Growth, serum biochemistry, complement activity, and liver gene expression responses of Pekin ducklings to graded levels of cultured aflatoxin B1.

A 14-d study was conducted to evaluate the effects of cultured aflatoxin B1 (AFB1) on performance, serum biochemistry, serum natural antibody and complement activity, and hepatic gene expression parameters in Pekin ducklings. A total of 144 male Pekin ducklings were weighed, tagged, and randomly allotted to 4 dietary treatments containing 4 concentrations of AFB1 (0, 0.11, 0.14, and 0.21 mg/kg) from 0 to 14 d of age (6 cages per diet; 6 ducklings per cage). Compared with the control group, there was a 10.9, 31.7, and 47.4% (P < 0.05) decrease in cumulative BW gain with 0.11, 0.14, and 0.21 mg of AFB1/kg of diet, respectively, but feed efficiency was not affected. Increasing concentrations of AFB1 reduced cumulative BW gain and feed intake both linearly and quadratically, and regression equations were developed with r(2) ≥0.73. Feeding 0.11 to 0.21 mg of AFB1/kg reduced serum glucose, creatinine, albumin, total protein, globulin, Ca, P, and creatine phosphokinase linearly, whereas serum urea N, Cl, alkaline phosphatase, and aspartate amino transferase concentrations increased linearly with increasing AFB1 (P < 0.05). Additionally, 0.11 to 0.21 mg of AFB1/kg diets impaired classical and alternative complement pathways in the duckling serum when tested by lysis of rabbit, human type O, and horse erythrocytes, and decreased rabbit and horse agglutinins (P < 0.05). Liver peroxisome proliferator activated receptor α (PPARα) expression was linearly downregulated by AFB1 (P < 0.01). Results from this study indicate that for every 0.10 mg/kg increase in dietary AFB1, cumulative feed intake and BW gain decrease approximately 230 and 169 g per duckling from hatch to 14 d; and that AFB1 at very low concentrations can significantly impair liver function and gene expression, and innate immune dynamics in Pekin ducklings.

The contrasting properties of 2 xenogenic erythrocyte-reactive natural antibodies in commercial ducks.

Sera obtained from commercial drakes on days 14 and 38 of age were tested by microtiter for the capacity to agglutinate and lyse rabbit (Rb) and human (HuO) erythrocytes. Three agglutination types, differing by strength, were recognized: HA1 (strong), HA2 (weak), and HA45 (very weak).Two degrees of lysis: L(100) (complete) and L(50) (partial), measured complement activity. Day 14 sera agglutinated Rb (average log(2) titers: HA1 = 1.5, HA2 = 4.1) and lysed Rb (average log(2) titers: L(100) = 2, L(50) = 2.5) but only 8/115 (~9%) agglutinated HuO (HA45 = 0.4) while most (>80%) lysed HuO (average log(2) titers: L(100) = 1.3, L(50) = 1.8). Both Rb and HuO agglutination and lysis titers were higher by d 38. At that age, all ducks lysed HuO and 50% of ducks acquired a capacity to agglutinate these cells with more strength. However, the quality of HuO agglutination could not be differentiated into HA1 or HA2 types. Average d 38 log(2) titers of all measures were Rb: HA1 = 4, HA2 = 8.4, L(100) = 3, and L(50) = 4; and HuO: HA = 2.8, L(100) = 3.9, and L(50) = 1.2. The quality of the Rb agglutination suggested the participation of both IgM and standard-sized IgY antibodies. Lysis of Rb may occur by both classic and alternate complement pathways. The HuO lysis appears to depend primarily on the alternate complement pathway. It is suggested that multiple measurement systems such as these offer a practical way of obtaining information on immunity in experiments where the chief interest lies elsewhere.

Marek's disease in immunosuppressed chickens: growth of a transplantable lymphoma and development of the disease by natural exposure.

Evidence indicated that treatment of chickens with Cytoxan at hatching (bursectomy) had little effect on the development of transplantable Marek's disease (md) lymphoma. Cytoxan treatment of chickens at 2 and 3 weeks of age, however, apparently reduced the incidence of visceral tumors in chickens naturally exposed to MD. Amelioration of MD in immunosuppressed chickens was possibly mediated through suppression of MD-induced T-cell proliferation.

Salmonella challenge affects the antibody isotype profile of bile in hens differing in metabolic efficiency.

Gel precipitation reactions determined antibody isotypes in bile from hens differing in dietary efficiency. Ouchterlony double diffusion employing alpha-chain specific goat-anti-chicken IgA, rabbit anti-chicken IgG, goat anti-chicken IgM, black turtle bean (BTB), and Jacalin lectins as precipitating reagents detected bile IgA, IgG, and IgM from Salmonella exposed and nonexposed hens. The IgA was present in 1 of 3 forms designated by reagent and frequency: IgAB (precipitated by BTB lectin) 100%; IgAA (precipitated by anti-alpha chain antibody) 98%, and IgAJ (precipitated by Jacalin) 97%. That both BTB and Jacalin precipitates contain IgA was confirmed by immuno-dot blots using affinity purified alpha-chain specific antibody, establishing each as IgA glycoforms. Three measurements of Ouchterlony precipitates were made; d1 and d2 indicate diffusion from sample or reagent wells, lambda indicates arc length. Mean values for lambda, estimating quantity, were IgAA (11.3 mm) and IgAB (11.6 mm) and IgAJ (8.3 mm). The crescent shape IgAJ arc and its slower diffusion (d1) suggested its molecular weight is greater than either IgAA or IgAB. Arc lengths of individual samples were not significantly correlated suggesting that these are independent components of bile. Oral Salmonella enteritidis challenge resulted in a highly significant difference in bile IgA profiles. The IgAJ arc lengths (lambda) in R- hens increased by 20% over those in nonchallenged R- hens. Conversely S. enteritidis challenge was associated with a decrease of 10% in IgAJ arc lengths in nonefficient (R+) hens. Salmonella enteritidis challenge was not associated with arc length differences in either IgAA or IgAB. The IgG was present in all specimens, and in 9 of 59 (15%) 2 forms were detected. The IgG quantity was unaffected by either efficiency type or S. enteritidis challenge. The IgM was detected in only 2 of 59 (3.4%) specimens. Our observations suggest IgA of bile is composed of multiple forms influenced both by diet efficiency status and S. enteritidis exposure. It appears that the latter resulted in an increased quantity of IgAJ in R- hens, and suggests the existence of functional differences among the various IgA types.

Natural anti-Gal and Salmonella-specific antibodies in bile and plasma of hens differing in diet efficiency.

Specific anti-Salmonella enteritidis (SE) and natural anti-alpha-gal epitope (Galalpha1-3Galbeta-1-4GlcNAc-R; anti-Gal) antibodies were measured in plasma sample pools and individual bile specimens obtained from hens differing in diet efficiency. More SE somatic (O) and flagellar (H) antibodies were found in plasma pools from efficient hens (R-) compared with nonefficient hens (R+) after oral challenge with live SE. Mean titers of somatic agglutinins in bile were 2.3 in R- hens and 1.9 in R+ hens (P = 0.06) following live challenge. Salmonella enteritidis antibodies were also found in bile of nonchallenged hens of both types but their levels were not significantly different. Flagellar (H) agglutinin scores were higher in SE-challenged hens compared with nonchallenged hens (3.1 vs. 2.1; P < or = 0.004) but efficiency types did not differ. Bile also contained high titers of the anti-Gal antibody indicated by the agglutination of glutaraldehyde-stabilized rabbit erythrocytes. The average titer of all SE-nonexposed hens was 9.0 corresponding to 1:5,120 when corrected for the initial dilution and expressed in conventional terms. Salmonella enteritidis exposure was associated with higher anti-Gal titers. The average anti-Gal titer for all SE-exposed hens was 10.0, corresponding to 1:10,240 in conventional terms; this difference was significant (P < or = 0.016). Diet efficiency type-associated differences in anti-Gal titers were not significant. Collectively, our data indicate that diet efficiency status is not associated with compromised Salmonella-specific immune responses. Rather it appears that the immune responses of diet efficient hens (R-) are also more efficient. This is because R- hens produced higher levels of O- and H-type antibody only as the result of direct exposure to living SE. On the contrary, R+ hens produced H-type antibody because of challenge with heat-killed SE, a circumstance that will not result in disease. Moreover, the hen type difference does not seem to occur at the expense of innate immunity as measured by anti-Gal antibody levels.

Directional selection for specific sheep cell antibody responses affects natural rabbit agglutinins of chickens.

Agglutination data from generations 8 through 19 indicate that bidirectional selection for specific SRBC antibody responses was successful in a line cross of ISA x Warren medium heavy layers. After 11 generations titers of the high SRBC selected line (H line) were nearly 1:32,000; those of the low SRBC selected line (L line) were less than 1:2, but titers of the randombred control line remained stable at 1:32. Directional SRBC selection also affected levels of a naturally occurring rabbit cell agglutinating antibody (RRBC), presumably the avian form of alpha-galactose antibody (anti-Gal). This indirect response was biphasic and opposite in direction to the SRBC responses through generation 14 after which anti-Gal titers of all 3 lines increased. At generation 19, line H had the highest agglutinin titers; of both types, control line was intermediate, and line L was lowest. The correlation between SRBC and RRBC titers was 0.43 (P = 0.0). Females had higher titers than males, but the difference was only significant for the SRBC antibody (P = 0.028). Qualitative changes in anti-Gal accompanied SRBC selection. Rabbit agglutinins of 4 types were recognizable: classic, granular, annular, and one negative or very weak reaction. The score type means in line L were highest, in the control line were intermediate, and in line H were lowest, suggesting avidity differences now exist among these lines. The results show integration of natural and acquired immune systems because selection for one temporarily affected the other. Given the importance of anti-Gal in primates, our results should stimulate further study of this antibody in poultry species.

The length of uncomplicated human gestation.

By retrospective exclusion of gestations with known obstetric complications, maternal diseases, or unreliable menstrual histories, we found that uncomplicated, spontaneous-labor pregnancy in private-care white mothers is longer than Naegele's rule predicts. For primiparas, the median duration of gestation from assumed ovulation to delivery was 274 days, significantly longer than the predicted 266 days (P = .0003). For multiparas, the median duration of pregnancy was 269 days, also significantly longer than the prediction (P = .019). Moreover, the median length of pregnancy in primiparas proved to be significantly longer than that for multiparas (P = .0032). Thus, this study suggests that when estimating a due date for private-care white patients, one should count back 3 months from the first day of the last menses, then add 15 days for primiparas or 10 days for multiparas, instead of using the common algorithm for Naegele's rule.

Kinetics of the wattle reaction to human plasma.

Human plasma produced cutaneous basophil hypersensitivity when injected into chicken wattles. The kinetics of the response development were affected by presensitization, whether or not complete Freund's adjuvant was used. Presensitized chickens developed their maximum response significantly sooner (12.6 hr earlier) than the controls.

Identification of Salmonella enteritidis from experimentally infected hens using a colorimetric DNA hybridization method.

Identification of Salmonella enteritidis from cloacally challenged commercial laying hens was studied by comparing bacterial isolations using conventional methods with detection by the use of the GENE-TRAK colorimetric DNA probe assay. More positive test results were obtained using the latter on days 14, 28, and 42 postchallenge, but the difference between the two methods was not statistically significant. Over the duration of the experiment, positive cloacal samples were statistically more frequent from a commercial strain of white leghorn hens when compared with a commercial brown egg-producing strain (28/60 vs. 9/57; chi-square 1 df = 12.9, P < 0.001). Eggs having various shell defects were produced by the infected hens only after Salmonella challenge. These defects included, in order of frequency, elongated shape, thin shells, off-white color (tints), small size, wrinkles, and pimples. No Salmonella could be recovered from 193 defective eggs, nor were positive isolates made from additional tests performed on 50 normal eggs. Proteus sp. was isolated from 10 eggs, however. Our observations demonstrate that the GENE-TRAK colorimetric method is comparable with conventional bacteriology for the identification of Salmonella in cloacal samples taken from laying hens. Moreover, the two methods demonstrate the existence of breed differences in susceptibility to S. enteritidis challenge.

The influence of thymectomy on Rous sarcoma regression.

The incidence of regression of wing-web tumors induced by Rous sarcoma virus was shown to be dependent on the quantity of thymus tissue remaining after neonatal thymectomy in chickens of inbred line 6. Frequency of metastasis was associated negatively with the amount of thymus tissue present. Tumor regression and metastasis restriction both appeared dependent on the quantity of thymic tissue present.

Analysis of chicken bile by gel precipitation reactions using a lectin in the place of antibody.

A lectin obtained from black turtle beans (BTB) was precipitated with IgA in chicken bile samples in various forms of agarose gel systems. Ouchterlony-type double-diffusion (ODD) precipitation patterns between the lectin, bile IgA, and heavy chain-specific antibody contained spurs of the type suggestive of partial immunologic identity. The immunoelectrophoresis precipitation patterns between the same three reactants were mirror images and fused on the cathodic side of the immunoelectrophoresis origin. In addition to use in ODD-type gels, BTB could also be incorporated into agarose gels suitable for Mancini (radial immunodiffusion) or Laurell-type rocket electrophoresis. Bile samples obtained from Cornell lines OS and C, broiler breeder males, and University of California-Davis congenic lines were investigated using BTB- and antibody-based methods. The results of this study indicated that IgA was the most frequently detected isotype in bile, occurring in 139 of 156 (89%) samples. Most bile samples (128/156; 82%) also contained IgG, whereas fewer (19/156; 12%) contained IgM. Cornell lines appeared to differ from broiler breeders, having a higher frequency of IgM-positive samples. Of the total bile samples studied, 11% (17/156) of samples from broiler breeders and the Cornell lines appeared to be devoid of IgA; the bile of one broiler breeder was found to be devoid of all three isotypes. Instances were found in which bile samples shown to be negative for IgA by antibody-ODD were shown to be positive by BTB-ODD. Thus BTB appears to be a suitable adjunct to antibody for the study of IgA.

Differential resistance to Staphylococcus aureus challenge in two related lines of chickens.

Trials were conducted to determine whether differential resistance to challenge with Staphylococcus aureus was characteristic of two related lines of New Hampshire chickens differing genetically in size of the bursa of Fabricius. Neonatal small bursa line (SBL) chicks were superior to the unselected Lester J. Dreesen (LJD) line in five of six trials employing intracardiac challenge (chi 2 = 6.3, .05 greater than P greater than .01). Older (7 to 12 wk) SBL chicks, challenged intravenously, had superior resistance than Line LJD chicks in three of four trials where a direct comparison was possible. The mortality rate in Line SBL was 34% in all trials compared with a 54% mortality rate (chi 2 = 11.7, P less than .001) in Line LJD. Moreover, the development of morbidity was more rapid in Line LJD. It is suggested that these lines can be of use in investigations of the nature of resistance to staphylococcal disease.

Differential resistance to Staphylococcus aureus challenge in major histocompatibility (B) complex congenic lines.

Ten inbred B-congenic Leghorn lines were challenged with two isolates of Staphylococcus aureus at 3 days and 6 wk of age. Significant differences in mortality were observed among such lines when challenged at 3 days with either S. aureus Isolate P4L (moderately pathogenic) or S. aureus Isolate 3727 (highly pathogenic). Line 331 (B2/B2 genotype) had lower mortality than either Line 004 (B17/B17, chi 2 = 4.13, P < .05) or Line 253 (B18/B18, chi 2 = 4.23, P < .05) challenged with Isolate P4L. The use of a susceptibility index allowed for the detection of additional differences among the various lines challenged by Isolate 3727. Line 336 (BQ/BQ) was more resistant than either Line 335 (B19/B19, P < .01) or Line 330 (B21/B21, P < .01). No significant differences were found among the lines challenged at 6 wk by either isolate. The results provide additional evidence for the importance of the B complex in genetically determined disease resistance, and further demonstrate the usefulness of congenic lines in such investigations.

B-complex associated immunity to Salmonella enteritidis challenge in congenic chickens.

Six trials were conducted during which a total of 12 congenic lines (University of California-Davis, UCD) homozygous for various B-complex haplotypes, were challenged as neonates by intraperitoneal injection with either of two isolates of Salmonella enteritidis. Because these B haplotypes were expressed on a common genetic background (highly inbred Line UCD 003), and mortality differences among lines were statistically significant in three of the six trials, and morbidity (body weight) differences were significant in another trial; it is suggested that B-complex alleles affect the degree of immunity to these isolates. When all lines and trials were compared, line 342 (BC/BC) emerged as particularly resistant, whereas lines 253 (B18/B18) and 254 (B15/B15) were more susceptible. The remainder of the lines were of neutral (intermediate) susceptibility. Sex did not appear to influence the results of the challenge, but more resistance was observed with an increase in the age at inoculation. Although the mechanism that determined this resistance is unknown it was present as early as 3 d of age, and it is suggested that complement proteins, which have a known role in protection from bacterial infections, and are encoded by genes located within the B-complex, or acute phase proteins, may account for these observations. The results provide additional evidence for the importance of the B-complex in determining immunity to Salmonella.

The delayed and saline wattle reactions in broilers challenged with bovine serum albumin.

Bovine serum albumin was used to study the development of a wattle reaction in broiler-type chickens. A strong response developed in both nonsensitized and sensitized chickens and the frequency of such was shown to be dependent on the method used for the sensitization. A similar swelling developed in a proportion of the wattles injected with saline and this too seemed to be influenced by the sensitization method. Moreover, both types of responses were shown to be somewhat unstable during the first 24 hr postchallenge.

Major histocompatibility (B) complex-associated differences in the delayed wattle reaction to staphylococcal antigen.

The influence of the major histocompatibility (B) complex on the delayed wattle reaction (DWR) to Staphylococcus aureus was studied in 109 segregants (B2/B2, B2/B5, and B5/B5) of a fourth generation cross between inbred Regional Poultry Research Laboratory lines 6(1) and 15(1). Chickens were sensitized at 6 weeks of age with S. aureus antigen. One week later, DWR was evaluated by injecting the right wattle with S. aureus antigen. Thickness measurements were taken 4, 24, 48, 72, and 96 hr after injection. A quadratic equation model was used for each bird to calculate maximum wattle thickness, hour of maximum response, and rate of response development and decline. In males, the maximum wattle thickness in response to S. aureus antigen was significantly greater in B2/B5 heterozygotes (1.77 +/- .07 mm) than in either homozygote, B2/B2 (1.36 +/- .13 mm) or B5/B5 (1.39 +/- .08 mm). Heterozygous males reached maximum response sooner and recovered more quickly than homozygous males but these differences were not statistically significant. In females, response developed later than in males but no B complex effect was detected in either rate of development or maximum response.

Detection of cellular immunity to rous tumors of chickens by the leukocyte migration inhibition reaction.

Peripheral blood leukocytes obtained from Rous sarcoma-bearing chickens were tested for their ability to be inhibited from migrating in vitro by soluble tumor extract. It was found that the inhibition of leukocytes from chickens with regressing Rous tumors was significantly greater than that of chickens with progressing tumors or non-tumor bearing controls.

Effects of feeding blends of grains naturally contaminated with Fusarium mycotoxins on production and metabolism in broilers.

Three hundred sixty, 1-d-old male broiler chicks were fed diets containing grains naturally contaminated with Fusarium mycotoxins for 56 d. The four diets included control (0.14 mg/kg deoxynivalenol, 18 mg/ kg fusaric acid, < 0.1 mg/kg zearalenone), low level of contaminated grains (4.7 mg/kg deoxynivalenol, 20.6 mg/kg fusaric acid, 0.2 mg/kg zearalenone), and high level of contaminated grains without (8.2 mg/kg deoxynivalenol, 20.3 mg/kg fusaric acid, 0.56 mg/kg zearalenone) and with (9.7 mg/kg deoxynivalenol, 21.6 mg/kg fusaric acid, 0.8 mg/kg zearalenone) 0.2% esterified-glucomannan polymer derived from Saccharomyces cerevisiae1026 (E-GM). Body weight gain and feed consumption responded in a significant quadratic fashion to the inclusion of contaminated grains during the finisher period. Efficiency of feed utilization, however, was not affected by diets. The feeding of contaminated grains in the finisher period also caused significant linear increases in blood erythrocyte count and serum uric acid concentration and a significant linear decline in the serum lipase activity. Dietary inclusion of contaminated grains resulted in a significant quadratic effect on serum albumin and y-glutamyltransferase activity. Blood hemoglobin and biliary IgA concentrations, however, responded in significant linear and quadratic fashions. Supplementation of E-GM counteracted most of the blood parameter alterations caused by the Fusarium mycotoxin-contaminated grains and reduced breast muscle redness. It was concluded that broiler chickens may be susceptible to Fusarium mycotoxicoses when naturally contaminated grains are fed containing a combination of mycotoxins.