First description of a natural infection with spleen and kidney necrosis virus in zebrafish

Zebrafish has become a popular research model in the last years, and several diseases affecting zebrafish research facilities have been reported. However, only one case of naturally occurring viral infections was described for this species. In 2015, infectious spleen and kidney necrosis virus (ISKNV) was detected in zebrafish from a research facility in Spain. Affected fish showed lethargy, loss of appetite, abnormal swimming, distention of the coelomic cavity and, in the most severe cases, respiratory distress, pale gills and petechial haemorrhages at the base of fins. Cytomegaly was the most relevant histopathological finding in organs and tissues, sometimes associated to degenerative and necrotic changes. ISKNV belongs to the relatively newly defined genus Megalocytivirus, family Iridoviridae, comprising large, icosahedral cytoplasmic DNA viruses. This is the first case of naturally occurring Megalocytivirus infection in zebrafish research facilities, associated with morbidity. The virus has been identified based on both pathologic and genetic evidence, to better understand the pathogenesis of the infection in zebrafish and the phylogenetic relationship with other iridoviruses. Given the ability of megalocytiviruses to cross‐species boundaries, it seems necessary to implement stringent biosecurity practices as these infections may invalidate experimental data and have major impact on laboratory and cultured fish.     

Changes in Gut Microbiota Linked to a Reduction in Systolic Blood Pressure in Spontaneously Hypertensive Rats Fed an Extra Virgin Olive Oil-Enriched Diet

Fat type in diet is responsible for specific changes in gut microbiota (GM). Extra virgin olive oil (EVOO) has been shown to be beneficial for blood pressure and to produce effects on GM. To analyze the cause-effect relationship between intestinal microbial changes and blood pressure, we studied the effect of EVOO on fecal microbiota and systolic blood pressure (SBP) levels in spontaneously hypertensive rats (SHR). SHR were fed either an enriched EVOO diet or a standard diet for a period of 12 weeks. At the end of the experimental period, the microbial profiles in the feces were studied in both groups by using PCR-denaturing gradient gel electrophoresis. Real-time PCR was used to quantify the selected bacterial groups. The results demonstrated significant differences when using Lactobacillus (p<0.05), clostridia XIV (p<0.01) and universal (p<0.05) primers. A significant (r=?0.475; p=0.04) inverse correlation between the abundance of clostridia XIV and SBP, which depends on the type of diet, was also observed. Finally, the results suggested an increase in the microbial diversity of the feces of the animals fed the EVOO diet. These results strongly connect the pattern of GM in SHR fed a diet enriched with EVOO to the lower levels of SBP observed in these animals at the end of the feeding period.     

Production of Hyaluronic Acid by Streptococcus zooepidemicus on Protein Substrates Obtained from Scyliorhinus canicula Discards

This work investigates the production of hyaluronic acid (H) by Streptococcus equi subsp. zooepidemicus in complex media formulated with peptones obtained from Scyliorhinus canicula viscera by-products. Initially, in batch cultures, the greatest productions were achieved using commercial media (3.03 g/L) followed by peptones from alcalase hydrolyzed viscera (2.32 g/L) and peptones from non-hydrolyzed viscera (2.26 g/L). An increase of between 12% and 15% was found in subsequent fed-batch cultures performed on waste peptones. Such organic nitrogen sources were shown to be an excellent low-cost substrate for microbial H, saving more than 50% of the nutrient costs.     

Effect of salicylic acid treatment on tomato plant physiology and tolerance to potato virus X infection

Salicylic acid (SA) is an inducer of systemic acquired resistance (SAR) and could be a potential candidate in the control of plant virus diseases. In this study we assayed under controlled conditions the potential effect of three doses of exogenous SA treatment on tomato plants infected with Potato virus X (PVX) and measured their effects on: different physiological parameters (gas exchange, stable isotopes, chlorophyll content), the activation of secondary metabolism, viral accumulation and induction of the expression of pathogenesis-related proteins (PRs) such as ß-1, 3-glucanase (PR2) and chitinase (PR3). SA treatment increased the expression of PR2, the activity of phenylalanine ammonia lyase (PAL) and the concentration of antioxidant compounds at 7 days post-treatment. Earlier expression of PR3 compared to PR2 was observed. SA treatment delayed the detection of PVX by ELISA in uninoculated leaves of mechanically infected tomato plants. Although the effect of PVX infection on physiological parameters was weak, moderate SA treatments showed enhanced photosynthesis, particularly for infected plants. The results obtained confirm that SA promotes major changes in the induction of resistance in tomato plants and suggest that treatment with exogenous SA could be considered to reduce the infections caused by PVX.     

Nomenclature and genetic relationships of apples and pears from Terceira Island

Heritage apple (Malus domestica Borkh. hybrids) and pear (Pyrus communis L. hybrid) trees grow in villages throughout Terceira Island, Azores, Portugal. Some of these pears have different names but similar morphology. The objective of this study was to determine synonymy, homology, and phylogeny of apples and pears from Terceira and to examine potential relationships of the island pears with standard apples and pears of Portuguese or American descent. Nine apple microsatellite markers were used to determine genetic relationships. Distance- and parsimony-based cluster analysis grouped these genotypes into separate apple and pear clades. The Terceira apples were divided into two clades: the maçā and the reineta-reinette. Among the 17 heritage apple genotypes, seven unique accessions were identified and four groups of synonyms, or possibly clones, were detected including: ‘Reineta Agosto’ and ‘Reineta Verde’ from Altares; ‘Reineta Castanha’ and ‘Reineta Verde Miuda’; ‘Maçā Pêra,’ ‘Maçā Calhau’, ‘Pêro Branco’ from Salga and from Terra-Chā and ‘Maçā Marmelo’; and the five genotypes ‘Maçā Sao Joao’, ‘Malápio Rosa’, ‘Maçā Gaspar’, ‘Maçā Branca’ and ‘Maçā Pato’. In addition, two homonyms were detected. ‘Pêro Vermelho’ from Terra Chā was a separate genotype from a tree from Doze Ribeiras of the same name, but Pêro Branco from Terra Chā appears to be a clone that can be distinguished by an additional allele at CH1F07a from a tree with that name from Salga. One pair of apple clones, ‘Reineta Agosto’ and ‘Reineta Verde’ from Altares appear to be derived from an unreduced gamete of ‘Golden Delicious.’ Another apple genotype ‘Maçā Acida’ could be a sibling of the ‘Maçā Pêra’ clonal group. Other tested standard apples from the US genebank were unrelated to Terceira genotypes. Of the seven heritage pears, five unique genotypes and one pair of synonyms were detected. ‘Pêra Papo Pintassilgo’ from Raminho and ‘Pêra Vermelha’ from the nursery of Serviço de Desenvolvimento Agario da Terceira (SDAT) were synonyms. ‘Passans du Portugal’ was related to ‘Pêra Cabaca’ but other standard pears from the US genebank were unrelated to Terceira genotypes. Future studies will include additional apple and pear cultivars from other Islands of the Azores and continental Portugal, and wild Asian species to further explore genetic relationships.     

Ixodid ticks parasitizing Iberian red deer (Cervus elaphus hispanicus) and European wild boar (Sus scrofa) from Spain: geographical and temporal distribution

Commercial hunting of Spanish wild ungulates has made them an important economic resource. Wild ungulates may have an important role in the maintenance of ixodid tick populations, and also as reservoirs of pathogens. We studied the ixodid ticks that parasitize Iberian red deer and European wild boar from Spain. Ixodid ticks (n=6,336) were collected from 431 Iberian red deer and 142 wild boar in different regions of Spain. We found 10 different ixodid tick species parasitizing Iberian red deer, mainly Hyalomma marginatum marginatum (63.7%), Rhipicephalus (Boophilus) annulatus (7.9%) and R. bursa (7.5%). R. (Boophilus) annulatus was only collected in the province of Cádiz (southern Spain). We found 8 ixodid tick species on the wild boar, mainly Hy. m. marginatum (68.7%), R. bursa (14.6%) and Dermacentor marginatus (9.3%). We found one adult Hy. marginatum rufipes and one adult Hy. anatolicum excavatum parasitizing wild boar from south-central Spain. Mean prevalence of ixodid ticks was 41.3+/-0.08% (n=475) and 31+/-0.09% (n=284) and intensity of parasitization was 13.9+/-0.2 (n=283) and 13.6+/-0.3 (n=130) ticks/animal for Iberian red deer and wild boar, respectively. Only 5 of the 13 ixodid tick species found were shared by Iberian red deer and wild boar. This finding could indicate a host preference when Iberian red deer and wild boar share common habitats. In both Iberian red deer and wild boar from south-central Spain the monthly relative frequencies of Hy. m. marginatum and R. bursa presented an inverse pattern. The highest Hy. m. marginatum relative frequencies coincided with the lowest R. bursa relative frequencies along the year. R. bursa and I. ricinus were present in areas from northern to southern Spain while Hyalomma sp. and D. marginatus were exclusively collected in the two southern thirds of Spain. Haemaphysalis sp. and D. reticulatus were collected in northern Spain. Hy. m. marginatum and R. bursa were present during the whole year in red deer and wild boar from south-central Spain, showing more than one life cycle per year. These results are important for understanding the role of wild ungulates in the maintenance of tick infestations and to improve tick control programmes.     

Assay conditions and validation of a new UV spectrophotometric method using microplates for the determination of polyphenol content

A new spectrophotometric assay for the quantification of polyphenolic content has been validated. It is based on Prussian Blue method and adapted to microplate spectrophotometry. Prussian Blue reaction is critically dependent on reaction time, but microplate spectrophotometry permits exact measures of many samples at the same time. This new method is precise, reproducible, repeatable and exact.     

Site-related influences on cone-borne inoculum and asymptomatic persistence of Diplodia shoot blight fungi on or in mature red pines

The shoot blight and canker pathogens Diplodia pinea and D. scrobiculata sporulate abundantly on cones of many pine hosts. Variation in incidence and abundance of potential inoculum from cones and frequency of asymptomatic persistence on or in shoots was examined for mature red pines in sites differing in dominant presettlement vegetation and soil type in Bayfield and Douglas counties in northern Wisconsin. Collections were made in each county from 6 plantations, 3 each in areas historically vegetated with jack pine and soils mapped as sands and three in areas historically vegetated with red pine with soils mapped as loamy sands. At each site, 5 cones were collected from each of 5 red pines and 10 shoots were collected from up to 5 red pines. Conidia from cones were quantified with a water wash and filtration technique. Diplodia species were cultured from surface-disinfested asymptomatic shoots. A species-specific PCR assay was used to identify the Diplodia species from cones and shoots. Although cones and asymptomatic shoots from each county yielded D. pinea and D. scrobiculata, D. pinea was detected more frequently. More conidia were obtained from cones from Douglas Co., where there is a history of severe shoot blight damage, than cones from Bayfield Co. In Douglas Co., more conidia were obtained from cones from plantations in areas of more sandy soil and presettlement jack pine dominance than cones from plantations in areas of less sandy soil and presettlement red pine dominance. The numbers of conidia and frequencies of cultural detection of Diplodia species from asymptomatic shoots at a site were positively correlated. These results provide evidence for site-related influences on abundance of pathogen inoculum and asymptomatic persistence on or in red pine crowns that may contribute to differences in frequency and severity of damage from Diplodia shoot blight.