Analysis of five polymorphic DNA markers for indirect genetic diagnosis of haemophilia A in the Brazilian population

Hemophilia A is an X-linked, inherited, bleeding disorder caused by the partial or total inactivity of the coagulation factor VIII (FVIII). Due to difficulties in the direct recognition of the disease-associated mutation in the F8 gene, indirect diagnosis using polymorphic markers located inside or close to the gene is used as an alternative for determining the segregation of the mutant gene within families and thus for detecting carrier individuals and/or assisting in prenatal diagnosis. This study characterizes the allelic and haplotype frequencies, genetic diversity, population differentiation and linkage disequilibrium of five microsatellites (F8Int1, F8Int13, F8Int22, F8Int25.3 and IKBKG) in samples of healthy individuals from Sao Paulo, Rio Grande do Sul and Pernambuco and of patients from Sao Paulo with haemophilia A to determine the degree of informativeness of these microsatellites for diagnostic purposes. The interpopulational diversity parameters highlight the differences among the analyzed population samples. Regional differences in allelic frequencies must be taken into account when conducting indirect diagnosis of haemophilia A. With the exception of IKBKG, all of the microsatellites presented high heterozygosity levels. Using the markers described, diagnosis was possible in 10 of 11 families. The F8Int22, F8Int1, F8Int13, F8Int25.3 and IKBKG microsatellites were informative in seven, six, five and two of the cases, respectively, demonstrating the effectiveness of using these microsatellites in prenatal diagnosis and in carrier identification in the Brazilian population.

Characterization of Equine Adipose Tissue-Derived Progenitor Cells Before and After Cryopreservation

In horses, stem cell therapies are a promising tool to the treatment of many injuries, which are common consequences of athletic endeavor, resulting in high morbidity and often compromising the performance. In spite of many advantages, the isolation of stem cells similar to human, from equine adipose tissue, occurred only recently. The aim of this study was to isolate equine adipose tissue-derived progenitor cells (eAT-PC), to characterize their proliferative potential, and to study their differentiation capacity before and after cryopreservation. The cells, isolated from horse adipose tissue, presented similar fibroblast-like cell morphology in vitro. Their proliferation rate was evaluated during 63 days (23 passages) before and after cryopreservation. After the induction of osteogenic differentiation, von Kossa staining and positive immunostaining studies revealed the formation of calcified extracellular matrix confirming the osteogenic potential of these cells. Adipogenic differentiation was induced using two protocols: routine and other one developed by us, while our protocol requires a shorter time (Oil Red O staining revealed significant accumulation of lipid droplets after 7 days). Chondrogenic differentiation was observed after 21 days of induced pellet culture, as evidenced by histological (toluidine blue) and immunohistochemistry studies. Our data demonstrate that eAT-PC can be easily isolated and successfully expanded in vitro while presenting significant proliferating rate. These cells can be maintained undifferentiated in vitro and can efficiently undergo differentiation at least into mesodermal derivates. These eAT-PC properties were preserved even after cryopreservation. Our findings classify eAT-PC as a promising type of progenitor cells that can be applied in different cell therapies in equines.

Canine population dynamics: the potential effect of sterilization campaigns

Objective. To analyze, through mathematical modeling, the potential ability of sterilization campaigns to reduce the population density of pet dogs. Methods. Mathematical models were constructed to simulate the canine population dynamics and project the results of control strategies based on several sterilization rates. Results. Even at high sterilization rates (for example, 0.80 year(-1)), it would take approximately 5 years to reduce density by 20%. Even so, other sources of population growth, such as the importing of dogs from other geographic areas, could outweigh the effects of a sterilization program. Conclusions. A program`s effectiveness is contingent upon not only on the sterilization rate, but also the rate of population growth. Sterilization campaigns may potentially reduce population density, but this reduction may not be immediately evident.

Population structure and mouse-virulence of Toxoplasma gondii in Brazil

Recent studies found that isolates of Toxoplasma gondii from Brazil were biologically and genetically different from those in North America and Europe. However, to date only a small number of isolates have been analysed from different animal hosts in Brazil. In the present study DNA samples of 46 T. gondii isolates from cats in 11 counties in Sao Paulo state, Brazil were genetically characterised using 10 PCR restriction fragment length polymorphism markers including SAG1, SAG2, SAG3, STUB, GRA6, c22-8, c29-2, L358, PKI and Apico. An additional marker, CS3, that locates on chromosome VIIa and has previously been shown to be linked to acute virulence of T. gondii was also used to determine its association to virulence in mice. Genotyping of these 46 isolates revealed a high genetic diversity with 20 genotypes but no clonal Type I, II or III lineage was found. Two of the 46 isolates showed mixed infections. Combining genotyping data in this study with recent reported results from chickens, dogs and cats in Brazil (total 125 isolates) identified 48 genotypes and 26 of these genotypes had single isolates. Four of the 48 genotypes with multiple isolates identified from different hosts and locations are considered the common clonal lineages in Brazil. These lineages are designated as Types BrI, BrII, BrIII and BrIV. These results indicate that the T. gondii population in Brazil is highly diverse with a few successful clonal lineages expanded into wide geographical areas. In contrast to North America and Europe, where the Type II clonal lineage is overwhelmingly predominant, no Type II strain was identified from the 125 Brazil isolates. Analysis of mortality rates in infected mice indicates that Type BrI is highly virulent, Type BrIII is non-virulent, whilst Type BrII and BrIV lineages are intermediately virulent. In addition, allele types at the CS3 locus are strongly linked to mouse-virulence of the parasite. Thus, T. gondii has an epidemic population structure in Brazil and the major lineages have different biological traits. (C) 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Isolation and genetic characterisation of Toxoplasma gondii from a red-handed howler monkey (Alouatta belzebul), a jaguarundi (Puma yagouaroundi), and a black-eared opossum (Didelphis aurita) from Brazil

Toxoplasma gondii isolates are highly diverse in domestic animals from Brazil. However, little is known about the genetics of this parasite from wild mammals in the same region. Reveal genetic similarity or difference of T. gondii among different animal populations is necessary for us to understand transmission of this parasite. Here we reported isolation and genetic characterisation of three T. gondii isolates from wild animals in Brazil. The parasite was isolated by bioassay in mice from tissues of a young male red handed howler monkey (Alouatta belzebul), an adult male jaguarundi (Puma yagouaroundi), and an adult female black-eared opossum (Didelphis aurita). The monkey and the jaguarundi had inhabited the Zoo of Parque Estadual Dois Irmaos, Pernambuco State, Northeastern Brazil, for 1 year and 8 years, respectively. The wild black-eared opossum was captured in Sao Paulo State, Southeastern Brazil, and euthanised for this study because it was seropositive for T. gondii (titre 1:100 by the modified agglutination test, MAT). Ten PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) markers, SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico, were used to genotype the isolates. T. gondii was isolated from the brain and heart homogenate of the monkey, the muscle homogenate of the jaguarundi, and the heart homogenate of the black-eared opossum. This was the first isolation of T. gondii from a neotropical fetid from Brazil. The isolate from the monkey (TgRhHmBr1) was not virulent in mice, whereas the isolates from the jaguarundi (TgJagBr1) and the black-eared opossum (TgOpBr1) were virulent in mice. The genotype of the isolate from the monkey has been identified in isolates from a goat and ten chickens in the same region of Brazil, suggesting that it may be a common lineage circulating in this region. The genotypes of the isolates from the jaguarundi and the black-eared opossum have not been previously reported. Although there are already 88 genotypes identified from a variety of animal hosts in Brazil, new genotypes are continuously being identified from different animal species, indicating an extremely high diversity of T. gondii in the population. (C) 2010 Elsevier B.V. All rights reserved.

Modeling the Dynamics of Viral Evolution Considering Competition Within Individual Hosts and at Population Level: The Effects of Treatment

We consider two viral strains competing against each other within individual hosts (at cellular level) and at population level (for infecting hosts) by studying two cases. In the first case, the strains do not mutate into each other. In this case, we found that each individual in the population can be infected by only one strain and that co-existence in the population is possible only when the strain that has the greater basic intracellular reproduction number, R (0c) , has the smaller population number R (0p) . Treatment against the one strain shifts the population equilibrium toward the other strain in a complicated way (see Appendix B). In the second case, we assume that the strain that has the greater intracellular number R (0c) can mutate into the other strain. In this case, individual hosts can be simultaneously infected by both strains (co-existence within the host). Treatment shifts the prevalence of the two strains within the hosts, depending on the mortality induced by the treatment, which is, in turn, dependent upon the doses given to each individual. The relative proportions of the strains at the population level, under treatment, depend both on the relative proportions within the hosts (which is determined by the dosage of treatment) and on the number of individuals treated per unit time, that is, the rate of treatment. Implications for cases of real diseases are briefly discussed.

Avian Infectious Bronchitis Virus in Brazil: A Highly Complex Virus Meets a Highly Susceptible Host Population

Infectious bronchitis (IB) is a highly aggressive disease for poultry in terms of symptoms and economic losses, and the control of this disease is difficult if flocks are not protected against type-specific challenges by the Avian infectious bronchitis virus (IBV). This article summarizes data presented by the author at the Workshop on Infectious Bronchitis 2009 on IB and IBV, including future developments on the field.

Periodontal attachment loss in an untreated isolated population of Brazil

Background: The aim of this study was to assess the prevalence, extent, and severity of clinical attachment loss (CAL) and to investigate the association of demographic, socioeconomic, and behavioral risk indicators with CAL in an untreated isolated population in Brazil. Methods: All subjects aged >= 12 years were identified by a census. Participants were submitted to a full-mouth clinical examination of six sites per tooth and were interviewed using a structured, written questionnaire. Results: Among the 214 subjects who were interviewed and examined clinically, CAL >= 5 mm in at least one site was observed in 8% of the 12- to 19-year-olds and in all dentate subjects >= 50 years of age; the age-dependent prevalence of CAL >= 7 mm in at least one site ranged from 5% among 12- to 19-year-olds to 83% among subjects >= 50 years old. Multivariate analysis identified plaque (odds ratio [OR] = 2.8), supragingival calculus (OR = 2.9 to 10.6), age >= 30 years (OR = 11.4), and smoking (OR = 2.4) as risk indicators for CAL >= 5 mm and smoking (OR = 8.2) as a risk indicator for CAL >= 7 mm. Conclusions: CAL is highly prevalent in this isolated population. The high occurrence of CAL in young age groups and the confirmation of traditional risk indicators for CAL in this study suggest that other factors, such as host susceptibility, may be needed to explain the high levels of CAL found. Age and behavioral factors were risk indicators associated significantly with the CAL found in this population and may be useful indicators of high-risk subjects for periodontal diseases.

Tooth loss prevalence and risk indicators in an isolated population of Brazil

Objective. The aim of this study was to assess the prevalence, extent, and risk indicators of tooth loss in an isolated population of Brazil. Material and methods. Two-hundred-and-forty-two subjects, ranging in age from 14 to 82 years (mean 36.2 years), were identified by census in an isolated population of Brazil. All consenting subjects received a full-mouth clinical (DFT index and information about missing teeth) and periodontal examination of 6 sites per tooth. Furthermore, they were interviewed using a structured written questionnaire in order to gather information about demographic, environmental, and biological variables. Results. Of the 200 subjects (80% response rate), 19 (9.5%) were edentulous, 90% had lost at least one tooth, and 39% had lost more than 8 teeth. The mean number of teeth lost was 9.5 (95% CI = 8.2-10.8). First mandibular molars were the most commonly missing teeth. In a multiple logistic regression analysis based on a theoretical hierarchical model of tooth loss, having more than 8 teeth lost was strongly associated with adult age (OR = 18.3-17.3, 95% CIs = 4.8-69.7 and 4.0-75.1) and female gender (OR = 5.9, 95% CI = 1.9-18.2) in the final model. Conclusions. Tooth loss was highly prevalent and extensive in this isolated population. Demographic and behavioral factors played an important role in tooth loss prevalence in this population.

Risk indicators for increased probing depth in an isolated population in Brazil

Background: The aim of this study was to assess the prevalence, extent, and severity of probing depth (PD) and to investigate the associations between demographic, socioeconomic, and behavioral risk indicators and PD in a periodontally untreated and isolated population in Brazil. Methods: The target population consisted of all individuals aged >= 12 years as identified by a census. Consenting participants were submitted to a full-mouth clinical examination of six sites per tooth and were interviewed using a structured written questionnaire. Results: Among the 214 subjects who were interviewed and clinically examined, PD >= 4 mm was observed in 54% to 83% of the subjects, depending on age, whereas the age-dependent prevalence of PD :6 mm ranged from 5% among 12- to 19-year-olds to 50% among 40- to 49-year-olds, decreasing to 40% among subjects >= 50 years of age. Multivariate analyses identified supragingival calculus (odds ratio [OR] = 5.4 to 10.3; 95% confidence intervals [CIs]: 2.5 to 11.6 and 4.0 to 26.2 for 20% to 50% and > 50% of the sites, respectively) as a risk indicator for PD A mm, whereas age :40 years (OR = 9.0; 95% CI: 1.7 to 48.5), being a moderate/heavy smoker (OR = 3.7; 95% CI: 1.4 to 10. 1), and having supragingival calculus in 20% to 50% of sites (OR = 6.8; 95% CI: 1.4 to 32.4) or in >50% of sites (OR = 15.3; 95% CI: 3.2 to 73.6) were risk indicators for PD >= 6 mm. Having undergone urgency dental treatment was a protective factor for PD A and >= 6 mm (OR = 0.4; 95% CI: 0.2 to 0.8). Conclusions: Increased PD is highly prevalent in this isolated population. Behavioral factors played a significant role as risk indicators for increased PD in this isolated population.

Development of a genetic method to estimate effective spawner numbers in the NPF and Queensland tiger prawn fisheries

OBJECTIVES: 1. To critically evaluate a variety of mathematical methods of calculating effective population size (Ne) by conducting comprehensive computer simulations and by analysis of empirical data collected from the Moreton Bay population of tiger prawns. 2. To lay the groundwork for the application of the technology in the NPF. 3. To produce software for the calculation of Ne, and to make it widely available.