Studies on cell fusion between Babesia rodhaini-infected mouse erythrocytes and baby hamster kidney cells.

Heterokaryons were formed by fusion of B. rodhaini-infected mouse erythrocytes and baby hamster kidney (BHK) cells, using Sendai virus. The erythrocyte membrane rapidly lysed inside the BHK cell cytoplasm releasing free parasites. There was no evidence that parasite multiplication occurred inside the BHK cells, nor that parasitized BHK cells were infective for mice.Transient erythrocyte homokaryons were observed in some preparations.The approach indicates a possible method for the in vitro cultivation of Babesia.     

Theileria parva: Effects of irradiation on a culture of parasitized bovine lymphoid cells

Aliquots of a culture of Theileria parva-infected bovine lymphoid cells were irradiated at 0, 300, 600, 900, and 1200 rads. The short-term effects of irradiation were evaluated on examination of Giemsa-stained smears and on autoradiography of cells labeled with [³H]thymidine. Irradiation inhibited cell division but parasite division did not appear to be inhibited and macroschizont nuclear particles increased in number, frequently to several hundred per schizont. There was no evidence of an increased percentage switch from macro- to microschizont. Apparently viable cells were still present in all cultures 4 days after irradiation.     

Characterization of the Theileria parva sporozoite proteome

East Coast fever is a lymphoproliferative disease caused by the tick-borne protozoan parasite Theileria parva. The sporozoite stage of this parasite, harboured and released from the salivary glands of the tick Rhipicephalus appendiculatus during feeding, invades and establishes infection in bovine lymphocytes. Blocking this initial stage of invasion presents a promising vaccine strategy for control of East Coast fever and can in part be achieved by targeting the major sporozoite surface protein p67. To support research on the biology of T. parva and the identification of additional candidate vaccine antigens, we report on the sporozoite proteome as defined by LC–MS/MS analysis. In total, 4780 proteins were identified in an enriched preparation of sporozoites. Of these, 2007 were identified as T. parva proteins, representing close to 50% of the total predicted parasite proteome. The remaining 2773 proteins were derived from the tick vector. The identified sporozoite proteins include a set of known T. parva antigens targeted by antibodies and cytotoxic T cells from cattle that are immune to East Coast fever. We also identified proteins predicted to be orthologs of Plasmodium falciparum sporozoite surface molecules and invasion organelle proteins, and proteins that may contribute to the phenomenon of bovine lymphocyte transformation. Overall, these data establish a protein expression profile of T. parva sporozoites as an important starting point for further study of a parasitic species which has considerable agricultural impact.     

Absolute Quantification of the Host-To-Parasite DNA Ratio in Theileria parva-Infected Lymphocyte Cell Lines

Theileria parva is a tick-transmitted intracellular apicomplexan pathogen of cattle in sub-Saharan Africa that causes East Coast fever (ECF). ECF is an acute fatal disease that kills over one million cattle annually, imposing a tremendous burden on African small-holder cattle farmers. The pathology and level of T. parva infections in its wildlife host, African buffalo (Syncerus caffer), and in cattle are distinct. We have developed an absolute quantification method based on quantitative PCR (qPCR) in which recombinant plasmids containing single copy genes specific to the parasite (apical membrane antigen 1 gene, ama1) or the host (hypoxanthine phosphoribosyltransferase 1, hprt1) are used as the quantification reference standards. Our study shows that T. parva and bovine cells are present in similar numbers in T. parva-infected lymphocyte cell lines and that consequently, due to its much smaller genome size, T. parva DNA comprises between 0.9% and 3% of the total DNA samples extracted from these lines. This absolute quantification assay of parasite and host genome copy number in a sample provides a simple and reliable method of assessing T. parva load in infected bovine lymphocytes, and is accurate over a wide range of host-to-parasite DNA ratios. Knowledge of the proportion of target DNA in a sample, as enabled by this method, is essential for efficient high-throughput genome sequencing applications for a variety of intracellular pathogens. This assay will also be very useful in future studies of interactions of distinct host-T. parva stocks and to fully characterize the dynamics of ECF infection in the field.     

Theileria parva: significance of leukocytes for infecting cattle

Using an artificial feeding technique, infective particles of Theileria parva were harvested in bovine blood in capillary tubes from prefed female Rhipicephalus appendiculatus over a 2-hr period. Inoculations of this blood feed pool invariably resulted in the establishment of patent East Coast fever in autogeneic or syngeneic cattle, i.e., the blood donors or their monozygotic twins, but not in unrelated animals. Mechanical passage of 4 × 10⁶ macroschizont-infected lymphoid cells, harvested from a heifer with East Coast fever, successfully induced patent theileriosis in the donor's monozygotic twin but not in a susceptible allogeneic bovid. The significance of parasite-cell association and histocompatibility of infected cells is discussed in relation to natural and mechanical transmission of Theileria parva.     

STUDIES ON CELL ADHESION : III. Adhesion of Baby Hamster Kidney Cells

Normal and transformed baby hamster kidney (BHK) cells attach to Falcon polystyrene with the same first order rate constant. The longer the cells are attached to the bottles, the more difficult they are to remove. Sulfhydryl (—SH) binding reagents inhibit both the attachment of BHK cells and the increase in adhesive strength of attached cells. Attached BHK cells bind fewer molecules of [1-¹⁴C]N-ethylamleimide (an —SH binding reagent) than do suspended cells. Incubation of cells with high concentrations of trypsin results in a reversible loss of cell adhesiveness. The recovery of adhesiveness of trypsin-treated cells is inhibited by cycloheximide.     

Growth of <Emphasis Type="Italic">Theileria parva</Emphasis>-infected Bovine Lymphoid Cells in Irradiated Mice

SEVERAL attempts to establish Theileria parva, the causative organism of East Coast fever of cattle, in laboratory animals^1–4 have failed. The recent successful establishment of T. parva macroschizont-infected bovine lymphoid cells in tissue culture⁵ provided a concentrated source of material which could be used in further attempts to infect laboratory animals.     

Using the natural evolution of a rotavirus-specific human monoclonal antibody to predict the complex topography of a viral antigenic site

Background

Immunity against the bovine protozoan parasite Theileria parva has previously been shown to be mediated through lysis of parasite-infected cells by MHC class I restricted CD8⁺ cytotoxic T lymphocytes. It is hypothesized that identification of CTL target schizont antigens will aid the development of a sub-unit vaccine. We exploited the availability of the complete genome sequence data and bioinformatics tools to identify genes encoding secreted or membrane anchored proteins that may be processed and presented by the MHC class I molecules of infected cells to CTL.

Results

Of the 986 predicted open reading frames (ORFs) encoded by chromosome 1 of the T. parva genome, 55 were selected based on the presence of a signal peptide and/or a transmembrane helix domain. Thirty six selected ORFs were successfully cloned into a eukaryotic expression vector, transiently transfected into immortalized bovine skin fibroblasts and screened in vitro using T. parva-specific CTL. Recognition of gene products by CTL was assessed using an IFN-γ ELISpot assay. A 525 base pair ORF encoding a 174 amino acid protein, designated Tp2, was identified by T. parva-specific CTL from 4 animals. These CTL recognized and lysed Tp2 transfected skin fibroblasts and recognized 4 distinct epitopes. Significantly, Tp2 specific CD8⁺ T cell responses were observed during the protective immune response against sporozoite challenge.

Conclusion

The identification of an antigen containing multiple CTL epitopes and its apparent immunodominance during a protective anti-parasite response makes Tp2 an attractive candidate for evaluation of its vaccine potential.     

Fibroblast to substratum contacts mediated by the different forms of fibronectin

Highly purified preparations of fibronectin from bovine or hamster plasma and conditioned medium or urea extracts of BHK cells facilitate spreading of BHK cells to plastic surfaces. The different forms of fibronectin have very similar specific activities.     

Theileria parva: isolation of macroschizonts from in vitro propagated lymphoblastoid cells of cattle

A method for purifying macroschizonts of Theileria parva from bovine lymphoblastoid cells, propagated in vitro, was developed. This method involved three steps. First, the macroschizonts were liberated by disrupting host cells suspended in growth medium at 4 × 10⁶ cells/ml at 300–400 psi, using the Stansted cell disrupter. This yielded 80–90% disrupted cells while causing minimum damage to the macroschizonts. Second, the host cell nuclei were separated by (a) centrifuging the lysate at 300g for 60 min, (b) resuspending the pellet in 0.02 times the volume of initial host cell suspension in Leibovitz's L15 growth medium, and (c) lysing the host cell nuclei by adding nucleus-lysing buffer (NLB, containing 0.14 M Tris, 0.1 M HCl, 0.12 M glucose, and 0.5 M NaCl adjusted with NaOH to pH 7) to 0.2 times the volume of initial host cell suspension. The resulting chromatin precipitate was removed by adding DE-52 cellulose equilibrated with NLB and allowing the precipitate to sediment. Lastly, the final suspension obtained in the second step was applied on a DE-52 cellulose column which was equilibrated with the elution buffer (NLB with 10% fetal, or newborn, bovine serum, pH 7). Macroschizonts free of intact host cells and naked host cell nuclei were collected in the eluate. The protein yield was 2.7 mg per 10⁹ starting undisrupted host cells, which was 1.7% of the total starting protein.     

Effect of Serum on the Yield of Lymphocytic Choriomeningitis Virus in Baby Hamster Kidney Cells

The yields of the Armstrong and WE strains of lymphocytic choriomeningitis virus in baby hamster kidney (BHK) cells cultivated in either bovine, calf, fetal bovine, or horse serum were investigated. Lines of BHK cells were established in these sera. When the infected cell lines were observed by immunofluorescence, the per cent fluorescing cells for a given virus strain did not vary. However, for both strains, the extracellular virus yields per cell were significantly greater in the fetal bovine-cell line than in the other serum-cell lines.     

The incorporation of radio-labelled nucleic acid precursors by Theileria parva in bovine blood and salivary glands of Rhipicephalus appendiculatus ticks

Irvin A.D., Boarer C.D.H., Kurtti T.J. and Ocama J.G.R. 1981. The incorporation of radio-labelled nucleic acid precursors by Theileria parva in bovine blood and salivary glands of Rhipicephalus appendiculatus ticks. International Journal for Parasitology11:451–456. The uptake of radio-labelled nucleic acid precursors by blood and tick salivary gland forms of Theileria pana was studied. Piroplasms took up tritiated purines, particularly hypoxanthine, but not pyrimidines. Similar uptake was recorded by T. parva, both in tick saliva and in salivary glands maintained in vitro. Intermediate parasite stages were those most readily labelled in glands; this reflected active nucleic acid synthesis associated with rapid parasite division. Radio-labelling of T. parva in tick salivary glands could be of value in procedures used for concentrating and purifying theilerial sporozoites.     

Entamoeba histolytica: cytopathogenicity, including serum effects on contact-dependent and toxin-induced lysis of hamster kidney cell monolayers

Some properties of toxin, isolated from extracts of axenically cultivated Entamoeba histolytica or excreted by intact amoebae, were investigated using a toxicity assay in microplates with monolayers of baby hamster kidney cells. Preparative isoelectric focusing showed that the highest cytotoxic activity was present in a fraction of antigen containing protein bands with an isoelectric point between 4.5 and 5. Activity of the toxin was stable between pH 4 and 10. Nonimmune rabbit serum and concanavalin A, coupled to Sepharose beads, were able to bind a large amount of toxin. Cytotoxicity of antigen was inhibited by specific immune IgG and by unknown factors in nonimmune serum with a molecular weight between 50,000 and 100,000. The toxin was inactivated by trypsin, but not by trypsin inhibitor. Its activity was thiol dependent. Serum also had a marked inhibitory effect on contact lysis of BHK cells induced by intact trophozoites. A considerable reduction of both contactdependent and toxin-induced Cytopathogenicity was observed when Diamond's TP-S-1 medium was used in the assay, in which the TP broth had been autoclaved. It is suggested that Entamoeba histolytica exocytozes toxin, which acts on adjacent cells during close contact.     

New antifungal agents that inhibit the growth of Candida Species: Dichlorinated 8-quinolinols

Five dichlorinated 8-quinolinols (2,5- 5,6-, 3,5-, 3,7-, and 4,5-dichloro-8-quinolinol) were tested against Candida albicans and C. Tropicalis in Sabouraud dextrosebroth with and without bovine serum. The 5,6-, 3,5-, and 3,7-dichloro-8-quinolinols proved to be more effective than the control, 5-fluorocytosine. In cytotoxicity tests employing baby hamster kidney (BHK) cells, all test agents proved to be more cytotoxic than the control. However, the minimum inhibitory concentration (MIC) of 3,5-dichloro-8-quinolinol to both fungi was only one tenth the cytotoxic dose,suggesting that the compound may be useful as a topical or systemic antifungal agent.This revised version was published online in October 2005 with corrections to the Cover Date.     

A locus conferring tolerance to Theileria infection in African cattle

East Coast fever, a tick-borne cattle disease caused by the Theileria parva parasite, is among the biggest natural killers of cattle in East Africa, leading to over 1 million deaths annually. Here we report on the genetic analysis of a cohort of Bos indicus (Boran) cattle demonstrating heritable tolerance to infection with T. parva (h² = 0.65, s.e. 0.57). Through a linkage analysis we identify a 6 Mb genomic region on bovine chromosome 15 that is significantly associated with survival outcome following T. parva exposure. Testing this locus in an independent cohort of animals replicates this association with survival following T. parva infection. A stop gained variant in a paralogue of the FAF1 gene in this region was found to be highly associated with survival across both related and unrelated animals, with only one of the 20 homozygote carriers (T/T) of this change succumbing to the disease in contrast to 44 out of 97 animals homozygote for the reference allele (C/C). Consequently, we present a genetic locus linked to tolerance of one of Africa’s most important cattle diseases, raising the promise of marker-assisted selection for cattle that are less susceptible to infection by T. parva.     

The N-terminal region of the murine coronavirus spike glycoprotein is associated with the extended host range of viruses from persistently infected murine cells

Although murine coronaviruses naturally infect only mice, several virus variants derived from persistently infected murine cell cultures have an extended host range. The mouse hepatitis virus (MHV) variant MHV/BHK can infect hamster, rat, cat, dog, monkey, and human cell lines but not the swine testis (ST) porcine cell line (J. H. Schickli, B. D. Zelus, D. E. Wentworth, S. G. Sawicki, and K. V. Holmes, J. Virol. 71:9499-9507, 1997). The spike (S) gene of MHV/BHK had 63 point mutations and a 21-bp insert that encoded 56 amino acid substitutions and a 7-amino-acid insert compared to the parental MHV strain A59. Recombinant viruses between MHV-A59 and MHV/BHK were selected in hamster cells. All of the recombinants retained 21 amino acid substitutions and a 7-amino-acid insert found in the N-terminal region of S of MHV/BHK, suggesting that these residues were responsible for the extended host range of MHV/BHK. Flow cytometry showed that MHV-A59 bound only to cells that expressed the murine glycoprotein receptor CEACAM1a. In contrast, MHV/BHK and a recombinant virus, k6c, with the 21 amino acid substitutions and 7-amino-acid insert in S bound to hamster (BHK) and ST cells as well as murine cells. Thus, 21 amino acid substitutions and a 7-amino-acid insert in the N-terminal region of the S glycoprotein of MHV/BHK confer the ability to bind and in some cases infect cells of nonmurine species.     

Interactions of a mammalian beta-galactoside-binding lectin with hamster fibroblasts

A beta-galactoside-binding endogenous lectin extracted from bovine heart binds to the surface of baby hamster kidney (BHK) cells. The binding to and agglutination of cells is reduced in certain ricin-resistant mutants (Ric cells) in parallel with the decreased number of binding sites for the selective agent, ricin, a galactose-specific plant lectin. However, clear differences in the binding specificities of bovine lectin and ricin are shown by the effect of neuraminidase. BHK cells and Ric mutant cells treated with neuraminidase bind similar amounts of the bovine lectin compared with untreated cells, and ricin binding is greatly increased. The mammalian lectin immobilised on inert glass mediates the attachment and spreading of normal BHK cells and agglutinates these cells in solution. Ricin-resistant mutant cells respond poorly. These results are consistent with a role of endogenous lectins in cellular adhesiveness and show that cell adhesion may be regulated by the density of specific surface receptors for lectins.     

Occurrence of heat-dissociable ribosomal RNA in insects: the presence of three polynucleotide chains in 26 S RNA from cultured Aedes aegypti cells

When RNA extracted from a mixture of cultured mosquito (Aedes aegypti) and hamster (BHK) cells is heated at 60 °C for five minutes the 26 S mosquito RNA but not the 28 S BHK RNA is converted to 18 S products. These products are not separable from each other or from pre-existent 18 S RNA on 2.4% acrylamide gels and have molecular weights near 0.7 × 10⁶. The large ribosomal RNA from insects belonging to ten different orders shows a similar conversion, although this property is absent in two species of aphid.A. aegypti 26 S RNA dissociates over a narrow temperature range. The reaction equilibrium favours dissociation and is dependent on ionic strength, showing a 6 deg. C change in T_m′ (the temperature of 50% dissociation) with tenfold change in salt concentration. Although the T_m of 26 S RNA from Drosophila melanogaster and A. aegypti is markedly different, reflecting the difference in base composition, the T_m′ of the two RNA species was virtually the same.High molecular weight ribosomal RNA from Escherichia coli, BHK cells and A. aegypti cells was terminally labelled with [³H]isonicotinic acid hydrazide. The specific activities of the large RNA species show the presence of one, two and three polynucleotide chains in 23 S, 28 S and 26 S RNA, respectively. A. aegypti 26 S RNA contains a small, heat-dissociable “IRNA” similar in relative amount and mobility to that found in BHK cells.     

Erpobdella (Dina) parva complex (Annelida: Hirudinea: Arhynchobdellida: Erpobdellidae): additional description of Erpobdella parva, E. dubia, and E. lahontana and taxonomic revision

Erpobdella (Dina) parva complex (E. parva, E. dubia, and E. lahontana) were analyzed by species distribution, pigmentation, size, number and position of the labial eyes, tubercle presence, annulus width, gonopore position, and male genital atrium shape. I concluded that E. parva and E. dubia are conspecific, with the priority name of E. parva. The Lahontan basin species (E. lahontana), was found to be distinct by gonopore position, and now includes two additional basins (Granite Springs and Winnemucca). Endemism occurred in the Relict Dace basin complex (Butte and Steptoe basins) with respect to one form of gonopore positions. The variation in pigmentation, eye position and number, gonopore position, and male genital organ shape were highly variable in the Great Basin. Examination of the annular widths did not give any guidance to the taxonomic problem of the genera of this complex. It was noted that the Erpobdella parva complex did have some similarities with Nephelopsis obscura.     

Characterization of some East African Theileria species isolates by isoenzyme analysis, with particular reference to T. parva

Eight different isolates of Theileria parva and one isolate of T. taurotragi, in the form of intra-lymphocytic schizonts and/or purified piroplasms, were subjected to isoenzyme analysis for 24 enzymes by both isoelectric focusing in agarose and electrophoresis in starch gel. Twelve enzymes distinguished between T. parva and T. taurotragi; five enzymes (HK, GPI, PEP1, LDH and SOD) showed variations within T. parva. The metabolism of the host cell was affected by schizont infection, which masked intraspecific variations. Piroplasms were of more potential value for characterization of T. parva.