catus /em /th th align=”left” rowspan=”1″ colspan=”1″ em F. in wildcats and feral cats. Due to their potential role as viral reservoirs data concerning the health status of these animal populations will be needed in a future risk analysis. 2.?Material and methods 2.1. Biological sampling Between 1992 and 2007, 50 samples of LTE and/or serum and 28 stools samples were collected from 32 wildcats and 21 feral domestic cats in continental Portugal (Fig. 1 ), Baloxavir either from live captured animals or from carcasses, after a standard necropsy procedure. Animals were identified according to morphological characters (Kitchener et al., 2005), available genetic information (Oliveira et al., 2008, Pierpaoli et al., 2003; Driscoll and Fernandes, data not published) and lifestyle. No data was available regarding four samples. Open in a separate window Fig. 1 Geographic distribution of the Baloxavir samples collected from domestic; unknown and wild cats. A proposed area of occurrence for the wildcat in Portugal in shown based on nonsystematic data collected from 1996 to 2007 (Fernandes, 2007). 2.2. Serological screening Serum and lung tissue extract (LTE) (Ferroglio et al., 2000), were used for FeLV p27, FIV antibodies (Ab) and FCoV Ab detection by Enzyme-linked immunoassay using commercial kits (Virachek Felv Aufel 1N; ViraCHEK? FIV ACFIV3M, Synbiotic, France and Ingezim Corona Felino 16.FCV.K.1, Spain) following the manufacturer instructions; CDV Ab were also screened by a commercial enzyme-linked immunoassay (Ingezim Moquillo IgG 15.CDG.K.1) using Protein A-Peroxidase from Staphylococcus aureus/horseradish (SigmaCAldrich), for detection of the primary Ab/Ag Baloxavir complex (Lindmark et al., 1983). 2.3. Detection of viral nucleic acids Viral RNA and DNA were simultaneously extracted from stool using the QIAmp? Minielute? Virus spin Kit (Qiagen, Germany) following the manufacturer instructions. Detection of FCoV RNA was performed according to Herrewegh et al. (1995); FPV DNA was screened according to Desario et al. (2005) and CDV RNA according to Frisk et al. (1999). The amplification products were visualized after electrophoresis Mouse monoclonal to GSK3B in a 2% agarose gel, stained by EtBr in an Image Master? VDS (Pharmacia Biotech). 3.?Results Considering the geographical distribution (Fig. 1), 16 animals were caught in Northern Portugal mainly in the natural areas of Peneda-Geres National Park; and also Montesinho and Douro Internacional natural parks; cats from the center of the country were mainly from Serra da Estrela Natural Park with the exception of one animal from Serra de S. Mamede Natural Park. The remaining samples (LTE/serum?=?37; stool?=?21) were from 41 animals caught in Alentejo and Algarve in the South, including the Vale do Guadiana Natural Park ( em n /em ?=?21) and the Natura 2000 area of Moura Barrancos ( em n /em ?=?5). FeLV p27 antigen was detected in 13/50 samples (26%), FCoV RNA was present in 6/28 samples (21.4%) and FPV DNA in 2/28 samples (7.1%) (Table 1 ). CDV RNA was not detected ( em n /em ?=?13). CDV antibodies were detected in 2/26 samples (7.7%); FIV ( em n /em ?=?50) and FCoV ( em n /em ?=?26) antibodies were not detected. Feral cats ( em F. s. catus /em ) showed a higher frequency of FeLV antigen (33.3%) and FPV DNA (20%), and wild cats ( em F. s. silvestris /em ) of FCoV RNA (33.3%). Table 1 Frequencies of Ag (FeLV), Ab (FIV; CDV, FCoV) and FCoV, CDV and FPV nucleic acid in biological materials from wild ( em Felis silvestris /em ) and feral cats ( em Felis catus /em ) with 95% confidence intervals. thead th.