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Development of immunohistochemistry for detection of Brucella suis type-2 in formalin-fixed tissues from pigs

Introduction The aim of this study is to develop an immunohistochemical method for detection Brucella suis type 2 and to compare this method with PCR detection and bacteriological isolation. Material and methods Testis and lymp hnodes embedded in paraffin blocks from 10 naturally infected pigs that were PCR and bacteriologically positive to Brucella suis type 2 were analyzed by immunohistochemistry (IHC). Demonstration of Brucella suis type 2 performed using polyclonal antibodies and provided by Dr. Silvio Spicic (Croatian Veterinary Institute, Zagreb, Croatia). Briefly, 5μm cuts were placed on silanized slides, section was deparafinized in xilen and rehydrated through different grades of ethanol. Endogenous peroxidase was blocked with 3% H202 in 0.5M/0.15M Tris/NaCl (TBS). Slides were pretreated with Proteinase K (Dako ® ) 3 min. on room temperature according manufacture instruction. Non-specific reaction was blocked with incubation 1 hour in 5% normal goat serum. Primary antibody was diluted 1/500 in 2% normal goat serum in TBS and incubated over night at +4°C. Anti-mouse secondary antibody marked with labeling polymer (Dako Envision) was incubated according with manufacture instruction. For visualization is used DAB chromogen, slides were counterstained with Mayer’s hematoxylin and then were dehydrated and mounted with DPX. On a tissue from clinically healthy pigs and PCR and bacteriologically negative and all IHC positive samples without administration primary antibody was preformed complete IHC procedure and was used as negative controls. Results In testis and lymph nodes of PCR and bacteriologically positive pigs Brucella suis type 2 was found in all diseased animals. In their testis samples large amounts of Brucella suis type 2 antigen were concentrated in the machrophages-like cells of the inflammatory foci in testis and in hystiocytes of lymph nodes stroma. In all negative control samples show no positive reaction.. Discussion An optimized IHC protocol was developed for the detection of Brucella suis type 2 in formalin-fixed, paraffin-embedded tissues and its results were compared with those determined by PCR and bacteriological isolation. The results obtained show that the use of IHC test is a reliable detection method. In the present study observed infection pattern is consistent with similar studies. When only formalin-fixed tissues are available, IHC would be useful diagnostic methods for the detection of Brucella suis type 2. The high specificity of IHC test for Brucella suis type 2 could be used in experimental studies of disease in pigs.

Brucella suis type 2; IHC

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