Agent Of Human Visceral Leishmaniasis Research Articles

Canine Leishmaniasis in Southern Brazil: Diagnosis and Clinical Features in Domestic Dogs

Leishmania infantum is a hemopathogen of importance for the health of domestic dogs (Canis lupus familiaris), causing canine leishmaniasis (CanL), and it is also the etiological agent of human visceral leishmaniasis (HVL). This parasite was not reported in southern Brazil until the early 2000s, but CanL and HVL were increasingly reported in the last 15 years, mainly in cities bordering Argentina. The present study aimed to detect L. infantum in domestic dogs and to determine the main clinical manifestations in infected animals from Uruguaiana, a city with a high incidence of CanL. Fifty-one dogs suspected of having CanL in the urban perimeter of the city were clinically examined by veterinarians and investigated for the occurrence of L. infantum with two immunoassays (rapid chromatography test and ELISA) and real-time PCR (polymerase chain reaction). Clinical signs were compared in positive and negative L. infantum animals. A total of 31 dogs (60.8%) were infected with L. infantum. The main clinical manifestations associated with CanL dogs were onychogryphosis and peeling (p < 0.05). L. infantum was frequently detected in urban dogs from Uruguaiana, highlighting the concerning situation regarding health in this city. The occurrence of some clinical signs (onychogryphosis/peeling) could help to detect CanL more frequently in the canine population.

In Vitro Drug Susceptibility of a Leishmania (Leishmania) infantum Isolate from a Visceral Leishmaniasis Pediatric Patient after Multiple Relapses.

The parasitic protozoan Leishmania (Leishmania) infantum is the etiological agent of human visceral leishmaniasis in South America, an infectious disease associated with malnutrition, anemia, and hepatosplenomegaly. In Brazil alone, around 2700 cases are reported each year. Treatment failure can occur as a result of drug, host, and/or parasite-related factors. Here, we isolated a Leishmania species from a pediatric patient with visceral leishmaniasis that did not respond to chemotherapy, experiencing a total of nine therapeutic relapses and undergoing a splenectomy. The parasite was confirmed as L. (L.) infantum after sequencing of the ribosomal DNA internal transcribed spacer, and the clinical isolate, in both promastigote and amastigote forms, was submitted to in vitro susceptibility assays with all the drugs currently used in the chemotherapy of leishmaniasis. The isolate was susceptible to meglumine antimoniate, amphotericin B, pentamidine, miltefosine, and paromomycin, similarly to another strain of this species that had previously been characterized. These findings indicate that the multiples relapses observed in this pediatric patient were not due to a decrease in the drug susceptibility of this isolate; therefore, immunophysiological aspects of the patient should be further investigated to understand the basis of treatment failure in this case.

Detection of Leishmania sp. kDNA in questing Ixodes ricinus (Acari, Ixodidae) from the Emilia-Romagna Region in northeastern Italy

To date, sand flies (Phlebotominae) are the only recognized biological vectors of Leishmania infantum, the causative agent of human visceral leishmaniasis, which is endemic in the Mediterranean basin and also widespread in Central and South America, the Middle East, and Central Asia. Dogs are the main domestic reservoir of zoonotic visceral leishmaniasis, and the role of secondary vectors such as ticks and fleas and particularly Rhipicephalus sanguineus (the brown dog tick) in transmitting L. infantum has been investigated. In the present paper, the presence of Leishmania DNA was investigated in questing Ixodes ricinus ticks collected from 4 rural areas included in three parks of the Emilia-Romagna Region (north-eastern Italy), where active foci of human visceral leishmaniasis have been identified. The analyses were performed on 236 DNA extracts from 7 females, 6 males, 72 nymph pools, and 151 larvae pools. Four samples (1.7%) (i.e., one larva pool, 2 nymph pools, and one adult male) tested positive for Leishmania kDNA. To the best of our knowledge, this is the first report of the presence of Leishmania kDNA in questing I. ricinus ticks collected from a rural environment. This finding in unfed larvae, nymphs, and adult male ticks supports the hypothesis that L. infantum can have both transstadial and transovarial passage in I. ricinus ticks. The potential role of I. ricinus ticks in the sylvatic cycle of leishmaniasis should be further investigated.

Isolation, typing, and drug susceptibility of Leishmania (Leishmania) infantum isolates from dogs of the municipality of Embu das Artes, an endemic region for canine leishmaniasis in Brazil.

The parasitic protozoa Leishmania (Leishmania) infantum is the etiological agent of human visceral leishmaniasis and canine leishmaniasis in South America, where Brazil is the most affected country. This zoonotic disease is transmitted by the bite of an infected phlebotomine sand fly and dogs constitute the main domestic reservoir of the parasite. In this study, we screened 2348 dogs of the municipality of Embu das Artes, Brazil, for antibodies against the parasite. Prevalence for canine leishmaniasis seropositivity was 2.81%, as assessed using a Dual-Path Platform rapid test for canine leishmaniasis. Twenty-five seropositive dogs were euthanized for parasite isolation and 14 isolates were successful obtained. Nucleotide sequencing of the internal transcribed spacer confirmed the isolates to be L. (L.) infantum, and very low sequence variability was observed among them. The in vitro susceptibility to miltefosine and paromomycin was assessed and moderate variation in paromomycin susceptibility was found among the isolates in the promastigote and intracellular amastigote stages. On the other hand, in vitro susceptibility to miltefosine of these isolates was homogenous, particularly in the amastigote stage (EC50 values from 0.69 to 2.07μM). In addition, the miltefosine sensitivity locus was deleted in all the isolates, which does not corroborate the hypothesis that the absence of this locus is correlated with a low in vitro susceptibility. Our findings confirm that the municipality of Embu das Artes is endemic for canine leishmaniasis and that isolates from this region are susceptible to paromomycin and miltefosine, indicating the potential of these drugs to be clinically evaluated in the treatment of human visceral leishmaniasis in Brazil.

Resistance to Experimental Visceral Leishmaniasis in Mice Infected With Leishmania infantum Requires Batf3

Unveiling the protective immune response to visceral leishmaniasis is critical for a rational design of vaccines aimed at reducing the impact caused by this fatal, if left untreated, vector-borne disease. In this study we sought to determine the role of the basic leucine zipper transcription factor ATF-like 3 (Batf3) in the evolution of infection with Leishmania infantum, the causative agent of human visceral leishmaniasis in the Mediterranean Basin and Latin America. For that, Batf3-deficient mice in C57BL/6 background were infected with an L. infantum strain expressing the luciferase gene. Bioluminescent imaging, as well as in vitro parasite titration, demonstrated that Batf3-deficient mice were unable to control hepatic parasitosis as opposed to wild-type C57BL/6 mice. The impaired microbicide capacities of L. infantum-infected macrophages from Batf3-deficient mice mainly correlated with a reduction of parasite-specific IFN-γ production. Our results reinforce the implication of Batf3 in the generation of type 1 immunity against infectious diseases.

Canine neutrophils cooperate with macrophages in the early stages of Leishmania infantum in vitro infection.

Leishmania infantum is the aetiological agent of human visceral leishmaniasis and canine leishmaniasis, both systemic and potentially fatal diseases. Polymorphonuclear neutrophils (PMN) are the first cells to phagocyte this parasite at the inoculation site, but macrophages (MØ) are the definitive host cells, ensuring parasite replication. The interaction between dog MØ, PMN and Linfantum promastigotes was in vitro investigated. It was observed that promastigotes establish contact with blood monocyte-derived MØ mainly by the tip of the flagellum. These cells, that efficiently bind and internalize parasites, underwent major morphological changes, produced nitric oxide (NO) and released histone H1 in order to inactivate the parasite. Transfer of intracellular parasites from PMN to MØ was confirmed by flow cytometry, using Linfantum expressing a green fluorescent protein. The interaction of MØ with Linfantum-infected PMN lead to NO production and release of extracellular traps, which may contribute to parasite containment and inactivation. This study highlights for the first time the diversity of cellular and molecular events triggered by the interaction between canine PMN and MØ, which can promote a reduction of parasite burden in the early phase of Linfantum infection.

Genetic polymorphism in Leishmania infantum isolates from human and animals determined by nagt PCR-RFLP

BackgroundLeishmania infantum is the causative agent of human visceral leishmaniasis (VL) and sporadic human cutaneous leishmaniasis (CL) in the Mediterranean region. The genetic variation of the Leishmania parasites may result in different phenotypes that can be associated with the geographical distribution and diversity of the clinical manifestations. The main objective of this study was to explore the genetic polymorphism in L. infantum isolates from human and animal hosts in different regions of Morocco.MethodsThe intraspecific genetic variability of 40 Moroccan L. infantum MON-1 strains isolated from patients with VL (n = 31) and CL (n = 2) and from dogs (n = 7) was evaluated by PCR-RFLP of nagt, a single-copy gene encoding N-acetylglucosamine-1-phosphate transferase. For a more complete analysis of L. infantum polymorphism, we included the restriction patterns of nagt from 17 strains available in the literature and patterns determined by in-silico digestion of three sequences from the GenBank database.ResultsMoroccan L. infantum strains presented a certain level of genetic diversity and six distinct nagt-RFLP genotypes were identified. Three of the six genotypes were exclusively identified in the Moroccan population of L. infantum: variant M1 (15%), variant M2 (7.5%), and variant M3 (2.5%). The most common genotype (65%), variant 2 (2.5%), and variant 4 (7.5%), were previously described in several countries with endemic leishmaniasis. Phylogenetic analysis segregated our L. infantum population into two distinct clusters, whereas variant M2 was clearly distinguished from both cluster I and cluster II. This distribution highlights the degree of genetic variability among the Moroccan L. infantum population.ConclusionThe nagt PCR-RFLP method presented here showed an important genetic heterogeneity among Moroccan L. infantum strains isolated from human and canine reservoirs with 6 genotypes identified. Three of the six Moroccan nagt genotypes, have not been previously described and support the particular genetic diversity of the Moroccan L. infantum population reported in other studies.

First identification of L. major in a dog in an endemic area of human cutaneous leishmaniasis in Iraq: molecular and phylogenetic studies.

Canine leishmaniasis (CanL) caused by Leishmania infantum (L. infantum) is considered as a zoonotic disease and within the last few decades, studies have identified the parasite as a major causative agent of human visceral leishmaniasis. However, in dogs, few recent studies have determined L. major as a cause of cutaneous manifestations and L. tropica as an etiological agent for cutaneous lesions involving mucosa. Interestingly, current study has found canine cutaneous lesions with mucosal involvement in a dog diagnosed with L. major, for the first time, in a focused area of human cutaneous leishmaniasis (CL) in the borderline between northern and central Iraq. Both molecular and phylogenetic studies showed that the dog L. major strain is closely related to that previously isolated from human CL in the same area. Moreover, serological study using rK39 identified IgG response against Leishmania, and the histological finding revealed the infiltration of inflammatory cells around the infection sites. These data will broaden our knowledge about CanL concerning the appearance of cutaneous clinical manifestations with mucocutaneous lesions caused by L. major. Further study on other animal reservoirs and vectors will shed the light on the epidemiology of this disease.

An investigation on vertical transmission of Leishmania infantum in experimentally infected dogs and assessment of offspring's infectiousness potential by xenodiagnosis

Dogs are the main reservoir host of Leishmania infantum, etiologic agent of human visceral leishmaniasis (HVL) and canine visceral leishmaniasis (CVL). Transmission of L. infantum to humans and dogs is mainly through the bite of infected sand flies. In the Western Mediterranean basin, Phlebotomus perniciosus is the main vector of L. infantum. However, occasional vertical transmission of L. infantum has been reported. This study investigated L. infantum vertical transmission in offspring of experimentally infected dogs. Among 14 surviving puppies from three female beagle dogs that developed CVL following an experimental infection with L. infantum, one was tested positive by indirect immunofluorescence antibody test, by PCR and by xenodiagnosis with a high parasite burden in the spleen at 14 months old. None of the remaining puppies were tested positive for L. infantum. These findings strongly suggest that infected puppies following vertical transmission can sustain infection and contribute in infecting sand flies with L. infantum. Any strategy for controlling CVL should take into consideration the vertical transmission of L. infantum.

New insights about cross-reactive epitopes of six trypanosomatid genera revealed that Crithidia and Leptomonas have antigenic similarity to L. (L.) chagasi

We investigated whether ELISA using crude antigens from insect and plant trypanosomatids, which are non-pathogenic and easily cultivated in large scale, has the same positivity data as Leishmania (Leishmania) chagasi, the etiological agent of human visceral leishmaniasis (VL) or canine leishmaniasis (CanL), or as Trypanosoma cruzi, the etiological agent of Chagas disease (CD). The antigens from Crithidia fasciculata, Crithidia luciliae, and Leptomonas seymouri showed 100% cross-reactivity with VL and CanL samples, with no statistically titers differences from L. (L.) chagasi, however, 34% (17/50) of VL samples revealed higher titers using the insect trypanosomatids than the homologous antigen. On the other hand, antigens from Strigomonas culicis, Angomonas deanei, and Phytomonas serpens showed low cross-reactivity with VL and CanL samples. The sera from patients with American tegumentary leishmaniasis showed low levels of cross-reactivity with all trypanosomatids investigated, even with L. (L) chagasi, without titers dissimilarity among them. These parasites were also worthless as antigen source for detection of CD cases, which required homologous antigens to reach 100% positivity. This study showed, by ELISA, that crude extract of Crithidia and Leptomonas have epitopes similar to L. (L.) chagasi, which supports the idea of using them as antigens source for the serodiagnosis of visceral leishmaniasis.

Prevalence of canine leishmaniasis in Beichuan County, Sichuan, China and phylogenetic evidence for an undescribed Leishmania sp. in China based on 7SL RNA

BackgroundLeishmaniasis is a vector-borne disease, which is still endemic in the west and northwest area of China. Canines are the major reservoirs of Leishmania, the etiological agent of human visceral leishmaniasis. Phlebotomus chinensis is the main transmission vector of zoonotic visceral leishmaniasis (ZVL).MethodsIn this study, rK39 dip-stick, ELISA and PCR methods were used to investigate the prevalence of canine leishmaniasis (CanL) in Beichuan County, Sichuan Province, China.ResultsAmong the 86 dogs which were included in the study, 13 dogs were positive using the dip-stick test (15.12%), while 8 dogs were positive using ELISA (9.30%) and 19 dogs were positive for PCR (22.03%). In total, 32 dogs were positive for one or more tests (37.21%). Interestingly, phylogenetic analysis based on the partial 7SL RNA fragment provided evidence that an undescribed Leishmania species, which is clearly a causative agent of CanL and human visceral leishmaniasis, does exist in China. This result is consistent with our previous study.ConclusionsOur work confirmed that canine leishmaniasis is still prevalent in Beichuan County. Further control is urgently needed, as canine leishmaniasis is of great public health importance. The phylogenetic analysis based on 7SL RNA segment provides evidence for the existence of an undescribed Leishmania sp. in China.

Disseminated leishmaniasis caused by Leishmania tropica in HIV-positive patients in the Islamic Republic of Iran

Both cutaneous and visceral forms of leishmaniasis are prevalent in some parts of the Islamic Republic of Iran [1,2]. The Mediterranean type of visceral leishmaniasis (VL) is also reported sporadically throughout the country [3] with canines being the main animal reservoir hosts [4]. The cutaneous form of leishmaniasis is seen in 2 forms: anthroponotic and zoonotic. Anthroponotic cutaneous leishmaniasis (ACL) is caused by Leishmania tropica and is prevalent in many largeand medium-sized cities in the country [1,5]. Zoonotic cutaneous leishmaniasis (ZCL) is caused by L. major and is endemic in many foci in the north, east and south of the country [6]. Some studies have implicated L. tropica as another agent of VL in humans and dogs in reports from the north-west and south of the country [4,7,8]. Few data are available, however, about host immunological response and parasite destruction when leishmaniasis is associated with immunosuppressants. At present, the majority of cases of HIV– leishmaniasis co-infection reported in the Mediterranean basin were caused by L. infantum [9]. Disseminated leishmaniasis caused by L. tropica in patients with HIV infection is uncommon. Here, we report on 2 patients with HIV–leishmaniasis co-infection caused by L. tropica with generalized and multiple skin lesions and visceral involvement. This is the first such report from the Islamic Republic of Iran.

Immunomodulatory principles of Pelargonium sidoides.

Extracts and isolated constituents (coumarins and phenols) of Pelargonium sidoides DC, a plant species used in folk medicine by the Southern African native population, were evaluated for their effects on nonspecific immune functions. Although this herbal medicine is also successfully employed in modern phytotherapy in Europe to cure infectious diseases of the respiratory tract, the scientific basis of its remedial effects is still unclear. Thus, functional bioassays including an in vitro model for intracellular infection with Leishmania parasites, an extracellular Leishmania growth assay, a fibroblast-virus protection assay (IFN activity), a fibroblast-lysis assay (TNF activity) and a biochemical assay for inorganic nitric oxides (iNO) were employed. None of the test samples revealed significant activity against extracellular, promastigote Leishmania donovani, the causative agent of human visceral leishmaniasis. In contrast, apart from the coumarin samples, all the Pelargonium extracts (EC(50) <0.1-3.3 microg/mL), gallic acid (EC(50) 4.4 microg/mL) and its methyl ester (EC(50) 12.5 microg/mL) significantly reduced the intracellular survival of L. donovani amastigotes within murine macrophages. These data indicate that the samples acted indirectly on Leishmania parasites, possibly by activating leishmanicidal macrophage functions. Macrophage activation was confirmed by detection of tumour necrosis factor (TNF-alpha) and inorganic nitric oxides (iNO) in supernatants of sample-treated macrophage cultures. Synthesis of iNO is a well-known effector mechanism of macrophages against microorganisms such as Leishmania. Interestingly, blocking iNO-synthase with L-NMMA had no substantial effect on sample-induced intracellular Leishmania kill. From bioassay-guided fractionation, gallic acid and its methyl ester present in large amounts in P. sidoides and in its active extracts, were identified as the prominent immunomodulatory principle for this herbal medicine. The results, when taken together with recent reported antibacterial activity, provide a rational basis for both the traditional and the present utilization of P. sidoides in the claimed conditions.

Chemiluminescence activity in whole blood phagocytes of dogs naturally infected withLeishmania infantum

Dogs are the domestic reservoir of Leishmania infantum, a vector-borne intracellular protozoan agent of human visceral leishmaniasis. The role of polymorphonuclear leukocytes (PMNs) in the immune defence against this parasite has been poorly studied. We have investigated the function of peripheral blood PMNs in naive beagle dogs that have been naturally exposed to phlebotomine vectors in an area highly endemic for canine leishmaniasis, and found infected by Leishmania at the end of the transmission season. Whole blood phagocyte oxidative metabolism was assessed by a rapid method that determines a luminol-amplified chemiluminescence (CL) emission. This was evaluated using either a soluble stimulant, phorbol mirystate acetate (PMA), or phagocytic stimuli, such as zymosan unopsonized (ZYM) or opsonized with autologous serum (OPZ). In blood samples taken 2 months after exposure to Leishmania transmission, data on CL emission revealed a significant decrease of reactive oxygen intermediates (ROI) production in the presence of both PMA and ZYM, compared with blood samples obtained from dogs before exposure. On the contrary, no variations in CL emission were detected in presence of OPZ. Our data indicate that immunological changes occur early in canine leishmaniasis and confirm that the role of PMNs and their products need to be clarified.

Inhibition of Leishmania donovani promastigote internalization into murine macrophages by chemically defined parasite glycoconjugate ligands

Leishmania donovani, the agent of human visceral leishmaniasis, is an intracellular parasite that must be recognized and internalized by host macrophages to complete its biological cycle. In a search for possible ligands for macrophage surface receptors, glycoconjugates were obtained from Leishmania promastigotes by aqueous, phenol-aqueous, and alkaline extraction. A fucose-mannose glycoproteic ligand, a lipopeptidephosphoglycan, and a phosphate mannogalactan ligand were purified from promastigotes and analyzed for their chemical contents, with special attention to their glycidic moieties. Sugars that were identified as components of these glycoconjugates were tested for their capacity to inhibit promastigote internalization by BALB/c peritoneal macrophages in vitro. Neutral hexoses showed little inhibitory activity; fucose, charged monosaccharides, and a mannose polymer showed the highest activity. Two of the glycoconjugates (fucose-mannose glycoproteic ligand and phosphate mannogalactan ligand) purified from promastigotes were potent inhibitors of internalization, 75% inhibition being obtained at concentrations of 6 to 10 micrograms/ml. The simultaneous presence of both ligands in low concentrations yielded an increase in inhibitory activity above that found for each ligand alone, indicating that promastigotes may use at least two receptor sites for penetration into macrophages. These ligands are specific inhibitors of L. donovani promastigote phagocytosis, since 10 micrograms of each ligand per ml interfered neither with internalization of yeast cells nor with phagocytosis of Leishmania adleri promastigotes.

Multiplication of a human parasite (Leishmania donovani) in phagolysosomes of hamster macrophages in vitro.

Leishmania donovani, the etiological agent of human visceral leishmaniasis, was grown in hamster peritoneal macrophages in vitro. By electron microscopy, using a lysosomal marker, these parasitic protozoa were seen to multiply within host cell phagolysosomes. The survival mechanism of this intracellular parasite is based apparently upon resistance to macrophage lysosomal enzymic digestion.