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Research


Research at Avian Biotech is directed towards a better understanding of some fundamental aspects of the biology and evolution of birds. In the past the most intensely investigated subjects have involved:

a) The structure and evolution of the avian W- and Z-sex chromosomes, especially as this relates to molecular, DNA-based sex identification of birds.

b) The phylogeny and the evolution of the Psittaciformes (parrots, parakeets) as derived from the sequences of selected sex-chromosomal DNA sequence elements.

More recently research has been directed towards

c) A better understanding of the mode of action of certain avian viruses, most notably the virus causing Psittacine Beak and Feather disease.


About Dr. Siwo de Kloet

RECENT PUBLICATIONS:

Siwo R. de Kloet, Dorrestein GM.
Presence of avian bornavirus RNA and anti-avian bornavirus antibodies in apparently healthy macaws.
Avian Diseases 2009 Dec;53(4):568-73.
ABSTRACT: Recently a novel avian bornavirus has been described that has been suggested to be the possible etiological agent for proventricular dilatation disease or macaw wasting disease. This article describes two macaws that shed avian bornaviral RNA sequences and demonstrated anti-avian bornavirus antibodies as revealed by reverse transcriptase polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and Western blot, yet are free of outward clinical signs of the disease.

Siwo R. de Kloet
Sequence analysis of four double-stranded RNA genomic segments reveals an orthoreovirus with a unique genotype infecting psittaciformes.
Avian Diseases 2008 Sep;52(3):480-6.
ABSTRACT: This paper describes the characterization of four double-stranded ribonucleic acid segments, S1, S2, S3, and S4, of a newly identified pathogenic reovirus from parrots. The four segments share a unique 5' terminus GCUUUUC. The amino-acid sequences of the conserved sigma A and sigma NS proteins show less than 60% sequence similarity, whereas those of the outer capsid proteins sigma B and sigma C have at most 47% sequence similarity to their counterparts in other bird or bat reoviruses. In a phylogenetic analysis of the amino-acid sequences, the proteins coded for by the S1 segment, P10, P17, and sigma C, group with their homologous proteins in other avian reoviruses, whereas the major capsid protein, sigma B, and the nonstructural protein, sigma NS, show more sequence similarity to their bat reoviral counterparts. The phylogenetic relationship of sigma A with the homologous avian and bat sequences is unresolved. The possibility that the parrot reovirus has evolved from an ancestral, more batlike reovirus is discussed. It is proposed to designate this unique virus as PsRV.

Rolf S. de Kloet, Siwo R. de Kloet
The evolution of the spindlin gene in birds: sequence analysis of an intron of the spindlin W and Z gene reveals four major divisions of the Psittaciformes.
Mol Phylogenet Evol. 2005 Sep;36(3):706-21.
ABSTRACT:The Psittaciformes (parrots, parakeets) are among the most widely held captive birds. Yet, their evolution and their phylogenetic relationships have been relatively little studied. This paper describes the phylogenetic relationships between a number of Psittaciformes as derived from the sequences of the third intron of the Z-chromosomal and W-chromosomal spindlin genes. The Z-chromosomal sequences of the kakapo (Strigops habroptilus), the kea (Nestor notabilis), and the kaka (Nestor meridionalis) from New Zealand form a cluster which is the sister group to all other Psittaciformes. The results show further that the Z-chromosomal sequences of the other species can be divided into two groups based on the occurrence of a sequence element ACCCT. The group with the insert (A) is mainly from species with an Australasian geographical distribution and includes such species as the Lories (Lorius, etc.), the budgerigar (Melospittacus undulatus), and the rosellas (Platycercus). It also includes the African lovebirds (Agapornidae), which are the only representative of group A outside Australasia. Group B, without the insert, includes the neotropical parrots and parakeets such as the amazons (Amazona, etc.), the macaws (Ara, etc.), and the conures (Aratinga, etc.), the Australian Cacatuini and the African species such as the African grey parrot (Psittacus erithacus) as well as Coracopsis vasa from Madagascar and Psittrichas fulgidus from New Guinea. The W-chromosomal sequence data show that another division of the Psittacidae is found in the replacement of a pyrimidine-rich segment occurring in many non-psittacines as well as the kakapo (S. habroptilus), the kea (N. notabilis), the kaka (N. meridionalis), and the Cacatuini by a microsatellite consisting of a variable number of TATTA monomers in the other Psittaciformes. The results support a Gondwanan origin of the Psittaciformes and the suggestion that paleogeographic events were a major force in psittacine divergence.

Edwin de Kloet, Siwo R. de Kloet
Analysis of the Beak and Feather Disease Viral Genome Indicates the Existence of Several Genotypes Which Have a Complex Psittacine Host Specificity
Arch Virol. 2004 Dec;149(12):2393-412. Epub 2004 Jul 15.
ABSTRACT: A study was made of the phylogenetic relationships between fifteen complete nucleotide sequences as well as 43 nucleotide sequences of the putative coat protein gene of different strains of Beak and Feather Disease virus obtained from 39 individuals of 16 psittacine species. The species included among others, cockatoos (Cacatuini), African grey parrots (Psittacus erithacus) and peach-faced lovebirds (Agapornis roseicollis), which were infected at different geographical locations, within and outside Australia, the native origin of the virus. The derived amino acid sequences of the putative coat protein were highly diverse, with differences between some strains amounting to 50 of the 250 amino acids. Phylogenetic analysis demonstrated that the putative coat gene sequences form six clusters which show a varying degree of psittacine species specificity. Most, but not all strains infecting African grey parrots formed a single cluster as did the strains infecting the cockatoos. Strains infecting the lovebirds clustered with those infecting such Australasian species as Eclectus roratus, Psittacula kramerii and Psephotus haematogaster. Although individual birds included in this study were, where studied, often infected by closely related strains, infection by highly diverged trains was also detected. The possible relationship between BFD viral strains and clinical disease signs is discussed.

Rolf S. de Kloet, Siwo R. de Kloet
Independent cessation of recombination of sex chromosomes at the spindlin locus in neognathous birds and tinamous, a palaeognathous avian family.

Genetica 199: 333-342, 2003.
ABSTRACT: Tinamous (Aves, Palaeognathae, Tinamiformes) are primitive birds, generally considered to be the sister group to the ratites. Tinamous possess a W sex-chromosome, intermediate in heterochromatization between the largely euchromatic W chromosome of the ratites and the highly condensed W chromosome of the neognathous birds. Of the four genes which are known to have diverged copies on the neognathous avian W and Z chromosome (ATP5A1, CHD1, PKC and SPIN) only the spindlin gene has W- and Z-chromosomal forms in the tinamiformes. This paper describes experiments which show that the sequences of these forms are more similar to each other and to the homologous undifferentiated spindlin gene sequences in the ratite genome than to the W or Z forms of the spindlin gene in other, neognathous species. This suggests that cessation of recombination at the spindlin locus of the ancestral W and Z chromosomes of the tinamiformes and the neagnathous avian species were independent events.

de Kloet, SR (2002)
Molecular sex identification of tinamous with PCR using primers derived from the spindlin gene.

Molecular Ecology Notes 2:465 - 466.

ABSTRACT: This paper describes results which show that the spindlin gene has different forms on the tinamid W and Z chromosome, providing a sensitive and accurate procedure for a molecular, PCR-based, procedure for sex-identification of tinamous.

de Kloet, SR. (2001c)
A repetitive DNA sequence on the W chromosome of owls (Strigiformes) with sequence similarity to the chicken W chromosomal repeat.

The Auk, submitted

de Kloet, SR (2001b).
Development of a CAPS (Cleaved Amplified Polymorphics Sequence) assay for sex identification of the emu (Dromaius novaehollandiae)
.
Molecular Ecology Letters 1: 273 - 275 .

ABSTRACT: Polymerase chain rection (PCR) based procedures that have been used to identify the sex of most birds cannot be used in ratites. This paper described the identification of a female (W-chromosomal) specific randomly amplified polymorphic (RAPD) 1.3 kb DNA fragment (ESEXW) in the emu (Dromaius novaehollandiae). Southern blot experiments and sequence analysis revealed that a related (96% similarity) sequence exists of the emu Z-chromosome (ESEXZ). The sequences of ESEXW and ESEXZ have been used for the development of a two-primer CAPS (cleaved amplified polymorphic sequence) assay for reliable sex identification of the emu.

de Kloet, SR. (2001a)
Loss of the gene of the alpha subunit of ATP synthase (ATP5A1) from the W chromosome of the African grey parrot (Psittacus erithacus)
.
Journal of Molecular Evolution 53: 135 - 143.

ABSTRACT: This study describes the results of an analysis using Southern blotting, the polymerase chain reaction, and sequencing which shows that the African grey parrot (Psittacus erithacus) lacks the W-chromosomal gene for the alpha subunit of mitochondrial ATP synthase (ATP5A1W). Additional evidence shows that in other psittacines a fragment of the ATP5A1W gene contains five times as many nonsynonymous nucleotide replacements as the homologous fragment of the Z gene. Therefore, whereas in these other psittacines the corresponding ATP5A1Z protein fragment is highly conserved and varies by only a few, moderately conservative amino acid substitutions, the homologous ATP5A1W fragments contain a considerable number of, sometimes highly nonconservative, amino acid replacements. In one of these species, the ringneck parakeet (Psittacula krameri), the ATP5A1W gene is present in an inactive form because of the presence of a nonsense codon. Other changes, possibly leading to an inactive ATP5A1W gene product, involve the substitution of arginine residues by cysteine in the ATP5A1W protein of the mitred conure (Aratinga mitrata) and the blue and gold macaw (Ara ararauna). The data suggest also that although the divergence of the psittacine ATP5A1W and ATP5A1Z genes preceded the origin of the psittacidae, this divergence occurred independently of a similar process in the myna (Gracula religiosa), the outgroup used in this study.

 

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