Assessment of inbreeding by DNA fingerprinting: development of a calibration curve using defined strains of chickens.

By analyzing DNA fingerprints of chickens from seven well-defined genetic groups, a calibration curve was established relating the degree of inbreeding with the average band frequency, allelic frequency and band sharing. The probe used was bacteriophage M13 DNA and digestion of the genomic DNA was carried out with the MspI restriction enzyme. The analysis also provided an estimate of the average allelic frequency at a hypervariable locus and the average mutation frequency per locus and generation. The values of 0.24 and 1.7 X 10(-3), respectively, are similar to the estimates for humans using other probes and hybridization protocols. It is suggested that the calibration curve established can be used for determining inbreeding not only in chickens, but also in other species.

Prediction of heterosis from DNA fingerprints in chickens.

To assess the value of DNA fingerprints for the prediction of heterosis in chickens, retrospective analyses of data from three crossbreeding experiments and DNA fingerprints (DEP) of parental strains were conducted using two minisatellite and one middle-repetitive DNA probes. DEP bands were assessed on pooled DNA samples of 10-15 individuals per parental genetic group. The number of DEP bands evaluated in the experiments ranged from 81 to 139. The probes varied in their predictive value, but predictability of heterosis generally increased with multiple probes. Highly significant correlations (0.68-0.87) between band sharing ratios (SH) and heterosis were found in 25 crosses of White Leghorns in the first egg production cycle for age at sexual maturity, egg production, and mature body weight: traits with heterosis of 10% or more of the means. Regressions on SH explained 78.4% of the variation in heterosis in age at sexual maturity, 60.2% in egg production and 46.4% in mature body weight. For "broiler" traits with heterosis of < 1%, none of the correlations, based on 13 crosses, were significant. It was concluded that multilocus probe DFP of pooled DNA samples show promise as predictors of heterosis.

Identification of markers associated with quantitative trait loci in chickens by DNA fingerprinting.

Three approaches for identifying VNTR alleles associated with quantitative traits in chickens are described. One approach is based on the comparison of well-defined selected and non-selected control strains. The second approach is based on analyzing chickens within a breeding population ranked according to specific traits and the third approach involves segregation analysis. In this latter approach a large number of offspring of a single male segregating for a quantitative trait are produced and tested for trait association of the male DNA fingerprinting bands. In all cases pooled DNA samples of birds, rather than individual samples, are analyzed and band intensity is assumed to reflect the relative frequency of an allele. Examples from the literature and from our laboratory indicate that these methods permit the identification of DNA fingerprinting bands associated with quantitative traits. After developing locus-specific probes for these bands it should ultimately be possible to detect and map quantitative trait loci.

Distribution of lymphoid leukosis virus and p27 group-specific antigen in tissues from laying hens.

In order to gain insight into transmission and pathogenesis of infection, specimens from laying hens that had been naturally exposed to lymphoid leukosis virus (LLV) were tested for group-specific antigen (gsa) of the virus by immunofluorescence (IF), complement fixation (CF), and the enzyme-linked immunosorbant assay (ELISA). Electron microscopic examinations determined the distribution of C-type virus particles in tissues, and the phenotypic-mixing test served as a biological assay for exogenous LLV. The IF gsa was found in all organs tested, and fluorescence was usually found where virus particles were concentrated. In the oviduct and intestine, IF gsa was frequently at the border of the lumina and in the connective tissue associated with basal membranes of glands. In skin, the antigen was detected in smooth muscle, in feather pulp, and in basal epidermal cells of developing feathers. Results of various tests on Ottawa strains of chickens were usually in agreement. For example, among hens that shed gsa into egg albumen, only the viremic hens were consistently positive for IF gsa in both spleens and oviducts. Geometric mean CF titers of antigen were respectively five- and 23-fold higher in spleens and oviducts from viremic hens than in those from nonviremic hens. These findings suggest that the gsa was associated with exogenous virus infection. In Cornell S strain hens that had not been exposed to LLV, gsa was detected in splenic tissue by CF and ELISA but not by the IF test. This gsa was presumed to be of endogenous origin.

Influence of Marek's disease virus strain AC-1 on cellular immunity in birds carrying endogenous viral genes.

The effects of Marek's disease virus (MDV) strain AC-1 on humoral and cellular immune responses were investigated in two lines of chickens segregating for the endogenous viral (ev) genes ev-6 and ev-12. All birds were vaccinated at 14 days of age against Newcastle disease virus (NDV). At 3 wk of age, 48 birds per line received an intraperitoneal injection of MDV (AC-1 isolate), and 24 were injected with saline. Birds of each group were killed on days 5, 7, 12, and 14 postinfection. Data were first analyzed for each day of testing. Results showed that, for variable measured, treatment effects were similar on days 5 and 7, and on days 12 and 14. Therefore, day 5 and day 7 data, and day 12 and day 14 data were pooled and analyzed. In MDV-infected chickens, proliferative lymphocyte responses to mitogens were suppressed (P < 0.001) after the first and second week of infection, whereas responses to NDV antigen were enhanced (P < 0.001) after the first week and then reduced (P < 0.01) by the end of the second week when compared to uninfected birds. The percentage of CD4+ T cells was higher (P < 0.01), and the percentage of cells expressing major histocompatibility complex (MHC) class II antigens was lower (P < 0.001), in MDV-infected chickens than in uninfected birds. The cytotoxic activity of natural killer (NK) cells was also enhanced (P < 0.001) in MDV-infected birds when compared to uninfected birds. Antibody responses to NDV were not different among groups, and the presence of ev-6 or ev-12 genes did not influence the immune response parameters measured in both infected and uninfected chickens. In conclusion, a marked increase in the CD4+ T lymphocyte population occurred in the early stage of MDV infection in all chickens regardless of the presence of ev genes, whereas the number of cells expressing MHC class II antigen was severely reduced.

Studies on genetic and vaccination-induced resistance of chickens to lymphoid tumor transplants 2. Transmissible lymphoid tumor of Olson.

Transmissible lymphoid tumor (TLT) was inoculated in wing webs of five-week-old chickens of 6 strains. About half of the chickens of each strain had been vaccinated with turkey herpesvirus (HVT) one week before challenge in the wing web with TLT. Tumors which developed at the site of inoculation usually reached maximum size within 2 weeks and then regressed. In some chickens, however, tumors developed in visceral organs and caused death in the 2nd through 5th weeks postinoculation. Comparisons among strains of chickens in Expt. 1 revealed no differences in mortality. Vaccination with HVT reduced mortality and also the incidence of wing-web tumors (WWT) in all strains of chickens. A lymphoid leukosis virus and a Marek's disease (MD) virus of low virulence were detected in preparations of TLT, and it is suggested that the immunity induced by vaccination may have been directed against tumor antigens associated with MD virus.

Studies on genetic and vaccination-induced resistance of chickens to lymphoid tumor transplants. 1. Marek's disease tumor transplant (JMV).

Six strains and 4 inbred lines of chickens that differed in susceptibility to Marek's disease (MD) were inoculated in the wing web with JMV when 5 weeks old. Wing web tumors (WWT) developed in all strains and lines inoculated with low-passage JMV (JMV-L) but were largest in Cornell Strain S (highly susceptible to MD). Of 3 strains inoculated with high-passage JMV (JMV-H), only Strain S had appreciable WWT development. Seventy-five percent of the unvaccinated S strain chickens challenged with JMV-L or JMV-H died during the experiments, and approximately half of this mortality occurred during the second week postinoculation. Inbred Line GC ranked next in susceptibility to Strain S and was more susceptible than other lines and strains, including the strain from which it originated. Vaccination with turkey herpesvirus one week before challenge protected against mortality and suppressed WWT development. The effect on WWT development was less, however, in Strains S and NH than in other strains. The transplantability of the tumor was investigated with the use of sex chromosomes as cell markers. Five to 7 days postinoculation of male Strain S chicks with JMV-L or JMV-H, most cells in metaphase from wing web or visceral tumors were of female origin. By 56 days, only male cells were found in visceral tumors. The interpretation was that early lesions were due to tumor transplantation and later lesions were induced by virus.

Role of the major histocompatibility complex in resistance to Marek's disease: restriction of the growth of JMV-MD tumor cells in genetically resistant birds.

B21 is associated with resistance to Marek's disease (MD). Forty populations of chickens from all over the world were examined for the presence of the B21 allele. B21 was found in twelve of these populations and it's presence was confirmed by GVH testing in all ten populations which were tested. The populations in which B21 was detected represent the extreme production types of the species and include the progenitor of the species, the Red Jungle Fowl. Our studies suggest that B21 may have strong survival value for the species. An allogeneic transplantable lymphoma of MD, the JMV tumor cell line, grows more slowly in MD resistant (B21/B21) chicks than in MD susceptible (B2/B2) chicks. This is the first direct evidence that genetic resistance to MD may involve an active (immunological?) restriction of tumor cell growth. JMV cells were further characterized as a transplant of B1 carrying lymphoblastoid cells, an allele which may be associated with susceptibility to MD.

Partial replacement of chicken semen by turkey semen in artificial insemination of chickens.

Chicken semen undiluted, diluted with a diluent containing fructose and/or mixed with turkey semen was used to inseminate Leghorn hens. In two of three experiments there was an improvement in fertility from insemination by mixed semen as compared to semen diluted to the same extent with the diluent.

A new diagnostic method for the detection of endogenous Rous-associated virus-type provirus in chickens.

A quick and simple method has been developed to detect the presence or absence of the endogenous Rous-associated virus (RAV) element ev1 in chickens. The procedure consists of a one-tube multiplex polymerase chain reaction (PCR) involving three oligonucleotide primers that are specific for the upstream flanking region, the long terminal repeat (LTR), and the downstream flanking region of the proviral insert, respectively. The multiplex reaction allows for the unambiguous discrimination between ev1+/ev1+ homozygote, ev1-/ev1- homozygote, and ev1+/ev1- heterozygote birds. The method works best with purified genomic DNA as substrate, but can also be used with rapidly prepared, "crude" DNA samples. The combination of speed with the safety of a nonradioactive procedure, and the ability to perform large numbers of assays by a semi-automated procedure, make this method attractive for large-scale screening projects. The ev1 locus has been used as a model system to demonstrate the feasibility of the PCR diagnostic approach. However the same principle should be applicable to the analysis of other RAV-type ev loci, as well as endogenous elements belonging to other families of viruses as sequence information for the flanking regions of these inserts becomes available.

Major histocompatibility complex class II DNA polymorphisms in chicken strains selected for Marek's disease resistance and egg production or for egg production alone.

The objective of this study was to investigate frequencies of major histocompatibility complex (MHC) class II restriction fragments in two groups of White Leghorn strains. Each group consisted of an unselected control, a strain selected for egg production traits, and a strain selected for egg production traits and Marek's disease (MD) resistance. PvuII-digested genomic DNA was hybridized with a chicken genomic MHC class II probe. The MHC class II DNA fragment frequencies in the selected strains differed from those in the related unselected control and in the strain selected using the same criteria from a different base population. Based on the sizes of the breeding populations, particularly those in the control strain and in the strain selected for egg production, it was considered unlikely that the observed changes of the MHC class II fragment frequencies were due to random genetic drift. The data suggested that some MHC class II bands are associated with production traits or with MD resistance, and that these associations tend to be unique to each genetic background. Hence, MHC class II genes are likely candidates for the investigation of quantitative trait loci in egg production and disease resistance traits such as those for which the studied strains were selected.

Relationship between activity at a young age and feed efficiency in chickens.

Feed conversions of three Ottawa strains of Leghorns were measured from hatch to 27 days and bird activity at 1, 2, and 4 weeks of age was assessed. Males were more active than females. Chicks of the more active strain consumed more feed and had poorer conversion than those from the least active strain. Ranking of the strains on feed conversion to 27 days was similar to their ranking on feed consumed per egg mass produced in an earlier study.

Genetic variation in body size, composition, temperature, and feed intake in mature chickens.

Genetic variation for more than 40 traits was assessed in 26 stocks of mature chickens reared together and fed ad libitum from hatching to slaughter at 507 days of age. There was greater genetic variation among males than among females. The intraclass correlation, t, was high (greater than .75) for measures of size and weight and moderate (.20 to .60) for most other traits (P less than .05) including a measure of lean distribution (.37). Three main categories of stocks were studied, viz. outbred Leghorns, medium-sized stocks, and contemporary heavy meat chickens. Differences in lean distribution were not associated with category, but differences of 40 to 50 g/kg lean in the breast of some stocks may be caused by a single gene. Lean:bone ratios were similar in medium and heavy stocks but were greater (P less than .05) in heavy meat-types compared with outbred Leghorns. Bone density was higher (P less than .01) in females compared with males and in outbred Leghorns compared with heavier stocks (P less than .01). Heavy meat-type males were leaner (P less than .01) and had proportionately less fat in the abdominal cavity than outbred Leghorns. Carcass fatness was similar among stocks of females, but abdominal fat was lower in Leghorns selected for high egg production compared with unselected Leghorns (P less than .05) and heavy stocks (P less than .001). One resistant and two Marek's disease-susceptible stocks were replicated in a specific pathogen-free (SPF) environment. Spleen weight was larger (P less than .001) in the conventional environment. Females were relatively smaller (P less than .05) than males in the conventional environment. Body temperature (t = .25, P less than .05) and feed intake were assessed in males. Heavy meat birds had a lower (P less than .01) body temperature than outbred Leghorns and medium-sized stocks. Differences among stocks for feed intake (t = .77) were significant (P less than .05); however, they were greatly reduced when feed intake was expressed as g/kg liveweight (LW) (t = .49) or g/kg LW.75 (t = .25). Outbred Leghorns ate less in absolute terms but significantly more (P less than .01) as a proportion of LW or LW.75 than the medium and heavy lines.

Research note: avian leukosis retroviral genes are not detected in geese.

Genomic DNA from four strains of geese was analyzed for the presence of endogenous viral elements using a probe that can detect over 20 Rous-associated endogenous viral genes (ev genes) in chickens, as well as a probe and protocol that detects endogenous avian viruses (EAV). Southern blot analysis of genomic DNA did not reveal any ev genes in DNA of 15 geese from Chinese, Synthetic, or two Embden goose strains. Even under low stringency conditions, using a probe that covered most of the polymerase (pol) gene of the Rous-associated virus (RAV) and that revealed EAV elements in a chicken without ev genes, no viral loci were evident in goose DNA.

Divergent selection for incidence of degenerative myopathy of the Musculus supracoracoideus of meat-type chickens.

The response to three generations of selection for incidence of degenerative myopathy of the Musculus supracoracoideus (DMS) in meat-type chickens was studied. From each of two genetic bases named E and F, which were derived from two commercial broiler stocks, a high incidence, low incidence, and a nonselected control strain were initiated. Selection of breeders to produce Generations 1 and 2 was based on the incidence of DMS in their parents after induced wing exercise; mass selection for plasma creatine kinase levels after induced wing exercise was used to produce Generation 3. Little genetic progress in selection occurred in Generations 1 and 2. There was a response in Generation 3, especially in Base E selected for low incidence. Heritability of incidence of DMS was estimated to be .48. Body weights at 8 weeks of age, 18 weeks of age, and breast angle at 18 weeks of age of Generation 3 were greater (P less than .05) for affected than nonaffected birds and positively correlated phenotypically with incidence of DMS. Genetic correlations were extremely variable. The higher incidence of DMS in birds with desirable traits suggests that the breeder must use a multitrait selection program to reduce incidence of DMS as well as improve production traits.

Incidence of degenerative myopathy among turkeys fed corn or wheat based rations.

Turkeys were fed either corn based or wheat based rations and necropsied between 28 and 118 weeks of age. Practically all birds affected with degenerative myopathy were detected among those killed between 77 and 85 weeks of age. The incidence of the disease was 0% among 125 males and 7.9% among 139 females. No significant difference in incidence was observed between birds fed corn based and birds fed wheat based rations.

Effect of selection for high egg production in chickens on shedding of lymphoid leukosis virus and gs antigen into eggs.

Lymphoid leukosis virus (LLV) and group specific (gs) viral antigen were detected less frequently in albumen of eggs from two strains of Single Comb White Leghorns that had been selected for high egg production than in corresponding random-bred control strains, which represented the original base population. In a third selected strain, for which no comparable control strain was available, the frequency with which LLV and gs antigen were detected was similar to the other two selected strains. The greatest contrast was between selected Strain 1 and control Strain 5 in which the percentage of eggs with LLV in albumen was 1.4 and 21.4, respectively, and the percentage with gs antigen was 1.2 and 19.9. These differences between control and selected strains of chickens were not related to genetic cellular resistance to virus infection, because inoculation of chorioallantoic membranes with Rous sarcoma virus of subgroups A and B revealed that the proportion of birds resistant to subgroups A and B viruses was not greater in the selected strains than in the control strains.

Endogenous viral genes: association with reduced egg production rate and egg size in White Leghorns.

Endogenous viral (ev) genes are DNA sequences residing permanently in the genome of most chickens that have a high degree of homology to avian leukosis viruses (ALV). Association of ev genes with production trait differences was studied in White Leghorns free of exogenous ALV. The Cornell Strain K and S chickens used in Experiment 1 had multiple ev genes. In each of four lines of chickens in Experiment 2, there was a 1:1 segregation of full-sibs free of ev genes and those carrying one ev gene: ev-12 that produces the complete endogenous virus, ev-3 or ev-6 that express certain viral antigens, or ev-1, a silent gene. In Experiment 1, the presence of genes ev-10 or ev-19, known to produce the complete virus, was associated with a 9% reduction in the annual egg production rate (P less than .05) in Strain S. Similarly, the presence of the virus-producing ev-12 in Experiment 2 was associated with an 8% reduction of annual egg production rate (P less than .05), a 2.2-g reduction in egg weight (P less than .01), and a .003 reduction in egg specific gravity (P less than .01). No significant effects of ev genes on age at first egg, Haugh unit score, percentage of eggs with blood spots, and body weight of hens were observed. It was concluded that ev genes producing complete endogenous virus are associated with production trait differences similar to those associated with subclinical infections with exogenous ALV.

Screening chickens for endogenous virus ev21 viral element by the polymerase chain reaction.

The molecular architecture of the sex-linked late-feathering region of the chicken genome is still poorly defined. Current evidence points to a strong association between the presence of the endogenous viral element ev21 and the late-feathering phenotype. However, analysis at the molecular level has demonstrated that this is not a simple case of insertional mutagenesis. Instead, the structure of the region of the chicken genome containing the feathering locus is complex and variable between and within lines of chickens. Significant clues to the molecular structure of this genomic region can be obtained by analyzing rare and revertant genotypes. However, searching for rare genotypes can only be carried out effectively using quick screen methodology. This paper describes a quick, polymerase chain reaction-based test for ev21 that facilities the search for rare genotypes.

Endogenous viral gene distribution in populations of meat-type chickens.

The present study was designed to document the complexity of endogenous viral (ev) genes and seek evidence for their association with production traits in selected and control strains of meat-type chickens. Three populations were studied, each consisting of a control strain and one to three strains selected for various production traits. The ev genes were revealed by digesting genomic DNA with restriction enzymes and detecting DNA fragments on Southern blots using radioactive probes from nucleotide sequences of the avian leukosis virus genome. A total of 31 polymorphic ev loci were identified in these populations from a SacI digest, with an average of 7.3 ev genes per bird. There were no significant differences in ev genes per bird between strains within populations or between selected and control strains overall. Thirty of 62 comparisons in the three populations indicated ev gene frequency differences (P less than .05). Within populations, 13 of 93 comparisons of ev gene frequencies between control and selected strains and 8 of 62 between three selected strains of a sire population showed such differences (P less than .05). Selection for body weight and feed efficiency had been observed to reduce gene frequencies of the slow-feathering gene, which usually contains the ev21 locus; however, these effects were not detected (.05 less than P less than .06) between strains of the dam population in the current study. Such differences suggested possible associations between ev genes and production traits in meat-type chickens.