小鹅瘟病毒的分离与初步鉴定

从死亡的具有典型小鹅瘟症状的7日龄雏鹅肝中分离到1株病毒．选用未免疫小鹅瘟疫苗的健康母鹅所产的12日龄鹅胚，进行鹅胚接种试验，雏鹅表现出典型鹅瘟临床症状和剖检特征；鸡胚致死率达到25％；不能凝集多种动物红细胞；能凝集黄牛精子，经琼脂扩散试验，用此株病毒感染的鹅胚液能与小鹅瘟病毒阳性血清发生特异性反应．综上所述，这株病毒初步鉴定为小鹅瘟病毒．     

抗犬细小病毒的单克隆抗体的制备

CPV YZ株的细胞培养液经蔗糖密度梯度离心纯化后作为免疫原免疫BALB/C小鼠 ,应用杂交瘤技术筛选出 8株能稳定分泌单克隆抗体的杂交瘤细胞 ,分别命名为F5G10、3F11F4、F11G6、6C5C2、6E4G3、6C2B11、F11D6、3D9C4株 ,各株单抗均为IgG型 ;中和试验表明 :F11G6、3D9C4、6E4G3、6C5B114株单抗有中和作用 ,Dot ELISA试验证明了 8株单抗仅针对病毒抗原决定簇的空间结构。     

表达猪细小病毒VP2蛋白的重组干酪乳杆菌的构建

利用Oligo设计合成一对引物P1、P2,分别含有BamHI和XhoI酶切位点,以猪细小病毒株LJL12的DNA为模板,采用PCR技术扩增VP2基因,克隆到pMD18-TSimple载体,并进行酶切、PCR鉴定和序列测定。将VP2基因分别亚克隆到干酪乳杆菌细胞表面表达型载体pPG1和分泌型表达载体pPG2的BamHI和XhoI酶切位点,电转化干酪乳杆菌Lactobacillus casei393,获得阳性重组菌株。成功构建了猪细小病毒VP2蛋白的干酪乳杆菌表达系统,命名为pPG1-VP2/L.casei393和pPG2-VP2/L.casei393。     

鹅细小病毒四平分离株VP3基因的克隆与序列分析

根据已发表的鹅细小病毒(GPV)核苷酸序列,设计并合成了1对引物,对吉林农业大学预防兽医学研究室分离鉴定的GPV四平株(SP)主要保护性抗原蛋白VP3基因进行扩增。所得到的PCR产物片段大小约1.6 kb,与预期片段大小相符。将该片段纯化后与T载体连接,转化到感受态大肠杆菌JM-109中进行克隆。对所提质粒进行快速鉴定、PCR鉴定以及限制性内切酶NheⅠ和BamHⅠ双酶切鉴定,筛选阳性重组质粒,对重组质粒测序并进行核苷酸序列分析。结果表明:GPV四平株VP3基因长1 605 nt,包含了完整编码GPV VP3蛋白阅读框,且与国内GPV分离株B,GD,HG5/82相应核苷酸序列和氨基酸序列的同源性较高,分别为96.2%,96.3%,96.4%和97.4%,98.7%,98.9%。据此认为,GPV四平株VP3基因可以作为构建具有普遍应用价值的基因工程疫苗的候选基因。     

抗CPV-2a单克隆抗体的制备及双抗体夹心ELISA检测方法的建立

犬细小病毒病是危害养犬业的重要传染病之一,患病犬难以治愈。单克隆抗体治疗此病效果明显,本文介绍了制备抗CPV-2a单克隆抗体的方法。用纯化的犬细小病毒（canine parvovirus,CPV）2a型分离株免疫新西兰大白兔和Balb/c小鼠制备抗CPV-2a多克隆抗体及单克隆抗体。经亚克隆得到1H9、2B5、2B7和2C7共4株单抗,Western blotting鉴定单抗的免疫反应性;间接ELISA方法检测单抗的特异性。为了快速对犬细小病毒病作出诊断,建立了CPV-2a双抗夹心ELISA方法。兔多抗作为捕获抗体,鼠单抗作为示踪抗体,辣根过氧化物酶标记羊抗鼠IgG作为检测系统;捕获抗体和示踪抗体最佳稀释度分别为1：800和1：2000;检测系统最佳稀释度为1：4000。结果表明：所得4株单抗与pET-32a-VP2蛋白发生特异性反应,且与狂犬病毒（RV）、犬温热病毒（CDV）不交叉反应;建立的双抗夹心ELISA方法对病毒的最低检出量为4.375μg/mL,与美国RB试剂盒相比,符合率为95%。单抗制备为犬细小病毒病的治疗奠定了基础;双抗夹心ELISA方法的建立为疑似粪便样本提供了简单、快速和可靠的检测手段。     

Intracranial meningioma causing partial amaurosis in a cat

Objective To describe a case of intracranial meningioma causing visual impairment in a cat, successfully treated by surgery. Procedures An adult neutered male domestic cat was referred with a 10‐month history of progressive visual impairment and altered behavior. Investigations included physical, ophthalmologic and neurological examinations as well as hematology, serum biochemistry and CT scan of the head. Results The menace response was absent in the left eye and decreased in the right eye. Electroretinograms were normal on both eyes, as was ophthalmic examination, ruling out an ocular cause and allowing a presumptive diagnosis of partial amaurosis due to a post‐retinal lesion. CT scan demonstrated a large sessile extra axial mass along the right parietal bone and thickening of the adjacent bone. Cerebrospinal fluid was not collected because high intracranial pressure represented a risk for brain herniation. A right rostrotentorial craniectomy was performed to remove the tumor. Ten days after surgery, vision was improved, neurological examination was normal and normal behavior was restored. Ten months after surgery, ophthalmological examination showed no visual deficit and CT scan did not reveal any sign of recurrence. Conclusion Advanced imaging techniques allow veterinarians to detect early cerebral diseases and to provide specific treatment when it is possible. In cases of feline amaurosis due to intracranial meningioma, the vital prognosis is good while the visual prognosis is more uncertain, but recovery of normal vision and normal behavior is possible as demonstrated in the present case.     

Tear-film osmolarity in normal cats and cats with conjunctivitis

Objective To compare the tear‐film osmolarity of normal cats and cats with conjunctivitis. Animal studied The population consisted of shelter, research, and privately owned cats. Procedures Cats were classified as normal or having conjunctivitis. An ophthalmic examination including Schirmer tear test (STT), fluorescein staining, tear‐film break‐up time (TFBUT), intraocular pressure (IOP), and slit‐lamp biomicroscopy of the anterior segment was performed. The severity of conjunctivitis was graded and assigned a numerical score. The Tear Lab^TM Osmolarity System was utilized to determine the tear‐film osmolarity. Unpaired t‐tests were used to compare tear‐film osmolarity, TFBUT, IOP, and STT of the two groups. Results A total of 93 cats (186 eyes) were examined. There were 37 normal cats (74 eyes) and 39 conjunctivitis cats (78 eyes). The mean age was 2.34 years. There was no statistical difference (P = 0.2065) between the median tear‐film osmolarity of normal cats (328.5 ± 17.94 mOsms/L) and conjunctivitis cats (325.0 ± 24.84 mOsms/L). Cats with conjunctivitis had an accelerated TFBUT (P < 0.0001) and lower IOPs (P < 0.0001) as compared to normal cats. No statistical difference was found between STT values (P = 0.1304). Conclusions The median tear‐film osmolarity of normal cats was 328.5 mOsms/L. Despite the accelerated TFBUT, conjunctivitis did not cause a statistically significant change in tear‐film osmolarity. The Tear Lab^TM Osmolarity System was easily used and well tolerated by the cats in the study.     

Comparison of the pig and feline models for full thickness corneal transplantation

Purpose The goal of this study was to report on the advantages and limitations of the pig and feline models for experimental in vivo corneal transplantation. Methods Ten healthy domestic pigs and ten healthy cats were used. Full thickness penetrating keratoplasty was performed using autologous (eight cases), allogeneic (seven cases) or human xenogeneic (three cases) tissue. In two other cases, the inflammatory response to partial thickness trephination (without transplantation) was evaluated. Eyes were assessed daily before and after surgery by slit‐lamp, pachymetry, and tonometry. A transparency score ranging from 0 (opaque graft) to 4 (clear graft) was used, based on the slit‐lamp examination. Optical coherence tomography, histology, and electron microscopy were performed postmortem. Results In the pig, the mean (±SD) transparency score for the eight full thickness grafts was 0.88 ± 0.99, ranging from 0 to 3. In the feline model, the mean transparency score for the seven uncomplicated grafts was 3.93 ± 0.19, ranging from 3.5 to 4. Both negative controls without endothelium remained opaque at all time. Intraoperative tendency for iris incarceration into the wound, rapid corneal swelling, suture cheese wiring, and postoperative intraocular inflammation were the main factors jeopardizing the functional success of the corneal transplant in the pig model. Conclusion Suboptimal functional results were obtained after full thickness corneal transplantation in the pig model, while in the feline model, the same protocol yielded uneventful surgeries and clear transplants, with functional results similar to those achieved in human subjects.     

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.     

Porphyrins are not present in feline ocular tissues or corneal sequestra

Objective To determine if feline lacrimal glands, glands of the third eyelid, corneas, and corneal sequestra contain porphyrins, which could be responsible for the brown/amber discoloration of corneal sequestra and tears in affected cats. Procedures Samples of grossly normal cornea, lacrimal gland, gland of the third eyelid, and sequestra obtained via keratectomy were collected. Porphyrin concentrations of the homogenate were determined by spectrofluorometry with protoporphyrin IX and coproporphyrin III dihydrochloride used as standards. A hamster harderian gland was used as a positive control. Results Normal tissues were harvested from one eye each of 14 nonclient owned, adult, mixed‐breed, short‐hair cats euthanized for reasons not associated with this study. Eighteen sequestra were acquired from cats undergoing unilateral lamellar keratectomies. Breeds of the affected cats included eight Himalayan, five domestic shorthair, and one each of four other breeds. Only the positive control and standards contained levels of porphyrins above background. All feline samples examined were histologically normal with no evidence of porphyrins. Conclusions Porphyrins are absent in normal feline lacrimal glands, corneas, and corneal sequestra. Porphyrins do not appear to be the cause of the brown/amber color of feline corneal sequestra.