Comparative off-host survival of larval Rocky Mountain Wood Ticks (Dermacentor andersoni) collected from ecologically distinct field populations.

Dermacentor andersoni (Ixodida: Ixodidae) Stiles, also known as the Rocky Mountain Wood Tick (RMWT), is found throughout the western United States and transmits pathogens of importance to human and animal health. The distributions and activity patterns of RMWTs are shaped by regional climatic variation. However, it is unknown if responses to climatic variation differ across the tick's geographical range. The objective of this narrow study was to test the hypothesis that the responses of RMWTs to abiotic conditions [e.g. temperature and RH (relative humidity)] vary among populations. We collected RMWTs from ecologically distinct field sites in the states of Montana and Oregon (USA). In the laboratory, we tracked weekly survival of tick larvae under four combinations of RH (75% and 98%) and temperature (26 and 32 °C) that reflected the range of conditions observed in the source habitats during spring-summer. For both populations, larval survival time decreased at the higher ambient temperature (50% mortality 1-2 weeks earlier). Differences in RH did not affect the survival time of larvae from Oregon. By contrast, the survival time of larvae from Montana decreased at the lower RH (50% mortality 1 week earlier). These data suggest that the tolerance limits for water stress may differ among populations of D. andersoni.

Hybridization in natural sympatric populations of Dermacentor ticks in northwestern North America.

Hybridization in ticks has been described in a handful of species and mostly as a result of laboratory experiments. We used 148 AFLP loci to describe putative hybridization events between D. andersoni and D. variabilis in sympatric populations from northwestern North America. Recently, D. variabilis has expanded its range westward into the natural range of D. andersoni. Using a sample of 235 D. andersoni and 62 D. variabilis, we identified 31 individuals as putative hybrids: four F2 individuals and 27 backcrosses to D. andersoni (as defined by NewHybrids). We found no evidence of hybrids backcrossing into D. variabilis. Furthermore, all hybrids presented 16S mtDNA signatures characteristic of D. andersoni, which indicates the directionality of the hybrid crosses: female D. andersoni × male D. variabilis. We also discovered 13 species-specific AFLP fragments for D. andersoni. These loci were found to have a decreased occurrence in the putative hybrids and were absent altogether in D. variabilis samples. AFLP profiles were also used to determine the levels of genetic population structure and gene flow among nine populations of D. andersoni and three of D. variabilis. Genetic structure exists in both species (D. andersoni, ΦST = 0.110; D. variabilis, ΦST = 0.304) as well as significant estimates of isolation by distance (D. andersoni, ρ = 0.066, P = 0.001; D. variabilis, ρ = 0.729, P = 0.001).

Dispersion and sampling of adult Dermacentor andersoni in rangeland in Western North America.

A fixed precision sampling plan was developed for off-host populations of adult Rocky Mountain wood tick, Dermacentor andersoni (Stiles) based on data collected by dragging at 13 locations in Alberta, Canada; Washington; and Oregon. In total, 222 site-date combinations were sampled. Each site-date combination was considered a sample, and each sample ranged in size from 86 to 250 10 m2 quadrats. Analysis of simulated quadrats ranging in size from 10 to 50 m2 indicated that the most precise sample unit was the 10 m2 quadrat. Samples taken when abundance < 0.04 ticks per 10 m2 were more likely to not depart significantly from statistical randomness than samples taken when abundance was greater. Data were grouped into ten abundance classes and assessed for fit to the Poisson and negative binomial distributions. The Poisson distribution fit only data in abundance classes < 0.02 ticks per 10 m2, while the negative binomial distribution fit data from all abundance classes. A negative binomial distribution with common k = 0.3742 fit data in eight of the 10 abundance classes. Both the Taylor and Iwao mean-variance relationships were fit and used to predict sample sizes for a fixed level of precision. Sample sizes predicted using the Taylor model tended to underestimate actual sample sizes, while sample sizes estimated using the Iwao model tended to overestimate actual sample sizes. Using a negative binomial with common k provided estimates of required sample sizes closest to empirically calculated sample sizes.

Reproductive compatibility of prairie and montane populations of Dermacentor andersoni.

Genetic analysis of prairie and montane populations of Dermacentor andersoni (Stiles) originating from Alberta (AB) and British Columbia (BC), Canada, respectively, indicated limited gene flow (Nm <1) and a large amount of genetic differentiation (Fst = 0.49) between the populations. The prairie population also had a greater level of genetic diversity. Mating experiments indicated that females of geographically heterogeneous crosses had similar engorgement and oviposition failure as homogenous crosses in the parental generation but that egg mass sterility was greatest for the ABfemale x BCmale cross, intermediate for the homogenous crosses, and lowest for the BCfemale x ABmale cross. The progeny of all crosses produced fertile eggs, and the only significant effect in the progeny generation was increased oviposition failure of the pure AB cross. Covariate analysis indicated that egg mass sterility was associated with BC males in the parental generation and that oviposition failure was associated with AB males and AB females in the progeny generation. The hazard of cumulative reproductive failure was increased with AB females in both generations, reduced for AB males in the parental generation, and increased with AB males in the progeny generation. Overall, heterogenous crosses had the greatest and least reproductive failure in the parental generation, but they were intermediate to the homogenous crosses in the progeny generation. The limited gene flow between the populations seems to have been sufficient to maintain reproductive compatibility.

A relapsing fever group spirochete transmitted by Ixodes scapularis ticks.

A species of Borrelia spirochetes previously unknown from North America has been found to be transmitted by Ixodes scapularis ticks. Infected ticks are positive for Borrelia spp. by DFA test but negative for Borrelia burgdorferi by polymerase chain reaction (PCR) using species-specific primers for 16S rDNA, outer surface protein A, outer surface protein C, and flagellin genes. A 1,347-bp portion of 16S rDNA was amplified from a pool of infected nymphs, sequenced, and compared with the homologous fragment from 26 other species of Borrelia. The analysis showed 4.6% pairwise difference from B. burgdorferi, with the closest relative being Borrelia miyamotoi (99.3% similarity) reported from Ixodes persulcatus in Japan. Phylogenetic analysis showed the unknown Borrelia to cluster with relapsing fever group spirochetes rather than with Lyme disease spirochetes. A 764-bp fragment of the flagellin gene was also compared with the homologous fragment from 24 other Borrelia species. The flagellin sequence of B. burgdorferi was 19.5% different from the unknown Borrelia and showed 98.6% similarity with B. miyamotoi. A pair of PCR primers specifically designed to amplify a 219-bp fragment of the flagellin gene from this spirochete was used to survey field-collected I. scapularis nymphs from five northeastern states (Connecticut, Rhode Island, New York, New Jersey, and Maryland). Positive results were obtained in 1.9-2.5% of 712 nymphs sampled from four states but in none of 162 ticks collected from Maryland. Transovarial transmission was demonstrated by PCR of larval progeny from infected females with filial infection rates ranging from 6% to 73%. Transstadial passage occurred from larvae through adults. Vertebrate infection was demonstrated by feeding infected nymphs on Peromyscus leucopus mice and recovering the organism from uninfected xenodiagnostic larvae fed 7-21 days later. Considering the frequency of contact between I. scapularis and humans, further work is needed to determine the potential public health significance of yet another zoonotic agent transmitted by this tick species.

Pathogenesis of African swine fever virus in Ornithodoros ticks.

African swine fever virus (ASFV) is the only known DNA arbovirus and the sole member of the family Asfarviridae. It causes a lethal, hemorrhagic disease in domestic pigs. ASFV is enzootic in sub-Saharan Africa and is maintained in a sylvatic cycle by infecting both wild members of the Suidae (e.g. warthogs) and the argasid tick Ornithodoros porcinus porcinus. The pathogenesis of ASFV in O. porcinus porcinus ticks is characterized by a low infectious dose, lifelong infection, efficient transmission to both pigs and ticks, and low mortality until after the first oviposition. ASFV pathogenesis in warthogs is characterized by an inapparent infection with transient, low viremic titers. Thus O. porcinus porcinus ticks probably constitute the most important natural vector of ASFV, although both the mammalian and tick hosts are probably required for the maintenance of ASFV in the sylvatic cycle. The mechanism of ASFV transmission from the sylvatic cycle to domestic pigs is probably through infected ticks feeding on pigs. In addition to O. porcinus porcinus, a number of North American, Central American and Caribbean species of Ornithodoros have been shown to be potential vectors of ASFV.

African swine fever virus replication in the midgut epithelium is required for infection of Ornithodoros ticks.

Although the Malawi Lil20/1 (MAL) strain of African swine fever virus (ASFV) was isolated from Ornithodoros sp. ticks, our attempts to experimentally infect ticks by feeding them this strain failed. Ten different collections of Ornithodorus porcinus porcinus ticks and one collection of O. porcinus domesticus ticks were orally exposed to a high titer of MAL. At 3 weeks postinoculation (p.i.), <25% of the ticks contained detectable virus, with viral titers of <4 log(10) 50% hemadsorbing doses/ml. Viral titers declined to undetectability in >90% of the ticks by 5 weeks p.i. To further study the growth defect, O. porcinus porcinus ticks were orally exposed to MAL and assayed at regular intervals p.i. Whole-tick viral titers dramatically declined (>1,000-fold) between 2 and 6 days p.i., and by 18 days p.i., viral titers were below the detection limit. In contrast, viral titers of ticks orally exposed to a tick-competent ASFV isolate, Pretoriuskop/96/4/1 (Pr4), increased 10-fold by 10 days p.i. and 50-fold by 14 days p.i. Early viral gene expression, but not extensive late gene expression or viral DNA synthesis, was detected in the midguts of ticks orally exposed to MAL. Ultrastructural analysis demonstrated that progeny virus was rarely present in ticks orally exposed to MAL and, when present, was associated with extensive cytopathology of phagocytic midgut epithelial cells. To determine if viral replication was restricted only in the midgut epithelium, parenteral inoculations into the hemocoel were performed. With inoculation by this route, a persistent infection was established although a delay in generalization of MAL was detected and viral titers in most tissues were typically 10- to 1,000-fold lower than those of ticks injected with Pr4. MAL was detected in both the salivary secretion and coxal fluid following feeding but less frequently and at a lower titer compared to Pr4. Transovarial transmission of MAL was not detected after two gonotrophic cycles. Ultrastructural analysis demonstrated that, when injected, MAL replicated in a number of cell types but failed to replicate in midgut epithelial cells. In contrast, ticks injected with Pr4 had replicating virus in midgut epithelial cells. Together, these results indicate that MAL replication is restricted in midgut epithelial cells. This finding demonstrates the importance of viral replication in the midgut for successful ASFV infection of the arthropod host.

African swine fever virus infection in the argasid host, Ornithodoros porcinus porcinus.

The pathogenesis of African swine fever virus (ASFV) infection in Ornithodoros porcinus porcinus was examined in nymphal ticks infected with the ASFV isolate Chiredzi/83/1. At times postinfection (p.i.) ranging from 6 h to 290 days, ticks or dissected tick tissues were titrated for virus and examined ultrastructurally for evidence of virus replication. The ASFV infection rate in ticks was 100% in these experiments, and virus infection was not associated with a significant increase in tick mortality. Initial ASFV replication occurred in phagocytic digestive cells of the midgut epithelium. Subsequent infection and replication of ASFV in undifferentiated midgut cells was observed at 15 days p.i. Generalization of virus infection from midgut to other tick tissues required 2 to 3 weeks and most likely involved virus movement across the basal lamina of the midgut into the hemocoel. Secondary sites of virus replication included hemocytes (type I and II), connective tissue, coxal gland, salivary gland, and reproductive tissue. Virus replication was not observed in the nervous tissue of the synganglion, Malpighian tubules, and muscle. Persistent infection, characterized by active virus replication, was observed for all involved tick tissues. After 91 days p.i., viral titers in salivary gland and reproductive tissue were consistently the highest detected. Successful tick-to-pig transmission of ASFV at 48 days p.i. correlated with high viral titers in salivary and coxal gland tissue and their secretions. A similar pattern of virus infection and persistence in O. porcinus porcinus was observed for three additional ASFV tick isolates in their associated ticks.

Multiplex polymerase chain reaction for detection of Dirofilaria immitis (Filariidea: Onchocercidae) and Wuchereria bancrofti (Filarioidea: Dipetalonematidae) in their common vector Aedes polynesiensis (Diptera: Culicidae).

In French Polynesia, Aedes polynesiensis (Marks) is the vector of the human filarial parasite Wuchereria bancrofti (Cobbold) and dog heartworm, Dirofilaria immitis (Leidy). A multiplex polymerase chain reaction (PCR) assay was designed to screen pools of field-collected Ae. polynesiensis for the presence of both parasites simultaneously using primers specific for each parasite. The sensitivity of detection on purified DNA was 1 and 10 pg, equivalent to 0.1 and 1 L3 larva per pool for W. bancrofti and D. immitis, respectively. Codetection was performed at an hybridization temperature of 58 degrees C to avoid competition between heterologous DNA and primers that was observed at 55 degrees C. In addition, D. immitis was detected by PCR in the blood of infected dogs.

Parasitism of Aedes aegypti and Ae. albopictus (Diptera: Culicidae) by Ascogregarina spp. (Apicomplexa: Lecudinidae) in Florida.

Aedes aegypti (L.) and Ae. albopictus (Skuse) from 40 sites in 17 counties of Florida were surveyed for gregarine parasites during the spring and autumn of 1993 and in July 1994. Larvae collected from containers (mainly tires) were dissected to determine the number of gregarine trophozoites present. Ascogregarina spp. were found at 70% of the sites and occurred as frequently in Ae. aegypti populations as in Ae. albopictus. Within sites, parasite distributions were highly variable and aggregated in host populations. Parasite loads ranged from 1 to 486 trophozoites per host. Mean parasite load was significantly higher in Ae. aegypti larvae (52.5 per host) than in Ae. albopictus (33.5 per host). Parasite prevalence was significantly higher in Ae. aegypti populations that were not sympatric with Ae. albopictus compared with allopatric Ae. albopictus or sympatric populations of either species. In sympatric populations, Ae. aegypti tended to have equal or higher parasite prevalence than the cohabiting Ae. albopictus. Ascogregarina taiwanensis (Lien & Levine) prevalence in Ae. albopictus was significantly higher in areas where these hosts have been present for at least 3 yr. These data contribute to the hypothesis that parasite-mediated competition may be a factor in the apparent displacement of Ae. aegypti by Ae. albopictus in Florida.

Polymerase chain reaction-based method for the detection of canine heartworm (Filarioidea: Onchocercidae) in mosquitoes (Diptera: Culicidae) and vertebrate hosts.

A polymerase chain reaction (PCR)-based test was developed to aid in identification of Dirofilaria sp. and for use in surveys for infected vectors of Dirofilaria immitis (Leidy). A set of PCR primers was designed based on the DNA sequence of a D. immitis surface antigen gene. The predicted product was a 378 base-pair DNA fragment. The target fragment was amplified from free D. immitis larvae (both L3 and microfilariae), individual infected mosquitoes, pools of 30 mosquitoes (including a single infected mosquito), infected mosquitoes stored desiccated at room temperature for 7 mo, and whole blood from a dog infected with D. immitis. These primers did not amplify a homologous fragment from a mermithid or from 4 other species of filarioid nematodes (including 1 other Dirofilaria species). Third-stage larvae from field-collected mosquitoes also were tested. Field-collected L3, identified tentatively as D. immitis, were confirmed by PCR. There was no amplification using the PCR test for L3 identified tentatively as Dirofilaria sp. (possibly D. tenuis Chandler). These data provide a strong indication of the specificity of these primers. The potential utility of this technique for detecting the presence of D. immitis in field populations of mosquitoes is discussed.

Cryopreservation of infective larvae of Onchocerca volvulus (Filarioidea: Onchocercidae).

Infective larvae (L3) of Onchocerca volvulus were procured in Liberia, West Africa, in the natural black fly vector, Simulium yahense. A cryobiological technique was developed to preserve L3 of O. volvulus that were fully viable after thawing. Larvae were treated before cooling with 4 cryoprotective compounds. Three compounds, dimethyl sulfoxide (DMSO), glycerol, and ethylene glycol, were prepared with distilled water. The fourth compound was DMSO prepared in different concentrations with 0.25 M sucrose. The treatment with DMSO + 0.25 M sucrose cryoprotectant resulted in the highest survival of infective larvae. Five cooling rates between 0.5 C/min and 20.0 C/min were applied. The highest survival of L3 was with the cooling rate of 1.0 C/min. Two-step cooling of L3 was applied. In the first step, L3's were frozen to 5 levels from -10.0 C to -20.0 C, -30.0 C, -40.0 C, -60 C, and -80.0 C, and in the second step, larvae were transferred into liquid nitrogen at -196 C for rapid cooling and storage. The survival was the highest when larvae were cooled to approximately -40 C prior to transfer into liquid nitrogen. Slow, gradual, and rapid thawing procedures were applied. The survival was the highest in rapid warming.

Assessment of Aedes sierrensis as a vector of canine heartworm in Utah using a new technique for determining the infectivity rate.

Both Aedes sierrensis and Dirofilaria immitis have recently become established in Utah. We evaluated the vector potential of this Aedes sierrensis strain using a new technique for detecting Dirofilaria immitis in individual mosquitoes. Survival of Aedes sierrensis females after bloodfeeding did not differ from that of Ae. triseriatus but infective Ae. sierrensis produced significantly more L3 nematodes. This observation and epidemiological data support the hypothesis that Ae. sierrensis is the vector of canine heartworm in Utah. Infectivity was determined by counting infective-stage parasites that migrated into the medium after individual mosquitoes were decapitated or crushed in the wells of tissue culture plates. Complete recovery of infective-stage nematodes was attained in 60-74% of the mosquitoes and 77-93% of all L3 were collected with this technique. There were few false negatives. High recovery rates (mean = 89%) were also obtained for mosquitoes treated en masse.

Influence of habitat and temperature on dispersal behaviour of two pteromalid parasitoids of houseflies during an inundative release at a dairy barn.

1. About 11,000 each of Muscidifurax raptor Girault and Saunders and Urolepis rufipes (Ashmead) were released weekly for 7 weeks at a commercial dairy farm in central New York state, U.S.A. Dispersal behaviour was monitored by parasitism rates of house fly, Musca domestica L., pupae placed in sentinel bags. 2. M. raptor, which was released inside the barn, parasitized fly pupae both inside and outside, and it achieved highest rates of parasitism in indoor straw calf-bedding and in outdoor manure and silage. 3. U. rufipes, which was released outside the barn, did not attack pupae inside the barn, and its highest rates of parasitism occurred in outdoor manure and silage. 4. M. raptor appeared to be more effective than U. rufipes in parasitizing pupae located at sites where natural fly-breeding occurred. 5. Interspecific competition did not appear to explain these distribution patterns. 6. There was no significant trend in parasitism by M. raptor as a function of distance from the release station. Furthermore, high rates of parasitism near open doorways and at an outdoor site 30 m away suggests that M. raptor dispersed throughout the barn and its immediate surroundings. 7. Air temperature was positively correlated to flight activity, but not to parasitization activity in natural fly-breeding substrates.

Evaluation of fly-electrocuting black light devices in caged-layer poultry facilities.

Insect-electrocuting black light devices were evaluated for their effectiveness in killing flies in caged-layer poultry facilities. Concurrently, the effect of the addition of the attractant muscalure (Z-9-tricosene) to these devices on their fly-killing efficiency was evaluated. An average of over 29,000 flies were killed per device per week in Facility 1 and 7,000 flies per device per week in Facility 2 over the 8-wk evaluation period. The addition of muscalure (25 to 200 mg/device) increased the number of flies killed by the devices by as much as 76% but only one of the increases was statistically significant. Both the house fly, Musca domestica, and the garbage fly, Ophyra spp. were apparently attracted to muscalure, as the number of both fly species killed was consistently higher in devices containing the attractant than in devices without the attractant.

Complete generation rearing of Simulium damnosum s.l. (Diptera: Simuliidae) in the laboratory.

A closed system of water circulation previously devised for rearing nearctic black flies was used for two cohorts of Simulium damnosum s.l. (Ghana strain). Complete generation development from field-collected eggs occurred in both instances with the production of F1 adults and F1 pupae respectively. Survival of larvae form the 3rd instar to pupation was extremely high for both parental and filial generations and ranged form 63-98%. In 3 of the 4 rearings (1P, 2P, 1F1), pupal survival ranged form 98-100%. Mating attempts were frequently seen but insemination rates were low (greater than 1%). Nulliparous females were anthropophilic and exhibited bloodfeeding rates ranging form 67.5-87.5%. Parous females also readily engorged on humans. Porcine and rabbit hosts proved less attractive while two membrane systems (Baudruche, chicken skin)using equine, bovine, or chicken blood elicited practically no engorgement. Gravid females readily deposited fertile and infertile eggs in an oviposition chamber designed originally for Simulium decorum.