Abstract DNA genotyping of Mycobacterium tuberculosis has been widely applied in the understanding of disease transmission in many countries. The purpose of this study was to genotype the strains of M. tuberculosis isolated in patients with new tuberculosis (TB) cases in Minas Gerais, as well as to compare the similarity, discriminatory power, and agreement of the clusters between the IS6110 Restriction Fragment Length Polymorfism (RFLP) and 12 loci Variable Number Tandem Repeat - Mycobacterial Interspersed Repetitive Units (MIRU-VNTR) techniques. It was observed that 32% (66/204) of the isolated strains in the RFLP-IS6110 and 50.9% (104/204) of the isolated strains in the MIRU-VNTR presented a similarity of equal to or above 85%. The RFLP-IS6110 and MIRU-VNTR proved to contain a high discriminatory power. The similarity index resulting from the RFLP showed no recent transmission. Good agreement was observed between the techniques when clusters were detected; however, the best epidemiological relationship was found when using the RFLP-IS6110.

Los receptores para el fragmento cristali-zable "Fc" de la inmunoglobulina de clase G (IgG) son el puente entre la respuesta inmune humoral y celular, estos receptores se dividen en: FcyRIA, FcyRIA; FcyRIlA, FcyRIIB; FcyRIIIA, FcyRIIIB. El receptor FcyRIIIB presenta un polimorfismo bialélico codominante que se expresa en neutrófilos y se denomina Antígeno Neutrofílico (NA), el cual tiene dos variantes NA1 y NA2. Los mismos han sido identificados en diferentes estudios como factores genéticos que influyen en la susceptibilidad a lupus eritematoso sistémico (LES) o nefropatía lúpica (NL). Existe una diferencia en las capacidades de unión a la porción Fc de las IgG por parte de los alelos NA1 o NA2, que pueden estar asociadas con la eliminación ineficiente de las células apoptóticas, y complejos inmunes. El presente trabajo buscó establecer la existencia de la asociación genética entre el polimorfismo NA1 y NA2 del receptor FcyRIIIB con la susceptibilidad a LES y NL. Se estudiaron 134 pacientes lúpicos y 150 controles sanos. Todos los participantes dieron su consentimiento informado para participar en el estudio. Se realizó la determinación de los polimorfismos FcyRIIIB mediante PCR con el uso de primers alelo-específico. Los resultados permitieron observar que las frecuencias de los genotipos NA1 /NA1, NA1/NA2 y NA2/NA2 son del 0.57, 0.39 y 0.04 para el grupo control; y 0.20, 0.73,0.07 para pacientes lúpicos respectivamente. Los pacientes lúpicos mostraron un predominio del genotipo NA1/NA2, siendo el alelo NA2 el que condiciona riesgo a LES. El genotipo NA1 /NA1 demostró asociación estadísticamente significativa de riesgo a NL (Odss ratio 2.52, p<0.001) . Se concluye que los individuos que portan el genotipo NA1/NA2 o que portan el alelo NA2 tienen más probabilidad de desarrollar LES y cuando el paciente lúpico porta el genotipo NA1/NA1 tienen mayor probabilidad de desencadenar NL.

Fcy Receptors for the crystallizable fragment "Fc" of immunoglobulin class G ( IgG) are the bridge between the humoral and cellular immune response, these receptors are divided into: FcyRIA, FcyRIB; FcyRIlA, FcyRIIB; FcyRIIIA, FcyRIIIB. FcyRIIIB receptor has bialilic codominant polymorphism that is expressed in neutrophils and is called Neutrophilic Antigen (NA), which has two variants NA1 and NA2. They have been identified in different studies as genetic factors that influence susceptibility to Systemic Lupus Erythematosus (SLE) or lupus nephritis (NL). There is a difference in binding capacities to the Fc portion of IgG by NA1 NA2 alleles, that may be asso-ciated with the inefficient removal of apoptotic cells and immune complexes. This study tried to establish the existence of genetic polymorfism association between NA1 and NA2 FcyRIIIB polymorphism with thesusceptibility to SLE and N L. 134 SLE patients and 150 healthy controls werestudied. All participantsgave the informed consent to participate in the study. The FcyRIIIB polymorphism was determined by PCR, using allele-specific primers. The results allowed to observe that the frequencies of the NA1/NA1, NA1/NA2 and NA2/NA2 genotypes are 0.57, 0.39 and 0.04 for the control group; and 0.20, 0.73, 0.07 respectively for SLE patients.TheSLE patients showed apredominant of genotype NA1 /NA2, being the NA2 allele a risk factor for SLE. The NA/NA1 genotype showed statistically significantassociation to NL risk (Odds ratio 2.52, p<0.001).We conclude that individuals carrying the NA1/NA2 genotype or carrying the NA2 allele are more likely to develop SLE and lupus patientwhen carrying the NA1/NA1 genotype are more likely to trigger N L.

Dry chili pepper Mirasol type plants (Capsicum annuum L.) exhibiting foliar yellowing, big bud, and rolled-up margins symptoms were collected from commercial pepper fields in Calera de Victor Rosales country, Zacatecas, México. They were examined to determine the presence of phytoplasms by nested polymerase chain reaction (PCR) using the universal primer pair P1/Tint, followed by the primer pair R16F2n/R16R2 and restriction fragment length polymorfism (RFLP) analysis of 16S rDNA sequences. The presence of two different RFPL patterns in clones from different samples indicated the presence of two phytoplasmas isolates. Each was classified as strains of ‘Candidatus Phytoplasma trifolii', that belong to group 16SrVI. The first ChZac4F3 phytoplasma sequence belongs to subgroup 16SrVI-A, while the second ChZac5F1 phytoplasma sequence was classified into a new subgroup (16SrVI-J). ChZac4F3 and ChZac5F1 phytoplasma sequences showed a similarity of 99.2 and 99.1 %, respectively, with the reference strain of ‘Candidatus Phytoplasma trifolii' (AY390261), when both were analyzed via the iPhyClassifier program. This is the first report of two new strains of ‘Candidatus Phytoplasma trifolii' associated to foliar yellowing, big bud and rolled-up margins symptoms in Mirasol chili pepper in Zacatecas, México.

Plantas de chile (Capsicum annuum L.) para secado tipo Mirasol que mostraban síntomas de yema grande, amarillamiento y enrollamiento foliar, fueron colectadas en parcelas comerciales en el municipio de Calera de Víctor Rosales, Zacatecas, México. Se analizaron mediante la reacción en cadena de la polimerasa (PCR) anidada para determinar la presencia de fitoplasmas, con el par de oligonucleótidos universales P1/Tint, seguido del par R16F2n/R16R2, y del análisis de fragmentos del ADNr 16S mediante la obtención de bandas polimórficas a partir de patrones de restricción (RFLP). La presencia de dos patrones de restricción diferentes indicó la existencia de dos nuevas cepas de ‘Candidatus Phytoplasma trifolii', grupo 16SrVI. La primera secuencia de fitoplasma (ChZac4F3) correspondió al subgrupo 16SrVI-A; en cambio la segunda secuencia de fitoplasma (ChZac5F1) se clasificó dentro de un nuevo subgrupo (16SrVI-J). Ambas secuencias mostraron una similitud de 99.2 y 99.1 %, respectivamente, al ser comparadas con la cepa de referencia de ‘Candidatus Phytoplasma trifolii' (AY390261) al analizarlas con el programa iPhyClassifier. Este es el primer reporte de dos nuevas cepas de ‘Candidatus Phytoplasma trifolii' asociadas con la sintomatología de yema grande, amarillamiento y enrollamiento foliar en chile Mirasol en Zacatecas, México.

In this study, PCR assays targeting different Leishmania heat-shock protein 70 gene (hsp70) regions, producing fragments ranging in size from 230-390 bp were developed and evaluated to determine their potential as a tool for the specific molecular diagnosis of cutaneous leishmaniasis (CL). A total of 70 Leishmania strains were analysed, including seven reference strains (RS) and 63 previously typed strains. Analysis of the RS indicated a specific region of 234 bp in the hsp70 gene as a valid target that was highly sensitive for detection of Leishmania species DNA with capacity of distinguishing all analyzed species, after polymerase chain reaction-restriction fragment length polymorfism (PCR-RFLP). This PCR assay was compared with other PCR targets used for the molecular diagnosis of leishmaniasis: hsp70 (1400-bp region), internal transcribed spacer (ITS)1 and glucose-6-phosphate dehydrogenase (G6pd). A good agreement among the methods was observed concerning the Leishmania species identification. Moreover, to evaluate the potential for molecular diagnosis, we compared the PCR targets hsp70-234 bp, ITS1, G6pd and mkDNA using a panel of 99 DNA samples from tissue fragments collected from patients with confirmed CL. Both PCR-hsp70-234 bp and PCR-ITS1 detected Leishmania DNA in more than 70% of the samples. However, using hsp70-234 bp PCR-RFLP, identification of all of the Leishmania species associated with CL in Brazil can be achieved employing a simpler and cheaper electrophoresis protocol.

In this study, a genotypification of Leishmaniawas performed using polimerase chain reaction-restriction fragment length polymorfism (PCR-RFLP) and sequencing techniques to identify species of Leishmaniaparasites in phlebotomine sand flies and dogs naturally infected. Between January-February of 2009, CDC light traps were used to collect insect samples from 13 capture sites in the municipality of Posadas, which is located in the province of Misiones of Argentina. Sand flies identified as Lutzomyia longipalpiswere grouped into 28 separate pools for molecular biological analysis. Canine samples were taken from lymph node aspirates of two symptomatic stray animals that had been positively diagnosed with canine visceral leishmaniasis. One vector pool of 10 sand flies (1 out of the 28 pools tested) and both of the canine samples tested positively for Leishmania infantumby PCR and RFLP analysis. PCR products were confirmed by sequencing and showed a maximum identity with L. infantum. Given that infection was detected in one out of the 28 pools and that at least one infected insect was infected, it was possible to infer an infection rate at least of 0.47% for Lu. longipalpisamong the analyzed samples. These results contribute to incriminate Lu. longipalpis as the vector of L. infantumin the municipality of Posadas, where cases of the disease in humans and dogs have been reported since 2005.

Isolados de Colletotrichum gloeosporioides (ISO-1, ISO-2, ISO-3, ISO-4, ISO-5 e ISO-6), agente causal da antracnose em frutos de mangueira, foram caracterizados por meio de padrões eletroforéticos de proteínas totais e esterase, em gel de poliacrilamida e produção de enzimas extracelulares, em substratos sólidos específicos. A análise eletroforética mostrou variação no número, intensidade de coloração e posição das bandas no gel, dentro dos dois sistemas estudados. Verificou-se polimorfismo em relação a esterase, mostrando maior diferença entre os isolados, enquanto que nas proteínas totais, observou-se comportamento aparentemente uniforme. Quanto à produção de enzimas extracelulares: amilase, lípase e protease, todos os isolados, embora variando em comportamento, apresentaram atividade para essas enzimas, sendo aparentemente mais acentuada à atividade proteolítica.

Isolates of Colletotrichum gloeosporioides (ISO-1, ISO-2, ISO-3, ISO-4, ISO-5 and ISO-6), the causal agent of anthracnose disease on mango fruits, were characterized by electrophoretic patterns of total proteins and esterase in polyacrylamida gel, and also, by production of extracellular enzymes on specific solid substrate. The electrophoretic analysis showed variation in number, intensity of coloration and position of the bands in the gel at each studied system tested. In contrast to the monomorphic behavior to total proteins, high esterase polymorfism was observed indicating difference among isolates. All isolates showed the activity of extracellular enzymes such as amylase, lipase, and protease with some variation among them. The proteolitic activity seemed to be more accentuated than the two other enzymes studied.

O presente trabalho teve por objetivo verificar o efeito de diferentes períodos de luz ultravioleta sobre a similaridade genética do fungo simbionte de formigas cortadeiras pertencentes às espécies Acromyrmex ambiguus, Acromyrmex crassispinus e Acromyrmex lundi, através da técnica AFLP. Os fungos foram inoculados em meio de cultura Pagnocca e mantidos em câmaras climatizadas à temperatura de 25 + 1°C e escotofase de 24h, por um período de 15 dias. Posteriormente, foram expostos à luz ultravioleta por períodos de: 0, 5, 10, 15, 20, 25 e 30 minutos. Aos 45 dias após o tratamento, foi realizada a extração do DNA e padronização, realizando-se as reações de PCR por meio de 24 combinações de primers AFLP, das quais quatro foram selecionadas por apresentarem maior polimorfismo e consistência. Os dados moleculares foram transformados em matriz binária e analisados pelo programa estatístico NTSYS v. 2.1. As quatro combinações de primers utilizadas geraram 152 bandas polimórficas. As populações de fungos originárias de diferentes espécies de Acromyrmex apresentaram uma similaridade genética média de 37%, sendo que a indução de mutação através da luz ultravioleta permitiu obter isolados mais dissimilares.

The present research was aimed at verifing the effect of different periods of ultraviolet light on the development of the symbiont fungus of leaf cutting ants belonging to the species Acromyrmex ambiguus, Acromyrmex crassispinus and Acromyrmex lundi, through the AFLP technique. The fungus were inoculated in Pagnocca's medium and maintained in climatized cameras at 25 + 1°C and 24 hours of darkness, for a period of 15 days. After that, they were exposed to ultraviolet light for 0, 5, 10, 15, 20, 25, and 30 minutes. At 45 days after the treatment, the extraction and standardization of DNA was performed. PCR reactions were tested using 24 AFLP primer combinations, from which four were selected because they presented larger polymorfism and consistence. The molecular data were transformed in binary mattrix and analyzed by the statistical program NTSYS v. 2.1. The four combinations of primers generated 152 polymorfic bands. The fungus populations from different Acromyrmex species presented a medium genetic similarity of the 37%, and the mutations induced by the ultraviolet light allowed to obtain more dissimilar isolates.

Verificou-se o efeito dos genótipos halotano homozigoto dominante e heterozigoto e dos sistemas de criação confinado sobre piso de cimento, confinado sobre cama de maravalha e ao ar livre sobre a qualidade da carne suína. Foram utilizados 96 suínos machos castrados selecionados através do exame de DNA genômico, utilizando-se a técnica de reação em cadeia de polimerase (PCR) para amplificar a região do receptor rianodina. A região amplificada foi clivada pela técnica polimorfismo do comprimento dos fragmentos de restrição (RFLP). Mediu-se o pH nos músculos Longissimus dorsi e Semimembranosus aos quarenta e cinco minutos e vinte e quatro horas após o abate. A capacidade de retenção de água foi avaliada em amostras do músculo Longissimus dorsi através das técnicas de perda de líquido por gotejamento, de cocção e de descongelamento. Escores para a cor e o grau de marmorização da carne foram atribuídos com auxílio de padrões fotográficos. A maciez da carne foi medida pela força de cisalhamento em equipamento Warner-Bratzler Shear. Suínos com gene halotano heterozigotos apresentaram valores inferiores de pH, menor capacidade de retenção de água e uma freqüência de carcaças com carne PSE três vezes maior. O sistema de criação não afetou os valores de pH inicial e final ou a capacidade de retenção de água da carne suína. A maior incidência de carne PSE foi observada nas carcaças dos suínos criados em sistema confinado sobre piso de concreto. Não houve efeito significativo da interação genótipo halotano e sistema de criação para as características avaliadas.

The effect of halothane genotypes (heterozygous and dominant homozygous) and intensive rearing systems (indoor, wood shavings bedding and outdoor) on pork quality were determinated. Ninety six castrated male pigs were used for the trial. Identification of the halothane genotype was determined in blood samples using the DNA-test, based on the polymerase chain reaction (PCR) amplification of the critical region of the ryanodine receptor and subsequent restricion of the amplifield fragment by the restriction fragment lengh polymorfism (RFLP) technique. The pH at 45 minutes and 24 hours after slaughter was measured on the Longissimus dorsi and Semimembranosus muscles. At the laboratory, the Longissimus dorsi muscle was used for the evaluation of the color, marbling, drip loss, defrosting loss and cooking loss and shear force. The muscle of heterozygous pigs for the halothane gene had lower pH and higher drip loss and the incidence of the PSE condition in this genotype was three times higher. The rearing system did not affect the initial and ultimate pH or water holding capacity. Indoor reared pigs had greater frequency of PSE carcass. The interaction between halothane genotypes and rearing systems had no significant effect on any trait studied.

O objetivo deste trabalho foi verificar o efeito dos genótipos halotano homozigoto dominante e heterozigoto e de três sistemas de criação (confinado sobre piso de cimento, confinado sobre cama de maravalha e ao ar livre) no desempenho produtivo e na qualidade da carcaça e da carne suína. Foram utilizados 96 suínos machos castrados selecionados pelo exame de DNA genômico no sangue utilizando-se a técnica de reação em cadeia da polimerase (PCR) para amplificar a região do receptor rianodina. A região amplificada foi clivada pela técnica do polimorfismo do comprimento dos fragmentos de restrição (RFLP). Nas fases de crescimento e terminação, avaliaram-se ganho de peso, consumo de ração e conversão alimentar. Após o abate, as carcaças foram pesadas e tipificadas eletronicamente. Vinte e quatro horas após o abate, foram separados e pesados o pernil e o carrê e medida a área de olho de lombo. A presença do gene halotano não afetou o desempenho dos suínos, mas aumentou a porcentagem de carne magra e diminuiu a quantidade de gordura na carcaça. Suínos criados no sistema intensivo ao ar livre apresentaram menor ganho de peso e pior conversão alimentar. No sistema intensivo de criação confinado sobre cama, os suínos produziram carcaças com menor quantidade de carne magra e maior quantidade de gordura. Não houve efeito significativo da interação genótipo halotano e sistema de criação para as características avaliadas.

The objective of this study was to evaluate the effect of the halothane genotypes (heterozygous and dominant homozygous) and intensive rearing systems (indoor, wood shavings bedding and outdoor) on growth performance, carcass and meat yield. Ninety six castrated male pigs were used for the trial. Identification of the halothane genotype was determined in blood samples using the DNA-test, based on the polymerase chain reaction (PCR) for amplification of the critical region of the ryanodine receptor and subsequent restriction of the amplified fragment by restriction fragment lengh polymorfism technique (RFLP). Growth and finishing performance were evaluated by weight gain, feed intake and feed efficiency. Carcass quality was evaluated by backfat and muscle depth, meat percentage through Hennessy Grading Probe and carcass weight. Twenty four hours after slaugther, ham and loin weight were recorded and the loin eye area was evaluated. The growth performance was similar for the two genotypes. The heterozygous pigs for the halothane gene had higher percentage of fat-free lean and less fat in the carcass. Outdoor reared pigs showed lower average daily gain and worst efficiency than pigs reared indoor. Carcasses of pigs reared under wood shavings bed had less fat-free lean and greater backfat thickeness. The interaction between halothane genotypes and rearing systems had no significant effect on any trait studied.

We have demonstrated that Leishmania spp. grown as promastigotes, are sensitive to the K+ channel inhibitors 4-aminopyridine and glibenclamide. Their host cells, the macrophages, are not affected by similar concentrations of the drugs. We have also initiated the molecular characterization of the mechanisms involved in the development of drug resistance to glibenclamide by the parasite. Therefore, we have selected experimentally and begun to characterize the Venezuelan Leishmania (Leishmania) strain, NR resistant to glibenclamide [NR(Gr)]. The analysis of genomic DNA evidenced the existence of a fragment which apparently is amplified in NR(Gr). The fragment recognized by the pgpA probe, related to the Leishmania P-glycoprotein family and which was originally isolated from L. tarentolae, showed a size polymorfism between the sensitive and the resistant strain. These results suggest that the development of resistance to glibenclamide in the strain NR(Gr) might be associated with the amplification of the ltpgpA or related gene(s)