Mammalian immune responses to myiasis

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Mammalian Immune Responses to Myiasis R.W. Baron and D.D. Colwell Myiasis is responsible for significant losses to the livestock indus~ry worldwide. Control programs have been successful in reducing the number of infested animals. However, serious concerns regarding the use of pesticides have prompted research into alternative strategies l~r pest control. In this article, Bob Baron and Doug Colwell discuss progress made towards the understanding of the immune response to Hypoderma spp and other myiasis-producing arthropods. Prospects for vaccination are discussed. The nature of the host-parasite interaction in myiasis varies in degree and intensity depending on the biology of the insect involved. Contrast the deep tissue invasion by cattle grubs (warble fly), Hypoderma bovis (Fig. I) and H. lineatum, with the rather superficial tissue invasion by the nose bots such as Oestns ovis and Cephenernyia spp. Likewise, contrast the relatively limited damage associated with cattle grub infestations with that of the screwworm Cochliornyia hominovorax, or Neobellieria citellivora, where host tissue dam~,ge is massive, often leading to death of the host. The mammalian immune response should exhibit a similar variability. Unfortunately, much of the recent research into host immune responses has focused on a limited number of species that are of significance to livestock production or on host-parasite models that mimic livestock systems and has not addressed the entire range of hostparasite interactions. The.. economic impact of the species studied certainly justifies the focus of effort. In some cases, mandatory control programs, such as those in place in parts of Europe ~, and sterile fly release programs in Canada and the USA 2 for Hypoderma spp, haw~ significantly reduced the number of infested cattle and have been shown to produce positive benefit-cost ratios 3. However, the long-term use of chemical control measures for these and other arthropod pests can potentially lead to serious consequences4, including residues in meat and milk, development of resistance, destruction of nontargets and environmental pollution. These concerns have stimulated research into alternative control strategies that are environmentally sustainable. Artificial ~) 199 I, Elsevier Science Publishers Ltd, ,IUK) 0169 4707/9 I/$0200

induction of host resistance is one such alternative but an understanding of the host immune response is critical if this approach is to proceed. Significant progress has been made in the past decade in our knowledge of the immune response to myiasis-producing arthropods s.

Protective Antigens The origin and nature of the antigenic substances produced by the parasitic larvae is a primary concern in understanding the immunological response of the host. The enzymatic secretions of first instar H. lineatum larvae have been isolated and biochemically characterized 6-9 but information on H. bovis is more limited. Nondenaturing electrophoretic resolution of H. lineatum and H. bovis first instar protein antigens ~° has identified ten protein bands in each species. Differences in the profiles suggest charge, mass or shape differences. However, molecular weights appear similar when resolved by denaturing polyacrylamide gel electrophoresis (PAGE). The principal soluble proteins of H. lineaturn include three serine proteases: hypodermin A 8, hypodermin B 7 and hypodermin C 6. All are both antigenic and immunogenic in infested and vaccinated cattle ~~'12 Western blot analysis of PAGE-resolved H. bovis proteins shows extensive crossreactivity between the two species I°, which is in agreement with previous serological work 13-~5. The most common shared epitope is associated with hypodermin C while hypodermin B is not very prominent. Hypodermin A is equally prominent in both species! °. Although the most commonly shared epitope between the two species is associated with hypodermin C I°, it has been shown that hypodermin A 16 and/or hypodermin B and C ~2 are potentially protective immunogens in H. Iineatum infestations. Although not as well characterized, it is clear that the major antigens of Lucilia cuprina, the causative agent of fly strike in sheep in the southern hemisphere, are similar to those described for cattle grubs. Larvae of L. cuprina release a number of very active proteases that degrade skin proteins and may have a

direct effect in the plasma inflammatory and coagulation cascades 17. These proteases have been characterized as having both tryptic and chymotryptic activities ~8. They are inhibited by sheep plasma protease inhibitors ~9, suggesting that inhibition of larval proteases can affect fly infection and that plasma enzyme inhibitors may have these effects in sheep that are resistant to infection. Sheep respond most dramatically to proteases located in salivary gland and 'visceral homogenate' preparations 2°. However, these authors reported little host response to third instar cuticular antigens. The nature and source of protective antigens in other types of myiasis have not been characterized although it may be surmised that secretory/excretory antigens of salivary gland and midgut origin are of primary importance.

Humoral Immunity O'Donnell and colleagues2~'22 described the development of IgG antibodies in sheep struck by L. cuprina and observed that repeated exposure to these larvae engendered at least partial resistance to reinfection. Antibodies to whole third instar extracts were present in previously struck sheep and the weal size observed in skin tests was negatively correlated with subsequent wound size and larval survival. Bowles and colleagues23 also reported the occurrence of hypersensitivity reactions in sheep vaccinated with crude extracts of second instar larvae and suggested the presence of IgE-type antibodies. Similarly, the development and evaluation of diagnostic tests have shown that humoral responses are mounted against Dermatobia hominis 24, H. (= Oedemagena) tarandi 25 and Oestris OViS26, although little other information is available on the nature of the response. The development of specific IgG responses in experimental murine hosts to infestation with the bot Cuterebra fontinella occurs rapidly and shows anamnestic responses to challenge27. Antibody levels in rats are generally higher than those in mice and rats become resistant to infestation after two exposures to the bot while mice remain susceptible after several exposures. Information on

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y

Pupation in soil

Adult female

Mature larvae

emerge

SPRING 'Warbles' in back

Eggs on host hair

SUMMER Larvae hatch

Migration

WINTER Larvae in connective tissue

/

Migration

Fig. I. The annual life cycle of Hypoderma bovis. The female fly lays her eggsand glues them to host hairs during the summer. Larvae hatch after about four days and burrow through host skin, migrating through intermuscular connective tissue. During the winter H. bovisis generally found near the spinal canal while H. lineatumdevelops in the connective tissueof the oesophagus. In the spring the larvae travel to subcutaneous tissuesof the back and break through the host skin to breathe through a posterior spiracle. This is the 'warble stage' which lasts for about 45 days. Mature larvae wriggle out of the cyst and fall to the ground where they burrow and pupate. Adult flies emerge about 35 days later during the summer.

the immunobiology of the rodent-bot model may contribute to our understanding of infestations in livestock. Models offer obvious advantages in the number of bots available for study, number of animals under investigation and the duration of the life cycle. Humoral reponses to Hypoderma spp have been studied in more detail in cattle; IgG antibodies to first instar antigens of H. lineatum are first evident in sera 4-6 weeks after infestation of na~ve cattle 28'29. Antibody levels increase for 3-4 weeks following detection, then decline and remain at relatively low levels until the fly larvae begin migration to the back of their host at about 25-28 weeks post-infestation 28. Antibodies against first instar antigens reach maximum levels I-2 weeks prior to appearance of the maximum number of grubs on the back; levels then decline sharply as the grubs begin to emerge from their host 3°. Reaginic antibodies have also been reported in calves infested with H. lineatum 31 and the

presence of these antibodies may play a role in the eosinophilia noted in calves following challenge infestations with either species of cattle grub (D.D. Colwell, unpublished).

Cellular Immunity Cattle develop acquired resistance after repeated exposure to Hypoderma spp larvae 32 . This resistance is recognized as an important factor in controlling grub populations and depends upon the number of previous exposures 32 and the number of larvae invading the host 33. Both the maximum number in the back and the total accumulative number of grubs that leave the host are significantly reduced in animals exposed to their third consecutive infestation32. There is a strong suggestion that cellular components may be involved in the development of acquired resistance to grubs. Antigen-specific proliferation of lymphocytes, in vitro, varies with the

specific phases of the infestation and correlates positively with host resistance 32. High mortality early in the infestation coincides with increased antigen-specific lymphocyte responsiveness of animals that have acquired resistance through previous exposure. In naive animals this responsiveness is observed late in a primary infestation and correlates with grub mortality within the host, ie. in the back. Additional evidence supporting a protective role for cellular mechanisms is the coincidence between macrophage migration-inhibition factor (MIF) activity, delayed skin reactions to antigens from live, newly hatched larvae and the duration of protective effects 34. It has also been recently shown that treatment with an immunomodulator such as monophosphoryl lipid A (MPL), which stimulates cellular responsiveness, can enhance antigen-specific responsiveness to H. lineatum antigens in animals undergoing a primary infestation ~2. This enhanced responsiveness also correlates well with parasite mortality. The lack of apparent response to antigen in nai've animals until late in the infestation suggests that acquired resistance is either slow to develop or subject to immunosuppression 32, Indeed, it is possible that mechanisms regulating host inflammation, which are possibly parasite-induced 3s, could enhance parasite survival. It has been shown that both hypodermins A and B can inactivate complement components 36, in particular C3 (Ref. 37). This inactivation, which occurs directly through degradation of the C3 molecule 3s, may thus curtail the inflammatory response, allowing the parasite to escape an early host defence reaction. It also appears that hypodermins A and B can affect the antigen-specific response in a similar manner 38 . A similar downregulation of host immune response has been described in rabbits experimentally infested with D. hominis 24, The presence of a ruthenium redpositive surface on first instars of H. lineatum cultured, in vitro 39, suggests the presence of a negatively charged surface coat on these larvae. A surface coat of this nature would tend to reduce activation of host inflammatory responses to migrating larvae and subsequently play a part in slowing the onset of the immune response. Acquired resistance to L. cuprina in sheep develops after four or five consecutive infections at two-week intervals and is associated with larger wounds, earlier onset of wound exudation and larger Arthus-type (4 h) skin

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reactivity to larval excre.tory/secretory products 4°. Significant mortality of larvae is observed in challenged sheep while growth retardation is seen when larvae are cultured, in vitro, in the presence of serum from previously infested sheep 4~.

Prospects for Vaccination The discovery that antigens derived from first instar larvae of H. lineatum stimulate resistance42 has spawned further research using these antigens. Reduction of infestations by both H. lineaturn and H. bovis were reported after immunization of cattle with either crude extracts or antigens released from larvae cultured, in vitro 42. Both maximum and accumulative grub numbers were significantly reduced as was the mean number of pupating larvae. In other early attempts 28, immunization with crude extracts reduced grub survival by 26%, compared to untreated nmve animals. Immunization with purified hypodermin A has been shown to be highly effective in reducing grub survival29. It is also superior to immun, ization with a mixture of hypodermins C and B ~6. Both treatments stimulate resistance. However, total larval mortality in the backs of cattle and lower production of viable pupae suggest greater efficacy with hypodermin A. Most recently, the influence of an antigen-specific cellular and humoral response stimulated by immunizations on the survival of a challenge infestation of H. lineatum has been studied ~2 Calves immunized with a purified combination of hypodermins A, B and C plus MPL developed a strong antigenspecific cellular immune response. Western blot analysis at the time of maximum grub counts showed that immunized calves responded to hypodermins A, B and C while infested controls only responded to B and C. The humoral response in vaccinated calves was also significantly elevated over that in controls. The level of protection (95%) stimulated by immunization certainly confirms the feasibility of this approach in controlling cattle grubs. It would appear that these purified proteins are hi~;hly protective when used alone 16 or in various combinations 12. Shared epitopes between H. bovis proteins and hypodermins A and C of H. lineatum suggest that this is a distinct possibility t° and efforts in this area are continuing. Similarly, discoveries of the host humoral response to antigens of L.

cuprina have stimulated vaccination attempts 23, These efforts provide only partial protection against fly strike and it has been noted that intranasal presentation of antigen produces a significant reduction in larval numbers while subdermal presentation does not. Disappointingly, neither the intranasal nor subdermaL vaccination route provides protection against a second challenge. Promising studies+3 suggest that vaccines based on novel antigens isolated from the larval gut may provide protection although at present only growth retardation of larvae is observed. It has also been shown that vaccines against bacterial species associated with fleece rot, such as Pseudomonas aeruginosa +4, can successfully reduce fly strike and fleece rot. An effective and economic approach to the production of vaccines against pests causing myiasis would require a readily available source of purified antigen. To date, in vitro culture has not been successful and the use of recombinant DNA technology could be essential. Preliminary research suggests that this is a distinct possibility for Hypoderma spp and efforts in this area are continuing. References

I Tarry, D.W. (1986) Parasitology Today 2, t11-116 2 Kunz, S,E., Drummond, R,O, and Weintraub, J. (1984) Prev, Vet, Med. 2, 523-527 3 Klein, K.K. and Jetter, E,P, (1987) Can. J. Agnc. Econ. 35, 289-303 4 Beesley, W,N. (1985) in Parasites, Pests and Predators (Gaafar, S.M., Howard, W.E, and Marsh, R.E., eds), pp 299 315, Elsevier 5 Nelson, W.A. (1986) Immune Responses in Parasitic infections, CRC Press 6 Lecroisey, A., Boulard, C, and Keil, B, (1979) Eur. j. Biochem. 101, 385-393 7 Lecroisey, A., Tong, NT. and Keil, B. (1983) Eur. J. Biochern, 134, 261-267 8 Tong, NT. et at. (1981 ) Biochirn. Biophys, Acta 658, 209-219 9 Schwinghammer, K.A., Pruett, J.H. and Temeyer, K,B. (1988)J, Econ. Entornol. 81, 549-554 10 Pruett, J,H,, Scholl, P.J. and Temeyer, K,B. (I 990)J. Parasitol. 76, 881-888 II Pruett, J,H,, Temeyer, K.B. and Burkett, B.K. (I 988) Vet, Parasitol, 29, 53-63 2 Baron, R,W. and Colwell, D,D, (1991) Vet. Parasitol. 38, 185-197 3 Boulard, C, and Weintraub, J. (1973) Int. J. Parasitot. 3, 379-386 4 Robertson, R.H. (1980) Can. J. Zool. 58, 245-25 I 5 Sinclair, I.J. and Wassal, D,A. (1983) Res. Vet. Sci. 34, 251-252 6 Pruett, J,H., Fisher, W.F. and Temeyer, K.B, (I 989) Southwest. Entomot, 14, 363-373 7 Bowles, V.M,, Carnegie, P.R, and Sandeman, R,M (1988) Aust. J. Biol. Res. 4 I, 269-278 8 Sandeman, R,M, et al. (1990)Int. J. Parasitol, 20, 1019-1023 9 Bowles, V.M., Feehan, J.F, and Sandeman, R.M. (1990) Int. J, Parasitol. 20, 169-174 20 Skelly, P.J. and Howells, A.J. (1987) Int. J. Parasitol. 17, 1081- 1087

21 O'Donnell, I.J, et al. (1980) Aust.j. Biol. Sci. 33, 27-34 22 O'Donnell, I.J, et al. (1981 ) Aust. J. Biol. Sci. 34, 411-417 23 Bowles, V,M., Carnegie, P,R. and Sandeman, R.M. (1987) Int. J. Parasitol. 17, 753-758 24 De Lello, E. and Boulard, C. (I 990) Med. Vet Entomol. 4, 303-309 25 Monfray, K. and Boulard, C, (I 990) Med. Vet. Entornol. 4, 297-302 26 Bautista-Ganfias, C.R., Angulo-Contreras, R.M. and Garay-Garzon, E. (1988) Med. Vet. Entornol. 2, 351-355 27 Pruett, J,H. and Barrett, C.C, (1983)j. Med, Entomol. 20, I 13- I 19 28 Pruett, J.H. and Barrett, C.C, (1985) Southwest. Entornol. I 0, 39-48 29 Pruett, J,H,, Barrett, C,C. and Fisher, W.F. (I 987) Southwest. Entornol. 12, 79-88 30 Colwell, D,D. and Baron, R.W. (1990) Med. Vet, Entomol. 4, 35-42 31 Eyre, P., Boulard, C. and Deline, TR. (1980) Vet. Parasitol. 7, 243-254 32 Baron, R.W. and Weintraub, J, (1987) Vet. Parasitol. 24, 285-296 33 Weintraub, J, (1980) in Research Highlights (Croome, G.C.R. and Wilson, D,B,, eds), pp 6345, Agriculture Canada 34 Gingrich, R.E, (1982) Vet. Parasitof. 9, 233-242 35 Baron, R,W, (1990)J. Med. Entornol. 27, 899-904 36 Boulard, C. and Bencharif, F, (1989) Parasite Irnrnunol. 6, 459-467 37 Boulard, C, (1989) Vet. Irnrnunol. Irnrnunopathol, 20, 387-398 38 Chaboudie, N., Villejoubert, C. and Boulard, C. (I 990) Bull. Soc. Ft. Parasitol, 8, 56 I 39 Colwell, D.D. (1991) J. Med. Entornot. 28, 86-94 40 Sandeman, R.M. et al. (I 986) Int, J. Parasitol. 16, 69-75 41 Eisemann, C,H, et at, (1990)Int, J. Parasitol. 20, 299-305 42 Baron, R.W. and Weintraub, J. (1986) Vet. Parasitol. 2 I, 43-50 43 Sandeman, R,M. (1990) Int, J, Parasitol. 20, 537-54 I 44 Burrell, D.H. and MacDiarmid, J.A, (1983) in Second National Symposium on Sheep Blowfly and Flystrike in Sheep (Raadsma, H.W., ed.), pp 124-129, New South Wales Department of Agriculture

Bob Baron and Doug Colwell are at the Livestock Sciences Section, Lethbridge Research Station, Agriculture Canada, PO Box 3000 Main, Lethbridge, Alberta, Canada TIJ 4BI.

Look O u t for Outlook From the January 1992 issue, this new section will carry summaries of papers appearing in the primary literature that are of parasitological interest.