NUCLEAR POLYHEDROSIS
Causal organism : Borrelina bombycis
It is a viral disease and in view of the fact that diseased larvae appear pale yellow in colour, it is
commonly called as jaundice or grasserie. It generally attacks the advanced stage worms after fourth
moult and accounts for more than 15 per cent of the loss of cocoon yield (Vaidya, 1961). However,
several workers (Savanuranath et al., 1992; Swamy and Nagaraj, 1992) have reported much higher
incidence of this disease.
In early stages of infection, it is difficult to differentiate between the diseased and healthy larvae.
However, at moulting phase, the diseased worms wander about without entering into moult. The
worms become restless, lose appetite, and exhibit shiny and stretched skin and thereafter, intersegmental membrances become swollen. While the exact reason of such swellings is not known,it is believed that large number of polyhedral bodies are formed in the epidermal cells which become highly abnormal resulting in the swollen intersegmental membranes and loss of clasping
power of legs except the pro-legs. Meanwhile, the skin loses its tension due to change in permeability
of epidermal cells. Due to these changes in the permeability of cells the skin ruptures and white milky
fluid containing large number of polyhedral bodies oozes out. These bodies are found in all the organs of diseased silkworms including the haemolymph.
The haemocytes of the jaundiced larvae show increased catalase activity when polyhedral bodies appear in the nuclei of haemocytes. Just before death, the catalase activity of the haemolymph of the diseased larvae has been found 40 times more than those of healthy larvae (Aruga,1994).
Mode of Infection
Polyhedral bodies are the main source of infection of this disease. Each polyhedral body, a type of cell inclusion is haxagonal, colourless, brittle and transparent measuring 5 to 10µ across and are highly refractile. However, recently tetragonal bodies have also been found which are considered to be mutant. Electron microscope study revealed that virus is rod shaped containing few circular grains(sub-units) which is probably the unit of virus (Aruga, 1994). The grains are composed of nucleic acid and proteins which undergo self multiplication. The viral grain also uses the host substance and produces polyhedral bodies. When treated with weak alkalies, polyhedral bodies release large number of viral grains.
The larvae infected with NPV have abnormal tissues and organs. Among various organs, the epidermal cells of the integument, fat body cells, haemocytes and walls of tracheae get mainly infected. The nuclei of glandular cells and malpighian tubules are branched and are not easily infected. In silk gland ceLls also, the polyhedral bodies do not form easily. But when such bodies are formed in silk gland cells, they are usually very large, almost twice the size of polyhedral bodies found in other tissues and organs (Aruga, 1994).
It is not clear whether grasserie virus is transmitted from parent to offspring. However, it has been
found that virus is transmitted in a latent state from parent to offspring and if the larvae of next
generation are provided optimal environmental conditions, the virus continues to remain latent or
otherwise it gets activated and infects the larva and others also when milky fluid containing polyhedral bodies oozes out of the infected larva which becomes source of infection for others.
Management
Bad food, poor ventilation and abnormal environmental conditions which result in physiological
disorders of the worm lead to outbreak of grasserie disease. Therefore, maintaining optimal rearing
conditions in the room helps in checking the incidence of the disease. Feeding advanced worms with
too succulent leaves is not advisable. It has been observed that feed from bush system of plantation
leads to incidence of grasserie as compared to that from tall plantation as the tree leaf is less succulent and does not become too charged with water sap after rains.
Evolving races resistant to NPV can also help in minimising the incidence of disease. Aizawa and
associates succeeded in isolating a strain of silkworm resistant to virus by means of cold treatment and selection after 13 generations. Similarly, Uzigawa and Aruga, 1966; Watanabe, 1967; Nomani et al., (1979) also obtained resistant strains of silkworms after 5,8 and 13 generations of selection,
respectively. There is a scope for development of silkworm races with multiple resistance to diseases
by genetic engineering (Gopinathan, 1992).
Use of some antibiotics like tetracyclin or gentamycin (0.1 per cent) have been found effective in
reducing the grasserie infection besides enhancing the economic characters of larvae and cocoons.
(Radha et al., 1980; Tayde et al., 1988; Beig et al., 1990; Dhanorkar et al., 1992; Palaniswamy and
Elankumaran, 1994; Sivaprakasam and Rabindra; 1995). Use of bed disinfectant like Reshemkeet
(mixture of paraformaldehyde, benzoic acid and lime in the ratio of 1 : 2 : 97) is also helpful in
preventing the contamination.
CYTOPLASMIC POLYHEDROSIS
Causal Organism : Smithia virus
Ishimori (1935) was the first to observe cytoplasmic polyhedrosis in the midgut cells of diseased
larvae. The disease occurs from the first to fifth larval instar, however, more pronounced in late fifth
instar. Worms get infection through ingestion of leaves contaminated with polyhedra of the virus or
induction. The polyhedra are normally hexagonal when reared at 18 to 25°C but have been found
irregular in shape at 30°C and are not formed at 35°C (Aruga, 1971). This indicates that silkworm
larvae increase their resistance to CPV when reared at 35°C. CPV particles having a 6 sided outline
with projections on the corners of the polygon have vertex-to-vertex diameter of 69 + 25 mu and a side to side diameter of 63 + 1.8 mµ. The diamter of the circular outline is 66 + 2.2 mµ (Aizawa, 1971).
Hosaka and Aizawa, 1964 also observed the size of CPV particles as 69 mµ. The particles with a
regular hexagonal outline have 6 sub units with projections at each corner and no sub unit between the corners.Internally, tubular structures connect the corners of the surface shell and those of
the inner membrane. At the base of the projection, an opening or gate have been observed in an empty CPV particle (Kawase and Miyajima, 1968; Miura et al., 1969) Hosaka (1964) considers the
projections to be the morphological apparatus for the release of the core substance. A thin section of a
cytoplasmic polyhedron shows that a CPV particle consists of an electron transparent membrane and
an electron dense core (Aizawa, 1971). The membraneis 100 to 160Å in thickness and the core 30 to
40 mµ in diametre. Small bits of dense material approximately 50Å in diameter are present in the core.
CPV particles consist of two parallel coats, both icosahedral in shape with subunit structures present
at each vertex (Hosaka and Aizawa, 1964). The cytoplasmic polyhedron contains polyhedron protein,
viral protein and viral nucleic acid (Kawase, 1971).
When the worms get infected, the virus attacks the cytoplasm of midgut epithelial cells forming
large number of polyhedra which are released into the gut lumen and voided with faeces. The release of polyhedra into the gut lumen results in poor growth and development of larvae, disproportionately
large and transparent cephalothorax, frequent defecation of whitish semi solid matter (white colour of
faeces is due to the presence of large number of polyhedra) and occasional rectal .(Plate 29),
At advanced stage of the disease, the midgut lumen becomes loaded with polyhedra,large number of
which are regurgitated by larvae. The worms shrink and finally die and contaminated faeces as well as mulberry leaves become major source of infection during rearing.
Mode of infection
Cytoplasmic polyhedrosis mainly spreads through oral infection. The ingested CPV apparently
panetrates into the midgut cell in a region posterior to seventh abdominal segment and then the virus
spreads to cephalic region of the midgut (Aruga, 1971). The polyhedra formed in the anterior portion of the midgut are larger than those formed in the posterior portion of the midgut. Numerous factors play role in the process from pre-oral infection to death such as virus activation by the midgut Juice,
mechanism of the adsorption of the virus particle to the midgut cell, role of the peritrophic membrane, mechanism of virus penetration into the midgut cell, virus multiplication and formation of polyhedra in the cytoplasm of midgut cylindrical cells and the synthesis of viral RNA (Aruga, 1971). Silkworms have been found to be more susceptible to CPV soon after ecdysis during which the peritrophic membrane has not been formed in the midgut. The polyhedra, a type of cell inclusion, is found usually in the cylindrical and rarely in goblet cells of the midgut but not in the nuclei, hence the name CPV. The cylindrical cells, containing large number of polyhedral bodies, degenerate releasing large number of polyhedra in the lumen of the intestine which are passed out along with the faeces, giving the faeces a whitish appearance. The distruction of the infected cells results in the differentiation of new cylindrical cells from regenerative cells which increase in number by normal cell division. The newly differentiated cells are also susceptile to the virus and get affected. However, the biochemical mechanism of virus replication, the enzymes involved in the process and the mechanism of secondary infection remain to be studied in detail (Kobayashi, 1971).
The virus, upon entering the cytoplasm of the cells, utilize the cellular contents, multiply and
synthesize a special type of protein to form the polyhedral bodies containing the cell inclusion
(Kawase, 1971).
Under natural conditions, the infection spreads when mulberry leaves come in contact with the
faeces or saliva of infected worms which are eaten by healthy larvae. Sometimes, the disease breaks out in the absence of inoculum in the rearing rooms probably due to fluctuations in the environmental
conditions, poor food quality and unhygienic conditions which lead to the physiological abnormalities in the worm. This type of infection is known as induction. Aruga, 1971 observed that the factors controlling the induction of CPV are genetic, environmental (too high temperature 29 to 30°C or too low 15°C accelerate the incidence of infection) nutritional (quality of food) and physiological (conditions of the midgut).
Nutrition, such as the quality of leaf is believed to be important in activating CPV infection in silkworms (Aruga, 1958). The incidence of CPV infection is higher in silkworm larvae fed on yellowish and unripe leaves or on the leaves produced in shade or stored for about a weak than those fed on good quality and fresh mulberry leaves. It has been observed that when young silkworms are fed on bad quality leaves, CPV infection develops after the mid fifth instar. Starvation for 12 hours after ecdysis increases the induction rate of CPV (Aruga and Arai,1959) Among various physical factors, temperature has received high attention so for its effect on the incidence of CPV is concerned. Yokokawa and Yamaguchi (1960) have observed that higher incidence of disease occurs when larvae are reared at 30°C than at 25°C.It has also been found by Miyajima and Kawase (1968) that extreme high temperature such as 35°C supresses the incidence of CPV. The resistance of the larvae to infection at high temperature is perhaps not associated with the failure of the CPV to invade the host, but is more likely due to the factors such as the destruction of the virus or the cessation of the virus multiplication of the occurrence of the immunity in the host responses.
Aruga (1971) also found that silkworms increase their resistance to CPV when reared 35°C.
Hukuhara (1962) believed that CPV is transmitted to the next generation through the egg in an
accult state and this generation to generation transmission involves a complicated genetic mechanism.
When the virus in an occult state gets activated with the abnormal factors, cytoplasmic polyhedrosis
breaks out. Resistance to the induction of CPV in silkworms is markedly affected by the characteristics of the cytoplasm transmitted from the mother to the egg (Aruga and Nagashima, 1962; Watanabe,1966).
It has also been found that if silkworm eggs are incubated under unfavorable conditions, especially
abnormal temperature, disease makes its appearance in the advanced stages. Inadequate conditions of the treatment of hibernating silkworm eggs with HCI to break the diapause also results in incidence of CPV in the fifth instar larvae (Yokokawa, 1964).
Management
The intensity and incidence of cytoplasmic polyhedrosis virus varies with the race and season of
silkworm rearing, being high in autumn, slightly lesser in summer and mild in spring. The difference
in the intensity and incidence of infections in different seasons is probably due to difference in quality
of mulberry leaves fed to the silkworms. In spring, quality of leaves is best suited to the physiology of the worm,hence the incidence of infection is mild. On the contrary, the leaves available in summer
and autumn are course hence not suited to the physiology of the worms and lead to cytoplasmic
polyhedrosis is. Besides, extreme high and low temperature also induces the disease. It has also been
observed that if advanced worms are fed with extremely succulent leaves or those inadequately exposed to sunlight, disease gets induced.
Therefore, the disease can be prevented by effective disinfection of the rearing rooms and
appliances as well as proper incubation of seed, providing optimal environmental conditions, feeding
worms on quality and fresh mulberry leaves. Development of strains resistant to CPV can help in
reducing the incidence of disease. Watanabe (1967) believes that polygenes are involved in resistance of silkworms to CPV. Use of antibiotics, as in the case of NPV, have also been found effective against
this disease.
Japanese have found that spraying of calcium hydroxide on mulberry leaves is highly effective in
supressing the incidence of cytoplasmic polyhedrosis. Studies have also revealed that calcium
hydroxide increases the resistance of the silkworm to the CPV by inactivating the virus or by
preventing its penetration and multiplication. Therefore, to restrict the occurrence of CPV, mulberry
leaves can be sprayed with calcium hydroxide and fed to the silkworm larvae Aruga, 1971.
