Genomic epidemiology of third-generation cephalosporin-resistant Escherichia coli from Argentinian pig and dairy farms reveals animal-specific patterns of co-resistance and resistance mechanisms.

Control measures are being introduced globally to reduce the prevalence of antibiotic resistance (ABR) in bacteria on farms. However, little is known about the current prevalence and molecular ecology of ABR in bacterial species with the potential to be key opportunistic human pathogens, such as Escherichia coli, on South American farms. Working with 30 dairy cattle farms and 40 pig farms across two provinces in central-eastern Argentina, we report a comprehensive genomic analysis of third-generation cephalosporin-resistant (3GC-R) E. coli, which were recovered from 34.8% (cattle) and 47.8% (pigs) of samples from fecally contaminated sites. Phylogenetic analysis revealed substantial diversity suggestive of long-term horizontal and vertical transmission of 3GC-R mechanisms. CTX-M-15 and CTX-M-2 were more often produced by isolates from dairy farms, while CTX-M-8 and CMY-2 and co-carriage of amoxicillin/clavulanate resistance and florfenicol resistance were more common in isolates from pig farms. This suggests different selective pressures for antibiotic use in these two animal types. We identified the ß-lactamase gene blaROB, which has previously only been reported in the family Pasteurellaceae, in 3GC-R E. coli. blaROB was found alongside a novel florfenicol resistance gene, ydhC, also mobilized from a pig pathogen as part of a new composite transposon. As the first comprehensive genomic survey of 3GC-R E. coli in Argentina, these data set a baseline from which to measure the effects of interventions aimed at reducing on-farm ABR and provide an opportunity to investigate the zoonotic transmission of resistant bacteria in this region. IMPORTANCE: Little is known about the ecology of critically important antibiotic resistance among bacteria with the potential to be opportunistic human pathogens (e.g., Escherichia coli) on South American farms. By studying 70 pig and dairy cattle farms in central-eastern Argentina, we identified that third-generation cephalosporin resistance (3GC-R) in E. coli was mediated by mechanisms seen more often in certain species and that 3GC-R pig E. coli were more likely to be co-resistant to florfenicol and amoxicillin/clavulanate. This suggests that on-farm antibiotic usage is key to selecting the types of E. coli present on these farms. 3GC-R E. coli and 3GC-R plasmids were diverse, suggestive of long-term circulation in this region. We identified the de novo mobilization of the resistance gene blaROB from pig pathogens into E. coli on a novel mobile genetic element, which shows the importance of surveying poorly studied regions for antibiotic resistance that might impact human health.

Lactococcus lactis subsp lactis CRL1655 and Schleiferilactobacillus perolens CRL1724 inhibit the adherence of common bovine mastitis pathogens to mammary gland cells, without causing histological changes in the mammary gland.

AIMS: The present work assessed the ability of two selected lactic acid bacteria (LAB) strains (Schleiferilactobacillus perolens CRL1724 and Lactococcus lactis subsp. lactis CRL1655) to inhibit the adherence of bovine mastitis pathogens to mammary epithelial cells (MAC-T) and their effects (if any) on the structure of the gland after intramammary inoculation at dry-off. METHODS AND RESULTS: Established bovine mammary epithelial cells (MAC-T) were used to assess the LAB strains' ability to inhibit the adherence of bovine mastitis pathogens. Monolayers of MAC-T cells were co-cultured with the LABs and then individual pathogen was added. Both strains prevented the adherence of S. aureus RC108, S. chromogenes, S. uberis UT102 and E. coli ATCC 35218. Adherence of the latter two pathogens was inhibited most strongly in vitro. To evaluate the effect of the LAB on the structure of the bovine udders, quarters were intramammary inoculated with the LAB mixture at dry-off. After slaughtering, the teats were dissected and histopathologically analysed. No modifications were identified post-inoculation in the structure of the epithelial, subepithelial and connective tissues of the mammary gland. CONCLUSIONS: Probiotic strains L. lactis subsp lactis CRL1655 and S. perolens CRL1724 were both able to inhibit the adherence of a number of bovine mastitis pathogens in vitro, and that the intramammary inoculation of these strains at the established dose and concentration did not cause significant alterations in the mammary epithelium nor had undesirable effects on tissues, and may therefore be considered harmless. SIGNIFICANCE AND IMPACT OF STUDY: The promising findings demonstrated in this work support the potential of probiotic micro-organisms as a natural and effective alternative to prevent bovine mastitis during the dry-off period.

[Identification of species of Mycoplasma and Ureaplasma diversum from Argentinian dairy herds]. / Identificación de especies de Mycoplasma y de Ureaplasma diversum en rodeos lecheros de Argentina.

Several species of Mycoplasma and Ureaplasma diversum can cause diseases in dairy cattle, which can be associated or not with clinical manifestations. In our country, the presence of Mycoplasma bovis, Mycoplasma californicum and Mycoplasma canadense has been detected, being the only mycoplasma species identified so far. The objective of this study was to identify other species of the Mycoplasmataceae family. Thirty-five Mycoplasma spp.-like isolates obtained from different samples from cattle, with or without clinical symptoms, from eight herds located in the provinces of Santa Fe, Cordoba, Buenos Aires and San Luis were utilized in the present study. Through the use of species-specific polymerase chain reactions (PCR) Mycoplasma bovigenitalium, Mycoplasma alkalescens, Mycoplasma bovirhinis and U. diversum were identified and through amplification and further sequencing of the 16-23S rRNA intergenic spacer regions, Mycoplasma arginine and M. californicum were identified. The identification of these species represents an important advance in knowledge in order to include these pathogens in the differential diagnosis of certain clinical and pathological entities of cattle from Argentina.

Detection of Mycoplasma canadense and Mycoplasma californicum in dairy cattle from Argentina.

Different species of Mycoplasma can affect bovine cattle, causing several diseases. PCR sequencing and further analysis of the 16S-23S rRNA ITS region have shown a significant interspecies variability among Mollicutes. Sixteen suspected isolates of Mycoplasma spp. obtained from milk samples from dairy herds were amplified (16S-23S rRNA ITS region). Fourteen out of those 16 suspected Mycoplasma spp. isolates were PCR-positive. To confirm the identity of Mycoplasma bovis, these 14 isolates were tested by another species-specific PCR. Seven of the isolates rendered a positive result. The products of 16S-23S rRNA ITS PCR from one isolate that was identified as M. bovis and from two other isolates, identified as non- M. bovis were randomly selected, sequenced and analyzed. The three sequences (A, B and C) showed 100% similarity with M. bovis, Mycoplasma canadense and Mycoplasma californicum respectively.

Detección de Mycoplasma canadense y Mycoplasma californicum en ganado lechero de Argentina / Detection of Mycoplasma canadense and Mycoplasma californicum in dairy cattle from Argentina

Diferentes especies del género Mycoplasma pueden afectar al ganado bovino y causar varias enfermedades. La técnica de PCR, secuenciación y posterior análisis de la región ITS 16S-23S ARNr ha mostrado que existe una importante variabilidad interespecies entre Mollicutes. Se realizó la amplificación (región ITS 16S-23S ARNr) de 16 aislamientos sospechosos de corresponder a alguna especie de Mycoplasma, que habían sido obtenidos de muestras de leche provenientes de rodeos lecheros. Catorce de esos aislamientos fueron PCR positivos. Para confirmar la identidad de Mycoplasma bovis, dichos aislamientos fueron evaluados por otra PCR especie-específica. Siete aislamientos dieron un resultado positivo. Los productos de la PCR de la ITS 16S-23S ARNr de un aislamiento identificado como M. bovis y de otros dos aislamientos identificados como no-M. bovis fueron seleccionados al azar, secuenciados y analizados. Las tres secuencias (A, B y C) mostraron 100 % de similitud con cepas de M. bovis, Mycoplasma canadense y Mycoplasma californicum, respectivamente

Different species of Mycoplasma can affect bovine cattle, causing several diseases. PCR sequencing and further analysis of the 16S-23S rRNA ITS region have shown a significant interspecies variability among Mollicutes. Sixteen suspected isolates of Mycoplasma spp. obtained from milk samples from dairy herds were amplified (16S-23S rRNA ITS region). Fourteen out of those 16 suspected Mycoplasma spp. isolates were PCR-positive. To confirm the identity of Mycoplasma bovis, these 14 isolates were tested by another species-specific PCR. Seven of the isolates rendered a positive result. The products of 16S-23S rRNA ITS PCR from one isolate that was identified as M. bovis and from two other isolates, identified as non- M. bovis were randomly selected, sequenced and analyzed. The three sequences (A, B and C) showed 100% similarity with M. bovis, Mycoplasma canadense and Mycoplasma californicum respectively

Detection of Mycoplasma canadense and Mycoplasma californicum in dairy cattle from Argentina. / Detection of Mycoplasma canadense and Mycoplasma californicum in dairy cattle from Argentina.

Different species of Mycoplasma can affect bovine cattle, causing several diseases. PCR sequencing and further analysis of the 16S-23S rRNA ITS region have shown a significant interspecies variability among Mollicutes. Sixteen suspected isolates of Mycoplasma spp. obtained from milk samples from dairy herds were amplified (16S-23S rRNA ITS region). Fourteen out of those 16 suspected Mycoplasma spp. isolates were PCR-positive. To confirm the identity of Mycoplasma bovis, these 14 isolates were tested by another species-specific PCR. Seven of the isolates rendered a positive result. The products of 16S-23S rRNA ITS PCR from one isolate that was identified as M. bovis and from two other isolates, identified as non- M. bovis were randomly selected, sequenced and analyzed. The three sequences (A, B and C) showed 100

similarity with M. bovis, Mycoplasma canadense and Mycoplasma californicum respectively.

Histological examination of non-lactating bovine udders inoculated with Lactobacillus perolens CRL 1724.

The effect of intramammary inoculation of Lactobacillus perolens CRL 1724 on bovine udders at drying off was evaluated through histological examination of the canal and cistern tissues. The persistence of the strain in the udder 7 d post inoculation was also determined. Lb. perolens CRL 1724 was recovered from all mammary quarters and no clinical signs or teat damage were observed after inoculation of 10(6) cfu/ml. The udders showed a normal structural aspect and there were no modifications of the milk appearance. Lb. perolens CRL 1724 cells were evidenced on the surface of the epithelial cells of the cistern without causing any morphological modifications or cell alterations. Lb. perolens CRL 1724 produces a mild inflammatory reaction, characterized by recruitment of neutrophils to the epithelial zone and a slight hyperaemia into blood vessels. This preliminary study provides important information for further studies directed towards the inclusion of Lb. perolens CRL 1724 in the design of probiotic products for preventing bovine mastitis in non-lactating dairy cows.

Effects of intramammary inoculation of Lactobacillus perolens CRL1724 in lactating cows' udders.

Bovine mastitis is the most important infectious disease on dairy farms. Conventional antibiotic therapy is often unsatisfactory and alternative treatments are continually under investigation. Lactobacillus (Lb.) perolens CRL 1724 and Lactobacillus plantarum CRL 1716 were previously isolated from milk of dairy cows and selected according to their potential probiotic properties. In the present work the in-vitro capacity of Lactobacillus strains to adhere to bovine teat canal epithelial cells (BTCEC) and to inhibit and co-aggregate 14 mastitis-causing pathogens (MCPs) was investigated. The effect of Lb. perolens CRL 1724 after intramammary inoculation in lactating cows was evaluated through determination of clinical signs of mastitis, milk appearance, somatic cell counts and Lb. perolens CRL 1724 recovery from milk. Lb. perolens CRL 1724 was able to inhibit 12 of 14 MCPs (85·7%) in vitro, especially those considered to be major pathogens. In addition, Lb. perolens CRL 1724 co-aggregated with all of them. Lb. plantarum CRL 1716 was able to inhibit 7 of 14 MCPs (50%) in vitro and showed co-aggregation ability similar to Lb. perolens CRL 1724. Lb. perolens CRL 1724 showed a higher efficacy of adhesion to BTCEC (values of percentage of adhesion and adhesion index of 75% and 14·4, respectively) than Lb. plantarum CRL 1716 (37% and 7·4, respectively). Lb. perolens CRL 1724 was recovered from all mammary quarters and no clinical signs or teat damage were observed after the inoculation of 106 cfu/ml. The udders presented a normal aspect and there were no changes in the appearance of the milk. The results obtained will serve as the basis for further trials to evaluate the potential of Lb. perolens CRL 1724 to be included in a non-antibiotic formulation for the prevention of bovine mastitis.

Efficacy of immunization against bovine mastitis using a Staphylococcus aureus avirulent mutant vaccine.

Bovine mastitis is responsible of major economic losses on dairy farms worldwide. In Argentine dairy herds, Staphylococcus aureus is the main causative agent of the disease. The ineffectiveness of some current practices to control S. aureus infections, often leads to a chronic and recurrent infection with persistent bacterial reservoir within a herd. Vaccination against S. aureus seems to be a rational approach for the control of the disease. In the present study, we investigate the response of dairy heifers after a combined immunization schedule with the avirulent mutant RC122 S. aureus vaccine. Vaccinated and non-vaccinated heifers were challenged 40 days after calving with the parental virulent strain. After challenge, and during the study period, milk bacterial recovery was significantly higher in non-vaccinated heifers than vaccinates. Importantly, inoculated bacteria could not be isolated from the milk of vaccinated heifers until 72 h after challenge, and the overall percentage of infected quarters in these animals was significantly lower. An increase in the level of specific IgG was observed in blood and milk of vaccinated heifers during the trial. At calving, IgG(2) was the main antibody isotype found in blood. Immune sera from vaccinated heifers increased phagocytosis over sera from non-vaccinated heifers and were able to opsonize heterologous S. aureus strains. Results demonstrated that immunization of dairy heifers with strain RC122 was able to elicit a significant opsonic antibody production in blood and milk and provides protection by a significant reduction in bacterial shedding after challenge.

Experimental trial in heifers vaccinated with Staphylococcus aureus avirulent mutant against bovine mastitis.

Staphylococcus aureus, is the most frequently isolated pathogen from cases of bovine mastitis. Vaccination against S. aureus seems to be a rational approach for the control of staphylococcal mastitis. In the present work we evaluate the response of heifers vaccinated with a S. aureus avirulent mutant to the intramammary challenge with a S. aureus virulent strain. Clinical signs, production of milk, shedding of S. aureus cells, somatic cell count (SCC) and antigen-specific IgG in blood and milk, were determined. Two subcutaneous doses of a culture of the mutant, used as vaccine, was administered to four pregnant heifers 30 and 10 days before calving. The vaccinated heifers and four non-vaccinated were challenged 10 days after calving with the homologous virulent S. aureus strain, which was inoculated by intramammary route into two quarters of each animal. No local tissue damage was observed due to the administration of the vaccine. A significantly increase of specific IgG to S. aureus RC122 was detected in blood and milk of vaccinate heifers as well as a slight increase in daily milk yield during the trial. No significant difference on shedding of bacteria in milk and SCC were found among groups. In conclusion, vaccination of heifers before calving by an avirulent mutant vaccine of S. aureus, induced specific and significant antibody responses and provide better post-challenge conditions in vaccinated heifers.

Bovine and rabbit models for the study of a Staphylococcus aureus avirulent mutant strain, RC122.

Staphylococcus aureus is the main etiological agent of bovine mastitis. Intramammary infections are difficult to cure and vaccination appears to be an alternative to prevent the disease. Research has focused on the development of mutants affected in the synthesis of pathogenicity determinants. We constructed a mutant strain (RC122) after chemical mutagenesis. In a mouse model, the strain was shown to be 1500 times less virulent, showed similar kinetics of disappearance in the kidney as its parental strain, and a good degree of protection against a challenge from homologous and heterologous strains. The objective of the present report was to study the avirulent RC122 S. aureus mutant strain in rabbit and bovine infection models. The results clearly show that RC122 was less virulent than its parental strain in a rabbit skin model, and was also correlated with its avirulence as an udder pathogen. These traits make the RC122 mutant strain interesting as a potential strain for an experimental vaccine trial in dairy herds.

Derechos en la tercera edad

La presente investigación es de tipo descriptiva. Se procedió a la revisión bibliográfica de la legislación vigente en nuestro país, donde se encontrasen derechos para la Tercera Edad.El objetivo de este estudiofue indagar sobre que tipo de derechos encontrariamos en la Legislación Nacional y para darle un caracter federal al enfoque adecuandonos a los principios que organizan una Nación Republicana Representativa y Federal, procedimos a extraer de las leyes, decretos y ordenanzas que rigen en nuestras respectivas provincias (tanto a nivel provincial como municipal) aquellas disposiciones que resulten relevantes en el estudio de la Tercera Edad y sus derechos. Observando que en las ultimas épocas, con los aportes del constitucionalismo social y las reformas de las constituciones provinciales, fueron incluidos artículos específicos sobre el derecho de la ancianidad, seguridad social, seguro social, regimen de salud.De la revisión efectuada se desprende la inegable existencia de plurales derechos dentro de nuestra Carta Magna, Codigo Civil y demás legislaciones vigente

Derechos en la tercera edad

La presente investigación es de tipo descriptiva. Se procedió a la revisión bibliográfica de la legislación vigente en nuestro país, donde se encontrasen derechos para la Tercera Edad.El objetivo de este estudiofue indagar sobre que tipo de derechos encontrariamos en la Legislación Nacional y para darle un caracter federal al enfoque adecuandonos a los principios que organizan una Nación Republicana Representativa y Federal, procedimos a extraer de las leyes, decretos y ordenanzas que rigen en nuestras respectivas provincias (tanto a nivel provincial como municipal) aquellas disposiciones que resulten relevantes en el estudio de la Tercera Edad y sus derechos. Observando que en las ultimas épocas, con los aportes del constitucionalismo social y las reformas de las constituciones provinciales, fueron incluidos artículos específicos sobre el derecho de la ancianidad, seguridad social, seguro social, regimen de salud.De la revisión efectuada se desprende la inegable existencia de plurales derechos dentro de nuestra Carta Magna, Codigo Civil y demás legislaciones vigente

Derechos en la tercera edad

La presente investigación es de tipo descriptiva. Se procedió a la revisión bibliográfica de la legislación vigente en nuestro país, donde se encontrasen derechos para la Tercera Edad.El objetivo de este estudiofue indagar sobre que tipo de derechos encontrariamos en la Legislación Nacional y para darle un caracter federal al enfoque adecuandonos a los principios que organizan una Nación Republicana Representativa y Federal, procedimos a extraer de las leyes, decretos y ordenanzas que rigen en nuestras respectivas provincias (tanto a nivel provincial como municipal) aquellas disposiciones que resulten relevantes en el estudio de la Tercera Edad y sus derechos. Observando que en las ultimas épocas, con los aportes del constitucionalismo social y las reformas de las constituciones provinciales, fueron incluidos artículos específicos sobre el derecho de la ancianidad, seguridad social, seguro social, regimen de salud.De la revisión efectuada se desprende la inegable existencia de plurales derechos dentro de nuestra Carta Magna, Codigo Civil y demás legislaciones vigente