鸭坦布苏病毒对雏鸭免疫系统的影响

为研究鸭坦布苏病毒(DTMUV)对雏鸭免疫系统的影响,本试验对5日龄雏鸭静脉接种DTMUV,并于接种后不同时间取雏鸭脾脏、胸腺、法氏囊进行组织病理学、抗原和凋亡检测。结果显示,DTMUV感染雏鸭的脾脏、胸腺、法氏囊均严重受损。接种DTMUV后4d,脾脏淋巴细胞减少,胸腺可见严重细胞崩解和大面积坏死区域,法氏囊滤泡轻度萎缩;接种后6d免疫器官病变最为严重,其中胸腺静脉严重栓塞,淋巴细胞变性坏死,空泡化也更加严重;接种后8d免疫器官病变均有所减轻,至16d,免疫器官结构基本恢复正常。通过免疫组织化学方法在感染雏鸭的脾脏、胸腺、法氏囊中均检测到DTMUV抗原;细胞凋亡试验显示DTMUV能够显著引起脾淋巴细胞凋亡。综上所述,DTMUV可严重损伤雏鸭免疫器官,且这种损伤常发生于感染早期,并于感染后8d出现好转。     

副猪嗜血杆菌对小鼠的致病性探究及感染模型的建立

为研究副猪嗜血杆菌(Haemophilus parasuis,HPS)对昆明系(KM)小鼠的致病性,同时探索建立HPS的KM小鼠感染模型,应用血清4型(SD-05、ZC-7、ND-21)、5型(BD-1、HB-08、PZ-26)菌株对KM小鼠进行毒力测定,然后以2种血清型最适菌株的最适剂量,腹腔注射KM小鼠开展攻毒试验,观察临床症状及剖检变化,并对死亡小鼠进行细菌分离以及PCR鉴定。结果显示:血清4型SD-05株、血清5型HB-08株毒力较强,对KM小鼠最适接种剂量分别为1.2×10~9及1.175×10~9 CFU;攻毒后小鼠出现颤抖、萎靡厌食等临床症状,剖检可见肺脏出血、胸腔积液等病理变化,并在小鼠组织器官中成功分离出HPS菌株。结果表明,HPS血清4型、5型菌株对KM小鼠存在较强致病性,且初步认定KM小鼠可作为HPS感染动物模型。本研究为HPS致病性研究及小鼠感染模型建立奠定了应用基础。     

口服蓖麻毒素对小鼠肠道及免疫器官的毒性作用

本试验通过灌胃小鼠纯化蓖麻毒素(1/5 LD50),对毒素在机体内对胃肠道及免疫器官的毒效应进行了分析.中毒初期,小鼠体增重及胸腺、脾脏相对重均明显下降,随着毒素的不断排出,小鼠的体征有恢复正常的迹象.病理切片、扫描电镜及透射电镜的结果表明,蓖麻毒素可引起一系列肠道病理反应,肠粘膜损伤极其严重;脾细胞发生明显的凋亡和坏死症状.     

鸡感染J亚群禽白血病病毒的免疫抑制机理

通过人工接种建立了雏鸡感染J亚群禽白血病病毒（ALV-J）后发生免疫抑制的病理模型。对免疫抑制的机理进行了初步研究。结果表明：ALV-J感染早期可诱导胸腺、法氏囊的淋巴细胞凋亡，这种早期的淋巴细胞凋亡是胸腺和法氏囊萎缩的重要原因之一。病理组织学动态观察表明。ALV-J主要引起骨髓的髓系细胞灶状或弥漫性增生，病变导致骨髓机能受损，使机体免疫机能下降。是引起免疫抑制的根本原因。病毒感染组免疫器官均发生了严重的实质萎缩性病变．这种病变的发生除与骨髓的病变及淋巴细胞凋亡有关外。还与中后期淋巴细胞的坏死有关．从而导致严重的免疫抑制。ALV-J感染可引起免疫器官及部分内脏器官中嗜酸性粒细胞样的瘤细胞浸润增生，这可以作为病毒感染早期的病理诊断指标。     

小鼠感染猪圆环病毒2型的超微结构病变观察

为探讨猪圆环病毒2型（PCV2）对小鼠超微结构的病理损伤及其病毒在细胞内的复制,20只6周龄的昆明小鼠随机平均分成2组（即A组和B组）,A组小鼠经腹腔注射PCV2细胞培养物0.1mL/只（含病毒1 000TCID50）,B组以同样的方式和剂量注射无菌细胞培养液作为对照。于PCV2感染后14d,处死所有小鼠,取其组织做电镜观察和PCV2PCR检测。结果显示,在电镜下,所有PCV2感染鼠的超微结构病变基本一致,主要表现为淋巴器官、心脏、肝脏、肺脏、肾脏、脑、肠等脏器实质细胞凋亡或坏死,细胞内线粒体水肿、内质网扩张,间质毛细血管淤血、炎症细胞浸润,并在脾脏、胸腺、淋巴结的淋巴细、巨噬细胞,以及肝细胞,肾足细胞,脑神经细胞的胞浆或胞核内发现病毒包涵体。同时通过PCR检测,所有PCV2感染鼠的组织均可检出PCV2DNA。B组（对照组）小鼠除在淋巴结可见极少数淋巴细胞凋亡外,其他组织均无任何超微结构病变;同时,所有组织也未检出PCV2DNA。由此说明PCV2可在昆明小鼠多脏器实质细胞内复制,并诱导细胞凋亡。     

副猪嗜血杆菌重组抗原蛋白对小鼠的免疫效果评价

为研制对不同血清型具有交叉保护作用的副猪嗜血杆菌(HPS)重组抗原蛋白,本研究将390只6周龄雌性昆明鼠随机分为13组,每组30只,分别为HPS血清型5型单个重组蛋白免疫组:外膜蛋白(D15)组、分泌蛋白(afuA)组、转铁结合蛋白(tbpB)组、细胞致死膨胀毒素(cdtC)组、PBS组,联合重组蛋白免疫组:HPS血清型4+5+13型D15、HPS血清型5型D15+tbpB、D15+cdtC、cdtC+tbpB、D15+afuA、D15+cdtC+tbpB、D15+afuA+tbpB及PBS组。每间隔2周经皮下注射免疫2次各组相应乳化蛋白,于一免后2周、二免后2周采集各组小鼠尾静脉血,利用间接ELISA检测小鼠血清抗体效价,并通过免疫鼠脾淋巴细胞增殖试验、细胞因子检测、血清杀菌试验及攻毒保护试验评价重组蛋白的免疫效果。结果显示,免疫组小鼠均能产生显著的T淋巴细胞增殖反应,其产生的血清抗体均能极显著抑制HPS生长(p0.001),且抗体水平均极显著高于对照组(p0.001);联合重组蛋白免疫组小鼠产生的IgG抗体水平、细胞因子IL-2、IL-4和IFN-γ的水平均略高于单个重组蛋白免疫组;其中,HPS血清型5型D15+afuA和血清型4+5+13型D15免疫组小鼠产生的IgG抗体水平略高于其余联合重组蛋白免疫组,各联合免疫组间细胞因子水平差异不显著;HPS血清型5型D15+afuA+tbpB和血清型4+5+13型D15对4型HPS感染的保护率均为71%,对5型菌株的保护率均为86%,对13型菌株的保护率为71%和86%,表明血清型5型D15+afuA+tbpB重组蛋白组合和血清型4+5+13型D15重组蛋白组合对HPS感染具有交叉保护的潜力。本研究为HPS亚单位疫苗的研制提供了实验依据。     

多子小瓜虫感染对草鱼免疫相关器官影响的病理学观察

为观察多子小瓜虫(Ichthyophthirius multifiliis)感染草鱼免疫相关器官的病理学变化,人工进行草鱼小瓜虫感染,酯酶染色方法检测血液中T、B淋巴细胞的百分比,然后采用常规染色(H.E)和透射电镜方法观察各组草鱼鳃、肝和脾脏的结构变化。结果显示:感染组T淋巴细胞百分率明显高于B淋巴细胞百分率;光学显微镜下:感染组草鱼鳃丝上皮网状结构消失,细胞变性、坏死,有小瓜虫虫体;肝脏的肝板结构紊乱,细胞变性、坏死,窦间隙充满红细胞;脾脏红髓和白髓结构紊乱,淋巴细胞变性、坏死;透射电镜下,感染组草鱼鳃丝超微结构遭受严重破坏,细胞内可见残存细胞结构碎片和小瓜虫虫体;肝板完整结构断裂,细胞核溶解或消失;脾脏淋巴细胞数量减少,核周间隙消失。说明:感染小瓜虫的草鱼免疫相关器官损伤严重,各器官细胞以变性、坏死为主要特征,酯酶染色结果揭示草鱼感染小瓜虫初期以T淋巴细胞免疫为主。     

缺锌对雏鸭免疫功能影响的研究

1日龄天府肉鸭 10 0只分为 2组 ,喂以缺锌 (Zn 2 2 9mg)和对照 (Zn 10 0mg)日粮 7周 ,观察缺锌对免疫功能的影响。缺锌组免疫器官的绝对重量、生长指数和淋巴细胞生长周期以及外周血T淋巴细胞ANAE+阳性率、血清免疫球蛋白含量和红细胞C3b受体花环、免疫复合物花环率显著降低 (P <0 0 1或P <0 0 5 ) ;病理组织学观察 ,免疫器官淋巴细胞显著减少并呈退行性变 ,网状细胞变性坏死 ,电镜下淋巴细胞胞核固缩或溶解 ,细胞器消失 ,网状细胞粗面内质网扩张 ,线粒体肿胀 ,出现大小不等的溶酶体、吞噬体和自噬体。结果表明 ,缺锌严重抑制免疫器官生长发育和外周血T淋巴细胞活化 ,显著降低血清免疫球蛋白含量和红细胞免疫功能 ,并对免疫器官造成明显的病理损伤 ,导致免疫功能下降。     

实验性热应激对肉仔鸡免疫器官的影响

对实验性热应激肉仔鸡的免疫器官的组织学变化、超微组织学变化进行了动态观察，对热应激造成的免疫器官的细胞凋亡及免疫反应能力进行了检测。结果表明，热应激可引起免疫器官胸腺、脾脏、法氏囊的发育分化不良，使法氏囊指数、脾脏指数明显下降，胸腺指数虽有下降，但幅度较小；热应激对免疫器官的组织结构有显著影响，尤其对法氏囊和脾脏的影响更明显，主要表现为实质细胞的萎缩、消失性病变，但无明显的实质细胞崩解坏死、炎性细胞浸润、炎性充血等病理变化，并随热应激时间的延长而逐渐加重，最后以实质细胞几乎完全消失、间质结缔组织增生而纤维化告终。热应激可显著影响试验鸡血清新城疫病毒抗体滴度，与对照相比，其抗体滴度的峰值低，维持时间短，下降速度快；通过电镜观察和细胞凋亡的原位检测，证实热应激时免疫器官的淋巴细胞和巨噬细胞有凋亡现象，这种凋亡现象在早期尤其显著。     

实验性热应激对肉仔鸡免疫器官的影响

对实验性热应激肉仔鸡的免疫器官的组织学变化、超微组织学变化进行了动态观察,对热应激造成的免疫器官的细胞凋亡及免疫反应能力进行了检测.结果表明,热应激可引起免疫器官胸腺、脾脏、法氏囊的发育分化不良,使法氏囊指数、脾脏指数明显下降,胸腺指数虽有下降,但幅度较小;热应激对免疫器官的组织结构有显著影响,尤其对法氏囊和脾脏的影响更明显,主要表现为实质细胞的萎缩、消失性病变,但无明显的实质细胞崩解坏死、炎性细胞浸润、炎性充血等病理变化,并随热应激时间的延长而逐渐加重,最后以实质细胞几乎完全消失、间质结缔组织增生而纤维化告终.热应激可显著影响试验鸡血清新城疫病毒抗体滴度,与对照相比,其抗体滴度的峰值低,维持时间短,下降速度快;通过电镜观察和细胞凋亡的原位检测,证实热应激时免疫器官的淋巴细胞和巨噬细胞有凋亡现象,这种凋亡现象在早期尤其显著.     

Interactions of Haemophilus parasuis and its LOS with porcine brain microvascular endothelial cells

Haemophilus parasuis is a swine pathogen that causes Gl?sser's disease, which is characterized by polyserositis and meningitis. The pathogenesis of the H. parasuis infection is poorly understood. To cause meningitis, H. parasuis has to cross the blood-brain barrier (BBB) to gain access to the central nervous system (CNS). We recently showed that H. parasuis adheres to and invades porcine brain microvascular endothelial cells (PBMEC). The aim of this study was to evaluate the role of H. parasuis lipooligosaccharide (LOS) in the adhesion to PBMEC and to determine if H. parasuis (and/or its LOS) is able to induce apoptosis and activation of PBMEC. Results showed that adhesion of H. parasuis to PBMEC was partially mediated by LOS. Moreover, H. parasuis induces caspase-3-mediated apoptosis of PBMEC in a time--and dose--dependent manner, but its LOS did not seem to be involved in such a process. Furthermore, H. parasuis and, to a lesser extent, its LOS, was able to induce the release of IL-8 and IL-6 by PBMEC. Field strains of H. parasuis serotypes 4 and 5 induced similar levels of these inflammatory mediators. Our data suggest that H. parasuis uses cellular adhesion, induction of apoptosis and up-regulation of inflammatory mediators as mechanisms to invade the CNS via the BBB, and that LOS would play a certain but limited role in such pathological process.     

Differences in susceptibility to Haemophilus parasuis infection in pigs

In animal breeding programs, deoxyribonucleic acid (DNA) markers can be used to identify sires that are less susceptible to disease. These DNA markers are typically discovered in populations that display differences in susceptibility. To find those differences, it was hypothesized that sires influence their offspring responses to infection with H. parasuis. To identify differences in susceptibility, colostrum-deprived pigs derived from 6 sires were inoculated with a virulent strain of H. parasuis serovar 5. Pigs were infected at 21-d of age and euthanized 1, 2, or 3 days post-infection. Rectal temperatures, bacterial detection, clinical signs, and lesions were measured by comparing disease susceptibility in the offspring from each sire. The effect of the sire on the severity of disease in the offspring was statistically analyzed using to a 2-way ANOVA with sire and test day as fixed effects. Significant differences among sires were found for lesions, rectal temperatures from days 0-1 and 0-2 (P < 0.05) and marginal effects for clinical signs (P = 0.08). On average, the offspring of sire H94 was the most susceptible to challenge. Responses to infection were categorized to determine the clinical responses and analyzed by Chi square. Overall, 10% of all pigs infected were fully resistant to H. parasuis infection. Boar H94 didn't produce any fully resistant offspring. Differences in susceptibility to H. parasuis were observed, and the results support the hypothesis that sires influence their offspring's response to infection. Tissues from this population could be used to identify DNA markers for genetic selection of sires that produce offspring more resistant to H. parasuis infection.     

Immunological Identification and Characterization of Extracellular Serine Protease-Like Protein Encoded in a Putative espP2 Gene of Haemophilus parasuis

Haemophilus parasuis is known to produce a group of virulence-associated autotransporter (AT) proteins, VtaAs; however, no other ATs have been characterized yet. On the basis of the reported sequence of a putative espP2 gene for extracellular serine protease (ESP)-like protein of H. parasuis, this putative AT gene was successfully amplified from H. parasuis serotype 5 field strain HPS0819, cloned and sequenced. The confirmed ORF sequence showed 100% identity with the reported putative espP2 gene. The recombinant ESP-like protein purified from Escherichia coli with a pET expression system was used for immunological characterization. An approximately 85 kDa antigen was detected in cultured H. parasuis by using antiserum to the purified ESP-like protein, and antibodies against the recombinant ESP-like protein were detected in a selected serum from pigs with experimental H. parasuis infection. The results indicated that H. parasuis could produce ESP-like protein in vitro and in vivo. In an immune protection study using guinea pigs, 6 out of 10 animals immunized with the recombinant ESP-like protein survived after challenge with 5 × 10(9) bacteria of strain HPS0819, whereas 7 out of 10 animals immunized with formalin-inactivated H0819 bacterin survived after challenge. The results suggest that ESP-like protein could be one of the vaccine antigen candidates for H. parasuis infection.     

我国部分地区副猪嗜血杆菌病的流行病学调查

采用细菌分离鉴定、结合PCR方法,对江苏、上海、安徽、广西、浙江、江西、山东、河南、湖北等省市2003年11月～2005年4月送检的159个患病猪场仔猪肺脏进行了副猪嗜血杆菌检测,结果阳性猪场为76个,阳性率48%,临床症状主要为呼吸困难和体温升高;病理变化主要有肺炎病变和淋巴结肿大或出血。通过数据统计分析,发现该病已在我国不同地区广泛流行,且多发于秋、冬季节。该研究为副猪嗜血杆菌的流行病学研究奠定了基础,也为该病的临床预防提供了理论依据。     

豚鼠副猪嗜血杆菌感染动物模型的建立

为建立副猪嗜血杆菌(Hps)的感染动物模型,本试验用Hps血清5型标准菌株(Nagasaki),以2.0×10~9CFU剂量腹腔感染豚鼠,观察豚鼠发病及死亡情况.取死亡豚鼠的主要器官组织,观察其病理和组织病理变化,与猪Classer's病痛变进行比较.并同时对死亡豚鼠进行细茵分离,分离菌经PCR鉴定.实验结果显示:在接种14 h后试验组豚鼠(5/8)出现死亡,死亡豚鼠剖检时出现了与猪Classer's病相似的病变;主要组织器官组织学变化以炎性细胞浸润、纤维蛋白和红细胞渗出等变化;并通过细茵分离培养,在豚鼠大脑、心血、肺、肝、脾和肾主要器官中分离到Hps血清5型茵.实验结果表明豚鼠可以作为Hps的感染动物模型.这一结果为研究其致病机制、诊断和免疫研究奠定基础.     

Comparative virulence of Haemophilus parasuis serovars 1 to 7 in guinea pigs.

Reference strains for Haemophilus parasuis serovars 1 to 7 were examined for virulence by inoculation of guinea pigs. Guinea pig response to intraperitoneal inoculation was similar for the 7 reference strains. However, apparent differences in virulence were detected after intratracheal inoculation. Cells of the references strains for serovars 1 and 5 were most invasive, causing moribundity or death at higher doses and a persistent septicemia at lower doses. Haemophilus parasuis could be isolated from respiratory and systemic sites; purulent bronchopneumonia, pericarditis, and pleuritis were apparent in infected guinea pigs. Inoculation of cells of the reference strains for serovars 2 and 6 also resulted in bronchopneumonia and moribundity or death in some guinea pigs; however, reisolation of H parasuis and microscopic lesions at necropsy were less pronounced than those observed with serovars 1 and 5. Inoculation of cells of serovars 3, 4 and 7 induced only transient clinical signs and minimal evidence of H parasuis infection at necropsy. The data from intratracheal inoculation of guinea pigs are similar to data from other investigations in swine, indicating differences in the pathogenic potential of H parasuis strains. Thus, guinea pigs may be useful as a laboratory animal model for examining cellular factors associated with virulence and immunogenicity of H parasuis.     

Comparison between Haemophilus parasuis infection in colostrums-deprived and sow-reared piglets

The aim of this study was to compare the development of Glasser's disease in sow-reared and colostrum-deprived piglets. Ninety piglets from a commercial pig farm in Spain were used. The farm was positive for Haemophilus parasuis. Fifty-two pigs were sow-reared (SR) and 38 were colostrum-deprived (CD) piglets. The animals were intratracheally inoculated with H. parasuis serovar 5 and sacrificed at 1, 2 and 3 days post-infection. To assess the development of disease, antibody titers, clinical signs, pathological lesions, microbiological isolation and PCR amplification were compared between the groups. Inoculation of SR pigs did not cause clinical signs or lesions of Glasser's disease. In SR pigs, H. parasuis isolation and specific PCR amplification from tissues showed a very low number of positive samples. In contrast, in CD pigs, inoculation resulted in the typical signs and lesions of Glasser's disease. Positive microbiological isolation and specific PCR products were obtained from the majority of the tissues tested, and no antibodies against H. parasuis were detected. The experimental infection using CD pigs describes a successful method to study this microorganism and confirms the important role that maternal antibodies play in protection against clinical signs and disease.     

Analyses of Dutch Haemophilus parasuis isolates by serotyping, genotyping by ERIC-PCR and Hsp60 sequences and the presence of the virulence associated trimeric autotransporters marker

In this study, 117 isolates of Haemophilus parasuis from organs and tissues from pigs showing clinical signs, were characterised and compared with 10 H. parasuis reference strains. The isolates were subjected to the 16S rRNA gene PCR and subsequently serotyped, genotyped by 60-kDa heat shock protein (Hsp60) gene sequences, the enterobacterial repetitive intergenic consensus (ERIC) PCR and a multiplex PCR for the detection of the vtaA virulence associated trimeric autotransporter genes. Serotyping revealed the presence of 13 H. parasuis serovars. Serovars 3 and 10 were not detected, and 16 of the 117 H. parasuis isolates could not be typed by specific antisera. All isolates were positive in the 16S rRNA gene specific H. parasuis PCR. ERIC-PCR revealed a very heterogeneous pattern with 61 clusters; based on a 90% agreement. In total, 46 different Hsp60 sequence types were detected. Using 98% sequence similarity, as threshold for separation, 22 separate Hsp60 sequence clusters were distinguished. There was no correlation between H. parasuis serovars and ERIC-PCR clusters or Hsp60 sequence types, but both the ERIC-PCR and the Hsp60 sequence typing are suited as markers for H. parasuis molecular-epidemiology studies. In total, 102 H. parasuis swine isolates corresponded to the virulence associated group 1 vtaA type. The group 1 vtaA was detected in 12 different serovars. Only four of the 46 Hsp60 sequence types were not associated with the group 1 vtaA. This study shows that Dutch H. parasuis isolates from pigs with clinical signs have both a high serovar and genotypic lineage diversity. A majority of the known serovars contain the group 1 vtaA.     

Interaction of Haemophilus parasuis with nasal and tracheal mucosa following intranasal inoculation of cesarean derived colostrum deprived (CDCD) swine.

Twenty-three cesarean derived, colostrum deprived pigs were obtained at 5 wk of age and inoculated intranasally with either 1.4 x 10(8) colony forming units of Haemophilus parasuis or sterile phosphate buffered saline. Pigs were euthanized at 4, 8, 12, 18, 26, or 36 h post-inoculation and tissues from the oropharynx and respiratory tract were obtained for qualitative bacterial culture, immunohistochemistry for H. parasuis antigens, and light and transmission electron microscopy. Haemophilus parasuis was consistently isolated from the nasal cavity (17/17, 100%) and trachea (13/17, 76%) and rarely isolated from the lung (3/17, 18%) and blood stream (1/17, 6%) of infected pigs. Antigens of H. parasuis were sporadically detected on the nasal mucosa (6/17, 35%) and trachea (8/17, 47%). Light microscopic lesions included submucosal and intraepithelial infiltrates of neutrophils and infrequent, patchy loss of cilia. Ultrastructural changes in nasal mucosal epithelial cells included cell protrusion, loss of cilia, and dilation of the cytocavitary network. Bacteria were infrequently identified and were either within an amorphous material at the apical surface of the cilia or were between individual cilia. These results suggest H. parasuis associates with the nasal mucosa and can induce a suppurative rhinitis with nasal mucosal epithelial cell degeneration. This process may represent an initial event in the pathogenesis of H. parasuis infection of swine.     

副猪嗜血杆菌分离鉴定及药敏试验

副猪嗜血杆菌能够引起猪的多发性浆膜炎、关节炎和脑膜炎等,是影响猪的最重要细菌之一,目前在所有的主要养猪国家均有存在。为了弄清河南省副猪嗜血杆菌病流行情况,2012年-2015年,从河南不同地区猪场送检的疑似病料,进行副猪嗜血杆菌分离和鉴定,共分离到5株细菌,通过细菌形态观察、培养特征鉴定、生化试验、PCR检测,鉴定为副猪嗜血杆菌,分别命名为A6-fei、C3-xin、C12-xin、D2-fei和E1-fei。采用纸片扩散法,对分离5株副猪嗜血杆菌进行药敏试验,其结果表明所分离的5株副猪嗜血杆菌的药物敏感性不尽相同,各分离菌株对头孢噻肟、氟苯尼考星最敏感,对复方新诺明敏感性最差,其中菌株C3-xin对复方新诺明、庆大霉素、卡那霉素、青霉素完全耐药。表明副猪嗜血杆菌病在河南省依然存在,并且不同地区菌株对常用药物的敏感性各不相同,应当引起养猪场重视。