Identification of 3 neutralizing linear epitopes on the VP8 outer capsid protein of group A equine rotavirus.

OBJECTIVE: To identify protective equine rotavirus group A (ERVA) VP8 epitopes and demonstrate that immunizing hens with synthetic peptides based on these epitopes would yield high-titered, neutralizing egg yolk antibodies for potential application in foals. ANIMALS: 26 rotavirus-positive, client-owned foals were included in the study. Five white leghorn hens were used for antibody production. METHODS: Chicken antibodies were raised against 3 synthetic epitope peptides from the VP8 protein of the common ERVA P-type, P4[12] using CD40-targeted streptavidin-peptide complexes. Antipeptide serum- and egg yolk antibodies were subject to ELISA and in vitro virus neutralization assays to evaluate binding and neutralization activities. Lyophilized anti-VP8 egg yolk antibodies were orally administered (30 g; q 24 h for 5 days) to foals with rotaviral diarrhea. Physical examinations were performed daily. The duration of diarrhea and any adverse effects were recorded. RESULTS: CD40-targeted vaccination of hens generated high titers of anti-VP8 serum and egg yolk antibodies after just 3 immunizations. These antibodies prevented in vitro infection of ERVA with titers of 128 in the serum and 94.5 in the yolk. Oral administration (30 g; q 24 h for 5 days) of lyophilized hyperimmune egg yolk to foals with rotaviral diarrhea did not reveal any adverse effects of the treatment. CLINICAL RELEVANCE: This study demonstrated that antibodies raised against neutralizing epitopes of the ERVA VP8 protein could prevent ERVA infection in vitro. Based on these results and previous work in other animals, in vivo evaluation of the therapeutic efficacy of anti-VP8 egg yolk antibodies is warranted.

Electron-Beam Inactivation of Human Rotavirus (HRV) for the Production of Neutralizing Egg Yolk Antibodies.

Electron beam (eBeam) inactivation of pathogens is a commercially proven technology in multiple industries. While commonly used in a variety of decontamination processes, this technology can be considered relatively new to the pharmaceutical industry. Rotavirus is the leading cause of severe gastroenteritis among infants, children, and at-risk adults. Infections are more severe in developing countries where access to health care, clean food, and water is limited. Passive immunization using orally administered egg yolk antibodies (chicken IgY) is proven for prophylaxis and therapy of viral diarrhea, owing to the stability of avian IgY in the harsh gut environment. Since preservation of viral antigenicity is critical for successful antibody production, the aim of this study was to demonstrate the effective use of electron beam irradiation as a method of pathogen inactivation to produce rotavirus-specific neutralizing egg yolk antibodies. White leghorn hens were immunized with the eBeam-inactivated viruses every 2 weeks until serum antibody titers peaked. The relative antigenicity of eBeam-inactivated Wa G1P[8] human rotavirus (HRV) was compared to live virus, thermally, and chemically inactivated virus preparations. Using a sandwich ELISA (with antibodies against recombinant VP8 for capture and detection of HRV), the live virus was as expected, most immunoreactive. The eBeam-inactivated HRV's antigenicity was better preserved when compared to thermally and chemically inactivated viruses. Additionally, both egg yolk antibodies and serum-derived IgY were effective at neutralizing HRV in vitro. Electron beam inactivation is a suitable method for the inactivation of HRV and other enteric viruses for use in both passive and active immunization strategies.

From crypts to enteroids: establishment and characterization of avian intestinal organoids.

Intestinal organoids (IO), known as "mini-guts", derived from intestinal crypts, are self-organizing three-dimensional (3D) multicellular ex vivo models that recapitulate intestine epithelial structure and function and have been widely used for studying intestinal physiology, pathophysiology, molecular mechanisms of host-pathogen interactions, and intestinal disease in mammals. However, studies on avian IO are limited and the development of long-term cultures of IO model for poultry research is lacking. Therefore, the objectives of this study were to generate crypt-derived organoids from chicken intestines and to optimize conditions for cell growth and enrichments, passages, and cryopreservation. Crypts were collected from the small intestines of birds at embryonic d-19 and ceca from layer and broiler chickens with ages ranging from d 1 to 20 wk, embedded in a basement membrane matrix, and cultured with organoid growth media (OGM) prepared in house. The crypt-derived organoids were successfully grown and propagated to form 3D spheres like structures that were cultured for up to 3 wk. Organoids were formed on d one, budding appeared on d 3, and robust budding was observed on d 7 and beyond. For cryopreservation, dissociated organoids were resuspended in a freezing medium. The characteristics of IO upon extended passages and freeze-thaw cycles were analyzed using reverse transcription (RT)-PCR, immunoblotting, and live cell imaging. Immunoblotting and RT-PCR using E-cadherin (the marker for epithelial cells), leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5, the marker for stem cells), chromogranin A (the marker for enteroendocrine cells), lysozyme (the marker for Paneth cells), and mucin (the biomarker for goblet cells) confirmed that IO were composed of heterogeneous cell populations, including epithelial cells, stem cells, enteroendocrine cells, Paneth cells, and goblet cells. Furthermore, OGM supplemented with both valproic acid and CHIR99021, a glycogen synthase kinase 3ß inhibitor and a histone deacetylase inhibitor, increased the size of the avian IO (P < 0.001). To the best of our knowledge, this is the first comprehensive report for establishing long-term, organoid culture models from small intestines and ceca of layer and broiler chickens. This model will facilitate elucidation of the mechanisms impacting host-pathogen interactions, eventually leading to the discovery of pathogen intervention strategies in poultry.

Brucella activates the host RIDD pathway to subvert BLOS1-directed immune defense.

The phagocytosis and destruction of pathogens in lysosomes constitute central elements of innate immune defense. Here, we show that Brucella, the causative agent of brucellosis, the most prevalent bacterial zoonosis globally, subverts this immune defense pathway by activating regulated IRE1α-dependent decay (RIDD) of Bloc1s1 mRNA encoding BLOS1, a protein that promotes endosome-lysosome fusion. RIDD-deficient cells and mice harboring a RIDD-incompetent variant of IRE1α were resistant to infection. Inactivation of the Bloc1s1 gene impaired the ability to assemble BLOC-1-related complex (BORC), resulting in differential recruitment of BORC-related lysosome trafficking components, perinuclear trafficking of Brucella-containing vacuoles (BCVs), and enhanced susceptibility to infection. The RIDD-resistant Bloc1s1 variant maintains the integrity of BORC and a higher-level association of BORC-related components that promote centrifugal lysosome trafficking, resulting in enhanced BCV peripheral trafficking and lysosomal destruction, and resistance to infection. These findings demonstrate that host RIDD activity on BLOS1 regulates Brucella intracellular parasitism by disrupting BORC-directed lysosomal trafficking. Notably, coronavirus murine hepatitis virus also subverted the RIDD-BLOS1 axis to promote intracellular replication. Our work establishes BLOS1 as a novel immune defense factor whose activity is hijacked by diverse pathogens.

Marinobufagenin levels in preeclamptic patients: a preliminary report.

Preeclampsia is a disorder resulting in significant fetomaternal complications with no definitive pharmacological intervention. A bufadienolide, marinobufagenin, has been implicated in the etiology of preeclampsia. We investigated both the blood and urine levels of marinobufagenin in preeclamptic and control subjects. Preeclamptic and normotensive pregnant women were recruited at various gestational age periods. Blood and urine specimens were obtained and analyzed for marinobufagenin levels and creatinine. The former determination was performed utilizing a new, novel chemifluorescent enzyme-linked immunosorbent assay. The marinobufagenin levels were higher in preeclamptics than in the controls in both serum and urine at various gestational age periods. Additionally, the mean level of marinobufagenin in the preeclamptic group was significantly greater than in controls in both blood and urine specimens ( P < 0.05). These data are consistent with a role for marinobufagenin in the etiology of preeclampsia. This study demonstrated comparable results in blood and urine samples. This suggests that subsequent studies on levels of marinobufagenin as a screening test for preeclampsia could be done utilizing urine samples, which are easier to obtain, less invasive, more cost-effective, and as accurate as the serological tests.

A chemifluorescent immunoassay for the determination of marinobufagenin in body fluids.

We describe here the development of a chemifluorescent competitive enzyme-linked immunosorbent assay (ELISA) that quantifies marinobufagenin (MBG) levels in biological fluids. Based on a polyclonal antibody raised against a novel MBG-bovine serum albumin conjugate, this assay achieved an MBG detection limit of less than 9 pg/mL. MBG levels in various rat urine and serum samples were effectively determined using this methodology. Interassay variability averaged 9.8%, while intra-assay variability averaged 1.9 and 2.5% in representative serum and urine samples, respectively. Recovery of exogenously added MBG averaged 106%, and parallelism data further established the accuracy of the assay. Employment of this assay to detect MBG abnormalities represents a powerful tool for the possible diagnosis, prevention and management of human hypertensive states, particularly preeclampsia.

Golden Gate assembly with a bi-directional promoter (GBid): A simple, scalable method for phage display Fab library creation.

Fabs offer an attractive platform for monoclonal antibody discovery/engineering, but library construction can be cumbersome. We report a simple method - Golden Gate assembly with a bi-directional promoter (GBid) - for constructing phage display Fab libraries. In GBid, the constant domains of the Fabs are located in the backbone of the phagemid vector and the library insert comprises only the variable regions of the antibodies and a central bi-directional promoter. This vector design reduces the process of Fab library construction to "scFv-like" simplicity and the double promoter ensures robust expression of both constituent chains. To maximize the library size, the 3 fragments comprising the insert - two variable chains and one bi-directional promoter - are assembled via a 3-fragment overlap extension PCR and the insert is incorporated into the vector via a high-efficiency one-fragment, one-pot Golden Gate assembly. The reaction setup requires minimal preparatory work and enzyme quantities, making GBid highly scalable. Using GBid, we constructed a chimeric chicken-human Fab phage display library comprising 1010 variants targeting the multi-transmembrane protein human CD20 (hCD20). Selection/counter-selection on transfected whole cells yielded hCD20-specific antibodies in four rounds of panning. The simplicity and scalability of GBid makes it a powerful tool for the discovery/engineering of Fabs and IgGs.

DNA aptamer-based rolling circle amplification product as a novel immunological adjuvant.

Several agonists to CD40 have shown to induce acquired immune responses. Here, we developed and evaluated the rolling circle amplification (RCA) products that are based on anti-CD40 DNA aptamers as a novel vaccine adjuvant. First, we developed DNA aptamers with specific binding affinity to chicken CD40 extra domain (chCD40ED). Next, we prepared the RCA products that consist of these aptamers to increase the spanning space and overall binding affinity to chCD40ED. Using 8 DNA aptamer candidates, 4 aptamer-based RCA products (aptamer RCAs) were generated, each consisting of two distinct aptamers. We demonstrated that all 4 aptamer RCAs significantly induced the signal transduction in chicken HD11 macrophage cell line (p < 0.05). Finally, we conjugated one of the aptamer RCAs (Aptamer RCA II) to M2e epitope peptide of influenza virus as a model hapten, and the immune complex was injected to chickens. Aptamer RCA II stimulated anti-M2e IgG antibody production to the level significantly higher as compared to the control (M2e epitope alone; p < 0.05). The results of our work suggest that aptamer RCA is a novel platform to boost the efficacy of vaccines, which might find broad applications to other antigens beyond M2e epitope evaluated in this study using chicken infection model.

Carboxypeptidase E, an essential element of the regulated secretory pathway, is expressed and partially co-localized with chromogranin A in chicken thymus.

Although the functions of hormones and neuropeptides in the thymus have been extensively studied, we still do not know whether these intra-thymic humoral elements are released in a stimulated manner via the regulated secretory pathway or in a constitutive manner. Carboxypeptidase E (CpE) and chromogranin A (CgA) are functional and structural hallmarks of the regulated secretory pathway in (neuro)endocrine cells. Whereas we have previously shown a CgA-positive neuroendocrine population in the chicken thymus, the current study assesses the expression of CpE in the thymus, both at the mRNA and the protein level. Our immunohistochemical studies provide evidence for the co-existence of CgA and CpE in identical neuroendocrine cells in the thymus. CpE and CgA dual-positive cells have primarily been found in the transition zone between the cortex and medulla of the thymus, an area known to contain numerous arterioles and to be innervated by the autonomic nervous system. Our findings suggest that the diffuse neuroendocrine system serves as a relay for nervous stimuli delivered by the sympathetic and/or parasympathetic nervous system. Thus, these newly defined neuroendocrine cells might play an important role in the immuno-neuro-endocrine cross-talk in the thymus, potentially enabling thymopoiesis to be fine-tuned via the regulated secretory pathway by a variety of physical and environmental factors.

CD205-positive, Sepharose-induced peritoneal exudate cells: a new resource for DC research in the chicken.

Dendritic cells (DC) are important antigen-presenting cells and are among the least characterized immune cells in the chicken. In order to obtain chicken DC, current protocols require isolation of bone marrow myeloid progenitor cells and induction of DC differentiation with supplemental cytokines or negative selection of splenic cell preparations. Chicken peritoneal exudate cells (PEC) have traditionally been a source of various immune cells for ex vivo studies, primarily to investigate heterophils and macrophages. In this study, we observe the presence of CD205+ PEC populations, a marker of DC, as an additional resource to isolate and study chicken primary DCs. A panel of monoclonal antibodies was developed against the chicken CD205 DC marker and used to isolate CD205+ DC from the PEC population using magnetic bead cell sorting. This study reports the development of new anti-CD205 monoclonal antibodies as a reagent for chicken DC research, as well as PEC as a potential source of CD205+ DC for ex vivo studies in the chicken.

Comparison Of Four Anti-Avian IgY Secondary Antibodies Used In Western Blot And Dot-Blot ELISA To Detect Avian Bornavirus Antibodies In Four Different Bird Species.

PURPOSE: This study evaluated the specificity of different avian secondary antibodies used in Western blot and dot-blot ELISA to detect avian bornavirus antibodies in bird plasma. METHODS: Plasma samples were collected from: two Blue and gold macaws, one positive and one negative for avian bornavirus by RT-PCR; a Cockatiel and a Monk parakeet prior to and following experimental infection; and, two Mallards, one positive and one negative for avian bornavirus by RT-PCR Samples were analyzed by Western blot and dot-blot ELISA that incorporated recombinant avian bornavirus nucleoprotein as the target analyte. Four species-specific anti-IgY secondary antibodies were used in the assays: goat anti-macaw IgY, goat anti-bird IgY, goat anti-duck IgY, and rabbit anti-chicken IgY. RESULTS: In the Western blot, anti-macaw IgY secondary antibody produced strong signals with Blue and gold macaw and Cockatiel positive plasma, but no signal with Mallard positive plasma. Anti-bird IgY secondary antibody produced strong signals with Blue and gold macaw, Cockatiel, and Mallard positive plasma. Anti-duck and anti-chicken IgY secondary antibody produced a strong and moderate signal, respectively, only with Mallard positive plasma. In the dot-blot ELISA, there was a distinct and significant difference (P<0.05) in the signal intensity between the different secondary antibodies within a bird species. Anti-macaw IgY secondary antibody produced significantly (P<0.05) stronger signals than the other secondary antibodies in Blue and gold macaw, Cockatiel, and Monk parakeet positive plasma, while anti-duck IgY secondary antibody produced significantly (P<0.05) stronger signals than the other secondary antibodies in Mallard positive plasma. CONCLUSION: In testing psittacines with immunoassays, and especially in assays that incorporate short incubation reaction times such as a dot-blot ELISA, species-specific anti-IgY secondary antibodies provided more accurate results.

Phage display selection and characterization of single-chain recombinant antibodies against Eimeria tenella sporozoites.

A single-chain antibody library against Eimeria tenella sporozoites was constructed by phage display. Antibody-displaying phage was selected in five panning rounds against cryopreserved E. tenella sporozoites. A 1000-fold increase in phage output and a 3000-fold enrichment were obtained after three rounds of panning, as the binding clones became the dominant population in the library. Ten clones were randomly selected from the last selection round, and their nucleotide sequences were aligned and compared to chicken germ-line sequences. Analysis of the light chain variable regions revealed possible donor pseudogenes which act as donors in gene conversion events, and contribute to the diversification of the V(L) immune repertoire. Possible somatic hypermutation events, a consequence of affinity maturation, were also identified. Soluble antibody was produced in a non-suppressor E. coli strain, purified by nickel affinity chromatography, and characterized by immunoblotting. In an immunofluorescence assay, this recombinant antibody showed specific binding to E. tenella sporozoites.

Crude Inactivated Influenza A Virus Adjuvated with a Bispecific Antibody Complex Targeting Chicken CD40 and AIV M2e Confers Protection Against Lethal HPAI Challenge in Chickens.

In vivo targeting an immunogen to the CD40 receptor expressed on professional antigen-presenting cells (APCs) dramatically enhances speed, magnitude, and quality of the immune response. Our previous evaluation of this strategy in poultry was limited to immunogenicity studies using CD40-targeted synthetic peptides, which demonstrated significant antigen-specific serum IgG and tracheal IgA levels <1 week after primary administration. In this study, this antibody-guided immunization strategy was modified to permit incorporation of inactivated highly pathogenic avian influenza virions (in lieu of short synthetic peptides) as the immunogen by simply mixing a bispecific antibody complex (anti-CD40/M2e) with crude inactivated virus before injection. Adjuvated avian influenza virus (AIV) induced significant hemagglutination inhibition titers up to 6 weeks postimmunization. In efficacy studies, administration of a single vaccine dose yielded 56%-64% survival against challenge with highly pathogenic H5N1, and 100% protection was achieved upon boosting. These results represent a feasible strategy to effectively target whole inactivated influenza A virus to chicken APCs, regardless of AIV clade and without phenotyping or purifying the virus from crude allantoic fluid. The data represent proof of principle for the unique prophylactic efficacy and versatility of a CD40-targeting adjuvation strategy that can in principle also be harnessed in other poultry vaccines.

Scarless and site-directed mutagenesis in Salmonella enteritidis chromosome.

BACKGROUND: A variety of techniques have been described which introduce scarless, site-specific chromosomal mutations. These techniques can be applied to make point mutations or gene deletions as well as insert heterologous DNA into bacterial vectors for vaccine development. Most methods use a multi-step approach that requires cloning and/or designing repeat sequences to facilitate homologous recombination. We have modified previously published techniques to develop a simple, efficient PCR-based method for scarless insertion of DNA into Salmonella enteritidis chromosome. RESULTS: The final product of this mutation strategy is the insertion of DNA encoding a foreign epitope into the S. enteritidis genome without the addition of any unwanted sequence. This experiment was performed by a two-step mutation process via PCR fragments, Red recombinase and counter-selection with the I-SceI enzyme site. First, the I-SceI site and kanamycin resistance gene were introduced into the genome of cells expressing Red recombinase enzymes. Next, this sequence was replaced by a chosen insertion sequence. DNA fragments used for recombination were linear PCR products which consisted of the foreign insertion sequence flanked by homologous sequences of the target gene. Described herein is the insertion of a section of the M2e epitope (LM2) of Influenza A virus, a domain of CD154 (CD154s) or a combination of both into the outer membrane protein LamB of S. enteritidis. CONCLUSION: We have successfully used this method to produce multiple mutants with no antibiotic gene on the genome or extra sequence except those nucleotides required for expression of epitope regions. This method is advantageous over other protocols in that it does not require cloning or creating extra duplicate regions to facilitate homologous recombination, contains a universal construct in which an epitope of choice can be placed to check for cell surface expression, and shows high efficiency when screening for positive mutants. Other opportunities of this mutational strategy include creating attenuated mutants and site-specific, chromosomal deletion mutations. Furthermore, this method should be applicable in other gram-negative bacterial species where Red recombinase enzymes can be functionally expressed.

A Fast and Inexpensive Protocol for Empirical Verification of Neutralizing Epitopes in Microbial Toxins and Enzymes.

In vivo targeting of peptides to antigen-presenting cells by use of agonistic anti-CD40 monoclonal antibodies has been used successfully as an immune response enhancing strategy. When tested in chickens, the antibody-guided platform was capable of inducing specific IgG production within 1 week postimmunization. However, use of this method beyond its initial conception as a vaccine delivery tool has not been fully exploited. In this study, Clostridium perfringens alpha-toxin was used as a model microbial toxin for epitope mapping by using the antibody-guided immunization method to generate a panel of antibodies against specific, regions of the toxin in an attempt to identify crucial determinants on the toxin which, once bound, would hinder downstream toxicity. Alpha-toxin, which possesses both hemolytic and phospholipase C (PLC) enzymatic activities, has long been known to be one of the key destructive etiological agents of necrotic enteritis disease in poultry. Previous attempts to identify crucial antigenic determinants on the toxin mediating its enzymatic activities have been performed using expensive and labor-intensive site-directed mutagenesis techniques. To create a panel of antibodies, 23 short candidate alpha-toxin peptide regions were selected in silico using B-cell epitope prediction algorithms in the public domain and were custom synthesized to load onto the antibody-guided complex for immunization in birds for antisera production. Peptide-specific antibody responses were generated against all candidate neutralizing epitopes and used for in vitro toxin neutralization tests. Antisera against all 23 peptides were able to neutralize the toxin's hemolytic activity, with neutralization titers ranging from 80 to 320, but none were effective in blocking PLC. The novel approach of antibody-guided immunization introduces a new, inexpensive method for polyclonal IgG production and de facto identification of neutralizing epitopes in microbial toxins and enzymes within 2 weeks from in silico analysis of a putative target sequence.

Immunomodulatory Effects of Saccharomyces cerevisiae Fermentation Product Supplementation on Immune Gene Expression and Lymphocyte Distribution in Immune Organs in Broilers.

A study was conducted to evaluate the molecular and cellular immunomodulatory effects of a Saccharomyces cerevisiae fermentation product (Original XPC, Diamond V) in broilers. Our lab has previously demonstrated that broilers fed XPC generate faster and stronger antigen-specific humoral immune responses to Newcastle disease virus (NDV) vaccination. This study aims at investigating the mechanism behind this increased immunocompetence. One-day-old broilers were randomly assigned to one of two treatments: 1.25 kg/ton S. cerevisiae fermentation product (XPC treatment group) or control diet. Birds were vaccinated against NDV on day 1 (B1 strain) and day 21 (LaSota strain) post-hatch. Innate and adaptive immune-related gene expression profiles in central (thymus and bursa of Fabricius) and peripheral (spleen) immune organs were investigated at 14 and 28 days of age by qPCR array. Fold changes larger than 1.2 (P < 0.05) between treated and control were considered significant. Lymphocyte subpopulations in central and peripheral immune organs and blood leukocytes were analyzed by flow cytometry at 14, 21, 28, and 42 days of age. In the spleen, Th1 immune responses and antiviral genes, such as IFN-γ, and its downstream genes signal transducer and activator of transcription (STAT4) and NFκB, were significantly upregulated in the treated group by 14 days of age. In the thymus, genes belonging to different functional groups were influenced at different time points. Cytokine genes associated with lymphocyte maturation, differentiation, and proliferation, such as IL-1R, IL-2, and IL-15 were significantly upregulated in the treated group by 28 days of age. Genes preferentially expressed in the medulla of the thymus and mature thymocytes, such as Myxovirus resistance gene 1, interferon regulatory factor-1, interferon regulatory factor-7, and STAT1, were upregulated in the birds supplemented with XPC. Birds supplemented with XPC had significantly higher percentages of CD3+, CD4+, and CD8+ T-cells in the thymus at day 28 of age, indicating production of more mature T-cells, which was consistent with gene expression results. Results suggest that XPC supplementation primes broilers to become more immunocompetent, without compromising growth performance.

BCG vaccination of guinea pigs modulates Mycobacterium tuberculosis-induced CCL5 (RANTES) production in vitro and in vivo.

CCL5 can attract and activate macrophages and Th1 lymphocytes, which are involved in eliciting a protective immune response against tuberculosis. In this study, the effects of BCG vaccination on CCL5 production in vitro and in vivo in the guinea pig model were examined. Splenocytes, alveolar, and resident peritoneal macrophages obtained from naïve and BCG-vaccinated animals were infected with Mycobacterium tuberculosis in vitro at various time points and analyzed for CCL5 mRNA and protein levels. All three leukocyte populations harvested from BCG-vaccinated guinea pigs and infected with M. tuberculosis produced elevated CCL5 mRNA and protein compared to infected cells from naïve animals. The kinetics of CCL5 production in vivo was evaluated by inducing tuberculous pleurisy in BCG-vaccinated guinea pigs and analyzing CCL5 in pleural effusions at daily intervals. Both CCL5 mRNA and protein levels increased to maximum levels at day 4 post-pleurisy induction. These data suggest that BCG-vaccination enhances CCL5 production in vitro and in vivo. The effect of neutralizing CCL5 with polyclonal anti-CCL5 IgG in vivo during tuberculous pleurisy resulted in a trend toward diminished levels of pro-inflammatory cytokine mRNA, although neutralizing CCL5 in vivo did not appear to alter the intensity of the histopathological response.

Comparison of the ability of the three endogenous GnRHs to stimulate release of follicle-stimulating hormone and luteinizing hormone in chickens.

It is well established that GnRH can stimulate the release of LH and FSH in mammals. Two GnRHs have been found in the chicken hypothalamus, cGnRH-I and -II. There is controversy as to whether either peptide can stimulate release of FSH in birds. The present studies compared the ability of cGnRH-I and -II to stimulate the release of FSH and LH in chickens. Lamprey (l) GnRH-III may be a specific-releasing factor for FSH, as it selectively stimulates FSH release in rodents and cattle, and has been detected in the hypothalamus of rodents, sparrows and chickens. Therefore, the ability of lGnRH-III to stimulate LH and FSH release was also examined. In our first experiment, the effects of cGnRH-I and -II were studied using 17-week prepubertal females. Intravenous injection of cGnRH-II at 1 and 10 microg/kg BW significantly increased LH secretion more than did cGnRH-I. Neither peptide significantly increased plasma FSH levels. In our second study, we administered cGnRH-I, -II or lGnRH-III to mature males maintained on a short photoperiod. cGnRH-II was again more potent than cGnRH-I in stimulating LH release, while lGnRH-III produced a modest LH rise. No GnRH peptide provided specific or potent stimulus to FSH secretion, although the high dose of cGnRH-II modestly enhanced FSH levels in the adult male (P < 0.05). Our results are not consistent with the view that lGnRH-III is a specific FSH-releasing hormone across multiple classes of vertebrates. We conclude that the mechanism by which independent release of FSH occurs in chickens remains unresolved.

Significant mucosal sIgA production after a single oral or parenteral administration using in vivo CD40 targeting in the chicken.

Many pathogens enter the host through mucosal surfaces and spread rapidly via the circulation. The most effective way to prevent disease is to establish mucosal and systemic immunity against the pathogen. However, current vaccination programs in poultry industry require repeated administrations of live-attenuated virus or large amounts (10 to 100µg) of antigen together with adjuvant to induce specific secretory IgA immune responses at the mucosal effector sites. In the present study, we show that a single administration of 0.4µg of oligopeptide complexed with an agonistic anti-chicken CD40 (chCD40) monoclonal antibody (Mab) effectively targets antigen-presenting cells of the bird's mucosa-associated lymphoid tissue in vivo, and induces peptide-specific secretory IgA (sIgA) in the trachea 7days post administration. Anti-chCD40 Mab-peptide complex was administered once to four-week old male Leghorns via various mucosal routes (orally, via cloacal drinking, or oculo-nasally) or via subcutaneous (s.c.) immunization. Immunization through any of the three mucosal induction routes induced significant peptide-specific mucosal sIgA responses 7 and 14days after immunization. Interestingly, s.c. injection of the complex also induced mucosal sIgA. Our data suggest in vivo targeting of CD40 as a potential adjuvant platform, particularly for the purpose of enhancing and speeding up mucosal vaccine responses in chickens, and potentially other food animals. This is the first study able to elicit specific sIgA immune responses in remote mucosal sites with a single administration of only 0.4µg of antigen.

Induction of Robust Immune Responses in Swine by Using a Cocktail of Adenovirus-Vectored African Swine Fever Virus Antigens.

The African swine fever virus (ASFV) causes a fatal hemorrhagic disease in domestic swine, and at present no treatment or vaccine is available. Natural and gene-deleted, live attenuated strains protect against closely related virulent strains; however, they are yet to be deployed and evaluated in the field to rule out chronic persistence and a potential for reversion to virulence. Previous studies suggest that antibodies play a role in protection, but induction of cytotoxic T lymphocytes (CTLs) could be the key to complete protection. Hence, generation of an efficacious subunit vaccine depends on identification of CTL targets along with a suitable delivery method that will elicit effector CTLs capable of eliminating ASFV-infected host cells and confer long-term protection. To this end, we evaluated the safety and immunogenicity of an adenovirus-vectored ASFV (Ad-ASFV) multiantigen cocktail formulated in two different adjuvants and at two immunizing doses in swine. Immunization with the cocktail rapidly induced unprecedented ASFV antigen-specific antibody and cellular immune responses against all of the antigens. The robust antibody responses underwent rapid isotype switching within 1 week postpriming, steadily increased over a 2-month period, and underwent rapid recall upon boost. Importantly, the primed antibodies strongly recognized the parental ASFV (Georgia 2007/1) by indirect fluorescence antibody (IFA) assay and Western blotting. Significant antigen-specific gamma interferon-positive (IFN-γ+) responses were detected postpriming and postboosting. Furthermore, this study is the first to demonstrate induction of ASFV antigen-specific CTL responses in commercial swine using Ad-ASFV multiantigens. The relevance of the induced immune responses in regard to protection needs to be evaluated in a challenge study.