Non-invasive imaging of plant roots in different soils using magnetic resonance imaging (MRI)

Background

Root systems are highly plastic and adapt according to their soil environment. Studying the particular influence of soils on root development necessitates the adaptation and evaluation of imaging methods for multiple substrates. Non-invasive 3D root images in soil can be obtained using magnetic resonance imaging (MRI). Not all substrates, however, are suitable for MRI. Using barley as a model plant we investigated the achievable image quality and the suitability for root phenotyping of six commercially available natural soil substrates of commonly occurring soil textures. The results are compared with two artificially composed substrates previously documented for MRI root imaging.

Results

In five out of the eight tested substrates, barley lateral roots with diameters below 300 µm could still be resolved. In two other soils, only the thicker barley seminal roots were detectable. For these two substrates the minimal detectable root diameter was between 400 and 500 µm. Only one soil did not allow imaging of the roots with MRI. In the artificially composed substrates, soil moisture above 70% of the maximal water holding capacity (WHC_max) impeded root imaging. For the natural soil substrates, soil moisture had no effect on MRI root image quality in the investigated range of 50–80% WHC_max.

Conclusions

Almost all tested natural soil substrates allowed for root imaging using MRI. Half of these substrates resulted in root images comparable to our current lab standard substrate, allowing root detection down to a diameter of 300 µm. These soils were used as supplied by the vendor and, in particular, removal of ferromagnetic particles was not necessary. With the characterization of different soils, investigations such as trait stability across substrates are now possible using noninvasive MRI.

     

Evaluation of two commercial enzyme-linked immunosorbent assays for detection of bovine viral diarrhoea virus in serum and skin biopsies of cattle

AIM: To assess the ability of two commercial bovine viral diarrhoea (BVD) virus (BVDV) antigen-capture enzyme-linked immunosorbent assays (ELISAs) to detect virus in serum and skin biopsies. METHODS: Thirty cattle persistently infected (PI) with BVDV were identified using routine diagnostic laboratory testing. Additional ear-notch skin biopsies and blood samples were collected from these animals to confirm the diagnosis, and from 246 cohorts, to determine their BVDV status. Skin biopsies were soaked overnight in buffer and the eluate collected. All sera and eluate were tested using two commercially available ELISAs for detecting BVDV antigen, and a subsample of positive and negative sera was tested using a polymerase chain reaction (PCR) test. A study was also performed to ascertain the risk of cross contamination occurring during the collection and processing of skin biopsies. RESULTS: Both serum and skin samples tested using either ELISA resulted in the detection of all cattle identified as PI and no non-infected cattle were incorrectly classified as infected using either method. Agreement between all assays (ELISAs, whether performed on serum or skin, and PCR) was 100%. No cross-contamination of skin samples between animals was evident using routine biopsy methods. CONCLUSIONS: Viraemic cattle infected with BVDV were accurately identified using either of the two commercial ELISAs evaluated on either serum or skin samples. CLINICAL RELEVANCE: Either skin biopsies or serum samples can be collected from cattle to determine their BVDV status. This should overcome problems in accurately identifying the infection status of young calves in which colostral antibodies might interfere with the antigen-capture ELISA.     

Comparison of serological tests and faecal culture for the detection of Mycobacterium avium subsp. paratuberculosis infection in cattle and analysis of the antigens involved

Three hundred and forty-one sera from cattle in Western Australia and 106 sera from Mycobacterium paratuberculosis faecal culture positive cattle were used to evaluate the performance of two absorbed enzyme-linked immunosorbent assays (ELISA) (one locally produced, the other a commercial test) and a complement fixation test (CFT) for the detection of Johne's disease in cattle. The diagnostic sensitivity (47.2%) of the local ELISA was significantly higher than that of the commercial ELISA (31.1%), and significantly higher than that for the complement fixation test (17.9%) and immunoblot (20.8%). Diagnostic specificity for the two ELISAs was 99.7% and 97.9% and similar for CFT and immunoblot (97.1% and 97.7%, respectively). The diagnostic sensitivity rose for both ELISAs and the CFT as the number of M. paratuberculosis isolated from the faeces increased. The ELISA antigen was characterised by polyacrylamide gel electrophoresis and electrophoretic immunoblotting and was found to consist mostly of a carbohydrate-type macromolecule of 32-42 kDa. This macromolecule was identified as lipoarabinomannan (LAM) by using a LAM-specific monoclonal antibody in immunoblots and purified LAM in absorption experiments. By applying more complex antigen preparations in immunoblots, serum antibodies against proteins of 47, 37, 30, 24 and 21 kDa, and against the 32-42 kDa carbohydrate component were frequently found in infected cattle, and of these the 47 kDa protein and the 32-42 kDa antigen were immuno-dominant. Pre-absorption of the sera with M. phlei sonicate indicated that the protein antigens contributed markedly to non-specific serological cross-reactions, while the 32-42 kDa non-protein macromolecule appeared to be specific.     

Chronic intestinal pseudo-obstruction in a dog

SUMMARY A three-year-old neutered female Border Collie was presented with in-appetence, vomiting and diarrhoea. Abdominal radiographs revealed an obstructive pattern but no physical obstruction was evident at laparotomy. A diagnosis of chronic intestinal pseudo-obstruction was made based on histopathological changes in intestinal biopsies. Treatment was unsuccessful and the dog deteriorated progressively until euthanased five weeks after presentation.     

Attempted definition by immunoblotting of the causes of reactivity in suspected false-positive sera in the Brucella ovis complement fixation test

Seventy-nine suspected false-positive sera, obtained over 1 year from routine submissions for Brucella ovis serological testing, were used in this study. These sera, which exhibited titres in the complement fixation test, but which because of their epidemiological history and their reactions in the enzyme-linked immunosorbent assay and gel diffusion test were suspected to be false positives, were further analysed by immunoblotting. In blots, using B. ovis antigens, rough lipopolysaccharide was identified as the major, immuno-reactive bacterial component. Antibodies against this macromolecule were present in 46.8% of the suspected false-positive sera. In order to find out if rough lipopolysaccharides from other bacterial species could be the possible cause for the suspected false positivity, 23 sera with highest complement fixation titres were reacted in blots with cell extracts from Escherichia coli, Yersinia enterocolitica, Yersinia pseudotuberculosis, Bortedella bronchiseptica, Actinobacillus seminis, Campylobacter fetus fetus, Campylobacter jejuni, Mycobacterium paratuberculosis, Mycobacterium phlei, Corynebacterium pseudotuberculosis and pure lipopolysaccharides from Escherichia coli and Salmonella typhimurium. Despite high frequencies of antibody reaction with proteins in most of these bacterial cell extracts, which reflect the presence of infections with these bacteria, immuno-staining in the rough lipopolysaccharide region was not observed.     

Performance characteristics of an enzyme-linked immunosorbent assay performed in milk for the detection of liver fluke (Fasciola hepatica) infection in cattle

AIM: To determine the performance characteristics of an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against liver fluke (Fasciola hepatica) in bovine milk. METHODS: Serum and milk from liver fluke infected and non-infected cattle was assayed in a commercially available enzyme-linked immunosorbent assay. Serum test results were used to determine the "gold standard" infection status of cattle and milk ELISA results assessed by ROC analysis. RESULTS: ROC analysis suggested changes to the ELISA protocol, arriving at milk dilutions assayed considerably higher than those suggested by the manufacturer. With those changes, the ELISA performed with high sensitivity and specificity, 95 and 98.2%, respectively, for individual bovine milks (relative to sera). For bovine tank milks, sensitivity was lower, with bulk milks only testing positive if 60% or more of cattle milking in the herd were infected. CONCLUSIONS: The analysis of the ELISA's performance when used on individual bovine milks demonstrated high sensitivity and specificity. ROC analyses optimised the assay conditions and cut-off point suggested by the manufacturer for this commercial diagnostic assay. This would help with the identification and control of fasciolosis, enabling simpler sample collection.     

Effect of clenbuterol on growth,carcase and skeletal muscle characteristics in broiler chickens

1. Male and female broiler chickens (144 in total) were given diets supplemented with clenbuterol (CB) at 0 (control) and at 1 mg/kg between 28 and 49 d of age to study the effect of CB on growth, carcase and skeletal muscle.

2. CB improved growth in males by increasing daily weight gain and final live weight and by lowering food conversion ratio. In females it changed the carcase composition by reducing abdominal fat pad and by increasing the proportion of protein. Consequently, carcase protein gain was increased in both sexes (11% and 16%, respectively).

3. Skeletal muscle weights were enhanced by between 6% and 22%. Muscle fibre diameters were increased in extensor hallucis longus (EHL) but not in gastrocnemius (GAS) muscle. This increase was more pronounced in females. EHL total muscle fibre number remained unchanged. The proportion of fast‐twitch glycolytic fibres was increased at the expense of fast‐twitch oxidative fibres in males only. Nuclear/cytoplasm and DNA/protein ratios tended to be decreased by CB.

4. From the elevated EHL muscle RNA/DNA, unchanged protein/RNA and translation activity it is suggested that CB stimulated protein synthesis at the pretranslational level. Reduced protein degradation is deduced from decreased neutral calcium‐dependent proteolytic activity.

5. It is concluded that broiler chickens respond to long‐term CB treatment as has been shown in various mammals. However, the sex‐specific response in growth, carcase composition and skeletal muscle cellularity is more clearly apparent in broiler chickens.