猪附红细胞体小鼠感染模型建立条件的优化

为探究猪附红细胞体感染特性,进而优化猪附红细胞体小鼠感染模型建立的条件,本试验设计了不同小鼠处理方式感染试验（A试验）、不同病原形式感染试验（B试验）、冻存病原感染试验（C试验）和小鼠模型血液再感染小鼠试验（D试验）。通过对每个试验中各组小鼠血液感染情况、感染首现时间、临床症状和平均感染时间的综合评估,分析不同小鼠处理方式、不同病原形式、冻存病原及小鼠模型血液再感染是否对小鼠模型的建立有影响。通过PCR检测、电镜观察和特异基因片段序列测定,进一步鉴定小鼠模型感染的病原与猪附红细胞体是否一致。结果显示,A试验中,昆明小鼠切除脾脏组感染效果最优;B试验中,猪附红细胞体阳性血液样本组感染效果最优;C试验中,阳性血液未冻存组感染效果优于冻存组;D试验中,猪阳性血液样本组、小鼠模型阳性血液样本组无明显差异。PCR检测、电镜观察和特异基因片段序列测定结果显示,小鼠模型感染的病原与猪附红细胞体一致。结果表明,不同小鼠处理方式、不同病原形式和病原的冻存等条件对猪附红细胞体小鼠感染模型的感染效果均有影响,通过对比,以猪阳性血液感染切除脾脏的昆明小鼠的方式建立小鼠感染模型效果最优。     

人工感染猪附红细胞体病的病理组织学研究

为观察昆明小鼠人工感染猪附红细胞体后的病理组织学变化。用常规病理学方法,对6只腹腔注射感染猪附红细胞体的小鼠进行病理解剖学和病理组织学研究,光镜下观察并描述小鼠红细胞感染附红细胞体后的形态变化。剖检可见主要器官和皮下瘀血,胸腹部皮下脂肪黄染,病理组织学变化的主要特征是心、肝、脾、肺、肾等主要脏器实质细胞变性、坏死,毛细血管扩张充血、出血,脾窦内含铁血黄素沉着。结果表明,猪附红细胞体感染小鼠模型的成功建立,镜下红细胞的形态特征及主要器官剖检和病理组织学变化与文献报道的猪附红细胞致病特征基本吻合,存在的差异可能与不同地域猪附红细胞体病原株致病性有关。     

Development of a diagnostic PCR assay based on novel DNA sequences for the detection of Mycoplasma suis (Eperythrozoon suis) in porcine blood

An efficient method of control of porcine eperythrozoonosis (PE) caused by Mycoplasma suis is eradication of infection by detection and removal of infected carrier animals. At present, only a few tests are available for the diagnosis of these latent M. suis infections in pigs. The objective of this study was to develop a PCR assay based on novel DNA sequences for the identification of M. suis-infected pigs. A 1.8 kb EcoRI DNA fragment of the M. suis genome was isolated from the blood of pigs experimentally infected with M. suis. Specificity of the DNA fragment was confirmed by DNA sequence analysis and PCR using primers directed against sequences contained in the 1.8 kb fragment. PCR products of 782 bp in size were amplified only from M. suis particles prepared from the blood of experimentally infected pigs but not from any controls, comprising blood from gnotobiotic piglets and a panel of bacteria including other porcine mycoplasmas. PCR results were confirmed by dot blot hybridisation. The applicability of the PCR assay to diagnose M. suis infections in pigs was evaluated by investigating blood samples from 10 symptomatic pigs with clinical signs typical of porcine eperythrozoonosis and blood samples from 10 healthy pigs. The M. suis-specific PCR product was amplified from all samples taken at episodes of acute disease as well as from samples taken during the latent stage of infection, thus demonstrating the suitability of the PCR assay for detecting latent infected carrier animals.     

Prevalence of Streptococcus suis types 1 and 2 in domestic pigs in Australia and New Zealand

Using an indirect fluorescent antibody test, 54 per cent of 734 palatine tonsils of conventional pigs slaughtered in Australia and New Zealand were found to be infected with Streptococcus suis type 1 and 73 per cent of 959 were infected with S suis type 2. Variations in the prevalence of infection in pigs from different herds were thought to be due to differences in the sample sizes rather than to real differences in the prevalence between herds. The prevalence of infection with S suis was similar in pigs of either sex and in different age groups. Streptococcus suis type 2 was detected in the blood of 3 per cent of apparently normal pigs slaughtered at a meat processing plant. The presence of this organism in edible tissue may pose a health risk to consumers and meat-workers. Both S suis types 1 and 2 were detected in the vaginas and uteri of slaughtered pigs and the female reproductive tract could be another site for the carriage of infection. Piglets from sows with vaginas infected with S suis type 2 became infected earlier than piglets from sows with uninfected vaginas. No infected male reproductive tracts were detected and venereal transmission of S suis therefore appears unlikely. Three specific pathogen free herds were found to be free from infection with both S suis types 1 and 2. It is concluded that hysterectomy derived piglets are delivered free from infection, whereas some piglets born to sows with uterine and vaginal infections are either born infected or become infected at, or soon after, birth.     

2308、M28、S1330三株不同种布鲁氏菌的毒力测定

为了测定牛、羊、猪三株不同种布鲁氏菌参考强毒株的毒力,选择了牛种2308、羊种M28和猪种S1330株,分别用雌性豚鼠（Hartley）和雌性小鼠（Balb/c）对其毒力进行测定。豚鼠测毒试验中,用含不同菌数的菌液腹股沟皮下注射5只豚鼠,测定2308、M28、S1330菌株的豚鼠最小感染量（MID）,结果显示以上3种毒株对豚鼠的最小感染量分别为9 CFU、10 CFU和30CFU。小鼠测毒实验中,将2308、M28和S1330菌液按1×105CFU/0.2 mL/只腹股沟皮下注射小鼠各5只,2周后分别剖杀小鼠,取脾脏测定含菌量,平均脾含菌量分别为1676971、314765、83811CFU/g脾脏。豚鼠和小鼠测毒均显示牛种2308株毒力最强,羊种M28株次之,猪种S1330毒力最弱。本研究首次用豚鼠和小鼠同时测定了布鲁氏菌2308、M28、S1330株的毒力,补充了布鲁氏菌参考强毒株的毒力数据。     

Development and evaluation of enzyme-linked immunosorbent assay based on recombinant inorganic pyrophosphatase gene antigen for the detection of Mycoplasma suis antibodies

The recombinant ppa protein of Mycoplasma suis migrated to 21 kDa. Using this antigen, an ELISA system to detect the antibody against M. suis infection in swine was established. The rELISA demonstrated 98.5% specificities among negative samples and 96.9% sensitivity among positive samples with M. suis infection. A comparison of this ELISA system with an indirect hemagglutination assay (IHA) test using 132 swine samples revealed that the positive rate was 34.0% in ELISA and 28.0% in IHA. Compared with IHA, the present rELISA system using recombinant ppa antigen significantly improves the specificity, sensitivity, and stability for serodiagnosis of M. suis infection in swine.     

猪链球菌2型感染昆明小鼠模型的建立及评价

为建立稳定的猪链球菌2型(Streptococcus suis type 2,SS2)人工发病模型,本试验以天津某发病猪场分离的SS2 Y15293株为研究对象,经腹腔注射感染昆明小鼠,测定其LD₅₀,确定造模感染剂量,通过观察感染后小鼠的临床症状和病理变化,测定试验期间各组小鼠体重和采食量的变化及细菌回归等试验对模型进行评估。结果显示,SS2 Y15293株致昆明小鼠的LD₅₀为6.7×10~7 CFU/mL。以1个LD₅₀的剂量攻毒,与对照组相比,试验小鼠主要发病表现为攻菌后24 h出现精神沉郁、被毛逆立、眼睛有分泌物,72～96 h出现头颈歪斜、翻滚、震颤等神经症状,死亡高峰在48～72 h。3次重复试验中,试验组小鼠的发病率均为100%,死亡率为50%～70%,神经症状小鼠出现比率20%～30%。与对照组相比,试验组小鼠采食量和体重显著下降(P<0.05),临床症状分值显著升高(P<0.05)。组织病理检查发现,试验组小鼠心脏、肺脏组织均有不同程度出血、炎性细胞浸润;脑膜炎,脑室出血,充满炎性细胞。细菌回归试验结果表明,试验组死亡小鼠脑、心脏、肝脏、脾脏、肺脏、肾脏均有细菌定植,且形态、染色和分子生物学特性与攻毒菌一致。以上结果证实,SS2 Y15293株能感染昆明小鼠并致其发病,感染动物发病规律性强,重复性好,临床症状典型,说明SS2 Y15293株感染昆明小鼠可作为SS2的候选人工感染模型。     

Vertical Transmission of Brucella abortus Causes Sterility in Pregnant Mice

The mechanisms of abortion and sterility induced by bacterial infection are largely unknown. In the present study, we found that Brucella abortus, a causative agent of brucellosis and facultative intracellular pathogen, caused sterility in pregnant mice. We have recently established a mouse model for abortion induced by B. abortus infection and high rates of abortion are observed for bacterial infection on day 4.5 of gestation, but not for other days. Infected newborn (first generation) mice showed poor growth compared with uninfected newborn mice and bacterial replication in the spleen of the former was observed over a long period. When infected first generation female mice were mated to infected first generation male mice, the number of fetuses was significantly less than that in uninfected first generation mice. These infected second generation mice also showed poor growth. These results suggest that vertical transmission of B. abostus causes sterility in pregnant mice and our mouse model would be useful for the investigating of brucellosis.     

猪致病性链球菌的分离鉴定

采集33例临床症状疑似猪链球菌感染猪的全血及内脏器官进行涂(触)片染色镜检,取有球菌感染的样品16份进行血琼脂平板分离纯化试验;根据血琼脂平板上菌落形态、溶血情况及对单个菌落涂片染色镜检结果,共筛选出12份可疑样品,进一步采用液体培养基对12份样品进行增菌及纯化培养;选取纯化培养菌进行链球菌生化试验,结果共鉴定出11株纯化的猪链球菌;11株猪链球菌均对小鼠表现出高致病力.     

猪链球菌2型的分离鉴定及致病性试验

从全国部分猪场采集疑似猪链球菌感染病例脑样品20份进行细菌分离,成功分离到10株细菌,通过猪链球菌及血清类型鉴定,最终确定其中一株为2型猪链球菌。应用猪链球菌7种主要毒力因子特异性基因扩增检测方法检测所分离到的2型猪链球菌的毒力因子分布情况,并应用小鼠攻毒试验对其致病性进行观察研究。结果表明:分离的2型猪链球菌具备7种毒力因子;动物试验表明SS2能引起小鼠的急性败血症及脑膜炎;细菌回归试验结果表明,试验组死亡小鼠的脑、心脏、肝脏、脾脏、肺脏、肾脏均有细菌定植,且能分离出攻毒菌。此分离菌株的研究为研制2型猪链球菌病疫苗奠定了基础。     

头孢噻呋混悬剂对猪链球菌Ⅱ型小鼠模型的治疗试验

建立小鼠链球菌病的模型,评价头孢噻呋混悬剂的疗效。选用小白鼠人工感染猪链球菌Ⅱ型建立疾病模型,并应用不同剂量头孢噻呋混悬剂进行治疗试验。结果表明,链球菌Ⅱ型感染的小鼠模型能表现出典型的临床症状及病理变化,且具有较好的重复性。5和10mg/kg组治愈率均为100%,2mg/kg组治愈率为70%。因此,头孢噻呋混悬剂作为注射剂治疗小鼠猪链球菌Ⅱ型感染以剂量5mg/kg为佳。     

抗菌肽NZ2114对多重耐药2型猪链球菌感染小鼠的治疗效果评价

为探究抗菌肽NZ2114对猪链球菌感染小鼠模型的体内治疗效果,本试验以NZ2114和2型猪链球菌CVCC 3928为研究对象,利用小鼠模型评价NZ2114治疗猪链球菌感染的效果。36只ICR雌鼠随机均分为6组：空白对照组（不感染,腹腔注射0.2 mL PBS）、阴性对照组（感染,腹腔注射0.2 mL PBS）、试验Ⅰ和Ⅱ组（感染,分别腹腔注射0.2 mL 2.5和5.0 mg/kg NZ2114）、阳性对照Ⅰ和Ⅱ组（感染,分别腹腔注射0.2 mL 7.5和15.0 mg/kg头孢曲松钠）。结果显示,空白对照组和5.0 mg/kg NZ2114试验组小鼠存活率均为100%,而15.0 mg/kg头孢曲松钠阳性对照组小鼠存活率仅为50%。治疗1 d后,5.0 mg/kg NZ2114试验组小鼠肺脏和肝脏荷菌量分别下降59.41%（P < 0.01）和69.19%（P < 0.01）;而15.0 mg/kg头孢曲松钠阳性对照组中小鼠肺脏和肝脏荷菌量分别下降43.94%（P < 0.01）和19.60%（P < 0.05）。与阴性对照组相比,2.5 mg/kg NZ2114治疗组中抑制炎性因子TNF-α和IL-1β的释放分别下降85.83%（P < 0.01）和56.20%（P < 0.05）,5.0 mg/kgNZ2114试验组分别降低84.02%（P < 0.01）和43.86%（P < 0.05）,而15.0 mg/kg头孢曲松钠阳性对照组TNF-α及IL-1β的下降率均不显著,分别为24.49%和8.82%。另外,5.0 mg/kg NZ2114试验组1 d后即可明显减轻小鼠肺组织间质弥漫性炎细胞浸润等炎症症状,抑制肝细胞产生肿胀及空泡性变化,促进脾小结恢复正常;治疗7 d后各器官组织临床症状评分基本恢复正常。上述结果表明,NZ2114能有效提高猪链球菌感染小鼠的存活率,显著降低病原菌在小鼠肺脏和肝脏的移位及血清中TNF-α和IL-1β的水平,并有效缓解肝脏、脾脏和肺脏的急性损伤,治疗效果优于头孢曲松钠,具有作为治疗临床猪链球菌病抗生素替代品的潜力。     

Evaluation of an enzyme-linked immunosorbent assay for detection of Eperythrozoon suis antibodies in swine.

An ELISA was developed and tested to detect antibodies to Eperythrozoon suis in swine. Results were compared with those of the indirect hemagglutination (IHA) test. Antigen isolated from swine heavily infected with E suis was used for both tests. Comparison of the ELISA with the IHA test revealed a significant (P less than 0.001) correlation between results. Of 114 samples obtained from 9 swine infected with E suis, 87.7% were seropositive (titer greater than or equal to 200) via the ELISA, and 80.7% were seropositive (titer greater than or equal to 20) via the IHA test. The sensitivity of the ELISA was greater than that of the IHA test. All blood samples obtained from specific-pathogen-free swine tested negative for E suis antibody. Cross-reactions were not observed between E suis antigen and antisera against various swine and cattle disease agents using ELISA. We concluded that the ELISA may be used for rapid and effective diagnosis of infection with E suis in swine.     

猪附红细胞体PCR检测方法的建立和初步应用

基于猪附红细胞体广东株16S rRNA基因的序列特点，设计合成种特异性引物，建立了猪附红细胞体PCR检测方法。该方法能特异性扩增523bp的猪附红细胞体16SrRNA基因片段，而对猪丹毒杆菌G4T10株、猪链球菌STl71株、多杀性巴氏杆菌E0630株、猪胸膜肺炎放线杆菌、猪肺炎支原体、鸡毒支原体和猫血巴尔通氏体CA株的基因组DNA没有扩增带出现。对猪附红细胞体基因组DNA的最小检测量为160pg。通过对38份临床样品的检测，8份为猪附红细胞体感染阳性，其余为阴性。结果表明，建立的PCR检测方法具有极高的敏感性和特异性，可用于急性猪附红细胞体病和临床健康带菌猪的诊断。     

酵母甘露聚糖肽对致病性大肠杆菌感染小鼠的保护作用

本试验旨在研究酵母甘露聚糖肽（MTY）对致病性大肠杆菌（PEC）感染小鼠的保护作用。选取无特定病原体（SPF级）昆明小鼠40只,随机分为4组,分别为空白组、模型组、阳性药物组、MTY组（每组10个重复,每个重复1只小鼠）,其中,空白组和模型组灌服生理盐水,阳性药物组灌服抗生素阿莫西林,MTY组灌服酵母甘露聚糖肽。4周后,对模型组、阳性药物组、MTY组昆明小鼠进行致病性大肠杆菌（PEC）攻毒试验,通过对攻毒试验前小鼠肠道微生物分析以及PEC感染后小鼠死亡率、脏器指数、肠道组织以及各器官切片组织病理分析,同时观察体内试验过程小鼠体质变化,综合评价MTY对PEC感染小鼠的保护效果。结果表明,MTY对PEC有抑制作用。与空白组相比,MTY组小鼠肠道微生物菌群有显著性差异（P<0.05）,主要表现为有益菌丰度增加,菌群多样性增加;与模型组相比,MTY处理能有效降低小鼠感染PEC后的死亡率;MTY显著提高了小鼠胸腺指数（P<0.05）,降低脾指数（P>0.05）,并极显著降低肝指数（P<0.01）;MTY抑制了小鼠空肠杯状细胞的减少（P>0.05）;MTY抑制了小鼠肠道中MUC2及ZO-1表达下调（P<0.05）;MTY对感染PEC后的小鼠肠道、肝、脾和肺等器官均起显著的保护作用。由此可见,灌服酵母甘露聚糖肽能减轻PEC对机体的损害,进而对小鼠起保护作用。     

猪链球菌2型感染普通仔猪模型的建立及其病理学观察

试验采用普通长白系仔猪建立猪链球菌2型动物模型,并进行详细的病理学观察。将8头仔猪随机分为试验组和对照组,用猪链球菌2型菌血静脉接种试验组,对照组接种生理盐水2 mL/头。攻毒猪5 d内全部死亡, 主要表现为脑膜炎、心外膜炎、关节炎和败血症等典型症状和病理变化,与自然感染情况相似。从感染死亡猪肺、脾、肝、心中均分离到与攻毒菌株相同的细菌。证明猪链球菌2型菌血静脉接种普通长白系仔猪可以复制出典型病例,成功建立了动物模型,对SS2致病性研究具有重要意义。     

一例貉大肠杆菌与血虫混合感染的诊断鉴定

为诊断吉林养殖场一例病死貉的病原,本试验运用细菌培养、染色镜检、生化鉴定、分子生物学鉴定和血液涂片镜检等方法对采集的病死貂的肺脏、肝脏、脾脏、肾脏、肠道及血液进行病原分离鉴定,并对分离到的病原菌进行致病性和药敏试验测定。结果显示,共分离出1株优势菌株,该分离菌株在光学显微镜下呈现为两端钝圆的杆状革兰氏阴性菌;生化试验结果显示,其能分解葡萄糖、乳糖、甘露醇、麦芽糖和蔗糖产酸产气,对于M-R试验、吲哚试验、淀粉水解试验结果为阳性;其16S rRNA序列与NCBI数据库中登录的已知大肠杆菌序列同源性达99%;动物致死性试验结果表明,该菌株对小鼠具有致病性;药敏试验结果显示,分离菌对喹诺酮类药物诺氟沙星和环丙沙星最敏感,对其他药物具有不同程度的耐药性;对病貉抗凝血进行涂片镜检证明有血虫感染,且红细胞感染率达98%以上。综上所述,该病貉感染的病原菌株为大肠杆菌且该大肠杆菌具有致病性,同时病貉患有血虫病,并导致了其生长发育迟缓。     

不同储存方法对鼠肉内旋毛虫幼虫感染性影响

旋毛虫阳性小鼠的肌肉经不同方法储存后,为观察其对小鼠感染性的变化,取健康小白鼠20只随即分作4组,每组分别喂食新鲜、4℃冷藏、及-18℃冷冻,及腌制＋冷藏一定时间的旋毛虫阳性的小鼠腿肌肉约1g。饲养1～2周,剖杀,取其膈肌、腿肌镜下观察是否呈旋毛虫囊包阳性。结果显示,喂食新鲜旋毛虫阳性鼠肉的小鼠和喂食经过冷藏的阳性鼠肉的小鼠都感染了旋毛虫,而喂食经冷冻的和经腌制＋冷藏的鼠肉的小鼠均未感染旋毛虫,可见,将肉类冷冻或严格冷藏储存一定时间可杀死其中的旋毛虫,减弱其致病性,降低因食用疫肉而患病的风险。     

Alteration of integrin-associated protein (CD47) on experimental porcine eperythrozoonosis

To evaluate the alteration of CD47 on RBCs of pigs infected with M. suis, we induced the experimental porcine eperythrozoonosis and collected the blood samples at the different time points. The result of analysis by flow cytometry after reaction with mouse-anti-human CD47 and caprine-anti-mouse IgG-FITC reagents indicated that the CD47 quantity on RBCs changed correlatively with the course of PE. The lowest value of M1 (percentage of the positive cells in fluorescence intensity) occurred on day 7 post inoculation at the peak of parasitemia and decreased 82% compared with the control sample. And then, the values of M1 on day 14 and 21 rised slowly but were still significantly different with the controls (p < 0.01). These suggested that the quantity of CD47 on RBCs altered progressively with the phases of the PE disease. The decrease of CD47 on RBCs maybe weaken the inhibitory CD47-SIRPalpha interaction and provide positive signals for phagocytosis of macrophages resulting in the removal of the RBCs from the circulation. In conclusion, CD47, a marker on RBCs, maybe play an important role on the mechanism of hemolysis caused by infection of M. suis.     

Distribution of rabies virus in infected mice,vaccinated and submitted to P. acnes as immunomodulator

The lethality and distribution of rabies virus were evaluated in swiss mice experimentally infected with street rabies virus, vaccinated and submitted to immunomodulation by P .acnes (formerly Corynebacterium parvum). The animals were sacrificed at different times,when the different tissues were collected and submitted to fluorescent antibody test (FAT) and mouse inoculation test (MIT). The group submitted to vaccination and P. acnes treatment presented a percentage of survival superior to that observed in infected mice only treated with P. acnes. Control infected animals had the lowest survival rates.The distribution of rabies virus in spleen of infected mice, vaccinated and submitted to P. acnes was superior to that verified in infected mice not treated with P.acnes. The increased survival correlated with the distribution of rabies virus in lymphoid tissues, could be interpreted as the consequence of P. acnes activity on macrophages. The results suggest the role of macrophages against rabies virus infection in mice and the importance of vaccination in the post expositive treatment of rabies.