A cross-sectional study on canine Leishmania (L.) infantum chagasi infection in Amazonian Brazil ratifies a higher prevalence of specific IgG-antibody response than delayed-type hypersensitivity in symptomatic and asymptomatic dogs

This was a cross-sectional study which analyzed the prevalence and the clinical and immunological spectrum of canine Leishmania (L.) infantum chagasi infection in a cohort of 320 mongrel dogs living in an endemic area of American visceral leishmaniasis in the Amazonian Brazil by using, mainly, the indirect fluorescence antibody test (IFAT-IgG) and the delayed-type hypersensitivity (DTH), and the parasite research by the popliteal lymph node aspiration. The IFAT and DTH reactivity recognized three different immune response profiles: (1) IFAT((+))/DTH((-)) (107 dogs), (2) IFAT((-))/DTH((+)) (18 dogs), and (3) IFAT((+))/DTH((+)) (13 dogs), providing an overall prevalence of infection of 43?% (138/320). Thus, the specific prevalence of IFAT( (+) )/DTH( (-) ) 33.4?% (107/320) was higher than those of IFAT( (-) )/DTH( (+) ) 5.6?% (18/320) and IFAT( (+) )/DTH( (+) ) 4.0?% (13/320). Moreover, the frequency of these profiles among 138 infected dogs showed that the IFAT( (+) )/DTH( (-) ) rate of 77.5?% (107/138) was also higher than those of 13.0?% (18/138) of IFAT( (-) )/DTH( (+) ) and 9.5?% (13/138) of IFAT( (+) )/DTH( (+) ) rates. The frequency of asymptomatic dogs (76?%-105) was higher than those of symptomatic (16.6?%-23) and oligosymptomatic ones (7.4?%-10). A total of 16 (11.6?%) L. (L.) i. chagasi isolates were obtained from infected dogs, all from the IFAT( (+) ) /DTH( (-) ) profile: 41?% (9/22) from symptomatic, 33.3?% (3/9) from oligosymptomatic, and 5.2?% (4/76) from asymptomatic dogs. These findings strongly suggested that despite the higher frequency of asymptomatic dogs (76?%-105), the majority (72.4?%-76) was characterized by the IFAT( (+) ) /DTH( (-) ) profile with a doubtful immunogenetic resistance against infection.     

A longitudinal study on the transmission dynamics of human Leishmania (Leishmania) infantum chagasi infection in Amazonian Brazil, with special reference to its prevalence and incidence

This was a longitudinal study carried out during a period over 2 years with a cohort of 946 individuals of both sexes, aged 1 year and older, from an endemic area of American visceral leishmaniasis (AVL) in Pará State, Brazil. The object was to analyze the transmission dynamics of human Leishmania (Leishmania) infantum chagasi infection based principally on the prevalence and incidence. For diagnosis of the infection, the indirect fluorescent antibody test (IFAT) and leishmanin skin test (LST) were performed with amastigote and promastigote antigens of the parasite, respectively. The prevalence by LST (11.2%) was higher (p < 0.0001) than that (3.4%) by IFAT, and the combined prevalence by both tests was 12.6%. The incidences by LST were also higher (p < 0.05) than those by IFAT at 6 (4.7% × 0.6%), 12 (4.7% × 2.7%), and 24 months (2.9% × 0.3%). Moreover, there were no differences (p > 0.05) between the combined incidences by both tests on the same point surveys, 5.2%, 6.3%, and 3.6%. During the study, 12 infected persons showed high IFAT IgG titers with no LST reactions: five children and two adults developed AVL (2,560–10,120), and two children and three adults developed subclinical oligosymptomatic infection (1,280–2,560). The combined tests diagnosed a total of 231 cases of infection leading to an accumulated prevalence of 24.4%.     

Leishmania (Viannia) naiffi sp. n., a parasite of the armadillo, Dasypus novemcinctus (L.) in Amazonian Brazil

A new leishmanial parasite, Leishmania (Viannia) naiffi sp. n., is described from the nine-banded armadillo, Dasypus novemcinctus (Edentata: Dasypodidae), from Para State, north Brazil. The parasite grows luxuriantly in Diffco blood-agar medium (B47), but poorly in the skin of intradermally inoculated hamsters. A comparison of isoenzyme profiles by starch gel electrophoresis separates the parasite from L. (V) braziliensis and L. (V.) guyanensis by the enzymes ASAT, ALAT, PGM, GPI, G6PD, PEP, MPI and GD, and from Leishmania (Leishmania) chagasi, L. (L.) amazonensis and L. (L.) deanei by ASAT, ALAT, PGM, GPI, MPI, G6PD, MDH, PEP and ACON. Finally, L. (V.) naiffi is serologically differentiated from L. (V.) braziliensis, L. (V.) guyanensis and L. (V.) panamensis on monoclonal antibodies specific for these parasites.     

Furazolidone is a selective in vitro candidate against Leishmania (L.) chagasi: an ultrastructural study

The current treatment for leishmaniasis is unsatisfactory due to toxic side effects, high cost, and problems with drug resistance. Various approaches have been used to identify novel drug candidates to treat Leishmania sp. parasites including the use of re-purposed drugs. Furazolidone is a nitrofuran derivative with antiprotozoal and antibacterial activity and is used for the treatment of giardiasis. In the present work, we determined the in vitro antileishmanial activity of furazolidone and its ability to induce ultrastructural alterations of parasites. Promastigotes of Leishmania (L.) chagasi, Leishmania (V.) braziliensis, Leishmania (L.) major, and Leishmania (L.) amazonensis were highly susceptible to furazolidone, with IC₅₀ values ranging between 0.47 and 0.73 μg/mL. Furazolidone was also very effective against L. chagasi intracellular amastigotes, and despite mammalian cytotoxicity, the selectivity index was 8.0 in human monocytes. The drug also had limited toxicity in mice erythrocytes. Furazolidone demonstrated specific activity against Leishmania, a potential consequence of the lack of macrophage nitric oxide activation. As determined by electron transmission microscopy, drug treatment induced severe damage to the parasite mitochondria and nucleus. This older oral drug is an effective agent for the treatment of L. (L.) chagasi in vitro and is a novel candidate for further experimental studies.     

Immunopathological characterization of human cutaneous leishmaniasis lesions caused by Leishmania (Viannia) spp. in Amazonian Brazil

Parasitology Research - American cutaneous leishmaniasis (ACL) is a chronic infectious disease caused by different protozoan species of Leishmania, and it is endemic in both tropical and...     

Epidemiology of Leishmania (L.) infantum, L. major and L. killicki in Tunisia: results and analysis of the identification of 226 human and canine isolates

The epidemiological situation of leishmaniasis in Tunisia is characterised by the co-existence in a very limited territory (165,000 km2, Sahara included), of 4 clinical forms: the infantile visceral leishmaniasis (VL) and 3 cutaneous leishmaniasis (CL) forms (sporadic, zoonotic and chronic). In addition to the useful epidemiological data, identification of the causative parasitic species is essential to determine the geographic distribution of each form and to select appropriate therapeutic procedure and suitable control measures. 226 Leishmania isolates, 135 human's coming from 59 VL cases and 76 CL cases and 91 canine's were identified by the isoenzyme electrophoresis reference technique. Results confirm the endemicity of the 4 forms mentioned above. The sporadic CL, confined to the North of the country is principally caused by L. infantum MON-24 (72.2%). VL which has reached the southern ridge in the central area of Tunisia, in the governorate of Kairouan (36 typed isolates), presents an unusual high proportion of L. infantum MON-24. In fact, this zymodeme, rather dermotropic is responsible for 47.2% of the cases vs 13% in the other regions of the country where L. infantum MON-1 remains predominant with 78.3% of typed isolates, the difference being statistically significant (P < 0.01). A third zymodeme, L. infantum MON-80 is sporadically pointed out during VL or in sporadic CL. Despite the high number of canine isolates (n=91) coming from 6 governorates, only the zymodeme L. infantum MON-1 was identified, letting hypothetic the reservoir of the 2 other zymodemes of the species identified in humans. Those absences may be related to cross infections, with a low sensitivity to L. infantum MON-24 leading to a selection of MON-1 at the time of culture passages. Hence it is important to develop molecular tools of direct identification on initial biological samples without going through cultures. Zoonotic CL remains the predominant cutaneous form in the central and southern area of Tunisia. However L. killicki, agent of the chronic CL, is confirming its presence out of its original focus of Tataouine in the southern-east of the country in both zoonotic CL and VL areas.     

Real-time PCR as a new tool for quantifying Leishmania infantum in liver in infected mice.

The parasitic loads of mouse livers experimentally infected with Leishmania infantum were determined using a double real-time quantitative PCR test targeted to the parasite DNA polymerase gene and to the mouse brain-derived neutrophic factor gene. The Leishmania DNA copy number was normalized to the number of mouse gene copies in order to quantify the former independently of liver weight. The correlation coefficient with the microtitration method was 0.66. This PCR assay can be considered for experimental pharmaceutical studies.     

Leishmania (Viannia) shawi sp. n., a parasite of monkeys, sloths and procyonids in Amazonian Brazil

Leishmania (Viannia) shawi sp. n., is described from the monkeys Cebus apella and Chiropotes satanus, the sloths Choloepus didactylus and Bradypus tridactylus, the procyonid Nasua nasua, and the phlebotomine sandfly Lutzomyia whitmani, all from primary forest in the State of Pará, north Brazil. L. (V.) shawi is variably distinguished from all other known species within the subgenus Viannia by a combination of biological, biochemical and serological characters, as revealed by studies on morphology, isoenzyme profiles, kDNA buoyant densities and monoclonal antibodies.     

Identification and frequency distribution of Leishmania (L.) major infections in sand flies from a new endemic ZCL focus in southeast Iran

Zoonotic cutaneous leishmaniasis (ZCL) is an important public health challenge in Iran. It is often caused by the protozoan parasite, Leishmania major. This pathogen is principally transmitted by the infectious bites of adult female phlebotomine sand flies which belong to the subgenus Phlebotomus (Phlebotomus) Rondani and Berte (Diptera: Psychodidae). A recent outbreak of clinical ZCL cases in the rural district of Jask, Hormozgan province, southern Iran prompted the identification of sand flies naturally infected with L. major using polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). A total of 8,123 wild sand flies were caught using sticky paper traps indoors and outdoors in seven villages of Jask from March 2007 to February 2008. About 120 trap-nights per month were carried out during peak seasonal density. Eight sand fly species of two distinct genera were morphologically identified. The most abundant species, Phlebotomus papatasi (≈60?%) and Phlebotomus salehi (17?%), had simultaneous population peaks in May and October. The anthropophilic index of P. papatasi was approximately twice that of P. salehi. PCR reactivity of L. major infections in P. salehi was weaker than those in P. papatasi. This is discussed with regard to their role in the natural transmission cycle of ZCL. This study provided the first PCR and ELISA evidence on P. salehi as a vector of L. major parasites in a new endemic region of ZCL in southeast Iran.     

PNA on human sperm: a new approach for in situ aneuploidy estimation

Peptide nucleic acids (PNAs) are a relatively new class of synthetic DNA mimics based on a peptide-like backbone. Since their introduction, PNA probes have become established as an efficient variation on the standard FISH procedure for chromosomal identification. In this report we have experimented with centromeric PNA probes on human sperm preparations. Both NaOH and DTT sperm decondensation procedures have been tested and comparative estimates of disomies X, Y and 1 have been performed in sperm from two donors using PNA, FISH and PRINS techniques. Similar results were obtained with the three methods, demonstrating the efficiency of PNA probes in the analysis of human sperm. The fast kinetics, stability and high specificity of PNA probes make PNA-based methodologies very valuable for in situ cytogenetic investigations.     

Activity of the julocrotine, a glutarimide alkaloid from Croton pullei var. glabrior, on Leishmania (L.) amazonensis

The antiproliferative effect of julocrotine, an alkaloid isolated from Croton pullei var. glabrior (Euphorbiaceae), was studied in the macrophage amastigote and promastigote stages of the protozoan Leishmania (L.) amazonensis, which causes cutaneous leishmaniasis in the New World. Julocrotine showed a dose-dependent effect against the amastigote and promastigote forms, where 79 μM julocrotine inhibited promastigote growth by 54%, with an IC50 of 67 μM. To analyze the antiamastigote activity of the drug, murine peritoneal macrophages infected with L. amazonensis promastigotes were treated with different concentrations of julocrotine. An 80% inhibition of amastigote development was observed using 79 μM julocrotine for 72 h, with an IC50 of 19.8 μM. In addition, ultrastructural observation of the parasites showed a significant reduction in the number of amastigotes in the parasitophorous vacuoles and morphological changes in promastigotes, such as swelling of the mitochondrion, chromatin condensation, presence of membranous structures near the Golgi complex, and some vesicle bodies in the flagellar pocket. A colorimetric assay (MTT), which measures cytotoxic metabolic activity, showed that macrophages maintain their viability after treatment with the drug. These results suggest that julocrotine effectively inhibits the growth of parasites and does not have any cytototoxic effects on the host cell.     

Helminth parasites of the eurasian badger (Meles meles L.) in Spain: a biogeographic approach

Eighty-five Eurasian badgers, Meles meles (Linnaeus, 1758), from four mainland biogeographic Spanish areas were analysed for helminths. Seventeen helminth species were found: Brachylaima sp., Euparyphium melis and Euryhelmis squamula (Trematoda), Atriotaenia incisa and Mesocestoides sp. (Cestoda) and Aelurostrongylus pridhami, Angiostrongylus vasorum, Aonchotheca putorii, Crenosoma melesi, Mastophorus muris, Molineus patens, Pearsonema plica, Physaloptera sibirica, Strongyloides sp., Trichinella sp., Uncinaria criniformis and Vigisospirura potekhina hugoti (Nematoda). In the Mediterranean area, Aonchotheca putorii, M. patens, Strongyloides sp., and U. criniformis were more prevalent in the occidental part, whereas Atriotaenia incisa and Mesocestoides sp. cestodes showed higher values on the continental slope. Metastrongyloid species (Aelurostrongylus pridhami, Angiostrongylus vasorum and Crenosoma melesi) were only detected in the occidental Mediterranean area. In contrast, spirurid species (Mastophorus muris and Vigisospirura potekhina hugoti) were almost restricted to the continental Mediterranean area. Helminthological differences between areas may result from the badger diet, abiotic factors and biocenosis present in each biogeographic area. Received: 4 July 2000 / Accepted: 14 August 2000     

Effect of human tumor antigens on the active E-rosette-forming test (ARFT): a new diagnostic method for cancer

The "active" T-cell test was adapted into an in vitro assay for response to human tumor antigens, especially gastric, lung, liver, esophageal, and colonic tumors. Peripheral blood lymphocytes were isolated from 229 cancer patients, 60 non-cancer patients, and 70 normal adults. Tumor antigen, incubated with lymphocytes from patients with the corresponding cancer, produced a significant increase in the ability of the lymphocytes to function as active rosette-forming cells (ARFC) when compared with lymphocytes cultured without antigen (P less than 0.001). This potentiating effect on ARFC remained unimpaired even after tumor antigen refrigeration. Tumor antigen incubated with lymphocytes from normal donors produced no increase in ARFC ability. This assay appears to be specific for the antigen corresponding to the cancer of the given patient; for example, gastric tumor antigen increased the ARFC of gastric cancer patients, but produced no increase in ARFC of breast cancer patients. No interference by the HLA complex was found in this assay. This technique provides a novel in vitro method for investigating cell-mediated immunity and also a new, specific method for cancer diagnosis.     

Distinct innate and acquired immune responses to Leishmania in putative susceptible and resistant human populations endemically exposed to L. (Viannia) panamensis infection

Mechanisms of constitutive and acquired susceptibility/resistance to Leishmania Viannia panamensis (L. (V ) p.) were investigated in endemically exposed human populations presenting either recurrent disease (putative susceptible) or subclinical infection (clinically resistant). Cutaneous delayed type hypersensitivity response to leishmanin was significantly lower among individuals experiencing recurrent leishmaniasis than among those whose skin test converted without developing the disease. Monocyte derived macrophages from individuals with recurrent disease were more permissive in vitro to the entry of parasites than macrophages from subclinically infected individuals. In vitro proliferation of CD4 and CD8 T lymphocytes in response to intracellular amastigotes was significantly lower among individuals with a history of recurrent disease compared with subclinically infected individuals. Linear regression analyses revealed a strong direct relationship between the production of interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-10 by peripheral blood mononuclear cells (PBMC) from resistant (subclinically infected) individuals and no correlation in the production of these cytokines by PBMC from individuals who experienced recurrent disease. The results provide evidence of differences in the innate and acquired responses to Leishmania according to the outcome of the natural infection. These findings support the feasibility of identifying the immunological bases of innate and acquired resistance through studies in naturally exposed human populations.     

Further morphological (freeze-fracture) evidence for an impulse generating function of Mauthner axon collaterals in the tench (Tinca tinca L.) spinal cord

The freeze-etching technique was applied to the tench spinal cord. Replicas of Mauthner axons revealed that the non-junctional axolemma at the tip of axon collaterals alone exhibits intramembranous E-face particles whose size, distribution and overall density are comparable with those at Ranvier nodes. Since typical Ranvier nodes are lacking along the stem of the heavily myelinated Mauthner giant axon, this finding contributes further evidence to our earlier observations suggesting that the spiny Mauthner axon collaterals represent true nodal equivalents and most likely the long-sought substrate of impulse propagation.     

Breakpoint characterization: a new approach for segregation analysis of paracentric inversion in human sperm

Paracentric inversions (PAI) are structural chromosomal rearrangements generally considered to be harmless. Nevertheless, cases of viable recombinants have been reported, indicating the interest of studying the meiotic behaviour of these chromosomal abnormalities. To date, the few studies reported have been performed using either the human-hamster fertilization system or fluorescence in situ hybridization with centromeric or telomeric DNA probes. In order to improve the assessment of meiotic segregation in PAI, we present a new strategy based on the use of bacterial artificial chromosome (BAC) probes which allow a precise localization of chromosome breakpoints and the identification of all meiotic products in human sperm. Sperm samples from carriers of an inv(5) and an inv(14) were used to test this new high-resolution procedure.