Each PCR plate contained target genes and 28S rRNA concentration standard in triplicate, a serial dilution of target-specific RNA to generate a standard curve, and a no-template negative control. The cycle at which the sample amplicon reporter dye concentration crossed a preset threshold was recorded as the cycle threshold Ct value. Fixed main effects included genetic line broiler, Leghorn, Fayoumi , postexposure cell harvest time 2 h, 4 h , and treatment S. There was no significant main effect of chicken genetic line or postexposure time of cell harvest on the cytokine mRNA levels.
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Enteritidis-exposed PBMC, whereas values were The proinflammatory response, which includes expression of IL-6 and CXCLi2 , is a key initial host immune defense against pathogens. Reduced mRNA expression may reduce protein expression, which would reduce the host inflammatory response during an in vivo infection. There are conflicting reports regarding proinflammatory cytokine responses to in vitro S. Enteritidis exposure. Enteritidis exposure Kogut et al. The 2- and 4-h cell harvest times of the present study were based upon the detectable levels of cytokines observed at these times in the latter study.
Upregulation of cecal CXCLi2 mRNA, along with other proinflammatory cytokines, has been observed as early as 6 h post-in vivo oral challenge with Salmonella Typhimurium Withanage et al. Swaggerty et al. This does not preclude IL-2 playing a role in host immune response to S. Enteritidis at other times or in other tissues or cells.
Enteritidis for a maximum of 4 h, was directed toward measuring an early innate immune response and not T-cell proliferative cytokines, such as IL Enteritidis exposure in primary CKC after 4 h of postexposure Kaiser et al. The conventional paradigm for inflammatory responses is for inverse expression of proinflammatory and antiinflammatory cytokines Stober et al.
In vitro S. Kogut et al. These varied findings underscore the usefulness of evaluating defined and diverse chicken genetic lines to understand the specific relationship of proinflammatory and antiinflammatory cytokine mRNA expression to pathogen exposure. They also suggest the value of measuring heterogeneous cell cultures in which cells produce cytokines within the context of the activity of other cell types.
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The current study suggests that the initial encounter with S. Enteritidis results in suppression of both proinflammation and antiinflammation immune response in PBMC. The cytokines measured in the present study were expressed by PBMC at detectable levels without bacterial exposure. This baseline expression has been observed in heterophil Kogut et al. It is not possible to determine, from these collective studies, if the cytokines are constitutively expressed by PBMC or if the expression is a result of the isolation and culture conditions.
Previous studies of cytokine mRNA expression in response to S. Enteritidis in vitro exposure used homogeneous cell cultures of isolated heterophils Kogut et al. The present study evaluated cytokine mRNA expression of a heterogeneous PBMC cell population, providing an assessment of the combined response of diverse types of host cells to S. Enteritidis exposure and thereby expanding our understanding of cell interactions and avian cytokine function in host immunity. Genetic line, postexposure time, and treatment effects on peripheral blood mononuclear cell cytokine expression after in vitro exposure to Salmonella enterica serovar Enteritidis S.
Enteritidis; P -value. In vitro Salmonella enteritidis treatment effect on peripheral blood mononuclear cell cytokine mRNA expression levels least square means.
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We thank H. Enteritidis phage type 13a bacteria and J. McElroy Iowa State University for helpful discussion.
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