Detection of adenovirus hexon sequence in a cat by polymerase chain reaction (short communication)

Adenoviral nucleic acid was detected by polymerase chain reaction (PCR) in pharyngeal and rectal swab samples of a cat seropositive for adenovirus and suffering from transient hepatic failure. The samples were taken at a one-year interval, and both faecal samples as well as the second pharyngeal sample were positive in PCR performed with general adenovirus primers. The size of the amplified products corresponded to that of the positive control. The identity of the amplicons was also confirmed by DNA sequencing. The 301 bp long hexon gene fragment was very similar to but distinguishable from the corresponding hexon sequence of human adenovirus type 2. This result suggests the possibility of persistent carrier status and shedding of adenovirus in cats.     

Monoclonal antibodies to canine adenoviruses 1 and 2 that are type-specific by virus neutralization and immunofluorescence

Monoclonal antibodies were produced against the Mirandola strain of canine adenovirus Type 1 (CAV-1) and the Manhattan strain of canine adenovirus Type 2 (CAV-2). The monoclonal antibodies were used in vitro in virus neutralization (VN) assays and in indirect fluorescent antibody (IFA) tests to examine several strains of each virus type. Out of 36 monoclonal antibodies produced against the Mirandola strain, 18 were type-specific for CAV-1 by IFA and 13 of those neutralized the virus in vitro. The other 18 antibodies bound both CAV-1 and CAV-2 by IFA; however, 7 of those specifically neutralized only CAV-1. The 160 monoclonal antibodies made against the Manhattan strain of CAV-2 yielded 77 type-specific antibodies by IFA, of which 39 neutralized only CAV-2 in vitro. The remaining 83 antibodies recognized both CAV-1 and CAV-2 by IFA, with 3 of those neutralizing both viral types. The hemagglutination inhibition (HI) test was performed on a selected monoclonal antibody from each specificity group. Although Type 1 CAV could be readily differentiated from Type 2 CAV by using type-specific monoclonal antibodies in the IFA or VN tests, strains within each type could not be differentiated. This is the first report of neutralizing monoclonal antibodies for a mammalian adenovirus.     

Genetic characterization of equine adenovirus type 1

Two known serotypes of equine adenovirus (EAdV), equine adenovirus type 1 (EAdV-1) and equine adenovirus type 2 (EAdV-2) have been isolated from horses. EAdV-1 is predominantly associated with upper respiratory tract infections while EAdV-2 appears to be associated with gastrointestinal infections in horses. In this report the EAdV-1 genome has been sequenced for the first time. The EAdV-1 genome encoded genes are characteristic of the Mastadenovirus genus such as protein V and IX. Unexpectedly, phylogenetic reconstructions also revealed a close relationship between EAdV-1 and two recently characterized bat adenoviruses. The results of this study suggest that EAdV-1 may share a common ancestor with canine and bat adenoviruses.     

Detection and Differentiation of CAV-1 and CAV-2 by Polymerase Chain Reaction

Canine adenovirus type 1 (CAV-1) and type 2 (CAV-2) can be categorized in the laboratory by haemagglutination and neutralization tests, but they are difficult to differentiate from each other in specimens, especially when infection occurs in the digestive tract. The object of this study was to develop a simple method of detecting and differentiating them. One pair of common primers was designed and synthesized according to the sequences of the E3 and flanking regions and a polymerase chain reaction (PCR) assay was established using these two primers to amplify the virus-specific DNA fragment from clinical specimens as well as from cell cultures. After electrophoresis, under the same amplification conditions, 508 bp and 1030 bp PCR products were observed for CAV-1 and CAV-2, respectively. These were further shown to be adenovirus specific by dot hybridization and sequencing. As only one pair of primers was involved in the PCR procedure, it was faster and easier to perform than any of the other assays used for detecting canine adenovirus, making it applicable in the rapid confirmation of diagnosis and differentiation of the two types of canine adenoviruses.     

犬2型腺病毒毒株纤突蛋白基因的比较分析

根据GenBank中犬腺病毒（canine adenovirus,CAV)纤突基因序列，设计合成2对引物。用PCR方法，对犬2型腺病毒沈阳分离株（CAV-2SY株）第5代强毒（SY-V5）、经蚀斑克隆驯化的第60代毒弱毒（SY-V60）、SY-V60感犬体上传5代的回收毒（SY-CP5）、美国疫苗毒（US-V20）等4个毒株的纤突基因进行了扩增，PCR产物经纯化后进行基因序列测定，测序结果经拼接后得到1个由1629个核苷酸组成的纤突蛋白全基因序列，编码543个氨基酸。犬2型腺病毒与GenBank中的标准的CAV-2强毒株（Toronto A26/61)纤突蛋白基因序列的比较结果表明：CAV-2SY株强毒株与Tornto A26/61株相同；SY-V60株与SY-CP5相同，与驯化前的SY-V5相比，在1134位发生碱基颠换，编码的氨基酸由原来的天冬酰氨（N）变为赖氨酸（K），并导致N-X-S/T潜在糖基化位点发生改变，US-V20该部位与发生同样的突变，本试测定和比较的CAV-2强毒株有5个潜在的N-联糖基化位点，弱毒株仅有4个位点；US-V20与TorontoA26/61差异较大，有11个碱基发生变化，导致8个氨基酸的变异。     

Molecular and pathological investigations of EHV-1 and EHV-4 infections in horses in Turkey

The aim of the present study was to investigate abortion storms that occurred in the Marmara region of Turkey in 2008-2009 using a real-time PCR. Two aborted foetuses were necropsied and histo-pathological findings reported herein. Ten lungs, 3 brains and one nasal swab from 10 aborted foetuses, 6 nasal swabs and 3 vaginal swabs from aborting mares were included in this study. EHV-1 was isolated from the lung, liver and brain of 1 aborted foetus. EHV-1 DNA was detected in the lungs, livers and spleens of 2 necropsied foetuses and in 3 lungs from 10 foetuses submitted for diagnosis. A brain from one of the aborted foetuses was also positive for EHV-1 DNA. EHV-4 DNA was detected only in a nasal swab of one of the tested foetuses. Neither EHV-1 nor EHV-4 DNA was detected in the swabs of aborting mares. Sequence analysis of the glycoprotein B of the strains was performed and a phylogenetic tree was generated. The results indicated that 4 of the 5 Turkish EHV-1 strains (TR02, TR03, TR04 and TR05) clustered together; the fifth strain (TR01) was slightly removed from the group and clustered with other EHV-1 from various origins. Single nucleotide polyporphism (SNP in ORF30) associated with neuropathogenesis was not detected in any of the strains. At necropsy, sub-milier focal necrosis in the liver and spleen was observed. Microscopically, focal coagulation necrosis and marked eosinophilic intranuclear and intracytoplasmic inclusion bodies in the hepatocytes localised around the necrotic areas in the liver. Severe coagulation necrosis in white pulp of the spleen was also observed.     

Severe enteric disease in an animal shelter associated with dual infections by canine adenovirus type 1 and canine coronavirus

An outbreak of dual infection in dogs with canine adenovirus type 1 (CAV-1) and canine coronavirus (CCV) infection is reported in an animal shelter that comprised approximately 200 adults stray dogs and 30 puppies. Twenty puppies died 7-8 days after the onset of the clinical signs (severe enteritis, leucopoenia, respiratory distress and dehydration). Both CAV-1 and CCV were isolated from tissue or swab samples. Antibodies to CCV and, at high levels, to CAV-1 also were detected in several puppies. The principal histological findings were atrophy of small intestinal villi, lymphoid depletion, hepatitis and bronchopneumonia. The persistence of CCV in the faeces, observed by the polymerase chain reaction assay, was longer than previously reported. Results demonstrated the serious consequences which may occur with dual infections by CAV-1 and CCV in assembled groups of dogs that are housed in poorly managed kennels with inadequate vaccination programmes.     

5种常见犬腹泻病毒基因芯片检测方法的建立

为建立一种简便、快速、高效的可同时区分犬腺病毒1型（canine adenovirus type 1,CAV-1）、犬腺病毒2型（canine adenovirus type 2,CAV-2）、犬冠状病毒（canine coronavirus,CCV）、犬瘟热病毒（canine distemper virus,CDV）、犬细小病毒（canine parvovirus,CPV） 5种常见犬腹泻病毒的基因芯片诊断方法,本研究以CAV-1、CAV-2、CCV、CDV、CPV 5种犬腹泻病毒为靶病毒,根据NCBI上收录的病毒基因序列在其保守区域内设计引物,在此基础上根据变异区域设计针对每种病毒的探针2~3条,优化检测体系的各反应条件,确定该检测方法的特异性与敏感性,建立可同时区分5种病毒的基因芯片检测方法。结果显示,建立的基因芯片检测方法可同时检测以上述5种犬腹泻病毒,其中PCR的退火温度为55℃、延伸时间为1 min 15 s;探针与PCR产物的杂交温度40℃、杂交时间2.5 h时,该方法对CAV-1、CAV-2、CCV、CDV和CPV 5种病毒标准品的检测限分别为0.2 fg/μL、2 fg/μL、2 fg/μL、20 pg/μL和0.02 fg/μL,具有较高的灵敏性;同时对犬副流感病毒进行特异性试验,发现无阳性信号出现,具有较强的特异性;对14份临床腹泻样品检测结果显示,基因芯片方法对CAV-1、CAV-2、CCV、CDV和CPV 5种病毒的阳性检出率分别为28.57%、50.00%、64.28%、14.28%和85.71%,并且基因芯片检测方法的敏感性较PCR要高10~100倍。以上结果表明,本研究建立的基因芯片检测方法具有特异、敏感等特点,对临床中犬类混合感染病毒检测具有一定的诊断意义。     

Isolation and identification of canine adenovirus type-2 from the upper respiratory tract of a dog

AIM: To report on the isolation and identification of canine adenovirus type-2 (CAV-2) from a greyhound dog with tracheitis/tonsillitis. METHODS: Virus isolation was performed with Madin and Darby canine kidney (MDCK) cell monolayers using standard virological techniques. The isolated virus was identified by haemagglutination inhibition and serum neutralisation tests. Viral DNA was extracted from infected MDCK cells and subjected to restriction endonuclease analysis using the endonuclease enzymes Bam HI, Bgl II, Eco RI and Hind III. RESULTS: A virus, designated 5 113-87, was isolated in MDCK cells yielding typical cytopathic effect. The virus could be neutralised with a CAV-2 specific reference antiserum and also showed some cross neutralisation with CAV-1 specific reference antiserum. The virus 5 113-87 had a high haemagglutination inhibition titre with CAV-2 antiserum using human group 0 red blood-cells and CAV-1 and CAV-2 reference antisera. This virus also had DNA restriction profiles identical to those of the reference CAV-2 (Toronto A26/61), whereas previously isolated strains of adenovirus from dogs in New Zealand had DNA restriction patterns identical to the prototype CAV-1 strain (Utrecht). CONCLUSION: The findings show that the virus 5 113-87 isolated from the upper respiratory tract of a dog in New Zealand is CAV-2.     

First Report on the Frequency and Molecular Detection of Neuropathogenic EHV-1 in Turkey

There has been an increase in outbreaks of neuropathogenic equine herpesvirus-1 (EHV-1) in the United States and Europe. However, the presence and frequency of neuropathogenic EHV-1 in Turkish horses are not known at present. This study aimed to investigate the frequency of EHV-1 and neuropathogenic strains of EHV-1 in the Marmara Region of Turkey. Samples were analyzed for the presence of EHV-1 and neuropathogenic EHV-1 by real-time PCR TaqMan probe assays. Overall detection rate of EHV-1 was 45.5% (51 of 112). The detection rates were 70.5% (24 of 34) in aborted fetuses, 53.3% (8 of 15) in neonatal deads, 66.6% (4 of 6) in foals, 40% (2 of 5) in dead mares, and 25% (13 of 52) in living mares. Overall detection rate of neuropathogenic EHV-1 was 7.8% (4 of 51), and the real-time PCR results were confirmed by sequencing. Neuropathogenic strains of EHV-1 were detected in the brain and lung of two mares with neurological disease but without a history of abortion, in the brain of a foal that died of respiratory disorder, and in the nasal swab from a mare with a history of abortion. On histopathology, nonpurulent meningoencephalitis, hemorrhages, and vasculitis were seen in the brain. In conclusion, results of this study indicated, for the first time, that the neuropathogenic EHV-1 is circulating in the Marmara Region of Turkey. The results of this study also show that the current risk for non-neuropathogenic strains is high, whereas risk for the neuropathogenic EHV-1-G₂₂₅₄ strain seems to be low. As outbreaks of EHV-1 continue in the Marmara region of Turkey, surveillance for neuropathogenic EHV-1 genotype should be maintained.     

Isolation of canine adenovirus-2 from the faeces of dogs with enteric disease and its unambiguous typing by restriction endonuclease mapping

Fifty-four faecal specimens obtained from kennelled dogs with diarrhoeal disease were used to inoculate a range of cell types in tissue culture. Particles resembling adenovirus virions were seen in three specimens and 22 stools yielded an adenovirus upon culture. Viral DNA from each isolate was digested with the restriction endonucleases Bam H1 and Pst 1. Agarose gel electrophoresis revealed identical restriction patterns for all isolates. One isolate, 9228, was selected as a prototype and was compared with reference strains of canine adenovirus-2 (CAV-2) (Manhattan and Toronto A26/61) and CAV-1. Isolate 9228 was clearly distinct from CAV-1 but identical to both Manhattan and Toronto (A26/61) strains of CAV-2. However, restriction site polymorphism was observed in 9228 following digestion with Hpa II. Isolate 9228 was similarly compared with all commercially available vaccinal isolates of CAV-2 and was shown to be clearly distinct from these.     

Rapid detection of group I avian adenoviruses by a loop-mediated isothermal amplification

A loop-mediated isothermal amplification (LAMP) assay was optimized for the rapid detection of Group I avian adenoviruses. A set of six primers was designed from the DNA sequences of hexon genes from Group I avian adenovirus. The assay was performed in a water bath for 60 min at 63 C, and the amplification result was visualized by adding a fluorescence dye reagent or by inspecting the white sediment. The results showed that the LAMP assay could detect all 12 serotypes of Group I avian adenovirus and nine Guangxi Group I avian adenovirus isolates. This avian adenovirus Group I-specific LAMP assay could detect 238 copies of avian adenovirus. No cross-reactions were detected using the LAMP assay with avian adenoviruses type II and III or with other avian viruses. The ability of LAMP to detect Group I avian adenovirus isolates was further evaluated with 184 cloacal swab samples from poultry. In total, 72 out of 184 cloacal swab samples from poultry were identified as positive by LAMP, whereas 45 out of 184 were identified as positive by conventional PCR test. The Group I avian adenovirus specific LAMP results were further confirmed by real-time PCR. This specific LAMP method holds promise as a rapid and specific diagnostic assay for detection of samples from birds suspected of adenovirus infection.     

Biological and molecular characterization of chicken anemia virus isolates from Slovenia

The presence of chicken anemia virus (CAV) in Slovenia was confirmed by inoculation of 1-day-old chickens without antibodies against CAV and isolation of the virus on the Marek's disease chicken cell-MSB1 line and by polymerase chain reaction (PCR). Experimental inoculation of 1-day-old chickens resulted in lower hematocrit values, atrophy of the thymus, and atrophy of bone marrow. CAV was confirmed by PCR in the thymus, bone marrow, bursa of Fabricius, liver, spleen, ileocecal tonsils, duodenum, and proventriculus. The nucleotide sequence of the whole viral protein (VP)1 gene was determined by direct sequencing. Alignment of VP1 nucleotide sequences of Slovenian CAV isolates (CAV-69/00, CAV-469/01, and CAV-130/03) showed 99.4% to 99.9% homology. The VP1 nucleotide sequence alignment of Slovenian isolates with 19 other CAV strains demonstrated 94.4% to 99.4% homology. Slovenian isolates shared highest homology with the BD-3 isolate from Bangladesh. Alignment of the deduced VP1 amino acids showed that the Slovenian isolates shared 100% homology and had an amino acid sequence most similar to the BD-3 strain from Bangladesh (99.6%) and were 99.1% similar to the G6 strain from Japan and the L-028 strain from the United States. The Slovenian isolates were least similar (96.6%) to the 82-2 strain from Japan. A phylogeneric analysis on the basis of the alignment of the VP1 amino acids showed that CAV isolates used in the study formed three groups that indicated the possible existence of genetic groups among CAV strains. The CAV isolates were grouped together independent of their geographic origin and pathogenicity.     

犬腺病毒纤突蛋白基因功能区的比较分析

根据GenBank中已发表的CAV纤突基因序列，设计合成4对8条引物，用已建立的PCR方法，对犬2型腺病毒沈阳分离株第5代强毒经蚀斑克隆驯化的第60代毒弱毒（SY-V60）和犬1型腺病毒长春犬株（CCC-V6）的纤突基因进行了扩增，PCR产物经纯化后进行基因序列测定，测定结果经拼接后得到一个由1632和1629个核苷酸组成的纤突蛋白全基因序列，分别编码543和542个氨基酸。犬2型腺病毒（SY-V60）与犬1型腺病毒（CCC-V6）的纤突基因的同源性达到80.48%。犬2型腺病毒（SY-V60）与犬1型腺病毒（CCC-V6）纤突基因的同源性达到80.48%；根据犬的腺病毒与人2型腺病毒纤突蛋白的序列同源性比较结果，推测了犬腺病毒纤突蛋白的尾（tail)、轴（shaft)，结（knob)三个功能区的氨基酸序列，2个型之间的尾区同源性为76.9%；轴区同源性为78.59%；其结区同源性为83.24%，而同型毒株之间结和尾区同源性较高，轴区同源性较低。     

Comparison of one-step RT-PCR and a nested PCR for the detection of canine distemper virus in clinical samples

OBJECTIVE: To develop a rapid and sensitive method for the detection of canine distemper virus (CDV) by nested PCR using clinical specimens. DESIGN: A nested PCR was developed, compared to a one-step RT-PCR and validated. PROCEDURE: Two sets of specific primers for a one-step RT-PCR and a nested PCR, targeting a 640 bp fragment and a 297 bp fragment, respectively, were selected from the highly conserved region of the nucleocapsid protein (NP) gene of CDV. The nested PCR and the one-step RT-PCR were used to amplify a part of the CDV NP gene of a CDV vaccinal strain and samples of urine, blood, nasal discharge and saliva from 29 dogs suspected of suffering CD. RESULTS: Both the one-step RT-PCR and the nested PCR reacted with the CDV vaccinal strain, but not with canine parvovirus. The expected 640 bp fragment of the NP gene was detected in 11/22 (50.0%) blood, 10/20 (50.0%) urine, 5/25 (20.0%) saliva and 6/27 (22.2%) nasal swab samples by one-step RT-PCR, whereas the nested PCR amplified an expected 297 bp fragment of the NP gene in 18/22 (81.8%) blood, 15/20 (75.0%) urine, 14/25 (56%) saliva and 19/27 (70.3%) nasal swab samples. CONCLUSION: The nested PCR detected CDV in blood, urine, nasal swab and saliva more frequently than did the one-step RT-PCR. Therefore, this assay should be a useful aid to antemortem diagnosis of CDV infections in dogs.     

Canine adenovirus type 1 infection of a Eurasian river otter (Lutra lutra)

A 10-year-old female Eurasian river otter (Lutra lutra) died after prolonged anorexia and weight loss in the Seoul Grand Park Zoo, Seoul, Republic of Korea. On necropsy, the liver was found to be swollen and friable with 1 lobe enlarged and necrotic. The other organs showed no significant alterations except for mild atrophy of the right kidney. Microscopically, there was multifocal hepatic necrosis. The hepatocytes around the necrotic areas were swollen and contained large basophilic intranuclear inclusions. Periportal infiltration by plasma cells and lymphocytes was also evident. Transmission electron microscopy revealed characteristic hexagonal virus particles sized approximately 70 nm in diameter in the nuclei of the hepatocytes, which were consistent with an adenovirus. Polymerase chain reaction of the formalin-fixed, paraffin-embedded liver sections was used to determine whether the virus was either the canine adenovirus type 1 (CAV-1), canine adenovirus type 2 (CAV-2), or some other viral agent. The results of these tests showed that the virus was CAV-1. To our knowledge, this is the first report on a CAV-1 infection in an otter.     

2株Ⅰ群禽腺病毒的分离鉴定及致病性分析

为了解山东省禽腺病毒（fowl adenovirus,FAdV）毒株的基因遗传演化情况及致病性,本试验对山东省两家疑似暴发鸡包涵体肝炎和心包积液综合征鸡场采集的病料（肝脏、脾脏）进行PCR鉴定,并将鉴定为FAdV的2份阳性病料提取病毒液,接种鸡肝癌细胞（LMH）进行毒株传代培养和细胞病变（CPE）观察、PCR检测、TCID₅₀测定、鸡胚致病性试验、SPF鸡回归试验、病毒hexon部分基因扩增及序列分析。结果显示,试验成功分离到2株FAdV,2株分离株细胞传第1代即可观察到CPE,PCR均可扩增出大小为500 bp的片段,且2株分离株细胞F5代TCID₅₀分别为10^-7.75/0.1 mL和10^-7.60/0.1 mL,均对7日龄SPF鸡胚有强致病性;第1分离株和第2分离株对21日龄SPF鸡攻毒死亡率分别为80%和15%。hexon基因核苷酸同源性分析结果显示,第1分离株与FAdV-4株同源性最高（99.57%）,第2分离株与FAdV-8b株同源性最高（99.18%）。这2株FAdV分离株均与Ⅰ群禽腺病毒同源,与火鸡出血性肠炎病毒（HEV）所在的血清Ⅱ群及减蛋综合征病毒（EDSV）所在的血清Ⅲ群禽腺病毒位于不同分支。第1分离株基因型为C型,血清型为4型,命名为FAdV-SDC4株;第2分离株基因型为E型,血清型为8b型,命名为FAdV-SDE8b株。综上所述,FAdV-SDC4和FAdV-SDE8b株属于近几年国内流行毒株。本研究结果可为鸡包涵体肝炎、心包积液综合征的防控工作和疫苗株的选择提供科学依据。     

Ⅰ群禽腺病毒12个血清型毒株Hexon蛋白全基因序列测定和酶切位点分析

利用3对引物(A、B、C)分别进行Ⅰ群禽腺病毒的12个血清型毒株的Hexon全基因序列PCR扩增,并进行序列测定和PCR-RFLP分析,通过DNAStar软件和MEGA软件进行序列分析并绘制遗传发育进化树。结果显示,Hexon基因全长为2 800左右个核苷酸,编码约940个氨基酸,不同血清型之间的同源性为75.2%～99.5%,变异范围是0.0%～24.3%。推导的氨基酸同源性为20.7%～98.8%,差异性为0.0%～24.2%。12个血清型之间Hexon全基因序列信息用来评价Ⅰ群禽腺病毒家族的种系发育关系,构建的进化树显示出5个主要的分支。选用限制性内切酶HaeⅡ,利用存在差异的Hexon序列片段D特异性可以有效区分大部分血清型。结果表明,获得了Ⅰ群禽腺病毒12个血清型不同毒株的Hexon全基因序列并进行了分子进化分析,同时结合12个血清型PCR-RFLP分析,为进一步分析鉴别Ⅰ群禽腺病毒不同血清型提供依据。     

奶牛腐蹄病坏死杆菌分离鉴定及白细胞毒素基因lktA1序列分析

为分离纯化奶牛腐蹄病坏死杆菌,分析其与其他菌株的亲缘关系,本研究利用坏死杆菌白细胞毒素特异性引物,对奶牛腐蹄病病牛蹄部拭子样品进行了PCR检测,利用厌氧培养基对PCR检测阳性样品进行了坏死杆菌的分离培养,以分离的坏死杆菌基因组DNA为模板,对白细胞毒素基因进行了克隆和序列分析。结果显示,9份奶牛腐蹄病病牛蹄部拭子样品PCR检测结果均为阳性,对其中一份样品中的坏死杆菌进行分离培养,获得了纯培养物,命名为bFR13-1。坏死杆菌bFR13-1菌株白细胞毒素基因测序结果显示,与GenBank已发表的H05、A25和B35菌株的白细胞毒素基因在核苷酸水平的同源性分别为98.40%、98.35%和90.79%,推导氨基酸的同源性分别为97.7%、97.6%和89.0%。进化树分析结果显示,坏死杆菌bFR13-1菌株白细胞毒素与H05菌株的同源性最高,bFR13-1菌株与H05菌株和A25菌株呈较近亲缘关系。结果表明,不同坏死杆菌分离株的白细胞毒素呈现一定的变异性,这种变化是否与坏死杆菌致病性相关,值得深入研究。     

Isolation,Identification and Sequence Analysis of Leukotoxin Gene lktA1 of Fusobacterium necrophorum of Cow Footrot

For separation and purification of Fusobacterium necrophorum of cow footrot, and analysis of genetic relationship with other strains, the hoof ministry swab samples were detected by PCR based on specific primers of leukotoxin gene, and genomic DNA were isolated from PCR positive samples of Fusobacterium necrophorum culturing in anaerobic medium.The genes of leukotoxin were cloned and sequenced.The results showed that nine of hoof ministry swab samples were all PCR positive samples, and we obtained Fusobacterium necrophorum pure culture from one of the samples which named bFR13-1.The gene sequencing results indicated that the homologies of leukotoxin gene nucleotide sequence of bFR13-1 strain compared with H05, A25 and B35 strains from GenBank were 98.40%, 98.35% and 90.79%, respectively, and the homologies of deduced amino acid sequence were 97.7%, 97.6% and 89.0%, respectively.Phylogenetic tree analysis results showed that leukotoxin gene of Fusobacterium necrophorum bFR13-1 and H05 had high homology and bFR13-1, H05 and A25 showed a close genetic relationship.The result indicated that leukotoxin showed variability between different Fusobacterium necrophorum isolated strains, and it was worth to study whether this change and pathogenicity of Fusobacterium necrophorum were related.