Genotypic and phenotypic characterization of a Salmonella Typhimurium strain resistant to pulsed electric fields

Pulsed Electric Fields (PEF) technology is regarded as one of the most interesting alternatives to current food preservation methods, due to its capability to inactivate vegetative microorganisms while leaving the product's organoleptic and nutritional properties mostly unchanged. However, many aspects regarding the mechanisms of bacterial inactivation by PEF are still not fully understood. The aim of this study was to obtain further insight into the mechanisms responsible for the increased resistance to PEF of a Salmonella Typhimurium SL1344 variant (SL1344-RS, Sagarzazu et al., 2013), and to quantify the impact that the acquisition of PEF resistance has on other aspects of S. enterica physiology, such as growth fitness, biofilm formation ability, virulence and antibiotic resistance. WGS, RNAseq and qRT-PCR assays indicated that the increased PEF resistance of the SL1344-RS variant is due to a higher RpoS activity caused by a mutation in the hnr gene. This increased RpoS activity also results in higher resistance to multiple stresses (acidic, osmotic, oxidative, ethanol and UV-C, but not to heat and HHP), decreased growth rate in M9-Gluconate (but not in TSB-YE or LB-DPY), increased ability to adhere to Caco-2 cells (but no significant change in invasiveness) and enhanced antibiotic resistance (to six out of eight agents). This study significantly contributes to the understanding of the mechanisms of the development of stress resistance in Salmonellae and underscores the crucial role played by RpoS in this process. Further studies are needed to determine whether this PEF-resistant variant would represent a higher, equal or lower associated hazard than the parental strain

Comparative study of real-time pcr (Taqman probe and sybr green), serological techniques (elisa, ifa and dat) and clinical signs evaluation, for the diagnosis of canine leishmaniasis in experimentally infected dogs

Canine leishmaniasis (CanL) diagnosis is not fully resolved. Currently, two specific methodologies are in continuous development, the detection of the parasite DNA or RNA in target organs and the detection of specific antibodies against Leishmania sp. For a correct diagnosis, it has been shown that the joint use of this type of test is necessary. In this work, a Sybr Green and a TaqMan Probe based on real time PCRs (qPCR) was performed for the detection of Leishmania sp. in order to correlate the results with clinicopathological and serological evaluations (IFA, ELISA and DAT) to propose an optimal biological sample to be used to detect the parasite in both early and late stages of the infection. A total of four samples were processed: conjunctival swabs, popliteal lymph node aspirates, bone marrow aspirates, and peripheral blood from experimentally infected dogs belonging to a larger study. Our results indicated that a single non-invasive sample (conjunctival swab) and the application of both types of qPCR would be reliable for determining Leishmania infection as well as the disease stage in dogs, thus avoiding bone marrow, lymph node aspirate or blood samples collection. © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/)

Identificación de parvovirus canino tipo 2C en cachorros de Nicaragua

Objetivo. Identificar los genotipos de parvovirus canino-circulantes en cachorros en dos municipios de Nicaragua. Materiales y métodos. Se recolectaron muestras por hisopado rectal de 45 cachorros con y sin antecedentes de vacunación, con menos 6 meses de edad, con y sin sintomatología compatible con parvovirosis. Las muestras y dos de las vacunas que se comercializan en Nicaragua (vacuna nº1 y vacuna nº2) fueron analizadas por Reacción en Cadena de la Polimerasa (PCR) convencional para un producto de ˜ 630 pb del gen VP2. Además, se secuenciaron en sentido inverso cuatro muestras de campo elegidas al azar y ambas cepas de vacunas. Resultados. El 28.9% (13/45) de las muestras analizadas fueron positivas en PCR. No se encontraron diferencias significativas en la detección por PCR del fragmento de VP2, respecto al estado de vacunación de los animales (p=0.05). Las cuatro muestras de campo secuenciadas fueron identificadas como genotipo CPV-2C y las dos cepas vacunales se identificaron como genotipo CPV-2A. Conclusiones. La inferencia evolutiva de las secuencias alineadas de cepas vacunales mostró alta divergencia evolutiva respecto a las cepas de campo. Este hallazgo lleva a replantear el tema sobre la eficacia de las vacunas analizadas en este trabajo y que son aplicadas en Nicaragua. Objective. To identify genotypes of canine parvovirus circulating in puppies in two municipalities of Nicaragua. Materials and methods. Rectal swab samples from 45 puppies less under 6 months of age were collected and processed for presence of parvovirus bur conventional PCR technique. Puppies might or not have been vaccinated and with or without parvovirus infection symptoms. Two commercially available parvovirus vaccines in Nicaragua (vaccine no1 and vaccine no2) were also analyzed by conventional Polymerase Chain Reaction (PCR) resulting in a product of approximate to 630 bp of the VP2 gene. In addition, Sanger sequences of four randomly chosen field samples and both vaccine strains were obtained. Results. 28.9% (13/45) of the analyzed samples were positive by PCR, for CPV VP2 gene. No statistically significant differences (p >= 0.05) were obtained in PCR detection between dogs with or without vaccination history. The four sequenced field samples were identified as CPV-2C genotype while both vaccine strains were identified as CPV-2A genotype. Conclusions. The aligned sequences showed high evolutionary divergence of filed strains with respect to vaccines strains, leading us to reconsider the efficacy of the analyzed vaccines commercially available in Nicaragua nowadays

A cross-sectional epidemiological study of domestic animals related to human leptospirosis cases in Nicaragua

Leptospirosis is one of the most extended zoonosis worldwide and humans become infected most commonly through contact with the urine of carrier animals, either directly or via contaminated water or soil. The aim in this study was to analyse the epidemiological behaviour of Leptospira spp., from domestic animals around the sites of human leptospirosis cases in Nicaragua, from 2007 through 2013. We report the results of a cross-sectional epidemiological study with a non-probability sampling of blood (n = 3050) and urine (n = 299) from Domestic Animals (DA) around the sites of human leptospirosis cases in Nicaragua. We analysed data obtained through Microscopic Agglutination Test (MAT), in-vitro culture, real time PCR and sequencing of lfb1 locus. Frequencies of 30.31% (95% CI: 28.66–31.95) and 15.38% (95% CI: 11.12–19.64) were obtained from serological test and from in-vitro culture, respectively. Although similar frequencies from serology test (P = 0.05) were found in DA species, in-vitro culture frequencies were significantly higher from bovine, equine and sheep (P < 0.05) in comparison with swine and canine species. Ten serogroups of pathogenic Leptospira spp. were encountered, with the highest presence of Icterohaemorrhagiae serogroup 34.65% (95% CI: 29.35–39.94). We identified 7 samples homologous to L. interrogans species Pyrogenes serovar and 3 samples as L. noguchii Louisiana or Panama serovars by analysis of lfb1 sequences. We were able to establish a temporal and spatial correlation from DA and cumulative incidence of human cases. Therefore an effective epidemiological surveillance should be implemented with a specific control program toward DA in order to reduce human leptospirosis incidence

Characterization of the spoilage microbiota of hake fillets packaged under a modified atmosphere (MAP) rich in CO2 (50% CO2/50% N2) and stored at different temperatures

The aim of this study was to characterize the spoilage microbiota of hake fillets stored under modified atmospheres (MAP) (50% CO2/50% N2) at different temperatures using high-throughput 16S rRNA gene sequencing and to compare the results with those obtained using traditional microbiology techniques. The results obtained indicate that, as expected, higher storage temperatures lead to shorter shelf-lives (the time of sensory rejection by panelists). Thus, the shelf-life decreased from six days to two days for Batch A when the storage temperature increased from 1 to 7 °C, and from five to two days—when the same increase in storage temperature was compared—for Batch B. In all cases, the trimethylamine (TMA) levels measured at the time of sensory rejection of hake fillets exceeded the recommended threshold of 5 mg/100 g. Photobacterium and Psychrobacter were the most abundant genera at the time of spoilage in all but one of the samples analyzed: Thus, Photobacterium represented between 19% and 46%, and Psychrobacter between 27% and 38% of the total microbiota. They were followed by Moritella, Carnobacterium, Shewanella, and Vibrio, whose relative order varied depending on the sample/batch analyzed. These results highlight the relevance of Photobacterium as a spoiler of hake stored in atmospheres rich in CO2. Further research will be required to elucidate if other microorganisms, such as Psychrobacter, Moritella, or Carnobacterium, also contribute to spoilage of hake when stored under MAP

Epidemiología molecular de Bartonella henselae en gatos callejeros y de albergue en Zaragoza, España

Fundamentos: Bartonella henselae produce la enfermedad del araña- zo del gato en las personas y se considera infradiagnosticada. El objetivo fue detectar y cuantificar la carga de ácido desoxiribonucleico (ADN) de B. henselae en muestras de sangre y orales de gatos callejeros y de albergue de Zaragoza, España y analizar su relación con factores epidemiológicos y clínicos. Métodos: Se estudiaron 47 gatos. El ADN de B. henselae,se detectó mediante reacción en cadena de la polimerasa en tiempo real (qPCR) en sangre y muestras orales. Se usó el paquete estadístico SPSS para analizar la positividad de las muestras pareadas y su relación con factores epidemioló- gicos (edad, sexo, origen, mes de muestreo, presencia de pulgas/garrapatas) y clínicos (estado de salud y presencia de lesiones orales). Se realizó un análisis de regresión logística para conocer la asociación entre la presencia en sangre y cavidad oral y el resto de las variables. Resultados: el 23,40% de las muestras de sangre y el 27,65% de las orales portaba el ADN de B. henselae. Se observó débil correlación de la positividad de las muestras pareadas (kappa= 0,33; p 0,05) entre la presencia de ADN de B. henselae en las muestras y los factores epidemiológicos y clínicos. Los gatos con lesiones orales portaban una carga más elevada de ADN (3,12/1x106 células) en la boca que los que no tenían lesiones (2,58 /1por106 células), (p=0,032). Conclusiones: La detección de ADN de B. henselae en sangre no pare- ce estar relacionada con su presencia en cavidad oral y viceversa. Los gatos positivos con lesiones orales pueden significar mayor riesgo de infección por B. henselae para las personas que los manejan

Estudio de la eficacia de una vacuna frente a la Lactococosis de la trucha arco iris (Oncorhynchus mykiss) a diferentes temperaturas.

Lactococcus garvieae es el agente etiológico de la Lactococosis, considerada actualmente comouna enfermedad emergente responsable de graves pérdidas económicas tanto en acuiculturacontinental como marina cuando la temperatura del agua supera los 16ºC. En la actualidad, lavacunación con vacunas inactivadas se ha establecido como el método más eficaz para controlarel proceso.En el presente trabajo hemos evaluado la eficacia de una vacuna comercial (Icthiovac-Lg) frentea una infección experimental con L. garvieae en trucha arco iris (Oncorhynchus mykiss) habiendovacunado los peces a diferentes temperaturas del agua.Dos grupos de 50 truchas de 30-35 g de peso fueron vacunadas a temperaturas de 8 y 15ºCrespectivamente. Fueron mantenidas en estas condiciones junto con otras 50 truchas más sinvacunar en cada grupo durante 30 días, momento en el cual se procedió a realizar una infecciónexperimental vía intraperitoneal con una cepa patógena de L. garvieae. Los peces se mantuvieronen observación durante 21 días más. Al final del estudio se obtuvo un valor de RPS (PorcentajeRelativo de Supervivencia) para el grupo vacunado a temperatura alta de 96% mientras quepara el grupo vacunado a 8ºC fue de 83%

Simultaneous Identification of DNA and RNA Viruses Present in Pig Faeces Using Process-Controlled Deep Sequencing

Background: Animal faeces comprise a community of many different microorganisms including bacteria and viruses. Only scarce information is available about the diversity of viruses present in the faeces of pigs. Here we describe a protocol, which was optimized for the purification of the total fraction of viral particles from pig faeces. The genomes of the purified DNA and RNA viruses were simultaneously amplified by PCR and subjected to deep sequencing followed by bioinformatic analyses. The efficiency of the method was monitored using a process control consisting of three bacteriophages (T4, M13 and MS2) with different morphology and genome types. Defined amounts of the bacteriophages were added to the sample and their abundance was assessed by quantitative PCR during the preparation procedure. Results: The procedure was applied to a pooled faecal sample of five pigs. From this sample, 69,613 sequence reads were generated. All of the added bacteriophages were identified by sequence analysis of the reads. In total, 7.7 % of the reads showed significant sequence identities with published viral sequences. They mainly originated from bacteriophages (73.9%) and mammalian viruses (23.9%); 0.8 % of the sequences showed identities to plant viruses. The most abundant detected porcine viruses were kobuvirus, rotavirus C, astrovirus, enterovirus B, sapovirus and picobirnavirus. In addition, sequences with identities to the chimpanzee stool-associated circular ssDNA virus were identified. Whole genome analysis indicates that this virus, tentatively designated as pig stool-associated circular ssDNA virus (PigSCV), represents a novel pi