Quantitation of reticulated platelets in healthy dogs and in nonthrombocytopenic dogs with clinical disease

Background — Thrombocytopenia is a common disorder in dogs and development of an objective diagnostic assay to measure platelets newly released from bone marrow into the blood would provide a noninvasive way to predict megakaryocytopoiesis. Reticulated platelets are newly released platelets with increased concentrations of RNA that can be detected by flow cytometric analysis of blood stained with thiazole orange (TO).
Objectives — The goals of this study were to establish a reproducible method to quantitate reticulated platelets in dogs, to establish a reference interval for reticulated platelet percentages in healthy dogs, and to determine whether the percentage of reticulated platelets was nonspecifically increased in nonthrombocytopenic dogs with clinical disease.
Methods — Blood samples were obtained from healthy dogs and from nonthrombocytopenic dogs presented for a variety of disorders. An aliquot of whole blood was stained with TO and a phycoerythrin-labeled monoclonal antibody to platelet CD61, then analyzed by flow cytometry.
Results — The coefficients of variation were 7.8% to 15.6% (intra-assay precision) and 6.1% to 19.5% (interassay precision). Overnight storage for 18 to 26 hours, under variable conditions, resulted in an increase in the percentage of platelets staining with TO. The reference interval for reticulated platelets in the healthy control group was 0–4.3% (0–12,095/μL). No significant differences were found in the mean percentage of reticulated platelets or absolute concentration of reticulated platelets between control and affected dogs.
Conclusions — These studies demonstrate a reliable, noninvasive diagnostic assay for measurement of reticulated platelets in whole blood and provide a baseline for assessment of the clinical utility of the assay.     

GnRH antagonist inhibition of gonadotropin and steroid secretion in boars in vivo and steroid production in vitro

The hormone GnRH has a stimulatory effect on gonadotropin synthesis and secretion. The objective of the first study was to evaluate concentrations of FSH and LH in plasma of boars after successive treatment with SB75, a GnRH antagonist. Thirteen boars greater than 1 yr of age (eight White Composite [WC] and five Meishan [MS]) were injected once daily with SB75 (10 microg/kg of body weight) for 4 d. Plasma concentrations of LH and testosterone (T) decreased after 1 h from the first dose of SB75. After 12 h of treatment, LH gradually returned to pretreatment concentrations, but T remained suppressed (< 2 ng/mL) until after the last injection of SB75. There was a modest, but significant, reduction in FSH during treatment with SB75. The prolonged inhibitory effect of SB75 on suppression of plasma T concentrations, in the presence of pretreatment concentrations of LH, implied direct effects of SB75 at the testis. In the second experiment, testicular tissue from adult boars was incubated in the presence of three doses of human chorionic gonadotropin (hCG; 0, .5, and 5 IU) with SB75 (250 ng/mL) or with Deslorelin, a GnRH agonist (500 ng/mL). Samples of media were collected every hour for 3 h, and concentrations of T and estrone (E1) were determined by RIA. Concentrations of T and E1 increased with time in response to treatment with hCG. Co-treatment with SB75 decreased media concentrations of T (P < .01) and E1 (P < .03) compared to controls (77.9 vs 85.7 +/- 2.0 and 4.7 vs 5.3 +/- .2 ng/g). In contrast, treatment with Deslorelin had no effect on the amount of T (P > .50) or E1 (P > .26) released with all dosages of hCG. These results indicate that a GnRH antagonist has a direct effect on the testis, decreasing amounts of T and E1 released from the Leydig cells; however, treatment with a GnRH agonist had no direct effect on release of these gonadal steroids. Thus, it remains unresolved whether the site of action of GnRH antagonist on testicular steroidogenesis is through a testicular GnRH receptor or through some other mechanism.     

Papillomavirus-associated basosquamous carcinoma in an Egyptian fruit bat (Rousettus aegyptiacus).

A 5-yr-old female Egyptian fruit bat (Rousettus aegyptiacus) had a small raised pigmented mass removed from the lateral canthus of the left eye. Six additional variably sized, raised, smooth to cauliflower-like skin masses were observed randomly distributed throughout the left wing membranes. Four masses were removed and diagnosed microscopically as basosquamous carcinomas and papillomas. Additional masses, removed 6 mo and 1 yr later, showed bony invasion and squamous differentiation. Immunohistochemistry detected positive intranuclear staining for bovine papillomavirus antibody in all samples. Polymerase chain reaction done on DNA extracts from formalin-fixed, paraffin-embedded tumor tissue amplified a 450 base-pair segment analogous to the L1 region of human papillomavirus types 96 and 5. Basic Local Alignment Search Tool analysis of sequenced amplicons suggests a novel chiropteran papillomavirus. To our knowledge, this is the first report of papillomavirus-associated carcinoma in a chiropteran species.     

Drought responses of six ornamental herbaceous perennials

Although low water use landscaping is becoming common in arid regions, little is known about drought tolerance and drought responses of many ornamental plants, especially herbaceous perennials. Drought responses were assessed for six herbaceous ornamental landscape perennials in a 38 l pot-in-pot system in northern Utah over a 2-year period. The first year was an establishment period. During the second year, drought responses were evaluated for established Echinacea purpurea (L.) Moench, Gaillardia aristata Pursh, Lavandula angustifolia P. Mill., Leucanthemum × superbum (J.W. Ingram) Berg. ex Kent, ‘Alaska’, Penstemon barbatus Roth var. praecox nanus rondo, and Penstemon × mexicali Mitch. ‘Red Rocks’. Plants were irrigated at frequencies of 1 (control), 2, or 4 weeks between June and September, simulating well-watered conditions, moderate drought, or severe drought. Osmotic potential (Ψ_s), gas exchange, visual quality, leaf area, and dry weight were assessed. In a confined root zone, P. barbatus showed the greatest tolerance to all levels of drought, avoiding desiccation by increasing root:shoot ratio and decreasing stomatal conductance as water became limiting. L. angustifolia and P. × mexicali showed tolerance to moderate drought conditions, but died after exposure to the first episode of severe drought. Neither G. aristata nor L. superbum were able to regulate shoot water loss effectively. Instead, both species displayed drought avoidance mechanisms, dying back when water was limiting and showing new growth after they were watered. Compared to control plants, G. aristata shoot dry weight was reduced by 50% and 84%, and L. superbum shoot dry weight was reduced by 47% and 99% for the 2- and 4-week irrigation intervals, respectively. Root dry weights were affected similarly for both species. E. purpurea exhibited poor visual quality at all irrigation intervals, in particular wilting severely in both drought treatments, but regaining turgor when watered again. P. barbatus is recommended for ornamental landscapes that receive little or no supplemental irrigation, while E. purpurea is not recommended for low water landscapes because of low visual quality under even mild drought.     

Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs

MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 μl of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression.     

南方菜豆花叶病毒(SBMV)两典型株系特异cDNA和RNA探针的制备及应用

南方菜豆花叶病毒（ＳＢＭＶ）主要有２株系：菜豆株系（ＳＢＭＶ－Ｂ）和豇豆株系（ＳＢＭＶ－Ｃ）,血清学方法不能予以区分。根据已报道的核酸序列设计了２对特异引物,进行ＲＴ－ＰＣＲ扩增并克隆到载体Ｂｌｕｅｓｃｒｉｐｔ中,序列测定予以证实后利用随机引物法合成放射性ｃＤＮＡ探针,再进行体外转译合成地高辛ＵＴＰ标记的ＲＮＡ探针。２种探针均可分别特异地检测Ｎｏｒｔｈｅｒｎｂｌｏｔ膜上的ＳＢＭＶ－Ｂ和ＳＢＭＶ－Ｃ。ＲＮＡ探针检测ＳＢＭＶ－Ｂ和ＳＢＭＶ－Ｃ灵敏度分别为：０．１μｇ和０．０１μｇ。     

收获沙棘振动器试验研究

该项研究的核心是研制振动器,通过振动果枝的方式来收获沙棘果实。2003到2004年间,基于我们的试验研究结果显示,沙棘品种＂印度夏季＂通过用频率为40Hz,振幅为15mm,10s为一振动周期的果枝振动器可有效获得果实。利用这个参数的振动可以有效获得90%的果实,且对果枝的损害很小,从果枝上下来的叶片、芽及木枝的碎片也很少。在2003年进行了最原始的研究,2004年测试发现这种方法存在一定缺陷。发电机利用机轴进行来回的旋转运动,利用这个原理进行各种振幅试验,但这对果树各部分会产生大量的压力。用1.5kW的电机可以在车间及没有农业机械的土地上进行试验,但这个过程需要人工看护。最原始的方法效率不高且过程复杂。2005年进行了设备的改进,通过凸轮产生的振动来驱动液压马达。尽管这种方法使用时间还很短,但是它的振动功能还是非常显著的。如果选在最佳采摘季节收获果实,收获的果实品质最佳,同时对果树的伤害也是最小的。如果在每个季节采摘500株或是更少果树的果实,这种振动收获果实的方法是一种最佳选择。     

Vaccination of Mixed and Full-Sib Families of Channel Catfish Ictalurus punctatus Against Enteric Septicemia of Catfish With a Live Attenuated Edwardsiella ictaluri Isolate (RE-33)

These studies were conducted to evaluate the efficacy of a live attenuated Edwardsiella ictaluri vaccine against enteric septicemia of catfish. In one study channel catfish fingerlings (72 d of age post hatch) were immersed for 30 min in water containing E. ictaluri RE-33 at dosages of 1 × 10⁶, 1 × 10⁷ and 2 × 10⁷ CFU/ML of water. No mortalities were observed following vaccination. Following exposure to virulent Edwardsiella ictaluri the cumulative mortality of fish vaccinated with dosages of at least 1 × 10⁷ CFU/mL were significantly lower than that of non-vacccinated fish in both laboratory and field challenges. Vaccination with 1 × 10⁶ CFU RE-33mL provided some protection during the laboratory challenge but failed to protect fish under field conditions. In a second study, vaccination of 6 full-sib families of channel catfish at a vaccine dosage of 1 × 10⁷ CFU/mL resulted in a relative percent survival among families ranging from 67.1 to 100%. Significant differences in mortality were found among families and between vaccinated and unvaccinated groups, but there was no family by vaccine interaction. Families with the highest mortality after vaccination were also shown to have the highest mortality without vaccination (r = 0.82; P = 0.04).     

Effect of Feeding Frequency and Romet-Medicated Feed on Survival, Antibody Response, and Weight Gain of Fingerling Channel Catfish Ictalurus punctatus after Natural Exposure to Edwardsiella ictaluri

Abstract The efficacy of restrictive feeding regimes and Romet®-medicated feed (formulated at 40.5-mg Romet-30 premix/kg feed) fed at 2.0% body weight was evaluated under field trial conditions for the treatment of naturally induced Edwardsiella ictaluri infections in fingerling channel catfish. After detection of an Edwardsiella ictaluri epizootic, fish were fed medicated and non-medicated feed every day, every other day and every third day for the duration of the experiment. In addition, experimental treatments also included fish that were completely withheld from feed and those provided Romet-medicated feed for five consecutive days followed by non-medicated feed fed daily. Feeding frequency and diet significantly affected survival and weight gain. Survival was greatest among fish that were completely withheld from feed or that were fed medicated feed every other or every third day. The use of non-medicated feed every other or every third day was equally as effective for reducing Edwardsiella ictaluri associated mortalities as feeding Romet-medicated feed on a daily basis. Feeding fish Romet-medicated feed for five consecutive days was not an effective treatment. Agglutinating antibody titers against Edwardsiella ictaluri were significantly lower in all groups of fish fed Romet-medicated feed for the duration of the experiment. These data indicate that feeding frequency is a primary factor affecting the severity of Edwardsiella ictaluri infection and that prolonged consumption of Romet-medicated feed may suppress antibody production.