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

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.

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

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

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

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.