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Genus and species of phlebotomine sand flies have been recorded and described in caves in Brazil, but no study has provided the food source used by sand flies in these environments. Herein, we identified the blood source used by sand fly species in caves located at “Quadrilatero Ferrifero” (QF), Minas Gerais state. Specimens were manually collected near or on anurans inside ferruginous caves in Serra do Gandarela National Park and Serra do Rola Moca State Park. Males and females were placed in vials with 70% alcohol and stored at −10°C. Females engorged, after specific identification, had DNA extracted and followed for PCR amplification using specific primers. Sequencing was analyzed in the GenBank and Barcode of Life. A total of 198 specimens were collected (107 females and 91 males), all of them belonging to species Sciopemyia aff. microps (88.89%), Sciopemyia sordellii (10.61%), or Martinsmyia oliveirai (0.50%). When it comes to the females, 89 were S. aff. microps and 18 S. sordellii. Nineteen engorged females of S. aff. microps were analyzed and most of them (n=18) presented blood from Bokermannohyla martinsi and one contained blood from Scinax fuscovarius. The blood present in engorged females of S. sordellii (n=4) was from B. martinsi. Sciopemyia genus specimens are commonly found in collections carried out inside natural caves, but this was the first study to prove that females of this genus feed on cold-blooded animals in nature. • Here we proved that sand flies feed in cold-blooded animals in in Brazilian caves. • Females of the Sciopemyia genus were for the first time found feeding in natural habitats. • Anurans of the family Hylidae were identified as source by molecular analyzes. • Insect bloodmeal identification can help assessing the fauna in several biomes. • This is the first record of S. aff. microps in caves of Brazil.

The understanding on the role of bats in the ecology of zoonotic diseases, especially its relevance as a carrier of pathogens, is important for the determination of preventive measures considering the One Health context. The present study aimed to investigate the presence of Brucella spp., Leptospira spp. and Salmonella spp. in blood (n = 163), liver (n = 35) and spleen (n = 62) samples from bats captured in Montes Claros, Minas Gerais, Brazil. Only Salmonella spp. was found in a blood sample of an insectivorous female bat of the species Lasiurus blossevilli, evidencing the capacity of this animal species to host this pathogen. In conclusion, our results in bats from Montes Claros indicate that they do not act as hosts for Brucella spp. and Leptospira spp., although being potential carriers of Salmonella spp. in a low prevalence.

In Brazil, the visceral leishmaniasis (VL) is caused by Leishmania infantum, while the tegumentary leishmaniasis (TL) etiological agents are mainly Leishmania braziliensis and Leishmania amazonensis. The canine visceral leishmaniasis (CVL) diagnosis is an important step of the VL control program in Brazil, which involves the elimination of infected dogs, the main urban VL reservoirs. The current serology-based diagnostic tests have shown cross-reactivity between these three species, whereas molecular diagnosis allows high sensitivity and specie identification. In the present study, 349 dogs of the metropolitan region of Belo Horizonte (Minas Gerais state) were screened by conjunctival swab and the samples analyzed by ITS-1 nested PCR. Thirty dogs (8.5%) tested positive. The RFLP of amplicons using HaeIII demonstrated that 17/30 samples presented a banding pattern compatible with L. infantum, 4/30 matched with L. amazonenis, 1/30 with L. braziliensis and 8/30 showed a mixed infection pattern. The samples that were distinct of L. infantum or presented a mixed pattern were submitted to RFPL with HaeIII and RsaI enzymes that confirmed the mixed pattern. Such patterns were also confirmed by Sanger Sequencing. The results pointed eight dogs with mixed infections and the establishment of TL causing species in the Belo Horizonte dog population. These findings highlight the need for more comprehensive epidemiological studies, since the TL transmission profile might be changing. This study also shows the potential of the ITS1-nPCR associated with RFLP for the proper Leishmania diagnosis and typing in the dog population.

Leishmania (Leishmania) major is an important agent of cutaneous leishmaniasis, having as a vector sandflies belonging to the genus Phlebotomus. Although this species has been described as restricted to the Old World, parasites similar to L. major have been isolated from South American patients who have never travelled abroad. These parasites were named “L. major-like”, and several studies have been carried out to characterise them biochemically, molecularly, and biologically. However, the phylogenetic origin of these isolates is still unknown. In the present study we characterised three L. major-like isolates, named BH49, BH121 and BH129, using comparative genomics approaches. We evaluated the presence of gene and segmental duplications/deletions and the presence of aneuploidies that could explain the differences in infectivity observed in the BH49 and BH121 isolates. All isolates presented a pattern of mosaic aneuploidy and gene copy number variation, which are common in the genus Leishmania. Virulence factors such as phosphatases and peptidases were found to have increased gene copy numbers in the infective isolate, which could explain the difference in infectivity previously observed between BH121 and BH49. Phylogenetic analyses revealed that BH49, BH121 and BH129 L. major-like grouped with L. major isolates, and suggest they were imported from the Old World in at least two independent events. We suggest that new epidemiological inquiries should also evaluate L. major infections in South America, to assess the epidemiological importance of this species in the New World.

Visceral leishmaniasis (VL) is a systemic parasitic disease that leads to high rates of morbidity and mortality in humans worldwide. There is a great need to develop new drugs and novel strategies to make chemotherapy for this disease more efficacious and well tolerated. Recent reports on the immunomodulatory effects and the low toxicity of the spherical carbon nanostructure fullerol led us to investigate in vitro and in vivo antileishmanial activity in free and encapsulated forms in liposomes. When assayed against intramacrophagic Leishmania amastigotes, fullerol showed a dose-dependent reduction of the infection index with IC50 of 0.042 mg/mL. When given daily by i.p. route for 20 days (0.05 mg/kg/d) in a murine model of acute VL, fullerol promoted significant reduction in the liver parasite load. To improve the delivery of fullerol to the infection sites, liposomal formulations were prepared by the dehydration-rehydration method. When evaluated in the acute VL model, liposomal fullerol (Lip-Ful) formulations given i.p. at 0.05 and 0.2 mg/kg with 4-days intervals were more effective than the free form, with significant parasite reductions in both liver and spleen. Lip-Ful at 0.2 mg/kg promoted complete parasite elimination in the liver. The antileishmanial activity of Lip-Ful was further confirmed in a chronic model of VL. Lip-Ful was also found to induce secretion of pro-inflammatory TNF-α, IFN-γ and IL-1β cytokines. In conclusion, this work reports for the first time the antileishmanial activity of fullerol and introduces an innovative approach for treatment of VL based on the association of this nanostructure with liposomes.

The treatment of dogs naturally infected with Leishmania infantum using meglumine antimoniate (MA) encapsulated in conventional liposomes (LC) in association with allopurinol has been previously reported to promote a marked reduction in the parasite burden in the main infection sites. Here, a new assay in naturally infected dogs was performed using a novel liposome formulation of MA consisting of a mixture of conventional and long-circulating (PEGylated) liposomes (LCP), with expected broader distribution among affected tissues of the mononuclear phagocyte system. Experimental groups of naturally infected dogs were as follows: LCP plus Allop, receiving LCP intravenously as 2 cycles of 6 doses (6.5 mg Sb/kg of body weight/dose) at 4-day intervals plus allopurinol at 30 mg/kg/12 h per os (p.o.) during 130 days (LCP+Allop); LC plus Allop, receiving LC intravenously as 2 cycles of 6 doses (6.5 mg Sb/kg/dose) plus allopurinol during 130 days (LC+Allop); Allop, treated with allopurinol only; and a nontreated control. Parasite loads were evaluated by quantitative PCR in liver, spleen, and bone marrow tissue and by immunohistochemistry in the ear skin, before treatment, just after treatment, and 4 months later. The LCP+Allop and LC+Allop groups, but not the Allop group, showed significant suppression of the parasites in the liver, spleen, and bone marrow 4 months after treatment compared to the pretreatment period or the control group. Only LCP+Allop group showed significantly lower parasite burden in the skin in comparison to the control group. On the basis of clinical staging and parasitological evaluations, the LCP formulation exhibited a more favorable therapeutic profile than the LC one, being therefore promising for the treatment of canine visceral leishmaniasis.

SUMMARYIn South America, visceral leishmaniasis is frequently caused byLeishmania infantumand, at an unknown frequency, byLeishmania amazonensis. Therefore, mixed infections with these organisms are possible. Mixed infections might affect the clinical course, immune response, diagnosis, treatment and epidemiology of the disease. Here we describe the clinical course of mixed infections withL. amazonensisandL. infantumin a hamster model. We show that mixed infections are associated with more severe clinical disease than infection withL. amazonensisorL. infantumalone. In spleens with mixed infections,L. infantumoutcompetedL. amazonensisin the tissue, but not in culture from tissue. We found increased levels of IgG in animals infected withL. infantum.Although more than 30 bands were revealed in a Western blot, the highest immunogenicity was observed with proteins having molecular masses of 95 and 90 kDa, whereas proteins with molecular masses of lower than 50 kDa were reactive frequently with serum from hamsters infected withL. amazonensis, and proteins with molecular masses of 80 and 70 kDa were reactive only with serum from hamsters infected withL. infantum. This finding has important implications regarding the biology ofLeishmaniaand humoral immune responses to infections with these organisms.

Universidade Federal de Minas Gerais. Instituto de Ci?ncias Biol?gicas. Departamento de Patologia Geral. Belo Horizonte, MG, Brazil. Funda??o Oswaldo Cruz. Centro de Pesquisas Ren? Rachou. Belo Horizonte, MG, Brazil. Universidade Federal de Minas Gerais. Instituto de Ci?ncias Biol?gicas. Departamento de Patologia Geral. Belo Horizonte, MG, Brazil. Universidade Federal de Minas Gerais. Instituto de Ci?ncias Biol?gicas. Departamento de Patologia Geral. Belo Horizonte, MG, Brazil. Universidade Federal de Minas Gerais. Instituto de Ci?ncias Biol?gicas. Departamento de Parasitologia. Belo Horizonte, MG, Brazil. Universidade Federal de Goi?s. Instituto de Patologia Tropical e Sa?de P?blica. Goi?nia, GO, Brazil. Universidade Federal de Goi?s. Instituto de Patologia Tropical e Sa?de P?blica. Goi?nia, GO, Brazil. Universidade Federal de Goi?s. Faculdade de Medicina. Instituto Goiano de Oncologia e Hematologia. Goi?nia, GO, Brazil. Universidade Federal de Goi?s. Faculdade de Medicina. Instituto Goiano de Oncologia e Hematologia. Goi?nia, GO, Brazil. Universidade Federal de Goi?s. Faculdade de Medicina. Instituto Goiano de Oncologia e Hematologia. Goi?nia, GO, Brazil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Universidade Federal de Uberl?ndia. Instituto de Ci?ncias Biom?dicas. Departamento de Imunologia, Microbiologia e Parasitologia. Minas Gerais, MG, Brazil. Funda??o Oswaldo Cruz. Centro de Pesquisas Ren? Rachou. Belo Horizonte, MG, Brazil Universidade Federal de Minas Gerais. Instituto de Ci?ncias Biol?gicas. Departamento de Patologia Geral. Belo Horizonte, MG, Brazil. American tegumentary leishmaniasis (ATL) is a neglected disease widely distributed in Latin America. In Brazil, it is caused by different Leishmania species belonging to the Subgenera Viannia and Leishmania. ATL diagnosis is routinely based on clinical, epidemiological, parasitological and immunological (delayed-type hypersensitivity skin test-DTH) evidences. The main objective of this work was to determine the efficacy of a previous immunohistochemical (IHC) method developed by our group. Seventy eight skin biopsies from patients with different ATL clinical forms and origins were evaluated. The method was previously standardized in ATL patients from the municipality of Caratinga, Minas Gerais, Brazil, all infected with Leishmania (V.) braziliensis. Here, it is evaluated in patients from the North, Southeast and Midwest regions of Brazil. Clinical, parasitological (biopsy PCR) and immunological (Montenegro skin test-MST) diagnosis were performed prior to IHC procedure. The IHC procedure detected 70.5% of the cases having a high agreement with MST diagnosis (kappa = 0.84). A distinguished contribution of this work is that IHC succeed in diagnosing some negative DTH patients. Those were infected with Leishmania (L.) amazonensis, commonly causing the anergic form of the disease. In conclusion, IHC succeed in detecting ATL caused by different Leishmania species from various geographic regions and clinical status. Although it was not able to detect ATL in all patients, it was better than MST providing an additional tool for the diagnosis of ATL patients. There was no significant correlation between clinical forms and histological features including the presence of necrosis.

The protozoan Trypanosoma cruzi has the ability to spontaneously secrete extracellular vesicles (EVs). In this paper, T. cruzi EVs derived from epimastigote forms were evaluated during interaction with triatomine bugs Rhodnius prolixus and Triatoma infestans. T. cruzi EVs were purified and artificially offered to the insects prior to infection with epimastigote forms. No effect of EVs was detected in the parasite counts in the guts of both vectors after 49-50 days. On the other hand, pre-feeding with EVs delayed parasite migration to rectum only in the gut in R. prolixus after 21-22 days. Those data suggest a possible role of T. cruzi EVs during the earlier events of infection in the invertebrate host.

American tegumentary leishmaniasis (ATL) samples obtained from the lesions of patients with typical (n = 25, 29%), atypical (n = 60, 69%) or both (n = 2%) clinical manifestations were analysed by multilocus enzyme electrophoresis, hsp70 restriction-fragment length polymorphism (PCR-RFLP), hsp70 sequencing and phylogenetics methods. The hsp70 PCR-RFLP analysis revealed two different profiles whose the most samples differed from those expected for Leishmania braziliensis and the other Leishmania species tested: of 39 samples evaluated, two (5%) had a restriction profile corresponding to L. braziliensis, and 37 (95%) had a restriction profile corresponding to a variant pattern. A 1300-bp hsp70 gene fragment was sequenced to aid in parasite identification and a phylogenetic analysis was performed including 26 consensus sequences from the ATL patient's samples and comparing to other Leishmania and trypanosomatids species. The dendrogram allowed to observe a potential population structure of L. braziliensis complex in the studied region, emphasizing that the majority of clinical samples presented a variant genetic profile. Of interest, the L. braziliensis diversity was associated with different clinical manifestations whose parasites with hsp70 variant profile were associated with atypical lesions. The results may be helpful to improve the diagnosis, treatment and control measures of the ATL in endemic areas.

Leishmania major is an Old World species causing cutaneous leishmaniasis and is transmitted by Phlebotomus papatasi and Phlebotomus duboscqi. In Brazil, two isolates from patients who never left the country were characterised as L. major-like (BH49 and BH121). Using molecular techniques, these isolates were indistinguishable from the L. major reference strain (FV1). OBJECTIVES; We evaluated the lipophosphoglycans (LPGs) of the strains and their behaviour in Old and New World sand fly vectors. METHODS: LPGs were purified, and repeat units were qualitatively evaluated by immunoblotting. Experimental in vivo infection with L. major-like strains was performed in Lutzomyia longipalpis (New World, permissive vector) and Ph. papatasi(Old World, restrictive or specific vector). FINDINGS: The LPGs of both strains were devoid of arabinosylated side chains, whereas the LPG of strain BH49 was more galactosylated than that of strain BH121. All strains with different levels of galactosylation in their LPGs were able to infect both vectors, exhibiting colonisation of the stomodeal valve and metacyclogenesis. The BH121 strain (less galactosylated) exhibited lower infection intensity compared to BH49 and FV1 in both vectors. MAIN CONCLUSIONS: Intraspecific variation in the LPG of L. major-like strains occur, and the different galactosylation levels affected interactions with the invertebrate host. Leishmania major é uma espécie do Velho Mundo causadora da leishmaniose cutânea e é transmitida por Phlebotomus papatasi e Phlebotomus duboscqi. No Brasil, dois isolados de pacientes que nunca saíram do país foram caracterizados como L. major-like (BH49 e BH121). Usando técnicas moleculares, esses isolados foram indistinguíveis da cepa de referência de L. major (FV1). OBJETIVOS; Nós avaliamos os lipofosfoglicanos (LPGs) das cepas e seu comportamento em vetores de flebotomíneos do Velho e do Novo Mundo. MÉTODOS: Os GLPs foram purificados e as unidades repetidas foram avaliadas qualitativamente por immunoblotting. A infecção experimental in vivo com cepas semelhantes a L. major foi realizada em Lutzomyia longipalpis (Novo Mundo, vetor permissivo) e Ph. papatasi (Velho Mundo, vetor restritivo ou específico). DADOS: Os GLPs de ambas as cepas eram desprovidos de cadeias laterais arabinosiladas, enquanto o GLP da cepa BH49 era mais galactosilado que o da cepa BH121. Todas as cepas com diferentes níveis de galactosilação em seus GLPs foram capazes de infectar ambos os vetores, apresentando colonização da válvula estomodal e metaciclogênese. A cepa BH121 (menos galactosilada) apresentou menor intensidade de infecção em relação a BH49 e FV1 em ambos os vetores. PRINCIPAIS CONCLUSÕES: Ocorre variação intraespecífica no GLP de cepas semelhantes a L. major, e os diferentes níveis de galactosilação afetaram as interações com o hospedeiro invertebrado.

© 2015 Elsevier Inc. The development of effective prophylactic strategies to prevent leishmaniasis has become a high priority. No less important than the choice of an antigen, the association of an appropriate adjuvant is necessary to achieve a successful vaccination, as the majority of the tested antigens contain limited immunogenic properties, and need to be supplemented with immune response adjuvants in order to boost their immunogenicity. However, few effective adjuvants that can be used against leishmaniasis exist on the market today; therefore, it is possible to speculate that the research aiming to identify new adjuvants could be considered relevant. Recently, Agaricus blazei extracts have proved to be useful in enhancing the immune response to DNA vaccines against some diseases. This was based on the Th1 adjuvant activity of the polysaccharide-rich fractions from this mushroom. In this context, the present study evaluated purified fractions derived from Agaricus blazei as Th1 adjuvants through in vitro assays of their immune stimulation of spleen cells derived from naive BALB/c mice. Two of the tested six fractions (namely F2 and F4) were characterized as polysaccharide-rich fractions, and were able to induce high levels of IFN-γ, and low levels of IL-4 and IL-10 in the spleen cells. The efficacy of adjuvant action against L. infantum was evaluated in BALB/c mice, with these fractions being administered together with a recombinant antigen, LiHyp1, which was previously evaluated as a vaccine candidate, associated with saponin, against visceral leishmaniasis (VL). The associations between LiHyp1/F2 and LiHyp1/F4 were able to induce an in vivo Th1 response, which was primed by high levels of IFN-γ, IL-12, and GM-CSF, by low levels of IL-4 and IL-10; as well as by a predominance of IgG2a antibodies in the vaccinated animals. After infection, the immune profile was maintained, and the vaccines proved to be effective against L. infantum. The immune stimulatory effects in the BALB/c mice proved to be similar when comparing the F2 and F4 fractions with a known Th1 adjuvant (saponin), though animals vaccinated with saponin did present a slight to moderate inflammatory edema on their hind footpads. In conclusion, the F2 and F4 fractions appear to induce a Th1-type immune response and, in this context, they could be evaluated in association with other protective antigens against Leishmania, as well as in other disease models., Instituto Nacional de Ciencia e Tecnologia em Nano-biofarmaceutica (INCT-Nanobiofar), FAPEMIG (CBB-APQ-00496-11 and CBB-APQ-00819-12), and CNPq (APQ-472090/2011-9, RHAE-456287/2012-4, APQ-482976/2012-8 and APQ-488237/2013-0).

Leishmaniasis is a neglected tropical disease caused by >20 species of the protozoan parasite Leishmania . Meglumine antimoniate (Glucantime) is the first-choice drug recommended by the World Health Organization for the treatment of all types of leishmaniasis. However, the mechanisms of action and toxicity of pentavalent antimonials, including genotoxic effects, remain unclear. Therefore, the mechanism by which meglumine antimoniate causes DNA damage was investigated for BALB/c mice infected by Leishmania ( Leishmania ) infantum and treated with meglumine antimoniate (20 mg/kg for 20 days). DNA damage was analyzed by a comet assay using mouse leukocytes. Furthermore, comet assays were followed by treatment with formamidopyrimidine-DNA glycosylase and endonuclease III, which remove oxidized DNA bases. In addition, the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) in the animals' sera were assessed. To investigate mutagenicity, we carried out a micronucleus test. Our data demonstrate that meglumine antimoniate, as well as L. infantum infection, induces DNA damage in mammalian cells by the oxidation of nitrogenous bases. Additionally, the antileishmanial increased the frequency of micronucleated cells, confirming its mutagenic potential. According to our data, both meglumine antimoniate treatment and L. infantum infection promote oxidative stress-derived DNA damage, which promotes overactivation of the SOD-CAT axis, whereas the SOD-GPx axis is inhibited as a probable consequence of glutathione (GSH) depletion. Finally, our data enable us to suggest that a meglumine antimoniate regimen, as recommended by the World Health Organization, would compromise GPx activity, leading to the saturation of antioxidant defense systems that use thiol groups, and might be harmful to patients under treatment.

The growing resistance of leishmaniasis to first-line drugs like antimonials in some regions limits the control of this parasitic disease. The precise mechanisms involved in Leishmania antimony resistance are still subject to debate. The reduction of intracellular SbIII accumulation is a common change observed in both laboratory-selected and field isolated resistant Leishmania strains, but the exact transport pathways involved in antimony resistance have not yet been elucidated. In order to functionally characterize the antimony transport routes responsible for resistance, we performed systematic transport studies of SbIII in wild-type and resistant strains of L. (Viannia) guyanensis and L. (V.) braziliensis. Those include influx and efflux assays and the influence of ABC transporters and metabolism inhibitors: prochlorperazine, probenecid, verapamil, BSO, and sodium azide. The mRNA levels of genes associated with antimony resistance (MRPA, GSH1, ODC, AQP1, ABCI4, and ARM58) were also investigated in addition to intracellular thiol levels. A strong reduction of Sb influx was observed in L. guyanensis resistant mutant (LgSbR), but not in L. braziliensis (LbSbR). Both mutants showed increased energy-dependent efflux of SbIII, when compared to their respective parental strains. In LgSbR, BSO and prochlorperazine inhibited antimony efflux and resistance was associated with increased MRPA and GSH1 mRNA levels, while in LbSbR antimony efflux was inhibited by probenicid and prochlorperazine in absence of resistance-associated gene modulation. Intracellular thiol levels were increased in both Sb-resistant mutants. An energy-dependent SbIII efflux pathway sensitive to prochlorperazine was clearly evidenced in both Sb-resistant mutants. In conclusion, the present study allowed the biophysical and pharmacological characterization of energy-dependent Sb efflux pathway apparently independent of MRPA, ABCI4, and ARM58 upregulation, in Leishmania (Vianna) mutant selected in vitro for resistance to SbIII. Prochlorperazine has also been identified as an effective chemosensitizer in both Sb resistant mutants, which acts through inhibition of the active efflux of Sb.

The aims of the study were to determine the blood feeding preferences of sandflies and to identify species of Leishmania that infected phlebotomines in Caxias, Maranhão, Brazil, an area that is highly endemic for leishmaniasis. Sandflies were captured in light traps located in the peridomiciliary environments of randomly selected houses in urban and rural settings between 1800 and 0600 hours on new moon days between March 2013 and February 2015. DNA extracts from 982 engorged female sandflies were submitted to fragment length polymorphism analysis to identify infecting species of Leishmania, and blood sources were identified for 778 of these specimens. Infection by Leishmania infantum was detected in Lutzomyia longipalpis, Lu. whitmani and Lu. termitophila; L. infantum/L. braziliensis in Lu. longipalpis, Lu. whitmani and Lu. trinidadensis; L. shawi in Lu. longipalpis; L. mexicana in Lu. longipalpis; L. braziliensis in Lu. longipalpis and Lu. whitmani; L. guyanensis in Lu. longipalpis and Lu. termitophila; L. amazonensis in Lu. longipalpis and L. lainsoni or L. naiffi in Lu. longipalpis, while Lu. longipalpis and Lu. trinidadensis were infected with unidentified Leishmania sp. Blood sources were identified in 573 individual phlebotomines and the preferred hosts were, in decreasing order, chicken, dog, rodent and human with lower preferences for pig, horse, opossum and cattle. Lu. longipalpis and Lu. whitmani performed mixed feeding on man, dog and rodent, while Lu. longipalpis was the most opportunistic species, feeding on the blood of all hosts surveyed, but preferably on dog/chicken, dog/rodent and rodent/chicken. Our findings reveal the concomitant circulation of Leishmania species that cause visceral leishmaniasis and tegumentary leishmaniasis in the study area, and explain the occurrence of autochthonous human cases of both clinical forms of leishmaniasis in Caxias, Maranhão. The results support our hypothesis that, in the municipality of Caxias, transmission of Leishmania occurs in close proximity to humans.

Feline leishmaniasis has been reported in various parts of the world in recent years, occurring mainly in areas where zoo notic visceral leishmaniasis (VL) is endemic. The purpose of this study was to assess the occurrence of natural infection by Leishmania spp. in domestic cats (Felis catus) in the municipality of Teresina, Piauí state, Brazil, an endemic area of VL in Brazil. The prevalence of infection by Leishmania spp. in the population under study was 4% (3/83) in the enzyme-linked immunosorbent assay (ELISA) and 4% (3/83) by smear observation and isolation in a culture medium, using popliteal lymphnode sample. Only one of the three infected cats was positive for ELISA, also being positive for feline immunodefi ciency virus. In the haematologic parameters, normocytic normochromic anaemia was the most common change, being present in the three infected animals. In the biochemical measurements also were observed alterations in infected animals. The Leishmania spp. strains isolated from the culture medium were characterized as L. infantum. The presence of L. infantum infection in cats in the city of Teresina, an area endemic for VL, suggests the possible involvement of these animals in the epidemiological chain of L. infantum in high-transmission areas. A leishmaniose felina tem sido relatada em várias partes do mundo nos últimos anos, ocorrendo principalmente em áreas onde a leishmaniose visceral zoonótica (LV) é endêmica. O objetivo deste estudo foi avaliar a ocorrência de infecção natural por Leishmania spp. em gatos domésticos (Felis catus) no município de Teresina, Piauí, Brasil, área endêmica de LV em Brasil. A prevalência da infecção por Leishmania spp. na população em estudo foi de 4% (3/83) na ensaio imunossorvente (ELISA) e 4% (3/83) por observação de esfregaço e isolamento em meio de cultura, usando poplíteo amostra de linfonodo. Apenas um dos três gatos infectados foi positivo para ELISA, sendo também positivo para o vírus da imunodeficiência felina. Nos parâmetros hematológicos, a anemia normocítica normocrômica foi a alteração mais comum, sendo presente nos três animais infectados. Nas medidas bioquímicas também foram observadas alterações em animais. A Leishmania spp. cepas isoladas do meio de cultura foram caracterizadas como L. infantum. A presença de infecção por L. infantum em gatos na cidade de Teresina, área endêmica para LV, sugere o possível envolvimento desses animais da cadeia epidemiológica de L. infantum em áreas de alta transmissão.

Naturally Leishmania infantum infected bitches were divided into oestrogenized (n = 11) and non-oestrogenized (n = 6) groups. Vaginal secretions were collected for polymerase chain reaction (PCR), and vulval, vaginal and uterine tissues were collected for the immunohistochemical (IHQ) identification of L. infantum. Parasite DNA was identified in vaginal secretions of non-oestrogenized (41.8%) and oestrogenized (18.2%) bitches (P

This study reports the first evidence of infection by Leishmania infantum in Equus caballus in Americas and the first mixed infection of L. infantum/Leishmania braziliensis on this mammalian species in the world. The diagnoses was based on presence of parasites in lesions and bone marrow aspirates, their identification by using specific primers for L. infantum and L. braziliensis complexes and also serological methods IFAT and ELISA. The analysis of the PCR products suggested mixed infection in three animals. Further studies involving equine leishmaniasis are carrying out in order to clarify the dynamic of Leishmania sp. in this mammalian specie and their role in the transmission of those parasites in urban endemic area of Belo Horizonte, Minas Gerais State, Brazil.

Continuing our screening program for novel anti-parasite compounds, we synthesized seven 1,4-naphthoquinones coupled to 1,2,3-triazoles, five nor-β-lapachone-based 1,2,3-triazoles and ten α-lapachone-based 1,2,3-triazoles. These and other naphthoquinonoid compounds were evaluated for their activity against promastigote forms of antimony-sensitive and -resistant strains of Leishmania infantum (syn. Leishmania chagasi) and Leishmania amazonensis. The toxicity of these compounds to mammalian cells was also examined. The substances were more potent than an antimonial drug, with IC50 values ranging from 1.0 to 50.7 μM. Nor-α-lapachone derivatives showed the highest antileishmanial activity, with selectivity indices in the range of 10–15. These compounds emerged as important leads for further investigation as antileishmanial agents. Additionally, one of these compounds exhibited cross-resistance in Sb-resistant Leishmania and could provide a molecular tool for investigating the multidrug resistance mechanisms in Leishmania parasites.

Species of the genus Spirometra are diphyllobothriid tapeworms with complex life cycles and are involved in human sparganosis, a neglected disease that affects individuals worldwide. Although some species were reported in wild felids and human cases of sparganosis were described in Brazil, the biology and taxonomy of these parasites are poorly understood. In the present study, samples of diphyllobothriids (eggs and/or proglottids) obtained from the stools of wild carnivores (Leopardus pardalis and Lycalopex vetulus) and plerocercoid larvae found in a snake (Crotalus durissus) from Brazil were analysed by amplifying a fragment of the gene cytochrome c oxidase subunit 1 (cox 1). The DNA sequences obtained here for the first time from the Spirometra spp. from Brazil were used to evaluate the phylogenetic relationships with other species. Molecular data identified two species in the Brazilian samples (evolutionary divergence of 17.8-19.2%). The species were identified as Spirometra sp. 1, found in Le. pardalis, and Spirometra sp. 2 found in Ly. vetulus and C. durissus, and they differed from Asian isolates of Spirometra erinaceieuropaei (17.5-20.2% and 12.2-15.6%, respectively), a species previously considered to be distributed worldwide. Moreover, Spirometra sp. 1 is genetically distinct from Sparganum proliferum from Venezuela (19.6-20.4%), while Spirometra sp. 2 is more closely related with the Venezuelan species (6.1-7.0%). Sequences of Spirometra sp. 2 revealed that it is conspecific with the Argentinean isolate of Spirometra found in Lycalopex gymnocercus (1.9-2.2%). Taxonomic and phylogenetic aspects related to New World species of Spirometra are briefly discussed.

The double stranded RNA (dsRNA) virus Leishmaniavirus (Totiviridae) was first described in Leishmania guyanensis and L. braziliensis (LRV1), and more recently from L. major and L. aethiopica (LRV2). Parasites bearing LRV1 elicit a higher pro-inflammatory profile, arising through activation of Toll like receptor 3 (TLR3) interacting with the viral dsRNA. LRV1 is most common in Leishmania from the Amazon region; however data for other regions of Brazil are more limited. Here we applied PCR tests with validated ‘universal’ LRV1 primers to search for LRV1 in 40 strains of cultured L. braziliensis from several locales within Minas Gerais State, including patients presenting with atypical lesion pathology. All strains were negative however. These data are in agreement with results from other areas of Southeastern Brazil that LRV1 is relatively uncommon O vírus de RNA de fita dupla (dsRNA) Leishmaniavirus (Totiviridae) foi descrito pela primeira vez em Leishmania guyanensis e L. braziliensis (LRV1), e mais recentemente de L. major e L. aethiopica (LRV2). Parasitas portadores de LRV1 provocam um perfil pró-inflamatório mais elevado, surgindo através da ativação do Toll like receptor 3 (TLR3) interagindo com o dsRNA viral. LRV1 é mais comum em Leishmania da região amazônica; porém os dados para outras regiões do Brasil são mais limitado. Aqui aplicamos testes de PCR com primers LRV1 ‘universais’ validados para procurar LRV1 em 40 cepas de L. braziliensis cultivadas em diversas localidades do Estado de Minas Gerais, incluindo pacientes que apresentam patologia de lesão atípica. No entanto, todas as estirpes foram negativas. Esses dados estão de acordo com resultados de outras áreas do Sudeste do Brasil que LRV1 é relativamente incomum

Identification of novel antigens is essential for developing new diagnostic tests and vaccines. We used DIGE to compare protein expression in amastigote and promastigote forms of Leishmania chagasi. Nine hundred amastigote and promastigote spots were visualized. Five amastigote-specific, 25 promastigote-specific, and 10 proteins shared by the two parasite stages were identified. Furthermore, 41 proteins were identified in the Western blot employing 2-DE and sera from infected dogs. From these proteins, 3 and 38 were reactive with IgM and total IgG, respectively. The proteins recognized by total IgG presented different patterns in terms of their recognition by IgG1 and/or IgG2 isotypes. All the proteins selected by Western blot were mapped for B-cell epitopes. One hundred and eighty peptides were submitted to SPOT synthesis and immunoassay. A total of 25 peptides were shown of interest for serodiagnosis to visceral leishmaniasis. In addition, all proteins identified in this study were mapped for T cell epitopes by using the NetCTL software, and candidates for vaccine development were selected. Therefore, a large-scale screening of L. chagasi proteome was performed to identify new B and T cell epitopes with potential use for developing diagnostic tests and vaccines.

The potential of Gafanhoto Park as an American cutaneous leishmaniasis (ACL) focus was evaluated by examination of sand fly vectors of the Leishmania parasite. This forest remnant is located in a periurban area of Divinopolis, Brazil, where autochthonous cases of ACL have been reported. Sand fly populations were monitored over a 2-yr period (2006–2008) by using light traps (HP and Shannon). During systematic collections with HP traps, 824 specimens in total (342 males and 482 females) of 21 species were captured. Most prevalent species were as follows: Brumptomyia brumpti (Larrouse), Lutzomyia aragaoi (Costa Lima), Lutzomyia lutziana (Costa Lima), Lutzomyia sordellii (Shannon & Del Ponte), and Lutzomyia whitmani (Antunes & Coutinho). Using Shannon traps, 257 specimens representing 15 species were collected (159 females and 98 males), with a high prevalence of L. whitmani and Lutzomyia neivai (Pinto), both vectors of Leishmania braziliensis (Vianna). To ascertain the level of natural infection, a sample of females captured in Shannon traps was assayed for the presence of Leishmania by using polymerase chain reaction-restriction fragment length polymorphism, where 39% of insects were positive. The most infected species was L. whitmani (29 sand flies; 18.2%), followed by L. neivai (21; 13.2%), Lutzomyia christenseni (Young & Duncan) (five; 3.1%), Lutzomyia pessoai (Coutinho & Barreto) (three; 1.9%), L. aragaoi (one; 0.6%), Lutzomyia fischeri (Pinto) (one; 0.6%), Lutzomyia lenti (Mangabeira) (one; 0.6%), L. lutziana (one; 0.6%), and Lutzomyia monticula (Costa Lima) (one; 0.6%). The finding of potential and incriminated vectors naturally infected with Leishmania reinforces the need of epidemiologic surveillance in the area.

The identification and characterization of Leishmania are relevant to diagnosis, treatment, eco-epidemiology studies, prophylactic measures and control of the disease. Two strains of Leishmania (MHOM/BR/1971/BH49 and MHOM/BR/1971/BH121), isolated from human cutaneous leishmaniasis, were studied using biological and molecular characteristics, in comparison with WHO reference strains. These studies are important because both strains were incorporated in a vaccine against American cutaneous leishmaniasis, and one of these strains has been used to prepare specific and sensitive antigen for serodiagnosis of visceral leishmaniasis in Brazil. Studies were made on the growth rates of promastigotes in Grace's insect medium, infectivity to C57BL/6 and BALB/c mice, electrophoresic mobility patterns of isoenzymes, random amplification of polymorphic DNA (RAPD), simple sequence repeat-anchored PCR amplification (SSR-PCR) and DNA fingerprinting profiles, infectivity to murine macrophages and cellular immune response. Infections of mice and macrophages were significantly different among the strains studied. Attempts to infect mice with culture promastigotes were unsuccessful with BH121, but BH49 infected BALB/c and C57BL/6 mice. Isoenzyme electrophoretic mobility patterns, RAPD and SSR-PCR using DNA amplified by polymerase chain reaction (PCR) with nine arbitrary primers, as well as DNA fingerprinting studies with a biotin-labeled 33.15 fingerprinting probe showed similar profiles to those of the Leishmania major WHO reference strain.

American visceral leishmaniasis is a zoonosis of the New World. Dogs are the main reservoir of the disease and there is much interest in the understanding of mechanisms implicated in protection against canine infection. Nevertheless, most studies in dogs have not been carried out in organs that are targets of infection. This work is first to report the profile of cytokines and parasite burdens, as determined by real-time PCR, in the lymph nodes of dogs naturally infected with Leishmania chagasi. With this purpose, 18 mongrel dogs were divided in three groups: control non-infected dogs (n=6) and naturally infected animals with L. chagasi, asymptomatic (n=6) and symptomatic (n=6). Parasite burden in lymph nodes was 73-fold greater in symptomatic than asymptomatic animals. Prescapular lymph nodes of asymptomatic dogs had the highest expression of IFN-gamma and TNF-alpha and low parasite burden, indicating that these cytokines play a role in protection against infection. Highest expression of IL-10 and TGF-beta and high parasite burden were observed in symptomatic dogs, suggesting a role for these cytokines in the progression of disease. Hence, the balance of expression of IFN-gamma and TNF-alpha (protective) and IL-10 and TGF-beta (disease progression) in lymph nodes determine parasite burden and clinical expression in naturally infected dogs.

The toxicity and antileishmanial effectiveness of a novel liposome formulation of meglumine antimoniate in mongrel dogs with visceral leishmaniasis (VL) obtained from a region where VL is endemic in Brazil have been investigated. Groups of 12 animals received by the intravenous route four doses (with 4-day intervals) of either liposomal meglumine antimoniate (group I [GI], 6.5 mg Sb/kg of body weight/dose), empty liposomes (GII), or isotonic saline (GIII). Evaluation of markers of hematopoietic, hepatic, and renal functions before and just after treatment showed no significant change. On the other hand, transitory adverse reactions, including prostration, defecation, tachypnea, and sialorrhea, were observed during the first 15 min after injections in GI and GII. Parasitological evaluation of sternal bone marrow 4 days after the last dose showed a significant reduction of parasite burden in GI, compared to the other groups. Immunocytochemical evaluations of the skin, bone marrow, cervical lymph nodes, livers, and spleens of dogs for parasites, 150 days after treatment, indicated significant parasite suppression (higher than 95.7%) in the lymph nodes, livers, and spleens of GI, compared to control groups. Feeding of Lutzomyia longipalpis phlebotomines on dogs from GI, 150 days after treatment, resulted in a significant reduction of sand fly infection efficiency, compared to feeding on animals from GII and GIII. This is the first report of both long-term parasite suppression and reduction of infectivity to sand flies in naturally infected dogs following treatment with a liposome-encapsulated drug. Importantly, this was achieved using a 20-fold-lower cumulative dose of Sb than is used for conventional antimonial treatment.

One hundred and sixty-nine fragments of heart muscle collected from goats in the State of Ceara, Brazil, were analyzed by mouse bioassay with the aim of isolating Toxoplasma gondii . Two T. gondii isolates, named G1 and G2, were obtained and were characterized by PCR-RFLP. In addition, their virulence was evaluated by experimental inoculation into BALB/c mice. The G1 isolate presented high virulence leading to 100% mortality of mice after inoculations with 10 1 , 10 2 , and 10 3 tachyzoites. The G2 isolate presented low virulence and none of the doses tested lead to mortality of mice. The PCR-RFLP analysis showed that the two isolates are recombinants of the types I/III. However, they differ in the haplotype combination for the genotypes analyzed.

Background The Lençóis Maranhenses National Park, located in Maranhão, Brazil, is a region of exceptional beauty and a popular tourist destination. The adjoining area has suffered from the impact of human activity and, consequently, has experienced outbreaks of leishmaniasis. This study aimed to evaluate the composition, abundance, species richness and seasonal distribution of sand flies in the region and to determine the constancy of the insect population. Methods The survey was conducted at three sites located in the municipalities of Barreirinhas and Santo Amaro between September 2012 and August 2013. Sampling was performed monthly using automatic light traps installed 1.5 m above the soil adjacent to 13 randomly selected rural dwellings. At each site, one trap was placed in the peridomicile near to animal enclosures and another (extradomicile) at 500 m from the peridomicile. Results A total of 4,474 individual sand flies were collected over the year with the highest abundance recorded during the rainy season (December to June). Nine species were collected: L. whitmani, L. longipalpis, L. lenti, L. sordellii, L. evandroi, L. flaviscutellata, L. wellcomei, L. termitophila and L. intermedia. Although peridomiciliary and extradomiciliary environments presented similar species richness, the Shannon diversity index was significantly lower in the former (H’ = 2.4) compared with the latter (H’ = 4.98). Lutzomyia whitmani and L. longipalpis were the most abundant species and were classified as constant (constancy index, CI = 100 %) along with L. lenti (CI = 58.3), L. evandroi (CI = 58.3) and L. sordellii (CI = 66.7). The remaining four species presented CI values between 25 and 50 % and were considered accessory. Conclusions The present results confirm the present of L. whitmani and L. longipalpis in the peridomicile of houses in Lençóis National Park. The abundance of these species could explain, respectively, the endemicity of cutaneous leishmaniasis and sporadic cases of visceral leishmaniasis in the study area. However, in the case of cutaneous leishmaniasis, the presence of other sand fly vectors (in addition to L. whitmani) cannot be neglected. Finally, this study emphasizes the need for a more effective and permanent supervision to control the expansion of these vectors and leishmaniasis outbreaks.

The richness, relative abundance and the monthly frequency of species of sandflies (Diptera,Psychodidae) in an urban area of the municipality of Codo, Maranhao, a state that records a highprevalence of visceral leishmaniasis, was studied. The insects were captured with CDC light traps,from 18 h to 6 h, twice per month from June 2012 to May 2013 and 5,722 specimens of 11 species, 10belonging to the genus Lutzomyia and one to the genus Brumptomyia were collected. The abundancewas higher outdoors (4,009 samples: 66% males and 34% females) than in households (1,713 samples:55.9% males and 44.1% females). The most abundant species was L. longipalpis (97.7%), whichtogether with L. evandroi, L. sordellii and L. termitophila were more constant throughout the year. Thefrequency of sandflies was higher in the dry season (57.9%), rising with increasing mean temperature(p=0.0156, r=0.1007) and showed a decrease with increasing relative humidity (p=0.0001, r=-0.1740).Vector control during the dry season, when sandflies are most abundant, is suggested.

Leishmania major, the causative agent of zoonotic leishmaniasis, is restricted to Old World countries. Molecular and biochemical techniques have been used to identify some L. major-like isolated in South America including Brazil. Here, two L. major-like strains, one virulent (BH49) and one non-virulent (BH121), were subjected to suppression subtractive hybridization (SSH) technique in order to identify differentially expressed genes. SSH technique identified nine cDNA fragments exhibiting high homology to previously sequenced L. major genes. Five cDNAs (four specific for BH49 and one for BH121) were confirmed by RT-PCR. Among those differentially expressed subtracted genes, some were involved in physiological processes including metabolism, translation and destination of proteins, production of energy, virulence factors and unknown functions. Western-blot analysis confirmed a higher expression level of β-1,3-galactosyl residues in L. major-like lipophosphoglycan (LPG). This molecular analysis opens the possibility for identification of potential virulence factors not only in different strains, but also in others species of Leishmania.

Background Leishmania enriettii is a species non-infectious to man, whose reservoir is the guinea pig Cavia porcellus. Many aspects of the parasite-host interaction in this model are unknown, especially those involving parasite surface molecules. While lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs) of Leishmania species from the Old and New World have already been described, glycoconjugates of L. enriettii and their importance are still unknown. Methods Mice peritoneal macrophages from C57BL/6 and knock-out (TLR2 −/−, TLR4 −/−) were primed with IFN-γ and stimulated with purified LPG and GIPLs from both species. Nitric oxide and cytokine production were performed. MAPKs (p38 and JNK) and NF-kB activation were evaluated in J774.1 macrophages and CHO cells, respectively. Results LPGs were extracted, purified and analysed by western-blot, showing that LPG from L88 strain was longer than that of Cobaia strain. LPGs and GIPLs were depolymerised and their sugar content was determined. LPGs from both strains did not present side chains, having the common disaccharide Gal(β1,4)Man(α1)-PO4. The GIPL from L88 strain presented galactose in its structure, suggestive of type II GIPL. On the other hand, the GIPL of Cobaia strain presented an abundance of glucose, a characteristic not previously observed. Mice peritoneal macrophages from C57BL/6 and knock-outs (TLR2 -/- and TLR4 -/-) were primed with IFN-γ and stimulated with glycoconjugates and live parasites. No activation of NO or cytokines was observed with live parasites. On the other hand, LPGs and GIPLs were able to activate the production of NO, IL-6, IL-12 and TNF–α preferably via TRL2. However, in CHO cells, only GIPLs were able to activate TRL2 and TRL4. In vivo studies using male guinea pigs (Cavia porcellus) showed that only strain L88 was able to develop more severe ulcerated lesions especially in the presence of salivary gland extract (SGE). Conclusion The two L. enriettii strains exhibited polymorphisms in their LPGs and GIPLs and those features may be related to a more pro-inflammatory profile in the L88 strain.

The frequency of Leishmania (Viannia) braziliensis infection among patients of Mato Grosso, Brazil was estimated by polymerase chain reaction-PCR, DNA hybridization and by isoenzyme electrophoresis. Analysis of DNA polymorphism was carried out using random amplified polymorphic DNA-PCR (RAPDPCR) with five different primers. The patients were attended from May 1997 to February 1998 at the Reference Ambulatory for American Tegumentary Leishmaniasis at Júlio Müller University Hospital of the Federal University of Mato Grosso, Brazil. In a first screening by PCR and DNA hybridization 94.1% of 68 patients, from whom parasites were isolated in culture medium, were found to be infected with species of the Le. braziliensis complex. Only four patients (5.9%) were infected with species of Le. mexicana complex. Thirty-three samples of Le. braziliensis complex and three of Le. mexicana complex were typed by isoenzyme analysis as Le. (V.) braziliensis sensu stricto and Le. (Leishmania) amazonensis, respectively. The predominant species was Le. (V.) braziliensis, although most of the patients of this study came from the northern area of Mato Grosso, which is part of the Amazonian region of Brazil, where other known species of both subgenus Viannia (Le. braziliensis complex) and Leishmania (Le. mexicana complex) are present. The results of RAPD showed higher genetic variability among the Le. (V.) braziliensis samples from Mato Grosso. The importance of these results concerning the taxonomic status of New World Leishmania, and their implications for both clinical and epidemiological data is discussed.

In an attempt to isolate and characterize Toxoplasma gondii from the State of Minas Gerais, Brazil, musculature samples from 72 pigs, 25 dogs, 28 free-range chickens and 50 chickens produced in industrialized farms were collected. Antibodies to T. gondii have not been detected in pigs, but were found in nine (40.9 %) out of 22 dogs, and in 15 (53.6 %) of 28 free range chickens. T. gondii was not isolated from pigs and industrialized chickens, but from eight dogs and 11 free range chickens. In order to determine T. gondii virulence, female BALB/c mice were inoculated with 10(3), 10(2), 10(1) and 10(0) tachyzoites of the 19 isolates. The strains RH (virulent) and ME49 (non-virulent) were used as references. Isolates were divided into three groups according to the virulence phenotype: five isolates were classified into virulent in mice, one into non-virulent and 13 into intermediate virulent. Nested-PCR of T. gondii SAG2 locus amplified DNA from 21 out of 22 DNA samples directly extracted from heart of free range chickens. These samples were genotyped through a PCR-RFLP assay. Seventeen (80.9 %) were classified into type I; one (4.8 %) into type III and three (14.3 %) into type I or II.

Taxic responses may play a role in development of Leishmania in their phlebotomine sand fly vectors. They are possibly responsible for movement of the parasites towards the anterior regions of the gut, from where they would be transmitted to the vertebrate host. A methodology capable to distinguish chemotaxic from osmotaxic responses was described and used to characterise taxic responses in Leishmania promastigotes. These were able to respond to chemotaxic as well as to osmotaxic stimuli. Like bacteria, promastigotes were capable to undergo "adaptation," a phenomenon by which they stop responding to a continuos stimulus. A model capable to explain how a relatively small number of different receptors works to perceive gradients in chemotaxic responses was proposed. According to this model, these receptors possess low specificity and a wide range of affinities varying from high to low. A low specificity makes the same receptor able to bind to a large number of different but structurally related molecules and; a wide range of affinities (considering a population of receptors), implies that the number of receptors "occupied" by attractant molecules along a gradient would go growing step by step.

Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) at eight independent loci was used to determine the types (I, II or III) lineage of 20 Toxoplasma gondii strains isolated from humans and animals in Brazil. RH (type I, highly virulent), ME49 (type II, avirulent) and VEG (type III, avirulent) were used as reference strains. Differently from expected frequencies, all Brazilian strains showed to have recombinant genotypes, with typical alleles of types I, II or III at almost all loci assessed. The cB21-4 locus, a microsatellite marker, showed a higher allelic polymorphism with seven alleles among strains under analysis. Data have also shown that many Brazilian T. gondii strains presented a new haplotype at the L363 locus. When results of the eight loci were combined, 14 schizodemes were characterized out of the 20 T. gondii strains isolated in Brazil. The phenogram representing PCR–RFLP data separated Brazilian strains into two distinct genetic groups associated with murine virulence phenotype, termed groups I-A and I-B. Strains from group I-A (AS28, BV and N) that were highly virulent in BALB/c mice, were clustered with RH reference strain. Only those strains presented the haplotype I at the L363 locus, suggesting that this could be a possible marker of highly virulent strains. Strains from group I-B (cystogenic strains) showed a more heterogeneous behavior regarding virulence: a few of them (EGS, RAR, SAF, D5 and D6) were virulent, others (C4, P and D8) avirulent and most of them (D1, D2, D3, D4, D7, EFP, CH1, CH2 and CH3) intermediate virulent in mice. A significant linkage disequilibrium was observed in the population surveyed. However, the role of sexual recombination in the population structure of T. gondii in Brazil seems to be more central than in Europe and North America, where most studies have been performed.

DNA hybridisation, using probes labelled with 32P, was used to type Leishmania samples isolated from patients living in endemic areas of Mato Grosso State (Brazil), and clinically diagnosed as having tegumentary leishmaniasis. kDNA cloned mini-circle probes specific for the Leishmania mexicana and Leishmania braziliensis complexes were used. The results showed that L. braziliensis is the predominant group infecting human patients in the state. Sixty-eight samples were typed, 64 samples (94.1%) belonging to the L. braziliensis complex and only four (5.9%) belonging to the L. mexicana complex. Accurate identification of the Leishmania permits better orientation of the medical follow-up, since clinical manifestations may vary depending on the complex to which the parasite belongs. The epidemiological information furnished by the identification of the Leishmania in given endemic area is also essential for the design of appropriate control measuresHibridização, utilizando sondas de DNA marcadas com 32P, foi utilizada para a tipagem de amostras de Leishmania isoladas de pacientes do estado do Mato Grosso (Brasil), diagnosticados clinicamente como portadores de leishmaniose tegumentar. Sondas de minicírculos clonados de kDNA, específicas para os complexos Leishmania mexicana e Leishmania braziliensis, foram utilizadas. Os resultados demonstraram que o complexo L. brasiliensis é o grupo predominante infectando pacientes humanos no estado do Mato Grosso. Foram tipadas 68 amostras: 64 (94,1%) foram identificadas como pertencentes ao complexo L. brasiliensis e somente 4 (5,9%) como pertencentes ao complexo L. mexicana. A tipagem de Leishmania é importante para um melhor acompanhamento médico, uma vez que as manifestações clínicas podem variar em função do complexo ao qual o parasita pertence. A informação fornecida pela identificação também é essencial para a definição das medidas de controle mais adequadas e compreensão da epidemiologia da doença.

The aim of the present study was to evaluate the impact of a multiple dose regimen of a liposomal formulation of meglumine antimoniate (LMA) on the pharmacokinetics of antimony in the bone marrow of dogs with visceral leishmaniasis and on the ability of LMA to eliminate parasites from this tissue. Dogs naturally infected with Leishmania chagasi received 4 intravenous doses of either LMA (6.5 mg antimony/kg body weight, N = 9), or empty liposomes (at the same lipid dose as LMA, N = 9) at 4-day intervals. A third group of animals was untreated (N = 8). Before each administration and at different times after treatment, bone marrow was obtained and analyzed for antimony level (LMA group) by electrothermal atomic absorption spectrometry, and for the presence of Leishmania parasites (all groups). There was a significant increase of antimony concentration from 0.76 microg/kg wet organ (4 days after the first dose) to 2.07 microg/kg (4 days after the fourth dose) and a half-life of 4 days for antimony elimination from the bone marrow. Treatment with LMA significantly reduced the number of dogs positive for parasites (with at least one amastigote per 1000 host cells) compared to controls (positive dogs 30 days after treatment: 0 of 9 in the LMA group, 3 of 9 in the group treated with empty liposomes and 3 of 8 in the untreated group). However, complete elimination of parasites was not achieved. In conclusion, the present study showed that multiple dose treatment with LMA was effective in improving antimony levels in the bone marrow of dogs with visceral leishmaniasis and in reducing the number of positive animals, even though it was not sufficient to achieve complete elimination of parasites.

Canine visceral leishmaniasis (CVL) is a zoonosis and a chronic systemic disease of the dog caused by a protozoan by the species Leishmania infantum in the Old World and Leishmania chagasi in the New World. Several methods are currently employed for the diagnosis of CVL including microscopic detection of the parasite in bone marrow and lymph node aspirates, demonstration of specific antibodies anti-Leishmania in sera from infected animals, and isolation of the parasite by in vitro culture or by inoculation of laboratory animals. However, a definitive diagnosis is based on the actual detection of the parasite, which is conventionally achieved by examining Giemsa-stained smears or histopathological sections stained with hematoxylin and eosin. These methods have a low sensitivity, and therefore, they are often inconclusive. This is particularly true in canine organs that have a low level of parasitism such as kidneys, lungs, central nervous system, and testis, or, in some cases, the skin. The technique for immunohistochemical detection of leishmanial amastigotes in canine tissues has been reported previously and has proved to be undoubtedly efficient for the diagnosis. In this paper, we describe a straightforward and inexpensive immunohistochemical approach for Leishmania detection in formalin-fixed paraffin-embedded canine tissues. Amastigote forms of Leishmania were easily observed within macrophages in several organs from naturally infected dogs using the streptavidin–biotin immunohistochemical method with canine hyperimmune serum as the primary antibody. In addition, the secondary antibody used was not specific to canine immunoglobulin, characterizing a cross-immune reaction. Our results indicate that this technique could be a useful tool for epidemiological, clinical, and histopathological studies.

We detected an outbreak of American cutaneous leishmaniasis in the Jequitinhonha River Valley, Minas Gerais, Brazil. Clinical and epidemiological aspects were studied for a period of two years. Data include results of physical examinations, Montenegro skin test and serology. In total 72 of the 299 individuals evaluated presented active lesions. Only one case out of these 72 patients showed the mucosal form of the disease. The precarious sanitary conditions, low educational level and low income found in the population studied demonstrated that, as with the other parasitic diseases, cutaneous leishmaniasis occurs with greater frequency in needy populations. A canine serological survey detected 20.3% (30/148) of dogs reactive to the Leishmania antigen. Lutzomyia intermedia was the predominant phlebotomine species and the majority of the specimens (84.9%) were captured in the peridomicile. Four samples from human and three from canine cases were isolated and characterised by PCR and isoenzymes as being Leishmania (Viannia) braziliensis. The peridomiciliary nature of the disease is discussed.

© 2014 Costa et al. The development of cost-effective prophylactic strategies to prevent leishmaniasis has become a high-priority. The present study has used the phage display technology to identify new immunogens, which were evaluated as vaccines in the murine model of visceral leishmaniasis (VL). Epitope-based immunogens, represented by phage-fused peptides thatimic Leishmania infantum antigens, were selected according to their affinity to antibodies from asymptomatic and symptomatic VL dogs' sera. Methodology/Main Findings: Twenty phage clones were selected after three selection cycles, and were evaluated by means of in vitro assays of the immune stimulation of spleen cells derived from naive and chronically infected with L. infantum BALB/c mice. Clones that were able to induce specific Th1 immune response, represented by high levels of IFN-c and low levels of IL-4 were selected, and based on their selectivity and specificity, two clones, namely B10 and C01, were further employed in the vaccination protocols. BALB/c mice vaccinated with clones plus saponin showed both a high and specific production of IFN-c, IL-12, and GM-CSF after in vitro stimulation with individual clones or L. infantum extracts. Additionally, these animals, when compared to control groups (saline, saponin, wild-type phage plus saponin, or nonrelevant phage clone plus saponin), showed significant reductions in the parasite burden in the liver, spleen, bone marrow, and paws' draining lymph nodes. Protection was associated with an IL-12-dependent production of IFN-c, mainly by CD8+ T cells, against parasite proteins. These animals also presented decreased parasite-mediated IL-4 and IL-10 responses, and increased levels of parasite-specific IgG2a antibodies. Conclusions/Significance: This study describes two phage clones that mimic L. infantum antigens, which were directly used as immunogens in vaccines and presented Th1-type immune responses, and that significantly reduced the parasite burden. This is the first study that describes phage-displayed peptides as successful immunogens in vaccine formulations against VL., Instituto Nacional de Cieˆncia e Tecnologia em Nanobiofarmaceutica (INCT-Nanobiofar), FAPEMIG (PRONEX APQ-0101909, CBB-APQ-00496-11 and CBB-APQ-00819-12), CAPES (Rede Nanobiotec/Brasil) and CNPq (APQ-472090/2011-9 and APQ- 482976/2012-8)

Knowledge of Leishmania virulence is essential for understanding how the contact between the pathogen and host cells can lead to pathogenesis. Virulence in two L. infantum strains was characterized using macrophages and hamsters. Next, we used difference gel electrophoresis (DIGE) and mass spectrometry to identify the differentially expressed proteins. A total of 63 spots were identified corresponding to 36 proteins; 20 were up-regulated, in which 16 had been previously associated with Leishmania virulence. Considering our results and what has been reported before, we suggest the hypothesis that L. infatum virulence could be a result of the increased expression of KMP-11 and metallopeptidase, associated with an improved parasite-host interacting efficiency and degradation of the protective host proteins and peptides, respectively. Other factors are tryparedoxin peroxidase and peroxidoxin, which protect the parasite against the stress response, and 14-3-3 protein-like, which can prolong infected host cell lifetime. Proteins as chaperones and endoribonuclease L-PSP can increase parasite survival. Enolase is able to perform versatile functions in the cell, acting as a chaperone or in the transcription process, or as a plasminogen receptor or in cell migration events. As expected in more invasive cells with high replication rates, energy consumption and protein synthesis are higher, with up-regulation of Rieske iron-sulfur protein precursor, EF-2, S-adenosylhomocysteine, and phosphomannomutase.

Entamoeba histolyticais a parasite which presents capacity to degrade tissues and therefore has a pathogenic behavior. As this behavior is not shown by all strains, there have been several studies investigating molecular basis of the cytotoxicity process. Using the suppression subtractive hybridization (SSH) technique, differential gene expressions of twoE. histolyticastrains, one virulent (EGG) and one nonvirulent (452), have been analyzed with the purpose of isolating genes which may be involved with amoebic virulence. Nine cDNA fragments presenting high homology withE. histolyticapreviously sequenced genes were subtracted. Of these, four genes were confirmed by RT-PCR. Two coding for hypothetical proteins, one for a cysteine-rich protein, expressed only in the virulent strain, EGG and another one, coding for grainin 2 protein, exclusive from 452 strain. This study provided new insight into the proteins differences in the virulent and nonvirulentE. histolyticastrains. We believe that further studies with these proteins may prove association of them with tissue damage, providing new perceptions to improve treatment or diagnosis of the invasive disease.

Genetic relationships between Eimeria parasites of domestic fowl, including three isolates of E. acervulina, one of E. maxima, four of E. mitis, six of E. praecox, one of E. tenella, and two of uncertain identity, were analyzed by random amplified polymorphic DNA (RAPD) and the unweighted pair-group method with arithmetic mean, using 12 primers. Each primer amplified 4-34 DNA fragments/isolate. The similarity coefficients and phenograms were calculated from RAPD products with 230-2,000 bp in silver-stained polyacrylamide gels. Some primers generated RAPD markers which were species-specific for E. acervulina, E. mitis, and E. praecox. The phenograms revealed six clusters, each corresponding to an individual Eimeria species. The pBP and pBP2 isolates, of uncertain identity, clustered with the E. acervulina isolates (100% bootstrap). The intraspecific relationships showed certain degree of genetic isolation of the Eimeria populations, and it was associated with broiler house and with geographic separation.

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.

Screening for digestive glycosidases in different parts of the gut and associated organs of Lutzomyia longipalpis is reported. Searches for the enzymes were made in blood-fed and non-blood-fed females and the enzymes were characterized as soluble or membrane-bound molecules. A total of four different activities were detected, corresponding to the following specificities: an alpha-glucosidase, an N-acetyl-beta-d-glucosaminidase, an N-acetyl-beta-d-galactosaminidase, and an alpha-l-fucosidase. Their possible role and importance for Leishmania development are discussed and the alpha-glucosidase enzyme was partially characterized. The pH inside the gut of non-blood-fed phlebotomines was measured with pH indicator dyes. The pH ranges obtained for crop, midgut, and hindgut were, respectively, higher than pH 6, pH 6, and lower than pH 6. A hypothesis concerning these data and Leishmania development is proposed.

Differences in virulence of strains of Entamoeba histolytica have long been detected by various experimental assays, both in vivo and in vitro. Discrepancies in the strains characterization have been arisen when different biological assays are compared. In order to evaluate different parameters of virulence in the strains characterization, five strains of E. histolytica, kept under axenic culture, were characterized in respect to their, capability to induce hamster liver abscess, erythrophagocytosis rate and cytopathic effect upon VERO cells. It was found significant correlation between in vitro biological assays, but not between in vivo and in vitro assays. Good correlation was found between cytopathic effect and the mean number of uptaken erythrocytes, but not with percentage of phagocytic amoebae, showing that great variability can be observed in the same assay, according to the variable chosen. It was not possible to correlate isoenzyme and restriction fragment pattern with virulence indexes since all studied strains presented pathogenic patterns. The discordant results observed in different virulence assays suggests that virulence itself may not the directly assessed. What is in fact assessed are different biological characteristics or functions of the parasite more than virulence itself. These characteristics or functions may be related or not with pathogenic mechanisms occurring in the development of invasive amoebic diseaseParâmetros de virulência na caracterização de Cepas de Entamoeba histolytica. A caracterização quanto a virulência das diferentes cepas de E. histolytica foi avaliada através de ensaios in vivo e in vitro. Discrepâncias nesta caracterização têm surgido quando se compara os diferentes ensaios. Avaliamos alguns parâmetros de virulência na caracterização de 5 cepas axênicas de E. histolytica. Estas cepas foram caracterizadas com relação à sua capacidade para induzir abscesso em fígado de hamster, eritrofagocitose e efeito citopático sobre células VERO. Encontramos correlação significante entre os ensaios biológicos in vitro, mas não entre estes com os ensaios in vivo. Boa correlação foi achada entre o efeito citopático e o número médio de eritrócitos ingeridos, mas não com a percentagem de amebas que fagocitaram eritrócitos, mostrando que grande variabilidade pode ser observada no mesmo ensaio, dependendo da variável escolhida. Não foi possível correlacionar isoenzima e padrão de fragmento de restrição com o índice de virulência desde que todas as cepas estudadas apresentaram padrão patogênico. Os resultados discordantes observados nos diferentes ensaios de virulência sugerem que a mesma não deve ser avaliada diretamente. O que de fato está sendo avaliado são as diferentes características biológicas ou funções do parasita, mais do que a própria virulência. Estas características ou funções podem estar relacionadas ou não com mecanismos patogênicos que ocorrem no desenvolvimeto de amebíase invasiva

In the search for Leishmania recombinant antigens that can be used as a vaccine against American Cutaneous Leishmaniasis, we identified a Leishmania (Leishmania) amazonensis recombinant protein of 33 kD (Larp33) which is recognized by antibodies and peripheral blood leukocytes (PBL) from subjects vaccinated with Leishvacin ®, Larp33 was expressed in Escherichia coli after cloning of a 2,2 kb Sau3A digested genomic fragment of L. (L.) amazonensis into the pDS56-6 His vector. Immunoblotting analysis indicated that Larp33 corresponds to an approximately 40-kD native protein expressed in promastigotes of L.(L.) amazonensis and L. (Viannia) braziliensis. Northern blots of total RNA also demonstrated that the gene coding for this protein is expressed in promastigotes of the major lineages of Leishmania causing American Cutaneous Leishmaniasis. Larp33 induced partial protection in susceptible mouse strains (BALB/c and C57BL/10) against L. (L.) amazonensis after vaccination using Bacille Calmette-Guerin (BCG) as adjuvant. In vitro stimulation of splenocytes from BALB/c protected mice with Larp33 elicited the secretion of IL-2 and IFN-g, suggesting that a Th1 cell-mediated protective response is associated with the resistance observed in these mice. As revealed by its immunogenic and antigenic properties, this novel recombinant antigen is a suitable candidate to compose a vaccine against cutaneous leishmaniasisA resposta imune induzida por uma proteína recombinante de Leishmania (Leishmania) amazonensis de 33 kD (Larp33) foi avaliada em linfócitos de indivíduos vacinados com a Leishvacin® e em camundongos através de vacinação. Larp33 foi expressa em Escherichia coli após clonagem de um fragmento genômico de L. (L.) amazonensis de 2,2 kb no vetor pDS56-6His. Larp33 foi reconhecida por anticorpos IgG presentes no soro de indivíduos vacinados com Leishvacin® e induziu proliferação em linfócitos desses indivíduos em níveis comparáveis ao antígeno total de Leishmania. A análise por imunoblot indicou que Larp33 corresponde a uma proteína de aproximadamente 40 kD expressa em promastigotas de L. (L.) amazonensis e L. (Viannia) braziliensis. Hibridização com sonda de DNA correspondente a parte do fragmento clonado, também demonstrou que o gene codificador desta proteína é expresso em promastigotas destas duas espécies. Larp33 em associação com BCG (Bacille Calmette-Guerin) foi capaz de induzir 75% de proteção em camundongos C75BL/10 e BALB/c, suceptíveis à infecção por L. (L.) amazonensis. Linfócitos dos camundongos protegidos produziram IL-2 e IFN-g em resposta a Larp33. Nossos resultados indicam que Larp33 é imunogênica para linfócitos de indivíduos vacinados com Leishvacin ® e protetora para camundongos contra a infecção por L. (L.) amazonensis

Novel trivalent antimony complexes with the nitrogen donor heterocyclic ligand 2,2′-bipyridine (bipy), 1,10-phenanthroline (phen) or dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq) have been synthesized by the reaction with SbCl 3 or PhSbCl 2 . The crystal structures of [Sb(phen)Cl 3 ] and [PhSb(phen)Cl 2 ]CH 3 COOH were determined and shown to adopt a distorted square pyramid geometry with a five-coordinated Sb center. Surprisingly, all the complexes, the ligands and PhSbCl 2 showed very high antileishmanial activities, with IC 50 in the nanomolar range against Sb III -sensitive and -resistant Leishmania infantum (syn. Leishmania chagasi ) and Leishmania amazonensis strains. These compounds were much more active against these Leishmania strains than the old trivalent drug potassium antimonyl tartrate. [PhSb(phen)Cl 2 ]CH 3 COOH complex was found to be the most active compound and the lack of cross-resistance of PhSbCl 2 suggests that the transport pathways of this compound across the cell membrane differ from those responsible for the resistance of Leishmania to Sb(OH) 3 . In the case of the complexes with PhSbCl 2 , our data supports the model that both ligand and metal contributed to the overall activity of the complex. Furthermore, among the complexes with SbCl 3 , only bipy showed an improved activity upon complexation. Cytotoxicity evaluations of these compounds against murine peritoneal macrophages showed high selective indexes in the range of 7–70 for [Sb(phen)Cl 3 ], [Sb(bipy)Cl 3 ] and [Sb(dpq)Cl 3 ] complexes, being much more selective than potassium antimonyl tartrate. In conclusion, this study presents a set of new antileishmanial agents including one of the most active Sb-based compounds ever reported, which can contribute to the development of new chemotherapeutic strategies against leishmaniasis including Sb-resistant cases.

The need for daily parenteral administration is an important limitation in the clinical use of pentavalent antimonial drugs against leishmaniasis. In this study, amphiphilic antimony(V) complexes were prepared from alkylmethylglucamides (L8 and L10, with carbon chain lengths of 8 and 10, respectively), and their potential for the oral treatment of visceral leishmaniasis (VL) was evaluated. Complexes of Sb and ligand at 1:3 (SbL8 and SbL10) were obtained from the reaction of antimony(V) with L8 and L10, as evidenced by elemental and electrospray ionization-tandem mass spectrometry (ESI-MS) analyses. Fluorescence probing of hydrophobic environment and negative-staining transmission electron microscopy showed that SbL8 forms kinetically stabilized nanoassemblies in water. Pharmacokinetic studies with mice in which the compound was administered by the oral route at 200 mg of Sb/kg of body weight indicated that the SbL8 complex promoted greater and more sustained Sb levels in serum and liver than the levels obtained for the conventional antimonial drug meglumine antimoniate (Glucantime [Glu]). The efficacy of SbL8 and SbL10 administered by the oral route was evaluated in BALB/c mice infected with Leishmania infantum after a daily dose of 200 mg of Sb/kg for 20 days. Both complexes promoted significant reduction in the liver and spleen parasite burdens in relation to those in the saline-treated control group. The extent of parasite suppression (>99.96%) was similar to that achieved after Glu given intraperitoneally at 80 mg of Sb/kg/day. As expected, there was no significant reduction in the parasitic load in the group treated orally with Glu at 200 mg of Sb/(kg day). In conclusion, amphiphilic antimony(V) complexes emerge as an innovative and promising strategy for the oral treatment of VL.

Fluorescent and colorimetric reporter genes are valuable tools for drug screening models, since microscopy is labor intensive and subject to observer variation. In this work, we propose a fluorimetric method for drug screening using red fluorescent parasites. Fluorescent Leishmania amazonensis were developed after transfection with integration plasmids containing either red (RFP) or green fluorescent protein (GFP) genes. After transfection, wild-type (LaWT) and transfected (LaGFP and LaRFP) parasites were subjected to flow cytometry, macrophage infection, and tests of susceptibility to current antileishmanial agents and propranolol derivatives previously shown to be active against Trypanosoma cruzi. Flow cytometry analysis discriminated LaWT from LaRFP and LaGFP parasites, without affecting cell size or granulosity. With microscopy, transfection with antibiotic resistant genes was not shown to affect macrophage infectivity and susceptibility to amphotericin B and propranolol derivatives. Retention of fluorescence remained in the intracellular amastigotes in both LaGFP and LaRFP transfectants. However, detection of intracellular RFP parasites was only achieved in the fluorimeter. Murine BALB/c macrophages were infected with LaRFP parasites, exposed to standard (meglumine antimoniate, amphotericin B, Miltefosine, and allopurinol) and tested molecules. Although it was possible to determine IC(50) values for 4 propranolol derivatives (1, 2b, 3, and 4b), all compounds were considered inactive. This study is the first to develop a fluorimetric drug screening test for L. amazonensis RFP. The fluorimetric test was comparable to microscopy with the advantage of being faster and not requiring manual counting.