奶牛MTT比色法的最佳条件探讨

为建立奶牛淋巴细胞增殖反应MTT比色法,对试验条件进行了研究。应用L16(45)正交试验,对影响MTT比色法的四个主要因素,包括ConA浓度、细胞浓度、培养液小牛血清浓度及培养时间进行了比较和探索。试验表明几个因素对其增殖反应都有显著影响(P<0.05),且最佳反应条件为:15μg/mL的ConA、1×106/mL细胞浓度、10%小牛血清及60h的培养时间;影响增殖反应的先后顺序为细胞浓度、ConA浓度、培养时间及血清浓度。     

Changes of serum vascular endothelial growth factor levels in a rat model of acute myocardial infarction

AIM and METHODS: To study the changes of serum vascular endothelial growth factor (VEGF) levels in a rat model of acute myocardial infarction (MI) and its significance. Eighty-eight male Sprague-Dawley rats were used in this study. MI was produced by left coronary arterial ligation in 80 animals, and eight rats undergoing thoracotomy but not coronary ligation served as controls (sham).Blood samples were drawn from the right atrium before (sham animals) and 1, 3, 6, 12, 24 hours and 2, 3, 5, 7, 14 days after MI(n=8, respectively). Serum VEGF concentrations were measured by a sensitive enzyme-linked immunosorbent assay with a rabbit polyclonal antibody specific for VEGF. RESULTS: In 8 sham animals, the concentration of serum VEGF was (66.99±17.83) pg/mL. Six hours after MI, the level of serum VEGF significantly increased to (125.68±28.07)pg/mL (P＜0.01 vs sham control), and reached a peak (240.61±70.63 pg/mL, P＜0.01 vs sham control) at 24 hours after ligation and then decreased gradually over the remaining 2 weeks. But the level remained significantly elevated for 14 days (107.64±30.31 pg/mL, P＜0.01 vs sham control).CONCLUSION: Serum VEGF levels markedly and permanently increase in the rat model of acute MI may play an important role in the angiogenesis associated with MI     

灰飞虱体内水稻条纹病毒的检测

为评价灰飞虱体内水稻条纹病毒检测方法的适用范围,利用已经制备的水稻条纹病毒(Rice stripe virus,RSV)的多克隆抗体SV21和单克隆抗体3B9,采用多孔板间接ELISA、DIBA和Western blotting等三种方法进行单头灰飞虱Laodelphax striatellus Fallén(SBPH)体内RSV检测。结果表明,检测灵敏度以多孔板间接ELISA最高,其次为DIBA,Western blotting最低;用单克隆抗体3B9检测灵敏度高于多克隆抗体SV21。RT-PCR、IC-RT-PCR和DB-RT-PCR三种方法中,RT-PCR对单头灰飞虱稀释到400倍可检测到病毒;IC-RT-PCR在多克隆抗体SV21稀释浓度大于500倍时检测不出RSV,单克隆抗体3B9稀释浓度大于800倍时检测不到RSV;DB-RT-PCR检测结果显示,单头灰飞虱在稀释400倍后均无阳性反应。     

禽呼肠孤病毒P17蛋白基因在大肠杆菌中的表达及其ELISA检测方法的建立

用设计合成的1对跨越禽呼肠孤病毒（ARV）P17非结构蛋白基因完整开放阅读框（ORF）的特异性引物，对ARVS1133株进行了RT-PCR扩增。扩增产物与pGEX-4T-1原核表达载体连接后，转化大肠杆菌BL21感受态细胞。经0．2mmol／LIPTG诱导，表达的融合蛋白分子质量为42.4ku，占茵体总蛋白的34％。表达产物P17蛋白经不同浓度尿素纯化后，纯度达到85％。Western-blotting显示，纯化的P17蛋白能与感染ARV的阳性血清反应，说明其具有抗原性。以此重组蛋白为包被抗原初步建立了用于鉴别检测ARV活病毒感染与灭活疫苗免疫的SPF鸡血清的ELISA。     

应用ELISA检测猪繁殖和呼吸综合征抗体

用ＥＬＩＳＡ法对青海省东部各县的部分猪群进行了猪繁殖和呼吸综合征抗征抗体检测。共检测了１２０６份猪血清,检出阳性血清２７８份,阳性率为２３．０５％,说明青海省的猪群中有可能存在ＰＲＲＳ。     

Faecal shedding detected earlier than immune responses in goats naturally infected with Mycobacterium avium subsp. paratuberculosis

Paratuberculosis was diagnosed in a goat herd that participated in a sanitation program against Mycobacterium avium subsp. paratuberculosis. The aim of this study was to characterise the development of gamma interferon (IFN-γ) and antibody responses as well as the occurrence of faecal shedding. Faecal culture appeared surprisingly sensitive as about 18% and 40% of the goats were positive at 9 and 15-17 months of age, respectively, and shedding was often seen prior to peripheral immune responses. Peripheral IFN-γ responses were not related to protection as clinical and high shedding goats often had high responses. An IFN-γ response usually preceded a humoral response. However, positive antibody titers could sometimes be seen simultaneously with, and even prior to, IFN-γ responses. In conclusion, faecal culture appeared as sensitive as IFN-γ testing. Furthermore, the antibody ELISA and the IFN-γ assay may perform equally well in an infected herd if surveillance is conducted annually.     

Prevalence of campylobacter in pigs during fattening; an epidemiological study

Summary

Numerous epidemiological reports implicate foods of animal origin as vehicles of human campylobacteriosis. Pigs are probably an important reservoir of campylobacter and a potential source of human infection. In order to improve our knowledge of the epidemiology of campylobacter in pigs, the prevalence of campylobacter and its contamination of feed were monitored in eight pig farms. Faeces samples of pigs aged 11 and 22 weeks, and samples of rectal, ileal and gastric content at a slaughterhouse were collected for bacteriological examination. On 5 farms, subsequent groups of pigs housed in the same stalls was sampled, too. A selection of the campylobacter isolates was characterized with a genetic typing method (RFLP). More than 85% of the sampled porkers were shown to be intestinal carriers of campylobacter at all stages of fattening. Subsequent groups of pigs housed in the same stalls were all carriers, too. The level of campylobacters in the faeces tended to decrease as the pigs got older. There was no difference in the frequency and level of infection with campylobacter between porkers on different farms. The feeding system (wet feed versus dry pellets) did not seem to influence the prevalence of campylobacter although wet feed gave lower counts of Enterobacteriaceae in the faeces.

RFLP‐typing showed a high diversity of campylobacter strains at each sampling on the farm. Similarities were seen between strains isolated during two subsequent samplings of the same group of pigs, but not between strains isolated on the same farm from subsequent groups of pigs housed in the same stall. This suggests that the piglets were already infected at a young age on the breeding farm.     

Evaluation of a competitive ELISA for antibody detection against avian influenza virus

Active serologic surveillance is necessary to control the spread of the avian influenza virus (AIV). In this study, we evaluated a commercially-available cELISA in terms of its ability to detect AIV antibodies in the sera of 3,358 animals from twelve species. cELISA detected antibodies against reference H1- through H15-subtype AIV strains without cross reactivity. Furthermore, the cELISA was able to detect antibodies produced following a challenge of the AIV H9N2 subtype in chickens, or following vaccination of the AIV H9 or H5 subtypes in chickens, ducks and geese. Next, we tested the sensitivity and specificity of the cELISA with sera from twelve different animal species, and compared these results with those obtained by the hemagglutination-inhibition (HI) test, the "gold standard" in AIV sera surveillance, a second commercially-available cELISA (IZS ELISA), or the agar gel precipitation (AGP) test. Compared with the HI test, the sensitivities and specificities of cELISA were 95% and 96% in chicken, 86% and 88% in duck, 97% and 100% in turkey, 100% and 87% in goose, and 91% and 97% in swine, respectively. The sensitivities and specificities of the cELISA in this study were higher than those of IZS ELISA for the duck, turkey, goose, and grey partridge sera samples. The results of AGP test against duck and turkey sera also showed significant correlation with the results of cELISA (R-value >0.9). In terms of flock sensitivity, the cELISA correlated better with the HI test than with commercially-available indirect ELISAs, with 100% flock sensitivity.