Isolation of Trypanosoma Theileri from the Blood of Two Cows,One Leukotic,One Exhibiting Lymphocytosis

Trypanosoma theileri was isolated by the blood culture method from a leukotic cow in extremis. The parasite could be maintained along with leukocytes with weekly changes of culture medium up to 6 months. In connection with a subsequent transmission experiment a cow, not inoculated with trypanosomes but kept in the same shelter as the inoculated one, commenced to be positive for T. theileri in its blood cultures. This cow had a long history of lymphocytosis but showed no signs of leukosis when slaughtered. The significance of the findings is discussed from the point of view of false positive diagnosis of bovine leukosis and the possible role of trypanosome in the etiology of leukosis.     

Targeting cattle-borne zoonoses and cattle pathogens using a novel trypanosomatid-based delivery system

Trypanosomatid parasites are notorious for the human diseases they cause throughout Africa and South America. However, non-pathogenic trypanosomatids are also found worldwide, infecting a wide range of hosts. One example is Trypanosoma (Megatrypanum) theileri, a ubiquitous protozoan commensal of bovids, which is distributed globally. Exploiting knowledge of pathogenic trypanosomatids, we have developed Trypanosoma theileri as a novel vehicle to deliver vaccine antigens and other proteins to cattle. Conditions for the growth and transfection of T. theileri have been optimised and expressed heterologous proteins targeted for secretion or specific localisation at the cell interior or surface using trafficking signals from Trypanosoma brucei. In cattle, the engineered vehicle could establish in the context of a pre-existing natural T. theileri population, was maintained long-term and generated specific immune responses to an expressed Babesia antigen at protective levels. Building on several decades of basic research into trypanosomatid pathogens, Trypanosoma theileri offers significant potential to target multiple infections, including major cattle-borne zoonoses such as Escherichia coli, Salmonella spp., Brucella abortus and Mycobacterium spp. It also has the potential to deliver therapeutics to cattle, including the lytic factor that protects humans from cattle trypanosomiasis. This could alleviate poverty by protecting indigenous African cattle from African trypanosomiasis.     

An experiment on the culture of Tilapia esculenta (Graham) and Tilapia zillii (Gervais) (Cichlidae) in fish ponds

Segregated populations of Tilapia zillii and Tilapia esculenta were kept in artificial ponds. Supplementary food was given and phytoplankton was encouraged by the addition of superphosphate and ammonium sulphate fertilisers. The fry were cropped regularly in an attempt to control population size. A control population of T. zillii was also established in which none of these procedures were carried out.
The growth of T. esculenta was found to be dependent upon the phytoplankton density of the pond whilst T. zillii successfully utilized the supplementary food and grew well. The T. esculenta population also produced more fry than the T. zillii population. The comparative advantages of planktrvorous/brooders and herbivorous/guarders in fish culture are considered.
Despite the fecundity of the species used a population of known composition was maintained by manual cropping of the fry.
The addition of ammonium sulphate to the ponds caused blooms of phytoplankton which in turn had effects upon the base reserves and pH of the water, and probably caused some reduction of oxygen concentration beneath surface scums. This last point is used to explain the changes in phosphate and calcium content of the water also observed.
The low oxygen concentration frequently noted in the mornings probably affected the feeding behaviour of the fishes, and was low enough to have affected metabolism directly.     

Transmission of Megatrypanum trypanosomes to Cervus dama by Tabanidae

Four fallow deer, Cervus dama, became infected with Trypanosoma (megatrypanum) sp. by oral application of triturated guts from tabanids collected in an area with deer but without any cattle; four control calves remained negative. Upon challenge with triturated guts from tabanids from an area with pastured cattle, the four calves became infected with Trypanosoma (M.) theileri. The prepatent period in deer was five days or less. Haematopota spp. and Tabanus spp. were identified as vectors of the deer trypanosomes. It is concluded that the trypanosomes of C. dama belong to a Megatrypanum species that is not identical with T. theileri.     

Immunohistochemical demonstration of Trypanosoma evansi in tissues of experimentally infected rats and a naturally infected water buffalo (Bubalus bubalis)

Trypanosoma evansi was demonstrated by an immunohistochemical technique in formalin-fixed paraffin-embedded tissues of experimentally infected rats. Trypanosoma evansi was visible readily, nuclei were stained darkly, the cytoplasm was stained moderately, and the cell membranes were delineated clearly. The parasites were present in small- to large-sized blood vessels of all organs, in extravascular spaces of ventricles and neuropil of the brain, and in interstitial tissues of the lung and testes. This method also stained nuclei but not cytoplasm or cell membranes of Trypanosoma congolense, and did not stain Trypanosoma theileri. In a water buffalo (Bubalus bubalis) with nonsuppurative meningoencephalitis, the presence of T. evansi could not be demonstrated by conventional histological stains. However, the trypanosomes were recognized readily in the Virchow-Robin spaces and neuropil of the brain by the immunohistochemical method.     

Altitudinal genetic and morphometric variation among populations of Culex theileri Theobald (Diptera: Culicidae) from northeastern Turkey

Enviromental conditions, including such important climatic variables as temperature and precipitation, change with altitude; thus, elevation plays a significant role in determining population and community structure in a variety of organisms. Using single nucleotide polymorphisms (SNPs) and geometric morphometrics, nine populations of Culex theileri Theobald occurring in different ecological subregions at altitudes between 808-2,130 m in northeastern Turkey were compared. The wing size and shape data indicate that there are significant phenotypic differences among them, while Cx theileri populations are not genetically differentiated in the northeast part of Turkey. The size and shape variation analysis of wings showed that there is a positive correlation between wing (body) size/shape and altitude.     

Biology of Acarophenax lacunatus (Cross & Krantz) (Prostigmata: Acarophenacidae) on Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) and Cryptolestes ferrugineus (Stephens) (Coleoptera: Cucujidae)

The aim of the present study was to assess the effect of six different temperatures on the development of Acarophenax lacunatus (Cross & Krantz) using eggs of Tribolium castaneum (Herbst) and Cryptolestes ferrugineus (Stephens) as hosts. The temperature affected the development of A. lacunatus. The largest values for the progeny (19 mites in T. castaneum and 15 mites in C. ferrugineus) were obtained at about 30 degrees C, as also observed for the net reproductive rate (Ro), which revealed that the A. lacunatus population increased 18 times in T. castaneum and 14 times in C. ferrugineus in a generation. The intrinsic rate of increase (r m) gradually increased with temperature, reaching the maximum value at 35 degrees C in T. castaneum (1,608) and C. ferrugineus (1,289). The generation time was negatively correlated with temperature, ranging from 1,60 to 4,85 days in T. castaneum and from 1,96 to 5,34 days in C. ferrugineus. These results suggest that the mite A. lacunatus may be used in programs of biological control of T. castaneum and C. ferrugineus in the tropics.     

Trypanosoma theileri Associated with T-lymphocytes Isolated from a Latently Infected Cow

. Trypanosoma theileri infection, latent in a mature Hereford cow, could not be demonstrated in routine blood smears or cultures. Throughout the 2-year period an intravenous injection of dexamethasone consistently produced parasitaemia which was detectable in peripheral blood mononuclear cell (PBMC) cultures. Fractionation techniques such as plastic adherence and Sephadex-G10 fractionation, designed to deplete monocytes and enrich T-lymphocytes, increased trypanosome-positive cultures from 25 to 100%. Removal of B-lymphocytes from Sephadex, non-adherent (SE-NA) cells did not reduce the percentage of positive cultures. Light and transmission electron microscopy of SE-NA PBMC cultured for 36 or 45 h revealed numerous trypanosomes and widespread T-lym-phocyte destruction. No trypanosomes were observed in 12-h cultures. A close association, either extra- or intracellular, of a parasitic stage of T. theileri with T-lymphocytcs is inferred.     

Trypanosoma theileri associated with T-lymphocytes isolated from a latently infected cow

Trypanosoma theileri infection, latent in a mature Hereford cow, could not be demonstrated in routine blood smears or cultures. Throughout the 2-year period an intravenous injection of dexamethasone consistently produced parasitaemia which was detectable in peripheral blood mononuclear cell (PBMC) cultures. Fractionation techniques such as plastic adherence and Sephadex-G10 fractionation, designed to deplete monocytes and enrich T-lymphocytes, increased trypanosome-positive cultures from 25 to 100%. Removal of B-lymphocytes from Sephadex, non-adherent (SE-NA) cells did not reduce the percentage of positive cultures. Light and transmission electron microscopy of SE-NA PBMC cultured for 36 or 45 h revealed numerous trypanosomes and widespread T-lymphocyte destruction. No trypanosomes were observed in 12-h cultures. A close association, either extra- or intracellular, of a parasitic stage of T. theileri with T-lymphocytes is inferred.     

Keratin and carcinoembryonic antigen (CEA) in human melanoma cells.

Human melanomas are known to contain vimentin intermediate filaments but there has been some dispute about their expression of cytokeratins. The cytoplasm of human M21 melanoma cells maintained in culture reacted with a rabbit anti-keratin antibody and two monoclonal anti-keratin antibodies AE1 and AE2. Cells derived directly from subcutaneous xenografts of M21 melanoma in nude mice, however, failed to express cytokeratins. The presence of keratin filaments in cultured M21 cells was confirmed by electronmicroscopic and immuno-electronmicroscopic examinations of cell extracts. Polyacrylamide gel electrophoresis (PAGE), revealed 46 KD keratin proteins in cultured M21 cells. Small amounts of these low molecular weight keratins were detected by PAGE in M21 melanoma xenografts even though immunofluorescence and immunoperoxidase assays failed to demonstrate keratin at the light microscopic level. Immunofluorescence revealed keratin and carcinoembryonic antigen (hitherto undetected in human melanomas) first on the 9th day of culture of xenograft-derived M21 cells. The appearance of keratin and CEA in M21 melanoma cells in vitro was not affected by inhibition of cellular proliferation or as a result of exposure to methotrexate or adriamycin. However, adriamycin altered the cytoplasmic distribution of keratin.     

Localization of beta-(1,3)-glucanase in the mycelium of Sclerotium rolfsii.

The role of the lytic enzyme beta-(1,3)-glucanase in cell wall synthesis and its distribution in the mycelium of the fungus Sclerotium rolfsii were studied. Enzyme activity was determined after enzyme extraction with Triton X-100 from a cell wall preparation. Specific zones of immunofluorescence appeared in the hyphal tips, clamp connections, new septa, and lateral branching when a specific antiserum was used with the indirect method of the fluorescent antibody staining. Enzymatic activity in the cell wall preparation was inactivated by diethylpyrocarbonate. However, 69% of the total enzymatic activity was present in a latent form which was not affected by the ester. This result suggests that most of the beta-(1,3)-glucanase was present along the hyphal cell walls in a "masked" form. An active enzyme appeared only in those regions which showed immunofluorescence. The activity of glucan synthetase, an enzyme essential for wall formation, was higher in the branching funus grown on L-threonine-supplemented synthetic medium than in the synthetic medium-grown fungus.     

Lipopolysaccharide O antigen chains mask IcsA (VirG) in Shigella flexneri

Shigella flexneri 2a strain 2457T lipopolysaccharide (LPS) has O antigen (Oag) chains with two modal lengths (S-type and VL-type), and has IcsA apparently located at one pole on its cell surface. Treatment of Y serotype derivatives of 2457T and RMA696 (2457T wzz(SF)) with Sf6 tailspike protein (TSP) resulted in hydrolysis of Oag chains, and an increase in detection of IcsA by indirect immunofluorescence staining on both the lateral and polar regions of the cell surface. Newly synthesised IcsA expressed from a pBAD promoter in a S. flexneri Y strain was also detected on both the lateral and polar regions of the cell when incubated with TSP prior to immunofluorescence staining. We conclude that IcsA is actually located on both lateral and polar regions of the S. flexneri cell surface, and that LPS Oag chains mask the presence of IcsA by hindering its detection with antibodies. These results have implications for the mechanism of IcsA export. They suggest that while IcsA export is predominantly targeted to the old cell pole, it can also occur on the lateral regions of the cell surface.     

Expression of the rat CD26 antigen (dipeptidyl peptidase IV) on subpopulations of rat lymphocytes.

The T cell activation antigen CD26 has been recently identified as the cell surface ectopeptidase dipeptidyl peptidase IV (DPP-IV). DPP-IV is found on many cell types, including lymphocytes, epithelial cells, and certain endothelial cells. The MRC OX61 monoclonal antibody (MAb) which specifically recognises rat DPP-IV was used to examine the expression of CD26/DPP-IV on rat lymphocytes. The molecular nature of the antigen was examined by immunoprecipitation from thymocytes, splenocytes, and hepatocytes. Analysis by one- and two-dimensional gel electrophoresis indicated that the native form of CD26 includes a 220-kDa homodimer. On tissue sections MRC OX61 MAb stained nearly all thymocytes and in the spleen and lymph nodes predominantly stained the T cell areas. However, in immunofluorescence experiments OX61 stained 80 to 87% of lymph node cells and 78 to 85% of spleen cells. Furthermore, two-colour immunofluorescence analysis of the CD4+, CD8+, and Ig+ lymphocyte subsets indicated that only 2 to 5% of each of these subsets lacked OX61 staining. Spleen cells and thymocytes of both CD4+ and CD8+ subsets stained much more intensely with OX61 after these cells were stimulated with phytohemagglutinin. These findings indicate that rat CD26 antigen expression is not confined to the T cell population as has been suggested, but also occurs on B cells, and is increased on T cells following their activation.     

Evolution of the leaf economics spectrum in herbs: Evidence from environmental divergences in leaf physiology across Helianthus (Asteraceae)

The leaf economics spectrum (LES) describes a major axis of plant functional trait variation worldwide, defining suites of leaf traits aligned with resource‐acquisitive to resource‐conservative ecological strategies. The LES has been interpreted to arise from leaf‐level trade‐offs among ecophysiological traits common to all plants. However, it has been suggested that the defining leaf‐level trade‐offs of the LES may not hold within specific functional groups (e.g., herbs) nor within many groups of closely related species, which challenges the usefulness of the LES paradigm across evolutionary scales. Here, we examine the evolution of the LES across 28 species of the diverse herbaceous genus Helianthus (the sunflowers), which occupies a wide range of habitats and climate variation across North America. Using a phylogenetic comparative approach, we find repeated evolution of more resource‐acquisitive LES strategies in cooler, drier, and more fertile environments. We also find macroevolutionary correlations among LES traits that recapitulate aspects of the global LES, but with one major difference: leaf mass per area is uncorrelated with leaf lifespan. This indicates that whole‐plant processes likely drive variation in leaf lifespan across Helianthus, rather than leaf‐level trade‐offs. These results suggest that LES patterns do not reflect universal physiological trade‐offs at small evolutionary scales.     

Lysophosphatidylcholine sensitizes lipid extracts of pulmonary surfactant to inhibition by serum proteins

Interactions between serum protein and lysophospholipid inhibitors of pulmonary surfactant were examined in vitro using a pulsating bubble surfactometer. In previous studies a particular batch of Lipid Extract Surfactant (LES) was observed to be unusually sensitive to inhibition by fibrinogen. This sample was found to contain an abnormally high concentration of lysophosphatidylcholine (lysoPC). Addition of exogenous lysophospholipid to LES at similar concentrations sensitized the surfactant to inhibition by fibrinogen. Sensitization to inhibition by lysoPC is also observed with fetal bovine serum. Under the conditions used, inhibition by bovine serum albumin was not affected. Whereas only small amounts of lysoPC (1 mol% added) maximally sensitize LES to inhibition by fibrinogen, co-addition of equal amounts of palmitic acid can partially offset this effect at low lysoPC concentrations (less than 5 mol%). Lipid Extract Surfactant was digested with phospholipase A2 to mimic the generation of endogenous lysoPC at the expense of surfactant lipids. Digestion of 2-3% of the phosphatidylcholine to lysophosphatidylcholine vastly sensitized the surfactant to inhibition by fibrinogen. These results suggest that the degradation of surfactant phospholipids by phospholipase A2 to lysophospholipids could contribute to the development and progression of adult and neonatal respiratory distress syndromes.     

Introduction of T cell receptor (TCR)-alpha cDNA has differential effects on TCR-gamma delta/CD3 expression by PEER and Lyon-1 cells

A TCR heterodimer composed of a TCR gamma-chain and a TCR delta-chain was found to be expressed in association with CD3 by a small population of human peripheral blood T cells, thymocytes, and certain leukemic T cell lines. The leukemic T cell lines PEER and Lyon-1 express such a TCR-gamma delta/CD3 complex at the cell surface. In addition, PEER and Lyon-1 cells transcribe a productively rearranged TCR-beta gene. Introduction of TCR alpha-chain cDNA of human or murine origin resulted in cell surface expression of a TCR-alpha beta/CD3 complex on PEER and Lyon-1 cells. The expression of the TCR-gamma delta/CD3 complex on PEER cells was not affected by introduction of TCR-alpha cDNA. In contrast, introduction of a TCR-alpha cDNA and expression of the TCR-alpha beta/CD3 complex in Lyon-1 cells resulted in the disappearance of the TCR-gamma delta/CD3 complex. These data were confirmed by indirect immunofluorescence, at the protein level and by gene expression analysis. Triggering of the TCR-alpha beta/CD3 complexes by anti-CD3 mAb or anti-TCR mAb resulted in increased internal Ca2+ levels, indicating that these receptors were functional in signal transduction. These results indicate that, besides TCR gene rearrangements, membrane expression of TCR-alpha beta heterodimers may be important in regulating TCR-gamma delta cell surface expression.     

Branchial chloride cells in sea bass (Dicentrarchus labrax) adapted to fresh water,seawater, and doubly concentrated seawater

Branchial chloride cells (CC) were studied in sea bass (Dicentrarchus labrax) maintained in seawater (SW: 35 per thousand) or gradually adapted to and subsequently maintained in fresh water (0.2 per thousand) or doubly concentrated seawater (DSW: 70 per thousand). Changes were observed in the location, number, and structure of CCs, that were discriminated by light, scanning, and transmission electron microscopy, as well as by immunofluorescence on the basis of their high Na(+)/K(+)-ATPase antigen content. The number of CCs increased in both fresh water and doubly concentrated seawater compared to control fish maintained in SW. In both experimental conditions, these cells were found on the gill filament (as in control fish) and even on the lamellae, especially in hypersaline conditions. Structural changes concerned the shapes and sizes of CCs and their apical outcrops and particularly the structures of their functional complexes (mitochondria, tubular system, and endoplasmic reticulum), which developed significantly in DSW adapted fish. The changes in the expression of the Na(+)/K(+)-ATPase were evaluated by assessing the enzyme's density at the ultrastructural level following immunogold labeling. This parameter was significantly higher in doubly concentrated seawater. The adaptative significance of the quantitative and morphofunctional changes in branchial chloride cells is discussed in relation to the original osmoregulatory strategy of this marine euryhaline teleost.     

Comparison of angiotensin II (Ang II) effects in the internal anal sphincter (IAS) and lower esophageal sphincter smooth muscles

Studies were performed to compare the actions of Ang II in the internal anal sphincter (IAS) vs. lower esophageal sphincter (LES) smooth muscles in vitro, in opossum and rabbit. Studies also were carried out in isolated smooth muscle cells. In opossum, Ang II produced no discernible effects in the IAS, but did produce a concentration-dependent contraction in the LES. Conversely, in the rabbit, while Ang II caused a modest response in the LES, it caused a significant contraction in the IAS. The contractile responses of Ang II in the opossum LES were mostly resistant to different neurohumoral antagonists but were antagonized by AT1 antagonist losartan. AT2 antagonist PD 123,319, rather than inhibiting, prolonged the contractile action of Ang II. The contractile actions of Ang II in the opossum LES were not modified by the tyrosine kinase inhibitors (genistein and tyrphostin 1 x 10(-6) M) but were partially attenuated by the PKC inhibitor H-7 (1 x 10(-6) M), Ca2+ channel blocker nicardipine (1 x 10(-5) M), Rho kinase inhibitor HA-1077 (1 x 10(-7) M) or p(44/42) MAP kinase inhibitor PD 98059 (5 x 10(-5) M). The combination of HA-1077 and H-7 did not cause an additive attenuation of Ang II responses. Western blot analyses revealed the presence of both AT1 and AT2 receptors. We conclude that Ang lI-induced contraction of sphincteric smooth muscle occurs primarily by the activation of AT1 receptors at the smooth muscle cells and involves multiple pathways, influx of Ca2+, and PKC, Rho kinase and p(44/42) MAP kinase.     

Role of pp60(c-src) and p(44/42) MAPK in ANG II-induced contraction of rat tonic gastrointestinal smooth muscles

We examined the role of mitogen-activated protein kinase (p(44/42) MAPK) in ANG II-induced contraction of lower esophageal sphincter (LES) and internal anal sphincter (IAS) smooth muscles. Studies were performed in the isolated smooth muscles and cells (SMC). ANG II-induced changes in the levels of phosphorylation of different signal transduction and effector proteins were determined before and after selective inhibitors. ANG II-induced contraction of the rat LES and IAS SMC was inhibited by genistein, PD-98059 [a specific inhibitor of MAPK kinases (MEK 1/2)], herbimycin A (a pp60(c-src) inhibitor), and antibodies to pp60(c-src) and p(120) ras GTPase-activating protein (p(120) rasGAP). ANG II-induced contraction of the tonic smooth muscles was accompanied by an increase in tyrosine phosphorylation of p(120) rasGAP. These were attenuated by genistein but not by PD-98059. ANG II-induced increase in phosphorylations of p(44/42) MAPKs and caldesmon was attenuated by both genistein and PD-98059. We conclude that pp60(c-src) and p(44/42) MAPKs play an important role in ANG II-induced contraction of LES and IAS smooth muscles.     

Widespread cellular distribution of MAP-1A (microtubule-associated protein 1A) in the mitotic spindle and on interphase microtubules

In the accompanying paper (Bloom, G.S., T.A. Schoenfeld, and R.B. Vallee, 1983, J. Cell Biol. 98:320-330), we reported that microtubule-associated protein 1 (MAP 1) from brain comprises multiple protein species, and that the principal component, MAP 1A, can be detected in both neuronal and glial cells by immunofluorescence microscopy using a monoclonal antibody. In the present study, we sought to determine the cellular and subcellular distribution of MAP 1A in commonly used cultured cell systems. For this purpose we used immunofluorescence microscopy and immunoblot analysis with anti-MAP 1A to examine 18 types of mammalian cell cultures. MAP 1A was detected in every culture system examined. Included among these were cells of mouse, rat, Chinese hamster, Syrian hamster, Potoroo (marsupial), and human origin derived from a broad variety of tissues and organs. Anti-MAP 1A consistently labeled mitotic spindles and stained cytoplasmic fibers during interphase in most of the cultures. These fibers were identified as microtubules by co-localization with tubulin in double-labeling experiments, by their disappearance in response to colchicine or vinblastine, and by their reorganization in response to taxol. The anti-MAP 1A stained microtubules in a punctate manner, raising the possibility that MAP 1A is located along microtubules at discrete foci that might represent sites of interaction between microtubules and other organelles. Verification that MAP 1A was, indeed, the reactive material in immunofluorescence microscopy was obtained from immunoblots. Anti-MAP 1A stained a band at the position of MAP 1A in all cultures examined. These results establish that MAP 1A, a major MAP from brain, is widely distributed among cultured mammalian cells both within and outside of the nervous system.