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Biomedical subjects

M D Gokhale

Publications and source records attributed to M D Gokhale.

At least 19 recordsLinked to original sources

Effect of temperature and insecticide stresses on Aedes aegypti larvae and their influence on the susceptibility of mosquitoes to dengue-2 virus.

Two major factors, higher temperatures and the application of insecticides, can drastically alter the genetic structure of a vector mosquito population. Due to these two stresses, the majority of the population gets wiped out, but the ones that withstand the stress and survive are likely to pass on survivability, and have an altered physiology. Our study shows that exposures to higher temperatures and DDT during the larval stage affects their susceptibility as adult mosquitoes to the DEN-2 virus. The overall transcription and translation status of heat shock protein (Hsp70) in virus high- and low-susceptible was the same as that in other batches. In the case of a DDT-resistant (R-7) strain two bands were obtained during RT-PCRs after heat shock. These two alleles were obtained only with HY-1 in which R-7 males were used for the crosses, suggesting that the second allele is probably male sex linked. The higher expression of Hsp70 may provide DDT-resistant strains a better chance of survival high temperature environments, particularly in homozygotes and hybrids. It was also interesting to note that these strains have a significantly lower susceptibility to the virus. Wide-spread DDT-resistance and a rise in temperature above the average temperature during summer may result in a population with a low susceptibility to the virus. Several families of heat shock proteins are known to be expressed in mosquitoes, and may have a cumulative role in determining susceptibility to the virus, which itself is governed by several genes.

Animals↗

The role of hemagglutinins in the midgut extracts of two lines of Aedes aegypti in their susceptibility to Dengue-2 virus.

Hemagglutinin activity (HA) was studied in the midgut extracts from highly (h) and lowly susceptible strains of Aedes aegypti mosquitoes to Dengue-2 virus (DEN-2). HA in the midgut extracts from these two isofemale strains of mosquitoes was high in as compared to (h) mosquitoes. HA was found to be higher with chicken red blood cells (RBCs) than with rabbit and human RBCs of O group. Larval midgut extracts showed higher activity than those from adult female mosquitoes. Exposure of midgut extracts to 100 degrees C for 10 mins destroyed the activity. The activity was observed between pH 6 and pH 10. HA in midgut extracts was also studied using twenty different carbohydrates; five of them showed an inhibition of HA. The inhibitory carbohydrates, when incorporated into DEN-2-infected bloodmeal, showed a reduction in the susceptibility of mosquitoes to the virus as compared to the control ones fed on the virus alone. Similarly, when these carbohydrates were incorporated in the DEN-2-infected inoculum, the inoculated mosquitoes showed a reduction in the susceptibility to the virus. HA in the virus-infected midgut extracts was higher than that in the uninfected controls. These results suggest that the presence of HA in the midgut may be one of the factors that affect the susceptibility of Ae. aegypti mosquitoes to DEN-2.

Aedes↗

Isolation of bovine viral diarrhea virus 1, a pestivirus from autopsied lamb specimen from Tamil Nadu, India.

An epizootic of febrile illness among the Madras red breed of sheep had occurred in 1994 in Verrapuram, Chennai, India. The epizootic was suspected as Rift Valley fever (RVF)-like sickness based on clinical features. However, its etiological agent could neither be isolated nor implicated conclusively. During the post-epizootic period a male lamb died of similar clinical features and the spleen was immediately collected. Inoculation of spleen suspension in infant mouse brain yielded a virus that was serially passaged in infant mice and rhabdomyosarcoma (RD) cells. Electron microscopic observations revealed virus particles resembling flaviviruses. RT-PCR performed on extracted total RNA from infected cells and mouse brains with flavivirus-specific or RVF-specific primers gave negative results. However, an amplicon of 280 bp was obtained with pestivirus-specific primers from the 5'-UTR. Further, a nested PCR yielded a product of 157 bp. Nucleotide sequencing of the 157 bp product showed 100% homology to BVDV-1. Western blot analysis with a flavivirus envelope protein-specific MAb revealed three proteins of 33 K, 45 K and 55 K. Further studies suggested that the 33 K and 55 K proteins were glycosylated. This is the first report of isolation of BVDV-1 from a lamb in India.

Animals↗

Experimental transmission of Chikungunya virus by Anopheles stephensi mosquitoes.

The Ades aegypti mosquito has been considered the principal vector of Chikungunya (CHIK) virus. As CHIK epidemics usually occur in urban regions and Anopheles stephensi is another highly endophilic and anthropophilic mosquito, there is a very high probability of this mosquito to feed on CHIK virus-infected patients, to pick up and transmit the virus. Therefore the present study was conducted to test the CHIK virus transmission capability for the A. stephensi mosquito. The obtained results showed that this mosquito species is capable of transmitting CHIK virus. It is surmised that during any epidemic of febrile illness CHIK virus isolation attempts should also be made from this mosquito species.

Alphavirus Infections↗

Development of monoclonal antibody based antigen capture ELISA to detect chikungunya virus antigen in mosquitoes.

BACKGROUND & OBJECTIVES: Chikungunya (CHIK) virus has caused numerous large outbreaks in India. No active or passive surveillance has been carried out since the last epidemic which occurred in 1971. For active surveillance, it is necessary to have a test, which can detect the virus from a large number of field-collected mosquitoes. METHODS: The present study describes the standardization of monoclonal antibody (MAb) based antigen capture ELISA to detect chikungunya virus antigen from the mosquitoes. CHIK virus antigen from suspension of experimentally infected mosquitoes and their progeny was captured on mouse polyclonal antibody, while biotinylated CHIK Mab was used as a probing antibody. CHIK virus antigen in the head squashes of virus inoculated mosquitoes was detected using indirect immunofluorescence antibody (IFA) test for confirmation of ELISA results. RESULTS: The ELISA test was sensitive enough to detect antigen even if a small fraction of a single infected mosquito homogenate was incorporated in the test. The IFA test failed to detect CHIK antigen in 10 and 25 microliters of suspension whereas with ELISA it was detected in all the samples. Progeny of Aedes aegypti and Ae. albopictus mosquitoes infected with chikungunya virus did not show the possibility of existence of transovarial transmission. INTERPRETATION & CONCLUSION: This test is rapid and simple since it can be completed in two days as compared to the conventional mosquito inoculation and IFA techniques, which require at least 10 days. There is an additional advantage with this test that a large number of samples can be processed, and the remaining homogenate of the mosquitoes can be used for screening other viruses. Experimental data raised using this test showed that transovarial transmission of this virus does not occur in these vector species.

Aedes↗

Study of the effect of the midgut bacterial flora of Culex quinquefasciatus on the susceptibility of mosquitoes to Japanese encephalitis virus.

Culex quinquefasciatus mosquitoes were collected from the field and microbial flora was isolated from their midgut. Isolates belonging to 13 different genera were obtained. Four most abundant isolates viz. Pseudomonas sp., Acinetobacterjunii, Staphylococcus epidermis and Aeromonas culicicola were used to study their effect on the susceptibility of mosquitoes to Japanese encephalitis virus (JEV). Incorporation of individual isolates in the mosquito bloodmeal resulted in an increased susceptibility of mosquitoes to the virus. However, only the effects of Pseudomonas sp. and Acinetobacterjunii could be statistically evaluated but they were found insignificant.

Animals↗

Highly-substrate active isoenzyme acetylcholinesterase-II, in rosy eye mutant of Aedes aegypti mosquito.

Insecticide bioassays were carried out on larvae and adults of rosy eye mutant and wildtype strains of A. aegypti. Both the strains were equally susceptible to DDT, malathion and deltamethrin. Biochemical assays showed an increase in acetylcholinesterase enzyme (AChE) activity in all the stages of mutant strain with both the substrates i.e. acetylthiocholine iodide and S-butyrylthiocholine iodide. However, there was no difference in the percent inhibition of enzyme activity with propoxur in these two strains. Polyacrylamide gel electrophoresis performed in native conditions on the homogenates of adults of rosy eye mosquitoes showed that AChE-II allele was highly active with the substrate acetylthiocholine iodide as compared to wildtype strain. Frequency of the highly active AChE-II allele in the mutant strain was about 68%, whereas it was about 5% in the wildtype strain.

Acetylcholinesterase↗

Isolation of chikungunya virus from Aedes aegypti mosquitoes collected in the town of Yawat, Pune District, Maharashtra State, India.

Chikungunya (CHIK) virus is prevalent throughout Southeast Asia and Africa. It has caused numerous large outbreaks in India. No active or passive surveillance has been carried out since the last epidemic occurring in 1971. During a recent outbreak of Dengue (DEN)-like illness in eastern India, Aedes aegypti mosquitoes collected from the affected area were positive for CHIK virus. Evidence of dual infection with CHIK and DEN typel virus was also obtained. A widely circulating low-virulent CHIK virus is a possible explanation for the epidemiological pattern of the CHIK virus disease in this region.

Aedes↗

Dengue virus and insecticide susceptibility status of Aedes aegypti mosquitoes from Belagola village, Mandya District, Karnataka state: during and post-epidemic investigations.

Aedes aegypti mosquitoes collected during and after dengue virus epidemic season were exposed to diagnostic dosages of insecticides. Mosquitoes collected during the epidemic showed resistance to DDT only, while mosquitoes collected after the control measures showed tolerance to malathion and low resistance to DDT. Since there was no indication of malathion resistance in the mosquitoes collected during the epidemic, the second batch was subjected to malathion selection pressure for 8 generations to find out the mechanism of resistance in this strain. There was 2-4 fold increase in resistance to malathion. Polyacrylamide electrophoresis showed increase in intensity of three esterase bands after selection. Quantitative enzyme assays showed an increase in esterase activity by 3-4 folds. Mosquitoes collected during the epidemic showed higher susceptibility to dengue-2 virus as compared to the laboratory strain, while the mosquitoes collected after malathion fogging showed further increase in the susceptibility to dengue virus. Virus susceptibility of the mosquitoes after eight generations of selection with malathion did not show any further significant increase.

Aedes↗

Putative chikungunya virus-specific receptor proteins on the midgut brush border membrane of Aedes aegypti mosquito.

Two proteins (putative receptors) of 60 and 38 kDa, for chikungunya (CHIK) virus were detected in the brush border membrane fraction (BBMF) of the normal population of Aedes aegypti mosquitoes. Mosquitoes were infected orally with CHIK virus and infectivity checked by testing the head squashes. BBMF was prepared from proved positive and negative mosquitoes. The receptor proteins were found to be present in both the proved genotypes. However, dot-b'ot assays showed that the CHIK virus binding activity of BBMF/mg protein was noticeably low in the proved negative mosquitoes as compared to the positives. BBMF from the larvae of the normal populations also showed the presence of the receptor proteins, binding to CHIK virus. Receptor proteins from larvae as well as the adults were found glycosylated. CHIK virus receptor proteins of 24, 45, 58, 60 and 62 kDa were also seen in the membrane fraction of the C6/36 cells.

Aedes↗

Inheritance of oral susceptibility of Aedes aegypti to Chikungunya virus.

A colony of rosy eye mutants of Aedes aegypti was established. This strain was refractory to Chikungunya virus by oral route of infection when compared with the wild-type parent strain. The refractoriness of this strain seems to be due to a mesentronal barrier, since both the mosquito strains supported the multiplication of virus after intrathoracic inoculation. The rosy eye strain was also found to be refractory to Sagiyama virus (Alphaviridae: Getah virus subtype) when compared with wild-type parent strain, but no such difference in the oral susceptibility was found with dengue-2 (Flaviviridae) virus. The rosy eye mutant appears to be closely linked to the gene(s) for refractoriness to alpha viruses and may be useful in future studies in understanding the genetic basis of vector competence of Ae. aegypti to arboviruses.

Aedes↗

Effect of sublethal dosages of insecticides on chikungunya virus susceptible and refractory strains of Aedes aegypti.

Three strains of Aedes aegypti mosquitos viz (i) CRS, refractory to Chikungunya (CHIK) virus by oral route of infection but susceptible to DDT (2) CSS, susceptible to CHIK virus and also susceptible to DDT (3) CSS-DDTR, susceptible to CHIK virus but resistant to DDT, were examined for the effect of sublethal dosages of DDT and deltamethrin on their fecundity. Biochemical analysis showed that there was an increase in glutathione s-transferase activity in the CSS-DDTR strain which was associated with DDT resistance. There was an increase in acetylcholinesterase activity in the CRS strain, however it was not associated with resistance to all the three insecticides tested. No significant differences in the fecundity of these three strains were observed, though there was some increase in the number of non layers in CSS-DDTR strain after the treatment of DDT and mean number of eggs laid by CSS and CRS strains was slightly reduced (0.5 > p < 0.1) after the treatment with deltamethrin.

Acetylcholinesterase↗

Biochemical basis of DDT-resistance in Aedes aegypti population from a dengue affected area in Shahjahanpur city.

Entomological studies conducted in Jalalnagar, Shahjahanpur city, Uttar Pradesh, India, during an outbreak of dengue in 1992, showed that Ae. aegypti mosquitoes were resistant to DDT and had some tolerance to malathion in the adults and the larvae. Biochemical analysis suggested that DDT resistance was related to elevated glutathione s-transferase and tolerance to malathion was due to a little increase in esterase activity. Crosses of DDT-resistant and susceptible strain suggested that resistance was codominant (metabolic type).

Aedes↗