Dynamics of African swine fever virus shedding and excretion in domestic pigs infected by intramuscular inoculation and contact transmission

African swine fever virus (ASFV) is a highly virulent swine pathogen that has spread across Eastern Europe since 2007 and for which there is no effective vaccine or treatment available. The dynamics of shedding and excretion is not well known for this currently circulating ASFV strain. Therefore, susceptible pigs were exposed to pigs intramuscularly infected with the Georgia 2007/1 ASFV strain to measure those dynamics through within- and between-pen transmission scenarios. Blood, oral, nasal and rectal fluid samples were tested for the presence of ASFV by virus titration (VT) and quantitative real-time polymerase chain reaction (qPCR). Serum was tested for the presence of ASFV-specific antibodies. Both intramuscular inoculation and contact transmission resulted in development of acute disease in all pigs although the experiments indicated that the pathogenesis of the disease might be different, depending on the route of infection. Infectious ASFV was first isolated in blood among the inoculated pigs by day 3, and then chronologically among the direct and indirect contact pigs, by day 10 and 13, respectively. Close to the onset of clinical signs, higher ASFV titres were found in blood compared with nasal and rectal fluid samples among all pigs. No infectious ASFV was isolated in oral fluid samples although ASFV genome copies were detected. Only one animal developed antibodies starting after 12 days post-inoculation. The results provide quantitative data on shedding and excretion of the Georgia 2007/1 ASFV strain among domestic pigs and suggest a limited potential of this isolate to cause persistent infection.     

ASVCP guidelines: quality assurance for point‐of‐care testing in veterinary medicine

Point‐of‐care testing (POCT) refers to any laboratory testing performed outside the conventional reference laboratory and implies close proximity to patients. Instrumental POCT systems consist of small, handheld or benchtop analyzers. These have potential utility in many veterinary settings, including private clinics, academic veterinary medical centers, the community (eg, remote area veterinary medical teams), and for research applications in academia, government, and industry. Concern about the quality of veterinary in‐clinic testing has been expressed in published veterinary literature; however, little guidance focusing on POCT is available. Recognizing this void, the ASVCP formed a subcommittee in 2009 charged with developing quality assurance (QA) guidelines for veterinary POCT. Guidelines were developed through literature review and a consensus process. Major recommendations include (1) taking a formalized approach to POCT within the facility, (2) use of written policies, standard operating procedures, forms, and logs, (3) operator training, including periodic assessment of skills, (4) assessment of instrument analytical performance and use of both statistical quality control and external quality assessment programs, (5) use of properly established or validated reference intervals, (6) and ensuring accurate patient results reporting. Where possible, given instrument analytical performance, use of a validated 1_3s control rule for interpretation of control data is recommended. These guidelines are aimed at veterinarians and veterinary technicians seeking to improve management of POCT in their clinical or research setting, and address QA of small chemistry and hematology instruments. These guidelines are not intended to be all‐inclusive; rather, they provide a minimum standard for maintenance of POCT instruments in the veterinary setting.     

Discovery and directed evolution of a glyphosate tolerance gene

The herbicide glyphosate is effectively detoxified by N-acetylation. We screened a collection of microbial isolates and discovered enzymes exhibiting glyphosate N-acetyltransferase (GAT) activity. Kinetic properties of the discovered enzymes were insufficient to confer glyphosate tolerance to transgenic organisms. Eleven iterations of DNA shuffling improved enzyme efficiency by nearly four orders of magnitude from 0.87 mM-1 min-1 to 8320 mM-1 min-1. From the fifth iteration and beyond, GAT enzymes conferred increasing glyphosate tolerance to Escherichia coli, Arabidopsis, tobacco, and maize. Glyphosate acetylation provides an alternative strategy for supporting glyphosate use on crops.     

Molecular markers for improving control of soil-borne pathogen <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> in sugar beet

Fusarium spp. cause severe damage in many agricultural crops, including sugar beet, with Fusarium oxysporum historically being considered as the most damaging of all species. Sugar beet needs to be protected from this class of soil-borne pathogens in order to ensure an optimal sugar yield in the field. Genetic control of the disease is crucial in managing these pathogens. Identification of single nucleotide polymorphism (SNP) markers linked to resistance can be a powerful tool for the introgression of valuable genes needed to develop Fusarium-resistant varieties. A candidate gene approach was carried out to identify SNP markers linked to putative Fusarium resistance sources in sugar beet. Five resistant analogue genes (RGAs) were screened by means of high resolution melting (HRM) analysis in a set of sugar beet lines, considered as resistant and susceptible to Fusarium oxysporum. HRM polymorphisms were observed in 80% of amplicons. Two HRM polymorphisms were significantly associated with Fusarium resistance (P < 0.05). The amplicons that showed association were sequenced and two SNPs were identified. The association was further validated on 96 susceptible and 96 resistant plants using competitive allele-specific PCR (KASPar) technology. The selected SNPs could be used for marker-assisted breeding of Fusarium resistance in sugar beet.     

Effect of Germination Initiation on Competitive Capacity of Trichoderma atroviride P1 Conidia

ABSTRACT Trichoderma biocontrol isolates are most effective as highly concentrated inocula. Their antagonism to other fungi may be a result of pregermination respiration. In a nutrient-rich medium, almost all Trichoderma atroviride P1 (P1) conidia initiated germination processes and increased respiration, even in dense suspensions. When 1 x 10(7) P1 conidia/ml were coinoculated with 1 x 10(5) Botrytis cinerea conidia/ml, dissolved oxygen fell to <1% within 2 h and the pathogen failed to germinate. More dilute P1 suspensions consumed oxygen slowly enough to allow coinoculated B. cinerea to germinate. On nutrient-poor media, fewer P1 conidia initiated germination. Oxygen consumption by the inoculum and inhibition of B. cinerea were enhanced when P1 conidia were nutrient activated before inoculation. Pregermination respiration also affected competitive capacity of the antagonist on solid substrates, where respiratory CO(2) stimulated germination rate and initial colony growth. These parameters were directly correlated with inoculum concentration (R(2) >/= 0.97, P < 0.01). After initiating germination, Trichoderma conidia became more sensitive to desiccation and were killed by drying after only 2 h of incubation on a nutrient-rich substrate at 23 degrees C. These results indicate that nutrient-induced changes preceding germination in Trichoderma conidia can either enhance or decrease their biological control potential, depending on environmental conditions in the microhabitat.     

Clinical characteristics and mode of inheritance of familial focal seizures in Standard Poodles

OBJECTIVE: To determine clinical characteristics and mode of inheritance of seizures in a family of Standard Poodles. DESIGN: Case series. ANIMALS: 90 Standard Poodles descended from the same maternal bloodline (30 with probable idiopathic epilepsy [PIE] and 60 without any history of seizures). PROCEDURES: Researchers contacted owners to determine whether dogs had ever had any seizures and, if so, the nature of any such seizures and any potential underlying causes. Dogs were considered to have PIE if they were between 6 months and 7.5 years old at the time of seizure onset and had no evidence of any underlying cause. To determine the mode of inheritance, segregation analyses were designed to allow the family to be analyzed as a whole, as opposed to as nuclear families. Competing models of inheritance were compared statistically for their ability to explain the data. RESULTS: Of the dogs with PIE, 28 (93%) had focal onset seizures with or without secondary generalization. Median age of onset was 3.7 years; 6 dogs were > 5 years old at the onset of seizures. Segregation analyses strongly suggested that PIE was inherited as a simple recessive autosomal trait with complete or almost complete penetrance. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that in this family of Standard Poodles, PIE was inherited as a simple recessive autosomal trait with complete or almost complete penetrance. Seizures often had focal, as opposed to generalized, onsets, and it was not uncommon for seizures to begin after 5 years of age.     

Observations on the Quality Characteristics of Waxy (Amylose‐Free) Winter Wheats

Previous investigations have suggested waxy (amylose‐free) wheats (Triticum aestivum L.) possess weak gluten properties and may not be suitable for commercial gluten extraction. This limitation could prevent the use of waxy wheat as a source of unique starch, because gluten is a by‐product of the wheat starch purification process. Fifty waxy wheat lines were used to determine the extent to which gluten protein and other grain quality related traits might vary and, consequently, allow the development of waxy wheat with acceptable gluten properties. Among the waxy lines, significant variation was observed for all measured quality traits with the exception of flour protein concentration. No waxy entries statistically equaled the highest ranking nonwaxy entry for grain volume weight, falling number, flour yield, or mixograph mix time. No waxy lines numerically exceeded or equaled the mean of the nonwaxy controls for falling number, flour yield, or mixograph mix time. For grain and flour protein related variables, however, many waxy lines were identified well within the range of acceptability, relative to the nonwaxy controls used in this study. Approximately 50% of the waxy lines did not differ from the highest ranking nonwaxy cultivar for grain and flour protein concentrations. Forty‐three (86%) of the tested waxy lines were not sig‐nificantly different from the nonwaxy line with the highest mixograph mixing tolerance, 22/50 (44%) of the waxy wheat lines did not differ from the highest ranking nonwaxy line in gluten index scores, and 17/50 (34%) did not differ from the highest ranking nonwaxy line in extracted wet gluten. All waxy experimental lines produced gluten via Glutomatic washing. The quality of the gluten, as measured both by mixograph and gluten index, varied widely among the waxy lines tested. These observations suggest that weak gluten is not a natural consequence of the waxy trait, and waxy cultivars with acceptable gluten properties can be developed.