Sequence and transcript expression of the super-kdr locus of the horn fly, Haematobia irritans.

In horn flies, Haematobia irritans irritans (Diptera: Muscidae) (Linnaeus, 1758), target site resistance to pyrethroids can be diagnosed by an allele-specific PCR that genotypes individual flies at both the super-kdr (skdr) and the knock down resistance (kdr) associated loci. When this technique uses genomic DNA as template, modifications, such as alternative RNA splicing and RNA editing are not specifically detected. Alternative splicing at the skdr locus has been reported in Dipterans; thus, the genomic DNA-based allele-specific PCR may not accurately reflect the frequency of the skdr mutation in horn fly field populations. To investigate if alternative splicing occurs at the skdr locus of horn flies, genomic DNA and cDNA sequences isolated from two wild populations and two laboratory-reared colonies with varying degrees of pyrethroid resistance were compared. There was no indication of alternative splicing at the super-kdr locus neither in the wild populations nor in the laboratory-reared colonies.

Equine infectious anaemia and mechanical transmission: man and the wee beasties.

There is no credible evidence that the lentivirus that causes equine infectious anaemia (EIA) replicates in invertebrates. The virus persistently infects its equid hosts and is often present in blood in significant quantities. Blood-feeding arthropods thus have the potential to transfer the virus between hosts, especially if their feeding on the first host is interrupted and immediately continued on a second host. The general details and dynamics of mechanical transmission are included in this paper, as this agent presents an excellent model. Mechanical transmission can be effectively controlled if the dynamics and interactions of the host, virus and vector populations are understood. Efficient transmission is proportional to the amount of agent found in the source material, the environmental survival of the agent, the number of vector feedings, the number of interrupted feedings, vector refeeding, the proximity of infected and naive hosts, host population density, and the length of time during which vectors and hosts are in contact. Establishing firm quantitative risk estimates for EIA is impossible, mainly because the virus content in blood can change exponentially from day to day. The EIA virus can be transmitted by horse flies for at least 30 minutes after feeding on a horse with acute signs of EIA, butthe probability of a horse fly being interrupted and completing its blood feeding on a second host at a distance of 50 m is very low, and the separation of infected and uninfected equids by 200 m breaks transmission. The statements above assume that human interactions are absent or do not contribute to the risk of virus transmission; however, the risk from human interventions, such as the too-often-used procedure of administering > 200 ml of plasma to foals, can easily be more than 10(7) times greater than the risk posed by a single horse fly. Controlling EIA depends upon the identification of persistently infected equids by serological testing because other methods of identifying infective virus orviral genetic material are less accurate or impractical.

Oral treatment of rodents with fipronil for feed-through and systemic control of sand flies (Diptera: Psychodidae).

The sand fly Phlebotomus papatasi Scopoli is the vector of Leishmania major (Yakimoff & Schokhor), which is maintained in populations of burrowing rodents. The purpose of this study was to conduct a laboratory study to determine the efficacy of oral treatment of rodents with fipronil for control of sand flies that feed on rodent feces as larvae or on rodent blood as adults. We determined through larval bioassays that fipronil was eliminated in feces of orally-treated hamsters at a level that was significantly toxic to sand fly larvae for 21 d after the hamsters had been withdrawn from a fipronil-treated diet. Through bloodfeeding bioassays, we also found that fipronil was present in the peripheral blood of hamsters at a concentration that was significantly toxic to bloodfeeding adult female sand flies for 49 d after the hamsters had been withdrawn from their treated diet. The results of this study suggest that fipronil acts as well as or better than feed-through or systemic insecticides that previously have been measured against sand flies, and is particularly promising because this single compound acts against both larvae and bloodfeeding adults. An area-wide approach using rodent baits containing a fipronil could suppress vector populations that originate in the vicinity of rodent reservoirs, and could be used to eliminate the most epidemiologically important part of the vector population: female sand flies that take bloodmeals on rodent reservoirs.

Laboratory evaluation of rubidium as a long-lasting marker for bloodfeeding sand flies (Diptera: Psychodidae).

The objective of this study was to evaluate the use of the trace element rubidium (Rb) as a long-lasting systemic biomarker for bloodfeeding females of the sand fly Phlebotomus papatasi Scopoli. Baits containing Rb chloride were found to be palatable to hamsters in this study. We were able to detect Rb using a portable X-ray fluorescence analyzer in all sand flies that fed on Rb-treated hamsters for at least 14 d postbloodmeal. We also detected Rb in sand flies that took a bloodmeal from hamsters up to 10 d after the hamsters were withdrawn from a Rb-treated diet. Results of this study constitute proof of concept for the incorporation of Rb chloride into rodent baits for marking bloodfeeding sand flies, and suggest that Rb marking could be used as a technique for evaluating rodent-targeted sand fly control methods and in ecological studies on sand flies.

Evaluation of three feed-through insecticides using two rodent and two sand fly species as models.

The efficacy of 3 rodent feed-through insecticides (novaluron, pyriproxyfen, and ivermectin) was determined against larvae of the sand flies Phlebotomus duboscqi and P. papatasi using Syrian hamsters (Mesocricetus auratus) and Mongolian gerbils (Meriones unguiculatus) as laboratory models. For each insecticide, there were no significant differences between the longevity or percentage survival of sand fly larvae that had been fed feces of treated rodents for each sand fly or rodent species pairing. The results of this study suggest that larvae of P. duboscqi and P. papatasi are equally susceptible to the concentrations of the rodent feed-through insecticides tested in this study and that these insecticides are pharmacologically compatible with different rodent/sand fly interactions.

Oral treatment of rodents with insecticides for control of sand flies (Diptera: Psychodidae) and the fluorescent tracer technique (FTT) as a tool to evaluate potential sand fly control methods.

In laboratory studies, insecticides (diflubenzuron, novaluron, methoprene and, pyriproxyfen) that have been incorporated into rodent diets were effective as feed-throughs against sand fly larvae. Novaluron also was effective against sand fly larvae at low concentrations and under simulated field conditions. Ivermectin has been shown to be effective as a systemic insecticide, killing 100% of blood-feeding sand flies for up to seven d after rodents were treated. The fluorescent tracer technique (FTT) is the use of certain fluorescent dyes (rhodamine B or uranine O) as feed-through transtadial biomarkers for phlebotomine sand flies, systemic biomarkers for blood-feeding sand flies, and permanent markers for nectar-feeding sand flies. The results of these laboratory studies provide proof of concept for the FTT and indicate that the FTT could be used to delineate specific foci with rodent/sand fly associations that would be susceptible to control by using feed-through or systemic insecticides, or foci where insecticide-treated sugar baits could be used against sand flies.

Evaluation of juvenile hormone analogues as rodent feed-through insecticides for control of immature phlebotomine sandflies.

The juvenile hormone analogues methoprene and pyriproxyfen were evaluated as rodent feed-through insecticides for control of immature stages of the sandfly Phlebotomus papatasi Scopoli (Diptera: Psychodidae). The development and survival of P. papatasi second-instar larvae fed faeces from Syrian hamsters, Mesocricetus auratus, that had been fed a diet containing methoprene (0, 9.788, 97.88 or 978.8 p.p.m.) or pyriproxyfen (0, 9.82, 98.2 or 982 p.p.m.) were evaluated. The faeces of methoprene-treated hamsters greatly reduced the percentage of larvae that pupated at all concentrations tested and prevented adult emergence at all but the lowest concentration (9.788 p.p.m.). Pyriproxyfen prevented both pupation and adult emergence at all concentrations tested. The results of this study suggest that a control strategy using rodent baits containing juvenile hormone analogues to control phlebotomine sandflies that live in rodent burrows and feed on rodent faeces may be possible. As rodent reservoirs and vectors of Leishmania major live in close association in many parts of the Middle East, control of the transmission of the agent of zoonotic cutaneous leishmaniasis may also be possible.

Detection of target site resistance to pyrethroids and organophosphates in the horn fly using multiplex polymerase chain reaction.

The horn fly, Haematobia irritans L., is an obligate blood-feeding fly and the primary insect pest parasitizing cattle in the United States. Pesticide resistance has become a substantial problem for cattle producers, and although several mechanisms of resistance are possible, target site resistance is the most important mechanism preventing control of this fly in the United States and possibly other countries. We developed a multiplex polymerase chain reaction assay to detect the known target site, pyrethroid resistance-associated mutation in the horn fly and a recently reported G262A mutation in the horn fly acetylcholinesterase, the target site for organophosphates. As expected, the pyrethroid resistance target site mutation was found in fly populations from Texas, Louisiana, Washington, Georgia, Mexico, and Brazil. This mutation was found to have a gender bias as it was more prevalent in females than males. The G262A acetylcholinesterase mutation was found in Texas, Louisiana, Washington, Georgia, and Mexico, but not Brazil. There was no gender bias in the occurrence of this mutation, and there was no correlation between the occurrence of the kdr and the G262A mutations. Unlike the case with the pyrethroid target site mutation, the presence of G262A did not appear to exclusively provide the level of resistance required to account for bioassay results. It is likely an additional mutation(s) occurs in the target site and/or a metabolic resistance mechanism exists in organophosphate-resistant horn fly populations.

Laboratory evaluation of the efficacy of fluorescent biomarkers for sugar-feeding sand flies (Diptera: Psychodidae).

The purpose of this study was to evaluate the use of four fluorescent dyes (rhodamine B, uranine O, auramine O, and erythrosin B) and two nonfluorescent dyes (carmoisine and indigotine) incorporated into sugar baits as biomarkers for phlebotomine sand flies. Each dye could be detected in sand flies fed baits with dye for 24 h when examined using bright field microscopy, although there was considerable variability in the marking produced; all sand flies that had ingested rhodamine B-treated sucrose solution were marked clearly. Sand flies that had ingested sucrose solution containing rhodamine B or uranine O at concentrations as low as 10 mg/L were consistently detected under fluorescence microscopy. None of the treatments in this study reduced the longevity of sand flies. All sand flies fed sucrose solution containing rhodamine B or uranine O were marked for at least 14 d, whereas only 20% of sand flies were marked 3 d after feeding on a carmoisine-treated solution. When rhodamine B and uranine O were combined in a single sucrose solution or when the dyes were fed sequentially to sand flies, both dyes could be detected in sand flies using fluorescence microscopy. We propose that rhodamine B- or uranine O-treated sucrose baits could be used in ecological studies or to identify portions of the adult sand fly population that could be targeted with insecticide-treated sugar baits.

Oral treatment of rodents with ivermectin for the control of Phlebotomus papatasi (Diptera: Psychodidae) under laboratory conditions.

The macrocyclic lactone ivermectin was evaluated as a rodent systemic and feed-through insecticide for control of adult and immature sand flies (Phlebotomus papatasi Scopoli). Syrian hamsters were fed diets containing 0 or 20 mg/kg ivermectin for 9 days, and sand flies were allowed to take bloodmeals from the hamsters 0, 3, 7, and 14 days after they were withdrawn from their diets. Ivermectin treatment of hamsters was 100% effective against bloodfeeding sand flies for up to 7 days after hamsters were withdrawn from ivermectin-treated diets. The survival and fecundity of sand flies that took bloodmeals from hamsters 14 days after they had been withdrawn from their ivermectin-treated diets were not significantly different from sand flies that took bloodmeals from control hamsters. Feces of the hamsters were collected 0, 3, 7, and 14 days after the hamsters had been withdrawn from their diets, and the feces were fed to 2nd instars of P. papatasi. All larvae that were fed feces of ivermectin-treated hamsters voided 0 days after being withdrawn from their diets died before pupation; larvae fed feces voided by ivermectin-treated hamsters 3 and 7 days after being withdrawn from their treated diets had significantly reduced survival. The results of this study suggest that oral ivermectin treatment of rodents could control immature and adult female sand flies that are closely associated with rodents.

Laboratory evaluation of novaluron as a rodent feed-through insecticide against sand fly larvae (Diptera: Psychodidae).

Experiments were conducted to evaluate novaluron as a feed-through larvicide to control immature phlebotomine sand flies (Phlebotomus papatasi Scopoli) (Diptera: Psychodidae). The minimum effective concentration of novaluron against sand fly larvae fed hamster feces treated with novaluron, or feces of hamsters fed a diet containing novaluron, was 9.88 and 9.88 x 10(-1) mg/kg, respectively. Feces of novaluron-treated hamsters were held under conditions simulating the inside of a rodent burrow for up to 30 d, and all larvae that consumed these feces died before pupation; a significant reduction in treated larval survival relative to control was observed when the feces were aged for up to 150 d. Novaluron was shown to be effective as a feed-though larvicide when novaluron-treated food made up only a portion of the diet of hamsters. The results of this study suggest that novaluron could be effective as a rodent feed-through insecticide in a field setting.

Detection of bluetongue virus RNA in field-collected Culicoides spp. (Diptera: Ceratopogonidae) following the discovery of bluetongue virus serotype 1 in white-tailed deer and cattle in Louisiana.

In November 2004, bluetongue virus (family Reoviridae, genus Orbivirus, BTV) serotype 1 (BTV-1) was detected for the first time in the United States from a hunter-killed deer in St. Mary Parish, LA. In 2005, sera surveys were conducted on three cattle farms near the area where the deer was found, and BTV-1-seropositive cattle were found on two of the three farms; in 2006, sera surveys from the cattle on the three farms did not detect any BTV-1-positive animals. The purpose of this study was to survey ceratopogonid populations at the three farms and test field-collected specimens for the presence of BTV and epizootic hemorrhagic disease virus (family Reoviridae, genus Orbivirus, EHDV). Miniature CDC light traps and New Jersey traps were used to capture ceratopogonids on the three farms from January 2006 through November 2007. In total, 3,319 ceratopogonids were captured, including 1,790 specimens of 10 different species of Culicoides. IR-RT-polymerase chain reaction (PCR) was performed to screen for BTV and EHDV in 264 pools representing 2,309 specimens collected at the farms. All positive samples were sequenced for serotype determination. Five pools of 275 (1.8%) were positive for BTV. Pools of four species of Culicoides were found to be positive: Culicoides crepuscularis (Malloch), Culicoides debilipalpis Lutz (two pools), Culicoides haematopotus Malloch, and Gulicoidesfurens (Poey). The amplicons of the positive specimens were sequenced and found to be identical to both BTV-17 and BTV-13. During our study, no BTV-1 transmission was detected in cattle, and no BTV-1 was detected in specimens of ceratopogonids.

Laboratory evaluation of insecticide-treated sugar baits for control of phlebotomine sand flies (Diptera: Psychodidae).

The purpose of this study was to evaluate the use of boric acid, imidacloprid, ivermectin, or abamectin incorporated into sugar baits as oral toxicants for adult phlebotomine sand flies. Variable toxicity of insecticide-sugar bait solutions to adult male and female sand flies was demonstrated, based on male female median lethal concentration values of 0.10-0.08, 6.13-9.53, and 9.03-18.11 mg/liter of imidacloprid, ivermectin, and abamectin, respectively. Complete control of sand flies could not be achieved with as high as 40 g/liter of boric acid in sugar bait solution; concentrations >40 g/liter were found repellent to the sand flies. Uranine O (a fluorescent tracer dye that can be used to measure the ingestion of sugar baits by sand flies) did not interact negatively with imidacloprid, ivermectin, or abamectin when it was combined with the insecticides in a sugar bait. Also, incorporation of imidacloprid, ivermectin, or abamectin into sugar baits did not reduce the effect whether adult male and female sand flies fed on these sugar baits. We propose that imidacloprid, ivermectin, or abamectin could be used to control adult sand fly populations with targeted use of insecticide-treated sugar baits.

Evaluation of rhodamine B as an orally delivered biomarker for rodents and a feed-through transtadial biomarker for phlebotomine sand flies (Diptera: Psychodidae).

The purpose of this study was to evaluate the use of rhodamine B as an orally delivered biomarker for rodents and a feed-through transtadial biomarker for phlebotomine sand flies (Diptera: Psychodidae). Rhodamine B-treated hamsters were visibly marked for up to 8 wk, and their feces were fluorescent when examined under a fluorescence microscope. The development and survival of sand fly larvae fed feces of rhodamine B-treated hamsters were not significantly different from control sand flies. Adult male and female sand flies, that had been fed as larvae the feces of rhodamine B-treated hamsters, were fluorescent when examined using fluorescent microscopy and could be distinguished from control sand flies. Adult female sand flies that took bloodmeals from rhodamine B-treated hamsters were fluorescent when examined immediately after feeding. Rhodamine B incorporated rodent baits could be used to detect adult male and female sand flies that fed on the feces of baited rodents as larvae, or adult female sand flies that have taken a bloodmeal from bait-fed rodents. This would allow the delineation of specific foci with rodent-sand fly associations that would be susceptible to control by using feed-through or systemic insecticides.

Control of horn flies (Haematobia irritans) and gastrointestinal parasites and its relation with cow-calf performance.

The objective of this study was to assess the effects of horn fly and gastrointestinal (GI) nematode control on cow-calf performance with a spring-calving system. A total of 508 cow-calf records from two locations in Louisiana were available for study over two trials (2005 and 2006). Cow and calf pairs were stratified by cow age, calf sex, calf age, calf sire breed and calf sire and randomly assigned to three replicate treatment groups across the two locations. The treatments were: (1) no horn fly or GI nematode control; (2) horn fly control administrated to cows with insecticidal ear tags applied in late-May (start of trial); (3) GI nematode control administrated to calves in late-May, mid-July and late-August (each calf received fenbendazole at the rate of 5 mg kg(-1) body weight); and (4) horn fly and GI nematode control as stated above. Length of the trials ranged from 115 to 124 days (late-May to weaning in late-September or early-October). Cows and calves were weighed at the start of the trial, mid-July and weaning. Weekly horn fly counts were conducted. Individual calf fecal samples were collected in late-May, mid-July, late-August, weaning and 10-14 days after late-May and late-August collections. Cows treated for horn flies had lower (P<0.05) fly counts compared with untreated cows for each trial; however, overall fly counts for the treated cows at one location exceeded over 70 flies per side. Calves treated with fenbendazole had lower (P<0.01) GI nematode fecal egg counts (FEC) for the 10-14 day post-treatment collections. Calves treated GI nematodes and whose dams were treated for horn flies had numerically higher or greater (P<0.05) total calf gains (8.6 kg and 11.8 kg) in both trials than untreated calves. Weight gain differences for fenbendazole-treated calves compared with untreated calves were of greater magnitude from mid-July to weaning than weight gain differences from late-May to mid-July in both trials. There were no treatment differences for any of the cow response traits. In conclusion, improvement in calf growth from both horn fly and GI nematode treatments were realized even with marginal levels of fly control and a greater response in calf growth resulted when deworming of spring-born calves occurred when they were 4 months of age or older.

Expressed cDNAS from embryonic and larval stages of the horn fly (Diptera: Muscidae).

We used an expressed sequence tag approach to initiate a study of the genome of the horn fly, Hematobia irritans (L.) (Diptera: Muscidae). Two normalized cDNA libraries were synthesized from RNA isolated from embryos and first instars from a field population of horn flies. Approximately 10,000 clones were sequenced from both the 5' and 3' directions. Sequence data from each library was assembled into a database of tentative consensus sequences (TCs) and singletons and used to search public protein databases and annotate the sequences. Additionally, the sequences from both the egg and larval libraries were combined into a single database consisting of 16,702 expressed sequence tags (ESTs) assembling into 2886 TCs and 1,522 singleton entries. Several sequences were identified that may have roles in the horn fly's resistance to insecticides. The availability of this database will facilitate the design of microarray and other experiments to study horn fly gene expression on a larger scale than previously possible. This would include studies designed to investigate metabolic-based insecticide resistance, identify novel antigens for vaccine-based control approaches, and discover new proteins to serve as targets for new pesticide development.

Ivermectin as a rodent feed-through insecticide for control of immature sand flies (Diptera: Psychodidae).

Ivermectin was evaluated as a potential rodent feed-through for the control of immature stages of Phlebotomus papatasi. The survival of sand fly larvae fed feces of Syrian hamsters (Mesocricetus auratus) that had been fed a diet containing 0, 2, 6, 10, 20, 60, or 100 ppm ivermectin was measured. Sand fly larvae fed the feces of ivermectin-treated hamsters had significantly reduced survival, with 100% mortality of larvae fed feces of hamsters fed a diet containing 20, 60, and 100 ppm ivermectin. The results of this study suggest that a control strategy using rodent baits containing ivermectin to control phlebotomine sand flies may be possible. Because rodent reservoirs and sand fly vectors of Leishmania major live in close association in many parts of the Middle East, the control of transmission of the agent of zoonotic cutaneous leishmaniasis also may be possible.

Evaluation of novaluron as a feed-through insecticide for control of immature sand flies (Diptera: Psychodidae).

The development and survival of sand fly Phlebotomus papatasi Scopoli (Diptera: Psychodidae) larvae fed feces of Syrian hamsters, Mesocricetus auratus, that had been fed a diet containing novaluron were evaluated. In total, six larval diets were used in sand fly larval bioassays. Four groups of larvae were fed feces of hamsters that had been maintained on a diet containing either 0, 9.88, 98.8, or 988 ppm novaluron. Two additional groups were fed a larval diet composed of equal parts composted rabbit feces and rabbit chow containing either 0 or 988 ppm novaluron. No pupation, hence no adult emergence, occurred when larvae were fed feces of hamsters that were fed diets containing novaluron. The mortality of sand flies fed feces of treated hamsters occurred during larval molts. The results of this study suggest that a control strategy using rodent baits containing novaluron to control phlebotomine sand flies and zoonotic cutaneous leishmaniasis may be possible.

Laboratory evaluation of diflubenzuron as a feed-through for control of immature sand flies (Diptera: Psychodidae).

The benzoylurea chitin synthesis inhibitor diflubenzuron was evaluated as a rodent feed-through for the control of immature stages of Phlebotomus papatasi Scopoli (Diptera: Psychodidae). The development and survival of second instars of P. papatasi larvae that were fed feces from Syrian hamsters, Mesocricetus auratus, that had been fed a diet containing 0, 8.97, 89.7, or 897 ppm diflubenzuron was evaluated. No pupation or adult emergence occurred when larvae were fed feces from hamsters that were fed diets containing diflubenzuron. The mortality of sand flies fed feces from treated hamsters was coincident with pupation of the controls, suggesting a specific effect on the larval-to-pupal molt. The results of this study suggest that a control strategy using rodent baits containing diflubenzuron for phlebotomine sand flies and zoonotic cutaneous leishmaniasis may be possible.

The influence of distance on movement of tabanids (Diptera: Tabanidae) between horses.

Two studies evaluated the potential use of spatial barriers to reduce the mechanical transmission of disease agents by tabanids in the Pantanal region of Brazil. Tabanids at stations separated by four different distances (5, 10, 25, and 50m) were marked. In the first study, tabanids were marked and allowed to feed until engorgement or natural interruption occurred and captured if they transferred to the other horse. A total of 2847 tabanids belonging to nine different species were marked. The percentage of tabanids that moved between horses was 10.5 at 5m, 6.8 at 10m, and 4.6 at 25m. In the second study, flies were marked, feeding was then interrupted, and the flies were released approximately 50cm from the host. A total of 1274 tabanids belonging to five different species were marked. The percentage of flies that moved between horses was 9.7 at 5m, 9.7 at 10m, and 4.6 at 25m. No tabanids transferred between animals separated by 50m in either experiment. The results of this study strongly support the recommendation that segregation of animals effectively prevents the mechanical transmission of pathogens by tabanids.