免疫组化法检测布氏杆菌在人工感染牛体内的分布

将布氏杆菌分不同剂量皮下注射接种于健康牛,于接种后45 d剖杀,采集相关的器官组织制备组织切片,应用免疫组化方法检查细菌在牛体内的分布。结果显示,肝脏、脾脏、肾脏、淋巴结出现强阳性信号;在高剂量注射的牛体内,肺脏、颌下淋巴结出现阳性细胞;心脏和健康牛组织呈阴性反应。阳性信号主要位于感染细胞的胞质中,偶尔出现在细胞核内。     

Changes in Lymphocyte Subsets in the Bronchus‐associated Lymphoid Tissue of Goats Naturally Infected with Different Mycoplasma Species

The distribution of cells containing lysozyme, S‐100 protein, CD3, CD4, CD8, major histocompatibility complex class II antigen and immunoglobulin G (IgG) was analysed in the bronchus‐associated lymphoid tissue (BALT) of goats naturally infected with three Mycoplasma species. This study included the immunohistochemical characterization of the pneumonic lesions of 18 goats (3–5 months old) infected with one of the following Mycoplasma species: M. mycoides ssp. mycoides, Large Colony type (goats no. 1–6), M. mycoides ssp. capri (goats no. 7–12) and M. capricolum ssp. capricolum (goats no. 13–18). Microscopically, infected animals showed a moderate bronchointerstitial pneumonia, characterized by lymphoid hyperplasia of the BALT and infiltration of mononuclear cells in the alveolar walls and airways. The main cellular type in the BALT was represented by CD3⁺ T lymphocytes, and the ratio of CD4⁺:CD8⁺ cells was >2. The BALT showed large germinal centres mainly composed of IgG⁺ B lymphocytes, with numerous S‐100⁺ follicular dendritic cells. The presence of follicular dendritic cells confirmed the high degree of organization of this lymphoid tissue. The immunohistochemical results showed that activated T lymphocytes, particularly in the CD4 subset, and IgG⁺ B cells, play a major role in the immune response of the caprine lung infected with these species of mycoplasmas.     

Demonstration of Listeria monocytogenes by Immunohistochemistry in Formalin‐Fixed Brain Tissues from Natural Cases of Ovine and Bovine Encephalitis

In the present work, evidence of Listeria monocytogenes antigens based on the avidin–biotin complex (ABC) immunoperoxidase technique was performed on formalin‐fixed central nervous system tissues (CNS) from a total of 23 natural cases of encephalitis (four ovine and 19 bovine). Listeria monocytogenes serotype 4 was isolated from 10 of 17 cultured specimens. Meningoencephalitis characterized by focal necrosis, microabscesses, perivascular cuffing, and gliosis with presence of macrophages and/or neutrophils was observed at histological examination. Positive L. monocytogenes antigens were successfully identified by immunohistochemistry (IHC) in the CNS of all 23 cases. Paraffin‐embedded tissues assayed were stored up for 17 years. Morbidity of the outbreaks was between 0.3–3% and 0.1–1% for ovine and bovine cases, respectively. In all the ovine cases, flocks involved were under extensive grazing conditions. In nine of the 19 bovine cases (47.3%), supplementation with corn silage was used. The ABC test can help as a practical tool for the diagnosis of natural cases of L. monocytogenes encephalitis on formalin‐fixed specimens from ovine and bovine.     

原位杂交技术用于虹彩病毒对美国青蛙侵染的诊断

蛙虹彩病毒（Rana grylio virus,RGV)能引起鱼类、两栖类和爬行类水生动物严重的系统性疾病，RGV是从我国患病蛙中分离到一种虹彩病毒。体外扩增的RGV经人工方法感染幼龄美国青蛙（Rana grylio)，运用原位杂交技术，分别对感染1-3d后的蛙心、肺、肾、肠、脾、肝等6种组织进行RGV分子定位和检测，结果显示，在幼蛙的肺和肠中有较强的阳性信号，在其他组织中也检测到RGV的存在。本试验探讨了RGV感染早期在宿主体内的增殖及在不同组织中的分布状况，建立了一种彩虹病毒的早期诊断方法，并为揭示RGV的致病机制奠定了基础。     

免疫组化法检测猪圆环病毒2型在人工感染猪体内的分布

将猪圆环病毒2型（PCV2）BF株经滴鼻接种28日龄健康普通仔猪,于接种后不同时间剖杀,采集相关的器官组织制备组织切片,使用免疫组化方法检测病毒在猪体内的分布。结果显示,淋巴结、脾脏、扁桃体等淋巴组织均出现阳性信号,心、肝、肺、肾、胃、十二指肠、大脑及小脑同样存在阳性细胞,其他组织呈阴性反应。阳性信号主要位于感染细胞的胞质中,偶尔出现在细胞核内。结果表明,PCV2确已造成猪体组织感染。     

Immunohistochemical detection of Mycoplasma agalactiae in formalin-fixed,paraffin-embedded tissues from naturally and experimentally infected goats

Samples from the mammary tissue of 14 lactating goats (12 naturally infected and two experimentally infected) were examined for the presence of Mycoplasma agalactiae. A monoclonal antibody (5G12) was applied to formalin-fixed, paraffin-wax-embedded sections and labelled by the avidin-biotin peroxidase complex (ABC) method. Histological examination of tissue sections revealed strong immunoreactivity in all animals included in the study. Mycoplasma agalactiae antigen was mainly detected in the cellular debris at the periphery of purulent exudates present within lactiferous sinuses, and lactiferous and interlobular ducts. In addition, M. agalactiae organisms appeared in the cytoplasm of the epithelium of ducts, and in infiltrating macrophages and neutrophils within the ducts, alveoli, interstitial tissue and regional lymph node sinuses. It is concluded that this monoclonal antibody-based immunohistochemical technique is an efficient and specific method for the post-mortem detection of M. agalactiae in cases of clinical mastitis as well as being a useful tool for the study of the route of infection and cellular types involved during mastitis caused by this organism.     

Impact of invA‐PCR and Culture Detection Methods on Occurrence and Survival of Salmonella in the Flesh,Internal Organs and Lymphoid Tissues of Experimentally Infected Pigs

This study evaluated the suitability of invA gene amplification by PCR as an effective means of detecting Salmonella species in pigs experimentally infected with S. Typhimurium DT104. A controlled infection study using 24 pigs was performed in order to compare efficacy, precision and detection rates of the invA‐based PCR method originally described by Rahn, K. De Grandis, S.A., Clarke, R.C., McEwan, S.A., Galan, J.E., Ginocchio, C., Curtiss, R. 3rd, C.L. Gyles, (Mol. Cell. Probes 1992; 6 : 271–279) as a new in‐house invA‐based PCR method for the specific detection of Salmonella spp. in pork and different tissue samples of slaughter pigs. Finally, PCR results were compared with culture detection rates obtained by isolation procedures following the ISO 6579:2000, the ‘gold standard’. After slaughtering, 14 different tissue samples of each pig were investigated to verify the usefulness of the two invA‐based PCR methods in different matrices of slaughter pigs. The results demonstrate that the application of the widely used invA‐based primer pair (139 + 141) may result in questionable products if samples gained from selective enrichment in the Rappaport–Vassiliadis medium were investigated. These questionable products can lead to false‐positive results, if no additional hybridization procedure is attached or if unspecialized persons use this method in routine laboratory practice. The newly developed in‐house PCR method used is based on the 3′‐prime region of invA, especially designed and harmonized for the detection of Salmonella in different matrices of slaughtered pigs after bacterial enriched broth culture. In this study, this PCR revealed no questionable products and, furthermore, the specificity of the amplificate could be tested by means of the restriction enzyme NdeI. In comparison with the culture detection procedure, the new PCR method has a sensitivity of 100% and a specificity of 96%. Thus, this method might be used as a meaningful tool in eliminating Salmonella‐positive carcasses at slaughterhouse level and thus, keeping them out of the food chain.     

Bovine Viral Diarrhea Virus in Swine: Characteristics of Virus Recovered from Naturally and Experimentally Infected Swine

A noncytopathogenic field strain of bovine viral diarrhea virus (BVDV) was isolated from an Iowa farm brood sow and from her hysterectomy-derived, colostrum-deprived (HDCD) piglets. This field isolant was fully virulent for a neonatal calf. The NADL strain of BVDV was passaged through a series of HDCD piglets with no resultant loss of virulence for neonatal calves. Most of the BVD viral isolants recovered from pigs had been changed from a cytopathogenic biotype to a noncytopathogenic biotype. Circumstantial evidence points to swine as “carrier” hosts of BVDV.     

用原位杂交技术对牛组织中FMDV RNA检测和定位

采用原位杂交技术对6头试验牛易感染组织中的FMDVRNA进行了检测和定位。结果显示，接毒后7～28d4头牛舌上皮组织、7～21d的喉咽部有强阳性染色；接毒后35d的牛及正常对照牛舌上皮没有阳性染色；接毒牛及对照牛的扁桃体、淋巴结和蹄叉等其他组织中均没有阳性染色，表明牛舌上皮和喉咽部上皮组织中感染了FMDV，本研究为进一步阐明FMDV在宿主体内的持续感染机理提供极有价值的线索。     

Evaluation of 3 Serological Tests for Early Detection Of Leptospira‐specific Antibodies in Experimentally Infected Dogs

Background

Leptospirosis in dogs is a disease of global importance. Early detection and appropriate therapeutic intervention are necessary to resolve infection and prevent zoonotic transmission. However, its diagnosis is hindered by nonspecific clinical signs and lack of rapid diagnostic tests of early infection. Recently, 2 rapid point‐of‐care tests (WITNESS Lepto [WITNESS Lepto, Zoetis LLC, Kalamazoo, MI, USA] and SNAP Lepto [SNAP Lepto, IDEXX Laboratories, Westbrook, ME, USA]) for detection of Leptospira‐specific antibodies in canine sera were developed.

Hypothesis

Immunoglobulin M‐based WITNESS Lepto containing multiple detection antigens can detect Leptospira‐specific antibodies to common leptospiral serovars earlier in the course of infection as compared to microscopic agglutination test (MAT) and SNAP Lepto.

Animals

Four groups of 8 6‐ to 8‐month‐old male Beagle dogs were used.

Methods

Thirty‐two healthy seronegative dogs were inoculated experimentally with serovars Canicola, Grippotyphosa, Icterohaemorrhagiae, and Pomona (8 dogs/serovar). Acute‐phase sera were collected at regular intervals and monitored for Leptospira‐specific antibodies by WITNESS Lepto, MAT, and SNAP Lepto.

Results

Seroconversion was detected in all dogs by day 10 by WITNESS Lepto and in 30 of 32 dogs by day 14 by MAT. The SNAP Lepto test detected seroconversion in 3 dogs during the 2 weeks postchallenge.

Conclusions

Immunoglobulin M‐based WITNESS Lepto detected immune responses specific to multiple leptospiral serovars early in the course of infection and identified seroconversion in all animals earlier than did the gold standard MAT. The SNAP Lepto test displayed considerably lower and inconsistent performance during the study period. At the point‐of‐care, WITNESS Lepto should be the test of choice for rapid and reliable screening of acutely ill dogs suspected to have leptospirosis.     

Presence of Maedi Visna Virus (MVV)‐Proviral DNA in the Genital Tissues of Naturally Infected Ewes

Maedi Visna virus (MVV) causes progressive degenerative inflammatory disease in multiple organs including the lungs (pneumonia, ‘maedi’), mammary gland, joints and nervous system (meningoencephalomyelitis, ‘visna’) in sheep. Maedi Visna Virus has been detected in macrophages of several tissues and epithelial cells in vivo: bone marrow, cells of the central nervous system, lung and bronchial tissues, milk epithelial cells recovered from milk samples and epithelial cells of mammary tissue. However, the presence of MVV in the genital tracts of naturally infected ewes has not previously been studied. The aim of this study was to use nested‐PCR, targeting the gag gene, to determine whether genital tissues (ovaries, oviducts and uterus) from 83 ewes originating from various breeding herds in the South‐East of France were positive for MVV‐proviral DNA. Peripheral blood mononuclear cells (PBMC) tested positive for MVV‐proviral DNA, using nested‐PCR analysis, in 57.8% of ewes (48/83). The provirus was also identified in 47% (78/166) of the ovaries, 38.6% (64/166) of the oviducts and 45.8% (38/83) of the uteri sampled. These findings clearly demonstrate, for the first time, that tissue samples from the genital tract of ewes (ovary, oviduct and uterus) can be infected with MVV. This suggests that there is a risk of vertical and/or horizontal transmission of MVV during embryo transfer from embryos produced in vivo or in vitro.     

实验感染肝片吸虫山羊和大鼠血清花生四烯酸代谢物的变化研究

实验一选择9只健康、6-10月龄雄性白山羊，经粪便学检查和Dot-ELISA检测，确认无肝片吸虫感染，随机分成感染组（n=5)和对照组（n=4)，试验组每只一次口服接种150个肝片吸虫囊蚴，每周定时从颈静脉采集感染前（0周）和感染后23周山羊血液一次，分离血浆，实验二将60只大鼠随机分成感染组（n=30)和对照组（n=30)，感染组大鼠一次口服25个囊蚴，对照组不感染，于感染前（0周）和感染后（1，3，5，7，9周）采集血样，分别测定试验组和对照组羊和鼠花生四烯酸代谢物6-酮-前列腺素F1α（6-keto-PGF1α）和血栓素B2（TXB2）的变化。结果表明肝片吸虫感染后，山羊血浆中TXB2水平变化，在1-3周与对照组相比无显著差异，从第5周开始到实验结束，试验组血浆中TXB2水平均显著或极显著地高于对照组，大鼠血栓素水平在感染后前5周逐步下降，以后急剧上升，至实验结束时显著高于对照组；而整个实验期间，两种动物血浆中6-keto-PGF1α水平变化不显著，提示肝片吸虫感染后，机体发生多种功能的改变，花生四烯酸代谢物参与了肝片吸虫病的发展过程。     

雏鹅新型病毒性肠炎病毒强毒在感染鸭胚体内形态发生学和宿主细胞超微病理学研究

将雏鹅新型病毒性肠炎病毒（NGVEV）强毒CH株经尿囊腔途径人工感染10日龄鸭胚,应用透射电镜和超薄切片技术研究病毒在宿主细胞内的形态发生及各组织器官的超微结构变化。结果表明：感染后不同时间剖杀及死亡鸭胚的尿囊膜、肠、心、肝、脑和肌胃组织中,均观察到60～70nm的病毒粒子。病毒粒子主要通过与细胞膜融合而进入细胞质内,然后在细胞核内进行复制和装配。最后病毒粒子通过核膜和细胞膜破裂的方式被释放。病毒侵害的主要靶细胞包括鸭胚尿囊膜上皮细胞、肠上皮细胞、肠道平滑肌细胞、成纤维细胞、肝细胞、肌胃黏膜上皮细胞和心肌细胞等,表现为细胞核内外膜间隙严重扩张,细胞质整体结构严重空化。病毒侵害的主要靶细胞器包括粗面内质网和线粒体,表现为粗面内质网扩张呈囊状;尿囊膜上皮细胞的线粒体出现固缩和异常聚集变化,而其他组织细胞的线粒体均表现为肿胀和嵴断裂、消失。本试验还发现NGVEV可诱导宿主细胞发生严重的细胞凋亡现象,表现为细胞皱缩,胞核内染色质密度增高,核固缩成一个或数个团块凝聚在核膜周边,胞质浓缩深染并形成凋亡小体。     

Detection of Anti-listeriolysin O and Listeria monocytogenes in Experimentally Infected Buffaloes (Bubalus bubalis)

The kinetics of antibody production against listeriolysin O (ALLO) and the recovery pattern of Listeria monocytogenes from bacteriological samples were studied following oral infection of buffalo calves with 3×10⁹ cells each of pathogenic L. monocytogenes. Antibodies to LLO appeared by 7–10 days post infection (PI), with a shallow peak between days 16 and 36 PI, when tested by indirect plate-ELISA. The titres of ALLO in all the animals then declined slowly but remained detectable up to day 70 PI. In dot-ELISA, ALLO could be detected by days 5 to 7 PI, and with higher titres than with the plate-ELISA. The pathogen was recovered at low rates as ALLO first appeared but was absent in the faecal, nasal and blood cultures as production of ALLO peaked.     

Parasitological and Clinico‐pathological Profiles in Friesian Cattle Naturally Infected with Theileria annulata in Saudi Arabia

Clinico‐pathological profiles were studied in adult and young Friesian cattle naturally infected with Theileria annulata in the Qassim Region, Saudi Arabia. Sixty‐two clinical cases of T. annulata infection in adult and young Friesian cattle were diagnosed during the period from August 1999 to July 2000. Symptoms observed were marked fever, swelling of superficial lymph nodes, inappetance, tachycardia, dyspnoea and weakness. The most prominent gross pathological features were jaundice, petechial and ecchymotic haemorrhages involving mucosal and serosal surfaces of many organs as well as body fat. A number of young and adult Friesian cattle undergoing lethal T. annulata infection developed lymphoma‐like lesions in a manner similar to that of T. parva. The main histological findings were necrosis and severe lymphocytic infiltration. The spleen, lymph nodes and Peyer's patches were devoid of typical lymph nodules.