2010-12-英文文章大肠杆菌感染

glucan and E coli infection Introduction Escherichia 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 Cheville and Arp 1978 Rodriguez Siek et al 2005 Several E coli isolates are commonly associated with colibacillosis in 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 evidence of their immunomodulating capabilities Guo et al 2003 Zhang et al 2008 Materials and methods Experimental Animals and Treatments A 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 37 C 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 into the trachea of the 1 d old chicks by using a feeding needle in a farm setting The 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 Conditions A 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 33 C during the first week of life and later on reduced gradually 2 C per week to 20 C by the age of 6 wk Light period was kept at 12 h throughout the trial Feed and water were provided ad libitum In 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 37 C to 100 mL of tryptose phosphate broth and incubating for 2 5 h in a 37 C shaking waterbath The culture was serially diluted and held overnight for 18 to 20 h at 4 C 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 Culture The 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 4 C 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 37 C 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 37 C 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 cotton buds and plated directly on EMB agar The plates were then incubated at 37 C for 16 to 18 h and the presence of E coli colonies designated EMB E coli was determined Scoring Scheme and Laboratory Procedures Clinical 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 37 C 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 broth to 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 37 C 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 against SRBC was estimated from the serum samples collected after 10 d of first exposure to SRBC whereas the secondary antibody titer was estimated from the sera taken at the day of termination of the experiment Cell Mediated Immune Response The PHA skin test for T cell mediated immunity was conducted in 41 d old chickens following the procedures of Grasman and Scanlon 1995 using a 0 1 mL dose of 1 mg mL of PHAP Sigma St Louis MO in PBS Feathers were plucked from both wing webs One wing was injected with PHA whereas the other received a placebo injection of PBS alone The thickness of each wing web was measured to the nearest 0 05 mm immediately before and 24 3 h after the injections using vernier caliper with the precision of 0 01 mm A stimulation index was calculated as the change