Rami A. Dalloul

Multi-platform next-generation sequencing of the domestic Turkey (Meleagris gallopavo): Genome assembly and analysis

The combined application of next-generation sequencing platforms has provided an economical approach to unlocking the potential of the turkey genome.

Recent Advances in Immunomodulation and Vaccination Strategies Against Coccidiosis

Abstract Coccidiosis is a ubiquitous intestinal protozoan infection of poultry seriously impairing the growth and feed utilization of infected animals. Conventional disease control strategies rely heavily on chemoprophylaxis, which is a tremendous cost to the industry. Existing vaccines consist of live virulent or attenuated Eimeria strains with limited scope of protection against an ever-evolving and widespread pathogen. The continual emergence of drug-resistant strains of Eimeria, coupled with the increasing regulations and bans on the use of anticoccidial drugs in commercial poultry production, urges the need for novel approaches and alternative control strategies. Because of the complexity of the host immunity and the parasite life cycle, a comprehensive understanding of host-parasite interactions and protective immune mechanisms becomes necessary for successful prevention and control practices. Recent progress in functional genomics technology would facilitate the identification and characterization of host genes involved in immune responses as well as parasite genes and proteins that elicit protective host responses. This study reviews recent coccidiosis research and provides information on host immunity, immunomodulation, and the latest advances in live and recombinant vaccine development against coccidiosis. Such information will help magnify our understanding of host-parasite biology and mucosal immunology, and we hope it will lead to comprehensive designs of nutritional interventions and vaccination strategies for coccidiosis.

EMBRYO VACCINATION AGAINST EIMERIA TENELLA AND E. ACERVULINA INFECTIONS USING RECOMBINANT PROTEINS AND CYTOKINE ADJUVANTS

Avian coccidiosis is an intestinal disease caused by protozoa of the genus Eimeria. To investigate the potential of recombinant protein vaccines to control coccidiosis, we cloned 2 Eimeria sp. genes (EtMIC2 and 3-1E), expressed and purified their encoded proteins, and determined the efficacy of in ovo immunization to protect against Eimeria infections. Immunogen-specific serum antibody titers, parasite fecal shedding, and body weight gains were measured as parameters of disease. When administered alone, the recombinant EtMIC2 gene product induced significantly higher antibody responses, lower oocyst fecal shedding, and increased weight gains compared with nonvaccinated controls following infection with E. tenella. Combined embryo immunization with the EtMIC2 protein plus chicken cytokine or chemokine genes demonstrated that all 3 parameters of vaccination were improved compared with those of EtMIC2 alone. In particular, covaccination with EtMIC2 plus interleukin (IL)-8, IL-16, transforming growth factor-β4, or lymphotactin significantly decreased oocyst shedding and improved weight gains beyond those achieved by EtMIC2 alone. Finally, individual vaccination with either EtMIC2 or 3-1E stimulated protection against infection by the heterologous parasite E. acervulina. Taken together, these results indicate that in ovo vaccination with the EtMIC2 protein plus cytokine/chemokine genes may be an effective method to control coccidiosis.

Intestinal Immunomodulation by Vitamin A Deficiency and Lactobacillus-Based Probiotic in Eimeria acervulina–Infected Broiler Chickens

Abstract SUMMARY. In a 2 × 2 factorial study, a broiler starter ration was amended for vitamin A (control, C; deficient, A) and probiotic status (−, P) to investigate their modulatory effects on the host immune system. Birds were inoculated orally with Eimeria acervulina (EA) oocysts, and disease susceptibility was evaluated by assessment of fecal oocyst shedding. Humoral and local cellular mediated immunity were assessed by evaluation of antibody and cytokine (interferon-γ [IFN-γ] and interleukin-2 [IL-2]) levels in sera and intestinal secretions on a 3-day interval after inoculation. Fecal oocyst shedding was highest (P < 0.05) in A− birds, followed by AP, C−, and CP birds. Feeding the probiotic reduced shed oocysts by 20% in A fed birds and by 26% in C fed birds. Intestinal IFN-γ was relatively constant in all treatment groups except for A−, where it declined steadily and was lower (P < 0.05) from day 6 on. Serum IFN-γ levels fluctuated within each treatment and over time were not revealing. Intestinal IL-2 was highest in CP birds at 3 and 9 days postinfection (DPI) and lowest in A− birds at 3, 9, and 12 DPI (P < 0.05); no difference between treatments was found at 6 DPI (P > 0.05). Eimeria-specific intestinal antibody (Ab) level was constant (P > 0.05) in C− birds but increased with time (P < 0.05) in A−, AP, and CP birds. Serum Ab levels were also constant in A− and CP birds but increased (P < 0.05) in C− and AP birds after 6 DPI. The data demonstrate for the first time a probiotic-enhanced immunity in vitamin A deficient birds. This study is also the first to demonstrate the probiotic effect on local cell-mediated immunity of chickens, best manifested by apparent lower intestinal invasion and development by EA, on the basis of higher IL-2 secretion and lower EA oocyst production.

Immune responses to dietary β-glucan in broiler chicks during an Eimeria challenge

Escalating consumer concerns regarding pathogen resistance have placed the poultry industry under mounting pressure to eliminate the use of chemotherapeutic agents as feed additives. One possible alternative receiving increased attention is the use of immunomodulators such as β-glucan. A study was conducted to investigate the effects of a yeast-derived β-glucan (Auxoferm YGT) on broiler chick performance, lesion scores, and immune-related gene expression during a mixed Eimeria infection. Day-old chicks were fed diets containing 0, 0.02, or 0.1% YGT. On d 8 posthatch, one-half of the replicate pens were challenged with a mixed inoculum of Eimeria acervulina, Eimeria maxima, and Eimeria tenella. Measurements were taken and samples collected on d 4, 10, 14, and 21 posthatch. Dietary supplementation had no effect on performance or mortality. On d 14, 3 birds per pen (n = 24/treatment) were scored for intestinal coccidia lesions. Gross lesion severity was significantly reduced in birds supplemented with 0.1% YGT. On d 10, inducible nitric oxide synthase (iNOS) expression was downregulated in the jejunum of challenged birds fed 0.1% YGT. Expression of iNOS in the ileum was downregulated in the nonchallenged birds, but upregulated in the challenged birds fed 0.1% YGT on d 14. Interleukin (IL)-18 was upregulated in the jejunum of 0.1% YGT-treated birds. Interferon (IFN)-γ expression was decreased in challenged and nonchallenged birds fed 0.1% YGT. The IL-4 expression was downregulated in the nonchallenged birds with 0.1% YGT diet supplementation. The IL-13 and mucin-1 levels were also reduced due to β-glucan supplementation. Mucin-2 expression was increased in the nonchallenged birds, but decreased in the infected birds fed 0.1% YGT. These results suggest that although Auxoferm YGT at doses of 0.02 and 0.1% does not influence performance, it significantly reduces lesion severity and is capable of altering immune-related gene expression profiles, favoring an enhanced T helper type-1 cell response during coccidiosis.

Performance and immune responses to dietary β-glucan in broiler chicks

During the first week posthatch, the avian immune system is immature and inefficient at protecting chicks from invading pathogens. Among immunomodulators, beta-glucans are known as biological response modifiers due to their ability to activate the immune system. Current research suggests that beta-glucans may enhance avian immunity; however, very little is known about their influence on regulation of immune function. A study was performed to evaluate the effects of dietary beta-glucan on growth performance, immune organ weights, peripheral blood cell profiles, and immune-related gene expression in the intestine. One-day-old chicks were fed a diet containing 0, 0.02, or 0.1% yeast beta-glucan (n = 30/treatment). On d 7 and 14 posthatch, body and relative immune organ weights were measured and small intestinal sections were collected to evaluate gene expression by quantitative real-time PCR. Peripheral blood samples were also collected to determine heterophil:lymphocyte ratios. Supplementation of beta-glucan did not significantly affect BW gains, and no significant differences were observed among groups for relative immune organ weights or heterophil:lymphocyte ratios. Compared with controls, expression of interleukin (IL)-8 was downregulated in the beta-glucan-treated groups on d 7 and 14. On d 14, beta-glucan inclusion resulted in increased inducible nitric oxide synthase expression. Expression of IL-18 was upregulated on d 7 but reduced on d 14 due to beta-glucan supplementation. On d 7, interferon-gamma and IL-4 expression decreased in the beta-glucan-treated groups. However, on d 14, IL-4 expression was upregulated in the supplemented groups. Intestinal expression of IL-13 was also downregulated in the beta-glucan-treated birds on d 7. These results suggest that dietary inclusion of beta-glucans altered the cytokine-chemokine balance; however, it did not elicit a robust immune response in the absence of a challenge, resulting in no deleterious effects on performance.

Effects of probiotics and application methods on performance and response of broiler chickens to an Eimeria challenge

Coccidiosis is an inherent risk in the commercial broiler industry and inflicts devastating economic losses to poultry operations. Probiotics may provide a potential alternative to the prophylactic use of anticoccidials in commercial production. This study evaluated the effects of probiotic applications (feed and water) on bird performance and resistance to a mixed Eimeria infection in commercial broilers. On day of hatch, 1,008 commercial male broilers (Cobb 500) were assigned to 1 of 6 treatments (8 replicate floor pens; 21 birds/pen), including noninfected negative control (NEG), Eimeria-infected positive control (POS), anticoccidial control (0.01% salinomycin, SAL), intermittent high-dose water-applied probiotic (WPI), continuous low-dose water-applied probiotic (WPC), and feed-supplemented probiotic (FSP). On d 15, all birds except those in NEG were challenged with a mixed inoculum of Eimeria acervulina, Eimeria maxima, and Eimeria tenella. Measurements were taken on d 7, 15, 21, 28, 35, and 42. Fecal samples were collected from d 20 to 24 for oocyst counts, and lesion scores were evaluated on d 21. Data were analyzed using the Fit Model platform in JMP Pro 10.0 (SAS Institute Inc.). Differences in experimental treatments were tested using Tukeys honestly significant difference following ANOVA with significance reported at P ≤ 0.05. Overall, NEG birds outperformed all other groups. For performance, the probiotic groups were comparable with the SAL-treated birds, except during the 6 d immediately following the Eimeria species challenge, where the SAL birds exhibited better performance. The WPC birds had lower duodenal and jejunal lesion scores, indicating a healthier intestine and enhanced resistance to Eimeria species compared with POS. Birds in the WPI treatment shed fewer oocysts in the feces, although this was not a trend for all of the probiotic treatment groups. The results of this study suggest probiotic supplementation without anticoccidials can enhance performance and help alleviate the negative effects of a mixed Eimeria infection.

Expression of Toll-like receptors and antimicrobial peptides during Eimeria praecox infection in chickens

Intestinal colonization of avian species by Eimeria parasites results in the enteric disease, coccidiosis. A study was carried out to assess the immunologic effects of Eimeria praecox infection on the gut of infected chickens. In Experiment 1, birds were orally gavaged with 50,000 E. praecox oocysts; in Experiment 2, an infection dosage of 500,000 E. praecox oocysts was used. Duodenal and jejunal intestinal sections were sampled consecutively on days 1-7 post-infection. Intestinal expression of innate immune gene transcripts was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Analysis of relative gene expression in Experiment 1 revealed an increase (P<0.05) in duodenal Toll-like receptor (TLR)3 expression on days 4 and 6 post-infection. TLR15 expression was significantly decreased in the duodenum of infected birds on day 2, and significantly increased on day 6 post-infection. In Experiment 2, TLR3 was significantly downregulated in the duodenum on day 7 post-infection; however, no significant results were observed in terms of TLR15 expression. TLR4 also exhibited decreased expression (P<0.05) on day 7 post-infection in both intestinal sections. Regarding antimicrobial peptide expression; in the first experiment, expression of liver-expressed antimicrobial peptide-2 (LEAP-2) in infected birds was significantly decreased in the duodenum on days 3 and 4, and in the jejunum on day 4. Similarly, Experiment 2 resulted in depression of LEAP-2 (P<0.05) on days 3-5 in the duodenum. In Experiment 1, cathelicidin antimicrobial peptide (CATHL3) was downregulated (P<0.05) in the jejunum of infected chickens on day 3 post-infection; however, CATHL3 results were non-significant in Experiment 2. Based on the differing results observed in each experiment, it was concluded that both TLR and antimicrobial peptide expression, and thus immunity may be dependent on infection load.

Molecular cloning and functional characterization of the avian macrophage migration inhibitory factor (MIF)

Macrophage migration inhibitory factor (MIF) is recognized as a soluble factor produced by sensitized T lymphocytes and inhibits the random migration of macrophages. Recent studies have revealed a more prominent role for MIF as a multi-functional cytokine mediating both innate and adaptive immune responses. This study describes the cloning and functional characterization of avian MIF in an effort to better understand its role in innate and adaptive immunity, and potential use in poultry health applications. The full-length avian MIF gene was amplified from stimulated chicken lymphocytes and cloned into a prokaryotic expression vector. The confirmed 115 amino acid sequence of avian MIF has 71% identity with human and murine MIF. The bacterially expressed avian recombinant MIF (rChMIF) was purified, followed by endotoxin removal, and then tested by chemotactic assay and quantitative real-time PCR (qRT-PCR). Diff-Quick staining revealed a substantial decrease in migration of macrophages in the presence of 0.01microg/ml rChMIF. qRT-PCR analysis revealed that the presence of rChMIF enhanced levels of IL-1beta and iNOS during PBMCs stimulation with LPS. Additionally, the Con A-stimulated lymphocytes showed enhanced interferon (IFN)-gamma and IL-2 transcripts in the presence of rChMIF. Interestingly, addition of rChMIF to the stimulated PBMCs, in the presence of lymphocytes, showed anti-inflammatory function of rChMIF. To our knowledge, this study represents the first report for the functional characterization of avian MIF, demonstrating the inhibition of macrophage migration, similar to mammalian MIF, and the mediation of inflammatory responses during antigenic stimulation.

Effects of a dietary antioxidant blend and vitamin E on growth performance, oxidative status, and meat quality in broiler chickens fed a diet high in oxidants

The aim of the study was to determine the effects of a dietary antioxidant blend (AB) and vitamin E on performance, oxidative status, and meat quality. Cobb 500 male broilers (n = 1,200, d 0) were randomly distributed into 6 treatments with 10 replicate pens. Treatments included 1) HO: high oxidant diet, vitamin E at 10 IU/kg, 3% oxidized soybean oil, 3% polyunsaturated fatty acid (PUFA) source; 2) VE: the HO diet with vitamin E at 200 IU/kg; 3) AOX: the HO diet with AB at 135 mg/kg; 4) VE+AOX: the HO diet with vitamin E at 200 IU/kg and AB at 135 mg/kg; 5) SC: standard control; and 6) PC: positive control, the SC diet with AB at 135 mg/kg. From d 0 through d 21, high oxidant diet treatment birds had greater BW, ADG, and ADFI than the SC birds; the AOX birds had better G:F on d 10 and 42, and from d 0 to 42 than SC birds (P < 0.05). The plasma TBA reactive substance level was lower in the AOX birds than the VE treatment birds in all phases (P < 0.05). High oxidant diet treatment birds had greater α-1-acid glycoprotein levels on d 10 than SC and PC birds (P < 0.05). The AOX, PC, and SC birds had a greater level of uric acid than the HO and VE+AOX birds on d 10. Superoxide dismutase expression in the liver was less with the HO treatment compared with the SC treatment on d 7 (P < 0.05). The vitamin E concentration in the breast muscle was greatest in the VE birds, whereas vitamin A concentration was greater in the PC birds compared with the SC birds on d 21 (P < 0.05). Compared with VE and AOX, the HO treatment had greater drip loss (P < 0.05). In conclusion, dietary addition of AOX was effective in improving growth, moderately restored the whole body antioxidant capability, and reduced drip loss.