Localization of Brugia malayi (sub-periodic) adults in different organs of Mastomys coucha and its influence on microfilaraemia and host antibody response

Lymphatic filariasis caused by nematode parasites Wuchereria bancrofti or Brugia malayi is a spectral disease and produces wide range of immune responses and varying levels ofmicrofilaraemia in infected individuals. The relationship between the immune response of host and the developmental stage of the parasite as well as the microfilariae (mf) density and specific location of the adult worms is yet to be understood. As an experimental model, B. malayi adapted in the experimental animal Mastomys coucha has been used widely for various studies in filariasis. The present study was to assess microfilaraemia as well as the humoral immune response of M. coucha during various stages of B. malayi development and their localization in different organs. The result showed that the density of mf in the circulating blood of the experimental animal depended upon the number of female worms as well as the location and co-existence of male and female worms. The mf density in the blood increased with the increase in the number of females. The clearance of inoculated infective stage (L3) or single sex infection or segregation of male and female to different organs of infected host resulted in amicrofilaraemic condition. With respect to antibody response, those animals cleared L3 after inoculation and those with adult worm as well as mf showed low antibody levels. But those with developmental fourth stage and/or adult worms without mf showed significantly higher antibody levels.

Suppression of Brugia malayi (sub-periodic) larval development in Aedes aegypti (Liverpool strain) fed on blood of animals immunized with microfilariae

Preliminary studies were carried out to investigate the role of filarial specific antibodies, raised in an animal model against the filarial parasite, Brugia malayi (sub-periodic), in blocking their early development in an experimental mosquito host, Aedes aegypti (Liverpool strain). In order to generate filarial specific antibodies, Mongolian gerbils, Meriones unguiculatus, were immunized either with live microfilariae (mf) of B. malayi or their homogenate. Mf were harvested from the peritoneal cavity of Mongolian gerbils with patent infection of B. malayi and fed to A. aegypti along with the blood from immunized animals. Development of the parasite in infected mosquitoes was monitored until they reached infective stage larvae (L3). Fewer number of parasites developed to first stage (L1) and subsequently to L2 and L3 in mosquitoes fed with blood of immunized animals, when compared to those fed with blood of control animals. The results thus indicated that filarial parasite specific antibodies present in the blood of the immunized animals resulted in the reduction of number of larvae of B. malayi developing in the mosquito host.

Toxicity of a mosquitocidal metabolite of Pseudomonas fluorescens on larvae & pupae of the house fly, Musca domestica.

BACKGROUND & OBJECTIVES: Biological control through the use of parasitoids and pathogens is one of the alternatives to the use of chemical pesticides for control of insects of public health importance. At the Vector Control Research Centre, a liquid formulation developed using the metabolite of a Pseudomonas fluorescens strain was found to be lethal to larvae as well as pupae of vector mosquitoes. The lethal fraction of the metabolite is a protein with a molecular mass of 44 kDa and toxicity studies showed that it is safe to mammals. In the present study, this formulation was evaluated against immatures of the common house fly, Musca domestica, to find out whether it could be developed into a potential biocontrol tool. METHODS: Early second instar larvae of house fly were introduced into rearing medium incorporated with the formulation at concentrations of 1, 5, 10, 15, 20 and 25 per cent, which were equivalent to respectively 1.13, 5.63, 11.25, 16.88, 22.50 and 28.13 microg of the toxic protein/ g of rearing medium. Mortality was monitored until the emergence of adult house fly. Net mortality of larvae and pupae were calculated and the LC50 and LC90 values were determined through probit regression analysis. RESULTS: Larval mortality was obtained from day 3 to 6 post-treatment. Net mortality of larvae was higher at the concentration of 20 than at 25 per cent. However, it was higher at 25 per cent on day 5 and continued to day 6 when there was no larval mortality at other concentrations. The net mortality of pupae was higher than that of larvae at all the concentrations except at 20 per cent. The LC50 and LC90 values calculated from the net mortality of larvae and pupae together, from day 1 to 12 post-treatment, were respectively, 8.25 and 51.79 microg protein/g of the fly rearing medium. INTERPRETATION & CONCLUSION: The formulation prepared from the exotoxin of P. fluorescens was toxic to the house fly. Pupae were more susceptible than larvae and the activity of the toxin might have been through cuticular absorption. The results are indicative of the possibility of development of the mosquitocidal metabolite for house fly control through appropriate field evaluations.

Oviposition response of the mosquito, Culex quinquefasciatus to the secondary metabolite(s) of the fungus, Trichoderma viride

Secondary metabolites produced by Trichoderma viride, a deuteromycetes fungus, under submerged culture condition were formulated and evaluated for oviposition attractancy against gravid females of Culex quinquefasciatus mosquito. At a concentration of 10 æg ml-1 the formulation showed remarkable attractancy with an oviposition active index (OAI) of +0.52. When the oviposition attractancy of the formulation was compared with a known oviposition attractant, p-cresol, both at 10 æg ml-1, the former was found to be more attractive to result in 70 percent egg laying than the later with 30 percent egg laying. Thin layer chromatography fractions of the secondary metabolites showed that a fraction with Rf value of 0.88 was highly active as oviposition attractant with an OAI of +0.65. Further work on identification of the active principle(s) of the microbial formulation might lead to an oviposition attractant useful in mosquito vector management

Enhanced recovery of fourth stage larvae of Wuchereria bancrofti from mongolian gerbil, Meriones unguiculatus & their in vitro maintenance.

Earlier attempts to produce different stages of W. bancrofti, such as fourth stage larvae (L4), in small animal models have yielded very low recovery rates. In order to enhance the recovery of L4, two routes of inoculating a small animal, M. unguiculatus, with infective larvae (L3) viz., intraperitoneal and intrathoracic routes, were compared. On day 17 post-inoculation, higher percentage (23-25%) of L4 were recovered from animals inoculated intrathoracically compared to that from animals inoculated intraperitoneally (2-8%). Also, comparatively higher proportion of worms (75-92%) remained within the intrathoracic region, unlike in the intraperitoneal region (50-80%). A few worms (1-4%) could be recovered even on 31 days post-inoculation from animals inoculated intrathoracically. When the L4 produced in animals were cultured in modified Frank's medium, all of them survived for 15 days and 50 per cent survived till the 25th day. The higher yield and ease of recovery from the thoracic cavity makes this route of inoculation a suitable method for production of L4. In vitro maintenance of L4 for prolonged period is significant with respect to excretory/secretory products or for drug screening.

Effect of temperature on different stages of Romanomermis iyengari, a mermithid nematode parasite of mosquitoes

The effect of temperature (20 degrees-35 degrees C) on different stages of Romanomermis iyengari was studied. In embryonic development, the single-cell stage eggs developed into mature eggs in 4.5-6.5 days at 25-35 degrees C but, required 9.5 days at 20 degrees C. Complete hatching occurred in 7 and 9 days after egg-laying at 35 and 30 degrees C, respectively. At 25 and 20 degrees C, 85-96 of the eggs did not hatch even by 30th day. Loss of infectivity and death of the preparasites occurred faster at higher temperatures. The 50 survival durations of preparasites at 20 and 35 degrees C were 105.8 and 10.6 hr respectively. They retained 50 infectivity up to 69.7 and 30.3 hr. The duration of the parasitic phase increased as temperature decreased. Low temperature favoured production of a higher proportion of females which were also larger in size. The maximum time taken for the juveniles to become adults was 14 days at 20 degrees C and the minimum was 9 days at 35 degrees C. Oviposition began earlier at higher temperature than at lower temperature. However, its fecundic period was shorter at 20 degrees C than at 35 degrees C indicating enhanced rate of oviposition at 20 degrees C. Fecundity was adversely affected at 20 degrees C and 35 degrees C. It is shown that the temperature range of 25 degrees-30 degrees C favours optimum development of R. iyengari.

In vitro cultivation of third stage larvae of Wuchereria bancrofti to fourth stage: influence of some physico-chemical factors.

It has been reported that third stage larvae (L3) of Wuchereria bancrofti strain from Jakarta, molted to the fourth stage (L4) in vitro, in a simple culture medium supplemented with 10% human serum. In the present study, this culture medium has been used to examine the effects of some physico-chemical parameters on larval growth, development and molting of Wuchereria bancrofti from India. Lymph at 10% concentration enhanced the in vitro survival time of larvae. Molting of larvae from L3 to L4 stage has been obtained using human fetal lung cells in cellular co-culture and as a source of conditioned medium. Given these improvements in the medium supplementation, it has been observed that the age of L3s (duration of L3s maintenance within the mosquitos) is one of the most important parameters for the development of L3s in vitro. No molting was observed when one day L3s were used whereas, molting occurred with one or two weeks old L3s. On the contrary, when more than 3 weeks old L3s were used molting failed to occur even though duration of survival of L3s was improved and in this case, most of the larvae were degenerated.

Observations on population density of Culex quinquefasciatus and transmission indices of Bancroftian filariasis during and after Integrated Vector Management strategy.

An Integrated Vector Management strategy, implemented as an alternative to the conventional control operations that include mainly chemical control in Pondicherry, South India, reduced very substantially the population density of Culex quinquefasciatus. This resulted in drastic decrease in the intensity of transmission of bancroftian filariasis transmitted by Culex quinquefasciatus and consequently the incidence of new infections in children of 0-5 age group was minimized. When the IVM strategy was withdrawn after five years of implementation and conventional control measures were re-adopted, resilience of Culex quinquefasciatus population was observed and human exposure to the risk of infection increased. The results suggest that maintenance of vector density at reduced levels for prolonged periods, is necessary to control infectious diseases like filariasis, which is difficult in the present day urban situations in developing countries. Hence the emphasis should be on chemotherapy to achieve control of lymphatic filariasis.

Infectivity of a mermithid nematode Romanomermis iyengari (Welch) in different conductivity levels under laboratory and field conditions.

Infectivity of R. iyengari was examined by exposing mosquito (Culex quinquefasciatus) larvae to the preparasite at different conductivity levels. The preparasite infected 63.5, 30, 11, 1.5 and 0.5% of the mosquito larvae respectively at 2000, 2500, 3000, 3300 and 3600 mu ho/cm. Although, 62-69% of the preparasite survived at 4000-5400 mu ho/cm, it did not infect. Application of preparasite to tree-holes resulted in 53-63% infection of Aedes albopictus larvae initially. On 6th day the infection level was 40% which decreased further to 7% by 15th day. The infection reappeared on 38th day indicating that R. iyengari has not only infected mosquito larvae as soon as they were applied to tree-holes in which the conductivity was 600-2800 mu ho/cm but also got established there.

An improved method of mass culturing of Romanomermis iyengari, a mermithid nematode parasite of mosquito larvae.

An attempt was made to develop an alternative method of mass culturing for R. iyengari instead of the usual sand culture method. Fifty pairs each of post-parasitic juveniles were seeded in moist sandbed and beakers containing tap water and distilled water and examined for exsheathing, egg-laying and egg-hatching. All post-parasitic juveniles in the moist sandbed had moulted by 7th day whereas in tap water and distilled water it lasted up to 11th day. Maximum numbers of eggs were observed on day 14 in sandbed (15/ml), day 16 in tap water (24/ml) and day 29 in distilled water (28/ml). The preparasites obtained from moist sandbed, tap water and distilled water did not exhibit any difference in their infectivity to mosquito larvae. The eggs of the nematode obtained from a culture in distilled water maintained at 30 +/- 2 degrees C for 60 days, when treated with CO2 (18 to 556 ppm) showed enhanced rate of egg hatching (73-98% compared with 11.5% in the untreated ones). CO2 treatment did not affect the infectivity of the preparasites that hatched from the CO2-treated eggs.

Effect of temperature and host-parasite ratio on sex differentiation of Romanomermis iyengari (Welch), a mermithid parasite of mosquitoes.

The effect of temperature and host-parasite ratio on the percentage infection and sex differentiation of R. iyengari was studied. Significant differences were observed in the percentage infection due to different host-parasite ratios and temperatures. At 25 degrees and 30 degrees C, the host parasite ratio of 1:3 resulted in 86-92% infection of Culex quinquefasciatus larvae. At 20 degrees and 35 degrees C, a higher host-parasite ratio was required to get this level of infection. More number of post-parasites per mosquito larva emerged at 20 degrees (1.5-5.8) and 25 degrees C (1.9-6.3) than at 30 degrees (1.5-3.9) and 35 degrees C (1.6-3.6). More than 50% of the post-parasites were females at 20 degrees and 25 degrees, 30 degrees and 35 degrees C at 1:1-1:10, 1:1-1:4 and 1:1-1:3 host-parasite ratios, respectively.