Descriptive epidemiology of African horse sickness in Zimbabwe

A study of the prevalence of African horse sickness in horses was conducted, using records from two private equine practices in Harare for the period 1998–2004. Results indicated a higher prevalence of the disease in horses in Zimbabwe in the late rainy season (March – May). Age of the horse was found to be a significant risk factor, with foals or yearlings appearing to be 1.80 times more likely to contract the disease compared with horses older than two years. The case fatality rate in foals or yearlings was also higher than in older age groups, but this difference was not significant. The vaccination status was an important risk factor, with vaccinated horses 0.12 times less likely to die from the disease compared with unvaccinated horses. Young, unvaccinated horses therefore seem to be the most susceptible to the disease and have greater chances of fatality. This study highlights the importance of adequately protecting horses against African horse sickness by providing immunisation through vaccination and discusses the need to review current vaccination strategies being practiced in Zimbabwe

The Pathogen-Occupied Vacuoles of Anaplasma phagocytophilum and Anaplasma marginale Interact with the Endoplasmic Reticulum

The genus Anaplasma consists of tick-transmitted obligate intracellular bacteria that invade white or red blood cells to cause debilitating and potentially fatal infections. A. phagocytophilum, a human and veterinary pathogen, infects neutrophils to cause granulocytic anaplasmosis. A. marginale invades bovine erythrocytes. Evidence suggests that both species may also infect endothelial cells in vivo. In mammalian and arthropod host cells, A. phagocytophilum and A. marginale reside in host cell derived pathogen-occupied vacuoles (POVs). While it was recently demonstrated that the A. phagocytophilum-occupied vacuole (ApV) intercepts membrane traffic from the trans-Golgi network, it is unclear if it or the A. marginale-occupied vacuole (AmV) interacts with other secretory organelles. Here, we demonstrate that the ApV and AmV extensively interact with the host endoplasmic reticulum (ER) in endothelial, myeloid, and/or tick cells. ER lumen markers, calreticulin, and protein disulfide isomerase, and the ER membrane marker, derlin-1, were pronouncedly recruited to the peripheries of both POVs. ApV association with the ER initiated early and continued throughout the infection cycle. Both the ApV and AmV interacted with the rough ER and smooth ER. However, only derlin-1-positive rough ER derived vesicles were delivered into the ApV lumen where they localized with intravacuolar bacteria. Transmission electron microscopy identified multiple ER-POV membrane contact sites on the cytosolic faces of both species\u27 vacuoles that corresponded to areas on the vacuoles\u27 lumenal faces where intravacuolar Anaplasma organisms closely associated. A. phagocytophilum is known to hijack Rab10, a GTPase that regulates ER dynamics and morphology. Yet, ApV-ER interactions were unhindered in cells in which Rab10 had been knocked down, demonstrating that the GTPase is dispensable for the bacterium to parasitize the ER. These data establish the ApV and AmV as pathogen-host interfaces that directly engage the ER in vertebrate and invertebrate host cells and evidence the conservation of ER parasitism between two Anaplasma species

THE MOLECULAR MECHANISMS OF TICK PATHOGEN TRANSMISSION

Ticks vector numerous diseases important to livestock and humans and are often indispensable to the life cycle and continued existence of these agents. However, little is known about their biology and interactions with the pathogens they transmit. This gap in knowledge limits development of interventions that can be limit tick borne disease burden.Anaplasma marginale establishes infection within Dermacentor spp. for weeks escaping the complex network of vacuolar peptidases responsible for digestion of the tick blood meal. How this prolonged maintenance of infectivity is effected has been unknown. In the first study we used the natural vector Dermacentor andersoni, and demonstrated that A. marginale-infected tick vacuoles (AmVs) concurrently recruit markers of the early (Rab5), recycling (Rab4 and Rab11) and late endosome (Rab7), are maintained near neutral pH, do not fuse with lysosomes, exclude the protease cathepsin L, and engage the endoplasmic reticulum and Golgi for up to 21d post infection. Maintenance of this safe vacuolar niche required active A. marginale protein synthesis; in its absence AmVs mature into acidic, protease-active phagolysosomes.In the second chapter we analysed the transcriptional response to infection by ticks. Pathogens co-evolved with ticks developing mechanisms to subvert tick biological processes to facilitate infection, replication and eventual transmission. we used Ion torrent high throughput RNA sequencing to compare the midgut transcripts of Dermacentor andersoni (Da) male ticks that are unfed to those fed on a blood meal that is either uninfected or infected with Anaplasma marginale (Am) St. Maries strain. Sequencing of tick midgut mRNA yielded 11.7M reads. After De novo assembly the remaining 11 716 transcripts were annotated and analyzed using K-means clustering. Global analysis of all transcripts shows high degree of transcription in response to the blood meal and differential transcription of key gene families between infected and uninfected ticks. Among annotated genes, were genes related to antioxidant process (catalase and cytochrome p450) and the immune system (peritrophin) that reflect response to both the pathogen and oxidative challenge. These findings suggest that specific differential transcription in the midgut in response to pathogens may play a role in tick transmission competency

Localization of β-Nerve Growth Factor in the Stallion Reproductive Tract

β-Nerve growth factor (β-NGF) is a protein produced in the reproductive tract of camelids (camels, llamas, and alpacas) that has been identified as the ovulation inducing factor in seminal plasma. β-NGF from seminal plasma deposited into the reproductive tract of the female camelid acts systemically to stimulate the secretion of luteinizing hormone (LH) from the anterior pituitary, which in turn induces follicle maturation and ovulation. The objectives of the present study were to determine if β-NGF is present in the reproductive tract of the stallion and identify the specific site(s) of production. The hypotheses were that β-NGF would be present in the stallion reproductive tract and would primarily be localized in Sertoli cells of the testes and the prostate gland. Immunohistochemistry on paraffin-embedded paraformaldehyde-fixed tissues was performed using a rabbit polyclonal anti-β-NGF antibody on a total of six male equine reproductive tracts, including a one-day old colt, a one-year-old colt, and four adult stallion tracts. Strong immunostaining was observed in the efferent ducts of the testes and the epithelial cells of the prostate, seminal vesicles, bulbourethral glands, and ampullae. Weaker β-NGF staining was noted in Leydig cells, Sertoli cells, and spermatogonia within the testes and in epithelial cells of the epididymis. In conclusion, immunohistochemistry revealed that β-NGF is present in the stallion reproductive tract, and the protein is primarily present in the efferent ducts of the testes and in all accessory sex glands

Deploying Elemental Iodine in a Vapor Form to Disinfect Water and to Clear Biofilms

Traditionally, iodine has been delivered as a solution, tablet or resin to disinfect water. In this study we evaluated the “I2 vapor infusion” (I2VP) technology which passes an airstream through a matrix containing elemental iodine (I2) to produce I2 vapor as an innovative method of iodine delivery for water disinfection. Pressured air was provided either by a compressor or hand pump. Testing was performed with water inoculated with either Gram-negative (Escherichia, Salmonella) or Gram-positive (Enterococcus) bacteria or with pre-formed Acinetobacter or Staphylococcus biofilms. Bacterial colony forming units were used to assess efficacy of the device. In distilled water all bacteria and biofilms were eliminated after brief exposures (<90 s). Culturable bacteria were also eliminated from pond and municipal sewer water, but the technology was mostly ineffective against dairy lagoon water with high turbidity and organic particulate. Longer duration infusion and higher air volumes used to overcome interference from organic matter were also associated with higher concentrations of residual iodine. We conclude that I2 vapor infusion has the potential to be useful for emergency water treatment and potentially for reducing microbiological contamination of some waste streams

An optimized live bacterial delivery platform for the production and delivery of therapeutic nucleic acids and proteins

AbstractThere is an unmet need for delivery platforms that realize the full potential of next-generation therapeutic and vaccine technologies, especially those that require intracellular delivery of nucleic acids. The in vivo usefulness of the current state-of-the-art delivery systems is limited by numerous intrinsic weaknesses, including lack of targeting specificity, inefficient entry and endosomal escape into target cells, undesirable immune activation, off-target effects, a small therapeutic window, limited genetic encoding and cargo capacity, and manufacturing challenges. Here we present our characterization of a delivery platform based on the use of engineered live, tissue-targeting, non-pathogenic bacteria (Escherichia coli strain SVC1) for intracellular cargo delivery. The SVC1 bacteria are engineered to specifically bind to epithelial cells via a surface-expressed targeting ligand, to escape the endosome upon intracellularization, and to have minimal immunogenicity. Here we report findings on key features of this system. First, we demonstrated that bacterial delivery of a short hairpin RNA (shRNA) can target and silence a gene in an in vitro mammalian respiratory cell model. Next, we used an in vivo mouse model to demonstrate that SVC1 bacteria are invasive to epithelial cells of various tissues and organs (eye, nose, mouth, stomach, vagina, skeletal muscle, and lungs) via local administration. We also showed that repeat dosing of SVC1 bacteria to the lungs is minimally immunogenic and that it does not have adverse effects on tissue homeostasis. Finally, to validate the potential of SVC1 bacteria in therapeutic applications, we demonstrated that bacterial delivery of influenza-targeting shRNAs to the respiratory tissues can mitigate viral replication in a mouse model of influenza infection. Our ongoing work is focused on further refining this platform for efficient delivery of nucleic acids, gene editing machinery, and therapeutic proteins, and we expect that this platform technology will enable a wide range of advanced therapeutic approaches.</jats:p

Deploying Elemental Iodine in a Vapor Form to Disinfect Water and to Clear Biofilms

Traditionally, iodine has been delivered as a solution, tablet or resin to disinfect water. In this study we evaluated the “I2 vapor infusion” (I2VP) technology which passes an airstream through a matrix containing elemental iodine (I2) to produce I2 vapor as an innovative method of iodine delivery for water disinfection. Pressured air was provided either by a compressor or hand pump. Testing was performed with water inoculated with either Gram-negative (Escherichia, Salmonella) or Gram-positive (Enterococcus) bacteria or with pre-formed Acinetobacter or Staphylococcus biofilms. Bacterial colony forming units were used to assess efficacy of the device. In distilled water all bacteria and biofilms were eliminated after brief exposures (&lt;90 s). Culturable bacteria were also eliminated from pond and municipal sewer water, but the technology was mostly ineffective against dairy lagoon water with high turbidity and organic particulate. Longer duration infusion and higher air volumes used to overcome interference from organic matter were also associated with higher concentrations of residual iodine. We conclude that I2 vapor infusion has the potential to be useful for emergency water treatment and potentially for reducing microbiological contamination of some waste streams.</jats:p

Reduced Infectivity in Cattle for an Outer Membrane Protein Mutant of Anaplasma marginale

ABSTRACT Anaplasma marginale is the causative agent of anaplasmosis in cattle. Transposon mutagenesis of this pathogen using the Himar1 system resulted in the isolation of an omp10 operon insertional mutant referred to as the omp10 :: himar1 mutant. The work presented here evaluated if this mutant had morphological and/or growth rate defects compared to wild-type A. marginale . Results showed that the morphology, developmental cycle, and growth in tick and mammalian cell cultures are similar for the mutant and the wild type. Tick transmission experiments established that tick infection levels with the mutant were similar to those with wild-type A. marginale and that infected ticks successfully infected cattle. However, this mutant exhibited reduced infectivity and growth in cattle. The possibility of transforming A. marginale by transposon mutagenesis coupled with in vitro and in vivo assessment of altered phenotypes can aid in the identification of genes associated with virulence. The isolation of deliberately attenuated organisms that can be evaluated in their natural biological system is an important advance for the rational design of vaccines against this species. </jats:p

Identification and functional analysis of a galactosyltransferase capable of cholesterol glycolipid formation in the Lyme disease spirochete Borrelia burgdorferi

Borrelia burgdorferi (Bb), the etiological agent of Lyme disease, produces a series of simple glycolipids where diacylglycerol and cholesterol serve as the precursor. The cholesterol-based glycolipids, cholesteryl 6-O-acyl-β-D-galactopyranoside (ACGal) and cholesteryl-β-D-galactopyranoside (CGal) are immunogenic and proposed to contribute to the pathogenesis of Lyme disease. Detailed studies of CGal and ACGal in Bb have been hampered by a lack of knowledge of their underlying biosynthetic processes. The genome of Bb encodes four putative glycosyltransferases, and only one of these, BB0572, was predicted to be an inverting family 2 glycosyltransferase (GT2 enzyme) capable of using UDP-galactose as a substrate and forming a β-glycosidic bond. Comparison of the 42 kDa BB0572 amino acid sequence from Bb with other Borrelia spp demonstrates that this protein is highly conserved. To establish BB0572 as the galactosyltransferase capable of cholesterol glycolipid formation in Bb, the protein was produced as a recombinant product in Escherichia coli and tested in a cell-free assay with 14C-cholesterol and UDP-galactose as the substrates. This experiment resulted in a radiolabeled lipid that migrated with the cholesterol glycolipid standard of CGal when evaluated by thin layer chromatography. Additionally, mutation in the predicted active site of BB0572 resulted in a recombinant protein that was unable to catalyze the formation of the cholesterol glycolipid. These data characterize BB0572 as a putative cholesterol galactosyltransferase. This provides the first step in understanding how Bb cholesterol glycolipids are formed and will allow investigations into their involvement in pathogen transmission and disease development.</jats:p