Gamma-interferon, interleukin-10 and neopterin levels of sheep diagnosed with Mycobacterium avium subspecies paratuberculosis infection

Paratuberculosis is a disease caused by Mycobacterium avium subspecies paratuberculosis (Map) and characterized by chronic granulomatous enteritis. Different tests are used in the diagnosis according to the stage of the disease. The purpose of the present research was to investigate paratuberculosis infection in sheep by interferon gamma (IFN-γ) ELISA, antibody ELISA, Ziehl-Neelsen (ZN) staining, polymerase chain reaction (PCR) and culture methods and to compare the antibody, IFN-γ, interleukin -10 (IL-10) and neopterin (NP) levels between animals determined as positive and negative for paratuberculosis. Two years and older sheep were included in the study. Ten sheep from each of 15 sheep farms were selected randomly in Burdur province and total 150 blood and fecal samples were collected. Blood serum samples were examined for antibodies against Map and plasma samples were analyzed by ELISA for IFN-γ. Stool samples were used for ZN staining, PCR and culture. After decontamination, stool samples were planted on HEYM and modified Middlebrook 7H10 agar media. The DNA samples extracted from the fecal samples were analyzed by classical and nested PCR using two Map specific primer pairs (P90/P91 and 150C/921). DNA samples obtained from stool samples and Map isolates were typed by restriction endonuclease analysis (REA) after PCR procedure for IS1311 gene region. The levels of antibody, IFN-γ, IL-10 and NP in 40 sheep determined as positive and 40 sheep determined as negative were compared based on the mean optical density (OD) values. Of the 150 sheep in the study, 73 were found to be infected. The type of Map was determined to be sheep type by PCR-REA. None of 73 sheep were positive for all of the tests and the highest rate of positivity was determined by ZN staining (27,33%) and PCR (26,66%). This was followed by IFN-γ ELISA (22%), antibody ELISA (10.67%) and culture (3.33%). According to the culture results; the sensitivity and specificity of the ZN staining, PCR, IFN-γ and antibody ELISA tests were calculated as 80-74%, 60-74%, 20-77%, 20-89% respectively. There was no statistically significant difference (p>0,05) between the age groups of animals determined as positive and negative for paratuberculosis, whereas the difference between diarrhea and body condition scores was significant (p<0,01). Although antibody, IFN-γ, IL-10 and NP levels of positive animals were higher than negative animals, statistically significant difference was determined in antibody, IFN-γ, and IL-10 levels (p<0,01). In positive animals, except IL-10 levels (p>0,05), the difference was determined in antibody, IFN-γ and NP levels (p<0,01) between animals groups constituted according to test results. As a result; It was concluded that paratuberculosis can be detected by IFN-γ ELISA, antibody ELISA, ZN, PCR or culture methods in sheep as early as 2 years old depending on the stage of the disease, but ZN staining or PCR methods with higher sensitivity and specificity can be used in the clinical stage of the disease. On the other hand, since IL-10 levels were found to be very different between positive and negative animals and measurable in serum at every period of the infection, it was thought that this cytokine could be used for diagnosis as IFN-γ.