No abstract

Latex agglutination assay based on monoclonal antibodies (MCAs) described in this communication may be useful for detection of Pebrine infection in silkworm. Four murine MCAs were produced against Nosema bombycis spore. In ELISA all 4 MCAs (IgM isotype) reacted with alkali treated Nosema spores and to variable extent with acetone precipitated surface protein. However, MA-310 and MA-542 showed a low degree of cross reactivity with BmNPV. In contrast, MA-503 and MA-515 were devoid of reactivity with BmNPV, B. thuringiensis, S. marcescens, Azotobactor, Rhizobium and normal hemolymph protein in ELISA. Latex beads sensitized with a combination of MA-503 and MA-515 (50 micrograms each per ml of 0.4% latex beads) could detect 1 x 10(5) Nosema spores per test. Sensitization of the latex beads with the cocktail of these two MCAs through protein-A bridge further led to a 10-fold increase in the sensitivity (1 x 10(4) spores/test) of the assay. No agglutination was observed in presence of BmNPV, Rhizobium, Azotobactor, E. coli, B. thuringiensis, S. marcescens and normal hemolymph protein indicating the specificity of the test. The results obtained by latex agglutination assay on hemolymph samples of infected as well as normal larvae collected from field, II instar larvae infected in the laboratory and from infected mother moth revealed 100% correlation with results by microscopic examination.

The symptomology of BmNPV infection in Bombyx mori L depends on the stages of infection. Discernible symptoms develop at later stages of infection, which leads to improper diagnosis and poor crop yield with sericulturists. In the present study development of direct and protein-A linked monoclonal antibody latex (PALMAL) agglutination test for the detection of BmNPV infection in silkworm is described. Latex beads were precoated with protein-A and then sensitised with monoclonal antibody MA-231 (125 micrograms/ml). PALMAL test could detect 1 x 10(5) nuclear polyhedra/test and is ten times more sensitive than the direct agglutination test employing purified polyclonal antibodies. No agglutination was observed in presence of B. thuringiensis, S. marcescens, N. bombycis, group-A streptococci, Staphylococcus aureus, E. coli and normal haemolymph protein indicating the specificity of the test. Fifty haemolymph samples collected from the field were evaluated by PALMAL test. Twenty one samples having infection other than BmNPV, failed to show positive agglutination. Twenty five samples having > or = 5 x 10(6) BmNPV/ml showed positive agglutination. However, 4 samples having < 5 x 10(6) BmNPV/ml failed to show positive agglutination thereby indicating the limit of sensitivity of the assay.

The immune system in insects involves in clearing the haemolymph of foreign pathogens by haemocytes and humoral factor. They can accomplish this in a couple of different ways. One is a cellular mediated response by physically clearing the haemolymph by phagocytosis, nodulation and encapsulation and the other is by humoral secretion of proteins. Pathogenic infection in silkworm, Bombyx mori L. is common and there is a possibility of differential response by haemocytes in different breeds to microbial infection. Hence, the differential response in the form of difference in Total haemocyte count (THC) and Differential haemocyte count (DHC) in susceptible and tolerant breeds were investigated under normal and Beauveria bassiana invasion conditions. Under normal condition, there was a gradual increase in THC as the age of the silkworm increases and high THC was recorded in Nistari, PM and NB4D2 (tolerant breeds) compared to susceptible breeds (NB7, NB18 and KA). There was a gradual increase in THC, gradual decrease in prohaemocyte and oenocyte counts and increase in plasmatocyte and granulocyte counts during the progressive infection by Beauveria bassiana.