Influence of Clinical Status and Parasite Load on Erythropoiesis and Leucopoiesis in Dogs Naturally Infected with Leishmania (Leishmania) chagasi

Background: The bone marrow is considered to be an important storage of parasites in Leishmania-infected dogs, although little is known about cellular genesis in this organ during canine visceral leishmaniasis (CVL). Methodology/Principal Findings: The aim of the present study was to evaluate changes in erythropoiesis and leucopoiesis in bone marrow aspirates from dogs naturally infected with Leishmania chagasi and presenting different clinical statuses and bone marrow parasite densities. The evolution of CVL from asymptomatic to symptomatic status was accompanied by increasing parasite density in the bone marrow. The impact of bone marrow parasite density on cellularity was similar in dogs at different clinical stages, with animals in the high parasite density group. Erythroid and eosinophilic hypoplasia, proliferation of neutrophilic precursor cells and significant increases in lymphocytes and plasma cell numbers were the major alterations observed. Differential bone marrow cell counts revealed increases in the myeloid:erythroid ratio associated to increased numbers of granulopoietic cells in the different clinical groups compared with non-infected dogs. Conclusions: Analysis of the data obtained indicated that the assessment of bone marrow constitutes an additional and useful tool by which to elaborate a prognosis for CVL

Alterações eritropoéticas e leucopoéticas na Leishmaniose visceral canina.

A medula óssea é considerada um importante reservatório de parasitos em cães infectados por Leishmania chagasi. Poucos trabalhos na literatura relatam o processo da gênese celular na medula óssea na Leishmaniose Visceral Canina (LVC) em diferentes fomas clínicas. Nesse contexto, o objetivo do presente estudo foi avaliar as alterações na leucopoese e eritropoese bem como investigar a carga parasitária em aspirados de medula óssea de cães naturalmente infectados por L. chagasi apresentando diferentes formas clínicas e padrões distintos de densidade parasitária na medula óssea. Dessa forma, filmes de medula óssea corados por Giemsa foram avaliados considerando três grupos clínicos: assintomático (CA, n=50), oligossintomático (CO, n=44) e sintomático (CS, n=65) comparados com cães não-infectados (CNI, n=28). A densidade parasitária foi avaliada na medula óssea e os resultados expressos como “Leishman Donovan Units” (LDU), e classificados em tercis como baixo (BP, n=51), médio (MP, n=51) ou alto (AP, n=48) parasitismo. Em um segundo grupo de 48 cães (CA, n=10; CO, n= 9; CS, n= 12; CNI, n= 17) o mielograma e hemograma completo foram realizados para investigar a associação entre a celularidade da medula óssea e do sangue periférico. Os resultados obtidos indicaram diferenças significativas em relação à eritropoese considerando proeritroblastos, eritroblastos basófilos, policromáticos e ortocromáticos resultando em hipoplasia eritróide principalmente nos grupos CA e CS. A leucopoese apresentou algumas alterações em cães infectados. Por exemplo, células da linhagem eosinofílica mostraram uma diminuição significativa nos diferentes grupos clínicos comparado com o grupo CNI. Entretanto, células da linhagem neutrofílica mostraram alterações nos diferentes grupos clínicos, tais como um aumento significativo nos diferentes grupos clínicos. Em relação às células mononucleares, foi observado aumento de linfócitos nos grupos CO e CS quando comparado com o grupo CNI. Resultados similares foram encontrados em plasmócitos mostrando clara tendência ao aumento gradual de acordo com a gravidade da infecção. Por outro lado, monócitos tiveram significativo aumento no grupo CO comparado ao grupo CA. A contagem diferencial de células da medula óssea demonstrou um aumento na razão mielóide: eritróide (M:E) devido ao aumento de células granulocíticas nos diferentes grupos clínicos comparado com o grupo CNI. A avaliação parasitológica mostrou maiores índices de LDU em CS comparado com CA. Além disso, os dados demonstraram uma correlação positiva entre status clínico (CA,CO,CS) e densidade parasitária (BP,MP,AP). A avaliação do impacto do parasitismo na medula óssea mostrou resultados similares quando se avaliou os cães classificados em diferentes formas clínicas, tais como, hipoplasia eritróide, hipoplasia eosinofílica, proliferação de células precursoras neutrofílicas, um significativo aumento no número de linfócitos e plasmócitos, destacando o grupo de cães com alto parasitismo com resultados mais significativos. Os resultados do grupo 2 mostraram que as alterações do sangue periférico foram relacionadas às alterações da medula óssea principalmente no grupo CS. De forma interessante, o grupo CS demonstrou diminuição de células da linhagem eritropoética e eritrócitos, hemoglobina e hematócrito caracterizando anemia periférica não-regenerativa. Além disso, foram observados aumento de neutrófilos e seus precursores, diminuição de bastonetes e segmentados eosinófilos e significativo aumento de linfócitos na medula óssea associada à leucopenia com desvio à esquerda, eosinopenia, linfopenia e monocitopenia no sangue periférico. O presente estudo mostrou que a evolução clínica da LVC em cães naturalmente infectados promove claras alterações na medula óssea e no sangue periférico. A progressão da doença da forma clínica assintomática para sintomática foi acompanhada de parasitismo intenso na medula óssea. Assim, os dados analisados em conjunto permitem concluir que a avaliação desses parâmetros constitui uma ferramenta adicional bastante útil no prognóstico da leishmaniose visceral canina, bem como no diagnóstico em duas situações específicas, a saber: a) esclarecer casos com forte suspeita de LVC, porém não confirmados por sorologia, utilizando-se do mielograma; e b) orientar exames complementares para elucidar suspeita de LVC, a partir de dados clínicos e do hemograma.The bone marrow is considered an important reservoir of parasites in sick Leishmania-infected dogs. Little is know about the process cellular genesis in bone marrow in Canine Visceral Leishmaniasis (CVL) presenting different clinical status. In this context, the aim of the present study was to evaluate leucopoiesis and erythropoiesis alterations as well as to investigate parasite load in aspirates of bone marrow dogs naturally infected by L. chagasi with different clinical status and distinct patterns of bone marrow parasite density. Herein, bone marrow (BM) smears stained by Giemsa were evaluated considering three clinical groups: asymptomatic (AD, n=50), oligosymptomatic (OD, n=44) and symptomatic (SD, n=65) compared with non-infected dogs (NID, n=28). Parasite density was performed in bone marrow and the results expressed as “Leishman Donovan Units” (LDU index), and classified into tertiles as low (LP, n=51), medium (MP, n=51) or high (HP, n=48) parasitism. A second group of 48 dogs (AD, n=10; OD, n= 9; SD, n= 12; NID, n= 17) was evaluated by bone marrow analysis and hemogram to investigate the association between bone marrow and periphreral blood parameters. The results obtained indicated significant differences in relation to erythropoiesis considering proerythroblasts, basophilic, polychromatic and orthochromatic erythroblasts resulting in an erythroid hypoplasia mainly in AD and SD groups. Leucopoiesis presented some alterations in infected dogs. For example, eosinophilic lineage cells number showed a significant decrease in the different clinical groups compared to NID group. However, neutrophilic lineage cells number showed alterations in the different clinical groups, such as a significant increase in the different clinical groups. Related to mononuclear cells, it was observed for lymphocytes number an increase in OD and SD groups when compared with NID group. Similar results were found for plasma cell number showing a clear tendency to a gradual increase according to the severity of the infection. By the other hand, monocytes cell number had significantly increase in OD group compared to AD group. Differential cell counts of bone marrow demonstrated an increase in the myeloid: erythroid (M:E) ratio due to the increased numbers of granulopoietic cells in the different clinical groups compared to NID group. Parasitological assessment showed higher LDU index in SD compared with AD. Moreover, our data demonstrated a positive correlation among clinical status (AD,OD,SD) and parasite density (LP,MP,HP). Assessment of the impact of parasite density on bone marrow showed similar results when the dogs were evaluated in different clinical stages such as erythroid hypoplasia, eosinophilic hypoplasia, proliferation of neutrophilic precursors cells, a significant increase in lymphocytes and plasma cells number, highlighting the high parasitism dogs group with the most significatives results. The results of second group showed that periphreral blood alterations were related to those shown by bone marrow, mainly in SD group. Indeed, SD group dogs demonstrated significant decrease in erythropoietic lineage cells number and erythrocytes, hemoglobin and hematocryt resulting in a nonregenerative anemia. Moreover, there were observed increase in neutrophils and their precursors, decrease in band eosinophils and eosinophils and significant increase in lymphocytes number in bone marrow associated to leukopenia with left shift, decrease in eosinophils, lymphocytes and monocytes number in peripheral blood. The present study showed that the clinical evolution of CVL in naturally infected dogs promotes clear alterations in the bone marrow and periphreral blood. The progression of the disease from asymptomatic to symptomatic clinical status was accompanied by intense parasitism in the bone marrow. So, the data analysed together allow to conclude that the assessment of these parameters constitute an useful and additional tool for CVL prognosis, as well as for diagnosis in two specific situations, namely: a) to clarify cases with a strong suspicion of CVL, however not confirmed by sorologic tests, by using bone marrow analysis; and b) to orientate complementary laboratory tests to investigate CVL suspicion, based on clinical and hemogram data

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

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

Maturation Index Assessment of bone marrow cellularity in dogs naturally infected by <i>L. chagasi</i> presenting different clinical statuses and parasite densities.

a<p>Values statistically significantly different (p<0.05) from those of the NID group. NID indicates non-infected dogs; AD, asymptomatic dogs; OD, oligosymptomatic dogs; SD, symptomatic dogs; LP, low parasitism; MP, medium parasitism; HP, high parasitism; M:E, myeloid:erythroid; I:M, immature:mature; I:Mg, immature:mature (granulopoiesis); I:Me, immature:mature (erythropoiesis); MMI, myeloid maturation index; and EMI, erythroid maturation index.</p

Profiles of the erythroblastic cell series in dogs naturally infected with <i>Leishmania chagasi</i>.

<p>Panel <b>A</b> - animals categorised according to their clinical status into asymptomatic (AD = white boxes with small points), oligosymptomatic (OD = white boxes with large points), symptomatic (SD = white boxes with squares) or non-infected (NID = white boxes) groups. Panel <b>B</b> - animals categorised according to bone marrow parasite density into low (LP = gray boxes), medium (MP = dark gray boxes) and high (HP = black boxes) parasite density and non-infected (NID = white boxes) groups. Absolute cell counts are presented in a box-plot format, the median, maximum and minimum values represents the interquartile range. Significant differences (p<0.05) with respect to the NID group are indicated by the letter ‘a’. Panel <b>C</b> - cellularity of the erythroblastic series observed in the bone marrow of dogs assessed in this study. Slides were stained with Giemsa; bar  = 10 µm. P indicates proerythroblast; BE, basophilic erythroblast; PE, polychromatic erythroblast; OE, orthochromatic erythroblast.</p

A killed Leishmania vaccine with sand fly saliva extract and saponin adjuvant displays immunogenicity in dogs.

A vaccine against canine visceral leishmaniasis (CVL), comprising Leishmania braziliensispromastigote protein, sand fly gland extract (SGE) and saponin adjuvant, was eval-uated in dog model, in order to analyse the immunogenicity of the candidate vaccine. The vaccine candidate elicited strong antigenicity in dogs in respect of specific SGE andLeishmania humoral immune response. The major saliva proteins recognized by serum from immunized dogs exhibited molecular weights of 35 and 45 kDa, and were related to the resistance pattern against Leishmaniainfection. Immunophenotypic analysis revealed increased circulating CD21 + B-cells and CD5 + T-cells, reflected by higher counts of CD4 + and CD8 + T-cells. The observed interac-tion between potential antigen-presenting cells (evaluated as CD14 + monocytes) and lymphocyte activation status indicated a relationship between innate and adaptive immune responses. The higher frequency in L. chagasi antigen-specific CD8 + T-lymphocytes, and their positive association with intense cell proliferation, in addition to the progressively higher production of serum nitric oxide levels, showed a profile compatible with anti-CVL vaccine potential. Further studies on immunological response after challenge with L. chagasi may provide important information that will lead to a better understanding on vaccine trial and efficacy

Profiles of the agranulocytic cell series in dogs naturally infected with <i>Leishmania chagasi</i>.

<p>Panel <b>A</b> - animals categorised according to their clinical status into asymptomatic (AD = white boxes with small points), oligosymptomatic (OD = white boxes with large points), symptomatic (SD = white boxes with squares) or non-infected (NID = white boxes) groups. Panel <b>B</b> - animals categorised according to bone marrow parasite density into low (LP = gray boxes), medium (MP = dark gray boxes) and high (HP = black boxes) parasite density and non-infected (NID = white boxes) groups. Absolute cell counts are presented in a box-plot format, the median, maximum and minimum values represents the interquartile range. Significant differences (p<0.05) with respect to the NID group are indicated by the letter ‘a’. Panel <b>C</b> - cellularity of the agranulocytic series observed in the bone marrow of dogs assessed in this study. Slides were stained with Giemsa; bar  = 10 µm. L indicates lymphocyte; PC, plasma cell; M, monocyte.</p

Calculation of Maturation Index Ratios for the numerical determination of cellularity in the bone marrow.

<p>M indicates myeloid; E, erythroid; I, immature; M, mature; I:Mg, immature: mature (granulopoiesis); MMI, myeloid maturation index; I:Me, immature: mature (erythropoiesis); EMI, erythroid maturation index.</p

A killed Leishmania vaccine with sand fly saliva extract and saponin adjuvant displays immunogenicity in dogs.

A vaccine against canine visceral leishmaniasis (CVL), comprising Leishmania braziliensispromastigote protein, sand fly gland extract (SGE) and saponin adjuvant, was eval-uated in dog model, in order to analyse the immunogenicity of the candidate vaccine. The vaccine candidate elicited strong antigenicity in dogs in respect of specific SGE andLeishmania humoral immune response. The major saliva proteins recognized by serum from immunized dogs exhibited molecular weights of 35 and 45 kDa, and were related to the resistance pattern against Leishmaniainfection. Immunophenotypic analysis revealed increased circulating CD21 + B-cells and CD5 + T-cells, reflected by higher counts of CD4 + and CD8 + T-cells. The observed interac-tion between potential antigen-presenting cells (evaluated as CD14 + monocytes) and lymphocyte activation status indicated a relationship between innate and adaptive immune responses. The higher frequency in L. chagasi antigen-specific CD8 + T-lymphocytes, and their positive association with intense cell proliferation, in addition to the progressively higher production of serum nitric oxide levels, showed a profile compatible with anti-CVL vaccine potential. Further studies on immunological response after challenge with L. chagasi may provide important information that will lead to a better understanding on vaccine trial and efficacy

Profiles of the granulocytic cell series in dogs naturally infected with <i>Leishmania chagasi</i> categorised according to clinical status.

<p>Panel <b>A</b> - animals categorised according to their clinical status into asymptomatic (AD = white boxes with small points), oligosymptomatic (OD = white boxes with large points), symptomatic (SD = white boxes with squares) or non-infected (NID = white boxes) groups. Absolute cell counts are presented in a box-plot format, the median, maximum and minimum values represents the interquartile range. Significant differences (p<0.05) with respect to the NID, AD and OD groups are indicated by the letters ‘a’ and ‘b’ and ‘c’ respectively. Panel <b>B</b> - cellularity of the granulocytic series observed in the bone marrow of dogs assessed in this study. Slides were stained with Giemsa; bar  = 10 µm. M indicates myeloblast; PR, promyelocyte; NM, neutrophilic myelocyte; NMM, neutrophilic metamyelocyte; BN band neutrophil; N, neutrophil; EM, eosinophilic myelocyte; E, eosinophil.</p