Resolution of a complex ionic mixture of an apparently homogenous protein preparation by preparative electrophoresis

Preparations of recombinant envelope glycoprotein E2 of hepatitis C virus (r-HCV E2), found to be homogeneous by N-terminal amino acid sequencing and mass spectrometry, resolved into multiple ionic species (isoforms) when analysed by isoelectric focusing (IEF) gel electrophoresis in the p1 range of 3-10. These isoforms possessed pI values in the range of 4.5-8.2. The major isoform with p1 value of approximately 7.1 was separated from the rest of them by employing a method developed on Gradiflow BF 200, a device based on preparative electrophoresis. This isoform was adjudged to be homogenous by IEF and by native polyacrylamide gel electrophoresis (PAGE).     

Clinical Toxoplasma gondii, Hammondia heydorni, and Sarcocystis spp. infections in dogs

Concurrent infections with coccidians Toxoplasma gondii, Sarcocystis spp., and a Hammondia heydorni-like parasite were identified in tissues of three littermate pups on a Kelpie dog breeding farm in Australia. In total, 20 pups in four litters had died following vaccination with an attenuated distemper virus vaccine. Toxoplasma gondii tachyzoites were identified immunohistochemically in tissues of two dogs. Sarcocystis sp. sporocysts were seen in the intestinal lamina propria of two dogs. Asexual and sexual stages of H. heydorni-like parasite were found in enterocytes of the small intestine of two dogs. Ultrastructural development of schizonts and gamonts of this parasite is described. None of the protozoa in these dogs reacted with antibodies to Neospora caninum. Feeding of uncooked tissue of sheep was considered to be the likely source of infection for these coccidians in dogs.     

Qualitative changes accompany memory T cell generation: faster, more effective responses at lower doses of antigen

The generation of memory T cells is critically important for rapid clearance and neutralization of pathogens encountered previously by the immune system. We have studied the kinetics of response and Ag dose requirements for proliferation and cytokine secretion of CD4+ memory T cells to examine whether there are qualitative changes which might lead to improved immunity. TCR Tg CD4+ T cells were primed in vitro and transferred into T cell-deficient hosts. After 6 or more weeks, the persisting T cells were exclusively small resting cells with a memory phenotype: CD44high CD62L+/- CD25-. Memory CD4 T cells showed a similar pattern of response as naive cells to peptide analogues with similar Ag dose requirements for IL-2 secretion. However, memory cells (derived from both Th2 and Th1 effectors) displayed faster kinetics of cytokine secretion, cell division, and proliferation, enhanced proliferation in response to low doses of Ag or peptide analogues, and production of IL-4, IL-5, and IFN-gamma. These results suggest there is a much more efficient response of CD4 memory T cells to Ag re-exposure and that the expanded functional capacity of memory cells will promote a rapid development of effector functions, providing more rapid and effective immunity.     

Muscular sarcocystosis in coyotes from Oklahoma

In a recent survey in Oklahoma (USA), 52 free-ranging coyotes were examined for the presence of sarcocysts. Two of these coyotes were found infected with sarcocysts in skeletal muscle. By light microscopy, the cyst wall was thin and smooth. Ultrastructurally, the cyst wall had minute villar protrusions. The sarcocysts were 14.4 to 50.4 microm wide and 46.8 to 99 microm long. This is the first report of Sarcocystis sp. sarcocysts in the skeletal muscle of coyotes.     

Acute sarcocystosis in a captive white-tailed deer in Virginia

A 19-mo-old female captive white-tailed deer in a public wild animal park in Richmond (Virginia, USA) was necropsied and evaluated histologically following spontaneous death after a 1 wk period of lethargy in a captive herd of 22 deer. An acute necrotizing pneumonia was associated with intraendothelial protozoal schizonts that were identified immunohistochemically as Sarcocystis spp. This is the first confirmed report of acute sarcocystosis in a wild ruminant.     

Isolation of Sarcocystis speeri Dubey and Lindsay, 1999 parasite from the South American opossum (Didelphis albiventris) from Argentina

Sarcocystis sporocysts from the intestines of 2 opossums (Didelphis albiventris) from Argentina were fed to gamma-interferon knockout (KO) and nude mice. Protozoal schizonts were seen in brain, liver, spleen, and adrenal glands of mice examined 33-64 days after feeding sporocysts. Sarcocysts were seen in skeletal muscles of KO mice 34-71 days after feeding sporocysts. Schizonts and sarcocysts were structurally similar to Sarcocystis speeri Dubey and Lindsay, 1999 seen in mice fed sporocysts from the North American opossum Didelphis virginiana from the United States.     

Strain typing of Toxoplasma gondii: comparison of antigen-coding and housekeeping genes

Molecular characterization of Toxoplasma gondii isolates is central for understanding differences in disease transmission and manifestations. Only 3 subgroups (lineages) have been discerned with subtle within-lineage variation, permitting low-resolution classification of isolates. Because proteins, coding sequences, and especially antigen-coding genes have been used extensively in previous studies, we focused on sequence variation in introns of housekeeping genes, which may be more informative for phylogenetic analysis because they evolve under lower selection. We compared sequence variation in introns of 5 housekeeping genes with 2 antigen-coding genes. Introns of housekeeping genes were slightly more polymorphic than coding and noncoding regions of antigen-coding genes and only the former showed intralineage variation. Intragenic linkage disequilibrium was complete, but intergenic linkage, although highly significant, was incomplete, suggesting that genes are partially uncoupled. Six of 7 substitutions found within the region coding for the tachyzoite surface antigen, SAG2, were nonsynonymous, indicating that diversifying selection acts on this locus. Typing isolates on the basis of housekeeping and antigen-coding genes was consistent, but the phylogenetic relationships among the resulting groups was inconsistent. A cougar isolate typed as lineage II using a restriction fragment length polymorphism assay possessed multiple unique polymorphisms, suggesting that it represents a new lineage. We concluded that introns of housekeeping genes are preferred markers for phylogenetic study, and that multilocus genotyping is preferred for typing parasites, especially from feral or unstudied environments.     

Biological and molecular characterizations of Toxoplasma gondii strains obtained from southern sea otters (Enhydra lutris nereis)

Toxoplasma gondii was isolated from brain or heart tissue from 15 southern sea otters (Enhydra lutris nereis) in cell cultures. These strains were used to infect mice that developed antibodies to T. gondii as detected in the modified direct agglutination test and had T. gondii tissue cysts in their brains at necropsy. Mouse brains containing tissue cysts from 4 of the strains were fed to 4 cats. Two of the cats excreted T. gondii oocysts in their feces that were infectious for mice. Molecular analyses of 13 strains indicated that they were all type II strains, but that they were genetically distinct from one another.     

Processing of viral envelope glycoprotein by the endomannosidase pathway: evaluation of host cell specificity

Endo-alpha-D-mannosidase is an enzyme involved in N-linked oligosaccharide processing which through its capacity to cleave the internal linkage between the glucose-substituted mannose and the remainder of the polymannose carbohydrate unit can provide an alternate pathway for achieving deglucosylation and thereby make possible the continued formation of complex oligosaccharides during a glucosidase blockade. In view of the important role which has been attributed to glucose on nascent glycoproteins as a regulator of a number of biological events, we chose to further define the in vivo action of endomannosidase by focusing on the well characterized VSV envelope glycoprotein (G protein) which can be formed by the large array of cell lines susceptible to infection by this pathogen. Through an assessment of the extent to which the G protein was converted to an endo-beta-N- acetylglucosaminidase (endo H)-resistant form during a castanospermine imposed glucosidase blockade, we found that utilization of the endomannosidase-mediated deglucosylation route was clearly host cell specific, ranging from greater than 90% in HepG2 and PtK1 cells to complete absence in CHO, MDCK, and MDBK cells, with intermediate values in BHK, BW5147.3, LLC-PK1, BRL, and NRK cell lines. In some of the latter group the electrophoretic pattern after endo H treatment suggested that only one of the two N-linked oligosaccharides of the G protein was processed by endomannosidase. In the presence of the specific endomannosidase inhibitor, Glcalpha1-->3(1- deoxy)mannojirimycin, the conversion of the G protein into an endo H- resistant form was completely arrested. While the lack of G protein processing by CHO cells was consistent with the absence of in vitro measured endomannosidase activity in this cell line, the failure of MDBK and MDCK cells to convert the G protein into an endo H-resistant form was surprising since these cell lines have substantial levels of the enzyme. Similarly, we observed that influenza virus hemagglutinin was not processed in castanospermine-treated MDCK cells. Our findings suggest that studies which rely on glucosidase inhibition to explore the function of glucose in controlling such critical biological phenomena as intracellular movement or quality control should be carried out in cell lines in which the glycoprotein under study is not a substrate for endomannosidase action.