Development of a One-Step Real-Time Polymerase Chain Reaction Assay for Diagnosis of Phytophthora ramorum

ABSTRACT Phytophthora ramorum is a recently described pathogen causing bleeding cankers, dieback, and leaf blight on trees and shrubs in parts of Europe and North America, where the disease is commonly known as sudden oak death. This article describes the development of a single-round real-time polymerase chain reaction (PCR) assay based on TaqMan chemistry, designed within the internal transcribed spacer 1 region of the nuclear ribosomal (nr)RNA gene for detection of P. ramorum in plant material. Unlike previously described methods for the molecular detection of P. ramorum, this assay involves no post amplification steps or multiple rounds of PCR. The assay was found to have a limit of detection of 10 pg of P. ramorum DNA, and could detect P. ramorum in plant material containing 1% infected material by weight within 36 cycles of PCR. The assay also was used to test DNA from 28 other Phytophthora spp. to establish its specificity for P. ramorum. A quick and simple method was used to extract DNA directly from host plant material, and detection of P. ramorum was carried out in multiplex with an assay for a gene from the host plant in order to demonstrate whether amplifiable DNA had been extracted. Amplifiable DNA was extracted from 84.4% of samples, as demonstrated by amplification of host plant DNA. The real-time protocol was used to test 320 plant samples (from 19 different plant species) from which DNA extraction had been successful, and was shown to give results comparable with a traditional isolation technique for diagnosis of P. ramorum in plant material from common U.K. hosts.     

A randomized, double-blind, placebo-controlled study to evaluate the effect of EFF1001, an Actinidia arguta (hardy kiwi) preparation, on CADESI score and pruritus in dogs with mild to moderate atopic dermatitis

Canine atopic dermatitis (AD) is common and new therapies are beneficial. This multicentric, randomized, double-blind, placebo-controlled study tested the efficacy of Actinidia arguta (hardy kiwi) (EFF1001) in dogs with mild/moderate AD. The study was divided into two stages. Stage 1 lasted 6 weeks. In the first 2 weeks prednisolone [days 1–3: 0.2 mg/kg twice daily (BID), days 4–14: 0.2 mg/kg every other day (EOD)] was administered. Responsive dogs were placed on prednisolone 0.2 mg/kg EOD + assigned test article [either placebo or EFF1001 (30 mg/kg)] once daily for 4 weeks. Stage 1 responders were advanced to stage 2, which involved 4 weeks of just EFF1001. Clinicians scored lesions using Canine Atopic Dermatitis Extent and Severity Index (CADESI) and owners scored pruritus using a Pruritus Visual Analogue Scale. Seventy-seven dogs were enrolled, 76 were randomized on day 14, and 57 (57/76 = 75%) completed stage 1 (27 in EFF1001 and 30 in placebo). At the end of stage 1, 35 of 57 dogs (35/57 = 61%) responded (18 in EFF1001 and 17 in placebo) and advanced to stage 2. At completion of stage 1, CADESI scores did not significantly differ between groups while pruritus decreased in EFF1001 group and approached significance. At completion of stage 2, 19 dogs (19/35 = 54%) responded (15/19 = 79% had received EFF1001 and 4/19 = 21% placebo in stage 1). After completing stage 2, dogs placed on EFF1001 throughout the study were 3.5 times more likely to either maintain or improve scores than those that started it in stage 2. It is concluded that EFF1001 is beneficial adjunctive therapy after prolonged use.     

Ascorbate Oxidase,Protein Disulfide Isomerase,Ascorbic Acid,Dehydroascorbic Acid and Protein Levels in Developing Wheat Kernels and Their Relationship to Protein Disulfide Bond Formation

Developing wheat kernels of three New Zealand wheat cultivars at one to nine weeks postanthesis (WPA) were analyzed for kernel weight, salt‐soluble protein, salt‐insoluble protein, ascorbate oxidase (AOX), protein disulfide isomerase (PDI), ascorbic acid (AA), and dehydroascorbic acid (DHA). Kernel weight and salt‐soluble protein increased from one to five or six WPA then declined. The amount of salt‐insoluble protein per kernel rapidly increased from two to five or six WPA, then remained constant. AOX activity increased from one to two or three WPA then declined to almost zero at seven WPA. PDI activity increased from one to three WPA, stayed constant to six WPA, then declined about 25%. DHA increased from one to two or three WPA then declined to zero at seven WPA. AA increased from one to three or five WPA then declined to zero at seven WPA. When DHA, at similar concentrations (≈1 mM) to that found in one to six WPA kernels, was mixed with reduced‐gluten proteins, it was reduced to AA. The results are interpreted as a scheme showing that, during the formation of protein‐disulfide bonds, electrons are transferred from nascent protein thiols, ultimately to oxygen, through a series of electron carriers including PDI, AA‐DHA, and AOX.     

Effects of Co‐stocking Smallmouth Buffalo,Ictiobus bubalus,with Channel Catfish,Ictalurus punctatus

Proliferative gill disease (PGD) in catfish is caused by the myxozoan Henneguya ictaluri. The complex life cycle requires Dero digitata as the oligochaete host. Efforts to control PGD by eradicating D. digitata have been unsuccessful. Smallmouth buffalo, Ictiobus bubalus, (SMB) are opportunistic bottom feeders and a putative option for controlling D. digitata. In 2011, 15 ponds (0.4 ha) were stocked with 5000 channel catfish, Ictalurus punctatus; 7 of these 15 ponds were also stocked with 300 SMB fingerlings. There were no differences in benthic invertebrate numbers or water quality variables between ponds with or without SMB. At harvest, there were no differences in percent survival, total weight, or catfish feed conversion ratio. In the second year, 18 ponds (0.4 ha) were stocked with 6000 channel catfish. Half the ponds were also stocked with 300 SMB. Sentinel fish were used to estimate disease severity, and pond water was collected for molecular estimation of H. ictaluri actinospore concentrations. Similar to the first year, there were no differences between treatments in any variable tested, including PGD severity in sentinel fish and parasite concentrations in pond water. Under these study conditions, presence of SMB did not have a measureable effect on PGD incidence, parasite density, or overall catfish production.     

Impacts of Bolbophorus damnificus (Digenea: Bolbophoridae) on Production Characteristics of Channel Catfish,Ictalurus punctatus,Raised in Experimental Ponds

Bolbophorus damnificus was recognized as a threat to channel catfish, Ictalurus punctatus, production in Louisiana and Mississippi in 1997 and 1999, respectively. Severe to moderate outbreaks (≥34% prevalence) are correlated to heavy losses, but impact of mild outbreaks with low prevalence (<33%) was unknown. Laboratory and field studies were conducted to evaluate sublethal acute (early stage) and chronic (late stage) trematode infection impacts on catfish production. Fish raised in ponds seeded with ram's horn snails from ponds harboring active B. damnificus outbreaks resulted in infection prevalence ranging from 1.6 to 20.4%. Compared to fish raised in ponds not seeded with infected snails (0% prevalence), fish biomass, number harvested, and total feed fed were significantly lower. These data agree with previous studies where mild infections reduced feed consumption, fish production, and economic returns. Infected fish were collected from ponds and used in a laboratory growth study. Once removed from the infection source, weight gain, growth rate, feed consumption, and feed efficiency were equal to or greater than trematode‐free fish. This suggests that although early B. damnificus infection (premetacercarial development) can severely impact production, infection has little impact on performance once metacercarial cysts are fully developed, and the infection source is removed.     

Bilateral intracorporeally sutured inguinal herniorrhaphy using 3-dimensional laparoscopy in a dog

A 7-month-old, intact male, mixed breed dog with bilateral inguinal hernias underwent general anesthesia for laparoscopic bilateral inguinal herniorrhaphy via a 3-port approach. A 3-dimensional laparoscopic system was used to perform the procedure immediately following prescrotal open castration. Intracorporeal suturing with polypropylene was performed, and 2 cruciate sutures were placed to close each inguinal ring. The caudal aspect of each inguinal ring was left slightly open so as not to disrupt the passage or patency of vessels and nerves. No intra- or post-operative complications occurred. One year after surgery, the dog has no evidence of recurrence of the inguinal hernias.Key clinical message:This case report demonstrates a novel minimally invasive approach to inguinal herniorrhaphy in a dog with no reported complications and a good long-term outcome. Intracorporeally sutured inguinal herniorrhaphy is feasible in dogs with good results, although additional cases are needed to gain experience with this technique in dogs with varying presentations of inguinal hernias.     

Comparison of anal sac cytological findings and behaviour in clinically normal dogs and those affected with anal sac disease

No previous study has explored the relationship between cytology and the frequency of behaviours associated with anal sac disease (ASD). The goals of the study were: (i) to compare the cytological findings between anal sac secretions from normal dogs with no history of ASD to those with non-neoplastic ASD; (ii) to determine whether anal sac cytological findings can be used to differentiate between normal dogs and dogs with ASD; (iii) to explore the correlation of anal sac cytology and behaviour between normal dogs and dogs with ASD; and (iv) to describe behaviours typical of ASD as reported by owners. Thirty dogs were selected for this study, based on their behavioural history as detailed in a questionnaire completed by their owners. Of the thirty dogs, ten were considered normal insofar as they had no history of ASD clinical signs. The remaining 20 dogs were characterized as having ASD, with a chronic history of perianal pruritus, but no other pruritus. All dogs had their anal sacs manually expressed, and the discharge was examined microscopically in a blinded manner. A total of 171 oil immersion fields (OIFs) were examined from normal dogs and 333 OIFs from dogs with ASD. The behavioural results for dogs with ASD revealed that scooting recurred with a median frequency of 3 weeks post-anal sac expression. There were no clinically statistically significant cytological differences between normal dogs and those with ASD, thereby leading to the conclusion that cytology is an ineffective tool for diagnosing ASD.     

Canine plasma and erythrocyte response to a docosahexaenoic acid-enriched supplement: characterization and potential benefits

Results of this study confirm that dietary supplementation in dogs with a natural source of omega-3 fatty acids (salmon oil), with a docosahexaenoic acid:eicosapentaenoic acid (DHA:EPA) ratio of 1.5:1, increases plasma and red blood cell levels of these fatty acids. Supplementation with this DHA-enriched oil improves the long-chain polyunsaturated fatty acid omega-6:omega-3 (n-6:n-3) ratio, which may benefit dogs of all ages. Studies describing some of the neurologic, renal, cardiovascular, immune, and musculoskeletal effects of elevated blood levels of n-3 fatty acids, especially DHA, are reviewed. The importance of providing an enriched source of DHA, instead of its shorter precursors, is emphasized.     

Assessment of obesity and fear of fatness among inner-city Dublin schoolchildren in a one-year follow-up study

BACKGROUND: Positive secular trends in adolescent obesity and an increased prevalence of fear of fatness, particularly among girls, have been documented world-wide. There is a lack of consensus about assessment criteria for childhood obesity and no standard exists for assessing Irish children. In 1990, the Irish National Nutrition Survey used body mass index (BMI) > or =26 kg m(-2) to describe the prevalence of overweight among Irish adolescents. OBJECTIVES: (1) To examine the range in classification of Dublin schoolchildren as overweight according to four standard assessment methods; (2) to assess changes in weight status, prevalence of fear of fatness and accompanying slimming practices in a one-year follow-up; and (3) to compare the prevalence of overweight with that documented in 1990 among adolescents of similar age. DESIGN: A one-year follow-up study of 199 healthy schoolchildren (90 boys and 109 girls; mean age of 11 years at baseline) attending seven fee-paying (six single- and one mixed-sex) and eight non-fee-paying (four single- and four mixed-sex) primary schools in Dublin city centre. MEASUREMENTS: Weight, height, waist circumference and triceps skinfold were measured and used in five definitions of overweight, including published cut-off points of BMI-for-age (Centers for Disease Control and Prevention BMI-for-age charts for boys and girls; BMI reference curves for the UK 1990; International Obesity Task Force age- and sex-specific BMI cut-offs), actual relative weight and BM > or =26 kg m(-2). Assessment of body image perceptions and satisfaction (using figure line drawings) was reported in a questionnaire specifically designed for this study. RESULTS: The prevalence of overweight within the total group differed between the four standard definitions of weight status, by 9% at baseline and 8% at follow-up. Accordingly, increasing trends over the year ranged from zero to 3%. Using the criterion BMI > or =26 kg m(-2), 6% of Dublin schoolchildren were overweight, compared with 1.9% of schoolchildren in 1990. Significantly more girls than boys were affected by fear of fatness and were trying to lose weight. CONCLUSION: A standard method for assessment of weight status is urgently needed for the evaluation of obesity prevention initiatives among Irish schoolchildren. Such initiatives need to be sensitive to the pervasiveness of fear of fatness among adolescent girls.