Process parameters optimization to produce the recombinant protein CFP10 for the diagnosis of tuberculosis.

The aim of this study was to evaluate the parameters that affect the production of the recombinant 10 kDa culture filtrate protein (CFP10), a promising reagent of high specificity for intradermoreaction and other antigen-based methods used in the diagnosis of tuberculosis. Conditions of Escherichia coli growth temperature, induction temperature and IPTG-inducer concentration were evaluated in shake flasks and dissolved O2 concentrations of 15 and 30% were evaluated in a bioreactor. The process parameters defined on small scale were: growth temperature between 30 and 37 °C, induction temperature of 26 °C and IPTG concentration of 0.12 mM. The process conducted with 15% dissolved O2 presented a recombinant protein yield of 78.6 mg g-1 biomass and a proportion of recombinant protein (insoluble fraction) in relation to total insoluble protein of 72%, at the time of maximum productivity. The operation with 30% dissolved O2 resulted in lower recombinant protein yields of 62.9 mg g-1 biomass and 20% in relation to total insoluble protein, but in higher overall concentration in the culture broth (69.2 mg L-1versus 48.3 mg L-1). The protein identity was confirmed by mass spectrometry, showing high similarity to CFP10, 10 kDa of Mycobacterium tuberculosis H37Rv (score 95), and the purified antigen presented reactivity by the Western blotting assay.

Cutaneous leishmaniosis in naturally infected dogs in Paraná, Brazil, and the epidemiological implications of Leishmania (Viannia) braziliensis detection in internal organs and intact skin.

Environmental changes have occurred over the years, altering the eco-epidemiological pattern of leishmaniosis in the State of Paraná, Brazil, involving the pillars of the cycle (parasite, vectors, reservoir, and environment) and their interaction. Much has been discussed about the dog's role as a reservoir of the Leishmania (Viannia) braziliensis Vianna, 1911 transmission cycle. However, this question remains unanswered. The purpose of this study was to investigate, using parasitological and molecular methods, different samples in eight naturally infected dogs from an endemic rural locality where only L. (V.) braziliensis is present, and where human cases have been previously notified. Blood and biopsied organ samples from naturally infected dogs were analyzed by culture media, PCR, random amplified polymorphic DNA and sequencing methodologies. Only skin lesions from all dogs yielded positive cultures and when PCR was performed, L. (V.) braziliensis DNA was amplified from intact skin, peripheral blood, bone marrow, spleen, liver and lymph nodes. RAPD was also applied to isolates from the skin lesions, exhibiting the genetic variability of the parasite identified. To confirm which species of Leishmania was amplified in PCR, the sequencing method was performed, verifying 100% similarity with the Viannia subgenus. This study showed that L. (V.) braziliensis can spread to other sites besides the ulcerous lesions, such as intact skin, peripheral blood and internal organs, making it possibility for dogs to serve as active sources of parasite transmission. For definitive proof, xenodiagnostic test on intact skin of infected dogs, should be done.