Selection for pro-inflammatory mediators produces chickens more resistant to Clostridium perfringens-induced necrotic enteritis.

We developed a novel selection method based on an inherently high and low phenotype of pro-inflammatory mediators and produced "high" and "low" line chickens. We have shown high line birds are more resistant to Salmonella enterica serovar Enteritidis and Eimeria tenella compared to the low line. Clostridium perfringens is the fourth leading cause of bacterial-induced foodborne illness, and is also an economically important poultry pathogen and known etiologic agent of necrotic enteritis (NE). The objective of this study was to determine if high line birds were also more resistant to NE than low line birds using an established model. Birds were reared in floor pens and challenges were conducted twice (high line = 25/trial, 50 birds total; low line = 26/trial, 52 birds total). Day-old chicks were provided a 55% wheat-corn-based un-medicated starter diet. A bursal disease vaccine was administered at 10× the recommended dose via the ocular route at 14-d-of-age. Birds were challenged daily for 3 d beginning at 16-d-of-age by oral gavage (3 mL) with 10(7) colony forming units (cfu) of C. perfringens/mL then necropsied at 21-d-of-age. All birds had sections of the intestine examined and scored for lesions while the first 10 necropsied also had gut content collected for C. perfringens enumeration. Chickens from the high line were more resistant to C. perfringens-induced NE pathology compared to the low line, as indicated by reduced lesion scores. Ninety percent of the high line birds had lesions of zero or one compared to 67% of the low line birds. Wilcoxon rank sum test showed significantly higher lesion scores in the low line birds compared to the high line (P < 0.0001). There were no differences in the C. perfringens recovered (P = 0.83). These data provide additional validation and support selection based on elevated levels of pro-inflammatory mediators produces chickens with increased resistance against foodborne and poultry pathogens.

Selection for pro-inflammatory mediators produces chickens more resistant to Eimeria tenella.

We recently developed a novel selection method based on identification and selection of chickens with an inherently high and low phenotype of pro-inflammatory mediators, including interleukin (IL)-6, CXCLi2, and CCLi2. The resultant high line of chickens is more resistant to Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) compared to the low line. In the current study, we sought to determine if the high line birds were also more resistant to the protozoan parasite Eimeria tenella. In three separate experiments, 14-day-old chickens from the high and low lines were challenged orally with 10×10(3) to 45×10(3) E. tenella oocysts. Birds were sacrificed 6 d postchallenge and the caeca was removed and scored for lesions and body weight gain compared to mock-infected controls. The high line birds were more resistant to intestinal pathology as demonstrated by lower lesion scores (P≤0.04) compared to the low line. There were no differences in body weight gain between the lines. The results from this study showed that in addition to enhanced resistance against Salmonella Enteritidis, high line chickens are also more resistant to the pathology associated with coccidial infections compared to the low line birds. Taken together with our initial study utilizing the high and low lines, selection based on increased pro-inflammatory mediator expression produces chickens that are more resistant to both foodborne and poultry pathogens, including cecal pathology associated with costly coccidial infections.

Loxoribine pretreatment reduces Salmonella Enteritidis organ invasion in 1-day-old chickens.

Young poultry exhibit a transient colonization by some food-borne pathogens, including Salmonella, during the first week of life that stems from immature innate and acquired defense mechanisms. Consequently, modulation of the hosts' natural immune response is emerging as an important area of interest for food animal producers, including the poultry industry. Toll-like receptor (TLR) agonists have been shown to boost the innate immune response in young chickens and increase their resistance to colonization by Salmonella enterica serovar Enteritidis. The objective of the present study was to determine if pretreatment with loxoribine, a TLR7 agonist and immune modulator, protects young chicks from Salmonella Enteritidis organ invasion. Loxoribine (0-100 µg) was administered intra-abdominally to 1-d-old broiler chicks, and 4 h later, the birds were challenged orally with Salmonella Enteritidis. Twenty-four hours postchallenge, birds were euthanized and the liver and spleen aseptically removed and cultured for Salmonella Enteritidis. This was carried out on 3 separate occasions using 26 to 50 chicks per dose per experiment. Pretreatment of chicks with loxoribine (6.25-25 µg) significantly (P ≤ 0.05) reduced liver and spleen organ invasion by Salmonella Enteritidis. Higher doses (50-100 µg) of loxoribine had no effect. The results obtained in this study indicate that there is a potential application for using loxoribine to increase protection of young chicks when they are most susceptible to infections with Salmonella.

Protein tyrosine kinase and mitogen-activated protein kinase signalling pathways contribute to differences in heterophil-mediated innate immune responsiveness between two lines of broilers.

Protein tyrosine phosphorylation mediates signal transduction of cellular processes with protein tyrosine kinases (PTKs) regulating virtually all signalling events. The mitogen-activated protein kinase (MAPK) super-family consists of three conserved pathways that convert receptor activation into cellular functions: extracellular response kinases (ERK), c-Jun N-terminal kinases (JNK) and p38. Previously conducted studies using two chicken lines (A and B) show line A heterophils are functionally more responsive and produce a differential cytokine/chemokine profile compared with line B, which also translates to increased resistance to bacterial challenges. Therefore, we hypothesize the differences between the lines result from distinctive signalling cascades that mediate heterophil function. Heterophils from lines A and B were isolated from 1-day-old chickens and total phosphorylated PTK and p38, JNK, ERK, and transcription factor (activator protein 1 (AP-1) and nuclear factor kappa B (NF-κB)) protein levels quantified following interaction with Salmonella Enteritidis (SE). Control and SE-treated heterophils from line A had greater (P≤0.05) PTK phosphorylation compared to line B with increased (P≤0.05) activation of p38. Conversely, line B heterophils activated JNK (P≤0.05). There were no differences in ERK between control and activated heterophils for either line. Defined signalling inhibitors were used to show specificity. The AP-1 and NF-κB transcription factor families were also examined, and c-Jun and p50, respectively, were the only members different between the lines and both were up-regulated in line A compared with line B. These data indicate that increased responsiveness of line A heterophils is mediated, largely, by an increased ability to activate PTKs, the p38 MAPK pathway and specific transcription factors, all of which directly affect the innate immune response.

Caecal transcriptome analysis of colonized and non-colonized chickens within two genetic lines that differ in caecal colonization by Campylobacter jejuni.

Campylobacter jejuni is one of the most common causes of human bacterial enteritis worldwide. The molecular mechanisms of the host responses of chickens to C. jejuni colonization are not well understood. We have previously found differences in C. jejuni colonization at 7-days post-inoculation (pi) between two genetic broiler lines. However, within each line, not all birds were colonized by C. jejuni (27.5% colonized in line A, and 70% in line B). Therefore, the objective of the present experiments was to further define the differences in host gene expression between colonized and non-colonized chickens within each genetic line. RNA isolated from ceca of colonized and non-colonized birds within each line was applied to a chicken 44K Agilent microarray for the pair comparison. There were differences in the mechanisms of host resistant to C. jejuni colonization between line A and line B. Ten times more differentially expressed genes were observed between colonized and non-colonized chickens within line B than those within line A. Our study supports the fact that the MAPK pathway is important in host response to C. jejuni colonization in line B, but not in line A. The data indicate that inhibition of small GTPase-mediated signal transduction could enhance the resistance of chickens to C. jejuni colonization and that the tumour necrosis factor receptor superfamily genes play important roles in determining C. jejuni non-colonization in broilers.

Broiler breeders with an efficient innate immune response are more resistant to Eimeria tenella.

In previous studies we characterized the innate immune response of 2 parental broiler lines (A and B) and compared their resistance against Salmonella, Enterococcus, and Campylobacter challenges. In all cases, line A was more responsive and more resistant than line B. In the present study, we sought to determine whether this trend was also observed following challenge with the protozoan parasite Eimeria tenella. In 3 separate experiments, 14-d-old chickens from lines A and B were challenged orally with 15 to 50 × 10(3) E. tenella oocysts. Birds were killed 6 d postchallenge and the ceca was removed and scored for lesions and weight gain compared with noninfected controls. Line A birds were more resistant to intestinal pathology as demonstrated by lower lesion scores compared with line B birds. As might be expected, the lower lesion scores in line A chickens were often accompanied by higher weight gain compared with line B chickens, thus reducing potential revenue loss associated with low carcass weights often observed with coccidia-infected birds. The results from this study showed that in addition to having enhanced resistance against bacterial infections, line A chickens were also more resistant to coccidial infections compared with line B birds. Taken together with all of our earlier studies using these lines of birds, an efficient innate immune response protects against a broad range of foodborne and poultry pathogens, including costly coccidial infections.

In ovo treatment with CpG oligodeoxynucleotides decreases colonization of Salmonella enteriditis in broiler chickens.

Induction of the innate immune response in newly hatched chickens is important for limiting infections with bacteria, such as Salmonella enterica serovar Enteriditis (SE). CpG oligodeoxynucleotides (CpG-ODN) can stimulate the innate immune response of young chickens. Therefore, we examined the effectiveness of CpG-ODN administered in ovo on intestinal colonization by SE and the ability to modulate the function of heterophils in young chickens. Heterophils were isolated from 2-day-old chickens and were stimulated with heat-killed SE (HK-SE) or PMA for oxidative burst and HK-SE or live SE for degranulation assays. CpG-ODN treatment had no effect on heterophil oxidative burst when stimulated with HK-SE or PMA. However, HK-SE and live SE increased degranulation (P<0.01) in heterophils from CpG-ODN-treated birds compared to PBS-treated controls. In a second experiment, chickens were orally infected with SE on day 10 post-hatch and cecal contents were collected 6 days later for assessment of SE intestinal colonization. CpG-ODN treatment reduced SE colonization by greater than 10-fold (P<0.001) compared to PBS-injected control birds. Overall, we show for the first time that CpG-ODN given in ovo stimulates innate immune responsiveness of chicken heterophils and increases resistance of young chickens to SE colonization.

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.

Selection for pro-inflammatory mediators produces chickens more resistant to Campylobacter jejuni.

Campylobacter spp. are the second leading cause of bacterial-induced foodborne illnesses with an estimated economic burden of nearly $2B USD per year. Most human illness associated with campylobacteriosis is due to infection by C. jejuni and chickens are recognized as a reservoir that could lead to foodborne illness in humans resulting from handling or consuming raw or undercooked chicken. We recently developed a novel breeding strategy based on identification and selection of chickens with an inherently high and low phenotype of pro-inflammatory mediators including IL-6, CXCLi2, and CCLi2, hereafter referred to as the high and low lines, respectively. We have shown the high line chickens are more resistant to the foodborne and poultry pathogens Salmonella enterica serovar Enteritidis, Eimeria tenella, and Clostridium perfringens-induced necrotic enteritis compared to the low line. The objective of this study was to determine whether the same trend of enhanced resistance in the high line birds was observed for C. jejuni. Birds were challenged at 2 d of age by oral gavage (0.5 mL) with 5 × 106 colony forming units (cfu) of C. jejuni/mL, necropsied 4 d post challenge, and cecal content collected to determine if there was a difference in C. jejuni resistance between the high and low line chickens. There were fewer (P = 0.01) chickens from the high line (28/40 = 71.8%) that were colonized by C. jejuni compared to the low line (37/39 = 94.9%). The amount of C. jejuni recovered from the ceca of infected birds was quantified; however, no differences were observed (P = 0.10). Since the high line birds were also more resistant to C. jejuni, it provides additional validation of selection based on pro-inflammatory mediators producing a line of chickens with increased natural resistance against diverse foodborne and poultry pathogens. The poultry industry is moving towards reduced therapeutics and, as such, our breeding strategy would be a viable method to incorporate into traditional poultry breeding programs.

Purified beta-glucan as an abiotic feed additive up-regulates the innate immune response in immature chickens against Salmonella enterica serovar Enteritidis.

Functionally, the innate immune system of immature chickens is inefficient during the first week posthatch. This immunological inefficiency enables pathogens such as Salmonella enterica serovar Enteritidis (SE) to invade and colonize the visceral organs of immature chickens. The objective of this study was to evaluate the effect of purified beta-glucan as an immunomodulator of the innate immune response. beta-glucan, as a feed additive, significantly provided protection against SE organ invasion in young chickens (P<0.05). The functional efficiency of heterophils isolated from neonatal chickens fed a beta-glucan ration was significantly (P<0.05) up-regulated when compared to heterophils isolated from chickens fed a control ration as determined with an array of functional assays. Phagocytosis, bactericidal killing, and oxidative burst were significantly increased in heterophils isolated from chickens fed the purified beta-glucan ration (P<0.05). To our knowledge, this is the first report of a purified beta-glucan feed additive significantly decreasing the incidence of SE organ invasion in immature chickens and up-regulating the functional abilities of heterophils isolated from immature chickens against an invading pathogen, SE.

An in vitro study of theaflavins extracted from black tea to neutralize bovine rotavirus and bovine coronavirus infections.

Crude theaflavin was extracted from black tea and then fractionated by HPLC into five components (initial peaks (IP), TF1, TF2A, TF2B, and TF3). The crude extract and the various fractions of theaflavin were collected and tested, individually and in combination, for antirotaviral activity. The mean effective concentration (EC50) was calculated and compared. Activity varied from the most active being the uncharacterized theaflavin-like initial peaks (IP) with an EC50 of 0.125 microgram/ml to the least active being theaflavin-3 monogallate (TF2A) with an EC50 of 251.39 micrograms/ ml. The combination of TF1 + TF2A + TF2B + TF3 was more active than the sum of the activities of these four fractions individually, indicating synergism among the peaks. Only the crude extract was assayed for activity against coronavirus; the EC50 was 34.7 micrograms/ml.

Expression profile of toll-like receptors within the gastrointestinal tract of 2-day-old Salmonella enteriditis-infected broiler chickens.

Salmonella enterica serovar Enteriditis (SE) causes a majority of foodborne illness in the U.S. A more productive avian innate immune response could reduce bacterial colonization and the incidence of infection in humans. However, quantification and comparison of the toll-like receptors (TLR), a component of the innate immune system that recognize bacterial pathogens, and their response to SE colonization across the avian gastrointestinal (GI) tract has not been reported. Therefore, we assessed these changes using real-time qRT-PCR to measure expression of TLR 1LA, 2A, 2B, 3, 4, 5, 7, 15, and 21 in the duodenum, jejunum, ileum, cecal tonsil, ceca, and large intestine of uninfected and SE-infected 2-day-old broiler chickens. Samples were collected soon after hatch to approximate natural SE exposure and to measure initial changes in the immune response to infection. All TLRs had measurable expression within the duodenum, jejunum, ileum, cecal tonsil, ceca, and large intestine. The general expression pattern, with the exception of TLR 21, showed distal GI segments had higher TLR mRNA expression than proximal segments. Infected chickens had increased expression of TLR 1LA, 2A, 4, and 15 in distal GI segments and upregulation of TLR 2B, 3, and 15 in proximal segments, including the duodenum. Interestingly, SE-infection caused downregulation of TLR 5, with no change in TLR 7 or 21. Overall, we provide a comprehensive report of mRNA expression profiles for the TLR family of innate immune receptors in the GI tract of 2-day-old broilers and their differential response to SE colonization.

SU proteins from virulent and avirulent EIAV demonstrate distinct biological properties.

Biologic activity of equine infectious anemia virus (EIAV) surface (SU) glycoprotein was assayed in a mouse model. Recombinant SU from virulent EIAV17 (SU17), administered intraperitoneally to mouse pups, induced dose-dependent diarrheal responses similar to those reported for SIV SU (Virology 277 (2000) 250). SU17 caused fluid accumulation without histological lesions in mouse intestinal loops, induced chloride secretory currents in Ussing chambers and increased inositol 1,4,5 triphosphate (IP3) levels in HT29 cells. An SU17 peptide, SU17(299-330), provoked a dose-dependent diarrheal response akin to enterotoxic peptides from SIV. In contrast, SU from an avirulent EIAV strain failed to induce a dose response in mouse pups and produced lower levels of activity than SU17 in Ussing chambers and IP3 assays. These results demonstrate that a mouse pup model is useful to monitor EIAV SU biologic activity, showing clear differences between the activities of SU derived from virulent and avirulent viruses, and may provide a useful screen of EIAV virulence.

Comparison of SIVmac239(352-382) and SIVsmmPBj41(360-390) enterotoxic synthetic peptides.

To characterize the active domain of the simian immunodeficiency virus (SIV) surface unit (SU) enterotoxin, peptides corresponding to the V3 loop of SIVmac239 (SIVmac) and SIVsmmPBj41 (SIVpbj) were synthesized and examined for enterotoxic activity, alpha-helical structure, and interaction(s) with model membranes. SIVmac and SIVpbj induced a dose-dependent diarrhea in 6-8-day-old mouse pups similar to full-length SU. The peptides mobilized [Ca(2+)](i) in HT-29 cells with distinct oscillations and elevated inositol triphosphate levels. Circular dichroism analyses showed the peptides were predominantly random coil in buffer, but increased in alpha-helical content when placed in a hydrophobic environment or with cholesterol-containing membrane vesicles that are rich in anionic phospholipids. None of the peptides underwent significant secondary structural changes in the presence of neutral vesicles indicating ionic interactions were important. These data show that the SIV SU enterotoxic domain localizes in part to the V3 loop region and interacts with anionic membrane domains on the host cell surface.

The envelope glycoprotein of simian immunodeficiency virus contains an enterotoxin domain.

By the use of a mouse model, the enteropathic effects of the simian immunodeficiency virus (SIV) surface unit (SU) envelope glycoprotein were explored. Purified SU (0.01-0.45 nmol) was administered intraperitoneally to 6- to 8-day-old mouse pups and induced a dose-dependent diarrheal response. Surgical introduction of SU into adult mouse intestinal loops revealed fluid accumulation without histological alterations and SU-treated unstripped intestinal mucosa induced chloride (Cl(-)) secretory currents in Ussing chambers. Similarly to rotavirus NSP4, the first described viral enterotoxin, SU induced a transient increase in intracellular calcium levels and increased inositol 1,4,5-triphosphate (IP(3)) levels in HT-29 cells. These data indicate the calcium response is mediated by IP(3). The presence of diarrhea and fluid accumulation within intestinal loops in the absence of histological alterations and induction of Cl(-) secretory currents demonstrate that SIV contains an enterotoxic domain localized within SU and is the second viral enterotoxin described.