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Acta Veterinaria Hungarica
Authors: László Makrai, Csaba Nemes, Anna Simon, Éva Ivanics, Zoltán Dudás, László Fodor, and Róbert Glávits

Enterococcus cecorum is the most frequently occurring enterococcal species in the intestine of chickens of over 12 weeks of age, and there are few reports on its isolation from the skeleton of broiler parent chicks. In the present study, observations on vertebral osteomyelitis and spondylolisthesis (‘kinky back syndrome’) showing high incidence in 8 broiler parent flocks in different parts of Hungary are summarised. Clinical signs were seen only in roosters between 5 and 13 weeks of age. Diseased birds were alert and remained sitting on their hocks with their feet slightly raised off the ground. Incidence of the disease among male birds ranged from 8% to 30% depending on flocks. Enlargement and distortion of the body of the 6th vertebra were seen as the main pathological lesions. The cavity of the spinal canal was constricted by the distorted vertebral bodies. Resorption of bone tissue and sequestrum formation, signs of increased osteoclast activity, proliferation of fibrotic tissues, infiltration with heterophils and formation of sclerotic layers were detected in the vertebral bodies. From all 24 samples collected from the vertebral lesions, Enterococcus cecorum was isolated and identified using metabolic fingerprinting as well as 16S rRNA gene sequencing. Demonstration of E. cecorum from the vertebral lesions in all examined broiler breeder roosters showing the same clinical and pathological findings in different flocks suggested the pathogenic role of this microorganism for the first time in Hungary.

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Genetic variation in the flint maize (Zea mays L. conv. indurata) gene pool has decreased significantly since the introduction of hybrid breeding into Europe in the 1950s, leading to greater genetic vulnerability. Landraces, stored in gene banks, offer a valuable source to broaden the genetic basis again. The objective of this study was the genetic characterization of 166 Swiss landrace accessions originating from 7 Swiss regions (alpine valleys). The material was fingerprinted using a set of ten SSRs (Simple Sequence Repeat Markers). The resulting cladogram showed three main clusters comprising 95, 22 and 49 accessions, respectively. The largest group of accessions, from the Rhine valley of St. Gallen (RT), was present in all three main clusters. However, the majority of RT accessions was found in the first main cluster, together with those from the western neighbouring region (Linthtal) and from the southwestern neighbouring region (Wallis). Those from Tessin (southern Switzerland) were found mainly in one sub-cluster within the third main cluster. This is a very encouraging first step in appraising the genetic differences among accessions from Swiss regions.

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Lake Hévíz is the largest natural thermal lake of Europe, harboring special bacterial communities. The aim of the present study was to gain information about the distribution and species diversity of the sediment microbiota, with special focus on Actinobacteria, by using cultivation-based and -independent molecular methods. Samples from two depths were taken in two different locations in October 2007. 245 strains were isolated, grouped to 85 OTUs by ARDRA, and identified by 16S rDNA sequencing. Most of the strains showed highest sequence similarity with Bacillus and related genera. Strains belonging to the phylum Actinobacteria were identified as members of Arthrobacter, Brachybacterium, Brevibacterium, Curtobacterium, Friedmanniella, Gordonia, Kocuria, Microbacterium, Micrococcus, Micromonospora, Mycobacterium, Rhodococcus, Streptomyces and Williamsia . Two clone libraries were constructed from H3M and H4M samples, providing 288 and 192 clones which were grouped to 150 and 125 OTUs, respectively, by ARDRA. The two most abundant group of the H4M library were OP8-related. The phylum Proteobacteria was represented mostly by δ -Proteobacteria, other relevant groups were Cyanobacteria, Bacteroidetes, Acidobacteria and β -Proteobacteria. The H3M library was dominated by Cyanobacteria, Verrucomicrobia, β -Proteobacteria, γ -Proteobacteria and δ -Proteobacteria. Chloroflexi, Bacteroidetes, Spirochetes and Firmicutes were scarce. Results from the clone libraries were compared to the length-heterogeneity-PCR fingerprints of the communities.

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Cereal Research Communications
Authors: A. shahnejat Bushehri, A. Salavati, B. yazdi Samadi, M. Hassani, and S. Shahnejat Bushehri

A collection of new and obsolete Iranian bread wheat cultivars were characterized for gliadins using acid polyacrylamide gel electrophoresis (A-PAGE). Extensive polymorphism (H) = 0.734 in gliadin patterns was found. A total of 26 band patterns including 13, 8 and 5 different mobility bands were identified, in the zones of ω-, β + γ- and α-gliadins, respectively. There were a few patterns specific to each region and some were common among all the regions. Patterns of α-gliadin C, β + γ-gliadins A, and ω-gliadins H, C and E patterns were significantly higher in temperate and tropical zones. β + γ-gliadin C and ω-gliadin Q were significantly higher in Caspian-cold regions. Variation was observed in gliadins patterns of cultivars grown in different regions in Iran. Individual cultivars showed unique gliadin fingerprints. There were larger variation in ω- and γ + β-gliadins than in α-gliadins. These results may provide complementary information for relating genetic diversity, and quality characterization of Iranian wheat cultivars.

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Plumbago zeylanica L. (PZ) is a significant medicinal plant in Ayurveda, so is used for the treatment of various disorders. The main active part of this plant is the root, and due to its inherent uses, it has been exploited hence becoming an endangered species. Dyerophytum indicum (Gibbs ex Wight) Kuntze (DI), mainly considered as a substitute of PZ, is also an important folklore medicine, used in many health problems. Both plants are much similar in their physical as well as chemical properties. However, an effective validation is required before declaration of substitution. In the present study, quantitative and qualitative estimations were performed on both plants with the help of modern analytical techniques. Simultaneous high-performance liquid chromatography (HPLC) and high-performance thin-layer chromatography (HPTLC) and quantitative determinations of β-sitosterol have been performed for comparing both plants, i.e., PZ and DI. HPTLC fingerprinting analysis was also performed comparatively in different plant parts of PZ and DI. Successive extracts from different plant parts were evaluated for TLC separation profile of secondary metabolites. A comparative polyphenolic content- and antioxidant screening was evaluated to check the free radical scavenging effect of both plants (leaf, stem, and root) in comparison with the standards gallic and ascorbic acid.

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This study was designed to develop a simple, sensitive, selective, and precise high-performance thin-layer chromatography (HPTLC) fingerprint and quantitative estimation method for the analysis of three phenolic compounds from nine Leucas species. The developed HPTLC method was validated according to the International Conference on Harmonization guidelines. The method permits reliable quantification of one important phenolic acid (gallic acid) and two hydroxycinnamic acids (caffeic and ferulic acids) in 50% hydroethanolic extracts on silica gel with toluene—ethyl acetate—formic acid 8:2:1 (v/v) as the mobile phase. The system showed good resolution and separation of caffeic, ferulic, and gallic acids (at R f values 0.48, 0.60, and 0.30, respectively) from the other constituents of the extract. Densitometric scanning was done at 300 nm in absorbance mode. All the results obtained by the developed method were statistically compared for validation parameters, for accuracy and good precision. Caffeic, ferulic, and gallic acids were estimated in all the aforesaid nine Leucas species, but the quantity varied from species to species. Further, these phenolics were reported from the aforesaid Leucas species for the first time, except for Leucas lanata and Leucas urticifolia. However, Leucas biflora, Leucas decemdentata, and Leucas stricta were documented for their chemical constituents for the first time. The proposed HPTLC method can be used for routine quality testing of Leucas species.

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A simple, rapid, and precise high-performance thin-layer chromatographic method has been established for quantitative analysis of myricetin in the stem bark of Myrica esculenta, family Myricaceae. The marker myricetin was separated from other components of stem bark extract on silica gel 60 F254 HPTLC plates with toluene-ethyl acetate-formic acid-methanol 3:3:0.6:0.4 (v/v) as mobile phase. Densitometry in absorbance-reflection mode at 268 nm was used for quantitative analysis of myricetin. The stem bark of M. esculenta was found to contain 0.225% (w/w) myricetin. The method was validated for linearity, accuracy, precision, and specificity in accordance with ICH guidelines. The calibration plot was linear between 0.4 and 2.0 μg per band for myricetin. The limits of detection and quantitation were 0.0939 and 0.2845 μg per band, respectively. The method can be used as a quality control-method for fingerprint profiling and quantitative analysis of the stem bark of Myrica esculenta.

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The leaves of Hibiscus sabdariffa L. are one of the sources of food and traditional medicine. A combination of high-performance thin-layer chromatography (HPTLC) bioautographic assay with mass spectrometry (MS) has been performed to screen and identify the antioxidant compounds in the leaves of H. sabdariffa L. The crude extract of H. sabdariffa L. was separated on silica gel 60 HPTLC plates in an automatic developing chamber (ADC2) with toluene–ethyl acetate–formic acid–methanol (6:6:1.6:1, v/v) as the mobile phase. Antioxidant bands were visualized by dipping in 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent. Five antioxidant compounds were identified as neochlorogenic acid (1), chlorogenic acid (2), cryptochlorogenic acid (3), rutin (4), and isoquercitrin (5), which could be the predominant contributors to the antioxidant activity of the leaves of H. sabdariffa L. Furthermore, principal component analysis (PCA) was carried out to discriminate ten accessions of H. sabdariffa L. using an image-processing software. This simple HPTLC fingerprint assisted by PCA can be used as a reliable method for the discrimination of different accessions of H. sabdariffa L.

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An ingredient of ‘Dasamoola’ and ‘Laghupanchamoola’ group of drugs, the source of ‘Brihati’ has been controversial. Although the dried root of Solanum anguivi is considered as the source of the drug ‘Brihati’ according to the Ayurvedic Pharmacopoeia of India, closely related and morphologically similar few species like Solanum torvum, Solanum melongena, Solanum incanum, and Solanum insanum are known as its substitutes. In the present study, a high-performance thin-layer chromatography (HPTLC) method was developed and validated for the chemoprofiling and quantitative estimation of glycoalkaloid solamargine from 5 species of the genus Solanum as well as market samples. The developed method was precise, accurate, robust, specific, and linear. The results showed that S. incanum has the highest content of solamargine, followed by S. insanum. Out of the 9 market samples analyzed, solamargine was detected only in 3 samples. Unsupervised pattern recognition techniques, such as principal component analysis and hierarchical cluster analysis, were used to analyze the complex fingerprint patterns and to predict the grouping of samples. The method clearly segregated the field and market samples. Our study is the first attempt to evaluate the drug ‘Brihati’ and the market samples using HPTLC.

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A rapid and reliable HPTLC method for analysis of aristolochic acids (AA) has been developed on the basis of a TLC procedure previously published by the German Drug Codex (DAC). The new method enables visual detection of the acids with certainty at very low levels (400 pg absolute of aristolochic acid A) in plant material and can therefore be used for screening Chinese drugs to ensure their safety on the basis of the absence of AA. Qualitatively Stephania tetrandra can be distinguished from various Aristolochia spp., Asarum spp. , and other drugs with similar Chinese names, on the basis of fingerprints.At least five different aristolochic acids can be detected in Aristolochia spp. The method is very specific and sensitive and is suitable for establishing and documenting adulteration of Stephania raw material with less than 1% Aristolochia fangji . By use of scanning densitometry quantitative determination of AA can be performed at low concentrations (1 ppm). Validation data (ICH) including linearity, limit of detection, precision, and accuracy are presented. The performance of the method is discussed on the basis of comparison with an HPLC-MS method proposed by the US FDA. HPTLC analysis is presented as a very useful complementary technique to the more sophisticated procedures used in legal cases.

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