Protozoan Disease
The protozoan disease of silkworm is called pebrine because of the characteristic pepper-like black spots appearing on the infected silkworm. The disease spreads very quickly and assumes epizootic importance. The entire silk industry of France would have been
wiped out, if not for the timely intervention of Pasteur, who discovered the cause of and remedy for this disease. In India, though the disease might have existed from very ancient days the first official record of it dates back to 1895. With the introduction of mother moth examination and supply of disease-free layings, the disease is at present under reasonable control. But chances of its appearance is possible by cross-infection from wild silkworms ang other lepidopterans whom the parasite infects and which act as its secondary hosts. The use of seeds from unlicensed seed producers is also a potential cause for the spread of this disease. In this connection, it may be mentioned that according to a recent report, in the southern states, more than 20% of the basic seeds reared at P3 and P2 stations were affected by this disease. Seasonal and annual variation in the incidence of this disease have also been recorded.
Causative Organism
The disease is caused by the infection of the Protozoan Nosema bombycis Nageli. Formerly it was believed to be a single strain with definite characters. With the application of advanced molecular biological and immunological techniques to the study of this parasite, it is now established that there are several strains in the species
Nosema bombycis. These strains differ in their morphological, pathological and antigenic characters. For example, one currently isolated strain of Nosema bombycis infects only the midgut cells while the normalstrain affects cells of many types of tissues of the host. Hence, this strain is not as virulent as the normal strain. Recently, using Antibody Sensitized Latex Particles (ASLP)technique, three strains of Nosema bombycis have been isolated in India and these are called NIK-2r, NIK-3h, and NIK-4m. These differ from each other in spore shape, spore size, specificity of tissues affected
and antigens present. They also differ in their ability to form spores. These discoveries have made it clear that the classical mother moth examination or examination of the tissues of the infected worm for the Presence of spores is not sufficient ' exclude pebrine. Observations have also to be made for the presencé or absence of the short polar tube of the spore.
Modes of Transmission
The disease may be transmitted in three different ways-oral, contact and transovarial.
1. Oral: Spores may be liberated into the rearing bed by the infected worms through their faeces or through their dead bodies and contaminate the leaves.When such leaves which are contaminated
with spores are eaten by silkworm it becomes infected. If the surface of the eggs are contaminated by spores from the parents during oviposition or after oviposition, they may enter the body of the newly hatched worm which eats a part of the chorion of the eggs and gets the infection.
2. Contact: If the rearing bed contains infected worms, spores liberated by them through their faeces or their dead bodies may enter a healthy worm through skin wounds and cause infection.
3. Transovarial: If infection occurs in the late V instar, the infected worm may spin normal cocoon and moths may also hatch out. But in the moths, the parasite grows and sporulates in the oocytes in the ovary and is passed on to the eggs. The eggs acquire the infection during laying itself.
Life Cycle of Nosema bombycis
The life cycle of the parasite is completed within a single host, the silkworm. The life cycle is completed within a week in temperate regions and in four days in tropical regions. There are two stages in the life cycle - 1) a resistant spore stage which is the infective stage and 2) a growing or vegetative stage.
The spore stage: The spore is also called sporont. The mature spore is oval (3.8 x 2.0) and refracts light. The spore has a protective thick tunic called spore capsule. Two large vacuoles present at the two poles restrict the protoplasm to a girdle-like structure in the middle. It is called sporoplasm and is binucleate.
At one end of the spore capsule is a bag-like structure called polar capsule. The bag extends to the other end through the interior of — the sporoplasm. A long polar filament (30 times as long as the body)is kept coiled inside the polar capsule. the polar filament opens to the exterior by means of a small opening called micropyle. When the spore capsule is destroyed, the polar capsule and the Polar filament are everted and the binucleate sporoplasm is liberated as an amoebula.
Vegetative stage: The spores eaten along with the leaves transform into the vegetative stage in the alimentary canal. The digestive juices digest the spore membrane. The polar capsule and
the polar filament are everted and discarded. The sporoplasm is set free in the form of a binucleate amoebula. The two nuclei fuse and the uninucleate motile structure is called a plasmont. It has no
limiting membrane outside its thin plasma membrane. It is about 0.5 to 1.5µ in size and multiplies by fission and penetrates through the gut epithelium, enters the haemocoel and infects various organs
like fat body, trachea, silk gland and reproductive organs. This is called autoinfection. Plasmonts are extracellular, motile and uninucleate and divide by binary fission and produce vegetative
plasmonts.
Some of the plasmonts enter into the cells of the tissues which they infect and become converted into meronts. Like plasmonts they are uninucleate but differ from them in the following respects. Meronts are intracellular, immotile and have a definite limiting membrane on the outside. The meronts are larger than the plasmonts and are ellipsoidal or cylindrical in shape. They grow and undergo multiple fission. The products are the spores. Spores are packed tightly in a beaded manner within the host cells. Cytoplasm of the host cell is reduced by the multiplication of the meronts and the formation of the spores. The nucleus of the host cell is rarely invaded. Ultimately, the cells die and liberate the spores.
When the plasmont infects the fat body, there is poor energy turnover and the growth of the larva is affected. In the silk gland, the infected cells form tumour-like pustules, thereby distending the
glands. The silk synthesis by the gland cells is affected and the worm spins weak cocoons. When the gonads are affected, germ cell formation is affected and fecundity is greatly reduced.
The course of changes undergone by spores entering the haemocoel directly through skin wounds or those that are passed on transovarially is not recorded. But when in vitro cultures of silkworm tissues in tissue culture are infected with spores, a similar course of events from the formation of binucleate amoebula, uninucleate plasmont and meront stages are observed. This suggests that silkworm tissues have enzymes capable of digesting spore membrane.
Symptoms: Infection of the larvae by transovarial transmission from the mother moth is called Primary infection. The percentage of hatching from the egg is very low and those few that hatch out invariably die before reaching the III instar. They serve as sources of secondary infection during their life time through faeces and after death through their dead bodies. Since these larvae are infected when they are in the II or III instars, they may enter the spinning stage and spin weak cocoons. These show the typical pebrine symptoms and may die during or after spinning. These serve as sources for tertiary infection of fifth instar larvae which may spin cocoons and may even live upto moth emergence. It is these moths that are the sources of primary infection.
Economic loss is negligible in primary infection as the larvae die before III instar, severe in secondary infection and unpredictable in tertiary infection.
Pebrine-affected eggs: The infected eggs have insufficient or irregularly deposited glue and hence get easily detached from the egg card. Overlapping of the eggs in card indicates pebrine infection and this is also due to improper deposition of the glue. Failure of eggs to hatch is also an indication of pebrine infection. The infected eggs are light yellow in colour in contrast to the bright yellow of healthy eggs.
Pebrine affected larva: Symptoms are difficult to detect in the initial stages of infection. Larvae affected by primary infection succumb to disease and die. Those affected by secondary and tertiary infection show the following symptoms - loss of appetite, slow, irregular growth resulting in the appearance of “unequals” in the rearing bed, irregular and sometimes incomplete moulting in the bed, and appearance of thin and weak worms, popularly called “lean worms”, in the bed due to slow and irregular growth. Appearance of black pepper-like spots on the body, particularly of IV and V instar larvae. Infected irregular brown patches appear. these patches indicate the presence of dead hypodermal cells due to pebrine infection. infected larvae pass soft faeces. spinning larvae spit and waste their silk without spinning. infected worms die after spinning without pupating.
Pebrine-affected pupa: Though infected cocoons appear normal, the pupa inside show symptoms of pebrine infection. Some normal cocoon may have dead pupa inside. Body of the pupa is swollen and black in colour. Black spots are present on the sides of the abdomen.
Pebrine-affected moth: Irregular, decolourised patches are seen on the body and wings. Scales fall off easily. Black spots may be seen on the abdomen. Deformed antennae, unstretched and deformed
wings, low fecundity and laying of eggs in irregular heaps are indications of pebrine-infected adults.
Detection Methods
1. Diseased or dead larvae/pupae/moth are ground in little KOH and a drop of the homogenate is smeared on a glass slide and examined under the microscope for the presence of refractile spores.
2. The faecal pellets from the rearing bed of crop showing signs of disease are collected and homogenized and the homogenate smeared and examined for the presence of spores. This method has the advantage that no worms are sacrificed.
3. Fluorescent antibody technique of Huange et al. (1983) is used to detect the presence of pebrine spore antigen in the body fluid / homogenate of diseased larvae. This is sensitive and can help in diagnosis even using small samples.
4. Immunoenzymatic method of Qian et al. (1986) uses similar principle but instead of fluorescent antibody uses antibody linked to specific enzyme to detect the antigen of pebrine. ‘
5. Immunoperoxidase staining method of Han et al. (1987) uses the enzyme peroxidase as the enzyme linking to the antibody for the detection of pebrine on smears.
6. Microsporidian spore-specific monoclonal antibody sensitized latex-method of Mike et al. (1989) and Ke et al. (1990) uses latex-coated antibodies raised by monoclonal antibody method for detection of pebrine spores.
7. Protein A Linked Latex Antisera Test (PALLAS) has been evolved by Baig et al. (1992) as a cheap and quick method for detecting pebrine.
It must, however, be admitted that detection of spores microscopically still remains the only method available for practical purposes.
Control Measures
Rearing only disease-free layings prepared in grainages after mother moth examination, removing diseased larvae from the rearing tray and burning them, and disinfecting the rearing room and rearing appliances with 4 to 5% formalin or bleaching powder after pebrine infection to kill any spore that may be present. Use of a higher concentration of the disinfectant is necessary because the spores are
not killed by the routine 2% formalin.
