AIM: A method was developed to separate contaminant-free viable Toxoplasma gondii cysts from brain samples of infected mice for molecular biology studies and reinfection.
MATERIALS AND METHODS: The mice brains were homogenized and washed with phosphate buffered saline (PBS) Tween 80 prior to fractionation using 19-22% dextran solution. Finally, the supernatant was purified by two-step membrane filtration (100-160 microm and < 10 microm) to obtain pure T. gondii cyst. The isolates were analyzed through microscopic observation, qPCR and by reinfection of new batch of mice.
RESULTS: T. gondii cysts were best isolated with 21% dextran solution and two step filtration.
CONCLUSIONS: The method was observed not to disrupt the integrity of the cysts containing bradyzoites. In addition, the isolated cysts in the filtrate were found to be contaminant-free, viable and able to infect healthy mice when introduced orally; which, mimics the natural infectivity pathway.
SAG2 is one of the major surface antigens of the intracellular protozoan parasite Toxoplasma gondii. In the present study, truncated recombinant SAG2(S) and full length recombinant SAG2(T) of T. gondii were optimally produced (approximately 15 mg/liter) in Pichia pastoris expression system using BMMY medium at pH 3, 25 degrees C in 0.5-1% methanol and a time-course of 1-2 days. The recombinant proteins were purified using a commercial gel filtration purification system obtaining approximately 33% recovery. The purified SAG2(S) and SAG2(T) showed molecular masses of 45 and 36 kDa by SDS-PAGE, respectively. The recombinant proteins were evaluated by Western blotting with patients' sera and demonstrated 90% sensitivity and 100% specificity for detection of toxoplasmosis. This study provided a means for large-scale expression and purification of SAG2, which should be useful for diagnosis of toxoplasmosis.
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.
Mice were chronically infected with cysts of ME49 strain of Toxoplasma gondii. At different periods post-infection, their spleens were removed and single cell suspensions were made. Lymphocyte transformation experiments were performed on the lymphocyte suspensions using three different kinds of antigens of ME49 strain of T. gondii, namely soluble, excretory/secretory and cystic forms. The results showed that the pattern of lymphocyte responsiveness was dependent on the kind of antigen employed for induction of the blastogenesis. Using soluble and cystic forms of the antigen, different periods of lymphocyte suppression and lymphocyte proliferation were demonstrated. However, with the use of excretory/secretory antigen, no significant suppression of lymphocyte stimulation was noted throughout the course of infection. Thus excretory/secretory antigen may be the best form of antigen for stimulation of the cell-mediated immune response and hence it appears to be a good candidate for vaccine in toxoplasmosis.
C57BL/6 mice were orally infected with different doses of cysts of ME49 strain of Toxoplasma gondii to produce groups of acutely and chronically infected mice. Sera were obtained at different periods post-infection. SDS-PAGE was ran with excretory/secretory antigens of ME49 and RH strains of T. gondii, followed by Western blot analyses using the above sera and anti- IgA, IgM, IgG as conjugates. The SDS-PAGE profiles of the two antigens were similar. However the antigenic bands showed variations in all blots, most evidently in IgA blots of chronic sera. IgG blots showed greatest similarities in reactive bands. In IgM blots, more common bands were shown in chronic sera than in acute sera. Variations and similarities in prominence of some bands and time of their appearance were also noted, especially in IgM and IgG blots of chronic sera. Thus antigenic variations and similarities are present in excretory/secretory products of different strains of T. gondii.
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.
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.
Toxoplasmosis is an important cause of congenital infection. The present study was performed to evaluate the usefulness of recombinant (r) GRA-7 cloned from nucleotides (n) 39-711 in discriminating between acute and chronic toxoplasmosis. First, commercial IgM, IgG and IgG avidity ELISAs were used to determine the serological profile of the sera. Serum samples were from 20 symptomatic patients with acute infection (low IgG avidity, IgM positive), 10 with chronic infection (high IgG avidity, IgM negative) and 10 with indeterminate IgG avidity (IgM positive) which were tested for IgG avidity status with an in-house developed IgG avidity Western blot using the rGRA-7 recombinant antigen. All 20 sera from cases of probable acute infection showed bands which either faded out completely or reduced significantly in intensity after treatment with 8 M urea, whereas the band intensities of the 10 serum samples from chronic cases remained the same. Of the 10 sera with indeterminate IgG avidity status, after treatment with 8 M urea the band intensities with six sera remained the same, two sera had completely faded bands and another two sera had significantly reduced band intensities. Discrimination between acute and chronic toxoplasmosis was successfully performed by the in-house IgG avidity Western blot.
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.
A truncated form of surface antigen 2 (SAG2) of the protozoan parasite Toxoplasma gondii was cloned and expressed in the methylotrophic yeast Pichia pastoris. This recombinant antigen, designated as recSAG2-N, contained only the N-terminal half of the native SAG2. The recSAG2-N was secreted by the Pichia pastoris into the culture supernatant, and it was harvested by using the trichloroacetic acid precipitation method. Specificity of recSAG2-N was evaluated in western blot assays. Fifty human serum samples, including 32 from confirmed cases of toxoplasmosis, were tested. Results from the assays showed that recSAG2-N reacted with sera from the toxoplasmosis cases only. In vivo experiments showed that serum from mice which received recSAG2-N reacted with the native SAG2 of T. gondii.
Toxoplasmosis is a widespread parasitic infection by Toxoplasma gondii, a parasite with at least three distinct clonal lineages. This article reports the whole genome sequencing and de novo assembly of T. gondii RH (type I representative strain), as well as genome-wide comparison across major T. gondii lineages. Genomic DNA was extracted from tachyzoites of T. gondii RH strain and its identity was verified by PCR and LAMP. Subsequently, whole genome sequencing was performed, followed by sequence filtering, genome assembly, gene annotation assignments, clustering of gene orthologs and phylogenetic tree construction. Genome comparison was done with the already archived genomes of T. gondii. From this study, the genome size of T. gondii RH strain was found to be 69.35Mb, with a mean GC content of 52%. The genome shares high similarity to the archived genomes of T. gondii GT1, ME49 and VEG strains. Nevertheless, 111 genes were found to be unique to T. gondii RH strain. Importantly, unique genes annotated to functions that are potentially critical for T. gondii virulence were found, which may explain the unique phenotypes of this particular strain. This report complements the genomic archive of T. gondii. Data obtained from this study contribute to better understanding of T. gondii and serve as a reference for future studies on this parasite.
Toxoplasma gondii is the etiologic agent of toxoplasmosis, a disease which can lead to morbidity and mortality of the fetus and immunocompromised individuals. Due to the limited effectiveness or side effects of existing drugs, the search for better drug candidates is still ongoing. In this study, we performed structure-based screening of potential dual-targets inhibitors of active sites of T. gondii drug targets such as uracil phosphoribosyltransferase (UPRTase) and adenosine kinase (AK). First screening of virtual compounds from the National Cancer Institute (NCI) was performed via molecular docking. Subsequently, the hit compounds were tested in-vitro for anti- T. gondii effect using cell viability assay with Vero cells as host to determine cytotoxicity effects and drug selectivities. Clindamycin, as positive control, showed a selectivity index (SI) of 10.9, thus compounds with SI > 10.9 specifically target T. gondii proliferation with no significant effect on the host cells. Good anti- T. gondii effects were observed with NSC77468 (7-ethoxy-4-methyl-6,7-dihydro-5H-thiopyrano[2,3-d]pyrimidin-2-amine) which showed SI values of 25. This study showed that in-silico selection can serve as an effective way to discover potentially potent and selective compounds against T. gondii.
Toxoplasma gondii, a ubiquitous pathogen that infects nearly all warm-blooded animals and humans, can cause severe complications to the infected people and animals as well as serious economic losses and social problems. Here, one local strain (TgPIG-WH1) was isolated from an aborted pig fetus, and the genotype of this strain was identified as ToxoDB #3 by the PCR RFLP typing method using 10 molecular markers (SAG1, SAG2, alternative SAG2, SAG3, BTUB, GRA6, L358, PK1, C22-8, C29-2 and Apico). A comparison of the virulence of this isolate with other strains in both mice and piglets showed that TgPIG-WH1 was less virulent than type 1 strain RH and type 2 strain ME49 in mice, and caused similar symptoms to those of ME49 such as fever in piglets. Additionally, in piglet infection with both strains, the TgPIG-WH1 caused a higher IgG response and more severe pathological damages than ME49. Furthermore, TgPIG-WH1 caused one death in the 5 infected piglets, whereas ME49 did not, suggesting the higher virulence of TgPIG-WH1 than ME49 during piglet infection. Experimental infections indicate that the virulence of TgPIG-WH1 relative to ME49 is weaker in mice, but higher in pigs. This is probably the first report regarding a ToxoDB #3 strain from pigs in Hubei, China. These data will facilitate the understanding of genetic diversity of Toxoplasma strains in China as well as the prevention and control of porcine toxoplasmosis in the local region.
Toxoplasmosis is a disease caused by the protozoan parasite Toxoplasma gondii (T. gondii). Human toxoplasmosis seroprevalence in Malaysia has increased since it was first reported in 1973 as shown in previous reviews of 1991 and 2007. However, over a decade since the last review, comprehensive data on toxoplasmosis in Malaysia is lacking. This work aimed at reviewing articles on toxoplasmosis research in Malaysia in order to identify the research gaps, create public awareness, and efforts made so far and proffer management options on the disease. The present review examines the available published research articles from 2008 to 2018 related to toxoplasmosis research conducted in Malaysia. The articles reviewed were retrieved from nine credible databases such as Web of Science, Google Scholar, ScienceDirect, PubMed, Scopus, Springer, Wiley online library, Ovid, and Cochrane using the keywords; Malaysia, toxoplasmosis, Toxoplasma gondii, toxoplasma encephalitis, seroprevalence, human immunodeficiency virus (HIV) patients, pregnant women, genotype strain, anti-toxoplasma antibodies, felines, and vaccine. The data highlighted seropositive cases from healthy community members in Pangkor Island (59.7%) and among migrant workers (57.4%) at alarming rates, as well as 42.5% in pregnant women. Data on animal seroprevalence were limited and there was no information on cats as the definitive host. Genetic characterization of Toxoplasma gondii from HIV patients; pregnant women, and domestic cats is lacking. This present review on toxoplasmosis is beneficial to researchers, health workers, animal health professionals, and policymakers. Therefore, attention is required to educate and enlighten health workers and the general public about the risk factors associated with T. gondii infection in Malaysia.