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Purpose of Review We pretend to highlight the most important advances reached in the last few years in the biology ofBrucella suisandBrucella melitensisand focus attention on new tools for the comprehension, prevention and control of these zoonotic agents. Recent Findings Important progress lately done in the field of pathogenesis is presented here. This involves current studies on proteins involved in the survival and antigenic structure ofBrucella, as well as the findings of howBrucellahas achieved modifications in order to adapt, replicate, survive and modulate the host´s immune system, improve the knowledge of the performance of these bacteria. Summary Consequently, better approaches for vaccinology and diagnostic techniques are developed. Relevant epidemiological issues based on changes in the highly conserved genome of these bacteria will be valuable tools to describe outbreaks and disease status, along with risk factors that would otherwise be difficult to analyze.

The occurrence of ectoparasites in wild nutria is poorly understood. Fifty-five livetrapped wild nutria (Myocastor coypus) from its indigenous region were examined for ectoparasites after capture from December 2013 to December 2014. The captures came from the Buenos Aires Province, by far the area of the country most densely populated by nutria, characterized as a temperate grassland, which are prime areas for sustained agriculture. Only one species of chewing lice (Pitrufquenia coypus, Marelli, 1932), one flea (Nosopsyllus fasciatus, Bosc, 1800) and one tick (Rhipicephalus sanguineus, Latreille, 1806) were collected. Fourteen percent of the animals were infested and P.coypus, an obligate parasite of the nutria, which was the most prevalent ectoparasite. N. fasciatus and R. sanguineus occurrence remains controversial as they may or may not be some accidental host species. To our knowledge, this is the first comprehensive and systematic survey of ectoparasites in wild nutria from the southern hemisphere, the indigenous region of this species., A ocorrência de ectoparasitas em nutria selvagem é pouco compreendido. Cinquenta e cinco nutria selvagem capturadas (Myocastor coypus) de sua região indígena foram examinados para os ectoparasitas após até captura a partir de dezembro de 2013 a dezembro de 2014. As capturas ocorreram no estado de Buenos Aires, a área mais densamente povoada do país por nutria, caraterizada como uma pastagem temperada, que se tornou área principal para a agricultura sustentável. Uma espécie de piolhos de mastigação (Pitrufquenia coypus, Marelli, 1932), uma pulga (Nosopsyllus fasciatus, Bosc, 1800) e um carrapato (Rhipicephalus sanguineus, Latreille, 1806) foram recolhidos. Catorze por cento dos animais foram infestadas pelo P.coypus, um parasita obrigatório do nutria, sendo o ectoparasita mais prevalente. A ocorrência de N. fasciatus e R. sanguineus continua controversa, pois podem ou não ser algumas espécies hospedeiras acidentais. Para nosso conhecimento, este é o primeiro estudo abrangente e sistemático de ectoparasitas em nutria selvagem do hemisfério sul, a região indígena desta espécie., Facultad de Ciencias Veterinarias, Comisión de Investigaciones Científicas de la provincia de Buenos Aires

Background: Basic pathologic characteristics for farmed minks were previously reportedworldwide. However, its status in the wild has not been studied in detail. Also, there is a lack ofsystematic investigations from South America.Objective: Serology and electrophoresis were carried out for evidence of exposure to 12 minkpathogens on two different locations.Animals and methods: Serology was done in 87 wild minks by reference techniques againstToxoplasma gondii, Encephalitozoon cuniculi, Neospora caninum, Brucella abortus, Mycobacteriumbovis, Leptospira interrogans, canine distemper virus (CDV), canine adenovirus (CAV), canineparvovirus (CPV), rabies virus (RV), Influenza A virus (FLUAV) and Aleutian disease virus (ADV). Forthe detection of hypergammaglobulinemia (the Aleutian disease main clinical feature),gammaglobulin concentrations were determined by conventional electrophoresis.Results: Seventy-one percent of the 87 sera had antibodies against one or more pathogens.ADV accounted for the highest seroprevalence (29%), followed by T. gondii (26%), L. interrogans(14%), M. bovis (12%), B. abortus (9%), N. caninum (3%), CPV (3%) and CDV (2%). Seroprevalencewas influenced by location but not sex or age. Additionally, 16% of the seropositive samples forADV had gammaglobulin levels >20% of the total protein concentration. Antibody titers forCDV and CPV were low and difficult to interpret as almost all these cases had borderlineconcentrations.Conclusion: A cautious interpretation of the results is urged as the epidemiological role ofthe wild mink is largely unexplored for most of these agents. Nevertheless, the informationmay be clinically relevant and may stimulate conservation organizations to supportQ4 research programs., Facultad de Ciencias Veterinarias

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.

Little information is present in the literature on diseases of the nutria (Myocastor coypus) in the wild. Serum samples obtained from 176 trapped animals were tested for antibody reactivity against 11 pathogens of significant zoonoses. Sera were positive against Leptospira (38.0 %, odds relative risk = 0.03, Taylor series 95 % confidence limits 0.01–0.06), Toxoplasma gondii (27.8 %, odds relative risk = 2.30, Taylor series 95 % confidence limits 1.23–4.31), Chlamydophila psittaci (21.0 % odds relative risk = 4.94, Taylor series 95 % confidence limits 2.75–8.89), Streptococcus equi subspecies zooepidemicus (15.9 % odds relative risk = 1.87, Taylor series 95 % confidence limits 1.04–3.37), and encephalomyocarditis virus (3.4 %, odds relative risk = 0.05, Taylor series 95 % confidence limits 0.02–0.12). Some of the rodents showed antibodies at high titers, mostly indicating recent exposure. Seroprevalence rates varied among the location and age groups, although not by season or gender. All samples were seronegative for Brucella spp., Francisella tularensis, vesicular stomatitis virus, rabies virus, foot-and-mouth disease virus, and Encephalitozoon cuniculi.

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.

The objective of the present study was to compare the performance of three serological tests for diagnosis of Brucella abortus infections in buffaloes (Bubalus bubalis). Serum samples collected from 696 adult females were submitted to the competitive enzyme-linked immunosorbent assay (ELISA-C), (I-ELISA), fluorescence polarization test (FPA), 2-mercaptoethanol test (2-ME) and complement fixation test (CFT). The gold standard was the combination of CFT and 2-ME, considering as positive the reactors in both CFT and 2-ME, and as negative those non-reactors. ROC analyses were done for C-ELISA, I-ELISA and FPA and the Kappa agreement index were also calculated. The best combinations of relative sensitivity (SEr) and relative specificity (SPr) and Kappa were given by C-ELISA (96.9%, 99.1%, and 0.932, respectively) and FPA (92.2%, 97.6 and 0.836, respectively). The C-ELISA and FPA were the most promising confirmatory tests for the serological diagnosis of brucellosis in buffaloes, and for these tests, cut-off values for buffaloes may be the same as those used for bovines.

Brucella suis has been recognized as the major etiological agent of human brucellosis in areas free from Brucella melitensis infection. However, with changes in swine management, the occurrence of swine brucellosis has decreased as has the human incidence of B. suis infection. A swine brucellosis outbreak within a herd from Jaboticabal (São Paulo, Brazil) was detected in July 2006. The herd comprised approximately 300 sows and 1,500 finishing animals. Many sows within this herd experienced abortions, while others exhibited vaginal discharge; three sows suffered posterior paralysis. Among 271 sows, 254 (93.7%) tested positive for brucellosis by complement fixation, and among 62 randomly bled finishing animals, 17 (27.4%) also tested positive. The B. suis biovar 1 was cultured from 14 aborted fetuses and six sows. Brucella was identified using routine methods. Fourteen farm workers were tested using agglutination tests, with three workers showing evidence of Brucella antibody titers. A 39-year-old woman, who worked with maternal pigs and had direct contact with aborted fetuses, presented an agglutinating titer of 480 IU/mL and displayed clinical signs of infection. Our findings suggest that despite a reduction of swine brucellosis throughout Brazil, B. suis infection still occurs, thereby posing a zoonotic risk.

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

The study compared the performance of three screening serological tests: buffered plate antigen (BPA), RoseBengal produced with 1119-3 Brucella abortus strain (RB1119-3), and Rose-Bengal produced with 99 Brucella abortus strain (RB99). Sera from 696 adult female animals were submitted to BPA, RB1119-3, RB99, 2mercaptoethanol test (ME), and complement fixation test (FC). The gold standard was the combination of CF and ME. The Kappa values for BPA, RB99, and RB1119-3 were 0.82, 0.74, and 0.70, respectively. The relative sensitivity and specificity for the same tests were 0.98 and 0.96, 0.92 and 0.94, and 0.95 and 0.92, respectively. These results indicate that BPA is a better screening test than RB for buffalo, regardless of the B. abortus strain in RB.

Between 2003 and 2012, 605 southern right whales (SRW; Eubalaena australis) were found dead along the shores of Península Valdés (PV), Argentina. These deaths included alarmingly high annual losses between 2007 and 2012, a peak number of deaths (116) in 2012, and a significant number of deaths across years in calves-of-the-year (544 of 605 [89.9%]; average = 60.4 yr-1). Post-mortem examination and pathogen testing were performed on 212 whales; 208 (98.1%) were calves-of-the-year and 48.0% of these were newborns or neonates. A known or probable cause of death was established in only a small number (6.6%) of cases. These included ship strike in a juvenile and blunt trauma or lacerations (n = 5), pneumonia (n = 4), myocarditis (n = 2), meningitis (n = 1), or myocarditis and meningitis (n = 1) in calves. Ante-mortem gull parasitism was the most common gross finding. It was associated with systemic disease in a single 1-2 mo old calf. Immunohistochemical labeling for canine distemper virus, Toxoplasma gondii and Brucella spp., and PCR for cetacean morbillivirus (CeMV), influenza A, and apicomplexan protozoa were negative on formalin-fixed, paraffin-embedded lung and brain samples from a subset of whales; PCR for Brucella spp. was positive in a newborn/neonate with pneumonia. Skin samples from whales with gull parasitism were PCR negative for CeMV, poxvirus, and papillomavirus. This is the first long-term study to investigate and summarize notable post-mortem findings in the PV SRW population. Consistent, significant findings within or between years to explain the majority of deaths and those in high-mortality years remain to be identified Inst. de Patobiología Fil: McAloose, DeniseWildlife Conservation Society Zoological Health Program; Estados Unidos. Southern Right Whale Health Monitoring Program; Argentina Fil: Rago, Virginia. Southern Right Whale Health Monitoring Program; Argentina. Wildlife Conservation Society Wildlife Health & Health Policy Program; Estados Unidos Fil: Di Martino, Matías. Southern Right Whale Health Monitoring Program; Argentina. Wildlife Conservation Society Wildlife Health & Health Policy Program; Estados Unidos Fil: Chirife, AndreaSouthern Right Whale Health Monitoring Program; Argentina Fil: Olson, Sarah. Wildlife Conservation Society Wildlife Health & Health Policy Program; Estados Unidos Fil: Beltramino, Lucas. Southern Right Whale Health Monitoring Program; Argentina Fil: Pozzi, Luciana Melina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Nacional Patagónico; Argentina. Southern Right Whale Health Monitoring Program; Argentina. Fundación Patagonia Natural; Argentina Fil: Musmeci, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Nacional Patagónico; Argentina. Southern Right Whale Health Monitoring Program; Argentina. Fundación Patagonia Natural; Argentina Fil: la Sala, Luciano Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Bahia Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biologia, Bioquimica y Farmacia. Catedra de Parasitologia Clinica; Argentina Fil: Mohamed, Nadia. Southern Right Whale Health Monitoring Program; Argentina Fil: Sala, Juan Emilio. Southern Right Whale Health Monitoring Program; Argentina Fil: Bandieri, Lucas. Southern Right Whale Health Monitoring Program; Argentina Fil: Andrejuk, Julian. Southern Right Whale Health Monitoring Program; Argentina Fil: Tomaszewicz, Ania. Wildlife Conservation Society Zoological Health Program; Estados Unidos Fil: Tomaszewicz, Ania. Wildlife Conservation Society Zoological Health Program; Estados Unidos Fil: Seimon, Tracie. Wildlife Conservation Society Zoological Health Program; Estados Unidos Fil: Sironi, Mariano. Southern Right Whale Health Monitoring Program; Argentina. Instituto de Conservación de Ballenas; Argentina Fil: Samartino, Luis Ernesto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina Fil: Rowntree, Victoria. Southern Right Whale Health Monitoring Program; Argentina. University of Utah. Department of Biology; Estados Unidos Fil: Uhart, Marcela M. Southern Right Whale Health Monitoring Program; Argentina. Wildlife Conservation Society Wildlife Health & Health Policy Program; Estados Unidos. University of California. School of Veterinary Medicine. One Health Institute; Argentina

This paper describes an indirect enzyme-linked immunosorbent assay (I-ELISA) and a fluorescence polarisation assay (FPA), each capable of detecting antibody in several species of hosts to smooth and rough members of the genus Brucella. The I-ELISA uses a mixture of smooth lipopolysaccharide (SLPS) and rough lipopolysaccharide (RLPS) as the antigen, and a recombinant protein A/G conjugated with horseradish peroxidase as the detection reagent. When using individually determined cutoff values, the SLPS/RLPS combined-antigen I-ELISA detected antibody in slightly more animals exposed to SLPS or to RLPS than did I-ELISA procedures using each individual antigen separately. Similarly, the assay using combined antigens detected antibody in slightly fewer animals not exposed to Brucella sp. When a universal cutoff of 10% positivity was used (relative to strongly positive control sera of each species), the overall performance index (percentage sensitivity plus percentage specificity) value decreased by 1.0 (from 199.4 to 198.4). In the FPA, it was not possible to use a universal cutoff without significant loss of performance. The overall sensitivity value for the FPA using the combined FPA antigen was 1.0% lower than using the O-polysaccharide (OPS) from SLPS and 9.1% higher than using the core antigen (CORE) from RLPS. When the combined antigen was used, the FPA specificity was slightly higher (1.2%) than from only the OPS, and considerably higher (12.6%) than the CORE. Overall, both the I-ELISA and the FPA with combined antigens were suitable as screening tests for all species of Brucella in the animal species tested.

A total of 2213 goat sera were tested by the indirect enzyme immunoassay (IELISA), the competitive enzyme immunoassay (CELISA), the fluorescence polarization assay (FPA), complement fixation test (CFT) and the buffered antigen plate agglutination test (BPAT) for their antibody against Brucella melitensis . Of these, 528 blood samples collected in EDTA were tested in the field by the FPA (bFPA). In addition, 249 goat milk samples were tested by IELISA (mELISA). The serum samples were classified based on their reactivity in the BPAT and CFT. A total of 716 sera were positive while 1497 gave negative reactions in both tests. Relative to the BPAT and CFT reactivity, the sensitivity and specificity of the assays were as follows: IELISA: 96.2 and 99.7%, CELISA: 93.6 and 99.4% and FPA: 88.7 and 98.9%, respectively. The bFPA gave sensitivity and specificity values relative to the BPAT and CFT of 92.3 and 61.6% while the mELISA, based on the serum BPAT and CFT of the paired animals gave a relative sensitivity value of 90.9% and a relative specificity value of 92.4%.

The indirect enzyme-linked immunosorbent assay (IELISA), the competitive enzyme-linked immunosorbent assay (CELISA) and the fluorescence polarisation assay (FPA) were evaluated with sera from sheep experimentally infected with Brucella melitensis and negative Canadian sheep. The sensitivity and specificity of the assays were as follows: IELISA: 91.7% and 97.6%, CELISA: 75.0% and 99.8% and FPA: 91.7% and 89.5%. Sera from the same experimental population were divided according to serological reaction in the rose bengal agglutination test (RBT) and the complement fixation test (CFT). Reactivity relative to the RBT positive and CFT positive sera were as follows: IELISA: 99.7%, CELISA: 93.2% and FPA: 99.1%. Since sera from goats with proven B. melitensis infection were not available, 699 sera from goats judged positive in the buffered antigen plate agglutination test (BPAT) and CFT and 982 BPAT/CFT negative Canadian goats were used. The sensitivity and specificity of the assays relative to the BPAT and CFT positive sera were: IELISA: 99.4% and 98.0%, CELISA: 95.4% and 97.1% and FPA: 92.7% and 99.8%.

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.

The aim of this study wasto describe the isolation of a pathogenic strain of Leptospira interrogans from the urine sample of a male human living in the rural area of the County of Cruz Alta, Rio Grande do Sul. An aliquot of each urine sample was sown in a Fletcher and Ellinghausen - McCullough - Johnson - Harris (EMJH) media. Samples in which there was growth of spirochetes were sent to the Leptospirosis Laboratory of the Institute of Pathobiology in the National Institute of Agricultural Technology, Buenos Aires, Argentina and were typified by the Multiple Locus of Variable Number Tandem Repeat technique (MLVA). Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 was isolated, and this is a very important finding that serves as a warning to characterize risk situation of leptospirosis epidemic by a pathogenic strain. Health professionals need to be more committed to the primary health care in Brazil and routinely apply actions of preventive medicine in rural communities in order to get success in the control of leptospirosis and other important zoonoses. O objetivo do presente estudo foi descrever um caso de isolamento de espécie patogênica de Leptospira interrogans em amostra de urina de um humano morador da zona rural do Município de Cruz Alta, Estado do Rio Grande do Sul. De cada amostra de urina, uma alíquota foi semeada nos meios Fletcher e Ellinghausen - McCullough - Johnson - Harris (EMJH). As amostras, nas quais houve crescimento de espiroquetas, foram encaminhadas para o Laboratório de Leptospirose do Instituto de Patobiologia do Instituto Nacional de Tecnologia Agropecuária Buenos Aires, Argentina e foram tipificadas pela técnica Multiple Locus ofVariableNumber Tandem Repeat(MLVA).De um residente do sexo masculino da área rural do município de Cruz Alta, foi isolada Leptospira interrogans sorovariedade Copenhageni cepa Fiocruz L1-130, uma descoberta muito importante e que serve como um alerta por caracterizar uma situação de risco de epidemia de leptospirose por uma cepa patogênica. Os profissionais de saúde precisam ser mais comprometidos com a atenção primária à saúde no Brasil e rotineiramente aplicar ações de medicina preventiva nas comunidades rurais, a fim de obter sucesso no controle da leptospirose e de outras importantes zoonoses. Fil: da Silva, Felipe Jorge. Universidade Estadual Paulista Julio de Mesquita Filho; Brasil Fil: Pinheiro Silva, Glaucenyra Cecília. Universidade Estadual Paulista Julio de Mesquita Filho; Brasil Fil: Grune Loffler, Sylvia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Brihuega, Bibiana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina Fil: Samartino, Luis Ernesto. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina Fil: Fernandez Alarcon, Miguel Frederico. Universidade Estadual Paulista Julio de Mesquita Filho; Brasil Fil: Pereira dos Santos, Carlos Eduardo. Universidade Federal do Mato Grosso do Sul; Brasil Fil: Mathias , Luis Antonio. Universidade Estadual Paulista Julio de Mesquita Filho; Brasil

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) for detection of antibody to Brucella abortus in individual milk samples was developed and validated. Samples from 190 cattle from which B. abortus was isolated; milk samples from cattle in herds infected with B. abortus (n = 1,086) and positive in the milk ring test (MRT), as well as milk samples from Canadian cattle (with no evidence of brucellosis, n = 2,974) were tested by the indirect enzyme immunoassay (IELISA) and the FPA. The sensitivity (based on samples from culture positive cattle) and specificity (based on Canadian milk samples) of the IELISA and the FPA were 100%. The relative sensitivity value obtained with milk from cattle of infected herds and the specificity values of the IELISA were 98.5 and 99.9%, respectively. The relative sensitivity and specificity of the FPA with the same samples were 82.2 and 99.4% using a cutoff value of 90 millipolarization units (mP). The low relative sensitivity value of the FPA was shown, by competitive enzyme immunoassay (CELISA), to be due to vaccinal antibody (assumed as vaccinal antibody against B. abortus Sl19 is excluded by the FPA and CELISA but not by the MRT and the IELISA), present in some of the milk samples. The FPA is a homogeneous assay which, unlike the MRT and the IELISA, may be used for testing in the field.

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.

A homogeneous fluorescence polarization assay (FPIA) for detection of bovine antibody to Brucella abortus was validated in Argentina Sera were defined based on their reactivity in the buffered antigen plate agglutination test (BPAT) and the competitive enzyme immunoassay (CELISA). Sera negative in these tests were collected from farms without evidence of brucellosis (n=733). Sera positive in the two tests were collected from cattle on farms from which B. abortus was isolated from at least one animal (n=1039). Sera from cattle vaccinated 26, 89, 240 and 272 days previously with B. abortus strain 19 were collected and tested. A cut-off value of 87 mP was determined for the FPIA, resulting in relative sensitivity and specificity values of 98.1 and 99.6%. The specificity for B. abortus strain 19 vaccinated cattle was 64.9% (26 days post vaccination, DPV), 92.1% (89 DPV), 98.6% (242 DPV) and 97.1% (272 DPV). These values were compared to those obtained with the BPAT, the CELISA, the indirect ELISA, the complement fixation test and the 2-mercaptoethanol agglutination test. Sera from 18 cattle which were vaccinated and revaccinated with B. abortus strain 19 were also tested by the same assays and the FPIA was found to be 100% specific. The use of the FPIA as a diagnostic test for brucellosis is discussed.

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.

To evaluate the fluorescence polarization assay (FPA) for the serological diagnosis of bovine brucellosis, 118 sera from cattle which were culture positive for Brucella abortus, 1751 sera from cattle from premises containing cattle infected with B. abortus, 1222 sera from cattle vaccinated with B. abortus strain 19 and 1199 sera from cattle with no evidence of brucellosis were tested in Argentina, Chile, Mexico and in the American states of Iowa, Missouri and Texas. Initial determination of serological positivity and negativity was based upon reactivity in currently used serological tests, consisting of a rapid screening test, the rose-bengal or the buffered plate antigen tests, followed by a second serological test, the complement fixation test. Sensitivity of the FPA (sera from culture positive animals) ranged from 87.5% to 100%. Serological positivity of cattle from infected premises ranged from 65.5% to 99.0% while the % negative cattle in herds without evidence of brucellosis was between 94.9 and 100%. Of B. abortus strain 19 vaccinated cattle which were positive in at least one in-use serological tests, 88.2% were negative in the FPA. In contrast, previous Canadian studies, sensitivity values were 99.0% and 100% and the specificity in both cases was 100%. This discrepancy was probably due to the use of less well characterized sera in the current study.

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

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T19:00:16Z No. of bitstreams: 1 S0103-84782010001000013.pdf: 102219 bytes, checksum: 4a3e09ab65cb31edc0d04c6a86000710 (MD5) Made available in DSpace on 2013-08-22T19:00:16Z (GMT). No. of bitstreams: 1 S0103-84782010001000013.pdf: 102219 bytes, checksum: 4a3e09ab65cb31edc0d04c6a86000710 (MD5) Previous issue date: 2010-10-01 Made available in DSpace on 2013-09-30T19:58:31Z (GMT). No. of bitstreams: 2 S0103-84782010001000013.pdf: 102219 bytes, checksum: 4a3e09ab65cb31edc0d04c6a86000710 (MD5) S0103-84782010001000013.pdf.txt: 29241 bytes, checksum: c6d163b6149343fe08dd579a62f8c983 (MD5) Previous issue date: 2010-10-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:15:14Z No. of bitstreams: 2 S0103-84782010001000013.pdf: 102219 bytes, checksum: 4a3e09ab65cb31edc0d04c6a86000710 (MD5) S0103-84782010001000013.pdf.txt: 29241 bytes, checksum: c6d163b6149343fe08dd579a62f8c983 (MD5) Made available in DSpace on 2014-05-20T13:15:14Z (GMT). No. of bitstreams: 2 S0103-84782010001000013.pdf: 102219 bytes, checksum: 4a3e09ab65cb31edc0d04c6a86000710 (MD5) S0103-84782010001000013.pdf.txt: 29241 bytes, checksum: c6d163b6149343fe08dd579a62f8c983 (MD5) Previous issue date: 2010-10-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Esta investigação teve por objetivo validar o teste de polarização fluorescente (TPF) para o diagnóstico sorológico da brucelose bovina, determinando a sensibilidade (SE) e a especificidade relativas (SP) e verificando a reprodutibilidade do teste em quatro laboratórios no Brasil. Foram selecionadas 1.389 amostras de soro sanguíneo, as quais foram inicialmente submetidas aos testes do antígeno acidificado tamponado (AAT) e mercaptoetanol (2-ME). As mesmas amostras foram submetidas à reação de fixação de complemento (RFC) e ao TPF. Para a avaliação do TPF, foi adotada a combinação dos resultados do AAT, da RFC e do 2-ME, utilizados como população de referência (padrão-ouro). Para a determinação do ponto de corte do TPF que proporciona a melhor combinação de sensibilidade e especificidade, foi usada a análise TG-ROC. A concordância entre os resultados dos quatro laboratórios foi determinada com base no indicador kappa e no coeficiente de correlação de Pearson. Os pontos de corte do TPF situaram-se entre 85,2 e 93,6 mP, conforme o laboratório. A sensibilidade variou de 91,7 a 97,3%, e a especificidade situou-se na faixa de 82,6 a 98,3%. Na comparação entre os resultados do TPF dos quatro laboratórios, o indicador kappa ficou entre 0,69 e 0,95, o que indica, na maioria das situações, reprodutibilidade excelente, e o coeficiente de correlação variou entre 0,76 e 0,99. Os resultados indicaram que o TPF apresentou bom desempenho, na maioria das situações, com sensibilidade e especificidade elevadas. em comparação com os testes convencionais, o TPF apresenta as vantagens de ser de execução mais rápida e mais fácil e não estar sujeito à ocorrência de prozona, como a RFC e o 2-ME, nem de atividade anticomplementar, como a RFC. The purpose of this research was the interlaboratorial validation of the polarization fluorescence assay (PFA) for the serodiagnosis of bovine brucellosis, verifying the relative sensitivity, the relative specificity and the reproducibility of the test in four Brazilian laboratories. Serum samples from 1,389 bovines were selected and submitted to the rose Bengal (RBT) and 2-mercaptoethanol tests in one of the laboratories. The same samples were tested by the complement fixation (CFT) test and by the PFA in the four laboratories participating of the research. The reference population (golden standard) used to evaluate the PFA was the combination of the results of RBT, CFT and 2-ME. TG-ROC analysis was used to obtain the cut-off that provided the best combination of sensitivity and specificity. The agreement between laboratories was obtained by the kappa statistic and Pearson correlation coefficient (r). The PFA cut-off values were from 85.2 to 93.6. The sensitivity of the PFA assay varied from 91.7% to 97.3%, and the specificity values varied from 82.6% to 98.3%. When comparing PFA results from the four laboratories, the kappa values was between 0.69 and 0.95, which indicates, in most situations, excellent reproducibility, and the correlation coefficient varied from 0.76 to 0.99. The results showed that the PFA had a good performance, with high sensitivity and specificity. Compared to the conventional tests, the PFA has the advantages of being easy and quick to perform, and it is not prone to the occurrence of prozone, as the CFT or the 2-ME, nor to the occurrence of anti-complementary effect, as the CFT. Universidade Estadual Paulista Faculdade de Ciências Agrárias e Veterinárias Departamento de Medicina Veterinária Preventiva e Reprodução Animal Universidade Federal do Rio Grande do Sul (UFRGS) Laboratório de Epidemiologia Veterinária Ministério da Agricultura, Pecuária e Abastecimento Instituto Biológico de São Paulo Instituto Nacional de Tecnologia Agropecuária Universidade Estadual Paulista Faculdade de Ciências Agrárias e Veterinárias Departamento de Medicina Veterinária Preventiva e Reprodução Animal

A second generation competitive enzyme immunoassay (CELISA) for detection of bovine antibody to Brucella abortus was developed to eliminate reagent variables in the assay. This assay was different from earlier CELISA formats in that it used recombinant protein A and protein G immunoglobulin receptors (PAG), labelled with horseradish peroxidase, thus eliminating the requirement for polyclonal anti-mouse-enzyme conjugate for detection. This allowed standardization of the assay. The CELISA uses a monoclonal antibody specific for a common epitope of the O-polysaccharide (OPS) of smooth lipopolysaccharide (SLPS) derived from B. abortus S1119.3. This antibody did not react with PAG. This monoclonal antibody was used to compete with antibody in the bovine test serum to the smooth lipopolysaccharide (SLPS) antigen. Reaction of bovine antibody was then measured directly with the PAG enzyme conjugate. In this case, development of colour in the reaction indicated a positive reaction. The performance characteristics of the new CELISA, sensitivity, specificity and exclusion of antibody of B. abortus S19 vaccinated animals, were very similar to those of the classical CELISA and to the indirect enzyme immunoassay (IELISA) when using sera deemed positive by isolation of the bacterium, either from individual animals or from some animals on the premises. All sera were tested by the buffered antigen plate agglutination test (BPAT) and the complement fixation test (CFT). Only samples positive on both BPAT and CFT were considered as positive and only samples negative on both tests were used considered negative. Sufficient samples from cattle, swine, sheep and goats to validate the test were included based on OIE guidelines suggesting inclusion of a minimum of 300 positive and 1000 negative samples.

Because some batch-to-batch variation in the preparation of rough lipopolysaccharide (RLPS) from Brucella ovis has been experienced, several protocols were tested to establish the most reliable method for detection of antibody in indirect enzyme immunoassay. An early version of the assay gave a performance index (PI=sum of optimum percent sensitivity and percent specificity, determined by receiver operator characteristic analysis) of 198.6. This assay used RLPS from B. ovis as the antigen and a monoclonal antibody specific for bovine IgG(1) heavy chain-enzyme conjugate for detection. This was not repeatable using other batches of antigen. Newer versions of the assay generally had decreased sensitivity values, giving PIs of 193. Use of a recombinant protein A/G-enzyme conjugate did not improve the PI (PI=190), giving reduced specificity and higher sensitivity. The final version used B. abortus RB51 RLPS as the antigen and protein A/G-enzyme conjugate for detection, giving a PI of 197. Because of the batch uniformity of the B. abortus RB51 RLPS and the versatility of the protein A/G-enzyme conjugate, the latter version appears to be the most useful for diagnostic serology.

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.