Detection of Wolbachia endobacteria in Culex quinquefasciatus by Gimenez staining and confirmation by PCR.

Background & objectives: Wolbachia are common intracellular bacteria that are found in arthropods and nematodes. These endosymbionts are transmitted vertically through host eggs and alter host biology in diverse ways, including the induction of reproductive manipulations, such as feminization, parthenogenesis, male killing and sperm-egg incompatibility. Since they can also move horizontally across species boundaries, Wolbachia is gaining importance in recent days as it could be used as a biological control agent to control vector mosquitoes or for paratransgenic approaches. However, the study of Wolbachia requires sophisticated techniques such as PCR and cell culture facilities which cannot be affordable for many laboratories where the diseases transmitted by arthropod vectors are common. Hence, it would be beneficial to develop a simple method to detect the presence of Wolbachia in arthropods. Method: In this study, we described a method of staining Wolbachia endobacteria, present in the reproductive tissues of mosquitoes. The reliability of this method was compared with Gram staining and PCR based detection. Results: The microscopic observation of the Gimenez stained smear prepared from the teased ovary of wild caught and Wolbachia (+) Cx. quinquefasciatus revealed the presence of pink coloured pleomorphic cells of Wolbachia ranging from cocci, comma shaped cells to bacillus and chain forms. The ovaries of Wolbachia (–) cured mosquito did not show any cell. Although Gram’s staining is a reliable differential staining for the other bacteria, the bacterial cells in the smears from the ovaries of wild caught mosquitoes did not take the stain properly and the cells were not clearly visible. The PCR amplified product from the pooled remains of wild caught and Wolbachia (+) Cx. quinquefasciatus showed clear banding, whereas, no banding was observed for the negative control (distilled water) and Wolbachia (–) Cx. quinquefasciatus. Interpretation & conclusion: The Gimenez staining technique applied, could be used to detect the members of the endobacteria Wolbachia easily, even in a simple laboratory without any special facilities or even in the field condition and for handling large number of samples in a shorter duration.

The effects of climatic factors on the distribution and abundance of japanese encephalitis vectors in Kurnool district of Andhra Pradesh, India.

Background & objectives: Climatic attributes have been associated with relative mosquito abundance and transmission of mosquito borne infections in many parts of the world, especially in warm and tropical climatic regions. The main objectives of this study were to assess the change in seasonal pattern of Japanese encephalitis (JE) vectors, their density, to elucidate whether the lagged climate variables (precipitation, temperature and humidity) are associated with JE vector density, and to determine if temperature and precipitation are similarly important for the rise in the number of potential mosquito vectors for JE virus in the temperate climate of Andhra Pradesh, India. Methods: Mosquito samples were collected from Kurnool district of Andhra Pradesh using hand catch and light-trap methods during 2002 to 2006. The type and abundance of recovered species were compared to ecological correlates. In each geographic area, temperature and precipitation are the two possible proxy variables for mosquito density, in conjunction with other seasonal factors for JE epidemics. Results: Out of the various mosquito species collected, Culex gelidus and Cx. tritaeniorhynchus were noticed in high numbers. There was considerably high prevalence of Cx. gelidus (68.05%) in urban area than in rural areas whereas, Cx. tritaeniorhynchus (57.51%) was found to be more in rural areas than in the urban area. It is noticed that the factors such as rainfall and temperature were found to be correlated with the per man hour (PMH) density, whereas the humidity was inversely correlated with the PMH. Interpretation & conclusion: The environmental and eco-climatic factors are assisting in enhancing the breeding of these mosquitoes in Kurnool district of Andhra Pradesh, India. Both Cx. tritaeniorhyncus and Cx. gelidus are quite adaptable to these environmental conditions and this necessitates immediate control measures in both rural and urban areas of Kurnool district.

Studies on community knowledge and behavior following a dengue epidemic in Chennai city, Tamil Nadu, India

In 2001, a major dengue outbreak was recorded in Chennai city, with 737 cases (90%) out of a total of 861 cases recorded from Tamil Nadu state. A KAP survey was carried out to assess the community knowledge, attitude and practice on dengue fever (DF), following the major dengue outbreak in 2001. A pre- tested, structured questionnaire was used for data collection. The multistage cluster sampling method was employed and 640 households (HHs) were surveyed. Among the total HHs surveyed, 34.5% of HHs were aware of dengue and only 3.3% of HHs knew that virus is the causative agent for DF. Majority of the HHs (86.5%) practiced water storage and only 3% of them stored water more than 5 days. No control measures were followed to avoid mosquito breeding in the water holding containers by majority of HHs (65%). Sixty percent of HHs did not know the biting behaviour of dengue vector mosquitoes. The survey results indicate that the community knowledge was very poor on dengue, its transmission, vector breeding sources, biting behavior and preventive measures. The lack of basic knowledge of the community on dengue epidemiology and vector bionomics would be also a major cause of increasing trend of dengue in this highly populated urban environment. There is an inevitable need to organize health education programmes about dengue disease to increase community knowledge and also to sensitize the community to participate in integrated vector control programme to resolve the dengue problem.

A study on chikungunya outbreak during 2007 in Kerala, south India.

Background & objectives: The first chikungunya outbreak occurred in Kerala during 2006 affecting 14 districts, followed by another during May 2007 affecting almost whole of the State. Four of the worst affected districts viz, Pathanamthitta, Idukki, Kottayam and Thrissur were surveyed during 2007 to understand the magnitude of the problem of chikungunya fever, particularly clinical signs and symptoms. Methods: A total of 1265 persons from 310 houses were surveyed door-to-door in 20 different localities representing four affected districts. The history and examination findings from 354 clinically diagnosed chikungunya cases were recorded. The symptoms recorded were fever, headache, myalgia, arthralgia, itch/rash, oedema, eye congestion, eye pain, oral ulcers, distaste, nausea, vomiting and haemorrhage. Results: The major symptoms were fever (100%), headache (97.5%), arthralgia (99.4%) and myalgia (99.4%). A significant difference was observed in oedema, distaste, nausea and headache among different age groups and these symptoms were reported to be lower (12.2-89.8%) in younger age group than in older age group (90.4-100%). No genderwise difference was observed for any of the symptoms. In clinically diagnosed chikungunya cases higher age group (>35 years) found with higher rate of severity with symptoms of oedema, distaste, nausea and headache when compared with lower age group (1-35 yr). Interpretation & conclusions: Chikungunya invaded Kerala State for the first time in 2006 and continues to be a major vector borne disease in the State. The clinical symptoms in affected cases highlighted high fever, sever myalgia and prolonged arthralgia, with occasional history of skin itch/rash (petechiae).

Natural vertical transmission of dengue viruses by Aedes aegypti in Chennai, Tamil Nadu, India.

BACKGROUND & OBJECTIVE: Dengue viruses are spread and maintained in an Aedes aegypti-human- Ae. aegypti cycle in urban areas of the tropics. Dengue viruses are also maintained in nature by vertical transmission by Ae. aegypti. A study was undertaken in Chennai, a known endemic city in south India, to comprehend the natural vertical transmission dynamics in Ae. aegypti and to assess its epidemiological importance. METHODS: Ae. aegypti males collected in resting and landing collections were tested for dengue virus infection by antigen-capture enzyme-linked immunosorbent assay (ELISA) and further examined by insect bioassay, Toxorhynchites splendens inoculation-indirect immunofluorescence technique (Toxo-IFA) using serotype-specific monoclonal antibodies (Mabs), if found positive by ELISA. RESULTS: Of the 509 pools of Ae. aegypti males (n=5408) screened, 15 pools, collected in April, June- July, November-December in 2003 and March, May in 2004, were found positive for dengue virus infection and the minimum infection rate (MIR) among adult males was high in June 2003 (28.0/ 1000). Three positive pools could be serotyped as dengue-2 (2 pools) and dengue-3 (1 pool). INTERPRETATION & CONCLUSION: Dengue virus isolations from wild caught males of Ae. aegypti indicate the occurrence of transovarial transmission. Vertical transmission was mainly observed in summer months when dengue infections in humans were low suggesting that dengue viruses adopt a novel strategy of surviving adverse climatic conditions.

Japanese encephalitis in India: an overview.

Japanese encephalitis (JE)-epidemics have been reported in many parts of the country. The incidence has been reported to be high among pediatric group with high mortality. The incidence of JE in recent times is showing an increasing trend. It appears that JE may become one of the major public health problems in India, considering the quantum of the vulnerable pediatric population, the proportion of JEV infections among the encephalitic children and wide scattering of JE-prone areas. JE burden can be estimated satisfactorily to some extend by strengthening diagnostic facilities for JE confirmation in hospitals and by maintenance of contact with the nearby referral hospitals to collect the particulars on JE cases. Vaccination proves to be the best to protect the individual against any disease. In the case of JE, it is essential to immunize the pigs (amplifying host) also to interrupt the transmission of the disease.

Efficacy of diethylcarbamazine-medicated salt for microfilaraemia of Brugia malayi.

BACKGROUND: Pilot studies in India and abroad have demonstrated the benefit of cooking salt fortified with diethylcarbamazine citrate (DEC) for the control of lymphatic filariasis. In India, DEC-medicated salt has been introduced on a commercial basis in the Cherthala region of Kerala, which is endemic for Brugia malayi (B. malayi). We studied the efficacy of DEC-medicated salt in the clearance of microfilaraemia of B. malayi. METHODS: Eighteen cases of microfilaraemia (11 men; 7 women) were selected; 14 in the experimental group (i.e. treated with 0.2% w/w DEC-medicated salt) and the rest as the control group who were given a placebo. The consumption of salt and the status of parasitaemia were monitored till all the microfilaria carriers became negative. RESULTS: There was a significant reduction in the density of microfilariae (intensity) among the treated individuals over a period of time. The duration required for the clearance of microfilariae ranged from 9 to 30 [mean (SD) 19.4 (1.7)] weeks. All microfilaraemics except one reported side-effects which were mild-to-moderate and persisted for a maximum period of one month. CONCLUSION: DEC-medicated salt effectively clears microfilariae within 30 weeks in parasitaemic individuals. No 'endemic normals' reported any side-effects, though these were common among the microfilaria carriers.

Virus isolation from wild-caught mosquitoes during a Japanese encephalitis outbreak in Kerala in 1996.

Out of 5357 wild-caught mosquitoes in 163 pools tested for virus using antigen capture ELISA and an insect-bioassay (inoculation into Toxorhynchites splendens larvae and identification by IFA using JE virus-specific monoclonal antibody), 16 flavivirus isolations were made of which 12 (75%) were identified as JE virus. Of the 12 JE virus isolations, 7 were from Culex tritaeniorhynchus, 3 from Mansonia uniformis and 1 each from Ma. indiana and Anopheles subpictus. Four isolations from Mansonia species for the first time reported here are noteworthy.

Mattancherry urban agglomeration, a diminishing focus of lymphatic filariasis in Kerala.

Parasitological survey conducted recently in Mattancherry, Kerala showed a low prevalence of microfilariae (mf), with a mf rate of 1.81%, compared to that of 1956 when the mf rate was 14.7%. No brugian infection was encountered in the present investigation, though both bancroftian and brugian infections were reported earlier. Culex quinquefasciatus, the vector of bancroftian filariasis was the most predominant (93.3%) mosquito species, and its infection and infectivity rates were 0.76% and 0.54% respectively. Mansonioides spp., the vectors of brugian filariasis constituted only 0.2% of the total mosquitoes and with no infection. The overall reduction in the prevalence of mf cases and vector infection rates over the years is attributed to the continued activities of the National Filaria Control Programme. The disappearance of brugian filariasis could mainly be due to the drastic reduction in the Mansonioides breeding habitats, as a result of rapid urbanization coupled with improved socio economic conditions.

Brackish water mosquito problem of Vypeen Island, Cochin, Kerala.

A preliminary study has shown that the marshy terrain and brackish water bodies associated with mangrove forests contributed profuse breeding of mosquitos in Vypeen island, causing a severe menace to the island population. A total of 14 species belonging to four genera viz, Aedes, Anopheles, Armigeres and Culex was recorded from different habitats. Culex sitiens was found to be the predominant mosquito in all the perennial breeding habitats. The extent of different habitats in the production of mosquitos, and its possible abatement, using environmental and/or biocontrol methods are discussed.

Observations on population density of Culex quinquefasciatus and transmission indices of Bancroftian filariasis during and after Integrated Vector Management strategy.

An Integrated Vector Management strategy, implemented as an alternative to the conventional control operations that include mainly chemical control in Pondicherry, South India, reduced very substantially the population density of Culex quinquefasciatus. This resulted in drastic decrease in the intensity of transmission of bancroftian filariasis transmitted by Culex quinquefasciatus and consequently the incidence of new infections in children of 0-5 age group was minimized. When the IVM strategy was withdrawn after five years of implementation and conventional control measures were re-adopted, resilience of Culex quinquefasciatus population was observed and human exposure to the risk of infection increased. The results suggest that maintenance of vector density at reduced levels for prolonged periods, is necessary to control infectious diseases like filariasis, which is difficult in the present day urban situations in developing countries. Hence the emphasis should be on chemotherapy to achieve control of lymphatic filariasis.

Infectivity of a mermithid nematode Romanomermis iyengari (Welch) in different conductivity levels under laboratory and field conditions.

Infectivity of R. iyengari was examined by exposing mosquito (Culex quinquefasciatus) larvae to the preparasite at different conductivity levels. The preparasite infected 63.5, 30, 11, 1.5 and 0.5% of the mosquito larvae respectively at 2000, 2500, 3000, 3300 and 3600 mu ho/cm. Although, 62-69% of the preparasite survived at 4000-5400 mu ho/cm, it did not infect. Application of preparasite to tree-holes resulted in 53-63% infection of Aedes albopictus larvae initially. On 6th day the infection level was 40% which decreased further to 7% by 15th day. The infection reappeared on 38th day indicating that R. iyengari has not only infected mosquito larvae as soon as they were applied to tree-holes in which the conductivity was 600-2800 mu ho/cm but also got established there.

Insecticidal activity of carbosulfan (OMS 3022) and pyraclofos (OMS 3040) against mosquitoes.

Carbosulfan (OMS 3022), a carbamate and pyraclofos (OMS 3040), an organophosphate were evaluated for their biological activity against vector mosquitoes, both in laboratory and field conditions. Carbosulfan (25% EC), was found to be active against adults and immatures of Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti. The LD values for adults were 6.65, 5.19 and 8.35 micrograms/cm2 and LC 50values for immatures were 5.4 X 10(-5), 1.6 X 10(-4 and 5.4) X 10(-4) 50mg/1 respectively. Pyraclofos (50%) showed neither larvicidal nor adulticidal activities. The residual effect of carbosulfan (25% WP) on cement, thatch and mud surfaces at 400 mg (ai)/m2 lasted for 3, 16 and 21 weeks respectively. In the field, carbosulfan at 2.00 kg (ai)/ha resulted in complete control of C. quinquefasciatus larvae for a period of one week in cement tanks and less than a week in other polluted water bodies, such as cesspits, cesspools and drains. Pyraclofos was not effective at a dose of 2.00 kg (ai)/ha in any of the habitats.