猪瘟病毒低毒力毒株FJFQ株的分离鉴定

从福建某猪场分离到 1 株病毒,其在PK 15细胞上的毒价为 106.5 TCID50/mL,该病毒能被猪瘟病毒高免血清所中和(效价为1∶8)。通过 RT -PCR 扩增出猪瘟病毒约250 bp的E2蛋白主要抗原编码区序列,其与几株已发表毒株序列的核苷酸及氨基酸同源性分别为79.9%~87.9%,77.7%~86.6%,与Alfort 株同属于基因二群。经本动物传3代均不表现明显的临床症状。用猪瘟兔化弱毒疫苗免疫后以此分离毒作强攻进行免疫保护相关实验,结果免疫组猪在攻毒前及攻毒后扁桃体 HCFA检测均为阴性,对照组猪扁桃体HCFA于攻毒后1周开始出现阳性结果,且一直持续到试验结束。用分离株免疫本动物后再攻石门毒, 2 头试验猪中 1 头死亡,1头出现临床症状。初步说明,所分离的病毒为猪瘟病毒(命名为CSFV- FJFQ株),可能是一株低毒力毒株,且其免疫原性不好。     

Isolation of Chlamydia psittaci from nasal and conjunctival exudate of a domestic cat

A feline strain of Chlamydia psittaci was isolated in tissue culture from nasal and conjunctival swabs from a free range domestic cat with bilateral conjunctivitis and rhinitis, living in the Liverpool area of the UK. The isolate was identified as C psittaci on the basis of its characteristic inclusion morphology in cell culture and by means of specific indirect immunofluorescence with known C psittaci specific antiserum. The isolate could be differentiated from other chlamydiae of non-feline origin by its amino acid nutritional requirements.     

上海地区猪源链球菌分离株的病原特性鉴定

1997－1999年从上海地区分离出15株猪源链球菌，结合培养特性、生化特性及血清定型等对其进行了鉴定。其形态、染色及培养特性基本符合链球菌的特点，大多具有α或β溶血，生化试验结果不稳定。用国际通用的链球菌A－G乳胶分型诊断液和猪链球菌1、2型标准阳性血清检测5株分离菌，4株为C群链球菌，1株为2型猪链球菌，1株为B群链球菌，1株为D群链球菌，1株与A群和C群有交叉反应，7株不在其检测范围。小鼠对15株分离菌的敏感性有所不同。本试验表明，上海地区猪链球菌病的病原已不再是单一的C群链球菌。对新出现的2型猪链球菌，应引起足够重视。     

伪狂犬病弱毒株的分离鉴定及生物学特性的研究

在流行病学调查中分离到1株病毒,经鉴定为伪狂犬病弱毒株,定名为F971株。分离病毒经克隆纯化后测得其毒价为10^7.59TCID50/ml,通过细胞中和试验表明分离病毒能也有效地被猪伪狂犬病毒闽A株阳性血清中和。病毒在电镜下可以清楚地观察到囊膜及外周纤突。分离株对3日龄乳鼠有一定的致病力,但对家兔、3日龄乳猪及妊娠母猪都有很高的安全性。用不同的剂量10^0、10^-1、10^-2肌肉注射3日龄乳猪后14天用10^5.7TCID50伪狂犬病强毒攻击,所有试验仔猪均得到保护。用分离株免疫母猪,其后代可获高滴度的母源抗体,15日龄的仔猪能抵御10^5.7TCID50强毒的攻击。用ELISA普查试剂盒测定免疫猪抗体,结果均为阳性,而用g^1-ELISA试剂盒测定抗体时,结果均为阴性。证明分离株具有缺损g^1糖蛋白的特性。综合上述特性,确定F971为1株g^1糖蛋白缺损的猪伪狂犬病弱毒株。     

牛病毒性腹泻病毒JN株的分离鉴定及E2基因的序列分析

本研究从疑似牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)感染牛的分泌物与排泄物中分离鉴定1株牛病毒性腹泻病毒,并进行E2基因序列分析。结果表明,分离株病毒命名为JN株;Reed-Muench法测定分离株病毒TCID₅₀为10^-7.5/0.1 mL;病毒中和试验结果表明,BVDV JN分离株可被BVDV阳性血清特异性中和,而不能被BVDV阴性血清中和;分离株病毒E2基因序列测序结果表明,该分离毒株属于BVDVⅠa亚型。     

解淀粉芽孢杆菌的分离鉴定及其对嗜水气单胞菌抑制作用研究

本试验从鲫鱼肠道中分离到1株芽孢杆菌,经生物学特性测定及16S rRNA测序分析,鉴定该分离菌株为高产蛋白酶的解淀粉芽孢杆菌(命名为JX001).对该分离菌进行嗜水气单胞菌体外抑菌试验,结果发现有明显的抑菌圈和抑菌带,说明该分离菌对嗜水气单胞菌具有较强的抑制作用,在养殖水中加入分离菌JX001可防止嗜水气单胞菌的暴发;同时分离菌JX001在生长代谢过程中产生大量的蛋白酶,可有效地促进蛋白质水解,用于蛋白质饲料的降解.     

伪狂犬病病毒弱毒株LY株的分离鉴定

从辽阳某猪场的10日龄仔猪中分离到1株病毒,经纯化后测得其毒价为107.29TCID50/mL.细胞中和试验表明,该病毒能被猪伪狂犬病病毒标准阳性血清所中和.电镜下可见到典型的疱疹病毒粒子,具有囊膜及外周纤突.所分离的病毒对氯仿、胰蛋白酶、乙醚敏感,在pH5.0～9.0下稳定,56℃ 30 min可以灭活.应用特异性引物,通过PCR能扩增出伪狂犬病病毒1 240 bp的gD基因.分离病毒对3日龄乳鼠有一定的致病力,但对家兔、3～5日龄仔猪及妊娠母猪都有很高的安全性.用不同剂量的病毒培养液肌肉注射于3～5日龄仔猪,14 d后用105.7TCID50伪狂犬病病毒强毒攻击,所有试验仔猪均可得到有效保护.用分离毒免疫母猪,其后代可获高滴度的母源抗体,15日龄的仔猪能抵抗105.7TCID50强毒的攻击.试验的结果初步说明,所分离的病毒为伪狂犬病病毒(命名为PRV LY株),并可能是一株弱毒株,而且具有很好的免疫保护作用.     

Cryptosporidium andersoni from a Danish cattle herd: identification and preliminary characterisation

In November 1997, Cryptosporidium andersoni, for the first time, was isolated from a Danish heifer. The isolate was characterised morphologically, molecularly, and furthermore inoculated into mice and one calf. Data on the distribution of cryptosporidia in the herd of origin were obtained at two separate visits in December 1997 and April 1998. C. andersoni was detected in 27 (19.0%) of 142 cattle examined at the first visit, whereas C. parvum was found in six (4.2%). At the following visit 42 (28.0%) of 150 cattle excreted C. andersoni, while 25 (16.7%) were positive for C. parvum. Oocysts of the Danish C. andersoni isolate were ovoid, 7.3(6.5-8.0) x 5.7(5.0-7.0) microm(2) (n=25), with smooth, colourless, single layer oocyst wall and distinct oocyst residuum. The length to width ratio was 1.27 (1.14-1.40, n=25). The identification was verified by sequencing of a 246bp fragment of the rDNA, which was identical to Cryptosporidium muris, the calf genotype (AF093496). The Danish C. andersoni isolate was not transmissible to mice, whereas oocysts were detected in the faeces of one experimentally infected calf from 25 days post-infection (DPI) and shed intermittently at low numbers until 165 DPI, the day of euthanasia. No macroscopic or microscopic changes that could be attributed to infection with C. andersoni were seen in the gastro-intestinal tract of the experimentally infected calf following necropsy and histological examination. This is to our knowledge the first report of C. andersoni in Scandinavia.     

猪伪狂犬病病毒的分离鉴定及其gE基因序列分析

从福建省某猪场疑似伪狂犬病的发病3日龄仔猪的脑、肺脏中分离到1株病毒.该病毒接种家兔出现了典型的伪狂犬病症状,接种PK-15细胞36 h后出现了圆缩、集聚、脱落等典型的细胞病变,猪伪狂犬病病毒阳性血清能特异性中和该分离病毒.根据已发表的伪狂犬病病毒(PRV)gE基因的序列,设计并合成了一对引物,采用PCR方法可扩增特异性298 bp的DNA片段.测序结果与GenBank中有代表性的6株参考毒株相应基因序列比较,核苷酸和氨基酸序列同源性分别为95.5%~99%和91.8%~98%.系统进化树结果表明分离株与湖北分离株Ea株亲缘关系最近.     

Experimental conjunctival infection of lambs with a strain of Chlamydia psittaci isolated from the eyes of a sheep naturally affected with keratoconjunctivitis

Five ram-lambs were inoculated into the left conjunctival sac with the 15R isolate of Chlamydia psittaci, recovered from a sheep with keratoconjunctivitis. A sixth ram-lamb was kept in contact with them. The five lambs developed varying degrees of acute conjunctivitis and 14 days later C psittaci could be recovered from the inoculated eyes, from which Branhamella ovis was also isolated. The eyes were examined regularly for four months; C psittaci could not be re-isolated but the eyes developed varying degrees of follicular conjunctivitis. After four months the sheep were treated with corticosteroids in an attempt to reactivate a latent chlamydial infection but no chlamydiae could be isolated. Five months after the start of the experiment the six lambs were inoculated with 15R into the left conjunctival sacs. Acute conjunctivitis developed which was not as severe as after the first inoculation, but C psittaci could only be recovered from the left eyes of three sheep three days after inoculation. The eyes remained chronically affected by follicular conjunctivitis. Six months after the start of the experiment the left eyes were again inoculated with 15R; on this occasion acute conjunctivitis did not develop and chlamydiae could not be isolated. Chronic follicular conjunctivitis persisted until the experiment was terminated three months later.     

猪细小病毒的分离与鉴定

本试验从福建省某猪场疑似猪细小病毒病死胎的淋巴结、肝脏中分离到1 株病毒。病料接种PK-15细胞36 h后出现了圆缩、集聚、脱落等细胞病变,猪细小病毒阳性血清能特异性地中和该分离病毒。根据已发表的细小病毒(PPV) VP2基因的序列设计并合成了一对引物,采用PCR方法可扩增531 bp DNA片段。测序结果表明,分离株VP2基因与NCBI公布的NADL-2株的同源性高达99.2％,证实分离的病毒株为猪细小病毒。为进一步开展该病毒致病机理、流行病学、诊断研究与疫苗免疫等奠定了基础。     

Three unique groups of spirochetes isolated from digital dermatitis lesions in UK cattle

Bovine digital dermatitis (BDD) is a severe infectious cause of lameness which has spread through dairy cattle populations worldwide, causing serious welfare and agricultural problems. Spirochetes are the main organisms implicated and have previously proven difficult to isolate. This study aimed to isolate and characterise the range of spirochetes associated with BDD in the UK. Twenty-three spirochete isolates were obtained from 30 BDD lesions, which by 16S rRNA gene and flaB2 gene analysis clustered within the genus Treponema as three phylogroups; groups 1 (Treponema medium/Treponema vincentii-like), 2 (Treponema phagedenis-like) and 3 (Treponema denticola/Treponema putidum-like). The treponemes displayed large genotypic and phenotypic diversity between phylogroups and differed from named treponeme species. A previously isolated contagious ovine digital dermatitis spirochete was located within one of the three phylogroups, group 3, and could also be identified within this group on the basis of phenotype testing, suggesting BDD and contagious ovine digital dermatitis may share the same aetiological agent. A strain isolated from a bovine interdigital dermatitis lesion, could be identified as part of BDD isolate group 2, suggesting bovine interdigital dermatitis and BDD may have the same causative agent. Two common enzyme activities, C4 esterase and C8 esterase lipase, were identified in all BDD associated treponemes suggesting common metabolic pathways for sharing this novel niche or even common virulence traits. Further studies are required to determine whether the three groups of novel treponemes are representative of new treponeme taxa and to delineate how they interact with bovine tissues to cause disease.     

致病性犬瘟热病毒CDV-JT1株的分离与鉴定

应用MDCK细胞从吉林某地犬瘟热(Canine distemper,CD)疑似发病犬肺脏组织中分离出1株病毒,该分离毒株经MDCK细胞传至第6代时出现典型的合胞体细胞病变(CPE)。经病毒形态观察、理化特性鉴定、RT-PCR和直接免疫荧光鉴定可知该分离毒株为犬瘟热病毒(CDV),命名为CDV-JT1;动物试验表明,试验犬接种CDV-JT1后21 d内都出现典型的犬瘟热症状;H基因序列分析表明,CDV-JT1株的H基因与中国分离株CDTaiChung株、TN株、SHLJ(07)1株、日本Hamamatsu株、Ueno株、Yanaka株和KDK-1株在基因型上同属于Asia-1型。CDV-JT1株含有包括CDV野毒株特有的309~311位在内的8个潜在的N-连接天冬酰氨糖基化位点。CDV-JT1强毒株的分离成功,对进一步开展疫苗研发、流行病学调查以及致病机理等方面的研究具有重要意义。     

Equine parvovirus: initial isolation and partial characterization.

A viral agent was isolated from the fetal liver of an aborted equine fetus. The isolate hemagglutinated red blood cells from guinea pig, rhesus monkey and rooster. By hemagglutination inhibition tests, the isolate was shown to be antigenically distinct from parvoviruses of bovine and canine origin. Specific hemagglutination inhibiting antibody against the viral isolate was exhibited by 26 of 136 horse sera tested. The isolated virus showed properties compatible with those of an autonomous parvovirus including size, morphology, stability to ether treatment and heating to 56 degrees C, the presence of a 5300 base DNA genome, characteristic protein composition and density (1.405 g/mL). The virus was classified as an equine parvovirus.     

Isolation of avian reovirus as a possible etiologic agent of osteoporosis ("brittle bone disease"; "femoral head necrosis") in broiler chickens

Avian reovirus was isolated from intestines of 3-to-7-day-old broiler chickens with enteritis from broiler houses where osteoporosis was a problem. The virus was purified in a cesium chloride gradient (buoyant density 1.37 gm/ml) and identified as a reovirus by electron microscopy. Specific-pathogen-free (SPF) chickens and commercial broiler chickens with anti-reovirus maternal antibodies inoculated at 1 day of age with the reovirus isolate developed lesions of femoral head fractures and/or osteoporosis; reovirus could be reisolated from the bone marrow and intestinal tracts of experimentally infected SPF birds. The reovirus isolate, although isolated from intestines, induced development fo tenosynovitis lesions in SPF and commercial broiler chickens.     

紫花苜蓿种带阪崎克罗诺杆菌的分离鉴定及其致病性初步研究

采用稀释平板法从6种表面消毒的紫花苜蓿种子中分离和纯化出3株阪崎克罗诺杆菌疑似分离物。选取代表分离物1gF3室内条件下通过菌悬液(≈10⁹ cfu·mL^-1)浸种紫花苜蓿种子,皿内发芽;腹腔注射洁净级昆明小白鼠试验;测定阪崎克罗诺杆菌疑似分离物对紫花苜蓿和小鼠的致病性。结果表明,阪崎克罗诺杆菌疑似分离物对紫花苜蓿不致病;小鼠在接种分离物1gF3 12 h后病理特征显示化脓性脑炎、心肌水肿扩张,局部心肌纤维有断裂、肝脏局部坏死、急性脾炎、肺部急性弥散性出血以及肾脏弥散性血管内凝血等症状,1gF3对小鼠具有致病性。发病小鼠血液涂板分离到阪崎克罗诺杆菌疑似分离物TM2。结合表型特征和16S rDNA鉴定,确定分离物1gF3和TM2为阪崎克罗诺杆菌。在此基础上,测定了菌株1gF3的生物学特性,该菌对数生长期为24~36 h,之后衰退;干旱胁迫加重其生长量线性下降;菌株1gF3能在较宽温度(10~41 ℃)和pH (3~11)范围内生长;该菌不需盐生长,NaCl浓度上升该菌的生长量受到抑制;黑暗交替的光照有利其生长。研究材料为具有动物和植物跨域寄主特点的一类病原菌资源,研究结果对隐藏于植物或动物材料中跨域病原菌侵染传播特征及相关检疫防控提供了理论基础。     

猪伪狂犬病病毒北京株的分离鉴定

从北京某猪场疑似猪伪狂犬病发病死亡仔猪脑及内脏中分离到1株病毒并进行了鉴定。该分离毒株接种ST细胞24 h后出现圆缩、聚集、脱落等典型的细胞病变(CPE);分离毒株能够被伪狂犬病病毒标准阳性血清中和;分离病毒接种家兔后,引起家兔出现奇痒等典型的伪狂犬病临床症状;同时根据GenBank公布的PRV的gD基因设计引物并扩增出特异性的目的片段,扩增产物经过测序比较,表明扩增产物序列为猪伪狂犬病病毒基因序列。以上结果证实该病毒为猪伪狂犬病病毒,依据分离地点命名为猪伪狂犬病病毒北京株。     

Characterization of Campylobacter spp. from wild birds

Bacteria of the genus Campylobacter were isolated from 28 Rooks (Corvus frugilegus), 1 Red Kite (Milvus milvus), 1 Lapwing (Vanellus vanellus), 1 Coot (Fulica atra), 1 Common Moorhen (Gallinula chloropus) and 1 Northern Mallard (Anas platyrhynchos). Altogether, C. jejuni biovar 1, was isolated 19x, C. jejuni biovar 2 8x and C. coli 5x. Among C. jejuni biovar 1 and 2 there were 5 isolates tolerating a content of 1.5% NaCl in the medium. H2S proof of 3 C. jejuni biovar 2 and 1 C. coli isolates resulted positive or negative dependent on incubation time of the used bacterial inoculum. Concerning Rooks the findings indicate that nestlings are more often infected with campylobacters than older birds. Only 1 campylobacter isolate could be recovered from altogether 54 birds of prey although 16 Buzzards (Buteo buteo) were investigated as nestlings.     

The Kümm isolate of Ehrlichia ruminantium: in vitro isolation,propagation and characterization

An effective culture system for Ehrlichia (Cowdria) ruminantium comb. nov. was first established in 1985 and many stocks were subsequently isolated and propagated in vitro. A notable exception, however, was the Kümm isolate that resisted all attempts at in vitro culture until the successful experiment described here. In one experiment white blood cells were harvested from heparinized blood derived from a sheep infected with the Kümm isolate. The cells were added to DH 82 cells and incubated at 37 degrees C. The high metabolic activity of the DH 82 cells necessitated that cell growth be retarded by the addition of cycloheximide. Colonies were first detected 19 days after culture initiation and, once the cultures were established, they could be passaged every 3 days. Bovine and sheep endothelial cells were readily infected with culture supernatant obtained from the infected DH 82 cells. In a further experiment another sheep was infected, using a higher dose of the same batch of Kümm stabilate, and we attempted to infect several different cell lines: these were DH 82 cells, bovine aorta (BA 886) cells, sheep brain endothelial (SBE 189) cells and sheep fibroblastoid cells (E2). Ten days after culture initiation only the E2 cells had become positive for E. ruminantium. Culture supernatant from the first cultured isolate (Kümm-1) was less virulent for mice than that of the second cultured isolate (Kümm-2) which killed all mice. Upon molecular characterization with E. ruminantium 16S probes we found that Kümm-1 hybridized with a Senegal 16S genotype probe, whereas Kümm-2 hybridized only with an Omatjenne 16S genotype probe. The original stabilate used to infect the sheep hybridized with both probes. These results clearly indicate that two different stocks had been isolated in culture.