Cryptosporidium parvum genotype IIa and Giardia duodenalis assemblage A in Mytilus galloprovincialis on sale at local food markets

https://doi.org/10.1016/j.ijfoodmicro.2013.11.022Get rights and content

Highlights

  • Shellfish filter large volumes of water and can retain several zoonotic protozoans.

  • Edible shellfish Mytilus galloprovincialis harbour type/subtypes of C. parvum and G. duodenalis.

  • Subgenotypes A15G2R1, IIaA15G2 and IIaA14G3R1 and assemblage A have never been reported in shellfish.

  • Eating contaminated mussels can be a risk for human health.

Abstract

To date, there has been no study to establish the genotypic or subgenotypic identities of Cryptosporidium and Giardia in edible shellfish. Here, we explored the genetic composition of these protists in Mytilus galloprovincialis (Mediterranean mussel) purchased from three markets in the city of Foggia, Italy, from May to December 2012. Samples from the digestive glands, gills and haemolymph were tested by nested PCR, targeting DNA regions within the 60 kDa glycoprotein (gp60) gene of Cryptosporidium, and the triose-phosphate isomerase (tpi) and β-giardin genes of Giardia. In total, Cryptosporidium and Giardia were detected in 66.7% of mussels (M. galloprovincialis) tested. Cryptosporidium was detected mostly between May and September 2012. Sequencing of amplicons showed that 60% of mussels contained Cryptosporidium parvum genotype IIa (including subgenotypes A15G2R1, IIaA15G2 and IIaA14G3R1), 23.3% Giardia duodenalis assemblage A, and 6.6% had both genetic types. This is the first report of these types in fresh, edible shellfish, particularly the very commonly consumed M. galloprovincialis from highly frequented fish markets. These genetic types of Cryptosporidium and Giardia are known to infect humans and thus likely to represent a significant public health risk. The poor observance of hygiene rules by vendors, coupled to the large numbers of M. galloprovincialis sold and the eating habits of consumers in Italy, call for more effective sanitary measures pertaining to the selling of fresh shellfish in street markets.

Introduction

Seafood is a major part of the culinary culture in many countries around the world, and the shellfish industry is of major economic importance in the Mediterranean area with the highest production along the coasts of Italy, Spain and France.

In Italy, current estimates indicate that 203,810 and 131,000 tonnes of fish and shellfish, respectively, are processed each year (Ismea, 2012). The most commonly farmed shellfish for human consumption is Mytilus galloprovincialis (Mediterranean mussel), followed by Ruditapes philippinarum (Manila clams). In 2011, mussel production was estimated at 98,000 tonnes (including mussels from natural benches), with > 80% of the production plants located in the southern Italian regions (Ismea, 2012). Forty-six percent of consumers prefer mussels to other types of shellfish, and they are sold in street markets also in the south of Italy.

Excreta from humans and other animals are a source of a plethora of microorganisms, which are dispersed directly or, for example, via rainfall-initiated run-off from agricultural, suburban and urban lands, wastewater into rivers, streams, estuaries and coastal waters, thus contaminating the sea and its inhabitants. Bivalves filter large volumes of water and consequently can accumulate and retain particles and microorganisms; some of these organisms can be pathogenic and thus represent a potential risk to human health, particularly if eaten raw (EFSA, 2012). Pathogens of most concern in shellfish are viruses (e.g., norovirus, hepatitis A virus), bacteria (e.g., pathogenic Escherichia coli, Campylobacter jejuni, Salmonella spp., Vibrio vulnificus, Vibrio cholerae and Vibrio parahaemoliticus) and protozoans (including Cryptosporidium, Cyclospora, Giardia and Toxoplasma), particularly in young, old and/or immuno-compromised or -suppressed people (WHO, 2010).

A number of studies have shown that Cryptosporidium and Giardia are present worldwide in shellfish farmed or naturally present in lagoons and other marine environments. Several edible and inedible shellfish have been found to carry Cryptosporidium parvum (Gómez-Bautista et al., 2000, Fayer et al., 2002, Fayer et al., 2003, Gómez-Couso et al., 2004, Gómez-Couso et al., 2006a, Gómez-Couso et al., 2006b, Miller et al., 2005, Giangaspero et al., 2005, Li et al., 2006, Graczyk et al., 2007, Molini et al., 2007), whereas Giardia duodenalis assemblage A has only been reported to occur in inedible shellfish species (Graczyk et al., 1999a), although its precise identity and link to enteric disease (outbreaks) in humans had not been unequivocally established at the time.

In spite of the major public health importance of these protists and their potential to cause zoonotic disease (Giangaspero et al., 2007, Xiao and Fayer, 2008), there has been no global study to establish the specific and/or genotypic identities of Cryptosporidium and Giardia found in shellfish. To date, Cryptosporidium hominis (subgenotypes IbA10G2R2, IbA9G3R2, IeA11G3T3R1), C. parvum (subgenotypes IIaA16G2R1, IIaA19G3R1, IIcA5G3R2) and G. duodenalis (assemblages A and B) have been reported to be commonly associated with human cryptosporidiosis (Xiao and Fayer, 2008, Jex et al., 2008, Jex and Gasser, 2010); these assemblages are identified mostly using PCR-based techniques employing various genetic markers (Chalmers et al., 2005, Plutzer and Karanis, 2009, Bouzid et al., 2010, Putignani and Menichella, 2010, Feng and Xiao, 2011). For Cryptosporidium, the SSU, hsp70 and/or 60 kDa glycoprotein (gp60) genes have been used for specific, genotypic and/or subgenotypic identification (Jex et al., 2007, Widmer, 2009, Nolan et al., 2010, Nolan et al., 2013). For Giardia, markers in the β-giardin, tpi, gdh and/or SSU genes are most commonly used for identification to species and/or assemblages (Giangaspero et al., 2007). In the present study, we employed PCR-based sequencing of gp60 and tpi and β-giardin to genetically classify Cryptosporidium and Giardia, respectively, from M. galloprovincialis from local markets in a locality in south-eastern Italy.

Section snippets

Samples, and isolation of genomic DNA from mussels

Mussels (M. galloprovincialis) were purchased at ten different time points from each of three markets (I, II, and III) in the city of Foggia (41°28′0″N 15°34′0″E) at intervals of 6–10 days from May to December 2012, a period of time, which corresponds to the mussel commercialization (www.coopmare.com/public/relazioni/041245_RelFinale_Miglioramento%20mitili.pdf) in Southern Italy. At each time point, 500 g of mussels was purchased from one market, refrigerated at 5 °C and taken to the laboratory

Results

Under the present PCR conditions, there was no evidence of an inhibitory effect of molluscan components on enzymatic amplification for any of the samples tested. Sixty batches of 15 mussels each were obtained for a total of 30 samplings. Overall, 40 of 60 (66.7%) samples were PCR test-positive for Cryptosporidium, Giardia, or both Cryptosporidium and Giardia (Table 1). Cryptosporidium was detected in 34 of 60 (56.7%) samples, and was significantly more common in markets II and III than market

Discussion

This is the first published report of C. parvum IIa (subgenotypes IIaA15G2R1, IIa15G2 and IIaA14G3R1) and G. duodenalis assemblage A in edible shellfish, particularly very commonly consumed M. galloprovincialis in highly frequented daily fish markets in a city context. These potentially zoonotic protists (Cryptosporidium and Giardia) were detected in 66.7% of the 60 samples of mussels overall tested, with genotype IIa, assemblage A and both being identified in 60%, 23.3% and 6.6% of these,

Acknowledgements

The authors wish to thank Antonio Narducci and Raffaella Terlizzi for their laboratory assistance and Professor Giovanni Normanno for the helpful discussions on food inspection. RBG's laboratory is currently funded by the Australian Research Council (ARC) and the National Health and Medical Research Council (NHMRC). Other support from Melbourne Water Corporation and the Alexander von Humboldt Foundation is gratefully acknowledged (RBG).

References (56)

  • M. Lebbad et al.

    From mouse to moose: multilocus genotyping of Giardia isolates from various animal species

    Vet. Parasitol.

    (2010)
  • X. Li et al.

    Cryptosporidium oocysts in mussels (Mytilus edulis) from Normandy (France)

    Int. J. Food Microbiol.

    (2006)
  • W.A. Miller et al.

    New genotypes and factors associated with Cryptosporidium detection in mussels (Mytilus spp.) along the California coast

    Int. J. Parasitol.

    (2005)
  • U. Molini et al.

    Temporal occurrence of Cryptosporidium in the Asian clam Ruditapes philippinarum in the Northern Adriatic Italian lagoons

    J. Food Prot.

    (2007)
  • M.J. Nolan et al.

    Molecular detection of Cryptosporidium cuniculus in rabbits in Australia

    Infect. Genet. Evol.

    (2010)
  • M.J. Nolan et al.

    Molecular-based investigation of Cryptosporidium and Giardia from animals in water catchments in southeastern Australia

    Water Res.

    (2013)
  • J. Plutzer et al.

    Genetic polymorphism in Cryptosporidium species: an update

    Vet. Parasitol.

    (2009)
  • P.R. Wielinga et al.

    Molecular epidemiology of Cryptosporidium in humans and cattle in The Netherlands

    Int. J. Parasitol.

    (2008)
  • L. Xiao et al.

    Molecular characterisation of species and genotypes of Cryptosporidium and Giardia and assessment of zoonotic transmission

    Int. J. Parasitol.

    (2008)
  • M. Alves et al.

    Microsatellite analysis of Cryptosporidium hominis and C. parvum in Portugal: a preliminary study

    J. Eukaryot. Microbiol.

    (2003)
  • ARPA, Puglia

    Relazione sullo Stato dell'Ambiente 2010 Regione Puglia

  • A. Baumgartner et al.

    Frequency of Cryptosporidium spp. as cause of human gastrointestinal disease in Switzerland and possible sources of infection

    Schweiz. Med. Wochenschr.

    (2000)
  • M. Bouzid et al.

    Multi-locus analysis of human infective Cryptosporidium species and subtypes using ten novel genetic loci

    BMC Microbiol.

    (2010)
  • R.M. Chalmers et al.

    Direct comparison of selected methods for genetic categorisation of Cryptosporidium parvum and Cryptosporidium hominis species

    Int. J. Parasitol.

    (2005)
  • C.L. Chappell et al.

    Cryptosporidium parvum: intensity of infection and oocyst excretion patterns in healthy volunteers

    J. Infect. Dis.

    (1996)
  • F. Del Chierico et al.

    Cases of cryptosporidiosis co-infections in AIDS patients: a correlation between clinical presentation and GP60 subgenotype lineages from aged formalin-fixed stool samples

    Ann. Trop. Med. Parasitol.

    (2011)
  • EFSA

    European Food Safety Authority

  • A.A. Escobedo et al.

    Giardiasis: the ever-present threat of a neglected disease

    Infect. Disord. Drug Targets

    (2010)
  • Cited by (33)

    • Occurrence of the protozoan parasites Toxoplasma gondii and Cyclospora cayetanensis in the invasive Atlantic blue crab Callinectes sapidus from the Lesina Lagoon (SE Italy)

      2022, Marine Pollution Bulletin
      Citation Excerpt :

      There, each sample was individually homogenized in 1 mL of distilled water, then sieved through a double layer of gauze and pelleted by centrifugation (1000g, 4 °C, 10 min); the pellet was then washed with TE buffer (one time at 1000g, 4 °C, 10 min and one time of 13,000g at 4 °C for 15 min). 500 mL of pellet was subjected to three cycles of 20 °C/15 min and 80 °C/15 min (Giangaspero et al., 2014). Subsequently, genomic DNA was isolated from individual samples using the Nucleospin tissue kit (Macherey Nagel, Netherlands), according to the manufacturer's instructions.

    View all citing articles on Scopus
    View full text