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.

Molecular Phylogeny and Evolution of Amazon Parrots in the Greater Antilles.

Amazon parrots (Amazona spp.) colonized the islands of the Greater Antilles from the Central American mainland, but there has not been a consensus as to how and when this happened. Today, most of the five remaining island species are listed as endangered, threatened, or vulnerable as a consequence of human activity. We sequenced and annotated full mitochondrial genomes of all the extant Amazon parrot species from the Greater Antillean (A. leucocephala (Cuba), A. agilis, A. collaria (both from Jamaica), A. ventralis (Hispaniola), and A. vittata (Puerto Rico)), A. albifrons from mainland Central America, and A. rhodocorytha from the Atlantic Forest in Brazil. The assembled and annotated mitogenome maps provide information on sequence organization, variation, population diversity, and evolutionary history for the Caribbean species including the critically endangered A. vittata. Despite the larger number of available samples from the Puerto Rican Parrot Recovery Program, the sequence diversity of the A. vittata population in Puerto Rico was the lowest among all parrot species analyzed. Our data support the stepping-stone dispersal and speciation hypothesis that has started approximately 3.47 MYA when the ancestral population arrived from mainland Central America and led to diversification across the Greater Antilles, ultimately reaching the island of Puerto Rico 0.67 MYA. The results are presented and discussed in light of the geological history of the Caribbean and in the context of recent parrot evolution, island biogeography, and conservation. This analysis contributes to understating evolutionary history and empowers subsequent assessments of sequence variation and helps design future conservation efforts in the Caribbean.

Parrot Genomes and the Evolution of Heightened Longevity and Cognition.

Parrots are one of the most distinct and intriguing groups of birds, with highly expanded brains [1], highly developed cognitive [2] and vocal communication [3] skills, and a long lifespan compared to other similar-sized birds [4]. Yet the genetic basis of these traits remains largely unidentified. To address this question, we have generated a high-coverage, annotated assembly of the genome of the blue-fronted Amazon (Amazona aestiva) and carried out extensive comparative analyses with 30 other avian species, including 4 additional parrots. We identified several genomic features unique to parrots, including parrot-specific novel genes and parrot-specific modifications to coding and regulatory sequences of existing genes. We also discovered genomic features under strong selection in parrots and other long-lived birds, including genes previously associated with lifespan determination as well as several hundred new candidate genes. These genes support a range of cellular functions, including telomerase activity; DNA damage repair; control of cell proliferation, cancer, and immunity; and anti-oxidative mechanisms. We also identified brain-expressed, parrot-specific paralogs with known functions in neural development or vocal-learning brain circuits. Intriguingly, parrot-specific changes in conserved regulatory sequences were overwhelmingly associated with genes that are linked to cognitive abilities and have undergone similar selection in the human lineage, suggesting convergent evolution. These findings bring novel insights into the genetics and evolution of longevity and cognition, as well as provide novel targets for exploring the mechanistic basis of these traits.

Preliminary Findings of Structure and Expression of Opioid Receptor Genes in a Peregrine Falcon ( Falco peregrinus), a Snowy Owl ( Bubo scandiacus), and a Blue-fronted Amazon Parrot ( Amazona aestiva).

To further knowledge of the physiology of opioid receptors in birds, the structure and expression of the µ-, δ-, and κ-opioid receptor genes were studied in a peregrine falcon ( Falco peregrinus), a snowy owl ( Bubo scandiacus), and a blue-fronted Amazon parrot ( Amazona aestiva). Tissue samples were obtained from birds that had been euthanatized for poor release prognosis or medical reasons. Samples were taken from the brain (telencephalon, thalamus, pituitary gland, cerebellum, pons, medulla oblongata, mesencephalon), the spinal cord and dorsal root ganglions, and plantar foot skin. Messenger RNA was recovered, and reverse transcription polymerase chain reaction (RT-PCR) was performed to generate complementary DNA (cDNA) sequences. Gene structures were documented by directly comparing cDNA sequences with recently published genomic sequences for the peregrine falcon and the blue-fronted Amazon parrot or by comparisons with genomic sequences of related species for the snowy owl. Structurally, the avian µ-opioid receptor messenger RNA (mRNA) species were complex, displaying differential splicing, alternative stop codons, and multiple polyadenylation signals. In comparison, the structure of the avian κ-receptor mRNA was relatively simple. In contrast to what is seen in humans, the avian δ-receptor mRNA structure was found to be complex, demonstrating novel 3-prime coding and noncoding exons not identified in mammals. The role of the δ-opioid receptor merits further investigation in avian species.

Founded: Genetic Reconstruction of Lineage Diversity and Kinship Informs Ex situ Conservation of Cuban Amazon Parrots (Amazona leucocephala).

Captive breeding is a widespread conservation strategy, yet such programs rarely include empirical genetic data for assessing management assumptions and meeting conservation goals. Cuban Amazon parrots (Amazona leucocephala) are considered vulnerable, and multiple on-island captive populations have been established from wild-caught and confiscated individuals of unknown ancestry. Here, we used mitochondrial haplotypic and nuclear genotypic data at 9 microsatellite loci to quantify the extent and distribution of genetic variation within and among captive populations in Zapata Swamp and Managua, Cuba, and to estimate kinship among breeders (n = 88). Using Bayesian clustering analysis, we detected 2 distinct clusters within the Zapata population, one of which was shared with Managua. Individuals from the cluster unique to Zapata possessed mitochondrial haplotypes with affinities to Cuban subspecies (A. l. leucocephala, A. l. palmarum); the shared cluster was similar, but also included haplotypes closely related to the subspecies restricted to Cayman Brac (A. l. hesterna). Overall mean kinship was low within each captive population (-0.026 to -0.012), with 19 and 11 recommended breeding pairs in Zapata and Managua, respectively, ranked according to mean kinship and informed by molecular sexing. Our results highlight the importance of understanding population history within ex situ management programs, while providing genetic information to directly inform Cuban parrot conservation.

Phylogeny of Amazona barbadensis and the Yellow-headed Amazon complex (Aves: Psittacidae): a new look at South American parrot evolution.

The Yellow-shouldered Amazon (Amazona barbadensis) is the sole parrot of the genus Amazona that inhabits only dry forests. Its population has been dropping; therefore it has been the topic of many studies and conservation efforts. However, the phylogenetic relationship of this species to potential relatives classified within the Yellow-Headed Amazon (YHA) complex are still not clear. Therefore, we used more extensive data sets, including the newly sequenced mitochondrial genome of A. barbadensis, to conduct phylogenetic analyses. Various combinations of genes and many phylogenetic approaches showed that A. barbadensis clustered significantly with A. ochrocephala ochrocephala from Colombia and Venezuela, which created the Northern South American (NSA) lineage, clearly separated from two other lineages within the YHA complex, the Central (CA) and South American (SA). Tree topology tests and exclusion of rapidly evolving sites provided support for a NSA+SA grouping. We propose an evolutionary scenario for the YHA complex and its colonization of the American mainland. The NSA lineage likely represents the most ancestral lineage, which derived from Lesser Antillean Amazons and colonized the northern coast of Venezuela about a million years ago. Then, Central America was colonized through the Isthmus of Panama, which led to the emergence of the CA lineage. The southward expansion to South America and the origin of the SA lineage happened almost simultaneously. However, more intensive or prolonged gene flow or migrations have led to much weaker geographic differentiation of genetic markers in the SA than in the CA lineage.

Complete mitochondrial genome of endangered Yellow-shouldered Amazon (Amazona barbadensis): two control region copies in parrot species of the Amazona genus.

Amazona barbadensis is an endangered species of parrot living in northern coastal Venezuela and in several Caribbean islands. In this study, we sequenced full mitochondrial genome of the considered species. The total length of the mitogenome was 18,983 bp and contained 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, duplicated control region, and degenerate copies of ND6 and tRNA (Glu) genes. High degree of identity between two copies of control region suggests their coincident evolution and functionality. Comparative analysis of both the control region sequences from four Amazona species revealed their 89.1% identity over a region of 1300 bp and indicates the presence of distinctive parts of two control region copies.

Granulomatous encephalomyelitis and intestinal ganglionitis in a spectacled Amazon parrot (Amazona albifrons) infected with Mycobacterium genavense.

An approximately 30-year-old male spectacled Amazon parrot (Amazona albifrons) was presented with a 2-week history of ataxia, head shaking, weight loss and seizures. Gross findings on necropsy examination included atrophy of the musculature, ruffled feathers and minimal epicardial and abdominal fat. Microscopically, there were perivascular cuffs of macrophages with fewer lymphocytes in the grey and white matter of the brain and spinal cord. These lesions were accompanied by gliosis and mild vacuolation of the white matter. In the small intestine, up to 70% of the intestinal ganglia were effaced by infiltrates of macrophages and fewer lymphocytes. The intestinal lamina propria contained multiple inflammatory aggregates of a similar nature. Ziehl-Neelsen staining revealed the presence of numerous bacilli within the cytoplasm of macrophages in the central nervous system (CNS) and enteric ganglia. Amplification of the DNAJ gene confirmed a mycobacterial infection and subsequent polymerase chain reaction (PCR) using a species-specific primer confirmed the aetiology as Mycobacterium genavense. Infection of the CNS with Mycobacterium spp. is uncommon and has not been previously reported in a parrot. This case is unusual in that the organism exhibited tropism for neural tissue.

Population genetic structure of the blue-fronted Amazon (Amazona aestiva, Psittacidae: Aves) based on nuclear microsatellite loci: implications for conservation.

The blue-fronted Amazon (Amazona aestiva) is a widely distributed Neotropical parrot and one of the most captured parrots in nature to supply the illegal trade of wild animals. The objectives of the present study were to analyze the genetic structure of A. aestiva to identify management units and support conservation planning and to verified if A. aestiva populations have undergone a recent bottleneck due to habitat loss and capture for the pet trade. The genetic structure was accessed by analyzing six microsatellite loci in 74 individuals of A. aestiva, including samples from the two subspecies (A. a. aestiva and A. a. xanthopteryx), from five populations: four in Brazil and one in Argentina. A significant genetic differentiation (theta = 0.007, p = 0.005) could be detected only between the most distant populations, Tocantins and Argentina, localized at the northeast and southwest limits of the sample sites, respectively. There was no evidence of inbreeding within or between populations, suggesting random mating among individuals. These results suggest a clinal distribution of genetic variability, as observed for variation in plumage color of the two A. aestiva subspecies. Bottleneck analysis did not show a recent reduction in population size. Thus, for the management and conservation of the species, the populations from Argentina and Tocantins should be considered as different management units, and the other populations from the center of the geographical distribution as another management unit.