The Kümm isolate of Ehrlichia ruminantium: in vitro isolation,propagation and characterization

An effective culture system for Ehrlichia (Cowdria) ruminantium comb. nov. was first established in 1985 and many stocks were subsequently isolated and propagated in vitro. A notable exception, however, was the Kümm isolate that resisted all attempts at in vitro culture until the successful experiment described here. In one experiment white blood cells were harvested from heparinized blood derived from a sheep infected with the Kümm isolate. The cells were added to DH 82 cells and incubated at 37 degrees C. The high metabolic activity of the DH 82 cells necessitated that cell growth be retarded by the addition of cycloheximide. Colonies were first detected 19 days after culture initiation and, once the cultures were established, they could be passaged every 3 days. Bovine and sheep endothelial cells were readily infected with culture supernatant obtained from the infected DH 82 cells. In a further experiment another sheep was infected, using a higher dose of the same batch of Kümm stabilate, and we attempted to infect several different cell lines: these were DH 82 cells, bovine aorta (BA 886) cells, sheep brain endothelial (SBE 189) cells and sheep fibroblastoid cells (E2). Ten days after culture initiation only the E2 cells had become positive for E. ruminantium. Culture supernatant from the first cultured isolate (Kümm-1) was less virulent for mice than that of the second cultured isolate (Kümm-2) which killed all mice. Upon molecular characterization with E. ruminantium 16S probes we found that Kümm-1 hybridized with a Senegal 16S genotype probe, whereas Kümm-2 hybridized only with an Omatjenne 16S genotype probe. The original stabilate used to infect the sheep hybridized with both probes. These results clearly indicate that two different stocks had been isolated in culture.     

Down-regulation of MHC class II receptors of DH82 cells, following infection with Ehrlichia canis

In order to evaluate whether infection with E. canis alters the expression of major histocompatibility complex (MHC) class I and/or MHC class II receptors, and by doing so alters the immune response to the organism, flow cytometry was performed on DH82 cells infected with Ehrlichia canis (90% infection) and on uninfected DH82 cells of the same passage, using anti-canine MHC class I and II antibodies. MHC class II expression was evident in 47.6 and 46.2% (mean 46.9%) of uninfected DH82 cells using two different anti-MHC class II antibodies, while no MHC class II expression was evident in DH82 cells infected with E. canis. The present results indicate that infection of DH82 macrophages with E. canis down-regulates their MHC class II receptors. These results suggest a possible mechanism by which E. canis evades the immune system.     

Human intravenous immunoglobulin (hIVIG) inhibits anti-CD32 antibody binding to canine DH82 cells and canine monocytes in vitro

The IgG receptors CD16 and CD32 (Fc_γRIII and Fc_γRII) link the humoral immune response to effector cell immune responses by binding immune complexes. Human intravenous immunoglobulin (hIVIG) consisting of immunoglobulin from pooled donors is reported to block Fc_γRs and has been used to treat a variety of canine autoimmune disorders. Fc_γRs have been poorly described for canine monocytes; therefore, the objectives of this study were to: (1) identify canine monocyte/macrophage Fc_γR (CD16 and CD32) expression and (2) demonstrate in vitro hIVIG binding to these receptors. The canine monocyte/macrophage-like cell line (DH82) and monocytes isolated from peripheral blood of healthy dogs were evaluated by flow cytometry (FACS) for CD16 and CD32 expression using commercially available anti-CD16 and anti-CD32 antibodies directed against the human isoforms. The mean percentage of cells expressing CD16 was 55% of DH82 cells and 13% of blood monocytes and the mean percentage of cells expressing CD32 was 85% of DH82 cells and 73% of blood monocytes. Immunoprecipitation of canine DH82 cells lysate using the same anti-CD16 or anti-CD32 antibodies suggested that these anti-human antibodies recognize the canine homologues. To demonstrate Fc_γR blockade, cells were incubated with increasing concentrations of hIVIG and then incubated with anti-CD16 or anti-CD32 antibodies. The percentage of CD32 expression decreased in a concentration dependent fashion in DH82 cells and blood monocytes after incubation with increasing concentrations of IVIG, suggesting that hIVIG was binding to CD32 and inhibiting anti-CD32 antibody binding. The same results were not demonstrated with anti-CD16 antibody. We believe this is the first report to demonstrate Fc_γ receptors CD16 and CD32 expression on canine monocytes and in vitro CD32 binding by human IgG, which may represent one of the immunomodulatory mechanisms of hIVIG.     

Adenosine and ATP affect LPS-induced cytokine production in canine macrophage cell line DH82 cells

Macrophages are essential for controlling the majority of infections, and are mediators of natural immunity. During infection, lipopolysaccharide (LPS) stimulates macrophages to produce pro-inflammatory cytokines. Adenosine and ATP released into the extracellular space by immunological stimuli have been shown to regulate various immune functions. More recently, it has been shown adenosine and ATP have a critical role on the physiological negative feedback mechanism for limitation and termination of tissue-specific and systemic inflammatory responses. It was useful and meaningful to gain information about interaction between LPS, which generates the inflammation, and adenosine and ATP, which terminate the inflammation. We evaluate effects of adenosine and ATP on the production of cytokines related to inflammation in canine macrophage cell line DH82 cells. Adenosine and ATP respectively increased the production of IL-10 without affecting the production of IL-6, TNF-α and IL-12 in DH82 cells. In addition, adenosine and ATP prevented the production of LPS-induced IL-6, TNF-α and IL-12 in DH82 cells. In contrast, adenosine and ATP potentiated LPS-induced IL-10 production in DH82 cells. Moreover, adenosine, but not ATP inhibited LPS-induced expression of TLR4 in DH82 cells. These results suggest that conditions related to increased adenosine and/or ATP may play an important role in the inflammatory reactions.     

Propagation of avian rotavirus in primary chick kidney cell and MA104 cell cultures

The Ch2 strain of avian rotavirus was propagated in primary chick kidney cell (CKC) and MA104 cell cultures in the presence of trypsin. Cultures were evaluated for the presence of rotavirus by an indirect fluorescent antibody (IFA) assay and by an indirect enzyme-linked immunosorbent assay (ELISA). After two passages, the viral titer was significantly higher in CKC than MA104 cell cultures. Also, the IFA assay was more sensitive than the indirect ELISA for detecting rotavirus-positive cultures.     

Passage-dependent morphological and phenotypical changes of a canine histiocytic sarcoma cell line (DH82 cells)

DH82 cells represent a permanent macrophage cell line isolated from a dog with histiocytic sarcoma (HS) and are commonly used in various fields of research upon infection and cancer, respectively. Despite its frequent use, data on cell surface antigen expression of this cell line are fragmentary and in part inconsistent. We therefore aimed at a detailed morphological and antigenic characterization of DH82 cells with respect to passage-dependent differences. Cellular morphology of early (≤13) and late (≥66) passages of DH82 cells was evaluated via scanning electron microscopy. Moreover, cells were labelled with 10 monoclonal antibodies directed against CD11c, CD14, CD18, CD44, CD45, CD80, CD86, MHC-I, MHC-II, and ICAM-1 for flow cytometric analysis. Early passage cells were characterized by round cell bodies with abundant small cytoplasmic projections whereas later passages exhibited a spindle-shaped morphology with large processes. The percentage of CD11c-, CD14-, CD18-, CD45-, and CD80 positive cells significantly decreased in late passages whereas the expression of CD44, CD86, MHC-I, MHC-II and ICAM-1 remained unchanged. DH82 cells represent a remarkably heterogeneous cell line with divergent antigenic and morphologic properties. The present findings have important implications for future studies, which should consider distinct characteristics with regard to the used passage.     

Characterization of a canine CD44 specific monoclonal antibody

Monoclonal antibodies (mAbs) were generated against a CD44 mRNA expressing (RT-PCR) macrophage/monocyte cell line (DH82) from a dog with malignant histiocytosis. The mAbs, that reacted with DH82 cells by FACS analysis were tested on formalin-fixed, paraffin-embedded tissues. Exclusively the incubation of DH82 cell pellets with mAbs from clone 2D10 resulted in a cell membrane associated immunoreaction. Immunoelectron microscopy specified, that the antibody bound exclusively to the cell membrane and processes of DH82 cells. The mAb was tested on a variety of normal canine tissues, including lymphoid, urinary, alimentary, respiratory, and endocrine organs, nervous tissues, liver, pancreas, skin, and muscles. Furthermore, tumour and inflamed tissues were tested for immunoreaction with the mAb. Immunohistologically, the 2D10 mAb reacted with macrophages/monocytes, subsets of lymphocytes, epithelial cells, and central nervous system white matter. FACS analysis of canine peripheral blood leukocytes showed, that a high proportion of lymphocytes and granulocytes were positive with this mAb. Western blot analysis revealed, that the 2D10 mAb bound to a protein with a molecular weight of about 85 kDa. The results of FACS and Western blot analyses, RT-PCR, immunohistology and immunoelectron microscopy strongly suggest that the antigen detected by the 2D10 mAb is most likely the canine equivalent of human CD44, a cell bound hyaluronan binding proteoglycan.     

Serum level of apoptosis inhibitor of macrophage in dogs with histiocytic sarcoma and its association with the disease

Histiocytic sarcoma (HS) is a rare neoplasm of macrophages or dendritic cells with a poor prognosis in dogs. As the apoptosis inhibitor of macrophage (AIM) is characteristically expressed in canine macrophages, we hypothesised that AIM is involved in the development or progression of HS in dogs. In this study, AIM expression in the tumour region and serum AIM levels in dogs with HS was assessed. Additionally, the effects of AIM overexpression on HS cell viability were investigated using a HS cell line that was selected from five validated HS cell lines. Immunohistochemistry showed that AIM expression was observed in the cytoplasm of the HS cells. CD36, a candidate AIM receptor, was also observed on the cell membrane of HS cells. When the serum AIM level was detected in 36 dogs with HS and 10 healthy dogs via western blot analysis, the AIM levels in the HS dogs were significantly higher than those in the controls. AIM mRNA expression in the 5 HS cell lines varied but was higher than that in the other tumour-derived lines. Among the five HS cell lines, DH82 originally had lower AIM and the highest CD36 expression. When AIM was overexpressed in DH82, therein cell growth speed and invasion, apoptosis inhibition and phagocytic activity were strongly upregulated. These data suggest that elevated intra-tumour expression of AIM could induce the progression of HS cells in dogs. Moreover, elevated serum AIM levels in dogs with HS could serve as a biomarker of HS.     

The growth of the canine glioblastoma cell line D‐GBM and the canine histiocytic sarcoma cell line DH82 is inhibited by the resveratrol oligomers hopeaphenol and r2‐viniferin

Vineatrol^®30 is a grapevine‐shoot extract, which contains resveratrol as well as considerable amounts of so‐called resveratrol oligomers such as hopeaphenol and r2‐viniferin. In this study, we analysed whether the two above‐mentioned resveratrol oligomers were able to inhibit the growth of the canine glioblastoma cell line D‐GBM and the canine histiocytic sarcoma cell line DH82, compared their potency to inhibit tumour cell growth with that of resveratrol and determined whether the induction of apoptosis via caspase 9 and 3/7 activation underlies the tumour cell growth‐inhibiting effect of hopeaphenol and r2‐viniferin. Vineatrol^®30, resveratrol, hopeaphenol and r2‐viniferin inhibited the growth of D‐GBM and DH82 cells in a concentration‐dependent manner, whereby hopeaphenol and r2‐viniferin were more potent than resveratrol itself in inhibiting the growth of the canine tumour cell lines. Moreover, the anti‐proliferative effect of both resveratrol oligomers in D‐GBM cells is based on their capacity to induce caspase 9 and 3/7 activation.     

Establishment of optimal conditions for long-term culture of erythrocytic stages of Theileria uilenbergi

OBJECTIVE: To establish optimal conditions for long-term culture of the erythrocytic stage of Theileria uilenbergi. SAMPLE POPULATION: Red blood cells from 3 splenectomized sheep experimentally infected with a blood stabilate of T uilenbergi. PROCEDURES: Cultures of T uilenbergi were initiated by use of blood from experimentally infected sheep collected when parasites were detected in Giemsa-stained thin blood smears. Different culture conditions were tested to optimize in vitro growth of the organisms. Subcultures were performed at a ratio of 1:2, 1:4, and 1:8 when the percentage of parasitized erythrocytes (PPE) was at least 1% or when the initial PPE was doubled. RESULTS: The optimal culture medium was HL-1 medium (a complete chemically defined medium) supplemented with 20% sheep serum and 0.75% chemically defined lipids. Optimal culture conditions included incubation in a humidified 2% O(2), 5% CO(2), and 93% N(2) atmosphere at 37 degrees C. Cultures of the merozoite stage of the parasite were continuously propagated in vitro for > 1 year. The PPE reached values of up to 3%. CONCLUSIONS AND CLINICAL RELEVANCE: Optimization of culture conditions to reach a high PPE seems worthwhile. The continuous propagation of T uilenbergi in culture allows the production of parasite material without infecting animals and provides a continuous laboratory source of parasites for further studies.     

Propagation of the rotavirus of neonatal calf diarrhea in fetal intestinal cell cultures.

Bovine fetal intestinal cells were successfully propagated in monolayer cultures for up to 21 passages. Infection of these cells with the rotavirus of neonatal calf diarrhea resulted in a cytopathic effect that was more obvious than in infected bovine fetal kidney cells.     

免疫抗体压力下PRRSVGD株Nsp2和GP5基因变异分析

以PRRSVGD-2007株为材料,在自然条件和抗体压力下连续传至40代后,分别命名为PRRSV—GD—f40和PRRSV—GDAb—f40,进行RT—PCR,扩增Nsp2基因部分序列和GP5基因全长,克隆并测序。应用序列分析软件将测序结果和已经发表的PRRSV毒株进行比对,结果显示：Nsp2基因扩增片段大小均为796bp,自然传代和抗体压力下传代后的新毒株和母源毒株的相似性分别为98．0％和97．9％;扩增的GP5基因大小为603bp,编码200个氨基酸,和母源毒株的相似性分别为99．2％和98．8％。     

Upregulation of major histocompatibility complex class II antigens in hepatocytes in Doberman hepatitis

Major histocompatibility complex (MHC) class II antigen expression in hepatocytes and its correlation with mononuclear cell infiltration into the liver were studied using immunohistochemical techniques in 38 Dobermans with Doberman hepatitis (DH). Liver biopsy samples were obtained from 18 dogs at the subclinical stage. Autopsy samples were taken from 6 DH dogs euthanized for a reason other than DH, from 14 dogs euthanized because of advanced liver failure and from 6 control Dobermans. Upon examination of the control liver samples, no expression of MHC class II antigens was detected in hepatocytes. By contrast, in 15 of the 18 DH biopsies (83%) and in all 20 DH autopsy liver samples, hepatocytes expressed MHC class II molecules. MHC class II expression was either cytoplasmic or membranous and occurred in conjunction with lymphocyte infiltration. A correlation between the inflammatory reaction and the expression of MHC class II in hepatocytes suggests that the aberrant expression of MHC class II in hepatocytes is induced by cytokines. Hepatocytes presenting a putative MHC class II molecule-associated autoantigen could thus become the target of an immune attack mediated by CD4+ T cells. In addition, corticosteroid treatment was observed to significantly decrease MHC class II expression in DH hepatocytes. Inappropriate MHC class II expression in hepatocytes and mononuclear cell infiltration are suggesting an autoimmune nature for chronic hepatitis in Dobermans.     

国家牧草种质中期库豆科植物种子监测和扩繁时间的判定

以38种1 153份豆科植物种子为研究对象,测定了其在中期库储存4~7年后种子发芽率的变化状况,判断是否需要继续监测或直接扩繁。结果表明:5年后无需监测直接扩繁的种子有饭豆、柱花草、田菁、小豆和银合欢等5种(同一草种,发芽率降低超过20%的份数占总数的百分比小于5%且发芽率升高的份数占总数的百分比大于70%);无需监测无需扩繁的种子有白三叶、杂三叶、黄花苜蓿、黄芪、野大豆等9种(发芽率降低超过20%的份数占总数的百分比大于60%);需要监测的种子有紫花苜蓿、沙打旺、草木樨、岩黄芪、红豆草等24种(发芽率变化在以上2个范围之外,则需要继续监测)。     

Cultivation of Ehrlichia canis in a continuous BALB/C mouse macrophage cell culture line.

This report describes the successful adaptation of the Israeli isolate of Ehrlichia canis on a continuous mouse macrophage cell line (J774.A1). Successful infection of the J774.AI cells was first judged by the direct immunofluorescence antibody test using an anti-E. canis-IgG:FITC conjugate. A particular property of infected J774.A1 cells was the ability to reestablish after harvesting of the monolayer by scaping. Infected cells were used as antigen for immunofluorescence antibody tests (IFA), and the results compared well with those of DH82 cells. It was concluded that the J774.A1 continuous cell line could serve as an alternate propagation cell line for E. canis organisms.     

A randomized comparative clinical trial of recombinant canine interferon-gamma (KT-100) in atopic dogs using antihistamine as control

Recombinant canine interferon-gamma (KT-100) or topical antihistamine (diphenhydramine: DH) was administered to dogs with atopic dermatitis (AD) for 4 weeks and their efficacies were compared using pruritus, excoriation, erythema and alopecia as evaluation criteria. Clinical studies on 92 atopic dogs (KT-100 group: 63, DH group: 29) were conducted at 18 animal hospitals in Japan. KT-100 was administered subcutaneously once a day three times a week on alternating days for 4 weeks. DH was administered topically twice daily for 4 weeks. The efficacy rates of the KT-100 group on day 28 were 72.1% for pruritus, 73.8% for excoriation, 75.4% for erythema and 60.7% for alopecia, which were significantly higher than those of the DH group (20.7% for pruritus, 27.6% for excoriation, 24.1% for erythema and 24.1% for alopecia).     

Pattern-recognition receptor mRNA expression and function in canine monocyte/macrophages and relevance to canine anal furunuclosis

Pattern-recognition receptors (PRRs) are important components of the innate immune system, enabling early detection of infection. Defective PRR function has been implicated in several infectious and immune-mediated diseases of human beings, including Crohn's disease (CD). Anal furunculosis (AF) is an immune-mediated disease which primarily occurs in German shepherd dogs (GSD) and could result from a similar type of PRR dysfunction. The aim of the current study was to investigate canine PRR responses in vitro and to test the hypothesis that these were altered in AF-affected GSD. The pattern-recognition receptors TLR1, TLR2, TLR4, TLR6, TLR9, NOD1 (nucleotide-binding oligomerisation domain) and NOD2 were evaluated in the DH82 canine monocyte/macrophage cell line. These cells were found to express mRNA for all the selected PRRs with TLR2 mRNA the most and TLR5 mRNA the least abundant. A similar pattern of expression was found in canine blood-derived monocyte/macrophages. Stimulation of DH82 cells and blood-derived monocyte/macrophages using specific PRR-ligands, resulted in expression of pro-inflammatory cytokine mRNA. Quantification of TNFalpha mRNA and protein secretion from stimulated cells demonstrated variable responses with lipopolysaccharide (TLR4 ligand) and PAM(3)CSK4 (TLR1/2 ligand) proving to be the most potent and CpG DNA (TLR9 ligand) the least potent. Comparing PRR responses in blood-derived monocyte/macrophages from healthy blood-donor dogs with those from AF-affected GSD showed a deficiency in the latter in response to LD-MDP (NOD2 ligand) at the mRNA level but not at the protein level. It is possible that dysfunctional NOD2 responses by cells of the monocyte/macrophage lineage are involved in the pathogenesis of AF.     

Selection for weaning weight in Targhee sheep in two environments. II. Correlated effects

Targhee sheep were selected for 120-d weight under irrigated pasture-drylot conditions at Davis (DW) and under range conditions at Hopland (HW). Unselected control lines were maintained in both environments (DC, HC1 and HC2). At Hopland, a line (DH) was maintained in which ewes were mated to Davis (DW) rams. Selection for 120-d weight was successful in both environments, with more improvement made in the drylot environment. The genetic improvement made in the drylot environment was expressed, although to a lesser degree, under range conditions. Correlated responses were analyzed. Birth weight increased significantly in all three selected lines; the increase was less in line DH than in the other two lines. In all selected lines, weights of ewes of all ages at mating increased significantly compared with their respective controls. Proportion of ewes lambing decreased (P less than .05) in line DH; the trend was negative but nonsignificant in line DW. Differences in litter size between lines within location were not significant. Lamb survival to weaning decreased in lines DW (P less than .05) and DH (P less than .01), compared with their respective controls; and the trend in HW was negative but nonsignificant. Fertility and survival data indicated that, under range conditions, the line selected under drylot conditions (DH) was less fit than the line selected under range conditions (HW). As a result of the decreases in lamb survival and fertility, none of the selected lines produced more total lamb weight weaned per ewe than the controls, in spite of the significant direct response to selection. Mature ewes of lines DH and DW produced less total lamb weight weaned per ewe (P less than .001 and P less than .05) than their respective controls. The results indicate that while single trait selection for growth rate to weaning results in heavier lambs, it does not increase and may decrease total lamb production per ewe.     

Syncytium-induction inhibition test with complement for detection of antibodies against bovine leukemia virus.

A modified syncytium-induction inhibition test which is more sensitive than the immunodiffusion test, was developed using rabbit complement. In this test, fetal lamb kidney cells continuously infected with bovine leukemia virus were used as effector cells, and the CC81 cat cells transformed with murine sarcoma virus, were used as indicator cells. The syncytium-induction inhibition effect of anti-bovine leukemia virus serum was enhanced significantly by the addition of rabbit complement. The syncytium-induction inhibition titers had a statistically significant correlation with the immunodiffusion titers and were four to 64 times higher than immunodiffusion titers. In 12 experimentally infected cattle, the syncytium-induction inhibition test detected the antibodies earlier than the immunodiffusion test and continuously detected them when immunodiffusion antibody changed to negative. In the 81 sera from naturally infected herds, 35 (43.2%) were positive by the immunodiffusion test and 55 (67.9%) by the syncytium-induction inhibition test.