Comparison of two serological diagnosis tests for bovine paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of paratuberculosis (PTBC), a chronic infectious granulomatous enteritis of ruminants. The PTBC diagnosis with commercial ELISA has limitations in sensitivity and specificity, and its results depend on the state of progress of the disease. This research aimed to evaluate two different ELISAs: (a) an "in-house" ELISA with a sonicated antigen obtained from a MAP I47 strain, and (b) a commercial ELISA. In total, the evaluated sample consisted of 394 bovine serum samples from 12 farms in Argentina with high (5-9%) and low (≤ 0.05%) prevalence of PTBC. The evaluation of the new antigen (2.5 µg/mL) was against a 1:50 dilution of the M. phlei faced sera. The cut-off point, sensitivity, and specificity determinations of both techniques were by ROC curve analysis. The area under the curve for the I47 ELISA was 0.9 (CI 95%, 0.93-0.97). With a cut-off point of 8.8%, the sensitivity was 84.3% and the specificity 96.6%. The agreement between both techniques was 0.7 (CI 95%, 0.6-0.8). These results indicate a high discriminative capacity to differentiate positive and negative bovine sera of MAP infection with the I47 ELISA. This result would represent an advantage to dispense with the imported kit.

Early-stage findings in an experimental calf model infected with Argentinean isolates of Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is an important pathogen that causes granulomatous enteritis known as Johne's disease or paratuberculosis (PTB). In this study an experimental model of calves infected with Argentinean isolates of MAP for 180 days was used to provide more data of the early PTB stages. Calves were challenged by oral route with MAP strain IS900-RFLPA (MA; n = 3), MAP strain IS900-RFLPC (MC; n = 2) or mock infected (MI; n = 2), and response to infection was evaluated through peripheral cytokine expression, MAP tissue distribution and histopathological early-stage findings. Specific and varied levels of IFN-γ were only detected at 80 days post-infection in infected calves. These data indicate that specific IFN-γ is not a useful indicator for early detection of MAP infection in our calf model. At 110 days post-infection, TNF-α expression was higher than IL-10 in 4 of the 5 infected animals and a significant decrease of TNF-α expression was detected in infected vs. non-infected calves. All calves challenged were identified as infected by mesenteric lymph node tissue culture and real time IS900 PCR. In addition, for lymph nodes samples, the agreement between these techniques was almost perfect (κ = 0.86). Colonization of tissues and levels of tissue infection varied between individuals. Evidence of early MAP dissemination to extraintestinal tissues such as the liver was detected by culture in one animal (MAP strain IS900-RFLPA). In both groups microgranulomatous lesions were observed predominantly in the lymph nodes, with giant cells present only in the MA group. In summary, the findings described herein may indicate that local MAP strains induced specific immune responses with particularities that could suggest differences in their biological behavior. Further studies should be carried out in order to obtain an in-depth understanding of the influence of MAP strains in host-pathogen interactions and the outcome of disease.

Campylobacter fetus releases S-layered and immunoreactive outer membrane vesicles

Abstract The study of outer membrane vesicles (OMVs) became relevant because of theirprobable important role in the transfer of virulence factors to host cells. Campylobacter fetusis mainly a mammal pathogen whose virulence characterization is still limited. The aim of thisstudy was to evaluate and to characterize the secretion of OMVs in this bacterium. By trans-mission electron microscopy, we confirmed the production of OMVs in all the strains assayed.Purified OMVs showed a spherical shape and variable size, although comparable to those ofother gram-negative bacteria. We also confirmed the presence of the S-layer on the surface ofthe OMVs of all the strains assayed with the exception of those derived from the NTCC referencestrain. In addition, we demonstrated their immunoreactivity by the dot-blot assay. Hence, C.fetus OMVs could contribute to the modulation of the host response and constitute a candidateto be evaluated as an adjuvant of current vaccines used in the veterinary field. This work rep-resents a platform to drive future studies towards the role of these subcellular structures in C.fetus-host interaction.

Resumen El estudio de las vesículas de membrana externa (VME) tomó un rol protagónico, yaque se las ha relacionado con la transferencia de factores de virulencia a la célula hospedadora.Campylobacter fetus es, principalmente, un patógeno de mamíferos cuya virulencia solo hasido caracterizada de forma limitada. El objetivo de este trabajo fue evaluar y caracterizar la secreción de VME en esta bacteria. Mediante microscopía electrónica de transmisión confir-mamos la producción espontánea de VME en todas las cepas estudiadas. Las VME purificadasmostraron una morfología esférica y un tama˜no variable, pero compatible con el reporte deotras bacterias gram negativas. Asimismo, hemos demostrado que estas vesículas conservanla capa S en todas las cepas, menos en la cepa de referencia NCTC y hemos confirmado suinmunorreactividad por dot-blot inmunoblot. Estas VME de C. fetus podrían contribuir a la mod-ulación de la respuesta del hospedador y constituir un buen candidato como adyuvante de lasactuales vacunas empleadas en el campo veterinario. Este trabajo representa una plataformapara impulsar estudios futuros en torno al rol de estas estructuras subcelulares en la interfaseC. fetus-hospedador.

Evidence of in utero infection by Mycobacterium avium subsp. paratuberculosis using Multiple-Locus Variable-number tandem-repeat Analysis: first report in Argentina / Evidência de infecção intra-uterina por Mycobacterium avium subsp. paratuberculosis usando Multiple-Locus Variable-number tandem-repeat Analysis: primeiro relato na Argentina

A pregnant heifer with an advanced clinical stage of paratuberculosis was reported in a herd in Argentina. Thus, the animal was euthanized and samples of organs of the cow and its fetus was taken and cultured for bacteriology in specific medium. Tissues were analyzed by histopathology (hematoxylin-eosin and Ziehl-Neelsen staining). Histopathological analysis of the cow's samples revealed the presence of lesions consistent with paratuberculosis, and Ziehl-Neelsen staining revealed the presence of acid-fast bacilli, whereas the fetal tissues showed absence of lesions but the presence of acid-fast bacilli by Ziehl-Neelsen staining. After growing in specific medium, colonies in tissues from both cow and fetus were positive for IS900-PCR, confirming the presence of Mycobacterium avium subsp. paratuberculosis (MAP). Finally, the isolates were typed by Multiple-Locus Variable-number tandem-repeat Analysis (MLVA), which confirmed the epidemiological link between them. This study is the first in Argentina to report the detection of MAP that shares an identical MLVA type in a pregnant cow and its fetus. The results of this study are consistent with previous reports and highlight the intra-uterine transmission of MAP as an important source of infection within herds.(AU)

Uma novilha prenha em estado clínico avançado de paratuberculose foi observada em um rebanho bovino na Argentina. O animal foi eutanasiado e foram colhidas amostras dos seus órgãos e dos órgãos feto as quais foram cultivadas para bacteriologia em meio específico. Os tecidos foram examinados por histopatologia (coloração de hematoxilina-eosina e Ziehl-Neelsen). Na histopatologia das amostras colhidas da novilha foram observadas lesões compatíveis com paratuberculose e a coloração de Ziehl-Neelsen revelou a presença de bacilos álcool-ácido resistentes, nos tecidos fetais não foram observadas lesões, porém a coloração de Ziehl-Neelsen revelou a presença de bacilos álcool-ácido resistentes. Após o crescimento em meio específico, as colônias foram positivas para o teste IS900-PCR nos tecidos de ambos, vaca e feto, confirmando a presença de Mycobacterium avium subsp. paratuberculosis. Por fim, os isolados foram tipados por Multiple-Locus Variable-number tandem-repeat Analysis, confirmando a relação epidemiológica entre eles. Este estudo relata a primeira detecção de Mycobacterium avium subsp. paratuberculosis na Argentina em que houve o compartilhamento de um tipo idêntico de MLVA em uma vaca prenhe e no seu feto. Os resultados deste estudo são consistentes com relatos anteriores e destacam a transmissão intra-uterina de Mycobacterium avium subsp. paratuberculosis como importante fonte de infecção nos rebanhos de bovinos.(AU)

Corynebacterium pseudotuberculosis biovar ovis: evaluación de la sensibilidad antibiótica in vitro / Corynebacterium pseudotuberculosis biovar ovis: Evaluation of antibiotics susceptibility in vitro

Resumen Los objetivos de este trabajo fueron estudiar la sensibilidad antibiótica de aislamientos de Corynebacterium pseudotuberculosis procedentes de pequeños rumiantes e investigar la presencia de integrones que contienen genes de resistencia. Se estudiaron 15 aislamientos de diferentes fuentes por los métodos de difusión y dilución. Por el método de difusión, amoxicilina-clavulánico, ampicilina, cefotaxima, cefoxitina, ciprofloxacina, cloranfenicol, eritromicina, estreptomicina, gentamicina, imipenem, kanamicina, norfloxacina, penicilina, rifampicina, tetraciclina, trimetroprima-sulfametoxazol y vancomicina fueron activos frente al 100% de los aislamientos, mientras que amicacina presentó resultados variables. En los aislamientos que desarrollaron frente a amicacina se investigó la presencia de integrones de clase 1. El resultado fue negativo, sugiriendo la ausencia del integrón. Utilizando el método de dilución, los antibióticos más activos correspondieron a los grupos de cefalosporinas, gluco-péptidos, macrólidos, quinolonas y tetraciclinas. Se demostró menor actividad de p-lactámicos y aminoglucósidos. No se registró variabilidad en los perfiles antibióticos en los aislamientos procedentes de diferentes fuentes.

Abstract The aims of this work were to study the antibiotic susceptibility in Corynebacterium pseudotuberculosis isolated from small ruminants and to determine the presence of integrons that contain resistance genes. Fifteen isolates of different sources were analysed using the diffusion and the dilution methods. When the diffusion method was performed, amoxicillin-clavulanic, ampicillin, cefotaxime, cefoxitin, ciprofloxacin, chloramphenicol, erythromycin, streptomycin, gentamicin, imipenem, kanamycin, norfloxacin, penicillin, rifampicin, tetracycline, trimethoprim-sulfamethoxazole and vancomycin were effective against the 100% of isolates, while amikacin showed variable results. The isolates that were able to grow with amikacin, were studied in relation to the presence of integron class 1. The result was negative, suggesting the absence of integron. Using dilution method, the antibiotics belonging to the cephalosporin, glycopeptide, macrolide, quinolone, and tetracycline groups were the most active ones for the C. pseudotuberculosis biovar ovis isolates. Less activity of p-lactam and aminoglycosides were observed. There was no observation of variability in the antibiotic patterns in the strains coming from different sources.

L-cysteine transporter-PCR to detect hydrogen sulfide-producing Campylobacter fetus.

Phenotypic differences between Campylobacter fetus fetus and C. fetus venerealis subspecies allow the differential diagnosis of bovine genital campylobacteriosis. The hydrogen sulfide production, for example, is a trait exclusive to C. fetus fetus and C. fetus venerealis biovar intermedius. This gas that can be biochemically tested can be produced from L-cysteine (L-Cys). Herein, we report a novel multiplex-PCR to differentiate C. fetus based on the evaluation of a deletion of an ATP-binding cassette-type L-Cys transporter that could be involved in hydrogen sulfide production, as previously described. A wet lab approach combined with an in silico whole genome data analysis showed complete agreement between this L-Cys transporter-PCR and the hydrogen sulfide production biochemical test. This multiplex-PCR may complement the tests currently employed for the differential diagnosis of C. fetus.

Current trends in bovine abortion in Argentina / Tendências atuais do aborto bovino na Argentina

Bovine abortion is an important cause of significant economic losses in beef and dairy herds. This syndrome is usually difficult to diagnose. The aim of this study was to characterize bovine abortion causes in Argentina by standard diagnosis procedures (histology, bacterial and viral isolation) and other diagnostic tests like direct fluorescent antibody test (DFAT), fetal serology, immunohistochemistry (IHC), and PCR, showing their specific advantages and limitations. Necropsies were performed in 150 aborted bovine fetuses submitted to the diagnostic laboratories of Instituto Nacional de Tecnología Agropecuaria (INTA) Balcarce, Argentina. Etiological diagnosis was confirmed in 78 fetuses (52% of the cases). Most causes of abortion were of infectious origin, being Neospora caninum (14.67%), Campylobacter fetus sp. (9.33%), Leptospira spp. (7.33%) and Brucella abortus (6.65%) the main microorganisms identified. Bovine viral diarrhea virus (BVDV) and bovine herpes virus (BHV) were diagnosed in 2 (1.33%) and 3 (2%) cases, respectively. This study showed a better characterization of bovine abortion compared with previous researches done on this topic.(AU)

O aborto bovino é uma causa importante de perdas econômicas significativas em rebanhos bovinos e leiteiros. Esta síndrome é geralmente difícil de diagnosticar. O objetivo deste estudo foi caracterizar o aborto bovino na Argentina por procedimentos diagnósticos de rotina (histologia, isolamento viral e bacteriana) e outros testes diagnósticos como ensaio directo de anticorpos fluorescentes (DFAT), sorologia fetal, imuno-histoquica (IHC), e PCR; mostrando suas vantagens e limitações específicas. As necropsias foram realizadas em 150 fetos bovinos abortados submetidos aos laboratórios de diagnóstico do Instituto Nacional de Tecnologia Agropecuária (INTA) de Balcarce, na Argentina. O diagnóstico etiológico foi confirmado em 78 fetos (52% dos casos). A maioria das causas de aborto foram de origem infecciosa, sendo Neospora caninum (14,67%), Campylobacter fetus sp. (9,33%), Leptospira spp. (7,33%) e Brucella abortus (6,65%) os principais microrganismos identificados. O vírus da diarréia viral bovina (BVDV) e o herpesvírus bovino (BHV) foram diagnosticados em 2 (1,33%) e 3 (2%) casos, respectivamente. Este estudo mostrou uma melhor caracterização do aborto bovino em comparação com pesquisas anteriores feita sobre este tema.(AU)

Mycobacterium paratuberculosis detection in cow's milk in Argentina by immunomagnetic separation-PCR

Abstract The aim of this study was to standardize a diagnosis procedure to detect Mycobacterium avium subsp. paratuberculosis (Map) DNA in raw cow milk samples under field conditions. A procedure that combines both immunomagnetic separation and IS900 -PCR detection (IMS-IS1 PCR) was employed on milk samples from 265 lactating Holstein cows from Map infected and uninfected herds in Argentina. IMS-IS1 PCR results were analyzed and compared with those obtained from milk and fecal culture and serum ELISA. The extent of agreement between both tests was determined by the Kappa test. IMS-IS1 PCR showed a detection limit of 101 CFU of Map/mL of milk, when 50:50 mix of monoclonal and polyclonal antibodies were used to coat magnetic beads. All of the 118 samples from the Map uninfected herds were negative for the set of the tests. In Map infected herds, 80 out of 147 cows tested positive by milk IMS-IS1 PCR (55%), of which 2 (1.4%) were also positive by milk culture, 15 (10%) by fecal culture, and 20 (14%) by serum ELISA. Kappa statistics (95% CI) showed a slight agreement between the different tests (<0.20), and the proportions of agreement were ≤0.55. The IMS-IS1 PCR method detected Map in milk of the cows that were not positive in other techniques. This is the first report dealing with the application of IMS-IS1 PCR in the detection of Map in raw milk samples under field conditions in Argentina.

Immunological findings associated with Argentinean strains of Mycobacterium avium subsp. paratuberculosis in bovine models.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of ruminant paratuberculosis. The aim of this study was to evaluate the biological behavior of different Argentinean strains of MAP in two bovine infection models: macrophage (in vitro) and calf (in vivo) through the evaluation of early immune responses at the peripheral and local levels. Two MAP strains (A and C) were selected taking into account the different patterns of TNF-α and IL-10 secretion displayed by infected bovine macrophages in vitro. Two groups of calves were infected with 250mg of total wet weight live MAP: strain A infected group (MA, n=3), strain C infected group (MC, n=2). Another group of animals was mock-infected (MI, n=3). Infection was confirmed by MAP culture of feces and microscopic observation of granulomatous lesions in the gut tissue. All infected calves showed positive results in the DTH skin test. A significant increase in peripheral CD4CD25(+) cells in MC group on day 150 was detected. The specific cellular immune response developed allowed the identification of the infection as early as 30days in the MA group. However, the percentage of CD8CD25(+) cells was significantly increased on day 120 in MC group. Significant differences between groups in proliferation and cellular responses were also detected in ileocecal lymph node samples. In summary, the strains of MAP employed herein induced differential immune responses in peripheral cells, in the proliferative responses and in cell functionality at the local level. Our findings support the hypotheses that the in vitro behavior displayed by macrophages could be a tool to identify differences among MAP strains infecting bovines and that the host-pathogen interactions occurring upon infection are dependent on the strain of MAP involved.

Eficiencia del cultivo bacteriológico y de la inmunofluorescencia en la detección de Campylobacter fetus en fluidos genitales bovinos / Efficiency of bacteriological culture and the immunofluorescent assay to detect Campylobacter fetus in bovine genital fluids

La campilobacteriosis genital bovina es una enfermedad reproductiva que afecta la producción bovina. Es causada por las subespecies de Campylobacter fetus, C. fetus fetus (Cff) y C. fetus venerealis (Cfv). El objetivo de este estudio fue identificar la presencia de C. fetus en fluidos genitales mediante cultivo bacteriológico e inmunofluorescencia directa (IFD) y comparar los resultados. Se conformaron 2 grupos de 6 vaquillonas y 5 toros cada uno. Uno se infectó con Cff (grupo Cff) y el otro con Cfv (grupo Cfv). Dos vaquillonas y 2 toros sin infectar conformaron el grupo control. Periódicamente se tomaron muestras de mucus cervicovaginal y fluido prepucial, las que se procesaron por cultivo e IFD. En el grupo Cff se infectó el 100 % de las vaquillonas y el 80 % de los toros, mientras que en el grupo Cfv se infectó el 50 y el 60 %, respectivamente. Los valores de concordancia (Kappa) obtenidos al comparar las técnicas diagnósticas fueron de 0,57 para las vaquillonas del grupo Cff y 0,52 para las del grupo Cfv, y para los toros fueron de 0,17 y 0,27, respectivamente. En las vaquillonas, la IFD arrojó más resultados positivos que el cultivo, un 5,6 % más para el grupo Cff y un 7,4 % más para el grupo Cfv. El menor porcentaje de resultados positivos por IFD en los toros, un 40 % menos que por cultivo para el grupo Cff y un 5,3 % menos para el grupo Cfv, podría deberse a un muestreo incorrecto. Los valores de Kappa indican una concordancia moderada en las vaquillonas y baja en los toros.

Bovine genital campylobacteriosis is a reproductive disease that affects cattle production. It is caused by Campylobacter fetus subspecies, C. fetus fetus (Cff) and C. fetus venerealis (Cfv). The aim of this study was to identify the presence of C. fetus in genital fluids by bacteriological culture and direct immunofluorescence (DIF) and to compare the results. Two groups of 6 heifers and 5 bulls, one infected with Cff (Cff group) and the other with Cfv (Cfv group) were formed. Two heifers and 2 bulls, all of them uninfected, made up the control group. Samples of cervicovaginal mucus and preputial fluid were processed by culture and DIF. In the Cff group, 100 % of the heifers and 80 % of the bulls were infected, while in the Cfv group, 50 % of the heifers and 60 % of the bulls were infected. The degree of agreement (Kappa values) from benchmarking diagnostic techniques were 0.57 for heifers in the Cff group and 0.52 for heifers in the Cfv group, whereas the values for bulls were 0.17 and 0.27, respectively. Heifers yielded more positive results in the DIF assay than in the culture, exhibiting 5.6 % increase in the Cff group and 7.4 % in the Cfv group. The lowest percentage of positive results for DIF in bulls, 40 % less for the Cff group and 5.2 % for the Cfv group, could be due to improper sampling. Kappa values showed moderate agreement for the heifers and low for the bulls.

Molecular typing of Argentinian Mycobacterium avium subsp. paratuberculosis isolates by multiple-locus variable number-tandem repeat analysis

Multiple-locus variable number-tandem repeat analysis (MLVA) of Mycobacterium avium subspecies paratuberculosis (MAP) isolates may contribute to the knowledge of strain diversity in Argentina. Although the diversity of MAP has been previously investigated in Argentina using IS900-RFLP, a small number of isolates were employed, and a low discriminative power was reached. The aim of the present study was to test the genetic diversity among MAP isolates using an MLVA approach based on 8 repetitive loci. We studied 97 isolates from cattle, goat and sheep and could describe 7 different patterns: INMV1, INMV2, INMV11, INMV13, INMV16, INMV33 and one incomplete pattern. INMV1 and INMV2 were the most frequent patterns, grouping 76.3% of the isolates. We were also able to demonstrate the coexistence of genotypes in herds and co-infection at the organism level. This study shows that all the patterns described are common to those described in Europe, suggesting an epidemiological link between the continents.

Neosporosis epidémica y endémica: descripción de dos eventos en bovinos para cría / Endemic and epidemic bovine neosporosis: description of two events in beef cattle

El objetivo de este trabajo es describir dos eventos producidos en la provincia de Buenos Aires en los cuales Neospora caninum estuvo asociado a la ocurrencia de abortos en bovinos de cría para carne. En uno de ellos se registraron 11 abortos en 57 vaquillonas durante 45 días, en este evento fue 5 veces más probable que una vaquillona que sufrió un aborto fuera seropositiva a N. caninum que una que no lo sufrió (odds ratio [OR] = 4,9 IC 1,2­19,9) (p<0,05). En el otro evento se registraron 14 abortos en 140 vacas, y no se observó asociación significativa entre los abortos y la seropositividad frente a N. caninum OR = 0,69 (0,06­7,31) (p>0,05). Se analizaron dos fetos de cada evento: estos resultaron negativos a otros patógenos de la reproducción, aunque presentaron anticuerpos específicos y lesiones histopatológicas compatibles con infecciones por N. caninum. Estos resultados sugieren dos posibles modalidades de presentación de abortos en bovinos causados por N. caninum: una epidémica, como la del primer evento aquí referido, y una endémica, como la del segundo

The aim of this study was to describe two events in which Neospora caninum was involved in bovine abortions in beef cattle. In the first event, 11 abortions in 57 heifers were recorded in 45 days. One aborted heifer was 5 times more likely to be seropositive than a non-aborted heifer (OR=4.9; IC 1.2­19.9) (p<0.05). In the second event, no association between serological results and abortions were observed (OR= 0,69; 0,06­7,31) (p>0.05). Neither antibodies nor isolation of other pathogens were achieved in any case. On the contrary, antibodies and pathognomonic histopathological lesions were observed in the four fetuses from both cases. Interestingly, the findings in the first event suggest the epidemic behavior of the disease. In contrast, in the second event it appears that few abortions were due to N. caninum, suggesting the presence of endemic neosporosis

Genotyping Mycobacterium bovis from cattle in the Central Pampas of Argentina: temporal and regional trends

Mycobacterium bovis is the causative agent of bovine tuberculosis (TB), a disease that affects approximately 5% of Argentinean cattle. Among the molecular methods for genotyping, the most convenient are spoligotyping and variable number of tandem repeats (VNTR). A total of 378 samples from bovines with visible lesions consistent with TB were collected at slaughterhouses in three provinces, yielding 265 M. bovis spoligotyped isolates, which were distributed into 35 spoligotypes. In addition, 197 isolates were also typed by the VNTR method and 54 combined VNTR types were detected. There were 24 clusters and 27 orphan types. When both typing methods were combined, 98 spoligotypes and VNTR types were observed with 27 clusters and 71 orphan types. By performing a meta-analysis with previous spoligotyping results, we identified regional and temporal trends in the population structure of M. bovis. For SB0140, the most predominant spoligotype in Argentina, the prevalence percentage remained high during different periods, varying from 25.5-57.8% (1994-2011). By contrast, the second and third most prevalent spoligotypes exhibited important fluctuations. This study shows that there has been an expansion in ancestral lineages as demonstrated by spoligotyping. However, exact tandem repeat typing suggests dynamic changes in the clonal population of this microorganism.

Association between spoligotype-VNTR types and virulence of Mycobacterium bovis in cattle.

Mycobacterium bovis is the causative agent of bovine tuberculosis, a disease that affects approximately 5% of Argentine cattle. The aim of this research was to study if it is possible to infer the degree of virulence of different M. bovis genotypes based on scorified observations of tuberculosis lesions in cattle. In this study, we performed association analyses between several parameters with tuberculosis lesions: M. bovis genotype, degree of progression of tuberculosis, and animal age. For this purpose, the genotype was determined by spoligotyping and the degree of bovine tuberculosis gross lesion was quantified with a score based on clinical observations (number, size, and location of granulomas along with histopathologic features). This study was performed with naturally infected cattle of slaughterhouses from three provinces in Argentina. A total of 265 M. bovis isolates were obtained from 378 pathological lesion samples and 192 spoligotyping and VNTR (based on ETR sequences) typing patterns were obtained. SB0140 was the most predominant spoligotype, followed by SB0145. The spoligotype with the highest lesion score was SB0273 (median score of 27 ± 4.46), followed by SB0520 (18 ± 5.8). Furthermore, the most common spoligotype, SB0140, had a median score of 11 ± 0.74. Finally, the spoligotype with the lowest score was SB0145 (8 ± 1.0). ETR typing of SB0140, SB0145, SB0273, and SB0520 did not subdivide the lesion scores in those spoligotypes. In conclusion, SB0273 and SB0520 were the spoligotypes with the strongest association with hypervirulence and both spoligotypes were only found in Río Cuarto at the south of Córdoba province. Interestingly, there is no other report of any of these spoligotyes in Latin America.

Respuesta humoral y consecuencias reproductivas en ovejas desafiadas con Brucella ovis al final de la gestación / Immune response and reproductive consequences in experimentally infected ewes with Brucella ovis during late pregnancy

La brucelosis ovina por Brucella ovis es una enfermedad de prevalencia alta en Argentina. Para evaluar la patogenicidad de B. ovis y la respuesta serológica durante el último mes de gestación, 6 ovejas se distribuyeron en dos grupos: G1, ovejas preñadas, n = 4 y G2, ovejas no preñadas, n = 2. Tres ovejas del G1 (15 días preparto) y una del G2 fueron inoculadas con B. ovis. Se analizaron muestras de suero mediante diferentes pruebas serológicas. Se realizó aislamiento y PCR a partir de mucus cérvico-vaginal (mcv), placenta y leche. En las muestras de placenta se realizó histopatología. Las hembras del G1 parieron corderos vivos; se detectaron anticuerpos en las ovejas desafiadas del G1 a partir de los 5 días posinoculación. El mcv de las ovejas desafiadas resultó negativo al aislamiento en ambos grupos. Las muestras de leche del G1 fueron positivas por cultivo y PCR a B. ovis. La técnica de PCR resultó positiva en las placentas de las ovejas desafiadas del G1. La histopatología reveló una placentitis necrótica supurativa en una de las ovejas desafiadas. El desafío con B. ovis preparto resultó en la invasión de la placenta y de la glándula mamaria, con la consecuente excreción de la bacteria por leche. La infección con B. ovis indujo una respuesta humoral temprana en las ovejas. La colonización de la placenta por B. ovis y la excreción de la bacteria por la leche sugieren un potencial riesgo de infección activa para los corderos y la posibilidad de que estos se comporten como portadores latentes de la infección.

Ovine brucellosis by Brucella ovis is a highly prevalent disease in Argentina. This study aimed to evaluate the pathogenicity of B. ovis and the serological response in ewes during late pregnancy and in their offspring. Six adult ewes were distributed in two groupsGI (pregnant females, n = 4) and G2 (nonpregnant females, n = 2). Three pregnant ewes at 15 days prepartum and one nonpregnant eve were inoculated with B. ovis. Sera of sheep and their offspring were analyzed by different serological tests. Samples of cervicovaginal mucus, placenta and milk were studied by bacteriology. A Brucella genus-specific PCR assay was carried out in placenta and milk samples. Placenta samples were hystopathologically processed. G1 females gave birth to live lambs, but one died hours postpartum. Serological techniques employed detected antibodies in serum of inoculated pregnant animal 5 days postchallenge. Sera of female controls G1 and G2 remained negative throughout the study. Cervicovaginal mucus of infected ewes in G1 and G2 yielded negative results to bacteriology, but B. ovis was isolated from milk. The PCR assay was positive for the placenta and milk from inoculated pregnant ewes. Histopathology revealed necrotic suppurative placentitis in one placenta. However, although results demonstrated that B. ovis can invade the placenta and mammary gland, this bacterium did not cause abortion when it was inoculated intravenously at 15 days prepartum. B. ovis infection induced an early humoral response in pregnant ewes, but their lambs remained seronegative, indicating that there was no transfer of antibodies in infancy. Placenta colonization and milk excretion of B. ovis involves a potential source of infection for lambs, which could play a role as latent carriers of infection.

Bovine response to lipoarabinomannan vaccination and challenge with Mycobacterium paratuberculosis

This study aimed to evaluate the immune response in bovines following immunization with a mycobaterial Lipoarabinomannan extract (LAMe) and the effect of Map challenge. LAMe vaccine induced specific antibody levels that diminished after the challenge and affected Map excretion at least for 100 days thereafter.

Bovine paratuberculosis: a review of the advantages and disadvantages of different diagnostic tests.

Paratuberculosis (PTB), or Johne's disease, is a chronic infectious granulomatous enteritis of ruminants, caused by Mycobacterium avium subspecies paratuberculosis (Map). It is characterized by diarrhea and progressive cachexia, which may cause the death of the animal. Calves are the most susceptible to infection. Infected animals excrete Map mainly by the feces. PTB is endemic worldwide, with high prevalence levels, strong economic impact and public health relevance because of its possible association with Crohn's disease. Although the current reference diagnostic test is identification of Map in the bacterial culture, there are different diagnostic tests to identify infected individuals and/or herds. The sensitivity and specificity of these tests vary according to the stage of the disease in the animals to be evaluated. The correct choice and application of each of these diagnostic tests will ensure their success and may allow to establish a control program. The aim of this work is to review and discuss the different diagnostic tests used in the detection of Map-infected animals, focusing on their advantages and disadvantages.

First isolation of Mycobacterium avium subsp paratuberculosis from commercial pasteurized milk in Argentina

Mycobacterium avium subsp paratuberculosis was isolated from two out of seventy samples (2.86 %) of pasteurized and ultra-pasteurized milk. The isolates were positives to IS900 PCR and showed a C17 RFLP pattern, the most prevalent in Argentina. The present study is the first report of Mycobacterium avium subsp paratuberculosis culture from pasteurized milk in Argentina.