Parasite burden, distribution and immunopathology of Leishmania Martiniquensis - infected BALB/c mice in different routes and time points

Abstract

Leishmaniasis is an emerging zoonotic vector-borne disease in Thailand transmitted by the bite of a tiny female phlebotomine sandfly. Autochthonous L. martiniquensis infection in Thai patients has been continuously reported since 2014 in patients co-infected with HIV, immunocompetent patient with or without underlying diseases and children. The objective of this study was to investigate the parasite burden, distribution and immunopathology in L. martiniquensis-infected BALB/c mice. Sixteen mice were intraperitoneally, i.p., intravenously, i.v., and subcutaneously, s.c., infected with 5x106 promastigotes of L. martiniquensis each. On 7, 14, 28 and 112 days post infection, dpi, 4 inoculated mice were euthanized from each inoculation group. Blood, livers, spleens, bone marrows, salivary glands and kidneys were collected at each time points. To detect the distribution of parasite, PCR targeting Leishmania-specific ITS1 genes was performed. DNA amplicons were detected from all BALB/c livers inoculated via i.v. and almost all mice via i.p. in all time points. For spleen, Leishmania DNAs were present in all time points from both i.v. and i.p. routes. Giemsa-stained impression smear of liver and spleen was conducted to analyse parasite burden in Leishman-Donovan unit (LDU). Via i.v. route, the highest parasite burden in liver was found on 7 dpi, 101 LDU, whereas it was shown in the spleen on 112 dpi, 3.1 LDU. To evaluate cell-mediated immune responses in liver, indirect immunohistochemical staining using L. martiniquensis-infected human serum was performed. The number of mature granuloma on 7, 14 and 28 dpi from i.v. route was found significantly superior than the non-infected control and other routes (p < 0.05). Reduction of immature and mature granuloma with development of involuting granuloma on 112 dpi in livers revealed the ability of BALB/c cellular immunity to control L. martiniquensis infection. In conclusion, this study is the first to reveal that L. martiniquensis is a causative agent for visceral leishmaniasis in BALB/c mice by experimental inoculation via i.v. and i.p. routes based on the presence of amastigotes as well as genomic DNAs of L. martiniquensis in important target organs.