ABSTRACT

Viral inactivated vaccines play a fundamental role in animal heath, both for individual protection and for the induction of antibodies that will be passively transmitted to the progeny. Depending on the field challenges, it is eventually necessary to combine more than one etiology. Inactivated polyvalent vaccines with appropriate combinations to ensure adequate and balanced responses may induce a satisfactory and long-lasting immune response. This study aimed to evaluate the antibody responses of a new oil-based inactivated pentavalent viral vaccine for intramuscular administration containing a newly developed immunomodulator, and compare it with conventional vaccines through antibody responses to the same antigens by ELISA in successive weeks after administration. Mortality rate, weight gain, egg production and hatching were also determined to evaluate safety. The results obtained revealed significant differences (p<0.05) between the group that received the new vaccine and the control group, presenting persistent and long-lasting IgG (IgY) antibodies in specific pathogen free (SPF) chickens for 146 days. In addition, there was a statistically significant (p<0.05) difference in the serological response of the new pentavalent vaccine in commercial poultry in relation to the monovalent commercial vaccines for infectious bronchitis, avian metapneumovirus, Newcastle disease, Gumboro, and reovirus fractions. There was no change in the productive parameters evaluated when compared to the conventional vaccine or the control.

Keywords:
Immunomodulator; breeders; serology; vaccine

INTRODUCTION

Vaccination of breeder hens with inactivated polyvalent viral vaccines is widely used in industrial poultry farming, as it results in two important benefits. It both contributes to the protection of the poultry due to the high levels of circulating antibodies generated, while also participating in the production and transfer of part of this immunity to progeny (maternal antibodies) (Marangon & Busani, 2007Marangon S, Busani L. The use of vaccination in poultry production. Revue Scientifique et Technique (International Office of Epizootics) 2007;26(1):265-74.).

Due to increases in field challenges and to produce a more complete maternal immunity, Brazilian breeder farmers have adopted broad immunoprophylactic programs (Gomes, 2022). Thus, they make use of combinations of inactivated polyvalent vaccines to carry out the desired immunoprophylactic program and achieve a satisfactory duration of immunity, leading to the need for multiple injectable administrations. Such application is usually performed intramuscularly and, in cases of double application, each side of the breast muscle of the poultry receives an injectable application. In addition to the labor intense activity of individual handling of poultry, it is extremely common for the injectable vaccine to produce lesions at the application site, causing stress to the birds. Thus, the concentration of several antigens in the same product is desirable, as it potentially reduces the multiple administrations of vaccines by intramuscular route (Droual et al., 1990Droual R, Bickford AA, Charlton BR, et al. Investigation of problems associated with intramuscular breast injection of oil-adjuvanted killed vaccines in chickens. Avian Diseases 1990;34(2):473-8. https://doi.org/10.2307/1591439
https://doi.org/10.2307/1591439... ).

Vaccines emulsified in oily adjuvants enhance and standardize the humoral response previously induced by live vaccines, thus reducing the workforce and stress resulting from frequent vaccinations (Schijns, 2000Schijns VE. Immunological concepts of vaccine adjuvant activity. Current Opinion Immunology, 2000;12(4):456-63. https://doi.org/10.1016/S0952-7915(00)00120-5
https://doi.org/10.1016/S0952-7915(00)00... ). In an ideal inactivated vaccine, an attempt is made to satisfactorily combine immunogenicity and harmlessness. This constant search for a balance between the ability to induce a solid immune response and the reduction or absence of adverse reactions has resulted in the development of new technologies for the production, formulation, emulsification, and enhancement of the immune response with the addition of immunomodulating components (Salem & Weiner, 2009Salem A, Weiner G. CpG oligonucleotides as immunotherapeutic adjuvants: Innovative applications and delivery strategies. Advanced Drug Delivery Reviews 2009;61(3):193-4. https://doi.org/10.1016/j.addr.2008.12.003
https://doi.org/10.1016/j.addr.2008.12.0... ). The addition of immunomodulators to vaccines can maximize their effectiveness, especially when they contain inactivated or highly purified microorganisms. This modulation of the immune response results in increased efficacy and allows for a reduction in the amount of injected antigen, a reduction in the number of doses of the vaccine program, and even additions of new antigens that make the vaccine more complete (Schijns & O’Hagan, 2006).

The objective of this study was to evaluate the serological response of a new inactivated pentavalent viral vaccine plus a new emulsion immunomodulator developed for use in breeder hens in Brazil, and compare it with an existing commercial vaccine. In addition, productive parameters such as mortality, weight gain, egg production, and hatching were also measured to assess the safety of the new product.

MATERIAL AND METHODS

Study 1 - Antibody response in SPF chickens

Poultry and experimental design

A group of 50 one-day-old White Leghorn Specific Pathogen Free (SPF) chickens were individually identified and housed in four HEPA-filtered isolators at the Zoetis Animal Facility in Paulínia (SP). Each isolator had a capacity of up to 15 animals. Prior to housing, cleaning and disinfection procedures were performed. In addition, blood samples were randomly collected from four chickens to determine the absence of maternal antibodies for all diseases investigated in this study. In this initial phase, the isolators were equipped with tubular feeders, bell drinkers, shaving litter, and lamp heating. The isolators also relied on environmental enrichment to promote animal welfare. When they reached 6 weeks of age, all chickens were transferred to 13 cages. Each cage had a maximum capacity of 4 animals. The distribution of the chickens was totally randomized, and there was at least one bird from each of the treatments in each cage. During the entire experimental period, chickens were provided with bran feed ad libitum and water by properly trained professionals, following lineage recommendations. The chickens received a wing washer for individual identification that remained until the end of their life, to allow for the individual serological evaluation of the animals in each treatment group.

All animals received the same live vaccines to primer the inactivated vaccine, according to the table 1:

This protocol was submitted to the Animal Use Ethics Committee (CEUA) of Zoetis in accordance with the rules of the National Council for Animal Experimentation (CONCEA) (Brazil, 2015). It was approved and registered under code AF 009/20 before the start of the activities.

Inactivated vaccine and vaccination process

In the 9^th week of life, only the T02 poultry received a dose of the inactivated vaccine intramuscularly, with an injected volume of 0.5 mL in the left chest musculature. The poultry of the control group (T01) did not receive any viral oily vaccines. The product used in the T02 treatment was one of the pilot batches of the new pentavalent viral vaccine (Poulvac ^® Maternavac^® Ultra 5) recently licensed by the Ministry of Agriculture and Supply (MAPA).

Each of the vaccines used is described in the table 2:

Sample collection

Blood samples from the 25 chickens/treatment were collected at the 9^th, 11^th, 13^th, 15th, 17^th, and 19^th weeks of life of the animals. The collection was performed by means of a standard procedure (puncture of the wing vein) that was always conducted by the same person. After the blood sample was collected, serum separation, freezing and storage were performed. At the end of the study, all individually identified samples were sent to the JFLAB animal pathology laboratory (Campinas, SP) to perform the Enzyme-Linked Immunosorbent Assay (ELISA) test with a commercial kit from Biochek.

Laboratory Analysis

The ELISA test performed was indirect for antibody detection of Gumboro diseases (IBD), Avian Metapneumovirus (aMPV), Newcastle Disease (NDV), Infectious Bronchitis (IBV), and Reovirus (REO). The result was read on a spectrophotometer and the percentage of light transmission was evaluated by a reader software and converted to OD (optical density). The results of each sample/chicken were expressed as titers and categorized as positive or negative according to a cutoff value determined by the manufacturer of the ELISA kit.

Statistical Analysis

Initially, we carried out the descriptive analysis of all variables with the estimation of means, medians, standard deviations, and interquartile ranges. Then, the Shapiro-Wilk test for normal distribution was performed and the non-parametric approach was chosen for the data, since not all data presented normal distribution (Shapiro-Wilk p-value <0.05). Differences between treatments were verified with the Kruskal-Wallys test (non-parametric) followed by Dunn’s test to adjust for multiple comparisons. For a better visualization of the results, boxplot charts were produced, differentiating between treatments at each moment. The difference in the percentage of positives between the groups was verified with the chi-square test and the intensity of the association was verified with the estimation of relative risk. The analyses were performed in SPSS 20.0 (IBM, 2012IBM. IBM SPSS statistics for windows. Version 21.0. Armonk: IBM; 2012.), and tests were considered significant when p<0.05.

Study 2 - Serological curve in commercial poultry

Poultry and experimental design

A total of 300 12-weeks-old commercial chickens of the Novogen White Light lineage were housed in 05 pens/treatment in the Veterinary Research Support Center (CAPEV) located in the municipality of Amparo - SP. The experimental design involved three treatments with 100 chickens each, being T01 - control/placebo, T02 - Poulvac^® Maternavac^® Ultra 5, and T03 - commercial pentavalent vaccine. 20 chickens and 2 roosters were housed in each pen (1:10). 4 chickens in each pen were also randomly assigned for blood collection, weighing and necropsy of the application site at the end of the study. Prior to housing, cleaning and disinfection procedures were performed to prevent any spread of infectious or vaccine agents to the poultry of the experiment.

In the initial phase (before 12 weeks), the poultry were raised in a commercial farm with gutter type feeders, nipple drinkers, shaving litter, and heating by campanulas. Throughout the experimental period, feed was provided following lineage recommendations. All animals received the same live vaccines to primer the inactivated vaccine according to the table 3.

This protocol was submitted to the Animal Use Ethics Committee (CEUA) of Zoetis and was in accordance with the rules of the National Council for Animal Experimentation (CONCEA) (Brazil, 2015). It was approved and registered under no. B1D94/21 before the start of the activities.

Inactivated vaccine and vaccination process

In the 14^th week of life, only the T02 and T03 poultry received a dose of the inactivated vaccine intramuscularly, with an injected volume of 0.5 mL in the left chest musculature. The T01 poultry (negative control group) received a placebo formulation containing the same excipients as the T02 oily vaccine, except for the antigenic fractions. The product used in the T02 treatment was one of the pilot batches of the new pentavalent viral vaccine (Poulvac^® Maternavac^® Ultra 5) recently licensed by the Ministry of Agriculture and Supply (MAPA).

Each of the vaccines used is described in the table 4.

Sample collection

Blood samples from 4 chickens/pen were collected on the day of vaccination with the inactivated vaccines or placebo (before application) and also 13, 20, 27, 34, 41, 48, 132, 139, and 146 days after vaccination. The collection was performed by means of a standard procedure (puncture of the wing vein), and the procedure was always conducted by the same person. After the blood sample collection, serum separation, freezing, and storage were performed. At the end of the study, all identified samples were sent to the JFLAB animal pathology laboratory (Campinas, SP) to perform the ELISA test.

Laboratory Analysis

The ELISA test performed was indirect for the detection of antibodies for Gumboro, Reovirus, and Newcastle disease with the IDEXX commercial kit, and of avian Metapneumovirus, and Infectious Bronchitis with the BIOCHEK commercial kit. The result was read on a spectrophotometer and the percentage of light transmission was evaluated by a reader software and converted to OD (optical density).

Mortalities and removals

Occurring mortalities as well as poultry removed and euthanized for welfare reasons (dying poultry) were recorded daily.

Weighing

Twenty chickens from each group, as determined by the randomization plan, were individually weighed weekly.

Egg Production

The hens started to lay eggs when they were around 17-18 weeks old. From this moment onwards, all the eggs of each pen were daily harvested and counted. Moreover, from 24 weeks of age onwards, all viable eggs (without cracks, shell problems, and/or deformations) of each pen were incubated once a week for hatching evaluation.

Evaluation of the Application Site

At the end of the study, after the animals were euthanized, the application site was evaluated for the presence of possible macroscopic lesions. For this evaluation, the same poultry randomized for the weighing and blood collection procedures (20 birds/group) were necropsied.

Statistical Analysis

The mortality percentage was calculated for each treatment, with the number of poultry allocated in each pen associated with the respective treatments being used as the denominator for the calculations. Mortality results were analyzed with a mixed generalized linear model for binomial distribution with logit link. The model included the fixed effect of the treatment, as well as the random effects of the block and its interaction with the treatment. Mortality was analyzed considering the total period of the study.

Body weight, egg production, percent of egg hatching and serological results were analyzed by a general linear mixed model for repeated measures. For this analysis, the fixed effects of treatment group, experimental moment and their interactions were considered. Regarding results collected at the animal level (body weight and serological results), the random effects of the block, the interaction between block and treatment, the animal within the block and treatment, the interactions between block, treatment and experimental moment, and the error were considered. For results collected at the pen level (egg production and egg hatching percentage), the random effects of block, the interaction of block and treatment, and the error were considered. Serological results were log transformed prior to analysis, and the egg hatching percentage was arcsine square root transformed prior to analysis. The analysis considered a significance level of 5% (p≤0.05). Least squares means, standard errors, and 95% confidence limits were re-transformed (BT LSmeans) as appropriate for each experimental time point. Minimum and maximum values were calculated for each experimental moment. When there was a significant effect of the treatment group or treatment group by experimental moment (P≤0.05), experimental groups T01, T02 and T03 were compared at each moment using contrasts.

Weight gain and total egg production considering the total study period were analyzed with a general linear mixed model for binomial distribution with logit link. The model included the fixed effect of the treatment. Regarding weight gain, the random effects included block, its interaction with the treatment, and error. For total egg production, the random effects included block and error.

RESULTS

Study 1 - Serological curve in SPF poultry

Figure 1 shows the boxplot graphs of the quantification of serology in SPF poultry for IBD, REO, NDV, IBV, and aMPV in each study treatment 1 on days 14, 28, 42, 56, 70 and 84 after vaccination (ELISA - IDEXX kit).

Study 2 - Serological curve in commercial poultry

Figure 2 shows the graphs of serology quantification in commercial poultry vaccinated with Poulvac^® Maternavac^® Ultra 5 compared with a conventional pentavalent vaccine for the IBD, REO, NDV, IBV and aMPV fractions on days 0, 13, 20, 27, 34, 41, 48, 132, 139 and 146 after vaccination (ELISA - IDEXX and BIOCHEK).

Study 2 - Mortality and production data

DISCUSSION

The development of high performance and safe veterinary vaccines requires the improvement of formulations and often the use of components that can modulate the immune response to induce better performance when compared to conventional adjuvants and formulations (Manuja et al., 2013Manuja A, Manuja BK, Kaushik J, et al. Immunotherapeutic potential of CpG oligodeoxynucleotides in veterinary species. Immunopharmacology Immunotoxicology 2013;35(5):535-44. https://doi.org/10.3109/08923973.2013.828743
https://doi.org/10.3109/08923973.2013.82... ). Adjuvants and immunomodulators potentiate the presentation of immunogens to cells of the immune system in order to initiate or enhance the response through affinity with the Toll-like receptor (Vollmer & Krieg, 2009Vollmer J, Krieg AM. Immunotherapeutic applications of CpG oligodeoxynucleotide TLR9 agonists. Advanced Drug Delivery Reviews 2009;61(3):195-204. https://doi.org/10.1016/j.addr.2008.12.008
https://doi.org/10.1016/j.addr.2008.12.0... ; Fehér, 2019Fehér K. Single stranded DNA Immune modulators with unmethylated cpg motifs: structure and molecular recognition by toll-like receptor 9. Current Protein Peptide Science 2019;20(11):1060-8. https:// doi.org/10.2174/1389203720666190830162149
https:// doi.org/10.2174/138920372066619... ). Immunomodulators are also able to redirect the immune response, making it more potent and even safer, as they can reduce the deleterious effects of some components (Cserep, 2009Cserep T. Vaccination: theory and practice. In: Hocking P.M. Biology of breeding poultry. London: CAB International; 2009. p.337-90. ISBN 13-9781845933753; Scheiermann & Klinman, 2014Scheiermann J, Klinman DM. Clinical evaluation of CpG oligonucleotides as adjuvants for vaccines targeting infectious diseases and cancer. Vaccine 2014;32(48):6377-89. https://doi.org/10.1016/j.vaccine.2014.06.065
https://doi.org/10.1016/j.vaccine.2014.0... ). The formulation of this new pentavalent inactivated vaccine includes Fortilyst^®, which has an immunomodulatory action that improves the response to viral antigens (IBD, REO, NDV, IBV and aMPV).

Studies have been conducted to measure the level of transfer of maternal antibodies and it is well established that the rate of transfer varies from disease to disease. In Gumboro disease, for example, there is a transfer of up to 70% of the mother’s antibody level to its progeny (Gharaibeh et al., 2008Gharaibeh S, Mahmoud K, Al-Natour M. Field evaluation of maternal antibody transfer to a group of pathogens in meat-type chickens. Poultry Science 2008;87(8):1550-5.). This maternal immunity is an important tool for the early protection of the progeny since there invariably are viral challenges during the first days of chicks’ lives. The greater the amount of antibodies produced by the inactivated vaccine in the breeder, the greater the level of antibodies transferred to the progeny tends to be (Ahmad & Akhter, 2003Ahmad Z, Akhter S. Role of maternal antibodies in protection against infectious bursal disease in commercial broilers. International Journal of Poultry Science 2003;2(4):251-5. https://doi.org/10.3923/ijps.2003.251.255
https://doi.org/10.3923/ijps.2003.251.25... ).

The genetic variability of the field challenge is also an important aspect in the protection of maternal immunity, and in Brazil, in addition to classical viruses, Gumboro variant viruses have also been also identified (Muniz et al., 2018Muniz EC, Verdi R, Jackwood DJ, et al. Molecular epidemiologic survey of infectious bursal disease viruses in broiler farms raised under different vaccination programs. Journal of Applied Poultry Research 2018;27(2):253-61. https://doi.org/10.3382/japr/pfx043
https://doi.org/10.3382/japr/pfx043... ; de Fraga et al., 2019Fraga AP, Gäf T, Coltro VP, et al. Phylodinamic analyses of Brazilian antigenic variants of infectious bursal disease virus. Infection, Genetics and Evolution 2019;73:159-66.). Thus, in addition to the amount of antibodies, their quality and diversity are also important factors in the protection of the progeny.

Inactivated vaccines for Gumboro disease aim to stimulate the production of high levels of antibodies in breeders. These should be used in poultry that have already been sensitized by live vaccines or even by the field virus, which can be evaluated serologically (Dey et al., 2019Dey S, Pathak DC, Ramamurthy N, et al. Infectious bursal disease virus in chickens: prevalence, impact, and management strategies. Veterinary Medicine 2019;10:85-97. https://doi.org/10.2147/VMRR.S185159
https://doi.org/10.2147/VMRR.S185159... ). Considering that the use of inactivated vaccines generally occurs from the 12^th week of life onwards, it is known that within a primary vaccination program with live vaccines, the evaluation of the serological curve is an important parameter of vaccine efficacy. In addition, the correlation between poultry protection and antibody levels has already been described in the literature (Maas et al., 2001Maas RA, Venema S, Ori HL, et al. Efficacy of inactivated infectious bursal disease (IBD) vaccines: comparison of serology with protection of progeny against IBD virus strains of varying virulence. Avian Pathology 2001;30(4):345-54. https://doi.org/10.1080/03079450120066359
https://doi.org/10.1080/0307945012006635... ; Bolis et al., 2003Bolis DA, Paganini FJ, Simon VA, et al. Gumboro disease: evaluation of serological and anatomopathological responses in vaccinated broiler chickens challenged with very virulent virus strain. Brazilian Journal of Poultry Research 2003;(5):2. https://doi.org/10.1590/S1516-635X2003000200008
https://doi.org/10.1590/S1516-635X200300... ).

In the construction of a vaccination program for breeding animals, it is extremely important to think not only about the protection of the breeders themselves, but also about the protection of the progeny. This is particularly true when talking about Gumboro disease, as chicks in their first weeks of life are dependent on maternal antibodies for effective protection against it (Van Den Berg et al., 2000). The booster effect of vaccination with Poulvac^® Maternavac^® Ultra 5 for the IBD fraction was measured and is shown in figure 1a. In the comparison between the two vaccines, after 13 days of vaccination, it was possible to observe higher average titers (p≤0.05) in the T02 group when compared to T01 and T03 groups (Figure 2a). The correlation of higher antibody titers in hens leading to higher antibody titers in the progeny is well established and, consequently, these chicks will be better protected.

Malabsorption syndrome and viral arthritis, both caused by the Reovirus, are challenges observed in Brazilian poultry farming that can cause great losses to producers and companies when neglected (Souza et al., 2018Souza SO, Carli S, Lunge VR, et al. Pathological and molecular findings of avian reoviruses from clinical cases of tenosynovitis in poultry flocks from Brazil. Poultry Science 2018;97(10):3550-5. https://doi.org/10.3382/ps/pey239
https://doi.org/10.3382/ps/pey239... ). During the first weeks of life, chicks are highly dependent on maternal antibodies to control Reovirus and Gumboro health challenges in the field (Edison et al., 1979Edison CS, Page RK, Fletcher OJ, et al. Vaccination of broiler breeders with a tenosyvitis virus and turkey herpesvirus. Poultry Science 1979;58:1490-7. https://doi.org/10.3382/ps.0581490
https://doi.org/10.3382/ps.0581490... ; Van Der Heide & Page, 1980; Jones & Georgiou, 1984Jones RC, Georgiou K. The temporal distribution of an arthrotropic reovirus in the leg of the chicken after oral infection. Avian Pathology 1984;14(1):75-85. https://doi.org/10.1080/03079458508436209.
https://doi.org/10.1080/0307945850843620... ; Jones & Nwajei, 1985; Martins & Resende, 2009Martins NRS, Resende JS. Adenoviroses, reoviroses, rotaviroses e viroses intestinais. In: Berchieri Júnior A. Silva EM, Di Fabio, J, editors. Doenças das aves. 2th ed. São Paulo: Facta; 2009. p.677-710.). Therefore, immunoglobulins transferred from breeders can avoid significant losses (particularly in chicks’ locomotor and digestive systems) if they present a high homology with field challenges (Rau et al., 1980Rau WE, Van Der Heide L, Kalbac M, et al. Onset if progeny immunity against viral arthritis/tenosynovitis after experimental vaccination of parent breeder chicken and cross-immunity against six reovirus isolates. Avian Diseases 1980;24(3):648-657.; Kant et al., 2003Kant A, Balk F, Born L, et al. Classification of dutch and german avian reoviruses by sequencing the sigma C protein. Veterinary Research 2003;34(2):203-12.; Liu et al., 2003Liu HJ, Lee LH, Hsu HW, et al. Molecular evolution of avian reovirus: Evidence for genetic diversity and reassortment of the S-class genome segments and multiple cocirculating lineages. Virology 2003;314:336-49. https://doi.org/10.1016/S0042-6822(03)00415-X
https://doi.org/10.1016/S0042-6822(03)00... ; Day & Pantin-Jackwood, 2007Day JM, Pantin-Jackwood MJ. Sequence and phylogenetic analysis of the S1 genome segment of turkey-origin reoviruses. Virus Genes 2007;35:235-42. https://doi.org/10.1007/s11262-006-0044-1
https://doi.org/10.1007/s11262-006-0044-... ; Jones, 2008; Sellers, 2017Sellers HS. Current limitations in control of viral arthritis and tenosynovitis caused by avian reoviruses in commercial poultry. Veterinary Microbiology 2017;(206):152-6. https://doi.org/10.1016/j.vetmic.
https://doi.org/10.1016/j.vetmic... ). In Figure 1b of study 1, it is possible to observe the booster effect related to the REO fraction from 28 days after vaccination onwards. In study 2, the mean titers were similar (p≤0.05) for the T02 and T03 groups and differed significantly from T01 (Fig 2b). These findings show that the Poulvac ^® Maternavac ^® Ultra 5 vaccine generates high titers in poultry and, consequently, presents greater potential for progeny production.

In Brazil, Newcastle disease was included in a National Contingency Plan in 2009, which included the determination of the sacrifice of affected poultry to keep the national poultry flock free from it (Brazil, 2009). It is also considered a notifiable disease and has an impact on the international marketing of poultry products (Brazil, 1994).

The evaluation of the efficacy of inactivated vaccines against Newcastle disease is based on the determination of circulating antibodies, and the induction of seroconversion in long-lived poultry has a fundamental role in the protection of the breeder, while also contributing to the progeny in their first weeks of life through passive immunity (Dimitrov et al., 2017Dimitrov KM, Afonso CL, Yu Q, et al. Newcastle disease vaccines-A solved problem or a continuous challenge? Veterinary Microbiology 2017;Jul,206:126-36. https//doi.org/10.1016/j.vetmic.2016.12.019.
https//doi.org/10.1016/j.vetmic.2016.12.... ). In Figure 2c of study 2, it is possible to observe that the average titers for NDV were similar (p≤0.05) for the T02 and T03 groups throughout the evaluation period. The result obtained 13 days after vaccination shows the booster effect. On the other hand, in Figure 1c of study 1 with SPF poultry, the induction of seroconversion of the group vaccinated with Poulvac^® Maternavac^® Ultra 5 was evident 42 days after vaccination, with the maintenance of the antibody plateau being observed after this age. This difference in the speed of seroconversion for NDV comparing SPF and commercial poultry may be related to the response of the two genetic strains used in the studies (Zou et al., 2020Zou A, Nadeau K, Wang PW, et al. Accumulation of genetic variants associated with immunity in the selective breeding of broilers. BMC Genetics 2020;21(1):5. https://doi.org/10.1186/s12863-020-0807-z.
https://doi.org/10.1186/s12863-020-0807-... ).

The etiological agent that causes avian infectious bronchitis belonging to the family Coronaviridae, genus Gammacoronavirus (group 3), subgenus Igacovirus, is characterized by having a positive and non-segmented sense, single RNA-stranded genome. Chicken infectious bronchitis is a highly contagious acute respiratory disease, which affects poultry of the Gallus species and has great economic importance. The most common symptoms include nasal discharge, lacrimation, cough, sneezing, rales, and lethargy. In addition to respiratory symptoms, the avian infectious bronchitis virus can cause reproductive changes and nephritis (Ignatovic & Sapatos, 2000Ignatovic J, Sapatos S. Avian Infectious bronchits virus. Revue Scientifique et Technicque 2000;9:493-508. https://doi.org/10.20506/rst.19.2.1228.
https://doi.org/10.20506/rst.19.2.1228... ; Cavanagh & Gelb, 2008Cavanagh D, Gelb JR J. Infectious bronchits. In: SAIF Y.M. Diseases of poultry. 12nd ed. Hoboken: Blackwell Publishing; 2008. p.117-8. ISBN 10- 9780813807188).

Cavanagh (2003Cavanagh D. Severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus. Avian Pathology 2003;32(6):567-82.) described that the efficacy of an inactivated vaccine for protection against IBV infections depends heavily on a suitable program with primary vaccinations with live vaccines. This characteristic could be observed in the study conducted by Santos et al. (2019Santos RM, Fernando FS, Montassier MFS, et al. Memory immune responses and protection of chickens against a enteropathogenic infectious bronchitis virus strain by combining live heterologous and inactivated homologous vaccines. The Journal of Veterinary Medical Science 2019;81(4):612-9. https://doi:10.1292/jvms.18-0065
https://doi:10.1292/jvms.18-0065... ), where the activation of immune memory after the use of a live attenuated vaccine (IBV-Mass) followed by the administration of an inactivated vaccine (inat IBV-BR1) conferred protection against the challenge with the IBV-BR1 strain. This protection against the challenge was only complete due to the use of the inactivated vaccine, since the live attenuated vaccine (IBV-Mass) would be unable to guarantee complete protection by itself.

In study 2, when comparing the two inactivated vaccines in commercial poultry, it was possible to observe higher averages (p≤0.05) for the fraction of IBV from day 13 post-vaccination onwards in the T02 group when compared to the T01 and T03 groups by the ELISA method (Figure 2d). Moreover, it was possible to observe the long-lasting effect of the vaccine response during the evaluation period (146 days post-vaccination), in which titers greater than 833 (reactivity threshold or kit cut-off) were observed. On the other hand, in study 1 with SPF poultry, we observed the induction of seroconversion from 28 days after vaccination onwards (Figure 1d).

Vaccination programs with live vaccines followed by inactivated vaccines are consolidated strategies and are widely used in industrial poultry production (Bhuiyan et al., 2021Bhuiyan MSA, Amin Z, Rodrigues KF, et al. Infectious Bronchitis Virus (Gammacoronavirus) in Poultry Farming: Vaccination, Immune Response and Measures for Mitigation. Veterinary Sciences 2021;8(11)273. https://doi.org/10.3390/vetsci8110273.
https://doi.org/10.3390/vetsci8110273... ) for various poultry diseases, including IBV. The high level of antibodies generated by vaccination with injectable inactivated products preceded by immunization with a live vaccine plays a decisive role in the protection of long-lived poultry, as this immunity is very effective to control systemic IBV infection, even in cases of nephropathogenic pathotypes (Landman et al., 2002; Santos et al., 2019Santos RM, Fernando FS, Montassier MFS, et al. Memory immune responses and protection of chickens against a enteropathogenic infectious bronchitis virus strain by combining live heterologous and inactivated homologous vaccines. The Journal of Veterinary Medical Science 2019;81(4):612-9. https://doi:10.1292/jvms.18-0065
https://doi:10.1292/jvms.18-0065... ).

It is known and reported in the literature and in field observations that aMPV is a weak inducer of seroconversion, but when there is high infection pressure, serology is the most practical method to perform risk monitoring and analysis (Park et al., 2011Park JE, Lee DW, Shin HJ. Serological survey of antibodies against avian metapneumovirus in Korean chicken flocks. Journal of Applied Poultry Research 2011;20(4):573-6. https://doi.org/10.3382/japr.2010-00185
https://doi.org/10.3382/japr.2010-00185... ). Furthermore, the OIE guide (2018) recommends that the evaluation of the efficacy of inactivated vaccines that have aMPV in their composition should be carried out by assessing the serological response of animals, given the great difficulty of reproducing challenge conditions for the evaluation of variables related to aMPV infection (Turpin et al., 2002Turpin EA, Perkins LEL, Swayne DE. Experimental infection of turkeys with avian pneumovirus and either newcastle disease virus or Escherichia coli. Avian Diseases 2002;46(2):412-22. https://doi.org/10.1637/0005-2086(2002)046[0412:eiotwa]2.0.co;2.
https://doi.org/10.1637/0005-2086(2002)0... ; Gough & Jones, 2008Jones RC. Viral arthritis. In: Saif YM. Diseases of poultry. Hoboken: Blackwell Publishing; 2008. p.309-10. ISBN 10- 9780813807188). Live vaccines, without the presence of challenge, do not result in high circulating antibody titers, and seroconversion is quite slow when compared to other diseases. In addition, aMPV is difficult to isolate, as it stays present for a short period of time, being detected only at the beginning of clinical signs in infected poultry (Cook & Cavanagh, 2002Cook JKA, Cavanagh D. Detection and differentiation of avian pneumoviruses (metapneumoviruses). Avian Pathology 2002;31(2):117-32. https://doi.org/ 10.1080/03079450120118603
https://doi.org/... ).

The greatest differences between the designs of the serological curves of the two studies were observed in the aMPV fraction. In study 1 (Figure 1e) with SPF poultry, a slow seroconversion was observed, consolidating in the last week, that is, 84 days after vaccination. On the other hand, in study 2 (Figure 2e) the result obtained as soon as 7 days after vaccination shows the booster effect of the Poulvac^® Maternavac^® Ultra 5 vaccine. Moreover, it was possible to verify the long-lasting effect of the vaccine response, with significantly high titers until 146 days after vaccination. The curve of study number 2 indicates that in addition to the effect of the vaccine, there may also have been a field challenge, since the poultry of the experiment were commercial and were recreated in a field environment with the possibility of being exposed to a field virus.

Studies published in Brazil have reported seroconversion in unvaccinated poultry, proving that the agent is present in industrial farms (Peres et al., 2006Peres MF, Carrijo AS, Higa JA, et al. Evidência sorológica de Pneumovírus aviário em lotes de frangos de corte em municípios de Mato Grosso do Sul. Pesquisa Veterinária Brasileira 2006;26(4):254-8. https://doi.org/10.1590/S0100-736X2006000400011
https://doi.org/10.1590/S0100-736X200600... ) and in free-range poultry (Sales et al., 2010Sales TS, Herval EFG, da Silva PS, et al. Frequência de anticorpos contra metapneumovírus aviário em criações industriais e de galinhas de quintal no polo avícola da Bahia. Ciência Animal Brasileira 2010;11(3):718-723. https://doi.org/10.5216/cab.v11i3.6800
https://doi.org/10.5216/cab.v11i3.6800... ). In any case, this serological profile observed in study 2 indicates the excellent ability of vaccines to produce protection, since the appearance of clinical signs of the disease was not observed at any time during the study. The intense seroconversion demonstrated that there was exposure to the agent, but the poultry were protected by the vaccination program with live and inactivated vaccines and the serological response was enhanced in T02 by the immunostimulant (Bode et al., 2011Bode C, Zhao G, Steinhagen F, et al. CpG DNA as a vaccine adjuvant. Expert Reviews of Vaccines 2011;10(4):499-511.).

The induction of early - and long - term antibodies and their duration in poultry vaccinated with the new inactivated pentavalent vaccine was observed for each of the antigenic fractions present in the formulation of the Poulvac^® Maternavac^® Ultra 5, considering the serological responses of the animals included in both studies. It was possible to establish the serological response curve of the neutralizing antibodies that are the basis of the evidence of efficacy of this type of product. Also, it is important to note that before the administration of the inactivated vaccine, the poultry in groups T02 and T03 were submitted to the administration of an extensive program of primary vaccines with live vaccines, which aimed to mimic the real field conditions in which the vaccine will be used when it is available on the market (Brazil, 2006).

The use of Fortilyst^® (immunomodulator) was shown to be effective to induce the appropriate antigenic immune response (Figures 1 and 2). In addition, it is presented as an alternative for the preparation of multivalent products with new antigenic fractions in the formulation of vaccines (Shirota & Klinman, 2014Shirota H, Klinman DM. Recent progress concerning CpG DNA and its use as a vaccine adjuvant. Expert Reviews of Vaccines 2014;13(2):299-312. https://doi.org/10.1586/14760584.2014.863715
https://doi.org/10.1586/14760584.2014.86... ; Shirota et al., 2015). Moreover, study 2 demonstrated that the administration of a single dose of the Poulvac ^® Maternavac ^® Ultra 5 vaccine in breeder hens did not cause clinical, behavioral or increased animal mortality changes (Table 5). In the macroscopic evaluation, no signs of tissue necrosis, edema, changes in muscle color, presence of nodules or hemorrhage were observed. In the three groups, poultry with neovascularization near the application site were observed, which is expected for inactivated vaccines in general.

Weight gain throughout the study was similar between the groups, with no negative impact on the weight of vaccinated poultry being verified, especially when compared with the negative control group (Table 6 and Figure 3). Body weights of the Poulvac^® Maternavac^® Ultra 5 group (T02) were significantly higher than those of negative control group (T01) from day 77 until the end of the study, although there were no significant differences in average daily weight gain. The production of eggs by poultry vaccinated with the tested vaccine was similar to that of the control groups (Negative - T01 and Positive - T03), considering the total average of eggs had no significant differences between groups (Table 7). The percentage of egg hatching was also similar between poultry vaccinated with Poulvac^® Maternavac^® Ultra 5 (T02) and the control groups (Negative - T01 and Positive - T03) (Figure 4), with no significant differences between groups. All these data prove the safety of the vaccine.

The oligonucleotide (ODN) called CpG has gained considerable prominence among the various types of existing immunomodulators. CpG oligonucleotides are small single-stranded DNA molecules of defined sequence and size containing nucleotide bases of Cytosine (C) followed by Guanine (G) (Krieg, 2002Krieg AM. From A to Z on CpG. Trends in Immunology 2002;23(2):64-5.; Krieg, 2012). This nucleotide sequence is widely found in the genetic material of viruses and bacteria, but rarely found in vertebrate genomes. One difference between the sequence of CpG oligonucleotides found in vertebrates and the CpG ODNs found in viruses and bacteria is that in vertebrates this sequence is methylated. The synthetic CpG ODN molecule mimics sequences naturally found in viruses and bacteria. Just as the DNA of a pathogen, synthetic CpG ODN has been shown to stimulate innate protection as well as adaptive immune responses, therefore serving as an immunomodulator (Klinman et al., 2009Klinman DM, Klaschik S, Sato T, et al. CpG oligonucleotides as adjuvants for vaccines targeting infectious diseases. Advanced Drug Delivery Reviews 2009;61(3):248-55.; Salem & Weiner, 2009Salem A, Weiner G. CpG oligonucleotides as immunotherapeutic adjuvants: Innovative applications and delivery strategies. Advanced Drug Delivery Reviews 2009;61(3):193-4. https://doi.org/10.1016/j.addr.2008.12.003
https://doi.org/10.1016/j.addr.2008.12.0... ; Bode et al., 2011Bode C, Zhao G, Steinhagen F, et al. CpG DNA as a vaccine adjuvant. Expert Reviews of Vaccines 2011;10(4):499-511.).

There are several publications describing and evaluating 3 different classes of CpGs, referred to as class A, B and C CpGs. Most studies in the literature have used class B CpGs as adjuvants in vaccines (Vollmer et al. 2004Vollmer J, Weeratna R, Payette P, et al. Characterization of three CpG oligodeoxynucleotide classes with distinct immunostimulatory activities. European Journal of Immunology 2004;34(1):251-62. https://doi.org/10.1002/eji.200324032
https://doi.org/10.1002/eji.200324032... ), but more recently, a new, different class has been developed. This new compound has undergone modifications to improve its immunostimulatory activity and is called ‘E-modified P-class CpG’ or ‘EP-CpG’ (Samulowitz et al, 2010Samulowitz U, Weber M, Weeratna R, et al. A novel class of immune-stimulatory CpG oligodeoxynucleotides unifies high potency in type I interferon induction with preferred structural properties. Oligonucleotides 2010;20(2):93-101. https://doi.org/10.1089/oli.2009.0210
https://doi.org/10.1089/oli.2009.0210... ).

Modified CpG ODNs associated with viral antigens in an oily emulsion enable better antigen presentation by stimulating Th1 cells via TLR-9, thereby improving the immune response with superior antibody production (McCluskie & Krieg, 2006McCluskie MJ, Krieg AM. Enhancement of infectious disease vaccines through TLR9-dependent recognition of CpG DNA. Current Topics on Microbiology and Immunology 2006;311:155-78. https://doi.org/10.1007/3-540-32636-7_6
https://doi.org/10.1007/3-540-32636-7_6... ; Jurk & Vollmer, 2007Jurk M, Vollmer J. Therapeutic applications of synthetic CpG oligodeoxynucleotides as TLR9 agonists for immune modulation. BioDrugs 2007;21(6):387-401.). The synthetic CpG ODN interacts with Toll-Like Receptor 9 receptors present in the innate immune system cells of poultry and act as agonists. The TLR-9 agonist activity of synthetic CpG ODN was shown to stimulate both innate protection and adaptive immune response (Donhauser et al., 2010Donhauser N, Helm M, Pritschet K, et al. Differential effects of P-class versus other CpG oligodeoxynucleotide classes on the impaired innate immunity of plasmacytoid dendritic cells in HIV type 1 infection. AIDS Research and Human Retroviruses 2010;26(2):161-71. https://doi.org/10.1089/aid.2008.0278.
https://doi.org/10.1089/aid.2008.0278... ; Meng et al., 2016Meng Z, Zhang X, Pei R, et al. Combination therapy including CpG oligodeoxynucleotides and entecavir induces early viral response and enhanced inhibition of viral replication in a woodchuck model of chronic hepadnaviral infection. Antiviral Research 2016;125:14-24. https://doi.org/10.1016/j.antiviral.2015.11.001
https://doi.org/10.1016/j.antiviral.2015... ).

Therefore, based on experimental studies in both SPF and commercial poultry, the Poulvac^® Maternavac^® Ultra 5 vaccine was shown to be safe and have proven efficacy in the protection against diseases caused by the IBD, REO, NDV, IBV and aMPV viruses through the induction of seroconversion when used in revaccination protocols in the target species, as recommended in the product package insert. Multivalent protection was demonstrated through seroconversion curves and was enhanced by Fortilyst^® (immunomodulator). The results showed that the addition of the immunomodulator was essential to induce adequate levels of seroconversion and contribute to the enhancement of the immune response, in line with previous literature reports on the immunomodulatory effect of this molecule.

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