Diethylcarbamazine citrate-fortified salt for lymphatic filariasis elimination in India

Lymphatic filariasis (LF) is a vector-borne neglected tropical disease, causing permanent disability. The disease is debilitating and widespread, leading to tremendous productivity and economic loss. The Government of India (GOI) prioritized the elimination of LF through the annual mass drug administration (MDA) programme in 2004 and continued with a single dose of diethylcarbamazine citrate (DEC), 6 mg/kg of body weight, plus albendazole annually over a period of 5-6 years. The GOI had set the target to achieve LF elimination by 2015 and now by 2030. The progress so far has been suboptimal. Much remains to be done as about 84 per cent of the total 328 endemic districts are still under MDA. The major challenge in implementing MDA is poor compliance. It is necessary to have a feasible alternative strategy addressing the above challenge to achieve the desired goal of LF elimination. At this juncture, a well-researched approach, i.e. the use of DEC-fortified salt, also advocated by the World Health Organization, as a unique form of MDA, is proposed. As per this strategy, a low dose of DEC (0.2% w/w) is added to the cooking salt at the manufacturing facility of iodized salt and consumed by the LF-endemic communities for about two years. Many examples of successful use of this strategy for LF elimination in small- and large-scale trials have been documented in India and several other endemic countries in the world. Implementing DEC–iodine-fortified salt is a safe, less expensive, more efficient and prompt approach for achieving the elimination of LF in India. Adverse effects are none or minor and self-limiting. The DEC-fortified salt strategy can easily piggyback on the existing countrywide deployment of iodized salt under the National Iodine Deficiency Disorders Control Programme (NIDDCP), which has achieved a great success in reducing iodine-deficiency disorders such as hypothyroidism. This existing robust programme can be leveraged to launch DEC-fortified salt for the community. If implemented appropriately, this strategy will ensure the complete cessation of LF transmission within two years from its introduction. If the said strategy is implemented in 2022, it is expected that India will be able to achieve the LF elimination by 2024, much before the global target of 2030.

An alternative strategy to generate coding sequence of macrophage migration inhibitory factor-2 of Wuchereria bancrofti.

Background & objectives: Different developmental stages of Wuchereria bancrofti, the major causal organism of lymphatic filariasis (LF), are difficult to obtain. Beside this limitation, to obtain complete coding sequence (CDS) of a gene one has to isolate mRNA and perform subsequent cDNA synthesis which is laborious and not successful at times. In this study, an alternative strategy employing polymerase chain reaction (PCR) was optimized and validated, to generate CDS of Macrophage migration Inhibitory Factor-2 (wbMIF-2), a gene expressed in the transition stage between L3 to L4. Methods: The genomic DNA of W. bancrofti microfilariae was extracted and used to amplify the full length wbMIF-2 gene (4.275 kb). This amplified product was used as a template for amplifying the exons separately, using the overlapping primers, which were then assembled through another round of PCR. Results: A simple strategy was developed based on PCR, which is used routinely in molecular biology laboratories. The amplified CDS of 363 bp of wbMIF-2 generated using genomic DNA splicing technique was devoid of any intronic sequence. Interpretation & conclusions: The cDNA of wbMIF-2 gene was successfully amplified from genomic DNA of microfilarial stage of W. bancrofti thus circumventing the use of inaccessible L3-L4 transitional stage of this parasite. This strategy is useful for generating CDS of genes from parasites that have restricted availability.

Outbreak of dengue virus serotype-2 (DENV-2) of Cambodian origin in Manipur, India - Association with meteorological factors.

Background & objectives: Dengue is a major health problem in many parts of India and its neighbouring countries. Dengue cases have not been reported from Manipur, a northeastern State of India till 2007. But, the sudden outbreak of fever with febrile illness during 2007 and 2008, suspected to be dengue/dengue haemorrhagic fever was investigated to detect the causative agent. Potential impact of climatic variables on dengue transmission has been documented and hence the association between climatic factors, entomological parameters and dengue cases was also analysed. Methods: Forty two and 16 blood samples were collected from patients suspected to have dengue infection in the year 2007 and 2008, respectively. Viral RNA was extracted from serum samples and subjected to multiplex one step RT-PCR assay. Dengue specific amplicons were sequenced and phylogenetic analysis was carried out. Multiyear trend analysis and ‘t’ test were performed for the comparison of different meteorological variables between the years 2000-2004 and 2005-2008. Results: The aetiological agent was found to be DENV-2 and the phylogenetic analysis showed that the isolate was similar to that of Cambodian isolate. There was a significant difference in minimum temperature (P<0.05), Relative humidity - morning hours (P<0.001), relative humanity - afternoon hours (P<0.01) and cumulative precipitation (P< 0.05) between the years 2000-2004 and 2005-2008. Interpretation & conclusions: The sudden outbreak of dengue fever in Manipur State occurred was possibly due to the increased temperature, relative humidity and decrease in cumulative precipitation. These climatic factors would have contributed to the Aedes mosquito abundance and increased virus transmission. Proper diseases surveillance system integrated with meteorological warning system and management of vector breeding sites will prevent such outbreaks in future.

Effect of a single dose of diethylcarbamazine, albendazole or both on the clearance of Wuchereria bancrofti microfilariae and antigenaemia among microfilaria carriers: A randomized trial.

Background. Lymphatic filariasis is a major vector-borne parasitic disease. The global programme to eliminate lymphatic filariasis was launched in 1997 and currently over 570 million people are covered under it in 48 countries. Mass annual single-dose drug administration of diethylcarbamazine (DEC), co-administrated with albendazole for 5–6 years and mass distribution of diethylcarbamazine-fortified salt are the two strategies for elimination of filariasis. Methods. Asymptomatic volunteers residing in Puducherry, India were screened for microfilaria (mf) by examining nocturnal thick blood smears. Those testing positive were randomly assigned to receive a single dose of DEC (6 mg/kg body weight) or albendazole 400 mg or both. Participants were hospitalized for 5 days. Membrane filtration count was used to assess microfilaraemia and ELISA (Og4C3) assay to measure circulating filarial antigens (CFA). Measurements were done before treatment and at 1, 2 and 3 years post-treatment. Viability of the adult worms was assessed by looking for the filarial dance sign (FDS) using ultrasound examination of the scrotum in men with hydrocele. Results. Fifty-four microfilaraemic individuals were studied. The mf prevalence started decreasing only by day 180 posttreatment in the DEC group but much earlier in the other two groups (day 30 in the albendazole and day 90 in the DEC with albendazole group). The decrease in mf was marginal (17.6%, 26.3% and 27.8%, respectively) by the end of year 1 posttreatment, but significant (96.7%, 78.6% and 93.3%, respectively) by the end of year 2 post-treatment (p<0.05). By the end of year 3, the level decreased to 80% in the DEC, 90% in the albendazole and to 100% in the DEC and albendazole groups. However, the mf intensity decreased © The National Medical Journal of India 2010 Vector Control Research Centre, Department of Health Research (ICMR), Indira Nagar, Puducherry 605006, India S. L. HOTI, S. P. PANI, P. VANAMAIL, K. ATHISAYA MARY, L. K. DAS, P. K. DAS Correspondence to S. L. HOTI; slhoti@yahoo.com significantly (by 39%; p<0.05) by day 7 post-treatment in both the DEC and DEC with albendazole groups, but only by day 30 in the albendazole group. In all the drug groups, the prevalence as well as intensity of CFA returned to pretreatment levels by the end of year 3 post-treatment. Conclusion. Annual single-dose administration of all the 3 drug regimens significantly reduced antigenaemia levels. There were no significant differences in the efficacy and overall pattern of CFA clearance between the 3 drug regimens.

An allele specific PCR assay for screening for drug resistance among Wuchereria bancrofti populations in India.

Background & objectives: Albendazole, a commonly used anthelminthic drug that targets the polymerization of α- and β-tubulin dimer is currently co-administered with the antifilarial drug, diethylcarbamazine citrate (DEC) in the ongoing Global Programme for Elimination of Lymphatic Filariasis (GPELF). The experience in veterinary field has shown that there can be a rapid development of resistance to this drug, which therefore, needs to be monitored regularly in GPELF. Hence, we investigated the nucleotide polymorphism in the albendazole-binding domain of the isotype 1 β-tubulin gene from several populations of Wuchereria bancrofti and developed an AS-PCR assay useful in screening for sensitive/resistance alleles among parasite populations and also evaluated its utility. Methods: For studying the polymorphism of isotype 1 β-tubulin gene, a 475 bp fragment spanning exon 5 and 6 of the gene was amplified and sequenced from the genomic DNA of W. bancrofti collected from six geographic regions of India. An allele specific (AS) PCR for screening albendazole sensitivity/resistance was developed and a total of 55 mf samples from blood smears on slides collected from Thiruvannamalai, Thanjavur and Puducherry were screened. Selective therapy with DEC was in place in three areas, mass drug administration (MDA) with DEC alone was implemented in four areas, while DEC plus albendazole was administered in one district. Results: The analysis of the nucleotide sequence of the fragment from 20 W. bancrofti populations showed the domain to be highly conserved. An allele-specific PCR assay developed was used to detect sensitive/ resistance alleles among 55 isolates of W. bancrofti and no albendazole resistance alleles were detected among the populations tested. Interpretation & conclusion: The drug-binding domain of isotype 1 β-tubulin gene of W. bancrofti from different geographical locations was highly conserved. The AS-PCR developed showed potential application as a tool for monitoring albendazole sensitivity/resistance alleles among W. bancrofti populations, in areas where combination therapy of DEC-albendazole is being mass administered in the LF elimination programme.

RT-PCR assay for the detection of infective (L3) larvae of lymphatic filarial parasite, Wuchereria bancrofti, in vector mosquito Culex quinquefasciatus.

BACKGROUND & OBJECTIVES: Periodic monitoring of vector population for infection and infectivity rates is central to the evaluation of the filariasis elimination strategies in endemic areas to monitor the success of MDA and also to establish endpoints for intervention. The main objective of this study was to develop a RT-PCR assay, based on L3 stage-specific primers to detect the presence of infective stage larvae of filarial parasite, Wuchereria bancrofti in the vector Culex quinquefasciatus. MATERIAL & METHODS: Subtracted probe development technique was employed for the identification of infective stage (L3) specific genes. The subtracted cDNA was labeled by non-radioisotopic method and used for screening cDNA library of L3 stage larvae of W. bancrofti constructed in UniZap XR. Recombinants were probed and identified from the library. The inserts of the recombinant clones were purified and sequenced. Primers were designed based on the sequence information of three recombinant clones for detecting L3 larvae of W. bancrofti in the vector by RT-PCR assay. Preliminary laboratory evaluation was carried out to assess the sensitivity and specificity of WbL31 RT-PCR assay. RESULTS: cDNA library of L3 stage of W. bancrofti constructed in UniZap XR vector, constituted 5 x 10(5) phages with 80-90% recombinant phages and the size of inserts varied from 0.1 to 1.0 kb. When subtracted cDNA was random prime labeled and used for screening cDNA library of L3 stage of W. bancrofti constructed in UniZap XR, 18 clones were identified from the library. Three genes were found up-regulated in the L3 stage, out of which WbL31 (cuticular collagen) was found to be useful in detecting L3 larvae of W. bancrofti in the vector by RT-PCR assay with high specificity and sensitivity (98-100%). CONCLUSION: Present paper marks first report on the development of an infective stage-specific RT-PCR assay (WbL31 RT-PCR assay) to detect L3 stage W. bancrofti in the vector. This assay will have potential application in assessing the transmission of infection and hence in decision-making related to elimination programme.

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.

Comparison of an immunochromatographic card test with night blood smear examination for detection of Wuchereria bancrofti microfilaria carriers.

BACKGROUND: At present, two diagnostic tests--Og4C3 ELISA and an immunochromatographic card test (ICT)--are available to detect circulating filarial antigens of Wuchereria bancrofti in serum/whole blood samples collected during the day. We aimed to assess the sensitivity of the new format card test 'NOW ICT Filariasis' in detecting microfilaria carriers of W. bancrofti in comparison with conventional microscopic techniques and Og4C3 ELISA. METHODS: A total of 200 persons were selected from two villages following a quota sampling design (100 in each village). The required number of houses was selected using a systematic sampling procedure with a random start of the first household. Blood samples were taken from all the available persons in each selected house until the quota of 100 was reached. The new format ICT test, Og4C3 ELISA and night blood smear examination for microfilaria were carried out following standard procedures. RESULTS: The sensitivity of the new format ICT test was 100% among microfilaria carriers (detected by both early and late readings). The kappa statistic measure of agreement between the two readings of all the samples (n =200) tested was 0.811 (p<0.05). The new format test also reported 25% of microfilaria-negative individuals as being positive for circulating filarial antigens. However, the diagnostic lines were not stable beyond 10 minutes (particularly in the case of amicrofilaraemic persons). Though there was an overall agreement between the results of ICT and Og4C3 tests (kappa =0.612; p< 0.05), the sensitivity of the Og4C3 test was lower than that of ICT. CONCLUSION: The new format ICT test is highly sensitive in detecting microfilaria carriers in endemic communities. Improvement in the format to provide stable diagnostic lines, specificity of the format and cost of the test kit are to be considered before its large-scale use.

Presence of Wolbachia endosymbionts in microfilariae of Wuchereria bancrofti (Spirurida: Onchocercidae) from different geographical regions in India

In view of the recent discovery of rickettsial endosymbionts, Wolbachia in lymphatic filarial parasites, Wuchereria bancrofti and Brugia malayi and subsequently of their vital role in the survival and development of the latter, antibiotics such as tetracycline are being suggested for the treatment of lymphatic filariasis, by way of eliminating the endosymbiont. But, it is essential to assess their presence in parasites from areas endemic for lymphatic filariasis before such a new control tool is employed. In the present communication, we report the detection of Wolbachia endosymbionts in microfilariae of W. bancrofti parasites collected from geographically distant locations of India, such as Pondicherry (Union Territory), Calicut (Kerala), Jagadalpur (Madhya Pradesh), Thirukoilur (TamilNadu), Chinnanergunam (TamilNadu), Rajahmundry (Andhra Pradesh), and Varanasi (Uttar Pradesh), using Wolbachia specific 16S rDNA polymerase chain reaction.

Detection of day blood filarial antigens by Og4C3 ELISA test using filter paper samples.

BACKGROUND: The launching of the global filariasis elimination programme has necessitated the use of highly sensitive and specific diagnostic tests. The Og4C3 monoclonal antibody-based ELISA test has been found to be highly specific and sensitive for the diagnosis of filariasis using night blood samples. However, it requires a serum sample which poses problems of transport and storage. Collection of blood samples on filter paper the will greatly circumvent these problems. Therefore, we evaluated the utility of the Og4C3 assay on filter paper samples collected during daytime. METHODS: Blood samples were collected from 63 microfilariae (mf) carriers during different time periods in a day on filter paper discs as well as venous blood for sera. The mf carriers and chronic (hydrocele n = 20; lymphoedema n = 120) and acute filariasis (adenolymphangitis n = 39) patients were from the endemic areas and the non-endemic normals were from Uthagamandalam district of Tamil Nadu, India. The filarial antigens in the samples were determined using the Og4C3 filarial antigen assay as per the manufacturer's instructions (JCU TrapBio, Australia). The sensitivity of the assay on sera and filter paper samples collected during night and also on filter paper samples collected during different time intervals of the day were compared with those of the membrane filtration technique, which was used as a gold standard. RESULTS: The geometric mean titre of the sera samples collected during night was 11 units/ml for non-endemic normals and 601.2 units/ml for mf carriers. The specificity of the assay on sera samples collected during night was 100% and the sensitivity 96.8% and the positive and negative values were 100% and 95.2%, respectively. The antigen positivity of the filter paper samples collected during morning hours was 93.3% while it was 76.6% and 86.7% for afternoon and evening hours. A significant association was observed between antigenaemia levels and mf density in the blood samples collected during the night. CONCLUSION: The samples collected on filter paper during the day can be used as an alternative to sera samples for detection of filarial antigens employing Og4C3 ELISA. Also, samples collected during morning hours yield a higher positivity. The assay when applied to serum samples will be useful especially when quantitative results are required.

Laboratory evaluation of Ssp I PCR assay for the detection of Wuchereria bancrofti infection in Culex quinquefasciatus.

BACKGROUND & OBJECTIVES: There is a need to delimit the areas of filariasis transmission in view of the Filariasis Elimination Programme launched in India. Infection rate in vectors is an important parameter in determining transmission and it is conventionally assessed by dissection and microscopy. A PCR assay based on Ssp I repeats of Wuchereria bancrofti has shown potential in the detection of infection in vectors. The aim of the present study was to evaluate the specificity and sensitivity of this assay on W. bancrofti and its vector, Culex quinquefasciatus, prevalent in India. METHODS: The DNA from pools of C. quinquefasciatus to which W. bancrofti microfilariae (mf) were added, was extracted by lysing with 0.1 M NaOH and 0.2 per cent sodium dodecyl sulphate (SDS), followed by silica absorption in the presence of guanidinium thiocyanate. The PCR assay of the DNA samples was carried out using NV-1 and NV-2 primers and the species specific SspI band was visualized on agarose gels stained with ethidium bromide. RESULTS: The Ssp I PCR assay was found to be highly species specific, as it did not detect the DNA of a closely related filarial parasite, Brugia malayi. The assay detected as little as 0.04 pg of W. bancrarofti DNA. Minimum number of parasite detectable in pools of mosquitoes was 1 mf. A pool size of 50 mosquitoes was found to be optimum for the PCR assay. INTERPRETATION & CONCLUSION: The Ssp I PCR assay was found to be highly specific and sensitive in detecting filarial parasite in pools of mosquitoes and therefore has potential application in rapid assessment of transmission of filariasis.

Association of the level of mosquito larvicidal activity with the growth & sporulation in Bacillus sphaericus H-5a5b strains.

The role of growth and sporulation in the production of mosquito larvicidal factors in B. sphaericus H-5a5b strains was investigated using 6 strains that differed in their larvicidal activity. Among these, strain B64 produced maximum biomass (15.5 g/l by 29th h) while B45 and B85 yielded the least (12.8 g/l by 41st and 37th h respectively). Strains B43 and B42 reached the peak of viable cell count (4-6 x 10(10) cells/ml) 4 h earlier than B64 and 12 h earlier than the rest of the strains. Strains B42 and B43 produced higher number of heat resistant spores (4 x 10(8) spores/ml), while strains B45 and B57 produced the lowest numbers (2-4 x 10(5) spores/ml). Mosquitocidal toxin synthesis was noticed as early as the 5th and 9th h in the cultures of the strains B42 and B64 respectively while in those of other strains it was detected by the 13th h or later. The results indicated that generally the highly and moderately toxic strains grew faster and sporulated better than the poorly toxic ones.

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.

Tropical vaginal hydroceles: are they all filarial in origin?

Hydrocele of the tunica vaginalis testis has been conventionally used as an absolute indicator of filarial disease in most clinical surveys. The prevalence of filarial etiology in 100 consecutive hydroceles was studied using clinical, parasitological, histopathological and immunological parameters. Filarial etiology could be proved in 57% of hydrocele cases using major criteria: presence of microfilaria in hydrocele fluid, presence of chyle in hydrocele fluid, demonstration of adult worm in tunica, ratio of fluid antibody titer to serum antibody titer more than 2 and presence of filarial antigen in hydrocele fluid. The results of other tests in these 57 cases were used to define the minor criteria. In the other 43 cases, based on the minor criteria, 12 hydroceles could be classified as likely to be due to filariasis and the rest were probably non-filarial. Thus only 69% of hydroceles were definitely or probably filarial.

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.

Microbial flora in gut of Culex quinquefasciatus breeding in cess pits.

The number and types of microorganisms in the gut of Culex quinquefasciatus larvae varied considerably from one site of collection to another. Larval gut, in general, contained enormous number of bacteria, a few fungi and negligible number of actinomycetes which belonged to 15 bacterial, 6 fungal and 4 actinomycete genera, respectively. Bacillus sp., Staphylococcus sp. and Pseudomonas sp. among bacteria, Aspergillus among fungi and Streptomyces sp. among actinomycetes were frequently encountered. Escherichia, Proteus, Aspergillus and Streptomyces were the most abundant genera. Isolates of Bacillus, Pseudomonas, Shigella and Staphylococcus caused 100% mortality during the early instar of larval development. None of the fungal isolates effected 100% mortality while Nocardiopsis sp. among actinomycetes gave 100% mortality. One of the Escherichia isolate suppressed the adult emergence completely while 27 others, belonging to most of the genera found, suppressed significantly. Isolates of Aspergillus, Alternaria and Streptomyces inhibited the emergence of adults completely.

Method for storing Lagenidium (Oomycetes: Lagenidiales).

Techniques for storing the mosquito pathogenic fungus, Lagenidium, were evaluated. A technique, which involves storage of fungal mycelia in sterile distilled water of pH 6-7 with 0.0025 M glucose at 30-35 degrees C, was found to be useful. When stored in this manner the fungus retained it's larvicidal activity for 190 days.

Evaluation of Bacillus sphaericus formulations against the vector of bancroftian filariasis.

Three different formulations of Bacillus sphaericus viz, Spherimos, Vectobac and Spherifix, were evaluated for their efficacy and residual activity against Culex quinquefasciatus breeding in polluted disused wells. Spherimos, a flowable concentrate formulation, exerted 96-100% control when treated at the dosage of 10 l/ha for 17 days, whereas the effective residual activity lasted up to 67 days at 15 l/ha. In the case of Vectolex, a granular formulation, the residual activity lasted up to 56 days with the dosage of 30 l/ha and up to 66-77 days with higher dosages of 45 and 60 l/ha. The residual activity of Spherifix, a floating controlled release formulation, lasted up to 67 days with a dosage of 10 kg/ha.

Characterization & larvicidal activity of indigenous isolates of Bacillus sphaericus from natural breeding habitats.

Soil, water and moribund/or dead mosquito larval samples collected from various mosquito breeding habitats of Pondicherry and Tamil Nadu were screened for the presence of mosquito pathogenic B. sphaericus isolates. From 1892 samples 21 isolates were obtained. All these isolates fell under 3 serogroups viz., H5a5b, H6 and H45, the latter two serotypes not reported hitherto as toxic to mosquito larvae and three phage-groups namely, phage group 2, phage group 3 and an unknown one. Twelve of the isolates were highly toxic and superior to the standard strains 1593, 2297 and 2362 supplied by Pasteur Institute, Paris when tested against Culex quinquefasciatus larvae. Out of these 12 strains 11 belonged to the serotype H5a5b and the phage-group 3 and 1 belonged to the serotype H45 and an unknown phage group.