Identification of Leishmania donovani amastigotes in canine tissues by immunoperoxidase staining

This paper describes the demonstration of Leishmania donovani amastigotes in canine tissues by immunoperoxidase staining. An indirect immunoperoxidase method was applied to the organs of 20 dogs in which leishmaniasis was previously diagnosed. Haemosiderin pigment was eliminated with 5 per cent oxalic acid. Amastigotes of L donovani appeared as dark brown stained bodies which contrasted with haematoxylin stained host cells. No positively stained amastigotes could be seen in any of the sections incubated with control serum. The organs which more frequently showed leishmanids were: skin (macrophages and fibroblasts), liver, spleen, lymph nodes and bone marrow. In a few cases amastigotes were seen in kidneys, gut, adrenal glands, eyes and testicles. This technique is simple to perform, gives consistent results and allows unequivocal histopathological diagnosis of canine leishmaniasis.     

High iNOS expression in macrophages in canine leishmaniasis is associated with low intracellular parasite burden

The expression of iNOS by macrophages in 33 dogs suffering from spontaneous leishmaniasis was analysed by immunohistochemistry in skin, liver and lymph nodes. A correlation study between the number of macrophages expressing iNOS and the number of macrophages containing leishmania amastigotes was carried out. Formalin-fixed paraffin-embedded tissue sections from the skin (28 cases), popliteal lymph nodes (8 cases) and liver (3 cases) of dogs of different age, sex and breed suffering from leishmaniasis were included in the study. Dogs were referred as positive for Leishmania spp by serology and the diagnosis was confirmed by demonstration of leishmania amastigotes within macrophages by histopathology. Tissue samples of skin (3 cases), popliteal lymph nodes (5 cases) and liver (3 cases) from dogs seronegative for leishmaniasis with no histopathological changes were included in the study as controls. The immunohistochemical study revealed that macrophages containing a high number of leishmania did not express iNOS. Correlation between the number of macrophages expressing iNOS and the number of macrophages containing leishmania amastigotes was assessed using the Spearman test. High expression of iNOS in macrophages was related with low number of leishmania amastigotes in macrophages in all cases (r=-0.47, p=0.002). These results suggest that iNOS expression by macrophages plays an important role during the control of Leishmania infection in dogs.     

Claw histopathology and parasitic load in natural cases of canine leishmaniosis associated with Leishmania infantum

Histological lesions and the presence of Leishmania spp. amastigotes in claw tissues were investigated in 40 dogs with leishmaniosis, with (16/40--group A) or without (24/40--group B) generalized onychogryphosis. Following euthanasia, the entire third phalanx with intact claw was amputated, formalin fixed, decalcified in a formic acid solution, embedded in paraffin, sectioned longitudinally and stained with haematoxylin and eosin, and acid orcein-Giemsa. Nested polymerase chain reaction (PCR) was used for the detection of Leishmania amastigotes. Lichenoid mononuclear infiltration (all dogs in group A, 21 of 24 dogs in group B), basal keratinocyte vacuolation (nine of 16 dogs in group A, 15 of 24 dogs in group B) and dermoepidermal clefting (13 of 16 dogs in group A, 18 of 24 dogs in group B) were the most prominent histopathological findings. There was no difference in the frequency and severity of these lesions between the two groups. Leishmania amastigotes could not be visualized in the dermis of any of the H&E sections, but their presence was demonstrated by nested PCR in three of 16 dogs in group A and two of 24 dogs in group B. However, the frequency of positive nested PCRs was not significantly different between the two groups. In conclusion, claw histopathology in symptomatic dogs with leishmaniosis, either with or without onychogryphosis is mainly characterized by mononuclear lichenoid dermatitis with or without interface dermatitis and dermoepidermal clefting, and is not accompanied by substantial local parasitism.     

Osteolytic osteomyelitis associated with visceral leishmaniasis in a dog

A dog was examined with a history of weight loss and lameness of the left hind limb. A painful response to examination of the left hip joint, and lymphadenopathy were noted. Amastigote forms of Leishmania sp. were observed by cytology in samples from the popliteus lymph node, and anti-Leishmania sp. antibodies at a titer of 1:640 were detected in serum by indirect immunofluorescence. Radiological changes included osteolysis and a periosteal proliferative reaction in the left femoral greater trochanter. These changes were histologically characterized as an osteolytic granulomatous osteomyelitis associated with amastigotes within macrophages. Non-decalcified fragments of the periosteum were processed for immunohistochemistry, observed with prominent immunolabelling of amastigotes of Leishmania sp. within macrophages. The diagnosis was further confirmed by positive PCR for Leishmania sp., belonging to the Leishmania donovani complex.     

Visceral leishmaniasis in a captive crab-eating fox Cerdocyon thous

Visceral leishmaniasis (VL) is a zoonotic disease with worldwide distribution. The crab-eating fox (Cerdocyon thous) is considered a wild reservoir of many zoonotical diseases, particularly VL. This study reported the presence of Leishmania infantum amastigotes in different organs of one captive C. thous found dead in a zoo. This animal was positive by the indirect fluorescence antibody test and had many clinical signs of VL. Intracellular amastigote forms of L. infantum were seen in neutrophils and macrophages in sample tissues from skin, lymph nodes (popliteal, submandibular, prescapular, and mesenteric), spleen, and liver. The numbers of positive cells and intracellular parasites were higher in macrophages than in neutrophils. In addition, polymerase chain reaction demonstrated extensive distribution of Leishmania DNA in C. thous tissues from multiple organs. The presence of intracellular amastigotes in neutrophils and macrophages as well as DNA of the parasite in tissues, specifically skin demonstrate that this crab-eating fox is an adequate host for L. infantum and reinforce the importance of VL for symptomatic wild canids kept in captivity in endemic areas.     

Clinicopathological study of canine transmissible venereal tumour in leishmaniotic dogs

Introduction : Canine transmissible venereal tumour is occasionally observed in leishmaniotic dogs, and Leishmania amastigotes can be harboured in canine transmissible venereal tumour cells. Objectives : The aim of this paper was to investigate the clinicopathological significance of the association of both diseases. Methods : Nineteen dogs affected by canine transmissible venereal tumour and canine leishmaniasis were studied retrospectively. Results : In these dogs, the tumour manifested a large size and often aggressive behaviour (42%) and no predictive sign of spontaneous regression was observed. Sporadic Leishmania amastigotes were found within the canine transmissible venereal tumour in three cases, probably transported by infected macrophages often infiltrating the tumour. A high Leishmania parasitisation of canine transmissible venereal tumour was observed in two other cases and verified by immunohistochemistry. Clinical Significance : Canine transmissible venereal tumour is a tumour of the dog able to harbour a large number of Leishmania parasites. Alternatively, the systemic disease (canine leishmaniasis) may lower the immune defence against malignancy (canine transmissible venereal tumour).     

Detection of Leishmania amastigotes in peripheral blood from four dogs--Short communication

The authors carried out microscopic examination of blood smears of 1438 dogs infected with Leishmania infantum. Unusual findings of leishmaniosis associated with circulating parasitised cells are described in four dogs. Most of the dogs presented severe illness, with lethargy, dysorexia, emaciation and alterations of the haematological pattern (anaemia, thrombocytopenia, neutrophilia and monocytosis). In three cases, leishmaniosis was associated with ehrlichiosis. On examination of peripheral blood smears, Leishmania sp. amastigotes were observed both in various circulating leukocytes (neutrophil, monocyte, macrophage) and free. In conclusion, parasites can rarely be detected in blood smears (in 0.28% of the animals examined); thus, the time-consuming microscopic search for amastigotes can make only a weak contribution to the conventional diagnosis of canine leishmaniosis.     

Leishmanicidal activity in vitro of Musa paradisiaca L. and Spondias mombin L. fractions

Visceral leishmaniasis (VL) is a zoonotic disease characterized by infection of mononuclear phagocytes by Leishmania chagasi. The primary vector is Lutzomyia longipalpis and the dog is the main domestic reservoir. The control and current treatment of dogs using synthetic drugs have not shown effectiveness in reducing the incidence of disease in man. In attempt to find new compounds with leishmanicidal action, plant secondary metabolites have been studied in search of treatments of VL. This study aimed to evaluate the leishmanicidal activity of Musa paradisiaca (banana tree) and Spondias mombin (cajazeira) chemical constituents on promastigotes and amastigotes of L. chagasi. Phytochemical analysis by column chromatography was performed on ethanol extracts of two plants and fractions were isolated. Thin layer chromatography was used to compare the fractions and for isolation the substances to be used in vitro tests. The in vitro tests on promastigotes of L. chagasi used the MTT colorimetric method and the method of ELISA in situ was used against amastigotes besides the cytotoxicity in RAW 264.7 cells. Of the eight fractions tested, Sm1 and Sm2 from S. mombin had no action against promastigotes, but had good activity against amastigotes. The fractions Mp1 e Mp4 of M. paradisiaca were very cytotoxic to RAW 264.7 cells. The best result was obtained with the fraction Sm3 from S. mombin with IC(50) of 11.26 μg/ml against promastigotes and amastigotes of 0.27 μg/ml. The fraction Sm3 characterized as tannic acid showed the best results against both forms of Leishmania being a good candidate for evaluation in in vivo tests.     

Subclinical leishmaniasis associated with infertility and chronic prostatitis in a dog

A stud dog was presented for acquired infertility. Haematospermia and teratozoospermia were found on two ejaculates 2 weeks apart. A presumptive diagnosis of prostatitis was made follo-wing ultrasound examination. An ultrasound-guided needle core biopsy was performed under general anaesthesia, revealing a mild chronic macrophagic and plasma cell prostatitis with intracytoplasmic amastigotes consistent with Leishmania spp. infection. Presence of Leishmania infantum, Leishmania donovani or Leishmania chagasi was confirmed by polymerase chain reaction in seminal plasma. Serology and serum protein electrophoresis confirmed the diagnosis of a subclinical active systemic leishmaniasis. A meglumine antimoniate and allopurinol treatment was given which clearly improved within 3 months both general condition and the quality of sperm. To the authors' knowledge, this is the first reported case of a prostatitis secondary to a Leishmania spp. infection. Subclinical systemic leishmaniasis should be considered in the differential diagnosis of infertility in dogs suffering from semen alterations.     

Cell mediated immunity and specific IgG1 and IgG2 antibody response in natural and experimental canine leishmaniosis

In the present study, we have followed up Leishmania infantum infection in dogs: (1) naturally infected; (2) experimentally infected with amastigotes; and (3) experimentally infected with culture promastigotes. The main objective was to evaluate the differences of the humoral and cellular immune responses of each group. Sera from 12 beagle dogs were analysed for total anti-leishmanial antibodies and IgG1 and IgG2 subclasses by enzyme-linked immunosorbent assay (ELISA). Lymphoproliferation to L. infantum antigen was also performed. All naturally infected animals were symptomatic with a marked humoral response. Dogs inoculated with amastigotes were asymptomotic and presented lower antibody titres than naturally infected. Dogs inoculated with culture promastigotes were asymptomotic with no significant humoral response. Strong proliferative responses to Leishmania antigen was observed in dogs inoculated with promastigotes. In our experimental model, IgG1 antibody levels presented a similar pattern in all infected animals, and IgG2 reactivity was high in naturally infected dogs.     

Visceral leishmaniasis with cardiac involvement in a dog: a case report

A dog presented with cutaneous nodules, enlarged lymph nodes and oedema in limbs, face and abdomen. The diagnosis of visceral leishmaniasis was established by identification of Leishmania amastigotes within macrophages from skin and popliteal lymph node biopsies. At necropsy, lesions were found in different organs, but it was particularly striking to observe large areas of pallor in the myocardium. Histological examination revealed an intense chronic inflammatory reaction in many organs, and numerous macrophages were found to contain amastigote forms of Leishmania. The inflammatory reaction was especially severe in the heart, where large areas of the myocardium appeared infiltrated with huge numbers of mononuclear immune cells, causing cardiac muscle atrophy and degeneration. Despite the severe inflammation, the number of parasitized macrophages was low in the myocardium, as revealed by immunohistochemical staining of Leishmania amastigotes. Because cardiac involvement is not usually described in this condition, this dog represents a very rare case of canine visceral leishmaniasis with affection of the myocardium.     

Axenic culture and identification of amastigotes from Sichuan human strain of Chinese Leishmania isolates

This work describes a simple method to yield large amounts of the isolate MHOM/CN/90/SC10H2 amastigotes-like forms in axenic cultures using promastigotes as the starting population. The isolate MHOM/CN/90/SC10H2, used in this study, belongs to an undescribed species of Leishmania endemic to hill foci in China. The method describes induced extracellular amastigote transformation of this isolate. The rounded parasite obtained in axenic culture was morphologically similar, even at the ultrastructural level, to intracellular amastigotes. Moreover, the axenic amastigotes remained viable as verified by the stage-specific genes (gp46 and p4 genes) with RT-PCR. A 70-80 kDa protein was recognized by polyclonal antibody HRP-IgG only in axenic-derived amastigotes and not in promastigotes.     

Canine visceral leishmaniasis: comparison of in vitro leishmanicidal activity of marbofloxacin, meglumine antimoniate and sodium stibogluconate

The control of canine leishmaniasis largely depends on the success of treatment. Drugs currently available to treat this disease are toxic and partially effective. The curative effect of marbofloxacin, a third-generation fluoroquinolone developed for veterinarian individual treatment, was evaluated in vitro in the presence of Leishmania infantum promastigotes and dog-monocyte-derived macrophages; meglumine antimoniate and sodium stibogluconate were used as comparative treatments. We observed that the killing of Leishmania promastigotes and intracellular amastigotes by marbofloxacin was dose-dependent. We demonstrated that successful treatment of canine infected macrophages for 48 h was possible with 500 microg/ml of marbofloxacin. Leishmanicidal activity acted through a TNF-alpha and nitric oxide pathway and correlated with the generation of nitric oxide (NO(2)) production by monocytes derived macrophages from infected (23+/-5 microM) or healthy (21+/-6 microM) dogs, in comparison with NO(2) concentration in infected/non-treated macrophages (< 3 microM, P<0.01). This significant induced parasiticidal effect correlated with extensive elimination of amastigotes by macrophages derived from infected (11+/-5) and healthy dogs (6+/-2), when compared to infected/non-treated macrophages (530+/-105 and 472+/-86 amastigotes, respectively, P< 0.01). Marbofloxacin was shown to be non-toxic at 500 microg/ml in vitro and no cell apoptosis was observed. The molecule was able to induce a parasitic process after significant elimination of amastigotes in leishmania-infected dog macrophages. We propose that marbofloxacin, compared to standard chemotherapeutic agents (meglumine antimoniate and sodium stibogluconate), could be an effective and pragmatic oral route alternative to treat canine leishmaniasis.     

Canine visceral leishmaniosis: a remarkable histopathological picture of one case reported from Brazil

This report describes a remarkable histopathological presentation of a symptomatic dog naturally infected with Leishmania (Leishmania) chagasi from Brazil. An intense inflammatory granulomatous reaction was observed in the liver and spleen associated with hypertrophy and hyperplasia of the mononuclear system (the classical histopathological picture of the disease). In addition, a spectrum of vascular lesions was observed in many organs. However, we did not find parasites (amastigotes of Leishmania) in any skin fragments of the ear, nose and or abdominal tissue. In fact, this animal had severe clinical signs, showed parasites in many organs, but no parasites in the skin. It appears that the presence or absence of parasites in the skin is not a good indicator of parasites in other organs or vice versa.     

Oxidative stress and non-enzymatic antioxidative status in dogs with visceral Leishmaniasis

Leishmaniasis is a potentially fatal chronic protozoan disease in human, canine and rodent species. The infection by Leishmania is endemic in the Mediterranean Sea region, Africa, Asia and South America. Canine visceral leishmaniasis (CanVL) is a systemic disease caused by Leishmania infantum and Leishmania chagasi from the Leishmania donovani complex group. The blood glutathione (GSH), plasma malondialdehyde (MDA), ascorbic acid (AA), beta-carotene, retinol and ceruloplasmin levels of dogs with CanVL were investigated to establish the status of the antioxidant defense mechanism in the infected animals. Dogs diagnosed as CanVL with amastigotes in lymph node smear examination and/or antibody titers > or = 128 were used as subjects, while those with no serological response against leishmaniasis were used as healthy controls. The glutathione and retinol amounts were decreased although not significantly (p > 0.05), but the MDA levels were significantly higher in dogs with VL, suggesting increased lipid peroxidation.     

Placentitis associated with leishmaniasis in a dog

A 1.5-year-old Coonhound from Maryland aborted 7 fetuses. Placenta and internal tissues of 1 fetus were examined histologically. The predominant lesion was placentitis characterized by necrosis and infiltration of mixed leukocytes. Numerous Leishmania spp amastigotes were identified in placental trophoblasts, and the diagnosis was confirmed by use of immunohistochemical staining with Leishmania-specific antibodies. Protozoa were not found in the fetal tissues. An indirect fluorescent antibody test yielded a serum titer of 1:100, and a recombinant K39 immunoassay of serum yielded positive results for the K39 Leishmania antigen.     

Cytologic patterns of lymphadenopathy in dogs infected with Leishmania infantum

BACKGROUND: Lymphadenopathy in canine leishmaniosis has been reported as reactive lymphoid hyperplasia or granulomatous (histiocytic) lymphadenitis. However, we are unaware of information on the effect of latent Leishmania infection on lymph node cytology compared with clinically affected dogs. OBJECTIVES: The aim of the present study was to investigate cytologic patterns of lymphadenopathy in dogs with clinical and subclinical forms of leishmaniosis and to correlate cytologic findings with the density of Leishmania amastigotes in fine needle aspiration (FNA) smears. METHODS: FNA cytology of prescapular or popliteal lymph nodes was evaluated on 32 dogs with clinical evidence of leishmaniosis (group A), 24 subclinically infected dogs (group B), and 17 clinically healthy noninfected dogs (group C); groups were based on the results of serologic and PCR tests for Leishmania sp. Differential nucleated cell counts (based on 300 cells) and amastigote density were determined microscopically. Cytologic findings were categorized and compared among groups. RESULTS: Cytologic abnormalities were found in 19 of 32 (59.4%) dogs in group A, 1 of 24 (4.2%) dogs in group B, and 2 of 17 (11.8%) dogs in group C and were significantly more frequent in group A than group B (P <.001) or C (P = .001). In group A, 68.7% of the dogs had lymphoid hyperplasia, 12.5% had lymphoid hyperplasia and histiocytic lymphadenitis, 6.3% had histiocytic lymphadenitis, and 3.1% had lymphoid hyperplasia and neutrophilic lymphadenitis. Lymphoid hyperplasia was also noted in 1 dog in group B, and lymphoid hyperplasia and eosinophilic lymphadenitis were each found in 1 dog in group C. Lymph node smears from 31 (96.9%) dogs in group A and 6 (25%) dogs in group B were positive for Leishmania amastigotes; however, no correlation was found between the density of amastigotes and cytopathologic patterns of lymphadenopathy. CONCLUSION: Abnormal lymph node cytology is much more common in dogs with clinical leishmaniosis than in dogs with subclinical infection, and primarily involves lymphoid hyperplasia. Despite finding no association between the density of amastigotes and type of lymphadenopathy, lymph node cytology still is a valuable diagnostic tool for diagnosing canine leishmaniosis.     

In situ hybridisation for the detection of Leishmania species in paraffin wax-embedded canine tissues using a digoxigenin-labelled oligonucleotide probe

The diagnosis of canine leishmaniosis (CanL) is currently predominantly achieved by cytological or histological identification of amastigotes in biopsy samples, demonstration of specific anti-Leishmania antibodies and PCR-based approaches. All these methods have the advantage of being sensitive and more or less specific; nevertheless, most of them also have disadvantages. A chromogenic in situ hybridisation (ISH) procedure with a digoxigenin-labelled probe, targeting a fragment of the 5.8S rRNA was developed for the detection of all species of Leishmania parasites in routinely paraffin wax-embedded canine tissues. This method was validated in comparison with traditional techniques (histology, PCR), on various tissues from three dogs with histological changes consistent with a florid leishmaniosis. Amastigote forms of Leishmania gave clear signals and were easily identified using ISH. Various tissues from 10 additional dogs with clinical suspicion or/and a positive serological test but without histological presence of amastigotes did not show any ISH signals. Potential cross-reactivity of the probe was ruled out by negative outcome of the ISH against selected protozoa (including the related Trypanosoma cruzi) and fungi. Thus, ISH proved to be a powerful tool for unambiguous detection of Leishmania parasites in paraffin wax-embedded tissues.     

Primary hypothyroidism associated with leishmaniasis in a dog

A case of primary hypothyroidism associated with leishmaniasis is described in a four-year-old, male Yorkshire terrier. Clinical diagnosis of hypothyroidism was confirmed by a low baseline serum tetraiodothyronine (T4), a reduced response to thyroid-stimulating hormone (TSH) stimulation, an increased serum TSH concentration, and scintigraphic thyroid gland examination. Examination of a thyroid biopsy showed many Leishmania amastigotes, both inside and outside of macrophages, together with signs of follicular atrophy.     

Feline leishmaniasis due to Leishmania (Leishmania) amazonensis in Mato Grosso do Sul State, Brazil

A case of leishmaniasis in a domestic cat (Felis domesticus) is described. The animal showed a single, nodular lesion on the nose and many nodules of different size on the ears and digital regions of all the paws. Diagnosis was made by microscopic detection of amastigotes in Giemsa-stained smears from the lesions. By monoclonal antibodies the aetiological agent was identified as Leishmania (Leishmania) amazonensis, one of the seven species implicated in human leishmaniasis in Brazil. The clinical signs in feline leishmaniasis are unspecific and similar to those observed in other diseases such as cryptococcosis and in sporotrichosis, commonly found in cats. Leishmaniasis should therefore, be added to the differential diagnosis by feline veterinary practitioners and adequate investigations should carried out for dermal leishmaniasis in the area where the feline infection is detected.