Neorickettsia risticii surface-exposed proteins: proteomics identification, recognition by naturally-infected horses, and strain variations

Neorickettsia risticii is the Gram-negative, obligate, and intracellular bacterial pathogen responsible for Potomac horse fever (PHF): an important acute systemic disease of horses. N. risticii surface proteins, critical for immune recognition, have not been thoroughly characterized. In this paper, we identified the 51-kDa antigen (P51) as a major surface-exposed outer membrane protein of older and contemporary strains of N. risticii through mass spectrometry of streptavidin-purified biotinylated surface-labeled proteins. Western blot analysis of sera from naturally-infected horses demonstrated universal and strong recognition of recombinant P51 over other Neorickettsia recombinant proteins. Comparisons of amino acid sequences for predicted secondary structures of P51, as well as Neorickettsia surface proteins 2 (Nsp2) and 3 (Nsp3) among N. risticii strains from horses with PHF during a 26-year period throughout the United States revealed that the majority of variations among strains were concentrated in regions predicted to be external loops of their β-barrel structures. Large insertions or deletions occurred within a tandem-repeat region in Ssa3. These data demonstrate patterns of geographical association for P51 and temporal associations for Nsp2, Nsp3, and Ssa3, indicating evolutionary trends for these Neorickettsia surface antigen genes. This study showed N. risticii surface protein population dynamics, providing groundwork for designing immunodiagnostic targets for PHF

BOVINE HERPESVIRUS 1 ENTRY BY A LOW PH-DEPENDENT, SPHINGOMYELIN-INDEPENDENT, ENDOCYTIC PATHWAY

Bovine herpesvirus (BoHV-1) is an alphaherpesvirus that poses a significant challenge to health and welfare in the cattle industry. We investigated the cellular entry route utilized by BoHV-1. We report that BoHV-1 enters Madin Darby bovine kidney (MDBK) cells, bovine turbinate cells, and African green monkey kidney (Vero) cells via a low pH-mediated endocytosis pathway that is independent of host cell sphingomyelin. Treatment of MDBK cells with hypertonic medium, which inhibits receptor-mediated endocytosis, prevented infection as measured by a beta-galactosidase reporter assay. Treatment of cells with noncytotoxic concentrations of the lysosomotropic agents ammonium chloride and monensin, which block the acidification of endosomes, inhibited BoHV-1 entry in a concentration-dependent fashion. The kinetics of endocytic uptake of BoHV-1 from the cell surface was rapid (t1/2 of ~ 5 min). Time-of-addition experiments indicated that the lysosomotropic agents acted at early times post-infection, consistent with entry. Inactivation of virions by pretreatment with mildly acidic pH is a hallmark characteristic of viruses that utilize a low pH-activated entry pathway. When BoHV-1 particles were exposed to pH 5.0 in the absence of target membrane, infectivity was markedly reduced. Treatment of MDBK cells with noncytotoxic concentrations of staphylococcus aureus-derived sphingomyelinase successfully depleted cellular surface-exposed sphingomyelin, but did not significantly inhibit BoHV-1 infection as measured by beta-galactosidase reporter assay. Treatment of MDBK cells with noncytotoxic concentrations of the functional inhibitors of acid sphingomyelinase imipramine and amitriptyline, which induce degradation of the cellular enzyme, also did not significantly inhibit BoHV-1 beta-galactosidase production. Depletion of viral envelope sphingomyelin significantly inhibited viral beta-galactosidase production. Together, these results support a model of BoHV-1 infection in which low endosomal pH is a critical host trigger for fusion of the viral envelope with an endocytic membrane, and necessary for successful infection of the target cell, and in which the virus does not require cellular sphingomyelin or acid sphingomyelinase, but does require viral envelope sphingomyelin

Bovine Herpesvirus 1 Entry by a Low-pH Endosomal Pathway

BoHV-1 is a ubiquitous pathogen affecting cattle populations worldwide. Infection can result in complicated, polymicrobial infections due to the immunosuppressive properties of the virus. While there are vaccines on the market, they only limit disease severity and spread but do not prevent infection. The financial and animal welfare ramifications of this virus are significant, and in order to develop more effective prevention and treatment regimens, a more complete understanding of the initial steps in viral infection is necessary. This research establishes the initial entry pathway of BoHV-1, which provides a foundation for future development of effective treatments and preventative vaccines. Additionally, it allows comparisons to the entry pathways of other alphaherpesviruses, such as HSV-1. Bovine herpesvirus 1 (BoHV-1) is an alphaherpesvirus that poses a significant challenge to health and welfare in the cattle industry. We investigated the cellular entry route utilized by BoHV-1. We report that BoHV-1 enters Madin Darby bovine kidney (MDBK) cells, bovine turbinate cells, and African green monkey kidney (Vero) cells via a low-pH-mediated endocytosis pathway. Treatment of MDBK cells with hypertonic medium, which inhibits receptor-mediated endocytosis, prevented infection as measured by a beta-galactosidase reporter assay. Treatment of cells with noncytotoxic concentrations of the lysosomotropic agents ammonium chloride and monensin, which block the acidification of endosomes, inhibited BoHV-1 entry in a concentration-dependent fashion. The kinetics of endocytic uptake of BoHV-1 from the cell surface was rapid (50% uptake by ∼5 min). Time-of-addition experiments indicated that the lysosomotropic agents acted at early times postinfection, consistent with entry. Inactivation of virions by pretreatment with mildly acidic pH is a hallmark characteristic of viruses that utilize a low-pH-activated entry pathway. When BoHV-1 particles were exposed to pH 5.0 in the absence of target membrane, infectivity was markedly reduced. Lastly, treatment of cells with the proteasome inhibitor MG132 inhibited BoHV-1 entry in a concentration-dependent manner. Together, these results support a model of BoHV-1 infection in which low endosomal pH is a critical host trigger for fusion of the viral envelope with an endocytic membrane and necessary for successful infection of the target cell. IMPORTANCE BoHV-1 is a ubiquitous pathogen affecting cattle populations worldwide. Infection can result in complicated, polymicrobial infections due to the immunosuppressive properties of the virus. While there are vaccines on the market, they only limit disease severity and spread but do not prevent infection. The financial and animal welfare ramifications of this virus are significant, and in order to develop more effective prevention and treatment regimens, a more complete understanding of the initial steps in viral infection is necessary. This research establishes the initial entry pathway of BoHV-1, which provides a foundation for future development of effective treatments and preventative vaccines. Additionally, it allows comparisons to the entry pathways of other alphaherpesviruses, such as HSV-1

Autochthonous Transmission of Coccidioides in Animals, Washington, USA

We report 5 cases of coccidioidomycosis in animals that were acquired within Washington, USA, and provide further evidence for the environmental endemicity of Coccidioides immitis within the state. Veterinarians should consider coccidioidomycosis in animals with compatible clinical signs that reside in, or have traveled to, south central Washington

Role of Sphingomyelin in Alphaherpesvirus Entry

Bovine herpesvirus 1 (BoHV-1) is an alphaherpesvirus that causes disease in cattle populations worldwide. Sphingomyelin (SM) is the most abundant sphingolipid in the mammalian cell membrane, where it preferentially associates with cholesterol to form lipid raft domains. SM is a substrate for the lysosome-resident enzyme acid sphingomyelinase, which plays a role in cell membrane repair following injury. Treatment of cells with noncytotoxic concentrations of -derived sphingomyelinase successfully reduced cell surface-exposed sphingomyelin but did not significantly inhibit BoHV-1 entry and infection, as measured by the beta-galactosidase reporter assay. Interestingly, entry of the porcine alphaherpesvirus pseudorabies virus (PRV) was inhibited by sphingomyelin-depletion of cells. Treatment of BoHV-1 particles with sphingomyelinase inhibited viral entry activity, suggesting that viral SM plays a role in BoHV-1 entry, while cellular SM does not. Treatment of cells with noncytotoxic concentrations of the functional inhibitors of host acid sphingomyelinase, imipramine and amitriptyline, which induce degradation of the cellular enzyme, did not significantly inhibit BoHV-1 entry. In contrast, inhibition of cellular acid sphingomyelinase inhibited PRV entry. Entry of the human alphaherpesvirus herpes simplex virus 1 (HSV-1) was independent of both host SM and acid sphingomyelinase, in a manner similar to BoHV-1. Together, the results suggest that among the alphaherpesviruses, there is variability in entry requirements for cellular sphingomyelin and acid sphingomyelinase activity. Bovine herpesvirus 1 (BoHV-1) is an ubiquitous pathogen affecting cattle populations worldwide. Infection can result in complicated, polymicrobial infections due to the immunosuppressive properties of the virus. Available vaccines limit disease severity and spread but do not prevent infection. The financial and animal welfare ramifications of BoHV-1 are significant. In order to develop more effective prevention and treatment regimens, a more complete understanding of the initial steps in viral infection is necessary. We recently identified a low pH endocytosis pathway for BoHV-1. Here, we examine the role of cellular factors responsible for membrane integrity and repair in alphaherpesviral entry. This study allows comparisons of the BoHV-1 entry pathway with those of other alphaherpesviruses (pseudorabies virus [PRV] and herpes simplex virus 1 [HSV-1]). Lastly, this is the first report of sphingomyelin and lysosomal sphingomyelinase playing a role in the entry of a herpesvirus. The results may lead to the development of more effective prevention and treatment regimens

Inflammatory Effects of Thickened Water on the Lungs in a Murine Model of Recurrent Aspiration

ObjectiveLiquid thickeners are commonly recommended in individuals with dysphagia and recurrent aspiration as a strategy for pneumonia prevention. The goal of this study was to examine the effects of small amounts of aspirated liquid thickener on the lungs.Study designAnimal model. Prospective small animal clinical trial.MethodsAdult Sprague Dawley rats (n = 19) were divided into two groups and underwent three intratracheal instillations of either xanthan gum-based nectar-thick water (0.1-0.25 mL/kg) or water-only control over the course of 8 days. Blood was collected from a peripheral vein on days 1 and 8 and submitted for complete blood count (CBC) analysis. Rats were euthanized 10 days after the last instillation, and the lungs were harvested. Histopathology was conducted on lung specimens by a blinded licensed veterinary pathologist and scored for evidence of lung injury and pneumonia.ResultsFifteen animals (8 nectar-thickener group, 7 control group) survived until the endpoint of the study (day 18). Serum CBC did not show abnormalities at any timepoint in either group. Histological evidence of lung inflammation and edema were significantly greater in the nectar-thick group compared to controls (P < .05). Signs of inflammation included aggregates of foamy macrophages, expansion of bronchiolar lymphoid tissue, and large numbers of eosinophilic intraalveolar crystals. Histiocytic and neutrophilic pneumonia was noted in one animal that received thickened liquids.ConclusionRecurrent aspiration of small amounts of thickened water resulted in significant pulmonary inflammation in a murine model of aspiration. Results of this study support the need for further investigation of liquid thickener safety and its efficacy in reducing the pulmonary complications of swallowing disorders.Level of evidenceNA Laryngoscope, 131:1223-1228, 2021

An immune deficient mouse model for mucopolysaccharidosis IIIA (Sanfilippo syndrome).

Mucopolysaccharidosis III (MPSIII, Sanfilippo syndrome) is a devastating lysosomal storage disease that primarily affects the central nervous system. MPSIIIA is caused by loss-of-function mutations in the gene coding for sulfamidase (N-sulfoglucosamine sulfohydrolase/SGSH) resulting in SGSH enzyme deficiency, a buildup of heparin sulfate and subsequent neurodegeneration. There is currently no cure or disease modifying treatment for MPSIIIA. A mouse model for MPSIIIA was characterized in 1999 and later backcrossed onto the C57BL/6 background. In the present study, a novel immune deficient MPSIIIA mouse model (MPSIIIA-TKO) was created by backcrossing the immune competent, C57BL/6 MPSIIIA mouse to an immune deficient mouse model lacking Rag2, CD47 and Il2rg genes. The resulting mouse model has undetectable SGSH activity, exhibits histological changes consistent with MPSIIIA and lacks T cells, B cells and NK cells. This new mouse model has the potential to be extremely useful in testing human cellular therapies in an animal model as it retains the MPSIIIA disease phenotype while tolerating xenotransplantation

Inflammatory Effects of Thickened Water on the Lungs in a Murine Model of Recurrent Aspiration.

ObjectiveLiquid thickeners are commonly recommended in individuals with dysphagia and recurrent aspiration as a strategy for pneumonia prevention. The goal of this study was to examine the effects of small amounts of aspirated liquid thickener on the lungs.Study designAnimal model. Prospective small animal clinical trial.MethodsAdult Sprague Dawley rats (n = 19) were divided into two groups and underwent three intratracheal instillations of either xanthan gum-based nectar-thick water (0.1-0.25 mL/kg) or water-only control over the course of 8 days. Blood was collected from a peripheral vein on days 1 and 8 and submitted for complete blood count (CBC) analysis. Rats were euthanized 10 days after the last instillation, and the lungs were harvested. Histopathology was conducted on lung specimens by a blinded licensed veterinary pathologist and scored for evidence of lung injury and pneumonia.ResultsFifteen animals (8 nectar-thickener group, 7 control group) survived until the endpoint of the study (day 18). Serum CBC did not show abnormalities at any timepoint in either group. Histological evidence of lung inflammation and edema were significantly greater in the nectar-thick group compared to controls (P < .05). Signs of inflammation included aggregates of foamy macrophages, expansion of bronchiolar lymphoid tissue, and large numbers of eosinophilic intraalveolar crystals. Histiocytic and neutrophilic pneumonia was noted in one animal that received thickened liquids.ConclusionRecurrent aspiration of small amounts of thickened water resulted in significant pulmonary inflammation in a murine model of aspiration. Results of this study support the need for further investigation of liquid thickener safety and its efficacy in reducing the pulmonary complications of swallowing disorders.Level of evidenceNA Laryngoscope, 131:1223-1228, 2021

Mucosal Vaccination with Cyclic Dinucleotide Adjuvants Induces Effective T Cell Homing and IL-17–Dependent Protection against Mycobacterium tuberculosis Infection

Tuberculosis consistently causes more deaths worldwide annually than any other single pathogen, making new effective vaccines an urgent priority for global public health. Among potential adjuvants, STING-activating cyclic dinucleotides (CDNs) uniquely stimulate a cytosolic sensing pathway activated only by pathogens. Recently, we demonstrated that a CDN-adjuvanted protein subunit vaccine robustly protects against tuberculosis infection in mice. In this study, we delineate the mechanistic basis underlying the efficacy of CDN vaccines for tuberculosis. CDN vaccines elicit CD4 T cells that home to lung parenchyma and penetrate into macrophage lesions in the lung. Although CDNs, like other mucosal vaccines, generate B cell-containing lymphoid structures in the lungs, protection is independent of B cells. Mucosal vaccination with a CDN vaccine induces Th1, Th17, and Th1-Th17 cells, and protection is dependent upon both IL-17 and IFN-γ. Single-cell RNA sequencing experiments reveal that vaccination enhances a metabolic state in Th17 cells reflective of activated effector function and implicate expression of Tnfsf8 (CD153) in vaccine-induced protection. Finally, we demonstrate that simply eliciting Th17 cells via mucosal vaccination with any adjuvant is not sufficient for protection. A vaccine adjuvanted with deacylated monophosphoryl lipid A (MPLA) failed to protect against tuberculosis infection when delivered mucosally, despite eliciting Th17 cells, highlighting the unique promise of CDNs as adjuvants for tuberculosis vaccines