**3. Economic size of the industry**

Autogenous vaccine market revenues in 2022 from across the world are estimated in to 129.6 million USD, and it is anticipated to reach to 231.6 million USD by the end of 2033, a growth of 5.4% compounded annual growth rate (CAGR) [29]. These estimations were based on the increased CAGR of 4.7% obtained between 2015 and 2022 and the increased rise in zoonotic disease incidence, rare infectious diseases, and variant emergence in livestock and companion animals [11, 29]. Furthermore, autogenous vaccines containing bacterial antigens help reduce the overall usage of antimicrobials in a complex [30] and are considered as alternatives to antibiotics in livestock [12]. This is important as judicious use of antibiotics and consumer demand for products without antibiotics such as "Raise without Antibiotics" (RWA) and "No Antibiotic Ever" (NAE) have led to a sharp decrease of antibiotics in clinical practice to limit the emergence of multidrugresistant bacteria [31]. Growth estimates of the autogenous vaccine market might decrease if the industry reaches critical mass for researching and developing a standard licensed product. One example would be that of Avian Reovirus. Variants of this virus expanded through North America and Europe in the last 10 years, and only in the US, it is estimated to have a cost of more than \$US90 million per year in the broiler industry in culls and mortality alone, while losses in the turkey industry are estimated at \$US33 million per year [32]. Thus, vaccine manufacturers are encouraged to fund and develop licensed vaccines, hence the newly developed inactivated avian reovirus vaccine including serotypes 1, 2, and 3 from a vaccine

manufacturer in the US [33], which correspond to genotypes 2, 4, and 5 under Kant classification (Dr. Sellers, personal communication).

## **4. General considerations on autogenous vaccines**

Before thinking about implementing an autogenous vaccine program in the field, the first step would be to study the characteristics of the field challenge (e.g., antigenic diversity, virulence markers), to evaluate if the current vaccination program is performed properly (e.g., review vaccination audits; ELISA titers, field and hatchery vaccination records), and to find out the presence of immunosuppression (e.g., aflatoxins, MDV, chicken anemia virus—CAV, IBDV), and whether the problem can be resolved by adjustments to existing vaccination programs with licensed vaccines. Also, it is paramount to understand the nature of the problem in the field - most of the times, a change in management can resolve the issue, make it more controllable, or synergize with other interventions, such as vaccination modifications or additions (e.g., autogenous vaccines). For instance, if having a Fowl Cholera challenge with a *Pasteurella multocida* from a serogroup different than the vaccines being used, the field veterinarian not only should review the vaccination schedule but also should evaluate the pest control program, as well as water sanitation, as possible sources of challenge. Review biosecurity and management, including but not limited to: downtime length, proper water sanitation, environment disinfection upon reception of baby chicks, proper disinfection of equipment, monitoring programs in place, and pest and insect control at the farm. After determining that the problem cannot be successfully controlled by management and/or modification of existing vaccination programs with licensed vaccines, an autogenous vaccine program should be considered for a company. Interestingly, there are some diseases that can be well managed with a program, such as Avian Reovirus and Fowl Adenovirus, but others that might or might not have success, perhaps due to unaddressed management/sanitary problems; limited immunity provided by inactivated vaccine; high number of serotypes in the field, which makes vaccine candidate selection difficult [34]; and/or high level of transfer of virulence genes (e.g., *Escherichia coli, Clostridium perfringens, Clostridium septicum*). Control of these agents might or might not benefit from an autogenous vaccine program, and in some cases, control by autogenous vaccines has been deemed as not a viable option for the industry [24]. Because of the high vaccine cost and high labor in most of North America and Europe, autogenous vaccines are most used in longlife birds, such as broiler & turkey breeders, layer breeders, and layers but not in broilers. The goal is to generate enough humoral immunity to protect the progeny during the first days of life. This is particularly useful in diseases in which infection early in life can cause important clinical signs later in life (e.g., Viral Arthritis, Inclusion Body Hepatitis, Infectious Bursal Disease). In countries with lower labor costs, for instance those in Latin America or Southeast Asia, the application of inactivated vaccines is still economically feasible in areas with need for control of fatal disease challenges (e.g., virulent Newcastle disease, Avian Influenza).

An estimate of autogenous vaccine order, personnel training, equipment investment, vaccination logistics, and monitoring costs, as well as the expected benefits from such program implementation, would have to be discussed with the upper management for a cost:benefit discussion of the program and final approval- including but not limited to: complex manager, live production

manager, broiler breeder manager, processing plant manager, and others. Upon approval of the program, the field veterinarian would find valuable the following considerations:

### **4.1 Vaccine candidate(s) selection**

A good monitoring program is necessary for proper selection of vaccine candidates. Such a program should contain comparable data across all levels of poultry production. Although most molecular classifications of poultry diseases are standardized and results are interchangeable between reference labs (i.e., serotyping of Fowl Adenovirus, *Pasteurella multocida*, *Escherichia coli*); some other pathogens can be classified using different systems among laboratories (i.e., Avian Reovirus); thus, caution should be exercised as to where clinical data should be generated and analyzed, or in the case such data can be easily converted from one system to another [35]. In case of food-borne diseases, such as *Salmonella enterica* colonizing the gut of chickens, it is recommended that a multiple strategy, considering licensed killed and live vaccines, should be used [36]. Federally licensed autogenous vaccines can be used to tailor the vaccination strategy to further decrease prevalence and level of contamination of the carcasses at the processing plant [37, 38]. As several types of salmonella can be present at different levels of the poultry production [39–41], it has been recommended to consider *Salmonella enterica* isolates/serovars found at processing plant monitoring as autogenous vaccine candidates [42]. This is because these isolates have successfully overcome the different control strategies already in place and are the most likely to find their way to human consumers and cause food-borne illness.

Another major problem when selecting the proper isolate in an ongoing autogenous vaccine program was the fact that isolation of the causative agents was extremely difficult once the program was in place. Because these agents could not be isolated, the isolates expired after 2 years of isolation and could not be used for the next batch. In the US, this problem was solved in the last Veterinary Services Memorandum (VSM) 800.69, and now isolates can be used up to 60 months from the point of isolation [43].

### **4.2 Production and implementation time**

Federally licensed autogenous vaccines would require time for being produced (6–18 months Canada/4–9 months the US) and, once available, 6–12 more months to be fully implemented in a parent stock [33, 35]. Time variation would depend on the type of autogenous vaccine. For instance, autogenous bacterins would require less time for development than autogenous viral vaccines due to more testing on the master viral seed (up to two more months), availability of raw material (e.g., specialized media, SPF eggs), regulatory requirements (e.g., state regulations, country regulations, export-import paperwork), shipping scheduling (refrigerated truck for large orders over long distances), etc. Although an autogenous vaccine can be available for use in an extremely short time when compared with classic licensed vaccines, it still requires important time to be fully implemented; thus, it is crucial to get isolates in the autogenous vaccine that are representative of the challenge in the field (**Figure 1**).

### **4.3 Vaccine reactions**

Birds react poorly to killed antigens (e.g., bacterial, viral) [44]. Thus, all killed vaccines in commercial poultry are adjuvanted for enhancing innate and adaptative *Autogenous Vaccines in the Poultry Industry: A Field Perspective DOI: http://dx.doi.org/10.5772/intechopen.110426*

### **Figure 1.**

*Timeline of avian reovirus autogenous vaccine design in Canada.*

immune responses. The most common adjuvants used are based on mineral oil and aluminum hydroxide [45]; mineral-oil-based adjuvants usually exhibit a strong reaction at the site of application and stimulate a (predominantly) robust humoral reaction with effects that can last for months but take longer to mount (~ 4 weeks); whereas aluminum-hydroxide-based adjuvants last for a shorter period of time (several weeks) but take less time to mount (~2 weeks) [45]. In essence, a mineral-oilbased adjuvant is an incomplete Freund's adjuvant and can be acquired commercially or produced in-house [46]. Because of its ability to produce strong humoral titers, lasting effects, and high cost, oil-based vaccines are predominantly used in parent stock vaccine programs across poultry. A common oil-based adjuvanted vaccine is constituted by two phases: (1) the oil phase, which is composed by the oil adjuvant plus emulsifiers and surfactants constituting two-thirds of the total volume of the vaccine and (2) the aqueous phase, which is composed by the harvest containing the antigen or antigens—also known as "fractions," which can be added from direct harvest or diluted in sterile media (e.g., 1X PBS, saline), and which constitutes the remaining third of the vaccine. These "phases" are processed and compounded in a specific way within parameters described in a document named as "Outline of Production," which has received government approval. In short, both phases are sheared and emulsified following standard protocols in specific tanks for a predetermined amount of time to reach an emulsion with the given particle size distribution. Because of different fractions, different compounding of the aqueous phase, and small modifications to manufacturing procedures, there might be some unwanted variations in the quality of the emulsion. In general terms, in the experience of the author, a non-reactive oil-based vaccine would be within the following quality parameters: less than 10 microSiemens/centimeter (μS/cm) conductivity in a WTW conductivimeter or the Drop Test as described by Aucouturier *et al.* (indicating a water-in-oil emulsion) [47, 48], and a monomodal particle size distribution of 95% within 0–10 μm measured by a microscope [49] or outsourcing for assessment with

a Mastersizer device (Malvern, Worcestershire, UK) [50]. Emulsions with particle size distribution of 95% larger than 10 μm would be reactive and less stable and would stratify after a short settling; thus, small-sized particles are considered more effective than larger ones [50–52]. Furthermore, particles smaller than 10 μM are appropriate for direct uptake by antigen-presenting cells (i.e., macrophages, dendritic cells) [53, 54]. Although rare, in the event of an adverse local vaccine reaction, it is important to rule out all other potential vaccine management errors, such as cold application of vaccine—causing cold shock in the surrounding tissues, contamination of vaccine, vaccine tube manifold, needles, blunt needles, and harsh vaccine application [45, 55]. It is recommended to keep a sample of the same vaccine sent to the farm in which the adverse reaction is observed or a bottle from the same batch number for particle size testing. Another common vaccine reaction is that of hemorrhagic hepatopathy, which has been described with commercial and autogenous bacterins containing *Salmonella enterica* serovars, with high levels of LPS in the vaccine [56]. In this scenario, unknown seeds might be responsible for a higher than usual generation of LPS, which adds another level of complexity to the issue.

### **4.4 "Antigenic dilution" and potency issues**

The term refers to "the more different antigens are included in the vaccine, the more diluted each individual antigen is within the serial" [33]. Autogenous vaccines are made from field strains that are not selected or optimized for the industrial propagation systems used for the vaccine manufacturing industry. One serial can include either bacterial or viral antigens. Both bacterins and viral autogenous vaccines contain at least one antigen; however, most include several (~2–5 antigens). Formulation examples for viral autogenous vaccines used in the field include: 2–4 antigens from different clusters of Virus A and 2–4 different serotypes from Virus B. For autogenous bacterins: 3–4 different serovars of Bacteria A (e.g., *S. enterica*) or 1–2 different serovars from two different bacteria (e.g., *Escherichia coli*, combined with *Riemerella anatipestifer*—usually used in ducks or with *Clostridium perfringens*—usually used in turkeys). An important concern from users is the limited space under the aqueous phase. Thus, the more different antigens are included in the vaccine, the more "diluted" each individual antigen is within the serial [33]. In short, most autogenous vaccines are produced in bottles of 0.5 L using an oil-based adjuvant at 0.25 mL per dose (2000 doses per bottle). This means that in 0.25 mL of dose per bird, two-thirds (~0.16 mL) would correspond to the oil phase (oil-based adjuvant plus emulsifier and surfactants) and one-third (~0.09 mL) would correspond to the aqueous phase, which contains the antigens. In this minuscule volume, antigens would have to be included at a proper antigenic concentration to elicit a satisfactory immune reaction, which most of the times require harvesting titers that might not be achieved by wild organisms because they are not adapted to industry propagation systems.

It is unclear what is the limit of antigens that can be delivered successfully at the same time. However, preliminary data show no significant negative effect on antibody levels when analyzing individual antigen vaccination versus application of all vaccines in a commercial broiler breeder program [57]. Thus, evidence suggest that antigenic level (potency) of an antigen is more relevant than the number of antigens in a particular vaccine. Although antigen at high concentrations in both viral and bacterial harvests can be diluted, only bacterial antigen can be cheaply concentrated. Viral antigen is more difficult to concentrate as it requires an ultracentrifuge that is labor-intensive and increases the costs of vaccine manufacturing. Therefore, most of

### *Autogenous Vaccines in the Poultry Industry: A Field Perspective DOI: http://dx.doi.org/10.5772/intechopen.110426*

the times, viral antigens are not concentrated, and in some cases, these field viruses do not propagate in high numbers on the factory/lab production systems (SPF eggs, cell culture) and are added undiluted to the vaccine. This quantity of antigen might not be enough to elicit the strong immunity required by the program, and usage of multiple antigens in the aqueous phase could further "dilute" the already low titers in one individual dose [58]. Because vaccine manufacturing companies rarely share production details with clients, such as the quantity of antigen of each fraction in each serial, the efficacy of the autogenous batch should be indirectly measured in the field. The most common method would be by ELISA serological monitoring. Gamble and Sellers recommend to evaluate sera ELISA titers at 3–4 weeks after completion of the priming vaccines, at 6–8 weeks after the completion of the inactivated booster series, and at the end-of-lay in broiler breeders to create a good complex-specific baseline for Avian Reovirus [33]. The opinion of the author is that this sampling strategy can be used for the monitoring of other diseases as well (e.g., *Salmonella enterica* serovar Typhimurium and Enteritidis, Fowl adenovirus, etc.), though it might be influenced not only by the antigenic content of the autogenous vaccine but also by a live challenge at the field, vaccination errors, immunosuppression of the birds, and others, and it will only provide indirect, subjective information about the antigen content of the fractions included in the vaccine. Nucleic acids can be recovered from oil-based inactivated vaccines by separating the aqueous phase from the oil phase [59], so molecular techniques (e.g., qPCR or qRT-PCR) [60] might be researched and developed as a tool to indirectly assess the amount of antigen fraction included in the autogenous vaccine.

Potency issues relate with the issue of "antigenic dilution" and can be found more frequently in viral vaccines than in bacterines, as some field isolates propagate better in embryonated eggs (e.g., Avian Reovirus), or specialized differentiated tissues like spleen (e.g., Hemorrhagic enteritis virus) rather than some of the more common production systems used for their licensed counterparts (chicken embryo fibroblasts— CEF for Avian Reovirus S1133; or MDTC-RP-19 for HEV). Because of this different propagation ability, or growth potential in medias in the case of bacterial isolates, and lack of potency studies, it is common to have important variability between different isolates harvest titers, which can be translated into different antigenic levels of the vaccine fractions within an autogenous serial, potentially under-stimulating the immunity against some serotypes over others within the same vaccine. Thus, autogenous vaccines, even when containing the same isolate (from different harvests), may not share the same efficiency [61]. Other consequences of this issue would be the limitations of monitoring between one batch of vaccine from another as even viruses from the same cluster or bacteria from the same serotype may elicit important ELISA titer differences in the field. Other factors might obscure the meaning of these serological monitoring, as it can depend on other factors (e.g., priming, homologous/ heterologous challenge, vaccination issues).

### **4.5 Order size of vaccine batch**

Multiple vaccine manufacturing company acquisitions in the last decade and the search for scale efficiencies in volume production have caused that the minimal order for an autogenous vaccine in poultry be of 200,000 doses. This order is limited by the emulsification tank batch capacity of 50 liters with a formulation of 2000 doses per 0.5 L bottle at 0.25 mL per dose. This is important as small operations (across all livestock industries and aquaculture, not only poultry) require lower number of dosages and would have to purchase a higher total order than the one required. This represents an important market opportunity for a new competitor created by the large mergers of vaccine manufacturer companies in the last two decades.

### **4.6 Pathogen evolution**

Multiple factors can influence the evolution of the agent in an operation with an autogenous vaccine program. These include but are not limited to: (a) agent mutation rate; (b) prevalence of the agent in the environment/resistance to disinfectants; (c) ability to prime and type of priming; (d) source of the challenge/reintroduction of pathogen; (e) level of agent shedding in vaccinated individuals or the progeny of vaccinated individuals.


lacking of proper live priming to accompany an autogenous vaccine program would be the lack of proper mucosal immunity [45, 76].

