Bulletin UASVM, Veterinary Medicine 67(2)/2010 ISSN 1843-5270; Electronic ISSN 1843-5378
Epidemiological Studies on some E. coli Strains in Broiler Chickens Ionica FODOR 1), Nicolae CATANA1), Viorel HERMAN1) 1)
Faculty of Veterinary Medicine, Banat University of Agricultural Sciences and Veterinary Medicine Timisoara, 119 Calea Aradului, Timisoara, Romania,
[email protected]
Abstract. Colisepticaemia is a frequently disease of poultry caused by Escherichia coli. The researches have been performed on 128 strains of E. coli isolated from broilers with different ages, from five farms, and from the shed air, only from one farm. The strains have been identified according to their biochemical characters, and then we have tested their hemolytic activity on agar with 5% sheep defibrined blood, fixed the Congo Red in agar TSA and the profile of resistance to antibiotics. This study was to confirm the association between Congo Red binding E coli and colisepticaemia in commercial broilers. Keywords: Escherichia coli, broilers, colisepticaemia, Congo Red
INTRODUCTION Avian colibacillosis is an infectious disease, extra-intestinal, caused by E.coli strains belonging to the pathotype APEC (Avian Pathogenic E. coli). More frequently, it develops in young poultry with septicemia, post-septicemia sequels and localized infections (Barnes et all. 2003, Ewers et all. 2003). The Escherichia coli strains, framed in the APEC patotype, are responsible for the extra intestinal infections, due to their invasive properties, having, very often their triggering point at the respiratory level. This prospectice cohort study was designed to confirm the association between Congo red binding E.coli (CREC) and colisepticaemia in commercial broilers. MATERIALS AND METHODS The researches have been performed on 128 strains of E. coli isolated from broilers with different ages, from five farms, and from the shed air, only from one farm. The bacteriological exam has been made on chicken bodies from the groups where the avian colibacillosis had evolved. The bodies have been anatomopathologically tested, and, depending on the existing injuries, samples have been taken (long bone), and used for the primary inseminations. The primary inseminations were made using the long bone (femur), using the usual methodology of working, in bouillon and agar. After 18-20 hours of incubation, at 37°C in aerobiosis conditions, the cultural characters have been appreciate again from the cultures with bacilar form bacteria, Gram negative, passages on the medium S-S have been made in order to mark out the fermentation of the lactose on the Levin medium as well. The biochemical properties have been marked out on multi test mediums, respectively on the TSI medium (Triple Sugar Iron) and on the MIU medium (Mobility, Indol, Urea).
74
Inseminations have been made on these mediums from positive lactose colonies, taken from the S-S and Levin mediums. After the insemination, the mediums have been incubated at thermostat for 18-20 hours. After that, the interpretation was carried out (Barnes et al. 2003). The fixing of the red Congo dye was made on TSA agar (Trypticase Soy Agar), with an additional 0,15% biliary salts and 0,03% dye, the inseminations having been made through exhaustion, with the bacteriological dowser, in order to obtain isolated colonies (McPeake et al 2005, Rodriguez et al. 2005). The strains have been identified according to their biochemical characters, and then we have tested their hemolytic activity on agar with 5% sheep defibrined blood, fixed the Congo Red in agar TSA and the profile of resistance to antibiotics (Ewers et al. 2003). Congo Red agar plates were prepared as described previously. The Congo Red agar plates were exposed in the broiler farm for 3 min by placing an open plate on top of each rack in the farms just prior to removing the chicks from the farms (Stebbins et al. 1992). RESULTS AND DISCUSSION The primary inseminations, we have isolated 128 strains (124 from bodies and 4 from air), which, according to their biochemical characters, were determined to be E. coli strains. After the insemination, made after the methodology described before, cultures characteristic of the Escherichia coli species were obtained. This way, a dense enough turbidity was noticed in bouillon, and on the agar, colonies type S with a 2-6 mm diameter opaque and with no pigmentation. On the S-S environment, the lactose positive cultures, namely Escherichia coli, have formed pink colonies, and on the Levin environment, the colonies had dark colors, with metallic gloss We mention that the isolated strains that could not reach these criteria, characteristic of the Escherichia coli species were excluded. Biochemical exams, carried out after the described methodology, have shown characters with phenotypical characteristics for the Escherichia coli. This way, on the TSI environment, on the right side, the fermentation of the glucose took place. It is characterized by the production of gas and yellow dye, and on lean surface, after the fermentation of the lactose, the color turned to yellow. The mobility was shown on the MIU environment, the production of indol and the hydrolysis of the urea. These biochemical characteristics were positive at all 121 isolated strains. This way their belonging to the Escherichia coli species was confirmed. Most isolated strains were sensible to florfenicol and ciprofloxacin, moderately sensible to colistin sulphate, spectinomycin and enrofloxacin and resistant to neomycin, tetracycline, doxycycline and erythromycin. The results achieved make evident the presence of the APEC strains in broilers; these strains may be transmitted through direct or indirect contacts with different sources within sheds. This fact was proved by the identification of 4 APEC strains within the shed air. The CREC colonies were identified by their dark red color and dry, wrinkled appearance. The Congo Red-negative E.coli colonies were mucoid and pale pink or white.
75
Tab. 1 The characteristics E. coli strains No. unit 1.
Characterizing Strains tested
2.
Fermentation of lactose
3. 4.
Hemolytic activity Congo Red in agar
S-S agar Levine agar
No
%
128 128 128 5 100
100 100 100 4 78
The fixation of Congo Red was present in 100 strains (78 %), and the hemolysis was present in five strains (tab. 1). Some researches even consider it as an epidemiological marker, destined to identify the APEC strains this character is associated with other properties of the APEC strains (Ewers et all 2003, Rodriguez et all 2005, Mellata et al. 2010). CONCLUSIONS
This study was to confirm the association between Congo red binding E coli and colisepticaemia in commercial broilers. The fixation of Congo Red was present in 100 strains (78 %), and the hemolysis was present in five strains. Antibiotic sensitivity tests showed that all isolates were sensible to: florfenicol and ciprofloxacin and resistant to neomycin, tetracycline, doxycycline and erythromycin.
Acknowledgments. This study has been financed by UEFISCU - Human Resources Program Projects Doctoral Research - Type PD. REFERENCES 1. Barnes, H. J., Vaillantcourt, J.P., Gross, W.B. (2003) Colibacillosis, in Diseases of Poultry, 11 th edition, editor in Chief, Saif, Y.M., Blackwell Publishing, Ames, Iowa, 631-657. 2. Ewers, Christa, Janben Traute, Wieler, L.H. (2003) Aviare pathogene Escherichia coli (APEQ), Munch. Tierarztl. Wschr., 116, 381-395. 3. McPeake, S. J. W., Smyth, J. A., Ball, H. J. (2005) Characterisation of avian pathogenic Escherichia coli (APEC) associated with colisepticaemia compared to faecal isolates from healthy birds, Veterinary Microbiology, 110, (3/4): 245-253. 4. Mellata Melha, Ameiss, K, Hua Mo, Curtiss R. (2010) Characterization of the Contribution to Virulence of Three Large Plasmids of Avian Pathogenic Escherichia coli _7122 (O78:K80:H9), Infection and immunity, 78 (4): 1528-1541. 5. Rodriguez-Siek, K. E., Giddings, C. W., Doetkott, C., Johnson, T. J., Fakhr, M. K., Nolan, L. K. (2005) Comparison of Escherichia coli isolates implicated in human urinary tract infection and avian colibacillosis, Microbiology (Reading), 151 (6): 2097-2110. 6. Stebbins, M E, Berkhoff, H A, Corbett, W T (1992) Epidemiological studies of Congo Red Escherichia coli in broiler chickens, Can J Vet Res. 56(3): 220–225.
76
