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.

Efficacy of 25-OH Vitamin D3 prophylactic administration for reducing lameness in broilers grown on wire flooring.

Bacterial chondronecrosis with osteomyelitis (BCO) is the most common cause of lameness in commercial broilers. Growing broilers on wire flooring provides an excellent experimental model for reproducibly triggering significant levels of lameness attributable to BCO. In the present study we evaluated the efficacy of adding HyD (25-OH vitamin D3) to the drinking water as a preventative/prophylactic treatment for lameness. Broiler chicks were reared on 5 x 10 ft flat wire floor panels within 6 environmental chambers. Three chambers were supplied with tap water (Control group) and the remaining chambers were supplied with HyD (HyD group: 0.06 mL HyD solution/L water; dosing based on the HyD Solution label to provide 33.9 µg 25-OHD3/L) from d 1 through 56. Feed was provided ad libitum and was formulated to meet or exceed minimum standards for all ingredients, including 5,500 IU vitamin D3/kg. Lameness initially was detected on d 28, and the cumulative incidence of lameness on d 56 was higher in the Control group than in the HyD group (34.7 vs. 22.7%, respectively; P = 0.03; Z-test of proportions; chambers pooled). The most prevalent diagnoses for lame birds were osteochondrosis and osteomyelitis (BCO) of the proximal femora (52%) and tibiae (79%), accompanied by minor incidences of tibial dyschondroplasia (0.33%), spondylolisthesis, or kinky back (0.67%), and twisted legs (1%). Broilers that survived to d 56 without developing lameness did not differ in BW when compared by group within a gender. The wire flooring model imposes a rigorous, sustained challenge that undoubtedly is much more severe than typically would be experienced by broilers under normal commercial conditions. Therefore the encouraging response to HyD supplementation in the present study supports the potential for 25-OH vitamin D3 to attenuate outbreaks of lameness caused by BCO in commercial broiler flocks.

Prophylactic administration of a combined prebiotic and probiotic, or therapeutic administration of enrofloxacin, to reduce the incidence of bacterial chondronecrosis with osteomyelitis in broilers.

Bacteria entering the bloodstream via translocation from the gastrointestinal tract spread hematogenously and can trigger bacterial chondronecrosis with osteomyelitis (BCO) by infecting osteochondrotic microfractures in the epiphyseal-physeal cartilage of the proximal femora and tibiae. In experiment 1, broilers were fed control feed or the same feed containing BacPack 2X, which includes the prebiotic IMW50 (a mannan oligosaccharide beta-glucan yeast cell wall product) plus the probiotic Calsporin (Bacillus subtilis C-3102). Broilers reared on wire flooring consistently developed higher incidences of BCO than hatchmates reared on wood shavings litter (≥24 vs. ≤4%, respectively; P=0.001). Adding BacPack 2X to the feed on d 1 through 56 delayed the age of onset and reduced the cumulative incidence of BCO on wire flooring when compared with broilers fed the control feed (24.0 vs. 40.7%, respectively; P=0.003). In experiment 2, broilers reared on wire flooring received tap water on d 1 through 62 (control group) or therapeutic levels of the potent fluoroquinolone antimicrobial enrofloxacin in the water on d 35 through 54 (enrofloxacin group). During enrofloxacin administration, half as many birds developed BCO in the enrofloxacin group when compared with the control group (8.1 vs. 19.5%, respectively, on d 35 through 54; P=0.001), whereas both groups had similar BCO incidences subsequent to withdrawing enrofloxacin on d 55 through 62 (14.8 vs. 18.2% for the enrofloxacin vs. control groups; P=0.386). Cumulative lameness incidences for d 1 through 62 were higher for the control group than for the enrofloxacin group (39.0 vs. 25.8%, respectively; P=0.003). These results demonstrate that wire flooring imposes a rigorous challenge that leads to high incidences of BCO that can be difficult to suppress, even with therapeutic doses of enrofloxacin. Prophylactically adding BacPack 2X to the feed reduced the incidence of BCO lameness by a proportion similar to that achieved with enrofloxacin, indicating that probiotics potentially can provide effective alternatives to antibiotics for reducing BCO lameness attributable to bacterial translocation and hematogenous distribution.

Evaluating portable wire-flooring models for inducing bacterial chondronecrosis with osteomyelitis in broilers.

Rearing broilers on flat or sloping wire flooring is an effective method for consistently triggering lameness attributable to bacterial chondronecrosis with osteomyelitis (BCO). Portable obstacles known as speed bumps (SB) also consistently trigger modest incidences of BCO when they are installed between feed and water lines in litter flooring facilities. Two experiments were conducted to determine the most effective broiler age for introducing the SB into litter flooring pens, and to evaluate alternative configurations of the traditional SB with the expectation that amplified mechanical challenges to the legs of broilers should increase the incidence of BCO. Broiler chicks obtained from commercial hatcheries (lines B and D in experiment 1, lines A and B in experiment 2) were reared in floor pens with ad libitum feed and water and a 23L:1D photoperiod. In experiment 1, the 5 floor treatments included wood shavings litter only (L), flat wire only (W), or litter plus SB installed at 14, 28, or 42 d of age. Line B was more susceptible to lameness than line D (25.9 vs. 15.3% for all treatments combined; P = 0.001). Both lines developed low incidences of lameness on L (11 to 13%), intermediate incidences on SB regardless of day of installation (12 to 23%), and high incidences on W (21 to 39%). In experiment 2, broilers were reared with 7 floor treatments, including L, W, SB with a 50% slope (SB50%); SB50% with a limbo bar installed over the apex; SB with a 66% slope and limbo bar; SB50% with a nipple water line suspended over the apex; and a pagoda-top SB. All SB were inserted on d 28. Line B was more susceptible to lameness than line A (20.2 vs. 16.1% for all treatments combined; P < 0.05), and for both lines combined the lameness percentages averaged 7.7 (L), 29.2 (W), 17.3 (SB50%), 16.2 (SB50% with a limbo bar), 21.5 (SB with a 66% slope and limbo bar), 20.8 (SB50% with a nipple water line), and 11.5% (pagoda-top). These studies demonstrate the portable SB can be effectively used to experimentally trigger BCO in broilers.

Bacterial chondronecrosis with osteomyelitis in broilers: influence of sires and straight-run versus sex-separate rearing.

Two experiments (E1, E2) were conducted to compare the influence of sires (sire A on dam C vs. sire B on dam C) and straight-run versus sex-separate rearing on the incidence of bacterial chondronecrosis with osteomyelitis (BCO) in broilers. Fertile eggs from commercial breeder flocks were incubated and hatched at the University of Arkansas Poultry Research Hatchery. Male and female chicks were reared together (straight-run) or separately (sex-separate) in 3 × 3 m pens on litter or flat wire flooring with 65 (E1) or 60 (E2) birds per pen. Necropsies revealed lesions that are pathognomonic for BCO in ≥98% of the birds that became lame. The SigmaStat Z-test was used to compare cumulative BCO incidences through 8 wk of age. For birds reared on litter, the incidences of BCO were low regardless of cross or sex (range: 1.7 to 5.1%; P ≥ 0.6). Within a cross and sex, rearing the broilers straight-run versus sex-separate on wire flooring did not significantly affect the incidence of BCO. Significant incidences of BCO did not develop until after d 40. Males from the sire A cross developed a higher incidence of BCO than males from the sire B cross in E1 (27 vs. 17%, respectively; P = 0.009) but not in E2 (28.5 vs. 22.6%, respectively; P = 0.141). In both experiments, males from the sire A cross developed higher incidences of BCO than females from the sire B cross (27 vs. 11.9%, in E1; 28.5 vs. 14.8%, in E2). With the sexes pooled, broilers from the sire A cross consistently developed higher incidences of BCO than broilers from the sire B cross (21.4 vs. 14.9%, P = 0.005 in E1; 26.5 vs. 18.7%, P = 0.003 in E2). High susceptibilities to both femoral head (all femoral head necrosis = 66 to 85% incidences) and tibial head (all tibial head necrosis = 81 to 96% incidences) BCO lesions were demonstrated in lame birds from both sexes and crosses. This study supports a sire influence on the susceptibility of broilers to BCO. Sire lines can be chosen to reduce BCO susceptibility when broilers are grown beyond 6 wk of age.

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.

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.

Maternal antibody transfer from dams to their egg yolks, egg whites, and chicks in meat lines of chickens.

Maternal antibodies are transferred from hens to the chicks via the egg. To gain insight into maternal antibody transfer and endogenous production of antibodies in broiler chicks, total IgY, IgA, IgM, as well as anti-Newcastle disease virus (NDV) and anti-infectious bronchitis (IBV) antibody levels were examined in the dams' plasma, egg yolks, egg whites, and chicks' plasma on d 3, 7, 14, and 21. Blood was collected from 39-wk-old breeder hens (line 1, n = 17; line 2, n = 21). Fertile eggs were used for antibody extraction from the egg yolks and egg whites (4 to 5 eggs/dam) and for hatching. Unvaccinated chicks (4 to 5 chicks/dam) were reared in a HEPA-filtered room and were bled on d 3, 7, 14 and 21. Based on ELISA methods, plasma levels of IgY and IgM were higher (P < 0.0001), and those of IgA were similar (P = 0.31), in line 2 compared with line 1. Egg yolk IgY and IgA, as well as egg white IgY, IgA, and IgM levels were higher in line 2 compared with line 1 (P < 0.0001). Independent of line of chicken, the percentage dam-to-chick (3 d) plasma transfer of IgY was estimated to be approximately 30%, with that for IgM and IgA less than 1%. Chicks synthesized IgM first, followed by IgA and IgY. Anti-NDV and anti-IBV antibodies were detected in the dams' plasma, egg yolks, and in the chicks' plasma on d 3 and 7, with line 2 having higher anti-IBV and lower anti-NDV levels than line 1 in all samples (P < 0.0001). In summary, IgY levels, total or antigen-specific, in the dams' plasma or eggs were found to be a direct indicator of maternal antibody transfer to the chicks' circulation, with an expected percentage transfer of approximately 30%. This knowledge, together with the observed time course of endogenous antibody production in broiler chicks, may find direct application in formulating strategies for protecting chicks, especially during the first few weeks of age when their immune system is not yet fully functional.

Immune response and disease resistance in chickens. II. Marek's disease and immune response to GAT.

Genetic control of resistance of susceptibility to Marek's disease in chickens was found to be associated with immune response to the amino acid polymer GAT. Thus, both B1B1 and B19B19 GAT low responders had significantly greater incidence of MD than the B1B1 and B19B19 GAT high responder counterparts. The results point to gene(s) controlling MD resistance mapping within the immune response region of the B complex, the major histocompatibility system of the chicken. Patterns of general mortality demonstrate that the low viability, observed over several years among B1B1 birds, likewise is associated with the immune response region of the B complex.

Subregions and functions of the chicken major histocompatibility complex.

The chicken major histocompatibility complex (MHC) exerts genetic influence over a variety of important biological functions including immune response, disease resistance, growth and development, aging, and reproduction. The chicken MHC possesses at least three subregions encoding distinct gene products. The B-G subregion encodes antigens unique to erythrocyte surfaces. The B-L and B-F subregions encode cell surface glycoproteins homologous to mammalian Class II and Class I antigens, respectively. Class I and Class II molecules are crucial for recognition of self vs. nonself and for cell communication, and therefore are fundamental for all immune responses. Studies of chromosomal recombinants have been particularly useful in eliciting the structure and function of subregions of the chicken MHC.

Immune response and disease resistance in chickens. I. Selection for high and low titer to Salmonella pullorum antigen.

A selection experiment for high and low anti-Salmonella pullorum antibody titer was carried out over four generations within the B1B1 blood group genotype in chickens. The study was aimed primarily at identifying different response patterns controlled by immune response genes linked to the B system, the major histocompatibility complex of the chicken. Maximal divergence was obtained in the third generation of selection when agglutination titers of 1/320 and 1/80 in B1B1 high and low responders, respectively, were reached. Immune response of S. pullorum was deduced to be controlled by polygenes. The B1B1 population, selected for high immune response to S. pullorum antigen, consistently had greater total mortality as well as greater susceptibility to challenge with Marek's disease virus compared with B1B1 population selected for low response. This, however, is believed to be a consequence of random drift of genes for disease resistance in the relatively small populations and not a direct consequence of selection for high or low S. pullorum titer.

B-complex genetic control of immune response to HSA, (T,G)-A--L, GT and other substances in chickens.

Immune response to poly-(L-tyrosine-L-glutamic acid)-poly-D,L-alanine-poly-L-lysine (T,G)-A--L), human serum albumin (HSA), and (L-glutamic acid50, L-tyrosine50)n (GT) was found to be linked to the B complex in an outbred line of Leghorns segregating for the B1, B2, and B19 alleles. Birds of the blood group genotypes B1B1, B2B2, and B19B19 were low, intermediate, and high responders, respectively to either (T,G)-A--L or HSA. Response to GT, however, differed, with the B2B2 genotype being the only responder. No real genotype differences in immune response to DNP-congugates and sheep red blood cells (SRBC) could be detected.

Recombination between genes coding for immune response and the serologically determined antigens in the chickenB system.

Evidence is presented for a crossover between the genes coding for the serologically determined (SD) antigens on erythrocytes and an immune response gene (Ir-GAT) controlling immune response to the synthetic polypeptide GAT within theB complex, the MHC of chickens. TheIr-GAT (1) andIr-GAT (19) alleles control low and high immune response to GAT, respectively. Both low and high responders were recovered as recombinants fromB (1) B (1) andB (19) B (19) birds. The low-responder haplotypes are homozygous for theIr-GAT (1) allele and the high-responder haplotypes carry theIr-GAT (19) allele. Mortality forB (1) B (1) nonresponder birds was 39%, compared with 19% for theB (1) B (1) high responders; this suggests the possibility that genes located within the immune response region of theB complex exert some genetic control over viability and survival.

Glyoxalase 1 (GLO) in the chicken: genetic variation and lack of linkage to the MHC.

Electrophoretic variation of glyoxalase 1 (GLO) has been detected in chicken red-cell lysates. Three phenotypes are shown to be inherited through a diallelic system, just as in humans and mice. The chicken GLO phenotype differ from their mammalian counterparts in that one of the homozygotes is devoid of GLO activity. The heterozygote produces two bands, while the other homozygote yields a single band of GLO activity with mobility equal to the faster of these two bands. In noninbred White Leghorn birds, the GLO2 allele occurred significantly more often in birds homozygous for the B1 allele at the chicken MHC than in those homozygous for B19, suggesting that the products of these loci may have population associations in the chicken. Absence of close linkage between the GLO and B loci was, however, demonstrated by appropriate test crosses.