Acquired resistance of sheep to larvae of Lucilia cuprina, assessed in vivo and in vitro

lnwrnorional Journalfor Prrnred in Grear Britain

Parastrology

Vol. 20, No. 3. pp. 299-305.

1990 0

OOZC-7519/90 $3.00 + 0.00 Pergamm Press plc Society forPorosirolog~v

I990 Auslralian

ACQUIRED RESISTANCE OF SHEEP TO LARVAE OF LUCILIA CUPRINA, ASSESSED IN VW0 AND IN VITRO H.

and J. D. KERR? *CSIRO Division of Tropical Animal Production, and tCSIR0 Division of Mathematics and Statistics. Long Pocket Laboratories, Private Bag No. 3, P.O., Indooroopiiiy, Queensland 4068, Australia IQueensland Department of Primary Industries, Animal Research Institute, 665 Fairfield Road, Yeerongpiiiy, Queensland 4105, Australia (Recejved 7 June 1989; accepted 15 November 1989)

AbStTaCt-EISEMANNC. H., JOHNSTON L. A. Y., BROADMEADOW M., O’SULLIVAN B. M., DONALDSON R. A,, PEARSON R. D., VUOCOLO T. and KERRJ. D. 1990. Acquired resistance of sheep to larvae of Lucifia cuprina, assessed in viva and in vitro. Z~~ernutionu~3our~ul~r Par~~ifolog~ 20: 299-305. The effect on subsequent larval survival of infesting sheep repeatedly with larvae of Luciliu cuprina was assayed in viva and in vitro. One in viva assay technique, in which implanted larvae were grown to third instar, indicated a significant reduction in larval survival; another in viva technique, in which larvae were allowed to develop to second instar in small aluminium ripgs attached to the sheep, indicated no reduction in larval growth or survival. Larvae of Lucifia cuprina grown in vitro on media containing sera from previously infested sheep were significantly retarded in growth after 20 h compared with controls; no difference was detected when larvae were allowed to develop to pupation on two changes of the same media. No significant differences in survival of larvae either to 20 h or to pupation were obtained between the two treatments. ELISA antibody levels

against crude soluble larval material were significantly higher for sera from infested sheep than for control sera, and the regression of antibody level on mean larval weight obtained after 20 h growth in virro was significant. The immunogiobuiin fraction isolated from sera of infested sheep significantly growth when incorporated with normal serum in growth media. These results are consistent specific anti-larval antibody produced by sheep in response to infestation. INDEX repeated

KEY WORDS: infestation.

Lucifia cuprina; sheep blowfly;

INTRODUCTION

IN a recent experiment, Sandeman,

Bowles, Stacey & Carnegie (1986) infested separate groups of sheep eight times with either 50 or 500 larvae of Luciliu cuprina at intervals of 2 weeks. No significant differences were found between numbers of larvae recovered from either group after eight infestations and from control animals. However, in the group which received 500 larvae, there was evidence of heterogeneity in the response; the five most ‘resistant’ animals at the eighth infestation had shown progressively decreasing larval returns after the fourth infestation, while the most ‘susceptible’ five, which showed relatively low returns at the first infestation, progressively increased their larval returns, at least up to the fifth infestation. An earlier experiment (Sandeman, Dowse & Carnegie, 1985) had also indicated the development of resistance in some repeatedly infested sheep. In both of these experiments, increased titres of antibodies against soluble larval materials were found following infestation. Several other reports exist of the production by sheep of antibodies against antigens of

myiasis;

response;

acquired

resistance;

L. cuprina larvae following infestation (Elliott, Pattie & Dobson, 1980. Proceedings of the Australian Society of Animal Production Vol. 13, p. 500; O’Donnell, Green, Connell & Hopkins, 1980; Skelly & Howells, 1987). The latter authors found that infested sheep produced antibodies against a range of components of al1 three larval instars, the strongest response with third instar larvae being against an extract of salivary glands, with excretory/secretory and midgut material also reacting strongly. Recently, Eisemann, Johnston & Kerr (1989) infested sheep for 20 h (larvae developed to second instar) with a total of 1500 larvae of L. cuprina without affecting larval survival or growth rate, as measured by a new in vivo technique. The present work was undertaken in an attempt to resolve the remaining uncertainty regarding acquisition of resistance by sheep to larvae of the Australian sheep blowfly. Two different in vivo techniques were used to assess the development of resistance to larval growth and survival following repeated infestation, while the effect of serum from repeatedly infested animals on the development of larvae of L. cuprina was also tested in vitro.

299

immune

retarded larval with an effect of

C. H. EISEMANN PI ul.

300 MATERIALS AND METHODS

~nfestution ofsheep and collection ofsera. Five groups of 10 Merino ewes were infested seven times at intervals of 1 week with 500 neonate first instar L. cuprina larvae per animal; larvae were allowed to grow on the sheep for at least 3 days. All larvae came from laboratory cultures which originated from Aystruck sheep and were maintained on liver or an artificial medium (Singh & Jerram, 1976) for up to 30 generations. The first infestations of the five groups were made I week apart, so that the sequence of infestations was staggered among groups. Implants t-4 and 6 on each sheep were performed as follows: a small area of skin and fleece on the mid-lower back region was wetted with 500 ml tap water; the skin of this area was then damaged slightly by applying a tuberculin testing instrument to it six times. This instrument lightly punctures the skin with 16 needles in a circular pattern 10 mm in diameter. The larvae, on a wetted swab, were placed directly on the prepared skin, the swab being held in place by the surrounding fleece, which was clamped at the tips by rubber bands. All implants were wetted with 200 ml water each morning for 2 days following implantation. The larvae which developed from implants l-4 and 6 were not collected. Samples of serum for bioassay were drawn from each animal both before its first infestation (pre-infestation) and 2 weeks after its seventh infestation (post-infestation). Serum samples (control) were also drawn from a different group of five previously uninfested sheep at the same time as the postinfestation sera were taken from each group of infested animals. In vivo assays of larvalsurvival. Implants 5 and 7 were used to assess the survival of larvae after 3 days’ growth on the sheep (Queensland Department of Primary Industries, QDPI method). They were made on the shoulder within a 140 cm* area of skin bearing long fleece, surrounded by a ring of closely-clipped skin. Twenty-four hours before the implant was made, one end of a tubular gauze sleeve of jersey fabric (22 cm in diameter, 30 cm long) was glued to the skin all around the clipped area. Once 500 first instar larvae had been implanted, the free end of the sleeve was knotted, preventing the escape of post-feeding larvae until they were retrieved for counting after 72 h on the sheep. Concurrently with implants 5 and 7 of each group, similar implants were made on five previously unused control animals. Two weeks after the seventh implant on each group of sheep had been completed, they, along with five new controls, were tested for resistance by the CSIRO method, which is described in detail in Eisemann et al. (1989). Fifty newly-hatched larvae from a laboratory culture (Eisemann et al., 1989) of L. cuprina were placed in each of six metal rings glued to the back of each sheep, and held there under covers to maintain humidity. After 20 h, by which time second instar had been reached, the larvae from each ring were counted and weighed. In vitro assays oflarvalgrowth andsurvival. Growth media for bioassays of sera were prepared by adding 1 part 4% Difco noble agar (m.p. approx. 38°C) dissolved in distilled water to 3 parts serum to which brewer’s yeast and potassium dihydrogen orthophosphate had been added (cf. Green & Connole, I98 1) to give respectively 2.0% and 0.5% w/v in the mixture. Streptomycin (C.S.L., Melbourne, Australia) (100 pg ml. ‘) and Mycostatin (E. R. Squibb & Sons, Noble Park, Victoria, Australia) (125 i.u. ml-‘) were also added. The 4% agar solution was cooled to about 45°C before addition to the serum, which had been warmed to 38-4OC. After mixing, the complete medium was dispensed as 1 ml or 4 ml aliquots in 20 ml plastic Coulter counter vials (Johns Professional Products, Clayton, Victoria, Australia) before solidification.

The vials containing growth medium were stored at 4°C for up to 24 h before the addition of larvae. Ten larvae in distilled water were pipetted onto a 4 mm diameter Millipore filter pad (Millipore Corp., Bedford, MA, U.S.A.); this was then inverted and placed in a vial on the surface of the medium, which had been deeply scored to facilitate penetration by the larvae. Vials were closed with fine nylon gauze. In Experiment 1, post-infestation and controi sera from the sheep of each group were prepared individually as larval growth media (control sera were from the controls used for the CSIRO in viva assay). Ten replicates, each with I ml of medium, were prepared for each serum and incubated with larvae for 20 h at 30°C and 95% relative humidity, after which time the larvae were removed to be counted and weighed. Sera from the five groups of sheep were assayed separately, using different batches of larvae. In Experiment 2, pre-infestation and post-infestation sera were pooled separately within each sheep group and formulated as larva1 growth media to give five 1 ml replicates each. These were incubated with larvae as in Experiment 1. The experiment was performed twice (parts a & b). For Experiment 3, postchallenge sera from three repeatedly infested sheep and serum from one uninfested control were formulated separately into growth media which were dispensed in both 1 ml and 4 ml aliquots. For each serum, larvae were allowed to grow in two series of ten 1 ml replicates at 3o’C and 95% relative humidity; after 20 h, the larvae in one series were counted and weighed, while after 30 h those in the other were transferred to new vials each containing 4 ml of the corresponding fresh medium and incubated as before for a further 18 h. The vials were then opened and placed individually in screened containers with a layer of sand to encourage pupation by larvae emerging from the growth medium. After a further 3 days at 30°C the pupae (along with a small number of unpupated larvae) were sieved from the sand, counted and weighed. For Experiment 4, sera were pooled into three samples of post-infestation and three of control sera, each pooled sample containing 5 ml from each of three or 3.75 ml from each of four animals, to total 15 ml. Samples 1 and 2 contained only serum from sheep groups 2 and 3, respectively, and sample 3 from groups 4 and 5. Pooling was necessary because of the small quantities of serum remaining. Five millilitres of serum from each pooled sample was formulated into diet and dispensed as described above, except that 2% Difco Yeastolatk yeast extract was substituted for brewer’s yeast. Immunoglobulin was isolated from 10 ml of serum remaining from each pooled group by precipitation in 50% saturated aqueous ammonium sulphate following precipitation ofother serum components in rivanol (2-ethoxy-6,9_diaminoacridine lactate) according to method E of Mostratos & Beswick (1969). The precipitated immunoglobulin fraction was concentrated to 1.0 ml by vacuum dialysis against 0.012 M-phosphate-buffered saline (pH 7.3). Each preparation was then supplemented with 4 ml of the same normal sheep serum to provide adequate nutrition for larval development, giving a total volume of 5 ml, which was formulated into diets and dispensed as for the corresponding sera. Larvae were allowed to grow on the diets for 20 h at 32’C. Assay of sheep serum antibody. An enzyme-linked immunosorbent assay @LISA) was performed on the postinfestation and control sera used in Experiment I to assay binding of antibody to an extract of second instar larvae of L. cup&a. This antigen was prepared by homogenizing early second instar larvae, reared on standard diet (Singh & Jerram, 1976), in extraction buffer (0.012 M-PBS at pH 7.3 with 5 mw-di-sodium EDTA and 2.5 mM-benzamidine

Acquired

resistance

of sheep to L. cuprina

Statistical analysis. Data were subjected to analysis of variance. For the QDPI assays, data from implants 5 and 7 were analysed separately, using data from the control animals tested at the same time. ELBA optical density data were analysed after square root transformation. Regression coefficients of O.D. on mean weight of larvae after 20 h growth on the same serum were calculated.

hydrochloride), sonicating the homogenate in 40 ml aliquots (180 W Rapidis Sonicator, 30 s at power setting 10 with 9 mm probe) and centrifuging it at 50,000 g (max.) for I h at 4°C.

The supernatant was filtered through a 0.5 pm Millipore filter and diluted to a protein concentration of 50 pg ml-’ (BioRad protein assay) in extraction buffer, before being stored at - IOO’C. The stock antigen solution was diluted after thawing to 2 pg ml-’ in coupling buffer (pH 9.6 : 1.59 g sodium carbonate plus 2.93 g sodium hydrogen carbonate made up to 1 litre with distilled water) and added to the wells of poly vinyl chloride microtitre plates, which were incubated for 18 hat 4°C. After being washed twice with PBS containing 0.05% Tween 20, the plates were blocked with 1% gelatin in coupling buffer for 2 h at room temperature. After six washes in PBS Tween 20, 1 in 4000 dilutions of the sera in PBS with 1% gelatin were added to the wells and incubated for 2 h at room temperature. The plates were again washed six times in PBS Tween 20, and a 1 in 2000 dilution of a horseradish peroxidase-labelled rabbit antibody against sheep IgG (Kirkegaard and Perry, Gaithersburg, MD, U.S.A) in PBS containing 1% normal rabbit serum was applied for 2 h at room temperature. After six more washes with PBS Tween 20, the plates were developed by adding 1 mg ml ’ 5aminosalicylic acid and I pl ml-’ hydrogen peroxide (30% w/ v) in substrate buffer (0.01 M-phosphate buffer, pH 6.8, with 0.1 mM-EDTA) and agitating for 30 min. Optical densities (O.D.) of the solutions in wells were measured at 492 nm in a Titertek Multiskan microtitre plate reader.

TABLE

I-MEAN

NUMBEROFTHIRDINSTAR

301

RESULTS In vivo assu~js Analysis of data from the QDPI assays (Table 1) showed a highly significant (P
Lucilia cuprina (+ s D.)RECOVEREDFROM

LARVAEOF

PREVlOUSLYlNFESTEDANDCONTROLSHEEPFOLLOWlNGQUANTlTATlVEIMPLANTS*

Group

Fifth implant

1 2 3 4 5 Treatment

means1

196 f 191 f 229 f nd. 334 f 238 f

28 53 40 78 67

Controlt 333 209 259 208 315 264

f f f f f f

Seventh implant

40 62 58 124 69 58

146 f n.d§ 287 f 268 f 270 f 243 f

Controlt

71

259 208 315 403 310 299

77 97 73 65

* Implants were of an initial 500 first instar larvae. t Controls were naive sheep assessed at the same time as the corresponding infested animals. $ Based on group means. 5 Gauze sleeves were torn open by sheep, allowing larvae to escape.

TABLE

~-MEAN

NUMBER

AND

WEIGHT

(+ SD.) OF SECOND

INSTAR

LARVAE

OF

repeatedly

Ludia

RECOVEREDFROMEACHMETALRlNGONEXPERlMENTALSHEEPAFTERCHALLENGEFOR

Sheep group no. I 2 3 4 5 Treatment

meanst

Mean no. of larvae recovered* Control

43.9 42.0 44.4 42.4 40.6 42.7

f f f f f f

Previously infested 4.9 6.7 3.6 6.1 6.8 1.5

* From an initial 50 larvae per ring. t Based on group means.

42.2 44.3 42.6 43.5 44.1 43.3

f f f f f f

4.7 4.6 6.5 5.2 5.5 0.9

f 58 f 124 f 69 ZIZ 42 f 93 * 73

cuprina 20h

Mean wt of larvae (mg) Control

1.63 1.03 1.58 1.03 1.19 1.29

f f f f f f

0.45 0.28 0.41 0.35 0.27 0.29

Previously infested 1.31 1.08 1.61 1.30 1.46 1.35

f f f f f f

0.45 0.31 0.57 0.34 0.40 0.20

C. H. EISEMANNet al.

302

In Experiment 3, post-infestation sera from three sheep, selected for having returned larvae substantially lighter in weight than their corresponding controls in Experiment 1, all returned significantly (P< 0.001) lighter larvae than did a control serum after 20 h growth (Table 5). This difference was no longer present after larvae had completed their growth on two changes of serum media and pupated. Serum from one animal (316) returned slightly lighter pupae than that from the others. Results of Experiment 4 (Table 6) showed that significant (PcO.001) retardation of larval growth occurred with both the pooled sera from repeatedly infested animals and the immunoglobuhn isolated from the same sera. The degree of growth retardation in comparison with the corresponding controls was similar for serum and isolated immunoglobulin preparations.

1, larvae grown on post-infestation sera for 20 h weighed si~i~cantly (P-C 0.001) less than those grown on control sera (Table 3). In Experiment 2, larvae grown on post-infestation sera pooled within groups were lighter in weight than those grown on the corresponding pooled pre-infestation sera. However, this difference was not statistically significant (0.1 > P> 0.05) when the results of both parts of the experiment were analysed together, owing to an unexplained increase in mean weight on postinfestation serum of group 5 in part a (Table 4). Insufficient pre-infestation serum was available for further testing.

TABLE 3-EXPERIMENT 1, MEAN SURVIVAL AND WEIGHT (* s.D.) OF LARVAL Lucifia cup&a AFTER 20 h GROWTH ON MEDIUM INCORWRATiNGSERUMFROMGROUPSOFSHEEPINFESTEDREPEATEDLYWITHLARVAEOF~.~~~~i~~ANDUNINFESTEDCONTROLSIiEEP.MEAN ELBA OPTICALDENSITlESOBTAINEDUSINGTHESESERAAREAtSOSHOWN

Mean no. of larvae recovered per vial*

Sheep group

Infested I 2 3 4 5 Treatment means? Standard error of difference

Control

9.6 f 0.3

9.7 f 0.1

9.5 9.6 9.5 9.5 9.5

9.5 9.3 9.4 9.5 9.5

f f * f f

0.3 0.3 0.3 0.3 0.1

+ 0.1 rfr 0.3 + 0.2 f 0.1 f 0.1

Mean wt of

Mean ELISA

larvae (mg)

-Infested 1.62 I.51 I.15 1.53 2.04 1.58

f i i f f f

O.D.

Control

0.34 0.28 0.12 0.24 0.38 0.32

2.22 2.09 1.51 1.81 2.65 2.06

f + f It f f

Infested

0.04 0.17 0.15 0.28 0.20 0.43

0.49 0.38 0.56 0.48 0.52 0.49

Control

zk 0.28 f 0.13 i 0.20 + 0.17 f 0.20 f 0.07

0.06

0.17 0.21 0.15 0.44 0.30 0.26 0.05

* From an initial IO larvae t From group means. TABLE ~-EXPERIMENT 2, ~EANSURVIVALANDW~IGHT~* SDJOFLARVAL tucilia cuprina AFTER 20 h GROWTHONMEDlUMCONTAiNINGPRE-ANDPOST-INFESTATIONSERAPOOLEDWITHINSHEEPGROuPS

Sheep group

Mean no. of larvae recovered Pre-infestation

per vial*

Post-infestation

Mean wt of larvae (mg) ~be-infestation

Post-infestation

Part a$ 1 2 3 4 5 Treatment means? Part bf

1 2 3 4 5 Treatment meanst

9.6 9.6 10.0 9.6 9.6 9.7

f f f f f f

0.5 0.6 0.0 0.6 0.5 0.2

9.4 9.8 10.0 10.0 10.0 9.8

f f f + * f

0.9 0.5 0.7 0.0 0.0 0.3

2.44 2.70 2.78 2.61 1.61 2.43

f f + f f f

0.24 0.12 0.34 0.27 0.11 0.47

2.20 2.01 2.09 2.28 1.98 2.11

f f i rt rt f

9.6 9.8 9.4 9.2 9.4 9.5

iz f f f f f

0.6 0.5 0.6 1.1 0.9 0.2

10.0 9.6 9.3 9.6 9.6 9.6

f f f f f f

0.0 0.6 1.5 0.6 0.6 0.3

1.45 1.34 1.46 1.55 1.18 1.39

i f + + f f

0.25 0.07 0.2 0.19 0.15 0.14

1.17 1.05 1.15 1.13 1.10 1.12

f 0.20 i 0.17 f 0.09 zk 0.14 f 0.13 zk 0.05

* From an initial 10 larvae per vial. t From group means. t Parts a and b are replicates of the same experiment,

performed

on different

days.

0.31 0.27 0.27 0.23 0.19 0.17

f * sr f k $I

0.02 0.06 0.02 0.22 0.13 0.12

Acquired resistance of sheep to L. cuprina

303

TABLE~--EXPERIMENT~,MEANSUR~I~ALANDWEIGHT~*S.D.~OFLARVAEAND~UPAEOF Luciliacuprina AFTERGROWTHFOR 20 h ANDTOPUPATIONONMEDlUMCONTAININGSERUMFROMREPEATEDLYINFESTEDANDCONTROLSHEEP

Mean no. of larvae per pupae recovered per vial*

Sheep no.

Mean wt of larvae or pupae (mg) Larvae

Larvae At pupation

At20h 9.1 8.8 9.1 9.3

290 (control) 316 (infested) 364 (infested) 369 (infested)

f 1.3 f 0.8 f 1.2 f0.8

7.2 6.8 6.3 6.8

f f f f

2.0 1.8 1.3 1.3

At pupation

At20h 1.46 f 1.10 f 1.05 f 1.05 f

26.18 f 23.26 f 26.40 f 26.36 f

0.11 0.10 0.15 0.17

1.76 1.76 2.65 1.95

* From an initial 10 larvae per vial. TABLET-EXPERIMENT~,MEANSURVIVALANDWEIGHT(+ S.D.)OFLARVALLuciliacuprina AFTER 20 h GROWTHONMEDIUM CONTAININGCONTROLANDPOST-INFESTATIONSERA,ANDTHEIRISOLATEDIMMUNOGLOBULIN

Pooled serum sample

Mean no. of larvae recovered per vial* Control

Control

9.8 f 0.5 9.8 f 0.5

9.8 f 0.8 10.2 f 0.5

10.0 f 0.0 9.9 f 0.1

10.0 f 0.0 10.0 f 0.2

1.85 f 0.36 2.23 f 0.18 2.23 f 0.28

2.91 f 0.28 2.83 f 0.18 2.74 f 0.19

2.10 f 0.22

2.83 f 0.09

10.2 f 0.5 9.4 f 0.6 9.6 f 0.9

1.88 f 0.21 2.04 f 0.18 1.86 f 0.15

2.91 f 0.23 2.84 f 0.21 2.82 f 0.16

9.7 f 0.4

1.93 f 0.10

2.86 f 0.05

Infested Whole serum

1 2 3

Means%

Mean wt of larvae (mg) Infested

Isolated immunoglobulin

1

10.0 f 0.0

2 3

9.8 f 0.5 9.8 f 0.5

Meanst Normal serurnt

9.9 f 0.1

2.82 f 0.29

10.0 f 0.0

* From an initial 10 larvae per vial.

t As used to supplement all isolated immunoglobulin preparations. $ From means for each preparation

Optical density data from the ELISA (Table 3) showed significantly (P
DISCUSSION

Results of the QDPI in vivo assay for the seventh implant support the conclusion of Sandeman et al. (1986) that sheep can acquire resistance to the development of blowfly larvae. The assessment of resistance by the CSIRO method, however, did not support this conclusion. The reasons for this inconsistency are not clear. The CSIRO technique gave more repeatable results and might have been expected to show resistance effects more clearly than the QDPI technique. The disappearance of acquired resistance in the repeatedly infested sheep during the 2 weeks between their seventh implant and the challenge employing the CSIRO method would appear improbable, especially in view of the results of Sandeman et al. (1986) which showed a decline in larval returns from the five ‘resistant’ sheep from the seventh to the eighth implants. It is possible, therefore, that the CSIRO method does not permit the expression of resistance acquired after infestation with larvae, either because of its short duration or because it prevents or mitigates the functioning of resistance mechanisms which can operate in the more ‘natural’ QDPI method. One possibility involves the development of

304

C. H. EJSEMANNet

hypersensitivity in sheep following repeated infestation. Increases in immediate and Arthus-type hypersensitivity responses of sheep to larval antigens following eight infestations with L. cuprina larvaewere indicated by tests performed by Sandeman rf al. (1986). Hy~rsensitivity may result in increased irritation to the host during subsequent infestation and a consequent increase in host grooming behaviour, such as biting and rubbing the source of irritation. A similar mechanism (Bennett, 1969; Koudstaal, Kemp & Kerr, 1978) has been shown to account for a large part of the acquired resistance of some breeds of cattle to the cattle tick ~~op~ifus micropius. The rigid covers used in the CSIRO technique may protect the larvae more effectively from injury by the host than the cloth sleeves used in the QDPI method, and thus prevent the greater mortality on previously infested sheep, which is obtained with the QDPI method. Without further work, it is not possible to decide which in vivo technique produced the more valid measure of acquired resistance to blowfly larvae. The closer approximation of the QDPI method and the implant technique used by Sandeman et al. (1985, 1986) to natural flystrike suggests that these reflect more accurately than the CSIRO method the situation which occurs under natural conditions. However, the CSIRO method has proved useful in assaying attempts to vaccinate sheep against larvae of L. cuprina (unpublished data) and may have the advantage of permitting this assessment to be performed without the confounding effects of resistance arising from any previous infestations with L. cupr~a. The in vitro results obtained after 20 h of larval development show a clear growth-retarding effect of sera from sheep which had been infested repeatedly with larvae of L. cuprina. However. this effect did not persist in larvae grown in pupation on three of these sera; possible reasons for this include progressive degradation of antibodies in growth media by feeding larvae and micro-organisms, feeding of larvae for a slightly longer time on post-infestation serum than on control to compensate for a reduced growth rate on the former, and stage-specific effects of the immune serum on larvae. The negative relationship obtained between ELISA optical density values and mean larval weights suggests that specific antibodies caused retardation of larval growth. More direct evidence that specific antibodies were mainly responsible for the observed growth retardation is provided by the results of Experiment 4. In this experiment, isolated immunoglobulin was concentrated, nominally to twice its concentration in serum, for the growth assays. The true concentration factor could not be determined, as the percentage yield of immunoglobulin from the isolation was unknown. Previous experience with immunoglobulin from immune sera of vaccinated animals has indicated that a nominal concentration factor of about two is necessary to match the growth-

al.

retarding effect of the corresponding whole serum on blowfly larvae, possibly owing to the loss of much immunoglobulin during the isolation procedure. The high ELISA O.D. values obtained with some control sera, especially in group 4, remain unexplain~, No experimental sheep were known to have suffered flystrike prior to or during the experiment, and although they were exposed to field flies for much of this time, it is considered unlikely that all sheep which showed elevated levels of antibodies against blowfly antigens could have suffered flystrikes which remained undetected, given the close supervision to which all animals were subjected. Although an overall negative correlation between ELISA O.D. value and weight of larvae returned in vitro has been established, this relationship did not hold in all individual cases. Both post-infestation and control sera which produced relatively high O.D. values did not necessarily return larvae of low weight in growth assays. These anomalies may well be related to the variety of antigens against which the measured antibody titres have been raised (Skelly & Howells, 1987) and the irrelevance of many of them to hostprotective mechanisms. Results of the 20 h in vitro growth assays were inconsistent with those of the CSIRO in viva assay. in that no retardation of growth was obtained in the iatter. One possible explanation is provided by a recent finding (Eisemann & Pearson, unpublished) that L. cuprina larvae grown on whole serum by the in vitro method described here contained, in most cases, substantially more functional immunoglobulin than those grown in vivo by the CSIRO method. Larval antigenic material which may be expected to gain entry to the host animal during myiasis would include excretory/secretory products of the gut and salivary glands, exuviae and material released by the death and decomposition of larvae in the area affected by myiasis. Skelly & Howells (1987) found that sheep which had been infested with larvae of t. cuprina produced strong antibody reactivity against excretory/secretory products from all instars and against extracts of third instar salivary glands and midgut, but little reactivity against haemolymph or cuticle extract. The demonstration in vitro of a growthretarding effect of antibody from repeatedly infested sheep suggests that protective antigens for vaccination may be found in the midgut and salivary glands of blowfly larvae. Acknowledgements-This Bett Trust Fund.

work was supported

by the

L. W.

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Acquired resistance of sheep to t. cuprina GREEN

P. E. & CONNOLEM. D. 1981. Screening of fungal metabolites for insecticidal activity against the sheep blowfly, Lucifia cuprinu (Wied.) and the cattle tick, Boophilus microplus (Can.). General and Applied

Journal of Biological Science 33: 21-34. SA~D~~ANR. M., DOWSEC. A. &CARNEGIEP. R. 1985. Initial characterisation of the sheep immune response to infections of Lucilia cuprina. International Journal for Parasitology 15: 18 1-185.

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& BESWICK T. S. L. 1969. Comparison of some simple methods of preparing y-globulin and antiglobulin sera for use in the indirect immunofluorescence technique. Journal of Pathology 98: 17-24.

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305

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M., STACEY I. N. &CARNEGIEP. R. 1986. Acquired resistance in sheep to infection with larvae of the blowfly, Lucilia cuprina. International Journal

for Parasitology 16: 69-75. SINGH P.

& JERRAME. M. 1976. Rearing housefly larvae in polythene bags. New Zealand Journal of Zoology 3: 57-58. SKELLYP. J. & HOWELLS A. J. 1987. The humoral immune response of sheep to antigens from larvae of the sheep blowfly (Lucilia cuprina). International Journal for Parasitology 17: 1081-1087.