This invention relates to "A virulence marker protein for pebrine disease of silkworm Bombyz mori".
Pebrine disease is caused by the parasitism of the microsporidian Nosema which belongs to protozoa. This disease causes great loss to Sericulture Industry due to complete mortality of silkworm Larvae in a rearing batch.This dreaded disease once completely wiped the Sericulture industry in all European countries.This was mainly due to lack of technology, in the production of quality disease free seeds. During the year 1865 -70, Louis Pasteur brought out a method of diagnosis by crushing the mother moth and observing under the microscope for the spores.This disease was also reported from different sericultural regions of India. The first report of such an occurrence was from Bengal in 1859 followed by a report on the epidemic of pebrine from Kashmir, during 1878. The disease occurrence was reported from Madras and Mysore provinces between 1925-1930. In Mysore seed area, the disease out break was reported during 1940-47. The Sericulture department has discovered that the pebrine outbreak occurs once in four or five years. It had occurred earlier in 1973-74, 1978-79, 1983-84, 1987-88, and 1991 (Indian Silk Dec. 91)
In India the identification of microsporidian parasite in silk moth has been based on the morphological characters observed under light microscopy by highly trained technicians as developed by Louis Pasteur in 1870. The recent discovery of morphologically distinct developmental sequences (dimorphism) in microsporidian

poses difficulties in identification. Baig et al. , (1992) developed slide agglutination test for microsporidian spores using an antibody sensitized latex beads. However, this technique has not proved to be totally satisfactory as there is no discreate difference between infected & uninfected silkworms and also the efficiency is very low.
I have identified the microsporidian infection -specific polypeptide in Bombyz mori.Polyclonal antibodies were raised for Nosema spore proteins in rabbit. The serological reaction with Nosema infected silkworm larvae and egg shows the presence of a 17 kDa polypeptide which seems to be specific for infection. The batch of eggs showing the 17 kDa polsrpeptide in western blot resulted in complete mortality in lab and field. This 17 kDa protein can be used as a virulence marker for the identification of microsporidian infection. This protein is useful not only to identify the infection in moth, but also in larval, pupal stages. The 17 kDa protein was noticed in some of the disease free layings collected from sericulture grainages, testifying the efficiency of the protein in detect ion. Even a very low level of the infection can be detected by using this protein.
Pebrine disease spreads in two ways (1) The spores present in the environment of the rearing houses by secondary contamination (2) From the mother moths through the egg to the next generation. Using this method identification of this pathogen is possible at any stage of silkworm life cycle. This would help the Sericulturists to identify a good batch of larvae for egg production. Normally the infection is realised

only after complete loss of the rearing batch after 20 days Adoption of this technique would save manpower and resources and at the same time would prevent the contamination of rearing room which is very difficult to eradicate. This would in turn improve the quality and quantity of silk yield.
As per Saratchandra (Indian silk, April 1992) occurance of the disease shall bring bad reputation or loss or both either to the Seed cocoon rearers or to the Seed producers or to the commercial rearer depending upon the stage at which it is detected. A system has to be evolved where either a seed cocoon grower or a seed producer is not put to hardship due to the detection of pebrine. This is possible only when such losses are compensated. If the fifth instar silkworms are identifled for pebrine by using this protein, then those seed cocoons can be used for reeling purpose thereby saving the loss.By careful screening of larva, moth & egg by westernblot method, we can eliminate pebrine completely in near future.The cost and time factors involved can be regulated by careful planning. This also provides an opportunity for the educated farmers to set up an enterprise.
The manner in which it has to be performed :
1. Homogenise the sample egg, larva, pupa or moth in bicarbonate buffer (0.2 M pH8.0)
2. Run a SDS-Polyacrylamide gel.
3. Transfer to nitrocellulose membrane.
4. React the membrane with primary antibody raised against microsporidian Nosema bombycis and secondary antibody.

5. Develop the membrane with suitable substrate.
6. Look for the presence of 17 kDa protein.If present discard that batch of sample as they are infected with the microsporidian ie it has pebrine disease.

I claim that that a 17 kDa virulence marker protein was identified for the first time for the early identification of pebrine infection. The presence of this marker would indicate infection and the loss to the seed or egg producer and also to the silkworm rearing farmers can be prevented by using this indicator protein. The subsequent rearing can also be protected from contamination which is otherwise very difficult to eradicate.