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PubMedDate not supplied

Suppression of chemiluminescence of eastern oyster (Crassostrea virginica) hemocytes by the protozoan parasite Perkinsus marinus.

Experiments were conducted to determine the ability of the protistan parasite, Perkinsus marinus, to inhibit chemiluminescence of hemocytes from the eastern oyster, Crassostrea virginica. Luminol-enhanced chemiluminescence (CL) was used to measure the production of reactive oxygen intermediates (ROI) generated by oyster hemocytes using zymosan as a stimulant. To determine whether P. marinus suppresses ROI evoked from zymosan-stimulated hemocytes, live or heat killed P. marinus in filtered estuarine water (YRW) (salinity = 20 ppt) were added to (1) zymosan-stimulated hemocytes after CL reached its peak, or (2) hemocytes at the same time as zymosan, and reduction of CL responses were recorded. In both tests, controls received only estuarine water. Live P. marinus meronts significantly suppressed ROI production by zymosan-stimulated hemocytes. The suppression of ROI production was dose dependent. Suppression of ROI production from zymosan-stimulated hemocytes by heat killed P. marinus was significantly less than by live P. marinus. Similarly, CL of hemocytes was reduced, though not significantly when hemocytes were exposed to YRW preincubated with P. marinus. When P. marinus meronts were used as a stimulant, no CL response was elicited. Results of this study suggest that P. marinus cells are able to suppress ROI release from oyster hemocytes, thus evading this component of the host's defense.

Animals↗
PubMedDate not supplied

In vitro interaction of Perkinsus marinus merozoites with eastern and Pacific oyster hemocytes.

This study compared hemocyte responses of eastern and Pacific oysters to Perkinsus marinus, in vitro. Except for the percentage of hemocytes associated with P. marinus there was little or no significant difference between eastern and Pacific oysters with regard to their hemocytic response to P. marinus. In phagocytosis assays, merozoites were bound to all hemocyte types but in unequal proportions, unlike zymosan which was found predominantly associated with granulocytes. The number of merozoites enlarging in Ray's fluid thioglycollate medium after incubation with hemocytes in plasma for one day was significantly lower than after incubation in plasma alone in both oyster species. Electron microscopy or merozoites indicated that the parasites were rapidly phagocytosed and that some of the merozoites showed signs of degeneration in less than 12 h. The results suggest that limited intracellular killing of P. marinus had occurred, but was probably not mediated by oxygen metabolites, since no increase in chemiluminescence was observed when hemocytes of either eastern or Pacific oysters were exposed to merozoites.

Animals↗
PubMed1979

A progress report on the status of a new disease of American cats: cytauxzoonosis.

A protozoon disease of domestic cats in the U.S.A. which resembled diseases of African ungulates caused by blood parasites of the family Theileridae and the genus Cytauxzoon was first described by scientists of the University of Missouri in 1976. The Plum Island Animal Disease Center of the U.S. Department of Agriculture (USDA) became involved in an effort to discover the relationship of the parasite to those of African species and to ascertain if the disease posed a threat to American livestock. More than 500 cats were infected and the disease studied experimentally by scientists of both institutions. Passage of the parasite was accomplished by parenteral administration of fresh or deep frozen blood or tissues from infected cats. With the exception of a single cat, immunized at PIADC, the experimental disease has terminated fatally about 20 days following exposure. Most cats died within 9-15 days after infection. Diagnosis of the disease is accomplished by study of blood smears, stained with Giemsa and by examination of sections from major visceral organs. Piroplasms are not always found in blood smears, but large masses of schizonts are always found in at least one of the following organs: lungs, spleen, liver or lymph nodes. Since the disease almost invariably has a fatal termination in cats, it is believed that the reservoir is in another species of animal. The new disease has now been reported from Missouri, Arkansas, Texas and Georgia, with new isolations of the parasite having been made in recent months.

Animals↗
PubMed1982

Lack of reactivity of sera from Theileria parva-infected and recovered cattle against cell membrane antigens of Theileria parva transformed cell lines.

Sera from Theileria parva infected, recovered and rechallenged cattle were tested in complement-dependent cytotoxicity, membrane immunofluorescence and antibody-dependent cellular cytotoxicity assays for the presence of antibodies against cell membrane antigens of T. parva transformed cell lines. In the complement-dependent antibody-mediated cytotoxicity assay, sera from lethally infected animals were negative. Some recovered cattle showed a positive reaction, but such reactions were also observed when an eland cell line infected with T. taurotragi, and bovine lymphoblastoid cells were used as targets. Reaction was less against Ig-negative peripheral blood lymphocytes. Evidence is presented that these reactions could be evoked by attachment of immune complexes to Fc-receptors. It is concluded that cattle exposed to T. parva infection do not develop antibodies against specific T. parva (or T. parva-induced) cell surface antigens.

Animals↗
PubMed1983

Complement activation and fibrinolysis during infection with Theileria parva (East Coast fever) in cattle.

The role played by complement and fibrinolysis in the pathogenesis of pulmonary oedema and the disseminated, subendothelial, petechial haemorrhages in cattle infected with Theileria parva was investigated. There was a marked reduction in total haemolytic complement in animals that died of this disease whereas the survivors showed only transient changes. The reduction in C3 levels in animals with severe disease, however, was equivalent to that in the survivors. Neither circulating soluble immune complexes nor free antibody against macroschizonts were detected in sera from cattle with severe infection. In addition to the complement changes, high levels of fibrinogen degradation products (FDPs) were detected in sera from the infected animals. The rate of production of FDPs and reduction in total haemolytic complement paralleled the clinical course of the disease. It is, therefore, suggested that the pulmonary oedema and disseminated petechial haemorrhages seen in East Coast fever may be the direct result of the combined effects of complement activation and consumption of fibrinogen and fibrin.

Animals↗
PubMed1985

Specific antibodies to Besnoitia besnoiti precipitated from serum of cattle by live parasites and by soluble antigen.

Specific precipitable antibodies of both IgG and IgM classes were detected in sera of cattle naturally infected with B. besnoiti. The amount of specific antibodies of the IgG class precipitated by soluble antigen was in the range of 17-50 micrograms/ml serum while that of the IgM class ranged between 5 and 24 micrograms/ml serum. Specific antibodies precipitated by live B. besnoiti parasites were in amounts of 10 to 22 micrograms/ml serum for IgG and 4 to 26 micrograms/ml serum for IgM. Different ratios of IgG/IgM were obtained by the two methods of precipitation. This might indicate that antibodies to B. besnoiti of the IgM class can be precipitated and detected in sera of naturally infected cattle in similar amounts either by live parasites or by solubilized antigen, whereas antibodies of the IgG class can be preferentially detected when solubilized antigen is used for precipitation.

Animals↗
PubMed1986

The immune response of a marine teleost, Pseudopleuronectes americanus, (winter flounder) to the protozoan parasite Glugea stephani.

G. stephani is an intracellular cyst-forming microsporidan parasite that is found in the intestine of winter flounder (WF) Pseudopleuronectes americanus. No detectable humoral response was seen in parasitized fish or in fish injected with either spores or spore homogenate from this parasite. Quantification of total immunoglobulin (Ig) levels showed a decrease in Ig levels rather than enhancement, 21 days after intramuscular (IM) injections of spores (3 X 10(6)/ml). When a second injection of spores was administered on day 21 and tested 3 weeks later, a further decrease in total serum Ig's occurred. A decrease in total IgM levels also occurred in WF that were simultaneously injected with G. stephani and the antigens, horse red blood cells (HRBC) or formalin-killed Klebsiella pneumonia (KP). The total Ig levels of fish injected with an antigen plus spores was not as low as those injected with the parasite alone. The Ig levels, as well as antibody titers to HRBC and KP were however, lower when compared to fish injected only with the HRBC or bacteria. Disrupted spore homogenate injected into winter flounder, showed a less marked decrease in Ig levels when compared with whole spores. When a single IM injection of spores was given, followed by two weekly injections of indomethacin (a drug that inhibits prostaglandin activity), no decrease in Ig levels occurred and levels were comparable to control (saline injected) fish.

Animals↗
PubMed1989

Infection of bovine monocyte/macrophage populations with Theileria annulata and Theileria parva.

Infection and transformation of cells of the bovine immune system by Theileria annulata and T. parva were compared. Preliminary experiments with mammary gland macrophages indicated that they were permissive to infection by T. annulata but only to a limited extent by T. parva. Further experiments involved several purified subpopulations of bovine cells including bovine monocytes, T cells and MHC class II positive and negative populations. These subpopulations were incubated with T. annulata or T. parva sporozoites in limiting dilution cultures. T. annulata preferentially infected macrophage type cells and also MHC class II positive cells, whereas the frequency of MHC class II negative cells infected by this parasite was negligible. T cells also showed a very low level of infection. In complete contrast, T. parva preferentially infected T cells and did not infect cells phenotypically defined as monocytes at all. These results suggested that class II expression was necessary for T. annulata infection and not necessary for, though not a barrier to T. parva infection. T. annulata infected cell lines all expressed class II molecules to varying degrees. Other available phenotypic markers were only expressed at very low levels or no longer expressed. The immunological significance of the different cell preferences and phenotypes of infected cell lines of T. annulata and T. parva is discussed.

Analysis of Variance↗
PubMed1991

Analysis of the reactivity of anti-bovine CD8 monoclonal antibodies with cloned T cell lines and mouse L-cells transfected with bovine CD8.

Mouse L-cells transfected with bovine CD8 and two Theileria parva-infected cloned T cell lines expressing bovine CD8 were used to screen the panel of ten monoclonal antibodies (mAbs) submitted to the workshop. Eight of the ten mAbs reacted with the transfectant and both the cloned T cell lines. However, two mAbs CC58 and BAT82A did not recognise the transfectant and only reacted with one of the T cell lines. Further biochemical studies indicated that the eight mAbs react with both homo- and heterodimeric forms of bovine CD8 whilst the two mAbs CC58 and BAT82A react with only heterodimeric forms. These data suggest that bovine DC8 is encoded by two genes as is the case in mouse and man.

Animals↗
PubMed1991

Cell surface phenotype of two cloned populations of bovine lymphocytes displaying non-specific cytotoxic activity.

Monoclonal antibodies specific for T cell differentiation antigens were tested on four cloned populations of lymphocytes derived from the peripheral blood mononuclear cells of an animal immunised with Theileria parva. The clones were defined functionally in terms of cytotoxic activity, MHC restriction and expression of messenger RNA for CD3 and T cell receptor (TCR). Two clones contained RNA transcripts for CD3, TCR-alpha and beta and were positive for CD2, CD5 and CD6; one of these was a typical CD4+ class II MHC-restricted non-cytotoxic clone while the other was a CD8+ class I MHC-restricted cytotoxic clone. By contrast, the remaining two clones had the characteristics of non-specific killer cells in that they exhibited moderate levels of non-MHC-restricted killing; they contained TCR-delta mRNA and a 1.2 kb truncated form of TCR-beta message, but they did not contain CD3 or TCR-alpha mRNA. One of these non-specific killer clones only expressed CD2 whereas the other clone only expressed CD8, but without the CD8 determinant recognised by monoclonal antibodies CC58 and BAT52. All four clones were negative for the WC1 antigen which is expressed on gamma/delta T lymphocytes.

Animals↗
PubMed1992

In vitro immune recognition of synthetic peptides from the Plasmodium falciparum CS protein by individuals naturally exposed to different sporozoite challenge.

The impact of duration and intensity of sporozoite challenge on the in vitro cell immune response to synthetic peptides of the circumsporozoite (CS) protein of Plasmodium falciparum was investigated in residents of a malaria endemic area in Burkina Faso (West Africa). Lymphocyte proliferation and interferon-gamma (IFN-gamma) production were used to assess immune recognition of synthetic peptides corresponding to the polymorphic Th2R and Th3R regions, to the conserved CS.T3 sequence and to NANP and degenerate NVDP repeats. Immune responses were measured in adults and children from a village where they received more than 100 sporozoite inoculations per year and in adults living in a town, exposed to a 10-100 times lower challenge. A lifetime intense exposure apparently increased the ability to proliferate in response to most peptides in the rural adults, who all produced antibodies to NANP repeats. Surprisingly, cell cultures from these subjects seldom contained appreciable levels of IFN-gamma. In the urban adults, possibly due to the moderate challenge they are exposed to, significant differences in the proliferative potentials of the peptides could be detected. The highest stimulation indices were obtained with the genetically unrestricted CS.T3 peptide. Remarkably, proliferative responses to Th2R and Th3R appeared to be correlated with the humoral response to the CS protein, indicating a T helper significance of the epitopes. The differing proliferative potential of the polymorphic epitopes in the urban adults suggests that polymorphism might delay the development of immune responsiveness under conditions of sporadic transmission. The children from the highly malarious village displayed the lowest proliferative scores, accompanied by a high prevalence of antibodies to NANP repeats. On the basis of these findings, the hypothesis is proposed that a pure B cell reactivity to NANP repeats could ontogenetically precede the mounting of a conventional T-B cooperative immune response.

Adult↗
PubMed1981

The enzymes of pyrimidine biosynthesis in a range of parasitic protozoa and helminths.

The activities of carbamoylphosphate synthase, aspartate transcarbamoylase, dihydroorotase, orotate phosphoribosyl transferase and orotidine-5'-phosphate decarboxylase, five of the six enzymes of pyrimidine biosynthesis, have been measured in Crithidia fasciculata, Trypanosoma cruzi, Leishmania major, Trichomonas vaginalis, Eimeria tenella, Toxoplasma gondii, Plasmodium berghei, Fasciola gigantica, Schistosoma mansoni, Hymenolepis diminuta, Nippostrongylus brasiliensis and Trichuris muris. The majority of organisms contained all five enzyme activities. However, in T. vaginalis only carbamoylphosphate synthetase activity and in E. tenella only orotate phosphoribosyl transferase and orotidine-5'-phosphate decarboxylase activities could be detected. It appears therefore that the ability to synthesise pyrimidines by the de novo route is probably both common and widespread amongst parasitic organisms.

Animals↗
PubMed1987

DNA probes detect genomic diversity in Theileria parva stocks.

Different stocks of Theileria parva were analysed for restriction fragment length polymorphisms by agarose gel electrophoresis, orthogonal-field-alternation gel electrophoresis (OFAGE) and Southern hybridization with DNA probes. Polymorphisms seen with DNA from purified piroplasms of different T. parva stocks, after digestion with restriction enzymes, were more clearly apparent with OFAGE than with standard agarose gel electrophoresis. Genomic differences between these theilerial parasites were investigated further using three DNA probes, which were selected from a genomic library of T. parva (Muguga) piroplasm DNA cloned in lambda gt11. All three clones hybridized to T. parva DNA in preparations from schizont-infected bovine lymphoblastoid cells and to DNA from intraerythrocytic piroplasms. These probes did not, however, hybridize under high stringency conditions to DNA prepared from uninfected bovine lymphoblasts, T. mutans piroplasms, or bovine lymphoblasts infected with T. annulata or T. taurotragi. The five Kenyan stocks of T. parva that were tested showed characteristic hybridization patterns with these DNA probes. Our results show that DNA probes can be used to distinguish selected stocks of T. parva by hybridization to DNA either from intraerythrocytic piroplasms taken from infected cattle, or from isolates of schizont-infected bovine lymphoblastoid cells that are maintained continuously in vitro.

Animals↗
PubMed1988

Theileria parva: genomic DNA studies reveal intra-specific sequence diversity.

Theileria parva parva piroplasm DNA was purified from 11 different infections of cattle with 6 different East African isolates of the parasite. Total DNA was also prepared from bovine lymphoblastoid cells infected with schizonts of one of the isolates. Two of the infections were with cloned parasites. The DNA was of high molecular weight and free from protein and RNA, but some of the samples contained a proportion of bovine DNA. The 6 samples least contaminated with bovine DNA had a mean 'melting' temperature (Tm) of 84 degrees C and a mean GC content of 31.3%. Reassociation kinetics gave an estimate of 1.2 x 10(7) base pairs for the genome of T. parva. Repetitive restriction fragments were cloned from two samples, separated from the recombinant vectors and used as probes to demonstrate RFLPs between isolates. Discrimination into five groups was achieved. Schizont and piroplasm DNAs from the same isolate gave identical RFLPs, and one of the cloned parasites appeared to be a sub-population selected from a mixed-infection field isolate. Comparison of RFLPs with monoclonal antibody profiles suggested that neither technique yet provides discrimination between all the isolates which may comprise a strain. The importance of DNA probes for studying the epidemiology of theileriosis and for control programs is discussed.

Animals↗
PubMed1989

Isolation and characterization of RNA from the intracellular parasite Theileria parva.

Using a rapid procedure to isolate schizonts of the intracellular parasite Theileria parva from infected bovine lymphocytes, we have prepared parasite RNA that is more than 90% pure. Characterization of this schizont RNA has revealed the presence of two ribosomal RNA species of 3.3 kb and 1.8 kb and a third non-adenylated abundant RNA species of 1.9 kb. In vitro translation of the isolated schizont mRNA has identified about 200 parasite specific polypeptides, only a few of which could be detected by translation of mRNA from infected host cells. By analysing the kinetics of liquid hybridization of schizont mRNA with its homologous complementary DNA the nucleotide sequence complexity of the abundant class of the parasite mRNA has been estimated to be 1.7 x 10(3) kb. Assuming a number average size of 2 kb per mRNA molecule this would represent 4000 transcripts for all abundance classes of the schizont mRNA. Using the same technique we estimate that approximately 10% of the mRNA isolated from infected lymphocytes were transcripts from the parasite genome. We conclude that the low number of parasite specific translation products in the mRNA from infected lymphocytes and the low number of parasite proteins detected in isolated schizonts reported previously is due to the low abundance of the parasite transcripts rather than a low number of expressed parasite genes.

Animals↗
PubMed1989

Characterization of surface polypeptides on different life-cycle stages of Theileria annulata.

We describe the characterisation of polypeptides located on the surface of Theileria annulata sporozoites, macroschizonts, piroplasms and infected lymphoblastoid cells using surface iodination techniques. The sporozoite stage exhibited a complex profile of surface polypeptides. However, using data from experiments with defined monoclonal antibodies, the sporozoite surface appeared to be composed of several distinct groups of related polypeptides. Analysis of the macroschizont detected seven surface polypeptides, while eight polypeptides were identified for the piroplasm stage. On the basis of molecular weight comparisons, one of the surface polypeptides appeared to be common to the sporozoite, macroschizont and piroplasm. Stage cross-reactive monoclonals failed to immunoprecipitate a surface-radiolabelled polypeptide, and this prohibited the characterisation of a stage common surface antigen. From the surface labelling studies of Theileria-infected and uninfected lymphoblastoid cell lines, we concluded that infection results in major changes at the surface of the host cell, including both the appearance and loss of specific polypeptides. By employing monoclonal antibodies which detect infection-associated determinants, and a polyclonal antiserum raised against a glycoprotein fraction of an infected cell lysate, surface-labelled polypeptides were specifically immunoprecipitated from extracts of infected cells. The polypeptide detected by monoclonal antibody 4H5 was characterised as an infection-associated glycoprotein which varies in molecular mass when immunoprecipitated from different infected cell lines. The identification of infection-associated glycoproteins on the surface of the lymphoblastoid cell suggests that these molecules may be recognised by the cytotoxic T cells of immune animals.

Animals↗
PubMed1989

Sequence and expression of a gene from Theileria annulata coding for a 70-kilodalton heat-shock protein.

A library consisting of randomly sheared Theileria annulata genomic DNA fragments in the vector lambda gt11 was screened with a Drosophila DNA segment containing the coding region of the 70-kDa heat-shock protein (hsp) gene. A positive recombinant was isolated and subjected to nucleotide sequence analysis. The DNA segment contains an open reading frame coding for a 71-kDa protein strongly homologous to hsp 70 from other organisms. Using this DNA as a probe, a homologous 2.5-kb RNA species was detected in sporozoites, piroplasms and a macroschizont-infected cell line, showing that this gene is constitutively expressed. The amount of this RNA increased following heat shock in the macroschizont-infected cell line. The T. annulata genome contains other sequences that hybridize weakly with this heat-shock gene.

Amino Acid Sequence↗