ABSTRACT:

This study detected Cryptosporidium spp. in cultivated oysters and the natural oyster stock of the state of Maranhão and determine the elective tissue(s) to examine this protozoan. For this purpose, 200 cultivated oysters were purchased from the municipality of Raposa and another 100 from Paço do Lumiar. Additionally, 100 oysters were extracted from the natural stock of the municipality of Primeira Cruz, thus making up a total of 400 oysters. They were grouped into 80 pools consisting of 5 oysters each. From each pool, the gills and visceral mass were removed to obtain 160 pools, 80 pools for the gill group and another 80 for the visceral mass group. Then, DNA was extracted from each pool using a commercial kit with modifications. Subsequently, the protozoan DNA was detected using nested polymerase chain reaction. With this technique, the DNA of the protozoan under investigation was detected in 2.5% (n = 2/80) of the pools containing gills, with 1.25% of the pools (n = 1/80) belonging to the cultivation group of oysters and the other 1.25% (n = 1/80) to the natural stock. With the results obtained in this study, it was concluded that the analyzed oysters of the genus Crassostrea, from cultivation and natural stock groups, found in the state of Maranhão, were contaminated by Cryptosporidium spp. and may become potential sources of infection in humans and other animals. In addition, the gills are the elective tissue for the study of Cryptosporidium spp. in oysters.

Key words:
bivalve mollusc; cryptosporidiosis; nested polymerase chain reaction

RESUMO:

Objetivou-se com o estudo detectar Cryptosporidium sp. em ostras de cultivo e estoque natural no estado do Maranhão e determinar o(s) tecido(s) eletivo(s) para pesquisa desse protozoário. Para a realização do estudo foram adquiridas 200 ostras de cultivo do município de Raposa e 100 de Paço do Lumiar, além de 100 ostras extraídas de estoque natural do município de Primeira Cruz, totalizando 400 ostras. Estas foram agrupadas em 80 pools constituídos por cinco animais. De cada pool, as brânquias e a massa visceral foram removidas totalizando 160 pools, sendo 80 para o grupo das brânquias e 80 para o grupo de massa visceral. Na sequência, procedeu-se à extração de DNA de cada pool com a utilização de kit comercial com modificações. Posteriormente, realizou-se a detecção do DNA do protozoário por meio da técnica de Nested-PCR. Com a técnica utilizada, foi detectado o DNA do protozoário pesquisado em 2,5% (n=2/80) pools apenas de brânquias, sendo 1,25% pools (n=1/80) oriundos de cultivo e os outros 1,25% (n=1/80) de estoque natural. Com os resultados obtidos nesse estudo, conclui-se que as ostras analisadas do gênero Crassostrea sp., oriundas de cultivo e estoque natural no estado do Maranhão, estavam contaminadas por Cryptosporidium sp. e podem se reverter em fontes potenciais para seres humanos e outros animais. Para a pesquisa de Cryptosporidium sp. em ostras, as brânquias são o tecido eletivo.

Palavras-chave:
molusco bivalve; criptosporidiose; nested-PCR

INTRODUCTION:

Cryptosporidium spp. is an obligate intracellular protozoan that completes its biological cycle in different types of vertebrate epithelial cells, especially those of the gastrointestinal tract (THOMPSON et al., 2016THOMPSON, A. R. C. et al. Cryptosporidium - What is it. Food and Waterborne Parasitology, v.4, p.54-61, 2016. Available from: <Available from: https://www.sciencedirect.com/science/article/pii/S2405676616300142 >. Accessed: Jul. 28, 2019. doi: 10.1016/j.fawpar.2016.08.004.
https://www.sciencedirect.com/science/ar... ; KVÁČ et al., 2016KVÁČ, M. et al. Cryptosporidium proliferans n. sp. (Apicomplexa Cryptosporidiidae): molecular and biological evidence of cryptic species within gastric Cryptosporidium of mammals. PLoS One, v.11, n.1, p.e0147090, 2016. Available from: <Available from: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0147090 >. Accessed: Jul. 28, 2019. doi: 10.1371/journal.pone.0147090.
https://journals.plos.org/plosone/articl... ). There are descriptions of 30 Cryptosporidium species and genotypes that affect birds, reptiles, amphibians, fish, and mammals (LI et al., 2015LI, X. et al. Cryptosporidium rubeyi n. sp. (Apicomplexa Cryptosporidiidae) in multiple Spermophilus ground squirrel species. Internacional Journal for Parasitology: Parasites and Wildlife, v.4, n.3, p.343-350, 2015. Available from: <Available from: https://www.sciencedirect.com/science/article/pii/S2213224415300110 >. Accessed: Mar. 28, 2021. doi: 10.1016/j.ijppaw.2015.08.005.
https://www.sciencedirect.com/science/ar... ; HOLUBOVÁ et al., 2016HOLUBOVÁ, N. et al. Cryptosporidium avium n. sp. (Apicomplexa Cryptosporidiidae) in birds. Parasitology Research, v.115, p.2243-2251, 2016. Available from: <Available from: https://link.springer.com/article/10.1007/s00436-016-4967-8 >. Accessed: Mar. 28, 2021. doi: 10.1007/s00436-016-4967-8.
https://link.springer.com/article/10.100... ). Of these, 17 species (C. parvum, C. hominis, C. canis, C. felis, C. muris, C. suis, C. meleagridis, C. andersoni, C. bovis, C. ryanae, C. baileyi, C. galli, C. molnari, C. scophithalmi, C. saurophilum, C. serpentis, and C. wrairi) and 2 genotypes (“Cervid” and “Monkey”) are related to human infection (SUNNOTEL et al., 2006SUNNOTEL, O. et al. Under the microscope, Cryptosporidium. Letters in Applied Microbiology, v.43, n.1, p.7-16, 2006. Available from: <Available from: https://sfamjournals.onlinelibrary.wiley.com/doi/epdf/10.1111/j.1472-765X.2006.01936.x >. Accessed: Mar. 28, 2021. doi: 10.1111/j.1472-765X.2006.01936.x.
https://sfamjournals.onlinelibrary.wiley... ; SMITH et al., 2007SMITH, H. V. et al. Cryptosporidium and Giardia as foodborne zoonoses. Veterinary Parasitology , v.149, p.29-40, 2007. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/17728067 >. Accessed: Jul. 28, 2019. doi: 10.1016/j.vetpar.2007.07.015.
https://www.ncbi.nlm.nih.gov/pubmed/1772... ; FAYER et al., 2008FAYER, R. et al. Cryptosporidium ryanae n. sp. (Apicomplexa Cryptosporidiidae) in cattle (Bos taurus). Veterinary Parasitology , v.156, n.3-4, p.191-198, 2008. Available from: <Available from: https://www.sciencedirect.com/science/article/abs/pii/S0304401708002781 >. Accessed: Mar. 28, 2021. doi: 10.1016/j.vetpar.2008.05.024.
https://www.sciencedirect.com/science/ar... ).

Cryptosporidium spp. infect via a fecal-oral transmission route through the ingestion of oocysts present in water (SMITH et al., 2007SMITH, H. V. et al. Cryptosporidium and Giardia as foodborne zoonoses. Veterinary Parasitology , v.149, p.29-40, 2007. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/17728067 >. Accessed: Jul. 28, 2019. doi: 10.1016/j.vetpar.2007.07.015.
https://www.ncbi.nlm.nih.gov/pubmed/1772... ). An infected host can excrete a large number of oocysts; however, it is assumed that the ingestion of only one oocyst of this protozoan is sufficient for the development of infection in immunodeficient individuals or in those with chronic or transient immunosuppression (ROSE et al., 2002ROSE, J. B. et al. Risk and control of waterborne cryptosporidiosis. FEMSMicrobiology Reviews , v.26, n.2, p.113-123, 2002. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/12069877/ >. Accessed: Mar. 28, 2021. doi: 10.1111/j.1574-6976.2002.tb00604.x.
https://pubmed.ncbi.nlm.nih.gov/12069877... ). In addition to being small and buoyant, oocysts are resistant to environmental conditions such as high temperature and salinity (SUTTHIKORNCHAI et al., 2016SUTTHIKORNCHAI, C. et al. Oyster is an effective transmission vehicle for Cryptosporidium infection in human. Asian Pacific Journal of Tropical Medicine, v.9, n.6, p.562-566, 2016. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/27262067 >. Accessed: Jul. 28, 2019. doi: 10.1016/j.apjtm.2016.04.018.
https://www.ncbi.nlm.nih.gov/pubmed/2726... ).

Inadequate disposal of domestic sewage and industrial effluents, in addition to the runoff of wastewater containing feces of domestic animals and humans, are potential causes of environmental contamination by Cryptosporidium spp. (FRANCO et al., 2012FRANCO, R. M. B. et al. Environmental parasitology: Methods for concentrating and detecting Cryptosporidium spp. and Giardia spp. in water samples. Revista de Patologia Tropical, v.41, n.2, p.119-135, 2012. Available from: <Available from: http://www.revistas.ufg.br/index.php/iptsp/article/view/19320 >. Accessed: Mar. 28, 2021. doi: 10.5216/rpt.v41i2.19320.
http://www.revistas.ufg.br/index.php/ipt... ). Therefore, the use of contaminated water and its natural resources (fish, crustaceans, and molluscs) are important points to be considered by public health authorities in the study of this protozoan (GIANGASPERO et al., 2014GIANGASPERO, A. et al. Cryptosporidium parvum genotype IIa and Giardia duodenalis assemblage A in Mytilus galloprovincialis on sale at local food markets. International Journal of Food Microbiology, v.171, p.62-67, 2014. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/24334090 >. Accessed: Jul. 28, 2019. doi: 10.1016/j.infoodmicro.2013.11.022.
https://www.ncbi.nlm.nih.gov/pubmed/2433... ; SUTTHIKORNCHAI et al., 2016SUTTHIKORNCHAI, C. et al. Oyster is an effective transmission vehicle for Cryptosporidium infection in human. Asian Pacific Journal of Tropical Medicine, v.9, n.6, p.562-566, 2016. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/27262067 >. Accessed: Jul. 28, 2019. doi: 10.1016/j.apjtm.2016.04.018.
https://www.ncbi.nlm.nih.gov/pubmed/2726... ).

Cosmopolitan distribution, sessility, and efficient filtering behavior make oysters capable of bioaccumulating pollutants and pathogens, such as protozoa present in water (SCHETS et al., 2007SCHETS, F. M. et al. Cryptosporidium and Giardia in commercial and non-commercial oysters (Crassostrea gigas) and water from the Oosterschelde, The Netherlands. International Journal of Food Microbiology , v.113, p.189-194, 2007. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/16973232 >. Accessed: Jul. 28, 2019. doi: 10.1016/j.infoodmicro.2006.06.031.
https://www.ncbi.nlm.nih.gov/pubmed/1697... ); therefore, it is important to use bioindicators in aquatic environments (PALOS LADEIRO et al., 2013PALOS LADEIRO, M. P. et al. Protozoa interaction with aquatic invertebrate: interest for water courses biomonitoring. Environmental Science and Pollution Research International, v.20, n.2, p.778-789, 2013. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/23001759 >. Accessed: Jul. 28, 2019. doi: 10.1007/s11356-012-1189-1.
https://www.ncbi.nlm.nih.gov/pubmed/2300... ).

Oysters are considered as delicacies worldwide and in Brazil and can be harvested from natural banks or cultivated. The state of Maranhão has a vast coastline (approximately 640 km long) that makes it suitable for oyster farming. Along the coast of Maranhão there are estuarine areas where oysters of the genus Crassostrea occur naturally (FRANÇA et al., 2013FRANÇA, V. L. et al. Selection of potential areas for the cultivation of native oyster, Crassostrea spp. and Sururu, Mytella falcata, in Raposa, Maranhão. Science of the Sea Archives, v.46, n.1, p.62-75, 2013. Available from: <Available from: http://www.periodicos.ufc.br/arquivosdecienciadomar/article/view/891 >. Accessed: Jul. 28, 2019. doi: 10.32360/acmar.v46i1.891.
http://www.periodicos.ufc.br/arquivosdec... ). In addition, the government of the state of Maranhão has developed public policies that promote the cultivation of this type of catch.

Therefore, considering that monitoring the occurrence of the oocysts of zoonotic disease-causing protozoa in oysters is important to determine whether their consumption poses a risk to human health, in addition to the fact that oysters can serve as bioindicators of water contamination. The present study detected Cryptosporidium spp. in cultivated and wild oysters in the state of Maranhão and determined the elective tissue(s) to study this protozoan.

MATERIALS AND METHODS:

Study site and oyster (Crassostrea spp.) collection

The samples used in the study were acquired from mariculturists in the municipalities of Paço do Lumiar (02º31′S and 44º06′W) and Raposa (02º25′S and 44º06′W) and from shellfish farmers in the municipality of Primeira Cruz (2°33′S and 43°21′W), state of Maranhão, Brazil. The selection of the study site was based on the presence of oyster crops and extraction sites (Figure 1).

Figure 1
Collection sites of the oysters of the genus Crassostrea in the state of Maranhão.

In the period from September to December 2018, 400 live oysters were acquired, 200 from the municipality of Raposa, 100 from Primeira Cruz, and 100 from Paço do Lumiar. These were grouped into 80 pools consisting of 5 animals each. In each pool, the oysters’ gills and the region of the digestive glands (called visceral mass in this study) were removed. This protocol resulted in the formation of 160 pools; of which, 80 pools belonged to the gill group and 80 pools belonged to the visceral mass group. When forming pools, it was taken care that the oysters from different municipalities and of different forms of production (cultivation and natural stock) were not mixed. The sampling design of the study followed the protocol proposed by ESMERINI et al. (2010ESMERINI, P. O. et al. Analysis of marine bivalve shellfish from the fish market in Santos city, São Paulo state, Brazil, for Toxoplasma gondii. Veterinary Parasitology, v.170, n.1-2, p.8-13, 2010. Available from: <Available from: https://www.sciencedirect.com/science/article/abs/pii/S0304401710000701?via%3Dihub >. Accessed: Jul. 28, 2019. doi: 10.1016/j.vetpar.2010.01.036.
https://www.sciencedirect.com/science/ar... ).

Sample processing

The oysters were transported alive to the Food and Water Physicochemistry Laboratory of the State University of Maranhão (UEMA) and refrigerated within 12 h after acquisition. In this laboratory, the outer surfaces of the oyster shells were washed with distilled water, and dirt [mud and barnacles (Cirripedia)] was removed with the aid of a brush and a sharp instrument.

Subsequently, the animals’ heights (dorsoventral axis) and lengths were measured using a manual caliper, and their total mass was determined using a digital scale (0.001 g precision). Groups of five animals were formed based on similar morphometric characteristics and numbered. Subsequently, the shells of the oysters were opened using a sharp instrument, and once the shells were removed, the oysters were weighed on a scale. Next, the gills and the portions of visceral mass were excised with scissors, their weights were measured, and the pools were separately stored in labeled 2.0-mL microtubes at −20 °C.

The samples were thawed and the tissues (gills and visceral mass) of the sample groups were macerated in a crucible using a scalpel. Thus, each sample consisted of gills or visceral mass of five animals. Then, aliquots of approximately 0.5 mL were transferred to sterile 2.0 mL microtubes and stored at −20 °C until DNA extraction was performed.

DNA extraction

DNA extraction and polymerase chain reaction (PCR) were performed at the molecular biology laboratory, a part of the multiuser graduate laboratory (LAMP) at UEMA. The Wizard^® genomic DNA purification kit (Promega) was used to extract DNA from the collected tissue samples (gills and visceral mass) according to the manufacturer’s instructions, with some modifications related to the amount of sample (approximately 100 mg) and the procedure to break the oocyst wall [0.5 mm glass spheres were used at a ratio of 1:1 and 400 μL of Tris-ethylenediaminetetraacetic acid (TE), followed by homogenization in a vortex mixer for 10 min at 2800 rpm].

The extracted DNA was quantified using a spectrophotometer; absorbance was measured at 260 nm, and the 260/280 nm ratio was used to determine the purity of the samples (SAMBROOK & RUSSEL, 2001SAMBROOK, J.; RUSSEL, D. W. Molecular Cloning. The Laboratory Manual. 3rd ed. New York: Cold Spring Harbor Laboratory Press, 2001. 2100p.). Next, the concentration of the samples was adjusted to approximately 200 ng/μL using TE (pH 8.0), and the DNA was stored at −20 °C until nested PCR (n-PCR) was performed.

n-PCR

Detection of Cryptosporidium spp. DNA was performed according to the protocol proposed by SILVA et al. (2013SILVA, S. O. S. 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. Experimental Parasitology , v.135, n.3, p.551-557, 2013. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/24036321 >. Accessed: Jul. 28, 2019. doi: 10.1016/j.exppara.2013.09.003.
https://www.ncbi.nlm.nih.gov/pubmed/2403... ). The amplification of fragments of the 18S rRNA subunit gene (611-bp fragment, GenBank number: AF108862) was performed using the n-PCR technique and the SHP1 (5′ ACC TAT CAG CTT TAG ACG GTA GGG TAT 3′) and SHP2 (5′ TTC TCA TAA GGT GCT GAA GGA GTA AGG 3′) primers in the first amplification and SHP3 (5′ ACA GGG AGG TAG TGA CAA GAA ATA ACA 3′) and SSU-R3 primers (5′ AAG GAG TAA GGA ACA ACC TCC A 3′) in the second amplification. In the first amplification, 25 μL of solution containing 3.0 μL of DNA sample, 12.5 μL of GoTaq^® colorless master mix (Promega^®), 7.5 μL of ultrapure water, and 10 pmol of each oligonucleotide were used.

Reactions were performed using the EP gradient thermal cycler (Eppendorf^®) under the following conditions: initial denaturation at 94 ^oC for 3 min, followed by 39 cycles at 94 ^oC for 45 s; 56 ^oC for 45 s; and 72 ^oC for 60 s, with final extension at 72 ^oC for 7 min. In the second amplification, the same conditions were used, replacing the oligonucleotides with the appropriate pair and using 3.0 μL of the product obtained from the first amplification. A positive control (DNA extracted from oocysts isolated from dog feces provided by the Universidade Estadual Paulista, Araçatuba Campus, from cultivation) and a negative control (water) were added to each amplification batch.

Electrophoresis and record of the amplification products

Aliquots of 5.0 μL of the samples amplified in n-PCR were homogenized with 5.0 µL of 5X sample buffer [glycerol (50%), bromophenol blue (0.125%), xylene cyanol (0.125%), and TE (pH 8.0)] and subjected to electrophoresis on 1.5% agarose gel containing 4 µL of 10 mg/mL ethidium bromide making up a final concentration of 0.4 mg/mL agarose gel. Electrophoresis was performed in a horizontal vat with 1X Tris-borate-ethylenediaminetetraacetic acid buffer solution (TBE), pH 8.0, for 45 min at 120 V. The bands were visualized under ultraviolet light, and digital images were recorded with the L-PIX Image EX software (Loccus Biotecnologia, Brazil).

RESULTS AND DISCUSSION:

To our knowledge, this is the first study conducted in the state of Maranhão for the detection of Cryptosporidium spp. DNA in oysters. Cryptosporidium spp. was detected in tissue homogenates from the gills alone after molecular analysis of 160 pools were conducted (Figure 2).

Figure 2
Agarose gel electrophoresis performed after n-PCR for determining the presence of Cryptosporidium spp. in the gill tissue samples obtained from Crassostrea spp. oysters of the state of Maranhão: Kb molecular weight marker of 1 kb; PC positive control; 1 and 2: samples demonstrating amplification of a product of 611 base pairs; NC negative control.

In total, two gill pools (2.5%) were positive for the protozoan, one from the municipality of Primeira Cruz (n = 1/80; 1.25%) and the other from Paço do Lumiar (n = 1/80; 1.25%). The oysters from the former were obtained from the natural bank and those from the latter were obtained from suspended longline cultivation.

OLIVEIRA et al. (2016OLIVEIRA, G. F. M. et al. Mussels (Leg Leg) as bioindicator of environmental contamination by Cryptosporidium species with zoonotic potential. International Journal for Parasitology: Parasites and Wildlife, v.5, p.28-33, 2016. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/26977402 >. Accessed: Jul. 28, 2019. doi: 10.1016/j.ijppaw.2016.01.004.
https://www.ncbi.nlm.nih.gov/pubmed/2697... ) evaluated 72 samples of mussels (Perna perna) from the municipality of Mangaratiba, state of Rio de Janeiro, and detected the DNA of Cryptosporidium spp. in 29.2% of the samples analyzed using n-PCR. According to ROBERTSON (2007ROBERTSON, L. J. The potential for marine bivalve shellfish to act as transmission vehicles for outbreaks of protozoan infections in humans: a review. International Journal of Food Microbiology , v.120, p.201-216, 2007. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/17928081 >. Accessed: Jul. 28, 2019. doi: 10.1016/j.infoodmicro.2007.07.058.
https://www.ncbi.nlm.nih.gov/pubmed/1792... ) and GUIGUET LEAL & FRANCO (2008GUIGUET LEAL, D. A.; FRANCO, R. M. B. Bivalve molluscs intended for human consumption as vectors of pathogenic protozoa: detection methodologies and control standards. Panamericana Journal of Infectology, v.10, p.48-57, 2008. Available from: <Available from: https://www.researchgate.net/publication/311264224_Moluscos_Bivalves_Destinados_ao_Consumo_Humano_como_Vetores_de_Protozoarios_Patogenicos_Metodologia_de_Deteccao_e_Normas_de_Controle >. Accessed: Jul. 28, 2019.
https://www.researchgate.net/publication... ), the occurrence of this parasite in edible bivalve species has already been documented in European countries (Portugal, Spain, Italy, France, the Netherlands, England, Ireland, and Northern Ireland), North America (USA and Canada), Oceania (New Zealand and Australia), and Africa (Egypt).

In Brazil specifically, contamination by Cryptosporidium spp. oocysts observed in the tissue homogenates of gills of C. rhizophorae and vongoles (Tivela mactroides) was first documented in 2008, with frequencies of 10% and 50%, respectively (GUIGUET LEAL et al., 2008LEAL, D. A.; FRANCO, R. M. B. Bivalve molluscs intended for human consumption as vectors of pathogenic protozoa: detection methodology and control standards. Revista Panamericana Infectologia, v.10, n.4, p.48-57, 2008. Available from: <Available from: https://www.researchgate.net/publication/311264224_Moluscos_Bivalves_Destinados_ao_Consumo_Humano_como_Vetores_de_Protozoarios_Patogenicos_Metodologia_de_Deteccao_e_Normas_de_Controle > Accessed: Jul. 28, 2019.
https://www.researchgate.net/publication... ).

Molecular techniques have been widely used for conducting research on the diagnosis and detection of Cryptosporidium spp. over the past 25 years (SMITH et al., 2006SMITH, H. V. et al. Tools for investigating the environmental transmission of Cryptosporidium and Giardia infections in humans. Trends in Parasitology, v.22, n.4, p.160-167, 2006. Available from: <Available from: https://www.sciencedirect.com/science/article/abs/pii/S1471492206000547 >. Accessed: Mar. 28, 2021. doi: 10.1016/j.pt.2006.02.009.
https://www.sciencedirect.com/science/ar... ; THOMPSON and ASH, 2015), and n-PCR is a very sensitive molecular assay based on the amplification of a sequence present in a previously amplified fragment (RIBEIRO et al., 2015RIBEIRO, L. A. et al. Detection of Toxoplasma gondii DNA in Brazilian oysters (Crassostrea rhizophorae). Genetics and Molecular Research, v.14, n.2, p.4658-4665, 2015. Available from: <Available from: http://dx.doi.org/10.4238/2015.May.4.25 >. Accessed: Jul. 17, 2021.
http://dx.doi.org/10.4238/2015.May.4.25... ).

The detection of Cryptosporidium spp. oocysts in environmental or animal tissue samples is considered a challenge although n-PCR is sensitive in terms of detecting this protozoan. According to DUMÈTRE & DARDÉ (2003DUMÈTRE, A.; DARDÉ, M. L. How to detect Toxoplasma gondii oocysts in environmental samples? FEMS Microbiology Reviews, v.27, n.5, p.651-661, 2003. Available from: <Available from: https://www.sciencedirect.com/science/article/abs/pii/S0168644503000718 >. Accessed: Mar. 28, 2021. doi: 10.1016/S0168-6445(03)00071-8.
https://www.sciencedirect.com/science/ar... ), there are a variety of techniques for breaking the walls of protozoan oocysts that include in vitro excystation, digestion with proteinase K, grinding with glass beads, and the use of thermal shocks. However, there is no standard protocol related to the storage temperature or number of freeze/thaw cycles to be employed. In the present study, glass spheres were used, a protocol that is similar to that used by OLIVEIRA et al. (2016OLIVEIRA, G. F. M. et al. Mussels (Leg Leg) as bioindicator of environmental contamination by Cryptosporidium species with zoonotic potential. International Journal for Parasitology: Parasites and Wildlife, v.5, p.28-33, 2016. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/26977402 >. Accessed: Jul. 28, 2019. doi: 10.1016/j.ijppaw.2016.01.004.
https://www.ncbi.nlm.nih.gov/pubmed/2697... ), with the exception of the use of thermal shocks. Differences in the procedures used for DNA extraction may influence the results obtained in different studies.

Several factors are related to the identification of Cryptosporidium spp. in bivalve molluscs, such as the tissue used (LEAL & FRANCO, 2008LEAL, D. A.; FRANCO, R. M. B. Bivalve molluscs intended for human consumption as vectors of pathogenic protozoa: detection methodology and control standards. Revista Panamericana Infectologia, v.10, n.4, p.48-57, 2008. Available from: <Available from: https://www.researchgate.net/publication/311264224_Moluscos_Bivalves_Destinados_ao_Consumo_Humano_como_Vetores_de_Protozoarios_Patogenicos_Metodologia_de_Deteccao_e_Normas_de_Controle > Accessed: Jul. 28, 2019.
https://www.researchgate.net/publication... ), the cultivation environment with the presence of wild and domestic animals (KVÁČ et al., 2016KVÁČ, M. et al. Cryptosporidium proliferans n. sp. (Apicomplexa Cryptosporidiidae): molecular and biological evidence of cryptic species within gastric Cryptosporidium of mammals. PLoS One, v.11, n.1, p.e0147090, 2016. Available from: <Available from: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0147090 >. Accessed: Jul. 28, 2019. doi: 10.1371/journal.pone.0147090.
https://journals.plos.org/plosone/articl... ), and rainfall index associated with scarcity of basic sanitation (GIANGASPERO et al., 2014GIANGASPERO, A. et al. Cryptosporidium parvum genotype IIa and Giardia duodenalis assemblage A in Mytilus galloprovincialis on sale at local food markets. International Journal of Food Microbiology, v.171, p.62-67, 2014. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/24334090 >. Accessed: Jul. 28, 2019. doi: 10.1016/j.infoodmicro.2013.11.022.
https://www.ncbi.nlm.nih.gov/pubmed/2433... ; SUTTHIKORNCHAI et al., 2016SUTTHIKORNCHAI, C. et al. Oyster is an effective transmission vehicle for Cryptosporidium infection in human. Asian Pacific Journal of Tropical Medicine, v.9, n.6, p.562-566, 2016. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pubmed/27262067 >. Accessed: Jul. 28, 2019. doi: 10.1016/j.apjtm.2016.04.018.
https://www.ncbi.nlm.nih.gov/pubmed/2726... ).

With regard to the tissue used, some studies describe the gill as the elective tissue for the investigation of protozoa in oysters because of the filter feeding mechanism that retains light particles in the gills (ESMERINE et al., 2010; PUTIGNANI et al., 2011), which was confirmed in the present study.

With regard to the cultivation environment, when the oysters were acquired it was possible to capture a photographic record of the proximity of the oyster extraction location to a breeding site of domestic animals (pigs) and with waste (Figure 3). In this sense, it is important to highlight that oysters reflect the conditions in which they live, which is why they are considered bioindicators of environmental contamination. The cohabitation of several species is a potential cause of environmental contamination by Cryptosporidium spp. through the drainage of wastewater with feces from these animals to the extraction and cultivation sites.

Figure 3
Environmental conditions of the oyster (Crassostrea spp.) cultivation site in the municipality of Paço do Lumiar, Maranhão. (A) movement of domestic animals and (B) deposition of solid waste.

It can be assumed that the marine environment where the sampled oysters were extracted and cultivated is contaminated with Cryptosporidium spp. oocysts and; therefore, the protozoan moves from the terrestrial environment to the marine environment. It is also speculated that the high rainfall in the state of Maranhão during the rainy season, associated with the scarcity of basic sanitation, is a source of infection by Cryptosporidium spp. in oysters in the municipalities of Primeira Cruz and Paço do Lumiar.

Although the viability of oocysts has not been confirmed through the isolation of Cryptosporidium spp. in mice, the consumption of raw or lightly cooked oysters of the genus Crassostrea, which is common on the coast of Maranhão, may be a source of transmission of Cryptosporidium spp. in humans. Moreover, there is a lack of regulatory apparatuses that regulate the maximum permissible values of pathogenic protozoa in oysters as a way to ensure the quality and safety of this catch for human consumption.

FAYER et al. (1999FAYER, R. et al. Cryptosporidium parvum in Oysters from Commercial Harvesting Sites in the Chesapeake Bay. Emerging Infectious Diseases, v.5, n.5, p.706-710, 1999. Available from: <Available from: https://wwwnc.cdc.gov/eid/article/5/5/99-0513_article >. Accessed: Mar. 28, 2021. doi: 10.3201/eid0505.990513.
https://wwwnc.cdc.gov/eid/article/5/5/99... ) conducted a study on the presence of Cryptosporidium parvum in oysters harvested from the Chesapeake Bay for human consumption and, in the three collection periods, found oocysts corresponding in size and shape to those of the investigated protozoan. With regard to the results of infectivity studies, the researchers reported that in 3 of the 16 tested sites, in the three collection periods, oocysts produced detectable infections in mice and concluded that the low rate of infectivity may reflect the small number of oocysts administered to each mouse or the lack of infectivity due to age or unknown environmental effects. Therefore, it is essential that oysters are correctly and completely cooked for ensuring the safety of the consumers of this delicacy.

CONCLUSION:

Under the conditions described in this experiment, DNA from Cryptosporidium spp. was detected in the analyzed oysters, thereby leading to the conclusion that cultivated oysters of the genus Crassostrea from those from the natural stock of the state of Maranhão may be contaminated by Cryptosporidium spp. Moreover, the gills were found to be the elective tissue for the study of this protozoan.

ACKNOWLEDGMENTS

The authors would like to thank the Dean of Research (PPG) of the State University of Maranhão for granting a scientific initiation scholarship to conduct this research (Process BIC-04052/18). This study was funded by the Foundation for Research and Scientific and Technological and Development of Maranhão (FAPEMA) , Process Universal-00885/18.

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