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Biomedical subjects

R L Witter

Publications and source records attributed to R L Witter.

At least 127 records · Page 7Linked to original sources
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Turkey herpesvirus infection in chickens: induction of lymphoproliferative lesions and characterization of vaccinal immunity against Marek's disease.

Chickens vaccinated at hatching with high doses of turkey herpesvirus (HVT) developed viremia that peaked in titer around the 12th day and gradually declined. HVT infection also induced mild microscopic lymphoproliferative lesions in the nerves and gonads. These lesions were most prominent around the 12th day and then regressed. The fact that such lesions were also induced by HVT in cyclophosphamide-treated chicks suggests that they were T-cell-dependent. Some of the cells in early HVT lesions appeared to have morphologic properties of neoplastic cells. HVT viremia and lesions were both dose-dependent and were less in chickens with maternal antibodies against Marek's disease virus (MDV). Sequential studies on chickens vaccinated with HVT and challenged with MDV showed that chickens were protected against the earliest detectable MD viremia and lymphoproliferative lesion response attributed to MD. Also, the transient necrobiotic lesions associated with productive infection of thymic lymphocytes by MDV were totally absent in vaccinated chickens. These data provide further insight on the mechanisms by which HVT protects against MD lymphoma induction. A limited oncogenic (transforming) potential of HVT as suggested by our data would provide the basis to assume that at least one component of HVT-induced immunity may be directed against tumor-specific antigens. On the other hand, our observations that HVT protects against productive MDV infection in the thymus and against cell-associated viremia are evidence for an anti-viral immune response. These hypotheses are not mutually exclusive.

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Studies on the etiology of lymphomas in turkeys: isolation of reticuloendotheliosis in virus.

Studies of tissues from 25 normal turkey flocks and of 15 selected turkey lymphoid tumors revealed no Marek's disease or lymphoid leukosis viruses, although reticuloendotheliosis viruses (REV) were isolated from 3 flocks (2 with a history of neoplastic disease). The REV isolates were culturally and antigenically similar to the prototype REV strain T but were of low pathogenicity. Attempts failed to transmit 6 lymphoid tumors of diverse origin by inoculation into 2 strains of recipient turkeys. These data support an etiological role for REV in some though not all forms of turkey neoplastic disease.

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A nonproducer T lymphoblastoid cell line from Marek's disease transplantable tumor (JMV).

A continuous lymphoblastoid cell line was established from a JMV tumor transplant related to Marek's disease (MD). It is designated RPL1 (JMV) lymphoblastoid cell line. This cell line contains DNA sequences complementary to MD virus DNA and has an antigen similar to MD-tumor-associated surface antigen (MATSA). However, it lacks any MD virus (MDV) rescuable in vivo or in vitro. The cell line has surface antigens typical of chicken thymus cells (T cells) and histocompatability antigens different from those of the host chicken.

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Lymphoid leukosis: interrelations among virus infections in hens, eggs, embryos, and chicks.

Hens from a commercial source were selected because they were infected with lymphoid leukosis virus (LLV). LLV was detected in vaginal swabs from 17 viremic hens and from 27 of 44 hens that were not viremic. All hens that were positive on the vaginal swab test (VST) produced one or more eggs with virus in albumen or in embryos, whereas in comparable tests, virus was detected only in eggs from 5 of 17 hens that were negative on VST. Congenital transmission of LLV was erratic and neither the VST nor tests for virus in egg albumen prior to incubating eggs identified all hens that transmitted infection. For example, 14 hens negative on VST produced 50 eggs negative for virus in albumen and yet one of the embryos from these eggs was infected. Eggs from other hens had infectious virus in albumen and about half of the embryos from these were infected. Tests for virus in cloacal swabs from one-day-old chicks were as sensitive as tests on embryos for detecting congenital transmission. Titers of LLV in the meconium of congenitally infected chicks were as high as 10(7) infectious units per ml. The cloacal swab test should be a valuable adjunct to the VST and tests on egg albumen in programs designed to eradicate lymphoid leukosis from chickens.

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Complement-fixation test for reticuloendotheliosis viruses: limits of sensitivity in infected avian cells.

A specific micro-complement-fixation procedure for assay of avian reticuloendotheliosis viruses (REV), designated by use as the COFAR test, was compared with an assay based on immunofluorescent (IF) antibody staining of infected chick embryo fibroblasts. Endpoint titrations in which REV strain T, chick syncytial virus, and spleen necrosis virus were used indicated that cultures infected with limiting dilutions of each strain were positive by both procedures within 6 days. Depending on cell density, infection of 2 to 9% of the cells cultured produced an unambiguous positive response (titer greater than or equal to 1:2) with COFAR test. When both tests were used in a study on the transmission and in vivo status of ducks infected with spleen necrosis virus, COFAR was no less, possibly more, sensitive than IF for detecting infection in cultures inoculated with plasma.

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Studies of flocks with high mortality from lymphoid leukosis.

Two pairs of commercial white-egg parent flocks were selected for study, because one of each pair was observed to be dying with lymphoid leukosis at a high rate. The proportion of each flock producing eggs with lymphoid leukosis virus in the albumen was studied. In one pair the rate of birds shedding was no different in the high- and low-mortality flocks. The total rate of shedding was lower in the other pair, but the rate was higher in the high-mortality flock. These data indicate that the rate of virus infection is not always proportional to the development of lymphoid leukosis, and that other environmental factors may play a role. Direct complement-fixation tests on the albumens which were positive for lymphoid leukosis virus showed that group-specific antigen could be detected in 83%. Therefore, the direct complement-fixation test can be used on albumens as a rough estimate of shedding rate.

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Duration of vaccinal immunity against Marek's disease.

Specific vaccinal immunity against Marek's disease (MD) virus was measured by a short-term challenge test in which chickens vaccinated with turkey herpesvirus (HVT) and control chickens at various ages up to 40 weeks were inoculated with virulent MD virus and evaluated 2 weeks later for microscopic lesions and MD viremia. Vaccinal immunity induced by high (2,900 PFU) and low (29 PFU) doses of HVT persisted without detectable reduction through the 40th week in line 151 chickens, thus indicating that resistance of older vaccinated chickens to MD may not be based solely on general age resistance (immunocompetence) or on subsequent exposure to field MD virus. The HVT viremia titers (number of buffy-coat cells inducing virus plaques in vitro) decreased with increasing age, but even chickens from which no HVT was isolated were usually immune to MD. The dose of vaccine appeared to influence, at least slightly, the magnitude of MD immunity and vaccine virus viremia.

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Biological diversity among serotype 2 Marek's disease viruses.

Selected biological characteristics were determined for 14 low-passage serotype 2 Marek's disease virus (MDV) isolates. Four of these isolates were also tested after extensive serial passage in chicken embryo fibroblast cultures. Observations were made on replication in vitro and in vivo, pathogenicity by in ovo inoculation, antigenicity, and protection against virulent MDV challenge. Among the low-passage isolates, there were some differences in pathogenicity after in ovo inoculation but relatively little difference in other characteristics, with the exception of the HN-1 strain, which replicated more rapidly in cell culture but produced generally lower in vivo responses than other isolates. After extended in vitro passage, isolates replicated much more readily in cell culture and produced lower pathologic responses in vivo than low-passage isolates, as has been reported for serotype 1 isolates. No antigenic differences among isolates were detected, but high-passage isolates induced lower levels of precipitating antibodies than low-passage isolates, indicating a possible reduction in A antigen production. The observed diversity associated with strain and passage level may be of value in the selection of optimum vaccine strains.

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Low incidence of lymphoid tumors in chickens continuously producing endogenous virus.

A flock of 258 male and 243 female chickens of a cross of Regional Poultry Research Laboratory lines 15B and 7(2) were kept in a filtered-air positive-pressure house and observed for tumors from 100 to more than 729 days of age. These birds produced high titers of a subgroup E endogenous virus from the middle of the embryonic incubation period through the end of the experiment. No neoplasms were observed in the males. The females had two neoplasms indistinguishable from lymphoid leukosis and three other neoplasms not involving lymphoid cells. No evidence was found of infection with exogenous lymphoid leukosis viruses, Marek's disease virus, reticuloendotheliosis virus, or adenovirus (isolated on the isolation farm). Inoculation of another sample of this cross with a lymphoid leukosis virus of subgroup A resulted in 88% mortality with neoplasms (mostly lymphoid leukosis) by 167 days of age. The conclusion is that high levels of spontaneously produced endogenous virus do not induce high levels of neoplasms in chickens susceptible to lymphoid leukosis.

Alpharetrovirus↗
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Vertical transmission of reticuloendotheliosis virus in breeder turkeys.

Epizootiological studies were conducted on a commercial turkey breeder flock naturally infected with nondefective reticuloendotheliosis (RE) virus. Although RE virus was isolated from 27 (46%) of the 59 hens studied, only 4 of the 45 hens tested transmitted RE virus to progeny during a 6-week observation period and the overall transmission rate was 1.8%. The transmitter hens were of two types: three hens were consistently viremic and antigenemic and lacked antibody; one hen was viremic but lacked detectable viral antigen and possessed antibody. Toms appeared to play no role in vertical transmission of the infection. Of several tests evaluated for detection of transmitter hens, the direct enzyme-linked immunosorbent assay on albumen was probably best, since it detected three of four transmitter hens, detected relatively few nontransmitter hens, and had the best consistency of any test. No significant differences in hatchability were found between eggs from viremic and non-viremic hens. These findings can be utilized in the development of programs for eradication of RE virus from turkey breeding flocks.

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New serotype 2 and attenuated serotype 1 Marek's disease vaccine viruses: comparative efficacy.

Attempts were made, through selection of optimum viral strains, to develop improved vaccines against Marek's disease (MD). Seven attenuated serotype 1 strains and 22 avirulent serotype 2 strains, both alone and in combination with the FC126 strain of serotype 3, were screened for protective efficacy against challenge with virulent and very virulent MD viral strains. The three viruses selected as most promising were evaluated alone and in various combinations and compared with commercially available vaccines, including FC126, bivalent (FC126 + SB-1), and CV1988/C, in 12 separate assays. Two of these new viruses--301B/1 (serotype 2) and Md11/75C/R2 (serotype 1)--were exceptionally protective compared with prototype vaccine strains. Four new monovalent and polyvalent vaccines based on these two isolates protected chickens better than FC126 alone or CV1988/C alone. Three of these new vaccines provided better protection than the bivalent (FC126 + SB-1) vaccine. Protective synergism was noted commonly between viruses of serotypes 2 and 3 but only sporadically between serotypes 1 and 2 or between serotypes 1 and 3. Strain CVI988/C was protective but was no better than FC126 alone, and it was less effective than bivalent (FC126 + SB-1) vaccine, even when used as a bivalent vaccine with FC126 or SB-1.

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New serotype 2 and attenuated serotype 1 Marek's disease vaccine viruses: selected biological and molecular characteristics.

Two new Marek's disease vaccine viruses, Md11/75C/R2 (serotype 1) and 301B/1 (serotype 2), were evaluated in chickens with maternal antibodies (ab+) or without maternal antibodies (ab-). Strain Md11/75C/R2 was mildly pathogenic in ab--chickens, but this pathogenicity was markedly reduced in ab+ chickens. Md11/75C/R2 spread less by contact and replicated better, both in vivo and in vitro, than CVI988/C, another serotype 1 vaccine virus. Strain 301B/1 was similar to SB-1, another serotype 2 vaccine virus: both were nonpathogenic for ab--chickens, spread readily by contact, and replicated well in vivo. In vitro, 301B/1 grew more rapidly and produced larger plaques than SB-1. Notable characteristics of strain CVI988/C included absence of pathogenicity, poor replicative ability, and the absence of one epitope detected by a common serotype-1-specific monoclonal antibody. All four viruses could be distinguished from each other by restriction enzyme analysis of viral DNA. We conclude that Md11/75C/R2, although exceptionally protective, may require further attenuation. On the other hand, 301B/1, which in other studies induced higher levels of protection than SB-1, is nonpathogenic and may be considered for use as a commercial vaccine.

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Monoclonal-antibody-mediated enzyme-linked immunosorbent assay for detection of reticuloendotheliosis viruses.

An enzyme-linked immunosorbent assay (ELISA) is described for the detection of reticuloendotheliosis viruses (REVs). The assay uses a mixture of monoclonal antibodies (MCAs) prepared against a 62-kilodalton REV envelope glycoprotein (gp62) to capture antigen, rabbit anti-REV serum as detection antibody, and peroxidase-conjugated anti-rabbit IgG as indicator antibody. The MCAs were reactive with REV strain T, chick syncytial virus, and duck infectious anemia virus but unreactive against Marek's disease and avian leukosis viruses. The ELISA was compared with complement fixation test and REV immunofluorescent assay of infected fibroblasts, plasmas, and egg albumen from infected chickens. The lower limit of gp62 detection was about 120 ng of REV protein. Limit dilution of infectious REV was detected after 7-8 days of cultivation of infected fibroblasts.

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Effects of chemically or virus-induced immunodepression on response of chickens to avian leukosis virus.

The effects of chemically or virus-induced immunodepression on the infection profile (development of viremia and antibody) and shedding of avian leukosis virus (ALV) were studied in progeny chickens of experimental or commercial breeder flocks. Chickens were infected with ALV subgroup A by contact at hatching and by oral inoculation at 4-5 weeks of age. In the first experiment, chickens were inoculated with a virulent strain of infectious bursal disease virus (IBDV) at 1 day or 6 weeks of age. In the second experiment, chickens were neonatally treated with cyclophosphamide (CY), or were inoculated with strain T of reticuloendotheliosis virus (REV) at hatching, or were inoculated with strain JM of Marek's disease virus (MDV) at 2 weeks of age. The infection profile and cloacal shedding of ALV in chickens exposed to ALV and inoculated with immunodepressive viruses or CY were compared with those in hatchmates exposed only to ALV. In two of four chicken lines tested in the first experiment, shedding of ALV, as determined by virological assays of cloacal swabs at 22 weeks of age, was significantly higher in chickens infected with IBDV at 1 day of age than in uninfected hatchmates. The rate of shedding of ALV in one of these two lines was also significantly higher in chickens infected with IBDV at 6 weeks of age than in uninfected chickens. Further, the frequency of ALV-antibody detection at 22 weeks of age was significantly lower in chickens of these two lines infected with IBDV at 1 day of age than in uninfected chickens. In the second experiment, neonatal treatment with CY significantly increased the frequency of viremic chickens of both experimental and commercial flocks. The frequency of ALV-viremic chickens at 22 weeks of age was considerably higher in the REV- and MDV-inoculated groups (54% and 44%, respectively) than in control hatchmates (29%), but only in chickens of the commercial line. These findings suggest that chemically or virus-induced immunodepression may lead to an increase in rates of viremia and shedding of ALV in chickens infected with virus after hatching, especially in certain genetic lines.

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Epidemiology of reticuloendotheliosis virus in broiler breeder flocks.

Six broiler breeder flocks from two companies in Mississippi were tested at intervals for reticuloendotheliosis (RE) virus infection. Virus was isolated and antibody demonstrated in all six flocks. Infection was first detected at ages ranging from 13 to 47 weeks. Studies showed that neither congenital transmission from grandparent flocks nor treatment with contaminated vaccines was a likely source of infection; thus, exposure to RE virus was assumed to come from the environment. Virus was isolated from litter samples from two of the flocks, but no specific environmental infection source was identified. Infection rates of flocks differed between the two companies. Although adequate controls were lacking, no performance problems due to RE virus infection were apparent in breeder or broiler progeny flocks. However, the RE viruses isolated from these flocks were immunosuppressive and oncogenic when inoculated into day-old chicks. A moderate (3-16%) incidence of neoplasms was induced by contact exposure to these field isolates in the laboratory.

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