Vern M. Bowles

Characterization of ES products involved in wound initiation by Lucilia cuprina larvae.

Excretory/ secretory (ES) products were collected up to 6 h after egg hatch and analysed by SDS-PAGE. The larvae produced a complex array of molecules, the pattern of which changed dramatically over the time of culture. When larvae were cultured on isolated sheep skin, skin degradation was found to occur immediately upon egg hatch with digestion of the available soluble skin molecules virtually complete within 6 h of culture. Proteolytic activity of the larval ES products was assessed by gelatine substrate SDS-PAGE gels and revealed numerous secreted proteases, the majority of which belong to the serine class of protease. Several of these proteases appeared to be developmentally regulated including a 28 kDa protease which was secreted only during the time of egg hatch. Metabolic labelling experiments indicated that culture conditions can influence the production of ES products with larvae cultured in the absence of sheep skin producing greater amounts and numbers of specific proteins.

Vaccination of sheep against larvae of the sheep blowfly (Lucilia cuprina)

Four first stage larval antigens from the sheep blowfly were identified using supernatants from cultures of antibody secreting cells. These partially purified larval antigens, when added to Montanide ISA-25 containing recombinant ovine IL-1 beta (rovIL-1 beta) were used to successfully vaccinate sheep against larvae of the sheep blowfly. Significantly less strikes were recorded on vaccinated sheep compared to controls (P < 0.033) with surviving larvae from vaccinated sheep up to 85% smaller than larvae from control sheep. RovIL-1 beta was found to be an important component of the vaccine. Vaccinated sheep showed both humoral and cellular immune responses to the larval antigens. Antibody levels generally correlated directly with delayed-type hypersensitivity (DTH) responses, but neither antibody nor DTH correlated positively with protection in vaccinated sheep. Skin sections removed from individual sheep immediately after challenge revealed aggregations of CD4+, gamma delta-TCR+ and CD1+ cells located directly under the epidermis in vaccinated sheep.

Production and in vivo use of recombinant ovine IL-1β as an immunological adjuvant

To determine the potential of ovine interleukin 1 (IL-1) as a vaccine adjuvant in sheep, we have expressed and purified recombinant ovine IL-1 beta (rovIL-1 beta) from bacterial cultures using a modified form of the ovine IL-1 beta cDNA. Adjuvant trials using the model protein avidin demonstrated that rovIL-1 beta when administered in association with a compound providing a slow-release mechanism, resulted in significant enhancement of specific serum antibody levels in both mice and sheep. In a dose-response experiment in sheep, intradermal immunization with avidin plus either 10 or 100 micrograms of rovIL-1 beta in aluminium hydroxide resulted in antibody levels four- to eightfold higher than immunizations without rovIL-1 beta. The addition of rovIL-1 beta also resulted in a more severe DTH response to avidin indicating that rovIL-1 beta is able to enhance both humoral and cell-mediated responses to avidin. The highest antibody titres were observed when sheep received rovIL-1 beta in both the primary and secondary immunizations although the addition of rovIL-1 beta in only one of the immunizations still resulted in a significant increase in antibody levels. Additional experiments showed that rovIL-1 beta and avidin must be administered in a site drained by the same lymph node for the adjuvant effect of rovIL-1 beta to be observed.

Characterisation of proteases involved in egg hatching of the sheep blowfly, Lucilia cuprina.

A number of proteases were identified in the egg shell washings (ESW) collected during the egg hatching of Lucilia cuprina (sheep blowfly). Characterization of these proteases indicated a pH optima in a similar pH range that was optimal for L. cuprina egg hatching. Mechanistic characterization of these proteases indicated that they were predominantly of the serine class. Several protease inhibitors were tested for their ability to inhibit L. cuprina egg hatching in vitro. Egg hatching was significantly (P<0.05) inhibited by PMSF (61%), 1,10-Phenanthroline (42%) and Pepstatin (29%). The inhibition of egg hatching by PMSF showed a strong concentration dependence, with its effects ranging from inhibition at high concentrations to enhancement of egg hatching at low concentrations. Addition of ESW to unhatched eggs, significantly (P<0.05) enhanced their rate of hatching above untreated control eggs. This enhancement of egg hatching was significantly (P<0.05) reversed by the protease inhibitors Elastatinal (40%), 1,10-Phenanthroline (40%) and PMSF (38%). These studies indicate a role for serine and/or metallo-proteases in facilitating L. cuprina egg hatch.

Local cell traffic and cytokine production associated with ectoparasite infection

This paper reviews recent advances in our understanding of changes in local cellular traffic and cytokine synthesis that occur as a result of infection of sheep with the ectoparasite Lucilia cuprina. Changes in the cellular composition and cytokine profile of infected skin and draining afferent and efferent lymph were assessed using standard approaches and, in addition, a variety of techniques that have only recently become available as a result of advances in ruminant cytokine biology. These include cytokine-specific immunoassay, reverse transcription PCR (RT-PCR) and immunohistology. The initial acute inflammatory response was characterised by the infiltration of polymorphonuclear cells followed by selected lymphocyte subsets into discrete areas adjacent to the site of infection. Analysis of cytokine expression in skin prior to and following infection provided a molecular basis for the observed cellular events. Both cellular and molecular events within the skin were reflected within draining afferent lymph providing a basis for the conclusion that events within the skin (other than antigen uptake and transport) may influence events within the draining node and thus the outcome of the immune response to the parasite. Analysis of cellular and molecular changes in efferent lymph during infection suggested initiation of antigen-specific immunity.

Proteases released by Lucilia cuprina during egg hatch

Abstract Products released by Luclia cuprina (sheep blowfly) during egg hatch and early larval development were found to contain a variable number of proteases, probably reflecting their different functions during these developmental phases. Moreover, a number of the developmentally regulated proteases produced only during egg hatch appeared to be strongly egg shell associated, indicating the egg shell proteins to be their specific substrates. Several proteases were tested for their ability to enhance the rate of egg hatch. The commercial proteases chymotrypsin and trypsin were able to significantly enhance ( p ≤0.01) egg hatch by 36% and 44%, respectively, when compared to an untreated control. Interestingly, fluids containing the egg shell associated proteases were able to significantly ( p ≤0.001) enhance L. cuprina egg hatch by up to 70%. This hatch enhancement activity could be significantly ( p ≤0.001) reversed by the addition of Phenylmethylsulphonyl fluoride (PMSF). Fractionation of the egg-associated proteases by gelatin affinity chromatography suggested that gelatin-binding molecules were responsible for the majority of the egg hatch enhancement activity in this preparation.

Cytokine mRNA expression in skin in response to ectoparasite infection

Cellular infiltration and local cytokine mRNA levels were examined during the first 48 h of infection of skin by larvae of the sheep blowfly Lucilia cuprina. At the cellular level the response involved a dramatic influx of leucocytes (CD45+ cells). Among these infiltrating cells were large numbers of granulocytes, including neutrophils and eosinophils, as well as macrophage‐like cells and lymphocytes. Many of the lymphocytes expressed cell surface markers characteristic of T cells including CD4, CD8 and the γδ TCR. The numbers of each of these cell types increased progressively as infection continued so that by 48 h the lesions were densely populated. Expression of mRNA for IL‐6 could be detected by Northern blot analysis while mRNA for other inflammatory cytokines including IL‐1α, IL‐1β, IL‐8 and TNFα was detected using the polymerase chain reaction. Coincident with the influx of granulocytes and other cells there was an increase in the level of mRNA for the cytokines IL‐lα, IL‐1β, IL‐6 and IL‐8. In the skin of the sheep there appeared to be constitutive expression of message for the cytokines IL‐1β, IL‐6 and TNFα, with the level of the latter not found to increase during the 48 h of infection examined. In situ hybridization was used to determine the location of IL‐6 and TNFα mRNA within resting and infected skin. During infection, fibroblasts, macrophage‐like cells and endothelium appeared to produce high levels of IL‐6 mRNA. Expression of the T cell dependent cytokines IL‐2 and IFN‐γ but not IL‐4, increased in expression as time progressed and the population of infiltrating cells, including T cells, expanded.

Biochemical aspects of egg hatch in endo- and ectoparasites: potential for rational drug design.

Control of parasites through rational drug design requires a thorough understanding of the parasites lifecycle encompassing the biochemical and physiological processes which contribute to normal parasite homeostasis. The hatching of parasite eggs for example, represents an important process in the development of a parasitic infection. Previous studies in helminths have indicated that secreted enzymes often facilitate successful endoparasite egg hatch. In contrast, there are relatively few examples demonstrating a role for secreted enzymes in the egg hatching process of insects. An analysis of this process in the ectoparasite Lucilia cuprina suggests a role for secreted enzymes in the hatching of sheep blowfly eggs. Characterisation of the proteases collected at the time of egg hatch indicates the presence of serine proteases. Further purification and characterisation of these proteases may enable the design of specific inhibitors to interfere with the egg hatch process and therefore provide a novel means of control.

Systemic immunization of sheep with surface antigens from Haemonchus contortus larvae

Sheep immunized systemically with a surface extract from third-stage H. contortus larvae showed high serum antibody reactivity against surface antigens and whole, viable larvae. After a first infection, no significant difference was found between the mean egg counts of the vaccinates and controls although most vaccinated sheep seemed to show an increased susceptibility to infection. The local abomasal response was stimulated by giving both vaccinated and control sheep a large, abbreviated infection cured after 11 days by drenching. Thereafter, a second challenge infection was given. This immunization regime resulted in seven of the nine vaccinated sheep showing clear protection against the second challenge infection.