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.     

Toxoplasmosis in Pallas' cats (Otocolobus felis manul) at the Denver Zoological Gardens.

In May 1996 the Denver Zoological Gardens obtained two male and two female Pallas' cats (Otocolobus felis manul) that were wild-caught in the Ukraine. These animals were part of a group of 16 wild-caught adults (eight male and eight female) imported to the United States and Canada between 1995 and 1996. The Denver Zoological Gardens cats were quarantined at the zoo hospital for approximately I mo. During the quarantine period they were immobilized for physical examination, and sera were obtained from them to evaluate for exposure to Toxoplasma gondii. All cats were positive for T. gondii antibodies by latex agglutination (titers from 1:512 to 1:1,024). After being paired for breeding, one pair produced two litters, and another pair produced four litters, a total of 17 kittens between 1997 and 2001. Four kittens and two young adults died from a disseminated granulomatous and necrotizing inflammation consistent with toxoplasmosis. Toxoplasma gondii infection was confirmed in all six deceased cats by polymerase chain reaction performed on formalin-fixed tissues. An additional five kittens disappeared and were not available for necropsy. The fatality rate from toxoplasmosis was 35.3% (6/17) for cats that were available for necropsy and could have been as high as 64.7% (11/17) if it were assumed that the disappeared kittens were also affected. The Pallas' kitten survival rate at the Denver Zoological Gardens was 35.3%. This article describes the clinical and pathologic features of toxoplasmosis in a group of Pallas' cats at the Denver Zoological Gardens.     

Low concentrations of zearalenone in diets of boars for a prolonged period of time

Thirty-two boars were fed diets containing 0, 3, 6 or 9 ppm zearalenone from 32 d of age up to 145 or 312 d of age. Rate of gain, feed intake and gain:feed were not significantly different among treatments. Feeding of diets containing 9 ppm zearalenone did not affect the libido in seven boars. There was a tendency for boars fed 9 ppm dietary zearalenone to produce lower total and gel free volumes of semen, with lower total motile sperm; however, only the percent motility of the sperm was reduced (P less than .05).     

Relationship between level of forage intake,blood flow and oxygen consumption by splanchnic tissues of sheep fed a tropical grass forage

Four Polwarth castrated male sheep (42 ± 4.4 kg live weight (LW) surgically implanted with chronic indwelling catheters into the mesenteric, portal and hepatic veins, housed in metabolism cages and offered Cynodon sp. hay at rates (g of dry matter (DM)/kg LW) of 7, 14, 21 or ad libitum, were used in a 4 × 4 Latin square experiment to evaluate the effect of the level of forage intake on blood flow and oxygen consumption by the portal‐drained viscera (PDV), liver and total splanchnic tissues (ST). The portal blood flow and the oxygen consumption by PDV linearly increased at increased organic matter (OM) intake. No effect of level of OM intake was obtained for the hepatic artery blood flow and oxygen consumption by liver. As a consequence, the level of OM intake only tended to directly affect hepatic blood flow and oxygen consumption by total ST. Oxygen consumption was linearly and positively related to blood flow across PDV, liver and total ST. The heat production by PDV and total ST, as proportion of metabolizable energy (ME) intake, decreased curvilinearly at increased ME intake. In conclusion, the oxygen consumption by PDV, but not by liver, was directly related to the level of forage intake by sheep. Moreover, when ingested at levels below maintenance, most of ME was spent as heat produced by ST.     

Disposition of the anti‐ulcer medications ranitidine,cimetidine, and omeprazole following administration of multiple doses to exercised Thoroughbred horses

The use of anti‐ulcer medications, such as cimetidine, ranitidine, and omeprazole, is common in performance horses. The use of these drugs is regulated in performance horses, and as such a withdrawal time is necessary prior to competition to avoid a medication violation. To the authors' knowledge, there are no reports in the literature describing repeated oral administrations of these drugs in the horse to determine a regulatory threshold and related withdrawal time recommendations. Therefore, the objective of the current study was to describe the disposition and elimination pharmacokinetics of these anti‐ulcer medications following oral administration to provide data upon which appropriate regulatory recommendations can be established. Nine exercised Thoroughbred horses were administered 20 mg/kg BID of cimetidine or 8 mg/kg BID of ranitidine, both for seven doses or 2.28 g of omeprazole SID for four doses. Blood samples were collected, serum drug concentrations were determined, and elimination pharmacokinetic parameters were calculated. The serum elimination half‐life was 7.05 ± 1.02, 7.43 ± 0.851 and 3.94 ± 1.04 h for cimetidine, ranitidine, and omeprazole, respectively. Serum cimetidine and ranitidine concentrations were above the LOQ and omeprazole and omeprazole sulfide below the LOQ in all horses studied upon termination of sample collection.     

Population genetic structure and landscape connectivity of the Eastern Yellowbelly Racer (<Emphasis Type="Italic">Coluber constrictor flaviventris</Emphasis>) in the contiguous tallgrass prairie of northeastern Kansas,USA

The tallgrass prairie of North America has undergone widespread habitat loss and fragmentation (<4% remains). The Flint Hills region of Kansas and Oklahoma is the largest tallgrass prairie remaining and therefore provides an opportunity to study the population genetic structure of grassland species in a relatively contiguous landscape and set a baseline for evaluating changes when the habitat is fragmented. We adopted a landscape genetics approach to identify how landscape structure affected dispersal, population genetic structure, and landscape connectivity of the Eastern Yellowbelly Racer (Coluber constrictor flaviventris) across a 13,500-km² landscape in northeastern Kansas, USA. The racer population had high allelic diversity, high heterozygosity, and was maintaining migration-drift equilibrium. Autocorrelation between genetic and geographic distance revealed that racers exhibited restricted dispersal within 3 km, and isolation-by-distance. Significant isolation-by-distance occurred at broad regional scales (>100 km), but because of sufficient gene flow between locations, we were unable to define discrete subpopulations using Bayesian clustering analyses. Resistance distance, which considers the permeability of habitats, did not explain significant variation in genetic distance beyond Euclidean distance alone, suggesting that racers are not currently influenced by landscape composition. In northeastern Kansas, racers appear to be an abundant and continuously distributed snake that perceives the landscape as well connected with no cover type currently impeding snake dispersal or gene flow.     

Acute and chronic hormone and metabolite changes in lambs fed the beta-agonist, cimaterol

The objective of this study was to determine if acute and chronic changes in circulating metabolic hormone and metabolite concentrations are associated with β-agonist-induced nutrient repartitioning in young growing lambs. Two groups of 12 Dorset and Dorset-Finn cross ram lambs weighing 36 or 33 kg live weight were assigned to 3- or 6-week treatment intervals, respectively, to achieve similar slaughter weights. Six lambs within each treatment interval were fed ad libitum a complete mixed high-concentrate diet containing either 0 or 10 ppm cimaterol. During the first 12 hr of cimaterol administration plasma somatotropin (ST), thyroxine (T₄), and triiodothyronine (T₃) concentrations were not altered by treatment, but plasma insulin, glucose, non-esterified fatty acids (NEFA) and glycerol concentrations were elevated 2 hr after ingestion. These acute responses suggest direct stimulation of glycogenolysis and lipolysis by cimaterol, which is characteristic of β-adrenergic alteration of carbohydrate and lipid metabolism. Chronic administration of cimaterol significantly decreased insulin concentrations by 36% and 52% at 3 and 6 weeks, respectively, while glucose concentrations remained unchanged. Serum IGF-I concentrations were not significantly altered by cimaterol. T₄ levels were reduced 22.1% after 3 weeks of cimaterol treatment. Although plasma NEFA concentrations were chronically elevated 56% to 65% in lambs fed cimaterol, plasma glycerol concentrations remained at baseline levels. The relative changes in plasma NEFA and glycerol concentrations are consistent with a decreased rate of lipogenesis, rather than an increase in lipolysis.     

The vitamin A requirement and the vitamin A status of growing cattle. 1. Studies of calves

Five experiments with 18 to 36 male calves each of the black and white dairy cattle breed (age: 14-21 days, initial live weight: approximately 45 kg per animal) were carried out in order to investigate the influence of various vitamin A supply (0-80,000 IU per 100 kg LW and day) on dry matter intake and weight gain as well as the vitamin A status of liver and blood plasma over 84 days. The calves consumed a diet free of carotene and vitamin A consisting of milk replacer, concentrate and chopped wheat straw. The calves were fed in three experiments for a longer time in order to observe the further vitamin A depletion. Nine animals consumed an unsupplemented ration, nine other one got 10,000 IU vitamin A per 100 kg LW and day. Biopsies of liver and plasma samples were taken from 4 animals per group every four weeks. The various vitamin A supplementation did not significantly influence the dry matter intake (Mean: 1.67; 1.48 to 1.80 kg DM per animal and day) and the weight gain of calves (Mean: 702, 599 to 770 g per animal and day). First vitamin A deficiency symptoms (reduced feed intake, decreased weight gain, diarrhoea etc.) were observed in animals of unsupplemented group after 100 days of experiments. After 84 days the vitamin A concentration of liver of animals of unsupplemented groups decreased to 1.3-32.2% compared with the begin of experiments (60.6-155.7 mumol/kg fresh matter). Up to 51% of initial concentration were found when 10,000 IU vitamin A per 100 kg LW and day were fed. About 25,000 IU vitamin A per 100 kg LW and day were required in order to keep the initial level of vitamin A concentration of liver. The plasma vitamin A concentration is unsuitable for estimation of vitamin A status of calves. The concentration of vitamin A of liver and plasma amounted to 114 mumol per kg and 0.25 mumol per litre at the begin of experiments. The vitamin A concentration of liver of unsupplemented group decreased to 20 mumol per kg, that of plasma increased to 0.28 mumol per 1 at the end. A strong vitamin A deficiency (liver concentration: less than 10 mumol/kg) may cause a decrease of vitamin A concentration of blood.     

Simultaneous intramammary and intranasal inoculation of lactating cows with bovine herpesvirus 4 induce subclinical mastitis

In this study, we examined whether an experimental bovine herpesvirus 4 (BHV4) infection can induce bovine mastitis, or can enhance bovine mastitis induced by Streptococcus uberis (S. uberis). Four lactating cows were inoculated intramammarily and intranasally with BHV4, and four lactating control cows were mock-inoculated. After 14 days, two of four cows from each group were inoculated intramammarily with S. uberis. No clinical signs were recorded in cows inoculated only with BHV4, and their milk samples showed no abnormal morphology, despite the fact that BHV4 replicated in inoculated quarters. Somatic cell count increased significantly in milk from three of six BHV4-inoculated quarters, compared to the non-inoculated quarters of the same cows (within-cow) and the quarters of mock-inoculated cows (control group) on days 8, 9 and 11 post-inoculation (pi). BHV4 was isolated from nasal swabs between days 2 and 9 pi. Clinical mastitis was observed in all four cows intramammarily inoculated with S. uberis. A preceding BHV4 infection did not exacerbate the clinical mastitis induced by S. uberis. S. uberis infections appeared to trigger BHV4 replication. From one quarter of each of two cows inoculated with BHV4 and S. uberis, BHV4 was isolated, and not from quarters inoculated with BHV4 only. In conclusion, BHV4 did not induce bovine clinical mastitis after simultaneous intranasal and intramammary inoculation. However, the BHV4 infection did induce subclinical mastitis in 50% of the cows and the quarters.