副猪嗜血杆菌弱毒疫苗的研制

根据本教研室分离得到的副猪嗜血杆菌,选择毒力强、生长性能和毒力都稳定的菌株作为原始毒株。通过化学诱导剂诱导原始菌株发生突变,再经过毒力试验,挑选出毒力有明显减弱的菌株。安全性试验发现,这些减毒菌株具有较高的安全性,为弱毒疫苗的研制奠定基础。     

鸡败血支原体鸡传染性鼻炎二联灭活疫苗菌种复壮鉴定试验

从国外引进的鸡败血支原体R株和副鸡嗜血杆菌221株和668株的冻干品,经我所扩增复壮和系统的鉴定试验证明:鸡败血支原体R株属典型的强毒株,副鸡嗜血杆菌221和668株分别属典型的A型和C型强毒株.经过免疫原性试验、毒力鉴定试验及效力试验证明,这三种菌株是制备鸡败血支原体、鸡传染性鼻炎二联疫苗最好的菌株.     

副猪嗜血杆菌4种疑似毒力因子多克隆抗体的制备与鉴定

在利用蛋白质非标记定量技术(Lable-free)分析副猪嗜血杆菌强毒株与无毒株差异蛋白的基础上,筛选出4种疑似毒力因子,通过基因扩增后克隆入原核表达载体p ET-32a,构建重组质粒,转入Rosetta菌株后成功表达了目标蛋白。重组蛋白经亲和层析纯化后免疫小鼠,制备多克隆抗体,并将其与菌体蛋白进行Western blot验证。结果表明,表达的4种蛋白免疫原性良好,制备的多克隆抗体免疫反应条带单一。该研究为从蛋白水平上探讨副猪嗜血杆菌毒力因子的功能奠定了基础。     

副猪嗜血杆菌国内流行血清型菌株的致病性比较研究

对我国最为流行的血清4、5、12、13和14型副猪嗜血杆菌进行了豚鼠和仔猪毒力试验的比较研究。豚鼠毒力试验结果表明,对豚鼠毒力最强的是12和14型菌株,其LD50均低于3.5×108 CFU;其次是5型,其LD50介于1.0×109¹.9×109 CFU之间;毒力最弱的是13型,其LD50介于4.0×1091.9×109 CFU之间;毒力最弱的是13型,其LD50介于4.0×109⁴.1×109 CFU之间。4型的毒力差异较大,强毒菌株与5型接近,弱毒菌株与13型接近。与我们前期报道的小鼠毒力试验结果相比,豚鼠接种副猪嗜血杆菌后具有更长的发病死亡周期,且具有纤维素性渗出和败血症等副猪嗜血杆菌病的病变特征,表明豚鼠较小鼠更适合作为该病的替代实验动物。进一步的仔猪毒力试验结果与豚鼠毒力试验结果相一致。剖检发现,所有5、12和14型菌株以及4型强毒菌株均能导致严重的败血症性病理变化,表明副猪嗜血杆菌强毒菌株能导致猪的系统感染和全身性疾病;而所有13型菌株和4型弱毒株则不能。这表明国内流行的5、12和14型副猪嗜血杆菌的毒力与国外已有报道一致,均为强毒菌株;而4和13型则不同,具有明显的地域特征。     

牛源荚膜A型多杀性巴氏杆菌疫苗候选株的筛选及鉴定

为了筛选生长快、毒力强、免疫原性好、副反应小的牛源荚膜A型多杀性巴氏杆菌（Pasteurella multocida，Pm）灭活疫苗菌株，本试验选取6株来自不同地区致犊牛肺炎死亡的牛源荚膜A型多杀性巴氏杆菌分离株，测定了培养基生长曲线、小鼠毒力、菌体脂多糖（LPS）含量及各菌株灭活菌苗免疫小鼠和家兔后的抗体效价，并进行了攻毒保护试验。结果显示，分离株Pm2、Pm3、Pm5生长速度较快、毒力较强、LPS含量较多，均含有与毒力和免疫相关的ptfA和fimA基因；免疫小鼠及家兔未发现明显不良反应，在二免后14 d血清抗体达1：64~1：128，强毒攻毒后全部存活，而PBS对照组全部死亡。本试验结果表明，Pm2、Pm3、Pm5均可作为多杀性巴氏杆菌灭活菌苗的候选菌株，其中Pm3作为首选株。     

仔猪不同部位副猪嗜血杆菌分离株的血清抗性检测

本研究应用血清抗性试验,检测了从仔猪不同部位分离的66株副猪嗜血杆菌的毒力,对比菌株对仔猪、兔、豚鼠的血清敏感性差异.试验结果显示:66株菌株中,35株是高抗血清的强毒力菌株,7株是中等抗血清的中等毒力菌株,24株是对血清敏感的无毒力菌株.说明不同分离部位的副猪嗜血杆菌对血清分别呈现血清敏感性和血清抗性,在致病力方面也...     

腐蹄病C型节瘤拟杆菌纤毛蛋白基因的克隆与表达

用 PCR从腐蹄病 C型节瘤拟杆菌扩增出具有免疫保护性抗原 0 .85 kb纤毛蛋白基因 (pili基因 ) ,并构建了该基因的表达载体。转化宿主细胞 PAK/2 pfs,在营养肉汤中进行 pili基因的表达。培养 18～ 2 4h后 ,收集培养液上清 ,加入 0 .1mol/L Mg Cl2 ,离心提取重组纤毛蛋白。用羊抗兔 C型节瘤拟杆菌抗血清与提取的纤毛蛋白进行对流免疫电泳试验 ,结果表达的重组纤毛蛋白有特异性 ;用 SDS- PAGE和 Western- blot证明表达的蛋白是 C型节瘤拟杆菌纤毛蛋白。     

节瘤拟杆菌K-凝集试验方法的建立

根据节瘤拟杆菌(D.nodosus)特异的表面抗原———K抗原具有凝集反应的特性,建立了检测抗节瘤拟杆菌抗体的方法———K-凝集试验方法。节瘤拟杆菌液体培养物接种兔,制备节瘤拟杆菌阳性血清;福尔马林灭活肉汤培养物制备抗原,结果表明,该方法使用方便、特异性强。     

抑制性消减杂交技术分析猪链球菌2型毒力相关基因

以猪链球菌2型四川分离强毒株458#为检测子,国际参考无毒菌株1330#为驱动子,用抑制性消减杂交方法寻找其基因组水平的差异.采用3种不同的限制性内切酶分别酶切458#与1330#基因组获得3套酶切片段,经消减杂交后构建猪链球菌2型强毒株458#特异DNA的差异文库,并对差异序列进行比较分析.结果表明,试验获得了3套差异文库共计42个特异性差异片段,所有差异片段均与已发表的猪链球菌2型05ZYH33株全基因组序列高度同源.构建了具有代表性的猪链球菌2型强毒株与国际无毒参考菌株基因组差异DNA文库,差异片段通过Blast比对,部分差异片段与已知功能基因如转座子,耐药基因、1型限制酶修饰系统、表面蛋白和毒力相关蛋白等有同源性,尚有许多差异片段属于未知功能蛋白或假定蛋白,其中可能包括与猪链球菌2型毒力相关的基因,为进一步分析猪链球菌2型可能的毒力相关基因奠定了基础.     

禽巴氏杆菌B_(26)-T_(1200)弱毒冻干苗的研究 Ⅰ.禽巴氏杆菌B_(26)- T_(1200)弱毒菌株的选育

从广西各地患禽巴氏杆菌病急性死亡的鸡、鸭肝脏分离出５６株禽巴氏杆菌强毒株，经培养特性和生化特性试验鉴定，从中选出１５株典型禽巴氏杆菌菌株。Ｃａｒｔｅｒ荚膜抗原分型鉴定，１５株菌均为荚膜Ａ型，间接血凝滴度为１∶１６０～１∶６４０。通过毒力和免疫原性测定，从中选择毒力较强、免疫原性较好的Ｂ２５、Ｂ２６、Ｂ２７３菌株进行人工致弱，其中Ｂ２６菌株在０．１％裂解全血马丁汤中，通过物理诱变方法致弱，即在传代过程中，把培养温度由３７℃逐渐提高到４５℃，每１２ｈ传１代而成功致弱，传至１２００代时，毒力显著减弱，且仍保持其良好的免疫原性和培养特性，从而获得了禽巴氏杆菌Ｂ２６－Ｔ１２００弱毒菌株     

The role of elastase in the differentiation of Bacteroides nodosus infections in sheep and cattle

Eighty-seven Bacteroides nodosus isolates were examined for elastase production by clearing of elastin particles in TAS agar medium. These included 54 ovine virulent isolates, 28 ovine benign isolates and five bovine isolates. In addition 22 ovine virulent, 16 ovine benign and two bovine isolates were examined for decline in proteolytic activity over a 13-day period in the degrading proteinase test using hide power-azure as substrate. There was a remarkable correlation between elastase production, relative stability of proteolytic activity in the hide powder-azure test and virulence of B nodosus. Ovine virulent isolates invariably produced elastase whereas ovine benign isolates and bovine isolates were elastase negative. Bovine isolates produced only mild lesions in the feet of challenged sheep.     

The virulence of Dichelobacter nodosus,measured by the elastase test,is an important predictor for virulent footrot diagnosis in New South Wales sheep flocks

Ovine footrot is a contagious bacterial disease that causes foot lesions, and depending on the virulence of the causative strains, may lead to severe underrunning of the hoof and lameness. Virulent footrot can be identified, treated and controlled more effectively than less virulent benign forms. The in vitro elastase test for virulence of the causative bacteria, Dichelobacter nodosus, has been used to support clinical diagnosis. However, not all laboratory-designated virulent D. nodosus strains cause clinical signs of virulent footrot. This study evaluated retrospectively how well the elastase test supported clinical footrot diagnosis in 150 sheep flocks examined for suspect footrot in New South Wales between August 2020 and December 2021. Flocks were included if measures of clinical disease, environmental conditions and the virulence of D. nodosus isolates were available. Variation in the elastase activity result between D. nodosus isolated from the same flock made bacterial virulence hard to interpret, but calculating the mean elastase rate for all isolates from the same flock made correlations between bacterial virulence and flock footrot diagnosis possible. Simplifying bacterial virulence into whether there were any elastase-positive D. nodosus isolates before 12 days increased the predictive value of elastase results for virulent diagnosis, compared with using the first day that any isolate was elastase positive or the percentage of elastase-positive isolates by 12 days, but not all clinically virulent flocks had isolates with elastase activity before 12 days. Logistic regression models were fitted to identify the minimum number of predictors for virulent footrot diagnosis, with models suggesting that virulent footrot diagnosis was best predicted by adding the elastase test result and environmental conditions to the prevalence of severe foot lesions (score 4 and 5). However, performing the same analysis with different breeds, ages of sheep and seasons might highlight other factors important in the diagnosis of virulent footrot.     

Characterisation of isolates of Pseudomonas aeruginosa from sheep

Fleece rot isolates of Pseudomonas aeruginosa were characterised in terms of biochemical reactions, serological typing and production of keratinase, lipase and the potentially dermal necrotic enzymes exotoxin A, protease, elastase phospholipase and lecithinase. The similarities generally obtained between these characteristics of isolates from sheep and man suggests a role for P. aeruginosa and mechanisms of pathogenesis in the development of the dermatitis associated with fleece rot.     

Biochemical and toxigenic characteristics of Aeromonas spp. isolated from diseased mammals, moribund and healthy fish

In this study we describe biochemical, toxigenic and surface characteristics of 33 motile Aeromonas isolated from diseased mammals, 3 from moribund marine mammals, 24 from healthy fish and 4 from moribund fish. Aeromonas hydrophila, A. caviae and A. sobria were isolated from both mammals and fish but at a different incidence. Aeromonas hydrophila was the predominant species isolated from clinical specimens; it was isolated from pneumonia, wound infections, septicemia and abortion in horses, cattle and pigs. Aeromonas sobria was isolated from one mammal and 11 healthy fish. Aeromonas caviae was isolated in 2 cases from healthy fish and in 9 cases from diseased mammals. Variations in some biochemical tests including sorbitol, amylase and citrate, were observed between isolates from different sources. However, these differences did not allow the differentiation of isolates from diseased mammals and healthy fish. The majority of A. hydrophila isolates produced different extracellular products; A. sobria isolates produced less exotoxin. With A. caviae isolates no hemolysin, protease, enterotoxin or elastase were detected. There was no quantitative difference in hemolysin, protease, enterotoxin or elastase production between isolates from mammals and fish. It is suggested that A. hydrophila could be a potential pathogen for domestic animals, and fish may represent a potential reservoir of infection.     

Serotypic and biochemical characterization of Bacteroides nodosus isolates from Oregon.

Ninety-seven Bacteroides nodosus isolates were characterized by the tube agglutination test. Fourteen serotypes were identified including isolates that were serologically similar to Australian serotypes A, B and C. One additional isolate remains untyped and possibly represents another serotype. The isolates were cultured from 20 different flocks. Multiple isolates were obtained from 15 of the flocks and 13 of these had two to seven different B. nodosus serotypes. Eleven B. nodosus isolates representing one Australian and ten Oregon serotypes were nonfermentative in various carbohydrates and did not produce indole. These isolates all exhibited proteolytic activity. The prototype strains of 12 of the 14 serotypes demonstrated virulence as assessed by an elastase production assay.     

Pheno- and genotyping of Rhodococcus equi isolated from faeces of healthy horses and cattle

The present study was designed to comparatively investigate 21 Rhodococcus equi isolates from the faeces of clinically healthy horses and cattle. The isolates were identified by cultural and biochemical properties and by PCR analysis. The latter, targeted to the gene coding for the 16S ribosomal RNA, revealed a species specific PCR product.The isolates were further characterised by serotyping with two typing systems, by haemagglutination tests and by plasmid and virulence protein profiling. Among the 21 cultures, four cultures contained plasmids, two of the four cultures expressed 15–17 kDa virulence proteins, no cultures contained 20 kDA virulence proteins. The 21 cultures were further analysed by DNA-finger-printing. This was performed by macrorestriction analysis of chromosomal DNA by pulsed-field gel electrophoresis (PFGE). The DNA-restriction patterns were different for most of the isolates indicating a clone heterogeneity among isolates from single farms.Serotyping, determination of virulence marker and PFGE analysis of R equi appeared to be useful for further characterisation of this species, possibly of importance for virulence estimation of single R equi isolates and for epidemiological studies.     

Pasteurella haemolytica bacteriophage: identification, partial characterization, and relationship of temperate bacteriophages from isolates of Pasteurella haemolytica (biotype A, serotype 1)

Pasteurella haemolytica (biotype A, serotype 1) isolates (n = 15) from the upper respiratory tract of clinically normal cattle, as well as from lung lesions from cases of fatal bovine pasteurellosis, were examined for the presence of bacteriophage after irradiation with UV light. Treatment of all P haemolytica isolates with UV irradiation resulted in lysis of bacteria due to the induction of vegetative development of bacteriophages. The extent of growth inhibition and bacterial lysis in irradiated cultures was UV dose-dependent. Bacterial cultures exposed to UV light for 20 s reached peak culture density between 60 and 70 minutes after irradiation; thereafter, culture density declined rapidly, so that by 120 minutes, it was approximately 60% of the original value. When examined ultrastructurally, lytic cultures from each isolate revealed bacteriophages with an overall length of approximately 200 nm and that appeared to have a head with icosahedral symmetry and a contractile tail. Cell-free filtrate from each noninduced bacterial isolate was inoculated onto the other bacterial isolates in a cross-culture sensitivity assay for the presence of phages lytic for the host bacterial isolates. Zones of lysis (plaques) did not develop when bacterial lawns grown from the different isolates were inoculated with filtrates from the heterologous isolates.     

Characterisation of avian adenoviruses associated with inclusion body hepatitis

Eleven avian adenoviruses were isolated in monolayer cultures of specific pathogen free chicken kidney cells which were inoculated with suspensions of liver, intestine or bursa obtained from 15 broiler flocks experiencing outbreaks of inclusion body hepatitis (10 isolates) and from five unaffected flocks (one isolate). Of the 11 isolates obtained, nine were identified by virus neutralisation tests as serotype 8, one as serotype 1 and one as serotype 12. Adeno-associated viruses were only observed in combination with adenoviral particles of the serotype 12 isolate which was derived from a relatively mild outbreak of inclusion body hepatitis. Only the serotype 1 isolate, obtained from the unaffected broiler flock, consistently caused the death of embryos with marked pathological changes. All of the isolates produced basophilic intranuclear inclusion bodies surrounded by clear halos in chicken kidney cell cultures. DNA preparations, obtained from six strains of serotype 8 avian adenovirus (two New Zealand isolates, three Australian isolates and the reference strain HVI) after digestion with the restriction enzymes EcoRI and BamHI, gave electrophoretic patterns showing the New Zealand isolates to be similar to one another and to strain HVI, but quite distinct from the Australian isolates.     

Effects of Moraxella bovis and culture filtrates on 51Cr-labeled bovine neutrophils

The cytotoxicity of Moraxella bovis whole cells and culture filtrates was studied, using 51Cr-labeled bovine and human blood neutrophils. The cytotoxicity of living M bovis was directly related to the concentration of bacteria in the neutrophil cultures, and was maximal at an approximate neutrophil to bacteria ratio of 1:10. Cytotoxicity was maximal by 30 minutes after living bacteria were added to the suspension of the 51Cr-labeled neutrophils. Expression of the cytotoxicity was dependent on the presence of Ca2+ in the media, and was independent of the presence of Mg2+. Cytotoxic activity was eliminated by inactivating M bovis in buffers containing formalin or sodium azide. Hemolytic and nonhemolytic isolates of M bovis were examined for cytotoxic activity. All 7 of the hemolytic isolates were cytotoxic for bovine neutrophils, but all 4 of the nonhemolytic isolates were devoid of cytotoxic activity. None of the 11 isolates were cytotoxic for human neutrophils. Sterile filtrates from 6-hour shaker cultures of a hemolytic M bovis isolate were cytotoxic for bovine neutrophils. Cytotoxicity of the filtrate was eliminated by heating, incubation with trypsin, or addition of EDTA to the media. Bacterial homogenates or sterile filtrates prepared from statistically incubated cultures of M bovis were not toxic for bovine neutrophils.