Search

Hypodermin A (HA) is a serine protease secreted by first-instar Hypoderma lineatum larvae (Oestridae, Diptera). It plays a crucial role in induced immunosuppression during hypodermosis. This report describes the production of recombinant HA in Drosophila melanogaster Schneider 2 cells, its purification and its characterization, and compares it with the protease extracted form parasite larvae. The recombinant protein and the native HA have similar biochemical and biological features. Activity of the recombinant protease on the lymphocyte proliferation inhibition and on membrane antigen cleavage was tested and shown to be similar to the native one. Tunicamycin treatment of the recombinant HA shows that the two putative glycosylation sites carry glycan residues. Unglycosylated recombinant HA has the same enzymatic activity as the fully glycosylated protein, indicating that glycosylation is not important for the protease activity of HA.

The protease hypodermin A (HA) is produced by the parasitic warble-fly larva and is implicated in the modulation of the bovine immune system. This study examines the effect of this enzyme on the cell surface markers of bovine lymphocytes. HA interfered with the binding of all anti-lymphocyte receptor antibodies tested. Anti-BoCD2 and CD5 staining was completely abolished. But the mean fluorescence intensity (MFI) only was diminished for antibodies against BoCD4, CD8 and CD18. On the contrary, the MFI for anti-MHC Cl I molecules staining was increased. This effect of HA began as early as one h, and was reversed by removal of HA. Heating or PMSF treatment, which both inhibit protease activity, abolished the action of HA on the surface antigens. The HA concentrations (100 micrograms/ml) needed to alter antibody binding were similar to those that inhibited phytohaemagglutinin (PHA)-induced proliferation. These results show that enzymatic activity of HA on lymphocyte surface markers may be implicated in the inhibition of lymphocyte proliferation.

A cattle herd from the experimental farm of INRA in Nouzilly has been treated for hypodermosis since October 1990. Additionally, a regional eradication scheme has been implemented in this area since autumn 1992. Bi-monthly warble counts were performed between March and July each year on an average of 200 animals. No warble was recorded in this herd from 1991 to 1994 with the exception of two dairy cows in 1993. In autumn 1994, therapeutic control measures were stopped. Serological surveys were performed in the autumn of each year from 1991 until 1995. Anti-Hypoderma antibodies were found in 25%, 27.2%, 4.3%, 3.2% and 0% of the animals respectively. An experimental low infestation was conducted in the summer of 1994. During the spring 1994, third instars of Hypoderma bovis were collected from naturally infested animals. From a total of 13 pupae, six adults (four males and two females) emerged and were released in the herd of Nouzilly on 24 June and 4 July. In October 1994 serological investigations revealed two animals seropositive for hypodermosis. This number increased to six in January 1995. The antibody kinetics of these six animals remained parallel throughout the next 6 months: the titres increased up to April and started to fall in May to return to negative values in August. Manual examinations of the animals at weekly intervals between April and July revealed the presence of four warbled animals with one, one, two and three warbles respectively. The two other seropositive animals remained warble free. One other animal showed antibody titre fluctuations between negative and low positive values, but was warble free in the spring. In October 1995 all the animals of the herd were seronegative. The interpretation and the value of a sensitive immunodiagnosis in a large eradication programme are discussed and compared with warble counts, especially in the case of a low level of infestation.

The larval stages of different economically important Oestridae species were studied for their antigenicity and cross reactivity, using ELISA and immunoblotting. The immune sera of cattle from Algeria, Belgium, France and Switzerland were compared for their capacity to recognize the stage-specific antigens of their specific parasites Hypoderma bovis and H lineatum, originating from different populations. This comparison was extended to other Hypoderminae species responsible for economic losses in European animal production: H diana in roe deer and H tarandi in reindeer. The specific host for each of these parasites recognized common epitopes of hypodermin C, a collagenolytic enzyme previously well characterized in the first instar of H bovis and H lineatum. No cross reactivity was found with other Oestridae, such as Oestrus ovis or Gasterophilus intestinalis. The specificity of hypodermin C was evaluated using sera from animals harbouring other arthropods such as ticks, helminths, including Fasciola hepatica and Haemonchus contortus, or protozoa such as Anaplasma sp, and no reaction was observed. The use of hypodermin C is therefore suggested as an antigen in the immuno-survey of economically-important Hypoderminae.

The cDNAs of hypodermins, enzymes secreted by the larvae of the parasitic fly Hypoderma lineatum, were sequenced. Four cDNA clones were isolated, one encoding hypodermin A (HA), one encoding hypodermin C (HC), and the two others encoding proteins related to hypodermin B (HB). The amino acid sequences deduced from the nucleotide sequences confirmed that these enzymes are serine proteases. HA and one of the HB proteins had potential N-linked glycosylation sites. Analysis of hypodermin protein, RNA and DNA at different larval stages indicated that protein overexpression is regulated transcriptionally for HA and HB, and by transcriptional and DNA amplification for HC.