In this study, Blastocystis was detected in 45% (9/20) of the produce using qPCR, including the three that were positive by microscopy. Of the 18 above ground samples eight (44%) were positive for Blastocystis. Of these, four were from produce with edible peel. Only one out of four (25%) root samples was positive. Sequences have been submitted to GenBank under accession numbers xxx-xxxx. Foodborne transmission of Blastocystis has been previously speculated. A study in Riyadh, Saudi Arabia reported the common occurrence of Blastocystis in leafy vegetables, most especially lettuce and parsley (Al-Megrin 2010). Similarly, the organism was detected in lettuce sold in markets of northern Brazil and in ready-to-eat (prewashed) packaged salads in Italy (Caradonna et al. 2017). In Iran, more than 20% of raw herbs (Heidar Nejadi 2021) and 16% of fresh vegetables (Isazadeh et al. 2020) were contaminated with Blastocystis. Our results revealed a higher percentage of Blastocystis in fresh produce than previous works elsewhere, likely due to the more sensitive nature of qPCR.
Of the Blastocystis positive samples, subtype (ST) 3 was the most common, comprising 78% (n = 7) of the total (Table 1). We have designated two of the sequences (22%) as unknown. In the blast search and in the phylogeny, both of these sequences group with subtype 30, however this placement is not entirely robust. Since there are only very few ST30 sequences in the database and the fragments generated in this study are relatively short, a longer fragment is necessary to conclusively determine the subtype. Subtype three is the most frequent and widely distributed globally having been found in humans, animals, and also the environment (Adamska 2020; Banaticla and Rivera 2011; Ii Lee et al. 2012; Ithoi et al. 2011; Javanmard et al. 2019; Jinatham et al. 2021, 2022; Koloren, Gulabi, and Karanis 2018; Leelayoova et al. 2008.) Previous studies with a similar focus used microscopic detection, hence it is not possible at this time to speculate whether ST3 is a common occurrence on vegetables.
Table 1
Occurrence of Blastocystis in vegetables bought at two street markets in Chiang Rai, Thailand. Asterisks denote samples that were sampled in the root and in the above ground part
| Vegetable samples | Blastocystis positive (ST) |
| Above ground |
| Beetroot | Beta vulgaris | ST3 | 44% |
| Cabbage | Brassica oleracea var. capitata | UNK |
| Carrot | Daucus carota | ST3 |
| Cauliflower | Brassica oleracea var. botrytis | - |
| Chinese Cabbage | Brassica pekinensis | ST3 |
| Coriander* | Coriandrum sativum | - |
| Cucumber | Cucumis sativus | - |
| False Pak Choi | Brassica chinensis | - |
| Galangal | Alpinia galanga | ST3 |
| Ginger | Zingiber officinale | - |
| Kale | Brassica alboglabra | ST3 |
| Lemongrass | Cymbopogon citratus | ST3 |
| Lettuce | Lactuca sativa | - |
| Long Coriander* | Eryngium foetidum | - |
| Onion | Allium cepa | - |
| Pumpkin | Cucurbita moschata | - |
| Tomato | Lycopersicon esculentum | UNK |
| Turmeric | Curcuma longa | - |
| Root |
| Asiatic Pennywort | Centella asiatica | - | 25% |
| Chinese Morning Glory | Ipomoea aquatica | ST3 |
| Coriander* | Coriandrum sativum | - |
| Long Coriander* | Eryngium foetidum | - |
The presence of Blastocystis on produce in this and other studies is surprising given the low tolerance of the organism to oxygen (Tsaousis et al. 2018). The shape and surface of vegetables has been associated with the degree of contamination. Micro-organisms are usually attached to green leafy vegetables with uneven surfaces, whereas their occurrence on vegetables with smooth surfaces is lower (Abougrain et al. 2010; Damen et al. 2007; El Said Said 2012). The multi-layering and roughness of vegetable stems could create micro niches encouraging Blastocystis attachment.
Parasite contamination of vegetables can occur at any point in the supply chain, both at the pre- and post-harvest phases. Its occurrence depends on several factors such as water source, soil supplementation and presence of animals (Ismail 2016; El Said Said 2012). In Thailand, crops are regularly fertilized with inadequately composted manure from animals that are known to carry Blastocystis. Produce such as galangal, carrot and beetroot grows near and/or under soil, hence it is possible that the organism was established pre-harvest in these foodstuffs. Soil was also recently proposed as a route of Blastocystis transmission (Jinatham et al. 2021). Alternatively, the organism could have been acquired following harvest. Vegetables in this study were washed/sprinkled with tap water available in the markets before being distributed to the customers. Tap water in the same study area was previously shown to be contaminated with Blastocystis (Jinatham et al. 2022). Regardless, these sources of transmission should be investigated using a combination of molecular and epidemiological approaches.
This pioneer pilot study provides the first molecular investigation of Blastocystis and its subtypes in vegetables. Our results pinpoint towards fresh produce being a transmission route of Blastocystis. These findings have important implications for the reduction or avoidance of contamination by parasites in vegetables in the various stages of the food supply chain ensuring food safety. Identification of another potential transmission route of Blastocystis highlights the need of a One Health approach in future large-scale investigations on the prevalence of this and other organisms.