Expression of Occludin in Canine Testis and Epididymis

Tight junctions (TJ) in inter-Sertoli junctional areas and epididymal epithelia are important for the formation of blood-testis barrier (BTB) and blood-epididymal barrier (BEB). In this study, the expression of occludin, an integral member of the TJ, was verified in canine testis and epididymis. Both low molecular weight (MW) (25-28 kDa) forms as well as high MW (68-72 kDa) forms of occludin were detected in the testis and epididymis using Western blot. The relative amount of the high MW forms of occludin vs low MW forms was higher in the testis than in the epididymis. Some difference in the composition of different MW forms of occludin was found along the segments of epididymis, suggesting the possible correlation between cellular composition of occludin proteins and paracellular permeability of epithelia along the epididymal tubule. In the testis, intense occludin immunoreactivity was found in the basally located inter-Sertoli junctional area. Diffused immunoreactivity of occludin was also found in the cytoplasm of Sertoli cells. A similar pattern of zonula occludens-1 immunoreactivity was found in the cytoplasm of Sertoli cells, suggesting that occludin was not confined to the inter-Sertoli junctional areas and that subcellular localization of occludin in the Sertoli cells was dynamically regulated during spermatogenesis in canine testis. In the epididymis weak immunoreactivity was found in the apical sides and cytoplasm of epithelial cells.     

The Ductus Epididymis of the Alpaca: Immunohistochemical and Lectin Histochemical Study

Our objective was to characterize epithelial cells lining the epididymal duct (caput, corpus, cauda) of the alpaca using AE1/AE3 cytokeratin antibodies and a battery of different lectins: Con-A, UEA-I, LTA WGA, GSA-II, GSA-IB4, SBA, PNA, ECA, DBA, MAL-II and SNA. Sialidase digestion and deglycosylation pre-treatments were also employed. The principal cells (PCs) along the epididymis showed differences in immunostaining patterns toward keratin antibodies. Lectin histochemistry demonstrated variations in the content and distribution of glycosidic residues of glycoconjugates in different epididymal regions. In particular, staining of the Golgi zone in the epithelial PCs was interpreted as evidence for synthesis and secretion of O- and N-linked oligosaccharides. In the caput, the apical mitochondria-rich cells contained mainly β-GalNAc, subterminal α-GalNAc, α-Gal and Neu5Ac α2,3Gal residues. Conversely, in the corpus they were particularly rich in α-GalNac and β-Gal-(1–3)- d -GalNAc linked to sialic acid moieties. Basal cells mainly expressed β-GalNAc and α-Gal in the caput, α-Gal in the corpus and α-Fuc and β-GalNAc in the cauda. The differences in immunostaining patterns and in lectin histochemistry in the alpaca epididymis reported in this investigation seem to be related to regional differences in function.     

Histochemical Identification of the Striated Muscle of the Canine Esophagus

Histochemistry on the cervical, thoracic, and abdominal esophageal muscle of immature, young, and adult normal dogs revealed type IIA striated musculature in contrast to some other species. No other types or subtypes were observed. This suggests that esophageal muscle type is established at birth and does not vary or that any variation has been completed by 4 weeks-of-age unlike some canine limb musculature.     

Histochemical Analysis of Glycoconjugates in the Skin of a Catfish (Arius Tenuispinis, Day)

A histochemical study using conventional carbohydrate histochemistry (periodic‐acid staining including diastase controls, alcian blue staining at pH 1 and 2.5) as well as using a battery of 14 fluorescein isothiocyanate (FITC)‐labelled lectins to identify glycoconjugates present in 10 different areas of the skin of a catfish (Arius tenuispinis) was carried out. The lectins used were: mannose‐binding lectins (Con A, LCA and PSA), galactose‐binding lectins (PNA, RCA), N‐acetylgalactosamine‐binding lectins (DBA, SBA, SJA and GSL I), N‐acetylglucosamine‐binding lectins (WGA and WGAs), fucose‐binding lectins (UEA) and lectins which bind to complex carbohydrate configurations (PHA E, PHA L). Conventional glycoconjugate staining (PAS staining, alcian blue at pH 1 and 2.5) showed that the mucous goblet cells contain a considerable amount of glycoconjugates in all locations of the skin, whereas the other unicellular gland type, the club cells, lacked these glycoconjugates. The glycoproteins found in goblet cells are neutral and therefore stain magenta when subjected to PAS staining. Alcian blue staining indicating acid glycoproteins was distinctly positive at pH 1, but gave only a comparable staining at pH 2.5. The mucus of the goblet cells therefore also contains acid glycoproteins rich in sulphate groups. Using FITC‐labelled lectins, the carbohydrate composition of the glycoproteins of goblet cells could be more fully characterized. A distinct staining of the mucus of goblet cells was found with the mannose‐binding lectins LCA and PSA; the galactosamine‐binding lectins DBA, SBA and GLS I; the glucosamine‐binding lectin WGA; and PHA E which stains glycoproteins with complex carbohydrate configurations. No reaction occurred with the fucose‐binding lectin UEA and the sialic acid‐specific lectin SNA. In addition, the galactose‐binding lectins PNA and RCA showed only a weak or completely negative staining of the mucus in the goblet cells. The specificity of the lectin staining could be proved by inhibiting binding of the lectins by competitive inhibition with the corresponding sugars. From these data, we can conclude that the mucus produced by the epidermal goblet cells of A. tenuispinis is rich in mannose, N‐acetylgalactosamine and N‐acetylglucosamine residues.     

犬场蚊子与犬细小病毒

２０００年４月，应用套式ＰＣＲ技术从犬场蚊子的血液样品检测出犬细小病毒。将分离到的犬细小病毒ＶＰ２－Ｙ３４基因片段克隆到ＰＭＤ１８－Ｔ载全，其病毒基因组ＣＰＶ－ＰＶ２－Ｙ３４序列测定结果显示与作者在ＧＥＮＥＢＡＮＫ发表的肺病变犬细小病毒ＣＰＶ－ＨＮ－１株有９９％同源性。     

Detection of Steroid Hormone Receptors in the Canine Circumanal Gland

The oestrogen receptor beta (ERß) is largely distributed in the ovary of many species but data for the bovine ovary are scare. Therefore, the expression of ERß mRNA in the different follicles of the bovine ovary was studied using in situ hybridization. Ovarian tissue sections of three cows with different plasma progesterone concentrations were used (cow 1: 3.50 ng/ml; cow 2: 1.00 ng/ml, cow 3: 0.35 ng/ml). A 602 bp fragment of ERß mRNA was cloned, sequenced and digoxigenin (DIG)-labelled. Subsequently, in situ hybridization was performed by incubating the sections with the DIG-labelled RNA anti-sense probe. For the semi-quantitative evaluation of ERß mRNA expression the ERß mRNA score (SER) was determined for the different follicular cell types using the formula: SER = 0.n0 + 1.n1+ 2.n2 + 3.n3 with n0, n1, n2, n3 indicating the percentage of cells exhibiting a staining intensity 0 (absent), 1 (weak), 2 (moderate) or 3 (strong), respectively. High ER mRNA levels were noticed in primordial and primary follicle cells, and suggest a role of ER mRNA in early folliculogenesis. A lower SER was observed in the granulosa cells of secondary and tertiary follicles. This significant difference in the SER of follicle cells during follicular growth may be associated with cell proliferation. In obliterative and cystic atretic follicles high SER were observed, although ERß mRNA levels in obliterative follicles showed much inter-individual variation. This is suggestive for ERß mediated oestrogen action in atretic follicles. In the corpora lutea moderate ERß mRNA levels were noticed. Our findings are in accordance with studies in the ewe in which corpora lutea cells synthesize estrogen. These preliminary findings will be further elaborated in a higher number of cows to examine the role of ERß in the ovary throughout the oestrus cycle.     

Colour-coded and Pulsed Doppler Sonography of the Canine Testis, Epididymis and Prostate Gland: Physiological and Pathological Findings

Two-dimensional ultrasound was used in combination with colour-coded and pulsed Doppler sonography to study the blood flow of the testes and prostate gland in a total of 30 male dogs. After detection of the vessels by colour-coded Doppler sonography, the blood flow patterns were determined by pulsed Doppler sonography measuring and describing the systolic and diastolic peak velocity (SPV, DPV), the end-diastolic velocity, the time-averaged maximum velocity (TAMAX), the pulsatility and resistance index, as well as the ratios of the systolic peak velocity and end-diastolic velocity and of the systolic and diastolic peak velocities. The blood flow of the testicular artery was measured within the pampiniform plexus and the marginal location. The prostatic blood supply was measured in the artery of the deferential duct (cranial), the prostatic artery outside (lateral) and within the gland (subcapsular). The physiological testicular flow pattern was monophasic with a high diastolic flow. Testes with neoplastic alterations showed a significant increase of SPV and TAMAX. The epididymal vessels could not be detected. Under physiological conditions the prostatic blood flow pattern was biphasic in the cranial and lateral location and monophasic in the subcapsular location. Benign prostatic hyperplasia was characterized by a significant increase of SPV, DPV and TAMAX. The results of the present investigation demonstrate that the colour-coded and pulsed Doppler sonography give additional valuable information which improves the andrological diagnostics in the dog.     

犬巴贝斯虫病检测研究

犬巴贝斯虫病是经蜱传播的血液寄生虫病,其临床症状是高热、贫血、血红蛋白尿和脾肿大。通过血涂片检验,血常规检验并结合临床特征,对西安市103只宠物犬进行了巴贝斯虫的感染情况检测。结果显示,犬血液中巴贝斯虫的感染数为16例,占总数的15.53%,其中5月份的感染犬最多,为13例,占感染数的81.25%(13/16)。     

核酸水平检测犬副流感病毒方法的建立

根据GenBank中与犬副流感(CPIV)同源性较近的SV5病毒N基因保守序列,利用DNAStar软件设计了一对特异性引物,能扩增265bp大小的片段,并以此建立了检测CPIV的RT-PCR方法,实验证明该对引物特异扩增CPIV;不扩增犬瘟热病毒、犬细小病毒、犬腺病毒和狂犬病病毒犬的四种病原的核酸。检测临床病料20份,其中2分为CPIV阳性。此法敏感性较高。是检测犬急性传染性呼吸道疾病(CIRD)中CPIV的有效的方法。     

Detection and Sequence Analysis of Canine Circovirus in Chongqing

The aim of this study was to investigate the prevalence of canine circovirus (CCV) in Chongqing in recent years,and to explore the characteristics of the field strains in Chongqing.In this study,100 dog serum samples collected in Chongqing in 2017 were tested for CCV nucleic acid by PCR,and the obtained CCV positive samples were amplified and sequenced with the full length genome,and the Chongqing CCV strain isolated was analyzed by MegAlign,Mega 6.0 and RDP4 software.Four positive sample were found,with a positive rate of 4%,and three full length CCV genomes (CQ76,CQ79,and CQ82) were obtained,with a total length of 2 062 nt.Compared with the previously reported genome of CCV with a length of 2 063 nt,one base was missing,which was located downstream of the stem ring structure in the 5'-intergenic region.The length of the 5'-intergenic region and 3'-intergenic region between the two ORFs of the three strains were 134 (1 929-2 062 nt) and 203 nt (913-1 115 nt),respectively.The homology of CQ76,CQ79,and CQ82 ranged from 99.8% to 100%,among which,CQ76 and CQ82 only had synonymous mutations at the 2 019 locus.The homology of genome sequences of the three Chongqing strains and other strains was 82.7% to 97.1%,among which,the degree of variation of ORF2 was greater than that of ORF1.Based on virus ORF2 sequences and genome-wide respectively build Neighbor-Joining in the evolutionary tree,CQ76,CQ79 and CQ82 all belonged to the same subgroup,which was close to the Chinese strain 204,which belonged to genotype Ⅱ,and the homology was 96.5% to 96.7%.RDP4 recombinant analysis indicated that the genomes of CQ76,CQ79 and CQ82 strains were all the recombinant sequences of 204 and 388 strains from Guangxi,and the recombinant region was located in ORF2.Above results enriched the epidemiological and genetic evolution information of CCV and provided further basic data for prevention and control research.     

应用电镜技术和血凝试验检测犬细小病毒和犬腺病毒

电镜技术和血凝试验是检测犬细小病毒和犬腺病毒快速而简便的方法。本试验结果表明,两种方法检验结果大部分相符,但各具特色。电镜技术的优势在于能够直观地显示目标病毒的生长发育、粒子的完整性以及有无非目标病毒、细菌、支原体等污染情况,这对于动物病毒疫苗种毒质量的检测是十分重要的。     

重庆地区犬圆环病毒检测及序列分析

本试验旨在调查近年来新发的犬圆环病毒（canine circovirus,CCV）在重庆地区的流行情况,探索重庆流行毒株的特性。本研究利用PCR方法对2017年采集于重庆地区的100份犬血清样品进行CCV核酸检测,对所得CCV阳性样品进行全基因组扩增与测序,并利用MegAlign、Mega 6.0和RDP4等软件对分离到的重庆CCV毒株进行分析。结果显示,重庆地区100份犬血清中共有4份为CCV阳性,阳性率为4%,从4份阳性样品中共获得3株不同的CCV基因组（CQ76、CQ79和CQ82）,全长基因组序列均为2 062 nt,与此前报道长度为2 063 nt的CCV基因组相比缺少了一个碱基,该碱基位于5'-基因间隔区内茎环结构的下游。3个毒株的两个ORF之间5'末端间隔区和3'末端间隔区长度分别为134（1 929-2 062 nt）和203 nt（913-1 115 nt）。CQ76、CQ79和CQ82的同源性在99.8%~100%之间,其中CQ76与CQ82仅在第2 019位点存在同义突变;所获得的3个重庆毒株与国内外报道的其他CCV基因组序列同源性为82.7%~97.1%,其中,ORF2的变异程度大于ORF1。基于病毒ORF2序列和全基因组分别构建NJ进化树中,CQ76、CQ79和CQ82均属于同一亚群,与属于基因Ⅱ型的中国毒株204株遗传距离较近,同源性为96.5%~96.7%。RDP4重组分析发现,CQ76、CQ79和CQ82毒株基因组均为广西毒株204株和388株的重组序列,重组区域坐落于ORF2。本研究为丰富CCV流行病学和遗传进化信息,以及进一步防控研究提供基础数据。     

ELISA法检测犬腹泻粪样中的犬冠状病毒

用ＦＥ细胞增殖犬冠状病毒（ＣＣＶ）参考株，分别免疫家兔和ＢＡＬＢ／ｃ小鼠制备ＣＣＶ多抗和单抗，建立了夹心ＥＬＩＳＡ及Ｄｏｔ－ＥＬＩＳＡ诊断方法。在检测的８４例犬腹泻粪样中，多抗、单抗夹心法显示ＣＣＶ阳性１６例，Ｄｏｔ－ＥＬＩＳＡ阳性１３例，后１３例包括在前１６例中。从８４例腹泻犬粪样中随机取３８例作ＣＣＶ、犬细小病毒（ＣＰＶ）双项检测，ＣＣＶ阳性１６例，ＣＰＶ阳性６例，ＣＣＶ、ＣＰＶ混合感染４例。结果显示，在南京地区流行的犬腹泻中，ＣＣＶ感染比例有超过ＣＰＶ的趋势。     

免疫金电镜技术检测犬细小病毒免疫球蛋白

本文应用免疫金电镜技术检测犬细小病毒免疫球蛋白（Ｉｇ）。检测结果证明驴抗犬细小病毒所产生的Ｉｇ对犬细小病毒具有较强的特异性反应。因此认为,第四军医大学动物保健品研制中心所制备的免疫球蛋白对犬细小病毒性肠炎和心肌炎具有良好的治疗和预防作用。     

应用电镜技术对犬腺病毒及其免疫球蛋白的检测

应用电镜技术及免疫金电镜技术检测犬腺病毒及驴抗犬腺病毒免疫球蛋白（Ｉｇ）。实验结果证明了由驴所产生的Ｉｇ对犬腺病毒具有较强的特异性反应,对犬肝炎、犬喉气管炎等疫病具有良好的治疗和预防作用。     

检测犬冠状病毒中和抗体的方法与应用

在２４孔组织培养板上应用从美国引进的犬冠状病毒（ＣＣＶ）参考株ＮＬ１８与猫肾传代细胞（ＣＲＦＫ）,采用固定病毒（１００ＴＣＩＤ５０）稀释血清法建立了ＣＣＶ中和试验。运用该方法测定了１４２条群养犬和３５条散养犬的中和抗体水平,以１∶４作为阳性血清的判定标准,群养犬与散养犬的血清阳性率分别为１００％和８２９％;测定了母犬的血清与乳汁、所产仔犬及仔犬脐带血的ＣＣＶ抗体效价,证实母犬可以通过胎盘及乳汁将抗体转移给仔犬,吮食乳汁是仔犬获得母源抗体的主要途径。本试验从抗体水平上证实我国的犬已发生ＣＣＶ感染。     

犬瘟热—犬冠状病毒双重PCR诊断方法的建立及初步应用

Two pairs of PCR primers were designed according to sequence of canine distemper virus （CDV） and canine coronavirus （CCV） from GenBank, respectively, which could amplify 550 bp fragment for CDV and 225 bp fragment for CCV. The products of PCR were cloned to pMD18-T for sequencing, which proved to be specific. Positive plasma were developed for standard DNA, and the sensitivity result of duplex PCR showed that the method could amplify 0.1 ng/μL nucleic acid for both of viruses. The result of rudimentary application showed that the method was specific, sensitive, efficient, and was a new detecting method for the mixed infection of CDV and CCV.     

犬圆环病毒检测和致病性的研究进展

犬圆环病毒(Canine circovirus,Canine CV)是2012年首次在美国报道的一种新型哺乳动物圆环病毒。之后,意大利、德国和中国陆续报道检测到该病毒。目前已证实Canine CV可引起犬发生坏死性血管炎和淋巴结肉芽肿,临床表现为出血性腹泻和呕吐等,但该病毒的致病机理尚不清楚。文中主要对Canine CV的检测和致病性研究进展进行综述,为Canine CV的流行病学调查和致病机理研究等提供参考。     

犬瘟热、犬细小病毒胶体金二联检测卡研制

本研究通过细胞融合、间接ELISA方法筛选出4株可稳定分泌犬瘟热病毒(canine distemper virus,CDV)和犬细小病毒(canine parvovirus,CPV)单克隆抗体的杂交瘤细胞株,以此为基础研制了一种可同时检测CDV和CPV的胶体金二联检测卡,并对其特异性、灵敏度及准确性进行了测试。结果显示：4株杂交瘤细胞可稳定传代,分泌的单克隆抗体纯度高,效价均在1:320以上。制备的胶体金二联检测卡特异性强,只与CDV和CPV产生特异性条带,而不与其他犬类病毒反应;灵敏度高,最低检测限可达到10²拷贝/μL;准确性好,与荧光定量PCR结果的符合率高于88.0%,与市面上单一病毒检测卡的符合率高于97.2%。结果表明,本研究制备的CDV、CPV胶体金二联检测卡特异性强、灵敏度高、准确性好,兼具操作简便、肉眼可判读、结果易保存和无需特殊仪器设备等优势,可用于两种疾病的临床快速诊断和大规模检测工作。     

PCR检测犬腺病毒方法的建立

利用人工合成的两条引物,对病犬肝脏和细胞培养物冻融上清中的1、2型犬腺病毒DNA进行多聚酶链式反应（PCR）检测;再将扩增产物进行同位素标记,与纯化病毒DNA进行打点杂交。扩增产物经电泳分析结果表明,其大小与设计的引物间的序列大小基本一致;扩增产物经标记后只与犬腺病毒DNA发生杂交反应,表明其为犬腺病毒特异性核酸片段。