Enhancement of protective efficacy of innate immunostimulant based formulations against yolk sac infection in young chicks.

This study was conducted to characterize and compare the protective effects of various innate immune stimulants against yolk sac infection (YSI) caused by an avian pathogenic Escherichia coli in young chicks. The immune stimulants were administered alone or in various combinations of unmethylated CpG oligodeoxynucleotides (CpG), polyinosinic:polycytidylic acid (Poly I:C), and avian antimicrobial peptides (AMPs). Routes included in ovo or in ovo followed by a subcutaneous (S/C) injection. CpG alone and in combination with Poly I:C, truncated avian cathelicidin (CATH)-1(6-26), avian beta defensin (AvBD)1, and CATH-1(6-26) + AvBD1, were administered in ovo to 18-day-old embryonated eggs for gene expression and challenge studies. Next, CpG alone and the potentially effective formulation of CpG + Poly I:C, were administrated via the in ovo route using 40 embryonated eggs. At 1 day post-hatch, half of each group also received their respective treatments via the S/C route. Four hours later, all chicks were challenged using E. coli strain EC317 and mortalities were recorded for 14 d. The first challenge study revealed that amongst the single use and combinations of CpG with different innate immune stimulants, a higher protection and a lower clinical score were offered by the combination of CpG + Poly I:C. The second challenge study showed that this combination (CpG + Poly I:C) provides an even higher level of protection when a second dose is administered via the S/C route at 1 day post-hatch. The current research highlights the efficacy of a combination of CpG + Poly I:C administered either in ovo or in ovo along with a S/C injection and its potential use as an alternative to antibiotics against yolk sac infection in young chicks.

Characterization of Dosage Levels for In Ovo Administration of Innate Immune Stimulants for Prevention of Yolk Sac Infection in Chicks.

Innate immune stimulants, especially toll-like receptor (TLR) ligands and agonists, are the main players in the initiation of innate immunity and have been widely studied as alternatives to antibiotics to control infection. In the present study, we characterized the dosage levels of various innate immune stimulants, including unmethylated cytosine-phosphate-guanosine dinucleotide -containing oligodeoxynucleotides (CpG ODN), polyinosinic-polycytidylic acid (poly I:C), cyclic polyphosphazene 75B (CPZ75B), avian beta-defensin 2 (ABD2), and combinations of these reagents given in ovo. Data derived from a series of animal experiments demonstrated that the in ovo administration of 10-50 µg CpG ODN/embryo (on embryonic day 18) is an effective formulation for control of yolk sac infection (YSI) due to avian pathogenic Escherichia coli (E. coli) in young chicks. Amongst the different combinations of innate immune stimulants, the in ovo administration of CpG ODN 10 µg in combination with 15 µg of poly I:C was the most effective combination, offering 100% protection from YSI. It is expected that the introduction of these reagents to management practices at the hatchery level may serve as a potential replacement for antibiotics for the reduction of early chick mortality (ECM) due to YSI/colibacillosis.

In Ovo Administration of Innate Immune Stimulants and Protection from Early Chick Mortalities due to Yolk Sac Infection.

Omphalitis or yolk sac infection (YSI) and colibacillosis are the most common infectious diseases that lead to high rates of early chick mortalities (ECMs) in young chicks. Out of numerous microbial causes, avian pathogenic Escherichia coli (APEC) or extraintestinal pathogenic E. coli infections are considered the most common cause of these conditions. YSI causes deterioration and decomposition of yolk, leading to deficiency of necessary nutrients and maternal antibodies, retarded growth, poor carcass quality, and increased susceptibility to other infections, including omphalitis, colibacillosis, and respiratory tract infection. Presently, in ovo injection of antibiotics, heavy culling, or after hatch use of antibiotics is practiced to manage ECM. However, increased antibiotic resistance and emergence of "super bugs" associated with use or misuse of antibiotics in the animal industry have raised serious concerns. These concerns urgently require a focus on host-driven nonantibiotic approaches for stimulation of protective antimicrobial immunity. Using an experimental YSI model in newborn chicks, we evaluated the prophylactic potential of three in ovo-administered innate immune stimulants and immune adjuvants for protection from ECM due to YSI. Our data have shown >80%, 65%, and 60% survival with in ovo use of cytosine-phosphodiester-guanine (CpG) oligodeoxynucleotides (ODN), polyinosinic:polycytidylic acid, and polyphosphazene, respectively. In conclusion, data from these studies suggest that in ovo administration of CpG ODN may serve as a potential candidate for replacement of antibiotics for the prevention and control of ECM due to YSI in young chicks.

Immune responses to in ovo vaccine formulations containing inactivated fowl adenovirus 8b with poly[di(sodium carboxylatoethylphenoxy)]phosphazene (PCEP) and avian beta defensin as adjuvants in chickens.

Inclusion body hepatitis (IBH) is one of the major viral infections causing substantial economic loss to the global poultry industry. The disease is characterized by a sudden onset of mortality (2-30%) and high morbidity (60-70%). IBH is caused by a number of serotypes of fowl adenovirus with substantially low levels of serotype cross protection. Thus far, there is no effective and safe vaccine commercially available in the North America for the control of IBH in chickens. Poly[di(sodium carboxylatoethylphenoxy)]phosphazene (PCEP) is a high molecular weight, biodegradable water soluble polymer that has been well characterized as a safe and effective adjuvant for a number of experimental veterinary vaccines. Similarly, host defence peptides, including ß-defensins, have also been shown to exhibit strong adjuvant potential. In this study, we evaluated the adjuvant activity of PCEP and avian beta defensin (ABD) in a vaccine formulation containing inactivated fowl adenovirus (FAdV) serotype 8b administered in ovo. Our data showed that a combination of PCEP and inactivated virus is capable of inducing a robust and long lasting antibody response. Moreover, significant enhancement of IFN-γ, IFN-α, IL-12(p40) and IL-6 gene expression under the influence of PCEP suggests that as an in ovo adjuvant PCEP has the ability to activate a substantial balanced immune response in chickens. To our knowledge, these are the first studies in which PCEP and ABD have been characterized as adjuvants for the development of an in ovo poultry vaccine. It is expected that these preliminary studies will be helpful in the development of safer and more effective in ovo vaccine against IBH and other infectious diseases affecting chickens.