Occurrence and genetic diversity of Enterocytozoon bieneusi (Microsporidia) in owned and sheltered dogs and cats in Northern Spain.

Enterocytozoon bieneusi is an obligate intracellular protist-like fungi parasite that infects numerous mammal hosts including humans, raising concerns of zoonotic transmission. There is little information available on the presence and diversity of E. bieneusi genotypes in companion animals. Here, we determined the occurrence and genetic diversity of E. bieneusi in domestic dogs and cats from Northern Spain. A total of 336 genomic DNA samples extracted from canine (n = 237) and feline (n = 99) faecal specimens were retrospectively investigated. The presence of E. bieneusi was assessed by PCR of the rRNA internal transcribed spacer (ITS) gene. The parasite was detected in 3.0% (3/99) and 0.8% (2/237) of the cats and dogs examined, respectively. All three feline positive samples were from stray cats living in an urban setting, whereas the two canine samples were from owned dogs living in rural areas. Sequence analysis revealed the presence of two genotypes in dogs, BEB6 and PtEb IX, and two genotypes in cats, D and Peru11. The identification of Peru11 in a cat and BEB6 in a dog constitutes the first report of those genotypes in such hosts as well as first report in Spain. This is also the first evidence of genotype D in cats and PtEb IX in dogs in Spain. Three out of the four genotypes, BEB6, D and Peru11, have been previously reported as human pathogens and are potentially zoonotic indicating that dogs and cats need to be considered potential sources of human infection and environmental contamination.

Detection and molecular diversity of Giardia duodenalis and Cryptosporidium spp. in sheltered dogs and cats in Northern Spain.

Domestic dogs and cats may act as natural reservoirs of a large number of zoonotic pathogens, including the enteric parasites Giardia duodenalis and Cryptosporidium spp., the most relevant protozoan species causing gastrointestinal disease worldwide. A cross-sectional epidemiological study aiming to assess the prevalence and molecular diversity of G. duodenalis and Cryptosporidium spp. was conducted in an animal rescue centre in the province of Álava (Northern Spain). A total of 194 and 65 faecal dropping samples from individual dogs and cats, respectively, were collected between November 2013 and June 2016. G. duodenalis cysts and Cryptosporidium spp. oocysts were detected by direct fluorescence microscopy and PCR-based methods targeting the small subunit ribosomal RNA gene of these parasites. Overall, G. duodenalis and Cryptosporidium spp. were detected in 33% (63/194) and 4.1% (8/194) of dogs, and 9.2% (6/65) and 4.6% (3/65) of cats, respectively. G. duodenalis and Cryptosporidium co-infections were observed in 1.5% (3/194) of dogs, but not in cats. No significant differences in infection rates could be demonstrated among dogs or cats according to their sex, age group, status, or geographical origin. Multi-locus sequence-based genotyping of the glutamate dehydrogenase and ß-giardin genes of G. duodenalis allowed the characterization of 19 canine isolates that were unambiguously assigned to sub-assemblages AII (n=7), BIII (n=1), and BIV (n=7), and assemblages C (n=3) and D (n=1). Two feline isolates were genotyped as assemblages A and F, respectively. No mixed assemblage or sub-assemblage infections were identified. C. canis (n=5) and C. hominis (n=1) were the Cryptosporidium species found in dogs, whereas C. felis (n=1) was identified in cats. The finding of G. duodenalis sub-assemblages AII, BIII, and BIV circulating in dogs (but not cats) may have zoonotic potential, although most of the AII and BIV isolates sub-genotyped corresponded to genetic variants not previously found in Spanish human populations. Dogs may also act as novel suitable hosts for C. hominis. We recommend to considerer companion animals as sentinel surveillance system for zoonotic giardiasis and cryptosporidiosis in order to minimize the risk of spreading of these parasitic diseases among the human population.

Genes and molecules involved in Aspergillus fumigatus virulence.

Aspergillus fumigatus causes a wide range of diseases that include mycotoxicosis, allergic reactions and systemic diseases (invasive aspergillosis) with high mortality rates. Pathogenicity depends on immune status of patients and fungal strain. There is no unique essential virulence factor for development of this fungus in the patient and its virulence appears to be under polygenetic control. The group of molecules and genes associated with the virulence of this fungus includes many cell wall components, such as beta-(1-3)-glucan, galactomannan, galactomannanproteins (Afmp1 and Afmp2), and the chitin synthetases (Chs; chsE and chsG), as well as others. Some genes and molecules have been implicated in evasion from the immune response, such as the rodlets layer (rodA/hyp1 gene) and the conidial melanin-DHN (pksP/alb1 gene). The detoxifying systems for Reactive Oxygen Species (ROS) by catalases (Cat1p and Cat2p) and superoxide dismutases (MnSOD and Cu, ZnSOD), had also been pointed out as essential for virulence. In addition, this fungus produces toxins (14 kDa diffusible substance from conidia, fumigaclavin C, aurasperon C, gliotoxin, helvolic acid, fumagilin, Asp-hemolysin, and ribotoxin Asp fI/mitogilin F/restrictocin), allergens (Asp f1 to Asp f23), and enzymatic proteins as alkaline serin proteases (Alp and Alp2), metalloproteases (Mep), aspartic proteases (Pep and Pep2), dipeptidyl-peptidases (DppIV and DppV), phospholipase C and phospholipase B (Plb1 and Plb2). These toxic substances and enzymes seems to be additive and/or synergistic, decreasing the survival rates of the infected animals due to their direct action on cells or supporting microbial invasion during infection. Adaptation ability to different trophic situations is an essential attribute of most pathogens. To maintain its virulence attributes A. fumigatus requires iron obtaining by hydroxamate type siderophores (ornitin monooxigenase/SidA), phosphorous obtaining (fos1, fos2, and fos3), signal transductional falls that regulate morphogenesis and/or usage of nutrients as nitrogen (rasA, rasB, rhbA), mitogen activated kinases (sakA codified MAP-kinase), AMPc-Pka signal transductional route, as well as others. In addition, they seem to be essential in this field the amino acid biosynthesis (cpcA and homoaconitase/lysF), the activation and expression of some genes at 37 degrees C (Hsp1/Asp f12, cgrA), some molecules and genes that maintain cellular viability (smcA, Prp8, anexins), etc. Conversely, knowledge about relationship between pathogen and immune response of the host has been improved, opening new research possibilities. The involvement of non-professional cells (endothelial, and tracheal and alveolar epithelial cells) and professional cells (natural killer or NK, and dendritic cells) in infection has been also observed. Pathogen Associated Molecular Patterns (PAMP) and Patterns Recognizing Receptors (PRR; as Toll like receptors TLR-2 and TLR-4) could influence inflammatory response and dominant cytokine profile, and consequently Th response to infec tion. Superficial components of fungus and host cell surface receptors driving these phenomena are still unknown, although some molecules already associated with its virulence could also be involved. Sequencing of A. fumigatus genome and study of gene expression during their infective process by using DNA microarray and biochips, promises to improve the knowledge of virulence of this fungus.

Molecular genotyping methods and computerized analysis for the study of Salmonella enterica.

Salmonella enterica is widely recognized as a major cause of foodborne diseases in humans and animals and has been isolated from environmental sources in increasing numbers worldwide. Conventional typing methods such as serotyping and phage typing have been and still are the mainstay in descriptive epidemiology of this microorganism. Nevertheless, limitations on the availability of phage reagents circumscribes the performance of such technique in reference laboratories. The resolving power of epidemiological typing has been expanded during recent years through the molecular analysis of microbial DNA. The broader availability of the reagents and equipment is accelerating their generalized use in clinical and public health laboratories. Important differences in the performance criteria of the genotyping techniques (typability, reproducibility, stability, and discriminatory power) and the convenience criteria (flexibility, accessibility, and ease of use) exist between them, and there is no ideal typing system for universal use. Most of these powerful strain-discriminative techniques are based on comparison of electrophoretic patterns or fingerprints, for which computer-assisted strategies and software packages have been developed to help in construction and analysis of microbial databases. Several initiatives, such as PulseNet (http://www.cdc.gov/pulsenet) or Harmony (http://www.phls.org.uk/inter/harmony), have arisen during recent years for international construction of such fingerprinting databases, which will allow the rapid detection of new strains and the spread of pathogenic clones of bacteria through different regions or countries. Nevertheless, complete consensus has not yet been achieved on the techniques to use or the criteria for interpretation of the results, but these goals may be reached soon.

Computerized restriction endonuclease analysis compared with O-serotype and phage type in the epidemiologic fingerprinting of Pseudomonas aeruginosa strains.

OBJECTIVE: To assess restriction endonuclease analysis (REA) of chromosomal DNA using SalI enzyme, low-concentration (0.4%) agarose gels and digitalized data management of the REA patterns obtained for the typing of clinical Pseudomonas aeruginosa isolates. METHODS: A group of 67 clinical unrelated isolates from 10 Spanish hospitals was used to study the discriminatory power, reproducibility and typeability of REA typing. RESULTS: A SalI REA pattern consisted of a variety (1--10) of restriction bands in the range between 12.2 and 48.5 kb and an unresolvable smear of low-molecular-weight bands. Forty different SalI REA patterns with an index of discrimination of 0.979 were obtained. Low typeability (91.04%) was the major limitation of REA typing. Analysis of blinded subcultures of eight Pseudomonas aeruginosa strains showed the reproducibility of REA typing to be 87.5%. Combined phenotypic typing (O-serotyping and phage typing) performed on the same group of strains showed comparable discrimination but much lower reproducibility. Isolates selected from five clusters of nosocomial infections in hospitals in the UK were typed by REA typing, and the results show high agreement when compared with conventional phenotypic typing methods in distinguishing between strains. CONCLUSIONS: These data underline the usefulness of REA typing enhanced with digitalized data management for the epidemiologic subtyping of clinical Pseudomonas aeruginosa isolates.

Gelatinas preparadas con lácteos: suplemento nutricional útilen centros geriátricos / Jellies prepared with milk products: a useful nutritional supplement in geriatric centres

Introducción El envejecimiento poblacional y el aumento de las enfermedades neurodegenerativas y vasculares hacen de la disfagia un síndrome creciente entre la población mayor, especialmente en los centros geriátricos, donde el número de demencias avanzadas es elevado. En estos pacientes resulta difícil conseguir una ingesta hídrica y energética suficiente y segura, siendo frecuentes las complicaciones de la disfagia como desnutrición, aspiración bronquial e infecciones respiratorias. La gelatina, utilizada principalmente como agua gelificada es junto a los líquidos espesados, una de las formas tradicionales de hidratación de los pacientes con disfagia. Pero, si sustituyéramos el agua por derivados lácteos podríamos mejorar tanto la hidratación como el aporte energético en esa toma. Objetivos Presentar un tipo de gelatinas preparadas con lácteos que puede ser útil como suplemento dietético en la alimentación de personas mayores institucionalizadas con disfagia. Métodos A la hora de elaborar el preparado nutricional se emplean los siguientes ingredientes: leche entera, yogur, fibra soluble y hojas de gelatina. El proceso de elaboración en la cocina de nuestro centro consiste en: 1º) Hidratación de la gelatina en agua fría; 2º Disolución con agua templada y leche; 3º) Mezcla de los yogures con la leche y la fibra. 4º) Añadir la gelatina hidratada a la mezcla anterior; homogeneizar y repartir en recipientes. 5º) Refrigeración hasta el consumo. Resultados Estos preparados son estables en el tiempo y microbiológicamente seguros para su consumo, aportando una media de 82.86 Kcal y 90.44 g de agua por cada unidad. Discusión Con el uso de lácteos gelatinizados, el profesional sanitario puede disponer de una alternativa más en el manejo de los pacientes con disfagia (AU)

Introduction The ageing of the population and the increase in neurodegenerative and vascular diseases mean that dysphagia is an increasingly common condition in the elderly, especially in those geriatric centres wich have a large number of patients with advanced dementia. It is difficult to achieve an adequate, safe liquid and energy intake in these patients and the complications of dysphagia, such as malnutrition, bronchial aspiration and respiratory infections, are frequently seen. The use of gelatine, mainly in the form of jellied water and thickened liquids, is one of the traditional forms of hydration in patients with dysphagia. However, if we were to replace the water with milk derivatives, we could both improve hydration and provide an energy source in the same intake. Objectives To introduce jellied milk products to intensify the elderly people diet with dysphagia who are in geriatrics centres. Methods Trough this study we present a jellied preparation whose ingredients are: whole milk, yoghourt, soluble fibre and commercial gelatine leaves. This is prepared in the kitchen of our centre by the following process: 1º) The gelatine is hydrated by soaking in cold water; 2º It is dissol ved in warm water with milk; 3º) The yoghourt is mixed with the milk and fibre; 4º) The hydra - ted gelatine is added to the mixture, homogenized and divided bet ween the containers; 5º) Refrigeration until it is consumed. Results These preparations are stable over time and microbiologically safe for consumption and provided 82.86 Kcal and 90.44 g of water per unit. Discussion With the use of jellied milk products, healthcare professionals have at their disposal one more alternative for the management of patients with dysphagia (AU)