Integrated histopathological and urinary metabonomic investigation of the pathogenesis of microcystin-LR toxicosis.

Patterns of change of endogenous metabolites may closely reflect systemic and organ-specific toxic changes. The authors examined the metabolic effects of the cyanobacterial (blue-green algal) toxin microcystin-LR by (1)H-nuclear magnetic resonance (NMR) analysis of urinary endogenous metabolites. Rats were treated with a single sublethal dose, either 20 or 80 µg/kg intraperitoneally, and sacrificed at 2 or 7 days post dosing. Changes in the high-dose, 2-day sacrifice group included centrilobular hepatic necrosis and congestion, accompanied in some animals by regeneration and neovascularization. By 7 days, animals had recovered, the necrotizing process had ended, and the centrilobular areas had been replaced by regenerative, usually hypertrophic hepatocytes. There was considerable interanimal variation in the histologic process and severity, which correlated with the changes in patterns of endogenous metabolites in the urine, thus providing additional validation of the biomarker and biochemical changes. Similarity of the shape of the metabolic trajectories suggests that the mechanisms of toxic effects and recovery are similar among the individual animals, albeit that the magnitude and timing are different for the individual animals. Initial decreases in urinary citrate, 2-oxoglutarate, succinate, and hippurate concentrations were accompanied by a temporary increase in betaine and taurine, then creatine from 24 to 48 hours. Further changes were an increase in guanidinoacetate, dimethylglycine, urocanic acid, and bile acids. As a tool, urine can be repeatedly and noninvasively sampled and metabonomics utilized to study the onset and recovery after toxicity, thus identifying time points of maximal effect. This can help to employ histopathological examination in a guided and effective fashion.

Ivermectin sensitivity in collies is associated with a deletion mutation of the mdr1 gene.

A subpopulation of collie dogs is extremely sensitive to neurotoxicity induced by ivermectin. The aim of this study was to determine the mechanistic basis for this phenomenon. The multi-drug-resistance gene (mdr1) encodes a large transmembrane protein, P-glycoprotein (P-gp), that is an integral part of the blood-brain barrier. P-gp functions as a drug-transport pump at the blood-brain barrier, transporting a variety of drugs from the brain back into the blood. Since ivermectin is a substrate for P-gp, we hypothesized that ivermectin-sensitive collies had altered mdr1 expression compared with unaffected collies. We report a deletion mutation of the mdr1 gene that is associated with ivermectin sensitivity. The 4-bp deletion results in a frame shift, generating several stop codons that prematurely terminate P-gp synthesis. Dogs that are homozygous for the deletion mutation display the ivermectin-sensitive phenotype, while those that are homozygous normal or heterozygous do not display increased sensitivity to ivermectin.

Immune reconstitution prevents continuous equine infectious anemia virus replication in an Arabian foal with severe combined immunodeficiency: lessons for control of lentiviruses.

Acute infection with equine infectious anemia virus (EIAV), a lentivirus of horses, results in a persistent high-level viremia in Arabian foals affected with severe combined immunodeficiency (SCID). This observation argues against the idea that the transient nature of acute lentiviral viremia is solely a function of viral population dynamics. To extend these studies, EIAV-specific immune reconstitution was attempted prior to EIAV challenge in two SCID foals, using adoptively transferred virus-stimulated lymphocytes derived from persistently EIAV-infected half sibling donors. Following transfer, lymphocyte engraftment occurred in one foal, and EIAV-specific cytotoxic T lymphocytes as well as neutralizing antibody activity developed. Following a brief period of plasma viremia in this foal, EIAV replication was controlled and plasma virus could not be detected by RT-PCR or culture. These results provide further direct evidence that a specific immune response is required for termination of plasma viremia in acute lentiviral infections.

Role of the proline-rich motif of bovine leukemia virus transmembrane protein gp30 in viral load and pathogenicity in sheep.

The cytoplasmic tail of bovine leukemia virus (BLV) transmembrane protein gp30 has multiple amino acid motifs that mimic those present in signaling proteins associated with B-cell and T-cell receptors. The proline-rich motif of gp30, PX(2)PX(4-5)P, is analogous to the recognition site of Src homology 3 (SH3) domains of signaling molecules. Using site-directed mutagenesis of an infectious molecular clone of BLV, point mutations were introduced which changed three of the prolines of the motif to alanines. The influence of these mutations on the pathogenicity of BLV was studied in sheep which received either (i) plasmid DNA with provirus containing proline-to-alanine mutations (pppBLV), (ii) plasmid DNA with wild-type provirus (wtBLV), or (iii) transfection reagent alone. Although all of the BLV-injected animals seroconverted at approximately the same time, viral loads at later time points were high in five of five of the wtBLV group and two of five of the pppBLV group but low in three of five of the pppBLV group, as determined by semiquantitative PCR. Viral expression was lower in the pppBLV-transfected sheep, as measured by p24 antigen enzyme-linked immunosorbent assay in cultured cells, and serologic titers were lower. Thirty-one months after transfection, four of four wtBLV-transfected sheep had died of leukemia and lymphoma, and all five of the pppBLV-transfected sheep were clinically healthy and had normal peripheral blood lymphocyte counts. These data indicate that the proline-rich motif of gp30 is not required for viral infectivity but is important for high viral load in vivo, suggesting that SH3-mediated gp30 interactions are critical for viral pathogenesis following infection. Absence of interactions with the proline-rich motif may prevent or delay tumorigenesis in sheep.

Functional cystic thyroid adenoma in a cat.

A 9-year-old cat with hyperthyroidism was referred for radioactive iodine treatment. The cat also had a ventral cervical mass that the owners reported had been present for several years and had increased in size during the past few weeks. On physical examination, the mass was found to have caused lateral displacement of the trachea, esophagus, jugular vein, and common carotid artery. The mass was aspirated and was determined to be cystic in nature. Concentrations of thyroid hormones in the cystic fluid were similar to serum concentrations, and nuclear scintigraphy revealed thyroactive tissue lining the cyst wall. Magnetic resonance imaging suggested that the cyst originated from the right lobe of the thyroid gland. The cat was treated with sodium iodide I 131 but died 4 days later, presumably as a result of aspiration of gastric or esophageal contents. A necropsy was not performed, but histologic examination of a biopsy specimen of the mass indicated that it was a cystic thyroid adenoma.

CD5 is dissociated from the B-cell receptor in B cells from bovine leukemia virus-infected, persistently lymphocytotic cattle: consequences to B-cell receptor-mediated apoptosis.

Bovine leukemia virus (BLV), a retrovirus related to human T-cell leukemia virus types 1 and 2, can induce persistent nonneoplastic expansion of the CD5(+) B-cell population, termed persistent lymphocytosis (PL). As in human CD5(+) B cells, we report here that CD5 was physically associated with the B-cell receptor (BCR) in normal bovine CD5(+) B cells. In contrast, in CD5(+) B cells from BLV-infected PL cattle, CD5 was dissociated from the BCR. In B cells from PL cattle, apoptosis decreased when cells were stimulated with antibody to surface immunoglobulin M (sIgM), while in B cells from uninfected cattle, apoptosis increased after sIgM stimulation. The functional significance of the CD5-BCR association was suggested by experimental dissociation of the CD5-BCR interaction by cross-linking of CD5. This caused CD5(+) B cells from uninfected animals to decrease apoptosis when stimulated with anti-sIgM. In contrast, in CD5(+) B cells from PL animals, in which CD5 was already dissociated from the BCR, there was no statistically significant change in apoptosis when CD5 was cross-linked and the cells were stimulated with anti-sIgM. Disruption of CD5-BCR interactions and subsequent decreased apoptosis and increased survival in antigenically stimulated B cells may be a mechanism of BLV-induced PL.

The use of crossreactive monoclonal antibodies to characterize the immune system of the water buffalo (Bubalus bubalis).

One of the major difficulties in studying the mechanisms of host defense in economically important species indigenous to Asia and the Middle East is the lack of monoclonal antibody (mAb) reagents that define the immune systems of species other than cattle, goats, sheep, and pigs. One strategy that could obviate this problem at minimal cost is to identify existing mAbs that recognize conserved epitopes on orthologous major histocompatibility complex (MHC) and leukocyte differentiation molecules. To explore the potential of this approach, we screened a large set of mAbs that recognize bovine MHC class I and II molecules and leukocyte differentiation molecules to identify mAbs that react with orthologous molecules in water buffalo. One hundred thirty eight were found that recognize conserved determinants on orthologous molecules. In addition to identifying a useful set of reagents, the study has provided insight into the composition of the immune system of the water buffalo (Bubalus bubalis).

Protective effects of a live attenuated bovine leukaemia virus vaccine with deletion in the R3 and G4 genes.

In this study the protective effects of a live attenuated bovine leukaemia provirus (pBLVDX) with deletion in the R3 and G4 genes were tested. Six out of six sheep appeared to resist challenge with parental BLV344. Two out of three animals transfected with pBLVDX were protected against challenge with bovine leukaemia virus (BLV) from a naturally infected cow. As a model for the protection against infection by members of the human T-lymphotropic virus/BLV group, these data provide evidence that a DNA-based vaccination with an attenuated provirus is able to protect against challenge infections.

Bovine leukemia virus transmembrane protein gp30 physically associates with the down-regulatory phosphatase SHP-1.

In B lymphocytes, the down-regulatory phosphatase SHP-1 associates with CD22 and CD32b (also known as FcgammaRIIB) and acts as a critical negative regulator of B-cell receptor signaling. Bovine leukemia virus, a retrovirus of the HTLV/BLV group, causes persistently increased numbers of peripheral blood B lymphocytes, known as persistent lymphocytosis (PL) and, in some animals, progression to B-cell leukemia and/or lymphoma. Here, we show that SHP-1 associates with the bovine leukemia virus transmembrane protein, gp30. This interaction is either direct or indirect. The interaction is dependent on tyrosine phosphorylation, and the interaction increases after cell stimulation with sodium pervanadate. The gp30-SHP-1 interaction is seen in all of the BLV-infected, PL animals tested, but is not seen in uninfected animals or in most BLV-infected, non-PL animals, which do not express significant quantities of gp30. However, one BLV-infected, non-PL animal expressed large quantities of gp30, yet no gp30-SHP-1 interaction was detected, suggesting that there may be other factors in cells from the PL animals that facilitate the gp30-SHP-1 interaction. The association of gp30 and SHP-1 suggests the hypothesis that gp30 may act as a decoy to sequester SHP-1, resulting in up-regulation of B-cell receptor signaling. The implication of this could be a novel mechanism of viral activation of lymphocytes by removal of a down-regulatory phosphatase.

Bovine leukemia virus-induced persistent lymphocytosis in cattle does not correlate with increased ex vivo survival of B lymphocytes.

Bovine leukemia virus (BLV) is an oncogenic retrovirus associated with B-cell lymphocytosis, leukemia, and lymphosarcoma in the ovine and bovine species. We have recently reported that in sheep, BLV protects the total population of peripheral blood mononuclear cells (PBMCs) from ex vivo spontaneous apoptosis. This global decrease in the apoptosis rates resulted from both direct and indirect mechanisms which allow extension of cell survival. Although sheep are not natural hosts for BLV, these animals are prone to develop virus-induced leukemia at very high frequencies. Most infected cattle, however, remain clinically healthy. This difference in the susceptibilities to development of leukemia in these two species might be related to alterations of the apoptotic processes. Therefore, we designed this study to unravel the mechanisms of programmed cell death in cattle. We have observed that PBMCs from persistently lymphocytotic BLV-infected cows were more susceptible to spontaneous ex vivo apoptosis than cells from uninfected or aleukemic animals. These higher apoptosis rates were the consequence of an increased proportion of B cells exhibiting lower survival abilities. About one-third of the BLV-expressing cells did not survive the ex vivo culture conditions, demonstrating that viral expression is not strictly associated with cell survival in cattle. Surprisingly, culture supernatants from persistently lymphocytotic cows exhibited efficient antiapoptotic properties on both uninfected bovine and uninfected ovine cells. It thus appears that indirect inhibition of cell death can occur even in the presence of high apoptosis rates. Together, these results demonstrate that the protection against spontaneous apoptosis associated with BLV is different in cattle and in sheep. The higher levels of ex vivo apoptosis occurring in cattle might indicate a decreased susceptibility to development of leukemia in vivo.

The conduction system in sudden death in Alaskan sled dogs during the Iditarod race and/or during training.

Using serial section examination, we studied the conduction system in five Alaskan sled dogs that died suddenly; four during the Iditarod race and one during training. We compared our findings with the conduction system of three sled dogs of similar age that died of natural causes unrelated to the cardiovascular system. The conduction system of sudden death dogs revealed marked fibrosis of the sinoatrial (SA) node and/or its approaches and narrowing of the SA nodal artery in 3 dogs, fibrosis and marked fatty infiltration in and around the AV node in all 5, total isolation and/or tenuous connection of the AV node with its approaches in 4, fat and fibrosis in the AV bundle and bundle branches to a varying degree in all, and focal fibrotic scars in the left ventricle with fat and/or some disarray in 3. The control group revealed mild fibro-fatty changes in the conduction system without fibrotic scar areas in the heart. These findings are similar to the pathological findings in and around the conduction system in cases of sudden death in humans, especially trained athletes. These changes may form an anatomical substrate for an arrhythmic event in susceptible dogs during an altered physiological state.

Analysis of monoclonal antibodies reactive with molecules upregulated or expressed only on activated lymphocytes.

Monoclonal antibodies potentially specific for antigens expressed or upregulated on activated leukocytes were selected for further analysis from the panel submitted to the third international workshop on ruminant leukocyte antigens. The kinetics of expression of these activation antigens on resting peripheral mononuclear cells (PBMC) and PBMC stimulated with concanavalin A or staphylococcal superantigen SECI for 4, 24 or 96 h were compared, as well as their appearance on various subsets of cells. For some of them, a molecular mass could be determined after immunoprecipitation from radio-labeled, lectin-stimulated cells. Based on the results from the clustering, kinetic studies and biochemical data, evidence was gathered for assigning two additional mAbs to cluster BoCD25 (IL-2 receptor) and two mAbs to cluster BoCD71 (transferrin receptor). Four mAbs recognized an early activation antigen predominantly expressed on gamma delta T cells in short-term cultures. A number of other activation antigens were further characterized.

Rapid differentiation of Mycobacterium avium and M. paratuberculosis by PCR and restriction enzyme analysis.

Mycobacterium avium subsp. avium (M. avium) and M. avium subsp. paratuberculosis (M. paratuberculosis), intracellular bacteria that can cause chronic granulomatous enteritis in cattle, are difficult to distinguish on the basis of growth and biochemical characteristics. We report the development of a PCR-based strategy for the rapid differentiation of isolates of M. avium from isolates of M. paratuberculosis. Restriction fragment length polymorphism was identified by PCR amplification and subsequent restriction enzyme digestion with PstI of a 960-bp fragment of the 65-kDa heat shock protein (hsp65) from 21 clinical isolates of M. paratuberculosis and 14 isolates of M. avium. These results indicate that a restriction fragment length polymorphism in the hsp65 gene can be used for the rapid differentiation of clinical isolates of M. paratuberculosis and M. avium.

Comparison of the antiviral efficacy of ribozymes and antisense RNA directed against bovine leukemia virus rex/tax.

Despite the theoretical attraction of ribozymes, which cleave their target RNA, as compared with antisense RNA, which only blocks translation, few studies have assessed the relative efficacy of ribozymes and antisense RNA directed against the identical target sequence. Previously, we described the efficacy of a hammerhead ribozyme targeted against rex/tax, a regulatory gene of bovine leukemia virus (BLV). In this study, we asked whether antisense RNA targeted against the same site would also be efficacious. BLV-infected bat lung cells were transfected with an antisense RNA-encoding plasmid under the control of sarcoma virus (RSV) promoter, and stable cell lines were selected. No inhibition of viral p24 expression was demonstrated in seven cell lines transfected with the antisense RNA targeted at the same site as the ribozyme or in three cell lines transfected with an antisense sequence targeted against the tax 5' initiation codon. Although in previous experiments antisense DNA oligonucleotides inhibited Tax expression in vitro, stably transfected plasmids encoding antisense RNA of the same sequence did not inhibit viral expression in BLV-infected cells in this study. These results suggest that in persistently infected cells producing high levels of BLV, the ribozyme is more effective than antisense RNA directed at rex/tax transcripts.

Ribozyme cleaves rex/tax mRNA and inhibits bovine leukemia virus expression.

Bovine leukemia virus (BLV) encodes at least two regulatory proteins, Rex and Tax. Tax, the transactivating protein, stimulates the long terminal repeat to promote viral transcription and may be involved in tumorigenesis. Rex is involved in the transition from early expression of regulatory proteins to later expression of viral structural proteins. We have targeted ribozymes against the mRNA encoding Rex and Tax. The ribozymes consist of the hammer-head catalytic motif flanked by antisense sequences that hybridize with the complementary rex/tax mRNA. To evaluate cleavage in a cell-free system, we transcribed portions of rex/tax mRNA and incubated them with synthetic RNA ribozymes. A ribozyme was identified that cleaves > 80% of the target RNA. Synthetic DNA encoding this ribozyme was cloned into the expression vector pRc/RSV and transfected into BLV-infected bat lung cells. Intracellular cleavage of rex/tax mRNA was confirmed by reverse transcriptase PCR. In cells expressing the ribozyme, viral expression was markedly inhibited. Expression of the BLV core protein p24 was inhibited by 61%, and reverse transcriptase activity in supernatant was inhibited by 92%. Ribozyme inhibition of BLV expression suggests that cattle expressing these sequences may be able to control BLV replication.