The effect of water source and soil supplementation on parasite contamination in organic vegetable gardens

O efeito da fonte de água e suplementação de solo na contaminação parasitária em hortas orgânicas

Fernanda Pinto Ferreira Eloiza Teles Caldart Roberta Lemos Freire Regina Mitsuka-Breganó Felipe Machado de Freitas Ana Carolina Miura Marcelle Mareze Felippe Danyel Cardoso Martins Mariana Ragassi Urbano Adilson Luiz Seifert Italmar Teodorico Navarro About the authors

Abstract

The objective of this study was to determine factors associated with vegetable contamination with zoonotic protozoan. Samples of water, soil and vegetables were collected from July/2014 to May/2016, totaling 83 samples, 21 properties of Londrina region, Paraná, Brazil. DNA amplification of Toxoplasma gondii, Cryptosporidium spp. and Giardia intestinalis in the samples was conducted using polymerase chain reaction (PCR). The PCR results were positive for T. gondii in 12.9% (8/62), Cryptosporidium spp. in 11.3% (7/62) and G. intestinalis in 25.8% (16/62) of the samples. DNA sequencing identified C. parvum in five samples and G. intestinalis Assemblage E in three. The statistical associations demonstrated greater probability of positive samples for T. gondii and for at least one of the three protozoa when the source of irrigation water was the river; a greater chance of positive samples for Cryptosporidium spp. when deer were present on the property; and a smaller chance of positive samples for at least one of the three etiologic agents when soil was supplemented with limestone. The results expose some critical contamination points, providing support for training farmers on good management practices during the production process.

Keywords:
Vegetables; water; environmental contamination; Cryptosporidium spp.; Toxoplasma gondii; Giardia intestinalis

Resumo

O trabalho teve como objetivo determinar os fatores associados à contaminação de vegetais por protozoários zoonóticos. Amostras de água, solo e vegetais foram coletadas de julho/2014 a maio/2016, totalizando 83 amostras de 21 propriedades da região de Londrina, Paraná, Brasil. A amplificação de fragmentos de DNA de T. gondii, Cryptosporidium spp. e Giardia intestinalis foi realizada por meio da reação em cadeia da polimerase (PCR). Os resultados da PCR foram positivos para T. gondii em 12,9% (8/62), Cryptosporidium spp. em 11,3% (7/62) e G. intestinalis. em 25,8% (16/62) das amostras. O sequenciamento de DNA identificou C. parvum em cinco amostras e G. intestinalis, Assemblage E em três amostras. As associações estatísticas evidenciaram maior probabilidade de amostras serem positivas para T. gondii ou para pelo menos um dos três protozoários quando a fonte de água de irrigação era o rio; uma maior chance de amostras positivas para Cryptosporidium spp. quando havia cervos na propriedade; e uma menor chance das amostras serem positivas para pelo menos um dos três agentes etiológicos quando o solo era suplementado com calcário. Os resultados expõem alguns pontos críticos de contaminação, fornecendo suporte para capacitar os agricultores em boas práticas de gestão durante o processo de produção.

Palavras-chave:
Legumes; água; contaminação ambiental; Cryptosporidium spp.; Toxoplasma gondii; Giardia intestinalis

Introduction

Vegetables, including all vegetables grown in gardens, are foods rich in vitamins, carbohydrates, fiber and minerals; are easy to digest; and offer high satiety, a high antioxidant content and a low caloric content. Due to their nutritional importance, vegetables are essential in human nutrition ( SINGH et al., 2001 Singh G, Kawatra A, Sehgal S. Nutritional composition of selected green leafy vegetables, herbs and carrots. Plant Foods Hum Nutr 2001; 56(4): 359-364. http://dx.doi.org/10.1023/A:1011873119620. PMid:11678441.
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).

In Brazil, vegetables are produced commonly by the conventional farming system; however, in recent years there was an important growth in organic cultivation. It is mainly related to the search for healthier food, with better quality and flavor, environmental preservation and pesticides’ free ( ARCHANJO et al., 2001 Archanjo LR, Brito KFW, Sauerbeck S. Alimentos orgânicos em Curitiba: consumo e significado. Rev Cad Debate 2001; 8: 1-6. ). The main differences between organic and conventional cultivation are the use, in the latter, of agricultural pesticides, chemical fertilizers and genetically modified organisms. Organic agriculture excludes these elements from the production process, using natural implements and predators ( BOURN & PRESCOTT, 2002 Bourn D, Prescott J. A comparison of the nutritional value, sensory qualities, and food safety of organically and conventionally produced foods. Crit Rev Food Sci Nutr 2002; 42(1): 1-34. http://dx.doi.org/10.1080/10408690290825439. PMid:11833635.
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; GARCIA & TEIXEIRA, 2017 Garcia JM, Teixeira P. Organic versus conventional food: A comparison regarding food safety. Food Rev Int 2017; 33(4): 424-446. http://dx.doi.org/10.1080/87559129.2016.1196490.
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; GOMIERO et al., 2011 Gomiero T, Pimentel D, Paoletti MG. Environmental Impact of Different Agricultural Management Practices: Conventional vs. Organic Agriculture. Crit Rev Plant Sci 2011; 30(1-2): 95-124. http://dx.doi.org/10.1080/07352689.2011.554355.
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).

Contamination of vegetables can occur during production, transportation, storage and commercialization; however, cultivation conditions involving the quality of irrigation water, fertilizer type, the presence of animals in the property and direct contamination from farm workers are the main risk points ( ALMEIDA et al., 2013 Almeida AJ, Lima VS, Rodrigues ABF, Di Filippo PA. Contamination with Cryptosporidium spp. in manure used as fertilizer in urban gardens. Rev Port Ciênc Vet 2013; 108(585-586): 23-27. ; AMAHMID et al., 1999 Amahmid O, Asmama S, Bouhoum K. The effect of waste water reuse in irrigation on the contamination level of food crops by Giardia cysts and Ascaris eggs. Int J Food Microbiol 1999; 49(1-2): 19-26. http://dx.doi.org/10.1016/S0168-1605(99)00058-6. PMid:10477066.
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; DIXON et al., 2013 Dixon B, Parrington L, Cook A, Pollari F, Farber J. Detection of Cyclospora, Cryptosporidium , and Giardia in Ready-to-Eat Packaged Leafy Greens in Ontario, Canada. J Food Prot 2013; 76(2): 307-313. http://dx.doi.org/10.4315/0362-028X.JFP-12-282. PMid:23433379.
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).

Toxoplasmosis is one of the most common parasitic zoonoses in the world, and it can affect all warm-blooded animals, presenting a wide variability in clinical outcomes ( DUBEY, 2010 Dubey JP. Toxoplasmosis of Animals and Humans. Maryland: CRC Press; 2010. ). Cryptosporidiosis and giardiasis are zoonoses and are described as important causes of diarrhea ( FAYER, 1997 Fayer R. Cryptosporidium and Cryptosporidiosis. Maryland: CRC Press; 1997. ; PEDROSO & AMARANTE, 2006 Pedroso RF, Amarante MK. Giardíase: aspectos parasitológicos e imunológicos. Biosaúde 2006; 8(1): 61-71. ). In an immunocompetent individual, these diseases may assume a self-limiting characteristic with clinical manifestations of low expression, but in an immunocompromised patient they may manifest in a severe and prolonged form ( MITSUKA-BREGANÓ et al., 2010 Mitsuka-Breganó R, Lopes-Mori FMR, Navarro IT. Toxoplasmose adquirida na gestação e congênita: vigilância em saúde, diagnóstico, tratamento e condutas. Londrina: Eduel; 2010. http://dx.doi.org/10.7476/9788572166768.
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; PEDROSO & AMARANTE, 2006 Pedroso RF, Amarante MK. Giardíase: aspectos parasitológicos e imunológicos. Biosaúde 2006; 8(1): 61-71. ; SHIKANI & WEISS, 2014 Shikani H, Weiss LM. Human cryptosporidiosis: a clinical perspective. In: Cacció SM, Widmer G. Cryptosporidium: parasite and disease. 1st ed. New York: Springer; 2014. p. 383-421. http://dx.doi.org/10.1007/978-3-7091-1562-6_9.
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). The description of these protozoa in food and waterborne outbreaks ( BERGER et al., 2010 Berger CN, Sodha SV, Shaw RK, Griffin PM, Pink D, Hand P, et al. Fresh fruit and vegetables as vehicles for the transmission of human pathogens. Environ Microbiol 2010; 12(9): 2385-2397. http://dx.doi.org/10.1111/j.1462-2920.2010.02297.x. PMid:20636374.
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; CHEUN et al., 2013 Cheun H-I, Kim C-H, Cho S-H, Ma D-W, Goo B-L, Na MS, et al. The first outbreak of giardiasis with drinking water in Korea. Osong Public Health Res Perspect 2013; 4(2): 89-92. http://dx.doi.org/10.1016/j.phrp.2013.03.003. PMid:24159537.
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; MAC KENZIE et al., 1994 Mac Kenzie WR, Hoxie NJ, Proctor ME, Gradus MS, Blair KA, Peterson DE, et al. Massive outbreak in Milwaukee of Cryptosporidium infection transmitted through the public water supply. N Engl J Med 1994; 331(3): 161-167. http://dx.doi.org/10.1056/NEJM199407213310304. PMid:7818640.
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; SILVA et al., 2005 Silva CGM, Andrade SAC, Stamford TLM. Occurrence of Cryptosporidium spp. and others parasites in vegetables consumed in natura, Recife, Brazil. Cien Saude Colet 2005; 10(Suppl): 63-69. http://dx.doi.org/10.1590/S1413-81232005000500009.
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; VAUDAUX et al., 2010 Vaudaux JD, Muccioli C, James ER, Silveira C, Magargal SL, Jung C, et al. Identification of an atypical strain of Toxoplasma gondii as the cause of a waterborne outbreak of toxoplasmosis in Santa Isabel do Ivai, Brazil. J Infect Dis 2010; 202(8): 1226-1233. http://dx.doi.org/10.1086/656397. PMid:20836703.
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; YEUNG et al., 2013 Yeung NW, Hayes K, Cowie RH. Effects of washing produce contaminated with the snail and slug hosts of Angiostrongylus cantonensis with three common household solutions. Hawaii J Med Public Health 2013;72(6 Suppl2): 83-86. PMid:23901391. ) highlights the importance of leafy vegetables, which are consumed in natura and facilitate the transmission of these protozoa and other pathogens.

Considering the health benefits of consuming vegetables, their greater inclusion in the diet of people worldwide and their risk as a source of zoonotic protozoa such as T. gondii , Cryptosporidium spp. and G. intestinalis , this work aimed to evaluate the contamination and hygienic-sanitary conditions of their production in organic gardens.

Materials and Methods

The study was carried out from July 2014 to May 2016 on 21 horticultural properties of the municipalities of Apucarana, Marilândia do Sul, Ortigueira, Rolândia and Londrina (District of Guaravera), in the state of Paraná ( Figure 1 ). As an inclusion criterion, small commercial vegetable-producing organic properties were assisted by the Organic Certification Project of the State University of Londrina. On eleven properties, water and vegetable samples were collected, and soil samples were included on the other properties.

Figure 1
Map of Paraná highlighting the municipalities where samples of vegetables, water and soil were collected and submitted for parasitological and microbiological research from 2014 to 2016, Paraná, Brazil.

Obtaining and analyzing samples of vegetables

Forty-two clumps of leafy vegetables were randomly collected, two per property (from property 1 to 21), packed in plastic bags and kept in refrigeration. For T. gondii , Cryptosporidium spp. and G. intestinalis detection, 50 g of the leaves were washed in 300 mL of 1% Tween 80 extraction solution in a plastic bag under manual shaking for 10 minutes and then filtered into 500 mL glass beakers through two layers of gauze. The wash was divided into conical tubes and subjected twice to centrifugation at 2100 x g for 10 minutes. The pellet was aliquoted into microtubes and stored at -20 °C until DNA extraction.

Obtaining and analyzing soil samples

For T. gondii, Cryptosporidium spp. and G. intestinalis detection, a total of 10 samples of approximately 10 g of soil, collected from the surface, one per property (from property 12 to 21) and stored in 50 mL conical tubes with 30 mL of 1M glycine, were homogenized with the aid of a stirrer for 30 minutes. They were then kept at rest for five minutes for sedimentation. After the pellet was discarded, the supernatant was centrifuged at 1500 x g for 15 minutes. The final concentrate was aliquoted into microtubes and stored at -20 °C until DNA extraction.

Obtaining and analyzing water samples

For the microbiological analysis by means of the chromogenic substrate technique ( APHA, 2005 APHA. Standard methods for the examination of water and wastewater . Washington, DC; 2005. ), 21 samples of water, one per property (from property 1 to 21), were collected from the irrigation tap of the vegetable gardens, as recommended by the protocol in Brazil ( BRASIL, 2013 Brasil. Ministério da Saúde, Fundação Nacional de Saúde, Departamento de Saúde Ambiental, Coordenação de Controle da Qualidade da Água. Manual prático de análise de água . [Online]. 4. ed. Brasília: FUNASA; 2013. [cited 2018 Jun 13]. Available from: http://www.funasa.gov.br/site/wp-content/files_mf/manual_pratico_de_analise_de_agua_2.pdf
http://www.funasa.gov.br/site/wp-conten...
).

For T. gondii, Cryptosporidium spp. and G. intestinalis detection, a total of 10 samples (from property 12 to 21) of 10 mL were collected in clean plastic bottles from irrigation tap. The water was filtered through a cellulose ester membrane with a 47 mm-diameter and 1.2 μm porosity (Millipore®, Billerica, Massachusetts, USA) in a filter holder system using a vacuum pump (4 L/min). After filtration, the material was eluted in 0.1% Tween 80 with the aid of flexible plastic loops (Thermo Fisher Scientific, Massachusetts, USA) ( BRANCO et al., 2012 Branco N, Leal DAG, Franco RMB. A parasitological survey of natural water springs and inhabitants of a tourist city in southeastern Brazil. Vector Borne Zoonotic Dis 2012; 12(5): 410-417. http://dx.doi.org/10.1089/vbz.2011.0679. PMid:22217166.
http://dx.doi.org/10.1089/vbz.2011.0679...
). The obtained material was concentrated by centrifugation twice at 1050 x g for 15 min at 4°C. The obtained pellet was stored in microtubes at -20 °C until DNA extraction.

Molecular analyses

The samples were previously submitted to freeze-thaw (5 cycles of freezing at -80 °C and thawing at 56 °C), then DNA extraction was performed using a commercial kit (NucleoSpin Tissue®, Macherey-Nagel, Düren, Germany) in accordance with the manufacturer's instructions and DNA was collected in a final volume of 100 µL. DNA extracted were stored at -20°C until polymerase chain reaction (PCR) processing.

PCR assays for T. gondii were performed as previously described by Homan et al. (2000) Homan WL, Vercammen M, De Braekeleer J, Verschueren H. Identification of a 200- to 300-fold repetitive 529 bp DNA fragment in Toxoplasma gondii, and its use for diagnostic and quantitative PCR. Int J Parasitol 2000; 30(1): 69-75. http://dx.doi.org/10.1016/S0020-7519(99)00170-8. PMid:10675747.
http://dx.doi.org/10.1016/S0020-7519(99...
, to amplify a fragment of 529 bp. Cryptosporidium spp. were detected using a nested-PCR reaction with primers described by Xiao et al. (1999) Xiao L, Escalante L, Yang C, Sulaiman I, Escalante AA, Montali RJ, et al. Phylogenetic analysis of Cryptosporidium parasites based on the small-subunit rRNA gene locus. Appl Environ Microbiol 1999; 65(4): 1578-1583. PMid:10103253. , which target a fragment of 18S rRNA gene between 826 and 840 bp. For G. intestinalis DNA detection, the samples were subjected to nested-PCR to amplify an approximately 300-bp fragment of the 18S rRNA gene from G. intestinalis ( COKLIN et al., 2007 Coklin T, Farber J, Parrington L, Dixon B. Prevalence and molecular characterization of Giardia duodenalis and Cryptosporidium spp. in dairy cattle in Ontario, Canada. Vet Parasitol 2007; 150(4): 297-305. http://dx.doi.org/10.1016/j.vetpar.2007.09.014. PMid:17964724.
http://dx.doi.org/10.1016/j.vetpar.2007...
), in addition to 530 bp of the TPI (triose-phosphate isomerase) gene with primers described by Sulaiman et al. (2003) Sulaiman IM, Fayer R, Bern C, Gilman RH, Trout JM, Schantz PM, et al. Triosephosphate Isomerase Gene Characterization and Potential Zoonotic Transmission of Giardia duodenalis. Emerg Infect Dis 2003; 9(11): 1444-1452. http://dx.doi.org/10.3201/eid0911.030084. PMid:14718089.
http://dx.doi.org/10.3201/eid0911.03008...
and 432 bp of the GDH (glutamate dehydrogenase) gene with primers described by Read et al. (2004) Read C, Monis P, Andrewthompson R. Discrimination of all genotypes of Giardia duodenalis at the glutamate dehydrogenase locus using PCR-RFLP. Infect Genet Evol 2004; 4(2): 125-130. http://dx.doi.org/10.1016/j.meegid.2004.02.001. PMid:15157630.
http://dx.doi.org/10.1016/j.meegid.2004...
in a semi-nested PCR.

PCR products were visualized by 1.5% agarose gel electrophoresis stained with SYBR Safe DNA (Invitrogen®, California, USA). In addition, when positive for Cryptosporidium spp. and for G. intestinalis, the PCR products were purified using the QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany), and the DNA was quantified by a Picodrop (Thermo Fisher Scientific, Wilmington, USA). DNA sequencing was performed with the BigDye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems, Carlsbad, USA) with the corresponding forward or reverse primers on a 3500 genetic analyzer (Applied Biosystems, Carlsbad, USA), according to the instructions of the manufacturer. The sequences obtained were examined with PHRED software ( EWING & GREEN, 1998 Ewing B, Green P. Base-calling of automated sequencer traces using phred. II Error probabilities. Genome Res 1998; 8(3): 186-194. http://dx.doi.org/10.1101/gr.8.3.186. PMid:9521922.
http://dx.doi.org/10.1101/gr.8.3.186 ...
) and inspected with CHROMAS software for quality analysis of chromatograms. Consensus sequences were determined by the CAP3 software ( http://asparagin.cenargen.embrapa.br/cgi-bin/phph/cap3.pl ), and their identities were compared to the sequences deposited in GenBank using BLAST software ( CAMACHO et al., 2009 Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, et al. BLAST+: architecture and applications. BMC Bioinformatics 2009; 10(1): 421. http://dx.doi.org/10.1186/1471-2105-10-421. PMid:20003500.
http://dx.doi.org/10.1186/1471-2105-10-...
). The nucleotide sequence data reported in this article were deposited in GenBank under the following accession numbers: Cryptosporidium parvum (MF353924 to MF353928) and Giardia intestinalis (MF425817 to MF425819). The phylogenetic relationships between G. intestinalis sequences of the present study and GenBank standard sequences were characterized by the alignment of 390 nucleotides of the GDH coding gene using the maximum likelihood method with 250 bootstraps in the MEGA 4.0 program. The tree was rooted with Giardia ardeae (AF069060) and the GenBank sequences used in this tree are standards of assemblages A1 (AY178735), A2 (AY178737), B (AY826193), D (U60986), E (AY178741) and F (AF069057).

Statistical Analysis

A semi-structured questionnaire was applied to all horticulturists participating in the study, who were questioned about the type of planting and fertilization, soil supplementation, presence of animals on the property, irrigation system, toilet and sewage characteristics. The program EpiInfo 3.5.4 ( DEAN et al., 1990 Dean AG, Dean JA, Burton AH, Discker RC. Epi-Info, Version 5: a word processing, database, and statistics programme for epidemiology on microcomputers. Atlanta, GA USD Inc.; 1990. ) was used to tabulate the variables together with the microbiological and molecular results found. Statistical analysis was performed using the EpiInfo programs 3.5.4 and R 3.3.2 ( R CORE TEAM, 2013 R Core Team. A language and environment for statistical computing [online]. 2013. [cited 2017 Dec 10] Available from: http//www.R-project.org
http//www.R-project.org...
) by means of the Chi-square test or Fisher's exact test, where appropriate. The analysis of these associations with a control of the confounding variables was performed applying simple and multiple logistic regression analysis ( HOSMER et al., 2013 Hosmer DW Jr, Lemeshow S, Sturdivant RX. Applied logistic regression. New Jersey: John Wiley & Sons; 2013. http://dx.doi.org/10.1002/9781118548387.
http://dx.doi.org/10.1002/9781118548387...
). Only the variables whose p-values were less than 0.10 in the screening analysis and had some biological meaning were included in the logistic regression models. The association measurement was obtained by the Odds Ratio (OR) calculation with a 95% confidence interval (CI). The multiple correspondence analysis was performed with program R 3.3.3 ( R CORE TEAM, 2013 R Core Team. A language and environment for statistical computing [online]. 2013. [cited 2017 Dec 10] Available from: http//www.R-project.org
http//www.R-project.org...
) through the FactoMineR ( HAIR et al., 2009 Hair JF, Black WC, Babin BJ, Anderson RE, Tatham RL. Análise multivariada de dados. Porto Alegre: Bookman Editora; 2009. ) package, an exploratory technique that does not rely on statistical tests but allows the visualization of the most important relationships of a large set of variables among each other ( HOFFMAN & FRANKE, 1986 Hoffman DL, Franke GR. Correspondence analysis: graphical representation of categorical data in marketing research. J Mark Res 1986; 23(3): 213-227. http://dx.doi.org/10.2307/3151480.
http://dx.doi.org/10.2307/3151480 ...
). In addition, it can help in visualizing the multivariate relationship between the categories of the different variables, and their geometric proximity in the graph, suggesting the possibility of associations between them.

Results

In total, 83 samples were collected, being 42 of vegetables, 31 of water (21 for microbiological analysis and 10 for parasitological analysis) and 10 of soil; from 21 properties of the municipalities of Apucarana (6/21), Marilândia do Sul (7/21), Ortigueira (6/21), Rolândia (1/21) and Londrina (District of Guaravera) (1/21). Among the leafy vegetables, 17 samples were of crisp lettuce (Lactuca sativa), seven of arugula (Eruca sativa), nine of chicory (Cichorium intybus and Cichorium endivia), five of chives (Allium fistulosum), two of purple lettuce, one spinach (Spinacia oleracea) and one chard (Beta vulgaris subsp. Vulgaris). On the first eleven properties, water was not submitted for molecular analysis, and no soil samples were collected.

Eighteen properties were organic cultivation certified, and three (14.3%) were in the certification phase. The organic fertilizer source was variable among the properties: 23.8% (5/21) used chicken manure, 14.3% (3/21) used mixed manure, 14.3% (3/21) used chicken litter, 14.3% (3/21) used commercial compounds and 33.3% (7/21) used bovine manure. Eleven (52.3%) of the properties underwent mineral supplementation on a regular basis, six with limestone (54.5%), three with a potassium supplement (27.2%) and one with mineral present in the fertilizer.

Wild birds and domestic animals, such as dogs, cats, horses, and cattle, were present on all the properties studied, and in five (23.8%) of them, the animals had access to the gardens. Wild animals, such as cervids, hare, armadillos, capybara, fox and cougar were present on 13 of the studied properties (61.9%). All the properties were washing their vegetables: 52.4% (11/21) of the properties in treated tap water and 47.6% (10/21) in rinsing tanks. Irrigation in 57.1% (13/21) was automated. The source of irrigation water on 57.1% (12/21) of the properties was spring, 14.3% (3/21) of the properties used untreated river water, and 28.6% (6/21) of the properties used artesian wells.

Of the water samples, 95.2% (20/21) presented total coliforms, with a variation of 1 to > 2419.6 CTNMP/100 mL; for CTT and Escherichia coli, 76.2% (16/21) of the samples were positive, ranging from 1 to 218.7 CTTNMP/100 mL.

Regarding the PCR results, 12.9% (8/62) of the samples were positive for T. gondii , 11.3% (7/62) for Cryptosporidium spp., and 25.8% (16/62) for G. intestinalis (two in TPI, seven in GDH, five in 18S rRNA and two in TPI and GDH) ( Table 1 ). It was possible to identify the parasite species by DNA sequencing in seven of the positive samples. C. parvum was the species present in three water samples and one soil sample sample showing 100% of similarity with other sequences from GenBank. G. intestinalis Assemblage E was present in two vegetable samples showing 99% of similarity with other sequences from GenBank. In order to confirm the G. intestinalis Assemblage, the phylogenetic analysis with the partial sequences of the GDH gene of G. intestinalis obtained was performed, a clustering of samples with standard sequences of Assemblage E ( Figure 2 ), commonly associated with giardiasis in cattle, was observed. In other PCR-positive samples, there was no success in sequencing due to the low amount of DNA.

Table 1
Vegetable, water and soil samples positive for the protozoa Toxoplasma gondii , Cryptosporidium spp. and Giardia intestinalis from organic crop properties, from 2014 to 2016 in Paraná, Brazil. Results are based on molecular analyses.
Figure 2
Phylogenetic relationships among G. intestinalis isolates characterized by the alignment of 390 nucleotides of the GDH coding gene using the maximum likelihood method with 250 bootstraps in the MEGA 4.0 program. The tree is rooted with Giardia ardeae (AF069060), and the GenBank sequences used in this tree are standards of the assemblies A1 (AY178735), A2 (AY178737), B (AY826193), D (U60986), E (AY178741) and F (AF069057).

Factors associated with the presence of DNA of zoonotic protozoa such as T. gondii , Cryptosporidium spp. and G. intestinalis in vegetables, soil and water are listed in Tables 2 , 3 and 4 .

Table 2
Characteristics of the organic vegetable producing properties of northern Paraná, Brazil, significantly associated with the occurrence of Toxoplasma gondii and Giardia intestinalis.
Table 3
Characteristics of the organic vegetable producing properties of northern Paraná, Brazil, significantly associated with the occurrence of Cryptosporidium spp.
Table 4
Characteristics of the organic vegetable producing properties of northern Paraná, Brazil, significantly associated with the occurrence of at least one of the protozoa ( Toxoplasma gondii, Cryptosporidium spp. or G. intestinalis).

The graph generated by the multiple correspondence analysis ( Figure 3 ) confirmed the main findings of this study related to the risk factors for the presence of zoonotic protozoa in vegetables, water and soil of properties with organic production systems. It is shown in dimension one of the graph (x-axis) that the categories of negative variables for T. gondii, Cryptosporidium spp. and G. intestinalis and at least one of the three zoonotic protozoa in question are on the left along with the categories of limestone supplementation, spring water source and absence of cervids. On the other hand, on the right side of the graph we have the categories of positive variables for T. gondii, G. intestinalis, Cryptosporidium spp. and at least one of the three zoonotic protozoa together with the categories of potassium and fertilizer supplementation, river water source and the presence of cervids. The interpretation of dimension two (y-axis) of the graph was not possible due to the lack of biological meaning.

Figure 3
Graph resulting from the multiple match analysis. Presence of cervids (NCERV, YCERV); positive results of T. gondii (NTGON, YTGON), Cryptosporidium spp. (NCRY, YCRY), Giardia spp. (NGIA, YGIA), and one of the three protozoa (NPRO, YPRO); water source (SPRING, WELL, RIVER) and supplement used in growing (LIMESTONE, NONE, POTASSIUM and FERTILIZER).

Discussion

Resolution 357/05 of CONAMA (National Council for the Environment) ( BRASIL, 2005 Brasil. Conselho Nacional do Meio Ambiente - CONAMA. Resolução nº 357 de 18 de março de 2005. Dispõe sobre a classificação dos corpos de água e diretrizes ambientais para o seu enquadramento, bem como estabelece as condições e padrões de lançamento de efluentes, e dá outras providências. [online]. Diário Oficial da República Federativa do Brasil, Brasília, 2005 [cited 2018 Feb 10]. Available from: http://www.mma.gov.br/port/conama/res/res05/res35705.pdf
http://www.mma.gov.br/port/conama/res/r...
) recommends that for the irrigation of vegetables and fruits with development on the ground, the Thermotolerant coliforms (CTT) count should not exceed 200/100 mL; Escherichia coli is the main species of the CTT group, whose exclusive habitat is the intestine ( BRASIL, 2005 Brasil. Conselho Nacional do Meio Ambiente - CONAMA. Resolução nº 357 de 18 de março de 2005. Dispõe sobre a classificação dos corpos de água e diretrizes ambientais para o seu enquadramento, bem como estabelece as condições e padrões de lançamento de efluentes, e dá outras providências. [online]. Diário Oficial da República Federativa do Brasil, Brasília, 2005 [cited 2018 Feb 10]. Available from: http://www.mma.gov.br/port/conama/res/res05/res35705.pdf
http://www.mma.gov.br/port/conama/res/r...
). E. coli was present in 76.2% (16/21) of the analyzed water samples, and of these, 18.7% (3/16), one of spring (209.8 CTNMP/100mL), one of river (328.2 CTNMP/100mL) and another of artesian well (201.2 CTNMP/100mL), with higher counts than that allowed by the resolution. The use of faecal indicators, particularly, E. coli, is a better practical approach to identify contaminated water sources or food ( ALLENDE & MONAGHAN, 2015 Allende A, Monaghan J. Irrigation water quality for leafy crops: a perspective of risks and potential solutions. Int J Environ Res Public Health 2015; 12(7): 7457-7477. http://dx.doi.org/10.3390/ijerph120707457. PMid:26151764.
http://dx.doi.org/10.3390/ijerph1207074...
; NATARO & KAPER, 1998 Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev 1998; 11(1): 142-201. PMid:9457432. ). Recent studies associate the presence of parasites such as G. intestinalis and Cryptosporidium spp. to the presence of CTT ( TOLEDO et al., 2017 Toledo RDS, Martins FDC, Ferreira FP, de Almeida JC, Ogawa L, Dos Santos HL, et al. Cryptosporidium spp. and Giardia spp. in feces and water and the associated exposure factors on dairy farms. PLoS One 2017; 12(4): e0175311. http://dx.doi.org/10.1371/journal.pone.0175311. PMid:28403147.
http://dx.doi.org/10.1371/journal.pone....
) in the water, indicating the necessity of monitoring parasite presence in vegetable gardens for the production of innocuous vegetables.

Regarding the PCR results, 12.9% (8/62) of the samples had DNA fragments compatible with T. gondii, and of these, 50.0% (4/8) were vegetables, 25.0% (2/8) were water and 25.0% (2/8) were soil. Lass et al. (2012) Lass A, Pietkiewicz H, Szostakowska B, Myjak P. The first detection of Toxoplasma gondii DNA in environmental fruits and vegetables samples. Eur J Clin Microbiol Infect Dis 2012; 31(6): 1101-1108. http://dx.doi.org/10.1007/s10096-011-1414-8. PMid:21948336.
http://dx.doi.org/10.1007/s10096-011-14...
studied T. gondii in 216 fruits and vegetables in Poland and observed a frequency of 9.7% positive samples. The eight positive samples came from six properties, of which 50.0% (3/6) had cats, all with access to the cultivation area, suggesting direct contamination of vegetables by definitive hosts on these properties ( FRENKEL et al., 1970 Frenkel JK, Dubey JP, Miller NL. Toxoplasma gondii in cats: fecal stages identified as coccidian oocysts. Science 1970; 167(3919): 893-896. http://dx.doi.org/10.1126/science.167.3919.893. PMid:4903651.
http://dx.doi.org/10.1126/science.167.3...
). In vegetable gardens that did not have cats (3/6), the source of irrigation water from the river may have influenced the contamination due to the flow during rains, when the river receives several fecal pathogens from domestic and wild animals ( WELLS et al., 2015 Wells B, Shaw H, Hotchkiss E, Gilray J, Ayton R, Green J, et al. Prevalence, species identification and genotyping Cryptosporidium from livestock and deer in a catchment in the Cairngorms with a history of a contaminated public water supply. Parasit Vectors 2015; 8(1): 66. http://dx.doi.org/10.1186/s13071-015-0684-x. PMid:25650114.
http://dx.doi.org/10.1186/s13071-015-06...
); its use in irrigation should be avoided, unless it is properly treated. Water was related to outbreaks of human toxoplasmosis previously in Panamá ( BENENSON et al., 1982 Benenson MW, Takafuji ET, Lemon SM, Greenup RL, Sulzer AJ. Oocyst-transmitted toxoplasmosis associated with ingestion of contaminated water. N Engl J Med 1982; 307(11): 666-669. http://dx.doi.org/10.1056/NEJM198209093071107. PMid:7110216.
http://dx.doi.org/10.1056/NEJM198209093...
), Canada ( BOWIE et al., 1997 Bowie WR, King AS, Werker DH, Isaac-Renton JL, Bell A, Eng SB, et al. Outbreak of toxoplasmosis associated with municipal drinking water. Lancet 1997; 350(9072): 173-177. http://dx.doi.org/10.1016/S0140-6736(96)11105-3. PMid:9250185.
http://dx.doi.org/10.1016/S0140-6736(96...
), Atlanta ( DUBEY et al., 1981 Dubey JP, Sharma SP, Juranek DD, Sulzer AJ, Teutsch SM. Characterization of Toxoplasma gondii isolates from an outbreak of toxoplasmosis in Atlanta, Georgia. Am J Vet Res 1981; 42(6): 1007-1010. PMid:7283229. ) and Brazil ( VAUDAUX et al., 2010 Vaudaux JD, Muccioli C, James ER, Silveira C, Magargal SL, Jung C, et al. Identification of an atypical strain of Toxoplasma gondii as the cause of a waterborne outbreak of toxoplasmosis in Santa Isabel do Ivai, Brazil. J Infect Dis 2010; 202(8): 1226-1233. http://dx.doi.org/10.1086/656397. PMid:20836703.
http://dx.doi.org/10.1086/656397 ...
). The use of treated water with regular microbiological analysis is essential to ensure its quality and safe use in irrigation.

From the analyzed samples, 11.3% (7/62) were positive for Cryptosporidium spp. Of these, 42,9% (3/7) water samples (two of artesian well and one of spring), 28.6% (2/7) soil sample and 28,6% (2/7) vegetable sample were identified as containing C. parvum , an important zoonotic species and the main cause of human cryptosporidiosis, which represents a threat to public health because it is associated with diarrhea and death in immunocompromised individuals ( SANTÍN, 2013 Santín M. Clinical and subclinical infections with Cryptosporidium in animals. N Z Vet J 2013; 61(1): 1-10. http://dx.doi.org/10.1080/00480169.2012.731681. PMid:23134088.
http://dx.doi.org/10.1080/00480169.2012...
). The main known reservoirs are production and wild animals ( RYAN et al., 2014 Ryan U, Fayer R, Xiao L. Cryptosporidium species in humans and animals: current understanding and research needs. Parasitology 2014; 141(13): 1667-1685. http://dx.doi.org/10.1017/S0031182014001085. PMid:25111501.
http://dx.doi.org/10.1017/S003118201400...
). In this study, the presence of cervids on the properties was associated with the presence of Cryptosporidium spp., which could explain environmental contamination by water drained. Wells et al. (2015) Wells B, Shaw H, Hotchkiss E, Gilray J, Ayton R, Green J, et al. Prevalence, species identification and genotyping Cryptosporidium from livestock and deer in a catchment in the Cairngorms with a history of a contaminated public water supply. Parasit Vectors 2015; 8(1): 66. http://dx.doi.org/10.1186/s13071-015-0684-x. PMid:25650114.
http://dx.doi.org/10.1186/s13071-015-06...
, when conducting a study in a water supply network in Scotland, detected C. parvum and related the high prevalence of the protozoan to cervids inhabiting the region studied. Ranjbar-Bahadori et al., (2013) Ranjbar-Bahadori S, Mostoophi A, Shemshadi B. Study on Cryptosporidium contamination in vegetable farms around Tehran. Trop Biomed 2013; 30(2): 193-198. PMid:23959484. suggested that plants may provide a route of infection by Cryptosporidium spp. for humans when they observed a frequency of 6.6% (33/496) positive samples with risk factors related to the type of vegetable and irrigation water in gardens of Tehran, Iran. Poulton et al. (1991) Poulton M, Colbourne J, Dennis PJ. Thames water’s experiences with Cryptosporidium. Water Sci Technol 1991; 24(2): 21-26. http://dx.doi.org/10.2166/wst.1991.0023.
http://dx.doi.org/10.2166/wst.1991.0023...
suggested that the fertilizer used in agriculture was one of the main sources responsible for the high levels of oocysts of Cryptosporidium spp. observed in the River Thames in the south of England. In this study, the type of fertilizers used on the positive properties were 50.0% (3/6) chicken litter, 16.7% (1/6) bovine manure and 33.3% (2/6) mixed manure (chicken and bovine), the type of fertilizer had no statistical association with positivity by Cryptosporidium spp., however the parasite was previously described in these animals ( AL MAWLY et al., 2015 Al Mawly J, Grinberg A, Velathanthiri N, French N. Cross sectional study of prevalence, genetic diversity and zoonotic potential of Cryptosporidium parvum cycling in New Zealand dairy farms. Parasit Vectors 2015; 8(240): 1-7. http://dx.doi.org/10.1186/s13071-015-0855-9. PMid:25896433.
http://dx.doi.org/10.1186/s13071-015-08...
; EWALD et al., 2017 Ewald MPC, Martins FDC, Caldart ET, Vieira FEG, Yamamura MH, Sasse JP, et al. The first study of molecular prevalence and species characterization of Cryptosporidium in free-range chicken (Gallus gallus domesticus) from Brazil. Rev Bras Parasitol Vet 2017; 26(4): 472-478. http://dx.doi.org/10.1590/s1984-29612017068. PMid:29185602.
http://dx.doi.org/10.1590/s1984-2961201...
; HELMY et al., 2017 Helmy YA, Krücken J, Abdelwhab ESM, von Samson-Himmelstjerna G, Hafez HM. Molecular diagnosis and characterization of Cryptosporidium spp. in turkeys and chickens in Germany reveals evidence for previously undetected parasite species. PLoS One 2017; 12(6): e0177150. http://dx.doi.org/10.1371/journal.pone.0177150. PMid:28575116.
http://dx.doi.org/10.1371/journal.pone....
).

G. intestinalis was found in 25.8% (16/62) of the analyzed samples, of which 12 samples were vegetables, two were water of artesian well and two were soil, on nine properties. The presence of cysts of G. intestinalis at any stage of the production of vegetables is of extreme concern, as this parasite is resistant in the environment, remaining viable for weeks or months ( ALMEIDA et al., 2010 Almeida A, Pozio E, Cacciò SM. Genotyping of Giardia duodenalis cysts by new real-time PCR assays for detection of mixed infections in human samples. Appl Environ Microbiol 2010; 76(6): 1895-1901. http://dx.doi.org/10.1128/AEM.02305-09. PMid:20080999.
http://dx.doi.org/10.1128/AEM.02305-09 ...
). The G. intestinalis samples from the present study showed similarity to the Assemblage E ( Figure 2 ), commonly described in cattle ( EY et al., 1997 Ey PL, Mansouri M, Kulda J, Nohýnková E, Monis PT, Andrews RH, et al. Genetic analysis of Giardia from hoofed farm animals reveals artiodactyl-specific and potentially zoonotic genotypes. J Eukaryot Microbiol 1997; 44(6): 626-635. http://dx.doi.org/10.1111/j.1550-7408.1997.tb05970.x. PMid:9435134.
http://dx.doi.org/10.1111/j.1550-7408.1...
; PLUTZER et al., 2010 Plutzer J, Ongerth J, Karanis P. Giardia taxonomy, phylogeny and epidemiology: facts and open questions. Int J Hyg Environ Health 2010; 213(5): 321-333. http://dx.doi.org/10.1016/j.ijheh.2010.06.005. PMid:20619729.
http://dx.doi.org/10.1016/j.ijheh.2010....
). Fertilization using bovine or mixed manure may facilitate environmental contamination when not properly processed. Taking into account that infected young animals can release up to 106 cysts per gram of feces ( GEURDEN et al., 2010 Geurden T, Vercruysse J, Claerebout E. Is Giardia a significant pathogen in production animals? Exp Parasitol 2010; 124(1): 98-106. http://dx.doi.org/10.1016/j.exppara.2009.03.001. PMid:19285075.
http://dx.doi.org/10.1016/j.exppara.200...
), the presence of bovine animals on a farm may lead to contamination of soil and water, since environmental contamination may precede the contamination of water and vegetables ( ALMEIDA et al., 2010 Almeida A, Pozio E, Cacciò SM. Genotyping of Giardia duodenalis cysts by new real-time PCR assays for detection of mixed infections in human samples. Appl Environ Microbiol 2010; 76(6): 1895-1901. http://dx.doi.org/10.1128/AEM.02305-09. PMid:20080999.
http://dx.doi.org/10.1128/AEM.02305-09 ...
). The three positive samples for non-zoonotic Assemblage E came from vegetables (two lettuces, one arugula) from two properties that did not have cattle, so the contamination could have occurred due to the use of bovine fertilizer on one of the properties or by contaminated irrigation water in both.

Statistically, there was a significant association between being positive for Cryptosporidium spp. and G. intestinalis, we suggest that this association may be due to the fact that both pathogens are known to be water-borne. Before the 1980s, diseases of bacterial and viral origin were associated with water, but with the inclusion of chlorination in the treatment process, there was an effective reduction of these microorganisms ( FURTADO et al., 1998 Furtado C, Adak GK, Stuart JM, Wall PG, Evans HS, Casemore DP. Outbreaks of waterborne infectious intestinal disease in England and Wales, 1992-5. Epidemiol Infect 1998; 121(1): 109-119. http://dx.doi.org/10.1017/S0950268898001083. PMid:9747762.
http://dx.doi.org/10.1017/S095026889800...
). In contrast, Cryptosporidium spp. and G. intestinalis have become the main etiological agents of water transport and the cause of outbreaks ( CHEUN et al., 2013 Cheun H-I, Kim C-H, Cho S-H, Ma D-W, Goo B-L, Na MS, et al. The first outbreak of giardiasis with drinking water in Korea. Osong Public Health Res Perspect 2013; 4(2): 89-92. http://dx.doi.org/10.1016/j.phrp.2013.03.003. PMid:24159537.
http://dx.doi.org/10.1016/j.phrp.2013.0...
; MAC KENZIE et al., 1994 Mac Kenzie WR, Hoxie NJ, Proctor ME, Gradus MS, Blair KA, Peterson DE, et al. Massive outbreak in Milwaukee of Cryptosporidium infection transmitted through the public water supply. N Engl J Med 1994; 331(3): 161-167. http://dx.doi.org/10.1056/NEJM199407213310304. PMid:7818640.
http://dx.doi.org/10.1056/NEJM199407213...
). They are of great importance in public health because of their low infective doses, resistance to conventional water treatment and adverse environmental conditions ( SLIFKO et al., 2000 Slifko TR, Smith HV, Rose JB. Emerging parasite zoonoses associated with water and food. Int J Parasitol 2000; 30(12-13): 1379-1393. http://dx.doi.org/10.1016/S0020-7519(00)00128-4. PMid:11113263.
http://dx.doi.org/10.1016/S0020-7519(00...
). Water, once contaminated, acts as an efficient carrier of pathogens, either because of its great dispersion capacity or because of its direct and indirect compulsory consumption ( FRANCO, et al., 2012 Franco RMB, Branco N, Leal DAG. Parasitologia Ambiental: Métodos de concentração e detecção de Cryptosporidium spp. e Giardia spp. em amostras de água. Rev Patol Trop 2012; 41(2): 119-135. http://dx.doi.org/10.5216/rpt.v41i2.19320.
http://dx.doi.org/10.5216/rpt.v41i2.193...
). Wild animals play an important role in the contamination of water by their aquatic activities or by feces flowing to the rivers and springs ( HAMNES et al., 2006 Hamnes IS, Gjerde B, Robertson L, Vikøren T, Handeland K. Prevalence of Cryptosporidium and Giardia in free-ranging wild cervids in Norway. Vet Parasitol 2006; 141(1-2): 30-41. http://dx.doi.org/10.1016/j.vetpar.2006.05.004. PMid:16797126.
http://dx.doi.org/10.1016/j.vetpar.2006...
; WELLS et al., 2015 Wells B, Shaw H, Hotchkiss E, Gilray J, Ayton R, Green J, et al. Prevalence, species identification and genotyping Cryptosporidium from livestock and deer in a catchment in the Cairngorms with a history of a contaminated public water supply. Parasit Vectors 2015; 8(1): 66. http://dx.doi.org/10.1186/s13071-015-0684-x. PMid:25650114.
http://dx.doi.org/10.1186/s13071-015-06...
).

Vegetables do not develop in soil with acidic pH, which is the case of Brazilian soil ( ERNANI et al., 2002 Ernani PR, Bayer C, Maestri L. Corn yield as affected by liming and tillage system on an acid Brazilian Oxisol. Agron J 2002; 94(2): 305-309. http://dx.doi.org/10.2134/agronj2002.0305.
http://dx.doi.org/10.2134/agronj2002.03...
). Acidic soils are neutralized, most of the times, by the application of limestone ( ALBUQUERQUE et al., 2003 Albuquerque JA, Bayer C, Ernani PR, Mafra AL, Fontana EC. Aplicação de calcário e fósforo e estabilidade da estrutura de um solo ácido. Rev Bras Ciênc Solo 2003; 27(5): 799-806. http://dx.doi.org/10.1590/S0100-06832003000500004.
http://dx.doi.org/10.1590/S0100-0683200...
; PRADO & FERNANDES, 2000 Prado RM, Fernandes FM. Escória de siderurgia e calcário na correção da acidez do solo cultivado com cana-de-açúcar em vaso. Sci Agric 2000; 57(4): 739-744. http://dx.doi.org/10.1590/S0103-90162000000400023.
http://dx.doi.org/10.1590/S0103-9016200...
). The use of this supplement was a factor associated with a lower chance of positive samples for at least one of the three parasitic agents studied, it is known that its use reduces the viability of helminth eggs ( CAPIZZI-BANAS et al., 2004 Capizzi-Banas S, Deloge M, Remy M, Schwartzbrod J. Liming as an advanced treatment for sludge sanitisation: helminth eggs elimination - Ascaris eggs as model. Water Res 2004; 38(14-15): 3251-3258. http://dx.doi.org/10.1016/j.watres.2004.04.015. PMid:15276741.
http://dx.doi.org/10.1016/j.watres.2004...
). Franco et al. (2012) Franco RMB, Branco N, Leal DAG. Parasitologia Ambiental: Métodos de concentração e detecção de Cryptosporidium spp. e Giardia spp. em amostras de água. Rev Patol Trop 2012; 41(2): 119-135. http://dx.doi.org/10.5216/rpt.v41i2.19320.
http://dx.doi.org/10.5216/rpt.v41i2.193...
have described that the use of alkaline reagents in water, such as calcium carbonate, can produce deformations in cysts of G. intestinalis and oocysts of Cryptosporidium spp. Transferring this description to this study, the direct contact of the limestone with the cysts and oocysts for a prolonged period may have led to their deformation, DNA exposure and degradation, thus diminishing the positive samples on these properties. Further studies are needed to clarify this result.

Among the analyzed gardens, 57.1% (12/21) presented at least one of the protozoa studied, and with respect to the samples, the frequency was 52.5% (22/42) in vegetables, 20.0% (2/10) in soil and 20.0% (2/10) in water. As in Falavigna et al. (2005) Falavigna LM, Freitas CBR, Melo GC, Nishi L, Araújo SM, Falavigna-Guilherme AL. Qualidade de hortaliças comercializadas no noroeste do Paraná, Brasil. Parasitol Latinoam 2005; 60(3-4): 144-149. , there was no significant difference between the types of leafy greens tested. Commonly used decontamination treatments, such as vinegar and sodium hypochlorite ( YUCEL SENGUN & KARAPINAR, 2005 Yucel Sengun I, Karapinar M. Effectiveness of household natural sanitizers in the elimination of Salmonella typhimurium on rocket (Eruca sativa Miller) and spring onion (Allium cepa L.). Int J Food Microbiol 2005; 98(3): 319-323. http://dx.doi.org/10.1016/j.ijfoodmicro.2004.07.011. PMid:15698693.
http://dx.doi.org/10.1016/j.ijfoodmicro...
), are not able to destroy the resistant forms of T. gondii, Cryptosporidium spp., and Giardia spp. ( HUNTER & THOMPSON, 2005 Hunter PR, Thompson RCA. The zoonotic transmission of Giardia and Cryptosporidium. Int J Parasitol 2005; 35(11-12): 1181-1190. http://dx.doi.org/10.1016/j.ijpara.2005.07.009. PMid:16159658.
http://dx.doi.org/10.1016/j.ijpara.2005...
). The morphological characteristics of leafy vegetables, such as compact structure and presence of multiple leaves facilitate a greater fixation by parasites, making manual mechanical cleaning difficult ( FALAVIGNA et al., 2005 Falavigna LM, Freitas CBR, Melo GC, Nishi L, Araújo SM, Falavigna-Guilherme AL. Qualidade de hortaliças comercializadas no noroeste do Paraná, Brasil. Parasitol Latinoam 2005; 60(3-4): 144-149. ). According to Guilherme et al. (1999) Guilherme AL, Araújo SM, Falavigna DL, Pupulim AR, Dias ML, Oliveira HS, et al. Prevalência de enteroparasitas em horticultores e hortaliças da Feira do Produtor de Maringá, Paraná. Rev Soc Bras Med Trop 1999; 32(4): 405-411. http://dx.doi.org/10.1590/S0037-86821999000400012. PMid:10495671.
http://dx.doi.org/10.1590/S0037-8682199...
, the clarification of food safety to producers is extremely important because it is a determining factor for the production of safe food.

The main forms of contamination of fruits and vegetables during production are fertilizer ( ALMEIDA et al., 2013 Almeida AJ, Lima VS, Rodrigues ABF, Di Filippo PA. Contamination with Cryptosporidium spp. in manure used as fertilizer in urban gardens. Rev Port Ciênc Vet 2013; 108(585-586): 23-27. ; ERDOGRUL & SENER., 2005 Erdogrul O, Sener H. The contamination of various fruit and vegetable with Enterobius vermicularis, Ascaris eggs, Entamoeba histolyca cysts and Giardia cysts. Food Control 2005; 16(6): 559-562. ), direct contamination from farm workers, access of wild animals ( WELLS et al., 2015 Wells B, Shaw H, Hotchkiss E, Gilray J, Ayton R, Green J, et al. Prevalence, species identification and genotyping Cryptosporidium from livestock and deer in a catchment in the Cairngorms with a history of a contaminated public water supply. Parasit Vectors 2015; 8(1): 66. http://dx.doi.org/10.1186/s13071-015-0684-x. PMid:25650114.
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) and domestic animals to gardens, and contaminated irrigation water ( BERALDO & FARACHE, 2011 Beraldo RM, Farache A Fo. Bacteriological quality of irrigation water from vegetable gardens in the Municipalities of Araraquara, Boa Esperança do Sul and Ibitinga, SP. Aliment Nutr 2011; 22(3): 345-350. ; CHAIDEZ et al., 2005 Chaidez C, Soto M, Gortares P, Mena K. Occurrence of Cryptosporidium and Giardia in irrigation water and its impact on the fresh produce industry. Int J Environ Health Res 2005; 15(5): 339-345. http://dx.doi.org/10.1080/09603120500289010. PMid:16416751.
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), the latter was a factor associated with the presence on the samples of at least one of the agents. River water, in turn, had a greater number of positive samples than expected; the use of this type of water for irrigation is a big problem in Brazil ( MAROUELLI & SILVA, 1998 Marouelli WA, Silva HR. Aspectos sanitários da água para fins de irrigação. [online]. Brasília: EMBRAPA; 1998. (Comunicado Técnico; vol. 5). [cited 2017 Dec 10]. Available from: https://ainfo.cnptia.embrapa.br/digital/bitstream/item/107307/1/Aspectos-sanitarios-da-agua-para-fins.pdf.
https://ainfo.cnptia.embrapa.br/digital...
), because a significant number of rivers are polluted, probably due to the extensive area of agriculture and livestock around the surface water sources and the contamination by untreated municipal effluents, mainly sewage, constituting another important vehicle of transmission and dissemination of pathogens ( AMORÓS et al., 2010 Amorós I, Alonso JL, Cuesta G. Cryptosporidium oocysts and Giardia cysts on salad products irrigated with contaminated water. J Food Prot 2010; 73(6): 1138-1140. http://dx.doi.org/10.4315/0362-028X-73.6.1138. PMid:20537274.
http://dx.doi.org/10.4315/0362-028X-73....
; BERALDO & FARACHE, 2011 Beraldo RM, Farache A Fo. Bacteriological quality of irrigation water from vegetable gardens in the Municipalities of Araraquara, Boa Esperança do Sul and Ibitinga, SP. Aliment Nutr 2011; 22(3): 345-350. ; DIXON et al., 2013 Dixon B, Parrington L, Cook A, Pollari F, Farber J. Detection of Cyclospora, Cryptosporidium , and Giardia in Ready-to-Eat Packaged Leafy Greens in Ontario, Canada. J Food Prot 2013; 76(2): 307-313. http://dx.doi.org/10.4315/0362-028X.JFP-12-282. PMid:23433379.
http://dx.doi.org/10.4315/0362-028X.JFP...
; MAROUELLI & SILVA, 1998 Marouelli WA, Silva HR. Aspectos sanitários da água para fins de irrigação. [online]. Brasília: EMBRAPA; 1998. (Comunicado Técnico; vol. 5). [cited 2017 Dec 10]. Available from: https://ainfo.cnptia.embrapa.br/digital/bitstream/item/107307/1/Aspectos-sanitarios-da-agua-para-fins.pdf.
https://ainfo.cnptia.embrapa.br/digital...
; SILVA et al., 2005 Silva CGM, Andrade SAC, Stamford TLM. Occurrence of Cryptosporidium spp. and others parasites in vegetables consumed in natura, Recife, Brazil. Cien Saude Colet 2005; 10(Suppl): 63-69. http://dx.doi.org/10.1590/S1413-81232005000500009.
http://dx.doi.org/10.1590/S1413-8123200...
). Toledo et al. (2017) Toledo RDS, Martins FDC, Ferreira FP, de Almeida JC, Ogawa L, Dos Santos HL, et al. Cryptosporidium spp. and Giardia spp. in feces and water and the associated exposure factors on dairy farms. PLoS One 2017; 12(4): e0175311. http://dx.doi.org/10.1371/journal.pone.0175311. PMid:28403147.
http://dx.doi.org/10.1371/journal.pone....
in a study in dairy farms in Paraná, Brazil, observed that the absence of vegetation and protective structures around the springs was associated with the frequency of positive water samples to Cryptosporidium spp. and G. intestinalis . The vegetation around the springs acts as a physical barrier, thus reducing the drainage of animal waste and other contaminants ( TOLEDO et al., 2017 Toledo RDS, Martins FDC, Ferreira FP, de Almeida JC, Ogawa L, Dos Santos HL, et al. Cryptosporidium spp. and Giardia spp. in feces and water and the associated exposure factors on dairy farms. PLoS One 2017; 12(4): e0175311. http://dx.doi.org/10.1371/journal.pone.0175311. PMid:28403147.
http://dx.doi.org/10.1371/journal.pone....
). Most of the springs visited in this study were protected, which may have been an important factor for a smaller number of positive samples than expected in this source.

Of the 12 properties with at least one positive sample for one of the three agents, 66.7% (8/12) produced their own fertilizer with bovine manure or chicken litter, and of these, 87.5% (7/8) did not respect the period of composting. The maturity of the compost is related to the complete microbiological decomposition and the transformation of the organic matter into humus, and this process occurs after approximately 90 days of tanning; correct composting can promote the inactivation of some pathogens ( DÉPORTES et al., 1998 Déportes I, Benoit-Guyod JL, Zmirou D, Bouvier MC. Microbial disinfection capacity of municipal solid waste (MSW) composting. J Appl Microbiol 1998; 85(2): 238-246. http://dx.doi.org/10.1046/j.1365-2672.1998.00484.x. PMid:9750296.
http://dx.doi.org/10.1046/j.1365-2672.1...
; KIEHL, 1998 Kiehl EJM. Manual de compostagem: maturação e qualidade do composto. Piracicaba; 1998. ; VINNERÅS et al., 2003 Vinnerås B, Björklund A, Jönsson H. Thermal composting of faecal matter as treatment and possible disinfection method - Laboratory-scale and pilot-scale studies. Bioresour Technol 2003; 88(1): 47-54. http://dx.doi.org/10.1016/S0960-8524(02)00268-7. PMid:12573563.
http://dx.doi.org/10.1016/S0960-8524(02...
). Inadequate fertilizer production, in addition to causing contamination of vegetables and water, prevents the inactivation of pathogens present in the raw material, making the fertilizer unfit for use in edible vegetables.

Conclusion

The control of parasitological contamination of raw vegetables is a major challenge, since contamination can occur in all the stages that precede arrival to the consumers' table. The results indicate that inadequate hygienic-sanitary conditions in organic gardens, as well as the risk of infection by protozoa are of public health importance. In addition, the results expose some of the points of contamination risk, such as the water source, soil supplementation and presence of animals on properties. Thus, there is a need to raise awareness among producers and consumers about the implementation of good handling practices during the cultivation process and before the time of consumption.

Acknowledgements

We would like to acknowledge the CNPQ (National Research Council) for granting the doctoral scholarship.

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Publication Dates

  • Publication in this collection
    30 Aug 2018
  • Date of issue
    Jul-Sep 2018

History

  • Received
    21 Mar 2018
  • Accepted
    12 June 2018
Colégio Brasileiro de Parasitologia Veterinária FCAV/UNESP - Departamento de Patologia Veterinária, Via de acesso Prof. Paulo Donato Castellane s/n, Zona Rural, , 14884-900 Jaboticabal - SP, Brasil, Fone: (16) 3209-7100 RAMAL 7934 - Jaboticabal - SP - Brazil
E-mail: cbpv_rbpv.fcav@unesp.br