首届兽药职业技能大赛参赛选手活菌计数能力分析

通过对首届兽药职业技能大赛62名参赛选手大肠杆菌活菌计数结果进行分析,了解我国兽用生物制品生产企业活菌计数能力的整体水平。每名参赛选手发放大肠杆菌菌液1支．要求参赛选手在规定时间内进行活菌计数并提交计数结果。40％参赛选手活菌计数检验结果满意（25／62）;60％检验结果不满意（37／62）。此次技能大赛体现出我国兽用生物制品生产企业检验人员的活菌计数水平参差不齐,部分检验人员活菌计数能力有待进一步提高。     

不同保存温度对布氏菌病活疫苗（S2株）细菌存活的影响

（《中华人民共和国兽用生物制品规程》规定布氏菌病活疫苗贮藏条件为2℃～8℃,有效期为一年。但在疫苗检验过程中,发现保存在2℃～8℃条件下的布氏菌病活疫苗活菌数下降的幅度较大,造成活菌数时有不达标的情况。为了保证疫苗在一年有效期内的活菌数量,常常采用增大附加量（计算头份时一般不少于30％的附加量）或低温冷冻条件保存（-15℃以下）的方法。     

布氏菌病活疫苗生产现状与质量分析

为了解布氏菌病活疫苗质量状况,对其活疫苗连续三年的生产现状和监督检验结果进行了统计分析,同时对疫苗生产和检验过程中存在的问题提出了改进建议。     

布氏菌病活疫苗（Rev.1株）冻干保护剂工艺的研究

布氏菌病活疫苗(Rev.1株)的冻干工艺借鉴、采纳布氏菌病活疫苗(S2株)成熟的冻干工艺及菌液的配比,对冻干保护剂的选择进行了优化。我们选择蔗糖明胶保护剂和蔗糖脱脂乳保护剂,保护剂与布鲁氏菌Rev.1株菌液以16的比例混合,冻干后测定其活菌含量。结果显示,3批使用蔗糖脱脂乳保护剂的不同配比比例的冻干疫苗活菌数分别为12×10^9CFUmL、12.44×10^9CFUmL、12.65×10^9CFUmL;使用蔗糖明胶再加入终浓度约1%硫脲的不同配比比例的冻干疫苗活菌数分别为12.34×10^9CFUmL、12.62×10^9CFUmL、12.91×10^9CFUmL;使用蔗糖明胶的不同配比比例的冻干疫苗活菌数分别为12.22×10^9CFUmL、12.64×10^9CFUmL、12.8×10^9CFUmL。3种冻干保护剂对冻干疫苗的活菌含量影响无明显差异,但使用蔗糖明胶保护剂制备冻干疫苗时加入适量硫脲可适当增加冻干疫苗的耐热性,因此我们选择10%明胶、20%蔗糖冻干保护剂与菌液以1∶6配比比例混合,再加入终浓度约为1%的硫脲作为布氏菌病活疫苗(Rev.1株)的冻干工艺。     

稳健统计方法及t检验法在活菌计数能力比对中的应用

为比较检验员活菌计数能力及常量法和微量法在活菌计数应用中是否存在差异,8名检验员于同时、同环境中对同一细菌培养物采用常量法和微量法进行活菌计数,采用稳健统计方法对不同人员相同方法的计数结果进行统计,将检验结果分为“满意”、“可疑”和“不满意”;采用t检验法对使用不同方法对同一样品的计数结果进行统计,将结果分为“差异显著”和“差异不显著”。稳健统计方法表明,用常量法进行检测,有6名检验员的检验结果全部满意,而微量法的结果中只有2名检验员获得了全部满意的结果;t检验法表明常量法和微量法之间存在显著差异。稳健统计方法及t检验法统计结果显示,不能用微量法代替常量法进行检验,个别检验员的活菌计数能力有待进一步提高。     

沙门氏菌类活疫苗活菌计数参考疫苗的研究

为研制沙门氏菌类活疫苗活菌计数的参考疫苗,以期更科学地评价活菌计数结果的有效性,研究制备了一批仔猪副伤寒活疫苗作为参考疫苗候选物,对其性状、纯粹检验、真空度、剩余水分进行检测,对其运输稳定性、均一性进行测定,组织协作标定对参考疫苗候选物进行赋值,用协作标定的方法统计参考疫苗候选物在18个月的保存期,将参考疫苗初步应用于仔猪副伤寒活疫苗活菌计数的检验。结果表明,参考疫苗的性状、纯粹检验、剩余水分和真空度测定均符合《中国兽药典》的规定,均一性试验结果显示10瓶参考疫苗候选物计数结果的变异系数小于10%,说明其均一性良好。协作标定统计出参考疫苗候选物的赋值范围为(3.0,4.0)×109CFU/头份,保存期试验结果显示参考疫苗候选物在-70℃以下保存18个月菌数稳定。参考疫苗的初步应用显示本参考疫苗不仅可以为沙门氏菌类活疫苗活菌计数提供参照物,而且还可用于控制普通琼脂培养基的质量。     

兽用生物制品活菌计数方法改进的研究

活菌计数是细菌类活疫苗质量标准中重要的检验参数,与疫苗的保护效果显著相关。现行兽用细菌类生物制品检验中的活菌计数方法部分步骤易产生误差,试验成立标准表述笼统,最终影响到细菌活疫苗质量评判。因此,从检验实际需求出发,参照国内外活菌计数相关规程,通过比对试验研究,对“活菌计数法”中平板干湿程度、菌液滴加方法、菌液分散方法进行改进,提出及验证以标准差(s)衡量结果的有效性并提供参考值。改进后的方法要求更明确,普适性更高,从而减少了操作误差,提升了“活菌计数法”的规范性、可操作性及检验结果可靠性。     

猪丹毒活疫苗活菌计数参考品的研究

为了更加客观、科学地评价猪丹毒活疫苗活菌计数检验结果,在活菌计数检验中引入了参考品。研制并标定了一批猪丹毒活疫苗活菌计数的参考品,从性状、纯粹性、均一性、加速稳定性、运输稳定性、剩余水分测定、真空度等方面对参考品进行了评价,并对参考品的保存期限进行了探索。结果显示:参考品的性状、纯粹性、活菌计数、剩余水分含量、真空度均符合《中国兽药典》中猪丹毒活疫苗质量标准。通过协作标定,对参考品赋值为每头份含活菌数为8.5×10~8～12.5×10~8 CFU。均一性检验变异系数分别为7%和6%。参考品在夏季和冬季由泡沫盒加冰袋的方式进行运输不超过48 h,计数结果稳定。对参考品的保存期进行探索,发现参考品于-70℃保存时可在18个月内保持稳定。表明猪丹毒活疫苗活菌计数参考品符合国家标准物质的要求,可应用于检验。     

猪链球菌病活疫苗在室温及高温环境中保存菌数存活试验

本试验对保存于室温(25℃)及高温(37℃)环境下的猪链球菌病活疫苗(ST171株)进行了活菌计数。结果表明,在室温中保存,14d菌数没有变化,150d菌存活率为60%;在高温中保存,3d菌数没有变化,7~14d菌存活率为50%,32d菌存活率仅为12%。     

多杀性巴氏杆菌类活疫苗活菌计数参考品的研究

为了研制多杀性巴氏杆菌类活疫苗活菌计数的参考品,以期更科学的评价活菌计数结果的有效性。制备一批猪多杀性巴氏杆菌病活疫苗为参考品候选物,对其性状、纯粹、真空度、剩余水分进行检测,对其运输稳定性、均一性进行测定,组织协作标定对参考品候选物进行定值,用协作标定的方法统计参考品候选物24个月的保存期,将参考品应用于多杀性巴氏杆菌类活疫苗活菌计数的检验。结果表明,参考品的性状、纯粹检验、剩余水分和真空度测定均符合《中国兽药典》的规定,均一性试验结果显示,10瓶参考品候选物计数结果的变异系数为5.4%,小于10%,说明其均一性良好。协作标定的赋值范围为(4.2～5.6)×10~8CFU/头份,保存期试验结果显示,参考品候选物在-70℃以下保存24个月菌数稳定。参考品的初步应用显示本参考品不仅可以为多杀性巴氏杆菌类活疫苗活菌计数提供参照物,而且可以用于控制多杀性巴氏杆菌类疫苗检验用马丁琼脂培养基的质量。     

Immunogenic and protective antigens of Brucella as vaccine candidates

Brucella is an intracellular pathogen that causes abortion in domestic animals and undulant fever in humans. Due to the lack of a human vaccine against brucellosis, animal vaccines play an important role in the management of animal and human brucellosis for decades. Strain 19, RB51 and Rev1 are the approved Brucella spp. vaccine strains that are most commonly used to protect livestock against infection and abortion. However, due to some disadvantages of these vaccines, numerous studies have been conducted for the development of effective vaccines that could also be used in other susceptible animals. In this review, we compare different aspects of immunogenic antigens that have been a candidate for the brucellosis vaccine.     

A simulation model of brucellosis spread in British cattle under several testing regimes

Brucellosis is a widespread, economically devastating and highly infectious zoonosis. In cattle, infection predominantly is caused by Brucella abortus, and is usually detected in pregnant females through abortions. Great Britain (GB) has been declared free from brucellosis (officially brucellosis free (OBF)) since 1993 and as such is required by European Union (EU) regulations to test > or =20% of both beef and dairy cattle >24 months old routinely. Currently, however, GB serologically tests more cattle than required and the issue of reducing the level of testing has come under consideration. We developed a simulation model to determine the rate of spread of brucellosis under a variety of testing regimes. For dairy herds, we found that reducing the level of testing would have a major effect on the rate of spread of infection, should it be imported. For beef herds, reducing the level of testing would have much less effect. We also found that abortion notification is a very-important additional means of surveillance. As a result of our predictions, policy-makers decided not to reduce the level of testing and actively to promote abortion notification.     

Intestinal torsion in cattle

Brucella abortus strain 19 vaccine became widely available in New Zealand in 1950. In 1966, vaccination was made compulsory and age restrictions were imposed. Surveys of the cattle population were carried out between 1966 and 1970 to establish the prevalence of brucellosis and to compare the performance of various diagnostic tests under New Zealand conditions. In 1969 and 1970 a voluntary test-and-slaughter scheme was introduced basedon the brucellosis card test as a screening test, the complement fixation test as the definitive test, and the milk ring test to screen accredited dairy herds. This scheme was extended to a compulsory national Brucellosis Eradication Scheme in September 1971, with the aim of bringing all eligible cattle under test by 1977.     

Brucellosis in the European Union and Norway at the turn of the twenty-first century

Control and eradication programs of brucellosis in cattle, sheep, goats and pigs have been more or less successfully implemented within the Member States (MS) of the European Union (EU) and Norway after Word War II. As a result, the epidemiological situation of animal brucellosis is extremely diverse among different MS or regions within a MS and among the different animal species. Some MS, mainly North European countries, and Norway are declared “officially bovine brucellosis free” and/or “officially ovine and caprine (Brucella melitensis) free”. The situation is less favorable in Southern European countries, particularly as far as sheep and goat brucellosis are concerned. This situation has important zoonotic consequences as reflected in the number of human brucellosis cases due to B. melitensis that are still encountered in those countries. Brucellosis in swine has re-emerged as a result of spillover from the wild boar brucellosis (Brucella suis biovar 2) reservoir, particularly in outdoor reared pigs. Besides the actual challenge to eradicate brucellosis, further issues have to be addressed: (1) the management of false positive serological results that occur in the course of brucellosis testing, particularly in cattle; (2) the impact of wildlife brucellosis, particularly wild boar brucellosis in domestic animals; and (3) the importance of B. melitensis infection in cattle that are in contact with infected sheep.     

An evaluation of Irish cattle herds with inconclusive serological evidence of bovine brucellosis

Since 1998, there has been a steady decline in herd restrictions and de-populations in Ireland due to bovine brucellosis. There is concern that the interpretation of laboratory results may become increasingly problematic, as brucellosis prevalence falls in Ireland. Therefore, the purpose of the current study was to evaluate the infection status of Irish herds and animals with inconclusive serological evidence of bovine brucellosis. During 12 months from September 1, 2004, laboratory and observational epidemiological data were collected from all Irish herds where animal testing identified at least one animal with a complement fixation test (CFT) reading greater than zero and/or a positive result to the indirect enzyme-linked immunosorbent assay (iELISA). Due to the observational nature of the study, we have robust estimates of the relative, but not the absolute, performance of the CFT, iELISA and brucellin skin test (BST). Herds were divided into three categories (Group A, B or C) on the basis of test results at initial assessment. A total of 639 herds were enrolled into the study, and observed for at least two years following enrolment. A rising CFT titre, with a CFT reading of 111 International CFT Units (IU) or greater at the subsequent blood test, was generally associated with herds where other evidence of infection was also available. Knowledge of the CFT reading at the initial and a subsequent blood test proved useful in distinguishing false-positive and true-positive brucellosis results. There was poor correlation between the CFT and iELISA results, and between the CFT and BST results. As a result of this study, national policy has been modified to include re-sampling of all animals with CFT readings of 20 IU or greater. This project has also led to a reduction in the number of herds restricted, as well as restriction duration. It has also contributed to a reduction in the number of herds listed for contiguous tests, and therefore the potential for contiguity testing of false positive results.     

Brucellosis in Argentina

Brucellosis has been recognized in Argentina since the 19th century. Several studies demonstrated the presence of the disease in most of the domestic species. Actually, the estimate of prevalence is that between 10 and 13% of the farm animals are infected with bovine brucellosis with an individual rate of 4–5%. The annual economical losses have been estimated at US$ 60,000,000. The control of bovine brucellosis began in 1932 and successive resolutions have been issued since then. The current resolution indicates that B. abortus S19 is mandatory in female calves between 3 and 8 months of age. The vaccine strain B. abortus RB51 was provisionally approved but only for cattle older than 10 months of age. The brucellosis control program consists principally of test and slaughter. This methodology has been successful mainly in the dairy farms that have the incentive due to increased pricing because of obtaining a low prevalence of the disease. Brucellosis has been found in porcine, caprine, ovine and canine species. All Brucella species have been found in the country. Human brucellosis is an important disease and a national coordinated diagnostic net has been formed to better control the disease in man.     

动物布鲁氏菌Rev.1疫苗研究进展

布鲁氏菌病（布病）是由布鲁氏菌引起的一种重要的人畜共患病,对公共卫生有着巨大的威胁。布鲁氏菌Rev.1疫苗研制于上世纪50年代中期,并在欧洲、中东和蒙古等地区被广泛应用于小反刍动物布病的防控,一度被认为是防控小反刍动物布病最为有效的疫苗。但是,由于Rev.1疫苗存在毒力偏强及毒力不稳定等现象,其安全性仍值得关注。本文主要从Rev.1疫苗背景及其菌株的生物学特性、疫苗使用情况以及存在的不足等几个方面进行阐述,以期为Rev.1疫苗的应用和布鲁氏菌相关疫苗的开发提供借鉴。     

Rabies and brucellosis immunization status and adverse reactions to rabies vaccines in veterinary students.

Rabies pre-immunization has been recommended for high risk professions, including veterinarians. Cell-cultured rabies vaccines have considerably reduced the risk of post-vaccination neurological reactions found in earlier vaccines. However, some adverse reactions have been reported with Human Diploid Cell Vaccines. 329 French veterinary students were surveyed about their rabies and brucellosis vaccination status, the occurrence of adverse reactions to rabies vaccine, and their antibody titer monitoring practices. Questions also were asked to determine if mandatory rabies pre-exposure immunization upon entry to veterinary school motivated students to maintain their rabies pre-exposure vaccination. The overall vaccination rate was 98.5% for rabies and 17% for brucellosis. 19% of the rabies vaccinated students reported some form of adverse reaction, whatever the vaccine brand used, but not experienced systemic allergic reaction. Adverse reactions were twice more frequent in female than male students and were more frequent after primary series than revaccination series (Relative Risk = 1.76). Despite the mild reactions encountered, rabies pre-exposure vaccination has been well-accepted by French veterinary students. In contrast, vaccination against brucellosis was not as well-accepted for prophylaxis.     

动物布鲁氏菌病活疫苗生产环节生物安全风险分析与对策

布鲁氏菌病（布病）活疫苗是世界各国防控布病的重要生物资源,我国目前主要生产和应用的布病活疫苗为S2株和A19株。生物安全是布病活疫苗生产过程中的必须优先考虑的重要环节,随着兽药GMP（2020年版）的即将实施,规范布病活疫苗的生产与检验,防控生产环节中的生物安全风险,成为迫切需要研究和解决的突出问题。本文通过对布病活疫苗生产检验、设施维修维护、生产人员个人防护等环节的生物安全风险点进行分析,对布病活疫苗生产线提出改进建议,为相关企业动物布病活疫苗生产提供参考。     

Brucellosis in domestic water buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>) of Trinidad and Tobago with comparative epidemiology to cattle

The water buffalo is an important domestic animal worldwide, and the local Buffalypso variety was developed in Trinidad to have improved beef qualities. Brucellosis was diagnosed in Trinidad and Tobago during 1998 in both cattle and domestic water buffalo (Bubalus bubalis) populations. Brucellosis in the latter species is caused by infection with Brucella abortus, similar to bovine brucellosis. Control of brucellosis is of paramount importance to preservation of the genetic diversity of these animals in Trinidad, and this has been complicated by differences in the epidemiology of water buffalo and bovine brucellosis. Some diagnostic tests do not have comparable accuracy between the two species, and the RB51 vaccine does not adequately protect against infection in water buffalo. The water buffalo in Trinidad may also be more resistant to infection than cattle. Development of effective vaccination protocols is key to brucellosis control in Buffalypso in Trinidad, and prohibitions on import of virulent B. abortus strains for vaccine efficacy studies has impeded progress in this area. These Trinidadian strains are of variable virulence; some might be effective for challenge in vaccine efficacy studies, while other, of lower virulence, may be vaccine candidates for use in water buffalo.