Immune response gene expression in spleens of diverse chicken lines fed dietary immunomodulators.

Vaccines, antibiotics, and other therapeutic agents used to combat disease in poultry generate recurring costs and the potential of residues in poultry products. Enhancing the immune response using alternative approaches such as selection for increased disease resistance or dietary immunomodulation may be effective additions to the portfolio of strategies the industry applies in poultry health management. The objective of this study was to characterize the effects of dietary supplementation with 3 immunomodulators [ascorbic acid, 1,3-1,6 ß-glucans from baker's yeast, and corticosterone] on cytokine gene expression in the spleen of 3 distinct genetic lines of chickens. Relative mRNA expression levels were determined using quantitative reverse transcriptase PCR for IL-1ß, IL-2, inducible nitric oxide synthase, and toll-like receptors 4 and 15, all of which play important roles in chicken immune function. Expression data were analyzed by mixed model analysis. The only significant effect detected was sex effect (P < 0.04) on expression of IL-1ß. The present findings suggest the need for further investigations into the effects of dietary immunomodulators on cytokine gene expression in chickens so as to generate a better understanding of the immunomodulation process.

Differential splenic cytokine responses to dietary immune modulation by diverse chicken lines.

Nutritional modulation of the immune system is an often exploited but poorly characterized process. In chickens and other food production animals, dietary enhancement of the immune response is an attractive alternative to antimicrobial use. A yeast cell wall component, beta-1,3/1,6-glucan, augments the response to disease in poultry and other species; however, the mechanism of action is not clear. Ascorbic acid and corticosterone are better characterized immunomodulators. In chickens, the spleen acts both as reservoir and activation site for leukocytes and, therefore, splenic gene expression reflects systemic immune function. To determine effects of genetic line and dietary immunomodulators, chickens of outbred broiler and inbred Leghorn and Fayoumi lines were fed either a basal diet or an experimental diet containing beta-glucans, ascorbic acid, or corticosterone from 56 to 77 d of age. Spleens were harvested, mRNA was isolated, and expression of interleukin (IL)-4, IL-6, IL-18, macrophage inflammatory protein-1beta, interferon-gamma, and phosphoinositide 3-kinase p110gamma transcripts was measured by quantitative reverse transcription PCR. Effects of diet, genetic line, sex, and diet x genetic line interaction on weight gain and gene expression were analyzed. At 1, 2, and 3 wk after starting the diet treatments, birds fed the corticosterone diet had gained less weight compared with birds fed the other diets (P < 0.001). Sex affected expression of IL-18 (P = 0.010), with higher levels in males. There was a significant interaction between genetic line and diet on expression of IL-4, IL-6, and IL-18 (P = 0.021, 0.006, and 0.026, respectively). Broiler line gene expression did not change in response to the experimental diet. Splenic expression of IL-6 was higher in Leghorns fed the basal or ascorbic acid diets, rather than the beta-glucan or corticosterone diets, whereas the opposite relationship was observed in the Fayoumi line. Expression of IL-4 and IL-18 responded to diet only within the Fayoumi line. The differential splenic expression of birds from diverse genetic lines in response to nutritional immunomodulation emphasizes the need for further study of this process.

Comparison of heterophil phagocytosis for heterophil-adapted Salmonella enteritidis (HASE) and wild-type Salmonella enteritidis (SE).

Serial passage of Salmonella enteritidis (SE) yields heterophil-adapted SE (HASE) strains that have resulted in decreased shedding of SE in feces and reduced egg contamination. Additionally, increasing the number of heterophil passages further reduced the number and frequency of fecal shedding. To evaluate SE and heterophil interaction, nine SE strains were fluorescein isothiocyanate-labeled when viable. There were six wild-types: SE TK 474, SE TK 584, SE TK 599, SE TK 600, SE TK 655, and SE TK 657; and three HASE strains: TK 499 heterophil adapted five times, TK 598 heterophil adapted six times, and TK 605 heterophil adapted 11 times. Trials were repeated seven times in duplicate with heterophils isolated from seven healthy chickens. Heterophils were incubated with the bacterial strains at 41 C for 15 min, and 10,000 heterophils were analyzed by flow cytometry. Percentage of phagocytosis and mean channel number of fluorescence were compared. Both parameters were significantly increased for all HASE-type strains compared with wild-type, nonadapted SE strains. Increased phagocytosis of HASE bacterial strains may be significant in processing and elimination of the HASE strains and may be related to the protective effect of HASE by decreased shedding of wild-type SE challenge strains.

Comparison of bronchoalveolar lavage fluid obtained from Mannheimia haemolytica-inoculated calves with and without prior treatment with the selectin inhibitor TBC1269.

OBJECTIVES: To determine effects of selectin inhibitor TBC1269 on neutrophil infiltration, and neutrophil-associated injury during pneumonia induced by Mannheimia haemolytica and concentration of antimicrobial anionic peptide (AAP) in bronchoalveolar lavage fluid (BALF) as well as antimicrobial activity of BALF from healthy (control) neonatal calves, neonatal calves with M haemolytica-induced pneumonia, neonatal calves with prior treatment with TBC1269, and adult cattle. ANIMALS: Eighteen 1- to 3-day-old calves and 9 adult cattle. PROCEDURE: Calves were inoculated with M haemolytica or pyrogen-free saline (0.14M NaCl) solution into the right cranial lung lobe, and BALF was collected 2 or 6 hours after inoculation. Thirty minutes before and 2 hours after inoculation, 4 calves received TBC1269. The BALF collected from 9 adult cattle was used for comparison of BALF AAP concentration and antimicrobial activity. Protein concentration and neutrophil differential percentage and degeneration in BALF were determined. An ELISA and killing assay were used to determine BALF AAP concentration and antimicrobial activity, respectively. RESULTS: Total protein concentration was significantly decreased in BALF from calves receiving TBC1269. Similar concentrations of AAP were detected in BALF from all calves, which were 3-fold higher than those in BALF from adult cattle. However, BALF from neonates had little or no anti-M haemolytica activity. CONCLUSIONS AND CLINICAL RELEVANCE: These results suggest that TBC1269 decreases pulmonary tissue injury in neonatal calves infected with M haemolytica. Although AAP is detectable in neonatal BALF at 3 times the concentration detected in adult BALF, neonatal BALF lacks antimicrobial activity for M haemolytica.

Effect of oral administration of excessive iron in adult ponies.

OBJECTIVE: To evaluate the potential of excess dietary iron to cause hepatic lesions similar to those described in horses with suspected iron toxicosis or hemochromatosis. DESIGN: Prospective study. ANIMALS: 6 adult male ponies. PROCEDURE: 4 ponies received 50 mg of iron/kg (22.7 mg/lb) of body weight each day by oral administration of ferrous sulfate, which contained 20% elemental iron; 2 ponies received only the carrier (applesauce). Complete blood counts, serum biochemical analyses, and hepatic tissue biopsies were performed, and serum iron concentrations were measured. Blood and tissue samples were obtained at days 0 and 2, and at the end of weeks 1, 3, 6, and 8 after administration of iron was initiated. Treatment was discontinued after 8 weeks, and hepatic iron concentrations were measured at 28 weeks. RESULTS: Hepatic iron concentrations, serum iron concentrations, percentage saturation of transferrin, and serum ferritin concentrations were increased, compared with baseline and control concentrations, by week 8. Adverse clinical signs or histologic lesions in the liver were not detected in any ponies. At 28 weeks, hepatic iron concentrations had decreased. CONCLUSIONS AND CLINICAL RELEVANCE: Histologic lesions were not seen in the hepatic biopsy specimens obtained from the ponies treated with ferrous sulfate. It was concluded that it would be unlikely for iron toxicosis to develop in adult ponies or horses during a period of < 8 weeks when food or water contained increased amounts of iron. It is suspected that previous reports of hepatopathies in animals with hemosiderin accumulation may represent a primary hepatopathy with secondary hemosiderin accumulation, especially if the only source of iron is via oral consumption.

Use of a nonionic detergent (Triton WR 1339) in healthy cats to assess hepatic secretion of triglyceride.

OBJECTIVE: To determine whether a nonionic detergent (Triton WR 1339) can be used in cats to assess hepatic secretion of triglyceride. ANIMALS: 28 healthy cats. PROCEDURE: Triton WR 1339 was administered IV according to the following schedule: 5, 50, 150, and 250 mg/kg of body weight. Control cats did not receive an injection or received 0.9% NaCl or PBS solutions at the same osmolarity and volume as the 250 mg/kg group. Blood samples were collected throughout the 48-hour period after administration for determination of triglyceride and cholesterol concentrations and for RBC morphology and osmotic fragility studies. RESULTS: Administration of Triton WR 1339 at 150 and 250 mg/kg caused profound hypertriglyceridemia. Triglyceride concentrations increased in a curvilinear fashion for the first 2 hours and remained increased for approximately 24 hours. Area under the time-concentration curve for triglyceride at 5 hours differed significantly among groups. At 12 and 24 hours, cholesterol was significantly higher in cats receiving 250 mg/kg. The most dramatic changes in osmotic fragility and RBC morphology were in cats receiving 250 mg/kg; 1 of these cats developed severe icterus and died 5 days later. Feeding rice and casein before administering Triton WR 1339 at 150 mg/kg did not appear to affect the hypertriglyceridemia response. CONCLUSIONS AND CLINICAL RELEVANCE: Triton WR 1339 can be administered IV to cats at a rate of 150 mg/kg to assess hepatic triglyceride secretion, although some cats may have increased RBC osmotic fragility. Higher dosages caused substantial adverse effects, whereas lower dosages did not alter plasma triglyceride concentration.

The effects of ascorbic acid on in vitro heterophil function.

As a feed additive, ascorbic acid has been shown to have a protective effect against bacterial and viral diseases and to reduce the impact of detrimental stress in chickens. This study examined the effect of ascorbic acid treatment on in vitro heterophil function by examining random migration and phagocytosis and bacterial killing of Staphylococcus aureus. Heterophils were evaluated in broiler chickens ranging from 5 to 16 wk of age, and age differences were seen. Significant increases in bacterial killing were found in heterophils treated with ascorbic acid, and this difference tended to be greater in chickens from 5 to 10.5 wk of age. No significant differences were found in phagocytosis or random migration, but ascorbic acid tended to decrease random migration. The most significant effect on in vitro heterophil function was an increase in bacterial killing.

Lawsonia intracellularis-like organism infection in a miniature foal.

A 7-month-old foal was admitted to the hospital with a history of lethargy, weight loss, mild diarrhea, and anorexia. A diagnosis of proliferative enteritis caused by Lawsonia intracellularis-like organisms was made after necropsy and histologic examination of the small intestine. Although infection with L intracellularis-like organisms is a rare cause of enteritis in foals, it should be considered in the differential diagnosis, especially if the foal was housed in the proximity of pigs or pig feces. Antemortem diagnosis remains challenging because isolation of the organism in fecal material requires cell culture, and histologic evaluation of intestinal biopsy specimens may be unrewarding because of the lack of information regarding the frequency and distribution of lesions in horses. Alternatively, use of immunochemical stain, dot-blot technique, and polymerase chain reaction provide specific diagnostic tests that can be performed on fecal material. Postmortem diagnosis relies on histologic examination of infected tissues and use of immunofluorescence and polymerase chain reaction.

Cytologic examination of exfoliative specimens obtained during endoscopy for diagnosis of gastrointestinal tract disease in dogs and cats.

OBJECTIVE: To determine whether cytologic examination of exfoliative specimens obtained during endoscopy was as useful as histologic examination of mucosal biopsy specimens for the diagnosis of gastrointestinal tract disease in dogs and cats and to compare the diagnostic accuracy of 2 techniques (brush or touch) in preparing specimens for cytologic examination. DESIGN: Prospective case series. ANIMALS: 85 dogs and 23 cats. PROCEDURE: Specimens for cytologic and histologic examination were obtained during routine endoscopic examination of the stomach, small intestine, and colon. A diagnosis was made on the basis of cytologic findings (graded objectively) and compared with the diagnosis on the basis of histologic findings. RESULTS: The diagnostic accuracy of cytologic examination was high for all 3 organs. Sensitivities, specificities, and predictive values of positive and negative results were > 90% in most instances. The diagnostic accuracy of the brush technique was equal or superior to that of the touch technique for 84% of specimens. The brush technique was most useful in detecting cellular infiltrates in the lamina propria, whereas the touch technique was more likely to detect acute mucosal inflammation. Percentages of false-positive (3.2%) and false-negative (6.9%) cytologic interpretations were low. CLINICAL IMPLICATIONS: Endoscopy is safe and requires little time to procure specimens for cytologic examination, which can be obtained concurrently with mucosal biopsy specimens. Cytologic examination of exfoliative specimens obtained during endoscopy is a useful and reliable adjunct to histologic examination of biopsy specimens in the diagnosis of gastrointestinal tract disease in dogs and cats.

Normal reference intervals and the effects of time and feeding on serum bile acid concentrations in llamas.

Fifty clinically healthy llamas, 0.5-13 years of age (22 intact males, 10 neutered males, 18 females), with no biochemical evidence of liver disease or hematologic abnormalities, were selected to establish serum bile acid reference intervals. Serum samples submitted to the clinical pathology laboratory were analyzed using a colorimetric enzymatic assay to establish bile acid reference intervals. A nonparametric distribution of llama bile acid concentrations was 1-23 micromol/liter for llamas >1 year of age and 10-44 micromol/liter for llamas < or = 1 year of age. A significant difference was found between these 2 age groups. No correlation was detected between gender and bile acid concentrations. The reference intervals were 1.1-22.9 micromol/liter for llamas >1 year of age and 1.8-49.8 micromol/liter for llamas < or = 1 year of age. Additionally, a separate group of 10 healthy adult llamas (5 males, 5 females, 5-11 years of age) without biochemical or hematologic abnormalities was selected to assess the effects of feeding and time intervals on serum bile acid concentrations. These 10 llamas were provided fresh water and hay ad libitum, and serum samples were obtained via an indwelling jugular catheter hourly for 11 hours. Llamas were then kept from food overnight (12 hours), and subsequent samples were taken prior to feeding (fasting baseline time, 23 hours after trial initiation) and postprandially at 0.5, 1, 2, 4, and 8 hours. In feeding trials, there was no consistent interaction between bile acid concentrations and time, feeding, or 12-hour fasting. Prior feeding or time of day did not result in serum bile acid concentrations outside the reference interval, but concentrations from individual llamas varied within this interval over time.

Clinical and clinicopathologic abnormalities in greyhounds with cutaneous and renal glomerular vasculopathy: 18 cases (1992-1994)

OBJECTIVE: To determine clinical signs and clinicopathologic abnormalities in Greyhounds with cutaneous and renal glomerular vasculopathy and to determine whether there were any differences between dogs with and without renal azotemia. DESIGN: Retrospective study. ANIMALS: 18 Greyhounds. PROCEDURE: Results of CBC, serum biochemical analyses, urinalyses, coagulation tests, tests of RBC morphology, bacterial culture of blood samples, and serologic tests for Rickettsia rickettsii, Ehrlichia canis, E platys, and Leptospira interrogans were reviewed. Glomerular filtration rates and urine protein:creatinine ratios were determined in most dogs. t-Tests and a test of equality of proportions were used to compare dogs that developed renal azotemia with dogs that did not. RESULTS: None of the dogs was bacteremic or had serologic evidence of infectious disease. Ten dogs had renal azotemia, 16 had anemia, 11 had hypoalbuminemia, and 18 developed thrombocytopenia. Compared with dogs without renal azotemia, dogs with renal azotemia had significantly lower mean platelet count, hematocrit, and serum albumin concentration and significantly higher mean neutrophil count and creatine kinase activity. All 10 dogs with renal azotemia died or were euthanatized; 7 of 8 dogs without azotemia survived. CLINICAL IMPLICATIONS: Greyhounds with cutaneous and renal glomerular vasculopathy that developed renal azotemia had evidence of more severe systemic disease than did dogs that did not have azotemia and, despite supportive treatment, had a poorer prognosis.

Comparison of the effect of different opsonins on the phagocytosis of fluorescein-labeled staphylococcal bacteria by chicken heterophils.

Heterophil phagocytosis of fluorescein-labeled staphylococcal bacteria was analyzed by flow cytometry. Opsonization with two types of normal pooled sera and staphylococcal antisera significantly increased bacterial phagocytosis compared to samples without an opsonin. The staphylococcal antisera did not significantly increase bacterial phagocytosis compared to the normal pooled sera. Opsonization appears to increase bacterial phagocytosis but specific antisera may not increase phagocytosis beyond that caused by pooled normal sera.

Atypical pneumonia associated with ryegrass staggers in calves.

A group of 6- to 8-month-old calves developed head tremors, stiff gait, and staggering after consuming ryegrass straw that contained 3,711 micrograms of lolitrem-B/ kg. Signs were consistent with ryegrass staggers syndrome. At necropsy, all calves examined had atypical interstitial pneumonia, with marked emphysema and bullae. Infectious organisms and pneumotoxins were not identified. Experimentally, feeding the same ryegrass straw to age-matched calves induced similar neurologic signs, but did not result in pneumonic lesions. The high concentration of lolitrem-B in the straw or other, undefined factors, such as feed changes, may have contributed to the atypical interstitial pneumonia in the naturally exposed calves.

The effects of haemolysis on serum chemistry measurements in poultry.

The effects of haemolysis on serum chemistry values in broiler and layer chickens, and in turkeys were determined using two types of serum chemistry analysers, a wet reagent analyser and a dry slide reagent analyser. The interfering effects of haemolysis were evaluated using eight levels of haemoglobin in serum analysed by the wet reagent instrument and six levels of haemoglobin in serum analysed by the dry slide reagent instrument. Nine serum chemistry analytical tests were performed on each analyser, including determination of glucose, total protein, albumin, creatine kinase, gamma-glutamyl transferase, aspartate aminotransferase, calcium, phosphorus and uric acid. The interfering effects of haemolysis varied depending on type of analyser, type of bird and the specific test. With the wet reagent chemistry analyser, the gamma-glutamyl transferase, phosphorus and uric acid analytes were most sensitive to haemoglobin interference, and the albumin, total protein and creatine kinase analytes were most resistant. With the dry slide reagent analyser, the gamma-glutamyl transferase, phosphorus, and albumin analytes were most sensitive to haemoglobin interference, and the glucose and aspartate aminotransferase analytes were most resistant. The effects of haemoglobin interference were not consistent from one type of chemistry analyser to another. The dry slide reagent analyser did not appear to resist the effects of haemoglobin interference better than the wet reagent analyser in this study. Our results suggest the need to construct interferographs for each chemistry analyser, species, and type of bird.

Multicentric malignant lymphoma in a pallid bat.

A 2.5-year-old, female pallid bat (Antrozous pallidus) was captured in Oregon (USA) in 1990 and later died in 1992. At the time of death, abdominal distension due to ascites, splenomegaly, and hepatomegaly, and a cheek mass were noted. Based on histologic examination of these tissues, a diagnosis of multicentric lymphoma was made. Retroviral particles were not found on electron microscopic examination.

Serum chemistry of bowhead whales (Balaena mysticetus).

Sera of 19 male and female bowhead whales (Balaena mysticetus) collected near Barrow, Alaska (USA) between 30 August and 13 October 1992 were evaluated for 18 serum chemistry values. Male bowhead whales had significantly greater creatinine and sodium concentrations, and significantly lower glucose concentrations than females. Pregnant females had greater triglyceride levels than non-pregnant females. The mean concentrations of creatinine, blood urea nitrogen, alkaline phosphatase, total bilirubin, total protein, sodium, potassium, chloride, phosphorus, and calcium were similar to those previously reported from bowhead whales. High aspartate aminotransferase and creatinine kinase levels were attributed to muscle damage associated with harpooning.

Chicken heterophil chemotaxis using Staphylococcus-generated chemoattractants.

Heterophil chemotaxis, in response to chemotactic factors generated by three different strains of staphylococcal bacteria, was measured using the modified Boyden-chamber technique. Heterophils were obtained from healthy 6-to-8-week-old broiler chickens. Each bacterial strain generated factors that were chemotactic for chicken heterophils. Factors generated by two pathogenic isolates of Staphylococcus aureus, however, induced significantly greater chemotaxis in chicken heterophils than those generated by a nonpathogenic Staphylococcus isolate.