Cutaneous leishmaniasis in a newly established treatment centre in the Lay Gayint district, Northwest Ethiopia

Background: Cutaneous leishmaniasis (CL) is a neglected tropical disease that primarily affects the most vulnerable populations. In Ethiopia, where this study took place, CL is an important health problem, however, the incidence of CL is poorly monitored. Objectives: This study took place in a recently established CL treatment centre, at Nefas Mewcha Hospital, Lay Gayint. This area was considered to be endemic for CL, however, no cases of CL from Lay Gayint had previously been officially reported to the Amhara Regional Health Bureau. Methods: Following a CL awareness campaign, a retrospective data review was performed of patients presenting to this centre between July 2019 and March 2021. Basic demographic and clinical data were collected by a nurse and recorded in the logbook of the CL treatment centre. Results: Two hundred and one patients presented for diagnosis and treatment. The age of the patients ranged from 2 to 75 years and 63.2% were males. Most patients were between 10- and 19-years-old. The majority (79.1%) of the patients presented with localised cutaneous leishmaniasis and 20.9% with mucocutaneous leishmaniasis. 98% of the patients tested positive for Leishmania parasites by microscopy. Conclusions: This work underpinned how CL is a major public health problem in the Lay Gayint district. It also shows that raising awareness about CL in the community and providing diagnosis and treatment encouraged patients to travel to seek diagnosis and treatment

Experimental infection of calves by two genetically-distinct strains of rift valley fever virus

Citation: Wilson, W. C., Davis, A. S., Gaudreault, N. N., Faburay, B., Trujillo, J. D., Shivanna, V., . . . Richt, J. A. (2016). Experimental infection of calves by two genetically-distinct strains of rift valley fever virus. Viruses, 8(5). doi:10.3390/v8050145Additional Authors: McVey, D. S.Recent outbreaks of Rift Valley fever in ruminant livestock, characterized by mass abortion and high mortality rates in neonates, have raised international interest in improving vaccine control strategies. Previously, we developed a reliable challenge model for sheep that improves the evaluation of existing and novel vaccines in sheep. This sheep model demonstrated differences in the pathogenesis of Rift Valley fever virus (RVFV) infection between two genetically-distinct wild-type strains of the virus, Saudi Arabia 2001 (SA01) and Kenya 2006 (Ken06). Here, we evaluated the pathogenicity of these two RVFV strains in mixed breed beef calves. There was a transient increase in rectal temperatures with both virus strains, but this clinical sign was less consistent than previously reported with sheep. Three of the five Ken06-infected animals had an early-onset viremia, one day post-infection (dpi), with viremia lasting at least three days. The same number of SA01-infected animals developed viremia at 2 dpi, but it only persisted through 3 dpi in one animal. The average virus titer for the SA01-infected calves was 1.6 logs less than for the Ken06-infected calves. Calves, inoculated with either strain, seroconverted by 5 dpi and showed time-dependent increases in their virus-neutralizing antibody titers. Consistent with the results obtained in the previous sheep study, elevated liver enzyme levels, more severe liver pathology and higher virus titers occurred with the Ken06 strain as compared to the SA01 strain. These results demonstrate the establishment of a virulent challenge model for vaccine evaluation in calves. © 2016 by the authors; licensee MDPI, Basel, Switzerland

Development of animal model, vaccines, and diagnostics for Schmallenberg virus

Doctor of PhilosophyDepartment of Diagnostic Medicine/PathobiologyJuergen RichtSchmallenberg virus (SBV) is a novel orthobunyavirus in the Simbu serogroup, genus Orthobunyavirus, family Peribunyaviridae, and order Bunyavirales. The virus emerged in late 2011 near the German/Dutch border region and was mostly associated with a mild transient disease of sheep and cattle. It is transmitted by biting midges (Culicoides species) and causes abortions, stillbirths, and congenital defects in naïve pregnant sheep and cattle. SBV has spread throughout most of Europe. However, to date, the initial introduction route of the virus to Europe is poorly understood. Consequently, SBV poses a threat to other countries like the US, where competent insect vector species and susceptible livestock populations exist and extensive trade with Europe is conducted. To this end, research work was conducted with the following three aims: (1) development of a large animal model for SBV infection; (2) subunit vaccine development for SBV; and (3) development and evaluation of Schmallenberg disease diagnostics. This dissertation contains four chapters. The first chapter provides a literature review on SBV emergence, taxonomy, replication, transmission, pathogenesis, diagnosis, and control. The remaining three chapters contain the research conducted to address the above-mentioned three study aims. In chapter two, the clinical, virological, and serological responses in two ruminant models, i.e., cattle and sheep, are presented. Infectious serum was shown to be the best inoculum in both species when compared to infectious cell culture supernatant and brain homogenate. The virological and serological responses to SBV were more apparent in cattle than in sheep. Thus, cattle are recommended as the better SBV infection model for the evaluation of vaccines and diagnostics. Chapter three presents the immunogenicity and efficacy of a baculovirus-expressed subunit vaccine composed of SBV glycoproteins C and N. After vaccination, SBV Gc-specific antibody response was detected in all vaccinated animals. Neither of the vaccines conferred protection against SBV challenge infection. Therefore, future studies should focus on better understanding of the difference in post-translational modifications on Gc protein in different expression systems and subsequent conformational and stability conditions that are crucial for its immunogenicity. Using the different reagents (sera, recombinant proteins, and tissues) generated in the SBV animal model development and vaccine experiments, we tried to develop and evaluate serological and molecular diagnostic assays for SBV. Among the different SBV proteins evaluated for SBV antibody detection, N and Gc were the most reactive antigens, followed by Gn. In the indirect ELISA experiments, the SBV-infected cell lysate was the least reactive. Among the three commercial nucleic acid extraction kits, the GeneReach total RNA extraction kit yielded approximately 10× more SBV in vitro transcribed RNA than the Qiagen and Applied Biosystems extraction kits. Multiplex RT-qPCR using in vitro transcribed SBV RNA resulted in favorable amplification of the L and M genes than the S gene. In order to facilitate full genome sequencing of SBV isolates for diagnostic purposes, we established a full genome sequencing approach for three different SBV isolates: FLI-serum, KSU-serum, and KSU-serum cell passaged, using Next Generation Sequencing. Overall, SBV genomes between each of the tested samples have over 99.9% homology, indicating a low level of molecular evolution at this level of investigation. Overall, these studies have highlighted (i) that cattle is the preferred animal model to evaluate SBV vaccines and diagnostics, (ii) the need for identifying the right protein expression system that ensures proper conformation and stability and, finally, (iii) that both M and L segment PCRs are more specific than the S segment PCR, which is relevant especially in areas where other Simbu serogroup viruses are endemic

Prevalence of Leishmania RNA virus in Leishmania parasites in patients with tegumentary leishmaniasis: A systematic review and meta-analysis.

BackgroundCutaneous leishmaniasis is caused by different protozoan parasites of the genus Leishmania. Leishmania RNA virus (LRV) was identified as the first Leishmania infecting virus in 1998. Different studies showed the presence and role of the LRV in Leishmania parasites causing cutaneous leishmaniasis (CL). However, there is limited data on the pooled prevalence of LRV in Leishmania parasites causing CL. Therefore, the aim of this systematic review and meta-analysis was to determine the pooled prevalence of LRV in Leishmania parasite isolates and/or lesion biopsies in patients with CL from the available literature globally.MethodologyWe retrieved the studies from different electronic databases. The studies were screened and identified based on the inclusion and exclusion criteria. We excluded studies exclusively done in experimental animals and in vitro studies. The review was conducted in line with PRISMA guidelines. The meta-analysis was performed with Stata software version 14 with metan command. The forest plot with random-effect model was used to estimate the pooled prevalence with 95% confidence interval. Inverse variance index (I2) was used to assess the heterogeneity among the included articles.Principal findingsA total of 1215 samples from 25 studies were included. Of these, 40.1% (487/1215) were positive for LRV. The overall pooled prevalence of LRV globally was 37.22% (95% CI: 27.54% - 46.90%). The pooled prevalence of LRV in the New World (NW) and Old World (OW) regions was 34.18% and 45.77%, respectively. Leishmania guyanensis, L. braziliensis, L. major, and L. tropica were the most studied species for the detection of LRV. The prevalence of LRV from Leishmania isolates and lesion biopsies was 42.9% (349/813) and 34.3% (138/402), respectively.ConclusionThis systematic study revealed that there is high prevalence of LRV in Leishmania parasites isolated from patients with CL. More comprehensive studies would be required to investigate the presence of the LRV in other Leishmania species such as L. aethiopica to fully understand the role of LRV in different clinical manifestations and disease pathology presented in CL patients