Restriction fragment analysis of fowlpox virus using Bgl I, Bam HI, Hha I and Sma I restriction endonucleases.

Fowlpox virus vaccine strain and two field isolates collected from out breaks of disease were purified from cell culture using sucrose gradient ultracentrifugation. The viral DNAs were digested with Bgl I, Bam HI, Hha I and Sma I restriction endonucleases and the fragment pattern was analysed on 0.7% agarose gel. Bgl I digestion produced 54 fragments of size ranging from 31.50 to 0.60 kb, having similar electrophoretic mobilities in both the vaccine strain and two field isolates. Only 9 well resolved and one unresolved or partially digested fragment were obtained after Bam HI digestion. A total number of 29 and 41 fragments were obtained with Hha I and Sma I respectively. Almost similar restriction fragment pattern was observed in vaccine strain and the field isolates. The total genomic size was calculated to be between 265.00 and 302.91 kb. The three viruses were found to be genetically similar.

Fowlpox virus structural protein immunogens and characterization of single band polypeptide.

A fowlpox virus isolate obtained from an outbreak of disease in a vaccinated poultry flock was propagated in chicken embryo fibroblast cell culture. Analysis of purified virus polypeptide on 7.5-15% gradient polyacrylamide gel revealed 45 structural polypeptides after Coomassie blue staining. The mol.wt. of polypeptides ranged between 225.53 and 10.50 kDa with total mol.wt. of 2650 kDa. Variable numbers of immunogenic virion polypeptides were detected in immunoblot with fowlpox virus infected chicken sera collected at different time intervals. A total of 29 polypeptides reacted with sera collected at 1st week post-infection and the number gradually declined to 27, 26, 20, 17 and 15 when reacted with 2nd, 3rd, 4th, 5th and 6th week post-infection sera, respectively. Reaction with fowlpox virus hyperimmune sera revealed 35 immunogenic polypeptides. A number of major and minor immunogens were detected. Antisera against seven major single band polypeptides including one double band polypeptide showed very low reactivity both in ELISA and serum neutralization test. Involvement of multigenic components in virus neutralization is indicated.

Comparative immunological and genomic characterization of fowlpox virus isolates.

Chickens infected with fowlpox virus (FPV) IVRI vaccine strain and two field isolates collected from clinical cases of disease (Bareilly isolate and Panchmahal isolate) produced humoral antibody response after 2nd week post-infection, with a noticeable variation in degree of immune response. Serum antibody titre peaked at 4th week post-infection with a titre of 25,600, 25,600 and 51,200 being detected in ELISA and neutralization index of 2.75, 2.43 and 3.12 in serum neutralization test (SNT) with IVRI vaccine strain, Panchmahal isolate and Bareilly isolate, respectively. Cellular immune response was detected as early as 1st week post-infection by leukocyte migration inhibition test (LMIT). Per cent migration inhibition too peaked at 4th week with a value of 40.30 +/- 3.45, 36.93 +/- 4.11 and 45.45 +/- 3.66 being detected with the three viruses respectively. The Hind III and Hae III restriction fragment profile of viral DNA showed almost similar pattern both in vaccine strain and two field isolates. Hind III digestion produced 47 well resolved fragments of sizes between 24.30 and 1.20 kb and the total genomic size was estimated to be between 305.81 and 306.06 kb. Hae III digestion revealed 34 well resolved fragments of sizes between 27.55 and 1.32 kb. The three viruses could not be differentiated on the basis of their genomic restriction pattern. However immunogenic and antigenic differences were noticed by ELISA and SNT tests.

Structural proteins of fowlpox virus vaccine strain and field isolates.

Structural polypeptides of IVRI vaccine strain and two field isoaltes of Fowlpox virus (Bareilly isolate and Panchmahal isolate) were analysed on SDS-PAGE and by immunoblotting technique. In 5%-20% gradient acrylamide gel 31, 29 and 31 polypeptide bands and in 7.5%-15% gradient gel 45, 37 and 39 polypeptide bands were detected after Coomassie blue staining respectively for Bareilly isolate, Panchmahal isolate and IVRI vaccine strain. The molecular weight (MW) of the polypeptides ranged from 226.10 to 10.30 kDa with total MW of 2650.12, 2259.50, and 2378.68 kDa respectively for the three viruses. The immunoblot revealed 35, 29 and 30 immunogenic polypeptides indicating most virion polypeptides to be immunogenic in nature. Although most polypeptides had similar electrophoretic pattern both in SDS-PAGE and immunoblotting, still the three viruses could be differentiated. The viruses were found to be antigenically different with Panchmahal isolate lacking the polypeptides 81.15, 76.33, 39.30, 37.50 and 29.35 kDa and the vaccine strain lacking 76.33, 37.50 and 29.35 kDa polypeptides as were present in Bareilly isolate.

Polyclonal B-lymphocyte activation of sheep by Mycobacterium phlei against sheep pox virus.

A trypsinized preparation of Mycobacterium phlei, non specific stimulator of immunity (NSI), and Sheep Pox Virus (SPV) were inoculated in different groups of sheep to activate B-lymphocytes and induce SPV neutralizing substance(s). NSI sensitized sheep B-lymphocytes in the presence of NSI or lymphokine elaborated SPV neutralizing substance(s). The SPV sensitized B-lymphocytes also mediated such neutralizing substance(s). Healthy control sheep B-lymphocytes failed to show any appreciable amount of viral neutralizing substance. However, a significant virus neutralizing substance(s) was detected when healthy sheep B-lymphocytes were cultured in presence of NSI antigen along with lymphokines.

Enhanced production of antibody with specific antigen.

On inoculation of nonspecific stimulator of immunity (NSI), prepared from Mycobacterium phlei (M. phlei), simultaneously along with sheep pox virus (SPV) in sheep, the recipient has exhibited appreciable level of SPV specific antibody as early as on 10th day which reached at peak level on 20th day and remained unaltered on 30th day of postimmunisation as evinced by serum neutralisation test (SNT), enzyme linked immunosorbant assay (ELISA) indirect, fluorescent antibody technique (FAT) indirect, counter immunoelectrophoresis (CIEP) and finally by virulent SPV challenge. On the contrary, sheep, when immunised with SPV only could not produce appreciable level of antibody on 10th day but did so on 20th day of inoculation. SPV and NSI immunised sheep produced enhanced protection against virulent SPV challenge in comparison with sheep immunised with SPV only. Healthy control sheep, however, could not resist challenge.

Induction of cell mediated immune response by nonspecific stimulator of immunity against antigenically unrelated oncogenic virus (Marek's disease virus).

An enzyme treated preparation of Mycobacterium phlei (NSI), induced strong cell mediated immune response (CMIR) against specific as well as against nonspecific oncogenic Marek's disease (MDV) in birds, as evinced by Lymphocyte migration inhibition, (LMIT) lymphocyte transformation test (LT) and Lymphokine (Lymphocyte migration inhibition factor) LyIF assay. Maximum CMIR could be observed towards third week post inoculation. All the three tests exhibited a positive correlation. Such phenomenon of CMIR induction by NSI, nonspecifically to unrelated viral/cancerous diseases (MD) in birds generates hopes for immunoprevention of these maladies by utilizing such phenomenon.

Nonspecific immunoprotection against Rous sarcoma tumor in chicks.

An enzyme treated preparation of saprophytic Mycobacterium phlei, referred as NSI, when administered intramuscularly has been found to protect the chicks against Rous Sarcoma Virus induced tumor. A protection level of 35.4%, 24.1% and 21.2% were observed when challenged on 10th, 20th and 30th day post NSI inoculation. The tumor growth inhibitory-activity of NSI was significant (P less than 0.01). Both, systemic and intralesional administration of NSI exhibited significant tumorostatic activity (P less than 0.05). NSI stimulated the cell mediated immune response to specific as well as to nonspecific Rous sarcoma antigen. These studies indicated the immunopreventive activity of NSI against Rous sarcoma tumor which had an immunogenic basis.