Investigation of routes of entry and dispersal pattern of RGNNV in tissues of European sea bass,Dicentrarchus labrax

Viral encephalopathy and retinopathy (VER) is a serious neuropathological fish disease affecting in the Mediterranean aquaculture mainly European sea bass, Dicentrarchus labrax. It is well known that betanodaviruses are neurotropic viruses that replicate in nerve tissues, preferentially brain and retina. However, routes of entry and progression of the virus in the central nervous system (CNS) remain unclear. The role of four tissues—eye, oesophagus, gills and skin—as possible gateways of a betanodavirus, the redspotted grouper nervous necrosis virus (RGNNV), was investigated after experimental challenges performed on European seabass juveniles. The dispersal pattern of Betanodavirus at primarily stages of the disease was also assessed, using a real-time qPCR assay. The development of typical clinical signs of VER, the presence of characteristic histopathological lesions in the brain and retina and the detection of viral RNA in the tissues of all experimental groups ascertained that successful invasion of RGNNV under all experimental routes was achieved. Transneuronal spread along pathways known to be connected to the initial site of entry seems to be the predominant scenario of viral progression in the CNS. Furthermore, viraemia appeared only after the installation of the infection in the brain.     

Effect of storage time on the urine protein: creatinine ratio in alkaline ovine urine

The urine protein:creatinine (UPC) ratio is considered the reference method to assess proteinuria. Its diagnostic value in ovine medicine needs further elucidation. In population monitoring and/or for research purposes, it is convenient to collect many samples simultaneously and store them for later analysis. However, analyte stability data are required to ensure reliable results. We used 15 of 90 urine samples collected from sheep to assess the effect of storage time on the UPC ratio. After centrifugation, the supernatant of each sample was divided into 6 aliquots. Urine protein and creatinine concentrations were determined immediately in one aliquot using the pyrogallol red and a modified Jaffè method, respectively. The other aliquots were stored at −18°C. Based on the absence of active sediment, alkaline urine pH, and UPC ratio ≥0.2, we included 15 samples in our study. The UPC ratio was determined in the stored aliquots 2, 7, 14, 21, and 60 d after collection. The data were analyzed with univariate ANOVA. No significant difference was observed in the urinary concentrations of protein, creatinine, and the UPC ratio (0.8 ± 0.84 in conventional units and 0.09 ± 0.095 in SI units) among different times (p > 0.05). The UPC ratio remained stable for 2 mo in ovine urine samples stored at −18°C.     

Social network analysis of sheep grazing different plant functional groups

Social Network Analysis (SNA) is used for the first time to investigate the relations between plant functional groups and social structure of grazing sheep. The research was conducted on a grassland in Crete's island Lefka Ori, Greece during 2016. A flock of 20 sheep of Sfakion race was analysed as a network and the sheep as nodes. The focal sampling technique was applied during four experimental periods of four consecutive days × eight hours/day. The plant species selected by sheep during grazing were categorized into four functional groups (grasses, broad‐leaved forbs, shrubs and trees), and the proximity and conflict relations that were developed among the flock members were recorded. The tools of SNA were defined and interpreted in the sheep flock, and the Visone software is used to calculate the network variables of proximity and conflicts relations of nodes. Correlations between the plant functional groups and network variables were examined by Spearman's bivariate correlation test. Sheep collectivity was enhanced while grazing grasses and simultaneously, a tendency for hierarchization within the flock was identified. Similarly, the proximity of sheep increased during forbs’ grazing while their individualism was strengthened. When grazing shrubs, the proximity of sheep was only in part maintained while the grazing of trees seems as the most deconstructive forage option of sheep proximity relations. In general, the grazing of all plant functional groups decreases conflicts among sheep except that of trees, which was insignificant for developing such relations.     

Isolation and Antimicrobial Resistance of Staphylococcus spp. in Freshwater Fish and Greek Marketplaces

The presence and antibiotic resistance of Staphylococcus spp. in freshwater fish and the environment of fish markets of Northern Greece were investigated. A total of 269 samples were examined, consisting of 71 rainbow trout (Oncorhynchus mykiss), 65 gibel carp (Carassius gibelio), and 133 environmental samples swabbed from various surfaces at fish markets.Staphylococcus spp. was isolated from 27% of the samples, and 16%, 9%, 0.7%, 0.4% and 0.4% were found to be positive for S. aureus, S. epidermidis, S. warneri, S. hominis-hominis, and S. haemolyticus, respectively. All isolates were examined for their susceptibility to 20 antibiotics. None of the S. aureus isolates were resistant to oxacillin and glycopeptides. Three S. epidermidis, two S. Warneri, and one S. haemolyticus isolates were resistant to oxacillin (methicillin resistant Staphylococcus, MRS). All isolates, except one, showed resistance to as many as 15 antibiotics. The population of Staphylococcus spp. in fish did not exceed 1.0 log CFU/g. Freshwater fish and the environments of retail fish markets were found to harbor multi-drug resistant staphylococci. Whether these findings present a real health risk for humans and to what extent needs to be evaluated.     

Detection of Cyprinid herpesvirus 2 in association with an Aeromonas sobria infection of Carassius carassius (L.), in Italy

Sixteen specimens of female crucian carp, Carassius carassius (L.), during the breeding season, were investigated for post‐mortem and full diagnostic examination during a mortality outbreak in a tributary stream of the Arno River in Tuscany in 2011. Necropsy highlighted the presence of a swollen anus and widespread haemorrhages in the body, fins, gills and eyes. Haemorrhages in internal organs and spleen granulomas were also observed. Bacteria isolated from the brain, kidney and spleen of affected fish were identified as A. sobria. Microscopic lesions observed in gills were characterized by necrosis of the secondary lamellae, congestion and multifocal lamellar fusion. The kidney showed necrosis, oedema, fibrin exudation and areas of haemorrhages, while in the spleen the main lesions were by multifocal necrosis of the lymphoid tissue. In the gills, transmission electron microscopy revealed herpesvirus‐like particles, subsequently identified as Cyprinid herpesvirus‐2 (CyHV‐2) with a nested PCR protocol. Although it was not possible to attribute a pathogenic role to CyHV‐2 in this mortality event, the identification of this herpesvirus in crucian carp increases the concern about its potential role in this species.     

THE DETERMINANTS OF CONSUMPTION FOR ORGANIC AQUACULTURE PRODUCTS—EVIDENCE FROM GREECE

This study investigates Greek consumers' behavior towards organic aquaculture products, identifying possible distinct consumer groups and examining possible linkages between consumer characteristics and marketing aspects of organic aquaculture. Using data from an in-person field survey, a two-step cluster analysis demonstrated different attitudes among consumers with different profiles and also identified two distinct consumer groups differentiated primarily by income: the low-potential consumers and the high-potential consumers, representing 34% and 66% of the total sample, respectively. This study provides evidence that consumers' socioeconomic and demographic characteristics are key factors determining the consumers' purchasing behavior for organic aquaculture products. Thus, a market segmentation strategy is needed to allow organic aquaculture production to take advantage of the significant potential for growth in the near future.     

Germplasm diversity and genetic relationships among walnut (Juglans regia L.) cultivars and Greek local selections revealed by Inter-Simple Sequence Repeat (ISSR) markers

Inter-Simple Sequence Repeat (ISSR) markers were used for the assessment of genetic diversity among walnut (Juglans regia L.) selections from Greek native populations in comparison to internationally cultivated walnut genotypes. Similarity coefficient values from 0.13 to 0.93 (with an average of 0.48) were found among the 56 accessions examined, which indicated the presence of a high degree of genetic variability. Most international cultivars were grouped together while most Greek native populations could not be placed into a distinct group. The Greek native population genotypes were found more diverse than the international cultivars. The mean similarity coefficient values for the former and latter were 0.44 and 0.56, respectively. In the cultivar group, two subgroups were distinguished; one consisted of genotypes involving ‘Payne’ and the other ‘Franquette’ in their pedigrees. Some cultivars and populations could not be grouped according to their pedigrees or collection area. Analysis of molecular variance (AMOVA) revealed that a larger part of the genetic variation exists among Greek walnut populations within a collection region (89%) than among the regions (11%). The pairwise regional PhiPT values indicated that the most geographically distant regions are the most genetically differentiated. The high variability existing in the Greek germplasm in combination with their valuable agro-morphological traits suggested that it would be beneficial to utilize this native germplasm pool in walnut breeding programs and germplasm management activities to maximize genetic diversity in cultivated walnut.     

Reliability of indicators of sheep welfare assessed by a group observation method

Assessments of animal-based outcomes form the core of routine veterinary clinical examinations and are being increasingly used as indicators of animal welfare. A method of group observation that did not require gathering and handling of individual sheep, was used to assess eight animal-based indicators of sheep welfare: demeanour, skin irritation, wool loss, excessive panting, coughing, lameness, and cleanliness of the ventral abdominal and 'breech' (perineum/gluteal/caudal hindlimb) areas. The inter-observer reliability of two or three observers who independently assessed these indicators was tested on 2406 adult sheep and growing lambs across 36 farms and the intra-observer reliability of an experienced, veterinary assessor--the 'test standard observer'--was assessed on 88 adult sheep during four on-farm assessments. Observer reliability was evaluated using Cronbach's alpha (α) comparison of the recorded proportion (%) of sheep affected by each welfare condition and by binomial logistic regression modelling. High levels of inter-observer reliability were identified for the assessment of group lameness (α 0.76-1.00) and cleanliness of the breech area (α 0.97-1.00). Excellent intra-observer reliability was determined for lameness (α 0.99), cleanliness of the breech area (α 0.97), demeanour (α 1.00) and wool loss (α 1.00). In addition, proportion data and logistic regression models identified few between-observer differences. The results suggest that welfare outcomes based on observations of the behaviour and physical appearance of individual animals within a group may offer a reliable and feasible measurement tool for the on-farm assessment of sheep welfare.