Toxoplasmosis in Animals in the Czech Republic – The Last 10 Years

In humans, clinical form of toxoplasmosis is rare in immunocompetent people, while it may leads to eye diseases, CNS or generalized infection in immunocompromissed individuals as well as interfere with the course or outcome of pregnancy. In Europe, T. gondii seroprevalence in humans ranges from 8% to 77% (Dubey 2010). In the Czech Republic, T. gondii antibodies were detected in 35% and 25% pregnant women by Sabin-Feldman Test (SFT) and Complement Fixation Test (CFT), respectively (Hejlicek et al. 1999). Repeated prevalence studies in humans in some European countries (France, Belgium, Sweden and Norway), revealed an evident trend of a decrease in T. gondii seroprevalence (Welton and Ades 2005). The same trend is observed in the Czech Republic. The prevalence of infection varies among ethnic groups due to sanitary and cooking habits. Consumption of raw or almost raw, dried, cured or smoked meat from domestic animals, unpasteurized goat milk or consumption of meat from wild animals may be associated with ingestion of the parasite (Kijlstra and Jongert 2008, Jones et al. 2009). Higher prevalence was found also in people who had frequent contact with animals and soil, such as abattoir workers, garbage handlers and waste pickers (Dubey and Beattie 1988). Children playing with dogs and cats can be infected by direct contact because animals can act as mechanical vectors (Etheredge et al. 2004).


Introduction
Toxoplasmosis is a significant zoonosis that affects humans and warm blooded animals. The definitive hosts of parasite Toxoplasma gondii are cats and other felids. Many species of domestic, wild or zoo animals may serve as intermediate hosts.
In humans, clinical form of toxoplasmosis is rare in immunocompetent people, while it may leads to eye diseases, CNS or generalized infection in immunocompromissed individuals as well as interfere with the course or outcome of pregnancy. In Europe, T. gondii seroprevalence in humans ranges from 8% to 77% (Dubey 2010). In the Czech Republic, T.
gondii antibodies were detected in 35% and 25% pregnant women by Sabin-Feldman Test (SFT) and Complement Fixation Test (CFT), respectively (Hejlicek et al. 1999). Repeated prevalence studies in humans in some European countries (France, Belgium, Sweden and Norway), revealed an evident trend of a decrease in T. gondii seroprevalence (Welton and Ades 2005). The same trend is observed in the Czech Republic. The prevalence of infection varies among ethnic groups due to sanitary and cooking habits. Consumption of raw or almost raw, dried, cured or smoked meat from domestic animals, unpasteurized goat milk or consumption of meat from wild animals may be associated with ingestion of the parasite (Kijlstra and Jongert 2008, Jones et al. 2009). Higher prevalence was found also in people who had frequent contact with animals and soil, such as abattoir workers, garbage handlers and waste pickers (Dubey and Beattie 1988). Children playing with dogs and cats can be infected by direct contact because animals can act as mechanical vectors (Etheredge et al. 2004).
In animals, T. gondii infection is a frequent cause of early embryonic death and resorption, fetal death and mummification, abortion, still birth and neonatal death. Thus, toxoplasmosis in domestic and farm animals is a disease of great importance for veterinary medicine and husbandry since it can cause productive and economic losses.
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Toxoplasmosis -Recent Advances 56 T. gondii antibodies have been found in animals worldwide. Seroprevalence to T. gondii varies among countries, within different areas of a country and within the same city. Dubey (2010) summarized the results of seroprevalence studies performed on different groups of animals from several countries.
In the Czech Republic, some important studies concerning T. gondii in animals were done in past years. The seroprevalence of T. gondii infection in domestic animals obtained by different serological methods is summarized in Table 1 In a group of game animals, a prevalence of 15% was found in wild boars by SFT (Hejlicek et al. 1997), 4 -31% in hares by SFT or Microprecipitation in Agar (MPA) (Havlik and Hubner 1958, Zastera et al. 1966, Vosta et al. 1981, Hejlicek et al. 1997, and 14 -58% prevalence in wild ruminants by SFT (Havlik and Hubner 1958, Zastera et al. 1966, Hejlicek et al. 1997).
These studies were performed by one or by a combination of methods such as SFT, CFT, MPA and Indirect Hemaglutination Assay (IHA). Nowadays, these methods are less frequently used; it is preferred to use Modified Agglutination Test (MAT), and/or Indirect Fluorescent Antibody Test (IFAT), and/or an Enzyme-Linked Immunosorbent Assay (ELI-SA), and/or a Latex Agglutination Test (LAT). This trend is also evident from a recent review summarized worldwide prevalence of T. gondii infection in animals and humans (Dubey 2010).
Based on the results of examination of different groups of animals in the State Veterinary Institute Prague in years 2003 -2006, it is evident that lethal toxoplasmosis in the Czech Republic is the most important in some species of zoo animals; while in domestic animals it was not proved . Contrary, the sera of cats and dogs were the most frequently examined. Insufficient attention is paid to small ruminants that can abort or have reproduction disorders due to toxoplasmosis with subsequent economic losses.
That is why during the last 10 years, our research team focused on T. gondii serosurveys in different groups of animals to obtain actual data and to evaluate which group of animals is the most affected by T. gondii infection. Following parts of chapter summarises the results of seroprevalence studies in domestic, game and zoo animals tested by using IFAT, ELISA and LAT with the possibility to compare the results with those obtained from other countries with the same methods used. The results of experimental studies and cases of clinical toxoplasmosis recorded in the Czech Republic are mentioned too.

Serological studies
During years 1995-2012, the samples of blood were collected from different groups of animals and examined for specific T. gondii antibodies. The animals tested for T. gondii antibodies were clinically healthy, no case of abortion or other symptoms of toxoplasmosis were recorded. The blood samples were collected by veterinarians on farms, zoo or during hunting seasons and sent to State Veterinary Institute Prague for routine examination.
In a group of domestic animals, in total 4254 animals were tested with the following number of animals used: 286 cats, 413 dogs, 547 sheep, 251 goats, 546 cattle, 551 pigs, 552 horses and 1108 poultry (217 chickens and 293 broilers, 60 turkeys, 178 geese and 360 ducks). The animals came from 2 -14 different districts of the Czech Republic ( Figure 1).  Sera of domestic animals were tested for T. gondii antibodies by an indirect fluorescent antibody test (IFAT), using the Sevatest Toxoplasma Antigen IFR (Sevac, Prague, Czech Republic) and specific conjugates, by an ELISA (Institut Pourquier, Montpellier, France), or by a latex agglutination test (Pastorex TM Toxo, Biorad, France). The data on the method and cut-off used, specific conjugate for IFAT and producer are summarized in Table 2.
In a group of domestic animals, T. gondii antibodies were found in 66% goats, 59% sheep, 44% cats, 36% pigs, 26% dogs, 23% horses, 12% poultry (43% goose, 14% ducks, and 0.3% in broiler; turkeys and chickens were negative) and 9.7% cattle. The results of serological examination including the number of samples tested, the method and cut-off used, the number and percentage of positive samples, titres or %S/P obtained in positive samples and reference about published data are summarized in Table 3.

Experimental studies
In the Czech Republic, two experimental studies were conducted on domestic poultry.
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The first study was conducted on chickens (Gallus domesticus) that were inoculated per os with two different doses of T. gondii oocysts (Sedlak et al. 2000b). Antibodies to T. gondii were detected by IFAT first on day 14 p.i.; all chickens were serologically positive on days 21 and 28 p.i. No clinical symptoms were recorded. Parasite T. gondii was isolated from heart, muscle, spleen and brain. In one case, no T. gondii was isolated from any organ. Based on this experiment chickens seems highly resistant to T. gondii infection.
The second experimental study was conducted on domestic ducks ( Bartova et al. 2004). Ducks were inoculated per os with different doses of T. gondii oocysts. Antibodies to T. gondii were detected in all ducks by IFAT on day 7 p.i. Antibody titres were found in the range of 20-640 depending on the infectious dose of the oocysts. From day 14 p.i., antibody titres increased to 80-20 480. Bioassay in mice revealed T. gondii in the breast and leg muscles, heart, brain, liver and stomach. The infected ducks showed no clinical symptoms, however, the results of bioassay indicate that, compared to gallinaceous birds, domestic ducks are relatively susceptible to T. gondii infection.

Comparison of data obtained
Cats Clinical signs of toxoplasmosis in cats include fever, anorexia, dyspnea, uveitis, pneumonitis and others. Kittens can develop acute toxoplasmosis and die from it. The seropositivity increases with the age of cat, indicating postnatal transmission of infection. T. gondii antibodies have been found worldwide (Dubey 2010). Seroprevalence varies among countries, within different areas of a country and within the same city. In Europe, the highest prevalence 76% was found by SFT in Turkey (Karatepe et al. 2008), while the lowest 17% in Israel by ELISA (Salant and Spira 2004). In the Czech Republic, we found 44% prevalence by IFAT. In the previous studies from the Czech Republic, 17% -91% prevalence was found by SFT, CFT and MPA. During the last 20 years, there is a trend of decreasing seroprevalence especially in cats staying at home and fed with commercial diet.

Dogs
T. gondii antibodies have been found in canine sera worldwide. Seroprevalence increases with age indicating postnatal infection, is higher in dogs from rural areas, in dogs housed exclusively outdoors, in dogs eating birds, small mammals, meat, viscera and home-cooked meals (Lopes et al. 2011b). In Europe, the highest prevalence 75% was found by SFT in Turkey (Aktas et al. 1998), while the lowest 5% in Sweden by ELISA (Lunden et al. 2002). In the Czech Republic, we found 26% prevalence by IFAT. In the previous studies from the Czech Republic, 4% -58% prevalence was found by SFT or CFT. The lower prevalence is recorded in dogs staying at home and fed with commercial diet.

Sheep
T. gondii has been recognized as one of the main cause of infective ovine abortion in New Zealand, Australia, the United Kingdom, Norway and the United States. In the Czech Please use Adobe Acrobat Reader to read this book chapter for free.
Just open this same document with Adobe Reader. If you do not have it, you can download it here. You can freely access the chapter at the Web Viewer here. Republic, not yet a case of toxoplasmic abortion has been recorded in sheep herds. Seroprevalence was shown to increase with age, suggesting that animals acquire infection postnatally, however transplacental transmission of T. gondii may be more common than previously believed. Antibodies to T. gondii have been found in sheep worldwide (Dubey 2010). There is no validation of any serological test for the detection of T. gondii infection in sheep; different methods and cut-off are used.
In Europe, the highest prevalence 96% was found by ELISA in Turkey (Mor and Arslan 2007), while the lowest 10% was found in Slovak Republic by SFT (Kovacova 1993). We found 59% prevalence by ELISA. In the Czech Republic, 4% -77% prevalence was found in past years.
Based on experimental studies, T. gondii was more frequently detected in brain and heart than in muscles; however T. gondii was detected also in milk (Camossi et al. 2011). Attention should be paid to meat or milk consumed without sufficient temperature treatment.

Goats
T. gondii antibodies have been found in goats worldwide (Dubey 2010). In Europe, the highest prevalence 91% was found by LAT in Netherland (McSporran et al. 1985), while no antibodies were found in Poland by IFAT (Gerecki et al. 2005). We found 66% prevalence by ELISA. In the Czech Republic, 20% -86% prevalence was found in past years.
Goats appear to be more susceptible to clinical toxoplasmosis compared to other domestic animals, and even adult goats could die of acute toxoplasmosis. In the Czech Republic, toxoplasmosis was diagnosed in two Angora goat herds in South Moravia with an outbreak of abortions and births of weak kids; the goats showed also iodine deficiency (Slosarkova et al. 1999). Based on several experimental studies conducted on goats, T. gondii was detected in liver, muscles, heart, diaphragm, brain, kidneys and could be excreted in semen and milk. Attention should be paid to raw goat meat and milk if consumed without sufficient temperature treatment.

Cattle
Serum antibodies to T. gondii have been found in cattle in many surveys worldwide (Dubey 2010). In Europe, the highest seroprevalence 92% was found by MAT in Italy (Avezza et al. 1993), while no antibodies were found in Slovak Republic (Pleva et al. 1997) and Turkey (Oz et al. 1995). Actual prevalence rates are likely to be lower than indicated because of problem with the specifity of the tests used. The SFT test gives false or erratic results with cattle sera; on the other hand a titer of 1:100 or higher in the MAT appears to be indicative of T. gondii infection in cattle (Dubey 2010). We found 9.7% seroprevalence by ELISA. In the previous studies in the Czech Republic, 2% -42% seroprevalence was found by SFT and DT.
There are no confirmed reports of clinical toxoplasmosis in adult cattle. In cattle, T. gondii can be transplacentally transmitted resulting in aborts; but it is probably a rare occurrence. There is more important parasite Neospora caninum leading to abortion in cattle. In the Czech Republic, there is very low prevalence of N. caninum in herds of cattle. The ingestion of beef Please use Adobe Acrobat Reader to read this book chapter for free.
Just open this same document with Adobe Reader. If you do not have it, you can download it here. You can freely access the chapter at the Web Viewer here. or dairy products is not considered important in the epidemiology of T. gondii because cattle are not a good host for this parasite. Attempts to isolate T. gondii from cattle tissues have been unsuccessful, that is why it does not present risk of infection for humans.

Pigs
Clinical manifestation of toxoplasmosis in pigs could include diarhea, encephalitis, pneumonitis, necrotic hepatitis and abortion. Surveys based on the presence of T. gondii antibodies in blood sera of pigs have been reported worldwide (Dubey 2010). In Europe, T. gondii prevalence declined in the last decade especially because of good management system. There is a different sensitivity and specifity of the assays used for serosurveys in the following order MAT, IHA, LAT and ELISA starting with the most sensitive one. Good correlation was obtained between ELISA and MAT. In Europe, the highest prevalence 64% was found by IFAT in Italy (Genchi et al. 1991), while only 1% prevalence was found by the same method used in Austria (Edelhofer 1994). In the Czech Republic, we found 36% prevalence by ELISA. In the previous studies from the Czech Republic, 0 -38% prevalence was found by SFT, CFT or MPA.
The higher prevalence is found among pigs from small backyard operations, while the prevalence among pigs from traditional large farms and modern large-scale farms is usually lower. Attention should be paid if pork meat is consumed nearly raw or without sufficient temperature treatment.

Horses
Horses have been shown to be susceptible to Toxoplasma infection (Tassi 2006) however there is no confirmed report of clinical toxoplasmosis. Serum antibodies to T. gondii have been found in horses in many surveys worldwide (Dubey 2010). In Europe, the highest prevalence 37% was found by SFT in Turkey (Gazayagci et al. 2011), while the lowest 1% in Sweden by DAT (Jakubek et al. 2006). In the Czech Republic, we found 23% by LAT. In the previous studies from the Czech Republic, 4 -11% prevalence was found by SFT or CFT. B y r e a s o n t h a t e q u i n e m e a t r e p r e s e n t s a n i m p o r t a n t s o u r c e o f f o o d i n m a n y h u m a n communities, infected equine meat could represent potential risk of T. gondii infection for humans.

Poultry
In general, there is a different sensitivity of birds to T. gondii infection. Owls and other predatory birds and domestic poultry seem to be resistant to T. gondii infection, while e.g. rock partridge (Alectoris graeca), pigeons and canaries are highly susceptible to toxoplasmosis. In Europe, there were some reports of birds (galliformes, columbiformes, psittaciformes and passeriformes) that died due to toxoplasmosis (Dubey 2010). Toxoplasmosis can also lead to drop in egg production and high mortality in embryonated eggs. In the Czech Republic, confirmed clinical toxoplasmosis has not been recorded in birds. Little is known concerning the validity of the serologic tests for the detection of T. gondii antibodies in avian sera. It is preferred to use MAT, nevertheless other methods such as SFT, CFT, ELISA and IFAT have been used worldwide. We found higher prevalence in water fowls (43% and 14% in goose and ducks, respectively) compared to gallinaceous poultry (0.3% in broiler; turkeys and chickens were negative). In Europe, higher prevalence 36% was found in chicken from Austria by MAT (Dubey et al. 2005), or 20% in turkeys by ELISA in Germany (Koethe et al. 2011).
T. gondii have been isolated from brain, heart and leg muscles, but not from the pectoral muscle and liver (Dubey et al. 1993).

Serological studies
In majority of game animals, the course of infection is subclinical. However, considering the high prevalence of T. gondii infection in game animals, they should be taken into account as the possible source of infection for human.
A total of 1618 game animals were tested, including 720 wild ruminants or ruminants living in reservations (377 red deer, 79 roe deer, 14 sika, 143 fallow deer, 105 mouflon and 2 reindeer), 565 wild boars and 333 hares. The animals came from 3 -11 districts of the Czech Republic ( Figure 1).
Sera of game animals were tested for T. gondii antibodies by an IFAT, using the Sevatest Toxoplasma Antigen IFR (Sevac) and specific conjugates (  In a group of game animals, T. gondii antibodies were detected in 32% wild ruminants (50% in sika, 45% red deer, 24% roe deer, 17% fallow deer, 9% mouflon, and in one reindeer), 26% wild boars and 20% hares. The results of serological examination including the number of samples tested, the method and cut-off used, the number and percentage of positive samples, titres obtained in positive samples and reference about published data are summarized in Table 5.

Experimental studies
In the Czech Republic, two experimental studies were conducted on game animals.
The first study was conducted on hares (Sedlak et al. 2000a). Hares were experimentally infected with T. gondii oocysts. Most infected hares demonstrated behavioural changes, and all of them died between 8 and 19 days. In all hares, parasitemia was demonstrated on days 7 and 12 p.i. T. gondii was isolated from liver, brain, spleen, kidney, lung, heart and skeletal muscles. Based on this result, hares seem to be very sensitive species to T.
The second study was conducted on gallinaceous game birds (Sedlak et al. 2000b). Partridges (Perdix perdix), chukars (Alectoris chukar), wild guineafowl (Numida meleagris) and wild turkeys (Meleagris gallopavo) were inoculated per os with two doses of T. gondii oocysts. Antibodies to T. gondii were detected in the birds by IFAT first on day 7 p.i. Two of five partridges fed 10 3 oocysts and six of eight partridges fed 10 5 oocysts died between day 6 and 16 p.i. No clinical symptoms were observed in surviving birds, however enteritis was the most striking lesion in partridges that died. Bioassay in mice revealed T. gondii in the brain, liver, spleen, heart and leg muscles of all partridges and chukars. These results indicate that partridges are highly susceptible to toxoplasmosis, while chukars, wild guineafowls and turkeys seem to be less susceptible.

Wild boars
In Europe, the highest seroprevalence 100% was found in wild boars from Portugal prevalence 8% was found in wild boars from Slovak Republic (Antolova et al. 2007). In the Czech Republic we found 26% prevalence by IFAT. This prevalence was higher compared to 0% -15% prevalence found by SFT in the previous studies from the Czech Republic.
The meat of wild boars may harbour tissue cysts of T. gondii and may represent a vehicle of human toxoplasmosis infection. Hejlicek et al. (1997) found tissue cysts in 2% examined wild boars from the Czech Republic, while in the neighbouring Slovakia, T. gondii was isolated from 31% of wild boars (Catar 1972). Hunters and their families consuming meat from wild boars should be aware of T. gondii infection and advised to take precautions. It is highly recommended to cook meat from wild boars thoroughly before human consumption.

Hares
There are several reports of T. gondii infection in hares from Europe (Dubey 2010). The highest seroprevalence 46% was found in hares from Germany (Frolich et al. 2003); in contrast no antibodies were detected in hares from Sweeden (Gustafsson and Uggla 1994). In the Czech Republic, we found 20% prevalence by IFAT. This result is comparable with 4% -31% prevalence found in previous studies by SFT or MPA. Based on the results of experimental infection, hares seem to be sensitive to T. gondii infection; T. gondii was isolated from liver, brain, spleen, kidney, lung, heart and skeletal muscles (Sedlak et al. 2000).

Wild ruminants
T. gondii infection in game animals is of epidemiological significance. Deer are strictly herbivores and that is why the high prevalence of T. gondii in deer suggests widespread contamination of the environment with T. gondii oocysts. In red deer, the highest seroprevalence 32% was found by SFT in Scotland (Williamson and Williams 1980), while the lowest 8% by DAT in Norway (Vikoren et al. 2004). We found relatively high prevalence 45% by IFAT in red deer from the Czech Republic. In roe deer, the highest prevalence 63% was found in Norway and Sweden by SFT (Kapperud 1978), while the lowest 13% prevalence was found in Austria by IHA (Edelhofer et al. 1989). In the Czech Republic, we found 24% prevalence by IFAT that was also in range 14% -58% prevalence found in our country in previous studies. In fallow deer, we found 17% prevalence that is comparable with 24% prevalence found in Spain by MAT (Gauss et al. 2006). In the Czech Republic, we found 9% prevalence in mouflon that is lower compared to 23% prevalence found in France (Aubert et al. 2010). In case of reindeer, only two animals were examined in the Czech Republic. This is very low number that is why it is not possible to compare it with 1% prevalence found in Norway by DAT (Vikoren et al. 20004).
Deer are popular game animals in several countries. The meat of deer may harbour tissue cysts of T. gondii and may represent a vehicle of human toxoplasmosis infection. Toxoplasmosis infection in men was documented after consummation of raw or nearly raw deer meat in USA (Sacks et al. 1983, Ross et al. 2001).
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Serological studies
In a group of zoo animals, 556 animals belonging to 114 species were tested (5 species of primates, 28 species of carnivores, 8 species of perissodactyla and 73 species of artiodactyla). The animals came from 12 zoo and 4 small private exotic centres in the Czech Republic.
Sera of zoo animals were tested for T. gondii antibodies by an IFAT, using the Sevatest Toxoplasma Antigen IFR (Sevac) and specific conjugates (  confirmed also by PCR with TGR1E and immunohistochemically. Toxoplasmic hepatitis and pneumonia were considered to be a primary cause of death. The other cases of fatal toxoplasmosis were recorded in year 2004 in seven Pallas cats in several zoos in the Czech Republic (Sedlak and Vodicka 2005).

Comparison of data obtained
There are many reports on toxoplasmosis in zoo animals. Marsupials, New World monkeys, hares and some small ruminants belong to the most sensitive to clinic toxoplasmosis. Fatal toxoplasmosis was also recorded e.g. in captive dik-dik and Pallas cats from zoo in USA (Riemann et al. 1974;Dubey et al. 2002), in lions from a zoo in Africa (Ocholi et al. 1989) and in a Siberian tiger from a zoo in Belgium (Dorny et al. 1989). In the Czech Republic, fatal toxoplasmosis was recorded in saiga and nilgais antelopes from Prague and Chomutov zoos ) and in Pallas cats (Sedlak and Vodicka 2005).
The potential source of T. gondii infection for carnivores is meat contaminated with T. gondii tissue cysts; herbivores can be infected by food contaminated with T. gondii oocysts and omnivorous animals by both ways. To prevent spreading of T. gondii infection among zoo animals, cats, including all wild felids should be housed in buildings separated from other animals, particularly the most sensitive marsupials and New World monkeys. There must be protection against free access of domestic cats to sources of food and water or into the buildings with animals, especially those that are the most sensitive to toxoplasmosis. Feline faeces should be removed daily to prevent sporulation of oocysts.

Further research
Further work should focus on serological studies in other animal groups that are neglected but may represent a risk of infection for humans in case of consumption of their meat or other products. Such animals include, for example, rabbits, ostriches, pigeons, pheasants and mallard ducks. In addition, rodents, wild birds and wild carnivores (foxes, marten and others) may play an important part in the circulation of T. gondii infection in nature and thus represent a risk of infection for wildlife, domestic animals and human people alike. Serological studies should be supplemented with an evaluation of the infection risk factors and with the use of molecular methods to detect T. gondii in animal products, as well as to characterize T. gondii genotypes circulating in animal populations in the Czech Republic.
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