Persistence of Brucella abortus lineages revealed by genomic characterization and phylodynamic analysis.

Brucellosis, caused by Brucella abortus, is a major disease of cattle and humans worldwide distributed. Eradication and control of the disease has been difficult in Central and South America, Central Asia, the Mediterranean and the Middle East. Epidemiological strategies combined with phylogenetic methods provide the high-resolution power needed to study relationships between surveillance data and pathogen population dynamics, using genetic diversity and spatiotemporal distributions. This information is crucial for prevention and control of disease spreading at a local and worldwide level. In Costa Rica (CR), the disease was first reported at the beginning of the 20th century and has not been controlled despite many efforts. We characterized 188 B. abortus isolates from CR recovered from cattle, humans and water buffalo, from 2003 to 2018, and whole genome sequencing (WGS) was performed in 95 of them. They were also assessed based on geographic origin, date of introduction, and phylogenetic associations in a worldwide and national context. Our results show circulation of five B. abortus lineages (I to V) in CR, phylogenetically related to isolates from the United States, United Kingdom, and South America. Lineage I was dominant and probably introduced at the end of the 19th century. Lineage II, represented by a single isolate from a water buffalo, clustered with a Colombian sample, and was likely introduced after 1845. Lineages III and IV were likely introduced during the early 2000s. Fourteen isolates from humans were found within the same lineage (lineage I) regardless of their geographic origin within the country. The main CR lineages, introduced more than 100 years ago, are widely spread throughout the country, in contrast to new introductions that seemed to be more geographically restricted. Following the brucellosis prevalence and the farming practices of several middle- and low-income countries, similar scenarios could be found in other regions worldwide.

Identification of Brucella abortus biovar 4 of bovine origin in Colombia / Identificación de Brucella abortus biovar 4 de origen bovino en Colombia

The objective of this study was to identify twelve Brucella abortus isolates of bovine origin from the department of Nariño in Colombia up to the biovar level. These isolates are included in the collection of the Germplasm Bank of Microorganisms of Animal Health Interest -Bacteria and Virus (BGSA-BV). The identification was carried out through conventional methods such as macro and microscopic morphological descriptions, enzymatic activity, biochemical profile, substrate use and sensitivity to dyes. Complementary genotypic characterization was carried out using multiplex PCR for B. abortus, Brucella melitensis, Brucella ovis, and Brucella suis-Erytritol (AMOS-ERY-PCR), RFLP-IS711, by southern blot hybridization, as well as by the multiple locus variable number of tandem repeat analysis (MLVA) using the ery gene and the insertion sequence IS711 and variable number of tandem repeats (VNTR) as molecular markers. The results of the phenotypic and molecular characterization allowed to identify twelve isolates as B. abortus biovar 4 as well as to differentiate field from vaccine strains. This is the first study on the phenotypic and molecular identification of B. abortus isolates in Colombia. It was concluded that the phenotypic and molecular identification of twelve isolates as B. abortus biovar 4 could be achieved using conventional and molecular techniques with enough resolution power. The identification of these isolates to the biovar level in taxonomic and epidemiological terms will allow the use of this genetic resource as reference strains in future research. This finding constitutes the basis for identifying biotypes not previously reported in the country that might be useful to support brucellosis survey programs in Colombia.

El objetivo de este estudio fue identificar 12 aislamientos de Brucella abortus de origen bovino procedentes del departamento de Narino, Colombia, hasta la descripción de biovar. Estos aislamientos conforman la colección del Banco de Germoplasma de Microorganismos de Interés en Salud Animal, Bacterias y Virus. La identificación se hizo mediante métodos convencionales, como la descripción morfológica macro y microscópica de actividad enzimática, de perfiles bioquímicos, de utilización de sustratos y de sensibilidad a colorantes. Se hizo una caracterización genotipica complementaria mediante PCR múltiple para Brucella abortus, Brucella melitensis, Brucella ovisy Brucella suis-eritritol (AMOS-ERY-PCR); RFLP-/S7II; hibridación Southern blot y análisis multi-locus de repeticiones en tándem de número variable (MLVA), empleando como marcadores moleculares el gen ery, la secuencia de inserción /S711 y el número variable de repeticiones en tándem (VNTR). Los resultados de la caracterización fenotípica y molecular permitieron identificar 12 aislamientos de campo como B. abortus biovar 4 y diferenciar cepas de campo de cepas vacunales. Este es el primer estudio de identificación fenotípica y molecular de aislamientos de B. abortus en Colombia. Por su importancia taxonómica y epidemiológica, la identificación de estos aislamientos hasta el nivel de biovar permitirá disponer de recursos genéticos que se pueden emplear como cepas de referencia en futuras investigaciones. Estos resultados pueden considerarse como una base para la identificación de biotipos no reportados en el país y podrán ser utilizados en programas de monitoreo y vigilancia de la brucelosis bovina en Colombia.

Identification of Brucella abortus biovar 4 of bovine origin in Colombia.

The objective of this study was to identify twelve Brucella abortus isolates of bovine origin from the department of Nariño in Colombia up to the biovar level. These isolates are included in the collection of the Germplasm Bank of Microorganisms of Animal Health Interest - Bacteria and Virus (BGSA-BV). The identification was carried out through conventional methods such as macro and microscopic morphological descriptions, enzymatic activity, biochemical profile, substrate use and sensitivity to dyes. Complementary genotypic characterization was carried out using multiplex PCR for B. abortus, Brucella melitensis, Brucella ovis, and Brucella suis-Erytritol (AMOS-ERY-PCR), RFLP-IS711, by southern blot hybridization, as well as by the multiple locus variable number of tandem repeat analysis (MLVA) using the ery gene and the insertion sequence IS711 and variable number of tandem repeats (VNTR) as molecular markers. The results of the phenotypic and molecular characterization allowed to identify twelve isolates as B. abortus biovar 4 as well as to differentiate field from vaccine strains. This is the first study on the phenotypic and molecular identification of B. abortus isolates in Colombia. It was concluded that the phenotypic and molecular identification of twelve isolates as B. abortus biovar 4 could be achieved using conventional and molecular techniques with enough resolution power. The identification of these isolates to the biovar level in taxonomic and epidemiological terms will allow the use of this genetic resource as reference strains in future research. This finding constitutes the basis for identifying biotypes not previously reported in the country that might be useful to support brucellosis survey programs in Colombia.

Reduced Susceptibility to Rifampicin and Resistance to Multiple Antimicrobial Agents among Brucella abortus Isolates from Cattle in Brazil.

This study aimed to determine the susceptibility profile of Brazilian Brucella abortus isolates from cattle to eight antimicrobial agents that are recommended for the treatment of human brucellosis and to correlate the susceptibility patterns with origin, biotype and MLVA16-genotype of the strains. Screening of 147 B. abortus strains showed 100% sensitivity to doxycycline and ofloxacin, one (0.68%) strain resistant to ciprofloxacin, two strains (1.36%) resistant to streptomycin, two strains (1.36%) resistant to trimethoprim-sulfamethoxazole and five strains (3.40%) resistant to gentamicin. For rifampicin, three strains (2.04%) were resistant and 54 strains (36.73%) showed reduced sensitivity. Two strains were considered multidrug resistant. In conclusion, the majority of B. abortus strains isolated from cattle in Brazil were sensitive to the antimicrobials commonly used for the treatment of human brucellosis; however, a considerable proportion of strains showed reduced susceptibility to rifampicin and two strains were considered multidrug resistant. Moreover, there was no correlation among the drug susceptibility pattern, origin, biotype and MLVA16-genotypes of these strains.

Different resistance patterns of reference and field strains of Brucella abortus.

The aim of this study was to evaluate the growth of the B. abortus reference strains and field isolates on media containing different inhibitor agents. Reference strains were seeded on tryptose agar containing: i-erythritol (1.0 mg/mL), fuchsin (20 µg/mL and 80 µg/mL), thionin (2.5 µg/mL and 10 µg/mL), rifampicin (200 µg/mL) and safranin O (200 µg/mL). Field isolates were tested only on media containing i-erythritol, rifampicin and thionin. Furthermore, each suspension was also inoculated on tryptose agar incubated in air, to test its ability to grow without CO 2 . Sensitivity to fuchsin was similar among reference strains evaluated. Growth of S19, 544 and 2308 but not RB51 were inhibited on media containing rifampicin. Medium with safranin O showed no inhibition for RB51, 544 and 2308, but it partially inhibited the S19 growth as well as medium containing i-erythritol. Treatment/control growth ratio for 2308 on tryptose agar containing thionin (2.5 µg/mL) was approximatelly 1.0, whereas S19 and RB51 showed 0.85 and 0.89 ratios, respectively. Growth of 544, S19 and RB51 but not 2308 was completely inhibited on medium with thionin (10 µg/mL). All field strains grew on medium containing i-erythritol, but were completelly inhibited by rifampicin. With exception of A1 ( B. abortus biovar 3) all field isolates grew on medium with thionin, although some strains showed a treatment/control growth ratio of 0.75-0.80 (10 µg/mL). These results showed that tryptose agar with thionin, i-erythritol or rifampicin could be useful for differentiating vaccine, challenge and field strains of B. abortus.

Different resistance patterns of reference and field strains of Brucella abortus

The aim of this study was to evaluate the growth of the B. abortus reference strains and field isolates on media containing different inhibitor agents. Reference strains were seeded on tryptose agar containing: i-erythritol (1.0 mg/mL), fuchsin (20 μg/mL and 80 μg/mL), thionin (2.5 μg/mL and 10 μg/mL), rifampicin (200 μg/mL) and safranin O (200 μg/mL). Field isolates were tested only on media containing i-erythritol, rifampicin and thionin. Furthermore, each suspension was also inoculated on tryptose agar incubated in air, to test its ability to grow without CO2. Sensitivity to fuchsin was similar among reference strains evaluated. Growth of S19, 544 and 2308 but not RB51 were inhibited on media containing rifampicin. Medium with safranin O showed no inhibition for RB51, 544 and 2308, but it partially inhibited the S19 growth as well as medium containing i-erythritol. Treatment/control growth ratio for 2308 on tryptose agar containing thionin (2.5 μg/mL) was approximatelly 1.0, whereas S19 and RB51 showed 0.85 and 0.89 ratios, respectively. Growth of 544, S19 and RB51 but not 2308 was completely inhibited on medium with thionin (10 μg/mL). All field strains grew on medium containing i-erythritol, but were completelly inhibited by rifampicin. With exception of A1 (B. abortus biovar 3) all field isolates grew on medium with thionin, although some strains showed a treatment/control growth ratio of 0.75–0.80 (10 μg/mL). These results showed that tryptose agar with thionin, i-erythritol or rifampicin could be useful for differentiating vaccine, challenge and field strains of B. abortus.

Different resistance patterns of reference and field strains of Brucella abortus

The aim of this study was to evaluate the growth of the B. abortus reference strains and field isolates on media containing different inhibitor agents. Reference strains were seeded on tryptose agar containing: i-erythritol (1.0 mg/mL), fuchsin (20 μg/mL and 80 μg/mL), thionin (2.5 μg/mL and 10 μg/mL), rifampicin (200 μg/mL) and safranin O (200 μg/mL). Field isolates were tested only on media containing i-erythritol, rifampicin and thionin. Furthermore, each suspension was also inoculated on tryptose agar incubated in air, to test its ability to grow without CO2. Sensitivity to fuchsin was similar among reference strains evaluated. Growth of S19, 544 and 2308 but not RB51 were inhibited on media containing rifampicin. Medium with safranin O showed no inhibition for RB51, 544 and 2308, but it partially inhibited the S19 growth as well as medium containing i-erythritol. Treatment/control growth ratio for 2308 on tryptose agar containing thionin (2.5 μg/mL) was approximatelly 1.0, whereas S19 and RB51 showed 0.85 and 0.89 ratios, respectively. Growth of 544, S19 and RB51 but not 2308 was completely inhibited on medium with thionin (10 μg/mL). All field strains grew on medium containing i-erythritol, but were completelly inhibited by rifampicin. With exception of A1 (B. abortus biovar 3) all field isolates grew on medium with thionin, although some strains showed a treatment/control growth ratio of 0.75–0.80 (10 μg/mL). These results showed that tryptose agar with thionin, i-erythritol or rifampicin could be useful for differentiating vaccine, challenge and field strains of B. abortus.(AU)

Pheno- and genotyping of Brucella abortus biovar 5 isolated from a water buffalo (Bubalus bubalis) fetus: First case reported in the Americas.

An isolate of Brucella spp. from an aborted water buffalo (Bubalus bubalis) fetus was characterized based on its pheno- and genotype. The phenotype was defined by carbon dioxide requirement, hydrogen sulfide production, sensitivity to thionin and basic fuchsin and agglutination with Brucella A and M monospecific antisera. The genotype was based on the amplification of the following genes: bcsp31, omp2ab, and eri and the species-specific localization of the insertion sequence IS711 in the Brucella chromosome via B. abortus-B. melitensis-B. ovis-B. suis (AMOS)-PCR. Unexpectedly, the isolate showed a phenotype different from B. abortus bv 1, the most prevalent strain in cattle in Argentina, and from vaccine strain 19, currently used in bovines and water buffaloes. Genotyping supported the phenotypic results, as the analysis of the omp2ab gene sequence showed an identical pattern to either B. abortus bv 5 or B. melitensis. Finally, the AMOS PCR generated a 1700-bp fragment from the isolate, different than those amplified from B. abortus bv 1 (498bp) and B. melitensis (731bp), confirming the presence of B. abortus bv 5. The OIE/FAO Reference Laboratory for Brucellosis confirmed this typing. This is the first report of B. abortus bv 5 from a water buffalo in the Americas.

Biotyping and genotyping (MLVA16) of Brucella abortus isolated from cattle in Brazil, 1977 to 2008.

Brucellosis is a worldwide distributed zoonosis that causes important economic losses to animal production. In Brazil, information on the distribution of biovars and genotypes of Brucella spp. is scarce or unavailable. This study aimed (i) to biotype and genotype 137 Brazilian cattle isolates (from 1977 to 2008) of B. abortus and (ii) to analyze their distribution. B. abortus biovars 1, 2 and 3 (subgroup 3b) were confirmed and biovars 4 and 6 were first described in Brazil. Genotyping by the panel 1 revealed two groups, one clustering around genotype 40 and another around genotype 28. Panels 2A and 2B disclosed a high diversity among Brazilian B. abortus strains. Eighty-nine genotypes were found by MLVA16. MLVA16 panel 1 and 2 showed geographic clustering of some genotypes. Biotyping and MLVA16 genotyping of Brazilian B. abortus isolates were useful to better understand the epidemiology of bovine brucellosis in the region.

Detección de un complejo clonal con el genotipo de Brucella abortus biovariedad 2 como fundador en aislamientos de B. abortus de Argentina / Detection of a clonal complex with Brucella abortus biovar 2 genotype as founder in B. abortus isolates from Argentina

Brucella abortus es el agente causal de la brucelosis bovina, enfermedad zoonótica que se encuentra ampliamente distribuida en el mundo. Actualmente existen ocho biovariedades de B. abortus. En Argentina se encuentra con mayor frecuencia la biovariedad 1, pero también se suele aislar la biovariedad 2, que es más patogénica que la anterior. Resulta necesario contar con métodos de tipificación que tengan la resolución suficiente para permitir el seguimiento epidemiológico de los brotes de brucelosis y de los programas de control de la enfermedad. Debido a la gran homogeneidad genética que existe entre las distintas especies del género Brucella, ha sido dificultoso el desarrollo de herramientas moleculares para realizar el análisis epidemiológico de los aislamientos. La publicación del genoma de varias especies de Brucella facilitó el diseño de estas herramientas. El objetivo del presente trabajo fue emplear un esquema de análisis multilocus de VNTR en aislamientos de Argentina obtenidos en nuestro laboratorio. De los 56 aislamientos analizados se obtuvieron 47 perfiles genotípicos diferentes. El empleo de este esquema permitió asignarles a dichos aislamientos la biovariedad correspondiente. A través del análisis goeBURST se pudo relacionar a todos los genotipos entre sí, y además, proponer al genotipo de la biovariedad 2 como fundador.

Brucella abortus is the causative agent of bovine brucellosis, a worldwide zoonosis. Up to date, eight biovars of B. abortus have been described. In Argentina, biovar 1 is the most frequently isolated. However, biovar 2, which is more pathogenic than biovar 1, is also found. Molecular methods for subtyping isolates are necessary for allowing epidemiological surveillance and control of eradication programs. Due to the genetic homogeneity of the genus Brucella, the development of molecular typing tools has been difficult. The publication of microorganism genomes facilitates the design of this approach. The aim of this work was to employ a Multiple Locus VNTR Analysis (MLVA) scheme for strains from Argentina isolated in our laboratory. From the 56 isolates analyzed, 47 different genotypic profiles were obtained. All the strains typed as biovar 2 showed the same profile. This scheme allowed assigning each isolate to the biovar it belongs to. All the genotypes were related using the goeBURST analysis and biovar 2 was proposed as founder.

Detección de un complejo clonal con el genotipo de Brucella abortus biovariedad 2 como fundador en aislamientos de B. abortus de Argentina / Detection of a clonal complex with Brucella abortus biovar 2 genotype as founder in B. abortus isolates from Argentina

Brucella abortus es el agente causal de la brucelosis bovina, enfermedad zoonótica que se encuentra ampliamente distribuida en el mundo. Actualmente existen ocho biovariedades de B. abortus. En Argentina se encuentra con mayor frecuencia la biovariedad 1, pero también se suele aislar la biovariedad 2, que es más patogénica que la anterior. Resulta necesario contar con métodos de tipificación que tengan la resolución suficiente para permitir el seguimiento epidemiológico de los brotes de brucelosis y de los programas de control de la enfermedad. Debido a la gran homogeneidad genética que existe entre las distintas especies del género Brucella, ha sido dificultoso el desarrollo de herramientas moleculares para realizar el análisis epidemiológico de los aislamientos. La publicación del genoma de varias especies de Brucella facilitó el diseño de estas herramientas. El objetivo del presente trabajo fue emplear un esquema de análisis multilocus de VNTR en aislamientos de Argentina obtenidos en nuestro laboratorio. De los 56 aislamientos analizados se obtuvieron 47 perfiles genotípicos diferentes. El empleo de este esquema permitió asignarles a dichos aislamientos la biovariedad correspondiente. A través del análisis goeBURST se pudo relacionar a todos los genotipos entre sí, y además, proponer al genotipo de la biovariedad 2 como fundador.(AU)

Brucella abortus is the causative agent of bovine brucellosis, a worldwide zoonosis. Up to date, eight biovars of B. abortus have been described. In Argentina, biovar 1 is the most frequently isolated. However, biovar 2, which is more pathogenic than biovar 1, is also found. Molecular methods for subtyping isolates are necessary for allowing epidemiological surveillance and control of eradication programs. Due to the genetic homogeneity of the genus Brucella, the development of molecular typing tools has been difficult. The publication of microorganism genomes facilitates the design of this approach. The aim of this work was to employ a Multiple Locus VNTR Analysis (MLVA) scheme for strains from Argentina isolated in our laboratory. From the 56 isolates analyzed, 47 different genotypic profiles were obtained. All the strains typed as biovar 2 showed the same profile. This scheme allowed assigning each isolate to the biovar it belongs to. All the genotypes were related using the goeBURST analysis and biovar 2 was proposed as founder.(AU)

Isolation of a field strain of Brucella abortus from RB51-vaccinated- and brucellosis-seronegative bovine yearlings that calved normally.

A study was carried out in Pichucalco, Chiapas (Mexico) to determine whether recently calved cows or those that aborted shed Brucella. Serological diagnosis of brucellosis was made in all animals (209). Six of the cows that calved normally and two that aborted underwent a bacteriological study of milk and vaginal exudate. Brucella abortus was isolated from vaginal exudate samples in two 3- to 4-year-old seronegative first-birth cows that had calved normally. This was confirmed through bacteriological identification and PCR as a field strain and smooth phenotypes. We conclude that seronegative cows vaccinated with RB51 which calved normally and shed B. abortus in the vaginal exudate after calving could be a serious problem because these cows are overlooked in routine diagnoses and are a source of Brucella infection.

[Detection of a clonal complex with Brucella abortus biovar 2 genotype as founder in B. abortus isolates from Argentina]. / Detección de un complejo clonal con el genotipo de Brucella abortus biovariedad 2 como fundador en aislamientos de B. abortus de Argentina.

Brucella abortus is the causative agent of bovine brucellosis, a worldwide zoonosis. Up to date, eight biovars of B. abortus have been described. In Argentina, biovar 1 is the most frequently isolated. However, biovar 2, which is more pathogenic than biovar 1, is also found. Molecular methods for subtyping isolates are necessary for allowing epidemiological surveillance and control of eradication programs. Due to the genetic homogeneity of the genus Brucella, the development of molecular typing tools has been difficult. The publication of microorganism genomes facilitates the design of this approach. The aim of this work was to employ a Multiple Locus VNTR Analysis (MLVA) scheme for strains from Argentina isolated in our laboratory. From the 56 isolates analyzed, 47 different genotypic profiles were obtained. All the strains typed as biovar 2 showed the same profile. This scheme allowed assigning each isolate to the biovar it belongs to. All the genotypes were related using the goeBURST analysis and biovar 2 was proposed as founder.

Detección de un complejo clonal con el genotipo de Brucella abortus biovariedad 2 como fundador en aislamientos de B. abortus de Argentina. / [Detection of a clonal complex with Brucella abortus biovar 2 genotype as founder in B. abortus isolates from Argentina].

Brucella abortus is the causative agent of bovine brucellosis, a worldwide zoonosis. Up to date, eight biovars of B. abortus have been described. In Argentina, biovar 1 is the most frequently isolated. However, biovar 2, which is more pathogenic than biovar 1, is also found. Molecular methods for subtyping isolates are necessary for allowing epidemiological surveillance and control of eradication programs. Due to the genetic homogeneity of the genus Brucella, the development of molecular typing tools has been difficult. The publication of microorganism genomes facilitates the design of this approach. The aim of this work was to employ a Multiple Locus VNTR Analysis (MLVA) scheme for strains from Argentina isolated in our laboratory. From the 56 isolates analyzed, 47 different genotypic profiles were obtained. All the strains typed as biovar 2 showed the same profile. This scheme allowed assigning each isolate to the biovar it belongs to. All the genotypes were related using the goeBURST analysis and biovar 2 was proposed as founder.

Pesquisa de anticorpus anti-brucella canis e anti-brucella abortus em cães (canis familiaris) da região metropolitana de Belém e Ilha do Marajó, estado do Pará

A brucelose canina caracteriza-se por ser uma doença infecto-contagiosa crônica que acomete várias espécies de animais domésticos, silvestres e também o homem. A infecção de cães, na maioria dos casos, é ocasionada pela Brucella canis, no entanto, a Brucella abortus pode estar envolvida. Este trabalho teve como objetivo identificar a ocorrência de anticorpos anti-Brucella canis e anti-Brucella abortus em cães da Região Metropolitana de Belém e Ilha de Marajó, estado do Pará, e descrever sua ocorrência em 3 municípios na Ilha do Marajó: Arari, Breves e Portel. Foram utilizadas 1.400 amostras de soros de cães, sendo 637 machos e 763 fêmeas. Provenientes de Clínicas Particulares (607), do Centro de Controle de Zoonoses (215), Ação Pet 2010 (199), Canis Particulares (107), Hospital Veterinário e Projeto Vida Digna (87), Abrigos (56) e da Região de ilha do Marajó (129), com padrão e sem padrão de raças definida; a idade dos animais variou de 1 à 17 anos e não apresentavam sintomatologia clínica da enfermidade. A colheita foi realizada no período de 2010 a 2012. Os métodos utilizados foram a soroaglutinação rápida com Antígeno Acidificado Tamponado (AAT) para a pesquisa de anti-Brucella abortus e Imunodifusão em Gel de Ágar (IDGA) para a pesquisa de anti-Brucella canis. Obtendo-se 17,1% de soropositividade para anti-Brucella abortus e 1,1% para anti-Brucella canis

Characterization of genomic island 3 and genetic variability of Chilean field strains of Brucella abortus.

One of the capabilities developed by bacteria is the ability to gain large fragments of DNA from other bacteria or to lose portions of their own genomes. Among these exchangeable fragments are the genomic islands (GIs). Nine GIs have been identified in Brucella, and genomic island 3 (GI-3) is shared by two pathogenic species, B. melitensis and B. abortus. GI-3 encodes mostly unknown proteins. One of the aims of this study was to perform pulsed-field gel electrophoresis (PFGE) on field isolates of B. abortus from Chile to determine whether these isolates are clonally related. Furthermore, we focused on the characterization of GI-3, studying its organization and the genetic conservation of the GI-3 sequence using techniques such as tiling-path PCR (TP-PCR) and restriction fragment length polymorphism-PCR (RFLP-PCR). Our results, after PFGE was performed on 69 field isolates of B. abortus from Chile, showed that the strains were genetically homogeneous. To increase the power of genetic discrimination among these strains, we used multiple locus variable-number tandem-repeat (VNTR) analysis with 16 loci (MLVA-16). The results obtained by MLVA-16 showed that the strains of B. abortus were genetically heterogeneous and that most of them clustered according to their geographic origin. Of the genetic loci studied, panel 2B was the one describing the highest diversity in the analysis, as well as locus Bruce19 in panel 2A. In relation to the study of GI-3, our experimental analysis by TP-PCR identified and confirmed that GI-3 is present in all wild strains of B. abortus, demonstrating the high stability of gene cluster GI-3 in Chilean field strains.

Multiplicación de Brucella abortus y producción de óxido nítrico en dos líneas celulares de macrófagos de distinto origen / Multiplication of Brucella abortus and production of nitric oxide in two macrophage cell lines of different origin

Brucella abortus es una bacteria que causa abortos e infertilidad en el ganado y fiebre ondulante en el hombre. Se multiplica en el citoplasma celular evadiendo los mecanismos de muerte intracelular. El óxido nítrico (NO) es importante en la regulación de la respuesta inmune. En el presente trabajo estudiamos la habilidad de tres cepas de B. abortus para sobrevivir intracelularmente en dos líneas celulares de macrófagos. La multiplicación de bacterias en ambas líneas celulares fue determinada a distintos tiempos en número de UFC/ml, también fue observada al microscopio de campo claro y de fluorescencia utilizando Giemsa y naranja de acridina, respectivamente. La tinción de ambas líneas celulares inoculadas con B. abortus mostró un resultado concordante con el encontrado en la determinación del número de UFC. Fue confirmada la presencia de B. abortus por microscopía electrónica. Para medir la producción de NO se utilizó el reactivo de Griess. La multiplicación de la cepa rugosa RB51 disminuyó en ambas líneas celulares y los niveles de NO fueron mayores en células inoculadas con dicha cepa que cuando fueron inoculadas con las cepas lisas (S19 y 2308). Estos resultados sugieren que probablemente la ausencia de cadena O en el lipopolisacárido afecta el crecimiento intracelular de B. abortus.

Brucella abortus is a bacterium which causes abortions and infertility in cattle and undulant fever in humans. It multiplies intracellularly, evading the mechanisms of cellular death. Nitric oxide (NO) is important in the regulation of the immune response. In the present work, we studied the ability of three B. abortus strains to survive intracellularly in two macrophage cell lines. The bacterial multiplication in both cell lines was determined at two different times in UFC/ ml units. Moreover the inoculated cells were also observed under light-field and fluorescence microscopy stained with Giemsa and acridine orange, respectively. The stain of both cellular lines showed similar results with respect to the UFC/ml determination. The presence of B. abortus was confirmed by electronic microscopy. In both macrophage cell lines inoculated with RB51, the multiplication diminished and the level of NO was higher, compared with cells inoculated with smooth strains (S19 and 2308). These results suggest that the absence of O-chain of LPS probably has affects the intracellular growth of B. abortus.

NRAMP1 3' untranslated region polymorphisms are not associated with natural resistance to Brucella abortus in cattle.

The NRAMP1 gene encodes a divalent cation transporter, located in the phagolysosomal membrane of macrophages, that has been associated with resistance to intracellular pathogens. In cattle, natural resistance against brucellosis has been associated with polymorphisms at the 3' untranslated region (3'UTR) of the NRAMP1 gene, which are detectable by single-strand conformational analysis (SSCA). This study aimed to evaluate the association between NRAMP1 3'UTR polymorphisms and resistance against bovine brucellosis in experimental and natural infections. In experimentally infected pregnant cows, abortion occurred in 42.1% of cows with a resistant genotype (SSCA(r); n = 19) and in 43.1% of those with a susceptible genotype (SSCA(s); n = 23). Furthermore, no association between intensity of pathological changes and genotype was detected. In a farm with a very high prevalence of bovine brucellosis, the percentages of strains of the SSCA(r) genotype were 86 and 84% in serologically positive (n = 64) and negative (n = 36) cows, respectively. Therefore, no association was found between the NRAMP1-resistant allele and the resistant phenotype in either experimental or naturally occurring brucellosis. To further support these results, bacterial intracellular survival was assessed in bovine monocyte-derived macrophages from cattle with either the resistant or susceptible genotype. In agreement with our previous results, no difference was observed in the rates of intracellular survival of B. abortus within macrophages from cattle with susceptible or resistant genotypes. Taken together, these results indicate that these polymorphisms at the NRAMP1 3'UTR do not affect resistance against B. abortus in cattle and that they are therefore not suitable markers of natural resistance against bovine brucellosis.

[Multiplication of Brucella abortus and production of nitric oxide in two macrophage cell lines of different origin]. / Multiplicación de Brucella abortus y producción de óxido nítrico en dos líneas celulares de macrófagos de distinto origen.

Brucella abortus is a bacterium which causes abortions and infertility in cattle and undulant fever in humans. It multiplies intracellularly, evading the mechanisms of cellular death. Nitric oxide (NO) is important in the regulation of the immune response. In the present work, we studied the ability of three B. abortus strains to survive intracellularly in two macrophage cell lines. The bacterial multiplication in both cell lines was determined at two different times in UFC/ ml units. Moreover the inoculated cells were also observed under light-field and fluorescence microscopy stained with Giemsa and acridine orange, respectively. The stain of both cellular lines showed similar results with respect to the UFC/ml determination. The presence of B. abortus was confirmed by electronic microscopy. In both macrophage cell lines inoculated with the rough strain RB51, the multiplication diminished and the level of NO was higher, compared with cells inoculated with smooth strains (S19 and 2308). These results suggest that the absence of O-chain of LPS probably affects the intracellular growth of B. abortus.

[Molecular characterization of Brucella abortus strain 19 and its application for controlling biologics]. / Tipificación molecular de Brucella abortus cepa 19 y su aplicación al control de biológicos.

Brucella abortus is the etiological agent of bovine brucellosis. The strain 19 used in vaccine elaboration can be identified through a deletion in the eri region associated with its susceptibility to erythritol. We optimized a PCR assay for specific characterization of this strain. The method described here is a rapid procedure that enables identification of B. abortus, and simultaneous differentiation of the strain 19 from other B. abortus biovar 1 strains. We applied the assay to detect the strain 19 in vaccines against B. abortus produced in Argentina. Thq results show this method could be used to follow vaccine seed cultures of this strain. The methodology could also contribute to reduce the risk of a laboratory-acquired infection and could be of great help as a routine test for confirmation of B. abortus in non related vaccines.