Evaluating Trends in Strangles Outbreaks Using Temperature and Precipitation Data in the United States of America for 2018-2022.

Strangles is a highly contagious upper respiratory infection of equids that is globally distributed. The causative agent of strangles, Streptococcus equi subspecies equi, can be spread through indirect contact with infected fomites, and studies have shown this microbe to live well in varying environmental conditions. The purpose of this study was to analyze strangles case numbers across the United States of America from 2018 to 2022 to investigate potential temporal or weather patterns associated with outbreaks. Diagnosed case records were obtained from the Equine Disease Communication Center, university databases, government agencies, or veterinary diagnostic labs, and geographic information systems (GISs) were used to map cases and to acquire relevant meteorological data from outbreak areas. These data were analyzed using logistic regression to explore trends that occur between outbreaks and changes in temperature and precipitation. Initial review of weather data suggested monthly changes in strangles case numbers corresponded with changing seasons. Logistic regression indicated that changes in monthly average temperature and minimum temperature were significantly associated with increased or decreased odds of strangles outbreaks, respectively. Future analyses should focus on weather data isolated within a smaller region or state to better resolve trends in strangles outbreaks throughout the continental USA.

Comparison of DNA Extraction and Amplification Techniques for Use with Engorged Hard-Bodied Ticks.

Tick-borne infections are a serious threat to humans, livestock, and companion animals in many parts of the world, often leading to high morbidity and mortality rates, along with decreased production values and/or costly treatments. The prevalence of the microbes responsible for these infections is typically assessed by the molecular identification of pathogens within the tick vectors. Ticks sampled from animals are often engorged with animal blood, presenting difficulties in the amplification of nucleic acids due to the inhibitory effects of mammalian blood on the enzymes used in polymerase chain reactions (PCRs). This study tested two tick preparation methods, three methods of DNA extraction, and four commercially available DNA polymerases to determine the most reliable method of extracting and amplifying DNA from engorged ticks. Our study found that the phenol-chloroform extraction method yielded the highest concentration of DNA, yet DNA extracted by this method was amplified the least successfully. Thermo Scientific's Phusion Plus PCR Master Mix was the best at amplifying the tick 16s rRNA gene, regardless of extraction method. Finally, our study identified that using the Qiagen DNeasy Blood & Tissues kit for DNA extraction coupled with either Phusion Plus PCR Master Mix or GoTaq DNA polymerase Master Mix is the best combination for the optimized amplification of DNA extracted from engorged ticks.

Creating Custom Digital Assistants for the Scientific Laboratory using the HelixAI Platform.

Voice technology and fully virtual digital assistants are becoming increasingly prevalent in many industries, including the scientific laboratory. This environment can greatly benefit from the use of hands-free digital assistants due to the fact that scientists regularly need access to tools and information while performing bench work. The use of a digital assistant in this environment has the potential to streamline laboratory work and reduce the chances of human error due to contamination and the context switching involved in moving between experiments and information storage media. Because the particular protocols and reagents used by each laboratory are often different, there is a need to create custom digital assistants for individual laboratories. In this technical brief we describe a custom software and web application, referred to as the HelixAI platform, that can be used to create digital assistants for individual scientific laboratories. Digital assistants created with this platform can be accessed through any Alexa-enabled smart speaker device. Here we describe the process by which labs can use this platform to create their own digital assistants, along with a description of the underlying technology. An assistant containing information from the scientific company New England Biolabs (NEB) has been created using this software and will serve as an example throughout this paper.

Adapting a Bacterial Unknowns Project to Online Learning: Using Microsoft PowerPoint To Create an Unknowns Identification Simulation.

The COVID-19 pandemic forced institutions to move to online learning abruptly during the spring 2020 semester. As a result of this swift change, many microbiology laboratory courses chose to switch all in-class learning activities to simulations. To provide students with a remote bacterial unknowns identification project, we developed an unknowns simulation using Microsoft PowerPoint. This simulation provides students with an assignment that allows them to utilize their knowledge in a cumulative project while attending classes remotely. Because this simulation does not require an internet connection and can be performed on any device that is capable of running Microsoft PowerPoint, it provides an accessible and equitable option for students in different learning environments. In addition, the simulation format is flexible enough to be easily customized for any course or assessment style. We provide here details and examples of how to create an unknowns simulation using Microsoft PowerPoint. In addition, a full template and an example simulation are available for download on GitHub at https://github.com/drhodes-berry-college/BacterialUnknowsSimulation.

Presence of Rickettsia Species in Ticks Collected from Companion Animals in Northeastern Georgia, United States.

Tick-borne diseases are a major threat to both humans and their pets; therefore, it is important to evaluate the prevalence of pathogens carried by ticks on companion animals. In this study, attached and unattached Ixodid ticks were removed from companion animals by a veterinary practice in Hall County, Georgia. DNA was extracted from unengorged adult ticks and each was screened for the presence of Rickettsia spp. by polymerase chain reaction (PCR) and sequenced to determine the species present. Two hundred and four adult hard-bodied ticks were identified to species and Rickettsia spp. were found in 19.6% (n = 38) of the 194 analyzed DNA extracts. Rickettsia montanensis was found in Dermacentor variablis (14.7%; n = 25), Amblyomma maculatum (33.3%; n = 2), and Rhipicephalus sanguineus s.l. ticks (25%; n = 4). One Amblyomma americanum tick contained Rickettsia amblyommatis, while Rickettsia felis was found in one Dermacentor variablis tick, serving as the first report of Rickettsia felis in a tick in this region and within this tick vector. This study suggests that there is a risk of companion animals contracting a species of Rickettsia from a tick bite in northeastern Georgia, indicating a need for more investigation and highlighting the importance of tick prevention on pets.

Development and optimization of OspC chimeritope vaccinogens for Lyme disease.

Experimental Outer surface protein (Osp) C based subunit chimeritope vaccinogens for Lyme disease (LD) were assessed for immunogenicity, structure, ability to elicit antibody (Ab) responses to divergent OspC proteins, and bactericidal activity. Chimeritopes are chimeric epitope based proteins that consist of linear epitopes derived from multiple proteins or multiple variants of a protein. An inherent advantage to chimeritope vaccinogens is that they can be constructed to trigger broadly protective Ab responses. Three OspC chimeritope proteins were comparatively assessed: Chv1, Chv2 and Chv3. The Chv proteins possess the same set of 18 linear epitopes derived from 9 OspC type proteins but differ in the physical ordering of epitopes or by the presence or absence of linkers. All Chv proteins were immunogenic in mice and rats eliciting high titer Ab. Immunoblot and enzyme linked immunosorbent assays demonstrated that the Chv proteins elicit IgG that recognizes a diverse array of OspC type proteins. The panel included OspC proteins produced by N. American and European strains of the LD spirochetes. Rat anti-Chv antisera uniformly labeled intact, non-permeabilized Borreliella burgdorferi demonstrating that vaccinal Ab can bind to targets that are naturally presented on the spirochete cell surface. Vaccinal Ab also displayed potent complement dependent-Ab mediated killing activity. This study highlights the ability of OspC chimeritopes to serve as vaccinogens that trigger potentially broadly protective Ab responses. In addition to the current use of an OspC chimeritope in a canine LD vaccine, chimeritopes can serve as key components of human LD subunit vaccines.

Genetic characterization and role in virulence of the ribonucleotide reductases of Streptococcus sanguinis.

Streptococcus sanguinis is a cause of infective endocarditis and has been shown to require a manganese transporter called SsaB for virulence and O2 tolerance. Like certain other pathogens, S. sanguinis possesses aerobic class Ib (NrdEF) and anaerobic class III (NrdDG) ribonucleotide reductases (RNRs) that perform the essential function of reducing ribonucleotides to deoxyribonucleotides. The accompanying paper (Makhlynets, O., Boal, A. K., Rhodes, D. V., Kitten, T., Rosenzweig, A. C., and Stubbe, J. (2014) J. Biol. Chem. 289, 6259-6272) indicates that in the presence of O2, the S. sanguinis class Ib RNR self-assembles an essential diferric-tyrosyl radical (Fe(III)2-Y(•)) in vitro, whereas assembly of a dimanganese-tyrosyl radical (Mn(III)2-Y(•)) cofactor requires NrdI, and Mn(III)2-Y(•) is more active than Fe(III)2-Y(•) with the endogenous reducing system of NrdH and thioredoxin reductase (TrxR1). In this study, we have shown that deletion of either nrdHEKF or nrdI completely abolishes virulence in an animal model of endocarditis, whereas nrdD mutation has no effect. The nrdHEKF, nrdI, and trxR1 mutants fail to grow aerobically, whereas anaerobic growth requires nrdD. The nrdJ gene encoding an O2-independent adenosylcobalamin-cofactored RNR was introduced into the nrdHEKF, nrdI, and trxR1 mutants. Growth of the nrdHEKF and nrdI mutants in the presence of O2 was partially restored. The combined results suggest that Mn(III)2-Y(•)-cofactored NrdF is required for growth under aerobic conditions and in animals. This could explain in part why manganese is necessary for virulence and O2 tolerance in many bacterial pathogens possessing a class Ib RNR and suggests NrdF and NrdI may serve as promising new antimicrobial targets.

Streptococcus sanguinis class Ib ribonucleotide reductase: high activity with both iron and manganese cofactors and structural insights.

Streptococcus sanguinis is a causative agent of infective endocarditis. Deletion of SsaB, a manganese transporter, drastically reduces S. sanguinis virulence. Many pathogenic organisms require class Ib ribonucleotide reductase (RNR) to catalyze the conversion of nucleotides to deoxynucleotides under aerobic conditions, and recent studies demonstrate that this enzyme uses a dimanganese-tyrosyl radical (Mn(III)2-Y(•)) cofactor in vivo. The proteins required for S. sanguinis ribonucleotide reduction (NrdE and NrdF, α and ß subunits of RNR; NrdH and TrxR, a glutaredoxin-like thioredoxin and a thioredoxin reductase; and NrdI, a flavodoxin essential for assembly of the RNR metallo-cofactor) have been identified and characterized. Apo-NrdF with Fe(II) and O2 can self-assemble a diferric-tyrosyl radical (Fe(III)2-Y(•)) cofactor (1.2 Y(•)/ß2) and with the help of NrdI can assemble a Mn(III)2-Y(•) cofactor (0.9 Y(•)/ß2). The activity of RNR with its endogenous reductants, NrdH and TrxR, is 5,000 and 1,500 units/mg for the Mn- and Fe-NrdFs (Fe-loaded NrdF), respectively. X-ray structures of S. sanguinis NrdIox and Mn(II)2-NrdF are reported and provide a possible rationale for the weak affinity (2.9 µM) between them. These streptococcal proteins form a structurally distinct subclass relative to other Ib proteins with unique features likely important in cluster assembly, including a long and negatively charged loop near the NrdI flavin and a bulky residue (Thr) at a constriction in the oxidant channel to the NrdI interface. These studies set the stage for identifying the active form of S. sanguinis class Ib RNR in an animal model for infective endocarditis and establishing whether the manganese requirement for pathogenesis is associated with RNR.

Assessment of the potential contribution of the highly conserved C-terminal motif (C10) of Borrelia burgdorferi outer surface protein C in transmission and infectivity.

OspC is produced by all species of the Borrelia burgdorferi sensu lato complex and is required for infectivity in mammals. To test the hypothesis that the conserved C-terminal motif (C10) of OspC is required for function in vivo, a mutant B. burgdorferi strain (B31::ospCΔC10) was created in which ospC was replaced with an ospC gene lacking the C10 motif. The ability of the mutant to infect mice was investigated using tick transmission and needle inoculation. Infectivity was assessed by cultivation, qRT-PCR, and measurement of IgG antibody responses. B31::ospCΔC10 retained the ability to infect mice by both needle and tick challenge and was competent to survive in ticks after exposure to the blood meal. To determine whether recombinant OspC protein lacking the C-terminal 10 amino acid residues (rOspCΔC10) can bind plasminogen, the only known mammalian-derived ligand for OspC, binding analyses were performed. Deletion of the C10 motif resulted in a statistically significant decrease in plasminogen binding. Although deletion of the C10 motif influenced plasminogen binding, it can be concluded that the C10 motif is not required for OspC to carry out its critical in vivo functions in tick to mouse transmission.

Disulfide-mediated oligomer formation in Borrelia burgdorferi outer surface protein C, a critical virulence factor and potential Lyme disease vaccine candidate.

Borrelia burgdorferi OspC is an outer membrane lipoprotein required for the establishment of infection in mammals. Due to its universal distribution among B. burgdorferi sensu lato strains and high antigenicity, it is being explored for the development of a next-generation Lyme disease vaccine. An understanding of the surface presentation of OspC will facilitate efforts to maximize its potential as a vaccine candidate. OspC forms homodimers at the cell surface, and it has been hypothesized that it may also form oligomeric arrays. Here, we employ site-directed mutagenesis to test the hypothesis that interdimeric disulfide bonds at cysteine 130 (C130) mediate oligomerization. B. burgdorferi B31 ospC was replaced with a C130A substitution mutant to yield strain B31::ospC(C130A). Recombinant protein was also generated. Disulfide-bond-dependent oligomer formation was demonstrated and determined to be dependent on C130. Oligomerization was not required for in vivo function, as B31::ospC(C130A) retained infectivity and disseminated normally. The total IgG response and the induced isotype pattern were similar between mice infected with untransformed B31 and those infected with the B31::ospC(C130A) strain. These data indicate that the immune response to OspC is not significantly altered by formation of OspC oligomers, a finding that has significant implications in Lyme disease vaccine design.