PubMed Health⌕ Search

Biomedical subjects

J M Wastling

Publications and source records attributed to J M Wastling.

At least 19 recordsLinked to original sources
PubMed2006

A preliminary proteomic survey of the in vitro excretory/secretory products of fourth-stage larval and adult Teladorsagia circumcincta.

The nature of the proteins which comprise the in vitro excretory/secretory products (ES) of the fourth-stage larva (L4) and adult Teladorsagia circumcincta are largely undefined, despite the fact that this nematode induces profound changes, in part related to parasite ES, in the cellular architecture of the glands lining the abomasal surface of infected sheep and goats. In this study, the protein components of L4 and adult ES were fractionated using 1D gel electrophoresis and the major protein bands, detected by Coomassie blue staining, excised from the gel and subjected to tryptic digest and subsequent mass spectrometric analysis. The resultant peptide mass fingerprints were used to identify 15 L4 and 13 adult ES proteins. Several proteins, such as globin and some metabolic enzymes, were present in both ES. L4 ES alone contained thioredoxin peroxidase, an enzyme that can detoxify free radicals resulting from host inflammatory responses to the parasite, a cysteine proteinase which may aid penetration of the gastric mucosa and 2 different galectins which may influence cell differentiation and morphogenesis. Adult ES contained a nucleoside diphosphate kinase homologue, an enzyme which has been linked to cellular changes and can affect liquid secretion and goblet cell degranulation.

Animals↗
PubMed2004

Characterization of a mitochondrion-like organelle in Cryptosporidium parvum.

Cryptosporidium parvum is a protozoan parasite that causes widespread diarrhoeal disease in humans and other animals and is responsible for large waterborne outbreaks of cryptosporidiosis. Unlike many organisms belonging to the phylum Apicomplexa, such as Plasmodium spp. and Toxoplasma gondii, there is no clinically proven drug treatment against this parasite. Aspects of the basic biology of C. parvum remain poorly understood, including a detailed knowledge of key metabolic pathways, its genome organization and organellar complement. Previous studies have proposed that C. parvum lacks a relic plastid organelle, or 'apicoplast', but that it may possess a mitochondrion. Here we characterize a mitochondrion-like organelle in C. parvum by (i) ultrastructural and morphological description (ii) localization of heterologous mitochondrial chaperonin antibody probes (iii) phylogenetic analysis of genes encoding mitochondrial transport proteins (iv) identification and analysis of mitochondrion-associated gene sequences. Our descriptive morphological analysis was performed by energy-filtering transmission electron microscopy (EFTEM) of C. hominis and C. parvum. The 'mitochondrion-like' organelle was characterized by labelling the structure with a heterologous mitochondrial chaperonin probe (hsp60) both in immunoelectron microscopy (IMEM) and immunofluorescence (IMF). Phylogenetic analysis of the mitochondrial import system and housekeeping components (hsp60 and hsp70-dnaK) suggested that the C. parvum mitochondrion-like organelle is likely to have descended from a common ancestral apicomplexan mitochondrion. We also identified a partial cDNA sequence coding for an alternative oxidase (AOX) gene, a component of the electron transport chain which can act as an alternative to the terminal mitochondrial respiratory complexes III and IV, which has not yet been reported in any other member of this phylum. Degenerate primers developed to identify selected mitochondrial genes failed to identify either cytochrome oxidase subunit I, or cytochrome b. Taken together, our data aim to provide new insights into the characterization of this Cryptosporidium organelle and a logical framework for future functional investigation.

Amino Acid Sequence↗
PubMed2002

Characterisation of global protein expression by two-dimensional electrophoresis and mass spectrometry: proteomics of Toxoplasma gondii.

The development of tools for the analysis of global gene expression is vital for the optimal exploitation of the data on parasite genomes that are now being generated in abundance. Recent advances in two-dimensional electrophoresis (2-DE), mass spectrometry and bioinformatics have greatly enhanced the possibilities for mapping and characterisation of protein populations. We have employed these developments in a proteomics approach for the analysis of proteins expressed in the tachyzoite stage of Toxoplasma gondii. Over 1000 polypeptides were reproducibly separated by high-resolution 2-DE using the pH ranges 4-7 and 6-11. Further separations using narrow range gels suggest that at least 3000-4000 polypeptides should be resolvable by 2-DE using multiple single pH unit gels. Mass spectrometry was used to characterise a variety of protein spots on the 2-DE gels. Peptide mass fingerprints, acquired by matrix-assisted laser desorption/ionisation-(MALDI) mass spectrometry, enabled unambiguous protein identifications to be made where full gene sequence information was available. However, interpretation of peptide mass fingerprint data using the T. gondii expressed sequence tag (EST) database was less reliable. Peptide fragmentation data, acquired by post-source decay mass spectrometry, proved a more successful strategy for the putative identification of proteins using the T. gondii EST database and protein databases from other organisms. In some instances, several protein spots appeared to be encoded by the same gene, indicating that post-translational modification and/or alternative splicing events may be a common feature of functional gene expression in T. gondii. The data demonstrate that proteomic analyses are now viable for T. gondii and other protozoa for which there are good EST databases, even in the absence of complete genome sequence. Moreover, proteomics is of great value in interpreting and annotating EST databases.

Animals↗
PubMed2001

Genetic and biological diversity among isolates of Neospora caninum.

Neospora caninum is a protozoan parasite that causes bovine abortion. The epidemiology of N. caninum is poorly understood and little is known about the genetic diversity of the parasite, or whether individual isolates differ in virulence. Such diversity may, among other factors, underlie the range of pathologies seen in cattle. In this study we analysed biological and genetic variation in 6 isolates of N. caninum originating from canine and bovine hosts by measurement of growth rate in vitro, Western blotting and random amplification of polymorphic DNA (RAPD). This comparative analysis of intra-species diversity demonstrated that heterogeneity exists within the species. The relative growth rate in vitro, as assessed by 3[H]uracil uptake, showed significant variation between isolates. However, no significant differences were detected between the antigenic profiles of each isolate by Western blotting. RAPD-PCR was performed on DNA from the 6 Neospora isolates; 3 strains of Toxoplasma gondii, Sarcocystis sp. and Cryptosporidium parvum were also analysed. Twenty-six RAPD primers gave rise to 434 markers of which 222 were conserved between all the Neospora isolates and distinguished them from the other Apicomplexa. An additional 54 markers were unique for Neospora but were polymorphic within the species and able to differentiate between the individual isolates. The RAPD data were subjected to pair-wise similarity and cluster analysis and showed that the Neospora isolates clustered together as a group, with T. gondii as their nearest neighbour. N. caninum isolates showed no clustering with respect either to host or geographical origin. The genetic similarity between Neospora isolates from cattle and dogs suggests that these hosts may be epidemiologically related, although further analysis of bovine and canine field samples are required. The genetic and biological diversity observed in this study may have important implications for our understanding of the pathology and epidemiology of neosporosis.

Animals↗
PubMed2000

The prevalence of anti-Toxoplasma gondii antibodies in Ghanaian sheep and goats.

The enzyme-linked immunosorbent assay (ELISA) was used to detect anti-Toxoplasma gondii antibodies in 1258 small ruminants (732 sheep and 526 goats) sampled from 28 different locations in the three ecological zones of Ghana. The animals sampled had an overall seroprevalence of 30.5% (384 of the total). Sheep had a higher overall prevalence (33.2%) compared to the goats (26.8%). Animals sampled from the Coastal Savannah and the Forest zones had prevalences of 39.4% and 39.1%, respectively, which were significantly higher (P<0.01) than the prevalence recorded for the drier Guinea Savannah zone (20%). Prevalence of antibodies in female animals (35.8%) was significantly higher (P<0.01) than that for males (21.1%). Significant differences were also observed between breeds and age groups. The ELISA was found to be both highly sensitive (92%) and specific (91%) when compared to the IFAT, which was used as a reference test.

Age Factors↗
PubMed2000

The seroprevalence of toxoplasmosis in pigs in Ghana.

A serological survey of toxoplasmosis in pigs in Ghana was carried out between October 1997 and April 1998 in the three ecological zones of Ghana: the Coastal Savannah, the Forest Belt and the Guinea Savannah. Antibody against Toxoplasma gondii was measured in pig serum using a microplate-ELISA which had a sensitivity and specificity of 90.2 and 92.3%, respectively when compared with IFAT. A national seroprevalence of 39% was obtained in pigs, with the ecological distribution being 43.9, 30.5 and 42.5% for the Coastal Savannah, the Forest Belt and the Guinea Savannah, respectively. The age of the animal, the breed, the environmental conditions and the management practices appeared to be the major determinants of prevalence of antibodies against T. gondii. The prevalence of anti-T. gondii antibodies was found to increase with age (P<0.05). Pigs from the two Savannah zones had a significantly higher (P<0.05) antibody prevalence than those sampled from the Forest belt. Antibody prevalence (46.8%) in crossbreed pigs was significantly higher (P<0.05) than that of the exotic Large White breed (38.8%).

Age Factors↗
PubMed2000

The seroprevalence of antibodies to toxoplasma gondii in domestic goats in Uganda.

Only limited epidemiological information is available on the seroprevalence of Toxoplasma gondii in domestic livestock in sub-Saharan Africa. In Uganda, goats are important to the local economy and are also popular food animals. A high incidence of T. gondii infection in goats would have implications both for animal production and for public health, but no data is available on Toxoplasma infection in these animals. In this study we estimated the seroprevalence of antibodies against T. gondii in goats located in both urban and rural environments and from different geographical regions within Uganda. Goat sera were collected using a random, two-stage clustering method. Of 784 samples analysed by antibody-ELISA from various districts in Uganda, 240 tested positive. The combined (cluster-adjusted) seroprevalence was 0.31 (31%) (95% confidence intervals 0.28, 0.34) indicating a substantial level of infection in these regions. Seroprevalence was significantly higher in goats from urban locations. A strong positive relationship between age and seroprevalence was demonstrated and a mathematical model based on continuous exposure proved generally accurate in predicting seroprevalence. Farm environments were identified as being suitable for oocyst survival and transmission, and the reported incidence of caprine abortion was high. The importance of toxoplasmosis to goat production in Uganda has yet to be determined, but the high seroprevalence detected in this study suggests that it may have a significant impact and that the consumption of goat meat may play a role in zoonotic transmission to humans.

Animals↗
PubMed1999

Towards evaluating the economic impact of bovine neosporosis.

In spite of the global importance of neosporosis as a cause of bovine abortion, there is very little information about its economic consequences. The economic costs are a product of estimations of the quantity of the effects attributable to Neospora infection, and the particular unit costs of those effects. In this brief review, which arose from a workshop on the economics of coccidiosis held at the COST 820 meeting, Toledo 1998, we discuss the possible effects of neosporosis which are of economic significance and summarise the available estimates of their magnitude to provide a basis for further economic analysis. Neospora infection has been associated with abortion, increased culling and reduced milk yield. In addition, it has been diagnosed in cases of stillbirth and neonatal mortality, it is likely to contribute to early foetal death and resorption and it is responsible for a reduction in the value of female breeding cattle. In quantifying the role of Neospora, it is important that epidemiologically based, case-controlled studies are conducted because, given the extreme efficiency with which bovine Neospora infection is vertically transmitted, demonstration of prevalence of infection in affected animals (including foetuses) is not a true indicator of the significance of this disease. Relatively few epidemiological studies have been conducted, but in investigations in the USA, Holland and Britain, infected cows have been shown to be about three times more likely to abort than non-infected cattle. In the UK this approach has been used to estimate the proportion of abortions in the national dairy population which may be attributable to Neospora caninum.

Abortion, Veterinary↗
PubMed1999

Mucosal mast cell responses and release of mast cell protease-I in infections of mice with Hymenolepis diminuta and H. microstoma: modulation by cyclosporin A.

The dynamics of intestinal mucosal mast cells and the major mucosal mast cell protease were followed during the course of laboratory infections of mice with Hymenolepis diminuta and H. microstoma. The effects of the drug cyclosporin A (CsA), which is both immunosuppressive and selectively anthelmintic depending upon dose regime, were determined. In H. diminuta infections worm expulsion occurred around day 9 and coincided with peak mastocytosis and peak mMCP-I concentrations in tissues and serum. Immunosuppressive treatment with CsA prevented worm expulsion, permitting some individuals to reach maturity, and abrogated mast cell proliferation and mMCP-I production and release. By contrast, H. microstoma infections persisted for 64 days in spite of a considerable mastocyosis in both intestine and bile duct tissues accompanied by a high level of mMCP-I in tissues and serum. A subimmunosuppressive regime of CsA had only limited effects on worms and mast cell numbers and activity. Together these data shed light on the variable mast cell response to gastrointestinal infections and on the potential significance of parasite location in evasion of mast cell action. Use of CsA reveals the contributions of both T cell-dependent mechanisms, including mast cell proliferation and activation, and T cell-independent events in regulating intestinal helminth infections.

Animals↗
PubMed1998

Histochemical and ultrastructural modification of mucosal mast cell granules in parasitized mice lacking the beta-chymase, mouse mast cell protease-1.

The soluble beta-chymases mouse mast cell protease-1 (mMCP-1) and rat mast cell protease-II are predominantly expressed by intestinal mucosal mast cells (IMMCs) and may promote mucosal epithelial permeability when released during intestinal allergic hypersensitivity responses. To study the function of these chymases, we generated mice with a homozygous null mutation of the mMCP-1 gene and investigated their response to infection with the intestinal nematode Nippostrongylus brasiliensis. Whereas mMCP-2, -4, and -5 were transcribed normally, there was no transcription of the mMCP-1 gene in null (-/-) mice, nor was mature mMCP-1 protein detected in (-/-) jejunal mucosa. In contrast, levels of mMCP-1 in wild-type (+/+) jejunal mucosa increased 200- to 350-fold from 0.66 microg mMCP-1/g wet weight in uninfected mice to 129 and 229 microg/g wet weight on days 8 and 10 of infection, respectively. The kinetics of IMMC recruitment differed in -/- mice compared with +/+ controls on days 8 (P < 0.05) and 10 (P < 0.03) of infection. The IMMCs in infected -/- mice stained poorly, if at all, for esterase with naphthol AS-D chloroacetate compared with the intense staining observed in +/+ controls. Ultrastructurally, the prominent crystal intragranular structures that are found in intraepithelial +/+ IMMCs were absent from -/- IMMCs. These data show that disruption of the mMCP-1 gene leads to profound histochemical and ultrastructural changes in IMMC granules.

Animals↗
PubMed1997

Constitutive expression of mouse mast cell protease-1 in normal BALB/c mice and its up-regulation during intestinal nematode infection.

Rodent intestinal mucosal mast cells (IMMC) store and secrete soluble granule serine proteases, the beta-chymases, which may promote epithelial permeability during intestinal hypersensitivity reactions. The beta-chymase mouse mast cell protease-1 (mMCP-1) is generally considered to be expressed late in the in vitro differentiation of mast cells. The purpose of this study was to determine the kinetics of mMCP-1 transcription and expression in vivo during nematode-induced IMMC hyperplasia. Concentrations of mMCP-1 in blood and jejunum of BALB/c mice were quantified by enzyme-linked immunosorbent assay before and at various stages after infection with the intestinal nematode Nippostronglyus brasilliensis. Mature mMCP-1 enzyme was detected in jejunal homogenate (194 ng/mg soluble protein) and in blood (8.3 ng/ml serum) from normal uninfected BALB/c mice. Maximal IMMC hyperplasia occurred 7-14 days post infection and was significantly correlated with increased levels of mMCP-1 in jejunum (r = 0.58, P < 0.001) and with raised concentrations of mMCP-1 in serum (r = 0.66, P < 0.001). Transcription of the mMCP-1 gene was detected by RNA blotting in normal, uninfected jejunum, but transcription was up-regulated after infection with maximal transcription occurring on days 7 and 14. In conclusion, mMCP-1 transcription, storage and secretion occur constitutively in normal BALB/c jejunum but this basal secretion is up-regulated during nematode infection, suggesting both a physiological and pathological function for this protease.

Animals↗
PubMed1995

Kinetics of the local and systemic antibody response to primary and secondary infection with S48 Toxoplasma gondii in sheep.

Vaccination of sheep with live tachyzoites of Toxoplasma gondii, strain S48, affords protection against subsequent challenge with the parasite, but the mechanisms of immunity have not been fully determined. To understand better the nature of the antibody response the kinetics of both local and systemic antibody production were monitored in vaccinated sheep by means of an enzyme-linked immunosorbent assay and Western blotting. Local specific IgG production was analysed in efferent lymph obtained from the cannulated pre-femoral lymph node draining the site of infection. Antibody in efferent lymph plasma and peripheral blood serum from animals vaccinated with S48 tachyzoites was monitored and compared with IgG production in vaccinated sheep given a secondary tachyzoite challenge. Secondary challenge resulted in a clear immunological memory response, antibody being detected in the lymph 3 to 4 days after infection as compared with 7 to 8 days after a primary infection. IgG production was dominated by antibody recognizing a protein with an apparent molecular weight of 30 kDa, but other antigens (32, 24 and 11 kDa) were also readily detected.

Animals↗
PubMed1995

Induction of CD4+ and CD8+ T cell responses in efferent lymph responding to Toxoplasma gondii infection: analysis of phenotype and function.

The kinetics of induction of T cell responses were examined in efferent lymph from a node draining the site of a primary inoculation of Toxoplasma gondii. The numbers of T cells increased after infection, due initially to an expansion of the CD4+ T cell population followed by an increase in the number of CD8+ T cells which coincided with the peak lymphoblast response. Proliferative responses of CD4+ T cells to T. gondii antigen were detectable from day six after infection and immune efferent lymph cells inhibited the intracellular multiplication of T. gondii in vitro. Optimum inhibition was achieved using CD8+ T cells restimulated in vitro, and the effector function appeared to be directed preferentially against the autologous rather than the allogeneic infected target cell. The results provide unique information on the induction of immune responses to T. gondii in vivo and provide evidence that both CD4+ and CD8+ T cells are necessary for the development of protective immunity induced by the S48 strain of T. gondii which is used as a live vaccine in sheep.

Acute Disease↗