DE19828322A1 - New coccidiocide vaccine comprises inactivated, immunogenic sporocytes of pathogenic Eimeria species - Google Patents

Abstract

A coccidiocide vaccine (I) comprising inactivated immunogenic sporocytes (II) of pathogenic Eimeria species, is new. An Independent claim is also included for preparing (I).

Description

To control coccidiosis are especially important in the factory farming of poultry Coccidiostatically effective feed additives during the rearing and fattening period mandatory. Through the permanent, long-term commitment of today Frequently used coccidiostatics of the type of polyether antibiotics have become Numerous polyether-resistant egg series strains developed over time. For Avoiding a longer lasting persistence of resistance today so-called rotation and shuttle programs applied, one mutual and temporarily limited use of a coccidiostat to the goal to have. The problems with the use of veterinary drugs in feed agricultural livestock, the associated residue problems, the Availability of only a few new active ingredients as well as the increasing Resistance problems of the commonly used polyether antibiotics play a role chemotherapy changed. An alternative is the control of coccidiosis via vaccination (Shirley (1992) B.vet J. 148, 479).

Surviving a coccidia infection induces a 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 recorded Oocysts.
• 2. partial immunity; after infection, oocysts are excreted, but occur no lesions.
• 3. Despite severe lesions in the affected section of the intestine, no clinical signs occur Appearances.

Immunity is largely species-specific; Cross immunity has been demonstrated between E. tenella, E. necatrix, E. acervulina and E. maxima, which, however, a maximum of half is as effective as against its own species (Boch and Supperer 1992). Of the Degree of immunity varies depending on the type of bucket, the severity of infection and is itself very variable within the population of a particular breed of chicken. In addition to the individual variability is also the strongly fluctuating susceptibility of animals from Meaning (1995, COST 820, Vaccines against animal coccidioses), whereby intraepithelial T lymphocytes (CD4 or CD8 subpopulations) of the Intestinal mucosa play a special role. T cell deficient animals are not in capable of immunity to Eimeria spp. to develop; however, can bursectomized animals still control infection. With a disease of Bursa's immunity training is significantly impaired; this indicates suggest that antibodies to a certain extent contribute to the structure of the Immunity (M. Wallach et al. 1995, Parasitology Today, vol. 11, no. 7). In particular, primarily invasive stages such as sporozoites or early ones Stages of development such as Merozoiten (S.Jeurissen et al. 1996, Veterinary Immunology and Immunopathology 54, 23 1-23 8) are newer Studies involved in building immunity. For immunoprophylaxis Currently 4 live vaccines are used, which are made up of vital oocysts put together. Contain live vaccines such as Coccivac® and Immucox® virulent strains of the most important pathogenic egg series, however, are in Paracox® and Livacox® contain pre-mature, attenuated Eimeria strains, which, depending on the species, lack the second, third or fourth generation of schizonts. The vaccine-Qocysts are applied via drinking water, but it does Disadvantage that the correct dosage is difficult because the oocysts are quick sediment. This can lead to clinical coccidiosis if individual animals have no resilient immunity due to an insufficient vaccine dose can build. Recently, therefore, attempts have been made to vaccine oocysts in Encapsulated to apply polymers encapsulated via feed (e.g. Immucox in the form of a "Gelwurst"). The production of live vaccines is labor intensive and expensive because an elaborate quality control especially the back mutation to the wild type  must exclude 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 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 relatively simple and inexpensive in the fermenter with high and constant Quality can be produced. A crucial disadvantage of such vaccines is the still lacking, effective and resilient immune protection. Due to the Complexity of parasite-host interactions combined with lack of Knowledge of the protective immune mechanisms that occur during a Expect infection is the availability of subunit vaccines in the next Years not expected.

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

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

The vaccine according to the invention can be encapsulated together with the sporocysts Contain adjuvants and / or protective adjuvants. Suitable adjuvants are adjuvants particularly common in immunology; have special meaning biodegradable carriers or microparticles (microparticles preferably with Diameter 0.2-10 µm), which with mucosal adjuvants (e.g. cholera toxin or subunit A or B of cholera toxin or heat-labile E.coli enterotoxin) loaded and / or coated. Preferred carriers or microparticles are e.g. B. built from biopolymers or synthetic biodegradable polymers; polyelectrolyte complexes, stearyl alcohol, docosanol, Behenic acid, poly-e-caprolactone or polylactide-co-glycolide. Suitable as excipients auxiliaries common in immunology; special importance as protective Auxiliaries have e.g. B. Protective proteins and / or carbohydrates. Particularly preferred are z. B. bovine serum albumin, ovalbumin, galactose or trahalose.

As encapsulation polymers are polymers that are used in the process of Spray drying to an enteric-coated and enteric-soluble encapsulation to lead. Particularly suitable products are the pH-controlled Eudragit Arcyl polymers (Eudragit®, manufacturer: Rhöm GmbH Darmstadt). Most notably preferred are the methacrylic acid copolymers Eudragit L100-55 (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 "), 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 is also used for Subject of the present invention is characterized in that Oocysts are cultured in the host animal, isolated from the feces of the infected animals be, surface sterilized and broken up and the obtained and Sporocysts can be inactivated or by spray drying in one gastro-juice-resistant and intestinal-soluble or body-juice-soluble, muco adhesive polymer are encapsulated, possibly together with the Sporocyst adjuvants and / or protective adjuvants are encapsulated.

The vaccine according to the invention is preferably used using cleaned and surface-sterilized oocysts. These can be done beforehand for example in domestic chicken, turkey, goose, rabbit or beef increased after the animals were appropriately infected. For Mass production of the qocysts is preferably by single oocyst Infected cloned, virulent Eimeria strains, whose identity is identified by Isoenzyme electrophoresis was determined. The propagation is preferably in Isolation stalls are carried out to prevent contamination with other types of buckets avoid. 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, washed and e.g. B. sporulated in potassium bichromate solution. Until use, the sporulated oocysts at low temperature, preferably about 4 ° C, kept. Then the oocysts are surface sterilized - for example using Clorox® (sodium hypochlorite solution) - and broken open, for example with the help of rotating glass beads. Cell debris and oocyst walls can be separated using prior art methods, for example using Percol® density gradient centrifugation. Highly pure, sterile sporocyst suspensions prepared.

The sporocysts obtained can be used to produce the actual vaccine be used. For encapsulation, they are made with suitable polymers  mixed (possibly dissolved in solvent or in a suitable, e.g. aqueous Suspension). The sporocysts can also be made using conventional methods inactivate and vaccine according to prior art methods process further.

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 become. To avoid severe denaturation of the parasite, it can be helpful to add additives to the mixture. The sporocysts of the Vaccines according to the invention can be used in veterinary medicine Vaccinology usual methods are inactivated; that is preferred Spray drying.

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

In particular through the process of spray drying, the parasites inactivated and are no longer infectious after application in the target animal; of the Proven immune protection proves that the inactivated parasites are their Maintain immunogenicity.

The vaccine according to the invention can be administered orally in the feed either alone or in Combination with other active ingredients can be administered. Preferred additional Active substances are chemotherapeutic agents against coccidia, those are particularly preferred Products Sacox® (salinomycin, manufacturer: Hoechst Roussel Vet GmbH, Wiesbaden) and / or Stenorol® (halofuginone). Furthermore, the invention Vaccine also parenterally via subcutaneous, intramuscular or intra peritoneal Application can be administered.  

The dead vaccine so produced is in a dose of 1-1000 ppm in the feed applied. The vaccine is used in buffer systems for parenteral application suspended, the pH of which is at least 1 pH unit below the pH Solubility profile of the polymer used is. The parenteral dose Application is 0.01-100 mg of the vaccine formulation per chicken.

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

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

The faeces of 100 animals are daily between the 4th and 10th day after infection collected in 4% potassium bichromate and in 2 L containers for 15 min to Homogeneity stirred. Then 350 g of NaCl are slowly added and so stirred that the salt dissolved in 15 min. Coarse components become removed by the homogeneous droppings suspension by vertically superimposed Filters with decreasing porosity (2 mm, 1 mm, 0.5 mm) are filtered. The droppings suspension is then centrifuged for 5 min at 2540 g. They floated Oocysts are suctioned off, filtered through gauze again and the identity of the Microscopically controlled oocysts. 10 ml of the oocyst suspension obtained is diluted with 40 ml of distilled water and at 2400g for 3 min centrifuged. The oocyst pellet is washed twice with distilled water washed 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, in Resuspend 200-300 ml of a sodium hypochlorite solution (Clorox) and 20-30 min incubated with moderate stirring. The oocyst suspension is then 3 min  2400g centrifuged and floating oocysts aspirated. 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 cleaning of the sporocysts (applies to all Eimeria spp.)

50 ml of the surface-sterilized oocyst suspension are 3 min at 2400g centrifuged and the resulting oocyst pellet 2 times with Hanks solution washed; the pellet is then resuspended in 15 ml Hanks solution. In thick-walled reagent tubes are rotating the oocyst walls Broken glass beads (0.7-1.2 µm diameter); the efficiency of the physical digestion is controlled microscopically. The freed ones Sporocysts are separated from the glass beads and then onto one 6b% iso-osmotic Percoll solution layered; the mixture is then at 39800g centrifuged for 20 min. The sporocyst band is isolated and Percoll removed by washing 3 times with Hanks solution (3 min, 2500g). The The purity of the sporocyst suspension is checked microscopically.

Example 4 Encapsulation of sporocysts via aqueous Eudragit dispersions (applies for 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 (MilliQWater quality). This suspension is mixed 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 pumping speed is 5% of the maximum output isolated.

Example 5 Encapsulation of sporocysts via methanolic Eudragit solution (applies for all Eimeria spp.)

2.5 g of Eudragit S 100 are dissolved in 50 ml of methanol. 1 ml a sporocyst suspension (108 sporocysts / ml). This dispersion is sprayed immediately using a mini spray dryer (Büchi 191). The Inlet temperature is set to 60 ° C and the aspirator to 70% of the maximum Power set. The nitrogen flow is 600 l / h and Pump speed 10% of the maximum output. There are 1.1 g encapsulated Material isolated.

Example 6 Encapsulation of sporocysts and protective protein via methanolic Eudragit solution (applies to all Eimeria spp.)

2.5g Eudragit S 100 are dissolved in 50 ml methanol. To 1 ml of a sporocyst Suspension (108 sporocysts / ml) is an aqueous 10% bovine serum albumin Solution added. The sporocyst suspension is then in the introduced methanolic Eudragit solution. This dispersion is immediately of a mini spray dryer (Büchi 191). The input temperature will rise to 60 ° C, the aspirator set to 70% of the maximum output. The nitrogen flow is 600 l / h and the pumping speed is 10% of the maximum output. It 1.1 g of encapsulated material are isolated.

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

An aqueous solution is added to 1 ml of a sporocyst suspension (108 sporocysts / ml) 10% bovine serum albumin solution is added, which is then mixed with 8.3ml of aqueous Eudragit dispersion L30 DSS (30% dry matter content) and 41.7 ml ultra pure water (MilliQWasser quality) can be 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 performance. The nitrogen flow is 600 l / h, the pumping speed 5% of the maximum power. 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.) An aqueous solution is added to 1 ml of a sporocyst suspension (108 sporocysts / ml) 10% bovine serum albumin solution was added, which was then mixed with microparticles (Diameter 1-10 µm), loaded with cholera toxin or subunit A or B from Cholera toxin is mixed. This mixture is again with 8.3 ml of aqueous Eudragit dispersion L30 DSS (30% dry matter content) and 41.7ml ultrapure water (MilliQ water quality) mixed, which then in a mini spray dryer (Büchi 191) is sprayed. The inlet temperature is set to 60 ° C, the aspirator to 70% of the maximum output. Of the Nitrogen flow is 600 l / h, the pump speed is 5% of the maximum output. 1.2g of encapsulated material are isolated.

Example 9 Proof of a protective effect after oral application of the Dead vaccine

Chicks of the Leghorn breed were coccidia-free at the age of 7 days vaccinated orally with the dead vaccine; the duration of the vaccination was 3 days. The The total dose was administered in 6 single doses via a throat tube; ever Two single doses were applied on the day of treatment. To check the The dead vaccine was inactivated from the 5th day to the 10th day after vaccination the faeces of the vaccinated animals are checked for oocysts 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. As a parameter for The protective effect was assessed individually Total excretion of oocysts on days 5 to 10 after infection in the Comparison compared to the non-vaccinated infection control. The reduction the oocyst excretion in the vaccinated group was after vaccination with Eudragit S 100 microcapsules, depending on the dose, 48 to 80%.  

The biological property is the broad protection against infections with pathogenic Eimeria spp. outstanding of the domestic chicken.

Claims (11)

1. Coccidia vaccine, characterized in that it is inactivated, immunogenic Sporocysts of pathogenic egg spp. contains. 2. Vaccine according to claim 1, characterized in that the sporocysts in a gastro-resistant and enteric or body juice-soluble, muco-adhesive polymer is encapsulated. 3. Vaccine according to one of claims 1 or 2, characterized in that the sporocysts have been inactivated by the spray drying process. 4. Vaccine according to one or more of claims 1 to 3, characterized characterized in that the sporocysts along 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 characterized in that the sporocysts together with protective adjuvants have been encapsulated. 6. Vaccine according to one or more of claims 1-5, characterized characterized in that it is formulated with adjuvants and / or customary auxiliaries has been. 7. A method for producing a vaccine according to one or more of the Claims 1-6, characterized in that oocysts are cultivated in the host animal are isolated from the faeces of the infected animals, surface sterilized and are broken open and the sporocysts obtained are inactivated or by spray drying in a gastric juice-resistant and intestinal-soluble or body juice-soluble, muco-adhesive polymer are encapsulated, where necessary   together with the sporocysts adjuvants and / or protective adjuvants be encapsulated. 8. Use of a vaccine according to one or more of claims 1-6 for the control and / or prophylaxis of coccidial infections. 9. Performance enhancing agent containing a vaccine according to one or several of claims 1-6, if appropriate, in addition to one or more further 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 the Claims 1-6 optionally with one or more other active ingredients in one appropriate dosage form are brought.