Determination of intracellular cytokines IFN-gamma and IL-4 in canine T lymphocytes by flow cytometry following whole-blood culture.

This report describes a whole-blood flow cytometric method for the determination of intracellular cytokines IFN-gamma and IL-4 in canine T lymphocyte subpopulations. Conjugated anti-cytokine antibodies and commercially available reagents for cell fixation and permeabilization were used. Canine peripheral blood was cultured with a combination of phorbol-12-myristate-13-acetate (PMA) and ionomycin to promote cytokine synthesis in each cell, along with monensin to increase the sensitivity of the method by retaining IFN-gamma and IL-4 within the cell to detectable levels. The optimum concentrations of PMA and ionomycin were determined. Maximum IFN-gamma expression from both CD4+ and CD8+ T lymphocytes was detected after 6 h of incubation of cell culture, while maximum IL-4 production took 6 h from CD4+ cells and 4 h from CD8+ cells. This method is a simple immunologic technique for measuring intracellular cytokines which could be of value in the investigation of canine immunological response mainly in various intracellular and extracellular infections, since IFN-gamma and IL-4 are considered key cytokines activating the cellular and humoral immunity, respectively.

Some microbiological aspects of rats captured alive at the port city of Piraeus, Greece.

The aim was to investigate the presence of leptospira infection and the antibiotic resistance of Enterobacteriaceae among wild rats living in a densely populated area. For this purpose 3-4 large traps were placed daily across the commercial port-front of Piraeus for the duration of two months. The rats captured were transferred in the laboratory alive, where samples were freshly collected. None of the 25 rats captured was found to be infected with leptospira. Only one rat was infected with Salmonella. Other predominant members of the family of Enterobacteriaceae were Escherichia coli, Proteus spp. and Klebsiella spp. Resistance to antimicrobial agents commonly used in veterinary treatment was very high for spectinomycin, erythromycin, neomycin and tetracycline in all tested species of Enterobacteriaceae. It appears that port rats living in the densely populated area of the city of Piraeus could become a source of infection for man and animals with microorganisms highly resistant to antimicrobial agents.

A cross-sectional study of Leishmania spp. infection in clinically healthy dogs with polymerase chain reaction and serology in Greece.

A total of 73 clinically healthy hunting dogs, experiencing an outdoor lifestyle and originating from an area where canine leishmaniasis is endemic, were included in the study. Polymerase chain reaction (PCR) and indirect immunofluorescent antibody test (IFAT) for Leishmania spp. were done on bone marrow and serum samples, respectively, obtained from all 73 dogs, just before the beginning of the sandfly season. PCR was found positive in 46/73 (63%) whereas, IFAT only in 9/73 (12.3%) of the dogs. The prevalence and the incidence of Leishmania infection by PCR were 61.9 and 47.1%, respectively. No association was found between the breed, age, sex, length of hair coat of the dog, urban or rural life and the presence of ample vegetation and water collections in the proximity of their living quarters, and the result of PCR. These findings clearly demonstrate that most of the dogs residing areas where leishmaniasis is endemic become infected but usually remain seronegative. Serological screening of the general canine population in these areas may result in an underestimation of the true prevalence of the infection rate.