Comparative evaluation of molecular diagnostic tests for Nucleospora salmonis and prevalence in migrating juvenile salmonids from the Snake River, USA

Nucleospora salmonis is an intranuclear microsporidian that primarily infects lymphoblast cells and contributes to chronic lymphoblastosis and a leukemia-like condition in a range of salmonid species. The primary goal of this study was to evaluate the prevalence of N. salmonis in out-migrating juvenile hatchery and wild Chinook salmon Oncorhynchus tshawytscha and steelhead O. mykiss from the Snake River in the U.S. Pacific Northwest. To achieve this goal, we first addressed the following concerns about current molecular diagnostic tests for N. salmonis: (1) nonspecific amplification patterns by the published nested polymerase chain reaction (nPCR) test, (2) incomplete validation of the published quantitative PCR (qPCR) test, and (3) whether N. salmonis can be detected reliably from nonlethal samples. Here, we present an optimized nPCR protocol that eliminates nonspecific amplification. During validation of the published qPCR test, our laboratory developed a second qPCR test that targeted a different gene sequence and used different probe chemistry for comparison purposes. We simultaneously evaluated the two different qPCR tests for N. salmonis and foundthat both assays were highly specific, sensitive, and repeatable. The nPCR and qPCR tests had good overall concordance when DNA samples derived from both apparently healthy and clinically diseased hatchery rainbow trout were tested. Finally, we demonstrated that gill snips were a suitable tissue for nonlethal detection of N. salmonis DNA in juvenile salmonids. Monitoring of juvenile salmonid fish in the Snake River over a 3-year period revealed low prevalence of N. salmonis in hatchery and wild Chinook salmon and wild steelhead but significantly higher prevalence in hatchery-derived steelhead. Routine monitoring of N. salmonis is not performed for all hatchery steelhead populations. At present, the possible contribution of this pathogen to delayed mortality of steelhead has not been determined.     

DIAGNOSIS OF URINARY BLADDER RUPTURE USING ULTRASOUND CONTRAST CYSTOGRAPHY: IN VITRO MODEL AND TWO CASE-HISTORY REPORTS

Because urinary bladder rupture can be life threatening, a simple, safe technique for evaluating patients is desirable. Current diagnostic protocols involve radiographic imaging, but ultrasound-based contrast techniques have not been methodically evaluated in veterinary patients with urologic trauma. Ultrasound contrast cystography (contrast cystosonography) involves infusion of microbubbled saline solution through a urinary catheter. It was performed in an in vitro model and in 2 dogs with naturally occurring urinary bladder rupture. A positive result consisted of visualizing microbubbles sonographically in fluid surrounding the bladder immediately after infusion of contrast into the urinary catheter. A positive result was obtained both in the in vitro model and in the 2 dogs, with radiographic and surgical confirmation of naturally occurring intraperitoneal urinary bladder rupture in the dogs. Based on the results of this study, ultrasound contrast cystography appears to be more sensitive than two-dimensional (2D) abdominal sonography for detecting naturally occurring urinary bladder rupture in dogs.     

Evaluation of a real-time polymerase chain reaction assay for rapid identification of methicillin-resistant Staphylococcus aureus directly from nasal swabs in horses

Screening for nasal colonization is an important aspect of many methicillin-resistant Staphylococcus aureus (MRSA) control programs. Real-time polymerase chain reaction (RT-PCR) is an attractive alternative to standard culture techniques because of the considerably shorter turnaround time. An assay has been validated for diagnostic purposes in humans, however this methodology has not been evaluated in horses. The purpose of this study was to compare an RT-PCR assay for rapid identification of MRSA directly from nasal swabs in horses to standard culture techniques. Nasal swabs collected from 293 horses were processed using a commercial RT-PCR assay (IDI-MRSA, GeneOhm Sciences, San Diego, CA) according to the manufacturer's instructions. The swabs were also cultured and MRSA was identified according to standard protocols. Initially only 176/293 samples yielded valid PCR results. Two of 176 and 167/176 samples were positive and negative, respectively, by both PCR and culture. Seven of 176 samples were positive by PCR and negative by culture, whereas 0/176 samples were negative by PCR and positive by culture. The kappa statistic was 0.35, which represented poor agreement between the tests. Of the remaining 117 samples, 105 samples were initially reported as "unresolved". Following one freeze-thaw cycle of the lysates, the recommended technique to resolve such samples, 61/110 (55%) samples remained unresolved. In this study, the IDI-MRSA assay was not a clinically practical screening test for horses harbouring nasal MRSA. Its agreement with culture was poor and the high unresolved rate (37%) also significantly decreased the clinical utility of the test.     

Acute pancreatitis following granulosa cell tumor removal in a mare

Acute pancreatitis is a rare disease in horses and is often associated with gastrointestinal disorders. Accurate diagnosis is challenging due to the presence of nonspecific clinical signs. This case represents the first documentation of acute pancreatitis in a horse following surgery of the reproductive tract.     

Carpal intra-articular blastomycosis in a Labrador retriever

A 6-month-old male castrated Labrador retriever was presented for coughing and forelimb lameness. Blastomyces dermatitidis was identified in cytology of sputum and synovial fluid. Repeat arthrocentesis 7 months later revealed resolution of septic arthritis. Fungal septic arthritis should be considered for cases of monoarthritis and may respond to oral itraconazole treatment.     

Incidence and diversity of phosphate-solubilising bacteria are linked to phosphorus status in grassland soils

The extent to which soil phosphorus (P) status affected the incidence of soil phosphate-solubilising bacteria (PSB) and their taxonomic abundance and diversity was examined at three long-term fertiliser trials (Whatawhata, Winchmore and Ballantrae) in New Zealand. Bacteria were isolated from rhizosphere (ryegrass and clover) and non-rhizosphere soils differing in P status. The P-solubilising phenotype was determined on agar supplemented with sparingly-soluble mineral phosphates (Ca₂OH(PO₄)₃ and CaHPO₄). The frequency of P-solubilisation in the bacterial population was significantly greater (P < 0.001) in soils of low-P status, demonstrating a selection pressure for this trait based on soil P availability. P-solubilising bacteria from high-P level soils and soils which had not received P fertiliser (nil-P soils) were identified based on 16S rRNA-gene sequence analysis. Across the samples, the P-solubilising community was very rich with 39 genera of PSB found, spanning 24 families and 4 phyla. At Ballantrae and Winchmore, the PSB composition differed (P < 0.05) across soil P status, which was associated with an alteration in abundance of Actinobacteria, Pseudomonadaceae and Moraxellaceae. The phylogenetic composition of PSB differed significantly (P < 0.05) between sites, however nearly half the families were common across all sites, constituting a ‘core community’ of P-solubilising bacteria for these New Zealand pasture soils. As the abundance and composition of P-solubilising bacteria are under strong selection pressure affected through farm management strategies, better understanding of their ecology provides the opportunity to increase the availability of soil P for plant-uptake.     

Virgibacillus halodenitrificans MSK-10P,a Potential Protease-producing Starter Culture for Fermented Shrimp Paste (kapi) Production

Fermented shrimp paste (Kapi) is traditionally produced using a high content of salt and mainly consumed as a food condiment by the Thai people. In order to select potential functional autochthonous starter culture, the protease-producing bacteria from Kapi were isolated, screened, and evaluated for technological and safety aspects. Among 195 isolates, five bacterial strains (MSK-3P, MSK-4P, MSK-5P, MSK-7P, and MSK-10P) exhibited the highest activity of protease using Anson method. All of them were identified as Virgibacillus halodenitrificans using 16S rRNA sequence analysis. All selected strains exhibited growth in the presence of NaCl up to 25%. Neither strain showed hemolytic activity nor biogenic amine formation. Among all selected strains, only strain MSK-10P had no ability to form a biofilm, while four other strains showed a weak ability. Strain MSK-7P exhibited multiple resistances to tetracycline and vancomycin, while strains MSK-3P, MSK-4P, and MSK-10P showed susceptibility to all antibiotics tested. According to the obtained results, this protease-producing V. halodenitrificans MSK-10P is a good candidate for further investigation for use in Kapi fermentation to assess its technological performance as a autochthonous starter culture.     

New Models of Hemostasis

Hemostasis is an essential protective mechanism that depends on a delicate balance of procoagulant and anticoagulant processes. The waterfall/cascade models of coagulation are useful for understanding several essential steps of coagulation in vitro. These have resulted in the creation of the plasma-based tests used commonly and the ability to identify deficiencies in the extrinsic, intrinsic, and common pathways of coagulation. The model was also essential in elucidating the role of several of the inhibitors of coagulation and is currently used to demonstrate coagulation as it occurs in plasma in a static environment that is devoid of endothelial interactions. The intrinsic pathway originally described by these models does not appear to be essential for in vivo hemostasis but may play a role in pathologic thrombosis. The waterfall/cascade models' lack of cellular elements sets the stage for the cell-based model of coagulation. The cell-based model of blood coagulation, which includes the varied, complicated network of factors necessary for appropriate in vivo coagulation to occur, was the next step in the evolution of our understanding of coagulation. Recently, researchers have focused on real-time, in vivo models of hemostasis and this research reveals unexpected phenomena.