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.

Biocontrol agents and oviposition attractants are promising alternate tools/adjuncts for mosquito control. Using oviposition attractants, monitoring of vector populations, especially Aedes species, could be carried out so as to plan control measures, or to attract gravid females to lay eggs at chosen sites and kill the emerging larvae by combining a larvicide. Microorganisms inhabiting mosquitobreeding sites (Androsov et al. 1986) play a major role in the decomposition of detritus present in the habitats leading to the production of several metabolites. Some of these metabolites are likely to act as oviposition attractant and/ or stimulants for mosquitoes. Therefore, in the present study culture filtrates containing metabolites of a few bacteria were examined for oviposition attractancy and the results are presented hereunder. Cultivation of bacteria -Nutrient broth (NB) containing (wt/v %) glucose (0.5), beef extract (0.5), sodium chloride (0.5), and peptone (0.5) in distilled water at a pH 7.5 was used to grow P. (Yousten et al. 1980). And to grow B. sphaericus the medium NYSM without glucose was used. For the cultivation of A. brasilense a medium containing (wt/v %) yeast extract, 1.0 in distilled water, pH 7.5 was used. A loop full of bacterial growth from an agar slope was transferred to 10 ml of growth medium (in a boiling tube) and incubated for 8 h on a rotatory shaker at 250 rpm and 28 ± 2 o C. The culture was then transferred to 50 ml of growth medium (in 250 ml capacity Erlenmeyer flask) and incubated, as stated above, for 10 h. Five ml of this inoculum was transferred to 500 ml of production medium (in 2 l flask) and incubated for 48 h. Then, the cell-mass from the culture was harvested by spinning at 10,000 rpm for 10 min. The cell-mass was discarded and the cell-free supernatant was used as test material for oviposition attractancy tests. MATERIALS AND METHODS Bacterial strains -Determination of optimum concentration of bacterial culture filtrates for oviposition attractancy testThree-day-old Culex quinquefasciatus females, obtained from a colony maintained at VCRC, were fed on fowl blood and maintained for two days on raisins at 28 ± 2 o C and 70-80% RH. Gravid females were used for determination of oviposition attractancy of various compounds. Different concentrations (5-3000 ppm) of the test materials were prepared in tap water. Two hundred ml of each test preparation held in disposable cups (capacity, 250 ml) was placed in a mosquito cage (size, 55 x 55 x 55 cm). Tap water was used as a control. One hundred fully gravid female mosquitoes were released into the cage. For each test, one cage was used and at any given time, not more than five disposable cups were kept on the floor of the cage. Four cups with four different concentrations of the culture filtrate were on four corners and the fifth cup with tap water (control) was at the center of the cage. The cages were kept at 28 ± 2 o C and 70-80% RH. Experiments were set up

Methanolic extracts of 20 medicinal plants were screened at 1-10 mg/ml for in vitro macrofilaricidal activity by worm motility assay against adult Setaria digitata, the cattle filarial worm. Four plant extracts showed macrofilaricidal activity by worm motility at concentrations below 4 mg/ml and an incubation period of 100 min. Complete inhibition of worm motility and subsequent mortality was observed at 3, 2, 1 and 1 mg/ml, respectively, for Centratherum anthelminticum, Cedrus deodara, Sphaeranthus indicus and Ricinus communis. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) reduction assay was carried out at 1 mg ml(-1) and 4-h incubation period, and the results showed that C. deodara, R. communis, S. indicus and C. anthelminticum exhibited 86.56, 72.39, 61.20 and 43.15% inhibition respectively in formazan formation compared to the control.

The culture supernatant of a strain of Bacillus subtilis subsp. subtilis isolated from mangrove forests of Andaman and Nicobar islands, India was found to kill larval and pupal stages of mosquitoes. A chloroform extract of the culture supernatant of the bacterium showed pupicidal effects at an LC(50) dose of 1 microg/ml. The mosquitocidal metabolite(s) produced by this strain were purified by gel permeation chromatography. The purified fraction was subjected to Fourier transform infrared (FTIR) spectroscopy and Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The FTIR spectrum of active fraction/CHCl3 residue showed strong band characteristic of peptides. MALDI-TOF spectrum of the sample showed well-resolved group of peaks at m/z values 1,030.6, 1,046.7, 1,044.6, 1,060.5, 1,058.6, 1,058.7, and 1,074.6. The results indicated production of different isoforms of surfactin, ranging from C13-C15. Further, the sfp gene responsible for the production of surfactin was amplified and sequenced. In conclusion, this study showed that the mosquito pupicidal metabolite(s), produced by B. subtilis subsp. subtilis is the cyclic lipopeptide, surfactin. The mode of action of surfactin on pupae of mosquitoes is discussed. This is the first report on the mosquito pupicidal activity of surfactin produced by B. subtilis subsp. subtilis.

Transferrin is a defence protein known to be up-regulated upon infection of parasites/pathogens in Aedes aegypti mosquito. However, no information is available on its up-regulation in Culex quinquefasciatus, the vector of bancroftian filarial parasite. In the present study, enhancement of transferrin in C. quinquefasciatus infected with Wuchereria bancrofti is demonstrated through amplification of the specific mosquito transcript, its sequencing, cloning, and expression. By using two oligonucleotide primers, a 950-bp polymerase chain reaction product was obtained from the first strand cDNA made from RNA of C. quinquefasciatus infected with W. bancrofti. A 707-bp sequence encoding the mature portion of transferrin was confirmed by sequencing the product. This is the first report of transferrin expression in C. quinquefasciatus. The deduced amino acid sequence shared 85% homology with A. aegypti transferrin precursor molecule. Western blot analysis of haemolymph proteins of infected C. quinquefasciatus with antibodies raised against recombinant transferrin protein showed binding to a 66-kDa protein, confirming its identity as transferrin. Hence, this molecule also could be added to the list of immune molecules of C. pipiens group, such as the defensin, gambicin, and cecropin, which are already known.

No abstract

Outbreaks of acute encephalitis syndrome (AES) with high fatality and disability, are reported every year in the Gorakhpur region of Uttar Pradesh, India, with the etiology of >60% of the cases being attributed to scrub typhus. In the present study, the prevalence of Orientia tsutsugamushi, the etiological agent of scrub typhus, was investigated among animal hosts and their ectoparasitic trombiculid mites prevalent in AES-reported areas of Gorakhpur. A total of 154 rodents/shrews were collected using 777 Sherman traps set in 12 study villages, and the overall trap rate was 19.8%. In total, 2726 trombiculid mites belonging to 12 species were collected from 154 rodents/shrews trapped. The shrew mouse Suncus murinus was the predominant animal species (78.6%) collected. The principal vector mite Leptotrombidium deliense was the predominant species (82.7%), and its index was 14.6 per animal. Of 114 rodent/shrew sera samples screened through the Weil-Felix test, 57% were positive for antibodies against O. tsutsugamushi. Of 128 blood samples tested by polymerase chain reaction (PCR), one rodent sample was positive for the gene encoding 56 kDa protein and 25 for 60 kDa. Among 2726 mite samples tested as 315 pools through nested PCR, seven pools were positive for 56 kDa gene. Phylogenetic analysis confirmed circulation of Gilliam, Karp, and TA678 serotypes of O. tsutsugamushi in Gorakhpur. The study clearly demonstrated natural infection of O. tsutsugamushi in both small-animal hosts and vector mites in the AES-reporting villages of Gorakhpur, which confirms transmission of the scrub typhus pathogen in this region. The high infestation rate of L. deliense with O. tsutsugamushi infection indicates that the people living in the rural villages of Gorakhpur are at risk of infection with scrub typhus, which might lead to AES.

Background & objectives:Human cases of scrub typhus are reported every year from Puducherry and adjoining areas in southern India. However, information on the presence of causative agent, Orientia tsutsugamushi, and its vectors is lacking. Hence, the objective of the study was to find out the vector as well as pathogen distribution in rodents and shrews present in the scrub typhus-reported areas in southern India.Methods:Trombiculid mites were collected by combing rats and shrews collected using Sherman traps and identified to species level following standard taxonomical keys. The serum samples of the animals were used for Weil–Felix test and the clots containing blood cells were used for DNA extraction and polymerase chain reaction (PCR).Results:A total of 181 animals comprising four rodent species and one shrew species were collected from 12 villages. High proportion of chiggers was collected from the shrew, Suncus murinus (79.1%) and Rattus rattus (47.6%). A total of 10,491 trombiculid mites belonging to nine species were collected. Leptotrombidium deliense, the known vector of scrub typhus pathogen, was the predominant species (71.0%) and the chigger (L. deliense) index was 41.1 per animal. Of the 50 animals screened for the pathogen, 28 showed agglutination against OX-K in Weil–Felix test indicating the presence of antibodies against O. tsutsugamushi, the causative agent of scrub typhus. PCR carried out with the DNA extracted from blood samples of two of the animals were positive for GroEl gene of O. tsutsugamushi.Interpretation & conclusions:L. deliense index was well above the critical limit of chigger load, indicating that all the villages were receptive for high risk of transmission of scrub typhus to human. Pathogen positivity was higher among animals collected from villages recorded for higher chigger indices due to active transmission between the chigger mites and reservoir host animals. The results are suggestive of routine vector/pathogen surveillance at hot spots to initiate timely preventive measures.