Neena Singla

Rodents as reservoirs of parasites in India.

We monitored the prevalence of endoparasitic infections of rodents in Punjab State, India, between January 2004 and December 2005. Three species of wild rodents, namely the house rat, Rattus rattus (n= 42), the lesser bandicoot rat, Bandicota bengalensis (n= 34) and the Indian gerbil, Tatera indica (n= 15), were live-captured from houses and crop fields. Examination of various organs revealed that the highest rates of endoparasitic infection occurred in R. rattus (40.5%), followed by B. bengalensis (35.3 %) and then T. indica (20.0%), with an overall infection rate of 35.2%. All three rodent species were found naturally infected with one or more species of helminths. Metacestodes (1-6) of Cysticercus fasciolaris (larvae of Taenia taeniaeformis) were found in all three rodent species (in the liver). In one male T. indica, numerous robust T. taeniaeformis metacestodes were found in oval sacs attached to the mesentery and the abdominal wall, an unusual site. The cauda epididymal fluid of the same gerbil was also found to be infected with a very rare species of strongylid nematode, which could not be identified to genus or species level. It is possible that this nematode is transmitted sexually and thus may affect the reproductive potential of gerbils. This appears to be the first report of this phenomenon. In one B. bengalensis individual, the intestine was found to be obstructed with an acanthocephalan, Moniliformis moniliformis, with concurrent infection with C. fasciolaris in the form of multiple cysts in the liver. Although no natural protozoan infection was found in these field rodents, experimental Trypanosoma evansi infection has been established in all three species with high pathogenicity, and the possibility of sexual transmission was supported by the presence of T. evansi in the cauda epididymal fluid of male rats.

Efficacy of acute and anticoagulant rodenticide baiting in sugarcane fields of Punjab, India.

The aim of this study was to recommend locality-specific rodent pest management techniques for sugarcane using acute and anticoagulant rodenticides during the months when maximum damage is inflicted on the crop in Punjab, India. Sugarcane crops were found to be infested with four rodent species namely: Bandicota bengalensis (Gray and Hardwicke); Mus booduga (Gray); Millardia meltada (Gray) and Golunda ellioti (Gray), with B. bengalensis being the most prevalent species (accounting for 69.45–83.36% of the total catch). Surveys in 11 villages of three districts of Punjab (India) revealed 19.12 ± 12.22% rodent damage to the sugarcane crop during the months of December–January. Rodenticide treatments were conducted in farmers fields in two districts of Punjab from December 2003 to January 2007. The results revealed that, to protect the sugarcane crop from rodent damage during the months of December–January, the rodenticide treatment may be applied either: (1) by double-baiting with 2% zinc phosphide followed by 0.005% bromadiolone after 15d at 1 kg/ha each, or (2) by single-baiting with 0.005% bromadiolone at 2 kg/ha. The impact of rodenticide treatment in canefields was also evident in the adjoining wheat crop fields where the incidence of rodent damage was less (0.97–3.24%) than in the fields surrounding untreated canefields (3.53–6.22%).

Effect of crude cottonseed oil containing gossypol on fertility of male and estrous cycle of female Bandicota bengalensis Gray and Hardwicke

Crude cottonseed oil (CCSO) containing 0.01% gossypol was evaluated in the present studies to record its effect on fertility of male and estrous cycle of female lesser bandicoot rat, Bandicota bengalensis Gray and Hardwicke. Laboratory feeding of bait (cracked wheat and powdered sugar in 98:2) containing 5 and 10% CCSO to female B. bengalensis for 13 days in no-choice feeding test, had no significant effect on the duration of the estrous cycle. However, laboratory feeding of bait containing 10% CCSO to male B. bengalensis for 15 and 30 days duration in a no-choice feeding test, significantly (P≤0.05) reduced sperm motility (%) and viability (%) in cauda epididymal fluid of treated rats compared to untreated ones. The weight of seminal vesicles of rats treated for 30 days was found to be significantly less (P≤0.05) than that for rats treated for 15 days. No significant effect of treatment was observed on the breeding success of rats, diameter of seminiferous tubules, cell count and association of various germ cells in testis. Further studies may be conducted by increasing the duration of treatment or by using CCSO containing a higher concentration of gossypol.

Potential of triptolide in reproductive management of the house rat, Rattus rattus.

Mature and healthy male house rats, Rattus rattus (n= 160) were fed on bait (cracked wheat: powdered sugar, 98:2) containing different concentrations of triptolide (0.1, 0.05, 0.025 and 0%) for 7 and 14 days in no-choice and bi-choice feeding tests in the laboratory. The objective of the study was to record the antifertility affects of triptolide after 30 and 60 days of termination of treatment. Results revealed no significant effect of triptolide treatment on weights of testis, epididymis, seminal vesicles and prostate gland of rats. Overall, sperm motility, live sperm count, sperm density and sperm morphology in the cauda epididymal fluid were found to differ significantly (P≤ 0.05) between untreated and treated groups of rats. The major effect of triptolide on sperm morphology was in the form of sperm head tail separation, which was up to 56.0% in rats treated for 14 days in no-choice and autopsied after 30 days. A significant effect (P≤ 0.05) of triptolide treatment was observed on the histomorphology of the testis, which included a dose-dependent decrease in diameter of seminiferous tubules, thickness of germinal epithelium and numbers of various spermatogenic cells. Cell associations in the seminiferous epithelial cycle were poorly developed in rats ingesting medium (4.7-5.1 mg/100 g bw) and high doses (6.9-7.2 mg/100 g bw) of triptolide than rats ingesting low doses (1.8-2.3 mg/100 g bw) and untreated rats. The cell stages affected had not recovered fully within the 60 day period following triptolide withdrawal. The present study suggests the potential of triptolide in reproductive management of Rattus rattus.

Potential of Eucalyptus Oil as Repellent against House Rat, Rattus rattus

Rodent repellents are chemicals which by taste or odour or possibly by both will prevent animal from feeding or gnawing. Such substances may be used in protecting an area from rodent infestation or in protecting packaged food, packing materials, electric cables, and other important vulnerable materials. Mature and healthy house rat, Rattus rattus of both sexes, was exposed to 5, 10, and 20% eucalyptus oil applied as spray in laboratory pens in bichoice tests. Each concentration was applied through three different modes of application, that is, daily, once, and alternatively in a week. Repellent effect of the oil was assessed based on food consumption from treated and untreated sides for four days. In overall, food consumption was significantly (P < 0.0001) low from treatment side compared to the untreated side indicating significant repellent effect of the oil at all the three concentrations tested. Repellent effect of the oil was, however, not found to differ significantly between the two sexes. Percent repellency in both male and female rats was apparently more with daily application of 5 and 10% eucalyptus oil. Present studies reveal the potential of eucalyptus oil in repelling away R. rattus; however, further studies may be conducted to enhance the persistence of repellent effect for longer period of time.

Effect of triptolide on reproduction of female lesser bandicoot rat, Bandicota bengalensis.

Abstract Triptolide has been reported to cause antifertility in male rats and mice. However, studies on female rats have been limited. Present study was aimed to evaluate the effects of triptolide on reproduction of wild female rodent pest species, Bandicota bengalensis. Feeding of bait containing 0.1, 0.15 and 0.2% triptolide for a period of 15 days in bi-choice resulted in per day ingestion of 17.37, 23.54 and 27.49 mg/kg body weight of triptolide, respectively. Examination of vaginal smear of all the rats revealed a significant (p ≤ 0.05) increase in duration of estrous cycle due to increase in durations of metestrous and diestrous stages in rats of treated groups. Autopsy of rats after 15 and 30 days of treatment withdrawal revealed significant (p ≤ 0.05) reduction in weights of uterus and ovaries, non-significant reduction in weights of liver and levels of estradiol and progesterone and significant (p ≤ 0.05) reduction in levels of urea and BUN and increase in levels of plasma proteins, ALT, AST, ALP, ACP and LDH in rats of treated groups compared to untreated group. There was no significant (p ≤ 0.05) effect of treatment on body weight. Triptolide treatment affected the histomorphology of uterus by causing a decrease in lumen and columnar cell height and number of uterine glands and ovary by increasing the number of atretic follicles and decreasing the number of developing follicles. The present study suggests triptolide to be a strong candidate affecting reproduction of female B. bengalensis.

Potential of citronella oil as rodent repellent measured as aversion to food

Repellents act by stimulating the primary or secondary defense mechanisms, causing the food to be rejected. Mature and healthy house rats, Rattus rattus, of both the sexes, were exposed to 5, 10, and 20% citronella oil applied as paint in laboratory pens in choice with no oil. Each concentration was applied through 3 different modes of application (daily, once and alternatively in a week). Repellent effect of oil was assessed by comparing food consumption from treated sides with those of untreated sides. The food consumption was recorded over a period of 4 days. In overall, food consumption was significantly (p≤ 0.05) lower from treatment side compared to the untreated side indicating significant repellent effect of the oil. Repellent effect of oil, however, did not differ significantly between the two sexes. Significant difference in average percent repellent effect between 5 and 10% concentrations was observed when oil was applied daily in both female and male rats with higher effect of treatment at 10%. The studies revealed higher potential of daily application of citronella oil as paint in repelling away rats of both sexes.

Histomorphological and biochemical changes induced by triptolide treatment in male lesser bandicoot rat, Bandicota bengalensis.

Mature and healthy male lesser bandicoot rats, Bandicota bengalensis (n = 40) were fed on bait (mixture of cracked wheat and powdered sugar in 98:2) containing different concentrations of triptolide (0, 0.15, 0.20 and 0.25% w/w) for 15 days in two-choice trials. Results revealed no significant effect of triptolide treatment on weights of vital organs after 30 and 60 days of treatment withdrawal. A significant (P ≤ 0.05) increase in plasma levels of TP, ALP, ACP, ALT and AST in response to stress induced in groups of rats treated with 0.20 and 0.25% triptolide was observed after 30 days of treatment withdrawal. No significant effect of treatment was observed on histomorphology of liver. A significant (P ≤ 0.05) effect of triptolide treatment was, however, observed on testicular function in the form of reduced diameter of seminiferous tubules and number of various spermatogenic cells indicating effect on spermatogenesis and spermiogenesis. The cell stages affected did not recover fully within 60 days period following treatment withdrawal. The present study suggests the potential of triptolide in the reproductive management of B. bengalensis by way of affecting testicular function.

Poultry egg components as cereal bait additives for enhancing rodenticide based control success and trap index of house rat, Rattus rattus.

OBJECTIVE To compare the acceptance and efficacy of cereal bait containing different concentrations of poultry egg components in laboratory and poultry farms to control house rat, Rattus rattus (R. rattus). METHODS Acceptance of cereal bait containing different concentrations (2%, 5% and 10%) of poultry egg components such as egg shell powder (ESP), egg albumin (EA) and crushed egg shell as bait additives were studied after exposing them to different groups of rats in bi-choice with bait without additive. Behaviour of rats towards cereal bait containing 2% concentration of different egg components was recorded in no-choice conditions through Food Scale Consumption Monitor. In poultry farm predominantly infested with R. rattus, acceptance and efficacy of 2% zinc phosphide bait containing 2% EA and ESP was evaluated. Trap success of single rat traps containing chapatti pieces smeared with 2% EA and 2% ESP was also evaluated in poultry farm. RESULTS In bi-choice tests, significantly (P<0.05) higher preference was observed for baits containing 2% and 5% ESP and all the three concentrations of EA compared to plain bait by female rats and that of baits containing 5% and 10% EA by male rats. In no-choice test, non-significantly higher consumption, number of bouts made and time spent towards bait containing 2% EA was found by rats of both sexes. In poultry farm, acceptance and efficacy of 2% zinc phosphide bait containing 2% EA and ESP was significantly (P<0.05) more than 2% zinc phosphide bait without additive. No significant difference was, however, found in trap success of single rat traps containing chapatti pieces smeared with 2% concentration of EA and ESP placed in the poultry farm. CONCLUSIONS Present data support the use of 2% egg albumin and egg shell powder in cereal bait to enhance acceptance and efficacy of 2% zinc phosphide bait against R. rattus. This may further help in checking the spread of rodent borne diseases to animals and humans.

Effect of Triptolide on Vital Organs, Blood Biochemical Parameters and Histomorphology of Testis in Male Bandicota Bengalensis

Present study reports the effect of triptolide in male Bandicota bengalensis. One group of rats was kept as vehicle control and the other three groups were fed on bait containing 0.15, 0.2 and 0.25% triptolide. Autopsy of rats immediately after 15 days of treatment period revealed no significant effect on weights of vital organs. A significant (P ≤ 0.05) increase in plasma levels of total proteins, alkaline and acid phosphatases and alanine and aspartate aminotransferases in response to stress induced by triptolide was observed in treated groups of rats. A significant (P ≤ 0.05) effect of treatment was also observed on histomorphology of testis in the form of decrease in seminiferous tubule diameter and number of various spermatogenic cells. Giant cells containing several round nuclei within a single cytoplasmic boundary were also observed in treated rats. Present studies suggest the antifertility potential of triptolide against male B. bengalensis. Introduction Rodents constitute the largest and most successful group of mammals worldwide. Among different rodent species, lesser bandicoot rat, Bandicota bengalensis Gray and Hardwicke has been reported as the most detrimental pest. It is the predominant species inhabiting irrigated crop fields and causing heavy losses in Punjab, India (Singla and Parshad 2010, Singla and Babbar 2010, 2012). The species also acts as reservoir of a number of parasites of zoonotic importance (Singla et al 2008, 2013). The economic losses and health problems associated with this pest species emphasize the need to develop techniques for its management. The use of rodenticides is the main approach being followed to tackle rodent problems all over the world. However, the excessive use of rodenticides have resulted in non target toxicity hazards (Brakes and Smith, 2005) in addition to development of resistance among animals (Pelz and Klemann 2004). Moreover, due to their high rate of reproduction, rodents rapidly rebuildup their population after a successful control operation with rodenticides (Shilova and Tchabovsky 2009). So the challenge is to develop strategies which can reduce the reproductive output of rodents and be effective in long term. Triptolide, a diterpenoid triepoxide obtained from Tripterygium wilfordii Hook, has several forms of pharmacological activities including anti-inflammatory, anti-fertility and anticancer activities (Zhang et al 2010). However, the clinical use of triptolide is known to present several practical disadvantages mainly due to its lower water solubility such as toxicity towards the heart, liver and kidney; reproductive dysfunction and hematopoietic dysfunction (Yang et al 2012, Zhang et al 2011). Earlier studies on antifertility effects of triptolide were conducted in laboratory rats and mice keeping in view the development of an oral human contraceptive (Lue et al 1998, Sinha Hikim et al 2000, Huynh et al 2000, Ni et al 2008, Liu et al 2010). Deng et al (2011) studied antifertility potential of oral doses of extracts of T. hypoglaucum containing triptolide against Mongolian gerbils keeping in view their management. In India, for the first time, Singla et al (2013) reported antifertility potential of triptolide fed in cereal based bait against house rat, Rattus rattus. Our previous studies (Dhar and Singla 2013) have reported complete inhibition of reproduction in female B. bengalensis paired with male rats treated with 0.2 and 0.25% triptolide in bait immediately after treatment withdrawal. Autopsy of rats immediately after termination of treatment have revealed significant effect of triptolide treatment on weights reproductive organs, sperm motility, viability, density and sperm morphology. Present studies report the effect of triptolide treatment on weights of vital organs, blood biochemical parameters and histomorphology of testes in male B. bengalensis. Material and Methods The present study was carried out in the Animal House Laboratory, Department of Zoology, Punjab Ag ricultural University, Ludhiana, India. Collection and maintenance of animals Mature and healthy male lesser bandicoot rat, B. bengalensis were live-trapped with multi-catch rat traps from crop fields in and around Ludhiana. In the laboratory, rats were acclimatized individually in cages for 10-15 days before the experiment with food and water provided ad libitum. Food consisted of a loose mixture of cracked wheat, powdered sugar and groundnut oil (WSO bait) at a ratio of 96:2:2. Proper hygienic conditions were maintained. Approval of the Institutional Animal Ethics Committee was obtained for the use of animals. Treatment Triptolide (molecular formula C20H24O6, molecular weight 360.41) was kindly supplied by Pidilite Indus tries, New Delhi, India. Rats (n = 20) were divided into 4 groups of 5 rats each. The average body weight of rats in different groups ranged from 248 to 311.2 g. There was no significant difference in average body weight of rats among the four groups. Rats of groups II–IV were fed on bait containing 0.15, 0.2 and 0.25% triptolide, respectively for 15 days in bi-choice with WSO bait. Treatment bait was prepared by mixing the de sired concentration of triptolide in a mixture of cracked wheat and powdered sugar (98:2). Triptolide was mixed in bait after being dissolved in vehicle (1% sodium carboxy methyl cellulose solution). Rats of group I, fed on WSO bait containing vehicle were kept as untreated control. Water was provided ad libitum throughout the treatment. Effect on weights of vital organs At autopsy of rats immediately after termination of treat-