First occurrence of an autochthonous canine case of Leishmania (Leishmania) infantum chagasi in the municipality of Campinas, State of São Paulo, Brazil

An autochthonous case of visceral leishmaniasis is reported in a dog (Canis familiaris) as an apparently natural infection in a non-endemic area. DNA obtained from spleen and liver samples produced the expected fragment in a Leishmania-specific rDNA-based nested-PCR assay. The PCR product, a 490 bp fragment, was sequenced and the nucleotide sequence was identical to that of Leishmania (Leishmania) infantum chagasi. These results are surprising since no autochthonous human or canine cases of visceral leishmaniasis have ever been reported in this municipality. This case suggests that natural transmission of this disease is occurring in this area.

In vitro activity of the clinical pulmonary surfactant Surfacen® against Leishmania amazonensis

Surfacen® is an exogenous natural lung surfactant, composed by phospholipids and hydrophobic proteins, which is applied successfully in Newborn Respiratory Distress Syndrome. In this paper, in vitro activity of Surfacen® against Leishmania amazonensis is described. The product showed activity against the amastigote form found in peritoneal macrophages from BALB/c mice, with an IC50 value of 17.9 ± 3.0 µg/mL; while no toxic effect on host cell was observed up to 200 µg/mL. This is the first report about the antileishmanial activity of Surfacen®.

Safety and skin delayed-type hypersensitivity response in vervet monkeys immunized with Leishmania donovani sonicate antigen delivered with adjuvants

In this study, we report on the safety and skin delayed-type hypersensitivity (DTH), responses of the Leishmania donovani whole cell sonicate antigen delivered in conjunction with alum-BCG (AlBCG), Montanide ISA 720 (MISA) or Monophosphoryl lipid A (MPLA) in groups of vervet monkeys. Following three intradermal injections of the inoculums on days 0, 28 and 42, safety and DTH responses were assessed. Preliminary tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) levels were also measured and these were compared with DTH. Only those animals immunized with alum-BCG reacted adversely to the inoculum by producing ulcerative erythematous skin indurations. Non-parametric analysis of variance followed by a post-test showed significantly higher DTH responses in the MISA+Ag group compared with other immunized groups (p < 0.001). The MPLA+Ag group indicated significantly lower DTH responses to the sonicate antigen compared with the AlBCG+Ag group. There was a significant correlation between the DTH and cytokine responses (p < 0.0001). Based on this study we conclude that Leishmania donovani sonicate antigen containing MISA 720 is safe and is associated with a strong DTH reaction following immunization.

Susceptibility of peritoneal macrophage from different species of neotropical primates to Ex vivo Leishmania (L.) infantum chagasi-infection

This study examined the susceptibility of peritoneal macrophage (PM) from the Neotropical primates: Callithrix jacchus, Callithrix penicillata, Saimiri sciureus, Aotus azarae infulatus and Callimico goeldii to ex vivo Leishmania (L.) infantum chagasi-infection, the etiological agent of American visceral leishmaniasis (AVL), as a screening assay for evaluating the potential of these non-human primates as experimental models for studying AVL. The PM-susceptibility to infection was accessed by the PM-infection index (PMI) at 24, 72 h and by the mean of these rates (FPMI), as well as by the TNF-α, IL-12 (Capture ELISA) and Nitric oxide (NO) responses (Griess method). At 24h, the PMI of A. azarae infulatus (128) was higher than those of C. penicillata (83), C. goeldii (78), S. sciureus (77) and C. jacchus (55). At 72h, there was a significant PMI decrease in four monkeys: A. azarae infulatus (128/37), C. penicillata (83/38), S. sciureus (77/38) and C. jacchus (55/12), with exception of C. goeldii (78/54). The FPMI of A. azarae infulatus (82.5) and C. goeldii (66) were higher than C. jacchus (33.5), but not higher than those of C. penicillata (60.5) and S. sciureus (57.5). The TNF-a response was more regular in those four primates which decreased their PMI at 24/72 h: C. jacchus (145/122 pg/mL), C. penicillata (154/130 pg/mL), S. sciureus (164/104 pg/mL) and A. azarae infulatus (154/104 pg/mL), with exception of C. goeldii (38/83 pg/mL). The IL-12 response was mainly prominent in A. infulatus and C. goeldii which presented the highest FPMI and, the NO response was higher in C. goeldii, mainly at 72 h. These findings strongly suggest that these New World primates have developed a resistant innate immune response mechanism capable of controlling the macrophage intracellular growth of L. (L.) i. chagasi-infection, which do not encourage their use as animal model for studying AVL.

Preliminary study towards a novel experimental model to study localized cutaneous leishmaniasis caused bY Leishmania (Leishmania) mexicana

There is not an experimental model of localized cutaneous leishmaniasis (LCL) caused by Leishmania (Leishmania) mexicana. The aim of the present study was to characterize the clinical and histological features of Peromyscus yucatanicus experimentally infected with L. (L.) mexicana. A total of 54 P. yucatanicus (groups of 18) were inoculated with 1x10(6) promastigotes of L. (L.) mexicana in the base of the tail. They were euthanized at three and six months post experimental infection. The control group was inoculated with RPMI-1640. The predominant clinical sign observed was a single ulcerated lesion in 27.77% (5/18) and in 11.11% (2/18) P. yucatanicus at three and six months respectively. The histological pattern described as chronic granulomatous inflammation with or without necrosis was found in 7/7 (100%) biopsies of euthanized P. yucatanicus at three (n = 5) and six (n = 2) months, respectively. These results resembled clinical and histological features caused by L. (L.) mexicana in humans, and support the possibility to employ P. yucatanicus as a novel experimental model to study LCL caused by this parasite.

USE OF THE POLYMERASE CHAIN REACTION FOR THE DIAGNOSIS OF ASYMPTOMATIC Leishmania INFECTION IN A VISCERAL LEISHMANIASIS-ENDEMIC AREA

The diagnosis of asymptomatic infection with Leishmania (Leishmania) chagasi has become more important over recent years. Expansion of visceral leishmaniasis might be associated with other routes of transmission such as transfusion, congenital or even vector transmission, and subjects with asymptomatic infection are potential reservoirs. Moreover, the identification of infection may contribute to the management of patients with immunosuppressive conditions (HIV, transplants, use of immunomodulators) and to the assessment of the effectiveness of control measures. In this study, 149 subjects living in a visceral leishmaniasis endemic area were evaluated clinically and submitted to genus-specific polymerase chain reaction (PCR), serological testing, and the Montenegro skin test. Forty-nine (32.9%) of the subjects had a positive PCR result and none of them developed the disease within a follow-up period of three years. No association was observed between the results of PCR, serological and skin tests. A positive PCR result in subjects from the endemic area did not indicate a risk of progression to visceral leishmaniasis and was not associated with a positive result in the serological tests.

THE SERO-CONVERSION AND EVALUATION OF RENAL ALTERATIONS IN DOGS INFECTED BY Leishmania (Infantum) chagasi

This study investigated the sero-conversion period in which dogs from endemic areas test positive for visceral leishmaniasis (VL) as well as the early post-infection period in which renal alterations are observed. Dogs that were initially negative for Canine Visceral Leishmaniasis (CVL) were clinically evaluated every three months by serological, parasitological and biochemical tests until sero-conversion was confirmed, and six months later a subsequent evaluation was performed. Samples of kidney tissues were processed and stained with Hematoxylin and Eosin (H&E), Periodic Acid Schiff (PAS) and Masson’s trichrome stain and lesions were classified based on the WHO criteria. Of the 40 dogs that initially tested negative for VL, 25 (62.5%) exhibited positive serological tests during the study period. Of these 25 dogs, 15 (60%) tested positive within three months, five (20%) tested positive within six months and five (20%) tested positive within nine months. The dogs exhibited antibody titers between 1:40 and 1:80 and 72% of the dogs exhibited clinical symptoms. The Leishmania antigen was present in the kidneys of recently infected dogs. We found higher levels of total protein and globulin as well as lower levels of albumin in the infected dogs when compared to the control dogs. Additionally, infected dogs presented levels of urea and creatinine that were higher than those of the uninfected dogs. Glomerulonephritis was detected in some of the dogs examined in this study. These data suggest that in Teresina, the sero-conversion for VL occurs quickly and showed that the infected dogs presented abnormal serum proteins, as well as structural and functional alterations in the kidneys during the early post-infection period.

BODY WEIGHT AS A DETERMINANT OF CLINICAL EVOLUTION IN HAMSTERS (Mesocricetus auratus) INFECTED WITH Leishmania (Viannia) panamensis

SUMMARY The clinical outcome of infection with Leishmania species of the subgenus Viannia in hamster model (Mesocricetus auratus) has shown to be different depending on experimental protocol. Body weight has been a relevant determinant of the clinical outcome of the infection in hamsters with visceral leishmaniasis but its importance as a clinical parameter in hamsters with cutaneous leishmaniasis is not known. In this study, the clinical evolution of infection with L. (V) panamensis was evaluated in juvenile and adult male hamsters during 11 weeks by comparing clinical parameters such as attitude, temperature, respiratory rate, appearance of the stool, and body weight between infected and non-infected groups. Results showed that body weight decreased in adult hamsters after infection by L. (V) panamensis; this observation supports the use of body weight as an additional parameter to define the management or treatment of cutaneous leishmaniasis in infected adult hamsters used as an animal experimental model for leishmaniasis.

DISTINCT CELLULAR MIGRATION INDUCED BY Leishmania infantum chagasi AND SALIVA FROM Lutzomyia longipalpis IN A HEMORRHAGIC POOL MODEL

Recruitment of a specific cell population after Leishmania infection can influence the outcome of the disease. Cellular migration in response to Leishmania or vector saliva has been reported in air pouch model, however, cellular migration induced by Leishmania associated with host's blood and vector saliva in this model has not been described. Herein we investigated cellular migration into air pouch of hamster after stimulation with combination of L. chagasi and host's blood and Lutzomyia longipalpis saliva. Migration induced by saliva was 3-fold more than those induced by L. chagasi alone. Additionally, L. chagasi associated with blood and saliva induced significantly even more leukocytes into air pouch than Leishmania alone. L. chagasi recruited a diverse cell population; however, most of these cells seem to have not migrated to the inflammatory exudate, remaining in the pouch lining tissue. These results indicate that L. chagasi can reduce leukocyte accumulation to the initial site of infection, and when associated with vector saliva in the presence of blood components, increase the influx of more neutrophils than macrophages, suggesting that the parasite has developed a strategy to minimize the initial inflammatory response, allowing an unlimited progression within the host. This work reinforces the importance of studies on the salivary components of sand fly vectors of leishmaniasis in the transmission process and the establishment of the infection.

EVIDENCE OF Leishmania (Leishmania) infantum INFECTION IN DOGS FROM JUIZ DE FORA, MINAS GERAIS STATE, BRAZIL, BASED ON IMMUNOCHROMATOGRAPHIC DUAL-PATH PLATFORM (DPP®) AND PCR ASSAYS

In Brazil, domestic dogs are branded as the primary reservoir for zoonotic visceral leishmaniasis, due to the clear positive correlation observed between human and canine infection rates. This study aimed to carry out a serological survey of canine visceral leishmaniasis (CVL) in dogs housed at a public kennel in the municipality of Juiz de Fora, Minas Gerais State, Brazil, using the immunochromatographic TR DPP® CVL rapid test. Additionally, conventional and/or real time PCR assay was used to detect and confirm L. infantum infection in the DPP positive dogs only. Of the 400 dogs studied, most did not present clinical signs for CVL (p < 0.05), and fifteen (3.8%) were seropositive in the DPP test. There was no statistically significant difference between the DPP seropositive dogs and the clinical signs of the disease (p > 0.05). Both conventional and real time PCR tests confirmed L. infantum infection in nine (75.0%) of the twelve DPP seropositive dogs that remained alive during the follow-up period. This study is the first seroepidemiologic survey of CVL held in the city of Juiz de Fora, and the results reinforce the idea that this disease is currently in a process of expansion and urbanization in Brazil. Furthermore, this study highlights the use of the DPP test as an alternative for diagnosing CVL in large and mid-sized cities, due to its ease of implementation.

MOLECULAR DETECTION OF Leishmania IN PHLEBOTOMINE SAND FLIES IN A CUTANEOUS AND VISCERAL LEISHMANIASIS ENDEMIC AREA IN NORTHEASTERN BRAZIL

Several phlebotomine sand fly species have been regarded as putative or proven vectors of parasites of the genus Leishmania in Brazil, but data for the northeastern region remains incipient. In this study, a total of 600 phlebotomine sand flies were grouped in pools of 10 specimens each and tested by a Leishmania genus-specific PCR and by a PCR targeting Leishmania (Leishmania) infantum. Fourteen out of 60 pools were positive by the genus-specific PCR, being five pools of L. migonei, seven of L. complexa, one of L. sordellii and one of L. naftalekatzi, which correspond to a minimal infection rate of 2.3% (14/600). Our results, associated with their known anthropophily and their abundance, suggest the participation of L. migonei and L. complexa as vectors of Leishmania in northeastern Brazil. Remarkably, this is the first time in this country that the detection of Leishmania DNA in L. sordellii and L. naftalekatzi has been reported, but future studies are necessary to better understand the significance of these findings.

DETECTION OF Leishmania (Viannia) IN Nyssomyia neivai AND Nyssomyia whitmani BY MULTIPLEX POLYMERASE CHAIN REACTION, IN SOUTHERN BRAZIL

Sandflies transmit pathogens of leishmaniasis. The natural infection of sandflies by Leishmania (Viannia) was assessed in municipalities, in the state of Paraná, in Southern Brazil. Sandflies were collected with Falcão and Shannon traps. After dissection in search of flagellates in digestive tubes and identification of the species, female sandflies were submitted to the Multiplex Polymerase Chain Reaction (multiplex PCR) for detection of the fragment of the kDNA of Leishmania (Viannia) and the fragment from the IVS6 cacophony gene region of the phlebotomine insects. The analysis was performed in pools containing seven to 12 guts from females of the same species. A total of 510 female sandflies were analyzed, including nine Migonemyia migonei, 17 Pintomyia fischeri, 216 Nyssomyia neivai, and 268 Nyssomyia whitmani. Although none of the females was found naturally infected by flagellates through dissection, the fragment of DNA from Leishmania (Viannia) was shown by multiplex PCR in one sample of Ny. neivai (0.46%) and three samples of Ny. whitmani (1.12%). It was concluded that Ny. neivai and Ny. whitmani are susceptible to Leishmania infection, and that multiplex PCR can be used in epidemiological studies to detect the natural infection of the sandfly vector, because of its sensitivity, specificity and feasibility.

IDENTIFICATION OF Leishmania infantum IN PUERTO IGUAZÚ, MISIONES, ARGENTINA

 The emergence of zoonotic visceral leishmaniasis (ZVL) in Latin America is a growing public health problem. The urbanization of ZVL has been observed in different countries around the world, and there are a growing number of reports drawing attention to the emergence of this infection in new locations, as well as its increase in previously established areas of endemicity. In the city of Posadas, Misiones province, Northeastern Argentina, the transmission of ZVL associated with canines and Lutzomyia longipalpis was first reported in 2006. In the city of Puerto Iguazú, also in Misiones province, the first human case of ZVL was reported in February 2014. From 209 surveyed dogs, 15 (7.17%) were identified as positive by serological and/or parasitological methods. Amplification was observed in 14 samples and in all cases the species implicated was Leishmania infantum. To the authors’ knowledge, this is the first molecular characterization of L. infantum from dogs in this area.

GENOTYPE CHARACTERIZATION OF Leishmania (Viannia) braziliensis ISOLATED FROM HUMAN AND CANINE BIOPSIES WITH AMERICAN CUTANEOUS LEISHMANIASIS

Introduction:American tegumentary leishmaniasis (ATL) can be caused by Leishmania (Viannia) braziliensis complex. The evolution of ATL initially results in lesions and can develop into disseminated or diffuse forms. The genetic diversity of L. (V.) braziliensis in some endemic areas of Brazil has been poorly studied, such as in the state of São Paulo. This study analyzed the genetic diversity of L. (V.) braziliensis isolates collected from patients and dogs with LTA from the state of São Paulo.Methods:Leishmaniasis diagnosis was determined by PCR. The 132 biopsies were collected in different regions of Sao Paulo State, Brazil (36 municipalities). The genetic characterization of L. (V.) braziliensis isolates was tested by RFLP-PCR using DNA extracted from biopsies. The primer set amplified a specific region of Leishmania internal transcribed spacers of the ribosomal DNA locus.Results:Of the 132 samples, 52 (40%) were completely genotyped by RFLP-PCR (44 from human patients and eight from dogs). The results showed nine distinct patterns. The majority of the genotyped samples were from Sorocaba (30), and the others were distributed among 14 other municipalities. The first pattern was more frequent (29 samples), followed by pattern 2 (nine samples) and pattern 3 (three samples). Patterns 4, 6, 7, 8 and 9 were composed of two samples each and pattern 5 of one sample.Conclusion:These results suggest that polymorphic strains of L. (V.) braziliensis circulate in the state of São Paulo. These data agree with studies from other regions of Brazil, showing great variability among the natural populations of endemic foci.

NEW PRIMERS FOR DETECTION OF Leishmania infantum USING POLYMERASE CHAIN REACTION

SUMMARY Leishmania infantum causes visceral leishmaniasis (VL) in the New World. The diagnosis of VL is confirmed by parasitological and serological tests, which are not always sensitive or specific. Our aim was to design new primers to perform a Polymerase Chain Reaction (PCR) for detecting L. infantum. Sequences of the minicircle kinetoplast DNA (kDNA) were obtained from GenBank, and the FLC2/RLC2 primers were designed. Samples of DNA from L. infantum, Leishmania amazonensis, Leishmania braziliensis, Leishmania guyanensis, Leishmania naiffi, Leishmania lainsoni, Leishmania panamensis, Leishmania major and Trypanosoma cruzi were used to standardize the PCR. PCR with FLC2/RLC2 primers amplified a fragment of 230 bp and the detection limit was 0.2 fg of L. infantum DNA. Of the parasite species assayed, only L. infantum DNA was amplified. After sequencing, the fragment was aligned to GenBank sequences, and showed (99%) homology with L. infantum. In the analysis of blood samples and lesion biopsy from a dog clinically suspected to have VL, the PCR detected DNA from L. infantum. In biopsy lesions from humans and dogs with cutaneous leishmaniasis, the PCR was negative. The PCR with FLC2/RLC2 primers showed high sensitivity and specificity, and constitutes a promising technique for the diagnosis of VL.