Parasite burden in hamsters infected with two different strains of leishmania (Leishmania) infantum: "Leishman Donovan units" versus real-time PCR

Abstract

To develop and test new therapeutics and immune prophylaxis strategies for visceral leishmaniasis (VL), understanding tissue parasitism evolution after experimental infection with Leishmania infantum is important. Experimental infection in a hamster model (Mesocricetus auratus) reproduces several typical aspects of canine and human VL that are closely related to the inoculum's route. We quantified the parasitism in the liver and spleen of hamsters experimentally infected by various routes (intradermal, intraperitoneal, and intracardiac [IC]) and different strains of L. infantum (MHOM/BR/74/PP75 and Wild) and compared two different methodologies to evaluate tissue parasitism (Leishman Donovan units [LDU] and real-time qPCR). In addition, the quantification of specific total-IgG in the serum of uninfected and infected hamsters was determined by ELISA. The animals were followed for 1, 3, 6 and 9 months post-infection for survival analysis. We found that infection with the Wild strain by the IC route resulted in higher mortality. Positive antibody (IgG) responses were detected with higher peaks at 6 and 9 months in the IC group inoculated with PP75 strain. However, in animals infected with the Wild strain the IgG levels were elevated in all infected groups during all the time evaluated. We also observed by LDU analysis that the IC route lead to higher parasitism in the liver and spleen with both strains. Furthermore, qPCR showed higher sensitivity for identifying animals with low parasitic burden. In conclusion, qPCR can be useful for assessing parasitism in the spleen and liver of a hamster model infected with L. infantum independent of the route of infection, and this technique may become an essential tool for assessing parasite density in the hamster model after experimental treatment or immunization with potential vaccine candidates.

Figure 1. Gel electrophoresis of PCR products from different DNA samples.

The PCR products were digested with Hae III restriction enzyme, and the digested samples were separated by electrophoresis in a 10% agarose gel to produce DNA fragments. Lane M: 25-base pair ladder; lane 1: L. amazonensis (MHOM/BR/1973/M2269); lane 2: L. braziliensis (MHOM/BR/1975/M2903); lane 3: L. infantum (WHO/MHOM/BR/74/PP75); lane 4: L. infantum (wild strain) and lane 5: negative control.

Figure 2. Cumulative survival in hamsters experimentally infected with two strains of L. infantum (PP75 and Wild) by different routes of inoculation assessed at 1, 3, 6, and 9 months after infection.

Control group (C; black circle) and experimentally infected animals with two strains of L. infantum (PP75 and Wild) by different routes of inoculation: intradermal (ID; black square); intraperitoneal (IP; black triangle), and intracardiac (IC; black diamond).

Figure 3. Evaluation of humoral immune response in hamsters experimentally infected with two strains of L. infantum (PP75 and Wild) by different routes of inoculation.

(A) Means of the Leishmania-specific IgG levels. (B) Correlation between IgG levels and months post-infection. (C) Correlation between IgG levels and experimental groups. IgG levels were determined by ELISA in sera of uninfected hamsters as a control group (C; white) and hamsters infected with L. infantum from intradermal (ID; light gray), intraperitoneal (IP; dark gray) and intracardiac (IC; black) routes. The results are expressed as mean ± standard deviation. The cut-off is represented by the line. Significant differences (p<0.05) between infection with the different routes of inoculation are represented by the connected lines. Spearman’s correlation indexes (r and P-values) are shown on the graphs and the connecting lines illustrate positive and negative correlation indexes.

Figure 4. Photomicrographs of liver and spleen smears in hamsters experimentally infected with two strains of L. infantum (PP75 or Wild) at 9 months after infection.

Presence of few amastigotes in animals infected with PP75 strain via the IC route (A) and infected with the Wild strain by the IP route (B–E) and IC (C). Intense splenic parasitism in animals infected with either PP75 (D) or Wild (F) by the IC route. Giemsa. Bar = 50 µm.

Figure 5. Parasite burden in hamsters experimentally infected with two strains of L. infantum (PP75 and Wild) by different routes of inoculation.

Number of amastigotes deduced from the qPCR data by the total weight of the liver (A) and spleen (B) in hamsters experimentally infected with two strains of L. infantum (PP75 or Wild) by different routes of inoculation: intradermal (ID, n = 80; light gray), intraperitoneal (IP, n = 80; dark gray), and intracardiac (IC, n = 80; black) after 1, 3, 6, and 9 months of infection. The results are expressed as mean ± standard deviation. Significant differences (p<0.05) between infection with the different routes of inoculation are represented by the letters “a” and “b” related to the group ID and IP, respectively.