Evaluation of a new qPCR test to identify the organisms causing high total bacterial count in bulk tank milk

Milk quality in bulk tank milk(BTM)is measured by flow cytometry technology as total bacterial count(TBC)and somatic cell count(SCC).To investigate SCC problems,culture or PCR can be used to identify mastitis causing bacteria,e.g.,Mastit 4,a commercially available qPCR test.TBC in BTM can be investigated further using culture-based methods such as standard plate count,laboratory pasteurization count,coliform count,and spore counts.To our knowledge,no qPCR addressing the bacteria involved in TBC has been commercially introduced.The aim of this study is to evaluate a recently introduced 3-h qPCR test,TBC 4.The TBC 4 qPCR detects four target groups,Pseudomonas,Streptococci,Enterobacteriacea/Enterococcus,and Bacillus/Clostridia.These target groups relate to problems on the farm such as cooling,mastitis,environment,and silage.We will continue with new research to compare the TBC 4 qPCR test with traditional culture.For this study,BTM samples from different TBC intervals were selected based on Bacto Count results found at routine payment investigation at Eurofins laboratory(Vejen,Denmark).These samples were analyzed using TBC 4 qPCR assay within 24 h.In total,346 BTM samples were divided into six different intervals of colony forming units(CFU).For all four targets in each of the different intervals of CFU,the percent of positive samples,the average C_t-value,the percent of positive samples with C_t30 and C_t25 were calculated.For Pseudomonas,Streptococci,and Enterobacteriacea/Enterococcus,the number of positive samples with lower C_t-values(high bacteria content)correlated with the CFU mL~(–1).We found Enterobacteriacea/Enterococcus,Pseudomonas,and Streptococci in high number of bacteria(C_t25)in 25,19 and 56%of samples with CFU mL~(–1) between 50 001–100 000 and 53,44,and 39%in samples with CFU mL~(–1)100 000.The TBC 4 qPCR test showed to be a strong and fast tool for farmers,advisors and service technicians to address problems with high TBC and ensuring the delivery of good quality milk to the dairy.     

RESPONSE OF LIME THYME TO SALINITY AND IONIC CONCENTRATION IN NUTRIENT SOLUTION

□ Plants from 60-day-old Lime Thyme (Thymus citriodorus) cuttings were potted in a medium of coconut fiber and peat moss and were treated with three different nutrient solutions: T1, T2, and T3. T1 was a standard nutrient solution; T2 was incremented with macronutrients up to an electrical conductivity (EC) of 2.8 dS m^?1; and T3 was the same as T1 but incremented up to an EC of 2.8 dS m^?1 with sodium chloride. The plants were then grown for 90 days in a greenhouse with natural daylight in Almería, Spain. Root growth was not affected by the treatments. The dry weight of the leaves and the total dry weight of the plants benefited from the salinity. The specific salinity of the sodium chloride negatively affected growth compared to the same salinity in the complete nutrient solution.     

Transition state stabilization by a catalytic RNA

The hairpin ribozyme catalyzes sequence-specific cleavage of RNA through transesterification of the scissile phosphate. Vanadate has previously been used as a transition state mimic of protein enzymes that catalyze the same reaction. Comparison of the 2.2 angstrom resolution structure of a vanadate-hairpin ribozyme complex with structures of precursor and product complexes reveals a rigid active site that makes more hydrogen bonds to the transition state than to the precursor or product. Because of the paucity of RNA functional groups capable of general acid-base or electrostatic catalysis, transition state stabilization is likely to be an important catalytic strategy for ribozymes.     

Effect of the Extrusion on Functional Properties and Mineral Dialyzability from <Emphasis Type="Italic">Phaseolus Vulgaris</Emphasis> Bean Flour

The effects of extrusion conditions on cooking degree, flour dispersion viscosity and mineral potential availability of extruded bean flour were studied. Phaseolus vulgaris beans of the agronomic cultivar “Flor de mayo” were ground and dehulled to obtain grits and then extruded at different temperatures (140, 160 and 180 °C) and moisture contents (17, 20 and 23%), according to a bifactorial experimental design. Degree of cooking was estimated by water solubility (WS) and specific mechanical energy (SME). The effect of variables on WS and SME were analysed by surface response methodology. Flour dispersion viscosity and mineral availability (estimated by in vitro dialyzability), were also evaluated on selected samples. Results showed that, within the ranges of the variables used for this study, only the effect of temperature was significant on the degree of cooking. No direct correlation was observed between water solubility and SME, although a maximum value of WS corresponded to a range of SME values of 400–500 J/g was observed. Dispersion viscosity decreases as WS increases, so if high calorie density is desired, for instance in order to produce a cream soup formula, bean grits should be extruded at high temperature and as low moisture as possible, in our case 180 °C and 17% moisture. On the other hand, the effects of extrusion variables on iron and zinc dialyzability were not much affected.     

云南咖啡锈菌小种鉴定

为明确云南咖啡锈菌小种类型,采用国际通用的 19 个咖啡锈菌生理小种鉴定寄主,利用人工接种鉴定的方法,对采自云南咖啡主产区 12 县（市）的 51 份咖啡锈菌进行鉴定,鉴定出 9 个小种,分别为 VIII（v2,3,5）、XXXIII（v5,7 or v5,7,9）、XXXIV（v2,5,7 or v2,5,7,9）、XXXVII（v2,5,6,7,9）、XLI（v2,5,8）、XLII（v2,5,7,8 or v2,5,7,8,9）、New race（v2,5,6,7）、New race（v1,2,5,7 or v1,2,5,7,9）、New race（v1,5,7 or v1,5,7,9）,这 9 个小种均为国内首次鉴定,其中小种 XXXIII、XXXIV、New race（v2,5,6,7）、New race（v1,5,7 or v1,5,7,9）、New race（v1,2,5,7 or v1,2,5,7,9）、XLI 和 XLII 均侵染 Catimor7963,小种 XXXVII 侵染 CatimorT5175。     

Levels of Renibacterium salmoninarum antigens in resident and anadromous salmonids in the River Ellidaár system in Iceland

In relation to stock enhancement programmes, wild salmon broodfish have been routinely screened for the presence of Renibacterium salmoninarum antigens (Rs‐Ag) for decades. A sudden increase in the prevalence of Rs‐Ag experienced caused extensive problems to this industry as eggs from positive fish are discarded. The prevalence and level of Rs‐Ag were examined in resident and anadromous salmonids in the River Ellidaár system and the progress of Rs‐Ag in a cohort of salmon followed. Both prevalence and Rs‐Ag levels were high in resident salmonids and emigrating salmon smolts in the river system. When the smolts re‐entered their home river as adults the following summer, they were almost free of Rs‐Ag, but the longer they stayed in the river, the more Rs‐Ag they acquired; the majority being positive at spawning. This study demonstrates a high level of Rs‐Ag in salmonids in the River Ellidaár system which significantly reduces in the salmon during its seawater phase. Accordingly, it seems ideal to sample salmon broodfish as soon as possible after ascending the river and subsequently transfer to Rs‐free environment for storage until stripping, which could result in lower Rs‐prevalence and minimize the problems that stock enhancement programmes have faced due to Rs‐positive wild broodfish.     

Partial characterization of digestive proteases of the three‐spot cichlid Cichlasoma trimaculatum (Günter 1867)

Partial characterization of digestive proteases in the three‐spot cichlid Cichlasoma trimaculatum juveniles was conducted. It was determined that there is higher alkaline proteases activity (3.95 ± 0.32 IU mg^?1 protein) compared to acidic proteases (2.01 ± 0.57 IU mg^?1 protein). Optimal temperature for alkaline proteases is 60 °C which resulted in more thermostability to temperature changes. On the other hand, optimal temperature for acidic proteases is 50 °C. Optimal pH for acidic proteases was pH 2, while for alkaline proteases, it was pH 10, which resulted in more stability in relation to pH changes than acidic protease. The use of specific inhibitors and the SDS‐PAGE electrophoresis analysis revealed seven types of bands for alkaline proteases, which make evident the main presence of serine proteases. In acidic proteases, more than 98 g kg^?1 of the activity was inhibited with pepstatin A inhibitor. Therefore, it is evident that C. trimaculatum digestion is composed by acidic and alkaline proteases; thus, it should be considered an omnivorous fish.     

Effect of diet on growth and body biochemical composition of juvenile four‐sided sea cucumber Isostichopus badionotus (Selenka, 1867)

The farming of sea cucumber is a growing industry that demands feeds that are tailored to the organism's nutritional needs. Therefore, in this study, the effect of diet composition, with and without additives, on the growth, survival, specific growth rate (SGR) and body biochemical composition of the four‐sided sea cucumber Isostichopus badionotus was evaluated over a period of 3 months. Four diets were used: D1 (Macrocystis sp. and Sargassum sp.), D2 (Macrocystis sp. and Spirulina sp.), D3 (Sargassum sp. and Spirulina sp.) and D4 (Macrocystis sp., Sargassum sp. and Spirulina sp.), and the same diets enriched with 0.22% of Algamac 2000^® (D1H, D2H, D3H and D4H) were also tested. The organisms fed the diets containing Spirulina sp. demonstrated the highest growth rates and significantly greater SGR (% per day) (p < .05), an effect that was enhanced by the food additive (D2 0.67%; D2H 0.78%; D3H 0.76%). Survival and body composition were not affected by diet type. Based on these results, the use of Spirulina sp. combined with supplements rich in PUFA content is recommended in the formulation of diets to promote the growth of I. badionotus juvenile.     

Growth,tissue metabolites and digestive functionality in Sparus aurata juveniles fed different levels of macroalgae,Gracilaria cornea and Ulva rigida

The aim of this study was to evaluate the inclusion of two macroalgae as dietary ingredients on growth, body composition, intermediate metabolism, intestinal enzymes and gut histology of Sparus aurata juveniles. Six isocaloric and isonitrogenous diets incorporating 5%, 15% or 25% of Gracilaria cornea or Ulva rigida meal were tested against a control diet. Duplicate groups of fifteen fish (14 g) were fed the experimental diets for 70 days. Growth performance showed an inverse linear relationship with the inclusion level of Gracilaria. Feed conversion ratio increased significantly in fish fed 25% Gracilaria. Ulva presented a quadratic relationship with the lowest SGR at 15% of inclusion. Fish fed 25% Ulva exhibited significantly lower lipid content than those fed control diet. Overall, metabolites did not show differences with specimens fed control diet, except for plasmatic glucose and muscle lactate. Intestinal proteolytic activity was reduced with the macroalgae inclusion level, but all fish showed the same enzyme pattern. The histological study revealed no negative effect on liver and intestine structure by the macroalgae inclusion. The present work demonstrated that these ingredients can be used in practical diets for sea bream, but a more comprehensive research is needed to determine the optimum dietary level in a longer feeding trial.