Bursa of Fabricius

The broilers were weighed, and feed intake was recorded on d 1, 21, and 42, and average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR) were calculated.
At the end of the third week and the experiment, 4 broilers were randomly selected from each cage and blood samples were collected from the jugular vein into a sterile syringe and stored at −4°C. Blood samples were then centrifuged at 3,000×g for 15 min and serum was separated. The levels of superoxide dismutase (SOD), total antioxidative capacity (T-AOC), malondialdehyde (MDA), glutathione peroxidase (GSH-PX), immunoglobulin G (IgG), interleukin 2 (IL-2), IL-6, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in the serum were analyzed using ELISA method (Jiancheng Biotechnology Institute, Nanjing, China) described previously [22 (link)]. The peripheral blood T lymphocyte proliferation and acid α-naphthylacetate esterase positive ratios (ANAE+) of the peripheral blood T lymphocytes was determined according to previous study [23 ]. Lysozyme activity was measured by the reduction speed of its substrate (Micrococcus lysodeikticus), which was reflected by the change in transmissivity at the wavelength of 530 nm following the manufacturer’s recommendation (Jiancheng Biotechnology Institute, China). The specific antibody titer of broilers (avian influenza H₅N₁ antibody) was determined by HA/HI method [24 ]. After blood collection, the same broilers were weighed individually, and then sacrificed by cervical dislocation. The liver, proventriculus, gizzard, pancreas, thymus, bursa of Fabricius, spleen, duodenum, jejunum and ileum were removed by trained personnel and weighed after flushing with saline. Organ size was expressed as a percentage of BW.
After weighing, the samples of small intestine tissues (ap proximately 2 cm from duodenum, jejunum, and ileum, respectively) were collected for determination of mucosal morphology. The tissues from duodenum, jejunum, and ileum, respectively, were cleaned with saline and then fixed in 10% neutral formalin. The fixed tissues were trimmed, embedded in paraffin for mucosal morphology and integrity. Thin sections (5 μm) were sliced and mounted on slide, and then stained with haematoxylin and eosin (H&E staining procedure) for histopathological examination by an optical microscope (Olympus, Tokyo, Japan) using the method described previously [25 (link)]. Intestinal morphological measurements included the following 3 indices: villus height (VH), crypt depth (CD), and VH/CD. These indexes were quantified according to the method described previously [26 (link)]. Mean values of VH, CD, and VH/CD within each segment (eight villi per bird) were calculated.

The clinical status of the ducks was observed daily and the signs of feed refusal, diarrhea, atypical behavior of birds (tonic immobility reaction or strutting behavior) and the presence of undigested feed in excreta were examined. On days 14, 28, and 42 five (d14 and 28) and eight (d42) ducks per treatment group were randomly selected, their individual body weight was measured, and blood samples of 1 mL per animal were collected from the wing vein (v. cutanea ulnaris). Serum was separated after centrifugation at 5500× g for 10 min, aspirated by pipette and transferred into 1.5 mL Eppendorf tubes and stored at −20 °C until further analysis. Selected ducks were terminally anaesthetized with carbon dioxide and the liver, spleen, and bursa of Fabricius were removed and weighed. The oropharyngeal cavity and esophagus of birds were inspected macroscopically. Relative organ weights were calculated as a percentage of the live body weight of animals measured before slaughter. For histological examination, samples of 10 g of the listed organs were fixed in a 10% buffered formaldehyde solution.

Concentrations of DON and ZEN in corn were determined using the commercially available ELISA kits RIDASCREEN™ DON and RIDASCREEN™ ZEN (R-Biopharm GmbH, Darmstadt, Germany). The clinical chemical analyses of serum samples (AST and ALT activity, concentrations of glucose, cholesterol, triglyceride, creatinine, and uric acid) were performed by Vet-Med-Labor Ltd. using colorimetric assay kits (Diagnosticum Co., Budapest, Hungary) based on spectrophotometric methods. Histopathological examinations were performed by Autopsy KKT (Budapest, Hungary). The liver, spleen, and bursa of Fabricius samples in formaldehyde solution were embedded in paraffin and 5 μm thick sections were stained with hematoxylin and eosin. Tissue morphology was observed under a light microscope. The mean histological score was derived from the grade and stage of histological lesions seen in the investigated organs of the affected animals. The listed lesions were characterized per animal (1 point = mild, 2 points = medium, 3 points = high-grade alterations) and then mean score values were calculated in the group. The extent of vacuolar degeneration of hepatocytes, solitary hepatocyte necrosis, individual cell deaths of the mononuclear phagocyte system (MPS), focal lymphocytic and histiocytic interstitial infiltrates and interstitial fibrosis in liver samples, as well as lymphocyte counts in spleen and bursa of Fabricius samples, were evaluated.