Production of Interesting Peptide Fractions by Enzymatic Hydrolysis of Tuna Dark Muscle By-Product Using Alcalase

ABSTRACT

A protein hydrolysate was prepared from proteins of tuna dark muscle by-product. The hydrolysis conditions (time, temperature, pH, and enzyme concentration) using Alcalase was optimized by response surface methodology (RSM). The regression coefficient close to 1.0, observed during experimental and validation runs, indicated the validity of the model. The hydrolysate produced under the optimum conditions determined by RSM has a low rate of peptide fraction of molecular weight of 4–1 kDa. Meanwhile, the results obtained by hydrolysis under optimal conditions determined by a complementary study (temperature 55°C, time 60 min, 1% enzyme concentration, and pH 8.5) show that the hydrolysate produced has a height rate of the peptide fraction of molecular weight of 4–1 kDa. The amino acid composition of the protein hydrolysate prepared proved to have the potential for application as an ingredient in balanced fish diets and as a source of nitrogen in microbial growth media.     

Coprological analyses on apparently healthy Alpine ibex (Capra ibex ibex) from two Swiss colonies

To provide baseline parasitological data for health surveillance in free-ranging Alpine ibex (Capra ibex ibex), we assessed the endoparasite population and level of parasitism in apparently healthy ibex. Faecal samples from 148 ibex were collected between 2006 and 2008 in two different Swiss ibex colonies. They were analysed by coprology, including combined sedimentation/flotation method, sedimentation method, Baermann funnel technique and Ziehl-Neelsen staining. Gastrointestinal parasites and lungworms were identified in 100% and 81.8% of the examined animals, respectively. Highest prevalences were recorded for gastrointestinal strongylids other than Nematodirus/Marshallagia spp. (100%), Eimeria spp. (100%), Muellerius spp. (79.8%) and Nematodirus/Marshallagia spp. (79.0%). We report for the first time Cryptosporidium sp. in free-ranging Alpine ibex and Cystocaulus spp. in free-ranging ibex from Switzerland. On average, ibex were infected with 3.9 different parasites taxa (range: 1-8). Parasite prevalence and diversity varied significantly between sexes, study sites and seasons. Parasite egg output was low in 95.7% and moderate in 5.3% of the samples. Overall, the results indicate that Alpine ibex are widely infected with endoparasites and suggest that multiple infections are very common in apparently healthy populations. Furthermore, our data underline the potential influence of factors such as sex, study site and season on parasitological findings.     

Interactions between mycorrhizal fungi and mycorrhizosphere bacteria during mineral weathering: Budget analysis and bacterial quantification

The impact of ectomycorrhizal fungi or rhizosphere bacteria on tree seedling growth and nutrient uptake is well known. However, few studies have combined those microorganisms in one experiment to clarify their relative contribution and interactions in nutrient acquisition. Here, we monitored the respective contributions of pine roots, two ubiquitous forest ectomycorrhizal fungi Scleroderma citrinum and Laccaria bicolor, and two S. citrinum-mycorrhizosphere bacterial strains of Burkholderia glathei and Collimonas sp., on mineral weathering, nutrient uptake, and plant growth. Pinus sylvestris plants were grown on quartz–biotite substrate and inoculated or not with combinations of mycorrhizal fungi and/or bacterial strains. Magnesium and potassium fluxes were measured and nutrient budgets were calculated. Both ectomycorrhizal fungi significantly increased Mg plant uptake. No significant effects of the two bacterial strains were detected on the K and Mg budgets, but co-inoculating the mycorrhizal fungus S. citrinum and the efficient mineral-weathering B. glathei bacterial strain significantly improved the Mg budget. Similarly, co-inoculating S. citrinum with the Collimonas sp. bacterial strain significantly improved the pine biomass compared to non-inoculated pine plants. In order to better understand this process, we monitored the survival of the inoculated bacterial strains in the quartz–biotite substrate, the pine rhizosphere, and the mycorrhizal niche. The results showed that the two bacterial strains harboured different colonization behaviours both of which depended on the presence of the ectomycorrhizal partner. The populations of the Burkholderia strain were maintained in all these environments with a significantly higher density in the mycorrhizal niche, especially of S. citrinum. In contrast the population of the Collimonas strain reached the detection level except in the treatment inoculated with S. citrinum. These results highlight the need for taking into account the ecology of the microorganisms, and more specifically the fungal–bacterial interactions, when studying mineral weathering and plant nutrition.     

Experimental study on the toxicity of imidacloprid given in syrup to honey bee (Apis mellifera) colonies

Two groups of eight honey bee colonies were fed with two different concentrations of imidacloprid in saccharose syrup during summer (each colony was given 1 litre of saccharose syrup containing 0.5 microg litre(-1) or 5 microg litre(-1) of imidacloprid on 13 occasions). Their development and survival were followed in parallel with control hives (unfed or fed with saccharose syrup) until the end of the following winter. The parameters followed were: adult bee activity (number of bee entering the hive and pollen carrying activity), adult bee population level, capped brood area, frequency of parasitic and other diseases, mortality, number of frames with brood after wintering and a global score of colonies after wintering. The only parameters linked to feeding with imidacloprid-supplemented saccharose syrup when compared with feeding with non-supplemented syrup were: a statistically non-significant higher activity index of adult bees, a significantly higher frequency of pollen carrying during the feeding period and a larger number of capped brood cells. When imidacloprid was no longer applied, activity and pollen carrying were re-established at a similar level for all groups. Repeated feeding with syrup supplemented with imidacloprid did not provoke any immediate or any delayed mortality before, during or following the next winter, whereas such severe effects are described by several French bee keepers as a consequence of imidacloprid use for seed dressing in neighbouring cultures. In any case, during the whole study, mortality was very low in all groups, with no difference between imidacloprid-fed and control colonies. Further research should now address several hypotheses: the troubles described by bee keepers have causes other than imidacloprid; if such troubles are really due to this insecticide, they may only be observed either when bees consume contaminated pollen, when no other sources of food are available, in the presence of synergic factors (that still need to be identified), with some particular races of bees or when colonies are not strong and healthy.     

Prophylaxis using paromomycin of natural cryptosporidial infection in neonatal kids

The chemoprophylactic effects of paromomycin sulfate against natural cryptosporidiosis in young kids were investigated. Two studies were carried out using two groups of 18 and 12 animals in two pens. In each pen, kids were allocated to treated or control groups. The treatment consisted of oral paromomycin given at 100 mg kg⁻¹ body weight day⁻¹ for 11 consecutive days from 2 days of age. All kids were weighed at 2, 6 and 10 days of age. Infection was monitored by collecting fecal samples and staining fecal smears every 3–4 days from days 4 or 5 to days 15 or 19. The results clearly showed the efficacy of paromomycin in reducing cryptosporidial oocyst output. Moreover, paromomycin prevented clinical signs and mortality.     

A novel isolation method of Brucella species and molecular tracking of Brucella suis biovar 2 in domestic and wild animals

Brucella suis biovar 2 is the most common aetiological agent of porcine brucellosis in Europe. B. suis biovar 2 is considered to have low zoonotic potential, but is a causative agent of reproductive losses in pigs, and it is thus economically important. The multilocus variable-number of tandem repeats genotyping analysis of 16 loci (MLVA-16) has proven to be highly discriminatory and is the most suitable assay for simultaneously identifying B. suis and tracking infections. The aim of this study was to investigate the relatedness between isolates of B. suis biovar 2 obtained during a brucellosis outbreak in domestic pigs and isolates from wild boars and hares collected from proximal or remote geographical areas by MLVA-16. A cluster analysis of the MLVA-16 data revealed that most of the isolates obtained from Switzerland clustered together, with the exception of one isolate. The outbreak isolates constituted a unique subcluster (with a genetic similarity >93.8%) distinct from that of the isolates obtained from wild animals, suggesting that direct transmission of the bacterium from wild boars to domestic pigs did not occur in this outbreak. To obtain a representative number of isolates for MLVA-16, alternative methods of Brucella spp. isolation from tissue samples were compared with conventional direct cultivation on a Brucella-selective agar. We observed an enhanced sensitivity when mechanical homogenisation was followed by host cell lysis prior to cultivation on the Brucella-selective agar. This work demonstrates that MLVA-16 is an excellent tool for both monitoring brucellosis and investigating outbreaks. Additionally, we present efficient alternatives for the isolation of Brucella spp.     

Effect of an inactivated paratuberculosis vaccine on the intradermal testing of goats for tuberculosis

The effect of an inactivated paratuberculosis vaccine on the diagnosis of tuberculosis (TB) in goats was investigated in a herd with a history of clinical paratuberculosis but which was free of TB. Cohorts of animals in 2006, 2008 and 2009, were vaccinated once at 1 month of age, and 50% of the 2006 cohort served as unvaccinated controls. The goats were aged 8 months, 20 months and 3.5 years old at the time of the survey. All animals were assessed using a single intradermal injection of bovine tuberculin purified protein derivative (PPD) (SID test), or using both bovine and avian PPD (CID test). An interferon (IFN)-γ assay using both bovine and avian PPD was carried out on the 2006 cohort and was interpreted according to three different 'cut-off' points. No unvaccinated (control) animals tested positive to any of the assays, confirming that the herd was TB-free. The SID test had a low specificity in vaccinated animals at 8 and 20 months of age, whereas the CID test demonstrated 100% specificity in animals ≥20 months-old. The specificity of IFN-γ assay was less than maximal for vaccinated animals 3.5 years old as small numbers of false positives were detected, although this depended on the chosen cut-off point. The study findings demonstrate that the use of an inactivated paratuberculosis vaccine in goats <1 month-old in a TB-free herd does not result in false positives to a CID test for TB when performed in animals ≥20 months-old.     

Babesiosis in free-ranging chamois (Rupicapra r. rupicapra) from Switzerland

Pathological examination of five adult chamois (Rupicapra r. rupicapra) found dead in two different regions from the Swiss Alps revealed pale mucous membranes and musculature, swollen spleen and haemoglobinuria. Histologically, haemosiderosis in the spleen and centrilobular hepatic necrosis were the predominant findings. On blood smears, small (approximately 0.84-1.47 microm), round to pyriform, peripherally located inclusions were present in the erythrocytes. PCR followed by sequencing of DNA extracted from blood or spleen of the infected animals revealed 99-100% identity of the amplified part of the 18S rRNA gene with GenBank entries attributed to Babesia divergens/Babesia capreoli. This is the first report of fatal Babesia infections in chamois raising the question of an emerging disease in this species.     

Quantitative determination of minerals in acid forest soils of granite

A procedure for quantifying minerals in particle size fractions of fine earth (< 2 mm) is presented. Mineral fractions of the acid forest soils are characterised by various transition products. Transitional minerals between illite and vermiculite or smectite, polyhydroxy-Al polymers interlayered in smectite and vermiculite, as well as poorly crystalline compounds are present. Normative calculations with standard minerals, generally applied to rocks, cannot be used to quantify precisely minerals of these soil samples. Conventional qualitative and quantitative chemical analyses are therefore completed by microanalyses of well-identifiable minerals. The calculation procedure is based mainly on a system of simultaneous linear equations. These are set up from the chemical contents of a particle size fraction on the one hand and from the corresponding element contents of single minerals on the other hand. The contribution of single minerals to overall parameters can be calculated by programmed algorithms. The methodology was adjusted to different particle size fractions.