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Confirmed diagnosis of alveolar echinococcosis (AE) is based on pathological criteria and molecular evidence. This parasite-borne disease, caused by the cestode Echinococcus multilocularis, sparingly involves humans as a dead-end host. In humans, the parasite mainly colonizes the liver but can colonize any organ and cause atypical forms, often difficult to characterize clinically. Moreover, molecular methods may be suitable to make the diagnosis of AE in cases of atypical forms, extra-hepatic localizations, or immunosuppressed patients. The aim of this study was to determine the most relevant published PCR techniques, for diagnosis of AE in patients and adopt the best strategy for molecular diagnosis depending on the nature of the tested sample. In this study, we evaluated nine end-point PCR assays and one real-time PCR assay (qPCR), targeting mitochondrial genes, using a total of 89 frozen or formalin-fixed paraffin-embedded (FFPE) samples from either 48 AE or 9 cystic echinococcosis patients. Targeted fragment-genes ranged from 84 to 529 bp. Six PCR assays were able to amplify the DNA of 100% of the frozen AE-samples and for one PCR, 69.8% of the FFPE AE-samples. The 16S rrnL PCR (84 bp) was positive in PCR for 77% of the AE samples and in qPCR for 86.5%. The sensitivity of the PCR assays was higher for fresh samples and FFPE samples stored for less than 5 years. The qPCR assay further increased sensitivity for the tested samples, confirming the need for the development of an Echinococcus spp. qPCR to improve the molecular diagnosis of echinococcoses.Diagnostic moléculaire de l’échinococcose alvéolaire chez les patients à partir d’échantillons de tissus congelés et fixés au formol et inclus en paraffine.La confirmation diagnostique de l’échinococcose alvéolaire (EA) est basée sur des critères anatomo-pathologiques et moléculaires. Cette maladie d’origine parasitaire, causée par le cestode Echinococcus multilocularis, implique sporadiquement l’homme, impasse parasitaire. Chez l’homme, le parasite colonise principalement le foie mais peut coloniser tout organe et causer des formes atypiques, souvent difficiles à caractériser cliniquement. En outre, les méthodes moléculaires permettent de réaliser le diagnostic de l’EA dans les formes atypiques, les localisations extra-hépatiques ou chez les patients immunodéprimés. Le but de cette étude était de déterminer les techniques PCR publiées les plus pertinentes, pour le diagnostic de l’EA chez les patients et adopter la meilleure stratégie par diagnostic moléculaire en fonction de la nature de l’échantillon testé. Dans cette étude nous avons évalué neuf PCR en point-final et une PCR-temps-réel (qPCR), ciblant des gènes mitochondriaux, utilisant 89 échantillons congelés ou fixés en paraffine (FFPE) de patients EA (n = 48) ou présentant une échinococcose kystique (n = 9). Les fragments de gènes ciblés allaient de 84 à 529 pb. Six tests PCR ont permis d’amplifier l’ADN de 100 % des échantillons EA congelés, et pour une PCR, 69,8 % des échantillons EA-FFPE. La PCR 16S rrnL (84 pb) était positive en PCR pour 77 % des échantillons EA et en qPCR pour 86,5 %. La sensibilité des tests PCR était plus importante pour les échantillons congelés et les FFPE stockés moins de 5 ans. Le test qPCR a permis d’augmenter la sensibilité pour les échantillons testés, confirmant le besoin de développement d’une qPCR Echinococcus spp. pour améliorer le diagnostic moléculaire des échinococcoses.

This article (work) gives information about production of methane from 11 samples of grass silage. Production was studied in two following levels:* specific methane production per 1 kg of organic total solids,* specific methane production per 1 kg of fresh material.These grass silage samples were at the same time analyzed from the point of view of feeding properties and quality: protein, digestible protein, fiber, acid-detergent fiber (ADF), neutral- detergent fiber (NDF), BNVL, lactic acid, acetic acid, butyric acid, starch values, metabolizable energy, total energy, net energy lactation, net energy for fattening, pH, acidity of the water leachate mg KOH, protein PDIA, protein PDIN, protein PDIE, Na, K, P, Mg, Ca and ash.Statistic dependence was searched between feeding quality and methane production of tested samples. As statistically significant parameters influencing methane production were: lactic acid, pH, acidity of the water leachate mg KOH and total solids. Change of concentration of remaining parameters had not adequately provable influence on methane production.

Two new species of Coccidia, Eimeria sparis sp. nov. and Goussia sparis sp. nov., were found in the intestine of Sparus aurata from different culture systems of Spain. These are the first coccidian species described from this host. E. sparis is distinguished from other Eimeria spp. from Perciformes in the size and shape of the sporocysts, which measure 6-9.7 (mean = 7.78, SD = 1.33) x 46.5 (5.3 ± 0.92) μm, and in the Stieda-like body. Mature oocysts, spherical or subspherical, measure 9.4-14.3 (11.88 ± 1.85) μm. G. sparis differs from other Goussia spp. from fish of the Mediterranean area in the size and shape of the oocysts and sporocysts as well as in the location. Mature oocysts measure 16-21 (17.4 ± 1.5) x 13-18 (14.4 ± 1.7) μm and sporocysts, 8.6-10.3 (9.5 ± 0.5) x 5.7-7.4 (mean 6.5 ± 0.5) μm. The different stages of merogony, gamogony, and sporogony of both species were examined at the light-microscope level in fresh material and histology specimens. Sporulation was endogenous in E. sparis and exogenous in G. sparis., This research was supported by research grants PTR-393-0073 from the Spanish CICYT and FAR/AQ/1.178 from the EC. We are grateful to Mr. J. Monfort and Ms. M. J. San Cleto for the histology processing. O. Palenzuela was the recipient of a grant from the Departamento de Educación, Universidades e Investigación del Gobierno Vasco.