鸡败血支原体、鸡传染性鼻炎二联苗的防病优势

用鸡毒支原体的MG—R或MG—S6株和副鸡嗜血杆菌标准毒株HPG221(A型)和HPG668(C型)分别经特定培养基扩增，鉴定、浓缩、灭活后，按适当比例配制，以司盘和吐温为乳化剂，白油为佐剂和适当浓度的硬脂酸铝为稳定剂，经乳化制成用于防制鸡支原体病(AM)和鸡传染性鼻炎(IC)的双价二联灭活疫苗(AM—     

混合感染中鸡毒支原体和副鸡嗜血杆菌的分离鉴定

从冀南地区某养鸡场患呼吸道疾病的病鸡群采集5份样品（气管和眶下窦分泌物）中，同时分离到鸡毒支原体和副鸡嗜血杆菌两种病原体。通过病原的分离培养、细菌L型检验、生化试验、血清学试验等鉴定，证明分离的鸡毒支原体与国际标准株S6的血清型一致，副鸡嗜血杆菌的血清型为A型，动物试验表明，分离的鸡毒支原体和副鸡嗜血杆菌均具有明显的致病性，说明该病鸡群同时混合感染了鸡毒支原体和副鸡嗜血杆菌。     

副鸡嗜血杆菌的分离鉴定及16SrRNA序列分析

从云南省和四川省规模化养鸡场鸡传染性鼻炎疑似病例鸡鼻窦分离到2株革兰氏阴性小杆菌,并对其进行生化鉴 定、PCR鉴定和16SrRNA序列比对鉴定.16SrRNA分析表明两分离株与GenBank中的其他副鸡嗜血杆菌(Haemophilus paragallinarum,HPg)参考株核苷酸同源性为94.2％～99.2％.两分离菌株鉴定为副鸡嗜血杆菌,分别命名为YNAN20120110和SCPZH20120120株.16SrRNA遗传进化关系表明:分离株与NAD非依赖型副鸡嗜血杆菌血清A型代表株GU951543(HP105strain)的16SrRNA序列位于一个分支上,遗传进化关系最近,它们之间的核苷酸同源性为99.2％;与副鸡嗜血杆菌血清A型参考株AY498867(0083株)核苷酸同源性为96.9％.致病性实验表明分离菌株对成年产蛋鸡有强致病性.抗生素药物敏感试验表明:分离菌株对头孢吡肟、头孢噻吩、氯霉素、卡那霉素和氧氟沙星高敏,对四环素中敏,对磺胺甲唑耐药.     

鸡传染性鼻炎平板染色诊断抗原的制备及应用

应用鸡副嗜血杆菌（HPG）A型（HPG221）和C型（HPG668）鸡传染性鼻炎（IC）的致病性血清型国际标准株,制备了IC平板凝集试验（SPA）染色诊断抗原。该抗原特异性强：不但与鸡新凤（ND）、鸡白痢、鸡支原体等9种鸡血清无交叉反应,而且可以进行鸡传染性鼻（IC）血清型的鉴定;敏感性高;SPA较琼脂免疫扩散试验（AGID）高2个滴度;稳定性好：4℃保存2年,检测效果不变;应用此抗原进行平板凝集反应检测IC抗体,操作简便,检测快速,适于普及。     

鸡副嗜血杆菌(HPG-A221、C668)对10个家系BWEL-SPF鸡胚的感染试验

用鸡副嗜血杆菌国际标准株（HPGA-221、C668）分别接种1-10个家系的BWEL-SPF鸡6日龄鸡胚,观察攻毒后16-30小时鸡胚死亡的情况。结果,第5、7、8家系死亡率为100％;1、4、6家系死亡率为90％;2、3、9、10家系死亡率为80％-85％,10个家系HPG平均死亡率为90.5％。对照组（HWL-SPF鸡胚）平均死亡率为90％。从而评估BWEL-SPF鸡群每个家系对鸡副嗜血杆菌（HPG-A221、C668）的敏感性。     

B型副鸡嗜血杆菌的分离鉴定

从辽宁某公司鸡场疑似鸡传染性鼻炎的病鸡眶下窦分离到一株副鸡嗜血杆菌，用Page程序和Kume程序对其进行血清型鉴定，确认为B型副鸡嗜血杆菌，这是首次在我国分离到B型副鸡嗜血杆菌。     

用单克隆抗体进行副鸡嗜血杆菌定型

抗副鸡嗜血杆菌血清A和C型株所制备的两个血清型单克隆抗体（MAbs），分别对副鸡嗜血杆菌血清型A、B、C中的各型参考株作HI和dot－blotting试验。一种MAb（E5C12D10）为抗血清型A代表株221，另一种MAb（F2E6）为抗血清型C代表株S1。在两种试验中，不同血清型的MAbs可与对应的血清型中的副鸡嗜血杆菌株血凝（HA）抗原反应，而与血清型B代表株91、147均无反应。故这些MAbs可用于dot－blotting或HI试验进行副鸡嗜血杆菌定型。     

A型副鸡嗜血杆菌(山东株)的分离与鉴定

2011年11月,山东某肉鸡场发生以肉鸡眼睑及眶下窦周围明显肿胀为主要特征的传染性疾病。从采集的发病鸡眶下窦中分离到1株细菌,根据发病鸡群的临床症状、细菌的分离培养、形态观察、过氧化氢酶试验、革兰氏染色、动物回归试验、免疫攻毒试验和PCR等方法初步鉴定为副鸡嗜血杆菌(Hpg)。此外通过分离菌株的水平传播试验结果表明,造成该鸡场Hpg的水平传播和鸡传染性鼻炎的反复发作与饲养密度有很大的关系。最后将分离菌株送匈牙利诗华研发中心进行分型鉴定,确定为A型副鸡嗜血杆菌。根据分离地点将分离菌株命名为A型副鸡嗜血杆菌(山东株)。另外通过与GenBank已发表的Hpg基因序列进行同源性比对分析,结果显示,该基因序列与参考株的基因核苷酸序列同源性达99.0%。     

鸡支原体,鸡传染性鼻炎双价二联油乳剂灭活疫苗的研究

应用鸡败血支原体国际标准株（ＭＧＲ）和鸡副嗜血杆菌（ＨＰＧ）国际标准株Ａ型（ＨＰＧ２２１）和Ｃ型（ＨＰＧ６６８）制备了抗鸡支原体病（ＡＭ）和鸡传染性鼻炎（ＩＣ）的双价二联油乳剂灭活疫苗。通过多批次ＳＰＦ鸡免疫试验,进行了疫苗安全性、抗体产生时间及抗体动态变化规律、攻毒保护率、免疫期及现地鸡群的中间试验。结果表明,疫苗接种后１０～１５天可相继产生ＭＧ和ＨＰＧ血清抗体;接种６个月内,强毒攻击的免疫保护率为９１％～１００％;各项试验的指标不低于各自的单苗,具有很好的推广应用价值。     

青岛地区鸡传染性鼻炎病原株的分离,鉴定与定型研究

1993年青岛市周围一些鸡场相继发生了传染性鼻炎，为确诊本病，从一些鸡场选发病典型鸡，进行了细菌分离和鉴定试验，获得副鸡嗜血杆菌4株，按Kume等人方法作血清学定型，所有分离株被定为C型。     

三株牛支原体的致病性比较

为检测从内蒙古发病牛场分离到的3株牛支原体(HS2019、HSZ2019、HSS2019)的致病性,对其进行本动物回归试验,通过观察攻毒后的临床症状、病理变化,以及应用实时荧光定量PCR确定组织器官中支原体载量,分析3株支原体的毒力。结果显示,3株牛支原体回归牛体后均使试验牛出现体温升高、咳嗽、呼吸困难等临床症状,解剖可观察到试验牛肺部损伤以及肺脏与胸腔粘连的病理变化,其中HS2019株较其他两株引起的症状与病变更为明显,组织脏器中的载菌量最高。结果表明,这3株支原体均有致病性,其中HS2019株致病性最强,可作为今后疫苗研制的预备菌株。本研究既为国内疫苗的研制提供了菌株资源,也为今后牛支原体的免疫攻毒试验提供了评价标准。     

Prevalence of hemoplasmas and Bartonella species in client-owned cats in Beijing and Shanghai,China

A year-round molecular epidemiological survey (2017 to 2018) was conducted on three hemoplasmas and two Bartonella species with zoonotic potential in client-owned cats in Beijing and Shanghai. Among 668 specimens, the overall hemoplasma-positive rate was 4.9% (3.4% for Candidatus Mycoplasma haemominutum, 0.9% for Mycoplasma haemofelis and 1.2% for Candidatus Mycoplasma turicensis). The overall Bartonella-positive rate was 8.5% (4.8% for B. henselae and 4.3% for B. clarridgeiae). Age, breed, ectoparasiticide use and stray history, but not city, season and gender, were significantly associated with the positive rates of one or more pathogens. This is also the first report on the prevalence of Candidatus Mycoplasma turicensis in cats in China.     

Adherence to various host cell lines of Mycoplasma bovis strains differing in pathogenic and cultural features

Mycoplasma bovis is known to be responsible for pneumonia and arthritis in calves, as well as mastitis in dairy cows. Despite clear evidence of its pathogenic potential, little is known about mechanisms of cytadherence and the molecular factors involved. The purpose of this work was to compare adherence rates of M. bovis field strains to different host cell lines and study the effects of cloning and sub-culturing M. bovis strains on their adherence properties. Eighteen metabolically labeled M. bovis strains isolated from different pathological backgrounds were examined in adherence trials using four different host cell lines, i.e. embryonic bovine lung (EBL), embryonic bovine trachea (EBTr), Madin Darby bovine kidney (MDBK) and rabbit kidney (RK) cells. Although large interstrain variations in adherence rates (3.4-19.1%) were measured they could not be correlated to the pathological background (pneumonia, arthritis or mastitis). Adherence rates to the fibroblast cell line (EBTr) were significantly lower than those to the three epithelial cell lines (EBL, MDBK and RK). The only non-pathogenic strain (221/89) exhibited lower adherence rates than three isolates from clinical mastitis. Interestingly, adherence rates were significantly reduced after in vitro passaging. In contrast, no effect of single cloning of strains on adherence was observed. There was no general correlation between expression of variable surface proteins (Vsps) as monitored by immunoblotting and adherence rates, although alterations in Vsp expression profiles were seen as a consequence of passaging. As there is probably a large number of adhesins, variable and non-variable, on the surface of M. bovis cells the issue is very complex, and the most active components have yet to be identified.     

Isolation of Mycoplasma capricolum-like strains from chickens

Members of the genus Mycoplasma infect a wide range of hosts, but individual Mycoplasma species tend to exhibit a considerable degree of host specificity. We characterized Mycoplasma strain 700, isolated from a kidney of a layer hen in Spain and Mycoplasma strains ULB-A and ULB-B, isolated from the air sac and from the bile of stunted broiler chickens in Slovenia. The serologic examination showed that these three strains are antigenically unrelated to all of the recognized Mycoplasma species of avian origin, but closely related to the ruminant mycoplasma Mycoplasma capricolum subspecies capricolum (M. capricolum). The comparison of their 16S rRNA gene sequences with the sequence of M. capricolum (California kid) revealed 99.66% sequence identity for the strain 700 and 99.59% identity for strains ULB-A and ULB-B. Moreover, the predicted DnaK sequences of the M. capricolum-like strains, isolated from chickens, were identical to DnaK sequences of M. capricolum. Comparison of their dnaK gene sequences with M. capricolum showed 99.64% sequence identity for strain 700 and 99.27% identity for strains ULB-A and ULB-B. In the flock from which M. capricolum-like strains ULB-A and ULB-B were isolated, the majority of chickens (83% of the chickens examined) raised antibodies reacting with M. capricolum antigens. Notably, the infection of chickens with M. capricolum-like strains represents an unusual exception to the range of Mycoplasma species host specificity.     

A Serologic Variant of Mycoplasma Hyorhinis Recovered from the Conjunctiva of Swine

In an examination of conjunctival samples from 40 piglets for mycoplasmas, 17 isolates were obtained. Eight could be identified as Mycoplasma hyorhinis, three as Mycoplasma flocculare, and one as Acholenlasma sp. Five strains were not readily identifiable, but together with two previously recovered strains they were found to represent a distinct serogroup. All seven strains were glucose and phosphatase positive. Incubation in a CO₂-enriched atmosphere led to enhancement of the growth on solid medium. The serogroup was serologically related to M. hyorhinis, but not to a number of other glucose fermenting species of mycoplasma, and it may therefore be regarded as a new subspecies of M. hyorhinis.     

旋毛虫T668基因真核表达载体的构建及其在BHK细胞中的表达

利用RT-PCR技术从中国河南猪旋毛虫(国际标准虫种编号为ISS534)新生幼虫得到T668基因,并克隆入pEGFP-N1真核表达载体中构建重组质粒,该重组质粒在脂质体介导下转染BHK细胞。EGFP标签证明质粒DNA成功转染到细胞中并得以表达,Western blotting鉴定,细胞裂解液样品中有1条约77 000的条带,可被绿色荧光抗体及猪感染旋毛虫阳性血清所识别,与预计大小一致,表明成功构建T668-pEGFP-N1真核表达质粒,并在BHK细胞融合表达,表达产物具有抗原性。     

Evaluation of PCR systems for the identification and differentiation of Mycoplasma agalactiae and Mycoplasma bovis: a collaborative trial

Diagnostic differentiation between the ruminant pathogens Mycoplasma agalactiae and Mycoplasma bovis is known to be problematic when only conventional serological and biochemical tests are used. The main reason for this is that both agents share a considerable number of related proteins and common epitopes. DNA-based detection methods offer advantages in terms of specificity and sensitivity. However, there is an urgent need to compare currently used PCR assays because they target different genomic regions and, therefore, may perform differently. In the present work, five laboratories, which use PCR routinely, evaluated the specificity of four different PCR systems for M. agalactiae and three systems for M. bovis on a total of 41 strains of the two Mycoplasma species including six previously unidentified strains. As the vast majority of PCR examinations (97.1% of all tests) correctly identified the strains the specificity of all seven detection systems appears to be high. In four cases, incorrect identification by conventional diagnostic methods was rectified by PCR. Isolates from non-typical hosts, i.e. three M. bovis strains from small ruminants and two M. agalactiae strains from cattle, were characterised by sequencing the 16S and part of the 23S ribosomal RNA genes.     

Mycoplasma Dispar As a Causative Agent in Pneumonia of Calves

Three field strains of Mycoplasma dispar were inoculated, by aerosol inhalation, into a total of eight naturally-born, colostrum-deprived calves. All three strains produced macroscopic pneumonia, each in one calf. Histopaithologically an exudative bronchitis accompanied by moderate interstitial cell proliferations was found.Reisolation studies indicated that the entire respiratory tract is the natural habitat of Mycoplasma dispar, which apparently does not spread via the blood stream.     

7株鸡滑液囊支原体四川分离株全基因组分析

旨在了解滑液囊支原体(Mycoplasma synoviae,MS)遗传多样性,本研究对7株MS四川分离株进行全基因组测序及生物信息学分析.采用Illumina HiSeq平台和PE文库结合方式全基因组测序,运用各数据库注释,用MEGAX软件将7株MS与NCBI收录的8株MS全基因比较.结果 显示,分离自同一鸡场的4株...     

牛支原体新疆株的分离鉴定

为调查新疆规模化奶牛场病牛死亡原因并确定病原,本研究无菌采集7份肺炎病死牛病变肺组织样,通过牛支原体液体培养基和固体培养基分离到1株支原体,采用形态学观察和生化试验鉴定该分离株,采用支原体特异性引物和牛支原体16S rRNA通用引物扩增基因序列并测序,使用DNAStar软件将分离菌株测序结果与GenBank中的标准株序列进行同源性比对,采用Mega 6.0软件中的邻接法(Neighbor-Joining,NJ)依据16S rRNA序列构建分离株系统进化树。结果显示,分离株菌落呈典型的"煎蛋样",菌落中心凹陷深入培养基,周边菲薄而透明,经Dienes染液染色后,菌落中心呈深蓝色。该分离株不分解葡萄糖、尿素、不水解精氨酸,血细胞吸附试验和溶血试验均呈阴性,氯化三苯基四氮唑还原反应呈阳性,产生膜和斑。PCR反应扩增出大小为1 911 bp的牛支原体特异性目的片段;分离株16S rRNA基因序列与牛支原体标准株PG45的序列同源性为99.8%,与牛支原体地方株(Mb NM2012、Mb HB0801、Mb Hubei-1、Mb Ningxia-1、Mb CQ-W70和Mb 08M)的同源性为99.3%～99.7%。系统进化树显示,分离株16S rRNA基因与Mb Ningxia-1株和Mb 08M株亲缘关系较近,处于同一分支。本研究结果证实了引起病牛死亡的病原为牛支原体,为新疆牛支原体病的防治提供了科学依据。