Ultrastructural localization of anaplasmal antigens (Pawhuska isolate) with ferritin-conjugated antibody.

Antibodies from a cow with an experimentally induced infection of the Pawhuska isolate of bovine anaplasmosis were conjugated with ferritin and used to label antigenic sites in preparations of parasitized erythrocytes. Intact erythrocytes did not label on the extracellular surface. Ferritin-conjugated antibody did not pass through the intact erythrocyte to label the parasite, probably due to the large molecular size of the antibody. Damage to erythrocytic plasmalemma and inclusion body in the hemolyzed erythrocytes and complement-fixation antigen allowed labeling of anaplasmal inclusion structures. The positively labeled structures were outer surface of the pellicle, chromatin of the initial body, and inclusion appendage. Unlabeled structures included inner organismic membrane of the initial body, inclusion membrane, fibrillar protoplasmic network of the initial body, and small electron-dense bodies derived from the initial body.     

Immunocytochemical labeling of Anaplasma marginale Theiler in Dermacentor andersoni Stiles with peroxidase- antiperoxidase technique

Colonies of Anaplasma marginale in midgut epithelial cells of adult ticks that had been infected as nymphs were specifically labeled, using the unlabeled antibody peroxidase-antiperoxidase method of immunocytochemistry. Visual comparison of infected and control tissue sections with the electron microscope demonstrated deposition of ring-like peroxidase-antiperoxidase complexes over organisms within the colonies. The intensity of labeling differed among organisms within a single colony, possibly as a result of varying antigenicity. The labeling observed on organisms in the colonies was similar to that seen on anaplasmal initial bodies in inclusions of infected bovine erythrocytes examined concurrently.     

Development of an ELISA system for detection of anti-Anaplasma marginale antibodies in cattle in Brazil

An ELISA test was developed for detecting antibodies against Anaplasma marginale in bovine sera. Four antigenic preparations were produced from infected red blood cells. Some aliquots of this preparation were stored at -70 degrees C with 30% DMSO in phosphate-buffered saline (PBS) and others were lysed with 0.9% NH4Cl and stored at -20 degrees C. Typical anaplasmal structures were seen by electron microscopy in the antigenic preparations containing the erythrocytes that had been stored with DMSO. The performance of the ELISA test was evaluated by testing 298 positive serum samples collected from immunized cattle, 39 negative serum samples collected from cattle imported from areas free of A. marginale and 50 samples collected from cattle naturally infected in the field. The test gave a specificity of 94.87% and a sensitivity of 100%.     

Development of an ELISA system for Detection of Anti‐Anaplasma marginale Antibodies in cattle in Brazil

An ELISA test was developed for detecting antibodies against Anaplasma marginale in bovine sera. Four antigenic preparations were produced from infected red blood cells. Some aliquots of this preparation were stored at ‐70°C with 30% DMSO in phosphate‐buffered saline (PBS) and others were lysed with 0.9% NH₄Cl and stored at ‐ 20°C. Typical anaplasmal structures were seen by electron microscopy in the antigenic preparations containing the erythrocytes that had been stored with DMSO. The performance of the ELISA test was evaluated by testing 298 positive serum samples collected from immunized cattle, 39 negative serum samples collected from cattle imported from areas free of A. marginale and 50 samples collected from cattle naturally infected in the field. The test gave a specificity of 94.87% and a sensitivity of 100%.     

Demonstration of the inclusion appendage of Anaplasma marginale in nymphal Dermacentor andersoni

Dermacentor andersoni nymphs were placed in stockinettes and allowed to feed on a splenectomized calf with experimentally induced anaplasmosis when the parasitemia was 3%-5%. Nymphs were selected on each of the 6 days of feeding and every 5 days from repletion through molting to the adult stage (25 days postrepletion); they were killed and midgut tissues were processed and examined by light and electron microscopies. No stages of A marginale were seen in tissues of feeding ticks. Visualization of individual components of gut contents was difficult owing to presence of the concentrated, electrondense blood meal containing hemoglobin. Inclusion appendages were observed in midgut tissues of nymphs at 5 and 10 days postrepletion, but not at 20 or 25 days. The morphology of the appendages was similar to that described for inclusion appendages commonly associated with anaplasmal inclusions in bovine erythrocytes. Some appendages were free in the lumen of the midgut and occurred either alone or with clusters of small vesicular particles. Occasionally, initial bodies like those generally found in bovine erythrocytes were seen with the appendage, but most of them were swollen and appeared to be degenerating. Frequently, inclusion appendages were observed attached to the luminal surface of the midgut cell membrane by a blunt, electron-dense attachment complex. The attachment of the appendage appeared to be extracellular, with the pointed end extending into the lumen. Often, small particles were observed immediately across the cell membrane from where the appendages were attached; the small particles appeared to be generated from the appendage itself and to have passed through the membrane of the midgut cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between Clostridium septicum alpha-toxin activity and binding to erythrocyte membranes

The activity of Clostridium septicum alpha-toxin was determined in erythrocytes of various animals, with sensitivities observed in the order of mouse, rat, canine, equine, rabbit, chicken, bovine, swine and ovine. Temperature and protease treatment affected the sensitivity of erythrocytes to alpha-toxin. Proteinase K treatment decreased the sensitivity of murine, canine, equine and bovine erythrocytes, but ovine erythrocytes did not change the sensitivity to alpha-toxin activity. On the other hand, the activity of alpha-toxin on swine erythrocytes increased after treatment with proteinase K, trypsin, chymotrypsin or lysyl endopeptidase. Toxin overlay assay showed that alpha-toxin bound to erythrocyte membrane proteins with a molecular mass of 30 to 45-kDa in mouse, equine, bovine, swine and chicken, whereas in rat erythrocyte membranes the toxin reacted with 100-kDa protein. The treatment of murine and swine erythrocyte membranes with phosphatidylinositol-specific phospholipase C resulted in liberation of the toxin-binding protein from the individual membranes in a native state. These results show that alpha-toxin associates with specific erythrocyte membrane proteins in any animal species, and are subsets of glycosylphosphatidylinositol-anchored proteins in various animal species. These results may reflect distinct characteristics of the hemolytic activity of alpha-toxin in response to various erythrocytes.     

Production and characterization of monoclonal bovine immunoglobulins G1, G2 and M from bovine × murine hybridomas

Three bovine × murine hybridomas, which secrete bovine IgG₁, IgG₂ and IgM, respectively, were derived by the fusion of normal bovine B lymphocytes to the murine cell line SP-2/0. The hybridomas were initially selected by testing the culture supernatant of individual clones with rabbit anti-bovine light chain antibody. The bovine nature of Ig secreted by the bovine × murine hybridomas was confirmed by the ability of bovine serum but not murine serum to inhibit specific adsorption by murine anti-bovine Ig coupled to Sepharose 4B and the inability of sheep anti-mouse Ig (all isotypes) to bind biosynthetically labelled Ig from the respective bovine × murine hybridomas or to react with bovine × murine hybridoma Ig in Ouchterlony analysis. The isotype of the bovine Ig produced by each hybridoma was determined by cRIA using bovine Ig isotype-specific murine mcab, Ouchterlony analysis with guinea pig anti-bovine Ig isotypes and molecular weight analysis of unreduced and reduced affinity purified Ig from the respective bovine × murine hybridomas. Even though the hybridomas in this study showed chromosome loss, they have continued to secrete bovine Ig in culture for over 16 months, indicating that it is possible to isolate and stabilize interspecific hybridomas retaining the chromosome, or at least the genes, encoding an Ig.These hybridomas will provide monoclonal bovine Ig for; 1) serological standards, 2) production of polyclonal and monoclonal antisera to bovine Ig isotypes, 3) sequencing studies, 4) serological and structural studies of bovine Ig classes and subclasses, and messenger RNA for the production of cDNA probes for the cloning of bovine Ig genes and for determining the organization of Ig genes in the bovine genome.     

Evidence for a relationship between bovine erythrocyte lipid membrane peculiarities and immune pressure from ruminal ciliates

Erythrocytes of bovines and other ruminants have a strikingly anomalous phospholipid composition, with low or absent phosphatidylcholine (PC) together with high sphingomyelin (SM) content. Here, we report the presence in normal bovine serum of high levels of anti-phospholipid antibodies of IgM isotype against, PC and the phosphono analogue of phosphatidylethanolamine, aminoethylphosphonolipid (AEPL), normally produced by rumen ciliates. In contrast, no antibodies were detected against SM or N-acyl-phosphatidylethanolamine (NAPE), the major components of bovine erythrocytes. In addition, we found that exposure of the ciliate Tetrahymena thermophila to bovine serum results in rapid lysis, an effect that was inhibited by adsorption of the serum with SM/AEPL liposomes. Furthermore, incubation with bovine serum had a similar effect on freshly obtained ruminal ciliates, and the lytic activity was eliminated by pre-adsorption of the serum with SM/PE liposomes. The ruminant mode of life with its concomitant ciliate fauna is hereby linked to the peculiar conformation of bovine erythrocyte membranes. We propose that the unique phospholipid composition of bovine erythrocytes appears as an evolutionary adaptation to tolerate the lytic effects of anti-phospholipid antibodies generated against AEPL, a membrane component of the huge mass of ruminal ciliates, necessary commensals of this group of mammals.     

The effect of macrophages on the erythrocyte oxidative damage and the pathogenesis of anemia in Babesia gibsoni-infected dogs with low parasitemia

The role of macrophages in the erythrocyte membrane oxidative damage and the pathogenesis of anemia in Babesia gibsoni-infected dogs with low parasitemia were investigated. Macrophages derived from peripheral blood monocytes (PBM) from B. gibsoni-infected dogs produced significantly higher chemiluminescent responses, indicating the release of reactive oxygen intermediates, than those from non-infected dogs when the cells were subjected to non-specific stimulation with phorbol 12-myristate 13-acetate (PMA) and opsonized zymosan (OZ), or infected dog erythrocyte membranes opsonized with infected dog serum. These results indicate that PBM of B. gibsoni-infected dogs with low parasitemia were highly activated compared to those of non-infected dogs. Furthermore, the membrane lipid peroxidation of normal dog erythrocytes incubated with PBM from B. gibsoni-infected dogs was significantly higher (p<0.05) than that of erythrocytes incubated with PBM from non-infected dogs when the PBM were stimulated with the opsonized membranes. These results suggest that the oxidative damage of erythrocytes observed in B. gibsoni-infected dogs with low parasitemia might be induced, in part, by reactive oxygen species released from the activated PBM. On the other hand, the present study also showed a significant increase (p<0.001) of IgG-bound erythrocytes in B. gibsoni-infected dogs compared with such erythrocytes in non-infected dogs. The increase of IgG-bound erythrocytes in infected dogs might reflect the increase of erythrocytes with oxidative damage induced by the infection with B. gibsoni. The results of the present study suggest that the increase of IgG-bound erythrocytes in the circulation of infected dogs induce a high degree of erythrocyte loss via immunological phagocytosis by activated macrophages, resulting in severe anemia in spite of low parasitemia.     

Bovine CD18 identified as a species specific receptor for Pasteurella haemolytica leukotoxin.

Ligand blotting of Pasteurella haemolytica leukotoxin (LKT) susceptible BL3 bovine lymphoma cell membranes with LKT detected two putative receptors with Mr of 95 and 100 kDa, whereas no LKT binding to membrane proteins was detected for LKT non-susceptible human leukemic cells. Anti-bovine CD18 and CD11a/CD18 mAb recognized 95 and 100kDa bands from BL3 cell membranes. CD18 isolated from BL3 cell membranes bound LKT. Pre-incubation of BL3 cells with anti-bovine CD18 or CD11a/CD18 mAb caused partial inhibition of LKT-induced leukolysis. Therefore, we propose that bovine CD18 acts as a species-specific leukocyte receptor for P. haemolytica LKT.     

Inhibition of passive systemic anaphylaxis in guinea pigs by Trichinella spiralis infection

Thirty of 33 guinea pigs experimentally infected with Trichinella spiralis survived passive systemic anaphylactic reactions which were fatal for 32 of 33 uninfected controls. Guinea pigs were infected with viable excysted trichinella larvae 20 to 32 days before they and uninfected controls were sensitized by intracardiac injection of rabbit anti-bovine serum albumin. Animals were challenged 24 hours later by intracardiac injection of bovine serum albumin. Respiratory anaphylaxis was observed in all challenged animals. Protection from passive anaphylaxis in parasitized guinea pigs was most likely due to preemption of mast cell receptors by parasite-induced antibody with consequent blockage of passive sensitization.     

A new hemolytic assay for bovine serum complement and its application during experimental bovine anaplasmosis

A new hemolytic assay for bovine complement is presented. Using this assay we found a significant reduction in bovine serum complement activity during the acute phase of anaplasmosis, and an increase in the sensitivity of the red blood cells (RBC) to bovine complement lysis in vitro. The new hemolytic test is performed with bovine RBC, rabbit anti-bovine RBC serum and bovine serum complement. An isotonic sucrose Tris-buffered saline solution of ionic strength 0.094 and pH 7.2 was found to be adequate for this test. The titres obtained with this new assay, which uses autologous RBC, are comparable with those obtained using the guinea pig RBC assay. The finding of a reduction in bovine serum complement during anaplasmosis may be suggestive of a mechanism responsible for the pathology of this disease.     

Alternative pathway fof bovine complement Immunochemical studies on factor B-like serum protein and its conversion product B gamma 2.

Rabbits produced antibodies to a factor B-like serum protein (factor Bbov), its conversion product B gamma 2 and some other bovine serum proteins after repeated immunization with zymosan which previously had been incubated with fresh bovine serum. Such antisera were used to monitor purification of B gamma 2 from fresh bovine sera incubated with zxymosan. Subsequently, antisera specific for factor Bbov and B gamma 2 were produced. Antiserum produced against B gamma 2 cross-reacted with factor Bbov. Functional assays for factor Bbov were carried out in a hemolytic system with guinea pig erythrocytes in EGTA buffer. Heat inactivation (56 degrees C/5 min) of bovine serum destroyed the antigenicity of factor Bbov but not that of B gamma 2. Factor Bbov had an apparent molecular weight of 95,000 and B gamma 2 a molecular weight of 40,000 daltons. Conversion of factor Bbov to B gamma 2 was determined qualitatively by immunoelectrophoresis and quantitatively by radial immunodiffusion. Conversion of factor Bbov to B gamma 2 in bovine serum, in the presence of zymosan or cobra venom factor (CoVF) required Mg++ but not Ca++, did not occur in heat inactivated (56 degrees C/5 min) serum and was maximal, but not complete, when fresh bovine serum was incubated with zymosan (20 mg/mL) at 37 for two hours.     

Trichomonas gallinae: a possible contact-dependent mechanism in the hemolytic activity

The in vitro hemolytic activity of Trichomonas gallinae was investigated. The parasite was tested against human erythrocytes of groups A, B, AB, and O, and against erythrocytes of six adult animals of different species (rabbit, rat, chicken, horse, bovine, and sheep). Results showed that T. gallinae lysed all human erythrocytes groups, as well as rabbit, rat, chicken, horse, bovine and sheep erythrocytes. No hemolysin released by the parasites could be identified. Hemolysis did not occur with trichomonad culture supernatants, with sonicated extracts of T. gallinae, or with killed organisms. The scanning electron microscopy (SEM) showed that the erythrocytes adhered to the parasite surface and were phagocytosed. These observations suggest that the contact between T. gallinae and erythrocytes may be an important mechanism in the injury caused to the erythrocytes. The hemolytic activity of T. gallinae may be an efficient means of obtaining nutrients for the parasite and allow the investigation of the mechanism used by T. gallinae to damage cellular membranes.     

Optimum conditions for the radioimmunoprecipitation assay for the major internal protein of bovine leukemia virus

The sensitivity of the radioimmunoprecipitation assay for the major internal protein (p24) of bovine leukemia virus (BLV) was examined. Conditions were varied for the assay by 1) using larger amounts of serum and 2) increasing the amount of second antibody. Sensitivity for the p24 radioimmunoprecipitation test was greatest when 10 microliter of test serum and 200 microliter of rabbit anti-bovine IgG were used. Under these conditions there were no discrepancies between the p24 radioimmunoprecipitation test and the radioimmunoprecipitation test for the surface glycoprotein (gp51) in 380 cattle from commercial dairy herds.     

Common membrane neoantigens on bovine papilloma virus-induced fibroma cells from cattle and horses.

Cultured cells from bovine papilloma virus (BPV)-induced fibroblastic tumors and normal dermis of cattle, horses, and hamsters were examined for cell membrane or internal neoantigens, using the indirect immunofluorescence technique. Sera from cattle and horses bearing BPV-induced fibromas cross reacted with cell membranes of tumor, but not with normal dermal cells of both species. The reaction could be blocked with homologous, but not heterologous, serum of these 2 species. Immunofluorescence was not detected with sera from hamsters bearing BPV-induced sarcomas if incubated with bovine, equine, or hamster cells. Internal neoantigens were not found in any of the acetone-fixed tumor cells, using sera from the 3 species. Both tumor and normal cells were all found free of BPV antigen, using direct immunofluorescence.     

Neutralization kinetics of bovine viral diarrhea virus by hyperimmune serum: one or multi-hit mechanism

The kinetics of bovine viral diarrhea (BVD) virus neutralization (VN) by hyperimmune serum followed first order kinetics at low dilutions (1:8 and 1:16) of hyperimmune bovine serum. A lag phase in the VN curve occurred when the serum was further diluted. Addition of rabbit anti-bovine IgG, but not anti-IgM, significantly increased the degree of VN after BVD virus was reacted with further diluted (1:256 dilution) anti-BVD bovine hyperimmune serum. Incubation of virus-hyperimmune serum (1:64 dilution) at 4 degrees C for 0 to 60 min, followed by incubation at 37 degrees C indicated VN occurred as a two-stage process: a binding (temperature-independent) phase that was followed by a triggering (temperature-dependent) phase. The data favor the thesis that neutralization of BVD virus occurs by a multi-hit mechanism and requires combination of at least two molecules of antibody with each virus.     

A Comparison Between the Active Cutaneous Arthus Reaction and Immune-mediated Enteroluminal Neutrophil Emigration in Pigs

The induction of neutrophil emigration into the intestinal lumen in bovine serum albumin immune and nonimmune pigs by mucosal exposure to bovine serum albumin was studied using a ligated intestinal loop technique. In order to compare the response in the skin to that in the intestine, test materials were inoculated intracutaneously as well as enterally. Several histochemical procedures were applied to the intestinal mucosa and skin for evaluation of responses.

In immune animals, mucosal exposure to bovine serum albumin evoked the emigration of neutrophils into the intestinal mucosa and lumen. The neutrophil emigration tended to occur focally. Denudation of a few epithelial cells occurred at emigration sites. Hemorrhage, thrombosis, and edema, quite obvious after intracutaneous inoculation were not apparent after enteroluminal inoculation of bovine serum albumin into immune animals.

Enteroluminal inoculation of bovine serum albumin or bovine serum albumin plus anti-bovine serum albumin into nonimmune animals did not elicit neutrophil emigration or any other pathological lesion in the intestine, whereas intracutaneous inoculation of bovine serum albumin plus anti-bovine serum albumin into the same animals elicited an Arthus reaction.

     

牛IRF7蛋白的原核表达及多克隆抗体的制备

本研究旨在通过原核表达系统表达牛干扰素调节因子7（interferon regulatory factor 7,IRF7）蛋白,并对其进行纯化,进而制备高纯度的牛IRF7兔多克隆抗体。使用生物信息学软件预测并分析牛IRF7潜在的生物学功能,参考GenBank已公布的牛IRF7基因序列（登录号：NM_001105040.1）设计引物,利用PCR技术从牛病毒性腹泻病毒（Bovine viral diarrhea virus,BVDV）感染的MDBK细胞中扩增牛IRF7基因,连接至pMD19-T克隆载体,提取质粒连接至原核表达载体pET-28a （+）,转化大肠杆菌DH5α感受态细胞,构建重组质粒pET-28a-IRF7。将重组质粒pET-28a-IRF7转化大肠杆菌BL21（DE3）感受态细胞,经IPTG诱导后,通过SDS-PAGE进行表达产物的分析与鉴定。通过Western blotting鉴定多克隆抗体的特异性,间接ELISA测定兔抗牛IRF7多克隆抗体效价,经BVDV感染细胞后,实时荧光定量PCR检测抗病毒因子IRF7的表达。生物信息学分析发现,IRF7蛋白不存在跨膜结构域,无信号肽,存在较多磷酸化位点,二级结构主要以无规则卷曲为主,与三级结构的三维模型一致,说明该蛋白可能存在很好的抗原潜力。PCR成功扩增出大小为1 497 bp的IRF7基因片段,成功表达牛IRF7蛋白,分子质量约为60 ku,间接ELISA测得兔抗牛IRF7多克隆抗体效价为1∶128 000,并可与牛IRF7蛋白发生免疫反应,感染BVDV毒株的MDBK细胞中IRF7表达量下降;BVDV感染过表达IRF7后的细胞发现,IRF7能显著促进干扰素-β（IFN-β）的表达（P<0.05）。综上,本研究成功表达纯化了牛IRF7蛋白,并制备兔抗牛IRF7多克隆抗体,为阐明牛IRF7在先天免疫抗病毒应答的分子机制提供了材料。     

Comparative studies on the validity of renal function tests in the experimentally-induced bovine glomerulonephritis.

To establish the usefulness of the bovine clinical renal function tests, experimental glomerulonephritis was experimentally induced in calves and some renal clearance tests were performed. Two (No. 1, 2) of three calves were injected intravenously with anti-bovine kidney rabbit serum (antiserum) and the other (No. 3) with normal rabbit serum (control serum). The early stage of proliferative glomerulonephritis was observed in the kidneys of Nos. 1 and 2. The degree of lesions in No. 1 was severer than that in No. 2. No remarkable change was observed in the kidneys of No. 3. Endogenous creatinine clearance value (CCRE), thiosulfate clearance value (CTHIO) and maximal tubular secretion of para-amino hippuric acid (TmPAH) of all calves did not show remarkable changes after the injection of antiserum or control serum. In the phenolsulfonphthalein (PSP) test, PSP excretion of Nos. 1 and 2 was disposed to delay after the injection, and in No. 3 there was no significant change after the injection. PAH clearance value (CPAH) of No. 1 decreased from 10.24 to 6.96 ml/min/kg (-32%). A small change was noted in the CPAH of Nos. 2 and 3. These results suggest that the simplified method for measuring CPAH performed in this study could assess the lesions formed in No. 1, which was pathologically diagnosed as the early stage of proliferative glomerulonephritis.