A novel method for estimating tree dimensions and calculating canopy volume using digital photography

Studies on plant phenology and browse capacity require effective methods to rapidly quantify plant dimensions such as tree height, height of maximum canopy diameter, height of first leaves, maximum canopy diameter, and diameter of trunk(s) at height of first leaves. Here we describe a method for estimating tree dimensions and calculating canopy volume using a measuring staff (for calibration), a digital camera and our VolCalc software. The method requires a photograph be taken of the measuring staff placed next to an object whose measurements are to be determined. The two objects must be adjacent to one another in the photograph. For rapid analysis, multiple photographs of different objects can be taken over a short period of time using the measuring staff. The method is not limited to plants and can be used to determine, for example, browser height, height at which browsers feed, and primate resource abundance. The method has been tested in the field and provides a fast and precise tree dimension parameter estimation option, where sampling time is of the essence. Test results compare well to alternative methods currently utilised, showing improved precision and faster field data collection times, which are important to researchers and ecologists.     

A novel method for estimating the adaptive ability of guide dogs using salivary sIgA

Salivary secretory immunoglobulin A (sIgA) concentrations of prospective guide dogs for the blind were determined to clarify whether salivary sIgA is useful in evaluating the potential suitability of guide dogs for the blind. Saliva was collected from 73 prospective guide dogs in the kennel on day 1 (the day of separation from volunteer puppy-raisers), 2, 3, 7 and 14 during the estimation period (at about 1 year old). We selected particularly suitable dogs (superior dogs) and unsuitable dogs (inferior dogs) on the basis of the trainers' estimation. All dogs were divided into two groups, those were acceptable dogs would become the guide dogs and rejected dogs could not become guide dogs. The sIgA concentrations in superior dogs gradually increased from day 1 to 14 and those in inferior dogs remained at low levels. Moreover, the sIgA concentrations on day 14 in the acceptable dogs were significantly higher than those in rejected dogs. The cut-off point of sIgA concentrations on day 14 using an ROC curve was 90 EU/ml, and the specificity of the estimation at this point (70.4%) was higher than that of trainers' estimation (50%). Moreover, parallel testing using both trainers' estimation and sIgA estimation, showed that specificity was further improved (79.5%). The present study showed that sIgA concentration was extremely useful in estimating the adaptive ability for guide dogs for the blind.     

A turbidimetric method for fibronectin assay in the dog

An immunoturbidimetric method, using spectrophotometry, for the assay of canine plasma fibronectin concentration was compared with the immunoelectrophoretic method. The spectrophotometric method (S) correlated positively (r = 0.7) and significantly (P less than 0.01) with the immunoelectrophoretic method (I). The regression equation was S = 0.37I + 53. Ninety-five percent confidence levels for the regression line were calculated to allow detection, by spectrophotometry, of plasma fibronectin concentrations outside the normal range.     

塞内卡病毒荧光定量RT-PCR检测方法的建立及初步应用

为建立塞内卡病毒(SVA)荧光定量RT-PCR(FQ-PCR)检测方法,本研究根据GenBank中SVA 3D基因保守区域序列设计了一对特异性引物和一条特异性探针,以SVA cDNA为模板,经优化反应条件,建立了SVA FQ-PCR检测方法,并对其进行了特异性、敏感性、重复性试验;利用所建立的方法对28份临床疑似SVA感染样品进行了检测,并与本实验室建立的SVA RT-PCR方法检测结果及测序结果比较分析。结果显示,该方法仅对SVA出现阳性扩增信号,对BHK-21正常细胞对照和口蹄疫病毒、猪繁殖与呼吸障碍综合征病毒、猪流行性腹泻病毒、猪圆环病毒Ⅱ型、猪伪狂犬病毒等5种病原均未出现扩增,特异性较强;该方法最低检测限为10.4拷贝/μL质粒标准品,比常规RT-PCR敏感性高10倍,敏感性较高;该方法批内及批间变异系数均小于4%,重复性好。利用该方法对28份疑似SVA感染样品检测显示,检出9份阳性样品,与测序结果一致,而常规RT-PCR仅检出6份阳性样品,其敏感性高于常规RT-PCR方法,两种方法的符合率为89.3%。本研究为SVA的早期检测提供了特异、敏感、快速的方法。     

Validation and method comparison for a point-of-care lateral flow assay measuring equine whole blood insulin concentrations

The Wellness Ready Test (WRT) is a lateral flow, stall-side assay that measures equine insulin in whole blood and requires validation before recommending clinical use. We evaluated intra- and inter-assay precision and linearity and compared the WRT with a radioimmunoassay (RIA). Tested concentrations ranged from <139 to >695 pmol/L (<20 to >100 μIU/mL). For 20 replicates at each insulin level, intra-assay CVs of the WRT for insulin were 13.3%, 12.9%, and 15.3% at low (139–278 pmol/L; 20–40 μIU/mL), intermediate (278–417 pmol/L; 40–60 μIU/mL), and high (>417  pmol/L; >60 μIU/mL) concentrations, respectively. For 10 replicates at each level (3 assay lots), inter-assay CVs were 15.9%, 11.0%, and 11.7%, respectively. In the weighted linear regression of 5 measured insulin concentrations against expected concentrations, R² = 0.98, slope = 1.02, and y-intercept = 14.4 pmol/L (2.08 μIU/mL). The Spearman correlation coefficient (r_s) was 0.90 (95% CI: 0.85–0.94) between the WRT and RIA; the WRT = f(RIA) Passing–Bablok regression yielded the fit, y = 1.005x + 24.3 pmol/L (3.50 μIU/mL). The WRT result averaged 10.4% higher than the RIA result, with targeted bias of 25.9, 26.1, and 26.7 pmol/L (3.74, 3.76, and 3.84 μIU/mL) for cutoffs used to diagnose insulin dysregulation of 312, 347, and 451 pmol/L (45, 50, and 65 μIU/mL). Assay clinical sensitivities, specificities, and accuracies determined at the 3 selected clinical cutoffs and using the RIA as gold standard were 87–95%, 92–96%, and 91–95%, respectively (n = 99 samples). Observed total error was 28.4–30.4%. The WRT had acceptable precision, excellent linearity, and good association with the RIA.     

A novel method for lifting weanling research pigs

It is possible to modify lifting techniques in small laboratory pigs to evoke less of a fear response, strengthen the human–animal bond, and improve welfare. The authors hypothesized that recently weaned pigs lifted with a ventral (belly) scoop method would show less fear of new humans and less fear during treatment than pigs lifted vertically by their hind limbs. To evaluate this hypothesis, 32 Yorkshire-cross pigs (age 3 weeks) were divided into 8 groups of 4. All pigs were acclimated to humans for 11 days and uniformly enriched prior to any lifting tests. Pigs were randomly assigned to 1 of 4 different combinations of ventral and/or vertical lifting techniques that varied by consecutive day (1 ventral-ventral; 2 ventral-vertical; 3 vertical-ventral; 4 vertical-vertical). Each day, data were collected regarding pigs’ aversion to lifting and willingness to be caught multiple times. Two hours later, their time to approach a new person and proportion of time spent hiding from the new person were also measured. Aversion scores were assigned based on duration of squealing, shaking, and freezing responses during lifting. Pigs that were lifted using alternate methods on consecutive days showed significantly less aversion when being scooped ventrally (P = 0.008) as compared to lifted vertically by their hind limbs. Additionally, behavioral trends were identified during the study. Based on standardized aversion scores, this research shows that the ventral scoop method induces less fear than the vertical lift method.     

A fluorescence polarization assay for the detection of antibodies to Mycobacterium bovis in cattle sera

A fluorescence polarization assay (FPA) utilizing fluorescein-labelled MPB70 protein as the antigen was developed and evaluated for its ability to detect antibodies to Mycobacterium bovis in cattle sera. Three panels of sera were examined in this study. These included: (A) sera (n=28) obtained from cattle from which M. bovis was cultured; (B) sera (n=5666) from Canadian field cattle which were presumed to be free from M. bovis; (C) sera (n=10) from cattle infected with Mycobacterium paratuberculosis and known to contain antibodies to this organism. Receiver operating characteristic (ROC) curve analysis of the results of panels A and B yielded an area under the curve value of 0.975 (95% confidence interval=0.971-0.979), which indicated that this FPA is an accurate indicator of M. bovis infection. At the cut-off point recommended by the ROC curve analysis, the FPA sensitivity and specificity estimates were 92.9% (95% confidence interval=76.5-98.9%) and 98.3% (95% confidence interval=97.9-98.6%) respectively. The FPA results were compared to the results of the single intradermal (SID) test for the 28 infected cattle. Fifteen of these animals were scored positive with the SID test (sensitivity=53.6%). The FPA detected 15/15 (100%) of the SID test-positive animals and 11/13 (84.6%) of the SID test-negative animals. Two of the culture-positive cattle were not detected by either test. None of the sera that were obtained from the M. paratuberculosis-infected animals cross-reacted in this assay.     

A quantitative real-time PCR assay for Ehrlichia ruminantium using pCS20

Heartwater is a tick borne disease that affects ruminants and wild animals in Africa south of the Sahara. It is caused by Ehrlichia ruminantium and transmitted by the tick Amblyomma hebraeum. The protocols currently used to detect heartwater take several days to complete. Here, we describe the development of a pCS20 quantitative real-time PCR TaqMan probe assay to detect E. ruminantium in livestock blood and ticks from the field. The assay is based on the conserved pCS20 gene region of E. ruminantium that contains two overlapping genes, rnc and ctaG [Collins, N.E., Liebenberg, J., De Villiers, E.P., Brayton, K.A., Louw, E., Pretorius, A., Faber, F.E., Van Heerden, H., Josemans, A., Van Kleef, M., Steyn, H.C., Van Strijp, M.F., Zweygarth, E., Jongejan, F., Maillard, J.C., Berthier, D., Botha, M., Joubert, F., Corton, C.H., Thomson, N.R., Allsopp, M.T., Allsopp, B.A., 2005. The genome of the heartwater agent Ehrlichia ruminantium contains multiple tandem repeats of actively variable copy number. PNAS 102, 838-843]. The pCS20 quantitative real-time PCR TaqMan probe was compared to the currently used pCS20 PCR and PCR/(32)P-probe test with regards to sensitivity, specificity and the ability to detect DNA in field samples and in blood from experimentally infected sheep. This investigation showed that the pCS20 quantitative real-time PCR TaqMan probe was the most sensitive assay detecting seven copies of DNA/mul of cell culture. All three assays, however, cross react with Ehrlichia canis and Ehrlichia chaffeensis. The pCS20 real-time PCR detected significantly more positive field samples. Both the PCR and pCS20 real-time PCR could only detect E. ruminantium parasites in the blood of experimentally infected sheep during the febrile reaction. The PCR/(32)P-probe assay, however, detected the parasite DNA 1 day before and during the febrile reaction. Thus, because this new quantitative pCS20 real-time PCR TaqMan probe assay was the most sensitive and can be performed within 2h it is an effective assay for epidemiological surveillance and monitoring of infected animals.     

A rapid agglutination assay for canine brucellosis using antigen coated beads

Brucella canis is the causative agent of canine brucellosis and facultative intracellular pathogen. The diagnosis of canine brucellosis is based on bacteriological examination and serological methods including agglutination and gel diffusion tests. In this study, crude antigens were extracted from B. canis using hot saline, coated on to latex beads and their usefulness in the serological diagnosis of canine brucellosis was examined. Mixing the antigen coated latex beads with the sera of dogs infected with B. canis produced clear agglutination, but this was not so for B. canis free dog sera. N-terminal amino acid sequence analysis of the crude hot saline extracts, showed that they contained copper-zinc superoxide dismutase, ribose ABC transporter and hypothetical protein of Brucella as antigens. A serological survey of canine serum samples conducted by means of an agglutination test using the antigen coated latex beads, showed that this method was more specific than the tube agglutination test using whole bacterial cell antigens. Although these results suggest that our method in which crude hot saline extracted antigens are coated on to latex beads would be useful in the serological diagnosis of canine brucellosis, we need further investigation using more serum samples to confirm the usefulness of our method.     

猪血浆中磺胺二甲嘧啶HPLC残留检测方法

试验采用高效液相色谱法(HPLC)测定猪血浆中的SM(2磺胺二甲嘧啶)的残留量。应用AgilentHP1100系统,AgilentODSC18(150mm×4.6×5)柱,流动相为乙腈-0.017mol/l磷酸(3+2),检测波长为267nm,进样量为20μl,柱温为室温,提取溶剂为二氯甲烷,检测限为0.01μg/ml,平均回收率在80%以上,变异系数均小于8.5%。该方法样品前处理简单,药物的线性关系好,相关系数达0.999以上。灵敏度高,结果准确、可靠。     

A novel method for preparing histology slides without a microtome

A study was undertaken to determine if it might be possible to prepare tissue sections on slides without the use of paraffin embedding, microtome sectioning, or cryostat sectioning which involve equipment and training not always available to scholars or professionals wishing to examine tissue microscopically. After evaluating many different reagents, cutting instruments and solid supports, we developed a method involving application of super glue to a slide, adhering a section of tissue to it, cutting the tissue with a disposable microtome blade, staining the tissue and removing the superglue with a commercially available product. The sections are similar to those sectioned on a microtome, but do not at this time equal their quality. However, histoarchitecture is preserved and individual cell morphology is usually good. We conclude that this is a viable method for preparing histology sections without the use of a microtome or cryostat, something long thought impossible. We have dubbed the method 'RAMP' (Rapid Adhesive-Mediated Procedure).     

应用高效液相色谱测定饲料中金霉素方法探讨

金霉素是由美国氰胺公司于 1 949年开发的四环素类广谱抗生素 ,对革兰氏阳性菌、阴性菌、螺旋体、立克次氏体、大型病毒均有抑制作用。本试验应用高效液相色谱法对饲料中金霉素含量测定方法进行探讨。添加量在 4 96mg/kg时 ,回收率为 88 3 % ;添加量在 1 9 97mg/kg时 ,回收率为89 5 % ;添加量在 39 37mg/kg时 ,回收率为 95 9% ;添加量在 60 2 6mg/kg时 ,回收率为 96 4% ;添加量为 79 1 9mg/kg时 ,回收率为 97 9% ;平均回收率为 93 6 %。本试验回收率范围在 88 3 %～ 97 9%。1　仪器设备1 1　高效液相色谱仪　日立 65 5A型 ,65 5A— 2 2…     

Technical note: time-resolved fluoro-immunometric assay for intact insulin in livestock species

Insulin levels in ruminants are often very low and hence are difficult to measure with commercially available RIA kits designed for use with human serum or plasma samples. Those assays may also have high cross-reactivity with nonintact insulin. An assay originally invented for human insulin and based on a pair of monoclonal antibodies binding to specific parts of the insulin molecule was further developed and validated for use with bovine or porcine plasma or serum. The assay is of the sandwich type, with the catching antibody coated to the solid phase of microtiter plate wells and with the detecting antibody labeled with europium, and measured as time-delayed fluorescence. The assay protocol includes an incubation step in which plasma samples of 50 microL are incubated with buffer and detecting antibody for 3 h in coated wells, followed by an enhancement step in which the fluorescence from the europium label is stabilized before measurement. This gives a sensitivity of 3 pmol/L and a possible working range up 16,700 pmol/L. There is no cross-reactivity with pro-insulin or IGF-I. Calibrators are prepared in heat-inactivated serum from the relevant species. Porcine and bovine insulin have different calibration curves; porcine insulin is more reactive and has a higher background than bovine insulin. Validation results show low CV values, parallel dilution of samples, and a recovery ratio close to unity. Comparison with a commercial RIA shows good agreement, except at low concentrations, at which the RIA determinations are inaccurate. Plasma samples from other domestic species (horse, sheep, goat, and mink) have also been assayed, but it is emphasized that calibrators should be prepared in heat-inactivated serum from the appropriate species, and preferably insulin from that species should be used for calibration.     

Quantitative determination of progesterone (P4) in canine blood serum using an enzyme-linked fluorescence assay

Progesterone (P4) measurement in the peripheral blood is an objective parameter for determination of reproductive functions in the bitch. This study evaluates an enzyme-linked fluorescence assay (ELFA) (Biomerieux, France) for the determination of progesterone validated for use in human. The ELFA is to be performed on the MiniVidas automated analyser which provides quantitative results within 45 min. Blood samples from a total of 27 female dogs of different breeds were used. To test the correctness of the ELFA 15 blood samples with a range of 0.3-40.0 ng/ml were compared to a radioimmunoassay (RIA) validated in the dog. The values obtained with the MiniVidas showed a high agreement (mean deviation 15%), deviations were in both directions and the correlation coefficient was 0.989. The coefficient of correlation according to Passing-Bablok test was 0.995. The intra-assay reproducibility in the MiniVidas system was tested on five samples (mean values 61.8, 6.8, 51.4, 43.7 and 1.1 ng/ml). The coefficients of variation (CV; 10-12 replicates) were 3.4%, 6.7%, 2.6%, 3.1% and 25.4%, respectively. Four serum samples (mean value 47.0, 15.1, 49.1 and 4.0 ng/ml) from different bitches were assayed singly in 10 separate series to test the inter-assay variability. The corresponding CV was 2.1%, 2.2%, 3.1% and 4.3% respectively. Samples from three dogs were used to test the accuracy of the assay. These samples were diluted (1/2, 1/4, 1/8 and 1/16) with charcoal-stripped human serum (Biomerieux, France) and tested in three runs. The expected values were met in a range of 60-75%. Measurement of progesterone for the detection of ovulation as well as prediction of parturition provided meaningful results. As a conclusion the use of the MiniVidas system for determination of P4 in peripheral blood of the bitch provides rapid and reliable results.     

A longitudinal study of a novel dot-enzyme-linked immunosorbent assay for detection of avian influenza virus

A monoclonal antibody (MAb)-based dot-enzyme-linked immunosorbent assay (ELISA) has been developed that detected the epitopes specifically associated with avian influenza virus (AIV). The dot-ELISA detected the antigens of AIV directly from clinical and field specimens. Data obtained from experimentally AIV-infected specific-pathogen-free chickens and also the 2001/02 AIV outbreak of serotype H7N2 positive flocks in Pennsylvania indicated that the mean sensitivity (Se) of the dot-ELISA ranged between 45% and 68% and the mean specificity (Sp), between 85% and 90%. The values were derived from various clinical and field specimens when compared with virus isolation with embryonating chicken eggs. On routine AIV surveillance samples, the dot-ELISA achieved a 92%-100% Sp on the basis of resting over 1500 AIV surveillance samples that were confirmed negative by virus isolation. The dot-ELISA detected AIV antigens with a 5-microl allantoic fluid sample that contained a concentration of 0.4 hemagglutinating units. Furthermore, the dot-ELISA retained its specificity for AIV because no cross-reactions were obtained with various other avian viruses. The findings in this study indicated that the dot-ELISA was highly sensitive and specific and comparable with the commercial Directigen test in the detection of AIV obtained from clinical and field specimens.     

Real-time PCR方法检测高脂饲喂小鼠肝脏CYP2A5的表达

为了建立非酒精性脂肪肝(NAFLD)模型小鼠肝组织中CYP2A5表达的实时荧光定量PCR(real-time fluorescence quantitative PCR,qRT-PCR)方法。高脂饮食诱导小鼠至8周,采用qRT-PCR方法检测小鼠肝脏中CYP2A5的表达水平,同时评价该方法的特异性。建立了小鼠肝组织中CYP2A5的SYBR Green实时荧光定量PCR检测方法,结果显示,该方法的溶解曲线为单峰,同时核酸电泳显示一条特异性条带。检测结果表明,高脂诱导的NAFLD小鼠肝组织中CYP2A5的表达水平极明显高于正常对照组(P0.01)。表明该实时荧光定量PCR方法,特异性好、灵敏度强,为进一步研究小鼠CYP2A5的表达提供了试验基础。     

口蹄疫病毒RT-LAMP检测方法的建立

为了建立检测口蹄疫的快速实用分子生物学技术，本研究借助新兴的环介导等温扩增技术，建立了一种灵敏、特异、快速的分子生物学检测方法-体外等温扩增检测方法。设计了6条针对FMDV3D基因的8个位点的特异性引物，建立了一套从核酸抽提到检测仅需要75min的快速检测技术。所建立的检测方法灵敏，与实时荧光RT-PCR灵敏度相当；且具有特异性，对其他水泡性疾病痛原体检测均为阴性；而且简单，不需要复杂的仪器，常规水浴锅在63．5℃即可进行，肉眼可直接观察检测结果。该方法是适合基层和现场检测而无需送至中心实验室的快速灵敏实用的分子生物学检测方法。