Psittacid herpesvirus 1 (PsHV-1) is the causative agent of Pacheco's disease, an acute, highly contagious, and potentially lethal respiratory herpesvirus infection in psittacine birds, while infectious laryngotracheitis virus (ILTV) is a highly contagious and economically significant avian herpesvirus which is responsible for an acute respiratory disease limited to galliform birds. The complete genome sequence of PsHV-1 has been determined and compared to the ILTV sequence, assembled from published data. The PsHV-1 and ILTV genomes exhibit similar structural characteristics and are 163,025 bp and 148,665 bp in length, respectively. The PsHV-1 genome contains 73 predicted open reading frames (ORFs), while the ILTV genome contains 77 predicted ORFs. Both genomes contain an inversion in the unique long region similar to that observed in pseudorabies virus. PsHV-1 is closely related to ILTV, and it is proposed that it be assigned to the Iltovirus genus. These two avian herpesviruses represent a phylogenetically unique clade of alphaherpesviruses that are distinct from the Marek's disease-like viruses ( Mardivirus ). The determination of the complete genomic nucleotide sequences of PsHV-1 and ILTV provides a tool for further comparative and functional analysis of this unique class of avian alphaherpesviruses.

Avian-specific toll like receptor 15 (TLR15) is functionally equivalent to a group of TLR2 family proteins that the mammalian innate immune system utilizes to recognize a broad spectrum of microbe-associated molecular patterns, including bacterial lipoproteins. In this study we examined the role of chicken TLR2 family members in the innate immune response to the avian pathogenic bacterium, Mycoplasma synoviae. We found that Mycoplasma synoviae, and specifically the N-terminal diacylated lipopeptide (MDLP) representing the amino-terminal portion of its mature haemagglutinin protein, significantly induces the expression of TLR15, but not TLR1 and TLR2 in chicken macrophages and chondrocytes. TLR15 activation is specific and depends on diacylation of the lipopeptide. Activation of TLR15 after stimulation with Mycoplasma synoviae and MDLP triggers an increase in the expression of transcription factor nuclear factor kappa B and nitric oxide production. Moreover, transfection of avian macrophage cells with small interfering RNA reduces the expression of TLR15 after stimulation with MDLP. This leads to decreased activation of the innate immune response, as measured by nitric oxide production. Additionally, pretreatment of cells with neutralizing anti-TLR15 antibody results in a notable attenuation of MDLP-driven release of nitric oxide. This positive correlation may constitute a mechanism for stimulating the innate immune response against avian mycoplasmas in chicken cells via TLR15.

A 150-kDa cytadhesin-like protein from Mycoplasma gallisepticum has been identified. A previously described 583-bp fragment (J.E. Dohms, L.L. Hnatow, P. Whetzel, R. Morgan and C.L. Keeler, Jr., Avian Dis. 37:380-388, 1993) was used to probe a genomic library of M. gallisepticum DNA. An 8.0-kb SacI fragment was identified, cloned, and partially sequenced. Analysis of the resulting 3,750-bp sequence revealed the presence of a 3,366-nucleotide open reading frame, mgc1. The 1,122-amino-acid protein encoded by this open reading frame, MGC1, has characteristics of a class I membrane protein and has homology with the MgPa cytadhesin of Mycoplasma genitalium (26.3%) and the P1 cytadhesin of Mycoplasma pneumoniae (28.7%). A portion of MGC1 was expressed as a glutathione S-transferase fusion protein and used to produce antiserum in rabbits. The antiserum recognizes a 150-kDa protein from M. gallisepticum. The protein is sensitive to trypsin, confirming that it is surface exposed. Primer extension analysis indicates that the mgc1 RNA starts within an upstream open reading frame, suggesting complex control of its expression. This is the first description of a functional gene from M. gallisepticum showing homology to cytadhesin genes from human mycoplasmas.

Background: Wild waterfowl, including ducks, represent the classic reservoir for low pathogenicity avian influenza (LPAI) viruses and play a major role in the worldwide dissemination of AIV. AIVs belonging to the hemagglutinin (H) 7 subtype are of epidemiological and economic importance due to their potential to mutate into a highly pathogenic form of the virus. Thus far, however, relatively little work has been conducted on elucidating the host-pathogen interactions of ducks and H7 LPAIVs. In the current study, three H7 LPAIVs isolated from either chicken, duck, or turkey avian species were evaluated for their comparative effect on the transcriptional innate immune response of ducks. Results: Three H7 LPAIV isolates, chicken-origin (A/chicken/Maryland/MinhMa/2004), duck-origin (A/pintail/Minnesota/423/1999), and turkey-origin (A/turkey/Virginia/SEP-67/2002) were used to infect Pekin ducks. At 3 days post-infection, RNA from spleen tissue was used for transcriptional analysis using the Avian Innate Immune Microarray (AIIM) and quantitative real-time RT-PCR (qRT-PCR). Microarray analysis revealed that a core set of 61 genes was differentially regulated in response to all three LPAIVs. Furthermore, we observed 101, 135, and 628 differentially expressed genes unique to infection with the chicken-, duck-, or turkey-origin LPAIV isolates, respectively. qRT-PCR results revealed significant (p<0.05) induction of IL-1β, IL-2, and IFNγ transcription, with the greatest induction observed upon infection with the chicken-origin isolate. Several key innate immune pathways were activated in response to LPAIV infection including the toll-like receptor and RIG-I-like receptor pathways. Conclusions: Pekin ducks elicit a unique innate immune response to different species-of-origin H7 LPAIV isolates. However, twelve identifiable genes and their associated cell signaling pathways (RIG-I, NOD, TLR) are differentially expressed regardless of isolate origin. This core set of genes are critical to the duck immune response to AI. These data provide insight into the potential mechanisms employed by ducks to tolerate AI viral infection.

Elucidating the complex microbial interactions in biological environments requires the identification and characterization of not only the bacterial component but also the eukaryotic viruses, bacteriophage, and fungi. In a proof of concept experiment, next generation sequencing approaches, accompanied by the development of novel computational and bioinformatics tools, were utilized to examine the evolution of the microbial ecology of the avian trachea during the growth of a healthy commercial broiler flock. The flock was sampled weekly, beginning at placement and concluding at 49 days, the day before processing. Metagenomic sequencing of DNA and RNA was utilized to examine the bacteria, virus, bacteriophage, and fungal components during flock growth. The utility of using a metagenomic approach to study the avian respiratory virome was confirmed by detecting the dysbiosis in the avian respiratory virome of broiler chickens diagnosed with infection with infectious laryngotracheitis virus. This study provides the first comprehensive analysis of the ecology of the avian respiratory microbiome and demonstrates the feasibility for the use of this approach in future investigations of avian respiratory diseases.

A second-generation 4,959 element cDNA microarray has been created and evaluated for its potential use in examining the avian innate immune response. The elements in this array were obtained from EST libraries of stimulated avian PMNC-derived monocytes/macrophages and supplemented by genes of interest from several specific innate immune pathways. The elements are spotted in triplicate resulting in 14,877 total spots per slide. The avian innate immunity microarray (AIIM) contains 25 avian interleukin, chemokine, and cytokine elements. The array also contains elements for several innate immune pathways, including genes involved in the Toll-like receptor (TLR) pathway (including six of the currently known avian TLR receptors), avian interferon/antiviral response pathway genes, and genes involved in apoptosis, antigen presentation and the oxidative burst. The AIIM can be used to evaluate global gene expression patterns in a number of immunologically relevant tissues and in chickens, turkeys and ducks. The array has also been evaluated for its ability to monitor the avian immune response to both bacterial (avian pathogenic Escherichia coli) and viral (avian influenza) avian pathogens.