Surface Antigen Gene Research Articles

Unwrap RAP1's Mystery at Kinetoplastid Telomeres.

Although located at the chromosome end, telomeres are an essential chromosome component that helps maintain genome integrity and chromosome stability from protozoa to mammals. The role of telomere proteins in chromosome end protection is conserved, where they suppress various DNA damage response machineries and block nucleolytic degradation of the natural chromosome ends, although the detailed underlying mechanisms are not identical. In addition, the specialized telomere structure exerts a repressive epigenetic effect on expression of genes located at subtelomeres in a number of eukaryotic organisms. This so-called telomeric silencing also affects virulence of a number of microbial pathogens that undergo antigenic variation/phenotypic switching. Telomere proteins, particularly the RAP1 homologs, have been shown to be a key player for telomeric silencing. RAP1 homologs also suppress the expression of Telomere Repeat-containing RNA (TERRA), which is linked to their roles in telomere stability maintenance. The functions of RAP1s in suppressing telomere recombination are largely conserved from kinetoplastids to mammals. However, the underlying mechanisms of RAP1-mediated telomeric silencing have many species-specific features. In this review, I will focus on Trypanosoma brucei RAP1's functions in suppressing telomeric/subtelomeric DNA recombination and in the regulation of monoallelic expression of subtelomere-located major surface antigen genes. Common and unique mechanisms will be compared among RAP1 homologs, and their implications will be discussed.

Molecular prevalence, associated risk factors and phylogenetic evaluation of Theileria lestoquardi in the blood samples of small ruminants.

Raising small ruminants is the main source of income for farmers in Pakistan especially in rural areas of Dera Ghazi Khan in Punjab. Despite having large sheep population, the prevalence of intra-erythrocytic protozoa, Theileria (T.) lestoquardi, has never been reported from this area. This study was conducted to fill this knowledge gap and 333 blood samples of apparently healthy small ruminants (168 sheep and 165 goats) along with their epidemiological data were collected from Dera Ghazi Khan district during August till November 2022. The polymerase chain reaction (PCR) analysis amplified a 785 base pair amplicon specific for the Merozoite surface antigen (ms 1-2) gene of T. lestoquardi in 2 out of the 168 (3.3%) sheep blood samples, while no goat blood sample out of 165 was found to be infected with T. lestoquardi. DNA sequencing confirmed the presence of Theileria lestoquardi in both samples and phylogenetic analysis revealed that these amplicon resembled the partial ms 1-2 gene sequences detected in small ruminants from Pakistan, India Iran and Egypt. All the studied epidemiological factors (age, sex, breed, size of herd, dogs with herd, composition of herd, size of herd and Tick burden on sheep) were not found associated with the prevalence of T. lestoquardi. In conclusion, this study reports a low prevalence of T. lestoquardi infection in the Dera Ghazi Khan District of Punjab, Pakistan. The data generated from this work will help pave the way for the prophylactic detection and control of ovine and caprine theileriosis in the region.

Detection and quantification of Babesia bovis and Babesia bigemina using different target genes

Molecular assays have been widely used for the detection and quantification of bovine babesiosis due to their high sensitivity and specificity. However, variations in the sensitivity of pathogen detection may occur depending on the selected target gene. Thus, this study aimed to compare the detection sensitivity (DS) of Babesia bovis and B. bigemina infection levels in artificially and naturally infected cattle using quantitative PCR (qPCR) and six target genes. For B. bovis, the merozoite surface antigen genes 2b and 2c (msa-2b and msa-2c), and the mitochondrial cytochrome b gene (cybmt) were used. For B. bigemina, the genes encoding the proteins associated with rhoptry 1c (rap-1c), rap-1a, and cybmt were used. Six bovines, free of babesiosis, were artificially infected with 1 × 10−8 red blood cells infected (iRBC) with B. bovis (n = 3) or 1 × 10−6B. bigemina iRBC (n = 3). The animals were evaluated daily until parasitemia was confirmed (≥ 2.0%). The quantity of iRBC present in each animal was determined by examining blood smears. Blood samples were then subjected to DNA extraction, serial dilution, and qPCR analysis to determine the DS of each target gene. In addition, 30 calves naturally infected by Babesia spp. were also evaluated using the same six target genes. Regarding the artificial infection, B. bovis cybmt showed 25-fold higher sensitivity than the msa-2b and msa-2c genes, while the B. bigemina cybmt exhibited 5-fold and 25-fold higher sensitivity than the rap-1a and rap-1c genes, respectively. The rap-1a gene was found to be 5 times more sensitive than the rap-1c gene, while the B. bovis msa-2b and msa-2c genes exhibited similar DS. The positive frequencies of naturally infected calves for the target cybmt, msa-2b, and msa-2c genes (B. bovis) were: 100%, 33.3% and 50%, while cybmt, rap-1a, and rap-1c genes (B. bigemina) were 90%, 83.3%, and 63.3%, respectively. This study may contribute to the selection of suitable genes for molecular monitoring of bovine babesiosis. Mitochondrial genes could be considered as an alternative to improve the sensitivity of B. bovis and B. bigemina detection using qPCR.

Cloning and periplasmic soluble expression of hepatitis B surface antigen gene in Escherichia coli.

The objective of this study was to clone and express the hepatitis B surface antigen gene (HBsAg) in Escherichia coli (E. coli), thereby aiming to develop potential local therapeutics for combating Hepatitis B virus (HBV) infection in the Pakistani community by producing HBsAg in E. coli. Blood serum samples were collected from hepatitis B-infected patients, and their genomic DNA was extracted. Real-time and nested polymerase chain reaction (PCR) was performed to amplify the HBsAg gene. The gene of interest was cloned into the pET20b expression vector and transformed into E. coli BL21 (DE3) using Isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. The gene's precise size was confirmed with gene-specific external and internal primers (681 bp and 400 bp, respectively). The HBsAg gene was successfully sequenced and submitted to GenBank, exhibiting 98% homology with targeted HBV sequences worldwide. The expression of HBsAg protein was confirmed through silver staining, Coomassie staining, western blot, and dot blot analysis. The expressed protein clones are now available for further development as a local recombinant DNA vaccine to prevent hepatitis B viral infection in the local community.

Prevalence and Risk Factors Associated with Theileria annulata Infection in Two Bovine Portuguese Autochthonous Breeds.

Tropical Bovine Theileriosis is an important tick-borne disease. This study aims to assess the occurrence of Theileria annulata infection in two indigenous Portuguese cattle breeds. A total of 843 blood samples collected from animals of Alentejana (n = 420) and Mertolenga (n = 423) breeds were analyzed. The detection of Theileria annulata was determined by amplification of a fragment of the merozoite-pyroplasm surface antigen gene with 319 base pairs (bp). The prevalence found (10.8%) is lower than that reported in previous studies (21.3%). A statistically significant difference was found for positivity between breeds (p < 0.05). There is also a higher probability of older animals being positive compared to younger ones (p < 0.05). The region where Mertolenga animals are located is shown to have a significant impact on positivity (p < 0.05). Thus, the development of sustainable T. annulata control strategies and their implementation, adapted to the epidemiological conditions of higher risk, will be extremely important.

Phylogenic study of toxoplasmosis in aborted women in Al- Diwnaniya province

The current study included the detection of Toxoplasma gondii from aborted women attented to the Maternity and Childhood Teaching Hospital to detected the rate of abortion due to toxoplasmosis using PCR technique, the samples which was used placenta and fetus, this study also identify the differentiation between it by phylogenic with SAG3 gene.&#x0D; A total of 60 ( placenta and fetus) samples were collected randomly from aborted women who received to the Maternity and Childhood Teaching Hospital.&#x0D; The present study include molecular method to detected small subunit ribosomal RNA ( ssRNA) using Nested Polymerase Chain Reaction ( nPCR) ,the results of the nPCR for ssRNA gene, the result showed that the rate of toxoplasmosis in the aborted women was Positive 60%. (There is a statistically significant between species in P &lt;0.05).&#x0D; The present study also detected for Surface Antigen (SAG3) gene to conform the nPCP result and to send the samples for sequences.&#x0D; The SAGA gene showed 100% positive results according to the ssRNA gene .&#x0D; T.gondii isolates surface Ag SAG3 gene with NCBI BLAST Toxoplasma gondii isolates, and the DNA nucleotides sequencing analysis of surface Ag SAG3 complete gene was show clear gentic variation between isolates from different host. &#x0D;

Expression of CD47 and its ligands in pregnant mice infected with Toxoplasma gondii during pregnancy

Expression of CD47 and its ligands in pregnant mice infected with Toxoplasma gondii during pregnancy

Multiplex PCR for rapid differential diagnosis of co-prevalent species of Theileria (Theileria annulata and Theileria orientalis) in cattle

Theileriosis is a tick-borne disease that causes enormous losses in the dairy industry. There are several species of Theileria that can infect bovines. Generally, more than one species are prevalent in any geographical area; thus, chances of co-infections are high. Differentiation of these species may not be possible by microscopic examination or serological tests. Therefore, in this study, a multiplex PCR assay was standardized and evaluated for rapid and simultaneous differential detection of two species of Theileria viz., Theileria annulata and Theileria orientalis. Species-specific primers were designed to target the merozoite piroplasm surface antigen gene (TAMS1) of T. annulata and the major piroplasm surface protein gene of T. orientalis, yielding specific amplicon of 229bp and 466bp, respectively. The sensitivity of multiplex PCR was 102 and 103 copies for T. annulata and T. orientalis, respectively. The simplex and multiplex PCRs were specific and showed no cross-reactivity with other hemoprotozoa for either primer. For comparative evaluation, blood samples from 216 cattle were tested by simplex and multiplex PCR for both species. Using multiplex PCR, 131 animals were found infected for theileriosis, of which 112 were infected with T. annulata, five were infected with T. orientalis, and 14 had mixed infections. This is the first report of T. orientalis from Haryana, India. Representative sequences of T. annulata (ON248941) and T. orientalis (ON248942) were submitted in GenBank. The standardized multiplex PCR assay used in this study was specific, sensitive, for the screening of field samples.

Theileria infection in bullfighting cattle in Thailand.

An apicomplexan protozoan parasite, namely, Theileria, primarily causes theileriosis in cattle worldwide. The virulence of the disease has been neglected because of it's low pathogenicity. However, the disease can have a substantial effect, depending on the virulence of the species, low host immunity, and coinfection. In Thailand, the molecular detection of Theileria infection in bullfighting cattle and its hematological alterations have not been reported. Thus, this study aimed to identify Theileria species in bullfighting cattle in Thailand. Blood samples were collected from bullfighting cattle presented at the Prince of Songkla University Animal Hospital and were determined on the basis of hematological evaluation and DNA extraction. Molecular detection using the 18s rRNA and merozoite surface antigen genes was conducted for Theileria spp. and Theileria orientalis, respectively. In addition, bidirectional sequencing of the positive samples was performed. Hematological alterations between Theileria infected and uninfected groups were statistically evaluated. The levels of Theileria spp. and T. orientalis infection in bullfighting cattle were 44.62% (58/130) and 41.54% (54/130), respectively. Theileria orientalis, Theileria sinensis, and Theileria spp. infections were identified in bullfighting cattle samples. Hematological evaluation indicated that the red blood cell (RBC) level was significantly lower in Theileria-infected cattle. This study was the first to use molecular techniques in the identification of Theileria infection in bullfighting cattle in Thailand, with nearly one-half of the study population infected. Theileria infection in bullfighting cattle altered the RBC level, resulting in anemia. Therefore, tick control measures should be promoted.

Species-Specific Duplication of Surface Antigen Genes in Paramecium.

Paramecium is a free-living ciliate that undergoes antigenic variation and still the functions of these variable surface antigen coats in this non-pathogenic ciliate remain elusive. Only a few surface antigen genes have been described, mainly in the two model species P. tetraurelia strain 51 and P. primaurelia strain 156. Given the lack of suitable sequence data to allow for phylogenetics and deeper sequence comparisons, we screened the genomes of six different Paramecium species for serotype genes and isolated 548 candidates. Our approach identified the subfamilies of the isogenes of individual serotypes that were mostly represented by intrachromosomal gene duplicates. These showed different duplication levels, and chromosome synteny suggested rather young duplication events after the emergence of the P. aurelia species complex, indicating a rapid evolution of surface antigen genes. We were able to identify the different subfamilies of the surface antigen genes with internal tandem repeats, which showed consensus motifs across species. The individual isogene families showed additional consensus motifs, indicating that the selection pressure holds individual amino acids constant in these repeats. This may be a hint of the receptor function of these antigens rather than a presentation of random epitopes, generating the variability of these surface molecules.

Anti-Inflammatory Effect of Korean Propolis on Helicobacter pylori-Infected Gastric Mucosal Injury Mice Model.

Propolis, a natural resinous substance obtained from a variety of buds and plants, has been reported to possess various biological functions. Several recent studies have demonstrated the inhibitory effects of propolis on the growth of Helicobacter&amp;nbsp;pylori (H. pylori) in vitro; however, current research efforts on Korean propolis (KP) remain insufficient especially in vivo. Our study aims to investigate the anti-inflammatory effect and molecular mechanism of KP on mouse gastric mucosa during H. pylori infection. We examined an in vivo H. pylori-induced gastric mucosal injury mice model. We found that KP inhibited the growth of H. pylori and attenuated the expression of H. pylori virulence factors such as cytotoxin-associated gene A, encoding urease A subunit, surface antigen gene and neutrophil-activating protein A. Moreover, KP reduced both gross lesions and pathological scores in H. pylori-challenged mice. In addition, KP markedly restrained the production of pro-inflammatory cytokines and nitric oxide levels compared with an untreated H. pylori-infected group. In particular, we found that KP repressed the phosphorylation of IκBα and NF-κB p65 subunit, and subsequently suppressed their downstream target genes. Taken together, these findings demonstrate the beneficial effects of KP on inflammation through the inhibition of NF-κB signaling as well as inhibition of H. pylori growth in a mouse model infected with H. pylori. This suggests the potential application of KP as a natural supplement for patient's suffering from gastric mucosal injury caused by H. pylori infection.

Comparative Performance and Evaluation of Two Molecular Assays and Conventional Detection of Theileria annulata in Bovines

Background: Theileria annulata is predominant and of utmost economical importance tick borne pathogen of bovines in the region which is routinely diagnosed based on the microscopic examination of Romanowsky stained thin blood smears. Present study was intended to evaluate and analyze the detection efficacy of a commercial polymerase chain reaction kit based assay in comparison to conventional PCR assay and classical microscopy for detection of T. annulata from blood samples of bovines from Punjab state. Methods: In this comparative study 360 bovine blood samples from various districts of agro-climatic zones in Punjab were first screened for T. annulata by Giemsa-stained thin blood smear (GSTBS) examination. The same panel of blood samples was tested for T. annulata by a commercial PCR kit Bovi-TheiDX Theileria annulata (Genext Genomics) (PCR1) and established conventional PCR assay targeting merozoite piroplasm surface antigen (Tams1) gene of T. annulata (PCR2). Result: Out of 360 samples screened, positivity of T. annulata by GSTBS was found to be 12.5% (45/360) with a sensitivity of 37.20% and specificity of 96.00% when compared with commercial kit (PCR1), the difference was statistically significant (p less than .0001). The detection prevalence by PCR1 29.70% (107/360) and PCR2 assay 33.90% (122/360) showed significant (p less than .0001) difference. The conventional PCR targeting the Tams1 gene (PCR2) was found to be more sensitive (84.6%) (p less than 0.001) than PCR1 (69.7%).

Applicability of oral fluid samples for tracking hepatitis B virus mutations, genotyping, and phylogenetic analysis.

Little is known about the usefulness of saliva samples for hepatitis B virus (HBV) genotyping and mutation analysis. The aim of this study was to evaluate the usefulness of oral fluid samples to determine HBV genotype distribution, S/polymerase mutations, and HBV subpopulation diversity among chronically HBV-infected individuals. Serum and oral fluid samples were obtained from 18 individuals for PCR and nucleotide sequencing of the HBV surface antigen gene. Biochemical analysis of liver enzymes (ALT, AST, GGT) and HBV, HCV, and HIV serological tests were also performed. All serum samples were HBsAg (+), anti-HBc (+), and anti-HBs (-); 55.6% were HBeAg (+)/anti-HBe (-), and 11.1% were anti-HIV (+). The mean HBV DNA viral load was 6.1 ± 2.3 log IU/mL. The HBV genotype distribution was as follows: A, 72.2%; D, 11.1%; E, 5.6%; F, 11.1%. A concordance of 100% in genotype classification and 99.8% in sequence similarity between paired oral fluid and serum samples was observed. HBsAg mutations were detected in all samples, but no resistance mutations were found in the polymerase gene. This study demonstrates that oral fluid samples can be used reliably for tracking HBV mutations, genotyping, and phylogenetic analysis. This could be important for molecular epidemiology studies with hard-to-reach populations.

Evaluation of sporozoite and macroschizont antigen (Spm2) of Theileria annulata for its diagnostic potential

Theileria are tick-borne apicomplexan parasites, which mainly infect ruminants in tropical and subtropical regions of the world. The present study was directed to investigate the serological methods for the diagnosis of theileriosis in crossbred cattle. Blood samples (n = 176) were collected from the regional cattle populations of Bihar state situated at the Gangetic plains of India. Microscopic examination of blood smears from the cattle revealed the presence of tick-borne infectious organisms (Theileria and Anaplasma) in the region. PCR-based detection of Tams1 (Theileria annulata merozoite surface antigen) gene and the sequencing of 18S rRNA amplicon from the blood samples confirmed T. annulata as the primary causative agent of theileriosis in cattle of the Bihar region. Similarly, the amplification of the msp5 gene confirmed Anaplasma marginale infection. For the large-scale epidemiological investigation, sporozoite and macroschizont (spm2) partial gene from T. annulata was cloned in pET-28a (+) vector and overexpressed in E. coli BL21 cells. Overexpressed recombinant-Spm2 (43 kDa) was purified by Ni-NTA affinity chromatography and was used for immunodetection of theileriosis in cattle serum samples. Sequence analysis of the cloned partial spm2 gene in this study showed multiple SNPs (single nucleotide polymorphisms) in T. annulata. Recombinant-Spm2 antigen was explicitly recognised by the immunoglobulins (IgG) of the cattle naturally infected with Theileria. Further, an indirect enzyme-linked immunosorbent assay (ELISA) was developed using partial r-Spm2 antigen that exhibited high sensitivity (100 %) and specificity (90.9 %). Thus, this study suggests that partial r-Spm2 can be used as a diagnostic antigen for seroepidemiological studies of T. annulata infection in crossbred cattle.

Variant analysis of the sporozoite surface antigen gene reveals that asymptomatic cattle from wildlife-livestock interface areas in northern Tanzania harbour buffalo-derived T. parva

Buffalo-derived Theileria parva can ‘break through’ the immunity induced by the infection and treatment vaccination method (ITM) in cattle. However, no such ‘breakthroughs’ have been reported in northern Tanzania where there has been long and widespread ITM use in pastoralist cattle, and the Cape buffalo (Syncerus caffer) is also present. We studied the exposure of vaccinated and unvaccinated cattle in northern Tanzania to buffalo-derived T. parva using p67 gene polymorphisms and compared this to its distribution in vaccinated cattle exposed to buffalo-derived T. parva in central Kenya, where vaccine ‘breakthroughs’ have been reported. Additionally, we analysed the CD8+ T cell target antigen Tp2 for positive selection. Our results showed that 10% of the p67 sequences from Tanzanian cattle (n = 39) had a buffalo type p67 (allele 4), an allele that is rare among East African isolates studied so far. The percentage of buffalo-derived p67 alleles observed in Kenyan cattle comprised 19% of the parasites (n = 36), with two different p67 alleles (2 and 3) of presumptive buffalo origin. The Tp2 protein was generally conserved with only three Tp2 variants from Tanzania (n = 33) and five from Kenya (n = 40). Two Tanzanian Tp2 variants and two Kenyan Tp2 variants were identical to variants present in the trivalent Muguga vaccine. Tp2 evolutionary analysis did not show evidence for positive selection within previously mapped epitope coding sites. The p67 data indicates that some ITM-vaccinated cattle are protected against disease induced by a buffalo-derived T. parva challenge in northern Tanzania and suggests that the parasite genotype may represent one factor explaining this.

Molecular detection of Theileria annulata infection in cattle by conventional PCR and quantitative real time PCR in India.

Theileria annulata (T. annulata) is a tick-borne apicomplexan parasite that affects bovine. It is endemic in many tropical and subtropics areas, including Odisha, India. The objective of this study is to identify T. annulata infection in the peripheral blood of cattle as a biological sample by conventional PCR (cPCR) and quantitative PCR (qPCR). The phylogenetic analysis was done using the T. annulata merozoite surface antigen (Tams 1) gene. Out of 552 samples of examined blood smears by microscopy, 454 (82.24%) animals were positive for Theileria species. Out of 454 samples, 96 samples were further examined by both cPCR and qPCR, 52 samples (54.16%) were found positive for T. annulata in both PCR methodologies. Phylogenetic analysis revealed that T. annulata Odisha isolate was closely related to T. annulata Uttarakhand, India isolate (KM061799) and Hyderabad, India isolate (MK034702) with Nucleotide sequence identity 95.36%, 95.25%, respectively. This is the first study to detect T. annulata by qPCR in Odisha and supported that both PCR techniques were equally effective for the detection of Tams 1 gene of T. annulata in cattle's blood.

Characterization of the variable merozoite surface antigen (VMSA) gene family of Babesia orientalis.

Due to its wide presence in apicomplexan parasites as well as high polymorphism and antigenic diversity, the variable merozoite surface antigen (VMSA) family in Babesia sp. has attracted increasing attention of researchers. Here, all the reported VMSA genes of Babesia spp. were obtained from GenBank, and multiple alignments were performed by using conserved regions to blast the Babesia orientalis genome database (unpublished data). Five MSA genes (named MSA-2a1, MSA-2a2, MSA-2c1, MSA-1, and MSA-2c2, respectively) were identified, sequenced, and cloned from B. orientalis, which were shown to encode proteins with open reading frames ranging in size from 266 (MSA-2c1) to 317 (MSA-1) amino acids. All the five proteins contain an MSA-2c superfamily conserved domain, with an identical signal peptide and glycosyl phosphatidyl inositol (GPI)-anchor for each of them. The five proteins were also predicted to contain B cell epitopes, with only three for BoMSA-2c1, the smallest protein in the BoVMSA family, while at least six for each of the others. Notably, BoMSA-2a1 has 2 identical copies, a specific phenomenon only present in B. orientalis. This research has determined the MSA genes of B. orientalis and provides a genetic basis for further research of functional genes in B. orientalis.

Genetic Diversity of Babesia bovis MSA-1, MSA-2b and MSA-2c in China.

The apicomplexan parasite Babesia bovis is a tick-borne intracellular hemoprotozoan parasite that is widespread across China. Genetic diversity is an important strategy used by parasites to escape the immune responses of their hosts. In our present study, 575 blood samples, collected from cattle in 10 provinces, were initially screened using a nested PCR (polymerase chain reaction) for detection of B. bovis infection. To perform genetic diversity analyses, positive samples were further amplified to obtain sequences of three B. bovis merozoite surface antigen genes (MSA-1, MSA-2b, MSA-2c). The results of the nested PCR approach showed that an average of 8.9% (51/575) of cattle were positive for B. bovis infection. Phylogenetic analyses of the predicted amino acid sequences revealed that unique antigen variants were formed only by Chinese isolates. Our findings provide vital information for understanding the genetic diversity of B. bovis in China.

Deep sequencing of hepatitis B surface antigen gene in the preserved umbilical cords in immunoprophylaxis failure against mother-to-child HBV transmission

BackgroundVaccine escape mutants (VEMs) are one of the causes of breakthrough infections in the mother-to-child transmission of hepatitis B virus (HBV). We hypothesized that VEMs existing as minor populations in the maternal blood are associated with breakthrough infections in children. We sought to determine whether VEMs exist as minor populations in the preserved umbilical cords of children with breakthrough infections.Case presentationTwo families (Family 1: three children, Family 2: two children) were enrolled. Despite immunoprophylaxis, a breakthrough infection occurred in two Family 1 children and two Family 2 children. Preserved umbilical cords, serum, and nails were used for the HBV DNA analysis. To detect VEMs, we performed direct and deep sequencing of hepatitis B surface antigen gene. The direct sequencing showed that there were no VEMs in the serum of the children or mother of Family 1 and family 2, but it identified a G145A mutant in the nails of the mother of Family 2. In Family 1, deep sequencing detected a T143S mutant as a minor population (1.7–2.0%) in the umbilical cords and serum of all three children and in the serum of the mother. A T126A mutant was also detected in the umbilical cord (9.2%) and serum (7.0%) of the first-born child of Family 1. In Family 2, the deep sequencing showed no VEMs in the umbilical cords, but it detected D144A (2.5%) and G145A (11.2%) mutants in the serum of the 2nd-born child.ConclusionsVEMs were present as minor populations in the preserved umbilical cords of children with breakthrough infections. The VEMs did not become major populations after the breakthrough infections. The evolution of VEMs from a minor form to a major form might not be a prerequisite for breakthrough infections in mother-to-child transmission.

Evaluation of immunogenicity and protective effect of DNA vaccine encoding surface antigen1 (SAG1) of Toxoplasma gondii and TLR-5 ligand as a genetic adjuvant against acute toxoplasmosis in BALB/c mice

AimsToxoplasma gondii is an obligate intracellular, protozoan that causes a high incidence of serious zoonotic parasitic disease in humans. In the present study the immune-protective efficacy of a DNA vaccine encoding SAG1 in combination with a gene sequence encoding FliC of Salmonella typhimurium (Toll-like receptor 5 agonist) was evaluated against acute T. gondii infection in mice. Methods and resultsNinety-nine female inbred BALB/c mice were divided into nine groups of 11 mice and were immunized intramuscularly three times at three-week intervals (days 0, 21 and 42) and challenged with virulent T. gondii RH strain 4 weeks later. The immunization of pVAX1-SAG1 administered with pVAX1-fliC in mice indicated specific humoral responses, with higher IgG antibody titers and a mixed IgG1/IgG2a response than in other groups (with a predominance of IgG2a over IgG2b and IgG1). Also, the cellular immune response elicited high levels of IFN-γ and IL-12 cytokines and low levels of IL-4 production compared to traditional adjuvants. Furthermore, the mice vaccinated with pVAX1-SAG1+pVAX1-fliC survived for slightly longer after the last immunization and challenge with the T. gondii. ConclusionThis investigation indicated that cocktail DNA vaccine encoded SAG1 gene of T. gondii and FliC can protect against acute toxoplasmosis.