Hyperimmune egg yolk antibodies developed against Clostridium perfringens antigens protect against necrotic enteritis.

Necrotic enteritis (NE) is a widespread infectious disease caused by Clostridium perfringens that inflicts major economic losses on the global poultry industry. Due to regulations on antibiotic use in poultry production, there is an urgent need for alternative strategies to mitigate the negative effects of NE. This paper presents a passive immunization technology that utilizes hyperimmune egg yolk immunoglobulin Y (IgY) specific to the major immunodominant antigens of C. perfringens. Egg yolk IgYs were generated by immunizing hens with 4 different recombinant C. perfringens antigens, and their protective effects against NE were evaluated in commercial broilers. Six different spray-dried egg powders were produced using recombinant C. perfringens antigens: α-toxin, NE B-like toxin (NetB; EB), elongation factor-Tu (ET), pyruvate:ferredoxin oxidoreductase, a mixture of 4 antigens (EM-1), and a nonimmunized control (EC). The challenged groups were either provided with different egg powders at a 1% level or no egg powders (EN). The NE challenge model based on Eimeria maxima and C. perfringens dual infection was used. In Experiments 1 and 2, the EB and ET groups exhibited increased body weight gain (BWG; P < 0.01), decreased NE lesion scores (P < 0.001), and reduced serum NetB levels (P < 0.01) compared to the EN and EC groups. IgY against NetB significantly reduced Leghorn male hepatocellular cytotoxicity in an in vitro test (P < 0.01). In Experiment 3, the protective effect of the IgYs mixture (EM-2) against C. perfringens antigens (NetB and EFTu) and Eimeria antigens (elongation factor-1-alpha: EF1α and Eimeria profilin: 3-1E) was tested. The EM-2 group showed similar body weight, BWG, and feed intake from d 7 to 22 compared to the NC group (P < 0.05). On d 20, the EM-2 group showed comparable intestinal permeability, NE lesion scores, and jejunal NetB and collagen adhesion protein levels to the NC group (P < 0.05). In conclusion, dietary mixture containing antibodies to NetB and EFTu provides protection against experimental NE in chickens through passive immunization.

Allium hookeri supplementation improves intestinal immune response against necrotic enteritis in young broiler chickens.

Three hundred birds (1 day old) were randomly assigned to 6 groups (n = 50 birds/treatment) and fed a basal diet (control) or basal diet supplemented with Allium hookeri (AH) root (1 or 3%). At day 14, half of the birds in each group were orally challenged with E. maxima 41A (1 × 104 cells/chicken), followed by C. perfringens infection (1 × 109 cfu/chicken) on day 18. Necrotic enteritis (NE)-associated infections and intestinal immune response were assessed by average body weight gain, lesion score, and oocyst shedding. The effect of dietary supplementation, AH, on transcript levels of pro-inflammatory cytokines, and tight junction proteins and mucin protein in the jejunum, were quantified by quantitative real-time (qRT)-PCR. At day 20, birds fed with diet supplementation (3% of AH) significantly weighted more than the control group. Although the NE-challenged had significantly reduced average body weight gain, there was no significance in the effect between diet × NE-challenge interactions on the average body weight gain. Among the NE-challenged groups, gut lesion score and oocyst shedding were significantly decreased in birds given AH (1 or 3%) compared to the control group. There was a correlation between diet and NE infection with regards to interleukin (IL)-17A, and inducible nitric oxide synthase (iNOS). The up-regulated transcript levels of cytokines IL-8, IL-17A, iNOS, and LITAF by NE challenged groups were significantly reduced by AH (1 or 3%) supplementation. Down-regulated expression levels of tight junction (TJ) proteins: junctional adhesion molecule 2 (JAM2), occluding, and intestinal mucin 2 (MUC2) by NE challenge, was up-regulated by the addition of AH (1 or 3%) supplementation. All TJ proteins (JAM2, ZO1, Ocluddin and MUC2) in the jejunum had a significant diet × NE-challenge interaction. These findings demonstrate that dietary supplementation of AH in chicken feed could be beneficially used to improve chicken health against NE.

Alternatives to antibiotics for maximizing growth performance and feed efficiency in poultry: a review.

With the increase in regulations regarding the use of antibiotic growth promoters and the rise in consumer demand for poultry products from 'Raised Without Antibiotics' or 'No Antibiotics Ever' flocks, the quest for alternative products or approaches has intensified in recent years. A great deal of research has focused on the development of antibiotic alternatives to maintain or improve poultry health and performance. This review describes the potential for the various alternatives available to increase animal productivity and help poultry perform to their genetic potential under existing commercial conditions. The classes of alternatives described include probiotics, prebiotics, synbiotics, organic acids, enzymes, phytogenics, antimicrobial peptides, hyperimmune egg antibodies, bacteriophages, clay, and metals. A brief description of the mechanism of action, efficacy, and advantages and disadvantages of their uses are also presented. Though the beneficial effects of many of the alternatives developed have been well demonstrated, the general consensus is that these products lack consistency and the results vary greatly from farm to farm. Furthermore, their mode of action needs to be better defined. Optimal combinations of various alternatives coupled with good management and husbandry practices will be the key to maximize performance and maintain animal productivity, while we move forward with the ultimate goal of reducing antibiotic use in the animal industry.

The Effects of Direct-fed Microbial Supplementation, as an Alternative to Antibiotics, on Growth Performance, Intestinal Immune Status, and Epithelial Barrier Gene Expression in Broiler Chickens.

The objective of this study was to investigate the effects of Bacillus subtilis-based probiotic supplementation in broiler chicken diets on growth performance, feed efficiency, intestinal cytokine, and tight junction (TJ) protein mRNA expression. Zero-day-old broiler chicks (n = 140) were randomly assigned to one of five dietary treatments: basal diet (CON); basal diet supplemented with either antibiotic bacitracin methylene disalicylate (BMD); or probiotics, namely, B. subtilis strain 1781 (PB1), a combination of B. subtilis strain 1104 + strain 747 (PB2), or B. subtilis strain 1781 + strain 747 (PB3). Body weight and feed intake were measured at 14 days of age, and the feed conversion ratio (FCR) was calculated. At 14 days of age, ileal samples were collected and used for intestinal cytokine, TJ protein, and mucin gene expression analysis using qRT-PCR. The chickens supplemented with antibiotic (BMD) and B. subtilis strain 1781 alone (PB1) had significantly higher body weights compared to controls of the same age. Dietary supplementation with antibiotic (BMD) or probiotics (PB1, PB2, PB3) significantly improved the feed efficiency as evidenced by decreased FCR compared to controls. No differences were observed in the expression of IL1ß, IL17F, IFNγ, and MUC2 gene among the different treatment groups. However, elevated expression of IL6 (BMD, PB1, PB2), IL8 (PB2), and TNFSF15 (PB1, PB2, PB3) compared to controls was observed in the ileum. IL2 and IL10 expression was upregulated in chicks in the PB2 and PB3 groups, and IL4 was elevated in the PB1 group. IL13 was elevated in all probiotic-fed groups (PB1, PB2, PB3). Probiotic supplementation was also shown to significantly increase the expression of TJ proteins JAM2, ZO1 (PB2, PB3), and occludin (PB1, PB2). Taken together, B. subtilis supplementation altered intestinal immune activity and influenced gut barrier integrity through increased tight junction gene expression.

Evaluation of chlorine dioxide based product as a hatchery sanitizer.

Formaldehyde is commonly used to overcome contaminants introduced by hatching eggs or water supply in the hatcher cabinets. However, health risks associated with its use make economical alternatives important. This project evaluated a chlorine dioxide based product (CDBP) (0.3% concentrate) as a hatchery sanitizer in decontaminating microbial populations on the shell surface of hatching eggs (>18 d old), as well as its impact on hatchability and chick performance. Hatchers (0.20 m2) designed to hold approximately 50 eggs and equipped with circulation fans, heaters, and thermostats were used for the evaluation. For each of the 2 trials conducted, 450 hatching eggs were obtained and incubated in a common setter. Eggs used in trial 1 were floor eggs whereas in trial 2 nest eggs were used. On d 18 of incubation, eggs were removed from the setter, and viable eggs were randomly allocated to 9 hatchers. Pre-treatment egg rinse samples (10 eggs per hatcher) were collected for initial microbial analysis. Three hatchers were treated with CDBP and 3 hatchers with a formaldehyde based product (FBP). Three untreated hatchers served as control (C). Prior to hatch, 10 eggs/incubator, not previously rinsed, were used for post treatment microbial counts. The hatched chicks were reared until d 21 in floor pens with a common starter diet. For the CDBP treated eggs, hatchability and chick performance (weight gains, mortality, and FCR on d 7 and d 21) were similar to the other treatments. The application rate of CDBP evaluated in this study was not an effective antimicrobial alternative to formaldehyde for sanitizing hatching eggs in hatcher cabinets prior to hatch.

Attenuation of a drug-sensitive strain of a turkey protozoan parasite Eimeria meleagrimitis by selection for precocious development.

An attenuated line of Eimeria meleagrimitis was established by repeated propagation of the parasite in 9-day old turkey poults and subsequent selection for precocious development. Following 20 passages, the prepatent period decreased from 120 to 104h. A series of experiments were conducted to evaluate the pathogenicity, immunogenicity and fecundity of the newly selected line. Judged by body weight gain, feed consumption and feed efficiency following infection, the attenuated line had appreciably reduced pathogenicity. Immunogenicity of the attenuated line was examined by infecting poults successively with incremental doses of 10(2), 10(3) and 10(4) oocysts at 0, 7, and 14 days of age respectively. No oocysts were detected following challenge with 5×10(2) oocysts, indicating that the attenuated line had retained immunogenicity. Fecundity was assessed by infecting two-week old birds with 5×10(2) oocysts of either parent or attenuated line. Oocyst production from 96 to 240h post-infection showed that the patent period of the attenuated line commenced earlier and was of shorter duration than the parent line.

Passive immunization with hyperimmune egg-yolk IgY as prophylaxis and therapy for poultry diseases--A review.

Passive immunization with pathogen-specific egg yolk antibodies (IgY) is emerging as a potential alternative to antibiotics for the treatment and prevention of various human and animal diseases. Laying hens are an excellent source of high-quality polyclonal antibodies, which can be collected noninvasively from egg yolks. The use of IgY offers several advantages in that it is environmentally friendly, nontoxic, and reduces the numbers of animals required for antibody production. This paper reviews the use of IgY antibodies in the treatment and prevention of enteric pathogen infections in poultry. Brief descriptions of the production, structure, and properties of IgY are also presented. Some limitations of the technology and future perspectives are discussed.

Molecular detection of field isolates of Turkey Eimeria by polymerase chain reaction amplification of the cytochrome c oxidase I gene.

Oocysts of Eimeria spp. were isolated from litter samples obtained from 30 commercial turkey farms. Genomic DNA was extracted from clean oocysts, and polymerase chain amplification of the species-specific cytochrome c oxidase subunit I (COI) gene was performed for five species of turkey Eimeria. The species tested were Eimeria adenoeides, Eimeria meleagrimitis, Eimeria meleagridis, Eimeria dispersa, and Eimeria gallopavonis. All DNA samples were positive for E. meleagrimitis, nine were positive for E. adenoeides, two were positive for E. dispersa, and none for E. meleagridis and E. gallopavonis. E. meleagrimitis occurred as a single species in 21 (70 %) of the farms while 9 (30 %) farms had a mixed species with E. meleagrimitis and E. adenoeides and 2 (7 %) were triple positive with E. meleagrimitis, E. adenoeides, and E. dispersa. This is the first account of the field prevalence of turkey Eimeria species using molecular methods.

Sericea Lespedeza has no anticoccidial effect when included in the diet of chickens infected with three species of Eimeria.

Anticoccidial effects of Sericea Lespedeza (SL) included in the diet at different levels were evaluated in chickens following oral infection with sporulated oocysts of either Eimeria acervulina, Eimeria maxima or Eimeria tenella. A series of experiments were conducted to determine the effect of SL upon the ability of the parasites to multiply in the intestine, and the effect on bodyweight gain, feed intake, and feed conversion following infection. Chicks infected with a low dose of oocysts (500 oocysts/bird) of E. acervulina or E. maxima did not show differences in the numbers of oocysts produced in the feces whether they were given 0, 1, 2, or 4% SL in the diet. There was no significant difference in the weight gain, feed intake, or FCR of birds infected with high doses of E. acervulina or E. maxima (200,000 or 100,000 oocysts/bird respectively) whether 0, 1, 2, or 4% SL was included in the feed. No significant difference in the numbers of oocysts in the feces, weight gain, feed intake, and FCR of birds infected with E. tenella (low dose of 500 oocysts; high dose of 50,000 oocysts per bird) whether 4% SL was included in the feed. The results of this study indicate that SL has no anticoccidial activity against Eimeria species in the chicken.

Acquisition of immunity to the protozoan parasite Eimeria adenoeides in turkey poults and cellular responses to infection.

Newly hatched turkey poults were infected with 10(2) oocysts of Eimeria adenoeides and subsequently reinfected with 10(3) and 10(4) oocysts at 6 and 12 d of age, respectively. Three peaks in oocyst production were observed in the feces of poults following this series of infections. A second group of poults given the same dosing regimen was challenged with 5 × 10(4) oocysts/poult at different times to evaluate the acquisition of immunity. Judging by weight gain and mortality, no protection had been acquired at 6 d of age, but partial protection was observed by 12 and 18 d of age. A third group of poults were also infected with 10(2) oocysts and subsequently reinfected with 10(3) and 10(4) oocysts at 6 and 12 d of age to evaluate cellular immune responses to infection. Sections of ceca from infected poults showed a significantly higher leukocyte infiltration on d 6, 10, 12, 16, and 18 after infection than uninfected controls. The percent area occupied by CD4+ and CD8+ lymphocytes in the ceca, as assessed by immunohistochemistry, was significantly elevated in infected poults on d 12, 16, and 18. The relative expression of chemokine CXCLi2, and cytokines IL1ß, IFNγ, IL10, IL13, IL2, IL12b, and IL18 was measured by real-time reverse-transcription PCR. The expression of CXCLi2 and IL10 was found to be elevated on d 12, and IFNγ on d 10, 12, and 16. Expression of IL13 and IL18 was increased on d 10 and IL2 on d 10 and 16, and that of IL12b on d 16 in infected poults. Increase in the infiltration of leukocytes, percent area occupied by CD4+ and CD8+ lymphocytes, and changes in the relative expression of cytokines in the ceca characterize the dynamics of immune responses in turkey poults infected with E. adenoeides early in life.

Cellular immune responses, chemokine, and cytokine profiles in Turkey poults following infection with the intestinal parasite Eimeria adenoeides.

Cellular immune responses, chemokine, and cytokine profiles were investigated in 20-d-old turkey poults following an oral infection with 12.5 × 10(3) oocysts of Eimeria adenoeides, a protozoan parasite of the genus Eimeria that develops in the ceca. Large numbers of oocysts were produced in the feces of infected birds from d 5 after infection followed by a rapid decline by d 7. Local immune activities were characterized by observing the extent of leukocyte infiltration in the ceca by histology, measuring subsets of the lymphocyte population by immunohistochemistry, and determining the relative expression of cytokines by real-time, reverse-transcription PCR. Inflammation, assessed by scoring the extent of cellular infiltration of leukocytes in sections of ceca, was significantly higher in infected poults compared with uninfected poults on d 4, 7, 9, and 11 following infection. The percent area occupied by CD4+ and CD8+ cells in the ceca was significantly greater on d 9 and 11 (CD4+) and d 11 (CD8+) in infected poults compared with uninfected controls. The relative expression of the chemokine CXCLi2 and the cytokines interleukin (IL) 1ß, interferon (IFN) γ, IL13, and IL10 was investigated in tissue samples taken from the ceca. Increased expression of CXCLi2 occurred on d 4 and 7. Increased expression of IL10 and IFNγ occurred on d 4 and of IL1ß and IL13 occurred on d 7 postinfection. The increased leukocyte infiltration in the ceca, alterations in the lymphocyte subpopulations, and changes in expression of chemokines and cytokines are an indication of the cell-mediated immune mechanisms occurring in the host as a result of exposure to E. adenoeides.

Acquisition of immunity to the protozoan parasite Eimeria adenoeides in turkey poults and the peripheral blood leukocyte response to a primary infection.

A primary infection of 12.5 x 10(3) oocysts of Eimeria adenoeides , given to 20-d-old turkey poults, resulted in depression of weight gain, and the production of large numbers of oocysts in the feces, compared with uninfected controls. Poults were raised under conditions to prevent possible reinfection to determine the ability of the primary infection to confer protective immunity against a challenge infection of 5 x 10(4) oocysts given at 34 d of age. Using weight gain and oocyst production after challenge as criteria for protection, the results indicated that immunity had developed. The concentration and proportions among white blood cell (WBC) populations in peripheral blood were determined at different times after the primary infection. The WBC concentration of infected poults was elevated on d 7 and 11, primarily due to elevated levels of lymphocytes and monocytes on d 7 and eosinophils on d 11. There were no differences in heterophil and basophil concentrations between infected and uninfected poults at any of the time points examined. With the exception of increased percentages of eosinophils on d 11, infection was not associated with alterations in the proportions among WBC populations. Comparison of CD4+ and CD8+ defined lymphocyte subpopulations in the blood of infected versus uninfected poults revealed higher concentrations of CD4+ lymphocytes on d 11, lower concentrations of CD8+ cells on d 4, and higher concentrations of CD8+ cells on d 11 of infection, as well as elevated ratios of CD4+:CD8+ lymphocytes in infected birds on d 4 and 11. These alterations in WBC profiles after primary E. adenoeides infection in turkey poults suggest initiation of both innate and adaptive cellular immune activities designed to effectively cope with a parasitic, intracellular pathogen.