S. L. Hoti

A review of the complexity of biology of lymphatic filarial parasites

There are about five more common, including Wuchereria bancrofti and Brugia malayi, and four less common filarial parasites infecting human. Genetic analysis of W. bancrofti populations in India showed that two strains of the species are prevalent in the country. The adult filarial parasites are tissue specific in the human host and their embryonic stage, called microfilariae (mf), are found in the blood or skin of the host, depending upon the species of the parasite. Three genetically determined physiological races exist in W. bancrofti and B. malayi, based on the microfilarial periodicity. They are the nocturnally periodic, nocturnally subperiodic and diurnally subperiodic forms. The susceptibility of a mosquito species to filarial infection depends on various factors, which could be genetic, physiological or physical. Survival analysis of Culex quinquefasciatus infected with W. bancrofti showed that the parasite load in the mosquito is a risk factor of vector survival. The extrinsic life cycle of the parasite is initiated when the mf are ingested by a mosquito vector during feeding on the host blood. On maturity, most of the infective L3 stage larvae migrate to the head and proboscis of the mosquito to get transmitted to the mammalian host during subsequent feeding. They develop to the adult L5 stage and the period of development and the longevity of the parasites varies according to the species of the nematode and the mammalian host. The rate of production of mf by the adult female was found to be stable at least for a period of five years. The life span of the mf has some influence on the dynamics of transmission of filariasis. Recent studies show that the endosymbiont, Wolbachia, plays an important role in the survival of filarial parasites. The possibility of in vitro and in vivo culture of filarial parasites is also reviewed.

Longevity and migration of Wuchereria bancrofti infective larvae and their distribution pattern in relation to the resting and feeding behaviour of the vector mosquito, Culex quinquefasciatus

The longevity, migration and distribution of infective larvae (L3) of Wuchereria bancrofti within the host mosquito were studied by feeding Culex quinquefasciatus on microfilaraemic human blood and allowing the microfilariae to develop to L3. The L3 were found to remain alive and active for 46-50 days, i.e. as long as the host mosquitoes survived. The larvae started their migration to the head of the mosquito soon after their development to L3, on day 13 after the initial, infective bloodmeal. Although more L3 were usually found in the head region of the mosquitoes than in the thorax or abdomen, the larvae showed an oscillatory pattern of movement between all three regions. This movement was significant in the mosquitoes fed only on microfilaraemic blood, but not in those that took a second bloodmeal on normal human blood on day 8 after the infective bloodmeal. The distribution of the L3 in the three regions of the mosquitoes was independent of parasite density. Observations on L3 movement over a 24-h period indicated that there were always more L3 in the head than in the thorax and abdomen and that the number of L3 in the head was maximal at 18.00 hours and minimal at 06.00 hours. When infective mosquitoes were fed on human blood through a Parafilm membrane, 45.2% of the L3 present in the mosquitoes were shed into the blood or on the membrane. All the L3 present in each mosquito migrated to its head during feeding.

Development of Lymphatic Filarial Parasite Wuchereria bancrofti (Spirurida: Onchocercidae) in Mosquito Species (Diptera: Culicidae) Fed Artificially on Microfilaremic Blood

Abstract The efficiency of laboratory colonies of mosquitoes such as Anopheles stephensi Liston, Aedes aegypti (L.) Liverpool strain, Ae. aegypti wild type, Aedes albopictus (Skuse), Culex tritaeniorhynchus Giles, Culex sitiens Wiedemann, and Armigeres subalbatus Coquillett in supporting the development of Wuchereria bancrofti (Cobbold) (Spirurida: Onchocercidae) microfilariae to infective larvae was investigated. The mosquitoes were fed on heparinized microfilaremic human blood by using a membrane-feeding unit with Parafilm as membrane. The rate of infection, parasite development, and parasite burden were compared with that in the known vector mosquito Culex quinquefasciatus Say. Cx. quinquefasciatus showed the highest percentage of infection, followed by Ae. aegypti Liverpool strain and An. stephensi. The rate of development of the parasite was more or less similar in all the three species, and infective larvae were found on day 13. When the larvae were harvested on day 17, Cx. quinquefasciatus yielded the highest numbers, followed by Ae. aegypti Liverpool strain and An. stephensi. The percentage of infection was low, and the development was slow in Cx. tritaeniorhynchus compared with the other susceptible species. The parasite developed to second-stage larvae only by day 22 and to infective larvae by day 28. When 2-wk-old Cx. tritaeniorhynchus were fed on microfilaremic blood, they could develop the parasite to infective larvae by day 13 postfeeding. All other species of mosquitoes tested were found to be refractory to parasite development. It is shown that Cx. quinquefasciatus is the most suitable mosquito host for the production of infective larvae. However, Ae. aegypti Liverpool strain, which is commonly used for Brugia malayi filarial parasite, also can be used for generation of W. bancrofti infective larvae to circumvent the problem of maintaining two mosquito species.

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

Abstract 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.

A report on the demonstration of microfilariae of Brugia malayi in the brain of an experimental animal host, Mastomys natalensis

The brain tissues of microfilaraemic animals, Mastomys natalensis, which were earlier inoculated (s.c.) with Brugia malayi infective larvae (100 each) were examined for the occurrence of Mf. This was done by staining squash preparations of the brain tissues which were cleared off from the vascular piamater. Animals with blood Mf count of 50 >/per 20 cu. mm were found to harbour Mf in the brain tissues. The Mf count in the brain varied from 5-86/81 cu. mm (sum of Mf detected in 3 tissue pieces, each of 27 cu. mm collected from 3 parts of the brain, viz., the cerebral hemispheres, cerebellum and medulla oblongata). Teh presence of Mf in the brain was confirmed by its detection in 20-micrometers-thick cryosections of the tissue. Also, fine needle aspirates of cerebral hemispheres of an animal showed live Mf.

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

AbstractnFilarial 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 100xa0µl 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 100xa0µl 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 (100xa0µl) 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 5xa0µl 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 15xa0µl serum is adequate for use in Og4C3 assay. So the test can be performed without losing its sensitivity even with 5xa0µl serum samples at high titre of antigen (titre group 8) and 15xa0µl 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.

Detection of filarial specific IgG4 antibodies in individuals residing in endemic areas using panLFRAPID test card

In order to achieve the goal of global programme for elimination of lymphatic filariasis (GPELF), chemotherapy programmes are underway to interrupt transmission of the disease. At this point, detection of exposure will be more appropriate to monitor the programme and to certify areas cleared of active transmission as disease-free. A recently available cassette form of rapid test, panLFRAPID is a filarial IgG4 antibody detection test that may be useful for the programme. Therefore, we carried out a preliminary test using this cassette test on various categories of serum samples. The result showed that the test appeared to have potential in monitoring the exposure to filarial infection in GPELF.