Isolation, characterization and cDNA sequencing of a Kazal family proteinase inhibitor from seminal plasma of turkey (Meleagris gallopavo)

The turkey reproductive tract and seminal plasma contain a serine proteinase inhibitor that seems to be unique for the reproductive tract. Our experimental objective was to isolate, characterize and cDNA sequence the Kazal family proteinase inhibitor from turkey seminal plasma and testis. Seminal plasma contains two forms of a Kazal family inhibitor: virgin (Ia) represented by an inhibitor of moderate electrophoretic migration rate (present also in the testis) and modified (Ib, a split peptide bond) represented by an inhibitor with a fast migration rate. The inhibitor from the seminal plasma was purified by affinity, ion-exchange and reverse phase chromatography. The testis inhibitor was purified by affinity and ion-exchange chromatography. N-terminal Edman sequencing of the two seminal plasma inhibitors and testis inhibitor were identical. This sequence was used to construct primers and obtain a cDNA sequence from the testis. Analysis of a cDNA sequence indicated that turkey proteinase inhibitor belongs to Kazal family inhibitors (pancreatic secretory trypsin inhibitors, mammalian acrosin inhibitors) and caltrin. The turkey seminal plasma Kazal inhibitor belongs to low molecular mass inhibitors and is characterized by a high value of the equilibrium association constant for inhibitor/trypsin complexes.     

FHF-2 in the turkey (Meleagris gallopavo)

A cDNA clone homologous to the fibroblast growth factor homologous factor (FHF-2) was isolated and sequenced from the turkey (Meleagris gallopavo). The DNA sequence of the turkey was almost identical to that of the chicken (99% similarity) differing at only 8 of 770 nucleotides in the coding region resulting in a single amino acid difference between these poultry species. The 3'UTR of the turkey FHF-2 gene was 445 nucleotides in length and included an imperfect CT microsatellite (ms) repeat. The sequence of the 3'UTR was amplified from genomic DNA of the chicken and found to be highly conserved differing at only three nucleotides when compared to the turkey. Length of the CT repeat was indifferent in a sample of 52 turkeys (monomorphic) however, the number of CT repeats was greater in the turkey than in the chicken. No inter-individual polymorphism was detected in multiple sequences of the 3'UTR of the FHF-2 gene in the turkey. Based on comparison of the turkey and chicken sequences, the mutation rate for coding and associated non-coding (3'UTR) regions of FHF-2 are approximately equal.     

In silco mapping of ESTs from the turkey (Meleagris gallopavo)

Sequence similarity was used to predict the position of expressed sequence tags (ESTs) in the genome of the turkey (Meleagris gallopavo). Turkey EST sequences were compared with the draft assembly of the chicken whole-genome sequence and the chicken EST database by BLASTN. Among the 877 ESTs examined, 788 had significant matches in the chicken genome sequence. Position of orthologous sequences in the chicken genome and the predicted position of the EST loci in the turkey genome are presented Genetic assignments suggest a high level of accuracy for the COMPASS predictions.     

Microsatellite loci for genetic mapping in the turkey (Meleagris gallopavo)

New microsatellite loci for the turkey (Meleagris gallopavo) were developed from two small insert DNA libraries. Polymorphism at these new loci was examined in domestic birds and two resource populations designed for genetic linkage mapping. The majority of loci (152 of 168) was polymorphic in domestic turkeys and informative in two mapping resource populations and thus will be useful for genetic linkage mapping.     

Genetic variation across the historical range of the wild turkey (Meleagris gallopavo)

Genetic differences within and among naturally occurring populations of wild turkeys (Meleagris gallopavo) were characterized across five subspecies' historical ranges using amplified fragment length polymorphism (AFLP) analysis, microsatellite loci and mitochondrial control region sequencing. Current subspecific designations based on morphological traits were generally supported by these analyses, with the exception of the eastern (M. g. silvestris) and Florida (M. g. osceola) subspecies, which consistently formed a single unit. The Gould's subspecies was both the most genetically divergent and the least genetically diverse of the subspecies. These genetic patterns were consistent with current and historical patterns of habitat continuity. Merriam's populations showed a positive association between genetic and geographical distance, Rio Grande populations showed a weaker association and the eastern populations showed none, suggesting differing demographic forces at work in these subspecies. We recommend managing turkeys to maintain subspecies integrity, while recognizing the importance of maintaining regional population structure that may reflect important adaptive variation.     

Preliminary linkage map of the turkey (Meleagris gallopavo) based on microsatellite markers

The turkey is an agriculturally important species for which, until now, there is no published genetic linkage map based on microsatellite markers--still the markers most used in the chicken and other farm animals. In order to increase the number of markers on a turkey genetic linkage map we decided to map new microsatellite sequences obtained from a GT-enriched turkey genomic library. In different chicken populations more than 35-55% of microsatellites are polymorphic. In the turkey populations tested here, 43% of all turkey primers tested were found to be polymorphic, in both commercial and wild type turkeys. Twenty linkage groups (including the Z chromosome) containing 74 markers have been established, along with 37 other unassigned markers. This map will lay the foundations for further genetic mapping and the identification of genes and quantitative trait loci in this economically important species. Genome comparisons, based on genetic maps, with related species such as the chicken would then also be possible. All primer information, polymerase chain reaction (PCR) conditions, allele sizes and genetic linkage maps can be viewed at http://roslin.thearkdb.org/. The DNA is also available on request through the Roslin Institute.     

Survey of a cDNA library from the turkey (Meleagris gallopavo).

Genome characterization and analysis is an imperative step in identifying and selectively breeding for improved traits of agriculturally important species. Expressed sequence tags (ESTs) represent a transcribed portion of the genome and are an effective way to identify genes within a species. Downstream applications of EST projects include DNA microarray construction and interspecies comparisons. In this study, 694 ESTs were sequenced and analyzed from a library derived from a 24-day-old turkey embryo. The 437 unique sequences identified were divided into 76 assembled contigs and 361 singletons. The majority of significant comparative matches occurred between the turkey sequences and sequences reported from the chicken. Whole genome sequence from the chicken was used to identify potential exon-intron boundaries for selected turkey clones and intron-amplifying primers were developed for sequence analysis and single nucleotide polymorphism (SNP) discovery. Identified SNPs were genotyped for linkage analysis on two turkey reference populations. This study significantly increases the number of EST sequences available for the turkey.     

Histomoniasis and reticuloendotheliosis in a wild turkey (Meleagris gallopavo) in North Carolina

A moribund wild turkey (Meleagris gallopavo) died shortly after it was discovered in Martin County, North Carolina (USA). The 4.3-kg female turkey appeared in good condition with no visible external lesions or evidence of injury. There were 2- to 5-mm yellow-white plaques on the mucosal surfaces of the oral cavity and mid-esophagus. The liver had large, multifocal, irregular pale areas on cut and uncut surfaces. The spleen contained multifocal, pale, hard, nodules. Microscopic changes in the liver consisted of large multifocal coalescing areas of necrosis. Occasional spherical 10 to 15 microns in diameter organisms consistent with Histomonas meleagridis were present in the necrotic areas. Viable hepatic parenchyma contained multifocal infiltrations of numerous mononuclear cells interpreted as neoplastic cells resembling lymphoblasts and plasma cells. Similar neoplastic cell infiltrates, consistent with the lymphoproliferative disease reticuloendotheliosis, were present in spleen, lung, and esophageal and oral mucosa. Reticuloendotheliosis virus, subtype 2, was isolated from samples of liver and spleen.     

Integration of microsatellite-based genetic maps for the turkey (Meleagris gallopavo).

Integration of turkey genetic maps and their associated markers is essential to increase marker density in support of map-based genetic studies. The objectives of this study were to integrate 2 microsatellite-based turkey genetic maps--the Roslin map and the University of Minnesota (UMN) map--by genotyping markers from the Roslin study on the mapping families of the UMN study. A total of 279 markers was tested, and 240 were subsequently screened for polymorphisms in the UMN/Nicholas Turkey Breeding Farms (NTBF) mapping families. Of the 240 markers, 89 were genetically informative and were used for genotyping the F2 offspring. Significant genetic linkages (log of odds > 3.0) were found for 84 markers from the Roslin study. BLASTn comparison of marker sequences with the draft assembly of the chicken genome found 263 significant matches. The combination of genetic and in silico mapping allowed for the alignment of all linkage groups of the Roslin map with those of the UMN map. With the addition of the markers from the Roslin map, 438 markers are now genetically linked in the UMN/NTBF families, and more than 1700 turkey sequences have now been assigned to likely positions in the chicken-genome sequence.     

Reticuloendotheliosis in a wild turkey (Meleagris gallopavo) from coastal Georgia.

An emaciated wild turkey (Meleagris gallopavo) exhibiting neurologic signs was found on Ossabaw Island, Chatham County, Georgia (USA) on 11 April 1989. The neurologic abnormalities observed included ataxia, drooping wings, head tremors, torticollis, and circling. At necropsy, discrete yellowish-white nodules, varying in size from 2 to 5 mm, were present in the spleen. White nodular lesions approximately 2 mm in diameter were observed beneath the mucosal surface of the distal esophagus. Histopathologic examination of the splenic nodules disclosed large numbers of primitive lymphoreticular cells with leptochromatic nuclei and abundant, slightly basophilic cytoplasms. The mitotic index in these cells was moderate to high. Similar neoplastic cells composed the masses observed in the esophagus. Multifocal, mild perivascular cuffing with mononuclear cells was found in the lumbar spinal cord, brain, and brain stem. Reticuloendotheliosis virus, subtype 3, was isolated from samples of the spleen and liver.     

Intestinal adaptation to diet in the young domestic and wild turkey (Meleagris gallopavo)

The short-term effects of diet on jejunal growth, alanine transport rate, and leucine aminopeptidase activity (LAP) were compared in the domestic and wild turkey poult. One-day-old poults of each strain were fed diets of high vs., low protein, with carbohydrate varied to maintain isocaloric conditions. Prior to feeding, relative jejunal mass and alanine transport rates were not significantly different in the two turkey strains, whereas LAP activity was 270% higher in wild poults. After feeding for 72 h, relative jejunal mass doubled in both turkey strains. In domestic turkeys, alanine transport rate and LAP activity were reduced by approximately 42% and 25%, respectively, in poults fed a 24% protein-69% carbohydrate diet vs. a 49% protein-35% carbohydrate diet. Analysis of the combined data from feeding experiments revealed that alanine transport rate was not correlated with total food, protein or lipid intake, but was negatively correlated with carbohydrate consumption (P<0.05). In wild turkeys, neither alanine transport rate nor LAP activity were altered by diet. These results reveal that domestic turkey hatchlings can modulate protein digestive and absorptive functions as protein/carbohydrate composition of the diet changes and suggest that high dietary carbohydrate down-regulates the intestinal alanine transporter.     

Effect of fasting on small intestinal antiperistalsis in the Nicholas turkey (Meleagris gallopavo)

Small intestinal refluxes (SIRs) were monitored in the gastrointestinal tract of Nicholas turkeys via implanted strain gage transducers and radiographic observations. It was determined that reflux of ingesta from the small intestine into the gizzard was a result of a single antiperistaltic contraction. Radiographic observation indicated that the antiperistaltic contraction originated in the ileum 88% of the time and the duodenum 12% of the time. Each antiperistaltic contraction was preceded by a single peristaltic contraction. No gastric contractions occurred during SIRs. Fasting for 12 to 24 hr significantly increased the frequency of occurrence of SIRs in the Nicholas turkey (P < 0.0001) but had no significant effect on the velocity of the peristaltic and antiperistaltic contractions or on recovery time to pre-SIR gastric contractile frequency. The pooled mean velocity of peristaltic contractions was determined to be 10.55 +/- 3.68 cm/sec, and that of antiperistaltic contractions was determined to be 12.12 +/- 3.69 cm/sec. The pooled mean recovery time to pre-SIR gastric contraction frequency was 28.31 +/- 8.25 sec. It has been suggested that the SIR may be a mechanism of maximizing nutrient utilization by the turkey.     

One hundred fifty-four genetic markers for the turkey (Meleagris gallopavo).

Identifying and selectively breeding for improved traits is one of the ultimate goals of genetic research in agriculturally important species. Genome characterization and analysis are important first steps in this process. Genetic linkage maps based on the linear order of polymorphic DNA markers are typically developed through statistical analysis of inheritance patterns in pedigreed families. To develop microsatellite markers for further improvement of the turkey genetic linkage map, small-insert genomic libraries were screened for tandem repeats. Oligonuclotide primers were designed to amplify 164 microsatellite-containing fragments from genomic DNA. Genetic polymorphisms at 154 markers were determined by genotyping the F(1) individuals of two resource populations. Markers determined as segregating in the University of Minnesota/Nicholas Turkey Breeding Farms (UMN/NTBF) reference population were used to genotype F(2) individuals and a two-point linkage analysis was performed.     

Chemical and biological evidence for presence of estrogens in the domestic turkey (Meleagris gallopavo)

Solvent partitioning, column chromatography on activated alumina, and Ittrich extraction of Kober chromogens were employed to isolate, identify and quantitate estrone, estradiol-17B and estriol from blood of individual turkey hens. The intravaginal tetrazolium method was used to estimate the biological potencies of the three fractions separated chromatographically. The data show that estrone is the main estrogen present in heart blood of laying turkeys, with lesser amounts of estradiol-17B and estriol. Data are presented on the accuracy, precision, sensitivity, and specificity of the methodology used.