Identification and Characterization of Anaplasma phagocytophilum Proteins Involved in Infection of the Tick Vector,Ixodes scapularis

Anaplasma phagocytophilum is an emerging zoonotic pathogen transmitted by Ixodes scapularis that causes human granulocytic anaplasmosis. Here, a high throughput quantitative proteomics approach was used to characterize A. phagocytophilum proteome during rickettsial multiplication and identify proteins involved in infection of the tick vector, I. scapularis. The first step in this research was focused on tick cells infected with A. phagocytophilum and sampled at two time points containing 10–15% and 65–71% infected cells, respectively to identify key bacterial proteins over-represented in high percentage infected cells. The second step was focused on adult female tick guts and salivary glands infected with A. phagocytophilum to compare in vitro results with those occurring during bacterial infection in vivo. The results showed differences in the proteome of A. phagocytophilum in infected ticks with higher impact on protein synthesis and processing than on bacterial replication in tick salivary glands. These results correlated well with the developmental cycle of A. phagocytophilum, in which cells convert from an intracellular reticulated, replicative form to the nondividing infectious dense-core form. The analysis of A. phagocytophilum differentially represented proteins identified stress response (GroEL, HSP70) and surface (MSP4) proteins that were over-represented in high percentage infected tick cells and salivary glands when compared to low percentage infected cells and guts, respectively. The results demonstrated that MSP4, GroEL and HSP70 interact and bind to tick cells, thus playing a role in rickettsia-tick interactions. The most important finding of these studies is the increase in the level of certain bacterial stress response and surface proteins in A. phagocytophilum-infected tick cells and salivary glands with functional implication in tick-pathogen interactions. These results gave a new dimension to the role of these stress response and surface proteins during A. phagocytophilum infection in ticks. Characterization of Anaplasma proteome contributes information on host-pathogen interactions and provides targets for development of novel control strategies for pathogen infection and transmission.     

Genomics Virtual Laboratory: A Practical Bioinformatics Workbench for the Cloud

Background

Analyzing high throughput genomics data is a complex and compute intensive task, generally requiring numerous software tools and large reference data sets, tied together in successive stages of data transformation and visualisation. A computational platform enabling best practice genomics analysis ideally meets a number of requirements, including: a wide range of analysis and visualisation tools, closely linked to large user and reference data sets; workflow platform(s) enabling accessible, reproducible, portable analyses, through a flexible set of interfaces; highly available, scalable computational resources; and flexibility and versatility in the use of these resources to meet demands and expertise of a variety of users. Access to an appropriate computational platform can be a significant barrier to researchers, as establishing such a platform requires a large upfront investment in hardware, experience, and expertise.

Results

We designed and implemented the Genomics Virtual Laboratory (GVL) as a middleware layer of machine images, cloud management tools, and online services that enable researchers to build arbitrarily sized compute clusters on demand, pre-populated with fully configured bioinformatics tools, reference datasets and workflow and visualisation options. The platform is flexible in that users can conduct analyses through web-based (Galaxy, RStudio, IPython Notebook) or command-line interfaces, and add/remove compute nodes and data resources as required. Best-practice tutorials and protocols provide a path from introductory training to practice. The GVL is available on the OpenStack-based Australian Research Cloud (http://nectar.org.au) and the Amazon Web Services cloud. The principles, implementation and build process are designed to be cloud-agnostic.

Conclusions

This paper provides a blueprint for the design and implementation of a cloud-based Genomics Virtual Laboratory. We discuss scope, design considerations and technical and logistical constraints, and explore the value added to the research community through the suite of services and resources provided by our implementation.     

Silencing of genes involved in Anaplasma marginale-tick interactions affects the pathogen developmental cycle in Dermacentor variabilis

Background  

The cattle pathogen, Anaplasma marginale, undergoes a developmental cycle in ticks that begins in gut cells. Transmission to cattle occurs from salivary glands during a second tick feeding. At each site of development two forms of A. marginale (reticulated and dense) occur within a parasitophorous vacuole in the host cell cytoplasm. However, the role of tick genes in pathogen development is unknown. Four genes, found in previous studies to be differentially expressed in Dermacentor variabilis ticks in response to infection with A. marginale, were silenced by RNA interference (RNAi) to determine the effect of silencing on the A. marginale developmental cycle. These four genes encoded for putative glutathione S-transferase (GST), salivary selenoprotein M (SelM), H+ transporting lysosomal vacuolar proton pump (vATPase) and subolesin.     

Characterization of Anaplasma marginale Isolated from North American Bison

Anaplasma marginale (Rickettsiales: Anaplasmataceae), a tick-borne pathogen of cattle, is endemic in tropical and subtropical regions of the world. Although serologic tests have identified American bison, Bison bison, as being infected with A. marginale, the present study was undertaken to confirm A. marginale infection and to characterize isolates obtained from naturally infected bison in the United States and Canada. Major surface protein (MSP1a and MSP4) sequences of bison isolates were characterized in comparison with New World cattle isolates. Blood from one U.S. bison was inoculated into a susceptible, splenectomized calf, which developed acute anaplasmosis, demonstrating infectivity of this A. marginale bison isolate for cattle. The results of this study showed that these A. marginale isolates obtained from bison were similar to ones from naturally infected cattle.     

Differential adhesion of major surface proteins 1a and 1b of the ehrlichial cattle pathogen Anaplasma marginale to bovine erythrocytes and tick cells

Anaplasma marginale is a tick-borne ehrlichial pathogen of cattle for which six major surface proteins (MSPs) have been described. The MSP1 complex, a heterodimer composed of MSP1a and MSP1b, was shown to induce a protective immune response in cattle and both proteins have been identified as putative adhesins for bovine erythrocytes. In this study the role of MSP1a and MSP1b as adhesins for bovine erythrocytes and tick cells was defined. msp1alpha and msp1beta1 genes from the Oklahoma isolate of A. marginale were cloned and expressed in Escherichia coli K-12 under the control of endogenous and tac promoters for both low and high level protein expression. Expression of the recombinant polypeptides was confirmed and localised on the surface of transformed E. coli. The adhesion properties of MSP1a and MSP1b were determined by allowing recombinant E. coli expressing these surface polypetides to react with bovine erythrocytes, Dermacentor variabilis gut cells and cultured tick cells derived from embryonic Ixodes scapularis. Adhesion of the recombinant E. coli to the three cell types was determined using recovery adhesion and microtiter haemagglutination assays, and by light and electron microscopy. MSP1a was shown by all methods tested to be an adhesin for bovine erythrocytes and both native and cultured tick cells. In contrast, recombinant E. coli expressing MSP1b adhered only to bovine erythrocytes and not to tick cells. When low expression vectors were used, single E. coli expressing MSP1a was seen adhered to individual tick cells while reaction of tick cells with the E. coli/MSP1a/high expression vector resulted in adhesion of multiple bacteria per cell. With electron microscopy, fusion of E. coli cell membranes expressing MSP1a or MSP1b with erythrocyte membranes was observed, as well as fusion of tick cell membranes with E. coli membranes expressing MSP1a. These studies demonstrated differential adhesion for MSP1a and MSP1b for which MSP1a is an A. marginale adhesin for both bovine erythrocytes and tick cells while MSP1b is an adhesin only for bovine erythrocytes. The role of the MSP1 complex, therefore, appears to vary among vertebrate and invertebrate hosts.     

An intraerythrocytic small piroplasm in wild-caught Pallas's cats (Otocolobus manul) from Mongolia

During the quarantine examination of four Pallas's cats (Otocolobus manul) imported from Mongolia in October and December 2000, intraerythrocytic piroplasms were detected on Wright-Giemsa stained blood films that were morphologically indistinguishable from other small piroplasms of felids. Further characterization of this unknown organism via polymerase chain reaction amplification, sequencing of a portion of the 18S nuclear small subunit rRNA gene, and comparisons with orthologous sequences from other piroplasms, revealed similarity to Cytauxzoon felis. This is the first report of naturally occurring erythroparasitemia in Pallas's cats and the first documented case of naturally occurring piroplasm infections in a free-ranging felid from Mongolia.     

Lipid peroxides and antioxidant enzymes in cisplatin-induced chronic nephrotoxicity in rats

Cisplatin (CDDP), an anticancer drug, induces remarkable toxicity in the kidneys of animals and humans and it has been well documented that reactive oxygen species and the renal antioxidant system are strongly involved in acute renal damage induced by CDDP. The aim of the present study was to investigate whether or not the renal antioxidant system plays also an important role in chronic renal damage induced by repeated doses of CDDP (1 mg/kg intraperitoneally twice weekly during 10 weeks in rats). In order to elucidate it, serum creatinine and urea levels, renal glutathione and thiobarbituric acid-reactive substances (TBARS) content, as well as renal superoxide dismutase and glutathione peroxidase activities were measured in the kidney homogenates of chronically CDDP-treated rats and additionally histological studies were performed in the rat kidneys. The chronic treatment with CDDP induced a significant increase in creatinine and urea levels in serum, but the other parameters mentioned above were not significantly modified as compared to the values in nontreated rats. Taking into account these results, we conclude that chronic CDDP administration induces also severe nephrotoxicity, in contrast to CDDP acute application, without any significant modification in the activity of relevant antioxidant enzymes such as superoxide dismutase and glutathione peroxidase, renal glutathione and lipid peroxides, by which the role of the antioxidant system in chronic nephrotoxicity induced by CDDP in rats is uncertain.     

Cardiac preconditioning with 4-h, 17 degrees C ischemia reduces [Ca(2+)](i) load and damage in part via K(ATP) channel opening

Brief ischemia before normothermic ischemia protects hearts against reperfusion injury (ischemic preconditioning, IPC), but it is unclear whether it protects against long-term moderate hypothermic ischemia. We explored in isolated guinea pig hearts 1) the influence of two 2-min periods of normothermic ischemia before 4 h, 17 degrees C hypothermic ischemia on cardiac cytosolic [Ca(2+)], mechanical and metabolic function, and infarct size, and 2) the potential role of K(ATP) channels in eliciting cardioprotection. We found that IPC before 4 h moderate hypothermia improved myocardial perfusion, contractility, and relaxation during normothermic reperfusion. Protection was associated with markedly reduced diastolic [Ca(2+)] loading throughout both hypothermic storage and reperfusion. Global infarct size was markedly reduced from 36 +/- 2 (SE)% to 15 +/- 1% with IPC. Bracketing ischemic pulses with 200 microM 5-hydroxydecanoic acid or 10 microM glibenclamide increased infarct size to 28 +/- 3% and 26 +/- 4%, respectively. These results suggest that brief ischemia before long-term hypothermic storage adds to the cardioprotective effects of hypothermia and that this is associated with decreased cytosolic [Ca(2+)] loading and enhanced ATP-sensitive K channel opening.     

Naturally occurring hepatozoonosis in coyotes from Oklahoma

Nine of 16 free-ranging coyotes (Canis latrans) from central Oklahoma (USA) had naturally acquired infections of Hepatozoon americanum. Infections were confirmed by recognition of tissue stages closely resembling H. americanum in skeletal and cardiac muscle. At the time coyotes were collected they were infested with a variety of ticks, including adult Gulf Coast ticks (Amblyomma maculatum). We propose that the high prevalence of H. americanum in this small sample of free-ranging coyotes and the ability of these same animals to harbor adult populations of A. maculatum is an important component of the epizootiology of canine hepatozoonosis in North America.