Eimeria acervulina: DNA cloning and characterization of recombinant sporozoite and merozoite antigens

Genes encoding antigens of Eimeria acervulina were cloned from cDNA expression libraries prepared from the sporozoite and merozoite stages in order to examine humoral and cellular immune responses to this protozoan parasite. Two clones expressing surface antigens were characterized by DNA hybridization studies to identify homologous genomic DNA fragments. The proteins they encode were identified by 125I-labeling, immunoblotting, immunofluorescence, and T-cell activation experiments. One, designated cSZ-1, encodes a 130-kDa beta-galactosidase fusion protein which represents a portion of a p240/p160 immunodominant sporozoite surface antigen. Immunofluorescence studies using anti-cSZ-1 sera and live or 1% paraformaldehyde-fixed E. acervulina sporozoites have confirmed this surface locale. Purified cSZ-1 fusion protein, which is not recognized by sera from E. acervulina-infected chickens, induced the activation of immune T lymphocytes in vitro. Another cDNA clone, designated cMZ-8, gives rise to a 150-kDa fusion protein and encodes a portion of a p250 immunodominant merozoite surface antigen. This was established by immunoblotting of 125I-labeled merozoite proteins with anti-cMZ-8 sera and immunofluorescence staining of live and 1% paraformaldehyde-fixed E. acervulina merozoites. Purified cMZ-8 is recognized by sera from E. acervulina-infected chickens and induces a significant activation of immune T lymphocytes in vitro.     

Growth of three established cell lines on glass microcarriers

Three established cell lines were examined for growth on a newly developed microcarrier which consists of glass beads. The cells were simultaneously exmined for growth on commercially available microcarriers made from DEAE-dextran and from plastic. Cell yields on the glass microcarriers were comparble to the cell yields on the commercially available products. Cells grown on the glass microcarriers were easily separated from the substratum by trypsinization (as were the cells grown on the plastic substratum) while the cells grown on the DEAE-dextran particles were much more trypsin resistant. After removal of cells from the glass microcarriers, the cells reattached and spread out in plastic flasks as readily as cells harvested from monolayer. Scanning electron microscopy revealed dramatic differences in the appearence of the cell grown on the glass microcarriers and cells grown on the DEAE-dextran microcarriers. On the glass microcarriers, cells attached to the substratum through lond, slender filopodia while on the DEAE-dextran microcarriers, the entire edge of the cell appeared to be in contact with the substratum. This dissimilarity in attachment could underly the difference in sensitivity to trypsin-mediated detachment. Finally, the glass microcarriers were washed after being used once and retested for their ability to support cell growth a second time. Nearly identical results were obtained with the reprocessed beads as with previously unused ones.     

Colonization factors of diarrheagenicE. coli and their intestinal receptors

WhileEscherichia coli is common as a commensal organism in the distal ileum and colon, the presence of colonization factors (CF) on pathogenic strains ofE. coli facilitates attachment of the organism to intestinal receptor molecules in a species- and tissue-specific fashion. After the initial adherence, colonization occurs, and the involvement of additional virulence determinants leads to illness. EnterotoxigenicE. coli (ETEC) is the most extensively studied of the five categories ofE. coli that cause diarrheal disease, and has the greatest impact on health worldwide. ETEC can be isolated from domestic animals and humans. The biochemistry, genetics, epidemiology, antigenic characteristics, and cell and receptor binding properties of ETEC have been extensively described. Another major category, enteropathogenicE. coli (EPEC), has virulence mechanisms, primarily effacement and cytoskeletal rearrangement of intestinal brush borders, that are distinct from ETEC. An EPEC CF receptor has been purified and characterized as a sialidated transmembrane glycoprotein complex directly attached to actin, thereby associating CF-binding with host-cell response. Three, additional categories ofE. coli diarrheal disease, their colonization factors and their host cell receptors are discussed. It appears that biofilms exist in the intestine in a manner similar to oral bacterial biofilms, and thatE. coli is part of these biofilms as both commensals and pathogens.Abbreviations CF colonization factor - CFA Colonization Factor Antigen - CS coli-surface-associated antigen - EAggEC enteroaggregativeE. coli - ECDD E. coli diarrheal disease - EHEC enterohemorrhagicE. coli - EIEC enteroinvasiveE. coli - EPEC enteropathogenicE. coli - ETEC enterotoxigenicE. coli - Gal galactose - GalNAc N-acetyl galactosamine - LT heat-labile toxin - NeuAc N-acetyl neuraminic acid - PCF Putative colonization factor - RBC red blood cells - SLT Shiga-like toxin - ST heat-stable toxin     

The prevalence of Fasciola hepatica in its snail intermediate host determined by DNA probe assay

Accurate snail intermediate host infection prevalence data have the potential to be extremely useful in determining seasonal transmission dynamics of Fasciola hepatica. Because the microscopic techniques currently used lack the sensitivity and specificity necessary to obtain meaningful infection prevalence data, we developed a highly accurate and efficient DNA probe assay. The assay has a sensitivity of 100%, a specificity of >99%, easily detects a single miracidia and does not cross-hybridize with DNA of Fascioloides magna, Paramphistomum liorchis or Heterobilharzia americana, trematodes that share the same intermediate host and enzootic range as Fasciola hepatica. Using this assay, we determined the prevalence of F. hepatica in its snail intermediate host, Fossaria cubensis, during the second year of a 2-year study on the epizootiology of Fasciola hepatica in Florida. The overall infection prevalence of snails assayed in this study (n = 5246) was 1.5% and ranged from 0.1% to 3.1% for individual cattle ranches. Additionally, infection prevalence differed significantly for successive size groupings of snails, varying from 0% for 1-mm snails to 18.5% for 9- and 10-mm snails. The accuracy and efficiency of the DNA probe assay reported here for determining snail infection prevalence offers an inexpensive alternative to tracer animal studies for determining the epizootiology of F. hepatica.     

Minimizing ELISA background in the diagnosis of swine trichinellosis.

After confirming that long-term serum storage (frozen at -20 C for greater than 3 mo) causes optical density to drift upward, several modifications of an enzyme-linked immunosorbent assay (ELISA) protocol were evaluated to identify a protocol that would reduce background in porcine sera tested for trichinellosis. Modifications evaluated included blocking the antigen-coated ELISA plate with sample diluent containing 10% bovine serum albumin (BSA) or 10% nonfat milk powder (bovine lacto transfer optimizer or BLOTTO), diluting sera in sample diluent containing 10% BSA or 10% BLOTTO, and preincubating samples in sample diluent containing 10% BSA or 10% BLOTTO. Overnight preincubation (approximately 12 hr at 2 C) of fresh sera diluted (1:10) in sample diluent containing 10% BLOTTO significantly reduced background and improved the detection of experimentally infected pigs by enhancing positive-negative discrimination. When testing stored sera, the modified protocol effectively reduced the effect of storage and the kit revealed specificity of 98.4%; there was no loss in sensitivity. The effect of long-term storage at -20 C must therefore be considered when testing swine sera for trichinellosis by ELISA and possibly also when conducting other immunoglobulin assays. The modification described here may prove useful if there is no alternative to using serum stored for greater than 3 mo at -20 C.     

Microengineered systems with iPSC-derived cardiac and hepatic cells to evaluate drug adverse effects

Hepatic and cardiac drug adverse effects are among the leading causes of attrition in drug development programs, in part due to predictive failures of current animal or in vitro models. Hepatocytes and cardiomyocytes differentiated from human induced pluripotent stem cells (iPSCs) hold promise for predicting clinical drug effects, given their human-specific properties and their ability to harbor genetically determined characteristics that underlie inter-individual variations in drug response. Currently, the fetal-like properties and heterogeneity of hepatocytes and cardiomyocytes differentiated from iPSCs make them physiologically different from their counterparts isolated from primary tissues and limit their use for predicting clinical drug effects. To address this hurdle, there have been ongoing advances in differentiation and maturation protocols to improve the quality and use of iPSC-differentiated lineages. Among these are in vitro hepatic and cardiac cellular microsystems that can further enhance the physiology of cultured cells, can be used to better predict drug adverse effects, and investigate drug metabolism, pharmacokinetics, and pharmacodynamics to facilitate successful drug development. In this article, we discuss how cellular microsystems can establish microenvironments for these applications and propose how they could be used for potentially controlling the differentiation of hepatocytes or cardiomyocytes. The physiological relevance of cells is enhanced in cellular microsystems by simulating properties of tissue microenvironments, such as structural dimensionality, media flow, microfluidic control of media composition, and co-cultures with interacting cell types. Recent studies demonstrated that these properties also affect iPSC differentiations and we further elaborate on how they could control differentiation efficiency in microengineered devices. In summary, we describe recent advances in the field of cellular microsystems that can control the differentiation and maturation of hepatocytes and cardiomyocytes for drug evaluation. We also propose how future research with iPSCs within engineered microenvironments could enable their differentiation for scalable evaluations of drug effects.     

Determination of acid α-naphthyl acetate esterase enzyme activity in peripheral blood leukocytes of gazelles (Gazella subgutturosa)

We examined gazelle peripheral blood leucocytes using the α-Naphthyl acetate esterase (ANAE) staining technique (pH 5.8). Our purpose was to determine the percentage of ANAE positive lymphocytes. The proportion of ANAE positive T-lymphocytes was 72%. T-lymphocytes showed an ANAE positive reaction, but eosinophilic granulocytes and monocytes also showed a positive reaction. By contrast, no reaction was detected in B-lymphocytes, neutrophil granulocytes or platelets. The reaction observed in T-lymphocytes was a red-brown coloration, usually 1–2 granules, but enough granules to fill the cytoplasm were detected rarely. As a result of ANAE enzyme staining, we concluded that the staining technique can be used as a cytochemical marker for gazelle T-lymphocytes.