Y. H. Hong

Differential gene expression profiles of β-defensins in the crop, intestine, and spleen using a necrotic enteritis model in 2 commercial broiler chicken lines

Changes in the expression levels of avian β-defensin (AvBD) mRNA were evaluated in necrotic enteritis (NE) disease model in 2 genetically disparate commercial broiler chicken lines: Ross and Cobb. The NE was initiated in the gut by a previously established co-infection model using oral Eimeria maxima infection followed by a Clostridium perfringens challenge. Among the 14 AvBD types examined, there was a tissue-specific expression of AvBD transcripts: AvBD1, AvBD7, and AvBD9 in the crop; AvBD8, AvBD10, and AvBD13; in the intestine and AvBD1 and AvBD7 in the spleen. The 2 different commercial broiler chicken lines showed differential gene expression patterns of AvBD transcripts following co-infection with E. maxima and C. perfringens, with R-line chickens generally showing higher expression levels than the C strain. Both chicken strains showed enhanced gene expression levels of proinflammatory cytokines, such as IL-1β, IL-6, IL-17F, and TNFSF15 in spleen, and TNFSF15 in intestine, whereas IL-17F was significantly increased only in the intestine of R-line chickens following NE infection. Although the exact nature of interactions between defensins and cytokines in determining the outcome of host innate immune responses to the pathogens of NE remains to be investigated, the differences in gene expression levels of β-defensins and proinflammatory cytokines in the intestine, crop, and spleen could explain the predisposed disease resistance and susceptibility to NE in the 2 commercial broiler chicken lines.

Immune-Related Gene Expression in Two B-Complex Disparate Genetically Inbred Fayoumi Chicken Lines Following Eimeria maxima Infection

To investigate the influence of genetic differences in the MHC on susceptibility to avian coccidiosis, M5.1 and M15.2 B-haplotype-disparate Fayoumi chickens were orally infected with live Eimeria maxima oocysts, and BW gain, fecal oocyst production, and expression of 14 immune-related genes were determined as parameters of protective immunity. Weight loss was reduced and fecal parasite numbers were lower in birds of the M5.1 line compared with M15.2 line birds. Intestinal intraepithelial lymphocytes from M5.1 chickens expressed greater levels of transcripts encoding interferon-gamma (IFN-gamma), interleukin-1beta (IL-1beta), IL-6, IL-8, IL-12, IL-15, IL-17A, inducible nitric oxide synthase, and lipopolysaccharide-induced tumor necrosis factor-alpha factor and lower levels of mRNA for IFN-alpha, IL-10, IL-17D, NK-lysin, and tumor necrosis factor superfamily 15 compared with the M15.2 line. In the spleen, E. maxima infection was associated with greater expression levels of IFN-gamma, IL-15, and IL-8 and lower levels of IL-6, IL-17D, and IL-12 in M5.1 vs. M15.2 birds. These results suggest that genetic determinants within the chicken MHC influence resistance to E. maxima infection by controlling the local and systemic expression of immune-related cytokine and chemokine genes.

In vitro effects of plant and mushroom extracts on immunological function of chicken lymphocytes and macrophages

1.u2003The present study was conducted to examine the effects of organic extracts from milk thistle (Silybum marianum), turmeric (Curcuma longa), reishi mushroom (Ganoderma lucidum), and shiitake mushroom (Lentinus edodes) on innate immunity and tumor cell viability. 2.u2003Innate immunity was measured by lymphocyte proliferation and nitric oxide production by macrophages, and the inhibitory effect on tumor cell growth was assessed using a non-radioactive assay. For measuring the cytokine levels in the HD11 macrophages which were treated with extracts of turmeric or shiitake mushroom, the levels of mRNAs for interferon-α (IFN- α), interleukin-1β (IL-1β), IL-6, IL-12, IL-15, IL-18, and tumor necrosis factor superfamily 15 (TNFSF15) were quantified by real time RT-PCR. 3.u2003In vitro culture of chicken spleen lymphocytes with extracts of milk thistle, turmeric, and shiitake and reishi mushrooms induced significantly higher cell proliferation compared with the untreated control cells. Stimulation of macrophages with extracts of milk thistle and shiitake and reishi mushrooms, but not turmeric, resulted in robust nitric oxide production to levels that were similar with those induced by recombinant chicken interferon-γ. All extracts uniformly inhibited the growth of chicken tumor cells in vitro at the concentration of 6·3 through 100 µg/ml. Finally, the levels of mRNAs encoding IL-1β, IL-6, IL-12, IL-18, and TNFSF15 were enhanced in macrophages that were treated with extracts of turmeric or shiitake mushroom compared with the untreated control. 4.u2003These results document the immunologically-based enhancement of innate immunity in chickens by extracts of plants and mushrooms with known medicinal properties in vitro. In vivo studies are being planned to delineate the cellular and molecular mechanisms responsible for their mechanism of action.

Clostridium perfringens α-Toxin and NetB Toxin Antibodies and Their Possible Role in Protection Against Necrotic Enteritis and Gangrenous Dermatitis in Broiler Chickens

SUMMARY. Necrotic enteritis (NE) and gangrenous dermatitis (GD) are important infectious diseases of poultry. Although NE and GD share a common pathogen, Clostridium perfringens, they differ in other important aspects such as clinical signs, pathologic symptoms, and age of onset. The primary virulence factors of C. perfringens are its four major toxins (&agr;, &bgr;, &egr;, &igr;) and the newly described NE B-like (NetB) toxin. While neutralizing antibodies against some C. perfringens toxins are associated with protection against infection in mammals, the serologic responses of NE- and GD-afflicted birds to these toxins have not been evaluated. Therefore, we measured serum antibody levels to C. perfringens &agr;-toxin and NetB toxin in commercial birds from field outbreaks of NE and GD using recombinant toxin-based enzyme-linked immunosorbent assay (ELISA). Initially, we used this ELISA system to detect antibody titers against C. perfringens &agr;-toxin and NetB toxin that were increased in birds experimentally co-infected with Eimeria maxima and C. perfringens compared with uninfected controls. Next, we applied this ELISA to field serum samples from flock-mated birds with or without clinical signs of NE or GD. The results showed that the levels of antibodies against both toxins were significantly higher in apparently healthy chickens compared to birds with clinical signs of NE or GD, suggesting that these antitoxin antibodies may play a role in protection against NE and GD.

Embryo vaccination of chickens using a novel adjuvant formulation stimulates protective immunity against Eimeria maxima infection

n Abstractn n Our previous study demonstrated that chickens immunized subcutaneously with an Eimeria recombinant profilin protein vaccine emulsified in a Quil A/cholesterol/DDA/Carbopol (QCDC) adjuvant developed partial protection against experimental avian coccidiosis compared with animals immunized with profilin alone. Because in ovo vaccination is presently used in commercial applications worldwide throughout the poultry industry, the current study was undertaken to investigate chicken embryo vaccination with profilin plus QCDC adjuvant. Eighteen day-old embryos were immunized with isotonic saline (control), profilin alone, QCDC alone, or profilin plus QCDC, and orally challenged with live Eimeria maxima at 7 days post-hatch. Body weight gain, fecal oocyst output, and intestinal cytokine transcript levels were assessed as measures of protective immunity. While immunization with profilin alone or QCDC alone did not alter body weight gain of infected chickens compared with the saline control group, vaccination with profilin plus QCDC increased body weight gain such that it was equal to the uninfected controls. Immunization with profilin plus QCDC also reduced fecal oocyst shedding compared with unimmunized controls, although in this case QCDC failed to provide an adjuvant effect since no difference was observed between the profilin-only and profilin/QCDC groups. Finally, increased levels of transcripts encoding IL-1β, IL-15, and IFN-γ were seen in the intestinal tissues of animals given profilin plus QCDC compared with the profilin-only or QCDC-only groups. In summary, this study demonstrates an adjuvant effect of QCDC on body weight gain and intestinal cytokine responses following in ovo vaccination of chickens with an Eimeria profilin vaccine.n n

Association of resistance to avian coccidiosis with single nucleotide polymorphisms in the zyxin gene.

Our previous genetic studies demonstrated that resistance to avian coccidiosis is linked with microsatellite markers LEI0071 and LEI0101 on chromosome 1. In this study, the associations between parameters of resistance to coccidiosis and single nucleotide polymorphisms (SNP) in 3 candidate genes located between LEI0071 and LEI0101 [zyxin, CD4, and tumor necrosis factor receptor super family 1A (TNFRSF1A)] were determined. The SNP were genotyped in 24 F(1) generation and 290 F(2) generation animals. No SNP were identified in the TNFRSF1A gene, whereas 10 were located in the zyxin gene and 4 in the CD4 gene. At various times following experimental infection of the F(2) generation with Eimeria maxima, BW, fecal oocyst shedding, and plasma levels of carotenoid, nitrite plus nitrate (NO(2)(-) + NO(3)(-)), and interferon-gamma (IFN-gamma) were measured as parameters of resistance. Single marker and haplotype-based tests were applied to determine the associations between the 14 SNP and the parameters of coccidiosis resistance. None of the CD4 SNP were correlated with disease resistance. However, by single marker association, several of the zyxin SNP were significantly associated with carotenoid or NO(2)(-) + NO(3)(-) concentrations. These were the SNP at nucleotide 149 associated with carotenoid at d 3 postinfection (PI), nucleotide 187 with carotenoid at d 6 and 9 PI, and nucleotide 159 with carotenoid between d 3 and 9 PI. In addition, the zyxin SNP at nucleotide 191 was significantly associated with increased levels of NO(2)(-) + NO(3)(-) at d 3 PI. By haplotype association, the zyxin SNP also were found to be highly associated with NO(2)(-) + NO(3)(-) at d 3 PI and increased IFN-gamma at d 6 PI. These results suggest that zyxin is a candidate gene potentially associated with increased resistance to experimental avian coccidiosis.

Differential immune-related gene expression in two genetically disparate chicken lines during infection by Eimeria maxima.

Two M5.1 and M15.2 B complex congenic lines of Fayoumi chickens were evaluated for body weight loss and faecal oocyst counts as parameters of avian coccidiosis. M5.1 chickens exhibited resistance to E. maxima compared with M15.2. To correlate the differential responses of the M5.1 and M15.2 lines to E. maxima infection with cellular immune responses, the expression levels of mRNAs encoding 14 immune-related molecules were measured by quantitative RT-PCR in intestinal intraepithelial lymphocytes (IELs) and splenocytes at 0, 3, 4, and 5 days following parasite infection. Intestinal IELs from M5.1 chickens expressed higher levels of transcripts encoding interferon gamma (IFNG), interleukin-lbeta (1L1B), IL6, IL8, IL12, IL15, IL17A, inducible nitric oxide synthase (iNOS), and lipopolysaccharide-induced tumour necrosis a factor (LITAF), and lower levels of mRNAs for IFNA, IL10, IL17D, NK-lysin (NKL), and tumour necrosis factor superfamily 15 (TNFSF15) at 3 days post infection, compared with the M15.2 line. In the spleen, E. maxima infection was associated with higher expression levels of IFNA, and IL15 and lower levels of IL6, IL17D, and IL12 in M5.1 compared to M15.2 birds. Using an intestinal IEL cDNA microarray, the differential dynamics of gene expression in the gut of M5.1 and M15.2 chickens following experimental coccidiosis were evident. In particular, the genes encoding lymphotactin and parathymosin were expressed at significantly higher levels in M5.1 compared with M15.2 line chickens. In conclusion, genetic determinants within the chicken major histocompatibility complex (MHC) B complex influence resistance to E. maxima infection by controlling the local and systemic expression of immune-related cytokine and chemokine genes.

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.

Quantitative genetic and functional genomics approaches to investigating parasite disease resistance and protective immune mechanisms in avian coccidiosis.

A major challenge for the commercial poultry production is controlling enteric disease-causing pathogens in the absence of prophylactic drugs. Although traditional genetic approaches have contributed to enhanced efficiency of poultry production and increased the ability of the industry to provide safe, high quality, and low cost meat products to consumers, economic losses due to enteric diseases remains a significant problem. Therefore, there is a need to develop alternative control strategies against poultry enteric pathogens of economic importance. Recent advances in molecular genetics and functional genomics are now allowing for rapid progress in understanding the molecular mechanisms of disease resistance against major infectious agents of poultry. It is anticipated that identifying the nature of host-pathogen interactions will facilitate the development of novel vaccines and therapeutics. In this paper, recent progress in identifying genes that influence resistance to avian coccidiosis, the most economically important disease of poultry, will be discussed. This work provides an excellent example of how the integration of new molecular genetic and functional genomics tools increases our knowledge of the mechanism of genetic resistance to complex diseases.

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.