Parasitology International 64 (2015) 299–302
Contents lists available at ScienceDirect
Parasitology International journal homepage: www.elsevier.com/locate/parint
Short communication
Survey for protozoan parasites in Eastern oysters (Crassostrea virginica) from the Gulf of Maine using PCR-based assays Nicholas D. Marquis a,b, Nicholas R. Record a, José A. Fernández Robledo a,⁎ a b
Bigelow Laboratory for Ocean Sciences, PO Box 380, 60 Bigelow Drive, East Boothbay, ME 04544-0380, USA Southern Maine Community College, Research Experience for Undergraduates (2014), National Science Foundation, USA
a r t i c l e
i n f o
Article history: Received 26 September 2014 Received in revised form 23 March 2015 Accepted 6 April 2015 Available online 15 April 2015
a b s t r a c t Protozoan pathogens represent a serious threat to oyster aquaculture, since they can lead to significant production loses. Moreover, oysters can concentrate human pathogens through filter feeding, thus putting at risk raw oyster consumers' health. Using PCR-based assays in oysters (Crassostrea virginica) from Maine, we expand the Northeast range in the USA for the protozoans Perkinsus marinus, Perkinsus chesapeaki, and Haplosporidium nelsoni, and report for the first time the detection of the human pathogens Toxoplasma gondii and Cryptosporidium parvum. Oysters hosting both P. marinus and P. chesapeaki were more than three times as likely to be infected by a non-Perkinsus than those free of Perkinsus infections. © 2015 Elsevier Ireland Ltd. All rights reserved.
Mollusk bivalves are key components of marine and estuarine environments because, as filter feeders, they play a critical role in maintaining water quality and ecosystem integrity [1]. Bivalves also are an abundant resource for the coastal inhabitants and in most places traditional local harvesting of the natural populations is being substituted worldwide by semi-intensive aquaculture initiatives [2]. According to the Food and Agriculture Organization of the United Nations (http:// www.fao.org/), the production of farmed clams, mussels, oysters, and scallops reached 14,297,010 metric tons with an estimated value of $13.7 bn (2010 data). Moving from harvesting natural beds to aquaculture initiatives has associated risk including the presence of protozoan pathogens capable of producing significant production loses and, derived from the condition of being filter feeders, the ability concentrate human pathogens that put at risk the consumers [3]. Protozoan parasites within the genera Perkinsus and Haplosporidium severely affect mollusk species commercially harvested or farmed around the world; bivalve trading and global warming have been proposed as two of the main causes for expansion of parasitic diseases in mollusks [4]. Indeed, Perkinsus marinus and Perkinsus olseni (“Dermo” disease) are under surveillance by the World Organization for Animal Health (OIE; http:// www.oie.int/) and Haplosporidium nelsoni (MSX: Multinucleated Sphere X) has also been associated to mass mortalities of oysters in the USA [5,6]. Oysters and clams feed on suspended phytoplankton by trapping it in the gills; simultaneously, the gills also filter pathogenic microorganisms from the water, concentrating them in the digestive system. If these pathogens are not cleared or inactivated by the bivalves, their ⁎ Corresponding author. Tel.: +1 207 315 2567x315; fax: +1 207 315 2329. E-mail addresses: [email protected] (N.D. Marquis), [email protected] (N.R. Record), [email protected] (J.A. Fernández Robledo).
http://dx.doi.org/10.1016/j.parint.2015.04.001 1383-5769/© 2015 Elsevier Ireland Ltd. All rights reserved.
consumption might pose a public health concern and impact the costal economies in the event of temporary or long-term closure of the affected areas [3,7]. Toxoplasma gondii and Cryptosporidium parvum are widely distributed pathogens of humans and animals that may retain their infectivity in raw or undercooked mollusks; their waterborne transmission has been largely demonstrated in both marine and freshwater systems where they interact with aquatic organisms [3]. T. gondii is a protozoan parasite whose final hosts are felines, including domestic cats; however, it can also affect humans and other mammals (e.g. pigs, sheeps); millions of cysts derived from both wild and domestic animals reach the shores through runoff and sewage [8]. Recent studies indicate that infected oysters Crassostrea virginica may serve as a source of T. gondii for marine mammals and humans, since oysters can readily acquire T. gondii oocysts from seawater, and T. gondii can survive for several months in oysters [9]. Toxoplasmosis has been recently in the public eye for being responsible for mass mortalities of West coast sea otters associated with the consumption of infected bivalves; moreover, a number of epidemiological studies link Toxoplasma to pathological and psychological conditions in humans [10]. C. parvum is a parasite of the mammalian intestinal tract; it causes self-limiting diarrhea in people with intact immune systems and it is particularly severe in immunocompromised individuals; the resistance stage (oocyst) has been detected in many oysters, mussels, and clams within both fecally contaminated and clean oyster/clam growing areas across the globe [3]. P. marinus is found in the Pacific coast, Gulf of Mexico, and East coast of North America [4] with Damariscotta River Estuary (Maine, USA) being the most northeast limit [11]. In Maine, H. nelsoni has been associated to outbreaks in the Piscataqua River Estuary in the 1990's [5] and in the Damariscotta River Estuary in 2010 [6]. To our knowledge, no surveys have been done for Cryptosporidium and Toxoplasma in mollusks from the Gulf of Maine. Increasing the production of shellfish from
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coastal inshore ecosystems and creating a more sustainable economic development would require understanding the risks of mollusk pathogens in different areas to identify the places for oyster leases with less risk of outbreaks happening, as well as guaranteeing a product that is free of human pathogens and/or that the human pathogens do not pose a threat to oyster's consumers. Linked to the increase in storm events and an increment in the temperature, most models for climate change predict an increase in the runoff from terrestrial ecosystems in
the estuaries [12]. In addition to the effect on primary production, runoff from terrestrial ecosystems might also affect pathogen abundance in the shores where oysters are commercially grown [3]. In this study, we surveyed during the summer of 2014 oysters from nine oyster leases and one wild population in the Gulf of Maine (Fig. 1A) for three protozoan parasites of molluscs and two protozoan parasites of humans and animals. From each individual oyster (n = 25/site) rectum, gill, and mantle tissue were collected and pooled (50–100 mg wet
Fig. 1. A. Sampling site locations and identified protozoan parasites. Adult eastern oysters (Crassostrea virginica) were collected between June and July 2014 from a natural oyster bed and from shellfish farmers from nine oyster leases along the coast of the Gulf of Maine (Northeast USA). B. Clustering of the study sites based on detected parasites. Sites were clustered based on the Manhattan distance metric (Σi5 = 1 |xi − yi| where xi and yi are the probabilities of the presence of parasite i at sites x and y respectively). Other distance metrics produced similar clustering. Colors show whether a site was above average (red) or below average (blue) with respect to the prevalence of a given parasite. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
N.D. Marquis et al. / Parasitology International 64 (2015) 299–302
weight of total tissues/pool) and DNA was extracted using a commercial kit (E.Z.N.A. Tissue DNA kit; Omega Bio-Tek, Norcross, GA, USA). PCRbased assays specific for P. marinus, Perkinsus chesapeaki, and H. nelsoni, T. gondii, and C. parvum were performed as reported elsewhere [13–17] using 200–400 ng/PCR, PCR master mix (EconoTaq Plus Green 2X, Lucigen, WI, USA), and the parasite specific primers in a final volume of 25 μl (Table 1). The latest large epizootiological survey for Perkinsus spp. in oysters in the East coast of the USA was conducted in 2002 with reported low prevalences for P. marinus (1%) and for P. chesapeaki (3%) [11]. Damariscotta River Estuary used to be a good source for hemocytes from parasite-free oysters for in vitro host-parasite interaction studies [18]; however, over the last 2–3 years, it has become difficult to find specimens negative for Perkinsus spp. (G.R. Vasta, IMET, pers. comm.). In our survey, oysters from Jones Cove (Damariscotta River Estuary) reached prevalences of 65.2% for P. marinus and 43.5% for P. chesapeaki, a 65–15 fold increase of the prevalence respectively over a period of 12 years [11]. We also found high prevalences for P. marinus in the rest of the sites sampled with oysters from Webhannet River and Weskeag River reaching the highest prevalence values of the survey (73.9% in both); the highest prevalence for P. chesapeaki was in Bagaduce River (80.0%) and Weskeag River (85.7%). Interestingly we found no P. chesapeaki in oysters from Maquoit Bay. The wild oyster population (Deer Meadow Brook), which is considered the northernmost native oyster population in the United States [19], although in the same range size as cultured oysters, was older than the oysters from leases based on analysis of the shell (data not shown). The prevalence was high for P. marinus (65.2%) but very low for P. chesapeaki (4.3%) suggesting that although salinity and temperature are suitable for oyster survival and growth, the heavy tidal action [20] might not favor P. chesapeaki transmission or, alternatively, that native populations have some resistance to P. chesapeaki. There are several hypotheses that would explain the high prevalence of Perkinsus spp. in the Gulf of Maine including the presence of new Perkinsus races or genetic variants reaching the area. Indeed, the presence of P. marinus “races” and genetic strains have been documented along the Atlantic and Gulf coasts of the USA based on phenotypic, genotypic characters, and sensitivity to drugs [21,22]. Another hypothesis is that the genetic background of the oyster used in commercial leases is most susceptible to Perkinsus spp. Indeed, 12 disease-resistance quantitative trait loci have been evaluated in relation to resistance in the Eastern oyster to both Dermo and MSX [23]. Testing these hypotheses would require more focused studies. Based on our 2014 sampling, we expanded the range of MSX in the Gulf of Maine [5,6] from two to five sites. In our study, the prevalence varied from moderate (26%) in Jones Cove to low in Webhannet River and Basket Island (9%); in addition, we reported the presence of MSX in oysters from Bagaduce River (17%) and Maquoit Bay (22%). Oysters from the Damariscotta River Estuary sampled in the summer of 2012 reported prevalences of 50% [6], which were higher than the prevalences
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reported in this work; this can be attributed to year to year variation (2012 versus 2014), to site location variation (Jones Cove versus upper Damariscotta River), or to oyster genetic background (wild populations versus farmed oysters). Perkinsus trophozoites are up-taken by the oyster hemocytes [18], which are the main defense mechanisms against pathogens; consequently, any oyster infected with Perkinsus spp. would be impaired to fight other protozoan pathogens including MSX. To assess if the infection of a parasites facilitates the infection by other parasites, we compared the frequency of co-occurrence of each pair of parasites with a random binomial null model [24] based on the likelihood of infection for each parasite and found a statistically significant deviation from the null (random) model. An oyster with P. marinus is 1.7 times as likely to be infected by a non-Perkinsus parasite (p = 0.049), and an oyster with both Perkinsus spp. is 3.2 times as likely to be infected by a nonPerkinsus parasite (p = 0.010). At this point, we do not know if new P. marinus/P. chesapeaki strains are responsible for the high prevalence observed, or if the same strains are affecting impaired oysters or oysters with different genetic background. Nevertheless, future surveys should take into account both Perkinsus spp. and Haplosporidium spp. to provide the baseline for the protozoan parasites, which is essential, especially in the event of die-offs, to ascertain the causes of the mortality. Here, we also report positives for the protozoan parasites C. parvum and T. gondii from oyster samples using PCR-based assays. Livestock, particularly cattle and sheep, are the most important reservoirs of zoonotic infections; however, wild animals can also carry multiple Cryptosporidium species or genotypes [25]. Maximum C. parvum prevalence corresponded to oysters from Weskeag River (39%); oysters from Nonesuch River and Webhannet River also tested positive, although with low prevalences, 17% and 13% respectively. Samples from the Bagaduce River and the Scarborough River tested positive for T. gondii with prevalences of 4% and 14% respectively. In our study, there is some spatial clustering of sites based on parasites common to each site (Fig. 1B), suggesting the possibility of hydrographic pathways. Yet there are exceptions as well; for example T. gondii was found at two widely separated sites. The actual public health threat posed by both T. gondii and C. parvum via consumption of raw oysters remains unresolved; nevertheless, it has been suggested to include in the analysis of oysters when outbreaks occurs and oysters have been consumed [3]. The presence of human pathogens is hardly controllable; however, knowing its presence could serve to provide rules about oyster trade and consumption of raw oysters, and also be useful for management and aquaculture planning. Our study represents the first snapshot in time for these protozoan parasites in bivalves from Maine; both ability of the oocysts to excysts and origin (fecal contamination from wildlife or run off from farmed land) are unknown. Our findings indicate that establishing a baseline of waterborne parasites in commercial shellfisheries together with defining high versus low risk areas would contribute to the development of a sustainable and safe aquaculture.
Table 1 Summary of the protozoan parasite prevalence in Crassostrea virginica from the Gulf of Maine using PCR-based assays. Prevalence (%) Site
Date
n
Length (mm)/STD
Weight (gr)/STD
P. marinus
P. chesapeaki
H. nelsoni
C. parvum
T. gondii
Bagaduce River Weskeag River Jones Cove Deer Meadow Brook⁎ West Bath Maquoit Bay Basket Island Nonesuch River Scarborough River Webhannet River
6/29/14 6/12/14 6/26/14 6/18/14 6/26/14 6/29/14 6/21/14 6/21/14 6/29/14 6/21/14
23 23 23 23 23 23 23 23 23 23
37.3/1.6 41.5/3.2 44.1/5.2 45.6/12.5 48.9/5.4 37.9/3.5 39.6/3.5 37.9/2.8 40.7/6.1 35.1/3.2
44.0/5.4 53.4/7.5 80.2/18.2 73.5/36.8 82.9/13.7 48.0/7.7 50.9/8.1 36.2/6.0 44.2/9.6 34.3/5.9
52.2 73.9 65.2 65.2 56.2 60.9 52.2 69.6 47.6 73.9
80.0 85.7 43.5 4.3 43.5 0.0 34.8 47.8 42.9 47.8
17.4 0.0 26.1 0.0 0.0 21.8 8.7 0.0 0.0 8.7
0.0 39.1 0.0 0.0 0.0 0.0 0.0 17.4 0.0 13.0
4.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 14.3 0.0
⁎ Natural bed.
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Acknowledgments This study was supported by institutional funds from Bigelow Laboratory for Ocean Sciences (132016 and 132018) and by the grant OCE0755142 (REU Program) awarded to D. M. Fields from the NSF. We thank Dr. P. Larsen (Bigelow Laboratory), D. Morse (Maine Sea Grant College Program and UMaine Cooperative Extension), and K. Rousseau (Maine Department of Marine Resources) for their advice in selecting the samples sites. We thank the oyster farmers who kindly donated their oysters for this project. Positive controls for the PCR assays were provided by V.B. Carruthers and J.P. Duvey (T. gondii), K. Miska (C. parvum), and T.J. Bowden (H. nelsoni). We also thank I. Gilg and Dr. J. Martínez Martínez for their advice and guidance on technical aspects of the project. References [1] Coen LD, Brumbaugh RD, Bushek D, Grizzle R, Luckenbach MW, Posey MH, et al. Ecosystem services related to oyster restoration. Mar Ecol Prog Ser 2007;341:303–7. [2] Dumbauld BR, Ruesink JL, Rumrill SS. The ecological role of bivalve shellfish aquaculture in the estuarine environment: a review with application to oyster and clam culture in West Coast (USA) estuaries. Aquaculture 2009;290:196–223. [3] Robertson LJ. The potential for marine bivalve shellfish to act as transmission vehicles for outbreaks of protozoan infections in humans: a review. Int J Food Microbiol 2007;120:201–16. [4] Fernández Robledo JA, Vasta GR, Record NR. Protozoan parasites of bivalve molluscs: literature follows culture. PLoS One 2014;9:e100872. [5] Barber BJ, Langan R, Howell TL. Haplosporidium nelsoni (MSX) epizootic in the Piscataqua River estuary (Maine/New Hampshire, U.S.A.). J Parasitol 1997;83:148–50. [6] Messerman NA, Johndrow KE, Bowden TJ. Prevalence of the protozoan parasite Haplosporidium nelsoni in the Eastern oyster, Crassostrea virginica, in the Damariscotta River Estuary, in Maine, USA in 2012. Bul Europ Assoc Fish Pathol 2014;34:54–62. [7] Lafferty KD, Harvell CD, Conrad JM, Friedman CS, Kent ML, Kuris AM, et al. Infectious diseases affect marine fisheries and aquaculture economics. Ann Rev Mar Sci 2015; 7:471–96. [8] Vanwormer E, Conrad PA, Miller MA, Melli AC, Carpenter TE, Mazet JA. Toxoplasma gondii, source to sea: higher contribution of domestic felids to terrestrial parasite loading despite lower infection prevalence. Ecohealth 2013;10:277–89. [9] Lindsay DS, Collins MV, Mitchell SM, Wetch CN, Rosypal AC, Flick GJ, et al. Survival of Toxoplasma gondii oocysts in Eastern oysters (Crassostrea virginica). J Parasitol 2004; 90:1054–7.
[10] Flegr J. How and why Toxoplasma makes us crazy. Trends Parasitol 2013;29:156–63. [11] Pecher WT, Alavi MR, Schott EJ, Fernández Robledo JA, Roth L, Berg ST, et al. Assessment of the northern distribution range of selected Perkinsus species in Eastern oysters (Crassostrea virginica) and hard clams (Mercenaria mercenaria) with the use of PCR-based detection assays. J Parasitol 2008;94:410–22. [12] Balch WM, Drapeau DT, Bowler BC, Huntington TG. Step-changes in the physical, chemical and biological characteristics of the Gulf of Maine, as documented by the GNATS time series. Mar Ecol Prog Ser 2012;450:11–35. [13] Marsh AG, Gauthier JD, Vasta GR. A semiquantitative PCR assay for assessing Perkinsus marinus infections in the Eastern oyster, Crassostrea virginica. J Parasitol 1995;81:577–83. [14] Coss CA, Robledo JAF, Ruiz GM, Vasta GR. Description of Perkinsus andrewsi n. sp. isolated from the Baltic clam (Macoma balthica) by characterization of the ribosomal RNA locus, and development of a species-specific PCR-based diagnostic assay. J Eukaryot Microbiol 2001;48:52–61. [15] Penna MS, Khan M, French RA. Development of a multiplex PCR for the detection of Haplosporidium nelsoni, Haplosporidium costale and Perkinsus marinus in the Eastern oyster (Crassostrea virginica, Gmelin, 1971). Mol Cell Probes 2001;15:385–90. [16] Mahittikorn A, Wickert H, Sukthana Y. Comparison of five DNA extraction methods and optimization of a B1 gene nested PCR (nPCR) for detection of Toxoplasma gondii tissue cyst in mouse brain. Southeast Asian J Trop Med Public Health 2005;36: 1377–82. [17] Yu JR, Lee SU, Park WY. Comparative sensitivity of PCR primer sets for detection of Cryptosporidium parvum. Korean J Parasitol 2009;47:293–7. [18] Tasumi S, Vasta GR. A galectin of unique domain organization from hemocytes of the Eastern oyster (Crassostrea virginica) is a receptor for the protistan parasite Perkinsus marinus. J Immunol 2007;179:3086–98. [19] Cowger J. Natural occurrence of the American Oyster, Crassostrea virginica, in Maine and its relevance to the Critical Areas Program. Planning report number 4, 04333. Augusta, ME: Maine State Planning Office; 1975 [21 pp.]. [20] Larsen PF, Wilson KA, Morse D. Observations on the expansion of a relict population of Eastern oysters (Crassostrea virginica) in a Maine estuary: implications for climate change and restoration. Northeast Nat 2013;20:N28–32. [21] Bushek D, Allen Jr SK. Races of Perkinsus marinus. J Shellfish Res 1996;15:103–7. [22] Alemán Resto Y, Fernández Robledo JA. Identification of MMV Malaria Box inhibitors of Perkinsus marinus using an ATP-based bioluminescence assay. PLoS One 2014;9: e111051. [23] Yu Z, Guo X. Identification and mapping of disease-resistance QTLs in the Eastern oyster, Crassostrea virginica Gmelin. Aquaculture 2006;254:160–70. [24] Rice JA. Mathematical statistics and data analysis. 3rd ed. Duxbury Press; 2007. [25] Ryan U, Fayer R, Xiao L. Cryptosporidium species in humans and animals: current understanding and research needs. Parasitology 2014:1–19.
Contents lists available at ScienceDirect
Parasitology International journal homepage: www.elsevier.com/locate/parint
Short communication
Survey for protozoan parasites in Eastern oysters (Crassostrea virginica) from the Gulf of Maine using PCR-based assays Nicholas D. Marquis a,b, Nicholas R. Record a, José A. Fernández Robledo a,⁎ a b
Bigelow Laboratory for Ocean Sciences, PO Box 380, 60 Bigelow Drive, East Boothbay, ME 04544-0380, USA Southern Maine Community College, Research Experience for Undergraduates (2014), National Science Foundation, USA
a r t i c l e
i n f o
Article history: Received 26 September 2014 Received in revised form 23 March 2015 Accepted 6 April 2015 Available online 15 April 2015
a b s t r a c t Protozoan pathogens represent a serious threat to oyster aquaculture, since they can lead to significant production loses. Moreover, oysters can concentrate human pathogens through filter feeding, thus putting at risk raw oyster consumers' health. Using PCR-based assays in oysters (Crassostrea virginica) from Maine, we expand the Northeast range in the USA for the protozoans Perkinsus marinus, Perkinsus chesapeaki, and Haplosporidium nelsoni, and report for the first time the detection of the human pathogens Toxoplasma gondii and Cryptosporidium parvum. Oysters hosting both P. marinus and P. chesapeaki were more than three times as likely to be infected by a non-Perkinsus than those free of Perkinsus infections. © 2015 Elsevier Ireland Ltd. All rights reserved.
Mollusk bivalves are key components of marine and estuarine environments because, as filter feeders, they play a critical role in maintaining water quality and ecosystem integrity [1]. Bivalves also are an abundant resource for the coastal inhabitants and in most places traditional local harvesting of the natural populations is being substituted worldwide by semi-intensive aquaculture initiatives [2]. According to the Food and Agriculture Organization of the United Nations (http:// www.fao.org/), the production of farmed clams, mussels, oysters, and scallops reached 14,297,010 metric tons with an estimated value of $13.7 bn (2010 data). Moving from harvesting natural beds to aquaculture initiatives has associated risk including the presence of protozoan pathogens capable of producing significant production loses and, derived from the condition of being filter feeders, the ability concentrate human pathogens that put at risk the consumers [3]. Protozoan parasites within the genera Perkinsus and Haplosporidium severely affect mollusk species commercially harvested or farmed around the world; bivalve trading and global warming have been proposed as two of the main causes for expansion of parasitic diseases in mollusks [4]. Indeed, Perkinsus marinus and Perkinsus olseni (“Dermo” disease) are under surveillance by the World Organization for Animal Health (OIE; http:// www.oie.int/) and Haplosporidium nelsoni (MSX: Multinucleated Sphere X) has also been associated to mass mortalities of oysters in the USA [5,6]. Oysters and clams feed on suspended phytoplankton by trapping it in the gills; simultaneously, the gills also filter pathogenic microorganisms from the water, concentrating them in the digestive system. If these pathogens are not cleared or inactivated by the bivalves, their ⁎ Corresponding author. Tel.: +1 207 315 2567x315; fax: +1 207 315 2329. E-mail addresses: [email protected] (N.D. Marquis), [email protected] (N.R. Record), [email protected] (J.A. Fernández Robledo).
http://dx.doi.org/10.1016/j.parint.2015.04.001 1383-5769/© 2015 Elsevier Ireland Ltd. All rights reserved.
consumption might pose a public health concern and impact the costal economies in the event of temporary or long-term closure of the affected areas [3,7]. Toxoplasma gondii and Cryptosporidium parvum are widely distributed pathogens of humans and animals that may retain their infectivity in raw or undercooked mollusks; their waterborne transmission has been largely demonstrated in both marine and freshwater systems where they interact with aquatic organisms [3]. T. gondii is a protozoan parasite whose final hosts are felines, including domestic cats; however, it can also affect humans and other mammals (e.g. pigs, sheeps); millions of cysts derived from both wild and domestic animals reach the shores through runoff and sewage [8]. Recent studies indicate that infected oysters Crassostrea virginica may serve as a source of T. gondii for marine mammals and humans, since oysters can readily acquire T. gondii oocysts from seawater, and T. gondii can survive for several months in oysters [9]. Toxoplasmosis has been recently in the public eye for being responsible for mass mortalities of West coast sea otters associated with the consumption of infected bivalves; moreover, a number of epidemiological studies link Toxoplasma to pathological and psychological conditions in humans [10]. C. parvum is a parasite of the mammalian intestinal tract; it causes self-limiting diarrhea in people with intact immune systems and it is particularly severe in immunocompromised individuals; the resistance stage (oocyst) has been detected in many oysters, mussels, and clams within both fecally contaminated and clean oyster/clam growing areas across the globe [3]. P. marinus is found in the Pacific coast, Gulf of Mexico, and East coast of North America [4] with Damariscotta River Estuary (Maine, USA) being the most northeast limit [11]. In Maine, H. nelsoni has been associated to outbreaks in the Piscataqua River Estuary in the 1990's [5] and in the Damariscotta River Estuary in 2010 [6]. To our knowledge, no surveys have been done for Cryptosporidium and Toxoplasma in mollusks from the Gulf of Maine. Increasing the production of shellfish from
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N.D. Marquis et al. / Parasitology International 64 (2015) 299–302
coastal inshore ecosystems and creating a more sustainable economic development would require understanding the risks of mollusk pathogens in different areas to identify the places for oyster leases with less risk of outbreaks happening, as well as guaranteeing a product that is free of human pathogens and/or that the human pathogens do not pose a threat to oyster's consumers. Linked to the increase in storm events and an increment in the temperature, most models for climate change predict an increase in the runoff from terrestrial ecosystems in
the estuaries [12]. In addition to the effect on primary production, runoff from terrestrial ecosystems might also affect pathogen abundance in the shores where oysters are commercially grown [3]. In this study, we surveyed during the summer of 2014 oysters from nine oyster leases and one wild population in the Gulf of Maine (Fig. 1A) for three protozoan parasites of molluscs and two protozoan parasites of humans and animals. From each individual oyster (n = 25/site) rectum, gill, and mantle tissue were collected and pooled (50–100 mg wet
Fig. 1. A. Sampling site locations and identified protozoan parasites. Adult eastern oysters (Crassostrea virginica) were collected between June and July 2014 from a natural oyster bed and from shellfish farmers from nine oyster leases along the coast of the Gulf of Maine (Northeast USA). B. Clustering of the study sites based on detected parasites. Sites were clustered based on the Manhattan distance metric (Σi5 = 1 |xi − yi| where xi and yi are the probabilities of the presence of parasite i at sites x and y respectively). Other distance metrics produced similar clustering. Colors show whether a site was above average (red) or below average (blue) with respect to the prevalence of a given parasite. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
N.D. Marquis et al. / Parasitology International 64 (2015) 299–302
weight of total tissues/pool) and DNA was extracted using a commercial kit (E.Z.N.A. Tissue DNA kit; Omega Bio-Tek, Norcross, GA, USA). PCRbased assays specific for P. marinus, Perkinsus chesapeaki, and H. nelsoni, T. gondii, and C. parvum were performed as reported elsewhere [13–17] using 200–400 ng/PCR, PCR master mix (EconoTaq Plus Green 2X, Lucigen, WI, USA), and the parasite specific primers in a final volume of 25 μl (Table 1). The latest large epizootiological survey for Perkinsus spp. in oysters in the East coast of the USA was conducted in 2002 with reported low prevalences for P. marinus (1%) and for P. chesapeaki (3%) [11]. Damariscotta River Estuary used to be a good source for hemocytes from parasite-free oysters for in vitro host-parasite interaction studies [18]; however, over the last 2–3 years, it has become difficult to find specimens negative for Perkinsus spp. (G.R. Vasta, IMET, pers. comm.). In our survey, oysters from Jones Cove (Damariscotta River Estuary) reached prevalences of 65.2% for P. marinus and 43.5% for P. chesapeaki, a 65–15 fold increase of the prevalence respectively over a period of 12 years [11]. We also found high prevalences for P. marinus in the rest of the sites sampled with oysters from Webhannet River and Weskeag River reaching the highest prevalence values of the survey (73.9% in both); the highest prevalence for P. chesapeaki was in Bagaduce River (80.0%) and Weskeag River (85.7%). Interestingly we found no P. chesapeaki in oysters from Maquoit Bay. The wild oyster population (Deer Meadow Brook), which is considered the northernmost native oyster population in the United States [19], although in the same range size as cultured oysters, was older than the oysters from leases based on analysis of the shell (data not shown). The prevalence was high for P. marinus (65.2%) but very low for P. chesapeaki (4.3%) suggesting that although salinity and temperature are suitable for oyster survival and growth, the heavy tidal action [20] might not favor P. chesapeaki transmission or, alternatively, that native populations have some resistance to P. chesapeaki. There are several hypotheses that would explain the high prevalence of Perkinsus spp. in the Gulf of Maine including the presence of new Perkinsus races or genetic variants reaching the area. Indeed, the presence of P. marinus “races” and genetic strains have been documented along the Atlantic and Gulf coasts of the USA based on phenotypic, genotypic characters, and sensitivity to drugs [21,22]. Another hypothesis is that the genetic background of the oyster used in commercial leases is most susceptible to Perkinsus spp. Indeed, 12 disease-resistance quantitative trait loci have been evaluated in relation to resistance in the Eastern oyster to both Dermo and MSX [23]. Testing these hypotheses would require more focused studies. Based on our 2014 sampling, we expanded the range of MSX in the Gulf of Maine [5,6] from two to five sites. In our study, the prevalence varied from moderate (26%) in Jones Cove to low in Webhannet River and Basket Island (9%); in addition, we reported the presence of MSX in oysters from Bagaduce River (17%) and Maquoit Bay (22%). Oysters from the Damariscotta River Estuary sampled in the summer of 2012 reported prevalences of 50% [6], which were higher than the prevalences
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reported in this work; this can be attributed to year to year variation (2012 versus 2014), to site location variation (Jones Cove versus upper Damariscotta River), or to oyster genetic background (wild populations versus farmed oysters). Perkinsus trophozoites are up-taken by the oyster hemocytes [18], which are the main defense mechanisms against pathogens; consequently, any oyster infected with Perkinsus spp. would be impaired to fight other protozoan pathogens including MSX. To assess if the infection of a parasites facilitates the infection by other parasites, we compared the frequency of co-occurrence of each pair of parasites with a random binomial null model [24] based on the likelihood of infection for each parasite and found a statistically significant deviation from the null (random) model. An oyster with P. marinus is 1.7 times as likely to be infected by a non-Perkinsus parasite (p = 0.049), and an oyster with both Perkinsus spp. is 3.2 times as likely to be infected by a nonPerkinsus parasite (p = 0.010). At this point, we do not know if new P. marinus/P. chesapeaki strains are responsible for the high prevalence observed, or if the same strains are affecting impaired oysters or oysters with different genetic background. Nevertheless, future surveys should take into account both Perkinsus spp. and Haplosporidium spp. to provide the baseline for the protozoan parasites, which is essential, especially in the event of die-offs, to ascertain the causes of the mortality. Here, we also report positives for the protozoan parasites C. parvum and T. gondii from oyster samples using PCR-based assays. Livestock, particularly cattle and sheep, are the most important reservoirs of zoonotic infections; however, wild animals can also carry multiple Cryptosporidium species or genotypes [25]. Maximum C. parvum prevalence corresponded to oysters from Weskeag River (39%); oysters from Nonesuch River and Webhannet River also tested positive, although with low prevalences, 17% and 13% respectively. Samples from the Bagaduce River and the Scarborough River tested positive for T. gondii with prevalences of 4% and 14% respectively. In our study, there is some spatial clustering of sites based on parasites common to each site (Fig. 1B), suggesting the possibility of hydrographic pathways. Yet there are exceptions as well; for example T. gondii was found at two widely separated sites. The actual public health threat posed by both T. gondii and C. parvum via consumption of raw oysters remains unresolved; nevertheless, it has been suggested to include in the analysis of oysters when outbreaks occurs and oysters have been consumed [3]. The presence of human pathogens is hardly controllable; however, knowing its presence could serve to provide rules about oyster trade and consumption of raw oysters, and also be useful for management and aquaculture planning. Our study represents the first snapshot in time for these protozoan parasites in bivalves from Maine; both ability of the oocysts to excysts and origin (fecal contamination from wildlife or run off from farmed land) are unknown. Our findings indicate that establishing a baseline of waterborne parasites in commercial shellfisheries together with defining high versus low risk areas would contribute to the development of a sustainable and safe aquaculture.
Table 1 Summary of the protozoan parasite prevalence in Crassostrea virginica from the Gulf of Maine using PCR-based assays. Prevalence (%) Site
Date
n
Length (mm)/STD
Weight (gr)/STD
P. marinus
P. chesapeaki
H. nelsoni
C. parvum
T. gondii
Bagaduce River Weskeag River Jones Cove Deer Meadow Brook⁎ West Bath Maquoit Bay Basket Island Nonesuch River Scarborough River Webhannet River
6/29/14 6/12/14 6/26/14 6/18/14 6/26/14 6/29/14 6/21/14 6/21/14 6/29/14 6/21/14
23 23 23 23 23 23 23 23 23 23
37.3/1.6 41.5/3.2 44.1/5.2 45.6/12.5 48.9/5.4 37.9/3.5 39.6/3.5 37.9/2.8 40.7/6.1 35.1/3.2
44.0/5.4 53.4/7.5 80.2/18.2 73.5/36.8 82.9/13.7 48.0/7.7 50.9/8.1 36.2/6.0 44.2/9.6 34.3/5.9
52.2 73.9 65.2 65.2 56.2 60.9 52.2 69.6 47.6 73.9
80.0 85.7 43.5 4.3 43.5 0.0 34.8 47.8 42.9 47.8
17.4 0.0 26.1 0.0 0.0 21.8 8.7 0.0 0.0 8.7
0.0 39.1 0.0 0.0 0.0 0.0 0.0 17.4 0.0 13.0
4.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 14.3 0.0
⁎ Natural bed.
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Acknowledgments This study was supported by institutional funds from Bigelow Laboratory for Ocean Sciences (132016 and 132018) and by the grant OCE0755142 (REU Program) awarded to D. M. Fields from the NSF. We thank Dr. P. Larsen (Bigelow Laboratory), D. Morse (Maine Sea Grant College Program and UMaine Cooperative Extension), and K. Rousseau (Maine Department of Marine Resources) for their advice in selecting the samples sites. We thank the oyster farmers who kindly donated their oysters for this project. Positive controls for the PCR assays were provided by V.B. Carruthers and J.P. Duvey (T. gondii), K. Miska (C. parvum), and T.J. Bowden (H. nelsoni). We also thank I. Gilg and Dr. J. Martínez Martínez for their advice and guidance on technical aspects of the project. References [1] Coen LD, Brumbaugh RD, Bushek D, Grizzle R, Luckenbach MW, Posey MH, et al. Ecosystem services related to oyster restoration. Mar Ecol Prog Ser 2007;341:303–7. [2] Dumbauld BR, Ruesink JL, Rumrill SS. The ecological role of bivalve shellfish aquaculture in the estuarine environment: a review with application to oyster and clam culture in West Coast (USA) estuaries. Aquaculture 2009;290:196–223. [3] Robertson LJ. The potential for marine bivalve shellfish to act as transmission vehicles for outbreaks of protozoan infections in humans: a review. Int J Food Microbiol 2007;120:201–16. [4] Fernández Robledo JA, Vasta GR, Record NR. Protozoan parasites of bivalve molluscs: literature follows culture. PLoS One 2014;9:e100872. [5] Barber BJ, Langan R, Howell TL. Haplosporidium nelsoni (MSX) epizootic in the Piscataqua River estuary (Maine/New Hampshire, U.S.A.). J Parasitol 1997;83:148–50. [6] Messerman NA, Johndrow KE, Bowden TJ. Prevalence of the protozoan parasite Haplosporidium nelsoni in the Eastern oyster, Crassostrea virginica, in the Damariscotta River Estuary, in Maine, USA in 2012. Bul Europ Assoc Fish Pathol 2014;34:54–62. [7] Lafferty KD, Harvell CD, Conrad JM, Friedman CS, Kent ML, Kuris AM, et al. Infectious diseases affect marine fisheries and aquaculture economics. Ann Rev Mar Sci 2015; 7:471–96. [8] Vanwormer E, Conrad PA, Miller MA, Melli AC, Carpenter TE, Mazet JA. Toxoplasma gondii, source to sea: higher contribution of domestic felids to terrestrial parasite loading despite lower infection prevalence. Ecohealth 2013;10:277–89. [9] Lindsay DS, Collins MV, Mitchell SM, Wetch CN, Rosypal AC, Flick GJ, et al. Survival of Toxoplasma gondii oocysts in Eastern oysters (Crassostrea virginica). J Parasitol 2004; 90:1054–7.
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