Santos Roseclea Chagas Dos, Pinho Flaviane Alves de, Passos Gabriela Porfírio, Larangeira Daniela Farias, Barrouin-Melo Stella Maria
Laboratory of Veterinary Infectious Diseases, Teaching Hospital of Veterinary Medicine, Federal University of Bahia (UFBA), Av. Adhemar de Barros 500, Salvador, BA, CEP: 40170-110, Brazil.
Laboratory of Veterinary Infectious Diseases, Teaching Hospital of Veterinary Medicine, Federal University of Bahia (UFBA), Av. Adhemar de Barros 500, Salvador, BA, CEP: 40170-110, Brazil; Department of Veterinary Anatomy, Pathology and Clinics of the School of Veterinary Medicine and Zootechny, UFBA, Salvador, BA, CEP: 40170-110, Brazil.
Vet Parasitol. 2018 Jun 15;257:10-14. doi: 10.1016/j.vetpar.2018.05.010. Epub 2018 May 22.
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 × 10 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 × 10/mL vs. NNN-R = 3.25 × 10/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.
从犬类生物样本中体外分离利什曼原虫属最常用的培养基是双相诺维-麦克尼尔-尼科尔（NNN）培养基，其固相用兔血制备。当首次从犬体内获取时，自然感染的婴儿利什曼原虫寄生虫在无共生体条件下对生长高度敏感且要求苛刻。本研究的目的是评估用鸡血（NNN-C）、牛血（NNN-O）、马血（NNN-H）或羊血（NNN-S）制备的NNN培养基（NNN试验）是否适用于从内脏利什曼病流行地区自然感染的犬只中分离寄生虫，该病由婴儿利什曼原虫引起。将先前经寄生虫学方法诊断为感染的6只犬的脾脏穿刺物同时接种到每种NNN试验培养基中，包括用兔血制备的传统培养基（NNN-R），并在光学显微镜下检查培养物三周。还通过定量PCR（qPCR）分析脾脏样本中的寄生虫载量。6只犬中有3只（50%）的培养物在至少一种NNN试验培养基中呈阳性：一个样本通过qPCR显示脾脏寄生虫载量最高（1.19×10个寄生虫/毫升），且在所有试验培养基中均呈阳性；第二个样本在所有接种培养基培养的第一周出现寄生虫分离，其中NNN-C培养基的平均寄生虫计数最高（NNN-C = 23.5×10/毫升，而NNN-R = 3.25×10/毫升）；第三个样本除传统对照NNN-R外仅在NNN-S培养基中呈阳性。其余3只犬的培养物在所有NNN培养基中均为阴性，包括对照和试验培养基；根据qPCR，这3只犬中有2只的脾脏寄生虫载量最低。结果表明，鸡、牛、马和羊的血液可用于制备从自然感染犬只样本中初次分离婴儿利什曼原虫的NNN培养基，必要时可作为兔血的替代品。
