Differentiation of Haemonchus placei from Haemonchus contortus by PCR and by morphometrics of adult parasites and third stage larvae

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Human Platelet Polymorphism can be a genetic marker associated with HIV/HCV coinfection

To evaluate the associations of HPA polymorphisms -1, -3, and -5 with HIV/HCV coinfection were included in this study 60 HIV/HCV-coinfected patients from the Sao Paulo State health service centers. Data reported by Verdichio-Moraes et al. (2009: J. Med Virol 81:757-759) were used as the non-infected and HCV monoinfected groups. Human Platelet Polymorphism genotyping was performed in 60 Patients co-infected with HIV/HCV by PCR-SSP or PCR-RFLP. HIV subtyping and HCV genotyping was performed by RT-PCR followed sequencing. The data analyses were performed using the χ2 test or Fisher's Exact Test and the logistic regression model. Patients coinfected with HIV/HCV presented HCV either genotype 1 (78.3%) or non-1 (21.7%) and HIV either subtype B (85.0%) or non-B (15%). The Human Platelet Polymorphism-1a/1b genotype was more frequent (P < 0.05) in HIV/HCV coinfection than in HCV monoinfection and the allelic frequency of Human Platelet Polymorphism-5b in the Patients coinfected with HIV/HCV was higher (P < 0.05) than in HCV monoinfected cases and non-infected individuals. These data suggest that the presence of specific HPA allele on platelets could favor the existence of coinfection. On the other hand, Human Platelet Polymorphism-5a/5b was more frequent (P < 0.05) in HIV/HCV coinfected and HCV monoinfected groups than in the non-infected individuals, suggesting that this platelet genotype is related to HCV infection, regardless of HIV presence. Results suggest that the Human Platelet Polymorphism profile in HIV/HCV coinfected individuals differs from the one of both HCV monoinfected and non-infected population. So, the Human Platelet Polymorphism can be a genetic marker associated with HIV/HCV coinfection.

Low frequency of Y anomaly detected in Australian Brahman cow-herds

Indicine cattle have lower reproductive performance in comparison to taurine. A chromosomal anomaly characterized by the presence Y markers in females was reported and associated with infertility in cattle. The aim of this study was to investigate the occurrence of the anomaly in Brahman cows. Brahman cows (n = 929) were genotyped for a Y chromosome specific region using real time-PCR. Only six out of 929 cows had the anomaly (0.6%). The anomaly frequency was much lower in Brahman cows than in the crossbred population, in which it was first detected. It also seems that the anomaly doesn't affect pregnancy in the population. Due to the low frequency, association analyses couldn't be executed. Further, SNP signal of the pseudoautosomal boundary region of the Y chromosome was investigated using HD SNP chip. Pooled DNA of non-pregnant and pregnant cows were compared and no difference in SNP allele frequency was observed. Results suggest that the anomaly had a very low frequency in this Australian Brahman population and had no effect on reproduction. Further studies comparing pregnant cows and cows that failed to conceive should be executed after better assembly and annotation of the Y chromosome in cattle.

Validation of absolute quantitative real-time PCR for the diagnosis of streptococcus agalactiae in fish

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Distribution of serotype-specific genotypes of Aggregatibacter actinomycetemcomitns in Brazilian patients with Down syndrome with different periodontal conditions

Fundação do Amparo à Pesquisa do Estado de São Paulo (FAPESP)

GUS expression in sweet oranges (Citrus sinensis L. Osbeck) driven by three different phloem-specific promoters

Huanglongbing (HLB) is associated with Candidatus Liberibacter spp., endogenous, sieve tube-restricted bacteria that are transmitted by citrus psyllid insect vectors. Transgenic expression in the phloem of specific genes that might affect Ca. Liberibacter spp. growth and development may be an adequate strategy to improve citrus resistance to HLB. To study specific phloem gene expression in citrus, we developed three different binary vector constructs with expression cassettes bearing the beta-glucuronidase (GUS) reporter gene (uidA) under the control of one of the three different promoters: Citrus phloem protein 2 (CsPP2), Arabidopsis thaliana phloem protein 2 (AtPP2), and Arabidopsis thaliana sucrose transporter 2 (AtSUC2). Transgenic lines of 'Hamlin', 'Pera', and 'Valencia' sweet oranges [Citrus sinensis (L.) Osbeck] were produced via Agrobacterium tumefaciens transformation. The epicotyl segments collected from in vitro germinated seedlings were used as explants. The gene nptII, which confers resistance to the antibiotic kanamycin, was used for selection. The transformation efficiency was expressed as the number of GUS-positive shoots over the total number of explants and varied from 1.54 to 6.08 % among the three cultivars and three constructs studied. Several lines of the three sweet orange cultivars analyzed using PCR and Southern blot analysis were genetically transformed with the three constructs evaluated. The histological GUS activity in the leaves indicates that the uidA gene was preferentially expressed in the phloem, which suggests that the use of the three promoters might be adequate for producing HLB-resistant transgenic sweet oranges. The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters. Key message The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters.

Strain-specific polyketide synthase genes of Aspergillus niger

In silico comparison of 34 putative pks genes in Aspergillus niger strain CBS 513.88 versus A. niger strain ATCC 1015 genome revealed significant nucleotide identity (>95% covering a minimum of 99% of the gene sequence) for 31 of these genes (approximately 91%). A. niger CBS 513.88 harbors three putative pks genes (An01g01130, An11g05940, and An15g07920), for which nucleotide identity was not found in A. niger ATCC 1015. To compare the results of the in silico analysis with the in vivo situation, experimental data were obtained for a large number of A. niger strains obtained from different substrates and geographical regions. Three putative Os genes that were found to be variable between the two A. niger strains using bioinformatics tools were in fact strain-specific genes based on experimental data. The PCR amplification signals for the An01g01130, An11g05940, and An15g07920 pks genes were detected in only 97%, 71%, and 26% of the strains, respectively. Southern blot analyses confirmed the PCR data. Because one of the strain-specific pits genes (An15g07920) is located in a putative ochratoxin cluster, we focused our investigation on that region. We assessed the ochratoxin production capability of the 119 A. niger strains and found a positive association between the presence of this pia gene and the capability of the respective strain to produce ochratoxin. (C) 2012 Elsevier B.V. All rights reserved.

Deregulation of apoptosis-related genes is associated with PRV1 overexpression and JAK2 V617F allele burden in Essential Thrombocythemia and Myelofibrosis

Background: Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are Chronic Myeloproliferative Neoplasms (MPN) characterized by clonal myeloproliferation/myeloaccumulation without cell maturation impairment. The JAK2 V617F mutation and PRV1 gene overexpression may contribute to MPN physiopathology. We hypothesized that deregulation of the apoptotic machinery may also play a role in the pathogenesis of ET and PMF. In this study we evaluated the apoptosis-related gene and protein expression of BCL2 family members in bone marrow CD34(+) hematopoietic stem cells (HSC) and peripheral blood leukocytes from ET and PMF patients. We also tested whether the gene expression results were correlated with JAK2 V617F allele burden percentage, PRV1 overexpression, and clinical and laboratory parameters. Results: By real time PCR assay, we observed that A1, MCL1, BIK and BID, as well as A1, BCLW and BAK gene expression were increased in ET and PMF CD34(+) cells respectively, while pro-apoptotic BAX and anti-apoptotic BCL2 mRNA levels were found to be lower in ET and PMF CD34(+) cells respectively, in relation to controls. In patients' leukocytes, we detected an upregulation of anti-apoptotic genes A1, BCL2, BCL-XL and BCLW. In contrast, pro-apoptotic BID and BIMEL expression were downregulated in ET leukocytes. Increased BCL-XL protein expression in PMF leukocytes and decreased BID protein expression in ET leukocytes were observed by Western Blot. In ET leukocytes, we found a correlation between JAK2 V617F allele burden and BAX, BIK and BAD gene expression and between A1, BAX and BIK and PRV1 gene expression. A negative correlation between PRV1 gene expression and platelet count was observed, as well as a positive correlation between PRV1 gene expression and splenomegaly. Conclusions: Our results suggest the participation of intrinsic apoptosis pathway in the MPN physiopathology. In addition, PRV1 and JAK2 V617F allele burden were linked to deregulation of the apoptotic machinery.

Plasmodium vivax: Reverse transcriptase real-time PCR for gametocyte detection and quantitation in clinical samples

The proportion of Plasmodium vivax-infected subjects that carry mature gametocytes, and thus are potentially infectious, remains poorly characterized in endemic settings. Here, we describe a quantitative reverse transcriptase (RI) real-time PCR (qRT-PCR) that targets transcripts of the mature gametocyte-specific pvs25 gene. We found mature gametocytes in 42 of 44 (95.4%) P. vivax infections diagnosed during an ongoing cohort study in northwestern Brazil. SYBR green qRT-PCR was more sensitive than a conventional RT-PCR that targets the same gene. Molecular detection of gametocytes failed, however, when dried bloodspots were used for RNA isolation and complementary DNA synthesis. Estimating the number of pvs25 gene transcripts allowed for examining the potential infectiousness of gametocyte carriers in a quantitative way. We found that most (61.9%) gametocyte carriers were either asymptomatic or had subpatent parasitemias and would have been missed by routine malaria control strategies. However, potentially undiagnosed gametocyte carriers usually had low-density infections and contributed a small fraction (up to 4%) to the overall gametocyte burden in the community. Further studies are required to determine the relative contribution to malaria transmission of long-lasting but low-density gametocytemias in asymptomatic carriers that are left undiagnosed and untreated. (C) 2012 Elsevier Inc. All rights reserved.

Real-time PCR assay targeting the actin gene for the detection of Cryptosporidium parvum in calf fecal samples

Cryptosporidium parvum infection is very important with respect to public health, owing to foodborne and waterborne outbreaks and gastrointestinal illness in immunocompetent and immunocompromised persons. In cattle, infection with this species manifests either as a subclinical disease or with diarrheal illness, which occurs more often in the presence of other infectious agents than when alone. The aim of this study was to develop a real-time polymerase chain reaction (PCR) assay for the detection of C. parvum in calf fecal samples and to compare the results of this assay with those of the method routinely used for the diagnosis of Cryptosporidium spp., nested PCR targeting the 18S rRNA gene. Two hundred and nine fecal samples from calves ranging in age from 1 day to 6 months were examined using real-time PCR specific for the actin gene of C. parvum and by a nested PCR targeting the 18S rRNA gene of Cryptosporidium spp. Using real-time PCR detection, 73.2% (153 out of 209) of the samples were positive for C. parvum, while 56.5% (118 out of 209) of the samples were positive for Cryptosporidium spp. when the nested PCR amplification method was used for the detection. The analytical sensitivity of the real-time PCR was approximately one C. parvum oocyst. There was no significant nonspecific DNA amplification of any of the following species and genotype: Cryptosporidium andersoni, Cryptosporidium baileyi, Cryptosporidium bovis, Cryptosporidium canis, Cryptosporidium galli, Cryptosporidium ryanae, Cryptosporidium serpentis, or avian genotype II. Thus, we conclude that real-time PCR targeting the actin gene is a sensitive and specific method for the detection of C. parvum in calf fecal samples.

Genetic diversity of Leishmania amazonensis strains isolated in northeastern Brazil as revealed by DNA sequencing, PCR-based analyses and molecular karyotyping

Abstract Background Leishmania (Leishmania) amazonensis infection in man results in a clinical spectrum of disease manifestations ranging from cutaneous to mucosal or visceral involvement. In the present study, we have investigated the genetic variability of 18 L. amazonensis strains isolated in northeastern Brazil from patients with different clinical manifestations of leishmaniasis. Parasite DNA was analyzed by sequencing of the ITS flanking the 5.8 S subunit of the ribosomal RNA genes, by RAPD and SSR-PCR and by PFGE followed by hybridization with gene-specific probes. Results ITS sequencing and PCR-based methods revealed genetic heterogeneity among the L. amazonensis isolates examined and molecular karyotyping also showed variation in the chromosome size of different isolates. Unrooted genetic trees separated strains into different groups. Conclusion These results indicate that L. amazonensis strains isolated from leishmaniasis patients from northeastern Brazil are genetically diverse, however, no correlation between genetic polymorphism and phenotype were found.

Frequency of human platelet antigens in oncohematological patients with thrombocytopenia and the probability of incompatibility to platelet transfusions

OBJECTIVE: The objective of this study was to evaluate the frequencies of human platelet antigens in oncohematological patients with thrombocytopenia and to analyze the probability of their incompatibility with platelet transfusions. METHODS: Platelet antigen genotyping was performed by sequence-specific primer polymerase chain reaction (SSP-PCR) for the HPA-1a, HPA-1b, HPA-2a, HPA-2b, HPA-3a, HPA-3b, HPA-4a, HPA-4b, HPA-5a, HPA-5b; HPA-15a, HPA-15b alleles in 150 patients of the Hematology Service of the Hospital das Clínicas (FMUSP). RESULTS: The allele frequencies found were: HPA-1a: 0.837; HPA-1b: 0.163; HPA-2a: 0.830; HPA-2b: 0.170; HPA-3a: 0.700; HPA-3b: 0.300; HPA-4a: 1; HPA-4b: 0; HPA-5a: 0.887; HPA-5b: 0.113; HPA-15a: 0.457 and HPA-15b: 0.543. CONCLUSIONS: Data from the present study showed that the A allele is more common in the population than the B allele, except for HPA-15. This suggests that patients homozygous for the B allele are more predisposed to present alloimmunization and refractoriness to platelet transfusions by immune causes. Platelet genotyping could be of great value in the diagnosis of alloimmune thrombocytopenia and to provide compatible platelet concentrates for these patients.

ERIC-PCR genotypic characterization of Haemophilus parasuis isolated from Brazilian swine

Haemophilus parasuis infection, known as Glässer’s disease, is characterized by fibrinous polyserositis, arthritis and meningitis in piglets. Although traditional diagnosis is based on herd history, clinical signs, bacterial isolation and serotyping, the molecular-based methods are alternatives for species-specific tests and epidemiologic study. The aim of this study was to characterize H. parasuis strains isolated from different states of Brazil by serotyping, PCR and ERIC-PCR. Serotyping revealed serovar 4 as the most prevalent (24 %), followed by serovars 14 (14 %), 5 (12 %), 13 (8 %) and 2 (2 %), whereas 40 % of the strains were considered as non-typeable. From 50 strains tested 43 (86%) were positive to Group 1 vtaA gene that have been related to virulent strains of H.parasuis. ERIC-PCR was able to type isolates tested among 23 different patterns, including non-typeable strains. ERIC-PCR patterns were very heterogeneous and presented high similarity between strains of the same animal or farm origin. The results indicated ERIC-PCR as a valuable tool for typing H. parasuis isolates collected in Brazil.

A quantitative TaqMan PCR assay for the detection of Ureaplasma diversum

Ureaplasma diversum in veterinary studies is an undesirable microbe, which may cause infection in bulls and may result in seminal vesiculitis, balanopostitis, and alterations in spermatozoids, whereas in cows, it may cause placentitis, fetal alveolitis, abortion, and birth of weak calves. U. diversum is released through organic secretions, especially semen, preputial and vaginal mucus, conjunctival secretion, and milk. The aim of the present study was to develop a TaqMan probe, highly sensitive and specific quantitative PCR (qPCR) assay for the detection and quantification of U. diversum from genital swabs of bovines. Primers and probes specific to U. diversum 16S rRNA gene were designed. The specificity, detection limit, intra- and inter-assay variability of qPCR to detect this ureaplasma was compared with the results of the conventional PCR assay (cPCR). Swabs of vaginal mucus from 169 cows were tested. The qPCR assay detected as few as 10 copies of U. diversum and was 100-fold more sensitive than the cPCR. No cross-reactivity with other Mollicutes or eubacteria was observed. U. diversum was detected in 79 swabs (46.42%) by qPCR, while using cPCR it was detected in 42 (25%) samples. The difference in cPCR and qPCR ureaplasma detection between healthy and sick animals was not statistically significant. But the U. diversum load in samples from animals with genital disorders was higher than in healthy animals. The qPCR assay developed herein is highly sensitive and specific for the detection and quantification of U. diversum in vaginal bovine samples.

A novel specific genetic translocation in epithelioid hemangioendotelioma, showing a fusion of the WWTR1 and CAMTA1 genes, supports the monoclonality of multifocal epithelioid hemangioendotelioma.

Like other vascular tumors, epithelioid hemangioendothelioma (EHE) is multifocal in approximately 50% of cases, and it is unclear whether the separate lesions represent multifocal disease or metastases. We hypothesized that the identification of an identical WWTR1-CAMTA1 rearrangement in different EHEs from the same patient supports the monoclonal origin of EHE. To test our hypothesis, we undertook a molecular analysis of two multicentric EHEs of the liver, including separate tumor samples from each patient. Matherial and Methods: We retrieved two cases of EHE with available tissue for molecular analysis. In both cases, fluorescence in situ hybridization (FISH) was performed to identify the presence of the WWTR1-CAMTA1 rearrangement to confirm the histologic diagnosis of EHE, as previously described. The reverse transcription-polymerase chain reaction (RT-PCR) products were analyzed by electrophoresis and the RT-PCR–amplified products were sequenced using the Sanger method. Results: FISH analysis revealed signal abnormalities in both WWTR1 and CAMTA1. Combined results confirmed the presence of the t(1;3)(1p36.23;3q25.1) translocation in both cases of EHE. Using RT-PCR analysis, we found that the size of the rearranged bands was identical in the different tumors from each patient. The sequence of the fusion gene confirmed a different WWTR1-CAMTA1 rearrangement in each patient, but an identical WWTR1-CAMTA1 rearrangement in the different lesions from each patient. Discussion: Because of its generally indolent clinical course, EHE is commonly classified as a multifocal, rather than metastatic, disease. In this study, we examined two cases of multifocal liver EHE and found an identical WWTR1-CAMTA1 rearrangement in each lesion from the same patient, but not between the two patients. These findings suggest that multifocal EHE arises from metastasis of the same neoplastic clone rather than from the simultaneous formation of multiple neoplastic clones, which supports the monoclonal origin of multifocal EHE.