Scope of detectability of circulating antigens of human lymphatic filarial parasite Wuchereria bancrofti with smaller amount of serum by Og4C3 assay: its application in lymphatic filariasis elimination programme.

Filarial antigen detection is an appropriate epidemiological indicator for mapping lymphatic filariasis and impact evaluation of filariasis elimination programme in view of low sensitivity of parasite detection. Monoclonal antibody-based Og4C3 immunological test requires 100 µl serum, which is difficult to collect by finger prick method during community based surveys. Hence, we tested lesser volume of serum compared to standard volume of 100 µl to compare its sensitivity and specificity in detecting the circulating filarial antigens. Blood samples were collected from individuals who tested positive [with titer groups 4 (border line positives), 6 (medium positives), and 8 (high positives)] and negative (titre group 3) for Og4C3 assay. Different volumes of serum samples were used to make-up required volume (100 µl) with appropriate dilutions and subjected to Og4C3 assay. The results showed that known negative samples tested negative at all the serum volumes tested. All positives (titer groups 6 and 8) showed positivity at all reduced volumes of serum sample. However one of the medium positive sample showed negative reaction in 5 µl volume of serum and two of the border line positives showed negative at all the serum volume tested. The results thus showed as less as 15 µl serum is adequate for use in Og4C3 assay. So the test can be performed without losing its sensitivity even with 5 µl serum samples at high titre of antigen (titre group 8) and 15 µl for other groups and this method has scope in programme evaluation.

Binding sites of mosquitocidal toxins of Pseudomonas fluorescens and Bacillus subtilis on pupae and larvae of Culex quinquefasciatus.

Two of the potential bacterial isolates, viz., Pseudomonas fluorescens (VCRC B-426) and Bacillus subtilis (VCRC B-471) whose toxins kill the mosquito pupae/larvae have been identified at our center. As the mode of action of these bacteria are not known, an attempt was made to find out the binding sites of the toxic proteins through immunological methods. Antibodies were raised in BALB/c mice and egg yolk system of chicken layers against the mosquitocidal proteins. The antibodies showed specific binding on to the cephalic and thoracic cuticle of the pupae as well as the paddles of the larvae, indicating the binding of the mosquitocidal proteins.

Hexamerin a novel protein associated with Bacillus sphaericus resistance in Culex quinquefasciatus.

Bacterial insecticides like, Bacillus sphaericus and Bacillus thuringiensis serovar israelensis, have been used for the control of nuisance and vector mosquitoes for more than two decades. For many years, it was assumed that the use of microbial larvicides based on B. sphaericus would not lead to resistance in mosquitoes. However, recent reports have shown that B. sphaericus toxins are not free from this problem. Therefore, the resistance of mosquito populations to be will seriously threaten the sustainability of current mosquito control programme using these microbial insecticides. In the present study, we have characterised a novel protein responsible for resistance development in the filariasis vector of Culex quinquefasciatus. Laboratory selection experiments with B. sphaericus against the larvae were carried out up to 17 generations, and the occurrence of resistance was reported (resistance ratio (RR) at lethal concentration (LC)50 and LC90 = 1,987 and 2,051 folds, respectively). The protein profiles of B. sphaericus-resistant and susceptible population have confirmed with the expression of a new polypeptide (80 kDa) in the resistant strain only. Sequence result revealed that the newly expressed protein was 'hexamerin', and this factor might conceivably be responsible for the inheritance of resistance. This study is therefore valuable for comprehending the underlining factor and management of B. sphaericus resistance problem in mosquito population.

A method for detecting microfilaraemia, filarial specific antigens and antibodies and typing of parasites for drug resistance and genotypes using finger prick blood sample.

Monitoring and evaluation of programme to eliminate lymphatic filariasis (LF) depends on epidemiological assessment using appropriate indicators. Minimum efforts using reliable tests are necessary to guide the programme managers in decision-making. Impact of Mass Drug Administration (MDA) towards filariasis elimination can be assessed by the detection of microfilariae (mf) or parasite DNA (infective), filarial antigens (infected) and antibodies (exposure). It is also important to monitor drug resistance and variation in genetic structure of parasite populations using molecular markers. We developed a method to carry out parasitological, molecular, immunological and genetic analysis from a minimum volume of blood sample (about 150 microl) drawn from finger tip of an individual residing in LF endemic area. The method involves separation of sera for immunological assays and isolation of mf of Wuchereria bancrofti from the blood clots for counting, which were then used for W. bancrofti specific PCR, screening for albendazole sensitivity/resistance alleles by AS-PCR, RAPD profiling and ITS 2 PCR for genotyping. A protocol is also suggested for the separation of sera for assays to detect antigen and antibodies and isolation of mf from clots for genetic analysis. The protocol developed has shown potential application in monitoring several immunological, parasitological and molecular parameters from a limited amount of blood sample collected by finger prick, in large-scale operations.

Monoclonal antibodies generated against excretory/secretory antigens of mammalian stage larvae of the lymphatic filarial parasite Wuchereria bancrofti.

Monoclonal antibodies (Mabs) against excretory/secretary (e/s) antigens of fourth stage (L4) larvae of Wuchereria bancrofti were raised and screened for their specificity and sensitivity and evaluated for their potential in detecting homologous e/s antigens in human blood samples. Five Mabs were obtained and, among them, Mab A7 showed high reactivity against e/s antigens of L4 and crude somatic antigens of microfilariae (mf) of W. bancrofti, and infective stage (L3) and adult stage larvae of Brugia malayi. It reacted strongly with sera of Mastomys coucha harbouring L4 stage of B. malayi moderately against sera of the animal having later stages of the parasite. But, it exhibited a low and negligible reactivity against the crude antigens of Setaria cervi and Ascaris lumbricoides, respectively. Another Mab, A6, showed very high reactivity against mf antigens of W. bancrofti and B. malayi and a moderate reactivity against antigens of S. cervi and A. lumbricoides. The two Mabs were tested for their reactivity against filarial antigens in human sera, whose microfilaraemic status was determined by membrane filtration of 1 mL blood sample collected during night. When Mab A7 was tested, 7 out of 22 serum samples (32.0%) from amicrofilaraemic normal individuals from filariasis endemic areas showed positive reactions for filarial antigens, indicating the presence of early stage (L4) of the parasite in them. It also reacted with 84% (n=19) mf positive samples and 11% of non endemic normal serum samples (n=17). Mab A6 showed high reactivity with 86% (n=26) of mf positive serum samples, but did not react with non-endemic normal serum samples (n=17). The results, thus, indicate that the Mab A7 has potential in the detection of e/s antigens of L4 stage larvae of filarial parasites in humans, enabling early diagnosis of filariasis. Mab A6 could be used in the diagnosis of patent infection with microfilaraemia. Western blotting with Mab A7 reacted with the 29.0 kDa protein band of L4 e/s antigens of W. bancrofti.

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.