The relation between the rabbit potency test and the response of sheep to sheep clostridial vaccines.

Six commercially available clostridial vaccines comprising one oil-emulsion, two alum-precipitated and three aluminum hydroxide adjuvanted preparations, each containing between two and seven antigenic components, were administered to groups of 10 rabbits and eight sheep in accordance with manufacturers' recommendations. Serum antitoxic values to Cl welchii beta, Cl welchii epsilon, Cl septicum, Cl oedematins and Cl tetani toxins were determined 14 days after completion of each vaccination course. The overall pattern of mean antitoxic values was found to be similar in sheep and rabbits, a vaccine eliciting a comparatively high antibody titre to any given antigen component in sheep also inducing a comparatively high titre in the corresponding group of rabbits. Similarly, comparatively poor responses in sheep were associated with poor responses in rabbits. The degree of variation in response within groups of animals was greater in sheep than in rabbits for all five antigenic components assayed. Sheep consistently developed higher titres than rabbits to Cl oedematins component but consistently lower titres to both Cl welchii beta and epsilon components irrespective of the type of vaccine used. The response of both species to Cl tetani antigen was similar in terms of serum antitoxic values. It was concluded that rabbits provide a suitable model for the assessment of potency of sheep clostridial vaccines.     

多头绦虫抗原特性的研究──应用IHA观察绵羊免疫及感染后的抗体消长规律

为了观察绵羊用多头蚴抗原免疫及感染后的抗体消长规律,为羊脑多头蚴病的免疫预防和免疫诊断提供依据,本试验应用多头蚴原头节可溶性抗原、囊壁可溶性抗原、囊液粗抗原致敏绵羊红细胞对绵羊免疫3次及虫卵攻击感染后的血清抗体进行间接血凝试验（IHA）检测。结果表明,原头节抗原免疫组、囊壁抗原免疫组、囊液抗原免疫组及原头节ES抗原免疫组在首次免疫后1周,抗体滴度迅速升高,第3次免疫后1周达到峰值,虫卵感染后开始下降,到感染后30周接近正常水平。多头蚴3种抗原对同种抗原免疫组血清检测敏感性、特异性优于其它抗原,原头节免疫组、囊壁免疫组、囊液免疫组抗体水平明显高于原头节ES抗原免疫组。     

Effect of Haemaphysalis punctata infestation on growing lambs and rabbits

Lambs infected with adult Haemaphysalis punctata and rabbits infected with nymphs developed a macrocytic normochromic anaemia during seven and six successive infestations, respectively. The anaemia was directly proportional to the degree of infestation but disappeared several days after the termination of infestation. A leucocytosis, due to neutrophilia, was seen in both lambs and rabbits. Rabbits developed a thrombocytosis and reticulocytosis. Infested lambs grew less rapidly than uninfested animals. Signs of tick toxicosis and several other clinical manifestations appeared in both infested sheep and rabbits. Circulating antibodies against salivary antigen of adult H punctata were demonstrated in the sera of infested lambs by the micro-ELISA test. Titres were first detected on day 3 after infestation and increased gradually as infestation progressed. No precipitating antibodies in either infested sheep or rabbits were detected.     

Improvements in the modified direct complement fixation test and its application in the detection of bluetongue antibodies in cattle and sheep sera.

In this study, improvements were made in the technique and the preparation of the antigen. It was possible to perform three extractions and elutions resulting in a soluble reactive preparation from each batch of infected mouse brain. This led to an appreciable increase in the yield of highly reactive antigen. The presence of bluetongue antibodies was not detected in 13,210 sheep sera. Of the 13,486 bovine sera tested, only three questionable reactions were obtained. It was possible to determine that two of these animals were imported. Various isolation methods, including transmission trials to susceptible sheep followed by serological tests on the sheep sera, failed to confirm the infection in the three reactors.     

Tick-borne fever: Leucocyte migration inhibition

When sheep were infected with Cytoecetes phagocytophila the in vitro migration of peripheral leucocytes was reduced even when antigen was not added. The inhibition of migration coincided with parasitaemia and was due to soluble factors which inhibited the migration of leucocytes from normal sheep. After the cessation of parasitaemia addition of antigen was necessary for migration inhibition to occur.     

Comparison of the immunogenic effects of covalent-bonded immune complexes in mice and sheep

Covalent antigen-antibody complexes containing the protein antigen ovo-transferrin primed both mice and sheep to give an enhanced antibody response to a subsequent single injection of soluble ovo-transferrin. Complexes prepared using horse, sheep or rabbit antibody had a priming effect in mice, although rabbit antibody-antigen complexes were the most effective. In sheep, only rabbit antibody-antigen complexes significantly enhanced antibody levels.     

Immunologic response of sheep to inactivated and virulent bluetongue virus

Humoral and cellular immune responses of sheep to inactivated and virulent bluetongue virus (BTV) were studied. All sheep inoculated with inactivated BTV developed BTV group-specific nonneutralizing antibodies, as determined by agar-gel immunodiffusion. The development of group-specific, nonneutralizing, complement-fixing antibodies was variable and appeared to be dependent on immunizing BTV serotype, sheep breed, and individual variation. Virus-neutralizing antibodies were never detected after inoculation with the inactivated BTV. In vitro lymphocyte stimulation to BTV soluble antigen was observed with cells from all inoculated Warhill sheep and with cells from 1 of 3 inoculated Suffolk cross sheep. Complement-fixation titers did not appear to correlate with the degree of protection observed, ie, duration of postchallenge-exposure viremia. The development of postchallenge-exposure neutralizing antibody titer was inversely correlated to protective immunity. The development of a response to BTV antigen in the lymphocyte-stimulation test associated most closely with protection. Warhill sheep were afforded better protection, by inoculation with inactivated BTV, to live virus challenge exposure than were the Suffolk cross sheep. Approximately 30% of the inoculated Suffolk cross sheep responded to challenge exposure with intensified clinical signs of blue-tongue, compared with the challenge-exposed control sheep of the same breed.     

Suppression of immunological responses in rabbits experimentally infected with bovine leukemia virus

Ten 2- to 4-month-old rabbits were inoculated subcutaneously with bovine leukemia virus (BLV)-infected bovine or sheep cells. By 6 weeks after inoculation all ten rabbits had converted to BLV antibody-positive, and BLV or BLV antigen was detected in lymphocytes from most of the rabbits tested, although there were few antigen-producing cells. Three rabbits showed continuous respiratory symptoms after infection and one died with pneumonia. Humoral immune responses against mouse serum were significantly suppressed in BLV-infected rabbits compared with non-infected control rabbits. The lymphocyte blastogenesis response was also suppressed in BLV-infected rabbits. At the time of necropsy, six rabbits showed pulmonary lesions; however, none of the BLV-infected rabbits had tumors during an observation period of over 1 year.     

犬细粒棘球绦虫粪抗原夹心ELISA检测方法的建立

为建立特异性和敏感性高的检验犬细粒棘球绦虫感染的方法。用细粒棘球绦虫(简称,Eg)成虫抗原分别免疫兔和绵羊,收集高免血清,纯化的高免抗体。依据抗体夹心ELISA工作原理,以兔抗体包被,检测感染Eg、不同犬带科绦虫的实验犬和空白犬粪样,绵羊抗体扑捉抗原,HRP标记兔抗绵羊IgG(1∶8 000)催化显色,用酶标仪测定OD 405nm吸光度,用以确定其特异性和敏感性。试验结果表明,敏感性为82.69%(43/52),特异性为85.88%(140/163);粪抗原在感染细粒棘球绦虫16d后可检出,最低抗原浓度为9.7ng/mL即犬感染5条成虫时可检测出阳性。该检测方法具有较好的特异性和灵敏性,为进一步研制检测细粒棘球绦虫虫体抗原ELISA检测试剂盒奠定了基础。     

节瘤拟杆菌K-凝集试验方法的建立

根据节瘤拟杆菌(D.nodosus)特异的表面抗原———K抗原具有凝集反应的特性,建立了检测抗节瘤拟杆菌抗体的方法———K-凝集试验方法。节瘤拟杆菌液体培养物接种兔,制备节瘤拟杆菌阳性血清;福尔马林灭活肉汤培养物制备抗原,结果表明,该方法使用方便、特异性强。     

绵羊细粒棘球蚴全血金标渗滤检测方法的建立

为建立一种快速、高效的诊断家畜细粒棘球蚴病方法,提取绵羊细粒棘球蚴包囊新鲜囊液,盐析囊液抗原,点样于硝酸纤维膜,以胶体金-驴抗羊IgG和胶体金-兔抗鼠IgG为检测标记物,采用垂直流渗滤装置检测绵羊与人工感染细粒棘球蚴小鼠血清和全血特异性抗体。患病绵羊阳性血清及全血检出率在90.91%~94.4%,细粒棘球蚴感染小鼠血清及全血检出率均为100%;细粒棘球蚴阴性羊血清和全血假阳性率为4.00%~4.59%;与脑多头蚴病血清交叉反应率为28.57%(2/7)。研究结果表明细粒棘球蚴全血金标渗滤法(DIGFA)可应用于绵羊棘球蚴病的诊断与检疫。     

西藏牦牛大肠埃希氏菌生物学特性的研究

本文对西藏牦牛大肠埃希氏菌进行培养特性、形态染色、鞭毛检查、血凝性、生化试验、血清型鉴定、抗原性、致病性等生物学特性研究,同时用大肠埃希氏菌标准菌种C83907做对照试验.结果表明,西藏牦牛大肠埃希氏菌在普通琼脂平板上可形成光滑型和粗糙型两种类型的菌落,在5%绵羊鲜血琼脂平板上无溶血现象;具有运动性,对家兔红细胞表现出强凝集,而对牦牛、绵羊、马、鸡、猪、鸭的红细胞不凝集,能发酵大多数糖类;其0抗原血清型为026、0142、0148和0158混合型;牦牛大肠埃希氏菌的培养物对家兔具有致病性,用牦牛大肠埃希氏菌油乳剂疫苗2次免疫家兔14 d后检测抗体效价,抗体效价≥2<8>.     

Studies on the antigenic structure of Bacteroides nodosus.

The antigenic mosaics of three Bacteroides nodosus isolates (198, 199 and 127) were studied to elucidate the nature of the protective immunogen. In vaccinated sheep the three isolates induced high homologous serum agglutinin titres but it was also apparent that 198 and 199 shared a major surface antigen not present on 217. This major cross-reacting antigen was not detected with rabbit antisera. The fimbriae, consisting predominantly of protein, induced high homologous titres in rabbits and represented the type-specific antigen. Lipopolysaccharide (LPS) from each of the isolates induced low agglutinin titres and high 2-mercaptoethanol-sensitive indirect haemagglutinating antibody titres. The heat-stable LPS contained at least two common carbohydrate O antigen determinants but no type-specific O antigens were detected.     

Anti-idiotype technique: an alternative approach for immunodiagnosis of bluetongue

In development of a bluetongue alternative immunodiagnostic rest, the polyclonal anti-idiotypic antibodies were generated by the sequential immunization of rabbits with three monoclonal antibodies to VP7 of bluetongue virus. The anti-idiotypic antibodies recognize the idiotypes that are located within or near the antigen-combining sites and are associated with both heavy and light chains of the antibodies to VP7 of bluetongue virus. The anti-idiotypic antibodies mimic the VP7 antigen by recognizing the anti-VP7 antibodies from cattle and sheep that were infected with various serotypes of bluetongue viruses. The results indicate that the rabbit anti-idiotypic antibodies may be used as surrogate antigen in serological assays to detect the antibodies from different species of animals infected with various serotypes of bluetongue viruses.     

A potential krimpsiekte vaccine

Krimpsiekte, a chronic form of cardiac glycoside poisoning, is an important plant-induced intoxication of small stock in South Africa. It is caused by cumulative, neurotoxic bufadienolides, such as cotyledoside. A cotyledoside-bovine serum albumin conjugate was synthesized to immunize animals. The efficacy of the cotyledoside-conjugate in inducing an immunological response was ascertained in rabbits (n = 4) and sheep (n = 4) by determining cotyledoside antibody titres with an ELISA using cotyledoside-hen ovalbumin as antigen. The formation of anticotyledoside antibodies was induced in both rabbits and sheep following immunization with the cotyledoside-protein conjugate. Protection provided by the vaccine was demonstrated by challenging sheep (n = 4) with repeated, daily doses of cotyledoside (0.015 mg/kg) administered intravenously, commencing 45 days after the initial vaccination. One control animal died on Day 3 of the challenge period and the other was severely affected after administration of the third cotyledoside dose. The immunized ewes (n = 2) remained clinically unaffected and the challenge was suspended following six daily injections. Vaccination as a means of preventing krimpsiekte seems to be quite feasible and deserves further investigation.     

Comparative study of the pharmacokinetics of alfentanil in rabbits, sheep, and dogs

The central arterial pharmacokinetics of alfentanil, a short-acting opioid agonist, were studied in rabbits, sheep, and dogs after short-duration infusion of the drug. Alfentanil was infused until a set end point (high-amplitude, slow-wave activity on the EEG) was reached. This required a larger alfentanil dose and a higher alfentanil arterial concentration in sheep, compared with rabbits and dogs. The plasma concentration-time data for each animal were fitted, using nonlinear regression, and in all animals, were best described by use of a triexponential function. In this study, differences in the disposition kinetics of alfentanil among the 3 species were found for only distribution clearance and initial distribution half-life. In dogs, compared with rabbits and sheep, the first distribution half-life was longer, probably because of pronounced drug-induced bradycardia (mean +/- SD, 48 +/- 21 beats/min). Distribution clearance was faster in sheep, compared with dogs, also probably because of better blood flow in sheep. Elimination half-life was similar in all species (rabbits, 62.4 +/- 11.3 minutes; sheep, 65.1 +/- 27.1 minutes; dogs, 58.3 +/- 10.3 minutes). This rapid half-life resulted from a small steady-state volume of distribution (rabbits, 908.3 +/- 269.0 ml/kg; sheep, 720.0 +/- 306.7 ml/kg; dogs, 597.7 +/- 290.2 ml/kg) and rapid systemic clearance (rabbits, 19.4 +/- 5.3 ml/min/kg; sheep, 13.3 +/- 3.0 ml/min/kg; dogs, 18.7 +/- 7.5 ml/min/kg). On the basis of these pharmacokinetic variables, alfentanil should have short duration of action in rabbits, sheep, and dogs. This may be beneficial in veterinary practice where rapid recovery would be expected after bolus administration for short procedures or after infusion for longer procedures.     

Antibodies to the CP24 protein of Brucella melitensis lack diagnostic usefulness in ovine brucellosis

The potential diagnostic usefulness of antibodies to the ribosome recycling factor of Brucella melitensis (CP24) was assessed in sheep by an indirect ELISA with purified recombinant CP24. Sera from uninfected animals from the UK (n=44) and from local flocks (n=42), from sheep naturally infected with B. melitensis (n=12) or B. ovis (n=12), and from lambs (n=7) or pregnant ewes (n=6) vaccinated with B. melitensis Rev-1, were assayed. High specific optical densities (OD(with antigen) - OD(without antigen)) were obtained with both the groups of normal sera, which resulted in high cut-off values (1.414 and 1.267, respectively). Only two infected sheep yielded specific OD higher than these cut-off values. No significant difference was found between mean specific OD from B. melitensis- or B. ovis-infected sheep (0.574 and 0.472, respectively), those from vaccinated animals (0.396 and 0.400 for pregnant ewes and lambs, respectively), and those from Brucella-free animals. An inhibition ELISA with soluble CP24 confirmed the specificity of the antibodies detected in normal sera by the indirect ELISA; these antibodies belonged to the IgG class as revealed by the use of a specific conjugate. Sera from infected sheep were all positive for antibodies against lipopolysaccharides and lumazine synthase from Brucella. These results show that anti-CP24 antibodies have no diagnostic role in ovine brucellosis.     

IgG precipitating and non-precipitating antibodies in rabbits repeatedly injected with soluble and particulate antigens

The immune response of precipitating antibodies and non-precipitating antibodies of high affinity (co-precipitating) of the IgG class was analyzed in rabbits repeatedly injected with egg albumin (as a soluble antigen) B. abortus-egg albumin and polymerized egg albumin (as particulate antigens). The results showed that the levels of anti-egg albumin non-precipitating antibodies induced by the soluble antigen were never higher than 10-15% of total antibodies throughout the experimental time. When particulate antigens were injected, the levels of non-precipitating antibodies increased up to 30-70% of the total antibody levels. This phenomenon is related to the way in which the antigen is available to the immune system (particle or aggregated), and is independent of the response induced by the particulate carrier. Components from the cell wall or bacterial membrane that could act as coadjuvants do not participate in this phenomenon. The results obtained seem to indicate that possibly there was a suppression of the synthesis of precipitating antibodies, and this would produce a relative increase in the non-precipitating antibodies.     

Development of enzyme-linked immunosorbent assays for the detection of Fusobacterium necrophorum antibody in animal sera

Enzyme-linked immunosorbent assays (ELISA) for the detection of Fusobacterium necrophorum antibody in the sera of rabbits, cattle, and sheep were developed, using a ribosome-rich extract (RRE) from F necrophorum as the antigen. Test character, including optimal antigen dilution and substrate incubation periods, was established, using rabbit, bovine, and ovine antisera produced against RRE from isolates of F necrophorum. Rabbit antisera produced against 7 other species of bacteria were used to test the specificity of the F necrophorum RRE antigen. Cross-reactivity was not detected. Sera from 50 feedlot cattle were examined with the bovine ELISA. Of the 50 samples, 43 (88%) were positive for F necrophorum antibody. The ELISA developed in this study were sensitive and specific and appear to be readily adaptable to serologic investigations of F necrophorum.     

Immunogenic and antigenic properties of recombinant soluble glycoprotein of rabies virus

Rabies virus glycoprotein is a type I transmembrane protein exposed on the surface on the mature virus particle that induces virus neutralizing antibodies. In the present study, 60 amino acid C-terminal hydrophobic anchor (transmembrane) and cytoplasmic domains of glycoprotein were deleted from full-length glycoprotein and fused with polyhistidine tag. The N-terminal viral signal peptide was also replaced with CD33 signal peptide for efficient secretion in mammalian cells. Following transfection of Madin Darby bovine kidney (MDBK) cells with plasmid encoding this soluble form of glycoprotein, polyclonal populations of stably transfected resistant cells were obtained after G418 selection. The protein was expressed as a glycosylated protein and secreted outside the cells utilizing N-terminal CD33 signal peptide. The secreted soluble glycoprotein was purified from cell culture supernatant by Ni--agarose affinity chromatography utilizing C-terminal polyhistidine tag. Like full-length glycoprotein, the expressed recombinant soluble glycoprotein was found to be immunogenic when injected in rabbits. In this study, we have assessed the potential of recombinant soluble glycoprotein as diagnostic antigen in ELISA and found that this recombinant protein can be used as diagnostic antigen in ELISA for detecting anti-glycoprotein antibodies in immunized host.