H9N2 influenza viruses possessing H5N1-like internal genomes continue to circulate in poultry in southeastern China

The transmission of H9N2 influenza viruses to humans and the realization that the A/Hong Kong/156/97-like (H5N1) (abbreviated HK/156/97) genome complex may be present in H9N2 viruses in southeastern China necessitated a study of the distribution and characterization of H9N2 viruses in poultry in the Hong Kong SAR in 1999. Serological studies indicated that H9N2 influenza viruses had infected a high proportion of chickens and other land-based birds (pigeon, pheasant, quail, guinea fowl, and chukka) from southeastern China. Two lineages of H9N2 influenza viruses present in the live-poultry markets were represented by A/Quail/Hong Kong/G1/97 (Qa/HK/G1/97)-like and A/Duck/Hong Kong/Y280/97 (Dk/HK/Y280/97)-like viruses. Up to 16% of cages of quail in the poultry markets contained Qa/HK/G1/97-like viruses, while about 5% of cages of other land-based birds were infected with Dk/HK/Y280/97-like viruses. No reassortant between the two H9N2 virus lineages was detected despite their cocirculation in the poultry markets. Reassortant viruses represented by A/Chicken/Hong Kong/G9/97 (H9N2) were the major H9N2 influenza viruses circulating in the Hong Kong markets in 1997 but have not been detected since the chicken slaughter in 1997. The Qa/HK/G1/97-like viruses were frequently isolated from quail, while Dk/HK/Y280/97-like viruses were predominately associated with chickens. The Qa/HK/G1/97-like viruses were evolving relatively rapidly, especially in their PB2, HA, NP, and NA genes, suggesting that they are in the process of adapting to a new host. Experimental studies showed that both H9N2 lineages were primarily spread by the aerosol route and that neither quail nor chickens showed evidence of disease. The high prevalence of quail infected with Qa/HK/G1/97-like virus that contains six gene segments genetically highly related to HK/156/97 (H5N1) virus emphasizes the need for surveillance of mammals including humans.     

Reticuloendotheliosis Virus Nucleic Acid Sequences in Cellular DNA

Reticuloendotheliosis virus 60S RNA labeled with (125)I, or reticuloendotheliosis virus complementary DNA labeled with (3)H, were hybridized to DNAs from infected chicken and pheasant cells. Most of the sequences of the viral RNA were found in the infected cell DNAs. The reticuloendotheliosis viruses, therefore, replicate through a DNA intermediate. The same labeled nucleic acids were hybridized to DNA of uninfected chicken, pheasant, quail, turkey, and duck. About 10% of the sequences of reticuloendotheliosis virus RNA were present in the DNA of uninfected chicken, pheasant, quail, and turkey. None were detected in DNA of duck. The specificity of the hybridization was shown by competition between unlabeled and (125)I-labeled viral RNAs and by determination of melting temperatures. In contrast, (125)I-labeled RNA of Rous-associated virus-O, an avian leukosis-sarcoma virus, hybridized 55% to DNA of uninfected chicken, 20% to DNA of uninfected pheasant, 15% to DNA of uninfected quail, 10% to DNA of uninfected turkey, and less than 1% to DNA of uninfected duck.     

Induction of cytochrome P4501A by highly purified hexachlorobenzene in primary cultures of ring-necked pheasant and Japanese quail embryo hepatocytes

Primary cultures of ring-necked pheasant (Phasianus colchicus) and Japanese quail (Coturnix japonica) embryo hepatocytes were used to compare the potencies of highly purified hexachlorobenzne (HCB-P), reagent-grade HCB (RG-HCB) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as inducers of ethoxyresorufin O-deethylase (EROD) activity, cytochrome P4501A (CYP1A4) messenger ribonucleic acid (mRNA) and CYP1A5 mRNA. HCB-P, RG-HCB and TCDD all induced EROD activity and up-regulated CYP1A4 and CYP1A5 mRNA. Induction was not caused by contamination of HCB with polychlorinated dibenzo-p-dioxins, dibenzofurans or biphenyls. Based upon a comparison of the EC(50) and EC(threshold) values for EROD and CYP1A4/5 concentration-response curves, the potency of HCB relative to TCDD was 0.001 in ring-necked pheasant and 0.01 in Japanese quail embryo hepatocytes. Differences in species sensitivity to HCB were found to be mainly dictated by differences in species sensitivity to TCDD rather than differences in the absolute potency of HCB. Consequently, ring-necked pheasant and Japanese quail embryo hepatocytes were found to be equally sensitive to HCB exposure. Species sensitivity comparisons were also made with chicken (Gallus gallus domesticus) and revealed that chicken embryo hepatocytes were less responsive to EROD induction (lower maximal response) by HCB compared to the embryo hepatocytes of pheasant and quail.     

The phylogenetic relationships of immunoglobulin allotypes and 7S immunoglobulin isotypes of chickens and other phasianoids (turkey,pheasant, quail)

Pheasants, quail and turkeys from different geographical locations were surveyed for the presence of eight 7S Ig and four IgM chicken allotypes. No IgM and only two 7S Ig allotypes were detected. Chicken 7S Ig allotypic specificity G-1.7 cross-reacted with pheasant and turkey isotypic specificities, and was absent in quail. The other determinant (G-1.9) cross-reacted with an allotype found only in turkeys and golden pheasants. These data suggest that G-1.7 and G-1.9 are probably phylogenetically ancient determinants and that polymorphism of chicken immunoglobulins arose after divergence of chickens from other phasianoid birds. Based on the allotypic and isotypic analysis of the 7S Ig antigenic determinants, turkey 7S Ig was as closely related to chicken 7S Ig as was pheasant 7S Ig. Jungle fowl, the ancestor of chickens, had most of the chicken 7S Ig and IgM allotypes present as polymorphic markers.     

Avian smooth muscle: Morphometric analysis and comparison of different types

Summary Smooth muscle from the pigeon gizzard and intestine, and quail gizzard were investigated using ultrastructural morphometric analysis and compared on the basis of volume percent mitochondria, dense bodies, sarcoplasmic reticulum, number of mitochondria/m², and fiber diameter. One-way analysis of variance tests showed significant differences in (1) volume percent mitochondria between pigeon intestine and quail gizzard and between pigeon and quail gizzards; (2) mitochondrial number between all three muscles; and (3) fiber diameter between pigeon gizzard and intestine and pigeon intestine and quail gizzard.     

The phylogenetic relationships of immunoglobulin allotypes and 7S immunoglobulin isotypes of chickens and other phasianoids (turkey, pheasant, quail)

Pheasants, quail and turkeys from different geographical locations were surveyed for the presence of eight 7S Ig and four IgM chicken allotypes. No IgM and only two 7S Ig allotypes were detected. Chicken 7S Ig allotypic specificity G-1.7 cross-reacted with pheasant and turkey isotypic specificities, and was absent in quail. The other determinant (G-1.9) cross-reacted with an allotype found only in turkeys and golden pheasants. These data suggest that G-1.7 and G-1.9 are probably phylogenetically ancient determinants and that polymorphism of chicken immunoglobulins arose after divergence of chickens from other phasianoid birds. Based on the allotypic and isotypic analysis of the 7S Ig antigenic determinants, turkey 7S Ig was as closely related to chicken 7S Ig as was pheasant 7S Ig. Jungle fowl, the ancestor of chickens, had most of the chicken 7S Ig and IgM allotypes present as polymorphic markers.     

Polymorphic microsatellites in Buff-throated partridge developed by cross-species amplification

Microsatellite loci for the buff-throated partridge (Tetraophasis szechenyii), an endemic pheasant species of China, are here described for the first time. Twenty-five microsatellite markers from chicken and Japanese quail were tested on buff-throated partridge DNA by means of cross-amplification. Twenty (80%) primers yielded specific products and polymorphisms were tested in a wild population of buff-throated partridge. Twelve (48%) proved to be polymorphic with an average of two alleles per locus. Current results of buff-throated partridge microsatellites loci could be employed in population genetic studies and on other endangered pheasant species.     

Karyotypic evolution in the Galliformes: an examination of the process of karyotypic evolution by comparison of the molecular cytogenetic findings with the molecular phylogeny

To define the process of karyotypic evolution in the Galliformes on a molecular basis, we conducted genome-wide comparative chromosome painting for eight species, i.e. silver pheasant (Lophura nycthemera), Lady Amherst's pheasant (Chrysolophus amherstiae), ring-necked pheasant (Phasianus colchicus), turkey (Meleagris gallopavo), Western capercaillie (Tetrao urogallus), Chinese bamboo-partridge (Bambusicola thoracica) and common peafowl (Pavo cristatus) of the Phasianidae, and plain chachalaca (Ortalis vetula) of the Cracidae, with chicken DNA probes of chromosomes 1-9 and Z. Including our previous data from five other species, chicken (Gallus gallus), Japanese quail (Coturnix japonica) and blue-breasted quail (Coturnix chinensis) of the Phasianidae, guinea fowl (Numida meleagris) of the Numididae and California quail (Callipepla californica) of the Odontophoridae, we represented the evolutionary changes of karyotypes in the 13 species of the Galliformes. In addition, we compared the cytogenetic data with the molecular phylogeny of the 13 species constructed with the nucleotide sequences of the mitochondrial cytochrome b gene, and discussed the process of karyotypic evolution in the Galliformes. Comparative chromosome painting confirmed the previous data on chromosome rearrangements obtained by G-banding analysis, and identified several novel chromosome rearrangements. The process of the evolutionary changes of macrochromosomes in the 13 species was in good accordance with the molecular phylogeny, and the ancestral karyotype of the Galliformes is represented.     

Comparative analysis of NADPH-diaphorase positive neurons in the rat, rabbit and pheasant thoracic spinal cord. A histochemical study.

The distribution of NADPH-diaphorase (NADPH-d) activity was investigated and compared in the rat, rabbit and pheasant thoracic spinal cord. The investigation of all spinal cord regions (laminae) in three experimental species revealed marked differences in the distribution of NADPH-d activity. Cross sectional analysis of the spinal cord of the rat, rabbit and pheasant confirmed differences in the shape of the gray matter in all examined species. More detailed investigation of Rexed's laminas showed similar distribution of NADPH-d activity in the spinal cord of the rat and rabbit, which were different when compared with the spinal cord of the pheasant. Ventral horn of the rat and rabbit showed no labelling whereas in pheasant this area possessed a number of scattered, intensively stained neurons. In the location of autonomic preganglionic neurons, differences were found as well. In the rat there was seen a number of densely packed, clearly dark blue coloured neurons. Similarly, these neurons were present in the rabbit spinal cord but they were less numerous. No staining was found in this region of pheasant. Pericentral area (lamina X) and intermediate zone (laminaVII) revealed the presence of NADPH-d positive neurons in all examined species although they differed in number and shape of their bodies. The dorsal horn showed the presence of NADPH-d staining in all three animals but its distribution was different in medio-lateral direction. It can be suggested that observed differencies in the presence and distribution of NADPH-d activity across the examined species may reflect different fylogenetic development.     

Cloning and sequencing of quail and pigeon prion genes

Quail and pigeon PrP genes were cloned and sequenced. Like mammalian PrP genes, quail and pigeon genes are encoded by a single exon of a single copy gene in the genome. All of the structural features of mammalian PrP genes were found in the quail and pigeon PrP gene. Compared with the nucleotide sequences of mammalian PrP, they display generally 30% similarity. When compared with chicken PrP's DNA sequence, they show a higher homology (90%), and an even higher homology (99%) when compared to each other. A phylogenetic tree was constructed to trace the evolution of the prion gene in animals.     

Is the "mammalian" brown fat-specific mitochondrial uncoupling protein present in adipose tissues of birds?

1. Mitochondria were isolated from the furcular, subcutaneous, abdominal, nape and lateral adipose tissue depots of five species of bird (pheasant, Japanese quail, pigeon, house sparrow and great tit) acclimatized to the Northern winter. 2. Mitochondrial proteins were separated by SDS-polyacrylamide gel electrophoresis, blotted onto nitrocellulose membranes, and probed for the presence of the 32,000-33,000 Mr uncoupling protein characteristic of "mammalian" brown adipose tissue, using an anti-(ground squirrel uncoupling protein)serum. 3. Immunoreactivity consistent with uncoupling protein was not detected in mitochondria from any of the avian adipose tissues. Immunoreactivity was, however, evident in mitochondria from perirenal or interscapular adipose tissue from a range of mammals--rats, mice, golden hamsters, Orkney voles, wood mice, pipistrelle bats, wood lemmings, and newborn lambs, cattle, reindeer and red deer. 4. These results provide biochemical evidence that "mammalian-like" thermogenic brown adipose tissue is absent from avian species; adipose tissues in birds appear to be functionally "white".     

Erythrocytes of Japanese Quail for Hemagglutination by Rubella Virus

Erythrocytes (RBC) of adult Japanese quail, Coturnix coturnix japonica, were found to be as sensitive as day-old chick RBC for rubella virus hemagglutination (HA). Various factors involved in the HA were studied with quail RBC as well as with adult pigeon and day-old chick RBC. Pigeon RBC, unlike chick and quail RBC, tended to show, without hemagglutinin, a pattern of sedimented RBC resembling the HA pattern by the virus at 4 C, to a lesser extent at room temperature, in 0.85% NaCl solution buffered with m/100 phosphate (PBS), and were prevented from doing so by addition of a small amount of bovine plasma albumin to the diluent. A small amount (about 10^?3 m ) of CaCL₂ in PBS gave higher HA titers. The HA titer was higher at 4 C than at room temperature and much reduced at 36 C with chick and quail RBC. With pigeon RBC, addition of bovine plasma albumin to the diluent tended to reduce HA titers. The HA titer was highest at pH 5.8 to 6.8, and was inversely proportional to the RBC concentration. Under the conditions established on the basis of these findings, chick and quail RBC gave similar HA titers, but pigeon RBC gave consistently higher titers. However, these types of RBC gave no significant difference in HA-inhibiting antibody titer when 4 units of hemagglutinin as determined with the homologous RBC was used.     

CLONING AND SEQUENCING OF QUAIL AND PIGEON PRION GENES

ABSTRACT

Quail and pigeon PrP genes were cloned and sequenced. Like mammalianPrP genes, quail and pigeon genes are encoded by a single exon of a single copy gene in the genome. All of the structural features of mammalian PrP genes were found in the quail and pigeon PrP gene. Compared with the nucleotide sequences of mammalian PrP, they display generally 30% similarity. When compared with chicken PrP's DNA sequence, they show a higher homology (90%), and an even higher homology (99%) when compared to each other. A phylogenetic tree was constructed to trace the evolution of the prion gene in animals.     

Complete nucleotide sequence of the Coturnix chinensis (blue-breasted quail) mitochondrial genome and a phylogenetic analysis with related species

Coturnix chinensis (blue-breasted quail) has been classically grouped in Galliformes Phasianidae Coturnix, based on morphologic features and biochemical evidence. Since the blue-breasted quail has the smallest body size among the species of Galliformes, in addition to a short generation time and an excellent reproductive performance, it is a possible model fowl for breeding and physiological studies of the Coturnix japonica (Japanese quail) and Gallus gallus domesticus (chicken), which are classified in the same family as blue-breasted quail. However, since its phylogenetic position in the family Phasianidae has not been determined conclusively, the sequence of the entire blue-breasted quail mitochondria (mt) genome was obtained to provide genetic information for phylogenetic analysis in the present study. The blue-breasted quail mtDNA was found to be a circular DNA of 16,687 base pairs (bp) with the same genomic structure as the mtDNAs of Japanese quail and chicken, though it is smaller than Japanese quail and chicken mtDNAs by 10 bp and 88 bp, respectively. The sequence identity of all mitochondrial genes, including those for 12S and 16S ribosomal RNAs, between blue-breasted quail and Japanese quail ranged from 84.5% to 93.5%; between blue-breasted quail and chicken, sequence identity ranged from 78.0% to 89.6%. In order to obtain information on the phylogenetic position of blue-breasted quail in Galliformes Phasianidae, the 2,184 bp sequence comprising NADH dehydrogenase subunit 2 and cytochrome b genes available for eight species in Galliformes [Japanese quail, chicken, Gallus varius (green junglefowl), Bambusicola thoracica (Chinese bamboo partridge), Pavo cristatus (Indian peafowl), Perdix perdix (gray partridge), Phasianus colchicus (ring-neck pheasant), and Tympanchus phasianellus (sharp-tailed grouse)] together with that of Aythya americana (redhead) were examined using a maximum likelihood (ML) method. The ML analyses on the first/second codon positions, the third codon positions, and amino acid sequence consistently demonstrated that blue-breasted quail and Japanese quail are in the same phylogenetic cluster.     

Isolation of a 97-kb minimal essential MHC B locus from a new reverse-4D BAC library of the golden pheasant

The bacterial artificial chromosome (BAC) system is widely used in isolation of large genomic fragments of interest. Construction of a routine BAC library requires several months for picking clones and arraying BACs into superpools in order to employ 4D-PCR to screen positive BACs, which might be time-consuming and laborious. The major histocompatibility complex (MHC) is a cluster of genes involved in the vertebrate immune system, and the classical avian MHC-B locus is a minimal essential one, occupying a 100-kb genomic region. In this study, we constructed a more effective reverse-4D BAC library for the golden pheasant, which first creates sub-libraries and then only picks clones of positive sub-libraries, and identified several MHC clones within thirty days. The full sequencing of a 97-kb reverse-4D BAC demonstrated that the golden pheasant MHC-B locus contained 20 genes and showed good synteny with that of the chicken. The notable differences between these two species were the numbers of class II B loci and NK genes and the inversions of the TAPBP gene and the TAP1-TAP2 region. Furthermore, the inverse TAP2-TAP1 was unique in the golden pheasant in comparison with that of chicken, turkey, and quail. The newly defined genomic structure of the golden pheasant MHC will give an insight into the evolutionary history of the avian MHC.     

A comparison of the protective action of added egg yolks from five avian species to the cryopreservation of bull sperm

Cryopreservation of domestic animal sperm has been widely used for artificial insemination (AI), and egg yolk is one of the most commonly used cryoprotectants during the freezing-thawing process. The objectives of this study were to compare the effectiveness of egg yolk from five avian species (domestic chicken, domestic duck, domestic goose, Japanese quail or domestic pigeon) and to optimize the concentration of egg yolk on the cryopreservation of bull sperm in terms of frozen-thawed sperm progressive motility and viability. The results were two-fold. First, they showed that pigeon egg yolk provided the best cryoprotective effects on the cryopreservation of bull sperm, compared with egg yolk of chicken, quail, goose or duck. Second, the best concentration of pigeon egg yolk in extender was 20% during cryopreservation among five concentrations of 5, 10, 20, 30 or 40%. The results suggest that pigeon egg yolk could be used as an alternative to chicken egg yolk in extender but requires further testing in fertility trials.     

Evolution of the lumbo-sacral neural crest in the avian embryo: Origin and differentiation of the ganglionated nerve of Remak studied in interspecific quail-chick chimaerae

Summary Isotopic and isochronic transplantation of fragments of quail neural tube into chick demonstrates that neural and glial cells of the entire ganglion of Remak (RG) arise from the lumbo-sacral level of the neural crest.The ganglioblasts first accumulate in the mesorectum (stage 24 of Hamburger and Hamilton, in the chick and I8 of Zacchei in the quail) and subsequently migrate cranially.Histochemical studies have been carried out on the rectal and cloacal parts of the quail RG at various stages of development. Cholinesterase activity can be detected as soon as the primordium is in place and the intensity of the reaction increases rapidly. During morphogenesis of the cloacal region the RG and the pelvic plexus become intimately associated. Catecholamine-containing cells are found first in the pelvic plexus, then in the cloacal part of the RG. Fluorescent cells are often grouped close to blood vessels and associated with non-fluorescent ganglia. Cranial to the level of the bursa of Fabricius, the RG is composed only of non-fluorescent neurons whatever the developmental stage considered (up to 1 day after hatching).The developmental capabilities of the RG of the 5-day quail have been tested by transplanting various parts of the hind-gut with the dorsal mesentery onto the chorio-allantoic membrane. Catecholamine-containing cells develop only in grafts including the cloacal region.By using quail-chick chimaerae in which the RG belongs to the quail while mesentery and gut are of chick origin, it was possible to show that neurons which develop in the graft (i.e. in the absence of preganglionic innervation), send nerve fibres into the gut wall. Moreover some neuroblasts located in the primordium of the RG migrate into the gut wall and give rise to some enteric ganglion cells. The contribution of the lumbo-sacral neural crest to the enteric ganglia, by this route, is discussed.List of Abbreviations in Text FIF formol-induced fluorescence - H & H Hamburger and Hamilton - Z Zacchei - CAM chorio-allantoic membrane - SIF small intensely fluorescent (cells)     

The genesis and evolution of H9N2 influenza viruses in poultry from southern China, 2000 to 2005

H9N2 influenza viruses have become established in terrestrial poultry in different Asian countries over the last 2 decades. Our previous study demonstrated that quail harbor increasingly diverse novel H9N2 reassortants, including both Chicken/Beijing/1/94 (Ck/Bei-like) and Quail/Hong Kong/G1/97 (G1-like) viruses. However, since 1999, the genesis and evolution of H9N2 viruses in different types of poultry have not been investigated systematically. In the present study, H9N2 viruses isolated from chickens, ducks, and other minor poultry species were characterized genetically and antigenically. Our findings demonstrate that Ck/Bei-like H9N2 viruses have been introduced into many different types of poultry in southern China, including quail, partridges, chukar, pheasant, guinea fowl, and domestic ducks, while G1-like viruses were commonly detected in quail, less frequently detected in other minor poultry species, and not detected in chickens and ducks. Genetic analysis revealed 35 genotypes of H9N2 viruses, including 14 novel genotypes that have not been recognized before. Our results also suggested that two-way interspecies transmission exists between different types of poultry. Our study demonstrates that the long-term cocirculation of multiple virus lineages (e.g., H5N1 and H9N2 viruses) in different types of poultry has facilitated the frequent reassortment events that are mostly responsible for the current great genetic diversity in H9N2 and H5N1 influenza viruses in this region. This situation favors the emergence of influenza viruses with pandemic potential.     

Development and characterization of expressed sequence tags for the turkey (Meleagris gallopavo) genome and comparative sequence analysis with other birds

Twenty-one randomly selected clones from a turkey (Meleagris gallopavo) pituitary complementary DNA (cDNA) library were sequenced to develop expressed sequence tags (ESTs) for this economically important avian species whose genome is among the least understood. Primers specific for the ESTs were used to produce amplicons from the genomic DNA of turkey, chicken (Gallus gallus), guinea fowl (Numidia meleagris), pigeon (Columba domestica), and quail (Corturnix japonica). The amplicons were sequenced and analyzed for sequence variation within- and similarity among-species and with GenBank database sequences. The proportion of shared bases between the turkey sequence and the consensus sequence from each of the other species ranged from 72% to 93% between turkey and pigeon and quail and between turkey and chicken, respectively. The total number of single nucleotide polymorphisms (SNPs) observed ranged from 3 in quail to 18 in chicken out of 4898 and 5265 bases analyzed, respectively. The most frequent nucleotide variation observed was a C-->T transition. Linkage analysis of one such SNP in the backcross progeny of the East Lansing reference DNA panel, localized TUS0005, the chicken sequence derived from primers specific for turkey TUT2E EST, to chromosome 4. The ESTs reported, as well as the SNPs may provide a useful resource for ongoing efforts to develop high utility genome maps for the turkey and chicken. The primers described can also be used as a tool in future investigations directed at further understanding the biology of the guinea fowl, pigeon and quail and their relatedness to the turkey.