Morphological changes in brucellosis in guinea pigs due to experimental infection with Brucella suis

Sixty-four guinea-pigs were infected with the germs of Brucella suis. biotype 2 via the vaginal, peroral, intranasal, conjunctival and subcutaneous routes. In the subcutaneous infection the length of the trial was 151 days and in the other methods of infection 56 days. As found, guinea-pigs are highly sensitive and brucellosis lesions were detected in all cases. Brucellosis was chronical and spread locally in the guinea-pigs. In the haematogenic spreading of the disease, lesions were constantly recorded in liver, spleen and lungs. In the cases of subcutaneous infection lesions were also observed in regional lymph nodes at the places of infection. The lesions had amorphous structure and regular borders. The lesions were characterized by pronounced exudation and the exudate was subject to caseous necrosis, followed by colliquation. Secondary central colliquation was regularly observed in older granulomas, so that colliquation is considered as a characteristic morphological trait of brucellosis process in guinea-pigs. Histiocytary granuloma constituted the microscopic basis of the lesions. In older granulomas the central necroses were wide and contained a large amount of nuclear detritus, owing to the karyorhexis of cells, mainly neutrophiles. In the necroses of caseous type lipoid droplets were found and calcification was observed on the 151st day. Big cells of Langhans type occurred sometimes in the histiocytary layer of granulomas; they were present most frequently in the lesions affecting lymph nodes. Interstitial pneumonia was a characteristic symptom in liver and big cells of Sternberg type were found in the granulomas. Follicular tumor was constantly observed in spleens. Morphologically detectable lesions first developed in liver after about 7 days from conjunctival infection and subcutaneous infection, after 21 days from vaginal and intranasal infection, and after 28 days from peroral infection. In the case of subcutaneous infection, granulomas were found in the regional lymph nodes on the 14th day. The most expressive lesions in organs, mainly liver, were found from the 49th to 56th day from infection.     

Evaluation of cattle for experimental infection with and transmission of Brucella suis biovar 4

OBJECTIVE: To determine whether cattle can become persistently infected with Brucella suis biovar 4, whether the organism can be transmitted vertically or horizontally, and whether tests for bovine brucellosis are diagnostic. DESIGN: Observational study. ANIMALS: 24 pregnant cows and their calves and 6 bulls. PROCEDURE: Cows and bulls were housed separately in groups of 6 with each group consisting of 3 cattle experimentally infected with B suis biovar 4 and 3 na?ve animals. Cattle were observed for clinical signs daily; blood samples were collected weekly. Clotted blood from each sample was submitted for bacterial culture. Serum was tested with an indirect ELISA and the standard tube agglutination test (STAT), buffered plate agglutination test, brucellosis card test (BCT), and complemen't fixation test (CFT). Tissues collected at necropsy were submitted for bacterial culture and histologic examination. RESULTS: All 15 inoculated cattle seroconverted on 2 or more serologic tests, and bacteria were isolated from 4 inoculated cows at necropsy. There was no bacteriologic evidence of vertical or horizontal transmission, and none of the cattle developed clinical abnormalities or gross or histologic lesions. Results of the indirect ELISA were positive for all inoculated cattle. The other tests gave variable results; the CFT, STAT, and BCT yielded negative results for at least 1 of the 4 cattle from which the organism was isolated. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that cattle-to-cattle transmission of B suis biovar 4 is unlikely. Serologic tests for bovine brucellosis should be used cautiously when attempting to identify cattle with rangiferine brucellosis, as they do not discriminate between the 2 diseases and vary in their ability to detect exposed cattle.     

Brucella suis biotype 1 infection in a dog

Brucella suis biotype 1 was isolated from the semen of a dog with hindlimb weakness and a large, firm, left epididymis. A semen sample was oligospermic, with many neutrophils, the numbers of which decreased in serial sampling. A card agglutination test for B abortus and a rapid slide agglutination test for B canis were positive. The modified 2-mercaptoethanol slide agglutination test for B canis and the agar gel immunodiffusion test, using B canis cell wall antigen, were negative. At necropsy, chronic granulomatous inflammation was found in, and B suis biotype 1 was isolated from, the left epididymis and prostate gland.     

猪布鲁菌感染对巨噬细胞泛素蛋白酶体功能的影响

巨噬细胞(macrophage,M_φ)是布鲁菌的主要宿主细胞,为了探明布鲁菌感染与M_φ泛素-蛋白酶体功能之间的关系,本试验在应用蛋白酶体抑制剂Lactacystin与促进剂IFN-γ的基础上,对M_φ感染猪种布鲁菌S2株(Brucella suis,B.suis)后细胞上清中泛素、蛋白酶体及胞内B.suis数量进行了检测。结果显示,IFN-γ与Lactacystin对M_φ泛素的表达均有明显的促进作用,且IFN-γ效果显著优于Lactacystin;其次IFN-γ有效促进了M_φ蛋白酶体的表达,而Lactacystin显著抑制了M_φ蛋白酶体的表达。在此基础上,将B.suis感染不同状态的M_φ,在0.5~24h分别进行胞内B.suis计数,研究表明,一方面B.suis的感染促进了M_φ泛素及蛋白酶体的表达;另一方面,泛素蛋白酶体系统功能的增强,显著降低了B.suis的早期感染,但对于B.suis感染中后期作用不明显,而感染后期抑制M_φ蛋白酶体功能,却可显著降低B.suis的胞内增殖能力。     

Experimental infection of rabbits (Oryctolagus cuniculus) with Brucella suis biovar 1 isolated from wild hares (Lepus europaeus)

Brucella suis biovar 1 is the causative agent of brucellosis in several domestic and wild animals and it is a common agent of human brucellosis. European hares (Lepus europaeus) have been shown to be infected by B. suis biovar 1 and the transmission to other animals has been suggested. In this work, experimental rabbits (Cuniculus orictolagus) were infected with B. suis biovar 1 isolated from wild hares. Infected rabbits showed high serological response in 2 weeks after discharge and typical granulomatous lesions (2mm diameter) were found in liver, spleen and kidneys after 50 days. B. suis biovar 1 was cultured from the lesion of the organs mentioned above as well as from urine, placenta and fetuses. These data suggest that hares are a potential source for horizontal transmission of B. suis biovar 1 to other mammalians.     

猪链球菌2型对家兔的试验感染及其病理学观察

利用猪链球菌2型四川分离强毒菌株458#,通过腹腔注射接种日本大耳白家兔,观察其临床症状和病理学变化。结果表明,猪链球菌2型强毒株458#可引起家兔发病或死亡,并能从发病家兔的组织样品中分离到所接种毒株。感染家兔的特征性组织病理学变化表现为典型的弥漫性血管内凝血和微血栓形成。猪链球菌2型感染家兔动物模型的建立,对于评价猪链球菌2型的毒力,探索其发病机制具有重要的意义。     

表达绿色荧光蛋白猪种布鲁菌的构建

为了更直观的观察猪种布鲁菌在宿主细胞/宿主体内的活动规律,本研究首先应用PCR技术扩增pEGFP-N1质粒的N1基因,与pMD18-T载体连接后,成功构建PMD18-N1质粒;进而应用XbalⅠ与SacⅡ限制性内切酶,将PMD18-N1质粒与pBBR1MCS-6质粒分别进行双酶切,并将N1片段反向连接于PBBR1MCS-6载体上,经菌液PCR鉴定与双酶切鉴定,证明成功构建了PBBR1MCS-N1质粒;在此基础上,将PBBR1MCS-N1质粒电转化至猪种布鲁菌感受态细胞,通过含氯霉素的TSA培养基筛选、PCR鉴定、荧光鉴定及菌株的传代稳定性试验与培养特性的观察,最终获得了可稳定表达绿色荧光的猪种布鲁菌。     

Trichuris suis population dynamics following a primary experimental infection

Trichuris suis population kinetics was studied by experimentally infecting 40 pigs with 5000 T. suis infective eggs. Six pigs were sacrificed every 2 weeks from 1 to 9 weeks post-inoculation (p.i.) and the remaining 10 pigs were sacrificed 11 weeks p.i. to estimate worm burdens. An equal number of uninfected control pigs were sacrificed at the same time points for comparison. Egg excretions from each pig were evaluated every week from 5 to 11 weeks p.i. Peripheral blood eosinophilia and basophilia were also evaluated every 2 weeks throughout the experimental period. After an initial almost 100% establishment of T. suis an expulsion phase followed approximately 9 weeks p.i., resulting in an aggregated distribution of the worms in the pig population, as it is characteristic for most helminth infections. By 11 weeks p.i. almost all worms had been expelled. Egg excretion peaked 7 weeks p.i. and a significant non-linear relationship between worm burdens and egg excretion was found. The predilection site for T. suis was the caecum and proximal colon and the relative distribution of worms in the large intestine did not change over time until expulsion. Peaking peripheral blood eosinophil and basophil levels were observed in the T. suis infected pigs 5 and 7 weeks p.i., respectively, reflecting the host activated immune response against the parasite. We here describe the course of a primary T. suis infection in pigs by detailed worm counts, demonstrating an effective expulsion that results in an almost 100% clearance of infection as previously indicated by monitoring faecal egg excretion.     

Comparison of experimental models for Streptococcus suis infection of conventional pigs

Four different experimental models for Streptococcus suis-induced disease were compared to find a model that closely mimics naturally occurring disease in conventional pigs. Fourteen, 2-week old pigs free of S. suis type 2 were used in 2 experiments. In experiment 1, 3 pigs were inoculated intravenously (IV) and 3 pigs intranasally (IN) with S. suis. Two out of 3 of the IV-inoculated pigs exhibited signs of severe central nervous system disease (CNS) and were euthanized. Streptococcus suis type 2 was isolated from whole blood, joints, and serosal surfaces of both pigs. No clinical signs and no growth of S. suis were detected in the IN-inoculated pigs. In experiment 2, 4 pigs were inoculated IV and another 4 were inoculated IN with the same isolate as in experiment 1. One hour before inoculation the IN-inoculated pigs were given 5 mL of 1% acetic acid intranasally (IN-AA). All the IV-inoculated pigs showed CNS disease and lameness, and 2 of the pigs became severely affected and were euthanized. All the IN-AA inoculated pigs exhibited roughened hair coats and 2 pigs developed severe CNS disease and were euthanized. Streptococcus suis was isolated from the joints and blood of 3 pigs in the IV-inoculated group. Streptococcus suis was isolated from blood of 2 pigs, meninges of 3 pigs, and joints of 1 pig in the IN-AA inoculated group. Natural exposure to S. suis most likely occurs by the intranasal route. The IN-AA model should serve as a good model for S. suis-induced disease, because the natural route of exposure is intranasal and the IN-AA model was effective in inducing disease that mimics what is observed in the field.     

小鼠阴道黏膜接种布鲁菌S_2疫苗株树突状细胞动力学变化

将25只小鼠随机分为5组,1~4组经阴道接种布鲁菌,第5组阴道滴注PBS(对照组),分别在处理后12、24、487、2 h摘取髂内淋巴结,用S-100抗体进行免疫组织化学染色,观察小鼠树突状细胞(Dendritic cells,DCs)在淋巴结的分布变化;同时收集外周血用ELISA法进行IFN-γ检测。用布鲁菌负载DCs,分别在2、4、6、8、10、12 h收集细胞悬液制备涂片,姬姆萨染色。结果显示:阴道接种布鲁氏菌疫苗12~48 h髂内淋巴结中DCs的数量明显增多,成群分布,呈现由浅层皮质向副皮质区迁移的趋势;72 h后大量DCs呈单在分布,分布范围增大;接种疫苗后血清中IFN-γ含量逐渐升高,至48 h,与对照组相比差异显著(P0.05),72 h后,血清中IFN-γ含量达到最大值,与对照组相比差异极显著(P0.01);布鲁菌负载DCs后,在4 h仅有少量DCs吞噬布鲁菌,至12 h吞噬布鲁菌的DCs数量明显增加。结果表明,DCs具有较强捕获布鲁菌的能力,并在抗原刺激下向引流淋巴结迁移,刺激淋巴结免疫细胞产生IFN-γ应答。     

Experimental infection with Tritrichomonas suis in heifers.

Nine heifers were intravaginally challenged with 9.3x10(6) Tritrichomonas suis reference strains. Vaginal mucus and serum samples were collected weekly 4 weeks post-inoculation. Vaginal mucus was cultured for T. suis and sera was tested by ELISA against whole cell antigens for T. suis and Tritrichomonas foetus. All vaginal mucus cultures were T. suis-negative during the experiment. ELISA values for both antigens were similar and differences were not significant (P>0.05). Positive control serum samples from one heifer vaccinated against T. foetus showed anti-T. suis ELISA values. We concluded that T. suis intravaginal inoculation induced a low level of serum immune response in heifers measured by ELISA and both protozoa probably share a common antigen. However, under the experimental conditions of this trial, colonization of the heifers' genital tract was not possible in any of the nine animals.     

猪附红细胞体实验动物模型的建立

为建立一种合适的猪附红细胞体病动物模型，利用摘除脾和／或注射地塞米松的昆明小白鼠，以腹腔注射方式人工感染猪附红细胞体，通过血液涂片镜检、PCR检测、临床症状观察及病理剖检对感染情况进行了鉴定。结果显示，猪附红细胞体能够经腹腔注射感染昆明小白鼠，且感染鼠表现出与猪附红细胞体病相似的临床症状。结果表明，猪附红细胞体实验动物模型已成功建立，并证实啮齿类动物在附红细胞体的传播过程中发挥着重耍作用。     

The porcine acute phase protein response to acute clinical and subclinical experimental infection with Streptococcus suis

The pig acute phase protein (APP) response to experimental Streptococcus suis (S. suis) infection was mapped by the measurement of the positive APPs C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin (Hp) and major acute phase protein (pig-MAP) and the negative APPs albumin and apolipoprotein (Apo) A-I. The aim was to elucidate the differences in the acute phase behaviour of the individual APPs during a typical bacterial septicaemic infection. Pigs were inoculated subcutaneously with live S. suis serotype 2 and blood was sampled before and on various days post inoculation (p.i.), until the pigs were killed and autopsied on day 14 p.i. Clinical signs (fever and lameness) were observed in four of the five inoculated pigs from day 2 p.i., and these pigs also had arthritic lesions at autopsy. CRP and SAA showed fast increases in serum concentrations, CRP being elevated from days 1 to 12 p.i. and peaking at 10 times the day 0-levels on day 1 p.i. SAA rose quickly to peak levels of 30-40 times the day 0-level on days 1-2 and returned to pre-inoculation level on day 5 p.i. Hp and pig-MAP showed slightly slower responses, both peaking around 5 days p.i. Hp was increased throughout the experiment with maximum levels around 10 times the day 0-levels, and pig-MAP was elevated on days 1-12 p.i. with peak levels of around seven times the day 0-levels. Apo A-I was decreased from days 1 to 8 and showed minimum levels of about 40% of day 0-levels around 1-2 days p.i. No clear pattern of changes in albumin levels could be identified. One pig, showing clinical signs on day 2 only, also showed an APP response, although of a relatively short duration, whereas three pigs presenting clinical signs for several days had a more protracted acute phase response. Remarkably, the one pig showing no clinical signs and no arthritic lesions showed an APP response comparable to that of the other, clinically affected pigs. Thus, both acute clinical and subclinical S. suis infection could be revealed by the measurement of one or more of the APPs CRP, SAA, Hp, pig-MAP and Apo A-I. The combined measurement of two or three APPs, including proteins with slow and fast kinetics, should be used to achieve the highest sensitivity for the detection of ongoing S. suis infection during a prolonged time period. A diagnostic tool based on such APP-measurements could considerably improve strategic control procedures for this important infection.     

Experimental infection of opossums with Brucella abortus

Thirteen opossums (Didelphis virginiana) trapped in east central Alabama were fed approximately 1.5 X 10(9) Brucella abortus colony forming units. Serologic responses to at least 1 of 3 tests developed in 8 of the 13 opossums. Brucella abortus was recovered from 18 of 159 blood samples from 4 of the 13 opossums and from 7 of 159 fecal samples from 6 of them. All culture-positive feces had been excreted within 4 days after exposure. Sixty-four urine, 123 saliva, and 78 vaginal samples were culture-negative. Eleven baby opossums, in their mothers' pouches at the time of capture, were culture-negative. Brucella abortus was isolated from 10 of 13 adult opossums. Ten additional opossums were trapped and tested for brucellosis. One had a tube agglutination titer of 1:25, and B abortus was isolated from the liver and spleen. Brucella abortus was isolated from lung and spleen of 8 seronegative opossums. The remaining 8 opossums were negative to all tests.     

Persistence of viral RNA in rabbits which overcome an experimental RHDV infection detected by a highly sensitive multiplex real-time RT-PCR

An internally controlled multiplex real-time RT-PCR using TaqMan probes and external standards for absolute RNA quantification was developed as a new diagnostic tool for the detection of rabbit haemorrhagic disease virus (RHDV). The test revealed a specificity of 100%, an analytical sensitivity of 10 copies/well and a linearity over a range from 10(1) to 10(10) copies. The viral loads in organs, leukocytes, sera and excretions of seropositive, convalescent rabbits which were overcoming an experimental infection with RHDV were determined using the validated assay. As a result, viral RNA was demonstrated and quantified for at least 15 weeks. Thus, a persistence of viral RNA after experimental infection of rabbits could be shown for the first time. In contrast, neither antigen nor infectious virus could be detected by antigen-ELISA, immunohistochemistry or experimental transmission. Therefore, further experiments are necessary to prove that the persistence of RNA is linked with the persistence of infectious virus particles.     

The pathology of experimental respiratory infection with Pasteurella multocida and Bordetella bronchiseptica in rabbits

Groups of female New Zealand White rabbits, 8-10 weeks old, were inoculated intranasally with three different Pasteurella multocida serotypes (A:3, A:4 and A:12) or one of three Bordetella bronchiseptica strains of rabbit origin. Seven out of 18 rabbits died of experimental infection with P. multocida. B. bronchiseptica killed 3 out of the 8 animals inoculated with it. Deaths occurred between 3 and 6 days postinoculation (PI). In the rabbits that died of P. multocida inoculation, necropsy and histology revealed severe pleuritis with the accumulation of a remarkable amount of fibrinopurulent exudate in the thoracic cavity, serous rhinitis and tracheitis, acute hepatitis with necrotic foci in the parenchyma, and atrophy of the lymphoid organs and tissues. Rabbits killed 10 days PI developed only subacute serous rhinitis and hyperplasia of the lymphoid tissues. Rabbits that died of B. bronchiseptica inoculation showed acute serous rhinitis, acute catarrhal-fibrinopurulent pneumonia and mild pleuritis. As opposed to P. multocida inoculated animals, hepatitis and atrophy of the lymphoid tissues were not characteristic of these rabbits. Rabbits killed 10 days PI developed subacute purulent and necrotic pneumonia with remarkable macrophage proliferation, involving all lobes, and hyperplasia of the lymphoid tissues.     

布鲁菌S2疫苗株免疫牛与牛种、羊种布鲁菌自然感染牛ELISA鉴别诊断方法的建立

为建立区分猪种布鲁菌S2疫苗株接种奶牛与布鲁菌自然感染奶牛,BLAST比对分析羊种、牛种、猪种、犬种、沙林鼠种和绵羊种6种布鲁菌基因序列,发现repA—related基因是猪种布鲁菌与牛种及羊种布鲁菌的差异基因。设计引物PCR扩增获得repA-related基因片段,克隆并原核表达得到了布鲁菌repA—related融合蛋白,以repArelated蛋白建立间接EI．IsA检测方法。用repA—related蛋白间接ELISA检测猪种s2疫苗株接种动物血清为阳性,检测牛种和羊种布鲁菌自然感染动物血清为阴性。repA—related蛋白间接EusA能从试管凝聚实验（SAT）及常规ELIsA检测阳性的奶牛血清样本中,区分出s2疫苗接种牛与牛种布鲁菌感染牛。