Identificación y caracterización bacteriológica y molecular de cepas de Mycobacterium avium subsp. paratuberculosis en productos lácteos y agua.

Tesis de doctorado para obtener el grado de Doctor en Ciencia Animal presentada en la Facultad de Ciencias Veterinarias, Universidad Nacional del Centro de la Provincia de Buenos Aires, 2015La paratuberculosis (PTBC) o enfermedad de Johne es una enteritis crónica producida por Mycobacterium avium subsp. paratuberculosis (MAP) que afecta a bovinos y otras especies. MAP ha sido vinculado en humanos a una inflamación crónica del intestino, denominada enfermedad de Crohn. La leche y sus subproductos sería una de las posibles fuentes de infección y se ha sugerido que esta micobacteria podría resistir las condiciones de pasteurización. El objetivo del trabajo fue identificar MAP en alimentos lácteos crudos y/o procesados y en agua, mediante cultivo y reacción en cadena de la polimerasa (PCR) para la caracterización de las diferentes cepas aisladas. Se evaluaron diferentes protocolos para identificar MAP en leche cruda y comercial a través de cultivo y PCR y se determinó el límite de detección de ambas técnicas. Para la identificación de MAP en leche cruda, el protocolo de descontaminación de la muestra con menor límite de detección fue el método que utiliza ácido oxálico al 5% (p/v). Para la identificación de esta micobacteria a través de PCR, la extracción de ADN fue más eficiente utilizando el kit comercial evaluado y las perlas inmunomagnéticas. Se evaluó la supervivencia de MAP, E. coli, and S. Enteritidis durante la elaboración y almacenamiento tradicional de yogur. MAP no fue detectado durante la preparación del yogur, pero fue recuperado a los 180 min luego de la inoculación y durante el almacenamiento. Los recuentos de E. coli y S. Enteritidis aumentaron durante las primeras 24 hs, seguido de un ligero descenso hacia el final del estudio. Estos resultados demostrarían que MAP, E. coli, y S. Enteritidis resistirían las condiciones ácidas generadas durante la preparación de yogur y las bajas temperaturas del almacenamiento. También se evaluó la supervivencia de MAP en quesos blandos elaborados con leche bovina artificialmente inoculada con dos niveles diferentes de unidades formadoras de colonias (UFC) de MAP. Esta micobacteria no fue recuperada desde las muestras tomadas en las primeras etapas de la elaboración. Sin embargo, en los quesos elaborados con baja concentración de MAP, fue aislado hasta los 2 días de almacenamiento y en los inoculados con alta concertación de MAP, hasta los 15 días. Esto podría sugerir una adaptación de la bacteria al medio y posterior desarrollo. Se evaluaron 384 muestras de leche comercializadas en cuenca lechera Mar y Sierras de diferentes marcas comerciales, porcentaje de grasa y tratamientos térmicos. A cada una de las muestras se le realizó cultivo para identificación de MAP y otras micobacterias, recuento de bacterias aerobias mesófilas totales (BAMT) y el test de fosfatasa alcalina (FA) utilizando un kit v comercial. La totalidad de las muestras resultaron negativas al cultivo de micobacterias. Sin embargo, el 1,56% (6/384) de las muestras de leche comercial fueron positivas a PCR para identificación de MAP y el 40,1% (154/384) de las muestras resultaron positivas a la coloración Ziehl Neelsen realizada sobre el barrido de los cultivos negativos. Todas las muestras fueron negativas al test de FA y un 4,95% (19/384) de las muestras, el recuento de BAMT fue superior al límite que establece el Código Alimentario Argentino. Deberían ser evaluadas las medidas necesarias para evitar la transmisión de MAP y otras micobacterias dentro de establecimiento lecheros y enfatizarse las medidas de control en cuanto a higiene, cadena de frío, etc., tanto en industrias lácteas como en los puntos de venta para asegurar la calidad de los productos que llegan al consumidor.Paratuberculosis (PTBC) or Johne´s disease is Chronic Enteritis disease caused by Mycobacterium avium subsp. paratuberculosis (MAP) which affects cattle and other animal species. MAP has been linked to Chronic Inflammatory Bowel disease in humans, named Crohn's disease. Milk and byproducts are possible sources of infection to humans as it was suggested that this mycobacterium could resist pasteurization conditions. The aim of this work was to isolate MAP in water and raw or processed dairy products by bacteriological culture and polymerase chain reaction (PCR) followed by molecular characterization of different strains isolated. In order to define the minimum concentration limit for detection of MAP in raw and commercial milk different culture and PCR protocols were evaluated. For the identification of MAP in raw milk, the decontamination protocol with lower detection limit was achieved by using oxalic acid 5% (w/v). DNA extraction methods were evaluated using the commercial kit and the immunomagnetic beads for PCR. During the traditional preparation and storage of yogurt, the survival of MAP, Escherichia coli, and Salmonella Enteritidis was evaluated. MAP was not detected during the preparation of yogurt, but was recovered 180 min after inoculation and during storage. The live bacterial counts of E. coli and S. Enteritidis increased during the first 24 hours of refrigerated storage, followed by a slight decrease at the end of the study. These results have demonstrated that MAP, E. coli and S. Enteritidis resist the acidic conditions generated during the preparation of yogurt and low temperature storage. MAP survival in cream cheese made from bovine milk artificially inoculated with two different concentrations of MAP was also evaluated. Viable MAP was not recovered from early stages of processing. However, during refrigerated storage, the cheese inoculated with a low concentration of MAP, the bacterium was isolated until the second day of storage while the cheese inoculated with a high concentration of MAP, and it was isolated until the 15th day of storage. These results suggest an initial adaptation of the bacteria to the environment with followed by a growth phase. Furthermore, 384 samples of commercial milk from Mar y Sierras area, representing different commercial brands, fat percentage and thermal treatments were evaluated. These samples were examined for MAP and other mycobacterium identification, count of total aerobic mesophilic bacteria (TAMB) count and alkaline phosphatase (AP) test using a commercial kit. All samples resulted negative to mycobacterium culture. Despite that, 1.56% (6/384) of the samples were positive to IS900 PCR and 40.1% (154/384) of the samples were positive for the vii Ziehl-Neelsen. All the samples were negatives to AP test and 4.95% (19/384) had TAMB counts higher than the limit established by the Argentine Food Code. In order to ensure the quality of dairy products to consumers, the necessary measures should be assessed to prevent the transmission of MAP and other mycobacteria in dairy establishment. Furthermore, an emphasize should be give to general measures of control such as hygiene, cold chain, etc. in dairy industries and retail stores.EEA BalcarceFil: Cirone, Karina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentin

Detection of Mycobacterium avium subsp. paratuberculosis and other mycobacteria in retail milk and dairy products in Argentina

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of paratuberculosis or Johne’s disease (JD), a chronic enteritis disease that affects cattle and other animal species and has been linked to Crohn’s disease, a chronic inflammatory bowel disease in humans. MAP could resist pasteurization conditions and has been isolated from retail milk in several countries. The aims were to identify MAP and other mycobacteria in retail milk and dairy products in Argentina and to assess the product quality through Mesophilic Total Aerobic Bacteria (TAB) count. Three hundred and eighty-four samples of retail milk were tested for 24 months. All samples were negative for mycobacteria growth. However, 1.56% of the samples were positive for MAP identification by IS900-PCR. The TAB count was higher than the limits established by the Alimentary Argentinian Code (AAC) in 4.95% of the samples. Mycobacteria other than MAP were not detected either by culture or PCR. The MAP positive PCR from retail milk samples would indicate that they could come from dairy farms with JD and suggests that pasteurized milk or dairy products are not significant sources of human exposure to MAP in Argentina. Some milk samples exceeded the limits established by the AAC for TAB counts, indicating that commercialized milk could be processed and stored incorrectly.Mycobacterium avium subesp. paratuberculosis (MAP) es el agente etiológico de la paratuberculosis o la enfermedad de Johne (JD), una enteritis crónica que afecta al ganado vacuno y otras especies animales que se la vincula con la enfermedad de Crohn, una afección intestinal inflamatoria crónica en humanos. MAP podría resistir las condiciones de pasteurización y se lo aisló de la leche al por menor en varios países. Los objetivos fueron identificar MAP y otras micobacterias en leche y productos lácteos al por menor en Argentina y evaluar la calidad de estos a través del conteo de bacterias aerobias totales mesófilas (TAB). Se analizaron 384 muestras de leche de venta minorista durante 24 meses. Todas las muestras resultaron negativas en cuanto al crecimiento de micobacterias. Sin embargo, el 1.56% de las muestras dieron positivas en la identificación de MAP por IS900-PCR. El recuento de TAB superó los límites establecidos por el Código Alimentario Argentino (CAA) en el 4.95% de las muestras. No se detectaron micobacterias distintas de MAP ni por cultivo ni por PCR. La PCR positiva en MAP resultante de muestras de leche al por menor indicaría que podrían provenir de granjas lecheras con JD y sugiere que la leche pasteurizada o los productos lácteos no son fuentes significativas de exposición humana a MAP en Argentina. Algunas muestras de leche excedieron los límites establecidos por la AAC para los recuentos de TAB, lo cual indica que la leche comercializada podría procesarse y almacenarse incorrectamente.EEA BalcarceFil: Cirone, Karina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina.Fil: Cirone, Karina Mariela. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; ArgentinaFil: Morsella, Claudia Graciela. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina.Fil: Mendez, Laura Luján. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Paolicchi, Fernando. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina.Fil: Paolicchi, Fernando. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina

Evaluación por ELISA en leche para la detección de anticuerpos frente a Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (MAP) es el agente causal de la paratuberculosis (PTBC). Afecta a rumiantes domésticos y especies silvestres causando enteritis granulomatosa. La prueba más utilizada para su diagnóstico es el ELISA (Enzime-linked ImmunoSorbent Assay) debido a su bajo costo y rápido tiempo de procesamiento, aunque la sensibilidad depende del estadio clínico del animal.Trabajo publicado en Cagliada, Maria del Pilar Lilia y Galosi, Cecilia Mónica (comps.). I Congreso de Microbiología Veterinaria. Libro de resúmenes. La Plata: Facultad de Ciencias Veterinarias, 2021.Facultad de Ciencias Veterinaria

Calidad bacteriológica de aguas de pozo del sudeste bonaerense en distintas estaciones climáticas del año

El objetivo del presente trabajo fue determinar la calidad bacteriológica del agua subterránea para consumo humano de acuerdo a los límites establecidos por el CAA. Al mismo tiempo, se investigó la presencia de Micobacterias en distintas estaciones climáticas del año.EEA BalcarceFil: Perez Escalante, Joaquín. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias – Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Unidad Integrada Balcarce; ArgentinaFil: Andreoli, Yolanda Elina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce - Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias. Unidad Integrada Balcarce; ArgentinaFil: Puricelli, Marino Marcelo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce - Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias. Unidad Integrada Balcarce; ArgentinaFil: Castellari, Claudia Carla. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias – Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Unidad Integrada Balcarce; ArgentinaFil: Cirone, Karina Mariela. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce - Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias. Unidad Integrada Balcarce; ArgentinaFil: Valle, Facundo Marcos. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias – Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Unidad Integrada Balcarce; Argentin

Reactivation and foetal infection in pregnant heifers infected with neospora caninum live tachyzoites at prepubertal age

Neospora caninum is recognised for causing cattle abortion, provoking severe economic losses in the livestock industry worldwide. The aim of the present study was to evaluate the reactivation and foetal infection in pregnant heifers inoculated with live N. caninum tachyzoites before puberty. A total of 15 30-month-old pregnant heifers were allocated into four groups: animals inoculated with live tachyzoites of NC-Argentina LP1 isolate before puberty and challenged with live tachyzoites of NC-1 strain at 210 days of gestation (DG) (Group A); animals mock inoculated before puberty and challenged with NC-1 strain at 210 DG (Group B), animals inoculated before puberty but not subsequently challenged (Group C); and noninfected and nonchallenged animals (Group D). The results of this study showed that 100% of animals infected before puberty (Groups A and C) suffered reactivation of the infection at the seventh month of gestation. In addition, in three and two calves from Groups A and C, respectively, congenital infection was confirmed. Interestingly, we provide evidence that the use of live N. caninum tachyzoites in young animals as a strategy to induce protection is neither safe nor effective.EEA BalcarceFil: Hecker, Yanina Paola. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina.Fil: Burucúa, Mercedes. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina.Fil: Fiorani, Franco. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina.Fil: Fiorani, Franco. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina.Fil: Maldonado Rivera, Jaime. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina.Fil: Maldonado Rivera, Jaime. Universidad de Cuenca. Facultad de Ciencias Agropecuarias; Ecuador.Fil: Cirone, Karina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina.Fil: Cirone, Karina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina.Fil: Dorsch, Matías. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina.Fil: Dorsch, Matías. Instituto Nacional de Investigación Agropecuaria (INIA). Estación Experimental La Estanzuela; UruguayFil: Cheuquepán Valenzuela, Felipe. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina.Fil: Campero, Lucía. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina.Fil: Cantón, Germán José. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina.Fil: Marín, Maia. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina.Fil: Ortega Mora, Luis. Universidad Complutense de Madrid. Facultad de Ciencias Veterinarias; EspañaFil: Moore, Dadin Prando. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina

Host innate immune responses and microbiome profile of neonatal calves challenged with Cryptosporidium parvum and the effect of bovine colostrum supplementation

IntroductionCalves are highly susceptible to gastrointestinal infection with Cryptosporidium parvum (C. parvum), which can result in watery diarrhea and eventually death or impaired development. With little to no effective therapeutics, understanding the host’s microbiota and pathogen interaction at the mucosal immune system has been critical to identify and test novel control strategies.MethodsHerein, we used an experimental model of C. parvum challenge in neonatal calves to describe the clinical signs and histological and proteomic profiling of the mucosal innate immunity and microbiota shifts by metagenomics in the ileum and colon during cryptosporidiosis. Also, we investigated the impact of supplemental colostrum feeding on C. parvum infection.ResultsWe showed that C. parvum challenged calves experienced clinical signs including pyrexia and diarrhea 5 days post challenge. These calves showed ulcerative neutrophil ileitis with a proteomic signature driven by inflammatory effectors, including reactive oxygen species and myeloperoxidases. Colitis was also noticed with an aggravated mucin barrier depletion and incompletely filled goblet cells. The C. parvum challenged calves also displayed a pronounced dysbiosis with a high prevalence of Clostridium species (spp.) and number of exotoxins, adherence factors, and secretion systems related to Clostridium spp. and other enteropathogens, including Campylobacter spp., Escherichia sp., Shigella spp., and Listeria spp. Daily supplementation with a high-quality bovine colostrum product mitigated some of the clinical signs and modulated the gut immune response and concomitant microbiota to a pattern more similar to that of healthy unchallenged calves.DiscussionC. parvum infection in neonatal calves provoked severe diarrheic neutrophilic enterocolitis, perhaps augmented due to the lack of fully developed innate gut defenses. Colostrum supplementation showed limited effect mitigating diarrhea but demonstrated some clinical alleviation and specific modulatory influence on host gut immune responses and concomitant microbiota

Hyperoxemia and excess oxygen use in early acute respiratory distress syndrome : Insights from the LUNG SAFE study

Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Concerns exist regarding the prevalence and impact of unnecessary oxygen use in patients with acute respiratory distress syndrome (ARDS). We examined this issue in patients with ARDS enrolled in the Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) study. Methods: In this secondary analysis of the LUNG SAFE study, we wished to determine the prevalence and the outcomes associated with hyperoxemia on day 1, sustained hyperoxemia, and excessive oxygen use in patients with early ARDS. Patients who fulfilled criteria of ARDS on day 1 and day 2 of acute hypoxemic respiratory failure were categorized based on the presence of hyperoxemia (PaO2 > 100 mmHg) on day 1, sustained (i.e., present on day 1 and day 2) hyperoxemia, or excessive oxygen use (FIO2 ≥ 0.60 during hyperoxemia). Results: Of 2005 patients that met the inclusion criteria, 131 (6.5%) were hypoxemic (PaO2 < 55 mmHg), 607 (30%) had hyperoxemia on day 1, and 250 (12%) had sustained hyperoxemia. Excess FIO2 use occurred in 400 (66%) out of 607 patients with hyperoxemia. Excess FIO2 use decreased from day 1 to day 2 of ARDS, with most hyperoxemic patients on day 2 receiving relatively low FIO2. Multivariate analyses found no independent relationship between day 1 hyperoxemia, sustained hyperoxemia, or excess FIO2 use and adverse clinical outcomes. Mortality was 42% in patients with excess FIO2 use, compared to 39% in a propensity-matched sample of normoxemic (PaO2 55-100 mmHg) patients (P = 0.47). Conclusions: Hyperoxemia and excess oxygen use are both prevalent in early ARDS but are most often non-sustained. No relationship was found between hyperoxemia or excessive oxygen use and patient outcome in this cohort. Trial registration: LUNG-SAFE is registered with ClinicalTrials.gov, NCT02010073publishersversionPeer reviewe

Control of paratuberculosis: who, why and how. A review of 48 countries

Paratuberculosis, a chronic disease affecting ruminant livestock, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). It has direct and indirect economic costs, impacts animal welfare and arouses public health concerns. In a survey of 48 countries we found paratuberculosis to be very common in livestock. In about half the countries more than 20% of herds and flocks were infected with MAP. Most countries had large ruminant populations (millions), several types of farmed ruminants, multiple husbandry systems and tens of thousands of individual farms, creating challenges for disease control. In addition, numerous species of free-living wildlife were infected. Paratuberculosis was notifiable in most countries, but formal control programs were present in only 22 countries. Generally, these were the more highly developed countries with advanced veterinary services. Of the countries without a formal control program for paratuberculosis, 76% were in South and Central America, Asia and Africa while 20% were in Europe. Control programs were justified most commonly on animal health grounds, but protecting market access and public health were other factors. Prevalence reduction was the major objective in most countries, but Norway and Sweden aimed to eradicate the disease, so surveillance and response were their major objectives. Government funding was involved in about two thirds of countries, but operations tended to be funded by farmers and their organizations and not by government alone. The majority of countries (60%) had voluntary control programs. Generally, programs were supported by incentives for joining, financial compensation and/or penalties for non-participation. Performance indicators, structure, leadership, practices and tools used in control programs are also presented. Securing funding for long-term control activities was a widespread problem. Control programs were reported to be successful in 16 (73%) of the 22 countries. Recommendations are made for future control programs, including a primary goal of establishing an international code for paratuberculosis, leading to universal acknowledgment of the principles and methods of control in relation to endemic and transboundary disease. An holistic approach across all ruminant livestock industries and long-term commitment is required for control of paratuberculosis

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

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