Identification of mosquito avian-derived blood meals by polymerase chain reaction-heteroduplex analysis

A polymerase chain reaction (PCR) heteroduplex assay (HDA) was developed to identify avian derived mosquito blood meals to the species level. The assay used primers amplifying a fragment of the cytochrome B gene from vertebrate but not invertebrate species. In Culex tarsalis fed on quail, PCR products derived from the quail cytochrome B gene were detected seven days post-engorgement. In an analysis of wild-caught mosquitoes, 85% of blood-fed mosquitoes produced detectable PCR products. Heteroduplex patterns obtained from bird-derived PCR products were found to permit the unambiguous identification of all species examined. No intraspecific variation in HDA patterns was found. The PCR-HDA was used to characterize blood meals in wild caught Cx. tarsalis. Of the 67 blood meals analyzed, 60% were derived from avian sources. Of the avian blood meals, 65% were derived from a single host, the common grackle.     

Molecular identification of blood-meal sources in Culiseta melanura and Culiseta morsitans from an endemic focus of eastern equine encephalitis virus in New York

Eastern equine encephalitis (EEE) virus perpetuates in an enzootic cycle involving ornithophilic mosquito vectors, principally Culiseta melanura (Coquillett) and avian amplification hosts. To better understand the role of Cs. melanura and Culiseta morsitans (Theobald) in the epizootiology of EEE virus, we collected blood-fed mosquitoes between 31 May and 15 October 2004 at two sites associated with an EEE virus focus in central New York and identified the source of vertebrate blood by nucleotide sequencing of polymerase chain reaction (PCR) products of the cytochrome b gene. Analysis of 484 Cs. melanura and 122 Cs. morsitans revealed that 94.2% and 86.9%, respectively, acquired blood solely from avian hosts. Blood meals derived exclusively from mammals were detected in 0.8% of Cs. melanura and 1.6% of Cs. morsitans. Individual mosquitoes containing mixed-blood meals from both avian and mammalian hosts were also detected in 5.0% of Cs. melanura and 11.5% of Cs. morsitans. Wood thrush constituted the most common vertebrate host for Cs. melanura (23.6%) and Cs. morsitans (30.9%), followed by American robin, song sparrow, ovenbird, red-eyed vireo, and common yellowthroat. Mammalian-derived blood meals were identified as white-tailed deer, horse, domestic cat, and eastern pipistrelle bat. There were three isolations of EEE virus from Cs. melanura and one from Cs. morsitans. These results suggest that wood thrush and a few other passerine birds may play key roles in supporting EEE virus transmission in the northeast and possibly throughout the geographic range of EEE in North America. The frequency of mammalian feedings also suggests that Cs. melanura and Cs. morsitans may play a role in the transmission of EEE virus to equines, in addition to maintaining enzootic transmission among avian hosts. We report the first isolation of arboviruses from mosquito vectors concomitant with the identifications of their blood meal sources.     

Avian host preference by vectors of eastern equine encephalomyelitis virus

An important variable in the amplification and escape from the enzootic cycle of the arboviral encephalitides is the degree of contact between avian hosts and mosquito vectors. To analyze this interaction in detail, blood-fed mosquitoes that were confirmed vectors of eastern equine encephalomyelitis (EEE) virus were collected in 2002 from an enzootic site in central Alabama during the time this virus was actively transmitted. Avian-derived blood meals were identified to the species level of the host, and the proportion derived from each species was compared with the overall composition of the avifauna at the study site. The EEE vector mosquito species fed significantly more on some bird species and less on other species than expected given the overall abundance, biomass, or surface area of the local avifauna. When viewed collectively, these data suggest that these mosquitoes are selectively targeting particular avian species.     

Host feeding patterns of Connecticut mosquitoes (Diptera: Culicidae).

Blood-engorged Coquillettidia perturbans, Psorophora ferox, Culex, Culiseta, and Aedes mosquitoes were collected principally by sweep net from salt marsh and woodland habitats in Connecticut. Of the 570 mosquitoes tested, precipitin tests identified the origins of 517 blood meals and revealed distinct host feeding patterns. Aedes mosquitoes fed chiefly on mammals; A. abserratus, A. cantator, and A. vexans showed selectivity for cattle and (or) horses. A. cantator also obtained blood from avian hosts and, in some instances, showed mixed passerine-mammal blood meals. These findings increase the vector potential of this salt marsh mosquito for eastern equine encephalomyelitis virus. Feedings on deer by A. abserratus suggest potential involvement of this mosquito in the transmission of certain subtypes of California encephalitis. Culex-pipiens, C. restuans, Culiseta melanura, and Cs. morsitans dyari acquired blood almost exclusively from passeriform birds.     

Virus-vector-host relationships of Aedes stimulans and Jamestown Canyon virus in a northern Indiana enzootic focus

Collections of hematophagous Diptera at the Kingsbury State Fish and Wildlife Area in northern Indiana between 1982 and 1984 yielded 118,972 mosquitoes from which 5 isolates of Jamestown Canyon virus and 3 isolates of trivittatus virus were obtained. All Jamestown Canyon isolates were from Aedes stimulans, including 1 from a pool of newly emerged males and 2 from pools of newly emerged females. These 3 isolates suggest that Jamestown Canyon virus is transovarially transmitted by Ae. stimulans. All isolates of trivittatus virus came from pools of Ae. trivittatus. No isolates were obtained from greater than 4,000 tabanids collected along with the mosquitoes those years. Transmission trials with field-collected newly emerged adult female Ae. stimulans demonstrated a mean midgut infection rate of 44%, a disseminated infection rate of 16%, and an oral transmission rate of 12% to suckling mice. Precipitin tests of field-collected bloodfed female mosquitoes indicated that white-tailed deer were the preferred host for numerous mosquito species including Ae. stimulans. The results of this study suggest that Ae. stimulans is a primary vector of Jamestown Canyon virus and that transovarial transmission is the probable overwintering mechanism for this California group virus.     

Nutritional stress affects mosquito survival and vector competence for West Nile virus

Most anautogenous female mosquitoes ingest plant carbohydrates for flight energy and survival, and they imbibe vertebrate blood for egg development. We evaluated the effect of different sucrose meals following a blood meal containing West Nile virus (WNV) on Culex pipiens pipiens survival, nutritional status, and susceptibility to viral infection and transmission. Ten days after blood feeding, no mosquitoes survived on distilled water, 55% survived on 2% sucrose, 61% on 10 and 20% sucrose meals, and over 70% survived on 40% sucrose. There was a positive correlation between sucrose meal concentration and detectable sugars, glycogen, and lipid in whole-body homogenates. Average sugar values increased from 0 microg per starved mosquito (range 0-1.0 microg) to an average of 392 microg per mosquito fed on 40% sucrose (85-1088 microg). Average glycogen values increased from 0 microg (0-5.7 microg) to an average of 620 microg (118-1421 microg). Average lipid values were identical for mosquitoes in the starved and 2% sucrose series (38 microg) and increased to 172 microg per mosquito fed on 40% sucrose (92-266 microg). Mosquitoes in all sucrose series were equally susceptible to WNV infection (p > 0.5), but mosquitoes with lower nutrient reserves as a result of lower sucrose meals were more likely to orally transmit virus (p < 0.05). We discuss how mosquito nutritional status influences probability of daily survival, susceptibility to infection, and vectorial capacity. We conclude that maintaining C. p. pipiens on standard 10% sucrose is justified in light of these results.     

Susceptibility of Ochlerotatus trivittatus (Coq.), Aedes albopictus (Skuse), and Culex pipiens (L.) to West Nile virus infection

The susceptibility of Ochlerotatus trivittatus (Coq.) to West Nile virus (WNV) was assessed by comparing it to the susceptibility of Aedes albopictus (Skuse), a likely bridge vector, and Culex pipiens (L.), a primary WNV amplifying species. The three species were infected with WNV (NY crow-1999) by feeding on 2-3-day-old chickens with serum virus titers ranging from 10(2.5) to 10(9.5) cell culture infective dose (CID) 50s/mL. The lowest infective titer for Oc. trivittatus and Cx. pipiens was 10(4.5) CID50s/mL. Thirteen percent (4/32) and 2% (1/45) of each species became infected postprandially. Infection rates of the two species increased to 43% (6/14) and 15% (6/40) after blood meals with a titer of 10(5.5) CID50s/mL. In contrast no infection was observed in nine Ae. albopictus that fed among three chickens with titers of 10(4.5) CID50s/mL nor in 41 Ae. albopictus that fed among three chickens with titers of 10(5.0) CID50s/mL. The infective dose 50s for Oc. trivittatus, Cx. pipiens and Ae. albopictus were 10(6.0), 10(6.2), and 10(6.6) CID50s/mL, respectively. Collectively these observations suggest that Oc. trivittatus and Cx. pipiens are more susceptible than Ae. albopictus to WNV when they feed on hosts with WNV titers of <10(7.5) CID50s/mL, but nearly as susceptible with blood meal titers of > or =10(7.5) CID50s/mL. Unpublished studies in our laboratory showed that cottontail rabbits fed on by WNV-infected Oc. trivittatus developed viremias as high as 10(5.5) CID50s/mL serum which exceeds 10 (4.2 (3.4-4.6)) CID50s/mL, the predicted ID10+/-95% CI of Oc. trivittatus. Consequently this mosquito, which also feeds on humans and birds has the potential to serve as a bridge vector and as a maintenance vector among mammals.     

Temporal analysis of feeding patterns of Culex erraticus in central Alabama

Host blood meals in seven mosquito species previously shown to be infected with eastern equine encephalitis virus at a site in the Tuskegee National Forest in southcentral Alabama were investigated. Of 1374 blood meals derived from 88 different host species collected over 6 years from these seven mosquito species, 1099 were derived from Culex erraticus. Analysis of the temporal pattern of Cx. erraticus meals using a Runs test revealed that the patterns of feeding upon avian and mammalian hosts from March to September of each year were not randomly distributed over time. Similarly, meals taken from the three most commonly targeted host species (yellow-crowned night heron, great blue heron, and white-tailed deer) were not randomly distributed. A Tukey's two-way analysis of variance test demonstrated that although the temporal pattern of meals taken from avian hosts were consistent over the years, the patterns of meals taken from the individual host species were not consistent from year to year.     

Detection of host virus-reactive antibodies in blood meals of naturally engorged mosquitoes

Although serosurvey in human or animals is a useful and straightforward strategy routinely used for public health, it often faces different types of impediments: ethics, beliefs, limitation by animal owners, hazard of access to wild animals. To survey virus circulation, we applied the enzyme-linked immunosorbent assay (ELISA) technique to detect Dengue and Japanese encephalitis (JE) virus-reactive antibodies in blood meals collected from mosquitoes without regard to the potential of mosquito species to be a virus vector. ELISA was performed on mosquito colonies and wild specimens collected from farms and urban areas. Blood meals from Aedes aegypti freshly fed on naturally infected volunteers showed the same levels of dengue immunoglobulin (Ig)G and IgM as the sera directly collected from volunteers. A significant clearance of antibodies during the digestion process started from 13 hours after blood meal, and a negative baseline was reached after 30 hours. The ELISA test performed on wild mosquitoes showed that 37% of Culex quinquefasciatus mosquitoes that engorged on humans in a dengue urban endemic area tested positive for dengue IgG, and in a JE virus-endemic area, 88% of Culex tritaeniorhynchus mosquitoes that engorged on pigs from a large pig farm tested positive for JE virus antibodies versus 11% in a small farm. The main limitation of the ELISA method is the antibody cross-reactivity among flaviviruses; also, sampling strategy should be adjusted to take into account that the actual host from which the blood meal was taken may not be determined. Nevertheless, ELISA performed on recently (1-2 days) engorged mosquito, or any other hematophagous arthropod species, could potentially be used as a "wild phlebotomist" to monitor the prevalence or emergence of a variety of pathogens, with less of the practical, ethical, or risk limitations due to direct blood collection from humans and wild or domestic animals.     

Experimental transmission of trivittatus virus (California virus group) by Aedes trivittatus.

The mosquito, Aedes trivittatus, when fed through a membrane a trivittatus virus dosage of 10(3.4) to 10(5.5) suckling mouse LD50/0.03 ml of blood, transmitted the virus to suckling mice. Virus multiplication indicative of a biological vector occurred in this species. When Aedes vexans and A. triseriatus ingested similar doses of trivittatus virus, both the infection and transmission rates were low and virus multiplication was poor. These results, added to evidence based on virus isolations from mosqito populations in nature, indicate that A. trivittatus is the primary vector of trivittatus virus in the north central United States.     

Host feeding patterns of established and potential mosquito vectors of West Nile virus in the eastern United States

An important variable in determining the vectorial capacity of mosquito species for arthropod-borne infections is the degree of contact of the vector and the vertebrate reservoir. This parameter can be estimated by examining the host-feeding habits of vectors. Serological and polymerase chain reaction based methods have been used to study the host-feedings patterns of 21 mosquito species from New York, New Jersey, and Tennessee, 19 of which previously have been found infected with West Nile virus. Mammalophilic mosquito species in New Jersey and New York fed primarily upon white-tailed deer, while those from Memphis, Tennessee, fed mainly upon domestic dogs. A total of 24 different avian host species were detected among the avian-derived blood meals. American Robin, Northern Cardinal, Northern Mockingbird, Tufted Titmouse, and Brown-headed Cowbird were common avian hosts, while blood meals derived from the American Crow were relatively rare. Although the majority of common host species were potentially among the most abundant birds at each location, the proportion of blood meals from the most commonly fed upon avian species was greater than was predicted based upon the likely abundance of these species alone. These findings suggest that vector species for West Nile virus may preferentially feed upon certain avian hosts.     

A comparision of West Nile Virus transmission by Ochlerotatus trivittatus (COQ.), Culex pipiens (L.), and Aedes albopictus (Skuse)

Transmission of West Nile virus (WNV) by Ochlerotatus trivittatus, Culex pipiens, and Aedes albopictus were compared 14 days after taking blood meals from viremic chickens with titers ranging from 10(2.5) to 10(9.5) cell infective dose (50)s (CID50s)/mL serum. Transmission occurred in one of four (25%) Oc. trivittatus and one of 25 (4%) Cx. pipiens that fed on chickens with titers of 10(5.5) CID50s/mL. No transmission occurred among two of 16 (13%) Oc. trivittatus or one of 25 (4%) Cx. pipiens that became infected after blood meals with titers of 10(5.0) and 10(4.5) CID50s/mL, the next lowest blood meal titers evaluated. Seventeen of 28 (61%) Ae. albopictus transmitted WNV after blood meals with titers of 10(7.0) CID50s/mL, but no infection or transmission was observed among 21 Ae. albopictus that fed on chickens with titers of 10(5.0) CID50s/mL, the next lowest titer evaluated. Transmission by all three species increased dramatically after blood meals with WNV titers of > or = 10(5.5) CID50s/mL. No significant differences occurred in dissemination and transmission rates of the three species after taking blood meals with titers of > 10(7.0) CID50s/mL. The cumulative mean +/- SE transmission rates of Oc. trivittatus, Cx. pipiens, and Ae. albopictus after blood meals with titers of > or = 10(7.0) CID50s/mL were 45.5 +/- 4.1%, 46.8 +/- 4.5%, and 72.4 +/- 5.5%. The cumulative mean dissemination rates of the three species were 78.3 +/- 6.7%, 74.8 +/- 2.6%, and 88.6 +/- 2.1%. The rates of transmission by the three species that developed disseminated infections after blood meals with titers of > or = 10(7.0) CID50s/mL were 58.8 +/- 4.4%, 62.6 +/- 5.8%, and 81.6 +/- 5.4%, respectively. In a previous study, we found that susceptibility of the three species to WNV was essentially the same when fed on chickens with WNV titers of > 10(7.0) CID50s/mL, but Oc. trivittatus and Cx. pipiens were more susceptible than Ae. albopictus to WNV at lower virus titers. The current study strongly suggests that Ae. albopictus is a more efficient vector than Oc. trivittatus and Cx. pipiens when fed blood meals with titers of > 10(7.0) CID50s/mL. However, Oc. trivittatus and Cx. pipiens might be more efficient as vectors when infected by blood meals with titers of < 10(7.0) CID50s/mL.     

三带喙库蚊和致倦库蚊经口感染乙型脑炎病毒疫苗SA14-14-2株的研究

目的通过实验，确定被SA14—14—2乙脑减毒活疫苗免疫的宿主动物和人在被媒介蚊虫叮咬后，是否存在感染和传播的可能。方法建立三带喙库蚊和致倦库蚊的实验室种群，用乙型脑炎SA14—14—2疫苗株病毒和乙脑野毒株经口感染两种库蚊，感染后不同时间取一定数量的蚊，研磨制成悬液，应用空斑试验方法检测感染后不同时间蚊体内的病毒存在情况和病毒滴度。结果在用6．0610gPFU／ml SA14～14—2病毒经口感染的两种库蚊中，没有检测到病毒空斑，即没发现蚊虫的感染；在用较高的6．1810gPFU／ml病毒经口感染的三带喙库蚊和致倦库蚊中，分别有一组蚊虫出现低滴度感染，空斑形成单位分别是1．24和1．11log10PFU／ml；在用野毒株经口感染的两种库蚊，共计19组蚊虫中，有14组发生感染，空斑形成单位在3．18～4．7910g10PFU／ml。结论作为乙脑主要传播媒介的三带喙库蚊和致倦库蚊对SA14—14—2疫苗株病毒的经口感染和病毒在体内的复制严重受限，当叮咬接种疫苗的人后，不具备发生乙脑病毒感染和传播的能力。     

Pathologic changes in the midgut of Culex tarsalis following infection with Western equine encephalomyelitis virus.

Midguts of two strains of the mosquito vector Culex tarsalis were examined by light and electron microscopy following infection with western equine encephalomyelitis virus. Infection of the highly susceptible Knight's Landing strain with high-titered blood meals resulted in pathologic changes in the midgut epithelium after 2-4 days of incubation; lesions included sloughing of epithelial cells into the lumen and necrosis of cells in situ. Infection of Knight's Landing strain mosquitoes with low-titered blood meals and infection of the less susceptible Fort Collins strain with high-titered blood meals did not result in a significant increase in detached luminal cells, with respect to uninfected controls. Sloughing of infected cells into the midgut lumen may contribute to modulation of the mosquito infection. Lesions in the midgut of Cx. tarsalis are inconsistent with traditional views that regarded arbovirus infections of mosquito vectors as non-pathologic. These findings demonstrate that mosquito pathology is not an oddity limited to the previously described interaction between Culiseta melanura and eastern equine encephalomyelitis virus, and suggest that alphaviruses in general may adversely affect their mosquito vectors in nature.     

Host Selection of Potential West Nile Virus Vectors in Puerto Barrios,Guatemala, 2007

The selection of vertebrate hosts by Culex mosquitoes relative to West Nile virus (WNV) transmission in neotropical countries such as Guatemala is not described. This study determined the feeding patterns of Cx. quinquefasciatus and Cx. nigripalpus and estimated the relative contribution of two common and frequently infected wild bird species, Turdus grayi and Quiscalus mexicanus, to WNV transmission. Engorged mosquitoes were collected from rural and urban habitats after the dry and wet seasons in the Department of Izabal in 2007. Host selection by Cx. nigripalpus varied significantly between urban and rural habitats. Both Cx. quinquefasciatus and Cx. nigripalpus fed predominantly on chickens and other domestic animals. Blood meals from wild birds were rare, accounting for 1.1% of blood meals identified from Cx. quinquefasciatus and 6.5% of blood meals from Cx. nigripalpus. Transmission of WNV by these two mosquito species may be dampened by extensive feeding on reservoir-incompetent hosts.     

Genetic influences on mosquito feeding behavior and the emergence of zoonotic pathogens

The feeding behavior of vectors influences the likelihood of pathogen invasion and the exposure of humans to vector-borne zoonotic pathogens. We used multilocus microsatellite genetic typing of an introduced mosquito vector and DNA sequencing of mosquito blood meals to determine the impact of hybrid ancestry on feeding behavior and the emergence of West Nile virus (WNV). The probability of ancestry of Culex pipiens mosquitoes from two bionomically divergent forms, form molestus and form pipiens, influenced the probability that they fed on humans but did not explain a late summer feeding shift from birds to humans. We used a simple model to show that the occurrence of pure form molestus mosquitoes would have decreased the likelihood of WNV invasion (R(0) in bird populations) 3- to 8-fold, whereas the occurrence of pure forms pipiens mosquitoes would have halved human exposure compared with the hybrids that are present. Data and modeling suggest that feeding preferences may be influenced by genetic ancestry and contribute to the emergence of vector-borne pathogens transmitted by introduced species, including malaria, and dengue, Chikungunya, yellow fever, and West Nile viruses.     

Identification of mosquito blood meals by enzyme-linked immunosorbent assay

A micro enzyme-linked immunosorbent assay for identifying mosquito blood meals is described. Antisera for the assay were prepared in rabbits and guinea pigs and purified by precipitation with cross-reacting blood sera. Positive blood source identifications could be made to the generic level using as little as 58 micrograms of ingested blood serum. The blood meals of 37 of 38 laboratory mosquitoes fed on seven host animals were correctly identified, including a mosquito that contained a double feed.     

Nonvascular delivery of Rift Valley fever virus by infected mosquitoes.

To determine whether virus-transmitting mosquitoes inoculate infectious particles extravascularly or directly into the vascular system, we permitted mosquitoes infected with Rift Valley fever virus to feed on the distal third of the tails of suckling mice. Amputation of the distal half of the tail within 5 min after their being bitten significantly increased mouse survival as compared with that of mice whose tails remained intact. Even when tails were amputated 10 or more min after mosquito feeding, the median time to death was significantly longer in the group with the amputated tails (53.5 hr) than in those mice with intact tails (46.0 hr). Mouse survival did not correlate with ingestion of blood by the infecting mosquito. We conclude that mosquitoes inoculate virus extravascularly, rather than directly into the vascular system, when feeding on a vertebrate host. Such extravascular delivery of virus by a transmitting mosquito may affect viral pathogenesis, antiviral activity, and vaccine efficacy.     

Host feeding pattern of Culex quinquefasciatus (Diptera: Culicidae) and its role in transmission of West Nile virus in Harris County, Texas

The vertebrate hosts of 672 blood-engorged Culex quinquefasciatus Say, collected in Harris County, Texas, during 2005, were identified by nucleotide sequencing PCR products of the cytochrome b gene. Analysis revealed that 39.1% had acquired blood from birds, 52.5% from mammals, and 8.3% were mixed avian and mammalian blood meals. Most frequent vertebrate hosts were dog (41.0%), mourning dove (18.3%), domestic cat (8.8%), white-winged dove (4.3%), house sparrow (3.2%), house finch (3.0%), gray catbird (3.0%), and American robin (2.5%). Results are interpreted in conjunction with concurrent avian and mosquito West Nile virus (WNV) surveillance activities in Harris County. We conclude that Cx. quinquefasciatus is an opportunistic feeder and principal mosquito vector of WNV in this metropolitan area; however, transmission by other mosquito species or by other modes of infection, such as ingestion, must account for the high WNV infection rates among local blue jays and American crows.