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.

Wolbachia endobacteria in a natural population of Culex quinquefasciatus from filariasis endemic villages of south India and its phylogenetic implication

Understanding Wolbachia mosquito interactions have been recognized as an important concept to develop novel vector control strategies. The prevalence of Wolbachia endobacteria in a natural population of the filariasis vector Culex quinquefasciatus was determined by the polymerase chain reaction method. Earlier workers had estimated the infection rates of Wolbachia with only one or very few individuals per species. In our study large number of specimens were assayed, and a total of 750 adult Culex quinquefasciatus mosquitoes were collected from three south Indian villages of Tirukoilur and Mugaiyur blocks, monthly for a period of five months (December 2006 to April 2007) and screened for the presence of Wolbachia. The percentage prevalence in adult males ranged from 88% to 96%; while in females from 84% to 100%. An overall prevalence of 91.2% was observed. There was no significant difference observed in the proportion of mosquitoes positive for Wolbachia between males and females, and also between different months of the survey; except during the month of February ‘07. The wsp gene sequence of the Wolbachia strain of Cx. quinquefasciatus detected was BLAST analysed and showed 99% sequence similarity with Wolbachia sp. of Culex pipiens isolated from different geographical regions. Phylogenetic analysis based on wsp gene fragments showed that the present Wolbachia isolate was closely related with Wolbachia from Culex pipens pipiens, Niphotettix virescens (Order: Hemiptera) and Cnaphalocrosis medinalis (Order: Lepidoptera).

Serological and entomological investigations of an outbreak of dengue fever in certain rural areas of Kanyakumari district, Tamil Nadu.

BACKGROUND AND OBJECTIVES: During the first week of July 2003, suspected cases of dengue fever were reported from three villages in Kanyakumari district in Tamil Nadu. Since the fever outbreak occurred for the first time in these villages, serological, virological and entomological investigations were carried out to confirm the aetiology of outbreak. METHODS: A total of 76 plasma samples were collected from suspected cases of dengue fever and screened for the presence of IgM antibodies by Pan Bio ELISA kit. Toxo-IFA system was used for the isolation of dengue virus from the plasma samples. Vector survey employing ovitraps and adult landing collection were carried out in the study villages. Pooled samples of Aedes mosquito were screened for dengue virus antigen by an in-house antigen capture ELISA test employing dengue virus specific monoclonal antibodies. RESULTS: Of the 76 samples tested, 15 (20%) were found positive for dengue virus specific IgM antibodies. Dengue virus serotype-3 was detected from a plasma sample by Toxo-IFA test using virus specific monoclonal antibodies. Entomological survey revealed the abundance of Aedes albopictus (Skuse) mosquitoes in the study area. One pool consisting of 12 Ae. albopictus males were found positive for dengue virus infection. INTERPRETATION AND CONCLUSION: Based on the IgM antibody capture ELISA results, it was evident that the current infection was caused by dengue virus in the affected areas. All the age groups were affected during this outbreak. Detection of dengue virus serotype-3 in plasma samples further confirmed the aetiology of this outbreak. The high prevalence of the mosquito vector Ae. albopictus (Skuse) was observed. Detection of dengue virus antigen in the male mosquitoes confirms that the virus is maintained in wild populations of Ae. albopictus in these areas.