Since the discovery of Toxoplasma gondii in 1908, it is estimated that one-third of the global population has been exposed to this ubiquitous intracellular protozoan. The complex life cycle of T. gondii has enabled itself to overcome stress and transmit easily within a broad host range thus achieving a high seroprevalence worldwide. To date, toxoplasmosis remains one of the most prevalent HIV-associated opportunistic central nervous system infections. This review presents a comprehensive overview of different vaccination approaches ranging from traditional inactivated whole-T. gondii vaccines to the popular DNA vaccines. Extensive discussions are made to highlight the challenges in constructing these vaccines, selecting adjuvants as well as delivery methods, immunisation approaches and developing study models. Herein we also deliberate over the latest and promising enhancement strategies that can address the limitations in developing an effective T. gondii prophylactic vaccine.
Schizophrenia is a pervasive neuropsychiatric disease of unknown cause. Previous studies have reported that toxoplasmosis may be a possible cause of schizophrenia. To ascertain possible relationship between Toxoplasma gondii and schizophrenia, a cross sectional study, employing an enzyme-linked immunosorbent assay (ELISA) was performed to study the seroprevalence of anti-T. gondii IgG antibody in schizophrenic patients. Furthermore, demographic data analysis from schizophrenic patients were analysed to associate toxoplasmosis with schizophrenia. A total of 288 serum samples from schizophrenic patients (n=144) and psychiatrically healthy volunteers (n=144) were recruited in this study. Interestingly, a significant result in the serointensity rate of anti-T. gondii IgG antibody (> 60 IU/mL) in schizophrenic patients (61.1%) was demonstrated as compared to psychiatrically healthy volunteers (40.8%) (X² = 4.236, p < 0.050). However, there was no significant difference between the seropositivity rate of anti-T. gondii IgG antibody between the two groups. Analysis from demographic data revealed that the seropositivity rate of anti-T. gondii IgG antibody in schizophrenic patients was significantly associated with age group of more than 40 years old (p=0.007) and between ethnic (p=0.046). Nevertheless, no significant association between seropositivity rate of anti-T. gondii IgG antibody with gender (p=0.897), duration of illness (p=0.344) and family history of schizophrenia (p=0.282) in these patients. Thus, this finding is essential as a preliminary data in Malaysia to establish the association between T. gondii and schizophrenia.
Toxoplasmosis can cause serious disease in immunocompromised patients and to congenitally infected foetuses. Appropriate laboratory investigations in potential cases of acute Toxoplasma infection are important. Excretory secretory antigen (ESA) is immunogenic during both human and experimental infections, therefore is considered as a good candidate for investigation into new infection markers. In this study, ESA was prepared from in vitro cultures of Toxoplasma gondii to identify T. gondii ESA antigenic component(s) that is/are most reactive with serum samples from probable acute cases of toxoplasmosis. Serum samples were obtained from several categories of individuals with the following Toxoplasma serology: Group I: IgM+ IgG+ (low IgG avidity) or IgM+ IgG- from sera of patients who had clinical query of toxoplasmosis (n=35). Group II: IgM- IgG+ (high IgG avidity) from chronically infected individuals (n=30). Group III: normal/healthy individuals with anti-Toxoplasma IgMIgG- (n=20). Group IV: individuals with other infections who had anti-Toxoplasma IgM- IgG- (n=10). The ESA was subjected to SDS-PAGE, followed by Western blot analysis using the above sera and probed with peroxidase conjugated anti-human IgM and IgA antibodies. The blots were then developed using chemiluminescence substrate. The selected antigenic band was excised from the gel after two dimensional electrophoresis and sent for mass spectrometry analysis using MALDI TOF-TOF. The most promising antigenic band was a 10 kDa protein which showed sensitivity of 80% in both IgM and IgA blots, and specificity of 96.7% with sera from other infections and healthy controls. The two best identifications for the 10 kDa band were ubiquitin (ribosomal protein CEP52 fusion protein) and polyubiquitin.
Flourescent antibody test (FAT) was applied to determine the cross-reactivities of monoclonal (mAb), polyclonal (pAb) antibodies to Neospora, Toxoplasma and Cryptosporidium and antisera from cattle naturally infected with Neospora canium against antigens from a number of sources. Both mAb and pAb to Neospora reacted strongly (FAT titre up to 2560) with the homologous antigens and demonstrated weak titre (80) or no reaction with both Toxoplasma and Cryptosporidium antigens. Also mAb and pAb to Toxoplasma gondii reacted at titres of 80 - 640 with homologous antigens and at titres of 10-40 with N. caninum. No cross-reactions with either mAb or pAb antibodies to N. caninum and T. gondii were observed with Cryptosporidium parvum. The same results were observed with C. parvum mAb when tested with both N. caninum and T. gondii antigens. Sera from cattle naturally infected with N. caninum had titres ranging from 80- 640 with N. caninum antigens, and 10- 40 with T. gondii and C. parvum antigens. At low dilutions, the complete surfaces of Neospora and Toxoplasma parasites were fluorescent, while in higher dilutions only dotted fluorescence appeared on the apical complex. These results indicated the presence of cross-reactivity between Neospora and Toxoplasma but not with Cryptosporidium. Accordingly the recommended cut-off antibody titre for diagnosis of neosporosis is 80.
Toxoplasma gondii is a public health risk in developing countries, especially those located in the tropics. Widespread infection may inflict a substantial burden on state resources, as patients can develop severe neurological defects and ocular diseases that result in lifelong loss of economic independence. We tested sera for IgG antibody from 493 eye patients in Malaysia. Overall age-adjusted seroprevalence was estimated to be 25% (95% CI: [21%, 29%]). We found approximately equal age-adjusted seroprevalence in Chinese (31%; 95% CI: [25%, 38%]) and Malays (29%; 95% CI: [21%, 36%]), followed by Indians (19%; 95% CI: [13%, 25%]). A logistic regression of the odds for T. gondii seroprevalence against age, gender, ethnicity and the occurrence of six types of ocular diseases showed that only age and ethnicity were significant predictors. The odds for T. gondii seroprevalence were 2.7 (95% CI for OR: [1.9, 4.0]) times higher for a patient twice as old as the other, with ethnicity held constant. In Malays, we estimated the odds for T. gondii seroprevalence to be 2.9 (95% CI for OR: [1.8, 4.5]) times higher compared to non-Malays, with age held constant. Previous studies of T. gondii seroprevalence in Malaysia did not explicitly adjust for age, rendering comparisons difficult. Our study highlights the need to adopt a more rigorous epidemiological approach in monitoring T. gondii seroprevalence in Malaysia.
Sera from 243 donors belonging to the four main ethnic groups in West Malaysia (Orang Asli, Malays, Chinese and Indians) were tested, using the indirect fluorescent antibody technique for the prevalence of antibodies to Sarcocystis. Almost 20% reacted positively at dilutions of 1:64 or higher and eight among the Orang Asli and Malays gave the highest titres of 1:256. Prevalence was highest in the Orang Asli and lowest in Chinese. 22 sera also reacted positively to Toxoplasma, whether due to polyparasitism or cross-reaction is, as yet, unknown.
Detection of Toxoplasma gondii in blood by means of the polymerase chain reaction (PCR) may facilitate early diagnosis of toxoplasmosis in different groups of patients. We evaluated this approach in 42 patients presenting with ocular or psychotic diseases by comparing the sensitivity and specificity of PCR after heat treatment using a microwave oven with a standard genomic DNA extraction method for paired serum and whole blood samples. The presence of serum IgM and IgG antibodies against T. gondii was detected using a standard commercial enzyme-linked immunosorbent assay and enzyme immunoassay for IgG avidity test. Of 42 whole blood samples, PCR after microwave treatment was positive in 8 samples with a sensitivity of 73% and specificity of 100% compared to 11 samples positive by the extraction method. Although none of 42 sera samples was PCR positive by the extraction method, 7 specimens were positive after microwave treatment. This is the first study to use a microwave heat treatment, which is simple, rapid and a promising alternative method, in detecting small amounts of T. gondii DNA in human blood. Furthermore, irradiation of blood samples with microwaves allows incorporation of PCR into a practical tool for routine clinical assessment of patients with Toxoplasma infection.
Numerous studies have reported the prevalence of toxoplasmosis among Malaysians and various domestic animals; but there is paucity of information on its prevalence among rodents which could potentially contribute to the transmission of Toxoplasma gondii in both domestic and sylvatic fauna. Five hundred twenty-six rodents were captured from six locations in Malaysia and identified to species. Serum samples were collected from these rodents and tested for T.gondii antibodies using an immunofluorescent antibody test (IFAT). T.gondii antibodies were found in 5.9% (31/526) of the tested samples. Most of the positive antibodies were from commensal rats: Rattus exulans (9/64, 14.0%), Rattus argentiventer (2/8, 25%), Rattus rattus diardii (10/166, 6.0%) and Rattus tiomanicus (6/215, 2.7%). Only two of the forest rats were positive: Maxomys rajah (1/9, 11.1%) and Rattus bowersi (1/12, 8.3%). Eighteen point one percent of ground squirrels (Tupaia glis) tested (2/11) were positive for antibodies. The highest antibodies titer (1:1024) was found in Rattus exulans followed by T.glis (1:256). Sabak Bernam, Selangor had the highest prevalence (10.8%) followed by Baling, Kedah (5.0%) and Bagan Terap, Selangor (4.0%). None of the serum samples of rodents collected from Gua Musang, Kelantan; Jasin, Malacca; or Labis, Johor were positive. Our study reports for the first time the serologic prevalence of T.gondii antibodies among rodents in Peninsular Malaysia. Further studies are needed to confirm T.gondii infection among wild rodents, such as a bioassay, to assess their potential role in transmission of the parasite.
The distribution of anti-toxoplasma antibodies among the aborigines in Malaysia and its association with other soil transmitted infections and eosinophilia were studied. A total of 415 serum samples were collected and tested by IFA test. Overall prevalence was 10.6%, lower than previously reported. The antibody titers showed a unimodal distribution peaking at 1:8 dilution. There was a higher proportion of high antibody titer (> 1:128) in the adult compared to the children with no significant difference in prevalence rate by sex. The pattern of infection does not differ from other soil transmitted infections and there was no association between raised Toxoplasma antibodies with eosinophilia.
A total of 736 sera collected from cord blood babies, children and adults of both sexes and of different age groups in Malaysia were tested using indirect fluorescent antibody technique for Toxoplasma antibodies. The RH strain of Toxoplasma gondii zoites were used as antigen. One hundred and twenty five sera which were reactive at 1:64 or high titres were tested with IgM specific conjugate. Results of the present studies showed that the prevalence of Toxoplasma antibody was highest among the Malays and lowest among children than among adults. The significance of Toxoplasma specific IgM was discussed.
Various studies on toxoplasmosis in Malaysia have shown that specific antibodies to Toxoplasma gondii are common among Malaysians. Among the ethnic groups, the Malays have the highest prevalence rate followed by Indians, Orang Aslis (aborigines) and Chinese. Antibody is acquired early in life and increases with age. There is no significant difference in the prevalence rate between males and females. The disease is apparently more prevalent among rural dwellers and those in the lower socioeconomic group. It appears that the prevalence rate is also influenced by environmental conditions, occupation, diet and cultural habits. Studies with animals have shown the presence of antibody to T. gondii, but this does not seem to be the source of infection since Malaysians normally cook their meat well.
Thirty in vitro serial passages of Toxoplasman gondii cultures in Vero cell line performed once in every five days had a mean increase in parasite count of 74.4 +/- 14.8 times from that of initial counts. Long term cultures in Vero cell line did not alter the virulence of the parasite. The good correlation (r = 0.99) between the IFA titer and ELISA OD values using the parasite antigens from in vitro sources indicates that long term maintenance of T. gondii in culture does not affect significantly the ability to recognize antibodies to surface and soluble antigens. The results also show that soluble antigens containing host cells can be directly used for immunodiagnostic purposes without purification. The in vitro maintenance of T. gondii is safer and cheaper when compared to the in vivo method.
Toxoplasmosis was found not to be an important cause of intrauterine infection in Malaysia as the rate of toxoplasma-specific IgM in 1,060 congenitally defective Malaysian children, 0 to 4 months old (0.4%) was lower than that in 405 normal children of the same age group (2.0%). A total of 8.2 intra-uterine toxoplasmic infections per 1,000 live births was detected of which one-third (2.7 per 1,000 live births) was overt, manifesting symptoms more of liver damage, than eye or brain damage. A comparison was made with the rates in U.S.A. and Europe. The role of toxoplasmosis in abortion needs to be studied.
The aim of this cross sectional case control study was to examine the serofrequency and serointensity of Toxoplasma gondii (Tg) IgG, IgM, and DNA among patients with schizophrenia. A total of 101 patients with schizophrenia and 55 healthy controls from Sungai Buloh Hospital, Selangor, Malaysia and University Malaya Medical Center (UMMC) were included in this study. The diagnosis of schizophrenia was made based on the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV). The presence of Tg infection was examined using both indirect (ELISA) and direct (quantitative real-time PCR) detection methods by measuring Tg IgG and IgM and DNA, respectively. The serofrequency of Tg IgG antibodies (51.5%, 52/101) and DNA (32.67%, 33/101) among patients with schizophrenia was significantly higher than IgG (18.2%, 10/55) and DNA (3.64%, 2/55) of the controls (IgG, P=0.000, OD=4.8, CI=2.2-10.5; DNA, P=0.000, OD=12.9, CI=2.17-10.51). However, the Tg IgM antibody between patients with schizophrenia and controls was not significant (P>0.005). There was no significant difference (P>0.005) in both serointensity of Tg IgG and DNA between patients with schizophrenia and controls. These findings have further demonstrated the strong association between the active Tg infection and schizophrenia.
Toxoplasmosis is an opportunistic infection caused by the protozoan parasite Toxoplasma gondii. T. gondii is widespread globally and causes severe diseases in individuals with impaired immune defences as well as congenitally infected infants. The high prevalence rate in some parts of the world such as South America and Africa, coupled with the current drug treatments that trigger hypersensitivity reactions, makes the development of immunotherapeutics intervention a highly important research priority. Immunotherapeutics strategies could either be a vaccine which would confer a pre-emptive immunity to infection, or passive immunization in cases of disease recrudescence or recurrent clinical diseases. As the severity of clinical manifestations is often greater in developing nations, the development of well-tolerated and safe immunotherapeutics becomes not only a scientific pursuit, but a humanitarian enterprise. In the last few years, much progress has been made in vaccine research with new antigens, novel adjuvants, and innovative vaccine delivery such as nanoparticles and antigen encapsulations. A literature search over the past 5 years showed that most experimental studies were focused on DNA vaccination at 52%, followed by protein vaccination which formed 36% of the studies, live attenuated vaccinations at 9%, and heterologous vaccination at 3%; while there were few on passive immunization. Recent progress in studies on vaccination, passive immunization, as well as insights gained from these immunotherapeutics is highlighted in this review.
Toxoplasmosis is caused by an obligate intracellular protozoan parasite; Toxoplasma gondii, which is one of the most important zoonotic parasite worldwide. In dogs, the sexual reproductive cycle of T. gondii is lacking, and the animals are not widely consumed as food, but they are vital in the mechanical transmission of the parasite. However, there is no present data on the exposure of stray dogs to T. gondii in Malaysia. The objective of this serological survey was to determine the prevalence of T. gondii antibodies (IgG) and associated factors in stray dogs in East and West Malaysia. Antibodies to T. gondii were determined in serum samples from 222 stray dogs from 6 different states in East and West Malaysia (Peninsular Malaysia) using an Indirect ELISA. The seroprevalence for T. gondii was 23.4% (Confidence interval: CI 17.8-29.2%). Stray dogs from Selangor and Kuala Lumpur had the highest seroprevalence (32.4%; CI 13.2-45.5%) and lowest in those from Penang and Kedah (12.5%; CI 1.3-23.5%). Gender and breed were not associated with T. gondii seropositivity. However, adult dogs were more likely to be seropositive for T. gondii (OR=2.89; CI 1.1-7.7) compared with younger dogs. These results revealed that T. gondii is prevalent in stray dogs in the studied areas in Malaysia, and indicative of the level of environmental contamination of this parasite especially in urban areas.
Toxoplasmosis in humans and other animals is caused by the protozoan parasite Toxoplasma gondii. During the process of host cell invasion and parasitophorous vacuole formation by the tachyzoites, the parasite secretes Rhoptry protein 8 (ROP8), an apical secretory organelle. Thus, ROP8 is an important protein for the pathogenesis of T. gondii. The ROP8 DNA was constructed into a pVAX-1 vaccine vector and used for immunizing BALB/c mice. Immunized mice developed immune response characterized by significant antibody responses, antigen-specific proliferation of spleen cells, and production of high levels of IFN-γ (816 ± 26.3 pg/mL). Challenge experiments showed significant levels of increase in the survival period (29 days compared with 9 days in control) in ROP8 DNA vaccinated mice after a lethal challenge with T. gondii. Results presented in this study suggest that ROP8 DNA is a promising and potential vaccine candidate against toxoplasmosis.
Toxoplasma gondii primary infection in pregnancy is associated with poor obstetric outcomes. This study aimed to determine the seroprevalence of Toxoplasma infection in pregnant migrant and refugee women from Myanmar attending antenatal care in Thailand. A random selection of 199 residual blood samples from first antenatal screen in 2014-2015 was tested for Toxoplasma IgG and IgM antibodies. Seroprevalence of Toxoplasma infection was 31.7% (95% confidence interval = 25.6-38.4). Avidity testing in the three positive IgM cases indicated all were past infections. Multiparity (≥ 3 children) was significantly associated with higher Toxoplasma seropositivity rates. Seroprevalence of T. gondii infection in this pregnant population is similar to the only other report from Myanmar, where multiparity was also identified as a significant association. Toxoplasma infection is important in pregnant women. Nevertheless, in this marginalized population, this infection may be given less priority, due to resource constraints in providing the most basic components of safe motherhood programs.