IE48464B1 - A method for the prevention or reduction of fly strike in sheep - Google Patents

Abstract

Sheep are protected against fly strike (e.g. by larvae of Lucilia spp. and/or Calliphora spp.) by injecting them with antigenic material obtained from the larvae of the fly responsible for the fly strike. The antigenic material may be conventionally made up into a vaccine.

Description

This invention relates to a vaccine effective in reducing or preventing the incidence of fly-strike in sheep, to methods for the preparation of the vaccine and to processes for reducing or preventing the incidence of fly-strike in sheep using the vaccine.

Primary fly strike in sheep in New Zealand and Australia is mainly caused by flies of the Lucilia spp. and to a lesser extent by flies of the Calliphora spp.

In New Zealand the major pest is Lucilia sericata and in Australia the major pest is Lucilia cuprina which is commonly known as the sheep blowfly. Gravid flies seek out areas of moist or soiled fleece in which to lay their eggs and the newly-hatched larvae move down the wool staple and penetrate the intact skin eventually causing serious lesions commonly referred to as a strike. Primary strike by flies of the Lucilia spp. may be supported by a number of related species of flies including Calliphora spp. and Chrysomia spp. and deep penetration may occur often resulting in the death of the animal if no preventative or remedial measures are taken.

There are six main forms of fly strike in sheep: crutch or breech strike; tail strike; body strike; head or poll strike; pizzle strike; and wound strike. Prior to the present invention the only effective method for the prevention of body strike has been applying to the sheep insecticides active against fly larVae. Treatment with insecticides is an attractive method for the prevention of fly strike in sheep as in addition to being the only method for the prevention of body strike it may also be used to prevent, breech, pizzle and head strike.

For the insecticide treatment to have practical utility in the prevention of fly strike the insecticide used must have a high level of activity against the larval stage of the fly, it must persist on the fleece maintaining the activity preferably for at least twelve - 3 weeks and it must have low mammalian toxicity. A number of insecticides have been found which when first introduced have proved very effective in preventing fly strike. However, reports indicate that the fly species responsible for fly strike in sheep are able to rapidly develop resistance to insecticides and in some countries this has resulted in the necessity to continue to introduce new insecticides. Therefore, it is evident that a new, non-insecticidal approach to the reduction or prevention of fly strike in sheep would be of great benefit to the sheep industry.

A completely novel approach to the control of fly strike in sheep has now been found in which sheep are vaccinated in order to immunize them against fly strike.

Accordingly the invention provides a method for the reduction or prevention of fly strike in sheep which process comprises treating said sheep with antigenic material effective in immunizing said sheep against fly strike.

The method of the present invention is completely novel and represents a radically new approach to the control of fly strike in sheep. It is completely unexpected to find that sheep can be protected from fly strike by vaccination and especially surprising in view of the fact that the flies/fly-larvae which cause fly strike are ectoparasites.

Although the following theories should not be construed as limiting, it is believed that the larvae of the flies responsible for primary fly strike in sheep either sensitize the skin causing exudation of plasma through the skin providing a source of food for the larvae or penetrate the skin by a combination of physical abrasion of the epidermis and the release of one or more proteolytic enzymes which act to dissolve or soften the skin, exposing softer more susceptible subsurface tissue. - 4 We have found that antigenic material effective in immunizing sheep against fly strike may conveniently be prepared from the larvae of the flies responsible for primary fly strike in sheep.

The antigenic material may be prepared from the fly larvae responsible for primary strike in sheep by a number of processes. For example, the antigenic material may be prepared: directly from the larvae; from the fluid produced by growing larvae on a range of natural and artificial nutrients; or from the culture of larval cells or organs such as the salivary glands.

Thus in a further embodiment the invention provides antigenic material effective in preventing or reducing the incidence of fly strike in sheep prepared directly from the larvae of flies responsible for primary fly strike in sheep, from the fluid produced by growing said larvae on a nutrient medium or from a culture of the cells or organs of said larvae.

The antigenic material is conveniently prepared by growing larvae of the fly or flies responsible for primary fly strike in sheep on a nutrient medium, separating the liquid produced and concentrating the liquid to give a solution of antigenic material.

Thus in yet a further embodiment the invention provides a process for the preparation of antigenic material effective in preventing or reducing the incidence of fly strike in sheep which process comprises growing on a nutrient medium larvae of the fly or flies responsible for primary fly strike in sheep, separating the liquid produced, and concentrating said liquid to give a solution of said antigenic material.

Preferred nutrient media include natural nutrient materials including animal tissue such as meat and liver and artificial mixtures of nutrient materials including mixtures of nutrients chosen from casein, agar, dried - 5 milk, yeast and glucose such as dried milk/yeast/agar, casein/agar, casein/agar/glucose and casein/agar/glucose/ yeast each mixture optionally containing added amino acids and/or vitamins.

In preferred processes for the preparation of antigenic material, freshly hatched fly larvae are grown on a suitable nutrient medium and the liquid produced is separated. The pH of the liquid is adjusted to a pH in the range from 6 to 8. On most nutrient media the larvae produce an alkaline liquid which is preferably adjusted to a pH in the range from 6 to 7 by the addition of a pharmaceutically acceptable mineral or organic acid.

Insoluble material is then separated from the pH adjusted liguid, preferably by centrifugation and/or filtration through a series of Millipore· (Trade Mark) filters, to give a solution. The solution is concentrated, preferably by ultrafiltration, to give a solution containing material of molecular weight of above about 20,000 and finally sterile filtered.

In the treatment of sheep to prevent or reduce fly strike the antigenic material is preferably formulated into a vaccine. Thus in still a further embodiment the invention provides a vaccine effective in preventing or reducing fly strike in sheep which comprises antigenic material prepared from the larvae of flies responsible for primary fly strike in sheep and a pharmaceutically acceptable vehicle therefor.

The vaccine may be prepared by direct formulation of the antigenid material or it may be prepared by the formulation of the antigenic material after it has been treated (attenuated or toxoided) with a suitable inhibitor (e.g. formaldehyde) which does not destroy its antigenic activity. The antigenic material may be used as prepared, that is an aqueous solution, and formulated into a vaccine optionally in combination with suitable , 48464 - 6 pharmaceutically acceptable adjuvants and/or preservatives and/or additional medical or therapeutic agents and stored, preferably at a tenperature in the range of from 0 to 4°C, ready for use. Alternatively, the antigenic material may be freeze-dried and the vaccine may be made up immediately prior to use by the addition of a suitable liquid medium, for example a sterile saline solution, and, optionally, suitable adjuvants.

Suitable adjuvants include potassium alum, prot10 amine, aluminium phosphate, aluminium hydroxide,, calcium phosphate, glycerol, sorbitol, propylene glycol, carboxyvinyl polymers available under the Trade Mark Carbopol and bearing the designation 934, 940 and 941, Freund's universal adjuvant, Freund's incomplete adjuvant, soluble diethylaminoethyl-dextran (DEAE-dextran), sodium chloride solution (saline solution), phosphate buffered saline (PBS), saponin, and the fixed oils and synthetic esters of higher fatty acids which are known to be effective adjuvants. Preferred adjuvants include Freund's in20 complete adjuvant, soluble DEAE-dextran and phosphate buffered saline.

Suitable preservatives include phenol, formaldehyde, propylene glycol, glycerol, esters of ]3hydroxybenzoic acid, benzoic acid and its sodium salt, hexachlorophene, quaternary germicides and thiomersal as such or in the form in which it is available under the Trade Mark Merthiolate.

The vaccine may be a simple monovalent vaccine or may be a complex multivalent vaccine comprising a number of antigenic components. In areas in which only one type of fly is responsible for'primary fly strike in sheep it may be necessary to use a vaccine comprising antigenic material prepared only from the larvae of that fly in order to protect sheep against fly strike. In areas where a number of types of fly are responsible for primary - 7 fly strike in sheep it may be necessary to use a vaccine comprising a mixture of antigenic materials prepared from the larvae of each of those flies in order to protect sheep against fly strike.

In most of the major sheep growing areas of the world the flies responsible for primary fly strike in sheep are flies of the Lucilia spp. and in particular Lucilia cuprina and/or Lucilia sericata. A vaccine which is particularly suitable for.use in the protection of sheep from fly strike in these areas is one comprising antigenic material prepared from the larvae of the flies Lucilia cuprina and Lucilia sericata. In some areas the fly Calliphora stygia is also responsible for primary fly strike in sheep. In these areas it may be necessary to use a vaccine comprising antigenic material prepared from the larvae of the flies Lucilia cuprina, Lucilia sericata and Calliphora stygia in order to protect sheep from fly strike.

The vaccine may also be used in combination with other therapeutic agents useful in the treatment of diseases in sheep, including anthelmintics such as, for example, levamisole and its acid salts, and vaccines such as, for example, the clostridial vaccines.

Treatment of sheep with antigenic material according to the process of the invention stimulates antibody production and the antibodies so produced inhibit the fly larvae from penetrating the epidermis of the sheep and establishing fly strike. Thus the vaccine comprising said antigenic material should be administered at a dose rate sufficient to promote the production of a blood antibody level which will inhibit larvae of the flies responsible for primary strike in sheep from penetrating the epidermis of the sheep and establishing fly strike. The amount of vaccine administered will depend, therefore, to a large extent on the relative activity of 8 4 6 4 - 8 the antigenic material. Preferably the vaccine is formulated in such a manner that sufficient antigen to protect a sheep from fly strike is contained in a volume of from 1 to 10 ml of vaccine and more preferably in from 1 to 5 ml of vaccine.

Preferably the antigenic material is administered parenterally in the form of a vaccine. The term parenterally is used herein to mean intravenous, intramuscular and subcutaneous injection.

The novel method for the control of fly strike in sheep provided by the invention represents a significant advance in the protection of sheep from fly strike. The. method of the invention offers a number of advantages over the conventional methods of treatment employing insecti15 cides. Such advantages include: simple labour-saving parenteral administration of the protectant; elimination of problem of external application of possibly toxic insecticides; elimination of the problem of the development of insecticide resistant strains of fly; and a long period of protection.

The period of protection offered by the method of the invention is particularly important. Protection for a period of at least 104 days is possible and it is expected that the period of protection provided by the process of the invention will be at least 200 days. Such a period of protection is particularly advantageous as it eliminates the need for repeated administration of a protectant when the season in which sheep are particularly prone to fly strike is extended or delayed by climatic conditions. 84 64 The invention is now illustrated by, but by no means limited to, the following examples.

Examples 1 to 11 illustrate the preparation of antigens and vaccines of the invention.

Example 1 Adult Lucilia sericata were induced to lay eggs on slices of moist raw sheep liver. The eggs were hatched and the first instar larvae were transferred to preserving jars containing diced sheep liver and covered with muslin covers. The jars were incubated at a temperature of 30°C and the larvae were allowed to grow for two days. The cultures were then strained and washed with saline solution to give about 1 litre total volume. The pH of the extract was adjusted from 8.4-8.5 to about 7 by the addition of 2 M HCl and the solution was filtered through celite and finally through 0.45μ Millipore filters. The extract was then concentrated by ultrafiltration to about 100 ml then centrifuged and refiltered. Thiomersal (0.02%) was added to the extract and then the extract was sterile filtered through 0.20μ Millipore filters. This preparation, hereinafter referred to as Antigen 1, was stored at a temperature in the range from 0 to 4°C prior to formulation.

Antigen 1 was formulated into Vaccine 1 using Freund's incomplete adjuvant. The vaccine, an homogenised oil-in-water emulsion, was prepared from the following components and stored at a temperature in the range from 0 to 4°C.

Oil Phase Drakeol 6VR Arlacel A Water Phase Saline (PBS) Antigen ml 4.5 ml ml 10 ml 8 4 6 4 - 10 Tween 80 0.5 ml Phenonip 0.2% v/v Formaldehyde 0.1% v/v Drakeol 6VR is a Trade name for a mineral oil.

Arlacel A is a Trade name for a sorbitan fatty acid ester.

Tween 80 is a Trade name for a sorbitan monooleate ethoxylated with 20 moles of ethylene oxide.

Drakeol, Arlacel and Tween are Trade Marks.

Example 2 Portion of the Antigen 1 preparation was attenuated by the addition of 0.2% formaldehyde (0.5% v/v formalin) and the incubation of the treated preparation for a period 2-3 days at a temperature of 37°C and pH 7-7.2. The preparation, hereinafter referred to as Antigen 2, was stored at a temperature of 0 to 4°C prior to formulation.

Antigen 2 was formulated into Vaccine 2 using Freund's incomplete adjuvant following the procedure des20 cribed in Example 1 for the formulation of Vaccine 1 and was stored at a temperature in the range from 0 to 4°C. Example 3 The procedure described in Example 1 for the preparation of Antigen 1 and Vaccine 1 was repeated using Lucilia sericata larvae grown on ox liver to prepare Antigen 3 and Vaccine 3. Both the antigen and the vaccine were stored at a temperature in the range from 0 to 4°C. Example 4 The procedure described in Example 2 for the preparation of Antigen 2 and Vaccine 2 from Antigen 1 was repeated using portion of the Antigen 3 preparation prepared as described in Example 3. The resulting antigen (Antigen 4) and vaccine (Vaccine 4) were stored at a temperature in the range from 0 to 4°C. - 11 Example 5 The larvae grown on sheep liver and separated from the growth medium as described in Example 1 were thoroughly washed with water and saline. Saline solution was added to the larvae (wet weight 100 g) and the mixture was homogenised at a temperature of 10°C using a ’’Silverson Mixer. The homogenised mixture (500 ml; pH 6.8) was treated with formaldehyde (0.2% to 0.5% v/v formalin) and the preparation was incubated for a period of 4 days at a temperature of 37°C. This preparation, hereinafter referred to as Antigen 5, was stored at a temperature in the range from 0 to 4°C.

Antigen 5 was formulated into Vaccine 5 using Freund's incomplete adjuvant following the procedure described in Example 1 for the formulation of Vaccine 1, and was stored at a temperature in the range from 0 to 4°C. Example 6 Antigen 6 was prepared from equal volumes of Antigen 3 prepared according to Example 3 and Antigen 5 prepared according to Example 5. vaccine 6 was prepared from Antigen 6 using Freund's incomplete adjuvant following the procedure described in Example 1 for the preparation of Vaccine 1. Both the Antigen and The Vaccine were stored at a temperature in the range from 0 to 4°C.

Example 7 The procedure described in Example 1 for the preparation of Antigen 1 was repeated with the exception that Lucilia sericata larvae were grown on bovine liver for a period of 24 hours. The antigen prepared (Antigen 7) was formulated into Vaccine 7 following the procedure described in Exanple 1 for the preparation of Vaccine 1.

Both the Antigen and Vaccine were stored at a temperature in the range from 0 to 4°C. - 12 Example 8 Antigen material prepared as described in Example 7 was formulated into Vaccine 8 following the procedure described in Example 1 for the formulation of Vaccine 1 with the exception that soluble DEAE - Dextran (5 percent of total volume) was substituted for Freund's incomplete adjuvant. The Vaccine was stored at a temperature in the range from 0 to 4°C.

Example 9 Antigen material prepared as described in Example was formulated into Vaccine 9 following the procedure described in Example 1 for the formulation of Vaccine 1 with the exception that soluble DEAE - Dextran (5 percent of total volume) was substituted for Freund's incomplete adjuvant. The Vaccine was stored at a temperature in the range from 0 to 4°C.

Example 10 The procedure described in Example 1 was repeated, using first instar Lucilia cuprina larvae with bovine liver as nutrient medium, to prepare Antigen 10, The antigenic material was formulated into Vaccine 10 following the procedure described in Example 1, with the exception that soluble DEAE - Dextran (5 percent of total volume) was used as an adjuvant in place of Freund's incomplete adjuvant. Both the Antigen and the Vaccine were stored at a temperature in the range from 0 to 4°C. Example 11 Lucilia cuprina eggs were placed in score marks on the surface of a 10% casein/1% agar gel containing phosphate buffered saline (PBS), glucose and dried yeast. The eggs were incubated at a temperature of 27°C. After hatching the larvae were allowed to feed on the casein/ agar nutrient medium at a temperature of 27°C for up to seven days. At harvest the liquid was strained from the culture medium and the remaining solids were 8 464 - 13 thoroughly washed with PBS and the washings added to the stained liquid. The pH of the liquid was adjusted to 6.0 to 6.5, the liquid was centrifuged and the supernatant was filtered through a series of Millipore filters. The titrate was concentrated by ultrafiltration to give a fraction containing material of molecular weight above 25,000, refiltered and finally sterile filtered. This preparation, hereinafter referred to as Antigen 11, was stored at a temperature in the range from 0 to 4 °C.

Vaccine 11 was prepared from Antigen 11 following the procedure described in Example 1 for the preparation of Vaccine 1 with the exception that soluble DEAE Dextran (5 percent of total volume) was used as an adjuvant in place of Freund's incomplete adjuvant. Both the Antigen and the Vaccine were stored at a temperature in the range from 0 to 4°C.

Example 12 Antigenic material prepared as described in Example 1 was formulated into Vaccine 12 following the procedure described in Example 1 with the exception that soluble DEAE - Dextran (5 percent of total volume) was substituted for Freund's incomplete adjuvant. The vaccine was stored at a temperature in the range from 0 to 4°C.

Example 13 This Example demonstrates the effectiveness of vaccines of the invention in stimulating the production of antibodies in sheep.

Vaccines 1 to 6 inclusive were administered to different Romney sheep by subcutaneous injection in the neck. The dosing regimen outlined in Table 1 below was followed and serum samples were taken on the 42 and 139 days after injection. 8 4 C> 4 - 14 TABLE 1 Days Post First Vaccination Volume Injected Comments 0 1 ml 7 1 ml - 22 2 ml - 42 - Blood sample taken 125 5 ml - 139 - Blood sample taken Serum samples from the vaccinated sheep and an unvaccinated control were run in conventional agar, geldiffusion plates against the larval antigen preparations 5 and against sheep and ox liver extracts which had not been in contact with larvae. The plates were run by placing a serum sample in a central well in the agar and placing the antigens and control liver extracts in wells spaced uniformly around the central well.

The results are presented in Table 2 below wherein line development between the serum sample and the antigen indicates the presence of antibodies in the serum sample and is coded as follows: Code Line Development 0 None + Weak ++ Medium Strong +++ - 15 TABLE 2 Vaccine No Day Serum Sampled Line Development Antigen No Control Liver Extract 1 2 3 4 5 6 Sheep Ox 1 42 + + + + + + 0 0 1 139 +++ +++ +++ +++ +++ +++ 0 0 2 42 0 0 0 0 0 0 0 0 2 139 ++ ++ ++ ++ ++ ++ 0 0 3 42 + + + + + 0 0 3 139 +++ +++ +++ +++ +++ +++ 0 0 4 42 0 0 0 0 0 0 0 0 4 139 ++ ++ ++ ++ ++ ++ 0 0 5 42 0 0 0 0 0 0 0 0 5 139 + + + + + + 0 0 6 42 0 0 0 0 0 0 0 0 6 139 + + + + + + 0 0 Control 42 0 0 0 0 0 0 0 0 Control 139 0 0 0 0 0 0 0 0 - 16 Example 14 This Example demonstrates the effectiveness of vaccines of the invention in preventing the incidence of fly-strike in sheep.

The sheep vaccinated with Vaccine 1 according to the dose regimen detailed in Example 13 and unvaccinated control sheep were subjected to larval implant challenge. First instar larvae of Lucilia sericata were implanted onto the backs of the vaccinated sheep and the control sheep 72 days after the first vaccination and again 139 days after the first vaccination. Three days post implant the implant sites were inspected for surviving larvae.

The vaccinated sheep showed no evidence of strike after either implantation whereas the unvaccinated control sheep showed strike after each implantation. Example 15 This example demonstrates the effectiveness of vaccines of the invention in preventing fly strike in sheep.

Romney sheep (25) which had not previously been treated with insecticide or struck by fly were selected and divided into five equal groups and tagged. Group A sheep were retained as controls and remained untreated throughout the trial. The remaining sheep were vaccinated by subcutaneous injection with 5 ml of test vaccine and the injection was repeated 21 days later.

The vaccines tested were as follows: Group B - Vaccine 8 Group C - Vaccine 9 Group D - Vaccine 7 Group E - Vaccine 3 - 17 After vaccination the sheep were allowed to graze as one mob together with the untreated controls. The sheep, both vaccinated and controls, were subjected to larval implant challenge 63, 70, 77 and 104 days after the first vaccination by implanting first instar larvae of Lucilia sericata onto one or more sites on the back of each sheep. Three days post implant the implant sites were inspected for surviving larvae.

The results are detailed in Table 3 below in 10 which the symbols have the following meanings S - Strike established N - No strike established (larvae dead) R - Restricted strike (larvae alive but stunted and no strike established) 8 4 6 4 TABLE 3 Group Sheep No RESULT OF CHALLENGE ON DAY 63 70 77 104 1 S R N S 2 S R R S A 3 S R N R 4 S S R . N 5 R S S N 1 R R R N 2 R R N N B 3 R N R N 4 R R R R 5 N N N N 1 N R N N 2 N N R N C 3 N N N . N 4 R R N R 5 N R N N 1 R R N N 2 R R N N D 3 R R R R 4 R R N R 5 R R R N 1 R R - R 2 R R - N E 3 RR 1 I N 4 S S N 5 S S ΐ N 8 4 6 4 - 19 Example 16 This Example demonstrates the effectiveness of a vaccine of the invention in the prevention of fly strike in sheep.

Romney sheep (67) which had not previously been treated with insecticide or struck by fly were selected and divided into two groups and tagged. Group B sheep (24 sheep) were retained as controls and remained untreated throughout the trial. Group A sheep (43 sheep) were vaccinated by subcutaneous injection with 5 ml of Vaccine 12 and the injection was repeated 21 days later.

After vaccination the vaccinated sheep and the controls were allowed to run together as one mob. The sheep, both vaccinated and controls, were subjected to larval implant challenge 104 days after the first vaccination by implanting first instar larvae of lucilia sericata onto one or more sites on the back of each sheep. Two days post implant the implant sites were inspected for surviving larvae.

The detailed results are presented in Table 4 below in which the symbols ahve the following meanings: S - Strike established N - No strike established (larvae dead) R - Restricted strike (larvae alive but stunted and no strike established) The summarised results are presented in Table 5. 8 4 6 4 - 20 TABLE 4 Group Sheep Number Result of Challenge on Day 104 A 84 R >1 78 R 11 48 R If 92 S II 55 N tl 69 S II 53 R « 45 N II 57 R tt 65 R II 227 N II 80 S II 74 N tf 52 N - II 66 N II 231 N II 81 R II 276 N tl 64 N 1 83 R ί j 71 N : ·’ J 6i N : ” ί 82 N ί ί 46 R I 54 R ” ! 97 R 1 225 R II 49 N II 205 R II 63 R 8 4 6 4 - 21 TABLE 4 Continued Group Sheep Number Result of Challenge on Day 104 A 67 R II 96 N II 98 R II 75 N II 95 R « 232 N tl 86 R If 79 R 59 N II 76 N II 224 N II 70 N II 56 R B 358 R II 381 S II 374 S II 378 S M 368 R II 399 S ir 386 S II 392 s ll 393 S II 385 R tl 394 R II 379 R It j 400 S II! 397 N i 1 ! 308 i R 1 n 366 R 8 4 () 4 - 22 TABLE 4 Continued Group Sheep Number Result of Challenge on Day 104 B 370 R H 384 S II 371 S II 372 S tt 377 R It 359 R II 353 R II 387 S TABLE 5 RESULTS OF CHALLENGE OK DAY 104 Symptom Group A Group B Number of Sheep % Number of Sheep s Strike 2 4.7 11 45.8 Restricted strike 21 48.8 12 50.0 No Strike 20 46.5 1 4.2 - 23 Example 17 This Example demonstrates the effectiveness of vaccines of the invention in stimulating the production of antibodies in sheep.

Merino sheep (200) which had not previously been treated with insecticide were selected, divided into 25 groups of 8 sheep, and tagged. The groups were treated as follows by subcutaneous injection over the ribs on day 0 and day 21 of the trial: TABLE 6 Group No Test or Control Material Injected Volume (ml) A and B Untreated Controls - C and D 5% DEAE - Dextran in PBS 10 E Alum Precipitate Suspended in PBS 5 F Vaccine 10 10 G Vaccine 10 5 H Vaccine 10 2 I Antigen 10 10 J Antigen 10 5 K Antigen 10 2 M and T Vaccine 11 10 N and U Vaccine 11 5 0 and V Vaccine 11 2 P and W Antigen 11 10 Q and X Antigen 11 5 R and Y Antigen 11 2 8 4 6 4 - 24 Serum samples from sheep from each group were taken on days 35 to 37 of the trial. Each serum sample was diluted one thousand fold with a solution of PBS containing 1% albumin and 0.05% Tween 20 and assayed for antibodies to Lucilia cuprina antigen using the following radioimmunosassay technique.

Antigenic material prepared from Lucilia cuprina larvae as described in Examples 10 and 11 were allowed to bind to poly (vinyl chloride) wells of multiwell plates and the wells were then washed with PBS. Diluted serum samples were added to separate wells and the plates were incubated for four hours. After incubation the wells were' washed with a 0.05% solution of Tween 20 in PBS. Suitably diluted iodine-125 labelled rabbit antisheep immunoglobulin antiserum was then added to each well- and the plates were incubated overnight. The wells were again washed and were then dried and the bound radioactivity was counted.

The radioactivity count gives and indirect measure of the amount of antibody to Lucilia cuprina antigen present in the sheep sera. The results are given in Table 7 and are expressed, after the subtraction of the background count given by sheep never exposed to fly strike, as a percentage of the antibody count given by a sheep very severely struck by Lucilia cuprina and which was used as standard throughout.

Serum samples from sheep from Groups A, G, N, and U were run in conventional agar gel-diffusion plates against Antigen 8 and Antigen 11 following the procedure described in Example 13.

The results are given in Table 8 wherein line - 25 development between the serum sample and the antigen indicating the presence of antibodies in the serum sample is coded as detailed in Example 13.

TABLE 7 Group No Antibody Count (%) in Serum Sample 1 2 3 4 A 37 6 27 23 B 6 -6 -3 24 C 15 21 - - D 4 37 -11 4 E 4 3 5 -1 F 49 106 88 68 G 70 67 70 35 H 74 38 32 25 X 8 5 5 -2 J 3 19 11 41 K -3 23 20 13 M 116 67 120 61 T 87 91 81 89 N 112 32 84 37 U 84 103 118 95 0 41 102 26 76 V 81 67 73 101 P 33 33 23 22 w 114 38 36 28 Q 21 11 25 -2 X 69 44 71 70 R 2 7 -1 30 y 27 13 22 84-6 4 - 26 TABLE 8 Day Line Development Serum No Sampled Antigen No 8 11 A 63 0 0 A 63 0 o 1 A 63 0 0 G 63 + +++ G 63 + +++ G 63 - +++ N 63 + ++ N 63 + +++ N 63 - ϋ 63 + +++ U 63 + ++

Claims (36)

1. A method for the prevention or reduction of fly strike in sheep which method comprises treating said sheep with an antigenic material effective in immunizing 5 said sheep against fly strike.

2. A method according to claim 1 wherein said antigenic material is prepared from the larvae of the fly or flies responsible for primary fly strike in sheep.

3. A method according to claim 2 wherein said 10 antigenic material is prepared from the fluid produced by growing said larvae or from a culture of the cells or organs of said larvae.

4. A method according to claim 2 or claim 3 wherein said antigenic material is prepared from the fluid pro15 duced by growing on a nutrient medium larvae of flies of the Lucilia spp., Calliphora spp. or a mixture of both of said species.

5. A method according to any one of claims 2 to 4 inclusive wherein said larvae are larvae of the fly 2o Lucilia sericata.

6. A method according to any one of claims 2 to 5 inclusive wherein said larvae are larvae of the fly 4 8 4 6 4 - 28 Lucilia cuprina.

7. A process for the preparation of antigenic material effective in preventing or reducing the incidence of fly strike in sheep which process comprises growing on a 5 nutrient medium larvae of the fly or flies responsible for primary fly strike in sheep, separating the liquid produced, and concentrating said liquid to give a solution of said antigenic material.

8. A process according to claim 7 which comprises: 10 growing freshly hatched fly larvae on a nutrient medium; separating the liquid formed and adjusting the pH of said liquid as required to give a liquid of pH in the range of from 6 to 8; removing the insoluble material from said pH adjusted liquid to give a solution; concentrating said 15 solution to give a solution containing material of molecular weight above 20,000; and sterile filtering said concentrated solution.

9. A process according to claim 7 or claim 8 which comprises: growing freshly hatched fly larvae on a 2o nutrient medium; separating the liquid formed and adjusting the pH of said liquid by the addition of a pharmaceutically acceptable mineral or organic acid to give a liquid of pH in the range of from 6 to 7; removing the insoluble material from said pH adjusted liquid by 25 centrifugation and/or filtration to give a solution; concentrating said solution by ultrafiltration to give a solution containing material of molecular weight above 20,000; and sterile filtering said concentrated solution.

10. A process according to any one of claims 7 to 9 30 inclusive wherein said nutrient medium comprises animal tissue. - 29

11. A process according to any one of claims 7 to 9 inclusive wherein said nutrient medium comprises an artificial mixture of nutrients.

12. A process according to any one of claims 7 to 10 inclusive wherein said nutrient medium comprises animal liver.

13. A process according to any one of claims 7 to 9 inclusive or claim 11 wherein said nutrient medium comprises an artificial mixture of nutrients chosen from dried milk/yeast/agar, casein/agar, casein/agar/glucose and casin/agar/glucose/yeast and wherein each mixture optionally comprises added amino acids and/or vitamins.

14. A process according to any one of claims 7 to 13 inclusive wherein said larvae are chosen fran the larvae of flies of the lucilia spp., Calliphora spp. or a mixture of both of said species.

15. A process according to any one of claims 7 to 13 inclusive wherein said larvae are larvae of the fly lucilia sericata.

16. A process according to any one of claims 7 to 13 inclusive wherein said larvae are larvae of the fly Lucilia cuprina.

17. A process according to any one of claims 7 to 13 inclusive wherein said larvae are larvae of the fly Calliphora stygia.

18. Antigenic material effective in preventing or reducing the incidence of fly strike in sheep which comprises homogenising the larvae of flies responsible for primary fly strike in sheep; separating and concentrating the fluid produced by growing said larvae on a nutrient medium; or homogenising a culture of 4 8 4 6 4 - 30 the cells or organs of said larvae.

19. Antigenic material prepared according to the process of any one of claims 7 to 17 inclusive.

20. Antigenic material according to claim 18 or claim 5 19 wherein said antigenic material has been freeze-dried.

21. Antigenic material according to any one of claims 18 to 20 inclusive wherein said antigenic material has been treated with an inhibitor to attenuate said antigenic material without destroying its antigenic 10 activity.

22. A vaccine effective in preventing or reducing fly strike in sheep comprising antigenic material as defined according to any one of claims 18 to 21 inclusive and a pharmaceutically acceptable vehicle therefor. 15

23. A vaccine effective in preventing or reducing the incidence of fly strike in sheep comprising antigenic material prepared according to the process of any one of claims 7 to 17 inclusive and a pharmaceutically acceptable vehicle therefor. 20

24. A vaccine according to claim 23 comprising a mixture of antigenic material prepared according to the processs of two or more of claims 15, 16 and 17.

25. A vaccine according to any one of claims 22 to 24 inclusive wherein said vehicle comprises at least one 25 adjuvant and at least one preservative.

26. A vaccine according to any one of claims 22 to 25 inclusive in the form of an oil-in-water emulsion said vehicle comprising Freund's incomplete adjuvant and phosphate buffered saline. - 31

27. A vaccine according to any one of claims 22 to 25 inclusive in the form of an aqueous solution comprising soluble diethylarainoethyl dextran and phosphate buffered saline.

28. A vaccine according to any one of claims 22 to 27 inclusive wherein said vaccine comprises in addition to said antigenic material another medicinal or therapeutic agent.

29. A process for the preparation of a vaccine according to any one of claims 22 to 28 inclusive which process comprises formulating antigenic material as defined according to any one of claims 18 to 21 inclusive with a pharmaceutically acceptable vehicle comprising adjuvants and/or preservatives and/or additional medicinal or therapeutic agents.

30. A method for the prevention or reduction of fly strike in sheep which method comprises administering to said sheep a therapeutically effective amount of a vaccine according to any one of claims 22 to 28 inclusive.

31. A method according to claim 30 wherein said vaccine is administered parenterally.

32. A method according to any one of claims 1 to 6 inclusive, 30 and 31 substantially as herein described with reference to any one of Examples 14 to 16 inclusive.

33. A process according to any one of claims 7 to 17 inclusive substantially as herein described with reference to any one of Examples 1 to 7 inclusive 10 and 11.

34. Antigenic material according to any one of claims - 32 18 to 21 inclusive substantially as herein described with reference to any one of Examples 1 to 7 inclusive 10 and 11.

35. A vaccine according to any one of claims 22 to 28 5 inclusive substantially as herein described with reference to any one of Examples 1 to 12 inclusive.

36. A process according to claim 29 substantially as herein described with reference to any one of Examples 1 to 12 inclusive.