Volume 2, 2014

avianinsight
With the advent of new generations of oils and surfactants, emulsion technology as applied to vaccines has undergone considerable improvement over the years. Depending on the types of oils and surfactants employed, their formulation, and the amount of energy added, several and different emulsion types can be designed for specific purposes.

A LO H M A N N A N I M A L H E A LT H N E W S B R I E F

Poultry Emulsion Vaccines

Background In 1796, one of the greatest achievements in science emerged when Edward Jenner discovered that human smallpox can be prevented through vaccination; giving origin to the term vaccine (1, 4). Although the concept of Maria Elena Vazquez, Director of R&D vaccination is more than two hundred years old and new discoveries in science and technology have been introduced, the basic aim of this practice remains the same: to prevent or reduce infection through the generation of an immune response in the host. Two concepts of vaccines are still current: the live attenuated and inactivated vaccines. In live attenuated vaccines the virulence of the antigen is reduced and, although it is still alive and it can replicate, the clinical signs in the host are minimized. Although the concept of live vaccine emerged with Jenner at the beginning of 19th century, the term attenuation was born with the empirical observation that the virulence of germs was reduced after several passages in other non-host animal species (lapinization). This procedure was utilized by Louis Pasteur in 1881 in the development of the swine erysipelas vaccine (1). Inactivated vaccines eliminate the virulence of the germ by inactivating it with a chemical or physical method thereby rendering it a non-live antigen. The first evidence of antigen inactivation appeared in 1948 when formaldehyde was utilized in attempts to improve the efficacy and safety of the Foot and Mouth Disease vaccines (1). But current inactivated vaccines could not exist without another important scientific contribution, adjuvants. In 1924, Gaston Ramon, a veterinarian at the Pasteur Institute and first Director General of the World Organization for Animal Health (OIE) pioneered the use of Aluminum Hydroxide to adjuvant tetanus and infantile diphtheria vaccines in Europe (1). The concept of emulsion arose in 1916, when a very unstable and viscous uid was prepared by Le Moignie and Pinoy (4). However the real introduction of emulsion technology emerged in 1936, when Jules Freund developed a water-oil emulsion containing killed mycobacteria; today known as complete Freund adjuvant (3). Poultry Emulsion Vaccines An emulsion is defined as droplets of a liquid, in what is referred to as the dispersed phase, suspended in a second liquid called the continuous phase (3, 5). With the advent of new generations of oils and surfactants, emulsion technology as applied to vaccines has undergone considerable improvement over the years. Depending on the types of oils and surfactants employed, their formulation, and the amount of energy added, several and different emulsion types can be designed for specific purposes. For poultry, the most usual type of vaccine emulsion is the water-in-oil (WO) formulation. In a WO formulation the antigen is in the aqueous phase and it is emulsified or dispersed in a high quality oil (oil phase). In an oil-in-water (OW)

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Poultry Emulsion Vaccines, p.1

Notes from the Director of Sales, p.4

emulsion the antigen is in the aqueous phase and the oil/ aqueous ratio is low. Despite of the excellent uidity of the OW emulsion they are not often employed for use in poultry vaccines due to their relatively short duration of the immunity. Lastly water-in-oil-in-water (WOW) emulsions are WO emulsions that are then suspended in an outer water phase. WOW emulsions can be prepared by two methods. One method is to conduct double emulsifications. In the first emulsification, a traditional WO emulsion is prepared and it is then re-emulsified in a second aqueous continuous phase. In the other WOW method, the double emulsion is conducted in one step using a well-balanced mixture of emulsifiers. The decision of the use of one method over the other depends on the vaccine manufacturer and the desired exibility at the moment that the vaccine is formulated. While each method has its advantages and disadvantages, both processes if performed properly can produce the expected quality in the vaccine. Emulsified vaccines are particularly well suited for poultry for the following reasons: Chickens seem to be more tolerant to the oil than other animal species Emulsion vaccines produce a longer duration of the immunity than other vaccine preparations (i.e. Al (OH)3 adjuvants) and therefore provide lifelong immunity for a chickens one year production cycle. Formulation versatility, viral and bacterial antigens and combinations of both can be included. Capability to prepare multivalent vaccines containing antigens to multiple different diseases thereby allowing immunization for all in one injection. Properties The emulsion vaccines induce the formation of deposits at the injection site (Figure 1); the

Fig. 1. Emulsion deposits in a chicken neck, after 2 weeks post SQ vaccination.

objective is that the oil and the antigen(s) are slowly released over time to stimulate antigenic presenting cells. The first antigen presentation is conducted by macrophages via endocytosis and then the lymph nodes (thymus in chickens) release antigen-specific lymphocytes (3, 4). Depending on the emulsion formulation and the oil quality, the kinetics of release of the antigen varies according to the host oil-tolerance and the degree of inammation at the injection site. Rapidly metabolized oils may be safer from an inammation standpoint but at the same time induce a weak or transient immune response. WOW emulsions are designed to have the same immunogenic characteristics as the WO formulations with the advantage of having minimal or no adverse reactions at the application site. Efficacy and Safety The efficacy and safety of emulsion vaccines are directly related to the emulsion quality. In most of the cases this quality is assured by

controlling all the steps in the manufacturing process, including the raw materials and the emulsification method used. Some quality control parameters frequently used as indicative of quality are: droplet dispersion in water, viscosity, syringeability, particle size and visual homogeneity of the uid after incubation at various temperatures. The droplet test determines the type (WO, OW, and WOW) of emulsion formed. A droplet of a WO emulsion into a petri dish of water will stay on the surface while an OW emulsion will disperse into the water (Figure 2). As the external phase of the WOW emulsions is water and the internal phase is a WO emulsion, the expected performance of the droplet test is a combination of the previous two: some fraction of the drop will be dispersed and some fraction will stay on the surface of the water.

Fig. 3. The emulsification process has a direct impact on the droplets size. The only difference between Process A and Process B is the emulsification process.

The viscosity and syringeability of the emulsions are linked to the HydrophilicLipophilic balance (HLB). An inadequate balance in the ratio of the surfactants and/or in the emulsion phases may result in a creamy, highly viscous emulsion. The balance of the surfactants, the emulsification process and the quality of the antigens has a direct impact in the emulsion particle size. It is generally believed that emulsions with smaller particle size and a

Fig. 2. Demonstration of WO type of emulsion. A drop of the test emulsion is collocated in a petri dish with DI water.

homogeneous distribution maintain the particles (micelles) in suspension for longer time and therefore are stable for a longer period of time (Figure 3).

Emulsion Properties Viscosity Syringeability Stability Antigen Release Duration of the Immunity Reactions in the Injection Site

OW Low Low Long Quick Short None

WO Medium Medium Long Slow Long Low-Strong

WOW Low Low Medium Quick and slow Medium-long None-low

propagated. For example, an emulsion with viral antigens propagated in cell culture will most likely be a different color than one made with bacterial antigen grown in broth medium. The intensity of the white color in the emulsion may also vary according of the type of the emulsion (WO-WOW). For instance, WO emulsions are little more viscose than WOW emulsions and therefore can appear whiter.
Fig. 4. Effect of the storage temperature in the emulsion particle size

The real-life stability of emulsions is generally determined at 4C, but in order to predict long term stability the emulsion may be stressed at 37C (Figure 4). Differences Between Emulsions A frequent question that poultry producers ask is why the same type of emulsion can have variation in colors or other characteristics. In most cases the color of the emulsion is triggered by the color of the antigen used in the formulation. Bacterial and viral antigens have different colors and intensities as a result of the medium in which they were

The Best Type of Emulsion for Poultry Defining the best type of emulsion for a poultry vaccine is a complex task. This decision must be based according to the specific requirements and challenges on the poultry farm, although sometimes this decision is based more on economic factors than on vaccine performance. Nevertheless, a good beginning is identifying the advantages and disadvantages that emulsion vaccines have to offer. Vaccine Efficacy and Duration of the Immunity in Poultry Emulsions Sometimes there are unrealistic expectations of the performance of the emulsions in the

field. These false expectations are originated by the misinterpretation of vaccine efficacy and duration of the immunity terms. Vaccine efficacy is determined or defined by the direct comparison between vaccinated and unvaccinated chickens in similar testing conditions. It is based by the evaluation of a protective indicator which could be the rate of infection, clinical signs, mortality or any other adequate variable; this study is usually conducted during licensing. For the other side, duration of the immunity may be defined as the period of time that the emulsion is able to induce in the chicken adequate immunity to reduce the incidence of disease. Lifelong immunity is not always provided by either natural infection or vaccination; therefore the duration of the immunity varies according the type of emulsion, quality of vaccine application, the breed of chicken, ock health status, climatic conditions, age, infectious challenges and combinations of the above. This is why the design and implementation of an adequate vaccination program according to the needs of the poultry complex plays a definitive role.

Summary A hundred years ago, Le Moignie and Pinoy introduced the first emulsion formulation and it still remains in use. Emulsions are generally used to enhance the immune response to poorly immunogenic antigens and/or to prolong the duration of the immune response to an antigen. Three types of vaccine emulsions are the most frequently used in poultry OW, WO and WOW. The choice of the emulsion type or its combinations should be based on the needs of the poultry operation. Some poultry breeds may be more sensitive than others to certain oils and formulations: broiler breeders vs. layers. Knowledge of the kind of microorganisms prevalent on the farm along with the level of bacterialviral challenge are also important variables to be considered to define the vaccination schedules and vaccine combinations. Specific diseases, endemic zones or areas with high challenges, a balance between side effects and duration of the immunity must be defined. Lifelong immunity is not a very realistic expectation; but with the development of an adequate vaccination program in conjunction with a good quality combination of vaccines, and the implementation of adequate

biosecurity in the farm, it is very likely that the chicken ocks will develop the best immune protection to cover the period of life when they will be the most vulnerable to acquire the disease. References 1. Lombard M., Pastoret P.P., and Moulin A.M. A brief history of vaccines and vaccination. 2007. Rev. sci. tech. Off. int. Epiz. 26(1):2948. 2. Petrovsky N. and Aguilar J.C. 2004. Vaccine adjuvants: Current state and future trends. Immunology and Cell Biology. 82: 488-496. 3. Aucouturier J., Dupuis L. and Ganne V. 2001. Adjuvants designed for veterinary and human vaccines. Vaccine 19: 266-2672 4. Marciani D.J. 2003. Vaccine adjuvants, role and mechanisms of action in vaccine immunogenicity. Drug discovery today. 8(20):934943. 5. Robins M.M., Watson A.D. and Wilde P.J. 2002. Emulsions creaming and rheology. Current opinion in Colloid and Interface Science. 7:419-425.

Notes from the Director of Sales

The production of vaccines to generate a safe and effective immune response is quite complex. Inactivated vaccines have and will continue to be a key part of the product line manufactured at the Winslow, Maine facility. The emulsion in the inactivated vaccines is as key an ingredient in the finished product as the antigen(s) used.
Tim Hopson Director of Sales, US & Canada Technology, quality, and systems Lohmann Animal Health used in the manufacturing of poultry International, Inc.

industry first looked to inactivated vaccines to improve bird health and to improve the health of the progeny. More recently these vaccines are identified as an intervention to improve food safety. The need for the bird to develop immunity to more antigens puts more pressure on having the best emulsion possible. That pressure is why Lohmann Animal Health puts tremendous focus, resources and systems in place to consistently produce and improve on quality emulsions and quality vaccines. To learn more about our emulsions or vaccines please visit our website at www.lahinternational.com or reach out to one of our Area Managers or Tech Service Manager. As always, we thank you for your business.

vaccines and emulsions must continue to advance to meet changing global needs. The poultry

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