GB2038627A - Process for producing fascioliasis vaccine - Google Patents

Abstract

Fascioliasis vaccine, especially for bovine administration, can be prepared by gamma irradiation of Fasciola gigantica metacercariae obtained by breeding Lymnaea natalensis snails successively in a series of tanks, infecting their descendants with Fasciola gigantica miracidia and growing the infected snails in cultivation tanks until they shed cercariae which encyst to become metacercariae. The vaccinated cattle are immunized against fascioliasis even when they graze outdoors in conditions where they are at risk of infection.

Description

SPECIFICATION Process for producing fascioliasis vaccine The present invention relates to a vaccine for combating fascioliasis, especially bovine fascioliasis.

Fascioliasis in cattle is a problem in the wetter areas of the continent of Africa and Asia, although not so severe as in sheep. The reason for the difference in severity of the disease between sheep and cattle would appear to lie basically in the fact that unlike sheep, cattle which have been previously infected with Fasciola gigantica acquire a degree of resistance to reinfection and also that the bovine liver appears to be able to tolerate the presence of very large numbers of developing immature parasites without the occurrence of the severe pathogenic effects seen in sheep. Outbreaks of acute and sub-acutefascioliasis comparable to those seen in the sheep are therefore rare.

Bovine fascioliasis can be considered mainly as a chronic disease of susceptible cattle, usually calves or yearlings, which have spent their time on infected pasture, and drinking infected water. Adult stock which have had previous experience of the parasite can acquire an immunity and therefore withstand a heavy challenge from infected pastures without showing clinical disease. They may however harbour a small population ofF. gigantica which can contribute to the epidemiology of the disease.

The clinical signs are similar to those seen in chronicfascioliasis in sheep, namely loss of condition, marked anaemia and in severe cases subman dibularoedema. Diarrhoea is not found in cases of chronicfascioliasis unless they are complicated by the presence of certain helminth spp, and thus results in a different syndrome.

The pathogenesis of the disease in cattle as in sheep is with a severe haemorrhagic anaemia, a marked eosinophilia, and a severe hypoalbuminaemia. F. gigantica eggs are found in the faeces. At post-mortem examination the liver is cirrhosed, with the ventral lobe again being the most severely affected and the bile ducts show typical distention and calcification. In these cases 200 or more adult parasites can be recovered from the bile ducts.

Sub-acute fascioliasis occurs in calves and yearlings after heavy ingestion of metacercariae and it appears clinically as an acute haemorrhagic anaemia with eosinophilia and hypoalbuminaemia but in these cases the disease is not so rapidly fatal and the effected animal may show clinical signs for one or two weeks prior to death. Large numbers of flukes are again present in an infected liver but the numbers are somewhat reduced from those found in cases of acute fluke though the parasites have developed further and a substantial proportion of the fluke population is now present as adults in the major bile ducts. This form of the disease may develop where the calves have ingested large numbers of metacercariae over a longer period or the number ingested at any one time has not been sufficlient to cause the acute form of the disease.Affected calves lose condition rapidly, become markedly anaemic with obvious pallor of their mucous membranes and may have a palpably enlarged liver and resent abdominal palpation. Submandibular oedema and ascites may be present.in some cases.

Outbreaks of chronic fascioliasis are seen in the herds living in water areas in the tropics and the disease is characterised by a progressive loss of condition and development of a severe anaemia and hypoalbuminaemia which terminally, clinically, results in emaciation, pallor of the mucous membranes, submandibular oedema and ascites. This form of the disease is the result of the infection picked up from drinking infected water and eating infected pasture which is now present as adult flukes in the bile ducts and over the ensuing months these flukes remove more blood from the circulation than the calves can replace and so the anaemia and hypoalbuminaemia become progressively worse the longer the parasites persist. Terminally these animals show a hypochromic macrocytic anaemia with an eosinophilia, a severe hypoalbuminaemia, and F.

gigantica eggs can be found in their faeces.

It is therefore of great interest to produce a vaccine to immunize cattle against bovine fascioliasis. This has now been achieved.

The discovery of bovine fascioliasis vaccine results from an intensive study of the biological life cycle of Fasciola gigantica, the causative agent of bovine fascioliasis in cattle. The life cycle of this parasite involves the use of Lymnaea natalensis, a fresh-water snail, as the immediate host, and mammals, e.g. cattle and sheep, as the definitive hosts. A developmental stage in this parasite's biological life cycle (METACERCARIAE) is formed afterthecer- cariae have become encysted on being shed by the fresh-water snail, Lymnaea natalensis. The life cycle of Fasciola gigantica (the common liverfluke) is as follows: The adult fluke normally lives in the bile ducts of its definitive hosts which are usually cattle, sheep, goats, etc.In the bile duct, the adult fluke lays large numbers of eggs which pass into the bowel and so escape from the body in the faeces of the host. Each egg hatches and releases a small larva (MIRACIDIUM) within seventeen days under suitable conditions of temperature (26"C) and moisture. The miracidium swims about in the water and can survive for about 8 hours in search of a freshwater snail known as Lymnaea natalensis which it penetrates to continue with further development.

The miracidium usually migrates to the pulmonary chamber of the snail where it becomes a sporocyst.

Each sporocyst produces several rediae in the portal system of the snail. The free rediae ultimately produce hundreds of cercariae which swim about in the water and encyst on grass, under-surface of leaves or on some other object. The encysted cercariae (metacercariae) can survive almostfortwelve months in the tropics. Once the encysted cercariae are swallowed by the host animal they excyst in the gut to juvenile flukes which penetrate the mucosa of the small intestine, and migrate through the body cavity to the liver. The juvenile flukes penetrate the liver through the liver capsule and move extensively into the parenchyma.After ten weeks of extensive migration and traumatic damage in the liversubstance, the flukes settle in the bileducts and start sucking blood from the host and producing eggs again to repeat the cycle in a period of thirteen weeks from the time of swallowing encysted cercariae (metacercariae).

An effective fascioliasis vaccine, particularly for bovine administration, can be produced by a process which comprises breeding snails of the species Lymnaea natalensis, infecting said snails with Fasciola gigantica miracidia, growing the infected snails until they shed Fasciola gigantica cercariae, which thereupon encystto become metacercariae, collecting said metacercariae, subjecting said metacercariae to gamma irradiation from a radicactive source to reduce substantially the pathogenic character of flukes excysting from said metacercariae in the gut of an animal to which metacercariae have been administered relative to flukes so excysting from non-irradiated metacercariae but without substantially altering the immunogenic character of said metacercariae, and collecting said irradiated metacercariae to constitute the effective ingredient of said vaccine.

The expression "substantially reduced pathogenic character" means thatthe flukes have no pathogenic character or pathogenic character only to such a degree that the animal to which the metacercariae is administered contracts fascioliasis in a mild form only. It is naturally preferred that the excysting flukes should possess no pathogenic character.

The radiation dosage to the metacercariae is conveniently in the range from 2500 to 3500 rads, for example in the range from 2800 to 3200 rads. Preferably, the metacercariae are subjected collectively to a uniform radiation dosage of about3000 rads at a temperature of about 20"C.

Preferably, the irradiation will be provided by a caesium-137 source. This has a high activity of 10,500 Ci (+5%) and a half-life of 30 years and is in general used with appropriate shielding by depleted uranium suitably enclosed in a steel case, its longevity of use, energy output and the versatility of the equipment making Cs-137 a superior radioactive source to Cobalt-60. In practice, irradiation is conveniently provided by a radiation machine having such a source and capable of delivering 200 Krd/h. Such machine should preferably have three sources of radiation.

In a particular embodiment, the vaccine is in capsule form, the metacercariae having been encapsulated prior to irradiation. Suitably each capsule contains about 1,000 metacercariae and appropriate adjuvants to form an injectable vaccine. Once formed, the vaccine should in general be stored at 4"C or below.

In order to achieve large-scale production of the vaccine it is necessary to have large-scale production of the metacercariae and this in turn requires large-scale breeding of the snails. It would of course be possible for collectors to collect an individual snail of this species from its natural habitat, to infect it and to collect the encysted cercariae when they are shed but such an individual procedure is clearly impractical on a large scale.

We have developed a method of manufacturing the metacercariae on a large-scale which is suitable for industriai realisation in the production of fascioliasis vaccine.

According to the present invention an industrialscale process for the manufacture of Fasciola gigantica metacercariae for use in producing fascioliasis vaccine, comprises breeding Lymnaea natalensis snail; successively in a series of breeder tanks until their rirst-generation descendants have been produced in each individual breedertank,transferring these young snails from their respective breeder tank to one or more ancillary cultivation tanks, infecting these young snails with F. gigantica miracidia, growing the infected snails in the cultivation tank or tanks until they begin to shed cercariae, which encystto become metacercariae, and collecting the metacercariae. These metacercariae may then be irradiated as explained above to from the vaccine.

Suitably at least 500, preferably 800-1200, fullygrown breeder snails are introduced into the first breeder tank, these breeder snails suitably being a field strain of the snails collected from their natural habitat, and surviving breeder snails from this tank can be transferred to the next breeder tank (and so on down the line) in 2-4 months, after the first generation of laboratory-bred snails has been produced. In the preferred embodiment these breeder snails are never infected. When all the breeder snails and laboratory-bred snails have been transferred from the first breedertankthis can be restocked with a fresh group of wild snails and the cycle recommenced. The breeder tanks suitably have a volume of about 50,000 cc if they are to accommodate 1,000 snails.

The tanks are suitably glass or perspex tanks placed on a bench above ground level. The bottom of each tank is suitably covered with sand obtained from a natural habitat of L. natalensis. Conveniently the rest of the tank is three-quarters filled with filtered pond water. Water lilies complete with their root systems from the same habitat are desirably sunk into the tanks, and their roots embedded in the sand, the leaves resting on the water surface. Aeration of the tanks can be obtained from their permanently open-tops and by artificially pumping air through the water using a 'Hy-Flo' model 'B' aeration pump. One or two leaves of lightly boiled lettuce are desirably added to each tank once a week to supplement algae, the main food supply for the snails.

The laboratory-bred snails from the breeder tanks are preferably distributed into several individual cultivation tanks as each breeder snail can be expected to produce on average about 100 first-generation descendants in each breeding period. Suitably there are more than 4, preferably 6 to 10, cultivation tanks associated with each breeder tank, although one cultivation tank may, if desired, be associated with more than one breeder tank. Each cultivation tank suitably accommodates several thousand snails, e.g.

8,000 to 12,000.

The infection of the laboratory-bred snails (which is conveniently carried out during their transfer between breeder tank and cultivation tank but can occur later), the cultivation of these snails and the collec tion of the metacercariae may be carried out in any convenient manner.

For the purpose of producing enough dosage of the bovine fascioliasis vaccine to cater for its African market, a possible outline of production is as follows: Each snail is a hermaphrodite individual capable of producing at least 100 eggs per day and a snail egg takes approximately six weeks for the young snail to hatch out and to reach egg-producing stage.

A minimum economic unit for industrial-scale production should contain approximately 1,000,000 breeding L. natalensis snails, which implies a need for 1,000 tanks.

It is the progeny of these snails that will be suitable for infection with suitable doses of miracidia under appropriate conditions of temperature, pressure and moisture. After an appropriate period of the infection maturation inside the snails, the metacercariae (mc) should be produced at the rate of approximately 10 mc per snail per day, so that the progeny population from the entire stock should be approximately 10,000,000 young snails per day. Assuming 10% of the egg population will survive to continue the generation, the metacercarial production could therefore be 100,000,000 mc per day, i.e. 3,000,000,000 per month. As each dose of the vaccine suitably contains about 1,000 gamma irradiated mc, dose production per month could be 3,000,000.

Tests have shown that cattle immunized by injection with a fascioliasis vaccine produced as described above have a strong resistance to reinfection with F. gigantica, not only under controlled conditions indoors (Example 3) but also under normal field conditions where they are naturally exposed to the risk of infection with F. gigantica (Example 4).

Further details concerning the performance of the invention in preferred embodiments are as follows: EXAMPLE 1 LYMNAEA NATALENSIS CULTURE A laboratory culture of L. natalensis is started by collecting a field strain of the snails from Ondiri swamp, Nairobi Dam, Sukari Dams or several other water catchments in and around Nairobi. The seed ing of specially prepared aquaria or breeding tanks is done by putting 1000 freshly collected L. natalensis from the natural habitat into a first breeder tank.

Identification of the snail is confirmed by its characteristic appearance. The shells are dextral and oblong. They are 8-25 mm. high and have 3 or4 whorls rapidly increasing in size, the ultimate whorl forming almost the entire shell. The collumellar margin of the aperture is twisted and the outer lip sharp. The spire is depressed and about half the height of the aperture. The shells are colourless, yel low or dark.

The aquaria systems are set close together but independent of one another as far as the working mechanisms are concerned. An aquarium essen tially consists of a glass tank of 60 cm. x 30 cm. x 30 cm. containing about 30,000 cc, of tap water con tinuouslyfiltered and maintained at 24 to 27"C. Each tank is kept under fluorescent light for 12 hours dur ing the day time only. The first breeder tank is sur rounded by 8 symmetrically arranged cultivation tanks. First generation snails from the first breeder tank are distributed into the surrounding cultivation tanks after infecting them with approximately 5 Fasciola gigantica miracidia per snail. Snail infection is done 3 months after initial seeding of the breeder tank.As first generation snails get infected with miracidia and transferred to the surrounding cultivation tanks, the surviving breeder snails are removed from the first breeder tank and transferred to a second breeder tank, similarly surrounded by cultivation tanks, and the process of breeding and infection started all over again. It is reckoned that one unit could contain 5 breeder tanks and associated cultivation tanks of the size mentioned above in a production plant unit measuring 10 x 5 metres and still leave adequate unoccupied space for carrying out other activities connected with breeding, infection of snails, production of metacercariae and storage of vaccine doses.

Considering the performance of the first breeder tank and its surrounding cultivation tank system we should be able to get a yield of 100,000 infected snails, each shedding 100 metacercariae per month, i.e. a yeild of 10,000,000 F. gigantica metacercariae or 10,000 doses of the vaccine, from one unit system of tanks. This rate of production can be repeated once a month and with as many doses as possible depending on how many tank units one can set up.

EXAMPLE2 SNAIL INFECTION WITHFASCIOLA GIGANTICA MIRA CIDIA F. gigantica eggs are obtained from the bile of affected cattle in the local abattoir. The bile is washed through a 60-mesh sieve (aperture: 250,am), using tap water, and the eggs collected on a 400-mesh sieve (aperture: 37,mum). The eggs are then washed off the 400-mesh sieve into a 500 ml. beaker with tap water until the beaker is three quarters full.

The beaker containing the egg-suspension is maintained at 26"C. The eggs will hatch out miracidia in 15-17 days. After 3 months of culture young L.

natalensis snails with a shell-height of 3-5 mm. are selected from the aquaria. Each snail is exposed to 5 miracidia suspended in a small volume of water in a watch-glass. The miracidia penetrate the body of the snail for further development.

Breeding and infected snails in their aquaria will be maintained on modified aquarian growth food.

Sixty days after infection, the snails will be shed ding hundreds of cercariae which immediately encyst and become metacercariae. These are col lected by putting a shedding snail into a single tube of No. 3/1 lined with Cellophane (Trade Mark) paper and three-quarters full of water. The tubes contain ing shedding snails are put in a tube holder and maintained at an ambient temperature of 22"C. to 27"C. in a room for 48 hours. The cercariae will encyst on the Cellophane usually at water level. The metacercariae produced are now adherent on the Cellophane paper. The Cellophane paper is now removed from the specimen tube and laid on a Petri dish before putting the Petri dish into a refrigerator at 4"C. The snail is transferred into another freshly prepared Cellophane-lined tube for 48 hours. This process of metacercarial collection and storage is repeated until the snail dies.

EXAMPLE 3 EFFECTIVENESS OF VACCINE Twenty-four male calves at the age- of 6 months and free from Fasciola gigantica were randomly sub-divided into six groups and subjected to the following treatment:-two groups received, on Day 0 and Day 42, two oral doses each of 1000 metacercariae ofFasciola gigantica which had been gamma irradiated at a dosage of 3000 rads. One of these groups was challenged, on Day 140, with 1000 infective non-irradiated metacercariae. Another three groups received 1000 infective metacercariae on Day 0,42 and 140 respectively, and the remaining group served as untreated control. Autopsies were made on Day 84 or 224, and the livers were checked for recovery of developed parasites and histological alterations.Ten weeks after challenge, three animals of each group were injected with t251-albumin and s9Fe as ferric citrate to determine the effects of the administration of metacercariae on albumin and iron turnover.

In the groups double-vaccinated with irradiated metacercariae, autopsy 84 days after challenge revealed five animals with no worm burden and no liver alterations, whereas two calves had a recovery of 1-2% of the challenge dose and slight bile duct thickening. An average recovery of 27% of the challenge dose was observed in the non-vaccinated groups, 182 and 84 days after challenge. Gross and microscopial lesions were found in the livers of these animals. Albumin turnover and catabolic rate was significantly increased in the non-vaccinated groups as a result of the infective challenge dose.

Iron metabolism was only slightly affected in the non-vaccinated groups as a result of the infective challenge dose. This is attributed to the fact that the animals were killed before anaemia had developed.

EXAMPLE4 FIELD TRIALS ON VA CCINE EFFECTIVENESS A herd of 60 six-month old bulk calves were castrated and set aside from the rest of the main herd on a ranch in Masindi, Uganda. They were randomly divided into six groups-of 10 animals in each group as shown in Table 1. All the experimental calves were kept running, grazing and watering under the same husbandry with the rest of the main herd which consisted of 400 calves.

The calves of group I were each double vaccinated with 1000 Cs-137 gamma-irradiated mc. ofF.g. at an interval of 4 weeks between the first and the second dose of vaccination. For attenuation of the metacercariae, irradiation of the mc. was done by giving them a total dose of 3000 rads or 3 Kr. The vaccinated calves were each challenged with 1000 nonirradiated metacercariae 4 weeks after administration of the second vaccinating dose. Twelve months after challenge the steers were slaughtered and their livers thoroughly examined forthe presence or absence of flukes and lesions.

Blood samples were taken weekly starting one week prior to infection. Faeces samples were taken weekly starting 12 weeks post inoculation.

The calves of groups II, III, IV and V were used as vaccine controls at different levels of vaccination and challenge. Calves of group Vl were used as controls of the entire system after artificial challenge of groups I and V calves. The calves of group VI were exposed to natural challenge from the field at the time all the calves of Groups I, IV and V were transferred to the highly endemic paddock and were exposed to drinking water from the stream that run through the paddock. The stream was heavily populated with infected and non-infected Lymnaea natalensis snails.

A similar type of field experiment was arranged on a farm at Sumaru, Zaria in Nigeria. Similar environmental conditions did obtain on this farm except that in addition to a stream which run through the challenge paddock, there was a big dam which was full ofLymnaea natalensis snails. A herd of 30 calves of the same age vide supra but of mixed sex were obtained and divided randomly into six groups of 5 calves in each group. They were treated likethose in Uganda vide supra.

At slaughter of the steers, note was taken of the weight of liver, portal lymph nodes, number and size of the flukes recovered. Fertility of the flukes recovered was also checked for by microscopic examination of individual flukes.

Tablel DIAGRAMMATIC LAY-OUT OF FIELD CATTLE VACCINATION EXPERIMENTS IN UGANDA GROUP NO. OF FIRST SECOND CHALLENGE KILL CALVES VACCINATION VACCINATION 4W 4W V 4W C 12 MONTHS 10 V V C K 11 10 C 4W K 4W III 10 C 4W K IV 10 V V 13 MONTHS K V 10 C 13 MONTHS V 10 C K VI 10 N 13 MONTHS K IN NIGERIA Group No. of First Second Challenge Kill Calves Vaccination Vaccination 4W 4W 12 MONTHS 5 V 4W V 4W C 12 MONTHS K V C II 5 C 4W K III 5 C 4W K 4W V 13 MONTHS IV 5 V 4W V 13 month K V 5 C 13 MONTHS K VI 5 N 13 MONTHS VI 5 N K KEY:: V = Vaccinate the calf with 1000 gamma-irradiated metacercariae ofFasciolagigantica C = Infect the calf with 1000 non-irradiated metacercariae of Fasciola gigantica K = Slaughter or autopsy the calf at the end of experiment N = Expose the calf to infection naturally in the field W = Week.

RESUL TS Survival All the calves of groups I and IV in Uganda and Nigeria survived the entire 14 months of the experiment and were killed at the end of this period. In Uganda and Nigeria, all the calves of groups II and Ill survived the 4 weeks post-inoculation but were killed at the end of that period. Calves of group V in Uganda died 6 months post-inoculation and exposure to natural infection whereas those in a similar group in Nigeria died 5 months post-inoculation and exposure to natural infection. Group VI steers in Uganda and Nigeria died of natural infection between 10 and 12 months after exposure to natural infection.

Fluke-recovery The recovery of flukes from all the calves after death in Uganda and Nigeria is to be considered together and it is recorded in the accompanyiny tables: Uganda Cattle Group Mean Parasitological Data Analysis Group Wt. ofPortal Fluke No. % Protection Lymph Nodes Recovered 280 g 5.1 99.83 II 221 g 601.7 39.93 lil 189 g 660.6 33.94 IV 263 g 0.7 99.97 V 230 g 566.cm 44.0 VI 211 g 263.8 Nigerian Cattle Group Mean Parasitological Data Analysis Group Wt. ofPcrtal Fluke No. %Protection Lymph Nodes Recovered I 267g 0 0 100 II 232 g 550.4 44.96 lil 203 g 477.6 52.24 IV 270 g 7 99.30 V 196g 534.2 46.58 VI 203g 529.8 Haematological Observations Blood samples were taken from all the calves throughout the period of two experiments for the purpose of determining the calves' packed-cellvolumes percentages (PVC) using a Hanksley mic rohaematocrit centrifuge and reader. Since the lay out and performance of the Uganda and Nigeria experiments were the same, it is found necessary to present results of the groups together.

Group I calves in Uganda and Nigeria behaved much in the same way as far as packed-cell-volume percentages are concerned. The mean values of the calves before vaccination were 40%, 4 weeks post vacination they were 38%, and finally the P.C.V. percentages were 40%. Calves of groups II, III and V started with a mean PCV of 35-40% but it declined to 28-32% by 4 weeks post infection when they were autopsied. The calves of group IV had their PCV behaving in the same way as that of Group I throughout the experimental period. Calves of Group VI started with a PCV of 35-37% before they were exposed to the infection. Five months after exposure their P.C.V. was running to between 25% and 20%.Their PCV continued to go down through out the experimental period and the calves became weaker and weaker until their mean PCV was about 9% at 12 months post exposure and they were autopsied.

Faecal Fasciola Egg Examination Generally the method of Bitakarmire (1967) was used to determine egg output. No Fasciola eggs were detected in the faeces of groupes I, II, III and IV calves in Uganda and Nigeria. Group V calves in Uganda became patent 14 weeks post infection whereas those in Nigeria became patent 13 weeks after infection. Faciola eggs per gram of faeces con tinued to be secreted in the faeces of all the calves of the group in Uganda and Nigeria, throughoutthe period of the experiment. Calves of G roup Vl became patent 14 weeks post exposure into the infective paddocks. Their mean faecal egg count started at 10 e.p.g. and rose up to approximately 500 e.p.g. over the 12 months of exposure, up to the time they were autopsied.

Pathological Findings No gross or microscopic lesions were seen in the livers of groups I or IV calves. The only abnormality constantly observed in these calves was gross enlargement of portal lymph nodes.

There was gross enlargement of portal lymph nodes in calves of groups II and Ill. There were haemorrhages and burrows in and underthe liver capsule and in the parenchyma of calves II and III groups. Their liver capsules had been punctured in several places and flukes could be seen crawling on the liver surface.

Portal lymph node gross enlargement in calves of groups V and VI was obvious. Their livers were chronically altered. The bile ducts were seriously enlarged, thickened and fibrosis was observed as if it radiated from their walls, and fibres extended into the parenchyma. Histopathologically several types of liver fibrosis and hyperplasticcholengitis could be seen. The fibrosis could be arranged as follows: (a) Postnecroticfibrosis (b) Ischaemic necrosis and fibrosis (c) Peribiliaryfibrosis (d) Perilobularfibrosis The first two types of fibrosis appeared to be the effect of migration of young flukes through the parenchyma. Peribiliary fibrosis was linked with the presence of parasites in the bile ducts. Perilobular or monolobularfibrosis described the situation where portal canals, were linked by fibrous tissue. This could have been the result of a phlebitis of the portal vein. The presence of lymphoblasts followed by eosinophilic cells could have been due to the presence of an immuno-mechanism. Another contribu tion to the possible presence of immuno-mechanism phenomena in the livers of these calves is the increasing population of plasma cells, lymphocytes, macrophages, eosinophilic cells, mast cells and globule leucoeytes in the biliary mucosa.

CONCL USION The results obtained from field experiments conducted in Uganda and Nigeria on vaccination of cattle against bovine fascioliasis using gammairradiated metacerca riae of Fasciola gigantica have encouraged us in an effort to try and produce "Bovine Fascioliasis Vaccine". There is every indication that the experiments were successfully conducted and the vaccinated cattle were protected from the disease.

Claims (8)

1. A process for producing a fascioliasis vaccine which comprises breeding Lymnaea natalensis snails successively in a series of breeder tanks until theirfirst-generation descendants have been produced in each individual breedertank,transferring these young snails from their respective breeder tank to one or more ancillary cultivation tanks, infecting these young snails with Fasciola gigantica miracidia, growing the infected young snails in the cultivation tank or tanks until they shed Fasciola gigantica cercariae, which encystto become metacercariae, collecting said metacercariae and subjecting them to gamma irradiation from a radioactive source to reduce substantially the pathogenic character of flukes excysting from said metacercariae in the gut of an animal to which said metacercariae have been administered relative to flukes so excysting from non-irradiated metacercariae but without substantially altering the immunogenic character of said metacercariae and collecting said irradiated metacercariae to constitute the effective ingredient of said vaccine.

2. A process as claimed in claim 1 wherein the metacercariae are subjected to a uniform gamma radiation dosage of from 2500 to 3500 rads to destroy the pathogenic character of flukes excysting from them in the gut of an animal to which they have been administered.

3. A process as claimed in claim 1 or 2 wherein the metacercariae are encapsulated in an administrable form in a sealed capsule priorto irradiation.

4. A process as claimed in any of claims 1 to 3 wherein the radioactive source is a cobalt-60 or caesium-137 source.

5. A process as claimed in claim 1 and substantially as hereinbefore described.

6. Fascioliasis vaccine produced by a process as claimed in any of claims 1 to 5.

7. A process for immunizing cattle aga inst fas- cioliasis wherein the cattle are injected with a fascioliasis vaccine produced by a process which comprises breeding snails of the species Lymnaea natalensis, infecting said snails with Fasciola gigantica miracidia, growing the infected snails until they shed Fasciola gigantica cercariae, which thereupon encystto become metacercariae, collecting said metacercariae, subjecting said metacercariae to gamma radiation from a radioactive source to reduce substantially the pathogenic character of flukes excysting from said metacercariae in the gut of an animal to which said metacercariae have been administered relative to flukes so excysting from non-irradiated metacercariae but without substantially altering the immunogenic character of said metacercariae, and collecting said irradiated metacercariae to constitute the effective ingredient of said vaccine, and are subsequently allowed to graze outdoors under conditions where they are exposed to the risk of Fasciola gigantica infection.

8. A process as claimed in claim 7 wherein a vaccine as claimed in claim 6 is used.