Development of a multiplex PCR assay for polymorphism analysis of Brucella suis biovars causing brucellosis in swine

Swine brucellosis is caused by the biovars 1, 2 and 3 of Brucella suis the identification of which up to now relies on microbiological tests lacking adequate specificity together with time consuming and expensive molecular procedures. Based on sequence variation of the omp2b gene, we have developed a four primer set multiplex PCR assay that was tested for polymorphism analysis of B. suis biovars causing brucellosis in swine. The assay exploits the single nucleotide polymorphisms found in omp2b gene of B. suis reference biovars which are conserved in 43 B. suis field isolates from different geographic origins and hosts. Three specific amplification patterns (S1, S2 and S3) were obtained for reference strains of B. suis biovars 1, 2 and 3, respectively. However, some B. suis field isolates identified as biovars 2 or 3 according AMOS-PCR, PCR-RFLP of omp31 and omp2 genes and classical bacteriological methods, resulted also in S1 patterns, limiting the typing usefulness of the method.     

PCR‐based prevalence of a fatal reovirus of the blue crab,Callinectes sapidus (Rathbun) along the northern Atlantic coast of the USA

There is a need for more information on the relationship between diseases and fluctuations of wild populations of marine animals. In the case of Callinectes sapidus reovirus 1 (CsRV1, also known as RLV), there is a lack of baseline information on range, prevalence and outbreaks, from which to develop an understanding of population‐level impacts. An RT‐qPCR assay was developed that is capable of detecting 10 copies of the CsRV1 genome. In collaboration with state, federal and academic partners, blue crabs were collected from sites throughout the north‐eastern United States to assess the northern range of this pathogen. In addition, archived crab samples from the Chesapeake Bay were assessed for CsRV1 by RT‐qPCR and histology. PCR‐based assessments indicate that CsRV1 was present at all but one site. Prevalence of CsRV1 as assessed by RT‐qPCR was highly variable between locations, and CsRV1 prevalence varied between years at a given location. Mean CsRV1 prevalence as assessed by RT‐qPCR was >15% each year, and peak prevalence was 79%. The wide geographic range and highly variable prevalence of CsRV1 indicate that more study is needed to understand CsRV1 dynamics and the role the virus plays in blue crab natural mortality.     

Evaluation and validation of a commercial ELISA for diazinon in surface waters

The performance of a commercially available microtiter plate ELISA kit for the determination of diazinon was evaluated for sensitivity, selectivity, intra-assay repeatability, accuracy, and matrix effects in fortified distilled water and filtered and unfiltered environmental surface water samples. Repeatability and reproducibility studies show that the kit satisfies current EPA criteria for the assessment of analytical methods. Mean recoveries from spiked samples averaged 80.3, 95.5, and 103.5% from distilled, unfiltered surface, and filtered surface waters, respectively. The experimentally determined method detection limit (MDL) for the commercial diazinon microtiter plate format (0.0159 microg L(-)(1)) was comparable to the least detectable dose (LDD) established by the manufacturer (0.022 microg L(-)(1)). Specificity studies indicate that the diazinon polyclonal antibody can readily distinguish the target compound from other structurally similar organophosphorus analogues, with the exception of diazoxon. Cross-reactivity with the oxon was approximately 29%, while reactivity with pirimiphos-methyl, pirimiphos-ethyl, and chlorpyrifos-ethyl was negligible. A slight matrix effect was discovered to be present in both filtered and unfiltered environmental water matrixes, but its effect on the immunoassays is insignificant within experimental error. For validation of the microtiter plate ELISA format, environmental surface and storm runoff water samples were collected, split, and analyzed directly by ELISA and by liquid-liquid extraction followed by GC (California State Department of Food and Agriculture method EM 46.0). Results of the two analytical methods were then compared statistically. A close correlation was found between methods for unspiked and untreated river water samples (r = 0.969) while a much less robust correlation was obtained for runoff waters (r = 0.728). Results from runoff waters exhibit a particularly high positive bias for the ELISA method relative to the GC method. Cross-reactivity of diazoxon and probably other unidentified cross-reacting components may be responsible for the exaggerated account of the target analyte in surface and runoff waters. While excellent for screening purposes, further study is required to elucidate and quantify the factors responsible for the consistent overestimation of ELISA results before the kit can be employed routinely for regulatory compliance monitoring.     

Clinical and epidemiological features of canine leptospirosis in North Queensland

OBJECTIVE: To evaluate the clinical signs, results of clinical pathology and serology tests, and treatment outcome of clinical leptospirosis in 40 dogs from North Queensland. DESIGN: Retrospective study from January 1995 to August 1999. PROCEDURE: Case records were reviewed for age, breed, sex, month of submission, geographical location and presenting clinical signs in 40 dogs with titres of > or = 200 for leptospirosis by the microscopic agglutination test. A biochemistry panel and complete blood count were performed on 18 dogs. RESULTS: Canine leptospirosis occurred most frequently during the summer and autumn particularly in the 'wet' tropical coastal areas of Mackay and Cairns. Fewer cases were seen in the Atherton Tablelands and 'dry' tropics around Townsville. Young and male dogs were more commonly affected. Most cases were caused by L australis (80%) and L zanoni (15%) with individual cases of L hardjo and L copenhageni. All dogs showed a distinctive multiorgan disease pattern including renal failure and cholestatic hepatopathy. Presenting clinical signs were related to these disease syndromes and included jaundice, vomiting, lethargy, inappetence, dehydration, pyrexia, abdominal pain and diarrhoea. Just over 50% of the dogs in the present study failed to recover. Clinical biochemical changes indicative of renal failure and cholestasis were significantly less severe in dogs that recovered compared with dogs that did not survive. CONCLUSIONS: Clinical pathology testing should be performed on all dogs presented for illness in the endemic areas so as to help make a presumptive diagnosis and assist in determining the prognosis as soon as possible. The presumptive diagnosis should be confirmed serologically.     

Localization and physical properties experiments conducted by Spirit at Gusev Crater

The precise location and relative elevation of Spirit during its traverses from the Columbia Memorial station to Bonneville crater were determined with bundle-adjusted retrievals from rover wheel turns, suspension and tilt angles, and overlapping images. Physical properties experiments show a decrease of 0.2% per Mars solar day in solar cell output resulting from deposition of airborne dust, cohesive soil-like deposits in plains and hollows, bright and dark rock coatings, and relatively weak volcanic rocks of basaltic composition. Volcanic, impact, aeolian, and water-related processes produced the encountered landforms and materials.     

Coastal land scalding in NSW,Australia: soil chemical characteristics and their implications for remediation of the scalded lands

Scalded lands are common in acid sulphate soil areas on the New South Wales coast, Australia. In this work, chemical characteristics of the scalded acid sulphate soils at nine sites along this coast were investigated. The investigated acid sulphate scalds are characterized by an extremely acidified topsoil layer (0–0.6 m) although they derive from the sediments of varying salinity and the metal sulphides contained in the soils have experienced different degrees of oxidation. Almost all of the investigated scalds occur in the areas that have a lower surface elevation than the surrounding areas. These hollows may act as sinks for acid sulphate materials and salts that are transported from the surrounding areas and the shallower watertables in such locations may enhance upward transport of acid and salt materials from the underlying oxidized sulphidic sediments. In general, the scalded acid sulphate soils have less organic matter and soluble phosphorus, and a greater salinity, soluble acidity, soluble Al, Mn and Zn concentrations, compared to the adjacent non‐scalded acid sulphate soils. These are most likely soil constraints for revegetation of the scalded lands and treatment will need to involve acid neutralization (e.g. application of lime) and addition of P fertilizers to reduce the soluble acidity, immobilize soluble Al, Mn and Zn, and increase P availability. The evidence also shows that the higher soluble Al concentration in the scalded soils, relative to the non‐scalded soils, is related to their lower organic matter content. Hence, rehabilitation of these scalded lands should involve the addition of organic matter to reduce soluble Al concentrations; it may also help reduce Mn and Zn toxicity, and salinity. Copyright © 2001 John Wiley & Sons, Ltd.