Iain J. East

Characterization of a Major Peritrophic Membrane Protein, Peritrophin-44, from the Larvae of Lucilia cuprina cDNA AND DEDUCED AMINO ACID SEQUENCES

The peritrophic membrane is a semi-permeable chitinous matrix lining the gut of most insects and is thought to have important roles in the maintenance of insect gut structure, facilitation of digestion, and protection from invasion by microrganisms and parasites. Proteins are integral components of this matrix, although the structures and functions of these proteins have not been characterized in any detail. The peritrophic membrane from the larvae of the fly Lucilia cuprina, the primary agent of cutaneous myiasis in sheep, was shown to contain six major integral peritrophic membrane proteins. Two of these proteins, a 44-kDa glycoprotein (peritrophin-44) and a 48-kDa protein (peritrophin-48) together represent >70% of the total mass of the integral peritrophic membrane proteins. Peritrophin-44 was purified and its complete amino acid sequence was determined by cloning and sequencing the DNA complementary to its mRNA. The deduced amino acid sequence codes for a protein of 356 amino acids containing an amino-terminal signal sequence followed by five similar but nonidentical domains, each of approximately 70 amino acids and characterized by a specific register of 6 cysteines. One of these domains was also present in the noncatalytic regions of chitinases from Brugia malayi, Manduca sexta, and Chelonus. Peritrophin-44 has a uniform distribution throughout the larval peritrophic membrane. Reverse transcriptase-polymerase chain reaction detected the expression of peritrophin-44 in all three larval instars but only trace levels in adult L. cuprina. The protein binds specifically to tri-N-acetyl chitotriose and reacetylated chitosan in vitro. It is concluded that the multiple cysteine-rich domains in peritrophin-44 are responsible for binding to chitin, the major constituent of peritrophic membrane. Peritrophin-44 probably has roles in the maintenance of peritrophic membrane structure and in the determination of the porosity of the peritrophic membrane. This report represents the first characterization of an insect peritrophic membrane protein.

Lucilia cuprina: Inhibition of larval growth induced by immunization of host sheep with extracts of larval peritrophic membrane

A culture system has been established to produce gram amounts of peritrophic membrane from larvae of the sheep blowfly, Lucilia cuprina. Peritrophic membrane obtained from this culture has been used to immunize sheep. The immunization produced an immune response which resulted in the average weight of larvae on immunized sheep being only 50% of that of larvae grown on control sheep (P < 0.05). Fractionation of the components of the peritrophic membrane followed by immunization trials showed that the protective antigen fraction comprised material that could only be solubilized by harsh agents such as 4 M-urea. Even after solubilization by 4 M-urea, the protective antigens were able to produce a protective immune response which reduced growth of larvae on immunized sheep to 55% of larvae grown on control sheep (P < 0.05). This immune response which reduced growth of the larvae did not cause gross morphological damage to the larvae.

Vaccination against Lucilia cuprina: The causative agent of sheep blowfly strike

The sheep blowfly, Lucilia cuprina, is responsible for over 80% of cases of blowfly strike in Australia and the tosses in production and sheep deaths due to flystrike exceed S200 million per annum. Traditional methods of control are becoming less effective because of the blowflys resistance to insecticides and thus other methods of control are necessary. In general, sheep develop very little immunity to flystrike even after repeated infestation, however vaccination against L. cuprina has shown considerable potential for controlling flystrike. The most successful sources of antigens have been the larvaes secreted proteases and several extracts from gut or peritrophic membrane. Immunization with these antigens results in retardation of larval growth and in some cases larval mortality. On sheep immunized with peritrophic membrane extracts, the growth retardation appears to be caused by a blocking of the peritrophic membrane which results in the larvae being starved of nutrients. The prospects for vaccine development and the remaining barriers to be overcome are discussed.

A single amino acid deletion in the antigen binding site of BoLA-DRB3 is predicted to affect peptide binding.

Two bovine MHC class II alleles, BoLA-DRB3*0201 and BoLA-DRB3*3301, contain a three base pair deletion which results in the deletion of a lysine (K beta 65) in the antigen recognition site (ARS). Modelling of BoLA-DRB3*0201 with the conserved lysine K beta 65 and BoLA-DRB3*0201 without K beta 65 indicated that this deletion altered the peptide specificity of the ARS, and may impact on the immune response. To test this hypothesis, the presence of K beta 65 was analysed in a sample of cattle vaccinated with the commercial cattle tick vaccine (TickGARD). Homozygous deletion of K beta 65 was significantly associated with high response to TickGARD (P<0.05). Screening of the TickGARD antigen identified a potential T cell epitope that is recognised better by animals that are homozygous for the K beta 65 deletion. This study provides evidence that changes in the ARS of MHC class II molecules may be associated with the well recognised animal to animal variation in magnitude of vaccine response.

Potent immunosuppression by secretory/excretory products of larvae from the sheep blowfly Lucilia cuprina.

Summary Secretory/excretory products (sec/ex) of parasitic larvae of the sheep blowfly Lucilia cuprina potently inhibited proliferation of peripheral blood leucocytes stimulated by mitogens in vitro. Suppression of proliferation was not due to irreversible damage because cells cultured for 24 h in high concentrations of sec/ex appeared viable (assessed by Trypan blue exclusion) and did not show impaired proliferation after washing. Furthermore, suppression induced by sec/ex could be overcome by increasing concentrations of mitogen. The inhibitory activity could be demonstrated in cultures where sec/ex was added at different times during the culture period. Inhibitory activities in sec/ex were heat‐labile and sensitive to treatment with trypsin. In addition to effects in vitro, sec/ex was strongly immunosuppressive in vivo. Sheep given combined injections of myoglo‐bin and sec/ex had markedly lower anti‐myoglobin antibody levels in sera than sheep that received injections of myoglobin alone. There was no significant antibody response to sec/ex itself. Immunosuppressive moieties in sec/ex produced by blowfly larvae may promote parasite survival by inhibiting the immune response of host sheep.

Vaccines against blowfly strike: The effect of adjuvant type on vaccine effectiveness

Vaccination of sheep with a partially purified extract of Lucilia cuprina larvae in some cases resulted in marked reduction of growth in larvae which fed on the sheep. Twelve adjuvants were assessed, in vitro and in vivo, to determine which induced the largest inhibitory effect on larval growth. The Freunds complete adjuvant and Quil A groups produced ELISA antibody levels significantly higher (P less than 0.05) than other groups. Seven adjuvants mediated an immune response which caused significant inhibition of larval growth (P less than 0.05). When the sheep were assessed by in vivo larval culture, only larvae feeding on sheep vaccinated with the antigen presented in Freunds complete adjuvant or dextran sulphate or a dextran sulphate/Freunds incomplete adjuvant mixture weighed significantly less (P less than 0.05) than larvae feeding on control sheep. The effect on larvae was monitored in vitro for 70 days after vaccination, by which time significant reduction in larval weight was no longer observed. The loss of larval growth inhibition was not associated with a corresponding reduction in overall antibody levels.

Mapping the T cell epitopes of the Babesia bovis antigen 12D3: implications for vaccine design

The Babesia bovis antigen 12D3 was analysed to identify potential T‐cell epitopes. Two predictive algorithms identified 13 possible sites but there was minimal agreement between the different predictive methods. Experimental determination of the T‐cell epitopes recognized by nine cattle was achieved using a panel of overlapping peptides which identified seven different epitopes, five of which were clustered together around residues 210–320 of the molecule. No T cell epitopes were located within the tightly disulphide bonded core of 12D3. Using a series of truncated peptides, the location of two of the epitopes was mapped to residues 35–43 and 266–275. The sequences of these two epitopes was compared with a database of previously described binding motifs for MHC II alleles and each epitope was found to contain three sequence motifs recognized by HLA‐DR alleles. The BoLA‐DRB3 alleles occurring in these cattle were determined by a sequence specific oligonucleotide hybridization assay. Within those cattle whose T cells proliferated in response to 12D3, there was a consistent pattern of epitope recognition and presence of particular DRB3 alleles. The implications for effective vaccine design are discussed.

Vaccination against Babesia bovis: T cells from protected and unprotected animals show different cytokine profiles

Vaccination of cattle against the haemoprotozoan parasite, Babesia bovis, with the recombinant antigen 11C5 resulted in 9 of 15 cattle being protected against challenge infection. The cellular immune responses of protected and unprotected cattle were compared in order to identify differences in response. No differences were observed in the pattern of change in various blood leukocyte populations throughout challenge infection. FACScan analysis revealed an increase in the proportion of cells bearing the CD2 marker in both protected and unprotected cattle over the course of infection. There were no observable differences in the frequency of various cell-surface markers between the unprotected and protected cattle. During the period of patent parasitaemia, in vitro cultures of peripheral blood mononuclear cells (PBMC) from protected cattle produced significantly more TNF-alpha (P < 0.05) than cultures from unprotected cattle. TNF-alpha concentrations remained at pre-challenge levels until day 10, when levels in the unvaccinated control and vaccinated/unprotected animals dropped. By peak parasitaemia, TNF-alpha production in vitro was significantly greater (P < 0.05) in cultures of PBMCs from protected cattle. Interferon production showed an initial peak at day 5 in all cattle, followed by a decrease and a second peak at days 10-13 in protected cattle only, which coincided with resolution of the infection.

Adjuvants for new veterinary vaccines

Many important diseases of domestic animals have been rendered impotent by the development of safe and effective vaccines to prevent or ameliorate infection. Up to now, veterinary vaccines consisted mainly of killed or attenuated whole organisms. Vaccines of this type that are registered for use in the state of Queensland, Australia are listed in Table 1.1 (63,161). While not including many of the vaccines found in other countries, the list is indicative of current vaccine technologies. Vaccines such as these, which mostly contain whole pathogens, are usually highly immunogenic and utilize either no adjuvant or a simple adjuvant, such as alum or oil, to prolong the effect of the vaccine. The term adjuvant was first defined by Ramon (117) in 1925 as a substance that, when used in combination with antigen, enhanced levels of immunity beyond those developed with the antigen alone. A synopsis of the adjuvants currently available for veterinary vaccines was recently published by Vanselow (152).

Vaccines for the Skin and Mammary Gland of Ruminants

For many infectious diseases of ruminants vaccination is a feasible and desirable alternative to therapy. Vaccines that result in elevated herd immunity and disease prevention have important advantages in terms of cost effectiveness, animal welfare, and reduction of the environmental contamination that occurs when animals are treated with drugs and chemicals. Currently on the market are many efficacious and successful vaccines for ruminant diseases, but there are very few vaccines available for diseases of the mammary gland and skin. Undoubtedly the diseases that are easiest to control by vaccination have already been conquered; it is equally true that at the time such vaccines were developed the process was not easy! In the 1990s we have at our disposal new technologies such as molecular engineering and peptide synthesis. These technologies have dramatically improved our ability to synthesize usable quantities of antigens, yet many economically important diseases of the skin and mammary gland remain intractable. In this chapter we review progress that has been made with vaccines against such diseases and try to identify areas that may be profitable for future research endeavours.