H.J.L. Maas

Some Virological and Serological Observations On Marek’s Disease: A Preliminary Report

SUMMARY Virological and serological investigations on Marek’s disease reported in this study have been based on the duck-embryo-fibroblast tissue-culture system. Cell-associated cytopathological agents could be isolated from cases of Marek’s disease, but also from apparently healthy individuals. Different isolates of the MD-agent show a considerable variability with respect to their pathogenicity. By means of an indirect fluorescent antibody technique the ubiquitous presence of MD-infection was clearly demonstrated. Adult chickens hatched and reared in strict isolation units appeared to be free of infection.

On epizootiology and control of lymphoid leukosis in chickens

Abstract Sera and organ extracts from ten different commercial stocks of layer chickens were examined for the presence of lymphoid leukosis (LL) viruses. Virus was recovered from 40.8% of the cockerels between three and six weeks of age. Their female hatch mates were examined at the age of 20 months. A mean of 11.3% of these laying hens was positive in the NP activation test. Lymphoid leukosis was successfully controlled in three inbred strains of White Leghorn chickens and in a commercial White Plymouth Rock line. All flocks were kept in a filtered air positive pressure (FAPP) house during the first two months of life and thereafter transferred to a conventional environment. The control method is based on three elements: • —from an infected flock, hens are selected in whose eggs no avian lymphoid leukosis viruses can be detected by examination of pooled extracts of groups of embryos; • —only eggs from hens that are shown not to shed congenitally virus in their eggs are used for the production of progeny. The offspring are reared in isolation until two months of age at which time the age-related resistance against tumour formation appears to be sufficiently developed; • —the chickens are subsequently intramuscularly inoculated with lymphoid leukosis viruses of subgroups A and B and transferred to a conventional chicken house. The inoculated birds become persistently viremic and resist horizontal virus exposure and intramuscular challenge infections. Horizontal virus transmission was observed to take place when virus-free non-vaccinated chickens were reared in isolation for two months and then exposed under field conditions. Efficiency of virus recovery was considerably improved when washed buffy coat cells were cocultivated with chick embryo fibroblasts or explant cultures were prepared from various tissues before testing with the NP activation test.

Age related resistance to avian leukosis virus. III. Infectious virus, neutralising antibody and tumours in chickens inoculated at various ages

Viraemia and neutralising antibodies were determined in chickens of six age-groups following inoculation with leukosis virus of subgroups A and B at the age of 1 day, and 2, 4, 6, 8 and 10 weeks respectively. The birds were kept in a filtered air positive pressure (FAPP) house. A seventh age-group, accommodated in a separate FAPP-house, was used as an untreated control. Serum samples, received at biweekly intervals between 1-17 weeks post-inoculation, from birds of the groups inoculated at 4, 6, 8 and 10 weeks of age, showed at 1 week post-inoculation a transient viraemia followed by neutralising antibodies at the later sampling times. Neutralising antibody to subgroup A virus was detected in nearly all birds tested; this was not so for antibody to subgroup B. In all four groups the average titre of the former antibody was higher than that of the latter. Midway through the laying period birds of each group inoculated with leukosis virus, and some of the uninoculated controls, were challenged by infection with either subgroup A or B virus. At termination of the experiment survivors from each group were tested for the presence of leukosis virus. The virus recovery was performed with plasma samples, white blood cell preparations and explant cultures of various organs. The plasma samples were all negative; the great majority of blood cell specimens received from birds inoculated early with leukosis virus were positive, whereas the majority of the preparations from the birds inoculated later remained negative. The organ explants from the two youngest age groups were mostly leukosis virus-positive, from the birds inoculated at 4 weeks of age the spleen and kidney explants contained leukosis virus whereas in the groups inoculated at 6, 8 and 10 weeks of age only the spleen explants of birds challenged with subgroup A virus In a subsidiary experiment, started 4 months after the challenge infection, four birds from each group (two challenged with leukosis virus of subgroup A and two with subgroup B) were accommodated in isolators. The birds were challenged again, this time with Rous sarcoma virus (RSV) of the homologous subgroup used for the previous challenge. The tests for virus just prior to the challenge showed leukosis virus only in the white blood cell preparations from the birds in the three youngest age groups; the birds from the older groups were virus-negative. The serological tests after challenge showed neutralising antibodies to both subgroups in birds of nearly all groups. Tumour formation at the site of injection was mainly observed in the chickens challenged with RSV of subgroup B. The virological and serological results as well as the tumour response show that the immune system of birds between 0-4 weeks of age is insufficiently developed to cope with a controlled exposure with leukosis virus, whereas in birds of 4-10 weeks of age an adequate immunological response has developed. The significance of the presence of leukosis virus in sera, plasma, white blood cell preparations and organ explant cultures is mentioned. In programmes for the control of lymphoid leukosis in reproductive stock the use of information on virus and neutralising antibodies is recommended.

Age-related resistance to avian leukosis virus. I. Influence of age at exposure on mortality and congenital transmission.

Groups of White Leghorn chickens were inoculated at 1 day and at 2, 4, 6 and 8 weeks of age respectively with a mixture of leukosis viruses of subgroups A and B. The five infected groups were kept in a filtered air positive pressure house. A sixth group was accommodated separately in a similar house as a control. All birds which died or were removed were subjected to pathohistological examination; diagnosis of lymphoid leukosis was made upon either gross lesions plus microscopical lesions or microscopical lesions only. The incidence of lymphoid leukosis in the infected groups appeared inversely proportional to age of infection, i.e. the mortality due to lymphoid leukosis decreased from 54.3% in the group infected at 1-day-old to 7.4% in the group infected at 8 weeks of age. Prevalence of leukosis in the latter group may be attributed to a small number of chicks already infected vertically with the virus. Congenital transmission of leukosis virus was demonstrated in embryos in the groups infected at 1-day-old, 2, 4 and 6 weeks of age. In the latter group congenital transmission was extremely low; from 214 pooled embryo extracts (1007 embryos) only 2 (0.9%) contained leukosis virus. In the group infected at 8 weeks of age no virus was detected in the embryos. Congenital transmission of leukosis virus appeared to be related to age of infection, i.e. early infection went parallel with a high rate of transmission. The pattern of congenital transmission was erratic and the number of hens shedding leukosis virus continually was small. From the results in this trial it is concluded that both lymphoid leukosis and congenital transmission occur rarely if chickens (born free of leukosis virus) are kept free from infection during the first 6-8 weeks of life and subsequently are injected with a relatively high dose of leukosis virus.

The control of lymphoid leukosis in a flock White Plymouth Rock chickens.

Summary Lymphoid leukosis (LL) was successfully controlled in a commercial basic breeding line of White Plymouth Rock chickens. The control method has been developed for breeder flocks and consists of three elements: - In the flock under study, homogenates of embryos from all eggs collected during a number of I4-day periods are tested for the presence of LL viruses. - Only eggs from hens that have been shown not to shed virus in their eggs are used for the production of progeny. The offspring are reared in isolation during the first two months of life, at which time the age-related resistance against tumour formation by LL viruses appears to be sufficiently developed. - The chickens are subsequently inoculated intramuscularly with LL viruses of subgroups A and B transferred to a conventional chicken house. The vaccination raises a solid immunity to horizontal LL virus exposure and, due to the age-related resistance, tumour formation does not follow. No excretion of LL viruses could be detected in three generations of White Plymouth Rock chickens to which the three elements of the control procedure were applied. Clinical disease was not observed in any of the chickens under notice.

Absence of Marek's disease virus antigen in the feather follicle epithelium of vaccinated chicks demonstrated by immunofluorescence

In chickens vaccinated with MDV strain CVI 988, HVT strain FC 126 or PB-THV 1, no MDV-specific antigens could be demonstrated in the feather follicle epithelium by immunofluorescence (IF). In chickens given virulent MDV strains, the epithelium of the feather follicle was positive in IF. In an experiment where chickens were vaccinated with strain CVI 988, positive IF was observed in the lung, bursa and pancreas, but not in the feather follicle epithelium, kidney, cloaca, or caecal tonsils. Absence of IF antigen in the skin may be indicative of avirulence of the strain of MDV.

Evaluation of resistance to Marek's disease in three white Plymouth rock unes and in two reciprocal crosses

Two White Plymouth Rock lines (WPR), both developed into lines resistant for Mareks disease (MD), and one commercial WPR line, supposedly susceptible to the disease, were mated in a number of combinations to evaluate resistance to MD in their pure- and crossbred progeny. For this purpose the pedigreed 1-day-old progeny chicks were exposed to 3-week-old spreader chicks which had been inoculated at 1-day-old with virulent MD virus (MDV) and reared with them for the whole experimental period of 15 weeks. Response to the challenge showed that resistance to MD in the cross-breds was intermediate to that of resistant and susceptible parental lines. It appeared that, besides a sex effect in the male progeny, the trait for MD resistance was most effectively transferred to the crossbred progeny by the resistant male parents. The rather high level of resistance to MD observed in the progeny of crossmatings between resistant males and susceptible females suggested that resistant males can be employed for upgrading MD resistance in (commercial) stock.

The development of two White Plymouth Rock lines resistant to Marek's disease by breeding from survivors.

A programme to develop resistance to Mareks disease (MD) in chickens in two non-inbred White Plymouth Rock (WPR) lines by breeding from survivors was initiated in 1968 and since then nine generations have been produced. In each generation only sires and dams which survived heavy exposure to virulent MD virus (MDV), Dutch strain K, either by inoculation (for the first two generations) or by contact-exposure (for the following seven generations) were used. In this long term trial a high level of resistance to MD was reached within five generations; thereafter no marked further increase in resistance could be obtained. Susceptibility to MD, reflected by MD mortality in each generation, was expressed as % incidence of MD. Statistical evaluation of MD susceptibility in the nine generations of the two lines revealed a significant interaction between MD mortality and two sources of variation, sex and generation. No overall line effect was found, but there was a significant line x generation interaction. The remaining interactions were not significant. Results indicate that breeding from survivors for development of resistance to MD is feasible. Exposure of birds to either artificial (by injection) or contact challenge with virulent strain K MDV changed lines of birds highly susceptible to MD into highly resistant lines in a few generations. Complete resistance to MD was not attained.

Hyperplasia of proventricular duct epithelium in broilers

Spontaneous non-leukotic tumours of the proventriculus are regarded as rare neoplasms of the digestive tract in domestic fowl. In a broiler flock, however, a high incidence of hyperplasia of glandular tissue in the proventriculus has been observed. The condition was correlated with a rather high mortality and condemnation rate. Macroscopically the proventriculi appeared swollen and had thickened, firm walls. Microscopically the epithelial cells lining the ducts of the proventricular glands showed a marked hyperplasia, and several ducts were conspicuously convoluted and formed prominent stellate lumina. Neither proliferation of glandular epithelial cells into the surrounding tissue nor metastases to other organs were detected, and the condition was classified as a hyperplasia of the duct epithelium. The aetiology of the hyperplasia and the striking mortality is unknown, although the presence of a toxic factor(s) in the litter of the chicken-house cannot be entirely ruled out.