Genetic Engineering of Trypanosoma (Dutonella) vivax and In Vitro Differentiation under Axenic Conditions

Trypanosoma vivax is one of the most common parasites responsible for animal trypanosomosis, and although this disease is widespread in Africa and Latin America, very few studies have been conducted on the parasite's biology. This is in part due to the fact that no reproducible experimental methods had been developed to maintain the different evolutive forms of this trypanosome under laboratory conditions. Appropriate protocols were developed in the 1990s for the axenic maintenance of three major animal Trypanosoma species: T. b. brucei, T. congolense and T. vivax. These pioneer studies rapidly led to the successful genetic manipulation of T. b. brucei and T. congolense. Advances were made in the understanding of these parasites' biology and virulence, and new drug targets were identified. By contrast, challenging in vitro conditions have been developed for T. vivax in the past, and this per se has contributed to defer both its genetic manipulation and subsequent gene function studies. Here we report on the optimization of non-infective T. vivax epimastigote axenic cultures and on the process of parasite in vitro differentiation into metacyclic infective forms. We have also constructed the first T. vivax specific expression vector that drives constitutive expression of the luciferase reporter gene. This vector was then used to establish and optimize epimastigote transfection. We then developed highly reproducible conditions that can be used to obtain and select stably transfected mutants that continue metacyclogenesis and are infectious in immunocompetent rodents

Complete Genome Characterisation of a Novel 26th Bluetongue Virus Serotype from Kuwait

Bluetongue virus is the “type” species of the genus Orbivirus, family Reoviridae. Twenty four distinct bluetongue virus (BTV) serotypes have been recognized for decades, any of which is thought to be capable of causing “bluetongue” (BT), an insect-borne disease of ruminants. However, two further BTV serotypes, BTV-25 (Toggenburg orbivirus, from Switzerland) and BTV-26 (from Kuwait) have recently been identified in goats and sheep, respectively. The BTV genome is composed of ten segments of linear dsRNA, encoding 7 virus-structural proteins (VP1 to VP7) and four distinct non-structural (NS) proteins (NS1 to NS4). We report the entire BTV-26 genome sequence (isolate KUW2010/02) and comparisons to other orbiviruses. Highest identity levels were consistently detected with other BTV strains, identifying KUW2010/02 as BTV. The outer-core protein and major BTV serogroup-specific antigen “VP7” showed 98% aa sequence identity with BTV-25, indicating a common ancestry. However, higher level of variation in the nucleotide sequence of Seg-7 (81.2% identity) suggests strong conservation pressures on the protein of these two strains, and that they diverged a long time ago. Comparisons of Seg-2, encoding major outer-capsid component and cell-attachment protein “VP2” identified KUW2010/02 as 26th BTV, within a 12th Seg-2 nucleotype [nucleotype L]. Comparisons of Seg-6, encoding the smaller outer capsid protein VP5, also showed levels of nt/aa variation consistent with identification of KUW2010/02 as BTV-26 (within a 9th Seg-6 nucleotype - nucleotype I). Sequence data for Seg-2 of KUW2010/02 were used to design four sets of oligonucleotide primers for use in BTV-26, type-specific RT-PCR assays. Analyses of other more conserved genome segments placed KUW2010/02 and BTV-25/SWI2008/01 closer to each other than to other “eastern” or “western” BTV strains, but as representatives of two novel and distinct geographic groups (topotypes). Our analyses indicate that all of the BTV genome segments have evolved under strong purifying selection

A prospective case series for a minimally invasive internal fixation device for anterior pelvic ring fractures

Background: External fixation is commonly used as a means of definitive fixation of pelvic fractures. Pin site infection is common, with some cases of osteomyelitis and inpatient nursing can be challenging. The aim of this study is to report the outcomes and complications of an alternative minimally invasive technique, known as INFIX, utilising spinal pedicle screws inserted into the supra-acetabular bone and connected by a subcutaneous rod. Methods: A single-centre prospective case series was performed. The primary outcome measures were fracture stability and displacement at time of implant removal and intra- and post-operative complications. Results: Twenty-one patients were recruited, with 85.7 % of fractures being lateral compression type. Mean follow-up was 342 days. Mean application time was 51 min (range 44–65). Nineteen were removed electively, with mean time to removal 109 days. All cases were stable with no displacement. Two cases were removed emergently, one due to wound infection and the other due to lateral femoral cutaneous nerve neuropathic pain. Twelve patients sustained a lateral femoral cutaneous nerve palsy, with 20/42 nerves being affected. Improvement in all lateral femoral cutaneous nerve symptoms were reported with removal. Nine patients developed asymptomatic heterotopic ossification, and there were three deep infections and one symptomatic due to the bar. Conclusions: Minimally invasive internal fixation with the INFIX for anterior pelvic ring fractures is an alternative to anterior external fixation. However, a higher rate of lateral femoral cutaneous nerve palsy is noted, and the implant is not well tolerated by all patients. Further studies are required to define fracture types and patients best suited to the technique and how LFCN complications may be minimised. Trial registration: ACTRN12616001421426. Registered 12 October 2016. Retrospectively registered