Real-time RT-qPCR assay to detect sequences in the Piscine orthoreovirus-1 genome segment S1 associated with heart and skeletal muscle inflammation in Atlantic salmon

During the last decade, Piscine orthoreovirus was identified as the main causative agent of heart and skeletal muscle inflammation (HSMI) in Atlantic Salmon, Norway. A recent study showed that PRV-1 sequences from salmonid collected in North Atlantic Pacific Coast (NAPC) grouped separately from the Norwegian sequences found in Atlantic Salmon diagnosed with HSMI. Currently, the routine assay used to screen for PRV-1 in NAPC water and worldwide cannot differentiate between the two groups of PRV-1. Therefore, this study aimed at developing a real-time polymerase chain reaction (RT-qPCR) assay to target the PRV-1 genome segments specific for variants associated with HSMI. The assay was optimized and tested against 71 tissue samples collected from different regions including Norway, Chile and both coast of Canada and different hosts farmed Atlantic Salmon, wild Coho Salmon and escaped Atlantic Salmon collected in British Columbia, West Coast of Canada. This assay has the potential to be used for screening salmonids and non-salmonids that may carry PRV-1 potentially causing HSMI.     

Exchange of conduction pathways between two related K+ channels

The structure of the ion conduction pathway or pore of voltage-gated ion channels is unknown, although the linker between the membrane spanning segments S5 and S6 has been suggested to form part of the pore in potassium channels. To test whether this region controls potassium channel conduction, a 21-amino acid segment of the S5-S6 linker was transplanted from the voltage-activated potassium channel NGK2 to another potassium channel DRK1, which has very different pore properties. In the resulting chimeric channel, the single channel conductance and blockade by external and internal tetraethylammonium (TEA) ion were characteristic of the donor NGK2 channel. Thus, this 21-amino acid segment controls the essential biophysical properties of the pore and may form the conduction pathway of these potassium channels.     

Xenobiotic metabolism in suspensions and primary cultures of isolated hepatocytes prepared from the caudate process of bovine liver

Isolated hepatocytes were prepared from 100- to 125-kg Holstein male calves (n = 10) by perfusion of the caudate process of the caudate lobe of the liver. The 11th or 12th rib on the right side was resected to provide exposure of the caudate process. Complete postsurgical recovery of the donor from partial lobectomy was confirmed by growth data and serum chemical and hematologic criteria. Hepatocytes were isolated under aseptic conditions, using a 2-step collagenase vascular perfusion procedure. Hepatocyte preparations averaged 85% viability, and the yield averaged 1.2 X 10(7) viable hepatocytes/g of (wet weight) liver. Morphologic characteristics of hepatocytes examined under light and scanning electron microscopy were considered normal, except for occasional surface blebs. Freshly isolated hepatocytes in suspension rapidly decreased in viability and xenobiotic metabolizing capacity (aldrin epoxidation and ethoxycoumarin 0-deethylation and 7-hydroxycoumarin glucuronidation and sulfation), and hepatocytes surviving the initial 2 to 3 hours appeared to undergo repair. As an alternative, primary monolayer cultures on collagen-coated plates were evaluated. Hepatocytes attached to the collagen surface within 4 hours and appeared flattened by 12 hours. Although metabolic activity decreased about 30% over 8 hours in culture, the pattern of ethoxycoumarin metabolites was relatively constant. It was not determined to what extent the apparent loss of metabolic capacity was caused by hepatocyte detachment from the collagen surface. Although complicated by the requirement for asepsis, primary cultures were superior to suspensions for xenobiotic metabolism studies in cattle.     

Measuring statistical agreement between four point of care (POC) lactate meters and a laboratory blood analyzer in cats

The use of blood lactate concentrations as a prognostic indicator and therapeutic gauge in feline medicine has been hindered by the inability to obtain values in a timely manner with minimal quantities of blood. Recently, hand-held point-of-care (POC) lactate meters have become commercially available. The objective of this prospective study was to determine if lactate values produced by three commercially available and one medical grade POC meter were in agreement with a laboratory blood analyzer. Blood samples from 47 cats were collected on presentation to an emergency service and processed on four POC meters and a Stat Profile Critical Care Xpress blood analyzer. The results were analyzed using the Bland-Altman method. The blood lactate values produced by the hospital grade POC meter and one of the commercially POC meters were in good agreement with the Critical Care Xpress blood analyzer. Other commercially available POC meters produced acceptable agreement.     

First report of Seville root-knot nematode (Meloidogyne hispanica) infecting tobacco in the Brazil

Journal of Plant Diseases and Protection - Tobacco (Nicotiana tabacum L.) is one of the major nonfood crops in the Southern of Brazil. During 2019/2020 and 2020/2021 crop seasons, Root Knot...     

Nitrogen and phosphorus availability from liquid dairy manure to potatoes

The effective use of manure nutrients requires an accurate assessment of their availability to the specific crops being grown. A fertilizer equivalence approach was used to evaluate the availability of nitrogen (N) and phosphorus (P) from liquid dairy manure to potatoes in field experiments conducted in northeast Wisconsin in 2000, 2001, and 2002. Crop responses to moderate and high rates of liquid dairy manure (93,500 and 187,000 L ha^?1) were compared to results obtained from N or P fertilizer applied at five rates (0 to 269 kg ha^?1). Availability estimates were made using the fertilizer equivalence method based on tuber yield, harvested tuber N or P concentration and uptake, petiole nitrate or total P concentration, and soil nitrate or extractable P phosphate levels. Results showed an apparent availability of manurial N from 10% to 40%, with an overall average across all 3 years and all parameters of 28.6% for the lower rate and 24.6% for the high rate. These values are slightly less than average availability measured where corn has been used as the test crop. Although all parameters did not show responses to P fertilizer additions in these trials, where estimates were possible, P availability ranged from 20% to 90% with an overall average across all 3 years of 55.4% for the low and 50.5% for the high manure rates. Despite potato being more shallow-rooted and somewhat less efficient in N and P use compared to corn, it appears that only minor adjustments to manurial nutrient availability estimates are warranted when potatoes are grown.     

Validation of Diagnostic Methods for European Foulbrood on Commercial Honey Bee Colonies in the United States

One of the most serious bacterial pathogens of Western honey bees (Apis mellifera Linnaeus [Hymenoptera: Apidae]) is Melissococcus plutonius, the cause of the disease European foulbrood. Because European foulbrood is highly variable, with diverse outcomes at both the individual and colony levels, it is difficult to diagnose through visual inspection alone. Common lab diagnostic techniques include microscopic examination and molecular detection through PCR. In 2009, a lateral flow device was developed and validated for field diagnosis of European foulbrood. At the time, M. plutonius was thought to be genetically homogenous, but we have subsequently learned that this bacterium exists as multiple strains, including some strains that are classified as ‘atypical’ for which the lateral flow device is potentially less effective. These devices are increasingly used in the United States, though they have never been validated using strains from North America. It is essential to validate this device in multiple locations as different strains of M. plutonius circulate in different geographical regions. In this study, we validate the field use of the lateral flow device compared to microscopic examination and qPCR on larval samples from 78 commercial honey bee colonies in the United States with visual signs of infection. In this study, microscopic diagnosis was more sensitive than the lateral flow device (sensitivity = 97.40% and 89.47%, respectively), and we found no false positive results with the lateral flow device. We find high concurrence between the three diagnostic techniques, and all three methods are highly sensitive for diagnosing European foulbrood.     

Plasticity of bacterial genomes: pathogenicity islands and the locus of enterocyte effacement (LEE)

Many bacterial virulence attributes, like toxins, adhesins, invasins, iron uptake systems, are encoded within specific regions of the bacterial genome. These in size varying regions are termed pathogenicity islands (PAIs) since they confer pathogenic properties to the respective micro-organism. Per definition PAIs are exclusively found in pathogenic strains and are often inserted near transfer-RNA genes. Nevertheless, non-pathogenic bacteria also possess foreign DNA elements that confer advantageous features, leading to improved fitness. These additional DNA elements as well as PAIs are termed genomic islands and were acquired during bacterial evolution. Significant G+C content deviation in pathogenicity islands with respect to the rest of the genome, the presence of direct repeat sequences at the flanking regions, the presence of integrase gene determinants as other mobility features,the particular insertion site (tRNA gene) as well as the observed genetic instability suggests that pathogenicity islands were acquired by horizontal gene transfer. PAIs are the fascinating proof of the plasticity of bacterial genomes. PAIs were originally described in human pathogenic Escherichia (E.) coli strains. In the meantime PAIs have been found in various pathogenic bacteria of humans, animals and even plants. The Locus of Enterocyte Effacement (LEE) is one particular widely distributed PAI of E coli. In addition, it also confers pathogenicity to the related species Citrobacter (C.) rodentium and Escherichia (E.) alvei. The LEE is an important virulence feature of several animal pathogens. It is an obligate PAI of all animal and human enteropathogenic E. coli (EPEC), and most enterohaemorrhegic E. coli (EHEC) also harbor the LEE. The LEE encodes a type III secretion system, an adhesion (intimin) that mediates the intimate contact between the bacterium and the epithelial cell, as well as various proteins which are secreted via the type III secretion system. The LEE encoded virulence features are responsible for the formation of so called attaching and effacing (AE) lesions in the intestinal epithelium. Due to its wide distribution in animal pathogens, LEE encoded antigens are suitable vaccine antigens. Acquisition and structure of the LEE pathogenicity island is the crucial point of numerous investigations. However, the evolution of the LEE, its origin and further spread in E. coli, are far from being resolved.