Janice M. Balog

Bacteriophage treatment of a severe Escherichia coli respiratory infection in broiler chickens.

Abstract SUMMARY. A bacteriophage to a serotype 02, nonmotile Escherichia coli was isolated from municipal waste treatment facilities and poultry processing plants. A study was conducted to determine the efficacy of multiple vs. single intramuscular (i.m.) injections of bacteriophage to treat a severe E. coli respiratory infection. The birds were challenged at 7 days of age by injection of 6 × 104 colony-forming units (cfu) of E. coli into the thoracic air sac followed by an i.m. injection into the thigh with either heat-killed or active bacteriophage. There were 16 treatments with three replicate pens of 10 birds. There were four control treatments, which included untreated birds, birds injected with either heat-killed or active bacteriophage, and birds challenged only with E. coli. In the remaining treatments, birds were injected with heat-killed or active bacteriophage either once immediately after E. coli challenge or immediately after challenge and at 8 and 9 days of age, once at 8 days of age or at 8, 9, and 10 days of age, and once at 9 days of age or at 9, 10, and 11 days of age. Mortality was significantly decreased from 57% to 13% in the birds given a single i.m. injection of bacteriophage immediately after E. coli challenge, and there was complete recovery in birds treated immediately after challenge and at 8 and 9 days of age, which was a significant improvement from the single injection treatment. There was a significant reduction in mortality from 57% to 10% in the birds treated with bacteriophage once at 8 days of age and those birds treated at 8, 9, and 10 days of age, with no difference between single or multiple treatments. The mortality in the single or multiple phage treated birds that started at 9 days of age was reduced from 57% to 28% and 27%, respectively, but was not statistically different from the control. These data suggest that bacteriophage can be an effective treatment when administered early in this experimental E. coli respiratory disease and that early multiple treatments are better than a single treatment. The efficacy of bacteriophage treatment diminishes as it is delayed, with no difference between single or multiple treatments. Bacteriophage may provide an effective alternative to antibiotics, but like antibiotic therapy, the effectiveness of phage to rescue animals decreases the longer treatment is delayed in the disease process.

Changes in Serum Ovotransferrin Levels in Chickens with Experimentally Induced Inflammation and Diseases

SUMMARY. A competitive enzyme immunoassay was developed to measure the changes in serum levels of ovotransferrin (OTF) during inflammation and infectious diseases in chickens. The assay is based on the competition of serum OTF with a fixed concentration of biotin-labeled OTF to bind to a rabbit anti-chicken transferrin antibody immobilized on microtiter wells. After several washing steps, the antibody-bound biotinylated OTF is probed with streptavidin–horseradish peroxidase conjugate (HRP) followed by a colorimetric detection of the HRP activity. The relative changes in the optical density of color are plotted against the competing concentrations of OTF with logarithmic regression to generate a standard curve that is used to determine the concentrations of OTF in unknown samples. Serum had no effect on the measurement of OTF. By this method, the time course changes of serum OTF levels in 4-wk-old male broiler chickens that were subjected to inflammation by croton oil injection were measured. The results showed croton oil–induced inflammation elevated serum OTF levels at 16 hr postinjection. OTF levels reached a peak by 72 hr, remained high through 120 hr, and returned to a basal level of olive oil–injected controls by 240 hr. There were no changes in serum OTF levels at any of the above time points in olive oil–injected control chickens. For studies with poultry diseases, specific-pathogen-free (SPF) male chickens were challenged with known bacterial and viral pathogens, and serum was collected at the height of the infection, i.e., 7 days after the challenge. Compared with uninjected controls, the SPF chickens challenged with Escherichia coli, fowl poxvirus, respiratory enteric orphan virus, infectious bursal disease virus, infectious bronchitis virus, or infectious laryngotracheitis virus had higher levels of OTF in serum. Inflammation-induced changes in serum OTF levels were also evident in the changes in the density of a 65-kD band protein corresponding to OTF. These results demonstrate that serum OTF may be a nonspecific clinical marker of inflammation associated with traumatic or infectious avian diseases.

Cell Death in Avian Tibial Dyschondroplasia

Tibial dyschondroplasia (TD) is a local defect of growth plates in fast-growing poultry where the transitional zone cartilage fails to resorb and persists as an avascular plug that prevents endochondral bone formation. We compared the differences in the cartilages from normal and TD-affected growth plates using the reduction of MTS to assess cartilage viability. Chondrocyte apoptosis was determined using biochemical measurement of DNA fragmentation, and in situ labeling of nuclei with fluorescein-dUTP using terminal deoxynucleotide transferase (TdT)-mediated nick end labeling (TUNEL) of isolated chondrocytes and growth plate sections. The TD-affected cartilage showed a significantly lower level of MTS reduction and a decrease in trichloroacetic acid (TCA)-precipitable DNA content. The TD cartilages had a higher percentage of fragmented DNA, which was also evident with agarose gel electrophoresis. A significantly higher number of chondrocytes isolated from TD-affected cartilages had condensed morphology, shrunken nuclei with little cytoplasm, and were TUNEL positive as identified by the incorporation of fluorescein-dUTP into the nuclei. In vivo results similarly showed a significant population of chondrocytes in transition zones undergoing condensation and apoptosis as determined by in situ TUNEL staining of growth plate sections. Normal growth plates, under similar conditions, showed no significant apoptosis of chondrocytes from hypertrophic and chondrolyzing zones. The condensation and apoptotic cell death may be responsible for the reduction of growth plate viability as well as the reduction in DNA content and increased DNA fragmentation. While the cause of the pathogenesis of TD is unknown, it appears that the aberrant death of chondrocytes in hypertrophic regions of growth plates may be responsible for the accumulation of cartilage and the arrest of endochondral bone formation.

Identification of transforming growth factor-beta and interleukin-6 in chicken ascites fluid.

Ascites fluids from chickens were analyzed for the occurrence of transforming growth factor-beta (TGF-beta) and interleukin-6 (Il-6) using the mink lung epithelial cell inhibition and B9 hybridoma proliferation assays, respectively. Both of these cytokines were significantly elevated in ascites fluids (TGF-beta, 0.129 +/- 0.017 ng/mg protein; Il-6, 0.054 +/- 0.011 ng/mg protein) relative to serum (TGF-beta, 0.005 +/- 0.003 ng/mg protein; Il-6, < 0.002 ng/mg protein) derived from the same individual birds. TGF-beta occurred in a latent form and required activation by heat or acid (heat, 100%; non-activated, 5.2 +/- 1.1%; acid-activated, 89.5 +/- 12.3%). Heat treatment destroyed Il-6 activity. Both TGF-beta and Il-6 activities could be neutralized by antibodies directed against the recombinant human counterpart of these cytokines. Increasing dilutions of ascites fluid caused proportionate decreases in cytokine activities. Il-6 activity was further characterized by gel filtration using high-pressure liquid chromatography, which yielded a peak of biological activity corresponding to an approximate molecular weight of 35,000. These data suggest that ascites fluid may be an interesting biological model and source for studying avian cytokines and their physiological relevance.

A longitudinal study of green-liver osteomyelitis complex in commercial turkeys.

Two flocks of Nicholas tom turkeys from separate farms with histories of above-average condemnations for turkey green-liver osteomyelitis complex (TOC) were studied throughout a 16-week growout. Fifty birds from each farm were necropsied each week for 15 weeks, and birds that had green livers, osteomyelitis in the proximal tibia, or swollen joints were cultured for aerobic bacteria along with an equal number of control birds. At processing, TOC lesions and green livers were obtained for bacterial culture and histopathology. Green-liver-associated TOC was not observed until the turkeys were 9 or 10 weeks of age. The incidence of TOC was higher on one farm, which also had a higher incidence of airsacculitis, higher early and weekly mortality, seroconversion to Newcastle disease virus and Mycoplasma meleagridis, and significantly higher average body weights, relative spleen weights, and relative liver weights. Both farms had a high incidence of intestinal lesions and infestation with Ascaridia dissimilis. Histological evaluation of green livers revealed hyperplasia of bile ducts, dilation of sinusoids, and pigment-containing Kupffers cells, some of which stained positive for iron. The bacterial isolates most frequently cultured from bones and livers were pleomorphic gram-variable coccobacilli, which grew visible colonies only after a series of subcultures and extended incubation.

The Effects of Water Supplementation with Vitamin E and Sodium Salicylate (Uni-Sol®) on the Resistance of Turkeys to Escherichia coli Respiratory Infection

Abstract The objective of this study was to determine the prophylactic efficacy of two commercial products, soluble vitamin E and soluble sodium salicylate (Uni-Sol®), in an Escherichia coli respiratory challenge. The drinking water of male turkey poults was non-supplemented or supplemented with either vitamin E or Uni-Sol or a combination of both at dosages recommended by the manufacturer. There were 110 birds in each of the four treatments, housed in four floor pens per treatment. At 5 wk of age, birds in half of the pens were challenged with an air sac inoculation of approximately 50 colony-forming units of E. coli. Water treatment commenced 5 days before challenge and continued for 2 wk after challenge, when birds were necropsied. All water treatments prevented the decrease in body weight due to E. coli challenge; however, either vitamin E or Uni-Sol alone, but not the combination of the two, decreased body weight in nonchallenged controls. Either vitamin E or Uni-Sol treatment alone, but not the combination of the two, significantly decreased mortality and air sacculitis scores of challenged birds, and all treatments decreased the isolation rates of E. coli from the liver. All treatments protected liver, spleen, and bursa weights (relative to body weight) from the effects of E. coli challenge, and Uni-Sol alone or vitamin E with Uni-Sol protected relative heart weights from the effect of challenge. Uni-Sol treatment alone increased the main effect mean total leukocyte counts and the number and percent of lymphocytes. Uni-Sol in combination with vitamin E increased the number of lymphocytes of challenged birds. Uni-Sol alone decreased the main effect mean heterophil/lymphocyte ratio (H/L) ratio, whereas vitamin E alone increased the H/L ratio of challenged birds. These results indicate that treatment of turkey poults with vitamin E or Uni-Sol prior to and during the stressful events that can lead to colisepticema may decrease disease incidence and mortality.

Effect of Spectinomycin on Escherichia coli Infection in 1-day-old Ducklings

Two challenge trials and one confirmation trial were conducted to evaluate the efficacy of spectinomycin in the treatment of 1-day-old ducklings infected with Escherichia coli. In the challenge trials, ducklings were injected in the right posterior thoracic air sac with 0.2 cm3 of broth containing 10(8) colony-forming units E. coli (strain O78, E38)/ml. Spectinomycin at dosage levels of 2.5 mg, 5.0 mg, and 10.0 mg of activity was injected subcutaneously 6 hours following infection. The confirmation trial was conducted to confirm the challenge trials; procedures were similar to those used in the challenge trials, except that only the 5.0 mg of activity dosage of spectinomycin was used. In both types of trials, spectinomycin-treated ducklings had significantly lower mortality and higher average weight gain, average daily gain, and feed consumption than infected unmedicated controls. These results indicate that spectinomycin is effective in treating ducks for experimentally induced colibacillosis caused by E. coli (strain O78, E38).