Christina L. Swaggerty

Differential cytokine mRNA expression in heterophils isolated from Salmonella-resistant and -susceptible chickens

We recently showed that increased in vitro heterophil functional efficiency translates to increased in vivo resistance to a systemic Salmonella enteritidis (SE) infection utilizing a parental pair of broiler chickens (lines A and B) and the F1 reciprocal crosses (C and D). Heterophils produce cytokines and modulate acute protection against Salmonella in young poultry. Therefore, we hypothesize that heterophils from SE‐resistant chickens (A and D) have the ability to produce an up‐regulated pro‐inflammatory cytokine response compared to that of heterophils from SE‐susceptible chickens (B and C). In this study, heterophils were isolated from day‐old chickens and treated with either RPMI‐1640 (as the control), or phagocytic agonists (SE, or SE opsonized with either normal chicken serum or immune serum against SE) and cytokine mRNA expression assessed using real‐time quantitative reverse transcription–polymerase chain reaction. Heterophils from SE‐resistant chickens (A and D) had significantly higher levels of pro‐inflammatory cytokine (interleukin (IL)‐6, IL‐8, and IL‐18) mRNA expression upon treatment with all agonists compared to heterophils from SE‐susceptible lines (B and C). Further, heterophils from SE‐resistant chickens had significantly decreased mRNA expression levels of transforming growth factor‐β4, an anti‐inflammatory cytokine, when compared to heterophils from SE‐susceptible chickens. These data indicate cytokine gene expression in heterophils may be a useful parameter in determining resistance to Salmonella, as indicated by our previous in vivo SE studies. Therefore, heterophil functional efficiency and cytokine production may be useful biomarkers for poultry breeders to consider when developing new immunocompetent lines of birds.

Heterophils isolated from chickens resistant to extra-intestinal Salmonella enteritidis infection express higher levels of pro-inflammatory cytokine mRNA following infection than heterophils from susceptible chickens

Previous studies showed differences in in vitro heterophil function between parental (A > B) broilers and F1 reciprocal crosses (D > C). Our objectives were to (1) determine if in vitro variations translate to differences in resistance to Salmonella enteritidis (SE) and (2) quantitate cytokine mRNA in heterophils from SE-infected chicks. One-day-old chicks were challenged and organs were cultured for SE. Chicks with efficient heterophils (A and D) were less susceptible to SE compared to chicks with inefficient heterophils (B and C). Heterophils were isolated from SE-infected chicks and cytokine mRNA expression was evaluated using quantitative real-time RT-PCR. Pro-inflammatory cytokine mRNA was up-regulated in heterophils from SE-resistant chicks compared to susceptible chicks. This is the first report to quantitate cytokine mRNA in heterophils from SE-infected chicks. These data show a relationship between in vitro heterophil function, increased pro-inflammatory cytokine mRNA expression, and increased resistance to SE in 1-day-old chicks.

Gene expression profiling in chicken heterophils with Salmonella enteritidis stimulation using a chicken 44 K Agilent microarray

BackgroundSalmonella enterica serovar Enteritidis (SE) is one of the most common food-borne pathogens that cause human salmonellosis and usually results from the consumption of contaminated poultry products. The mechanism of SE resistance in chickens remains largely unknown. Previously, heterophils isolated from broilers with different genetic backgrounds (SE-resistant [line A] and -susceptible [line B]) have been shown to be important in defending against SE infections. To dissect the interplay between heterophils and SE infection, we utilized large-scale gene expression profiling.ResultsThe results showed more differentially expressed genes were found between different lines than between infection (SE-treated) and non-infection (control) samples within line. However, the numbers of expressed immune-related genes between these two comparisons were dramatically different. More genes related to immune function were down-regulated in line B than line A. The analysis of the immune-related genes indicated that SE infection induced a stronger, up-regulated gene expression of line heterophils A than line B, and these genes include several components in the Toll-like receptor (TLR) signaling pathway, and genes involved in T-helper cell activation.ConclusionWe found: (1) A divergent expression pattern of immune-related genes between lines of different genetic backgrounds. The higher expression of immune-related genes might be more beneficial to enhance host immunity in the resistant line; (2) a similar TLR regulatory network might exist in both lines, where a possible MyD88-independent pathway may participate in the regulation of host innate immunity; (3) the genes exclusively differentially expressed in line A or line B with SE infection provided strong candidates for further investigating SE resistance and susceptibility. These findings have laid the foundation for future studies of TLR pathway regulation and cellular modulation of SE infection in chickens.

Functional comparison of heterophils isolated from commercial broiler chickens

Heterophils from two pure lines (A and B) of commercial broiler chickens were isolated on days 1, 4, and 7 post-hatch to evaluate their ability to (1) phagocytose Salmonella enteritidis (SE) (2) degranulate when exposed to immune-IgG opsonized SE, and (3) produce an oxidative burst. On days 1 and 4, heterophils from line A were functionally more efficient compared to heterophils from line B (p<0.05). By 7 days post hatch, heterophil functions for both lines were comparable. To further study the inheritance of heterophil functional efficiency, F1 reciprocal crosses (line C=male B×female A; line D=male A×female B) were evaluated for functional activity and compared with the immunologically efficient (A) and non-efficient (B) parent lines. Heterophils from line D had a more efficient heterophil function (p<0.05) when compared to heterophils from C. These results suggest that heterophil function and efficiency can be genetically transferred to progeny. Moreover they indicate that heterophil function is sex-associated and genetically controlled by the rooster since progeny of line A males maintained immunologically efficient characteristics whereas heterophils from the progeny of line B roosters remained immunologically inefficient. To our knowledge, this is the first report to describe a functional relationship between pure and F1 reciprocal crosses of broiler chickens with regard to heterophils and the innate immune response.

Gene Expression Profiling of the Local Cecal Response of Genetic Chicken Lines That Differ in Their Susceptibility to Campylobacter jejuni Colonization

Campylobacter jejuni (C. jejuni) is one of the most common causes of human bacterial enteritis worldwide primarily due to contaminated poultry products. Previously, we found a significant difference in C. jejuni colonization in the ceca between two genetically distinct broiler lines (Line A (resistant) has less colony than line B (susceptible) on day 7 post inoculation). We hypothesize that different mechanisms between these two genetic lines may affect their ability to resist C. jejuni colonization in chickens. The molecular mechanisms of the local host response to C. jejuni colonization in chickens have not been well understood. In the present study, to profile the cecal gene expression in the response to C. jejuni colonization and to compare differences between two lines at the molecular level, RNA of ceca from two genetic lines of chickens (A and B) were applied to a chicken whole genome microarray for a pair-comparison between inoculated (I) and non-inoculated (N) chickens within each line and between lines. Our results demonstrated that metabolism process and insulin receptor signaling pathways are key contributors to the different response to C. jejuni colonization between lines A and B. With C. jejuni inoculation, lymphocyte activation and lymphoid organ development functions are important for line A host defenses, while cell differentiation, communication and signaling pathways are important for line B. Interestingly, circadian rhythm appears play a critical role in host response of the more resistant A line to C. jejuni colonization. A dramatic differential host response was observed between these two lines of chickens. The more susceptible line B chickens responded to C. jejuni inoculation with a dramatic up-regulation in lipid, glucose, and amino acid metabolism, which is undoubtedly for use in the response to the colonization with little or no change in immune host defenses. However, in more resistant line A birds the host defense responses were characterized by an up-regulation lymphocyte activation, probably by regulatory T cells and an increased expression of the NLR recognition receptor NALP1. To our knowledge, this is the first time each of these responses has been observed in the avian response to an intestinal bacterial pathogen.

Profiling pro-inflammatory cytokine and chemokine mRNA expression levels as a novel method for selection of increased innate immune responsiveness

Previous studies using F1 reciprocal crosses and two parental lines of broilers show the sire is instrumental in determining the in vitro leukocyte function and cytokine/chemokine profile. Since the innate immune response is the primary means young chickens have to protect themselves, we hypothesize utilizing a novel genomics approach to select sires based on an elevated pro-inflammatory cytokine and chemokine profile. By identifying sires with increased pro-inflammatory cytokine (interleukin [IL]-1beta and IL-6) and chemokine (CXCLi2 and CCLi2) mRNA expression levels, we expect the progeny will also have elevated profiles. We characterized the pro-inflammatory cytokine and chemokine profile of 119 sires using quantitative real-time RT-PCR (qRT-PCR) and identified two populations with inherently high and low mRNA expression levels of IL-1beta, IL-6, CXCLi2, and CCLi2. Select high and low sires were then used to produce progeny for the second phase of the trial. Blood samples were collected from 214 progeny and the cytokine and chemokine mRNA expression levels determined. Progeny from high sires had significantly (P<or=0.02) higher cytokine (IL-1beta and IL-6) and chemokine (CXCLi2 and CCLi2) mRNA expression levels compared to progeny from low sires. We have identified a broiler population of sires with higher and lower than average pro-inflammatory cytokine/chemokine mRNA expression levels and used them to produce progeny with similar profiles.

The avian heterophil.

Heterophils play an indispensable role in the immune defense of the avian host. To accomplish this defense, heterophils use sophisticated mechanisms to both detect and destroy pathogenic microbes. Detection of pathogens through the toll-like receptors (TLR), FC and complement receptors, and other pathogen recognition receptors has been recently described for the avian heterophil. Upon detection of pathogens, the avian heterophil, through a network of intracellular signaling pathways and the release and response to cytokines and chemokines, responds using a repertoire of microbial killing mechanisms including production of an oxidative burst, cellular degranulation, and production of extracellular matrices of DNA and histones (HETs). In this review, the authors describe the recent advances in our understanding of the avian heterophil, its functions, receptors and signaling, identified antimicrobial products, cytokine and chemokine production, and some of the effects of genetic selection on heterophils and their functional characteristics.

Selection of Broilers with Improved Innate Immune Responsiveness to Reduce On-Farm Infection by Foodborne Pathogens

Economic pressure on the modern poultry industry has directed the selection process towards fast-growing broilers that have a reduced feed conversion ratio. Selection based heavily on growth characteristics could adversely affect immune competence leaving chickens more susceptible to disease. Since the innate immune response directs the acquired immune response, efforts to select poultry with an efficient innate immune response would be beneficial. Our laboratories have been evaluating the innate immune system of two parental broiler lines to assess their capacity to protect against multiple infections. We have shown increased in vitro heterophil function corresponds with increased in vivo resistance to Gram-positive and Gram-negative bacterial infections. Additionally, there are increased mRNA expression levels of pro-inflammatory cytokines/chemokines in heterophils isolated from resistant lines compared to susceptible lines. Collectively, all data indicate there are measurable differences in innate responsiveness under genetic control. Recently, a small-scale selection trial was begun. We identified sires within a broiler population with higher and/or lower-than-average pro-inflammatory cytokine/chemokine mRNA expression levels and subsequently utilized small numbers of high-expressing and low-expressing sires to produce progeny with increased or decreased, respectively, pro-inflammatory cytokine/chemokine profiles. This novel approach should allow us to improve breeding stock by improving the overall immunological responsiveness. This will produce a line of chickens with an effective pro-inflammatory innate immune response that should improve resistance against diverse pathogens, improve responses to vaccines, and increase livability. Ongoing work from this project is providing fundamental information for the development of poultry lines that will be inherently resistant to colonization by pathogenic and food-poisoning microorganisms. Utilization of pathogen-resistant birds by the poultry production industry would significantly enhance the microbiological safety of poultry products reaching the consumer.

The Paternal Effect of Campylobacter jejuni Colonization in Ceca in Broilers

Campylobacter jejuni is one of the most common causes of acute enteritis worldwide. Chickens are believed to be the main reservoir of C. jejuni. The role that host genetics play in resistance/susceptibility to C. jejuni colonization in broilers is still not clear. Day-old broilers from 2 parental lines (A and B) and their F(1) reciprocal crosses (C and D) were challenged orally with 10(5) cfu of C. jejuni to address the role of genetics in determining resistance/susceptibility to C. jejuni colonization in broilers. Cloacal swabs were collected on 6, 10, and 13 d postinoculation (dpi), and cecal contents cultured for C. jejuni on 7 and 14 dpi. The number of C. jejuni colonies in the cloacal swabs and cecal contents of each bird were recorded at each time point. Significantly fewer bacteria were found in the cecal contents from line A than B (P < 0.05) and cross D (A male x B female) when compared with cross C (A female x B male) at both 7 and 14 dpi. There was a significant correlation between C. jejuni counts in cloacal swabs and those in cecal contents. The results indicated that a paternal effect might be one of the important genetic factors influencing resistance to C. jejuni colonization in broilers.

A comparative study on invasion, survival, modulation of oxidative burst, and nitric oxide responses of macrophages (HD11), and systemic infection in chickens by prevalent poultry Salmonella serovars.

Poultry is a major reservoir for foodborne Salmonella serovars. Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Heidelberg, Salmonella Kentucky, and Salmonella Senftenberg are the most prevalent serovars in U.S. poultry. Information concerning the interactions between different Salmonella species and host cells in poultry is lacking. In the present study, the above mentioned Salmonella serovars were examined for invasion, intracellular survival, and their ability to modulate oxidative burst and nitric oxide (NO) responses in chicken macrophage HD11 cells. All Salmonella serovars demonstrated similar capacity to invade HD11 cells. At 24 h post-infection, a 36-43% reduction of intracellular bacteria, in log(10)(CFU), was observed for Salmonella Typhimurium, Salmonella Heidelberg, Salmonella Kentucky, and Salmonella Senftenberg, whereas a significantly lower reduction (16%) was observed for Salmonella Enteritidis, indicating its higher resistance to the killing by HD11 cells. Production of NO was completely diminished in HD11 cells infected with Salmonella Typhimurium and Salmonella Enteritidis, but remained intact when infected with Salmonella Heidelberg, Salmonella Kentucky, and Salmonella Senftenberg. Phorbol myristate acetate-stimulated oxidative burst in HD11 cells was greatly impaired after infection by each of the five serovars. When newly hatched chickens were challenged orally, a high rate (86-98%) of systemic infection (Salmonella positive in liver/spleen) was observed in birds challenged with Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Heidelberg, and Salmonella Kentucky, while only 14% of the birds were Salmonella Senftenberg positive. However, there was no direct correlation between systemic infection and in vitro differential intracellular survival and modulation of NO response among the tested serovars.