Skip to main content
NCBI home page
PLOS One logoLink to PLOS One
. 2021 Aug 16;16(8):e0256199. doi: 10.1371/journal.pone.0256199

Detection of Cryptosporidium spp. and Giardia duodenalis in small wild mammals in northeastern Brazil

Hllytchaikra Ferraz Fehlberg 1,¤,*,#, Cássia Matos Ribeiro 1,#, Pedro de Alcântara Brito Junior 1, Bruno César Miranda Oliveira 2, Camila Albano dos Santos 1, Martín Roberto del Valle Alvarez 3, Tatiane Vitor Harvey 1, George Rêgo Albuquerque 1
Editor: Maria Stefania Latrofa4
PMCID: PMC8366977  PMID: 34398925

Abstract

This study investigated the occurrence of Giardia duodenalis and Cryptosporidium spp. in rodents and marsupials from the Atlantic Forest in southern Bahia, northeastern Brazil. Two hundred and four fecal samples were collected from different forest areas in the municipalities of Ilhéus, Una, Belmonte, and Mascote. Identifications were performed using PCR and nested PCR followed by sequencing of the gdh and tpi genes for G. duodenalis, and the gp60 and Hsp-70 genes for Cryptosporidium. The total frequency of positive PCR samples for both G. duodenalis and Cryptosporidium spp. was 5.4% (11/204). Giardia duodenalis occurred in 2.94% (4/136) of rodents and 2.94% (2/68) of marsupials. The prevalence of Cryptosporidium in rodents and marsupials was 1.47% (2/136) and 4.41% (3/68), respectively. In the areas sampled, the frequency of parasitism was 50% (7/14), while the Mascote region alone had no parasitized animals. The G. duodenalis subgenotype AI was identified in the rodent species Hylaeamys laticeps, Oecomys catherinae, Oligoryzomys nigripes and Akodon cursor, and in the marsupials Gracilinanus agilis and Monodelphis americana. In the rodents Rhipidomys mastacalis, H. laticeps and in the marsupial Marmosa murina the protozoa Cryptosporidium fayeri, Cryptosporidium parvum and Cryptosporidium ubiquitum with subtypes IIa and IVg by the gp60 gene were found. In conclusion, this study provides the genetic characterization of Giardia and Cryptosporidium species and genotypes in rodents and marsupials. And, these findings reinforce that the rodent and marsupial species mentioned above play a role as new hosts for Giardia and Cryptosporidium.

Introduction

Small mammals such as rodents (Rodentia, Cricetidae) and marsupials (Mammalia, Didelphimorphia) transmit pathogens to humans and domestic animals; however, the consequent risk to public health is poorly understood [1,2]. Environmental disruption due to human activity influences the occurrence and spread of zoonotic and parasitic diseases (e.g., giardiasis and cryptosporidiosis) in these animals, affecting the wildlife species balance [3].

Giardia Kunstler, 1882 and Cryptosporidium Tizzer, 1907 are protozoa known worldwide for causing severe gastroenteric disease in humans, as well as domestic and wild animals [2,4,5]. These protozoa cause infections from cysts or oocysts found in environmental and water contaminations [4,6].

The role of wild animals in human giardiasis and cryptosporidiosis epidemiology is uncertain. However, molecular studies have allowed the identification of several species of Giardia and Cryptosporidium in wild animals [6,7–9].

Molecular techniques have successfully determined and supported the understanding of epidemiological processes [9] by using several genes to identify distinct species of Giardia and Cryptosporidium. Additionally, they reveal genotypes and subgenotypes, of which some are specific to humans and others to animals [6].

To determine Cryptosporidium spp. genotypes and subgenotypes, coding genes stand out as small subunit 18S ribosomal rRNA (SSu-rRNA) [10]. Both gp60 and Hsp-70 demonstrate a high polymorphism in different species [11,12]. In addition, wall-protein coding genes (COWPs), actin, acetyl-CoA synthetase, and internal space transcribed from rDNA (rDNA ITS 1) are also used [13,14].

To detect the genotype and subgenotype of the Giardia duodenalis species, genes of SSu-rRNA [15,16], glutamate dehydrogenase (gdh), triose-phosphate isomerase (tpi), and beta-giardin (bg) coding genes are used [16–18].

Molecular studies to detect Giardia and Cryptosporidium in wildlife reported the presence of these protozoa in different species of small mammals. However, in northeastern Brazil, no studies have employed molecular genotyping to identify G. duodenalis and Cryptosporidium spp. Thus, the objective of this study was to identify, through a molecular technique at the level of genotypes and subgenotypes, G. duodenalis and Cryptosporidium spp. in fecal samples of rodents and marsupials captured in agroforestry areas (Cabruca) and the Atlantic Forest in southern Bahia, northeastern Brazil.

Material and methods

Collection area

Within the study area, 14 forest areas, distributed in four municipalities in the southern region of the State of Bahia, were sampled. These included three cocoa agroforestry areas located in the rural area of Ilhéus (areas 1–3), and 11 forest areas located in the municipalities of Una, Mascote and Belmonte (areas 4–14) (Fig 1). The study region is characterized by a hot and humid tropical climate, with an average relative humidity of 89–90% and an average temperature of 24–25°C, predominantly covered by tropical forest vegetation and an agroforestry system, which preserves native forest [19]. In the region, it rains 150 days a year on average, with precipitation reaching 2,000 mm/year. The dry seasons are not well defined; occasionally, one to three months receive less than 100 mm of rain [20]. Elevation of the sampled areas ranged from 42–100 m above sea level and were georeferenced with a Global Positioning System (GPS).

Fig 1. Map depicting the capture and collection areas, of fecal samples from rodents and marsupials in southern Bahia, northeastern Brazil.

Fig 1

Open in a new tab

Geographic coordinates of the collection points. 01: 14°38’15.8”S39°12’02.3”W; 02: 14°42’11.2”S 39°15’34.8”W; 03: 14°45’04.0”S 39°11’51.2”W; 04: 15°09’57.8”S 39°13’10.1”W; 05: 15°12’35.9”S 39°08’37.4”W; 06: 15°14’53.1”S 39°09’34.3”W; 07: 15°16’54.5”S 39°10’54.2”W; 08: 15°14’59.0”S 39°04’41.0”W; 09: 15°20’53.0”S 39°02’43.5”W; 10: 15°42’53.6”S 39°21’52.6”W; 11: 15°43’40.9”S 39°22’56.7”W; 12: 15°48’01.9”S 39°30’23.8”W; 13: 15°53’40.4”S 39°14’19.2”W; 14: 15°54’03.0”S 39°13’40.4”W.

Capturing animals and obtaining biological material

The capture period ranged from June 2015 to December 2016. The animals were captured using Sherman (23 × 8 × 9 cm), Tomahawk (50 × 17 × 17 cm), and pitfall traps. Each area was divided into three plots, with for a total of 24 traps per plot and 72 traps per area. The study was approved by the Biodiversity Authorization and Information System (SISBIO) under number 17131–4 from the Brazilian Institute for the Environment and Renewable Natural Resources (IBAMA) and by the Council for the Ethical Use of Animals of the State University of Santa Cruz (CEUA-UESC; Case No. 003/2013).

After identification of the species, fecal samples were collected with subsequent release of the animals at the places of origin (Table 1). Fecal samples were stored in 1.5 mL microtubes, kept refrigerated and delivered to Laboratory of Veterinary Parasitology of the State University of Santa Cruz (LAPVET-UESC), weighed, and standardized between 180 and 200 mg.

Table 1. Species of marsupials and wild rodents captured in the Atlantic Forest and Cabruca areas in southern Bahia, northeastern Brazil, and positivity of infected animals.

Area N*/Positives Molecular diagnosis (Nested/PCR)
ORDER DIDELPHIMORPHIA
Family Didelphidae Cryptosporidium Giardia
Marmosa murina (Linnaeus, 1758)
3;4;6;7;8;9;10;11;12;13;14 26/3 3 0
Marmosa incanus (Lund, 1840) 11; 13 7/0 0 0
Marmosa demerarai (Thomas, 1905) 4;7;8 9/0 0 0
Monodelphis americana (Müller, 1776) 3;4;14 8/1 0 1
Gracilinanus agilis (Burmeister, 1854) 12;14 10/1 0 1
Didelphis aurita (Wied-Neuwied, 1826) 7;8 8/0 0 0
TOTAL 68/5 3 2
ORDER RODENTIA
Family Cricetidae
Hylaeamys laticeps (Lund, 1840)
1;2;3;4;5;8 81/2 1 1
Akodon cursor (Winge, 1887) 1;2;3;11;14 13/1 0 1
Rhipidomys mastacalis (Lund, 1840) 1;2;3;5;8;12;13 11/1 1 0
Thaptomys nigrita (Lichtenstein, 1829) 1;5;8;13;14 9/0 0 0
Oecomys catherinae (Thomas, 1909) 5;7;8;13 5/1 0 1
Calomys expulsus (Lund, 1841) 12 2/0 0 0
Cerradomys subflavus (Percequillo et al., 2008) 1;2;11 4/0 0 0
Oligoryzomys nigripes (Olfers, 1818) 1;2;5;7;8;12 7/1 0 1
Euryoryzomys russatus (Wagner, 1848) 1;3;13 4/0 0 0
TOTAL 136/6 2 4
GRAND TOTAL 204/11 5 6
Open in a new tab

DNA extraction and molecular characterization

The fecal samples were washed with sterile PBS (pH 7.2) and subjected to genomic DNA extraction using the QIAamp DNA Stool Mini kit® (Qiagen), according to manufacturer’s instructions. After adding the lysis buffer, the samples were subjected to five cycles of heating (96°C) and freezing (-196°C), with 3 minutes of heating and 5 minutes of freezing, then homogenized in a vortex for 5 minutes with 0.2 g of glass beads (0.5 mm), following the kit’s guidelines thereafter. The amount of extracted genomic DNA was established using a NanoDrop 2000 (Thermo Scientific, USA), stored in boxes, and placed in a freezer at -20°C.

To detect the presence of G. duodenalis and Cryptosporidium spp., each isolated DNA sample was subjected to nested PCR. For the amplification of Giardia fragments, gdh [16] and tpi coding genes [17] were used. Cryptosporidium fragments were amplified using gp60 [12] and Hsp-70 [11] genes (Table 1).

The tests were carried out in a Proflex PCR system thermocycler (Applied Biosystems) using the Platinum Taq DNA polymerase kit (Invitrogen) for the mix. Positive fecal samples from Giardia cysts and isolates from the Veterinary Parasitology Laboratory at UESC were used as positive controls. Cryptosporidium (isolates 13F and 13C) from the Laboratory of Clinical Analysis (LAC) of the State University of Feira de Santana, Bahia [21] and ultrapure water were used as negative controls. The PCR products were subjected to 1% agarose gel electrophoresis, developed with SYBR® Safe, purified using the PureLink PCR Purification kit (Invitrogen), and sent for sequencing.

Sequencing was performed using capillary electrophoresis (modified Sanger sequencing) on the ABI 3500XL Genetic Analyzer platform (Applied Biosystems) in both directions. Chromatogram analysis was performed using the FinchTV 1.4.0 software. Amplicons were Sanger-sequenced in both directions. DNA sequences were deposited in GenBank under accession numbers MW202351, MW202352, MW202353, MW202354, MW202355, MW202356, MW202357, MW202358, MW202359, MW202360, MW202361, MW202362, MW202363, MW202364, MW202365, MW202366 and MW202367.

Statistical analysis

To verify the association between the positivity of the samples with the catch area (agroforestry and forest areas), statistical analysis was performed using Fisher’s exact test with 95% confidence intervals using the Epi Info ™ 7.2.0.1 software.

Results

Out of 204 fecal samples collected, 5.4% (11/204) tested positive (Table 1). The occurrence of G. duodenalis was 2.94% (6/204) for rodents 2.94% (4/136), and marsupials 2.94% (2/68) (Table 2). For Cryptosporidium, the combined positivity was 2.45% (5/204), with 1.47% (2/136) and 4.41% (3/68) for rodents and marsupials, respectively (Table 3). In the collection areas, the frequency of parasitism was 50% (7/14) and there were no parasitized animals in the municipality of Mascote (Fig 1). The agroforestry areas had the highest frequency of infected animals, although the differences between the positivity in capture areas were not statistically significant (p> 0.05).

Table 2. Species of Giardia per parasitized host caught in forest and Cabruca areas in southern Bahia, northeastern Brazil.

Hosts PCR marker Subgenotypes
Species Order TPI GDH
Gracilinanus agilis Didelmorphia Gd Gd AI*
Monodelphis americana Didelmorphia Gd Gd AI
Oecomys catherinae Rodentia Gd Gd AI
Oligoryzomys nigripes Rodentia Gd Gd AI
Hylaeamys laticeps Rodentia Gd Gd AI
Akodon cursor Rodentia Gd Gd AI
Open in a new tab

Abbreviations: Gd: Giardia duodenalis.

*Subgenotype.

Table 3. Species of Cryptosporidium per parasitized host caught in forest and Cabruca area in southern Bahia, northeastern Brazil.

Hosts PCR marker Gp60 subgenotype family
Species Order HSP-70 Gp60
Marmosa murina Didelmorphia Cp Cp IIa*
M. murina Didelmorphia Cr Cf IVg*
M. murina Didelmorphia Cr Cp IIa
Rhipidomis mastacalis Rodentia Cp Cp IIa
Hylaeamys laticeps Rodentia Cu
Open in a new tab

Abbreviations: Cp: Cryptosporidium parvum; Cf: Cryptosporidium fayeri; Cr: Cryptosporidium sp.; Cu: Cryptosporidium ubiquitum.

* Subgenotype.

The analysis of the tpi and gdh gene sequences demonstrated 100% genetic similarity with the G. duodenalis species of the subgenotype AI (Table 2). The genetic analysis of Cryptosporidium identified C. parvum, C. ubiquitum, and C. fayeri, and subtypes that belong to the IIa and IVg allelic families. No subtype found for C. ubiquitum (Table 3).

Discussion

The present study investigated, for the first time, the presence of the protozoa Giardia and Cryptosporidium in rodents and marsupials captured in the northeast region of Brazil. The southern region of Bahia includes an extensive area of the Atlantic Forest with a richness of fauna and flora species, being an important area for the conservation of global biodiversity [20]. In addition to having areas of cocoa agroforestry, providing shade for planting and preserving native forests [22].

Giardia duodenalis infection has been described in wild animals, such as rodents and marsupials, with a prevalence ranging from 2% to 12% [3,23–27]. This defines a low prevalence in forest areas, compared to that in urban areas with rodents having a higher prevalence ranging from 24.4% to 64.3% [2,23,28]. In the present study, the frequency of positive animals was 5.4%, and such low positivity may be related to the sampling site, which has rich and abundant flora, low anthropization, and the presence of some arboreal animal species, such as G. agilis and O. catherinae, which have herbivorous and insectivorous diet, respectively [26,29,30] reducing contact with the pathogen.

The subgenotype AI found in this study is commonly found in humans [31], which characterizes these animals as participants in the epidemiology of human Giardia infection [25]. Vermeulen et al. [25], Caccio and Ryan [32], Karim et al. [33], and Garcia et al. [34] identified the same subgenotype in the gdh and tpi genes in animals. Marsupials and rodents, especially those which are terrestrial, such as the marsupials M. murina and M. americana, and the rodents O. nigripes, H. laticeps, A. cursor, and R. mastacalis, become infected through contaminated water, food, and fomites, thus playing an important role in the evolution of this protozoan [29]. Additionally, this brings the parasite into contact with humans, presenting a risk to public health [31,35].

The gdh and tpi genes demonstrated good sensitivity, allowing the generated sequences to identify the G. duodenalis species and the subgenotype AI in the six isolates. Because it has conserved regions, characterization of these genes can identify all genotypes and subgenotypes of G. duodenalis [36–38].

The Cryptosporidium frequency was 1.47% and 4.41% in rodents and marsupials, respectively, similar to that described by Santos [24]. The literature describes this protozoan infecting a variety of small mammal species [3,24,39–44]. Studies in urban areas also show a greater degree of parasitism of this protozoan in synanthropic rodents [2,28,41,42]. The presence of this protozoan may be associated with anthropic action and the presence of domestic animals provides an interaction between humans and wild fauna, favoring its dissemination [45].

Cryptosporidium parvum is responsible for the majority of human enteric infections worldwide [44]. The subgenotype IIa obtained in this study is frequently found in humans and animals [43,44,46–48]. Cryptosporidium fayeri is common in marsupial species [40,44,49,50] despite has also been identified in humans [44,51,52]. Its pathogenicity is unknown, but it often causes asymptomatic infections in marsupials [40]. The subgenotype IVg has been identified in marsupials (Macropus giganteus) [44].

Cryptosporidium ubiquitum was found in Hylaeamys laticeps, the first finding in wild rodents captured in Brazil. This species has low specificity and is commonly reported in animals, including rodents, marsupials, and other host species [35,41,43,53,54]. Cases in humans have shown that [55,56] the most common route of C. ubiquitum transmission is through water [56].

The two genes assessed, gp60 and Hsp-70, have satisfactory sensitivity and can be used in studies to identify Cryptosporidium and verify its genetic diversity [45,53,57,58]. Using more than one gene provides a more detailed understanding of the protozoan’s genetic variability and abiotic factors in the study population [59].

In this study, the occurrence of protozoa in small mammals was similar in the Atlantic Forest (Una and Belmonte) and agroforestry (Ilhéus) environments. The difference in the number of positive animals between capture areas was not statistically significant, demonstrating that agroforestry areas maintain low contamination due to the continued diversity of fauna and flora, despite greater anthropic action and transit of domestic animals that threaten the diversity of wild animals [60].

The close human relationship with wildlife as a result of disorderly urban occupation, illegal trade in wild animals, or the maintenance of these animals as pets, are some of the factors that enhance the transmission of zoonotic diseases between species, thus threatening both conservation of biodiversity, and public health [61,62]. Thus, surveillance and monitoring of wildlife pathogens is necessary for the detection, mitigation and prevention of diseases with zoonotic potential.

Conclusion

Results herein obtained pioneer Giardia and Cryptosporidium identification in rodents and marsupials from southern Bahia, northeastern Brazil, showing the present technique as sensitive enough to identify the subgenotypes of Giardia and Cryptosporidium through the gdh and tpi, and Hsp-70 and gp60 genes, respectively.

Acknowledgments

The authors would like to thank we thank Prof. Aristeu Vieira da Silva (State University of Feira de Santana—UEFS) for the strains of C. parvum (13F and 13C), to the Fiocruz Technological Platform Network for the use of its Sequencing facility at FIOCRUZ-Bahia and to Professor Giovanni Widmer, from Tufts University, for their help in sequencing.

Data Availability

The data that support the findings of this study are openly available in NCBI BLAST database at [https://blast.ncbi.nlm.nih.gov/Blast], reference numbers MW202351, MW202352, MW202353, MW202354, MW202355, MW202356, MW202357, MW202358, MW202359, MW202360, MW202361, MW202362, MW202363, MW202364, MW202365, MW202366 e MW202367.

Funding Statement

Coordination for the Improvement of Higher Education Personnel - Brazil (CAPES) - Financial Code 001, Foundation for Research Support of Bahia (FAPESB) (scholarship PNE0001 / 2014) and National Council for Scientific and Technological Development (CNPQ) (grant) 306308 / 2015-0). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  • 1.De Seixas Filho JT, Santana AC, Mesquita EDFM. Parasitism in thewild animals of tthe atlantic rainforest biome used as hunting meat. Semioses, 8 (1) (2014), pp. 69–70. [Google Scholar]
  • 2.Perec-Matysiak A, Buńkowska-Gawlik K, Zaleśny G, Hildebrand J. Small rodents as reservoirs of Cryptosporidium spp. and Giardia spp. in south-western Poland. Ann Agric Environ Med. 22 (1) (2015). doi: 10.5604/12321966.1141359 [DOI] [PubMed] [Google Scholar]
  • 3.Lallo MA, Pereira A, Araújo R, Favorito SE, Bertolla P, Bondon EF. Ocorrência de Giardia, Cryptosporidium e microsporídios em animais silvestres em área de desmatamento no Estado de São Paulo, Brasil. Cienc. Rural. 39 (5) (2009), pp.1465–1470. 10.1590/S0103-84782009005000085. [DOI] [Google Scholar]
  • 4.Thompson RCA, Ash A. Molecular epidemiology of Giardia and Cryptosporidium infections. Infect Genet Evol. 40 (2016), pp. 315–323. 10.1016/j.meegid.2015.09.028 [DOI] [PubMed] [Google Scholar]
  • 5.Siqueira-Castro ICV, J. Greinert-Goulart A, Bonatti TR, Yamashiro S, Franco RMB. First report of predation of Giardia sp. cysts by ciliated protozoa and confirmation of predation of Cryptosporidium spp. oocysts by ciliate species. Environ Sci Pollut Res. 23 (11) (2016), pp. 11357–11362. doi: 10.1007/s11356-016-6689-y [DOI] [PubMed] [Google Scholar]
  • 6.Xiao L, Fayer R. Molecular characterisation of species and genotypes of Cryptosporidium and Giardia and assessment of zoonotic transmission. Int J Parasitol. 38 (11) (2008), pp. 1239–1255. 10.1016/j.ijpara.2008.03.006 [DOI] [PubMed] [Google Scholar]
  • 7.Heitman TL, Frederick LM, Viste JR, Guselle NJ, Morgan UM, Thompson RCA, et al. Prevalence of Giardia and Cryptosporidium and characterization of Cryptosporidium spp. isolated from wildlife, human, and agricultural sources in the North Saskatchewan River Basin in Alberta, Canada. Can. J. Microbiol., 48 (2002), pp. 530–541. 10.1139/w02-047 [DOI] [PubMed] [Google Scholar]
  • 8.Zhou L, Fayer R, Trout JM, Ryan UM, Schaefer FW, Xiao L. Genotypes of Cryptosporidium species infecting fur-bearing mammals differ from those of species infecting humans. Appl Environ Microbiol. 70 (12) (2004), pp. 7574–7577. doi: 10.1128/AEM.70.12.7574-7577.2004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Appelbee AJ, Thompson RC, M. Olson EC. Giardia and Cryptosporidium in mammalian wildlife–current status and future needs. T parasitology., 21 (8) (2005), pp. 370–376. 10.1016/j.pt.2005.06.004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Xiao L, Escalante L, Yang C, Sulaiman I, Escalante AA, Montali RJ, Lal AA. Phylogenetic analysis of Cryptosporidium parasites based on the small-subunit rRNA gene locus. Appl Environ Microbiol. 65 (4) (1999), pp. 1578–1583. doi: 10.1128/AEM.65.4.1578-1583.1999 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Khramtsov NV, Tilley M, Blunt DS, Montelone BA, Upton SJ. Cloning and analysis of a Cryptosporidium parvum gene encoding a protein with homology to cytoplasmic form Hsp70. J Eukaryot Microbiol. 42 (4) (1995), pp. 416–422. 10.1111/j.1550-7408.1995.tb01605.x [DOI] [PubMed] [Google Scholar]
  • 12.Peng M. M., Matos O., Gatei W., Das P., Stantic- Pavlinic M. I. R. J. A. N. A., Bern C., Xiao L.. A comparison of Cryptosporidium subgenotypes from several geographic regions. J Eukaryot Microbiol. 48 (2001), pp. 28s–31s. 10.1111/j.1550-7408.2001.tb00442.x [DOI] [PubMed] [Google Scholar]
  • 13.Widmer G, Lin I, Kapur V, Feng X, Abrahamsen MS. Genomics and genetics of Cryptosporidium parvum: the key to understanding cryptosporidiosis. Microbes Infect. 4 (10) (2002), pp. 1081–1090. 10.1016/S1286-4579(02)01632-5 [DOI] [PubMed] [Google Scholar]
  • 14.Monis PT, Thompson RCA. Cryptosporidium and Giardia-zoonoses: fact or fiction? Infect Genet Evol. 3 (4) (2003), pp. 233–244. 10.1016/j.meegid.2003.08.003 [DOI] [PubMed] [Google Scholar]
  • 15.Hopkins RH, Meloni BP, Groth DM, Wetheralll JD, Reynoldson JA, Thompson RA. Ribosomal RNA sequencing reveals differences between the genotypes of Giardia isolates recovered from humans and dogs living in the same locality. J parasitol. 83 (1997), pp. 44–51. 10.2307/3284315. [DOI] [PubMed] [Google Scholar]
  • 16.Cacciò SM, Beck R, Lalle M, Marinculic A, Pozio E. Multilocus genotyping of Giardia duodenalis reveals striking differences between assemblages A and B. Int J parasitol., 38 (2008), pp. 1523–1531. 10.1016/j.ijpara.2008.04.008 [DOI] [PubMed] [Google Scholar]
  • 17.Sulaiman IM, Fayer R, Bern C, Gilman RH, Trout JM, Schantz PM, Xiao L. Triosephosphate isomerase gene characterization and potential zoonotic transmission of Giardia duodenalis. Emerg Infect Dis. 9 (11) (2003), pp. 1444–1452. doi: 10.3201/eid0911.030084 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Lalle M, Pozio E, Capelli G, Bruschi F, Critti D, Cacciò SM. Genetic heterogeneity at the β-giardin locus among human and animal isolates of Giardia duodenalis and identification of potentially zoonotic subgenotypes. Int J Parasitol. 35 (2) (2005), pp. 207–213. 10.1016/j.ijpara.2004.10.022 [DOI] [PubMed] [Google Scholar]
  • 19.Sambuichi RHR. Fitossociologia e diversidade de espécies arbóreas em cabruca (Mata Atlântica raleada sobre plantação de cacau) na região sul da Bahia, Brasil. Acta Bot Bras. 16 (1) (2002), pp. 89–101. 10.1590/S0102-33062002000100011. [DOI] [Google Scholar]
  • 20.Sambuichi RHR. Estrutura e dinâmica do componente arbóreo em área de cabruca na região cacaueira do sul da Bahia, Brasil. Acta Bot Bras. 20 (4) (2006), pp. 943–954. 10.1590/S0102-33062006000400018. [DOI] [Google Scholar]
  • 21.Fehlberg HF, Maciel BM, Albuquerque GR. Identification and discrimination of Toxoplasma gondii, Sarcocystis spp., Neospora spp., and Cryptosporidium spp. by righ-resolution melting analysis. Plos one, 12 (3) (2017), pp. e0174168. 10.1371/journal.pone.0174168 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Palomo KGS. Vulnerabilidade da Mata Atlântica no Sul da Bahia frente à expansão da fronteira econômica. J. Soc., Technol. Technol. Environ. Sci. 4 (2) (2015), pp. 70–82. [Google Scholar]
  • 23.Thompson RCA, Smith A, Lymbery AJ, Averis S, Morris KD, Wayne AF. Giardia in western Australian wildlife. Vet. Parasitol. 170 (3–4) (2010), pp. 207–211. 10.1016/j.vetpar.2010.02.012 [DOI] [PubMed] [Google Scholar]
  • 24.Santos RCF. Importância de mamíferos neotropicais na epidemiologia de protozooses: diagnóstico, caracterização molecular e aspectos ecológicos da infecção por Giardia e Cryptosporidium. Tese. Universidade de São Paulo 2011. 10.11606/D.10.2011.tde-05102012-150543.
  • 25.Vermeulen ET, Ashworth DL, Eldrigde MDB, Power ML. Investigation into potential transmission sources of Giardia duodenalis in a threatened marsupial (Petrogale penicillata. Infect Genet Evol. 33 (2015), pp. 277–280. 10.1016/j.meegid.2015.05.015 [DOI] [PubMed] [Google Scholar]
  • 26.Lima VFS, Ramos RAN, Lepold R, Borges JCG, Ferreira CD, Rinaldi L, et al. Gastrointestinal parasites in feral cats and rodents from the Fernando de Noronha Archipelago, Brazil. Rev Bras Parasitol Vet. 26 (4) (2017), pp. 521–524. 10.1590/s1984-29612017066 [DOI] [PubMed] [Google Scholar]
  • 27.Melo TF. Ocorrência de endoparasitas em pequenos mamíferos em um fragmento de floresta atlântica e em uma plantação de eucaliptos no Nordeste do Brasil. Tese. Universidade Federal de Pernambuco, 2015. https://repositorio.ufpe.br/handle/123456789/24875.
  • 28.Hillman AE, Lymbery AJ, Elliot AD, Thompson ARC. Urban environments alter parasite fauna, weight and reproductive activity in the quenda (Isoodon obesulus). Sci Total Environ. 607 (2017), pp. 1466–1478. 10.1016/j.scitotenv.2017.07.086 [DOI] [PubMed] [Google Scholar]
  • 29.Paglia AP, Da Fonseca GA, Rylands AB, Hermann G, Aguiar LM, Chiarello AG, et al. Lista Anotada dos Mamíferos do Brasil 2ª Edição/Annotated Checklist of Brazilian Mammals. Occas pap cons biol., 6 (2012), pp. 1–82. [Google Scholar]
  • 30.Bandouk AC, Danola CBC, Masi E, Diz FAC, Gladyston CV, Bica IM, et al. Programa de Vigilância e Controle de Leptospirose e Roedores do município de São Paulo. (2013), pp. 16–18. [Google Scholar]
  • 31.Colli CM, Bezagio RC, Nishi L, Bignott TS, Ferreira EC, Falavigna-Guilherme AL, et al. Identical assemblage of Giardia duodenalis in humans, animals and vegetables in an urban area in southern Brazil indicates a relationship among them. PLoS One. 10 (3) (2015), pp. e0118065, 10.1371/journal.pone.0118065 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Cacciò SM, Ryan U. Molecular epidemiology of giardiasis. Mol Biochem Parasitol., 160 (2) (2008), pp. 75–80, 10.1016/j.molbiopara.2008.04.006 [DOI] [PubMed] [Google Scholar]
  • 33.Karim MR, Wang R, Yu F, Li T, Dong H, Li HD, et al. Multi-locus analysis of Giardia duodenalis from nonhuman primates kept in zoos in China: geographical segregation and host-adaptation of assemblage B isolates. Infect Genet Evol. 30 (2015), pp. 82–88, 10.1016/j.meegid.2014.12.013 [DOI] [PubMed] [Google Scholar]
  • 34.Garcia–r JC, French N, Pita A, Velathanthiri N, Shrestha R, Hayman D. Local and global genetic diversity of protozoan parasites: spatial distribution of Cryptosporidium and Giardia genotypes. PLoS Negl Trop Dis., 11 (7) (2017), pp. e0005736, 10.1371/journal.pntd.0005736 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Sahraoui L, Thomas M, Chevillot A, Mammeri M, Polack B, Vallèe I, et al. Molecular characterization of zoonotic Cryptosporidium spp. and Giardia duodenalis pathogens in Algerian sheep. Vet Parasitol Reg Stud Reports. 16 (2019), pp. 100280, 10.1016/j.vprsr.2019.100280 [DOI] [PubMed] [Google Scholar]
  • 36.Siripattanapipong S, Leelayoova S, Mungthin M, Thompson RA, Boontanom P, Saksirisamphant W, et al. Determination of discriminatory power of genetic markers used for genotyping giardia duodenalis. Southeast Asian J Trop Med Public Health. 42 (4) (2011), pp. 764–771, http://www.tm.mahidol.ac.th/seameo/2011-42-4/02-52. [PubMed] [Google Scholar]
  • 37.Yaoyu F, Xiao L. Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev. 24 (1) (2011), pp. 110–140, 10.1128/CMR.00033-10 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Marques ARL. Aplicação do MLTS (multilocus sequence typing) na Genotipagem de Giardia Lamblia. Dissertação. Universidade de Coimbra, 2015, http://hdl.handle.net/10316/32320.
  • 39.Dall’olio AJ, Franco RMB. Ocorrência de Cryptosporidium spp. em pequenos mamíferos silvestres de três áreas serranas do Sudeste brasileiro. Arq. Bras. Med. Vet. Zootec., 56 (1) (2004), pp. 25–31, 10.1590/S0102-09352004000100005. [DOI] [Google Scholar]
  • 40.Ryan UM, Power M, Xiao L. Cryptosporidium fayeri n. sp. (Apicomplexa: Cryptosporidiidae) from the Red Kangaroo (Macropus rufus). J Eukaryot Microbiol. 55 (1) (2008), pp. 22–26, 10.1111/j.1550-7408.2007.00299.x [DOI] [PubMed] [Google Scholar]
  • 41.Murakoshi F, Fukuda Y, Matsubara R, kato Y, Sato R, Sasaki T, et al. Detection and genotyping of Cryptosporidium spp. in large Japanese field mice, Apodemus speciosus. Vet parasitol. 196 (1–2) (2013), pp. 184–188, 10.1016/j.vetpar.2013.02.011 [DOI] [PubMed] [Google Scholar]
  • 42.Silva SOS, Richtzenhain LJ, Barros IN, Gomes AM, Silva AV, Kozerski ND, et al. A new set of primers directed to 18S rRNA gene for molecular identification of Cryptosporidium spp. and their performance in the detection and differentiation of oocysts shed by synanthropic rodents. Exp. Parasitol. 135 (3) (2013), pp. 551–557 10.1016/j.exppara.2013.09.003 [DOI] [PubMed] [Google Scholar]
  • 43.Danišová O, Valenčáková A, Stanko M, Luptáková L, Hatalová E, Čanády A. Rodents as a reservoir of infection caused by multiple zoonotic species/genotypes of C. parvum, C. hominis, C. suis, C. scrofarum, and the first evidence of C. muskrat genotypes I and II of rodents in Europe. Acta Trop., 172 (2017), pp. 29–35, 10.1016/j.actatropica.2017.04.013 [DOI] [PubMed] [Google Scholar]
  • 44.Zahedi A, Monis P, Gofton AW, Oskam CL, Ball A, Bath A, et al. Cryptosporidium species and subtypes in animals inhabiting drinking water catchments in three states across Australia. Water Res. 134 (2018), pp. 327–340, doi: 10.1016/j.watres.2018.02.005 [DOI] [PubMed] [Google Scholar]
  • 45.Fiocruz ZC. Diagnóstico Urbanístico do Setor 1 da Colônia Juliano Moreira. FIocruz. 2004.
  • 46.Xiao L. Molecular epidemiology of cryptosporidiosis: an update. Exp parasitol. 124 (1) (2010), pp. 80–89, 10.1016/j.exppara.2009.03.018 [DOI] [PubMed] [Google Scholar]
  • 47.Feng Y, Torres E, Li N, Wang L, Bowman D, Xiao L. Population genetic characterisation of dominant Cryptosporidium parvum subtype IIaA15G2R1. Int J parasitol. 43 (14) (2013), pp.1141–1147, 10.1016/j.ijpara.2013.09.002 [DOI] [PubMed] [Google Scholar]
  • 48.Helmy YA, Krücken J, Nöcklerd K, Samson-Himmelstjernac G, Zessinb KH. Molecular epidemiology of Cryptosporidium in livestock animals and humans in the Ismailia province of Egypt. Vet Parasitol. 193 (1–3) (2013), pp.15–24, 10.1016/j.vetpar.2012.12.015 [DOI] [PubMed] [Google Scholar]
  • 49.Xiao L, Escalante L, Yang C, Sulaiman I, Escalante AA, Montali RJ, Lal AA. Host adaptation and host-parasite co-evolution in Cryptosporidium: implications for taxonomy and public health. Int J Parasitol. 32 (14) (2002), pp. 1773–1785, 10.1016/S0020-7519(02)00197-2 [DOI] [PubMed] [Google Scholar]
  • 50.Power ML, Ryan UM. A new species of Cryptosporidium (Apicomplexa: Cryptosporidiidae) from eastern grey kangaroos (Macropus giganteus). J. Parasitol., 94 (5) (2008), pp. 1114–1117, 10.1645/GE-1508.1 [DOI] [PubMed] [Google Scholar]
  • 51.Waldron LS, Cheung-Kwok-Sang C, Power ML. Wildlife-associated Cryptosporidium fayeri in human, Australia. Emerg. Infect. Dis. 16 (12) (2010), pp. 2006, doi: 10.3201/eid1612.100715 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Koehler AV, Whipp M, Hogg G, Haydon SR, Stevens MA, Jex AR, et al. First genetic analysis of Cryptosporidium from humans from Tasmania, and identification of a new genotype from a traveller to Bali. Electrophoresis. 35 (18) (2014), pp. 2600–2607, 10.1002/elps.201400225 [DOI] [PubMed] [Google Scholar]
  • 53.Fayer R, Santin M, Macarisin D. Cryptosporidium ubiquitum n. sp. in animals and humans. Vet parasitol., 172 (1–2) (2010), pp. 23–32, 10.1016/j.vetpar.2010.04.028 [DOI] [PubMed] [Google Scholar]
  • 54.Li N, Xiao L, Alderisio K, Elwin K, Cebelinski E, Chalmers R, Feng Y. Subtyping Cryptosporidium ubiquitum, a zoonotic pathogen emerging in humans. Emerg Infect Dis. 20 (2) (2014), pp. 217, doi: 10.3201/eid2002.121797 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Feng Y, Alderisio KA, Yang W, Blancero LA, Kuhne WG, Nadareski CA, Xiao L. Cryptosporidium genotypes in wildlife from a New York watershed. Appl Environ Microbiol. 73 (20) (2007), pp. 6475–6483, doi: 10.1128/AEM.01034-07 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Feng Y, Ryan UM, Xiao L. Genetic diversity and population structure of Cryptosporidium. Trends Parasitol., 34 (11) (2018), pp. 997–1011, 10.1016/j.pt.2018.07.009 [DOI] [PubMed] [Google Scholar]
  • 57.Da Silva AJ, Cacciò S, Williams C, Won KY, Nace EK, Whittier C, et al. Molecular and morphologic characterization of a Cryptosporidium genotype identified in lemurs. Vet parasitol., 111 (4) (2003), pp. 297–307, 10.1016/S0304-4017(02)00384-9 [DOI] [PubMed] [Google Scholar]
  • 58.De Carvalho TTR. Estado atual do conhecimento de Cryptosporidium e Giardia. J Trop Pathol., 38 (1) (2009), pp. 01–16. [Google Scholar]
  • 59.Santín M. Clinical and subclinical infections with Cryptosporidium in animals. N Z Vet J. 61 (1) (2013), pp. 1–10, 10.1080/00480169.2012.731681 [DOI] [PubMed] [Google Scholar]
  • 60.Dos Santos CLA, Silva AP, Santos SB, Pardini R, Cassano CR. Dog invasion in agroforests: the importance of households, roads and dog population size in the surroundings. Perspect Ecol Conser., 15 (3) (2017), pp. 221–226, 10.1016/j.pecon.2017.08.001. [DOI] [Google Scholar]
  • 61.Bezerra-Santos MA, Mendoza-Roldan JA, Thompson RCA, Dantas-Torres F, Otranto D. Legal versus Illegal Wildlife Trade: Zoonotic Disease Risks. Trends in Parasitology, 37 (5) (2021), pp. 360–361, 10.1016/j.pt.2021.02.003 [DOI] [PubMed] [Google Scholar]
  • 62.Shivaprakash KN, Sen S, Paul S, Kiesecker JM, Bawa KS. Mammals, wildlife trade, and the next global pandemic. Current Biology, 31 (1–7) (2021), 10.1016/j.cub.2021.06.006. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are openly available in NCBI BLAST database at [https://blast.ncbi.nlm.nih.gov/Blast], reference numbers MW202351, MW202352, MW202353, MW202354, MW202355, MW202356, MW202357, MW202358, MW202359, MW202360, MW202361, MW202362, MW202363, MW202364, MW202365, MW202366 e MW202367.

Articles from PLoS ONE are provided here courtesy of PLOS

RESOURCES