Toxoplasma gondii in edible fishes captured in the Mediterranean basin

Abstract The issue of whether market fish can be involved in the transmission of Toxoplasma gondii in the marine environment is highly debated since toxoplasmosis has been diagnosed frequently in cetaceans stranded along the Mediterranean coastlines in recent times. To support the hypothesis that fishes can harbour and effectively transmit the parasite to top‐of‐the‐food‐chain marine organisms and to human consumers of fishery products, a total of 1,293 fishes from 17 species obtained from wholesale and local fish markets were examined for T. gondii DNA. Real‐time PCR was performed in samples obtained by separately pooling intestines, gills and skin/muscles collected from each fish species. Thirty‐two out of 147 pooled samples from 12 different fish species were found contaminated with T. gondii DNA that was detected in 16 samples of skin/muscle and in 11 samples of both intestine and gills. Quantitative analysis of amplified DNA performed by both real‐time PCR and digital PCR (dPCR) confirmed that positive fish samples were contaminated with Toxoplasma genomic DNA to an extent of 6.10 × 10−2 to 2.77 × 104 copies/ml (quantitative PCR) and of 1 to 5.7 × 104 copies/ml (dPCR). Fishes are not considered competent biological hosts for T. gondii; nonetheless, they can be contaminated with T. gondii oocysts flowing via freshwater run‐offs (untreated sewage discharges, soil flooding) into the marine environment, thus acting as mechanical carriers. Although the detection of viable and infective T. gondii oocysts was not the objective of this investigation, the results here reported suggest that fish species sold for human consumption can be accidentally involved in the transmission route of the parasite in the marine environment and that the risk of foodborne transmission of toxoplasmosis to fish consumers should be further investigated.

cycle involving an intermediate host, virtually all warm-blooded animals, wherein the parasite multiplies by asexual reproduction leading to intracellular cysts developing in muscles and other organs and a definitive host (wild and domestic felids) in which sexual reproduction results in the shedding through faeces of countless infective oocysts in the environment. As a result, the risk factors for human and animal infection include consuming infected raw or undercooked meat; ingestion of contaminated water, soil, vegetables or anything contaminated with oocysts shed in faeces; blood transfusion or organ transplants; intrauterine or transplacental transmission; and drinking infected unpasteurized milk (Aguirre et al., 2019).
In the past, oocysts-acquired infections have been deemed as less important than foodborne infections although they are considered more severe clinically in intermediate hosts than those related to the ingestion of tissue cysts (Hill & Dubey, 2002). Despite the consumption of raw or undercooked meat harbouring T. gondii cysts is still considered the most likely source of infection, in the last 40 years' toxoplasmosis outbreaks associated with exposure to oocysts-contaminated water have been reported in Panama (Benenson, Takafuji, Lemon, Greenup, & Sulzer, 1982), in British Columbia (Bowie et al., 1997), in Brazil (De Moura et al., 2006 and in India (Balasundaram, Andavar, Palaniswamy, & Venkatapathy, 2010) with the latter two episodes resulting from the ingestion of municipal drinking water.
Furthermore, the majority (78%) of congenital toxoplasmosis cases from four epidemics in North America originated from oocysts exposure, though only 49% of these cases could be confirmed as foodborne (Aguirre et al., 2019). always be linked to typical risk factors, such as consumption of cysts-contaminated meat, involved with toxoplasmosis and that a different route needed to be sought. Finally, the development of more sensitive analytical methods for the detection of oocysts in soil and water samples has added further evidences of the spread of oocysts shed in the environment whose role is no longer seen as secondary to tissue cysts-based transmission.
Environmental pollution with T. gondii oocysts can be accounted for the contamination of both freshwater and marine aquatic environments where Toxoplasma infection is being frequently diagnosed in several sea mammals (Fayer, Dubey, & Lindsay, 2004) that are at the top of the aquatic food chain. Field surveys suggest that as many as eight different families of marine mammals are susceptible to T. gondii infection (Dabritz et al., 2007) including the endangered southern sea otter (Enhydra lutris nereis) in which toxoplasmosis is responsible for high mortality and slow rate of population recovery (Conrad et al., 2005). Cetaceans are also susceptible to T. gondii infection that is often found in individuals stranded in the Mediterranean coastlines due to infection with viral and bacterial pathogens (Morbillivirus, Herpesvirus and Brucella spp) (Di Guardo & Mazzariol, 2016) with toxoplasmosis more likely to be secondary to concurrent immunosuppression.
Anthropogenic coastline pollution with sewage or surface runoff of freshwater contaminated with the T. gondii oocysts is likely the key factor in the epidemiology of toxoplasmosis in the marine environment with feral and domestic cats playing as the sole source of oocysts. Following infection, a single cat can shed millions of oocysts within 1 week. Also cats' defecation behaviour, such as faeces burial in shady areas, increases the survival of oocysts that can persist in the environment longer than 1 year. Flood-related natural events could also drive the transmission of terrestrial pathogens like T. gondii especially in estuarine environments.
Many gaps remain to be filled in the transmission pathways and pathogenesis of toxoplasmosis in aquatic mammals. For those cetaceans exclusively from offshore waters, contact with oocysts-contaminated wastewaters discharged from ships has been advocated (Di Guardo et al., 2010). Recently novel routes of oocysts transmission involving a complex interaction of suspended bioparticles, biofilms, small invertebrates and gastropods have been hypothesized whereas mammals can also become infected nearby the coast prior to stranding or via consumption of fishes or mussels carrying the parasite.
In the search for potential hosts involved in the transmission of the parasite between terrestrial and marine environments where it is known to trigger the infection in competent hosts like sea mammals, we searched for T. gondii DNA in fishes belonging to small to Impacts • Toxoplasma gondii is an ubiquitous parasite isolated from cetaceans with meningoencephalitis stranded in the Mediterranean basin.
• Extracellular polymeric substances (EPS), surface-scraping mollusks, shellfish and fish are potentially involved in the transmission pathways of T. gondii in the marine environment.
• Market fish contaminated with T. gondii could be of a public health concern for workers involved on fish manipulation and consumers besides a marker of environmental pollution. medium-size species positioned at different levels of the aquatic food webs. The main goal of the study was to assess whether contaminated fishery products could be a further risk factor of foodborne toxoplasmosis for human consumers and also an occupational hazard for fishing and fish processing operators considering that the fish species tested are among those most commonly harvested and sold for human consumption in the Mediterranean area. to a minimum of a single unit for the European conger (Conger conger), the thornback ray (Raya clavata) and the cow bream (Sarpa salpa).

| MATERIAL S AND ME THODS
Since the main goal of the study was to evaluate the extent to which T. gondii in fish can be a food safety issue under everyday life habits of relatively small quantities of fresh fish purchased for consumption, we decided to include in the survey also fish species for which few units were collected because of limited availability in markets at the time of sampling. Detailed description of sampling is shown in Table 1.
In order to process and analyse samples of similar size, fishes were not individually tested but each species collected from the market was divided in groups of 3, 5, 6, 8 or 10 units according to fish size and weight. From each fish, intestine, gills and multiple aliquots of skin-skeletal muscles complex (to a maximum weight of 10 g) were split in asepsis, separately. Guts were examined to determine the fishes' capacity to retain oocysts as it was previously documented (Massie, Ware, Villegas, & Black, 2010). Likewise, gills were analysed to determine whether they can trap oocysts filtered from seawater. Skin and muscle were not separated prior to analysis as it was considered unrealistic to avoid potential DNA cross-contamination from specimen to specimen contact during processing and also to evaluate the extent to which ingestion of skin and meat from  was applied on sampled tissues (Marino et al., 2017). PCR was performed in a 20 μl final volume containing 10 μl of master mix 2×, added to nuclease-free water (4 μl According to validation performed in compliance with OIE and Codex Alimentarius guidelines (Marino et al., 2017), the overall sensitivity and specificity of both applied qualitative and qPCR methods was 100%. The limit of detection threshold was 0.01 pg/μl.
As a further step for the quantification of T. gondii DNA, a digital PCR (dPCR) protocol was employed. A notable benefit of dPCR over qPCR is that this analysis technique requires no standard, calibration or information about the molecular weight distribution of the template molecules (Sanders, Mason, Foy, & Huggett, 2013  Quantification of amplified DNA by both quantitative real-time PCR and dPCR confirmed that positive fish samples were contaminated with Toxoplasma genomic DNA to an extent of 6.10 × 10 −2 to 2.77 × 10 4 copies/ml (qPCR) and of 1-5.7 × 10 4 copies/ml (dPCR).

| RE SULTS
The estimated heaviest parasitic load was found in pooled samples of B. boops intestines (qPCR) and in gills samples of M. merluccius (dPCR). Detailed results of quantitative analyses are reported in Table 3. Our findings that fishery products are somehow exposed to T. gondii are consistent with reports suggesting that T. gondii can survive in the sea (Arkush et al., 2003;Lindsay et al., 2003Lindsay et al., , 2004 and Due to their sticky properties, EPS can mediate transportation of soil-derived pathogens, including T. gondii, in the marine ecosystem either by incorporating into marine macroaggregates (e.g., marine snow) or through direct adhesion to biofilms coated on the surface of seaweeds or other benthic organisms (Wotton, 2004).

| D ISCUSS I ON
Both mechanisms are likely to increase the chance of pathogens entry into the marine food web through aggregate-consuming invertebrates such as bivalves or surface-scraping molluscs such as snails (Shapiro et al., 2014) (Mazzillo, Shapiro, & Silver, 2013). Similarly, EPS attached to sediment particles have also been proved to be ingested by deposit-feeding marine organisms (Hoskins, Stancyk, & Decho, 2003). These studies provide a novel transmission route for T. gondii in the marine Fishes are not considered competent biological hosts for T. gondii although experimental studies indicate that common commercial species like anchovies and sardines are able to pass T. gondii oocyst through their intestine following exposure to oocysts-spiked sea water (Massie et al., 2010).

No. of samples per tissue
Strictly speaking of toxoplasmosis, only in vitro infection has been documented for fishes (Omata et al., 2005;Sanders et al., 2015). Following intraperitoneal inoculation of T. gondii bradyzoites in zebrafish (Danio rerio), tachyzoites were detected through histological examination in several tissues including skeletal muscle (Sanders et al., 2015). A similar study carried out in goldfishes (Carassius auratus) showed that T. gondii tachyzoites can persist up to 3 days post-infection (intramuscular inoculation) as evidenced by PCR analysis of inoculated tissues and by bioassay in mice intraperitoneally inoculated with tissue homogenate from experimentally infected fishes (Omata et al., 2005). Also in vitro infection of oviduct epithelial cells of goldfish showed active intracellular reproduction of T. gondii tachyzoites within 6 hr of inoculation (Omata et al., 2005).
However, a temperature of 37°C was an absolute limiting factor of the in vitro studies, since at lower temperatures T. gondii tachyzoites do not appear to be able to penetrate and multiply in the somatic host cells (Omata et al., 2005;Sanders et al., 2015). Therefore, the TA B L E 3 Quantitative PCR and digital PCR analysis of Toxoplasma gondii DNA detected in positive fish pooled samples existence of a T. gondii biological cycle in fish similar to that occurring in mammals is highly unlikely and consequently trophically transmitted infection in marine mammals should be incidental and limited to the predation of sea organisms mechanically carrying oocysts.
The issue of the viability and availability of oocysts dispersed in the marine environment is also highly debated and whether oocysts-carrying fishes are able to mechanically transmit viable parasites to larger predators at the top level of the food chain, including human consumers, remains controversial. Oocyst viability, assessed through bioassay or molecular diagnosis of toxoplasmosis on spf mice fed with infected tissues from sardines exposed to T. gondii oocysts, was confirmed for fishes exposed to high parasite load (at least 100,000 oocysts/L of seawater) (Massie et al., 2010).
Additionally filter-feeding fishes exposed to oocysts for 2 hr are able to retain oocysts in the alimentary canal for at least 8 hr (anchovies) although oocysts viability has been proven only for contaminated sardine tissues (Massie et al., 2010). As filter feeders specialized in eating microparticles (Kucas, 1986), both anchovies and sardines are naturally suited to consume T. gondii oocysts as they enter the nearshore environment; as the fishes migrate offshore, they could then serve as biotic vectors for T. gondii into the greater marine environment (Massie et al., 2010). should also be trained to follow proper hygiene practices and required to wear protective clothing and boots that must be cleaned regularly in order to minimize the risk of fish contamination with environmental T. gondii oocysts.
The risk of T. gondii occurrence in fishes should therefore be taken into account also as an occupational issue. Operators working in the fishing industry should be aware of the risk of exposure to the parasite both in the primary production stages (fishing and harvesting) and during raw fish handling for the production of processed fishery products like salt cured anchovies. This issue should be addressed especially for female workers that make up the largest part of employees in small enterprises in the south Mediterranean countries where fish processing largely relies on manual labour. Good manufacturing practices must therefore be implemented to reduce the risk for exposed workers namely for pregnant women. This procedure does not ensure food security since it has been documented that oocysts can retain viability in aqueous 2% sulphuric acid for several years at 4°C (Dumètre & Dardé, 2003;Dumètre et al., 2008).
Although there are no documented evidences of T. gondii infection through the consumption of fresh raw or undercooked fish, findings of this study suggest that fishes should be regarded as liable of accidental contamination with the parasite and therefore deemed as a potential source of infection for marine mammals and humans alongside shellfish that are known to be able to capture and concentrate T. gondii oocyst from sea water (Aksoy et al., 2014;Esmerini, Gennari, & Pena, 2010). Hence, the guidelines suggested for the safe storing, handling and cooking of meat and meat products should also apply to raw fish that must always be cooked to a temperature likely to inactivate the parasite (at least 66°C) or deep frozen at −21°C for 1 or 28 days (for unsporulated or sporulated oocysts, respectively) (Dumètre & Dardé, 2003) since storing in household refrigerators (4°C for 6-11 weeks) does not prevent the development of oocysts infectivity (Lindsay, Blagburn, & Dubey, 2002).
Additional fields investigations are of course recommended to improve our understanding of the role of market fish as potential vector for T. gondii whereas the issue of oocysts viability remained to be assessed to unravel the parasite transmission pathways in the sea environment and to evaluate the risk of foodborne toxoplasmosis for human consumers.

ACK N OWLED G EM ENTS
The authors disclosed receipt of the following financial support for the research, authorship and/or publication of this article: Grant funding for this study was awarded by the Italian Ministry of Health (Ricerca Corrente 2015 IZS SI 02/15).

CO N FLI C T S O F I NTE R E S T S
The authors declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.