Incursions of Japanese encephalitis (JE) virus into northern Queensland are currently monitored using sentinel pigs. However, the maintenance of these pigs is expensive, and because pigs are the major amplifying hosts of the virus, they may contribute to JE transmission. Therefore, we evaluated a mosquito-based detection system to potentially replace the sentinel pigs. Single, inactivated JE-infected Culex annulirostris Skuse and C. sitiens Wiedemann were placed into pools of uninfected mosquitoes that were housed in a MosquitoMagnet Pro (MM) trap set under wet season field conditions in Cairns, Queensland for 0, 7, or 14 d. JE viral RNA was detected (cycling threshold [CT] = 40) in 11/12, 10/14, and 2/5 pools containing 200, 1,000, and 5,000 mosquitoes, respectively, using a TaqMan real-time reverse transcription-polymerase chain reaction (RT-PCR). The ability to detect virus was not affected by the length of time pools were maintained under field conditions, although the CT score tended to increase with field exposure time. Furthermore, JE viral RNA was detected in three pools of 1,000 mosquitoes collected from Badu Island using a MM trap. These results indicated that a mosquito trap system employing self-powered traps, such as the MosquitoMagnet, and a real-time PCR system, could be used to monitor for JE in remote areas.

We describe the first documented field transmission of West Nile (WN) virus by a North American mosquito. WN was first detected in northern Florida in 2001. An intensive mosquito trapping and surveillance program was conducted in this region for four nights to assess mosquito transmission of WN. Four mosquito traps, each with a single sentinel chicken, were placed at five different locations on each of four nights. A total of 11,948 mosquitoes was collected, and 14 mosquito pools were found to contain WN, giving a minimum infection rate between 1.08 and 7.54 per 1,000. Only one of the 80 sentinel chickens seroconverted to WN, demonstrating a single mosquito transmission event during the study and a mosquito transmission rate of between 0.8 and 1 per 1,000. Culex nigripalpus Theobald was responsible for WN transmission to the sentinel chicken, although both Cx. nigripalpus and Culex quinquefasciatus Say were found infected with WN. Mosquito transmission rates are reported in this study for the first time for a WN outbreak. This information is essential to determine risk of human and animal infection.

The surface ultrastructure of all larval instars of Chrysomya rufifacies (Macquart) is described by means of scanning electron microscopy (SEM). Morphological changes were greatest from the first to the second instar, but less from the second to the third instar. Most of these changes involved the structure of the anterior spiracle, posterior spiracle, integument of the body, and mouthhooks. Modification of the mouthhooks, especially in the third instar, are helpful in explaining the ferocious feeding ability of the older maggots. The common name of “hairy-maggot” for C. rufifacies is only appropriate for the second and third instars because of their elongated tubercles along the body, whereas this name is not descriptive of the first instar that lack tubercles.

The lake from Porto-Primavera hydroelectric power station inundated an area of 2,200 km² at the border of São Paulo and Mato-Grosso do Sul States, Brazil. Infestations by ticks were evaluated on 135 marsh deer, Blastocerus dichotomus (Illiger), captured before and after inundation. Ticks were collected for identification, and infestation level of animals was assessed by scoring. Deer were divided into four groups according to capture location and temporal relation to the inundation. Groups 1, 2, and 3 were captured before inundation. Group 4 was captured after inundation. Four tick species were found: Amblyomma cajennense (F.), Amblyomma triste Koch, Anocentor nitens (Neumann), and Boophilus microplus (Canestrini). Groups 1, 2, 3, and 4 had 30, 45, 100, and 96%, respectively, of animals carrying B. microplus ticks. A. triste was observed on 16, 22, 22, and 88% of animals from groups 1, 2, 3, and 4, respectively. A. nitens and A. cajennense were observed only on group 4, on 32 and 16% of the animals, respectively. Groups 1 and 2 had only 4.8 and 6.1% of animals with high infestation levels, respectively, and no ticks on 46.8% and 45.5% of the animals, respectively. Conversely, groups 3 and 4 lacked noninfested animals and had high infestation levels on 77.8 and 50% of deer, respectively. Marsh area shrinkage was blamed for higher infestation levels on deer from groups 3 and 4. The widespread presence of A. triste on marsh deer, a Neotropical tick species, raises the possibility of a natural host–parasite relationship.

Three common insect repellents (N,N-diethyl-m-toluamide [DEET], Pyranha, and Repel X) were tested to determine whether they affected Africanized honey bee attack behavior. Eight Africanized honey bee (Apis mellifera L.) colonies were exposed in an alternating series to the test repellents or blank controls delivered in a stream of air directed toward the colony entrances. The response generated by the repellents and the controls was measured as the number of attacking honey bees recorded with an electronic temper tester. Neither a citronella-based repellent (Pyranha) nor DEET had any effect on colony behavior; however, Repel X consistently caused a greater attack response after exposure.

An entomological study was conducted in a village of Sudano-Guinean savanna in Senegal, during the rainy season from July to November 2001, to investigate the biology and the involvement of each anopheline species in malaria transmission. Mosquitoes were captured when landing on human volunteers and by pyrethrum spray catches. Twelve anopheline species were captured. Four species amounted to 97% of human-bait sampling: Anopheles gambiae molecular form S, An. arabiensis, An. funestus, and An. nili s.s. All An. gambiae and An. nili females were fed on human, whereas the anthropophilic rate was 94.5% for An. funestus and 88.9% for An. arabiensis. Plasmodium falciparum was the only malaria parasite found, and infecting only An. gambiae, An. arabiensis, An. funestus, and An. nili. The circumsporozoite rate was 4.5% for An. gambiae, 1.6% for An. arabiensis, 3.9% for An. funestus, and 2.1% for An. nili. During the period of study, the entomological inoculation rate was estimated to 264 infected bites. An. gambiae, An. arabiensis, An. funestus, and An. nili were responsible respectively of 56, 3, 20, and 21% of malaria transmission. This study shows for the first time the implication of An. nili in malaria transmission in this area and the complexity of the malaria vectorial system that should be taken into account for any malaria control strategy.

Because of their impact on human and veterinary health, there is considerable interest in understanding how Culicoides use olfactory cues in host location. The adequate chemical stimulus for sensilla located on the maxillary palps was determined for several species of female Culicoides. Electrophysiological studies identified and characterized the sensory neurons on Culicoides maxillary palps that responded to stimulation with low concentrations of CO₂. The concentration response function in different background concentrations of CO₂ was established for C. furens (Poey), C. stellifer (Coquillet) and C. mississippiensis Hoffman. Comparisons were made to previously studied CO₂-sensitive neurons in mosquitoes. Understanding what sensory signals the host releases and how they are detected may lead to the development of strategies aimed at controlling these insects.

Optically active (1S, 2′S)-2-methylpiperidinyl-3-cyclohexen-1-carboxamide (SS220) is a new synthetic arthropod repellent. A three-step synthesis based on a chiral Diels-Alder reaction and diastereomeric resolution of 2-methylpiperidine was developed to prepare the compound. Quantitative laboratory assays using human volunteers compared the effectiveness of SS220 with the commonly used repellents Deet and Bayrepel against Aedes aegypti (Linnaeus) and Anopheles stephensi Liston mosquitoes. In two experiments using Aedes aegypti, one using a single identical dose and one with varying doses used to develop a dose–response curve, SS220 was as effective as Deet and both compounds were more effective than Bayrepel. The three compounds were equally effective against An. stephensi. Based on the ease of its synthetic preparation and its repellent efficacy, we surmise that SS220 is a candidate to serve as a new and effective alternate repellent for protection against arthropod disease vectors.

Antibody titers against St. Louis encephalitis virus (SLE) measured by a plaque reduction neutralization test (PRNT) decreased rapidly in house finches (Capodacus mexicanus) after initial infection, whereas antibodies measured by enzyme immunoassay (EIA) remained detectable in all birds for the length of the experiment, indicating long-term persistence and greater assay sensitivity of the EIA. After 52 wk, birds were challenged by subcutaneous inoculation with the same strain of SLE virus. Virus was not detected for 1–4 d postchallenge in blood samples tested by plaque assay and RT-PCR or by xenodiagnosis in Culex tarsalis fed concurrently and then held for 11 d at 26°C. Virus was detected by all three methods in control birds infected concurrently for the first time. Challenge with SLE produced a rapid and marked ananmestic rise in both neutralizing and EIA antibody titers that exceeded the primary response in the same birds or in concurrently inoculated control birds. At necropsy 4 wk postchallenge, 3 of 7 challenged and 1of 2 positive control birds were chronically infected, with viral RNA detected by RT-PCR in brain, spleen, lung, and/or kidney tissues. Our results indicated that persistence of protective antibody prevents reinfection during the following season and may prevent the recrudescence of infectious virus in chronically infected birds.

Experimental studies evaluated the vector competence of Ochlerotatus taeniorhynchus (Wiedemann), Culex cancer Theobald, Culex pseudes (Dyar and Knab), Culex taeniopus Dyar and Knab, and a Culex (Culex) species, probably Culex quinquefasciatus Say, and Culex nigripalpus Theobald from Chiapas, Mexico, and Tocoa, Honduras, for epizootic (IC) and enzootic (IE) strains of Venezuelan equine encephalomyelitis (VEE) virus. Culex pseudes was highly susceptible to infection with both the IC and IE strains of VEE (infection rates >78%). Patterns of susceptibility to VEE were similar for Oc. taeniorhynchus collected in Mexico and Honduras. Although Oc. taeniorhynchus was highly susceptible to the epizootic IC strains (infection rates ≥95%, n = 190), this species was less susceptible to the enzootic IE strain (infection rates ≤35%, n = 233). The Culex (Culex) species were refractory to both subtypes of VEE, and none of 166 contained evidence of a disseminated infection. Virus-exposed Cx. pseudes that refed on susceptible hamsters readily transmitted virus, confirming that this species was an efficient vector of VEE. Although Oc. taeniorhynchus that fed on hamsters infected with the epizootic IC strain transmitted VEE efficiently, only one of six of those with a disseminated infection with the enzootic IE virus that fed on hamsters transmitted virus by bite. These data indicate that Cx. pseudes is an efficient laboratory vector of both epizootic and enzootic strains of VEE and that Oc. taeniorhynchus could be an important vector of epizootic subtypes of VEE.

We investigated the experimental vector competence of Ixodes pacificus Cooley and Kohls and Ixodes spinipalpis Hadwen and Nuttall, and the reservoir competence of the dusky-footed woodrat (Neotoma fuscipes Baird) and the deer mouse (Peromyscus maniculatus [Wagner]), for Borrelia bissettii Postic, Marti Ras, Lane, Hendson, and Baranton. Both rodent species are capable reservoirs for B. bissettii; infection rates for I. pacificus or I. spinipalpis nymphs fed as larvae on infected animals ranged from 50 to 57%. Moreover, both I. pacificus and I. spinipalpis are efficient vectors of B. bissettii. Viable infections were recorded from all rodents known to be exposed to one or more infected nymphs of I. spinipalpis (seven deer mice, two white mice) or I. pacificus (seven deer mice). In contrast, none of four New Zealand white rabbits fed upon by 90 I. pacificus nymphs with a probable B. bissettii infection rate of >50% became infected or seroconverted. The attachment and feeding success of laboratory-reared nymphs similarly confined with deer mice in muslin-covered wire-mesh cages for 24 h ranged from 0% for I. pacificus to 17–73% for I. spinipalpis. Notably, the I. pacificus nymphs were physiologically capable of feeding; nymphs failing to attach to rodents fed readily when placed in feeding capsules upon rabbits. We conclude that the dusky-footed woodrat and the deer mouse are capable experimental reservoir hosts of B. bissettii, and that both I. spinipalpis and I. pacificus are efficient experimental vectors of B. bissettii. However, the reluctance of I. pacificus nymphs to feed on certain rodents may limit its importance as an enzootic vector of B. burgdorferi sensu lato spirochetes.

Two forms of leishmaniasis are endemic to the Jenin district in the northern region of the West Bank. Visceral leishmaniasis (VL), caused by Leishmania infantum, mainly affects infants. Cutaneous leishmaniasis (CL) affects a broader age group and is probably caused by L. tropica. Although the Jenin district is the most important focus of leishmaniasis in the West Bank, the sand fly fauna of the area has never been studied in a systematic manner. We collected base-line data on sand fly species, their distribution, and their feeding preferences to facilitate risk assessments for contracting leishmaniasis. Light traps, sticky traps, insecticide knockdown collections, aspirator, and human-landing collections were used. A total of 4,082 sand flies was collected in foci of confidence limits and/or VL between June and December 1998. Nine Phlebotomus species representing seven subgenera were identified: P. (Larroussius) perfiliewi transcaucasicus Perfil’ev, P. (La.) tobbi Adler & Theodor, P. (La.) mascitti canaaniticus Adler & Theodor, P. (La.) mascitti mascitti Grassi, P. (La.) syriacus Adler & Theodor, P. (Phlebotomus) papatasi Scopoli, P. (Synphlebotomus) s.p., P. (Paraphlebotomus) sergenti Parrot, P. (Par.) jacusieli Theodor, P. (Adlerius) halepensis Theodor. Two other Phlebotomus subspecies, P. (La.) major major Annandale, P. (La.) neglectus Tonnoir, require confirmation. In addition, four species of the closely related genus, Sergentomyia were also found: S. (Sergentomyia) theodori Parrot, S. (S.) fallax Parrot, S. (Sintonius) tiberiadis Adler, Theodor & Lourie, S. (Sin.) christophersi Sinton. Among five species of sand flies collected on human bait, P. papatasi constituted ≈90% followed by P. major syriacus (8%) and P. mascitti (2%). Sand fly human-biting activity occurred through the night and it was highest between 2400 and 0300 hours. P. papatasi. P. perfiliewi, P, major and P.tobbi were the more endophilic species constituting 93% of all flies caught indoors. Seven Phlebotomus spp. constitute potential vectors of leishmaniasis but the most probable ones are as follows: P. papatasi the main human-biting species, a recognized vector of L. major (CL), P. sergenti, L. tropica (CL) and P. syriacus, L. infantum (VL).

We developed a multiplex polymerase chain reaction (PCR) assay that simultaneously detects three types of flea-associated microorganisms. Targets for the assay were sequences encoding portions of the gltA, a 17-kDa antigen, and pla genes of Bartonella spp. Strong et al., Rickettsia spp. da Rocha-Lima, and Yersinia pestis Yersin, respectively. A total of 260 flea samples containing bloodmeal remnants were analyzed from fleas collected from abandoned prairie dog (Cynomys ludovicianus) burrows at the site of an active plague epizootic in Jefferson County, CO. Results indicated that 34 (13.1%) fleas were positive for Bartonella spp., 0 (0%) were positive for Rickettsia spp., and 120 (46.2%) were positive for Y. pestis. Twenty-three (8.8%) of these fleas were coinfected with Bartonella spp. and Y. pestis. A second group of 295 bloodmeal-containing fleas was collected and analyzed from abandoned burrows in Logan County, CO, where a prairie dog die-off had occurred 2–4 mo before the time of sampling. Of these 295 fleas, 7 (2.4%) were positive for Bartonella spp., 0 (0%) were positive for Rickettsia spp., and 46 (15.6%) were positive for Y. pestis. Coinfections were not observed in fleas from the Logan County epizootic site. The multiplex PCR also was used to identify Y. pestis and Bartonella in prairie dog blood and tissues. This report represents the first identification of Bartonella from prairie dogs and their fleas. Prairie dog fleas were tested with PCR, and the Bartonella PCR amplicons produced were sequenced and found to be closely related to similar sequences amplified from Bartonella that had been isolated from prairie dog blood samples. Phylogenetic analyses indicate that the sequences of bartonellae from prairie dogs and prairie dog fleas cluster tightly within a clade that is distinct from those containing other known Bartonella genotypes.

Seven fresh animal carcasses were monitored throughout decomposition in a mixed flatwood forest in East Baton Rouge Parish, LA from 1 April to 1 July 1999. Succession patterns of necrophilous insects were documented for the following: one Louisiana black bear (threatened species), two white-tailed deer, two alligators, and two swine as the experimental reference. Our results suggest variation in the species composition of necrophilous insects among animal carcass types. A total of 93 arthropod species, from 46 families and three classes, were manually collected from the seven carcasses. Only 19 insect species were collected on all four animal types and were represented by eight families: Coleoptera: Histeridae, Nitidulidae, Silphidae, Staphylinidae; Diptera: Calliphoridae, Muscidae, Piophilidae, Sepsidae. Eleven of the 46 families were not collected at either alligator site but were observed at bear, deer, and swine carrion: Coleoptera: Cleridae, Dermestidae, Geotrupidae, Scarabaeidae; Diptera: Micropezidae, Sarcophagidae, Syrphidae; Hymenoptera: Apidae; Lepidoptera: Nymphalidae; and Odonata: Libellulidae. Residency and succession patterns of necrophilous insects are presented for each animal type with particular emphasis on selected fly (Calliphoridae, Muscidae, Piophilidae, Stratiomyidae) and beetle species (Cleridae, Dermestidae, Histeridae, Nitidulidae, Silphidae, Staphylinidae).

Two cell lines, ABADRL-Cs-W3 (W3) and ABADRL-Cs-W8A (W8), were developed from a field population of Culicoides sonorensis Wirth & Jones. The cell lines were characterized by isozyme phenotyping and the ability to support the replication of bluetongue virus (BLU) and epizootic hemorrhagic disease virus (EHDV) (Orbivirus, Reoviridae). Comparison of isozymes found in the cell lines with those found in adult C. sonorensis colony insects confirmed that the cell lines were of C. sonorensis origin. There was, however, sufficient isozyme variation present in the cell lines to construct a unique isozyme profile for each cell line. Although both cell lines supported BLU and EHDV replication to the same level, one-step growth curves for BLU indicated that virus replication was faster and attained a peak titer earlier in the W3 cell line than in the W8 cell line. Viral proteins and RNA were detected earlier in the W3 cell line as well. The accelerated virus growth kinetics observed in the W3 cell line and the adherent nature of the cells makes it more suitable for certain Orbivirus studies.

Adult female Culicoides occidentalis Wirth and Jones were collected in a coastal salt marsh habitat in southern California over a period of 15 mo using CO₂-baited suction traps. Adults were active year-round. Adult females overall were large based on wing length (1.86 mm). Because of coastal influences, daily and seasonal changes in air temperatures were greatly buffered, and mean temperatures ranged from 10 to 15°C in winter to 19–21°C in summer. Wing lengths varied inversely with air temperatures, with an average of 2.13 mm (February) to 1.6 mm (July). Parity was consistently low (3.6% overall) and may reflect poor host availability in this isolated salt marsh habitat.

Wolbachia are cytoplasmically inherited, endosymbiotic bacteria known to infect a wide variety of arthropods. Polymerase chain reaction (PCR) amplification of the Wolbachia surface protein (wsp) gene was used to assay the infection of geographically disparate populations of Aedes albopictus (Skuse) by Wolbachia. Nine North American, four South American, one Hawaiian, and four Old World populations of A. albopictus were all doubly infected with both the wAlbA and wAlbB strains of Wolbachia. A 365-bp region of the wAlbA wsp gene was sequenced from seven geographically disparate host populations, and all sequences were identical. Similarly, a 474-bp region of the wAlbB wsp gene was sequenced from the same populations, and all sequences were identical. These results suggest a role for Wolbachia infection in causing the previously established pattern of low mitochondrial DNA variability, but average nuclear gene diversity, within and among populations of A. albopictus.

More than 30,000 mosquitoes in 22 species or species groups were collected from the Florida Keys, Monroe County, FL, USA, in dry ice-baited light and gravid traps. Dry ice-baited traps collected more mosquitoes than did gravid traps. West Nile virus was detected in pools of Anopheles atropos Dyar & Knab, Deinocerites cancer Theobald, and Ochlerotatus taeniorhynchus (Wiedemann).

We describe a procedure for the introduction of Borrelia burgdorferi, the spirochetal agent of Lyme disease, into larvae of the tick vector Ixodes scapularis. Internalized spirochetes were observed in larvae examined after 15 or 45 min immersion at 32°C in liquid culture suspensions of low passage B. burgdorferi strain B31. Larval ticks immersed in low passage strain B31 were able to feed to repletion on white-footed mice. Midguts of larvae contained many spirochetes 1 wk postengorgement, while larvae incubated with high passage strain B31 were free of detectable spirochetes at the same interval. Larvae incubated with low passage strain B31 were competent to transmit the pathogen to mice, as shown by serology, reisolation of B. burgdorferi from mice, and xenodiagnosis. Ticks maintained the infection transstadially to the nymphal stage and transmitted the infection to naive mice, replicating an essential aspect of natural infection. This method requires no special equipment and allows artificial infection of large numbers of ticks at the larval stage. It will facilitate studies of the contribution of specific B. burgdorferi genetic loci to tick colonization.

Bacterial infections were investigated in midguts of insectary and field-collected Anopheles albimanus Weidemann from southern Mexico. Serratia marcescens, Enterobacter cloacae and Enterobacter amnigenus 2, Enterobacter sp., and Serratia sp. were isolated in field samples obtained in 1998, but only Enterobacter sp. was recovered in field samples of 1997 and no bacteria were isolated from insectary specimens. These bacteria were offered along with Plasmodium vivax infected blood to aseptic insectary An. albimanus, and the number of infected mosquitoes as well as the oocyst densities assessed after 7d. Plasmodium vivax infections in mosquitoes co-infected with En. amnigenus 2, En. cloacae, and S. marcensces were 53, 17, and 210 times, respectively, lower than in control mosquitoes, and the mean oocyst density in mosquitoes co-infected with En. cloacae was 2.5 times lower than in controls. Mortality was 13 times higher in S. marcensces-infected mosquitoes compared with controls. The overall midgut bacterial infection in mosquito field populations may influence P. vivax transmission, and could contribute to explain the annual variations in malaria incidence observed in the area.