Edward D. Walker

Reemergence of Anopheles funestus as a vector of Plasmodium falciparum in western Kenya after long-term implementation of insecticide-treated bed nets.

Historically, the malaria vectors in western Kenya have been Anopheles funestus, Anopheles gambiae s.s., and Anopheles arabiensis. Of these species, An. funestus populations declined the most after the introduction of insecticide-treated bed nets (ITNs) in the 1990s in Asembo, and collections of An. funestus in the region remained low until at least 2008. Contrary to findings during the early years of ITN use in Asembo, the majority of the Anopheles collected here in 2010 and 2011 were An. funestus. Female An. funestus had characteristically high Plasmodium falciparum sporozoite rates and showed nearly 100% anthropophily. Female An. funestus were found more often indoors than outdoors and had relatively low mortality rates during insecticide bioassays. Together, these results are of serious concern for public health in the region, indicating that An. funestus may once again be contributing significantly to the transmission of malaria in this region despite the widespread use of ITNs/long-lasting insecticidal nets (LLINs).

Associations of passerine birds, rabbits, and ticks with Borrelia miyamotoi and Borrelia andersonii in Michigan, U.S.A.

BackgroundWild birds contribute to maintenance and dissemination of vectors and microbes, including those that impact human, domestic animal, and wildlife health. Here we elucidate roles of wild passerine birds, eastern cottontail rabbits (Sylvilagus floridanus), and Ixodes dentatus ticks in enzootic cycles of two spirochetes, Borrelia miyamotoi and B. andersonii in a region of Michigan where the zoonotic pathogen B. burgdorferi co-circulates.MethodsOver a four-year period, wild birds (n = 19,631) and rabbits (n = 20) were inspected for tick presence and ear tissue was obtained from rabbits. Samples were tested for Borrelia spirochetes using nested PCR of the 16S-23S rRNA intergenic spacer region (IGS) and bidirectional DNA sequencing. Natural xenodiagnosis was used to implicate wildlife reservoirs.ResultsIxodes dentatus, a tick that specializes on birds and rabbits and rarely bites humans, was the most common tick found, comprising 86.5% of the 12,432 ticks collected in the study. The relapsing fever group spirochete B. miyamotoi was documented for the first time in ticks removed from wild birds (0.7% minimum infection prevalence; MIP, in I. dentatus), and included two IGS strains. The majority of B. miyamotoi-positive ticks were removed from Northern Cardinals (Cardinalis cardinalis). Borrelia andersonii infected ticks removed from birds (1.6% MIP), ticks removed from rabbits (5.3% MIP), and rabbit ear biopsies (5%) comprised twelve novel IGS strains. Six species of wild birds were implicated as reservoirs for B. andersonii. Frequency of I. dentatus larval and nymphal co-feeding on birds was ten times greater than expected by chance. The relatively well-studied ecology of I. scapularis and the Lyme disease pathogen provides a context for understanding how the phenology of bird ticks may impact B. miyamotoi and B. andersonii prevalence and host associations.ConclusionsGiven the current invasion of I. scapularis, a human biting species that serves as a bridge vector for Borrelia spirochetes, human exposure to B. miyamotoi and B. andersonii in this region may increase. The presence of these spirochetes underscores the ecological complexity within which Borrelia organisms are maintained and the need for diagnostic tests to differentiate among these organisms.

Incidence of malaria among mosquito collectors conducting human landing catches in western kenya

The human landing catch (HLC) has long been the gold standard for estimating malaria transmission by mosquitoes, but has come under scrutiny because of ethical concerns of exposing collectors to infectious bites. We estimated the incidence of Plasmodium falciparum malaria infection in a cohort of 152 persons conducting HLCs and compared it with that of 147 non-collectors in western Kenya. Participants were presumptively cleared of malaria with Coartem™ (artemether-lumefantrine) and tested for malaria every 2 weeks for 12 weeks. The HLC collections were conducted four nights per week for six weeks. Collectors were provided chemoprophylaxis with Malarone™ (atovaquone-proguanil) during the six weeks of HLC activities and one week after HLC activities were completed. The incidence of malaria was 96.6% lower in collectors than in non-collectors (hazard ratio = 0.034, P < 0.0001). Therefore, with proper prophylaxis, concern about increased risk of malaria among collectors should not be an impediment to conducting HLC studies.

Pyrethroid resistance in Anopheles gambiae s.s. and Anopheles arabiensis in western Kenya: phenotypic, metabolic and target site characterizations of three populations.

Field and laboratory investigations revealed phenotypic, target site and metabolic resistance to permethrin in an Anopheles gambiae s.s. (Diptera: Culicidae) population in Bungoma District, a region in western Kenya in which malaria is endemic and rates of ownership of insecticide‐treated bednets are high. The sensitivity of individual An. gambiae s.l. females as indicated in assays using World Health Organization (WHO) test kits demonstrated reduced mortality in response to permethrin, deltamethrin and bendiocarb. Estimated time to knock‐down of 50% (KDT50) of the test population in Centers for Disease Control (CDC) bottle bioassays was significantly lengthened for the three insecticides compared with that in a susceptible control strain. Anopheles arabiensis from all three sites showed higher mortality to all three insecticides in the WHO susceptibility assays compared with the CDC bottle assays, in which they showed less sensitivity and longer KDT50 than the reference strain for permethrin and deltamethrin. Microplate assays revealed elevated activity of β‐esterases and oxidases, but not glutathione‐S‐transferase, in An. gambiae s.s. survivors exposed to permethrin in bottle bioassays compared with knocked down and unexposed individuals. No An. arabiensis showed elevated enzyme activity. The 1014S kdr allele was fixed in the Bungoma An. gambiae s.s. population and absent from An. arabiensis, whereas the 1014F kdr allele was absent from all samples of both species. Insecticide resistance could compromise vector control in Bungoma and could spread to other areas as coverage with longlasting insecticide‐treated bednets increases.

Molecular characterization of a cold-active recombinant xylanase from Flavobacterium johnsoniae and its applicability in xylan hydrolysis

A novel xylanase gene, xyn10A, was cloned from Flavobacterium johsoniae, overexpressed in a flavobacterial expression system, the recombinant enzyme purified by Ni-affinity chromatography, and enzyme structure and activity analyzed. Xyn10A was found to be a modular xylanase with an Fn3 accessory domain on its N-terminal and a catalytic region on the C-terminal. The optimum pH and temperature for Xyn10A was 8.0 and 30 °C, but Xyn10A retained 50% activity at 4 °C, indicating that Xyn10A is a cold-active xylanase. A Fn3-deletion xylanase had relative activity ca. 3.6-fold lower than the wild-type, indicating that Fn3 promotes xylanase activity. The Fn3 region also contributed to stability of the enzyme at elevated temperatures. However, Fn3 did not bind this xylanase to insoluble substrates. The enzyme hydrolyzed xylo-oligosaccharides into xylobiose, and xylose with xylobiose as the main product, confirming that Xyn10A is a strict endo-β-1,4-xylanase. Xyn10A also hydrolyzed birchwood and beechwood xylan to yield mainly xylose, xylobiose and xylotriose.

Terrestrial vegetation and aquatic chemistry influence larval mosquito abundance in catch basins, Chicago, USA

BackgroundAn important determinant of mosquito-borne pathogen transmission is the spatial distribution of vectors. The primary vectors of West Nile virus (WNV) in Illinois are Culex pipiens Linnaeus (Diptera: Culicidae) and Culex restuans Theobald. In urban environments, these mosquitoes commonly oviposit in roadside storm water catch basins. However, use of this habitat is inconsistent, with abundance of larvae varying significantly across catch basins at a fine spatial scale.MethodsWe tested the hypothesis that attributes of the biotic and abiotic environment contribute to spatial and temporal variation in production of mosquito vectors, characterizing the relationship between terrestrial vegetation and aquatic chemistry and Culex abundance in Chicago, Illinois. Larvae were sampled from 60 catch basins from June 14 to October 3, 2009. Density of shrubs and 14 tree genera surrounding the basins were quantified, as well as aquatic chemistry content of each basin.ResultsWe demonstrate that the spatial pattern of Culex abundance in catch basins is strongly influenced by environmental characteristics, resulting in significant variation across the urban landscape. Using regression and machine learning techniques, we described landscape features and microhabitat characteristics of four Chicago neighborhoods and examined the implications of these measures for larval abundance in adjacent catch basins. The important positive predictors of high larval abundance were aquatic ammonia, nitrates, and area of shrubs of height <1 m surrounding the catch basins, whereas pH and area of flowering shrub were negatively correlated with larval abundance. Tree density, particularly of arborvitae, maple, and pear, also positively influenced the distribution of Culex during the fruit-bearing periods and early senescent periods in August and September.ConclusionsThis study identifies environmental predictors of mosquito production in urban environments. Because an abundance of adult Culex is integral to efficient WNV transmission and mosquitoes are found in especially high densities near larval habitats, identifying aquatic sites for Culex and landscape features that promote larval production are important in predicting the spatial pattern of cases of human and veterinary illness. Thus, these data enable accurate assessment of regions at risk for exposure to WNV and aid in the prevention of vector-borne disease transmission.

Diversity of Fish-Associated Flavobacteria of Michigan

Flavobacteriosis poses a serious threat to wild and propagated fish stocks alike, accounting for more fish mortality in Michigan and its associated state fish hatcheries than all other pathogens combined. Although this consortium of fish diseases has primarily been attributed to Flavobacterium psychrophilum, F. columnare, and F. branchiophilum, herein we describe a diverse assemblage of Flavobacterium and Chryseobacterium spp. isolates recovered from diseased as well as apparently healthy wild, feral, and farmed fish of Michigan. Among 254 fish-associated flavobacterial isolates recovered from 21 fish species during 2003-2010, 211 were identified as Flavobacterium spp., whereas 43 were identified as Chryseobacterium spp. according to ribosomal RNA partial gene sequencing and phylogenetic analysis. Although F. psychrophilum and F. columnare were indeed associated with multiple fish mortality events, many previously uncharacterized flavobacteria were recovered from systemically infected fish showing overt signs of disease, and in vitro protease assays demonstrated that these isolates were highly proteolytic to multiple substrates that comprise host tissues. Indeed, the majority of the isolates either (1) were most similar to recently described fish-associated Flavobacterium and Chryseobacterium spp. that have never before been reported in North America (e.g., F. oncorhynchi, F. araucananum, C. viscerum, C. piscicola, and C. chaponense) or (2) did not cluster with any described species and most likely represent novel flavobacterial taxa. This study highlights the extreme diversity of flavobacteria that are potentially associated with flavobacteriosis in Michigan.

Divergent host preferences of above- and below-ground Culex pipiens mosquitoes and their hybrid offspring

Culex pipiens form pipiens and Cx. pipiens form molestus (Diptera: Culicidae) belong to a cosmopolitan taxonomic group known as the Pipiens Assemblage. Hybridization between these forms is thought to contribute to human transmission of West Nile virus (WNV) in North America. Complementary choice and no‐choice landing assays were developed to examine host acceptance by North American Cx. pipiens in the laboratory. Populations collected from above‐ and below‐ground sites in suburban Chicago were identified as forms pipiens and molestus using a polymerase chain reaction‐based assay. Avian and human host acceptance was then quantified for the two populations, as well as for their hybrid and backcross offspring. No‐choice tests were used to demonstrate that both the pipiens and molestus forms were capable of feeding on human and avian hosts. Choice tests were used to demonstrate that form pipiens females were strongly avian‐seeking; an individuals probability of accepting the chick host was 85%. Form molestus females were more likely to accept the human host (87%). Rates of host acceptance by F1 and backcross progeny were intermediate to those of their parents. The results suggest that host preferences in Cx. pipiens are genetically determined, and that ongoing hybridization between above‐ and below‐ground populations is an important contributor to epizootic transmission of WNV in North America.

Dispersal of Adult Culex Mosquitoes in an Urban West Nile Virus Hotspot: A Mark-Capture Study Incorporating Stable Isotope Enrichment of Natural Larval Habitats

Dispersal is a critical life history behavior for mosquitoes and is important for the spread of mosquito-borne disease. We implemented the first stable isotope mark-capture study to measure mosquito dispersal, focusing on Culex pipiens in southwest suburban Chicago, Illinois, a hotspot of West Nile virus (WNV) transmission. We enriched nine catch basins in 2010 and 2011 with 15N-potassium nitrate and detected dispersal of enriched adult females emerging from these catch basins using CDC light and gravid traps to distances as far as 3 km. We detected 12 isotopically enriched pools of mosquitoes out of 2,442 tested during the two years and calculated a mean dispersal distance of 1.15 km and maximum flight range of 2.48 km. According to a logistic distribution function, 90% of the female Culex mosquitoes stayed within 3 km of their larval habitat, which corresponds with the distance-limited genetic variation of WNV observed in this study region. This study provides new insights on the dispersal of the most important vector of WNV in the eastern United States and demonstrates the utility of stable isotope enrichment for studying the biology of mosquitoes in other disease systems.

Elizabethkingia anophelis: Molecular Manipulation and Interactions with Mosquito Hosts

ABSTRACT Flavobacteria (members of the family Flavobacteriaceae) dominate the bacterial community in the Anopheles mosquito midgut. One such commensal, Elizabethkingia anophelis, is closely associated with Anopheles mosquitoes through transstadial persistence (i.e., from one life stage to the next); these and other properties favor its development for paratransgenic applications in control of malaria parasite transmission. However, the physiological requirements of E. anophelis have not been investigated, nor has its capacity to perpetuate despite digestion pressure in the gut been quantified. To this end, we first developed techniques for genetic manipulation of E. anophelis, including selectable markers, reporter systems (green fluorescent protein [GFP] and NanoLuc), and transposons that function in E. anophelis. A flavobacterial expression system based on the promoter PompA was integrated into the E. anophelis chromosome and showed strong promoter activity to drive GFP and NanoLuc reporter production. Introduced, GFP-tagged E. anophelis associated with mosquitoes at successive developmental stages and propagated in Anopheles gambiae and Anopheles stephensi but not in Aedes triseriatus mosquitoes. Feeding NanoLuc-tagged cells to A. gambiae and A. stephensi in the larval stage led to infection rates of 71% and 82%, respectively. In contrast, a very low infection rate (3%) was detected in Aedes triseriatus mosquitoes under the same conditions. Of the initial E. anophelis cells provided to larvae, 23%, 71%, and 85% were digested in A. stephensi, A. gambiae, and Aedes triseriatus, respectively, demonstrating that E. anophelis adapted to various mosquito midgut environments differently. Bacterial cell growth increased up to 3-fold when arginine was supplemented in the defined medium. Furthermore, the number of NanoLuc-tagged cells in A. stephensi significantly increased when arginine was added to a sugar diet, showing it to be an important amino acid for E. anophelis. Animal erythrocytes promoted E. anophelis growth in vivo and in vitro, indicating that this bacterium could obtain nutrients by participating in erythrocyte lysis in the mosquito midgut.