Isolation and characterization of the promoter sequence of a cassava gene coding for Pt2L4, a glutamic acid-rich protein differentially expressed in storage roots.

Cassava is one of the most important tropical food crops for more than 600 million people worldwide. Transgenic technologies can be useful for increasing its nutritional value and its resistance to viral diseases and insect pests. However, tissue-specific promoters that guarantee correct expression of transgenes would be necessary. We used inverse polymerase chain reaction to isolate a promoter sequence of the Mec1 gene coding for Pt2L4, a glutamic acid-rich protein differentially expressed in cassava storage roots. In silico analysis revealed putative cis-acting regulatory elements within this promoter sequence, including root-specific elements that may be required for its expression in vascular tissues. Transient expression experiments showed that the Mec1 promoter is functional, since this sequence was able to drive GUS expression in bean embryonic axes. Results from our computational analysis can serve as a guide for functional experiments to identify regions with tissue-specific Mec1 promoter activity. The DNA sequence that we identified is a new promoter that could be a candidate for genetic engineering of cassava roots.

Whole-Genome Sequence of Leptospira interrogans Serovar Hardjo Subtype Hardjoprajitno Strain Norma, Isolated from Cattle in a Leptospirosis Outbreak in Brazil.

Leptospirosis is caused by pathogenic bacteria of the genus Leptospira spp. This neglected re-emergent disease has global distribution and relevance in veterinary production. Here, we report the whole-genome sequence and annotation of Leptospira interrogans serovar Hardjo subtype Hardjoprajitno strain Norma, isolated from cattle in a livestock leptospirosis outbreak in Brazil.

Is there a relationship between antipsychotic blood levels and their clinical efficacy? An analysis of studies design and methodology.

There are now more than 50 studies concerning neuroleptic blood levels and clinical outcome relationships. Haloperidol, the most studied, is the only antipsychotic permitting some conclusions. A number of authors suggest that the striking lack of agreement between different studies results from heterogeneity of their quality. Here, we have used a scoring system for assessing the quality of those studies. According to this system, none (0/14) of the studies having a score < 0.60 was able to show a therapeutic window, as compared to 53% (10/19) of those having a score > or = 0.60 (p = 0.002, Fisher exact test). Also, the studies able to identify the presence of a therapeutic window during haloperidol treatment were those having sample size > 20 (p = 0.06) and those whose patients were treated with fixed doses (p = 0.02). The diagnosis of schizophrenia in the studies seems not to be an exclusive condition, as compared with those also including schizophreniform and schizoaffective disorders (p = 0.12). Our qualitative analysis of haloperidol blood level publications seem to indicate that an upper limit may exist for haloperidol efficacy; values above this limit seem not to provide any supplementary clinical improvement and may even reduce therapeutic effect.

Effect of gamma irradiation on the behavioral properties of crotoxin.

Crotoxin has been detoxified with gamma radiation in order to improve crotalic antiserum production. Nevertheless, present knowledge of the biological characteristics of irradiated crotoxin is insufficient to propose it as an immunizing agent. Crotoxin is known to increase the emotional state of rats and to decrease their exploratory behavior (Moreira EG, Nascimento N, Rosa GJM, Rogero JR and Vassilieff VS (1996) Brazilian Journal of Medical and Biological Research, 29: 629-632). Therefore, we decided 1) to evaluate the effects of crotoxin in the social interaction test, which has been widely used for the evaluation of anxiogenic drugs, and 2) to determine if irradiated crotoxin induces behavioral alterations similar to those of crotoxin in the social interaction, open-field and hole-board tests. Male Wistar rats (180-220 g) were used. Crotoxin (100, 250, and 500 micrograms/kg) was injected intraperitoneally 2 h before the social interaction test. Similarly, irradiated crotoxin (2000 Gy gamma radiation from a 60Co source) was administered at the doses of 100, 250, and 500 micrograms/kg for the hole-board test, and at the doses of 1000 and 2500 micrograms/kg for the open-field and social interaction tests. ANOVA complemented with the Dunnett test was used for statistical analysis (P < 0.05). Crotoxin decreased the social interaction time(s) at the doses of 100, 250 and 500 micrograms/kg (means +/- SEM) from 51.6 +/- 4.4 to 32.6 +/- 3.7, 28.0 +/- 3.6 and 31.6 +/- 4.4, respectively. Irradiated crotoxin did not induce behavioral alterations. These results indicate that 1) crotoxin may be an anxiogenic compound, and 2) in contrast to crotoxin, irradiated crotoxin was unable to induce behavioral alterations, which makes it a promising compound for the production of crotalic antiserum.

Evaluation of an ELISA-PPD for the diagnosis of bovine tuberculosis in field trials in Brazil.

Bovine tuberculosis is a major health problem in Brazil. The intradermal tuberculin test is the standard test for its detection, but it can lack both sensitivity and specificity. The purpose of this study was to evaluate a bovine enzyme-linked immunosorbent assay- (ELISA - PPD) under field conditions in Brazil. A total of 1632 animals from 13 dairy farms were tested with the intradermal tuberculin test (ITT). Two hundred and seven cows gave a positive reaction, which represents 12.7 per cent of the cattle studied. The sensitivity and specificity rates to ITT were 87.7 per cent and 95.2 per cent, respectively. From the 1632 animals 15 per cent of each herd (220 in total) were selected to be tested by the ELISA. Differences between mean optical density (OD) of the control group, ITT -positive and ITT -negative groups were all significant (P<0.01). The sensitivity rates to ELISA - PPD were 86.7 per cent, while specificity was 90.6 per cent. The use of ELISA - PPD is suggested for situations where the investigation of the whole herd is more important than the individual testing of each cow. In addition, the ELISA - PPD can also be helpful when a collective diagnosis is desired to elucidate clinical suspicions of disease, or in the first steps of a control program, for identification of foci.

Molecular characterization by LSSP-PCR and DNA sequencing of a pathogenic isolate of Leptospira interrogans from Brazil.

We report the initial characterization of a leptospiral isolate, Leptospira interrogans, serogroup Sejroe, serovar Hardjo, genotype Hardjoprajitno, strain Norma, and its relatedness with L. interrogans, serogroup Sejroe, serovar Hardjo, genotype Hardjoprajitno, strain Hardjo and Leptospira borgpetersenii, serogroup Sejroe, serovar Hardjo, genotype Hardjobovis, strain Sponselee. The Norma strain singled out during a leptospirosis outbreak in cattle immunized with antigens from the reference strain Hardjoprajitno (OMS). By applying a microscopic agglutination serological test (MAT) to cattle (n = 2966) with symptoms of leptospirosis between 2003 and 2007, more than 50% of sera were found positive for one of the following serotypes: Hardjoprajitno (31-21%), Hardjo Norma (46-40%), Hardjo hardjobovis (18-10%), Mini (8-4%) and Wolffi (7-4%). In immunization trials using six isolates plus Norma isolate, the remission of MAT in these isolates was observed following 6 months of the initial vaccination. To provide molecular ground for the high MAT Norma frequency found in these isolates, a DNA polymorphic analysis was conducted by comparing the Norma isolate with reference strains Hardjoprajitno and Sponselee. The polymorphic analysis in secY showed five base changes in Norma relative to Hardjoprajitno strain, corresponding to 98% identity, while Sponselee displayed 49 polymorphic sites relative to the Hardjoprajitno strain, representing 80% identity. The alignment of secY translated sequences shows no differences between Hardjoprajitno and Norma, and eight polymorphisms between genotype hardjoprajttno and strain Sponselee. Three-dimensional modelling located these variations within the loop region connecting helices 7 and 8 from secY which is less conserved. DNA sequencing of 23S ribosomal conserved fragment revealed a single polymorphism between Hardjoprajitno and Norma, and 13 polymorphisms between strains Sponselee, Hardjoprajitno and Norma. The differences between Hardjo and Norma were confirmed by low stringency single-specific primer polymerase chain reaction (LSSP-PCR) signature experiments with the primer G2, using as template the 285 bp fragment initially amplified with G1/G2 primers.

Mordedura canina e atendimento antirrábico humano em Minas Gerais / Dog bite and human anti-rabies healthcare, Minas Gerais

O objetivo do presente trabalho foi descrever e avaliar a mordedura canina e o atendimento antirrábico humano em Minas Gerais, de 1999 a 2004, correlacionando fontes de informação e áreas de risco predeterminadas para raiva humana transmitida por cão. Realizou-se um estudo observacional descritivo retrospectivo, utilizando-se, de forma adaptada, a análise exploratória de prontuários dos atendimentos da Superintendência de Epidemiologia da Secretaria de Estado da Saúde de Minas Gerais (339.012 de atendimentos), do Sistema de Informação de Notificação de Agravos, do Sistema de Informações sobre Mortalidade, do Sistema de Informações Hospitalares e do Programa Nacional de Imunizações (132.452 fichas). Para a classificação dos agravos, usou-se o Código Internacional de Doenças (10ª revisão). Os dados foram armazenados e analisados com auxílio dos softwares Epi-Info, Tab-Win e Office®. Verificou-se que o tratamento antirrábico humano é excessivo nas áreas de baixo e médio risco para raiva e, ao contrário, reduzido nas áreas de alto risco. O perfil do paciente é estudante masculino, menor de 14 anos, residente em área urbana de baixo risco para raiva humana transmitida por cão, com mordedura única nos membros, provocada por cão sadio e observável. Os sistemas de informação não oferecem a confiabilidade necessária ao médico responsável para a prescrição do tratamento antirrábico adequado. A profilaxia da raiva deve ter um aspecto multicêntrico, com interfaces na atenção tanto à saúde humana quanto à animal, o que não tem ocorrido, propiciando falhas na vigilância e no atendimento do agravo.

The objective of the present paper is describe and evaluate dog bite and some aspects of anti-rabid human care in Minas Gerais during five years, correlating the sources of information and epidemiological risk areas defined for human rabies transmitted by dogs in the State. We performed an observational retrospective study by adapted exploration form analysis, from 1999 to 2004, using databases of Epidemiology of Minas Gerais and National Information Systems of reportable disease, Immunization, Mortality, Hospitalization, International Code of Diseases (10th revision). The areas of risk for human rabies transmitted by dogs were pre determined. The dog bite is still the main complaint that leads to care. The profile of the patient is a male student, under 14 years of age, with a single wound in members, resident in an urban low risk area for human rabies transmitted by dogs, and is healthy and observable. The treatment is excessive in areas of low and medium risk. In high-risk areas, there is a low indication of treatment. Information systems do not offer the reliability required by the doctor responsible for prescribing the appropriate anti-rabies treatment. Rabies prophylaxis should have a multi-centre aspect, with interfaces in attention to health and veterinary, which has not occurred, providing surveillance failures.

Caracterização da movimentação de bovinos com o uso de um modelo tipo centro-periferia / Characterization of the bovine movement using a core-periphery model

Verificou-se a influência da movimentação de bovinos na configuração de uma estrutura do tipo centro-periferia. Foram aplicadas técnicas das redes sociais - modelo centro/periferia (C/P) contínuo - para inferir sobre a semelhança entre os padrões de movimentação registrados no estudo com uma estrutura C/P perfeita. Os resultados mostram alta correlação, e o poder discriminatório do algoritmo permite identificar a existência de uma estrutura tipo C/P. Este resultado perfila a metodologia proposta como uma ferramenta analítica nos processos de caracterização produtiva e a regionalização de medidas de intervenção sanitária. O trabalho considera a aplicação de conceitos intuitivos sobre a definição de estruturas C/P condizentes com os determinantes de circuitos pecuários.

A degree in which bovine movement patterns configures a core-periphery structure was studied. Social networks analysis was used - a core-periphery (C/P) continuous model - estimating high correlation and an identification of a core with a discrimination power that outline this model as a tool for the characterization of livestock forms of production and regionalization of measures of sanitary intervention in animal health. This work considers an application of intuitive concepts about the definition of C/P structures that are according to the determinants of the livestock market circuits.

Distribuição temporal e espacial da leishmaniose visceral em humanos e cães em Belo Horizonte-MG, 1993 a 2007 / Temporal and spatial distribution of leishmaniasis in humans and dogs from Belo Horizonte-MG, 1993-2007

O objetivo desta pesquisa foi descrever as análises da série cronológica e determinar a tendência da leishmaniose visceral em humanos e cães para o município de Belo Horizonte, de 1993 a 2007. De 1994, quando surgiram os primeiros casos da doença, até 2007, foram registrados 994 casos humanos autóctones com 116 óbitos. De 1993 a 2007, foram analisadas 1.492.401 amostras de sangue canino com percentual de positividade de 5,9 por cento, utilizando-se os testes de enzyme-linked immunosorbent assay (ELISA) como triagem e de imunofluorescência indireta (IFI) como confirmatório. As análises indicaram que a leishmaniose visceral apresentou tendência crescente dos coeficientes de incidência em humanos e de prevalência em cães, e observaram-se arquétipos distintos dessa tendência, retratando o momento histórico de transição endêmica. A leishmaniose visceral em cães não apresentou características de distribuição sazonal, e não foi possível concluir a existência ou não de ciclicidade do fenômeno. A leishmaniose visceral em humanos e cães tem ampla distribuição espacial nas nove regionais administrativas com risco diferenciado de acometimento da doença, apesar da presença das fontes de infecção e dos susceptíveis em todo o município.

The chronological series analysis and the tendencies of both human and canine visceral leishmaniasis were determined in Belo Horizonte city, from 1993 to 2007. From 1994, when the first cases of the disease were reported, until 2007, 994 human cases were recorded, which led to 116 deaths. From 1993 to 2007, 1,492,401 samples of canine blood were analyzed by the enzyme-linked immunosorbent assay (ELISA) as screening test and by the Indirect Immunofluorescence (IFI) as confirmatory test. The rate of positive results was 5.9 percent. The analyses indicated that the visceral leishmaniasis presents a tendency of increasing the coefficient of incidence in humans and prevalence in dogs and different archetypes of this tendency were observed portraying historical moment of endemic transition. Canine visceral leishmaniasis did not show a seasonal distribution, and it was not possible to determine whether the phenomenon was cyclic or not. Human and canine visceral leishmaniasis showed a broad spatial distribution in the nine administrative sectors with a differentiated risk of illness, in spite of the presence either of the sources of infection or the susceptible individuals in the whole county.

Molecular typing of Mycobacterium bovis isolates from south-east Brazil by spoligotyping and RFLP.

The identification of 163 strains of Mycobacterium bovis by polymerase chain reaction (PCR) and microbiological tests was carried out on 252 tuberculous-like lesions (TLLs) collected from slaughtered cattle in south-east Brazil. This study compared the usefulness of three genotyping techniques, IS6110-restriction fragment length polymorphism (RFLP), polymorphic guanine-cytosine-rich sequence (PGRS)-RFLP and direct repeat (DR)-spoligotyping, as applied to M. bovis isolates. Based on IS6110-RFLP genotyping we selected a group of 23 isolates containing more than one IS6110 copy, along with 16 samples containing one IS6110 copy from different geographical areas, evenly distributed among dairy (eight) and beef cattle (eight). These selected isolates were analysed by PGRS-RFLP and DR-spoligotyping genotyping. Dairy cattle (17%) display a higher frequency of multiple IS6110 copies than beef cattle (10%). A comparison between the genotype data obtained fails to show a correlation between the main clusters found by the three techniques. However, the clustering of each genotyping procedure revealed that the majority of strains are closely related. The RFLP-PGRS patterns showed a sizable group (20.5%) containing a 5.5 kb fragment and the predominant spoligotype is similar to that from the BCG vaccine strain. Unexpectedly, four strains (2.4%) showed drug resistance to 0.2 microg/ml isoniazid and 20 microg/ml ethionamide, but none of them was resistant to rifampicin or other antibiotics tested.

Caracterização demográfica e epidemiológica de cães e gatos domiciliados em Barbacena, MG / Demographic and epidemiologic characterization of dogs and cats domiciled in Barbacena-MG, Brazil

The characterization of demographic and epidemiologic aspects of dogs and cats domiciled in households in Barbacena, Minas Gerais, Brazil, is imperative to understand the dynamics of animal populations for the purpose of planning actions in public health and zoonosis control. A total of 840 semi-structured questionnaires were applied. Respondents were randomly selected owners from urban and rural areas. The results revealed 0.22 per capita dog, 0.051 per capita cat, 0.81 dog per house, and 0.19 cat per house. Dogs were present in 51.3% of the properties, distributed as 77.9% in rural and 48.6% in urban areas (47.3% in houses and 16.5% in apartments). The average age was 4.47 years for dogs, with odds male/female of 1.58. For the cats, the average age was 2.9 years and odds male/female of 0.91. The mortality rate before weaning was 13.4% for dogs and 22.8% for cats. The breeding purpose rate was 68.3% of dogs while cats as pets were 99.4%. The main form of animals acquisition was by donation (65.8%) and only 4.14% came from other cities. About the type of feeding, 55.9% of dogs and 46.3% of cats ate manufactured products. Only 29.6% of dogs and 16.5% of cats had some type of veterinary care. The rabies vaccination covers 89.2% and 68.1% of dogs and cats, respectively. Castration or use of contraceptive was found in 18.7% of dogs and 17.9% of cats. These numbers could be used for measures of control in dogs and cats population of the municipality, as well in programs for the control of zoonotic diseases.

[Hypochondriasis by proxy in children: report of two cases] / Hipocondria por procuração em crianças: relato de dois casos.

OBJECTIVE: To emphasize important aspects for the diagnosis and approach of patients with history of child abuse, related with the Munchausen Syndrome by proxy, and its variant, Hypochondriases by proxy. METHODS: The authors describe two cases followed in the Pediatric Service in a terciary hospital of Salvador, Bahia, in which they found aspects of the medical history and physical examination of these children and in the mothers' behavior compatible with Munchausen by proxy (Factitious disorder by proxy). RESULTS: In the two cases presented, we observed a standard in the clinical history, described as recurrent inpatient and outpatient medical support, through the last three years, without the identification of abnormal results. These facts do not decreased the certainty of the mothers about their children's disease (hypochondriases by proxy). Furthermore we observed cooperation on the part of the children by incorporating the role of the sick one, which made their school and social adaptation more difficult. CONCLUSIONS: The pediatrician must be aware of the diagnosis of Munchausen Syndrome by Proxy and its variation, hypochondriases by proxy, in those cases of unsuitable and recurrent disease, lack of symptoms and signs in the absence of parents, and diagnostic methods with meaningless results. When absolutely certain about diagnosis, the physician must establish the risk for the child (increased mortality in these cases) and suggest psychotherapy for the mother or perpetrator.

A comparison of three DNA extractive procedures with Leptospira for polymerase chain reaction analysis.

Three DNA extraction methods were evaluated in this study: proteinase K followed by phenol-chloroform; a plant proteinase (E6870) followed by phenol-chloroform; and boiling of leptospires in 0.1 mM Tris, pH 7.0 for 10 min at 100 degrees C, with no phenol treatment. Every strain treated with proteinase K or E6870 afforded positive polymerase chain reaction (PCR) reaction. On the other hand, from five strains extracted by the boiling method, three did not feature the 849 bp band characteristic in Leptospira. We also evaluated by RAPD-PCR, DNAs from serovars isolated with proteinase K and proteinase 6870 with primers B11/B12. Each of the DNA samples provided PCR profiles in agreement with previous data. Moreover, the results with E6870 showed less background non-specific amplification, suggesting that removal of nucleases was more efficient with E6870. The limit for detection by PCR using Lep13/Lep14 was determined to be 10(2) leptospira, using the silver stain procedure.

Detection of Mycobacterium bovis in milk by polymerase chain reaction.

A simple method was developed for DNA extraction of Mycobacterium bovis in milk and further detection of the bacterium by polymerase chain reaction (PCR). Milk previously seeded with M. bovis was used as the starting material. The procedure involved overnight digestion of a milk sample with proteinase K at 56 degrees C and phenol extraction, followed by ethanol precipitation and PCR. The amplification pattern obtained was analyzed with primers BW8-BW9 which amplify a 248 bp in strains of M. bovis. By using the BW8-BW9 primers, 10(3) CFU were detected on silver-stained PAGE gels. The procedure was validated by PCR analysis of milk in tuberculin-positive animals. It is anticipated that this method can be used for routine diagnosis of M. bovis in milk samples.

Comparison between a gamma-IFN assay and intradermal tuberculin test for the diagnosis of bovine tuberculosis in field trials in Brazil.

Bovine tuberculosis is a major problem in Brazil. The intradermal tuberculin test is the standard test for detection of bovine tuberculosis in Brazil but can lack both sensitivity and specificity. The purpose of this study was to compare a bovine gamma-IFN assay with the tuberculin test under field conditions in Brazil. A total of 1632 animals from 13 dairy farms were tested using the single cervical tuberculin test (SCTT). Among those animals, about 15% of each herd, 220 in total, represented a high-risk group and were selected to be tested using the gamma-IFN test. Of the 1632 animals tested, 207 presented significant reactions representing 12.7% of the cattle studied. In the selected group the number of animals positive by the gamma-IFN assay was 126/220 (57.3%) and the total number of reactive cows on SCTT was 106/220 (48.2%). The real number of infected cattle, following standards, was 120/220 (54.5%). From these results the relative sensitivity rate of gamma-IFN test was 100% including the false-positive results and 88.3% for the SCTT--a significant (P < 0.01) difference in favour of the gamma-IFN test of 11.7%. The gamma-IFN assay also identified some positive animals 60-120 days earlier than the SCTT. In conclusion, we believe that the gamma-IFN assay can be used alone or in combination with the SCTT, as a valuable tool for the control of bovine tuberculosis in the Brazilian national herd.

Characterization of a novel bacteriocin-encoding plasmid found in clinical isolates of Staphylococcus aureus.

Plasmids specifying bacteriocin production and immunity to its action were found in three clinical isolates of Staphylococcus aureus obtained in different hospitals located in Rio de Janeiro. These plasmids (pRJ28, pRJ29 and pRJ30) of 8.0 kb were found to generate identical restriction fragment patterns upon digestion with several enzymes, although the range of strains susceptible to the respective bacteriocin varied among the producer strains, when different Gram-positive bacteria were used as indicators, pRJ29 was then chosen for further characterization in order to compare it with pRJ6 and pRJ9, two small bacteriocin-encoding plasmids previously described in strains isolated from food. pRJ29 was found to code for a bacteriocin with chemical properties (sensitivity to proteases, heat resistance, activity under anaerobiosis, and estimated molecular weight) similar to those of pRJ6-encoded bacteriocin, conferring cross-immunity to it. However, its restriction map differed from those of pRJ6 and pRJ9. These studies together with hybridization, incompatibility, and mobilization analyses using a derivative of pRJ29 tagged with Tn917-lac suggest that pRJ29 is a mosaic composed of genetic determinants found on pRJ6 and pRJ9, and that IS257 was not involved in the recombination events which gave rise to pRJ29.

Variação da contagem de células somáticas em vacas leiteiras de acordo com patógenos da mastite / Somatic cell counts variation in dairy cows according to mastitis pathogens

Avaliou-se o efeito de patógenos da mastite sobre a contagem de células somáticas (CCS) em leite. Foram coletadas 3.987 amostras de leite de 2.657 animais oriundos de 24 rebanhos leiteiros localizados nos estados do Rio de Janeiro e Minas Gerais. As amostras de leite foram usadas para CCS e identificação de patógenos da mastite. Estatísticas descritivas, teste T para amostras independentes e modelo linear generalizado foram usados para análise dos dados. O modelo linear generalizado identificou os efeitos de rebanho, animal dentro de rebanho, ordem de parto, estação do ano e infecção intramamária causada por Streptococcus agalactiae e Streptococcus spp. que não S. agalactiae como significativos na variação da CCS. O efeito de animal dentro de rebanho foi maior que o efeito de rebanho. S. agalactiae foi o patógeno responsável pelo maior aumento da CCS em vacas e apresentou em média 1.520.000 células/mL. Foi observado efeito específico dos patógenos na variação da CCS.

The influence of mastitis pathogens on variation of milk somatic cell count (SCC) was evaluated. Three thousand nine hundred eighty-seven milk samples were colected from 2,657 dairy cows in 24 herds located in the states of Minas Gerais and Rio de Janeiro. The milk samples were used to SCC and identification of mastitis pathogens. Descriptive statistics, T test for independent samples, and generalized linear model were used to data analysis. The generalized linear model identified the effects of herd, animal within herd, parity, year season, intramammary infection, and infection caused by Streptococcus agalactiae and Streptococcus spp. except S. agalactiae as significant on SCC variation. The effect of animal within herd was higher than the effect of herd. S. agalactiae was the pathogen responsible for higher SCC increasing and presented the average of 1,520,000 cells/mL. The specific effect on SCC variation was observed in the study.