Randomly primed,strand-switching,MinION-based sequencing for the detection and characterization of cultured RNA viruses

RNA viruses rapidly mutate, which can result in increased virulence, increased escape from vaccine protection, and false-negative detection results. Targeted detection methods have a limited ability to detect unknown viruses and often provide insufficient data to detect coinfections or identify antigenic variants. Random, deep sequencing is a method that can more fully detect and characterize RNA viruses and is often coupled with molecular techniques or culture methods for viral enrichment. We tested viral culture coupled with third-generation sequencing for the ability to detect and characterize RNA viruses. Cultures of bovine viral diarrhea virus, canine distemper virus (CDV), epizootic hemorrhagic disease virus, infectious bronchitis virus, 2 influenza A viruses, and porcine respiratory and reproductive syndrome virus were sequenced on the MinION platform using a random, reverse primer in a strand-switching reaction, coupled with PCR-based barcoding. Reads were taxonomically classified and used for reference-based sequence building using a stock personal computer. This method accurately detected and identified complete coding sequence genomes with a minimum of 20× coverage depth for all 7 viruses, including a sample containing 2 viruses. Each lineage-typing region had at least 26× coverage depth for all viruses. Furthermore, analyzing the CDV sample through a pipeline devoid of CDV reference sequences modeled the ability of this protocol to detect unknown viruses. Our results show the ability of this technique to detect and characterize dsRNA, negative- and positive-sense ssRNA, and nonsegmented and segmented RNA viruses.     

Insecticidal potency of novel compounds on multiple insect species of medical and veterinary importance

BACKGROUND: Vector‐borne diseases continue to present significant threats to human, animal and plant health. Mosquitoes, houseflies, sand flies and stable flies are well‐known vectors of several human and animal pathogens. The toxicity of selected semiochemicals with molecular structures indicative of insecticidal activity was determined against these insect species with the aim of developing novel insecticides toxic to multiple insect species. RESULTS: Three semiochemicals, namely beta‐damascone, cyclemone A and melafleur, showed remarkable toxicity to three mosquitoes, Aedes aegypti L., Ae. albopictus (Skuse) and Anopheles quadrimaculatus Say, the housefly, Musca domestica L., the stable fly, Stomoxys calcitrans L., and the sand fly, Lutzomyia shannoni (Dyar). The chemicals were equally toxic to several field‐collected permethrin‐tolerant housefly strains. When formulated as 500 mL L^?1 emulsifiable concentrates, the chemicals demonstrated stability and toxicity on filter paper and camouflage military fabrics, with persistence up to 8 days under laboratory conditions. The chemicals were equally effective under field conditions when evaluated on unpainted plywood panels, although a higher dosage was required under field conditions to achieve similar efficacy. CONCLUSION: Laboratory quantification of LC₅₀ values and field efficacy of three semiochemicals as formulated compounds on mosquitoes, houseflies, stable flies and sand flies showed that these semiochemicals could serve as potent insecticides for multiple insect species. Copyright © 2010 Society of Chemical Industry     

Lipoprotein electrophoresis differentiation of chylous and nonchylous pleural effusions in dogs and cats and its correlation with pleural effusion triglyceride concentration

Pleural effusions from 23 dogs and 25 cats hospitalized between November 1986 and April 1987 at the Veterinary Hospital of the University of Pennsylvania (VHUP) were classified as chylous (chylomicrons present) or nonchylous (chylomicrons absent) by the presence or absence, respectively, of a chylomicron band on lipoprotein electrophoresis gels of the effusions. Triglyceride concentrations, cholesterol concentrations, and cholesterol/triglyceride (C/T) ratios were compared between the chylous and nonchylous groups in each species. Cholesterol concentrations were not significantly different between chylous and nonchylous effusions in both dogs and cats. Cholesterol/triglyceride ratios of less than 1 were present in all dogs and cats with chylous effusions; however, 12% of dogs with nonchylous effusions and 50% of cats with nonchylous effusions also had a C/T ratio less than 1. Triglyceride concentrations accurately classified all effusions as chylous or nonchylous in both dogs and cats. Pleural effusions with triglyceride concentrations greater than l00 mg/dl were chylous in all cases, whereas, effusions with triglyceride concentrations less than l00 mg/dl were nonchylous in all cases.     

Endotoxn-induced nonthyroidal illness in dogs

OBJECTIVE: To determine the effects of endotoxin administration on thyroid function test results and serum tumor necrosis factor-alpha (TNF-alpha) activity in healthy dogs. ANIMALS: 6 healthy adult male dogs. PROCEDURES: Serum concentrations of thyroxine (T4), 3,5,3'-triiodothyronine (T3), 3,3'5'-triiodothyronine (rT3), free T4 (fT4), and endogenous canine thyroid stimulating hormone (TSH), and TNF-alpha activity were measured before (day-1; baseline), during (days 0 to 3), and after (days 4 to 24) IV administration of endotoxin every 12 hours for 84 hours. RESULTS: Compared with baseline values, serum T3 concentration decreased significantly, whereas rT3 concentration increased significantly 8 hours after initial endotoxin administration. Serum T4 concentration decreased significantly at 8 and 12 hours after initiating endotoxin administration. Serum T4 concentration returned to reference range limits, then decreased significantly on days 6 to 12 and 16 to 20. Serum fT4 concentration increased significantly at 12, 24, and 48 hours after cessation of endotoxin treatment, compared with baseline values. Serum rT3 concentration returned to reference range, then decreased significantly days 5 and 7 after stopping endotoxin treatment. Serum TNF-alpha activity was significantly increased only 4 hours after initial endotoxin treatment, compared with baseline activity. CONCLUSIONS AND CLINICAL RELEVANCE: Endotoxin administration modeled alterations in thyroid function test results found in dogs with spontaneous nonthyroidal illness syndrome. A decrease in serum T4 andT3 concentrations and increase in serum rT3 concentration indicate impaired secretion and metabolism of thyroid hormones. The persistent decrease in serum T4 concentration indicates that caution should be used in interpreting serum T4 concentrations after resolution of an illness in dogs.     

Use of a polymerase chain reaction assay to detect bovine herpesvirus type 2 DNA in skin lesions from cattle suspected to have pseudo-lumpy skin disease

Beef cattle from a herd in north Alabama were examined because of an outbreak of nonfatal skin disease characterized by discrete circumscribed areas of inflammation that developed on the skin from the neck to the hips. Areas of inflammation, which tended to be superficial, underwent necrosis and scabbed over. The scabs eventually dropped off leaving discrete, round, whitish, hairless lesions that were 1.2 to 2.5 cm diameter. Because clinical signs were consistent with those expected with pseudo-lumpy skin disease (PLSD) caused by bovine herpesvirus type 2 (BHV-2), samples from 16 representative animals were submitted for BHV-2 testing. All 16 animals were seropositive for BHV-2, but the virus could not be isolated from skin biopsy specimens or buffy coat samples. Results of a polymerase chain reaction assay incorporating primers designed to amplify 2 DNA sequences from BHV-2 were positive for 3 of the 10 cattle, suggesting that skin lesions in these cattle were a result of PLSD. Our findings suggest that PLSD may be more common and widespread in the United States than suggested by the frequency with which BHV-2 has been isolated from cattle with PLSD-like skin lesions.     

Ameloblastic odontoma in the mandible of a llama

A 4-year-old llama had an aggressive, multiloculated, expansile bone lesion involving the rostral mandible. The mandibular lesion was imaged using radiography and computed tomography. Antemortem diagnosis of an ossifying fibroma was made histologically. Postmortem findings showed the lesion to be limited to the mandible. Final diagnosis was ameloblastic odontoma.     

Using Unmanned Helicopters to Assess Vegetation Cover in Sagebrush Steppe Ecosystems

Evaluating vegetation cover is an important factor in understanding the sustainability of many ecosystems. Remote sensing methods with sufficient accuracy could dramatically alter how biotic resources are monitored on both public and private lands. Idaho National Laboratory (INL), in conjunction with the University of Idaho, evaluated whether unmanned aerial vehicles (UAVs) are sufficiently accurate and more efficient than the point-frame field method for monitoring vegetative cover and bare ground in sagebrush steppe ecosystems. These values are of interest to land managers because typically there are limited natural resource scientists and funding for comprehensive ground evaluations. In this project, unmanned helicopters were used to collect still-frame imagery to determine vegetation cover during June and July 2005. The images were used to estimate percent cover for six vegetative cover classes (shrub, dead shrub, grass, forbs, litter, and bare ground). Field plots used to collect imagery and on-the-ground measurements were located on the INL site west of Idaho Falls, Idaho. Ocular assessments of digital imagery were performed using SamplePoint, and the results were compared with field measurements collected using a point-frame method. The helicopter imagery evaluation showed a high degree of agreement with field cover class values for grass, litter, and bare ground and reasonable agreement for dead shrubs. Shrub cover was often overestimated, and forbs were generally underestimated. The helicopter method took 45% less time than the field method. This study demonstrates that UAV technology provides a viable method for monitoring selective types of cover on rangelands and could save time and resources.     

Investigation of the distance to slaughterhouses and weather parameters in the occurrence of porcine reproductive and respiratory syndrome outbreaks in U.S. swine breeding herds

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important diseases affecting the swine industry. The main objective of this study was to assess whether sow farm distance to slaughterhouses and meteorological variables were associated with PRRS outbreaks. This case-control study paired 104 sow farms with or without a reported PRRS outbreak (N = 208) during the same period. Data on the distance to the closest slaughterhouse, swine density, presence of an air filtration system, or a neighboring farm, and weather conditions were collected, and a multivariable conditional logistic regression model was created to investigate the association between variables of interest and the occurrence of a PRRS outbreak.Swine density, presence of an air filtration system, presence of a neighboring farm, and PRRS herd-level status before the outbreak were associated with the occurrence of PRRS outbreaks. Farms in areas with higher swine density and nearby swine farms had increased odds of reporting an outbreak compared to farms in low swine density areas and farms having no neighbors. Under the conditions of this study, none of the meteorological variables or the distance to the closest slaughterhouse were associated with occurrence of PRRS outbreaks.