Molecular identification of Leishmania in free-ranging black and gold howler monkeys (Alouatta caraya) in northeastern Argentina.

Parasitological surveys of non-human primates provides an important opportunity to better understand the epidemiology, transmission dynamics and emergence risk of anthropozoonoses such as leishmaniasis, which affect human populations in several regions accross South America. Our study area, in northeastern Argentina, can be considered a southern marginal region for the presence of leishmaniases and includes the habitat of black and gold howler monkeys, Alouatta caraya. To evaluate if A. caraya serve as potential hosts in the Leishmania cycle, we used molecular methods to examine infection by Leishmania spp. in 109 howler monkeys of different ages captured between July and August 2010. External ear tissue samples were subjected to PCR amplification for the Leishmania ribosomal internal transcribed spacer (ITS-1) and a RFLP assay with the Hae III restriction enzyme, and finally confirmed by sequencing. Nine howler monkeys (8.3%) were infected with Le. braziliensis (2.8%), Le. amazonensis (2.8%) and/or Le. infantum (3.7%). The results also suggest a case of co-infection between Le. braziliensis and Le. amazonensis. Further, we report the first observation of Le. amazonensis in the northeastern region of Argentina. The detection of Leishmania spp. in free-ranging howler monkeys gives rise to questions about the actual prevalence of the parasite in the wild, as well as if the number of infected wild monkeys detected may present a risk of leishmaniasis emergence in surronding human populations. Anyway, the presence of Leishmania spp. in A. caraya suggests the possible importance of these monkeys in the sylvatic and periurban transmission.

Multiscale environmental determinants of Leishmania vectors in the urban-rural context.

BACKGROUND: In South America, cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) are emerging diseases, expanding in the border area of Argentina, Brazil and Paraguay. Outbreaks of CL were reported since the 1990s, with Nyssomyia whitmani as the main vector in this region. Regarding VL, urban reports started in 2010 with Lutzomyia longipalpis as the main vector. The aim of this study was to evaluate environmental determinants related to the main vectors of leishmaniasis, to contribute to the prevention and control response to the emergence of VL and CL in the Argentina-Brazil-Paraguay border region. METHODS: The cross-sectional survey includes two cities and two close rural areas in the Argentinean Northeast Region, between November 2014 and January 2015, with a total of 95 sampling sites. REDILA-BL traps were set for three consecutive nights, and a total of 68 meso- and microscale environmental and landscape characteristics were surveyed. The association between vector abundance with different variables was evaluated using a generalized linear model with zero-inflated negative binomial distribution. We analyzed females for detection of Leishmania DNA. RESULTS: The analysis for Lu. longipalpis indicates an excess of absences when the mean NDWI around the sites were higher. The abundance of Lu. longipalpis at mesoscale level was higher when more urban services were present, and when blood sources such as chickens or dogs at the microscale level were present. For Ny. whitmani, no variable was found to be associated with the absences, while its abundance increased in association with the following variables: percentage of tree cover, presence of garbage collection service, hosted people and, at microscale, the presence of poultry. Leshmania infantum DNA was detected in 2/49 (4%) Lu. longipalpis. CONCLUSIONS: The abundance of both species is influenced by variables at different scales, their influence probably has a hierarchy and they are acting on different aspects of the biology of these vectors. The urban spatial segregation of Lu. longipalpis and the peri-urban and rural segregation of N. whitmani increase the risk of VL and CL. The selection of the better variables for each scale will allow the design of appropriate control strategies depending on species.

Twice upon a time: The progression of canine visceral leishmaniasis in an Argentinean city.

Canine Visceral Leishmaniasis (CVL) prevalence, spatial distribution and associated factors were assessed in four locations in Iguazú department in 2014 and in Puerto Iguazú city again in 2018. The city areas were divided into a grid of 400x400m cells. All cells were sampled in 2014 and a random subsampling was developed in 2018. In each cell, five dogs clustered in a 'critical scenario' (prone to have vectors) were sampled. A rapid immunochromatographic dipstick was used to detect antibodies against Leishmania infantum, confirming by lymph node smears observation and PCR. For Puerto Iguazú, Generalized Linear Models (GLMs) were constructed considering environmental, dog and clinical variables. Pearson's Chi square and Fisher's exact tests were employed to evaluate the association between CVL, dog clinical signs and infestation with other parasites. Cartographic outputs were made and Moran's I indices were calculated as spatial autocorrelation indicators. CVL prevalence rates were 26.18% in 2014 and 17.50% in 2018. No associations were established in environmental models, but dog age and repellent use were significant when running 2014 dog models. Clinical models showed significant associations between seropositive dogs and ophthalmological, dermal signs and onychogryphosis in 2014. In 2018, only adenomegaly was associated. The results of global Moran´s I were not significant but regarding local analysis, six sites in 2014 and one in 2018 presented autocorrelation with neighboring sites. The decrease in CVL prevalence may be associated to transmission stabilization, which could explain the lack of associations with dog-related variables. Further, spatial distribution of CVL is a poor evidence for design of transmission control measures but could be important in case of intensive parasite circulation or when the first autochthonous cases appear. For control success, sensitivity of diagnostic methods, political will and adequate material resources remain critical. Modeling of multiple variables will be required to identify factors that drive disease stabilization/destabilization.

Molecular characterization of trypanosomatid infections in wild howler monkeys (Alouatta caraya) in northeastern Argentina.

The transmission of Trypanosoma cruzi by vectors is confined to the Americas, and the infection circulates in at least two broadly defined transmission cycles occurring in domestic and sylvatic habitats. This study sought to detect and characterize infection by T. cruzi and other trypanosomes using PCR strategies in blood samples from free-ranging howler monkeys, Alouatta caraya, in the northeastern Argentina. Blood samples were collected at four sites with variable levels of habitat modification by human activity. PCR was conducted using primers for kinetoplast DNA, satellite DNA and ribosomal DNA of the trypanosomatid parasites. Ribosomal and satellite DNA fragments were sequenced to identify the trypanosomatid species and to characterize the discrete typing units (DTUs) of T. cruzi. Overall, 46% (50/109) of the howlers were positive according to the kDNA-PCR assay, but only 7 of the howlers were positive according to the SatDNA-PCR protocol. We sequenced the amplicons of the satellite DNA obtained from five specimens, and the sequences were 99% and 100% similar to T. cruzi. A sequence typical of DTU T. cruzi I was found in one howler monkey from the "remote" site, while sequences compatible with DTUs II, V, and VI were found in howlers from the "remote", "rural" and "village" sites. We detected 96% positive samples for RibDNA-PCR, 9 of which were sequenced and displayed 99% identity with Trypanosoma minasense, while none showed identity with T. cruzi. The results demonstrated the presence of T. cruzi and a species closely related to T. minasense in blood samples from free-ranging A. caraya, belonging to different T. cruzi DTUs circulating in these howler monkey populations. The results obtained in this study could help evaluate the role of A. caraya as a reservoir of T. cruzi in regions where Chagas disease is hyper-endemic and where the human-wildlife interface is increasing.

Performance of light-emitting diode traps for collecting sand flies in entomological surveys in Argentina.

The performance of two light-emitting diode traps with white and black light for capturing phlebotomine sand flies, developed by the Argentinean Leishmaniasis Research Network (REDILA-WL and REDILA-BL traps), were compared with the traditional CDC incandescent light trap. Entomological data were obtained from six sand fly surveys conducted in Argentina in different environments. Data analyses were conducted for the presence and the abundance of Lutzomyia longipalpis, Migonemyia migonei, and Nyssomyia whitmani (106 sites). No differences were found in presence/absence among the three types of traps for all sand fly species (p>0.05). The collection mean of Lu. longipalpis from the REDILA-BL didn´t differ from the CDC trap means, nor were differences seen between the REDILA-WL and the CDC trap collection means (p>0.05), but collections were larger from the REDILA-BL trap compared to the REDILA-WL trap (p<0.05). For Mg. migonei and Ny. whitmani, no differences were found among the three types of traps in the number of individuals captured (p>0.05). These results suggest that both REDILA traps could be used as an alternative capture tool to the original CDC trap for surveillance of these species, and that the REDILA-BL will also allow a comparable estimation of the abundance of these flies to the CDC light trap captures. In addition, the REDILA-BL has better performance than the REDILA-WL, at least for Lu. longipalpis.