Detection and molecular analysis of Toxoplasma gondii and Neospora caninum from dogs with neurological disorders

Introduction: Toxoplasma gondii and Neospora caninum are related Apicomplexa parasites responsible for systemic diseases in many species of animals, including dogs. Methods: This study aimed to determine the occurrence of T. gondii and N. caninum infections in 50 dogs with neurological signs that were admitted to the Veterinary Hospital of Universidade Estadual Paulista, City of Botucatu, Brazil. All animals were screened for antibodies using an immunofluorescent antibody test for both parasites. Tissues of positive animals were bioassayed in mice (T. gondii) and gerbils (N. caninum), and DNA was analyzed using the polymerase chain reaction (PCR). Positive samples for T. gondii by PCR were typed using restriction fragment length polymorphism-PCR for 11 markers: SAG1, SAG2 (5'-3'-SAG2 and alt.SAG2), SAG3, Btub, GRA6, L358, c22-8, c29-6, PK1 and Apico, and CS3 marker for virulence analysis. Results: Specific antibodies were detected in 11/50 (22%; 95% confidence interval (CI95%), 12.8-35.3%) animals for T. gondii and 7/50 (14%; CI95%, 7.02-26.3%) for N. caninum. In the bioassay and PCR, 7/11 (63.6%; CI95%, 34.9-84.8%) samples were positive for T. gondii and 3/7 (42.9%; CI95% I, 15.7-75.5%) samples were positive for N. caninum. Three different genotypes were identified, but only 1 was unique. Conclusions: These data confirm the presence of T. gondii and N. caninum in dogs from Brazil, indicating the importance of this host as a sentinel of T. gondii for human beings, and the genotypic variation of this parasite in Brazil.

Genotypic characterization of Toxoplasma gondii in sheep from Brazilian slaughterhouses: New atypical genotypes and the clonal type II strain identified

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

Detection of the single nucleotide polymorphism (rs2227307) in the human interleukin 8 gene using a pcr-rflp assay

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

Genetic relationships among Arachis species based on AFLP

Amplified Fragment Length Polymorphism (AFLP) was used to establish the genetic relationships among 20 species from seven of the nine sections of genus Arachis. The level of polymorphism among nine accessions of the cultivated peanut, A. hypogaea L., was also evaluated. Three combinations of primers were used to amplify the AFLPs. The fragments were separated in 6% denaturing acrylamide gels. A total of 408 fragments were analyzed. An average of 135.3 fragments per primer combination were scored, and the largest number of fragments was 169 using primer combination Eco RI - ACC / Mse I - CTG, while the lowest was 108, with Eco RI - ACT / Mse I - CTT. In general, the genetic relationships established using AFLPs agreed with the classification established using morphology and crossability data. The results indicated that AFLPs are good markers for establishing the relationships among Arachis species. The polymorphism detected in A. hypogaea by this method was higher than the one found with other markers, like RAPDs and RFLPs. However, our data suggest that the polymorphism detected be using AFLP with only three primer combinations is still too low to be used for any kind of genetic study in this species.

Identification of the shark species Rhizoprionodon lalandii and R-porosus (Elasmobranchii, Carcharhinidae) by multiplex PCR and PCR-RFLP techniques

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

IL-1ra anti-inflammatory cytokine polymorphism is associated with risk of gastric cancer and chronic gastritis in a Brazilian population, but the TNF-beta pro-inflammatory cytokine is not

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

Differentiation of Acidithiobacillus ferrooxidans and A-thiooxidans strains based on 16S-23S rDNA spacer polymorphism analysis

Restriction fragment length polymorphism (RFLP) and sequence analyses of the PCR-amplified 16S-23S rDNA intergenic spacer (ITS) were used for differentiating Acidithiobacillus thiooxidans strains from other related acidithiobacilli, including A. ferrooxidans and A. caldus. RFLP fingerprints obtained with AluI, DdeI, HaeIII, HinfI and MspI enabled the differentiation of all Acidithiobacillus reference strains into species groups. The A. thiooxidans strains investigated (metal mine isolates) yielded identical RFLP patterns to the A. thiooxidans type strain (ATCC 19377(T)), except for strain DAMS, which had a distinct pattern for all enzymes tested. Fourteen A. ferrooxidans mine strains were assigned to 3 RFLP groups, the majority of which were grouped with A. ferrooxidans ATCC 23270(T). The spacer region of one representative strain from each of the RFLP groups obtained was subjected to sequence analysis, in addition to eleven additional A. thiooxidans strains isolated from sediment and water samples, and A. caldus DSM 8584(T). The tRNA(IIe) and tRNA(Ala) genes, present in all strains analyzed, showed high sequence similarity. Phylogenetic analysis of the ITS sequences differentiated all three Acidithiobacillus species. Inter- and infraspecific genetic variations detected were mainly due to the size and sequence polymorphism of the ITS3 region. Mantel tests showed no significant correlation between ITS sequence similarity and the geographical origin of strains. The results showed that the 16S-23S rDNA spacer region is a useful target for the development of molecular-based methods aimed at the detection, rapid differentiation and identification of acidithiobacilli. (C) 2004 Elsevier SAS. All rights reserved.

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.

Polymorphism of the ITS-2 region of the ribosomal DNA of the Triatominae Rhodnius domesticus, R-pictipes, R-prolixus and R-stali

The polymerase chain reaction-restriction fragment length polymorphism technique (PCR-RFLP) was used to compare Rhodnius domesticus (Neiva & Pinto), R. pictipes (Stal), R. prolixus (Stal) and R. stali (Lent; Jurberg & Galvao) (Hemiptera: Reduviidae). The enzyme BstUI differentiated R. donzesticus, R. pictipes and R. prolixus, and HhaI differentiated R. domesticus, R. pictipes and R. stali. With the fingerprinting analysis generated by these two enzymes, it was possible to clearly identify all four species in the study.

Primary myiasis in dog caused by Phaenicia eximia (Diptera: Calliphoridae) and preliminary mitochondrial DNA analysis of the species in Brazil

A case of primary myiasis in a dog caused by Phaenicia eximia (Robineau-Desvoidy) in Brazil is presented. A young and healthy female dog, Canis familiaris, approximate to 10 d old and still under maternal care, was found to have several eggs and Is: instars larvae in its abdomen and urogenital regions. Samples were collected in Campinas, São Paulo, and transferred to the laboratory for rearing and identification. A comparative analysis of the mitochondrial DNA (mtDNA) with 12 restriction enzymes in 2 sampled populations of P. eximia collected in different hosts (live dog and bovine carcass) and in the same locality revealed that 4, EcoRI, EcoRV, HaeIII, and MspI were suitable for detecting mtDNA markers in the 2 populations.

Analysis of the association of polymorphism in the osteoprotegerin gene with susceptibility to chronic kidney disease and periodontitis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Molecular genetic maps of the group 6 chromosomes of hexaploid wheat (Triticum aestivum L. em. Thell.)

Restriction fragment length polymorphism (RFLP) maps of chromosomes 6A, 6B, and 6D of hexaploid wheat (Triticum aestivum L. em. Thell.) have been produced. They were constructed using a population of F 7-8 recombinant inbred lines derived from a synthetic wheat X bread wheat cross. The maps consist of 74 markers assigned to map positions at a LOD ≥ 3 (29 markers assigned to 6A, 24 to 6B, and 21 to 6D) and 2 markers assigned to 6D ordered at a LOD of 2.7. Another 78 markers were assigned to intervals on the maps. The maps of 6A, 6B, and 6D span 178, 132, and 206 cM, respectively. Twenty-one clones detected orthologous loci in two homoeologues and 3 detected an orthologous locus in each chromosome. Orthologous loci are located at intervals of from 1.5 to 26 cM throughout 70% of the length of the linkage maps. Within this portion of the maps, colinearity (homosequentiality) among the three homoeologues is strongly indicated. The remainder of the linkage maps consists of three segments ranging in length from 47 to 60 cM. Colinearity among these chromosomes and other Triticeae homoeologous group 6 chromosomes is indicated and a consensus RFLP map derived from maps of the homoeologous group 6 chromosomes of hexaploid wheat, tetraploid wheat, Triticum tauschii, and barley is presented.

Genetic structure of populations of Rhizoctonia solani AG-3 on potato in eastern North Carolina

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was developed to identify and differentiate genotypes of Rhizoctonia solani anastomosis group 3 subgroup PT (AG-3 PT), a fungal pathogen of potato. Polymorphic co-dominant single-locus PCR-RFLP markers were identified after sequencing of clones from a genomic library and digestion with restriction enzymes. Multilocus genotypes were determined by a combination of PCR product and digestion with a specific restriction enzyme for each of seven loci. A sample of 104 isolates from one commercial field in each of five counties in eastern North Carolina was analyzed, and evidence for high levels of gene flow between populations was revealed. When data were clone-corrected and samples pooled into one single North Carolina population, random associations of alleles were found for all loci or pairs of loci, indicating random mating. However, when all genotypes were analyzed, the observed genotypic diversity deviated from panmixia and alleles within and between loci were not randomly associated. These findings support a model of population structure for R. solani AG-3 PT on potato that includes both recombination and clonality.

Strong selection against hybrids at a hybrid zone in the Ensatina ring species complex and its evolutionary implications

The analysis of interactions between lineages at varying levels of genetic divergence can provide insights into the process of speciation through the accumulation of incompatible mutations. Ring species, and especially the Ensatina eschscholtzii system exemplify this approach. The plethodontid salamanders E. eschscholtzii xanthoptica and E. eschscholtzii platensis hybridize in the central Sierran foothills of California. We compared the genetic structure across two transects (southern and northern Calaveras Co.), one of which was resampled over 20 years, and examined diagnostic molecular markers (eight allozyme loci and mitochondrial DNA) and a diagnostic quantitative trait (color pattern). Key results across all studies were: (1) cline centers for all markers were coincident and the zones were narrow, with width estimates of 730 m to 2000 m; (2) cline centers at the northern Calaveras transect were coincident between 1981 and 2001, demonstrating repeatability over five generations; (3) there were very few if any putative F1s, but a relatively high number of backcrossed individuals in the central portion of transects: and (4) we found substantial linkage disequilibrium in all three studies and strong heterozygote deficit both in northern Calaveras, in 2001, and southern Calaveras. Both linkage disequilibrium and heterozygote deficit showed maximum values near the center of the zones. Using estimates of cline width and dispersal, we infer strong selection against hybrids. This is sufficient to promote accumulation of differences at loci that are neutral or under divergent selection, but would still allow for introgression of adaptive alleles. The evidence for strong but incomplete isolation across this centrally located contact is consistent with theory suggesting a gradual increase in postzygotic incompatibility between allopatric populations subject to divergent selection and reinforces the value of Ensatina as a system for the study of divergence and speciation at multiple stages. © 2005 The Society for the Study of Evolution. All rights reserved.

Variabilidade genética intrapopulacional em Myracrodruon urundeuva Fr. All. por marcador AFLP

Levels of genetic variability for in situ and ex situ genetic conservation were estimated in a population of Myracrodruon urundeuva using the PCR (polymerase chain reaction) technique with the AFLP (Amplified fragment-length polymorphism) genetic marker. Seeds for progeny tests were collected from 30 open-pollination trees (matrices) at Paulo de Faria Ecological Station - SP. From this genetic material, three progeny tests were installed on the Teaching and Research Farm of Ilha Solteira Faculty of Engineering - University of São Paulo State (UNESP), which is located in Selvlria - MS, Brazil. The analysis by genetic marker was conducted with three combinations of different starters EcoRl-Msel, resulting in a total number of 137 polymorphic bands, thus forming a table of binary data. These data were used for the analysis of genetic divergence and distance between progenies. High levels of genetic divergence were observed among families. Based on the Analysis of Molecular Variance (AMOVA), it was shown that 16.2% of genetic diversity is found among progenies and 83.8% within progenies, which suggests deviances of random matings. The grouping of progenies, based on genetic distances, suggests that progenies deriving from trees which are close to each other tend to be more similar. This, in turn, indicates that the population originating the seeds may be genetically structured.