WO1999066953A2 - Coccidiosis vaccine, method for the production thereof, and the use thereof - Google Patents

Abstract

The invention relates to a vaccine containing inactivated, immunogenic sporocysts of pathogenic Eimeria spp. which is suited for combating coccidiosis.

Description

Coccidia vaccine, process for its preparation and its use

To control coccidiosis, coccidiostatic feed additives are essential during the rearing and fattening period, especially in the factory farming of poultry. Due to the permanent, long-term use of the coccidiostatics of the polyether-antibiotic type that are frequently used today, numerous polyether-resistant egg series strains have developed over time. So-called rotation and shuttle programs are used today to avoid a persistence of resistance for a longer period of time, the aim of which is the mutual and temporarily limited use of a coccidiostat. The problems with the use of veterinary drugs in the feed of farm animals, the associated residue problems, the availability of only a few new active substances and the increasing

Resistance problems in the commonly used polyether antibiotics have changed the role of chemotherapy. An alternative is the control of coccidiosis via vaccination (Shirley (1992) B.vet J. 148, 479). Surviving a coccidia infection induces significant immune protection in the host compared to subsequent infections. The level of immunity can be divided into 3 levels:

1. Complete immunity; there is no development of ingested oocysts.

2. partial immunity; oocysts are excreted after infection, but no lesions occur.

3. Despite severe lesions in the affected part of the intestine, there are no clinical signs. Immunity is largely species-specific; Cross immunity has been demonstrated between E.tenella, E.necatrix, E.acervulina and E.maxima, but is at most half as effective as against its own species (Boch and Supperer 1992). The level of immunity varies depending on the type of bucket, the level of infection and is very variable even within the population of a particular breed of chicken. In addition to individual variability, the strongly fluctuating susceptibility of the animals is also important (1995, COST 820, Vaccines against animal coccidioses), with intraepithelial T-lymphocytes (CD4 or CD8 subpopulations) of the intestinal mucosa playing a special role. T cell deficient animals are unable to immunity to Eimeria spp. to develop; however, bursectomized animals can still control infection. In the case of bursa disease, immunity training is considerably impaired; this indicates that antibodies are also involved to some extent in building immunity (M. Wallach et al. 1995, Parasitology Today, vol. 11, No. 7). In particular, primarily invasive stages such as sporozoites or early ones

Developmental stages such as Merozoiten (S.Jeurissen et al. 1996, Veterinary Immunology and Immunopathology 54,23 1-23 8) are involved in the build-up of immunity according to recent studies. 4 live vaccines, which are composed of vital oocysts, are currently used for immunoprophylaxis. Live vaccines such as Coccivac® and Immucox® contain virulent strains of the most important, pathogenic egg series, however, in Paracox® and Livacox®, attenuated Eimeria strains are selected for early maturation, which, depending on the species, lack the second, third or fourth generation of schizones. The vaccine oocysts are applied via the drinking water, but has the disadvantage that the correct dosage is difficult because the oocysts sediment quickly. This can lead to the onset of clinical coccidiosis if individual animals cannot build up resilient immunity due to an insufficient vaccine dose. For this reason, attempts have recently been made to apply vaccine oocysts encapsulated in polymers via feed (for example Immucox in the form of a "gel sausage"). The production of live vaccines is labor intensive and expensive, since a complex quality control must rule out the return mutation to the wild type, particularly in the case of attenuated strains. These disadvantages are associated with a inadequate economy compared to the performance promotion of the polyether antibiotics during the fattening period are responsible for the low acceptance in the fattening poultry industry.

Dead vaccines such as genetically engineered subunit vaccines are currently not available. This type of vaccine is based on protective, recombinant proteins that can be produced relatively easily and inexpensively in the fermenter with high and constant quality. A crucial disadvantage of such vaccines is the still lacking, effective and resilient immune protection. Due to the complexity of the parasite-host interactions combined with a lack of knowledge of the protective immune mechanisms that take place during an infection, the availability of subunit vaccines is not expected in the next few years.

The disadvantages described in combating coccidiosis can be overcome by using the vaccine according to the invention which inactivates immunogenic sporocysts of pathogenic Eimeria spp. contains. The vaccine according to the invention can be used to combat parasites in various animals; it is preferably used for immunoprophylaxis against coccidiosis in domestic chickens, turkeys, goose, rabbits and cattle. The parasites particularly suitable according to the present invention are selected from the following group: pathogenic Eimeria spp. of domestic chicken (E.tenella, E. acervulina, E. maxima, E. brunetti, E. mitis, E. necatrix, E. practecox) turkey (E. adenoides, E. meleagrimitis, E. meleagridis, E. dispersa, E.gallopavonis), goose (E. truncata, E.anseris, E. nocens, E. kotlani), rabbit (E. flavescens, E. intestinalis, E. magna, E. stidea) and bovine (E. bovis, E.zuemii, E. alabamensis).

A preferred embodiment of the vaccine according to the invention is characterized in that the sporocysts are encapsulated in an enteric and intestinal juice-soluble or body juice-soluble and mucoadhesive polymer, which can dissolve depending on the pH of the environment and a targeted, local release of the inactivated but enables immunogenic parasites to trigger relevant immune mechanisms, preferably in the intestine.

The vaccine according to the invention can contain, together with the sporocysts, encapsulated adjuvants and / or protective auxiliaries. Suitable adjuvants are, in particular, adjuvants customary in immunology; Biodegradable carriers or microparticles (microparticles, preferably with a diameter of 0-2-10 μm), which are loaded and / or coated with mucosal adjuvants (e.g. cholera toxin or subunit A or B of cholera toxin or heat-labile E. coli enterotoxin) are of particular importance. Preferred carriers or microparticles are e.g. constructed from biopolymers or synthetic biodegradable polymers; polyelectrolyte complexes, stearyl alcohol, docosanol, behenic acid, poly-e-caprolactone or polylactide-co-glycolide are particularly suitable. Auxiliaries customary in immunology are suitable; are particularly important as protective auxiliary substances, e.g. Protective proteins and / or carbohydrates are particularly preferred e.g. Bovine serum albumin, ovalbumin, galactose or trahalose.

Suitable encapsulation polymers are polymers which, in the process of spray drying, lead to an encapsulation which is enteric and enteric-soluble. Particularly suitable products are the pH-controlled Eudragit

Arcyl polymers (Eudragit®, manufacturer: Rhöm GmbH Darmstadt). The methacrylic acid copolymers Eudragit L1OO-55 (methacrylic acid / ethyl acrylate copolymer, USP / NF: "methacrylic acid copolymer type C"), Eudragit L100 (methacrylic acid methyl methacrylate, USP / NF: "methacrylic acid copolymer type A" are very particularly preferred. ), Eudragit S100 (methacrylic acid-methyl methacrylate, USP / NF: "Methacrylic Acid Copolymer Type B", Eudragit L100 / S100, Eudragit L30 D-55 (aqueous dispersion of a methacrylic acid-ethyl acrylate copolymer, USP / NF: "Methacrylic Acid Copolymer Type C ").

The method for producing the vaccine according to the invention, which also belongs to the subject of the present invention, is characterized in that Oocysts are cultivated in the host animal, isolated from the faeces of the infected animals, surface sterilized and broken up and the obtained and sporocysts are inactivated or spray-dried in an enteric acid-soluble and intestinal juice-soluble or body-juice-soluble, muco-adhesive polymer, where appropriate together with the Sporocyst adjuvants and / or protective adjuvants are encapsulated.

The vaccine according to the invention is preferably produced using cleaned and surface-sterilized oocysts. These may have previously been propagated, for example, in the domestic chicken, turkey, goose, rabbit or cattle after the animals have been infected accordingly. For the mass production of the oocysts, monocular, virulent Eimeria strains are preferably used by single oocyst infection, the identity of which was determined by isoenzyme electrophoresis. The propagation is preferably carried out in isolation houses to avoid contamination with other types of buckets. Depending on the age of the animals, the infection dose is between 100-1000 oocysts per animal. The oocysts excreted during the patent period are preferably enriched from the faeces using flotation processes, washed and e.g. sporulated in potassium bichromate solution. Until use, the sporulated oocysts are stored at a low temperature, preferably about 4 ° C. Then the oocysts are surface sterilized - for example using Clorox® (sodium hypochlorite solution) - and broken up, for example with the help of rotating glass beads. Cell debris and oocyst walls can be separated using methods known in the art, for example using Percoll® density gradient centrifugation. Highly pure, sterile sporocyst suspensions are preferably produced.

The sporocysts obtained can be used to produce the actual vaccine. For encapsulation, they are mixed with suitable polymers (if appropriate dissolved in solvent or in a suitable, for example aqueous suspension). The sporocysts can also be inactivated by conventional methods and further processed into vaccines by the prior art methods.

It may be advantageous to add adjuvants to the vaccine according to the invention. This can be done by admixing the adjuvants with the sporocysts obtained. To avoid severe denaturation of the parasite, it can be helpful to add additives to the mixture. The sporocysts of the vaccine according to the invention can be inactivated by methods customary in veterinary vaccinology; spray drying is preferred.

The mixture obtained as described above is preferably in turn encapsulated with inactivated or - preferably not inactivated - sporocysts, preferably by mixing with organic solutions or aqueous suspensions of suitable polymers, preferably Eudragit-acrylic polymers, and spraying in a spray drying system.

The parasites are inactivated in particular by the process of spray drying and are no longer infectious after application to the target animal; the proven immune protection proves that the inactivated parasites maintain their immunogenicity.

The vaccine according to the invention can be administered orally in the feed either alone or in combination with other active ingredients. Preferred additional active substances are chemotherapeutic agents against coccidia, the products Sacox® (salinomycin, manufacturer: Hoechst Roussel Vet GmbH, Wiesbaden) and / or Stenorol® (halofuginone) are particularly preferred. Furthermore, the vaccine according to the invention can also be administered parenterally via subcutaneous, intramuscular or intra peritoneal application. The dead vaccine so produced is applied in a dose of 1 - 1,000 ppm in the feed. For parenteral administration, the vaccine is suspended in buffer systems whose pH is at least 1 pH unit below the pH solubility profile of the polymer used. The dose for parenteral administration is 0.01-100 mg of the vaccine formulation per chicken.

The present invention is intended to be explained in more detail by the following exemplary embodiments and by the content of the patent claims.

Example 1: Mass production of Eimeria oocysts (applies to all Eimeria spp.)

The faeces of 100 animals are collected daily in the 4% potassium bichromate between the 4th and 10th day after infection and stirred in 2 L containers for 15 min until homogeneous. Then 350 g of NaCl are slowly added and the mixture is stirred so that the salt has dissolved in 15 minutes. Coarse constituents are removed by filtering the homogeneous droppings suspension through vertically superimposed sieves with decreasing porosity (2 mm, 1 mm, 0.5 mm). The droppings suspension is then centrifuged for 5 min at 2540 g. The floated oocysts are suctioned off, filtered through gauze again and the identity of the oocysts is microscopically checked. 10 ml of the oocyst suspension obtained is diluted with 40 ml of distilled water and centrifuged at 24OOg for 3 min. The oocyst pellet is washed twice with distilled water and finally resuspended in 4% potassium bichromate. For sporulation, the oocyst suspension is aerated for 72 hours at room temperature. It can then be stored at 4 ° C.

Example 2: Sterilization of sporulated oocysts (applies to all Eimeria spp.) 50 ml of the oocyst suspension is centrifuged for 3 min at 2400 g, resuspended in 200-300 ml of a sodium hypochlorite solution (Clorox) and incubated for 20-30 min with moderate stirring. The oocyst suspension is then centrifuged at 24OOg for 3 min and floating oocysts are suctioned off. 10 ml of the floated Oocyst suspension are diluted with 40 ml of distilled water and centrifuged; the oocyst pellet is washed 3 times with water.

Example 3: Extraction and purification of the sporocysts (applies to all Eimeria spp.)

50 ml of the surface-sterilized oocyst suspension are centrifuged for 3 min at 24OOg and the resulting oocyst pellet is washed twice with Hanks solution; the pellet is then resuspended in 15 ml Hanks solution. The oocyst walls are broken up with rotating glass beads (0.7-1.2 μm diameter) in thick-walled reagent tubes; the efficiency of the physical digestion is checked microscopically. The released sporocysts are separated from the glass beads and then layered on a 65% isoosmotic Percoll solution; the mixture is then centrifuged at 39800 g for 20 min. The sporocyst band is isolated and Percoll removed by washing 3 times with Hanks solution (3 min, 25OOg). The purity of the sporocyst suspension is checked microscopically.

Example 4: Encapsulation of sporocysts via aqueous Eudragit dispersions (applies to all Eimeria spp.)

1 ml of a sporocyst suspension (10 ⁸ sporocysts / ml) is mixed with 8.3 ml of the aqueous Eudragit dispersion L30 D55 (30% dry matter content) and 41.7 ml of ultrapure water (Milli Q water quality). This suspension is sprayed using a mini spray dryer (Büchi 191). The inlet temperature is set to 40-80 ° C, the aspirator to 70% of the maximum output. The nitrogen flow is 600 1 / h and the pump speed is 5% of the maximum output. 820 mg of encapsulated material are isolated.

Example 5: Encapsulation of sporocysts via methanolic Eudragit solution (applies to all Eimeria spp.) 2.5 g of Eudragit S 100 are dissolved in 50 ml of methanol. 1 ml of a sporocyst suspension (10 ⁸ sporocysts / ml) is dispersed in this solution. This dispersion is sprayed immediately using a mini spray dryer (Büchi 191). The input temperature is set to 60 ° C and the aspirator to 70% of the maximum output. The nitrogen flow is 600 l / h and the pump speed is 10% of the maximum output. 1.1 g of encapsulated material are isolated.

Example 6: Encapsulation of sporocysts and protective protein via methanolic Eudragit solution (applies to all Eimeria spp.) 2.5 g of Eudragit S 100 are dissolved in 50 ml of methanol. An aqueous 10% bovine serum albumin solution is added to 1 ml of a sporocyst suspension (10 ⁸ sporocysts / ml). The sporocyst suspension is then introduced into the methanolic Eudragit solution. This dispersion is sprayed immediately using a mini spray dryer (Büchi 191). The inlet temperature is set to 60 ° C, the aspirator to 70% of the maximum output. The nitrogen flow is 600 l / h and the pump speed is 10% of the maximum output. 1.1g of encapsulated material are isolated.

Example 7: Encapsulation of sporocysts and protective protein via an aqueous Eudragit dispersion (applies to all Eimeria spp.)

An aqueous 10% bovine serum albumin solution is added to 1 ml of a sporocyst suspension (10 ⁸ sporocysts / ml), which is then mixed with 8.3 ml of the aqueous Eudragit dispersion L30 D55 (30% dry matter content) and 41.7 ml of ultrapure water (MilliQWasser quality) ) are mixed. This suspension is sprayed using a mini spray dryer (Büchi 191). The

The input temperature is set to 40-80 ° C, the aspirator to 70% of the maximum output. The nitrogen flow is 600 l / h, the pump speed 5% of the maximum output. 1g of encapsulated material are isolated.

Example 8: Encapsulation of sporocysts, protective protein and mucosal adjuvants via aqueous Eudragit dispersion (applies to all Eimeria spp.) WO 99/66953 ^< | Q PCT / EP99 / 04368

An aqueous 10% bovine serum albumin solution is added to 1 ml of a sporocyst suspension (10 ⁸ sporocysts / ml), which is then loaded with microparticles (diameter 1-1 μm), loaded with cholera toxin or the subunit A or B of cholera toxin, is mixed. This mixture is again mixed with 8.3 ml of the aqueous Eudragit dispersion L30 D55 (30% dry matter content) and 41.7 ml of ultrapure water (MilliQ water quality), which is then sprayed in a mini spray dryer (Büchi 191). The inlet temperature is set to 60 ° C, the aspirator to 70% of the maximum output. The nitrogen flow is 600 l / h, the pump speed 5% of the maximum output. 1.2g of encapsulated material are isolated.

Example 9: Detection of a protective effect after oral application of the dead vaccine

Chickens of the Leghorn breed, free from coccidia, were vaccinated orally with the dead vaccine at the age of 7 days; the duration of the vaccination was 3 days. The total dose was administered in 6 single doses via gavage; 2 single doses were applied per day of treatment. In order to check the inactivation of the dead vaccine, the faeces of the vaccinated animals were checked for oocysts from the δ day to the 10 day after vaccination in order to rule out that immunity is built up by a natural infection. 3 weeks after the vaccination, the animals were infected with sporulated oocysts from Eimeria tenella; a non-vaccinated animal collective served as infection control. The individual total oocyst excretion on days 5 to 10 after infection was assessed as a parameter for assessing the protective effect

Comparison compared to the non-vaccinated infection control. The reduction in oocyst excretion in the vaccinated group after vaccination with Eudragit S 100 microcapsules was 48 to 80% depending on the dose. The biological property is the broad protection against infections with pathogenic Eimeria spp. outstanding of the domestic chicken.

Claims

WO 99/66953 "| - | PCT / EP99 / 04368 patent claims 1. Coccidia vaccine, characterized in that it inactivated, immunogenic sporocysts of pathogenic egg spp. contains. 2. Vaccine according to claim 1, characterized in that the sporocysts are encapsulated in a gastric juice-resistant and intestinal juice-soluble or body-juice-soluble, muco-adhesive polymer. 3. Vaccine according to one of claims 1 or 2, characterized in that the sporocysts have been inactivated by the process of spray drying. 4. Vaccine, according to one or more of claims 1 to 3, characterized in that the sporocysts together with one with mucosal Adjuvants coated carrier or microparticles have been encapsulated. 5. vaccine, according to one or more of claims 1 to 4, characterized in that the sporocysts have been encapsulated together with protective auxiliaries. 6. Vaccine according to one or more of claims 1-5, characterized in that it has been formulated with adjuvants and / or customary auxiliaries. 7. A process for the preparation of a vaccine according to one or more of claims 1-6, characterized in that oocysts are cultivated in the host animal, isolated from the faeces of the infected animals, surface sterilized and broken up and the sporocysts obtained are inactivated or by spray drying in a gastric juice-resistant and intestinal juice-soluble or body juice-soluble, muco-adhesive polymer, where necessary encapsulated with the sporocysts adjuvants and / or protective adjuvants. 8. Use of a vaccine according to one or more of claims 1-6 for combating and / or prophylaxis of coccidial infections. 9. Performance-enhancing agent, containing a vaccine according to one or more of claims 1-6, optionally in addition to one or more other active ingredients. 10. A method for producing a performance-promoting agent according to claim 9, characterized in that a vaccine according to one or more of claims 1-6, if appropriate, are brought into a suitable dosage form with one or more further active ingredients.