The effects of diet supplemented with Lactobacillus rhamnosus on tissue parameters of rainbow trout,Oncorhynchus mykiss (Walbaum)

This study was carried out to establish the effects of a 6 week treatment with the diet supplemented with L. rhamnosus in concentrations of 10⁷ CFU g^?1 (G1 group) and 10⁸ CFU g^?1 (G2 group) on the condition expressed by condition factors (Fulton's, Clark's and B), intestinal microbiology, haematological, histological and selected antioxidative parameters of rainbow trout. A significantly higher condition factors were found in G1 group indicating that higher concentration of probiotic (10⁸ CFU g^?1) did not result in the better condition. Cholesterol and urea levels were significantly higher in both G1 and G2 groups, albumin in G1 and creatinine in G2 group with respect to control. A significantly higher liver TBARS level was observed in G2 group. The feeding with supplemented probiont apparently changed the resident microbiota. Three weeks after withdrawal of the supplemented feed, the microflora mostly reverted to the control composition, although L. rhamnosus in faecal matter of fish remained inherent. The epithelial structure of the proximal and distal intestine revealed the increased absorptive area in both treated groups, as well as the increase in the mucin‐secreting goblet cells. The L. rhamnosus‐treated groups demonstrated the capacity for the augmentation of the innate host defence.     

Comparative study on the antioxidant and biological activities of carvacrol, thymol, and eugenol derivatives

Four derivatives of thymol, carvacrol, and eugenol were synthesized: 4-(hydroxymethyl)-5-isopropyl-2-methylphenol, 4,4'-methylenebis(5-isopropyl-2-methyl)phenol, 4-allyl-6-(hydroxymethyl)-2-methoxyphenol, and 4-(hydroxymethyl)-2-isopropyl-5-methylphenol. The obtained derivatives showed remarkably better antioxidative properties according to 1,1-diphenyl-2-picrylhydrazyl assay (50% inhibitory concentrations = 4-156 microg/mL) and Rancimat assay (protection factors = 1.55-5.84) when compared with parent compounds and values similar to or better than those of butylated hydroxytoluene and vitamin C. At concentrations of 10 mM carvacrol derivatives had no toxic effect on viability of Escherichia coli K-12 (determined by minimum inhibitory concentrations). Other phenol derivatives showed reduced cytotoxic effect on E. coli K-12 at concentrations of 2-5 mM on the basis of 50% lethal dose measurements. In comparison with the parent compounds, phenol derivatives showed reduced cytotoxic effect for Saccharomyces cerevisiae cells (determined by yeast colony reduction). On the other hand, the majority of synthesized compounds had dose-dependent antiproliferative effects on human uterine carcinoma cells (HeLa), which makes them potentially interesting for the adjuvant experimental cancer treatments. The 4,4'-methylenebis(5-isopropyl-2-methyl)phenol derivative of carvacrol showed lower inhibiting capacity also for the HeLa cells, which makes this particular derivative attractive as an efficient antioxidant with negligible cytotoxic effects.     

Synthetic Zeolite A as Zinc and Manganese Fertilizer in Calcareous Soil

The Leccino olive cultivar was grown in pots contained calcareous rendzina soil amended with NPK fertilizer (control treatment) or with further addition of Zeolite A (ZA) containing either zinc (Zn²⁺) or manganese (Mn²⁺) (Zn-ZA and Mn-ZA). After one year, vegetative growth and root morphological parameters were measured and elemental analysis was performed on the root, stem, and leaves. Compared to other treatments, the Zn-ZA fertilizer enhanced Zn concentration and total quantity in the root, stem, and leaves, as well as the root diameter. The root volume was greater using Zn-ZA treatment than control treatment. The total quantity of Mn in root and the root length were enhanced when using Mn-ZA compared to control or Zn-ZA treatment. According to the results of this study, it appears that zeolites containing Zn provide favorable conditions for Zn uptake in calcareous soils, while both Zn and Mn zeolites have a positive impact on olive root morphology.     

Fatty acid and proximate composition of bluefin tuna (Thunnus thynnus) muscle with regard to plasma lipids

The composition of tail muscle fatty acids from wild and cultured bluefin tuna reared on a diet based on herring and sardine, along with the plasma lipid profile of the farmed individuals, was determined. The total lipid content of farmed bluefin in this study was 0.922 g/100 g or 3.49 g of saturated fatty acids (SAFA), 4.48 g of monounsaturated fatty acids (MUFA), 2.58 g polyunsaturated fatty acids (PUFA) n‐3 and 0.37 g of PUFA n‐6 fatty acids; for wild specimens, it was 0.920 g/100 g, or 2.85 g of SAFA, 4.82 g of MUFA, 2.78 g PUFA n‐3 and 0.27 g of PUFA n‐6 fatty acids. The major fatty acids in this study were 16:0; 16:1, n‐7; 18:1, n‐9 and DHA 22:6, n‐3 acids. The sum of these major components accounted for more than 57% and 80% of the total fatty acids in all the samples of farmed and wild tuna respectively. No significant differences in the proximate composition were demonstrated between farmed and wild samples, except for the energy value, in favour of the farmed tuna. Statistically, glucose tends to increase together with cholesterol (CHOL) and plasma triglyceride, as for these pairs, it showed positive correlation coefficients and P>0.05. Some measured tuna metabolites demonstrated strong mutual correlations, especially GLU, CHOL and TRIG, which are crucial factors in the lipid profile of animals.     

Spreading of West Nile virus infection in Croatia

West Nile virus (WNV) is an emerging zoonotic pathogen with rapid global expansion. The virus circulation is confirmed in many countries of Mediterranean Basin and Southern and Central Europe. In our study detection of specific WNV antibodies was performed in horses and cattle sera samples collected from October 2010 to April 2011. Serum samples were randomly taken from different parts of Croatia and tested by IgG and IgM ELISA. Positive serological results were confirmed by virus neutralization assay (VN-assay) and plaque reduction neutralization test (PRNT). Results showed that WNV antibodies were present in 72 out of 2098 horse sera (3.43%) and 3 of 2695 cattle sera (0.11%). The highest seroprevalence was found in Eastern Croatia in counties next to Hungarian, Serbian and Bosnia and Herzegovinian state borders. In Adriatic part of Croatia positive animals were found only in the westernmost county, near Slovenian and Italian borders. Geographic distribution and number of positive horses indicated that WNV is highly present in Croatia and spreading from East to West. However, positive horses in westernmost part of country indicate possible second origin of spreading. Location of serological positive cattle supports the hypothesis that seropositive cattle could be indicators of high WNV activity in the respective geographic regions.     

Geochemistry of stream sediments within the reclaimed coastal floodplain as indicator of anthropogenic impact (River Neretva,Croatia)

Purpose

This study used multi-element signatures of stream sediments to assess both natural and human-induced impact on fluvial system in the River Neretva delta receiving environment over time. The river basin actually comprises several sub-catchments, and the geochemical features of major elements, trace metals radiometric and mineralogical characterisation of river bed sediments were used to assist the interpretation of the environment of deposition and its subsequent modifications caused by various anthropogenic pressures within the river basin.

Materials and methods

Five sites were chosen for sediment sampling at key locations within the study area with assumed undisturbed, continuous sedimentation process. At each of the sites, three representative cores were taken by scuba divers. Sample sites were selected in order to reflect the influence of different sub-catchments they belong to and the land-use pattern of the surrounding area. Samples were analysed for pH, redox-potential, granulometry, mineralogy, thermogravimetry, major and trace element concentrations and radionuclide activities. The univariate and multivariate statistics were applied. The geochemical normalisation of data was done using Al, the procedure based on calculation of the regression line of the metal on the normaliser followed by testing the ratios metal/normaliser on all data-points.

Results and discussion

All studied sediments are classified as silt, ranging from clayey silt to silt and sandy silt. Mineralogically, the sediments were dominated by carbonates and quartz. The chemical contaminant data are generally of good quality, mostly below guideline levels. Sedimentation rates were estimated using vertical distribution of ¹³⁷Cs activities. Normalisation of TMs done by using Al shows strong R ² _adj values for the regressions of Al and V, Al and Cr, and Al and Ni. However, Al cannot fairly explain the fluctuation of the concentrations of Cd, Cu, Pb and Zn in sediment cores from all of the sampling sites. Generally, inorganic scavengers such as clay minerals followed by Fe and Mn oxides and S (pyrite and gypsum) seem to be dominant factors controlling TMs in studied sediments.

Conclusions

Although the River Neretva delta occupies a rather small area, the geochemical features of major and trace elements and ¹³⁷Cs activities show complex sediment provenances. Each of the sampling sites reflect exactly different effects of anthropogenic intervention that particularly refer to the changes in river morphology and ecology, along with the altered flow regimes within the catchment on sediment loads and quality.

     

Investment Potential of Private Forests in Croatia

This paper presents a comprehensive financial analysis of the investment potential of seven private forest categories in the Republic of Croatia (total study area of 268,072 ha). It covers the period from 2018 to 2048 and includes forest purchase costs, management simulation, timber transport to mills, timber processing and veneer production, and finally the selling of sawmill, veneer and forest products. Data from the Croatian National Forest Inventory and forest habitats map were used as a basis for analyses. Spatial data on private forests, roads and timber process plants at the national level were also integrated into the analyses. For the forest management simulation, the MOSES 3.0 simulator was used, and the QGis 3.4 software was used for spatial analyses of forests, roads and mils. Based on data from several world stock exchanges that cover companies of the forestry sector, a real (inflation-free) discount rates were used. The financial analyses showed which forest categories have positive investment potential and under which conditions. Results pointed out that multi-aged European beech forests have the highest internal rate of return (8.45%) and are the only one which would meet the expectations of a financially rational investor using criteria of a risk-adjusted inflation-free discount rate of 8%.     

Trichinellosis in wolves from Croatia

The aim of the present study was to investigate the prevalence of Trichinella infection in wolves (Canis lupus) in a 17,468 km² area in Croatia. Muscle samples were collected from 67 wolves between 1996 and 2007 and analyzed by artificial digestion. Muscle larvae were detected in 21 wolves (31%) and genotyped by multiplex PCR. Trichinella britovi was the predominant species confirmed in 90% (19 wolves) while Trichinella spiralis was detected in 9% (2 wolves). The presence of the so called “domestic” Trichinella species was a surprise since, to date, only T. britovi had been reported in wild animals in this region. The larval burdens in infected animals ranged from 0.3 to 45.9 larvae per gram. The prevalence of infected animals varied by geographic region; infected animals were found in the region of Gorski Kotar (20%) which has very similar environment to the region of Lika, where almost all wolves were found infected. Interestingly, this is the first report of infected wolves in Dalmatia.     

Implementation of immunohistochemistry on frozen ear notch tissue samples in diagnosis of bovine viral diarrhea virus in persistently infected cattle

Background

Bovine viral diarrhea is a contagious disease of domestic and wild ruminants and one of the most economically important diseases in cattle. Bovine viral diarrhea virus belongs to the genus Pestivirus, within the family Flaviviridae. The identification and elimination of the persistently infected animals from herds is the initial step in the control and eradication programs. It is therefore necessary to have reliable methods for diagnosis of bovine viral diarrhea virus. One of those methods is immunohistochemistry. Immunohistochemistry on formalin fixed, paraffin embedded tissue is a routine technique in diagnosis of persistently infected cattle from ear notch tissue samples. However, such technique is inappropriate due to complicated tissue fixation process and it requires more days for preparation. On the contrary, immunohistochemistry on frozen tissue was usually applied on organs from dead animals. In this paper, for the first time, the imunohistochemistry on frozen ear notch tissue samples was described.

Findings

Seventeen ear notch tissue samples were obtained during the period 2008-2009 from persistently infected cattle. Samples were fixed in liquid nitrogen and stored on -20°C until testing. Ear notch tissue samples from all persistently infected cattle showed positive results with good section quality and possibility to determinate type of infected cells.

Conclusions

Although the number of samples was limited, this study indicated that immunohistochemistry on formalin fixed paraffin embedded tissue can be successfully replaced with immunohistochemistry on frozen ear notch tissue samples in diagnosis of persistently infected cattle.     

Vaccine failure caused an outbreak of equine influenza in Croatia

In April 2004 an outbreak of equine influenza occurred at the Zagreb hippodrome, Croatia. Clinical respiratory disease of the same intensity was recorded in vaccinated and non-vaccinated horses. The equine influenza vaccine used in Croatia at the time of the outbreak contained the strains A/equine/Miami/63 (H3N8), A/equine/Fontainebleau/79 (H3N8) and A/equine/Prague/56 (H7N7). At the same time, the usual strains in vaccines used in Europe were, in accordance with the recommendation of the World Organisation for Animal Health (OIE) Expert Surveillance Panel on equine influenza, A/equine/Newmarket/1/93 (H3N8) and A/equine/Newmarket/2/93 (H3N8). At the same time, some current vaccines in the USA contained A/equine/Kentucky/97 (H3N8). Genetic characterization of the HA1 portion of the haemagglutinin (HA) gene of virus isolated from the outbreak indicated that the isolate (A/equine/Zagreb/04) was an H3N8 strain closely related to recent representative viruses of the American lineage Florida sub-lineage. In comparison with both H3N8 vaccine strains used in horses at the Zagreb hippodrome, A/equine/Zagreb/04 displayed amino acids changes localised to 4 of the 5 described antigenic sites (A-D) of subunit protein HA1. Comparison of the amino acid sequence of the HA1 subunit protein of the outbreak strain with that of A/equine/Newmarket/1/93 displayed three amino acids changes localised in antigenic sites B and C, while antigenic sites A, D and E were unchanged. The Zagreb 2004 outbreak strain had the same amino acids at antigenic sites of the HA1 subunit protein as the strain A/equine/Kentucky/97. Amino acid changes in antigenic sites between HA1 subunit of the outbreak strain and the strains used in the vaccines likely accounted for the vaccine failure and the same clinical signs in vaccinated and unvaccinated horses. Use of a recent strain in vaccines should limit future outbreaks.