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Molecular detection of Apicomplexa protozoa in tissues from Alouatta guariba clamitans

Detecção molecular de protozoários Apicomplexa em tecidos de Alouatta guariba clamitans

ABSTRACT:

The brown howler monkey (Alouatta guariba clamitans) is a primate species widely distributed in South America. Infections by protozoa are common in primates. However, studies on protozoa in primates in Brazil are scarce, so the goal of this study was to investigate DNA from the apicomplexan protozoa Neospora caninum, Sarcocystis spp. and Toxoplasma gondii in tissues of A. guariba clamitans. DNA extraction was performed on tissue samples from the heart, brain, liver, spleen, lung and intestine of six A. guariba clamitans from Santa Maria, Central Region of Rio Grande do Sul, Brazil. Conventional PCR was performed using 18S rRNA gene general primers for Apicomplexa and also specific primers to amplify Neosporaspp. and Toxoplasma gondii DNA. All animals were positive in the 18S PCR and the genetic sequencing confirmed the presence of Sarcocystis spp. DNA in the tissues of four animals belonging to at least two species (S. neurona and S. gigantea) and T. gondii DNA in the other two animals. One positive sample for T. gondii was genotypically characterized as atypical by the restriction fragment length polymorphism technique. N. caninum DNA was not detected in the tested samples. The presence of Apicomplexa protozoan DNA in the tissues of the six animals tested in this study highlights the importance of howler monkeys as maintainers of these pathogens in nature.

INDEX TERMS:
Molecular detection; Apicomplexa; protozoa; Alouatta guariba clamitans; brown howler monkey; Sarcocystis; Toxoplasma; genotyping; wildlife animals

RESUMO:

O bugio ruivo (Alouatta guariba clamitans) é uma espécie de primata amplamente distribuída na América do Sul. As infecções por protozoários são comuns em primatas. Entretanto, estudos sobre protozoários em primatas no Brasil são escassos, portanto o objetivo deste estudo foi pesquisar DNA dos protozoários Apicomplexa Neospora caninum, Sarcocystisspp. e Toxoplasma gondii em tecidos de A. guariba clamitans. A extração de DNA foi realizada em amostras de tecido do coração, cérebro, fígado, baço, pulmão e intestino de seis A. guariba clamitans oriundos de Santa Maria, Região Central do Rio Grande do Sul, Brasil. Foi realizada PCR convencional utilizando primers geral do gene 18S rRNA para Apicomplexa e também primers específicos para amplificação de DNA de Neospora spp.e Toxoplasma gondii. Todos os animais foram positivos no PCR geral para Apicomplexa e no sequenciamento genético confirmou-se a presença de DNA de Sarcocystis nos tecidos de quatro animais pertencentes a pelo menos duas espécies (S. neurona e S. gigantea), e DNA de T. gondii foi detectado nos outros dois animais. Uma amostra positiva para T. gondii foi caracterizada genotipicamente como atípico pela técnica de polimorfismo do comprimento do fragmento de restrição. Não foi detectado DNA de N. caninum nas amostras testadas. A presença de DNA de protozoários apicomplexa nos tecidos dos seis animais testados neste estudo destaca a importância dos bugios ruivos como mantenedores desses patógenos na natureza.

TERMOS DE INDEXAÇÃO:
Detecção molecular; protozoários; Apicomplexa; Alouatta guariba clamitans; bugio ruivo; Sarcocystis; Toxoplasma; genotipagem; animais selvagens

Introduction

The Apicomplexa phylum comprises several genera of pathogenic parasites (Adl et al. 2007Adl S.M., Leander B.S., Simpson A.G.B., Archibald J.M., Anderson O.R., Bass D., Bowser S.S., Brugerolle G., Farmer M.A., Karpov S., Kolisko M., Lane C.E., Lodge D.J., Mann D.G., Meisterfeld R., Mendoza L., Moestrup Ø., Mozley-Standridge S.E., Smirnov A.V. & Spiegel F. 2007. Diversity, nomenclature, and taxonomy of protists. Syst. Biol. 56(4):684-689. <https://dx.doi.org/10.1080/10635150701494127> <PMid:17661235>
https://doi.org/10.1080/1063515070149412...
), such as Toxoplasma gondii, Neospora caninum, and Sarcocystis spp. These apicomplexan parasites have a heteroxenous life cycle infecting multiple hosts and their transmission is facilitated by predator-prey relationships. In the intermediate hosts (often herbivorous or omnivorous), infectious stages are formed by tissue cysts that can be ingested by the definitive host (usually a carnivorous predator) (Sibley 2003Sibley D.L. 2003. Recent origins among ancient parasites. Vet. Parasitol. 115(2):185-198. <https://dx.doi.org/10.1016/s0304-4017(03)00206-1> <PMid:12878422>
https://doi.org/10.1016/s0304-4017(03)00...
).

Protozoal infections in nonhuman primates are common and native populations of primates host an amazing diversity of parasites and infectious agents, reinforcing the role of wild animals as reservoirs for zoonosis (Nunn & Altizer 2005Nunn C.L. & Altizer S.M. 2005. The global mammal parasite database: an online resource for infectious disease records in wild primates. Evol. Anthropol. 14(1):1-2. <https://dx.doi.org/10.1002/evan.20041>
https://doi.org/10.1002/evan.20041...
, Silva et al. 2006Silva A.V., Moraes G.B.S., Langoni H. & Bagagli E. 2006. Study of Toxoplasma infection in Brazilian wild mammals: serological evidence in Dasypus novemcinctus Linnaeus, 1758 and Euphractus sexcinctus Wagler, 1830. Vet. Parasitol. 135(1):81-83. <https://dx.doi.org/10.1016/j.vetpar.2005.08.013> <PMid:16188389>
https://doi.org/10.1016/j.vetpar.2005.08...
).

Primates from genus Alouatta are the most widely distributed New World monkeys and Alouatta guariba clamitans (brown howler monkey) is endemic in the Atlantic Forest in Brazil and Argentina (Meireles et al. 1999Meireles C.M., Czelusniak J., Ferrari S.F., Schneider M.P.C. & Goodman M. 1999. Phylogenetic relationships among brazilian howler monkeys, genus Alouatta (Platyrrhini, Atelidae), based on γ1 -globin pseudogene sequences. Genet. Mol. Biol. 22(3)337-344. <https://dx.doi.org/10.1590/S1415-47571999000300009>
https://doi.org/10.1590/S1415-4757199900...
). Because of this wide distribution, howler monkeys are a potential parasite carrier of great zoonotic importance.

There are few studies on endoparasites in primates in Brazil. Therefore, this study aimed to evaluate the presence of T. gondii, Sarcocystis spp., and N. caninum DNA in different tissues of A. guariba clamitans, to further knowledge regarding protozoan infection in local primates.

Materials and Methods

Samples. Tissue samples of brown howler monkeys (Alouatta guariba clamitans) were obtained from dead animals sent for necropsy at the “Núcleo de Estudos e Pesquisas em Animals Silvestres” from the “Laboratório Central de Diagnóstico de Patologias Aviárias”, “Universidade Federal de Santa Maria”. All animals came from a wild fauna maintainer located near the city Santa Maria (29°41’52.2”S 53°55’11.0”W), in the Central Region of Rio Grande do Sul State, Southern region of Brazil. The animals died at different times and there is no information about the place of birth, the time they lived in the reserve, clinical signs, or previous health conditions. Samples from the brain, heart, liver, lung, spleen, and intestine (n=35, since the brain of one animal was not provided) were collected and stored at -20°C until processed.

DNA extraction. A total of 20 to 50mg of collected tissues was macerated for DNA extraction using the Wizard Genomic DNA Purification kit (Promega, Madison/WI USA), following the instructions of the manufacturer with modification in the lysis step (carried out overnight at 55°C) according to a previous report (Moré et al. 2011Moré G., Abrahamovich P., Jurado S., Bacigalupe D., Marin J.C., Rambeaud M., Venturini L. & Venturini M.C. 2011. Prevalence of Sarcocystis spp. in Argentinean cattle. Vet. Parasitol. 177(1/2):162-165. <https://dx.doi.org/10.1016/j.vetpar.2010.11.036> <PMid:21168276>
https://doi.org/10.1016/j.vetpar.2010.11...
). Conventional polymerase chain reaction (PCR) was performed for molecular detection of DNA from each protozoan.

Molecular detection of Toxoplasma gondii.T. gondii DNA detection was performed using a 529bp genetic marker that repeats approximately 300 times in the T. gondii genome, consequently showing high sensitivity (Homan et al. 2000Homan W.L., Vercammen M., Braekeleer J. & Verschueren H. 2000. Identification of a 200- to 300-fold repetitive 529 bp DNA fragment in Toxoplasma gondii, and its use for diagnostic and quantitative PCR. Int. J. Parasitol. 30(1):69-75. <https://dx.doi.org/10.1016/s0020-7519(99)00170-8> <PMid:10675747>
https://doi.org/10.1016/s0020-7519(99)00...
). The primers used were: forward (Tox4) 5’-CGCTGCAGGGAGGAAGACGAAAGTTG-3’ and reverse (Tox5) 5’-CGCTGCAGACACAGTGCATCTGGATT-3’. The reaction was run in a final volume of 25μl mixture containing 2.5μl 10x PCR Buffer with MgCl2, 10mM dNTP, 10pmol of each primer, 0.7U JumpStart Taq DNA Polymerase (Sigma Aldrich, St. Louis/MO, USA), and 100ng DNA. DNA from T. gondii (RH strain) and ultrapure water were used as positive and negative controls, respectively. Amplification was conducted in a thermal bath with initial denaturation for 5 min at 94°C, followed by 35 cycles of 30 s at 95°C, 1 min at 55°C, 1 min at 72°C and a final extension of 5 min at 72°C. Amplification products were visualized in a transilluminator after 1.5% agarose gel electrophoresis using gel red as a DNA dye.

Toxoplasma gondii genotype was determined by restriction fragment length polymorphism (PCR-RFLP) using six genetic markers, SAG1, SAG3, BTUB, GRA6, L358, and PK1, as previously described (Su et al. 2006Su C., Zhang X. & Dubey J.P. 2006. Genotyping of Toxoplasma gondii by multilocus PCR-RFLP markers: a high resolution and simple method for identification of parasites. Int. J. Parasitol. 36(7):841-848. <https://dx.doi.org/10.1016/j.ijpara.2006.03.003> <PMid:16643922>
https://doi.org/10.1016/j.ijpara.2006.03...
, Su et al. 2010Su C., Shwab E.K., Zhou P., Zhu X.Q. & Dubey J.P. 2010. Moving towards an integrated approach to molecular detection and identification of Toxoplasma gondii. Parasitology 137(1):1-11. <https://dx.doi.org/10.1017/S0031182009991065> <PMid:19765337>
https://doi.org/10.1017/S003118200999106...
). DNA target sequences were first amplified by PCR multiplex using external primers of all markers, followed by a nested PCR using internal primers individually for each marker. The nested PCR products were then cleaved by restriction enzymes under specific temperature and time conditions. The primer sequences and enzymes are described in a previous study (Su et al. 2010Su C., Shwab E.K., Zhou P., Zhu X.Q. & Dubey J.P. 2010. Moving towards an integrated approach to molecular detection and identification of Toxoplasma gondii. Parasitology 137(1):1-11. <https://dx.doi.org/10.1017/S0031182009991065> <PMid:19765337>
https://doi.org/10.1017/S003118200999106...
). Positive controls of type I (GTI), II (PTG), and III (CTG) strains were used in the reactions. Further, samples were subjected to 2.5-3% agarose gel electrophoresis and were visualized using an ultraviolet light transilluminator. The images were compared with controls for genotype determination.

Molecular detection of Neospora caninum. PCR was performed using a primer set for specific amplification of the N. caninum Nc-5 gene: forward (Np21), 5’-CCCAGTGCGTCCAATCCTGTAAC-3’; and reverse (Np6), 5’-CTCGCCAGTCAACCTACGTCTTCT-3’ with a 328bp expected product according to Yamage et al. (1996)Yamage M., Flechtner O. & Gottstein B. 1996. Neospora caninum: specific oligonucleotide primers for the detection of brain “cyst” DNA of experimentally infected nude mice by the polymerase chain reaction (PCR). J. Parasitol. 82(2):272-279. <https://dx.doi.org/10.2307/3284160> <PMid:8604096>
https://doi.org/10.2307/3284160...
. The reaction was conducted in 25μl final volume using 0.7U JumpStart Taq DNA Polymerase (Sigma Aldrich, St. Louis/MO, USA); 2.5μl 10x PCR Buffer with MgCl2; 5pmol from each primer, 6mM dNTPs, and 50ng template DNA. For amplification of the genetic material in a thermal cycler, a protocol was performed using 35 cycles, with initial denaturation at 95°C for 5 min, denaturation at 95°C for 1 min, annealing at 50°C for 1 min, extension at 72°C for 1 min, followed by a final extension at 72°C for 10 min. Amplification products were visualized in a transilluminator after 1.5% agarose gel electrophoresis. DNA from N. caninum NC1 and ultrapure water were used as positive and negative controls, respectively.

Amplification and sequencing of apicomplexan 18S rRNA. The presence of Sarcocystis spp. and potentially other apicomplexans were detected by amplification of 18S ribosomal RNA gene (18S rRNA). Nested PCR of 18S rRNA was performed using the general primers for Apicomplexa, external primers Tg18s48F(5’-CCATGCATGTCTAAGTATAAGC-3’) and Tg18s359R (5’-GTTACCCGTCACTGCCAC-3’), and internal primers Tg18s58F (5’-CTAAGTATAAGCTTTTATACGGC-3’) and Tg18s348R (5′-TGCCACGGTAGTCCAATAC-3’) (Silva et al. 2009Silva R.C., Su C. & Langoni H. 2009. First identification of Sarcocystis tenella (Railliet, 1886) Moulé, 1886 (Protozoa: Apicomplexa) by PCR in naturally infected sheep from Brazil. Vet. Parasitol. 165(3/4):332-336. <https://dx.doi.org/10.1016/j.vetpar.2009.07.016> <PMid:19647370>
https://doi.org/10.1016/j.vetpar.2009.07...
). The PCR mix contained 25mM of external primers or 50mM of internal primers. It also contained 0.7U JumpStart Taq DNA Polymerase: 2.5μl 10x PCR Buffer with MgCl2, 10mM dNTP, and 50ng DNA. Amplifications were conducted with initial denaturation for 5 min at 94°C, followed by 35 cycles of 45 s at 94°C, 45 s at 55°C, 45 s at 72°C and a final extension of 3 min at 72°C. Amplification products were visualized in a transilluminator after 1.5% agarose gel electrophoresis. Amplification can generate distinct amplicon sizes, such as about 290bp product for Sarcocystis neurona, N. caninum, Hammondia hammondi, and T. gondii, and 310bp for other Sarcocystis spp. As this test is not species-specific, the nested PCR amplicons were subjected to sequencing for genus confirmation. The amplified DNA product was purified using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions and subjected to DNA sequencing by ACTGene - Sequencing Service (Alvorada/RS, Brazil) with internal forward and reverse primers. The sequences obtained were filtered by quality using Staden Package (Staden et al. 2000Staden R., Beal K.F. & Bonfield J.K. 2000. The Staden package, 1998. Methods Mol. Biol. 132:115-130. <https://dx.doi.org/10.1385/1-59259-192-2:115> <PMid:10547834>
https://doi.org/10.1385/1-59259-192-2:11...
), and the identity of the sequences was confirmed using blastn (Johnson et al. 2008Johnson M., Zaretskaya I., Raytselis Y., Merezhuk Y., McGinnis S. & Madden T.L. 2008. NCBI BLAST: a better web interface. Nucleic Acids Res. 36(Web Server issue):W5-W9. <https://dx.doi.org/10.1093/nar/gkn201> <PMid:18440982>
https://doi.org/10.1093/nar/gkn201...
) comparing to the Genbank nucleotide collection. Sequences identified as Sarcocystis spp. were aligned using MUSCLE (Edgar 2004Edgar R.C. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32(5):1792-1797. <https://dx.doi.org/10.1093/nar/gkh340> <PMid:15034147>
https://doi.org/10.1093/nar/gkh340...
) and the alignment was visualized in Genedoc 2.7 (Nicholas et al. 1997Nicholas K.B., Nicholas H.B.J. & Deerfield D.W.I. 1997. Genedoc: a tool for editing and annotating multiple sequence alignments. Embnewnews 4:14.).

Results and Discussion

The most common parasite found in New World primates are nematodes, followed by protozoa. Among primates, Alouatta is one of the most studied genera, although parasitological knowledge is still limited. Therefore, parasitological screenings, both by morphological and molecular analysis, are crucial to improve our understanding in this field (Solórzano-García & Pérez-Ponce de León 2018Solórzano-García B. & Pérez-Ponce de León G. 2018. Parasites of neotropical primates: a review. Int. J. Primatol. 39:155-182. <https://dx.doi.org/10.1007/s10764-018-0031-0>
https://doi.org/10.1007/s10764-018-0031-...
). In this study, we performed molecular detection of three protozoan genera in six different tissues from six brown howler monkeys. By macroscopic analysis during necropsy no significant lesions were found, so it was not possible to define the causes of the death of the animals. However, in all animals, DNA was detected in at least one of the protozoa, indicating that the brown howler monkeys in the region served as parasite reservoirs. The PCR results are shown in Table 1, and the genetic sequencing analysis is shown in Table 2.

Table 1.
Results of PCR for detection of Toxoplasma gondii marker and 18S rRNA gene in different tissues of six specimens of brown howler monkey (Alouatta guariba clamitans)
Table 2.
Result of blastn searches of sequenced samples of six specimens of brown howler monkey (Alouatta guariba clamitans) against the GenBank

Neospora caninum DNA was not detected in any of the samples tested in this study. There is little information in the literature regarding the occurrence and importance of N. caninum in primates. However, experimental infection in non-human primates by Neospora has been confirmed (Barr et al. 1994Barr B.C., Conrad P.A., Sverlow K.W., Tarantal A.F & Hendrickx A.G. 1994. Experimental fetal and transplacental Neospora infection in the nonhuman primate. Lab. Invest. 71(2):236-242. <PMid:8078303>, Ho et al. 1997Ho M.S., Barr B.C., Tarantal A.F., Lai L.T., Hendrickx A.G., Marsh A.E., Sverlow K.W., Packham A.E. & Conrad P.A. 1997. Detection of Neospora from tissues of experimentally infected rhesus macaques by PCR and specific DNA probe hybridization. J. Clin. Microbiol. 35(7):1740-1745. <https://dx.doi.org/10.1128/jcm.35.7.1740-1745.1997> <PMid:9196184>
https://doi.org/10.1128/jcm.35.7.1740-17...
), and a case of natural infection in Night Monkey (Aotus azarae infulatus) has been reported (Costa et al. 2018Costa T.L.C., Iglesias G.A., Rosa J.M.A., Bento H.J., Rondelli L.A.S., Furlan F., Morgado T.O., Dutra V. & Corrêa S.H.R. 2018. Detecção molecular de Neospora caninum em macaco-da-noite (Aotus azarae) de vida livre no estado do Mato Grosso: relato de caso. Arq. Bras. Med. Vet. Zootec. 70(4):1227-1232. <https://dx.doi.org/10.1590/1678-4162-9900>
https://doi.org/10.1590/1678-4162-9900...
). The differential diagnosis is relevant because of the similarity of tachyzoites, the close phylogenetic relationship between T. gondii and N. caninum (Dubey & Lindsay 1996Dubey J.P. & Lindsay D.S. 1996. A review of Neospora caninum and neosporosis. Vet. Parasitol. 67(1/2):1-59. <https://dx.doi.org/10.1016/s0304-4017(96)01035-7> <PMid:9011014>
https://doi.org/10.1016/s0304-4017(96)01...
), and the lack of information of the consequences of neosporosis in primates.

Toxoplasma gondii DNA was found in one animal (Animal 4) with the specific primers and in two (Animals 4 and 5) with the 18S rRNA marker. The identity of T. gondii sequences was confirmed using blastn. Although both genes are repeated in the genome, differences in amplification sensitivity among primers are expected if a low amount of parasite DNA is present in the sample (Jones et al. 2000Jones C.D., Okhravi N., Adamson P., Tasker S. & Lightman S. 2000. Comparison of PCR detection methods for B1, P30, and 18S rDNA genes of T. gondii in aqueous humor. Investig. Ophthalmol. Vis. Sci. 41(3):634-644. <PMid:10711675>, Cresti et al. 2001Cresti S., Ciacci C., Donati E., Giordano I., Tordini G. & Barberi A. 2001. Evaluation of PCR methods for 5S-rDNA and p30 genes to detect Toxoplasma gondii in blood and other clinical samples. New Microbiol. 24(2):171-174. <PMid:11346301>).

Toxoplasma gondii is widespread in the world, affecting humans and many warm-blooded animals (Dubey 1986Dubey J.P. 1986. Toxoplasmosis. J. Am. Vet. Med. Assoc. 189(2):166-170. <PMid:3528098>). In this study, this parasite was found in two brown howler monkeys. New World Monkeys, such as howler monkeys, are very susceptible to toxoplasmosis (Nery-Guimarães & Franken 1971Nery-Guimarães F. & Franken A.J. 1971. Toxoplasmose em primatas não humanos: II - Tentativas de infecções experimentais em Macacca mulatta, Cebus apella e Callithrix jacchus; e pesquisa de anticorpos em várias espécies de platyrrhinus. Mem. Inst. Oswaldo Cruz. 69(2):97-111. <https://dx.doi.org/10.1590/S0074-02761971000200005> <PMid:5002530>
https://doi.org/10.1590/S0074-0276197100...
, Molina et al. 2014Molina C.V., Catão-Dias J.L., Ferreira Neto J.S., Vasconcellos S.A., Gennari S.M., Valle R.D., Souza G.O., Morais Z.M., Vitaliano S.N., Strefezzi R.F. & Bueno M.G. 2014. Sero-epidemiological survey for brucellosis, leptospirosis, and toxoplasmosis in free-ranging Alouatta caraya and Callithrix penicillata from São Paulo State, Brazil. J. Med. Primatol. 43(3):197-201. <https://dx.doi.org/10.1111/jmp.12112> <PMid:24646216>
https://doi.org/10.1111/jmp.12112...
, Santos et al. 2014Santos S.V., Strefezzi R.F., Pissinatti A., Kanamura C.T., Takakura C.F.H., Duarte M.I.S. & Catao-Dias J.L. 2014. Detection of Toxoplasma gondii in two southern Wooly spider monkeys (Brachyteles arachnoides Geoffroy, 1806) from the Rio de Janeiro primate center, Brazil. J. Med. Primatol. 43(2):125-129. <https://dx.doi.org/10.1111/jmp.12093> <PMid:24757733>
https://doi.org/10.1111/jmp.12093...
), and the disease is often acute and fatal (Epiphanio et al. 2003Epiphanio S., Sinhorini I.L. & Catão-Dias J.L. 2003 Pathology of toxoplasmosis in captive new world primates. J. Comp. Pathol. 129(2/3):196-204. <https://dx.doi.org/10.1016/s0021-9975(03)00035-5> <PMid:12921726>
https://doi.org/10.1016/s0021-9975(03)00...
). The presence of infected herbivores indicates environmental contamination by oocysts. In the natural environment, wild felids (definitive hosts) cohabit with other animals and may spread T. gondii to the wild population (Dubey & Jones 2008Dubey J.P. & Jones J.L. 2008. Toxoplasma gondii infection in humans and animals in the United States. Int. J. Parasitol. 38(11):1257-1278. <https://dx.doi.org/10.1016/j.ijpara.2008.03.007> <PMid:18508057>
https://doi.org/10.1016/j.ijpara.2008.03...
).

Toxoplasma gondii genotypic analysis of Animal 4 suggested an atypical genotype because different markers indicated distinct genotypes: SAG1 = type 1; SAG3 = type 1; PK1 = type 1; BTUB = type 1; GRA6 = type 1; L358 = type 3. This finding is consistent with previous studies that show a large circulation of atypical genotypes in Brazil, different from those found in North America and Europe (Pena et al. 2008Pena H.F.J, Gennari S.M., Dubey J.P. & Su C. 2008. Population structure and mouse-virulence of Toxoplasma gondii in Brazil. Int. J. Parasitol. 38(5):561-569. <https://dx.doi.org/10.1016/j.ijpara.2007.09.004> <PMid:17963770>
https://doi.org/10.1016/j.ijpara.2007.09...
, Dubey & Su 2009Dubey J.P. & Su C. 2009. Population biology of Toxoplasma gondii: what’s out and where did they come from. Mem. Inst. Oswaldo Cruz 104(2):190-195. <https://dx.doi.org/10.1590/s0074-02762009000200011> <PMid:19430643>
https://doi.org/10.1590/s0074-0276200900...
, Shwab et al. 2014Shwab E., Zhu X-Q., Majumdar D., Pena H.F.J., Gennari S.M., Dubey J.P. & Su C. 2014. Geographical patterns of Toxoplasma gondii genetic diversity revealed by multilocus PCR-RFLP genotyping. Parasitology 141(4):453-461. <https://dx.doi.org/10.1017/S0031182013001844> <PMid:24477076>
https://doi.org/10.1017/S003118201300184...
, Silva et al. 2014Silva L.A., Andrade R.O., Carneiro A.C.A.V. & Vitor R.W.A. 2014. Overlapping Toxoplasma gondii genotypes circulating in domestic animals and humans in Southeastern Brazil. PLoS One 9(2):e90237. <https://dx.doi.org/10.1371/journal.pone.0090237> <PMid:24587295>
https://doi.org/10.1371/journal.pone.009...
). Recently, fatal toxoplasmosis cases caused by atypical T. gondii, in valley quail (Callipepla californica) (Casagrande et al. 2015Casagrande R.A., Pena H.F.J., Cabral A.D., Rolim V.M., Oliveira L.G.S., Boabaid F.M., Wouters A.T.B., Wouters F., Cruz C.E.F. & Driemeier D. 2015. Fatal systemic toxoplasmosis in Valley quail (Callipepla californica). Int. J. Parasitol. Parasites Wildl. 4(2):264-267. <https://dx.doi.org/10.1016/j.ijppaw.2015.04.003> <PMid:26101744>
https://doi.org/10.1016/j.ijppaw.2015.04...
) and an outbreak in chickens (Vielmo et al. 2019Vielmo A., Pena H.F.J., Panziera W., Bianchi R.M., Lorenzo C., Oliveira S., Alves B.F., Gennari S.M., Pavarini S.P., Barros C.S.L. & Driemeier D. 2019. Outbreak of toxoplasmosis in a flock of domestic chickens (Gallus gallus domesticus) and guinea fowl (Numida meleagris). Parasitol. Res. 118(3):991-997. <https://dx.doi.org/10.1007/s00436-019-06233-w> <PMid:30725180>
https://doi.org/10.1007/s00436-019-06233...
) have been reported in Rio Grande do Sul (Brazil). An atypical genotype also seems to be responsible for the recent largest Brazilian outbreak of human toxoplasmosis in Santa Maria city (Minuzzi et al. 2020Minuzzi C.E., Portella L.P., Bräunig P., Sangioni L.A., Ludwig A., Ramos L.S., Pacheco L., Silva C.R., Pacheco F.C., Menegolla I.A., Farinha L.B., Kist P.P., Breganó R.M., Nino B.S.L., Martins F.D.C., Monica T.C., Ferreira F.P., Britto I., Signori A., Medici K.C., Freire R.L., Garcia J.L., Navarro I.T., Difante C.M. & Vogel F.S.F. 2020. Isolation and molecular characterization of Toxoplasma gondii from placental tissues of pregnant women who received toxoplasmosis treatment during an outbreak in southern Brazil. PLoS One 15(1):e0228442. <https://dx.doi.org/10.1371/journal.pone.0228442> <PMid:31999785>
https://doi.org/10.1371/journal.pone.022...
). Moreover, atypical genotypes have been reported in primates in Argentina (Pardini et al. 2015Pardini L., Dellarupe A., Bacigalupe D., Quiroga M.A., Moré G., Rambeaud M., Basso W., Unzaga J.M., Schares G. & Venturini M.C. 2015. Isolation and molecular characterization of Toxoplasma gondii in a colony of captive black-capped squirrel monkeys (Saimiri boliviensis). Parasitol. Int. 64(6):587-590. <https://dx.doi.org/10.1016/j.parint.2015.08.009> <PMid:26299363>
https://doi.org/10.1016/j.parint.2015.08...
).

Sarcocystis spp. DNA was detected in four animals, as confirmed by blastn of the amplified fragment of the 18S rRNA gene. At least two different species of Sarcocystis were detected. Sequencing results from Animals 1, 2, and 3 showed 99.4% similarity with S. gigantia and Animal 6 showed 99.3% similarity with S. neurona (Table 2). The alignment of the sequences clearly indicated the difference in size and composition sequence of the Animal 6 sequence and the others (76% identity) (Fig.1). The 18S rRNA was previously confirmed as a powerful tool for species-specific differentiation of the ovine Sarcocystis species (Heckeroth & Tenter 1999Heckeroth A.R. & Tenter A.M. 1999. Development and validation of species-specific nested PCRs for diagnosis of acute sarcocystiosis in sheep. Int. J. Parasitol. 29(8):1331-1349. <https://dx.doi.org/10.1016/s0020-7519(99)00111-3> <PMid:10576582>
https://doi.org/10.1016/s0020-7519(99)00...
). Thus, we can state the presence of S. gigantia and S. neurona or very closely related species infecting A. guariba clamitans.

Fig.1.
Alignment of Sarcocystis spp. sequences amplified from different tissue samples of brown howler monkey (Alouatta guariba clamitans) visualized in Genedoc 2.7. The order of samples is: Animal 6/ brain, Animal 1/brain, Animal 2/heart, and Animal 3/brain. Every 10 positions in alignment are shown by the number or * and black shade indicates identical nucleotides in all sequences. Polymorphic positions were identified in the sequences from Animals 1 and 2 (position 90, R = A or G; position 129, R = A or G) and Animal 3 (position 90, R = A or G; position 112, Y = C or T).

Sarcocystis spp. have already been detected in many wildlife species, and primates may serve as both intermediate and definitive hosts, depending on the species of Sarcocystis. Several studies have reported Sarcocystis spp. cysts found in primates (Hernández-Jáuregui et al. 1983Hernández-Jáuregui P., Silva-Lemoine E. & Girón-Rojas H. 1983. A case of sarcocystic myocarditis in a rhesus monkey. Light and electron microscopic study. Arch. Invest. Med., Mexico, 14(2):139-144. <PMid:6416199>, Mandour 1969Mandour A.M. 1969. Sarcocystis nesbitti n. sp. from the rhesus monkey. J. Protozool. 16(2):353-254. <https://dx.doi.org/10.1111/j.1550-7408.1969.tb02281.x> <PMid:4978784>
https://doi.org/10.1111/j.1550-7408.1969...
, Karr & Wong 1975Karr S.L. & Wong M.M. 1975. A survey of Sarcocystis in nonhuman primates. Lab. Anim. Sci. 25(5):641-645. <PMid:810620>, Mehlhorn & Heydorn 1978Mehlhorn H. & Heydorn A.O. 1978. The sarcosporidia (Protozoa, Sporozoa): life cycle and fine structure. Adv. Parasitol. 16:43-91. <https://dx.doi.org/10.1016/s0065-308x(08)60572-2> <PMid:103377>
https://doi.org/10.1016/s0065-308x(08)60...
, Kan et al. 1979Kan S.P., Prathap K. & Dissanaike A.S. 1979. Light and electron microstructure of a Sarcocystis sp. from the Malaysian long-tailed monkey, Macaca fascicularis. Am. J. Trop. Med. Hyg. 28(4):634-642. <PMid:111569>, Kimura et al. 1987Kimura T., Ito J., Suzuki M. & Inokuchi S. 1987. Sarcocystis found in the skeletal muscle of common squirrel monkeys. Primates 28:247-255. <https://dx.doi.org/10.1007/BF02382574>
https://doi.org/10.1007/BF02382574...
, Lane et al. 1998Lane J.H., Mansfield K.G., Jackson L.R., Diters R.W., Lin K.C., MacKey J.J. & Sasseville V.G. 1998. Acute fulminant sarcocystosis in a captive-born rhesus macaque. Vet. Pathol. 35(6):499-505. <https://dx.doi.org/10.1177/030098589803500604> <PMid:9823591>
https://doi.org/10.1177/0300985898035006...
, Yang et al. 2005Yang Z-.Q., Wei C-.G., Zen J-S., Song J-.L., Zuo Y-.X., He Y-.S., Zhang H-.F., Attwood S.W., Chen X-.W., Yang G-.C., Zhou X., Quan X., Li C-.Y., Han D., Liu A-.W. & Lin P. 2005. A taxonomic re-appraisal of Sarcocystis nesbitti (Protozoa: Sarcocystidae) from the monkey Macaca fascicularis in Yunnan, PR China. Parasitol. Int. 54(1):75-81. <https://dx.doi.org/10.1016/j.parint.2004.12.004> <PMid:15710555>
https://doi.org/10.1016/j.parint.2004.12...
) however, to the best of our knowledge, this is the first report of Sarcocystis spp. infection in primates in Brazil.

Felids are the definitive hosts of S. gigantea, and the known intermediate hosts are small ruminants (Levine & Tadros 1980Levine N.D. & Tadros W. 1980. Named species and hosts of Sarcocystis (Protozoa: Apicomplexa: Sarcocystidae). Syst. Parasitol. 2:41-59. <https://dx.doi.org/10.1007/BF00015094>
https://doi.org/10.1007/BF00015094...
); therefore, this is the first report of molecular detection of this species in primates. Macroscopic cysts of S. gigantea were previously detected in sheep in southern Brazil (Minuzzi et al. 2019Minuzzi C.E., Cezar A.S., Bräunig P., Portella L.P., Rodrigues F.S., Sangioni L.A. & Vogel F.S.F. 2019. Occurrence of Sarcocystis gigantea macrocysts and high frequency of S. tenella microcysts in sheep from southern Brazil. Vet. Parasitol. Reg. Stud. Rep. 15:100256. <https://dx.doi.org/10.1016/j.vprsr.2018.12.002> <PMid:30929933>
https://doi.org/10.1016/j.vprsr.2018.12....
) confirming the circulation of this protozoan in the region. In our study, S. gigantea DNA was detected in three animals but no injuries or macrocysts were found during necropsy.

Molecular analysis confirmed S. neurona in one animal. Didelphis sp. opossums are the definitive hosts of S. neurona, and several intermediate and/or accidental hosts are known, such as domestic cats, raccoons, seals, otters, armadillo and horses (Elsheikha 2009Elsheikha H.M. 2009. Has Sarcocystis neurona Dubey et al., 1991 (Sporozoa: Apicomplexa: Sarcocystidae) cospeciated with its intermediate hosts? Vet Parasitol. 163(6):307-314. <https://dx.doi.org/10.1016/j.vetpar.2009.03.019> <PMid:19375231>
https://doi.org/10.1016/j.vetpar.2009.03...
). In horses, it causes myeloencephalitis; this condition has also been reported in a rhesus monkey (Klumpp et al. 1994Klumpp S.A., Anderson D.C., McClure H.M. & Dubey J.P. 1994. Encephalomyelitis due to a Sarcocystis neurona-like protozoan in a rhesus monkey (Macaca mulatta) infected with simian immunodeficiency virus. Am. J. Trop. Med. Hyg. 51(3):332-338. <https://dx.doi.org/10.4269/ajtmh.1994.51.332> <PMid:7943553>
https://doi.org/10.4269/ajtmh.1994.51.33...
). Positive serology for S. neurona has also been reported in other primates (Yabsley et al. 2007Yabsley M.J., Jordan C.N., Mitchell S.M., Norton T.M. & Lindsay D.S. 2007. Seroprevalence of Toxoplasma gondii, Sarcocystis neurona, and Encephalitozoon cuniculi in three species of lemurs from St. Catherines Island, GA, USA. Vet. Parasitol. 144(1/2):28-32. <https://dx.doi.org/10.1016/j.vetpar.2006.09.020> <PMid:17052854>
https://doi.org/10.1016/j.vetpar.2006.09...
).

Alouatta spp. are herbivores, their diet is basically composed of leaves, fruits, and flowers (Bicca-Marques & Calegaro-Marques 1994Bicca-Marques J.C. & Calegaro-Marques C. 1994. Feeding behavior of the black howler monkey (Alouatta caraya) in a seminatural forest. Acta Biol. Leopoldensia 16(2):69-84.); thus, they become infected by ingesting oocysts present in water or food contaminated with definitive host feces. Additionally, geophagy has already been reported in these animals (Bicca-Marques & Calegaro-Marques 1994Bicca-Marques J.C. & Calegaro-Marques C. 1994. Feeding behavior of the black howler monkey (Alouatta caraya) in a seminatural forest. Acta Biol. Leopoldensia 16(2):69-84.), and this habit can increase the contact of animals with feces contaminated with oocysts. It is important to note that the animals from this study lived in a wild fauna maintainer, in which there were also several other species of animals, including wild felids. This environment might have provided the source of infection for brown howler monkeys.

The natural habitat of howler monkeys is fragmented and as cities have expanded, contact between these animals and humans has increased which contributes to the transmission of many diseases (Wolfe et al. 1998Wolfe N.D., Escalante A.A., Karesh W.B., Kilbourn A., Spielman A. & Lal A.A. 1998. Wild primate populations in emerging infectious disease research: the missing link? Emerg. Infect. Dis. 4(2):149-158. <https://dx.doi.org/10.3201/eid0402.980202> <PMid:9621185>
https://doi.org/10.3201/eid0402.980202...
). Toxoplasmosis and sarcocystosis are zoonoses and our results suggest that brown howler monkeys in this region may serve as reservoirs for Apicomplexa. Therefore, studies focused on the presence and consequences of these protozoa in primates are important to understand the role of brown howler monkeys in the epidemiology of T. gondii, N. caninum and Sarcocystis spp.

Conclusions

Our analyses detected the presence of Sarcocystis spp. and Toxoplasma gondii DNA in samples of brown howler monkeys from the Central Region of Rio Grande do Sul. Even with the small sample size and the lack of information about the animal’s history, our findings are relevant because they confirm the role of brown howler monkeys as hosts of protozoa with zoonotic potential and reinforce the circulation of atypical T. gondii genotypes in southern Brazil.

Further studies should be conducted to confirm the epidemiological role of Alouatta guariba clamitans in the life cycle of these protozoans.

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Publication Dates

  • Publication in this collection
    17 May 2021
  • Date of issue
    2021

History

  • Received
    27 Oct 2020
  • Accepted
    09 Dec 2020
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