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.

Avian antimicrobial peptides: in vitro and in ovo characterization and protection from early chick mortality caused by yolk sac infection.

Increasing antibiotic resistance is a matter of grave concern for consumers, public health authorities, farmers, and researchers. Antimicrobial peptides (AMPs) are emerging as novel and effective non-antibiotic tools to combat infectious diseases in poultry. In this study, we evaluated six avian AMPs including 2 truncated cathelicidins, [CATH-1(6-26) and CATH-2(1-15)], and 4 avian ß-defensins (ABD1, 2, 6 and 9) for their bactericidal and immunomodulatory activities. Our findings have shown CATH-1(6-26) and ABD1 being the two most potent avian AMPs effective against Gram-positive and Gram-negative bacteria investigated in these studies. Moreover, CATH-1(6-26) inhibited LPS-induced NO production and exhibited dose-dependent cytotoxicity to HD11 cells. While, ABD1 blocked LPS-induced IL-1ß gene induction and was non-toxic to HD11 cells. Importantly, in ovo administration of these AMPs demonstrated that ABD1 can offer significant protection from early chick mortality (44% less mortality in ABD1 treated group versus the control group) due to the experimental yolk sac infection caused by avian pathogenic Escherichia coli. Our data suggest that in ovo administration of ABD1 has immunomodulatory and anti-infection activity comparable with CpG ODN. Thus, ABD1 can be a significant addition to potential alternatives to antibiotics for the control of bacterial infections in young chicks.

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.

Administration of Poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP) and Avian Beta Defensin as Adjuvants in Inactivated Inclusion Body Hepatitis Virus and its Hexon Protein-Based Experimental Vaccine Formulations in Chickens.

Inclusion body hepatitis (IBH) is one of the major infectious diseases adversely affecting the poultry industry of the United States and Canada. Currently, no effective and safe vaccine is available for the control of IBH virus (IBHV) infection in chickens. However, based on the excellent safety and immunogenic profiles of experimental veterinary vaccines developed with the use of new generation adjuvants, we hypothesized that characterization of vaccine formulations containing inactivated IBHV or its capsid protein hexon as antigens, along with poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP) and avian beta defensin 2 (ABD2) as vaccine adjuvants, will be helpful in development of an effective and safe vaccine formulation for IBH. Our data demonstrated that experimental administration of vaccine formulations containing inactivated IBHV and a mixture of PCEP with or without ABD2 as an adjuvant induced significantly higher antibody responses compared with other vaccine formulations, while hexon protein-based vaccine formulations showed relatively lower levels of antibody responses. Thus, a vaccine formulation containing inactivated IBHV with PCEP or a mixture of PCEP and ABD2 (with a reduced dosage of PCEP) as an adjuvant may serve as a potential vaccine candidate. However, in order to overcome the risks associated with whole virus inactivated vaccines, characterization of additional viral capsid proteins, including fiber protein and penton of IBHV along with hexon protein in combination with more new generation adjuvants, will be helpful in further improvements of vaccines against IBHV infection.

CpG-ODNs induced changes in cytokine/chemokines genes expression associated with suppression of infectious bronchitis virus replication in chicken lungs.

The process of virus replication in host cells is greatly influenced by the set of cytokines, chemokines and antiviral substances activated as a result of host-virus interaction. Alteration of cytokines profiles through manipulation of the innate immune system by innate immune stimulants may be helpful in inhibiting virus replication in otherwise permissive cells. The aim of present studies was to characterize innate immune responses capable of inhibiting infectious bronchitis virus (IBV) replication in chicken lungs after in ovo administration of CpG ODN. In our experiments, CpG ODN 2007 or PBS solution was injected on 18th embryonic day (ED) via the chorioallontoic route. CpG ODN and PBS inoculated embryos were challenged with virulent IBV on the 19th ED. Lung tissue samples from experimental chicks were analysed for cytokines/chemokines gene expression at 24h, 48h, and 72h, post infection. Our data showed significant differential up-regulation of IFN-γ, IL-8 (CXCLi2) and MIP-1ß genes and suppression of IL-6 gene expression being associated with inhibition of IBV replication in lungs tissue retrieved from embryos pre-treated with CpG ODN. It is expected that understanding of the innate immune modulation of target tissues by the virus and innate immune stimulants will be helpful in identification of valuable targets for development of novel, safe, effective and economical control strategies against IBV infection in chickens.

Pathotypic and molecular characterization of a fowl adenovirus associated with inclusion body hepatitis in Saskatchewan chickens.

Inclusion body hepatitis (IBH) is one of the major global disease problems, causing significant economic losses to poultry industry of the United States and Canada. The disease is characterized by its sudden onset and high mortalities. Amongst different serotypes of fowl adenoviruses (FAdVs) associated with IBH, serotype 8 of group I FAdV has been isolated from majority of IBH cases. In present studies, we isolated a FAdV from morbid liver of a 17-day-old broiler from a Saskatchewan broiler farm. This newly isolated virus was designated as IBHV(SK). However, based on the sequence analysis of the L1 region of the hexon gene, the IBHV(SK) may be classified as FAdV 8b strain 764. These studies describe for the first time the complete hexon gene sequence of FAdV serotype 8b. Experimental infection of 2-day-old (n = 48) and 2-wk-old (n = 56) chicks caused 83% and 43% mortalities, respectively. Determination of the complete hexon gene sequence of IBHV(SK) with establishment of a disease model in chickens will facilitate the development of type-specific diagnostic reagents and assays for the evaluation of potential experimental vaccines against pathogenic FAdV infections.

Administration of poly[di(sodium carboxylatoethylphenoxy)]phosphazene (PCEP) as adjuvant activated mixed Th1/Th2 immune responses in pigs.

The selection of an optimal adjuvant to enhance the potency and longevity of antigen specific immune responses has always been imperative for the development of more effective and safer vaccines. A balanced type of immune enhancing ability of a new adjuvant known as polyphosphazene (PCEP) has been demonstrated in mice. In the present study we have compared the humoral and cellular immune responses to vaccine formulations containing Actinobacillus pleuropneumoniae outer membrane antigen (OmlA) with PCEP or Emulsigen as adjuvants. Our data showed a significant improvement and a shift toward more balanced immune responses when OmlA antigen was formulated with PCEP and CpG ODN. Moreover, in contrast to Emulsigen, immunization with PCEP did not result in local injection site reactions highlighting its potential as a safe and effective adjuvant for pigs. Further, the ease of formulation, administration and relatively low per dose cost of PCEP make it a promising adjuvant for pigs.

All three classes of CpG ODNs up-regulate IP-10 gene in pigs.

The analysis of CpG ODN induced innate immune responses in different animal species has shown substantial similarities and differences in levels and types of induced cytokines profile. The objectives of these studies were to identify innate immune biomarkers activated by three classes of CpG ODNs in pigs. For this purpose, we investigated the kinetics of innate immune responses in immune cells from pigs following in vitro and in vivo stimulation with CpG ODNs. The mRNA expression of cytokine and chemokine genes were assayed by SYBR green based quantitative real time PCR. A-class CpG ODN induced significant but transient levels of IFN-gamma, IL-12 (P40), IL-6, IL-4 and TNF-alpha mRNA, C-class CpG ODN induced significant level of IFN-gamma, IFN-alpha and IL-12 mRNA and the lowest level of IL-4 (Th-2 type) mRNA. A very low level of some cytokines stimulation was observed by GC ODNs. It is noteworthy, that IL-12 (P35) mRNA was significantly stimulated by B-class GpC ODN 7909. Interestingly, all classes of CpG ODNs induced significant level of IP-10 at 12h post stimulation. These in vitro and in vivo observations suggest that interferon-gamma inducible protein 10 (IP-10) may be a reliable biomarker for immune activity induced by CpG ODNs in pigs.

CpG oligodeoxynucleotides activate innate immune response that suppresses infectious bronchitis virus replication in chicken embryos.

The understanding of innate immune modulation by pathogens and immune-modulating agents, including synthetic oligodeoxynucleotides (CpG ODNs), has offered several new approaches to improve prophylactic and therapeutic strategies against infectious diseases in humans and animals. However, in this regard not much work has been done in avian medicine. In the present study, we analyzed the kinetics of interferon (IFN), cytokine, and chemokine mRNA expression in chicken embryonic spleen at 6 hr, 24 hr, 48 hr, and 72 hr after administration of CpG ODN 2007 (B-class) in 18-day-old chicken embryos. Our data showed enhanced expression of IFN-gamma; interleukin (IL)-1 beta, IL-6, and IL-8; and oligoadenyl synthetase A mRNA after CpG ODN administration. In addition, CpG ODN administration to chicken embryos 24 hr before the challenge with infectious bronchitis virus (IBV) was capable of limiting IBV propagation in different embryonic tissues. Based on the kinetics and type of cytokines induced after in ovo administration of CpG ODN, it may be speculated that in ovo administration of CpG ODNs may enhance resistance from viral infection in neonatal chicks and that CpG ODNs may contribute toward the development of more effective and safer poultry vaccines including in ovo vaccines.

Oligodeoxynucleotides containing CpG motifs (CpG-ODN) predominantly induce Th1-type immune response in neonatal chicks.

Earlier, we demonstrated that intramuscular administration of oligodeoxynucleotides containing CpG motifs (CpG-ODN) induces protection in neonatal chicks against a lethal challenge of Escherichia coli. However, the mechanism of induction of the protection was not clear. In an attempt to elucidate the mechanism of induced protection, we determined the kinetics of expression of cytokines/chemokines in the spleen and bursa of Fabricius of newly hatched chicks that had received intramuscular administration of CpG-ODN or non-CpG ODN compared to saline-treated controls. SyBr green, real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis of the RNA demonstrated increased expression of IL-1beta, IL-6, IL-8, IL-10, IL-18, IFN-gamma and MIP-3alpha mRNAs in the spleen and; IL-10 and IFN-alpha in bursa of Fabricious of chicks that had received CpG-ODN. However, non-CpG ODN failed to induce any of the cytokine. The increased level of IL-18 and IFN-gamma but not IL-4 mRNA suggests that the administration of CpG-ODN elicits a Th1 biased immune response, which may be important in inducing protection against infections in neonatal chicks. To our knowledge, this is the first report evaluating the induction of cytokines/chemokines in neonatal chicks following administration of CpG-ODN.

Attenuated cytokine responses in porcine lymph node cells stimulated with CpG DNA are associated with low frequency of IFNalpha-producing cells and TLR9 mRNA expression.

The immune stimulatory effects of synthetic CpG DNA, on porcine peripheral blood mononuclear cells (PBMC) have been reported, but little is known about CpG-induced responses in other lymphoid tissues of pigs. We investigated innate immune responses induced by CpG DNA in cells from blood, lymph nodes (LN) and spleens of pigs. Porcine PBMC and lymph node cells (LNC) were stimulated in vitro with three classes (A-, B- and C-class) of CpG oligodeoxynucleotides (ODNs), and a non-CpG control ODN. All three classes of CpG ODNs induced significant production of IFNalpha, TNFalpha, IL-1, IL-6 and IL-12 in PBMC. In contrast, in LNC, only IL-12 was stimulated by all three classes of CpG ODNs, while IFNalpha, and IL-6 were induced by A- and C-class ODNs. No TNFalpha was induced in LNC by any of the ODNs. Significant lymphocyte proliferation was induced in PBMC by all three classes of CpG ODNs and non-CpG control. However, in LNC, B- and C-class ODNs induced significant proliferation, while no proliferation was seen with A-class and non-CpG control ODN. All three classes of ODNs induced NK-like cytotoxicity in PBMC and spleen cells, but were less effective in inducing NK cytotoxicity in LNC. We then investigated the reasons for the relatively poor CpG-induced responses in LNC. Our investigations revealed that LNC had a lower frequency of IFNalpha-secreting cells and expressed low levels of TLR9 mRNA compared to PBMC. We conclude that the lower number of IFNalpha-secreting cells and receptor expression may contribute to the attenuated responses in LNC following stimulation with CpG ODN.

Innate immune responses induced by classes of CpG oligodeoxynucleotides in ovine lymph node and blood mononuclear cells.

CpG ODN signal through Toll-like receptor 9 (TLR9) and trigger a cascade of events that lead to activation of innate and adaptive immune responses. Our current understanding of the immunobiology of host responses to CpG is based largely on studies on peripheral blood mononuclear cells (PBMC) and splenocytes. Little is known regarding CpG-induced responses in other lymphoid tissues. In the present study, we investigated responses induced by CpG in both PBMC and lymph nodes. Cells were isolated from the superficial cervical lymph node (LNC) and blood and then stimulated with CpG ODN (either A-, or B- or C-class ODN). Cytokine production was assayed by ELISA, and lymphocyte proliferation was determined by (3)H-thymidine incorporation. NK-like cytotoxicity was analyzed by lysis of (51)Cr-labelled target cells. All three classes of CpG induced IFNalpha and IFNgamma in LNC. In contrast, only A and C-class ODN induced IFNalpha and IFNgamma in PBMC. Moreover, the IFN levels in LNC were 20-40-fold higher than in PBMC. Furthermore, all classes of ODN induced higher IL-12 levels in LNC (five- to six-fold) than in PBMC. Both B and C-class ODN induced good proliferative responses in PBMC and LNC, but the A-class ODN did not induce proliferation of PBMC and only induced moderate proliferation of LNC. A-class ODN induced significant NK-like activity in LNC. Thus, all three classes of CpG ODN induced similar responses in LNC, and these responses were consistently higher than in PBMC. These observations indicate that CpG ODN-induced responses differ between blood and lymph nodes, and suggest that the functional classification of CpG ODN based on PBMC responses may not be directly applicable to cells from other immune tissues.

Immune mechanisms and therapeutic potential of CpG oligodeoxynucleotides.

Unmethylated CpG motifs in bacterial DNA and synthetic oligodeoxynucleotides activate immune cells that express Toll-like Receptor 9. Activation through this receptor triggers cellular signaling that leads to production of a proinflammatory and a Th1-type, antigen-specific immune response. The immunostimulatory effects of CpG oligodeoxynucleotides confer protection against infectious disease, allergy and cancer in animal models, and clinical trials have been initiated. However, CpG oligodeoxynucleotides may exacerbate disease in some situations. We will review current concepts in the mechanisms of activating Toll-like Receptor 9 with CpG oligodeoxynucleotides and highlight opportunities for using large animal models to better determine the mechanisms of action.

Transcriptional analysis of avian embryonic tissues following infection with avian infectious bronchitis virus.

Avian infectious bronchitis virus (IBV) infection is one of the major viral respiratory diseases of chickens. Better understanding of the molecular basis of viral pathogenesis should contribute significantly towards the development of improved prophylactic, therapeutic and diagnostic reagents to control infections. In the present investigation, transcriptional profiles were analyzed by using RNA recovered from the lung tissue of IBV infected 18-day-old chicken embryos at 6, 24, 48 and 72 h post IBV infection. This microarray analysis was completed using avian cDNA arrays comprised of fragments of 1191 unique chicken and turkey gene transcripts. These arrays were generated from normalized cDNA subtraction libraries that were derived from avian pneumovirus (APV) infected chicken embryo fibroblast (CEF) cultures and tissues obtained from APV infected turkeys subtracted with their respective uninfected cultures and tissues. Of the 1191 unique genes represented on the array, the expression of a total of 327 genes (27% of total) were altered by two-fold or more from 6 through 72 h post-infection. A comparative analysis of IBV regulated genes with genes previously reported to change in expression following infection with other avian respiratory viruses revealed both conserved and unique changes. Real-time qRT-PCR was used to confirm the regulated expression of genes related to several functional classes including kinases, interferon induced genes, chemokines and adhesion molecules, vesicular trafficking and fusion protein genes, extracellular matrix protein genes, cell cycle, metabolism, cell physiology and development, translation, RNA binding, lysosomal, protein degradation and ubiquitination related genes. Microarray analysis served as an efficient tool in facilitating a comparative analysis of avian respiratory viral infections and provided insight into host transcriptional changes that were conserved as well as those which were unique to individual pathogens.

Sequence analysis of the matrix (M2) protein gene of avian pneumovirus recovered from turkey flocks in the United States.

We here report the comparative sequence and phylogenetic analysis of the avian pneumovirus subgroup C (APV C) matrix (M2) gene of cell culture-adapted isolates and clinical samples. Limited heterogeneity was observed among the M2 sequences, suggesting that diagnostic tests and vaccines against APV C are likely to exhibit broad cross-reactivity.

A single subtype of avian pneumovirus circulates among Minnesota turkey flocks.

The recent emergence of avian pneumovirus (APV) infection among US turkey flocks has resulted in a major economic threat to the turkey industry. In order to elucidate the molecular epidemiology of APV, comparative sequence analysis of the fusion (F) protein gene of APV was performed for 3 cell culture-adapted isolates and 10 APV positive clinical samples recovered from US turkey flocks. Relatively modest levels of nucleotide and amino acid sequence divergence were identified, suggesting the prevalence of a single lineage of APV among US turkey flocks. Additionally, numerous polymorphisms were identified that were only represented in the clinical samples but not in the in vitro propagated isolates of APV. Phylogenetic analyses confirm that the subtype of APV circulating in the upper Midwestern United States is evolutionarily related to, but distinct from, European APV subgroups A and B. Overall, the results of the present investigation suggest that there has been only a single recent introduction of APV into US turkey populations in the upper Midwestern United States.