Bat trypanosomatids (first report of T. wauwau) in Triângulo Mineiro, Brazil

In this study, trypanosomatids commonly found in bats, including Trypanosoma cruzi marinkellei, T. dionisii, and Leishmania braziliensis, were identified. Additionally, T. wauwau was identified in one specimen of Anoura caudifer, and represents the first report of this parasite from the Central West region of Brazil. T. wauwau was previously identified by other researchers in the North of the country, in only three species of bats in the genus Pteronotus: P. parnellii (Pará and Rondônia states), and P. personatus and P. gymnonotus (Rondônia). The identification of T. wauwau indicates how different trypanosomatids are able to adapt to new host species of bats. This is owing to bats’ high mobility, wide geographic distribution, social behavior, and ability to coexist in large colonies. These characteristics may facilitate the transmission of infectious agents in nature, which are responsible for outbreaks of some zoonoses. Therefore, health authorities should focus on both vertebrates and vectors associated with the environments where these bats are found. Author summary The prevalence of Trypanosoma in bats is high, with T. cruzi, T. cruzi marinkellei, and T. dionisii as the most prevalent infective species. This study reports for the first time the presence of T. wauwau in the southeast region of Brazil in the bat Anoura caudifer. Although this species of Trypanosoma has been found in bats of the genus Pteronotus, it was not detected in any other genus, including in the bats that share the same shelter with Pteronotus. The species T. wauwau was found infecting bats only in Brazil. Its occurrence was restricted to the northern region of the country, in the states of Pará, infecting the species P. parnellii and in Rondônia infecting P. personatus, P. gymnonotus as well as P. parnellii. Although its morphology is similar to that of T. cruzi, little is known about the development of T. wauwau, both in its vertebrate host and the existence of a plausible invertebrate vector. Its characteristics include its inability to develop in mammalian cells and its non-infectiousness in mice and triatomine insects. Further research, through molecular studies, may provide important and valuable data for understanding the origin, evolution, and global distribution of, and the association between the different species of Trypanosoma and their hosts.


Introduction
Until now, more than up to 30 species of trypanosomatids have been isolated from bats [1][2][3][4]. The most frequently reported species of Trypanosoma in bats are T. cruzi, T.
Given the above observations and concerns, the objective of the present study was to evaluate the presence and identity of the possible trypanosomatid species (Trypanosoma spp. and Leishmania spp.) in bats of the Triângulo Mineiro Region of Brazil.

Area of study and capture of bats
This study was carried out in the city of Ituiutaba, Minas Gerais (MG), Brazil, located in the Triângulo Mineiro mesoregion (lat 18º58'08" S, lon 49º27'54" W; altitude: 544 m; area: 2595.2 km 2 ), west of MG, Brazil.
Bats were captured from November 2014 to September 2015, at night between 18h00 and midnight (0h00) using mist nets, and during the day in shelters using manual 4 nets. The identification of bats species was done based on taxonomic keys to the family [17], genus, and species levels [18][19].

Blood collection
A total of 216 bats were collected, and 0.5 to 1.0 mL of blood was collected from each specimen by cardiac puncture. Of these samples, 25 µL was used to estimate microhematocrit, while the remaining blood was stored in EDTA V/V and guanidine solution (6 M Guanidine-HCl and 0.2 M disodium) at 4°C until further use.

Identification of trypanosomatid species
DNA extraction was performed using a GeneJET Genomic DNA Purification® kit from Thermo Scientific, according to the manufacturer's instructions.
Electrophoresis was performed on 6% polyacrylamide gel, stained with silver.
Sequencing was performed at ACTGene Análises Moleculares (Brazil). Chromatograms were analyzed using ChromasPro 2.1.4, and consensus sequences were generated. The Phred threshold value was set at > 20. Sequences were aligned using SeaView 4.5.2, with the Muscle algorithm. Maximum Likelihood trees using Neighbor-Joining methods were generated using MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for 5 bigger datasets [22], with 1000 bootstrap iterations, and including reference sequences from trypanosomatids retrieved from GenBank.

Statistical analyses
The Chi-square test was used to assess the possible association between the dietary habits of bat hosts and the positivity of their PCR tests for both Leishmania and Trypanosoma, using the TIBCO® Statistica TM program [23], with a significance level of 5% (P < 0.05).

Ethical considerations
Bats were captured and manipulated according to the recommendations of the The species of bats with the highest infection rates were Desmodus rotundus, Anoura caudifer, and Glossophaga soricina (25.00%, 11.11%, and 6.83%, respectively).
The species of trypanosomes identified were T. cruzi marinkellei and T. dionisii in various bat species, and Trypanosoma wauwau in Anoura caudifer (Table 2, Figure 1).

Identification of Leishmania species
Of the samples analyzed, nine (4.46%) were positive for Leishmania spp. (Table   1). The only species of Leishmania identified was L. braziliensis, in the bats Anoura caudifer and Phyllostomus discolor (Table 2). 8

Discussion
Some species of trypanosomatids have already been found to naturally infect bats [4,[7][8][9][10][24][25][26][27]. Many of these trypanosomatids are responsible for important zoonoses, but most of those found in bats remain poorly known. In this work, we investigated the presence of Leishmania spp. and Trypanosoma spp. in bats from the city of Ituiutaba, Triângulo Mineiro, Brazil, an area previously showing endemism for Chagas disease.
In this study, the use of the direct parasitological method of microhematocrit revealed frequent positive results for trypanosomatids, as has been demonstrated by other studies [4,28]. It is worth highlighting that the microhematocrit method, in all the field studies carried out by our group, especially in small animals, has shown greater sensitivity or, in certain cases, similar behavior to that of traditional blood culture methods [29].
Regarding PCR positivity, the prevalence of trypanosomatids was similar to that observed by other studies in Brazil [3][4], as was the result that in terms of dietary habits most of these bats were nectarivores. It was also demonstrated in this study that the two most common species that circulate in bats in this region are T. c. marinkellei and T.
The only species of Leishmania found was L. braziliensis, in Anoura caudifer and Phyllostomus discolor. This is the first report of this species of Leishmania in these species of bats. In this region, the species L. infantum, L. amazonensis, and L. braziliensis were previously identified from the bat species G. soricina and M. molossus [3].
In this study, it was possible to identify T. wauwau in only one specimen of T. wauwau is phylogenetically associated with Australian trypanosomes, possibly constituting one more piece of evidence that T. cruzi may have evolved from the recent dispersion of an ancestral bat host across several continents [9]. The finding of bats positive for a disease at aplace where that disease is not endemic, rather is introduced from neighboring areas, demonstrates the considerable mobility of these animals, often involving migrations over long distances, including to urban areas, thereby acting as potential agents of zoonoses.