Toxoplasmosis in Animals and Humans - Its Diagnosis, Epidemiology and Control

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1 Page1 Toxoplasmosis in Animals and Humans - Its Diagnosis, Epidemiology and Control Mahendra Pal,* 1 Biruk Alem*, Getachew Gari 2 and Getachew Tuli 2 1 Department of Microbiology, Immunology and Public Health, College of Veterinary Medicine and Agriculture, Addis Ababa University, P.B. Box No.34,Debre Zeit, Ethiopia 2 National Animal Health Diagnostic and Investigation Center, P.O.Box 4, Sebeta, Ethiopia *Corresponding author: palmahendra2@gmail.com Rec. Date: Feb 11, :14 Accept Date: Mar 01, :25 Abstract Toxoplasmosis, caused by the protozoan parasite Toxoplasma gondii, is one of the most common global parasitic zoonoses of great public health and economic importance. Nearly one-third of human population has been exposed to this parasite. In most adults, it does not produce serious illness, but it can cause blindness and mental retardation in congenitally infected children and devastating disease in immunocompromised individuals. Toxoplasmosis ranks high on the list of diseases that lead to the death of patients with AIDS. Among the food animals, T. gondii infections are more prevalent in pigs, sheep, and goats than in cattle. Toxoplasmosis causes considerable economic losses to the sheep industry worldwide. Clinical signs of toxoplasmosis are non-specific and are not sufficiently characteristic for a definite diagnosis. Diagnosis of toxoplasmosis is made by biological, serological, histological, molecular methods, or by some combination of the above. To prevent food-borne horizontal transmission of T. gondii to humans, meat and other edible parts of animals should not be consumed raw or undercooked. Pregnant women, especially, should avoid contact with cats, soil and raw meat. Serological screening of pregnant women is an effective strategy to prevent prenatal infections of T. gondii to their children. Extreme care should be used in handling litter boxes used by cat. The development of a simple, sensitive, rapid and cheap method for the detection and identification of T.gondii is highly imperative for the diagnosis and epidemiological studies of toxoplasmosis. In addition, health education of the public about source of infection, the mode of transmission, severity of disease, and preventive measures is also emphasized. Key words: Cat, Control, Diagnosis, Epidemiology, Toxoplasmosis, Women, Zoonosis Introduction Toxoplasmosis, a protozoan zoonosis of global significance, is caused by an obligate intracellular protozoan parasite Toxoplasma gondii that affects a wide range of hosts and results into high morbidity and mortality (Pal, 2007; Dubey, 2010). The parasite is of significant medical and veterinary importance worldwide. T. gondii has infected 500 million people, and the pathogen account for 30 % CNS infection in ADIS patients (Pal, 2005). It has been estimated that up to one third of the world human population is infected with this parasite (Tenter et al., 2000).

2 Page2 Probably all warm-blooded animals including most livestock and humans act as intermediate hosts. Definitive hosts are members of the family Felidae, for example domestic cats. The life cycle of T. gondii includes asexual multiplication in various tissues of intermediate hosts and sexual reproduction in the intestine of definitive hosts (Hill and Dubey, 2002). There are three infectious stages in the life cycle of T. gondii, i.e. tachyzoites, bradyzoites contained in tissue cysts, and sporozoites contained in sporulated oocysts. All three stages are infectious for both intermediate and definitive hosts. Its life cycle may continue indefinitely by transmission of tissue cysts between intermediate hosts (even in the absence of definitive hosts) and also by transmission of oocysts between definitive hosts (Innes, 2010). People usually acquire infection via ingestion of tissue cysts in undercooked meat, consuming food and water that has been contaminated with sporulated oocysts, or by accidentally ingesting oocysts from the environment, or vertically by transplacental transmission of tachyzoites. Transfusion or organ transplantation from an infected person can also transmit the organism (Pal, 2007; Dubey and Jones, 2008). Once infected, humans may remain infected for the entire life. Most cases of T. gondii infections in immunocompetent humans are asymptomatic. Occasionally, various mild symptoms may be observed of which lymphadenopathy is the most significant clinical manifestation. However, there are now several recorded cases in which development of ocular symptoms, such as retinitis and retinochoroiditis, was associated with acquired toxoplasmosis in humans (Tenter et al., 2000). Congenital toxoplasmosis generally occurs when a woman is newly infected with T. gondii during pregnancy, although rare exceptions have been reported in which women were infected just before pregnancy. In addition, in immunosuppressed women reactivation of an infection acquired before pregnancy can lead to congenital toxoplasmosis (Dubey, 2010). Infection with T. gondii during pregnancy can result in fetal death, neonatal death or various congenital defects, such as hydrocephalus, central nervous system abnormalities and chorioretinitis (Zewdu et al., 2013). In immunocompromised humans, a previously acquired latent infection can lead to reactivated toxoplasmosis with encephalitis. Toxoplasmosis ranks high on the list of diseases that lead to the death of patients with AIDS. In most AIDS patients, the disease occurs from reactivation of latent T. gondii infection because of immunosuppressive effects of the HIV infection. Clinically, patients may have headache, disorientation, drowsiness, hemiparesis, reflex changes, and convulsions, and many become comatose (Dubey, 2010). Non-clinically manifested disease in food-producing animals is not a veterinary health problem, due to its low health significance. On the other hand, food producing animals may represent a real risk for

3 Page3 transmission of the disease to humans, either directly or through farming (Cenci-Goga et al., 2011). Among the food animals, T. gondii infections are more prevalent in pigs, sheep, and goats than in cattle (Dubey, 2010). Toxoplasmosis causes considerable economic losses to the sheep industry worldwide. T. gondii induced abortion can occur in ewes of all ages, although maiden ewes are most affected (Edwards and Dubey, 2013). This communication focuses on diagnosis, epidemiology and control of toxoplasmosis in animals as well as in humans. Diagnosis of Toxoplasmosis Clinical signs of toxoplasmosis are not pathognomonic for a definite diagnosis. Hence, diagnosis is confirmed by biological, serological, histological, or molecular methods, or by some combination of the above (Hill and Dubey, 2002; Pal, 2007). Examination of cat feces for Toxoplasma gondii oocysts Oocysts can be detected by examination of cat faeces, though for epidemiological surveys, detection of T. gondii oocysts in cat feces is not practical. Concentration method (flotation in high-density sucrose solution) is often used because the number of T. gondii oocysts in cat faeces may be too few to be detected by direct smear. For definitive identification, T. gondii oocysts should be sporulated and then bioassayed in mice to distinguish them from other related coccidians. Bioassays of tissues in mice The present trend, for obvious ethical reasons and animal welfare, it is limited or avoided to use biological testing for diagnosis. However, the possibility of inoculating or feeding Toxoplasma free laboratory animals (mice and cats) to later demonstrate T. gondii in organs and tissues should be held in consideration, especially for the high sensitivity and specificity which make this biological testing the gold standard (Cenci-Goga et al., 2011) The choice of inoculum will depend upon the circumstances. Secretions, excretions, body fluids, and tissues taken by biopsy, such as lymph nodes or muscle tissue, are possible specimens from which to attempt isolation. Cerebral spinal fluid from a child with possible congenital infection and encephalitis or lymph node material from a person with lymphadenopathy are good sources of T. gondii (Dubey, 2010). Immunological techniques Detection of humoral antibodies The finding of antibodies to T. gondii in one serum sample only establishes that the host has been infected at some time in the past. It is best to collect paired samples from the same individual, the second collected

4 Page4 2 4 weeks after the first. A 16-fold higher antibody titer in the second sample indicates an acute infection. A high antibody titer sometimes persists for months after infection. A rise in antibody titer may not be associated with clinical symptoms because, as indicated earlier, most infections in humans are asymptomatic (Hill and Dubey, 2002). There are numerous serological procedures available for the detection of IgG and IgM antibodies; these include the Sabin Feldman dye test (DT), the indirect hemagglutination assay (IHA), the indirect fluorescent antibody assay (IFA), modified agglutination test (MAT), the latex agglutination test (LAT), the enzyme-linked immunosorbent assay (ELISA), and complement fixation test (CFT) (Pal, 2007). Of these, IFA, ELISA, and MAT have been modified to detect IgM antibodies (Dubey, 2010). Avidity tests The binding (avidity) of T. gondii antigen to specific antibodies can change during the course of infection. During the early (acute) stage of infection, avidity values are low and increase with duration of infection. In this test, sera are run with or without treatment with urea (or other protein denaturing agents) and the difference in titers can be used to determine recency of infection. The test can be used with IgG, IgA, and IgE antibodies using different serological procedures, most often ELISA (Dubey, 2010). The IgG avidity test was developed to help discriminate between past and recently acquired infection. Results are based on the measurement of the avidity (functional affinity) of Toxoplasma-specific IgG antibodies. Depending on the method used, the avidity tests currently available are helpful primarily to rule out that a patient s infection occurred within the prior 4 to 5 months. This is most useful in pregnant women in their first months of gestation who have a positive test for both IgG and IgM Toxoplasma antibodies. Molecular methods for the detection and identification Molecular methods rely on PCR for the specific detection or analysis of T. gondii DNA. These methods have proved to be simple, sensitive (it can detect DNA from one tachyzoite), reproducible and costeffective, and have been applied to a variety of clinical samples from animals and humans (Su et al., 2010). Molecular methods can be divided into 2 groups. The first group focuses on specific detection of T. gondii DNA in biological samples. The conventional PCR, nested PCR (n-pcr) and quantitative realtime PCR (qpcr) of repetitive DNA sequences belong to this group. The second group of molecular methods focuses on a high resolution identification of T. gondii isolates. The multilocus PCR-RFLP, microsatellite, and multilocus sequence typing (MLST) of single copy DNA sequences belong to this group (Su et al., 2010).

5 Page5 The PCR has been successfully used to diagnose congenital and ocular toxoplasmosis and toxoplasmosis in immunocompromised patients. For this purpose PCR with amniotic fluid, placental and brain tissues, whole blood, cerebrospinal fluid, urine, vitreous fluid, aqueous humor, bronchoalveolar lavage fluid, and pleural and peritoneal fluids has proved of value. The most common use of PCR is for prenatal diagnosis of the congenital infection using amniotic fluid (Remington et al., 2004). Real-time PCR has recently been introduced for the diagnosis of toxoplasmosis. It combines the steps of amplification and PCR product detection in a single phase, thereby shortening the turn-around time from 24 to 48 h to less than 4 h. Real-time PCR uses a fluorescence-labeled oligonucleotide probe, which eliminates the need for post-pcr processing. DNA extraction methods have also recently been automated. Using magnetic-bead technology, DNA can be purified from a variety of clinical samples, thereby eliminating the need for centrifugation, vacuum pumps, and other steps with high risk for contamination (Loeffler et al., 2002). Immunohistochemical (IHC) Staining Formalin-fixed, paraffin-embedded tissues can be used in this technique. Although T. gondii antigen can be detected even 1 year after fixation in 10% formalin, fixation for short periods (24 hr) is recommended. The IHC technique can be used to distinguish tachyzoites from bradyzoites. An antibody produced in rabbits against BAG1 (formerly BAG5) antigen of T. gondii -896 has proved very useful in tracing the development of bradyzoites in tissues. This anti-bag1 antibody is against the developmental stage, but not to the parasite and non-specific. It does not react with tachyzoites but will react with bradyzoites; but also reacts with Besnoitia, Neospora and Sarcocystis (Dubey, 2010). Epidemiology of Toxoplasmosis Distribution of Toxoplasma gondii It has been 100 years since the discovery and naming of T. gondii. The parasite was first found in laboratory animals. Its medical importance remained unknown until 1939 when T. gondii was identified conclusively in tissues of a congenitally-infected infant in New York City, USA, and its veterinary importance became known when it was found to cause abortion storms in sheep in 1957 in Australia (Dubey and Jones, 2008). T. gondii infection in man and animals is widespread throughout the world, but varies in different geographical areas of a country, among different geographical areas within one country, and among different ethnic groups living in the same area (Tenter et al., 2000). Causes for these variations are not yet known. Environmental conditions may determine the degree of natural spread of T. gondii infection.

6 Page6 Infection is more prevalent in warm climates and in low-lying areas than in cold climates and mountain regions and in humid areas than in dry areas. This is probably related to conditions favoring sporulation and survival of oocysts in the environment (Dubey, 2010). In 1939, Sabin first proved that Toxoplasma isolates from humans and those previously obtained from animals belonged to the same species. In 1948, the introduction of the methylene blue dye test by Sabin and Feldman enabled sero-epidemiological studies in humans as well as a broad range of animal species which provided evidence for a wide distribution and high prevalence of T. gondii in many areas of the world. Since then, it has been estimated that up to one third of the world human population has been exposed to the parasite (Tenter et al., 2000). When comparing seroprevalence data for infections with T. gondii, it should be taken into account that the different serological methods used to obtain these data are not standardized. In the 1990s seroprevalences in Central European countries, such as Austria, Belgium, France, Germany, and Switzerland, have been estimated to range between 37 and 58% in women of childbearing age with no obstetric history. Comparable seroprevalences have been observed in similar populations in Croatia, Poland, Slovenia, Australia, and Northern Africa. Seroprevalences are higher in several Latin-American countries, including Argentina, Brazil, Cuba, Jamaica, and Venezuela (51-72%), and in West African countries on the Gulf of Guinea, i.e. Benin, Cameroon, Congo, Gabon, and Togo (54-77%). Lower seroprevalences have been reported from women of childbearing age in Southeast Asia, China, and Korea (4-39%). Seroprevalences are also low in areas with a cold climate, such as the Scandinavian countries (11-28%).In one review on importance of zoonoses in public health, Pal (2007) mentioned that 37 % of AIDS patients in France have evidence of T.gondii encephalitis at autopsy. The protozoan affects more than 3500 newborns in USA every year. The infection has been reported from many countries of the world including India and Ethiopia (Dhume et al., 2007; Zewdu et al.,2013). However, there is no doubt that overall T. gondii infections are highly prevalent in adult human populations throughout the world (Tenter et al., 2000). Epidemiological risk factors for infection Currently, there are no tests which can discriminate between oocyst ingestion and tissue cyst ingestion as the infection route. Available evidence for the oocyst infection route is based upon epidemiological surveys. For example, in certain areas of Brazil, approximately 60% of 6 8-year old children have antibodies to T. gondii linked to the ingestion of oocysts from an environment heavily contaminated with T. gondii oocysts. Infections in aquatic mammals indicate contamination and survival of oocysts in sea water. The largest outbreak of clinical toxoplasmosis in humans was epidemiologically linked to drinking

7 Page7 water from a municipal water reservoir in British Columbia, Canada. This water reservoir was thought to be contaminated with T. gondii oocysts excreted by cougars (Felis concolor) (Hill and Dubey, 2002). Recent outbreaks of acute toxoplasmosis in humans in various regions of the world demonstrate that the sources of infection vary greatly in different human populations with differences in culture and eating habits. In Canada, an outbreak of congenital toxoplasmosis in a settlement of Inuits in northern Quebec was associated with frequent consumption of caribou meat, in addition to skinning of fur animals, while seropositivity in pregnant women living in the same settlement was associated with consumption of dried seal meat, seal liver, and raw caribou meat. In Australia, an outbreak of acute and congenital toxoplasmosis was associated with rare kangaroo meat and undercooked lamb satay which were consumed during a cocktail party in Queensland (Tenter et al., 2000). The ingestion of undercooked lamb meat is probably an important source of T. gondii infection in many countries, especially Europe. Very little lamb is often consumed in the United States. The ingestion of undercooked meat (mainly beef) was considered as the main risk for T. gondii infection in humans in Serbia (Dubey, 2010). In Israel, the rate of infection for various populations of ground feeding animals that likely become infected by ingesting oocysts contaminating the environment has been recently examined. Amongst ground-feeding animals, 8.9% 4 6 months old slaughtered sheep from the south of the country and 37.5% sheep of ages 5 months to 2 years and raised in the central region were T. gondii seropositive (Salant et al., 2013). The role of cat in the epidemiology of T. gondii infections The domestic cat is the only domestic animal that is used as a definitive host by T. gondii, and thus appears to play a key role in the epidemiology of T. gondii infections. Domestic cats and other feline species may become infected with T. gondii either by ingesting infectious oocysts from the environment or by ingesting tissue cysts from intermediate hosts (Tenter et al., 2000). After primary infection with T. gondii, cats that are kept inside houses may shed large numbers of oocysts into the household, thereby putting their owners at risk of infection. Stray cats or cats that are roaming on farms may contaminate the environment with oocysts which may infect livestock that will later be slaughtered for human consumption. However, oocysts shed by cats are unsporulated and, thus, are not immediately infectious (Tenter, 2009).

8 Page8 Infections with T. gondii in cats are usually asymptomatic, and vertical transmissions occur only infrequently. However, latent infections with T. gondii are common in domestic cats and wild felines throughout the world (Tenter et al., 2000). Prevention and Control To prevent infection of human beings by T. gondii, people handling meat should wash thoroughly their hands with soap and water before they begin other tasks. All cutting boards, sink tops, knives, and other materials coming in contact with uncooked meat should be washed with soap and water because the stages of T. gondii in meat are killed by water (Hill and Dubey, 2002:Pal,2007). Meat of any animal should be cooked thoroughly until internal temperature has reached 66 o C before human or animal consumption, and tasting meat while cooking or seasoning home-made sausages should be avoided. Cooking times will vary with the thickness and the type of the cut of meat. Microwave cooking is unreliable for killing T. gondii (Dubey, 2010). Toxoplasma in tissue cysts are also killed by exposure to 0.5 kilorads of gamma irradiation (Hill and Dubey, 2002). Freezing meat to an internal temperature of 12 o C is effective in killing tissue cysts; freezing meat overnight in a household freezer is effective in killing most tissue cysts. Salting, curing, smoking, and the addition of products to meat to enhance color and taste (enhancing solutions) can have deleterious effect on the viability of T. gondii in meat, but there is too much variability in standards for these procedures to make a safety recommendation (Dubey, 2010). Pregnant women, especially, should avoid contact with cats, soil and raw meat. The cat litter box should be emptied every day (to prevent sporulation of oocysts), a task to be avoided by pregnant women. Gloves should be worn while gardening, while changing cat litter, and while handling soil potentially contaminated with cat feces. Owners may also be advised to keep dogs away from the cat litter box to prevent ingestion of and passage through of oocysts. Vegetables should be washed thoroughly before eating because they may have been contaminated with cat faeces (Hill and Dubey, 2002; Pal, 2007; Dubey, 2010). Serological screening of pregnant women is an effective strategy to prevent prenatal infections with T. gondii in their children (Tenter et al., 2000). Extreme care should be taken to prevent infection in seronegative pregnant women from congenital toxoplasmosis or those women who acquire a primary infection with T. gondii, as reflected in maternal seroconversion during pregnancy should be immediately treated. In some cases, education programmes aimed at reducing the risk of primary maternal infection during pregnancy also have been successful in decreasing maternal infection rates. Pregnant women should be aware of the dangers of toxoplasmosis (Pal, 2007).

9 Page9 To prevent infection in cats, they should never be fed uncooked meat, viscera, or bones, and efforts should be made to keep cats indoors to prevent hunting (Pal, 2007). Pet cats should be fed only dry, canned, or cooked food. However, if a choice has to be made, frozen meat is less infective than fresh meat, and beef is less likely to contain T. gondii than is horse meat, pork, or mutton. Because cats cannot utilize plant sources of vitamin A, some owners feed raw liver to improve the coat of their cat. This practice should be discontinued because T. gondii tissue cysts frequently are found in the liver of food animals. Trash cans also should be covered to prevent scavenging. Cats should be spayed to control the feline population on farms (Dubey, 2010). Sheep that have aborted due to toxoplasmosis usually do not have recurrent toxoplasmic abortions, and thus can be saved for future breeding. Fetal membranes and dead fetuses should not be handled with bare hands and should be buried or incinerated to prevent infection of felids and other animals on the farm. Cats should not be allowed near pregnant sheep and goats. Grain should be kept covered to prevent contamination of T.gondii cysts (Dubey, 2010).It is pertinent to educate the public particularly the cat handler, women, butcher about the source of infection, mode of transmission, nature of disease, personal hygiene and hazards of eating raw or undercooked meat (Pal,2007). References 1. Cenci-Goga, B. T., Rossitto, P. V., Sechi, P., McCrindle, C. M.E. and Cullor, J. S Toxoplasma in Animals, Food, and Humans: An Old Parasite of New Concern. Foodborne Pathogens and Disease 8: Dhume, M., Segupta,C., Kadival, G., Rathinaswamy, A. and Velimani,A.2007.National seroprevalence of Toxoplasma gondii in India. Journal Paraitology 99: Dubey, J.P Toxoplasmosis of Animals and Humans. CRC Press, Boca Rotan, USA. 4. Dubey, J.P. and Jones, J.L Toxoplasma gondii infection in humans and animals in the United States. International Journal for Parasitology 38: Edwards, J. F. and Dubey, J.P Toxoplasma gondii abortion storm in sheep on a Texas farm and isolation of mouse virulent atypical genotype T. gondii from an aborted lamb from a chronically infected ewe. Veterinary Parasitology 192: Hill, D. and Dubey, J. P Toxoplasma gondii: transmission, diagnosis and prevention. Clinical Microbiology and Infectious Diseases 8: Innes, E. A A Brief History and Overview of Toxoplasma gondii. Zoonoses and Public Health 57: Loeffler, J.,Schmidt, K., Hebart, H.,Schumacher,U. and Einsele,H Automated extraction of genomic DNA from medically important yeast species and filamentous fungi by using the MagNA Pure LC system. Journal of Clinical Microbiology 40: Pal, M Importance of zoonoses in public health. Indian Journal of Animal Sciences 75: Pal, M Zoonoses.2 nd Ed. Satyam Publishers, Jaipur, India. 11. Remington, J. S., Thulliez, P. and Montoya, J. G Recent developments for diagnosis of toxoplasmosis.journal of Clinical Microbiology 42:

10 Page Salant, H., Hamburger, J., King, R. and Baneth, G Toxoplasma gondii prevalence in Israeli crows and Griffon vultures. Veterinary Parasitology 191: Su, C., Shwab, E. K., Zhou, P., Zhu, X.Q. and Dubey, J. P Moving towards an integrated approach to molecular detection and identification of Toxoplasma gondii. Parasitology 137: Tenter A.M Toxoplasma gondii in animals used for human consumption. Mem Inst Oswaldo Cruz 104: Tenter, A.M., Heckeroth, A.R.and Weiss, L.M Toxoplasma gondii: from animals to humans. International Journal of Parasitology 30: Zewdu, E. G., Hailu, A., Tessema, T.S., Desta, K. T., Medhin, G., Vitale, M., Di Marco, V., Cox, E.and Dorny, P Seroepidemiology of Toxoplasma gondii infection in women of childbearing age in central Ethiopia. BMC Infectious Diseases 13:101.