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Toxoplasmosis, caused by Toxoplasma gondii (T. gondii), an obligate intracellular parasite, is a globally distributed zoonosis. It is estimated that one third of population has been infected by this parasite worldwide. In some regions of Europe, the serum antibodies against T. gondii were detected in more than 80% of the examined population. In China, T. gondii was isolated for the first time from a rabbit in Fujian Province in 1954,［1］ and the first case of human toxoplasmosis was reported in Jiangxi Province in 1964.［2］ Later, in 1978, the first epidemic survey on toxoplasmosis was carried out in Guangxi Province. In 1982, the first national survey on toxoplasmosis supported by the National Natural Sciences Foundation of China was carried out and the result was published in 1988. Since that, many surveys on toxoplasmosis were reported in China. These results indicated that T. gondii infection in this country is very common and in some circumstances, the infection causes serious disease. For instance, T. gondii infection in pregnant women causes abortion, stillbirth, abnormal embryo and thus, it has been considered as the most commonly infectious agent of “TORCH syndrome” (TORCH syndrome refers to infection of a developing fetus or newborn by any of a group of infectious agents. “TORCH” is an acronym meaning toxoplasmosis, other agents, rubella, cytomegalovirus, and herpes simples). In immunocompromised patients, toxoplasmosis is also one of the most frequent complications. Because of the medical importance of T. gondii, more and more Chinese researchers are interested in this parasite.
Aetiology
Infected animals are the most important source for T. gondii infection, although humans can also be taken as the infective source for this disease particularly the vertical transmission from pregnant woman to fetus through the placenta. T. gondii widely distributes in variant animals from mammals to birds. At least 190 species of animals displayed serologically positive to T. gondii and 141 species of these animals pathogenically infected by T. gondii have been confirmed in China.［3］
For human beings, felines particularly the domestic cats are the most important infective source. These animals are not only the definitive host, but also the intermediate host of T. gondii. It has been estimated that one cat can discharge approximately 10 million of oocysts per day and which can last continually for two weeks. Evidence indicated that T. gondii oocysts were detected in 29 of 67 cat faeces collected in Nanchang.［4］ The seropositive rates for toxoplasma in the people who frequently contacting the cats are much higher than those people who have seldom contacted with cats.［5］ Therefore, well control of cats especially their faeces is one of the important ways to prevent T. gondii infection.
In China, pigs are considered another important source for the infection of T. gondii. Early in 1959, T. gondii was isolated from a pig in China, but it was not attended until 1977, when this parasite was isolated from a large number of pigs suffered from “obscure high fever” in Shanghai.［6］ The prevalence of T. gondii in pigs from 11 cities, provinces and autonomous regions is summarized in Table 1 . So far, over 20 strains of T. gondii have been isolated from pigs in China.［7］ Further study indicated that the prevalence of T. gondii in the pigs with fever (14 of 118, 11.9%) was ten times higher than the normal pigs (6 of 570, 1.1%) indicating that some sick pigs might be infected by T. gondii. In China, pork is the most popular meat for most people. Thus, swine toxoplasmosis not only affects the development of livestock industry, but also plays an important infective source for human toxoplasmosis. Therefore, one of the most effective ways to limit human infection by T. gondii is to avoid eating undercooked or raw pork.
In addition, the prevalences of T. gondii in avian and livestock in 14 provinces of China were also reported by Lu et al［8］ in 1994 ( Table 2 ). Their results showed that T. gondii infection was quite frequent in 9 species of animals examined with a prevalent rate from 4.1% to 16.5%.
Toxoplasma tachyzoites can infect all kinds of tissues and organs of mammals including humans. Thus T. gondii can be transmitted from person to person through the blood or organ transplantation. It was reported that 6 of 638 blood samples donated displayed positive for IgM and IgA against T. gondii, and 3 of the 6 IgM and IgA positive samples also showed circulating antigen (CAg) positive,［9］ indicating that some donators were in the acute stages of T. gondii infection. Similar result from 264 blood samples was also reported in Mudanjiang county.［10］ The acute stage is a highly risk stage in which T. gondii can be transmitted from the donor to the recipient and thus it was suggested that the screen of T. gondii infection should be listed as a routine examination item for the donors.
In general, many warm-blooded animals can be the infective sources for human toxoplasmosis. Among them, cats and pigs are the most important species. Although most T. gondii infections in these animals are considered to be asymptomatic, they can be a health carrier for transmission of this parasite to human. Therefore, these animals play a significant role in the epidemiology of toxoplasmosis in this country.
So far, many strains of T. gondii were isolated from human and animals in different areas of China since the first strain of this parasite was isolated from rabbit in 1954. Among these strains, ZS1, ZS2, SH1, PP, CN, RT and GG were widely used in different purposes by the Chinese researchers ( Table 3 ).［11-18］ In addition, some strains of T. gondii such as RH, BEV and FUK were imported from abroad, and were also widely used in different scientific purposes in this country.
Epidemiology
In 1994, the nationwide prevalent status of T. gondii in 19 cities, provinces and autonomous regions of China was reported.［19］ In this survey, 81968 people were examined by indirect hemagglutination (IHA) method and the average prevalence of T. gondii infection was 5.17%. The prevalent rate in different provinces ranged from 0.3% to 11.8% ( Table 4 ). These data showed that the seroprevalence of T. gondii infection in humans was common in China, but it was relatively lower than that in many European countries.
Table 4 also shows the prevalent status of T. gondii infection of 51282 people among 15 different professions and of 40848 people among 13 nationalities in China.［20］ Results demonstrated that some professions such as veterinarians and slaughters were higher infected with T. gondii than others. Therefore it is necessary to make the related rules to against the infection of this parasite among these professions.
Manifestation of toxoplasmosis
Abortion and abnormal embryo
It is well known that toxoplasma infection is a high risk of abortion and abnormal embryo. In 1999, 4420 women were detected for T. gondii infection, and 282 of them had the specific IgG antibodies against T. gondii. With the follow-up study, abnormal embryo was found in 17 of the 282 (6.0%) cases, while abnormal embryo was only detected in 28 of 4138 cases without T. gondii infection (0.7%).［21］ It was evidenced that the earlier infection occurred in the pregnancy, the more abnormal embryos were observed. If T. gondii infection occurs in the late stage of the pregnancy (the last three months of pregnancy) the infants may not have obviously clinical signs but some symptoms such as neurological deficiencies, convulsions, and mental retardation may appear several years later after born. Some reports indicated that the T. gondii infection in children could cause lower intelligence quotient (IQ),［22］ although the exact mechanism is not clear. Results showed that the positive rates of CAg, IgM and IgG in these children were 16.7%, 19.7% and 37.8%, respectively, which were significantly higher than those without T. gondii infection (2.9%, 2.9% and 11.7% of CAg, IgM and IgG respectively). This study shows that T. gondii infection may affect the development of brain and further affects the intellect of the infected children. Interestingly, it was reported that the specific antibodies against T. gondii was found in 27% of 140 patients with sterility, which was much higher than the prevalence of T. gondii in normal people.［23］ It was suggested that there might be some relationships between T. gondii infection and sterility.
Mental diseases
In early 1959, Buentell suggested that the T. gondii infection might be one of the causes of psychosis. In fact, many epidemiological evidences from China also indicated that there had a close relationship between T. gondii infection and psychosis.［24,25］ It was reported that the seropositive rate of T. gondii was 29.3% in 58 patients with mental diseases in hospital, which was much higher than the patients without mental diseases (6.9%).［26］ In another report, the seropositive rate of T. gondii in 134 patients with mental diseases was 28.3%, which was significantly higher than the control group (8.3%).［24］ More and more investigation results showed that T. gondii infection in the patients with mental diseases was significantly higher than the normal people and the pathogenesis of such mental disorder might be due to the pathogenicity of T. gondii infection in the brain.［27,28］ However, the reason of the patients with mental diseases often showing a higher infection rate with T. gondii is still unknown. But it is possible that these patients have less hygiene and more opportunities to contact with water and food contaminated with oocysts of this parasite.
Immunocompromised complications
Many cases of toxoplasmosis were found in AIDS patients from European and North American as well as other countries. But in China, most T. gondii infections were found from pregnant women and infants. This might be the reason that AIDS was a rare disease in this country before the 1990's and few people worked on it. However, this situation has been quickly changed after the first AIDS patient died in 1991 in this country［29］ since T. gondii was found in the cerebellum of this corpse. As a matter of fact, toxoplasmosis is obviously one of the serious problems in AIDS patients because it is one of most common parasites and causes severe diseases such as encephalitis in these patients. In addition, as an opportunistic pathogen, T. gondii often causes severe complications in the patients who are receiving immunosuppressive therapy, such as malignant tumor or organ transplantation. Result from a study indicated that 34.4% of 93 patients with malignant tumor was found to be infected with T. gondii, which was much higher than the control group with only 6.1% positive of T. gondii infection.［30］ Many other investigations also showed that the prevalence of T. gondii in malignant tumor patients was significant higher than the control people.［31,32］
Diagnosis
Clinically, the diagnosis of toxoplasmosis is difficult because the clinic signs and symptoms are varied and mimic those of a variety of other diseases. So far, like in other countries, the laboratory diagnosis of toxoplasmosis in China is mainly relied on the pathogenic, serological and molecular biological methods. Owing to the lower sensitivity and time consuming, pathogenic diagnosis is seldom to be used in clinic. Although molecular biological diagnosis, such as PCR and DNA hybridization, are highly sensitive and specific, the application of these methods has been limited because of their higher pseudo-positive rate and requirement of the special equipments.
Immunological techniques, such as dye test (DT), IHA, latex agglutination test (LAT), immuno-fluorescence assay (IFA) and enzyme linked immunosorbent assay (ELISA) are commonly used in the investigation and diagnosis of T. gondii infection in this country. In comparison with pathogenic method, serological analysis is faster, more sensitive and specific. Because the latent infection of T. gondii is relatively high and only the patients with obvious clinic symptoms and signs need to be treated, it is important to distinguish the present infection from the past and latent infection. However, some serological methods, such as IHA and IFA, which have been used to detecte IgG antibodies, can be used in the epidemiological investigations but not suitable for the clinical diagnosis. At the present in China, both IgG and IgM or IgG and CAg are usually used in clinic to diagnose the present infection of T. gondii.
Most current antigens used for diagnosis are prepared from tachyzoites that are usually collected from cell cultures or infected mice. Owing to the different sources or different collecting and purification methods, the quality of antigens is often varied. Thus, the diagnostic systems developed with such antigens are different in their efficiencies. The efficiency of five commercial detective kits for T. gondii antibodies were evaluated and found that the sensitivities of these kits were only 60%, 60%, 30%, 60%, and 70%, with specificities of 35%, 90%, 75%, 54% and 87% respectively.［33］ It was considered that it might be one of the reasons that the prevalence of T. gondii appears significant difference in different investigations because of the different kits used. DNA recombination and gene engineering techniques have provided a novel way to produce high quality of diagnostic antigens. As a matter of fact, many Chinese researchers are working on these fields. Chen and colleagues reported that they had expressed SAG1 gene of T. gondii in different expression systems, including in Escherichia coli, yeast and baculovirus.［34,35］ A truncated SAG1 fragment has been successfully expressed in E. coli in a soluble form and the purified recombinant antigen has a specific immunoactivity with the human sera against T. gondii infection. The monoclonal antibody against rSAG1 has also been developed and the preliminary test using this McAb to detect the circulated SAG1 is successful.［36］ The CAg of T. gondii in the urine of the infected mice were detected by a SPA-ELISA based on the rabbit against T. gondii antibodies.［37］ The specific CAg in urine of all infected mice (with 10（4）, 10（6）, 10（8） tachyzoites of T. gondii RH strain, respectively) was detected 6 days post infection (Chen et al, unpublished). The higher dose of T. gondii was administrated, the earlier CAg was detected in urine. The earliest time of CAg occurred in urine was 3 days post infection. CAg is a marker of acute infection of T. gondii, it is more useful than antibody detection in clinic.
PCR, as a fast, sensitive and specific molecular diagnostic method, has been widely used in the detection of parasite DNA. In the past years, many PCR detective systems specifically amplifying toxoplasma DNA had been reported in China.［38,39］ Although PCR technique used for diagnosing toxoplasmosis especially the patients in the situation of immunocompromise is highly sensitive and specific, the results are affected by many factors including the inhibitors in the samples, laboratory contamination and the skill of the operators. Owing to the potentially high pseudopositive rate, PCR method had been prohibited to be used in clinic as a routine diagnosis method since 2000 in China.
Prevention and treatment
The combined therapy with sulfonamide and pyrimethamine is widely used in the treatment of toxoplamosis in China. These compounds are synergistic in combination and are effective against tachyzoites, but less effective against tissue cysts of this parasite and its side effects were observed in some patients, especially the pregnant women. Recent studies displayed that azithromycin may be the most effective drug for treatment of toxoplasmosis. It not only can effectively kill the tachyzoites but also destroys the cysts.［40,41］
Most Chinese traditional medicines are directly from plants and their side effects are usually less. Artesunate is a derivative of artemisinin, originally used against malaria parasites. Result from a study demonstrated that artesunate not only can affect the invasion of T. gondii into the cells, but also inhibits the proliferation of the parasite in vitro.［42］ Electronic micrography demonstrated that the membrane, mitochondria and other organelles of T. gondii appeared in pathogenic changes including disruption of the tachyzoites after exposure to the artesunate. Other results also support that artesunate has a significant effect to kill T. gondii in vitro and the dosage of artesunate at 3 μg/ml displayed a more effect than that of acetylspiramycin at 25 μg/ml to against T. gondii.［43］ However, it is interesting to know that the effect of artesunate against T. gondii in vivo is not as good as it is in vitro. Both small dose (100 mg/kg) and larger dose (250 mg/kg) of artesunate could only delay the death of the infected mice but failed to avoid their death from the challenge infection.［44］ These results indicated that the average survival time of the infected mice treated with artesunate was 10.5 days, significantly shorter than those treated with sulfonamide (30.5 days). It was suggested that the effect of artesunate on T. gondii in vivo should be further evaluated with various dose schedules. Artemether, another derivative of artemisinin, has also been used to treat toxoplasmosis. A preliminary observation demonstrated that artemether could prolongate significantly the survival of the mice infected with T. gondii RH strain, but the effect was not as good as the combination of sulfamethoxazole and trimetnoprin.［45］ Interestingly, however, artemether showed a more effect when it was used to treat the infection of T. gondii B36 strain.［46］
To our knowledge, over 100 species of Chinese herbs have been screened for the effect against T. gondii through the in vitro cultural systems and four of them (Atractylodes chinensis, Castor seed, Magnolia officinalis and Fructus psoraleae) showed promising effect against T. gondii.［47］ Further study indicated that the extracts of these four herbs can significantly prolongate the mice infected with T. gondii when they were orally administrated with the extract at 0.5 ml·mice（-1）·day（-1） for 14 days. Comparatively, the effect of Castor seed is the best and the next is Fructus psoraleae.
Theoretically, the best measure to prevent T. gondii infection is to immunize humans and sensitive animals with an efficient vaccine, however, this vaccine is far away from the clinic use. In the recent years, subunit vaccines and DNA vaccines have been studied to protect animals from T. gondii infection. The recombinant SAG1 of T. gondii was used to immunize mice and result showed that the specific humoral and cellular immunity against T. gondii were stimulated so that the immunized mice were partially protected from the challenge infection with T. gondii RH strain.［34］ In addition, the mice immunized by combined SAG1 and ROP1 gene survived longer than the control mice after challenged by RH strain infection.［48］ A similar result was also observed when the mice were genetically immunized with pcDNA3/T.g-R1 and then challenged infection with 100 RH strain tachyzoites.［49］ Because there are several stages in the life cycle of T. gondii and the antigens in different stage are also different, thus the immunity stimulated by a single immunogen is usually not complete. To solve this problem, a multi-epitopes gene fragment including 5 epitopes of SAG1, ROP2 and GRA1 was designed and synthesized and successfully expressed in E. coli and in tomato.［50,51］ The further immunization and evaluation are in process in our laboratory. It should be noted that why the protective rate of the reported vaccines against T. gondii was seldom complete. One of the reasons might be the challenge infection was usually taken with the RH strain via intraperitoneal injection, which is highly toxic to mice. We consider that the suitable stage for toxoplasma challenge should be cyst or oocyst and the infective route is by oral, because these are the natural infective ways by this parasite.
Acknowledgement: The authors thank Prof. John Kusel for his critical review on this manuscript.
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