英文文章大肠杆菌感染.doc

-glucan and E. coli infectionIntroductionEscherichia coli is commonly found in the avian gastrointestinal tract and other mucosal surfaces. Although most of the strains are commensals, a separate group, designated avian pathogenic E. coli, has the ability to cause extraintestinal disease in poultry, collectively called colibacillosis (Kariyawasam et al., 2006; Bonnet et al., 2009). Serotypes O1, O2, and O78, and to some extent O15 and O55, are the most common serotypes associated with colibacillosis found in chickens (Gomis et al., 1997; Raji et al., 2007). They commonly cause airsacculitis, pericarditis, perihepatitis, peritonitis, salpingitis, and subsequently the most acute form, septicemia, resulting in sudden death (Mellata et al., 2003; Ask et al., 2006). The poultry industries worldwide suffer great financial losses every year because of the high morbidity and mortality rates caused by colibacillosis. Treatment strategies include the control of environmental factors and the use of antibiotics. However, concerns exist regarding the emergence of antibiotic resistance of normal microflora and pathogenic bacteria, which may in turn threaten human health through transfer f drug resistance genes to zoonotic bacteria (Food and griculture Organization of the United Nations, World ealth Organization, and World Organization for Animal ealth, 2003).Avian colibacillosis, a disease caused by a group of bacteria called avian pathogenic Escherichia coli (APEC) in chickens, turkeys, and other avian species, is an infectious disease that often causes severe mortality and subsequently results in economic losses to the poultry industry ( Gibbs et al., 2004). The disease is associated with a complete set of syndromes including septicemia, airsaculitis, pericarditis, and swollen head syndrome (Chevilleand Arp, 1978; Rodriguez-Siek et al., 2005). Several E.coli isolates are commonly associated with colibacillosisin poultry, and the serogroups O1, O2, and O78 have been recognized as the predominant sources involved in this disease (Whittam and Wilson, 1988; McPeake et al., 2005). A high raA high rate of antibiotic resistance was observed while testing these serogroups, which probably originates from the extensive use of antibiotics in the poultry industry (Allan et al., 1993), as well as by acquisition of R plasmids (Johnson et al., 2005b; Skyberg et al., 2006). Numerous concerns about the use of antibiotics in the poultry industry have been raised including the further selection of drug-resistant strains (Franklin, 1999; Angulo et al., 2004). There are also human health issues involved due to the potential transfer of E. coli from animals via the food chain (Angulo et al., 2004; Johnson et al., 2005a). This has attracted considerable attention from researchers who are seeking alternatives for control and treatment of colibacillosis in animals.One promising alternative to antibiotics is the use of virulent bacteriophage against E. coli serogroups O1, O2, and O78, a well-established approach that phages for these serogroups are able to be isolated and used in phage therapy against bacterial cells. Bacteriophages are a class of viruses that live and replicate in bacteria (Ackermann, 2000) and have the ability to attack a single species or subset of a species of bacterium, making them potential antibacterial agents. -Glucans have been well studied in human and animal subjects, and their immune-enhancing effects have been well noted (Volman et al., 2008). Due to their ability to augment the immune response, -glucans have been termed biological response modifiers.-Glucans are structural components of the cell wall of many bacteria, fungi, and yeast, as well as cereal grains such as oat and barley. -Glucans from fungal and yeast sources have been widely studied and shown to be most effective in enhancing protective immunity against infectious agents (Soltanian et al., 2009). Though the immune-enhancing capabilities of -glucans have been proven in mammals, limited reported research is available for poultry, with mixed results in terms of performance and immune response. Some studies have shown that -glucan supplementation improves BW (Zhang et al., 2008), whereas other groups have found no significant effects (Chae et al., 2006). Huff et al. (2006) reported contradictory results in which -glucan supplementation was detrimental to BW in a nonchallenge setting but was found to be beneficial during an Escherichia coli challenge. These varying results indicate that more research needs to be carried out to determine the optimal dosage and proper usage of -glucans to obtain consistent results. -Glucans have beneficial effects on both the innate and adaptive immune systems. When exposed to -glucans in vitro, chicken macrophages and splenocytes have been shown to experience enhanced proliferation and improved phagocytic capabilities (Chen et al., 2003; Guo et al., 2003). In terms of the adaptive immune response, -glucans magnify plasma IgG and IgA levels, indicating an upregulation of the humoral immune response (Zhang et al., 2008). The T-lymphocyte subpopulations are also affected, with higher CD4+, CD8+, and CD4+:CD8+ T-cell populations found in chickens supplemented with -glucan (Chen et al., 2003; Chae et al., 2006). Furthermore, -glucans have demonstrated the ability to augment the secretion of several cytokines to aid in pathogen elimination. Macrophages isolated from birds fed -glucans demonstrated enhanced interleukin (IL)-1 (Guo et al., 2003), IL-2, and interferon (IFN)- levels (Zhang et al., 2008). Dietary -glucan has also been shown to increase the size of the primary and secondary lymphoid organs, providing further evidenceof their immunomodulating capabilities (Guo et al., 2003; Zhang et al., 2008).Materials and methods Experimental Animals and TreatmentsA 3-wk experiment was conducted to determine the efficacy of bacteriophage EC1 in treating respiratory infection in birds caused by E. coli O78:K80. A total of 480 one-day-old male broiler chicks (Ross 308) were obtained from a commercial hatchery. The chicks were assigned randomly to 4 treatment groups, each with 4 pens of 30 chicks per pen. Water and broiler feed (antibiotic free) were provided ad libitum throughout the experimental period. The 4 treatment groups were group I (control), in which untreated, unchallenged birds were administered 0.2 mL of PBS only (0.14 M NaCl, 0.0027 M KCl, 0.01 M Na2HPO4, 0.0018 M KH2PO4; pH 7.4); group II (control), in which unchallenged birds were treated with 0.2 mL of bacteriophage EC1 (1011 pfu/ mL); group III, in which birds were challenged with 0.2 mL of a 5-h-old E. coli O78:K80 culture (grown in Luria-Bertani broth at 37C and shaken at 180 rpm) containing 109 cfu of bacterial cells/mL, followed by 0.2 mL of bacteriophage EC1 (1011 pfu/mL) at 2 h postchallenge; and group IV, in which birds were challenged with 0.2 mL of a 5-h-old E. coli O78:K80 culture containing 109 cfu of bacteria cells/mL only. The time point at which to inoculate the bacteriophage (2 h postchallenge) was selected based on the results of a preliminary trial showing that E. coli O78:K80 had colonized the lungs and that the bacteria had spread to other organs, such as the liver and heart, 2 h after the birds were challenged with the pathogen (data not shown). All the materials were inoculated directly intothe trachea of the 1-d-old chicks by using a feeding needle in a farm settingThe BW of live birds were taken weekly. Sampling was carried out on d 0 (before inoculation of E. coli or bacteriophage EC1), 1, 2, 3, 7, 14, and 21 from 3 of the pens of each treatment group. The last pen was used for the observation of mortality rate. On each sampling day, 6 birds from each group (2 randomly selected from each of the 3 sampling pens) were weighed and killed by CO2 inhalation for laboratory examination. Birds that died on the sampling day were also dissected and subjected to the same laboratory examinations. All animal management and sampling procedures complied with the guidelines of the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (Federation of Animal Science Societies, 1999).Animals and Housing ConditionsA total of 144 specific pathogen-free (SPF) chickens (Valo; Lohmann-Tierzucht, Cuxhaven, Germany) hatched at the Clinic for Avian, Reptile and Fish Medicine, University of Veterinary Medicine, Vienna, Austria, were used in the study. The chickens were distributed randomly and kept under controlled conditions in sterilized isolation units (Montair Andersen B.V. HM 1500, Sevenum, the Netherlands; size: 1.2 m2) with the airflow of 30 to 32 m3/h. The temperature was adjusted at 33C during the first week of life and later on reduced gradually (2C per week) to 20C by the age of 6 wk. Light period was kept at 12 h throughout the trial. Feedand water were provided ad libitumIn experiments, birds were kept under incandescent lighting on a light schedule consisting of 23 h light and 1 h dark. They were provided ad libitum access to water and an unmedicated standard corn and soybean broiler starter diet that met or exceeded the NRC recommended allowances (National Research Council, 1994), and which contained 3,000 kcal of ME/kg and 21.0% CP. Birds were fed an unsupplemented diet or the same diet supplemented with a low level (LL) of 500g/tonne (1lb/ton) or a high level (HL) of 1,000 g/tonne (2 lb/ton) of a standardized yeast extract feed supplement (Alphamune). Individual bird weights and feed consumption by pen were determined weekly.The temperature of the control room was maintained according to established standard operating procedures. Brooders were set at 32.3C for the first week, after which room temperature was maintained at 24.8C 1.5 and RH at 63 2.3% for the remainder of the study using an automated air handling system.At 7 d of age, during the cold-stress treatment, birds were challenged by coarse spray inoculation of eyes and nares with approximately 3 to 4 108 cfu of a nonmotile serotype O2 strain of E. coli that had originally been isolated from chickens with colisepticemia and has been used to reproduce turkey osteomyelitis complex (Huff et al., 1998, 2000). The inoculum was prepared by adding 2 loops of a fresh 18-h culture that was grown on Columbia sheep blood agar at 37C to 100 mL of tryptose phosphate broth and incubating for 2.5 h in a 37C shaking waterbath. The culture was serially diluted and held overnight for 18 to 20 h at 4C while a standard plate count was made and counted.Mortality data were collected twice each day after challenge and birds were weighed and examined for lesions of airsacculitis. The following key, modified from that described by Piercy and West (1976), was used to score lesions of airsacculitis and pericarditis observed in both mortalities and at necropsy: 0 = no inflammation; 1 =opacity and thickening of the inoculated air sac; 2 = mild airsacculitis and mild pericarditis; 3 = moderate airsacculitis or pericarditis with spread to liver or abdominal cavity(perihepatitis or peritonitis); 4 = severe fibrinous airsacculitis and severe pericarditis; and 5 = severe airsacculitis or pericarditis with spread to liver or abdominal cavity.E. coli Challenge CultureThe E. coli used in these studies were initially isolated from the blood of chickens with colisepticemia (Bayyari et al., 1997; Huff et al., 1998). This E. coli strain is serotype 02, nonmotile, and lactose negative. The E. coli culture was prepared by inoculation of tryptose phosphate broth (Sigma Chemical Co., St. Louis, MO) that was incubated in a shaking water bath for 2.5 h. The culture was removed from the water bath and held at 4C. The culture was enumerated by making duplicate 10-fold serial dilutions of the culture and by spread-plating the appropriate dilutions in duplicate on tryptose phosphate agar plates, which were enumerated after overnight incubation at 37C. The challenge cultures were made by diluting this E. coli stock culture, and verified with serial dilutions of the challenge culture and enumeration by spread plating.Preparation of Pathogenic EC. Pathogenic EC serotype O2:K1 was cultured overnight in nutrient broth at 37 C. The culture was centrifuged for 15 min at 3400 g, washed, and resuspended in PBS (pH 7.4). Bacterial concentration was measured by a spectrophotometer (570nm). Each chick received 0.1 mL of bacterial suspension (1 1010 cfu/mL in PBS).Laboratory Examinations Gross Lesion Examinations. Macroscopic examinations of the air sac, liver, and heart of slaughtered birds were carried out. Opacity or thickening of the air sac and the presence of tissue lesions or fibrinous exudates on the liver and heart were considered indicative of airsacculitis, perihepatitis, and pericarditis, respectively.Organ Weight. At necropsy, the lung, liver, heart, and spleen were excised aseptically and weighed. The weights of the organs were reported as the percentage relative to BW (organ weight/BW 100%; Huff et al., 2006a).Isolation of E. coli from Lungs (Quantitative Analysis). The lungs of birds were removed aseptically, weighed, diluted 10 in Maximum Recovery Diluent (Merck KGaA, Darmstadt, Germany), and homogenized. The homogenates were then serially diluted before plating on eosin methylene blue (EMB) agar (Merck KGaA). The EMB agar plates were incubated overnight at 37C, after which the metallic green sheen colonies of E. coli (designated EMB + E. coli) were counted to determine the number of E. coli (cfu/g) colonizing the lungs. The populations of EMB + E. coli in lung samples from birds in the different treatment groups were then compared to determine the severity of infection.Isolation of E. coli from Organs and Blood. Blood samples of birds were collected by cardiac puncture and cultured on EMB agar. The liver, heart, and spleen of each bird were cut open, and the inner parts of these organs were swabbed 3 to 4 times with sterile cottonbuds and plated directly on EMB agar. The plates were then incubated at 37C for 16 to 18 h, and the presenceof E. coli colonies (designated EMB + E. coli) was determined.Scoring Scheme and Laboratory ProceduresClinical Scores. The health status of the birds was scored from 0 to 4 on the basis of following criteria: 0 = animal active with no clinical symptoms; 1 = slightly weak, dropping wings, diarrhea; 2 = depressed with swollen crop; 3 = weak with ruffled feathers, reluctant to walk, and apathy; and 4 = animal unable to move or stand, eyes closed, and intense breathing. The health status was scored daily from day of inoculation to the day of termination of experiment.Gross Pathological Lesion Score. Tissue lesions from liver and heart were scored according to Mellata et al. (2003). The scoring scale for different organs was as follows: (i) Liver: 0 = normal; 1 = slight amounts of fibrinous exudate; 2 = marked perihepatitis. (ii) Heart and pericardium: 0 = normal; 1 = vascularization, opacity, cloudy fluid in the pericardial cavity; 2 = acute pericarditis. (iii) Lung: 0 = normal; 1 = edema; 2 = edema and hyperaemia; 3 = edema, hyperemia and fibrin in air sacs. (iv) Spleen: 0 = normal; 1 = swollen 2 = fibrinated bedding.Bacteriological Examinations of Tissues (Qualitative Examination). The presence of the bacterial strain used for infection was determined qualitatively by streaking the samples from liver, lung, heart, and spleen directly on McConkey agar plates. The plates were incubated overnight at 37C for 24 h and observed for the presence of E. coli.Bacterial Recovery (Quantitative Examination). Liver, heart, lung, and spleen (100 to 200 mg) were homogenized in 1 to 2 mL of PBS and 100 L of serial dilutions of the homogenate were spread on McConkey agar plates for bacterial quantification. Moreover, 1 mL of the homogenate was incubated overnight in LB brothto investigate the presence of E. coli in the tissue samples given above.Hematology and Clinical Biochemistry. Hematological investigations were performed on heparinized blood samples taken from birds during euthanization. Erythrocyte counts and PCV were measured following Swarup et al. (1986), whereas granulocytes were counted using eosinophil unopette method (Campbell, 1995). For erythrocyte counts the blood was diluted (1:200) in Natt and Herrick (1952) solution, and for granulocyte count it was diluted (1:20) in unopette solution, which stains only heterophils and eosinophils, the number of cells were counted in 9-mm area in a Neubauer chamber. For clinical biochemistry, plasma was separated by centrifuging blood at 3,380 g for 15 min, and GOT, LDH, ALP, total protein, and albumin were measured on automated clinical chemistry analyzer Hitachi 911 (Roche Diagnostics, Mannheim, Germany) with reagent test kits supplied by Roche. Globulin was determined as a difference between total protein and albumin (Varley, 1975).Humoral Immune Response. Antibodies against SRBC were measured by quantifying total antibody titer in addition to mercaptoethanol sensitive IgM and mercaptoethanol resistant IgG using microagglutination assay (Delhanty and Solomon, 1966). Briefly, 2-fold serial dilutions of serum were prepared in PBS in microtiter plates; later, an equal volume of 1% SRBC in PBS was placed in all wells. Plates were shaken for 1 min and incubated for 1 h at 37C for total antibody titer. The agglutination titer was expressed as log2 of the highest dilution of sera giving visible agglutination. For IgG the test was performed exactly in the same manner except that the plasma was incubated with equal volume of 0.2 M of 2-mercaptoethanol for 1 h at room temperature before making 2-fold dilutions. The IgM was calculated as a difference of total immunogloubin and IgG titer. Primary antibody titer aga