Development and characterization of rifampicin-resistant mutants from high virulent strains of Flavobacterium columnare.

Flavobacterium columnare is divided into three genetic groups or genomovars, genomovar II being highly virulent for channel catfish. A modified live vaccine is currently available to prevent columnaris disease under the licensed name Aquavac-Col(®) . The strain of F. columnare used to generate the avirulent rifampicin-resistant mutant used in Aquavac-Col(®) belonged to genomovar I, the less virulent group towards channel catfish. In this study, we describe the generation and characterization of rifampicin-resistant mutants from genomovar II strains. A total of 13 new mutants were obtained, and eight of them (two from each parent strain) were genetically and phenotypically characterized. Highly conserved regions within the ribosomal operons were identical between parent and mutant strains. Genetic differences between mutants and their parent strains were revealed by amplified fragment length polymorphism (AFLP). Genetic changes were distinctive among different mutants. Analysis of the lipopolysaccharide (LPS) showed that while some mutants lacked a few molecular bands of the LPS, some exhibited the same LPS profiles as their parent strains. Comparison between immunogenic proteins from mutants and parents was carried out by immunoblot analysis and further confirmed the uniqueness of individual mutants. A complete set of rifampicin-resistant mutants with different genetic and immunogenic properties from the highly virulent genomovar II has been created. These mutants may have the potential of becoming vaccine candidates against columnaris disease.

Estabilidad temporal de mepivacaína al 1.5% alcalinizada / Alkalinized 1.5% mepivacaine stability over time

Objetivos: Estudiar la estabilidad en el tiempo de la mepivacaína alcalinizada en cuanto a valores de pH y formación de precipitados, ya que apenas existe información en la literatura acerca de la estabilidad temporal de este anestésico, y la información disponible aconseja alcalinizarla justo antes de su empleo. Material y métodos: Fueron preparadas tres jeringas (A, B y C) de 20 mL conteniendo mepivacaína al 1.5%. La jeringa A fue utilizada como control de pH, y las jeringas B y C fueron alcalinizadas adicionando 2 mL de bicarbonato al 8.4%. Fue medido el pH y calculado el porcentaje de base libre asociado en cada jeringa previamente a la alcalinización y posteriormente cada 10 minutos hasta completar una hora, salvo para la jeringa C en los últimos 30 minutos, que permaneció cerrada como control de fuga de CO2. Las soluciones fueron inspeccionadas visualmente durante todo el procedimiento para identificar eventuales precipitados macroscópicos y, tras la hora de estudio, fueron filtradas para indagar sobre la formación de precipitados microscópicos. Tras el filtrado, el pH de cada solución anestésica fue medido de nuevo. Resultados: Tras la alcalinización de la mepivacaína al 1.5% se produjo un aumento inmediato y significativo de los valores de pH y del porcentaje calculado de base libre en las jeringas alcalinizadas B y C con respecto a la de control A, y en todas las jeringas el pH permaneció muy estable durante una hora. Además, a los 60 minutos apenas existieron diferencias entre los valores de pH de las jeringas B y C, lo que indica que no se produjo fuga significativa de CO2. En este tiempo no hubo sospecha de formación de precipitados por inspección visual, y las mínimas diferencias encontradas entre los pesos secos de los filtros indican que no hubo formación de precipitados significativa (…) (AU)

PurposeIt is recommended to alkalinize mepivacaine just before employing it. However, data regarding alkalinized mepivacain estability over time are very scarce. The aim of this work was to investigate for pH stability and precipitation of alkalinized mepivacaine. Materials and Methods: Three syringes (A, B, C) containing 20mL of 1.5% mepivacaine each one were prepared. Syringe A served as pH control, and syringes B and C were alkalinized by adding 2mL of8.4% sodium bicarbonate solution. pH was measured into each syringe before alkalinization and every ten minutes lasting for one hour after. Associated free base percentage was calculated, except for the last 30 minutes for syringe C, which remained closed to serve as CO2 leakage control. Solutions were examinated by naked eyes looking for macroscopic precipitates, filtered after the procedure and the filters were weighed in order to looking for microscopic precipitates. After filtration pH was again measured. Results: Alkalinization resulted in immediate and significant increases in pH values and associated free base percentage of syringes B and C compared to syringe A. After that and for one hour pH values remained very stable. Besides, pH values between syringes B and C were very similar at 60 minutes, indicating no-significative leakage of CO2. The procedure was completed without evidence of precipitation by visual inspection, and differences between dry filters weights were minimalindicating no significative formation of microprecipitates. Conclusions: 1.5% mepivacaine solutions can be alkalinized and stored at room temperature in closed syringes for at least one hour before administration. In our opinion previous alkalinizationis much more convenient to daily clinical practice that actual recommendation of alkalinization just before use (AU)

Use of suppressive subtractive hybridization to identify Flavobacterium columnare DNA sequences not shared with Flavobacterium johnsoniae.

AIMS: To identify specific sequences in the fish pathogen Flavobacterium columnare not shared by Flavobacterium johnsoniae. METHODS AND RESULTS: Suppressive subtractive hybridization (SSH) was used to selectively amplify and clone F. columnare-specific sequences. A highly virulent strain of F. columnare was used as tester and the type strain of F. johnsoniae was used as driver. After library construction, 192 clones were selected and sequenced. From those, 110 clones contained unique F. columnare-specific sequences that were verified using dot blot hybridization. Sequence sizes ranged from 55 to 872 bp with 45,363 bp sequenced in total. CONCLUSIONS: Specific F. columnare sequences representing all but one (motility related) functional categories were annotated. Several putative virulence factors were identified in F. columnare such as a collagenase, a chondroitinase, proteases, as well as drug resistance and iron transport-related genes. SIGNIFICANCE AND IMPACT OF THE STUDY: Suppressive subtractive hybridization is a cost-effective method for identifying genetic differences between Flavobacterium spp. The number of sequences available from F. columnare has been doubled.

Molecular typing of Streptococcus agalactiae isolates from fish.

The genetic variability among Streptococcus agalactiae isolates recovered from fish was characterized using single-stranded conformation polymorphism (SSCP) analysis of the intergenic spacer region (ISR), and amplified fragment length polymorphism (AFLP) fingerprinting. A total of 46 S. agalactiae cultures isolated from different fish species and geographic origins as well as related reference strains were included in the study. ISR-SSCP divided the S. agalactiae isolates analysed into five distinct genotypes. Genotype 1 grouped all Kuwait isolates while genotype 4 clustered the majority of non-Kuwait isolates (USA, Brazil and Honduras). AFLP analysis offered a higher resolution level by dividing the isolates into 13 different genotypes. Two different AFLP profiles were identified within the Kuwait isolates. When data from both ISR-SSCP and AFLP were combined through a multidimensional analysis (MDS), a good correlation between geographical origin and genotypes was observed. Both AFLP and ISR-SSCP revealed genetic differences between S. agalactiae isolates from fish. While AFLP offered a higher resolution, ISR-SSCP also provided valid information being a simpler and faster method.

Flavobacterium columnare genomovar influences mortality in channel catfish (Ictalurus punctatus).

Flavobacterium columnare, causal agent of columnaris disease, is pathogenic to many species of freshwater fish throughout the world. The United States channel catfish (Ictalurus punctatus) aquaculture industry is severely impacted by columnaris disease. The majority of the F. columnare isolates recovered from diseased channel catfish belonged to either genomovars I or II. The objective of the present study was to determine if differences existed in the ability of these genomovars to induce mortality in channel catfish. Single strand conformation polymorphism analysis (SSCP) was used to ascribe the isolates used in this study to the appropriate genomovar. Immersion challenge experiments (15min immersion exposure to approximately 5x10(5) to 1x10(6) CFU/mL) were carried out to assess virulence of genomovar I and II isolates to channel catfish. The results demonstrated that genomovar II (n=4) isolates were significantly (P<0.05) more virulent to channel catfish fry (92-100% mortality) than genomovar I (n=3) isolates (0-46% mortality). In vivo adhesion of the genetically characterized F. columnare also correlated (r2=0.73) to increased mortality in the challenged fry. In fingerling channel catfish, significantly higher mortality (P<0.05) resulted with genomovar II isolates ALM-05-182 and ALG-00-530 as compared to all the genomovar I isolates (n=3). Mortality of genomovar II isolate BGFS-27 with similar to genomovar II isolate (ALG-00-530) and two genomovar I isolates (ALM-05-53 and 140). The results suggest that although both genomovars are present in the aquatic environment, genomovar II appears to be more pathogenic for channel catfish.

Protocolo para el control de la Vasectomía / Guide about the assessment of semen samples after vasectomy

Recientemente, hemos diseñado en nuestro laboratorio unprotocolo para el control de la vasectomía basado en la guía depráctica clínica publicada por la Sociedad Británica de Andrología(BAS). Esta guía fue editada para ayudar a los profesionalesdel laboratorio en la estandarización del análisis seminalpara el control de la vasectomía y en el informe de los resultados,y sigue las recomendaciones de la Organización Mundialde la Salud (OMS) para la recogida de semen. En la parte superiorde nuestro protocolo, se incluyen las instrucciones preanalíticasadecuadas para recoger el eyaculado y, en la inferior, uncuestionario de calidad preanalítica (CCPA) compuesto por 12preguntas. En primer lugar, hay unas cuestiones relativas a losdatos demográficos y clínicos del paciente; en segundo lugar,hay una serie de puntos acerca de la información que ha recibidoprevia a la realización de la prueba que nos ayuda a conocerla calidad de esa muestra, la difusión y la comprensión de nuestroprotocolo, y a concienciar al paciente de la importancia deseguir las recomendaciones que se le han dado

Recently, in our laboratory we designed a guide about theassessment of semen samples after vasectomy, based uponthis of the British Andrology Society (BAS). These guidelineswere published to give guidance to laboratory staff to ensurestandarisation of seminal analysis protocol and reporting ofresults, and follow the World Health Organization reccomendationfor semen collection. In the upper part of the form thereare instructions to collect the ejaculate and, in the lower partof the form a questionnaire composed of 12 questions. First,there are some questions about identifiers and clinical data ofthe patients; secondly, there are some questions about theinformation that they received previous to the test that mayhelp us to know about sample quality and the diffusion of ourguide, and to make them conscious about the importance offollow our instructions

Cristales de colesterol en líquido ascítico / Cholesterol crystals in ascitic fluid

Se presenta un caso de un paciente de 44 años cuyas manifestacionesclínicas fueron diversos derrames pleurales y ascíticosen cuyo examen microscópico se evidenció la presencia decristales de colesterol. Tras descartar posibles etiologías comoel LES, mixedema u origen tumoral, el paciente fue diagnosticadode tuberculosis con afectación extrapulmonar, respondiendoeficazmente al tratamiento.El interés de este caso es resaltar la aparición de cristalesde colesterol en un líquido ascítico en un caso de tuberculosisextrapulmonar, hecho escasamente documentado en la literaturamédica

We report the case of a male aged 44 years, with severalepisodes of pleural and ascitic effusion whose microscopycalstudy showed cholesterol crystals. Systemic lupus erythematosus,mixedema or tumoral etiologies were ruled out and thepatient was diagnosed of extra pulmonary tuberculosis and theantituberculosis therapy was successful.The relevant finding in the case was the presence of cholesterolcrystals in the ascitic effusion in a case of extra pulmonarytuberculosis which has been rarely reported in the medicalliterature

Electrochemical protoplast fusion in citrus.

We report here the development of a novel protoplast fusion method for citrus somatic hybridization. This new procedure, which we have named electrochemical protoplast fusion, is based on chemically induced protoplast aggregation, using a low concentration of polyethylene glycol, and DC pulse-promoted membrane fusion. Based on the results of nucleus and mitochondria molecular analyses, we were successful in using this method to regenerate both symmetric somatic hybrids and cybrids. Various parameters, including pulse intensity, pulse length, and composition of the fusion media, were tested, and the optimum fusion condition selected consisted of two 100-micros pulses of 1,500 V cm(-1). Our conclusion is that electrochemical fusion is a reliable and reproducible method that combines the best features of both the chemical and electrical methods, thereby promoting cell division and high embryogenesis rates of the fused cells. It represents a new approach to citrus somatic hybridization. Various interesting features of this new approach are presented and discussed.

Green fluorescent protein as a visual marker in somatic hybridization.

Using a transgenic citrus plant expressing Green Fluorescent Protein (GFP) as a parent in somatic fusion experiments, we investigated the suitability of GFP as an in vivo marker to follow the processes of protoplast fusion, regeneration and selection of hybrid plants. A high level of GFP expression was detected in transgenic citrus protoplasts, hybrid callus, embryos and plants. It is demonstrated that GFP can be used for the continuous monitoring of the fusion process, localization of hybrid colonies and callus, and selection of somatic hybrid embryos and plants.