Fast analysis of sugars, fruit acids, and vitamin C in sea buckthorn (Hippophaë rhamnoides L.) varieties

A fast, one-step gas chromatographic method was developed to analyze trimethylsilyl (TMS) derivatives of sugars, fruit acids, and ascorbic acid in sea buckthorn (Hippoha? rhamnoides L.) berries. The method was applied to berry press juice of sea buckthorn of different origins grown in Finland during the 2003 and 2004 seasons. The method gave reliable results for D-fructose, D-glucose, ethyl-D-glucose, and malic, quinic, and ascorbic acids, which are the major sugars and acids in sea buckthorn juice. For the first time in sea buckthorn and evidently in any berry, the presence of ethyl beta-D-glucopyranoside is reported. The structure of ethyl glucose was verified by high-performance liquid chromatography (HPLC), gas chromatography (GC), MS, and NMR analyses of both the isolated and the synthesized compounds. In the GC method, vitamin C was analyzed as ascorbic acid only, and dehydroascorbic acid was thus not taken into account.     

64,XX, SRY-, and ZFY-Negative Icelandic Horse Likely to Be True Hermaphrodite

A 4-year-old Icelandic horse, considered to be a mare, showed stallion-like behavior in a group of mares. On clinical examination, the horse turned out to have an enlarged erectable phallic clitoris. Ultrasound examination showed a normal-sized left ovary covered with numerous small follicular cysts and a compact testis-like tissue in place of the right ovary. The karyotype was normal for a mare (64,XX), and the horse was found to be negative for the Y chromosome–specific markers SRY, ZFY, and EIF1AY. This case indicates that the intersexual phenotype may be caused by autosomal recessive mutation, resulting in defects in cortisol biosynthesis rather than transferal of Y chromosome male–specific genes. This is the first report of an intersexual phenotype in an Icelandic horse that is likely to be a true hermaphrodite because of female sex chromosomes and a mixture of female and male gonads and external genitals.     

Immunopathogenesis and immunotherapy of Culicoides hypersensitivity in horses: an update

Culicoides hypersensitivity (CH) is a strongly pruritic dermatitis of horses, characterised histologically by mixed perivascular to diffuse cellular infiltrates of mononuclear cells and eosinophils, and caused by helper T cell (Th)2, immunoglobulin (Ig)E-mediated reactions to antigens present in the saliva of biting Culicoides midges. As treatment of CH is still not satisfactory, a better knowledge of the pathogenesis of CH is needed for the development of new therapeutic modalities. Accordingly, availability of pure Culicoides allergens instead of crude whole body extracts is essential for the improvement of in vitro diagnosis of CH and development of a more effective allergen immunotherapy (AIT). Beside the Th2 cytokines interleukin (IL)-4 and IL-13, recent studies have shown the involvement of thymic stromal lymphopoietin, IL-5 and IL-31 in the pathogenesis of CH. The importance of IL-5 and IL-31 has been confirmed through targeting these cytokines by active immunisation of CH patients, resulting in a significant decrease of CH lesion scores. Thirty Culicoides salivary allergens have been described over the last 10 years and produced as pure recombinant (r-) proteins. Use of a protein microarray has allowed the identification of the most relevant Culicoides r-allergens for CH in central and northern Europe. An immunisation protocol has been developed for preventive AIT against CH. Small amounts of r-Culicoides allergens injected into the submandibular lymph nodes with Alum/MPLA as adjuvants seem to induce a suitable immune response for both preventive and therapeutic AIT. The next years will reveal whether these recent advancements will translate into an improved diagnosis, treatment and prevention of CH.     

Generation of equine TSLP-specific antibodies and their use for detection of TSLP produced by equine keratinocytes and leukocytes

Allergic horses react to innocuous environmental substances by activation of Th2 cells and production of allergen-specific IgE antibodies. The mechanisms leading to Th2 differentiation are not well understood. In humans and mice, epithelial cell-derived thymic stromal lymphopoietin (TSLP) plays a central role in this process. Little is known about equine TSLP (eqTSLP) and its role in allergic diseases and our current knowledge is limited to the assessment of TSLP mRNA expression. In order to be able to study eqTSLP at the protein level, the aim of the present study was to produce recombinant eqTSLP protein and generate TSLP-specific antibodies. EqTSLP was cloned from a skin biopsy sample from a horse with chronic urticaria and eqTSLP protein was expressed in E.coli and in mammalian cells. Recombinant proteins were designed to include C-terminal Histag, which allowed subsequent purification and detection by Histag-specific Ab. Polyclonal and monoclonal eqTSLP-specific Ab were generated after immunization of mice with E.coli-expressed TSLP. Their specificity was tested by western blotting and ELISA. In addition, a commercially available polyclonal human TSLP-specific antibody was tested for cross-reactivity with eqTSLP. Expression of TSLP protein was confirmed by western blotting using Histag-specific Ab. E.coli-expressed TSLP appears as a band of ～13 kDa, whereas mammalian cell-expressed TSLP showed several bands at 20-25 kDa, probably representing several glycosylation forms. Polyclonal and monoclonal antibodies generated in this study, as well as commercially available human TSLP-specific Ab reacted with both E.coli- and mammalian cell-expressed TSLP in western blotting and ELISA. A capture ELISA was established to quantitate TSLP in cell supernatants and validated using supernatants from primary equine keratinocytes and peripheral blood leukocytes (PBL). Increased TSLP concentrations were found after stimulation of keratinocytes with PMA+ionomycine and with Culicoides extract. Similarly, increased TSLP concentrations were detected in PBL after stimulation with ConA, Culicoides extract, or IgE cross-linking. In conclusion, recombinant TSLP proteins and TSLP-specific antibodies produced in this study will allow further studies of the role of TSLP in equine allergic diseases.     

Application of prospective probionts at early stages of Atlantic cod (Gadus morhua L.) rearing

This work aimed at validating the use of two prospective probionts (Arthrobacter sp. and Enterococcus sp.) at early stages of cod (Gadus morhua L.) rearing. Ova at late post‐fertilized stage and larvae during their first 4 weeks of life were bathed with both probionts, isolated previously from the cod‐rearing environment. This treatment was compared with groups fed rotifers supplemented with a commercial probiotic (Remus^®) and those untreated. Microbiological analyses (total viable counts, presumptive Vibrio and lactic acid bacteria) were performed in rearing systems and larval survival, growth and development were assessed. Larval development was evaluated by proteolytic activity of larval lysates and immunological analysis of important proteins: apolipoprotein A‐I, haemoglobin, C‐reactive protein, C3 and cod serum proteins. Bacterial bathing led to a significantly higher larval weight, length and culturable microbial load in larval gastrointestinal (GI) tract when compared with the control and Remus groups. Development occurred earlier in bathed larvae. However, their survival was negatively affected compared with the control group, but was significantly higher than for the Remus group. The non‐pathogenicity of both probionts was demonstrated by intraperitoneal injection of 13 g cod juveniles. The results suggest that Arthrobacter and Enterococcus probionts affected the larval GI microbiota and contributed to growth, development and digestion, either directly or indirectly.     

Microsatellite analysis of Icelandic populations of the poplar fungal pathogen Melampsora larici-populina shows evidence of repeated colonization events

The basidiomycete Melampsora larici-populina causes foliar rust on Populus species from the sections Aigeiros and Tacamahaca, causing reduction in biomass production and economic losses. In the present study, samples of Icelandic M. larici-populina were collected for analysis of genetic diversity and population structure. A total of 439 isolates, collected at 15 locations, and analysed using 22 microsatellite markers were compared to data from French M. larici-populina populations. Twenty-one of the loci analysed were polymorphic, with an average of 3.4 alleles per locus. The mean observed and expected heterozygosities for all populations were 0.35 and 0.38. Evidence was found for a substructure within the Icelandic population with three subpopulations being the most likely scenario with low levels of gene flow. The population structure seen here is most likely shaped by both isolation and genetic drift as well as repeated events of colonization. In the future it can therefore be expected that regional poplar rust genotypes in Iceland change by two different modes; on one hand by transport of spores within the country and on the other hand by repeated colonization events. The results reported here underline the importance of closely monitoring the development of fungal diseases in Iceland, and to carefully select for resistance in Icelandic plant breeding programs.