Gene B1 Research Articles

Stress-associated β -glucan administration stimulates the TLR – MYD88 – NFKB1 signaling pathway in Nile tilapia (Oreochromis niloticus)

There is evidence that the administration of β-glucan can effectively activate several defense mechanisms, such as the Tlr-Myd88-Nfkb1 pathway that induces the expression of immune cytokines. Thus, the objective of this work was to evaluate whether β-glucan acts on the mechanisms of gene transcription via the Tlr-Myd88-Nfkb1 pathway in Nile tilapia under stress after challenge with Streptococcus agalactiae. Therefore, we evaluated the expression of immune system genes such as toll-like receptors 1 (tlr1), toll-like receptors 2 (tlr2), primary myeloid differentiation response gene (myd88) and nuclear factor kappa B1 (nfkb1). A total of 408 fish were distributed in 24 polyethylene boxes and randomly divided into eight groups with 3 replications each: C15: Tilapias received a control diet (free of β-glucan) for 15 days and were sampled after the 15th day of the experiment; C15D: Tilapias received a control diet (free of β-glucan) for 15 days, were challenged on the 14th day and were sampled at the 15th day of the experiment; β15: Tilapias received experimental diet (1g kg−1 of β-glucan) for 15 days and were sampled after 15 days; β15D: Tilapias received an experimental diet (1g kg−1 of β-glucan) for 15 days, were challenged on the 14th day and were sampled at the 15th day of the experiment; C30: Tilapias received a control diet (free of β-glucan) for 30 days and were sampled on the 30th day of the experiment; C30D: Tilapias received a control diet (free of β-glucan) for 30 days, were challenged on the 29th day and were sampled at the 30th day of the experiment; β30: Tilapias received experimental diet (1g kg-1 of β-glucan) for 30 days and were sampled after 30 days and β30D: Tilapias received experimental diet (1g kg−1 of β-glucan) for 30 days, were challenged on the 29th day and were sampled at 30 of the experiment. In the fish sampled at 15 and 30 days of the experiment, after being anesthetized and killed by brain section, cranial kidney and spleen were collected for gene expression analysis. The analyzes showed that the association of β-glucan and stressful management modulated the immune system, using the Tlr-Myd88-Nfkb1 signaling pathway, indicating that this compound can be used to promote early defense and protect fish against diseases.

Detection of DNA from Toxoplasma gondii oocysts in water for reuse.

The absence of a standardized method for detecting oocysts in water samples makes it difficult to characterize them, including in water for reuse. This study aimed to detect Toxoplasma gondii oocysts using two extraction methods. Using method 1693/2014 USEPA, 30 L of water for reuse from two wastewater treatment plants (WWTPs) in the city of São Paulo, Brazil, was concentrated, totaling 20 samples. The supernatant generated from the immunomagnetic separation (IMS) step was collected for detection of T. gondii oocysts. For DNA extraction, two techniques were used: the commercial kit DNeasy PowerSoil Kit® optimized with the enzyme Zymolyase® and with freeze-thaw steps. DNA quantification was performed with the target sequence of gene B1. From 16 samples submitted to enzymatic extraction, four were positive. In freeze-thaw extraction, no DNA was detected. DNA extraction was the essential step for oocyst detection given the resistant nature of their wall.

Genetic Features of Triticale-Wheat Hybrids with Vaviloid-Type Spike Branching.

Vaviloid spike branching, also called sham ramification, is a typical trait of Triticum vavilovii Jakubz. and is characterized by a lengthening of the spikelet axis. In this article, we present the results of a study of three triticale–wheat hybrid lines with differences in terms of the manifestation of the vaviloid spike branching. Lines were obtained by crossing triticale with hexaploid wheat, T. aestivum var. velutinum. The parental triticale is a hybrid of synthetic wheat (T. durum × Ae. tauschii var. meyrei) with rye, S. cereale ssp. segetale. Line 857 has a karyotype corresponding to hexaploid wheat and has a spike morphology closest to normal, whereas Lines 808/1 and 844/4 are characterized by the greatest manifestation of vaviloid spike branching. In Lines 808/1 and 844/4, we found the substitution 2RL(2DL). The karyotypes of the latter lines differ in that a pair of telocentric chromosomes 2DS is detected in Line 808/1, and these telocentrics are fused into one unpaired chromosome in Line 844/4. Using molecular genetic analysis, we found a deletion of the wheat domestication gene Q located on 5AL in the three studied hybrid lines. The deletion is local since an analysis of the adjacent gene B1 showed the presence of this gene. We assume that the manifestation of vaviloid spike branching in two lines (808/1 and 844/4) is associated with a disturbance in the joint action of genes Q and AP2L2-2D, which is another important gene that determines spike morphology and is located on 2DL.

Profiles of Bacillus spp. Isolated from the Rhizosphere of Suaeda glauca and Their Potential to Promote Plant Growth and Suppress Fungal Phytopathogens.

Members of the genus Bacillus are known to play an important role in promoting plant growth and protecting plants against phytopathogenic microorganisms. In this study, 21 isolates of Bacillus spp. were obtained from the root micro-ecosystem of Suaeda glauca. Analysis of the 16S rRNA genes indicated that the isolates belong to the species Bacillus amyloliquefaciens, Bacillus velezensis, Bacillus subtilis, Bacillus pumilus, Bacillus aryabhattai and Brevibacterium frigoritolerans. One of the interesting findings of this study is that the four strains B1, B5, B16 and B21 are dominant in rhizosphere soil. Based on gyrA, gyrB, and rpoB gene analyses, B1, B5, and B21 were identified as B. amyloliquefaciens and B16 was identified as B. velezensis. Estimation of antifungal activity showed that the isolate B1 had a significant inhibitory effect on Fusarium verticillioides, B5 and B16 on Colletotrichum capsici (syd.) Butl, and B21 on Rhizoctonia cerealis van der Hoeven. The four strains grew well in medium with 1-10% NaCl, a pH value of 5-8, and promoted the growth of Arabidopsis thaliana. Our results indicate that these strains may be promising agents for the biocontrol and promotion of plant growth and further study of the relevant bacteria will provide a useful reference for the development of microbial resources.

Genetic analysis of oviposition deterrence to orange wheat blossom midge in spring wheat.

A major QTL for oviposition deterrence to orange wheat blossom midge was detected on chromosome 1A in the Canadian breeding line BW278 that was inherited from the Chinese variety Sumai-3. Orange wheat blossom midge (OWBM, Sitodiplosis mosellana Géhin, Diptera: Cecidomyiidae) is an important insect pest of wheat (Triticum aestivum L.) that reduces both grain yield and quality. Oviposition deterrence results in a reduction of eggs deposited on spikes relative to that observed on a wheat line preferred by OWBM. Quantification of oviposition deterrence is labor-intensive, so wheat breeders require efficient DNA markers for the selection of this trait. The objective of this study was to identify quantitative trait loci (QTL) for oviposition deterrence in a doubled haploid (DH) population developed from the spring wheat cross Superb/BW278. The DH population and check varieties were evaluated for OWBM kernel damage from five field nurseries over three growing seasons. QTL analysis identified major effect loci on chromosomes 1A (QSm.mrc-1A) and 5A (QSm.mrc-5A). Reduced kernel damage was contributed by BW278 at QSm.mrc-1A and Superb at QSm.mrc-5A. QSm.mrc-1A mapped to the approximate location of the oviposition deterrence QTL previously found in the American variety Reeder. However, haplotype analysis revealed that BW278 inherited this oviposition deterrence allele from the Chinese spring wheat variety Sumai-3. QSm.mrc-5A mapped to the location of awn inhibitor gene B1, suggesting that awns hinder OWBM oviposition. Single-nucleotide polymorphisms (SNPs) were identified for predicting the presence or absence of QSm.mrc-1A based upon haplotype. Functional annotation of candidate genes in 1A QTL intervals revealed eleven potential candidate genes, including a gene involved in terpenoid biosynthesis. SNPs for QSm.mrc-1A and fully awned spikes provide a basis for the selection of oviposition deterrence to OWBM.

Genetic analysis reveals four interacting loci underlying awn trait diversity in barley (Hordeum vulgare)

Barley (Hordeum vulgare) awns contribute to grain yield, but the genetic basis of awn development remains largely unclear. Five barley lines differing in awn traits and row types were used to create four F2 populations. Genetic analyses revealed that four pairs of genes were involved in awn development: A/a (awnless/awned), B/b (awnless/awned), H/h (hooded/straight), and L/l (long/short). Of these four loci, A, H and L functioned on both central rows (CR) and lateral rows (LR) of the barley spikes, while B exhibited effect only on LR. A and B had duplicate effects on LR, and both showed dominant epistasis to loci H and L, whereas H was epistatic to L. Meanwhile, A and B were found to be genetically linked, with a row-type locus V located between them. The genetic distances of A-V and B-V were estimated to be 9.6 and 7.7 cM, respectively. Literature search suggested that A, H and V may correspond to the reported Lks1, Kap1 and Vrs1, respectively, whereas B is a novel gene specifically controlling awn development on LR, designated as Lsa1 for lateral spikelet awnless 1. The only barley homolog of wheat awn inhibitor gene B1, HORVU2Hr1G077570, is a potential candidate of Lsa1.

Detection and genotypes of Toxoplasma gondii DNA in feces of domestic cats in Colombia.

The high prevalence of Toxoplasma gondii in the human population in Colombia has been linked to the existence of a high density of urban stray cats, exposing the whole population to a high density of oocysts. The goal of this study was to determine the DNA prevalence of T. gondii by conventional PCR and to phylogenetically analyze ROP18 sequences from positive samples in domestic cat (Felis catus) fecal samples in the city of Armenia, Quindío. Fecal samples from 140 cats were collected from 10 districts around the city. Samples were concentrated using Ritchie’s method and analyzed through optical microscopy. Concentrates were used for DNA extraction followed by nested PCR amplification for T. gondii gene B1. PCR for ROP18 was performed on all B1 positive samples; the ROP18 sequences obtained were related to the Archetype I Brazilian and Chinese strains. No oocysts were detected by optical microscopy; however, 17.8% (25/140) B1 and 24% (6/25) ROP18 PCR-positive samples were detected. Phylogenetic analyses showed that isolates clustered into a single group. We assessed whether associations existed between T. gondii positive fecal samples and survey variables such as cat healthcare and socioeconomic characteristics of owners, but no statistically significant associations were found. The presence of T. gondii in cat feces is an important factor contributing to the high prevalence in the human population of this city.

Detection of Toxoplasma gondii in Crassostrea spp. oysters cultured in an estuarine region in eastern Amazon

The aim of the present study was to detect DNA of Toxoplasma gondii in Crassostrea spp. oysters cultured in the state of Pará, Brazil. A total of 400 oysters were directly collected from a fixed rack system. Gills, gastrointestinal tract (GIT) and intervalvular liquid were separated and grouped into pool samples of 10 animals, resulting in 40 samples each of gills, GIT and intervalvular liquid. DNA extraction was performed using a commercial kit, and T.gondii DNA was detected by nested PCR using the primers Toxo3 and Toxo4, which produced an amplification product of 155bp of the T.gondii gene B1. Nucleotide sequencing was performed for positive samples, and the obtained sequences were identified by comparison with sequences in GenBank. The DNA of T.gondii was detected in 5.8% (7/120) of the pool samples, of which 7.5% (3/40) was in the GIT, 5% (2/40) in the gills, and 5% (2/40) was in the intervalvular liquid. The obtained sequences presented 100% identity and overlap with T.gondii DNA sequences. This is the report of detection of T.gondii DNA in oysters from genus Crassostrea spp. originating from the state of Pará, eastern Amazon.

Comparative Study in detection of Toxoplasma Gondii on Soil Sample from Baghdad and Kut Cities by Using PCR

Toxoplasma gondii caused a widely distributed protozoan disease called Toxoplasmosis. “The infective stage of T. gondii is capable of infecting variety of vertebrates including humans, domestic animals and wild felids are capable of serving as definitive hosts and T. gondii Oocyst are excreted in their feces”. “Toxoplasmosis is caused by ingestion T. gondii Oocyst from contaminated soil or water or foodstuff or by consuming T. gondii tissue cysts from infected hosts”. “The present study was designed to develop methods for detection of Oocyst from soil by nested PCR using Toxoplasma gene B1 primers”. The results in this study noted that the contamination was highest prevalence in gardens with T. gondii, schools and “backyards of homes” that is in Baghdad city (21.42%, 17.4%, 16.66%) respectively and in the Kut city is (6%, 5.5%, 4%) respectively. rural area infection with T. gondii was highest in the two cities Baghdad and Kut.

Utility of the cytochrome c oxidase subunit I gene for the diagnosis of toxoplasmosis using PCR

Toxoplasmosis is caused by the protozoan parasite Toxoplasma gondii, which belongs to the phylum Apicomplexa. Since this parasite causes severe clinical symptoms in immunocompromised patients, early diagnosis of toxoplasmosis is essential. PCR is currently used for early diagnosis, but there is no consensus regarding the most effective method for amplifying Toxoplasma DNA. In this study, we considered the utility of the cytochrome c subunit I (cox1) gene, which is encoded in the mitochondrial DNA of this parasite, as a novel target of PCR for the diagnosis of toxoplasmosis. To do this, we compared its copy number per haploid nuclear genome and the detection sensitivity of cox1-PCR with the previously reported target genes B1 and 18S rRNA and the AF146527 repeat element. We found that the copy number of cox1 was high and that the PCR using cox1 primers was more efficient at amplifying Toxoplasma DNA than the other PCR targets examined. In addition, PCR using clinical samples indicated that the cox1 gene would be useful for the diagnosis of toxoplasmosis. These findings suggest that use of cox1-PCR would facilitate the diagnosis of toxoplasmosis in clinical laboratories.

The influence of ecophysiological factors on growth, aflR gene expression and aflatoxin B1 production by a type strain of Aspergillus flavus

Maize is prone to infection by Aspergillus flavus, which can contaminate the product with aflatoxins. The objective of this study was to examine the impact that interactions between water activity (aw) and temperature may have on growth, the expression of a biosynthetic regulatory gene (aflR) and aflatoxin B1 (AFB1) production by a strain of A. flavus on a maize-based medium. Results showed that there were some differences between lag phases and growth rates of A. flavus. The optimum growth rate for A. flavus was at 30 °C and 0.99 aw. No growth occurred at 0.90 aw and 20 °C. Both temperature and aw had an influence on the relative aflR gene expression and AFB1 production by A. flavus; however, the results for AFB1 production were not consistent with the effects on gene expression or growth. These findings provide data that are useful to better understand the conditions which represent higher risks from AFB1 production. However, the aflR expression was not a good indicator of AFB1 production alone. Thus, further molecular studies of other AF-related genes should be done. These results are discussed in the context of harvesting and storage of maize and the prevailing environmental conditions to minimise AFB1 contamination.

Cloning gene fragment containing sequences of B gene activates anthocyanin biosynthesis in local sticky corn cultivars (Zea mays subsp. ceratina (Kuelshov) Zhuk)

Sticky corn (Zea mays subsp. ceratina (Kuelshov) Zhuk) is one of the widely grown plants in Vietnam, especially in mountainous regions. The local cultivars play an important role for people living in regions with water limitation and mountain slopes. Anthocyanin is considered a sign of the stress caused by drought. The expression of the structural genes and enzymes involved in anthocyanin biosynthesis in parts of maize require the presence of transcription factors belonging to MYB, Myc (or bHLH) and WD40. Genes of the MYB family regulating anthocyanin biosynthesis have been studied including C1 (Colored Aleurone 1), P1 (Purple), Pl (Purple leaf). Similarly, genes of Myc family (or bHLH) have been studied including B, R, Sn and Lc (Leaf color). Locus B (or b1) consist of alleles B- I, B- Peru, B-Bolivia. These proteins activate structural genes CHS, CHI, F3H, DFR, ANS, Bz, in which CHS, DFR and F3H are key genes of the anthocyanin biosynthesis. This paper analyzed the sequence of a segment of B gene that activates anthocyanin biosynthesis. We have cloned and determined the sequence of a part of genes B from NH and BS1 maize cultivars. The sequence consists of 801 nucleotides encoding 267 amino acids. The deduced protein of segments of gene B has 9 amino acid position changes among NH, BS1 and b1 gene on GenBank. The sequence of a segment of B gene in NH and BS1 had high similarity with genes b1, B1 and B- Peru (98.8%, 99.1% and 98.8%).

Detection of T. gondii infection in blood donors in Alexandria, Egypt, using serological and molecular strategies

Background Toxoplasmosis is a widespread disease caused by the opportunistic parasite Toxoplasma gondii, with variable overall prevalence according to the different geographical areas. Blood donors pose as possible contributors for transfer of infection. Objective The aim of this study was to estimate the prevalence of exposure to Toxoplasma in blood donors using sensitive techniques in a cross-sectional study. Materials and methods An aggregate of 150 blood donors from the blood donation center of Alexandria University participated in this study. The blood samples were tested for the presence of T. gondii immunoglobulin (Ig) G antibody and target gene B1 using enzyme-linked immunosorbent assay and real-time PCR, respectively. Results Of 150 participants, 65.3% tested were positive for anti-Toxoplasma IgG, and 10% showed parasitemia as B1 gene was successfully amplified in nine seropositive samples and in six seronegative samples. Conclusion The recorded IgG seropositivity in this selected group of individuals may be considered an indication of the general prevalence of toxoplasmosis in Alexandria. Detected parasitemia using real-time PCR draws attention to the possibility of transmission through blood transfusion even from seronegative donors and emphasizes the importance of specialized Toxoplasma DNA screening before donation of blood.

QTL mapping of flag leaf traits in common wheat using an integrated high-density SSR and SNP genetic linkage map

Photosynthesis of carbohydrates is the primary source of grain yield in wheat. Photosynthetic organs, especially flag leaves and awns play important roles in wheat growth and development. Genetic analysis of flag leaf posture, size and shape and presence/absence of awns was conducted using a set of 269 recombinant inbred lines (RILs) derived from Yanda1817 × Beinong6. Six agronomic traits comprising flag leaf angle (FLAN), flag leaf width (FLW), flag leaf length (FLL), the ratio of length/width of flag leaf (FLR), flag leaf area (FLA) and presence/absence of awns were evaluated in Shijiazhuang (2011, 2012 and 2013) and Beijing (2012). Using the available high-density single nucleotide polymorphism and simple sequence repeats (SSR) genetic linkage map, a total of 61 putative quantitative trait loci (QTL) for FLAN, FLW, FLL, FLR and FLA were detected on 16 of the 21 wheat chromosomes excluding 1D, 4B, 5D, 6A and 7A, with single QTL in different environments explaining 2.49–42.41 % of the phenotypic variation. Among the identified QTL, 17 were for FLAN, 11 for FLW, seven for FLL, 13 for FLR and 13 for FLA. Twenty-five (41 %) QTL were detected in at least two environments, while four QTL for FLW were detected in all environments. Thirty QTL were associated with higher number of flag leaf traits originated from Yanda1817 alleles, whereas the remaining 31 QTL were derived from Beinong6. In addition, pleiotropic effects were detected for QTL on chromosomes 2D, 3B, 4A, 4D, 5A, 5B, 6B, 6D and 7D that could serve as target regions for fine mapping and marker-assisted selection in wheat breeding programs. Genetic analysis revealed that the presence/absence of awns in the RIL population is controlled by the awn-inhibitor gene B1 linked to SSR marker Xgwm291 on the long arm of chromosome 5A. Our results also suggest that physiological traits FLL, FLW and FLA were significantly and positively correlated to spike length (SL), grain weight per spike and grain number per spike. FLR was significantly and positively related to SL but negatively related to grain width and grain thickness (GT). In addition, the awn trait was strongly and positively correlated to thousand grain weight, grain length and GT.

Usefulness and limitations of polymerase chain reaction in the etiologic diagnosis of neurotoxoplasmosis in immunocompromised patients

The objective of the present study was to assess the performance and the best indication of the polymerase chain reaction (PCR) for the detection of Toxoplasmosis gondii DNA in cerebrospinal fluid (CSF) from patients with suspected neurotoxoplasmosis. CSF samples were collected from 79 patients for amplification of the T. gondii genome (gene B1) by two PCR techniques (nested and real time). Twenty-seven of the 79 patients were classified as probable cases of neurotoxoplasmosis on the basis of clinical criteria, neuroimaging and therapeutic response. PCR showed high sensitivity (86.6%) when performed in CSF samples which were collected up to the seventh day of specific toxoplasmosis treatment. There was no positive test after 1week of treatment. These results suggest the usefulness of PCR for the diagnosis of cerebral toxoplasmosis, and support the first week as the window for the best performance of toxoplasmosis PCR in CSF.

A novel cassette method for probe evaluation in the designed biochips.

A critical step in biochip design is the selection of probes with identical hybridisation characteristics. In this article we describe a novel method for evaluating DNA hybridisation probes, allowing the fine-tuning of biochips, that uses cassettes with multiple probes. Each cassette contains probes in equimolar proportions so that their hybridisation performance can be assessed in a single reaction. The model used to demonstrate this method was a series of probes developed to detect TORCH pathogens. DNA probes were designed for Toxoplasma gondii, Chlamidia trachomatis, Rubella, Cytomegalovirus, and Herpes virus and these were used to construct the DNA cassettes. Five cassettes were constructed to detect TORCH pathogens using a variety of genes coding for membrane proteins, viral matrix protein, an early expressed viral protein, viral DNA polymerase and the repetitive gene B1 of Toxoplasma gondii. All of these probes, except that for the B1 gene, exhibited similar profiles under the same hybridisation conditions. The failure of the B1 gene probe to hybridise was not due to a position effect, and this indicated that the probe was unsuitable for inclusion in the biochip. The redesigned probe for the B1 gene exhibited identical hybridisation properties to the other probes, suitable for inclusion in a biochip.

Association of ABCB1 gene polymorphisms and haplotypes with therapeutic efficacy of glucocorticoids in Chinese patients with immune thrombocytopenia

Resistance to glucocorticoids (GCs) remains a tricky problem complicating the therapy of ITP. Recently, ATP binding cassette gene B1 gene (ABCB1) was reported to be correlated with susceptibility and therapeutic efficacy of autoimmune diseases through P-glycoprotein (Pgp). We investigated three single nucleotide polymorphisms (SNPs) of ABCB1 and their haplotypes by PCR–RFLP (restriction fragment length polymorphism) method in 471 ITP patients and 383 healthy controls, patients were further assigned into GCs-responsive and -non-responsive group according to the therapeutic effects of GCs. We observed a remarkable difference in genotypes of G2677T/A between GCs-responsive and non-responsive group, but not between patients and controls. A frequently expression of T/A allele within G2677T/A was recorded in GCs-responsive group. Furthermore, we found that some haplotypes (CGC, CTC/CAC, CTT/CAT, TGC, TGT, TTC/TAC and TTT/TAT, in the order of position 1236-2677-3435) were presented significantly differences between non-responsive and responsive group. No difference of C1236T and C3435T polymorphisms was observed between ITP and controls, and between the GCs-responsive and -non-responsive group. Our findings suggest that ABCB1 polymorphisms, as well as haplotypes derived from C1235T, G2677T/A and C3435T, are associated with inter-individual differences of GCs treatment in ITP.

Expression profiling of genes modulated by minocycline in a rat model of neuropathic pain.

BackgroundThe molecular mechanisms underlying neuropathic pain are constantly being studied to create new opportunities to prevent or alleviate neuropathic pain. The aim of our study was to determine the gene expression changes induced by sciatic nerve chronic constriction injury (CCI) that are modulated by minocycline, which can effectively diminish neuropathic pain in animal studies. The genes associated with minocycline efficacy in neuropathic pain should provide insight into the etiology of neuropathic pain and identify novel therapeutic targets.ResultsWe screened the ipsilateral dorsal part of the lumbar spinal cord of the rat CCI model for differentially expressed genes. Out of 22,500 studied transcripts, the abundance levels of 93 transcripts were altered following sciatic nerve ligation. Percentage analysis revealed that 54 transcripts were not affected by the repeated administration of minocycline (30 mg/kg, i.p.), but the levels of 39 transcripts were modulated following minocycline treatment. We then selected two gene expression patterns, B1 and B2. The first transcription pattern, B1, consisted of 10 mRNA transcripts that increased in abundance after injury, and minocycline treatment reversed or inhibited the effect of the injury; the B2 transcription pattern consisted of 7 mRNA transcripts whose abundance decreased following sciatic nerve ligation, and minocycline treatment reversed the effect of the injury. Based on the literature, we selected seven genes for further analysis: Cd40, Clec7a, Apobec3b, Slc7a7, and Fam22f from pattern B1 and Rwdd3 and Gimap5 from pattern B2. Additionally, these genes were analyzed using quantitative PCR to determine the transcriptional changes strongly related to the development of neuropathic pain; the ipsilateral DRGs (L4-L6) were also collected and analyzed in these rats using qPCR.ConclusionIn this work, we confirmed gene expression alterations previously identified by microarray analysis in the spinal cord and analyzed the expression of selected genes in the DRG. Moreover, we reviewed the literature to illustrate the relevance of these findings for neuropathic pain development and therapy. Further studies are needed to elucidate the roles of the individual genes in neuropathic pain and to determine the therapeutic role of minocycline in the rat neuropathic pain model.

Direct genotyping of Toxoplasma gondii from amniotic fluids based on B1 gene polymorphism using minisequencing analysis

BackgroundBecause some Toxoplasma gondii genotypes may be more virulent in pregnant women, discriminating between them appears valuable. Currently, the main genotyping method is based on single copy microsatellite markers, which limit direct genotyping from amniotic fluids (AFs) to samples with a high parasitic load. We investigated whether the multicopy gene B1 could type the parasite with a higher sensitivity. To estimate the amplifiable DNA present in AFs, we first compared three different PCR assays used for Toxoplasma infection diagnosis: the P30-PCR, targeting the single copy gene P30; the B1-PCR, targeting the repeated B1 gene; and RE-PCR, targeting the repeated element.ResultsOf the 1792 AFs analyzed between 2008 and 2011, 73 were RE-PCR positive. Of those, 49 (67.1%) were P30-PCR and B1-PCR positive, and 14 (19.2%) additional AFs were B1-PCR positive only.All 63 BI-positive AFs (France n = 49; overseas n = 14) could be genotyped based on an analysis of eight nucleotide polymorphisms (SNPs) located within the B1 gene. Following high-resolution melting (HRM) analysis, minisequencing was carried out for each of the eight SNPs. DNA from six reference strains was included in the study, and AFs were assigned to one of the three major lineages (Types I, II, and III). In total, 26 genotypes were observed, and the hierarchical clustering distinguished two clades in lineages II (IIa, n = 30 and IIb, n = 4) and III (IIIa n = 23 and IIIb n = 6). There was an overrepresentation of overseas isolates in Clade IIb (4/4, 100%) and Clade IIIa (8/22; 36.4%) (p <0.0001), whereas medical interruption and fetal death were overrepresented in Clade IIb (2/4, 50%) and Clade IIIa (4/23, 17.4%) (p = 0.049).ConclusionsAlthough the current genotyping system cannot pretend to replace multilocus typing, we clearly show that targeting the multicopy B1 gene yields a genotyping capacity of AFs around 20% better than when single copy targets are used. The present genotyping method also allows clear identification of genotypes of potential higher virulence.

Serologic and molecular diagnostic and bioassay in mice for detection of Toxoplasma gondii in free ranges chickens from Pantanal of Mato Grosso do Sul

The aim of this study was to investigate the occurrence of Toxoplasma gondii and compare the results obtained in the Modified Agglutination Test (MAT), Polimerase Chain Reaction (PCR) and bioassay in mice. In order to accomplish this, 40 free-range chickens from eight farms in neighboring areas to the Pantanal in Nhecolândia, Mato Grosso do Sul, were euthanized and blood samples, brain and heart were collected. The occurrence of anti-T. gondii antibodies found in chickens was 67.5% (27 samples), considering as a cutoff point the dilution 1:5. Among the samples analyzed, 7 (25.9%) were positive in the dilution 1:5, 3 (11.1%) in 1:10, 2 (7.4%) in 1:20, 3 (11.1%) in 1:320, 1 ( 3.7%) in 1:640, 3 (11.1%) in 1:1280, 2 (7.4%) in 1:2560, 4 (14.8%) in 1:5120 and 2 (7.4%) in 1:10.240. From the mixture of tissue samples (brain and heart) from the chickens analyzed, 16 (40%) presented electrophoretic bands compatible with T. gondii by PCR (gene B1). In the comparison of techniques, 59.26% positivity in PCR was revealed among animals that were seropositive in MAT (cutoff 1:5). From 141 inoculated mice, six (4.44%) died of acute toxoplasmosis between 15 and 23 days after inoculation. Surviving mice were sacrificed at 74 days after inoculation, and a total of 28 cysts were found in the brains of 10 distinct groups. From the seropositive hens, 27 bioassays were performed and 11 (40.7%) isolates were obtained. A greater number of isolations happened in mice that were inoculated with tissues from chickens that had high titers for anti-T. gondii antibodies. Chronic infection in mice was observed in nine groups (33.3%) from five different properties. Among the surviving mice, 25.6% were positive for T. gondii in MAT (1:25). From mice positive in PCR, 87.5% were also positive in MAT. Among the PCR-negative mice, 5.2% were positive for T. gondii in MAT. It can be concluded through this study that the occurrence of infecton by T. gondii in the rural properties studied was high, that PCR directed to gene B1 does not confirm the viability of the parasite, but it can be used as a screening method for the selection of chickens infected by T. gondii, that the animals with titer greater than 10 must be prioritized for the selection of animals for bioassay, since for them, the chances of isolating the parasite are greater and that seroconversion in experimentally infected mice is not a good indicator for isolating the agent.