Data analysis workflow for the detection of canine vector-borne pathogens using 16 S rRNA Next-Generation Sequencing.

BACKGROUND: Vector-borne diseases (VBDs) impact both human and veterinary medicine and pose special public health challenges. The main bacterial vector-borne pathogens (VBPs) of importance in veterinary medicine include Anaplasma spp., Bartonella spp., Ehrlichia spp., and Spotted Fever Group Rickettsia. Taxon-targeted PCR assays are the current gold standard for VBP diagnostics but limitations on the detection of genetically diverse organisms support a novel approach for broader detection of VBPs. We present a methodology for genetic characterization of VBPs using Next-Generation Sequencing (NGS) and computational approaches. A major advantage of NGS is the ability to detect multiple organisms present in the same clinical sample in an unsupervised (i.e. non-targeted) and semi-quantitative way. The Standard Operating Procedure (SOP) presented here combines industry-standard microbiome analysis tools with our ad-hoc bioinformatic scripts to form a complete analysis pipeline accessible to veterinary scientists and freely available for download and use at https://github.com/eltonjrv/microbiome.westernu/tree/SOP . RESULTS: We tested and validated our SOP by mimicking single, double, and triple infections in genomic canine DNA using serial dilutions of plasmids containing the entire 16 S rRNA gene sequence of (A) phagocytophilum, (B) v. berkhoffii, and E. canis. NGS with broad-range 16 S rRNA primers followed by our bioinformatics SOP was capable of detecting these pathogens in biological replicates of different dilutions. These results illustrate the ability of NGS to detect and genetically characterize multi-infections with different amounts of pathogens in a single sample. CONCLUSIONS: Bloodborne microbiomics & metagenomics approaches may help expand the molecular diagnostic toolbox in veterinary and human medicine. In this paper, we present both in vitro and in silico detailed protocols that can be combined into a single workflow that may provide a significant improvement in VBP diagnostics and also facilitate future applications of microbiome research in veterinary medicine.

Can the Mechanism of Injury Impact the Location of a Mandibular Fracture? A Systematic Review.

Purpose: Mandible fractures are the second most common fractures of the facial skeleton because of the prominent position of the lower jaw. The purpose of this study was to calculate the prevalence of mandibular fractures based on their causes and locations. Materials and Method: A systematic search of 3 electronic databases from January 2010 and January 2020 was conducted following the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines. There were 359 articles identified for screening against selection criteria. The search identified 39 articles to be included in our analysis. Results: A total of 20,135 patients with 31,468 mandible fractures in this review. There was a 76% male predominance. The third decade was the most common age group (21-30). Motor vehicle accidents (39.89%) were the leading cause of mandible fractures, followed by falls (27.72%) and violence (25.35%). Condylar fractures are the most common cause of MVA and fall (33.11%, 50% respectively). Mandible body fractures are the second most common type of MVA injury (17.06%). When it came to violence, the angle of the mandible was the most common site (31.73%). Conclusions: The prevalence of mandible fractures was higher in male patients in the current study, particularly in the second and third decades of life. Road traffic accidents were the most common cause, and the condylar process of the mandible was the most frequently affected region. Demographic data such as age, gender, and mechanism of injury can help surgeons predict and identify specific areas of mandibular fracture.

Bartonella rochalimae Detection by a Sensitive and Specific PCR Platform.

Bartonella rochalimae is an emerging zoonotic pathogen present in the United States, South America, and Europe. The molecular detection of B. rochalimae frequently relies on polymerase chain reaction (PCR) assays that target the genus Bartonella coupled with DNA sequencing for species determination. However, the presence of other Bartonella spp. in the sample being tested may result in false-negative results for B. rochalimae, especially when Sanger sequencing is used. We developed a sensitive and specific quantitative PCR platform for B. rochalimae by targeting the intergenic transcribed spacer, gltA, and rpoB genes, which are recommended for subtyping characterization. This PCR platform achieved the limit of detection between five and 10 genomic equivalents per reaction and did not amplify DNA from other Bartonella species or selected hosts. This PCR platform is a fast and cost-effective option to be used in epidemiological evaluations of reservoirs and vectors and in detecting and quantifying B. rochalimae infection in humans.

Immunotherapies: Exploiting the Immune System for Cancer Treatment.

Cancer is a condition that has plagued humanity for thousands of years, with the first depictions dating back to ancient Egyptian times. However, not until recent decades have biological therapeutics been developed and refined enough to safely and effectively combat cancer. Three unique immunotherapies have gained traction in recent decades: adoptive T cell transfer, checkpoint inhibitors, and bivalent antibodies. Each has led to clinically approved therapies, as well as to therapies in preclinical and ongoing clinical trials. In this review, we outline the method by which these 3 immunotherapies function as well as any major immunotherapeutic drugs developed for treating a variety of cancers.

Molecular Characterization of Tandem Repeat Protein 36 Gene of Ehrlichia canis Detected in Naturally Infected Dogs from Peru.

Ehrlichia spp. are emerging infectious pathogens, especially in the Americas. Although Ehrlichia canis is primarily a parasite of dogs, polymerase chain reaction-confirmed human infections have been reported from Mexico, Venezuela, and Costa Rica. This study reports the presence of E. canis DNA in 13.7% of 205 dogs from urban areas in Peru and of those, five were analyzed for phylogenetic variation using the Tandem Repeat Protein 36 (TRP36) gene. The use of the TRP36 gene for such analysis was validated against 16S rRNA and heat shock protein genes using Shannon's entropy bioinformatic approach. When compared with other E. canis strains previously reported, three unique and novel E. canis strains were detected. In addition, the TRP36 amino acid tandem repeat sequences of the Peruvian strains share close similarity to an E. canis strain detected from four human blood bank samples in Costa Rica. This study reports for the first time domestic dogs infected with E. canis strains closely related to a zoonotic strain, which may be of public health concern as dogs can be chronically infected with this pathogen.

Emergency preparedness of veterans and nonveterans.

This study examined statistical differences in levels of disaster preparedness between former members of the US Armed Forces (veterans) and civilians (nonveterans). It was hypothesized that veterans would exhibit a higher degree of disaster preparedness as compared to their nonveteran counterparts as a consequence of their training and life experience. Furthermore, if this were proven to be valid, the finding would identify this cohort as an ideal target audience for emergency and disaster preparedness education efforts. A four-page survey consisting principally of closed-ended questions about emergency preparedness was written to measure these differences. Most of the questions required respondents to rank their answers according to a five-step Likert Scale. The survey could be completed either in hard copy or online from September 2014 to January 2015. Ultimately, 113 surveys were returned for evaluation. Of those respondents, 62 were veterans and 51 were nonveterans. The responses were analyzed using one-way analysis of variance tests for statistical significance using the 95 percent confidence standard for each tested value. The results support that veterans are more prepared for domestic emergencies than nonveterans. In addition, veterans were more willing to provide leadership and direction to others in an effort to assist emergency managers in responding to domestic disasters. It is for these reasons that emergency managers should consider targeting veterans for disaster preparedness training to help ensure effective and efficient responses to emergencies.