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Biomedical subjects

J M Sharma

Publications and source records attributed to J M Sharma.

At least 127 records · Page 7Linked to original sources
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Embryo vaccination of specific-pathogen-free chickens with infectious bursal disease virus: tissue distribution of the vaccine virus and protection of hatched chickens against disease.

Vaccination of specific-pathogen-free chickens as 18-day embryos with the BVM isolate of infectious bursal disease virus (IBDV) resulted in extensive replication of the vaccine virus in the embryonic tissues. The virus was recovered from lung, thymus, proventriculus, liver, kidney, and spleen of embryos 1 day postvaccination, and recoverable virus persisted for at least 7 days. Replication and spread of the vaccine virus in chickens vaccinated as 18-day embryos was compared with that in chickens vaccinated at hatch. Distribution of the virus in tissues was more extensive, virus levels in tissues were generally higher, and detectable virus persisted longer in chickens vaccinated as 18-day embryos than in those vaccinated at hatch. Effective vaccine response could be initiated with 6.2 median embryo lethal doses, the lowest dose tested. Chickens immunized as embryos developed neutralizing antibody against IBDV and resisted challenge with pathogenic IBDV at 4, 6, 8, and 10 weeks of age.

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Effect of infectious bursal disease virus on protection against Marek's disease by turkey herpesvirus vaccine.

Chickens inoculated at hatch with pathogenic infectious bursal disease virus (IBDV) and the turkey herpesvirus (HVT) vaccine were poorly protected against challenge at 1 week with virulent Marek's disease virus (MDV). However, if vaccination with HVT preceded IBDV inoculation such as by embryonal vaccination with HVT and exposure to IBDV at hatch, or by vaccination at hatch and IBDV exposure 5 days or longer postvaccination, then the vaccinal efficacy of HVT was not influenced. Further, the inhibitory effect of pathogenic IBDV was transient: chickens that received IBDV and HVT at hatch were poorly protected against challenge with virulent MDV at 1 week but not against challenge at 2 weeks of age. Pathogenic IBDV routinely interfered in vaccine efficacy of HVT in chickens lacking maternal antibody to IBDV but not in chickens that had such antibodies at the time of exposure to IBDV. In certain experiments, pathogenic IBDV caused a high rate of mortality in newly hatched chicks. Chickens receiving IBDV and MDV had a lower incidence of Marek's disease than those receiving MDV alone.

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Development and characterization of a Marek's disease transplantable tumor in inbred line 72 chickens homozygous at the major (B) histocompatibility locus.

A serially transplantable Marek's disease (MD) tumor, designated MDCT-RP-3, was developed from an MD-virus-induced lymphoma (GA strain) in a pedigreed female chicken of the inbred B-histocompatible (B2/B2), line 72, and is the first MD tumor transplant to be developed in chickens both syngeneic and selected for susceptibility to MD. The MDCT-RP-3 tumor maintained its female karyotype through at least 80 passages in male 72 chickens. High doses of tumor cells caused progressively growing tumors at 5 days postinoculation and death of young 72 chicks in 7-10 days, whereas allogeneic chicks of other lines were less susceptible. Tumors frequently regressed when doses of tumor cells were low or older chickens were used. MD virus was rescued from MDCT-RP-3 cells in cell culture, and chickens surviving the early transplant response sometimes developed MD lymphomas. The tumor cells expressed MD-tumor-associated surface antigen (MATSA) and T-cell surface antigens. A serum raised in rabbits against MDCT-RP-3 cells and absorbed with normal 72 cells appeared to be reactive against MATSA on all MD tumor cells tested and is probably monospecific. Sera raised against MDCT-RP-3 in chickens also contained MATSA antibodies reactive against heterologous but not homologous MD tumor cells. Protection against transplantation of MDCT-RP-3 cells was not afforded by immunization with turkey herpesvirus vaccine. Some unvaccinated chickens that regressed MDCT-RP-3 transplant appeared to be partially immune to later development of MD lymphomas.

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Natural killer cell activity in chickens exposed to Marek's disease virus: inhibition of activity in susceptible chickens and enhancement of activity in resistant and vaccinated chickens.

Chickens of 2 genetic lines (lines P and N) were inoculated with a pathogenic strain of Marek's disease (MD) virus (MDV) and chronologically examined for disease response and natural killer (NK) cell expression. The NK cell reactivity was assayed in an in vitro cytotoxicity assay in which effector cells from the spleen of test chickens were reacted with 51Cr-labeled LSCC-RP9 target cells. Chickens of line P developed progressive debilitating disease and a high incidence of gross tumors and death. The NK cell reactivity of line-P chickens infected with MDV was significantly lower than that of uninfected control hatchmates. In contrast, NK cell levels were significantly elevated in MDV-inoculated line-N chickens that were resistant to MD and in chickens of lines P or N that had been inoculated with herpesvirus of turkeys (HVT). NK cell levels were also elevated in line P if chickens were vaccinated with HVT before infection with MDV. Inhibition of NK reactivity in susceptible chickens and elevation of reactivity in naturally resistant or vaccinated chickens may indicate a role for the NK cell system in regulating resistance to MD.

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Induction of lymphomas and tumor antigen by Marek's disease virus in turkeys.

Pathogenic Marek's disease (MD) virus (MDV) induced a high incidence of mortality and gross and microscopic lesions in turkey poults. Inoculated turkey poults became persistently viremic with MDV, although the levels of detectable circulating MDV were generally lower in turkeys than in similarly inoculated chickens. The early lytic phase of MD characterized by lymphoid cell destruction and the appearance of viral antigen in lymphoid organ was not as prominent in turkeys as in chickens. Gross MD lesions in turkeys were most prevalent in liver and spleen; peripheral nerves were involved infrequently. MD tumors in turkeys contained cells that reacted with antiserum prepared against chicken MD-tumor-associated surface antigen (MATSA); this result indicated that MDV induced cellular transformation in turkeys and the tumor-associated antigen in chicken and turkeys cross-reacted. Several in vitro-propagating B-cell lines were developed from the turkey lymphomas. As in chickens, MD in turkeys also resulted in immunodepression. Circulating lymphocytes from turkeys that eventually died of MD or had gross tumors at the end of the experiment were deficient in mitogenic response to Concanavalin A.

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Resistance to Marek's disease at hatching in chickens vaccinated as embryos with the turkey herpesvirus.

Chickens vaccinated with herpesvirus of turkey (HVT) as 18-day embryos or at hatching were challenged as neonates with pathogenic Marek's disease (MD) virus (MDV). Embryonally vaccinated chickens had much greater resistant to challenge than chickens vaccinated post-hatch. Embryos became readily infected with HVT regardless of whether the vaccine was deposited into the body of the embryo or extraembryonally, such as in the amniotic sac. Embryonally vaccinated chickens were viremic with HVT at hatching and remained persistently viremic through the duration of the experiment. The titer of recoverable virus was higher in the embryonally vaccinated chickens than in the chickens vaccinated post-hatch. Embryonal vaccination did not affect hatchability. Vaccination at any stage of embryonation tested protected better against neonatal challenge than did vaccination at hatching. Protection against an early challenge was greatest when the embryos were 17 or 18 days old at the time of vaccination. Lower protection in chickens vaccinated as 11-day embryos was not due to humoral immunologic tolerance. Chickens vaccinated at the 11th day of embryonation were poorly protected against MDV challenge at three or eight days of age but were well protected if the challenge was delayed until the 14th day of age.

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Influence of maternal antibody on efficacy of embryo vaccination with cell-associated and cell-free Marek's disease vaccine.

Vaccination with turkey herpesvirus (HVT) of 18-day-old chicken embryos from a commercial source or from a cross (15 X 7) of two inbred lines induced better protection against early post-hatch challenge with virulent Marek's disease virus (MDV) than vaccination at hatch, despite the presence in embryos of maternally derived antibodies to HVT or to HVT and MDV. However, 50%-protective-dose (PD50) assays revealed that maternal antibodies in embryos reduced vaccine efficacy. The PD50 assays were conducted by vaccinating 15 X 7 embryos with serial dilutions of HVT at the 18th day of incubation. Embryonally vaccinated and unvaccinated chicks were challenged with MDV on the day of hatch. In the absence of maternal antibodies, the PD50 values in plaque-forming units for cell-associated and cell-free HVT were 57 and 328, respectively. In the presence of maternal antibodies, PD50 values for cell-associated and cell-free HVT were 105 and greater than 4,000, respectively.

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Embryo vaccination against Marek's disease with serotypes 1, 2 and 3 vaccines administered singly or in combination.

Marek's disease virus (MDV) vaccines of serotypes 1 and 2 administered in 18-day-old embryonated eggs induced better protection against post-hatch challenge at 3 days with virulent MDV than vaccines given at hatch. Embryonal vaccination with a polyvalent vaccine containing equal quantities of serotypes 1 and 2 of MDV and serotype 3 virus (turkey herpesvirus, HVT) was also significantly more effective than post-hatch vaccination. These and earlier results indicate that protective efficacy of single or combined Marek's disease vaccine serotypes against post-hatch challenge at 3 days can be substantially improved if the vaccines are injected into 18-day embryos rather than at hatch. Injection of vaccines of serotypes 1 or 2 into embryonated eggs or hatched chicks did not cause detectable gross or microscopic lesions in chickens. Vaccine viruses of serotypes 1 and 2 could be isolated from spleen cells of chickens 1 week post-vaccination, and the titer of recoverable viruses was higher in chickens that received the vaccines at the 18th day of embryonation than in chickens vaccinated at hatch. Although embryo vaccination with HVT usually provided better protection than post-hatch vaccination against early post-hatch challenge with variant pathotypes of MDV, the protection was poor regardless of vaccination protocol. If challenge with variant pathotypes of MDV was delayed until embryonally or post-hatch HVT-vaccinated chickens were 21 days of age, protection of chickens by HVT was not enhanced. Thus, resistance induced by embryonal vaccination with HVT was qualitatively similar to that induced by post-hatch vaccination with this virus.

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Field trials to test the efficacy of polyvalent Marek's disease vaccines in broilers.

The efficacies of trivalent (Md11/75C + SB-1 + HVT), bivalent (SB-1 + HVT), and turkey herpesvirus (HVT) vaccines against Marek's disease (MD) were compared in commercial broiler flocks in four trials involving 11 farm locations and 486,300 chickens. In all four trials, chickens receiving polyvalent vaccines had lower leukosis (MD) condemnation rates than chickens vaccinated with HVT alone; when data were summarized for each vaccine type in each trial, condemnation rates for the bivalent- or trivalent-vaccinated groups were 56-96% (mean 78%) lower than those for HVT-vaccinated chickens. Polyvalent vaccination was clearly mor efficacious than HVT in 8 of 11 individual farms, although it did not always reduce leukosis condemnations to acceptable levels. Body weights of chickens vaccinated with polyvalent vaccines did not differ consistently from those vaccinated with HVT. Chickens inoculated with the trivalent vaccine had slightly lower overall leukosis condemnation rates (0.24%) than those inoculated with the bivalent vaccine (0.45%) in trials 1-3, where direct comparisons were made. Bivalent vaccines containing either 1,500 or 200 plaque-forming units of SB-1 virus were equally effective; thus, HVT may need to be supplemented with only small amounts of SB-1 to obtain the benefits of protective synergism. SB-1 virus did not appear to carry over from polyvalent-vaccinated flocks to subsequent HVT-vaccinated flocks in the same houses, even when old litter was used.

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Cryopreservation of avian lymphoid cells.

Conditions were standardized for optimum cryopreservation of avian lymphoid cells. Several factors that influenced cryopreservation were examined. The optimum procedure was as follows: a maximum of 50 x 10(6) cells/ml were suspended in the freezing medium, which contained 5-10% dimethyl sulfoxide (DMSO), and the cell suspension was frozen slowly at a rate of -1 C degree/min. The frozen cells were kept at -196 C. Cryopreserved cels were thawed rapidly in a 37 C water bath until the last ice crystal had thawed. The method of diluting out DMSO from thawed cells was critical, and results were best if dilutions were made at room temperature (20-25C) with diluent prewarmed to room temperature. Dilution was begun by adding 0.1 ml diluent to 1.0 ml freshly thawed cell suspension. Thereafter, diluent was added by doubling the volume after each 1-min interval until 12.7 ml of diluent had been added over 6 min. The recovery of viable cells from cryopreserved cells varied from 51.2% to 98.3% (mean 86.0%) for lymphoblastoid line cells and from 24.8% to 87.2% (mean 50.6%) for spleen cells obtained from normal chickens. Viable cryopreserved cells were reactive in a 4-hr Cr-release cytotoxicity assay and responded vigorously to phytohemagglutinin. The standardized method of freezing and thawing avian lymphoid cells may facilitate preservation of large stocks of standard reference cells with predetermined functions for laboratory studies, particularly those involving in vitro assays of cellular immunity.

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Response of specific-pathogen-free turkeys to vaccines derived from marble spleen disease virus and hemorrhagic enteritis virus.

Tissue-culture-propagated marble spleen disease virus (MSDV-TC) and two preparations of spleen homogenate (MSDV-SH and MSDV-SH-TC) were compared as anti-hemorrhagic enteritis virus (HEV) vaccines in specific-pathogen-free turkeys. Both types of vaccines spread horizontally among turkeys, induced anti-HEV antibodies, and protected turkeys against challenge with virulent HEV. Antibody development and horizontal spread of virus occurred earlier in turkeys given MSDV-SH or MSDV-SH-TC than in those given MSDV-TC. Virulent HEV was serially passed in MDTC-RP19 cells. The 30th passage virus (HEV-P30) was nonpathogenic for turkeys but was immunogenic. Turkeys exposed to HEV-P30 had viral antigen in the spleen, developed neutralizing antibodies, and resisted virulent HEV. The principal difference between MSDV-TC and HEV-P30 vaccines was that MSDV-TC caused well-defined splenomegaly in turkeys, whereas HEV-P30 protected turkeys without causing spleen enlargement.

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Flow cytometric analysis of B cell and T cell subpopulations in specific-pathogen-free chickens infected with infectious bursal disease virus.

Lymphocytes obtained from the blood, spleen, and bursa of normal chickens and of chickens infected with infectious bursal disease virus (IBDV) were analyzed for phenotypic expression of CT4, CT8, and immunoglobulin cell surface markers. Single-cell suspensions were stained with monoclonal antibodies by an indirect immunofluorescent assay, and percent staining was quantitated by flow cytometry. Although an appreciable decline from control levels in the percentage of lymphocytes expressing IgM was detected in the spleen and bursa of infected chickens, the relative proportions of lymphocytes expressing CT4 and CT8 in peripheral blood and spleen remained unchanged following infection. These results suggest that whereas humoral immune depression by IBDV may be associated with lysis of antibody-producing B cells, cellular immune depression is not associated with a detectable change in the proportion of helper or cytotoxic/suppressor subpopulations of T lymphocytes.

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Presence of lesions without virus replication in the thymus of chickens exposed to infectious bursal disease virus.

Specific-pathogen-free (SPF) chickens were exposed to the IM and VA isolates of virulent infectious bursal disease virus (IBDV). Both viruses induced rapidly progressing lymphoid cell depletion in the bursa. The bursal lesions persisted through the observation period of 16 days. The virus-exposed birds also had histologic lesions in the thymus. Thymic lesions peaked at 3-4 days postinoculation (PI) and then subsided. Immunofluorescence (IF) and antigen-capture enzyme-linked immunosorbent assay (ELISA) detected abundant viral antigen in the bursa, but not in the thymus, of chickens during the first week after infection with IM-IBDV or VA-IBDV. This result indicated that the presence of histologic lesions in the thymus was not associated with active infection and replication of the virus in thymic cells. Inoculation of homogenates of bursal and thymic tissues from virus-exposed chickens into embryonated chicken eggs revealed the presence of infectious virus from both tissues. We speculated that the virus recovered from thymus may have been contributed by virus-infected cells that were circulating through the thymus at the time when this tissue was homogenized.

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Protection against hemorrhagic enteritis and Newcastle disease in turkeys by embryo vaccination with monovalent and bivalent vaccines.

The feasibility of embryo vaccination against Hemorrhagic enteritis (HE) and Newcastle disease (ND) in specific-pathogen-free turkey embryos was studied. Turkey eggs were injected with marble spleen disease virus (MSDV) at embryonation day (ED) 24, and tissues of poults hatching from virus-injected eggs were examined for MSDV. The virus was detected in spleen, intestine, liver, and bursa between 4 to 10 days postinoculation (PI). The peak titer of MSDV was present in the spleen at 6 days PI. Poults hatching from eggs injected with MSDV produced antibodies to the virus and resisted a challenge with virulent hemorrhagic enteritis virus (HEV) at 4 weeks of age. The B1 strain of NDV (NDV-B1) injected in turkey eggs at ED 24 killed the embryos. NDV-B1 modified by treatment with ethylmethane sulfonate (NDV-B1-EMS) was not lethal for turkey embryos. Poults hatching from eggs injected at ED 24 with NDV-B1-EMS developed antibodies to NDV and were protected from challenge exposure with virulent NDV at 4 weeks of age. Poults from eggs inoculated at 24 ED with a bivalent vaccine containing MSDV and NDV-B1-EMS developed antibodies to both viruses and were resistant to challenge with both virulent viruses. The study showed that SPF turkeys may be immunized by in ovo injection of live viral vaccines.

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Embryo vaccination of chickens with infectious bronchitis virus: histologic and ultrastructural lesion response and immunologic response to vaccination.

Chicken embryos 18 days of age and newly hatched chicks were vaccinated with an infectious bronchitis virus (IBV) vaccine (V-IBV) or with an IBV vaccine that had been serially passaged 40 times in chick kidney tissue culture (P-IBV). Immunologic and pathologic changes in the chicks were compared at selected intervals until the 35th day. Pathologic changes were evaluated by light, transmission, and scanning electron microscopy. Immunologic changes were assayed by a constant virus-diluting serum plaque-reduction test in chicken cell cultures, by 51Cr-release cytotoxicity assays, and by phytohemagglutination (PHA) responses. Embryos vaccinated with P-IBV and 1-day-old chicks vaccinated with V-IBV had similar transient lesions that were confined primarily to the trachea. Embryo vaccination and posthatch vaccination induced similar primary and secondary antibody responses in chicks. It was concluded that neither vaccination technique consistently influenced PHA response of whole blood cells or natural killer cell reactivity of spleen effector cells. Additionally, effector cells cytotoxic to IBV-infected target cells were not detected in chicks vaccinated as embryos or at hatch. The pathologic and immunologic effects of vaccination with P-IBV were comparable to those induced by conventional vaccination of chicks.

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An antiviral effect of nitric oxide: inhibition of reovirus replication.

We have previously shown that macrophages from chickens infected with avian reovirus are primed to produce nitric oxide (NO) in response to T cell cytokines and bacterial lipopolysaccharide (LPS). We now show that NO exerts potent antireovirus effects. Reovirus replication was substantially reduced in a chicken macrophage cell line, HD11, induced to make NO by stimulation with LPS or conditioned medium from concanavalin A-stimulated spleen cells. The use of a competitive inhibitor of nitric oxide synthase, NG-monomethyl-L-arginine, reduced the antiviral effect of LPS-stimulated HD11 cells. Cytostatic effects were concurrent with the observed antiviral effects of NO. Among these cytostatic effects were reduction in DNA synthesis, protein synthesis, and mitochondrial metabolism. These results indicated that a potential consequence of macrophage priming following virus infection is the protection of cells against virus-induced replication and cytopathic effects, and this protection may be mediated by the cytostatic effects of NO on the host cell.

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