禽用活疫苗外源病毒污染情况的调查研究

为对禽用活疫苗污染外源病毒的情况进行彻底摸底调查,抽取了目前使用量大、覆盖面广的禽用活疫苗重点品种共8种28批,涉及18家国内生产企业、7家国外企业,疫苗生产用SPF鸡胚来自国内8家企业。按照《中国兽药典》2010年版三部鸡检查法和《欧洲药典》对抽检的禽病活疫苗进行了外源病毒污染和禽腺病毒污染检验。结果显示所抽检的禽用活疫苗均无外源病毒污染和禽腺病毒污染,表明我国禽用活疫苗的质量控制和安全性良好。     

鸡痘病毒活疫苗污染禽网状内皮组织 增生症病毒的间接免疫荧光法检测研究

通过向无外源病毒污染的鸡痘病毒活疫苗中添加不同剂量的禽网状内皮组织增生症病毒(REV),然后用间接免疫荧光法(IFA)进行检测,确定了该IFA方法的最低检出量为每500羽份疫苗中污染20 TCID_(50)的REV。使用该方法对国内16家企业生产的60批鸡痘病毒活疫苗进行了检验,结果显示2个企业生产的4批疫苗REV检测为阳性。随机选取5批IFA检测阴性样品和4批IFA检测阳性样品,按鸡检查法进行外源病毒检验,结果两种方法对REV污染的检测结果的符合率为100%。     

浅析禽用活毒疫苗污染外源病毒问题

疫苗的预防接种是目前有效控制家禽疫病的重要手段之一。然而被污染的疫苗不仅不能控制疾病,反而会加速疾病的传播。在许多地方,现在仍然有一些疾病通过污染的疫苗进行传播。由于疫苗生产原材料如组织、细胞基质、血清和胰蛋白酶等来源于动物,因此疫苗产品存在外源病毒污染的风险。通过分析过去禽用活毒疫苗污染外源病毒的事件,发现禽用活毒疫苗中污染禽白血病病毒、网状内皮组织增生症病毒和鸡传染性贫血病毒较为多见。本文就禽用活毒疫苗污染外源病毒产生的危害、历史与现状及疫苗外源病毒检测技术的优点和局限性等进行了综述,并提出了减少禽用活毒苗外源病毒污染的建议措施。     

猪瘟活疫苗外源病毒BVDV不同检测方法之比较

对于猪瘟细胞活疫苗的外源病毒检测,我国的《中国兽药典》有严格的要求和详细的检测方法,《中国兽药典》规定采用细胞培养法判定外源因子感染情况,应无外源病毒污染。但随着PCR技术的发展,许多农业学校、研究所、兽药监测机构常用PCR方法检测猪瘟细胞活疫苗中的BVDV(牛病毒性腹泻病毒)外源病毒。我们同时采用外源病毒检验的细胞培养法(法定的)和PCR方法(流行的)选用了一些猪瘟细胞苗做了BVDV检测的比较试验,结果显示:细胞培养法除了能检测到BVDV,还可检测蓝舌病病毒、细小病毒,对外源病毒检测更全面;PCR方法经常会有假阳性,如果对阳性结果不做基因测序,可能出现BVDV假阳性的结果,导致结果偏差,我们的试验结果希望引起同行的注意。     

猪瘟活疫苗外源病毒BVDV细胞培养与PCR检测的比较

采用细胞培养法和PCR方法,一次性对中股份江西生物药厂生产的18批猪瘟活疫苗(细胞源)进行了外源病毒BVDV(牛病毒性腹泻病毒)两种方法的检测,并进行了比较。结果显示:细胞检验外源病毒方法除了能用荧光抗体检测BVDV外,还能通过致细胞病变检查和红细胞吸附试验检测蓝舌病毒、细小病毒等其它外源病毒;而PCR方法检测的是病毒核酸,并不能鉴别病毒活性,另外,由于PCR灵敏度高,容易出现假阳性,如果不对DNA测序,可能出现假阳性而导致结果误判,给生产企业造成巨大的经济损失。     

影响非禽源外源病毒检验的主要因素

本文以《中华人民共和国兽药典》2015版三部非禽源外源病毒检验方法为依据,总结了检测过程中细胞状态、样品处理、接种与继代培养、荧光抗体检查等对外源病毒检验的影响,为企业提高外源病毒检验的准确度和检出率提供参考。     

部分禽源生物制品外源病毒的检测

进行了部分禽用生物制品外源病毒检测的鸡胚检查法、细胞检查法和鸡检查法。鸡胚检查法应用SPF鸡胚检验;细胞检查法主要通过鸡红细胞吸附试验、禽白血病病毒ELISA和禽网状内皮组织增生症病毒IFA检验疫苗是否含有外源鸡红细胞吸附因子、禽白血病病毒和禽网状内皮组织增生症病毒,并提出疫苗检验的判定标准,为各兽用生物制品生产企业新城疫疫苗的外源病毒检验提供参考。     

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。     

猪瘟疫苗质量的替代检验方法研究进展

国标的猪瘟疫苗效力检验、支原体检验、外源病毒检验等质量检验方法均存在试验周期长、操作繁琐、敏感性较低等缺点,随着现代免疫学与分子生物学技术的快速发展,猪瘟疫苗质量检验的新兴替代检验方法日臻发展和完善。论文就近年来针对猪瘟病毒的病毒含量测定、猪瘟疫苗效力检验、支原体检验、外源病毒检验等免疫学与分子生物学新兴检测方法的研究进展做一综述,以期为提高猪瘟疫苗的质量检验水平、保障猪瘟疫苗质量提供参考依据。     

肉食兽细小病毒阳性血清的制备与应用

为了研制用于检验肉食兽细小病毒相关制品的阳性血清，试验采用猫细小病毒(FPV)-A株对健康易感狐狸进行基础免疫，再用具有致病性的水貂肠炎病毒(MEV)-ZJ1株攻毒免疫的狐狸进行加强免疫，攻毒后第14天无菌采集血液，分离血清，并参照《中华人民共和国兽药典》中的方法对该血清进行细菌、支原体和外源病毒检验及中和抗体效价测定。通过观察细胞病变情况和进行免疫荧光检测测定中和指数，并用阳性血清对3个不同批次的水貂犬瘟热和细小病毒性肠炎二联活疫苗进行外源病毒检验和鉴别检验。结果表明：所制备的血清无细菌、支原体和外源病毒污染。对不同动物来源的肉食兽细小病毒均具有良好的中和作用，中和抗体效价为1∶2 330。对不同动物来源的肉食兽细小病毒的中和指数均大于1×10^4.7。3个批次的水貂犬瘟热和细小病毒性肠炎二联活疫苗顺利通过复合检验。说明试验制备的阳性血清能够用于肉食兽细小病毒相关生物制品的外源病毒检验和鉴别检验。     

Evaluation of the gene for inducible nitric oxide synthase as a radiosensitizer under hypoxic and oxic conditions

The radiosensitizing effect of inducible nitric oxide synthase (iNOS) was evaluated, in vitro, in a feline vaccine‐associated sarcoma (VAS) cell line and a canine osteosarcoma cell line (D17). The gene encoding the human iNOS was cloned into an expression plasmid under the control of a cytomegalovirus immediate early promoter. Transient transfections were performed in feline VAS cells and D17 cells. Nitric oxide was measured in the supernatant media 48 h later as an indirect measurement of iNOS expression. Cells were irradiated using cobalt‐60 under hypoxic or oxic conditions, and clonogenic assays were used to evaluate the effects of gene transfer on the sensitivity of cells to radiation. The results demonstrated that iNOS had no significant effect on improving the radiosensitivity of cells under oxic conditions. However, under hypoxic conditions, iNOS gene transfer significantly improved radiosensitization in osteosarcoma cells. These results demonstrate the feasibility of improving the outcome of radiotherapy in dogs with large bulky tumours using iNOS gene therapy.     

Prevalence of rotavirus and coronavirus antigens in the feces of normal cows.

The prevalence of rotavirus and coronavirus shedding by adult cows was investigated using capture enzyme-linked immunosorbent assays. Fecal samples from 121 cows in a single herd were tested for the presence of rotavirus and coronavirus, either free or complexed with immunoglobulin. Free rotavirus was not detected in any samples while rotavirus-immunoglobulin complexes were detected in 53 of 121 (44%) samples tested. In contrast, free coronavirus was detected in six (5%) samples and coronavirus-immunoglobulin complexes were detected in 85 (70%) of the samples tested. Thus it appears that subclinical infection of cows by either of these viruses is common, possibly providing a source for infection of the neonate. These assays may therefore provide important information regarding the epidemiology of enteric virus infections and suggest means of improving management to prevent epidemics of neonatal diarrhea.     

Application of real-time PCR and ELISA assays for equine influenza virus to determine the duration of viral RNA shedding and onset of antibody response in naturally infected horses

During the equine influenza (EI) outbreak, two assays were used in parallel to diagnose the disease, to demonstrate freedom from infection in disease control zones and ultimately to demonstrate that EI virus had been eliminated from the Australian horse population. A longitudinal study of a population of naturally infected horses was established to determine the performance characteristics of these assays.     

细胞因子的检测及其在鸡免疫抑制病中的应用

细胞因子的检测方法分免疫学检测法、分子生物学检测法和生物学活性检测法。感染鸡的多种病毒引起的免疫抑制与细胞因子的分泌水平和表达异常有关。因此,对细胞因子的检测为鸡病毒性免疫抑制病的发病机理和防制方法的研究提供了重要依据。     

The prevalence of three species of feline haemoplasmas in samples submitted to a diagnostics service as determined by three novel real-time duplex PCR assays

Three distinct species of feline haemoplasmas are recognised: Mycoplasma haemofelis (Mhf), Candidatus Mycoplasma haemominutum (CMhm) and Candidatus Mycoplasma turicensis (CMt). These species differ in pathogenicity as Mhf and CMt can be associated with anaemia whereas CMhm usually results in few clinical signs. The purpose of this study was to develop quantitative real-time PCR assays for the detection of all three feline haemoplasma species combined with an endogenous internal control and to determine the prevalence of infection, using these assays, in 1592 EDTA blood samples submitted to Langford Veterinary Diagnostics, University of Bristol for haemoplasma testing. Primers and TaqMan probes were designed against published 16S rDNA sequences. These assays were combined with a feline 28S rDNA-specific assay to produce three duplex assays. The assays detected 1-10 copies of a sequence-specific plasmid per PCR. None of the assays showed cross-reactivity with 10(6) copies of a sequence-specific plasmid from the non-target haemoplasma species. Real-time PCR was performed on all samples using the three assays. Seven samples were negative for feline 28S rDNA and were excluded from the study. Of the remaining 1585 samples, 45 (2.8%), 177 (11.2%) and 27 (1.7%) samples were positive for Mhf, CMhm and CMt, respectively, including 11 Mhf/CMhm, 10 CMhm/CMt and two Mhf/CMt dual infections and two triple infections. The results of this study demonstrate the utility of these new duplex PCR assays for the detection of haemoplasma infections. CMhm was the most common infection and CMt infections were often associated with co-infection with other haemoplasma species, especially CMhm.     

The role of molecular biology in veterinary parasitology

The tools of molecular biology are increasingly relevant to veterinary parasitology. The sequencing of the complete genomes of Caenorhabditis elegans and other helminths and protozoa is allowing great advances in studying the biology, and improving diagnosis and control of parasites. Unique DNA sequences provide very high levels of specificity for the diagnosis and identification of parasite species and strains, and PCR allows extremely high levels of sensitivity. New techniques, such as the use of uniquely designed molecular beacons and DNA microarrays will eventually allow rapid screening for specific parasite genotypes and assist in diagnostic and epidemiological studies of veterinary parasites. The ability to use genome data to clone and sequence genes which when expressed will provide antigens for vaccine screening and receptors and enzymes for mechanism-based chemotherapy screening will increase our options for parasite control. In addition, DNA vaccines can have desirable characteristics, such as sustained stimulation of the host immune system compared with protein based vaccines. One of the greatest threats to parasite control has been the development of drug resistance in parasites. Our knowledge of the basis of drug resistance and our ability to monitor its development with highly sensitive and specific DNA-based assays for 'resistance'-alleles will help maintain the effectiveness of existing antiparasitic drugs and provide hope that we can maintain control of parasitic disease outbreaks.     

Evaluation of five diagnostic methods for the detection and quantification of Myxobolus cerebralis.

Diagnostic methods were used to identify and quantify Myxobolus cerebralis, a myxozoan parasite of salmonid fish. In this study, 7-week-old, pathogen-free rainbow trout (Oncorhynchus mykiss) were experimentally infected with M. cerebralis and at 7 months postinfection were evaluated with 5 diagnostic assays: 1) pepsin-trypsin digest (PTD) to detect and enumerate spores found in cranial cartilage, 2) 2 different histopathology grading scales that provide a numerical score for severity of microscopic lesions in the head, 3) a conventional single-round polymerase chain reaction (PCR), 4) a nested PCR assay, and 5) a newly developed quantitative real-time TaqMan PCR. There were no significant differences (P > 0.05) among the 5 diagnostic assays in distinguishing between experimentally infected and uninfected control fish. The 2 histopathology grading scales were highly correlated (P < 0.001) for assessment of microscopic lesion severity. Quantification of parasite levels in cranial tissues using PTD and real-time TaqMan PCR was significantly correlated r = 0.540 (P < 0.001). Lastly, 104 copies of the 18S rDNA gene are present in the M. cerebralis genome, a feature that makes this gene an excellent target for PCR-based diagnostic assays. Also, 2 copies of the insulin growth factor-I gene are found in the rainbow trout genome, whose detection can serve both as an internal quality control for amplifiable DNA and as a basis to quantify pathogen genome equivalents present in quantitative PCR assays.     

提高牦母牛繁殖性能的研究

采用不同剂量生殖激素处理3.5岁的青年牦母牛,结果发情率提高5.84和23.26个百分点,繁殖率分别提高34.69和41.21个百分点。采用不同剂量生殖激素处理和怀孕牦母牛围产期补饲的措施,促进产后成年牦母牛发情配种,结果发情率分别提高42.42,54.67和37.51个百分点,繁殖率提高43.93,48.48和20.10个百分点。生殖激素处理和围产期补饲对提高牦牛繁殖性能有较大的促进作用,高剂量生殖激素的促进效果较明显。     

Rhodococcus equi: equine neutrophil chemiluminescent and bactericidal responses to opsonizing antibody

The opsonic capacity of serum containing R. equi-specific antibody was compared with antibody-deficient sera using luminol-dependent chemilumenscence (LDCL) and bactericidal assays. These assays incorporated peripheral blood polymorphonuclear neutrophilic leukocytes (PMNL) exposed to R. equi opsonized with neonatal equine pre-colostral serum (control) or serum from foals with R. equi infections (principal). All sera were complement inactivated at 56 degrees C for 30 min. Bacteria were obtained from the lung of a foal with R. equi pneumonia. Neutrophils were obtained from one adult horse for LDCL and another for bactericidal assays. Chemiluminescence of PMNL exposed to R. equi opsonized with control or principal sera was measured in a liquid scintillation counter. Mean peak LDCL within 1 h was significantly (P less than 0.01) higher with principal sera (2.4 X 10(5) cpm) than with control sera (0.018 X 10(5) cpm). A radioisotope bactericidal assay was used to determine the effect of control or principal sera on PMNL capacity to kill R. equi. Mean peak percent kill of R. equi by PMNL within 2 h was significantly (P less than 0.01) higher with principal sera (95.2%) than with control sera (54.6%). Enzyme-linked immunosorbent assay (ELISA) values for R. equi-specific antibody were determined on all sera. Mean ELISA values were significantly (P less than 0.01) higher for principal sera (71.8) than for controls (0.0). This investigation documents the presence and biological effectiveness of opsonic activity in complement-inactivated sera from foals with R. equi infections and R. equi-specific antibody.     

The requirement for early exposure of Haemonchus contortus larvae to Bacillus thuringiensis for effective inhibition of larval development

The potential for a nematocidal Bacillus thuringiensis (Bt) to target the free-living larval stages of Haemonchus contortus was examined using in vitro larval development and migration assays. Bt toxicity in larval development assays decreased as the time period between egg hatch and initial exposure to the Bt was increased; a time lag of 48 h resulted in a 350-fold increase in the IC(50) (from 2.6 ng/ml to 910 ng/ml). The effects on larval migration largely paralleled the effects on larval development, indicating that the larvae which reached the infective stage after exposure to Bt were generally as 'fit' as control worms in terms of migration ability. However, a comparison of the two assays also showed the presence of a level of Bt exposure which showed significantly more toxicity in migration assays than development assays, indicating that, in some cases, fully developed Bt-exposed larvae were less able to migrate than controls, and hence may be compromised in their ability to infect sheep. The rapid decrease in toxicity when exposure to the Bt is delayed highlights a significant issue concerning the use of Bt for control of the free-living larval stages of animal-parasitic nematodes. Targeting the larvae by delivering bacterial spores to the faeces through the host animal's digestive tract would require the spores to germinate upon defecation, grow through a vegetative phase, to then produce crystal toxin protein upon subsequent sporulation. This period of bacterial development will introduce a time lag between worm egg hatching and initial exposure of the larvae to the Bt, which, as demonstrated in the present study, may allow the worm larvae to develop to late larval stages which are relatively insensitive to the toxin.