Genetically manipulated vectors of human disease: a practical overview.
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Biomedical subjects
E D Walker
Publications and source records attributed to E D Walker.
Assessment of hepatitis B virus DNA and hepatitis C virus RNA in the common bedbug (Cimex lectularius L.) and kissing bug (Rodnius prolixus).
OBJECTIVE: Historical clinical studies suggest the potential for insect-borne transmission of human hepatitis viruses. Studies of hepatitis B virus (HBV) persistence in insects were performed before the advent of molecular techniques, and studies to assess possible insect-borne transmission of hepatitis viruses have not yet been performed. The aim of this study was to determine, using molecular techniques, whether HBV and hepatitis C virus (HCV) persist in and are excreted in the feces of the bedbug Cimex lectularius L. and kissing bug Rodnius prolixus after an infectious meal. METHODS: Blood-feeding insects from the insect order Hemiptera (Cimex lectularius L. and Rhodnius prolixus) were fed on blood from infected patients with high titers of HBV, HCV, and control uninfected patients. Insects and insect excrement were collected at weekly intervals and tested for HBV DNA and HCV RNA using the polymerase chain reaction. RESULTS: HBV DNA was detected in bedbugs and excrement up to 6 wk after feeding on an infectious meal. HBV DNA was also detected in most kissing bugs and excrement up to 2 wk after feeding. HCV RNA was not detected in bedbugs at any time after feeding. CONCLUSIONS: We did not detect HCV RNA in bedbugs after feeding on an infectious meal. Our data provide molecular evidence to suggest that HBV may persist in Hemiptera. Additional studies are ongoing to determine whether this viral persistence is capable of infection.
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Expression of the p20 gene from Bacillus thuringiensis H-14 increases Cry11A toxin production and enhances mosquito-larvicidal activity in recombinant gram-negative bacteria.
Experimental analyses with recombinant Escherichia coli and Pseudomonas putida transformed with plasmids bearing genes coding for the Cry11A toxin and P20 protein from Bacillus thuringiensis H-14 showed that cells producing both proteins were more toxic when fed to third-instar Aedes aegypti larvae than were cells expressing cry11A alone; the 50% lethal concentrations were in the range of 10(4) to 10(5) cells/ml. Western blots revealed a higher production of Cry11A when the p20 gene was coexpressed. Cry11A was detected primarily in insoluble form in recombinant cells. Cry11A was not detected in P. putida when P20 was not coproduced, and these recombinants were not toxic to larvae, whereas P. putida recombinants producing both proteins were toxic at concentrations similar to those for E. coli. A coelution experiment was conducted, in which a p20 gene construct producing the P20 protein with an extension of six histidines on the C terminus was mixed with the Cry11A protein. The results showed that Cry11A bound to the P20(His(6)) on a nickel chelating column, whereas Cry11A produced without the P20(His(6)) protein was washed through the column, thus indicating that Cry11A and P20 physically interact. Thus, P20 protein either stabilizes Cry11A or helps it attain the folding important for its toxic activity.
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Stercorarial shedding and transtadial transmission of hepatitis B virus by common bed bugs (Hemiptera: Cimicidae).
Transtadial persistence and stercorarial shedding of hepatitis B virus (HBV) in common bed bugs, Cimex lectularius L., was studied by using experimental infectious blood feedings, infectious intrathoracic inoculations, and virus detection by polymerase chain reaction and Southern hybridization. Results showed that HBV persisted after an infectious blood meal in bed bug bodies for up to 35 d after the infectious blood meal. It was passed transtadially through one molt regardless of instar, was shed in fecal droplets for up to 35 d after the infectious blood meal, but was not passed transovarially. In bugs inoculated intrathoracically, HBV was detected for 21 d postinoculation. Previous studies detected the hepatitis B surface antigen found on both infectious and noninfectious particles in bed bugs. In this study, the presence of nucleic acids amplified from a conserved core region of the viral genome in bodies and feces of C. lectularius suggests that the HBV virus may be mechanically transmitted in feces or when bugs are crushed, during feeding.
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Bacterial and fungal biomass responses to feeding by larval Aedes triseriatus (Diptera: Culicidae).
We investigated the effect of different densities (0, 20, or 40) of developing larval Aedes triseriatus (Say) on bacterial abundance, bacterial productivity, and leaf fungal biomass in a microcosm experiment. Larvae in the low-density treatment developed normally, but larvae at the high density were significantly slower to develop. Both bacterial abundance (direct microscopic counts) and bacterial productivity (3H-leucine incorporation rates) on leaf material were significantly lower in the presence of larvae. Bacterial abundance in the water column did not change significantly with treatment, but bacterial productivity varied with time and declined significantly at both larval densities. Bacteria on the walls and bottom of the containers also were less abundant and significantly less productive in the presence of larvae. Aside from presence/absence effects, there was no clear evidence that larval impacts were density-dependent. Leaf-associated fungal biomass, as measured by ergosterol levels, varied with time but was not significantly affected by any treatment, suggesting most fungal tissue was incorporated in the leaf matrix and unavailable to larvae. Based upon estimated biomass accrual and respiration of larvae, it appears that bacterial biomass and production were insufficient to account for carbon demands of growing larvae. Because fungal biomass and leaf mass likely contributed little to gross larval demands, other carbon sources (e.g., protozoa and extracellular microbial components) were probably used by larvae. Although apparently insufficient for all larval carbon demands, bacterial and leaf fungal biomass may be adequate for other larval nutritional needs (i.e., nitrogen and essential lipids).
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Canine surveillance system for Lyme borreliosis in Wisconsin and northern Illinois: geographic distribution and risk factor analysis.
A seroprevalence survey for Borrelia burgdorferi was conducted among the healthy canine pet population in selected counties of Wisconsin and northern Illinois to determine the distribution of Lyme disease and associated risk factors. Information obtained for each dog included place of residence, Lyme disease vaccination status, history of travel and tick exposure, signalment, and medical history. Serum samples were screened by enzyme-linked immunosorbent assay and confirmed by an immunoblot procedure. Seroprevalence by county ranged 0-40%, with the highest estimates from west-central Wisconsin. The spatial pattern was significantly correlated with human incidence of Lyme disease and with abundance of the tick vector, Ixodes scapularis. A geographic information system (GIS) was used to integrate environmental data with the location of the residences of the dogs to determine environmental risk factors. Seropositivity among dogs was positively associated with increased tick exposure and time spent outdoors and negatively associated with vaccination against Lyme disease. Seropositivity was also associated with living in forested and urban areas, and on sandy, fertile soils. A canine surveillance system is a useful method for assessing the geographic distribution of Lyme disease, and in combination with a GIS, it can be effective in determining environmental factors associated with I. scapularis endemicity.
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Probable autochthonous Plasmodium vivax malaria transmission in Michigan: case report and epidemiological investigation.
In September 1995, a Michigan resident with no history of international travel was diagnosed with Plasmodium vivax infection, and local mosquito-borne transmission was suspected. An epidemiological investigation did not identify additional cases of local transmission, and there was no apparent link to the 12 imported malaria cases detected in the region. Potential sites of nighttime outdoor exposure included a campground in a swampy area, close to a racetrack frequented by international travelers, some of whom were known to come from countries with malaria transmission. Entomological investigation identified Anopheles spp. larvae and adults near the campsite. Summer temperatures 4.2 degrees C above average would have contributed to shortened maturation time of P. vivax within the insect vector, increasing the likelihood of infectivity. These investigations indicated that this patient probably acquired P. vivax infection through the bite of a locally infected Anopheles spp. mosquito. Physicians need to consider malaria as a possible cause of unexplained febrile illness, even in the absence of international travel, particularly during the summer months.
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Research ethics. Managing risks of arthropod vector research.
Field research with vectors is an essential aspect of vector biology research and vector-borne disease prevention and control. This type of research, which brings experimental vector manipulations into endemic areas, can present risks to human populations. This paper seeks to stimulate a full discussion within the medical entomology community of the risks associated with vector field research. Such discussions will promote development of a consensus, among investigators, sponsoring agencies and the communities within which the work is done, so that appropriate steps can be taken to minimize and manage the risks, and adequate oversight can be maintained.
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Land use change alters malaria transmission parameters by modifying temperature in a highland area of Uganda.
As highland regions of Africa historically have been considered free of malaria, recent epidemics in these areas have raised concerns that high elevation malaria transmission may be increasing. Hypotheses about the reasons for this include changes in climate, land use and demographic patterns. We investigated the effect of land use change on malaria transmission in the south-western highlands of Uganda. From December 1997 to July 1998, we compared mosquito density, biting rates, sporozoite rates and entomological inoculation rates between 8 villages located along natural papyrus swamps and 8 villages located along swamps that have been drained and cultivated. Since vegetation changes affect evapotranspiration patterns and, thus, local climate, we also investigated differences in temperature, humidity and saturation deficit between natural and cultivated swamps. We found that on average all malaria indices were higher near cultivated swamps, although differences between cultivated and natural swamps were not statistically significant. However, maximum and minimum temperature were significantly higher in communities bordering cultivated swamps. In multivariate analysis using a generalized estimating equation approach to Poisson regression, the average minimum temperature of a village was significantly associated with the number of Anopheles gambiae s.l. per house after adjustment for potential confounding variables. It appears that replacement of natural swamp vegetation with agricultural crops has led to increased temperatures, which may be responsible for elevated malaria transmission risk in cultivated areas.
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Quantitative approach for the serodiagnosis of canine Lyme disease by the immunoblot procedure.
Serum samples obtained from healthy, asymptomatic dogs in areas of Wisconsin and northern Illinois where Lyme disease is endemic or nonendemic were assayed for antibodies to Borrelia burgdorferi by enzyme-linked immunosorbent assay (ELISA), and positive results were confirmed by immunoblot assay. We found that 56.9% (562 of 1,077) of the samples were positive by ELISA and 82.0% (461 of 562) were positive by immunoblotting. A logistic regression model was developed to distinguish between nonvaccinated dogs naturally infected with B. burgdorferi from areas where the disease is endemic and dogs from areas where the disease is nonendemic that were vaccinated against Lyme disease. Of the 18 protein bands analyzed, 8 were found to be significantly different (P<0.05) between the two groups. p93, p34, p31, and p28 occurred with increased frequency in vaccinated dogs, while p58, p37, p35, and p30 occurred more frequently in naturally infected dogs. The logistic regression equation obtained was used to determine the probability of natural infection among vaccinated dogs residing in areas where the disease is endemic. Of 125 samples, 87.2% had a very low probability of natural infection and only 2.4% were highly likely to be infected. Logistic regression is a useful method for distinguishing between vaccinated and naturally infected dogs and predicting the serological status of vaccinated dogs from areas where Lyme disease is endemic.
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Early warning of malaria epidemics in African highlands using Anopheles (Diptera: Culicidae) indoor resting density.
Several highland regions of Africa recently have suffered malaria epidemics. Because malaria transmission is unstable and the population has little or no immunity, these highlands are prone to explosive outbreaks when densities of Anopheles exceed critical levels and conditions favor transmission. If an incipient epidemic can be detected early enough, control efforts may reduce morbidity, mortality, and transmission. Here we present three methods (direct, minimum sample size, and sequential sampling approaches) that could be used to determine whether the household indoor resting density of Anopheles gambiae s.I. has exceeded critical levels associated with epidemic transmission. Data on Anopheles density before, during, and after a malaria epidemic (December 1997-July 1998) in the highlands of southwestern Uganda were evaluated to demonstrate the application of these three approaches. During this epidemic, a density of 0.25 Anopheles mosquitoes per house was associated with epidemic transmission, whereas 0.05 mosquitoes per house was chosen as a normal level expected during nonepidemic months. The direct approach to calculating mean Anopheles density with an allowable error of 20-50% of the mean would require the sampling of 102-16 houses, respectively. In contrast, with only seven houses, the minimum sample size approach could be used to determine whether Anopheles density had exceeded the critical level. This method, however, would result in an overestimation of the risk of an epidemic at low Anopheles density. Finally, a sequential sampling plan could require as many as 50 houses to conclude that risk of an epidemic existed, but this disadvantage is offset by the ability to preset the probabilities of concluding that risk of an epidemic exists at both the critical and normal Anopheles densities. Our study illustrated that it is feasible, and probably expedient, to include monitoring of Anopheles density in highland malaria epidemic early warning systems.
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Effects of larval mosquitoes (Aedes triseriatus) and stemflow on microbial community dynamics in container habitats.
The dynamics of the microbial food sources for Aedes triseriatus larvae in microcosms were found to be strongly influenced by larval presence. The total abundance of bacteria in water samples generally increased in response to larvae, including populations of cultivable, facultatively anaerobic bacteria. Additionally, a portion of the community shifted from Pseudomonaceae to Enterobacteriaceae. Bacterial abundance on leaf material was significantly reduced in the presence of actively feeding larvae. Principle-component analysis of whole community fatty acid methyl ester (FAME) profiles showed that larvae changed the microbial community structure in both the water column and the leaf material. Cyclopropyl FAMEs, typically associated with bacteria, were reduced in microcosms containing larvae; however, other bacterial fatty acids showed no consistent response. Long-chain polyunsaturated fatty acids characteristic of microeukaryotes (protozoans and meiofauna) declined in abundance when larvae were present, indicating that larval feeding reduced the densities of these microorganisms. However, presumed fungal lipid markers either increased or were unchanged in response to larvae. Larval presence also affected microbial nitrogen metabolism through modification of the physiochemical conditions or by grazing on populations of bacteria involved in nitrification-denitrification. Stemflow primarily influenced inorganic ion and organic compound concentrations in the microcosms and had less-pronounced effects on microbial community parameters than did larval presence. Stemflow treatments diluted concentrations of all inorganic ions (chloride, sulfate, and ammonium) and organic compounds (total dissolved organic carbon, soluble carbohydrates, and total protein) measured, with the exceptions of nitrite and nitrate. Stemflow addition did not measurably affect larval biomass in the microcosms but did enhance development rates and early emergence patterns of adults.
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Components of the vectorial capacity of Aedes poicilius for Wuchereria bancrofti in Sorsogon province, Philippines.
Components of vectorial capacity (biting density, survival, and host preferences) of a population of Aedes (Finlaya) poicilius, the principal vector of Wuchereria bancrofti in the Philippines, were studied in 1994-1995 in Sorsogon province. Aedes poicilius comprised 20.7% of 3243 mosquitoes of 24 species taken in 42 nights of human-biting collections, and 1.4% of 7586 mosquitoes of 27 species taken in 18 concurrent nights of carabao-trap collections. There were, on average, 16 bites by Ae. poicilius per person per night. As there was no relationship between body size of the female Ae. poicilius and parity status, body size was not a variable affecting survivorship of Ae. poicilius. The probability of daily survival was estimated to be 0.85, and the human blood index was 0.67. None of the 862 Ae. poicilius examined contained W. bancrofti larvae, probably because of distribution of diethylcarbamazine to microfilaraemic people prior to the study. The vectorial capacity of the Ae. poicilius population under study was estimated to be 2.4 new cases per primary case per day. However, the basic reproductive rate (i.e. the number of new cases of W. bancrofti infection generated from a single infective case) was estimated to be very low because of inefficiency in parasite transmission.
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Midgut bacteria in Anopheles gambiae and An. funestus (Diptera: Culicidae) from Kenya and Mali.
Field studies in Kenya and Mali investigated the prevalence of bacteria in the midguts of malaria vectors, and the potential relationship between gram-negative bacteria species and Plasmodium falciparum sporozoites. Midguts were dissected from 2,430 mosquitoes: 863 Anopheles funestus Giles and 1,037 An. gambiae s.l. Giles from Kenya, and 530 An. gambiae s.l. from Mali. An. funestus had a higher prevalence of gram-negative bacteria (28.5%) compared with An. gambiae collected in Kenya and Mali (15.4 and 12.5%, respectively). Twenty different genera of bacteria were identified by gas chromatography from 73 bacterial isolates from mosquito midguts. Pantoea agglomerans (Enterobacter agglomerans) was the most common species identified. There was no association between gram-negative bacteria in the midgut and P. falciparum sporozoites in field-collected An. gambiae s.l. and An. funestus. However, An. funestus females that harbored gram positive bacteria were more likely to be infected with sporozoites compared with those with no cultivable bacteria or gram negative bacteria in their midguts. Habitat-related variation in the prevalence of diverse types of bacteria in mosquitoes could influence malaria parasite development in mosquitoes and corresponding sporozoite prevalence.
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Geographic distribution of ticks (Acari: Ixodidae) in Michigan, with emphasis on Ixodes scapularis and Borrelia burgdorferi.
A 12-yr (1985-1996) passive survey in Michigan based upon tick submissions from citizens yielded 4,755 ticks of 21 species, 16 of which were probably indigenous in the state. Three species of Dermacentor [most common, D. variabilis Say and D. albipictus (Packard)]; 2 species of Amblyomma [most common, A. americanum (L.)]; and 12 species of Ixodes (most common, I. cookei Packard and I. scapularis Say), as well as Haemaphysalis leporispalustris (Packard), Rhipicephalus sanguineus Latreille, and the soft ticks Ornithodoros kelleyi Cooley & Kohls, and Otobius megnini (Duges) were submitted. New state records were I. kingi Bishopp, I. texanus Banks, I. sculptus Neumann, and I. baergi Cooley & Kohls. Examination of gut smears from dissections of 1,037 ticks of 13 species by indirect immunofluorescent assay, using murine monoclonal H9724 as the primary antibody, revealed that 11 of 175 I. scapularis were infected with Borrelia spp. All positive I. scapularis were from Menominee County in the upper peninsula of the state, which also provided 79.8% of all submitted I. scapularis. Surveys for ticks on 5,449 hunter-killed white-tailed deer were conducted from 1988 to 1990, encompassed deer taken from 65 of the state's 83 counties, and showed that although D. albipictus was distributed widely in the northern part of the state, I. scapularis occurred only on deer taken from southern townships of Menominee County. Of 1,218 canine sera tested for antibodies to B. burgdorferi in 1992 and 1993, 25 of 299 (8.0%) from Menominee County were positive but only 1 of 919 sera submitted from 5 counties in the lower peninsula was positive.
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Bacterial density and survey of cultivable heterotrophs in the surface water of a freshwater marsh habitat of Anopheles quadrimaculatus larvae (Diptera: Culicidae).
We examined surface water samples collected in September and October 1994 from a freshwater marsh habitat containing larval Anopheles quadrimaculatus mosquitoes. Bacterial densities in direct microscopic counts ranged from 9.7 x 10(5) to 1.3 x 10(7) cells/ml. Densities of cultivable bacteria on trypticase soy agar medium ranged from 1.0 to 1.5 x 10(5) cells/ml. The majority of 888 isolates were gram-positive rods (41%) followed by gram-negative rods (28%). Analysis of the cellular fatty acid profiles of 824 isolates using gas chromatography and Microbial Identification Systems TSBA (Rev. 3.60) library software grouped the bacteria into Bacillus spp. (35%), other gram-positive bacteria (16%), pseudomonads (15%), other gram-negative bacteria including mainly Enterobacteriaceae and Vibrionaceae (21%), and profiles not recognized (13%). Among 33 genera within these groups, the most common were Bacillus, Pseudomonas, Aeromonas, and Arthrobacter.
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Expanding a career progression program.
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Career Mobility↗
Borrelia burgdorferi-infected Ixodes scapularis (Acari: Ixodidae) and Peromyscus leucopus in northeastern Wisconsin.
Populations of the blacklegged tick, Ixodes scapularis Say, are established in western and central Wisconsin in the upper midwestern United States, but appear to be expanding geographically there. Here, we report a previously unknown population in northeastern Wisconsin. Questing I. scapularis nymphs and adults were collected by flagging vegetation from a riverine site in Marinette County, Wisconsin, in spring of 1993 and 1994. Dissection and culture of tick guts in modified Barbour-Stoenner-Kelley II medium showed that some of the ticks were infected with Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwald & Brenner, causative agent of Lyme disease. Fifteen of 30 white-footed mice, Peromyscus leucopus (Rafinesque), live-trapped at the site on 23-24 August 1994 were infested with immature I. scapularis, and ear-punch biopsies yielded B. burgdorferi cultures from 2 of the mice. However, none of 50 white-tailed deer, Odocoileus virginianus (Zimmermann), shot by hunters in Marinette County in November 1994 had I. scapularis on them, probably because no deer were shot at the same site where the tick population is located. These findings document existence of an established population of I. scapularis and a focus of Lyme disease in northeastern Wisconsin.
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