Efficacy of the entomopathogenic fungus Metarhizium brunneum in controlling the tick Rhipicephalus annulatus under field conditions.

High infectivity of entomopathogenic fungi to ticks under laboratory conditions has been demonstrated in many studies. However, the few reports on their use under field conditions demonstrate large variations in their success, often with no clear explanation. The present study evaluated the factors affecting the efficacy of the fungus Metarhizium brunneum against the tick Rhipicephalus (Boophilus) annulatus. It demonstrates how environmental conditions and ground cover affect the efficiency of the fungus under field conditions. During the summer, 93% of tick females exposed to fungus-contaminated ground died within 1 week, whereas during the winter, only 62.2% died within 6 weeks. Nevertheless, the hatchability of their eggs was only 6.1% during the summer and 0.0% during winter. Covering the ground with grass, leaves or gravel improved fungal performance. Aside from killing female ticks, the fungus had a substantial effect on tick fecundity. Fungal infection reduced the proportion of female ticks laying full-size egg masses by up to 91%, and reduced egg hatchability by up to 100%. To reduce the negative effect of outdoor factors on fungal activity, its conidia were mixed with different oils (olive, canola, mineral or paraffin at 10% v/v) and evaluated in both laboratory and field tests for efficacy. All tested oils without conidia sprayed on the sand did not influence tick survival or weight of the laid eggs but significantly reduced egghatchability. Conidia in water with canola or mineral oil spread on agarose and incubated for 18 h showed 57% and 0% germination, respectively. Comparing, under laboratory conditions, the effects of adding each of the four oils to conidia in water on ticks demonstrated no effect on female mortality or weight of the laid egg mass, but the percentage of hatched eggs was reduced. In outdoor trials, female ticks placed on the ground sprayed with conidia in water yielded an average of 175 larvae per female and there was no hatching of eggs laid by females placed on ground sprayed with conidia in water with canola or mineral oils.

Temporal association of ambient temperature and relative humidity with Spirocerca lupi infection of Onthophagus sellatus: a 14-year longitudinal study.

The parasitic nematode Spirocerca lupi causes major morbidity and mortality in dogs. The scarab beetle Onthophagus sellatus is its major intermediate host in Israel. We investigated the prevalence of beetle infection by S. lupi in different years between 1994 and 2008. The average monthly maximum and minimum relative humidity (RH) and ambient temperature (AT) throughout the study period were calculated based on daily meteorological data. The infection prevalence decreased over the study period, possibly due to a chronological change resulting from increased preventive treatment of dogs against S. lupi, or climate change. Multivariate analysis was performed for these two hypotheses. Under the first hypothesis, chronological change was forced into the model, and environmental variables were inserted stepwise. The final model included beetle-collection date, minimum RH (RH min) during the month preceding beetle collection, its interaction with maximal AT (AT max) during that same month, and the interaction of maximal RH (RH max) and AT max, during the month of beetle collection. Under the second hypothesis, chronological change was not forced. The final model included RH max during the month of beetle collection, average RH (RHave) during the month preceding beetle collection, and its interaction with AT max during the latter month. The results suggest that under both hypotheses, RH and AT during the month preceding beetle collection influence S. lupi's ability to develop and survive in O. sellatus, and may be used to predict the risk to dogs of S. lupi infection.

On-host control of the brown dog tick Rhipicephalus sanguineus Latreille (Acari: Ixodidae) by Metarhizium brunneum (Hypocreales: Clavicipitaceae).

Ticks are obligatory blood-sucking arthropods. Their life cycle includes a relatively short period of feeding on a vertebrate host and a long off-host period spent in the upper layer of the soil. Entomopathogenic fungi are known to be highly effective tick pathogens and the on-host application of these fungi may be a promising economic approach for tick control. In this study, we evaluated the tick control provided by spraying Metarhizium brunneum onto the tick's vertebrate host, specifically gerbils (Meriones tristrami) and rabbits (Oryctolagus cuniculus). The efficacy of the fungal treatment was not limited to a direct effect on the mortality of feeding ticks, but continued during molting (off host) and, in the case of female ticks, the treatment reduced the production of eggs and their hatching rate. The direct control of the on-host stages was relatively low (from 19 to 38%); whereas the effects of the applied fungus on subsequent tick development reduced the yield of the following engorged stages up to 30-63%. Engorged females that dropped from rabbits sprayed with M. brunneum laid 21.5% fewer eggs than the control females. Moreover, these ticks transmitted conidia by contact to the eggs which they laid, resulting a 3-fold reduction in the rate of hatching relative to the control. Based on theoretical cumulative calculations, these results suggest that if the progeny of each unfed stage feed on fungus-sprayed hosts, there will be a 92% reduction in the tick population within one generation. Two spray formulations, one based on mineral oil and another based on a starch-sucrose mixture, significantly enhanced on-host tick control, in comparison with an unformulated conidial suspension. The reduction in the number of nymphs that fed on the treated host and later developed into unfed adults was 54.9% for unformulated conidia, 70.4% for the oil formulation and 86.4% for the starch-sucrose formulation. Increasing the environmental humidity around the gerbils while the ticks fed on them to 90% RH significantly improved the control of the on-host developmental stages, reducing the number of engorged ticks that dropped from fungus-sprayed gerbils 3-fold in comparison with the same animals kept at 30-60% RH. There was no difference between the efficacy of the observed tick control at an ambient temperature of 21°C and that observed at 28°C.

Control of tick populations by spraying Metarhizium anisopliae conidia on cattle under field conditions.

Conidia of the entomopathogenic fungus, Metarhizium anisopliae, in oil/water formulation (1 × 10(8) conidia/ml) were sprayed at 3 weekly intervals on Rhipicephalus evertsi evertsi and Rhipicephalus (Boophilus) decoloratus ticks while feeding on Afrikana bulls grazing in paddocks for a period of 1 year. The fungus reduced the on-host tick populations by 83% 3 month after commencement of the experiment. The formulation by itself had only minimal effect on the tick population. Tick populations and fungal efficacy were highest at the peaks of rainfall and relative humidity or soon thereafter. Fed and unfed adult R. e. evertsi and R. (B.) decoloratus collected at the end of the experiment from the fungus-sprayed and from the control cattle and incubated in the laboratory exhibited a mortality of 93% in oil formulated conidia and 14% in oil control. The corresponding mortality in R. (B.) decoloratus was 100% in fungus and 11% in oil control. Ticks on the fungus-sprayed groups had significantly higher mortality (P < 0.05) than on the control groups. Furthermore, no significant difference (P < 0.05) was observed in fungus-induced mortality between the two tick species. Mortalities induced by Triton X-100 (0.05%), sunflower oil (20%) and water alone were low, suggesting that they were non-toxic to ticks at the concentrations used and no significant difference was observed among them. No physical or behavioral abnormalities were observed in the fungus-sprayed cattle at any time during the course of the experiment. All groups of cattle gained weights during the experimental period.

Experimental transmission of field Anaplasma marginale and the A. centrale vaccine strain by Hyalomma excavatum, Rhipicephalus sanguineus and Rhipicephalus (Boophilus) annulatus ticks.

The cattle rickettsia Anaplasma marginale is distributed worldwide and is transmitted by about 20 tick species, but only Rhipicephalus simus, a strictly African tick species, has been shown to transmit the vaccine strain of A. centrale. The aim of the present study was to examine transmission of field strains of A. marginale and of the vaccine strain of A. centrale by three tick species -Hyalomma excavatum, Rhipicephalus sanguineus and Rhipicephalus (Boophilus) annulatus - to susceptible calves. Two genetically distinct Israeli field strains of A. marginale, tailed and non-tailed (AmIsT and AmIsNT, respectively), were efficiently transmitted by R. sanguineus, whereas H. excavatum transmitted only the tailed isolate, and R. (Boophilus) annulatus did not transmit A. marginale. None of the three tick species transmitted A. centrale. By means of msp1a primers in PCR assays, amplicons of similar sizes were obtained from either A. marginale-infected calves that were used for acquisition feeding, from R. sanguineus fed on the infected calves, or from calves to which anaplasmosis had been successfully transmitted by these ticks. Although an A. centrale-specific fragment was amplified from salivary glands of R. sanguineus, no transmission to susceptible cattle occurred during 3 months of observation, and anaplasmosis was not induced in splenectomized calves that were subinoculated with blood from calves on which R. sanguineus had fed.

Protection of Metarhizium anisopliae conidia from ultra-violet radiation and their pathogenicity to Rhipicephalus evertsi evertsi ticks.

Metarhizium anisopliae conidia were formulated in water or in olive oil containing 3% commercial sunscreens (Everysun or E45 Sun Block 50) and exposed to an artificial UV source for up to 5 hours. Survival of conidia after 5 h of exposure to UV in oil formulation was 29% when protected with Everysun, 40% when protected with E45, and 4% in control. In comparison, survival of conidia formulated in water was 13% when protected with Everysun, 24% when protected with E45, and 0% in control. Furthermore, the influence of sunscreens on conidia viability and virulence to Rhipicephalus evertsi evertsi larvae and unfed adult ticks was evaluated. Adding these compounds to the conidial formulations did not reduce the viability of the conidia. Larval mortality was 95 and 100%, while unfed adult mortality was 90 and 97% after being exposed to unprotected conidia formulated in water or in oil, respectively. Conidia protected by Everysun or E45 formulated in water, induced 88 and 83% mortality in larvae, and 92 and 90% mortality in unfed adults, respectively. Conidia suspended in oil and protected by Everysun or E45 induced 94 and 91% mortality in larvae, and 83 and 81% in unfed adults, respectively. These observations indicate that olive oil and the two sunscreens confer protection to conidia against damages by UV radiation without interfering with their pathogenicity to ticks.

Biological control of ticks.

Ticks have numerous natural enemies, but only a few species have been evaluated as tick biocontrol agents (BCAs). Some laboratory results suggest that several bacteria are pathogenic to ticks, but their mode of action and their potential value as biocontrol agents remain to be determined. The most promising entomopathogenic fungi appear to be Metarhizium anisopliae and Beauveria bassiana, strains of which are already commercially available for the control of some pests. Development of effective formulations is critical for tick management. Entomopathogenic nematodes that are pathogenic to ticks can potentially control ticks, but improved formulations and selection of novel nematode strains are needed. Parasitoid wasps of the genus Ixodiphagus do not typically control ticks under natural conditions, but inundative releases show potential value. Most predators of ticks are generalists, with a limited potential for tick management (one possible exception is oxpeckers in Africa). Biological control is likely to play a substantial role in future IPM programmes for ticks because of the diversity of taxa that show high potential as tick BCAs. Considerable research is required to select appropriate strains, develop them as BCAs, establish their effectiveness, and devise production strategies to bring them to practical use.

Entomopathogenic nematodes for the biocontrol of ticks.

Entomopathogenic steinemematid and heterorhabditid nematodes are increasingly used to control insect pests of economically important crops. Laboratory and field simulation trials show that ticks are also susceptible to these nematodes. The authors review the potential of entomogenous nematodes for the control of ticks.

Factors affecting the virulence of entomopathogenic nematodes to engorged female Boophilus annulatus ticks.

The laboratory trials rank the virulence of entomopathogenic nematode strains (3 heterorhabditids and 6 steinernematids) to engorged female Boophilus annulatus ticks according to 3 parameters of the infection process: the effect of exposure time on tick mortality, the quantity of nematodes that penetrate ticks, and the rate of tick mortality after the injection of 1, 2, or 3 nematodes. Exposure of the ticks to heterorhabditid strains for 6 hr resulted in >80% mortality, but only 20 or 65% mortality after exposure to most steinernematids. The quantity of nematodes recovered per tick exposed to nematodes for 6 days averaged from 16 to 141. For steinernematids, a negative correlation was obtained between tick mortality and the average quantity of nematodes recovered. Injecting 1 infective juvenile from 1 of 2 heterorhabditid strains into each tick resulted in close to 100% mortality. Increasing the quantity of nematodes injected into each tick had little or no additive effect on tick mortality.

Transmission of hepatozoon canis to dogs by naturally-fed or percutaneously-injected Rhipicephalus sanguineus ticks.

Hepatozoon canis is an apicomplexan protozoan parasite of dogs, prevalent in Asia, Africa, and southern Europe. Experimental transmission of H. canis to dogs was performed with laboratory-reared Rhipicephalus sanguineus nymphs that fed on a naturally infected dog or were percutaneously injected with canine blood containing H. canis gamonts. Dogs were inoculated by oral ingestion of adult ticks containing H. canis oocysts. Transstadial transmission of H. canis was recorded, whereas transovarial transmission could not be demonstrated. Oocysts were detected in 85% of the adult ticks that had engorged as nymphs on an infected dog and in 61% of the adult ticks resulting from nymphs injected percutaneously with blood from the same dog. Nine of 12 dogs (75%) inoculated with naturally fed or percutaneously injected ticks became parasitologically positive, and all showed seroconversion. Meronts were initially detected in the bone marrow 13 days postinoculation and gamonts 28 days after infection. The variation in the time of initial detection of parasitemia among infected dogs and the rapid appearance of gamonts in dogs immunosuppressed with corticosteroids suggest that immune mechanisms play an important role in controlling H. canis parasitism. The artificial acquisition of Hepatozoon parasites by percutaneous injection of ticks, demonstrated here for the first time, may serve as a useful tool for studies on transmission, vector-host relationships, and the immunology of infection with Hepatozoon species.

Arthropods as predators of ticks (Ixodoidea).

The existing information on arthropods as predators of ticks is based mainly on sporadic observations and their role in reducing tick populations and in most cases is still not clear. Some reports suggest that in certain ecological habitats arthropods play an important role in the control of the tick population. This publication reports on some 100 relevant publications that appeared between 1906 and 1999. Ants, beetles, and spiders seem to be the major arthropods preying on ticks. In general, engorged ticks are more often preyed upon by arthropods than are unfed or feeding ticks.

Pathogenicity of entomopathogenic fungi to different developmental stages of Rhipicephalus sanguineus (Acari: Ixodidae).

The pathogenicity of 4 species of entomopathogenic fungi (Hyphomycetes species: Beauveria bassiana, Metarhizium anisopliae, Metarhizium flavoviride, and Paecilomyces fumosoroseus) to various developmental stages of Rhipicephalus sanguineus (Acari: Ixodidae) ticks was compared under laboratory conditions. The most virulent isolate, M. anisopliae-108, caused 92-96% mortality to unfed larvae and nymphs on day 7 postinfection (PI) and 100% mortality to unfed adults and engorged females on day 21 PI. The pathogenicity of M. anisopliae-108 to engorged larvae and nymphs was lower--82.6 and 60%, respectively. All tested B. bassiana, M. flavoviride, and P. fumosoroseus isolates were significantly less virulent (P < 0.05) or avirulent toward most life stages of R. sanguineus. The M. anisopliae and M. flavoviride isolates also prevented or reduced the ability of the ticks to lay eggs several days before their deaths. Female ticks infected by the fungi achieved only 11.3-60.8% of their egg-laying capacity compared with the controls.

Mortality rate of adult ticks due to infection by entomopathogenic nematodes.

The mortality of Boophilus annulatus, Hyalomma excavatum, Rhipicephalus bursa, and Rhipicephalus sanguineus adult ticks was recorded after their exposure in petri dishes to 5 entomopathogenic nematode strains. The strains used were DT and Mexican of Steinernema carpocapsae, Hb HP88 of Heterorhabditis bacteriophora, and IS-3 and IS-5 of Heterorhabditis sp. The most rapid killing rates (LT50 and LT90) were observed for B. annulatus ticks (0.8-5.0 days). Most unfed males died 0.3-2.8 days after the unfed females, whereas even more time (0.8-8.0 days) passed before engorged females died. In most bioassays, the IS-3 and IS-5 strains of Heterorhabditis sp. nematodes appear to be the most pathogenic for ticks and, in most cases, killed ticks several days before the other 3 nematode strains. Unfed adult ticks exposed to nematodes died within less time than it takes for adults to complete their prefeeding period after molting.

Antigenic analysis of gamonts of Hepatozoon canis purified from leukocytes.

Hepatozoon canis is a tick-borne apicomplexan parasite of dogs that infects neutrophils and parenchymal tissues. To study the antigenic characteristics of this parasite, a technique was devised for the purification of gamonts from peripheral blood neutrophils. White blood cells were separated on Ficoll-Hypaque density gradients and the gamonts were released from the host neutrophils by nitrogen cavitation. The blood used for purification originated from dogs with natural or experimental infections of H. canis with a parasitemia of 1.4-33%. The number of parasites collected ranged from 1.5 X 10(6) to 4.2 X 10(7). Portions of purified gamonts were separated and examined under phase and scanning electron microscopy, and the remaining purified parasites were then used as a source of antigens to characterize the humoral immune response by western blot analysis. Serum antibodies from infected dogs recognized more than 15 gamont antigens, and the antigenic patterns observed with sera from naturally and experimentally infected dogs were nearly similar. Four immunodominant protein bands of relative molecular weights of 107, 88, 63, and 28 kDa were recognized by all of the sera examined. The technique applied here for the isolation of host cell-free gamonts will facilitate studies on antigenic composition and immune responses against H. canis and on antigenic relationships between Hepatozoon from different host species and geographic regions.

Biocontrol of ticks.

The increasing resistance of arthropodes to pesticides, their high price, and the growing public demand for safer food and a cleaner environment are obliging animal growers to minimize the use of pesticides by introducing alternative means for tick control. The development of a biological tick control method has been neglected as compared to the control of plant pests or dipterous insects harmful to men and animals. There are abundant observations, but only a few studies have as yet been conducted on pathogens, parasitoides, and predators of ticks. A first attempt at tick biocontrol was made with the introduction of tick-parasitic wasps from France to the USA and Russia. During the past decade, interest in developing antitick biocontrol agents such as birds (Brazil, Kenya, and Zimbabwe), parasitoides (Kenya and USA), entomopathogenic nematodes (Egypt, Israel, Guadeloupe, and USA), entomopathogenic fungi (Brazil, Cuba, Israel, Kenya, and USA), and bacteria (Brazil) has gained momentum. The reintroduction of oxpecker birds in some areas of Zimbabwe remains up to now the only known successful attempt at tick biocontrol.

Laboratory evaluation of pathogenicity of entomogenous nematodes to African tick species.

Five strains of entomogenous nematodes, Steinernema carpocapsae strains DD, Mexican, SR, and Heterorhabditis bacteriophora strains 1S5 and HP88, were tested for their pathogenicity to various developmental stages of five African tick species namely; Rhipicephalus appendiculatus, R. evertsi, Amblyomma variegatum, A. gemma, and Boophilus decoloratus. In engorged female R. appendiculatus, all nematodes at a concentration of 1,000 infective juveniles (IJ)/dish, except S. carpocapsae Mexican strain, induced high mortalities (56-100%), whereas in engorged female R. evertsi, only S. carpocapsae DD and H. bacteriophora HP88 induced high mortalities (78% and 56%, respectively). In engorged B. decoloratus, S. carpocapsae DD, Mexican, SR and H. bacteriophora HP88 (100 IJ/dish) induced mortalities of 85%, 65%, 80%, and 100%, respectively. In all cases, except for S. carpocapsae Mexican strain, a higher concentration (5,000 IJ/dish) did not result in higher mortality than occurred with 1,000 IJ/dish. Unfed females and immature stages of ticks were found to be generally resistant to the nematodes. The feasibility of using entomogenous nematodes for biological control of African tick species are briefly discussed.

Biocontrol of ticks by entomopathogenic nematodes. Research update.

Entomopathogenic nematodes (EPNs) are lethal to ticks even though they do not use their normal propagation cycle within tick cadavers. The tick Boophilus annulatus was found to be far more susceptible to EPNs than Hyalomma excavatum, Rhipicephalus bursa, or Rhipicephalus sanguineus. Ticks seem to be less susceptible to nematodes when feeding on a host. Preimaginal tick stages were less susceptible to nematodes than adult ticks. The mortality rate of unfed females was highest, followed by unfed males, and engorged females. The virulence of nematodes to ticks varied greatly among different nematode strains. In most cases, the Heterorhabditis sp. strains were the most virulent strains tested in petri dishes. In buckets containing sandy soil sprayed with 50 nematodes/cm2 and engorged B. annulatus females, the LT50 of the ticks was less than five days. The addition of manure to soil or a manure extract to petri dishes reduced nematode virulence. Since ticks spend most of their life cycle in the upper humid layer of the ground, and many nematode strains share this same ecological niche, the use of EPNs for biocontrol of ticks appears promising.

Efficacy of entomopathogenic nematode strains against engorged Boophilus annulatus females (Acari: Ixodidae) under simulated field conditions.

The biocidal efficacy of entompathogenic nematodes against engorged females of Boophilus annulatus (Say) was evaluated in soil-filled buckets in a greenhouse, where conditions resemble nature. The 9 tested nematode strains differed markedly in their effect upon tick mortality. The Mexican strain of Steinernema carpocapsae was most efficient, inducing 100% tick mortality at a concentration of 50 nematodes per square centimeter. An increase in the concentration of the S. carpocapsae DT strain to > 200 IJ/cm2 failed to kill more ticks. It appears that entomopathogenic nematodes show promise as tick control agents.

Interaction between ticks (Acari: Ixodidae) and pathogenic nematodes (Nematoda): susceptibility of tick species at various developmental stages.

The virulence of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) to tick species under laboratory conditions is reported. The susceptibility of larval, nymphal, and adult stages of the ticks Hyalomma excavatum (Koch), Rhipicephalus bursa (Canestrini & Fanz), and R. sanguineus (Latereille) to 2 strains of Steinernema carpocapsae and 3 strains of Heterorhabditis bacteriophora were compared in laboratory assays. Preimaginal stages of ticks were found to be more resistant to the nematodes than were adult ticks which exhibited 80-100% mortality in a dish containing 5,000 infective juveniles of H. bacteriophora IS-3 or IS-5 strains isolated in Israel. These 2 strains were found to be much more virulent to unfed adult ticks than were the other isolates. No marked difference was found between engorged ticks and unfed adults of R. sanguineus or H. excavatum in terms of mortality, whereas engorged males and unfed females of R. bursa were significantly more susceptible than unfed males or engorged females.