Immunology is the branch of biology and biomedical sciences that studies cells, tissues and organs responsible for detecting foreign agents (antigens) and triggering a defensive action.

In natural immune events we find two main protagonists, the immune system and the antigen, while in induced immunity we have to take into account the immune system and vaccines. However, these agents are not uniform or static throughout life evolution, so it is necessary to carefully describe them and their interrelationship.

All this will be exposed as follows:

I. Animal immune system

I.1 Immune system of unicellular animals

I.2 Immune system of invertebrate multicellular animals

I.3 Immune system of vertebrate multicellular animals

II. Types of antigen

III. Types of vaccines

IV. Design, development, control, distribution and application of vaccines

V. Main avian veterinary vaccines

 

I. Animal Immune System

 

I.1 Immune system of unicellular animals

Protozoa are unicellular animals that, from an immune point of view, have a primitive immune defense line based on the cell membrane.

This membrane is composed of a double layer of phospholipids crossed by globular protein formations whose function is to exchange nutrients with the outside. On the surface of the protozoal cell membrane there are glycolipid formations (of oligosaccharides), whose mission is to detect external attacks and mobilize calcium reserves to neutralize the aggressor agent. Some authors described that another mechanism of primitive defense, the production of melanin, which helps to defend the organism against sunrays, can be adopted by unicellular animals as a defense mechanism against biological attacks, since melanin presents a certain degree of toxicity against microbial agents.

In summary, the immune system of unicellular animals has:

1. Barrier mechanism composed of its cell membrane
2. Chemical neutralization mechanism by mobilization of calcium reserves
3. Defense mechanism for the synthesis of toxic substances for microorganisms such as melanin

These mechanisms do not disappear in more complex animals, so each new evolutionary step incorporates new mechanisms without eliminating the previous ones. For example, the calcification process of lesions in organs of birds and mammals, original from the neutralization mechanisms of unicellular organisms, as well as the melanic pigmentation of lesions, which can be found in the production of melanin for the protection against sunrays, adapted from the defense mechanism against biological aggressions in unicellular animals.

 

I.2 Immune system of invertebrate multicellular animals

Invertebrates are multicellular animals without a backbone or notochord, but with an articulated internal skeleton, that constitute the most numerous branch of the animal kingdom that includes: Arthropoda (arachnids, insects, myriapods, crustaceans); Mollusca (clams, squid, octopus, snails); Porifera (sponges); Coelenterata (coral jellyfish, polyps); Echinodermata (starfish, sea urchins); Platyhelminthes (flatworms and parasites); Nematoda (roundworms); Annelids (earthworms, leeches).

To explain the complexity and immunological variability of invertebrates, we must consider their origin: the appearance of multicellular animals is the result of the association of several types of cells; with this, specialization appears and, among specializations, cells that are specialized in defensive functions appear, which are known as coelomocytes, if the invertebrate has a celomic cavity, or hemocytes if it does not. Therefore, coelomocytes and hemocytes are the phylogenetic novelty of the invertebrate immune system.

Similarly, some mechanisms present in protozoa are maintained in invertebrates (calcification and melanization) and new substances appear, such as pentraxins, which can be considered novel in invertebrates and phylogenetic precursors of vertebrate immunoglobulins.

Physicochemical barriers

1. Arthropod exoskeleton composed of a chitin polysaccharide, a polymer formed by straight and simple chains (unbranched) of N-acetyl-2-D-glucosamine, a monosaccharide that includes nitrogen in its composition. This layer is secreted by the hypodermis and can also contain calcium carbonate (crustaceans, crabs, lobsters) and deposits of excretion products that sometimes have an antimicrobial function as in the case of cecropin and drosomycin, isolated in insects.
2. gelatinous such as the mucous secretions of annelids and mollusks composed of mucopolysaccharides that may also contain antibacterial peptides, enzymes (lysozyme), glycoproteins.

Cellular response

1. Phagocytosis based on the activity of the cells or hemocytes already mentioned above. These cells act through 3 processes: chemotaxis (approximation), recognition (adherence) and activation of hydrolases (digestion in their lysosomes). This activity is transmitted throughout the evolution to the macrophages of vertebrates.
2. Encapsulation of pathogens by the formation of a multicellular wrap of coelomocytes and melanin deposits (recall of the defense mechanism of unicellular protozoa).
3. Nodule formation as an evolution of encapsulation.
4. Recognition of own and foreign components exerted by a subpopulation of coelomocytes or hemocytes that would be the precursors of lymphocytes (B and T) in vertebrate multicellular animals
5. Cytotoxicity derived from the lysate of the transformed cells by the coelomocytes or hemocytes. This activity would be the precursor of natural killer (NK) cells in vertebrate multicellular animals.

Preserologic response of soluble compounds

1. Opsonins that cover foreign agents and facilitate phagocytosis.
2. Agglutinins that produce the agglutination of foreign agents. For example, tachilepsin present in the blood serum of Limulus polyphemus.
3. Lysins, including lysozyme, that break foreign structures.
4. Cyclic and linear peptides.
5. Pentraxin-type proteins that can be considered the ancestors of vertebrate immunoglobulins

 

I.3 Immune system of vertebrate multicellular animals

Vertebrates are a subfile of chordates that comprise animals with a backbone or spine composed of vertebrae.

There are seven classes of living vertebrates: Agnathan or fish without jaw (cyclostomes), Chondrichthyan or cartilaginous fish (shark), Osteichthyes or bony fish (trout), Amphibians (urodeles = salamander and anurans = frogs), reptiles (anapsids = turtles, euryapsidans = snakes and archosaurs = crocodiles), birds and mammals (monotremes, marsupials and placentals). In this group, it maintains all the previous mechanisms (protozoa and invertebrates), such as physicochemical barriers and cellular responses, and also a specific response, both with cellular (T and B lymphocytes) and humoral components (antibodies).

 

Physicochemical barriers

Physicochemical barriers are basically constituted by the external epithelium or skin covered with scales, feathers or hairs and the internal epithelia of the respiratory and digestive systems.

Both external and internal epithelia can be provided with cells for the elaboration of defensive secretions such as saliva, mucus, sweat and gastric juice that may be accompanied by enzymes, melanin and acids.

Within these barriers, we can mention the temperature in the birds and the fever in the mammals as a defense reaction against bacterial and viral agents.

 

Nonspecific cell response

Based on the activity of specific cells with phagocytic capacity with increasing specialization in the form of macrophages, eosinophils, basophils, mastocytes, dendritic cells (interdigitating and follicular) and NK cells.

 

Nonspecific serological response

Based on the same preserological responses of invertebrates.

1. Opsonins that cover foreign agents and facilitate phagocytosis (C-reactive protein).
2. Agglutinins that cause the agglutination of foreign agents.
3. Lysins, including lysozyme, that breaks foreign structures.
4. Vertebrates’ molecules such as interleukins and chemokines that promote cell differentiation and chemotaxis.

 

Specific cellular response: tissues, lymphoid organs and lymphocytes

Vertebrate multicellular animals are the only existing animals that, as a consequence of the evolution and cell specialization, have cells capable of recognizing, remembering and responding to the presence of specific antigens, so that the second exposures produce specific responses.

As a consequence of this specialization, vertebrates have developed T and B lymphocytes that are differentiated from coelomocytes or hemocytes by the presence of receptors on their membrane formed by surface immunoglobulins.

Vertebrates have developed specific tissues and organs throughout their phylogeny that synthesize these specialized cells. The first vertebrate stages, the Agnathan (fish without jaw) and bone fish, have lymphoid tissues in the intestinal mucosa; the following stage, cartilaginous fish, maintain lymphoid tissues and develop special organs for the production of T lymphocytes (thymus) and the spleen.

Urodele amphibians maintain lymphoid tissues in the intestinal mucosa, the spleen and the thymus, and develop lymphoid tissues in the bone marrow for the synthesis of B lymphocytes. Anuran amphibians incorporate the lymph nodes that contain B and T lymphocytes. Reptiles maintain the same immune mechanism, but, with the emergence of the thermoregulatory center, animals need to increase their immunological level, since viruses and bacteria develop better in the constant temperature of homeotherms than in the irregular one of poikilotherms.

At this point a bifurcation between birds and mammals occurs:

Birds have body temperatures between 40 and 42°C, standing at the upper end of the mesophilic microorganisms, exerting an antimicrobial effect that makes their immune system evolve differently. For this reason, their primary lymphoid organ is the bursa of Fabricius.

Mammals adopt a body temperature between 36 and 39°C, enough for the control of fungal growth, but ideal for mesophilic bacteria. For this reason, mammals need to develop a general and regional lymphatic system, more complex than that of birds, formed by the structures described before for reptiles and the incorporation of the lumbar aortic lymph node chain, from which, through the suspensor ligaments, specific branches of lymph node chains reach each organ.

Thanks to these new structures, the immune response is faster (essential against mesophilic microorganisms) and more efficient (essential against specific microorganisms of each organic system).

 

Specific serological response

Vertebrates are the only existing animals capable of producing, as said with the cellular response, a specific serological response in the form of specific antibodies synthetized by B lymphocytes. Fish, amphibians and reptiles have structurally simple antibodies called immunoglobulins M (produced only by immature lymphocytes) that are maintained in birds together with new and more complex antibodies, called immunoglobulins A. On the other hand, mammals also produce immunoglobulins D (except rabbits and pigs), E and G.

 

In conclusion

The immune system, formed by physicochemical barriers, cellular responses (nonspecific and specific) and serological responses (nonspecific and specific), is the most complex system of the animal organism since, with each new group of animals (from protozoa to mammals), a new defense mechanism is added as a complement to the previous ones. The knowledge of the phylogeny of the immune system helps to understand the variability of the response and justifies the different specific vaccines developed for fish, birds or mammals.

 

II. Types of antigen

Together with the immune system, antigens are the main characters of the immunological events throughout evolution.

The antigen can be defined as any foreign or own substance that can be recognized by the immune system. Therefore, the concept antigen is relative and depends on the immune system’s reaction.

Antigens are chemically composed of proteins or polysaccharides. This includes all parts of bacteria (capsule, cell wall, flagella, fimbriae, and even their toxins), viruses, other microorganisms and tissues. Lipids or nucleic acids alone are not an antigen, but they become one when they are combined.

The most complex form of immune response consists in the synthesis of antibodies, protein substances capable of reacting specifically, although there are many types of immune reaction, as it was described in the section I of this conference.

This conference will focus in the six most important antigens in relation to vaccines manufacturing:

1. Non-modified live antigens
2. Attenuated live antigens
3. Modified or recombinant live antigens
4. Inactivated antigens
5. Subunits
6. Synthetic antigens
7. Anti-idiotypic antigens

Non-modified live antigens

Biological units that are administered with their original structure. Actually, the administration of these antigens pretends to cause the diseases, but in a controlled way.

This type of antigen is often used in antiprotozoal vaccines and avian viral vaccines. In the former, effectiveness depends on the amount and increasing rate of application. An example of these are the vaccines used in the prevention of avian coccidiosis, caused by Eimeria spp. Herpes Turkey Virus (VHP) vaccines and the ones to vaccinate chickens against Marek’s Disease are two examples of the latter.

 

Live attenuated antigens

Biological units that have been modified to decrease their pathogenicity by successive passes in animals or by cultivating them in microorganisms under dysgenetic conditions.

Successive passes are made in species other than the ones that are to be immunized. The degree of attenuation is directly proportional to the number of passes.

These attenuation methods are mainly used with viruses such as avian infectious bronchitis (ovoculture) and swine fever virus (lapinizated).

Attenuation of live antigens is also achieved by culturing them at temperatures slightly below those normally required, or by exposing them to inactivating substances at concentrations slightly lower than lethal. These attenuation methods are generally applied to bacteria such as Bacillus anthracis (agar culture with 50% serum in a CO₂-rich environment)

Modified or recombinant live antigens

Genetically modified biological units with the aim of preventing them from expressing their own protein or to make them express some foreign protein.

In the former, irreversibly attenuated live antigens and/or antigens that do not have parts that interfere with diagnostic techniques are obtained (e.g. Aujeszky disease).

It is very unlikely that modified live antigens become pathogen in the short term.

Recombinant live antigens have advantages, from an industrial point of view, when using bacteria or yeasts that grow faster and easier than the original donor.

The rabbit myxomatosis vaccine that has received genetic material from the viral hemorrhage virus is an example of a recombinant vaccine.

Inactivated antigens

Biological units, including certain toxins, that have been killed by the action of physical and/or chemical agents. They retain part of the chemical structure capable of causing an immune response. This type of antigen corresponds to the so-called bacterins and toxoids. It is the safest type of antigen.

 

Antigen subunits

Fractions of biological units that, due to their chemical composition, can cause an immune response. Usually, these subunits are subsequently inactivated. The components eliminated from these subunits can interfere with the immune response or cause a response that does not protect the animal.

This type of antigen is common in vaccines for colibacillosis, porcine atrophic rhinitis, meningococci and Hemophilus.

 

Synthetic antigens

Units or subunits obtained by chemical synthesis. The difficulty of using these is that, even though the antigen is chemically identical, the three-dimensional arrangement of aminoacids is different to the original antigen and, therefore, neither is the immune response (e.g. malaria).

 

Anti-idiotypic antigens

Biological units that replace the original antigen. They are antibodies of the original antibody and counteract the deficiencies of synthetic antigens using biological means.

The original antigen is inoculated and the antibodies that are produced are the negative of the template (idiotype). By inoculating this idiotype in another animal, antibodies (anti-idiotype) are obtained, which are identical to the original antigen, in matters of their three-dimensional structure.

Their elaboration is expensive, but has the advantage of acting immunologically as a living microorganism without being a microorganism (e.g. non-Hodgkin lymphoma, multiple myeloma).

 

In conclusion

To elaborate the safest and most effective vaccines, it is necessary to understand the immune system of the animal species to be protected and the most appropriate type of antigen.

From an industrial point of view, environmental safety is guaranteed with the development of inactivated vaccines and vaccines with anti-idiotypes.

Rafael Castejón and Martínez de Arizala and Ángel Sánchez Franco, teachers of veterinary immunology, developed pioneering techniques in the development of diagnostic reagents for typhoid and brucellas, anti-sera and vaccines against Clostridium, salmonellosis, erysipelas, pasteurellosis and classical swine fever, as well as anti-erysipelas and anti-tetanus sera. Their techniques inspired many others.

 

III. Types of vaccines

Along with the different types of antigens described in the previous section, there are other technological components used in the production of vaccines. By combining different types of antigens and different technological components, several types of vaccines can be produced, which are described below:

 

Main technological components different from antigens

Adjuvants

Chemical substances, materials of microbiological origin or mixtures that, administered together with the antigen, contribute to the production of a greater immune response:

• Aluminum and calcium salts

Delay the release of the antigen from the point of inoculation and allow prolonged immune responses.The most commonly used salts are aluminum phosphate and hydroxide and calcium phosphate.

• Immunostimulants

Increase or restore immune defenses. They can be materials of biological or chemical origin:

• Bacterial fractions:
  • Corynebacterium
  • Bacillus Calmette-Guérin (BCG strain of Mycobacterium tuberculosis)
  • Bordetella
• Vegetable substances:
  • Ellagic Acid from Emblica oficinalis
  • Tinospora from Tinospora cordiofolia
• Chemicals:
  • Fatty acid salts
  • Isoprinosine

Emulsifiers

Freund’s Complete Adjuvant, an emulsion of water-oil plus complete mycobacteria or a fraction of these, called muramyl dipeptide.

Liposomes

Inactivators

Substances that eliminate the pathogenic capacity of the antigens, used alone or in combination with the action of physical agents, such as heat.

Formalin

It acts on the amino and amide groups of proteins and on the amido groups that do not create hydrogen bonds with the pyric and pyrimidine bases of nucleic acids.

Formalin is the most commonly used inactivator in the manufacturing of anatoxins. Its maximum quantity in the final product is limited by Pharmacopoeia, to avoid its action on animal tissues.

Alkylating agents

Substances that give rise to the formation of transverse bonds between nucleic acid chains. Since they react with the microorganism’s surface proteins, they keep their antigenic power intact.

The most commonly used alkylating agents are ethylene oxide, beta-propiolactone and ethylenediamine.

 

Preservatives

Liquid or solid diluents that allow to achieve the concentration of active ingredients (antigens) in the final product, so that they is easily dosed.

Fenol

It is the most used preservative in the production of inactivated vaccines. Its content in the final product is limited by the monographs of each vaccine described in Pharmacopoeia.

Excipients

Liquid or solid diluents that allow to achieve the concentration of active ingredients (antigens) in the final product, so that they is easily dosed.

Physiological serum

Skim milk

 

Main types of vaccines

Depending on their composition the preparation method, the following types of vaccines can be distinguished:

Live vaccines

Those made with unmodified live antigens, live attenuated antigens or modified live antigens.

These vaccines confer rapid immunity, but are only suitable in contaminated media and poses a risk to a possible return to virulence and immunosuppressive effect.

Inactivated vaccines

Made with inactivated antigens. They confer slow immunity and many, although not all, require the incorporation of adjuvants. They are indicated in healthy environments, because they are safe.

Design vaccines

They are those made with subunits; synthetic antigens and anti-idiotypes.

 

In conclusion

The development of vaccines requires knowledge of the two main factors, the immune system of the animal species and the antigen, together with the technological components necessary to achieve safety, the immune effect and the adequate duration of the response. Safety factors must also be taken into account.

 

IV. Design, development, control, distribution and application of vaccines

 

Development and control of veterinary vaccines

The development of a veterinary vaccine is a complex process based on numerous, variable steps, depending on the type of antigen used. The work processes must follow the international recommendations on GMP and, the quality of the raw materials, the monographs described in European Pharmacopoeia, National Pharmacopoeia or recognized technical sheets.

The facilities and equipment for the manufacturing of veterinary vaccines must be in accordance with international and state regulations.

In 2008, the OIE published the principles of veterinary vaccine production set out in chapter 1.2.8 of its manual of the same year.

The Directive 81/851 / EEC and the Commission’s Directive 2009/9/EC of February 10^th, 2009, constitute the regulatory body in the European Union regarding the requirements that must be met by these facilities and the procedures for their preparation.

 

As for the constructions, the following will be taken into account:

1. The circulation of the vaccine components can be carried out in a direction that prevents the crossing of ingredients, finished and intermediate products.
2. The movement of people and their clothing should also be oriented in a certain direction in order to avoid crossings.
3. Air circulation and quality should avoid contamination from the outside. The packaging should be done under sterile conditions and positive pressure.
4. Facilities cannot have any convex right angles that difficult the cleaning process. It is advisable to build half-pipe corners. As for the materials, the following will be taken into account:
   1. Non-porous, to prevent the infiltration of microorganisms and organic matter.
   2. Easily washable.

The facilities will be divided into:

• Raw materials warehouse, including a microorganism warehouse (“cepario”)
• Intermediate products manufacturing area
• Mixing zone
• Packaging
• Quarantine warehouse
• Laboratorial analysis
• Labeling area for finished products and shipping area.

 

Equipment

The equipment used will be made of glass or stainless steel and will be easily accessible and cleaning.

 

Processing, control and storage procedures

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

a. Preparation of manufacturing data sheet:

A sheet that contains the quantities and identification codes of all the materials used in the preparation of the batch. This sheet must accompany the ingredients during the whole process route.

b. Preparation of starting materials:

In the raw material warehouse, with products that are already analyzed and declared suitable for their adaptation to the characteristics indicated for them in the European Pharmacopoeia or their own technical data sheet. In the case of vaccines, there is an exceptional starting material called “working seed”. It is a culture prepared from the master seed of the cepario or microorganism warehouse.

c. Preparation of intermediate products:

Intermediates are defined as those mixtures of starting materials that are part of the preparation of the finished product.

The suspension of microorganisms constitutes the main intermediate product in the production of bacterins. These are microorganisms produced by fermentation from the inoculum of the working seed to large fermenters. The content of these fermenters is concentrated by tangential filtration to obtain a highly concentrated suspension of microorganisms.

In the case of anatoxins, another intermediate product is the concentrated solution by molecular filtration of toxins secreted by the producing microorganism.

Both the suspension of microorganisms and the solution of toxins are inactivated by the use of inactivating products already described (formalin and others) and stored in refrigeration until used in the elaboration of the final product.

d. Preparation of the finished product:

It is done by mixing several intermediate products (microorganisms and toxins) with starting materials such as adjuvants, preservatives and excipients. The obtained product is packaged in a sterile environment in sterile bottles that are capped, sealed and stored in a quarantine area.

e. Quarantine

Once the final mixture is manufactured, it must be analyzed before being released to the market. For this, warehouses equipped with refrigeration (4-8ᵒC) will be available.

f. Quality control of finished products

The controls of the finished product must be carried out in accordance with the monograph of the product described in the European Pharmacopoeia, the National Pharmacopoeia or the internationally recognized data sheet.

Although there are particularities, the finished product must basically undergo the following controls:

1. Physicochemical characteristics
2. Power: essentially, it consists in administering a dose of vaccines to a group of animals and subsequently performing a confrontation with the same microorganisms of the antigen, but live and pathogenic. If the vaccine has an adequate power, the animals will survive.
3. Safety and abnormal toxicity: it consists in the administration, to a batch of animals, of a dose of vaccine higher than the recommended one. Animals should not show symptoms of disease or local reaction during a specific observation period.
4. Direct validity and validity once reconstituted: this determination is mandatory during the registration process of the product, and it is recommended to be performed sporadically in manufacturing batches or when, for technological reasons, some manufacturing process or material has been modified. The validity tests are used to determine how long a certain vaccine stored under specific temperature and light conditions maintains its effectivity.
5. Other determinations: phenol, formaldehyde, safety and absence of foreign pathogens.

g. Labeling

Once the suitability of the product has been established through the analysis described in another section, the product is labeled and provided with the informational material (box and leaflet) that allows its correct application.

The analytical and manufacturing performance data are incorporated into the manufacturing sheet and, these, together with the samples, are stored at least up to 3 months after the expiration date of the manufactured batch. The finished product is sent to the finished product warehouse until its distribution.

h. Warehouse of finished product and shipping area:

Note that both areas must be equipped with refrigeration and a computer or manual system that allows to know when, how and where each batch of manufacturing has been sent. This information is very valuable in case of detecting adverse reactions of the product once released into the market.

 

Distribution

Heat and sunlight negatively affect the vaccine efficacy. For this reason, distribution is as important as the development of a vaccine.

The distribution of veterinary vaccines must have the adequate local premises and the services of a technical director.

Refrigeration between 2-8ᵒC is usually necessary, but should not be frozen except in a specific vaccine in which freezing in liquid nitrogen or dry ice is necessary (Marek’s disease).

In addition to the corresponding cold chain, it is necessary to track the inputs and outputs of the different batches of products through the corresponding documentary records.

 

Administration

Veterinary vaccines are administered by various routes depending on the type of antigen and the breeding system of the target animals.

Oral administration

This route of administration is usually for live vaccines intended for birds. This practice allows mass vaccination in a few hours and with little labor when performed by suspending the antigen in drinking water. Minimal precautions are required, such as restricting drinking water 10-12 hours before, using disinfectant-free water and diluting skim milk powder with the antigen to improve its viability.

Airway administration (nebulization)

It is common in live viral and bacterial vaccines for birds and pigs. This route allows mass vaccination, but requires adequate facilities and equipment to produce nebulization. The size of the drops is an important factor, as the vaccine should stay in the air long enough for the animals to breathe it.

Parenteral administration

It is used for the administration of all types of antigens, especially of the inactivated ones. This route requires more staff and time than the previous ones and causes discomfort to the animals. Despite this, it guarantees the most appropriate administration to each animal.

Syringes should not be disinfected with alcohol or disinfectants that can inactivate live antigens.

Particular attention should be paid to make sure that the vaccine is applied to the appropriate tissue. The most commonly used parenteral routes are subcutaneous and intramuscular, although some vaccines are administered by specific routes such as intradermal (ecthyma and anthrax) and intravenous (canine distemper) routes.

Topic administration

This route is used to administer live viral antigens in nasal mucosa and ocular conjunctiva. It is the most expensive and most annoying route for animals, but produces the fastest local immune response in the respiratory and ocular mucosae.

 

Factors related to the organism to be immunized

Before the administration

A good planning of the animal handling is desired to minimize stress. It is also important to know the pathological history of the population or individuals to be vaccinated, as the administration of vaccines is contraindicated in sick animals, with febrile processes, treated with corticosteroids or receiving immunosuppressive medications.

Try to avoid vaccination of pregnant females during the first month of pregnancy or birds at the maximum of laying.

Vaccinating with herpes virus strains can be dangerous due to their latent infectious power: it can be apathogenic for an age, but trigger disease in younger animals.

 

During administration 

 

After the administration

Keep animals in good environmental conditions.

 

V. Main avian veterinary vaccines

 

Introduction

Nowadays, there is a wide range of biological products for the control of avian diseases, and other research projects are conducted to develop new vaccines. The same happens with the application methods and vaccination plans.

The diseases currently controlled by vaccination are the following:

Virus

• Newcastle disease
• Infectious bronchitis
• Marek’s disease
• Chickenpox
• Encephalomyelitis
• Gumboro disease
• Laryngotracheitis

Bacteria and Mycoplasmas

• Mycoplasmosis
• Avian cholera
• Fowl typhoid
• Colibacillosis
• Infectious coryza
• Infectious toxic hepatoenteritis

Protozoa

• Coccidia

 

Vaccines against viruses

The following are ordered from most to least used:

Newcastle disease

The vaccine against Newcastle disease is the most used, not only once, but several times in a single flock of birds in their different presentations and forms of application. Newcastle disease viruses are classified, according to their aggressiveness, into three types: lentogens, mesogens and velogens. Based on these data, the main strains of Newcastle are:

ICPI: index of intracerebral pathogenicity in 1-day-old chicks

IVPI: intravenous pathogenicity index in 6-week-old birds

EM: embryonic mortality

The most commonly used are the lentogen strains, among which are B1 and LaSota. Some examples of the mesogenic strains are The Roakin, Haifa (Komarov), which are more aggressive, their use is not as widespread and their application is usually intradermal.

Vaccines arrive prepared in different forms, but we can distinguish two types: live vaccines, which, as the name indicates, have the original virus with modifications or attenuations, and inactivated vaccines with their different adsorbents or adjuvants, such as aluminum hydroxide, B-propiolactone or others based on essential oils for a longer immunological activity.

Before talking deeper about them, some facts need to be taken into account:

The decision between inactivated and live vaccine should be based on the bird species that will receive it and the available labor. Another interesting fact is the administration route, the available ones for live vaccines are ocular (gout in the eye), oral (in drinking water), airway (spray) or intradermal (wing or feather follicles) administration.

Acquired immunity is another factor to keep in mind, particularly when vaccinating chicks immediately after birth or birds that have been repeatedly vaccine, as birds with acquired immunity will not achieve a better protection but, because of their high protective level, the “immunity break” will take place, which means that the birds will not be protected (the effect will be the opposite of the expected one). Therefore, live vaccines should only be administered when the antibody titers are adequate or when there is the need for viral interference, and one should be very cautious when recommending live vaccination.

Inactivated vaccines do not have the disadvantages of live vaccines, but they are slower in stimulating the organism for the production of antibodies.

For all of the above, it is essential to carefully study the conditions of every area, even those of every farm, to choose the vaccination program that best adapts to each of them.

Live vaccines, as mentioned above, can be administered through different routes:

• Ocular: the most effective route, because each bird receives the proper dose of viral particles, despite it is a slow method (regarding the application).
• Oral: faster administration, but the distribution of viral particles is irregular and, in addition, there is a risk of destruction, to a greater or lesser extent, of the viral particles, particularly if they are in contact with the environment for a long time and depending on the very different conditions of the waters used as the vaccine vehicle. Therefore, it is very important to use waters that do not decrease the vaccine efficacy.
• Airway or spray: broadly used, especially in farms with a large number of birds (thousands), because they are easily and quickly applied. The most important factor to take into account is the size of the drops: smaller size allows better vaccination, although it entails a serious risk of suffering diseases caused by latent infections by Mycoplasma or coli, because of the special anatomy of the respiratory system in birds or the poor hygienic conditions in the fam. For all of this, very specific conditions are required for the success of these type of vaccines.
• Intradermic: rarely used, only in case of layers or breeders, once, because it gives long-term immunity, because the virus in these vaccines is mesogenic (more aggressive than lentogenic virus). The disadvantages of these vaccines are that their application is slow and they are more aggressive and diffusible.

Inactivated vaccines are always administered parenterally, subcutaneously or intramuscularly, being the second the preferred route, because subcutaneous vaccines are easily administered and it avoids the risk of lameness (when applied in the tigh) or encystment (pectoral muscles), that affect the most valued part of the bird.

Immunity against Newcastle disease virus is achieved with live vaccines almost immediately by cell blockage, at the level of respiratory and digestive mucous membranes and, in the longer term, by the synthesis of antibodies.

In inactivated vaccines, immunity response is slower but lasts longer, and avoids post-vaccinal reactions and immune breakages, as we pointed out further back.

In any vaccination, it should be taken into account that any other disease (whether viral, bacterial or parasitic), can interfere with vaccination and reduce its effectiveness by creating fewer specific antibodies. The most common diseases that interfere with antibody synthesis against Newcastle disease are infectious bronchitis, Gumboro disease and intestinal parasitosis, such as coccidiosis.

 

Infectious Bronchitis

Infectious bronchitis is a widespread disease. There are two live virus strains available against this disease since the 60s, the Massachusetts strain and the Connecticut strain, which were used initially, although subsequent investigations pointed out that the Massachusetts strains conferred cross-immunity to the Connecticut strain, which is why, virtually, all current vaccines are based on the Massachusetts strain.

There are two variations of this strain that consist in the characteristics of the virus, because the more passes they receive through the embryo, the more attenuated they become towards the chick and, therefore, they become more pathogenic for the mentioned embryo. This way, two variations of the same vaccine can be manufactured, a more attenuated one (for young birds) and a more aggressive one (for birds before starting laying).

Application before the laying is due to the fact that these viruses have a high selectivity for the oviduct tissue, as they can seriously and irreversibly damage it, causing the so-called false layers or strong deformations in the shell of the eggs and loss of their interior quality, with molten albumen.

For some time now, cases of infectious bronchitis have increased despite the vaccination plans based on Massachusetts strain, so it seems that there is a field virus that does not match, antigenically speaking, the virus of the current vaccines. This means more attention should be paid to this disease, especially in countries or regions with migratory birds from north to south in October and from south to north in March in the northern hemisphere and on the contrary in the southern hemisphere.

The routes of application for this vaccine are oral or ocular, and it can be combined with live vaccines against Newcastle disease, although it is necessary to take into account that vaccination against Newcastle is always impaired, from the immunological point of view.

If vaccination is administered in association with other, the most attenuated B-1 virus should be used; and if they are administered separately, there should be an interval of 2 to 3 weeks between each vaccination.

Attempts have been made to manufacture inactivated infectious bronchitis vaccines, alone or associated with inactivated Newcastle disease vaccines, but, currently, results are very few.

To control these two diseases, from the immunological point of view, I there are several laboratory methods to detect the presence of antibodies in the blood serum, and the most commonly used in daily practice are the following two:

Inhibition of hemagglutination, for the diagnosis of the immune status against Newcastle disease, taking advantage of the ability that Newcastle virus has to produce hemagglutination of chicken red blood cells and fact that the serum has antibodies to inhibit hemagglutination.

Regarding infectious bronchitis, the test of choice is the so-called serum neutralization, which is the ability of serum with infectious bronchitis antibodies to neutralize the pathogenic virus.

These two diagnostic methods can be of great help when programming vaccinations, because they indicate the immune status of the birds.

 

Marek’s disease

Marek’s disease has been one of the last diseases controllable by vaccination methods, specifically by live virus vaccines. This happened in the 70s.

The viruses used are from the herpes virus family, and their origin is that of turkeys, because it was found that turkey virus is not a pathogenic for hens and has practically no diffusion power. It is a heterologous vaccine, that is, it comes from a different specie to which we want to vaccinate. This vaccine is known as HVT (Herpes Virus Turkey). It was designed by, what could be called, the American school.

The Dutch school, on the other hand, worked with a strain of chicken virus that is apathogenic for chickens, a homologous vaccine, that is, it comes from the same specie to be vaccinated.

Regarding the Turkey Herpes Virus vaccine, there are two important variations, from the point of view of storage and transport:

• One of them is made from live cells infected with the vaccine virus, its conservation and transport must be in liquid nitrogen, so that temperatures of 180ᵒC below zero are reached and it is used as a liquid form in which cells are in suspension.
• The other way in which it can be manufactured is from this same process, but in this case cells in which the virus developed are separated from the viral particles. With this method, the virus is lyophilized and, thus, storage and transport can be simplified, as there is no need for special packaging or storage conditions.

It is remembering that any vaccine needs a minimum care in its storage and transport, and not doing it by means of a simple refrigerator of insulating material, is to go against the effectiveness of the subsequent vaccination.

The Marek vaccine is usually applied in the incubation room itself; where the chicks that have been vaccinated were born, the route is subcutaneous (under the skin of the neck) by means of automatic syringes, with exact graduation, which allows calculating the amount of Plaque Forming Units according to the concentration of the commercial vaccine

The Plate Forming Units are cells that have been infected by the virus during the manufacturing process in the cell culture. They can form plates in their subsequent growth to check the infectious capacity of the vaccine material. This property is used to calculate vaccination titers.

It is considered that with a minimum of 700 to 800 PFU, it is sufficient, per bird to one day of life, to protect it against Marek’s disease throughout his life. Most vaccines are sold between 2000 and 3000 PFU, for each dose of 0.20 ml.

The second point, which also serves another type of vaccine in which its application is parenteral, is the hygienic conditions of its application.

How many times we will have seen the few conditions that the syringes meet without being disassembled or disinfected, which favors the nesting of germs inside, especially considering the material so easily contaminated that it is retained inside. This aspect is important to take into account and not miss any opportunity to repeat it, to guide who should perform this operation, to apply vaccines parenterally.

To conclude with this point, we only have to talk about immunity against Marek’s paralysis.

This issue of immunity has been much discussed and there has been talk of antibodies on one hand and interference on the other. In any case, in the creation of resistance against Marek’s Disease, it seems that the Fabrizio’s bursa plays a very important role, so any aggression against this bursa is detrimental to the creation of adequate resistance, so I want to state that a Gumboro disease virus attack, especially if it occurs at an early age, will create serious problems with the implantation of the vaccine virus against Marek’s Disease.

Likewise, the high vitamin B needs of modern strains (30% higher than those considered in the international nutritional needs tables) may influence the sciatic nerve weakness. So, as we see, there are many points to consider when assessing the good or bad result of a vaccination, which can fail because only one of the several factors that must be taken into account is not met.

 

Diphtheria – Smallpox

Diphtheria-smallpox in poultry farming and its vaccination is one of the first that was carried out, although, over the years and first due to an effective vaccine barrier and better sanitary and accommodation hygienic conditions, in which birds are currently exploited, its importance in frequency and intensity has diminished. In any case, I believe that this situation can be harmful if its use is abandoned, as is currently happening.

This vaccine is prepared from live viruses that have two origins. One of them is the live virus from chicken, and these vaccines are called Homologous vaccines. The other origin of the virus is from the pigeon, so these vaccines are called Heterologous vaccines; being, in the Homologous vaccine, its application usually intradermal (in the fold of the wing), being able to be associated in many cases to the Roakin strain against Newcastle Disease.

This diphtheria-smallpox vaccine produces a postvaccination reaction and after a few days a variola pustule appears at the place of its application (having to check this fact, after a few days of the vaccine, to verify that the vaccination has taken perfectly).

As for the application of the Heterologous vaccine, it is applied to the feather follicles, usually in the thigh area, by plucking a surface of 1 cm² and then, by brush impregnated with a vaccine, these follicles are rubbed. This type of vaccination produces little reaction, although the conferred immunity is less durable than if the vaccine used is the Homologous.

 

Encephalomyelitis

Encephalomyelitis is another disease that can be controlled through adequate vaccination, which is done through live virus vaccines, since inactivated vaccines have not been successful in practice.

This vaccination is only interesting for farms that are engaged in reproduction, since the disease itself, in adult birds, has very little economic action. Where it really affects in a serious way is in the results of incubation and in the subsequent mortality of chickens born to mothers who recently suffered an outbreak of this disease.

The application of the vaccine is always done orally; years ago, it was done with cannula directly to the mouth and about a % of the pack, so that they spread the virus among the rest of the pack.

At present it is carried out as a normal vaccination in drinking water, to the whole flock in general, to avoid the problem that a certain number of birds have to spread the virus, especially considering that the birds within a farm have a certain area to live.

Another aspect to take into account is the most appropriate time to carry out such vaccination, since what is really causing is the true disease, this must happen during the breeding period and is not excessively close to the period of onset of put, because if so in the first incubations some anomalies could appear in it, with subsequent casualties in the chicks born.

The immunity acquired by vaccinated breeders is transmitted through the egg to the embryos and the chick. This fact is used to measure immunity, through tests on embryonated eggs, from these breeders, these embryos are inoculated with a pathogenic virus, Van Roekel strain and if they come from well-vaccinated mothers, they resist that test well or not if the mothers do not have the antibodies against said disease.

It is a practical test that helps a great deal in deciding the fate of the production of breeders whose vaccination program is unknown.

 

Gumboro disease and Laryngotracheitis

I include here these two diseases to end the group of controllable diseases, caused by viruses, against which there are currently prepared vaccines.

In Gumboro disease its use is widespread, while there seems to be some unequal opinion on whether or not to extend its use. The disease was first discovered in Gumboro, Delaware in 1962. It is economically important for the poultry industry worldwide because of the increased susceptibility to other diseases and negative interference with effective vaccination. In recent years, very virulent strains of IBDV (vvIBDV), causing high mortality in chickens, have emerged in Europe, Latin America, Southeast Asia, Africa and the Middle East. This aggressiveness has led to the use of “hot” strains that act by viral interference rather than by the creation of antibodies. This usual practice in South Asia creates the need to continue vaccinating permanently.

The use of oily vaccines in 1-day birds presents the previously described difficulty of breaking immunity.

There are two serotypes: serotype 1 and serotype 2, which can be differentiated by immunization; birds that are immunized against serotype 1 are not immunized against serotype 2 and vice versa, there is a clear difference between them by using serology. Leaving aside serotype 2 that has only been isolated in turkeys, ducks and rarely in chickens, but has no clinical importance so far. On the other hand, serotype 1 is the one that has the greatest importance.

In serotype 1 it exists in two major types of strains:

• the standard or classic type strains, which were the first to appear when the disease was first isolated in the mid-50s, and which has now been determined to have different degrees of pathogenicity: low, intermediate, high and very virulent
• the other subtype of strains is known as antigenic variants, it is called that precisely because there was a change in the immunogenic characteristics of the viruses, which caused the birds previously immunized against the standard strains to be susceptible to being infected by these variants. antigenic, discovered in the 80s and that are highly immunosuppressive.

Regarding Laryngotracheitis Disease, there are two types of live vaccines on the market: one of chicken embryos origin and one of cell cultures origin. Those of chicken embryos origin are generally more invasive than those of cell cultures origin, but have greater residual pathogenicity.

One or two vaccinations are usually sufficient to induce immunity for life, but the level of it may vary. In outbreaks of infectious laryngotracheitis, an emergency vaccination by eye drop can stop the further spread of the virus in an already infected flock. Originally the vaccine against laryngotracheitis was done by brushing of the vaccine in the mucosa of the cloaca, which after a few days appears swollen and edematous, indicating that the vaccine has taken perfectly after nine days of vaccination, it is considered that birds are already sufficiently protected. Today there are forms of applications standardized in the industry (eye and spray). We believe that you should be very cautious when introducing this vaccine into an area, since it may create the need to continue vaccinating indefinitely.

 

Vaccines against bacteria and mycoplasmas

In this group of diseases caused by bacteria, initially included in group B, we highlight:

• Mycoplasmas
• Avian Cholera
• Colibacillosis
• Typhosis
• Infectious coryza
• Infectious Toxic Hepatoenteritis

With the use of antibiotics, vaccinations against these diseases have very little interest, except in the vaccination against Mycoplasmas (for the resistance of some strains of M. synoviae and infectious toxic hepatoenteritis (for the settlement of microorganisms in the bile ducts)

Mycoplasmas

It is a widespread disease for some years, it is caused by Mycoplasma gallisepticum that is responsible for Chronic Respiratory Disease (CRD), although in the field it always presents with colibacillar type complications and as triggers we have two fundamental viruses and driving conditions.

In the fight against this disease, apart from antibiotics, two ways have been tried in recent times:

• the first was to get lots of birds free of this germ, which got birds called PPLC, but these birds and their offspring, to keep them free, it was necessary to exploit them in exceptional isolation conditions and under strict control, so it has become very difficult to obtain generally good results, except in a few cases.
• as this path is very difficult, research has been followed towards the creation of a Mycoplasma vaccine and lately we have a vaccine that meets the appropriate conditions to be effective and practical to use.

Said vaccine is made with live germs of a strain of Mycoplasma gallisepticum that displaces the pathogenic Mycoplasma vaccinated in the organism of the birds, conferring the adequate resistance to new invasions of said pathogenic germ, while maintaining the vaccine pattern and on the other hand the one the vaccine germ does not believe in the vaccinated organism the presence of agglutinins, this gives the possibility of carrying out agglutination reactions in the vaccinated herds, to detect the presence of pathogenic carriers.

With this vaccine method it is possible to keep birds free of PPLO, without having to resort to such a difficult method of control and isolation. However, this vaccine does not protect against M. sinoviae, which creates a second problem.

The application of the vaccine is carried out by three methods: the most complicated aerosol, by eye drop, or that of drinking water, which because it is highly practical is the one usually used; This vaccination can be carried out in association with any other vaccine against Newcastle Disease or Infectious Bronchitis, which simplifies even more the already overloaded vaccination programs.

For its application, the following factors must be taken into account to obtain a good vaccination result. These factors are the following:

The water that will serve as a vaccine vehicle, must have an adequate saline concentration, to avoid osmosis phenomena, between the water and the inside of the germ through its cell membrane, for this, a concentration of 85 gr of CINa (Common salt) per liter of water is recommended, which is achieved by an isotonic medium that will not harm mycoplasmas in suspension.

The water will be free of chlorine or any other disinfectant, as it is a general rule for any vaccination in drinking water.

As the vaccine to be applied is from live germs and these germs have specific sensitivity to a group of medicinal products including tylosin, erythromycin, spiramycin, it will prevent them from being present in the feed consumed by the birds to be vaccinated or in form of other treatments, at least 5 days before vaccination and 5 days after it has been performed.

If the application of the vaccine is carried out by aerosol in the incubation room, it will be taken into account that formaldehyde gases are harmful to said germ, so this fact must be considered.

The recommended vaccination schedule varies greatly depending on the circumstances and the type of bird exploited, but as a rule, a good result is achieved applying said vaccine every 7 or 8 weeks and thereby keeping the birds free of PPLO. To their offspring should be followed an appropriate vaccination schedule, depending on the destination of them.

 

Avian cholera, typhosis and colibacillosis

We encompass these three diseases for having several points in common and for being their vaccination today almost null.

In Avian Cholera, the causative germ is Pasteurella multicide, which is normally used and, in a particular way, an inactivated vaccine (bacterin) that can be manufactured from the isolated germ in a specific case and only in the same farm , that is, it is a self-vaccination, since as there are different serological types, it is impossible to prepare a vaccine for general use.

In Avian Typhosis, the causative germ is Salmonella gallinarum, and the above can be applied in the case of cholera.

In Colibacillosis, the problem is even more intricate, because there is such a wide range of serological types that it makes it impossible for it to have a good result, even with polyvalent vaccines.

So, among the few results obtained by these vaccines, and the existence of medicated products that act well in these diseases, the use of vaccines is reduced to concrete and very specific cases.

 

Infectious coryza

Vaccination against Infectious Coryza is based on the application of a bacterium based on Haemophilus gallinarum (serotypes A, B, C) with good results in laying and breeding birds. Regarding the latter, an increase in the agglutination rate against Mycoplasma sinoviae has been detected, after the Infectious coryza vaccination. Finally, we must point out that the immunity lasts between 6 and 9 months according to tested flocks.

 

Infectious toxic hepatoenteritis

This disease caused by SH₂-producing enterobacteria has extended notably in recent years to the point of being one of the etiologies that produces the most economic losses. Protected by the loss of interest in the eradication of S. pullorum, these microorganisms (Salmonella Enteritidis, Proteus, Pseudomonas, Klebsiella and E. coli) have settled in the anatomical complex bile ducts-ovary-intestine and their consequences are the increase of vitellus retention, typhlitis, ovarian infections and hepatitis along with a toxigenic process.

Four ways of secretion of toxic substances in gram-negative bacteria have been reported:

• in type I secretion, exoproteins pass directly to the extracellular medium (proteases and alpha hemolysin).
• type II secretion pathway, also called general secretion pathway, is responsible for the secretion of most extracellular proteins (elastase, lipase, exotoxin A, phospholipases and alkaline phosphatase).
• type III secretion system translocate proteins directly from the bacteria to the bird’s cell (exotoxins S and T).
• type IV secretion system gives rise to tense-active compounds, with highly toxic detergent properties, composed of a glycolipid containing one or two molecules of rhamnose (rhamnolipids).

These toxic secretions are genetically linked to the production of SH2 and pyocyanin (greenish blue pigment that gives the color to embryos and chicks’ vitelli).

 

Vaccines against parasites

Among the diseases caused by protozoa, coccidiosis should be highlighted. The protozoan that causes this disease in poultry belongs to the genus Eimeria spp., which includes many species, although the most important are: E. tenella, E. necatrix and E. acervulina, not only because of their frequent presence in birds, but also because the losses they cause in the farms.

The fight against this disease has been raised in several fields that could fundamentally be summarized in three:

• the first and most widespread is the research and implementation of a wide range of pharmaceutical products systematically incorporated in feed to provide protection, up to certain limits.
• the second is the application of management and hygiene standards, but they are difficult to apply and require a lot of resources.
• the third is the one that enters fully into the topic at hand, because it depends on vaccination against coccidiosis. Back in 1965, the use of vaccines based on live and attenuated oocysts began against 6 of the most important species of coccidia. This vaccination was incorporated into the drinking water at 10 days of life and, then, a coccidiostat and litter irrigation program was initiated to create the appropriate conditions for sporulation of the oocysts expelled by the feces of the vaccinated bird to create the desired resistance to future invasions of protozoa by successive reinfections at the level of the cells of the digestive system.

The system just exposed did not have the desired success, as it was a highly technical vaccination and led to a series of failures due to a bad handling of the litter and its irrigation. On the other hand, this system had the inconvenience of having to introduce new forms of oocysts into the farm through the vaccine that did not exist before, for which reason such vaccinations were abandoned until their total disappearance.

However, in 1982 its practice was returned, again, adding the oocysts to the feed with acceptable results at the level of breeding birds.

Today, there are vaccines with multiple strains of Eimeria in the same vaccine that can be applied by air and even superficial protein fractions administered orally.