Risk factors and presence of antibodies to Toxoplasma gondii in dogs from the coast of São Paulo State, Brazil

Toxoplasmosis is caused by the obligate intracellular protozoan parasite Toxoplasma gondii and affects warm-blooded vertebrates, including pets and man. Dogs are epidemio-logically important since they act as sentinels for the infection in humans. The present study aimed to determine the presence of antibodies to T. gondii in 205 serum samples from dogs in Ubatuba, Sao Paulo state, Brazil, through indirect immunofluorescence reaction (IFAT), as well as the risk factors related to toxoplasmosis in the animals such as breed, age, sex, access to outdoors, homemade food ingestion, access to untreated water, and contact with rodents. Toxoplasmosis-positive samples accounted for 52/205 (25.4%), with titers ranging from 16 to 256. The serological results presented significant association (P<0.05) with homemade food ingestion (45/118; 38.1%; CI95% 29.9%-47.2%) (OR=7.0; CI95% 3.0-16.6), and with access to outdoors where those that do not have access to the street were prevalent (37/121; 30.6%; CI95% 23.1%-39.3%) (OR=0.5; CI95% 0.2-1.0). These results show that toxoplasmosis in this region is related to problems of sanitary education, mainly concerning the appropriate cooking of foods, since most positive animals did not show significant association with the presence of rodents or untreated water consumption but showed, instead association with ingestion of homemade food. Thus, toxoplasmosis is a public health problem in the studied region, and sanitary measures are needed to control the infection due to the strict relationship between man and dog and the presented risk factors

Identification and application of bile metabolites to assess the exposure of fish to pharmaceuticals in the environment

Thousands of tons of pharmaceuticals are consumed yearly worldwide. Due to the continuous and increasing consumption and their incomplete elimination in wastewater treatment plants (WWTP), pharmaceuticals and their metabolites can be detected in receiving waters, although at low concentrations (ng to low μg/L). As bioactive molecules the presence of pharmaceuticals in the aquatic environment must be considered potentially hazardous for the aquatic organisms. In this thesis, the biotransformation and excretion of pharmaceuticals in fish was studied. The main biotransformation pathways of three anti‐inflammatory drugs, diclofenac, naproxen and ibuprofen, in rainbow trout were glucuronidation and taurine conjugation of the parent compounds and their phase I metabolites. The same metabolites were present in fish bile in aquatic exposures as in fish dosed with intraperitoneal injection. Higher bioconcentration factor in bile (BCFbile) was found for ibuprofen when compared to diclofenac and naproxen. Laboratory exposure studies were followed by a study of uptake of pharmaceuticals in a wild fish population living in lake contaminated with WWTP effluents. Of the analyzed 17 pharmaceuticals and six phase I metabolites, only diclofenac, naproxen and ibuprofen was present in bream and roach bile. It was shown, that diclofenac, naproxen and ibuprofen excreted by the liver can be found in rainbow trout and in two native fish species living in the receiving waters. In the bream and roach bile, the concentrations of diclofenac, naproxen and ibuprofen were roughly 1000 times higher than those found in the lake water, while in the laboratory exposures, the bioconcentration of the compounds and their metabolites in rainbow trout bile were at the same level as in wild fish or an order of magnitude higher. Thus, the parent compounds and their metabolites in fish bile can be used as a reliable biomarker to monitor the exposure of fish to environmental pharmaceuticals present in water receiving discharges from WWTPs.

Transfer of passive immunity and serum proteinogram in the first six months of life of Criollo Lageano and black and white holstein calves

The objective of this study was to evaluate and compare the transfer of passive immunity and the proteinogram in Criollo Lageano (CL) and Black and White Holstein (BWH) calves. Two groups were utilized with 13 Criollo Lageano and 10 BWH calves. Blood samples were collected for the measurement of total serum protein, electrophoresis of serum proteins, activity of the gamma glutamyl transferase, and concentration of IgG by the method of the zinc sulfate turbidity in periods between 24 and 36 hours of life, 15, 30, 60, 90, 120, 150 and 180 days. Statistical analysis was performed by ANOVA and Tukey test at 5% significance level, and correlations between variables were calculated. Variations of serum proteins followed a pattern of physiological behavior over the first six months of life and production of immunoglobulins was active earlier in BWH calves and slower in the Criollo Lageano, without causing any impact on their health. Gamma globulin in the first days of life (24-36h) was correlated with IgG (r=0.87 for CL and r=0.89 for BWH), PTS (r=0.91 for CL and r=0.92 for BWH), Glob (r=0.99 for CL and r=0.98 for BWH) and GGT (r=0.14 for CL and r=0.83 for BWH). It was concluded that there was no failure in the transfer of passive immunity in Criollo Lageano calves but this failure occurred in the BWH calves. IgG values estimated by the zinc sulfate turbidity and serum proteins were considered good indicators of the transfer of passive immunity in calves between 24 and 36 hours of life.

Sperm morphological and morphometric evaluation in captive collared peccaries (Pecari tajacu)

The aim of this study was to compare different staining methods for the evaluation of sperm morphology by light microscopy and also to describe the morphometry of the entire sperm in collared peccaries (Pecari tajacu). Semen from 10 males was obtained by electroejaculation and evaluated for sperm motility, vigor, and concentration. Semen smears were prepared through three different staining methods: Bengal rose, brome-phenol blue, and eosin-nigrosin. Smears were evaluated under light microscopy and sperm morphologic alterations were determined in percentage. In addition, sperm morphometric analysis was conducted by light microscopy coupled to image analyzer software. The smears stained with Bengal Rose provide the best results for the visualization of the sperm tail, midpiece, and head. The use of eosin-nigrosin stain did not allow an adequate impregnation, and some sperm presented a few contrasts with the background. A higher incidence of bent coiled tails was verified in the use of brome-phenol blue staining (P<0.05). Through morphometric evaluation, it was observed that the tail occupies the greatest proportion (89%) of the sperm which presents a discretely elongated head. According to the results, the use of the Bengal Rose stain is recommended for the morphologic evaluation of the collared peccary sperm.

In vitro somatic embryogenesis and adventitious root initiation have a common origin in eggplant (Solanum melongena L.)

Somatic embryogenesis was induced from cotyledon explants of eggplant cultured on MS medium supplemented with 54 µM NAA. Anatomical analysis of somatic embryo initiation and development was performed during the first four weeks. Proembryo formation was observed after the second day of culture, directly from perivascular cells or via pro-embryogenic masses derived from indeterminate meristematic masses (IMMs) originated in the vascular tissue. Those IMMs also gave rise to root primordia after 10 days of culture. The origin of embryos is discussed as well as the similarities between somatic embryogenesis and adventitious root formation.

Molecular characteristics of Machado-Joseph disease mutation in 25 newly described Brazilian families

Machado-Joseph disease (MJD) is a form of autosomal dominant spinocerebellar ataxia first described in North-American patients originating from the Portuguese islands of the Azores. Clinically this disorder is characterized by late onset progressive ataxia with associated features, such as: ophthalmoplegia, pyramidal and extrapyramidal signs and distal muscular atrophies. The causative mutation is an expansion of a CAG repeat in the coding region of the MJD1 gene. We have identified 25 unrelated families segregating the MJD mutation during a large collaborative study of spinocerebellar ataxias in Brazil. In the present study a total of 62 family members were genotyped for the CAG repeat in the MJD1 gene, as well as 63 non-MJD individuals (126 normal chromosomes), used as normal controls. We observed a wide gap between the size range of the normal and expanded CAG repeats: the normal allele had from 12 to 33 CAGs (mean = 23 CAGs), whereas the expanded alleles ranged from 66 to 78 CAGs (mean = 71.5 CAGs). There were no differences in CAG tract length according to gender of affected individuals or transmitting parent. We observed a significant negative correlation between age at onset of the disease and length of the CAG tract in the expended allele (r = -0.6, P = 0.00006); however, the size of the expanded CAG repeat could explain only about 40% of the variability in age at onset (r2 = 0.4). There was instability of the expanded CAG tract during transmission from parent to offspring, both expansions and contractions were observed; however, there was an overall tendency for expansion, with a mean increase of +2.4 CAGs. The tendency for expansion appeared to the greater in paternal (mean increase of +3.5 CAGs) than in maternal transmissions (mean increase of +1.3 CAGs). Anticipation was observed in all transmissions in which ages at onset for parent and offspring were known; however, anticipation was not always associated with an increase in the expanded CAG repeat length. Our results indicate that the molecular diagnosis of MJD can be confirmed or excluded in all suspected individuals, since alleles of intermediary size were not observed.

Enterococcus gallinarum carrying the vanA gene cluster: first report in Brazil

In 2000, Enterococcus faecalis resistant to vancomycin was first reported at a tertiary hospital in Porto Alegre, southern Brazil. The resistance spread to other hospitals and surveillance programs were established by hospital infection committees to prevent the spread of vancomycin-resistant enterococci. In February 2002, an isolate initially identified at the genus level as Enterococcus was obtained by surveillance culture (rectal swab) from a patient admitted to a hospital for treatment of septic arthritis in the shoulder. The isolate proved to be resistant to vancomycin by the disc diffusion method and confirmed by an E-test resulting in a minimal inhibitory concentration of > or = 256 µg/ml. This isolate was sent to a reference laboratory (Laboratório Especial de Bacteriologia e Epidemiologia Molecular, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, USP) for further study and proved to be an E. gallinarum by the polymerase chain reaction (PCR) using specific primers for the species. Due to the phenotype of unusually high vancomycin resistance, the isolate presumably had the resistance genes (vanA and vanB) and this was confirmed by PCR, which indicated the presence of the vanA gene. A 10.8-kb Tn1546-related transposon was also identified by long-PCR. Interspecies transfer of the vancomycin-resistance gene from the donor E. gallinarum was performed in a successful conjugation experiment in vitro, using E. faecium GE-1 and E. faecalis JH22 as receptors. This is the first report of the detection of a vanA determinant naturally acquired by E. gallinarum in Brazil, indicating the importance of characterizing VRE by both phenotype and genotype methods.

Human prophylactic vaccine adjuvants and their determinant role in new vaccine formulations

Adjuvants have been considered for a long time to be an accessory and empirical component of vaccine formulations. However, accumulating evidence of their crucial role in initiating and directing the immune response has increased our awareness of the importance of adjuvant research in the past decade. Nevertheless, the importance of adjuvants still is not fully realized by many researchers working in the vaccine field, who are involved mostly in the search for better target antigens. The choice of a proper adjuvant can be determinant for obtaining the best results for a given vaccine candidate, but it is restricted due to intellectual property and know-how issues. Consequently, in most cases the selected adjuvant continues to be the aluminum salt, which has a record of safety, but predominantly constitutes a delivery system (DS). Ideally, new strategies should combine immune potentiators (IP) and DS by mixing both compounds or by obtaining structures that contain both IP and DS. In addition, the term immune polarizer has been introduced as an essential concept in the vaccine design strategies. Here, we review the theme, with emphasis on the discussion of the few licensed new adjuvants, the need for safe mucosal adjuvants and the adjuvant/immunopotentiating activity of conjugation. A summary of toxicology and regulatory issues will also be discussed, and the Finlay Adjuvant Platform is briefly summarized.

Microglia and neurons in the hippocampus of migratory sandpipers

The semipalmated sandpiper Calidris pusilla and the spotted sandpiper Actitis macularia are long- and short-distance migrants, respectively. C. pusilla breeds in the sub-arctic and mid-arctic tundra of Canada and Alaska and winters on the north and east coasts of South America. A. macularia breeds in a broad distribution across most of North America from the treeline to the southern United States. It winters in the southern United States, and Central and South America. The autumn migration route of C. pusilla includes a non-stop flight over the Atlantic Ocean, whereas autumn route of A. macularia is largely over land. Because of this difference in their migratory paths and the visuo-spatial recognition tasks involved, we hypothesized that hippocampal volume and neuronal and glial numbers would differ between these two species. A. macularia did not differ from C. pusilla in the total number of hippocampal neurons, but the species had a larger hippocampal formation and more hippocampal microglia. It remains to be investigated whether these differences indicate interspecies differences or neural specializations associated with different strategies of orientation and navigation.

Study of carbohydrates present in the cladodes of Opuntia ficus-indica (fodder palm), according to age and season

Types and content of carbohydrates were evaluated by chemical (spectrophotometric assay) and physicochemical (Thin Layer Chromatography - TLC and High Performance Liquid Chromatography - HPLC) methods in some Opuntia ficus-indica varieties according to age and season. The samples comprised four varieties of palm (giant, copena F1, clone 20, and round palm). The results demonstrated that the four varieties of palm contain a good quantity of neutral and acid sugars in both summer and winter seasons. However, samples collected in the summer presented a higher content of carbohydrates specially glucose, fructose, galactose, xylose, and arabinose. The tertiary cladode (old cladode) presented almost the double content of sugar found in the quaternary cladodes (young cladodes). Pectic polysaccharides were sequentially extracted with water at 60 ºC, and EDTA at 60 ºC solution resulting in Water-Soluble Pectin (WSP) and a Chelating-Soluble Pectin (CSP) respectively. Galacturonic acid was detected in the fractions WSP and CSP. However, the fraction CSP presented the highest content of sugar acid.

Application of exploratory factor analysis to assess fish consumption in a university community

The objective of this research was to use the technique of Exploratory Factor Analysis (EFA) for the adequacy of a tool for the assessment of fish consumption and the characteristics involved in this process. Data were collected during a campaign to encourage fish consumption in Brazil with the voluntarily participation of members of a university community. An assessment instrument consisting of multiple-choice questions and a five-point Likert scale was designed and used to measure the importance of certain attributes that influence the choice and consumption of fish. This study sample was composed of of 224 individuals, the majority were women (65.6%). With regard to the frequency of fish consumption, 37.67% of the volunteers interviewed said they consume the product two or three times a month, and 29.6% once a week. The Exploratory Factor Analysis (EFA) was used to group the variables; the extraction was made using the principal components and the rotation using the Quartimax method. The results show clusters in two main constructs, quality and consumption with Cronbach Alpha coefficients of 0.75 and 0.69, respectively, indicating good internal consistency.

Phenolic content and antioxidant capacity in organically and conventionally grown eggplant (Solanum melongena) fruits following thermal processing

AbstractThermal processing and production practices used in vegetables can cause changes in their phytochemical contents. Eggplant is characterized by its high antioxidant content. The objective of this work was to determine levels of anthocyanins, polyphenols, and flavonoids and antioxidant capacity in organically and conventionally grown eggplant prepared fresh or subjected to one of three thermal preparation methods: boiling, baking or steaming. The soluble and hydrolyzable polyphenols and flavonoids content were quantified by Folin-Ciocalteu and Aluminum chloride methods, respectively. Anthocyanins were quantified according to the pH differential method. Antioxidant capacity was determined by DPPH and ORAC methods. The results showed differences between organic and conventional eggplant for some variables although cultivation method did not have a consistent effect. Hydrolysable polyphenol content was greater, and soluble and hydrolysable antioxidant capacities were higher in organically grown eggplant, while anthocyanin content was greater in conventionally grown eggplant. Fresh eggplant produced under conventional cultivation had a much greater content of anthocyanins compared to that of other cultivation method-thermal treatment combination. In general, steamed eggplant contained higher total polyphenol and flavonoid levels as well as greater antioxidant capacity. Steamed eggplant from both conventional and organic systems also had high amounts of anthocyanins compared to other thermal treatments.

Attention focus and balance control in young and older adults

This thesis investigated attention focus and balance control in eighteen healthy young adults and eighteen healthy older adults. All participants performed sixteen consecutive trials of a balance task which involved standing for 30-s on an unstable platform that could rotate only in the roll direction. There were no attention focus instructions provided on any of the sixteen trials. Following the completion of the initial and final attempt in the series, participants reported "where" their attention had been focused when performing the task. The results showed differences in balance between young and older adults and improvements in balance with practice in both young and older adults. However, there were no differences in attention focus strategies between young and older adults. Both age groups directed attention to multiple sources during the balance task. An equal focus on internal (i.e., feet, trunk, and other body parts) and external (i.e., the platform) sources with little focus on events not related to the task dominated on the first attempt of the balance task. Focus on internal sources was maintained and focus on events not related to the task increased at the expense of focus on external sources on the final attempt of the balance task. Following the series of sixteen trials to establish "natural" attention focus, participants performed three randomly presented trials, each with specific attention focus instructions (i.e., think about minimizing movements of the feet, the trunk, or the platform). The results showed that, in contrast to the literature, instructions to focus on an internal source, the trunk, actually augmented control of the task as reflected in reduced trunk sway whereas instructions to focus on an internal source, the feet, or an external source, the platform, did not benefit performance on the task. Thus, the distance fi-om the interaction point of the body with the external source is critical and may not depend on whether the source is internal or external. Thus, a global attention focus instruction may not be beneficial and the nature of the task should be considered when adopting attention focus instructions for young and older adults.

Involvement of fimbriae in host-mycoparasite recognition

Extracellular, non-flagellar appendages, termed fimbriae are widespread among fungi. Fungal fimbriae range in diameter from 6-10 nm and exhibit lengths of up to 30 ~m. Fungal fimbriae have been implicated in several functions: adhesion, conjugation and flocculation. A possible role of fimbriae in host-mycoparasite interactions was the focus of this study . Using electron microscopy, fimbriae were observed on the surfaces of Mortiere lla cande labrum, Mortie re lla pusi lla and Phascolomyces articulosus with diameter means of 9.1±0.4 nm, 9.4±0.5 nm and 8.6±0.6 nm, respectively, and lengths of up to 25 ~m. Fimbriae were not observed on the surface of the mycoparasite, Piptocephalis virginiana. Polyclonal antiserum (AU) prepared against the fimbrial protein of Ustilago violacea cross-reacted with 60 and 57 kDa M. candelabrum proteins. In addition, AU cross-reacted with 64 kDa proteins from both M. pusilla and P. articulosus. The proteins that cross-reacted with AU were electroeluted from polyacrylamide gels and were shown to subsequently form fibrils. The diameter means for the electroeluted fibrils were: for M. candelabrum 9.7±0.3 nm, M. pusilla 8.4±0.6 nm and P articulosus 9.2±0.5 nm. Finally, to ascertain the role of fimbriae in host-mycoparasite interactions, AU was incubated with P. virginiana and M. pusilla (mycoparasite/susceptible host) and with P. virginiana and P . articulosus (mycoparasite/ resistant host). It was observed that AU decreased significantly the level of contact between P. virginiana and M. pusilla and between P. virginiana and P. articulosus in comparison to prelmmune serum treatments. Thus, it was proposed that fimbriae might play recognition and attachment roles in early events of mycoparasitism.

Implementación de la técnica de hibridación in situ con fluorescencia (fish) en el laboratorio de hematología clínica para el diagnóstico y seguimiento de pacientes con leucemia granulocítica crónica

Tesis (Especialidad en el Laboratorio de Hematología) UANL