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.

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.

No abstract

No abstract

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 ll 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 ll 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 ll) 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 ll 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 ll serum is adequate for use in Og4C3 assay. So the test can be performed without losing its sensitivity even with 5 ll serum samples at high titre of antigen (titre group 8) and 15 ll for other groups and this method has scope in programme evaluation.

Key words: Mastomys coucha -Brugia malayi -adult worm -clearance -microfilariae -antibody levels Lymphatic filariasis is one of the major tropical diseases and is caused by filarial nematodes such as Wuchereria bancrofti, Brugia malayi, and Brugia timori. About 120 million people are either infected or with lymphatic pathology worldwide and among them India accounts for 42.8% of the global burden of the disease (WHO 2002). Information on the parasite build-up and host immune responses against different life stages of the filarial parasite are important to find out the ways and means of diagnosing the filarial infection and for other related studies.Mastomys coucha is the known laboratory animal model of B. malayi sub-periodic strain and 71-81% of this animal could develop the inoculated infective stage (L3) to the adult worms (Petranyi et al. 1975, Murthy et al. 1983. Despite the development of the worms, the status of host microfilaraemia, i.e., the presence and density of microfilariae (mf) in the circulating blood, is determined by various factors. For example, segregation of male and female due to their localization in different organs, or all the worms developing to single sex lead to non-production of mf (Paily et al. 1995). Also, these animals can exhibit a variety of immunological response depending upon the presence or absence of mf, their developmental stages, adults or their products (Rao & Klei 1997, Athisaya Mary et al. 2005 with filarial parasites but not progressing to microfilaraemic stage (Dreyer et al. 1996) and the reason for this could be the same as stated above.In the present study, M. coucha inoculated with L3 of B. malayi sub-periodic strain were examined for mf, organ-wise adult worm localization, and immunological status to find out whether there is any relationship between these factors.

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.

(Ottesen et al. 1997). However, in order to attain the goal of filariasis elimination, it is desirable to have more than one control tool. Recently, great deal of interest has been generated in the possibility of blocking the development and transmission of parasites by vectors of diseases such as leishmaniasis (Tonui 1999), schistosomiasis (Capron et al. 1992), and Lyme disease (de Silva et al.1996). In the case of malaria, transmission-blocking vaccines have reached advanced stage of development (Kaslow 1997, Carter 2001. But there is only one such study on the antibodies regulating the development of filarial parasites (Fuhrman et al. 1992), which reports that a monoclonal antibody, MF1, could degrade the chitin containing structures of B. malayi mf in the vector mosquito. In the present paper, results of a preliminary study on the role of anti-filarial polyclonal antibodies in suppressing the development of early larval stages of the parasite in the mosquito host are presented.Mongolian gerbils, Meriones unguiculatus, were immunized by intra-peritoneal (i/p) inoculation of microfilariae (mf) of B. malayi (sub-periodic strain). obtained by lavaging the peritoneal cavity of B. malayi infected Mongolian gerbils. The animals were 6-8 weeks old and the mf were harvested on 120 days post-inoculation of infective larvae to ensure that the mf were identical with respect to their age and viability. The harvested mf were purified by Percoll gradient centrifugation (Chandrashekar et al.1984), and were used for immunization as well as feeding the mosquitoes. Either single dose of 1000 live mf or their homogenate (20 µg of protein) per animal was given in sterile phosphate buffered saline (PBS, pH 7.4). Homogenate of mf was prepared in PBS containing a cock-tail of protease inhibitors (Maizles et al. 1999) such as, EDTA-1M, EGTA-1M, NEM-1M, Pepstatin-1 mM, PMSF-0.33 Mm, and TPCK-0.1 M at 5 µl level/ml of the parasite extract. The animals were immunized in duplicate for each antigen type and an equal numbers were left as controls with only PBS inoculation. Considering the ethical issues involved in the usage of laboratory animals and the duration involved, the number of animals used in this experiment was restricted to only two in each group. The antibody titre in each animal was determined two weeks after immunization by direct enzyme linked immunosorbent assay (ELISA) as per Voller et al. (1976), using mf homogenate antigen. Briefly, crude antigen of B. malayi was coated (1 µg/well) onto 96 well polysterene plates (Nunc-US) and incubated overnight at 4 o C. Sera samples from the experimental and control animals diluted to 1:100 with 0.75% bovine serum albumin (BSA) in PBS/ T were reacted for 2 h after blocking the wells with 1% BSA in PBS/T. Anti-mouse peroxidase conjugate (Sigma Chem. Co., US) was allowed to react with the antigenantibody complex, followed by the addition of θ-Phenylenediamine dihydrochloride (Sigma Chem. Co., US) as substrate in phosphate citrate buffer (pH 5.0). The reac- tion was arrested with 5 N ...