Development and application of a novel triplex protein microarray method for rapid detection of antibodies against avian influenza virus, Newcastle disease virus, and avian infectious bronchitis virus.

Avian influenza virus (AIV), Newcastle disease virus (NDV), and avian infectious bronchitis virus (IBV) inflict immense damage on the global poultry industry annually. Serological diagnostic methods are fundamental for the effective control and prevention of outbreaks caused by these viruses. In this study, a novel triplex protein microarray assay was developed and validated for the rapid and simultaneous visualized detection of antibodies against AIV, NDV, and IBV in chicken sera. The AIV nuclear protein (NP), NDV phosphoprotein (P), and IBV nonstructural protein 5 (nsp5) were produced in a prokaryotic expression system, purified, and immobilized onto an initiator integrated poly(dimethylsiloxane) (iPDMS) film as probes to detect antibodies against these viruses in chicken sera. After optimization of the reaction conditions, no cross-reactivity was detected with infectious bursal disease virus, avian leukosis virus subgroup J and chicken anemia virus antisera. The lowest detectable antibody titers in this assay corresponded to hemagglutination inhibition (HI) titers of 24 and 21 for AIV and NDV, respectively, and to an IDEXX antibody titer of 103 for IBV, using the HI assay and IDEXX commercial ELISA kit as the reference methods. When156 serum samples were tested using the new assay, the HI test and the IBV IDEXX ELISA kit, the assay showed 96.8% (151/156), 97.4% (152/156) and 99.4% (155/156) diagnostic accuracy for detection of AIV, NDV and IBV antibody, respectively. The current study suggests that the newly developed triplex microarray is rapid, sensitive, and specific, providing a viable alternative assay for AIV, NDV, and IBV antibody screening in epidemiological investigations and vaccination evaluations.

A novel protein chip for simultaneous detection of antibodies against four epidemic swine viruses in China.

BACKGROUND: At present, pig industry in China is faced with the complex situation of mixed infection caused by multiple pathogens. It is urgent to develop some new high-throughput molecular diagnosis assays to simultaneously detect pathogens or antibodies. Biochip array technology has made it possible to screen thousands of samples simultaneously; it has been twice named as one of the top 10 scientific and technological breakthroughs. Studies have reported encouraging results using protein biochips for detecting antibodies against avian infectious bronchitis virus and ruminant bluetongue virus, but the research of this technology for the diagnosis of swine diseases is still sparse. RESULTS: In this study, a novel protein chip was developed that can simultaneously detect the antibodies of four important swine viruses as follow, classical swine fever virus (CSFV), porcine parvovirus (PPV), Japanese encephalitis virus (JEV), and porcine reproductive and respiratory syndrome virus (PRRSV). Four prokaryotic expression plasmids pET-32a-E2 of CSFV, -VP2 of PPV, -EDIII of JEV, and -N of PRRSV were induced by IPTG (Isopropyl ß-D-1-Thiogalactopyranoside) and overexpressed in E.coli, respectively. The purified proteins were identified by Western blotting and then printed on epoxy-coated glass slides. The optimized parameters of this diagnostic chip showed that the spotting concentrations of E2、VP2、EDIII、N proteins were 0.2, 0.4, 0.4, and 0.4 mg/mL. The optimal primary and secondary antibody dilutions were 1:50 and 1: 600. Compared with the commercial ELISA (Enzyme-linked immunosorbent assay) kits, the positive and negative coincidence rates of this chip were 95.8% ~ 100 and 86.2% ~ 100%, as well as, no cross-reaction. CONCLUSION: This protein chip provided a fast, specific, and sensitive method for simultaneous detection of antibodies in clinical serum samples. Compared with traditional methods, this protein chip can monitor very small amount of serum.

Use of meat juice and blood serum with a miniaturised protein microarray assay to develop a multi-parameter IgG screening test with high sample throughput potential for slaughtering pigs.

BACKGROUND: Serological screening of pig herds at the abattoir is considered a potential tool to improve meat inspection procedures and herd health management. Therefore, we previously reported the feasibility of a miniaturised protein microarray as a new serological IgG screening test for zoonotic agents and production diseases in pigs. The present study investigates whether the protein microarray-based assay is applicable for high sample throughput using either blood serum or meat juice. MATERIAL AND METHODS: Microarrays with 12 different antigens were produced by Abbott (formerly Alere Technologies GmbH) Jena, Germany in a previously offered 'ArrayTube' platform and in an 'ArrayStrip' platform for large-scale use. A test protocol for the use of meat juice on both microarray platforms was developed. Agreement between serum and meat juice was analysed with 88 paired samples from three German abattoirs. Serum was diluted 1:50 and meat juice 1:2. ELISA results for all tested antigens from a preceding study were used as reference test to perform Receiver Operating Characteristic analysis for both test specimens on both microarray platforms. RESULTS: High area under curve values (AUC > 0.7) were calculated for the analysis of T. gondii (0.87), Y. enterocolitica (0.97), Mycoplasma hyopneumoniae (0.84) and Actinobacillus pleuropneumoniae (0.71) with serum as the test specimen and for T. gondii (0.99), Y. enterocolitica (0.94), PRRSV (0.88), A. pleuropneumoniae (0.78) and Salmonella spp. (0.72) with meat juice as the test specimen on the ArrayStrip platform. Cohens kappa values of 0.92 for T. gondii and 0.82 for Y. enterocolitica were obtained for the comparison between serum and meat juice. When applying the new method in two further laboratories, kappa values between 0.63 and 0.94 were achieved between the laboratories for these two pathogens. CONCLUSION: Further development of a miniaturised pig-specific IgG protein microarray assay showed that meat juice can be used on microarray platforms. Two out of twelve tested antigens (T. gondii, Y. enterocolitica) showed high test accuracy on the ArrayTube and the ArrayStrip platform with both sample materials.

Novel protein microarray for the detection of avian influenza virus antibodies and simultaneous distinction of antibodies against H5 and H7 subtypes.

Avian influenza virus (AIV) can cause serious zoonotic disease, thereby threatening the poultry industry and human health. An efficient and rapid detection approach is crucial to prevent and control the spread of avian influenza. In this study, a novel protein microarray was developed. Haemagglutinin proteins of H5 and H7 subtypes and nucleoprotein (NP) were purified and spotted onto the initiator-integrated poly-(dimethylsiloxane) as antigens. Monoclonal antibodies with inhibition effect were screened and utilized for the synchronous detection of three avian influenza antibodies in different species. In the protein microarray, the cut-off values were 40%, 50% and 30% inhibition for H5 antibody detection; 50%, 50% and 20% for NP antibody detection; 40%, 50% and 40% for H7 antibody detection in chicken, peacock and duck sera, respectively. The 95 serum samples were detected by microarray, and results were compared with the findings of AIV antibody test enzyme-linked immunosorbent assay (ELISA) or haemagglutination inhibition (HI) test. NP antibody detection in the microarray showed 100% (55/55) agreement ratio in chicken using ELISA. Compared with HI, H5 antibody detection in the microarray showed 100% (95/95) agreement ratio in chicken, peacock and duck, whilst those of H7 displayed 98.18% (54/55) agreement in chicken, 100% (20/20) in peacock and 90% (18/20) in duck. In conclusion, this novel protein microarray is a high-throughput and specific method for the detection of AIV antibodies and simultaneous distinction of antibodies against H5 and H7 subtypes. It can be applied to the serological diagnosis and epidemiological investigation of AIV. RESEARCH HIGHLIGHTS A novel protein microarray method has been developed. The microarray can detect AIV antibodies and distinguish between H5 and H7 subtypes. The study lays the foundation for simultaneous identification of multiple pathogens.

Novel protein chip for the detection of antibodies against infectious bronchitis virus.

BACKGROUND: Infectious bronchitis (IB) caused by the IB virus (IBV) can cause acute damage to chickens around the world. Therefore, rapid diagnosis and immune status determination are critical for controlling IBV outbreaks. Enzyme-linked immunosorbent assays (ELISAs) have been widely used in the detection of IBV antibodies in the early infection and continuous infection of IB because they are more sensitive and quicker than other diagnostic methods. RESULTS: We have developed two indirect microarray methods to detect antibodies against IBV: a chemiluminescent immunoassay test (CIT) and a rapid diagnostic test (RDT). IBV nonstructural protein 5 (nsp5) was expressed, purified from Escherichia coli, and used to spot the initiator integrated poly(dimethylsiloxane), which can provide a near "zero" background for serological assays. Compared with the IDEXX IBV Ab Test kit, CIT and RDT have a sensitivity and specificity of at least 98.88% and 91.67%, respectively. No cross-reaction was detected with antibodies against avian influenza virus subtypes (H5, H7, and H9), Newcastle disease virus, Marek's disease virus, infectious bursal disease virus, and chicken anemia virus. The coefficients of variation of the reproducibility of the intra- and inter-assays for CIT ranged from 0.8 to 18.63%. The reproducibility of RDT was consistent with the original results. The application of the IBV nsp5 protein microarray showed that the positive rate of the CIT was 96.77%, that of the nsp5 ELISA was 91.40%, and that of the RDT was 90.32%. Furthermore, the RDT, which was visible to the naked eye, could be completed within 15 min. Our results indicated that compared with nsp5 ELISA, the CIT was more sensitive, and the RDT had similar positive rates but was faster. Furthermore, the two proposed methods were specific and stable. CONCLUSIONS: Two microarray assays, which were rapid, specific, sensitive, and relatively simple, were developed for the detection of an antibody against IBV. These methods can be of great value for the surveillance of pathogens and monitoring the efficiency of vaccination.

How does temperature influences the development of lactococcosis? Transcriptomic and immunoproteomic in vitro approaches.

Lactococcus garvieae is the aetiological agent of lactococcosis, a haemorrhagic septicaemia that affects marine and freshwater fish, with special incidence and economic relevance in farmed rainbow trout. Water temperature is one of the most important predisposing factors in the development of lactococcosis outbreaks. Lactococcosis in trout usually occur when water temperatures rise to about 18 °C, while fish carriers remain asymptomatic at temperatures below 13 °C. The aim of this work was to analyse the differences in the complete transcriptome response of L. garvieae grown at 18 °C and at 13 °C and to identify the immunogenic proteins expressed by this bacterium at 18 °C. Our results show that water temperature influences the expression of L. garvieae genes involved in the lysis of part of the bacterial cell population and in the cold response bacterial adaptation. Moreover, the surface immunogenic protein profile at 18 °C suggests an important role of the lysozyme-like enzyme, WxL surface proteins and some putative moonlighting proteins (proteins with more than one function, usually associated with different cellular locations) as virulence factors in L. garvieae. The results of this study could provide insights into the understanding of the virulence mechanisms of L. garvieae in fish.

Lymphocytic, cytokine and transcriptomic profiles in peripheral blood of dogs with atopic dermatitis.

BACKGROUND: Canine atopic dermatitis (cAD) is a common chronic and pruritic skin disease in dogs. The development of cAD involves complex interactions between environmental antigens, genetic predisposition and a number of disparate cell types. The aim of the present study was to perform comprehensive analyses of peripheral blood of AD dogs in relation to healthy subjects in order to determine the changes which would be characteristic for cAD. RESULTS: The number of cells in specific subpopulations of lymphocytes was analyzed by flow cytometry, concentration of chosen pro- and anti-inflammatory cytokines (IL-4, IL-10, IL-13, TNF-α, TGF-ß1) was determined by ELISA; and microarray analysis was performed on RNA samples isolated from peripheral blood nuclear cells of AD and healthy dogs. The number of Th cells (CD3(+)CD4(+)) in AD and healthy dogs was similar, whereas the percentage of Tc (CD3(+)CD8(+)) and Treg (CD4(+)CD25(+) Foxp3(+)) cells increased significantly in AD dogs. Increased concentrations of IL-13 and TNF-α, and decreased levels of IL-10 and TGF-ß1 was observed in AD dogs. The level of IL-4 was similar in both groups of animals. Results of the microarray experiment revealed differentially expressed genes involved in transcriptional regulation (e.g., transcription factors: SMAD2, RORA) or signal transduction pathways (e.g., VEGF, SHB21, PROC) taking part in T lymphocytes lineages differentiation and cytokines synthesis. CONCLUSIONS: Results obtained indicate that CD8(+) T cells, beside CD4(+) T lymphocytes, contribute to the development of the allergic response. Increased IL-13 concentration in AD dogs suggests that this cytokine may play more important role than IL-4 in mediating changes induced by allergic inflammation. Furthermore, observed increase in Treg cells in parallel with high concentrations of TNF-α and low levels of IL-10 and TGF-ß1 in the peripheral blood of AD dogs point at the functional insufficiency of Treg cells in patients with AD.

The early immune response to infection of chickens with Infectious Bronchitis Virus (IBV) in susceptible and resistant birds.

BACKGROUND: Infectious Bronchitis is a highly contagious respiratory disease which causes tracheal lesions and also affects the reproductive tract and is responsible for large economic losses to the poultry industry every year. This is due to both mortality (either directly provoked by IBV itself or due to subsequent bacterial infection) and lost egg production. The virus is difficult to control by vaccination, so new methods to curb the impact of the disease need to be sought. Here, we seek to identify genes conferring resistance to this coronavirus, which could help in selective breeding programs to rear chickens which do not succumb to the effects of this disease. METHODS: Whole genome gene expression microarrays were used to analyse the gene expression differences, which occur upon infection of birds with Infectious Bronchitis Virus (IBV). Tracheal tissue was examined from control and infected birds at 2, 3 and 4 days post-infection in birds known to be either susceptible or resistant to the virus. The host innate immune response was evaluated over these 3 days and differences between the susceptible and resistant lines examined. RESULTS: Genes and biological pathways involved in the early host response to IBV infection were determined andgene expression differences between susceptible and resistant birds were identified. Potential candidate genes for resistance to IBV are highlighted. CONCLUSIONS: The early host response to IBV is analysed and potential candidate genes for disease resistance are identified. These putative resistance genes can be used as targets for future genetic and functional studies to prove a causative link with resistance to IBV.

Salmonella enterica Typhimurium infection causes metabolic changes in chicken muscle involving AMPK, fatty acid and insulin/mTOR signaling.

Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) infection of chickens that are more than a few days old results in asymptomatic cecal colonization with persistent shedding of bacteria. We hypothesized that while the bacterium colonizes and persists locally in the cecum it has systemic effects, including changes to metabolic pathways of skeletal muscle, influencing the physiology of the avian host. Using species-specific peptide arrays to perform kinome analysis on metabolic signaling pathways in skeletal muscle of Salmonella Typhimurium infected chickens, we have observed key metabolic changes that affected fatty acid and glucose metabolism through the 5'-adenosine monophosphate-activated protein kinase (AMPK) and the insulin/mammalian target of rapamycin (mTOR) signaling pathway. Over a three week time course of infection, we observed changes in the phosphorylation state of the AMPK protein, and proteins up and down the pathway. In addition, changes to a large subset of the protein intermediates of the insulin/mTOR pathway in the skeletal muscle were altered by infection. These changes occur in pathways with direct effects on fatty acid and glucose metabolism. This is the first report of significant cellular metabolic changes occurring systemically in chicken due to a Salmonella infection. These results have implications not only for animal production and health but also for the understanding of how Salmonella infection in the intestine can have widespread, systemic effects on the metabolism of chickens without disease-like symptoms.

Early mucosal responses in blue catfish (Ictalurus furcatus) skin to Aeromonas hydrophila infection.

Bacterial pathogens are well-equipped to detect, adhere to, and initiate infection in their finfish hosts. The mucosal surfaces of fish, such as the skin, function as the front line of defense against such bacterial insults that are routinely encountered in the aquatic environment. While recent progress has been made, and despite the obvious importance of mucosal surfaces, the precise molecular events that occur soon after encountering bacterial pathogens remain unclear. Indeed, these early events are critical in mounting appropriate responses that ultimately determine host survival or death. In the present study, we investigated the transcriptional consequences of a virulent Aeromonas hydrophila challenge in the skin of blue catfish, Ictalurus furcatus. We utilized an 8×60K Agilent microarray to examine gene expression profiles at key early timepoints following challenge (2 h, 12 h, and 24 h). A total of 1155 unique genes were significantly altered during at least one timepoint. We observed dysregulation in a number of genes involved in diverse pathways including those involved in antioxidant responses, apoptosis, cytoskeletal rearrangement, immunity, and extracellular matrix protein diversity and regulation. Taken together, A. hydrophila coordinately modulates mucosal factors across numerous cellular pathways in a manner predicted to enhance its ability to adhere to and infect the blue catfish host.

Protein microarray allergen profiling in bronchoalveolar lavage fluid and serum of horses with asthma.

BACKGROUND: The diagnostic value of allergen-specific immunoglobulin E (IgE) in horses with asthma is uncertain. A recently developed protein microarray detected abnormally high latex-specific IgE concentrations in the serum of horses with severe asthma. OBJECTIVES: The main objective was to characterize the IgE profiles of asthmatic horses in Switzerland using a protein microarray platform in serum and bronchoalveolar lavage fluid (BALF). The secondary objective was to determine whether serological and BALF allergen-specific IgE concentrations correlated. ANIMALS: Forty-four asthmatic and 39 control horses ≥5 years of age. METHODS: This prospective cross-sectional study investigated the sensitization profiles of horses with asthma compared with environmentally matched healthy controls. Both serum and BALF were analyzed using the protein microarray. Partial least square-discriminant analysis (PLS-DA) was used to identify and rank the importance of the allergens for class detection (ie, asthma vs control), with a variable influence on the projection (VIP) >1 considered significant. RESULTS: The allergens that best discriminated (VIP >1) asthmatic horses from controls were proteins derived from fungi (Aspergillus fumigatus), insects (Culicoides spp.), and latex (Hevea brasiliensis). The serological model predictive ability was markedly inferior (area under the curve [AUC] 0.585, 95% confidence interval [CI]: 0.454-0.747) to that of the BALF (AUC 0.751, 95% CI: 0.582-0.866). The two models shared nine allergens, of which eight showed significant weak to moderate correlations. CONCLUSION AND CLINICAL IMPORTANCE: The concentrations of several allergen-specific IgE were higher in asthmatic horses. The protein microarray performed better on BALF than serum for detection of asthma. Serological IgE concentrations do not closely correlate with BALF concentrations and should be interpreted with caution.

Microarray analysis revealed that immunity-associated genes are primarily regulated by roxarsone in promoting broiler chicken (Gallus gallus domesticus) growth.

Addition of roxarsone can significantly improve the growth of broiler chickens (Gallus gallus domesticus). Nevertheless, this application will lead to the contamination of the environment as well as animal products. Understanding the response of genes to roxarsone may bring about the discovery of new, safer substitutes. In this study, we monitored the expression of 8,935 genes in chicken breast muscle using microarrays. Analysis showed that 30 genes, such as the interleukin 3 regulated nuclear factor (NFIL3), the regulatory factor X-associated ankyrin-containing protein (RFXANK), the cleavage and polyadenylation-specific factor 3 (CPSF3), and the FK506 binding protein 9 (FKBP9), have consistently up or downregulated (fold change ≥1.5 or ≤0.6, P < 0.05, false discovery rate ≤0.05) throughout the medication periods. The results from microarray analysis were validated by real-time quantitative PCR. Further functional investigation showed that 13 of the identified genes are well documented, and surprisingly, 11 (85%) of these are related to immunity (5 are immunity and defense related, 4 are immunodeficiency disease related, 2 are immunosuppressive drug related), and the remaining 2 are energy metabolism related. These findings may suggest that supplement of roxarsone can improve the immunity of chickens through regulating the expression of associated genes, and as a result contribute to the growth promotion. Further research on the encoded proteins of the differentially expressed genes should provide more evidence for the potential mechanism.

Effects of yeast cell wall-derived mannan-oligosaccharides on jejunal gene expression in young broiler chickens.

The use of mannan-oligosaccharides (MOS) as alternatives to antibiotic growth promoters (AGP) has gained in popularity in recent years due to regulatory restrictions of using AGP in food animal production. Benefits of MOS usage include improvement on animal performance, feed efficiency, and gastrointestinal health. The molecular mechanisms of these functions however are not clear. The goal of the current study was to use a transcriptomics approach to investigate the effects of MOS on the intestinal gene expression profile of young broilers and characterize biological gene pathways responsible for the actions of MOS. One hundred and twenty 1-d-old Cobb 500 broiler chicks were randomly divided into 2 groups and were fed either a standard wheat-soybean meal-based (control) diet or the same diet supplemented with 2.2 g/kg of MOS (Bio-Mos, Alltech, Nicholasville, KY) for 3 wk, followed by jejunal gene expression profiling analysis using chicken-specific Affymetrix microarrays. Results indicated that a total of 672 genes were differentially expressed (P < 0.01 and fold change >1.2) in the jejunum by MOS supplementation. Association analysis indicated that differentially expressed genes are involved in diverse biological functions including energy production, cell death, and protein translation. Expression of 77 protein synthesis-related genes was differentially regulated by MOS in the jejunum. Further pathway analysis indicated that 15 genes related to oxidative phosphorylation were upregulated in the jejunum, and expression of genes important in cellular stress response, such as peroxiredoxin 1, superoxide dismutase 1, and thioredoxin, were also increased by MOS. Differential expression of genes associated with cellular immune processes, including lysozyme, lumican, ß 2-microglobin, apolipoprotein A-1, and fibronectin 1, were also observed in MOS-fed broilers. In summary, this study systematically identified biological functions and gene pathways that are important in mediating the biological effects of MOS in broilers.

Gene expression in breast muscle associated with feed efficiency in a single male broiler line using a chicken 44K microarray. II. Differentially expressed focus genes.

Global RNA expression in breast muscle obtained from a male broiler line phenotyped for high or low feed efficiency (FE) was investigated using microarray analysis. Microarray procedures and validation were reported previously. By using an overlay function of a software program (Ingenuity Pathway Analysis, IPA) in which canonical pathways are projected onto a set of genes, a subset of 27 differentially expressed focus genes were identified. Focus genes that were upregulated in the high FE phenotype were associated with important signal transduction pathways (Jnk, G-coupled, and retinoic acid) or in sensing cell energy status and stimulating energy production that would likely enhance growth and development of muscle tissue. In contrast, focus genes that were upregulated in the low FE muscle phenotype were associated with cytoskeletal architecture (e.g., actin-myosin filaments), fatty acid oxidation, growth factors, or ones that would likely be induced in response to oxidative stress. The results of this study provide additional information on gene expression and the cellular basis of feed efficiency in broilers.

Gene expression in breast muscle associated with feed efficiency in a single male broiler line using a chicken 44K oligo microarray. I. Top differentially expressed genes.

Global RNA expression in breast muscle obtained from a male broiler line phenotyped for high or low feed efficiency (FE) was investigated. Pooled RNA samples (n = 6/phenotype) labeled with cyanine 3 or cyanine 5 fluorescent dyes to generate cRNA probes were hybridized on a 4 × 44K chicken oligo microarray. Local polynomial regression normalization was applied to background-corrected red and green intensities with a moderated t-statistic. Corresponding P-values were computed and adjusted for multiple testing by false discovery rate to identify differentially expressed genes. Microarray validation was carried out by comparing findings with quantitative reverse-transcription PCR. A 1.3-fold difference in gene expression was set as a cutoff value, which encompassed 20% (782 of 4,011) of the total number of genes that were differentially expressed between FE phenotypes. Using an online software program (Ingenuity Pathway Analysis), the top 10 upregulated genes identified by Ingenuity Pathway Analysis in the high-FE group were generally associated with anabolic processes. In contrast, 7 of the top 10 downregulated genes in the high-FE phenotype (upregulated in the low-FE phenotype) were associated with muscle fiber development, muscle function, and cytoskeletal organization, with the remaining 3 genes associated with self-recognition or stress-responding genes. The results from this study focusing on only the top differentially expressed genes suggest that the high-FE broiler phenotype is derived from the upregulation of genes associated with anabolic processes as well as a downregulation of genes associated with muscle fiber development, muscle function, cytoskeletal organization, and stress response.

Expression analysis of global gene response to chronic heat exposure in broiler chickens (Gallus gallus) reveals new reactive genes.

The process of heat regulation is complex and the exact molecular mechanism is not fully understood. To investigate the global gene response to chronic heat exposure, a breast muscle cDNA library and a liver tissue cDNA library from Silkie fowl were constructed and analyzed in bioinformatics. A total of 8,935 nonredundant EST were identified from and used for gene expression analysis. Microarray assay revealed that in breast muscle of broiler chickens (Gallus gallus), 110 genes changed expression levels after 3 wk of cycling heat stress. Ubiquitin B (UBB); ubiquitin C (UBC); tumor necrosis factor receptor-associated factor 3-interacting Jun amino-terminal kinase activating modulator (TRAF3IP3); eukaryotic translation initiation factor 3, subunit 6 (EIF3S6); poly(A) binding protein, cytoplasmic 1 (PABPC1); and F-box only protein 11 (FBXO11) were the only genes that have been reported to be involved in heat regulation; the majority of the other genes were shown to be related for the first time. The finding of new heat-reactive genes [mitogen-activated protein kinase activating protein PM20/PM21; suppressors of cytokine signaling (SOCS) box-containing protein 2 (ASB2); ubiquitin-specific proteinase 45 (USP45); and TRK-fused gene (TFG)] suggests that the mitogen-activated protein kinase pathways as well as the ubiquitin-proteasome pathways and the nuclear factor κB pathways play important roles in heat regulation. This study provides new information on the regulation of heat stress, though the mechanism is far from being understood. Further in-depth research on the newly discovered heat-reactive genes is required to fully understand their molecular functions in thermoregulation.

Reorganisation of the caecal extracellular matrix upon Salmonella infection--relation between bacterial invasiveness and expression of virulence genes.

Interactions of Salmonella (S.) outer membrane structures with extracellular matrix (ECM) of host tissues seem to be crucial for bacterial adhesion and invasion. To evaluate the relationship between the ECM and bacterial invasiveness, the reorganisation of fibronectin, tenascin-C and laminin after Salmonella exposure in vivo, the Salmonella adhesiveness to ECM proteins in vitro and the virulence gene expression upon co-cultivation of salmonellae and ECM proteins were elucidated for two Salmonella strains with different capabilities to enter the intestinal mucosa. Immunohistochemistry and confocal microscopy showed that the infection of day-old chicks using either the highly invasive S. Enteritidis (SE) or the nearly non-invasive S. Infantis (SINF) strain was associated with an invasion-dependent reorganisation of fibronectin and tenascin-C in the caecal wall. Compared to SINF, clustered formations of SE were localised within and attached to the fibronectin and tenascin-C scaffold in the lamina propria indicating a relevance of ECM for bacterial dissemination in lower regions of the mucosa. In adhesion assays, SE was, indeed, significantly more adhesive to the matrix proteins than SINF. The attachment was accompanied by an increased fliC mRNA expression in SE demonstrated by microarray analysis as well as quantitative real-time RT-PCR. The data suggest a relationship between the capability of Salmonella serovars to interact with matrix proteins and to disseminate in gut mucosa perhaps in consequence of a matrix-mediated upregulation of the Salmonella motility gene fliC.

Expression microarrays in equine sciences.

Microarrays have become an important research tool for life science researchers. Expression microarrays are capable of profiling the gene expression pattern of tens of thousands of genes in a single experiment. It appears to be the platform of choice for parallel gene expression profiling. Various equine-specific gene expression microarrays have been generated and used. However, homologous microarrays are not yet commercially available for the horse. An alternative is the use of heterologous microarrays, mainly microarrays specific for mice or humans. Although the use of microarrays in equine research is still in its infancy, gene expression microarrays have shown their potential in equine research. This review presents the previous, current and potential use of expression microarrays in equine research.

Microarray analysis of differentially expressed genes from Peyer's patches of cattle orally challenged with bovine spongiform encephalopathy.

The most likely route of entry of infection following oral exposure to transmissible spongiform encephalopathies (TSE) is via the immunologically active Peyer's patches (PP). These secondary lymphoid organs appear to be the potential route for prion neuroinvasion. However, the molecular mechanisms involved in the uptake of the infectious prion agent and progression of disease remain still unclear. This investigation examined the changes in gene expression in PP following oral exposure of cattle to bovine spongiform encephalopathy (BSE) agents. The gene expression patterns in PP from cows 12 mo after BSE challenge were compared with controls using a microarray platform containing 24,000 oligonucleotides representing 16,846 unique gene loci and 5943 Expressed Sequence Tag (EST) from bovine genome. Between the challanged and control animals, 90 genes and 16 EST were identified as significantly differentially, expressed (>2.0-fold change): 36 were upregulated and 70 were downregulated. Of these genes, five were found to be related to immune function. Major histocompatibility complex (MHC) class II, MHC class II DQ alpha, L-RAP, and two hypothetical proteins. Differentially expressed genes related to cellular and metabolic processes including development and maturation of cells in the PP were also identified. In this context, the potential impacts of these gene expression changes in PP on BSE development are discussed.

Sequential microarray to identify timing of molecular responses to Haemonchus contortus infection in sheep.

Anthelmintics are currently the most common method of worm control. The emergence of worms with multiple-drug resistance and issues of residues in the food chain make alternative parasite control measures a priority. To develop improved and sustainable methods for controlling Haemonchus contortus such as genetic selection of resistant sheep, a better understanding of the host-parasite relationship is required. A trial was undertaken using sheep surgically implanted with abomasal fistulas to enable sequential biopsy of the abomasal mucosa during trickle infection with two strains of H. contortus. These were ivermectin-resistant CAVR and ivermectin-sensitive McMaster. From a gross parasitology perspective, this approach enabled the effect of developing immunity to be observed on both the establishment and maturation of two CAVR doses within and between groups. Since the only difference in parasite treatment between the groups was the staggering of the two CAVR doses, microarray results from biopsies taken on the same day in different groups were combined and compared between different biopsy dates to observe differential gene transcription over time. Differential gene transcription was detected by comparing transcription in our array data between different biopsy dates using a low P value screen (P<0.01) and by compiling a list of 82 immunoparasitology-related genes and examining transcription in this list with a higher P value screen (P<0.05). Our microarray data were validated in silico by comparison with intelectin 2, trefoil factor 3, calcium activated chloride channel and mucin 5 from other gene transcription studies and with phenotypic data such as the response by gammadelta T cells and immunoglobulins to H. contortus. The first four genes are involved in non-specific responses to infection and mucosal healing. These were upregulated at the early time points and intelectin 2 remained prominent throughout the trial. As the trial progressed, immunoglobulin genes became strongly upregulated. These included IgCgamma IgG2a heavy chain constant region, IGHE immunoglobulin heavy constant epsilon and IGHM immunoglobulin heavy constant mu.