异硫氰酸荧光素标记鳗弧菌条件优化及应用条件对荧光信号的影响

为了实现对目标细菌的有效示踪,实验以异硫氰酸荧光素(FITC)为标记物,分别设置FITC标记浓度0.2、2、20、50、100、200μg/mL,标记时间1、15、30、60、120、240 min,研究了FITC标记浓度与标记时间对鳗弧菌标记效果的影响,同时对在不同温度(25、4、-20、-70℃)及不同时间(24、48、96、192 h)存储条件下鳗弧菌标记荧光的稳定性进行了比较,对标记鳗弧菌经甲醛灭活后的荧光变化进行了观察,研究了不同光照条件(0、150、1 500、25 000 lx)对标记鳗弧菌的荧光衰减影响,以及在-20和-70℃条件下反复冻融对标记鳗弧菌荧光强度的影响。结果表明,FITC的最适标记浓度范围为20~100μg/mL,最适标记时间范围60~120 min,储藏在-20和-70℃条件下,标记后的鳗弧菌荧光信号比储藏在4和25℃条件下稳定,标记后的鳗弧菌经甲醛灭活后的荧光信号稳定性优于对照组,不同强度自然光照对标记鳗弧菌的荧光衰减效果不明显,标记细菌经3次冻融,发现在-70℃条件下冻存的细菌荧光信号稳定性保持良好。本研究结果为FITC用于标记海洋细菌提供了实用的技术参数。     

Intraocular melanoma in an alpaca

A case of primary intraocular melanoma in an alpaca is reported. This patient also presented with a mild normocytic, normochromic anemia and hypoproteinemia. These can be markers of an immunodeficiency syndrome associated with llamas. Although this patient did not appear to have clinical signs of this syndrome, its presence needed to be ruled out as animals affected with this syndrome are subject to recurrent severe debilitating diseases and death. To our knowledge, intraocular melanoma has not been previously reported in an alpaca.     

Seed viability determination in parasitic broomrapes (Orobanche and Phelipanche) using fluorescein diacetate staining

Fluorescein diacetate (FDA) was investigated as a potential viability stain for seeds of holoparasitic broomrapes Orobanche and Phelipanche (Orobanchaceae), using four weedy and two non-weedy taxa. FDA viabilities were compared with the currently used 2,3,5-triphenyl tetrazolium chloride (TTC) test, and in vitro germination induced by the strigolactone analogue GR-24. Viable FDA-stained seeds were characterised by bright yellow–green fluorescence under 450–490 nm blue light. These viable seeds could be discriminated from non-viable seeds, in which low levels of fluorescence, similar to non-stained control seeds were observed. FDA viabilities were not significantly different from those obtained using the TTC stain; however, viability values for both stains were consistently higher than levels of artificially induced germination. Positive TTC-staining showed continuous variation that was difficult to interpret. Nevertheless, the TTC test does not involve grinding seeds, and therefore probably remains a practical alternative to FDA for screening soil samples contaminated with Orobanche and Phelipanche seeds, which indicates different applications for the two viability tests. Interestingly, the strigolactone analogue GR-24 only induced germination in O. crenata , P. ramosa and O. minor , suggesting a high specificity towards this germination inducer among the species investigated. We suggest that FDA-staining provides a potential alternative to the currently used TTC test for seed viability assays for Orobanche and Phelipanche , and other parasitic weeds which are an obstacle to crop cultivation.     

The in vitro effects of proxymetacaine, fluorescein, and fusidic acid on real-time PCR assays used for the diagnosis of Feline herpesvirus 1 and Chlamydophila felis infections

Purpose To investigate the possible inhibition of qPCR assays used for the diagnosis of ocular infections in cats by proxymetacaine, fluorescein, and fusidic acid, which are commonly used in veterinary ophthalmology. Methods Fluorescein, proxymetacaine, and fusidic acid were tested for possible inhibition of a triplex qPCR assay designed to detect Chamydophila felis, Feline herpesvirus 1 (FHV‐1), and the feline 28S ribosomal DNA (28S rDNA) gene by comparing threshold cycle (C_t) values of samples with and without the three products. A second experiment was carried out to measure the effects of various dilutions of fusidic acid. Results No statistically significant differences were detected between the C. felis, FHV‐1, and 28S rDNA C_t values with and without proxymetacaine or fluorescein. However, there was a statistically significant increase in FHV‐1 (P < 0.01), C. felis (P < 0.01), and 28S rDNA (P < 0.05) C_t values when fusidic acid was used. When dilutions of fusidic acid were tested, the results revealed that only the 1:2 dilution caused a statistically significant increase (P < 0.01) in the FHV‐1 Ct values. Conclusion Proxymetacaine and fluorescein did not interfere with our qPCR assays for the detection of C. felis and FHV‐1. The presence of fusidic acid caused a small inhibitory effect of doubtful clinical significance. In vivo studies are required to establish the clinical relevance of this study and to confirm our findings.     

In vitro effect of recombinant human tumor necrosis factor-alpha on canine neutrophil apoptosis

Neutrophils (polymorphonuclear leukocytes: PMNs) are essential for the host defense against various infections and are often injurious to the host, causing inflammatory diseases where tumor necrosis factor-alpha (TNF-alpha) is suggested to play an important role. Since an effect of TNF-alpha on canine PMN apoptosis has not been studied, canine PMNs were stimulated with recombinant human (rh)TNF-alpha in the present study to investigate the effect of TNF-alpha on canine PMN apoptosis. PMN apoptosis and function to produce ROS were assessed by flow cytometry. Delayed apoptosis was observed in the PMNs treated with rhTNF-alpha at 100 ng/ml, accompanied by retention of capability to produce ROS. However, PMN apoptosis was accelerated by rhTNF-alpha combined with cycloheximide. Therefore, it is indicated that TNF-alpha is able to activate anti- and pro-apoptotic pathways in PMNs and that the inhibition of PMN apoptosis by TNF-alpha requires protein synthesis in the PMNs.     

Comparative fluorescein angiography of the normal sheep and goat ocular fundi

Fluorescein angiography without sedative or anesthetic agents was evaluated in 20 normal goats and 20 normal sheep. All of the angiographic phases were observed using 20 mg/kg fluorescein IV in both species. Fundus fluorescein angiography results revealed wide stars of Winslow in the tapetal fundus, central or marginal flow during the first part of the arterial phase, delayed filling of the focal areas in the choroid near the optic disc that often coincided with others in the disc, and lack of evidence of the 'striate area' in the tapetal fundi. In goats, the angiographic times were 6.54+/-1.25 s for the arterial phase (TA), 7.80+/-1.37 s for the arterio-venous phase (TAV), and 14.13+/-2.01 s for the venous phase (TV). I1: 1.30+/-0.30 s (time elapsing between TA and TAV), and I2: 6.20+/-1.60 s (time elapsing between TAV and TV). In sheep, times were 9.54+/-2.18 s TA, 11.73+/-2.10 s TAV, and 20.86+/-2.74 s TV. I1: 2.04+/-0.75 s and I2: 8.98+/-2.47 s, respectively. Due to the large size of the fundic vessels in sheep and goats, fluorescein angiography of the retinal vasculature can facilitate the study of the different vascular diseases in these species.     

Fluorangiographic study of the ocular fundus in normal horses

Purpose:  To describe the protocol and appearance of fluorescein angiography (FA) in normal horses.
Animals:  A total of 25 healthy horses aged between 5 and 15 years.
Materials and Methods:  The horses were sedated with 15 µg/kg detomidine and 50 µg/kg butorphanol and dilated with topical tropicamide 1%. All angiograms were recorded after intravenous bolus injection of 10 mg/kg of fluorescein sodium solution.
Results:  Two successive angiographic phases could be discerned: the choriopapillary phase, starting at 46.95 ± 9.48 s, and the retinal vascular phase, starting at 47.79 ± 10.38 s. The retinal vascular phase was divided in three parts: filling phase , maximum fluorescence point , and fading phase . During the filling phase, the dye progressed into the retinal vessels, obtaining maximum fluorescence at 59.79 ± 10.39 s, termed the maximum fluorescence point. The fading phase started immediately following the maximum fluorescence point. During this phase, vascular fluorescence decreased to complete reduction at 74.76 ± 9.81 s. Also, areas of delayed choroidal filling, the presence of short retinal vessels in the ventral region of the optic disc, and a particular filling of the optic disc were also observed.
Conclusions:  The normal angiographic sequence was described in horses. FA may be a useful method for studying the integrity of the blood–retinal barriers in horses.     

原发性开角型青光眼患者眼底荧光血管造影及血液流变学改变与中医证型关系的研究

目的 探讨原发性开角型青光眼的发病机理。方法 将85例原发性开角型青光眼患者分为高眼压型组、正常眼压型青光眼组,并与24例正常人进行对照,对其眼底荧光血管造影和血液流变学指标的改变及与中医辨证分型的关系进行研究。结果 原发性开角型青光眼高眼压型患者和正常眼压型青光眼患者与正常组相比,高（VH）、中（VM）、低（VI）切变率下全血表观粘度值、红细胞压积升高,差异均有显著统计学意义（P<0.01）。原发性开角型青光眼中医辨证分型各组与正常组比较,均表现为红细胞压积升高（P<0.01）。其中肝郁气滞证和肝肾亏虚证组分别与正常组相比,高（VH）、中（VM）、低（VI）切变率下全血表观粘度值、红细胞压积升高;痰湿泛目证组与正常组相比,红细胞压积比值差异有统计学意义（P<0.05）。原发性开角型青光眼高眼压型患者和正常眼压型青光眼患者与正常组相比,眼底荧光血管造影中臂-脉络膜充盈时间（A-CT）、臂-视网膜动脉充盈时间（A-AT）、视网膜动-静脉充盈时间（A-VT）延长差异均有统计学意义（P<0.01）。原发性开角型青光眼中医辨证分型各组与正常组比较,均表现为A-CT、A-AT、A-VT延长。其中肝郁气滞证、肝肾亏虚证组与痰湿泛目证组与正常组相比差异有统计学意义（P<0.05）。结论 原发性开角型青光眼高眼压型患者和正常眼压型青光眼患者均存在明显的血液呈现高凝状态的血瘀病理改变,而正常对照组的血瘀改变不明显。在中医证型中,这种血瘀病理以肝郁气滞证最明显,肝肾亏虚证次之,痰湿泛目证最轻,呈现肝郁气滞证 >肝肾亏虚证 >痰湿泛目证的趋势。     

荧光标记技术在增殖放流中的应用现状

正标志技术是用来区分不同个体或种群的常用方法。自Petersen等[1]利用标志方法估算封闭水体中鱼类的利用率及种群大小后,标志技术在渔业和水产养殖研究中得到广泛的发展和应用,标记技术也不断更新。荧光标记技术是渔业和水产养殖研究中常用标志技术手段之一,其通过特定技术方法在标志对象上形成荧光标记进而通过肉眼或借     

Functional and structural assessment of retinal sheet allograft transplantation in feline hereditary retinal degeneration

Purpose  To investigate whether sheets of fetal retinal allografts can integrate into the dystrophic Abyssinian cat retina with progressive rod cone degeneration.
Methods  Fetal retinal sheets (cat gestational day 42), incubated with BDNF microspheres, were transplanted to the subretinal space of four cats at an early disease stage. Cats were studied by fundus examinations, bilateral full-field flash ERGs, and indocyanine green and fluorescein angiograms up to 4 months following surgery. E42 donor and transplanted eyes were analyzed by histology and immunohistochemistry for retinal markers.
Results  Funduscopy and angiography showed good integration of the transplants in two of four cats, including extension of host blood vessels into the transplant and some scarring in the host. In these two, transplants were found in the subretinal space with laminated areas, with photoreceptor outer segments in normal contacts with the host retinal pigment epithelium. In some areas, transplants appeared to be well-integrated within the host neural retina. Neither of these two cats showed functional improvement in ERGs. In the other two cats, only remnants of donor tissue were left. Transplants stained for all investigated cellular markers. No PKC immunoreactivity was detected in the fetal donor retina at E42, but developed in the 4-month-old grafts.
Conclusions  Fetal sheet transplants can integrate well within a degenerating cat retina and develop good lamination of photoreceptors. Functional improvement was not demonstrated by ERG in cats with well-laminated grafts. Transplants need to be further evaluated in cat host retinas with a more advanced retinal degeneration using longer follow-up times.