Factors Influencing Vertical Transmission of Psittacine Bornavirus in Cockatiels (Nymphicus hollandicus).

The transmission of parrot bornavirus is still not fully understood. Although horizontal transmission through wounds can be one route, vertical transmission is still discussed. PaBV RNA and PaBV antigen were detected in psittacine embryos, but isolation of the virus failed, raising doubts about this route. In this study, cockatiels were infected either as adults (adult group) or during the first 6 days after hatching (juvenile group) and raised until sexual maturity to breed and lay eggs. A total of 92 eggs (adult group: 49, juvenile group: 43) were laid and incubated until day 17. The embryos and yolk samples were examined by RT-PCR for PaBV RNA and by infectivity assay for infectious virus. In the adult group, 14/31 embryos (45.2%) and 20/39 (51%) of the yolk samples demonstrated PaBV RNA in the PCR. Isolation of PaBV was not possible in any embryo of this group, but it was achieved in six yolk samples from one female. Anti-PaBV antibodies were detected in the yolk samples after seroconversion of all female parents. In the juvenile group, 22/29 embryos (74.9%) were positive for PaBV RNA. In 9/21 embryos (42.9%), PaBV isolation was possible. PaBV RNA was detected in 100% and infectious virus in 41% of the yolk samples. Anti-PaBV antibodies were detected in all yolk samples. For the first time, successful vertical transmission of PaBV was proven, but it seems to depend on the age when the parent birds are infected. Therefore, the age of the bird at time of infection may be an important factor in the occurrence of vertical transmission.

Tissue Distribution of Parrot Bornavirus 4 (PaBV-4) in Experimentally Infected Young and Adult Cockatiels (Nymphicus hollandicus).

Proventricular dilatation disease (PDD) caused by parrot bornavirus (PaBV) infection is an often-fatal disease known to infect Psittaciformes. The impact of age at the time of PaBV infection on organ lesions and tissue distribution of virus antigen and RNA remains largely unclear. For this purpose, tissue sections of 11 cockatiels intravenously infected with PaBV-4 as adults or juveniles, respectively, were examined via histology, immunohistochemistry applying a phosphoprotein (P) antibody directed against the bornaviral phosphoprotein and in situ hybridisation to detect viral RNA in tissues. In both groups of adult- and juvenile-infected cockatiels, widespread tissue distribution of bornaviral antigen and RNA as well as histologic inflammatory lesions were demonstrated. The latter appeared more severe in the central nervous system in adults and in the proventriculus of juveniles, respectively. During the study, central nervous symptoms and signs of gastrointestinal affection were only demonstrated in adult birds. Our findings indicate a great role of the age at the time of infection in the development of histopathological lesions and clinical signs, and thus provide a better understanding of the pathogenesis, possible virus transmission routes, and the development of carrier birds posing a risk to psittacine collections.

Experimental Infection of Embryonic Cells and Embryonated Eggs of Cockatiels (Nymphicus hollandicus) with Two Parrot Bornavirus Isolates (PaBV-4 and PaBV-2).

Parrot bornavirus (PaBV) might be transmitted vertically. Cockatiel embryonic brain cells and embryonated eggs of cockatiels (ECE) were infected with PaBV-2 and PaBV-4. In embryonic brain cells, PaBV-2 and PaBV-4 showed no differences in viral spread despite the slower growth of PaBV-2 compared with PaBV-4 in CEC-32 cells. ECE were inoculated with PaBV-4 and 13-14 dpi, organs were sampled for RT-PCR, immunohistochemistry/histology, and virus isolation. In 28.1% of the embryos PaBV-4-RNA and in 81.3% PaBV-4-antigen was detected in the brain. Virus isolation failed. Division of organ samples and uneven tissue distribution of the virus limited the results. Therefore, 25 ECE were inoculated with PaBV-4 (group 1) and 15 ECE with PaBV-2 (group 3) in the yolk sac, and 25 ECE were inoculated with PaBV-4 (group 2) and 15 eggs with PaBV-2 (group 4) in the chorioallantoic membrane to use the complete organs from each embryo for each examination method. PaBV-RNA was detected in the brain of 80% of the embryos in groups 1, 2, 3 and in 100% of the embryos in group 4. In 90% of the infected embryos of group 1, and 100% of group 2, 3 and 4, PaBV antigen was detected in the brain. PaBV antigen-positive brain cells were negative for anti-neuronal nuclear protein, anti-glial fibrillary acidic protein, and anti S-100 staining. Virus was not re-isolated. These results demonstrated a specific distribution pattern and spread of PaBV-4 and PaBV-2 in the brain when inoculated in ECE. These findings support a potential for vertical transmission.

Characterization and Comparative Analysis of Complete Mitogenomes of Three Cacatua Parrots (Psittaciformes: Cacatuidae).

Cacatua alba, Cacatua galerita, and Cacatua goffiniana are parrots of the family Cacatuidae. Wild populations of these species are declining with C. alba listed by the International Union for the Conservation of Nature and Natural Resources (IUCN) as Endangered. In this study, complete mitogenomes were sequenced for a comparative analysis among the Cacatua species, and a detailed analysis of the control region. Mitogenome lengths of C. alba,C. galerita, and C. goffiniana were 18,894, 18,900, and 19,084 bp, respectively. They included 13 protein coding genes, two ribosomal RNA genes, 24 transfer RNA genes, three degenerated genes, and two control regions. Ten conserved motifs were found in three domains within each of the two control regions. For an evolution of duplicated control regions of Cacatua, domain I and the 3' end of domain III experienced an independent evolution, while domain II and most of the regions of domain III was subjected to a concerted evolution. Based on a phylogenetic analysis of 37 mitochondrial genes, the genus Cacatua formed a well-supported, monophyletic, crown group within the Cacatuidae. Molecular dating results showed that Cacatua diverged from other genera of Cacatuinae in the middle of Miocene.

Assessment of avian sperm DNA fragmentation using the sperm chromatin dispersion assay.

Herein we report a simple method for assessing avian sperm DNA fragmentation (SDF) using the sperm chromatin dispersion test (SCDt). The presence of sperm DNA damage was confirmed indirectly by correlating results of the SCDt determined in three bird species with results of a corresponding neutral comet assay (r=0.99; P<0.005). Frozen-thawed spermatozoa of each species were also incubated at 37°C for 5h and the within- and between-species variation of SDF, as an indicator of sperm DNA longevity, examined. The dynamic assessment of SDF using the SCDt revealed species and individual bird (rooster and turkey) differences in sperm DNA longevity.

Phylogeography of the iconic Australian red-tailed black-cockatoo (Calyptorhynchus banksii) and implications for its conservation.

Advances in sequencing technologies have revolutionized wildlife conservation genetics. Analysis of genomic data sets can provide high-resolution estimates of genetic structure, genetic diversity, gene flow, and evolutionary history. These data can be used to characterize conservation units and to effectively manage the genetic health of species in a broad evolutionary context. Here we utilize thousands of genome-wide single-nucleotide polymorphisms (SNPs) and mitochondrial DNA to provide the first genetic assessment of the Australian red-tailed black-cockatoo (Calyptorhynchus banksii), a widespread bird species comprising populations of varying conservation concern. We identified five evolutionarily significant units, which are estimated to have diverged during the Pleistocene. These units are only partially congruent with the existing morphology-based subspecies taxonomy. Genetic clusters inferred from mitochondrial DNA differed from those based on SNPs and were less resolved. Our study has a range of conservation and taxonomic implications for this species. In particular, we provide advice on the potential genetic rescue of the Endangered and restricted-range subspecies C. b. graptogyne, and propose that the western C. b. samueli population is diagnosable as a separate subspecies. The results of our study highlight the utility of considering the phylogeographic relationships inferred from genome-wide SNPs when characterizing conservation units and management priorities, which is particularly relevant as genomic data sets become increasingly accessible.

Museum specimens provide reliable SNP data for population genomic analysis of a widely distributed but threatened cockatoo species.

Natural history museums harbour a plethora of biological specimens which are of potential use in population and conservation genetic studies. Although technical advancements in museum genomics have enabled genome-wide markers to be generated from aged museum specimens, the suitability of these data for robust biological inference is not well characterized. The aim of this study was to test the utility of museum specimens in population and conservation genomics by assessing the biological and technical validity of single nucleotide polymorphism (SNP) data derived from such samples. To achieve this, we generated thousands of SNPs from 47 red-tailed black cockatoo (Calyptorhychus banksii) traditional museum samples (i.e. samples that were not collected with the primary intent of DNA analysis) and 113 fresh tissue samples (cryopreserved liver/muscle) using a restriction site-associated DNA marker approach (DArTseq™ ). Thousands of SNPs were successfully generated from most of the traditional museum samples (with a mean age of 44 years, ranging from 5 to 123 years), although 38% did not provide useful data. These SNPs exhibited higher error rates and contained significantly more missing data compared with SNPs from fresh tissue samples, likely due to considerable DNA fragmentation. However, based on simulation results, the level of genotyping error had a negligible effect on inference of population structure in this species. We did identify a bias towards low diversity SNPs in older samples that appears to compromise temporal inferences of genetic diversity. This study demonstrates the utility of a RADseq-based method to produce reliable genome-wide SNP data from traditional museum specimens.

Reference genes for quantitative gene expression studies in multiple avian species.

Quantitative real-time PCR (qPCR) rapidly and reliably quantifies gene expression levels across different experimental conditions. Selection of suitable reference genes is essential for meaningful normalization and thus correct interpretation of data. In recent years, an increasing number of avian species other than the chicken has been investigated molecularly, highlighting the need for an experimentally validated pan-avian primer set for reference genes. Here we report testing a set for 14 candidate reference genes (18S, ABL, GAPDH, GUSB, HMBS, HPRT, PGK1, RPL13, RPL19, RPS7, SDHA, TFRC, VIM, YWHAZ) on different tissues of the mallard (Anas platyrhynchos), domestic chicken (Gallus gallus domesticus), common crane (Grus grus), white-tailed eagle (Haliaeetus albicilla), domestic turkey (Meleagris gallopavo f. domestica), cockatiel (Nymphicus hollandicus), Humboldt penguin (Sphenicus humboldti), ostrich (Struthio camelus) and zebra finch (Taeniopygia guttata), spanning a broad range of the phylogenetic tree of birds. Primer pairs for six to 11 genes were successfully established for each of the nine species. As a proof of principle, we analyzed expression levels of 10 candidate reference genes as well as FOXP2 and the immediate early genes, EGR1 and CFOS, known to be rapidly induced by singing in the avian basal ganglia. We extracted RNA from microbiopsies of the striatal song nucleus Area X of adult male zebra finches after they had sang or remained silent. Using three different statistical algorithms, we identified five genes (18S, PGK1, RPS7, TFRC, YWHAZ) that were stably expressed within each group and also between the singing and silent conditions, establishing them as suitable reference genes. In conclusion, the newly developed pan-avian primer set allows accurate normalization and quantification of gene expression levels in multiple avian species.

Identification of protected avian species using a single feather barb.

We report on the unambiguous identification of protected avian species from as little as one barb of a feather. Many avian species are protected by international agreements and national legislation, yet they are traded illegally because of their high value. Two sections of the avian mitochondrial genome were chosen to identify bird species, being a 561-bp section of ND2 gene and a 921-bp section of the ND5 gene. Two different DNA extraction methods were compared for their ability to reliably isolate sufficient DNA to be detected in a subsequent PCR. Using a commercial kit supplied by QIAGEN, a complete sequence was obtained from one barb for the ND2 gene, whereas two barbs were required to reliably sequence the 921-bp section of the ND5 gene. The process worked on all species tested using feathers from archival museum specimens, resulted in minimal damage to the specimen and can readily be adopted by a forensic science laboratory.

Egg forensics: an appraisal of DNA sequencing to assist in species identification of illegally smuggled eggs.

Psittaciformes (parrots and cockatoos) are charismatic birds, their plumage and capacity for learning make them highly sought after pets. The illegal trade in parrots and cockatoos poses a serious threat to the viability of native populations; in addition, species transported to non-endemic areas may potentially vector disease and genetically 'pollute' local native avifauna. To reduce the logistical difficulties associated with trafficking live birds, smugglers often transport eggs. This creates a problem for authorities in elucidating accurate species identification without the laborious task of incubation and hand rearing until a morphological identification can be made. Here, we use 99 avian eggs seized from carriers coming into and within Australia, as a result of suspected illegal trade. We investigate and evaluate the use of mitochondrial DNA (mtDNA) to accurately identify eggs to family, genus or species level. However, Identification of a species based on percentage mtDNA similarities is difficult without good representations of the inter- and intra-levels of species variation. Based on the available reference database, we were able to identify 52% of the eggs to species level. Of those, 10 species from eight genera were detected, all of which belong to the parrot (Psittacidae) and cockatoo (Cacatuidae) families. Of the remaining 48%, a further 36% of eggs were identified to genus level, and 12% identified to family level using our assignment criteria. Clearly the lack of validated DNA reference sequences is hindering our ability to accurately assign a species identity, and accordingly, we advocate that more attention needs to be paid to establishing validated, multi locus mtDNA reference databases for exotic birds that can both assist in genetic identifications and withstand legal scrutiny.

Application of STR markers in wildlife forensic casework involving Australian black-cockatoos (Calyptorhynchus spp.).

Parrots and cockatoos are highly prized aviary birds and the demands for such species has fuelled their illegal trade and harvest from the wild. Here we report on three forensic case studies involving black-cockatoos (Calyptorhynchus spp.) endemic to Australia. These cases involve suspected poaching and illegal killing of endangered red- and white-tailed black-cockatoos. Through the prior development of 20 polymorphic microsatellite loci and population databases for white- and red-tailed black-cockatoos, the tools are available to conduct high-resolution paternity and individual identity testing. In one case, we matched a red-tailed black-cockatoo nestling to a tree hollow from which it was poached through the use of DNA from eggshell recovered from the nest. For the second case, we utilized our provenance population database (nest sites), and identified the kinship and geographic origin of a white-tailed black-cockatoo, which was illegally harvested from the wild. The third case determined the number individual white-tailed black-cockatoos allegedly shot at a fruit grower's orchard from body part remains. These genetic investigations highlight the significance and statistical confidence of DNA profiling and associated databases for endangered taxa, such as exotic birds. Our cockatoo population databases are the first of their kind in Australia, and demonstrate the efficacy of such approaches to identify such illegal activity. With a robust set of genetic markers and methodologies in place, we aim to broaden our population databases to include other cockatoo species of conservation concern.

The evolutionary history of cockatoos (Aves: Psittaciformes: Cacatuidae).

Cockatoos are the distinctive family Cacatuidae, a major lineage of the order of parrots (Psittaciformes) and distributed throughout the Australasian region of the world. However, the evolutionary history of cockatoos is not well understood. We investigated the phylogeny of cockatoos based on three mitochondrial and three nuclear DNA genes obtained from 16 of 21 species of Cacatuidae. In addition, five novel mitochondrial genomes were used to estimate time of divergence and our estimates indicate Cacatuidae diverged from Psittacidae approximately 40.7 million years ago (95% CI 51.6-30.3 Ma) during the Eocene. Our data shows Cacatuidae began to diversify approximately 27.9 Ma (95% CI 38.1-18.3 Ma) during the Oligocene. The early to middle Miocene (20-10 Ma) was a significant period in the evolution of modern Australian environments and vegetation, in which a transformation from mainly mesic to xeric habitats (e.g., fire-adapted sclerophyll vegetation and grasslands) occurred. We hypothesize that this environmental transformation was a driving force behind the diversification of cockatoos. A detailed multi-locus molecular phylogeny enabled us to resolve the phylogenetic placements of the Palm Cockatoo (Probosciger aterrimus), Galah (Eolophus roseicapillus), Gang-gang Cockatoo (Callocephalon fimbriatum) and Cockatiel (Nymphicus hollandicus), which have historically been difficult to place within Cacatuidae. When the molecular evidence is analysed in concert with morphology, it is clear that many of the cockatoo species' diagnostic phenotypic traits such as plumage colour, body size, wing shape and bill morphology have evolved in parallel or convergently across lineages.

The use of DNA identification in prosecuting wildlife-traffickers in Australia: do the penalties fit the crimes?

The use of genetic identification techniques in wildlife forensic investigations has increased significantly in recent years. The utilization of DNA is especially important when species identification using other methods are inconclusive. Australia has strict laws against illegal importation of wildlife as well as laws to protect its unique biodiversity from pests and diseases of quarantine concern. Two separate case studies in which genetic identification was essential for species identification are presented-the first involved illegally held shark fins, the second illegally imported live bird eggs. In the latter case genetic identification enabled charges to be laid for illegal importation of CITES Appendix I species. Australian laws allow for some of the highest penalties for illegal trade of wildlife compared to other countries, however only a fraction of cases are prosecuted and penalties applied to date have been lower than the maximum permitted. Both of the reported cases resulted in fines, and one in imprisonment of the offender, which provides a persuasive precedent for future prosecutions.