Christopher Marcq

Effects of feed supplementation with specific hen egg yolk antibody (immunoglobin Y) on Salmonella species cecal colonization and growth performances of challenged broiler chickens

Anti-Salmonella spp. egg yolk antibodies (IgY) simultaneously directed against Salmonella Enteritidis and Salmonella Typhimurium were tested to determine if their inclusion in feed decreased Salmonella spp. cecal colonization in experimentally infected broiler chickens. Egg yolk powder (EYP) was obtained by freeze-drying egg yolks containing anti-Salmonella spp. Immunoglobin Y was included in feed at 5 levels of concentration (0 to 5%). Feeds were formulated to similar nutrient levels and provided for ad libitum intake from d 1 to 28. Three days after initiation of feed treatments (d 4), chickens were co-challenged with equal numbers of Salmonella Enteritidis and Salmonella Typhimurium (2x10(6) cfu/bird). Cecal samples were recovered weekly over the experimental period (d 7 to 28) to enumerate Salmonella spp. The effect of anti-Salmonella spp. IgY feed supplementation on growth performance of infected chickens was also evaluated during the same period. In comparison with the positive control treatment (PC), treatments involving EYP (T1, T2, T3, T4, and T5), whether containing anti-Salmonella spp. IgY or not, significantly improved (P<0.05) the growth performance of challenged chickens, but without reaching the performance levels of nonchallenged chickens (NC1 and NC2). However, no link can be established between the enhancement in growth performance of challenged birds and their contamination levels by Salmonella because in-feed incorporation of EYP had no significant effect on cecal colonization by Salmonella. Furthermore, the comparison of the 5 anti-Salmonella spp. IgY concentration levels in feed did not reveal any anti-Salmonella spp. IgY concentration effect on growth performance and Salmonella cecal colonization. These results suggest that anti-Salmonella spp. IgY would undergo denaturation and degradation after their passage through the animal gastrointestinal tract and reveal that components of EYP other than specific antibodies have a beneficial effect on growth performance.

Salmonella Typhimurium oral challenge model in mature broilers: Bacteriological, immunological, and growth performance aspects

In this study, Salmonella enterica serovar Typhimurium challenge models were tested to identify the best conditions under which to perform the experimental infection of 3-wk-old broilers. Such a model would be useful to study the efficiency of therapeutic treatments that could take place at the end of the grow-out period. Salmonella-free chicks were obtained from a breeder flock vaccinated with Salmonella. Intestinal maternal immunity was monitored by ELISA analyses at 2, 9, and 16 d of age. Data indicated that protection of maternal origin was not maintained over time and was drastically reduced at 9 d of age (P < 0.01). At 21 d of age, chickens were orally inoculated with Salmonella Typhimurium. The effects of the oral challenge dose (0, 3 × 10(3), 3 × 10(6), and 3 × 10(9) cfu/bird) and vancomycin pretreatment (no administration or 25 mg/bird) on intestinal immune responses, growth performance, and Salmonella colonization of chickens were investigated. After infection, the mucosal immune response was rapid, with increased (P < 0.01) anti-Salmonella Typhimurium IgA titers measured at 8 d postinfection in intestinal contents. A linear relationship (P < 0.05) existed between specific IgA levels in intestinal and cecal contents and the challenge dose inoculated. None of the challenge protocols caused mortality or clinical symptoms after infection. Nevertheless, the experimental infection induced a significant deterioration of growth performance. The pretreatment with 25 mg of vancomycin at 3 h before Salmonella inoculation was able to establish stable infection rates among the population of 3-wk-old infected chickens. Nevertheless, Salmonella shedding was not stable over the rearing period, and the bacteria seemed to be naturally eliminated from most birds at 22 d postinfection. This natural clearance of the gut, which was related, at least in part, to the intestinal immune response, should limit the usability of the created mature challenge model within 1 to 2 wk after inoculation.

Adhesion and Growth Inhibitory Effect of Chicken Egg Yolk Antibody (IgY) on Salmonella enterica Serovars Enteritidis and Typhimurium In Vitro

The protective effects of powder preparation of egg yolk immunoglobulin Y (IgY), specific to Salmonella Enteritidis and Salmonella Typhimurium outer membrane proteins (OMP), against these two Salmonella sp. serovars were investigated in vitro in two different assays: adhesion-prevention and growth-inhibition. The adhesion-prevention assay was conducted using polarized monolayers of the human intestinal epithelial Caco-2 cell line. First, the conditions of Salmonella adherence to Caco-2 cells were optimized, and interferences of bacteria with the transepithelial electrical resistance (TER) of fully differentiated Caco-2 cell monolayers and the lactate dehydrogenase release upon exposure of the cells to Salmonella were evaluated. Both Salmonella sp. serovars were able to adhere to Caco-2 cells and decreased TER. Results from the adhesion-prevention assay demonstrated that specific IgY reduced the decrease in TER of the infected Caco-2 cell monolayers and blocked the Salmonella sp. adhesion in a concentration-dependent manner (p < 0.05). Nonspecific IgY also exhibited an inhibitory effect on these two parameters, but to a lesser extent than that of the specific IgY (p < 0.05). The protective effect of nonspecific IgY could be attributed to the low-density lipoprotein component of the water-soluble fraction of egg yolks that may not have been eliminated during ultrafiltration. The growth-inhibition assay revealed that specific IgY had an inhibitory effect on the bacterial growth, markedly during the late exponential phase, whereas nonspecific IgY failed to do so. Taken together, these results suggest that the in vitro growth inhibitory effect of specific IgY on Salmonella spp. resulted from the specific binding activity of these IgY to Salmonella sp. OMP. Passive immunization with Salmonella sp. OMP-specific IgY could thus be useful to prevent Salmonella colonization in broiler chickens and the subsequent carcass contamination during processing.

Improving adjuvant systems for polyclonal egg yolk antibody (IgY) production in laying hens in terms of productivity and animal welfare

The antibody production in the egg yolks of immunized laying hens is seen as a way of improving animal welfare compared with conventional production by mammals. Immunoglobulin Y (IgY) technology, however, has still to address welfare issues linked to the widespread use of an adjuvant in vaccines. Currently, Freunds adjuvants, complete (FCA) or incomplete (FIA), remain the standard. This study sought to evaluate various approaches used to enhance egg yolk antibody production in terms of both productivity and avian welfare. The outer membrane protein (OMP) of Salmonella Typhimurium was used as the prototype antigen. At 20 weeks of age, 56 ISA Brown hens, with specific-Salmonella-free status, were divided into seven groups (n=8) and received an initial intramuscular immunization. Hens in the two negative control groups received phosphate buffered saline (PBS) or FIA alone. Hens in the other groups received 80μg of Salmonella OMP emulsified with one of the following adjuvants: 200μl of FIA alone (T1); 200μl of FIA supplemented with 8μg of C-phosphate-guanosine oligodeoxynucleotides (CpG-ODN) (T2); and 280μl of Montanide ISA 70 VG (T4). Birds in the T3 group received the antigen in emulsion with FIA and were given the tested immunostimulatory component (l-carnitine) via their feed (100mg/kg). A positive control group (PC) received FCA for the first and final immunizations and FIA for the other boosters. Immunization was repeated after 20, 46, 82 and 221 days. Eggs were collected regularly until 242 days after the first immunization and the anti-Salmonella Typhimurium activities in the yolk were determined by ELISA. After 242 days, the birds were euthanized and the injection sites were evaluated for gross and microscopic lesions. Among the tested immunostimulatory approaches, supplementation of FIA with CpG-ODN led to a significant and long-lasting enhancement of the specific antibody response. This treatment was even higher than the positive benchmark using FCA in the first immunization. The study results showed that a clinical examination of injection sites is insufficient for drawing conclusions about the local tolerance of vaccines. Tissue damage was noticeable in all treatment groups. The birds receiving the Montanide adjuvant, however, had fewer and less severe lesions. Given these limited side-effects, Montanide ISA 70 VG could provide the depot effect needed to ensure the immunomodulatory efficiency of CpG-ODN. The association of these two adjuvants could prove a promising alternative to Freunds adjuvants (FA).