UA3/3/1 Comments on Activities at the Western Kentucky State College Farm

WKU Farm report emphasizing ongoing research projects, crops and livestock yields and physical facilities

Studies on perispawning mortalities in brown trout (Salmo trutta L.) from Loch Leven, Kinross, Scotland

Investigations into perispawning mortalities in the brown trout (Salmo trutta L) population of Loch Leven, Kinross, revealed that death occurred as a result of infection with a particular species of Saprolegnia diclina Humphrey type 1. Increased surface area of infection was correlated with a decrease in ion and protein content of the blood and was further reflected by changes in the electrocardiogram pattern. These changes were essentially a widening of the QRS complex with inflection of the RS component, changes consistent with a decrease in certain ions in mammals. Histological changes associated with sexual maturity and fungal infection are described and compared with changes resulting from the administration of androgens to brown trout

Thermal-structural panel buckling tests

The buckling characteristics of a titanium matrix composite hat-stiffened panel were experimentally examined for various combinations of thermal and mechanical loads. Panel failure was prevented by maintaining the applied loads below real-time critical buckling predictions. The test techniques used to apply the loads, minimize boundary were shown to compare well with a finite-element buckling analysis for previous panels. Comparisons between test predictions and analysis for this panel are ongoing

Comparative evaluation of sequence analysis of 16S rRNA and rpoB genes for identification of aquatic mycobacteria

The nucleotide sequences of partial 16S rRNA and bacterial RNA polymerase ß-subunit (rpoB) genes for 57 mycobacterial strains were determined. Compared to the 16S rRNA gene sequences, variable regions were scattered along the whole fragment sequence, indicating that therpoBgene is more polymorphic. Unlike 16S rRNA sequences, species variation was observed within M. fortuitum strains. The topology of therpoB-based phylogenetic tree was almost the same as that of the 16S rRNA sequence analysis. These results suggest that therpoBgene is a highly conserved gene, and taxonomical studies based on this gene may be comparable with similar studies based on the 16S rRNA gene. The overall mean distance forrpoB-gene?based sequences was 2.5 times greater than that of the 16S rRNA gene for all 57 mycobacterial strains examined. However, some slowly growing mycobacteria could not be differentiated based onrpoBgene sequences. Moreover, a bootstrap value above 70% was observed for 13 nodes, while this value was 14 nodes in the case of 16S rRNA sequences. To the best of our knowledge, this is the first investigation evaluating the use of 16S rRNA andrpoBsequence analyses for identification of aquatic mycobacteria obtained from diverse geographical locations

Titanium Honeycomb Panel Testing

Thermal-mechanical tests were performed on a titanium honeycomb sandwich panel to experimentally validate the hypersonic wing panel concept and compare test data with analysis. Details of the test article, test fixture development, instrumentation, and test results are presented. After extensive testing to 900 deg. F, non-destructive evaluation of the panel has not detected any significant structural degradation caused by the applied thermal-mechanical loads

Comparative evaluation of Polymerase Chain Reaction - Restriction Enzyme Analysis (PRA) and Sequencing of Heat shock protein 65 (hsp65) gene for identification of aquatic mycobacteria

Traditional identification of mycobacteria based on cultural and biochemical tests can take several weeks and may fail to provide a precise identification. Polymerase Chain Reaction-restriction analysis (PRA) of the gene encoding heat shock protein 65kDA (hsp65) gene has been proposed as a rapid and inexpensive alternative approach. Despite being widely used for differentiation of mammalian mycobacteria, this method has only been applied in the identification of a small number of aquatic mycobacteria. The present study aimed to evaluate the potential use of PRA of hsp65 for the identification of aquatic mycobacteria compared with sequence analysis. Seventy one mycobaterial isolates including, 10 type/reference strains and the remainder field isolates, were subjected to PRA of a 441 bp fragment of this gene. For 68 representative isolates, sequence analysis was performed. All rapidly and slow growing mycobacteria had best matches with 99.3% to 100% similarity with their corresponding species in the databanks. PRA proved to be a simple and rapid method for identifying aquatic mycobacteria. However, the incidence of similar or identical restriction patterns for some species of mycobacteria, and in particular, identification of new species of mycobacteria is a major problem using such a method. In contrast, the nucleic acid sequencing of the hsp65 gene yielded unambiguous results

Identification of aquatic mycobacteria based on sequence analysis of the 16S-23S rRNA internal transcribed spacer region

Purpose. Mycobacteria are common causative agents of bacterial infections in many species of freshwater and marine fish. Identification of mycobacteria to the species level based on phenotypic tests is inappropriate and time consuming. Molecular methods such as partial or entire gene sequence determination in mycobacteria have been employed to resolve these problems. The objective of this study was to assess the use of sequence analysis of the mycobacterial 16S–23S internal transcribed spacer (ITS) region for the identification of different aquatic mycobacteria species. Methodology. Using published primers, the ITS sequences of 64 field and reference strains were determined. Results/Key findings. The identity of all isolates previously identified as Mycobacterium marinum by RFLP was confirmed as M. marinum by sequence analysis. With the exception of five rapidly growing mycobacteria isolates, all other mycobacteria were easily identified by sequencing of the ITS region. Using this spacer region, it was possible to differentiate between slowly growing and rapidly growing mycobacteria, even before sequence analysis, by the size of the PCR product, although species identification could not be made by size alone. Conclusion. Overall, direct sequencing of this genetic element following PCR has been shown to be useful in the identification of aquatic mycobacteria species. With regard to the variability of the ITS region for different mycobacteria isolates, this may be a useful tool in epidemiological studies

Alternative cell line for the isolation of salmonid alphavirus-1

Salmonid alphavirus (SAV) has recently become an economically important pathogen in salmonid aquaculture in Europe. Subtype SAV-1 causes salmon pancreas disease (SPD) in Atlantic salmon in Scotland and Ireland, and was first isolated on Chinook salmon embryo-214 (CHSE-214) cells in 1995 in Ireland; several established cell lines have since been tested for viral growth, although the ability of these cell lines to support primary virus isolation has not being examined. In the present study, CHSE-214, Chum salmon heart -1 (CHH-1) and Salmon head kidney -1 (SHK-1) cell lines were evaluated for isolation of SAV-1 from kidney samples of experimentally infected Atlantic salmon (Salmo salar). The presence of infection in these samples was confirmed both by cell culture and reverse transcription polymerase chain reaction (RT-PCR). Homogenates of kidney from fish 3 days post-infection (p.i.) were inoculated onto the three cell lines and the development of a cytopathic effect (CPE) recorded. The CHH-1 cells produced a rapid CPE from Day 6 p.i., while the CHSE-214 cells showed the presence of a CPE from Day 10 p.i. In comparison, a CPE developed much later in the SHK-1 cells, from Day 20 p.i. The virus was successfully isolated on all three cell lines in subsequent passages, indicating that CHSE-214, CHH-1, and SHK-1 cells can be used for the isolation and culture of SAV-1. The CHH-1 cell line, however, has proven the most useful, since the CPE developed the quickest in this cell line

Environmental conditions influence susceptibility of striped catfish Pangasianodon hypophthalmus (Sauvage) to Edwardsiella ictaluri

Over the last 20 years the production farmed Vietnamese striped catfish (Pangasianodon hypophthalmus) has increased significantly and in 2016, over 1.2 million tonnes of catfish were farmed and sold globally. Bacterial disease outbreaks due to Edwardsiella ictaluri continue to be one of the biggest threats to the sector, however, little is known on how the environmental conditions affect the survival of the fish during disease outbreaks. Growth of 14 Edwardsiella ictaluri strains recovered from natural disease outbreaks occurring in 4 provinces in Vietnam between 2002 and 2011 was investigated in vitro under different pH and salt concentrations. The results showed that a pH value of 6.5, NaCl concentration of 0.5% was optimal for the growth of the bacteria in vitro. The effect of varied pH and salt concentrations on the susceptibility of striped catfish to E. ictaluri infection was also studied in vivo following an immersion bacterial challenge (1.1 x 107 cfu ml−1 E. ictaluri for 30 s). The cumulative mortality of striped catfish in water at pH 5.5 and pH 6.5 was significantly higher than that of fish maintained in more alkaline water (p < .05). The cumulative mortality of the striped catfish maintained in 0.5% NaCl was significantly lower than those kept in 0%, 1% and 1.5% NaCl (p < .05). This study identified the effect of pH and salinity changes on the susceptibility of striped catfish to E. ictaluri infections

Pathogenesis of experimental salmonid alphavirus infection in vivo: an ultrastructural insight

Salmonid alphavirus (SAV) is an enveloped, single-stranded, positive sense RNA virus belonging to the familyTogaviridae. It causes economically devastating disease in cultured salmonids. The characteristic features of SAV infection include severe histopathological changes in the heart, pancreas and skeletal muscles of diseased fish. Although the presence of virus has been reported in a wider range of tissues, the mechanisms responsible for viral tissue tropism and for lesion development during the disease are not clearly described or understood. Previously, we have described membrane-dependent morphogenesis of SAV and associated apoptosis-mediated cell death in vitro. The aims of the present study were to explore ultrastructural changes associated with SAV infection in vivo. Cytolytic changes were observed in heart, but not in gill and head-kidney of virus-infected fish, although they still exhibited signs of SAV morphogenesis. Ultrastructural changes associated with virus replication were also noted in leukocytes in the head kidney of virus-infected fish. These results further describe the presence of degenerative lesions in the heart as expected, but not in the gills and in the kidney