从血凝块中提取DNA的快捷方法

将7 mL全血去血清后的血凝块用携带不锈钢均质头的均质器裂解后,加入红细胞裂解液处理至无血凝块存在,得到1 mL左右的白细胞提取物,可较完全地去除红细胞成分。白细胞提取物提取的DNA成分经凝胶电泳分析,可得到较清晰的DNA条带。用牛特异性引物从所有血凝块扩增出271 bp的牛特异性条带,用血凝块处理后的细胞沉淀添加病毒悬液提取的DNA作BHV1病毒gB-PCR检测,结果显示添加BHV1的血凝块样品均出现478 bp的BHV1-gB特异性条带。     

A simple method for isolating leucocytes from bovine blood and their separation into lymphocyte and granulocyte fractions.

A method for the separation of leucocytes from bovine blood and its separation into lymphocyte and granulocyte fractions is described. The method, involving flash lysis of the erythrocyte population, was found to yield large numbers of viable cells suitable for maintenance in tissue culture medium and hence of value in immunological studies.     

一种简易高效的柞蚕血DNA提取方法

以柞蚕血淋巴为提取材料,进行了柞蚕DNA提取方法的改进试验。结果发现,柞蚕血淋巴经过12 000rpm条件下离心8min后,沉淀经SDS提取液震荡、混匀后,提取基因组的质量明显提高。且提取时间短,操作方便,适合于野外工作环境及实验室快速提取。     

A simple and rapid method for isolation of caliciviruses from liver of infected rabbits

Rabbit Haemorrhagic Disease Virus (RHDV), a member of the Caliciviridae family, is the etiologic agent of Rabbit Haemorrhagic Disease (RHD); this viral disease is highly contagious and kills more than 90% of infected adult rabbits. Research on experimental calicivirus infection uses inocula obtained from livers of rabbits dying from calicivirus infection. This implies that caliciviruses have to be purified from liver homogenates. Current methods to isolate caliciviruses from rabbit livers are time consuming. We propose here a new procedure for fast purification of rabbit caliciviruses from liver homogenates that uses centrifugation through an iodixanol gradient. This method offers in approximately 2 h a sample with a high degree of calicivirus purity, as shown by its biochemical and immunocytochemistry analysis, which is also able to kill adult rabbits from RHD within 48 h of inoculation.     

A rapid, non-invasive method for measuring total respiratory impedance in the horse

Total respiratory impedance was measured rapidly and noninvasively in conscious horses over the frequency range 3 to 40 Hz by the forced random noise method. The shape of curve of impedance versus frequency in horses was markedly different from that of humans. Respiratory resistance was readily found as the real part of impedence and both its absolute value and frequency dependence are useful indices of pulmonary function. It was difficult to obtain meaningful results in intubated animals with the method because of the mechanical properties of the endotracheal tube itself.     

A simple method for rapid identification of Sphaerophorus necrophorus isolates.

A hemagglutination inhibition test for the rapid identification of Sphaerophorus necrophorus is described. Erythrocytes from six species of animals were tested and human cells were found to be the best agglutination indicators. Antiserum prepared in rabbits was found to be specific for S. necrophorus hemagglutinins when tested against 20 isolates of S. necrophorus and 117 other bacteria belonging to 22 genera. The possibility of using a hemagglutination inhibition test for the detection of bovine necrobacillosis was explored.     

A rapid microtechnique for in vitro stimulation of canine lymphocytes using whole blood

A simple, rapid microtechnique utilizing whole blood has been developed for evaluating the in vitro stimulation of canine lymphocytes. The test uses 5 ul. of heparinized whole blood per culture in a microtiter plate and requires no serum supplement. Cultures are harvested by an automated multiple sample cell harvester. This technique permits large numbers of replicate samples to be tested on individual animals with minimal amounts of blood and minimal technician time.     

Stimulation by phytohaemagglutinin of peripheral blood lymphocytes from horse,pig, sheep and man

Optimal conditions for stimulation by phytohaemagglutinin (PHA) were established for equine, porcine, ovine and human lymphocytes in MEMS medium. Optimal thymidine concentration was determined for assay of cell transformation. With all species tested horse serum gave highest thymidine incorporation. Homologous serum was not more appropriate for lymphocytes of man, pig and sheep. Optimal stimulation was achieved at 20, 0.5–5, 5, and 10–40 μg PHA per 10⁶ cells for human, equine, porcine and ovine lymphocytes, respectively.     

A whole blood method for measuring mitogen-induced transformation of sheep lymphocytes

A simple and reproducible micromethod for determination of in vitro mitogenic responses of sheep peripheral blood lymphocytes is described. The test uses (i) whole blood diluted in RPMI 1640 medium to give a cell count of 0.5 x 106-1 x 106 lymphocytes/ml, (ii) mitogens in the range of 5-20 micrograms of phytohaemagglutinin/ml, 20-80 micrograms of lipopolysaccharide/ml or 20-80 microliters of poke weed mitogen/ml, and (iii) a stimulation time of 42 to 90 h. A considerable variation in mitogenic response was observed both between animals and on different occasions in the same animal. Because of the large periodic variation it was suggested that the test should be repeated using blood drawn at different times in order to determine the mitogenic response of an animal.     

A rapid and simple method to obtain canine peripheral blood-derived macrophages

Macrophages play an important role in a variety of situations, including pathogen elimination, inflammation, and tissue repair. However, these cells are not fully studied in dogs, in part, due to the difficulty of efficiently isolating and culturing them in vitro. In this study, we cultured canine peripheral blood mononuclear cells (PBMCs) with 10 ng/ml of phorbol 12-myristate-13-acetate (PMA) for 5 days to obtain macrophages. A high number of round-adherent cells were obtained without the addition of any cytokine. These cells showed active phagocytic activity and a cell surface antigen profile different from dendritic cells. Our method facilitates a high yield of macrophages in a short cultivation period compared with previous studies. This method might be a powerful tool to study macrophage functions in dogs.     

A simple, rapid and reliable method for selecting or assessing the number of replicates for animal experiments

A simple approach was developed for determining the number of replicates needed per treatment group to provide experiments of known power and sensitivity, where power equals the probability that a treatment effect would not go undetected if an effect existed and sensitivity equals the minimal treatment response that will be detectable. This approach, in turn, was used to construct reference tables, applicable across scientific disciplines, from which researchers may read replication requirements directly with ease, speed and reliability. To use the tables, one need only furnish a reliable estimate of the coefficient of variability expected among replicates, which may be obtained from prior observations on similar populations. The tabular data also enable a rapid, reliable assessment of the actual power and sensitivity of completed experiments, such as those contained within the published literature.     

A method for separation of bovine blood leukocytes for in vitro studies.

Various methods have been developed for separation of the cellular components of blood. Size and density differences in cellular blood elements of various animal species necessitate the use of specific separative procedures. This study describes a method which combines isopycnic density-gradient centrifugation with erythrocyte aggregation as a means of isolating leukocytes from bovine blood. The procedure yields a viable population of mixed leukocytes which has proven useful for cell culture and in vitro tests of cell-mediated immunity.     

A simple, rapid, and effective method for the extraction of Mycobacterium paratuberculosis DNA from fecal samples for polymerase chain reaction.

Diagnosis of paratuberculosis (Johne's disease) is stymied by the lack of 1 diagnostic tool that can be used to detect both subclinically and clinically infected animals. At present, fecal culture remains the single diagnostic test that can detect infection in both disease states provided the animals actively shed Mycobacterium paratuberculosis in their feces. Yet, fecal culture has a disadvantage associated with the protracted incubation period of 8-16 weeks before results are available. Detection of nucleic acids specific to M. paratuberculosis in fecal samples is a technique that can circumvent the culture method. This study describes a rapid, simple, and effective method to extract DNA from fecal samples and modification of a polymerase chain reaction assay for optimal sensitivity of detection. An evaluation of 1,000 well-characterized fecal samples was performed by the Colorado Department of Agriculture (Denver, CO) and the National Animal Disease Center (Ames, IA) to determine the sensitivity, specificity, and reproducibility of the new method. Results from this study show that the sensitivity of detection was highly dependent on the load of bacteria in the fecal sample with 81% detection of samples containing >70 colony-forming units (cfu)/g of feces and a 45% detection rate for samples containing less than 1 cfu/g. Similarly, reproducibility of the technique between the 2 laboratories (n = 250 samples) was much higher (75%) for the fecal samples containing high levels of M. paratuberculosis and reduced to 25% for samples with less than 1 cfu/g. An overall specificity of 83% was obtained for known negative samples. The method described here is rapid, simple, and inexpensive compared with other techniques. In addition, this method can detect animals that are shedding less than 1 cfu/g.