A disinfectant composition against silkworm pathogens

Abstract

The invention provides disinfectant composition that is effective against silkworm pathogens. The disinfectant composition comprises a peroxy sodium percarbonate in a concentration range of about 1% (w / v) to about 2% (w / v) and trisodium phosphate in a concentration range of about 1% (w / v) to about 2% (w / v).

Description

Description:The invention generally relates to a disinfectant composition and particularly to a disinfectant composition against silkworm pathogens. BACKGROUNDSericulture is one of the important sources of income for the farming fraternity. It provides stable income and employment opportunity to many, across globe. However, diseases due to microbial infestation have been a serious threat to sericulture from a long time. It attributes to major loss in this sector. Microbial infestation includes but is not limited to viral, fungal and bacterial. Frequently encountered major diseases in silkworm include but are not limited to Grasserie (viral disease), Muscardine (fungal disease), Pebrine (microsporidian infection) and flacherie, a syndrome inflicted by non-occluded viruses or bacteria. It is imperative to appropriately manage and prevent disease for a successful harvest of cocoon crop. Disease management approaches in sericulture industry involves primarily the prophylactic measures such as utilization of disease free silkworm eggs, disinfection of rearing room and rearing appliances, application of silkworm rearing seat / bed disinfectants and maintenance of personal hygiene. Amongst the above enlisted measures, a very important role in diminishing the degree of disease incidence is achieved through disinfection of rearing room and rearing appliances before the commencement and upon completion of each silkworm rearing crop followed by application of rearing seat / bed disinfectants during the course of rearing. Destruction of disease causing pathogens is termed as `disinfection’ and it is accomplished by diverse processes, but the most efficient method is by chemical means employing chemicals as disinfectants. Proper disinfection of rearing room, rearing appliances and the adjoining areas before the commencement of rearing ascertains an environment devoid of pathogens, which is conducive to silkworm rearing. Pathogen load accumulated over the larval rearing period, if any, is sterilized by disinfection upon the completion of rearing. In view of the fact that physical factors including temperature and humidity also play a significant role in pathogen multiplication, the importance of silkworm rearing room disinfection becomes more prominent across the tropics.The ideal characteristics of a disinfectant includes extensive antimicrobial spectrum, efficiency in the presence of organic matter, non-toxicity to the silkworm, eco-friendly and user-friendly nature, non-corrosive, non-hazardous, stable upon storage, odorless or with a pleasant odor, easy solubility in water, effective across wide range of pH, rapid action, ease of availability of raw materials and cost effective. Most of the disinfectants developed and used in sericulture are either formaldehyde or chlorine or phenol based formulations and each one has their own advantages and disadvantages. In India, 5% bleaching powder and chlorine dioxide based liquid spray disinfectants are widely employed for disinfection of rearing room and rearing appliances. Though 5% bleaching powder and chlorine dioxide are effective against major silkworm pathogens, they have certain disadvantages which include corrosive nature, less solubility in water, hazardous to the users besides being non-friendly to the environment. Therefore, there is a pressing need for developing an ecological and user friendly disinfectant, which is effective and efficient to inactivate silkworm pathogens. BRIEF DESCRIPTION OF DRAWINGSSo that the manner in which the recited features of the invention can be understood in detail, some of the embodiments are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.Fig. 1a shows cellular morphology of Bacillus thuringiensis in control, according to an embodiment of the invention.Fig.1b shows effect of disinfectant composition on the cellular morphology of Bacillus thuringiensis, according to an embodiment of the invention.Fig.2a shows cellular morphology of Staphylococcus spp. in control, according to an embodiment of the invention.Fig.2b shows effect of disinfectant composition on the cellular morphology of Staphylococcus spp., according to an embodiment of the invention.Fig.3a shows cellular morphology of Beauveria bassiana in control, according to an embodiment of the invention.Fig.3b shows the effect of disinfectant composition on the cellular morphology of Beauveria bassiana, according to an embodiment of the invention.Fig.4a shows cellular morphology of BmNPV Polyhedra in control, according to an embodiment of the invention.Fig.4b shows effect of disinfectant composition on the cellular morphology of BmNPV Polyhedra, according to an embodiment of the invention. Fig.5a shows cellular morphology of Nosema bombycis in control, according to an embodiment of the invention.Fig.5b shows effect of disinfectant composition on the cellular morphology of Nosema bombycis, according to an embodiment of the invention. SUMMARY OF THE INVENTIONOne aspect of the invention provides a disinfectant composition that is effective against silkworm pathogens. The disinfectant composition comprises a peroxy sodium percarbonate in a concentration range of about 1% (w / v) to about 2% (w / v) and trisodium phosphate in a concentration range of about 1% (w / v) to about 2% (w / v).DETAILED DESCRIPTION OF THE INVENTIONVarious embodiment of the invention provides a disinfectant composition that is effective against silkworm pathogens. The disinfectant composition comprises a peroxy compound and an alkaline detergent. The peroxy compound in the composition is sodium percarbonate and the alkaline detergent is trisodium phosphate. The sodium percarbonate is used in the concentration range of about 1% to 2%. In one example, about 2% (w / v) of sodium percarbonate is used in the composition. The trisodium phosphate is present in the concentration range of 1% to 2%. In one example, the concentration range is about 2% (w / v). In one embodiment of the invention, the composition is effective for disinfecting silkworm rearing house and rearing appliances. It is an aqueous solution that does not generate any toxic residue and is environment friendly. The composition has high water solubility that helps in rapid preparation.According to embodiment of the invention, the disinfectant composition has a wide spectrum. The composition inhibits the growth of pathogenic bacteria, fungi, microsporidia and viruses. Pathogenic bacteria include but are not limited to Staphylococcus sp., and Bacillus thuringiensis. Pathogenic fungi include but are not limited to Beauveria bassiana. Pathogenic viruses include but are not limited to BmNPV, BmDNV1, BmIFV. The composition is also effective against microsporidian including but not limited to Nosema bombycis.In one embodiment of the invention, the disinfectant composition is dissolved in water to obtain a disinfectant solution. The active concentration of the disinfectant composition is in the range of 1% (w / v) to 2% (w / v). In one example, for preparation of 100 litres of disinfectant solution, 2 kg of the disinfecting composition is dissolved in 100 litres of water. The disinfectant solution is sprayed on the appliances and surroundings for disinfestations. In one example, the disinfectant solution is sprayed using power sprayer. Sodium percarbonate decomposes into hydrogen peroxide and sodium carbonate in water, thereby bringing about the sterilizing effect against microbial pathogens. The key advantage employing sodium percarbonate is that it breaks down into oxygen, water and natural soda, which are environment friendly. Trisodium phosphate forms an extremely basic solution in water, which disrupts the membrane integrity of the pathogens. The hydrogen peroxide released as a result of dissolution of sodium percarbonate is stabilized by phosphates from trisodium phosphate thereby increasing the stability of the disinfectant solution. Moreover, the efficacy of hydrogen peroxide is elevated under alkaline conditions. Efficacy studies:The efficacy of the disinfectant composition has been studied against pathogenic bacteria, fungi, viruses and microsporidians. The pathogenic bacteria in the present efficacy study include Staphylococcus sp. and Bacillus thuringiensis. The pathogenic fungus in the present efficacy study is Beauveria bassiana and the pathogenic virus is BmNPV. The microsporidian in the present study is Nosema bombycis. All the above mentioned pathogenic organisms utilized in the efficacy study are maintained at Central Sericultural Research & Training Institute, Central Silk Board, Govt. of India, Berhampore-742101, West Bengal, India. The stock inoculums are prepared employing the below mentioned procedures and the same are utilized to prepare working dilutions or known concentrations. Bacteria: Non-sporulating, Gram positive Staphylococcus sp. and Gram positive, sporulating, toxin producing Bacillus thuringiensis are cultured in nutrient broth (g l-1: peptone, 5; beef extract, 1.5; yeast extract, 1.5; NaCl, 5) and are incubated at 30+2°C for about 36 hours. Bacterial enumeration is determined by serial dilution technique.Fungi: Beauveria bassiana causes white muscardine in silkworms. They are cultured on potato dextrose broth (g l-1: potato infusion, 200; dextrose, 20; pH 5.6±0.2) and incubated at 30+2°C for 72 hours. The conidial concentration is determined by using Neubauer haemocytometer. Viruses: BmNPV: The haemolymph of grasserie infected larvae is collected in sterile distilled water, incubated overnight at room temperature and centrifuged at 10,000 rpm for 10 minutes. The pellet is washed for 4 to 5 times with distilled water followed washing with sterile saline solution twice. The BmNPV Polyhedra are suspended in distilled water and further purified by gradient centrifugation. The polyhedral count is determined using Neubauer haemocytometer and kept for further use as stock BmNPV inoculums in sterile distilled water. Nosema bombycis: Pebrine inoculated larvae are collected, macerated in K2CO3 solution (0.6%; 3ml / g fresh weight) and filtered through muslin cloth. The filtrate is centrifuged at 2500 rpm / 10 minutes and the pellet containing the microsporidian spores are collected, washed thrice with saline followed by sterile water. The isolated spores are purified employing iso-density equilibrium centrifugation with Percoll. The spore count is determined using Neubauer haemocytometer and the N. bombycis spores are stored in the refrigerator as stock inoculum.Treatment of the inoculums stock:Post the preparation of stock inoculums, about 1ml of the pathogen suspensions containing Staphylococcus sp. & B. thuringiensis at a concentration range of 1.7 x 107 cfu / ml, B. bassiana at a concentration range of 2 x 107conidia / ml, N. bombycis at 1 x 107 spores / ml, BmNPV at 1x107 Polyhedra / ml are centrifuged at 10,000 rpm for about 10 minutes and the pellets are suspended in 1ml of disinfectant solution. The pathogens are exposed to the disinfectant solutions for a period of 10 minutes at room temperature (25±1°C). Upon the completion of inactivation exposure, the pathogen-disinfectant suspensions are centrifuged and the supernatants are discarded. The pellets are washed thrice with sterile distilled water to remove the traces of disinfectants. Finally, the pellets are suspended in 1ml sterile distilled water and used for further experimentation. Pathogens treated with distilled water are maintained as control in the experimentation. Microscopic examination of the inactivated pathogensUpon treatment, the suspension of bacterial, fungal, BmNPV Polyhedra, microsporidian spore suspensions are transferred on to a clean glass slide individually and a glass cover slip is placed over the solution. The samples are observed under light microscope at 600X magnification to record whether any changes are inflicted in the cellular morphology of pathogens upon treatment with disinfectant composition. The disinfectant composition results in variation in cellular morphology of the pathogens. Fig. 1a shows morphology of B. thuringiensis in a control, according to an embodiment of the invention. Under the treatment of disinfectant composition, B. thuringiensis shows aggregation indicating variations in the cellular morphology of pathogens, as shown in Fig.1 b. Similarly Fig. 2b shows distortion of bacterial cells observed in Staphylococcus as compared to its control shown in Fig.2a. In case of B. bassiana conidial spores, the conidial shape is distorted and structural integrity is lost. Fig. 3 a shows the morphology of B. bassiana conidial spores in control and Fig.3b shows the impact of disinfectant composition on the B. bassiana morphology according to an embodiment of the invention. Fig. 4b shows BmNPV polyhedral bodies completely dissolved without any polyhedra compared with the control in Fig.4.a. Nosema bombycis spores structural integrity is not noticed and disintegrated spore remains are recorded as shown in Fig. 5b in comparison with the control in Fig.5a. In one example of the invention, silkworm rearing rooms and rearing appliances are used to study the efficacy of the disinfectant composition. Two silkworm rearing rooms and the rearing appliances are selected for the study. One rearing house along with rearing appliances is disinfected with 5% bleaching powder and the other rearing house along with rearing appliances with the present disinfectant composition. The overall rearing performance including the cocoon harvest details is recorded. The harvested cocoons are evaluated for post-cocoon parameters and the important reeling traits are recorded as shown in table 1.Table1: Pre and Post-cocoon parameters and the important reeling traitsParameter 5% Bleaching Powder Disinfectant composition P value Sig. @ 5%ERR by No. 8303 8293 0.93 NSERR by wt. (kg) 12.28 12.61 0.02 *Single Cocoon Wt. (g) 1.453 1.501 0.25 NSSingle Shell Wt. (g) 0.244 0.253 0.23 NSShell Ratio (%) 16.79 16.85 0.87 NSFilament Length (m) 638 648 0.87 NSDenier 2.63 2.62 0.62 NSReelability (%) 73 73 1 NSThe present disinfectant composition is effective in the management of silkworm rearing across all the important pre- and post cocoon parameters recorded, which is on par with the existing formulation, 5% Bleaching Powder. The composition is effective for disinfecting silkworm rearing house and rearing appliances. This is a cost effective solution. It is easily soluble in water aiding in rapid preparation. Further, the composition is also very stable, non-corrosive and simple to prepare.The foregoing description of the invention has been set merely to illustrate the invention and is not intended to be limiting. Since modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur to person skilled in the art, the invention should be construed to include everything within the scope of the appended claims and equivalents thereof.

Claims

Claims:We Claim:

1. A disinfectant composition against silkworm pathogens having sodium percarbonate in a concentration range of about 1% (w / v) to about 2% (w / v); andtrisodium phosphate in a concentration range of about 1% (w / v) to about 2% (w / v).

2. The disinfectant composition as claimed in claim 1, wherein the pathogen is a bacterium, a fungus, a virus and a microsporidian.

3. The disinfectant composition as claimed in claim 1, wherein the bacterium is Staphylococcus sp. and Bacillus thuringiensis.

4. The disinfectant composition as claimed in claim 1, wherein the fungus is Beauveria bassiana.

5. The disinfectant composition as claimed in claim 1, wherein the viruses is BmNPV, BmDNV1 and BmIFV.

6. The disinfectant composition as claimed in claim 1, wherein the microsporidian is Nosema bombycis.