PROSPECTS FOR THE CONTROL OF SHEEP BLOWFLY STRIKE BY VACCINATION R. M. SANDEMAN School of Agriculture, La Trobe University, Bundoora, Victoria 3083, Australia R.M. 1990. Prospects for the control of sheep blowfly strike by vaccination. Parasitology UI: 537-541. Research into vaccination against @trike is aimed at either controlling the predisposing condition, fleece rot, or direct control of the fly maggots. A vaccine against the major bacterial species found in fleece rot lesions, ~se~dorno~ aerugfnosa, is undergoing field trials and results suggest that this vaccine may reduce fleece rot incidence. Problems to be investigated include the existence of variants of P. ueruginosa in the field and the involvement of ofher species of bacteria in fleece rot. Strategies for direct vaccination include immnnization with larval products involved in wound formation and larval nu~tionand immuni~tion against novel antigens usually from the gut of first instar larvae, Both methods have resulted in si~ifi~nt inhibition of larval growth. Analysis of larvai products has revealed a number of active proteases which degrade skin proteins such as collagen. Inhibition of these enzymes with plasma enzyme inhibitors also affects larval growth in vitro. Antibodies raised against these enzymes are being tested for inhibitory effects against larvae and used to isolate cDNA clones from Lucilia euprina libraries. Antigens from the gut are able to induce antibodies inhibitory to larval growth both in vitro and in viva. Isolation of these antigens is proceeding in a number of laboratories. Problems still to be analysed include whether growth inhibition produces effective protection in the field and whether sufficient antibody will have early access to the larvae to significantly affect them. A~S~TC~-SANDEMAN
International Journalfor
INDEX KEY WORDS: Blowfly strike; my&is; va~ination; sheep; Lucilia cuprino; fleeu: rot.
BLOWFLY strike, or infection of sheep skin with fly maggots, is the major ectoparasitic disease of sheep in Australia. Sheep deaths have been estimated at up to 3 million per year (Brideoake, 1979) or about 2% of the national flock. Costs due to these losses and the expenses of current control measures are around $200 million per annum (Beck, Moir & Meppem, 1985). The Ay primarily responsible is Lucilia cup&u which initiates over 90% of strikes (Watts, Muller, Dyce & Norris, 1976; Dallwitz, Roberts & Kitching, 1983). Although not an obligate parasite, I;. cuprina reproduces mainly on sheep and is less successful on carrion or other food sources Waterhouse, 1947; O’Sullivan, Perkins, Vokes, Suter & Hopkins, 1983). Merino sheep seem particularly susceptible to fly attack because of their fleece and skin characteristics and this, together with favourable weather conditions in spring and autumn over large areas of Australia, contribute to the size of the problem and to the difficulty of controlling strike outbreaks in individual flocks. Several conditions predispose sheep to Ay attack. These include (a) warm, humid weather, particularly after rain; (b) pathological changes such as bacterial dermatitis or fleece rot; (c) skin wounds or infections
such as foot rot and pizzle rot; (d) skin ir~tation from faecal or urine staining; and (e) sweating, especially around the horns in rams. All these factors provide ideal conditions for survival of the larvae and are attractive to female flies which oviposit in the wool near the affected sites. However, only wet wool is an absolute requirement for successful infections (Sandeman, Collins & Carnegie, 1987). Once hatched, the first instar larvae move on to the skin and cause local in~ammation and sloughing of the epidermal cells (Sandernan ef al., 1987). This leads to exudation from the skin which not only provides a nutrient source but also maintains the high moisture level required for larval survival (Hafl, Martin & McDonell, 1980). The maggots grow rapidly, moulting through three instars in 48-72 h and cause the formation of a puffy oedamatous wound which can cover large areas of the animal’s skin. At third instar, the larvae grow to about 15 mm long and often have their heads buried deep in the dermis, feeding on whole blood (Sandeman et al., 1987). During heavy infections sheep can die within 5 days and show symptoms consistent with severe toxaemia (Broadmeadow, Gibson, Dimmock, Thomas & O’Sullivan, 1984). The maggots drop off the sheep after about 5 days and pupate in the ground.
537
538
R.M. CURRENT
SANDEMAN
CONTROLS
VACCINATION-FLY
Control of infections is required soon after the larvae hatch in order to avoid major wound formation and resultant secondary fly infections. Currently, persistent chemicals such as cyromazine (Vetrazin, Ciba-Geigy Aust. Ltd.) protect sheep by killing the young larvae for up to 12 weeks after treatment (Hart, Cavey, Ryan, Strong, Moore, Thomas, Boray & von Orelli, 1982), though the protective period of the chemical depends on the level of rain exposure, faecal and urine staining or other leaching factors. Fly resistance is also an important factor with the older chemicals. Organo-phosphates and organo-chlorines are less effective for prophylactic protection but are still used to kill existing infections. In association with chemical control, a range of intensive management procedures are used including mulesing, crutching and regular checks for the treatment of sheep during fly wave conditions. As a result, an average grazier spends around $1500 per year on strike control (Beck et al.. 1985). In contrast to these current control measures, vaccination offers the possibility of a once a year injection giving long-term protection. The prerequisite is to induce an immune response in the sheep which can significantly affect larval development within the first 24 h following hatching, when the larvae are most susceptible and the skin wound is relatively contained (Sandeman et al., 1987). The increasing interest in vaccines against flystrike stems from recent research which suggests that this is an attainable and realistic goal. VACCINATION-FLEECE
ROT
One of the first approaches attempted for controlling flystrike was to vaccinate against the predisposing condition, fleece rot. This dermatitis is caused by bacterial proliferation on the skin which results in local skin inflammation, occlusion of wool follicles and a protein exudate in the wool (Watts & Merritt, 1981). The exudate and skin lesion are highly attractive to blowflies. Fleece rot lesions usually contain a number of bacterial species but the organism most often isolated from active lesions is Pseudomonas aeruginosa (Merritt&Watts, 1978). A vaccine against P. aeruginosa has been developed and initial trials suggest that it reduces the incidence of both fleece rot and flystrike (Burrell & MacDiarmid, 1983). Currently, the vaccine is undergoing field trials. Problems yet to be overcome include the existence of a number of serotypes of P. aeruginosa and whether the vaccine is cross-protective against these. However, even if totally effective, this vaccine will only affect fleece rot caused by P. aeruginosa, having little impact on other species of Pseudomonas or other bacteria important in fleece rot (London & Griffith, 1984; MacDiarmid & Burrell, 1986). In addition, it will not affect flystrike which occurs independently of fleece rot.
STRIKE
Immune responses A direct approach to vaccination against flystrike was first considered when it was shown that sheep produced antibodies against L. cuprina antigens after an infection (O’Donnell, Green, Connell & Hopkins, 1980). The same group also found that animals immunized with larval homogenates, although not protected against infection, did produce antibodies which could inhibit larval growth in in vitro cultures (O’Donnell, Green. Connell & Hopkins, 1981). At about the same time, ICI, Australia initiated a vaccine programme after work by N. Wynne-Jones and R. Shaw of ICI, New Zealand suggested that Romney sheep could be protected against L. serricata by vaccination with the excretory-secretory antigens of the fly larvae (Montague, unpublished). Despite many trials with a number of L. cuprina preparations in Merinos, no positive effects were obtained and the research was stopped. However, the results did suggest that some sheep could be made resistant and that antigens varied in their efficacy between fly strains, especially between ‘lab-adapted’ and ‘wild-type’ strains (Montague, unpublished). More recent research has suggested that the major antigens recognized by sheep during infections are from the gut and salivary glands (Skelly & Howells, 1987). Taken together. these results all indicate that the flystrike lesion is a fairly strong stimulus to the immune system of sheep and that some sheep may be able to mount effective anti-larval reactions. The possibility that sheep may be able to acquire an immune-based resistance to infection had been suggested from observations of resistance in older sheep (Watts, Murray & Graham, 1979). However, initial analysis showed that at second infection sheep were usually more sensitive than at first infection, both in terms of wound size and larval survival (O’Donnell et al., 1980). Later studies found that, if infections were continually given to a group of sheep, this initial sensitivity passed and some animals did develop a resistance which was characterized by reduced larval burdens (Sandeman, Dowse & Carnegie, 1985; Sandeman. Bowles, Stacey & Carnegie, 1986). The resistance developed after four or five consecutive infections at 2-week intervals and was expressed in half of the animals infected. Resistance was also associated with larger wounds per maggot recovered, earlier onset of wound exudation and larger Arthus-type (4 h) skin reactivity to larval excretory-secretory products. Recent experiments have confirmed that resistance develops in some sheep but only when infections were given at 2- and not 4-week intervals (Sandeman, Chandler. Feehan & O’Meara, unpublished). In addition, the resistant state did not persist beyond three or four infections which implies that it may not be due to acquired immunity but rather to changes in inflammation reactions or other non-memory responses.
Vaccination against flystrike
Whether in~ammation potentiates or inhibits larval survival is still equivocal. Initially, it was found that ~tamethasone inhibited larval survival (O’Donnell et al., 1980) and Broadmeadow (Abstract, Australian Society for Parasitology, AGM, Sydney, 1986) suggested that sheep with larger skin responses to larval products were more susceptible to infection. Our own work has also found positive correlations between skin histamine sensitivity and wound size at primary infections (Sandeman et al., 1985). However, we have also consistently found negative correlations between skin inflammation to larval products and larval survival (Sandeman et al., 1985, 1986; Bowles, Carnegie & Sandeman, 1987). In fact, recent experiments have confirmed and extended this finding, especially work on the flocks at the N.S.W. Department of Agriculture’s Trangie Research Station. These flocks have been bred since 1975 for resistance or susceptibility to natural and experimental fleece rot and natural flystrike (Raadsma, 1987). When these flocks are skin tested with L. cuprina larval excretorysecretory products, responses are significantly and consistently larger in the resistant flock in both previously infected and naive animals (O’Meara, Saville, Nesa & Sandeman, unpublished). It is obvious from these findings and work currently in progress that sheep produce different types of hy~rsensitivity responses to larval products and that the larvae also directly induce skin inflammation. The larval-induced response occurs with a l-2 h maximum weal size after product injection (Broadmeadow, 1986, abstract cited above; Collins, G’Meara, Seaton & Sandeman, unpublished). The major sheep response has a maximum weal size at 2-4 h after injection or later depending on the degree of sensitization to larval products (Collins, O’Meara, Seaton & Sandernan, unpublished). This response, which is consistent with an Arthus-type hypersensitivity, becomes the major reaction after only one or two injections of products or the same number of infections with larvae and can produce massive weals in the skin. Immediate anaphyiactic responses have not been confirmed because of the difficulty in carrying out PCA reactions with the toxic larval products and the lack of an antibody against sheep IgE. Despite this, it is clear that inflammation plays a central role in wound formation and exudate leakage and that the timing and specific nature of inflammatory events may be critical to larval establishment. Larval products Studies on the nature of the larval products that induce inflammation and wound formation are central, not only to understanding these processes, but also to one of the strategies for vaccination against flystrike. The larvae release a large range of products which include a number of very active protease enzymes. These enzymes are able to degrade skin proteins and may have direct effects in the plasma
539
in~ammatory and coagulation cascades (Bowles, Carnegie & Sandeman, 1988). The proteases are inhibited by several sheep plasma protease inhibitors including ~-macroglobulin (&M), cr, proteinase inhibitor and anti-Thrombin III (ATIII) (Bowles, Feehan & Sandeman, 1990), and two of these (%M and ATIII) also inhibit larval growth in vitro. This suggests not only that inhibition of larval proteases can affect fly infections, but also that plasma enzyme inhibitors may have these effects in those sheep which show some resistance to flystrike. The enzymes and other molecules released by the larvae are being isolated and early results suggest that antibodies against at least one of them have inhibitory activity against larvae in vitro (Sandeman, Farrell&Chandler, unpublished). Other prospects for vaccine antigens in the products of the larvae undoubtedly exist, particularly those molecules concerned with wound initiation and larval-induced inflammation. Novel antigens The finding by O’Donnell et al. (1981) that sera from sheep injected with larval homogenates were inhibitory to larval growth in vitro has led to the second major strategy of vaccine development; the search for a novel antigen vaccine. The early findings were foilowed by M. Broadmeadow (unpublished) who found that gut and salivary gland extracts induced antibody in sheep which inhibited larval growth in vitro and that whole gut and gut membrane extracts also inhibits larval establishment in vivo. These results, together with the development of a novel antigen vaccine against the cattle tick, ~~QF~il~ microplus (Johnston, Kemp & Pearson, 1986), have led to a number of laboratories using this approach. Most recent results suggest that molecules will be isolated from the larval gut which can induce inhibitory responses both in vivo and in vitro. These effects have already reached a 70% inhibition of larval growth with some preparations (I.East, unpublished; Sandeman, unpublished), however, the effects have not included larval killing. As a result, field trials will be necessary before the full significance of growth inhibition can be gauged on natural infections. However, it seems probable that inhibition of growth at first instar would also inhibit wound formation and, as a result, would lead to starvation and/or desiccation of the larvae. In addition to gut antigens, a number of other proteins are being tested as novel antigens in vaccine trials. These include developmental enzymes such as dopa-decarboxylase (A.J. Howells, unpublished) and phenyloxidase (Barrett & Trevalle, 1989). A problem in the development of these anti-larval gut vaccines is the peritrophic membrane in the midgut which protects the gut cells by gritting only smaller proteins to pass through. However, the current practice of ignoring this im~diment seems to be justified by the results to date. The use of novel antigens usually found in the haemolymph or other
540
R. M. SANDEMAN
internal sites also faces the problem of obtaining access for at least Fab portions of the antibody molecule. Little work has been carried out on either antibody access through the peritrophic membrane, or across the gut as a whole. The major problem with the current scenarios of vaccine action is ensuring access of adequate levels of antibody to the larvae as soon as possible after skin contact. In fact, the variability in this response between sheep suggests that some animals would be more easily vaccinated than others; for example, the Trangie resistant flock. In fact, effective vaccination of an outbred flock may require manipulation of the inflammation response, for example, antigens which induce specific hypersensitivity reactions could be included in the vaccine to ensure a rapid response. However, we have also found that antibody is the pr~ominant protein present in exudates from struck sheep, especially in the first 24 h of infection (O’Meara & Sandeman, unpublished). Thus, if the correct vaccine formulation and immunization schedule can be found to induce high levels of specific antibody in the skin, then other measures may not be required. CONCLUSIONS Overall, the prospects for vaccination against flystrike have improved markedly over the last 10 years. It is almost certain that the next few years will see field trials of a num~r of putative vaccines. If the results from these trials are as promising as results to date, then vaccines may be available to control flystrike early in the next century. In addition, of course, the research currently in progress will give a clearer understanding, not only of flystrike but also generally of immune and inflammatory responses to skin pathogens and ectoparasites. REFERENCES
Sheep (Edited by RAADSMAH. W.), pp. 7-21. New South _-._. Walds Department of Agriculture, Sidney. BROADMEA~WM.. GIBXIN J. E.. DIMM~CKC. K.. THDMAQ R. J. & O%LLI;AN B. M. 1984. The pathogenesis of Rystrike in sheep. Gyool Technology and Sheep Breeding 32: 28-32. BURRELLD. H. & MACDIARMIDJ. A. 1983. The role of Pseudomonas aeruginosa in pathogenesis of fleece rot and indirect control of body strike by immunization. In: Second National Symposium on Sheep Blowfly and Flystrike in Sheep (Edited by RAADSMAH. W.), pp. 283-291. New South Wales Department of Agriculture, Sydney. DALLWITZ R., ROBERTS J. A. & KITCHINC R. L. 1983. Blowflies visiting struck sheep and survival of their eggs and larvae in strikes. In: Second National Symposium on Sheep BlowpV and Flystrike in Sheep (Edited by RAADSMAH. W.), pp. 124-129. New South Wales Department of Agriculture, Sydney. HALL C. A., MARTIN I. C. A. & MCDONELL P. A. 1980. The effect of a drying agent (B26) on wool moisture and blowfiv strike. Research in Vet&nary Science 29: 186189. ‘.__’ HART R. J.. CAVEYW. A.. RYAN K. J.. STRONGM. B., Men= ,-..B., THOMASP. L., BORAYJ. C. & ORELLI M. VON1982. A new sheep blowfly insecticide. Australian Vererinary Journal59: 104-109. JOHNSTON L. A. Y., KEMP D. H. & PEARSON R. D. 1986. Immunization of cattle against Boophilus microplus using extracts derived from adult females: effects of induced immunity on tick populations. Internarional Journal for Parasitology 16: 27-34. L~HDONC. J. & GRIFFITII I. P. 1984. Characterization of P~udomonads isolated from diseased fleece. Applied and Environmental ~icrobio1og.v 47: 993-997. MACDIARM~DJ. A. & BURRELLD. H. 1986. Characterization of Ps~domonas maitophi~ia isolates from fleece rot. Applied and environmental Microbiology 51: 24&348. MERRITT G. C. & WATTS J. E. 1978. An in vitro technique for studying fleece rot and lly strike in sheep. Australian Veterinary Journal 54: 5 13-5 16. O’DONNELL I. J., GREENP. E., CONNELLJ. A. &HOPKINS P. S. 1980. lmmunoglobulin G antibodies to the antigens of Lucilia cuprina in the sera of flystruck sheep. Australian Journal of Biological Sciences 33: 27-34. O’DONNELL I. J.. GREEN P. E.. CONNELLJ. A.&HOPKINS P _. $ 1981. Immunization ofsheep with larval antigens of Lucilia cuprina. Australian Journal of Biological Sciences 34: 41 l417. O’SULLIVANB. M., PERKINSI., VOKESL., SUTERG. &HOPKINS P. S. 1983. The ecology of Lucifia cuprina (Weidemann) in western Queensland. In: Second National Symposia on Sheep Blowfy and Flystrike in Sheep (Edited by RAADSMA H. W.), pp. 96-99. New South Wales Department of Agriculture, Sydney. RAADSMA H. W. 1987. Flystrike control, an overview of management and breeding options. Wool Technology and Sheep Breeding 35: 174-l 86. SANDEMANR. M., DOWSEC. A. &CARNEGIE P. R. 1985. Initial characterization of the sheep immune response to infection with Lucilia cuprina. the sheep blowfly. International Journalfor Para&logy 15: 181-185. SANDEMANR. M., BOWLESV. M., STACEYI. N. &CARNEGIE .- P_ R. 1986. Acquired resistance in sheep to infection with larvae of the blowfly, Lucilia cuprina. Internationa~Joarna~ for Para.~jtology 16: 69-75. SANDEMA~;R. M.. COLLINSB. J. & CARNEGIE P. R. 1987. A scanning electron microscope study of Lucifia cuprina ll..l
.”
..I
BARRETT M. & TREVALLE W. 1989. The immune response of the sheep popliteal lymph node to a purified phenofoxidase from larval cuticle of the sheep ectoparasite, Luciiia cuprina. Journal ofparasito~ogy 75: 70-75. BECKT., MOIRB. & MEPPEMT. 1985. The costs ofparasites to the Australian sheep industry. Quarterly Review of the Rural Economy 7: 33&343. BOWLESV. M., CARNEGIEP. R. & SANDEMAN R. M. 1987. Immunization of sheep against infection with larvae of the blowfly, Lucilia cuprina. InternaGonal .Journal for Parasitology 17: 759-765. BOWLESV. M., CARNEGIEP. R. & SANDEMAN R. M. 1988. Characterization ofcollagenolytic and proteolytic enzymes from the larvae of Lucilia cuprina, the sheep blowfly. Australian Journal of Biological Research 41: 269-278. BOWLES V. M., FEEHAN J. P. & SANDEMAN R. M. 1990. Sheep plasma protease inhibitors influencing protease activity and growth of Lucilia cuprina larvae in vitro. international Jou~ai~r Par~iio~ogy 20: 16% I 74. BRIDEOAKE B. R. 1979. The estimated cost of blowfly control in the Australian sheep industry: 1969-70 to 1975576. In: National Symposium on Sheep Blou$y and Ffystrike in
Vaccination against flystrike
541
larvae and the development of flystrike on sheep. International Journalfor Parasitology II: 153-758. SKELLYP. J. & HOWELLS A. J. 1987. The humoral immune
WAITSJ. E., MULLERM. J., DYCE A. L. & NORRISK. R. 1976. The species of tlies reared from struck sheep in south-eastern Australia. Australian Veterinary Journal 52: 488489.
response of sheep to antigens from larvae of the sheep blowfly (Lucilia cuprina). International Journalfor Paras-
WANTSJ. E., MURIUYM. D. & GRAHAMN. P. H. 1979. The blowfly strike problem in New South Wales. Australian
itology 17: 1081-1087.
WATERHOUSE D. F. 1947. The relative importance of live sheep and of carrion as breeding grounds for the Australian sheep blowfly, Lucilia cuprina. Bulletin of the Council for ScientiJic and Industrial Research, No. 21 I.
Veterinary Journal 55: 325-334.
WATIXJ. E. & MERRII-~G. C. 1981. The leakage of plasma proteins on to the skin surface of sheep during the development of fleece rot and body strike. Australian Veterinary Journal 57: 98-99.
International Journalfor
INDEX KEY WORDS: Blowfly strike; my&is; va~ination; sheep; Lucilia cuprino; fleeu: rot.
BLOWFLY strike, or infection of sheep skin with fly maggots, is the major ectoparasitic disease of sheep in Australia. Sheep deaths have been estimated at up to 3 million per year (Brideoake, 1979) or about 2% of the national flock. Costs due to these losses and the expenses of current control measures are around $200 million per annum (Beck, Moir & Meppem, 1985). The Ay primarily responsible is Lucilia cup&u which initiates over 90% of strikes (Watts, Muller, Dyce & Norris, 1976; Dallwitz, Roberts & Kitching, 1983). Although not an obligate parasite, I;. cuprina reproduces mainly on sheep and is less successful on carrion or other food sources Waterhouse, 1947; O’Sullivan, Perkins, Vokes, Suter & Hopkins, 1983). Merino sheep seem particularly susceptible to fly attack because of their fleece and skin characteristics and this, together with favourable weather conditions in spring and autumn over large areas of Australia, contribute to the size of the problem and to the difficulty of controlling strike outbreaks in individual flocks. Several conditions predispose sheep to Ay attack. These include (a) warm, humid weather, particularly after rain; (b) pathological changes such as bacterial dermatitis or fleece rot; (c) skin wounds or infections
such as foot rot and pizzle rot; (d) skin ir~tation from faecal or urine staining; and (e) sweating, especially around the horns in rams. All these factors provide ideal conditions for survival of the larvae and are attractive to female flies which oviposit in the wool near the affected sites. However, only wet wool is an absolute requirement for successful infections (Sandeman, Collins & Carnegie, 1987). Once hatched, the first instar larvae move on to the skin and cause local in~ammation and sloughing of the epidermal cells (Sandernan ef al., 1987). This leads to exudation from the skin which not only provides a nutrient source but also maintains the high moisture level required for larval survival (Hafl, Martin & McDonell, 1980). The maggots grow rapidly, moulting through three instars in 48-72 h and cause the formation of a puffy oedamatous wound which can cover large areas of the animal’s skin. At third instar, the larvae grow to about 15 mm long and often have their heads buried deep in the dermis, feeding on whole blood (Sandeman et al., 1987). During heavy infections sheep can die within 5 days and show symptoms consistent with severe toxaemia (Broadmeadow, Gibson, Dimmock, Thomas & O’Sullivan, 1984). The maggots drop off the sheep after about 5 days and pupate in the ground.
537
538
R.M. CURRENT
SANDEMAN
CONTROLS
VACCINATION-FLY
Control of infections is required soon after the larvae hatch in order to avoid major wound formation and resultant secondary fly infections. Currently, persistent chemicals such as cyromazine (Vetrazin, Ciba-Geigy Aust. Ltd.) protect sheep by killing the young larvae for up to 12 weeks after treatment (Hart, Cavey, Ryan, Strong, Moore, Thomas, Boray & von Orelli, 1982), though the protective period of the chemical depends on the level of rain exposure, faecal and urine staining or other leaching factors. Fly resistance is also an important factor with the older chemicals. Organo-phosphates and organo-chlorines are less effective for prophylactic protection but are still used to kill existing infections. In association with chemical control, a range of intensive management procedures are used including mulesing, crutching and regular checks for the treatment of sheep during fly wave conditions. As a result, an average grazier spends around $1500 per year on strike control (Beck et al.. 1985). In contrast to these current control measures, vaccination offers the possibility of a once a year injection giving long-term protection. The prerequisite is to induce an immune response in the sheep which can significantly affect larval development within the first 24 h following hatching, when the larvae are most susceptible and the skin wound is relatively contained (Sandeman et al., 1987). The increasing interest in vaccines against flystrike stems from recent research which suggests that this is an attainable and realistic goal. VACCINATION-FLEECE
ROT
One of the first approaches attempted for controlling flystrike was to vaccinate against the predisposing condition, fleece rot. This dermatitis is caused by bacterial proliferation on the skin which results in local skin inflammation, occlusion of wool follicles and a protein exudate in the wool (Watts & Merritt, 1981). The exudate and skin lesion are highly attractive to blowflies. Fleece rot lesions usually contain a number of bacterial species but the organism most often isolated from active lesions is Pseudomonas aeruginosa (Merritt&Watts, 1978). A vaccine against P. aeruginosa has been developed and initial trials suggest that it reduces the incidence of both fleece rot and flystrike (Burrell & MacDiarmid, 1983). Currently, the vaccine is undergoing field trials. Problems yet to be overcome include the existence of a number of serotypes of P. aeruginosa and whether the vaccine is cross-protective against these. However, even if totally effective, this vaccine will only affect fleece rot caused by P. aeruginosa, having little impact on other species of Pseudomonas or other bacteria important in fleece rot (London & Griffith, 1984; MacDiarmid & Burrell, 1986). In addition, it will not affect flystrike which occurs independently of fleece rot.
STRIKE
Immune responses A direct approach to vaccination against flystrike was first considered when it was shown that sheep produced antibodies against L. cuprina antigens after an infection (O’Donnell, Green, Connell & Hopkins, 1980). The same group also found that animals immunized with larval homogenates, although not protected against infection, did produce antibodies which could inhibit larval growth in in vitro cultures (O’Donnell, Green. Connell & Hopkins, 1981). At about the same time, ICI, Australia initiated a vaccine programme after work by N. Wynne-Jones and R. Shaw of ICI, New Zealand suggested that Romney sheep could be protected against L. serricata by vaccination with the excretory-secretory antigens of the fly larvae (Montague, unpublished). Despite many trials with a number of L. cuprina preparations in Merinos, no positive effects were obtained and the research was stopped. However, the results did suggest that some sheep could be made resistant and that antigens varied in their efficacy between fly strains, especially between ‘lab-adapted’ and ‘wild-type’ strains (Montague, unpublished). More recent research has suggested that the major antigens recognized by sheep during infections are from the gut and salivary glands (Skelly & Howells, 1987). Taken together. these results all indicate that the flystrike lesion is a fairly strong stimulus to the immune system of sheep and that some sheep may be able to mount effective anti-larval reactions. The possibility that sheep may be able to acquire an immune-based resistance to infection had been suggested from observations of resistance in older sheep (Watts, Murray & Graham, 1979). However, initial analysis showed that at second infection sheep were usually more sensitive than at first infection, both in terms of wound size and larval survival (O’Donnell et al., 1980). Later studies found that, if infections were continually given to a group of sheep, this initial sensitivity passed and some animals did develop a resistance which was characterized by reduced larval burdens (Sandeman, Dowse & Carnegie, 1985; Sandeman. Bowles, Stacey & Carnegie, 1986). The resistance developed after four or five consecutive infections at 2-week intervals and was expressed in half of the animals infected. Resistance was also associated with larger wounds per maggot recovered, earlier onset of wound exudation and larger Arthus-type (4 h) skin reactivity to larval excretory-secretory products. Recent experiments have confirmed that resistance develops in some sheep but only when infections were given at 2- and not 4-week intervals (Sandeman, Chandler. Feehan & O’Meara, unpublished). In addition, the resistant state did not persist beyond three or four infections which implies that it may not be due to acquired immunity but rather to changes in inflammation reactions or other non-memory responses.
Vaccination against flystrike
Whether in~ammation potentiates or inhibits larval survival is still equivocal. Initially, it was found that ~tamethasone inhibited larval survival (O’Donnell et al., 1980) and Broadmeadow (Abstract, Australian Society for Parasitology, AGM, Sydney, 1986) suggested that sheep with larger skin responses to larval products were more susceptible to infection. Our own work has also found positive correlations between skin histamine sensitivity and wound size at primary infections (Sandeman et al., 1985). However, we have also consistently found negative correlations between skin inflammation to larval products and larval survival (Sandeman et al., 1985, 1986; Bowles, Carnegie & Sandeman, 1987). In fact, recent experiments have confirmed and extended this finding, especially work on the flocks at the N.S.W. Department of Agriculture’s Trangie Research Station. These flocks have been bred since 1975 for resistance or susceptibility to natural and experimental fleece rot and natural flystrike (Raadsma, 1987). When these flocks are skin tested with L. cuprina larval excretorysecretory products, responses are significantly and consistently larger in the resistant flock in both previously infected and naive animals (O’Meara, Saville, Nesa & Sandeman, unpublished). It is obvious from these findings and work currently in progress that sheep produce different types of hy~rsensitivity responses to larval products and that the larvae also directly induce skin inflammation. The larval-induced response occurs with a l-2 h maximum weal size after product injection (Broadmeadow, 1986, abstract cited above; Collins, G’Meara, Seaton & Sandeman, unpublished). The major sheep response has a maximum weal size at 2-4 h after injection or later depending on the degree of sensitization to larval products (Collins, O’Meara, Seaton & Sandernan, unpublished). This response, which is consistent with an Arthus-type hypersensitivity, becomes the major reaction after only one or two injections of products or the same number of infections with larvae and can produce massive weals in the skin. Immediate anaphyiactic responses have not been confirmed because of the difficulty in carrying out PCA reactions with the toxic larval products and the lack of an antibody against sheep IgE. Despite this, it is clear that inflammation plays a central role in wound formation and exudate leakage and that the timing and specific nature of inflammatory events may be critical to larval establishment. Larval products Studies on the nature of the larval products that induce inflammation and wound formation are central, not only to understanding these processes, but also to one of the strategies for vaccination against flystrike. The larvae release a large range of products which include a number of very active protease enzymes. These enzymes are able to degrade skin proteins and may have direct effects in the plasma
539
in~ammatory and coagulation cascades (Bowles, Carnegie & Sandeman, 1988). The proteases are inhibited by several sheep plasma protease inhibitors including ~-macroglobulin (&M), cr, proteinase inhibitor and anti-Thrombin III (ATIII) (Bowles, Feehan & Sandeman, 1990), and two of these (%M and ATIII) also inhibit larval growth in vitro. This suggests not only that inhibition of larval proteases can affect fly infections, but also that plasma enzyme inhibitors may have these effects in those sheep which show some resistance to flystrike. The enzymes and other molecules released by the larvae are being isolated and early results suggest that antibodies against at least one of them have inhibitory activity against larvae in vitro (Sandeman, Farrell&Chandler, unpublished). Other prospects for vaccine antigens in the products of the larvae undoubtedly exist, particularly those molecules concerned with wound initiation and larval-induced inflammation. Novel antigens The finding by O’Donnell et al. (1981) that sera from sheep injected with larval homogenates were inhibitory to larval growth in vitro has led to the second major strategy of vaccine development; the search for a novel antigen vaccine. The early findings were foilowed by M. Broadmeadow (unpublished) who found that gut and salivary gland extracts induced antibody in sheep which inhibited larval growth in vitro and that whole gut and gut membrane extracts also inhibits larval establishment in vivo. These results, together with the development of a novel antigen vaccine against the cattle tick, ~~QF~il~ microplus (Johnston, Kemp & Pearson, 1986), have led to a number of laboratories using this approach. Most recent results suggest that molecules will be isolated from the larval gut which can induce inhibitory responses both in vivo and in vitro. These effects have already reached a 70% inhibition of larval growth with some preparations (I.East, unpublished; Sandeman, unpublished), however, the effects have not included larval killing. As a result, field trials will be necessary before the full significance of growth inhibition can be gauged on natural infections. However, it seems probable that inhibition of growth at first instar would also inhibit wound formation and, as a result, would lead to starvation and/or desiccation of the larvae. In addition to gut antigens, a number of other proteins are being tested as novel antigens in vaccine trials. These include developmental enzymes such as dopa-decarboxylase (A.J. Howells, unpublished) and phenyloxidase (Barrett & Trevalle, 1989). A problem in the development of these anti-larval gut vaccines is the peritrophic membrane in the midgut which protects the gut cells by gritting only smaller proteins to pass through. However, the current practice of ignoring this im~diment seems to be justified by the results to date. The use of novel antigens usually found in the haemolymph or other
540
R. M. SANDEMAN
internal sites also faces the problem of obtaining access for at least Fab portions of the antibody molecule. Little work has been carried out on either antibody access through the peritrophic membrane, or across the gut as a whole. The major problem with the current scenarios of vaccine action is ensuring access of adequate levels of antibody to the larvae as soon as possible after skin contact. In fact, the variability in this response between sheep suggests that some animals would be more easily vaccinated than others; for example, the Trangie resistant flock. In fact, effective vaccination of an outbred flock may require manipulation of the inflammation response, for example, antigens which induce specific hypersensitivity reactions could be included in the vaccine to ensure a rapid response. However, we have also found that antibody is the pr~ominant protein present in exudates from struck sheep, especially in the first 24 h of infection (O’Meara & Sandeman, unpublished). Thus, if the correct vaccine formulation and immunization schedule can be found to induce high levels of specific antibody in the skin, then other measures may not be required. CONCLUSIONS Overall, the prospects for vaccination against flystrike have improved markedly over the last 10 years. It is almost certain that the next few years will see field trials of a num~r of putative vaccines. If the results from these trials are as promising as results to date, then vaccines may be available to control flystrike early in the next century. In addition, of course, the research currently in progress will give a clearer understanding, not only of flystrike but also generally of immune and inflammatory responses to skin pathogens and ectoparasites. REFERENCES
Sheep (Edited by RAADSMAH. W.), pp. 7-21. New South _-._. Walds Department of Agriculture, Sidney. BROADMEA~WM.. GIBXIN J. E.. DIMM~CKC. K.. THDMAQ R. J. & O%LLI;AN B. M. 1984. The pathogenesis of Rystrike in sheep. Gyool Technology and Sheep Breeding 32: 28-32. BURRELLD. H. & MACDIARMIDJ. A. 1983. The role of Pseudomonas aeruginosa in pathogenesis of fleece rot and indirect control of body strike by immunization. In: Second National Symposium on Sheep Blowfly and Flystrike in Sheep (Edited by RAADSMAH. W.), pp. 283-291. New South Wales Department of Agriculture, Sydney. DALLWITZ R., ROBERTS J. A. & KITCHINC R. L. 1983. Blowflies visiting struck sheep and survival of their eggs and larvae in strikes. In: Second National Symposium on Sheep BlowpV and Flystrike in Sheep (Edited by RAADSMAH. W.), pp. 124-129. New South Wales Department of Agriculture, Sydney. HALL C. A., MARTIN I. C. A. & MCDONELL P. A. 1980. The effect of a drying agent (B26) on wool moisture and blowfiv strike. Research in Vet&nary Science 29: 186189. ‘.__’ HART R. J.. CAVEYW. A.. RYAN K. J.. STRONGM. B., Men= ,-..B., THOMASP. L., BORAYJ. C. & ORELLI M. VON1982. A new sheep blowfly insecticide. Australian Vererinary Journal59: 104-109. JOHNSTON L. A. Y., KEMP D. H. & PEARSON R. D. 1986. Immunization of cattle against Boophilus microplus using extracts derived from adult females: effects of induced immunity on tick populations. Internarional Journal for Parasitology 16: 27-34. L~HDONC. J. & GRIFFITII I. P. 1984. Characterization of P~udomonads isolated from diseased fleece. Applied and Environmental ~icrobio1og.v 47: 993-997. MACDIARM~DJ. A. & BURRELLD. H. 1986. Characterization of Ps~domonas maitophi~ia isolates from fleece rot. Applied and environmental Microbiology 51: 24&348. MERRITT G. C. & WATTS J. E. 1978. An in vitro technique for studying fleece rot and lly strike in sheep. Australian Veterinary Journal 54: 5 13-5 16. O’DONNELL I. J., GREENP. E., CONNELLJ. A. &HOPKINS P. S. 1980. lmmunoglobulin G antibodies to the antigens of Lucilia cuprina in the sera of flystruck sheep. Australian Journal of Biological Sciences 33: 27-34. O’DONNELL I. J.. GREEN P. E.. CONNELLJ. A.&HOPKINS P _. $ 1981. Immunization ofsheep with larval antigens of Lucilia cuprina. Australian Journal of Biological Sciences 34: 41 l417. O’SULLIVANB. M., PERKINSI., VOKESL., SUTERG. &HOPKINS P. S. 1983. The ecology of Lucifia cuprina (Weidemann) in western Queensland. In: Second National Symposia on Sheep Blowfy and Flystrike in Sheep (Edited by RAADSMA H. W.), pp. 96-99. New South Wales Department of Agriculture, Sydney. RAADSMA H. W. 1987. Flystrike control, an overview of management and breeding options. Wool Technology and Sheep Breeding 35: 174-l 86. SANDEMANR. M., DOWSEC. A. &CARNEGIE P. R. 1985. Initial characterization of the sheep immune response to infection with Lucilia cuprina. the sheep blowfly. International Journalfor Para&logy 15: 181-185. SANDEMANR. M., BOWLESV. M., STACEYI. N. &CARNEGIE .- P_ R. 1986. Acquired resistance in sheep to infection with larvae of the blowfly, Lucilia cuprina. Internationa~Joarna~ for Para.~jtology 16: 69-75. SANDEMA~;R. M.. COLLINSB. J. & CARNEGIE P. R. 1987. A scanning electron microscope study of Lucifia cuprina ll..l
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BARRETT M. & TREVALLE W. 1989. The immune response of the sheep popliteal lymph node to a purified phenofoxidase from larval cuticle of the sheep ectoparasite, Luciiia cuprina. Journal ofparasito~ogy 75: 70-75. BECKT., MOIRB. & MEPPEMT. 1985. The costs ofparasites to the Australian sheep industry. Quarterly Review of the Rural Economy 7: 33&343. BOWLESV. M., CARNEGIEP. R. & SANDEMAN R. M. 1987. Immunization of sheep against infection with larvae of the blowfly, Lucilia cuprina. InternaGonal .Journal for Parasitology 17: 759-765. BOWLESV. M., CARNEGIEP. R. & SANDEMAN R. M. 1988. Characterization ofcollagenolytic and proteolytic enzymes from the larvae of Lucilia cuprina, the sheep blowfly. Australian Journal of Biological Research 41: 269-278. BOWLES V. M., FEEHAN J. P. & SANDEMAN R. M. 1990. Sheep plasma protease inhibitors influencing protease activity and growth of Lucilia cuprina larvae in vitro. international Jou~ai~r Par~iio~ogy 20: 16% I 74. BRIDEOAKE B. R. 1979. The estimated cost of blowfly control in the Australian sheep industry: 1969-70 to 1975576. In: National Symposium on Sheep Blou$y and Ffystrike in
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