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.     

A rapid method for the determination of liquid fumigant residues in food commodities using automated headspace analysis

An automated gas chromatographic headspace method capable of detecting halogenated compounds in cereals, nuts and seeds at levels down to μg kg^?1 or less has been developed and evaluated. Commodities were analysed directly without extraction or clean-up and a simple calibration technique offers a rapid screening quantitative analytical method. Comparison of results obtained using an established solvent extraction method with those obtained using the method described showed good quantitative agreement. Minimum detectable quantities were lower using the headspace method for every fumigant residue/commodity studied. The recommended analytical conditions are suitable for examination of further commodities for the fumigant residues examined and for other volatile halocarbons.     

Photodynamic therapy for the treatment of intranasal tumors in 3 dogs and 1 cat

Three dogs and 1 cat with intranasal tumors were treated with pyropheophorbide-a-hexyl ether-based photodynamic therapy (PDT). PDT was well tolerated by all the animals, and no adverse effects from photosensitizer injection, such as cutaneous photosensitization, were observed. Facial swelling was observed in all animals after each PDT treatment but resolved spontaneously within 72 hours after treatment. All animals had a decrease in severity of epistaxis, frequency of sneezing, and amount of nasal discharge after PDT. Clinical signs were controlled for variable time, although long-term responses were comparable with radiation therapy in 2 animals. This small case series demonstrates another application for PDT in veterinary medicine. On the basis of these findings. further studies are warranted to define the role of PDT in the management of intranasal tumors in dogs and cats.     

Treatment of dogs with oral melanoma by hypofractionated radiation therapy and platinum-based chemotherapy (1987-1997)

This retrospective study in 39 dogs with incompletely resected oral melanoma examined the efficacy of hypofractionated radiation therapy and platinum-containing chemotherapy. All dogs were completely staged, with the majority of dogs classified as stage 1. Dogs received 6 weekly fractions of 6-gray (Gy) megavoltage irradiation with a cobalt-60 unit or a 4-MeV (megaelectron volts) linear accelerator. Dogs received cisplatin (10-30 mg/m2 IV) or carboplatin (90 mg/m2 IV) chemotherapy 60 minutes before radiation delivery. Durations of local control, metastasis-free survival time, and overall survival time were recorded. By the Kaplan-Meier method, 15% of the dogs had local recurrence within a median time of 139 days. Fifty-one percent of the dogs developed metastatic disease within a median time of 311 days (range, 24-2, 163 days). Median survival time for all 39 dogs was 363 days. The combined use of chemotherapy and radiation therapy in this protocol provided local control consistent with previous studies. Low-dose chemotherapy was used with the intent of enhancing radiation therapy for the local control of an incompletely excised tumor. Survival times were longer than previously reported for dogs with oral malignant melanoma. Additional studies are required to determine whether these results were due to the effects of chemotherapy on microscopic disease or the enhanced local control provided by chemoradiation therapy.     

Risk factors and prevalence of injuries in horses during various types of steeplechase races

OBJECTIVE: To identify the types of injuries sustained by horses that competed in steeplechase races and determine the prevalence of and risk factors for those injuries. DESIGN: Prospective study. ANIMALS: 2,680 horses that competed in various types of steeplechase races from 1996 through 2000. PROCEDURE: Data regarding races; environment; equipment problems; the number of horses that entered, started, and finished races; the number of riders that fell; and the number of horses that were slowed or stopped by the rider, ran off the course, fell, and sustained injuries or physical abnormalities during races were collected on a standard form by the official veterinarian who attended each meet. Data from all meets were not recorded; however, in recorded meets, data from every race were reported. RESULTS: Data for 197 hurdle, 65 timber, 76 flat, and 8 mixed races were recorded. Nine (3.4/1,000 horses that started in races) horses died or were euthanatized, and 7 of those were associated with catastrophic musculoskeletal injury. Seven fractures were recorded. Four fractures involved forelimbs, 1 involved a hind limb, and 2 involved the cervical portion of the vertebral column. All horses with fractures were euthanatized. Deep or hard course conditions were associated with an increased risk of breakdown injuries. CONCLUSIONS AND CLINICAL RELEVANCE: Successful development and implementation of strategies to prevent injuries and death in horses in steeplechase races depend on a clear understanding of the types and prevalence of injuries involved and risk factors associated with those injuries.     

Liquid chromatography-mass spectrometry (LC-MS) investigation of the thiobarbituric acid reactive substances (TBARS) reaction

The thiobarbituric acid reactive substances (TBARS) assay is a commonly used method for the detection of lipid peroxidation. Malondialdehyde is formed as a result of lipid peroxidation and reacts with thiobarbituric acid to form a pink pigment that has an absorption maximum at 532 nm. Other compounds also react with thiobarbituric acid to form colored species that can interfere with this assay, but little is known about these interfering species. This is the first investigation using LC-MS and MS-MS to study the structures of the pink adduct as well as a common unstable yellow interference compound, which absorbs at 455 nm. Also, the presence of barbituric acid impurities in the thiobarbituric acid reagent was found to produce 1:1:1 thiobarbituric acid/malondialdehyde/barbituric acid and 2:1 barbituric acid/malondialdehyde adducts that absorbed at 513 and 490 nm, respectively, indicating that thiobarbituric acid should be purified before use.