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Changes in the acute phase protein concentrations in the peritoneal fluid during experimental peritonitis in horses

Grant number: 09/00289-5
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): July 01, 2009
Effective date (End): June 30, 2010
Field of knowledge:Agronomical Sciences - Veterinary Medicine - Animal Clinics and Surgery
Principal researcher:Luiz Claudio Nogueira Mendes
Grantee:Marina Gonzales de Carvalho
Home Institution: Faculdade de Odontologia (FOA). Universidade Estadual Paulista (UNESP). Campus de Araçatuba. Araçatuba , SP, Brazil

Abstract

Abdominal pain (colic) in the horse is one of the most acute problems facing equine practitioners. Peritonitis is considered to be an important complication of colic cases. Despite advances in diagnostic methods, advances in anesthetic methods, advances in surgical techniques, and the immediate institution of intense medical therapy in the postoperative period, mortality rates remain high. The purpose of this study was to determine, by means of SDS-PAGE, whether peritoneal fluid protein concentrations (fibrinogen, haptoglobin, transferring, a1-antitrypsin and ceruloplasmin) were altered in horses with experimentally induced peritonitis, establish reference intervals for these acute phase proteins in the peritoneal fluid of horses with experimentally induced peritonitis in order to consider their possible application as inflammatory markers in the abdominal cavity of horses with experimentally induced peritonitis. The following protocol was used: fifteen adult horses were randomly divided into five equal groups (G1, G2, G3, G4 e G5) of three animals each and the groups were submitted to one of the following treatments: G1 = 1´109 colony-forming units (CFU)/5 mL of E. coli + 5 g of hemoglobin; G2 = 1´109 CFU/5mL of Bacteroides fragilis + 5 g of hemoglobin; G3 = 1´109 CFU/5 mL of E. coli + 1´109 CFU/5 mL of B. fragilis + 5 g of hemoglobin; G4 = 5 g of hemoglobin and G5 (control) = 500 mL of 0.9% saline. Abdominal fluid was collected from all animals before the inoculation (time 0) and at 2, 4, 6, 8, 10, 12, 24, 36, 48, 60, 72, 120, 168 and 216 hours after inoculations (HAI) and samples were frozen at -20o C to be used in the present study. Peritoneal fluid concentrations of acute phase proteins will be determined by means of SDS-PAGE. Instrumentation to be used in this analysis will consist of an 8 x 8-in vertical gel electrophoresis system and a power supply programmed at 35 mA while samples are in the stacking gel and increased to 50 mA when samples move into the running gel. Gels will be stained for 15 minutes in 200 mL of Coomassie blue, and destained in 7% acetic acid until the gel background is completely clear. Concentration of protein fractions will be determined by use of computer-assisted videodensitometry. Proteins will be identified by use of reference markers with molecular weights of 29,000, 45,000, 66,000, 97,400, 116,000 and 205,000 Da and by comparison with electrophoretic mobility of purified albumin, fibrinogen, transferrin, haptoglobin, and a1-antitrypsin. Data will be analyzed by use of ANOVA for repeated measures. A value of P < 0.05 will be considered significant.

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