Flies caught in homes in a rural village in Guerrero, Mexico, between November 1994 and August 1995 were assessed for their role in the transmission ofTaenia soliumL. Most (99%) of the trapped flies wereMusca domesticaL. None of the 1,187 guts or 1,080 legs of the flies containedT. soliumeggs. Pigs roam freely in this village consuming human fecal material immediately after defecation, thereby limiting fly contact withT. soliumeggs.

We modified polymerase chain reaction (PCR)-based forensic DNA profiling for field studies on the feeding behavior ofAedes aegypti,the principal mosquito vector of dengue virus. Human DNA was extracted from oral swabs of human subjects and from blood-engorged mosquitoes, DNA was quantified by slot blot, and alleles at variable number tandem repeats and three short tandem repeats loci were amplified by PCR. Alleles were separated electrophoretically and then visualized by silver staining. A custom software program was written to match DNA fingerprints of potential human hosts to allelic profiles detected in engorged mosquitoes, and to calculate error rates for identification of human hosts of single and multiple-host blood meals. At 29°C in the laboratory, human DNA recovered from mosquito blood meals declined an average of 67% 8 h after feeding and 90% after 24 h. We obtained complete allelic profiles from seven of 10 mosquitoes collected after 24 h. In a field trial, complete DNA profiles were obtained successfully for 43 people living in a rural village in south central Thailand and for 20 of 100Ae. aegyptithat contained blood and were collected in those peoples’ homes. Blood imbibed from more than one person was detected in 45% (9 of 20) of the meals. Sixty-five percent of the meals contained blood from nonresidents of the house in which the mosquito was collected or from people who were not profiled; data consistent with the hypothesis that human movement is important for the spread of dengue virus within and among communities. When using alleles at four loci, all of the Thais and nine members spanning three generations of a Chinese-American family had unique allelic profiles. Error rates from classifying possible multiple-host meals as single-host meals were low (1–8%), with the highest error associated with closely related people. Results from our laboratory and field studies indicated that DNA profiling can be used to study the details and epidemiological implications ofAe. aegyptiblood-feeding behavior.

The activity of pyriproxyfen in the blood diet was investigated for its efficacy against adult, egg, and larval stages of the cat flea,Ctenocephalides felis(Bouché). Adult fleas were housed in plastic cages and fed treated bovine blood using an artificial membrane system that allows fleas to feed ad libitum through a parafilm membrane. Control fleas received blood without pyriproxyfen. Results showed that ingested pyriproxyfen was relatively nontoxic to adult fleas over a period of 10 d at concentrations as high as 100 ppm. These findings are in sharp contrast to earlier studies that showed that residues of pyriproxyfen on filter paper or dog hair were highly toxic to adult cat fleas at concentrations as low as 12.5 ppm. Fleas obviously fed on blood containing pyriproxyfen because they produced large numbers of eggs. However, none of the eggs hatched. Also, larvae of untreated fleas failed to develop to adults when they were fed fecal blood excreted by pyriproxyfen-treated fleas. The results indicate that although ingested pyriproxyfen was relatively nontoxic to adult fleas, enough chemical was absorbed through the gut wall to cause ovisterilant activity, while the remainder was excreted.

Temporal and spatial changes in the enzootic activity of western equine encephalomyelitis (WEE) and St. Louis encephalitis (SLE) viruses were monitored at representative wetland study sites in the Coachella, San Joaquin, and Sacramento valleys of California from 1996 to 1998 using three methods: (1) virus isolation from pools of 50 host-seekingCulex tarsalisCoquillett females, (2) seroconversions in flocks of 10 sentinel chickens, and (3) seroprevalence in wild birds collected by mist nets and grain baited traps. Overall, 74 WEE and one SLE isolates were obtained from 222,455Cx. tarsalisfemales tested in 4,988 pools. In addition, 133 and 40 seroconversions were detected in 28 chicken flocks, and 143 and 27 of 20,192 sera tested from 149 species of wild birds were positive for antibodies to WEE and SLE, respectively. WEE was active in all three valleys, whereas SLE only was detected in Coachella Valley. Seroconversions in sentinel chickens provided the most sensitive indication of enzootic activity and were correlated with seroprevalence rates in wild birds. Avian seroprevalence rates did not provide an early warning of pending enzootic activity in chickens, because positive sera from after hatching year birds collected during spring most probably were the result of infections acquired during the previous season. Few seroconversions were detected among banded recaptured birds collected during spring and early summer. Age and resident status, but not sex, were significant risk factors for wild bird infection, with the highest seroprevalence rates among after hatching year individuals of permanent resident species. Migrants (with the exception of mourning doves) and winter resident species rarely were positive. House finches, house sparrows, Gambel’s quail, California quail, common ground doves, and mourning doves were most frequently positive for antibodies. The initial detection of enzootic activity each summer coincided closely with the appearance of hatching year birds of these species in our study areas, perhaps indicating their role in virus amplification. Bird species most frequently positive roosted or nested in elevated upland vegetation, sites whereCx. tarsalishost-seeking females hunt most frequently. These serosurveys provided important background information for planned host competence and chronic infection studies.

A recombinant single-chain variable-region human antibody fragment (scFv) was expressed inEscherichia coli,extracted in hypertonic sucrose, mixed directly with blood and fed toAnopheles gambiaeGiles mosquitoes. WhenE. colicontaining the phagemids that encode these scFv were included in bloodmeals, phagemids could be recovered from the mosquito midgut for up to 3 d after feeding. Furthermore, large arrays of such gene-tagged scFv-containing bloodmeals could be fed to cages of mosquitoes using microtiter plates. Arrays of phagemids with and without an antibody insert were fed to single cages of mosquitoes to test whether individual mosquitoes fed from single wells of such arrays. Phagemids were recovered from 95% of blood-fed females and >80% of these phagemids were monoclonal. Therefore, it is possible to feed multiple sample arrays of recombinant proteins to single cages of mosquitoes and to recover the genetic material that encodes for only one of the array elements from individual mosquitoes. This demonstration indicates that multiple-sample feeding and recovery strategies are feasible and may represent a viable strategy for future rapid screening of biologically active genes, gene products or microorganisms in live arthropods.

A previously untreated field population ofCulex quinquefasciatusSay, collected near Bakersfield, CA, was subjected to intensive laboratory selection with the bacterial insecticideBacillus sphaericusNeide (strain 2362) at a level producing 95% mortality. Resistance rapidly appeared and resistance levels increased such that fourth instars of generation 12 were able to survive a concentration ofB. sphaericusthat was 7,000 times higher than the median lethal concentration (LC₅₀) of the susceptible reference colony. Similar resistance levels were detected in first instars. Cross-resistance in the selected colony was detected towardB. sphaericusstrains 1593 and 2297, but little or no cross-resistance was observed towardB. sphaericusstrains IAB59 or ISPC5 (=WHO 2173). Cross-resistance also was not detected toward the bacterial insecticideBacillus thuringiensissubsp.israelensis,toward a recombinant strain expressing bothB. thuringiensissubsp.israelensisandB. sphaericus(strain 1593) toxins, toward individual or multiple toxins fromB. thuringiensissubsp.israelensis,or toward conventional synthetic insecticides. Genetic analysis revealed thatB. sphaericusresistance was inherited as a recessive trait and controlled by a single major locus. These data are discussed in relation to cases of field resistance toward this biopesticide in theCx. pipiens(L.) complex.

The resting behavior ofAedes aegypti(L.) adults was investigated in 14 districts of Panama City, Panama, in relation to ground ultra-low volume (ULV) applications of malathion. Adults primarily rested inside the premises (75.1%) at a distance >6 m from the street (86.4%). Both sexes rested mainly in bedrooms, living rooms, and bathrooms. The small ULV aerosol droplets (2–4 μm) penetrated all indoor resting sites ofAe. aegypti,but in low amounts. Because of the low amount of insecticide reaching the primary resting sites within the premises, limited control of theAe. aegyptiwas obtained. This limited the potential effectiveness of ground vehicle ULV applied insecticide as a dengue epidemic control method.

The northernmost focus forOnchocerca volvulusLeuckhart (Nematoda: Onchocercidae), the causative agent of human onchocerciasis, is found along the Nile near the town of Abu Hamed in Sudan. The vector forO. volvulusat this focus is a single monomorphic population ofSimulium (Edwardsellum) damnosumTheobald. This black fly population is limited to a small area between the fourth and fifth cataracts of the Nile River that is isolated geographically from all other populations ofS. damnosumsensu lato. Phylogenies produced from cytological analyses and sequence data derived from the NADH dehydrogenase subunit 4 and 16S rRNA genes indicate that Abu Hamed black flies are similar to, but distinct from, the savanna-dwelling sibling species ofS. damnosums.l.,Simulium (Edwardsellum) damnosumsensu strictu Theobald, andS. (Edwardsellum) sirbanumVajime & Dunbar. The DNA sequence and the cytological data support the hypothesis that the black fly population present in Abu Hamed may represent a new sibling species ofS. damnosums.l. We propose that this population be informally designated as the hamedense form of theSimulium damnosumcomplex.

TheCulex vishnuisubgroup includes three important vectors of Japanese encephalitis virus,Culex tritaeniorhynchusGiles,Cx. pseudovishnuiColless, andCx. vishnuiTheobald, all of which occur in the Ryukyu Archipelago, Japan. Although these three species have been shown to be vectors of JE virus in many areas of Southeast Asia, it is not yet known what role each plays in the transmission of the virus in this region. Reliable identification of adult, field-collected specimens is a critical component in epidemiological studies of virus transmission. Mosquitoes in theCx. vishnuisubgroup can be reliably identified in the larval stage. However, because females of these species are very similar, it is difficult to distinguish among them using morphology. We developed a polymerase chain reaction (PCR) assay for the identification of these species. Three species-specific primers were developed for the PCR assay based on a comparative analysis of the nucleotide sequence of the first internal transcribed spacer (ITS1) in the ribosomal DNA gene array. The primers, CT2REV, CP1REV, and CV1REV were designed to amplify a single DNA fragment each fromCx. tritaeniorhynchus,Cx. pseudovishnui,andCx. vishnui,respectively, when paired with a single forward primer that is complementary to the highly conserved 18S rDNA gene. The amplified fragments were separated easily and identified on an agarose gel to facilitate species identification.

Aedes triseriatus(Say) population density patterns and La Crosse encephalitis virus infection rates were evaluated in relation to a variety of habitat parameters over a 14-wk period. Ovitraps and landing collections were used in a La Crosse virus-enzootic area in Nicholas County, WV. Study sites were divided into categories by habitat type and by proximity to the residences of known La Crosse encephalitis cases. Results demonstrated thatAe. triseriatuspopulation densities were higher in sugar maple/red maple habitats than in hemlock/mixed hardwood habitats or in a site characterized by a large number of small red maple trees. Sites containing artificial containers had higher population densities than those without. La Crosse virus minimum infection rates in mosquitoes collected as eggs ranged from 0.4/1,000 to 7.5/1,000 in the 12 study sites, but did not differ significantly among sites regardless of habitat type or proximity to human case residences. La Crosse virus infection rates in landingAe. triseriatusmosquitoes ranged from 0.0/1,000 to 27.0/1,000. La Crosse virus was also isolated from host-seekingAe. canadensis(Theobald) in two study sites, at rates similar to those found in theAe. triseriatuspopulations. TheAe. triseriatusoviposition patterns and La Crosse virus infection rates suggest that this mosquito species disperses readily in the large woodlands of central West Virginia. The La Crosse enzootic habitats in Nicholas County, WV, are contrasted with those studied in other geographic regions where La Crosse virus is found.

Larvae of the cat flea,Ctenocephalides felis(Bouché), are the target of numerous growth regulators. This study explores the development of an assay that tests the susceptibility of cat flea larvae to a wide range of compounds. Different rearing media and containment units were tested that would facilitate optimization. Larvae of various ages were compared, and 7-d-old larvae were found to be optimal because they were the most uniform in size and age and exhibited a need to feed. The assay could be used to distinguish insecticides from growth inhibitors. The insecticides chlorpyrifos and carbaryl caused 100% larval mortality in 24 h at 10 ppm, and cythioate and fipronil killed the larvae at concentrations of ≥100 ppm within 24 h. The insect growth regulators methoprene and pyriproxifen caused molt delay at concentrations of 100 ppm and bioallethrin delayed molt at 1,000 ppm. This assay can be used to identify compounds that are specific to cat flea larvae that may be useful in the control of cat flea infestations.

The mouthparts and antennae of the fourth-instar larvae of four sand fly species were studied using scanning electron microscopy. The morphology of the clypeus, labrum, mandible, maxilla, mentum, and antennae were compared forPhlebotomus argentipesAnnandale & Brunetti,P. papatasin(Scopoli),Sergentomyia babu babu(Annandale), andS. bailyi(Sinton). Most of structures exhibited species-specific features, particularly the characteristics of the antennae.P. papatasinlarvae had heart-shaped antennae, a long mandible, stout maxilla, and a heavy mentum with large teeth. In contrast,P. argentipeslarvae had dumbbell-shaped antennae and a singular club-shaped labrum. The antennae ofS. b. babuwere ovoid, whereas those ofS. bailyiwere elliptical. The labrum ofS. b. babuwas lanceolate, whereas that ofS. bailyiwas rounded and exhibited a small, thick projection with several folds. The teeth of the mentum of bothSergentomyiaspecies were shorter than those of thePhlebotomusspecies. Species-specific differences in the morphology of larval mouthparts and antennae indicate that it may not always be necessary to rely on adult morphology to identify sympatric phlebotomines.

We developed a nitrocellulose-based, dipstick circumsporozoite (CS)-enzyme immunoassay [ELISA] for the simultaneous detection ofPlasmodium falciparumandP. vivax−210 CS protein. The assay had a detection threshold of <250P. falciparumor 400P. vivaxsporozoites per sample, gave results concordant with dissection of salivary glands and CS-ELISA, but was slightly less sensitive than the CS-ELISA in microtiter plates. The assay consistently detected one infected mosquito in a pool of 10 or 20 mosquitoes, and was 100% specific in discriminating between species ofPlasmodiumwhen mosquito suspensions were spiked with sporozoites. The assay could be completed in 1 h, required no specialized equipment, and therefore was useful for field applications.

A ‘4-poster’ device that attracts white-tailed deer to a bait source, and as they feed, allows a self-application of a pesticide to the head, ears, and neck to control ticks was designed, constructed, and tested. The device consists of a central bin containing bait to attract deer and two feeding and application stations. These stations each have one bait port and two vertical pesticide-impregnated applicator rollers. This design allows unrestricted vertical retraction of the head to minimize injury to the deer or damage to the posts supporting the pesticide application rollers. Observations using deer demonstrated ready acceptance and repeated use by both antlered and antlerless deer. Results of an initial trial indicate that control values for lone star ticks,Amblyomma americanum(L.), exceeded 92–97% on deer that used the device regularly.

To continue monitoring the prevalence and distribution ofEhrlichia chaffeensis(Rickettsiales: Rickettsiaeceae) in southern Indiana, a total of 498Amblyomma americanum(L.) ticks (262 adults and 292 nymphs) was collected from five southern Indiana counties during May and June 1998. Ticks were pooled and examined for the presence ofE. chaffeensisusing nested polymerase chain reaction and primers specific for the 16S rRNA gene ofE. chaffeensis.The average minimum infection rate for adult ticks collected in 1998 was 3.8% (ranging from 0 to 7.7% in various counties) as compared with previous average minimum infection rates of 1.6% in 1995 and 4.9% in 1997. None of the pools ofA. americanumnymphs tested positive. In addition, blood samples were collected from 325 white-tailed deer taken in Indiana and 327 taken in Ohio in November 1998. Serum samples were tested for the presence ofE. chaffeensis-like organisms reactive to antibodies using an indirect immunofluorescence assay (IFA). Antibodies were found in deer from six Indiana counties where infection rates ranged from 42.6 to 66.7% and in four Ohio counties where infection rates ranged from 4.4 to 25%. The results of this study reconfirm thatE. chaffeensisis well established in southern Indiana and also provide the first evidence ofE. chaffeensis-like organisms infecting white-tailed deer in Ohio, suggesting the need to survey Ohio ticks for the presence ofEhrlichia.

This investigation compared the effects of repeated infestations to immunization of dogs with tick salivary gland or midgut extracts on the feeding and fecundity performances of femaleRhipicephalus sanguineus(Latrielle). In each immunized group, three tick-naive dogs were immunized three times with tick salivary gland or midgut extracts, and twice challenged at 21-d intervals by allowing 80 female and 40 male adult ticks to feed on each host. The repeated infestation group of three naive dogs was infested five times at 21-d intervals by the same numbers of ticks. The repeated infestation group showed a trend of reduced tick performance after the third infestation, but some of the tick performance parameters had recovered by the fifth infestation. Tick attachment was reduced by immunization with either tick salivary gland or midgut extract. Immunization with tick salivary gland extract had the greatest impact on the feeding period and engorgement weight of the female ticks. Immunization with tick midgut extract resulted in the greatest reduction of tick fecundity parameters, which included preoviposition, oviposition, and egg-incubation periods in addition to reduced egg production and egg viability. These results confirm that dogs can become resistant toR. sanguineus,and demonstrate that immunization with tick salivary gland or midgut extract has different effects on tick feeding and fecundity.

Separate black-tailed prairie dog,Cynomys ludovicianus(Ord), towns on the Rocky Mountain Arsenal National Wildlife Refuge, Colorado, were treated with technical pyriproxyfen (Nylar) spray, powder, and oral bait. The treatments were applied to reduce relative abundance of the plague vectorOropsylla hirsuta(Baker). Because pyriproxyfen is a juvenile hormone analog, we were also concerned with the effects of the treatments on nontarget arthropods, which is the focus of this study. Pitfall traps and sweep net sampling were used to measure relative abundance of arthropod populations pre- and posttreatment. Nontarget arthropod sampling produced a large number of statistical comparisons that indicated significant declines (P< 0.05) in relative arthropod abundance. Many of the significant declines were probably because of natural fluctuations in arthropod populations rather than treatment effects. Because arthropod populations appeared to fluctuate randomly, we only made inferences about highly significant (P< 0.001) declines. In doing so, we hoped to abate some of the confusion created by the natural fluctuation in arthropod abundance and increase our chance of correctly attributing a population reduction to a treatment effect. Only Homoptera at the pyriproxyfen powder site exhibited highly significant reductions that appeared to be attributed to the treatments. Pyriproxyfen spray treatments did not significantly reduce relative arthropod abundance.

We provide evidence ofEhrlichia risticiiHolland, the agent of Potomac horse fever, in trematode stages found in aquatic insects collected from a pasture stream in northern California, using nested polymerase chain reaction (PCR) amplification and sequence analyses of the 16S rRNA, 51 kDa major antigen andgroELheat shock protein genes.E. risticiiwas detected in metacercariae found in the immatures and adults of the following insects: caddisflies (Trichoptera), mayflies (Ephemeroptera), damselflies (Odonata, Zygoptera), dragonflies (Odonata, Anisoptera), and stoneflies (Plecoptera). The prevalence ofE. risticiiwas 31.9% (n= 454 individuals) in aquatic insects (13 of 17 species were positive). Prevalence within orders was as follows: 43.5% (n= 207) in caddisflies, 15.2% (n= 92) in mayflies, 13.9% (n= 115) in damselflies, 10.0% (n= 10) in dragonflies, and 80.0% (n= 30) in stoneflies. This study demonstrates a broad intermediate host range for trematodes that act as vector forE. risticii.Insects are likely to play an important role in the epidemiology of this disease.

Four general frequencies of human St. Louis encephalitis (SLE) virus (epidemic, focal, sporadic, and no transmission) occurred in Florida between 1990 and 1999. An epidemic with 226 clinical cases and 11 deaths was reported from 28 Florida counties between July 1990 and January 1991. During the autumn of 1993, a focal outbreak was reported from Lee (5 cases) and Collier (3) Counties in southwest Florida. During the autumn of 1997, sporadic transmission to nine humans was reported from five Florida counties (Brevard [1 case], Polk [3], Charlotte [1], Lee [2], and Palm Beach [2]). Human infection with SLE virus depends on a number of variables that drive virus transmission. These include vector, virus, and avian host abundance, and meteorological events, especially rainfall. We monitored the abundance and serological status of wild avian amplification hosts, virus isolations fromCulex nigripalpusTheobald females, and SLE virus transmission to sentinel chickens during 1990, 1993, and 1997. The epidemic of 1990 was characterized by conditions that produced an unusual abundance of vector mosquitoes and avian amplification hosts early in the year. We propose that epidemics may result when a specific combination of biotic and abiotic conditions favor SLE virus minimum field infection rates that approach 1:1,000 inCx. nigripalpusvectors.

Three experiments were conducted on cats to evaluate precocity and duration of the first blood meal ofCtenocephalides felis felis(Bouché). Percentage of engorged fleas was calculated for fleas held on cats for 5, 15, 30, and 60 min. Duration of first blood meal was also measured for individual fleas confined on cats. When fleas are free in the hair coat, 24.9% are engorged after 5 min and 97.2% are engorged after 1 h. Fleas confined to a vial on the cats fed significantly sooner; 60% of females were engorged within 5 min. The mean delay between deposition and biting for fleas, which began feeding within 15 min, was 24 s ± 31 s for females and 23 s ± 44 s for males. The mean duration of meals was 25 ± 18 min for females and 11 ± 8 min for males.

Proboscis amputation has facilitated the study of mosquito behavior. Using humans as a host is very important in the study of mosquito attractants, repellents, and host preference. However, mosquito bites cause potential medical problems because of hypersensitivity and perhaps secondary bacterial infection, even using laboratory mosquitoes. Moreover, once a normal female mosquito bites and feeds on human blood, it cannot be used in subsequent probing tests. These problems were resolved by proboscis amputation. Variation of attraction among humans was examined effectively without bites using proboscis-amputatedAedes albopictusSkuse. Proboscis-amputated and normal mosquitoes also showed equal repellency against 1% L-lactic acid. Although the mosquitoes lacked the tip of the labium and some sensilla, they alighted on human forearms in the same way as normal mosquitoes. Because proboscis-amputated mosquitoes continued to probe avidly, they could be used repeatedly, thereby reducing the number of mosquitoes required for experimentation. The use of proboscis-amputated mosquitoes would promote various studies of mosquito attraction or repellency with no risk of hypersensitivity and secondary bacterial infection by mosquito bites.

Amblyomma javanense(Supino) was collected from a Malayan pangolin (Manis javanicaDesmarest) and a wild boar (Sus scrofaL.) from Tak province on the western boundary of Thailand along the Myanmar (Burma) border. To date, this tick species has not been recorded from this area and from a wild boar.

When isolating dengue virus (DEN) from mosquitoes collected in endemic areas, pools may contain both anti-dengue antibodies from freshly engorged females and virus from DEN infected females. To determine if these antibodies may interfere with virus isolation, we simulated the isolation procedure usingAedes aegypti(L.) that we infected with the 16681 strain of dengue type 2 virus by intrathoracic inoculation. At 7 d postinfection, we allowed females to engorge on immunized or normal mouse blood. Virus in a mixture of anti-dengue-2 antibodies and dengue-2 virus became inactive after incubation at 37°C for 1 h, but remained infective without incubation. Therefore, at ambient conditions antibodies would not interfere with virus isolation from field-collectedAe. aegyptifrom endemic areas. In addition, DEN antibodies enhanced virus replication when inoculated intoAe. aegypti,but not C6/36 cells. The mechanism for this in vitro antibody enhancement of infection remains unclear.