Evaluation of microbial culture techniques for the isolation of Pythium insidiosum from equine tissues.

The purpose of this study was to evaluate the effects of sample handling, storage, and culture techniques on the isolation of Pythium insidiosum from infected equine tissues. Tissue and kunker samples obtained immediately posteuthanasia from a horse with subcutaneous pythiosis were used to assess the effects of sample type (kunkers vs. tissues), media type (selective vs. nonselective), storage technique, and storage time on P. insidiosum isolation rate. Overall, isolation rates were higher from fresh kunkers (94.6%) and stored kunkers (76.4%) than from fresh tissues (8.3%) or stored tissues (4.6%). Isolation of P. insidiosum also occurred more often on antibiotic-containing media than on nonselective media for both fresh and stored samples. For samples that were stored for 1-3 days prior to culture, P. insidiosum isolation rates were highest for the following techniques: kunkers stored at room temperature and plated on selective media (100%), kunkers stored at 4 C and then plated on either nonselective (91.7%) or selective (95.8%) media, kunkers stored on cold packs and then plated on either nonselective (93.8%) or selective (100%) media, kunkers stored in ampicillin solution and plated on selective media (100%), and kunkers stored in ampicillin/gentocin solution and plated on selective media (87.5%). For samples stored for 4-5 days, P. insidiosum isolation rates were highest for kunkers stored at 4 C and then plated on either nonselective (81.3%) or selective (87.5%) media, kunkers stored in ampicillin solution and then plated on selective media (87.5%), and kunkers stored in ampicillin/gentocin solution and plated on selective media (87.5%). Results of this study suggest that optimal isolation rates of P. insidiosum from infected equine tissues are achieved by culturing fresh kunkers on selective media. For samples that cannot be processed immediately, acceptable handling techniques include storage at room temperature for up to 3 days, refrigeration for up to 5 days, shipping on cold packs, and storage in antibiotic solution, each combined with subsequent inoculation on selective media.     

Validation of endogenous reference genes for expression profiling of RAW264.7 cells infected with Mycobacterium avium subsp. paratuberculosis by quantitative PCR

Reference genes are frequently used to normalize between different biological samples the levels of mRNA measured using quantitative PCR (qPCR). The expression level of many commonly used reference genes has been shown to vary between tissues or cells, or following exposure to various treatments including infection with microbes. The selection of an appropriate reference gene for an individual experiment is therefore a crucial step in the process of accurately determining changes in gene expression. For this purpose, we analyzed the expression of nine commonly used reference genes in a murine macrophage cell line, RAW264.7, for their potential use in the analysis of differential gene expression by quantitative polymerase chain reaction (qPCR) following experimental infection with Mycobacterium avium subsp. paratuberculosis. Only one of nine putative reference genes tested, casc3a, was found to be suitable, and combinations of two or more reference genes were disadvantageous. Based on data from the study, we recommend an approach for selection of reference genes, conducting assays with technical replicates in duplicate rather than triplicate, determining decision-limit quality control criteria for technical replicates and assessing the significance of gene expression fold differences using DeltaDeltaC(t) based on knowledge of the variation in the reference gene.     

Modelling the introduction and transmission of Campylobacter in a North American chicken flock

Campylobacter is the second leading cause of foodborne illness in the United States. Although many food production animals carry Campylobacter as commensal bacteria, consumption of poultry is the main source of human infection. Previous research suggests that the biology of Campylobacter results in complete flock colonization within days. However, a recent systematic review found that the on-farm prevalence of Campylobacter varies widely, with some flocks reporting low prevalence. We hypothesized that the low prevalence of Campylobacter in some flocks may be driven by a delayed introduction of the pathogen. The objectives of this study were to (a) develop a deterministic compartmental model that represents the biology of Campylobacter, (b) identify the parameter values that best represent the natural history of the pathogen in poultry flocks and (c) examine the possibility that a delayed introduction of the pathogen is sufficient to replicate the observed low prevalence examples documented in the literature. A deterministic compartmental model was developed to examine the dynamics of Campylobacter in chicken flocks over a 56-day time period prior to movement to the abattoir. The model outcome of interest was the final population prevalence of Campylobacter at day 56. The resulting model that incorporated a high transmission rate (β = 1.04) was able to reproduce the wide range of prevalence estimates observed in the literature when pathogen introduction time is varied. Overall, we established that the on-farm transmission rate of Campylobacter in chickens is likely high and can result in complete colonization of a flock when introduced early. However, delaying the time at which the pathogen enters the flock can reduce the prevalence observed at 56 days. These results highlight the importance of enforcing strict biosecurity measures to prevent or delay the introduction of the bacteria to a flock.     

Inheritance of yield components and yield in relation to evidence for heterosis in F1 barley hybrids

Summary Yield components and yield were studied in F₁ barley hybrids produced by hand pollination or male sterility. Grain number exhibited only partial dominance but grain weight showed dominance or overdominance and contributed to the heterotic situation particularly in 2×6-row crosses. For the commercial exploitation of heterosis it is essential that hybrids should be found which show greater dominance for high grain number.     

Morphological variation within collections of Moroccan almond clones and Mediterranean and North American cultivars

Summary Cultivated almonds (Prunus dulcis (Miller) D.A. Webb) in Morocco are still propagated from seeds by farmers to overcome transplant failure of grafted trees. Almond collections in southern Morocco conducted since 1975 have resulted in the selection of clones planted at 3 experimental stations. Principal component analysis (PCA) was used to compare kernel, nut, leaf, and growth habit characteristics among 67 selected Moroccan clones and 14 introduced cultivars. Clustering of clones from similar countries and collection areas would suggest the existence of different almond populations. The Moroccan clones did not cluster separately from the foreign cultivars. Three Moroccan clones had exceptionally large nuts and kernels while 7 selections had high yield potential due to high spur density. Moroccan selections tended to be characterized by small leaves in comparison to foreign cultivars. No evidence was found to suggest the existence of separate populations within the Moroccan almond germplasm.     

Morphological variation within a sour cherry collection

Summary Morphological traits of 28 full-sib sour cherry (Prunus cerasus L.) families developed with pollen from European sour cherry selections were evaluated with principal component (PC) analysis. The traits which loaded on the first PC were size characters such as lateral length, leaf area, fruit weight, and trunk diameter increase. These character loading on the first PC could be interpreted as representing gradations between morphologies characteristic of the 2 presumed progenitor species, sweet cherry (P. avium L.) and ground cherry (P. fruticosa Pall.). Mean family differences in trunk diameter increase, lateral length, leaf area, and fruit weight varied approximately 12, 3.7, 2.5, and 2 fold, respectively. These results suggest that it may be possible to select sour cherry hybrids approaching the tree and fruit size of either progenitor species. The results are discussed in reference to germplasm collection and the potential of certain cultivars and hybrids as parents.     

Re-test of Rhinocyllus conicus host specificity, and the prediction of ecological risk in biological control

Biological control is proposed as an ecological strategy to manage the threat of invasive plants, especially in natural areas. To pursue this strategy, we need to know that the host specificity criteria used to evaluate ecological risk with deliberate introduction of an exotic insect for biocontrol are sufficient to predict potential impact on native species. Host specificity is defined by adult feeding and oviposition preferences and larval development. One way to evaluate the criteria is to re-examine case histories where ecological effects are recorded, such as that of Rhinocyllus conicus Frölich. This flower head weevil, released in North America in 1968 to control exotic thistles like Musk thistle (Carduus nutans L), is now reducing seed production by multiple native North American thistle species (Cirsium spp.), and local population density of Platte thistle (Cirsium canescens Nutt.). We hypothesized that host specificity of R. conicus has changed since pre-release testing, providing an explanation for the unexpected magnitude of the documented ecological effects. Instead, when we re-tested host specificity of weevils naturalized over 28 generations, we found that host specificity has not changed. Naturalized adults of R. conicus showed strong feeding and oviposition preference for Musk thistle over Platte thistle. In addition, larval development by these weevils was faster and more successful (to larger size) on Musk thistle than on Platte thistle. Thus, our results indicate that a change in host specificity cannot explain the unexpectedly large build-up of R. conicus and significant ecological effect on Platte thistle. We conclude that accurate prediction of the potential level of impact on native host plants in the field requires further ecological information in addition to host specificity.     

Competitive ability of selected Cyclopia Vent. rhizobia under glasshouse and field conditions

This study tested the competitive ability of three locally isolated Cyclopia rhizobia and strain PPRICI3, the strain currently recommended for the cultivation of Cyclopia, a tea-producing legume. Under sterile glasshouse conditions, the three locally isolated strains were equally competitive with strain PPRICI3. In field soils, the inoculant strains were largely outcompeted by native rhizobia present in the soil, although nodule occupancy was higher in nodules growing close to the root crown (the original inoculation area). In glasshouse experiments using field soil, the test strains again performed poorly, gaining less than 6% nodule occupancy in the one soil type. The presence of Cyclopia-compatible rhizobia in field soils, together with the poor competitive ability of inoculant strains, resulted in inoculation having no effect on Cyclopia yield, nodule number or nodule mass. The native rhizobial population did not only effectively nodulate uninoculated control plants, they also out-competed introduced strains for nodule occupancy in inoculated plants. Nonetheless, the Cyclopia produced high crop yields, possibly due to an adequate supply of soil N.     

8S globulin of mungbean [Vigna radiata (L.) Wilczek]: cloning and characterization of its cDNA isoforms, expression in Escherichia coli, purification, and crystallization of the major recombinant 8S isoform

Three isoforms of the cDNA of the major 8S globulin of mungbean, 8Salpha, 8Salpha', and 8Sbeta, were isolated, cloned, and characterized. The cDNA sequences of 8Salpha, 8Salpha', and 8Sbeta had open reading frames of 1362, 1359 or 1362, and 1359 bp, respectively, which code for 454, 453 or 454, and 453 amino acids corresponding to molecular weights of 51 973, 51 627 or 51 758, and 51 779, respectively. Homology in terms of cDNA and amino acid sequences was 91-92% between 8Salpha and 8Salpha', 87% between 8Salpha and 8Sbeta, and 86-88% between 8Salpha' and 8Sbeta. The signal peptide was found to be 1-25, 1-24 or 25, and 1-23 for 8Salpha, 8Salpha', and 8Sbeta, respectively, using the signalP website (Nielsen, H.; Engelbrecht, J.; Brunak, S.; von Heijne, G. Protein Eng. 1997, 10, 1-6). The propeptide was determined to be IVHREN. A single site for glycosylation (N-X-S/T) was observed about 90 amino acids from the C terminus. Homology between mungbean 8S isoforms and other 7-8S proteins ranged from 45 to 68% within members of the legume family and 29 to 34% for crops of different species. The major isoform 8Salpha was expressed in Escherichia coli and purified by successive ammonium sulfate fractionation, hydrophobic interaction, and Mono Q column chromatography. The recombinant 8Salpha, but not the native form, was successfully crystallized producing rhombohedral crystals.