Clinical forms of canine visceral Leishmaniasis in naturally Leishmania infantum-infected dogs and related myelogram and hemogram changes

Abstract

Hematological analysis has limited applications for disease diagnosis in Leishmania infantum-infected dogs, but it can be very important in evaluating the clinical forms of the disease and in understanding the evolution of canine visceral leishmaniasis (CVL) pathogenesis. Recently, we demonstrated that alterations in leucopoiesis and erythropoiesis are related to clinical status and bone marrow parasite density in dogs naturally infected by L. infantum. To further characterize these alterations, we evaluated the association between the hematological parameters in bone marrow and peripheral blood alterations in groups of L. infantum-infected dogs: asymptomatic I (AD-I: serum negative/PCR+), asymptomatic II (AD-II: serum positive), oligosymptomatic (OD), and symptomatic (SD). Results were compared with those from noninfected dogs (NID). The SD group was found to present a decrease in erythropoietic lineage with concomitant reductions in erythrocytes, hemoglobin, and hematocrit parameters, resulting in anemia. The SD group also had increased neutrophils and precursors and decreased band eosinophils and eosinophils, leading to peripheral blood leucopenia. In the AD-II group, lymphocytosis occurred in both the peripheral blood and the bone marrow compartments. The SD group exhibited lymphocytosis in the bone marrow, with lymphopenia in the peripheral blood. In contrast, the AD-I group, showed no significant changes suggestive of CVL, presenting normal counts in bone marrow and peripheral blood. Our results showed for the first time that important changes in hematopoiesis and hematological parameters occur during ongoing CVL in naturally infected dogs, mainly in symptomatic disease. Taken together, our results based on myelogram and hemogram parameters enable better understanding of the pathogenesis of the anemia, lymphocytosis, and lymphopenia, as well as the leucopenia (eosinopenia and monocytopenia), that contribute to CVL prognosis.

Figure 1. Profiles of the erythroblastic cell series in dogs naturally infected by Leishmania infantum.

(A) Animals categorized according to their clinical status into asymptomatic I (AD-I, n = 6), asymptomatic II (AD-II, n = 7), oligosymptomatic (OD, n = 8), symptomatic (SD, n = 12), or noninfected (NID, n = 7) groups. Significant differences at p<0.05 are identified by the letters a, b, and d in comparison to NID, AD-I, and OD, respectively. The results are expressed on graphs as scattering of individual values and are also shown as median and interquartile range values. (B) Correlation between erythroblastic series cells and hematological parameters (erythrocytes [RBC], hemoglobin [Hb], and hematocrit [Ht]) considering all groups of dogs infected by L. infantum. The results are expressed as scattering of individual values. Spearman’s rank correlation (r _s and p values) are shown in the graphs.

Figure 2. Profiles of the precursors of granulocytic lineage cells in dogs naturally infected by Leishmania infantum categorized according to clinical status.

Animals categorized according to their clinical status into asymptomatic I (AD-I, n = 6), asymptomatic II (AD-II, n = 7), oligosymptomatic (OD, n = 8), symptomatic (SD, n = 12), or noninfected (NID, n = 7) groups. Significant differences at p<0.05 are identified by the letters a, b, and c in comparison to NID, AD-I, and AD-II respectively. The results are expressed in graphics as scattering of individual values and are also shown as median and interquartile range values. Spearman’s rank correlation (r _s and p values) are also shown.

Figure 3. Correlation between bone marrow (precursors of granulocytic lineage cells) and peripheral blood (with cells) parameters.

(A) Myeloblast versus total leucocytes and promyelocyte versus total leucocytes. (B) Neutrophilic myelocyte versus total neutrophils, neutrophilic metamyelocyte versus total neutrophils, band neutrophilic versus band neutrophils, and segmented neutrophilic versus neutrophils segmented. (C) Eosinophilic myelocytes, eosinophilic metamyelocytes, band eosinophils, and segmented eosinophils versus total eosinophils of peripheral blood. Correlation between peripheral blood and bone marrow of the granulocyte series. The results are expressed as scattering of individual values. Spearman’s rank correlation (r _s and p values) are also shown in the graphics.

Figure 4. Profiles of the agranulocytic cell series in bone marrow of dogs naturally infected by Leishmania infantum.

(A) Animals categorized according to their clinical status as asymptomatic I (AD-I, n = 6), asymptomatic II (AD-II, n = 7), oligosymptomatic (OD, n = 8), symptomatic (SD, n = 12), or noninfected (NID, n = 7) groups. Significant differences at p<0.05 are identified by the letter a and d in comparison to NID and OD, respectively. The results are expressed on graphs as scattering of individual values and are also shown as median and interquartile range values. (B) Correlation between peripheral blood and bone marrow of the lymphocytes in AD-II and SD groups. The results are expressed as scattering of individual values. Pearson correlation indexes (r and p values) are shown on the graphs.

Figure 5. Canine bone marrow cytology.

The bone marrow smears derived from the NID dogs are demonstrated (A–C). The SD group demonstrated plasma cell multiplication (arrow-head) and Mott cells (MC) (D). Emperipolesis, the penetration of hematopoietic cells into the cytoplasm of megakaryocytes (arrow) (E). Increase of precursor leucopoetic lineage cells (F) and decrease of all erythropoietic lineage cells (G) in the SD group. Symptomatic dogs presented an increase in neutrophilic lineage cells (H) and erythroblastic series cells in mitotic cell division (I) (arrow). Slides were stained with Giemsa; bar = 10 µm.