Abstract: Snail Lymnaea acuminata is the intermediate host of liver fluke Fasciola gigantica, which cause endemic fasciolosis among cattle population of eastern Uttar Pradesh. Control of snail population by molluscicides is one of the effective methods to control fasciolosis. In the present study molluscicidal activity of tertiary combination of freeze-dried urine of different Indian breeds of cow Sahiwal, Geer and Tharparkar with Annona squamosa seed powder, Ferula asafoetida root latex, Azadirachta indica oil and Camellia sinensis leaves have been tested against Lymnaea acuminata. It was noted that the toxicity of tertiary combination (1:1:5) of cow urine kept for 15 days in sunlight or laboratory condition with different plant products were highly toxic against snail L. acuminata. 96 h LC50 of tertiary combinations with Sahiwal urine kept for 15 days in sunlight with A. squamosa, F. asafoetida, A. indica oil and C. sinensis were 35.47 mg L-1, 37.13 mg L-1, 33.66 mg L-1, respectively higher than the Geer and Tharparkar. The toxicity of Sahiwal urine kept for 15 days in laboratory condition with A. squamosa and C. sinensis (96 h LC50 28.28 mg L-1) was more potent than the all other combinations. Cow urine in combination with plant product can be used for effective control of snail.
INTRODUCTION
Fasciolosis is one of the emerging or re-emerging diseases in recent time (Mas-Coma et al., 2005). Fasciola hepatica and F. gigantica are the causative agents of the fasciolosis in cattle and human population (Singh and Agarwal, 1981; Mas-Coma et al., 2009; Robinson and Dalton, 2009). According to WHO (2006) 2.4 million people are infected with Fasciola species and a further 180 million are at risk of infection. The snail Lymnaea acuminata is the intermediate host of the liver flukes F. gigantica (Singh and Agarwal, 1981; Agarwal and Singh, 1988). Extensive use of synthetic molluscicides in aquatic environment is not safe to the non target organism, sharing the same habitat with snails. Alternatively, use of native plants and animal products as molluscicide will be safer and user friendly. Plants molluscicides contain several active compounds that are more effective than their synthetic counterpart (Singh et al., 1996). In Indian system of aurveda cow urine has been used in treatment of various diseases (Shastri, 1998). Recently, Kumar et al. (2011a, b) have reported that urine obtained from different Indian breeds of cow and their formulations are very effective in killing the snails. In the present study, molluscicidal activity of cow urine of native breed and their tertiary combination with plants products against the snail L. acuminata have been studied to explore the use of safe indigenous product to control the fasciolosis.
MATERIALS AND METHODS
Three to four years old Indian breeds of cow i.e., Sahiwal, Geer and Tharparkar were selected in local live-stock keepers. The urine was collected sterilized bottle for experimental purpose.
Preparation of Freeze-dried cow urine (FCU): Sahiwal, Geer and Tharparkar cow urine were kept for 15 days in sunlight (8 h days-1) or in laboratory condition. Thereafter, cow urine preparations were freeze dried.
Tertiary combinations (1:1:5) of plant products such as Annona squamosa seed powder, Ferula asafoetida root latex powder, Azadirachta indica oil with Camellia sinensis leaves and freeze-dried cow urine (FCU) were prepared by the method of Shukla et al. (2005).
Plants used: Plants A. squamosa and F. asafoetida were collected locally and identified at the herbarium of the Botany Department, DDD Gorakhpur University, Gorakhpur where voucher herbarium specimens (No. 3228 for A. squamosa and No. 3815 for F. asafoetida) are on deposit. A. indica oil was obtained from Indian Herbs Saharanpur, UP, India. A. squamosa seed powder was prepared by the method of Singh and Singh (2001).
Test animal: The adult snail L. acuminata (2.60±0.30 cm in length) were collected locally from ponds, pools, lakes and low-lying submerged areas located almost adjacent to DDU Gorakhpur University campus. These snails were attached on the ventral surface of the leaves of aquatic plants. The collected snails were acclimatized to laboratory condition in dechlorinated tap water for 72 h and then used for toxicity study.
Experiment: Toxicity experiments were done by the method of Singh and Agarwal (1984). Ten experimental animals were kept in a glass aquarium containing 3 L of de-chlorinated tap water at 22 to 24°C. The pH of the water was 7.1-7.3 and dissolved oxygen, free carbon dioxide and bicarbonate alkalinity were 6.5-7.2, 5.2-6.3 and 102.0-105.0 mg L-1, respectively. Snails were exposed continuously for 96 h to different concentration of cow urine formulations with plant products, separately. Six aquaria were set up for each concentration. The control animals were kept in the equal amount of water under similar conditions without treatment. Mortality of snail was recorded at interval of 24 h each up to 96 h The dead animals were removed immediately to avoid contamination in aquarium water.
Statistical analysis: The LC50 value, lower and upper confidence limits (LCL and UCL), slope value, t-ratio, g-value and heterogeneity factors were calculated with the help of POLO computer software programme of Robertson et al. (2007). This program uses the heterogeneity factor as a correction factor when the value of Pearson’s chi-square statistic is significant at p<0.05. The regression coefficient between exposure time and different values of LC50 was determined by the method of Sokal and Rohlf (1973).
RESULTS
The fresh urine of different Indian breeds of cow was usually pale yellow in colour. It becomes dark brown when kept in sunlight (8 h day-1) for 15 days. Colour of urine kept in laboratory condition for 15 days was light brown. The toxicity of tertiary combination of plant molluscicides and lyophilized cow urine powder of different breeds such as Sahiwal, Geer and Tharparkar against L. acuminata was time and concentration dependent. Toxicity of tertiary combination of Sahiwal cow urine kept for 15 days in sunlight+A. squamosa seed powder+C. sinensis leaves/ F. asafoetida+C. sinensis/ A. indica oil+C. sinensis (24 h LC50 90.76, 129.47 and 78.59 mg L-1) was more effective than the Geer (24 h LC50 100.76, 135.08 and 88.08 mg L-1) and Tharparkar (24 h LC50 97.18, 140.13 and 90.33 mg L-1), respectively (Table 1).
Tertiary combination of Sahiwal cow urine kept for 15 days in laboratory condition with+A. squamosa seed powder+C. sinensis leaves, F. asafoetida+C. sinensis, A. indica oil+C. sinensis (24 h LC50 72.33, 99.89 and 82.87 mg L-1) was more potent than the Geer (24 h LC50 80.63, 109.17 and 91.08 mg L-1) and Tharparkar formulations (24 h LC50 82.34, 113.98 and 96.51 mg L-1), respectively (Table 2).
The slope values were steep and separate estimation of LC50 based on each of the six replicates was found to be within 95% confidence limits of LC50. The t-ratio was greater than 1.96 and the heterogeneity factor is less than 1.0. The p value was less than 0.5 at all probability levels (90, 95 and 99).
DISCUSSION
It is evident from the present study that tertiary formulation (1:1:5) of cow urine with A. squamosa seed powder/ F. asafoetida root latex/ A. indica oil and C. sinensis leaves are potent molluscicide against L. acuminata. Tertiary combination of Sahiwal cow urine kept for 15 days in sunlight (8 h day-1) or laboratory condition with A. squamosa seed powder/ F. asafoetida root latex/ A. indica oil+C. sinensis leaves were more potent than the Geer and Tharparkar. Cow urine is considered to be the effective animal product used in general health improvement (Khanuja et al., 2005). It contains various volatile and non volatile compounds which has significant antimicrobial activity (Shaw et al., 2007; Sathasivam et al., 2010). Uric acid noted in cow urine has a strong antibacterial activity (Kumar et al., (2011a, b). Cow urine distilled has been patented as an activity enhancer and availability facilitator for bio molecules including anti-infective and anti-cancer agents (Khanuja et al., 2002-US Patent No 6410 059/2002). Probably, in the present study cow urine act as bio-enhancer with plant products, therefore higher titer of active molluscicidal components of plants reaches at active site in snail body. In Ayurvedic system cow urine is widely used in combination with drugs and pharmaceuticals.
| Table 1: | Toxicity of freeze-dried cow urine powder (FCU) , urine kept for 15 days in sunlight of different breeds cow Sahiwal, Geer, Tharparkar and its tertiary combination (1:1:5), A. squamosa, F. asafoetida and A. indica oil with C. sinensis against L. acuminata at different exposure period |
| Six batches of 10 snails were exposed different concentration of the above molluscicides with different breeds of cow urine. Significant negative regression (p<0.05) was observed between exposure time and LC50 of treatments. Ts-testing significant of the regression coefficient: Sh.+A.S.+C.S.-14.17+; Gr.+A.S.+C.S.-15.88+; Th.+A.S.+C.S.-10.21+; Sh.+F.A.+C.S.-15.62++; Gr.+F.A.+C.S.-6.54++; Th.+F.A.+CS-7.60++; Sh.+A.I.+C.S.-9.47++; Gr.+A.I.+C.S.-44.15+; Th.+A.I.+C.S.-13.79++. +: linear regression between x and y, ++: non–linear regression between log x and log y. Abbreviations; A.I:.Azadirachta indica, A.S: Annona squamosa, C.S: Camellia sinensis, F.A:.Ferula asafoetida, Gr:Geer, Sh: Sahiwal, Th:Tharparkar, LCL: Lower confidence limit UCL: upper confidence limit | |
Cow urine contains few essential component such as estrogen, nitrogen, phosphorus, pheromone, potassium, chloride, calcium and urinary protein (Biddle et al., 2007; Yan et al., 2007; Bravo et al., 2003). Carbolic acid, which is mixture of phenol and cresol in cow urine, has sufficient disinfection activity (Kelly, 1997; Khanuja et al., 2002). Annonaceous acetogenins are cytotoxic and it has antitumor activity (Fang et al., 1993). Kumar et al., (2011a, b) have reported that binary combination of cow urine with A. squamosa seed/ F. asafoetida root latex/ A. indica oil is toxic against L. acuminata. Sublethal exposure of cow urine and their formulations with plant products significantly block the reproductive capacity of snail L. acuminata (Tripathi et al., 2010). Higher toxicity of cow urine formulations at 96 h exposure periods, may be due to accumulation of higher titer of molluscicidal components on active site or degradation of plant products in more toxic form in 96 h. The toxicity of tertiary combination of different breeds of cow urine and plant molluscicides may be due to action of three components at three different sites in the snail body, which ultimately resulted higher toxicity against the snail L. acuminata.
It is evident that the steep slope value that a small increase in the tertiary combination of plant derived molluscicides such as A. squamosa seed powder, A. indica oil, F. asafoetida root latex and C. sinensis leaves with freeze dried powder of different Indian breeds cow urine kept for 15 days laboratory and sunlight (8 h days-1) was causes a higher mortality in snail. A t-ratio value greater than 1.96 indicated that the regression is significant. Heterogeneity factor value is less than 1.0 denoted that in the replicate test of random sample, the concentration response line would fall within 95% confidence limits and thus the model fits our data adequately.
| Table 2: | Toxicity of freeze-dried cow urine powder (FCU) , urine kept for 15 days in laboratory condition of different breeds cow Sahiwal, Geer, Tharparkar and its tertiary combination (1:1:5), A. squamosa, F. asafoetida and A. indica oil with C. sinensis, against L. acuminata at different exposure period |
| Six batches of 10 snails were exposed different concentration of the above molluscicides with different breeds of cow urine. Significant negative regression (p<0.05) was observed between exposure time and LC50 of treatments. Ts-testing significant of the regression coefficient: Sh.+A.S.+C.S.-8.61++; Gr.+A.S.+C.S.-22.86+; Th.+A.S.+C.S.-9.12+; Sh.+F.A.+C.S.-15.40++; GrCU+FA+CS-15.23++; ThCU+FA+CS-10.12+; ShCU+AI+CS-7.30++; GrCU+AI+CS-15.28+; h CU+AI+CS-7.10++.: inear regression between x and y; ++: non–linear regression between log x and log y. A.I: Azadirachta indica, A.S: Annona squamosa, C.S: Camellia sinensis, F.A: Ferula asafoetida, Gr: Geer, Sh-Sahiwal, Th: Tharparkar, LCL: Lower confidence limit UCL: uper confidence limit | |
The index of significant of protein estimation p-value indicates that the value of mean is within the limits at all probability level of 90, 95 and 99%.
CONCLUSION
Conclusively, it can be stated that the urine of Indian cow breeds and their formulations with different native plants can be used as potent molluscicides. These products are easily available and ecologically and culturally more acceptable to live-stock keepers and farmers of this region than their synthetic molluscicides. Toxicity of Sahiwal urine formulations is more potent than Geer and Tharparkar.
ACKNOWLEDGMENT
One of the authors (Shiv Kumar) is thankful to Rajiv Gandhi National Fellowship (RJNF), University Grants Commission, New Delhi letter No. F. 14-2 (SC)/2010 (SA-III) for financial assistance.