In the face of reducing reliance on in-feed antibiotics, a greater emphasis has been placed on the development of vaccines. Adjuvants play important role in vaccine refinement. Apart from conventional adjuvants, others, like cytokines and toll-like receptors (TLR), have been evaluated experimentally. For example, Pam3CSK4, Polyinosinic-polycytidylic acid are used as immunostimulatory agents which upregulate the expression of type 1 IFNs against avian influenza. The use of TLR4 (lipopolysaccharide; LPS), TLR7 (Loxoribine) and TLR21 (CpG-ODN) agonists have shown good results when used as adjuvants with the avian influenza vaccine. The LPS (TLR4) was used as an adjuvant and liposome as intranasal antigen delivery system for Newcastle's disease (NDV) antigen, which induced significant increases in secretory IgA in tracheal lavage fluid and serum IgG levels. In a challenge study it provided 80% protection against virulent challenge. When resiquimod (TLR7) was used as an adjuvant with inactivated NDV, this potentiated the protective response to 100% against virulent NDV challenge, while protection was only 80% in the vaccine alone group. When 64CpG-plasmids (TLR21) were co-administered with inactivated AIV H5N2 vaccine in chickens, it led to a higher HI titre at two and four weeks of age, and provided 100% protection against virulent challenge of avian influenza. The TLR 3 agonist (poly I:C) showed good results against NDV and avian reovirus. Other TLR agonists and cytokines used against NDV include LPS, loxoribine, CpG-ODN and ChIFN-α, IFN- γ (with an in ovo vaccine). Immunostimulatory adjuvants like LPS, chicken TLR21, have been tried with Marek's disease vaccine, and TLR 5 agonist was used against Salmonella enteritidis and Eimeria tenella. Although the study of cytokines and TLRs in avian species is still in its infancy when compared to that of mammals, huge strides have been made in recent years. In this article, recent studies involving the assessment of immunostimulatory adjuvants in poultry are reviewed.

Oligodeoxynucleotides (ODNs) containing cytosine-phosphate-guanosine sequence (CpG) is considered as an immune stimulator when it is fed to animals. These synthetic molecules mount different immune responses in the animals including mice, chickens, ducks, dogs and horses. CpG ODNs induce specific antigenic immunity against co-administered vaccines and are well tolerated in healthy individuals and are capable of stimulating toll-like receptors (TLRs) such as TLR-9 to activate innate immunity. The CpG ODNs can be used as an adjuvant in different vaccines synthesised specifically for poultry diseases caused by viruses and bacteria. In chickens, CpG ODNs stimulate TLRs involved in humoral immunity. CpG ODNs have been used as mucosal vaccine adjuvants against several pathogens, including avian influenza and Newcastle disease. The CpG ODNs function to protect the chickens from Newcastle disease by producing plasma dendric cells (pDCs) which ultimately produce interferons (INFs). The inoculation of CpG ODNs along with the cationic microparticles and DNA vaccine for infectious bursal disease virus result in the influx of T cells and a reduction of antigen load. When CpG ODNs are used against avian leucosis, they result in significantly higher antibody titres. In many other vaccines e.g., infectious laryngotracheitis, infectious bronchitis, herpes, viral enteritis, Marek's disease virus, E. coli and Salmonella spp. including CpG ODNs exhibit immunostimulatory effects. In conclusion, CpG ODNs may be used as effective adjuvants in viral, bacterial and parasitic vaccines in poultry.

Toll-like receptors (TLRs) play a principle role in distinct pathogen recognition and in the initiation of innate immune responses of the intestinal mucosa. Activated innate immunity interconnects downstream with adaptive immunity in complex feedback regulatory loops. Intestinal disease might result from inappropriate activation of the mucosal immune system driven by TLRs in response to normal luminal flora.