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Bone-marrow aspirate material is commonly considered as one of the most sensitive tissues for a reliable diagnosis of leishmaniasis. The procedure herein described may permit less experienced veterinarians to be familiar with a quick and safe assessment method for leishmaniasis diagnosis in their patients. Animals are positioned in right lateral re...
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... Spleen samples were collected from each dog using a fine needle aspiration method under sedation (Barrouin-Melo et al. 2006). Bone marrow aspirates were collected from the sternum manubrium (Paparcone et al. 2013;Varjão et al. 2021). Spleen and bone marrow aspirates were used in qualitative PCR tests to confirm the diagnosis of natural infection by L. infantum. ...
Canine leishmaniasis (CanL) caused by Leishmania infantum commonly progresses with renal and ophthalmic lesions associated with active systemic disease. As chronic inflammation related to immune complex deposits is a pathophysiological factor in the development of both glomerulonephritis and uveitis, we aimed to evaluate renal and ocular histopathological lesions and analyze whether they were related to each other and the clinical degree of the disease. For that, we evaluated 15 dogs from CanL-endemic areas. L. infantum PCR-positive dogs were studied according to disease severity into two different groups: Group-1 (G1) had data from seven dogs with mild to moderate CanL and no history of treatment, and G2 was formed with eight dogs with severe to terminal disease that had not responded to CanL treatment. Histopathological analysis of kidneys showed higher frequencies and intensities of glomerular basement membrane thickening (p = 0.026), deposits in glomeruli (p = 0.016), epithelial necrosis (p = 0.020), tubular dilatation (p = 0.003) and interstitial fibrosis (p = 0.04) in G2 dogs than in G1 dogs. Surprisingly, the histopathology of eye bulbs showed a higher frequency and intensity of retinitis (p = 0.019) in G1 dogs than in G2 dogs. The comparative analysis showed that there was no correspondence between histopathological findings in kidneys versus eyes in milder or more severe CanL. Our findings suggested that (1) clinically undetectable eye alterations can be more precocious than those in kidneys in the development of CanL, and (2) the lower frequency of eye lesions and higher frequency of renal lesions in dogs with terminal disease even after treatment indicate that therapy may have been effective in reducing CanL-associated ophthalmic disease but not proportionally in reducing kidney disease.
... The diagnosis of CanL mainly relies on several serological techniques that determine antibody levels, PCR-based tests to detect and amplify Leishmania DNA in blood and tissues, and the direct visualization of amastigotes via bone marrow cytology [60]. In veterinary medicine, the collection of bone marrow samples is advisable for diagnosing and monitoring CanL [61] and mostly for the diagnosis of hematologic disorders [62]. It is therefore not recommended as an adequate diagnostic tool for HWD in dogs. ...
We report a rare and interesting case of mixed infection with Leishmania infantum and Dirofilaria immitis associated with the incidental finding of microfilariae in the bone marrow of a 9-year-old, intact, male Bullmastiff which was seropositive to L. infantum. Clinical signs showed progressive weakness, pale mucosae membranes, and a very low body condition score. Laboratory abnormalities included moderate, normocytic, normochromic, non-regenerative anemia; mild leukocytosis, neutrophilia, monocytosis, and eosinopenia; low platelet count; elevated C reactive protein; mild hyperkalemia, hypoalbuminemia, and hyperbeta-2-globulinemia; and a low A/G ratio. Hypoadrenocorticism, euthyroid sick syndrome, and alteration in the fibrinolytic phase of hemostasis were also detected. Microfilariae were incidentally found in bone marrow cytology aspirate in the absence of clinical features indicative of co-infection with D. immitis. PCR confirmed the identification of the Dirofilaria species. It is assumed that the microfilariae may have left the microcirculation and migrated to bone marrow tissues by crossing the vessel wall. To the best of our knowledge, only one such case has been previously reported in dogs.
... Bone marrow and lymph nodes are the most used sites for collecting samples 5 aimed at direct parasitological diagnosis of canine visceral leishmaniasis (CVL), especially in asymptomatic animals 6,7 . The most common clinical signs of the disease include onychogryphosis, skin lesions, lymphadenomegaly, splenomegaly, low body score [8][9][10][11] , and anemia 7,10,12-14 . ...
... The most common clinical signs of the disease include onychogryphosis, skin lesions, lymphadenomegaly, splenomegaly, low body score [8][9][10][11] , and anemia 7,10,12-14 . Signs such as epistaxis, increased blood clotting time, hematuria, medullary aplasia, and thrombocytopenia are also observed 7,15,16 . ...
... As no difference was observed between the bone marrow of the femur and sternal manubrium in terms of parasite intensity, both organs are useful for CVL diagnosis. In this context, Paparcone et al. 7 also found no significant differences in the cellularity and the degree of parasitism in the bone marrow collected from different sites for direct diagnosis of CVL, which was further ratified by Xavier et al. 12 in a comparative study of diagnostic techniques for the disease. ...
As the bone marrow is one of the most organs affected by canine visceral leishmaniasis (CVL), samples from this are frequently taken for parasitological tests, with occurrence of myelodysplastic changes, with consequent anemia, leukopenia, and thrombocytopenia. Thus, this study aimed to investigate the histological and immunohistochemical changes in the bone marrow of the femur and sternal manubrium of dogs reactive for leishmaniasis by DPP® and ELISA tests. For this, thirteen canines from the epidemiological routine for CVL carried out by the Directorate of Zoonosis Surveillance of Goiânia (DVZ), GO, Brazil, were subjected to anatomopathological examination. 46.2% of bone marrow samples from the femur showed a higher proportion of the red series, and 53.9% of bone marrow of the sternal manubrium evidenced a higher proportion of the red series. Also, there were varied macrophage hyperplasia, hemosiderosis, and megakaryocytic emperipolesis. Amastigote forms of Leishmania spp. in the bone marrow of the femur and sternal manubrium to histopathological and immunohistochemical evaluations were observed, with good agreement them, but without difference in the parasite intensity between the bone marrow of these anatomical sites. It was concluded that bone marrow of the femur and sternal manubrium of dogs reactive for leishmaniasis by DPP® and ELISA tests has histological changes resulting from the disease, regardless of the parasite presence or intensity, with macrophage hyperplasia, hemosiderosis, and emperipolesis being the main medullary changes in these animals. Also, the bone marrow of the femur and sternal manubrium are useful anatomical sites for the diagnosis of CVL by direct methods.
Keywords: amastigotes; canine visceral leishmaniasis; histopathology; immunostaining; medullary changes
... A medula óssea e os linfonodos são os locais mais utilizados para a colheita de amostras 5 voltadas ao diagnóstico parasitológico direto da leishmaniose visceral canina (LVC), principalmente em animais assintomáticos 6,7 . Os sinais clínicos mais comuns da doença incluem onicogrifose, lesões cutâneas, linfadenomegalia, esplenomegalia, baixo escore corporal 8-11 e anemia 7,10,12-14 . ...
... Os sinais clínicos mais comuns da doença incluem onicogrifose, lesões cutâneas, linfadenomegalia, esplenomegalia, baixo escore corporal 8-11 e anemia 7,10,12-14 . Adicionem-se, ainda, sinais como epistaxe, aumento do tempo de coagulação sanguínea, hematúria, aplasia medular e trombocitopenia 7,15,16 . ...
... Como não foi observada diferença entre a medula óssea do fêmur e do manúbrio esternal quanto à intensidade parasitária, pondera-se que ambos compreendem órgãos úteis ao diagnóstico da LVC. Nesse âmbito, Paparcone et al. 7 também descrevem que não há diferenças significativas na celularidade e no grau de parasitismo na medula óssea colhida de diferentes locais para diagnóstico direto da LVC, o que foi ainda ratificado por Xavier et al. 12 em estudo comparativo de técnicas de diagnóstico da doença. ...
Resumo Como a medula óssea é um dos órgãos mais acometidos pela leishmaniose visceral canina (LVC), amostras desta são frequentemente colhidas para exames parasitológicos, sendo possível a ocorrência de alterações mielodisplásicas, com consequente anemia, leucopenia e trombocitopenia. Assim, este estudo teve como objetivo investigar alterações histológicas e imunoistoquímicas na medula óssea do fêmur e manúbrio esternal de cães reativos para leishmaniose aos testes DPP® e ELISA. Para isso, 13 caninos da rotina epidemiológica para LVC realizada pela Diretoria de Vigilância de Zoonoses de Goiânia (DVZ), GO, Brasil, foram submetidos ao exame anatomopatológico. 46,2% e 53,9% das amostras de medula óssea do fêmur e do manúbrio esternal apresentaram maior proporção da série vermelha, respectivamente. Além disso, havia variados graus de hiperplasia macrofágica, hemossiderose e emperipolese megacariocítica. Formas amastigotas de Leishmania spp. na medula óssea do fêmur e do manúbrio esternal às avaliações histopatológicas e imunoistoquímicas foram observadas, com boa concordância entre essas, mas sem diferença na intensidade parasitária entre a medula óssea desses sítios anatômicos. Conclui-se que a medula óssea do fêmur e do manúbrio esternal de cães reativos para leishmaniose aos testes DPP® e ELISA apresenta alterações histológicas decorrentes da doença, independente da presença ou intensidade do parasito, sendo hiperplasia de macrófagos, hemossiderose e emperipolese as principais alterações medulares nesses animais. Além disso, a medula óssea do fêmur e do manúbrio esternal compreendem sítios anatômicos úteis ao diagnóstico de LVC por métodos diretos.
... The sternum is reported to be the most accessible site for bone marrow aspiration (Defarges et al. 2013). Bone marrow from the sternum can be used for diagnosis of many diseases, peripheral blood abnormalities, Canine Leishmaniasis (Paparcone et al. 2013), chronic kidney disease (Borin-Crivellenti et al. 2014), Bone marrow aspirate is rich in progenitors and stem cells, which is a golden key in regenerative medicine. Bone marrow aspirates from the sternum in the dog; however, it is not recommended to be used for regenerative purposes. ...
... It has been reported that some dogs undergone bone marrow aspiration (5%) failed to get from the first attempt, however, conducted by expertise (Paparcone et al. 2013). This failure could be due to wrong needle insertion or needle obstruction by bone spicules and tissues. ...
... Bone marrow was aspirated from different places from the sternum of dogs as manubrium, 3 rd and 5 th sternebra under the effect of sedation using xylazine (Xylaject, Adwia) in a dose of 1-2 mg/kg; animals were positioned at right lateral recumbency in case of aspiration from the 2 nd or 3 rd sternebra (Paparcone et al. 2013). However, bone marrow aspiration from manubrium sternal positioning (sitting position) is preferred (Guillot et al. 2011, Defarges et al. 2013. ...
Advanced diagnostic technologies such as ultrasound and computed tomography (CT) could not provide any information about the sternebrae cortex thickness and medullary cavity dimensions. Herein, we aim to provide relevant anatomy of the sternum and more sophisticated measurements to ensure safe needle insertion during bone marrow aspiration in dogs.
This study revealed that the space between skin and cortex measures an average of 0.8 cm, which increases towards the xiphoid, as shown by ultrasound. Meanwhile, the sagittal section of the sternum showed that the cortex measured as thin as one millimeter. The medullary space of sternebrae 2nd – 7th was 1.5 cm2, while the manubrium measured 3.8 cm2.
In conclusion, knowing the exact sternebrae dimensions helped the students to master the bone marrow aspiration technique and become sensitive to needle advancement once it reaches the cortex.
... Fine-needle aspiration cytology (FNAC) from mucocutaneous lesions or enlarged lymph node in dogs with clinical signs and/or laboratory abnormalities potentially consistent with CanL is an easy, cheap, and reliable diagnostic method to demonstrate the presence of L. infantum amastigotes [14]. Conversely, it should be noted that lymphoid tissue material could be difficult to obtain in sufficient amount when palpable nodes are not enlarged [15] and that lymph node enlargement is a not always evident clinical sign, becoming appreciable only several months after L. infantum infection [13,16]. Bone marrow FNAC is considered as one of the most sensitive techniques for a reliable diagnosis of CanL even though it is not a complication-free procedure, as it can cause pain, haemorrhage, and infection [14]. ...
Conjunctival swabs (CS) are the most promising non-invasive samples for the diagnosis and the regular screening of Leishmania infantum infection in dogs although knowledge on their diagnostic performance is still inconclusive. This study evaluates CS real time-PCR (qPCR) analysis for the diagnosis of canine leishmaniosis (CanL) and its prognostic value in seropositive dogs from an endemic area. In October 2020 (T0), 26 dogs were enrolled, divided in two groups according to anti-L. infantum antibody titres (n = 13, group low titre (LT) and n = 13, group high titre (HT)), and followed-up in August 2021. At both timepoints, animals underwent clinical examination, complete blood count and biochemical analyses, and serological (indirect fluorescent antibody test) and molecular (CS and peripheral blood qPCR) testing. At T0, 10 out of 26 enrolled dogs were positive at CS qPCR, with the number of positive animals significantly higher in group HT than in LT. After 10 months, only 5 out of 21 dogs that completed the trial still tested CS qPCR positive, and none of them developed an active CanL based on clinical score and antibody titre. None of the dogs required any leishmanicidal and/or leishmaniostatic treatments. This prospective study showed unsatisfying diagnostic and prognostic performances of CS qPCR analysis in L. infantum seropositive asymptomatic dogs from an endemic area.
... The diagnosis of CanL mainly relies on several serological techniques that determine antibody levels, PCR-based tests developed to detect and amplify Leishmania DNA in blood and tissues, and the demonstration of parasites by direct visualization with BMC [9,10]. Bone marrow biopsy is the gold standard for the diagnosis of leishmaniasis, as well as for ruling out other causes of pancytopenia [40]. Early diagnosis of CanL is crucial for the treatment and outcome of the infection. ...
Dogs with lymphoma are at risk of developing clinical complications due to immunosuppression and side effects of chemotherapy. Clinical reports of concurrent lymphoma and leishmaniasis are rare and confined to single cases of comorbidity at presentation. Herein, we describe a case of lymphoma during maintenance chemotherapy in which bone marrow cytology showed myelodysplasia associated with leishmaniasis. The dog was a seven-year-old intact female Parson Russel Terrier with a two-week history of generalized lymphadenopathy. Diagnosis of multicentric high-grade B-cell lymphoma stage Va was carried out with cytological and cytofluorimetric assays of external lymph nodes, abdominal ultrasound, chest radiology, and lymphoid blasts blood smear examination. The dog lived and had traveled in endemic areas of Leishmania with uninterrupted prevention against sand fly bites by an insecticide-impregnated collar and presented seronegativity to Leishmania at presentation. Chemotherapy for lymphoma was successful and the patient achieved complete remission. Approximately eight months after the diagnosis, a persistent pancytopenia was assessed. Unexpectedly, Leishmania amastigotes were identified in the bone marrow. Combined treatment rounds were administered with antileishmanial and antineoplastic drugs for approximately eight months. Eventually, lymphoma relapsed and became unresponsive to chemotherapy, and the dog was euthanatized. Canine lymphoma overlapping with subsequent Leishmania infection as a complication is rare and lacks specific clinical manifestations. A delayed diagnosis of leishmaniasis may occur. We suggest considering leishmaniasis as part of the differential diagnosis of persistent pancytopenia in dogs with lymphoma, particularly in dogs who reside or travel to endemic areas, when treatment fails or abnormal laboratory findings are present.
... However, no data were found in the literature to ratify or rectify the result relative to these organs. Nevertheless, Paparcone et al. (35) compared different bone marrow collection sites for CanL diagnosis and also observed no difference regarding parasite intensity. Despite this, the authors mentioned that although the lymph node is equally rich in parasites, especially in symptomatic dogs, the access to the lymphoid tissue in this organ can be limited, particularly when there is no increase in volume, which commonly occurs in animals at early stage of the disease or in chronic asymptomatic animals. ...
Canine leishmaniasis (CanL) is a zoonosis caused by the protozoan of the species Leishmania infantum. The spleen and lymph nodes undergo morphological changes during CanL. This research aimed to perform an anatomopathological and immunohistochemical study of these organs in dogs reactive to leishmaniasis in the Dual-path Platform chromatographic immunoassay (DPP®) and Enzyme Immunoabsorption Assay (ELISA). Twenty-seven dogs were evaluated for anatomopathological examination with 92.6% showing changes at gross evaluation, specially splenomegaly and lymphadenomegaly. All dogs showed changes in the spleen unrelated to the parasitic load, with granulomatous splenitis being the most severe change. Diffuse cortical and paracortical hyperplasia, and hyperplasia and hypertrophy of the medullary cords were observed in the lymph node. Amastigote forms of Leishmania spp. were found in the spleen and lymph node at histopathological and immunohistochemical evaluations, with good agreement between these evaluations (k = 0.55, p = 0.00124), but no difference was observed in the parasitic intensity of these organs at immunohistochemistry (p = 0.23). It was concluded that spleen and lymph node from dogs reactive to leishmaniasis on the DPP® and ELISA tests show histomorphological changes resulting from the disease, independent to the parasitic load, as well as these organs show similar parasitic load at immunohistochemical test.
... Urine aliquots were collected for analyses and quantification of urinary protein and creatinine (UPC). Bone marrow aspirates were performed to quantify the parasitic load of L. infantum at T0, T6, and T12, as described by Paparcone et al. (2013). ...
Clinical improvement of dogs treated for canine leishmaniasis (CanL) requires reducing Leishmania infantum loads, which depend on intracellular oxidant compounds to destroy the parasite. However, oxidative species’ excess and antioxidants consumption can culminate in oxidative stress, resulting in increased, widespread inflammation. We aimed to evaluate if early or late addition of nutritional adjuvants (NAs) – omega-3 polyunsaturated fatty acids and B vitamins – to anti-Leishmania drugs (ALDs) in the treatment of CanL would be clinically beneficial. For that, serum biomarkers including oxidative stress parameters were analyzed during 12 months in dogs allocated to two treatment groups: (G1) NAs administered from 30 days prior to the beginning of ALDs; and (G2) NAs administered from 61 days after the beginning of ALDs. Both G1 and G2 continued to receive NAs until the 12th month. The ALDs administered were metronidazole associated with ketoconazole (40 days), followed by allopurinol from day 41 until the 12th month. G1 exhibited superior inflammation control, with reduced globulins (p = 0.025), specific anti-Leishmania immunoglobulins (p = 0.016), total protein (p = 0.031), and an increased serum albumin/globulin ratio (p = 0.033), compared to G2. The early use of NAs associated with ALDs is clinically beneficial in treating dogs with CanL.
... 10 After sedation and before the surgical procedure, fine-needle aspiration sampling of BM from the sternum was performed according to a previously described technique. 11 Samples were processed using the cellblock technique to prepare cytoinclusions as previously described. 5,12 with modifications to comply with current standardization. ...
... BM sampling from the sternum is a straightforward and minimally invasive process in dogs that does not require general anesthesia. 11,21,22 The processing required for samples in the present study using the cell-block technique was simple and provided a high material yield for routine histology. Additionally, BM cytoinclusions as prepared by cellblock can be used in immunohistochemical and molecular diagnostic tests. ...
Studies using the cell-block technique for bone marrow (BM) analysis are limited in the veterinary literature. This work assessed whether the histopathological analysis of canine BM was feasible using cell-block cytoinclusions prepared from fine-needle sternal aspirate samples. Eight clinically healthy young-to-middle-aged dogs underwent both fine-needle sternal aspiration for BM cell-block (BM-Cb) processing and iliac-crest BM core biopsy (BM-B). Histopathologic parameters were compared between the two methods. There were no statistically significant histopathological differences between hematopoietic tissue areas (P = 0.6294) in the BM-Cb and BM-B sections, and they had similar microscopic characteristics and microarchitecture. Cellularity and reticulin-fiber staining were equivalent in the BM-Cb and BM-B preparations in 87.5% (7/8) and 100% (8/8) of the sections, respectively. However, the quantitative results of the megakaryocytic series differed between BM-Cb and BM-B in 37.5% (3/8) of the sections, and the myeloid:erythroid (M:E) ratios differed between the two methods in 25% (2/8). These preliminary data indicate that cell-block preparations made from sternal fine-needle aspiration samples warrant continued evaluation in a larger number of animals, including those with various diseases affecting the bone marrow.
