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El presente trabajo pretende ampliar los conocimientos de los aceites esenciales como antibióticos, se recolectaron 140 muestras de caninos con enfermedad periodontal y se determinó in vitro el efecto antimicrobiano de los aceites esenciales mediante la utilización de pruebas de sensibilidad. The present work aims to expand the knowledge of essential oils as antibiotics, 140 samples of canines with periodontal disease were collected and the antimicrobial effect of essential oils was determined in vitro by using sensitivity tests.

Esta investigación se realizó en San Gerardo para determinar la prevalencia de Brucella abortus, se tomó sangre de 184 bovinos lecheros, el resultado se obtuvo con la técnica de ELISA indirecto resultando una prevalencia de 7.07% de animales positivos. This research was carried out in San Gerardo to determine the prevalence of Brucella abortus, blood was taken from 184 dairy cattle, the result was obtained with the indirect ELISA technique resulting in a prevalence of 7.07% of positive animals.

En el siguiente documento encontramos un trabajo que se dedicó a calcular la prevalencia de muermo “Burkholderia mallei” en equinos en el cantón Cuenca y sus alrededores. Los resultados fueron una prevalencia del 0% y un caso negativo de alta reactividad, lo que sugiere una sospecha sin embargo se deberá probar con una muestra más grande. In the following document we find a work that was dedicated to calculating the prevalence of glanders "Burkholderia mallei" in equines in the canton of Cuenca and its surroundings. The results were a prevalence of 0% and a negative case of high reactivity, which suggests a suspicion, however, it should be tested with a larger sample.

El presente trabajo experimental tiene el objetivo de determinar la eficiencia de métodos tradicionales de extracción de ADN de bacterias gram negativo (Escherichia coli y Klebsiella pneumoniae) mediante la comparación con un kit comercial. The aim of this experimental work is to determine the efficiency of traditional methods of DNA extraction of gram-negative bacteria (Escherichia coli and Klebsiella pneumoniae) by comparison with a commercial kit.

Se analizó la eficiencia de las cinéticas de inactivación a las bacterias Escherichia coli ATCC 8739 y Enterococcus sp, mediante pruebas repetibles y comparables, así como la cinética de fotoreactivación y su relación con la dosis en un sistema UV, se encontró que la turbidez tiene gran influencia, mientras mayor sea, menor grado de eficiencia hay. The efficiency of the inactivation kinetics of Escherichia coli ATCC 8739 and Enterococcus sp bacteria was analyzed through repeatable and comparable tests, as well as the photoreactivation kinetics and its relationship with the dose in a UV system, it was found that turbidity has great influence, the greater it is, the lower degree of efficiency there is.

The increasing prevalence of antibiotic resistance genes (ARGs) in animal manure has attracted considerable attention because of their potential contribution to the development of multidrug resistance worldwide. Insect technology may be a promising alternative for the rapid attenuation of ARGs in manure; however, the underlying mechanism remains unclear. This study aimed to evaluate the effects of black soldier fly (BSF, Hermetia illucens [L.]) larvae conversion combined with composting on ARGs dynamics in swine manure and to uncover the mechanisms through metagenomic analysis. Compared to natural composting (i.e. without BSF), BSFL conversion combined with composting reduced the absolute abundance of ARGs by 93.2 % within 28 days. The rapid degradation of antibiotics and nutrient reformulation during BSFL conversion combined with composting indirectly altered manure bacterial communities, resulting in a lower abundance and richness of ARGs. The number of main antibiotic-resistant bacteria (e.g., Prevotella, Ruminococcus) decreased by 74.9 %, while their potential antagonistic bacteria (e.g., Bacillus, Pseudomonas) increased by 128.7 %. The number of antibiotic-resistant pathogenic bacteria (e.g., Selenomonas, Paenalcaligenes) decreased by 88.3 %, and the average number of ARGs carried by each human pathogenic bacterial genus declined by 55.8 %. BSF larvae gut microbiota (e.g., Clostridium butyricum, C. bornimense) could help reduce the risk of multidrug-resistant pathogens. These results provide insight into a novel approach to mitigate multidrug resistance from the animal industry in the environment by using insect technology combined with composting, in particular in light of the global “One Health” requirements. This work was funded by the National Natural Science Foundation of China (41977279), Fundamental Research Funds for the Central Universities (2662020SKPY002 and 2662022SKYJ006), Open Funding Project of the State Key Laboratory of Biocatalysis and Enzyme Engineering (SKLBEE2020013), and Key Technology R&D Program of Hubei Province (2021BBA258).

La presente investigación se enfocó en identificar la prevalencia de Anaplasma marginale en ganado bovino mediante el método ELISA competitivo, analizando un total de 188 muestras considerando edad, sexo y procedencia, dando un 53% (100/188) de muestras positivas para Anaplasma marginale; mientras que el 47% (88/188) fueron negativas. The present investigation focused on identifying the prevalence of Anaplasma marginale in cattle through the competitive ELISA method, analyzing a total of 188 samples considering age, sex and origin, giving 53% (100/188) of positive samples for Anaplasma marginale; while 47% (88/188) were negative.

Este artículo se encuentra disponible en la siguiente URL: https://onlinelibrary.wiley.com/doi/epdf/10.1111/tbed.13737 En este artículo de investigación también participan: Marta Cerdà Cuéllar, Teresa Ayats, Jaume Jordá, Clara Marín y Santiago Vega. This is the pre-peer reviewed version of the following article: Sevilla-Navarro, S., Cerdà-Cuéllar, M., Ayats, T., Jordá, J., Marin, C., & Vega, S. (2021). Characterization of "Salmonella Frintrop" isolated from dromedary camels (Camelus dromedarius). Transboundary and Emerging Diseases, vol. 68, i. 2 (mar.), pp. 742?746, which has been published in final form at https://doi.org/10.1111/tbed.13737. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. Este es el pre-print del siguiente artículo: Sevilla-Navarro, S., Cerdà-Cuéllar, M., Ayats, T., Jordá, J., Marin, C., & Vega, S. (2021). Characterization of "Salmonella Frintrop" isolated from dromedary camels (Camelus dromedarius). Transboundary and Emerging Diseases, vol. 68, i. 2 (mar.), pp. 742?746, que se ha publicado de forma definitiva en https://doi.org/10.1111/tbed.13737. Different studies have reported the prevalence and antibiotic resistance of Salmonella in dromedaries’ camels and its role in camelid-associated salmonellosis in humans, but little is known about the epidemiology of Campylobacter in dromedaries. Here we investigate the prevalence, genetic diversity and antibiotic resistance of Campylobacter and Salmonella in dromedary camels (Camelus dromedarius). A total of 54 individuals were sampled from two unique dromedary farms located in Tenerife (Canary Islands, Spain). Whilst all the samples were Campylobacter-negative, Salmonella prevalence was 5.5% (3/54) and the only serovar isolated was S. Frintrop. The pulsed field gel electrophoresis analysis revealed a low genetic diversity, with all isolates showing a nearly identical pulsotype (similarity > 95%). Our results indicate that dromedaries’ camels could not be a risk factor for Campylobacter human infection, but seems to be a reservoir for Salmonella transmission. Since camel ride has become one of the main touristic attractions in several countries and its popularity has considerably risen in the last years, a mandatory control, especially for zoonotic pathogens, such as Campylobacter and Salmonella should be implemented. Preprint

La investigación se realizó en la parroquia de Cumbe, tuvo como objetivo determinar la prevalencia de Enterobacterias en cobayos mediante diagnóstico microbiológico. Las muestras fueron sembradas en Agares diferenciales y selectivos. Se caracterizó mediante tinción de Gram y se tipificó mediante pruebas bioquímicas. The research was carried out in the parish of Cumbe, its objective was to determine the prevalence of Enterobacteria in guinea pigs through microbiological diagnosis. The samples were seeded in differential and selective agars. It was characterized by Gram stain and typified by biochemical tests.

Este artículo se encuentra disponible en la siguiente URL: https://www.mdpi.com/2076-2615/12/16/2065 Este artículo de investigación pertenece a la colección "Current Advances in Poultry Research". Campylobacter is recognised as one of the most important foodborne bacteria, with a worldwide health and socioeconomic impact. This bacterium is one of the most important zoonotic players in poultry, where efficient and fast detection methods are required. Current official culture methods for Campylobacter enumeration in poultry usually include >44 h of culture and >72 h for identification, thus requiring at least five working shifts (ISO/TS 10272-2:2017). Here, we have assembled a portable sequencing kit composed of the Bento Lab and the MinION and developed a workflow for on-site farm use that is able to detect and report the presence of Campylobacter from caecal samples in less than five hours from sampling time, as well as the relationship of Campylobacter with other caecal microbes. Beyond that, our workflow may offer a cost-effective and practical method of microbiologically monitoring poultry at the farm. These results would demonstrate the possibility of carrying out rapid on-site screening to monitor the health status of the poultry farm/flock during the production chain.

En el presente trabajo de investigación se seleccionó a 200 caninos entre hembras y machos para un muestreo en sangre evaluando la prevalencia de Brucela spp. El muestreo fue realizado en una zona ganadera como es la parroquia Chorocopte de la ciudad de Cañar. Donde se realizó una prueba de laboratorio (ELISA de bloqueo) en el suero sanguíneo analizando los resultados mediante estadística descriptiva y representando lo obtenido en gráficos. In this research work, 200 pathways between females and males were selected for blood sampling evaluating brucella spp.The sampling was carried out in a livestock area of Chorocopte parish in the city of Cañar. Where a laboratory test (blockage ELISA) was performed on the blood serum analyzing the results using descriptive statistics and represented what was obtained in graphs.

La investigación se realizó para medir antígenos para Coxiella Burnetii, de un total de 112 sujetos estudiados, para ello se empleó punción directa y luego se procedió a centrifugar las muestras para la obtención del plasma sanguíneo. Se realizó pools de 10 pacientes cada uno, las muestras fueron analizadas mediante ELISA indirecto multiespecies. The research was carried out to measure antigens for Coxiella Burnetii, out of a total of 112 subjects studied, for this direct puncture was used and then the samples were centrifuged to obtain blood plasma. Pools of 10 patients each were made, the samples were analyzed by means of indirect multispecies ELISA.

En Ecuador hay un déficit sanitario insuficiente que requiere generar conocimiento específico de patógenos zoonóticos, entre estos la fiebre Q que en cobayos no se han realizado estudios de manera específica, se analizó con ELISA indirecto la prevalencia en Paute, para detectar anticuerpos en suero, con esto se llegó a una prevalencia de 0% indicando que los cobayos del experimento no tuvieron fiebre Q. Ecuador there is an insufficient sanitary deficit that requires generating specific knowledge of zoonotic pathogens, among them Q fever, which in guinea pigs have not specifically conducted studies, the prevalence in pattern was analyzed with indirect ELISA, to detect antibodies in serum, with This reached a prevalence of 0% indicating that the guinea pigs in the experiment did not have Q fever.

Bovine respiratory disease (BRD) is an important viral and/or bacterial disease that mainly affects feedlot calves. The involvement of Mycoplasma bovis in BRD can lead to chronic pneumonia poorly responsive to antimicrobial treatment. Caseonecrotic bronchopneumonia is a pulmonary lesion typically associated with M. bovis. In Spain, M. bovis is widely distributed in the feedlots and circulating isolates are resistant to most antimicrobials in vitro. However, the role of this species in clinical respiratory disease of feedlot calves remains unknown. Furthermore, available data are relative to a fixed panel of antimicrobials commonly used to treat BRD, but not to the specific set of antimicrobials that have been used for treating each animal. This study examined 23 feedlot calves raised in southeast Spain (2016–2019) with clinical signs of respiratory disease unresponsive to treatment. The presence of M. bovis was investigated through bacteriology (culture and subsequent PCR), histopathology and immunohistochemistry. The pathogen was found in 86.9% (20/23) of the calves, mainly in the lungs (78.26%, 18/23). Immunohistochemistry revealed M. bovis antigens in 73.9% (17/23) of the calves in which caseonecrotic bronchopneumonia was the most frequent lesion (16/17). Minimum inhibitory concentration assays confirmed the resistance of a selection of 12 isolates to most of the antimicrobials specifically used for treating the animals in vivo. These results stress the importance of M. bovis in the BRD affecting feedlot calves in Spain.

Esta tesis doctoral pertenece al Programa en Tecnología y Ciencias de la Salud de CEINDO - CEU Escuela Internacional de Doctorado. Esta tesis doctoral está basada en cuatro trabajos de investigación: 1. Characterization of livestock-associated Methicillin-resistant "Staphylococcus aureus" isolates obtained from commercial rabbitries. 2. Characterization of Methicillin-resistant "Staphylococcus aureus" in wild Iagomorphs located in high density areas. 3. Phenotypic and genotypic study of the SCCmec cassette element in MRSA and MSSA strains isolated from rabbits. 4. Effect of selection by growth rate and vitrification of embryos on the rabbit (Oryctolagus cuniculus) immune system and its response after a "Staphylococcus aureus" experimental infection. Tesis - Universidad CEU Cardenal Herrera (Valencia), Facultad de Veterinaria, Departamento de Producción y Sanidad Animal, Salud Pública Veterinaria y Ciencia y Tecnología de los Alimentos , leída el 26-11-2021. En este trabajo, se ha estudiado el patógeno Staphylococcus aureus con especial atención a las cepas resistentes a meticilina. Para ello, se ha evaluado la presencia de S. aureus en granjas de conejos y en conejos silvestres. A continuación, se ha evaluado un hospedador frecuente de este patógeno, el conejo (Oryctolagus cuniculus). En primer lugar, se encontró una cantidad inesperada de cepas de MRSA de lesiones poco frecuentes conejos de granjas. Además, es la primera vez que se han descrito cepas mecC-MRSA aisladas de granjas de conejos. En segundo lugar, se detectó un alto porcentaje de portadores de S. aureus en conejos y liebres silvestres, siendo la oreja el principal nicho ecológico. Tras la secuenciación del elemento genético móvil SCCmec, dos tipos no descritos anteriormente fueron encontrados: uno que contiene el gen mecC y otro que contiene simultáneamente los genes mecA y mecC. Dos de estas cepas tenían además el SCCmec escindido del genoma. Finalmente, se observó que la selección genética por ganancia diaria promedio no afectó la capacidad de las hembras de conejos de generar una respuesta inmune y además favoreció la capacidad de respuesta del sistema inmunológico cuando éste se enfrenta a un desafío infeccioso con S. aureus.

La brucelosis es una enfermedad zoonótica, que no la toman mucho en consideración, en lo que en este trabajo de investigación se le ha considerado en cobayos; en el que el tipo de alimentación es herbívora, debido que los pastos son compartidos con otras especies que son portadoras de la enfermedad; se procedió en esta investigación a determinar anticuerpos presentes y la prevalencia de la enfermedad en esta especie. Brucellosis is a zoonotic disease, which is not taken much into consideration, in what in this research work it has been considered in guinea pigs; in which the type of feeding is herbivorous, because the grasses are shared with other species that are carriers of the disease; This investigation proceeded to determine the antibodies present and the prevalence of the disease in this species.

Simple Summary Knowing which bacteria dominate vaginal microbiota and its variation throughout the cycle is important to study how to prevent reproductive diseases. In women, vaginal microbiota is dominated by Lactobacillus but this does not happen in other animals. Little is known about equine vaginal microbiota. The aim of this study was to describe the dynamics of equine vaginal microbiota during the ovarian cycle. Eight healthy adult Arabian mares were used to characterize vaginal microbiota by standard microbiologic and metagenomic procedures. The abundance of Lactobacillus was < 2% by both methods, meaning that equine vaginal microbiota was not dominated by these bacteria. Dominant bacteria included other genera such as Porphyromonas and Campylobacter among others. No changes in vaginal microbiota composition were found, suggesting that equine vaginal microbiota was stable throughout the ovarian cycle. Abstract Lactic acid bacteria (LAB) dominate human vaginal microbiota and inhibit pathogen proliferation. In other mammals, LAB do not dominate vaginal microbiota, however shifts of dominant microorganisms occur during ovarian cycle. The study objectives were to characterize equine vaginal microbiota in mares by culture-dependent and independent methods and to describe its variation in estrus and diestrus. Vaginal swabs from 8 healthy adult Arabian mares were obtained in estrus and diestrus. For culture-dependent processing, bacteria were isolated on Columbia blood agar (BA) and Man Rogosa Sharpe (MRS) agar. LAB comprised only 2% of total bacterial isolates and were not related to ovarian phases. For culture-independent processing, V3/V4 variable regions of the 16S ribosomal RNA gene were amplified and sequenced using Illumina Miseq. The diversity and composition of the vaginal microbiota did not change during the estrous cycle. Core equine vaginal microbiome consisted of Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria at the phylum level. At the genus level it was defined by Porphyromonas, Campylobacter, Arcanobacterium, Corynebacterium, Streptococcus, Fusobacterium, uncultured Kiritimatiaellae and Akkermansia. Lactobacillus comprised only 0.18% of the taxonomic composition in estrus and 0.37% in diestrus. No differences in the relative abundance of the most abundant phylum or genera were observed between estrus and diestrus samples.

Simple Summary There is a lack of knowledge about the impact that phages present in the environment may have against certain Salmonella serovars. Thus, an improved understanding of Salmonella phage diversity will provide a better insight into the role of phages in Salmonella ecology and diversity. The results of this study showed that the poultry farm environment could represent a valuable source of Salmonella phages, which are more varied in broiler than in layer farms. Abstract The exploration of novel nonantibiotic interventions in the field, such as the use of bacteriophages, is necessary to avoid the presence of Salmonella. Bacteriophages are a group of viruses widely distributed in nature, strictly associated with the prokaryotic cell. Researchers have demonstrated the success of phage therapy in reducing Salmonella counts in poultry products. However, the impact that phage concentration in the environment may have against certain Salmonella serovars is not well understood. Therefore, the aim of this study was to assess Salmonella phage prevalence in commercial poultry farms in terms of the production type: layers or broilers. The most prevalent Salmonella serovars isolated in poultry production were used for phage isolation. Salmonella specific phages were isolated from 141 layer and broiler farms located in the Valencia region during 2019. Analysis of the samples revealed that 100% presented Salmonella phages, the most prevalent being against serovar S. Enteritidis (93%), followed by S. Virchow (59%), S. Typhimurium (55%), S. Infantis (52%) and S. Ohio (51%). These results indicate that poultry farms could represent an important source of Salmonella phages. Nevertheless, further studies are needed to assess the epidemiology of phages against other serovars present in other countries and their diversity from the point of view of molecular studies.

Submitted by Ana Cláudia Calchi (ana.calchi@unesp.br) on 2020-08-09T17:46:32Z No. of bitstreams: 1 Dissertação_Ana Claudia Calchi.pdf: 3933150 bytes, checksum: de4445fe3d0f3a08d00016e695975f68 (MD5) Approved for entry into archive by Adriana Helena Vitta Zoratti (adriana.zoratti@unesp.br) on 2020-08-10T17:23:08Z (GMT) No. of bitstreams: 1 calchi_ac_me_jabo.pdf: 3933150 bytes, checksum: de4445fe3d0f3a08d00016e695975f68 (MD5) Made available in DSpace on 2020-08-10T17:23:08Z (GMT). No. of bitstreams: 1 calchi_ac_me_jabo.pdf: 3933150 bytes, checksum: de4445fe3d0f3a08d00016e695975f68 (MD5) Previous issue date: 2020-07-08 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Os agentes Anaplasmataceae são α-proteobactérias Gram-negativos intracelulares obrigatórias que são transmitidos principalmente por vetores artrópodes. Embora os mamíferos da Superordem Xenarthra (preguiças, tamanduás e tatus) tenham sido implicados como reservatórios de vários agentes zoonóticos, apenas poucos estudos procuraram detectar agentes Anaplasmataceae neste grupo de mamíferos. Este estudo teve por objetivo investigar a ocorrência e diversidade genética de Anaplasma spp. e Ehrlichia spp. em amostras de sangue e baço de Xenarthra de vida livre de quatro estados no Brasil (São Paulo, Mato Grosso do Sul, Rondônia, e Pará). Foram efetuados ensaios de triagem por meio de nPCR e cPCR para detectar os genes rrs e dsb de Anaplasma spp. e Ehrlichia spp., respectivamente. Os ensaios foram positivos em 27,57% (91/330) para Anaplasma spp. e 24,54% (81/330) para Ehrlichia spp. Das 91 amostras positivas para Anaplasma spp., 56,04% foram positivas num ensaio de PCR convencional visando a região intergênica 23S-5S. Análises filogenéticas e de distância baseadas no gene rrs alocaram as sequências de Anaplasma de preguiças (Bradypus variegatus, Bradypus sp., Choloepus spp. e C. didactylus) capturadas nos estados de Rondônia e Pará em um único clado, que estava estreitamente relacionado com o clado de A. marginale, A. ovis, e A. capra. As sequências detectadas nos tamanduás-mirins (Tamandua tetradactyla) de São Paulo foram alocadas em um clado filogeneticamente relacionado com as sequências de Anaplasma spp. detectadas em Nasua nasua, Leopardus pardalis, e Cerdocyon thous no Brasil. Estas sequências foram posicionadas perto das sequências de A. odocoilei. A análise do genótipo corroborou as descobertas anteriores e demonstrou a circulação de dois genótipos distintos de Anaplasma em animais do norte e sudeste do Brasil. O primeiro genótipo era novo. O segundo foi previamente detectado em N. nasua, no estado de Mato Grosso do Sul. As análises da região intergênica também demonstraram dois genótipos distintos de Anaplasma. As sequências detectadas em Xenarthra dos estados do Pará e Rondônia estavam relacionadas com as de A. marginale, A. ovis, e A. capra. Enquanto que as sequências de Anaplasma spp. detectadas em Xenarthra de São Paulo foram alocadas em um clado irmão ao de A. phagocytophilum. As análises baseadas no gene dsb agruparam as sequências de Ehrlichia spp. de São Paulo, Mato Grosso do Sul, e Pará com sequências de E. canis e as de Rondônia e Pará com E. minasensis. Os dados indicam a ocorrência de E. canis e E. minasensis e duas possíveis novas espécies de Anaplasma spp. em mamíferos de vida livre da Superordem Xenarthra no Brasil. Anaplasmataceae agents are obligatory intracellular Gram-negative α-proteobacteria that are transmitted mostly by arthropod vectors. Although mammals of the Superorder Xenarthra (sloths, anteaters, and armadillos) have been implicated as reservoirs for several zoonotic agents, only few studies have sought to detect Anaplasmataceae agents in this group of mammals. This study aimed to investigate the occurrence and genetic diversity of Anaplasma spp. and Ehrlichia spp. in blood and spleen samples of free-living Xenarthra from four different states in Brazil (São Paulo, Mato Grosso do Sul, Rondônia, and Pará). Nested and conventional PCR screening assays were performed to detect the rrs and dsb genes of Anaplasma spp. and Ehrlichia spp., respectively. The assays were positive in 27.57% (91/330) of the Anaplasma spp. and 24.54% (81/330) of the Ehrlichia spp. Of the 91 positive Anaplasma spp. samples, 56.04% were positive in a conventional PCR assay targeting the 23S-5S intergenic region. Phylogenetic and distance analyses based on the rrs gene allocated Anaplasma sequences from sloths captured in Rondônia and Pará states in a single clade, which was closely related to the A. marginale, A. ovis, and A. capra clades. The sequences detected in southern anteaters from São Paulo were allocated in a clade closely related to sequences of Anaplasma spp. detected in Nasua nasua, Leopardus pardalis, and Cerdocyon thous in Brazil. These sequences were positioned close to A. odocoilei sequences. Genotype analysis corroborated previous findings and demonstrated the circulation of two distinct Anaplasma genotypes in animals from north and southeast Brazil. The first genotype was new. The second was previously detected in N. nasua in Mato Grosso do Sul state. The intergenic region analyses also demonstrated two distinct genotypes of Anaplasma. The sequences detected in Xenarthra from Pará and Rondônia states were closely related to those in A. marginale, A. ovis, and A. capra. Anaplasma spp. sequences detected in Xenarthra from São Paulo and were allocated close to those in A. phagocytophilum. The analyses based on the dsb gene grouped the Ehrlichia spp. sequences with sequences of E. canis (São Paulo, Mato Grosso do Sul, and Pará) and E. minasensis (Rondônia and Pará). The data indicate the occurrence of E. canis and E. minasensis and two possible new Candidatus species of Anaplasma spp. in free-living mammals of the Superorder Xenarthra in Brazil. FAPESP: 2018/13838-6

Simple Summary The use of intravaginal devices may generate retention of vaginal secretions and abnormal discharges at device withdrawal, which may create changes in the normal vaginal bacterial microbiota. These changes in vaginal microbiota could allow the growth of opportunistic pathogenic bacteria which can require antibiotic treatment. The addition of antibiotics on sponges poses a risk of milk contamination. In dairy cows, it has been observed that the use of lactic acid bacteria (LAB) confers a health benefit to the reproductive tract against bacterial infections. However, LAB use in intravaginal sponges in sheep has not been reported. The aims of this study were to describe the dynamics of the ewes’s vaginal cultivable microbiota during a classic long-term estrous synchronization protocol and to develop a model of probiotic infusion in vaginal sponges in order to study their influence in ewe’s vaginal microbiota, general health status, fertility and prolificity. The present results suggest that LAB infusion in the ewe’s vagina do not affect general health status and did not reduce fertility during natural mating. Abstract Sheep estrous synchronization is mainly based on progestagen-impregnated sponges which could cause vaginitis. Several species of Lactobacillus used as probiotics are commonly used in the treatment or prevention of urogenital infections in humans. However, no studies have been performed to analyze the potential use of probiotics to prevent urogenital infections in sheep. A randomized controlled clinical trial was conducted with 21 one-year-old ewes to develop a model of probiotic infusion in vaginal sponges in order to study their influence in ewe’s vaginal microbiota, general health status, fertility and prolificity. Synchronization of estrus was based on intravaginal sponges for 14 days. Bacterial communities (Enterobacteriaceae and lactic acid bacteria) were highly fluctuating over time and between animals. The safety of probiotic infusion (mix of Lactobacillus spp. 60% L. crispatus, 20% L. brevis and 20% L. gasseri) in the vagina of healthy ewes was firstly confirmed. Neutrophils were observed in 80% (8/10) of the control ewes compared to 36% (4/11) of the ewes in the probiotic group 2 days after sponge removal (p = 0.056). Fertility in the control and probiotic groups was 60% (6/10) and 91% (10/11), respectively p = 0.097. These results suggest that Lactobacillus spp. infusion in the ewe’s vagina does not affect general health status or fertility.

[EN] This study was conducted to demonstrate the potential hazards of cross-infection and cross-contamination of embryos during storage in liquid nitrogen biobanks. For the harmless and successful cryopreservation of embryos, the vitrification method must be chosen meticulously to guarantee not only a high post-thaw survival of embryos, but also to reduce the risk of disease transmission when those embryos are in storage for long periods. In recent decades, gamete and embryo cryopreservation have become routine procedures in livestock and human assisted reproduction. However, the safe storage of germplasm and the prevention of disease transmission continue to be potential hazards of disease transmission through embryo transfer. This study aimed to demonstrate the potential risk of cross-infection of embryos from contaminated liquid nitrogen, and cross-contamination of sterile liquid nitrogen from infected embryos in naked and closed devices. Additionally, we examined the effects of antibiotic-free media on culture development of infected embryos. The study was a laboratory-based analysis using rabbit as a model. Two experiments were performed to evaluate both cross-infection (liquid nitrogen to embryos) and cross-contamination (embryos to liquid nitrogen) of artificially inoculated Salmonella Typhimurium, Staphylococcus aureus, Enterobacter aerogenes, and Aspergillus brasiliensis. Rapid cooling through vitrification was conducted on rabbit embryos, stored for a year, thawed, and cultured. In vivo produced late morulae-early blastocyst stages (72 h) embryos were used (n = 480). Embryos were cultured for 1 h in solutions with and without pathogens. Then, the embryos were vitrified and stored in naked and closed devices for one year in two liquid nitrogen biobanks (one pathogen-free and the other artificially contaminated). Embryos were warmed and cultured for a further 48 h, assessing the development and the presence of microorganism (chromogenic media, scanning electron microscopy). Embryos stored in naked devices in artificially contaminated liquid nitrogen became infected (12.5%), while none of the embryos stored in closed devices were infected. Meanwhile, storage of artificially infected embryos incurred liquid nitrogen biobank contamination (100%). Observations by scanning electron microscopy revealed that all the microorganisms were caught in the surface of embryos after the vitrification-thawed procedure. Nevertheless, embryos cultured in antibiotics and antimycotic medium developed to the hatched blastocyst stage, while artificially infected embryos cultured in antibiotic-free medium failed to develop. In conclusion, our findings support that both cross-contamination and cross-infection during embryo storage in liquid nitrogen biobanks are plausible. So, to ensure biosafety for the cryogenic storage, closed systems that avoid direct contact with liquid nitrogen must be used. Moreover, it seems essential to provide best practice guidelines for the cryogenic preservation and storage of gametes and embryos, to define appropriate quality and risk management procedures., This research was supported by the Ministry of Economy, Industry and Competitiveness (MICINN), through a Spanish research project (AGL2017-85162-C2-1-R).

La investigación se realizó en Cuenca, se determinó la prevalencia de Salmonella spp. en huevos de gallina tipo criollo en mercados locales, con el Sistema 3M Petrifilm Salmonella Express. Se analizaron 144 muestras (3 yemas), obteniendo 14 muestras positivas, dando una prevalencia del 9,72% de Salmonella spp. en huevos de gallina tipo criollo. The research was conducted in Cuenca, the prevalence of Salmonella spp. in Creole chicken eggs in local markets, with the 3M Petrifilm Salmonella Express System. 144 samples (3 yolks) were analyzed, obtaining 14 positive samples, giving a prevalence of 9.72% Salmonella spp. in Creole chicken eggs.

Background Mycoplasma bovis is an important pathogen for the cattle industry worldwide causing significant economic losses. Several transmission routes, including those related to reproduction, have been described. Indeed, the pathogen can colonize the female reproductive tract after artificial insemination (AI) with contaminated semen. Lactobacillus spp.-based probiotics have been used for vaginal dysbiosis treatment in women and cows although their role in controlling cervico-vaginal infections due to M. bovis is unknown. The objective of the present work is to assess the viability of M. bovis (PG45, NCTC 10131) in experimentally contaminated cervical mucus after the addition of Lactobacillus spp. at different concentrations as a competing agent and pH acidifier. Results The addition of probiotic at a concentration higher than 108 colony forming units (CFU/mL had a detrimental effect (P P M. bovis survival was not affected and there was no significant LAB growth despite the drop of pH from 8.4 to 6.73 (P Conclusion The addition of concentrations higher than 108 CFU/mL of Lactobacillus spp. negatively affects M. bovis viability in bovine cervical mucus under in vitro conditions. Although the effect observed on the pathogen viability seems to be related to the pH decrease after LAB proliferation in cervical mucus, further studies are necessary to elucidate if other factors are implicated. Nevertheless, the administration of Lactobacillus spp.-based probiotics might be used in the future to control M. bovis proliferation in the cervico-vaginal tract of cows.

Este artículo se encuentra disponible en la siguiente URL: https://www.frontiersin.org/articles/10.3389/fvets.2020.00371/full#h1 The vaginal microbiota plays an important role in the health of dairy cattle, and it could be manipulated for the prevention and treatment of reproduction-related infections. The present study profiles and compares the vaginal microbiota of healthy dairy heifers during the estrous cycle focusing the results in follicular (estrus) and luteal (diestrus) phases using 16S rRNA sequencing of the V3–V4 hypervariable region. Twenty 13–16-months-old virgin dairy heifers from a single farm were included in this study. Vaginal swabs and blood samples were obtained during estrus (6–8 h before artificial insemination) and diestrus (14 days after insemination). Estrus was evaluated by an activity monitoring system and confirmed with plasma progesterone immunoassay. Results showed that the taxonomic composition of the vaginal microbiota was different during the follicular and luteal phases. At the phylum level, the most abundant bacterial phyla were Tenericutes, Firmicutes, and Bacteroidetes which comprised more than 75%of the vaginal microbiota composition. The next more abundant phyla, in order of decreasing abundance, were Proteobacteria, Actinobacteria, Fusobacteria, Epsilonbacteraeota, and Patescibacteria. Together with Tenericutes, Firmicutes, and Bacteroidetes represented more than 96% of the bacterial composition. Ureaplasma, Histophilus, f_Corynebacteriaceae, Porphyromonas, Mycoplasma, Ruminococcaceae UCG-005, were the most abundant genera or families. The results also showed that the vaginal microbiota of dairy heifers was non-lactobacillus dominant. The genus Lactobacillus was always found at a low relative abundance during the estrous cycle being more abundant in the follicular than in the luteal phase. Despite more research is needed to explore the potential use of native vaginal microbiota members as probiotics in dairy heifers, this study represents an important step forward. Understanding how the microbiota behaves in healthy heifers will help to identify vaginal dysbiosis related to disease.

Antimicrobial resistance is a current and important issue to public health, and it is usually associated with the indiscriminate use of antimicrobials in animal production. This study aimed to evaluate the antimicrobial susceptibility profile in bacterial isolates from pigs with clinical respiratory signs in Brazil. One hundred sixty bacterial strains isolated from pigs from 51 pig farms in Brazil were studied. In vitro disk-diffusion method was employed using 14 antimicrobial agents: amoxicillin, penicillin, ceftiofur, ciprofloxacin, enrofloxacin, chlortetracycline, doxycycline, oxytetracycline, tetracycline, erythromycin, tilmicosin, florfenicol, lincomycin, and sulfadiazine/trimethoprim. The majority of isolates were resistant to at least one antimicrobial agent (98.75%; 158/160), while 31.25% (50/160) of the strains were multidrug resistant. Streptococcus suis and Bordetella bronchiseptica were the pathogens that showed higher resistance levels. Haemophilus parasuis showed high resistance levels to sulfadiazine/trimethoprim (9/18=50%). We observed that isolates from the midwestern and southern regions exhibited four times greater chance of being multidrug resistant than the isolates from the southeastern region studied. Overall, the results of the present study showed a great level of resistance to lincomycin, erythromycin, sulfadiazine/trimethoprim, and tetracycline among bacterial respiratory pathogens isolated from pigs in Brazil. The high levels of antimicrobial resistance in swine respiratory bacterial pathogens highlight the need for the proper use of antimicrobials in Brazilian pig farms.

Staphylococcal mastitis is a major health problem in humans and livestock that leads to economic loss running in millions. This process is currently one of the main reasons for culling adult rabbit does. Surprisingly, the two most prevalent S. aureus lineages isolated from non-differentiable natural clinical mastitis in rabbits (ST121 and ST96) generate different immune responses. This study aimed to genetically compare both types of strains to search for possible dissimilarities to explain differences in immune response, and to check whether they showed similar virulence in in vitro tests as in experimental intramammary in vivo infection. The main differences were observed in the enterotoxin gene cluster (egc) and the immune-evasion-cluster (IEC) genes. While isolate ST121 harboured all six egc cluster members (seg, sei, selm, seln, selo, selu), isolate ST96 lacked the egc cluster. Strain ST96 carried a phage integrase Sa3 (Sa3int), compatible with a phage integrated into the hlb gene (β-haemolysin-converting bacteriophages) with IEC type F, while isolate ST121 lacked IEC genes and the hlb gene was intact. Moreover, the in vitro tests confirmed a different virulence capacity between strains as ST121 showed greater cytotoxicity for erythrocytes, polymorphonuclear leukocytes and macrophages than strain ST96. Differences were also found 7 days after experimental intramammary infection with 100 colony-forming units. The animals inoculated with strain ST121 developed more severe gross and histological mastitis, higher counts of macrophages in tissue and of all the cell populations in peripheral blood, and a significantly larger total number of bacteria than those infected by strain ST96.

Este artículo se encuentra disponible en la siguiente URL: https://bvajournals.onlinelibrary.wiley.com/doi/epdf/10.1136/vr.105558 This is the pre-peer reviewed version of the following article: Sevilla-Navarro, S., Marin, C., Cortés, V., García, C., & Catalá-Gregori, P. (2020). Campylobacter prevalence and risk factors associated with exceeding allowable limits in poultry slaughterhouses in Spain. Veterinary Record, vol. 186, i. 13 (apr.), p. 415, which has been published in final form at https://doi.org/10.1136/vr.105558. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. Este es el pre-print del siguiente artículo: Sevilla-Navarro, S., Marin, C., Cortés, V., García, C., & Catalá-Gregori, P. (2020). Campylobacter prevalence and risk factors associated with exceeding allowable limits in poultry slaughterhouses in Spain. Veterinary Record, vol. 186, i. 13 (apr.), p. 415, que se ha publicado de forma definitiva en https://doi.org/10.1136/vr.105558. Background: Campylobacter is the main pathogen involved in zoonotic gastrointestinal diseases. Last year, the European regulation 2017/1495 on Campylobacter in broiler carcasses came into force. In this context, the aim of this study was to assess the potential risk factors associated with exceeding the 1,000 CFU/g criterion set by the European Commission in several slaughterhouses in Spain. Methods: Information relating to 12 factors were collected using questionnaires. Samples were collected from 12 Spanish abattoirs during June, July and August 2017 (n=1,725) and were analysed following ISO/TS 10272-2:2006 method. Results: The proportion of Campylobacter-positive samples was 23.7% (n=409). Analysis of the flock age (41-50 days) revealed a significantly increased OR in Campylobacter enumeration (OR=7.41). Moreover, scalding temperature (51.9.54 ºC) was positively associated with an increase in OR (OR=2.75). Time in transit to slaughter (1-1.5h), showed a significant OR decrease (OR=0.25). However, when processed for more than 2 hours, presented an increase in OR (OR=4.44). Regarding carcass weight, the range from 3.21-3.58 presented a decrease in OR (OR=0.01). Conclusion: The outcomes of this study suggest that although most chickens are contaminated by the bacterium, the prevalence that exceeds the limit of 1,000 CFU/ is not so high as we thought.

Mycoplasma bovis is an important etiologic agent of bovine mycoplasmosis in cattle. Different transmission routes have been described, including those related to reproduction. The presence of mycoplasma in semen has led to its appearance in infection-free areas through artificial insemination (AI). Semen was recently reported to be the initial source of two M. bovis mastitis outbreaks in two closed dairy herds in Finland. This questions the effectiveness of the antimicrobials currently used in semen extenders to control the pathogens in contaminated semen. They should be re-evaluated, or alternative measures to antimicrobials should be tested to obtain M. bovis-free semen. This in vitro study aimed to assess different strategies to reduce the risk of transmission of M. bovis through AI technologies. The viability of M. bovis (PG45, NCTC 10131) in bull semen diluted (DS) in a Tris-citrate-fructose solution was tested, after the addition of enrofloxacin, doxycycline or a Lactobacillus spp.-based probiotic. The data show the susceptibility of the pathogen to the addition of 0.125 &mu, g/mL of enrofloxacin or 0.0625 &mu, g/mL of doxycycline and to the addition of the probiotic at a concentration of 3.24 &times, 106 colony forming units (CFU)/mL or 3.24 &times, 108 CFU/mL in DS. The Tris-citrate-fructose medium negatively affected the viability of M. bovis, although this effect was lower than that observed after the addition of the probiotic and antimicrobials (p &lt, 0.05). Our results may support new strategies for reducing the risk of M. bovis transmission through AI.

Porcine Corynebacterium infection is still poorly studied, even though the pig has been described as an asymptomatic carrier of Corynebacterium species, including the zoonotic species C. ulcerans, C. confusum and C. amycolatum. Here we present the identification, molecular and antimicrobial susceptibility characterization of coryneform bacteria isolated from sows with urinary tract infection. C. diphtheriae, C. confusum and C. amycolatum were isolated from sows with urinary infection and metritis. Corynebacterium species were identified by MALDI-TOF MS and confirmed by 16S rRNA and rpoB sequencing. All porcine C. diphtheriae strains were further characterized as non-toxigenic (tox-). SE-AFLP genotyping was also performed and enabled not only Corynebacterium species differentiation but also the assessment of C. amycolatum genetic heterogeneity. All studied porcine Corynebacterium strains presented alarming resistance profiles with high MIC values for macrolides/lincosamide, tetracyclines and quinolones, which can be related with high usage in both veterinary and human medicine. Isolation of zoonotic Corynebacterium species from commercial swine is important for assessing the potential zoonotic risk for farmers and further implication for both human and animal treatment.

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No. of bitstreams: 1 cruz_nrn_dr_jabo.pdf: 1363922 bytes, checksum: 505ba896a191561eeb1cb6b46bb8293f (MD5) Previous issue date: 2019-12-09 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) A hemoplasmose dos suínos, causada pelo Mycoplasma suis, caracteriza-se como doença geograficamente cosmopolita, atinge animais de diversas faixas etárias, frequentemente associada à anemia hemolítica moderada a grave e predispõe os animais a imunossupressão, infertilidade, diminuição do ganho de peso, aumento de natimortos, abortos e retorno ao cio, ou seja, impacta diretamente as granjas de suínos e leva a imensuráveis perdas econômicas. Do ponto de vista epidemiológico, animais assintomáticos são considerados como foco da hemoplasmose por serem portadores e apresentarem melhora clínica sem a eliminação do parasita e alguns autores reportam a micoplasmose hemotrófica como uma enfermidade de potencial zoonótico. A prevalência nos rebanhos brasileiros é discutível, especialmente por que o diagnóstico tradicional pela observação do M. suis no esfregaço sanguíneo tem baixa sensibilidade. Objetivou-se com o presente trabalho caracterizar a taxa de prevalência e correlacionar a quantidade do Mycoplasma suis quantificado por qPCR com alterações nos parâmetros hematológicos em suínos em diferentes fases de criação de granjas tecnificadas de ciclo completo do Estado de São Paulo. Foram avaliadas 20 diferentes granjas suinícolas de ciclo completa situadas em território paulista, foram coletadas 501 amostras de sangue total em EDTA-K2 para análise molecular de qPCR para Mycoplasma suis e realização de hemograma e pesquisa de hemoparasitas em esfregaço sanguíneo. No primeiro capítulo realizou-se uma revisão de literatura sobre o Mycoplasma suis, a patobiologia do agente bem como os sinais clínicos nos animais. No capítulo 2 relatou-se a ocorrência do Mycoplasma suis nas granjas tecnificadas de ciclo completo no Estado de São Paulo e foi possível determinar uma prevalência total de animais infectados de 31,93% (161/201) e 95% (19/20) de granjas infectados e por fase de criação foram: creche (30,47%); recria (31,29%), teminação (26,18%) e frigorífico (40,25%). No terceiro capítulo, correlacionou-se os resultados dos parâmetros hematológicos com a bacteremia de M suis, sendo que, na comparação dos hemogramas de animais positivos e negativos não houve diferença estatística e ambos os grupos não apresentavam valores alterações em relação aos valores referenciais adotados, situação na qual evidencia-se a condição de animais portadores, aparentemente sadios que auxiliam na permanência e propagação do agente nos rebanhos. Nos animais positivos foram observadas correlação entre hemoglobina (y = 9,978 + 0,186 x, p = 0,02, r²=0,354), plaquetas (y = 582,522 – 16,190 x, p = 0,04, r² = 0,286) e neutrófilos (y = 9,77 – 0,307 x, p = 0,05, r² = 0,276) com a bacteremia de M suis, esses resultados indicam a presença de hemólise, dano vascular e consumo plaquetário inerente aos animais portadores da micoplasmose hemotrófica. Porcine hemoplasmosis caused by Mycoplasma suis, is characterized as a geographically cosmopolitan disease. It affects animals of various age groups, often associated with moderate to severe hemolytic anemia and predisposes the animals to immunosuppression, infertility, decreased weight gain, increased stillbirths, miscarriages and return to heat, that is, directly impacts pig farms and leads to immeasurable economic losses. From an epidemiological point of view, asymptomatic animals are considered as the focus of hemoplasmosis because they are carriers and present clinical improvement without the elimination of the parasite and some authors report hemotrophic mycoplasmosis as a zoonotic potential disease. The prevalence in Brazilian herds is debatable, especially since the traditional diagnosis by observing M suis in the blood smear has low sensitivity. The objective of this study was to characterize the prevalence rate and correlate the quantity of Mycoplasma suis quantified by qPCR with changes in hematological parameters in pigs in different phases of rearing of full-cycle farms in the State of São Paulo. Twenty different complete cycle swine farms located in the state of São Paulo were evaluated, 501 whole blood samples were collected in EDTA-K2 for molecular analysis of qPCR for Mycoplasma suis and blood count and blood smear hemoparasites. In the first chapter there was a literature review on Mycoplasma suis, the pathobiology of the agent as well as clinical signs in animals. In Chapter 2, the occurrence of Mycoplasma suis was reported in the full-cycle technicated farms in the State of São Paulo and it was possible to determine a total prevalence of infected animals of 31.93% (161/201) and 95% (19/20) infected farms and by growth stages were: nursery (30.47%); growing (31.29%), finishing (26.18%) and slaughter (40.25%). In Chapter 3, the results of the hematological parameters were correlated with the M suis bacteremia, and in the comparison of the blood counts of positive and negative animals there was no statistical difference and both groups did not present alterations values in relation to the adopted reference values. This situation evidences the condition of apparently healthy carriers that help in the permanence and propagation of the agent in the herds. In positive animals, a correlation was observed between hemoglobin (y = 9.978 + 0.186 x, p = 0.02, r² = 0.354), platelets (y = 582.522 - 16.190 x, p = 0.04, r² = 0.286) and neutrophils (y = 9.77 - 0.307 x, p = 0.05, r² = 0.276) with M suis bacteremia, these results indicate the presence of hemolysis, vascular damage and platelet consumption inherent in animals with hemotrophic mycoplasmosis. Keywords: Mycoplasmose, swine, haemoplasma, prevalence, bacteremia, growth stages FAPESP: 16/14838-4

[EN] Wild birds have repeatedly been found to be involved in the dissemination of enteric bacterial pathogens in the environment. The aim of this study was to determine the occurrence of Salmonella and Campylobacter as well as the antimicrobial resistance in wild Bonelli's eagles nestlings in Eastern Spain. In addition, we compared the efficiency of two sampling methods (fresh faecal samples from nest and cloacal swabs from nestlings) for detection of both bacteria. A total of 28 nests with 45 nestlings were analysed. In the nest, Salmonella occurrence was 61 +/- 9.2%, while Campylobacter occurrence was 11 +/- 5.8% (p < 0.05). In the nestlings, Salmonella occurrence was 36 +/- 7.1%, while Campylobacter occurrence was 11 +/- 4.7% (p < 0.05). Eight Salmonella serovars were identified, and the most frequently isolated were S. Enteritidis, S. Typhimurium, S. Houston, and S. Cerro. Only one Campylobacter species was identified (C. jejuni). Regarding antimicrobial resistance, the Salmonella strains isolated were found to be most frequently resistant to ampicillin and to tigecycline; however, the sole Campylobacter strain recovered was multidrug resistant. In conclusion, this study demonstrated that wild Bonelli's eagles nestlings are greater carriers of Salmonella than of Campylobacter. Both Salmonella and Campylobacter isolates exhibited antimicrobial resistance. In addition, faecal samples from nests were most reliable for Salmonella detection, while cloacal swab from nestlings were most reliable for Campylobacter detection., We wish to thank the Ministry of Infrastructures, Territory and Environment (Regional Government/Generalitat Valenciana), the research group "Improvement of Production System-related Food Safety and End Products" research group (Veterinary Faculty, University CEU-Cardenal Herrera) and GEMAS (Study Group on Wildlife Medicine and Conservation) for their technical support. Moreover, we want to thank University CEU-UCH (Consolidacion de Indicadores INDI 18/19 and IDOC 18/12) for the financial support. The English text version was revised by N. Macowan English Language Service.

This study aimed to determine the occurrence of Mannheimiahaemolytica, Pasteurella multocida and Mycoplasma spp., in relation to clinical signs of respiratory disease. Tracheobronchial lavage samples were collected from 96 (healthy and unhealthy) cattle in the State of São Paulo, Brazil. Mycoplasma spp. (12.5 %) and Pasteurellamultocida (15.50 %) were the most prevalent species. Bacillus sp., Staphylococcus sp., Escherichia coli, Klebsiella oxytoca, Pseudomonas aeruginosa and Klebsiella pneumoniae were also isolated. Mollicutes (70.83 %), Mycoplasmabovis (2.94 %) and Mycoplasma dispar (38.23 %) were identified using conventional PCR. Submassive sound on acoustic percussion of the thorax was associated with the absence of Mollicutes (P=0.025). Whistling (P=0.076) and coarse crackle (P=0.046) were associated with the absence of Mycoplasma dispar. Clear sound on acoustic percussion of the thorax was associated with the absence of Mycoplasmabovis (P=0.007). Coughing was associated with the presence of Pasteurellamultocida [P=0.035; confidence interval (CI), 1.12-26.89], but its absence was associated with mucopurulent (P=0.0215; CI, 1.55-34.5) and mucoid nasal discharge (P=0.068; CI, 19-28.5), submassive sound (P=0.031; CI, 1.23-75.5), fine crackle (P=0.058; CI, 1.23-20.1) and coarse crackle (P=0.046; CI, 2.38-70.8). The high prevalence of Pasteurella multocida and Mycoplasma spp. in unhealthy calves increases the importance of these micro-organisms in the pathogenesis of respiratory diseases. This study increases the information about the role of Mycoplasma dispar in respiratory diseases. Differences in some species in relation to clinical signs can be applied as a presumptive diagnosis.

Mycoplasma bovis is an important bovine pathogen causing pneumonia, mastitis, and arthritis and is responsible for major economic losses worldwide. In the absence of an efficient vaccine, control of M. bovis infections mainly relies on antimicrobial treatments, but resistance is reported in an increasing number of countries. To address the situation in Spain, M. bovis was searched in 436 samples collected from beef and dairy cattle (2016&ndash, 2019) and 28% were positive. Single-locus typing using polC sequences further revealed that two subtypes ST2 and ST3, circulate in Spain both in beef and dairy cattle, regardless of the regions or the clinical signs. Monitoring of ST2 and ST3 isolates in a minimum inhibitory concentration (MIC) to a panel of antimicrobials revealed one major difference when using fluoroquinolones (FQL): ST2 is more susceptible than ST3. Accordingly, whole-genome sequencing (WGS) further identified mutations in the gyrA and parC regions, encoding quinolone resistance-determining regions (QRDR) only in ST3 isolates. This situation shows the capacity of ST3 to accumulate mutations in QRDR and might reflect the selective pressure imposed by the extensive use of these antimicrobials. MIC values and detection of mutations by WGS also showed that most Spanish isolates are resistant to macrolides, lincosamides, and tetracyclines. Valnemulin was the only one effective, at least in vitro, against both STs.

A pesquisa de Staphylococcus spp. coagulase negativa (CNS) em alimentos não é prevista na legislação, entretanto, estas bactérias têm emergido como patógenos oportunistas e sua capacidade enterotoxigênica já foi documentada. O queijo destaca-se entre os principais derivados lácteos associados a intoxicações alimentares e a presença de cepas enterotoxigênicas do gênero Staphylococcus neste alimento representa um risco ao consumidor. O queijo colonial, tradicionalmente consumido pelos gaúchos, não possui regulamento técnico específico e poucos são os estudos relacionados ao risco do consumo desse alimento, bem como, à identificação de micro-organismos presentes nessa matriz. Sendo assim, os objetivos do presente estudo foram: (i) identificar as espécies de Staphylococcus spp. coagulase negativa, presentes em queijo colonial inspecionado; (ii) pesquisar a presença de genes codificadores de enterotoxinas clássicas (SE), bem como de resistência à penicilina e à meticilina nas cepas isoladas desta matriz. Para tanto, no período de novembro de 2014 a maio de 2015, foram analisadas 205 amostras de queijos coloniais inspecionados, sendo 121 adquiridas em Feiras Modelo e 84 em bancas do Mercado Público de Porto Alegre, compreendendo 17 marcas distintas. O isolamento inicial de Staphylococcus spp. foi realizado de acordo com o protocolo ISO 6888-1:1999, adicionado da triagem fenotípica para CNS. A identificação genotípica dos isolados foi realizada pela amplificação da região V1-V2 do gene 16S rRNA, seguida de sequenciamento e comparação das sequências obtidas no GenBank. A pesquisa de genes de enterotoxinas clássicas foi realizada por amplificação dos genes sea, seb, sec, sed e see. A determinação de resistência à penicilina foi avaliada a partir da amplificação do gene blaZ. Para resistência à meticilina, foi realizado teste de triagem frente à cefoxitina, e confirmação pela pesquisa do gene mecA. O armazenamento sob refrigeração foi observado em quase 90% das amostras coletadas. Entre as 179 colônias retiradas do ágar Baird-Parker, 59 apresentaram-se fenotipicamente compatíveis com CNS e foram identificadas genotipicamente. Treze espécies foram identificadas, sendo Macrococcus caseolyticus (40%) a mais frequente. Das 35 cepas confirmadas como CNS, S. equorum e S. vitulinus foram as espécies predominantes seguidas de S. hyicus, S. saprophyticus e S. epidermidis. O gene blaZ foi detectado em cinco cepas de CNS e em uma cepa de M. caseolyticus, sendo relativamente mais frequente em S. hyicus e S. epidermidis. O gene mecA não foi detectado. Oito cepas de CNS amplificaram algum gene para SE, sendo seb o mais frequente, seguido por sed, sea e see. O gene para enterotoxina C não foi detectado. Onze cepas apresentaram pelo menos um dos genes investigados, das quais, seis cepas apresentaram genes para SE e blaZ, concomitantemente. Os perfis seb/blaZ (n=4) e blaZ (n=3) foram os mais frequentes. Foi possível confirmar a diversidade de Staphylococcus spp. coagulase negativa em queijos coloniais inspecionados, além da baixa frequência de cepas carreadoras de genes para enterotoxinas clássicas e resistência à penicilina e à meticilina. The investigation of coagulase-negative Staphylococcus spp. (CNS) is not included in food monitoring; although these bacteria have emerged as significant opportunistic pathogens and their toxigenic capacity has been documented. Among the dairy products, cheese features as one of the most involved in food poisoning outbreaks. The presence of enterotoxigenic strains of Staphylococcus in this food therefore represents a hazard for the consumers. Colonial cheese, which is a traditionally consumed cheese type in Rio Grande do Sul, does not have a specific technical regulation, and there are few studies targeting the risk for consumers, or aiming to identify its typical microbiota. Thus, the objectives of this study were: (i) to identify coagulase-negative Staphylococcus spp. species in inspected colonial cheese (ii) to investigate the presence of genes encoding classical enterotoxins (SE), and resistance to penicillin and methicillin in strains obtained from this food. For this purpose, from November 2014 to May 2015, 205 cheese samples were analyzed, 121 of which were acquired in street fairs and 84 in Central Market. The samples belonged to 17 different brands. The isolation of Staphylococcus spp. was performed according to the ISO 6888-1: 1999 protocol, followed by the phenotypic screening of CNS. The genotype identification of the isolates was performed by amplification of the V1-V2 region of the 16S rRNA gene, followed by sequencing and the sequences comparison with the GenBank database. Classical enterotoxin genes were investigated by amplification of sea, seb, sec, sed and see genes. The determination of penicillin resistance was evaluated by the amplification of blaZ gene. For methicillin resistance, a screening test with cefoxitin was conducted followed by confirmation through the mecA amplification. The majority (89,7%) of the collected samples were stored under refrigeration. Among the 179 atypical colonies obtained from Baird-Parker agar, 59 were phenotypically compatible with CNS and were further subjected to genotyping. Thirteen bacterial species were identified, being Macrococcus caseolyticus the most frequent (40%). Thirty-five strains were confirmed as CNS, being S. equorum and S. vitulinus the most prevalent followed by S. hyicus, S. saprophyticus and S. epidermidis. The gene blaZ was detected in five strains of CNS and in one strain of M. caseolyticus, being relatively more frequent in S. hyicus and S. warneri. The mecA gene was not detected. Eight CNS strains amplified SE gene: SEB was the most frequent, followed by SED, SEA and SEE. There was no enterotoxin C gene detected. Eleven strains carried at least one of the genes investigated; six strains presented genes for SE and blaZ, concomitantly. The profiles SEB/blaZ (n = 4) and blaZ (n = 3) were the most frequent. The diversity of CNS in inspected colonial cheeses was confirmed. In addition, the low frequency of strains carrying genes for enterotoxins and resistance to penicillin and methicillin was observed.

A população de bubalinos estimada no Brasil é de aproximadamente 3 milhões de animais, encontrando-se distribuídos em todos os Estados brasileiros, com crescimento médio anual de 12%. Apesar disso, os trabalhos realizados buscando os avanços na bubalinocultura são escassos. Em função da complexidade etiológica da diarreia em bubalinos e da falta de informações recentes nesta área, o presente estudo teve como objetivo avaliar a ocorrência dos principais agentes bacterianos e parasitários envolvidos na diarreia de bezerros búfalos lactentes, de explorações leiteiras semi-intensivas e intensivas em regiões dos estados de São Paulo e Paraná. De março de 2010 a junho de 2011, foram colhidas 53 amostras para exame coproparasitológico e 46 amostras para o exame bacteriológico de animais com quadro de diarreia nos municípios paulistas de São João da Boa Vista, Dourado, Pirassununga, Registro, Pariquera Açu, Pilar do Sul e uma propriedade no estado do Paraná, município de Santana do Itararé. No exame parasitológico, 45,28% (24) foram positivos para Eimeria spp., 26,42% (14) para Strongyloidea e 1,88 (1) para Toxocara vitulorum. No exame bacteriológico, 97,83%, (45) das amostras foram positivas para E. coli, contudo, somente duas foram consideradas patogênicas (E. coli STEC). Em uma amostra (2,17%) isolou-se Klebsiella pneumoniae; já a presença de Salmonella spp. não foi constatada. Para o presente estudo, a presença de endoparasitas foi bastante relevante, principalmente os casos Eimeria spp., sendo a higiene das instalações e falhas de manejo fatores importantes na ocorrência de diarreia em bezerros búfalos no estado de São Paulo. There are about 3 million buffalos in Brazil, spread through all of the Brazilian states; with mean annual growth of 12%. In spite of that, to the best of our knowledge few studies looking for advances in the industry have been done. Due to the etiological complexity of buffalo diarrhea and the lack of information in this area, this aimed at developing a clinical evaluation on the causes of buffalo calves bacterial and parasitical diarrhea in dairy farms of the states of São Paulo and Paraná. The survey was done in farms located in the cities of São João da Boa Vista, Dourado, Pirassununga, Registro, Pariquera Açu, Pilar do Sul (SP), and Santana do Itararé (PR). From March, 2010, to June, 2011, 53 diarrhea samples were collected and screened for endoparasite and bacteria; 45.28% (24) were positive for Eimeria spp.; 26.42% (14) had Strongyloidea; and 1.88% (1) had Toxocara vitulorum. In the bacteriological test, 97.83% (45) had E. coli, but only two were considered pathogenic (E. coli STEC); 2.17% had Klebsiella pneumonia and none presented Salmonella spp. In this study, the mainly causative agent of buffalo diarrhea was Eimeria spp., and the poor hygiene in installations and breeding failure are important factors on this diarrhea occurrence.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior A dissertação foi dividida em dois estudos. Com o primeiro, objetivou-se avaliar a ocorrência de microrganismos presentes na região vulvovaginal de vacas que receberam dispositivos intravaginais de progesterona durante os programas de inseminação artificial em tempo fixo (IATF), e correlacionar os resultados com as taxas de prenhez. As amostras foram coletadas da região vulvovaginal de 30 vacas de corte Guzerá e 30 vacas leiteiras mestiças, e também dos dispositivos intravaginais, aleatoriamente. Das 120 amostras coletadas das vacas, 60 corresponderam ao período anterior à introdução do dispositivo (D0) e 60 ao posterior à retirada do mesmo (D9); obteve-se 100% de crescimento bacteriano, sendo que, na maioria das amostras, encontrou-se mais de um isolado. No D0, o agente de maior frequência foi a Escherichia coli (52%), e no D9, Proteus spp e E.coli foram os mais encontrados (32% e 28%, respectivamente). Em relação aos dispositivos intravaginais de progesterona, no D0 foram isolados 37 microrganismos, sendo predominantes os do gênero Bacillus (35%); já no D9 foram isoladas 41 unidades formadoras de colônias (UFC), sendo que 36,6% corresponderam ao Proteus spp. Para a análise do perfil antimicrobiano, realizou-se antibiograma por difusão do disco em ágar, e foram selecionadas as vacas que não emprenharam após a IATF, a título de tratamento futuro. Houve resistência de 100% à penicilina, e sensibilidade de, aproximadamente, 90% à gentamicina, tanto para os isolados obtidos das amostras das vacas de corte quanto para os obtidos das vacas de leite. Em relação à taxa de prenhez, das 30 vacas de corte, 11 foram diagnosticadas prenhes (36,7%), sendo 4 (36,4%) tratadas com dispositivos intravaginais reutilizados e 7 (63,6%) com dispositivos novos, que se mostraram mais eficazes. Das 30 vacas de leite, 15 (50%) emprenharam, sendo que 8 (53,3%) foram implantadas com dispositivos reutilizados e 7 (46,7%) com dispositivos novos, não havendo diferenças significativas entre as taxas de prenhez. Por se tratar de uma pesquisa, as fêmeas foram escolhidas ao acaso, e fatores como escore corporal, manejo nutricional e sanitário não foram prioridade. Com o segundo estudo, objetivou-se analisar a similaridade entre cepas, realizada pela técnica Random Amplified Polymorphic DNA (RAPD-PCR). A presença de E.coli e a ausência de prenhez foram os critérios de seleção utilizados. Diante dos resultados, observou-se que a maioria dos isolados não foi filogeneticamente semelhante, uma vez que apresentaram similaridade inferior a 85%. O estudo ressaltou a importância da E. coli na microbiota vulvovaginal de vacas e a presença de caracteres fenotípicos e genotípicos dessa bactéria em possíveis problemas reprodutivos. The dissertation was divided in two studies. With the first, aimed to evaluate the occurrence of microorganisms present in the vulvovaginal region of cows that received intravaginal progesterone devices during the fixed-time artificial insemination (FTAI) programs, and correlate the results with pregnancy rates. Samples were collected from vulvovaginal region of 30 beef cows Guzerá and 30 crossbred dairy cows, and also intravaginal devices, randomly. Of the 120 samples of cows, 60 corresponded to the collections of the period prior to the introduction device (D0) and 60 to the subsequent withdrawal of it (D9); it yielded 100% of bacterial growth, whereas, in most samples, it was found more than one isolated. In D0, the most frequent agent was Escherichia coli (52%), and in D9, Proteus spp and E. coli were the most frequent (32% and 28%, respectively). Regarding intravaginal progesterone devices, in D0 were isolated 37 microorganisms, being predominant those of the genus Bacillus (35%); in D9, 41 colony forming units (CFU) were isolated, of which 36.6% corresponded to Proteus spp. For the analysis of the antimicrobial profile, susceptibility testing was performed by diffusion agar disk, and cows that did not became pregnant after FTAI program were selected, as a future treatment. There resistance 100% to penicillin, and sensitivity, approximately, 90% to gentamicin, both isolates obtained from samples of beef cows and obtained of dairy cows. Regarding pregnancy rate, the 30 beef cows, 11 were diagnosed pregnant (36.7%), 4 (36.4%) treated with reused devices and 7 (63.6%) with new devices, which showed more effective. Of the 30 dairy cows, 15 were pregnant (50%), 8 (53.3%) were implanted with reused devices and 7 (46.7%) with new devices, with no significant differences in pregnancy rates. Because it is a research, females were chosen at random, and factors such as body condition, nutritional management and health weren't priority. With the second study, aimed to analyze the similarity between strains, conducted by technical Random Amplified Polymorphic DNA (RAPD -PCR). Presence of E. coli and the absence of pregnancy were selection criteria used. From the results, it was observed that most of the isolates wasn't phylogenetically similar, since they showed lower than 85% similarity. The study stressed the importance of E. coli in vulvovaginal microbiota of cows and the presence of phenotypic and genotypic characters of this bacterium on possible reproductive problems. Dissertação (Mestrado)

Introdução: A utilização do tubo endotraqueal (TE) e da traqueia respiratória (TR) é fundamental na prática cirúrgica veterinária e a reutilização dos mesmos é também uma prática recorrente de forma a obter uma diminuição de custos. A avaliação da contaminação microbiológica dos mesmos está amplamente documentada em Medicina Humana. O mesmo não se sucede em Medicina Veterinária, pois devido aos altos custos de manutenção, torna-se impraticável realizar estudos microbiológicos de rotina. Material e Métodos: O presente estudo foi realizado num Hospital de Medicina Veterinária na zona de Lisboa, com o objectivo de determinar a contaminação bacteriana e fúngica nestes equipamentos. Procedeu-se à avaliação clínica e radiográfica de todos os pacientes. Foram recolhidas amostras microbiológicas a partir de 30 TEs e 30 TRs reutilizados, escolhidos aleatoriamente, antes do procedimento cirúrgico e cultivadas em meios de cultura para bactérias e fungos. Este procedimento foi realizado independentemente do sexo, raça e idade do animal, tipo e tempo de cirurgia. Seguido de determinação, caracterização e identificação microbiológica. Resultados: Verificou-se crescimento bacteriano e/ou fúngico total de 73% (44 amostras positivas em 60 amostras). Por fim, correlacionaram-se os resultados com o aparecimento de afecções respiratórias. Conclusão: O estudo de fontes de contaminação em ambientes hospitalares veterinários é uma actividade em crescimento, sendo necessário o desenvolvimento de programas de controlo mais específicos e eficazes, que tenham como pilares básicos um sistema de vigilância efectivo combinado com uma rotina de análises microbiológicas., Background: The use of the endotracheal tube (ET) and the tubing of breathing circuit systems (tBCS) is fundamental for the practice of the veterinary surgery and reusing them is also a recurring way of reducing costs. The evaluation of the microbiological contamination is widely documented in Human Medicine. In Veterinary Medicine however, due to high maintenance costs, it is impossible to perform routine microbiological studies. Materials and methods: The current study was done in a Veterinary Medicine Hospital in the Lisbon area, with the objective of defining the bacteriological and fungi contamination of these medical devices. All patients underwent a clinical and radiographic evaluation. Microbiological samples were taken from 30 ET and 30 tBCS reused randomly chosen and were collected before initiating the surgical procedure and cultured for bacteria and fungi. This procedure was performed regardless of the sex, race and age of the animal, type and time of surgery. Followed by determination, characterization and microbiological identification. Results: Bacterial and/or fungal growth was found to be 73% (44 positive samples in 60 samples). In the end the results were correlated with the appearance of respiratory disorders. Conclusion: The study of the sources of contamination in veterinary hospital environments is a growing activity, creating the need to develop more specific and efficient control programs, which will be supported by a basis of a surveillance system combined with a routine of microbiological analysis., Orientação: Pedro Faísca

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Staphylococcus aureus e Streptococcus agalactiae são patógenos contagiosos associados à forma subclínica e persistente da mastite bovina, principal doença da pecuária leiteira. Neste trabalho avaliou-se a presença de dois patógenos em amostras de leite CMT positivas coletadas durante seis meses de animais pertencentes a um rebanho da Zona da Mata de Minas Gerais. Foi encontrada uma alta prevalência dos patógenos contagiosos o que indica possível falta de higiene nos procedimentos de rotina de ordenha ou falha no tratamento da doença. A análise de multilocus de repetições em tandem de número variável (MLVA) revelou apenas um genótipo de S. aureus e seis genótipos de S. agalactiae, alguns dos quais foram associados a infecções persistentes e ao número de parições do animal. A maioria das bactérias isoladas foi considerada forte produtora de biofilme e no caso de S. aureus, os isolados apresentaram uma variada atividade hemolítica. É possível que esses fenótipos contribuam com a persistência da infecção. No capítulo 2, estirpes de S. aureus isoladas de animal com manifestação de mastite persistente ou não persistente foram contrastadas quanto a características importantes para a patogênese bacteriana e virulência in vivo. Genotipagem por eletroforese em campo pulsado (PFGE) revelou que as bactérias possuíam uma similaridade de 90%. As duas estirpes contrastadas apresentaram os mesmos genes de virulência. Porém, elas diferiram significativamente na produção de hemolisina e biofilme, na capacidade de invasão e persistência em células do epitélio mamário bovino (MAC-T). Nos ensaios in vivo realizados com o modelo Galleria mellonella, a estirpe SAU 302 revelou-se mais virulenta do que a SAU 322. Uma forte reação do sistema imune do inseto foi observada mediante infecção com as bactérias. Os resultados mostraram variações fenotípicas entre as estirpes geneticamente relacionadas que podem contribuir positivamente para o desenvolvimento da doença e devem ser cuidadosamente exploradas. Conclui-se que bactérias geneticamente relacionadas podem apresentar uma variação fenotípica não revelada por técnicas de fingerprint do DNA e que esta variação pode ter uma implicação na progressão da mastite bovina. Sugere-se a definição de um conjunto de ensaios fenotípicos relevantes para a doença a fim de caracterizar bactérias isoladas de animais com manifestação conhecida. Staphylococcus aureus and Streptococcus agalactiae are contagious pathogens associated with subclinical and persistent bovine mastitis, the main disease of dairy cattle. In this work we investigated the presence of these pathogens in samples of milk CMT-positive collected during six months from animals belonging to a herd from the Zona da Mata of Minas Gerais. A high prevalence of the contagious pathogens was found indicating a possible lack of hygiene in the milking routine procedures or failure in the treatment of the disease. The multilocus variable-number tandem repeat analysis of (MLVA) revealed one genotype of S. aureus and six genotypes of S. agalactiae, some of them associated with persistent infections and calving number. Most bacterial isolates was considered strong biofilm producer and for S. aureus the isolates showed a variation in hemolytic activity. These phenotypes may contribute to the persistence of the infection. In Chapter 2, we analyzed the in vivo virulence and phenotypic traits believed to be important for the pathogenicity of bovine S. aureus. In this study the strains SAU 302 and SAU 322 isolated from animals with persistent or non-persistent mastitis, respectively, were contrasted. Genotyping by pulsed field gel electrophoresis (PFGE) revealed a genetic relatedness of 90% between bacteria. The two strains showed the same set of virulence genes investigated. However, significant differences were seen in the production of hemolysin and biofilm, in invasiveness and persistence in the bovine mammary epithelial cells (MAC-T). In vivo tests carried out with the Galleria mellonella model showed that SAU 302 was more virulent than the SAU 322. A strong reaction of the insect immune system was observed upon infection with both bacteria. Despite their genetic proximity, the strains showed a phenotypic variation undetected by DNA-based methods that can contribute to the development of the disease and should be explored further. Our present study suggests that a set of phenotypic assays relevant to bovine mastitis should be defined and used to analyze a collection of bacteria for which information on the nature of the clinical manifestation is available.

Made available in DSpace on 2016-06-07T17:12:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-04-28. Added 1 bitstream(s) on 2016-06-07T17:16:47Z : No. of bitstreams: 1 000858080_20160630.pdf: 190100 bytes, checksum: 47dd9e01e892e41f2f90965bdb032201 (MD5) Bitstreams deleted on 2016-07-01T13:02:23Z: 000858080_20160630.pdf,. Added 1 bitstream(s) on 2016-07-01T13:03:19Z : No. of bitstreams: 1 000858080.pdf: 604042 bytes, checksum: 52a63eb5887518214744f20b5b57d70e (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Trueperella pyogenes são bactérias oportunistas caracterizadas por infecções piogênicas em animais, em geral refratárias aos tratamentos com antimicrobianos convencionais. Recentemente, os genes que codificam a exotoxina piolisina (plo), e fatores relacionados à adesão de T. pyogenes às células do animal susceptível, como fímbrias (fimA, fimC, fimE, fimG), neuraminidases (nanH, nanP), e proteína ligada ao colágeno (cbpA) têm sido associados a virulência do patógeno. O presente estudo investigou a ocorrência de multirresistência dos isolados aos antimicrobianos e a presença dos genes plo, fimA, fimC, fimE, fimG, nanH, nanP e cbpA em 41 linhagens de T. pyogenes isoladas de diferentes afecções em bovinos, caprinos, ovinos, equino e cão. T. pyogenes foi identificado predominantemente em casos de mastite (46,3%), abscessos (19,5%), pneumonia (9,8%), metritie (9,8%), linfadenite (7,3%), seguidos em menor frequência por encefalite (4,9%) e orquite (2,4%). A maior sensibilidade dos isolados foi observada para florfenicol (97,6%), azitromicina (97,6%), tetraciclina (95,2%), ceftiofur (92,7%), penicilina (92,7%), ampicilina (92,7%), gentamicina (90,3%) e eritromicina (87,8%). Em contraste, alta frequência de resistência dos isolados foi observada para bacitracina (46,3%), neomicina (31,7%), lincomicina (31,7%) e sulfametoxazole/trimetoprim (24,3%). O índice de resistência múltipla aos antimicrobianos - IRMA (>0,3) foi encontrado em 13 (31.7%) isolados, particularmente nos casos de mastite bovina. Dentre os isolados multirresistentes a três ou mais grupos de antimicrobianos, 12 (92,3%) foram identificados na espécie bovina, notadamente em infecções mamárias. Os genes mais frequentes detectados nos isolados foram: plo (40/41=97,6%), fimA (36/41=87,9%), nanP (34/41=82,9%), fimE (32/41=78,0%), nanH (28/41=62,3%), fimC (23/41=56,0%) e cbpA (5/41=12,2%). As principais associações de genes nos isolados foram observadas entre... Trueperella pyogenes are opportunistic bacterium characterized by suppurative infections in domestic animals, commonly refractory to conventional therapy. Recently, genes which encode exotoxin pyolysin (plo), and factors that promote adhesion of T. pyogenes to host cells, such as fimbriae (fimA, fimC, fimE, fimG), neuraminidases (nanH, nanP), and collagen-binding protein (cbpA) have been associated to virulence of pathogen. The aim of present study was investigate occurrence of multi-drug resistance of isolates, as well the presence of genes plo, fimA, fimC, fimE, fimG, nanH, nanP, and cbpA in 41 T. pyogenes strains obtaned from bovine, goats, sheep, horses, and dog isolated among different clinical manifestations. T. pyogenes was identified predominantly in mastitis (46.3%), abscesses (19.5%), pneumonia (9.8%), metritis (9.8%), lymphadenitis (7.3%), followed by encephalitis (4.9%) and orchitis (2.4%). The strains showed major in vitro sensitivity to florfenicol (97.6%), azithromicin (97.6%), tetracycline (95.2%), ceftiofur (92.7%), penicillin (92.7%), ampicillin (92.7%), gentamicin (90.3%), and erythromycin (87.8%). In contrast, highest frequency of resistance among isolates was observed to bacitracin (46.3%), neomycin (31.7%), lincomycin (31.7%), and trimethropim/sulfamethoxazole (24.3%). Antimicrobial multiple resistance index - AMRI (>0.3) was found in 13 (31.7%) isolates, particularly in bovine mastitis cases. Among multi-drug resistant isolates to 3 or more group of antimicrobials, 12 (92.3%) were identified in bovine, predominantly in mammary infections. The most common genes detected among isolates were: plo (40/41=97.6%), fimA (36/41=87.9%), nanP (34/41=82.9%), fimE (32/41=78.0%), nanH (28/41=62.3%), fimC (23/41=56.0%), and cbpA (5/41=12.2%). The major associations between genes of isolates were plo, fimA, fimE, nanH, and nanP (10/41=24.4%), and plo, fimA, fimE, fimC, nanH, and nanP (8/41=19.5%), mainly in bovine mastitis. To ...

Tesis - Universidad CEU Cardenal Herrera, Facultad de Veterinaria, Departamento de Producción Animal, Sanidad Animal y Ciencia y Tecnología de los Alimentos, leída el 29-06-2015.

A mastite um dos grandes problemas relacionados com criação de ovinos de leite devido às perdas econômicas decorrentes da redução na produção, gastos com medicamento e perdas de animais. A Contagem de Células Somáticas (CCS) e o isolamento bacteriano são ferramentas diagnósticas indicativas da saúde da glândula mamária que pode ser determinada através de método automatizado e por método manual, através de contagem celular em microscópio, sobre o qual existem discussões quanto à coloração adequada ao leite de ovelhas devido a celularidade diferenciada nesta espécie. Esta tese compreende três estudos que visam realizar o diagnóstico de mastite subclínica em ovelhas leiteiras do Estado Rio Grande do Sul – Brasil em um total de 315 amostras de leite oriundos de casos desta enfermidade em ovelhas de leite. [Capítulo 2] Foram utilizadas 116 amostras com o objetivo de identificar e validar uma metodologia de CCS microscópica adequada à espécie ovina. Assim foram confeccionadas lâminas de esfregaços de 10 μL de leite em 1 cm2, onde foram testados os fixadores de xilol e solução de Carnoy e as colorações de May-Grünwald (MG), Broadhurst- Paley (BP), Wrigth (W) e Panótico (P). Elegeu-se a solução de Carnoy, pois esta permitiu uma melhor fixação do filme lácteo às lâminas de microscopia, e o corante BP, pois permitiu uma boa coloração e visualização das células, bem como a diferenciação dos corpúsculos citoplasmáticos. Os valores de CCS variaram de 2,6x104 a 4,4x106 células.mL-1 e de 1,9x104 a 8,1x106 células.mL-1, respectivamente, pelos métodos automático e microscópico. [Capítulo 3] O objetivo do presente capítulo foi determinar a ocorrência de mastite subclínica em rebanho ovino leiteiro, através do isolamento bacteriano e da CCS. Determinou-se, ainda, a composição (gordura, lactose, proteína e sólidos totais) do leite e a relação entre estes parâmetros e a presença de mastite. Para tanto, coletaram-se 315 amostras de leite, individualizadas por teto, das quais 85 (27%) foram identificadas como mastite subclínica, sendo Staphylococcus coagulase negativos o agente prevalente (64,7%), seguido por Staphylococcus coagulase positivas (19,1%). A CCS variou de 2,5x103 a 9,3x106. Verificou-se CCS significativamente maior (p

Tratamentos de descontaminação pós-processamento podem ser adotados como medida complementar às Boas Práticas de Fabricação e Análise de Perigos e Pontos Críticos de Controle no processo de abate para diminuir a presença de bactérias patogênicas. O objetivo deste estudo foi testar a eficácia antimicrobiana do ozônio aplicado em carcaças suínas durante o armazenamento na câmara fria. Foi conduzido um experimento em cinco blocos consecutivos que incluíram dois tratamentos: grupo controle (T1) constituído por oito carcaças suínas submetidas ao resfriamento (16 horas a 3°C); grupo tratamento (T2) oito carcaças suínas submetidas a dois períodos de quatro horas de aplicação de até 5 ppm de ozônio durante o período de resfriamento (16 horas a 3°C). Para geração do ozônio foi utilizado um equipamento comercial (Alvap®) com cronômetro e medidor de concentração acoplados. A aplicação do ozônio foi iniciada logo após o fechamento da câmara fria. A superfície das carcaças alocadas em cada um dos grupos de tratamento foi amostrada antes e após o período de 16 horas de resfriamento. As amostras foram analisadas quanto à contagem de mesófilos aeróbios totais e presença de Escherichia coli, Salmonella sp. e Listeria sp. Isolados de Salmonella sp. e Listeria monocytogenes foram caracterizados por Eletroforese de Campo Pulsado. As médias de mesófilos aeróbios totais não diferiram significativamente (p>0,05) no grupo T1, antes e depois do resfriamento; enquanto no grupo T2 houve redução significativa (p0,05) no grupo T2 após o tratamento. Os isolados de Salmonella sp. foram identificados como pertencentes aos seguintes sorovares: S. Derby (7), S. Typhimurium (2), S. Agona (10) e Salmonella O:4,5 (1). Todos os sorovares, exceto o último, foram encontrados tanto no grupo T1 quanto no grupo T2. Pulsotipos de S. Agona e S. Derby foram encontrados em carcaças pertencentes ao mesmo bloco do grupo T1, antes e após o resfriamento. Não houve diferença significativa (p>0,05) entre os tratamentos e períodos amostrados na análise de Listeria sp. Nas carcaças positivas, duas espécies foram identificadas: L. inoccua (5) e L. monocytogenes (10). Três pulsotipos foram identificados entre os isolados de L. monocytogenes, sendo que o pulsotipo mais frequente incluiu oito isolados identificados em carcaças do grupo T1 amostradas antes e após o resfriamento. Diante dos resultados obtidos no presente estudo, conclui-se que a aplicação de ozônio na câmara fria, no protocolo testado, foi capaz de reduzir o número de mesófilos aeróbios totais em carcaças suínas, entretanto não foi eficaz na redução de Salmonella sp., Escherichia coli e Listeria sp. Decontamination post-processing treatments can be adopted as a complementary measure to Good Manufacturing Practices and Hazard Analysis and Points of Critical Control Point in slaughter process to reduce the presence of pathogenic bacteria. The objective of this study was to test the antimicrobial effectiveness of ozone applied in pig carcasses during storage in the cooler. The experiment comprised five consecutive blocks that included two treatments: control group (T1) made up of eight pig carcasses subjected to cooling (16 hours at 3°C); treatment group (T2) with eight pig carcasses subjected to two periods of four hours of application of up to 5 ppm of ozone during the cooling period (16 hours at 3°C). For ozone generation, a commercial equipment (Alvap®) with timer and monitor of concentration coupled was used. The application of ozone was initiated immediately after the closing of the cooler. The surface of the animal carcasses allocated to each treatment group was sampled before and after the 16 hour cooling. The samples were analyzed for total aerobic mesophilic count and presence of Escherichia coli, Salmonella sp. and Listeria sp. Isolates of Salmonella sp. and Listeria monocytogenes were characterized by Pulsed Field Gel Electrophoresis. The average of total aerobic mesophilics did not significantly differ (p>0,05) before and after cooling in T1; while in the T2 group it was significantly reduced (p0,05) in T2 after treatment. Salmonella sp. isolates were identified as belonging to the following serovars: S. Derby (7), S. Typhimurium (2), S. Agona (10) and Salmonella O:4.5 (1). All serotypes, except Salmonella O: 4.5 (1), were found in both groups. Pulsotypes of S. Agona and S. Derb were found in carcasses from a same batch of T1, before and after cooling. There was no significant difference (p>0,05) between treatments and periods of sampling regarding Listeria sp. In positive carcasses, two species were identified: L. inoccua (5) and L. monocytogenes (10). Three pulsotypes were identified among the isolates of L. monocytogenes, whereas the most frequent pulsotype included eight isolates identified in group T1 carcasses sampled before as well as after cooling. It was concluded that the tested protocol of ozone application during the cooling step is able to reduce the number of total aerobic mesophilic on pig carcasses, whereas it is not effective in reducing Salmonella sp., Escherichia coli and Listeria sp.

Tesis - Universidad CEU Cardenal Herrera (Valencia), Facultad de Veterinaria, Departamento de Producción y Sanidad Animal, Salud Pública Veterinaria y Ciencia y Tecnología de los Alimentos, leída el 07-05-2015. La estafilococia es una enfermedad ampliamente distribuida en cunicultura industrial que causa grandes pérdidas económicas, debido a la gran variedad de lesiones que genera. Entre estas lesiones, se incluye la mastitis supurativa crónica, que afecta principalmente a las hembras reproductoras. Sin embargo, todavía no se conoce con exactitud la patogénesis de esta lesión, por ello los objetivos de este trabajo fueron:(1) Caracterizar y relacionar las respuestas inmunes local y periférica, en casos naturales de mastitis estafilocócica (2) Establecer una relación entre las respuestas inmunes local y periférica, en casos naturales de mastitis estafilocócica, con las cepas de S. aureusque las provocan (3) Desarrollar un protocolo de infección experimental en glándula mamaria a bajas dosis infectivas (4) Describir las lesiones provocadas por S. aureus en glándula mamaria tras una infección experimental (5) Caracterizar y relacionar las respuestas inmunes local y periférica, en casos experimentales de mastitis estafilocócica y (6) Comparar las respuestas inmunes (local y periférica) así como las lesiones desarrolladas por conejas infectadas de forma natural y experimental. Para conseguir tales objetivos, se realizó un estudio de citometriá de flujo de la sangre de 204 conejas con mastitis estafilocócica crónica con el fin de poder ver la respuesta inmune a nivel periférico, estudiándose las poblaciones de linfocitos B,T(CD5+, CD4+,CD8+ y CD25+), macrófagos y granulocitos. Para poder estudiar la respuesta inmune local, se realizaron estudios de inmunohistoquímica en 89 mamas de las 204 glándulas mamarias afectadas. Las mastitis observadas en los 204 casos se clasificaron según el espectro lesional que mostraron siguiendo los criterios establecidos por Viana et al (2011). Además, se realizaron un estudio microbiológico para saber la cepa de la bacteria que aparecia en cada lesión. También se realizó una infección experimental, a bajas dosis infectivas, para poder estudiar la respuesta inmune de la lesión en las primeras fases infectivas dependiendo de la cepa inoculada. Los resultados estadísticos obtenidos, revelaron que existían diferencias en el número de las células estudiadas (monocitos, granulocitos y linfocitos) dependiendo del tipo de lesión histológica que mostraban las mastitis a nivel tanto periférico como local. La respuesta inmune periférica también varió dependiendo del tipo de cepa que causaba la mastitis, tanto en mastitis naturales como experimentales. Además se observó la exixtencia de una conexión entre la respuesta inmune local y periférica, sobre todo entre la población de monocitos de la sangre y granulocitos del tejído. Tras evaluar los resultados se pudo concluir que: (1) Las poblaciones celulares estudiadas en las mastitis naturales, variaron de forma cuantitativa en función del patrón histomorfológico que presentaban, siendo los linfocitos T de la sangre y los macrófagos del tejido, las células más numerosas en las mastitis supurativas con patrón lobulillar. En cambio, los valores de linfocitos T, tanto a nivel sistémico como local, fueron minoritarios para el grupo de los abscesos compactos. El tipo de fluctuación en el recuento de linfocitos T, que aparece entre las mastitis supurativas con patrón lobulillar y el grupo de los abscesos, hace pensar que forman parte de un mismo proceso lesional. (2) Existe una correlación entre la respuesta inmune local y periférica. (3) El aislado de las cepas bacterianas reveló que en nuestro estudio la cepa ST121 de S. aureus fue la más prevalente seguido de la cepa ST96 con un 88,7% frente a un 4,9%, respectivamente.(4) Las dos cepas más prevalentes producen en condiciones de infección natural, una respuesta inmune diferente, de modo que las cepas pertenecientes al linaje clonal ST96 mostraban en sangre más del doble de linfocitos B (P

The aim of this study was to evaluate the repeatability and performance of 4 methods of extracting DNA from Staphylococcus aureus (SAU) and the gene encoding bovine mitochondrial cytochrome B (BMCB) in milk samples from cows with subclinical mastitis for use in amplification by real-time polymerase chain reaction. Two milk samples were obtained from cows naturally infected with S. aureus and subjected to the following extraction methods: Qiagen DNA extraction kit; Axyprep DNA extraction kit; in silica column boil and in silica column method. After extraction in duplicate, eluates were subjected to purification and precipitation to determine purity (A₂₆₀/A₂₈₀ ratio) and concentration (μg/μL) by spectrophotometry and amplification by real-time polymerase chain reaction of target genes (SAU and BMCB). There was no effect of the DNA extraction method on DNA concentration and threshold cycle for BMCB and SAU. The purity ratio (A₂₆₀/A₂₈₀ ) was higher when using Qiagen DNA extraction (1.76 ± 0.136) compared to the other methods tested. Our results indicate that the DNA extraction kit from Qiagen produces samples of the highest purity ratio compared to other methods.

Staphylococcus pseudintermedius é uma bactéria comensal da pele e mucosa do cão, sendo o principal agente etiológico de piodermite bacteriana superficial e uma das principais causas para prescrição antibiótica em clínica de pequenos animais. O presente estudo teve como objetivo a determinação do perfil de resistência aos antibióticos recomendados pelas linhas de orientação da International Society for Companion Animal Infectious Disease (ISCAID), num grupo de isolados de S. pseudintermedius suscetíveis e resistentes à meticilina, provenientes de cães com piodermite superficial. Avaliou-se, igualmente, a incidência de multirresistência e a aplicabilidade das linhas de orientação da ISCAID neste grupo de isolados. Este constitui um estudo prospetivo, tendo sido utilizados 55 isolados de S. pseudintermedius, testados para a suscetibilidade à oxacilina pela técnica de Kirby-Bauer e, posteriormente, divididos em dois grupos: S. pseudintermedius resistente à meticilina (MRSP, 25/55, 45%) e S. pseudintermedius suscetível à meticilina (MSSP, 30/55, 55%). No grupo MRSP, foi observado 96% de resistência a ambas clindamicina e eritromicina; 92% à amoxicilina-ácido clavulânico, trimetoprim-sulfametoxazol e pradofloxacina; 88% à cefalotina, enrofloxacina e gentamicina; 80% à tetraciclina; 40% à minociclina; 12% ao cloranfenicol e rifampicina; e 8% à amicacina. No grupo MSSP, verificou-se 70% de resistência à tetraciclina; 67% à minociclina; 47% à eritromicina; 43% à clindamicina; 27% ao trimetoprim-sulfametoxazol; 23% ao cloranfenicol; 10% à gentamicina; 7% a ambas enrofloxacina e pradofloxacina; 3% à rifampicina e amicacina; e 0% de resistência à amoxicilina-ácido clavulânico e cefalotina. Foi possível, ainda, verificar que 66% dos isolados demonstraram-se multirresistentes, sendo que 67% eram MRSP e 33% MSSP; 33% demonstraram-se não multirresistentes, dos quais 95% eram MSSP e apenas 5% MRSP. Verifica-se, assim, a existência de níveis elevados de resistência a antibióticos de primeira escolha, muitas vezes utilizados de forma empírica e sem realização de antibiograma prévio. Este facto limita a sua utilização por parte do Médico Veterinário em isolados MRSP, apesar de no grupo MSSP ainda se detetar evidentes suscetibilidades. O número de isolados multirresistentes é, também, alarmante, diminuindo o número de alternativas antibióticas disponíveis em casos cujas opções antibióticas são limitadas, o que ocorre frequentemente em clínica de referência., Staphylococcus pseudintermedius is part of the normal microbiota of canine skin and mucosa. It is also the main etiological pathogen of canine superficial pyoderma, in addition to being a major cause for antimicrobial prescription in small animal practice. This study aimed to determine the resistance of the antibiotics recommended by the guidelines of the International Society for Companion Animal Infectious Disease (ISCAID) guidelines, in a group of S. pseudintermedius isolates susceptible and resistant to methicillin, from dogs with superficial pyoderma. The incidence of multidrug resistance and the applicability of the ISCAID guidelines in this group of isolates was also evaluated. This was a prospective study, where 55 isolates of S. pseudintermedius were tested for oxacillin susceptibility by the Kirby-Bauer technique and, then, divided into two groups: methicillin resistant S. pseudintermedius (MRSP, 25/55, 45%) and methicillin susceptible S. pseudintermedius (MSSP, 30/55, 55%). In the MRSP group, 96% of the isolates evidenced resistance to both clindamycin and erythromycin; 92% to amoxicillin-clavulanic acid, trimethoprim-sulfamethoxazole and pradofloxacin; 88% to cephalothin, enrofloxacin and gentamicin; 80% to tetracycline; 40% to minocicline; 12% to chloramphenicol and rifampicin; and 8% to amikacin. In the MSSP group, 70% of the isolates showed resistance to tetracycline; 67% to minocicline; 47% to erythromycin; 43% to clindamycin; 27% to trimethoprim-sulfamethoxazole; 23% to chloramphenicol; 10% to gentamicina; 7% to both enrofloxacine and pradofloxacin; 3% to rifampicin and 3% to amikacin; and 0% to amoxicillin-clavulanic acid and cephalothin. It was also determined that 66% of the isolates were multiresistant, in which 67% were MRSP and 33% MSSP; 33% were non multiresistant, with 95% of the isolates being MSSP and only 5% MRSP. The results reveal the existence of high levels of resistance to first tier antibiotics, often used empirically and without previous susceptibility testing. This limits their use by the Veterinarian in MRSP isolates, although there are still obvious susceptibilities in the MSSP group. The number of multiresistant isolates is also alarming, decreasing the number of antibiotic alternatives available in cases whose options are limited, which often occurs in reference practice., Orientação: Margarida Alves ; co-orientação: Ana Oliveira

Tesis - Universidad CEU Cardenal Herrera (Valencia), Facultad de Veterinaria, Departamento de Producción y Sanidad Animal, Salud Pública Veterinaria y Ciencia y Tecnología de los Alimentos, leída el 19-06-2015. Salmonella and Campylobacter have been widely recognized as most important zoonotic pathogens with economic impact in animals and humans. Handling or consumption of contaminated poultry meat is considered the most common sources of both human infections. In this sense, the legislators have been working to limit Salmonella and Campylobacter presence; however, high number of salmonellosis and campylobacteriosis are continued declare. In order to lower the risk for human infections, most studies have been focused on the farm level to reduce the number of flocks colonized with these bacteria. Nevertheless, it has been reported that, after processing, carcasses are contaminated even when the flocks are Salmonella or Campylobacter negative at the farm. Therefore, it is necessary to introduce additional treatments to control or inhibit the survival and potential growth of both pathogens in chicken meat. In this context, the aim of this Thesis is to study the Salmonella and Campylobacter contamination of poultry carcasses during the slaughter process and evaluate different modified atmospheres packaging against the main strain isolated at slaughter level.