Control of the sheep blowfly in Australia and New Zealand--are we there yet?

The last 50 years of research into infections in Australia and New Zealand caused by larvae of the sheep blowfly, Lucilia cuprina, have significantly advanced our understanding of this blowfly and its primary host, the sheep. However, apart from some highly effective drugs it could be argued that no new control methodologies have resulted. This review addresses the major areas of sheep blowfly research over this period describing the significant outcomes and analyses, and what is still required to produce new commercial control technologies. The use of drugs against this fly species has been very successful but resistance has developed to almost all current compounds. Integrated pest management is becoming basic to control, especially in the absence of mulesing, and has clearly benefited from computer-aided technologies. Biological control has more challenges but natural and perhaps transformed biopesticides offer possibilities for the future. Experimental vaccines have been developed but require further analysis of antigens and formulations to boost protection. Genetic technologies may provide potential for long-term control through more rapid indirect selection of sheep less prone to flystrike. Finally in the future, genetic analysis of the fly may allow suppression and perhaps eradication of blowfly populations or identification of new and more viable targets for drug and vaccine intervention. Clearly all these areas of research offer potential new controls but commercial development is perhaps inhibited by the success of current chemical insecticides and certainly requires a significant additional injection of resources.

The immunobiology of myiasis infections--whatever happened to vaccination?

The current state of myiasis vaccine technologies are reviewed mainly in the primary research genera of Lucilia and Hypoderma. The importance of myiasis flies as primary causes of morbidity and mortality in agricultural species and man has not diminished despite the existence of good control strategies. However, the development of vaccines against myiasis infections has been relatively quiescent for more than 10 years despite the rapid development of genomic and proteomic analysis and of skills in data interpretation. The value of vaccine research in an era of chemical primacy is analysed. In fact, recent findings of drug resistance and the impact of animal welfare concerns should mean a renewed interest in alternative controls. The reasons that this has not been true to date are explored and new possibilities discussed.

Prevalence of infestation with gastrointestinal nematodes in Pony Club horses in Victoria.

BACKGROUND: The aims of the present study were to identify the common gastrointestinal nematodes, to assess the prevalence of infestation with gastrointestinal nematodes, and to assess some potential indirect determinants of nematode parasitism in Victorian Pony Club horses. METHODS: A total of 106 horses from five Pony Clubs from outer Melbourne and Geelong, Victoria, Australia, were enrolled in a cross-sectional study. Fresh faecal samples were collected and faecal egg counts (FECs) performed on site within 2 h of collection. Potential determinants of the FEC were analysed using logistic and negative binomial regression. RESULTS: FECs ranged from 0 to 3750 eggs per gram (epg), with an average of 422 epg. Eggs were detected in the faeces of 52% of horses (55/106) and the average count was 813 epg. Counts were 500 epg or greater in 27% (29/106) of horses. Pony Club, season and sex of the horse were not associated with the FEC. Among horses treated with anthelmintics 8 weeks or less prior to sampling, FECs were commonly ≥50 epg, and high FECs were relatively common. CONCLUSIONS: The results indicate that treatment efficacy is commonly low and/or rapid re-infection after treatment is common, and show that management practices for internal parasite control are often inadequate for preventing high FECs among Pony Club horses in Victoria.

Identification of auto-antigens in skin scrapings from scabies-infected pigs.

Sarcoptes scabiei continues to cause major health and economic problems in a large range of animals and humans. Although the inflammatory response to the mite and its antigens is known to cause the main pathology, little work has been carried out on this response at the site of infection. This report presents an initial analysis of the proteins found in skin scrapings and their antigenic responsiveness in pigs. Skin scrapings and mite extracts were isolated from chronically infected sows while infected and uninfected sera were isolated from pigs with confirmed infections or mange-free pigs, respectively. Electrophoresis and sequencing confirmed the main components of both the skin and mite extracts to be serum proteins. Immunoblotting then suggested that transferrin was the major antigen recognised by pooled infected sera in the skin and the mite extracts. Immunoassays confirmed that a majority of infected pigs produced antibodies to transferrin while mange-free pigs did not. A pool of IgG from infected dogs was then used to isolate another antigen from pig skin scrapings which was shown to be haptoglobin. This was also found to induce high titres of antibody in infected pigs as compared with mange-free pigs. The use of albumin as a control antigen showed no reactivity in either group of sera. The finding of two iron-binding molecules as strong auto-antigens in pig scabies has implications for the importance of iron during this infection and may help to explain the persistence and magnitude of the host inflammatory response.

Management of myiasis: current status and future prospects.

The management of myiasis in livestock has been an example of the success of modern chemical approaches for parasite control, yet in some cases remains extremely intractable, requiring the development of novel strategies. In addition, the growing and urgent need to develop integrated strategies that enhance the sustainability of livestock production systems drives the search for new techniques [see Int. J. Parasitol. 29 (1999) 7].The following summary represents a synthesis of a symposium presented at the 19th International Conference of the World Association for the Advancement of Veterinary Parasitology, New Orleans,USA, 10­14 August 2003. The coverage began with a review of the need for more subtle economic analysis of the impact of myiasis based on the use of the sterile insect technique (SIT) for control of bovine hypodermosis in North America. This was followed by a review of the status of chemical control with particular emphasis on the macrocyclic lactones. The outcome of the use of these compounds in a regulated control program for eradication of bovine hypodermosis in EU was surveyed. Similarly, the success of the screwworm eradication program, using the sterile insect technique has shown how effective this approach can be given the appropriate target. Several aspects of the development of newer approaches were surveyed in discussion of newer chemical control products, development of vaccines, use of host genetics, use of predictive simulation modelling and trapping for monitoring and control and the development of new diagnostic approaches for occult infestations. Finally, use of the latest molecular tools for identification of larvae causing myiasis and their use for the identification of species coming from different and distant geographical areas to colonize regions where they have been eradicated was reviewed.

Inhibition of trypsin and chymotrypsin by anti-inflammatory triterpenoids from Compositae flowers.

Taraxastane, oleanane, ursane, lupane, taraxane, cycloartane, dammarane and tirucallane triterpenoids isolated from flowers of Compositae plants have been previously reported to exhibit anti-inflammatory effects and are variously competitive and non-competitive inhibitors of the serine proteases trypsin and chymotrypsin. The general features of those triterpenoids found to be protease inhibitors are having a hydroxy group and an appropriate side chain in the region of the molecule distal to the 3-hydroxy group. However, fatty acid esterification of the triterpenoid 3-hydroxy group can have a marked effect on inhibitor effectiveness. This suggests a possible means of rapid alteration of the plant defensive complement in vivo and of the bioactivity of these anti-inflammatory compounds.

Antibody-dependent cell-mediated cytotoxicity to newly excysted juvenile Fasciola hepatica in vitro is mediated by reactive nitrogen intermediates.

Passive intraperitoneal transfer of sera from Fasciola hepatica-infected sheep, cattle or rats can protect naive rats from F. hepatica infection, suggesting a parasite killing mechanism within the peritoneal cavity that is dependent on the presence of parasite-specific antibody. We investigated antibody-dependent cell-mediated cytotoxicity by resident peritoneal lavage cell populations, containing large numbers of monocytes/macrophages, as a potential host resistance mechanism by which juvenile flukes could be killed within the peritoneal cavity of naive rats. Comparative studies were conducted using cell populations containing large numbers of monocytes/macrophages from sheep. The results demonstrate that monocyte/macrophage-rich lavage cell populations from rat and sheep differ substantially in their ability to generate nitric oxide. Only resident rat peritoneal lavage cells were able to mediate antibody-dependent cell-mediated cytotoxicity against newly excysted juvenile liver fluke. The mechanism of cytotoxicity was dependent on, and directly proportional to, the production of nitric oxide and required attachment of effector cells to the newly excysted juvenile liver fluke tegument, which occurred following the addition of sera from F. hepatica-infected animals. This is the first report demonstrating a mechanism of cell-mediated cytotoxicity to newly excysted juvenile liver fluke.

Parasites, parasitology and parasitologists.

The science of parasitology is one of the many new disciplines of the twentieth century, as such it is a dynamic and rapidly evolving science which encompasses an increasing number of sub-disciplines and technologies. However, will the fragmentation involved in current methods of scientific enquiry and the competition for funding mean the decline of certain areas of parasitology or perhaps the complete loss of the discipline. This paper attempts to address these questions by considering the development of the discipline of parasitology especially within Australia and by considering the mechanisms of attaining funding for science. Technological change and its impact on parasitology is also considered, and requirements for maintenance of the discipline and its practitioners are suggested.

Inhibition of serine proteases by anti-inflammatory triterpenoids.

The lupane triterpenoid lupeol, the ursane triterpenoid alpha-amyrin and esters of these compounds are present in the bark of roots of Alstonia boonei (Apocynaceae) and have anti-inflammatory properties. alpha-Amyrin is a competitive inhibitor of bovine trypsin and chymotrypsin (Ki values 29 microM and 18 microM, respectively). Lupeol linoleate, lupeol palmitate and alpha-amyrin linoleate are non-competitive inhibitors of trypsin (Ki values 7 microM, 10 microM and 16 microM, respectively). alpha-Amyrin linoleate is also a non-competitive inhibitor of chymotrypsin (Ki value 28 microM). Lupeol is a competitive inhibitor of both trypsin and chymotrypsin (Ki values 22 and 8 microM, respectively). alpha-Amyrin palmitate is a potent non-competitive inhibitor of chymotrypsin (Ki 6 microM). Lupeol, alpha-amyrin and the palmitic and linoleic acid esters of these compounds are ineffective or very weak as inhibitors of porcine pancreatic elastase and of Lucilia cuprina and Helicoverpa punctigera leucine aminopeptidases. These hydrophobic triterpenoids represent further examples of anti-inflammatory triterpenoids that are PKA inhibitors as well as being selective protease inhibitors.

Aminopeptidases as potential targets for the control of the Australian sheep blowfly, Lucilia cuprina.

A series of experiments were carried out to investigate the role of proteinase enzymes in the growth of larvae of the sheep blowfly, Lucilia cuprina. First, instar larvae were incubated on an artificial growth media in the presence of various concentrations of inhibitors of all the major proteinase classes. Inhibitors of serine proteinases and aminopeptidases were found to cause significant growth inhibition and in some cases death of the larvae within 24 h, suggesting that these enzymes were the major classes involved in protein digestion in the gut of the insect. A second group of experiments analysed the effects of two inhibitors from the same or different proteinase classes in the growth media. Synergistic inhibition of larval growth was observed with the incorporation of inhibitors of serine proteinases and aminopeptidases. The results suggest that these classes of proteinases are both central to protein digestion in this insect, probably in the gut, and that the inhibition of both types of activity leads to an almost complete blockade of digestion. Testing in vivo gave similar results with infections on sheep skin inhibited by either serine proteinase or aminopeptidase enzyme inhibitors and the combination of both stopped the infection process. The role of aminopeptidases in larval metabolism and as potential targets for blowfly control agents is examined.

Fasciola hepatica: characterization and cloning of the major cathepsin B protease secreted by newly excysted juvenile liver fluke.

Proteolytic activity present in the excreted/secreted (ES) material of newly excysted juvenile (NEJ) Fasciola hepatica was biochemically analyzed. By gelatin substrate SDS-PAGE, only one region of activity was observed in the NEJ ES material at a molecular mass of 29 kDa. Both the secreted cathepsin L from adult fluke and the 29-kDa proteolytic activity of NEJ ES show a common pH optimum of 7.5, a cysteine protease inhibition profile, and preference for the N-benzyloxycarbonyl (Z)-Phe-Arg-NHMec fluorogenic substrate over Z-Arg-Arg-NHMec and Z-Arg-NHMec. In vitro analysis revealed that the NEJ protease activity digested sheep immunoglobulin heavy chain and bovine serum albumin but not bovine hemoglobin. Amino-terminal protein sequence analysis of the 29-kDa NEJ protease band revealed two sequences with homology to the cathepsin B family of proteases. Using degenerate oligonucleotides designed from the N-terminal sequence, reverse transcriptase polymerase chain reaction with NEJ RNA amplified a cDNA sequence encoding the first 236 amino acids of mature cathepsin B. Using this cDNA fragment an overlapping cDNA was isolated from a LambadaZAP cDNA library constructed with poly(A)+ RNA from immature 5-week-old liver fluke. Together with the N-terminal sequence, these cDNAs predict a mature cathepsin B sequence of 254 amino acids which shows 48-51% sequence identity to mammalian and Schistosoma mansoni cathepsin B. We conclude that, in contrast to the major proteases released by adult fluke, the major secreted protease of NEJ of F. hepatica is of the cathepsin B class.

Effect of immunisation against leukaemia inhibitory factor on the establishment of pregnancy in sheep.

Leukaemia inhibitory factor (LIF), a pleiotropic cytokine, is implicated in blastocyst implantation in mice and maintains the development of ovine embryos in culture. Previously, LIF mRNA and protein were demonstrated in the endometrium throughout the oestrous cycle and early pregnancy in the ewe. In this study pregnant ewes were passively immunised against human recombinant LIF with polyclonal antibodies raised in cows by active immunisation. Ewes were immunised during two stages of early pregnancy: blastocyst development to hatching, and blastocyst elongation to implantation. Only animals passively immunised against LIF showed detectable LIF antibodies in their sera and in uterine lumina flushings by radioimmunoassay and Western blot analysis. Pregnancy was confirmed by ultrasound on day 55 and a 33.5% non-significant decrease in pregnancy rate of anti-LIF treated animals was observed, when compared to animals in control groups (untreated or treated with bovine anti-keyhole limpet hemocyanin). Cows actively immunised with recombinant human LIF and exhibiting high levels of LIF antibodies in their sera at the time of blastocyst implantation also showed a reduced pregnancy rate in comparison to control animals. Although LIF may not be obligatory for implantation in ruminants it does appear to have a role during the establishment of pregnancy.

Leukaemia inhibitory factor in endometrium during the oestrous cycle, early pregnancy and in ovariectomized steroid-treated ewes.

Leukaemia inhibitory factor (LIF), a pleiotropic cytokine, is essential for blastocyst implantation in mice and maintains the development of ovine embryos in culture. The expression of LIF was examined by northern blot analysis in endometrial tissue from cyclic (days 4-16) and pregnant (days 4-20) ewes, and the corresponding protein was immunolocalized. Expression of mRNA encoding LIF remained relatively constant throughout the oestrous cycle and was present during early pregnancy. A decrease in mRNA encoding LIF was observed during early pregnancy (on days 12-14) and expression was highest on days 16-20. Immunoreactive LIF was present in the cellular compartments of the endometrium throughout the oestrous cycle and early pregnancy, with maximal immunostaining in the caruncular and intercaruncular luminal epithelium, and moderate staining in the glandular epithelium and intercaruncular stroma. Immunoreactive LIF was also detected in the trophoblast cells of day 17 blastocysts. Separately cultured endometrial epithelial and stromal cells from pregnant animals both expressed mRNA encoding LIF. Ovariectomized steroid-treated ewes were studied to establish whether steroid hormones had a role in regulating endometrial LIF. Ewes treated with oestradiol alone showed lower concentrations of immunoreactive LIF in the endometrium in comparison to ovariectomized, control animals, while treatment of ovariectomized animals with both oestradiol and progesterone had a greater inhibitory effect on LIF immunolocalization. These studies demonstrate the presence of mRNA encoding LIF and protein throughout the oestrous cycle and early pregnancy and suggest that steroid hormones may be involved in their regulation.

Antibody responses to Lucilia cuprina in sheep selected for resistance or susceptibility to L. cuprina.

Sheep bred for resistance (R) or susceptibility (S) to fleece rot and myiasis (blowfly strike) have been shown to differ in inflammatory response to intradermal administration of blowfly (Lucilia cuprina) antigens and artificial challenge. The current paper describes analysis of antibody responses to L. cuprina antigens in the R and S animals. Serum antibody titres and specificities to larval antigens were examined and the specificity of wound exudate antibodies was also investigated in animals artificially challenged with L. cuprina. Titres of L. cuprina specific serum IgA, IgM, IgG2 and IgG1 were measured by ELISA, while specificities were examined on two-dimensional immunoblots of larval homogenates. Exposure to L. cuprina stimulated the production of specific antibody in both R and S animals, however antibody titres did not differ between the R and S animals. There was large variation in antibody specificity between individual animals and some L. cuprina proteins appear to be more frequently recognized by sera from either resistant or susceptible animals, however the recognition of a specific protein could not be solely attributed to the resistance status of the animal. It appears that resistance in these animals may be independent of serum antibody and is likely to be an innate response. Despite high levels of IgG in wound exudates, this antibody recognized few antigens in comparison with serum from the same animal, suggesting that exudate contains little functional antibody in comparison to serum.

Fasciola hepatica: irradiation-induced alterations in carbohydrate and cathepsin-B protease expression in newly excysted juvenile liver fluke.

Irradiation has been successful in the attenuation of infective stage parasites for use as vaccines against a number of parasites including Fasciola spp. The mechanisms of action of irradiation-attenuated vaccines, however, are not clearly understood. In this study, we examined the effect of 3, 10, and 40 krad of gamma-irradiation on the expression of carbohydrates and cathepsin-B by newly excysted juvenile Fasciola hepatica (NEJ). Following irradiation of metacercariae, the expression of concanavalin A (ConA)-specific sugars was decreased on the surface of NEJ and the expression of wheat germ agglutinin (WGA)-specific sugars was increased in the gut and reduced on the surface of NEJ. Cathepsin proteases are a major component of liver fluke excretory/secretory material (ES) and can cleave host immunoglobulin (Ig). Cathepsin-B protease was localized in nonirradiated NEJ to the gut lumen and to secretory granules within the gut epithelia. Irradiation of fluke with 3, 10, and 40 krad of gamma-rays significantly reduced the tissue expression of cathepsin-B at 8 hr postirradiation in an apparently dose-dependent manner. After a further 24 hr culture tissue expression of cathepsin-B was significantly reduced in 10- and 40-krad-irradiated NEJ. Protease activity of ES samples collected over a 24-hr period from irradiated and nonirradiated NEJ cultured in vitro were tested using a rabbit Ig cleavage assay. The proteolytic activity of ES from 10- and 40-krad-irradiated NEJ was reduced during the initial 6 hr in culture and between 12 and 24 hr when compared to ES from nonirradiated controls. Biosynthetic labeling experiments using [35S]methionine and [35S]cysteine indicated that ES material was actively synthesised during 48 hr in vitro culture. Therefore, from this study, we conclude that gamma-irradiation of NEJ alters expression of cathepsin-B protease and WGA- and ConA-specific sugars which may be detrimental to parasite invasion and contribute to the protective immune responses generated in the host by irradiation-attenuated metacercariae of Fasciola spp.

IgE, TNF alpha, IL1 beta, IL4 and IFN gamma gene polymorphisms in sheep selected for resistance to fleece rot and flystrike.

Investigation of restriction fragment length polymorphisms (RFLPs) associated with immunoglobulin E (IgE) and cytokine genes in the sheep genome revealed polymorphisms in the IgE constant heavy chain, interferon gamma and interleukin 4 genes. No polymorphisms were found in interleukin 1 beta or tumour necrosis factor alpha. PstI and BamHI RFLPs in the IgE gene showed differences in frequency between animals selected for resistance or susceptibility to fleece rot and blowfly strike.

Fasciola hepatica: localisation of glutathione S-transferase isoenzymes in adult and juvenile liver fluke.

Four cDNA clones (GST-1, -7, -47, and -51) encoding isoenzymes of the detoxification enzyme glutathione S-transferase (GST) have previously been identified and characterised from Fasciola hepatica. In the present study, antisera were generated to synthetic peptides of regions unique to each of the four GST proteins predicted by the cDNAs. The antisera were characterised, and two were found to distinguish GST-1 from GST-7, GST-47, and GST-51 as a group. These two antisera were used to localise different GSTs in adult and newly excysted juvenile F. hepatica. The antiserum to GST-1 was specific and localised GST-1 to the parenchyma of adult fluke but not to the lamellae of the intestinal caeca. The antiserum to a GST-51 peptide, which cross-reacted with GST-7 and GST-47 but not GST-1, localised the other GSTs not only to the parenchyma but also to the intestinal lamellae of adult fluke. This appears to be the first evidence of tissue-specific expression of GST isoenzymes in trematodes. In contrast to adult fluke, immunolocalisation of the GSTs in juvenile F. hepatica revealed the binding of both the GST-1 and GST-51 antisera to the parenchymal cytoplasm, to cytoplasmic extensions of the parenchyma cells in the subtegumental area, as well as the excretory ducts. No labeling was observed in the intestinal epithelium of the juvenile fluke. These results demonstrate that adult F. hepatica, in contrast to juvenile flukes, contain a GST, which is not GST-1, associated with the lamellae of the gut and suggest that GSTs in adult fluke may play a role in the absorptive function of the adult gut.

Attempted immunisation of sheep against Fasciola hepatica using gamma-irradiated metacercariae.

The potential of gamma-irradiated Fasciola hepatica metacercariae to vaccinate sheep against fascioliasis was examined. The effect of the size of the inocula of irradiated metacercariae and the level of gamma-irradiation on the recovery of non-irradiated fluke was assessed following homologous challenge. Groups of Merino wethers were vaccinated with a single infection of either 500 or 2000 metacercariae, previously exposed to either 30, 100 or 400 Gy of gamma-irradiation. No significant reduction of fluke burdens were observed in any group, although a nonsignificant 20% reduction was observed in sheep vaccinated with 2000 metacercariae irradiated with 100 Gy. A second trial was conducted in which groups of sheep were vaccinated with 2 doses, given 4 weeks apart, of 2000 metacercariae, previously irradiated at either 70, 100 or 150 Gy. In both trials parasite viability was severely affected by doses of gamma-irradiation of 30 Gy or greater and no mature flukes were recovered from control sheep given metacercariae attenuated with 70 Gy or greater. A strong humoral immune response to somatic F. hepatica antigens was observed in all sheep. Only sera from sheep receiving 70 Gy irradiated metacercariae recognised the 2 candidate liver fluke vaccine molecules, F. hepatica glutathione S-transferase and cathepsin-L proteases. No reduction was observed in either the number of flukes or the production of fluke eggs in any vaccinated group. Vaccination appeared to affect the development of the challenge fluke population, resulting in reduced hepatic damage during migration, as measured by levels of serum glutamate dehydrogenase, and an increase in mean fluke weight.

Antibody degradation in wound exudates from blowfly infections on sheep.

Sheep were immunised with ovalbumin and then infected with the sheep blowfly, Lucilia cuprina in order to study immunoglobulin and specific antibody degradation at the wound site. Serum and wound exudates were collected over the infection period and the dry weight and protein content of the exudates were determined. Exudates were analysed by SDS-PAGE and immunoblotting for IgG degradation. Levels of IgG and specific anti-ovalbumin antibodies in the exudates were measured by ELISA. The total weight of exudates increased over the whole period of the infection, while protein content increased in the first 24 h and then remained relatively constant. Immunoglobulin was present 6 h after infection and levels increased with protein content. However, the levels of IgG measured were quite different depending on the secondary antibody used in the ELISA. A monoclonal antibody measured mainly intact IgG while a polyclonal anti-IgG measured intact and degraded IgG. This allowed an estimation that approximately 60% of the IgG in exudates was degraded from 6 h after infection. Assays in vitro showed that L. cuprina larval enzymes degraded sheep antibody. However, measurement of specific anti-ovalbumin levels in exudates suggested that although high levels of antibody were degraded this did not necessarily decrease the level of antigen binding. As a result, IgG degradation may assist and not hinder vaccine development by allowing antibody fragments to penetrate the peritrophic membrane and access gut cell antigens.

A comparison of inflammatory exudates released from myiasis wounds on sheep bred for resistance or susceptibility to Lucilia cuprina.

Sheep bred for resistance (R) or susceptibility (S) to fleece rot and myiasis (blowfly strike) were experimentally infected with L. cuprina larvae. Exudates released from the wound site were collected during the infection at 6, 12, 18 and 24 h. The exudates were separated using two-dimensional gel electrophoresis, and proteins were silver stained and identified by immunoblotting with specific antibody and by their isoelectric points and molecular weights. Comparisons of exudate composition were made over time and between R and S sheep. Between 6 and 12 h post larval implantation the exudate was rich in IgG and fibrinogen, which is before extensive tissue damage and suggests that the exudate is not simply tissue haemorrhage but the result of an inflammatory response by the sheep to L. cuprina. The exudate grew in complexity between 12 and 18 h and contained a maximum of 74 distinct peptide spots by 24 h. Exudate from wounds on resistant sheep contained many more peptides in the first 12 h of infection, suggesting a more rapid inflammatory response. The source of proteins from the exudate remains speculative; it appears to be composed of many acute-phase proteins, large amounts of immunoglobulin G and proportionally low levels of serum albumin. Exudate composition is likely to be influenced by the local synthesis of acute-phase proteins and perhaps immunoglobulins, selective transport to the infection site and also enzymic degradation by L. cuprina larval enzymes. The more rapid exudation of acute-phase and serum proteins at infection sites on R sheep may allow the inhibition of the establishment of fleece rot bacteria or L. cuprina larvae under natural challenge.