VECTOR-BORNE AND ZOONOTIC DISEASES Volume XX, Number XX, 2019 ª Mary Ann Liebert, Inc. DOI: 10.1089/vbz.2018.2433

Trypanosoma Found in Synanthropic Mammals from Urban Forests of Parana´, Southern Brazil

Ricardo Nascimento Drozino,1 Fla´vio Haragushiku Otomura,2 Janaina Gazarini,3 Moˆnica Lu´cia Gomes,1 and Max Jean de Ornelas Toledo1

Abstract

Trypanosoma cruzi is a parasitic protozoan that infects a diversity of hosts constituting the cycle of enzootic transmission in wild environments and causing disease in humans (Chagas disease) and domestic animals. Wild mammals constitute natural reservoirs of this parasite, which is transmitted by hematophagous kissing bugs of the family Reduviidae. T. cruzi is genetically subdivided into six discrete typing units (DTUs), T. cruzi (Tc)I to TcVI. In Brazil, especially in the state of Parana´, TcI and TcII are widely distributed. However, TcII is less frequently found in wild reservoirs and triatomine, and more frequently found in patients. The goal of this study was to investigate the natural occurrence of T. cruzi in wild synanthropic mammals captured in urban forest fragments of the Atlantic Forest of Parana´, southern Brazil. In this way, 12 opossums and 35 bats belonging to five species were captured in urban forest parks of the city of Maringa´, Parana´, an area considered endemic for Chagas disease. PCR-kinetoplast DNA molecular diagnostic reveals Trypanosoma sp. infection in 12 (100%) Didelphis albiventris and 10 (40%) Artibeus lituratus. In addition to demonstrating the presence of Trypano- soma in the two groups of mammals studied, we obtained an isolate of the parasite genotyped as TcII by amplification of the cytochrome oxidase II gene by PCR, followed by restriction fragment length polymorphism with AluI, and confirmed by PCR of rDNA 24Sa. This is the first record of the encounter in wild mammals of Trypanosoma DNA (in A. lituratus) and T. cruzi DTU TcII (in D. albiventris) in the state of Parana´.

Keywords: Trypanosoma cruzi II, Didelphis albiventris, Artibeus lituratus, natural infection, Parana´ endemic area

Introduction Atlantic Forest biome, TcII is the most frequently isolated (95%) DTU in human patients with chronic infection (Gas- rypanosoma cruzi (Kinetoplastida, Trypanosomati- parim et al. 2018), as well as in the triatomine species Pan- Tdae), the etiologic agent of Chagas disease or American strongylus megistus and Triatoma sordida, but not in wild

Downloaded by SENCKENBERG/ZEITSCHRIFTEN from www.liebertpub.com at 07/23/19. For personal use only. trypanosomiasis, is a parasitic protozoan capable of infecting mammals (Abolis et al. 2011). In this state, TcI has been a diversity of hosts, constituting a wild enzootic transmission isolated, in these same species of triatomines, both in pure cycle through vectors in humans and domestic animals. The and mixed infections with TcII, as well as the marsupial species is subdivided into discrete typing units (DTUs), from Didelphis albiventris. T. cruzi (Tc)I to TcVI, as well as Tcbat (Marcili et al. 2009, Mammals of the orders Chiroptera and Didelphimorphia Zingales et al. 2009, 2012, Lima et al. 2015). Another re- are well-known reservoirs of T. cruzi and other trypanoso- cently proposed classification system separates T. cruzi into matids. With ample population abundance, accentuated sy- just three groups, from mtTcI to mtTcIII, based on mito- nanthropism, and high mobility, these animals play an chondrial genes (Barnabe´ et al. 2016). All DTUs are found in important role in the distribution of diverse species of path- a diverse array of mammalian hosts, representing seven dif- ogens (De Oliveira et al. 2010, Hamilton et al. 2012, Brook ferent orders, and in triatomine insects (Reduviidae, Triato- and Dobson 2015, da Costa et al. 2015). These mammals’ minae) throughout all states and biomes in Brazil (Jansen species demonstrate the highly complex ecological interac- et al. 2015). However, in the state of Parana´, which is 97.8% tions found in the T. cruzi transmission cycle, being necessary

1Department of Basic Health Sciences, Universidade Estadual de Maringa´, Maringa´, Brazil. 2Biological Sciences Sector, Universidade Estadual do Norte do Parana´, Bandeirantes, Brazil. 3College of Biological and Environmental Sciences, Universidade Federal da Grande Dourados, Dourados, Brazil.

1 2 DROZINO ET AL.

the conduction of studies that evaluate its dynamics in the Wild mammal capture context of the One Health concept, triad that encompasses The campaigns were held once a month in the following humans, domestic animals, wildlife, and the changing eco- periods: from September 2014 to December 2015. Bats systems in which they live (Thompson 2013, Pinazo and were captured with mist nets, conditioned in cloth sacs, and Gascon 2015). identified at the species level. Opossums of the species In this study, a preliminary investigation into the presence D. albiventris were captured using Tomahawk Live Traps Trypanosoma of in free-range wild mammals was accom- baited with fruits and peanut butter. These methods were plished in Atlantic Forest remnants situated in urban areas, an approved by the Instituto Chico Mendes de Conservac¸a˜oda ´ environment not still explored in the state of Parana. Biodiversidade—Ministe´rio do Meio Ambiente, Brazil (no. 42881) and by the Ethics Committee in Animal Experi- Materials and Methods mentation (CEUA) at Universidade Estadual de Maringa´ Study area (UEM) (no. 023/2014). Wild mammals were captured in two urban forests frag- Blood collection and hemocultures ments, in 2325¢39¢ of latitude south and 5155¢51† of lon- gitude west, and in 2323¢27¢ of latitude south and 5156¢34† Opossums and bats were anesthetized with 4.0 mg/kg of longitude west. Both, remnants of the Atlantic Forest bi- chloridrate of xylazine and 20.0 mg/kg body weight chlori- ome of the city of Maringa´, located in the northeast of Parana´, drate of ketamine for blood collection. Approximately southern Brazil, and an area considered endemic for Chagas 0.5 mL of whole blood obtained by cardiac puncture in the disease (Fig. 1). bats and from the tail medium vein in the opossums was Downloaded by SENCKENBERG/ZEITSCHRIFTEN from www.liebertpub.com at 07/23/19. For personal use only.

FIG. 1. Map highlighting the two capture points located in the urban area of the Municipality of Maringa´, state of Parana´, Southern Brazil. TRYPANOSOMA IN WILD MAMMALS OF URBAN FOREST 3

placed in Novy, McNeal, and Nicolle medium, and covered PCR of rDNA 24Sa with an overlay of liver infusion tryptose (LIT) medium PCR amplification of rDNA 24Sa using D71 (5¢-AAGGT containing 10% fetal bovine serum. Another 0.2 mL was GCGTCGACAGTGTGG-3¢) and D72 (5¢-TTTTCAGAATG stored in microtubes containing 400 lL of 0.2 M ethylene- GCCGAACAGT-3¢) primers as previously described by diaminetetraacetic acid (EDTA) and 6.0 M guanidine solu- Souto et al. (1996). Amplified fragments were resolved on tion for the extraction and amplification of blood DNA 6% polyacrylamide gel, stained with silver, and analyzed according to Miyamoto et al. (2006). according to Sa´ et al. (2016).

Molecular analysis for detection of Trypanosoma DNA Results DNA was extracted by the phenol/chloroform method and Detection of the kDNA minicircle in Trypanosoma precipitated with addition of ethanol and sodium acetate (Ma- cedo et al. 1992). PCR amplification was performed with oli- PCR-kDNA revealed that all samples of captured gonucleotides number 121 (5¢-AAATAATGTACGGG(T/G) D. albiventris (n = 12, 100%) exhibited a fragment of GAGATGCATGA-3¢) and number 122 (5¢-GGTT CGATTG 330 bp kDNA minicircle from Trypanosoma.Ofthe35 GGGTTGGTGTAATATA-3¢) according to the protocol of examined bat species, PCR-kDNA also detected this Gomes et al. (1998) modified by Miyamoto et al. (2006). These same fragment in 10/25 (40%) Artibeus lituratus caught. sequences amplify a specific fragment of *330 base pairs (bp) In other species of captured bats, Sturnira lilium (n = 4), of kinetoplast DNA (kDNA) from Trypanosoma sp. The am- Carollia perspicillata (n = 3), Pygoderma bilabiatum plification products were analyzed using 4.5% polyacrylamide (n = 2), and Platyrrhinus lineatus (n = 1), this fragment gel electrophoresis, stained with silver, and digitally recorded. was not detected (Table 1). This high rate of positivity in PCR was not observed in hemocultures, and in only 1/12 (8.3%) opossums was positive in this technique, allowing Isolation and genotyping of T. cruzi to obtain a T. cruzi isolate (Table 1). Parasites from a unique positive hemoculture, with low manipulation to avoid clonal selection, were amplified in LIT Genotyping of DTU of T. cruzi isolate medium until the exponential phase. This material was The isolate was genotyped by analysis of mitochondrial centrifuged and washed with Krebs-Ringer-Tris/pH 7.2 so- COII gene polymorphisms. The PCR/RFLP-COII showed lution buffer three times. The obtained mass was then re- two bands, the first at *80 bp referring to the species T. cruzi suspended in 500 lL of lysis solution (0.5 M EDTA, 5 M and the second at 250 bp, which consistently classifies the sodium chloride and 1% sodium dodecyl sulfate) supple- isolate as TcII (Fig. 2A). mented with 10 mg/mL of proteinase K (Invitrogen) at 37C The result of PCR amplification of the rDNA 24Sa showed overnight. DNA extraction was performed by the phe- a band of *125 bp compatible with the TcII and TcVI ge- ´ nol/chloroform method after Sa et al. (2016). notypes. Although this marker is not able to distinguish these two genotypes, it was used to confirm the DTU of the isolate PCR/restriction fragment length polymorphism (Zingales et al. 2009, 2012). The profiles generated by the cytochrome oxidase II genetic analyzes of COII and rDNA 24Sa together, allowed to classify the T. cruzi isolate obtained as TcII (Fig. 2B). Differentiation of T. cruzi isolates (TcI to TcVI) was done by amplification of the cytochrome oxidase II (COII) gene by Discussion PCR, followed by restriction fragment length polymorphism (RFLP) with AluI (de Freitas et al. 2006, Abolis et al. 2011). Studies of the dynamic population of T. cruzi in Parana´, The primers used were the Tcmit-10 (5¢-CCATATATTG located in the Southern Region of Brazil, an endemic area for TTGCATTATT-3¢) and Tcmit-21 (5¢-TTGTAATAGGAGT Chagas disease (Gasparim et al. 2018), reveal TcII to be the CATGTTT-3¢) primers with Taq DNA polymerase (Plati- principal DTU isolated from chronic phase patients residents num, Invitrogen) and the restriction enzyme AluI (New in this state (Zalloum et al. 2005, Abolis et al. 2011). In con- Downloaded by SENCKENBERG/ZEITSCHRIFTEN from www.liebertpub.com at 07/23/19. For personal use only. England BioLabs) in NEB 4 buffer. The PCR amplicons were trast, a wild host of this DTU has yet to be registered in the resolved on 6% polyacrylamide gel and products sizes ana- state. TcII was surprisingly found in this central urban forest lyzed as described by Sa´ et al. (2016). (Atlantic Forest biome) of the city of Maringa´ in the northwest

Table 1. Wild Mammals Captured and Examined for the Presence of Trypanosoma cruzi (By Detection of Kinetoplast DNA PCR and Hemoculture) in Two Atlantic Forest Urban Parks of Maringa´ City, Parana´, Southern Brazil

Species No. of specimens Prevalence (%) Hemoculture (%) Didelphis albiventris 12 100.0 8.3 Artibeus lituratus 25 40.0 0.0 Sturnira lilium 4 0.0 0.0 Carollia perspicillata 3 0.0 0.0 Pygoderma bilabiatum 2 0.0 0.0 Platyrrhinus lineatus 1 0.0 0.0 Total 47 46.8 2.1 4 DROZINO ET AL. Downloaded by SENCKENBERG/ZEITSCHRIFTEN from www.liebertpub.com at 07/23/19. For personal use only.

FIG. 2. Amplification and restriction fragment profiles of PCR/RFLP-COII in silver-stained 6% polyacrylamide gel used for genotyping Trypanosoma cruzi isolate (MDID/BR/2015/DALB01) obtained from Didelphis albiventris (A). Schematic figure to discriminating and typing T. cruzi DTUs based in PCR RFLP-COII (de Sa´ et al. 2013) and rDNA 24Sa (Souto et al. 1996), and adapted to Sa´ et al. (2016) (B). *TcV and TcVI discriminating to second step (rDNA 24Sa); **does not amplify T. rangeli DNA. COII, cytochrome oxidase II; DTUs, discrete typing units; RFLP, restriction fragment length polymor- phism; Tc, Trypanosoma rangeli.

of the state, in D. albiventris, a wild reservoir generally con- counter with TcII being a rarity. When encountered, TcII is sidered uncommon for harboring this DTU. generally associated with mixed infections of TcI, TcV, or All previous attempts to associate T. cruzi populations with TcVI (Abolis et al. 2011, Jansen et al. 2015, 2018). However, specific mammalian host species have led to controversial the ability of Didelphis spp. to rapidly control TcII infections results (Jansen et al. 2015). Even without an association and reduce parasitemia to undetectable levels (Legey et al. between DTUs and mammalian reservoirs, a majority of 2003, Jansen et al. 2015) is a likely variable affecting isola- isolates from D. albiventris are genotyped as TcI, an en- tion efficiency of TcII from these animals. TRYPANOSOMA IN WILD MAMMALS OF URBAN FOREST 5

D. albiventris is characterized as a highly synanthropic taining parasite isolates could be directly related to these species, adaptable, opportunistic, and omnivorous, as well as mammals’ capacity to incubate pathogens (Brook and potential disseminators of disease and one of the main res- Dobson 2015), or still to the presence of Trypanosoma spp. ervoirs of T. cruzi and potentially other Trypanosoma spp. infection not grown in axenic medium (Dario et al. 2017, (De Oliveira et al. 2010, Jansen et al. 2015, Dario et al. 2017), Jansen et al. 2018). In the same way, D. albiventris has which justifies our single encounter of TcII. Jansen et al. already been found to be opportunistically predating on (2015) reported the finding of TcII infecting wild animals in A. lituratus (Gazarini et al. 2008), which may be the tip of the all Brazilian biomes, including the Caatinga in the Northeast iceberg for a semiopen cycle or spillover between niches in Region of Brazil, where that species of marsupial has been urban and periurban areas. Whereas bats weakened by con- found harboring TcII, in both pure and mixed infections with comitant infections, together with poor dietary supply, may TcV and TcVI. However, its discovery was never recorded in be the source of infection for this didelphid. The encounter of Parana´, a state that has 97.8% of its area inserted in the A. lituratus infected by Trypanosoma sp., a species that Atlantic Forest biome (Muylaert et al. 2018). This is the first presents high population densities and high synanthropism, registration of T. cruzi genotyped as TcII isolated from reinforces the importance of studies that focus on synan- D. albiventris in the state of Parana´. thropic species in the wild cycle of T. cruzi. This register The high positivity of PCR-kDNA for Trypanosoma spp. contrasts with the finding of a migratory bat (Tadarida bra- in D. albiventris and the nonencounter of triatomines in the siliensis) found infected with TcV in the southern United area of study could be related to the complexity of parasitic States (Nichols et al. 2019). The authors report that even cycles in the wild environment. Nouvellet et al. (2013) states with a low prevalence, T. brasiliensis may contribute to fu- that the probability of transmission after contact with an in- ture enzootic expansion of T. cruzi, playing a unique role in fected triatomine is 6.0 · 104, confirming that transmission is the epidemiology of T. cruzi through its annual migrations still more difficult in free-range wild mammals were thick, (Nichols et al. 2019). dense skin impedes contact with the metacyclic forms of the PCR-RFLP that targets the COII gene restricted by the parasite (Jansen et al. 2015). The same holds true for infected enzyme AluI is able to accurately separate the T. cruzi ge- A. lituratus specimens, in addition to the large diversity of notypes into five groups: TcI, TcII, TcIII, TcIV, and Trypanosoma spp. with which they are subject to becoming TcV/TcVI (de Sa´ et al. 2013, Sa´ et al. 2016). In its turn, the infected (Dario et al. 2017, Jansen et al. 2018, Lourenc¸o et al. TcV/TcVI pattern can be differentiated using a second ge- 2018). netic marker based on the 24Sa gene sequence of Trypano- Although no triatomine was found in the study area, the soma ribosomal DNA (de Sa´ et al. 2013). These markers TcII encounter with the high positivity of D. albiventris for together still allow identification of T. rangeli (a very com- Trypanosoma sp. suggest an active wild cycle. Opossums and mon species and frequently isolated from the wild environ- bats are reservoirs of T. cruzi that are frequently found in the ment), and the separation of its major genotypes: KP1+ and peridomicile (Abolis et al. 2011, Jansen et al. 2015). There- KP1- (Sa´ et al. 2016). This pair of techniques, therefore, fore, surveillance work is required that encompasses synan- allows to classify stocks of T. cruzi within the six genetic thropic animals in urban and periurban forest areas to avoid lineages previously proposed with a high resolution, hence its reintroduction or a spillover of T. cruzi to the domestic cycle use being of great value in population studies with obtaining in controlled areas for Chagas disease (Pinazo and Gascon isolates. This pair of techniques used allowed the isolate 2015, Gasparim et al. 2018). The encounter of TcII in obtained from D. albiventris to be genotyped as DTU TcII D. albiventris, the main isolate obtained in chronic patients in (Fig. 2). Parana´, suggests that surveillance actions in these areas with In previous studies, T. cruzi isolates obtained from humans a focus on synanthropic wild animals should be considered. living in Parana´, all in the chronic phase of infection, were PCR-kDNA analysis of T. cruzi is used in many experi- genotyped as TcI, TcII, and TcIII, with TcII being the most mental studies, both for proving the infection and for moni- prevalent DTU (Abolis et al. 2011). In contrast, TcII had toring the cure of treated animals due to its high sensitivity previously been found in triatomines in genetically pure or and specificity to T. cruzi (Teston et al. 2013, Margioto et al. mixed infections with TcI, whereas the isolates obtained from

Downloaded by SENCKENBERG/ZEITSCHRIFTEN from www.liebertpub.com at 07/23/19. For personal use only. 2016, Zanusso Junior et al. 2018). This is why it has also been marsupial D. albiventris presented only TcI profiles (Zalloum used in the laboratory diagnosis of American trypanosomi- et al. 2005, Abolis et al. 2011). These data allow the first asis in chronic human patients (Ramı´rez et al. 2015). Despite record of TcII isolate from a wild D. albiventris from an the high sensitivity, its use is limited to the genus Trypano- urban forest fragment of the state of Parana´. soma, when applied to the parasite’s detection in wild ani- mals. This is because wild animals are exposed to a wide Conclusion range of Trypanosoma spp., some of which have not yet been cataloged or even recently discovered (Lopes et al. 2018), PCR-kDNA, despite detecting the presence of kDNA re- requiring the use of several molecular markers to identify stricted to the Trypanosoma genus, is shown as an alternative these species unequivocally. However, the use of more than for the evaluation of groups of animals that act as reser- one molecular marker for blood samples obtained from wild voirs in areas with potential of transmission. Because it is a animals becomes infeasible, since the variety of Trypanoso- low-cost technique and requires a small amount of blood, ma spp. that can infect didelphids and chiroptera is immense PCR-kDNA has proved to be of great value in the monitoring (Dos Santos et al. 2018, Jansen et al. 2018, Lourenc¸o et al. of infection, allowing more efficient isolation strategies to be 2018). used in places where active cycles of Trypanosoma spp. are In bats, the presence of Trypanosoma infection detected detected. This strategy may potentiate the number of isolates through PCR-kDNA accompanied by the difficulty in ob- obtained in forest areas yet to be explored as to the genetic 6 DROZINO ET AL.

diversity of the parasites. The results show a relation of the Gomes ML, Macedo AM, Vago AR, Pena SD, et al. Trypano- synanthropic mammals sampled with persistent infections by soma cruzi: Optimization of polymerase chain reaction for one T. cruzi DTU (TcII), or even by Trypanosoma spp., detection in human blood. Exp Parasitol 1998; 88:28–33. which may be circulating in the wild environment of the Hamilton PB, Teixeira MM, Stevens JR. The evolution of region, but are more difficult to detect or isolate by more Trypanosoma cruzi: The ‘bat seeding’ hypothesis. Trends conventional techniques, such as hemoculture. Infection by Parasitol 2012; 28:136–141. Trypanosoma spp. was recorded in A. lituratus that inhab- Jansen AM, Xavier S, Roque ALR. Trypanosoma cruzi trans- its and forages urban forest areas and their surrounds in the mission in the wild and its most important reservoir hosts in city of Maringa´. The TcII genotype was also observed in Brazil. Parasit Vectors 2018; 11:502. 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