Diagnóstico de criptosporidiose em amostras fecais de bezerros por imunofluorescência direta e microscopia de contraste de fase

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

Quantitative real-time PCR identifies a critical region of deletion on 22q13 related to prognosis in oral cancer

Quantitative real time PCR was performed on genomic DNA from 40 primary oral carcinomas and the normal adjacent tissues. The target genes ECGFB, DIA1, BIK, and PDGFB and the microsatellite markers D22S274 and D22S277, mapped on 22q13, were selected according to our previous loss of heterozygosity findings in head and neck tumors. Quantitative PCR relies on the comparison of the amount of product generated from a target gene and that generated from a disomic reference gene (GAPDH-housekeeping gene). Reactions have been performed with normal control in triplicates, using the 7700 Sequence Detection System (PE Applied Biosystems). Losses in the sequences D22S274 (22q13.31) and in the DIA1 (22q13.2-13.31) gene were detected in 10 out of 40 cases (25%) each. Statistically significant correlations were observed for patients with relative copy number loss of the marker D22S274 and stages T3-T4 of disease (P=0.025), family history of cancer (P = 0.001), and death (P = 0.021). Relative copy number loss involving the DIA1 gene was correlated to family history of cancer (P<0.001), death (P=0.002), and consumption of alcohol (P=0.026). Log-rank test revealed a significant decrease in survival (P=0.0018) for patients with DIA1 gene loss. Relative copy number losses detected in these sequences may be related to disease progression and a worse prognosis in patients with oral cancer.

Diagnosis of canine brucellosis: Comparison between serological and microbiological tests and a PCR based on primers to 16S-23S rDNA interspacer

A pair of primers directed to 16S-23S rDNA interspacer (ITS) was designed directed to Brucella genetic sequences in order to develop a polymerase chain reaction (PCR) putatively capable of amplifying DNA from any Brucella species. Nucleic acid extracts from whole-blood from naive dogs were spiked with decreasing amounts of Brucella canis RM6/66 DNA and the resulting solutions were tested by PCR. In addition, the ability of PCR to amplify Brucella spp. genetic sequences from naturally infected dogs was evaluated using 210 whole-blood samples of dogs from 19 kennels. The whole-blood samples collected were subjected to blood culture and PCR. Serodiagnosis was performed using the rapid slide agglutination test with and without 2-mercaptoethanol. The DNA from whole blood was extracted using proteinase-K, sodium dodecyl sulphate and cetyl trimethyl ammonium bromide followed by phenol-chloroform purification. The PCR was capable of detecting as little as 3.8 fg of Brucella DNA mixed with 450 ng of host DNA. Theoretically, 3.8 fg of Brucella DNA represents the total genomic mass of fewer than two bacterial cells. The PCR diagnostic sensitivity and specificity were 100%. From the results observed in the present study, we conclude that PCR could be used as confirmatory test for diagnosis of B. canis infection.

Natural infection with zoonotic subtype of Cryptosporidium parvum in Capybara (Hydrochoerus hydrochaeris) from Brazil

A total of 145 capybara (Hydrochoerus hydrochaeris) fecal samples from the state of Sdo Paulo, Brazil, were screened for Cryptosporidium spp. oocysts using the malachite green method. Eight samples (5.52%) showed positive results and were further submitted to nested PCR reaction for amplification of fragments of 18S rRNA gene and 60-kDa glycoprotein gene for determination of species, alleles and subtypes of Cryptosporidium. Sequencing of the PCR products of the 18S rRNA gene fragments and 60-kDa glycoprotein gene fragments showed that for both genes all Cryptosporidium isolates from capybara were respectively 100% genetically similar to a bovine isolate of C. parvum and to C parvum subtype IIaA15G2R1. To the best of our knowledge this is the first report of Cryptosporidium infection in this rodent. The finding of zoonotic C parvum infection in a semi-aquatic mammal that inhabits anthroponotic habitats raises the concern that human water supplies may be contaminated with zoonotic Cryptosporidium oocysts from wildlife. (c) 2007 Elsevier B.V. All rights reserved.

A SIMPLE TECHNIQUE FOR ISOLATION OF DNA SUITABLE FOR PCR AMPLIFICATION FROM CYTOGENETIC PREPARATIONS

In order to rescue molecular information from chromosome preparations, we describe a rapid procedure to obtain DNA from cytogenetic preparations in microscope slides, stored for one to live years at room temperature. This technique was modified from previously described procedures and the DNA obtained was shown to be suitable for PCR amplification.

Detecção de herpesvírus bovino 5 (BoHV-5) em bovinos do Sudeste Brasileiro

This paper reports the detection of bovine herpesvirus type 5 (BoHV-5) by a specific nested PCR assay. Samples were collected from the central nervous system (CNS) of cattle from Minas Gerais and São Paulo States, Brazil. All animals died presenting neurological symptoms. Nineteen frozen CNS samples analyzed had been previously tested by fluorescence antibody test for rabies virus and showed negative results. Three paraffin-embedded brain tissue samples were examined by histopatology and the observed alterations suggested nonsuppurative meningoencephalitis. BoHV-5 was detected in five (22.7%) among 22 tested samples. The occurrence of BoHV-5 infection is reported in the Southeast region of Brazil, indicating that epidemiological studies should be carried out.

A novel PCR-RFLP assay for the detection of the single nucleotide polymorphism at position+1440 in the human CXCR2 gene

We designed a novel PCR-RFLP assay to genotype for the CXCR2 +1440 (G/A) single nucleotide polymorphism, which provides a simple, low-cost, practical, and reproducible method. Allele frequencies in healthy Brazilian individuals were found to be 0.65% for allele A and 0.35% for allele G.

Sequencing wasp venom peptides by endopeptidase digestion and nested collision-induced dissociation/post-source decay methods

A method incorporating nested collision-induced dissociation/post-source decay (CID/PSD) combined with endopeptidase digestion is described as an approach to determine the sequence of N-terminally modified peptides. The information from immonium and related ions observed in the CID/PSD spectrum was used for the selection of a suitable endopeptidase for the digestion of peptides. Rapid and reliable assignment of peptide sequence was performed by the comparison of CID/PSD spectra of both intact and endopeptidese-digested peptide fragments, since the assignments of the observed fragment ions to either N- or C-terminal ions can thus be carried out unambiguously. This nested CID/PSD method was applied to the sequence determination of two peptides from the solitary wasps Anoplius samariensis and Batozonellus maculifrons (pompilid wasps), which could not be sequenced by the Edman method due to N-terminal modification. Copyright (C) 2002 John Wiley Sons, Ltd.

Immunization of bovines using a DNA vaccine (pcDNA3.1/MSP1b) prepared from the jaboticabal strain of Anaplasma marginale

Anaplasma is a tick-borne ehrlichial pathogen of cattle that causes the disease, anaplasmosis. In the present study, a total of 11 Anaplasma marginale seronegative calves were assigned into two groups: one immunized (G1, n = 6) and one nonimmunized-control (G2, n = 5). Six calves were immunized by using a DNA vaccine containing the gene of a major surface protein, MSP1b, encoded by the plasmid identified as pcDNA3.1/MSPIb. Calves received three intramuscular inoculations of 100 mug of pcDNA3.1/MSP1b at a 20-day interval. The control group received buffer phosphate at the same schedule as the experimental group. The immune response elicited by immunization with pcDNA3.1/MSP1b was evaluated in mice and calves. Twenty days following initial immunization, specific serum antibody from four BALB/c mice bound MSP1b in inummoblots. Sixty days after the last immunization, all calves were challenged with cryopreserved A. marginale at a dose of 10(4) parasites/mL/animal by intravenous injection. Results of packed cell volume (PCV) and detection of infected erythrocytes in all experimental groups revealed that the decrease of PCV and detection of infected erythrocytes occurred at 28 to 42 days after challenge. Mean temperature values did not increase over 39.85degreesC. Antibodies developed by immunized bovines from G2 were detected 14 days after challenge. MSP1b was characterized during the immunization period and MSP2 was the most predominant polypeptide at the challenge period. DNA of A. marginale was detected in all groups just after challenge by nested PCR assay. It can be concluded that all immunized bovines were partially protected against homologous challenge.

Use of PCR-RFLP (Polymerase Chain Reaction-Restricted Fragment Length Polymorphism) in the gene of the enzyme Stearoyl-CoA-Desaturase in Bubalus bubalis

The milk is an important food because it contents Conjugated Linoleic Acids (CIA). These fatty acids are synthesized in mammary gland under action of the enzyme Stearoyl CoA-Desaturase (SCD) and have showed some positive effects in human disease prevention and treatments. A variation of CLA in milk fat exists and can be partially explained by the different levels of expression of SCD. The aim was to study part of the encoding regions of SCD's gene using PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism). Genomic DNA was extracted from lactating Murrah females. After this, PCR reactions were made by using primers Z (sic) (sic) D1 that encloses exon I, II and intron I. The fragments amplified are composed by 938 pb. Then, RFLP techniques were applied in the fragments using the restriction enzymes Pst I and Sma I. The enzyme Pst I has generated fragments of 788pb and 150bp and the Sma I has generated fragments of 693pb and 245pb. All the animals showed the same migration standard for both enzymes, characterizing a genetic monomorphism for this region of SCD gene. The analysis determined that there aren't genetic differences between these animals in the studied regions by using Pst I and Sma I enzymes.

A single multiplex PCR and SNaPshot minisequencing reaction of 42 SNPs to classify admixture populations into mitochondrial DNA haplogroups

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

Galectin-3 Overexpression in Invasive Laryngeal Carcinoma, Assessed by Computer-assisted Analysis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)