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Soluble fractions of Hypoderma lineatum third instar fat body, haemocytes and haemolymph were formulated with Quil A and used to immunize four groups of calves while a fifth group remained untreated. Calves received two subcutaneous injections of the soluble fractions, or adjuvant only delivered two weeks apart. Two weeks after the last injection the calves were exposed to 50 newly hatched larvae of H. lineatum which were placed on the skin and allowed to penetrate. Survival of larval stages was monitored by weekly palpation and collection of emergent third instars. Antibody responses to the immunogens were evaluated by immunoblots and following infestation antibody responses to first instar antigens were evaluated by an ELISA. Non-immunized calves and calves injected with adjuvant were all palpation positive for cattle grubs. In groups immunized with fat body, haemocyte and haemolymph components 100%, 33% and 33% were palpation positive for grubs respectively. First instar mortality, as reflected in palpable grubs, was high in the groups receiving injections with tissue components (99.3%, 95.1%, 95.8%, 83.9 and 80.4% mortality for those groups receiving fat body, haemocyte, haemolymph, adjuvant or control respectively). Second and third instar mortality was also higher in the immunized groups (100.0%, 91.7%, 91.7% for fat body, haemocyte, and haemolymph respectively) in comparison to the adjuvant only (14.0%) and unvaccinated (33.3%) groups. No viable flies emerged from pupae originating from larvae emergent from any of the immunized groups. Calves receiving the tissue extracts developed antibodies to several protein components following the second immunization which were still present 13 weeks post-infestation. Several proteins appeared to be common among the three tissue extracts and were recognized by antibodies from the immunized calves. All groups of calves became positive for antibodies to first instar antigens, although in some immunized calves the antibodies were transient., (Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.)

During the 2003-2005 hunting seasons, a total of 120 Cervidae, including 39 red deer (Cervus elaphus hispanicus) and 81 fallow deer (Dama dama), were examined for subcutaneous myiasis. Animals were shot from January to June in southern Spain. Specific antibodies against Hypodermatinae (Diptera: Oestridae) were detected by indirect enzyme-linked immunosorbent assay (iELISA) using a crude larval extract (CLE) and a purified antigen [hypodermin C (HC)] obtained from first instars of Hypoderma lineatum (De Villers) (Diptera: Oestridae). Hypoderma actaeon Brauer was the only species detected in this study, which represents the first confirmation of this species in fallow deer from Spain. The overall prevalence of animals presenting subcutaneous larvae (14.2%) was considerably lower than the prevalences determined by iELISA with CLE (43.3%) and HC (40.0%). Red deer showed a higher prevalence of Hypoderma than fallow deer. The concordance between larval examination during the hunting season and iELISA using both antigens was low, whereas the concordance between the CLE and HC ELISAs was good. Larval antigens obtained from H. lineatum constitute a good tool for the diagnosis of H. actaeon in Cervidae, especially when the hunting season does not coincide with the maximum presence of larvae on the back., (© 2010 The Authors. Medical and Veterinary Entomology © 2010 The Royal Entomological Society.)

An indirect ELISA test using as antigen a recombinant parasite protein, hypodermin C, was developed to measure Hypoderma-specific antibodies in cattle sera and compared with natural hypodermin C. To evaluate the field efficacy of the ELISA test, 334 serum samples were collected from cows raised at farms in Galicia for a serological survey. Compared with an ELISA based on natural parasite antigen, the recombinant hypodermin C gave excellent results, with a sensitivity of 95.8% and a specificity of 95.7%. Considering the cut-off point, with the recombinant hypodermin C, 70.9% of the animals had positive levels of antibodies to Hypoderma and with natural hypodermin C, 73.6%. Recombinant hypodermin C appears to be a useful alternative to the natural parasite antigen for the serodiagnosis of Hypoderma sp in cattle.

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.

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.

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.

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.

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.