Vector bionomics and malaria transmission along the Thailand-Myanmar border: a baseline entomological survey.

Baseline entomological surveys were conducted in four sentinel sites along the Thailand-Myanmar border to address vector bionomics and malaria transmission in the context of a study on malaria elimination. Adult Anopheles mosquitoes were collected using human-landing catch and cow-bait collection in four villages during the rainy season from May-June, 2013. Mosquitoes were identified to species level by morphological characters and by AS-PCR. Sporozoite indexes were determined on head/thoraces of primary and secondary malaria vectors using real-time PCR. A total of 4,301 anopheles belonging to 12 anopheline taxa were identified. Anopheles minimus represented >98% of the Minimus Complex members (n=1,683), whereas the An. maculatus group was composed of two dominant species, An. sawadwongporni and An. maculatus. Overall, 25 Plasmodium-positive mosquitoes (of 2,323) were found, representing a sporozoite index of 1.1% [95%CI 0.66-1.50]. The transmission intensity as measured by the EIR strongly varied according to the village (ANOVA, F=17.67, df=3, P<0.0001). Our findings highlight the diversity and complexity of the biting pattern of malaria vectors along the Thailand-Myanmar border that represent a formidable challenge for malaria control and elimination.

Comparison of the Performances of Five Primer Sets for the Detection and Quantification of Plasmodium in Anopheline Vectors by Real-Time PCR.

Quantitative real-time polymerase chain reaction (qrtPCR) has made a significant improvement for the detection of Plasmodium in anopheline vectors. A wide variety of primers has been used in different assays, mostly adapted from molecular diagnosis of malaria in human. However, such an adaptation can impact the sensitivity of the PCR. Therefore we compared the sensitivity of five primer sets with different molecular targets on blood stages, sporozoites and oocysts standards of Plasmodium falciparum (Pf) and P. vivax (Pv). Dilution series of standard DNA were used to discriminate between methods at low concentrations of parasite and to generate standard curves suitable for the absolute quantification of Plasmodium sporozoites. Our results showed that the best primers to detect blood stages were not necessarily the best ones to detect sporozoites. Absolute detection threshold of our qrtPCR assay varied between 3.6 and 360 Pv sporozoites and between 6 and 600 Pf sporozoites per mosquito according to the primer set used in the reaction mix. In this paper, we discuss the general performance of each primer set and highlight the need to use efficient detection methods for transmission studies.

A Comparison of Attractants for Sampling Stomoxys calcitrans (Diptera: Muscidae) on Dairy Farms in Saraburi Province, Thailand.

The efficacy of different stable fly attractants was evaluated at four dairy cattle farms in Muak Lek district, Saraburi province, Thailand. Dry ice, octenol, a mixture of cow dung and urine, a combination of dry ice plus octenol, and no attractants (control) were tested with Vavoua traps. In total, 7,000 individuals of Stomoxys species were collected between July 2013 to September 2014, of which 1,058, 867, 1,274, and 3,801 were trapped on farms 1­4, respectively. Four species of Stomoxys were identified: Stomoxys bengalensis Picard, 1908, Stomoxys calcitrans (L., 1758), Stomoxys indicus Picard, 1908, and Stomoxys sitiens Rondani, 1873. S. calcitrans was the predominant species, comprising 99% of all the samples collected. The number of male and female S. calcitrans collected differed significantly by attractant type. Significantly more S. calcitrans were attracted to dry ice or a combination of dry ice plus octenol-baited traps than to unbaited or octenol-baited traps. The Vavoua traps baited with dry ice alone or a combination of dry ice plus octenol were effective attractants for S. calcitrans.

Assessment of geraniol-incorporated polymers to control Aedes albopictus (Diptera: Culicidae).

Effective control of mosquito borne diseases has proven extremely difficult with both vector and pathogen remaining entrenched and expanding in many disease endemic areas. When lacking an effective vaccine, vector control methods targeting both larval habitats and adult mosquito populations remain the primary strategy for reducing risk. Aedes albopictus from Thailand was used as a reference baseline for evaluation of natural insecticides incorporated in polymer disks and pellets and tested both in laboratory and field conditions. In laboratory and field tests, the highest larval mortality was obtained with disks or pellets containing IKHC (Insect Killer Highly Concentrate) from Fulltec AG Company. This product is reputed to contain geraniol as an active ingredient. With pellets, high mortality of Ae. albopictus larvae (92%) was observed in presence of 1 g of pellets per 500 ml of water at day 1st, and the mortality was 100% at day 1st for larvae in presence of 5 or 10 g of pellets. Fulltec AG Company has not accepted to give us the exact composition of their IKHC product. Therefore, we cannot recommend it, but the principle of using monoterpenes like geraniol, incorporated into polymer disks or pellets as natural larvicide needs more attention as it could be considered as a powerful alternative in mosquito vector control.

Diversity of Stomoxys spp. (Diptera: Muscidae) and diurnal variations of activity of Stomoxys indicus and S. calcitrans in a farm, in Wang Nam Khiao District, Nakhon Ratchasima Province, Thailand.

A study of species diversity of Stomoxys spp. and diurnal variations of activity of the most abundant was performed during a one year period at a local dairy cattle farm in Wang Nam Khiao District, Nakhon Ratchasima Province, Thailand. Four species of stomoxyine flies were morphologically identified, including Stomoxys indicus Picard 1908, S. calcitrans (Linnaeus 1758), S. sitiens Rondani 1873 and S. uruma Shinonaga and Kanao 1966. The most common species were S. indicus (50.2%) and S. calcitrans (49.5%). S. sitiens and S. uruma were found in small proportions (< 1%). The number of flies captured was significantly different among the three seasons with the greatest number in the rainy season (mean = 66%; df = 2, P < 0.05). The variations of diurnal activity were observed during different period of times (06:00 to 18:00) during three seasons. Both sexes of S. indicus and males of S. calcitrans showed unimodal activity pattern in cool and summer seasons. But a bimodal activity pattern was recorded in rainy season. For females S. calcitrans, a unimodal peak of activity was observed in cool season and a constant variation of activity all along the day in summer and rainy seasons, with an increase from the morning to the evening. A better understanding of stomoxyine fly behavior, especially the daily flight activity, can assist in prioritization and design of appropriate vector prevention and control strategies.

Review on global co-transmission of human Plasmodium species and Wuchereria bancrofti by Anopheles mosquitoes.

Malaria and lymphatic filariasis are two of the most common mosquito-borne parasitic diseases worldwide which can occur as concomitant human infections while also sharing common mosquito vectors. This review presents the most recent available information on the co-transmission of human Plasmodium species and Wuchereria bancrofti by Anopheles mosquitoes. Important biological and epidemiological aspects are also described including the lifecycle of each parasite species and their specificities, the geographical biodiversity of each pathogen and their vectors where the parasites are co-endemic, and biological, environmental and climatic determinants influencing transmission. The co-transmission of each disease is illustrated from both a global perspective and a country level using Thailand as a study case. Different diagnostic methods are provided for the detection of the parasites in biological samples ranging from traditional to more recent molecular methods, including methodologies employing concomitant detection assays of W. bancrofti and Plasmodium spp. parasites. The relevant issues of combined malaria and Bancroftian filariasis control strategies are reviewed and discussed.

Biochemical studies of insecticide resistance in Aedes (Stegomyia) aegypti and Aedes (Stegomyia) albopictus (Diptera: Culicidae) in Thailand.

Biochemical analysis was performed on field caught Aedes (Stegomyia) aegypti and Aedes (Stegomyia) albopictus (Diptera: Culicidae) mosquitoes to determine activities of enzymes including mixed function oxidases (MFO), nonspecific esterases (alpha- and beta-), glutathione-S-transferases (GST), and insensitive acetylcholinesterase (AChE). Biochemical tests were performed on F1 generation of Ae. aegypti field caught mosquitoes, while in Ae. albopictus F2 progenies were used. Twenty-six samples of Ae. aegypti mosquito were collected from areas across different parts of Thailand including Bangkok (central), and the provinces of Chiang Rai (north), Nakhon Sawan (north-central), Nakhon Ratchasrima (northeast), Chonburi (east), Chanthaburi (east), and Songkhla (south). Eight wild caught samples of Ae. albopictus were from Songkhla, Nakhon Sawan, Nakhon Ratchasrima and Kanchanaburi (west) provinces. The susceptibility to pyrethroids (deltamethrin, permethrin), organophosphate (fenitrothion) and carbamate (propoxur) insecticides were revealed in these samples. The biochemical test results were compared with those of the susceptible Bora (French Polynesia) strain. There was significant enhancement of MFO in pyrethroid resistant Ae. aegypti samples, except those from Songkhla and Hauykwang district in Bangkok. Biochemical assay results suggested that nonspecific esterases conferred fenitrothion resistance in Ae. aegypti in Nakhon Sawan, while insensitive AChE and/or nonspecific esterases could play role in fenitrothion resistance in Nakhon Ratchasrima. There was no consistent association of GST with pyrethroid resistance in Ae. aegypti. Low enzyme activities found in Ae. aegypti in Songkhla and in Ae. albopictus corresponded to their insecticide susceptibility status. The increased enzyme activity in field samples reflecting local history of insecticide employment was discussed.

Insecticide-induced behavioral responses of Anopheles minimus, a malaria vector in Thailand.

This study was designed to determine the behavioral responses of 2 test populations of Anopheles minimus females to DDT at 2 g/m2, deltamethrin at 0.0625 g/m2, and lambdacyhalothrin at 0.0369 g/m2 using an improved excito-repellency escape chamber. One test population was colonized in 1993 and referred to as a young colony. The 2nd field test population was collected from Ta-Soa County, Tri-Yok District, Kanchanaburi Province. in western Thailand and referred to as a wild population. Results showed that females of both young and wild test populations rapidly escaped from direct contact with DDT, deltamethrin, and lambdacyhalothrin. Lambdacyhalothrin exhibited the strongest irritant effect on female mosquitoes, followed by DDT and deltamethrin. Fewer females escaped from test chambers without direct contact with treated surfaces but the response was significantly different from that of the controls (P < 0.05). The noncontact response is indicative of a noncontact repellent action. Both contact irritancy and noncontact repellency are involved in An. minimus escape responses. Experimental hut studies that include monitoring of house-entering populations of An. minimus are needed for a meaningful assessment of noncontact repellent actions.

Effects of nutritional and physiological status on behavioral avoidance of Anopheles minimus (Diptera: Culicidae) to DDT, deltamethrin and lambdacyhalothrin.

The monitoring of behavioral responses of mosquitoes to insecticides are critical to the understanding of how chemicals function in the control of disease transmission. The excito-repellency avoidance responses of laboratory-reared Anopheles minimus females exposed to diagnostic concentrations of DDT (2 g/m2), deltamethrin (0.0625 g/m2), and lambdacyhalothrin (0.0369 g/m2) were observed using an excito-repellency escape chamber. Insecticide contact (measuring irritancy) and non-contact (measuring repellency) behavioral assays were conducted on non-blood-fed (unfed), sugar-fed, early blood-fed (recently engorged) and late blood-fed mosquitoes. Rates of escape from the contact and non-contact chambers, regardless of chemical compounds, were most dramatic in unfed mosquitoes compared to other nutritional states (P < 0.05). In general, across all 3 chemicals, slower escape response was observed in sugar-fed and early blood-fed specimens, whereas late blood-fed showed an intermediate response. Relative suppression of escape flight response in comparison to matched non-insecticide treated controls and the unfed condition is likely the result of normal reduced flight activity among recent blood and sugar-engorged mosquitoes. We conclude that nutritional states and physiological conditions of mosquitoes as a result of blood feeding can dramatically influence excito-repellency test results. Therefore, for interpretive purposes, studies on chemical irritancy and repellency must account and control for the inherent variability of avoidance responses to insecticides influenced by nutritional and physiological conditions of the mosquitoes at the time of test.

Status of malaria in Thailand.

Despite decades of control success and a competent network of country-wide health infrastructure, malaria remains an important health threat in rural Thailand. All 4 known human malaria parasites have been reported present, with Plasmodium falciparum and Plasmodium vivax predominant. The expansion and intensity of multi-drug resistant Plasmodium falciparum is the most serious development to occur the last several decades. Members of 3 anopheline species complexes, Anopheles dirus, Anopheles minimus, and Anopheles manculatus, are considered to be primary malaria vectors in the country. Representatives within all 3 taxa are difficult or impossible to separate morphologically from one another, and insufficient information exists about population genetics between sibling species and vector status. Vector control in Thailand has been the primary means of malaria control, mainly by the use of routine residual insecticide spray inside houses. The use of DDT in vector control has resulted in measurable successes to interrupt malaria transmission in many parts of the country. Since 1949, DDT has been the predominant compound used: however, its public health use has continued to decline as a result of perceived operational difficulties, political issues and environmental concerns. The increased use of pyrethroids to impregnate bednets and for intradomiciliary spraying are generally more accepted by rural populations and are rapidly replacing the use of DDT. Organized malaria control activities have reduced malaria morbidity from 286/1,000 population in 1947 to 1.5/1,000 population by 1996. Despite encouraging trends in dramatically reducing malaria, the rates of disease may be re-emerging in the country as evidence from an increased annual parasite index from 1.78/1,000 in 1997 to 2.21 in 1998. The possible reasons for the apparent increase in incidence are discussed in terms of the technical, operational and social obstacles in malaria control in Thailand.

Comparison of isozyme patterns of Aedes aegypti populations collected from pre- and post-Bacillus thuringiensis israelensis treatment sites in Thailand.

Isozyme patterns of 13 field-collected populations of Aedes aegypti from Thailand were compared using starch gel electrophoresis. Three populations were collected before the Bacillus thuringiensis var. israelensis, (B.t.i.) application was initiated. The other 10 populations were collected after the B.t.i. treatment. Results revealed that the number of polymorphic loci were lower in the B.t.i. treated populations as compared to controls. In addition, lower genetic variability was found in populations collected from B.t.i. treated sites (Mae Ka Sa [KS] and Mae Kud Luang [KL] village). These results are most likely due to a genetic bottleneck produced by the B.t.i. treatment. Heterozygosity increased in the months following B.t.i. treatment, probably because of immigration when the control program was withdrawn. However, the anticipated reduction in the expected heterozygosity was only observed in the KS site. This may be due to preexisting low heterozygosity in the KL population. No fixed differences in alleles were detected among the 13 populations.

Pesticide avoidance behavior in Anopheles albimanus, a malaria vector in the Americas.

The behavioral responses of 4 populations of Anopheles albimanus females to DDT, permethrin, and deltamethrin were characterized in excito-repellency tests. One test population (ST) from El Salvador has been maintained as a colony for 20 years. A second population (ES) from Guatemala was colonized in 1992. Third and fourth populations consisted of field-caught specimens from Toledo District (TO) of southern Belize in 1994 and Corozal District (CO) of northern Belize, respectively. Females of ES, TO, and CO populations rapidly escaped from direct contact with treated surfaces for each of the 3 insecticides. Similarities in escape responses of insecticide-resistant (ES) versus insecticide-susceptible populations (TO, CO) suggest that there is no relationship between physiological and behavioral responses of An. albimanus populations to DDT, permethrin, and deltamethrin. Females from all but the ST colony escaped in greater numbers from chambers without direct contact with treated surfaces than from control chambers (P < 0.05). Few females from the ST colony escaped from test chambers, regardless of which insecticide was used or whether contact was allowed, indicating that the ST colony has lost its capability to respond to insecticides. Repellent responses were significant; but they were not pronounced in 30-min exposures, and they were very pronounced in 4-h exposures. We conclude that irritant and repellent responses of malaria vectors to insecticides are important components of malaria control operations.

Methods of testing and analyzing excito-repellency responses of malaria vectors to insecticides.

A new test system that includes an excito-repellency test box, test procedures, and statistical treatment of data is described. The method consists of enclosing 25 mosquitoes in an exposure chamber lined with insecticide-treated or untreated (control) test papers. Each chamber has a single portal for mosquitoes to escape to a receiving cage, and numbers escaping are manually recorded at 1-min intervals. The exposure chamber accommodates a screened, 2nd chamber that, when placed in the exposure chamber, prevents the mosquitoes from making physical contact with test papers. A full assay utilized one exposure chamber that permits physical contact with insecticide-treated papers, one chamber that permits physical contact with control papers, one chamber that prevents physical contact with insecticide-treated papers, and a 4th chamber that prevents contact with control papers. After insecticide exposure, test populations are held for observations on 24-h mortalities. A survival analysis approach is described for estimating mosquito escape rates and for comparing differences in mosquito escape rates, with or without physical contact with insecticide, among populations, insecticides, and doses of insecticide.