zoonotic protozoa: from land to sea

Zoonotic protozoa: from land to seaRonald Fayer1, Jitender P. Dubey2 and David S. Lindsay3

1Environmental Microbial Safety Laboratory, Animal and Natural Resources Institute, Agricultural Research Service,

United States Department of Agriculture, Beltsville, MD 20705, USA2Animal Parasitic Diseases Laboratory, Animal and Natural Resources Institute, Agricultural Research Service,

United States Department of Agriculture, Beltsville, MD 20705, USA3Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology,

Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, 1410 Prices Fork Rd, Blacksburg, VA 24061-0342, USA

Attention to worldwide pollution of the coastal marine

environment has focused primarily on toxic algal

blooms and pathogenic bacteria that multiply in

nutrient-rich waters. However, massive but unseen

amounts of feces from humans, their pets, and their

domesticated animals are discharged, dumped, or

carried in runoff, bringing encysted zoonotic protozoan

parasites to estuaries and coastal waters. Here, they

contaminate bathing beaches, are filtered and concen-

trated by shellfish eaten by humans and marine

mammals, and infect a wide range of marine animal

hosts, resulting in morbidity and mortality to some

populations. This review addresses the extent of

contamination and the animals affected by three genera

of important zoonoticprotozoa:Giardia,Cryptosporidium

and Toxoplasma.

Giardia, Cryptosporidium and Toxoplasma are foundworldwide and are among the most frequently reportedparasites of humans and animals. All are widely dispersedand have been associated with outbreaks of infectionresulting from drinking contaminated surface water.Giardia cysts and Cryptosporidium oocysts are trans-mitted by the fecal–oral route from humans and animals.Toxoplasma gondii oocysts are transmitted from cats tohundreds of warm-blooded vertebrate species. Althougheach infected person or animal often excretes largenumbers of these encysted parasites, relatively few arerequired to initiate an infection. Furthermore, they aresmall, buoyant, resistant to most commonly used disin-fectants, and can survive in moist environments for weeksor months, even when exposed to a wide range oftemperatures and salinities. These traits can contributeto rapid and widespread dispersal, especially after rainfall[1]. Rainfall-initiated runoff from urban, suburban andrural landscapes, and wastewater outfalls, can carryencysted protozoa into untreated waters used for agricul-ture, recreation and, in some locations, for drinking.Recent evidence indicates that these encysted protozoanpathogens are also in coastal waters, having contaminateda variety of shellfish and infected many species of marinemammals. These findings raise concern for the health ofanimals in coastal waters and for humans who eat rawshellfish and who use these waters for recreation.

Corresponding author: Ronald Fayer ([email protected]).Available online 3 September 2004

www.sciencedirect.com 1471-4922/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved

Species and hosts

Giardia duodenalis, the species that infects humans,consists of seven assemblages (genotypes). Among them,assemblage AI is found in humans, livestock, dogs, cats,beavers and other animals; assemblage AII is found onlyin humans [2]; and assemblage B is found in humans,beavers, dogs, muskrats and other animals [2,3]. Trans-mission from humans to beavers, dogs and muskratssuggests some Giardia are zoonotic, and similar genesequences among isolates support this possibility. Assem-blages C and D are found primarily in canids, andassemblages E, F and G, found primarily in hoofedlivestock, cats and rats, respectively, have not beenfound in human infections.

Several Cryptosporidium species infect humans:Cryptosporidium hominis (formerly Cryptosporidiumparvum genotype 1), C. parvum, Cryptosporidium melea-gridis, Cryptosporidium felis, Cryptosporidium canis,Cryptosporidium muris, cervine genotype and pig geno-type I [4]. Most infections are associated with C. hominisand C. parvum, with other species found predominantly inother hosts. Cryptosporidium hominis has been detectedin a few monkeys, sheep and a marine mammal (dugong),whereas C. parvum has been found in numerous cattle,sheep and deer, as well as in mice and pigs [4].

Three routes of transmission of T. gondii includetransmission through oocysts shed in the feces of wildand domesticated felids, ingestion of tissue cysts frommeat, and congenital transmission. The degree of trans-mission to humans and animals by each of these routes isnot always entirely clear and, although the oocyst mightappear the most likely source of infection for non-carnivores, the extent of vertical transmission in mosthost species is unknown and should not be underesti-mated [5]. All methods of transmission are possible, butoocysts are the only stage that enter the environment andremain infectious for up to a year under moist temperateconditions while being widely dispersed in large numbers.Within the species T. gondii, strains are based onpathogenicity in mice, and specific genetic markers haveconfirmed a clonal population with three dominantgenotypes (types I, II and III) evolved from sexualrecombination in felid definitive hosts [6–8]. Additionalgenotypes are known but appear to be less common. TypesI and II have been identified in human infections but thefull genetic diversity of isolates in humans is unknown.

Review TRENDS in Parasitology Vol.20 No.11 November 2004

. doi:10.1016/j.pt.2004.08.008

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Waterborne outbreaks and prevalence in humans

Giardia cysts have been detected in O2350 samples ofsurface water from eight countries at nearly five cysts perliter and in 21–100% of the samples examined [9]. In theUSA, from 1984 to 1994, 18 drinking-water-associatedoutbreaks of giardiasis affected 3994 individuals [10],whereas an estimated 2.8!108 cases occur globally eachyear [11].

Worldwide, Cryptosporidium oocysts have been foundin ground water, lakes, rivers, estuaries and ocean water[12]. In over 3700 surface water samples from 11countries, 0–100% of specimens contained 0–252.7 oocystsper liter [9]. Of 69 cryptosporidiosis outbreaks from 1983to 1999 in North America, the UK and Japan, 45 wererelated to contaminated drinking water. The largestoutbreak, from contaminated lake water, affected anestimated 403 000 individuals [12]. Cryptosporidiosishas been reported in over 50 countries. Seroprevalencerates of 25–35% in industrialized countries increase to64% in Latin America, and 42% and 57.5% in China andBrazil, respectively, where water purification and sani-tation standards are lower [13]. A 44% seroprevalenceamong dairy farmers versus 24% in others suggests azoonotic link [13].

A toxoplasmosis outbreak in 31 military troops from theUSA was associated with drinking water from a junglepond in Panama [14]. A municipal water system that usedunfiltered, chloraminated surface water was the likelysource of a community-wide outbreak of toxoplasmosisinvolving O100 individuals in Canada [15]. The epidemiccurve appeared bimodal, with peaks preceded byincreased rainfall and turbidity in the implicated reser-voir. Toxoplasmosis has been reported worldwide. In theUSA and UK, an estimated 16–40% of people acquireT. gondii infections, whereas, in Central and SouthAmerica and continental Europe, infection estimatesreach 50–80% [16–18].

Fecal production and fecal pathogens in the

environment

Pathogenic protozoa enter the environment in feces fromland-dwellers – both human and non-human hosts. Propermanagement, treatment and dispersal of human feces andanimal manures are essential for clean and safe drinkingand recreational water. Recreational water also includesestuaries and coastal waters. The enormity of the problemof keeping water clean and safe might seem overwhelmingwhen one considers the volume of feces that enters theenvironment. Although, from space, the Earth appears tobe covered in large part by water and is seen as a brightblue sphere, closer inspection would reveal human andanimal feces spread over much of the planet. On average,each of the current 6.35 billion human inhabitantsproduces w200 g of feces daily, bringing the annual globaldeposit of human feces to w232 million metric tons (Mg).Most is disposed with little or no treatment in under-developed countries before disposal into rivers or coastalwaters, whereas most is aerobically digested in developedcountries before such dispersal.

Worldwide domesticated livestock populations, esti-mated at 1.5 billion cattle and buffalo, 878 million pigs,

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and 1.7 billion sheep and goats, annually produce w3.6billion Mg, 228 million Mg and 408 million Mg of manure,respectively [19]. Most is deposited on fields with little orno treatment except in places that enforce manuremanagement programs. Other estimates of livestockmanure production reach 5.45 billion Mg per year, withbeef cattle accounting for w55%.

Cyst excretion can approach 107 Giardia cysts per gramof feces [9]. The median infectious dose is thought to bebetween 25 and 100 cysts, but volunteers became infectedafter ingesting as few as ten cysts [9]. Zoonoticassemblage A has been found in dairy cattle manure[20] and, in some farms, this constitutes as much as45% of Giardia isolates [20].

Infected persons and neonatal calves can excrete109–1010 C. parvum oocysts over approximately a week.Yet, in one study, as few as 30 oocysts initiated an infectionin a human volunteer, and the median infective dose was132 oocysts [21]. Although Cryptosporidium oocysts arefrequently identified by microscopy in feces from neonatalruminants, including cattle, sheep, goats and farmed deer,gene sequencing of oocysts from nearly 1000 dairy calvesdemonstrated that most pre-weaned calves had thezoonotic species C. parvum, whereas virtually all post-weaned calves had other non-zoonotic genotypes [22].

The worldwide population of felids, the only source ofT. gondii oocysts, is unknown, but it is estimated that w60million pets and 40 million free-ranging cats live in theUSA. These and other felid species such as bobcats,jaguars, lions and tigers are found worldwide. Although adomesticated cat might defecate only 25 g of feces per day,and the shedding period for Toxoplasma oocysts might lastonly a few days during the life of a cat, tens of millions ofoocysts can be excreted per day and as few as oneoocyst has been infective for mice and pigs [23]. Theminimum infectious dose for humans is unknown butthought to be low.

All other wild and domesticated animals contributeadditional trillions of kg to the global accumulation offeces. This results in a sum accumulation of an incalcul-able number of pathogens.

Contamination of food from polluted fresh and sea

water

Of waters used to irrigate vegetables in the USA andseveral Central American countries, 60% tested positivefor Giardia cysts and 36% tested positive for Crypto-sporidium oocysts [24]. Giardia cysts have been found oncoriander, carrots, mint, radishes and potatoes irrigatedwith wastewater [11], and outbreaks related to contami-nated fruits and vegetables have been reported. Crypto-sporidium oocysts were found on cilantro, lettuce,radishes, tomatoes, cucumbers and carrots in CostaRica, with the highest contamination during the rainyseason when irrigation water from rivers received indus-trial, domestic and agricultural wastewater [25]. Simi-larly, in Peru, Cryptosporidium oocysts were detected on14.5% of 110 fresh vegetables collected at 13 markets, andlater at 16.5% of 62 vegetables from 15 markets [26]. Anoutbreak of cryptosporidiosis was related to eating freshgreen onions. Surface water from many agricultural sites


Review TRENDS in Parasitology Vol.20 No.11 November 2004 533

eventually enters estuaries and coastal waters. Giardiacysts and Cryptosporidium oocysts have been found inshellfish in fresh, estuarine and coastal waters worldwide(Table 1). However, despite widespread and heavy con-tamination of shellfish in some areas, no related outbreaksor individual cases have been reported.

Toxoplasma gondii provides the most direct evidencethat encysted protozoa enter the marine environmentfrom terrestrial sources. Toxoplasmosis can be acquiredfrom ingestion of oocysts or from tissue cysts in raw orundercooked meat. The link between T. gondii oocystscontaminating the marine environment and humantoxoplasmosis acquired as a food-borne pathogen isfound in Inuit women of child-bearing age in northernCanada [27]. Nearly 50% of the women tested wereserologically positive for T. gondii. Of 22 responders to aquestionnaire, those seropositive to T. gondii were fourtimes more likely to have eaten dried seal meat and sixtimes more likely to have eaten seal liver than seronega-tive women. Toxoplasma gondii has been detected in sealsfrom several geographic areas (Table 2).

Giardia, Cryptosporidium and Toxoplasma in marine

waters

Encysted protozoans originating in human and animalfeces are transported in runoff from agricultural, sub-urban and urban land surfaces, wastewater dischargesand other sources to rivers and streams, which carrycontaminated sediments to estuaries and eventually tocoastal waters. Despite the fact that many countriesdispose of raw sewage and dredged sediments fromshipping lanes in coastal waters, there are no mandatedlocal or national monitoring programs for protozoan

Table 1. Cryptosporidium detected in contaminated shellfish

Location Species (common name)

Sligo area, Ireland Mytilus edulis (Common mussel)

Chesapeake Bay, MD, USA Crassostrea virginica (Virginia or Eastern oy

Chesapeake Bay, USA Ischadium recurvum (Bent mussel)

Chesapeake Bay, USA C. virginica

Galicia, Spain Dosinia exoleta (Rayed artemis)

Venerupis (Ruditapes) rhomboideus

(Carpet shell clam)

Venerupis (Ruditapes) pullastra (Venus clam

Venus verrucosa (Mediterranean common c

Mytilus galloprovincialis (Mediterranean m

Ostrea edulis (Native, common or flat oyste

Italy Venerupis (Ruditapes) phillipinarum

(Japanese littleneck)

UK O. edulis

Galicia, Spain M. galloprovincialis

Cerastoderma edule (Common cockle)

St Lawrence River, Canada Dreissena polymorpha (Zebra mussel)

Chesapeake Bay, USA C. virginica

Atlantic coast, USA C. virginica

Portugal C. edule

Scrobicularia plana (Soft shell clam)

M.edulis

Venerupis (Tapes) decussates (Carpet shell

Donax sp. (Donax clam)aAbbreviations: B, bioassay in mice; IFA, immunofluorescence microscopy; M, molecul


parasites in estuaries and coastal waters where they caninfect marine wildlife, enter the food chain and potentiallyinfect humans during recreational activities. However, afew studies indicate they are present.

Marine waters near sewage outfalls in Mamala Bay afew kilometers from Waikiki bathing beach in Hawaii [28]and off the coast of Panama [9] were found to containnumerous Giardia cysts and Cryptosporidium oocysts.High numbers of Cryptosporidium oocysts were detectedin marine waters off the coast of San Juan, Puerto Rico[29]. Tidal regions of the Georges River, which isimportant for recreation and shellfish production forSydney, Australia, had elevated concentrations of Giardiaand Cryptosporidium from rainfall and sewage overflowsafter wet weather events [30]. Some areas of Hong Kongdischarge large quantities of sewage into surroundingmarine waters mostly after preliminary or primarytreatment [31]. Of the Hong Kong beaches found suitablefor bathing, 41% of water samples contained Giardia cystsand, of beaches found unsuitable for bathing, 64% hadGiardia cysts, all of which is significantly linked to sewagepollution [31]. Toxoplasma gondii oocysts have not beenreported in coastal waters.

Longevity of encysted protozoa in sea water

Giardia cysts have not been experimentally examined todetermine their longevity in sea water, but infections inmarine mammals suggest they are somewhat resistant atleast to exposure at low salinities (Table 3). Cryptospor-idium oocysts can survive in sea water under a variety oflaboratory conditions for long periods of time. Bioassays inmice indicated that oocysts remained infectious afterbeing held at 10 parts per thousand (ppt) salinity at

No. positive/no. examined Detection

methoda

Ref.

1/3 sites IFA [57]

ster) 6/6 noncommercial sites;

5-18/30 oysters per site

IFA [34]

2/2 sites IFA [58]

5/7 commercial sites;

0-29/30 oysters/site

IFA [59]

15/29 shellfish IFA [60]

)

lam)

ussel)

r)

5/8 clams IFA [60]

1/1 oyster IFA [60]

2/9 sites IFA, B, M [61]

1/9 sites IFA, B, M [61]

514 pooled mussels;

positive finding

IFA, M [62]

7 sites, 8 collections/site;

all sites positive at each collection

IFA, B, M [1]

24/37 sites IFA, B, M [38]

4/26 cockles IFA [63]

1/10 clams IFA [63]

3/32 mussels IFA [63]

clam) 3/18 clams IFA [63]

2/5 clams IFA [63]

ar methods (detected Cryptosporidium parvum).


Table 2. Prevalence of Toxoplasma gondii antibodies in marine mammals

Species

(common name)

Source No. tested No. positive % positive

(MATR1:25a)

Ref.

Enhydra lutris (sea otter)

Dead CA, USA 100 82 82.0 [42]

CA, USA 107 66 62.0b [44]

WA, USA 15 7 47.0 [42]

Live AK, USA 65 0 0b [49]

CA, USA 116 49 42.0b [44]

CA, USA 80 29 36.0b [45]

WA, USA 30 18 60.0 [42]

WA, USA 21 8 38.0b [45]

Odobenus rosamarus (Pacific walrus) AK, USA 53 3 5.6 [42]

Zalophus californianus (Californian sea lion) CA, USA 27 8 29.6 [42]

Phoca vitulina (Harbor seal) AK, USA 311 51 16.4 [42]

Canada 34 3 8.8 [50]

WA, USA 380 29 7.6 [51]

Cystophora cristata (Hooded seal) Canada 60 1 1.7 [50]

Phoca hispida (Ringed seal) AK, USA 32 5 15.6 [42]

Canada 34 3 8.8 [50]

Halichoerus grypus (Grey seal) Canada 122 11 9.0 [50]

Erignathus barbatus (Bearded seal) AK, USA 8 4 50.0 [42]

Phoca groenlandica (Harp seal) Canada 112 0 0.0 [50]

Phoca largha (Spotted seal) AK, USA 9 1 11.1 [42]

Phoca fasciata (Ribbon seal) AK, USA 14 0 0 [42]

Tursiops truncatus (Bottlenose dolphin) Spain 7 4 57.1 [52]

CA, USA 94 91 96.8 [42]

FL, USA 47 47 100.0 [42]

Stenella coeruleoalba (Striped dolphin) Spain 36 4 11.1 [52]

Delphinus delphis (Common dolphin) Spain 4 2 50.0 [52]

aAbbreviation: MAT, modified agglutination test.bIndirect fluorescent antibody test.


208C for 12 weeks and up to 35 ppt at 188C for 40 days [12].Because T. gondii oocysts must become sporulated to beinfectious, their ability to sporulate and survive in seawater was tested. Of unsporulated oocysts placed in 15and 32 ppt artificial sea water, 75–80% sporulated withinthree days and were infectious to mice. Sporulated oocystsstored in 15 ppt sea water at 48C or room temperature forsix months also infected mice [32].

Protozoan parasites in contaminated shellfish

Giardia duodenalis genotype A was identified by molecu-lar tests in Macoma clams in the Rhode River [33]. Thesetiny clams have no economic value but serve as biologicalindicators of water contamination.

Cryptosporidium oocysts placed in sea water weredetected in experimentally exposed oysters [34,35], mus-sels [36] and clams [35]. Oocysts retained for as long as 30days without initiating infection in shellfish were infectivewhen recovered from artificially and naturally contami-nated shellfish [37]. Cryptosporidium has been detected innaturally contaminated mussels, oysters, clams andcockles worldwide (Table 1). The highest prevalence ofcontamination was found in seven Chesapeake Baylocations after a major rainfall event [1]. However,even during a period of low rainfall, Cryptosporidiumwas detected in oysters in 11 out of 13 Atlantic coaststates [38].

Mice became infected when fed tissues from oysters sixdays after exposure of oysters to T. gondii oocysts in a seawater aquarium [39]. Toxoplasma gondii-specific smallsubunit ribosomal RNA (ssrRNA) could be detected for upto 21 days post-exposure in artificially exposed mussels


that were positive by bioassay for only three days post-exposure [40]. Toxoplasma gondii oocysts have not beendetected in shellfish in nature.

Protozoan parasites in marine mammals

Giardia cysts have been detected in feces from a Californiasea lion, from ringed seals in arctic Canada, and fromharp, grey and harbor seals in the Gulf of St Lawrence,Canada (Table 3). Cryptosporidium has been detected inthe dugong, in Australia, in California sea lions and inseals in arctic Canada (Table 3). Toxoplasma gondii hasbeen isolated from a Pacific harbor seal [41]. It has alsobeen histologically identified in seals, dolphins, sea lions, amanate and a Beluga whale from the USA, Italy, Spain,Canada and Australia, without confirmation by isolationof organisms or serology [42]. The finding of T. gondiiantibodies in sera from marine mammals suggests world-wide contamination of the marine environment (Table 2).

Toxoplasmosis in sea otters is a concern [42–47]. Themain sea otter colonies are in California, Washington andAlaska. Mortalities in California have been studied themost [47], where T. gondii has been isolated from brains orhearts of 22.3% of 67 [43] and 32% of 75 [44] California seaotters. Initially, all isolates were identified as genotype II[43], but a recent study [46] found two genotypes in seaotter beachcast carcasses: 40% were Type II and 60% werea unique genotype called Type x. Comparison betweenisolates from sea otters and other marine mammals withisolates from terrestrial animals will hopefully clarifyroutes and mechanisms of land-to-sea transmission.Serological surveys further indicate exposure of manysea otters to T. gondii (Table 2). Although antibodies to


Table 3. Giardia and Cryptosporidium detected in marine mammals

Parasite Host (common name) Location No.

infected

Detection method Ref.

Giardia Phoca hispida (Ringed seal) Arctic Canada 3 Microscopy [53]

Giardia Phoca groenlandica (Harp seal) Gulf of St Lawrence 15 Microscopy [54]

Halichoerus grypus (Grey seal) 4

Phoca vitulina (Harbor seal) 1

Giardia P. hispida Ungava Bay, Canada 43 Flow cytometrya b

Giardia Zalophus californianus (California sea lion) Humboldt Bay, USA 1 Microscopy [55]

Cryptosporidium P. hispida Ungava Bay, Canada 2 Flow cytometrya b

Cryptosporidium Z. californianus Humboldt Bay, USA 1 Microscopy [55]

Cryptosporidium Dugong dugon (Dugong) Queensland, Australia 1 Histology [56]

aFluorescence labeling followed by flow cytometry.bB. Dixon et al., unpublished.

Review TRENDS in Parasitology Vol.20 No.11 November 2004 535

T. gondii were not found in Alaskan sea otters, antibodieswere found in other Alaskan marine mammals.

Grey seals that were fed T. gondii oocysts developedinfection [48], but how marine mammals become infectedin nature has not been well studied. Although mostmarine mammals do not prey on warm-blooded animals,which are potential sources of tissue cysts of T. gondii,congenital transmission has been reported in dolphins.Fresh water runoff into coastal waters poses risk for seaotters [45]. Ingesting oocysts from sea water or eatinganimals that have ingested oocysts are likely sources.Although sea otters eat shellfish, their role as paratenichosts for T. gondii is suspected but unproven. Dolphinsbecome infected (Table 2), but they drink little or no waterand derive their nutrition from fish, squid and other cold-blooded sea animals that do not develop tissue cysts ofT. gondii.

Conclusions

There are many indicators of widespread fecal contami-nation from land to sea, including: (i) recorded outbreaksof giardiasis, cryptosporidiosis and toxoplasmosis associ-ated with drinking water; (ii) findings of Giardia andCryptosporidium in surface waters used to irrigate freshvegetables; (iii) detection of Giardia and Cryptosporidiumin marine mammals and shellfish in fresh, estuarine andcoastal waters worldwide, and in marine waters used forrecreation; and (iv) the global presence of antibodies toT. gondii in many species of marine mammals. Someinfections might be indirect, through vertical trans-mission or transport hosts, and wild terrestrial animalsmight be sources of infectious organisms; however, manyorganisms originate from humans, their companionanimals and domesticated animals. Most shellfish con-taminated with Cryptosporidium have been found tocontain oocysts of C. parvum, the zoonotic speciesprevalent in young domesticated ruminants and inhumans. Cryptosporidium hominis, the only speciesidentified in a marine mammal (a dugong), is the mostprevalent species in human infections. Infection of marinemammals with Giardia has not been identified bymolecular methods and molecular data for T. gondii hasbeen reported only for sea otters. Hopefully, moremolecular data will become available from parasitesisolated from marine mammals and will be comparedwith data from terrestrial animals. Such data will clarifyour understanding of the complex relationships among


hosts, and will help to identify the routes and mechanismsof land to sea transmission. Nevertheless, practices ofwastewater disposal, drinking water purification, runoffcontrol and farm manure management must improve.Otherwise, as human and animal populations grow, so willthe negative impact of fecal contamination on publichealth and ultimately on marine life.

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