Introduction

Enterocytozoon bieneusi, Cryptosporidium, and Giardia are environmentally ubiquitous enteropathogens that cause serious human and animal diarrheal diseases (Ryan and Caccio 2013; Ryan et al. 2014; Santín 2015).

Cryptosporidiosis is one of the most prevalent parasitic infections in domesticated, caged, and wild birds worldwide (O’Donoghue 1995; Sréter and Varga 2000; Wang et al. 2014). More than 30 avian species has been reported infected with Cryptosporidium (Ryan 2010). So far, four Cryptosporidium species have been identified in birds, C. baileyi, C. meleagridis, C. galli, and C. avium (Slavin 1955; Current et al. 1986; Ryan et al. 2003; Holubová et al. 2016). Each of them can infect many avian species, but they differ in their host range, infection sites, and symptomatology associated with infection. In addition to these four species, 13 Cryptosporidium genotypes have been described in birds worldwide, including avian genotypes I–VI, goose genotypes I–V, black duck genotype, and Eurasian woodcock genotype (Nakamura and Meireles 2015; Chelladurai et al. 2016). Among them, only C. meleagridis is known to also infect humans and has public health significance (Xiao 2010).

Giardia have been reported in wild and captive birds worldwide (Erlandsen et al. 1991; McRoberts et al. 1996; Abe et al. 2012; Papini et al. 2012; Reboredo-Fernández et al. 2015). Two species of Giardia are responsible of avian giardiasis, G. ardeae and G. psittaci (Ryan and Caccio 2013). In addition to those two avian species, Giardia duodenalis has also been reported in birds. Thus far, zoonotic assemblages A and B as well as nonzoonotic assemblages D and F have been found in birds (Reboredo-Fernández et al. 2015; Majewska et al. 2009).

Enterocytozoon bieuneusi is an emerging opportunistic human pathogen included in the phylum Microsporidia. Besides humans, E. bieneusi has been found in a broad range of domestic and wild animals (Santín and Fayer 2011). Genetic studies have demonstrated the presence of more than 200 genotypes, some have been found only in humans or only in animal hosts, but several have been found in both humans and animals, suggestion zoonotic potential (Santín 2015). Multiple E. bieneusi genotypes, including those with zoonotic potential, have been identified in birds worldwide (Müller et al. 2008; Kasicková et al. 2009; Galvan-Díaz et al. 2014; da Cunha et al. 2016; Zhao et al. 2016).

Although it has been indicated that captive birds could play an important role in the transmission of zoonotic parasites Cryptosporidium, E. bieneusi, and Giardia for humans and other animals (Kasicková et al. 2009; Majewska et al. 2009; Papini et al. 2012; Pirestani et al. 2013), there is limited information in the literature on the presence of these parasites in captive birds worldwide. The present study was designed to determine the presence of species and genotypes of E. bieneusi, Cryptosporidium, and Giardia in captive birds from the state of Minas Gerais (Brazil).

Material and methods

Samples

From October 2013 to September 2014, a total of 85 fecal specimens were collected from captive birds at the Brazilian Institute of the Environment and Renewable Natural Resources (IBAMA) (n = 15), a private aviary (n = 6), a school of falconry (n = 10), and nine local markets (n = 54) in Uberlândia and Belo Horizonte in the state of Minas Gerais, Brazil. The birds included 9 orders and 33 species (Table 1). At the time of sampling, all birds examined were apparently in good health and no diarrhea was observed. To collect the samples, the animals were placed in individual sanitized cages and fresh feces were collected from the bottom of the cages. Fecal specimens were placed into sterile polystyrene tubes with records of the date, location, identification number, and species, and transferred in isothermal boxes to the Parasitology Laboratory of Federal University of Uberlândia (UFU) and held at −20 °C until DNA extraction.

Table 1 Prevalence and genotyping results for Cryptosporidium, Giardia, and Enterocytozoon bieneusi in captive birds in Uberlândia and Belo Horizonte in the state of Minas Gerais (Brazil)
Full size table

DNA extraction, PCR, and sequencing

Genomic DNA was extracted directly from each individual fecal specimen using the QIAamp Stool Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instruction with minor modifications. Modifications included the addition of 0.3 g of zirconia beads (Stratech Scientific, Luton, UK) to 0.2 g of feces and 1.4 ml lysis buffer (McLauchlin et al. 1999); then, the mixture was heated at 95 °C for 5 min followed by vigorous shaking (two rounds of 15 min) to facilitate the parasite rupture. The nucleic acid was eluted in 150 μl of AE buffer to increase the quantity of DNA recovered.

Polymerase chain reaction protocols for amplifying gene fragments for Cryptosporidium (SSU rRNA), Giardia (SSU rRNA), and E. bieneusi (SSU rRNA, ITS, and LSU rRNA) have been previously described (Hopkins et al. 1997; Xiao et al. 1999; Buckholt et al. 2002). Negative and positive controls were included in all PCR sets. Secondary PCR products were detected and visualized by electrophoresis in the QIAxcel Advanced System (Qiagen, Valencia, CA). All positive PCR products were purified using Exonuclease I/Shrimp Alkaline Phosphatase (Exo-SAP-ITTM) (USB Corporation, Cleveland, OH) and sequenced in both directions using the same PCR primers used in the secondary PCR in 10 μl reactions using Big Dye™ chemistries and an ABI 3130 sequencer analyzer (Applied Biosystems, Foster City, CA). Nucleotide sequences obtained in this study were aligned, examined, and compared with reference sequences from GenBank using SeqMan™ (DNAstar Inc., Madison, WI). The nucleotide sequences obtained in this study have been deposited in GenBank under accession numbers KY012352–KY012356.

Results

Of the 85 bird specimens, 3 (3.5%), 2 (2.3%), and 1 (1.2%) were PCR-positive for E. bieneusi, Cryptosporidium, and G. duodenalis, respectively (Table 1). For E. bieneusi, the three positive birds were a swan goose from market 3 in Uberlândia and two rock pigeons from market 7 in Belo Horizonte. Crypstoporidium was found in a swan goose and a mandarin duck from markets 2 and 3 in Uberlândia, respectively. Giardia was identified in a toco toucon from a private aviary in Uberlândia. All birds examined from the School of Falconry, IBAMA, and markets 1, 4, 5, 6, 8, and 9 were negative for the three parasites. E. bieneusi was found in birds from Uberlândia and Belo Horizonte while Cryptosporidium and Giardia were only identified in birds from Uberlândia.

All PCR positive samples were successfully sequenced, and no mixed infections were observed (Table 1). For E. bieneusi, nucleotide sequence analysis of the ITS revealed two distinct genotypes, genotype D in a swan goose and Peru 6 in two rock pigeons. For Cryptosporidium, C. baileyi was found in a swan goose and Duck genotype in a mandarin duck sample. For G. duodenalis, assemblage A was identified in a toco toucon.

Discussion

This study demonstrated the presence of E. bieneusi, Cryptosporidium, and G. duodenalis in captive birds in Brazil. This is the first report of E. bieneusi in captive birds in Brazil where, so far, it only been reported in chickens, free-living pigeons, and exotics birds (Lallo et al. 2012; da Cunha et al. 2016). Of the three parasites studied, E. bieneusi was the most frequent with a prevalence of 3.2%. The prevalence was similar to the one previously reported in free-living pigeons and exotic birds in Brazil (5.6%), but lower than the one reported in chickens in Brazil (15.9%) and those reported in various birds in studies in Portugal (29%), China (22.2%), Czech Republic (12.5%), and Spain (9.7%) (Haro et al. 2006; Lobo et al. 2006; Kasicková et al. 2009; Lallo et al. 2012; da Cunha et al. 2016; Zhao et al. 2016). In this study, E. bieneusi was identified in a swan goose and in two pigeons expanding the host range of this parasite in Brazil as it is the first time this parasite was found in birds of the order Anseriformes in this country. In the order Anseriformes, there is only another report of E. bieneusi in farmed domestic geese (Anser domestica) and domestic duck (Anas platyrhynchos domesticus) in China (Zhao et al. 2016). However, E. bieneusi has been commonly detected in pigeons with reports in Brazil, China, Iran, Netherlands, Spain, Poland, and Portugal (Haro et al. 2006; Lobo et al. 2006; Bart et al. 2008; Lallo et al. 2012; Pirestani et al. 2013; Stodkowicz-Kowalska et al. 2013; Zhao et al. 2016).

The nucleotide sequence analysis of the ITS region of the E. bieneusi isolates revealed the presence of two genotypes, D in the swan goose and Peru 6 in the two rock pigeons. Both genotypes belong to group 1 which predominately infects humans and has zoonotic importance (Santín and Fayer 2011). Genotypes D and Peru 6 have been widely reported in humans as well as in a wide range of animal hosts worldwide (Santín 2015). In Brazil, genotype D was found in humans and cattle (Feng et al. 2011; da Silva Fiuza et al. 2016) and Peru 6 in chickens (da Cunha et al. 2016). Genotype D has previously identified in avian hosts of the orders Falconiformes in Abu Dhabi (Müller et al. 2008), Gruiformes in China (Zhao et al. 2016), Columbiformes in Iran (Pirestani et al. 2013), and Galliformes in Brazil (da Cunha et al. 2016). Similarly, Peru 6 has been previously found in orders Psittaciformes and Columbiformes in Portugal (Lobo et al. 2006), Galliformes in Brazil (da Cunha et al. 2016), and Gruiformes in China (Zhao et al. 2016).

Cryptosporidium has been reported in a wide range of domestic, wild and captive avian hosts in worldwide (Ryan 2010; Nakamura and Meireles 2015). In Brazil, it has been previously reported in domestic, wild, exotic, and captive birds (Huber et al. 2007; Nakamura et al. 2009, 2014; Sevá et al. 2011; Gomes et al. 2012; Holsback et al. 2013; da Cunha et al. 2016). In this study, Cryptosporidium was detected in 2 (2.1%) of the 85 fecal samples examined. A similar prevalence was reported in domestic pigeons in Iran (2.94%). However, it was higher than the prevalence found in pigeons from China (0.8%) and a lower than the one reported in domestic and captive birds in studies in Brazil and other countries (Papini et al. 2012; Nakamura et al. 2014; Nakamura and Meireles 2015; Li et al. 2016). Molecular characterization identified C. baileyi in a swan goose and the Duck genotype in a mandarin duck. C. baileyi is considered the most common avian Cryptosporidium species worldwide (Nakamura et al. 2009; Ryan 2010). In Brazil, it has been previously reported in wild, captive, and domestic birds of the orders Passeriformes, Anseriformes, Galliformes, and Cathartiformes (Huber et al. 2007; Nakamura et al. 2009, 2014; Sevá et al. 2011). However, this is the first time that the Duck genotype has been reported in South America, until now, it was only reported twice in North America in Canada geese (Jellison et al. 2004; Zhou et al. 2004).

Although Giardia has been widely documented in mammals, there is limited number of studies in birds with no reports of Giardia in birds in Brazil. The presence of Giardia in a toco toucan constitutes not only the first report of Giardia in birds in Brazil but also the first report of Giardia in a bird from the order Piciformes. This parasite has been identified in various orders of captive and wild birds (Papini et al. 2012; Reboredo-Fernández et al. 2015; Cano et al. 2016). The overall Giardia prevalence was low (1.1%) and similar to prevalences reported in pet birds (1.6%) and zoo birds (3.6%) in Italy (Papini et al. 2012), and wild birds in Spain (2.1%) (Reboredo-Fernández et al. 2015). Molecular characterization of the toco toucan isolate identified G. duodenalis assemblage A. This assemblage is known to be infectious for humans and for a wide range of mammals. In birds, assemblage A has previously been reported in wild birds in Spain (Reboredo-Fernández et al. 2015), domestic birds in Ivory Coast (Berrilli et al. 2012), and captive birds in Italy (Papini et al. 2012). G. duodenalis assemblages B, D, and F have also been identified in wild and aquatic birds in Spain (Reboredo-Fernández et al. 2015; Cano et al. 2016) and domestic birds in Ivory Coast (Berrilli et al. 2012).

In conclusion, this study confirmed the occurrence of zoonotic parasites in captive birds in Brazil raising questions on potential zoonotic transmission of G. duodenalis and E. bieneusi from captive birds to humans. All positive birds were apparently healthy and might serve as asymptomatic carriers of these parasites. Birds keep in captivity are in close contact with humans and have to be periodically handled for feeding and sanitation, and all those activities could expose handlers to infection by these and others zoonotic parasites. In addition, if waste is not properly handled, it could contaminate water and food and contribute to the indirect transmission of these parasites.