Evaluation of nine microsatellite loci and misidentification paternity frequency in a population of Gyr breed bovines

Erros de identificação de paternidade são prejudiciais por reduzir o ganho genético anual e comprometer um programa eficiente de melhoramento genético. O objetivo principal deste trabalho foi avaliar o potencial de uso de nove microssatélites em testes de paternidade e investigar a freqüência de erro de identificação de famílias de um rebanho de animais da raça Gir. No experimento foram utilizadas amostras de sangue de quarenta famílias (touro/ vaca/ bezerro) de animais da raça Gir, Puros de Origem e registrados na Associação Brasileira dos Criadores de Zebu (ABCZ). A maior parte dos microssatélites avaliados neste trabalho são recomendados, para Testes de Paternidade em bovinos, pela Sociedade Internacional de Genética Animal (ISAG). As regiões microssatélites TGLA122, TGLA126, BM1824, BMS2533, SPS115, ETH3, ETH10, ETH225 e POTCHA foram amplificadas por meio da técnica de PCR. Os produtos da amplificação foram separados por eletroforese em gel de poliacrilamida desnaturante. A partir dos dados obtidos foram calculadas as freqüências alélicas, diversidade gênica, conteúdo de polimorfismo informativo e probabilidade de exclusão para cada microssatélite. Também foram calculadas as freqüências genotípicas, heterozigosidade, probabilidade de exclusão combinada e probabilidade de Paternidade nas famílias consideradas. A probabilidade de exclusão combinada para todos os microssatélites estudados foi de 0,9789. Os resultados dos testes de paternidade acusaram erro de identificação em onze das 40 famílias estudadas, ou seja, 27,5% da amostra. A probabilidade de paternidade variou entre 0,8691 e 0,9999, com valor médio de 0,9512.

Pesquisa de suínos portadores renais de leptospiras pelo isolamento microbiano e reação em cadeia pela polimerase em amostras de rins de animais sorologicamente positivos e negativos para leptospirose

Com o intuito de se estudar a presença de suínos portadores renais de leptospiras foram colhidas 131 amostras sanguíneas e os respectivos rins de animais durante o abate em abatedouro da região de Botucatu-SP. Pela prova de Soroaglutinação Microscópica obteve-se 48 amostras sorológicas positivas para um ou mais sorovar de Leptospira spp., com uma taxa de ocorrência de anticorpos anti-leptospira de 36,64%, e maior importância para o sorovar icterohaemorrhagiae. Para a pesquisa do agente nos rins, das 88 amostras renais submetidas a cultura em meio de EMJH e analisadas pela prova de PCR, foi isolado e detectado o agente em uma única amostra renal, pertencente a um animal soropositivo. Embora não tenha sido possível a comparação estatística, em termos de sensibilidade e especificidade das duas provas de detecção do agente a partir de amostras renais, a PCR mostrou-se mais rápida e prática na pesquisa de portadores renais. Pelo isolamento obtido, ressalta-se a importância desses animais como possíveis transmissores da doença para trabalhadores de abatedouro e inspetores de carne.

Nucleotide sequence, genomic organization and chromosome localization of 5S rDNA in two species of Curimatidae (Teleostei, Characiformes)

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

Duffy blood group gene polymorphisms among malaria vivax patients in four areas of the Brazilian Amazon region

Background: Duffy blood group polymorphisms are important in areas where Plasmodium vivax predominates, because this molecule acts as a receptor for this protozoan. In the present study, Duffy blood group genotyping in P. vivax malaria patients from four different Brazilian endemic areas is reported, exploring significant associations between blood group variants and susceptibility or resistance to malaria.Methods: the P. vivax identification was determined by non-genotypic and genotypic screening tests. The Duffy blood group was genotyped by PCR/RFLP in 330 blood donors and 312 malaria patients from four Brazilian Amazon areas. In order to assess the variables significance and to obtain independence among the proportions, the Fisher's exact test was used.Results: the data show a high frequency of the FYA/FYB genotype, followed by FYB/FYB, FYA/FYA, FYA/FYB-33 and FYB/FYB-33. Low frequencies were detected for the FYA/FY(X), FYB/FY(X), FYX/FY(X) and FYB-33/FYB-33 genotypes. Negative Duffy genotype (FYB-33/FYB-33) was found in both groups: individuals infected and non-infected (blood donors). No individual carried the FY(X)/FYB-33 genotype. Some of the Duffy genotypes frequencies showed significant differences between donors and malaria patients.Conclusion: the obtained data suggest that individuals with the FYA/FYB genotype have higher susceptibility to malaria. The presence of the FYB-33 allele may be a selective advantage in the population, reducing the rate of infection by P. vivax in this region. Additional efforts may contribute to better elucidate the physiopathologic differences in this parasite/host relationship in regions endemic for P. vivax malaria, in particular the Brazilian Amazon region.

Antigenic similarities to Paracoccidioides brasiliensis in thermo-dependent dimorphic fungi isolated from soil in Botucatu, SP, Brazil

We compared the antigenic characteristics of two thermo-dependent dimorphic fungi isolated from soil in Botucatu, an endemic area of paracoccidioidomycosis (PCM) and Paracoccidioides brasiliensis. The soil isolates grew as cerebriform colonies at 37 degrees C (yeast form) and as cottonous colonies at 25 degrees C (mycelial form). No pathogenicity for ddY mice or hamsters were observed. In immunodiffusion test, there were precipitation bands between the 2 soil isolates and pooled PCM patient sera. There were also common precipitation bands at 21, 50 and 58 kDa between the soil isolates antigens and PCM patient sera by Western-blotting, but no gp43 kDa band. No gene for gp43 kDa protein was detected in the soil isolates by PCR. The fact that these isolates were obtained from an endemic area of PCM and there were some antigenic similarities between the soil isolates and P. brasiliensis in immunodiffusion test and Western-blotting may have some importance in epidemiological surveys done with paracoccidioidin as well interfering with the immune response of the exposed population.

Ribosomal DNA ITS-1 intergenic spacer polymorphism in triatomines (Triatominae, Heteroptera)

The length polymorphism of ribosomal DNA ITS-1 intergenic spacer was analyzed in eight species of triatomines belonging to Triatoma, Rhodnius, and Panstrongylus genera. The analyzed species were Rhodnius domesticus, R. neivai, R. robustus, Triatoma brasiliensis, T. infestans, T. vitticeps, Panstrongylus megistus, and P. herreri. These insects are vectors of Chagas' disease, one of the most prominent public health problems among South American countries. This work allowed the differentiation between species of the Triatomini and Rhodniini tribes through the analysis of ITS-1 length polymorphism by PCR and RFLP techniques. The species of the Triatoma and Panstrongylus genera presented an amplified ITS-1 fragment between 600 and 1000 bp, whereas Rhodnius presented a less variable ITS-1 length fragment, around 300 bp, which could reflect the monophyletic origin of the Rhodniini tribe. Species belonging to this genus were further differentiated by RFLP with HaeIII and AluI endonucleases. Our results corroborate the hypothesis of polyphyletic origin in this group of insects and contribute to knowledge about evolutionary relationships in triatomines.

Candidatus Liberibacter americanus, associated with citrus huanglongbing (greening disease) in São Paulo State, Brazil

Symptoms of huanglongbing (HLB) were reported in São Paulo State (SPS), Brazil, in March 2004. In Asia, HLB is caused by 'Candidatus Liberibacterasiaticus'and in Africa by 'Candidatus Liberibacter africanus'. Detection of the liberibacters is based on PCR amplification of their 16S rRNA gene with specific primers. Leaves with blotchy mottle symptoms characteristic of HLB were sampled in several farms of SIPS and tested for the presence of liberibacters. 'Ca. L. asiaticus' was detected in a small number of samples but most samples gave negative PCR results. Therefore, a new HLB pathogen was suspected. Evidence for an SPS-HLB bacterium in symptomatic leaves was obtained by PCR amplification with universal primers for prokaryotic 16S rRNA gene sequences. The amplified 16S rRNA gene was cloned and sequenced. Sequence analysis and phylogeny studies showed that the 16S rRNA gene possessed the oligonucleotide signatures and the secondary loop structure characteristic of the alpha-Proteobacteria, including the liberibacters. The 16S rRNA gene sequence phylogenetic tree showed that the SPS-HLB bacterium clustered within the a-Proteobacteria, the liberibacters being its closest relatives. For these reasons, the SPS-HLB bacterium is considered a member of the genus 'Ca. Liberibacter'. However, while the 16S rRNA gene sequences of 'Ca. L. asiaticus' and 'Ca. L. africanus' had 98-4% similarity, the 16S rRNA gene sequence of the SPS-HLB liberibacter had only 96(.)0% similarity with the 16S rRNA gene sequences of 'Ca. L. asiaticus'or'Ca. L. africanus'. This lower similarity was reflected in the phylogenetic tree, where the SPS-HLB liberibacter did not cluster within the 'Ca. L asiaticus'/'Ca. L. africanus group', but as a separate branch. Within the genus 'Candidatus Liberibacter' and for a given species, the 16S/23S intergenic region does not vary greatly. The intergenic regions of three strains of 'Ca. L. asiaticus', from India, the People's Republic of China and Japan, were found to have identical or almost identical sequences. In contrast, the intergenic regions of the SPS-HLB liberibacter, 'Ca. L. asiaticus' and 'Ca. L. africanus' had quite different sequences, with similarity between 66(.)0 and 79(.)5%. These results confirm that the SPS-HLB liberibacter is a novel species for which the name 'Candidatus Liberibacter americanus' is proposed. Like the African and the Asian liberibacters, the 'American' liberibacter is restricted to the sieve tubes of the citrus host. The liberibacter could also be detected by PCR amplification of the 16S rRNA gene in Diaphorina citri, the psyllid vector of 'Ca. L. asiaticus', suggesting that this psyllid is also a vector of 'Ca. L. americanus' in SPS. 'Ca. L. americanus' was detected in 216 of 218 symptomatic leaf samples from 47 farms in 35 municipalities, while 'Ca. L. asiaticus' was detected in only 4 of the 218 samples, indicating that 'Ca. L. americanus' is the major cause of HLB in SIPS.

Structure, genomic DNA typing, and kinetic characterization of the D allozyme of placental alkaline phosphatase (PLAP/ALPP)

The D allozyme of placental alkaline phosphatase (PLAP) displays enzymatic properties at variance with those of the common PLAP allozymes. We have deduced the amino acid sequence of the PLAP D allele by PCR cloning of its gene, ALPP Two coding substitutions were found in comparison With the cDNA of the common PLAP F allele, i.e., 692C>G and 1352A>G, which translate into a P209R and E429G substitution. A single nucleotide primer extension (SNuPE) assay was developed using PCR primers that enable the amplification of a 1.9 kb PLAP fragment. Extension primers were then used on this PCR fragment to detect the 692C>G and 1352A>G substitution. The SNuPE assay on these two nucleotide substitutions enabled us to distinguish the PLAP F and D alleles from the PLAP S/I alleles. Functional studies on the D allozyme were made possible by constructing and expressing a PLAP D cDNA, i.e., [Arg209, Gly429] PLAP, into wildtype Chinese hamster ovary cells. We determined the k(cat) and K-m, of the PLAP S, F. and D allozymes using the non,physiological substrate p-nitrophenylphosphate at an optimal pH (9.8) as well as two physiological substrates, i.e., pyridoxal-5'-phosphate and inorganic pyrophosphate at physiological pH (7.5). We found that the biochemical properties of the D allozyme of PLAP are significantly different from those of the common PLAP allozymes. These biochemical findings suggest that a suboptimal enzymatic function by the PLAP D allozyme may be the basis for the apparent negative selective pressure of the PLAP D allele. The development of the SNuPE assay will enable us to test the hypothesis that the PLAP D allele is subjected to intrauterine selection by examining genomic DNA from statistically informative population samples. Hum Mutat 19:258-267, 2002. (C) 2002 Wiley-Liss, Inc.

Hemoglobin and HFE gene polymorphisms in Brazilian populations in regions endemic for malaria

Our objective was to determine how the distribution of red blood cell diseases is related to malaria occurrence in north Brazil, a region endemic for malaria. We evaluated the incidence of two mutations in the HFE gene, H63D and C282Y, in two study groups: a control blood donor group, with no indication of malaria infection, and a group constituted of malaria patients of four states of the Amazonian region. The hemoglobin polymorphisms were obtained by HPLC and classical laboratory methodologies, and the two mutations in the HFE gene were assayed by PCR-RFLP. We found a high frequency of alpha thalassemia, but there were no significant differences between blood donors and malaria patients. There were also no significant differences in the frequencies of HbA(2); however, the frequency of HbF was significantly different in individuals with malaria from Para and Rondonia. The mean number of reticulocytes was significantly reduced in the blood donors from the northern region, suggesting an adaptive strategy of these populations to parasitic attack by Plasmodium. Most individuals were heterozygous for the H63D allele of the HFE gene in both study groups. In the blood donors group, the greatest frequency of the H63D allele was found in Caucasians of all the states. In the malaria patients group in Rondonia, there was a high frequency of the H63D allele among the non-Caucasians. In the other states, and in the malaria patients group, the H63D allele was the most frequent among the Caucasians. Based on our results, we suggest that the maintenance of polymorphism of the mutations in the gene HFE can be explained by selective factors other than malaria, or it is due to simple allelic oscillation and by the constant gene flow among the populations in Brazil.

Polymorphisms of DNA repair genes XRCC1 and XRCC3, interaction with environmental exposure and risk of chronic gastritis and gastric cancer

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Identification of two subfamilies of micropia transposable element in species of the repleta group of Drosophila

The occurrence, number of insertion sites and antisense RNA expression of micropia transposable element were studied in 26 species that belong to three subgroups (mercatorum, mulleri and hydei) of repleta group of Drosophila. Under high specific PCR, micropia sequences were detected in 11 species, but under less stringent condition, this retrotransposon was detected in all species. The widespread distribution of micropia suggests that this element was already present at the common ancestor of the repleta group of Drosophila. Southern blot analysis showed a variation from 0 to 17 different insertion sites and the occurrence of male-specific sequences. We found that the expression of the 1.0 kb micropia antisense RNA is variable among the species and tissues (soma and testis), which suggests that more than one mechanism regulates transposition in these species. Variation of amplification by PCR and of antisense RNA expression, as well as divergence of nucleotide sequences among the species allow us to suggest that at least two subfamilies of micropia transposable element are harbored by the genome of this species group.

Plasmodium vivax infection among Duffy antigen-negative individuals from the Brazilian Amazon region: an exception?

We present evidence for Plasmodium vivax infection among Duffy blood group-negative inhabitants of Brazil. The P. vivax identification was determined by both genotypic and non-genotypic screening tests. The Duffy blood group was genotyped by PCR/RFLP and phenotyped using a microtyping kit. We detected two homozygous FY*B-33 carriers infected by P vivax, whose circumsporozoite protein genotypes were VK210 and/or P. vivax-like. Additional efforts are necessary in order to clarify the evidence that P. vivax is being transmitted among Duffy blood group-negative patients from the Brazilian Amazon region. (C) 2007 Published by Elsevier Ltd on behalf of Royal Society of Tropical Medicine and Hygiene.

Exfoliative cytology of the oral mucosa in type II diabetic patients: morphology and cytomorphometry

Background: In recent years, important advances have occurred in the determination of diagnostic criteria for the disease diabetes mellitus and in new strategies for its treatment. The purpose of this research was to develop a new method for diabetes diagnosis by microscopic and cytomorphometric analyses of the oral epithelium. Methods: the smears were obtained from three distinct oral sites: buccal mucosa (cheek), tongue dorsum, and floor of the mouth in 10 control individuals and 10 type II diabetic patients. The oral smears were stained with Papanicolaou EA-36 solution. The nuclear (NA) and cytoplasmic (CA) areas were evaluated from 50 integral cells predominant in each oral site by the use of the KS 300(TM) image analysis system (Carl Zeiss, Germany), by which the cytoplasmic/nuclear ratio (C/N) was calculated. Results: the results showed that: (i) the epithelial cells of the diabetic group exhibited figures of binucleation and occasional karyorrhexis in all layers; (ii) the NA was markedly higher (P<0.05) in the diabetic group; (iii) the CA did not exhibit a statistically significant difference (P>0.05) between these two groups; and (iv) the C/N mean was 37.4% lower in the type II diabetic group. Conclusions: These results associated with clinical observations suggest that diabetes mellitus can produce alterations in oral epithelial cells, detectable by microscopy and cytomorphometry, which can be used in the diagnosis of this disease.

Molecular organization of 5S rDNA in fishes of the genus Brycon

There are few reports on the genomic organization of 5S rDNA in fish species. To characterize the 5S rDNA nucleotide sequence and chromosomal localization in the Neotropical fishes of the genus Brycon, 5S rDNA copies from seven species were generated by PCR. The nucleotide sequences of the coding region (5S rRNA gene) and the nontranscribed spacer (NTS) were determined, revealing that the 5S rRNA genes were highly conserved, while the NTSs were widely variable among the species analyzed. Moreover, two classes of NTS were detected in each species, characterized by base substitutions and insertions-deletions. Using fluorescence in situ hybridization (FISH), two 5S rDNA chromosome loci that could be related to the two 5S rDNA NTS classes were observed in at least one of the species studied. 5S rDNA sequencing and chromosomal localization permitted the characterization of Brycon spp. and suggest a higher similarity among some of them. The data obtained indicate that the 5S rDNA can be an useful genetic marker for species identification and evolutionary studies.

Brain and lung cryptococcoma and concurrent corynebacterium pseudotuberculosis infection in a goat: a case report

A four-year-old male goat with a history of neurological disorder was euthanized. It presented uncommon nodules in the brain and lungs associated with multiple abscesses, predominantly in the spleen and liver. Histological examination of brain and lung sections revealed yeast forms confirmed to be Cryptococcus gattii after a combination of isolation and polymerase chain reaction (PCR) procedures. Moreover, Corynebacterium pseudotuberculosis infection was diagnosed by PCR of samples from the lung, spleen and liver. The present report highlights the rare concurrent infection of C. gatti and C. pseudotuberculosis in an adult goat from São Paulo state, Brazil, and indicates the necessity of surveillance in the treatment of goats with atypical pulmonary infections associated with neurological disorders.