Optimisation and validation of a PCR to detect viable Tenacibaculum maritimum in salmon skin tissue samples.

A PCR protocol was optimised and validated for the detection of viable Tenacibaculum maritimum cells in salmon skin tissue. Viability conventional (vPCR) and quantitative PCR (v-qPCR) assays both had a limit of detection of 103 CFU mL-1 viable cells. The v-qPCR assay showed a linear quantification over 4 log units. Conventional vPCR showed complete signal suppression when only dead cells were present at concentrations lower than 106 CFU mL-1. While the v-qPCR did not result in complete suppression when only dead cells were present, a method was developed to determine if viable cells were present based on the % Δ in cycle threshold (Ct) value. The procedure was validated for high-throughput processing and an enrichment protocol was validated to reliably detect low concentrations of viable cells both with and without a high background of dead cells. Performing this protocol on naturally infected tissues showed that vPCR and v-qPCR reduced the potential for false positives compared to using conventional PCR and qPCR. The optimised protocol developed for this study provides an efficient, reliable and robust alternative for the detection of viable T. maritimum in skin tissue.

New Zealand juvenile oyster mortality associated with ostreid herpesvirus 1-an opportunistic longitudinal study.

During the 2010-11 summer outbreak of ostreid herpesvirus 1 (OsHV-1) in New Zealand, an opportunistic longitudinal field study was conducted. OsHV-1 PCR-negative oyster spat (Crassostrea gigas) were relocated to an OsHV-1 PCR-positive area of the North Island of New Zealand that was experiencing juvenile oyster mortalities. Over a period of 13 d, spat were monitored for mortality, sampled for histopathology, and tested for the presence of OsHV-1 using real time PCR and Vibrio culture. Histopathology showed some evidence of tissue pathology; however, no consistent progressive pathology was apparent. Field mortalities were evident from Day 6 on. After 5 and 7 d of exposure, 83 and 100% of spat, respectively, tested positive for the virus by real time PCR. Vibrio species recovered during the longitudinal study included V. splendidus and V. aestuarianus. This study offers insight into the rapidity of onset and virulence of the virus in naïve oyster spat in New Zealand waters.

Development and validation of a real-time PCR assay for the detection of Aeromonas salmonicida.

A real-time PCR assay using a molecular beacon was developed and validated to detect the vapA (surface array protein) gene in the fish pathogen, Aeromonas salmonicida. The assay had 100% analytical specificity and analytical sensitivities of 5 ± 0 fg (DNA), 2.2 × 10(4) ± 1 × 10(4) CFU g(-1) (without enrichment) and 40 ± 10 CFU g(-1) (with enrichment) in kidney tissue. The assay was highly repeatable and proved to be robust following equivalency testing using a different real-time PCR platform. Following analytical validation, diagnostic specificity was determined using New Zealand farmed Chinook salmon, Oncorhynchus tshawytscha (Walbaum), (n = 750) and pink shubunkin, Carassius auratus (L.) (n = 157). The real-time PCR was run in parallel with culture and all fish tested were found to be negative by both methods for A. salmonicida, resulting in 100% diagnostic specificity (95% confidence interval). The molecular beacon real-time PCR system is specific, sensitive and a reproducible method for the detection of A. salmonicida. It can be used for diagnostic testing, health certification and active surveillance programmes.

Development and validation of real-time PCR for the detection of Yersinia ruckeri.

Yersiniosis (enteric red mouth disease) is a contagious bacterial disease caused by Yersinia ruckeri, which primarily affects salmonids. A real-time PCR assay using a molecular beacon has been developed and validated to improve the detection of the causative biotypes of Y. ruckeri. The assay, which targets the glnA (glutamine synthetase) gene, proved to have 100% analytical specificity and analytical sensitivities of 5 fg and 3 × 10(3) CFU g(-1) for DNA and seeded kidney tissue, respectively. The assay was highly repeatable with low % CV for intra- and inter-run experiments, and the optimized parameters transferred easily between different real-time PCR platforms. Following analytical validation, diagnostic specificity was determined using New Zealand farmed Chinook salmon (n = 750) from 10 farms during 2007/08. The real-time PCR was run in parallel with the bacterial culture detection method, and all fish tested were found to be negative by both methods for Y. ruckeri, resulting in 100% diagnostic specificity (95% confidence interval). The molecular beacon real-time PCR system is specific, sensitive, reproducible and a rapid method for the detection of Y. ruckeri and has the potential to be used for routine diagnostic testing, health certification and active surveillance programmes.

Outbreaks of pleuritis and peritonitis in calves associated with Pasteurella multocida capsular type B strain.

CASE HISTORY: Three dairy calf-rearing properties experienced high mortality in calves during 2008 and 2009. Affected calves were aged 13-18 weeks (Farm I), 6 months (Farm II), and 2-11 weeks (Farm III), and the mortality rate was 22/175 (13%), 5/80 (6%), and 60/900 (7%), respectively. CLINICAL AND LABORATORY FINDINGS: Affected calves rapidly became moribund, were in respiratory distress, and had a fever (40-41°C). Post-mortem examination of nine calves revealed fibrinopurulent pleuritis, pericarditis, and peritonitis. This was confirmed histopathologically on tissues from three calves, one from each farm; aggregates of small Gram-negative coccobacilli were evident on Gram stain. Pasteurella multocida was cultured from tissues from affected calves on the three farms, and PCR of DNA extracted from tissue samples amplified cap-sular type B-specific DNA. Multi-locus sequence typing (MLST) demonstrated that all capsular type B isolates belonged to the same sequence type (ST), ST62, but did not belong to serotype B:2, the only B serotype classified as causing haemorrhagic septicaemia by the Office International des Epizooties (OIE). DIAGNOSIS: Pleuritis and peritonitis due to infection with P. multocida capsular type B strain. CLINICAL RELEVANCE: Haemorrhagic septicaemia was excluded as a cause of disease from the three farms, however P. multocida was the primary agent in the affected calves. It is possible the agent has been present in New Zealand for some time but not reported, as there had been no transfer of animals between affected farms. Emergence of the syndrome could potentially be a result of factors other than just the presence of the organism, such as changing management. The syndrome described may be of increasing importance in the future.

Metals tolerance in moderately thermophilic isolates from a spent copper sulfide heap, closely related to Acidithiobacillus caldus, Acidimicrobium ferrooxidans and Sulfobacillus thermosulfidooxidans.

Selective enrichments enabled the recovery of moderately thermophilic isolates with copper bioleaching ability from a spent copper sulfide heap. Phylogenetic and physiological characterization revealed that the isolates were closely related to Sulfobacillus thermosulfidooxidans, Acidithiobacillus caldus and Acidimicrobium ferrooxidans. While isolates exhibited similar physiological characteristics to their corresponding type strains, in general they displayed similar or greater tolerance of high copper, zinc, nickel and cobalt concentrations. Considerable variation was found between species and between several strains related to S. thermosulfidooxidans. It is concluded that adaptation to metals present in the bioleaching heap from which they were isolated contributed to but did not entirely explain high metals tolerances. Higher metals tolerance did not confer stronger bioleaching performance, suggesting that a physical, mineralogical or chemical process is rate limiting for a specific ore or concentrate.

The discovery and synthesis of highly potent, A2a receptor agonists.

A series of N6,2-disubstituted adenosine analogues have been synthesized and their functional activity measured against A2a and A1 receptors. Examples of compounds with both a lipophilic N6-substituent and amino-functionalized 2-position were highly active at the A2a receptor on the human neutrophil.

The squalestatins: decarboxy and 4-deoxy analogues as potent squalene synthase inhibitors.

Squalestatins without either the hydroxy group at C-4 or the carboxylic acid at C-3 or C-4 were prepared and evaluated for their ability to inhibit rat liver microsomal squalene synthase (SQS) in vitro. These modifications were well tolerated for compounds with the 4,6-dimethyloctenoate ester at C-6 (S1 series). However in analogues without the C-6 ester (H1 series), removal of the C-4 hydroxy group gave compounds with reduced potency, whereas decarboxylation at C-3 resulted in a dramatic loss of SQS inhibitory activity. In comparison with S1 1, C-4 deoxyS1 3 and C-3 decarboxyS1 10 have shorter in vivo durations of action on the inhibition of hepatic cholesterol biosynthesis in rats. C-4 deoxyS1 3 retains good serum cholesterol-lowering ability in marmosets, while C-3 decarboxyS1 10 showed only a marginal effect even at high dose.

Inhibitors of cholesterol biosynthesis. 1. 3,5-Dihydroxy-7-(N-imidazolyl)-6-heptenoates and -heptanoates, a novel series of HMG-CoA reductase inhibitors.

3,5-Dihydroxy-7-(N-imidazolyl)heptanoates 4 and the corresponding heptenoates 5 were synthesized as novel classes of potent HMG-CoA reductase (HMGR) inhibitors in which members of the latter series possess enzyme inhibitory activity greater than that of lovastatin 1 and pravastatin 2. Structure-activity studies show that the 7-(N-imidazolyl)heptenoates 5 are more active than the corresponding heptanoates 4. For both imidazolyl series, the 4-fluorophenyl group is preferred at C-5, and a broad range of aryl substituents which promote widely different lipophilicities is tolerated at C-4. While the CF3 group is preferred at C-2 in the heptanoate series, the 2-(1-methylethyl) substituent is optimal in the heptenoate series. The 2-(1-methylethyl) and 5-(4-fluorophenyl) groups can be interchanged in the latter series as exemplified by 5ab. Enzyme inhibitory activity resides principally in the 3R,5S series. These potent HMGR inhibitory activities by members of the heptenoate series translated well into whole cell activities in HepG2 cells. X-ray crystallographic studies on the active enantiomer 28 reveal noncoplanarity of the heptenoate C-C double bond with the imidazole ring; this finding provides an explanation for the high acid stability of the heptenoate series.