Occurrence and molecular characterization of Cryptosporidium spp. isolated from domestic animals in a rural area surrounding Atlantic dry forest fragments in Teodoro Sampaio municipality, State of São Paulo, Brazil

The aim of this study was to assess the occurrence of Cryptosporidium in domestic animals in rural properties surrounding rain forest fragments within the municipality of Teodoro Sampaio, southeastern Brazil. Conventional sucrose flotation method followed by molecular characterization of the parasites by sequencing PCR products amplified from SSU rRNA gene were used. Stool samples were collected from domestic animals raised as pets and livestock in all rural properties surrounding three forest fragments. Samples from cattle (197), equine (63), pigs (25), sheep (11), and dogs (28) were collected from 98 rural properties. The frequency of occurrence of Cryptosporidium within each animal species was 3.0% (6/197) among cattle and 10.7% (3/28) among dogs. Cryptosporidium was not detected in stool samples from equine, sheep, and pigs. All sequences obtained from the six samples of calves showed molecular identity with Cryptosporidium andersoni while all sequences from dog samples were similar to C. canis. The frequency of occurrence of Cryptosporidium in these domestic animal species was low. The absence of C. parvum in the present study suggests that the zoonotic cycle of cryptosporidiosis may not be relevant in the region studied. The presence of Cryptosporidium species seldom described in humans may be, otherwise, important for the wild fauna as these animals are a source of infection and dissemination of this protozoan to other animal species. The impact and magnitude of infection by C. andersoni in wild ruminants and C. canis in wild canids have to be assessed in future studies to better understand the actual importance of these species in this region.

Molecular characterization of Cryptosporidium spp. from HIV infected patients from an urban area of Brazil

Cryptosporidium spp. are important cause of enteric disease in humans, but may also infect animals. This study describes the relative frequency of several Cryptosporidium species found in human specimens from HIV infected patients in the São Paulo municipality obtained from January to July 2007. Sequence analysis of the products of nested-PCR based on small subunit rRNA and Cryptosporidium oocyst wall protein coding genes revealed 17 (63.0%) isolates of C. hominis, four (14.8%) C. parvum, five (18.5%) C. felis and one (3.7%) C. canis. These findings suggest that, in urban environments of Brazil, the cat adapted C. felis may play a potential role in the zoonotic transmission of cryptosporidiosis whereas the anthroponotic transmission of cryptosporidiosis caused by C. hominis seems to predominate.

Caracterização molecular do vírus da raiva isolado de Desmodus rotundus capturados no Estado do Rio de Janeiro

Caracterizou-se filogeneticamente o vírus da raiva, isolado de morcegos hematógafos (Demodus rotundus). Cento e noventa e nove D. rotundus foram capturados em cinco abrigos, no Norte e Noroeste do Estado do Rio de Janeiro e sul do Espírito Santo. Sete deles foram positivos para a raiva. Amostras desses vírus foram sequenciadas e comparadas com sequências provenientes de diversos estados brasileiros. As sequências de vírus da raiva isoladas, na região norte do Estado do Rio de Janeiro, mostraram características que as distinguem de amostras de vírus isoladas em outras regiões do país, no entanto foram idênticas às isoladas de bovinos no noroeste do Rio de Janeiro.

Molecular detection of Hepatozoon canis and Babesia canis vogeli in domestic dogs from Cuiabá, Brazil

The objective of this study was to report for the first time infection by Hepatozoon spp. and Babesia spp. in 10 dogs from the city of Cuiabá, State of Mato Grosso, central-western Brazil. A pair of primers that amplifies a 574 bp fragment of the 18S rRNA of Hepatozoon spp., and a pair of primers that amplifies a 551 bp fragment of the gene 18S rRNA for Babesia spp. were used. Six dogs were positive for Babesia spp., and 9 were positive for Hepatozoon spp. Co-infection of Babesia spp. and Hepatozoon spp. was seen in 5 dogs. Sequenced samples revealed 100% identity with B. canis vogeli, and H. canis. This is the first molecular detection of H. canis in domestic dogs from Cuiabá. Additionally, it is described for the first time the presence of B. canis vogeli circulating among dogs in Cuiabá.

Recovery and upgrading bovine rumen protein by extrusion: effect of lipid content on protein disulphide cross-linking, solubility and molecular weight

Bovine rumen protein with two levels of residual lipids (1.9 per cent or 3.8 per cent) was subjected to thermoplastic extrusion under different temperatures and moisture contents. Protein solubility in different buffers, disulphide cross-linking and molecular weight distribution were determined on the extrudates. After extrusion, samples with 1.9 per cent residual lipids content had a higher concentration of protein insoluble by undetermined forces, irrespective of feed moisture and processing temperature used. Lipid content of 3.8 per cent in the feed material resulted in more protein participating in the extrudate network through non-covalent interactions (hydrophobic and electrostatic) and disulphide bonds. A small dependency of the extrusion process on moisture and temperature and a marked dependency on lipid content, especially phospholipid, was observed, Electrophoresis under non-reducing conditions showed that protein extrusion with low feed moisture promoted high molecular breakdown inside the barrel, probably due to intense shear force, and further protein aggregation at the die end

Gender identification of five genera of stingless bees (Apidae, Meliponini) based on wing morphology

Currently, the identification of pollinators is a critical necessity of conservation programs. After it was found that features extracted from patterns of wing venation are sufficient to discriminate among insect species, various studies have focused on this structure. We examined wing venation patterns of males and workers of five stingless bee species in order to determine if there are differences between sexes and if these differences are greater within than between species. Geometric morphometric analyses were made of the forewings of males and workers of Nannotrigona testaceicornis, Melipona quadrifasciata, Frieseomelitta varia, and Scaptotrigona aff. depilis and Plebeia remota. The patterns of males and workers from the same species were more similar than the patterns of individuals of the same sex from different species, and the patterns of both males and workers, when analyzed alone, were sufficiently different to distinguish among these five species. This demonstrates that we can use this kind of analysis for the identification of stingless bee species and that the sex of the individual does not impede identification. Computer-assisted morphometric analysis of bee wing images can be a useful tool for biodiversity studies and conservation programs.

Searching for molecular markers in head and neck squamous cell carcinomas (HNSCC) by statistical and bioinformatic analysis of larynx-derived SAGE libraries

Background: Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignancies in humans. The average 5-year survival rate is one of the lowest among aggressive cancers, showing no significant improvement in recent years. When detected early, HNSCC has a good prognosis, but most patients present metastatic disease at the time of diagnosis, which significantly reduces survival rate. Despite extensive research, no molecular markers are currently available for diagnostic or prognostic purposes. Methods: Aiming to identify differentially-expressed genes involved in laryngeal squamous cell carcinoma (LSCC) development and progression, we generated individual Serial Analysis of Gene Expression (SAGE) libraries from a metastatic and non-metastatic larynx carcinoma, as well as from a normal larynx mucosa sample. Approximately 54,000 unique tags were sequenced in three libraries. Results: Statistical data analysis identified a subset of 1,216 differentially expressed tags between tumor and normal libraries, and 894 differentially expressed tags between metastatic and non-metastatic carcinomas. Three genes displaying differential regulation, one down-regulated (KRT31) and two up-regulated (BST2, MFAP2), as well as one with a non-significant differential expression pattern (GNA15) in our SAGE data were selected for real-time polymerase chain reaction (PCR) in a set of HNSCC samples. Consistent with our statistical analysis, quantitative PCR confirmed the upregulation of BST2 and MFAP2 and the downregulation of KRT31 when samples of HNSCC were compared to tumor-free surgical margins. As expected, GNA15 presented a non-significant differential expression pattern when tumor samples were compared to normal tissues. Conclusion: To the best of our knowledge, this is the first study reporting SAGE data in head and neck squamous cell tumors. Statistical analysis was effective in identifying differentially expressed genes reportedly involved in cancer development. The differential expression of a subset of genes was confirmed in additional larynx carcinoma samples and in carcinomas from a distinct head and neck subsite. This result suggests the existence of potential common biomarkers for prognosis and targeted-therapy development in this heterogeneous type of tumor.

SELECTION OF TRICHOGRAMMA PRETIOSUM LINEAGES FOR CONTROL OF GRAPHOLITA MOLESTA IN PEACH

Grapholita molesta (Lepidoptera: Tortricidae) is one of the main pests of peach trees in Brazil, causing fruit losses of 3-5%. Among possible biological control agents, Trichogramma pretiosum (Hymenoptera: Trichogrammatidae) has been found in peach orchards. Our objectives were to study the rearing of T pretiosum in eggs of G. molesta and Anagasta kuehniella (Lepidoptera: Pyralidae), and select lineages of this parasitoid that have the potential to control G. molesta. Selection of best lineages was made from 5 populations of T pretiosum collected from organically-cultivated peach orchards. The study was done under controlled temperature (25 +/- 2 degrees C), relative humidity (70 +/- 10%) and 14:10 h (light:dark) photoperiod conditions. Grapholita molesta eggs were found to be adequate hosts for the development of T pretiosum, and the parameters for number of parasitized eggs, percent parasitized eggs, and sex ratio were similar to those for A. kuehniella eggs. The highest rate of parasitism of G. molesta eggs occurred in eggs with up to 48 h of embryonic development. Among the lineages of T pretiosum that were collected, HO8, PO8, PEL, and L3M showed the best biological performance and are therefore indicated for semi-field and field studies for biological control of oriental fruit moth.

Genome wide scan for quantitative trait loci affecting tick resistance in cattle (Bos taurus x Bos indicus)

Background : In tropical countries, losses caused by bovine tick Rhipicephalus (Boophilus) microplus infestation have a tremendous economic impact on cattle production systems. Genetic variation between Bos taurus and Bos indicus to tick resistance and molecular biology tools might allow for the identification of molecular markers linked to resistance traits that could be used as an auxiliary tool in selection programs. The objective of this work was to identify QTL associated with tick resistance/susceptibility in a bovine F2 population derived from the Gyr (Bos indicus) x Holstein (Bos taurus) cross. Results: Through a whole genome scan with microsatellite markers, we were able to map six genomic regions associated with bovine tick resistance. For most QTL, we have found that depending on the tick evaluation season (dry and rainy) different sets of genes could be involved in the resistance mechanism. We identified dry season specific QTL on BTA 2 and 10, rainy season specific QTL on BTA 5, 11 and 27. We also found a highly significant genome wide QTL for both dry and rainy seasons in the central region of BTA 23. Conclusions: The experimental F2 population derived from Gyr x Holstein cross successfully allowed the identification of six highly significant QTL associated with tick resistance in cattle. QTL located on BTA 23 might be related with the bovine histocompatibility complex. Further investigation of these QTL will help to isolate candidate genes involved with tick resistance in cattle.

Molecular basis for porcine parvovirus detection in dead fetuses

Reproductive failures are still common grounds for complaint by commercial swine producers. Porcine parvovirus (PPV) is associated with different clinical reproductive signs. The aim of the present study was to investigate PPV fetal infection at swine farms having ongoing reproductive performance problems. The presence of virus in fetal tissues was determined by nested-polymerase chain reaction assay directed to the conserved NS1 gene of PPV in aborted fetuses, mummies and stillborns. Fetuses show a high frequency of PPV infection (96.4%; N = 28). In 60.7% of the fetuses, PPV were detected in all tissue samples (lung, heart, thymus, kidney, and spleen). Viral infection differed among fetal tissues, with a higher frequency in the lung and heart (P < 0.05). Fetuses with up to 99 days of gestational age and from younger sows showed a higher frequency of PPV (P < 0.05). No significant difference in the presence of PPV was detected among the three clinical presentations. The results suggest that PPV remains an important pathogenic agent associated with porcine fetal death.

Genetic trend estimates of meat quality traits in a male broiler line

The present research was conducted to estimate the genetic trends for meat quality traits in a male broiler line. The traits analyzed were initial pH, pH at 6 h after slaughter, final pH, initial range of falling pH, final range of falling pH, lightness, redness, yellowness, weep loss, drip loss, shrink loss, and shear force. The number of observations varied between 618 and 2125 for each trait. Genetic values were obtained by restricted maximum likelihood, and the numerator relationship matrix had 107,154 animals. The genetic trends were estimated by regression of the broiler average genetic values with respect to unit of time (generations), and the average genetic trend was estimated by regression coefficients. Generally, for the traits analyzed, small genetic trends were obtained, except for drip loss and shear force, which were higher. The small magnitude of the trends found could be a consequence of the absence of selection for meat quality traits in the line analyzed. The estimates of genetic trends obtained were an indication of an improvement in the meat quality traits in the line analyzed, except for drip loss.

High-throughput SNP genotyping in the highly heterozygous genome of Eucalyptus: assay success, polymorphism and transferability across species

Background: High-throughput SNP genotyping has become an essential requirement for molecular breeding and population genomics studies in plant species. Large scale SNP developments have been reported for several mainstream crops. A growing interest now exists to expand the speed and resolution of genetic analysis to outbred species with highly heterozygous genomes. When nucleotide diversity is high, a refined diagnosis of the target SNP sequence context is needed to convert queried SNPs into high-quality genotypes using the Golden Gate Genotyping Technology (GGGT). This issue becomes exacerbated when attempting to transfer SNPs across species, a scarcely explored topic in plants, and likely to become significant for population genomics and inter specific breeding applications in less domesticated and less funded plant genera. Results: We have successfully developed the first set of 768 SNPs assayed by the GGGT for the highly heterozygous genome of Eucalyptus from a mixed Sanger/454 database with 1,164,695 ESTs and the preliminary 4.5X draft genome sequence for E. grandis. A systematic assessment of in silico SNP filtering requirements showed that stringent constraints on the SNP surrounding sequences have a significant impact on SNP genotyping performance and polymorphism. SNP assay success was high for the 288 SNPs selected with more rigorous in silico constraints; 93% of them provided high quality genotype calls and 71% of them were polymorphic in a diverse panel of 96 individuals of five different species. SNP reliability was high across nine Eucalyptus species belonging to three sections within subgenus Symphomyrtus and still satisfactory across species of two additional subgenera, although polymorphism declined as phylogenetic distance increased. Conclusions: This study indicates that the GGGT performs well both within and across species of Eucalyptus notwithstanding its nucleotide diversity >= 2%. The development of a much larger array of informative SNPs across multiple Eucalyptus species is feasible, although strongly dependent on having a representative and sufficiently deep collection of sequences from many individuals of each target species. A higher density SNP platform will be instrumental to undertake genome-wide phylogenetic and population genomics studies and to implement molecular breeding by Genomic Selection in Eucalyptus.

Pharmacogenetics of OATP Transporters Reveals That SLCO1B1 c.388A > G Variant Is Determinant of Increased Atorvastatin Response

Aims: The relationship between variants in SLCO1B1 and SLCO2B1 genes and lipid-lowering response to atorvastatin was investigated. Material and Methods: One-hundred-thirty-six unrelated individuals with hypercholesterolemia were selected and treated with atorvastatin (10 mg/day/4 weeks). They were genotyped with a panel of ancestry informative markers for individual African component of ancestry (ACA) estimation by SNaPshot (R) and SLCO1B1 (c.388A>G, c.463C>A and c.521T>C) and SLCO2B1 (-71T>C) gene polymorphisms were identified by TaqMan (R) Real-time PCR. Results: Subjects carrying SLCO1B1 c.388GG genotype exhibited significantly high low-density lipoprotein (LDL) cholesterol reduction relative to c.388AA+c.388AG carriers (41 vs. 37%, p = 0.034). Haplotype analysis revealed that homozygous of SLCO1B1*15 (c.521C and c.388G) variant had similar response to statin relative to heterozygous and non-carriers. A multivariate logistic regression analysis confirmed that c.388GG genotype was associated with higher LDL cholesterol reduction in the study population (OR: 3.2, CI95%: 1.3-8.0, p < 0.05). Conclusion: SLCO1B1 c.388A>G polymorphism causes significant increase in atorvastatin response and may be an important marker for predicting efficacy of lipid-lowering therapy.

Modification of composition of a nanoemulsion with different cholesteryl ester molecular species: Effects on stability, peroxidation, and cell uptake

Purpose: Use of lipid nanoemulsions as carriers of drugs for therapeutic or diagnostic purposes has been increasingly studied. Here, it was tested whether modifications of core particle constitution could affect the characteristics and biologic properties of lipid nanoemulsions. Methods: Three nanoemulsions were prepared using cholesteryl oleate, cholesteryl stearate, or cholesteryl linoleate as main core constituents. Particle size, stability, pH, peroxidation of the nanoemulsions, and cell survival and uptake by different cell lines were evaluated. Results: It was shown that cholesteryl stearate nanoemulsions had the greatest particle size and all three nanoemulsions were stable during the 237-day observation period. The pH of the three nanoemulsion preparations tended to decrease over time, but the decrease in pH of cholesteryl stearate was smaller than that of cholesteryl oleate and cholesteryl linoleate. Lipoperoxidation was greater in cholesteryl linoleate than in cholesteryl oleate and cholesteryl stearate. After four hours' incubation of human umbilical vein endothelial cells (HUVEC) with nanoemulsions, peroxidation was minimal in the presence of cholesteryl oleate and more pronounced with cholesteryl linoleate and cholesteryl stearate. In contrast, macrophage incubates showed the highest peroxidation rates with cholesteryl oleate. Cholesteryl linoleate induced the highest cell peroxidation rates, except in macrophages. Uptake of cholesteryl oleate nanoemulsion by HUVEC and fibroblasts was greater than that of cholesteryl linoleate and cholesteryl stearate. Uptake of the three nanoemulsions by monocytes was equal. Uptake of cholesteryl oleate and cholesteryl linoleate by macrophages was negligible, but macrophage uptake of cholesteryl stearate was higher. In H292 tumor cells, cholesteryl oleate showed the highest uptakes. HUVEC showed higher survival rates when incubated with cholesteryl stearate and smaller survival with cholesteryl linoleate. H292 survival was greater with cholesteryl stearate. Conclusion: Although all three nanoemulsion types were stable for a long period, considerable differences were observed in size, oxidation status, and cell survival and nanoemulsion uptake in all tested cell lines. Those differences may be helpful in protocol planning and interpretation of data from experiments with lipid nanoemulsions.

Long Terminal Repeat Sequence Analysis of HTLV-2 Molecular Variants Identified in Southern Brazil

In Brazil, human T-lymphotropic virus type 2 (HTLV-2) is endemic in Amerindians and epidemic in intravenous drug users (IDUs). The long terminal repeat (LTR) is the most divergent genomic region of HTLV-2, therefore useful to characterize subtypes. Nucleotide sequence and restriction fragment length polymorphism (RFLP) analysis of LTR genomic segments of fourteen HTLV-2 strains isolated from HIV-infected patients of Londrina, Southern Brazil, were carried out. Molecular analysis disclosed that all HTLV-2 strains belonged to 2a subtype, and RFLP detected the presence of the a4, a5, and a6 subgroups according to Switzer's nomenclature. RFLP correlated with nucleotide sequence, and phylogenetic analysis clustered HTLV-2 sequences of IDUs into subgroups a5 and a6. HTLV-2 sequences from individuals of sexual risk factor clustered into the a4 subgroup. These results extend the knowledge of the genetic diversity of HTLV-2 circulating in Brazil and provide insights into HTLV-2 transmission and virus movement in this geographic area.