Waterborne toxoplasmosis – Recent developments

Waterborne toxoplasmosis – Recent developments

Experimental Parasitology 124 (2010) 10–25 Contents lists available at ScienceDirect Experimental Parasitology journal homepage: www.elsevier.com/lo...

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Experimental Parasitology 124 (2010) 10–25

Contents lists available at ScienceDirect

Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr


Waterborne toxoplasmosis – Recent developments J.L. Jones a,*, J.P. Dubey b a

Division of Parasitic Diseases, National Center for Zoonotic, Vectorborne and Enteric Diseases, Coordinating Center for Infectious Diseases, Centers for Disease Control and Prevention, 4770 Buford Highway, MS: F22, Chamblee, GA 30341, USA Animal Parasitic Diseases Laboratory, United States Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, BARC-East, Building 1001, 10300 Baltimore Avenue, Beltsville, MD 20705-2350, USA b

a r t i c l e

i n f o

Article history: Received 31 October 2008 Received in revised form 10 March 2009 Accepted 17 March 2009 Available online 24 March 2009 Keywords: Toxoplasma gondii Toxoplasmosis Oocyst Pathogenesis Biology Diagnosis Epidemiology Toxoplasma Waterborne Parasite Protozoa

a b s t r a c t Humans become infected with Toxoplasma gondii mainly by ingesting uncooked meat containing viable tissue cysts or by ingesting food or water contaminated with oocysts from the feces of infected cats. Circumstantial evidence suggests that oocyst-induced infections in humans are clinically more severe than tissue cyst-acquired infections. Until recently, waterborne transmission of T. gondii was considered uncommon, but a large human outbreak linked to contamination of a municipal water reservoir in Canada by wild felids and the widespread infection of marine mammals in the USA provided reasons to question this view. The present paper examines the possible importance of T. gondii transmission by water. Published by Elsevier Inc.

Disclaimer: The findings and conclusions in this report are those of the author(s) and do not necessarily represent the views of the Department of Health and Human Services, the Centers for Disease Control and Prevention or the US Department of Agriculture. 1. Introduction Infection with the protozoan Toxoplasma gondii is one of the most common parasitic infections of man and other warm-blooded animals (Dubey and Beattie, 1988; Tenter et al., 2000; Hill et al., 2002). In most adults it does not cause serious illness, however, blindness and mental retardation can occur in congenitally infected children, severe disease occurs in those with depressed immunity, and ocular disease can occur from acute infection after birth. Toxoplasmosis, until recently, was not often considered a waterborne zoonosis. However, a major outbreak of toxoplasmosis in humans in Canada in 1994 (Bowie et al., 1997) was associated with T. gondii in municipal waters. Recently, T. gondii has been reported in many marine mammals, suggesting the possibility that the contamination of seawater with T. gondii may be more com-

* Corresponding author. Fax: +1 770 488 7761. E-mail address: [email protected] (J.L. Jones). 0014-4894/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.exppara.2009.03.013

mon than realized (Conrad et al., 2005; Dubey and Jones, 2008). This review focuses on the waterborne aspects of T. gondii. 2. Life cycle Toxoplasma gondii is a coccidian parasite where felids are the definitive hosts and warm-blooded animals are intermediate hosts (Frenkel et al., 1970). It is among the most common of parasites of animals and T. gondii is the only known species. Coccidia in general have complex life cycles. Although most are host-specific, and only transmitted by a fecal-oral cycle, T. gondii can also be transmitted transplacentally and by carnivorism. There are three stages of T. gondii that are infectious for all hosts: tachyzoites, bradyzoites, and oocysts. The tachyzoite is often crescent-shaped and 2  6 lm in size. It enters the host cell by active penetration of the cell membrane and becomes surrounded by a parasitophorous vacuole that protects it from host defense mechanisms. The tachyzoite multiplies asexually by repeated endodygeny until the host cell ruptures. After an unknown numbers of divisions, T. gondii tachyzoites give rise to another stage called a tissue cyst. Tissue cysts grow and remain intracellular. They vary in size from 5 to 70 lm and contain a few to several hundred bradyzoites (Dubey et al.,


J.L. Jones, J.P. Dubey / Experimental Parasitology 124 (2010) 10–25 Table 1 Wild felids as definitive host for T. gondii.a Definitive host

Oocyst shedding Expt.


African wild cat (Felis lybica) Amur leopard cat (Felis euptilurus) Asian leopard (Felis bengalensis)

Yesb No Yes Yesb No Yesb No Yesb No Yesb No No No No Yesb No Yesb No Yesb No No No Yesb No No No

No Yesb No No Yesb No Yesb No Yesb No Yesb Yesc Yesb Yesb No Yesd No Yesb No Yes Yesb Yesd No Yesc Yesb Yesb

Bobcat (Lynx rufus) Cheetah (Acinonyx jubatus) Cougar (Felis concolor) Cougar (Felis concolor vancouverensis) Geoffroy’s cat (Oncifelis geoffroyi) Iriomote cat (Felis iriomotensis) Jaguarundi (Felis yagouaroundi) Lion (Panthera leo) Mountain lion (Felis concolar) Ocelot (Felis pardalis) Pallas cat (Felis manul)

Pampas cat (Oncifelis colocolo) Siberian tiger (Panthera tigris altaica) Wild cat (Felis silvestris) a b c d


Polomoshnov (1979) Lukešová and Literák (1998) Janitschke and Werner (1972) Miller et al. (1972) Marchiondo et al. (1976) Miller et al. (1972) Marchiondo et al. (1976) Polomoshnov (1979) Marchiondo et al. (1976) Miller et al. (1972) Aramini et al. (1998) Pizzi et al. (1978) Lukešová and Literák (1998) Akuzawa et al. (1987) Jewell et al. (1972) Ocholi et al. (1989) Polomoshnov (1979) Marchiondo et al. (1976) Jewell et al. (1972) Patton et al. (1986) Basso et al. (2005) (Dubey et al. 1988) Polomoshnov (1979) Pizzi et al. (1978) Dorny and Fransen (1989) Lukešová and Literák (1998)

From Dubey (2009a). Confirmed by bioassay in mice. Confirmed by bioassay in pigs. Immunohistochemical post mortem examination.

1998). Although tissue cysts may develop in visceral organs, including lungs, liver, and kidneys, they are more prevalent in muscular and neural tissues, including the brain, eye, skeletal, and cardiac muscle. The tissue cyst wall is elastic, thin (<0.5 lm), and may enclose hundreds of crescent-shaped slender bradyzoites each measuring 7  1.5 lm. Intact tissue cysts are probably harmless and can persist for the life of the host (Dubey et al., 1998). Upon ingestion by cats, the wall of the tissue cyst is digested by the proteolytic enzymes in the stomach and small intestine, and bradyzoites are released. Some penetrate the lamina propria of the intestine and multiply as tachyzoites. Within a few hours, T. gondii may disseminate to extra-intestinal tissues. The bradyzoites that remain in the epithelial cells of the small intestine initiate the development of numerous generations of T. gondii (Dubey and Frenkel, 1972). Five morphologically distinct asexual types of T. gondii develop in intestinal epithelial cells before the sexual cycle begins. These stages are designated types A–E instead of generations because there are several generations within each T. gondii type. These asexual stages in the feline intestine are structurally distinct from tachyzoites that also develop in the lamina propria. The entroepithelial stages (types A–E, gamonts) are formed in the intestinal epithelium. Occasionally, type B and C schizonts develop within enterocytes that are displaced beneath the epithelium into the lamina propria. Types C, D, and E schizonts multiply by schizogony. In schizogony, the nucleus divides two or more times without cytoplasmic division. The sexual cycle starts 2 days after ingestion of tissue cysts by the cat. The origin of gamonts has not been determined, but the merozoites released from schizont types D and E probably initiate gamete formation. Gamonts occur throughout the small intestine but most commonly in the ileum, 3–15 days after inoculation. The male gamete has two flagellae and swims to and enters the female gamete. After the female gamete is fertilized by the male gamete, oocyst wall formation begins around the fertilized gamete. When oocysts are mature, they are

discharged into the intestinal lumen by the rupture of intestinal epithelial cells. Toxoplasma gondii persists in intestinal and extraintestinal tissue of cats for at least several months, and possibly for the life of the cat. Oocysts of T. gondii are formed only in cats, including both domestic and wild felids (Table 1). Cats shed oocysts after ingesting tachyzoites, bradzyoites, or oocysts. However, less than 50% of cats shed oocysts after ingesting tachyzoites or oocysts whereas nearly all shed oocysts after ingesting tissue cysts. Oocysts in freshly passed feces are unsporulated (non-infective), subspherical to spherical in shape, and 10  12 lm in diameter. Sporulation occurs outside the cat and within 1–5 days, depending upon aeration and temperature. Sporulated oocysts contain two ellipsoidal sporocysts. Each sporocyst contains four sporozoites. The sporozoites are 2  6–8 lm in size (Dubey et al., 1998). Hosts, including felids can acquire T. gondii by ingesting either tissues of infected animals, food and drink contaminated with sporulated oocysts, or by transplacental transmission. After ingestion, bradyzoites released from tissue cysts or sporozoites from oocysts penetrate intestinal tissues, transform to tachyzoites, multiply locally, and are disseminated in the body via blood or lymph. After a few multiplication cycles, tachyzoites give rise to bradyzoites in a variety of tissues. Toxoplasma gondii infection during pregnancy can lead to infection of the fetus. Congenital toxoplasmosis in humans, sheep, and goats can kill the fetus. 3. Estimating oocyst contamination in the environment 3.1. Oocyst shedding during primary and secondary infections Under laboratory conditions domestic cats shed millions of oocysts after feeding on one T. gondii-infected mouse (Dubey and Frenkel, 1972). In one study, cats fed as few as one bradyzoite shed


J.L. Jones, J.P. Dubey / Experimental Parasitology 124 (2010) 10–25

Table 2 Shedding of T. gondii oocysts by cats during primary infection and after challenge with I. felis.a Cat No.

No. of oocysts shed Primary infection

After challenge with I. felis

3 4 5 6 7 8 9 10 11 12

108.19 107.38 107.27 107.50 107.86 108.13 107.14 108.13 107.43 107.15

107.22 106.57 107.17 106.24 106.69 106.34 104 None 106.17 105.21


in Table 2. Isospora felis is a common coccidium of cats. It does not harm the cat but can initiate T. gondii oocyst shedding from a cat chronically infected with T. gondii. How often this happens in nature is not known but we wish to illustrate that many oocysts can be shed by cats. 3.2. Prevalence of oocysts in feces of naturally infected domestic cats

Modified from Dubey (1976).

millions of oocysts, indicating the high reproductive potential of this parasite (Dubey, 2001). Oocysts were shed for less than 3 weeks, mostly for 1 week. Under laboratory conditions, cats acquire good immunity to reshedding of oocysts, but in one study 4 of 9 cats reshed oocysts when they were fed tissue cysts 6 years after primary infection (Dubey, 1995). Concomitant infections, malnutrition, and immunosuppression might also affect reshedding of oocysts (Dubey, 1995, 2009). An example of this is given

How often cats shed oocysts in nature is unknown. Coprological surveys are unrewarding because at any given time less than 1% of cats were found shedding oocysts. Published reports for the last 20 years are summarized in Table 3. Detection of oocysts in feces has several technical problems, including the sensitivity of the methods used to detect and identify T. gondii oocysts in feces. Oocysts of Hammondia hammondi (and possibly other coccidians) shed in cat feces are morphologically similar to T. gondii. Additionally, microscopic examination is likely to miss a low density (less than 1000 oocysts per gram) of oocysts. Detection of DNA from T. gondii oocysts may present additional problems because of inhibitors in fecal matter and difficulty of releasing DNA from the oocysts (Schares et al., 2008a; Salant et al., 2007; Dabritz et al., 2007b). Salant et al. (2007) were able to detect DNA from five T. gondii oocysts in feces experimentally contaminated with oocysts. However, Dabritz et al. (2007b) did not find DNA in feces of three naturally infected cats that contained T. gondii-like oocysts (Table 3). From a

Table 3 Prevalence of T. gondii-like oocysts in feces of cats (principally 1988–2008). Country

Argentina Austria Belgium Brazil Colombia Czec Republic France Germany

India Iran Israel Japan Mexico Mexico New Zealand Nigeria Panama People’s Republic of China Singapore Spain Taiwan Turkey USA

a b c d e

No. of cats

50 1368 30 237 18 143 390 322 264 70a 24,106 2473 2472 441 9 50 100 122 335 200 200 63 52 383 26 722 382 592 96 72 274b 206 450 326 263 34

Grams of feces tested


Litters of kittens from farms. Cats from pig farms. Confirmed by bioassay in mice. Oocysts not found by microscopic examination. Negative by PCR.

Methods Micro



Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes NS Yes Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes

Yes No No Yes No Yes Yes No No No No No No No Yes No No No Yes Yes No No No Yes Yes No No No No No Yes No No No No Yes

No No No Yes No No No No No No Yes No No No No No No Yes No No No No No No No No No No No No No No No Noe No No

No. pos. (%)


1 (2.0)c 27 (2) 0 3 (1.2) 12 (66.6) 0 0 0 0 (17.1) 26 (0.11) 22 (0.9) 26 (1.0) 3 (0.7) 1 (11) 0 0 11 (9)d 1 (0.3) 14 (7.0) 0 0 0 2 (0.5) 0 0 0 0 0 0 5 (1.8) 0 3 (0.7) 3 (0.9) 3 (1.1) 0

Venturini et al. (1992,1997a) Edelhofer and Aspöck (1996) Vanparijs et al. (1991) Pena et al. (2006) Londoño et al. (1998) Dubey et al. (2006) Svobodová et al. (1998) Afonso et al. (2006) Knaus and Fehler (1989) Beelitz et al. (1992) Schares et al. (2008a) Barutzki and Schaper (2003) Epe et al. (1993) Epe et al. (2004) Shastri and Ratnaparkhi (1992) Hooshyar et al. (2007) Sharif et al. (2009) Salant et al. (2007) Oikawa et al. (1990) de Aluja and Aguilar (1977) Guevara Collazo et al. (1990) Langham and Charleston (1990) Umeche (1990) Frenkel et al. (1995) Dubey et al. (2007a) Chong et al. (1993) Miró et al. (2004) Montoya et al. (2008) Lin et al. (1990) Karatepe et al. (2008) Dubey et al. (1995) Hill et al. (2000) Rembiesa and Richardson (2003) Dabritz et al. (2007b) Victor Spain et al. (2001) de Camps et al. (2008)

J.L. Jones, J.P. Dubey / Experimental Parasitology 124 (2010) 10–25 Table 4 Toxoplasma gondii oocysts per gram (opg) of feces in naturally infected cats. Cat type Felis catus

No. of cats 2 1 26 1

Felis concolor


Opg or total 6


7.6  10 1.2  106 1  105 Up to 13  106 106

Ito et al. (1974)

12.5  106 2.4  105

Aramini et al. (1998)

Dubey et al. (1986) Schares et al. (2008a) Oikawa et al. (1990)

public health viewpoint it is necessary to distinguish T. gondii oocysts from oocysts of a related coccidium, H. hammondi present in cat feces. Hammondia hammondi is non-pathogenic (Frenkel and Dubey, 1975). Bioassays, the only definitive way to detect viable oocysts of these parasites currently, are expensive and only a few laboratories in the world have facilities to do them. Although DNA detection is considered highly specific, cross reactivity has been observed between T. gondii and H. hammondi (Schares et al., 2008b). There is little information on the number of oocysts shed by naturally infected cats. Although data in Table 3 indicate that only a few oocysts may be present at any given time, on occasion high numbers of oocysts were detected in cat feces (Table 4).


and drier (rain 20 mm). Rarity of rodents in the area (due to regular rodent poison control), and feeding of cats by people were considered as possible factors for this reduced prevalence. Economic underdevelopment and poor hygiene in a country were not the determining factors for low seroprevalence of T. gondii antibodies in cats in Durango, Mexico (Alvarado-Esquivel et al., 2007; Dubey et al., 2009a). Seroprevalence was 21% of 105 cats among those sampled in 2007, and only 9.3% in 150 cats sampled from the same sources a year later (Table 5); all the cats were tested at a cut-off of 1:25 in MAT and all sera were tested by the same person. This low seroprevalence was attributed to low T. gondii prevalence in the local small animal population; T. gondii was not isolated by bioassays of 249 rats, 127 mice, 69 squirrels, and 66 opossums (Dubey et al., 2009a). In conclusion, factors affecting the prevalence of T. gondii in cats are not fully understood and need further investigation. 3.4. Oocyst shedding by wild felids Wild felids can also shed T. gondii oocysts (Table 1). Serological prevalence in various wild felids is given in Table 6. It should be noted that many wild felids in zoos (and also free-ranging) were found to be seropositive and probably already had shed oocysts. These oocysts can be a source of T. gondii infections not only to the zoo animals but also to zoo workers and visitors, especially children.

3.3. Seroprevalence of T. gondii in domestic cats 3.5. Land and sea contamination by T. gondii oocysts Epidemiological data indicate that most cats become infected with T. gondii after birth by eating T. gondii-infected tissues (Dubey and Beattie, 1988). Nearly all cats fed tissue cysts shed oocysts and most seroconverted by 4 weeks post-inoculation (Dubey and Frenkel, 1972; Dubey and Thulliez, 1989). It is most likely that seropositive cats have already shed oocysts. In our opinion seroprevalence provides a good estimate of environmental contamination with oocysts. Data in Table 5 indicate that up to 80% of cats worldwide had T. gondii antibodies. Although it is not possible to compare results in various surveys because of the sample size, age of cats, and the serological tests used, they do provide a guide of the widespread nature of this infection. Prevalence of T. gondii infection varies according to the life style of cats. It is higher in feral cats that hunt for their food than in domestic cats. Seroprevalence of antibodies to T. gondii varied among countries, within different areas of a country, and within the same city (Table 5). The reasons for these variations are many, no generalizations should be made. For example, low seroprevalence (7.3–11%) in Bangkok, Thailand was attributed to the lifestyle of the cats surveyed. Most (95%) people in Thailand are Buddhist, and killing any pet (or any life) is sinful. There are approximately 15,000 cats (30  500 temples) that live permanently around these Buddhist temples, and these cats are fed by the public or monks, mainly a diet of cooked fish and rice (Sukthana et al., 2003). A somewhat similar situation exists in India. There are only limited data on T. gondii infection in cats from India. Most of the population is vegetarian, and very few people keep cats as pets. In addition, there are many stray dogs in India that keep the stray cat population in check. One of us (JPD) for many years has unsuccessfully tried to obtain samples from stay cats in India, because trapping a cat is considered a sin. We are not aware of any data on T. gondii infection in cats from Africa, except two surveys from Egypt that are approximately 20 years old. Afonso et al. (2006) conducted an excellent study of T. gondii antibody seroprevalence in an urban population of domestic cats in Lyon, France. Unexpectedly, the seroprevalence was only 18.6% of 301 cats, approximately half the prevalence in other surveys in Europe (see Table 5). Prevalence was the highest when the weather was hot (>32 °C) and moist (rain >22 mm), or moderate

Cats are everywhere, except the frozen arctic (Dubey, 2009). For example, approximately one-third of households in the USA own a cat and this number is steadily increasing. There are approximately 78 million domestic cats and 73 million feral cats in the US (Conrad et al., 2005). The fate of cat feces disposed of in the toilet or in domestic trash destined for landfills is unknown. It is anticipated that the heat generated and lack of oxygen in landfills will kill some or all oocysts, depending on the conditions. It is likely that oocysts are carried into our homes on shoes contaminated with oocysts on street pavements. It is probable that nearly every farm in the USA has cats. In one study of pig farms in Illinois, 366 cats were trapped on 43 farms, a mean of 8.5 cats per farm, with a mean of six seropositive cats on each farm (Weigel et al., 1999). Toxoplasma gondii oocysts were detected in cat feces, feed, soil or water samples on six farms (Dubey et al., 1995; Weigel et al., 1999). Thus, there is strong potential for T. gondii transmission in rural settings. If one assumes a 30% seropositivity of 151 (78 domestic and 73 feral) million cats and a conservative shedding of 1 million oocysts per cat then there will be enormous numbers of oocysts (50 million  1 million) in the environment. Dabritz et al. (2007a,b) estimated an annual burden of 94–4671 oocysts/m2 in California. The role of wild felids in contamination of the environment is difficult to assess. The bobcat (Lynx rufus) and cougar (Felis concolor) are the two main wild cats in the continental USA. The number of bobcats in the USA is thought to be in the millions and one study estimated thousands of cougars (Conrad et al., 2005). In a recent survey in Pennsylvania, 83% of 131 bobcats were found to have T. gondii antibodies (Mucker et al., 2006) and viable T. gondii was isolated from five of six bobcats from Georgia (Dubey et al., 2004a). Young and weak white-tailed deer and small mammals are common prey for bobcats and 60% of white-tailed deer in Pennsylvania, USA were seropositive for T. gondii (Dubey and Jones, 2008). In addition to live prey, eviscerated tissues from hunted deer and black bears would be a source of infection for wild cats. Thus, a sylvatic cycle of T. gondii in rural USA is feasible and appears to be efficient.


J.L. Jones, J.P. Dubey / Experimental Parasitology 124 (2010) 10–25

Table 5 Seroprevalence of T. gondii antibodies in domestic cats (1988–2008). Location



No. exam

% Pos.

Cut-off titer


Fernández et al. (1995) Venturini et al. (1995) Venturini et al. (1997a,b)


169 68 100

19.5 25 27

32 8 8

Australia Tasmania Melbourne

Milstein and Goldsmid (1997) Sumner and Ackland (1999)


18 103

50 39

64 NS


Edelhofer and Aspöck (1996)






Samad et al. (1997)





Belgium (Ghent)

Dorny et al. (2002) de Craeye et al. (2008)


346 567

70.2 24.9

40 50

Cavalcante et al. (2006b) Garcia et al. (1999) Dubey et al. (2004) Netto et al. (2003) Mendes-de-Almeida et al. (2007) Lucas et al. (1999) Silva et al. (2002) Meireles et al. (2004) Pena et al. (2006) da Silva et al. (2002)


63 163 58 41 118 248 502 100 237 100

87.3 73 84.4 24.4 72 17.7 26.3 40 35.4 19

25 16 20 NS 16 16 20 NS 25 16

Canada (Ontario)

Quesnel et al. (1997)





Chile Valdivia city

Stutzin et al. (1989) Ovalle et al. (2000)


27 97

85.2 33

16 16


Londoño et al. (1998) Dubey et al. (2006)


28 170

89.3 35.8

NS 20

Czech Republic

Svoboda et al. (1988) Svobodová et al. (1998) Sedlak and Bartova (2006,2007)


620 390 286

48.7 61.5 44.1

4 10 40

Egypt Cairo Gharbia

Aboul-Magd et al. (1988) Abu-Zakham et al. (1989)


177 114

58.8 18.4

64 64

France (Lyon)

Afonso et al. (2006)





Galapagos (Isabela Island)

Levy et al. (2008)






Knaus and Fehler (1989) Tenter et al. (1994) Hecking-Veltman et al. (2001)


267 306 300

59.9 45 65.6

16 NS NS

Guatemala (Petén region)

Lickey et al. (2005)






Hornok et al. (2008)





Haddadzadeh et al. (2006) Hooshyar et al. (2007) Sharif et al. (2009)


100 50 100

63 86 40

32 20 1

Israel (Jerusalem)

Salant and Spira (2004)





Italy Veneto Verona

Natale et al. (2007) D’Amore et al. (1997)


189 490

69.3 33.3

200 64

Maruyama et al. (2003) Khin-Sane-Win et al. (1997) Fujinami et al. (1983) Furuya et al. (1993) Kimbita et al. (2001) Huang et al. (2002) Huang et al. (2004) Oikawa et al. (1990) Nogami et al. (1998) Maruyama et al. (1998)


1447 231 171 726 193 192 179 335 800 471

5.4 19 47.1 10.5 20.7 21.9 18.4 25.7 6 8.7

64 64 64 32 NS 64 64 4 32 64

Afonso et al. (2007)





Sohn and Nam (1999) Kim et al. (2008)


198 174

13.1 8.1

200 32

Lee et al. (2008)




Chandrawathani et al. (2008)




Brazil Amazon Paraná Rio de Janeiro São Paulo

Iran Kashan Sari

Japan Horraido and Okinawa Hyogo Kanto Saitama Saporo and Tokyo Saitama Saitama Various areas

Kerguelen Islands (French territory) Korea (South)


200 (continued on next page)


J.L. Jones, J.P. Dubey / Experimental Parasitology 124 (2010) 10–25 Table 5 (continued) Location



No. exam

% Pos.

Cut-off titer

Garcia-Márquez et al. (2007) Alvarado-Esquivel et al. (2007) Dubey et al. (2009a) Galván Ramirez et al. (1999) Besné-Mérida et al. (2008)


80 105 150 24 169

28.8 21 9.3 70.8 21.8

NS 25 25 NS NS


Shahzad et al. (2006)





Panama (Panama City)

Frenkel et al. (1995)





Yu et al. (2006) Yu et al. (2008) Chen et al. (2005) Yuan et al. (2004) Chen (2001) Lu et al. (1997) Fu et al. (1995) Dubey et al. (2007a) Shen et al. (1990)


128 335 114 75 105 142 200 34 47

14.1 14.9 23.7 57.3 31.4 38.1 46 79.4 2.1

NS NS NS NS NS 64 64 20 64


S´mielewska-Łos´ and Pacon´ (2002) Michalski and Platt-Samoraj (2004)


200 17

52.5 70.6

64 20

Portugal (northeastern)

Lopes et al. (2008)





Puerto Rico (Mona Island)

Dubey et al. (2007b)






Chong et al. (1993)





Slovak Republic

Ondrejka et al. (2007)






Miró et al. (2004) Montoya et al. (2008) Gauss et al. (2003) Millán et al. (2009)


585 592 220 25

32.3 26 45 502

80 80 25 25


Uggla et al. (1990) Ljungström et al. (1994)


244 60

42 46.6



Lin et al. (1990) Tsai et al. (1997)


117 202

7.7 5.5


Thailand Bangkok

Sukthana et al. (2003) Sriwaranard et al. (1981) Jittapalapong et al. (2007)


315 40 592

7.3 57.5 11

16 256 64

Inci et al. (1996) Özkan et al. (2008) Karatepe et al. (2008)


65 99 72

43 40.3 76.4

16 4 16

Dabritz et al. (2007a) Miller et al. (2008) Hill et al. (2000) Luria et al. (2004) Lappin et al. (1992) Lappin et al. (1989) Danner et al. (2007) Dubey et al. (1995) Smith et al. (1992) Hill et al. (1998) Witt et al. (1989) de Camps et al. (2008) Nutter et al. (2004) Dubey et al. (2002) Rodgers and Baldwin (1990) Dubey et al. (2009b) DeFeo et al. (2002) Vollaire et al. (2005)


194 194 5 206 553 124 188 67 391 74 20 585 34 176 275 618 210 200 12628

24.2 36.1 60 23.6 10.8 74.2 60.7 37.7 68.3 41.9 80 15.2 29.4 50.6 48 22 19.5 42 31.6

64 16 320 64 NS NS NS 64 25 32 32 32 25 25 25 16 25 25 NS

Asthana et al. (2006) Dubey et al. (2009c) Moura et al. (2007) Dubey et al. (2009d)


40 176 106 96

35 28.9 84.9 73.9

25 25 25 10

Mexico Colima Durango Guadalajara Mexico City

People’s Republic of China Beijing Guangzhou Hebei Hubei Shanghai Shandong Guangzhou Guangdong

Barcelona Andalusia

Turkey Ankara Nigde USA California

Colorado Florida Florida, Georgia, Ohio Georgia Hawaii Illinois Iowa Maryland Midwestern zoos North Carolina Ohio Oklahoma Pennsylvania Rhode Island Nationwide West Indies Grenada St Kitts

DT, dye test; ELISA, enzyme-linked immunosorbent assay; IFA, indirect fluorescent antibody; IHA, indirect hemagglutination; LAT, latex agglutination test; MAT, modified agglutination test; PCR, polymerase chain reaction; NS, not stated; WB, Western blot.

The role of cougars in the sylvatic cycle of T. gondii has not been established. As stated earlier, a large waterborne outbreak of toxo-

plasmosis in humans was epidemiologically linked to oocyst contamination of a water reservoir in British Columbia, Canada (Bowie


J.L. Jones, J.P. Dubey / Experimental Parasitology 124 (2010) 10–25

Table 6 Seroprevalence of T. gondii antibodies in wild felids. Species




No. exam

% Pos.

Panthera spp. P. tigris (tiger)

Brazil Thailand USA Florida Various areas

Silva et al. (2001a) Thiangtum et al. (2006)


2 18

100 27.8

Cut-off titer 20 64

Lappin et al. (1991) Spencer et al. (2003)


4 11

75 63.6

64 50

P. t. altaica (Amur tiger)

USA - Mid western zoos

de Camps et al. (2008)





P. leo (lion)

Botswana Brazil Southern Africa

Penzhorn et al. (2002) Silva et al. (2001a) Cheadle et al. (1999) Spencer and Morkel (1993) Penzhorn et al. (2002) Thiangtum et al. (2006)


53 27 41 66 42 7

92 51.8 90.2 98 100 14.3

20 20 50 NS 20 64

Lappin et al. (1991) de Camps et al. (2008) Spencer et al. (2003) Penzhorn et al. (2002) Hove and Mukaratirwa (2005)


2 22 10 21 26

100 54.5 80 100 92.3

64 25 50 20 25

Silva et al. (2001a) Penzhorn et al. (2002) Cheadle et al. (1999) Penzhorn et al. (2002) Thiangtum et al. (2006)


3 1 4 7 19

100 100 75 86 15.8

20 20 50 20 64

Spencer et al. (2003) Lappin et al. (1991) de Camps et al. (2008)


1 3 1

100 66.6 100

50 64 25

Thailand USA Florida Mid western zoos Various areas Zimbabwe P. pardus (leopard)

P. onca (jaguar)

P. uncia (snow leopard, panthera snow)

Lynx spp. L. rufus (bobcat)

Brazil Botswana Southern Africa South Africa Thailand USA California Florida Mid western zoos French Guiana Brazil Thailand USA Various areas Mid western zoos

Demar et al. (2008) Silva et al. (2001b) Thiangtum et al. (2006)


1 212 3

100 63.2 33.3

4000 20 100

Spencer et al. (2003) de Camps et al. (2008)


2 1

100 100

50 25

USA Mid western zoos Thailand

de Camps et al. (2008) Thiangtum et al. (2006)


14 1

35.7 100

25 64

Labelle et al. (2001) Kikuchi et al. (2004)


10 6

40 66.6

25 64

Kikuchi et al. (2004) Riley et al. (2004) Miller et al. (2008) Dubey et al. (2004) Smith and Frenkel (1995) Mucker et al. (2006) Spencer et al. (2003)


52 25 3 6 2 131 3

50 88 100 83.3 50 83 333

64 64 320 25 8 25 50

Labelle et al. (2001)





Spencer et al. (2003)





Silva et al. (2001a) Philippa et al. (2004) Labelle et al. (2001) Ryser-Degiorgis et al. (2006)


1 5 106 207

100 20 44 75

20 NS 25 40

Sobrino et al. (2007) Roelke et al. (2008) Millán et al. (2009)


27 57 26

81.5 44 80.7

25 64, 25 25

Spencer et al. (2003) de Camps et al. (2008)


1 4

100 50

50 25

Cheadle et al. (1999) Thiangtum et al. (2006)


23 1

43.4 100

50 64

Spencer et al. (2003)



77.7 50 (continued on next page)

Canada Québec Mexico USA California

Georgia Kansas, Missouri Pennsylvania Various areas L. canadiensis (lynx)

L. lynx (Eurasian lynx)

Canada Québec USA California Brazil Canada Sweden

L. pardinus (Iberian lynx, polecat)


L. caracal (Caracal caracal)

USA California Mid western zoo

Acinonyx spp. Acinonyx jubatus (cheetah)

Southern Africa Thailand USA Various areas


J.L. Jones, J.P. Dubey / Experimental Parasitology 124 (2010) 10–25 Table 6 (continued) Species

Location Florida Mid western zoos

Felis spp. F. concolor (cougar, puma, or Florida panther, mountain lion)

Brazil Canada Central and South America Mexico USA California

Florida Mid western zoos Various areas F. c. vancouverensis

Canada Vancouver Island



No. exam

% Pos.

Stover et al. (1990) de Camps et al. (2008)


16 22

68.7 27.3

Cut-off titer 64 25

Silva et al. (2001b) Kikuchi et al. (2004) Philippa et al. (2004) Kikuchi et al. (2004) Kikuchi et al. (2004) Kikuchi et al. (2004) Paul-Murphy et al. (1994) Spencer et al. (2003) Miller et al. (2008) Roelke et al. (1993) Lappin et al. (1991) de Camps et al. (2008) Spencer et al. (2003)


172 23 15 83 12 320 36 42 26 38 6 8 5

48.3 34.8 7 32.5 16.7 19.1 58 25.5 92.3 9 83.3 62.5 60

20 64 NS 64 64 64 32 50 40 48 64 25 50

Stephen et al. (1996) Aramini et al. (1998)


5 12

100 92

40 25

F. chaus (jungle cat)


Silva et al. (2001a)





F. euptilurus (Amur leopard cat)

USA Midwestern zoos

de Camps et al. (2008)





F. margarita (sand cat)

United Arab Emirates

Pas and Dubey (2008b)


6 4

100 75

25 64

F. manul (Otocolobus manul), Pallas cat

Austria USA Colorado Midwestern zoos Ohio Oklahoma Wisconsin

Basso et al. (2005)





Kenny et al. (2002) de Camps et al. (2008) Swanson (1999) Ketz-Riley et al. (2003) Dubey et al. (1988)


4 5 14 6 3

100 20 79 100 67

32 25 NS 2048 64

F. lynx

USA Alaska

Zarnke et al. (2001)





F. silvestris (wild cat)

UK Spain

Yamaguchi et al. (1996) Sobrino et al. (2007)


45 6

62 50

16 25

F. s. gordoni (Gordon’s cat)

United Arab Emirates

Pas and Dubey (2008a)





F. serval (Leptailurus serval, serval)

Brazil USA Florida Various areas

Silva et al. (2001a)





Lappin et al. (1991) Spencer et al. (2003)


2 3

50 33.3

64 50


Thiangtum et al. (2006)





USA – Mid western zoos Thailand

de Camps et al. (2008) Thiangtum et al. (2006)


4 27

25 22.2

25 64

Bolivia Chaco Brazil

Fiorello et al. (2006) Silva et al. (2001b)


8 12

25 75

NS 20

O. colocolo (Pampas cat)


Silva et al. (2001b)





Leopardus spp. L. pardalis (ocelot)

Bolivian Chaco Brazil USA – Alabama

Fiorello et al. (2006) Silva et al. (2001b) Spencer et al. (2003)


10 168 1

100 57.7 100

NS 20 50

L. tigrinus (oncilla)


Silva et al. (2001b)





L. wiedii (margay)


Silva et al. (2001b)





Neofelis nebulosa (clouded leopard)

USA – California Mid western zoos Thailand

Spencer et al. (2003) de Camps et al. (2008) Thiangtum et al. (2006)


2 7 16

50 14.3 12.5

50 25 64

Herpailurus yogouaroundi (jaguarundi)

Brazil USA – Florida

Silva et al. (2001b) Lappin et al. (1991)


99 1

45.9 100

20 64

F. temmincki (asian golden cat, golden cat) F. viverrinus (fishing cat)

Oncifelis spp. O. geoffroyi (Geoffroy’s cat)

DT, dye test; ELISA, enzyme-linked immunosorbent assay; IFA, indirect fluorescent antibody; IHA, indirect hemagglutination; LAT, latex agglutination test; MAT, modified agglutination test; NS, not stated.

et al., 1997). Although oocysts were not detected in drinking water taken from the reservoir after the outbreak (Isaac-Renton et al.,

1998), viable oocysts were detected in rectal contents (sample A) of a wild trapped cougar (Table 4) and in a fecal pile (sample B) in


J.L. Jones, J.P. Dubey / Experimental Parasitology 124 (2010) 10–25

the vicinity of the reservoir (Aramini et al., 1998). It is noteworthy that many oocysts were present in feces of cougars (Table 4). Dabritz et al. (2007a,b) considered that the level of land contamination stated above is likely to be high enough for oocysts to reach marine waters (Dabritz et al., 2007a,b), where mammals such as sea otters can become infected. Sea otters eat approximately 25% of their body weight in invertebrate prey each day (Conrad et al., 2005). Cold blooded animals, including fish, are not hosts for T. gondii. With respect to oocysts, it is not known if the sporozoite excysts after ingestion by cold blooded animals. Mollusks, however, can act as transport hosts for T. gondii oocysts (Arkush et al., 2003; Lindsay et al., 2004; Miller et al., 2008). In addition, sea otters might ingest oocysts directly from marine water, but how much marine water is cycled through the gut of sea otters in a day is unknown (it is likely to be large quantities). Antibodies to T. gondii were found in a variety of marine mammals including sea otters, dolphins, seals and walruses. Prevalence of T. gondii antibodies in sea otters was high but varied between 47% and 100%, depending on the category (live versus dead), source (California versus Washington), and the serological test used (see Dubey and Jones, 2008). A very high seroprevalence of T. gondii antibodies was found in the bottlenose dolphin (Tursiops truncatus) from California, Florida, North Carolina and South Carolina, indicating that marine mammals on both coasts of the USA are exposed to T. gondii.

4. Direct detection of T. gondii oocysts in water Detection of T. gondii oocysts in environmental samples (soil, water, feed) is technically difficult. Additionally, T. gondii oocysts are highly infective for humans. Dubey (2009) has described procedures for recovering oocysts from cat feces and safety precautions for handling samples potentially contaminated with T. gondii. There is no rapid detection method for T. gondii oocysts in water or other environmental samples. Traditionally, detection of protozoa in water required their concentration from large volumes of water by filtration or centrifugation, isolation from concentrated particulates by immunomagnetic separation (IMS) or other methods, and detection by immunofluorescence microscopy, infection of cultured cells, biochemistry, bioassays, molecular techniques or combinations of these (Dumètre and Dardé, 2003; Zarlenga and Trout, 2004). For T. gondii oocysts, there are no commercially available IMS techniques, no widely available immunofluorescent staining reagents, and no standardized cultivation protocols. Identification of oocysts from environmental samples has included differential floatation and mouse inoculation (Isaac-Renton et al., 1998), filtration through micropore filters, and bioassays (de Moura et al., 2006). Recently, IMS techniques have been developed for the isolation of T. gondii oocysts and sporocysts in water (Dumètre and Dardé, 2005, 2007). Both the oocyst and sporocyst IMS assays, however, had poor specificity because antibodies cross-reacted with water debris and the sporocyst wall of H. hammondi, Hammondia heydorni, and Neospora caninum (Dumètre and Dardé, 2007). Schares et al. (2008b) recently developed new primers to distinguish H. hammondi and T. gondii oocyst DNA. PCR is becoming a favored technique for detection of T. gondii oocysts in water (Jones et al., 2000; Kourenti and Karanis, 2004, 2006; Schwab and McDevitt, 2003; Sroka et al., 2007; Villena et al., 2004) over the conventional mouse bioassay (Isaac-Renton et al., 1998; Villena et al., 2004), as it reduces the detection time from weeks to 1–2 days. Although they have been developed for detection of T. gondii in clinical specimens (Switaj et al., 2005), no real-time PCR assays have been adapted for detection of oocysts in water samples, possibly because of expected high concentra-

tions of PCR inhibitors and low numbers of T. gondii oocysts in environmental samples (Villena et al., 2004). There are several unresolved issues regarding the effectiveness of PCR detection of T. gondii oocysts in water. The most readily available method for isolation of T. gondii oocysts from water samples is flocculation or sucrose floatation prior to DNA extraction (Kourenti and Karanis, 2004, 2006; Sroka et al., 2007; Villena et al., 2004). Because sucrose flotation and flocculation result in oocyst losses, the recovery rate of using these methods is poor. For DNA extraction, the phenol–chloroform method or QIAamp(r) mini kit is frequently used (Dumètre and Dardé, 2007; Kourenti and Karanis, 2004, 2006; Schwab and McDevitt, 2003; Villena et al., 2004). When oocysts are recovered from water either by the EPA Information Collection Rule (ICR) method (USEPA, 1995) or EPA Method 1623 (USEPA, 2001) without purification by IMS, neither the conventional phenol chloroform DNA extraction nor the QIAamp(r) mini kit are effective at removing PCR inhibitors (Jiang et al., 2005; Villena et al., 2004; Xiao et al., 2000). Borchardt et al. (2009) recently reported a continuous separation channel centrifugation for concentrating T. gondii and Cyclospora cayetanensis oocysts from surface water and drinking water.

5. Environmental resistance of oocysts Toxoplasma gondii oocysts are highly resistant to environmental influences, including freezing (Tables 7 and 8). Oocysts are not killed by chemical and physical treatments currently applied in water treatment plants, including chlorination, ozone treatment, and ultraviolet rays. 6. Epidemiology Toxoplasma gondii has been known to be spread by food and soil for many years. Oocysts can survive for years in the soil (Frenkel et al., 1975), and it makes intuitive sense that oocysts can wash into bodies of water from the soil. Toxoplasma gondii oocysts survive up to 54 months in cold water (Dubey, 1998), so drinking unfiltered water contaminated with T. gondii can lead to infection. Toxoplasma gondii oocysts are about 10–12 lm in size, and parasites this size or smaller (for example Cryptosporidium spp. or Giardia spp.) should be filtered out by most normally operating municipal water filtering systems in developed countries that use coagulation, flocculation, and settling prior to filtration (Betancourt and Rose, 2004), but not necessarily by filters in small water systems or malfunctioning water filters. Water has been implicated in outbreaks of toxoplasmosis for many years. In 1979, an outbreak occurred in US Army soldiers during a 3-week training exercise in Panama (Benenson et al., 1982). Drinking unfiltered-, iodine treated-, water from streams contaminated by jungle cats was implicated as the source of infection; for food, the battalion used only canned rations supplemented by occasional fruit. As noted above in the section on contamination by oocysts, unfiltered water was implicated as the source of a large toxoplasmosis outbreak in the Victoria area of British Columbia in 1995 (Bowie et al., 1997; Burnett et al., 1998). Of the 100 persons who met the outbreak case definition, 19 had retinitis, 51 had lymphadenopathy, 4 had other symptoms consistent with toxoplasmosis, 7 had other symptoms, and 18 were symptom free but were identified as case-patients by serologic methods (for 1 case-patient, symptoms were not ascertained). Mapping (Eng et al., 1999) and case-control studies implicated one of the reservoirs supplying water to the Greater Victoria area. Peaks in coliform counts and turbidity in the reservoir water were associated with excessive rainfall and runoff into the reservoir, and each peak preceded a peak in the epidemic curve. Based on


J.L. Jones, J.P. Dubey / Experimental Parasitology 124 (2010) 10–25 Table 7 Effect of disinfectants on T. gondii oocysts.e Reagent

Concentration (%)

Duration of treatment



Formalin Sulfuric acid + dichromate

10 63/7 63/7 95/5 95/5 5.0 5.0 6.0 0.1 0.1 0.1 5.5 5.5 2.0 2.0 7.0 33

48 h 30 min 24 h 1h 24 h 10 min 30 min 24 h 24 h 24 h 24 h 1h 3h 10 min 3h 10 min 24 h 24 h 24 h 11 days 2 days 1h 3h 24 h 48 h 24 h 12 min 20 min

No No Yes No Yes No Yes No No No No No Yes No Yes Yes No No No Yes No No Yes No Yes No No No Yes/No Yes/No

Ito et al. (1975) Dubey et al. (1970)

Ethanol + acetic acid Ammonium hydroxide Sodium hyporchlorite (Purex) Sodium lauryl sulfate Cetyl trimethyl ammonium Tween 80 Ammoniaa, liquid Tincture of iodine

Aldesolb Tincture of Hibiseptc Izosan-Gd Drying at relative humidity Lomasept Neo Kurehasol Paracetic acid Chlorination of water Ozone treatment of water Ultravoilet irradiation a b c d e

0.02 19 0 1 5 5 100 mg/L 6 mg/L 9.4 mg/L >500 mJ/cm2 40 mJ/cm2

Dubey et al. (1970) Dubey et al. (1970) Dubey et al. (1970) Dubey et al. (1970) Dubey et al. (1970) Dubey et al. (1970) Dubey et al. (1970) Frenkel and Dubey (1972) Frenkel and Dubey (1972) Frenkel and Dubey (1972) Frenkel and Dubey (1972) Frenkel and Dubey (1972) Kuticˇic´ and Wilkerhauser (1993) Kuticˇic´ and Wilkerhauser (1993) Kuticˇic´ and Wilkerhauser (1993) Frenkel and Dubey (1972) Frenkel and Dubey (1972) Ito et al. (1975) Ito et al. (1975) Ito et al. (1975) Ito et al. (1975) Wainwright et al. (2007a) Wainwright et al. (2007a) Dumetre et al. (2008) Wainwright et al. (2007b) Dumetre et al. (2008)

Undiluted household ammonia. Aldesol, a solution for disinfection, contains 5 g benzalchoniumchloride, 6 g glutaraldehyde, and 8 g gloxal in 100 g of solution. Hibisept tincture contains 0.5 g chorhexidine gluconate in 70% ethanol in 100 ml of tincture. Izosan-G granulate contains 99 g of sodium dichloroizicyanurate-dihydrate in 100 g granulate. Modified from Dubey (2004).

Table 8 Effect of environmental influences on T. gondii oocysts. Temperature (°C) Indoors


Duration of treatment



21 4 4 10 15 20–22 30 35 40 40 45 50 52 55

Water 2% H2SO4 Water Water Water Water Water Water Water Water Water Water Water Water

60 Texas 6.5–37.5 Kansas 20–35 Costa Rica 15–30

Native feces Native feces Native feces

28 days 578 days 1620 days 200+ days 200+ days 548 days 107 days 32 days 28 days 24 h 3 days 60 min 5 min 1 min 2 min 1 min 334–410 days 548 days 56–357 days

No No No No No No No No Yes No No No No No Yes Yes No No No

Frenkel and Dubey (1973) Dubey (1977) Dubey (1998) Dubey (1998) Dubey (1998) Hutchison (1967) Dubey (1998) Dubey (1998) Dubey (1998) Dubey (1998) Dubey (1998) Dubey et al. (1970) Dubey (1998) Dubey (1998) Dubey (1998) Dubey (1998) Yilmaz and Hopkins (1972) Frenkel et al. (1975) Frenkel et al. (1975)

the rates of infection in screened pregnant women and persons selected as potential controls, an estimated 2894–7718 individuals in the Greater Victoria area were infected with T. gondii (Bowie et al., 1997). It was hypothesized that domestic cats (Felis catus) or cougars (Felis concolor) contaminated the reservoir (Aramini et al., 1999). An investigation of the Victoria watershed determined the presence of an endemic T. gondii cycle. Domestic cats and cougars

were observed in the watershed, both showed serologic evidence of T. gondii infection, and cougars were found to be shedding T. gondii oocysts (Aramini et al., 1998, 1999). As a result of this investigation, researchers developed a method for detection of T. gondii oocysts in water based on the US Environmental Protection Agency method for detection of Cryptosporidium oocysts (Isaac-Renton et al., 1998). However, they were not able to detect


J.L. Jones, J.P. Dubey / Experimental Parasitology 124 (2010) 10–25

oocysts in the reservoir water. The reservoir was drained after the outbreak. Since the 1990s waterborne T. gondii has been implicated in numerous outbreaks. In Santa Isabel do Ivai, Brazil, waterborne T. gondii was thought to be responsible for an outbreak involving 155 persons served by an underground tank reservoir delivering unfiltered water (de Moura et al., 2006). It was postulated that runoff contaminated with T. gondii oocysts entered the reservoir because a cat with a litter of kittens lived on top of the underground tank reservoir, which was not adequately sealed. Water from the reservoir that was stored in a school holding tank was found to be contaminated with T. gondii identified by bioassay and PCR. As a result of the investigation, the reservoir was closed and a new reservoir constructed. Further epidemiological studies indicated that toxoplasmosis was endemic in cats in that area. Toxoplasma gondii antibodies were found in 84% of 58 cats trapped in the town of Santa Isabel do Ivai, and viable T. gondii was isolated from 73.4% of them (Dubey et al., 2004b, see Table 5). Waterborne toxoplasmosis has been suggested or implicated in numerous other investigations in many areas of the world, including: Brazil (Heukelbach et al., 2007 [homemade ice]; Amendoeira et al., 2003; Cavalcante et al., 2006a; Bóia et al., 2008), Columbia (López-Castillo et al., 2005), India (Palanisamy et al., 2006; Hall et al., 1999), Poland (Paul, 1998), Turkey (Ertug et al., 2005), Cuba (Martínez Sáchez et al., 1991), Granada (Asthana et al., 2006), Taiwan (Hung et al., 2007), and China (Lin et al., 2008). Water has also been implicated as a source of endemic T. gondii infection in Brazil and Guatemala. Drinking unfiltered water was associated with an increase in T. gondii antibody prevalence in both lower and middle socio-economic groups in Rio de Janeiro state, Brazil (Bahia-Oliveira et al., 2003), and drinking unfiltered well water was found to be associated with a higher T. gondii prevalence in rural Guatemalan children (Jones et al., 2005). In addition, in a Polish farm population, there was a positive correlation between the consumption of unboiled well water and the presence of T. gondii antibodies (Sroka et al., 2006). Toxoplasma gondii was identified by both microscopic and PCR testing in some of the samples collected from wells on the Polish farms. Although studies in the literature have examined drinking water as a risk factor for T. gondii infection, one wonders if recreational exposure to contaminated rivers, lakes, streams, or ponds (with inadvertent swallowing of water) also periodically results in infection. The results of some epidemiologic studies in industrialized countries examining meat and soil-related risk factors for T. gondii infection indicated that a large proportion of human infections cannot be explained by contaminated food or soil exposure (Cook et al., 2000; Boyer et al., 2005). It is possible that ingestion of inadequately treated water or inadvertent ingestion of recreational water from streams, lakes, or ponds could explain some human infections. 7. Clinical toxoplasmosis in humans Water contaminated with T. gondii has the potential to spread infection to a large number of people, as illustrated by the outbreaks in Canada (Bowie et al., 1997) and Brazil (de Moura et al., 2006) described above. Acute infection in a previously uninfected pregnant woman can lead to congenital disease with retinochoroiditis, neurologic, or systemic disease occurring in the infant. Acute T. gondii infection can also lead to ocular lesions in up to 2% of those infected (Holland, 2003), some resulting in loss of vision. In addition, T. gondii leads to a chronic infection that can reactivate causing severe or even fatal encephalitis or other systemic disease in immunocompromised persons (Luft et al., 1983, 1984). Some outbreaks with water implicated as a mode of transmission have led to high attack rates, suggesting that the dosage of oocysts can be quite high (or the organism was particularly virulent)

(Benenson et al., 1982). The elimination of T. gondii contamination of water can potentially result in prevention of significant morbidity and mortality. 8. Prevention and control Although the level of contamination is not usually known, several measures can help prevent T. gondii infection from water. People should avoid ingesting untreated water from streams, lakes, rivers, or ponds, even during recreational activities. If untreated surface water is to be consumed or it is the main source of drinking water, filtering (absolute 1 lm filter) or boiling will eliminate T. gondii. Chlorine treatment will not inactivate T. gondii, however tincture of iodine (2%) will inactivate T. gondii with a long (3 h) exposure (Table 7). When possible, access of cats to the areas around drinking water tanks or reservoirs should be limited. In developed countries, cat feces should not be flushed into the municipal sanitation system, but rather bagged and disposed in trash that will be deposited in a landfill. In addition, public water supplies that could be contaminated by T. gondii or other parasites should be filtered as part of the treatment process sufficiently to eliminate Cryptosporidium spp. (3–5 lm), which should also eliminate T. gondii oocysts (10–12 lm). Travelers should also avoid drinking untreated or inadequately treated water and be aware that in some areas of the world municipal water in not adequately treated or filtered. Where possible, cat owners should be encouraged to keep their cats indoors in order to reduce hunting for rodents and birds, which leads to feline infection and oocyst shedding. In addition, cats should not be feed raw or undercooked meat. For further information, general guidelines for prevention of T. gondii infection have been published (Lopez et al., 2000). To help prevent environmental contamination with oocysts, future studies could focus on optimal methods to filter T. gondii in municipal water and methods to inactivate T. gondii in cat litter or bind the feces so they do not disperse in the environment. Another area for further study is the possible impact of recreational water use and inadvertent swallowing of water on acquisition of T. gondii infection. Finally, newer approaches to water treatment (for example, improved filtering methods, or new methods of chemical inactivation) could be explored further. Acknowledgment We thank Dr. L. Xiao for his help with methods to detect oocysts in environmental samples. References Aboul-Magd, L.A., Tawfik, M.S., Arafa, M.S., El-Ridi, A.M.S., 1988. Toxoplasma infection of cats in Cairo area as revealed by IFAT. Journal of the Egyptian Society of Parasitology 18, 403–409. Abu-Zakham, A.A., El-Shazly, A.M., Yossef, M.E., Romela, S.A., Handoussa, A.E., 1989. The prevalence of Toxoplasma gondii antibodies among cats from Mahalla ElKobra, Gharbia Governorate. Journal of the Egyptian Society of Parasitology 19, 225–229. Afonso, E., Thulliez, P., Gilot-Fromont, E., 2006. Transmission of Toxoplasma gondii in an urban population of domestic cats (Felis catus). International Journal for Parasitology 36, 1373–1382. Afonso, E., Thulliez, P., Pontier, D., Gilot-Fromont, E., 2007. Toxoplasmosis in prey species and consequences for prevalence in feral cats: not all prey species are equal. Parasitology 134, 1963–1971. Akuzawa, M., Mochizuki, M., Yasuda, N., 1987. Hematological and parasitological study of the Iriomote cat (Prionailurus iriomotensis). Canadian Journal of Zoology 65, 946–949. Alvarado-Esquivel, C., Liesenfeld, O., Herrera-Flores, R.G., Ramírez-Sánchez, B.E., González-Herrera, A., Martínez-García, S.A., Dubey, J.P., 2007. Seroprevalence of Toxoplasma gondii antibodies in cats from Durango City, Mexico. Journal of Parasitology 93, 1214–1216. Amendoeira, M.R.R., Sobral, C.A.Q., Teva, A., de Lima, J.N., Klein, C.H., 2003. Inquérito sorológico para a infecção por Toxoplasma gondii em ameríndios

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