic identification of previously undescribed avian viruses is important to further our understanding of their epidemiology, facilitating management of biosecurity aspects of the domestic and international bird trade, and conservation efforts of vulnerable species.