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Induction of intradermal skin reactions in the bovine by fractionated proteins of Hypoderma lineatum.

Protein species found in crude extracts of Hypoderma lineatum (Villers) 1st-instar gullet-stage larval homogenates were fractionated by chromatography and further identified by conventional polyacrylamide gel electrophoresis (PAGE) and SDS/PAGE techniques. Seven major fractions were resolved by ion exchange chromatography. Conventional PAGE of the crude antigen preparation revealed a minimum of 14 protein species, while SDS/PAGE revealed 3 major protein species. The ability to elicit cutaneous reactivity by isolated proteins was determined by the intradermal skin test. An immediate-type hypersensitivity reaction was elicited in the skin of vaccinated and previously infested animals by the components of the 4 protein fractions tested. Only one protein fraction, fraction 4, elicited an apparent delayed reaction at 48 h in vaccinated and previously infested animals. A non-specific background reaction was elicited in control animals by the fractionated proteins and was most notable between 1- and 4-h post-injection.

Animals↗

Effect of dose and route of application on the action of decamethrin against cattle grubs.

The efficacy of pour-on or orally administered decamethrin against bovine hypodermosis was investigated on naturally infested yearlings. Treatment at dose rates of 1, 2 or 10 mg per kg body weight were ineffective, but the highest dose had a long-term lethal effect on warbles. The severe side effects which followed the high dose suggest that this drug is unsatisfactory for systemic grub control. Investigations on the dynamics of circulating anti-Hypoderma antibodies in the post-treatment period showed that there is some sub-lethal activity of decamethrin on the larval physiology even at the lowest doses.

Administration, Oral↗

New methods of applying drugs for the control of ectoparasites.

A brief history of the development of methods used to apply drugs (commonly called insecticides or acaricides) to domesticated animals for the control of ectoparasites is presented. The focus is on methods used in the United States for treating cattle, sheep and goats, and swine. Details of the evolution of treatment methods are presented for lice, flies, cattle grubs, keds, ticks, and other arthropods. In general, treatment methods have undergone some change over the years; while, in contrast, treatment materials have changed considerably. Three trends in treatment methods become obvious - (1) Gradual reduction in the amount of treatment material applied dermally from spray or dip to pouron, to spoton, and to insecticide-impregnated ear tags, (2) Increasingly wider use of animal systemic insecticides administered orally or percutaneously to control a variety of ectoparasites, and (3) The development of new methods (sustained release devices) to lengthen the residual effectiveness of treatments. Unfortunately, lengthened residual effectiveness may lead to resistance of important ectoparasites to the treatment materials.

Animals↗

Immunization of cattle against hypodermatosis (Hypoderma lineatum (deVill.) and H. bovis (L.)) using H. lineatum antigens.

The feasibility of immunizing calves against Hypoderma lineatum and H. bovis with a crude H. lineatum larval extract or culture-derived antigen was investigated. Larval survival was followed through pupation. Grub appearance in the backs of both of the immunized groups was found to be 50% of that in the control groups. First appearance of H. bovis was significantly retarded in both immunized groups; however, H. lineatum was unaffected. Duration of H. bovis dropping was also shorter in animals receiving crude extract than in controls while duration of dropping of H. lineatum was not influenced by immunization. Survival of H. lineatum larvae to pupation was significantly reduced in animals immunized with crude larval extract, but not in those receiving culture-derived antigen. H. bovis survival was reduced by both treatments.

Animals↗

Lymphocyte responsiveness in cattle previously infested and uninfested with Hypoderma lineatum (de Vill.) and H. bovis (L.) (Diptera: Oestridae).

Cattle exposed to their third consecutive warble (Hypoderma lineatum and H. bovis) infestation had significantly reduced apparent and accumulative grub populations and produced significantly fewer grubs than animals exposed to their first infestation. These resistant animals had enhanced reactivity to mitogens (concanavalin A (con A) and pokeweed) upon reinfestation and a strong antigen-specific response at 2 months post-infestation. Their responsiveness to con A and antigen was also enhanced at 7 months post-infestation when grubs appeared in the backs, and the response to antigen continued at 10 months post-infestation. Responsiveness to mitogens in previously uninfested animals was similar to the control response throughout the infestation apart from the suppression of the con A response at 7 months post-infestation. These animals finally responded to antigen at 10 months post-infestation. These results suggest that acquired resistance to hypodermosis has a cellular basis with participation of both B- and T-cell components.

Animals↗

Antigenicity and immunogenicity of Hypoderma lineatum soluble proteins in the bovine host.

Protein species found in soluble crude extracts of Hypoderma lineatum (common cattle grub) 1st-instar larvae (HL1) were separated by non-denaturing and denaturing polyacrylamide gel electrophoresis (PAGE) and analyzed for antigenicity by Western blotting using serum from H. lineatum-infested and vaccinated cattle. All HL1 proteins resolved by non-denaturing PAGE were found to be antigenic in the infested bovine host. Treatment of the proteins with sodium dodecyl sulfate and 2-mercaptoethanol destroyed the ability of hypodermin B and the Peak 2 proteins from DEAE-ion exchange HPLC to be bound by antibody. The principal proteins, hypodermin A and hypodermin C (collagenase), appear to be the most immunogenic of the larval proteins. Although having similar amino acid composition, hypodermin A did not appear to share an antigenic epitope with the most prevalent protein, hypodermin C. These results may allow for the selection of proteins to be used in vaccine trials and studies of protective immunological mechanisms associated with acquired resistance to H. lineatum infestation in the bovine host.

Animals↗

Effect of ivermectin treatment on anti-hypodermin C titers of Asturiana cattle naturally infected with Hypoderma lineatum.

The collagenase (hypodermin C) from soluble crude extracts of Hypoderma lineatum 1st-instar larvae was purified by reverse-phase HPLC and used in a new indirect ELISA test. This pure protein had several advantages over the use of crude larval extracts allowing a much better discrimination between infested and non-infested cattle. The anti-hypodermin C titers of 19 Asturiana cattle were estimated over the course of a natural H. lineatum infestation cycle, in which the effect of ivermectin treatment was also investigated. The results showed differences in the onset and ending of the infestation with respect to those described for other European countries. The ivermectin treatment proved to be very effective and treated animals had relatively low anticollagenase titers.

Animals↗

Use of pooled serum or milk samples for the epidemiological surveillance of bovine hypodermosis.

An enzyme-linked immunosorbent assay (ELISA) was used on pooled serum and milk samples to determine whether hypodermosis could be detected where a larger sero-epidemiological survey was required. This study was undertaken to assess the potential of this assay for testing sera on milk samples, pooled from 10 cows, and determining the period of the year when detection was optimal. The sensitivity of the assay was determined by increasingly diluting a positive serum with pooled negative sera, from 1:10 to 1:100. The diagnostic lower limit of the assay requires at least two serological reactors within a herd of 100. The kinetic development and depletion of anti-Hypoderma antibody of individual and pooled sera or milk from 30 cows was evaluated from November to July. Anti-Hypoderma antibody levels of two groups of 8 calves, one control and one teated with ivermectin (Ivomec), were tested from October to June. These preliminary results indicate that an ELISA assay on serum or milk samples pooled from 10 cows can be used between February and April to evaluate the prevalence of hypodermosis within cattle herds in France, demonstrating the feasibility of using pooled serum already collected for bovine leucosis testing.

Animals↗

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.

Animals↗

Enhanced resistance to cattle grub infestation (Hypoderma lineatum de Vill.) in calves immunized with purified hypodermin A, B and C plus monophosphoryl lipid A (MPL).

The influence of an antigen-specific cellular and humoral immune response, stimulated by immunization, on survival of a challenge infestation of Hypoderma lineatum was investigated. Calves immunized with a purified combination of hypodermin A, B and C plus monophosphoryl lipid A (MPL) developed a strong antigen-specific cellular immune response by completion of the immunization schedule which persisted to 12 weeks post-infestation. Responsiveness of peripheral blood lymphocytes to the mitogens concanavalin A and pokeweed was also elevated at 4 and 12 weeks post-infestation. Western blot analysis at the time of maximum grub counts demonstrated that immunized calves responded to hypodermin A, B and C while those receiving only MPL or infested controls responded only to hypodermin B and C. The antigen-specific antibody response as measured by ELISA at maximum grub count was significantly higher in vaccinated calves than in infested controls while the response in calves receiving only immunostimulator was also significantly elevated. Immunized (antigen plus MPL) calves produced 5.0 +/- 6.9 grubs per animal which successfully pupated while those receiving MPL alone produced 16.4 +/- 6.1 and infested controls produced 32.2 +/- 10.9 grubs per animal.

Animals↗

Moxidectin: systemic activity against common cattle grubs (Hypoderma lineatum) (Diptera: Oestridae) and trichostrongyle nematodes in cattle.

Moxidectin, a systemic insecticide, was evaluated for its efficacy against the migrating first instars of the common cattle grub, Hypoderma lineatum, and against nematode egg production in beef cattle. It was observed that all three levels (0.1, 0.2 and 0.4 mg moxidectin kg-1) were 100% effective against cattle grubs when administered as a s.c. injection. The same levels of treatment were very effective (90-100%) in reducing trichostrongyle nematode egg production. However, there was a slight indication that at least one species, Cooperia oncophora, was not completely eliminated, as it was observed that small numbers of eggs began to appear after 2 weeks post-treatment when there had been no opportunity for reinfection.

Animals↗

Oral and parenteral administration of ivermectin to reindeer.

The anti-parasitic effect of the orally administered paste formulation of ivermectin (Ivomec) in reindeer was evaluated by means of a trial designed to compare the efficacies of orally and s.c. administered ivermectin at the same dosage (0.2 mg kg-1 body weight) in naturally infected adult reindeer (n = 92). Both formulations were 100% effective against larvae of the warble fly, Oedemagena tarandi, while oral treatment was less efficacious than s.c. injection against parasitic nematodes. Both formulations, but particularly the injectable ivermectin treatment, increased the weight gain of pregnant females compared to that of those not treated.

Administration, Oral↗

Efficacy of doramectin against warble fly larvae (Hypoderma bovis).

The efficacy of doramectin in the treatment of cattle harbouring naturally acquired infestations of first, second and third instar larvae of Hypoderma bovis was determined in two studies carried out in the Burgundy region of France. In the first study, cattle on six farms with a history of H. bovis infestations were treated during October 1989 with either doramectin at a dose of 200 micrograms kg-1 liveweight (186 animals) or with an equivalent volume of saline (157 animals). During the following March and April, all animals were examined for the presence of warbles. In the second study, cattle on four farms with warbles present in their backs were treated during March 1990, with either doramectin or saline (as before). The viability of larvae within each warble on all the animals was then assessed every 2 days for 14 days. In Study 1, no warbles were present in any of the doramectin-treated cattle at any time, whereas warbles were found in 135 saline-treated animals. In Study 2, all larvae in warbles on the backs of the doramectin-treated cattle were dead by Day 14 after treatment, whereas viable larvae were still present in warbles in the backs of all saline-treated cattle. No adverse reaction to doramectin treatment was observed in any animal at any time. It was concluded that doramectin is both safe and 100% efficacious in the treatment of first, second and third instar H. bovis infections of cattle.

Analysis of Variance↗

The efficacy of moxidectin 0.5% pour-on against Hypoderma bovis in naturally infested cattle: parasitological and serological data.

The field efficacy of moxidectin 0.5% pour-on formulation was assessed in heifers naturally infested with Hypoderma bovis. After their first grazing season in an infested area 34 heifers were selected on the basis of results of an ELISA test performed in January 1992. Twenty-five animals were treated with moxidectin 0.5% pour-on at a dose of 0.5 mg kg-1 bodyweight and nine heifers were left untreated. No local or general side-effects associated with the treatment were noticed. In April, seven of the nine untreated heifers were affected by warble fly, and in May all were infested; none of the treated animals harboured warble fly at any time during the trial. All the extracted larvae were identified as Hypoderma bovis. On the basis of the warble counts, efficacy was 100%. The antibody kinetics, studied on blood samples collected on Days 0 (14 January), 6, 27, 48, 90, 119 and 150, showed that in the untreated animals, the antibody titres increased until May (Day 119) and then declined sharply, whereas in the treated animals there was a drop from Day 30 onwards. The mean ELISA titres of the control and treated groups became significantly different from Day 48 onwards (P < 0.05). In conclusion, moxidectin 0.5% pour-on formulation at a dose of 0.5 mg kg-1 bodyweight can effectively treat cattle naturally infested with Hypoderma bovis first instar larvae.

Administration, Topical↗

Comparison of the use of secretory and somatic antigens in an ELISA for the serodiagnosis of hypodermosis.

An antigen was prepared from metabolic products which were produced by maintaining first instar larvae of Hypoderma lineatum in RPMI tissue culture medium for 48 h. Three major proteins were identified in the secretory products and were characterised in terms of their molecular weights and iso-electric points. The antigen compared favourably with a soluble extract of larvae when used in an enzyme-linked immunosorbent assay (ELISA) against a panel of control sera and 2000 bovine sera collected from farms in Andalucia, Spain.

Animals↗

Doramectin systemic activity against cattle grubs, Hypoderma lineatum and H. bovis (Diptera: Oestridae), and cattle lice, bovicola bovis (Mallophaga: Trichodectidae), Linognathus vituli and Solenopotes capillatus (Anoplura: Linognathidae), and Haematopinus eurysternus (Anoplura: Haematopinidae), in Wyoming.

Seven individual trials were conducted in Wyoming to evaluate the therapeutic efficacy of doramectin administered subcutaneously at a dosage of 200 micrograms kg-1 against multiple, natural infestations of cattle grubs or cattle lice. Insect species present and the number of trials that included each species were: Hypoderma lineatum, 2; Hypoderma bovis. 1; Bovicola bovis, 5; Haematopinus eurysternus, 1; Linognathus vituli, 5; and Solenopotes capillatus, 3. Examinations for lice were performed prior to treatment and either weekly or bi-weekly thereafter for 28 days. Examinations for cattle warbles were performed either weekly or every 4 to 5 weeks from time of first appearance through last appearance in the backs of the cattle. No H. lineatum, H. bovis, H. eurysternus, L. vituli, or S. capillatus were found on doramectin-treated animals at any time following treatment. By 28 days following treatment, the number of B. bovis was reduced between 58 and 98%. Treatments applied later in the season, i.e. in March, were more efficacious against B. bovis than those applied in January or February.

Animals↗

Detection of antibodies to Hypoderma lineatum in cattle by Western blotting with recombinant hypodermin C antigen.

The cDNA encoding the entire mature hypodermin C (HC) of Hypoderma lineatum was cloned and expressed in Escherichia coli as a glutathione S-transferase fusion protein using pGEX vector. The recombinant HC protein (rHC) was tested by Western blotting to detect antibodies to H. lineatum in cattle. Western blotting with rHC as antigen clearly differentiated between H. lineatum-infested cattle sera and normal cattle sera. Forty-six out of forty-eight serum samples from cattle in Central Mongolia were positive, whereas all 30 serum samples from cows in Hokkaido, Japan, were negative by Western blotting. The result of Western blotting was identical to that of a previously developed enzyme-linked immunosorbent assay. These data demonstrated that Western blotting, with rHC expressed in E. coli, might be a useful method for the diagnosis of cattle hypodermosis.

Animals↗

Stage specific mortality and humoral immune responses during pulse and trickle infestations of the common cattle grub, Hypoderma lineatum (Diptera: Oestridae).

Stage specific survival of larvae and pupae of the common cattle grub, Hypoderma lineatum de Villers, was compared in cattle artificially infested with larvae delivered by subcutaneous injection as a single infestation of 100 larvae, in trickle infestations of either two doses of 50 larvae, separated by 15 days, or in four doses of 25 larvae, separated by 9, 6, and 6 days, respectively. The duration of the migratory and 'warble' phases were also compared between treatments. The kinetics of the antibody response was monitored throughout the larval development by ELISA. No difference was noted in the level of antibodies among the three treatments. Mortality of first-instars differed among the infestation groups with mortality in the single pulse infestation group<two dose trickle infestation<four dose trickle infestation. A similar trend was seen in second and third instar mortality. These data suggest that host immune responses are activated by the first larvae to enter the animal and that this activation negatively affects the later arriving larvae. This may have consequences for the population biology of cattle grubs and suggests that synchronization of the adult stages is important to the survival of larval stages in the hosts.

Animals↗