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' vacuoles that corresponded to areas on the vacuoles' 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.

Antiserum to the Borrelia burgdorferi arthritis-related protein, Arp, has been shown to prevent or reduce arthritis in immunodeficient mice. To directly investigate the requirement for this lipoprotein in the generation of Lyme arthritis, we utilized targeted deletion to generate a B. burgdorferi clone that lacked only the arp gene locus. Infection of Lyme disease-susceptible C3H/ HeN mice with the arp deletion mutant demonstrated significantly reduced tibiotarsal joint swelling during the first 6 weeks of infection compared to a wild-type control. The severity of joint swelling was restored to wild-type levels in mice infected with an arp mutant clone complemented in cis. Interestingly, the reduced swelling of joint tissues exhibited by mice infected with the arp deletion mutant did not directly correspond to reduced underlying arthritis. Histopathology data at 2 weeks postinfection showed some reduction in arthritis severity caused by the arp mutant clone; however, by 8 weeks, no significant difference was observed between joint tissues infected by the wild-type or arp mutant clones. The spirochete load in the joint tissues of mice infected with the arp mutant was found to be greater than that exhibited by the wild-type control. Our findings demonstrate that this lipoprotein contributes to the generation of early-onset joint swelling and suggests that arp expression has a negative secondary effect on total spirochete numbers in joint tissues.

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.A naplasma marginale is a tick-associated bacterium and the etiologic agent of bovine anaplasmosis, a disease that causes considerable losses to both dairy and beef industries worldwide (1, 2). Although organisms of this species are principally pathogenic to cattle, they are also found in other ruminants, such as water buffalo and deer (3).The transmission cycle of A. marginale has been well documented and indicates that the success of this pathogen depends on its ability to adapt to its invertebrate and vertebrate hosts. In the tick, during its transit from the midgut to the salivary glands, A. marginale has to overcome different tissue barriers and defense mechanisms in order to ensure its transmission to the vertebrate host (4-7). In cattle, A. marginale replicates within mature erythrocytes, producing an acute disease characterized by hemolytic anemia. However, one of the most important features of the biology of these bacteria is the lifelong persistent infection of its ruminant host, achieved by evasion of the immune system using a mechanism of antigenic variation in which different variants of outer membrane proteins Msp2 and Msp3 are expressed. These persistently infected cattle remain a reservoir of A. marginale organisms for continued tick transmission (8-11).The ability of A. marginale to thrive in such diverse environments is mediated by differential gene transcription (12). Hence, the identification and characterization of these genes using recombinant DNA technologies is not only central to understanding the biology and pathogenesis of these organisms but also for the development of drug therapies and vaccines for the control of anaplasmosis. Recently, the use of transposon mutagenesis in the A. marginale Virginia strain to create insertional mutations was demonstrated (13). Delivery of a plasmid containing the Himar1 transposon and the A7 transpo...

Efforts to develop effective and safe drugs for treatment of tuberculosis require preclinical evaluation in animal models. Alongside efficacy testing of novel therapies, effects on pulmonary pathology and disease progression are monitored by using histopathology images from these infected animals. to compare the severity of disease across treatment cohorts, pathologists have historically assigned a semi-quantitative histopathology score that may be subjective in terms of their training, experience, and personal bias. Manual histopathology therefore has limitations regarding reproducibility between studies and pathologists, potentially masking successful treatments. This report describes a pathologist-assistive software tool that reduces these user limitations, while providing a rapid, quantitative scoring system for digital histopathology image analysis. The software, called 'Lesion Image Recognition and Analysis' (LIRA), employs convolutional neural networks to classify seven different pathology features, including three different lesion types from pulmonary tissues of the C3HeB/FeJ tuberculosis mouse model. LIRA was developed to improve the efficiency of histopathology analysis for mouse tuberculosis infection models, this approach has also broader applications to other disease models and tissues. the full source code and documentation is available from https://Github. com/TB-imaging/LIRA.

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.