First report of Cryptosporidium sp. (coccidia, apicomplexa) oocysts in the black mussel (Mytilus galloprovincialis) reared in the Mali Ston Bay, Adriatic Sea.

ABSTRACT Cryptosporidium spp. are obligate intracellular apicomplexan parasites that infect epithelial cells of the gastrointestinal systems of a wide range of vertebrate hosts, including humans. Its importance as a serious public health threat was recognized only since the HIV pandemic. Because of the particular filter feeding behavior of bivalves, these marine organisms are susceptible to the accumulation of Cryptosporidium spp. oocysts from the environment and their retention for a certain time, acting as potential zoonotic reservoirs. To preliminary evaluate the presence of Cryptosporidium spp. oocysts in cultured bivalves from the Mali Ston Bay, Adriatic Sea, we have analyzed individuals from a black mussel (Mytilus galloprovincialis) population by immunofluorescence, over a one year period at four different locations. Overall one-year prevalence of the Cryptosporidium spp. was 16.8%, and was correlated with the presumptive number of E. coli in the shellfish and seawater and abiotic factors (temperature, salinity, oxygen), suggesting the necessity for the updating of existing sanitary control measures in Croatia.

KEY WORDS: Cryptosporidum oocysts, black mussel, Mytilus galloprovincialis, Mali Ston Bay, Adriatic Sea

INTRODUCTION

Cryptosporidium spp. are obligate intracellular apicomplexan parasites that infect epithelial cells of the gastrointestinal systems of a wide range of vertebrate hosts, including humans. The infective stages are four naked sporozoites enclosed in a spherical, thick wall oocyst (measuring 4-6 [micro]m and lacking the sporocyst), which enables the parasite to survive for months outside the host (Fayer et al. 1998).

So far 16 species have been recognized (Xiao et al. 2004), whereas C. parvum is the major species responsible for clinical disease in humans and domestic animals, detected in about 80 different mammalian species including cattle, horse, sheep, goat, and pigs. However, the use of molecular tools with a greater capacity to detect and differentiate strains has resulted in the identification of other human pathogens: C. felis, C. meleagridis, C. canis and C. muris (see Caccio et al. 2002). It has been recognized that two genotypes exist within C. parvum; genotype 1 of H (human) and genotype 2 or C (cattle, infecting both humans and cattle) (McLaughlin et al. 2000). Recently however, these two genotypes, both of major interest for medicine, have been proposed as two separate species; zoonotic C. parvum and anthroponotic C. hominis (Morgan-Ryan et al. 2002), although other species as well contribute to human gastroenteritis (Xiao & Ryan 2004, Caccio et al. 2005).

Because the HIV pandemic, cryptosporidiosis has been recognized as a serious public health threat whose pathogenicity and etiology have become the subject of intensive research. The epidemiology of the infection in various subject groups has been reviewed in detail (Franzen & Muller 1999, Hunter & Nichols 2002, Hunter & Thompson 2005, Sunnotel et al. 2006). Generally, C. parvum elicits mild and self-limiting diarrhea in immunocompetent individuals, whereas in ah immunocompromised population (e.g., elderly, children, HIV patients, and patients ongoing immunosuppressive therapy) it has chronic and life threatening affects (Chen et al. 2005). Prevalence rates in patients with diarrhea have varied from as low as 1% in Europe and North America to up to 30% in other countries (Franzen & Muller 1999).

Etiology of cryptosporidiosis is mostly associated with waterborne outbreaks (e.g., ingestion of contaminated water or food, swimming in contaminated pools), as well as direct person/animal-to-person contacts (Franzen & Muller 1999). Massive waterborne infections have been described in different parts of the world (Hayes et al. 1989, MacKenzie et al. 1994, Baker et al. 1998, Kramer et al. 1998, Veverka et al. 2001, Jennings & Rhatigan 2002), pointing out the necessity to set up new regulations and standards for water quality control. Even though the EU Council has recognized the importance of this pathogen and new legislations are in preparation, only the UK so far has stated the maximum permitted number of Cryptosporidium spp. oocysts in the drinking water (less then 1/10 L) (Anonymous 1990, Anonymous 1995, Anonymous 1999).

In marine environments, C. parvum is commonly found in areas that are impacted by sewage overflow or increased urban and agricultural runoffs (Johnson et al. 1995, Gomez-Couso et al. 2006a). Bivalves are susceptible to accumulate the parasitic oocysts from the environment and retain them for a certain length of time because of their filter feeding behavior, acting as potential zoonotic reservoirs or accidental parathenic hosts (Ayres et al. 1978). A number of studies reported C. parvum in wide range of commercially important bivalve species worldwide including Mediterranean (Chalmers et al. 1997, Fayer et al. 1998, Fayer et al. 1999, Graczyk et al. 1999, Tamburrini & Pozzio 1999, Freire-Santos 2002, Freire-Santos et al. 2002, Li et al. 2006), highlighting the potential risk of consuming raw or light-cooked seafood. Even though it is wildly accepted that C. parvum can be transmitted through water and seafood, the role that bivalves may play in regard to the public health threat remains controversial, and at our knowledge there is no data directly linking C. parvum infection with the shellfish consumption. However, C. parvum oocysts can remain viable in the seawater for 1 y (Tamburrini & Pozio 1999) and after 30 days in two experimentally contaminated molluscan species, are still infective to newly born mice (Freire-Santos et al. 2002). This suggests that the contamination of molluscs by C. parvum is likely to be a possible source of infection to humans. For a detailed review on zoonotic aspects of cryptosporidiosis see Hunter & Thompson (2005) and Robertson (2007).

The Mali Ston Bay is the most productive and economically important area for bivalve aquaculture in the eastern Adriatic, where currently two bivalve species are commercially cultured --the European flat oyster (Ostrea edulis) and the black mussel (Mytilus galloprovincialis). The aim of this study was to preliminary assess spatial and temporal presence of Cruyptosporidium sp. oocysts in the black mussel from the Mali Ston Bay, which is an attractive shellfish species from the fishery and aquaculture point of view, harvested through the year and consumed lightly cooked. Because the identification method of our choice was direct immunoflorescence that detects oocysts of Cryptosporidium genus (Gomez-Couso et al. 2006b), we were notable to discern between species. To assess the environmental impact on the prevalence of the protozoan, we evaluated the correlation of the Cryptosporidium sp. prevalence with the presence of E. coli in shellfish and seawater as well as different abiotic parameters, respectively.

METHODS

Fifteen to twenty cultured black mussels (Mytilus galloprovincialis) were seasonally sampled in 2005 (February, May, August, and December) from a single site in the Mali Ston Bay, Sutvid, Mali Ston, Usko, and Bistrina (not sampled in February) (Fig. 1). Abiotic factors (sea temperature, salinity, and oxygen saturation) were measured at sampling sites with WTW multiline hydrographic probe. Values of abiotic factors per site and season are given in Table 1. In total 230 samples of harvest size bivalves were collected in plastic bags, shipped in refrigerated conditions and transported within hours in the laboratory, whereas prevalence was calculated from 184 analyzed samples. Bivalve length was measured using Vernier calipers to the 0.1 mm (mean shell length = 66.8 [+ or -] 6.7 mm). Collected specimens were individually put in Mg[Cl.sub.2] solution to relax the adductor muscles and central part of digestive gland was cut out with sterile blade and processed for routine histology. Briefly, the tissue was fixed in modified Davidson solution, dehydrated in increasing alcohol concentration, embedded in paraffin block and cut at sections 5-[micro]m thick. Four consecutive sections were loaded on a single slide.

[FIGURE 1 OMITTED]

For immunohistochemical identification of Cryptosporidium sp., monoclonal mouse antiCrypto FITC-conjugated antibodies were incubated on black mussel digestive gland tissue following producer protocol (Cypress Diagnostics, Belgium). The specificity of monoclonal antibodies to Cryptosporidium sp. is combined with the sensitivity of the direct immunofluorescent assay format. One section in each sample was incubated with PBS as a negative control. Commercially available suspension of C. parvum ([10.sup.4] purified oocysts, MeriFluor C/G) was smeared on a microscopic slide and used asa positive control. Samples were analyzed by Olympus BX-50 epifluorescent microscope under the green filter (SWB) at 400 x and x 1,000, according to guidelines of Fayer et al. (1998).

Additional samples of 30 black mussels and seawater were taken simultaneously at the same sampling sites for the measurement of the most probable number (MPN) of presumptive Escherichia coli according to international ISO standards (1990, 2000). Briefly, presumptive E. coli in the shellfish was determined by most probable number (MPN) method according to ISO 9308-2 (1990). Minerals modified glutamate medium (MMGB) was used as media for the presumptive test. Inoculated tubes were incubated at temperature 37 [+ or -] 0.5[degrees]C for 24 (48) hours. After incubation for 24 h, all MMGB culture tubes that provided a positive reading (acid and gas in Durham tubes) were transferred into test tubes containing tryptophan broth for the confirmation test. Confirmation test cultures were incubated at temperature 44[degrees]C for 24 h. The confirmation procedure was repeated after 48 h with all culture tubes containing MMBG broth, which had become positive in the 24 to 48 h interval. After the period of incubation, tryptophan broth tubes were examined for the production of indole by adding Kovac's reagent. MPN value was calculated from MPN tables.

E. coli in seawater was enumerated by membrane filtration (MF) method according to ISO 9308-1 (2000) (rapid test). After filtration the membrane filters were placed on double layer plates consisting of tryptone soy agar (TSA) and tryptone bile agar (TBA) and incubated at 36 [+ or -] 2[degrees]C for 4.5 h followed by incubation at 44.0 [+ or -] 0.5[degrees]C for 19.5 h. After incubation, membranes were placed on filter pads saturated with indole reagent (p-Dimethylaminobenzaldehyde dissolved in concentrated, hydrochloric acid) and irradiated with an UV lamp (wavelength 254 nm). All red colonies on the membrane filter are counted as E. coli.

Cryptosporidium sp. prevalence and its confidence limits (CL) at 95% were calculated according to Bush et al. (1997). Briefly, prevalence is the number of hosts infected with one or more individuals of a particular parasite species, divided by the number of hosts examined for that parasite species, expressed as a percentage.

To compare prevalence values among seasons and sampling sites, nonmetric multivariate analyses using the PRIMER statistical package was performed (Clarke & Warwick 1994). Because data did not display a normal distribution, PERMANOVA software was also used (Anderson 2005). Data were presented as presence/absence of the parasite in each analyzed mussel. All analyses were conducted using Bray-Curtis similarity matrix on untransformed parasite prevalence data. Nonmetric multidimensional scaling (nMDS) was used to obtain a graphic representation of the results; points that are close together represent individual black mussel samples that are very similar in parasite prevalence values, points that are far apart correspond to very different prevalence values. PERMANOVA (Anderson 2005) was used to test for significant differences among the different factors of the two-way crossed design. In this case of independent factors, the sites (Sutvid, Mali Ston, Usko & Bistrina) and seasons (spring, summer, autumn, winter) were considered as fixed factors. Results were considered significant at *p = 0.01 and **p = 0.001, respectively (Table 2).

A two-tailed non parametric Spearman's correlation analysis (95 % confidence levels) was applied to determine the degree of association between the prevalence of the parasite, MPN of presumptive of E. coli in the shellfish and seawater, and the measured environmental factors (temperature, salinity, and oxygen saturation).

RESULTS

Epifluorescence showed small, spherical bodies with visible membrane, emitting bright green fluorescence and embedded in digestive tract of the bivalve (Fig. 2). The same structures were not present in the consecutive sections of the same sample incubated with PBS instead of antibodies.

[FIGURE 2 OMITTED]

The overall prevalence of Cryptosporidim sp. identified by immunofluorescence in the black mussels cultured in the Mali Ston Bay area was 16.8% (CL = 11.3-2.2) in 2005 (Table 1). Qualitatively comparing the four sampling sites throughout the year, Mali Ston had the highest total prevalence (20%, CL = 7.8-31.1), whereas Bistrina had the lowest level of total prevalence (13.3%, CL = 0.6-25.2). On two occasions, at the site Usko in summer and Bistrina in autumn 2005, no Cryptosporidium sp. was observed in samples from the cultured black mussel.

PERMANOVA detected that the prevalence differed significantly among seasons (P < 0.01), but not between the sampled "Sites" or in the interaction of "Sites x Seasons." Pair-wise comparison of the factor "Season" indicated significant difference between spring-winter (P < 0.01) and spring-autumn (P < 0.001). The two-dimensional MDS confirmed seasonal separation, especially for spring (Fig. 3); graphical representation showed that mainly points belonging to spring time clustered apart from the rest of the samples.

At Sutvid sampling site, significant correlation was found between the prevalence of the parasite and E. coli in shellfish ([r.sup.2] = 0.791, P < 0.0001) and E. coli in seawater ([r.sup.2] = 4).305, P 0.018), as well as between prevalence and the geographic average of oxygen ([r.sup.2] = 0.82, P < 0.0001) and prevalence and geographic average of temperature ([r.sup.2] = 0.77, P < 0.0001). At Mali Ston, significant correlation was shown between parasite prevalence and E. coli in seawater ([r.sup.2] = 0.791, P < 0.0001), but not in shellfish. Significant correlations were detected between prevalence and geographic averages of salinity ([r.sup.2] = 0.63, P = 0.018), oxygen ([r.sup.2] = 0.76, P < 0.0001) and temperature ([r.sup.2] = 0.69, P < 0.0001). In Usko, parasite prevalence correlated with almost all measured parameters (E. coli in shellfish [r.sup.2] = 0.81, P < 0.0001, seawater [r.sup.2] = 0.71, P < 0.0001, oxygen [r.sup.2] = 0.85, P < 0.0001 and temperature [r.sup.2] = 0.68, P < 0.0001). In Bistrina correlations were found between parasite prevalence and geographic average of abiotic factors (salinity [r.sup.2] = 0.69, P < 0.0001, oxygen [r.sup.2] = 0.505, P < 0.0001, temperature [r.sup.2] = 0.616, P < 0.0001), but not with E. coli in the shellfish or seawater.

DISCUSSION

Several studies undertaken in recent years experimentally evaluated the potential role of oysters and mussels in the transmission of cryptosporidiosis (Tamburrini & Pozio 1999, Fayer et al. 1997) and this is a first report of the parasite in the cultured Adriatic Sea black mussels. Whereas transmission of infection via molluscs has been reported in many instances, the true incidence of diseases transmitted by shellfish (or any other food) is controversial and remains unknown. Robertson (2007) argues that for various reasons, which vary between and within countries, there is a bias in reporting, which results in ah under-estimation of the extent of these infections, largely because neither the patient nor the physician is aware of the etiological role of the food. The black mussel is traditionally eaten by the local people lightly cooked, and the presence of Cryptosporidium sp. suggests that this bivalve may be a good bioindicator of the environment contamination, because the parasite is not a natural component of the marine fauna, but is found in areas impacted by sewage overflow or increased urban and agricultural runoffs (Johnson et al. 1995, Gomez-Couso et al. 2006a). A recent study has shown that C. parvurn oocysts ate easily transmitted from one bivalve to coexisting individuals of the same of different species (Gomez-Couso et al. 2003). This is because of the major concentration of oocytes in the bivalve gastrointestinal tract, from where the parasite is easily expelled in the environment as feces (Tamburrini & Pozio 1999, Li et al. 2006). Moreover for C. parvum, the process of shellfish depuration was shown to be ineffective, sometimes even facilitating spread of the parasite (Freire-Santos et al. 2001). In the Mali Ston Bay, the black mussel is usually cocultivated with the European flat oyster, indicating potential accumulation of Cryptosporidium sp. oocysts in the oyster as well. As both are highly praised when consumed raw or lightly steamed, to secure the aspect of public health safety, it is necessary to develop ah appropriate monitoring tool and a reliable diagnostic method in both cultured species.

Our results showed seasonal distribution of Cryptosporidium sp. infection levels; the parasite peaks in spring and slightly decreases in the summer time. This is in accordance with previous studies, because the highest levels of Cryptosporidium sp. are usually associated with rainy seasons when the persistent or strong rains flush the parasite from pastures into the sea environment. Similarly, Li et al. (2006) showed that in mussels (Mytilus edulis) reared in Normandy, oocysts rates were highest in winter and autumn, correlating with hibernal heavy rains. Another aspect that triggers elevated Cryptosporidium sp. appearance is the agglomeration of population in the coastal area during the summer time, which can explain why in our case, high infection levels were maintained in summer months. The lack of statistical difference in the parasite prevalence between sites that ate geographically in proximate vicinity, suggest that all localities are probably subjected to similar source of parasite oocysts, even though a larger number of samples would reinforce this statement.

Even though some authors suggest that the presence of Cryptosporidium sp. can be a good bioindicator of fecal contamination, studies had shown different results in respect to the correlation between parasite presence and fecal coliforms, enteric viruses or Salmonella sp. In most cases correlations were not found (Fayer et al. 1997, Fayer et al. 1998, Hernroth et al. 2002, Lemarchand & Lebaron 2003, Gomez-Couso et al. 2004), whereas in others correlations were statistically significant (Freire-Santos et al. 2000). In the Mali Ston Bay however, in the majority of sites we have found significant correlations between parasite prevalence and the presumptive number of E. coli in the bivalve and seawater over the year, suggesting relationship between the fecal contamination and elevated presence of the parasite at certain locations.

[FIGURE 3 OMITTED]

Oocysts of all species of Cryptosporidium can be detected by IFA, but the polymerase chain reaction (PCR) techniques allow the identification of specific species (Gomez-Couso et al. 2006b). Even though the immunofluorescence method we used revealed genus specificity and it is notable to identify species level, literature data suggested that it tends to strongly underestimate both prevalence and abundance of oocysts in the investigated bivalve, as the cut surface is small and it weakly represents other sites of oocysts accumulation like the gills and mantle (Miller et al. 2006). This for, we argue that the real prevalence of Cryptosporidium sp. in cultured black mussel is much higher, but the exact species still needs to be identified.

CONCLUSION

The identification of Cryptosporidium sp. in the black mussel cultured in the Adriatic Sea suggests that one of the most productive shellfish aquaculture areas is exposed to seasonal parasite shedding, mostly in correlation with the presumptive number of E. coli in the shellfish and seawater. Better public education, increased awareness for cryptosporidiosis and specific identification by molecular tools along with new legislation policies for monitoring of the parasite in surface water, livestock and wildlife, are necessary to control and limit the possibility of parasite transmission to humans in Croatia.

ACKNOWLEDGMENTS

The authors thank the shellfish farmers for the indispensable technical support and valuable samples, as well as to the anonymous referee whose constructive remarks and suggestions improved greatly our study.

LITERATURE CITED

Anderson, M. J. 2005. PERMANOVA: a FORTRAN computer program for permutational multivariate analysis of variance. Department of Statistics, University of Auckland, New Zealand.

Anonymous. 1990. Cryptosporidium in water supplies. Report of the group of experts. Department of the environment. Department of health, London, UK.

Anonymous. 1995. Cryptosporidium in water supplies. Second report of the group of experts. Department of the environment. Department of health, London, UK.

Anonymous. 1999. Standard operating protocol for the monitoring of Cryptosporidium parvum oocysts in treated water supplies to satisfy water supply (water quality) (amendment) regulations. SI no. 1524. Her Majesty's stationery office, London, UK.

Ayres, P. A., H. W. Burton & M. L. Cullum. 1978. Techniques for the study of mixed populations. In: D.W. Lovelock & R. Davies, editors. Sewage pollution and shellfish. Soc. Appl. Bacteriol. Tech. Ser. 11, London: Academic Press. pp. 51-62.

Baker, M., N. Russell, C. Roseveare, J. O'Hallahan, S. Palmer & A. Bichan. 1998. Outbreak of cryptosporidiosis linked to Hutt Valley swimming pool. N.Z. Public Health Rep. 5:41-45.

Bush, A. O., K. D. Lafferty, J. M. Lotz & A. W. Shostak. 1997. Parasitology meets ecology on its own terms: Margolis et al. revisited. J. Parasitol. 83:575-583.

Caccio, S., E. Pinter, R. Fantini, I. Mezzaroma & E. Pozio. 2002. Human infection with Cryptosporidium felis: case report and literature review. Emerg. Inf. Dis. 8:85-86.

Caccio, S. M., R. C. Thompson, J. McLauchlin & H. V. Smith. 2005. Unraveling Cryptosporidium and Giardia epidemiology. Trends Parasitol. 21:430-437.

Chalmers, R. M., A. P. Sturdee, P. Mellors, V. Nicholson, F. Lawlor & P. Timpson. 1997. Cryptosporidium parvum in environmental samples in the Sligo area, Republic of Ireland: a preliminary report. Lett. Appl. Microbiol. 25:380-384.

Chen, X. M., S. P. O'Hara, B. Q. Huang, P. L. Splinter, J. B. Nelson & N. F. LaRusso. 2005. Localized glucose and water influx facilitates Cryptosporidium parvum cellular invasion by means of modulation of host-cell membrane protrusion. Proc. Natl. Acad. Sci. USA 102:6338-6343.

Clarke, K. R. & R. M. Warwick. 1994. Change in Marine Communities: ah approach to statistical analysis and interpretation. Natural Environment Research Council UK, ISBN 1 85531 140 2. 144 pp.

Fayer, R., C. A. Farley, E. J. Lewis, J. M. Trout & T. K. Graczyk. 1997. Potential role of the eastern oyster, Crassostrea virginica, in the epidemiology of Cryptosporidium parvum. Appl. Environ. Microbiol. 63:1070-1074.

Fayer, R., J. M. Trout & M. C. Jenkins. 1998. Infectivity of Cryptosporidium parvurn oocysts stored in water at environmental temperatures. J. Parasitol. 84:1165-1169.

Fayer, R., E. J. Lewis, J. M. Trout, T. K. Graczyk, M. C. Jenkins, J. Higgins, L. Xiao & A. A. Lal. 1999. Cryptosporidium parvum in oysters from commercial harvesting sites in the Chesapeake Bay. Emerg. Infect. Dis. 5:706-710.

Franzen, C. & A. Muller. 1999. Cryptosporidia and Microsporidia waterborne diseases in the immunocompromised host. Diagn. Microbiol. Infect. Dis. 34:245-262.

Freire-Santos, F., A; M. Oteiza-Lopez, C. A. Vergara-Castiblanco, E. Ares-Mazas, E. Alvarez-Suarez & O. Garcia-Martin. 2000. Detection of Cryptosporidium oocysts in bivalve mollusks destined for human consumption. J. Parasitol. 86:853 854.

Freire-Santos, F., A. M. Oteiza-Lopez, J. A. Castro-Hermida, O. Garcia-Martin & M. E. Ares-Mazas. 2001. Viability and infectivity of oocysts recovered from clams, Ruditapes philippinarum, experimentally contaminated with Cryptosporidium parvum. Parasitol. Res. 87:428-430.

Freire-Santos, F. 2002. Cryptosporidium in aquatic organisms. Environmental factors influential on the oocyst viability. PhD thesis. University of Santiago de Campostela, Spain.

Freire-Santos, F., H. Gomez-Couso, M. R. Ortega-Inarrea, J. A. Castro-Harmida, A. M. Oteiza-Lopez, O. Garcia-Martin & M. E. Ares-Mazas. 2002. Survival of Cryptosporidium parvum oocyst recovered from experimentally contaminated oyster (Ostrea edulis) and clams (Tapes decussates). Parasitol. Res. 88:1039-1042.

Gomez-Couso, H., F. Freire-Santos, M. R. Ortega-Inarrea, J. A. Castro-Harmida & M. E. Ares-Mazas. 2003. Environmental dispersal of Cryptosporidium parvum oocysts and cross transmission in cultured bivalve mollusca. Parasitol. Res. 90:140-142.

Gomez-Couso, H., F. Freire-Santos, J. Martinez-Courtaza, O. Garcia-Martin & M. E. Ares-Mazas. 2004. Detection of Cryptosporidium and Giardia in molluscan shellfish by multiplex nested-PCR. Int. J. Food Microbiol. 91:279-288.

Gomez-Couso, H., F. Mendez-Hermida, J. A. Castro-Hermida & E. Ares-Mazas. 2006a. Cryptosporidium contamination in harvest areas of bivalve mollusks. J. Food Prot. 69:185-190.

Gomez-Couso, H., F. Mendez-Hermida & E. Ares-Mazas. 2006b. Levels of detection of Cryptosporidium oocysts in mussels (Mytilus galloprovincialis) by IFA and PCR methods. Vet. Parasitol. 141:60-55.

Graczyk, T. K., R. Fayer, E. J. Lewis, J. M. Trout & C. A. Farley. 1999. Cryptosporidium oocysts in Bent mussels (Ischadium recurvum) in the Chesapeak Bay. Parasitol. Res. 85:518-521.

Hayes, E. B., T. D. Matte, T. R. O'Brien, T. W. McKinley, G. S. Logsdon, J. B. Rose, B. L. Ungar, D. M. Word, P. F. Pinsky & M. L. Cumings. 1989. Large community outbreak of cryptosporidiosis due to contamination of a filtered public water supply. N. Engl. J. Med. 320:1372-1376.

Hernroth, B. E., A. C. Conden-Hansson, A. S. Rehnstam-Holm, R. Girones & A. K. Allard. 2002. Environmental factors influencing human viral pathogens and their potential indicator organisms in the Blue mussel, Mytilus edulis: the first Scandinavian report. Appl. Environ. Microbiol. 68:4523-4533.

Hunter, P. R. & G. Nichols. 2002. The epidemiology and clinical features of Cryptosporidium infection in immune-compromised patients. Clin. Microbiol. Rev. 15:145-154.

Hunter, P. R. & R. C. A. Thompson. 2005. The zoonotic transmission of Giardia and Cryptosporidium. In t. J. Parasitol. 35:1181-1190.

ISO 9308-2. 1990. Water quality--Detection and enumeration of coliform organisms, thermotolerant coliform organisms and presumptive Escherichia coli--Part 2: Multiple tube (most probable number) method.

ISO 9308-1. 2000. Water quality--Detection and enumeration of Escherichia coli and coliform bacteria--Part 1: Membrane filtration method.

Jennings, P. & A. Rhatigan. 2002. Cryptosporidiosis outbreak in Ireland linked to public water supply. Eurosurv. Wkly. 5:205-213.

Johnson, D. C., K. A. Reynolds, C. P. Gerba, I. L. Pepper & J. B. Rose. 1995. Detection of Giardia and Cryptosporidium in marine waters. Water Sci. Technol. 31:439-442.

Kramer, M. H, B. L. Herwaldt, G. F. Craun, R. L. Calderon & D. D. Juranek. 1998. Surveillance for waterborne-disease outbreaks United States, 1993-1994. MMWR Morb. Mortal. Wkly. Rep. 45:1-33.

Lemarchand, K. & P. Lebaron. 2003. Occurrence of Salmonella spp. and Cryptosporidium spp. in French coastal watershed: relation with fecal coliforms. FEMS Microbiol. Lett. 218:203-209.

Li, X., K. Guyot, E. Dei-Cas, J.-P. Mallard, J. J. Ballet & P. Brasseur. 2006. Cryptosporidium oocysts in mussels (Mytilus edulis) from Normandy (France). Int. J. Food Microbiol. 108:321-325.

MacKenzie, W. R., M. D. Neil, N. J. Hoxie, M. S. Mary, M. E. Proctor, M. S. Gradus, K. A. Blair, D. E. Peterson, J. J. Kazmierczak, D. G. Addiss, K. R. Fox, J. B. Rose & J. P. Davis. 1994. A massive outbreak in Milwaukee of cryptosporidium infection transmitted through the public water supply. N. Engl. J. Med. 331:161-167.

McLaughlin, J., C. Amar, S. Pedraza-Diaz & G. L. Nichols. 2000. Molecular epidemiological analysis of Cryptosporidium spp. in the United Kingdom: Results of genotyping Cryptosporidium spp. in 1705 fecal samples from humans and 105 samples from livestock animals. J. Clin. Microbiol. 38:3984-3990.

Miller, W. A., I. A. Gardner, E. R. Atwill, C. M. Leutenegger, M. A. Miller, R. P. Hedrick, A. C. Melli, N. M. Barnes & P. A. Conrad. 2006. Evaluation of methods for improved detection of Cryptosporidium parvum spp. in mussels (Mytilus californianus). J. Mierobiol. Methods 65:367-379.

Morgan-Ryan, U. M., A. Fall, L. A. Ward, N. Hijjawi, I. Sulaiman, R. Fayer, R. C. Thompson, M. Olson, A. Lal & L. Xiao. 2002. Cryptosporidium hominis n. sp. (Apicomplexa: Cryptosporidiidae) from Horno sapiens. J. Eucar. Microbiol. 49:433-440.

Robertson, L. J. 2007. The potential for marine bivalve shellfish to act as transmission vehicles for outbreaks of protozoan infections in humans: A review, Int. J. Food Microbiol. 120:201-216.

Sunnotel, O., C. J. Lowery, J. E. Moore, J. S. G. Dooley, L. Xiao & B. C. Millar. 2006. Cryptosporidium. Lett. Appl. Microbiol. 43:7-16

Tamburrini, A. & E. Pozio. 1999. Long term survival of Cryptosporidium parvum oocysts in seawater and in experimentally infected mussels (Mytilus galloprovineialis). Int. J. Parasitol. 29:711-715.

Veverka, F., N. Shapiro, M. K. Parish, S. York, W. Becker, F. Smith, C. Allensworth, T. Baker, P. Iwen & T. Safranek. 2001. Protracted outbreaks of cryptosporidiosis associated with swimming pool use-Ohio and Nebraska, 2000. Morb. Mortal. Wkly. Rep. 50:406-410.

Xiao, L. & U. Ryan. 2004. Cryptosporidiosis: an update in molecular epidemiology. Curr. Opin. Infect. Dis. 17:483-490.

Xiao, L., R. Fayer, U. Ryan & S. J. Upton. 2004. Cryptosporidium taxonomy: recent advances and implications for public health. Clin. Microbiol. Rey. 17:72-97.

IVONA MLADINEO, * ZELJKA TRUMBIC, SLAVEN JOZIC AND TAN JA SEGVIC

Institute of Oceanography & Fisheries, Laboratory of Aquaculture, Setaliste Ivana Mestrovica 63, 21000 Split, Croatia

* Corresponding author. E-mail: [e-mail]

TABLE 1.
Temporal and spatial distribution of Cryptosporidium sp. prevalence
(P) at different sampling sites per season (w--winter; sp--spring;
s--summer s--autumn).

             N       C[degrees]

                       w      sp       s       a

Sutvid        60     11.8    17.2    21.8    14.8
Mali Ston     45      8.5    19.7    20.8    13.4
Usko          49     12      17.9    20.6    16.1
Bistrina      30       *     18      20.8    16
Total (%)     184

             [per thousand]

               w      sp       s       a

Sutvid       38.3    36.6    37.1    38
Mali Ston    34.8    36.5    37.6    34
Usko         37.9    36.7    37.6    38
Bistrina      *      37.3    37.6    37.9
Total (%)

             %

               w      sp       s       a

Sutvid        97      143     115     91
Mali Ston     98      160     117     89
Usko          98      117     110     86
Bistrina       *      153     119     82
Total (%)

             P                               Total

               w      sp       s       a      (%)

Sutvid        6.3    30.8    18.8    13.3    16.7
Mali Ston    12.5    36.4    50.0     7.1    20.0
Usko         12.5    41.7     0.0     7.1    16.3
Bistrina       *     28.6    25.0     0.0    13.3
Total (%)    10.4    34.9    20.0     6.9    16.8

C[degrees] = temperature; [per thousand] - salinity; % = oxygen;
N = total number of bivalves analyzed, * missing data.

TABLE 2.
PERMANOVA results and seasonal pair-wise comparison
of the Cryptosporidium sp. prevalence in the black mussel
from the Mali Ston Bay.

PERMANOVA

Source             df           MS        Pseudo-F
Site                3          19.867      0.13505
Season              3         762.19       5.1811
Site x
  season **         8          72.738      0.49445
Res               169         147.11
Total             183

                          Pair-wise test
                           for "season"
PERMANOVA
                 Groups          t        P (perm)
Source          w x sp *      2.8581       0.005
Site            w x s         0.474993     0.683
Season
Site x          w x a         0.20236      0.85
  season **    sp x s         1.5355       0.113
Res            sp x a **      3.5517       0.001
Total           s x a         1.4241       0.174

* Significant at P = 0.01, ** p = 0.001. w-winter, sp-spring
s-summer, a-autumn.