An optimised protocol for molecular identification of Eimeria from chickens.

Molecular approaches supporting identification of Eimeria parasites infecting chickens have been available for more than 20 years, although they have largely failed to replace traditional measures such as microscopy and pathology. Limitations of microscopy-led diagnostics, including a requirement for specialist parasitological expertise and low sample throughput, are yet to be outweighed by the difficulties associated with accessing genomic DNA from environmental Eimeria samples. A key step towards the use of Eimeria species-specific PCR as a sensitive and reproducible discriminatory tool for use in the field is the production of a standardised protocol that includes sample collection and DNA template preparation, as well as primer selection from the numerous PCR assays now published. Such a protocol will facilitate development of valuable epidemiological datasets which may be easily compared between studies and laboratories. The outcome of an optimisation process undertaken in laboratories in India and the UK is described here, identifying four steps. First, samples were collected into a 2% (w/v) potassium dichromate solution. Second, oocysts were enriched by flotation in saturated saline. Third, genomic DNA was extracted using a QIAamp DNA Stool mini kit protocol including a mechanical homogenisation step. Finally, nested PCR was carried out using previously published primers targeting the internal transcribed spacer region 1 (ITS-1). Alternative methods tested included sample processing in the presence of faecal material, DNA extraction using a traditional phenol/chloroform protocol, the use of SCAR multiplex PCR (one tube and two tube versions) and speciation using the morphometric tool COCCIMORPH for the first time with field samples.

Enzymic and in sacco methods to estimate rumen degradation of food protein in cattle.

BACKGROUND: Two factorial studies compared enzymic and in sacco methods to estimate degradation of ruminant foods. Enzyme degradation (in vitro = enzyme) was determined from the release of leucine-equivalent amino acid (LA) crude protein (CP) from sunflower meal (SF), maize gluten meal (MG), distillers' dark grain (DG) and field beans (FB) after their separate incubations with Streptomyces griseus enzyme for 0-24 h. In sacco crude protein (CP) degradation of these foods was estimated during washing (0 h) and rumen incubations in fistulated cows for 2-24 h. The LA data were expressed as g LA per either kg of CP (LACP) or acid-hydrolysable LA (HLA) of each food and compared with in sacco data. RESULTS: These methods showed comparable degradation with time (P < 0.01). The in sacco and HLA were greater than LACP for all foods except MG where in sacco value was either lower or equal to LACP depending upon the incubation time (P > 0.05 or P < 0.05). Conversely, HLA was significantly (P < 0.01) greater than LACP from 2 h onwards. At 0 h, in sacco values were significantly greater than those of enzyme for SF, DG and FB (P < 0.05) but not for MG. The foods differed significantly for degradation constants (a, b, c) in each method (P < 0.05). CONCLUSIONS: Despite variations between in sacco and enzyme estimates for different foods, the relationships between these estimates suggest that the HLA enzyme method has the potential to estimate food degradation. Copyright © 2007 Society of Chemical Industry.

Relative fibrolytic activities of anaerobic rumen fungi on untreated and sodium hydroxide treated barley straw in in vitro culture.

The fibrolytic activities of rumen fungi were studied in terms of dry matter loss, plant cell wall degradation and enzyme (cellulase and xylanase) activities, when grown in vitro on either untreated or sodium hydroxide treated stems of barley straw over a 12 day period. Changes in fungal growth, development and overall biomass were followed using chitin assay and scanning electron microscopy. Treatment with sodium hydroxide resulted in a decrease in the NDF content together with the disruption of cuticle and the loosening and separation of the plant cells within the straw fragments. The enzyme activities of the anaerobic fungi have a high positive correlation (R(2)=0.99) with their biomass concentration assessed by chitin assay indicating that chitin is a valuable index for the estimation of the fungal biomass in vitro. The anaerobic fungi produced very extensive rhizoidal systems in these in vitro cultures. After incubation with rumen fungi, dry matter losses were, respectively, 35% and 38% for the untreated and treated straw samples and the overall fungal biomass, determined by chitin assay, was significantly higher in the treated samples. In vitro degradation of cellulose and hemicellulose was also higher in the treated than that of untreated cultures. Although, comparatively, xylanase activity was higher than that of cellulase, the cellulose fraction of the straw was degraded more than hemicellulose in both treated and untreated straw.