New Saccharomyces cerevisiae-Kluyveromyces marxianus fusant shows enhanced alcoholic fermentation performance.

Kluyveromyces marxianus has a promising enological potential because of strong extracellular pectinase activity and the copious production of specific higher alcohols and esters. However, K. marxianus is unable to complete alcoholic fermentation on its own. For this reason it is only used in co- or sequential fermentation in conjunction with Saccharomyces spp. Here we applied the protoplast fusion technique to two commercial Saccharomyces cerevisiae strains (Lalvin EC1118® and Lalvin QA23®) and K. marxianus isolate IWBT Y885, to obtain fusants that possess pre-selected, enologically relevant features of K. marxianus, but with improved fermentation performance. After an extensive selection process, performed using non-auxotrophic markers, we identified one fusant, BF2020, that shows enhanced fermentation performance compared to the parental strain K. marxianus Y885, but that still exhibits strong pectinase activity and may be suitable for industrial production. The genome of the selected hybrid was further investigated and characterized by RAPD-PCR analysis and whole genome sequencing.

Raw milk and fecal microbiota of commercial Alpine dairy cows varies with herd, fat content and diet.

The factors that influence the diversity and composition of raw milk and fecal microbiota in healthy commercial dairy herds are not fully understood, partially because the majority of metataxonomic studies involve experimental farms and/or single factors. We analyzed the raw milk and fecal microbiota of 100 healthy cows from 10 commercial alpine farms from the Province of Trento, Italy, using metataxonomics and applied statistical modelling to investigate which extrinsic and intrinsic parameters (e.g. herd, diet and milk characteristics) correlated with microbiota richness and composition in these relatively small traditional farms. We confirmed that Firmicutes, Ruminococcaceae and Lachnospiraceae families dominated the fecal and milk samples of these dairy cows, but in addition, we found an association between the number of observed OTUs and Shannon entropy on each farm that indicates higher microbiota richness is associated with increased microbiota stability. Modelling showed that herd was the most significant factor affecting the variation in both milk and fecal microbiota composition. Furthermore, the most important predictors explaining the variation of microbiota richness were milk characteristics (i.e. percentage fat) and diet for milk and fecal samples, respectively. We discuss how high intra-herd variation could affect the development of treatments based on microbiota manipulation.

Evaluation of virulence factors profiles and antimicrobials resistance of Escherichia coli isolated from bulk tank milk and raw milk filters.

Data on the presence of pathogenic Escherichia coli in bulk tank milk (BTM) and raw milk filters (RMF) are not available in Italy and there are few studies worldwide. Therefore, a study under field condition was conducted to assess the presence of E.coli pathogenic and commensal (CoEC) strains in BTM and RMF samples and their associated AMR pattern. One hundred forty-nine E.coli isolates were characterized. Among all the isolates, 53 (35.6%) were classified as pathogenic while the other ones were classified as CoEC. Among the pathogenic ones, 23 (54.7%) were classified as enterotoxigenic E.coli (ETEC), 6 (11.3%) as enteroinvasive E.coli (EIEC), 2 (3.8%) as enteroaggregative E.coli (EAEC), 12 (22.6%) harboured virulence factors (VF) common to ETEC+EIEC, and 2 (3.8%) common to ETEC+EAEC. To our knowledge, it is the first time that ETEC isolates harboring VF associated with EAEC or EIEC are observed in raw milk. These data support the presence of transmission of VFs genes among isolates. None of the isolates showed resistance to three or more antimicrobials. The CoEC role as a vector of AMR was confirmed by the presence of 18% ampicillin- and cephalexin-resistant isolates. The presence of AMR in CoEC supports the role of these bacteria as source of resistance genes. Monitoring raw milk by either BTM or RMF analysis, and the relatively cheap procedure applied to identify E.coli pathotypes can be useful to identify hazards related to the spread of enteric diseases and antimicrobial resistance.

Effects of herd and physiological status on variation of 16 immunological and inflammatory parameters in dairy cows during drying off and the transition period.

During drying off and transition period, cows are subject to changes in endocrine status, metabolic stressors and altered immune functions, which could lead to an increased risk of disease. To expand our knowledge on the immune/inflammatory status and to identify markers to define cow status during this interval, the pattern of 9 different cellular parameters, 5 cytokines, 2 enzymes and 3 cellular ratios in blood samples were assessed in 15 primiparous cows belonging to three different dairy herds in Lombardy. Our data showed that the variation of almost all parameters was influenced by the physiological period in which the samples were collected, except for apoptosis, IL-1ß, IL-6, lysozyme and granulocyte/monocyte ratio. Several markers were directly correlated either to the herd alone (IL-1ß, IL-6, lysozyme, granulocyte/lymphocyte ratio and granulocyte/monocyte ratio) or in association with the sampling time (white blood cell count, necrosis, lymphocytes count, CD4+ lymphocytes proportion). Hierarchical cluster analysis identified three herd-associated sample clusters showing different frequency along the follow-up period. The results of this field study highlight the importance of the herd factor in the immune/inflammatory response. Furthermore, these results suggest that cellular parameters are probably the most suitable markers to define cow status during drying-off and the peripartum period.

A new integrated approach to analyze bulk tank milk and raw milk filters for the presence of the E. coli serogroups frequently associated with VTEC status.

We optimized a combination of microbiological and molecular methods to quickly identify the presence of the O157 and the six non-O157 serogroups (O26, O45, O103, O111, O121 and O145) most frequently associated with VTEC status, at herd level. The lower detection limit of this methodology is 101CFU/ml for each of the serogroups tested. We tested 67 bulk tank milk (BTM) and raw milk filters (RMF) derived from dairy herds located in Lombardy and Trentino Alto Adige. We identified 3 positive samples and 20 positive samples out of 67 respectively in the BTM and RMF. Interestingly, several samples showed positivity for more than one serogroups at the same time. We also identified the presence of E. coli O45 and O121 for the first time in raw milk and raw milk filters. Once screened the seven serogroups of interest in our samples, we evaluated the real pathogenicity of our positive, non-O157 samples through two parallel molecular biology methods: virulence gene research by PCR, and HRMA and sequencing. The most frequently isolated serogroups in milk were O157 (2.64%), O103 (2.11%), and O145 (1.06%), while in RMF the frequencies were, respectively 14.92%, 4.48%, and 2.98%. Moreover, this is the first published report in Italy of positive recovery of O45 and O121 serogroups in milk and milk filters. The new diagnostic approach proposed investigate the presence of the O157 and big six non-O157 serogroups at farm level and not to identify VTEC hazard only once the product is processed and/or is ready to be consumed.

High-resolution melting analysis of gyrA codon 84 and grlA codon 80 mutations conferring resistance to fluoroquinolones in Staphylococcus pseudintermedius isolates from canine clinical samples.

Staphylococcus pseudintermedius is an opportunistic pathogen of dogs and cats. A high-resolution melting analysis (HRMA) protocol was designed and tested on 42 clinical isolates with known fluoroquinolone (FQ) susceptibility and gyrA codon 84 and grlA codon 80 mutation status. The HRMA approach was able to discriminate between FQ-sensitive and FQ-resistant strains and confirmed previous reports that the main mutation site associated with FQ resistance in S. pseudintermedius is located at position 251 (Ser84Leu) of gyrA. Routine, HRMA-based FQ susceptibility profiles may be a valuable tool to guide therapy. The FQ resistance-predictive power of the assay should be tested in a significantly larger number of isolates.

Identification of Ixodes ricinus blood meals using an automated protocol with high resolution melting analysis (HRMA) reveals the importance of domestic dogs as larval tick hosts in Italian alpine forests.

BACKGROUND: In Europe, Ixodes ricinus L. is the main vector of a variety of zoonotic pathogens, acquired through blood meals taken once per stage from a vertebrate host. Defining the main tick hosts in a given area is important for planning public health interventions; however, until recently, no robust molecular methods existed for blood meal identification from questing ticks. Here we improved the time- and cost-effectiveness of an HRMA protocol for blood meal analysis and used it to identify blood meal sources of sheep tick larvae from Italian alpine forests. METHODS: Nine hundred questing nymphs were collected using blanket-dragging in 18 extensive forests and 12 forest patches close to rural villages in the Province of Trento. Total DNA was either extracted manually, with the QIAamp DNA Investigator kit, or automatically using the KingFisher™ Flex Magnetic Particle Processors (KingFisher Cell and Tissue DNA Kit). Host DNA was amplified with six independent host group real-time PCR reactions and identified by means of HRMA. Statistical analyses were performed in R to assess the variables important for achieving successful identification and to compare host use in the two types of forest. RESULTS: Automating DNA extraction improved time- and cost-effectiveness of the HRMA protocol, but identification success fell to 22.4% (KingFisher™) from 55.1% (QIAamp), with larval hosts identified in 215 of 848 questing nymphs; 23 mixed blood meals were noted. However, the list of hosts targeted by our primer sets was extended, improving the potential of the method. Host identification to species or genus level was possible for 137 and 102 blood meals, respectively. The most common hosts were Rodentia (28.9%) and, unexpectedly, Carnivora (28.4%), with domestic dogs accounting for 21.3% of all larval blood meals. Overall, Cetartiodactyla species fed 17.2% of larvae. Passeriformes (14.6%) fed a significantly higher proportion of larvae in forest patches (22.3%) than in extensive forest (9.6%), while Soricomorpha (10.9%) were more important hosts in extensive forest (15.2%) than in forest patches (4.3%). CONCLUSIONS: The HRMA protocol for blood meal analysis is a valuable tool in the study of feeding ecology of sheep ticks, especially with the cost- and time- reductions introduced here. To our knowledge, we show for the first time that domestic dogs are important larval hosts in the Alps, which may have possible implications for tick-borne disease cycles in urbanized areas.

Identifying the last bloodmeal of questing sheep tick nymphs (Ixodes ricinus L.) using high resolution melting analysis.

The sheep tick, Ixodes ricinus L., is an important hematophagous vector of zoonotic disease of both veterinary and public health importance in Europe. Risk models for tick-borne diseases can be improved by identifying the main hosts of this species in any given area. However, this generalist tick stays on a host for only a few days a year over its life cycle, making the study of its feeding ecology difficult. In contrast, ticks can easily be collected from vegetation when they are questing. Molecular methods have proved to be a reliable alternative to field observation, but most current methods have low sensitivity and/or low identification success (i.e. hosts are only identified to taxonomic levels higher than species). In this study we use Real-time PCR coupled with High Resolution Melting Analysis (HRMA) to identify the source of the last bloodmeal in questing tick nymphs. Twenty of the most important tick hosts were grouped taxonomically and six group-specific primer sets, targeting short mitochondrial DNA regions, were designed de novo. Firstly, we show that these primers successfully amplify target host DNA (from host tissue or engorged ticks), and that HRMA can be used to reliably identify hosts to species (or genera in the case of Sorex and Apodemus). Secondly, the new protocol was tested on field-collected questing nymphs. Bloodmeal source was identified in 65.4% of 52 individuals. In 83.3% of these, the host was identified to species or genera using HRMA alone. Moreover, the primer sets designed here can unequivocally identify mixed bloodmeals. The combination of sensitivity and identification success together with the closed-tube and single step approach that minimizes contamination, make Real-time HRMA a good alternative to current methods for bloodmeal identification.

Rapid differentiation of Dirofilaria immitis and Dirofilaria repens in canine peripheral blood by real-time PCR coupled to high resolution melting analysis.

Dirofilaria immitis and D. repens are the principal causative agents of canine filariosis and, although the number of dogs subjected to specific prevention is increasing, the prevalence of these parasites remains high in many areas of the world. The discrimination between the two Dirofilaria species using the classical diagnostic methods can be difficult and may lead to misdiagnosis especially on samples from areas where both Dirofilaria are present. Over the last years, several molecular methods with higher sensitivity and specificity compared to classical microscopy and ELISA assays were designed. Nevertheless, a need for simple, rapid, and cost-effective molecular protocols to accurately discriminate between D. immitis and D. repens still remains. High resolution melting analysis coupled to real-time PCR (real-time PCR-HRMA) is a widely used technique to target sequence polymorphisms of the same gene in different species without the need to perform DNA sequencing or to use species-specific probes. In this work, a fast and cost-effective real-time PCR-HRMA protocol to detect and differentiate simultaneously and unequivocally D. immitis and D. repens microfilarial DNA extracted from peripheral dog blood samples is described. The present method is simpler to use than most other DNA-based methods and provides comparable discrimination between the two sibling species.

Detection of Dirofilaria immitis in mid-western arid Argentina.

Dirofilariosis, caused by Dirofilaria immitis and D. repens, is (re-) emerging worldwide. Dogs are the main reservoirs, while human infection has recently become an important focus of interest and attention. In Argentina, canine D. immitis infection has been described in eastern and northern subtropical and temperate humid regions, but never reported in mid-western arid regions so far. In this research note we report for the first time the occurrence of autochthonous human and canine D. immitis infection in the region.

The expression ratio of miR-17-5p and miR-155 correlates with grading in canine splenic lymphoma.

In dogs as in humans, microRNAs (miRNAs) play a key role in normal and neoplastic hematopoiesis regulation. The general miRNA expression framework varies among different stages of development and differentiation of tumors, and miRNAs are widely investigated as new molecular tools for cancer diagnosis and classification. Canine lymphomas are currently classified according with the WHO classification, but a comprehensive grading study of clinical samples is still lacking, and molecular tools for quick grading are not yet available. In the present work, a retrospective study of the expression profile of a panel of miRNAs in canine primary splenic lymphomas was performed. The formalin fixed, paraffin embedded (FFPE) lymphoma samples were accurately classified according with the WHO classification, and were analyzed for miRNA expression using stem-loop TaqMan real time RT-PCR. For each miRNA investigated, relative and absolute quantification were performed after selecting the best housekeeping genes using the NormFinder and geNorm algorithms. The results of this study show a diversity in miRNA expression in low (L) grade lymphomas compared to intermediate-high (I-H) grade lymphomas. The molar ratio between miR-17-5p and miR-155 correlated with WHO grading. These results highlight the potential use of miR-17-5p/miR-155 molar ratio as a new molecular tool for grading of canine splenic lymphomas. The data here reported further support the utility of monitoring miRNA expression in canine hematopoietic malignancies diagnosis and prognosis.

Epidural dirofilariosis in a paraparetic cat: case report of Dirofilaria immitis infection.

A 6-year-old neutered female cat was examined for chronic and progressive pelvic limb ataxia that progressed to non-ambulatory paraparesis over 1 month. Haematological and serum analyses were mainly within normal ranges. Thoracic and abdominal radiographs did not reveal any morphological abnormalities. Magnetic resonance imaging investigation of the thoraco-lumbar spine demonstrated a well-defined, extradural mass that extended into the epidural space from the L2 to L3 vertebral bodies and expanded in the L2 to L3 left intervertebral foramen. During surgery, a long, narrow, white parasite which was weakly adherent to the phlogistic epidural fat tissue was gently removed from the spinal canal. Histological examination of the pathological tissue supported a diagnosis of epidural steatitis surrounding a female adult Dirofilaria immitis. This is a novel case of natural D immitis infection with spinal localisation in a cat, well documented with magnetic resonance investigation, and cytological and histological examinations, introducing a novel differential diagnosis for extradural spinal masses in cats.

Escherichia coli lipopolysaccharides and Staphylococcus aureus enterotoxin B differentially modulate inflammatory microRNAs in bovine monocytes.

MicroRNAs (miRNAs) are a family of regulatory molecules involved in many physiological processes, including activation of cells of the immune system. This study investigated the effect of Escherichia coli lipopolysaccharide (LPS) and Staphylococcus aureus enterotoxin B (SEB) on the expression of five miRNAs involved in the inflammatory response, including miR-9, miR-125 b, miR-155, miR-146 a and miR-223, in bovine CD14(+) cells (monocytes). Incubation of monocytes with SEB induced down-regulation of miR-155, miR-223 and miR-125 b, but not the anti-inflammatory miRNA miR-146 a. Conversely, incubation with LPS upregulated both miR-155 and miR-146 a. In vitro incubation of isolated CD14(+) bovine monocytes with LPS and SEB elicited different and opposite expression of miRNAs reportedly involved in inflammatory reactions.