Leishmania infantum-specific IFN-? production in stimulated blood from cats living in areas where canine leishmaniosis is endemic

Priolo, Vito; Martínez-Orellana, Pamela; Pennisi, Maria Grazia; Masucci, Marisa; Prandi, David; Ippolito, Dorotea; Bruno, Federica; Castelli, Germano; Solano-Gallego, Laia

Feline IFN-γ concentrations were determined using a specific DuoSet ® ELISA (Development Sistems R&D ™ , Abingdon, UK) according to the manufacturer's instructions with some modifications. The standard curve for IFN-γ started with 4000 pg/ml and serial 2-fold dilutions were made until a concentration of 31.25 pg/ml was reached. Duplicates of supernatants obtained from whole blood culture were tested in the ELISA plates. The optical density was measured with an ELISA reader (Anthos 2020, Cambridge, UK) at wavelength of 450 nm. The Table 1 Age class, sex, breed, lifestyle and clinical status of cats studied according to their geographical distribution a Indoor and outdoor columns concern the lifestyle of pet cats 103 (91) 10 (9) 7 (6) 26 (23) 80 (71) 89 (79) 24 (21) All cats (N = 179; 100) 35 (19) 130 (73) 14 (8) 84 (47) 95 (53) 168 (94) 11 (6) 11 (6) 71 (40) 97 (54) 119 (66) 60 (34) standard curve was calculated using a computer generating four parameters logistic curve-fit with the program MyAssays (http://www.myass ays.com/). Cats were classified as IFN-γ producers (IFNγ-p) when the L. infantumspecific IFN-γ concentration, after subtracting the value obtained with supernatant of unstimulated cell cultures, was higher than the last detectable dilution of the standard curve (31.25 pg/ml). Similarly, cats were classified as IFN-γ non-producers (IFNγ-np) when the L. infantumspecific IFN-γ concentration, after subtracting the value obtained with supernatant of unstimulated cell cultures, was less than 31.25 pg/ml or at undetectable levels. The same cat classification was made for IFN-γ production obtained by ConA stimulation of whole blood cultures.