Occurrence of Leishmania infantum in the central nervous system of naturally infected dogs: Parasite load, viability, co-infections and histological alterations.

Zoonotic visceral leishmaniasis is caused by the protozoan Leishmania infantum and little is known about the occurrence and pathogenesis of this parasite in the CNS. The aims of this study were to evaluate the occurrence, viability and load of L. infantum in the CNS, and to identify the neurological histological alterations associated with this protozoan and its co-infections in naturally infected dogs. Forty-eight Leishmania-seropositive dogs from which L. infantum was isolated after necropsy were examined. Cerebrospinal fluid (CSF) samples were analyzed by parasitological culture, quantitative real-time PCR (qPCR) and the rapid immunochromatographic Dual Path Platform test. Brain, spinal cord and spleen samples were submitted to parasitological culture, qPCR, and histological techniques. Additionally, anti-Toxoplasma gondii and anti-Ehrlichia canis antibodies in serum and distemper virus antigens in CSF were investigated. None of the dogs showed neurological signs. All dogs tested positive for L. infantum in the CNS. Viable forms of L. infantum were isolated from CSF, brain and spinal cord in 25% of the dogs. Anti-L. infantum antibodies were detected in CSF in 61% of 36 dogs. Inflammatory histological alterations were observed in the CNS of 31% of the animals; of these, 66% were seropositive for E. canis and/or T. gondii. Amastigote forms were associated with granulomatous non-suppurative encephalomyelitis in a dog without evidence of co-infections. The highest frequency of L. infantum DNA was observed in the brain (98%), followed by the spinal cord (96%), spleen (95%), and CSF (50%). The highest L. infantum load in CNS was found in the spinal cord. These results demonstrate that L. infantum can cross the blood-brain barrier, spread through CSF, and cause active infection in the entire CNS of dogs. Additionally, L. infantum can cause inflammation in the CNS that can lead to neurological signs with progression of the disease.

T lymphocyte immunophenotypes in the cerebrospinal fluid of dogs with visceral leishmaniasis.

Visceral leishmaniasis (VL) is a disease causing several clinical manifestations in dogs, including neurological disorders. Nevertheless, there are few studies related to the evaluation of the brain alterations during VL. Evidences of the involvement of cerebral barriers in infected dogs was reported, including the presence of brain inflammatory infiltrate, with a predominance of CD3+ T cells. Therefore, the aim of this study was to determine the immunophenotypes of T lymphocytes in the cerebrospinal fluid (CSF), as well as in peripheral blood, and to correlate with brain alterations in dogs with VL. We detected elevated percentages of double negative (DN) and double positive (DP) T cells in the CSF, with a predominance of TCRαb. In the histopathological analysis, we observed a predominance of lymphoplasmacytic infiltrate, mainly in leptomeninges, ranging from mild to intense, and we observed a positive correlation between the intensity of inflammation in the subependymal area and the DN T cells of the CSF. Thus, the DN T cells seem be acting as villains of the immune system through pro-inflammatory mechanisms. Further, the proportion of the different population of CSF T cells did not differ from those observed in the blood, which provides us with more evidence of blood-CSF barrier breakdown. Together, the results provide more explanation to the inflammation observed in the brain of dogs with VL, which the DN T cells contribute to the origin and progression of the neurological disease. This study provides insight into the immunophenotypes of T lymphocytes in the CSF during canine visceral leishmaniasis.

Intramedullary spinal cord mass presumptively associated with leishmaniasis in a dog.

CASE DESCRIPTION: A 9-year-old male Miniature Poodle was evaluated because of progressive severe right hemiparesis, right forelimb lameness, and signs of cervical pain. CLINICAL FINDINGS: A low body condition score (2/9) and popliteal lymphadenopathy were detected. Results of a CBC, serum biochemical analyses, urinalysis, cytologic examination of bone marrow and popliteal lymph node aspirates, and serum ELISA were consistent with systemic leishmaniasis. Magnetic resonance imaging of the cervical spinal cord revealed an intramedullary mass extending from the caudal aspect of the C5 vertebral body to the C5-6 intervertebral disk space with a contrast medium-enhanced pattern that had 3 zones (central contrast medium-enhanced core, intermediate isointense zone, and peripheral contrast medium-enhanced ring). Surgical biopsy of the mass was performed by means of a right C5-6 dorsal hemilaminectomy. Results of PCR assays for detection of Leishmania DNA in CSF and tissue biopsy samples were positive. TREATMENT AND OUTCOME: Treatment for systemic leishmaniasis was initiated. Two months later, body condition, neurologic signs, and gait of the dog had substantially improved; the dog had mild right forelimb paresis at that time. Results of follow-up MRI indicated resolution of the cervical spinal cord lesion. Four months after diagnosis, the dog's neurologic condition was stable. CLINICAL RELEVANCE: To the authors' knowledge, this report is the first in which clinical findings, clinicopathologic data, and MRI characteristics of an intramedullary inflammatory spinal cord lesion presumptively attributable to leishmaniasis in a dog have been reported, and the first report of CNS leishmaniasis in a dog with MRI resolution and a successful clinical response to treatment.

[Visceral leishmaniasis in two children after vacation in Southern Europe]. / Visceral leishmaniasis hos to børn efter ferie i Sydeuropa.

Pancytopenia, fever and splenomegaly are frequent causes for referrals to paediatric haematology departments, on the suspicion of acute leukaemia. We report two cases of Danish children with the tropical disease visceral leishmaniasis (VL) contracted on short vacations in Southern Europe. One of the patients developed secondary haemophagocytic lymphohistocytosis (HLH). Both children were successfully treated with liposomal amphotericin B. In Denmark, VL is a rare but important differential diagnosis to acute leukaemia and HLH, and should be ruled out after journeys to endemic areas, including Southern Europe.

Leishmania amastigotes in the central nervous system of a naturally infected dog.

A 4-year-old male Labrador Retriever dog was presented with a 10-day history of tetraplegia, depression, and absent postural reflexes. The cerebrospinal fluid was positive for Leishmania spp. DNA. At necropsy, a 2-cm long mass was observed adhered to C(7) and C(8) left spinal nerves. Microscopically, nerve fiber destruction together with mixed inflammatory infiltration was observed in the spinal nerves. Cervical spinal cord sections showed multifocal, diffuse granulomatous inflammation in the white matter. In the brain, perivascular infiltrates were observed in some areas together with subtle pallor of the parenchyma. Immunohistochemistry for Leishmania infantum confirmed the presence of amastigotes in the spinal nerves, spinal cord, brain parenchyma, and choroid plexuses. The current study describes the presence of Leishmania amastigotes in nervous tissue inciting radiculoneuritis, myelitis, and mild meningoencephalitis, suggesting a likely route by which L. infantum amastigotes reach and affect the central nervous system parenchyma.

Glial reactivity in dogs with visceral leishmaniasis: correlation with T lymphocyte infiltration and with cerebrospinal fluid anti-Leishmania antibody titres.

Visceral leishmaniasis is a multisystemic zoonotic disease that can manifest with several symptoms, including neurological disorders. Because glial cells are extensively associated with the immune response within the brain, we evaluated the morphology of astrocytes and microglia of dogs naturally infected with Leishmania chagasi. We used immunohistochemical and lectin-histochemical techniques for morphological analyses and we also examined the glial correlation with lymphocyte infiltration of the brain and with the presence of anti-Leishmania antibodies within the cerebrospinal fluid of the dogs. Although we did not detect a shared morphological pattern in the astrocytes or microglia in the brain tissue, these cells were more intensely labelled in infected dogs than in the control group. The density of microglia was increased in the ependymal/subependymal area, thus demonstrating a strong correlation with the presence of T lymphocytes and with cerebrospinal fluid antibody titres. Thus, our results indicate a pro-inflammatory state in the brains of dogs naturally infected with L. chagasi and strongly suggest that microglia and astrocytes are involved in the pathogenesis of the neurological disorders of visceral leishmaniasis in dogs.

Anti-leishmania antibodies in cerebrospinal fluid from dogs with visceral leishmaniasis.

Visceral leishmaniasis in Brazil is caused by Leishmania (Leishmania) chagasi and the dog is its most important reservoir. The clinical features in dogs include loss of weight, lymphadenopathy, renal failure, skin lesions, fever, hypergammaglobulinemia, hepatosplenomegaly, anemia, and, rarely, neurological symptoms. Most infected animals develop active disease, characterized by high anti-leishmania antibody titers and depressed lymphoproliferative ability. Antibody production is not primarily important for protection but might be involved in the pathogenesis of tissue lesions. An ELISA test was used to determine if there is an association between neurological symptoms and the presence of anti-L. chagasi antibodies in cerebrospinal fluid (CSF). Thirty serum and CSF samples from symptomatic mixed breed dogs (three with neurological symptoms) from a region of high incidence of visceral leishmaniasis in Brazil were examined for antibody using total parasite antigen and anti-dog IgG peroxidase conjugate. A high level of L. chagasi antibodies was observed in sera (mean absorbance SD, 1.939 0.405; negative control, N = 20, 0.154 0.074) and CSF (1.571 0.532; negative control, N = 10, 0.0195 0.040) from all animals studied. This observation suggests that L. chagasi can cause breakdown of filtration barriers and the transfer of antibodies and antigens from the blood to the CSF compartment. No correlation was observed between antibody titer in CSF and neurological symptoms.

Migration of Leishmania donovani amastigotes in the cerebrospinal fluid.

A 10-year-old boy had been suffering from kala-azar (visceral leishmaniasis) for two and a half years. He failed to respond to all known anti-leishmanial treatment regimens. Even the drastic step of splenectomy failed to cure him. In November 1991, he presented with symptoms of meningitis. A diagnostic lumbar puncture revealed leishmanial amastigotes in his cerebrospinal fluid. The patient was finally cured with a course of amphotericin B, a drug known to cross the blood-brain barrier.