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Here, we report the first complete genomes of three cultivable treponeme species from bovine digital dermatitis (DD) skin lesions, two comparative human treponemes, considered indistinguishable from bovine DD species, and a bovine gastrointestinal (GI) treponeme isolate. Key genomic differences between bovine and human treponemes implicate microbial mechanisms that enhance knowledge of how DD, a severe disease of ruminants, has emerged into a prolific, worldwide disease. Bovine DD treponemes have additional oxidative stress genes compared to nearest human-isolated relatives, suggesting better oxidative stress tolerance, and potentially explaining how bovine strains can colonize skin surfaces. Comparison of both bovine DD and GI treponemes as well as bovine pathogenic and human non-pathogenic saprophyte Treponema phagedenis strains indicates genes encoding a five-enzyme biosynthetic pathway for production of 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, a rare di-N-acetylated mannuronic acid sugar, as important for pathogenesis. Bovine T. phagedenis strains further differed from human strains by having unique genetic clusters including components of a type IV secretion system and a phosphate utilisation system including phoU, a gene associated with osmotic stress survival. Proteomic analyses confirmed bovine derived T. phagedenis exhibits expression of PhoU but not the putative secretion system, whilst the novel mannuronic acid pathway was expressed in near entirety across the DD treponemes. Analysis of osmotic stress response in water identified a difference between bovine and human T. phagedenis with bovine strains exhibiting enhanced survival. This novel mechanism could enable a selective advantage, allowing environmental persistence and transmission of bovine T. phagedenis. Finally, we investigated putative outer membrane protein (OMP) ortholog families across the DD treponemes and identified several families as multi-specific adhesins capable of binding extra cellular matrix (ECM) components. One bovine pathogen specific adhesin ortholog family showed considerable serodiagnostic potential with the Treponema medium representative demonstrating considerable disease specificity (91.6%). This work has shed light on treponeme host adaptation and has identified candidate molecules for future diagnostics, vaccination and therapeutic intervention., Author summary Bovine digital dermatitis (DD) is a severe infectious disease causing cattle lameness, which is now endemic in many countries across the world. This lameness results from inflamed lesions between the heel bulbs and is very painful resulting in poor animal welfare and substantially reduced production. There remains no single cure for DD and whilst topical antibiotic treatment enables some healing, lesions frequently reappear. Current evidence implicates bacteria known as Treponema in the pathogenesis of DD. Here we characterise the genomes of several bovine DD treponeme species as well as related bacteria from humans and the bovine gastrointestinal tract. Comparative analyses demonstrate that production of a novel mannuronic acid sugar is a key feature of bovine pathogens and several survival mechanisms were identified which likely enable the bovine pathogens to inhabit the skin surface and be transmitted within the farm environment. Studies investigating putative outer membrane proteins which are potential vaccine candidates identified that the majority have a role in host attachment, with one family of proteins exhibiting particular promise as serodiagnostic antigens. This increased understanding of the considered causal pathogens of bovine DD, together with the genomic and proteomic resources produced by this study should underpin future diagnostic, vaccination and therapeutics studies to combat this severe disease of ruminants.

Despite the importance of bovine neosporosis, relevant knowledge gaps remain concerning the pathogenic mechanisms of Neospora caninum. Infection of the placenta is a crucial event in the pathogenesis of the disease, however, very little is known about the relation of the parasite with this target organ. Recent studies have shown that isolates with important variations in virulence also show different interactions with the bovine trophoblast cell line F3 in terms of proliferative capacity and transcriptome host cell modulation. Herein, we used the same model of infection to study the interaction of Neospora with these target cells at the proteomic level using LC-MS/MS over the course of the parasite lytic cycle. We also analysed the proteome differences between high- (Nc-Spain7) and low-virulence (Nc-Spain1H) isolates. The results showed that mitochondrial processes and metabolism were the main points of Neospora-host interactions. Interestingly, Nc-Spain1H infection showed a higher level of influence on the host cell proteome than Nc-Spain7 infection.

The identification and characterization of the function of proteins in Toxoplasma gondii has been a focus of many research groups since the development of techniques that enabled the study of individual genes and molecules. Proteomic studies provide complementary data to those obtained by transcriptional “omics” approaches. Proteomics data for T. gondii has also been helpful for improving genome annotation. Improved methods to purify subproteomes and the use of biotin identification (and similar techniques) are dramatically expanding our understanding of the structures formed by this organism and their relationship to its host cell. This chapter reviews the current status of proteomic studies on T. gondii and the role of various posttranslational modifications in the biology of this pathogen.

When transmitted through the oral route, Toxoplasma gondii first interacts with its host at the small intestinal epithelium. This interaction is crucial to controlling initial invasion and replication, as well as shaping the quality of the systemic immune response. It is therefore an attractive target for the design of novel vaccines and adjuvants. However, due to a lack of tractable infection models, we understand surprisingly little about the molecular pathways that govern this interaction. The in vitro culture of small intestinal epithelium as 3D enteroids shows great promise for modeling the epithelial response to infection. However, the enclosed luminal space makes the application of infectious agents to the apical epithelial surface challenging. Here, we have developed three novel enteroid-based techniques for modeling T. gondii infection. In particular, we have adapted enteroid culture protocols to generate collagen-supported epithelial sheets with an exposed apical surface. These cultures retain epithelial polarization, and the presence of fully differentiated epithelial cell populations. They are susceptible to infection with, and support replication of, T. gondii. Using quantitative label-free mass spectrometry, we show that T. gondii infection of the enteroid epithelium is associated with up-regulation of proteins associated with cholesterol metabolism, extracellular exosomes, intermicrovillar adhesion, and cell junctions. Inhibition of host cholesterol and isoprenoid biosynthesis with Atorvastatin resulted in a reduction in parasite load only at higher doses, indicating that de novo synthesis may support, but is not required for, parasite replication. These novel models therefore offer tractable tools for investigating how interactions between T. gondii and the host intestinal epithelium influence the course of infection.

© 2018, The Author(s). The in vitro 3D culture of intestinal epithelium is a valuable resource in the study of its function. Organoid culture exploits stem cells’ ability to regenerate and produce differentiated epithelium. Intestinal organoid models from rodent or human tissue are widely available whereas large animal models are not. Livestock enteric and zoonotic diseases elicit significant morbidity and mortality in animal and human populations. Therefore, livestock species-specific models may offer novel insights into host-pathogen interactions and disease responses. Bovine and porcine jejunum were obtained from an abattoir and their intestinal crypts isolated, suspended in Matrigel, cultured, cryopreserved and resuscitated. ‘Rounding’ of crypts occurred followed by budding and then enlargement of the organoids. Epithelial cells were characterised using immunofluorescent staining and confocal microscopy. Organoids were successfully infected with Toxoplasma gondii or Salmonella typhimurium. This 3D organoid model offers a long-term, renewable resource for investigating species-specific intestinal infections with a variety of pathogens.

Infectious bronchitis is a highly contagious respiratory disease of poultry caused by the coronavirus IBV. It was thought that coronavirus virions were composed of three major viral structural proteins, until investigations of other coronaviruses showed that coronavirus virions also include viral non-structural and group specific proteins as well as host cell proteins. To study the proteome of IBV virions, virus was grown in embryonated chicken eggs and purified by sucrose gradient ultracentrifugation and analysed by mass spectrometry proteomic. Analysis of three preparations of purified IBV yielded the three expected structural proteins plus thirty-five additional virion-associated host proteins. Virion-associated host proteins had a diverse range of functional attributions, being involved in cytoskeleton formation, RNA binding and protein folding pathways. Some of these proteins were unique to this study, whilst others were found to be orthologous to proteins identified in SARS-CoV virions, and also virions from a number of other RNA and DNA viruses. Together these results demonstrate that coronaviruses have the capacity to incorporate a substantial variety of host protein, which may have implications for the disease process.

Recently, three‐dimensional small intestinal organoids (enteroids) have been developed from cultures of intestinal stem cells which differentiate in vitro to generate all the differentiated epithelial cell types associated with the intestine and mimic the structural properties of the intestine observed in vivo. Small‐molecule drug treatment can skew organoid epithelial cell differentiation towards particular lineages, and these skewed enteroids may provide useful tools to study specific epithelial cell populations, such as goblet and Paneth cells. However, the extent to which differentiated epithelial cell populations in these skewed enteroids represent their in vivo counterparts is not fully understood. In this study, we have performed label‐free quantitative proteomics to determine whether skewing murine enteroid cultures towards the goblet or Paneth cell lineages results in changes in abundance of proteins associated with these cell lineages in vivo. Our data confirm that skewed enteroids recapitulate important features of the in vivo gut environment, confirming that they can serve as useful models for the investigation of normal and disease processes in the intestine. Furthermore, by comparison of our mass spectrometry data with histology data contained within the Human Protein Atlas, we identify putative novel markers for goblet and Paneth cells.

The parasitic protozoan Toxoplasma gondii becomes encysted in brain and muscle tissue during chronic infection, a stage that was previously thought to be dormant but has been found to be active and associated with physiological effects in the host. Dysregulation of catecholamines in the CNS has previously been observed in chronically-infected animals. In the study described here, the noradrenergic system was suppressed with decreased levels of norepinephrine in brains of infected animals and in infected neuronal cells in vitro. Expression of dopamine β-hydroxylase (DBH), essential for synthesis of norepinephrine from dopamine, was the most differentially-expressed gene in infections in vitro and was down-regulated in infected brain tissue, particularly in the prefrontal cortex and dorsal locus coeruleus/pons region. The down-regulated DBH expression in infected rat catecholaminergic and human neuronal cells corresponded with decreased norepinephrine and increased dopamine. As the DBH suppression was observed in vitro, this effect is not caused by neuroinflammation. Silencing of DBH expression was specific for T. gondii infection and was not observed with CMV infection. The noradrenergic-linked behaviors of sociability and arousal were altered in chronically-infected animals, with a high correlation between DBH expression and infection intensity. These findings together provide a plausible mechanism to explain prior discrepancies in changes to CNS neurotransmitters levels with infection. The suppression of norepinephrine synthesis observed here may, in part, explain behavioural effects of infection, associations with mental illness, and neurological consequences of infection such as the loss of coordination and motor impairments associated with human toxoplasmosis.

Toxoplasma gondii can grow and replicate using either glucose or glutamine as the major carbon source. Here, we have studied the essentiality of glycolysis in the tachyzoite and bradyzoite stages of T. gondii, using transgenic parasites that lack a functional hexokinase gene (Δhk) in RH (Type-1) and Prugniaud (Type-II) strain parasites. Tachyzoite stage Δhk parasites exhibit a fitness defect similar to that reported previously for the major glucose transporter mutant, and remain virulent in mice. However, although Prugniaud strain Δhk tachyzoites were capable of transforming into bradyzoites in vitro, they were severely compromised in their ability to make mature bradyzoite cysts in the brain tissue of mice. Isotopic labelling studies reveal that glucose-deprived tacyzoites utilise glutamine to replenish glycolytic and pentose phosphate pathway intermediates via gluconeogenesis. Interestingly, while glutamine-deprived intracellular Δhk tachyzoites continued to replicate, extracellular parasites were unable to efficiently invade host cells. Further, studies on mutant tachyzoites lacking a functional phosphoenolpyruvate carboxykinase (Δpepck1) revealed that glutaminolysis is the sole source of gluconeogenic flux in glucose-deprived parasites. In addition, glutaminolysis is essential for sustaining oxidative phosphorylation in Δhk parasites, while wild type (wt) and Δpepck1 parasites can obtain ATP from either glycolysis or oxidative phosphorylation. This study provides insights into the role of nutrient metabolism during asexual propagation and development of T. gondii, and validates the versatile nature of central carbon and energy metabolism in this parasite.

Background: Giardia is a protozoan parasite of public health relevance that causes gastroenteritis in a wide range of hosts. Two genetically distinct lineages (assemblages A and B) are responsible for the human disease. Although it is clear that differences in virulence occur, the pathogenesis and virulence of Giardia remain poorly understood. Results: The genome of Giardia is believed to contain open reading frames that could encode as many as 6000 proteins. By successfully applying quantitative proteomic analyses to the whole parasite and to the supernatants derived from parasite culture of assemblages A and B, we confirm expression of ∼1600 proteins from each assemblage, the vast majority of which are common to both lineages. To look for signature enrichment of secreted proteins, we considered the ratio of proteins in the supernatant compared with the pellet, which defined a small group of enriched proteins, putatively secreted at a steady state by cultured growing trophozoites of both assemblages. This secretome is enriched with proteins annotated to have N-terminal signal peptide. The most abundant secreted proteins include known virulence factors such as cathepsin B cysteine proteases and members of a Giardia superfamily of cysteine-rich proteins that comprise variant surface proteins, high-cysteine membrane proteins, and a new class of virulence factors, the Giardia tenascins. We demonstrate that physiological function of human enteric epithelial cells is disrupted by such soluble factors even in the absence of the trophozoites. Conclusions: We are able to propose a straightforward model of Giardia pathogenesis incorporating key roles for the major Giardia-derived soluble mediators. Publisher PDF

This protocol provides an efficient and standardized way to prepare samples ready for mass spec analysis.

This protocol provides an efficient and rapid way to concentrate proteins present in supernatants using Vivaspin columns (3,000 MWCO).

This protocol provides an efficient way to observe proteins in samples.

Methods for TransEpithelial Electrical Resistance (TEER) and short circuit current measurement of the effects of soluble mediator on intestinal epithelial cells.

This protocol provides an inexpensive and fast way to cleanse the supernatant of Giardia trophozoites' culture from bovine proteins and any other protein digests present in TYI-S-33.

Giardia is a protozoan parasite of public health relevance that causes gastroenteritis in a wide range of hosts. Two genetically distinct lineages (assemblages A and B) are responsible for the human disease. Although it is clear that differences in virulence occur, pathogenesis and virulence of Giardiaremainspoorly understood. The genome of Giardia is believed to contain ORFs which could encode as many as 6,000 proteins. By successfully applying quantitative proteomic analyses to the whole parasite and to the supernatants derived from parasite culture of assemblages A and B, weconfirmexpression of ~1,600 proteins from each assemblage, the vast majority of which being common to both lineages. To look for signature enrichment of secreted proteins, we considered the ratio of proteins in the supernatant compared with the pellet which defined a small group of enriched proteins, putatively secreted at a steady state by cultured growing trophozoites of both assemblages. This secretome is enriched with proteins annotated to have N-terminal signal peptide. The most abundant secreted proteins include known virulence factors such as cathepsin B cysteine proteases and members of a Giardia superfamily ofcysteine richproteins which comprises VSPs, HCMPs and a new class of virulence factors, the Giardia tenascins. We demonstrate that physiological function of human enteric epithelial cells is disrupted by such soluble factors even in the absence of the trophozoites. We are able to propose a straightforward model of Giardia pathogenesis incorporating key roles for the major Giardia derived soluble mediators.

Background: Giardiasis is a common intestinal infection caused by the flagellated intestinal protozoan Giardia duodenalis. Several methods are available for the laboratory diagnosis of Giardia, ranging from the microscopic identification of the parasite trophozoite and cyst stages, to immunodiagnosis and PCR. Giardia has unique metabolic pathways resulting from its lack of mitochondria, making it an ideal target for volatile organic compound (VOC) profiling. Aim: To characterise the VOC profile of stool infected with Giardia to detect differences from those found in samples of diarrhoea without Giardia or other infections. Method: Stool was obtained from patients with confirmed Giardia infection and controls with diarrhoea but no identifiable infection. Faecal headspace gas extraction and gas chromatography-mass spectrometry were used to extract and identify VOCs. Results: More than 100 VOCs were identified when control and Giardia groups were combined, of which 24 showed significant differences between the two groups (p

This article explores the nature of calls for risk-based policy present in expert discourse from a cultural theory perspective. Semi-structured interviews with professionals engaged in the research and management of livestock disease control provide the data for a reading proposing that the real basis of policy relating to socio-technical hazards is deeply political and cannot be purified through ‘escape routes’ to objectivity. Scientists and risk managers are shown calling, on the one hand, for risk-based policy approaches while on the other acknowledging a range of policy drivers outside the scope of conventional quantitative risk analysis including group interests, eventualities such as outbreaks, historical antecedents, emergent scientific advances and other contingencies. Calls for risk-based policy are presented, following cultural theory, as ideals connected to a reductionist epistemology and serving particular professional interests over others rather than as realistic proposals for a paradigm shift.

Toxoplasma gondii and Neospora caninum are closely related intracellular protozoan parasites and tissue cyst-forming Coccidia of the phylum Apicomplexa. There are remarkable similarities between the morphology, genomes and transcriptomes of both parasites. Toxoplasma is zoonotic, with a wide host range and is mainly transmitted horizontally between its definitive host, the cat, and its intermediate hosts. Neospora causes disease within a narrow host range and with reduced virulence potential to the hosts. The dog is the definitive host of Neospora and its epidemiology in cattle mainly depends on vertical transmission. What causes these biological differences is not well understood. Since these parasites secrete an array of secretory proteins, including kinases, during infection to manipulate host cell responses. Host-parasite interactions due to phosphorylation of host cell proteins by T. gondii kinases enhance virulence and maintenance of infection. In this study, proteome-wide phosphorylation events of host cell proteins were investigated in response to infection with T. gondii and N. caninum using phosphoproteomic analyses, followed by pathway analysis on host signalling pathways. A few interesting differences in host responses at both the qualitative and quantitative levels were identified between the two infections; about one third of the phosphoproteomes, approximately 21% of the phospho-motifs and several pathways such as glycolysis/gluconeogenesis and mTOR pathways of the host cell were found differentially enriched between infection with these parasites. Identifying the differences in host-parasite interactions represents a promising step forward for uncovering the biological dissimilarities between both parasites.

Neospora caninum is one of the main causes of transmissible abortion in cattle. Intraspecific variations in virulence have been widely shown among N. caninum isolates. However, the molecular basis governing such variability have not been elucidated to date. In this study label free LC-MS/MS was used to investigate proteome differences between the high virulence isolate Nc-Spain7 and the low virulence isolate Nc-Spain1H throughout the tachyzoite lytic cycle. The results showed greater differences in the abundance of proteins at invasion and egress with 77 and 62 proteins, respectively. During parasite replication, only 19 proteins were differentially abundant between isolates. The microneme protein repertoire involved in parasite invasion and egress was more abundant in the Nc-Spain1H isolate, which displays a lower invasion rate. Rhoptry and dense granule proteins, proteins related to metabolism and stress responses also showed differential abundances between isolates. Comparative RNA-Seq analyses during tachyzoite egress were also performed, revealing an expression profile of genes associated with the bradyzoite stage in the low virulence Nc-Spain1H isolate. The differences in proteome and RNA expression profiles between these two isolates reveal interesting insights into likely mechanisms involved in specific phenotypic traits and virulence in N. caninum. Significance The molecular basis that governs biological variability in N. caninum and the pathogenesis of neosporosis has not been well-established yet. This is the first study in which high throughput technology of LC-MS/MS and RNA-Seq is used to investigate differences in the proteome and transcriptome between two well-characterized isolates. Both isolates displayed different proteomes throughout the lytic cycle and the transcriptomes also showed marked variations but were inconsistent with the proteome results. However, both datasets identified a pre-bradyzoite status of the low virulence isolate Nc-Spain1H. This study reveals interesting insights into likely mechanisms involved in virulence in N. caninum and shed light on a subset of proteins that are potentially involved in the pathogenesis of this parasite.

The invasion of Theileria sporozoites into bovine leukocytes is rapidly followed by the destruction of the surrounding host cell membrane, allowing the parasite to establish its niche within the host cell cytoplasm. Theileria infection induces host cell transformation, characterised by increased host cell proliferation and invasiveness, and the activation of anti-apoptotic genes. This process is strictly dependent on the presence of a viable parasite. Several host cell kinases, including PI3-K, JNK, CK2 and Src-family kinases, are constitutively activated in Theileria-infected cells and contribute to the transformed phenotype. Although a number of host cell molecules, including IkB kinase and polo-like kinase 1 (Plk1), are recruited to the schizont surface, very little is known about the schizont molecules involved in host-parasite interactions. In this study we used immunofluorescence to detect phosphorylated threonine (p-Thr), serine (p-Ser) and threonine-proline (p-Thr-Pro) epitopes on the schizont during host cell cycle progression, revealing extensive schizont phosphorylation during host cell interphase. Furthermore, we established a quick protocol to isolate schizonts from infected macrophages following synchronisation in S-phase or mitosis, and used mass spectrometry to detect phosphorylated schizont proteins. In total, 65 phosphorylated Theileria proteins were detected, 15 of which are potentially secreted or expressed on the surface of the schizont and thus may be targets for host cell kinases. In particular, we describe the cell cycle-dependent phosphorylation of two T. annulata surface proteins, TaSP and p104, both of which are highly phosphorylated during host cell S-phase. TaSP and p104 are involved in mediating interactions between the parasite and the host cell cytoskeleton, which is crucial for the persistence of the parasite within the dividing host cell and the maintenance of the transformed state.

Viral pathogenesis in the infected cell is a balance between antiviral responses and subversion of host-cell processes. Many viral proteins specifically interact with host-cell proteins to promote virus biology. Understanding these interactions can lead to knowledge gains about infection and provide potential targets for antiviral therapy. One such virus is Ebola, which has profound consequences for human health and causes viral hemorrhagic fever where case fatality rates can approach 90%. The Ebola virus VP24 protein plays a critical role in the evasion of the host immune response and is likely to interact with multiple cellular proteins. To map these interactions and better understand the potential functions of VP24, label-free quantitative proteomics was used to identify cellular proteins that had a high probability of forming the VP24 cellular interactome. Several known interactions were confirmed, thus placing confidence in the technique, but new interactions were also discovered including one with ATP1A1, which is involved in osmoregulation and cell signaling. Disrupting the activity of ATP1A1 in Ebola-virus-infected cells with a small molecule inhibitor resulted in a decrease in progeny virus, thus illustrating how quantitative proteomics can be used to identify potential therapeutic targets.

UNLABELLED: Profiling of protein species is important because gene polymorphisms, splice variations and post-translational modifications may combine and give rise to multiple protein species that have different effects on cellular function. Two-dimensional gel electrophoresis is one of the most robust methods for differential analysis of protein species, but bioinformatic interrogation is challenging because the consequences of changes in the abundance of individual protein species on cell function are unknown and cannot be predicted. We conducted DIGE of soleus muscle from male and female rats artificially selected as either high- or low-capacity runners (HCR and LCR, respectively). In total 696 protein species were resolved and LC-MS/MS identified proteins in 337 spots. Forty protein species were differentially (P

Graphical abstract Highlights ► Treatment failure in toxoplasmosis implies the possibility of drug resistance. ► In vitro resistance to sulfadiazine detected in three strains of Toxoplasma gondii. ► Comparison of sensitive and sulfadiazine resistant strains by DIGE. ► Thirty one proteins differentially expressed between sensitive and resistant strains., Treatment options for toxoplasmosis in humans are generally limited to the use of sulfonamide and/or pyrimethamine-based compounds. However, there is increasing evidence for clinical therapy failures in patients suggesting the existence of drug resistance in these classes of drug. In vitro resistance to sulfadiazine has been detected in three strains of Toxoplasma gondii isolated from clinical cases. In order to begin to understand the mechanisms of resistance, we undertook a difference-gel electrophoresis (DIGE) approach combined with mass spectrometry to identify proteins that are differentially expressed in sulfadiazine-resistance strains of the parasite. Naturally resistant strains TgA 103001 (Type I), TgH 32006 (Type II) and TgH 32045 (Type II variant) were compared to sensitive strains RH (Type I) and ME-49 (Type II) using DIGE and the modulated proteins analyzed using LC–MS/MS. In total, 68 differentially expressed protein spots were analyzed by mass spectrometer and 31 unique proteins, including four hypothetical proteins, were identified. Among the differentially expressed proteins, 44% were over-expressed in resistant strains and 56% were over-expressed in sensitive strains. The virulence-associated rhoptry protein, ROP2A, was found in greater abundance in both naturally resistant Type II strains TgH 32006 and TgH 32045 compared to the sensitive strain ME-49. Enolase 2 and IMC1 were found to be in greater abundance in sensitive strains RH and ME-49, and MIC2 was found to be more abundant in the sensitive strain ME-49. Proteins regulation of ROP2, MIC2, ENO2, IMC1 and GRA7 were confirmed by Western blot analysis. In addition, gene expression patterns of ROP2, MIC2, ENO2 and IMC1 were analyzed with qRT-PCR. This study provides the first proteomics insights into sulfadiazine resistance in T. gondii resistant strains isolated from clinical cases.

We report the proteomes of four life-cycle stages of the Apicomplexan parasite Eimeria tenella. A total of 1868 proteins were identified, with 630, 699, 845 and 1532 found in early oocysts (unsporulated), late oocysts (sporulated), sporozoites and second-generation merozoites, respectively. A multidimensional protein identification technology shotgun approach identified 812 sporozoites, 1528 merozoites and all of the oocyst proteins, whereas 2-D gel proteomics identified 230 sporozoites and 98 merozoite proteins. Comparing the invasive stages, we find moving junction components RON2 in both, whereas AMA-1 and RON4 are found only in merozoites and AMA-2 and RON5 are only found in sporozoites, suggesting stage-specific moving junction proteins. During early oocyst to sporozoite development, refractile body and most "glideosome" proteins are found throughout, whereas microneme and most rhoptry proteins are only found after sporulation. Quantitative analysis indicates glycolysis and gluconeogenesis are the most abundant metabolic groups detected in all stages. The mannitol cycle "off shoot" of glycolysis was not detected in merozoites but was well represented in the other stages. However, in merozoites we find more protein associated with oxidative phosphorylation, suggesting a metabolic shift mobilising greater energy production. We find a greater abundance of protein linked to transcription, protein synthesis and cell cycle in merozoites than in sporozoites, which may be residual protein from the preceding massive replication during schizogony.

Secretion of microneme proteins is essential to Plasmodium invasion but the molecular composition of these secretory organelles remains poorly defined. Here, we describe the first Plasmodium microneme proteome. Purification of micronemes by subcellular fractionation from cultured ookinetes was confirmed by enrichment of known micronemal proteins and electron microscopy. Quantitation of electron micrographs showed >14-fold microneme enrichment compared to the intact ookinete, such that micronemes comprised 85% of the identifiable organelles in the fraction. Gel LC-MS/MS of the most abundant protein constituents of the fraction identified three known micronemal proteins chitinase, CTRP, SOAP, together with protein disulphide isomerase (PDI) and HSP70. Highly sensitive MudPIT shotgun proteomics described a total of 345 proteins in the fraction. M1 aminopeptidase and PDI, the former a recognised target of drug development, were both shown to have a micronemal location by IFA. We further identified numerous proteins with established vesicle trafficking and signaling functions consistent with micronemes being part of a regulated secretory pathway. Previously uncharacterised proteins comprise the largest functional group of the microneme proteome and will include secreted proteins important to invasion.

In spite of 40 years of control efforts, onchocerciasis (river blindness) remains one of the most important neglected tropical diseases, with 17 million people affected. The aetiological agent, Onchocerca volvulus, is a filarial nematode with a complex lifecycle involving several distinct stages in the definitive host and blackfly vector. The challenges of obtaining sufficient material have prevented high-throughput studies and the development of novel strategies for disease control and diagnosis. Here, we utilise the closest relative of O. volvulus, the bovine parasite Onchocerca ochengi, to compare stage-specific proteomes and host-parasite interactions within the secretome. We identified a total of 4,260 unique O. ochengi proteins from adult males and females, infective larvae, intrauterine microfilariae, and fluid from intradermal nodules. In addition, 135 proteins were detected from the obligate Wolbachia symbiont. Observed protein families that were enriched in all whole body extracts relative to the complete search database included immunoglobulin-domain proteins, whereas redox and detoxification enzymes and proteins involved in intracellular transport displayed stage-specific overrepresentation. Unexpectedly, the larval stages exhibited enrichment for several mitochondrial-related protein families, including members of peptidase family M16 and proteins which mediate mitochondrial fission and fusion. Quantification of proteins across the lifecycle using the Hi-3 approach supported these qualitative analyses. In nodule fluid, we identified 94 O. ochengi secreted proteins, including homologs of transforming growth factor-β and a second member of a novel 6-ShK toxin-domain family, which was originally identified from a model filarial nematode (Litomosoides sigmodontis). Strikingly, the 498 bovine proteins identified in nodule fluid were strongly dominated by antimicrobial proteins, especially cathelicidins. This first high-throughput analysis of an Onchocerca spp. proteome across the lifecycle highlights its profound complexity and emphasises the extremely close relationship between O. ochengi and O. volvulus The insights provided here provide new candidates for vaccine development, drug targeting and diagnostic biomarkers.

Toxoplasma gondii maintains its intracellular life cycle using an extraordinary arsenal of parasite-specific organelles including the inner membrane complex (IMC), rhoptries, micronemes, and dense granules. While these unique compartments play critical roles in pathogenesis, many of their protein constituents have yet to be identified. We exploited the Vicia villosa lectin (VVL) to identify new glycosylated proteins that are present in these organelles. Purification of VVL-binding proteins by lectin affinity chromatography yielded a number of novel proteins that were subjected to further study, resulting in the identification of proteins from the dense granules, micronemes, rhoptries and IMC. We then chose to focus on three proteins identified by this approach, the SAG1 repeat containing protein SRS44, the rhoptry neck protein RON11 as well as a novel IMC protein we named IMC25. To assess function, we disrupted their genes by homologous recombination or CRISPR/Cas9. The knockouts were all successful, demonstrating that these proteins are not essential for invasion or intracellular survival. We also show that IMC25 undergoes substantial proteolytic processing that separates the C-terminal domain from the predicted glycosylation site. Together, we have demonstrated that lectin affinity chromatography is an efficient method of identifying new glycosylated parasite-specific proteins.

Soil-transmitted nematodes, including the Strongyloides genus, cause one of the most prevalent neglected tropical diseases. Here we compare the genomes of four Strongyloides species, including the human pathogen Strongyloides stercoralis, and their close relatives that are facultatively parasitic (Parastrongyloides trichosuri) and free-living (Rhabditophanes sp. KR3021). A significant paralogous expansion of key gene families - families encoding astacin-like and SCP/TAPS proteins - is associated with the evolution of parasitism in this clade. Exploiting the unique Strongyloides life cycle, we compare the transcriptomes of the parasitic and free-living stages and find that these same gene families are upregulated in the parasitic stages, underscoring their role in nematode parasitism.

Decision making for zoonotic disease management should be based on many forms of appropriate data and sources of evidence. However, the criteria and timing for policy response and the resulting management decisions are often altered when a disease outbreak occurs and captures full media attention. In the case of waterborne disease, such as the robust protozoa, Cryptosporidium spp, exposure can cause significant human health risks and preventing exposure by maintaining high standards of biological and chemical water quality remains a priority for water companies in the UK. Little has been documented on how knowledge and information is translated between the many stakeholders involved in the management of Cryptosporidium, which is surprising given the different drivers that have shaped management decisions. Such information, coupled with the uncertainties that surround these data is essential for improving future management strategies that minimise disease outbreaks. Here, we examine the interplay between scientific information, the media, and emergent government and company policies to examine these issues using qualitative and quantitative data relating to Cryptosporidium management decisions by a water company in the North West of England. Our results show that political and media influences are powerful drivers of management decisions if fuelled by high profile outbreaks. Furthermore, the strength of the scientific evidence is often constrained by uncertainties in the data, and in the way knowledge is translated between policy levels during established risk management procedures. In particular, under or over-estimating risk during risk assessment procedures together with uncertainty regarding risk factors within the wider environment, was found to restrict the knowledge-base for decision-making in Cryptosporidium management. Our findings highlight some key current and future challenges facing the management of such diseases that are widely applicable to other risk management situations. Keywords: Catchment management, Drinking water, Knowledge transfer, Public health, Waterborne outbreaks, Disease risk

Uncertainty is an inherent feature of strategies to contain animal disease. In this paper, an interdisciplinary framework for representing strategies of containment, and analysing how uncertainties are embedded and propagated through them, is developed and illustrated. Analysis centres on persistent, periodic and emerging disease threats, with a particular focus on cryptosporidiosis, foot and mouth disease and avian influenza. Uncertainty is shown to be produced at strategic, tactical and operational levels of containment, and across the different arenas of disease prevention, anticipation and alleviation. The paper argues for more critically reflexive assessments of uncertainty in containment policy and practice. An interdisciplinary approach has an important contribution to make, but is absent from current real-world containment policy.

We report a substantial prevalence study in symptomatic pet dogs of important zoonotic parasitic enteric infections. A total of 4526 dogs which had a faecal sample submitted to a diagnostic laboratory in the UK between 2003 and 2005 were included in the study. The most common parasite was Giardia spp., which was found in 380/4526 dogs (8.4%, 95% CI 7.6-9.2%). Surprisingly, Cryptosporidium spp. infection was detected in only 29/4526 (0.6%, 95% CI 0.4-0.9%). Toxocara canis was found in 63/4526 dogs (1.4%; 95% CI 1.1-1.8%). Prevalence of Giardia (P < 0.001) was significantly higher in dogs

To investigate how intracellular parasites manipulate their host cell environment at the molecular level, we undertook a quantitative proteomic study of cells following infection with the apicomplexan parasiteToxoplasma gondii. Using conventional two-dimensional electrophoresis, difference gel electrophoresis (DIGE), and mass spectrometry, we identified host proteins that were consistently modulated in expression following infection. We detected modification of protein expression in key metabolic pathways, including glycolysis, lipid and sterol metabolism, mitosis, apoptosis, and structural-protein expression, suggestive of global reprogramming of cell metabolism by the parasite. Many of the differentially expressed proteins had not been previously implicated in the response to the parasite, while others provide important corroborative protein evidence for previously proposed hypotheses of pathogen-cell interactions. Significantly, over one-third of all modulated proteins were mitochondrial, and this was further investigated by DIGE analysis of a mitochondrion-enriched preparation from infected cells. Comparison of our proteomic data with previous transcriptional studies suggested that a complex relationship exits between transcription and protein expression that may be partly explained by posttranslational modifications of proteins and revealed the importance of investigating protein changes when interpreting transcriptional data. To investigate this further, we used phosphatase treatment and DIGE to demonstrate changes in the phosphorylation states of several key proteins following infection. Overall, our findings indicate that the host cell proteome responds in a dramatic way toT. gondiiinvasion, in terms of both protein expression changes and protein modifications, and reveal a complex and intimate molecular relationship between host and parasite.

Background A Lactobacillus-dominated cervicovaginal microbiota (VMB) protects women from adverse reproductive health outcomes, but the role of L. iners in the VMB is poorly understood. Our aim was to explore the association between the cervicovaginal L. iners and L. crispatus proteomes and VMB composition. Methods The vaginal proteomes of 50 Rwandan women at high HIV risk, grouped into four VMB groups (based on 16S rDNA microarray results), were investigated by mass spectrometry using cervicovaginal lavage (CVL) samples. Only samples with positive 16S results for L. iners and/or L. crispatus within each group were included in subsequent comparative protein analyses: Lactobacillus crispatus-dominated VMB cluster (with 16S-proven L. iners (ni) = 0, and with 16S-proven L. crispatus (nc) = 5), L. iners-dominated VMB cluster (ni = 11, nc = 4), moderate dysbiosis (ni = 12, nc = 2); and severe dysbiosis (ni = 8, nc = 2). The relative abundances of proteins that were considered specific for L. iners and L. crispatus were compared among VMB groups. Results Forty Lactobacillus proteins were identified of which 7 were specific for L. iners and 11 for L. crispatus. The relative abundances of L. iners DNA starvation/stationary phase protection protein (DPS), and the glycolysis enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glucose-6-phosphate isomerase (GPI), were significantly decreased in women with L. iners-containing dysbiosis compared to women with a L. iners-dominated VMB, independent of vaginal pH and L. iners abundance. Furthermore, L. iners DPS, GAPDH, GPI, and fructose-bisphosphate aldolase (ALDO) were significantly negatively associated with vaginal pH. Glycolysis enzymes of L. crispatus showed a similar negative, but nonsignificant, trend related to dysbiosis. Conclusions Most identified Lactobacillus proteins had conserved intracellular functions, but their high abundance in CVL supernatant might imply an additional extracellular (moonlighting) role. Our findings suggest that these proteins can be important in maintaining a Lactobacillus-dominated VMB. Functional studies are needed to investigate their roles in vaginal bacterial communities and whether they can be used to prevent vaginal dysbiosis.

The evolution of parasitism is a recurrent event in the history of life and a core problem in evolutionary biology. Trypanosomatids are important parasites and include the human pathogens Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp., which in humans cause African trypanosomiasis, Chagas disease, and leishmaniasis, respectively. Genome comparison between trypanosomatids reveals that these parasites have evolved specialized cell-surface protein families, overlaid on a well-conserved cell template. Understanding how these features evolved and which ones are specifically associated with parasitism requires comparison with related non-parasites. We have produced genome sequences for Bodo saltans, the closest known non-parasitic relative of trypanosomatids, and a second bodonid, Trypanoplasma borreli. Here we show how genomic reduction and innovation contributed to the character of trypanosomatid genomes. We show that gene loss has "streamlined" trypanosomatid genomes, particularly with respect to macromolecular degradation and ion transport, but consistent with a widespread loss of functional redundancy, while adaptive radiations of gene families involved in membrane function provide the principal innovations in trypanosomatid evolution. Gene gain and loss continued during trypanosomatid diversification, resulting in the asymmetric assortment of ancestral characters such as peptidases between Trypanosoma and Leishmania, genomic differences that were subsequently amplified by lineage-specific innovations after divergence. Finally, we show how species-specific, cell-surface gene families (DGF-1 and PSA) with no apparent structural similarity are independent derivations of a common ancestral form, which we call "bodonin." This new evidence defines the parasitic innovations of trypanosomatid genomes, revealing how a free-living phagotroph became adapted to exploiting hostile host environments.

Vaginal microbiome (VMB) dysbiosis is associated with increased acquisition of HIV. Cervicovaginal inflammation and other changes to the mucosal barrier are thought to have important roles but human data are scarce. We compared the human cervicovaginal proteome by mass spectrometry of 50 Rwandan female sex workers who had previously been clustered into four VMB groups using a 16S phylogenetic microarray; in order of increasing bacterial diversity: Lactobacillus crispatus-dominated VMB (group 1), Lactobacillus iners-dominated VMB (group 2), moderate dysbiosis (group 3), and severe dysbiosis (group 4). We compared relative protein abundances among these VMB groups using targeted (abundance of pre-defined mucosal barrier proteins) and untargeted (differentially abundant proteins among all human proteins identified) approaches. With increasing bacterial diversity, we found: mucus alterations (increasing mucin 5B and 5AC), cytoskeleton alterations (increasing actin-organizing proteins; decreasing keratins and cornified envelope proteins), increasing lactate dehydrogenase A/B as markers of cell death, increasing proteolytic activity (increasing proteasome core complex proteins/proteases; decreasing antiproteases), altered antimicrobial peptide balance (increasing psoriasin, calprotectin, and histones; decreasing lysozyme and ubiquitin), increasing pro-inflammatory cytokines, and decreasing immunoglobulins immunoglobulin G1/2. Although temporal relationships cannot be derived, our findings support the hypothesis that dysbiosis causes cervicovaginal inflammation and other detrimental changes to the mucosal barrier.

Toxoplasma gondii is an important protozoan parasite that infects all warm-blooded animals and causes opportunistic infections in immuno-compromised humans. Its closest relative, Neospora caninum, is an important veterinary pathogen that causes spontaneous abortion in livestock. Comparative genomics of these two closely related coccidians has been of particular interest to identify genes that contribute to varied host cell specificity and disease. Here, we describe a manual evaluation of these genomes based on strand-specific RNA sequencing and shotgun proteomics from the invasive tachyzoite stages of these two parasites. We have corrected predicted structures of over one third of the previously annotated gene models and have annotated untranslated regions (UTRs) in over half of the predicted protein-coding genes.We observe distinctly long UTRs in both the organisms, almost four times longer than other model eukaryotes. We have also identified a putative set of cis-natural antisense transcripts (cis-NATs) and long intergenic non-coding RNAs (lincRNAs). We have significantly improved the annotation quality in these genomes that would serve as a manually curated dataset for Toxoplasma and Neospora research communities.

Giardia duodenalis is a major cause of infectious gastroenteritis worldwide, and it is diversified into eight genetic assemblages (A to H), which are distinguishable only by molecular typing. There is some evidence that the assemblages infecting humans (assemblages A and B) may have different transmission routes, but systematically acquired data, combining epidemiological and molecular findings, are required. We undertook a case-control study with Giardia genotyping in North West England, to determine general and parasite assemblage-specific risk factors. For people without a history of foreign travel, swimming in swimming pools and changing diapers were the most important risk factors for the disease. People infected with assemblage B reported a greater number of symptoms and higher frequencies of vomiting, abdominal pain, swollen stomach, and loss of appetite, compared with people infected with assemblage A. More importantly, keeping a dog was associated only with assemblage A infections, suggesting the presence of a potential zoonotic reservoir for this assemblage. This is the first case-control study to combine epidemiological data with Giardia genotyping, and it shows the importance of integrating these two levels of information for better understanding of the epidemiology of this pathogen.

Monoclonal antibodies (mabs) were generated against whole sonicated Neospora caninum tachyzoites as immunogen. Initial ELISA screening of the reactivity of hybridoma culture supernatants using the same antigen and antigen treated with sodium periodate prior to antibody binding resulted in the identification of 8 supernatants with reactivity against putative carbohydrate epitopes. Following immunoblotting, mab6D12 (IgG1), binding a 52/48-kDa doublet, and mab6C6 (IgM), binding a 190/180-kDa doublet, were selected for further studies. Immunofluorescence of tachyzoite-infected cultures localized the corresponding epitopes not to the surface, but to interior epitopes at the apical part of N. caninum tachyzoites. During in vitro tachyzoite to bradyzoite stage conversion, mab6C6 labeling translocated toward the cyst periphery, while for mab6D12 no changes in localization were noted. Upon extraction of tachyzoites with the nonionic detergent Triton-X-100, the 52-kDa band recognized by mab6D12 was present exclusively in the insoluble, cytoskeletal fraction of both N. caninum and Toxoplasma gondii tachyzoites. Tandem mass spectrometry analysis identified this protein as N. caninum beta tubulin. The 48-kDa band labeled by mab6D12 was a Vero cell protein contamination. The protein(s) reacting with mab6C6 could not be conclusively identified by mass spectrometry. Immunofluorescence consistently failed to label T. gondii tachyzoites, indicating that beta tubulin in T. gondii and N. caninum could be differentially modified or that the reactive epitope in T. gondii is masked. Immunogold TEM of isolated apical cytoskeletal preparations and dual immunofluorescence with antibody to tubulin confirmed that mab6D12 binds to the anterior part of apical complex-associated microtubules. The sodium periodate sensitivity of the beta tubulin associated epitope was confirmed by immunoblotting and ELISA, and treatment of N. caninum cytoskeletal proteins with sialidase prior to mab6D12 labeling resulted in a profound loss of antibody binding, suggesting that mab6D12 reacts with sialylated beta tubulin.

We have undertaken 2-DE and MS to identify proteins associated with arsenical drug resistance in Trypanosoma brucei. This parasite causes sleeping sickness in humans, and arsenical drug resistance is a significant potential problem. Comparative analysis of approximately 2000 spots resolved by 2-DE in the soluble proteomes of drug-sensitive and drug-resistant isogenic lines of T. brucei identified a protein spot whose absence associated with resistance to the arsenical drug, Cymelarsan. MS matched this protein to an identical pair of tandem genes Tb09.211.0120 and 0130 that encode a putative nascent polypeptide associated complex subunit. This protein also occurs as an isoform located in both resistant and sensitive lines at a similar molecular weight, but different pI. The difference between isogenic lines was confirmed by Western blot using an antibody against recombinant protein. Both genes were identical in sequence between drug-sensitive and drug-resistant lines and both were transcribed as determined by RT-PCR. We postulate that the missing protein isoform arose due to the lack of a PTM.

Neospora caninum is a recently recognized protozoan parasite which has been described as causing a neuromuscular paralysis in dogs and is emerging as a major cause of bovine infertility and abortion worldwide. The parasite is known to infect a range of warm blooded animals but the disease predominates in dogs and cattle. It is not yet known if N. caninum can infect and cause disease in people. The dog has recently been identified as the definitive host and the parasite may be transmitted through the ingestion of oocysts or congenitally from mother to fetus. N. caninum is known to infect red foxes (Vulpes vulpes) and coyotes (Canis latrans) and the role of wildlife species as reservoirs of infection requires further investigation. Little is known about the range of parasite genotypes within the environment or the variation in virulence between different strains. RAPD-PCR analysis of geographically distinct bovine and canine isolates has revealed little genetic variation. Epidemiological studies from different areas of the world have investigated the importance of N. caninum as an abortifacient agent and longitudinal studies have shown the high rate (approximately 80%) of congenital transmission within infected herds. Information on the rates of repeat abortion due to neosporosis are less well defined however current estimates put this at 5% suggesting that cattle may develop some form of protective immunity against N. caninum-induced abortion. Diagnosis of the disease is based upon detection of the parasite in the tissues, most commonly using immunohistochemistry with additional information provided by serology. However, although positive fetal serology is a strong indicator of exposure to the parasite, care should be taken in the interpretation of maternal serology. As we understand more about the epidemiology of neosporosis we are also better able to interpret the results of diagnostic tests. The mere presence of the parasite does not necessarily infer that this was the primary cause of abortion. CD4+ T-cells, interferon gamma and macrophages have all been found to significantly inhibit multiplication of N. caninum tachyzoites. The nature of a protective immune response and its modulation in the pregnant animal is discussed.

Cryptosporidium parvumis 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 asPlasmodiumspp. andToxoplasma gondii, there is no clinically proven drug treatment against this parasite. Aspects of the basic biology ofC. parvumremain poorly understood, including a detailed knowledge of key metabolic pathways, its genome organization and organellar complement. Previous studies have proposed thatC. parvumlacks a relic plastid organelle, or ‘apicoplast’, but that it may possess a mitochondrion. Here we characterize a mitochondrion-like organelle inC. parvumby (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) ofC. hominisandC. 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 theC. parvummitochondrion-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 thisCryptosporidiumorganelle and a logical framework for future functional investigation.

Motivation: Large-scale functional genomics analysis is now feasible and presents significant challenges in data analysis, storage and querying. Data standards are required to enable the development of public data repositories and to improve data sharing. There is an established data format for microarrays (microarray gene expression markup language, MAGE-ML) and a draft standard for proteomics (PEDRo). We believe that all types of functional genomics experiments should be annotated in a consistent manner, and we hope to open up new ways of comparing multiple datasets used in functional genomics. Results: We have created a functional genomics experiment object model (FGE-OM), developed from the microarray model, MAGE-OM and two models for proteomics, PEDRo and our own model (Gla-PSI—Glasgow Proposal for the Proteomics Standards Initiative). FGE-OM comprises three namespaces representing (i) the parts of the model common to all functional genomics experiments; (ii) microarray-specific components; and (iii) proteomics-specific components. We believe that FGE-OM should initiate discussion about the contents and structure of the next version of MAGE and the future of proteomics standards. A prototype database called RNA And Protein Abundance Database (RAPAD), based on FGE-OM, has been implemented and populated with data from microbial pathogenesis. Availability: FGE-OM and the RAPAD schema are available from http://www.gusdb.org/fge.html, along with a set of more detailed diagrams. RAPAD can be accessed by registration at the site.

The apicomplexan parasite Theileria annulata is the only intracellular eukaryote that is known to induce the proliferation of mammalian cells. However, as the parasite undergoes stage differentiation, host cell proliferation is inhibited, and the leukocyte is eventually destroyed. We have isolated a parasite gene ( SuAT1 ) encoding an AT hook DNA binding polypeptide that has a predicted signal peptide, PEST motifs, nuclear localization signals, and domains which indicate interaction with regulatory components of the higher eukaryotic cell cycle. The polypeptide is localized to the nuclei of macroschizont-infected cells and was detected at significant levels in cells that were undergoing parasite stage differentiation. Transfection of an uninfected transformed bovine macrophage cell line, BoMac, demonstrated that SuAT1 can modulate cellular morphology and alter the expression pattern of a cytoskeletal polypeptide in a manner similar to that found during the infection of leukocytes by the parasite. Our findings indicate that Theileria parasite molecules that are transported to the leukocyte nucleus have the potential to modulate the phenotype of infected cells.

Cryptosporidium parvum is an intracellular protozoan parasite that infects the gastrointestinal tract of humans and other mammals. It has significant economic importance as a pathogen of livestock and, as there is no effective treatment or vaccine available, understanding transmission routes and identifying sources of infection is key to preventing future outbreaks and controlling this disease. In this study we have determined the multilocus genotype (MLG) of 240 C. parvum Type 2 (bovine) isolates using a combination of seven micro- and minisatellite markers. These isolates were collected over a period of 19 months and are from three different geographical locations within Scotland and three different host species. The results of this study have enabled us to address questions concerning C. parvum population genetics in relation to host, temporal and geographical sub-structuring. We identified 48 multilocus genotypes within the Type 2 C. parvum isolates and found no evidence to support geographic or temporal sub-structuring of the populations. However host sub-structuring was identified within the human Type 2 population highlighting the potential use of such a typing system in understanding the epidemiology of this parasite in addition to raising interesting questions with regard to its population genetic structure. We also isolated two C. parvum 'monkey type' isolates from two separate human cases indicating that this genotype is not restricted to monkey hosts with the multilocus genotypes of these isolates distinguishing them from all other isolates.