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Leptospirosis is an infectious disease of wide global distribution, which is endemic in Argentina. The objective of this study was to obtain the genetic profiles of Leptospira spp. strains isolated from clinical cases of dogs in the province of Buenos Aires by the multiple-locus variable-number tandem repeat analysis (MLVA). Eight isolated canine strains were genotyped by MLVA, obtaining the identical profile of Leptospira interrogans serovar Canicola Hond Utrecht IV in the strains named Dogy and Mayo. The strains named Bel, Sarmiento, La Plata 4581 and La Plata 5478 were identical to the profile of the genotype of L. interrogans serovar Portlandvere MY 1039.The strain named Avellaneda was identical to the genotype profile of L. interrogans serovar Icterohaemorrhagiae RGA and the strain named SB had the same profile as the L. interrogans serovar Pomona Baires genotype and was similar to the profile of serovar Pomona Pomona genotype. It would be useful to include a larger number of isolates from different dog populations in various provinces of Argentina and to characterize the genetic profiles of the strains circulating in the country. The information obtained will be useful for the control of leptospirosis in the dog population.

ResumenBrucella 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.AbstractBrucella 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.

Leptospirosis outbreaks occur regularly in Argentina and other South American countries, but little is known about their epidemiological relationships. Application of new molecular tools, such as the Multiple-Locus Variable-number tandem repeat Analysis (MLVA) is limited by scant available data on regional strains. We have analyzed the genetic diversity of a collection of 31 strains of Leptospira interrogans isolated in Argentina during the past five decades from humans and animals, including a strain from an environmental source and another isolated from an opossum. Genotyping was performed by MLVA using the loci VNTR4, VNTR7, VNTR9, VNTR10, VNTR19, VNTR23 and VNTR31, as described by Majed et al. [Identification of variable-number tandem-repeat loci in Leptospira interrogans sensu stricto. J Clin Microbiol 2005;43:539-45 [1]]. Clustering analysis revealed eight distinct MLVA genotypes, with a dominant one, genotype A. Strains with this genotype were consistently isolated since 1960, representing 55% of the total strains and spanning an extensive geographical distribution. Other seven genotypes were less frequent, and only genotypes A and Hond Utrecht IV were isolated during the last decade. Different kinds of repeat blocks for each VNTR locus were identified by sequence analysis. VNTR copy number differences among genotypes always involved only one of these blocks. MLVA patterns obtained reveal the genetic diversity and relationships between strains, and constitute the framework for the genotyping of leptospires in the region. © 2010 Elsevier Ltd. Fil: Pavan, María Elisa. Biochemiq S.A.; Argentina Fil: Cairo, Fabian Martin. Biochemiq S.A.; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; Argentina Fil: Pettinari, María Julia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Samartino, Luis Ernesto. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina. Universidad del Salvador; Argentina Fil: Brihuega, Bibiana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina. Universidad del Salvador; Argentina

A recombinant protein combining the immunoglobulin binding sites of Proteins A and G, conjugated with horseradish peroxidase was used as a universal detection reagent for the assessment of antibodies against Brucella spp. The reagent was applied in an indirect enzyme immunoassay for detection of antibodies to smooth lipopolysaccharide antigen in sera from Brucella spp. exposed and non-exposed cattle, sheep, goats and pigs and to antibodies to rough lipopolysaccharide in sheep, dogs and cattle. The results were similar to those obtained when murine monoclonal antibody-enzyme conjugates were used. An added advantage was that a universal cut-off for all tests using the proteins A and G detection reagent could be established, simplifying diagnostic interpretation of the data.

Brucellosis has been recognized in Argentina since the 19th century. Several studies demonstrated the presence of the disease in most of the domestic species. Actually, the estimate of prevalence is that between 10 and 13% of the farm animals are infected with bovine brucellosis with an individual rate of 4–5%. The annual economical losses have been estimated at US$ 60,000,000. The control of bovine brucellosis began in 1932 and successive resolutions have been issued since then. The current resolution indicates that B. abortus S19 is mandatory in female calves between 3 and 8 months of age. The vaccine strain B. abortus RB51 was provisionally approved but only for cattle older than 10 months of age. The brucellosis control program consists principally of test and slaughter. This methodology has been successful mainly in the dairy farms that have the incentive due to increased pricing because of obtaining a low prevalence of the disease. Brucellosis has been found in porcine, caprine, ovine and canine species. All Brucella species have been found in the country. Human brucellosis is an important disease and a national coordinated diagnostic net has been formed to better control the disease in man.

A fluorescence polarization assay (FPA) was used to test whole blood samples prepared by mixing blood cells from cattle without exposure to Brucella abortus (B. abortus) with sera from animals with confirmed (bacteriologically) infection. A cut-off value between negative and positive values was initially established to be 87.2mP. This value was changed to 95mP to increase assay specificity without loss of sensitivity when testing blood samples from negative animals. The FPA technology was applied to whole blood samples in the field and to stored whole blood samples using two diluent buffers. Relative sensitivity and specificity values for the FPA performed in the field, based on buffered antigen plate agglutination test and competitive enzyme immunoassay results were 95.3 and 97.3%, respectively. However, to obtain maximum sensitivity and specificity, a cut-off value of 105mP was determined for fresh whole blood samples. The relative sensitivity and specificity values of the FPA when testing stored whole blood samples were 100% each using a 95mP cut-off.The usefulness of the FPA for testing whole blood samples in the field was demonstrated.

The purpose of this study was to evaluate the performance of the indirect enzyme immunoassay (IELISA) and the competitive enzyme immunoassay (CELISA) for the diagnosis of bovine brucellosis in comparison to conventional serological tests routinely used in Argentina. Serum samples (n = 3500), from Brucella-free herds, from vaccinated cattle and from naturally infected cattle, were tested by the following tests: buffered antigen agglutination test (BPAT), rose bengal test (RBT), 2-mercaptoethanol test (2-ME), complement fixation test (CFT), IELISA and CELISA. Sensitivity and specificity of the BPAT, RBT, IELISA and CELISA were determined relative to the 2-ME and the CFT. The CELISA was considered suitable for eliminating most serological reactions of vaccinated animals and was more specific than the other tests. The results indicate the potential use of the CELISA as a complementary assay in the brucellosis control and eradication program in Argentina and other countries, where Brucella abortusstrain 19 vaccination is mandatory.

Sera from Canadian pigs (brucellosis free, n = 14 037) and sera from pigs infected with Brucella suis (n = 401) were tested by the buffered antigen plate agglutination test, the complement fixation test, an indirect and a competitive enzyme immunoassay and a fluorescence polarization assay. The results were analysed and assay sensitivity and specificity estimates were calculated. The sensitivity and specificity of the tests were as follows: the buffered antigen plate agglutination test, 77.1 and 96.9%; the complement fixation test (considering anticomplementary sera as negative), 93.3 and 95.5%; the complement fixation test (considering anticomplementary sera as positive), 58.1 and 99.9%; the indirect enzyme immunoassay, 94.0 and 97.9%; the competitive enzyme immunoassay, 90.8 and 96.6%; and the fluorescence polarization assay, 93.5 and 97.2%; respectively. It was concluded that the fluorescence polarization assay was a valuable asset to the diagnosis of porcine brucellosis because of its accuracy, ease of performance and relative cost.

Leptospirosis is the most widespread zoonosis in the world and significant efforts have been made to determine and classify pathogenic Leptospira strains. This zoonosis is maintained in nature through chronic renal infections of carrier animals, with rodents and other small mammals serving as the most important reservoirs. Additionally, domestic animals, such as livestock and dogs, are significant sources of human infection. In this study, a multiple-locus variable-number tandem repeat analysis (MLVA) was applied to genotype 22 pathogenic Leptospira strains isolated from urban and periurban rodent populations from different regions of Argentina. Three MLVA profiles were identified in strains belonging to the species Leptospira interrogans (serovars Icterohaemorrhagiae and Canicola); one profile was observed in serovar Icterohaemorrhagiae and two MLVA profiles were observed in isolates of serovars Canicola and Portlandvere. All strains belonging to Leptospira borgpetersenii serovar Castellonis exhibited the same MLVA profile. Four different genotypes were isolated from urban populations of rodents, including both mice and rats and two different genotypes were isolated from periurban populations. Fil: Grune Loffler, Sylvia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina Fil: Pavan, María Elisa. Biochemiq SA; Argentina Fil: Vanasco, Norma Bibiana. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorios e Instituto de Salud "Dr. C. G. Malbran". Instituto Nacional de Enfermedades Respiratorias; Argentina Fil: Samartino, Luis Ernesto. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina. Universidad del Salvador; Argentina Fil: Suarez, Olga Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Ecología, Genética y Evolución; Argentina Fil: Auteri, Carmelo. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina Fil: Romero, Graciela. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina Fil: Brihuega, Bibiana. Universidad del Salvador; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina

Outbreaks of leptospirosis occur regularly in Argentina, but little is known about their epidemiological relationships. We have analyzed the genetic diversity of a collection of 16 strains of Leptospira interrogans serovar Pomona isolated from animals and humans in Argentina during the past 45 years. Genotyping was performed by multiple-locus variable-number tandem repeat analysis (MLVA) using the loci VNTR4, VNTR7, VNTR9, VNTR10, VNTR19, VNTR23 and VNTR31, as described by Majed et al. [Identification of variable-number tandem-repeat loci in Leptospira interrogans sensu stricto. J Clin Microbiol 2005;43:539-45]. Clustering analysis revealed four new distinct MLVA genotypes, with a dominant one. Strains with this genotype were consistently isolated since 1960 to the present, mainly from cows and pigs, but also from humans, representing 75% of the total strains studied. These strains coexisted temporally and geographically with isolates presenting the other new genotypes. VNTR4 locus, with four different alleles, presented the highest diversity between the VNTR loci analyzed. MLVA patterns obtained will be useful for future diagnostic and epidemiological tracing analysis.

Rough lipopolysaccharide (RLPS) antigens were prepared from cultures of Brucella abortus RB51, B. ovis, and B. canis. The preparations were standardized by weight and tested with sera from cattle immunized with B. abortus RB51, sheep infected with B. ovis, and dogs infected with B. canis. Populations of unexposed animals of each species were also tested. The tests used were the indirect enzyme immunoassay (IELISA) using RLPS and the fluorescence polarization assay (FPA) using RLPS core fractions, labeled with fluorescein isothiocyanate. The IELISA using B. abortus RB51 RLPS antigen resulted in sensitivity and specificity values of 94.8% and 97.3%, respectively, when testing bovine sera, 98.5% and 97.8% when testing ovine sera, and 95.8% and 100% when testing dog sera. The IELISA using B. ovis RLPS antigen gave sensitivity and specificity values of 80.5% and 91.7%, respectively with bovine sera, 98.9% and 93.8% with sheep sera, and 70.8% and 79.8% with dog sera. The IELISA using B. canis RLPS antigen resulted in sensitivity and specificity values of 97.0% and 97.4%, respectively, with bovine sera, 96.2% and 96.3% with sheep sera, and 95.8% and 98.8% with dog sera. Labeling RLPS core from B. ovis and B. canis with fluorescein was not successful. B. abortus RB51 core labeled with fluorescein resulted in sensitivity and specificity values of 93.5% and 99.8%, respectively, with bovine sera and 78.1% and 99.0% with sheep sera. It was not possible to test the dog sera in the FPA.

Objective—To develop a novel oral vaccine delivery system for swine, using the rough vaccine strain of Brucella abortus. Animals—56 crossbred pigs from a brucellosis-free facility. Procedure—In 3 separate experiments, pigs were orally vaccinated with doses of 1 × 109 to > 1 × 1011 CFU of strain RB51 vaccine. The vaccine was placed directly on the normal corn ration, placed inside a whole pecan, or mixed with cracked pecans and corn. Results—Oral vaccination of pigs with vaccine strain RB51 resulted in a humoral immune response to strain RB51 and short-term colonization of the regional lymph nodes. Conclusions and Clinical Relevance—A viscous liquid such as Karo corn syrup in association with pecans that scarify the oral mucosa are necessary when placing the live vaccine directly onto corn or other food rations. Doses of > 1 × 1011 CFU of RB51 organisms/pig in this mixture ensures 100% colonization of regional lymph nodes via the oral route. This method may allow an efficient and economical means to vaccinate feral swine for brucellosis. (Am J Vet Res 2001;62:1328–1331)

One hundred and seven pregnant cows, which had been calfhood vaccinated with Brucella abortus strain 19 (S-19) were revaccinated with either S-19 or strain RB51 (S-RB51). All S-19-revaccinated animals seroconverted, while none of the RB51-revaccinated animals seroconverted. Two out of 25 (8%) S-19-revaccinated animals aborted, while none of the 57 RB51-revaccinated group aborted. Four of the S-19-revaccinated animals shed S-19 in the milk for at least 7 days, while only 1 cow shed S-RB51 for at least 3 days (but

The ability of Brucella to infect and grow within extraplacentomal chorioallantoic explants (CAMs) derived from early and late gestational cattle was compared. Following inoculation of CAMs with equal numbers of strain 2308 B. abortus, the infectivity was approximately the same in CAMs from both ages, however, bacterial replication was significantly greater in late gestational CAMs than in early gestational CAMs. Co-culture of both early and late gestation CAMs or culture of both types of CAMs in the presence of tissue culture media collected from either early or late B. abortus inoculated CAMs failed to alter B. abortus growth rates and/or cytopathic effects.

Bovine brucellosis is a major disease of cattle characterized by abortion during the last trimester of gestation. During many years important pieces of research have been done looking for a better understanding of this particular phenomenon. Yet, the fact that the abortion takes place in the last period of gestation result in a fascinating interrogant for such a unique event. The present review includes most of the information available regarding to this matter. Emphasis is done in the interaction of Brucella abortus with the trophoblastic cells of the bovine placenta.