Fascioliasis Control: In Vivo and In Vitro Phytotherapy of Vector Snail to Kill Fasciola Larva

Snail is one of the important components of an aquatic ecosystem, it acts as intermediate host of Fasciola species. Control of snail population below a certain threshold level is one of the important methods in the campaign to reduce the incidence of fascioliasis. Life cycle of the parasite can be interrupted by killing the snail or Fasciola larva redia and cercaria in the snail body. In vivo and in vitro toxicity of the plant products and their active component such as citral, ferulic acid, umbelliferone, azadirachtin, and allicin against larva of Fasciola in infected snail Lymnaea acuminata were tested. Mortality of larvae were observed at 2 h, 4 h, 6 h, and 8 h, of treatment. In in vivo treatment, azadirachtin caused highest mortality in redia and cercaria larva (8 h, LC50 0.11, and 0.05 mg/L) whereas in in vitro condition allicin was highly toxic against redia and cercaria (8 h, LC50 0.01, and 0.009 mg/L). Toxicity of citral was lowest against redia and cercaria larva.


Introduction
Fascioliasis is a worldwide zoonotic disease caused by Fasciola hepatica and Fasciola gigantica (family fasciolidae) [1]. F. hepatica has worldwide distribution but predominates in temperate zones while F. gigantica is found primarily in tropical regions [2][3][4]. The definite host is very broad and includes many herbivorous mammals including humans. Human fascioliasis has been reported in 51 different countries from five continents [4]. Fascioliasis is now recognized as an emerging human disease. World health organization has estimated that 2.4 million people are infected with Fasciola and a further 180 million are at risk of infection [1]. Singh and Agarwal [5] reported that 94% of buffaloes slaughtered in local slaughtered house in Gorakhpur district are infected with F. gigantica. In northern India Lymnaea acuminata is the intermediate hosts of the Fasciola species [2]. Although control of snail population below a threshold level is one of the important methods for effective control of fascioliasis [6][7][8][9], yet snails are one of the important components in the aquatic ecosystem. Release of molluscicides in aquatic system for snail control also affects the other nontarget organism. The Fasciola larval stage sporocyst, redia, and cercaria in the snail body are in division phase of F. gigantica. If these larvae will be destroyed by plant molluscicides at sublethal concentration in the snail body, the rate of infection can be reduced without killing the snail. Different plants-derived molluscicides and their active component such as citral, ferulic acid, umbelliferone, azadirachtin, and allicin [8,[10][11][12] were tested against Fasciola larva in in vivo and in vitro condition. There are new approaches to reduce incidence of the fascioliasis without killing the intermediate host snail.

Animals. Adult
Lymnaea acuminata (2.6 ± 0.20 cm in length) were collected locally, and cercaria shedding infected and uninfected snails were separated in two groups. The snails were allowed to acclimatize for 24 hours in laboratory condition. Each infected snail was dissected in a glass petri dish containing 10 mL of dechlorinated water at 22 • C-24 • C. The pH of the water was 7.1-7.3, and dissolved oxygen, free carbon dioxidez and bicarbonate alkalinity were 6.5-7.2 mg/L, 5.2-6.3 mg/L, and 102.0-105.0 mg/L, respectively.
After dissection redia and cercaria were separated in a different petri dish containing 10 mL of dechlorinated water.

In Vitro.
In vitro toxicity of plant products were performed in the petri dish. Ten redia and cercaria larva of Fasciola were separated in different petri dishes containing 10 mL dechlorinated tap water. Treatment of different plant products and their active components was made directly in the petri dish, containing 10 redia/cercaria. Mortality of redia and cercaria were observed after 2 h, 4 h, 6 h, and 8 h of treatment. Counting of larvae was performed with help of a microscope. Lethal value (LC 50 ), low and upper confidence limits (LCL and UCL), slope values, t-ratio, g-value, and heterogeneity factor were calculated with the help of POLO computer programmed by Robertson et al. [13]. One-way ANOVA and product moment correlation coefficient were applied by the method of Sokal and Rohlf [14].

Results
In vivo and in vitro larvicidal activity of Z. officinale, F. asafoetida, A. indica, A. sativum, and their active molluscicides components against the redia and cercaria larva of F. gigantica is time and concentration dependent (Tables 2-5). In in vivo and in vitro treatment azadirachtin and allicin caused highest toxicity against redia and cercaria larva. 8 h LC 50 of azadirachtin against redia/cercaria larva in in vivo treatment was 0.11 mg/L/0.09 mg/L, respectively (Tables 2   and 3). In in vitro treatment 8 h LC 50 of allicin against redia and cercaria was 0.01 and 0.009 mg/L, respectively (Tables  4 and 5). Toxicity of citral against both the larval stage was lowest (Tables 2-5). Significant (P < 0.05) negative regression was observed between exposure period and LC 50 of different plant products.
The slope values were steep, and separate estimation of LC based on each six replicatewas found with in the 95% confidence limit of LC 50 . The t-ratio was greater than 1.96 and the heterogeneity less than 1.0. The g-value was less than 0.5 at all probability levels (90, 95, and 99 resp.,); Tables 2, 3, 4, and 5).

Discussion
Results of the present study clearly indicate that the Zingiber officinale (citral), Ferula asafoetida (ferulic acid, umbelliferone), Azadirachta indica oil (azadirachtin), and Allium sativum (allicin) have sufficient larvicidal activity against different larva of Fasciola gigantica in in vivo and in vitro treatments. The alcoholic extract of A. sativum bulb has also shown moderate in vitro anthelmintic activity against human Ascaris lumbricoides [15]. A. sativum has been reported to be effective in dysentery and also acts as vermifuge [16,17]. Oil of A. sativum has also been reported to possess anthelmintic activity [18][19][20] and discards all injurious parasites in the intestine [16]. In vitro toxicity of allicin against redia (8 h, LC 50 , 0.01 mg/L) and cercaria (8 h, LC 50 , 0.009 mg/L) is highest in in vitro condition. The steep slope value indicates that a small increase in the concentration of different larvicide caused higher larval mortality. A t-ratio value greater than 1.96 indicates that the regression is significant. Heterogeneity factor value less than 1.0 denote that in the replicate test of random sample the concentration response limits and, thus, the model fits the data adequately. The index of significance of the potency estimation g indicates that the value of mean is within the limit at all probability level (90, 95, and 99, resp.) since it is less than 0.5.
Zingiber officinale is a perennial plant and is considered to be the universal medicine in Ayurveda. Significant anthelmintic activity of ethanolic extracts of rhizomes of Zingiber officinale against Ascaris lumbricoides has been reported [15,21]. Goto et al., [22] observed the in vitro lethal effect of Zingiber officinale on Anisakis larvae. The antifilarial effect of Z. officinale against Dirofilaria immitis has been reported by Datta and Sukul [23]. Adewunmi et al. [24] and Singh et al. [12] have reported the molluscicidal activity of Z. officinale.
Ferulic acid and umbelliferone are (Ferula asafoetida) potent molluscicides against L. acuminata [7,8]. Although the antioxidant, anticarcinogenic, antispasmodic, antihelminthic activity of F. asafoetida extract and ferulic acid were reported by various workers [25][26][27][28], yet there was no report in in vivo and in vitro larvicidal activity of these active components against (redia and cercaria larva). Azadirachtin, an active component of Azadirachta indica inhibits the motility of Haemonchus contortus larva [29], Singh et al. [6] observed that A. Indica have sufficient molluscicides activity against L. acuminata. In in vivo treatment of azadirachtin caused highest toxicity against redia (8 h, LC 50 , 0.11 mg/L) and cercaria (8 h, LC 50 , 0.05 mg/L). Present study clearly demonstrates that different larval stages in snail body as well as outside of snail body can be killed without killing the snails. Earlier it has been reported that citral (24 h, LC 50 -68.95 mg/L), ferulic acid (24 h, LC 50 -2.21 mg/L), umbelliferone (24 h, LC 50 -3.43 mg/L), azadirachtin (24 h, LC 50 -0.35 mg/L), and allicin (24 h, LC 50 -6.34 mg/L) are active molluscicides against L. acuminata [7, 10-12] 8 h LC 50 of these plants against redia and cercaria larva is many time low that is used to kill in intermediate host L. acuminata. The concentrations that were used to kill redia and cercaria are not toxic to snails, even in 24 h exposure period. Consequently, phytotherapy of snails by these plants and their active component kills the redia and cercaria of F. gigantica, without killing the host snail. Snails are a crucial component of an aquatic ecosystem. In vivo and in vitro killing of redia and cercaria of F. gigantica is beneficial as it kills the target larva of F. gigantica. Generally active components, that is, citral, ferulic acid, umbelliferone, azadirachtin, and allicin, inhibit activity of acetylcholinesterase, acid/alkaline phosphates, and ATPase in the nervous tissue of L. acuminata [8,11,30]. In cercaria larva acetylcholinesterase (AChE) in nervous functioning and cytochrome oxidase system in electron transport is well developed for efficient release of energy in active cercaria [31][32][33]. To elucidate the mechanism of the larvicidal activity of active components against larval stages of F. gigantica, their effect on AChE and cytochrome oxidase in larva is required for further investigation.

Conclusion
It can be concluded from the present study that sublethal treatment of active molluscicidal components citral, ferulic acid, umbelliferone, azadirachtin, and allicin, kill the redia and cercaria larva of F. gigantica inside the body of snail L. acuminata. Phytotherapy of infected snails by these active components is one of the new method to control the fascioliasis without killing the vector snail, an important components of the aquatic ecosystem.