Goudarz Molaei

Host Feeding Patterns of Culex Mosquitoes and West Nile Virus Transmission, Northeastern United States

Culex salinarius is a bridge vector to humans, while Cx. pipiens and Cx. restuans are more efficient enzootic vectors.

Vector host-feeding preferences drive transmission of multi-host pathogens: West Nile virus as a model system

Seasonal epizootics of vector-borne pathogens infecting multiple species are ecologically complex and difficult to forecast. Pathogen transmission potential within the host community is determined by the relative abilities of host species to maintain and transmit the pathogen and by ecological factors influencing contact rates between hosts and vectors. Increasing evidence of strong feeding preferences by a number of vectors suggests that the host community experienced by the pathogen may be very different from the local host community. We developed an empirically informed transmission model for West Nile virus (WNV) in four sites using one vector species (Culex pipiens) and preferred and non-preferred avian hosts. We measured strong feeding preferences for American robins (Turdus migratorius) by Cx. pipiens, quantified as the proportion of Cx. pipiens blood meals from robins in relation to their abundance (feeding index). The model accurately predicted WNV prevalence in Cx. pipiens at three of four sites. Sensitivity analysis revealed feeding preference was the most influential parameter on intensity and timing of peak WNV infection in Cx. pipiens and a threshold feeding index for transmission was identified. Our findings indicate host preference-induced contact heterogeneity is a key mediator of vector-borne pathogen epizootics in multi-species host communities, and should be incorporated into multi-host transmission models.

Identification of avian- and mammalian-derived bloodmeals in Aedes vexans and Culiseta melanura (Diptera: Culicidae) and its implication for West Nile virus transmission in Connecticut, U.S.A

Abstract To evaluate the host-feeding patterns of Aedes vexans (Meigen) and Culiseta melanura (Coquillett) as secondary vectors of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) in Northeastern United States, we identified the source of vertebrate bloodmeals by sequencing portions of the cytochrome b gene of mitochondrial DNA. Analysis of polymerase chain reaction products from a total of 119 Ae. vexans revealed that 92.4% of individuals acquired blood solely from mammalian and 2.5% from avian hosts. Mixed bloodmeals from both avian and mammalian hosts were detected in 5% of individuals of this species. Ae. vexans obtained vertebrate bloodmeals most frequently from white-tailed deer (80%) followed by domestic horse, American robin, eastern cottontail, and domestic cat. In contrast, Cs. melanura fed predominantly on avian species (89.6%) but exhibited some inclination for mammalian blood (4.2%). Individual mosquitoes containing mixed bloodmeals were also identified in 6% of Cs. melanura. American robin was the most common source of vertebrate blood for Cs. melanura (23%), followed by wood thrush and gray catbird. American crow represented only 2% of the bloodmeals identified in Cs. melanura, as was similarly found with other recognized Culex vectors of WNV in the northeast. These findings support the view that Ae. vexans is likely to be a relatively important “bridge vector” to large mammals, including deer and horse, whereas Cs. melanura likely plays a secondary role in enzootic transmission of WNV among free-ranging birds in more rural environs.

Genetic variation associated with mammalian feeding in Culex pipiens from a West Nile virus epidemic region in Chicago, Illinois.

Mosquitoes of the Culex pipiens complex are important vectors of West Nile virus in the United States. We examined the genetic variations of Cx. pipiens mosquitoes from Chicago, Illinois that were determined to be principally ornithophilic but exhibited a relatively higher inclination for mammalian hosts including humans. Microsatellite analysis of 10 polymorphic markers was performed on 346 engorged Cx. pipiens specimens with identified avian or mammalian blood meals. Our results indicated that there were no significant differences in allelic richness, the pattern of conformity to Hardy-Weinberg equilibrium, and linkage disequilibrium, nor was there overall genetic differentiation between specimens with avian- and mammalian-derived blood meals. However, Cx. pipiens form pipiens with mammalian- (including human-) derived blood meals had significantly higher ancestry (p < 0.001) and proportion of hybrids (p < 0.01) from the Cx. pipiens form molestus (population from New York City) than did those with avian-derived blood meals. By contrast, there were no significant differences in the ancestry (p > 0.05) and the proportion of hybrids (p > 0.05) from Cx. quinquefasciatus (population from Harris Country, Texas). No temporal genetic variation was detected in accordance with the observation that there was no shift in blood feeding from birds to mammals. The results of this study in conjunction with regional host-feeding behavior suggest that the probability of genetic ancestry from Cx. pipiens f. molestus may predispose mosquitoes to feed more readily on mammals; however, the genetic mechanisms are unknown.

Human bloodfeeding by the recently introduced mosquito, Aedes japonicus japonicus, and public health implications.

Abstract Knowledge of the host-feeding behavior and extent of interactions with human hosts are important in evaluating the role and vector potential of invasive mosquitoes in transmission of native arboviruses. We collected blood-engorged females of the recently established exotic species Aedes japonicus japonicus from sites in New Jersey during 2000 to 2007 and identified the sources of vertebrate blood meals by sequencing portions of the cytochrome b gene of mitochondrial DNA. Over 1/3 (36%, n  =  36) of the engorged mosquitoes acquired blood meals from humans. Other mammalian hosts included white-tailed deer (53%), fallow deer (5%), horse (3%), and Virginia opossum (3%). No avian, amphibian, reptilian, or mixed blood meals were identified. Our detection of a comparatively high prevalence of human bloodfeeding in Ae. j. japonicus in association with its local abundance, vector competence, and repeated detection of West Nile virus from field-collected specimens illustrates the potential for this invasive mosquito to serve as a “bridge” vector in transmission of West Nile and other mosquito-borne viruses in North America.

Reexamination of Culex pipiens Hybridization Zone in the Eastern United States by Ribosomal DNA-Based Single Nucleotide Polymorphism Markers

Mosquitoes in the Culex pipiens complex are important vectors of several disease-causing pathogens, including West Nile virus. In North America, the complex consists of Cx. pipiens pipiens form pipiens, Cx. pipiens pipiens form molestus, Cx. pipiens quinquefasciatus, and their hybrids that exhibit substantial diversity in physiology, behavior, and geographic range. Hybridization among these mosquitoes is of concern because of potential implications for disease transmission. Currently, several morphological and molecular markers exist for differentiating members of the Cx. pipiens complex; however, these markers have specific limitations. We report here two highly reliable ribosomal DNA-based single nucleotide polymorphism (SNP) markers, CxpG2T and CxpA2d, for detecting Cx. pipiens complex mosquitoes containing Cx. p. quinquefasciatus alleles. Both CxpG2T and CxpA2d contain one allele that is present in all members of the Cx. pipiens complex, and the other allele is specific to Cx. p. quinquefasciatus. Testing of field populations from the eastern United States showed that these two SNP markers are capable of identifying a south to north gradient of Cx. p. quinquefasciatus and hybrids. The northern limit of detection of Cx. p. quinquefasciatus alleles in this study was in Fort Totten, NY (40.79°N), whereas the southern boundary was determined between Atlanta, GA (33.81°N) and Gainesville, FL (29.64°N). CxpG2T and CxpA2d were more accurate than the ACE-2 marker, and they may conceivably provide comparable resolution with microsatellite markers for detecting Cx. p. quinquefasciatus alleles.

Avian Communal Roosts as Amplification Foci for West Nile Virus in Urban Areas in Northeastern United States

West Nile virus (WNV) perpetuates in an enzootic transmission cycle involving Culex mosquitoes and virus-competent avian hosts. In the northeastern United States, the enzootic vectors, Cx. pipiens and Cx. restuans, feed preferentially on American robins (Turdus migratorius), suggesting a key role for this bird species in the WNV transmission cycle. We examined the role of American robin communal roosts as virus amplification foci in greater New Haven, Connecticut. Robin communal roosts were located by radio tracking. After mid-August, when most robins were using the roosts, Cx. pipiens and Cx. restuans fed often on robins and were significantly more infected with WNV at communal roosts than at non-roosting sites. We also identified 6.4% human-derived blood meals in Aedes vexans in communal roosts. Our results indicate that communal roosts act as late-season amplification foci facilitating transmission to humans because of high infection rates, high abundance, and feeding patterns of enzootic and bridge vectors.

Vector-Host Interactions and Epizootiology of Eastern Equine Encephalitis Virus in Massachusetts

Eastern equine encephalitis (EEE) virus is a highly pathogenic mosquito-borne zoonosis that is responsible for outbreaks of severe disease in humans and equines, resulting in high mortality or severe neurological impairment in most survivors. In the northeastern United States, EEE virus is maintained in an enzootic cycle involving the ornithophilic mosquito, Culiseta melanura (Coquillett) and passerine birds in freshwater swamp habitats. To evaluate the role of Cs. melanura and Culiseta morsitans (Theobald) in recent episodes of EEE virus activity in Massachusetts, we collected blood-fed mosquitoes between June, 2007, and October, 2008, from virus foci in 6 counties, and identified the source of blood meals by PCR amplification of mitochondrial cytochrome b gene and sequencing. Analysis of 529 Cs. melanura and 25 Cs. morsitans revealed that nearly 99% and 96% of mosquitoes, respectively, acquired blood meals solely from avian hosts. American Robin, Turdus migratorius Linnaeus was identified as the most common vertebrate host for Cs. melanura (21.7%, n=115), followed by Tufted Titmouse, Baeolophus bicolor (L.) (8.7%, n=46), Black-capped Chickadee, Poecile atricapillus (L.) (8.5%, n=45), Scarlet Tanager, Piranga olivacea (Gmelin) (6.8%, n=36), Field Sparrow, Spizella pusilla (Wilson) (6.2%, n=33), Northern Cardinal, Cardinalis cardinalis (L.) (5.7%, n=30), and other mostly Passeriformes birds. Mammalian-derived blood meals were identified as white-tailed deer, Odocoileus virginianus Zimmermann, domestic cow, Bos taurus L., and human, Homo sapiens L. There were 4 isolations of EEE virus, West Nile virus, and Highland J virus from Cs. melanura. Our results in conjunction with other lines of evidence, including reservoir competency, prevalence of antibody, and infection in nature, suggest that the American Robin, Tufted Titmouse, Black-capped Chickadee, and a few other passerine birds may play key roles in supporting EEE virus transmission in Massachusetts. Infrequent blood feeding of Cs. melanura on mammalian hosts, including humans, also indicates that this mosquito may occasionally contribute to epidemic/epizootic transmission of EEE virus in this region.

Identification of bloodmeals in Anopheles quadrimaculatus and Anopheles punctipennis from eastern equine encephalitis virus foci in northeastern U.S.A.

The host‐feeding patterns of Anopheles quadrimaculatus Say and Anopheles punctipennis (Say) were examined in order to evaluate their potential contributions to the transmission of eastern equine encephalitis virus (EEEv) and other arboviruses in the northeastern U.S.A. Engorged mosquitoes of the two species were collected from EEEv foci in central New York (NY) and throughout New Jersey (NJ), and their bloodmeals were identified using a polymerase chain reaction (PCR)‐based assay and sequencing portions of the mitochondrial cytochrome b gene. Analysis of 131 An. quadrimaculatus and 107 An. punctipennis from NY revealed that 97.7% and 97.2%, respectively, had acquired blood solely from mammalian hosts. Similarly, examination of 288 An. quadrimaculatus and 127 An. punctipennis from NJ showed 100% and 96.0%, respectively, contained mammalian‐derived bloodmeals. Mosquitoes containing mixed bloodmeals from both avian and mammalian hosts were detected in 1.6% of An. quadrimaculatus from NY, and 2.8% and 4.0% of An. punctipennis from NY and NJ, respectively. White‐tailed deer (Odocoileus virginianus) constituted the most common vertebrate host for these anopheline mosquitoes, accounting for 85.8–97.7% of all bloodmeals identified. The predominance of white‐tailed deer as a source of bloodmeals supports enzootic amplification of deer‐associated arboviruses in this region, including Jamestown Canyon, Cache Valley and Potosi viruses. One horse‐ and two human‐derived bloodmeals were also detected in An. quadrimaculatus collected in NJ. Limited avian‐derived bloodmeals were detected from mourning dove (Zenaida macroura), sharp‐shinned hawk (Accipiter striatus) and house finch (Carpodacus mexicanus), mostly in mixed bloodmeals. Occasional feeding on avian hosts suggests that these mosquitoes may participate as epizootic–epidemic bridge vectors of EEEv from viraemic birds to mammalian hosts of concern, including horses and humans. An isolate of EEEv was recovered from the head and thorax of an An. punctipennis mosquito collected in NY.

Dynamics of Vector-Host Interactions in Avian Communities in Four Eastern Equine Encephalitis Virus Foci in the Northeastern U.S.

Background Eastern equine encephalitis (EEE) virus (Togaviridae, Alphavirus) is a highly pathogenic mosquito-borne zoonosis that is responsible for occasional outbreaks of severe disease in humans and equines, resulting in high mortality and neurological impairment in most survivors. In the past, human disease outbreaks in the northeastern U.S. have occurred intermittently with no apparent pattern; however, during the last decade we have witnessed recurring annual emergence where EEE virus activity had been historically rare, and expansion into northern New England where the virus had been previously unknown. In the northeastern U.S., EEE virus is maintained in an enzootic cycle involving the ornithophagic mosquito, Culiseta melanura, and wild passerine (perching) birds in freshwater hardwood swamps. However, the identity of key avian species that serve as principal virus reservoir and amplification hosts has not been established. The efficiency with which pathogen transmission occurs within an avian community is largely determined by the relative reservoir competence of each species and by ecological factors that influence contact rates between these avian hosts and mosquito vectors. Methodology and principle findings Contacts between vector mosquitoes and potential avian hosts may be directly quantified by analyzing the blood meal contents of field-collected specimens. We used PCR-based molecular methods and direct sequencing of the mitochondrial cytochrome b gene for profiling of blood meals in Cs. melanura, in an effort to quantify its feeding behavior on specific vertebrate hosts, and to infer epidemiologic implications in four historic EEE virus foci in the northeastern U.S. Avian point count surveys were conducted to determine spatiotemporal host community composition. Of 1,127 blood meals successfully identified to species level, >99% of blood meals were from 65 avian hosts in 27 families and 11 orders, and only seven were from mammalian hosts representing three species. We developed an empirically informed mathematical model for EEE virus transmission using Cs. melanura abundance and preferred and non-preferred avian hosts. To our knowledge this is the first mathematical model for EEE virus, a pathogen with many potential hosts, in the northeastern U.S. We measured strong feeding preferences for a number of avian species based on the proportion of mosquito blood meals identified from these bird species in relation to their observed frequencies. These included: American Robin, Tufted Titmouse, Common Grackle, Wood Thrush, Chipping Sparrow, Black-capped Chickadee, Northern Cardinal, and Warbling Vireo. We found that these bird species, most notably Wood Thrush, play a dominant role in supporting EEE virus amplification. It is also noteworthy that the competence of some of the aforementioned avian species for EEE virus has not been established. Our findings indicate that heterogeneity induced by mosquito host preference, is a key mediator of the epizootic transmission of vector-borne pathogens. Conclusion and significance Detailed knowledge of the vector-host interactions of mosquito populations in nature is essential for evaluating their vectorial capacity and for assessing the role of individual vertebrates as reservoir hosts involved in the maintenance and amplification of zoonotic agents of human diseases. Our study clarifies the host associations of Cs. melanura in four EEE virus foci in the northeastern U.S., identifies vector host preferences as the most important transmission parameter, and quantifies the contribution of preference-induced contact heterogeneity to enzootic transmission. Our study identifies Wood Thrush, American Robin and a few avian species that may serve as superspreaders of EEE virus. Our study elucidates spatiotemporal host species utilization by Cs. melanura in relation to avian host community. This research provides a basis to better understand the involvement of Cs. melanura and avian hosts in the transmission and ecology of EEE virus and the risk of human infection in virus foci.