An evaluation of a recombinant multiepitope based antigen for detection of Toxoplasma gondii specific antibodies.

BACKGROUND: The inefficiency of the current tachyzoite antigen-based serological assays for the serodiagnosis of Toxoplasma gondii infection mandates the need for acquirement of reliable and standard diagnostic reagents. Recently, epitope-based antigens have emerged as an alternative diagnostic marker for the achievement of highly sensitive and specific capture antigens. In this study, the diagnostic utility of a recombinant multiepitope antigen (USM.TOXO1) for the serodiagnosis of human toxoplasmosis was evaluated. METHODS: An indirect enzyme-linked immunosorbent assay (ELISA) was developed to evaluate the usefulness of USM.TOXO1 antigen for the detection of IgG antibodies against Toxoplasma gondii in human sera. Whereas the reactivity of the developed antigen against IgM antibody was evaluated by western blot and Dot enzyme immunoassay (dot-EIA) analysis. RESULTS: The diagnostic performance of the new antigens in IgG ELISA was achieved at the maximum values of 85.43% and 81.25% for diagnostic sensitivity and specificity respectively. The USM.TOXO1 was also proven to be reactive with anti- T. gondii IgM antibody. CONCLUSIONS: This finding makes the USM.TOXO1 antigen an attractive candidate for improving the toxoplasmosis serodiagnosis and demonstrates that multiepitope antigens could be a potential and promising diagnostic marker for the development of high sensitive and accurate assays.

Experimental identification and characterization of 97 novel npcRNA candidates in Salmonella enterica serovar Typhi.

We experimentally identified and characterized 97 novel, non-protein-coding RNA candidates (npcRNAs) from the human pathogen Salmonella enterica serovar Typhi (hereafter referred to as S. typhi). Three were specific to S. typhi, 22 were restricted to Salmonella species and 33 were differentially expressed during S. typhi growth. We also identified Salmonella Pathogenicity Island-derived npcRNAs that might be involved in regulatory mechanisms of virulence, antibiotic resistance and pathogenic specificity of S. typhi. An in-depth characterization of S. typhi StyR-3 npcRNA showed that it specifically interacts with RamR, the transcriptional repressor of the ramA gene, which is involved in the multidrug resistance (MDR) of Salmonella. StyR-3 interfered with RamR-DNA binding activity and thus potentially plays a role in regulating ramA gene expression, resulting in the MDR phenotype. Our study also revealed a large number of cis-encoded antisense npcRNA candidates, supporting previous observations of global sense-antisense regulatory networks in bacteria. Finally, at least six of the npcRNA candidates interacted with the S. typhi Hfq protein, supporting an important role of Hfq in npcRNA networks. This study points to novel functional npcRNA candidates potentially involved in various regulatory roles including the pathogenicity of S. typhi.

Epitope-based vaccine as a universal vaccination strategy against Toxoplasma gondii infection: A mini-review.

Despite the significant progress in the recent efforts toward developing an effective vaccine against toxoplasmosis, the search for new protective vaccination strategy still remains a challenge and elusive goal because it becomes the appropriate way to prevent the disease. Various experimental approaches in the past few years showed that developing a potential vaccine against the disease can be achievable. The combination of multi-epitopes expressing different stages of the parasite life cycle has become an optimal strategy for acquiring a potent, safe, and effective vaccine. Epitope-based vaccines have gained attention as alternative vaccine candidates due to their ability of inducing protective immune responses. This mini-review highlights the current status and the prospects of Toxoplasma gondii vaccine development along with the application of epitope-based vaccine in the future parasite immunization as a novel under development and evaluation strategy.

Immunogenicity of Multiepitope Vaccine Candidate against Toxoplasma gondii Infection in BALB/c Mice.

BACKGROUND: Toxoplasma gondii is a widely prevalent intracellular protozoan parasite which causes serious clinical and veterinary problems. Development of an effective vaccine for controlling toxoplasmosis is an extremely important aim. In the present study, the protective efficacy of recombinant multiepitope antigen (USM.TOXO1) expressing nine potential epitopes identified from SAG1, GRA2, and GRA7 of Toxoplasma gondii was evaluated in BALB/c mice. METHODS: Mice were immunized subcutaneously with three doses of USM.TOXO1 antigen (10 µg/ml). Following the immunization, the IgG antibody, IgG subclass, IFN-γ and IL-4 production were evaluated using ELISA, the study was conducted at Animal Research and Service Center (ARASC), USM Health Campus in 2016. RESULTS: Mice immunized with USM.TOXO1 significantly induced a mixed Th1/Th2 response polarized toward the IgG1 antibody isotype. While the cytokine analysis revealed a significant release of IFN-γ cytokines. CONCLUSION: USM.TOXO1 is a potential vaccine candidate that elicits strong immunity in BALB/c mice. The proven immunogenicity of the generated antigen can serve as a premise for further use of epitope-based vaccine in the immunoprevention of human and animal toxoplasmosis.

Occurrence of Intestinal Parasitic Contamination in Select Consumed Local Raw Vegetables and Fruits in Kuantan, Pahang.

Intestinal parasitic infections are one of the most common causes of human diseases that result in serious health and economic issues in many developing and developed countries. Raw vegetables and fruits play an important role in transmitting parasites to humans. Hence, the aim of this study was to investigate the parasitological contamination of select commonly consumed local leafy vegetables and fruits in Kuantan, Malaysia. One kilogram of locally consumed raw vegetables and fruits were collected randomly from the Kuantan wet market (Pasar Tani) during the monsoon season (November 2014-January 2015) and the dry season (February 2015-April 2015). A standard wet mount procedure and modified Ziehl-Neelsen staining were used for the detection of parasites. In the present study, the examination of vegetables revealed five different parasite species. The vegetable samples collected from Kuantan's wet market were positive for both helminthes and protozoa. However, the fruits samples were negative for parasitic contamination. Pegaga was the most contaminated leafy vegetable in this study, and Strongyloides was the parasite found most frequently. Furthermore, there was a high diversity in the type of parasites observed during the dry season compared to the monsoon season. Therefore, further action should be taken to reduce the occurrence of parasitic contamination in vegetables by implementing the principles of good agricultural practice and improving water treatment efficacy.

Design and evaluation of a recombinant multi-epitope antigen for serodiagnosis of Toxoplasma gondii infection in humans.

BACKGROUND: Serological investigation remains the primary approach to achieve satisfactory results in Toxoplasma gondii identification. However, the accuracy of the native antigen used in the current diagnostic kits has proven to be insufficient as well as difficult to standardize, so significant efforts have been made to find alternative reagents as capture antigens. Consequently, multi-epitope peptides are promising diagnostic markers, with the potential for improving the accuracy of diagnostic kits. In this study, we described a simple, inexpensive and improved strategy to acquire such diagnostic markers. The study was aimed at producing novel synthetic protein consisting of multiple immunodominant epitopes of several T. gondii antigens. FINDINGS: To accomplish our goals, a single synthetic gene of approximately 456 bp, which encodes potential epitopes of T. gondii antigens, was successfully constructed using gene assembly PCR. The constructed gene was cloned into a pET32a expression vector and transformed into BL21 E. coli. The entire protein was successfully expressed and purified. Subsequently, the preliminary diagnostic performance of expressed protein was evaluated by developing IgG enzyme-linked immunosorbent assay (ELISA) and Western blot analysis using human sera. The results showed 100 % sensitivity and specificity. CONCLUSION: A purified protein expressing multi-immunodominant epitopes of T. gondii was generated. Further studies are required to evaluate the immunogenicity in animal models and to verify the immuno-reactivity of USM.TOXO1 as a diagnostic antigen.