Blastocystis sp. of Subtypes 2, 4 and 9 in Selected Avian Species, Brazil, 2009-2018ABSTRACT
Avian species of the metropolitan region of Belo Horizonte, Brazil, submitted to laboratory routine due to a variety of morbid conditions, were diagnosed as carriers of Blastocystis sp. All samples were examined under light microscopy and those with structures suggestive of Blastocystis sp. were tested by PCR. The total DNA was purified from feces and/or intestinal mucosa and used as a template for PCR, targeting the small subunit (SSU) rRNA gene, and the products were sequenced. The sequences detected in two chickens, one passerine (Saltator similis, Thraupidae) and a golden parakeet (Guaruba guarouba, Psittacidae) were phylogenetically characterized and shown to cluster with sequences of subtypes 2, 4 or 9. The occurrence of Blastocystis sp. in domestic and local native avifauna is described and the perspective of a complex local interspecies epidemiology is considered of risk, including to humans.
Keywords:
Stramenopiles; Gallus gallus domesticus; Guaruba guarouba; Saltator similis; Sicalis flaveola
INTRODUCTION
Blastocystis sp. is polymorphic and existing in different forms, including amoeboid, cyst, granular, vacuolar and others. It occurs in more than one billion humans, with certain subtypes (ST) considered pathogenic (Parija et al., 2013). The microorganism is a member of Stramenopiles, a group containing 21 classes and more than 100.000 species of eukaryotic organisms, mainly represented by photosynthetic algae. However, five classes are devoid of chloroplasts, including the etiology of a potato disease (Phytophthora infestans HERB-1), described in the great famine in Ireland when potato cultures were destroyed between 1845-1852 (Yoon et al., 2009).
A very wide range of species has been described as hosts for Blasto-cystis sp., in phyla Arthropoda, Chordata and Mollusca, including classes Amphibia, Aves, Insecta, Mammalia and Reptilia [Stenzel & Boreham, 1996; Tan, 2008]. Blastocystis sp. was initially considered commensal, and is known to be widespread in humans. It is especially prevalent in suboptimal public health conditions, while also acting opportunistically in cases of immunodeficiency and being associated to gastrointestinal diseases (Wawrzyniak et al., 2013). However, Blastocystis may possibly have a balancing role towards a healthy microbiome (Kodio et al., 2019). Regarding human intestinal microbiome health, clinically normal Malian (West Africa) children with intestinal colonization by Blastocystis had a higher diversity and evenness of bacterial species, with a higher count of beneficial bacteria (Kodio et al., 2019; Aykur et al., 2024). The celiac disease and colorectal cancer have been associated with low bacterial diversity and increased Bacteroides (De Palma et al., 2010). Subtypes ST1, ST3, and ST4 may increase the diversity of the gut microbiome and promote an anti-inflammatory state in the intestinal mucosa (Aykur et al., 2024).
At least 46 subtypes (ST) of Blastocystis have been described (Koehler at al., 2024), and the genetic diversity was initially proposed despite the identical phenotypes (Clark, 1997). The genetic diversity of Blastocystis ST in stool samples of human patients with chronic urticaria revealed ST3 (allele 34) as the most prevalent, with a significant total IgE associated with the presence of ST2 and ST3 (Aykur et al., 2022). The ST3 infection in humans was described as restricted to one of four mitochondrion-like organelles (MLO) clades, ST4 almost exclusively to one of two SSU rRNA clades, and only five multilocus sequence types (MLST) were detected among 50 ST4 of clade 1 (Stensvold et al., 2012). The occurrence of Blastocystis, mostly ST7 or ST9, was associated with irritable bowel syndrome and a history of contact with pigs and poultry (Stensvold et al., 2009).
In Italy, isolates were subtyped from fallow deer (Dama dama), goats, and pigs (ST5), pheasants (ST6), chickens (ST7), sheep (ST10) and water buffaloes (ST14), suggesting animal-to-human spillover for ST6 and ST7 (Gabrielli et al., 2021). The organism was detected in 1.3% of humans of Rio de Janeiro, along with cestodes, nematodes and protists, and was identified as ST1 (42%), ST2 (7%), ST3 (49%), ST4 (2%) and mixed infections (14%) (Valença Barbosa et al., 2017). Also in Brazil, Blastocystis sp. was visualized in feces of poultry (chicken 22.9-42.9%, mallard 37.1-55.3% and quail 5,5-42,9%) in live markets (Bomfim et al., 2013).
The marine fauna has been studied in the Northeastern Atlantic (France), detecting six different ST with three potentially new ones, with a prevalence of 3.5% in fish and 13.8% in mammals (Gantois et al., 2020). Zoo animals were also investigated in France, and the organism was detected in 37.9% of species, suggesting avian and artiodactyl species as reservoirs to humans, but not mammalian carnivores, insects or reptiles (Cian et al., 2017). Reptiles of 28 species were investigated in Singapore, and Blastocystis sp. was detected in three snake species, three tortoises, one lizard (iguana), and one crocodile species (Teow et al., 1992).
We describe the investigation of Blastocystis in exotic and native avian species received for diagnosis of diverse and unrelated morbid conditions.
MATERIALS AND METHODS
Samples
Intestinal mucosal scrapings were obtained at necropsy of one Pionopsitta pileata (Psittaciformes), five Nymphicus hollandicus (Psittaciformes), one Guaruba guarouba (Psittaciformes), one Saltator similis and one Sicalis flaveola (Passeriformes), one Aix sponsa (Anseriformes), and chickens of the family poultry (n=10) and layer industry (n=10). Most samples were taken by scraping off the duodenum/jejunum mucosa, except for sampling of ceca of the industrial layer chickens (Table 1 and Figure 1).
A. Blastocystis sp. in Pionopsitta pileata (Psittaciformes) case 932. Note the diversity in diameters. Each division = 10 µm. 40X. B. Blastocystis sp. in the small intestine of free-range chickens. Each division = 10 µm. 100X. C. Blastocystis sp. in free-range chicken, case 458. Note the apparent uniformity of diameters. 100X. D. Blastocystis sp. in Nymphicus hollandicus (Psittaciformes). Note the apparent uniformity of diameters. Each division = 10 µm. 400X. E. Blastocystis sp. in the cecum of an industrial layer chicken. Note the two large round central cells (arrows). 400X. F. Blastocystis sp. in the cecum of an industrial layer chicken. Note the two large round cells (arrows). Groups of Sarcina sp. (Clostridiaceae) are at left (hollow arrow). 400X. G. Blastocystis sp. in the small intestine of Sicalis flaveola (Passeriformes). 400X. H. Blastocystis sp. in the cecum of Aix sponsa (Anseriformes) case 543. A. Note the two round cells (blue arrow). Groups of Sarcina sp. (Clostridiaceae) are above the ruler at 50 (hollow arrow). 100X. I. Blastocystis sp. in the small intestine of Guaruba guarouba (Psittaciformes), case 804. 400X. J. Typical Blastocystis sp. vacuolate form in the small intestine of chicken. Cell with large vacuole formation with cytoplasm (*) and nuclei (arrows) are indicated. 1000x.
After the demonstration of typical Blastocystis spp. structures at microscopy, the total DNA was extracted and used as template in a PCR targeting the SSU rRNA gene. The amplified products were sequenced and four obtained sequences were aligned with sequences published in the GenBank.
Microscopy
Samples were investigated fresh under optical microscopy for cyst structures typical of Blastocystis sp. at 100 and 400-time magnifications (Olympus CBB, São Paulo, Brazil), and positive specimens were submitted to further molecular studies.
DNA extraction and PCR
After the microscopic demonstration of typical cystic Blastocystis sp. cells, the positive samples were submitted to DNA extraction. The total DNA was extracted and employed as a template for the PCR. The total DNA was extracted using 6M sodium iodide (Vogelstein & Gillespie, 1979; Boom et al., 1990) and adsorption to silicon dioxide (Boom et al., 1990), and the concentration and purity were determined (NanoVue®, GE, Healthcare, UK).
The PCR reactions were performed in a thermocycler (Axygen-Maxygene, USA) for the detection of the small subunit (SSU) of the rRNA encoding gene of Blastocystis sp., using the oligonucleotides Blastocystis Ext rRNA Fwd 5’-GGA ATC CTC TTA GAG GGA CAC TAT ACA T-3’ and Blastocystis Ext rRNA Reve 5’-TTA CTA AAA TCC AAA GTG TTC ATC GGA C-3’ for the amplification of a 310 bp product. PCR conditions were as previously described (Stensvold et al., 2006) (Table 2).
The electrophoresis of products was performed in 1% agarose gel at 100V/40 min (Life Technologies, Gaithersburgh, MD, USA), revealed by GelRed® (Biothium, CA, USA) and visualized in a UV transilluminator (Hoefer, San Francisco, CA, USA). The molecular mass of products was estimated by comparing with a 100 bp ladder (Promega, Fitchburg, WI, USA).
Sequencing and phylogeny
The obtained PCR amplification products were submitted to sequencing (Sanger et al., 1977), and sequences were phylogenetically analyzed. To obtain the products for sequencing, the previously described primers were employed in a reaction using BigDye™ Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher, Brazil; https://www.thermofisher.com/br/en/home.html), and sequences were determined bidirectionally. The forward and reverse consensus sequences were obtained using BioEdit v. 7.7.1.0 (Hall, 1999; https://bioedit.software.informer.com/7.7/). The alignment of sequences was determined by ClustalW, and the evolutionary history was inferred using the Minimum Evolution method (Rzhetsky & Nei, 1992), both in MEGA11 (Tamura et al., 2021; https://www.megasoftware.net/).
RESULTS
Typical Blastocystis structures were detected in species of Anseriformes, Galliformes, Passeriformes and Psittaciformes (Table 2 and Figure 1). The Aix sponsa individual was gravely affected by a fatal traumatic disease. In the chicken, mostly in family poultry cases, other gastrointestinal parasites, such as cestodes (Davainea spp., Hymenolepis spp., Raillietina spp.), nematodes (Ascaridia galli, Heterakis gallinarum), and protists (Eimeria spp.) were eventually present; and in a few cases, such as case 479 (Table 2), were concomitant with avian botulism. The S. similis (microscopy not shown) and S. flaveola cases were associated with a fatal disease caused by Isospora sp. in a triage center. Among the psittacines of the commercial and conservation institutions, the P. pileata had cachexia and vascular congestion of the ingluvius and the Guaruba guarouba had cachexia, dark feces adhered to the cloaca, and a dark spot in the gizzard. The cockatiel evaluated was not fatally affected and had altered to pasty fecal consistency.
New hosts for Blastocystis sp. are described in this study, including the captive native passerine species S. similis and S. flaveola (Passeriformes) and the captive native psittacine species G. guarouba and Pionopsitta pileata (Psittaciformes). The avian species were examined due to a diversity of morbid conditions, which may also favor an enhanced infection, replication and transmission of Blastocystis sp. In chickens, the intestinal presence of nematodes, cestodes and coccidia was common. In the native passerines, such as in Saltator similis and Sicalis flaveola, cases involved coccidiosis by Isospora sp.
The domestic avian species were chickens of the family or industrial poultry and pet cockatiels, and the native avifauna species were captive in commercial, conservation or triage facilities. Captivity, proximity and intensification are considered conditions of infection risk, which typically favor prevalence and transmission. However, none of the native species here described were previously investigated in a previous study in Brazil (Maloney et al., 2020).
The microscopy of feces and/or the intestinal mucosa at the routine laboratory diagnosis in the investigated species enabled the demonstration of typical Blastocystis sp. cyst structures (Figure 1). The four obtained sequences of PCR products (small subunit rRNA) were aligned with sequences of the GenBank and the identities to subtypes ST2, ST4 and ST9 were demonstrated (Figure 2). In humans in Brazil, studies revealed ST3 to have the highest occurrence, followed by ST1 and ST2 (Melo et al., 2021).
Sequences of the small subunit (SSU) rRNA of Blastocystis sp. were detected in two chickens, one passerine (S. similis) and one psittacine (G. guarouba) of the metropolitan region of Belo Horizonte, and analyzed phylogenetically. The amplified products were sequenced and four obtained sequences were aligned with sequences published in the GenBank. The sequences detected in one chicken (MK880049.1) and one G. guarouba (MK880050.1) were grouped with a subtype 2 (ST2) sequence of Coturnix japonica (AF408426.2) described in Japan. The sequence detected in S. similis (MK880048.1) was clustered with a ST4 strain (AY135411.1) detected in Meleagris gallopavo in France. Another sequence found in Gallus gallus domesticus (MK880051.1) was clustered with a ST9 sequence of Homo sapiens sapiens (AF408426.2) of Japan. All STs have been previously described in humans in Brazil (Malheiros et al., 2011; Valença Barbosa et al., 2017; Melo et al., 2021). The percentage of replicate trees in which the associated taxa are clustered together in the bootstrap test (1000 replicates) are shown next to the branches (Felsenstein, 1985). The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al., 2004) and are in the units of the number of base substitutions per site. The ME tree was searched using the Close-Neighbor-Interchange (CNI) algorithm (Nei & Kumar, 2000) at a search level of 1. The Neighbor-joining algorithm [Saitou & Nei, 1987] was used to generate the initial tree. The evolutionary analyses were conducted in MEGA11 (Tamura et al., 2021)(Figure 2).
We describe the detection of ST2 and ST4, both previously detected in humans in Brazil, and additionally describe ST9 for the first time. The avian species with the detected STs may be participating in a complex and potentially significant epidemiology. The occurrence of ST1 to ST9 were previously described in humans elsewhere (Skotarczak, 2018).
The biological samples have chronological differences. ST2 was detected in chicken in 2011 (MK880049.1) strain 479, and G. guarouba in 2017 (MK880050.1) strain 1564. ST4 was detected in S. similis in 2009 (MK880048.1) strain 117, and ST9 was detected in free-range chicken in 2017 (MK880049.1) strain 1565.
ST2 was previously detected in humans (7%), in Brazil (Rio de Janeiro), as well as ST1 (42%), ST3 (49%), ST4 (2%) and mixed infections (14%) (Valença-Barbosa et al., 2017), along with cestodes, nematodes and protists. In a previous study in humans, ST3 was detected with the highest positivity, followed by ST1 and ST2 (Melo et al., 2021). ST4 was previously detected in humans in Rio de Janeiro (Valença Barbosa et al., 2017), although it was not detected in an indigenous group of the Tapirapé ethny in the Amazon basin (Mato Grosso) (Malheiros et al., 2011). A previous investigation in feces of poultry revealed the occurrence in chicken (22.9-42.9%), mallard (37.1-55.3%) and quail (5,5-42,9%) in live markets (Bomfim et al., 2013). ST1 was described to occur in swine in Uberaba (Brazil) (Moura et al., 2018). A previous local study through the microscopy of feces in cracids has revealed the occurrence of an untyped Blastocystis sp. in Aburria jacutinga (Marques et al., 2013). Our unpublished routine diagnosis results suggest that Blastocystis sp. might be widespread in local domestic avian species, especially prevalent in free-range chicken, with some flocks acting as potential reservoirs.
Poultry has been suggested as reservoirs for Blastocystis sp. (Gabrielli et al., 2021). A very wide range of host species has been described to harbor Blastocystis sp., including most classes of Animalia (Liu et al., 2022; Stenzel & Boreham, 1996; Tan, 2008). In Italy, STs were identified in fallow deer, goat, and pig (ST5), pheasant (ST6), chicken (ST7), sheep (ST10) and water buffalo (ST14), and animal-to-human spillover was suggested for ST6 and ST7, with a high similarity (99-100%) when comparing human to other animal isolates (Gabrielli et al., 2021). The natural occurrence was described in the marine fauna of the Northeastern Atlantic ocean (France), and six different subtypes (ST) were detected in fish and mammals (Gantois et al., 2020). The occurrence was previously investigated in France, suggesting avian and artiodactyl species as reservoirs (Cian et al., 2017); and reptilian host species were investigated in Singapore (Teow et al., 1992). In humans, the occurrence of Blastocystis, mostly ST7 or ST9, has been associated with irritable bowel syndrome and the history of contact with pigs and poultry (Stensvold et al., 2009). A very wide range of species has been described as hosts for Blastocystis sp. (Stenzel & Boreham, 1996; Tan, 2008), which are known to be widespread in humans and associated to gastrointestinal diseases, especially in immunocompromised patients (Wawrzyniak et al., 2013). Such diversity of host taxa encourages broadening local investigation, as it is possible that additional subtypes are occurring in other avian hosts.
In this study, Blastocystis sp. was detected by microscopy and by PCR, along with additional untyped strains demonstrated only at microscopy. The detection of Blastocystis in local chicken hosts may have a higher epidemiological and public health significance due to its socioeconomic importance, large populations and for being a popular protein source. It may be cause for concern as potential risk to immunocompromised humans. The subtypes ST2, ST4 and ST9 may be also be locally relevant to different animal species, such as swine (Moura et al., 2018), in addition to humans.
CONCLUSION
Our study has shown the occurrence of three subtypes (ST2, ST4 and ST9) of Blastocystis sp. in different domestic and wild avian species in Brazil (Belo Horizonte). The obtained subtypes have chronological (2009-2017), host, and, potentially, pathogenicity differences. Blastocystis subtypes may have been circulating among different avian host species in the local environment.
REFERENCES
-
Aykur M, Camyar A, Türk BG, et al. Evaluation of association with subtypes and alleles of Blastocystis with chronic spontaneous urticaria. Acta Tropica 2004;231:106455. https://doi.org/10.1016/j.actatropica.2022.106455
» https://doi.org/10.1016/j.actatropica.2022.106455 -
Aykur M, Malatyali E, Demirel F, Cömert-Koçak B, et al. Blastocystis: a mysterious member of the gut microbiome. Microorganisms 2024;12(3):461. https://doi.org/10.3390/microorganisms12030461
» https://doi.org/10.3390/microorganisms12030461 -
Barbosa CV, Barreto MM, Andrade RD, et al. Intestinal parasite infections in a rural community of Rio de Janeiro (Brazil): prevalence and genetic diversity of Blastocystis subtypes. Public Library of Science One 2018;13(3):e0193860. https://doi.org/10.1371/journal.pone.0193860
» https://doi.org/10.1371/journal.pone.0193860 -
Cian A, El Safadi D, Osman M et al. Molecular epidemiology of Blastocystis sp. in various animal groups from two French zoos and evaluation of potential zoonotic risk. Public Library of Science One 2017;12(1):e0169659. https://doi.org/10.1371/journal.pone.0169659
» https://doi.org/10.1371/journal.pone.0169659 -
De Palma G, Nadal I, Medina M, et al. Intestinal dysbiosis and reduced immunoglobulin-coated bacteria associated with coeliac disease in children. BMC Microbiology 2010;10:1-7. https://doi.org/10.1186/1471-2180-10-63
» https://doi.org/10.1186/1471-2180-10-63 - Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 1985;39:783-91.
-
Gabrielli S, Palomba M, Furzi F, et al. Molecular subtyping of Blastocystis sp. isolated from farmed animals in southern Italy. Microorganisms 2021;9(8):1656. https://doi.org/10.3390/microorganisms9081656
» https://doi.org/10.3390/microorganisms9081656 -
Gantois N, Lamot A, Seesao Y, et al. First report on the prevalence and subtype distribution of Blastocystis sp. in edible marine fish and marine mammals: a large scale-study conducted in Atlantic Northeast and on the coasts of Northern France. Microorganisms 2020;8(3):460. https://doi.org/10.3390/microorganisms8030460
» https://doi.org/10.3390/microorganisms8030460 - Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 1999;41:95-8.
-
Kodio A, Coulibaly D, Koné AK, et al. Blastocystis colonization is associated with increased diversity and altered gut bacterial communities in healthy Malian children. Microorganisms 2019;7(12). https://doi. org/10.3390/microorganisms7120649
» https://doi. org/10.3390/microorganisms7120649 -
Koehler AV, Herath HD, Hall RS, et al. Marked genetic diversity within Blastocystis in Australian wildlife revealed using a next generation sequencing-phylogenetic approach. International Journal for Parasitology: Parasites and Wildlife 202;23:100902. https://doi.org/10.1016/j.ijppaw.2023.100902
» https://doi.org/10.1016/j.ijppaw.2023.100902 -
Liu L, Sun H, Wang Y, et al. Blastocystis is widely distributed in mammals and aves. Durham: Research Square; 2022. https://doi.org/10.21203/rs.3.rs-1467419/v1
» https://doi.org/10.21203/rs.3.rs-1467419/v1 -
Malheiros AF, Stensvold CR, Clark CG, et al. Molecular characterization of Blastocystis obtained from members of the indigenous Tapirapé ethnic group from the Brazilian Amazon region, Brazil. The American Journal of Tropical Medicine and Hygiene 2011;85(6):1050. https://doi.org/10.4269/ajtmh.2011.11-0481
» https://doi.org/10.4269/ajtmh.2011.11-0481 -
Maloney JG, Molokin A, Cunha MJ da, et al. Blastocystis subtype distribution in domestic and captive wild bird species from Brazil using next generation amplicon sequencing. Parasite Epidemiology and Control 2020;9:e00138. https://doi.org/10.1016/j.parepi.2020.e00138
» https://doi.org/10.1016/j.parepi.2020.e00138 -
Marques MV, Junior FC, Assis AD de, et al. Serologic, parasitic, and bacteriologic assessment of captive cracids (Aves: Galliformes: Cracidae) in Brazil. Journal of Zoo and Wildlife Medicine 2013;44(1):27-34. https://doi.org/10.1638/1042-7260-44.1.27
» https://doi.org/10.1638/1042-7260-44.1.27 -
Melo GB, Bosqui LR, Costa IN, et al. Current status of research regarding Blastocystis sp., an enigmatic protist, in Brazil. Clinics 2021;76:e2489. https://doi.org/10.6061/clinics/2021/e2489
» https://doi.org/10.6061/clinics/2021/e2489 -
Moura RG, Oliveira-Silva MB, et al. Occurrence of Blastocystis spp. in domestic animals in Triângulo Mineiro area of Brazil. Revista da Sociedade Brasileira de Medicina Tropical 2018;51:240-3. https://doi.org/10.1590/0037-8682-0484-2016
» https://doi.org/10.1590/0037-8682-0484-2016 - Nei M, Kumar S. Molecular evolution and phylogenetics. Oxford: Oxford University Press; 2000.
-
Rzhetsky A, Nei M. A simple method for estimating and testing minimum evolution trees. Molecular Biology and Evolution 1992;9:945-67. https://doi.org/10.1093/oxfordjournals.molbev.a040771
» https://doi.org/10.1093/oxfordjournals.molbev.a040771 -
Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 1987;4:406-25. https://doi.org/10.1093/oxfordjournals.molbev.a040454
» https://doi.org/10.1093/oxfordjournals.molbev.a040454 -
Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences 1977;74(12):5463-7. https://doi.org/10.1073/pnas.74.12.5463
» https://doi.org/10.1073/pnas.74.12.5463 -
Skotarczak B. Genetic diversity and pathogenicity of Blastocystis. Annals of Agricultural and Environmental Medicine 2018;25(3):411-6. https://doi.org/10.26444/aaem/81315
» https://doi.org/10.26444/aaem/81315 - Stensvold CR, Alfellani M, Clark CG. Levels of genetic diversity vary dramatically between Blastocystis subtypes. Infection, Genetics and Evolution 2012;12(2):263-73.
-
Stensvold CR, Lewis HC, Hammerum AM et al. Blastocystis: unravelling potential risk factors and clinical significance of a common but neglected parasite. Epidemiology & Infection 2009;137(11):1655-63. https://doi.org/10.1017/S0950268809002672
» https://doi.org/10.1017/S0950268809002672 -
Stensvold R, Brillowska-Dabrowska A, Nielsen HV, et al. Detection of Blastocystis hominis in unpreserved stool specimens by using polymerase chain reaction. Journal of Parasitology 2006;92(5):1081-7. https://doi.org/10.1645/GE-840R.1
» https://doi.org/10.1645/GE-840R.1 -
Tamura K, Nei M, Kumar S. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proceedings of the National Academy of Sciences (USA) 2004;101:11030-11035. https://doi.org/10.1073/pnas.0404206101
» https://doi.org/10.1073/pnas.0404206101 -
Tamura K, Stecher G, Kumar S. MEGA 11: molecular evolutionary genetics analysis version 11. Molecular Biology and Evolution 2021; 38(7):3022-7. https://doi.org/10.1093/molbev/msab120
» https://doi.org/10.1093/molbev/msab120 -
Tan KSW. Blastocystis in humans and animals: new insights using modern methodologies. Veterinary Parasitology 2004;126(1-2):121-44. https://doi.org/10.1016/j.vetpar.2004.09.017
» https://doi.org/10.1016/j.vetpar.2004.09.017 -
Teow WL, Ng GC, Chan PP, et al. A survey of Blastocystis in reptiles. Parasitology Research 1992;78:453-5. https://doi.org/10.1007/BF00931705
» https://doi.org/10.1007/BF00931705 -
Valença Barbosa C, Batista RJ, Igreja RP, et al. Distribution of Blastocystis subtypes isolated from humans from an urban community in Rio de Janeiro, Brazil. Parasites & Vectors 2017;10:1-9. https://doi.org/10.1186/s13071-017-2458-0
» https://doi.org/10.1186/s13071-017-2458-0 -
Wawrzyniak I, Poirier P, Viscogliosi E et al. Blastocystis, an unrecognized parasite: an overview of pathogenesis and diagnosis. Therapeutic Advances in Infectious Disease 2013;1(5):167-78. https://doi.org/10.1177/2049936113504754
» https://doi.org/10.1177/2049936113504754 -
Yoon HS, Andersen RA, Boo SM, et al. Stramenopiles. In: Schaechter M, editor. Encyclopedia of microbiology. 3rd ed. London: Academic Press; 2009. p.721-31. https://doi.org/10.1016/B978-012373944-5.00253-4
» https://doi.org/10.1016/B978-012373944-5.00253-4
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Funding
The authors thank CAPES, CNPq and FAPEMIG for scholarships, and to FEPE (Fundação de Apoio ao Ensino Pesquisa e Extensão, Escola de Veterinária, UFMG) for administrative support.
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Data availability statement
Data will be available upon request.
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Disclaimer/Publisher’s Note
The published papers’ statements, opinions, and data are those of the individual author(s) and contributor(s). The editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions, or products referred to in the content.
Data availability
Data will be available upon request.
Publication Dates
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Publication in this collection
01 Nov 2024 -
Date of issue
2024
History
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Received
15 Aug 2023 -
Accepted
30 May 2024
