The effects of a perch, dust bath, and nest box, either alone or in combination as used in furnished cages, on the welfare of laying hens.

This experiment examined the welfare-related effects of individual furniture items alone or in combination in a factorial experiment using Hy-Line Brown hens housed in 8-bird furnished cages. Welfare was assessed during two 8-wk sampling periods commencing at 29 and 59 wk of age. Measurement of stress, immunology, feather, foot and claw condition, and behavior were taken, and bone strength was measured at the end of the experiment. With the exception of the positive effects of a perch on bone strength, any effects of furniture items were relatively small, even though the furniture was extensively used. Although there were changes in behavior and small changes in feather, foot, and claw condition, it is unclear whether these changes have any meaningful implications for welfare. In this experiment there were 2 additional external control treatments for a small study that examined the effects of increasing space per bird (8 birds in single- and double-width cages) and the effects of group size (8 and 16 birds in double-width cages); using similar methodologies, these treatments showed differences in egg corticosterone concentrations and evidence of immunosuppression. Together, these data suggest that although furniture when present was well-used, any effects of furniture on hen welfare measured by physical and physiological traits, other than the benefit of a perch on bone strength, were smaller than effects of group size and space allowance.

The use of RNAi and transgenics to develop viral disease resistant livestock.

The possibility of genetically engineering poultry to make them resistant to avian influenza is attracting attention and has now become a real possibility with improved methods for genetic modification and the emergence of RNAi as an antiviral strategy. In order to test this possibility, we have generated transgenic mice that express RNAi molecules targeting a conserved region of the influenza A NP gene and are testing these mice for resistance to influenza infection. Transgenes were initially developed that express short hairpin RNAs (shRNAs) targeting multiple influenza A viral genes. The shRNAs were tested for inhibition of H1N1 PR8 virus in vitro. Two potent shRNAs that target the NP and PA genes were chosen for lentiviral mediated generation of transgenic mice. Transgenic founders for the NP shRNA construct and also a negative control shRNAtargeting EGFP were generated. The constitutive expression of the shRNA molecules in a range of tissue types including lung, was confirmed and so far stable transmission of the RNAi transgenes from the F0 to F3 generation has been observed. Resistance to influenza infection in these transgenic mice is now being confirmed.

ELISPOT and intracellular cytokine staining: novel assays for quantifying T cell responses in the chicken.

The measurement of T cell responses in chickens, not only for quantitative aspects but also for the qualitative nature of the responses, becomes increasingly important. However, there are very few assays available to measure T cell function. Therefore, we have developed enzyme-linked immunosorbent spot assay (ELISPOT) and an intracellular cytokine staining (ICCS) assay. ELISPOT assay for the detection of chicken interferon-gamma (ChIFN-gamma) production was set up and shown to be reproducible for both polyclonal and antigen-specific stimuli such as Newcastle disease virus (NDV). However, the ELISPOT assay lacks the ability to identify individual cytokine-producing cells. Separation of CD4+ and CD8+ T cell populations gave additional information, but appeared to have the disadvantage of a loss of cell interactions during stimulation. In a further refinement, individual cells were identifiable by ICCS, which gives the possibility to characterize for multiple characteristics, such as cytokine production and phenotype of the cell. Using ICCS, ChIFN-gamma production was evaluated. Although cells were detected at only low frequencies, polyclonal stimulation of peripheral blood mononuclear cell (PBMC) or spleen cells resulted in a significant increase in ChIFN-gamma production by CD4+ and CD8+ cells.

Manipulation of small RNAs to modify the chicken transcriptome and enhance productivity traits.

In recent years there has been a revolution in our understanding of genes and how they come to control the physical outcomes of development. Central to this has been the understanding of the cellular processes of RNA interference (RNAi), for which the Nobel Prize for Physiology or Medicine was awarded in 2006. Coupled with this has been the recognition that microRNAs are key mediators of this process within cells. RNAi whether mediated exogenously by synthetic oligonucleotides or vector-delivered double stranded RNA or endogenously by microRNAs can have a profound and specific effect on gene expression. Elucidating and understanding these processes in the chicken will provide critical information to enable more precise control over breeding strategies for improvement of traits in production poultry, either by direct or indirect means. It will also provide alternative strategies for the control and prevention of important avian diseases.

Avian genomics and the innate immune response to viruses.

Viral diseases pose a significant threat to the poultry industry. However, there is currently a lack of antivirals and suitable vaccine adjuvants available to the poultry industry to combat this problem. The innate immune system is now recognised to be essential in the response to viral infection. However, in contrast to mammals, the innate immune response in chickens is relatively uncharacterised. The release of the full chicken genome sequence has accelerated the identification of genes involved in the immune response. The characterisation of these genes, including Toll-like receptors and cytokines has led to the identification of potential alternate antivirals and adjuvants.

High-level production of recombinant chicken interferon-gamma by Brevibacillus choshinensis.

Cytokines, such as interferon-gamma have been shown to have adjuvant and growth promoting activity in poultry and livestock and have the potential to be used as alternatives to antibiotics. We have developed an efficient system for commercial-scale synthesis of recombinant chicken interferon-gamma (ChIFN-gamma) using Brevibacillus choshinensis as the host for protein production. The ChIFN-gamma expression vector, pNCIFN, was constructed using the novel Escherichia coli-B. choshinensis shuttle vector, pNCMO2. ChIFN-gamma expression was optimized by investigating different culture conditions and different host B. choshinensis mutants. The highest level of production was observed using the B. choshinensis HPD31-MB2 strain grown at 30 degrees C, where ChIFN-gamma was produced at approximately 300-500 mg/L. ChIFN-gamma was also produced as a His-tagged fusion protein by using the pNCHis-IFN expression vector, a derivative of pNCMO2. The protein was constitutively secreted into the culture supernatant and could be partially purified in a single step using a Ni-nitrilotriacetic acid column. This recombinant His-ChIFN-gamma was shown to have the same biological activity as native ChIFN-gamma.

Immune status assessment by abundance of IFN-alpha and IFN-gamma mRNA in chicken blood.

Avian diseases, including such viral infection as infectious bursal disease, infectious anemia, and Marek's disease, often cause immunosuppression, leading to more severe infection, problems with secondary infection, and inadequate responses to vaccination. Immunosuppression thus causes serious economic losses in commercial poultry production. To date, methods for assessing immune status have been too slow to be of practical help. Reasoning that immunosuppression should be reflected by reduced production of interferons (IFN) in response to a viral antigen, we have developed competitive nucleic acid hybridization microtiter plate assays for chicken IFN-alpha (ChIFN-alpha) and ChIFN-gamma mRNA. To evaluate the assay, chickens were challenged with inactivated Newcastle disease virus (iNDV). Whole blood samples were collected at various times subsequently and preserved with a cationic detergent. Later, total RNA was extracted, and mRNA for both ChIFN-alpha and ChIFN-gamma was measured. Both rose from undetectable levels to reach a peak by 4 h, remained high for about 3 days, and fell to undetectable levels by day 5. Results were similar in chickens aged between 1 and 28 days. In later experiments, blood was collected 4 h after viral challenge. When chickens were immunosuppressed by administering 4-5 mg cyclophosphamide (CY) daily for 3 days and challenged with iNDV, they transcribed less ChIFN-alpha and ChIFN-gamma mRNA, and their antibody response was impaired. Our results suggest that suspected immunosuppression in a commercial flock could be assessed within 2-3 days by challenging birds with iNDV and measuring the amounts of ChIFN-alpha and ChIFN-gamma mRNA in blood obtained 2-4 h later.

Structural and functional homology between duck and chicken interferon-gamma.

The Duck interferon gamma (DuIFN-gamma) cDNA was cloned from a phytohaemaglutinin-stimulated duck spleen cDNA library screened using a chicken IFN-gamma (ChIFN-gamma) cDNA probe. The DuIFN-gamma cDNA is 1392 nt long and shows 99% and 80% sequence identity with another cloned DuIFN-gamma cDNA, and with ChIFN-gamma cDNA, respectively. The cDNA contains a 495 bp ORF that encodes a putative 164 amino acid (AA) protein that shares 67% identity with ChIFN-gamma, but only 30-35% identity with mammalian IFN-gamma. The predicted three-dimensional (3D) structures of DuIFN-gamma and ChIFN-gamma are similar when analysed by comparative protein modelling. Culture supernatant collected from COS cells transfected with DuIFN-gamma cDNA was able to activate nitrite secretion from a chicken macrophage cell line (HD11) in a dose-dependent fashion. This activity could not be neutralised by an anti-ChIFN-gamma monoclonal antibody (Mab 85) that was able to neutralise the activity of ChIFN-gamma. Recombinant DuIFN-gamma (rDuIFN-gamma) protein was expressed in E. coli as an N-terminally His-tagged protein and was purified on a nickel affinity column. The eluted protein, which was detected as a approximately 18 kDa band with a purity of >90%, was also detected by Western blot using the anti-ChIFN-gamma monoclonal antibody (Mab 9.1). The rDuIFN-gamma was shown to activate nitrite secretion by HD11 cells in a dose-dependent fashion with a specific activity that was approximately 16-fold lower than a rChIFN-gamma control. Two rabbit antisera raised against rDuIFN-gamma were able to neutralise COS cell-expressed DuIFN-gamma activity; one of these also neutralised ChIFN-gamma activity. These findings indicate that DuIFN-gamma shares structural and functional identity with ChIFN-gamma, which is consistent with our previous results which demonstrated cross reactivity with other lymphokines from the two species.

Measurement of lymphokine receptors.

One of the most widely studied lymphokine systems is the T lymphocyte growth factor interleukin 2 (IL-2). This unit describes two basic methods for the quantitation and biochemical characterization of IL-2 receptors. The first method employs the radioreceptor assay. Support protocols to this technique describe quantification of data via calculations of association and dissociation rates and the Scatchard plot analysis. The second approach detects cell-surface lymphokine receptors by covalently cross-linking IL-2 to its receptor. This method employs the chemical disuccinimidyl suberate (DSS) to achieve irreversible cross-linking of IL-2 to IL-2R.

Delivery of avian cytokines by adenovirus vectors.

A fowl adenovirus serotype 8 (FAV-8) recombinant was constructed by inserting an expression cassette consisting of the FAV major late promoter/splice leader sequences (MLP/SL), the chicken interferon-gamma (ChIFN-gamma) gene and SV40 polyA into sites in the right hand end of the FAV-8 genome. One recombinant (A3-13) was constructed by an insertion of ChIFN-gamma into a 1.3 kilobase pair (kbp) deletion which removed a putative open reading frame (ORF) with identity to the CELO (FAV serotype 1) 36 kDa homologue. A second recombinant (S4) removed a further 0.9 kbp and a third recombinant (AA1) was constructed in a small 50 base pair (bp) SpeI deletion. The recombinants displayed differing growth characteristics in CK monolayers. A3-13 grew slowly and only attained a titre of 10(5) pfu/ml, S4 had intermediate growth and AA1 showed wild type growth kinetics. These differing growth properties indicated that removal of the 36 kDa homologue had an effect on growth in vitro. Supernatants from CK monolayers infected with the recombinant virus were assayed for the production of ChIFN-gamma. Detectable levels of ChIFN-gamma were observed in supernatants as early as 24 h post infection (p.i.), peaked at 48 h p.i. and this level was maintained for at least 10 days. The level of production of ChIFN-gamma correlated with each recombinant's growth characteristics in vitro. Chickens treated with rFAV-ChIFN-gamma showed increased weight gains compared to controls and suffered reduced weight loss when challenged with the coccidial parasite Eimeria acervulina.

Avian cytokines - the natural approach to therapeutics.

While the effective use of antibiotics for the control of human disease has saved countless lives and has increased life expectancy over the past few decades, there are concerns arising from their usage in livestock. The use of antibiotic feed additives in food production animals has been linked to the emergence in the food chain of multiple drug-resistant bacteria that appear impervious to even the most powerful antimicrobial agents. Furthermore, the use of chemical antimicrobials has led to concerns involving environmental contamination and unwanted residues in food products. The imminent banning of antibiotic usage in livestock feed has intensified the search for environmentally-friendly alternative methods to control disease. Cytokines, as natural mediators and regulators of the immune response, offer exciting new alternatives to conventional chemical-based therapeutics. The utilisation of cytokines is becoming more feasible, particularly in poultry, with the recent cloning of a number of avian cytokine genes. Chickens offer an attractive small animal model system with which to study the effectiveness of cytokine therapy in the control of disease in intensive livestock. In this report we will review the status of avian cytokines and focus on our recent studies involving the therapeutic potential of chicken interferon gamma (ChIFN-gamma) as a vaccine adjuvant and a growth promoter.

Cytokine therapy: a natural alternative for disease control.

Disease control in food production animals is normally mediated through the use of vaccines, chemicals and antibiotics. However, the extensive use of antibiotics and chemicals in livestock has resulted in environmental and human health concerns, particularly with regard to the emergence of drug-resistant bacteria in the food chain. In fact, the World Health Organisation (WHO) has now urged meat producers to use environmentally-friendly alternative methods to control disease. Cytokines, as natural mediators of the immune response, offer exciting alternatives to conventional therapeutics. The utilisation of cytokines is becoming more feasible with the recent cloning of a number of cytokine genes. Since the chicken's immune system is similar to that of mammals, they offer an attractive model system with which to study the effectiveness of cytokine therapy in the control of disease in intensive livestock. In this report we will review our recent studies on the therapeutic potential of chicken interferon gamma (ChIFN-gamma) as a vaccine adjuvant and a growth promoter.

Recombinant chicken IFN-gamma expressed in Escherichia coli: analysis of C-terminal truncation and effect on biologic activity.

Interferon-gamma (IFN-gamma) possesses potent immunostimulatory properties, and it has recently been shown to have potential therapeutic properties. Recombinant protein technology is frequently used for commercial production of therapeutics, such as IFN. Biologically active recombinant chicken IFN-gamma (rChIFN-gamma) constructs bearing an N-terminal poly-His tag were expressed in Escherichia coli. Preparations of rChIFN-gamma contained varying ratios of a full-length and a truncated protein species (18 and 16 kDa, respectively). Amino acid sequence analysis of the full-length protein corroborated the sequence previously predicted from the cDNA sequence. Full-length rChIFN-gamma contains two cysteine residues at the C-terminus, and these were labeled by reduction and subsequent specific alkylation with fluorescent tag (5-I-AEDANS) to distinguish between full-length and C-terminally truncated forms of rChIFN-gamma. Comparative peptide mapping, amino acid sequencing, and mass spectrometry revealed that the 16 kDa protein was truncated at Lys133. It was also observed that the 18 kDa rChIFN-gamma protein was infrequently contaminated with small quantities of protein truncated at Arg141. A truncated recombinant construct (His1-Lys133) was also expressed in E. coli and had biologic activity comparable with that of the full-length construct. The 3-D structure of rChIFN-gamma was deduced by comparative modeling with bovine and human IFN-gamma crystallographic structures. Analysis of sequences and comparison of structures have revealed that the 3-D structure of rChIFN-gamma is similar to those of bovine and human molecules despite an overall amino acid identity of only 32%.

Coadministration of IFN-gamma enhances antibody responses in chickens.

The development of new generation vaccines has focused on the use of natural immunologic adjuvants that are capable of enhancing a protective immune response. The use of cytokines as immunomodulators in livestock animals, particularly poultry, is becoming more feasible with the recent cloning of several cytokine genes and the progression of new delivery technologies, such as live vectors and DNA delivery. Given that chickens are reared under intensive conditions that are conducive to infection by opportunistic pathogens, the primary mechanism for disease control in poultry is early and effective vaccination. However, many poultry vaccines offer only short-term protection or give nonuniform responses within flocks. We have developed a model system with which to measure the adjuvant potential of cytokines in chickens. This involves measuring antibody levels following coadministration of chicken interferon-gamma (Ch-IFN-gamma) with sheep red blood cells (SRBC). Groups of SPF and commercial broiler birds were injected with two different doses of SRBC with and without coadministration of Ch-IFN-y. Three weeks later, all birds were boosted with SRBC alone. Sera were collected weekly and anti-SRBC antibody titers (total Ig and IgG) were determined by hemagglutination. Priming Ch-IFN-gamma resulted in enhanced primary and secondary (IgG) antibody responses that persisted at higher levels when compared with birds that received SRBC alone. Second, coadministration of Ch-IFN-y allowed a 10-fold lower dose of antigen to be as effective as a high dose of antigen that was given without Ch-IFN-gamma. Third, treatment with Ch-IFN-y resulted in an increase in the proportion of birds responding to antigen challenge. These results suggest the potential use for Ch-IFN-gamma as a vaccine adjuvant.

Potential use of cytokine therapy in poultry.

Newly hatched chickens are highly susceptible to infection during the first 2 weeks of life. The utilisation of cytokines as therapeutic agents in livestock animals, in particular poultry, has become more feasible with the recent cloning of cytokine genes and the progression of new technologies such as live vectors. We have constructed a live recombinant fowlpox virus (FPV) that expresses chicken myelomonocytic growth factor (fp/cMGF). Administration of fp/cMGF to chicks resulted in a marked and sustained increase in the number of circulating blood monocytes as well as an increase in their state of activation, as measured by enhanced phagocytic activity and elevated production of nitric oxide. We have recently cloned the gene for chicken interferon-gamma (ChIFN-gamma). Recombinant ChIFN-gamma was capable of protecting chick fibroblasts from undergoing virus-mediated lysis and induced nitrite secretion from chicken macrophages in vitro. Preliminary vaccination trials have indicated that co-administration of ChIFN-gamma with antigen (sheep red blood cells) resulted in enhanced secondary (IgG) antibody responses and allowed a 10-fold lower dose of antigen to be used. Furthermore, administration of ChIFN-gamma resulted in enhanced weight gain in chicks and improved their resistance to disease challenge. The ability of cytokines to combat infection and enhance vaccine efficacy makes them excellent candidates as a therapeutic agents and adjuvants.

Chicken interferon types I and II enhance synergistically the antiviral state and nitric oxide secretion.

This report shows that chicken type I and type II interferons (IFNs), like their mammalian counterparts, act synergistically such that a mixture of the two has much greater activity than that expected from the separate contribution of each type. The degree of antiviral synergy was measured by virus plaque reduction and cytopathic effect (CPE) inhibition in both primary and secondary chicken embryo cell cultures. Mixtures of the two Ch-IFNs produced antiviral effects 3-10 times greater than that expected from strict additivity of each IFN acting alone. At high concentrations of IFN mixtures there was a qualitative shift to an exponential IFN action does-response curve that revealed synergistic effects greater than 100-fold. Synergy resulted even with mixtures containing less than 1 U/ml of either type of Ch-IFN. The antiviral effects developed more rapidly with mixtures than when type I or II Ch-IFN was used alone. Mixtures of the two types of Ch-IFN synergistically potentiated nitric oxide secretion in cells of the HD11 chicken macrophage line. Molecular mechanisms are cited that may account for synergy between type I and type II IFNs, and speculation is offered on the epidemiologic and therapeutic implications of synergy in vivo.

Expression of the inhibitor of T-cell apoptosis (ita) gene in hen ovarian follicles during development.

It is now well established that within the hen ovary, preovulatory follicles rarely become atretic and that granulosa cells from preovulatory follicles are relatively resistant to undergoing apoptosis in vitro. By comparison, prehierarchal (< or = 8-mm diameter) follicles are highly susceptible to becoming atretic in vivo, and approximately 70% of granulosa cells collected from 3- to 8-mm-diameter follicles rapidly undergo apoptosis when incubated for as little as 6 h in vitro in defined medium. The present studies were conducted to characterize expression of an inhibitor of apoptosis (iap) gene, inhibitor of T-cell apoptosis (ita), within hen follicle tissues at various stages of follicle development. The ita gene product has recently been shown to share homology within both the baculovirus repeat sequences of the N-terminus and the zinc ring-finger motif from the C-terminus and was originally determined to be expressed in chicken cells of T-lymphocyte lineage. In the present studies, highest levels of ita mRNA within the granulosa cell layer were found in preovulatory (atresia-resistant) follicles, with significantly lower levels detected in prehierarchal follicles. After 24 h of primary culture, ita mRNA levels increased in granulosa cells from preovulatory follicles by 3.2-fold as compared to those in freshly collected cells and were elevated by 8.9-fold in those granulosa cells from 6- to 8-mm follicles that successfully formed a primary culture monolayer. Moreover, ita mRNA levels were significantly increased in 6- to 8-mm-follicle granulosa cells after only 2 h of suspension culture, and this increase could be prevented by actinomycin D. This spontaneous increase in ita expression may serve to protect from cell death the relatively small population of prehierarchal follicle granulosa cells that survive in vitro. It is concluded from these data, taken together, that patterns of ita mRNA expression during follicle development are consistent with a potential role for this gene in protecting granulosa cells from apoptosis and thus maintaining follicle viability.

In vivo effects of chicken interferon-gamma during infection with Eimeria.

Newly hatched chickens are highly susceptible to infection by opportunistic pathogens during the first 1 or 2 weeks of life. The use of cytokines as therapeutic agents has been studied in animal models as well as in immunosuppressed patients. This approach has become more feasible in livestock animals, in particular poultry, with the recent cloning of cytokine genes and the development of new technologies, such as live delivery vectors. We have recently cloned the gene for chicken interferon-gamma (Ch-IFN-gamma). Poly-HIS-tagged recombinant Ch-IFN-gamma was expressed in Escherichia coli, was purified by Ni chromatography, and was found to be stable at 4 degrees C and an ambient temperature for at least several months and Several weeks, respectively. Ch-IFN-gamma was capable of protecting chick fibroblasts from undergoing virus-mediated lysis, induced nitrite secretion from chicken macrophages in vitro, and enhanced MHC class II expression on macrophages. Administration of recombinant Ch-IFN-gamma to chickens resulted in enhanced weight gain over a 12-day period. Furthermore, the therapeutic potential of Ch-IFN-gamma was assessed using a coccidial challenge model. Birds were treated with Ch-IFN-gamma or a diluent control and then infected with Eimeria acervulina. Infected birds treated with Ch-IFN-gamma showed improved weight gain relative to noninfected birds. The ability of Ch-IFN-gamma to enhance weight gain in the face of coccidial infection makes it an excellent candidate as a therapeutic agent.