Selection for pro-inflammatory mediators yields chickens with increased resistance against Salmonella enterica serovar Enteritidis.

Salmonella is a leading cause of foodborne illness and can be transmitted through consumption of contaminated poultry; therefore, increasing a flock's natural resistance to Salmonella could improve food safety. Previously, we characterized the heterophil-mediated innate immune response of 2 parental broiler lines and F1 reciprocal crosses and showed that increased heterophil function and expression of pro-inflammatory mediators corresponds with increased resistance against diverse pathogens. A preliminary selection trial showed that individual sires had varying inherent levels of pro-inflammatory mediators and selection based on a high or low phenotype was passed onto progeny. Based on these results, we hypothesized selection of broilers for higher levels of the pro-inflammatory mediators IL-6, CXCLi2, and CCLi2 would produce progeny with increased resistance against Salmonella Enteritidis. Peripheral blood leukocytes were isolated from 75 commercial broiler sires, screened, and 10 naturally high and low expressing sires were selected and mated to randomly selected dams to produce the first generation of "high" and "low" progeny. The mRNA expression of CXCLi2 and CCLi2 were significantly (P ≤ 0.02) higher in the high progeny and were more resistant to liver and spleen organ invasion by Salmonella Enteritidis compared with low progeny. Production of the second generation yielded progeny that had differences (P ≤ 0.03) in all 3 mediators and further improved resistance against Salmonella Enteritidis. Feed conversion ratio and percent breast meat yield were calculated and were equal, whereas the high birds weighed slightly, but significantly, less than the low birds. These data clearly demonstrate that selection based on a higher phenotype of key pro-inflammatory mediators is a novel means to produce broilers that are naturally more resistant to Salmonella, one of the most important foodborne pathogens affecting the poultry industry.

Systemic response to Campylobacter jejuni infection by profiling gene transcription in the spleens of two genetic lines of chickens.

Campylobacter jejuni (C. jejuni) is a leading cause of human bacterial enteritis worldwide with poultry products being a major source of C. jejuni contamination. The chicken is the natural reservoir of C. jejuni where bacteria colonize the digestive tract of poultry, but rarely cause symptoms of disease. To understand the systemic molecular response mechanisms to C. jejuni infection in chickens, total splenic RNA was isolated and applied to a whole genome chicken microarray for comparison between infected (I) and non-infected (N) chickens within and between genetic lines A and B. There were more total splenic host genes responding to the infection in resistant line A than in susceptible line B. Specifically, genes for lymphocyte activation, differentiation and humoral response, and Ig light and heavy chain were upregulated in the resistant line. In the susceptible line, genes for regulation of erythrocyte differentiation, hemopoiesis, and RNA biosynthetic process were all downregulated. An interaction analysis between genetic lines and treatment demonstrated distinct defense mechanisms between lines: the resistant line promoted apoptosis and cytochrome c release from mitochondria, whereas the susceptible line responded with a downregulation of both functions. This was the first time that such systemic defensive mechanisms against C. jejuni infection have been reported. The results of this study revealed novel molecular mechanisms of the systemic host responses to C. jejuni infection in chickens that warrant further investigation.

Differential mRNA expression of the avian-specific toll-like receptor 15 between heterophils from Salmonella-susceptible and -resistant chickens.

Pattern recognition receptors (PRRs) are essential for recognition of conserved molecular constituents found on infectious microbes. Toll-like receptors (TLRs) are a critical component of the PRR repertoire and are coupled to downstream production of cytokines, chemokines, and antimicrobial peptides by TLR adaptor proteins. Our laboratory previously demonstrated a role for TLR function in the differential innate response of two lines of chickens to bacterial infections. The aim of the present study was to elucidate the role of TLRs in the differential innate responsiveness by measuring differences between lines A (resistant) and B (susceptible) in heterophil mRNA expression of selected TLRs (TLRs 4, 5, and 15) and TLR adaptor proteins (MyD88, TRIF, and TIRAP) in response to stimulation with Salmonella enterica serovar Enteritidis (SE). Although heterophils from both lines had significantly increased expression of TLR 15 mRNA in response to stimulation with SE, heterophils from chickens resistant to infection with SE had significantly greater levels of TLR 15 mRNA expression prior to and following stimulation with SE than heterophils from chickens susceptible to infection with SE. No significant differences were noted between lines in nonstimulated levels of TIRAP, but upon SE stimulation, line A birds had higher levels of expression than B birds. No significant differences were found in heterophils between lines for mRNA expression of TLRs 4 and 5 nor MyD88 and TRIF. These data indicate that differences in the gene expression of TLR 15 by heterophils likely accounts for some of the observed differences between the lines in their susceptibility to infection.

Selection for increased nitric oxide production does not increase resistance to Marek's disease in a primary broiler breeder line.

Two primary broiler breeder lines, A and B, were examined for their potential to produce nitric oxide (NO) after stimulating splenocytes from 20-day-old embryos with lipopolysaccharide and interferon-gamma. Significant differences were found between lines A and B. Overall, line A had a higher response than line B, but line A also had a large degree of variation between individual sire families. Selection for high and low responders within line A resulted in the segregation of high- and low-responder sire families. Offspring from sire families selected for high and low NO responses and from a nonselected control group from line A were challenged with RB-1B Marek's disease (MD) virus to determine whether these differences could be used to select for improved resistance to MD. Virus isolation rates at 6 and 10 days postinfection were not significantly different, but unexpectedly, the MD incidence in the high-responder group was significantly higher than in the other two groups.

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.

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

BACKGROUND: Salmonella 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. RESULTS: The 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. CONCLUSION: We 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.

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

Toll-like receptor agonists stimulate differential functional activation and cytokine and chemokine gene expression in heterophils isolated from chickens with differential innate responses.

Heterophils isolated from distinct broilers (lines A and B) differ in function and cytokine gene expression profiles. Nothing is known about Toll-like receptor (TLR) expression nor functional activation and cytokine/chemokine gene expression of line A and B heterophils when stimulated with TLR agonists. We found that line A and B heterophils express the same range of TLRs. All the bacterial TLR agonists, peptidoglycan, the synthetic lipoprotein Pam3CSK4, ultra-pure lipopolysaccharide, and flagellin all induced significantly greater functional activation of heterophils from line A compared to B. Only stimulation with the guanosine analog, loxoribine, (LOX) induced a significantly greater functional response in B over A. Additionally, all heterophils from line A stimulated with the bacterial TLR agonists had dramatic upregulation of pro-inflammatory cytokine and chemokine mRNA expression, whereas heterophils from line B had little or no upregulation of these genes. However, stimulation of all heterophils from line B with the bacterial TLR agonists and LOX induced a significant upregulation of IFN-alpha, with little transcription of this cytokine gene in line A heterophils. These findings suggest that the difference in heterophil functional efficiency between these parent lines is due to recognition of pathogens and activation of signaling pathways that induce innate cytokine and chemokine responses.

An investigation into blood microbiota and its potential association with Bacterial Chondronecrosis with Osteomyelitis (BCO) in Broilers.

Bacterial chondronecrosis with osteomyelitis (BCO) is a common cause of lameness in commercial broiler chickens worldwide. BCO represents substantial production loss and welfare issues of chickens. The bacterial species or communities underlying BCO pathogenesis still remain to be fully characterized. To gain insights on blood microbiota in broilers and its potential association with BCO, blood samples collected from healthy (n = 240) and lame (n = 12) chickens were analyzed by deep sequencing of 16S RNA genes. The chicken blood microbiota were dominated by Proteobacteria (60.58% ± 0.65) followed by Bactroidetes (13.99% ± 0.29), Firmicutes (11.45% ± 0.51), Actinobacteria (10.21% ± 0.37) and Cyanobacteria (1.96% ± 0.21) that constituted 98.18% (± 0.22) of the whole phyla. The bacterial communities consist of 30-40 OTUs in the blood of broiler chickens, regardless of ages and other environmental or host conditions, and the blood microbiomes of BCO chickens were largely distinct from those of healthy chickens. In addition, Linear discriminant analysis (LDA) effect size (LEfSe) method revealed that Staphylococcus, Granulicatella, and Microbacterium were significantly enriched in BCO chickens as compared to healthy chickens. The results from this study have significant implications in understanding blood microbiota present in broiler chickens and its potential role in BCO pathogenesis.

Heterophils are associated with resistance to systemic Salmonella enteritidis infections in genetically distinct chicken lines.

Heterophils mediate acute protection against Salmonella in young poultry. We evaluated susceptibility of genetically distinct lines of broilers to systemic Salmonella enteritidis (SE) infections. SE was administered into the abdomen of day-old chickens (parental lines [A and B]; F1 reciprocal crosses [C and D]) to assess modulation of leukocytes and survivability of chickens. Line A was more resistant to SE than line B; likewise cross D was more resistant than cross C. Significantly more heterophils migrated to the abdominal cavity post-infection in the resistant lines. These data indicate that increased heterophil influx to the infection site contributes to increased resistance against systemic SE infections in neonatal chickens.

Molecular survey of bacterial communities associated with bacterial chondronecrosis with osteomyelitis (BCO) in broilers.

Bacterial chondronecrosis with osteomyelitis (BCO) is recognized as an important cause of lameness in commercial broiler chickens (meat-type chickens). Relatively little is known about the microbial communities associated with BCO. This study was conducted to increase our understanding of the microbial factors associated with BCO using a culture-independent approach. Using Illumina sequencing of the hyper-variable region V6 in the 16S rRNA gene, we characterized the bacterial communities in 97 femoral or tibial heads from normal and lame broilers carefully selected to represent diverse variations in age, line, lesion type, floor type, clinical status and bone type. Our in-depth survey based on 14 million assembled sequence reads revealed that complex bacterial communities exist in all samples, including macroscopically normal bones from clinically healthy birds. Overall, Proteobacteria (mean 90.9%) comprised the most common phylum, followed by Firmicutes (6.1%) and Actinobacteria (2.6%), accounting for more than 99% of all reads. Statistical analyses demonstrated that there are differences in bacterial communities in different types of bones (femur vs. tibia), lesion types (macroscopically normal femora or tibiae vs. those with pathognomonic BCO lesions), and among individual birds. This analysis also showed that BCO samples overrepresented genera Staphylococcus, whose species have been frequently isolated in BCO samples in previous studies. Rarefaction analysis demonstrated the general tendency that increased severities of BCO lesions were associated with reduced species diversity in both femoral and tibial samples when compared to macroscopically normal samples. These observations suggest that certain bacterial subgroups are preferentially selected in association with the development of BCO lesions. Understanding the microbial species associated with BCO will identify opportunities for understanding and modulating the pathogenesis of this form of lameness in broilers.

Gene Expression Analysis of Toll-Like Receptor Pathways in Heterophils from Genetic Chicken Lines that Differ in Their Susceptibility to Salmonella enteritidis.

Previously conducted studies using two chicken lines (A and B) show that line A birds have increased resistance to a number of bacterial and protozoan challenges and that heterophils isolated from line A birds are functionally more responsive. Furthermore, when stimulated with Toll-like receptor (TLR) agonists, heterophils from line A expressed a totally different cytokine and chemokine mRNA expression pattern than heterophils from line B. A large-scale gene expression profile using an Agilent 44K microarray on heterophils isolated from line A and line B also revealed significantly differential expression in many immune-related genes following Salmonella enteritidis (SE) stimulation, which included genes involved in the TLR pathway. Therefore, we hypothesize the differences between the lines result from distinctive TLR pathway signaling cascades that mediate heterophil function and, thus, innate immune responsiveness to SE. Using quantitative RT-PCR on mRNA from heterophils isolated from control and SE-stimulated heterophils of each line, we profiled the expression of all chicken homologous genes identified in a reference TLR pathway. Several differentially expressed genes found were involved in the TLR-induced My88-dependent pathway, showing higher gene expression in line A than line B heterophils following SE stimulation. These genes included the TLR genes TLR4, TLR15, TLR21, MD-2, the adaptor proteins Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP), Tumor necrosis factor-receptor associated factor 3 (TRAF3), the IκB kinases transforming growth factor-ß-activating kinase 1 (TAK1), IKKε and IKKα, the transcription factors NFkB2 and interferon regulatory factor 7, phosphatidylinositol-3 kinase (PI-3K), and the mitogen-activated protein kinase p38. These results indicate that higher expression of TLR signaling activation of both MyD88-dependent and TRIF-dependent pathways are more beneficial to avian heterophil-mediated innate immunity and a complicated regulation of downstream adaptors is involved in stronger induction of a TLR-mediated innate response in the resistant line A. These findings identify new targets for genetic selection of chickens to increase resistance to bacterial infections.

The feathering gene is linked to degranulation and oxidative burst not cytokine/chemokine mRNA expression levels or Salmonella enteritidis organ invasion in broilers.

In the past, we showed differences in heterophil function between parental broilers (A [fast feathering] > B [slow feathering]) and their F1 reciprocal crosses (D [fast feathering] > C [slow feathering]). In the present study, we evaluated the linkage of the feathering gene to heterophil function, pro-inflammatory cytokine/chemokine mRNA expression levels, and resistance to Salmonella enteritidis organ invasion. Heterophils were isolated from 2-day-old chickens (C and D) separated into males and females - slow males and females (SM and SF), and fast males and females (FM and FF). Heterophil functions of degranulation and oxidative burst were measured. Heterophils from FF chickens (183+/-8.9) released more (P < 0.05) beta-d-glucuronidase (microM) than heterophils from SF chickens (149+/-3.7); FF heterophils (4.6 x 10(4)) generated a significantly (P < 0.05) greater oxidative burst (mean relative fluorescent units) compared with SF heterophils (4.2 x 10(4)). Interleukin-6, CXCLi2, and interferon-alpha mRNA expression levels were quantitated by real-time quantitative reverse transcriptase-polymerase chain reaction. No differences were observed between SM and FM or between SF and FF heterophils. Finally, 1-day-old chickens were administered S. enteritidis and liver/spleen organ invasion was quantitated. No differences were observed between the number of S. enteritidis-positive FF and SF chickens, but FM were significantly (P < 0.05) more resistant to S. enteritidis organ invasion than SM chickens. The data indicate degranulation and oxidative burst were linked with the feathering gene; however, interleukin-6, CXCLi2, and interferon-alpha mRNA expression levels were not. Furthermore, susceptibility to in vitro S. enteritidis organ invasion was not linked to the feathering gene.

Heterophil cytokine mRNA profiles from genetically distinct lines of chickens with differential heterophil-mediated innate immune responses.

Previously we demonstrated that increased in-vitro heterophil function translates to increased in-vivo resistance to Salmonella enteritidis infections in broilers (line A > B). Heterophils produce cytokines and modulate acute protection against Salmonella in neonatal poultry. We hypothesized that heterophils from S. enteritidis-resistant chickens produce an up-regulated pro-inflammatory cytokine/chemokine response compared with S. enteritidis-susceptible chickens. In this study, heterophils were isolated 1, 14, and 28 days post-hatch, treated with RPMI or phagocytic agonists, and the cytokine/chemokine mRNA expression assessed using quantitative real-time reverse transcriptase-polymerase chain reaction. At all time-points, heterophils from S. enteritidis-resistant chickens (line A) had higher levels of pro-inflammatory cytokine/chemokine mRNA expression upon stimulation compared with heterophils from S. enteritidis-susceptible chickens (line B). Furthermore, heterophils from line A chickens had decreased mRNA expression of transforming growth factor-beta4, an anti-inflammatory cytokine, compared with line B. These data indicate a relationship between cytokine/chemokine mRNA expression by heterophils and determining overall immune competence. Therefore, heterophil functional efficiency, accompanied by evaluating cytokine/chemokines produced by heterophils, may be useful biomarkers for breeders to consider when developing new immunocompetent lines.

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.

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-beta4, 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.

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 Bxfemale A; line D=male Axfemale 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.

Association between in vitro heterophil function and the feathering gene in commercial broiler chickens.

We recently showed that in vitro heterophil functional efficiency in commercial broiler chickens is genetically controlled and may be a sex-associated trait. To further characterize the genetic mechanism(s) of heterophil functional efficiency, we wanted to determine whether the feathering gene, present on the Z sex chromosome, contributes to heterophil functional efficiency. Heterophils from two pairs of broiler lines were evaluated; each pair contained a fast feather (FF) (lines A and X) and a slow feather (SF) line (lines B and Y). On days 1 and 4 post-hatch, heterophils isolated from two sets of pure line broilers (A and B, and X and Y) were evaluated for their ability to (1) phagocytize Salmonella enteritidis, and (2) exhibit bactericidal activity against S. enteritidis. On days 1 and 4 post-hatch, heterophils isolated from the FF lines were statistically (P < or = 0.02) more proficient at phagocytizing S. enteritidis than heterophils from SF lines. Bactericidal activity was also statistically (p < or = 0.02) greater on day 1 post-hatch in the heterophils isolated from FF lines compared to heterophils isolated from SF lines. These data indicate that the presence of the FF gene locus on the Z sex chromosome contributes to heterophil function and may contribute to the early innate immune competence of a flock.