Controle da leptospirose em bovinos de leite com vacina autógena em Santo Antônio do Monte, Minas Gerais

Um surto de leptospirose foi observado em bovinos leiteiros em Santo Antônio do Monte, Minas Gerais. O rebanho apresentava reações positivas anti-leptospira sorovar Hardjo no teste de microaglutinação (MAT) e havia sido vacinado anteriormente com vacina experimental contendo a sorovariedade Hardjo. O MAT revelou 48,06% dos bovinos positivos para sorovariedade Hardjo genótipo Hardjobovis, 36,82% para sorovariedade Hardjo genótipo Hardjoprajitno. Os animais apresentavam aborto e mastite com presença de sangue no leite. A presente pesquisa teve como objetivos isolar as sorovariedades existentes a partir da urina de vacas sorologicamente positivas, elaborar uma vacina experimental com as sorovariedades isoladas no rebanho, avaliar a eficiência do programa de vacinação por um período de dois anos por meio da sorologia do rebanho. Foi isolada Leptospira spp. a partir da urina de duas vacas com sinais sugestivos da doença. As amostras isoladas foram identificadas pela sorologia com anticorpos monoclonais e seqüenciamento do gene 16S rRNA como pertencentes à espécie Leptospira interrogans, sorogrupo Sejroe, sorovariedade Hardjo e genótipo Hardjoprajitno. O uso da vacina autógena foi eficaz no controle da leptospirose no rebanho no período de dois anos. Os resultados da sorologia revelaram ausência de animais positivos na última prova realizada no rebanho.

Ocorrência da infecção por Cryptosporidium spp. em cabritos (Capra hircus)

O presente estudo teve como objetivo determinar a ocorrência da infecção por Cryptosporidium spp. em cabritos de Quixadá, Ceará, Brasil. Participaram do estudo 400 cabritos, com idade entre três e 360 dias, de ambos os sexos, com e sem padrão racial definido, procedentes de 25 estabelecimentos rurais distribuídos em três circuitos. As fezes foram cadastradas de acordo com o aspecto e cor, distribuídas em tubos tipo "eppendorf®" e congeladas in natura a -20°C, até o momento das extrações de DNA genômico do parasito com auxílio de kit comercial. Para amplificação de fragmentos da subunidade 18S do RNA ribossômico (rRNA) foi utilizada a "Nested"-PCR. A ocorrência de Cryptosporidium spp em cabritos de Quixadá foi de 7,50% (30/400). A frequência no período seco e no chuvoso foi de 9,55% (19/199) e 5,47% (11/201), respectivamente (χ²=2,39 e P>0,05). Amostras positivas foram identificadas em 64,00% (16/25) das propriedades estudadas e dessas amostras 50,00% (15/30) e 70,00% (21/30) tinham as fezes com aspecto e cor normais, respectivamente, sugerindo que cabritos assintomáticos estão eliminando oocistos. Não foi observada positividade para Cryptosporidium spp. em animais com 301 a 360 dias, demonstrando que animais mais velhos apresentam menos possibilidade de se infectarem com o parasito.

Research of Klebsiella pneumoniae in dairy herds

Klebsiella pneumoniae is a common environmental agent of clinical and subclinical mastitis affecting dairy herds, and may be present in the final product decreasing its quality. Mastitis caused by K. pneumoniae is even more severe due to its poor response to antibiotic therapy, rapid evolution to toxic shock and death of the animal. This paper aimed to study the prevalence of this pathogen among dairy herds in ten farms located in different municipalities of São Paulo State based on size and use of milking technology. All mammary glands of all lactating cows were screened using the California Mastitis Test (CMT) and a strip cup. A single aseptic milk sample (20mL) was collected from all CMT-positive quarters and bulk tanks, whereas swab samples were collected from feces, hind limbs of the animals, bedding and milking parlor. Identification of K. pneumoniae was performed using conventional microbiology culture, biochemical assay and Polimerase Chain Reaction. The primers were designed and tested at the Laboratory of Molecular Biology applied to Zoonoses (FMVZ, Unesp-Botucatu) targeting the 16S rRNA gene. This study included 1067 animals. Six cases of intramammary infection by K. pneumoniae were detected in six different cows in two farms. Moreover, K. pneumoniae was isolated in 77 swabs (34 from bedding in 9 farms, 7 from waiting rooms in 5 farms, 6 from milking parlors in 4 farms, 11 from rectums in six farms, and 19 from hindlimbs in 7 farms. Molecular analysis confirmed the agent was K. pneumoniae. At least one strain of the agent was identified in a certain site in all farms, showing the need of maintaining the hygiene in dairy farms.

Histophilus somni-induced thrombotic meningoencephalitis in cattle from northern Paraná, Brazil

Thrombotic meningoencephalitis (TME) is a fatal neurological disease of cattle, predominantly from North America, that is caused by Histophilus somniwith sporadic descriptions from other countries. This manuscript describes the occurrence of spontaneous TME in cattle from northern Paraná, Brazil. Most cattle had acute neurological manifestations characteristic of brain dysfunction. Hematological and cerebrospinal fluid analyses were not suggestive of bacterial infections of the brain. Histopathology revealed meningoencephalitis with vasculitis and thrombosis of small vessels that contained discrete neutrophilic and/or lymphocytic infiltrates admixed with fibrin at the brainstem, cerebral cortex, and trigeminal nerve ganglion of all animals. All tissues from the central nervous system used during this study were previously characterized as negative for rabies virus by the direct immunofluorescence assay. PCR and RT-PCR assays investigated the participation of infectious agents associated with bovine neurological disease by targeting specific genes of H. somni, Listeria monocytogenes, bovine herpesvirus -1 and -5, bovine viral diarrhea virus, and ovine herpesvirus-2. PCR and subsequent sequencing resulted in partial fragments of the 16S rRNA gene of H. somni from brain sections of all animals with histopathological diagnosis of TME; all other PCR/RT-PCR assays were negative. These findings confirmed the participation of H. somni in the neuropathological disease observed in these animals, extend the geographical distribution of this disease, and support previous findings of H. somni from Brazil.

The nucleolus: a paradigm for cell proliferation and aging

The nucleolus is the cellular site of ribosome biosynthesis. At this site, active ribosomal DNA (rDNA) genes are rapidly transcribed by RNA polymerase I (pol I) molecules. Recent advances in our understanding of the pol I transcription system have indicated that regulation of ribosomal RNA (rRNA) synthesis is a critical factor in cell growth. Importantly, the same signaling networks that control cell growth and proliferation and are deregulated in cancer appear to control pol I transcription. Therefore, the study of the biochemical basis for growth regulation of pol I transcription can provide basic information about the nuclear signaling network. Hopefully, this information may facilitate the search for drugs that can inhibit the growth of tumor cells by blocking pol I activation. In addition to its function in ribosome biogenesis, recent studies have revealed the prominent role of the nucleolus in cell senescence. These findings have stimulated a new wave of research on the functional relationship between the nucleolus and aging. The aim of this review is to provide an overview of some current topics in the area of nucleolus biology, and it has been written for a general readership.

PCR-based ribotyping of Staphylococcus aureus

Genotyping techniques are valuable tools for the epidemiologic study of Staphylococcus aureus infections in the hospital setting. Pulsed-field gel electrophoresis (PFGE) is the current method of choice for S. aureus strain typing. However, the method is laborious and requires expensive equipment. In the present study, we evaluated the natural polymorphism of the genomic 16S-23S rRNA region for genotyping purpose, by PCR-based ribotyping. Three primer pairs were tested to determine the size of amplicons produced and to obtain better discrimination with agar gel electrophoresis and ethidium bromide staining. The resolution of the typing system was determined using sets of bacteria obtained from clinical specimens from a large tertiary care hospital. These included DNA from three samples obtained from a bacteremic patient, six strains with known and diverse PGFE patterns, and 88 strains collected over a 3-month period in the same hospital. Amplification patterns obtained from samples from the same patient were identical, and PFGE from samples known to be different produced three genotypes. Amplification of DNA from 61 methicillin-resistant isolates produced only one pattern. Methicillin-sensitive strains yielded a diversity of patterns, pointing to a true polyclonal distribution throughout the hospital (22 unique patterns from 27 strains). Computer-based software can be used to differentiate among identifiable strains, given the low number of bands and good characterization of PCR products. PCR-based ribotyping can be a useful technique for genotyping methicillin-sensitive S. aureus strains, but is of limited value for methicillin-resistant strains.

Association of Bartonella spp bacteremia with Chagas cardiomyopathy, endocarditis and arrythmias in patients from South America

Infection with Bartonella spp may cause cardiac arrhythmias, myocarditis and endocarditis in humans. The aim of the present study was to evaluate a possible association between Bartonella spp bacteremia and endocarditis, arrhythmia and Chagas cardiomyopathy in patients from Brazil and Argentina. We screened for the presence of bacterial 16S rRNA in human blood by PCR using oligonucleotides to amplify a 185-bp bacterial DNA fragment. Blood samples were taken from four groups of subjects in Brazil and Argentina: i) control patients without clinical disease, ii) patients with negative blood-culture endocarditis, iii) patients with arrhythmias, and iv) patients with chronic Chagas cardiomyopathy. PCR products were analyzed on 1.5% agarose gel to visualize the 185-bp fragment and then sequenced to confirm the identity of DNA. Sixty of 148 patients (40.5%) with cardiac disease and 1 of 56 subjects (1.8%) from the control group presented positive PCR amplification for Bartonella spp, suggesting a positive association of the bacteria with these diseases. Separate analysis of the four groups showed that the risk of a Brazilian patient with endocarditis being infected with Bartonella was 22 times higher than in the controls. In arrhythmic patients, the prevalence of infection was 45 times higher when compared to the same controls and 40 times higher for patients with Chagas cardiomyopathy. To the best of our knowledge this is the first report of the association between Bartonella spp bacteremia and Chagas disease. The present data may be useful for epidemiological and prevention studies in Brazil and Argentina.

Unusual association of NDM-1 with KPC-2 and armA among Brazilian Enterobacteriaceae isolates

We report the microbiological characterization of four New Delhi metallo-β-lactamase-1 (blaNDM-1)-producing Enterobacteriaceae isolated in Rio de Janeiro, Brazil. blaNDM-1 was located on a conjugative plasmid and was associated with Klebsiella pneumoniae carbapenemase-2 (blaKPC-2) or aminoglycoside-resistance methylase (armA), a 16S rRNA methylase not previously reported in Brazil, in two distinct strains of Enterobacter cloacae. Our results suggested that the introduction of blaNDM-1 in Brazil has been accompanied by rapid spread, since our isolates showed no genetic relationship.

Enhanced production and organic solvent stability of a protease fromBrevibacillus laterosporus strain PAP04

A bacterial strain (PAP04) isolated from cattle farm soil was shown to produce an extracellular, solvent-stable protease. Sequence analysis using 16S rRNA showed that this strain was highly homologous (99%) to Brevibacillus laterosporus. Growth conditions that optimize protease production in this strain were determined as maltose (carbon source), skim milk (nitrogen source), pH 7.0, 40°C temperature, and 48 h incubation. Overall, conditions were optimized to yield a 5.91-fold higher production of protease compared to standard conditions. Furthermore, the stability of the enzyme in organic solvents was assessed by incubation for 2 weeks in solutions containing 50% concentration of various organic solvents. The enzyme retained activity in all tested solvents except ethanol; however, the protease activity was stimulated in benzene (74%) followed by acetone (63%) and chloroform (54.8%). In addition, the plate assay and zymography results also confirmed the stability of the PAP04 protease in various organic solvents. The organic solvent stability of this protease at high (50%) concentrations of solvents makes it an alternative catalyst for peptide synthesis in non-aqueous media.

Screening and kinetics of glutaminase and glutamate decarboxylase producing lactic acid bacteria from fermented Thai foods

L-glutaminase and glutamic acid decarboxylase (GAD) catalyzes the hydrolysis of L-glutamine and glutamate, respectively. L-glutaminase widely used in cancer therapy along with a combination of other enzymes and most importantly these enzymes were used in food industries, as a major catalyst of bioconversion. The current investigation was aimed to screen and select L-glutaminase, and GAD producing lactic acid bacteria (LAB). A total of 338 LAB were isolated from fermented meat, fermented fish, fermented soya bean, fermented vegetables and fruits. Among 338 isolates, 22 and 237 LAB has been found to be positive for L-glutaminase and GAD, respectively. We found that 30 days of incubation at 35 ºC and pH 6.0 was the optimum condition for glutaminase activity by G507/1. G254/2 was found to be the best for GAD activity with the optimum condition of pH 6.5, temperature 40 ºC and ten days of incubation. These LAB strains, G507/1 and G254/2, were identified as close relative of Lactobacillus brevis ATCC 14869 and Lactobacillus fermentum NBRC 3956, respectively by 16S rRNA sequencing. Further, improvements in up-stream of the fermentation process with these LAB strains are currently under development.

Suun alueen pigmentoivat Prevotel/a-Iajit äideillä

Suuren osan hampaiston ja suun limakalvopintojen anaerobimikrobistosta muodostaa gram- negatiiviset Prevotella-suvun lajit. Viime vuosina Prevotella-ryhmässä on tapahtunut laajoja taksonomisia muutoksia ja Prevotella-sukuun on liitetty useita uusia lajeja. Näiden lisäksi on kantoja, jotka eivät asetu mihinkään jo tunnistettuun lajiin (Mario ym., 2011). Tässä tutkimuksessa käytimme materiaalina kahdesta eri väitösaineistosta peräisin olevia bakteeri-isolaatteja (Könönen 1994, Gürsoy 2012). Suun bakteeri-isolaatit oli eristetty synnyttäneiltä naisilta. Tutkimuksen päätavoitteena oli aiemmin biokemiallisesti tunnistettujen Prevotel/a-isolaattien lajitason tunnistus nykyaikaisin tunnistusmenetelmin. Tämän lisäksi tavoitteena oli vertailla niiden esiintyvyyttä äitien sylki- ja plakkinäytteissä. Fenotyyppisen tunnistuksen luotettavuuden arvioimiseksi tunnistusmenetelmänä käytettiin 16S rRNA osasekvenointia (n. 500 emäsparia) valikoiduille bakteeri-isolaateille pigmentoivien Prevotel/a-Iajien sekä mahdollisesti kokonaan uusien aiemmin tunnistamattomien lajien identifiointiin.

Coevolution of Heliconius spp. and Passiflora spp. : a phylogenetic comparison

Although a substantial amount of research has been done on all aspects ofHeliconius biology and their ecological interactions with Passiflora, there has not hitherto been a phylogenetic examination of this association for coevolution. To test the HeliconiuslPassilfora association for coevolutionary congruence, phylogenies for each group were established and compared. The phylogeny for 14 species ofHeliconiinae from Costa Rica was based on combined sequence data from rRNA ITS 2 and partial EF-1a gene regions. For the Passifloraceae, 17 host plant species were utilized to establish a phylogeny based on tRNALeucine and ITS 1/5.8S1 ITS 2 sequence data. The phylogenies for both groups were largely in agreement with current classification (for Passifloraceae) and previously established phylogenies. Associations with the large subgenera Passiflora and Decaloba correspond with the two major Advanced Radiation groups in Heliconius. Although strict congruence above subgenus level was not observed, broad scale congruence was evident. One main host shift as well as other possible explanations for lack of strict congruence are suggested.

Isolation and analysis of a corprinus cinereus DNA fragment showing homology to a fimbrial cDNA from ustilago violacea

Surface proteinaceous fibrils, termed fimbriae, were first identified on gram negative bacteria in the 1940s. Fungal fimbriae, discovered some 25 years later, are found on members of all fungal classes. In the present study, polyclonal antiserum raised against the fimbrial proteins of U. vio/acea were used in order to identify antigenically related proteins from Coprinus cinereus and Schizophy//um commune. Two polypeptides with molecular masses of 37 and 39 kDa from C. cinereus were observed and confirm earlier results. A single previously unidentified 50 kDa polypeptide in S. commune crossreacted with the antiserum. The 50 kDa protein was found to consist of 3 isoforms with isoelectric points ranging from 5.6 to 5.8. A fimbrial cDNA derived from U. vio/acea was used to identify DNA restriction fragments from C. cinereus and S. commune showing homology to the fimbrial transcript of U. vio/acea. Heterologous hybridization with this cDNA was used in order to screen a C. cinereus genomic DNA library. A single clone, A2-3A, with a 14 kbp insert showed strong homology to the pfim3-1 cDNA. The region of homology, a 700 bp Xba I fragment, was subcloned into pUG19. This plasmid was refered to as pXX8. DNA sequence determinations of pXX8 and adjacent fragments from A2-3A suggested that the cloned DNA was a portion of the rONA repeat encoding the small subunit rRNA. DNA sequence analysis of pfim3-1 yielded an incomplete open reading frame. The predicted amino acid sequence codes for a 206 amino acid, 22 kDa polypeptide which contains a domain similar to a transmembrane domain from rat leukocyte antigen, GDS3. As well, an untranslated 576 nucleotide domain showed 81 % homology to pXX8 and 830/0 homology to the 188 rRNA sequence of Ustilago maydis. This sequence was found adjacent to a region of adenine-thymine base pairs presumed to represent the polyadenylation sequence of the fimbrial transcript. The size and extent of homology is sufficient to account for the hybridization of pfim3-1 to rDNA. It is suggested that this domain represents a completely novel regulatory domain within eukaryotes that may enable the observed rapid regeneration of fimbriae in U. violacea.

Représentation et recherche de motifs cycliques et structuraux d’ARN connus dans les structures secondaires

L'acide désoxyribonucléique (ADN) et l'acide ribonucléique (ARN) sont des polymères de nucléotides essentiels à la cellule. À l'inverse de l'ADN qui sert principalement à stocker l'information génétique, les ARN sont impliqués dans plusieurs processus métaboliques. Par exemple, ils transmettent l’information génétique codée dans l’ADN. Ils sont essentiels pour la maturation des autres ARN, la régulation de l’expression génétique, la prévention de la dégradation des chromosomes et le ciblage des protéines dans la cellule. La polyvalence fonctionnelle de l'ARN résulte de sa plus grande diversité structurale. Notre laboratoire a développé MC-Fold, un algorithme pour prédire la structure des ARN qu'on représente avec des graphes d'interactions inter-nucléotidiques. Les sommets de ces graphes représentent les nucléotides et les arêtes leurs interactions. Notre laboratoire a aussi observé qu'un petit ensemble de cycles d'interactions à lui seul définit la structure de n'importe quel motif d'ARN. La formation de ces cycles dépend de la séquence de nucléotides et MC-Fold détermine les cycles les plus probables étant donnée cette séquence. Mon projet de maîtrise a été, dans un premier temps, de définir une base de données des motifs structuraux et fonctionnels d'ARN, bdMotifs, en terme de ces cycles. Par la suite, j’ai implanté un algorithme, MC-Motifs, qui recherche ces motifs dans des graphes d'interactions et, entre autres, ceux générés par MC-Fold. Finalement, j’ai validé mon algorithme sur des ARN dont la structure est connue, tels que les ARN ribosomaux (ARNr) 5S, 16S et 23S, et l'ARN utilisé pour prédire la structure des riborégulateurs. Le mémoire est divisé en cinq chapitres. Le premier chapitre présente la structure chimique, les fonctions cellulaires de l'ARN et le repliement structural du polymère. Dans le deuxième chapitre, je décris la base de données bdMotifs. Dans le troisième chapitre, l’algorithme de recherche MC-Motifs est introduit. Le quatrième chapitre présente les résultats de la validation et des prédictions. Finalement, le dernier chapitre porte sur la discussion des résultats suivis d’une conclusion sur le travail.

Étude de la diversité génétique d’isolats québécois de Mycoplasma hyopneumoniae provenant d’infections simples ou mixtes et caractérisation de leur sensibilité aux antimicrobiens

Mycoplasma hyopneumoniae est l’agent causal de la pneumonie enzootique. On le retrouve dans plusieurs élevages de porcs à travers le monde. Même si ce micro-organisme est présent dans plusieurs troupeaux canadiens, peu d’informations sont présentement disponibles sur les isolats québécois. Un total de 160 poumons de porcs possédant des lésions de pneumonie ont été récupérés à l’abattoir, mis en culture et testés par PCR pour M. hyopneumoniae et Mycoplasma hyorhinis. D’autres pathogènes bactériens communs du porc et les virus du syndrome reproducteur et respiratoire porcin (VSRRP), de l’influenza et le circovirus porcin de type 2 (CVP2) ont été également testés. Quatre-vingt-dix pourcent des échantillons étaient positifs pour M. hyopneumoniae et 5.6% l’étaient seulement pour M. hyorhinis. Dans ces échantillons positifs pour M. hyopneumoniae, la concentration de ce mycoplasme variait de 1.17 x 105 à 3.37 x 109 génomes/mL. Vingt-cinq poumons positifs en culture ou par PCR en temps réel pour M. hyopneumoniae ont été sélectionnés, parmi ceux-ci 10 étaient en coinfection avec Pasteurella multocida, 12 avec Streptococcus suis, 9 avec CVP2 et 2 avec le VSRRP. Les analyses des nombres variables de répétitions en tandem à de multiples loci (MLVA) et PCR-polymorphisme de longueur de fragments de restriction (PCR-RFLP) de M. hyopneumoniae ont démontré une forte diversité des isolats de terrain. Par contre, il semble y avoir plus d’homogénéité à l’intérieur d’un même élevage. L’analyse MLVA a également démontré que près de la moitié des isolats possédaient moins de 55% d’homologie avec les souches vaccinales et de référence utilisées dans la présente étude. L’absence d’amplification du locus 1 de M. hyopneumoniae en MLVA a été significativement associée à une baisse de la concentration de bactérie et de la sévérité des lésions. Pour tous les isolats de M. hyopneumoniae, des concentrations minimales inhibitrices (CMI) de faibles à intermédiaires ont été obtenues envers tous les antimicrobiens testés. Les isolats possédant des CMI intermédiaires envers les tétracyclines, les macrolides et les lincosamides ont été testés pour la présence des gènes de résistance tetM, ermB et pour des mutations ponctuelles dans les gènes des protéines L4, L22 et de l’ARNr 23S. Aucun de ces gènes n’a été détecté mais la mutation ponctuelle G2057A a été identifiée. Cette mutation est responsable de la résistance intrinsèque de M. hyopneumoniae face aux macrolides à 14 carbones. Ces résultats indiquent qu’il ne semble pas y avoir de résistance acquise aux antimicrobiens parmi ces isolats. En conclusion, cette recherche a permis d’obtenir de nouvelles données scientifiques sur les isolats québécois de M. hyopneumoniae.