- Email: [email protected]
Long-term survival of Cryptosporidium parvum oocysts in seawater and in experimentally infected mussels (Mytilus galloprovincialis)
International Journal for Parasitology 29 (1999) 711±715
Research note
Long-term survival of Cryptosporidium parvum oocysts in seawater and in experimentally infected mussels (Mytilus galloprovincialis) A. Tamburrini, E. Pozio * Laboratory of Parasitology, Istituto Superiore di SanitaÁ, viale Regina Elena 299, 00161 Rome, Italy Received 1 February 1999; received in revised form 10 March 1999; accepted 10 March 1999
Abstract Transmission of infectious oocysts of Cryptosporidium parvum via surface- and drinking-water supplies has been reported and many surface waters ¯ow into the sea, potentially causing runo of animal-infected faeces. Eating raw mussels is a common practice in many countries, increasing the public's risk of acquiring enteric pathogens. The aims of the present study were to estimate how long C. parvum oocysts remain infectious in arti®cial seawater, to determine if the oocysts are retained in mussel tissues (Mytilus galloprovincialis), and how long they maintain their infectivity. Oocysts were incubated in arti®cial seawater at 6±88C under moderate oxygenation and the infectivity of oocysts was tested ®ve times, over a 12 month period after incubation in seawater, in BALB/c mice. Each pup was inoculated per os with 105 oocysts and killed 5 days p.i. Oocysts remained infectious for 1 year. Forty mussels held in an aquarium containing arti®cial seawater ®ltered out more than 4108 oocysts in a 24 h period. Oocysts were detected in the gill washing up to 3 days p.i., in the haemolymph up to 7 days p.i., and in the intestinal tract up to 14 days p.i. Oocysts collected from the gut of mussels 7 and 14 days p.i. were observed to have infected mice. These results suggest that C. parvum oocysts can survive in seawater for at least 1 year and can be ®ltered out by benthic mussels, retaining their infectivity up to 14 days, so seawater and molluscs are a potential source of C. parvum infection for humans. # 1999 Australian Society for Parasitology. Published by Elsevier Science Ltd. All rights reserved. Keywords: Cryptosporidium parvum; Oocysts; Seawater; Mussels; Mytilus galloprovincialis; Survival; Paratenic host
Cryptosporidium parvum is a small protozoan capable of causing self limited enteritis in immunocompetent humans and animals and severe intractable diarrhoea in immunosuppressed hosts [1]. Within the past 15 years, with world-
* Corresponding author. Tel: +39 06 4990 2304; fax: +39 06 4938 7065; e-mail: [email protected]
wide recognition of the signi®cance of waterborne cryptosporidiosis, attention has increasingly focused on the viability and survival of C. parvum oocysts in the environment and paratenic hosts. Marine waters worldwide have been found to be contaminated with domestic sewage from outfalls and storm water runo [2] and oocysts of C. parvum have been found in marine water near a bathing beach in Hawaii [3]
0020-7519/$20.00 # 1999 Australian Society for Parasitology. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 0 - 7 5 1 9 ( 9 9 ) 0 0 0 3 3 - 8
712
A. Tamburrini, E. Pozio/International Journal for Parasitology 29 (1999) 711±715
and in the Mediterranean sea (Pozio E., unpublished data). Recent studies have demonstrated the potential role of molluscs (eastern oyster, Crassostrea virginica; freshwater benthic clam, Carbicula ¯uminea; and mussel, Mytilus edulis) as paratenic hosts of C. parvum [4±6]. These ®ndings demonstrate that bivalves in seawater and fresh water can harbour infectious C. parvum oocysts. Eating raw mussels is a common practice in many countries and has signi®cantly increased the public health risk of enteric pathogen infections. The present study was conducted to determine how long C. parvum oocysts remain infective in arti®cial seawater and if mussels (Mytilus galloprovincialis) can act as a paratenic host and thereby constitute a source of cryptosporidiosis when ingested raw by humans. Cryptosporidium parvum oocysts were collected from faeces of experimentally infected calves and puri®ed as previously described [7]. Oocysts were suspended in 0.1 M-PBS, pH 7.2 and stored at 48C for less than 2 weeks before use. Ten million puri®ed oocysts each were incubated in 10 ¯asks of 500 ml; ®ve ¯asks contained arti®cial seawater (33 ppt of Coral reef red sea salt, made by industrial free trade zone Eilat) and ®ve contained PBS (control). Flasks were stored at 6±88C under moderate oxygenation in the dark. Oocysts from one ¯ask containing seawater and from another containing PBS were collected by centrifugation at 4500 g5 min at 1, 3, 6, 9 and 12 months of incubation. They were washed twice in PBS, counted, and resuspended in 100 ml of PBS. One-hundred and ten mussels (M. galloprovincialis) with a 5±8 cm shell length were purchased at a seafood market and placed in two aquariums (aquarium A and aquarium C; 50 mussels/ aquarium) of 30 L of arti®cial seawater at 6±88C. Ten mussels were examined immediately after purchase to detect any natural infection with C. parvum. Mussels were fed with 1 ml of alga paste three times a week. The ®lter system had been o for 7 h at the moment of addition of algae to the aquariums. Mussels of aquarium A were acclimated for 10 days before testing. The seawater of acquarium A was contaminated with 400106 C. parvum puri®ed oocysts (8106 oocysts/mussel).
The ®lter system was turned o for 24 h. The mussels were then moved from aquarium A to aquarium B, which contained non-infected seawater. Water (500 ml) from aquarium A was collected and centrifuged at 4500 g5 min; the pellet was analysed to detect oocysts. At 1, 3, 7 and 14 days p.i., 10 mussels were examined to determine if oocysts were ®ltered and still present in the body or if they were phagocyted by haemocytes. At the same time, 10 mussels from aquarium C were examined as controls. The shell was opened by gently inserting a scalpel below the adductor muscles. The gill washing and haemolymph were aspirated with a pipette and the gastrointestinal tract was excised with forceps. The gill washing from 10 mussels was centrifuged at 4500 g5 min and resuspended in 1 ml of PBS; the haemolymph was collected as it was, and the gastrointestinal tract was homogenised for 1 min at 2500 rpm in 1 ml of PBS in a Potter apparatus. For each sample, 10 ml were analysed with the Meri¯uor Cryptosporidium/ Giardia kit test (Meridian Diagnostic) to detect oocysts. BALB/c mice (5 days old) were used to test the infectivity of C. parvum oocysts. Each pup was inoculated by a 24-gauge gavage needle with 100 ml of PBS containing 105 oocysts collected from seawater or with the intestinal tract collected from infected mussels. An equal number of mice were infected with oocysts (105/mouse) maintained in PBS under the same conditions described above or with the intestinal tract of unexposed mussels (controls). Litters were killed 5 days p.i. by CO2 overexposure to collect the ileum. The parasite load was assessed in the ileum homogenate from ®ve mice and in histological sections from two mice. The ileum was isolated and measured to express the parasite load (N) as Cryptosporidium stages per centimetre of ileum (N = n100/cm) (`n' is the total number of parasites counted in a 10 ml smear, see below) [8]. The ileum of each animal was homogenised separately in 1 ml of PBS for 30 sec at 300 rpm in a Potter apparatus. An amount of 10 ml for each homogenate, diluted 1/10 in PBS, was smeared onto a glass slide (2 cm2). The Meri¯uor
A. Tamburrini, E. Pozio/International Journal for Parasitology 29 (1999) 711±715
713
Table 1 Cryptosporidium parvum parasites per cm of ileum of BALB/c mice after dierent periods of incubation in seawater and in PBS Time of incubation (months)
Mean values2S.D. of the parasite load103 Seawater
PBS
1 3 6 9 12
1225.3 14.526.2 11.327.7 10.728.5 4.529.1
13.22 4.4 15.62 4.7 10.42 5.9 8.329.3 3.1210.2
Mean values2S.D. of the parasite load were from ®ve pups.
Cryptosporidium/Giardia mAb ¯uorescent test was used to detect and to count C. parvum parasites. The whole area was examined under an epi¯uorescent microscope. For histology, the ileum was ®xed in 10% formalin (pH 7,0), paran embedded, cut into 5 mm thick sections and stained with H and E. Histological sections were examined only when the ileum homogenate resulted negative at the ¯uorescent test. BALB/c mice inoculated with C. parvum oocysts that were incubated for 1, 3, 6, 9 and 12 months in arti®cial seawater and BALB/c mice inoculated with oocysts of control (incubated in PBS for the same length of time) were positive for C. parvum at dierent developmental stages in enterocytes; control BALB/c mice of the same litters which were not inoculated remained negative. Data reported in Table 1 show that the infectivity of oocysts held in seawater was higher than that of oocysts held in PBS.
The 10 mussels examined immediately after purchase to detect a possible natural infection with C. parvum remained negative. Twenty-four hours after the contamination of aquarium A with oocysts, the mussels were transferred to aquarium B, and the water in aquarium A was analysed; no oocysts were detected, suggesting that all 4108 oocysts were ®ltered from the 40 mussels in a 24 h period. As shown in Table 2, C. parvum oocysts were detected in the gill washing up to 3 days p.i., in the haemolymph up to 7 days p.i., and in the intestinal tract up to 14 days p.i. Oocysts collected from the intestinal tract of infected mussels 7 and 14 days p.i. were observed to have infected BALB/c mice. Overall, the mussel mortality rate was 20%. At the end of the experiment, when all mussels in aquarium B were examined, the ®lter of the aquarium was removed and examined for oocysts by immuno¯uorescence. The ®lter was positive for C. parvum
Table 2 Mean number of Cryptosporidium parvum oocysts detected in tissues and ¯uids of 10 mussels (Mytilus galloprovincialis) at dierent days post-infection Days postinfection 1 3 7 14 a
Mean number of oocysts2S.D. Gill washing 1028.2 327.5 0 0
These oocysts infected neonatal BALB/c mice.
Hemolymph 402 27.9 552 31.4 82 12.22 0
Gut 27223.5 71221.7 43a 234.5 10a 231.4
714
A. Tamburrini, E. Pozio/International Journal for Parasitology 29 (1999) 711±715
oocysts, suggesting that some oocysts were released from mussels via faeces in the water and that they were captured by the ®lter. Oocysts collected from the ®lter were able to infect BALB/c mice. Previous studies have shown that oocysts of C. parvum held in arti®cial seawater stored at 48C remain viable for over a month [9] and survive at dierent salinity and temperatures up to 12 weeks [10]. In the present study, C. parvum oocysts maintained their infectivity for up to 1 year of incubation in arti®cial seawater at 6±88C under moderate oxygenation. Furthermore, mice infected with oocysts recovered after 9 and 12 months of incubation in arti®cial seawater showed an infection level higher than that observed in mice infected with oocysts incubated in PBS for the same period of time. Consequently, seawater, especially near the mouth of rivers and canals and near the outlet of sewer systems, can represent an important reservoir of C. parvum oocysts, which can contaminate swimmers and seafood. These data and those from the literature suggest that C. parvum can now be numbered among other infectious pathogens related to the consumption of raw seafood (i.e. Vibrio, Campilobacter jejuni, hepatitis A virus, Norwalk virus, etc.) [11]. In the last two years, several studies have evaluated the potential role of oysters and clams in the transmission of cryptosporidiosis [4, 5, 10]. Mussels (Mytilus edulis) from the Ireland cost of the Atlantic ocean were found naturally infected with Cryptosporidium oocysts [6]. In Italy, C. parvum oocysts were found in the Adriatic sea (Pozio E, unpublished data) where mussels are bred and frequently eaten raw. In Italy, 150106 kg of mussels are eaten per year and a portion of them are eaten raw, especially in Southern Italy, representing a serious health problem, particularly for immunosuppressed persons. About 130106 kg of mussels are bred along the Italian coast every year and frequently these breeding sites are located near human settlements where the seawater is highly contaminated. The survival of oocysts in seawater and in mussels for a long period of time enhances the potential exposure to humans and animals. To date, no human or ani-
mal C. parvum infections have been attributed to the consumption of raw mussels or to contact with seawater. This could be related to the long (up to 9 days) incubation period of the disease, according to experimental infection in volunteers [12], suggesting diculties in correlating the exposure to seawater or the consumption of raw mussels with this infection. A case-control study would permit evaluation of the potential risk of acquiring cryptosporidiosis by the ingestion of seawater or by the consumption of raw molluscs in nature.
Acknowledgements We are grateful to Giuseppe La Rosa for his help in breeding mussels in the aquarium. This work was supported by MURST `Sottoprogetto 6ÐSalute umana' of the PRISMA 2 project, grant no. B28.
References [1] Fayer R. Cryptosporidium and cryptosporidiosis. Boca Raton: CRC Press, 1997. [2] Fleisher JM, Kay D, Salmon RL, Jones F, Wyer MD, Godfree AF. Marine waters contamined with domestic sewage: nonenteric illness associated with bather exposure in the United Kindom. Am J Public Health 1996;86:1228±34. [3] Johnson DC, Reynolds KA, Gerba CP, Pepper IL, Rose JB. Detection of Giardia and Cryptoporidium in marine waters. Water Sci Technol 1995;31:505±8. [4] Fayer R, Lewis EJ, Trout JM, Graczyk TK. Potential role of the eastern oyster, Crassostrea virginica, in the epidemiology of Cryptosporidium parvum. Appl Environ Microbiol 1997;63:2086±8. [5] Graczyk TK, Fayer R, Cran®eld MR, Conn DB. Recovery of waterborne Cryptosporidium parvum oocysts by freshwater benthic clams (Carbicula ¯uminea). Appl Environ Microbiol 1998;64:427±30. [6] Chalmers RM, Sturdee AP, Mellors P, Nicholson V, Lawlor F, Kenny F, Timpson P. Cryptosporidium parvum in environmental samples in the Sligo area, Republic of Ireland: a preliminary report. Letters Appl Microbiol 1997;25:380±4. [7] Rossi P, Pozio E, Besse MG, Gomez Morales MA, La Rosa G. Experimental cryptosporidiosis in hamsters. J Clin Microbiol 1990;28:356±7.
A. Tamburrini, E. Pozio/International Journal for Parasitology 29 (1999) 711±715 [8] Buraud M, Kapel N, Benhamou Y, Savel J, Gobert JG. A high-yield outbred suckling mouse model of cryptosporidiosis. Parasite 1995;2:81±4. [9] Robertson LJ, Campbell AT, Smith HV. Survival of Cryptosporidium parvum oocysts under various enviromental pressures. Appl Environ Microbiol 1992;58:3494± 500. [10] Fayer R, Graczyk TK, Lewis EJ, Trout JM, Farley CA. Survival of infectious Cryptosporidium parvum oocysts in seawater and eastern oysters (Crossostrea virginica) in the Chesapeake Bay. Appl Environ Microbiol 1998;64:1070± 4.
715
[11] Cromeans T, Nainan OV, Fields HA, Favorov MO, Margolis HS. Hepatitis A and E viruses. In: Huy YH, Gorham JR, Murrel KD, Cliver DO, editors. Foodborne disease handbook; diseases caused by viruses, parasites, and fungi. New York: Marcel Dekker, Inc, 1994;2:1±56. [12] DuPont HL, Chappel CL, Sterling CR, Okhuysen PC, Rose JB, Jakubowski W. The infectivity of Cryptosporidium parvum in healthy volunteers. N Engl J Med 1995;332:855±9.
Research note
Long-term survival of Cryptosporidium parvum oocysts in seawater and in experimentally infected mussels (Mytilus galloprovincialis) A. Tamburrini, E. Pozio * Laboratory of Parasitology, Istituto Superiore di SanitaÁ, viale Regina Elena 299, 00161 Rome, Italy Received 1 February 1999; received in revised form 10 March 1999; accepted 10 March 1999
Abstract Transmission of infectious oocysts of Cryptosporidium parvum via surface- and drinking-water supplies has been reported and many surface waters ¯ow into the sea, potentially causing runo of animal-infected faeces. Eating raw mussels is a common practice in many countries, increasing the public's risk of acquiring enteric pathogens. The aims of the present study were to estimate how long C. parvum oocysts remain infectious in arti®cial seawater, to determine if the oocysts are retained in mussel tissues (Mytilus galloprovincialis), and how long they maintain their infectivity. Oocysts were incubated in arti®cial seawater at 6±88C under moderate oxygenation and the infectivity of oocysts was tested ®ve times, over a 12 month period after incubation in seawater, in BALB/c mice. Each pup was inoculated per os with 105 oocysts and killed 5 days p.i. Oocysts remained infectious for 1 year. Forty mussels held in an aquarium containing arti®cial seawater ®ltered out more than 4108 oocysts in a 24 h period. Oocysts were detected in the gill washing up to 3 days p.i., in the haemolymph up to 7 days p.i., and in the intestinal tract up to 14 days p.i. Oocysts collected from the gut of mussels 7 and 14 days p.i. were observed to have infected mice. These results suggest that C. parvum oocysts can survive in seawater for at least 1 year and can be ®ltered out by benthic mussels, retaining their infectivity up to 14 days, so seawater and molluscs are a potential source of C. parvum infection for humans. # 1999 Australian Society for Parasitology. Published by Elsevier Science Ltd. All rights reserved. Keywords: Cryptosporidium parvum; Oocysts; Seawater; Mussels; Mytilus galloprovincialis; Survival; Paratenic host
Cryptosporidium parvum is a small protozoan capable of causing self limited enteritis in immunocompetent humans and animals and severe intractable diarrhoea in immunosuppressed hosts [1]. Within the past 15 years, with world-
* Corresponding author. Tel: +39 06 4990 2304; fax: +39 06 4938 7065; e-mail: [email protected]
wide recognition of the signi®cance of waterborne cryptosporidiosis, attention has increasingly focused on the viability and survival of C. parvum oocysts in the environment and paratenic hosts. Marine waters worldwide have been found to be contaminated with domestic sewage from outfalls and storm water runo [2] and oocysts of C. parvum have been found in marine water near a bathing beach in Hawaii [3]
0020-7519/$20.00 # 1999 Australian Society for Parasitology. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 0 - 7 5 1 9 ( 9 9 ) 0 0 0 3 3 - 8
712
A. Tamburrini, E. Pozio/International Journal for Parasitology 29 (1999) 711±715
and in the Mediterranean sea (Pozio E., unpublished data). Recent studies have demonstrated the potential role of molluscs (eastern oyster, Crassostrea virginica; freshwater benthic clam, Carbicula ¯uminea; and mussel, Mytilus edulis) as paratenic hosts of C. parvum [4±6]. These ®ndings demonstrate that bivalves in seawater and fresh water can harbour infectious C. parvum oocysts. Eating raw mussels is a common practice in many countries and has signi®cantly increased the public health risk of enteric pathogen infections. The present study was conducted to determine how long C. parvum oocysts remain infective in arti®cial seawater and if mussels (Mytilus galloprovincialis) can act as a paratenic host and thereby constitute a source of cryptosporidiosis when ingested raw by humans. Cryptosporidium parvum oocysts were collected from faeces of experimentally infected calves and puri®ed as previously described [7]. Oocysts were suspended in 0.1 M-PBS, pH 7.2 and stored at 48C for less than 2 weeks before use. Ten million puri®ed oocysts each were incubated in 10 ¯asks of 500 ml; ®ve ¯asks contained arti®cial seawater (33 ppt of Coral reef red sea salt, made by industrial free trade zone Eilat) and ®ve contained PBS (control). Flasks were stored at 6±88C under moderate oxygenation in the dark. Oocysts from one ¯ask containing seawater and from another containing PBS were collected by centrifugation at 4500 g5 min at 1, 3, 6, 9 and 12 months of incubation. They were washed twice in PBS, counted, and resuspended in 100 ml of PBS. One-hundred and ten mussels (M. galloprovincialis) with a 5±8 cm shell length were purchased at a seafood market and placed in two aquariums (aquarium A and aquarium C; 50 mussels/ aquarium) of 30 L of arti®cial seawater at 6±88C. Ten mussels were examined immediately after purchase to detect any natural infection with C. parvum. Mussels were fed with 1 ml of alga paste three times a week. The ®lter system had been o for 7 h at the moment of addition of algae to the aquariums. Mussels of aquarium A were acclimated for 10 days before testing. The seawater of acquarium A was contaminated with 400106 C. parvum puri®ed oocysts (8106 oocysts/mussel).
The ®lter system was turned o for 24 h. The mussels were then moved from aquarium A to aquarium B, which contained non-infected seawater. Water (500 ml) from aquarium A was collected and centrifuged at 4500 g5 min; the pellet was analysed to detect oocysts. At 1, 3, 7 and 14 days p.i., 10 mussels were examined to determine if oocysts were ®ltered and still present in the body or if they were phagocyted by haemocytes. At the same time, 10 mussels from aquarium C were examined as controls. The shell was opened by gently inserting a scalpel below the adductor muscles. The gill washing and haemolymph were aspirated with a pipette and the gastrointestinal tract was excised with forceps. The gill washing from 10 mussels was centrifuged at 4500 g5 min and resuspended in 1 ml of PBS; the haemolymph was collected as it was, and the gastrointestinal tract was homogenised for 1 min at 2500 rpm in 1 ml of PBS in a Potter apparatus. For each sample, 10 ml were analysed with the Meri¯uor Cryptosporidium/ Giardia kit test (Meridian Diagnostic) to detect oocysts. BALB/c mice (5 days old) were used to test the infectivity of C. parvum oocysts. Each pup was inoculated by a 24-gauge gavage needle with 100 ml of PBS containing 105 oocysts collected from seawater or with the intestinal tract collected from infected mussels. An equal number of mice were infected with oocysts (105/mouse) maintained in PBS under the same conditions described above or with the intestinal tract of unexposed mussels (controls). Litters were killed 5 days p.i. by CO2 overexposure to collect the ileum. The parasite load was assessed in the ileum homogenate from ®ve mice and in histological sections from two mice. The ileum was isolated and measured to express the parasite load (N) as Cryptosporidium stages per centimetre of ileum (N = n100/cm) (`n' is the total number of parasites counted in a 10 ml smear, see below) [8]. The ileum of each animal was homogenised separately in 1 ml of PBS for 30 sec at 300 rpm in a Potter apparatus. An amount of 10 ml for each homogenate, diluted 1/10 in PBS, was smeared onto a glass slide (2 cm2). The Meri¯uor
A. Tamburrini, E. Pozio/International Journal for Parasitology 29 (1999) 711±715
713
Table 1 Cryptosporidium parvum parasites per cm of ileum of BALB/c mice after dierent periods of incubation in seawater and in PBS Time of incubation (months)
Mean values2S.D. of the parasite load103 Seawater
PBS
1 3 6 9 12
1225.3 14.526.2 11.327.7 10.728.5 4.529.1
13.22 4.4 15.62 4.7 10.42 5.9 8.329.3 3.1210.2
Mean values2S.D. of the parasite load were from ®ve pups.
Cryptosporidium/Giardia mAb ¯uorescent test was used to detect and to count C. parvum parasites. The whole area was examined under an epi¯uorescent microscope. For histology, the ileum was ®xed in 10% formalin (pH 7,0), paran embedded, cut into 5 mm thick sections and stained with H and E. Histological sections were examined only when the ileum homogenate resulted negative at the ¯uorescent test. BALB/c mice inoculated with C. parvum oocysts that were incubated for 1, 3, 6, 9 and 12 months in arti®cial seawater and BALB/c mice inoculated with oocysts of control (incubated in PBS for the same length of time) were positive for C. parvum at dierent developmental stages in enterocytes; control BALB/c mice of the same litters which were not inoculated remained negative. Data reported in Table 1 show that the infectivity of oocysts held in seawater was higher than that of oocysts held in PBS.
The 10 mussels examined immediately after purchase to detect a possible natural infection with C. parvum remained negative. Twenty-four hours after the contamination of aquarium A with oocysts, the mussels were transferred to aquarium B, and the water in aquarium A was analysed; no oocysts were detected, suggesting that all 4108 oocysts were ®ltered from the 40 mussels in a 24 h period. As shown in Table 2, C. parvum oocysts were detected in the gill washing up to 3 days p.i., in the haemolymph up to 7 days p.i., and in the intestinal tract up to 14 days p.i. Oocysts collected from the intestinal tract of infected mussels 7 and 14 days p.i. were observed to have infected BALB/c mice. Overall, the mussel mortality rate was 20%. At the end of the experiment, when all mussels in aquarium B were examined, the ®lter of the aquarium was removed and examined for oocysts by immuno¯uorescence. The ®lter was positive for C. parvum
Table 2 Mean number of Cryptosporidium parvum oocysts detected in tissues and ¯uids of 10 mussels (Mytilus galloprovincialis) at dierent days post-infection Days postinfection 1 3 7 14 a
Mean number of oocysts2S.D. Gill washing 1028.2 327.5 0 0
These oocysts infected neonatal BALB/c mice.
Hemolymph 402 27.9 552 31.4 82 12.22 0
Gut 27223.5 71221.7 43a 234.5 10a 231.4
714
A. Tamburrini, E. Pozio/International Journal for Parasitology 29 (1999) 711±715
oocysts, suggesting that some oocysts were released from mussels via faeces in the water and that they were captured by the ®lter. Oocysts collected from the ®lter were able to infect BALB/c mice. Previous studies have shown that oocysts of C. parvum held in arti®cial seawater stored at 48C remain viable for over a month [9] and survive at dierent salinity and temperatures up to 12 weeks [10]. In the present study, C. parvum oocysts maintained their infectivity for up to 1 year of incubation in arti®cial seawater at 6±88C under moderate oxygenation. Furthermore, mice infected with oocysts recovered after 9 and 12 months of incubation in arti®cial seawater showed an infection level higher than that observed in mice infected with oocysts incubated in PBS for the same period of time. Consequently, seawater, especially near the mouth of rivers and canals and near the outlet of sewer systems, can represent an important reservoir of C. parvum oocysts, which can contaminate swimmers and seafood. These data and those from the literature suggest that C. parvum can now be numbered among other infectious pathogens related to the consumption of raw seafood (i.e. Vibrio, Campilobacter jejuni, hepatitis A virus, Norwalk virus, etc.) [11]. In the last two years, several studies have evaluated the potential role of oysters and clams in the transmission of cryptosporidiosis [4, 5, 10]. Mussels (Mytilus edulis) from the Ireland cost of the Atlantic ocean were found naturally infected with Cryptosporidium oocysts [6]. In Italy, C. parvum oocysts were found in the Adriatic sea (Pozio E, unpublished data) where mussels are bred and frequently eaten raw. In Italy, 150106 kg of mussels are eaten per year and a portion of them are eaten raw, especially in Southern Italy, representing a serious health problem, particularly for immunosuppressed persons. About 130106 kg of mussels are bred along the Italian coast every year and frequently these breeding sites are located near human settlements where the seawater is highly contaminated. The survival of oocysts in seawater and in mussels for a long period of time enhances the potential exposure to humans and animals. To date, no human or ani-
mal C. parvum infections have been attributed to the consumption of raw mussels or to contact with seawater. This could be related to the long (up to 9 days) incubation period of the disease, according to experimental infection in volunteers [12], suggesting diculties in correlating the exposure to seawater or the consumption of raw mussels with this infection. A case-control study would permit evaluation of the potential risk of acquiring cryptosporidiosis by the ingestion of seawater or by the consumption of raw molluscs in nature.
Acknowledgements We are grateful to Giuseppe La Rosa for his help in breeding mussels in the aquarium. This work was supported by MURST `Sottoprogetto 6ÐSalute umana' of the PRISMA 2 project, grant no. B28.
References [1] Fayer R. Cryptosporidium and cryptosporidiosis. Boca Raton: CRC Press, 1997. [2] Fleisher JM, Kay D, Salmon RL, Jones F, Wyer MD, Godfree AF. Marine waters contamined with domestic sewage: nonenteric illness associated with bather exposure in the United Kindom. Am J Public Health 1996;86:1228±34. [3] Johnson DC, Reynolds KA, Gerba CP, Pepper IL, Rose JB. Detection of Giardia and Cryptoporidium in marine waters. Water Sci Technol 1995;31:505±8. [4] Fayer R, Lewis EJ, Trout JM, Graczyk TK. Potential role of the eastern oyster, Crassostrea virginica, in the epidemiology of Cryptosporidium parvum. Appl Environ Microbiol 1997;63:2086±8. [5] Graczyk TK, Fayer R, Cran®eld MR, Conn DB. Recovery of waterborne Cryptosporidium parvum oocysts by freshwater benthic clams (Carbicula ¯uminea). Appl Environ Microbiol 1998;64:427±30. [6] Chalmers RM, Sturdee AP, Mellors P, Nicholson V, Lawlor F, Kenny F, Timpson P. Cryptosporidium parvum in environmental samples in the Sligo area, Republic of Ireland: a preliminary report. Letters Appl Microbiol 1997;25:380±4. [7] Rossi P, Pozio E, Besse MG, Gomez Morales MA, La Rosa G. Experimental cryptosporidiosis in hamsters. J Clin Microbiol 1990;28:356±7.
A. Tamburrini, E. Pozio/International Journal for Parasitology 29 (1999) 711±715 [8] Buraud M, Kapel N, Benhamou Y, Savel J, Gobert JG. A high-yield outbred suckling mouse model of cryptosporidiosis. Parasite 1995;2:81±4. [9] Robertson LJ, Campbell AT, Smith HV. Survival of Cryptosporidium parvum oocysts under various enviromental pressures. Appl Environ Microbiol 1992;58:3494± 500. [10] Fayer R, Graczyk TK, Lewis EJ, Trout JM, Farley CA. Survival of infectious Cryptosporidium parvum oocysts in seawater and eastern oysters (Crossostrea virginica) in the Chesapeake Bay. Appl Environ Microbiol 1998;64:1070± 4.
715
[11] Cromeans T, Nainan OV, Fields HA, Favorov MO, Margolis HS. Hepatitis A and E viruses. In: Huy YH, Gorham JR, Murrel KD, Cliver DO, editors. Foodborne disease handbook; diseases caused by viruses, parasites, and fungi. New York: Marcel Dekker, Inc, 1994;2:1±56. [12] DuPont HL, Chappel CL, Sterling CR, Okhuysen PC, Rose JB, Jakubowski W. The infectivity of Cryptosporidium parvum in healthy volunteers. N Engl J Med 1995;332:855±9.