Characterization of four endogenous viral genes in semi-congenic lines of meat chickens.

Restriction fragment length polymorphism analyses were used to examine endogenous viral genes (ev genes or ALVE genes) of the avian leukosis viral (ALV) family in semi-congenic lines of meat chickens. The Generation 6 lines examined in this study were semi-congenic in that each contained birds with either zero or with one ALVE gene in hemizygous state plus some solitary long terminal repeat (LTR) elements. Using four restriction enzymes on chicken genomic DNA and two probes, one representing the entire ALV retroviral genome and one with only a small part plus the LTR, four ALVE genes were characterized. Each seemed to be complete with no detectable deletions. None appeared to be similar to known ALVE genes of White Leghorns, whereas two of the four may be the same as ALVE genes reported by others in White Plymouth Rock chickens.

Characterization of eight endogenous viral (ev) genes of meat chickens in semi-congenic lines.

Restriction fragment length polymorphism analysis was conducted for a set of eight different meat chicken-derived endogenous viral genes (ev genes) of the avian leukosis viral (ALV) family. Each viral element was first isolated into a separate single-element line by selective breeding. Genomic DNA from the founder male for each semi-congenic, single-element line was digested with each of four restriction enzymes, and the resulting Southern blots were each hybridized with up to four probes representing different portions of the ALV retroviral genome. Among the eight elements, there was one that represents the broiler equivalent of locus ev3 of White Leghorn chickens. A second broiler element showed a SacI-specific junction fragment similar to that of ev8. The remainder appeared to be different from any of the 21 ev genes previously described for White Leghorn chickens. Four of the eight elements examined were essentially complete, but the rest have sustained internal deletions.

Influence of the alv6 recombinant avian leukosis virus transgene on production traits and infection with avian tumor viruses in chickens.

The biological costs of the alv6 recombinant transgene that in chickens induces dominant resistance to the subgroup A avian leukosis virus (ALV), in terms of effects on production traits, were studied. Four generations of White Leghorn chickens of Line TR, segregating for alv6 but free of endogenous viral genes, as well as two generations of crosses between TR and Ottawa Line WG (WGTR) were tested under a specific-pathogen-free environment. In the birds studied, the transgene appeared unchanged compared to the original alv6: No major changes in alv6 DNA were detected by restriction analysis, the transgene did not express the group-specific antigen of ALV, and its presence was associated with absence of immune response to ALV. In most test years, and both TR and WGTR genomic backgrounds, alv6 was associated with delayed sexual maturity by 4 to 6 d, reduced egg production to 497 d of age by 20 to 46 eggs, and a 3.6 to 15% decline in egg production rate. No consistent effects on other traits, including mortality, were detected. When inoculated with the AC-1 isolate of Marek's disease virus in a separate experiment, TR birds with alv6 had a significantly lower body weight gain to 10 d of age than their sibs without the transgene. Thus, transgenesis has biological costs that have to be assessed against desirable effects of transgenes.

A novel molecular fingerprint probe based on the endogenous avian retroviral element (EAV) of chickens.

We have developed a novel molecular probe that is useful for DNA fingerprint analysis in chickens. The probe is based on the middle-repetitive, chicken endogenous retroviral (EAV) element. It consists of 1503 bp of the 3' portion of the EAV element, extending from the down-stream end of the envelope gene to the beginning of the downstream long terminal repeat (LTR). Unlike other probes that are currently in use for fingerprint analysis with chicken DNA, the EAV-based probe works well at normal levels of stringency, and with standard hybridization buffers. Digestion of chicken genomic DNA with a variety of restriction enzymes routinely yields up to 30 resolvable bands per bird in the 500 bp to 20 kbp range. In order to test the efficacy of the EAV-based fingerprint probe, we have used it to estimate the degree of inbreeding in the inbred WG strain of White Leghorns. We find that the estimates derived with the EAV probe are very similar to those reported previously for the WG strain. These results suggest that molecular probes based on endogenous retroviruses and other middle-repetitive DNA elements should be useful for fingerprint analysis in chickens, and in vertebrates in general.

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.

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.

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.

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.

Influence of selection for egg production and Marek's disease resistance on the incidence of endogenous viral genes in White Leghorns.

The influence of selection on the frequencies of endogenous viral (ev) genes related to the avian leukosis virus was studied in two genetically distinct sets of White Leghorn strains. Each set consisted of four strains: an unselected control strain, two strains selected for egg production traits, and a strain selected for Marek's disease (MD) resistance as well as egg production traits. Eight different ev genes were observed in Set I and seven in Set II, four being common to both sets. Selection for egg production traits resulted in significant changes of the frequency of four ev genes in both sets. In Set I, increased frequencies were observed for ev-4, ev-7, and ev-8; a decreased frequency for ev-9 was observed. The ev-9 gene expresses the viral envelope protein, whereas the others are transcriptionally silent, with the possible exception of ev-7. In Set II, increased frequencies were observed for the transcriptionally silent ev-8 and for ev-15, a gene which consists of a solitary long terminal repeat. Decreased frequencies were observed for ev-18, which codes for infectious endogenous virus, and for a second ev gene of unknown phenotype. In the resistance-selected strains the frequencies of the ev genes were intermediate between those of the control strains and the strains selected for egg production traits with the exception of ev-6, which expresses the viral envelope protein, and ev-3, which expresses internal viral proteins as well as the envelope protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Semen traits and fertility of White Leghorn males shown to be positive or negative for lymphoid leukosis virus in semen and feather pulp.

1. Males from strains selected for high egg production (and other economic traits) and from unselected control strains were used to determine the frequency of shedding of lymphoid leukosis virus (LLV) into semen. The effect of the male's LLV status on semen production, fertility and hatchability was also examined in males of the unselected control strains. 2. The frequency of detection of exogenous LLV in semen by the phenotypic mixing test, and high concentrations of the viral group specific antigen in feather pulp by the complement fixation test, were both higher in control strains than in strains selected for high egg production. 3. Semen production was not reduced in LLV-shedding males. 4. Significant associations of LLV shedding with higher incidence of abnormal spermatozoa and reduced fertility were found in some populations but not in others. No significant effect of LLV shedding on hatchability was detected. 5. Tests for group specific antigen in feather pulp proved useful in identifying males that shed LLV in semen.

Feather pulp organ cultures for assessing host resistance to infection with avian leukosis-sarcoma viruses.

The purpose of this study was to improve in vitro procedures for detecting cellular resistance to the avian leukosis-sarcoma group of viruses. Four feather pulp organ cultures (FPOC) were prepared from each chicken by placing pulp squeezed from feathers in wells of microtitre plates that contained culture medium. Two of the four FPOC were inoculated with Rous sarcoma virus (RSV) of subgroup A and 5 to 6 days later the fluids from all four cultures were assayed for virus by inoculating chicken embryo fibroblasts (CEF) and examining for development of foci of transformed cells. Prior to the second assay of culture fluids, quail cells transformed by envelope-defective RSV [R(-)Q cells] were added to some RSV-inoculated and uninoculated FPOC. The R(-)Q cells produce infectious RSV when infected with avian leukosis virus (ALV), and hence made it possible to detect ALV in FPOC. Status of host infection was also assessed by tests for virus neutralising antibody and the enzyme-linked immunosorbent assay for group specific viral antigen. In one experiment FPOC from chickens not exposed to ALV were susceptible to RSV throughout the 140-day test period. In contrast, FPOC from ALV-inoculated chickens were usually infected with ALV and were resistant to RSV. FPOC from chickens reared in contact with the inoculated group for 121 days were free of ALV and were unexpectedly resistant to RSV. Two other experiments supported the observation that genetically susceptible chickens acquire cellular resistance to RSV as a result of persistent or transient ALV-infection. In Cornell K strain chickens there was close agreement between cellular susceptibility based on tests on FPOC prepared prior to inoculation of chickens with ALV and for antibody following inoculation with ALV. A New Hampshire strain showed a high degree of genetic cellular resistance by these test procedures.

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.

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.

Lymphoid leukosis virus infection: effects on production and mortality and consequences in selection for high egg production.

Lymphoid leukosis (LL) is virus-induced, lymphoblastic malignancy of chickens that can be congenitally transmitted. Mortality from LL is generally low. Effects of LL virus (LLV) on production and mortality were investigated in approximately 2000 Leghorn pullets in each of two consecutive years. The pullets were from nine strains developed in Ottawa, of which three were unselected control strains and six were strains under selection for up to 27 generations for high egg production and a complex of related commercially important traits. The overall frequency of birds shedding LL virus of gs antigen into eggs (LL-S) was significantly lower in the selected strains (3.9%) than in the control strains (18.5%), indicating that LLV may have negative effects on production and cause elimination of LL-S birds by selection. Such significant effects were indeed detected: the LL-S pullets produced to 497 days of age in 1976 and 1977, respectively, 30 and 25 eggs less per hen-housed than the nonshedders. The LL-S birds matured sexually later, produced smaller eggs at a lower rate, and their eggs had a lower specific gravity, indicating thinner shells. Mortality from all causes to 497 days was significantly higher in LL-S birds (+14.8%) in 1976. In 1977 the increase (+5.5%) did not reach statistical significance. In both years the mortality from LL itself remained very low. In another study, eggs from one of the control strains were incubated and hatched from the dams were 291 and 483 days old. The eggs from LL-S dams had 2.4% lower fertility and 12.4% lower hatchability. The effects on hatchability were more pronounced in the older dams. Since the lower production of LL-S birds results in a lower frequency of such birds in strains selected for high egg production, it is suggested that a part of the difference between the performance of the selected and control strains (delta S) is due to reduction in the frequency of LL-S birds (delta L) rather than due to true genetic gain. In this study, the size of delta L relative to delta S was estimated at 4 to 14% for egg production and 3 to 7% for egg weight. The negative effects of LLV infection on egg production, mortality, hatchability, and genetic gains show the desirability of producing chickens free of LLV infection.

Complete endogenous RNA tumour virus production by inbred and non-inbred chickens.

Nine- to 11-day embryos of five outbred and 12 inbred stocks were assayed for complete endogenous subgroup E virus. About one-third of the embryos produced a subgroup E virus. The percent virus production was higher in the inbred than in the outbred embryos, but wide variation among the inbred lines suggested that this was a chance phenomenon and probably not due to inbreeding per se. There was a reduced incidence of subgroup E virus production in the strains selected for economic traits when compared with the incidence in the corresponding random-bred control stocks. Further experimental work is required to determine if this trend is a real and not a chance phenomenon.

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.

Genetic resistance to lymphoid tumor transplants.

Day-old chicks of Ottawa strains 4 and 5 and Cornell strains K and S, with or without maternal antibody against antigens related to Marek's disease (MD), were challenged with serial dilutions of MD tumor transplants (JMV-L or JMV-H) or transmissible lymphoid tumor (TLT) of Olson in three consecutive experiments. Thirty chicks were tested in most combinations of strain, dilution and antibody status for a total of approximately 2000 chicks per experiment. Two subsequent experiments, with a total of more than 1900 chickens, investigated the influence of age at challenge on the resistance of strains 4, K and S to JMV-L and the objective of the sixth experiment was to determine the chromosomal sex of the tumor cells. In terms of mortality up to 16 or 17 days after inoculation, the response to challenge of day-old chicks with JMV-L did not appear to be influenced by the presence of antibody against MD related antigens but differed among strains of chickens. The estimated dose (LD50) that would kill 50% of a challenged population of Strain 4 was approximately 10,000 live JMV-L cells, while the corresponding estimates for strains 5 and K were approximately 600 cells and for strain S it was 50 cells. JMV-H and TLT were highly lethal to all four strains and all estimates of LD50 at 16 or 17 days post challenge were less than 10 cells. Resistance to JMV-L increased rapidly with age at challenge. Strains 4 and K were approaching complete resistance by 7 days of age and the susceptible strain S, when challenged at 14 days, was more resistant than day-old chicks of strains 4 and K. The chromosomal sex of the JMV-L, JMV-H and TLT tumor cells was female and this marker was used to confirm their transplantability.

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.

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.