Some phenomena associated with the development of Trypanosoma brucei rhodesiense infections in the tsetse fly, Glossina morsitans.

Immature salivary gland (SG) infections averaging 10(3) parasites per fly can apparently develop into mature gland infections averaging 10(5) parasites per fly in as little as 4 days. Frequently flies which extrude parasites in their saliva prove to have no parasites in the SG, but often show trypanosomes in the esophagus, cibarial pump, and proboscis. In some instances, SG infections have cleared, resulting in a loss of infectivity. Results of studying numbers of parasites regurgitated upon feeding or probing have shown that number to be highly variable and not necessarily related either to previous feeding status or the total number of parasites in the glands. Cloning of metacyclics in mice has been achieved, indicating that the minimum effective dose is one parasite. To date, no infections in mice have resulted from inoculation of extraglandular parasites. Histological and dissection studies support both the classical route and an alternate route of infection development in flies. No SG-infected flies have been found which did not also have proventricular and anterior and posterior midgut (AMG and PMG) infections. Although the AMG is where the heaviest MG infections occur, the PMG seems to support the last survivors in a moribund MG infection. No parasites have been found in the hindgut.

Circumsporozoite antibody as a serologic marker of Plasmodium falciparum transmission.

In a longitudinal study of a malaria-endemic village in southeastern Thailand, circumsporozoite (CS) antibody to sporozoites of Plasmodium falciparum was measured by an enzyme-linked immunosorbent assay to determine its usefulness as a seroepidemiologic marker of malaria transmission. The CS anti-(NANP)n antibody level and prevalence during a 25-month period paralleled the pattern of seasonal transmission consistent with conventional parasitologic and entomologic measurements. The prevalence and level of antibody decreased during the non-transmission wet season, and increased over a 1-2-month transition period between the end of monsoon rains and the onset of dry conditions, an interval of maximum vector activity. Antibody increased with age in the population. The prevalence of antibody to the asexual blood stage as measured by conventional indirect fluorescent antibody assay did not coincide with changes in transmission and was sustained throughout the study period. Thus, CS antibody appeared to reflect the relative population exposure to mosquito inoculation of P. falciparum sporozoites and provided a useful measure of malaria transmission dynamics.

Trypanosoma brucei rhodesiense: enhancement of infection rates in the tsetse fly, Glossina morsitans, by feeding artificial bloodmeal mixtures.

Low mature salivary gland (SG) infection rates (6%) in less than 24-hour-old flies fed on blood containing bloodform trypanosomes can be significantly enhanced by feeding flies an artificial mixture containing procyclic forms in a red cell: culture medium mixture (procyclic mixture, SG rate = 21.0%). However, enhancement is not solely a function of the use of procyclic forms since blood forms fed to flies in the same red cell: culture medium mixture produce SG rates (15.4%) intermediate to those of blood forms in blood and procyclic mixtures. Use of these artificial mixtures produces a similar result in 24- to 48-hour-old flies and also tends to equalize their infection rates with those found in less than 24-hour-old flies. The possible relationships between the different infection rates observed and digestive proteinases in the tsetse fly are discussed.

African sleeping sickness: new evidence that mature tsetse flies (Glossina morsitans) can become potent vectors.

Starved mature male tsetse flies (21 to 25 days old) are capable of developing salivary gland (SG) infections of Trypanosoma brucei rhodesiense at rates nearly comparable to teneral males less than 24 hours old when given an infective meal containing parasites, horse red cells and culture medium. Although the over-all SG infection rate for mature males starved for three, four or five days before infection was about half that for teneral males less than 48 hours old (8.0% v. 15.6%), males starved for four days developed infection rates (12.3%) that were comparable to those of teneral flies less than 24 hours old (11.8%). It is suggested that the acquisition of infection by mature flies should be considered when evaluating factors contributing either to maintenance of endemic infections or perhaps even epidemic infections of human sleeping sickness.

Comparison of antibody responses to the circumsporozoite protein repeat region and to intact sporozoites during acute falciparum malaria.

Most acute falciparum malaria patients mount an antibody response to the circumsporozoite (CS) protein which contains a dominant B-cell epitope. In order to investigate whether antibodies against other epitopes on the sporozoite surface may be important during a particular phase of infection or convalescence, we longitudinally studied the antibody responses of 13 Thai patients with acute falciparum malaria. Antibody comparisons were made using intact Plasmodium falciparum sporozoites in an indirect fluorescent antibody test and the recombinant peptide, R32tet32, as capture antigen in an enzyme-linked immunosorbent assay. Antibody response curves derived using the 2 methods were similar, and adsorption with R32tet32 greatly (greater than 95%) diminished anti-sporozoite activity in sera. Thus, peptide constructs containing the CS repeat region epitope, (NANP)n, can be used with confidence to assay anti-sporozoite antibodies, independent of both the time of infection and prior malaria history.

Hyperendemic malaria in a Thai village: dependence of year-round transmission on focal and seasonally circumscribed mosquito (Diptera: Culicidae) habitats.

In a longitudinal study of hyperendemic malaria in a village in eastern Thailand (from October 1985 to November 1987), man-biting anopheline mosquitoes were collected for 16 man-nights per month in 22 of 26 mo. Mosquitoes were separated according to collection sites (inner, central, more populated; outer, peripheral, more forested), biting period, and parity, and then they were tested for sporozoite antigen using an enzyme-linked immunosorbent assay (ELISA). Abundance of Anopheles dirus Peyton & Harrison was greater in outer than inner village sites, with bimodal peaks in the postmonsoon (October-November) and early rainy (April or May) seasons. Parity rates at both sites were high in postmonsoon seasons (means about 67%), low in cool dry (December-January, less than 56%) and monsoon (June-September, less than 60%) seasons, and variable in other seasons. Of 1,861 An. dirus collected, 16 (0.9%) were positive for Plasmodium falciparum (PF) and nine (0.4%) for P. vivax (PV), whereas of 386 An. minimus, one (0.3%) and three (0.8%) were PF- and PV-positive, respectively. Entomological inoculation rates (EIR) were higher in outer (means PF = 0.91, PV = 0.34) than inner village sites (means = 0.01 for PF and PV). The EIR of PF appeared bimodal, high in postmonsoon (October-November) and early rainy (April or May) seasons, low in monsoon seasons, and variable in other seasons. The vectorial capacity of An. dirus was higher than that of An. minimus, indicating that the two species were primary and secondary vectors, respectively. Human malaria prevalence data indicated that transmission depended greatly on the higher year-round vector abundance in outer than in inner village sites.

Japanese encephalitis virus in Bangkok: factors influencing vector infections in three suburban communities.

An unexpected outbreak of Japanese encephalitis (JE) in Bangkok in 1985 led us to investigate the vector ecology of urban JE from January 1986 to June 1987 at three suburban sites that displayed a wide range of factors imputed to influence JE transmission. Culex tritaeniorhynchus Giles and Cx. gelidus Theobald, suspected vectors, comprised 71-96% of all mosquitoes collected by CO2-baited CDC traps at the three sites. Mean of mosquito abundance per two trap-nights per month ranged from 28 to 5,728 mosquitoes at the sites of lowest and highest abundance, respectively. Cx. tritaeniorhynchus yielded more JE isolates (n = 16) than Cx. gelidus (n = 7), but the minimum infection rates of the two species (number of JE isolates per 1,000 mosquitoes tested; MIR, 0.17 and 0.47, respectively) were comparable and covaried with vector abundance. Moreover, the proportion of sentinel pigs that had JE antibodies generally increased proportionately with vector abundance at the sites. Vector abundance was high in monsoon (May-October), moderate in transition (March-April and November-December), and low in dry (January-February) seasons. Mosquitoes collected in monsoon seasons yielded 96% of the JE isolates, whereas 4 and 0% of the isolates were obtained from transition and dry season collections, respectively. More pigs seroconverted in monsoon and transition seasons than in dry seasons. Indices of JE transmission activity (vector abundance, pig seroconversions, and MIRs) increased proportionately with rainfall. Despite higher indices at the site of greatest vector abundance than elsewhere, the risk of human infection appeared greatest at the site with moderate vector abundance because of its greatest human population density.

Some entomological observations on malaria transmission in a remote village in northwestern Thailand.

Anopheline mosquitoes and their relation to malaria transmission were studied during the months of March, May and July in the Karen village of Mae Tha Waw located in the northwestern mountains of Tak Province. Thirteen species were captured on human bait during 80 man-nights of collecting. Four additional species were collected during routine larval surveys. Anopheles minimus and An. maculatus comprised 92.5% of the specimens captured biting man. Anopheles minimus and An. nivipes were implicated as vectors based on the detection of sporozoite infections using enzyme-linked immunosorbent assays for Plasmodium falciparum and P. vivax. Anopheles dirus was rarely encountered and probably played little part in transmission in Mae Tha Waw during the period of study. Information is provided on nightly biting activity, incidence of disease, infectivity and larval bionomics.

Relative repellency of two formulations of N,N-diethyl-3-methylbenzamide (deet) and permethrin-treated clothing against Culex sitiens and Aedes vigilax in Thailand.

Field tests were conducted to compare the effectiveness of 2 repellent formulations of N,N-diethyl-3-methylbenzamide (deet) in combination with permethrin-impregnated military uniforms against Culex sitiens and Aedes vigilax in Thailand. Repellency was determined during a 2 h crepuscular period using volunteers who had been treated with repellents 6, 8, 10, and 12 h prior to the end of each test period. An extended-duration repellent formulation (EDRF) containing 35% deet repelled significantly more Ae. vigilax than 75% deet in ethanol. Although not statistically significant, the EDRF also resulted in fewer biting attempts by Cx. sitiens. Neither formulation provided complete protection against either species 4-12 h post-application, but both provided greater overall protection against Ae. vigilax. Volunteers who wore treated uniforms without repellents were attacked by significantly fewer mosquitoes than controls.

A longitudinal study of Japanese encephalitis in suburban Bangkok, Thailand.

A one-year study of Japanese encephalitis (JE) in a small focus of transmission was conducted in suburban Bangkok in 1985. Monthly data were collected on weather, vector density, sentinel pig and chick JE antibody seroconversions, and epidemiology as related to human JE cases. The primary vector species were found to be Culex gelidus and Culex tritaeniorhynchus; from which one isolate each was obtained in March and June, respectively. Pig JE antibody seroconversion peaked in April (the hottest month), with secondary peaks following in July and December. Chick seroconversions were found only in June and July. Human cases (7) in the primary focus occurred from May-July, and started 2 months following the finding of the first JEV isolate in mosquitoes and 1 month following mass JEV seroconversion in pigs. Overall, the attack rate in the focus (0.83/10(5] was greater than 4 times that of the rest of Bangkok (0.19/10(5]. Attack rates were highest in 0-9 and 10-19 year-old groups, respectively. Indications are that JEV is transmitted to humans in Bangkok at least 10 out of 12 months per year, but that cases are concentrated in the May to July period.

Trypanosoma rhodesiense blood forms express all antigen specificities relevant to protection against metacyclic (insect form) challenge.

Monoclonal antibodies were used to demonstrate the expression of four distinct metacyclic (infective insect form) trypanosome antigens on blood forms of T. rhodesiense. Metacyclic antigens were consistently expressed on the blood forms on days 4 and 5 of the first parasitemia after metacyclic infection of C57BL/6 mice. In different mice examined, the percent of blood forms expressing metacyclic antigens ranged from 46 to 85%. Immunization with irradiated day-5 blood form trypanosomes was protective against metacyclic challenge, indicating that all antigen specificities relevant to protective immunization against metacyclic challenge are expressed on blood form trypanosomes. Blood forms, in contrast to metacyclic forms, can be isolated in quantities sufficient for purification of antigens and genetic cloning.

Genetic linkage relationships of eight enzyme/electromorph loci in Anopheles minimus.

Two laboratory stocks of Anopheles minimus, each fixed for variant electromorphs of esterases, aspartate aminotransferase, hydroxyacid dehydrogenase, phosphogluconate dehydrogenase, mannose phosphate isomerase and glycerol dehydrogenase were used to assess linkage relationships between presumed gene loci controlling this variation. The two F1, which had been obtained from crossing the stocks, were backcrossed to a parental stock. Three loci controlled the esterases and one locus each of the other enzymes. Mpi is sex-linked. The rest are autosomal and suggested relationships are: Pgd 2.3% recombination from Aat and unlinked to any other loci; Est-1-33.8%-Est-3-31.5%-Est-2-21.0%-Had. Gcd is unlinked to any other locus. There was evidence of strong interaction between the X chromosome of one stock and autosomes of the other in which individuals bearing the X chromosome of the one suffered relatively greater mortality and had delayed development with respect to other genotypic classes.

Protection against Japanese encephalitis by inactivated vaccines.

Encephalitis caused by Japanese encephalitis virus occurs in annual epidemics throughout Asia, making it the principal cause of epidemic viral encephalitis in the world. No currently available vaccine has demonstrated efficacy in preventing this disease in a controlled trial. We performed a placebo-controlled, blinded, randomized trial in a northern Thai province, with two doses of monovalent (Nakayama strain) or bivalent (Nakayama plus Beijing strains) inactivated, purified Japanese encephalitis vaccine made from whole virus derived from mouse brain. We examined the effect of these vaccines on the incidence and severity of Japanese encephalitis and dengue hemorrhagic fever, a disease caused by a closely related flavivirus. Between November 1984 and March 1985, 65,224 children received two doses of monovalent Japanese encephalitis vaccine (n = 21,628), bivalent Japanese encephalitis vaccine (n = 22,080), or tetanus toxoid placebo (n = 21,516), with only minor side effects. The cumulative attack rate for encephalitis due to Japanese encephalitis virus was 51 per 100,000 in the placebo group and 5 per 100,000 in each vaccine group. The efficacy in both vaccine groups combined was 91 percent (95 percent confidence interval, 70 to 97 percent). Attack rates for dengue hemorrhagic fever declined, but not significantly. The severity of cases of dengue was also reduced. We conclude that two doses of inactivated Japanese encephalitis vaccine, either monovalent or bivalent, protect against encephalitis due to Japanese encephalitis virus and may have a limited beneficial effect on the severity of dengue hemorrhagic fever.