Inhibition of growth of Lucilia cuprina larvae using serum from sheep vaccinated with first-instar larval antigens

ELSEVIER

International Journal for Parasitology 28 (1998) 439-450

Inhibition of growth of Lucilia cuprina larvae using serum from sheep vaccinated with first-instar larval antigens R.L. Tellam*

and C.H. Eisemann

CSIRO Tropical Agriculture, CSIRO Prk’ate Mail Bag 3, Indooroopilly 4068, Qld. Australia Received 18 September 1997; received in revised form 3 November 1997; accepted 4 November 1997

Abstract Whole first-instar Lucilia cuprina larvae were homogenised and sequentially extracted with a series of buffers of progressively more severe solubilising power. The final extract, using a buffer containing 6 M-urea, was fractionated by preparative isoelectric focussing. At each step in this process, protein fractions were tested in sheep vaccination trials for their ability to induce immune responses affecting the growth of L. cuprina larvae which fed on the sera from vaccinated sheep. One isoelectric focussing fraction (pH 5.9-6.7) containing a number of larval proteins induced an immune response which inhibited the growth of larvae by a mean of 84 f 7% in an in zho feeding bioassay. The recovery of larvae after feeding on sera from sheep vaccinated with this fraction was significantly reduced by 35 f 13%. This antilarval effect was shown to be mediated by ingested ovine antibodies. Immunofluorescence and immunogold localisations showed that the immune response was directed at proteins from the larval peritrophic membrane, larval cuticle and, to lesser extent, basement membranes and microvilli of digestive epithelial cells. Electron microscopic examination of larvae feeding on sera from sheep vaccinated with this fraction showed that the normally semi-permeable peritrophic membrane was blocked on the luminal side by an electron-lucent layer of undefined composition. It is postulated that this layer prevents nutrients from moving from the gut to the underlying digestive epithelial cells, thereby starving the larvae. The sera from sheep vaccinated with another isoelectric focussing fraction (pH 3.4-5.5) reduced the mean larval weight by 56 + 13% without significant effects on larval survival. 0 1998 Australian Society for Parasitology. Published by Elsevier Science Ltd. Key words: Lucilia cuprina; Blowfly strike; Myiasis; Control; Vaccine: Antigen; Peritrophic membrane

1. Introduction Blowfly strike in sheep is caused by the larvae of the fly are considerable production industry as a result of this increasing concerns relating

a cutaneous myiasis Lucilia cuprina. There losses in the sheep condition, as well as to the use of insec-

*Corresponding author. Tel: 61 732142724; 732142882: e-mail: Ross.Tellam@;‘tag.csiro.au.

Fax:

61

ticidal and surgical strategies used to control this pest [l-3]. One possible alternative control strategy is the development of a vaccine against this ectoparasite. In general, infestations of livestock with an ectoparasite do not result in the development of strong naturally acquired resistance in the host [461. In some animals a weak resistance develops which is often associated with a hypersensitivity response to the parasite or proteins excreted or secreted by the parasite [7]. This immune response in livestock animals such as sheep and cattle is

SOO20-7519/98 $19.00+0.00 0 1998 Australian Society for Parasitology. Published by Elsevier Science Ltd. Printed in Great Britain PZZ; SOO20-7519(97)00197-5

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generally associated with undesirable side effects, which can lead to decreased productivity [4, 5, 81. Repeated infestations of sheep with blowfly larvae induce strong humoral immune responses [912] and cellular responses typical of an acute inflammatory response [13-l 51, but these provide little or no protection of sheep against subsequent larval infestations [9-l 1, 15-171. Therefore, any vaccine strategy which is designed to duplicate this form of natural immune responseis unlikely to be effective. One approach to the formulation of an effective anti-larval vaccine is to use “concealed antigens”, i.e. parasite antigens not normally seen by the host immune surveillance system during natural parasite infestations, but which when used to vaccinate the host induce a protective immune response[ 181.The selection for antigens of this nature generally relies on efficacy in vaccination trials as the assay for antigen identification, i.e. there are no preconceived ideas as to what defines a protective antigen either in a structural, functional or locational sense. This paper describes the results of vaccination experiments using protein fractions from first-instar larvae of L. cuprina. It is demonstrated that a specific protein fraction induces a humoral immune response which inhibits the growth of larvae that feed on sera from vaccinated sheep.

flystrike and were maintained in pens on a diet of lucerne pellets. Sheep were assigned randomly to various treatment groups. Each protein fraction tested was homogenised with an equal volume of Freund’s incomplete adjuvant. The first injection was i.m., given half into each rear leg. The second injection (28days later) was also i.m., but in the neck region. A control group was given only adjuvant and PBS on both occasions. All animals were bled from the jugular vein prior to each injection and 2 weeks after the second injection. Each vaccination group contained four sheep. The effect of vaccinating sheep with a larval fraction was assessed by feeding newly hatched first-instar larvae at 34°C on an agar-based medium containing 75% serum from vaccinated and control sheep[l 11.Five replicate vials, each containing 10 larvae growing on 1ml of diet medium, were used for each serum. The number of surviving larvae and their weights were measured after 20 h. In addition to the control group, results were also obtained using pre-vaccination serum from each sheep. Antibody responseswere assessedby ELISA as described by Eisemann et al. [ 111.The antigen used on the ELISA plate was 0.2 pg of the 6 M-urea extract from firstinstar larvae. The urea contained in this buffer was sufficiently diluted such that it did not interfere with binding of the antigen to the ELISA plate.

2. Materials and methods

2.3. Antibody reconstitution

2.1. Maintenance of a L. cuprina colon)

Total immunoglobulin (lg) was isolated and purified [20] from the sera of vaccinated and control sheepand then reconstituted into the samevolume of normal sheepserum. The weights of larvae grown on these modified sera were then measured using the in vitro larval growth bioassay.

Laboratory populations of L. cuprina, which had originated from flystruck sheep, were maintained on an artificial medium for up to 10 generations. Eggs were collected by placing small trays of minced liver covered with fine nylon gauze inside cages of adult L. cuprina for 45 h. The eggs were then incubated overnight at 16’C and 100% r.h. before being transferred to 34°C until hatching. Larvae of L. cuprina were reared on a milk-powder growth medium [19]. 2.2. Vaccination trials Experimental animals were 612-month-old merino ewes.These animals had not previously suffered

2.4. Immunofluorescencelocalisations Sera from a sheep vaccinated with isoelectric focussing pool C antigen (group C) and corresponding pre-vaccination sera were diluted (l/ 1000) in PBS and incubated (7°C) overnight with whole peritrophic membrane dissectedfreshly from thirdinstar larvae. After four washes in PBS (over 1h at room temperature), the sampleswere incubated with a 1: 100 dilution of fluorescein isothiocyanate-

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labelled rabbit anti-sheep Ig serum in PBS for 2 h at room temperature. This was followed by another four washes in PBS (over 1 h at room temperature) and then the mounted samples were examined in a fluorescence microscope and photographed. 2.5. Immunogold

localisations

Frozen sections (3-pm thick) from first-instar larvae were fixed in 4% paraformaldehyde in 0.1 Msodium cacodylate buffer and embedded in BioRad LR White resin. Thin sections cut through the LR White-embedded sections were placed on copper grids. Immunogold localisations were performed, essentially as described by Elvin et al. [21]. A representative serum from a sheep in group C, diluted 1:250, was used as the primary antiserum. This was followed, after extensive washing, by a donkey antisheep antibody conjugated to 5-nm diameter colloidal gold particles (Biocell Research Laboratories, Cardiff, U.K.), diluted 1:lOO and examined in a Philips 400 T TEM. 2.6. Measurement trophic membrane

of the permeability

of the peri-

Newly-hatched larvae of L. cuprina were allowed to grow for 20 h at 33°C on 75% serum medium containing the most effective anti-larval serum from vaccination group C. The larvae were then transferred to a similar medium but also containing colloidal gold particles of approximately 6nm and 15 nm diameters [22], and allowed to feed for a further 2 h at the same temperature. They were then dissected open, fixed as before and embedded in epon-araldite. Larvae were also grown initially for 5 h on control sheep serum and then processed in the same manner. The reduced initial growth period for the control larvae was required to enable direct comparisons between larvae in the same developmental stage (i.e. first instar). Thin sections through the midgut of the larvae were cut and processed for EM as described by Eisemann et al. [24]. 2.7. Protein fractionation First-instar L. cuprina larvae (600 g, derived from 8 000000 larvae) which were initially frozen at

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-70°C were thawed in 1 litre of 50mM-Hepes, 4 mM-EDTA, 200mM-sucrose, pH 7.8, 2 mMphenylmethanesulphonyl fluoride (PMSF) (buffer A). The buffer contained EDTA and PMSF to inhibit the strong proteolytic activity present in the guts of these larvae [23]. The larvae were homogenised (Ultraturrax, 3min, full speed, 4°C) and then the extract was centrifuged (15 OOOg, 30min, 4C). The resulting pellet was resuspended in 600 ml of 50 mM-Hepes, 0.15 M-NaCl, 1 mM-EDTA, pH 7.5, 2mM-PMSF, 3% Zwittergent 3-14 (buffer B) and stirred at 4°C for 1 h. The extract was then centrifuged as above and the subsequent pellet reextracted with 600 ml of buffer B and recentrifuged. The resulting detergent-washed pellet was resuspended in 900ml of 20mM-Tris-HCl, 50mMNaCl, 1 mM-EDTA, pH 7.5, 1 mM-PMSF and 6 M-urea (buffer C) for 4 h at 4°C and then 1 h at room temperature. The extract was centrifuged as described above and the supernatant recovered. The soluble 6 M-urea extract was concentrated and subjected to preparative isoelectric focussing (IEF) in a Pharmacia Biotech Multiphor II system using pH 3-10 pharmalytes at 4°C. After lOOOOVh-‘, each fraction from the IEF was harvested by elution of the IEF-Sephadex with lOm1 of 6M-urea. The pH, protein concentration and SDS-PAGE profile of each fraction were determined. The IEF fractions were pooled into five groups (A-E) and 75% of the protein in each pool was used to vaccinate each group of four sheep as described above. The abilities of these protein fractions to induce immune responses which inhibited growth of L. cuprina larvae which subsequently fed on the sera from these vaccinated sheep were then measured. At each step in the protein fractionation, a vaccination trial in sheep was performed to identify fractions capable of inducing anti-larval immune responses. Protein concentration determinations were made using the Pierce BCA kit with BSA as a standard. Samples were diluted sufficiently such that buffer constituents did not interfere with the protein determinations. Reduced protein samples were analysed by SDS-PAGE on 618% gradient gels. All gels included mol. wt standards (Pharmacia) and were stained with silver using the method described by Morrissey [24]. Each sample contained the equivalent of 0.4% of the total protein in each pool.

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Table 1 Characteristics

3. Results

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439450

isoelectric

focussing

3.1. Protein fractionation Whole first-instar L. cuprina larvae were homogenised in buffer A and the pellet obtained after centrifugation was washed with a buffer containing 3% Zwittergent 3-14 (buffer B) and then extracted with a buffer containing 6M-urea (buffer C). At each step in this fractionation process a vaccination trial was performed in sheep to ascertain whether any of the solubilised protein or the corresponding pellet at that stage could induce an anti-larval immune response in the sera from vaccinated sheep. This was measured using an in vitro feeding bioassay which measured the growth and survival of larvae feeding on an agar-based medium containing serum from each sheep. The results of these multiple vaccination trials demonstrated that there was larval growth inhibitory activity in the sera of sheep vaccinated with the residual larval pellet after successive extraction of the homogenised larvae with buffer A and then buffer B. Activity was subsequently solubilised with buffer C (results not shown). The soluble material obtained from this last extraction was subjected to preparative IEF over a pH range 3-10. Most (N 70%) of the protein focussed between pH 4 and 7.5, reflecting the acidic nature of the proteins in this extract. A total of 31 fractions was obtained which were grouped into

Pool

Fraction

PH

Total w

A B C D E

l-10 1 l-15 1624 25-28 29-3 1

11.2-8.1 7.9-7.1 6.9-5.9 5.8-5.5 5.4-3.4

940 2010 3930 713 514

antigen protein

pools /pool

five vaccination pools (A-E, Table 1). Analytical SDS-PAGE of these fractions is shown in Fig. 1. There was very little protein in fractions l-10 (pH 11.2-8. l), but complex mixtures of proteins in most other fractions. The latter fractions (11-31) typically contained a substantial quantity of proteins of M, < 20 000, as well as a number of poorly focussed proteins of high mol. wt (M,> 100000). it would be expected that a 6M-urea extract of the larvae would solubilise proteins as well as glycosaminoglycans and proteoglycans. The latter are often characterised on SDSSPAGE as very high mol. wt poorly focussed bands. 3.2. Vaccination

trials

The IEF pools (A-E) were tested in a sheep vaccination trial to determine whether the proteins in

kDa 94 67 43 30

-

S 1 3 5

7 9 11 13 15 17 17 19 21 2325

Fig. 1. SDS-PAGE profile of 6 M-urea-extracted larva1 proteins separated by preparative weight standards; lanes l-31. fractions 1-31. respectively. Only every second lane is labelled. 0.4% of the total protein contained within that fraction. All samples were reduced.

27 29 31 isoelectric focussing. The protein loading

Lane S, molecular for each sample was

R.L. Tellam and C.H. Eisemann 1 International Journalfor Parasitology 28 (1998) 439450

any of these pools could induce an immune response which affected the growth and survival of L. cuprina larvae which subsequently fed on sera from the vaccinated sheep. All of the sheep vaccinated with these Lucilia antigens produced strong antibody responses. giving ELISA 0.D.s ranging between approximately 0.4 and 1.I at a primary

1.2

serum dilution of 1:4000 (Fig. 2(a)). In contrast, sera from all control animals which received only adjuvant and PBS gave 0.D.s of less than 0.1 at the same dilution. There was some animal-to-animal variation within each vaccination group in the magnitude of the ELISA 0.D.s (e.g., pool D). Figure 2(b) shows the mean weight of L. cuprina

a

1.0 1 0.0 d d La 0.6 .z

w 0.4 t

0

lb3.64~0.24mg

- I 5

3.80+0.16mg

OSk0.24mg

a

b

2.81 +2.02mg

il

1.62t0.47mg

ac

c

90.0t12.6%

93.5*1.9x

T

a

100 90 a0 ‘0 60 50

3.01 i0.41mg

a

a

96.5i1.97.

g E g g

443

97.OKl.6X

a

62.0+12.4X

b

a

a

Tr

= 40 +j 30 ..3 20 10 0 Conlrol A Fig. 2. Vaccination of sheep with IEF pools A-E. Sheep were vaccinated with IEF pools A-E and the immune responses were measured by ELISA and an in vitro larval growth bioassay. (a) ELBA optical densities for each serum diluted l/4000; (b) the mean weight of larvae growing on each serum for 20 h; (c) the percentage of larvae recovered in each in uitro feeding bioassay. Vaccination groups (AE) each consisting of four sheep are indicated on the horizontal axis. Error bars represent 1 S.D. The numbers above each group in (b) and (c) are group meansf 1 SD. Group means having a common letter below them are not significantly different from the control group (P-cO.05). The results for the control group are shaded more heavily than those for the vaccination groups,

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larvae after feeding for 20 h on a diet containing serum from each vaccinated sheep. The mean larval weight obtained from group C (four sheep injected with pool C IEF proteins) was only 16 + 7% of that of the control group (significant at P
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2.5 s

E - 2.0 r’ .po g 1.5 F f

1.0

2 2 0.5 0

a

b

C

d

Fig. 3. Transfer of Ig from a serum showing anti-larval activity to normal sheep serum. Total Ig was isolated and purified from both the serum of a sheep vaccinated with the IEF pool C antigen and from the prevaccination serum from that sheep. A sufficient quantity of Ig was then transferred into normal sheep serum to give a concentration of additional Ig which was equivalent to that present in the original serum. The effects of the modified normal sheep serum on larval growth were measured by an in vitro larval growth bioassay. (a) Unmodified prevaccination serum (control); (b) unmodified post-vaccination serum from a sheep vaccinated with IEF pool C; (c) normal sheep serum containing additional total Ig isolated from the prevaccination serum (control); (d) normal sheep serum containing total Ig isolated from the sheep vaccinated with the IEF pool C antigen.

difficult to solubilise except by using strong denaturants such as 6M-urea. To further characterise the nature of the proteins in IEF pool C, immunolocalisations were performed with various tissuesfrom first-instar larvae. Figure 4 shows the immunofluorescence localisation of antigens from IEF pool C on the peritrophic membrane from L. cuprina larvae. There was virtually no fluorescence associated with the control serum (panel a), but serum from a sheep vaccinated with the IEF pool C proteins showed strong and uniform fluorescence (panel b) indicative of the presenceof theseantigens in the peritrophic membrane which lines the midgut of these larvae. Sections through various structures in first-instar larvae were examined by immunogold labelling and TEM using serum raised to IEF pool C (Fig. 5). Panel b of this figure indicates that there was strong gold localisation on the peritrophic membrane.This result is consistentwith the immunofluorescence localisation shown in Fig. 4, although

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Fig. 4. Immunofluorescence localisation (a), control using prevaccination serum;

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of antigens from IEF pool C on whole panel (b). serum from a sheep vaccinated

panel b of Fig. 5 shows a preferential labelling of the endo-peritrophic surface. Interestingly, much of the gold was localised on an electron-lucent layer well separated from the electron-dense peritrophic membrane. This layer may represent a peritrophic membrane “glycocalyx”. There was also labelling of the microvilli of the digestive epithelial cells (panel d) and larval procuticle (panel f). The labelling of the microvilli is apparently on the external surface of these structures possibly associated with a glycocalyx. This location is consistent with the harsh extraction procedure required to solubilise the IEF pool C proteins. Likewise, the presence of cuticle proteins in the same antigen pool also would be expected. In this case the gold was localised to the internal procuticle region and was not associated with the external surface of the larvae. In addition to these tissues, there was also weak gold labelling of the basal lamina of the trachea (panel h). In all of these tissue localisations there was relatively little or no gold labelling of any larval structure with prevaccination serum (panels a, c, e and Ed.

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peritrophic membrane from L. cuprina larvae. with IEF pool C. Scale bars = 25 pm.

3.4. Reduced permeability

of peritrophic

Panel

membrane

Sections through the midgut of control larvae, when examined by EM, revealed that 6-nm colloidal gold passed readily through the peritrophic membrane and reached the underlying microvilli of the midgut epithelium, whereas 15-nm colloidal gold did not (Fig. 6(a)). This result demonstrates that the peritrophic membrane is permeable to 6nm particles but not to the larger 15-nm particles. Other studies indicate that the exclusion limit of the L. cuprina peritrophic membrane is approximately IO-nm (unpublished results). The peritrophic membrane, in general is thought to partition macromolecules between the ecto- and endo-peritrophic membrane spaces, depending on the size of the macromolecules [25]. In larvae fed on serum from a sheep vaccinated with IEF pool C, however, neither size of colloidal gold passed through the peritrophic membrane, indicating a reduced permeability of this structure (Fig. 6(b)). In addition, a densely packed layer of undetermined composition was found attached to the luminai side of the peritrophic

b

C

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d

a,. EESPS ‘, ~I / .:. .-*:,.. ‘. I

Fig. 6. Antibody-mediated blockage of the permeability of the sheep serum (a) or serum from a sheep vaccinated with IEF pool particle sizes (6 nm and 15 nm). PM = peritrophic membrane; space; ENPS = endoperitrophic space; L = densely-packed layer indicate 15-nm and 6-nm gold particles, respectively.

membrane. This electron-lucent layer excluded most of the colloidal gold in the gut lumen. Apart from this layer and the smaller size of the larvae feeding on this serum, there were no indications of other abnormalities in the larvae.

4. Discussion The approach taken in the current study does not rely on the immune response associated with natural larval infestation to identify candidate vac-

peritrophic membrane to colloidal gold. Larvae were fed on normal C antigen (b). In each case the serum contained colloidal gold of two MV = microvilli of midgut epithelial cells; ECPS = ectoperitrophic on luminal side of peritrophic membrane. The large and small arrows

tine antigens from L. cuprina larvae. Rather, candidate antigen fractions were identified directly by their efficacy in vaccination trials. In particular, it was demonstrated that antigens isolated from structural tissues of the larvae, like basement membranes, peritrophic membrane and cuticle (and also the external surface of the microvilli of the digestive epithelial cells), induced an immune response in sheep which resulted in strong inhibition of growth of larvae and, in some instances, reduced larval recovery. It was also demonstrated that this larval growth inhibition was mediated by Ig antibodies.

t Fig. 5. Immunogold localisations. Serum from a sheep vaccinated with IEF pool C was used to localise various larval tissues. Panels (a) and (b). peritrophic membrane; panels (c) and (d), microvilli of digestive (e) and (f), cuticle of body wall; (g) and (h), basal lamina and cuticular wall of tracheae, respectively. controls which used prevaccination serum. Panels (b), (d), (0 and (h) used serum from a sheep PM = peritrophic membrane; MV = microvilli; EC = epicuticle; BL = basal lamina: TC = cuticle of resentative gold particles. Scale bars = 100 nm.

antigens from this fraction in epithelial cells lining the gut; Panels (a), (c), (e) and (g) are vaccinated with IEF pool C. trachea. Arrows indicate rep-

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The lack of correlation between the Ig titre and larval growth inhibition may be expected because of the complexity of proteins in each pool and the different proteins present in each pool. Presumably, the antigens inducing an anti-larval immune response in some groups, particularly group C, represent a subset of the total protein complement in that pool. The strong anti-larval effects observed in the serum from sheep vaccinated with pool C are not due to non-specific effects. A large number of protein extracts from L. cuprina larvae, Boophilus microplus adults, B. microplus larvae, Escherichia coli and cultured insect cells (Sf9 cells) have been tested in similar vaccination trials and have shown no anti-larval effects. In addition, a number of vaccination trials were performed with several purified proteins from a wide diversity of species, including L. cuprina, and these also showed no anti-larval immune responses (results not shown). One serum from group D also reduced larval recovery, but the significance of this result in the context of considerable animal-to-animal variation within this group is not clear. Reduced numbers of recovered larvae may reflect, in part, the increased difficulty of finding very small larvae amongst the remaining diet medium. However, sufficient numbers of dead larvae were found in individual vials from group C to demonstrate that reduced larval recoveries reflected increased larval mortality. These results suggest that it may be feasible to vaccinate sheep against the larvae of L. cuprina using individual antigens isolated from the IEF pool C proteins. The mechanism whereby ovine antibodies to these heterogeneous structural antigens in IEF pool C affect larval growth is not entirely clear. There is no obvious sign of visible damage to the small larvae feeding on sera from the vaccinated sheep. This information, in combination with the reduced growth rates of the larvae, suggest that there is a general anti-nutritional effect in larvae feeding on these sera. It is noteworthy that the semi-permeable peritrophic membrane, which is thought to be intimately involved in the digestive processes in the larva, is exposed to ingested ovine antibodies and appears to be completely clogged in larvae feeding on serum from sheep vaccinated with the IEF pool C proteins. The immunolocalisation studies indicate that at least some of the antigens in the IEF

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pool C were derived from the peritrophic membrane. A virtually identical effect is observed on the peritrophic membrane of larvae feeding on antibodies to peritrophic membrane proteins [26, 271 and larvae feeding on specific lectins which also bind to peritrophic membrane proteins [21,22]. The apparently impervious layer of material lining the gut lumen side of the peritrophic membrane in these larvae is too thick (1 pm in some places) to be a simple antibody layer. One possibility is that this layer is caused by a biophysical effect involving the gelation of semi-digested proteins on the surface of the normally semi-permeable peritrophic membrane after the pores in the membrane are clogged by antibodies binding to intrinsic peritrophic membrane proteins or to oligosaccharides attached to these proteins. The peritrophic membrane, in this instance, is then unable to act as an ultrafilter and prevents movement of nutrients from the gut lumen to the underlying digestive epithelial cells and movement of digestive proteolytic enzymes in the reverse direction. The consequence of this effect is that there is a general anti-nutritional effect (starvation), which directly results in inhibition of larval growth. Another explanation for the anti-larval effects is that ovine antibodies gain access to internal larval structures and cause damage to these structures. However, it is difficult to envisage how antibodies localised to the microvilli of digestive epithelial cells (which are protected by the peritrophic membrane), internal basement membrane or procuticle could exert anti-larval effects when there is normally very little access of exogenous or ingested ovine antibodies to these tissues ([28]; Eisemann and Tellam, unpublished observations). Further, the apparently impervious layer of material on the gut lumen side of the peritrophic membrane would even further prevent ingested antibodies from gaining access to these underlying tissues. It is also unclear as to how antibodies, even given free access to these robust internal larval structures, could disrupt the functions of these structural tissues and thereby decrease the larval growth rate and survival. Exogenous antibodies bathing the external surface of the larvae would not have access to cuticle proteins, because the latter are embedded in a chitinous matrix which is overlain by an impermeable outer wax-like pro-

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cuticle composed of a cyclic or cross-linked structure consisting largely of methylene groups [29]. In general, larvae which were less than 2630% of the weight of control larvae had difficulty surviving [30]. Larvae with moderate growth inhibition (- 50%) however, did survive and eventually underwent normal pupation (results not shown). Thus, the larvae are very resilient to major variations in their growth rates and once they have progressed into the second larval instar they have largely escaped from this form of immunological control. Presumably, this escape reflects the delicate balance between the quantity of ingested ovine antibody specific to the relevant larval antigens (particularly peritrophic membrane antigens), the rate of inactivation of these antibodies by digestive proteases, the rate of growth of larvae, and the rate of growth of the peritrophic membrane. This conclusion suggests that an effective immunological anti-larval control strategy will need to exert its major effects on first-instar larvae. Further progress towards the development of an anti-larval vaccine for use in sheep to protect them from blowfly strike will require the identification and purification of specific, effective larval antigens and the production of these antigens as recombinant proteins. The antibody-mediated anti-larval effect potentially caused by the blockage of the peritrophic membrane may have more general application to the control of other insect pests feeding on mammalian tissues, tissue fluids or blood. The ability to produce recombinant antibodies in transgenie plants [31] raises the interesting possibility that this potential immuno-control strategy may also have application to the control of insects feeding on plants. Acknowledgements-We thank R.A. Donaldson, R. Pearson and L.C. Cadogan for expert technical assistance. We also thank the Bett Trust and Australian wool growers through the International Wool Secretariat for financial support.

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Beck T, Moir B, Meppem T. The cost of parasites to the Australian sheep industry. Q Rev Econ 1985;7:336343. [2] Arundel JH, Sutherland AK. Animal health in Australia. In: Arundel JH. Sutherland AK, editors. Ectoparasite dis-

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