Calvin L. Keeler

Transcriptional response of chicken macrophages to Salmonella enterica serovar enteritidis infection.

The transcriptional profiles of chicken macrophages (HD11) infected with Salmonella enterica serovar Enteritidis (SE) were analyzed by using an avian macrophage microarray and real time RT-PCR. Out of 4906 array elements interrogated, 269 genes exhibited a 2 fold change (P < 0.001) over a 24 h time-course. Genes coding for proinflammatory cytokines, CC and CXC chemokines, and chemokine ligand were upregulated; whereas genes associated with transcription, cell adhesion and proliferation were downregulated. Most transcriptional changes occurred at 5 hours post-inoculation (hpi), with more genes downregulated than upregulated.At 5 hpi, the levels of gallinacin 1, lymphotactin, RhoA, and MHCIB2M transcripts were significantly decreased. In contrast, the levels of Cdc42 and MHCIIBLB2 mRNA were elevated. Infection of HD11 cells with mutant SE strains carrying an inactivated type three secretion system (TTSS1 or TTSS2) induced significantly higher levels of CCL4, K203, lymphotactin, and RhoA than wild type SE. In conclusion, chicken macrophage genes belonging to diverse functional classes were transcriptionally modulated by SE and selective modulation of host innate responses involved the effectors of TTSS1/2.

A functional genomics approach to the study of avian innate immunity

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.

Construction and application of an avian intestinal intraepithelial lymphocyte cDNA microarray (AVIELA) for gene expression profiling during Eimeria maxima infection

Intestinal intraepithelial lymphocytes (IELs) are the primary immune effector cells in the gut and play a critical role in eliciting protective immunity to enteric pathogens such as Eimeria, the etiologic agent of avian coccidiosis. In this study, a microarray of genes expressed by intestinal IELs from Eimeria-infected chickens was constructed using the expressed sequence tag (EST) strategy. The avian intestinal IEL cDNA microarray (AVIELA) contained duplicates of 9,668 individual ESTs (6,654 known genes and 3,014 unique singletons of unknown identity) and was used to analyze gene expression profiles during primary and secondary Eimeria maxima infections. Following primary inoculation with E. maxima, the expression levels of 74 genes were significantly altered more than two-fold over the 3-day infection period (51 up-regulated, 23 down-regulated). Following secondary infection, the expression levels of 308 genes were significantly altered (62 up-regulated, 246 down-regulated). Pathway gene analysis indicated that many of the modulated genes were related to apoptosis, JAK/STAT, MAPK, interleukin, and TLR signaling pathways, and involving innate and adaptive immune responses. This chicken IEL microarray will provide a valuable resource for future transcriptional profiling of the genes involved in protective immunity to chicken enteric pathogens.

Gene expression profiles of two B-complex disparate, genetically inbred Fayoumi chicken lines that differ in susceptibility to Eimeria maxima

This study was conducted to compare the gene expression profiles, after Eimeria maxima infection, between 2 B-complex congenic lines of Fayoumi chickens that display differences in disease resistance and innate immunity against avian coccidiosis using cDNA microarray. When compared with uninfected controls using a cutoff of >2.0-fold alteration (P < 0.05), M5.1 demonstrated altered expression of 1 (downregulated), 12 (6 up, 6 down), and 18 (5 up, 13 down) mRNA at 3, 4, and 5 d postinfection, respectively. In the M15.2 line, altered expression was observed in 6 (3 up, 3 down), 29 (11 up, 18 down), and 32 (8 up, 24 down) transcripts at the 3 time points, compared with uninfected controls. Comparison of the expression levels between M5.1 and M15.2 chickens after E. maxima infection revealed alterations in 32 (10 up, 22 down), 98 (43 up, 55 down), and 92 (33 up, 59 down) mRNA at the 3 time points. Functional analysis using gene ontology categorized the genes exhibiting the different expression patterns between 2 chicken lines into several gene ontology terms including immunity and defense. In summary, transcriptional profiles showed that more gene expression changes occurred with E. maxima infection in the M15.2 than the M5.1 line. The most gene expression differences between the 2 chicken lines were exhibited at d 4 and 5 after E. maxima infection. These results demonstrate that differential gene expression patterns associated with the host genetic difference in coccidiosis resistance provide insights into the host protective immune mechanisms and present a rational basis to target specific genes and gene products to bolster host defenses against avian coccidiosis.

Characterization of the assembly and processing of infectious laryngotracheitis virus glycoprotein B.

Infectious laryngotracheitis virus (ILTV) is an alpha-herpesvirus that causes severe upper respiratory infections in chickens. Although ten putative ILTV glycoprotein genes have been identified by sequence analysis, no ILTV glycoprotein has been extensively characterized. In order to delineate the synthesis and processing pathway of ILTV glycoprotein B (gB), rabbit polyclonal antibodies were raised against a Cro-gB-beta-galactosidase fusion protein. Through immunoprecipitation analysis of ILTV-infected chicken embryo liver cells it was determined that ILTV gB is initially synthesized as a 110 kDa monomeric precursor protein which rapidly assembles into homodimers composed of 100 kDa subunits. The dimer form of ILTV gB is rapidly cleaved to form two disulphide-linked species of 58 kDa. The apparent reduction in mass (from 110 to 100 kDa) of the mature form of gB during processing in the Golgi apparatus appears to be a common feature of avian herpesvirus gB proteins.

Diacylated lipopeptide from Mycoplasma synoviae mediates TLR15 induced innate immune responses

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.

Comparison of global transcriptional responses to primary and secondary Eimeria acervulina infections in chickens.

In the current study, we compared chicken gene transcriptional profiles following primary and secondary infections with Eimeria acervulina using a 9.6K avian intestinal intraepithelial lymphocyte cDNA microarray (AVIELA). Gene Ontology analysis showed that primary infection significantly modulated the levels of mRNAs for genes involved in the metabolism of lipids and carbohydrates as well as those for innate immune-related genes. By contrast, secondary infection increased the levels of transcripts encoded by genes related to humoral immunity and reduced the levels of transcripts for the innate immune-related genes. Because the observed modulation in transcript levels for gene related to energy metabolism and immunity occurred concurrent with the clinical signs of coccidiosis, these results suggest that altered expression of a specific set of host genes induced by Eimeria infection may be responsible, in part, for the observed reduction in body weight gain and inflammatory gut damage that characterizes avian coccidiosis.

Transposon mutagenesis of Mycoplasma gallisepticum

There are few systems available for studying the genetics of the important avian respiratory pathogen, Mycoplasma gallisepticum. These techniques are needed to develop a mechanism to study the molecular pathogenesis of M. gallisepticum. Tn916 has the ability to transpose into the M. gallisepticum genome by both transformation and conjugation. In this study, PEG-mediated transformation was employed for the transfer of Tn916 into M. gallisepticum and create a transposon mutant library. Transformants were obtained at a frequency of approximately 5 x 10(-8) per recipient CFU. A total of 424 MG/Tn916 mutants were constructed and sequence data from the transposon junctions of 71 mutants was obtained and used to identify transposon insertion sites. Insertions were found throughout the genome in nearly all of the major gene categories, making this the first extensive characterization of a transposon mutant library of M. gallisepticum. Transposon stability was also examined, and it was determined that for two mutants the element was stably maintained in vivo in the absence of selective pressure.

Analysis of global transcriptional responses of chicken following primary and secondary Eimeria acervulina infections

BackgroundCharacterization of host transcriptional responses during coccidia infections can provide new clues for the development of alternative disease control strategies against these complex protozoan pathogens.MethodsIn the current study, we compared chicken duodenal transcriptome profiles following primary and secondary infections with Eimeria acervulina using a 9.6K avian intestinal intraepithelial lymphocyte cDNA microarray (AVIELA).ResultsGene Ontology analysis showed that primary infection significantly modulated the levels of mRNAs for genes involved in the metabolism of lipids and carbohydrates as well as those for innate immune-related genes. By contrast, secondary infection increased the levels of transcripts encoded by genes related to humoral immunity and reduced the levels of transcripts for the innate immune-related genes. The observed modulation in transcript levels for gene related to energy metabolism and immunity occurred concurrent with the clinical signs of coccidiosis.ConclusionsOur results suggest that altered expression of a specific set of host genes induced by Eimeria infection may be responsible, in part, for the observed reduction in body weight gain and inflammatory gut damage that characterizes avian coccidiosis.

Comparison of Transcriptional Changes Associated with E. acervulina and E. maxima Infections using cDNA Microarray Technology

Enhanced understanding of host-pathogen interactions at local sites of infection will extend our knowledge of disease pathogenesis and will facilitate the development of novel preventive methodologies against many infectious diseases of economic importance. In the current study, a 9.6K avian intestinal intraepithelial lymphocyte cDNA microarray (AVIELA) was developed to compare the local transcriptional profiles following primary and secondary infections with two major Eimeria parasites, E. acervulina (EA) and E. maxima (EM), which infect the intestinal duodenum and jejunum, respectively. Gene Ontology analyses showed that EAinfection primarily induced genes associated with lipid metabolism and intracellulartrafficking whereas EM infection upregulated the genes involved in protein biosynthesis and metabolism, and downregulated apoptosis related genes. Following primary EA infection, there was a significantly enhanced expression of genes involved in the signal pathway of T cell activation and cytoskeletal regulation. Thus, the AVIELA array provides a valuable tool for investigating host-pathogen interactions in avian coccidiosis and allows for the comparison of the transcriptional regulations induced by species of Eimeria that infect different areas of the intestine.