S. M. Hegazy* and Y. Adachi,1

Transcription

1 Comparison of the Effects of Dietary Selenium, Zinc, and Selenium and Zinc Supplementation on Growth and Immune Response Between Chick Groups that Were Inoculated with Salmonella and Aflatoxin or Salmonella S. M. Hegazy* and Y. Adachi,1 *Department of Animal Nutrition, Faculty of Veterinary Medicine, Tanta University, Kafr El-Sheikh, Egypt, and Animal Health Laboratory, School of Agriculture, Ibaraki University, Ami-cho, Ibaraki , Japan ABSTRACT The effects of four diets (basal diet, Se, Zn, enriched diets fed to the T1 and T2 groups. The antibody and Se- and Zn-enriched diets) fed to chicks that were administered one of three treatments [Salmonella and aflatoxin inoculation (T1), Salmonella inoculation (T2), or uninoculated (T3)] were investigated for growth and immune responses. We found a significant improvement in growth performance represented by relative body gain (RBG) and feed efficiency (FE), for the Zn- and Se + Znimmune response was significantly improved for the Se enrichment diet in the T1 and T2 groups. The weight of the bursa and thymus, which relate to the level of the immune response, showed significant decreases, whereas the spleen had a significantly increased relative weight (RW) in the T1 group. The variable dietary trace elements supplement increased the thymic RW in the T2 group. (Key words: selenium, zinc, aflatoxin, salmonella, chick) 2000 Poultry Science 79: INTRODUCTION The effect of aflatoxins on poultry with infectious agents are obvious yet enigmatic in many regards. Aflatoxin reduces resistance to paratyphoid infections (Wyatt and Hamilton, 1975), infectious bursal disease (Chang and Hamilton, 1982), Marek s disease (Edds et al., 1973), and Emeria tenella (Wyatt et al., 1975). Antibody titers formed during the primary immune response were decreased in fowl cholera infection during aflatoxicosis (Hegazy et al., 1991), and peak titer was delayed in a dose-related fashion in chickens (Thaxton et al., 1974). The production of immunoglobulins is depressed by aflatoxins in otherwise healthy chickens (Giambrone et al., 1978). Newberne et al. (1987) and Lei et al. (1990) have reported the ameliorating effect of Se supplementation on aflatoxin toxicity and carcinogenicity. Less extensive investigations have been done on the effect of other trace minerals on aflatoxin toxicity and carcinogenicity. There are no data available on the effect of trace mineral supplementation immunity against Salmonella infections during aflatoxicosis. Recently, Larsen et al. (1997) reported the effects of dietary Se on the response of stressed and unstressed chickens to an Escherichia coli challenge and antigens. Their data indicate that the incidence of death or lesions Received for publication April 26, Accepted for publication September 30, To whom all correspondence should be addressed: adachi@ipc. ibaraki.ac.jp. is reduced from 86 to 21% at the optimal dose of Se (0.4 mg/kg, resulting in a feed concentration of 0.45 mg/kg). Furthermore, the dietary addition of Se of between 0.1 and 0.8 mg/kg resulted in an antibody titer increase from 2.2 to 3.9 log 2 against sheep erythrocytes. After the chickens were stressed by chilling, Se was ineffective against E. coli. The antibody titer response was reduced from 4.9 to 2.4 log 2 against sheep erythrocytes. Larsen et al. (1997) speculated that this reduction could be prevented with dietary additions of Se between 0.1 and 1.2 mg/kg. However, Lesourd (1997) speculated that supplementation with a high pharmacological dose of a single nutrient (Zn or vitamin E) might be useful in improving the immune responses of self-sufficient elderly people living at home. Therefore, we focused on the influence of the micronutrients Zn and Se. The objectives of the current study were to determine whether dietary supplements of Se, Zn, or Se + Zn would enhance the immunity against chick salmonellosis and alleviate the suppressing effect of aflatoxin on the immune response and growth performance during Salmonella infection. MATERIALS AND METHODS Chickens and Feed One-day-old specific pathogen-free White Leghorn chicks were used in this experiment. The birds were allo- Abbreviation Key: FE = feed efficiency; NA = nalidixic acid; RBG = relative body gain; RW = relative weight. 331

2 332 HEGAZY AND ADACHI cated to 12 groups of two replicates; each group contained 12 chicks. Each chick in each group was individually marked with phenol red and brilliant green solubilized with ethanol. The chicks were individually weighed and assigned to electrically heated, isolated pens of a battery brooder under continuous illumination by fluorescent light. The pens, which were in a plastic isolator (a device for filtering the air the chicks breathed) and the experimental animal room were sterilized by formalin fumigation before use. Feed and water were given ad libitum throughout the 15-d trial. The basal diet was a commercial starter ration 2 containing 22% crude protein, 3,000 kcal ME, 0.15 ppm Se, and 40 ppm Zn and was without any drugs. Four variable diets were used during the present experiment: 1) Se- and Zn-fortified diet (basal diet supplemented with 1 and 60 ppm of Se and Zn, respectively), 2) Se-fortified diet (basal diet supplemented with 1 ppm of Se 3 ), 3) Zn-fortified diet (basal diet supplemented with 60 ppm of Zn 4 ), and 4) the basal diet (control). Feed consumption was determined daily for each group by subtracting the amount of the estimated feed that had been consumed by dead birds before they died. Bacteria and Infection Salmonella typhimurium L , which was sensitive to nalidixic acid (NA), was previously isolated from a chick. In this experiment, an NA-resistant mutant derived from this organism was used, allowing easy detection of Salmonella in feces with a selective medium containing NA. For specific pathogen-free chicks, the other organisms were never grown on selective medium with NA. The NAresistant mutant was prepared as follows. The NA-sensitive organism was grown in 200 ml trypticase soy broth 6 at 37 C for 18 h, centrifuged at 5,000 rpm for 20 min, spread on DHL agar 7 containing 25 µg NA/mL, and incubated at 37 C for 18 h. One colony growing on the agar plate with NA was taken, subcultured three times, and grown in trypticase soy broth at 37 C for 18 h. The culture was diluted 1/100 and used as inoculum. Then, 0.5 ml of the inoculum was intragastrically inoculated into each chick on Day 5 after feeding of each of the fortified feeds. The inoculum contained cells/0.5 ml. Aflatoxin and Inoculation Aflatoxin B 1 8 was solubilized with dimethyl sulfoxide and was used for the induction of aflatoxicosis by intraperitoneal injection at a dose of 2.6 mg/kg of body weight on Day 3 after inoculation with Salmonella. To prevent a human hazard during feeding, we chose to inject the aflatoxin B1 into the peritoneal cavity. 2 All Japan Agriculture Coop., Chiyoda-ku, Tokyo, Japan. 3 Wako Pure Chemical Industries, Chyuou-ku, Osaka, Japan. 4 Kanto Chemical Co., Chuou-ku, Tokyo, Japan. 5 National Institute of Animal Health, Tsukuba, Ibaraki, Japan. 6 Becton Dickinson, Cockeyville, MD Eiken, Bunkyou-ku, Tokyo, Japan. 8 Sigma Chemical Co., St. Louis, MO TABLE 1. Effect of Se, Zn, or Se + Zn dietary enhancement on relative body gain (RBG) and feed efficiency (FE) of chicks during Salmonella or Salmonella and aflatoxin inoculation 1 Chick group parameters 2 RBG FE RBG FE RBG FE RBG FE T ± 3.60 b,y 0.60 ± 0.04 b,x ± 8.25 a,x 0.53 ± 0.11 a,b,y ± a,b,y 0.56 ± 0.11 a,b,y ± a,y 0.46 ± a,y T ± 8.70 b,x,y 0.64 ± 0.08 a,x ± 7.61 a,y 0.60 ± 0.08 a,x ± 7.60 b,x,y 0.64 ± 0.14 a,x ± 8.12 a,y 0.62 ± 0.11 a,x T ± 5.25 a,x 0.66 ± 0.06 a,x ± 5.10 a,x 0.67 ± 0.05 a,x ± 5.18 a,x 0.68 ± 0.15 a,x ± 5.44 a,x 0.65 ± 0.09 a,x a,b Values within rows with no common superscript differ significantly (P < 0.05). x,y Values within columns with no common superscript differ significantly (P < 0.05). 1 Values are mean ± SD. 2 T1 = Salmonella and aflatoxin inoculated, T 2 = Salmonella inoculated and T 3 = uninoculated.

3 EFFECT OF SELENIUM AND ZINC TO AFLATOXICOSIS AND SALMONELLOSIS 333 TABLE 2. Reisolation rate of Salmonella typhimurium from cloaca swabs of the chicks on Days 2, 4, and 8 postinoculation Chick 1 Chicks, (d) group no T (2) (2) 9(1) (1) T (2) (1) T T1 = Salmonella and aflatoxin inoculated, T2 = Salmonella inoculated, and T3 = non-inoculated. 2 Number of living chicks used for Salmonella reisolation. 3 Values in parenthesis indicate the number of dead chicks. Experimental Design A completely randomized 4 3 factorial design was used to study the effect of the trace-element dietary supplement on the three chick treatments: aflatoxin and Salmonella infection (T1), Salmonella infection (T2), and no infection (T3). Each chick treatment contained two replicates of four groups of chicks. One of the four experimental diets was fed to a group in each chick treatment. On Day 5 after feeding one of the four variable diets, S. typhimurium was inoculated into the chicks. On Days 2, 4, and 8 after inoculation, cloaca swabs were taken using a cotton swab and were spread on DHL agar containing 25 µg NA/mL. On Day 15 after the start of the experimental diets, all living chicks were individually weighed, bled, and then necropsied. The bursa of Fabricious, thymus, and spleen were weighed, and their RW/100 g was calculated. The RBG and FE were determined (Brody, 1945). Serum was obtained and kept at 4 C before use. The serum was serially diluted twofold from 1/10 to 1/320 for a serum agglutination test. The agglutination titers were expressed as the geometric mean of the reciprocal of the maximum dilution of the serum to give complete agglutination. Statistical Analysis Analysis of variance (Snedecor and Cochran, 1989) was used for data analysis. Data obtained as percentage were TABLE 3. Effect of Se, Zn, or Se + Zn dietary enhancement on geometric mean of agglutination titers against Salmonella typhmurium during Salmonella or Salmonella and aflatoxin inoculation Chick group 1 T ;a,b,x b,z a,b,x a,x T a,y b,y a,y a,y T a,x a,x a,x 6.80 a,x a,b Values within rows with no common superscript differ significantly (P < 0.05). x,y,z Values within columns with no common superscript differ significantly (P < 0.05). 1 T 1 = Salmonella and aflatoxin inoculated, T 2 = Salmonella inoculated, and T 3 = noninoculated. 2 Agglutination titers were expressed as the geometric mean of the reciprocal of maximum dilution of the serum to give complete agglutination. subjected to arc sine transformation prior to statistical analysis. All statements of significance were based on the probability of no difference between means being less than P = RESULTS Table 1 shows the effect of the dietary treatments on RBG and FE during the experiment period. A significant improvement in RBG in the Zn and Se + Zn dietary enrichment groups was observed in the T1 and T2 groups. The RBG was significantly decreased with all diets fed to the T1 groups and with basal and Se-supplemented diets fed to the T2 groups, as compared with the T3 groups. No significant effect of the diets was observed in RBG for the T3 groups. A significant improvement in FE was confirmed in the Se + Zn-supplemented diet groups, as compared with those of the other groups in the T1 treatment. A significant decrease in FE was observed among groups with basal, Se-, or Zn-fortified diets in the T1 treatment, as compared with the T2 and T3 treatments. However, the FE data demonstrated no significant differences among the three treatment groups for the Se + Zn-enrichment diet. Table 2 shows the rate of reisolation of Salmonella from the cloaca of the chicks that were inoculated. All of the chicks harbored Salmonella in the T1 and T2 groups. There were no significant differences among these groups. Table 3 shows the serum agglutination titers for the Salmonella-inoculated chick treatments for the evaluation of the effects of the variable dietary supplementation. The antibody response to the infection with Salmonella was significantly lower in the T1 group, as compared with the T2 group, for all dietary treatments. However, the response in the T1 group was higher than that in T3 group. Furthermore, there was a significant increase in the agglutination titer in the T1 and T2 groups with the Se-fortified diet as compared with the other diets in the same chick treatment. Table 4 shows the RW of bursa, thymus, and spleen. The RW of all the organs relating to immunity demonstrated a significant difference between the groups in T1 and those of the other two treatments. In the T1 group, bursal and thymic RW were decreased, but the spleen RW was increased. The RW of these organs between dietary

4 334 HEGAZY AND ADACHI TABLE 4. Effect of Se, Zn, or Se + Zn dietary enhancement on bursa of Fabricious (F), thymus (T), and spleen (S) relative weight during Salmonella or Salmonella and aflatoxin inoculation 1 Chick group 2 F T S F T S F T S F T S T ± a,y ± a,y ± a,y ± a,y ± a,y ± a,y ± a,y ± a,y ± a,y ± a,y ± a,y ± a,y 2 T ± a,x ± b,x ± a,x ± a,x ± b,x ± a,x ± a,x ± b,x ± a,x ± a,x ± a,x ± a,x 3 T ± a,x ± a,x ± a,x ± a,x ± a,x ± a,x ± a,x ± a,x ± a,x ± a,x ± a,x ± a,x a,b Values within rows with no common superscript differ significantly (P < 0.05). x,y Values within columns with no common superscript differ significantly (P < 0.05). 1 Values are mean ± SD. 2 T1 = Salmonella and aflatoxin inoculated, T 2 = Salmonella inoculated, and T 3 = uninoculated. treatment groups were not different within each chick treatment. DISCUSSION There was a significant toxic effect of aflatoxin in all of the chick groups of T1 with respect to RBG and FE. Regardless of Zn or Se supplementation, there was a significant decrease in RBG and FE due to aflatoxin in the T1 groups compared with the T3 groups. Such results have been previously reported by Campbell et al. (1983) and Miller and Wilson (1994). The RBG was significantly decreased by Salmonellosis in the T2 chick groups when they were fed the basal or Se-fortified diets, relative to the uninfected chick treatment groups. The Zn-fortified and Se + Zn-fortified diets resulted in significant improvement in RBG and FE in chicks exposed to either Salmonella inoculation only, or the combination of Salmonella and aflatoxin inoculation, relative to the other groups of the same chick treatment. This result may be due to an increased plasma concentration of insulin-like growth factor-1, which depends mainly on an adequate level of Zn in the blood. This level may be altered by the Salmonella infection and aflatoxin treatments. Heindl et al. (1993) concluded that the effect of Zn deficiency reduced the level of plasma insulin-like growth factor-1, which may have been responsible for the retarded growth. The effect of infectious stress on plasma Zn concentration has been studied by Chirase et al. (1994). They found that the body weight and plasma Zn concentration decrease, usually observed with infectious bovine rheinotrachitis virus stress, did not occur when a free-choice dietary Zn supplement was available. The pattern of the agglutination titer clearly demonstrated the immunosuppressive effect of aflatoxin, suggesting that the aflatoxin is an immunosuppressive agent. Hegazy et al. (1991) has reported a lag or decrease in the antibody titer response (agglutinin and hemagglutinin) or a decrease in certain Ig responses (IgG and IgA). Moreover, the results of the present experiment show a significant improving effect of the Se-fortified diet on the level of the agglutination titer in the T1 and T2 groups as compared with that of the other diets in the respective chick treatments. Panda and Rao (1994) reported that there was an enhancement of the immune response following Se and vitamin E supplementation in chicks infected with infectious bursal disease virus. Also, Se had a significant effect on the proliferative response to antigens in an in vitro lymphocyte function evaluation (Pollock et al., 1994). The aflatoxin had an immunosuppressive effect on the bursa and thymus, the primary initiators of immunity in chicks, which resulted in their significant RW decrease. This decrease may correlate with the finding of a low antibody titer in the T1 groups. Campbell et al. (1983) found that the RW of bursa and its follicle count were reduced by aflatoxin. Bursal and thymic RW were significantly reduced, depending on the dose of aflatoxin (Thaxton et al., 1974; Pier et al., 1979). The thymic RW increase

5 EFFECT OF SELENIUM AND ZINC TO AFLATOXICOSIS AND SALMONELLOSIS 335 in groups consuming the diets enriched for Se, Zn, or both in the T2 treatment, relative to that of the respective basal diet, indicated that these elements have a significant effect during Salmonella infection. Larsen et al. (1997) reported that dietary addition of Se between 0.1 and 0.8 mg/kg resulted in an antibody titer increase from 2.2 to 3.9 log 2 against sheep erythrocytes in White Leghorn type chickens, whereas after stress by chilling, antibody titer response was reduced from 4.9 to 2.4 log 2. Furthermore, they found that the incidence of death or lesions was reduced from 86 to 21% at the dose of 0.4 mg Se. They speculated that this titer reduction could be prevented with dietary additions of Se between 0.1 and 1.2 mg/kg. Contrary to the effects on the bursa and thymus, the spleen RW in Salmonella infected chicks was significantly increased following aflatoxin inoculation. This response may be due to salmonellosis. Chang and Hamilton (1982) found a significant increase in spleen RW in the chicks with infectious bursal disease virus, which may be enhanced by synergism with aflatoxicosis. These data strongly suggest that Zn status is an important determinant of growth performance. However, Se is also useful for the improvement of immune responses during salmonellosis, with or without aflatoxicosis. REFERENCES Brody, S., Bioenergetic and Growth, Reinhold Publ., Crop, New York, NY. Campbell, M. L., J. D. May, W. E. Huff, and J. A. Doer, Evaluation of immunity of young broiler chicken during simultaneous aflatoxicosis and ochratoxicosis. Poultry Sci. 62: Chang, C., and P. B. Hamilton, Increased severity and new symptoms of IBD during aflatoxicosis in broiler chicken. Poultry Sci. 61: Chirase, N. K., D. P. Hutcheson, G. B. Thompson, and J. W. Spears, Recovery rate and plasma zinc and copper concentration of steer calves fed organic and inorganic Zn and Mn sources with or without injectable Cu and challenged with infectious rhinotracheitis virus. J. Anim. Sci. 72: Edds, G. T., K.P.C. Nair, and C. F. Simpson, Effect of aflatoxin B1 on resistance in poultry against cecal coccidiosis and Marek s disease. Am. J. Vet. Res. 34: Giambrone, J. J., D. L. Ewert, R. D. Wyatt, and C. S. Eideson, Effect of aflatoxin on humoral and cell mediated immune system of chicken. Am. J. Vet. Res. 39: Hegazy, S. M., A. Azzam, and M. A. Gabal, Interaction of naturally occurring aflatoxin in poultry feed and immunization against fowl cholera. Poultry Sci. 70: Heindl, U., M. Kirchgessner, and D. Scams, The effect of zinc deficiency and application of recombinant bovine growth hormone on plasma growth hormone and insulinlike growth factor-1 of calves. J. Anim. Physiol. Anim. Nutr. 70: Larsen, C. T., F. W. Pierson, and W. B. Gross, Effect of dietary selenium on the response of stressed and unstressed chickens to Escherichia coli challenge and antigen. Biol. Trace Elem. Res. 58: Lei, D. N., L. Q. Wang, B. H. Rubner, D.P.H. Hsieh, B. F. Wa, C. R. Zhu, and M. J. Du, Effect of Se on aflatoxin hepatocarcinogenesis in rat. Biomed. Environ. Sci. 3: Lesourd, B. M., Nutrition and immunity in the elderly: Modification of immune response with nutritional treatments. Am. J. Clin. Nutr. 66: Miller, D. N., and D. M. Wilson, Page 347 in: The Toxicology of Aflatoxin. Acad. Press Inc., Tokyo, Japan. Newberne, P. M., P. Punyarit, J. Decamargo, and V. Suphakakarn, The role of necrosis in hepatocellular proliferation and tumors. Arch. Toxicol. Suppl. 10: Panda, S. K., and A. T. Rao, Effect of a vitamin E-selenium combination on chickens infected with infectious bursal disease virus. Vet. Rec. 134: Pier, A. C., J. L. Richard, and J. R. Thruston, The influence of mycotoxins on resistance and immunity. Pages in: Interactions of Mycotoxins in Animal Production. Natl. Acad. Sci., Washington, DC. Pollock, J. M., J. McNair, S. Kennedy, D. G. Kennedy, D. M. Walsh, E. A. Goodall, D. P. Mackie, and A. D. Crockard, Effects of dietary vitamin E and selenium on in vitro cellular immune responses in cattle. Res. Vet. Sci. 56: Snedecor, G. W., and W. G. Cochran, Statistical Methods. 8th ed. Iowa State Univ. Press, Ames, IA. Thaxton, J. P., H. T. Tung, and P. B. Hamilton, Immunosuppression in chicken by aflatoxin. Poultry Sci. 53: Wyatt, R. D., and P. B. Hamilton, Interaction between aflatoxicosis and natural infection of chickens with Salmonella. Appl. Microbiol. 30: Wyatt, R. D., M. D. Ruff, and R. K. Page, Interaction of aflatoxin with Emeria tenella infection and monensin in young broiler chickens Avian Dis. 19: