First Detection of Toxoplasma gondii DNA in the Fresh Leafs of Vegetables in South America

Abstract

Despite the high dispersion of Toxoplasma gondii oocysts in the environment, there are few studies investigating their presence in vegetables consumed by the general population. This has led us to investigate its occurrence in raw vegetables. A total of 238 samples of vegetables were collected, including crisp lettuce, regular lettuce, chicory, rocket, and parsley, both organic and nonorganic, locally in northwestern Parana, Southern Brazil. Each sample (50 g) was washed and filtered separately. A PCR was performed to detect the parasite DNA from the sediment of each sample, using B1 (B22–23) and Toxo4–5 primers. We found contamination in 3.8% of the samples, 0.8% with the primer Toxo4–5 and 2.9% with B22–B23. The results were positive in 0.6% (1/62) of the samples of smooth lettuce, 3.7% (4/106) of crisp head lettuce, 5.0% (2/40) of chicory, 14.3% (1/7) of rocket, and 20% (1/5) of parsley. These data show the contamination by T. gondii in raw vegetables directly from production sites and stores, in both organic and nonorganic samples.

Introduction

T oxoplasma gondii is probably the most widespread parasite between human and animal populations (Dubey 2010). Severe forms of toxoplasmosis can be observed in immunocompromised individuals (Dubey 2010) and in neonates with congenital infections (Montoya and Remington 2008). The postnatal infection is an important cause of eye disease and it is a leading cause of retinal inflammation in immunocompetent patients in Brazil (Grigg et al. 2015).

Infections caused by the ingestion of oocysts are considered common (Gotteland et al. 2014) and more severe than the ingestion of tissue cysts (Jones and Dubey 2010). The oocysts are evolutionary forms and resistant to moist conditions and temperature (Jones and Dubey 2010). Despite the high dispersion in the environment, there are only a few records of instances of infection through the consumption of raw vegetables, probably due to technical difficulties. Lass et al. (2012), in Poland, was the first to report the presence of T. gondii in radish, carrot, and lettuce, with an approximate contamination rate of 10%. Considering their high consumption and the importance of vegetables in a balanced diet, we assessed the occurrence of T. gondii in raw vegetables.

Materials and Methods

Sampling

A total of 238 samples of vegetables, including crisp lettuce and regular lettuce (Lactuca sativa), chicory (Cichorium intybus), rocket (Eruca sativa), and parsley (Petroselinum crispum), were collected from sales outlets and production sites. These species of vegetable, normally consumed raw, were chosen because they have a pleasant taste, low cost, and are widely used in salads in domiciles and restaurants. In addition, they were selected for their ease and quantity of production and the possibility of contamination by water and polluted soil.

Sampling was done for convenience, which is suitable and commonly used for generating ideas in exploratory research (Oliveira 2001) and they were acquired in places of greater circulation of consumers. From these samples, 77 were from open fairs, 81 from producers’ fairs, where the producers sell their own products, and 80 from community fairs, which are cultivated in gardens by low-income residents who rely on technical support from the municipal agriculture offices. All the samples were collected in northwest Paraná, Brazil, between January and July 2015. In total 149 were organic and 89 were nonorganic.

Processing of samples

Each vegetable sample (50 g) was packaged in a new plastic bag and washed using manual stirring with 100 mL of 1% Tween 80 for 1 min. The resulting liquid was filtered through a cellulose ester membrane (47 mm diameter, 0.3 μm pore size; Millipore) with the aid of a negative pressure vacuum pump (10–15 mmHg). The membrane was then manually scraped as described by Colli et al. (2015). The material was concentrated by centrifugation; the supernatant was discarded and the sediment was resuspended in 500 μL of distilled water (Colli et al. 2015).

DNA extraction

A 250 μL sample was taken from the final volume of each sample and frozen in liquid nitrogen (−196°C) for 5 min, followed by thawing in a dry bath at 65°C for 5 min. This cycle was repeated five times. Extraction was performed using a commercial kit (Axy Prep Blood Genomic DNA, Axygen Biosciences^®).

Experimental control

To evaluate the efficiency of oocysts, the presence of T. gondii oocysts in 50 g of vegetables previously washed in 1% Tween solution was examined. The samples were then infected with known concentrations of oocysts, 10, 10², 10³, and 10⁴, in 2000 mL of distilled water. The samples were processed as described by Colli et al. (2015) and DNA was extracted with a commercial kit (Axy Prep Blood Genomic DNA, Axygen Biosciences) following the manufacturer's instructions.

Polymerase chain reaction

The samples were analyzed by PCR using the primers B1 (B22–B23) to amplify 115 base pairs (bp) (Burg et al. 1989) and Toxo4–5 to amplify a fragment of 529 bp (Homan et al. 2000). In each reaction, a negative control (mixture without DNA) and a positive control (DNA extracted from Me-49 strain) were processed. The amplified products were observed on 4.5% polyacrylamide gels, revealed by silver, and digitally recorded.

Results

In the experimental control, T. gondii DNA was detected in the lettuce samples experimentally infected with a concentration of 10 oocysts per microliter with the primer B1 (B22–B23). With the marker Toxo4–5, a positive result was observed in contaminated samples with ≥100 oocysts per microliter.

Using Toxo4–5, two samples with T. gondii DNA were found, a sample of regular lettuce and chicory (Table 1). The first sample came from an organic farm and was collected at the production site; the second sample was inorganic and collected at an open fair market (Table 1). By using the marker B1 (B22–B23), we found seven samples containing DNA from T. gondii, three from production sites, two from community gardens, and two from open fairs (Table 1). From these samples, five were organic and two were nonorganic, highlighting that one of them was hydroponically grown (Table 1).

Table 1.

Detection of Toxoplasma gondii dna in Fresh Leafy Vegetables

Leaf vegetables	Collection site	Type of crop	PCR/B22–23	PCR/Toxo4–5
Crisp lettuce	Local production	Organic	D	—
Crisp lettuce	Local production	Organic	D	—
Parsley	Local production	Organic	D	—
Crisp lettuce	Community gardens	Organic	D	—
Chicory	Street market	Nonorganic	D	—
Rocket	Street market	Nonorganic (hydroponic)	D	—
Crisp lettuce	Community gardens	Organic	D
Regular lettuce	Local production	Organic	—	D
Chicory	Street market	Nonorganic	—	D

D, detected DNA fragments of T. gondii.

Overall, 3.8% of vegetables were contaminated, with 0.8% found with Toxo4–5 and 2.9% with B22–B23. The results were not converged among the positive samples.

For each type of vegetable analyzed, there was contamination of 0.6% (1/62) in smooth lettuce, 3.7% (4/106) in crisp lettuce samples, 5.0% (2/40) in chicory, 14.3% (1/7) in rocket, and 20% (1/5) in parsley.

Discussion

This is the first report in America of evidence of the presence of T. gondii DNA on raw vegetables and one of the few in the literature (Lass et al. 2012). Some data indicate that environmental contamination (oocysts) could be the main source of transmission (Gotteland et al. 2014). Contamination by T. gondii was found in samples of chicory, lettuce, parsley, and rocket derived from organic or nonorganic production. During collections, we observed that in general producers show good hygiene, using protective screens and animal restraint fences. However, the contaminated leaf vegetables came from community gardens, markets in which the producers sell their own products, and fairs, demonstrating that regardless of the management/production or trade style of the vegetables, there may still be environmental dispersion of parasitic structures. For instance, contamination was found in a vegetable from hydroponic cultivation, where the plant has less contact with soil and irrigation is distributed in the bud, with no spraying on the leaves.

B22-23 PCR with primers that amplify B1 gene were more sensitive when compared to use of primers Toxo 4–5. The B1 gene consists of a highly conserved region of the genome of the parasite and is repeated 35 times (Mesquita et al. 2010) while the gene amplified by 4–5 Toxo repeated in the genome of T. gondii 200–300 times and corresponds to a non-coding region (Homan et al. 2000).

The primer B1 be a minor fragment (115 bp) compared to Toxo 4–5 (529 bp), it amplifies 4.6 times faster, in this way it is easier to happen the complementarity of the enzyme and this action is enhanced making the reaction more stable. Mesquita et al. (2010) also observed an improved sensitivity with primer B1 in biological samples.

Other authors have also noted that samples were not always positive for both primers (Mesquita et al. 2010). It is worth remembering that oocysts are highly dispersed in the environment and their detection in high concentrations is unlikely. In Brazil, a high genetic diversity in T. gondii from isolates from animals and some human biological material has been observed (Grigg et al. 2015, Silveira et al. 2015). Thus, a specific combination of primers is important for optimizing diagnostic sensitivity (Ajzenberg et al. 2004).

Conclusion

This study shows the contamination by T. gondii DNA in raw vegetables grown both organically and nonorganically in different locations, indicating that these vegetables can be a source of T. gondii infection.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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