Enhanced mucosal immunity against Eimeria acervulina in broilers fed a Lactobacillus-based probiotic.

The effect of feeding a Lactobacillus-based probiotic on intestinal intraepithelial lymphocyte (IEL) subpopulations and subsequent protection against coccidiosis was investigated in broiler chickens. Day-old male broilers were fed standard rations without control (CONT) or with a commercial probiotic (PROB) Primalac. Differences in IEL subpopulations were assessed by flow cytometry at 21 d postprobiotic treatment. At 25 d of age, a group of randomly selected birds from each diet was inoculated orally with 10,000 (per bird) sporulated oocysts of Eimeria acervulina and kept on the same diets. Fecal material, sera, and intestinal washes were collected 10 d postchallenge with E. acervulina. Birds on the PROB diet had more IEL expressing the surface markers CD3, CD4, CD8, and alphabetaTCR than those of the CONT diet. The probiotic-fed chickens produced less oocysts (P < 0.0001) compared to the untreated, control group (368 x 10(6) in CONT vs. 89 x 10(6) in PROB). The interferon-gamma levels in both serum and intestinal secretions were not significantly different between the two groups. However, CONT group showed higher antibody levels against a recombinant coccidial antigen in the intestinal secretions than the PROB group. No significant difference was found in serum antibody levels against the same antigen. These results dearly indicate that the probiotic bacteria impacted the local immune response as characterized by altered IEL subpopulations and increased the birds' resistance to E. acervulina as reflected by reduced oocyst shedding.

Effect of vitamin A deficiency on host intestinal immune response to Eimeria acervulina in broiler chickens.

The effects of vitamin A (VitA) deficiency on the host intestinal immune response and disease susceptibility to coccidiosis were investigated in broiler chickens following oral infection with Eimeria acervulina (EA). Day-old male broilers were fed milo-soybean meal diets either with 8,000 IU VitA/kg feed (CONT) or without added VitA (A-DEF). At 25 d, a group of randomly selected birds from each treatment was inoculated orally with EA-sporulated oocysts. Intestinal immune response was assessed by the changes in the duodenum intraepithelial lymphocyte (IEL) subpopulations using flow cytometry at 35 d in in fected and noninfected birds. Concanavalin A (ConA)-induced spleen lymphocyte proliferation was tested using dimethylthiazol diphenyltetrazolium bromide colorimetric assay. Whether challenged or not with EA, A-DEF birds had fewer IEL expressing the surface markers CD3, CD4, CD8, alphabetaTCR, and gammabetaTCR. Without EA challenge, A-DEF birds had more surface IgA-expressing cells than CONT birds. Upon challenge, A-DEF chickens showed lower CD4+ IEL than CONT chickens. Following EA infection, CD8+ IEL increased in the CONT group, whereas no change was found in CD8+ IEL of A-DEF birds. A higher number of EA oocysts was recovered from A-DEF birds than from CONT birds (9.2 x 10(8) vs 5.4 x 10(8), respectively; P < or = 0.05). Serum samples taken 10 d post challenge showed higher antibody level against a recombinant coccidial antigen in A-DEF birds than in CONT birds. The A-DEF birds showed depressed ConA-induced lymphoproliferation response and produced lower serum interferon-gamma than CONT birds. These data show that VitA deficiency compromised local immune defenses of challenged birds, as reflected in lymphocyte profiles, oocyst shedding, and interferon-gamma levels in A-DEF birds.

Effectiveness of different types of clay for reducing the detrimental effects of aflatoxin-contaminated diets on performance and serum profiles of weanling pigs.

Three trials were conducted with recently weaned pigs (n = 198) to determine the effects of feeding different types of clay in conjunction with aflatoxin-contaminated diets. In Trial 1, pigs (n = 54; trial length 4 wk) were assigned to either an uncontaminated treatment (NC), 800 ppb of aflatoxin from contaminated corn (AC), or AC with one of four clays. In Trial 2 (n = 81; trial length 5 wk), pigs were assigned to NC, AC (500 ppb of aflatoxin from rice starch), or AC with one of seven types of clay. In both trials, pigs fed AC had decreased ADG and gain:feed ratios (P < .05) compared with controls. The clays differed in their ability to produce gains similar to those of controls. The clays did reduce changes in the serum measurements normally affected by aflatoxin, including albumin, total protein, gamma glutamyltransferase (GGT), and alkaline phosphatase (ALP) levels, in a manner similar to their effect on ADG. In Trial 3, pigs (n = 63) were assigned to one of seven diets for 4 wk: NC, AC (800 ppb of aflatoxin) with no clay, AC with one of four levels of a treated Ca bentonite (.25, .5, 1, and 2%), or AC and .5% hydrated sodium calcium aluminosilicate. The addition of treated Ca bentonite to AC improved ADG (P < .05) and ADFI (P < .01) linearly. Gain:feed ratios were not affected by treatments. The inclusion of treated Ca bentonite to the AC diet linearly decreased aspartate aminotransferase (AST) levels and quadratically decreased ALP and GGT levels (P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Mycotoxin interactions in poultry and swine.

Mycotoxins are toxic compounds produced by fungi. When one mycotoxin is detected, one should suspect that others also are present in a contaminated feed ingredient or finished feeds. The toxicity and clinical signs of observed in animals when more than one mycotoxin is present in feed are complex and diverse. Some mycotoxins, such as the combination of aflatoxin with either ochratoxin A or T-2 toxin, interact to produce synergistic toxicity in broiler chicks. The effects observed during multiple mycotoxin exposure can differ greatly from the effects observed in animals exposed to a single mycotoxin. For example, fatty livers in poultry are used for presumptive diagnostic identification of aflatoxicosis. However, simultaneous presence of ochratoxin A prevents fatty livers. Of the mycotoxin combinations that have been investigated in poultry and swine, the aflatoxin + ochratoxin A and aflatoxin + T-2 toxin interactions appear to be the most toxic.

Increased sensitivity to staphylococcal beta hemolysins of erythrocytes from chickens during aflatoxicosis.

The size of the zones of beta-hemolysis surrounding staphylococcal colonies on blood agar was found to be related to the level of dietary aflatoxin consumed by the chickens donating the blood. Zone sizes on blood from chickens fed the highest level of aflatoxin (10 micrograms/g of diet) were about six-fold larger than those on blood from control birds. The percentage of staphylococcal isolates displaying beta-hemolysis was increased from about 15% in normal blood to about 90% in blood from chickens fed aflatoxin (10 micrograms/g) whereas the time required for detection of beta-hemolysis was decreased by about one-half. The hemolytic activity of purified staphylococcal beta-hemolysin against suspensions of washed erythrocytes increased as the level of aflatoxin consumed by the donor chickens increased. These data imply a new mechanism for enhanced susceptibility of animals to infectious agents during mycotoxicoses whereby the animal is made more sensitive to the virulence factors of pathogens.

Age-related changes in testicular development and reproductive endocrinology associated with aflatoxicosis in the male chicken.

A study was conducted to investigate endocrine and testicular changes in the male chicken associated with the ingestion of 10 or 20 ppm aflatoxin at 3 different stages of development. Weekly body weight gain, absolute and relative combined testes weights, and plasma testosterone concentrations were reduced in aflatoxin-fed males as compared to controls, with the greatest differences seen at 12 wk of age. The effect of dietary aflatoxin on levels of plasma luteinizing hormone (LH) was dependent on age at exposure. Concentrations of plasma LH in 6-wk-old control males were significantly higher than in aflatoxin-fed birds, whereas no treatment differences were observed in older males. Additionally, few changes were observed in static levels of monoamines in any of the brain regions assayed, regardless of age at exposure. The delay in peak levels of LH, as well as the suppression of plasma testosterone and testicular weight, indicate a possible delay in the onset of sexual maturation associated with aflatoxicosis in this species.

Relative importance of dietary aflatoxin and feed restriction on reproductive changes associated with aflatoxicosis in the maturing White Leghorn male.

A study was conducted to investigate the relative contribution of dietary aflatoxin and feed restriction on reproductive and endocrine changes associated with aflatoxicosis. Pair-feeding was used to factor out nutritional and toxicological effects of aflatoxin on reproduction in the maturing White Leghorn male. Birds were randomly assigned to treatment groups at 9 weeks and fed experimental diets for 3 weeks. Experiment 1 involved the partitioning of toxicological and nutritional effects of aflatoxin on both physiological and reproductive parameters. Aflatoxin-fed males had significantly greater relative liver weights and liver lipid content than control or pair-fed males; no differences in testicular weights were found among treatments. Plasma testosterone levels in controls were significantly higher than in pair-fed birds; aflatoxin-treated males exhibited the lowest concentrations. Pituitary and testicular responsiveness to synthetic luteinizing hormone-releasing hormone (LHRH) were monitored in Experiment 2. Plasma luteinizing hormone (LH) levels in aflatoxin-fed males were significantly higher than in control or pair-fed males prior to LHRH administration. Additionally, the LH response of pair-fed birds to exogenous LHRH was qualitatively different from that observed in the other two treatments; this indicates that hormonal changes observed during aflatoxicosis and feed restriction are a result of different physiological mechanisms. Plasma testosterone also increased after LHRH injection. This response was significantly greater in controls than in pair-fed males, and nonexistent in aflatoxin-treated birds.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of dietary copper on litter microbial population and broiler performance.

Proprietary broiler diets, containing added copper (125 mg/kg) as sulphate, were fed to broilers in 4 trials (32 pens each) over a one year period without a change of litter. Mould counts in the litter of pens containing birds fed the standard diets decreased to 2 X 10(3) propagules per g in trial 4. Those in the pens with birds fed the diets containing supplemental copper decreased to 6 X 10(2) propagules per g. Litter bacterial counts (10(7) organisms/g) were not affected by dietary copper. Litter copper concentrations in pens where the birds were fed supplemented diets increased significantly to more than 600 mg/kg in trial 4. Dietary copper sulphate addition significantly increased broiler weight gains at 7 weeks in trials 3 and 4 (P less than 0.05) and the efficiency of food utilisation was significantly improved in trial 4. The copper content of the chicks' livers remained unchanged. It is suggested that broiler performance may be independent of dietary copper content. Litter copper concentrations and litter microbial alterations may be important factors.

The individual and combined effects of aflatoxin and ochratoxin A on various processing parameters of broiler chickens.

Male broilers (Hubbard X Hubbard) were placed at hatching into a completely randomized 2 X 2 X 2 factorial experimental design with the treatments consisting of 0 and 2.5 micrograms aflatoxin/g of feed (ppm) and 0 and 2.0 ppm ochratoxin A. In Trial 1, there were four replicate pens of 10 broilers per replicate at each treatment level. At 3 weeks of age two replicate pens were randomly selected and placed on control feed (0 to 3 weeks), the remaining two replicate pens were maintained on treatment (full-term). In Trial 2, there were three replicates of 38 broilers per replicate at each treatment level. At 3 weeks of age 13 broilers from each replicate pen were randomly selected and placed in separate housing and fed control diets (0 to 3 weeks). The remaining 25 broilers were kept on mycytoxin treatments until the conclusion of this trial (full-term). Data collected in these trials indicate that body weights of broilers were significantly (P less than .05) decreased by aflatoxin, ochratoxin A, and the combination treatments of the full term feeding regimen. Broilers partially recovered body weight in the 0 to 3-week feeding regimen; however, recovery was less rapid during ochratoxicosis compared to aflatoxicosis. Body weights of broilers in the combination treatment remained significantly (P less than .05) depressed throughout the trials. As body weight decreased, parts weights decreased accordingly. Carcass yield was significantly (P less than .05) decreased by all treatments, primarily by a decrease in breast yield.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of immunity of young broiler chickens during simultaneous aflatoxicosis and ochratoxicosis.

A 2 X 2 factorial experimental design consisting of the treatments 0 and 2.5 micrograms/g aflatoxin and 0 and 2.0 micrograms/g ochratoxin A with 12 replicates of 10 birds per treatment level was used to evaluate the effects of these mycotoxins on various aspects of immunity. Male chicks (Hubbard X Hubbard) were maintained on these treatments from one day of age to 3 weeks of age at which time six replicate pens per treatment were sacrificed and various parameters measured. The additional six replicate pens per treatment were maintained on toxin feed beyond 3 weeks of age, and at 4 weeks of age, three replicate pens were immunized with sheep red blood cells (SRBC) and Brucella abortus. Antibody titers were measured up to 10 days postimmunization. Aflatoxin and ochratoxin A, individually, significantly (P less than .05) decreased body weight, and a synergistic toxicity was evident by a significant (P less than .05) decrease in body weight. Antibody titers and phagocytic activity of heterophils were not significantly (P less than .05) altered by any treatments. The relative weight of the bursa of Fabricius and the number of follicles for a given area of the folds of the bursa of Fabricius were significantly (P less than .05) decreased only by the interaction treatment. Complement activity was significantly (P less than .05) decreased by aflatoxin and the combination of aflatoxin and ochratoxin A and depressed, although not significantly (P less than .05), by ochratoxin A.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of low level chronic aflatoxicosis in broiler chickens.

A completely random design consisting of three replicates of 25 broiler chickens (Hubbard x Hubbard) at each of four treatments was used to evaluate the effects of low level, chronic aflatoxicosis on performance and various processing parameters. The treatments in Trial 1 were control, .075, .225, and .675 and in Trial 2 control, .3, .9, and 2.7 micrograms/g toxin in feed (ppm). The chickens were maintained on these treatments from day-old to 7 weeks of age with feed and water available ad libitum. All aflatoxin dose levels in Trial 1 significantly (P less than .05) decreased live, dressed, and chilled eviscerated weight, whereas only 2.7 ppm significantly (P less than .05) decreased live and dressed weight in Trial 2, with chilled eviscerated weight being significantly (P less than .05) decreased at .3 and 2.7 ppm in Trial 2. Parts weights and dimension measurements reflected the aflatoxin-induced decrease in dressed weight. Breast yield (%) was significantly (P less than .05) decreased by aflatoxin while back, wing, drum, and thigh yields were significantly increased by aflatoxin. No effect of aflatoxin was seen on the incidence of crooked keel, feather follicle infection, breast blisters, or conformation. A hypocarotenoidemia and hepatic hyperlipemia were clearly a result of chronic aflatoxicosis in these broiler chickens. These data demonstrate that the toxicity of aflatoxin is dependent on the environment in which broiler chickens are exposed.(ABSTRACT TRUNCATED AT 250 WORDS)

Individual and combined effects of aflatoxin and ochratoxin A on bruising in broiler chickens.

A 2 x 2 factorial design with treatments of 0 and 2.5 microgram/g aflatoxin and 0 and 2.0 microgram/g ochratoxin A was used to evaluate the individual and combined effects of these mycotoxins on bruising and blood thigh syndrome in broiler chickens. Trial 1 consisted of four replicate pens of 10 broilers per replicate, which were maintained on these dietary treatments from 0 to 3 weeks of age. At 3 weeks of age two replicate pens per treatment were randomly selected and placed on toxin-free feed with two replicate pens remaining on toxin feed until they were 6 weeks of age when the trial was concluded. In Trial 2 three replicate pens of 38 broilers per treatment were maintained on toxin feed from 0 to 3 weeks of age. At 3 weeks of age 13 broilers per replicate were placed on toxin-free feed with 25 broilers per replicate remaining on toxin until they reached 7 weeks of age when Trial 2 was concluded. These data indicate that a synergistic toxicity exists between aflatoxin and ochratoxin A to significantly (P less than .05) decrease body weight. Body weights of broilers on aflatoxin or ochratoxin A diets for only 3 weeks partially recovered by 6 or 7 weeks of age; however, the body weights of broilers on the interaction treatments for only 3 weeks remained significantly (P less than .05) depressed at 6 and 7 weeks of age.(ABSTRACT TRUNCATED AT 250 WORDS)

Aflatoxicosis and Intrinsic Coagulation function in broiler chickens.

Aflatoxin was fed (0, .625, 1.25, 2.5, 5.0, and 10.0 microgram/g) to broiler chickens from day-old to 3 weeks of age when the birds were bled and intrinsic coagulation parameters measured. Clotting times of whole blood were increased by aflatoxin (2.5, 5.0, and 10.0 microgram/g) and decreased in blood samples activated by contact with the surface of crushed glass. There was no interaction of aflatoxin with the contact phenomenon. Partial thromboplastin times were significantly (p less than .05) prolonged by 5.0 and 10.0 microgram/g aflatoxin. Intrinsic activity as judged by whole blood thromboplastin generation was reduced nearly 40% by those two levels of aflatoxin. Activity analogous to human factor VIII was depressed by the two highest doses, but factor IX was significantly (P less than .05) reduced by only the highest dose fed, 10 microgram/g. These data suggest that chickens possess an intrinsic coagulation mechanism that is sensitive to aflatoxin but that the factor or factors responsible for the contact response are refractory to this important mycotoxin.

Synergism between aflatoxin and ochratoxin A in broiler chickens.

A 2 X 2 factorial experimental design consisting of four treatments (0, 2.5 microgram/g aflatoxin, 2.0 microgram/g ochratoxin A, and 2.5 microgram/g aflatoxin + 2.0 microgram/g ochratoxin A) with six replicates of 10 birds each was used to evaluate the synergism between aflatoxin and ochratoxin A. The chicks (Hubbard X Hubbard) were maintained on these dietary treatments from hatching until they reached 3 weeks of age, when the experiment was terminated. The size of the liver, spleen, pancreas, and proventriculus was significantly (P less than .05) altered by the individual toxins; however, a synergistic effect on the size of these organs was not observed. The kidney and gizzard were sensitive to the coincident exposure to these mycotoxins and were significantly (P less than .05) enlarged. The kidney was the most sensitive organ to the combined toxicity of aflatoxin and ochratoxin A, and nephropathy was the most important characteristic of this interaction. The synergism between aflatoxin and ochratoxin A significantly (P less than .05) decreased growth rate and numerically increased mortality, demonstrating the enhanced toxicity of cocontaminated feed. Liver lipid levels were significantly (P less than .05) increased by aflatoxin and decreased by ochratoxin A. The interaction of both mycotoxins on this parameter was significant (P less than .05) and the combined effect demonstrates that ochratoxin A inhibited lipid accumulation normally induced by aflatoxin. The data show that toxicity-enhancing synergisms exist between mycotoxins and that symptom patterns are altered during multiple mycotoxicoses. The data also demonstrate that nephropathy is the primary effect of this interaction and, thus, is of diagnostic importance.

Experimental ochratoxicosis in turkey poults.

Graded concentrations of ochratoxin A (0, 1, 2, 4, and 8 micrograms/g of feed) incorporated into the diet of turkey poults from hatching until 3 weeks of age resulted in a decreased growth rate, enlarged proventriculus and gizzard, and a regressed thymus (all at 4 and 8 micrograms/g) and the sizes of liver, spleen, pancreas, kidneys, bursa of Fabricius were unaffected. Feed conversion ratio increased from 1.63 (control value) to 2.07 (8 micrograms/g). Mortality was increased significantly (P less than .05) at 8 micrograms/g. Water consumption and plasma uric acid were increased at 4 and 8 micrograms/g. Plasma glucose and the dry weight of the kidneys decreased significantly at 8 micrograms/g while total plasma proteins, prothrombin time, plasma carotenoids, and phenol red clearance rate were unaltered. A leucocytopenia, which was primarily a lymphocytopenia, was observed at 4 and 8 micrograms/g. Heterophils decreased while basophils increased at 8 micrograms/g. The oral LD50 for day-old and 3-week-old poults was 4.63 +/- .31 and 7.84 +/- .94 mg/kg, respectively. Intraperitoneally, the value for day-old birds was .16 +/- .03 +/- mg/kg and for 3-week-old birds was .34 +/- .09 mg/kg. These data suggest that ochratoxin is a potent nephrotoxin in turkeys, but that ochratoxicosis in turkeys differs markedly from the disease in other species.

New evidence for intrinsic blood coagulation in chickens.

Coagulation of blood in chickens is considered the result of an extrinsic clotting system initiated, as in mammals, by tissue thromboplastin released from injured tissues. Blood coagulation in mammals depends principally on an intrinsic mechanism in which thromboplastin is generated from blood itself. Only a negligible role, if any, has been ascribed to an intrinsic system in chickens. A reevaluation of intrinsic coagulation in chickens was undertaken in this study. Whole blood of chickens was found to clot over 30% faster when contacted by suitable surface activators such as kaolin or glass than when such contact was omitted. Plasma recalcification times were significantly (P less than .02) shortened by contact activators. Clotting functions were measurable both by partial thromboplastin time and activated partial thromboplastin time, tests that bypass extrinsic factors. Intrinsic thromboplastin could be generated from dilute whole chicken blood although at a slower rate than that reported for human blood. Modification of whole blood thromboplastin generation techniques permitted measurement of activities that seem analogous to human intrinsic factors VIII and IX but not XI or XII. These data provide evidence of a functioning intrinsic clotting mechanism in chickens. A complete description and role for this mechanism remains to be defined.

Delayed reproductive development resulting from aflatoxicosis in juvenile Japanese quail.

Aflatoxicosis was induced in young Japanese quail. In the first experiment five replicates of 30 birds per treatment were fed a soy-corn basal ration containing 0, 5, or 10 microgram aflatoxin per gram of feed from 1 to 3 weeks of age. At 3 weeks, the animals were sacrificed and mesurements taken. In the second experiment, 0 to 10 microgram aflatoxin per gram of diet were fed from either 1 to 3 weeks of age or 2 to 4 weeks of age. At 3 weeks of age body weights were significantly (P < .05) reduced and relative liver weights were significantly (P < .05) increased. Testicular weights relative to body weight were depressed by up to 50%. Ovary wet weights, but not relative weights, were reduced. Testicular development (weight) was impaired through 6 weeks of age. Ovarian development, determined both by weight and number of developing follicles, was delayed as long as 3 weeks following withdrawal of aflatoxin from the diet. Radioimmunoassay for circulating androgens revealed that aflatoxin suppressed both the onset of production and the final concentratin of male hormone. The data demonstrate that aflatoxin can exert a deleterious inhibition of sexual development in quail with subsequent impairment of reproductive capacity. These findings raise the implication of potential reproductive failure in economically important species such as broiler breeders.