Detection of antibodies to ovine lentivirus using recombinant capsid and transmembrane proteins

The coding sequences of the capsid protein p25 and transmembrane protein of Maedi-Visna virus were amplified using polymerase chain reaction and cloned into the plasmid expression vector pRSET-B. Both DNA constructs expressed proteins tagged with polyhistidine. The recombinant proteins were purified using Ni-NTA agarose and used in immunoblot to detect antibodies against Maedi-Visna virus. A total of 260 ovine serum specimens was analysed. The total number of p25-positive sera was 111 (42.7%). Higher sensitivity was achieved with rTM antigen, which detected antibodies in 118 (45.4%) sera. The combination of both recombinant proteins as antigens resulted in higher sensitivity of serological detection compared to whole virus antigen.     

Lesions and retroviruses associated with naturally occurring ovine pulmonary carcinoma (sheep pulmonary adenomatosis)

Five sheep with ovine pulmonary carcinoma were markedly dyspneic and had sporadic coughing; two had copious watery nasal exudate. In four, lesions consisted of multifocal nodules of neoplastic cuboidal epithelial cells in acinar or papillary patterns. Electron microscopically, cells had microvilli, tight junctions, and cytoplasmic lamellar bodies typical of alveolar type II cells. One sheep had a single lung tumor of nonciliated bronchiolar epithelial cells. Vacuolated alveolar macrophages surrounded adenomatous foci. One sheep had a metastatic lesion in the caudal mediastinal lymph node. All sheep had histologic lesions of lymphoid interstitial pneumonia (LIP, ovine progressive pneumonia) consisting of peribronchiolar and interstitial lymphoid hyperplasia, and fibromuscular proliferation; all had serum precipitating antibodies to ovine lentivirus. Lung fluids or tumor homogenates contained a 26-kd peptide that crossreacted with a primate-derived type D retrovirus as detected by immunoblotting or interspecies competition radioimmunoassay. Ovine lentivirus was isolated from concentrated lung fluids or tumor tissues of four sheep tested and from tumor cell DNA of one animal transfected into ovine muscle cells. These studies document the presence of type D-related retrovirus antigen in ovine pulmonary carcinoma (OPC) in the United States and indicate that lentivirus-induced LIP is a lesion frequently associated with this disease.     

Detection and quantitation of a type D retrovirus gag protein in ovine pulmonary carcinoma (sheep pulmonary adenomatosis) by means of a competition radioimmunoassay

A heterologous competition radioimmunoassay (RIA) which consisted of 125I-labeled langur retrovirus major gag protein and goat anti-squirrel monkey retrovirus serum was used to detect a type D retrovirus-associated antigen in tumor cell homogenates, lung fluid, and cell culture supernatant fluids of naturally occurring and experimentally-induced ovine pulmonary carcinoma (OPC, sheep pulmonary adenomatosis). In this assay, there was no cross reactivity between structural proteins of the type D retrovirus and an ovine lentivirus, which frequently co-infects OPC-affected sheep. The sensitivity of the assay was similar to an immunoblotting assay using antiserum to Mason-Pfizer monkey virus major gag protein which had been used previously to detect the OPC retrovirus antigen in tumor homogenates and lung fluids of OPC-affected sheep. All unconcentrated samples of lung fluid collected from five sheep with naturally occurring OPC or six sheep with experimentally induced OPC competed in the competition RIA. The competition RIA titers of the type D retrovirus antigen in lung fluids of lambs with induced OPC were relatively higher than the titers of this antigen in the naturally occurring OPC cases. The competition RIA detected the retrovirus antigen associated with OPC in the culture fluids of four out of five primary lung cultures from OPC sheep tested between 1 and 56 days after culture initiation. Because this RIA is appropriate for the quantitation of OPC-associated antigen, it will provide a means for determination of the target cell type for OPC virus replication in vitro.     

Altered expression of beta-catenin, E-cadherin, cycloxygenase-2, and p53 protein by ovine intestinal adenocarcinoma cells

Around 1.6% of sheep in New Zealand develop small-intestinal adenocarcinomas. These neoplasms typically develop widespread metastases. The common development of these neoplasms and their subsequent behavior suggests that sheep could be a useful animal model of human colonic cancer. However, for an animal model of human disease to be relevant, similar genetic mutations should be present. Genetic mutations within human colonic cancers frequently result in expression of cycloxygenase-2 (COX-2), loss of membranous expression of beta-catenin and E-cadherin, and accumulation of p53 protein within the neoplastic cells. Immunohistochemistry was used to investigate the presence of these 4 proteins within 26 ovine intestinal adenocarcinomas. Loss of membranous beta-catenin reactivity was observed in 14 of 26 ovine intestinal adenocarcinomas (54%). The loss of membranous beta-catenin reactivity was accompanied by cytoplasmic and nuclear reactivity in 2 neoplasms. Loss of E-cadherin was observed within 8 of 26 neoplasms (31%). Neoplastic cell expression of COX-2 was observed in 12 of 26 neoplasms (46%), whereas cells within 3 of 26 neoplasms (11%) contained visible p53 protein. In conclusion, all 4 proteins that commonly have altered expression in human colonic cancers were also altered in a proportion of the ovine intestinal adenocarcinomas. These results provide additional evidence that sheep could be useful for the study of human colonic cancer.     

Alveolar type II cells expressing jaagsiekte sheep retrovirus capsid protein and surfactant proteins are the predominant neoplastic cell type in ovine pulmonary adenocarcinoma

Ovine pulmonary adenocarcinoma is caused by jaagsiekte sheep retrovirus. To gain insight into the histogenesis and viral pathogenesis of this neoplasm, the tumor cell phenotypes and differentiation state were correlated with the distribution of jaagsiekte sheep retrovirus capsid protein in neoplastic and normal cells of the lung in nine naturally occurring and 12 experimentally induced cases of ovine pulmonary adenocarcinoma. Overall, 82% of tumor cells had ultrastructural features consistent with alveolar type II cells, 7% of tumor cells had features of Clara cells, and 11% of tumor cells were insufficiently differentiated to classify. The proportion of the neoplastic cell phenotypes varied within tumors, and no tumor consisted of a morphologically uniform cell population. To further characterize the neoplastic cell population, sections of tumors were immunostained with antibodies to surfactant protein A, surfactant protein C, and Clara cell 10-kd protein. Overall, surfactant proteins A and C were expressed in 70% and 80% of tumor cells, respectively, whereas Clara cell 10-kd protein was expressed in 17% of tumor cells. Jaagsiekte sheep retrovirus capsid protein was detected in 71% of tumor cells and in macrophages (5/21 tumors examined) and in nonneoplastic alveolar and bronchiolar cells (6/14 tumors). Expression of this viral protein in neoplastic cells, classified morphologically and by immunophenotyping primarily as of the alveolar type II lineage, implies an important role for specific virus-cell interactions in the pathogenesis of ovine pulmonary adenocarcinoma.     

Comparison of dot enzyme-linked immunosorbent assay (Dot-ELISA), passive haemagglutination test (PHT) and thin layer immunoassay (TIA) in the diagnosis of natural or experimental Fasciola hepatica infections in sheep

The dot enzyme-linked immunosorbent assay (Dot-ELISA) was compared with the passive haemagglutination test (PHT) and thin layer immunoassay (TIA) for the detection of antibodies against Fasciola hepatica in naturally and experimentally infected sheep. The infected animals gave titres from 1:25,000 to 1:204,000, while control animals gave titres of from 1:100 to 1:800. The titres of the infected sheep obtained by Dot-ELISA were 1000-2000 times higher than the ones obtained using TIA or PHT. Due to its high sensitivity, this technique could be very useful for the diagnosis of ovine fascioliasis.     

Survey of ovine and caprine toxoplasmosis by IFAT and PCR assays in Sardinia, Italy

During the period 1999-2002, we have analyzed 9639 serum samples and 815 aborted samples (670 fetuses and 145 placenta) from 964 ovine and caprine farms distributed over all Sardinia island. After abortion notification, sera collected at random from adult animals were examined to detect simultaneously IgG and IgM antibodies specific to Toxoplasma gondii by indirect immunofluorescence assay, whereas fetuses and placenta were analyzed by a single tube nested PCR assay. Specific IgG antibodies were detected in 2048 (28.4%) sheep and 302 (12.3%) goats, specific IgM antibodies were found in 652 (9%) sheep and 139 (5.6%) goats. From a total of 2471 ovine and 362 caprine fetal samples including muscle, liver, abomasum, spleen, brain and placenta, 271 (11.1%) ovine and 23 (6.4%) caprine samples were T. gondii PCR-positive. Although T. gondii DNA was amplified from different types of tissues, placenta was the tissue with the highest detection rate. On the one hand, these results indicate that the seroprevalence of T. gondii infection in sheep and goats is relatively high, on the other PCR results demonstrate that T. gondii has a significant role in ovine and caprine abortion. Adequate management might be useful and essential to control the toxoplasmosis in the sheep and goats herds of Sardinia.     

Enzyme-linked immunosorbent assay for detection of antibodies to Rift Valley fever virus in ovine and bovine sera

An enzyme-linked immunosorbent assay (ELISA) was developed for the rapid detection of antibodies to Rift Valley fever virus (RVFV) in ovine and bovine sera. Conditions to reduce nonspecific reactions were optimized. The ELISA results correlated with those of a plaque-reduction neutralization test, revealing 100% specificity and 90.7% sensitivity. In sera from sheep and cattle inoculated against RVFV, the hemagglutination-inhibition test in combination with the ELISA provided a better indication of response to killed RVFV vaccine than did either test alone.     

Antigenic profiles of recombinant proteins from Mycobacterium avium subsp. paratuberculosis in sheep with Johne's disease

Methods to improve the ELISA test to detect Mycobacterium avium subsp. paratuberculosis have been explored over several years. Previously, selected recombinant proteins of M. avium subspecies paratuberculosis were found to be immunogenic in cattle with Johne's disease. In the present study, antibody responses of infected and healthy sheep were evaluated using 18 purified recombinant proteins in an ELISA-based format for the serodiagnosis of ovine paratuberculosis. These selected recombinant proteins represent heat shock proteins, hypothetical proteins and cell surface proteins of M. avium subsp. paratuberculosis. Whereas, Map0862 (a gene uniquely present in M. avium subspecies paratuberculosis) and Map3786 encoded protein solicited the strongest antibody response in infected sheep. The protein encoded by Map2116c showed the weakest antibody response among the animals tested. Although none of the recombinant proteins detected all 11 infected sheep singly, antibodies to Map0862 were detected in 9 of 11 (81%) infected sheep. Furthermore, ovine responses to these selected antigens were assessed temporally over the course of 1 year during which we found a spiking effect rather than an incremental increase of antibody reactivity. This study evaluated multiple M. avium subsp. paratuberculosis recombinant proteins in an ELISA-based format for sheep.     

藏羊AMPK、ACC和GS酶活分布及饥饿应激对其酶活的影响

为分析AMPK等酶对机体代谢的影响,采用酶联免疫分析法（ELISA）,分别测定正常饲养和饥饿48 h后藏羊各组织的AMPK、ACC和GS的活性.结果表明：AMPK、ACC和GS分布于藏系绵羊各组织中,且各组织器官中3种酶活性差异显著（P＜0.05）;饥饿应激导致骨骼肌、肝脏、肾脏和小肠组织中AMPK活性升高,骨骼肌、肝脏、脾脏、肺脏、肾脏和小肠组织中ACC活性下降,骨骼肌、肝脏、脾脏、肺脏、肾脏和小肠组织中GS活性下降.因此,AMPK、ACC和GS在藏系绵羊的各组织器官中分布广泛;饥饿应激可能通过激活AMPK表达,下调ACC和GS表达而调节藏羊的应激代谢.     

绵羊β3肾上腺素能受体基因在脂肪组织中的表达研究

旨在对绵羊β3肾上腺素能受体基因在脂肪组织中的表达进行研究。本研究通过real-time PCR和免疫组化的方法检测了2个绵羊群体皮下脂肪、大网膜、小网膜、腹膜后脂肪、肠系膜和肾周等6种脂肪组织中ADRB3基因mRNA及其蛋白的表达量与分布情况。结果表明:ADRB3蛋白位于脂肪细胞的细胞膜中。ADRB3基因mRNA及其蛋白在皮下脂肪组织的表达丰度最小(0.159和0.139),在腹膜后脂肪组织的表达丰度最大(2.911和2.225),深层脂肪组织中ADRB3基因mRNA表达量要显著高于皮下脂肪组织(P<0.05),表明皮下脂肪组织的脂肪分解率要低于深层脂肪组织。品种对ADRB3基因mRNA的表达没有显著影响,但对于ADRB3蛋白的表达影响显著。不同脂肪组织中ADRB3表达丰度的差异反映了山西肉用绵羊的遗传稳定性较差。本研究的结果与已知的ADRB3调节脂肪分解和产热的功能是一致的,为利用ADRB3基因作为候选基因进行绵羊新品种的培育提供理论依据。     

New types of virus infections of domestic animals in the German Democratic Republic. 3. Seroepizootiologic and experimental studies of herpesvirus infections in sheep--the etiology of pulmonary adenomatosis

Neutralisation tests for antibodies against ovine herpesvirus were applied to 848 sera which had been sampled from different sheep herds across the GDR. Between six and 20 percent of sheep in the herds tested exhibited neutralising antibodies, notwithstanding their pulmonary adenomatosis status. Incidence and titre distribution of antibodies against ovine herpesviruses in pulmonary adenomatosis herds were identical with those recorded from unsuspected herds. From among 21 sheep with pathomorphologically secured pulmonary adenomatosis, six animals exhibited antibody titres just as high as those recorded from responders of all herds examined. Lambs were obtained by hysterectomy and raised without mothers and were experimentally infected with Herpesvirus ovis. All of these animals responded to infection by clearly rising titres (between 1:2 and 1:32). Adenomatous pulmonary lesions were not recordable from any of them. One lamb, following experimental ovine herpesvirus infection, exhibition, exhibited subclinical interstitial pneumonia. Herpesvirus ovis has been widespread in sheep herds across the GDR. The authors' serological and experimental investigations do not support the assumption of an aetiological relationship between ovine herpesvirus infection and incidence of pulmonary adenomatosis.     

Prion protein in sheep urine.

The misfolded form of cellular prion protein (PrP(C)) is the main component of the infectious agent of transmissible spongiform encephalopathies and the validated biomarker for these diseases. The expression of PrP(C) is highest in the central nervous system and has been found in peripheral tissues. Soluble PrP(C) has been detected in cerebrospinal fluid, urine, serum, milk, and seminal plasma. In this study, attempts were made to characterize prion protein in urine samples from normal and scrapie-infected sheep. Urine samples from scrapie-infected sheep and age-matched healthy sheep were collected and analyzed by Western blot following concentration. A protease K-sensitive protein band with a molecular weight of approximately 27-30 kDa was visualized after immunoblotting with anti-PrP monoclonal antibodies to a C-terminal part of PrP(C), but not after immunoblotting with monoclonal antibodies to an N-terminal epitope of PrP(C) or with secondary antibodies only. The amount of PrP(C) in the urine of 49 animals (control group: n = 16; naturally scrapie-infected group: n = 33) was estimated by comparison with known amounts of ovine recombinant PrP in the immunoblot. Background concentration of PrP(C) in urine was found to be 0-0.16 ng/ml (adjusted to the initial nonconcentrated volume of the urine samples). Seven out of 33 naturally scrapie-infected animals had an elevated level (0.3-4.7 ng/ml) of PrP(C) in urine. The origin of PrP(C) in urine and the reason for the increased level of PrP(C) in scrapie-infected sheep urine has yet to be explored.     

A comparison of whole virus and recombinant transmembrane ELISA and immunodiffusion for detection of ovine lentivirus antibodies in Italian sheep flocks

Sera from two sheep experimentally infected with ovine lentivirus (OLV) and from 186 sheep selected from flocks with known high or low prevalence of infection or on the basis of virological or histopathological examination were simultaneously tested by whole virus (WV) ELISA, recombinant transmembrane (r-TM) ELISA and AGID assay. Antigens for both the WV ELISA and AGID were prepared from an Italian field isolate; recombinant antigen was derived from the N-terminal region of the transmembrane envelope protein of strain K1514. The WV ELISA detected the highest number of seropositives, followed by the r-TM ELISA and AGID test. The sensitivity and specificity of the r-TM ELISA relative to the WV ELISA were 0.66 and 0.95, respectively. Immunoblot analysis of 14 WV ELISA-positive and r-TM ELISA-negative sera showed that the major core protein was immunodominant on WV antigen. It is concluded that the r-TM ELISA was more sensitive than the AGID test but less sensitive that the WV ELISA, particularly for detecting antibodies in the early stages of infection.Abbreviations AGID agar gel immunodiffusion - bp base pair - ELISA enzyme-linked immunosorbent assay - FBS fetal bovine serum - IB immunoblot - kD kilodalton - OLV ovine lentivirus - MEM minimal essential medium - p.i. post-infection - r recombinant - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TM transmembrane - WV whole virus     

新疆南疆和田羊、卡拉库尔羊毛囊KAP7基因的原核表达及多克隆抗体制备

本研究旨在制备新疆南疆平原型和田羊、山区型和田羊与卡拉库尔羊3个地方品种（系）绵羊毛囊角蛋白关联蛋白7（keratin associated protein 7,KAP7）基因多克隆抗体,为探索毛囊KAP7基因对半粗毛羊羊毛产量与质量的影响奠定基础。以3个品种绵羊的皮肤毛囊为试验材料,提取总RNA,反转录得到cDNA,PCR扩增获得绵羊毛囊KAP7基因,构建pMD19-T-KAP7克隆质粒,对重组质粒进行PCR、酶切鉴定和序列分析,再构建pET-28a（+）-KAP7原核表达重组质粒,并在大肠杆菌BL21（DE3）感受态细胞中表达,SDS-PAGE电泳检测原核表达的绵羊毛囊KAP7蛋白,经提取、纯化、回收得到目的蛋白,用其免疫家兔得到绵羊毛囊KAP7多克隆抗体血清,并用间接ELISA法检测其抗体效价。结果表明,与美利奴羊比对,平原型与山区型和田羊KAP7基因序列的同源性达99%,但平原型和田羊KAP7基因第22位碱基由G变为C,造成其KAP7蛋白第8位氨基酸由Gly变为Arg;山区型和田羊第70位碱基由A变为C,造成其24位氨基酸由Thr变为Ala;卡拉库尔羊毛囊KAP7基因与美利奴羊同源性达100%,其KAP7蛋白氨基酸序列与美利奴羊没有差异。绵羊毛囊KAP7基因原核表达重组质粒pET-28a（+）-KAP7可以在大肠杆菌BL21（DE3）感受态细胞中表达目的蛋白,其大小约为10 ku。利用绵羊毛囊KAP7蛋白免疫家兔得到的多克隆抗体阳性血清具有免疫活性和特异性,其抗体效价达到1：10 000。