Isolation of naturally infecting Leishmania infantum from canine samples in Novy-MacNeal-Nicolle medium prepared with defibrinated blood from different animal species

Abstract

The most commonly used culture medium for the in vitro isolation of Leishmania spp. from canine biological samples is biphasic Novy-MacNeal-Nicolle (NNN) medium, whose solid phase is prepared using rabbit blood. Leishmania infantum parasites from natural infections are highly sensitive and demanding for growth in axenic conditions when firstly obtained from the dog’s body. The objective of this study was to evaluate whether NNN medium (NNN-test) prepared with chicken blood (NNN-C), ox blood (NNN-O), horse blood (NNN-H) or sheep blood (NNN-S) was viable for the isolation of parasites from naturally infected dogs, in an endemic area for visceral leishmaniasis caused by L. infantum. Spleen aspirates from six dogs previously diagnosed as infected by parasitological methods were simultaneously inoculated in each NNN-test medium, including the conventional medium prepared with rabbit blood (NNN-R), and the cultures were examined for three weeks under optic microscopy. Spleen samples were also analyzed for parasite loads by quantitative PCR (qPCR). Cultures from three of the six dogs (50%) were positive in at least one of the NNN-test media: one sample presented the highest spleen parasite load by qPCR (1.19 × 104 parasites/mL) and was positive in all test media; the second sample presented parasitic isolation in the first week of culture in all inoculated media, of which the NNN-C medium had the highest mean parasite count (NNN-C = 23.5 × 104/mL vs. NNN-R = 3.25 × 104/mL); the third sample was positive only in the NNN-S medium besides the conventional control NNN-R. Cultures from the three remaining dogs were negative in all NNN media, including the control and test media; of those three dogs, two presented the lowest spleen parasitic loads according to qPCR. Blood from chicken, ox, horse and sheep shown to be viable for the preparation of NNN culture medium for the primary isolation of L. infantum from samples of naturally infected dogs and can be considered as an alternative to rabbit blood when necessary.