Serologically defined Rhipicephalus (Boophilus) microplus larval antigens in BmLF3, a partially pure Sephacryl S-300 fraction of crude larval proteins.

This report is designed to provide additional information regarding larval soluble proteins toward the planned development of a comprehensive database of Rhipicephalus (Boophilus) microplus proteins that elicit a humoral immune response in cattle as a result of natural ectoparasite infestation. Larval proteins of R. microplus are complex and the protein profile is not dominated by any major proteins. This report focuses upon an S-300 Sephacryl (molecular sieve) column fraction, fraction 3 (BmLF3). With the use of SDS-PAGE (without-2ME) and Western blotting with a composite pool of pre- and post-R. microplus larval infestation antiserum BmLF3 was found to contain 7 apparent common ixodid major antigens (207.3, 171.9, 98.0, 86.5, 65.7, 58.9, and 38.0 kDa), those potentially shared with other ixodid species, and 2 apparent R. microplus specific antigens evidenced by low-level antibody binding in crude BmLF3 (149.4 kDa) and HPLC peak 8 of BmLF3 (116.0 kDa). In addition, BmLF3 contains potent inhibitors of trypsin activity. However, these inhibitors of trypsin did not appear to elicit host antibodies as a result of natural ectoparasite exposure, as defined by Western blotting of reduced and denatured trypsin binding proteins purified by affinity chromatography.

Identification and partial purification of serologically defined Boophilus microplus larval antigens by natural ectoparasite exposure.

In an effort to identify life-stage specific Boophilus microplus proteins that elicit a humoral response in cattle, soluble proteins were extracted from 10- to 14-day-old larvae and subsequently fractionated by size-exclusion chromatography and reverse-phase high pressure liquid chromatography. Several antigens were identified by Western blotting as potentially shared with other ixodid tick species since antibodies to these proteins were present in sera of calves not previously exposed to B. microplus. Six putative B. microplus-specific antigens were identified by antibodies in the sera of calves repeatedly exposed to B. microplus larvae. One of the antigens, a 19.1 kDa protein, was used in the development of a diagnostic kELISA for previous exposure to B. microplus. The 19.1 kDa protein did not have tryptic protease activity or inhibit bovine trypsin activity, but appeared to be allergenic in that a partially pure fraction elicited immediate-type hypersensitivity responses in calves previously exposed to B. microplus.

Biochemical diagnosis of organophosphate-insensitivity with neural acetylcholinesterase extracted by sonication from the adult tick synganglion.

A sonication method for the homogeneous extraction of acetylcholinesterase (AChE) from the synganglia of adult ticks is described. The method provides for the extraction of sufficient AChE for multiple assays of enzyme activity in the presence of specific organophosphate (OP) inhibitors for the rapid diagnosis of OP-insensitivity and assignment of homozygous susceptible (SS), heterozygous resistant (RS), and homozygous resistant (RR) genotypes to individual ticks. A single synganglion from adult ticks of either gender and various stages of feeding can successfully be used for AChE extraction. The study presents the results of diagnostic screening of four Boophilus microplus strains for OP-insensitivity. The extraction method and these findings should find utility in support of researchers involved in the mitigation of acaricide resistance in tick populations worldwide, and in particular, the Cattle Fever Tick Surveillance and Quarantine Program maintained by USDA-APHIS/Veterinary Services along the south Texas border that prevents reentry of Boophilus spp. into the United States from endemic populations in Mexico.

Factors that influence the prevalence of acaricide resistance and tick-borne diseases.

This manuscript provides a summary of the results presented at a symposium organized to accumulate information on factors that influence the prevalence of acaricide resistance and tick-borne diseases. This symposium was part of the 19th International Conference of the World Association for the Advancement of Veterinary Parasitology (WAAVP), held in New Orleans, LA, USA, during August 10-14, 2003. Populations of southern cattle ticks, Boophilus microplus, from Mexico have developed resistance to many classes of acaricide including chlorinated hydrocarbons (DDT), pyrethroids, organophosphates, and formamidines (amitraz). Target site mutations are the most common resistance mechanism observed, but there are examples of metabolic mechanisms. In many pyrethroid resistant strains, a single target site mutation on the Na(+) channel confers very high resistance (resistance ratios: >1000x) to both DDT and all pyrethroid acaricides. Acetylcholine esterase affinity for OPs is changed in resistant tick populations. A second mechanism of OP resistance is linked to cytochrome P450 monooxygenase activity. A PCR-based assay to detect a specific sodium channel gene mutation that is associated with resistance to permethrin has been developed. This assay can be performed on individual ticks at any life stage with results available in a few hours. A number of Mexican strains of B. microplus with varying profiles of pesticide resistance have been genotyped using this test. Additionally, a specific metabolic esterase with permethrin-hydrolyzing activity, CzEst9, has been purified and its gene coding region cloned. This esterase has been associated with high resistance to permethrin in one Mexican tick population. Work is continuing to clone specific acetylcholinesterase (AChE) and carboxylesterase genes that appear to be involved in resistance to organophosphates. Our ultimate goal is the design of a battery of DNA- or ELISA-based assays capable of rapidly genotyping individual ticks to obtain a comprehensive profile of their susceptibility to various pesticides. More outbreaks of clinical bovine babesisois and anaplasmosis have been associated with the presence of synthetic pyrethroid (SP) resistance when compared to OP and amidine resistance. This may be the result of differences in the temporal and geographic patterns of resistance development to the different acaricides. If acaricide resistance develops slowly, herd immunity may not be affected. The use of pesticides for the control of pests of cattle other than ticks can affect the incidence of tick resistance and tick-borne diseases. Simple analytical models of tick- and tsetse-borne diseases suggest that reducing the abundance of ticks, by treating cattle with pyrethroids for example, can have a variety of effects on tick-borne diseases. In the worst-case scenario, the models suggest that treating cattle might not only have no impact on trypanosomosis but could increase the incidence of tick-borne disease. In the best-case, treatment could reduce the incidence of both trypanosomosis and tick-borne diseases Surveys of beef and dairy properties in Queensland for which tick resistance to amitraz was known were intended to provide a clear understanding of the economic and management consequences resistance had on their properties. Farmers continued to use amitraz as the major acaricide for tick control after the diagnosis of resistance, although it was supplemented with moxidectin (dairy farms) or fluazuron, macrocyclic lactones or cypermethrin/chlorfenvinphos.

Distribution of horn flies on individual cows as a percentage of the total horn fly population.

Twenty-three mixed-breed herd cows were phenotyped for their ability to serve as a suitable host for Haematobia irritans, the horn fly. Based upon consistent observations within the lower quartile or upper quartile of individual fly counts, four cows were phenotyped as low carriers and five cows were phenotyped as high carriers of horn flies. The cows designated as low carriers consistently carried levels of flies below the economic threshold. However, during a period of fly population explosion, low carriers harbored flies well above the economic threshold. Although the number of flies counted on these low carrying cattle increased as the population increased, the relative percentage of the population that they carried changed very little. A hypothesis is proposed to explain this observation, and future studies are suggested.

Isolation and identification of an esterase from a Mexican strain of Boophilus microplus (Acari: Ixodidae).

A strain of Mexican Boophilus microplus (Cz) collected near Coatzacoalcos, Veracruz, Mexico, exhibits a moderate, but significant, level of permethrin resistance. Unlike other highly permethrin resistant strains, the Cz strain does not have a mutation within the sodium channel gene that results in target-site insensitivity. However, the Cz strain possesses a substantial increase in general and permethrin esterase activity relative to highly permethrin resistant and control strains suggesting the involvement of a metabolic esterase(s) in the expression of permethrin resistance. We report the isolation of a 62.8 kDa protein from Cz strain larvae that we think is the esterase previously reported as Cz EST9. In addition, internal amino acid sequence data obtained from the 62.8 kDa protein suggest that it is the gene product of a previously reported B. microplus carboxylesterase cDNA. We propose that the 62.8 kDa protein (Cz EST9) has permethrin hydrolytic activity and as a result plays an important role in Cz strain resistance to permethrin.

Variation in general esterase activity within a population of Haematobia irritans (Diptera: Muscidae).

Control of the horn fly, Hematobia irritans (L.), is generally dependent on chemical insecticides. However, the biology and behavior of the horn fly favors rapid development of insecticide resistance. To prolong the effectiveness of the insecticide option, information is required regarding the mechanisms of insecticide resistance. Metabolic hydrolysis of insecticides by esterases is a detoxification mechanism in many insect species. Measurement of general esterase activity within populations of horn flies may provide a diagnostic tool for resistance management. In this study we evaluated the amount of variation in general esterase activity within female and male horn fly samples from a population that had not been exposed to insecticides for 8 yr. We found considerable variation in general esterase activity within samples of each sex, with females demonstrating the greater variation. The observed variation is thought to be the result of age-structure dynamics within the population. The amount of inherent variation makes it difficult to detect small mean differences between populations, thus limiting the utility of general esterase assays. Thus, effective diagnosis of esterase-mediated resistance mechanisms can only be achieved by the identification of specific detoxification esterases and the design of assays, either biochemical or molecular, for their detection and measurement.

Myiasis due to Hypoderma lineatum infection mimicking the hypereosinophilic syndrome.

Myiasis is the infestation of live humans with larvae of Diptera (true flies). This report describes a protracted illness caused by infestation with Hypoderma lineatum, resembling the hypereosinophilic syndrome. A 35-year-old man had a 9-month multisystemic illness with pronounced eosinophilia, pleuritis, pericarditis, and myositis. Treatments including glucocorticoids did not alter the disease. Diagnostic studies included computed tomography, 2-dimensional echocardiography, leukocyte count, surgical biopsy of skin and muscle, blood immunoglobulin levels, and blood chemistry. Myiasis was recognized when a worm emerged from the patient's skin; after a second worm emerged, the patient's symptoms disappeared rapidly. Other determinations included IgE and IgG levels specific for H lineatum, Western blot, and immunofluorescence for eosinophil major basic protein; IgG antibodies to H lineatum decreased after emergence of the worms. The patient's symptoms mimicked the hypereosinophilic syndrome but resolved when the myiasis became apparent. Specific serologic analyses can identify infected patients, and ivermectin may be useful as treatment.

Evaluation of horn flies (Diptera: Muscidae) from a pyrethroid susceptible colony for general and permethrin esterase activities.

In this study we describe a nonradioactive single-fly microassay for permethrin hydrolysis. We used this assay with a microplate assay for general esterase activity to evaluate the permethrin hydrolyzing and general esterase activities of aging pyrethroid-susceptible male and female horn flies, Haematobia irritans (L.). We found substantial gender- and age-related differences regarding general esterase activity, permethrin sensitivity, and permethrin hydrolyzing activity within the colony. Extracts of female flies collected 48 h after receiving their first blood meal yielded significantly greater esterase activity than male extracts. Aging female flies were more tolerant of permethrin than were male flies. In addition, a positive correlation was found to exist between the general esterase activity of aging females and their ability to hydrolyze permethrin.

Identification and purification of a 16-kDa allergen from Psoroptes ovis (Acarina: Psoroptidae).

Psoroptes ovis, (Hering), the sheep scab mite, is the causative agent of an allergic dermatitis of sheep and cattle. Recent studies of the host immune response to this ectoparasite have provided information that suggests control may be achieved by immune intervention. A significant effector in protection of the host from clinical lesions is host behavioral grooming. Host grooming is believed to be intensified by a pruritic immediate hypersensitivity response to mite allergens. Knowledge of potential P. ovis allergens is limited. This article reports on the identification and SDS-PAGE continuous elution purification of a 16-kDa polypeptide that elicits immediate type hypersensitivity in calves and has sequence homology with known group II mite allergens, Lep d 2 of Lepidoglyphus destructor (Schrank), and Der f II of Dermatophagoides farinae (Hughes). This P. ovis allergen appears to be a good vaccine candidate for further study and cloning.

Immunological control of arthropod ectoparasites--a review.

The control of arthropod ectoparasites of livestock by systemically delivered chemicals was introduced in the 1950s. Their low cost, ease of use, and high level of efficacy ensured dependence upon them for ectoparasite control. However, current societal and scientific concerns regarding dependency upon chemicals have emphasised the need for the evaluation of environmentally safe alternatives for ectoparasite control. Immunological intervention for the control of ectoparasite populations, either through the selection of animals with resistant genotypes or vaccination, is consistent with principles of sustainable agriculture. Unlike the activity of chemicals, currently available ectoparasite vaccines do not induce a rapid knockdown of the parasite population and they do not protect the individual from parasitism. However, if these vaccines are used in an integrated pest-management programme, they have the potential to reduce parasite populations over successive generations and reduce or eliminate the need for chemical application.

Evaluation of natural Psoroptes ovis (Acarina: Psoroptidae) soluble proteins as candidate vaccine immunogens.

In this study potential vaccine candidate immunogens were identified and evaluated in a vaccine challenge trial. Calves vaccinated with a partially purified fraction of Psoroptes ovis-soluble proteins had 8 of 14 calves free of palpable lesions 8 wk after a challenge infestation. A self-grooming behavioral response elicited by a pruritic immediate-type allergic reaction was believed to be an effector in protecting the vaccinated calves from a clinical P. ovis infestation.

Thermal requirements for Hypoderma lineatum (Diptera:Oestridae) egg development.

This study was designed to define lower thermal limits for common cattle grub, Hypoderma lineatum (Villers), egg development. The data collected in this study suggest that embryonic development was prolonged as temperature was lowered. No hatch was observed when eggs were incubated at a constant temperature of 20 degrees C. Measurements of cattle skin temperature and ovipositional behavior of the gravid female fly suggest that eggs are oviposited in a suitable thermal environment for successful embryonic development. Eggs oviposited on cattle held in an unheated barn hatched within 1 wk confirming that cattle can be successfully laboratory infested in unheated barns during winter in central Texas.

Warble stage development of third instars of Hypoderma lineatum (Diptera: Oestridae).

Hypoderma lineatum (Villers), the common cattle grub, is an insect parasite that resides in a warble in the subcutaneous tissues of the backs of cattle during a portion of their life cycle. Inside the warble, the larva undergoes 2 molts to the 3rd instar. In this study, the development of the posterior spiracular plates of the 3rd instar of H. lineatum was observed in situ. Larvae were observed to molt to the 3rd-instar phase 1 stage of development 28.6 +/- 3.9 d (+/- SD) after digesting a breathing hole in the backs of previously uninfested calves. Development of the spiracular plates through each of the various recognizable stages occurred on a 5-6-d interval. It took 54.2 +/- 5.1 d in the back for larvae to develop to the phase 3 stage, the stage reached before larvae exit the host. The average elapsed time from the 3rd-instar phase 3 stage to exit from the host was 5.5 +/- 2.9 d. Of 22 larvae that were followed from arrival in the back to pupariation, the elapsed time was 59.4 +/- 6.1 d. Most larval mortality occurred in the back during the 1st and 2nd instar. Of larvae surviving to the 3rd instar, 86.7% successfully exited from the host. Of 3rd instars surviving to the phase 2 goldplate or phase 3 stage, 93.3% exited successfully from the host.

Development of resistance to Hypoderma lineatum (Diptera: Oestridae) within a cattle herd.

Three experimental infestations of a herd of 27 cattle with the common cattle grub, Hypoderma lineatum, are described during a 4-yr period. The mean percentage of survival of larvae during internal migration was 67.7% in the initial infestation. In the 2nd infestation 1 yr later, the mean percentage of survival of larvae decreased to 40.5% during internal migration. Although fewer larvae survived to reach the tissues in the back in the 2nd infestation, more larvae in the back tissues survived (27.2%) and 53 more mature larvae (potential adults) were produced during the 2nd than the 1st infestation. The 3rd infestation resulted in no further decrease in larval survival to the back tissues (43%), but significant larval mortality in the back (5.7% survival) reduced the number of mature larvae. After 2 infestations, larval survival to the back tissues had stabilized at approximately 40%, whereas the significant decrease in larval survival in the back tissues during the 3rd infestation indicated that resistance manifested at this stage of the parasite life cycle may be important for H. lineatum population control. We can conclude that development of herd resistance through H. lineatum exposure may require several infestation cycles.

Effects of adjuvants on bovine humoral and cellular responses to hypodermin A.

Hypodermin A, a serine protease of the first-instar larva of the common cattle grub, Hypoderma lineatum (Villers), when formulated with complete Freund's adjuvant and administered to naive calves, will elicit protective immunity defined by an increase in in vivo larval mortality. This study evaluated two veterinary acceptable adjuvants, alhydrogel and amphigen (alone and in combination), for suitability as an adjuvant for hypodermin A. Complete Freund's adjuvant (CFA) is not an acceptable adjuvant for use because of adverse reactions at the injection site. The veterinary acceptable adjuvants were not as effective as CFA in inducing an antibody response as detected in the peripheral circulation. Of the adjuvants evaluated, the mixture of alhydrogel and amphigen induced the highest serum antibody response to hypodermin A. All adjuvants evaluated induced comparable immediate-type skin test responses, and the mixture of alhydrogel and amphigen was most comparable with CFA in terms of delayed-type skin reaction and resultant cellular infiltration at the reaction site. Although the mixture of alhydrogel and amphigen, when compared with CFA, did not elicit comparable levels of responsiveness in all parameters tested, the overall performance of the mixture suggests it to be worthy of further efficacy investigation in a vaccine formulation with hypodermin A.

Proteolytic cleavage of bovine IgG by hypodermin A, a serine protease of Hypoderma lineatum (Diptera: Oestridae).

Cattle grub larvae are able to survive a lengthy 8 1/2-mo internal migration within the naive bovine host connective tissues. Larval survival is observed even in the presence of host immunological responses to principal parasite proteins. In this study, data are presented that suggest that a digestive enzyme, hypodermin A, of the first-instar larvae effectively cleaves bovine immunoglobulin G in vitro. A (fab)'2 fragment is produced, and the heavy-chain fragments associated with the cleaved fc piece remain intact. Hypodermin A is not specific for bovine IgG as other species' IgGs are also degraded as are other bovine proteins. Destruction of the biological activity of bovine immunoglobulin G by hypodermin A suggests a potential parasite escape mechanism that may contribute to the survival of first-instar larvae within the bovine host.

Recombinant vaccine development: a novel approach to ectoparasite control. A review of development of a recombinant vaccine for hypodermosis based on hypodermin A.

Cattle grubs (Hypoderma lineatum and H. bovis) are obligate parasites of cattle for most of their one year life cycle. Previously exposed animals become resistant to productive reinfestation, presumably as a result of immune system involvement, suggesting potential control by vaccination. Research progress towards development and utilization of a recombinant subunit vaccine for hypodermosis is described.

Kinetic development and decline of antiscrewworm (Diptera: Calliphoridae) antibodies in serum of infested sheep.

Sheep infested with screwworms, Cochliomyia hominivorax (Coquerel), produced specific serum antibodies detectable by ELISA. Significant antibody levels were found beginning at 1 wk after infestation and persisting for 2 mo, with peak levels at 3 wk after infestation. Mean response values and corresponding larval survivorship were similar over a range of 25 to 200 larvae per individual sheep. Anomalous seropositive responses in some noninfested sheep suggested the possibility of cross-reactivity between antigens of screwworm and those of related myiasigenic blowflies.

Antigen-specific lymphocyte proliferative responses in vaccinated and Hypoderma lineatum-infested calves.

Cattle infested with the common cattle grub, Hypoderma lineatum (Villers) develop specific humoral antibodies and a cellular immune reaction, defined by delayed-type hypersensitivity, to purified H. lineatum proteins. This investigation was designed to study the antigen-specific bovine lymphocyte response to hypodermin A (HyA), a serine protease of larval first-instar H. lineatum. Calves were vaccinated with either native or denatured HyA, and challenge-infested with H. lineatum. The kinetic development of a cellular immune response to HyA was monitored during vaccination and infestation. The HyA-specific responses were highly variable and weak during vaccination and infestation. Although HyA-specific lymphocyte blastogenic responses were observed, no correlation was noted between the magnitude of antigen-specific, peripheral lymphocyte proliferation and larval mortality. In striking contrast to responses observed during infestation, intense HyA-specific lymphocyte responses were observed with 3 calves 6 months after recovery from infestation. In addition, those responses were further heightened by a 250 micrograms booster injection of pure HyA.