1. Definition of vaccine

Immune protection against animal diseases can be obtained actively by applying vaccines or passively by the use of serum. The application of vaccines needs the organic reaction which is conditioned by the characteristics of the vaccine, the application method and organic circumstances of the immunized animal.

Vaccines are prepared antigens to be used to prevent diseases caused by bacteria, viruses, fungi, mycoplasma, protozoa and parasites.

The type of vaccine is established depending on the type of antigen, which determines the method of elaboration.

In this article we will describe the aspects that influence on vaccine characteristics (antigens, inactivators, preservatives, excipients, adjuvants and elaboration and distribution processes), application (route and contraindications) and factors related to the animal organism that receives the vaccine.

2. Components and types of vaccines

• Components
  

  Veterinary vaccines are composed of active ingredients (antigens) and other components which affect the velocity of liberation (adjuvants) or are used to obtain proper forms (inactivators, preservatives and excipients).

  • Antigens
    

    The essential component of any vaccine is the antigen which consists of biologic units, usually proteins or infectious agents, and produce, when in contact with animals, the formation of antibodies, substances of protein nature, capable to react especifically against its antigen.

    • Non-modified live vaccines: They are biological units whose structure has not been modified. They basically provoke the disease under control. The use of this kind of antigen is frequent in antiprotozoal vaccines and avian viral vaccines. The former’s efficacy depends on the quantity and the frequency of application. One example of this type of vaccines is the one used to prevent avian coccidiosis caused by Eimeria. An example of the latter are the vaccines with turkey herpesvirus used to vaccinate hens against Marek’s disease.
    • Attenuated live vaccines: They are biological units that have been intervened by reducing their pathogenicity by successive passes in animals or cultivating microorganisms in swine reproductive conditions. The degree of attenuation is directly and proportionally related to the number of attenuation passes. These attenuation methods are mainly used on viruses such as avian infectious bronchitis (ovoculture) and swine plague virus (lapinized).
    • Modified or recombined live vaccines: They are genetically modified biological units to avoid the expression of some proteins or to express others that otherwise would not be expressed. In the first case, the objective is to obtain irreversibly attenuated live antigens , fractions that do not interfere with diagnostic techniques, or both characteristics at the same time (Aujeszky disease virus).Modified live antigens do not present, in the short-term, the possibility to become pathogenic, but, in the long-term, they can become pathogens by natural recombination. The recombined live antigens present advantages from the industrial point of view as they use bacteria and yeasts that are easier to cultivate than the donor microorganism, which generally have a viral origin. The vaccine with myxomatosis virus of rabbit, which has received genetic material from the haemorrhagic enteritis virus, is an example of a recombined vaccine.
    • Inactivated vaccines: They are biological units, with some toxins, that were killed by the action of physic, chemical agents, or both at the same time. They preserve part of the chemical structure that can cause an immune response. Bacterins and toxoids correspond to this type of antigen, which is the most secure type of antigen.
    • Subunits: They are fragments of biological units that, due to their composition, can provoke immune response. Generally, these subunits are subsequently inactivated. Some components in these subunits are eliminated despite activating theimmune response, as they can interfere with the subunit. This kind of antigen is frequent in vaccines for swine atrophic rhinitis and colibacillosis.
    • Synthetics: They are units or subunits obtained by chemical synthesis. Their disadvantage is that the three-dimensional disposition of aminoacids is not identical to the original antigen; therefore their immune response isn’t identical either.
    • Anti-idiotypes: They are obtained by biological mediums due to they lack synthetic antigens. They are biological units obtained from the secondary antibody created from the primary antibody of the antigen. The original antigen is inoculated, and the produced antibodies are called idiotypes. When this idiotypes are inoculate in another animal, some antibodies are obtained (anti-idiotype), whose three-dimensional structure is identical to the one of the natural antigens. Their elaboration is difficult, but has the advantage of acting immunologically as alive microorganism, but it is not a microorganism.

  • Adjuvants
    

    They are chemical substances, materials of microbiological origin or mixtures that, administered together with the antigen, contribute to the production of a greater immune response.

    • Aluminium and calcium salts: They delay the release of antigens to obtain longer immune responses. The most common salts are calcium phosphate, aluminium phosphate and hydroxide.
    • Immunostimulants: They increase the function of the immune system or help to recover it. They can be made of chemical or biological materials.
      • Bacterial fractions
      • Vegetable substances
      • Chemicals
    • Freund’s adjuvant is an emulsion of water-oil and complete mycobacterium or a fraction called muramyl dipeptide.
    • Liposomes

  • Inactivators
    

    Substances that eliminate the pathogenic capacity of the antigens. They can be used alone or combined with the action of physical agents, such as heat.

    • Formalin: The most frequently used inactivator in the elaboration of anatoxins. Its maximum quantity in the final product is limited by the Pharmacopoeia to avoid negative effects on animal tissues.
    • Alkylation: Substances that lead to the formation of bonds between nucleic acid. As they react with the superficial proteins of the microorganism, this one keeps its antigenic power. The most common alkylating substances are ethylene oxide, beta-propiolactone and ethyleneimine.

  • Preservatives

    Substances that, incorporated to inactivated vaccines, avoid contaminations during the handling in the farm.

    • Phenol is the most common preservative in the elaboration of inactivated vaccines. Its content in the final product is limited by the monographies of the vaccines described in Pharmacopoeia.

  • Excipients
    

    Liquid or solid diluents that allow to obtain the concentration of antigens in the final product  to simplify the doses.

    • Physiologic serum
    • Skimmed milk

• Types of vaccines
  

  • Live vaccines
    

They are elaborated with non-modified live antigens, attenuated live antigens and modified live antigens. This kind of vaccine provides fast immunity, but its use is only recommended in contaminated environments.

• • Dead vaccines

They are elaborated with inactive antigens. They cause slow immune reactions  and most of them require the incorporation of a coadjuvant. They are indicated in healthy environments because they are safe.

• • Design vaccines

They are elaborated with subunits, synthetic antigens and anti-idiotypes.

3. Elaboration and control of veterinary vaccines

The elaboration of a veterinary vaccine is a complex procedure based on many steps and variables depending on the antigen used. The work procedures must be adjusted to the GMP international recommendations and the quality of raw materials described by European Pharmacopoeia, National Pharmacopoeia and the recognized technical profile.

The construction and materials of facilities and equipment used in the elaboration of veterinary vaccines must follow the European and National rules. The rule 81/851/CEE and the Real Decree 109/1995 constitute the existing legal body regulator of the requirements that these facilities must accomplish.

Regarding the constructions, the following must be considered:

• The circulation of vaccine components must go in a direction that avoids the contact between ingredients, intermediate products and final products.
• The circulation of people and their clothing must also go in a determinate direction to avoid crossings.
• Air circulation and quality must avoid the entrance of pollution from the outside. The packing is must be done under sterile conditions and positive pressure.
• Cleaning and desinfection must avoid the formation of convex right angles of difficult access.

Regarding the materials, the following must be considered:

• To avoid the filtration of microorganisms and organic matter, material cannot be porous.
• Easily washable.

Facilities will be divided into the following parts: raw material warehouse, area for the elaboration of intermediate products, mixing area, packing, quarantine warehouse, laboratory of analysis, labelling area, final product warehouse and expedition area. Equipment must be made of glass or stainless steel and be of easy access and cleaning.

As didactic purposes, we will briefly describe the steps to produce a bacterin or an inactivated bacterial vaccine:

• Elaboration of manufacturing sheet: it consists of a sheet with the quantities and identification codes of all materials used . This sheet must accompany the ingredients during the entire process.
• Preparation of starting materials: it takes place in the raw material warehouse with products that have already been analyzed and declared suitable because they adjust to the characteristics mentioned in the European Pharmacopoeia and their technical profile. In the case of vaccines, there is an exceptional material called working seed. It is a culture prepared in the microorganism warehouse.
• Elaboration of intermediate products: intermediate products can be defined as those mixtures of starting materials that constitute parts of the elaboration of the final product. The suspension of microorganisms constitutes the main intermediate product in the elaboration of a bacterin. It consists of microorganisms produced by fermentation from the inoculum of the working seed in large fermenters. The content of these fermenters is concentrated through tangential filtration. Both the suspension of the microorganisms and the solution of toxins are inactivated using the inactivator products mentioned above (formalin and others) and storaged under refrigeration until they are used in the elaboration of thefinal product.
• Elaboration of the final product: it is done by mixing of several intermediate products (microorganisms and toxins) with starting materials such as adjuvants, preservatives, and excipients. The product obtained is packaged under a sterile atmosphere, in sterile flasks that are blocked, sealed and stored in the quarantine area.
• Once the final mixture is done, it must be analyszd before its release to the market. For this reason, a refrigerated warehouse (4-8ÂşC) will be necessary.
• Quality control for final products: the controls on final products must be conduced according to the monograph of the product described in the European Pharmacopoeia, National Pharmacopoeia and the internationally recognized technical profile. Despite of the existence of particularities, the final product must be subjected to the following general controls:
• Physico-chemical characteristics.
• Power: it consists of administering vaccine doses to a group of animals and, subsequently, confront it with the microorganisms of the antigen, but the latter are alive and pathogen. If the vaccine has the necessary power, animals will survive.
• Safety and abnormal toxicity: it consists of administering a dose of vaccine higher than recommended to a batch of animals. Animals should not present symptoms of disease and local reactions during a period of observation should be determined.
• Direct validity and validity after reconstitution: this process is compulsory and takes place during the process of registration of the specialty, but it is also advisable to perform it sporadically in manufacturing batches or when, because of technologic reasons, some stage or material has been modified. The validity tests are used to determine for how long vaccines can maintain the power under determinate temperature and light conditions.
• Security and absence of strange pathogenic agents.

4. Labeling of veterinary vaccines

Once  the aptitude of the product has been confirmed by the analysis described in early chapters, the product is labelled and provided with informative material for  its right application. The analytic and performance data of the manufacturing process are added to the manufacturing sheet. The manufacturing sheet, together with the tests, is stored for at least 3 months after the expiry date of the manufactured batch. The final product is sent to the warehouse of final products until its distribution.

5. Final product warehouse and expedition area

We can underline that both areas must count with refrigeration and there should be an automatic or manual system to know when, how and where every single manufactured batch has been sent. This information is very valuable in case of detecting adverse reactions of the product once it has been released to the market.

6. Vaccine distribution

Heat and sunlight make vaccines lose efficacy. The distribution of veterinary vaccines is regulated by the Real decree 109/1995, which obliges to dispose of adequate places as well as the services of a pharmaceutical technical director.

Refrigeration within 2-8ºC is usually necessary, but they cannot be frozen, except for some specific vaccines where the freezing in liquid nitrogen or dry ice (Marek’s disease) is necessary.

Apart from the corresponding cold chain, it is necessary to monitor the inputs and outputs of the product batches through the corresponding document registrations.

7. Administration

Veterinary vaccines are administered in different routes, depending on the type of antigen and the breeding system of the targeted animals.

• Oral route

This route is used in live vaccines intended for birds. This practice permits a massive vaccination, as it is really fast and does not need many personnel to add the antigen to the drinking water. Only a few precautions are necessary, such as having drinking water restricted for 10-12 hours prior to antigen adminsitration, using water without disinfectants, and dissolving skimmed milk in powder with the antigen in order to increase its viability.

• Air route

It is frequent in viral and bacterial live vaccines intended for birds and swine. It permits a massive vaccination, although it requires proper premises and equipment in order to produce nebulization. The size of the droplets is important, as they must be suspended enough time in the air to be breathed in by animals.

• Intradermal route

It is used for any type of administration of antigens, especially for inactivated ones. This route requires more personnel and time than the above-mentioned routes, and causes discomfort to animals. On the contrary, it guarantees the most adequate administration to every animal. Syringes used must not be disinfected with alcohol or other disinfectants, as they may inactivate live antigens. We must pay particularly attention in vaccinations without needle, to make sure they are properly administered in the tissue. Among the most used, we can find the subcutaneous and intramuscular although some vaccines are administered by specific ways, such the intravenous one.

• External route

This way of administration is used for live viral antigens in nasal mucosa and eye conjunctiva.

8. Factors related to the organism that may affect immunization

• Before the administration

It is necessary to plan the manipulation of animals in order to avoid unnecessary discomfort. It is also necessary to know the pathological antecedents of population or individuals who will be vaccinated. Vaccination of pregnant females during the first month or birds during of the laying peak must be avoided.Administration of vaccines is not intended for ill animals, with fever, treated with corticosteroids or those who are receiving immunosuppressor medicines. Vaccines with strains of herpesvirus are dangerous because of their latent infectious power, as one vaccine can be apathogenic for a determinate age but can trigger a disease in younger animals.

• During the vaccination

The vaccine must be properly administered. The following factors should be considered: dose, administration route, and homogeneity (all animal must receive the same dose of the vaccine).

• After the vaccination

Keep the animals in mild environmental conditions.