Immunohistochemical labeling of Giardia trophozoites spp. in formalin fixed paraffin embedded tissues.

An immunoperoxidase method using rabbit anti human Giardia lamblia serum for the demonstration of giardia in paraffin embedded intestinal animal tissue is described. Specificity was tested against other protozoal parasites.     

Demonstration of bovine virus diarrhoea virus antigen in formalin fixed, paraffin embedded tissue using a streptavidin/biotin technique

The detection of bovine virus diarrhoea virus (BVDV) antigen in sections from formalin fixed, paraffin embedded tissue is described. Pre-digestion of the sections with 0.02 per cent protease XIV for 18 hours at 4 degrees C is necessary to unmask formalin fixed antigen. A hyperimmune antiserum prepared in a pig, using a combination of BVDV and hog cholera virus inoculations, linked to a biotinylated anti-pig/streptavidin peroxidase detection system demonstrated antigen in a wide range of tissues from cases of mucosal disease and persistently viraemic animals. The inclusion of a monoclonal anti-pig immunoglobulin linked to a biotinylated anti-mouse/streptavidin peroxidase detection system greatly reduced non-specific staining.     

Peroxidase-antiperoxidase labelling of Leishmania amastigotes in formalin fixed and paraffin embedded tissue sections.

A modified PAP-Method (peroxidase-antiperoxidase) is established for laboratory use to demonstrate Leishmania spp. amastigotes in paraffin sections of experimentally and naturally infected dogs. The results demonstrate that the PAP technique is a valuable tool to detect Leishmania amastigotes in tissue sections with low parasite density.     

Kinetics of antibody production and clinical profiles of calves experimentally infected with Listeria monocytogenes

Clinical and serum antibody profiles were studied during oral Listeria monocytogenes infection of calves. No clinical signs, except for pyrexia with mild diarrhoea and staggering gait, were observed in the infected calves. Specific antibodies to listeriolysin O (LLO) appeared as early as day 8 of an oral infection and peaked by days 16-32 of infection. Antibodies to LLO were observed to persist over the period of 126 days observed in the study. LLO being a major virulence factor and capable of inducing a humoral response could therefore be used as an antigen for development of an immunoassay for diagnosis of Listeria monocytogenes infections in animals.     

单核细胞增多性李斯特菌人工感染小鼠致其脑炎模型的建立及优化

采用不同剂量单核细胞增多性李斯特菌(Listeria monoc ytogenes,LM)LM90SB2腹腔注射感染小鼠219只,观察小鼠临床症状,纪录死亡情况,在感染后不同时间采集小鼠脑组织,进行脑组织中细菌回收、鉴定及病理组织学观察.结果显示,不同感染剂量感染小鼠,其临床症状和脑组织病理变化基本一致.但是随着感染剂量的增加,其临床症状出现时间逐渐缩短,死亡率和脑组织中细菌分离的时间明显不同.1/2LD50剂量组小鼠未出现死亡,2/3LD50组和LD50组死亡率分别为12.5％和63.3％;另外1/2LD50组感染后24 h开始从脑组织中离到LM,96 h所有感染小鼠脑组织均能分离到LM,2/3LD50组感染后12h开始能分离到LM,72 h所有脑组织均能分离到LM.从小鼠临床症状及死亡率、脑组织细菌的回收情况和脑组织病理组织学变化综合考虑,以2/3LD50 LM LM90SB2 腹腔感染小鼠为动物感染模型.     

Light and electron microscopic study of the livers of pregnant mice infected with Listeria monocytogenes.

Listeria monocytogenes cells were observed in the hepatic cell cytoplasm or in the phagosome at 24 and 48 hours but not at 72 hours after inoculation in pregnant mice. The presence of bacteria initially in a membrane-bound vesicle indicates that the bacteria enter the hepatic cells by endocytosis, resulting in eventual destruction of hepatic cells. Characteristic lesions of the liver at 24 and 48 hours after inoculation consist of multiple focal areas of necrosis. The initial neutrophilic reaction seems to give way to a mononuclear reaction (listeriomas) at 72 hours after inoculation. Dilation of rough endoplasmic reticulum and release of many of the bound ribosomes with a relative increase in the number of free ribosomes was observed. Hepatic lesions were not observed in control (nonpregnant) mice.     

Molecular analysis of bovine leukemia virus in early epidemic phase in Japan using archived formalin fixed paraffin embedded histopathological specimens

Bovine leukemia virus (BLV) is an important pathogen associated with enzootic bovine leukosis. In this study, we performed PCR and sequencing analysis to characterize BLVgp51 sequences from formalin-fixed paraffin-embedded (FFPE) specimens made from 1974 to 2000 and successfully obtained BLV proviral genome sequences from 94% of the analyzed samples. Furthermore, from these samples, we reconstructed eight full-length and nearly full-length BLVgp51 sequences. These sequences were classified as BLV genotype 1, implying that genotype1 has already been circulating in Japan since the 1970s. In our results, the proviral DNA was detected in the 1970s, 1980s, and 1990s in the same manner, indicating that the detection of BLV proviral genome depends on storage conditions rather than storage period. The sequences obtained in this study provide direct insights into BLV sequences before 2000, which serves as a good calibrator for inferring ancient BLV diversity.     

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.     

The role of nitric oxide in Listeria encephalitis of ruminants and in rats intracisternally infected with Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular bacteria which infects a wide range of hosts. In ruminants, infection with L. monocytogenes frequently causes encephalitis, which is usually fatal in sheep and goat, while cattle often recover with antibiotic therapy. Since the role of NO in the control of Listeria is controversial, we have studied the expression of iNOS in the brains of cattle, sheep and goats which had succumbed to listeria encephalitis. iNOS was demonstrated in decreasing intensity in the M phi of microabscesses from cattle, sheep and goat. iNOS expression was accompanied by NT in the microabscesses of cattle, but was only present to a low degree in sheep and was absent in goats. This is indirect evidence for differences in the ability to produce NO in the three species. Presence of iNOS and NT were inversely correlated with the numbers of bacteria. While microabscesses of goats contained high amounts of L. monocytogenes they occurred only rarely in cattle. To corroborate our hypothesis that NO is involved in the control of listeria encephalitis a new animal model was developed. Eleven day old infant rats were infected intracisternally with a low dose of L. monocytogenes. This resulted in a transient meningoencephalitis with moderate clinical signs and low mortality. Listeria proliferated strongly in the inflammatory lesions during the first days of infection, reached a peak at day 4 and were eliminated until day 7. The presence of bacteria was closely accompanied by high numbers of iNOS-expressing M phi and the formation of NT. Administration of the iNOS inhibitor L-NIL or the radical scavenger PBN resulted in rapid death of the treated animals. However, the increase in bacterial numbers was one order of magnitude higher for animals treated with PBN compared with L-NIL administration. This shows that NO plays an important role in the control of a brain infection with Listeria, but suggests that reactive oxidants other than NO are also involved. In conclusion, our findings point to a possible involvement of the differences in the ability to express iNOS and subsequent NO production in the different clinical outcome of listeria encephalitis in cattle and small ruminants.     

Interferon-gamma expression and infectivity of Toxoplasma infected tissues in experimentally infected sheep in comparison with pigs

Livestock animals are a potential risk for transmission of toxoplasmosis to humans. Sheep and pigs still remain an important source because their meat is often eaten undercooked which has been regarded as a major route of infection in many countries. Moreover, porcine tissues are processed in many food products.In the current study, the IFN-gamma (T-helper 1 cells), IL-4 (Th2 cells) and IL-10 mRNA (Treg cells) expression by blood mononuclear cells, and the serum antibody response against Toxoplasma gondii total lysate antigen, recombinant T. gondii GRA1, rGRA7, rMIC3 and rEC2, a chimeric antigen composed of MIC2, MIC3 and SAG1, was studied in sheep the first two months after a T. gondii infection and compared with these responses in pigs. At the end of this period, the parasite distribution in heart, brain and two skeletal muscles in sheep was compared with this in pigs.Whereas the parasite distribution was similar in sheep and pigs, the antibody response differed considerably. In sheep, antibodies appeared against all tested T. gondii antigens, but mainly against rGRA7, rMIC3₂₃₄₃₀₇ and TLA whereas in pigs only rGRA7-specific antibodies could be demonstrated. Also, the cytokine response differed. Both in sheep and pigs an IFN-gamma response occurred which seemed to be a slightly more pronounced in sheep. In sheep, also IL-10 and IL-4 mRNA expression showed an increase, but later than IFN-gamma and with more variation. However, in pigs no such increase was seen.As concerning diagnosis, results indicate that serum antibodies against GRA7 in live sheep and pigs and heart tissue for bioassay and qPCR in slaughtered animals are the best targets to demonstrate presence of T. gondii infection.     

Induction of acquired cellular resistance in mice with viable and macrophage-processed Listeria monocytogenes

Intracutaneous immunization of mice with 10(5) or 10(6) viable listeria resulted in acquired cellular resistance (ACR) of short duration (7 days). The period during which viable Listeria monocytogenes had to be present in order to induce ACR was estimated by killing the listeria at different times after immunization by injecting the bactericidal antibiotic amoxycillin. The killing of listeria within 6 h after injection prevented the induction of A CR completely, between 6 and 12 h partially, while survival of listeria within animals for at least 18 h was required for the induction of complete protection. To determine whether multiplication of viable listeria was a prerequisite for the induction of ACR, the bacteriostatic antibiotic minocycline was injected for four days after immunization. Induction of ACR was only possible if the dose of viable listeria was large enough to permit a proportion of the listeria to escape bacteriostasis. Interaction of peritoneal macrophages of normal mice and viable listeria yielded a supernatant which induced specific ACR in normal recipient mice. No ACR could be induced with supernatant obtained from normal macrophages after digestion of killed listeria. A reduced level of ACR was obtained with supernatant collected after interaction of macrophages from immune mice and viable listeria. The immunogenic material present in the supernatant of normal macrophages after interaction with viable listeria is thermolabile, has a molecular weight of over 300,000, and is not affected by treatment with DNase, RNase, or trypsin.     

Demonstration of schizogonous stages of Sarcocystis gigantea in experimentally infected sheep

Sarcocystis-free lambs were orally dosed with 1 X 10(6) sporocysts of Sarcocystis gigantea. Schizonts were found in endothelial cells of capillaries and arterioles of the brain, lung and kidney of lambs 7 and 14 days post-inoculation (d.p.i.). Between 21 and 35 d.p.i. there was extensive multi-focal encephalitis; however no organisms were detected in association with these lesions.     

Demonstration of feline foamy virus in experimentally infected cats by immunohistochemistry

Foamy viruses (FV) are complex retroviruses which are commonly isolated from cats, cattle and non-human primates. The infection is persistent and infected animals have a sustained antibody response. The role of FV in diseases remains unclear, in cats, a possible association with uncharacterized renal symptoms remains to be confirmed. To demonstrate feline FV (FFV) in tissues of experimentally infected cats three polyclonal monospecific antisera from rabbits against three different viral proteins, the structural Gag and the non-structural Bel 1 and Bet proteins were tested for their applicability in immunohistochemistry with paraffin sections. Only the Bet antiserum allowed detection of FFV-specific proteins, the antibodies against Gag and Bel 1 did not work even after pre-treatment of the slides with proteinase K or cooking in a pressure cooking pot. The Bet-reactive antibodies were detected using a commercial streptavidin kit and revealed Bet in the cytoplasm of cells from different lymphoid tissues like lymphnodes, tonsils, thymus and spleen. The method described opens new ways to explore the in vivo replication and tissue specificity of FFV and its possible role in disease.     

猪附红细胞体人工感染小鼠的病理学试验

附红细胞体是寄生于人、猪以及其他动物的红细胞表面、血浆以及骨髓内的一群多形态微生物.猪附红细胞体病缺乏特征性临床症状和病理变化,使发病率和死亡率大大升高,给养殖户造成很大经济损失.本试验旨在研究猪附红细胞体病对小鼠血液生理指标的影响,及对小鼠各脏器的致病程度,为附红细胞体病的诊断及深入研究提供基础试验依据.     

Detection of Neorickettsia (Ehrlichia) risticii in tissues of mice experimentally infected with cercariae of trematodes by in situ hybridization

Neorickettsia (Ehrlichia) risticii was demonstrated to occur in cercariae developing in Juga yrekaensis snails by experimental transmission, genetic detection and histopathology. Cercariae were isolated from the digestive glands of snails collected in a fresh stream water area of Siskiyou County, CA, and inoculated into CF1 mice. Mice developed clinical signs, splenomegaly and histopathologic abnormalities. The agent was maintained by serial passages of whole blood in CF1 mice. A 527-bp product of the 16S rRNA gene of N. risticii was serially detected by nested PCR in blood, feces, salivary gland, suprarenal gland, spleen, intestine and bone marrow of inoculated mice. N. risticii DNA was detected by in situ hybridization with DIG-labeled probe in PCR-positive salivary gland, intestine and spleen tissue sections of experimental mice on day 30 after inoculation. Infection in mice was established when cercariae were inoculated by either IP or SC routes but not established following intraoral route. N. risticii was detected by PCR in spleen, intestine and bone marrow even after 73 days post-inoculation whereas blood from the same animals became negative at 58 days. N. risticii was observed by in situ hybridization in salivary gland, spleen and intestine of mice infected by IP or SC inoculation. This ISH protocol should aid investigations on the host range of the Neorickettsiosis and pathogenesis of neorickettiosis in vector, animal or human.