POPULATION STRUCTURE AND BARTONELLA QUINTANA IN HEAD AND BODY LICE IN POKHARA, NEPAL (ANOPLURA: PEDICULIDAE).

This study examined the population structure of head and body lice infesting a random sample of people in Pokhara, Nepal during 2003, 2004, and 2005. A total of 106 participants (6 to 72 yr old, median = 12) volunteered to have lice collected from their heads and clothing. Most participants (70%) harbored only head lice, some (15%) had only body lice, and some (15%) had concurrent infestations of head and body lice (dual infestations). A total of 1,472 lice was collected. Significantly more nymphs were collected than adult lice. Louse populations were generally small (geometric mean = 8.8 lice per person) but a few participants harbored larger louse populations (maximum = 65 lice). People with dual infestations harbored significantly more lice than people with single infestations; however, there was no difference in the infestation intensities between people infested with head lice only vs. those infested with body lice only. Male participants harbored significantly more lice than did females. There were no significant differences in infestation intensity due to participant age or their socioeconomic level. The sex ratio of adult lice was increasingly female biased with increasing adult louse density. Infection of lice with Bartonella quintana was low (ca. 1.5%). Pediculosis is a common problem in urban Nepal.

Bird species define the relationship between West Nile viremia and infectiousness to Culex pipiens mosquitoes.

The transmission cycle of West Nile virus (WNV) involves multiple species of birds. The relative importance of various bird species to the overall transmission is often inferred from the level and duration of viremia that they experience upon infection. Reports utilizing in vitro feeding techniques suggest that the source and condition of blood in which arboviruses are fed to mosquitoes can significantly alter the infectiousness of arbovirus to mosquitoes. We confirmed this using live hosts. A series of mosquito feedings with Culex pipiens was conducted on WNV-infected American robins and common grackles over a range of viremias. Mosquitoes were assayed individually by plaque assay for WNV at 3 to 7 days after feeding. At equivalent viremia, robins always infected more mosquitoes than did grackles. We conclude that the infectiousness of viremic birds cannot always be deduced from viremia alone. If information concerning the infectiousness of a particular bird species is important, such information is best acquired by feeding mosquitoes directly on experimentally infected individuals of that species.

Survival and Fecundity of Anopheles stephensi and Anopheles albimanus Mosquitoes (Diptera: Culicidae) After Ingesting Bovine Blood Containing Various Veterinary Systemic Parasiticides.

Systemic parasiticides in livestock can control zoophilic malaria vectors that contribute to residual malaria transmission. Membrane feeding techniques were used to screen seven systemic parasiticidic drugs currently in veterinary use for livestock and dogs. Drugs were tested in two laboratory strains of zoophilic Anopheles - A. stephensi (South Asian vector) and A. albimanus (Central American vector). To assess the relative potentials of these drugs, the resultant LC-50 for each drug was compared with what is known about the pharmacokinetic of the drug. Drugs with LC-50 values below the reported maximum plasma concentration of treated animals were considered as showing the most promise for use in the field. Ivermectin and fipronil showed the greatest promise for use in cattle against A. stephensi. Fipronil showed the greatest promise for use in cattle against A. albimanus. Both fluralaner and afoxolaner were highly effective against both mosquito species but pharmacokinetic data for these drugs in cattle are lacking. Eprinomectin, moxidectin and abamectin showed marginal to no promise for either mosquito species. At sublethal doses, ivermectin, fipronil, and afoxolaner (but not fluralaner) significantly reduced the larval production of surviving A. stephensi and A. albimanus. Further testing of candidate systemic parasiticides, including their product formulations, in livestock against field-collected populations of Anopheles is the next logical step toward full implementation of this strategy to manage zoophilic vectors.

Oral susceptibility to ivermectin is over fifty times greater in a wild population of Anopheles albimanus mosquitoes from Belize than the STECLA laboratory reference strain of this mosquito.

BACKGROUND: The STECLA strain of Anopheles albimanus has been in continuous colony for many years and is the reference strain on which genomic studies for the species are based. Recently, the STECLA strain was demonstrated to be much less susceptible to ivermectin ingested in a blood meal (4-day LC50 of 1468 ng/ml) than all other Anopheles species tested to-date (LC50 values range from 7 to 56 ng/ml). The ability of An. albimanus to survive ingestion of ivermectin at concentrations far beyond that typically found in the blood of ivermectin-treated people or livestock (i.e., 30-70 ng/ml) could invalidate the use of ivermectin as a malaria vector control strategy in areas where An. albimanus is a primary vector. METHODS: To investigate this, host-seeking An. albimanus were captured in northern Belize and used in membrane feeding bioassays of ivermectin, employing the same methods as described earlier with the STECLA strain. RESULTS: Field-collected An. albimanus in Belize were 55 times more susceptible to ingested ivermectin than were the STECLA reference strain. Oral susceptibility to ivermectin in wild An. albimanus from Belize (4-day LC50 of 26 ng/ml) was equivalent to that of other Anopheles species tested. CONCLUSIONS: Contrary to initial assessments using a highly inbred strain of mosquito, laboratory studies using a field population indicate that ivermectin treatment of livestock could reduce An. albimanus populations in areas of Central America and the Caribbean where malaria transmission may occur. Toxicity screening of ivermectin and other systemic parasiticides for malaria control should examine wild populations of the vector species being targeted.

Pre-existing Microfilarial Infections of American Robins (Passeriformes: Turdidae) and Common Grackles (Passeriformes: Icteridae) Have Limited Impact on Enhancing Dissemination of West Nile Virus in Culex pipiens Mosquitoes (Diptera: Culicidae).

Microfilariae (MF) are the immature stages of filarial nematode parasites and inhabit the blood and dermis of all classes of vertebrates, except fish. Concurrent ingestion of MF and arboviruses by mosquitoes can enhance mosquito transmission of virus compared to when virus is ingested alone. Shortly after being ingested, MF penetrate the mosquito's midgut and may introduce virus into the mosquito's hemocoel, creating a disseminated viral infection much sooner than normal. This phenomenon is known as microfilarial enhancement. Both American Robins and Common Grackles harbor MF-that is, Eufilaria sp. and Chandlerella quiscali von Linstow (Spirurida: Onchocercidae), respectively. We compared infection and dissemination rates in Culex pipiens L. mosquitoes that fed on birds with and without MF infections that had been infected with West Nile virus (WNV). At moderate viremias, about 107 plaque-forming units (pfu)/ml of blood, there were no differences in infection or dissemination rates among mosquitoes that ingested viremic blood from a bird with or without microfilaremia. At high viremias, >108.5 pfu/ml, mosquitoes feeding on a microfilaremic Grackle with concurrent viremia had significantly higher infection and dissemination rates than mosquitoes fed on viremic Grackles without microfilaremia. Microfilarial enhancement depends on the specific virus, MF, and mosquito species examined. How virus is introduced into the hemocoel by MF differs between the avian/WNV systems described here (i.e., leakage) and various arboviruses with MF of the human filarid, Brugia malayi (Brug) (Spirurida: Onchocercidae) (i.e., cotransport). Additional studies are needed to determine if other avian species and their MF are involved in the microfilarial enhancement of WNV in nature.

An inverse latitudinal gradient in infection probability and phylogenetic diversity for Leucocytozoon blood parasites in New World birds.

Geographic variation in environmental conditions as well as host traits that promote parasite transmission may impact infection rates and community assembly of vector-transmitted parasites. Identifying the ecological, environmental and historical determinants of parasite distributions and diversity is therefore necessary to understand disease outbreaks under changing environments. Here, we identified the predictors and contributions of infection probability and phylogenetic diversity of Leucocytozoon (an avian blood parasite) at site and species levels across the New World. To explore spatial patterns in infection probability and lineage diversity for Leucocytozoon parasites, we surveyed 69 bird communities from Alaska to Patagonia. Using phylogenetic Bayesian hierarchical models and high-resolution satellite remote-sensing data, we determined the relative influence of climate, landscape, geography and host phylogeny on regional parasite community assembly. Infection rates and parasite diversity exhibited considerable variation across regions in the Americas. In opposition to the latitudinal gradient hypothesis, both the diversity and prevalence of Leucocytozoon parasites decreased towards the equator. Host relatedness and traits known to promote vector exposure neither predicted infection probability nor parasite diversity. Instead, the probability of a bird being infected with Leucocytozoon increased with increasing vegetation cover (NDVI) and moisture levels (NDWI), whereas the diversity of parasite lineages decreased with increasing NDVI. Infection rates and parasite diversity also tended to be higher in cooler regions and higher latitudes. Whereas temperature partially constrains Leucocytozoon diversity and infection rates, landscape features, such as vegetation cover and water body availability, play a significant role in modulating the probability of a bird being infected. This suggests that, for Leucocytozoon, the barriers to host shifting and parasite host range expansion are jointly determined by environmental filtering and landscape, but not by host phylogeny. Our results show that integrating host traits, host ancestry, bioclimatic data and microhabitat characteristics that are important for vector reproduction are imperative to understand and predict infection prevalence and diversity of vector-transmitted parasites. Unlike other vector-transmitted diseases, our results show that Leucocytozoon diversity and prevalence will likely decrease with warming temperatures.

Fipronil and ivermectin treatment of cattle reduced the survival and ovarian development of field-collected Anopheles albimanus in a pilot trial conducted in northern Belize.

BACKGROUND: Most malaria vector control programmes rely on indoor residual spraying of insecticides and insecticide-treated bed nets. This is effective against vector species that feed indoors at night and rest inside the house afterwards. In Central America, malaria vectors have different behaviours and are typically exophagic (i.e., bite outdoors), exophilic (i.e., remain outdoors after feeding), and zoophagic (i.e., as likely to feed on non-humans as humans). Thus, malaria elimination in Central America may require additional tactics. This pilot study investigated whether commercially-available products used to treat livestock for ticks could also be used to kill and/or sterilize zoophagic malaria vectors that feed on treated cattle in Belize. METHODS: Cattle were treated with either a pour-on formulation of 1% fipronil (3 heifers) or injection of 1% ivemectin (1 heifer). Control heifers (n = 2) were left untreated. Field-collected Anopheles albimanus contained in screen-top cages were strapped onto cattle at 2, 5, 7, and 14 days after treatment. Mosquito mortality was monitored once a day for 4 successive days. Surviving mosquitoes were dissected to assess blood meal digestion and ovarian development. RESULTS: A total of 1078 female An. albimanus mosquitoes were fed and monitored for mortality. Both fipronil and ivermectin significantly reduced survivorship of An. albimanus for up to 7 days after treatment. By 14 days, efficacy had declined. The ivermectin treatment completely lost its effectiveness and even though the fipronil-treated heifers were still killing significantly more mosquitoes than the untreated heifers, the amount of mosquito killing had diminished greatly. Both treatments significantly reduced ovary development in mosquitoes fed on treated cattle for the duration of the 2-week trial. CONCLUSIONS: Treatment of cattle in northern Belize with topical fipronil and injectable ivermectin had significant lethal and sublethal effects on wild An. albimanus females. These results suggest that efforts towards eliminating residual transmission of malaria by zoophagic vectors in Central America may benefit by the judicious, targeted treatment of livestock with mosquitocidal compounds, such as fipronil or ivermectin.

Differential susceptibilities of Anopheles albimanus and Anopheles stephensi mosquitoes to ivermectin.

BACKGROUND: Vector control is a crucial element of anti-malaria campaigns and works best when there is a thorough knowledge of the biology and behaviour of the Anopheles vector species responsible for transmitting malaria within a given locale. With the push to eradicate malaria stronger than ever, there is a growing need to develop and deploy control strategies that exploit the behavioural attributes of local vector species. This is especially true in regions where the vectors are exophagic (i.e., prefer to bite outdoors), exophilic (i.e., prefer to remain outdoors), and zoophagic (i.e., as likely to feed on non-humans as humans). One promising strategy targeting vectors with these behavioural traits is the administration of avermectin-based endectocides, such as ivermectin, to humans and livestock. When ingested in a blood meal, ivermectin has been shown to reduce mosquito survivorship and fecundity in a number of Anopheles species. In this study, the relative toxicity of ivermectin was compared between two zoophagic, exophilic malaria vectors-Anopheles albimanus and Anopheles stephensi. RESULTS: Toxicity of ivermectin was assessed using membrane feedings, intrathoracic injections, and mosquito feedings on treated mice. When ingested in a blood meal, ivermectin was much less toxic to An. albimanus (4-day oral LC50 = 1468 ng/ml) than to An. stephensi (4-day oral LC50 = 7 ng/ml). However when injected into the haemocoel of An. albimanus, ivermectin was much more toxic (3-day parenteral LC50 = 188 ng/ml). Because the molecular targets of ivermectin (i.e., glutamate-gated chloride channels) reside outside the midgut in nerves and muscles, this suggests that ingested ivermectin was not readily absorbed across the midgut of An. albimanus. In contrast, ivermectin was considerably more toxic to An. stephensi when ingested (4-day oral LC50 = 7 ng/ml) than when injected (3-day parenteral LC50 = 49 ng/ml). This suggests that metabolic by-products from the digestion of ivermectin may play a role in the oral toxicity of ivermectin to An. stephensi. Blood meal digestion and subsequent oviposition rates were significantly hindered in both species by ingested ivermectin but only at concentrations at or above their respective oral LC50 concentrations. To test mosquitocidal activity of ivermectin in a live host system, two groups of three mice each received subcutaneous injections of either ivermectin (600 µg/kg BW) or saline (control). One day after injection, the ivermectin-treated mice (n = 3) exhibited significant mosquitocidal activity against both An. stephensi (85% mortality vs 0% in control-fed) and, to a lesser degree, An. albimanus (44% mortality vs 11% in control-fed). At 3 days, the mosquitocidal activity of ivermectin-treated mice waned and was effective only against An. stephensi (31% mortality vs 3% in control-fed). CONCLUSIONS: Ivermectin was not uniformly toxic to both Anopheles species. Previous studies indicate that ivermectin is a good choice of endectocide to use against malaria vectors in southeast Asia and Africa. However, these data suggest that ivermectin may not be the optimal endectocide to use in Central America or the Caribbean where An. albimanus is a major malaria vector species. If endectocides are to be used to help eradicate malaria, then additional efficacy data will be needed to define the activity of specific endectocides against the major malaria vector species of the world.

Potential of a Northern Population of Aedes vexans (Diptera: Culicidae) to Transmit Zika Virus.

Zika virus is an emerging arbovirus of humans in the western hemisphere. With its potential spread into new geographical areas, it is important to define the vector competence of native mosquito species. We tested the vector competency of Aedes vexans (Meigen) from the Lake Agassiz Plain of northwestern Minnesota and northeastern North Dakota. Aedes aegypti (L.) was used as a positive control for comparison. Mosquitoes were fed blood containing Zika virus and 2 wk later were tested for viral infection and dissemination. Aedes vexans (n = 60) were susceptible to midgut infection (28% infection rate) but displayed a fairly restrictive midgut escape barrier (3% dissemination rate). Cofed Ae. aegypti (n = 22) displayed significantly higher rates of midgut infection (61%) and dissemination (22%). To test virus transmission, mosquitoes were inoculated with virus and 16-17 d later, tested for their ability to transmit virus into fluid-filled capillary tubes. Unexpectedly, the transmission rate was significantly higher for Ae. vexans (34%, n = 47) than for Ae. aegypti (5%, n = 22). The overall transmission potential for Ae. vexans to transmit Zika virus was 1%. Because of its wide geographic distribution, often extreme abundance, and aggressive human biting activity, Ae. vexans could serve as a potential vector for Zika virus in northern latitudes where the conventional vectors, Ae. aegypti and Ae. albopictus Skuse, cannot survive. However, Zika virus is a primate virus and humans are the only amplifying host species in northern latitudes. To serve as a vector of Zika virus, Ae. vexans must feed repeatedly on humans. Defining the propensity of Ae. vexans to feed repeatedly on humans will be key to understanding its role as a potential vector of Zika virus.

Brugia malayi microfilariae transport alphaviruses across the mosquito midgut.

Concurrent ingestion of microfilariae (MF) and arboviruses by mosquitoes can enhance mosquito transmission of virus compared to when virus is ingested alone. Within hours of being ingested, MF penetrate the mosquito midgut and introduce virus into mosquito hemocoel, creating a disseminated viral infection much sooner than normal. How virus is actually introduced is not known. In this report, we present experimental evidence that suggests that certain alphaviruses may adhere or otherwise associate with sheathed Brugia malayi MF in the blood of a dually-infected host and that the virus is carried into the mosquito hemocoel by the MF during their penetration of the mosquito midgut. The mechanism of MF enhancement may be more complex than simple leakage of viremic blood into the hemocoel during MF penetration. The affinity of arboviruses to adhere to or otherwise associate with MF may depend on the specific combination of the virus and MF involved in a dual host infection. This in turn may determine the relative importance that MF enhancement has within an arbovirus transmission system.

Real-time PCR detection and phylogenetic relationships of Neorickettsia spp. in digeneans from Egypt, Philippines, Thailand, Vietnam and the United States.

Neorickettsia (Rickettsiales, Anaplasmataceae) is a genus of obligate intracellular bacterial endosymbionts of digeneans (Platyhelminthes, Digenea). Some Neorickettsia are able to invade cells of the digenean's vertebrate host and are known to cause diseases of domestic animals, wildlife, and humans. In this study we report the results of screening digenean samples for Neorickettsia collected from bats in Egypt and Mindoro Island, Philippines, snails and fishes from Thailand, and fishes from Vietnam and the USA. Neorickettsia were detected using a real-time PCR protocol targeting a 152bp fragment of the heat shock protein coding gene, GroEL, and verified with nested PCR and sequencing of a 1853bp long region of the GroESL operon and a 1371bp long region of 16S rRNA. Eight unique genotypes of Neorickettsia were obtained from digenean samples. Neorickettsia sp. 8 obtained from Lecithodendrium sp. from Egypt; Neorickettsia sp. 9 and 10 obtained from two species of Paralecithodendrium from Mindoro, Philippines; Neorickettsia sp. 11 from Lecithodendrium sp. and Neorickettsia sp. 4 (previously identified from Saccocoelioides lizae, from China) from Thailand; Neorickettsia sp. 12 from Dicrogaster sp. Florida, USA; Neorickettsia sp. 13 and SF agent from Vietnam. Sequence comparison and phylogenetic analysis demonstrated that the forms, provisionally named Neorickettsia sp. 8-13, represent new genotypes. We have for the first time detected Neorickettsia in a digenean from Egypt (and the African continent as a whole), the Philippines, Thailand and Vietnam based on PCR and sequencing evidence. Our findings suggest that further surveys from the African continent, SE Asia, and island countries are likely to reveal new Neorickettsia lineages as well as new digenean host associations.

LONGITUDINAL EVALUATION OF MALARIA EPIDEMIOLOGY IN AN ISOLATED VILLAGE IN WESTERN THAILAND: I. STUDY SITE AND ADULT ANOPHELINE BIONOMICS.

This is the first in a series of papers describing the epidemiology of malaria in an isolated village in western Thailand. The study site was the village of Kong Mong Tha, located in Sangkhla Buri District, Kanchanaburi Province, Thailand. In this paper we present an overview of the study site and results from our adult anopheline mosquito surveillance conducted over 56 consecutive months from June 1999 until January 2004. The collection site, indoor/outdoor location, parity, biting activity and Plasmodiumfalciparum (Pf) and P. vivax (Pv) infection rates were used to calculate seasonal entomological inoculation rates for the predominant four Anopheles species. A total of 21,566 anophelines representing 28 distinct species and 2 groups that were not identified to species were collected using human bait, with almost 95% of the collection consisting of Anopheles minimus, An. maculatus, An. sawadwongporni and An. barbirostris/campestris. Mosquitoes generally peaked during the wet season, were collected throughout the night, and were collected most often outside (ca. 75%) versus inside (ca. 25%) of houses. Approximately 50% of collected mosquitoes were parous. Overall Plasmodium infection rates were 0.27%, with a total of 16 and 42 pools of Pf- and Pv-positive mosquitoes, respectively. Annual EIRs were 2.3 times higher for Pv than for Pf, resulting in approximately 5.5 and 2.6 infective bites per person per year, respectively. The results suggest An. minimus and An. maculatus are the primary and secondary vectors of Pf and Pv transmission in Kong Mong Tha, while An. sawadwongporni and An. barbirostris/campestris also appear to play a role based on the presence of circumsporozoite protein (CSP) in the head/thorax of the specimens tested.

Germs within Worms: Localization of Neorickettsia sp. within Life Cycle Stages of the Digenean Plagiorchis elegans.

Neorickettsia spp. are bacterial endosymbionts of parasitic flukes (Digenea) that also have the potential to infect and cause disease (e.g., Sennetsu fever) in the vertebrate hosts of the fluke. One of the largest gaps in our knowledge of Neorickettsia biology is the very limited information available regarding the localization of the bacterial endosymbiont within its digenean host. In this study, we used indirect immunofluorescence microscopy to visualize Neorickettsia sp. within several life cycle stages of the digenean Plagiorchis elegans Individual sporocysts, cercariae, metacercariae, and adults of P. elegans naturally infected with Neorickettsia sp. were obtained from our laboratory-maintained life cycle, embedded, sectioned, and prepared for indirect immunofluorescence microscopy using anti-Neorickettsia risticiihorse serum as the primary antibody. Neorickettsiasp. was found within the tegument of sporocysts, throughout cercarial embryos (germ balls) and fully formed cercariae (within the sporocysts), throughout metacercariae, and within the tegument, parenchyma, vitellaria, uteri, testes, cirrus sacs, and eggs of adults. Interestingly, Neorickettsia sp. was not found within the ovarian tissue. This suggests that vertical transmission of Neorickettsia within adult digeneans occurs via the incorporation of infected vitelline cells into the egg rather than direct infection of the ooplasm of the oocyte, as has been described for other bacterial endosymbionts of invertebrates (e.g.,Rickettsia and Wolbachia).

Laboratory maintenance of the bacterial endosymbiont, Neorickettsia sp., through the life cycle of a digenean, Plagiorchis elegans.

The Digenea (Platyhelminthes: Trematoda) are a diverse and complex group of internal metazoan parasites. These parasites can serve as hosts to obligate intracellular bacteria belonging to the genus Neorickettsia (Family: Anaplasmataceae). Neorickettsiae persist within all stages of the fluke life cycle and thus are maintained through vertical transmission. However, the low prevalence of Neorickettsia in nature limits study of their transmission biology at different steps of digenean life cycles. To resolve this dilemma, we have developed for the first time a laboratory model allowing to maintain Neorickettsia sp. through the whole life cycle of a digenean, Plagiorchis elegans. The laboratory life cycle of P. elegans consists of a snail first intermediate host, Lymnaea stagnalis, an aquatic arthropod second intermediate host, Culex pipiens (mosquito larva), and a vertebrate definitive host, Mesocricetus auratus (Syrian hamster). This paper focuses on the development of the laboratory life cycle, as well as outlines its potential uses in studying the transmission biology of Neorickettsia and its evolutionary relationship within its digenean host.

The Western progression of lyme disease: infectious and Nonclonal Borrelia burgdorferi Sensu Lato populations in Grand Forks County, North Dakota.

Scant attention has been paid to Lyme disease, Borrelia burgdorferi, Ixodes scapularis, or reservoirs in eastern North Dakota despite the fact that it borders high-risk counties in Minnesota. Recent reports of B. burgdorferi and I. scapularis in North Dakota, however, prompted a more detailed examination. Spirochetes cultured from the hearts of five rodents trapped in Grand Forks County, ND, were identified as B. burgdorferi sensu lato through sequence analyses of the 16S rRNA gene, the 16S rRNA gene-ileT intergenic spacer region, flaB, ospA, ospC, and p66. OspC typing revealed the presence of groups A, B, E, F, L, and I. Two rodents were concurrently carrying multiple OspC types. Multilocus sequence typing suggested the eastern North Dakota strains are most closely related to those found in neighboring regions of the upper Midwest and Canada. BALB/c mice were infected with B. burgdorferi isolate M3 (OspC group B) by needle inoculation or tick bite. Tibiotarsal joints and ear pinnae were culture positive, and B. burgdorferi M3 was detected by quantitative PCR (qPCR) in the tibiotarsal joints, hearts, and ear pinnae of infected mice. Uninfected larval I. scapularis ticks were able to acquire B. burgdorferi M3 from infected mice; M3 was maintained in I. scapularis during the molt from larva to nymph; and further, M3 was transmitted from infected I. scapularis nymphs to naive mice, as evidenced by cultures and qPCR analyses. These results demonstrate that isolate M3 is capable of disseminated infection by both artificial and natural routes of infection. This study confirms the presence of unique (nonclonal) and infectious B. burgdorferi populations in eastern North Dakota.

Plasmodium falciparum: genetic diversity and complexity of infections in an isolated village in western Thailand.

Genetic diversity of Plasmodium falciparum is intimately associated with morbidity, mortality and malaria control strategies. It is therefore imperative to study genetic makeup and population structure of this parasite in endemic areas. In Kong Mong Tha, an isolated village in western Thailand, the majority of P. falciparum infections are asymptomatic. In this study we investigated complexity of infections and single nucleotide polymorphisms (SNPs) in the P. falciparum population of Kong Mong Tha, and compared results with those previously obtained from Mae Sod, in northwestern Thailand, where the majority of infections were symptomatic. Using PCR-based determination of the 5' merozoite surface protein 1 gene (msp1) recombinant types, we found that 39% of 59 P. falciparum isolates from Kong Mong Tha had multiple 5' recombinant types with a mean number of 1.54. These values were much lower than those obtained from Mae Sod: 96% for multiple infections and with a mean number of 3.61. Analysis of full-length sequences of two housekeeping genes, the P-type Ca(2+)-transporting ATPase gene (n=33) plus adenylosuccinate lyase gene (n=33), and three vaccine candidate antigen genes, msp1 (n=26), the circumsporozoite protein gene, csp (n=30) and the apical membrane antigen 1 gene, ama 1 (n=32), revealed that in all of these genes within-population SNP diversity was at similar levels between Kong Mong Tha and Mae Sod, suggesting that the extent of MOI and clinical manifestations of malaria are not strongly associated with genetic diversity. Additionally, we did not detect significant genetic differentiation between the two parasite populations, as estimated by the Wright's fixation index of inter-population variance in allele frequencies, suggesting that gene flow prevented the formation of population structuring. Thus, this study highlights unique features of P. falciparum populations in Thailand. The implications of these finding are discussed.

Survey of ticks (Acari: Ixodidae) and tick-borne pathogens in North Dakota.

Ticks were sampled at nine locations throughout North Dakota during early summer of 2010, using flagging techniques and small mammals trapping. In total, 1,762 ticks were collected from eight of the nine locations. The dominant species were Dermacentor variabilis (Say) (82%), found throughout the state, and Ixodes scapularis Say (17%), found in northeastern counties. A few nymphal and adult I. scapularis tested positive for Borrelia burgdorferi (3%) and Anaplasma phagocytophilum (8%). This is the first report of I. scapularis and associated pathogens occurring in North Dakota and provides evidence for continued westward expansion of this important vector tick species in the United States.

Molecular identification of vertebrate and hemoparasite DNA within mosquito blood meals from eastern North Dakota.

To understand local transmission of vector-borne diseases, it is important to identify potential vectors, characterize their host feeding patterns, and determine if vector-borne pathogens are circulating within the region. This study simultaneously investigated these aspects of disease transmission by collecting engorged mosquitoes within two rural study sites in the central Red River Valley of North Dakota. Mosquitoes were identified, midguts were excised, and the blood was expelled from the midguts. DNA was extracted from blood meals and subjected to PCR and direct sequencing to identify the vertebrate origin of the blood. Using different primer sets, PCR was used to screen for two types of vector-borne pathogens, filarioid nematodes and hemosporidian parasites. White-tailed deer were the primary source of blood meals for the eight aedine mosquito species collected. None of the 288 deer-derived blood meals contained filarioid or hemosporidian DNA. In contrast, 18 of 32 Culex tarsalis and three of three Cx. pipiens blood meals contained avian blood, representing eight different species of birds. Of 24 avian-derived blood meals examined, 12 contained Plasmodium DNA, three of which also contained Leucocytozoon DNA (i.e., dual infection). Potential confounding effects resulting from parasite acquisition and development from previous blood meals (e.g., oocysts) were eliminated because host blood had been removed from the midguts prior to DNA extraction. Thus, specific parasite lineages/species could be unequivocally linked to specific vertebrate species. By combining mosquito identification with molecular techniques for identifying blood meal source and pathogens, a relatively small sample of engorged mosquitoes yielded important new information about mosquito feeding patterns and hemosporidia infections in birds. Thorough analyses of wild-caught engorged mosquitoes and other arthropods represent a powerful tool in understanding the local transmission of vector-borne and zoonotic diseases.

Transmission rates of the bacterial endosymbiont, Neorickettsia risticii, during the asexual reproduction phase of its digenean host, Plagiorchis elegans, within naturally infected lymnaeid snails.

BACKGROUND: Neorickettsia are obligate intracellular bacterial endosymbionts of digenean parasites present in all lifestages of digeneans. Quantitative information on the transmission of neorickettsial endosymbionts throughout the complex life cycles of digeneans is lacking. This study quantified the transmission of Neorickettsia during the asexual reproductive phase of a digenean parasite, Plagiorchis elegans, developing within naturally parasitized lymnaeid pond snails. METHODS: Lymnaea stagnalis snails were collected from 3 ponds in Nelson County, North Dakota and screened for the presence of digenean cercariae. Cercariae were identified to species by PCR and sequencing of the 28S rRNA gene. Neorickettsia infections were initially detected using nested PCR and sequencing of a partial 16S rRNA gene of pooled cercariae shed from each parasitized snail. Fifty to 100 single cercariae or sporocysts were isolated from each of six parasitized snails and tested for the presence of Neorickettsia using nested PCR to estimate the efficiency at which Neorickettsia were transmitted to cercariae during asexual development of the digenean. RESULTS: A total of 616 L. stagnalis were collected and 240 (39%) shed digenean cercariae. Of these, 18 (8%) were Neorickettsia-positive. Six Neorickettsia infections were selected to determine the transmission efficiency of Neorickettsia from mother to daughter sporocyst and from daughter sporocyst to cercaria. The prevalence of neorickettsiae in cercariae varied from 11 to 91%. The prevalence of neorickettsiae in sporocysts from one snail was 100%. CONCLUSION: Prevalence of Neorickettsia infection in cercariae of Plagiorchis elegans was variable and never reached 100%. Reasons for this are speculative, however, the low prevalence of Neorickettsia observed in some of our samples (11 to 52%) differs from the high prevalence of other, related bacterial endosymbionts, e.g. Wolbachia in Wolbachia-dependent filariid nematodes, where the prevalence among progeny is universally 100%. This suggests that, unlike the Wolbachia-filaria relationship, the Neorickettsia-digenean relationship is not obligatory mutualism. Our study represents the first quantitative estimate of the Neorickettsia transmission through the asexual phase of the digenean life cycle.

Theoretical potential of passerine filariasis to enhance the enzootic transmission of West Nile virus.

Vertebrate reservoirs of arboviruses are often infected with microfilariae (MF). Laboratory studies have shown that MF can enhance the infectivity of arboviruses to mosquitoes. Soon after being ingested, MF penetrate the mosquito midgut. If the host blood also contains virus (i.e., vertebrate is dually infected), penetrating MF may introduce virus into the hemocoel. This can transform otherwise virus-incompetent mosquito species into virus-competent species and simultaneously accelerate viral development, allowing mosquitoes to transmit virus sooner than normal. This phenomenon is termed microfilarial enhancement of arboviral transmission. The prevalence of MF is very high in many passerine populations in North America. Therefore, we investigated if microfilarial enhancement could have facilitated the establishment and rapid spread of West Nile virus (WNV) across the mid-western United States. Our investigations revealed that mosquitoes, WNV, and passerine MF do interact in nature because; 1) 17% of 54 common grackles (Quiscalus quiscula L.), 8% of 26 American robins (Turdus migratorius L.), and 33% of three eastern kingbirds (Tyrannus tyrannus L.) were concurrently microfilaremic and seropositive to WNV; 2) feeding activities of mosquitoes overlapped temporally with the appearance of MF in the blood of common grackles; 3) mosquitoes fed on common grackles and American robins in nature; and 4) mosquito ingestion of two taxonomically distant species of passerine MF (i.e., Chandlerella quiscali and Eufilaria spp.) resulted in penetration of mosquito midguts. To estimate the theoretical effect that MF enhancement could have on WNV transmission in areas of high MF prevalence, vectorial capacity values were calculated for Culex mosquitoes feeding on common grackles, whereby MF enhancement was either invoked or ignored. For Cx. pipiens, vectorial capacity increased over three-fold when potential effects of MF were included in the calculations. For Cx. tarsalis, the effect was less (i.e., 1.4-fold increase). Closer attention should be paid to the potential of MF to enhance mosquito transmission of arboviruses.