24 research outputs found
Molecular and biological characterization of the Capripoxviruses and the development of molecular diagnostocs assays
No full textSheeppox (SPP), Goatpox (GTP) und Lumpy Skin Disease (LSD) sind drei wichtige Pockenviruserkrankungen bei Schafen, Ziegen und Rindern. Eine Reihe von Lebendinpfstoffen steht als Präventivmaßnahme zur Verfügung. Um zwischen dem Lebendimpfstoffstamm gegen SPPV (LAV-SPPV) und dem wild-typ SSPV (WT-SSPV) unterscheiden zu können, wurde zunächst eine 84 Nukleotid lange Deletion zwischen dem Ligasegen und dem Vriolavirus B22r homologen Gen, die in LAV-SPPV identifiziert wurde heramngezogen. Die von uns dafür enwickelte gelbasierte PCR-Methode konnte in weiterer Folge jedoch nicht zwischen WT-SPPV und WT-GTPV, beziehungsweise WT-LSDV. Daher wurde eine hochauflösende „High Resolution Melting“-Methode (HRM) entwickelt, die eine genaue Differenzierung der einzelnen Stämme ermöglichte. Beide Untersuchingsmethoden sind wichtige und wertvolle Werkzeuge für epidemologische Untersuchungen während der Impfkampagnen. Wir untersuchten LSDVs aus Ostafrika, die zwischen 2007 und 2012 isoliert wurden. Wir entdeckten, dass LSDV Embu/B338/2011, ein Isolat aus Kenia, eine Mischung aus dem WT-LSDV und dem dazugehörigen Lebendimpfstoffstamm darstellt. Diese Erkenntnis zeigt, wie wichtig ein ständiges Monitoring der im Umlauf befindlichen Virusstämme ist.Um mehr Einblick in die Epidemologie zu erlangen wurden molkularbiologische Untersuchungen anhand des GPCR-Gens von GTPV durchgeführt. „Alignment“- und „Alignment free“ basierte Methodenerlauben die Segregation von GTPV zu verfolgen, und so die Quellen neuer Ausbrüche zu identifizieren, wie auch voraus zu sehen.Bezüglich besseren Verständnisses der immunologischen Wechselwirkung zwischen Virus und Wirt wurden 13 Gene, die maßgeblich an der angeborenen Immunantwort beteiligt sind, in „Peripheral blood mononuclear cells“ (PBMC) aus Schafen mittels quantitativer PCR analysiert. Es zeigte sich, dass vier der analysierten Gene (RIG-1, TNFA, IL-15, IL-10) sowohl in WT-SPPV als auch in LAV-SPPV deutlich hochreguliert waren und ein Gen (RIG-1) in WT-SPPV im Vergleich zu LAV-SPPV nochmals deutlich erhöht war. Diese und weitere Erkenntnisse über protektive Immunantwort werden dazu beitragen effektivere und sichere Impfstoffe gegen SPPV und GTPV zu entwickeln.Sheeppox (SPP), goatpox (GTP) and lumpy skin disease (LSD) are three important pox diseases of sheep, goat, and cattle caused by SPP virus (SPPV) and GTP virus (GTPV), and LSD virus (LSDV), respectively. Live attenuated vaccines (LAV) are available for the control of these viruses. However, the efficacy and safety are not always satisfactory; therefore, studies are necessary to understand the immunology of SPPV infections, and tools are required to distinguish vaccinated from infected animals, characterize virus variants and trace the source of infections. To differentiate LAV SPPV from WT SPPV, we firstly identified an 84-nucleotide deletion between the DNA ligase gene and the variola virus B22R homolog gene of LAV SPPV and developed a gel-based PCR method. Furthermore, we designed a high-resolution melting (HRM) method exploiting specific markers in the CaPV B22R gene, which was successful in differentiating LAV SPPV from WT SPPV, GTPV, and LSDV. These two assays are a valuable tool for epidemiological investigations during vaccination programs. Secondly, we investigated LSDVs from East Africa collected between 2007 and 2012. We discovered that LSDV Embu/B338/2011, a field isolate from Kenya, presented mixed characteristics of the LSDV vaccine and WT isolates, highlighting the need for constant monitoring of LSDV genetic variability. Thirdly, we studied the GPCR gene of GTPVs using an alignment-based and an alignment-free method. This work enabled us to follow the segregation of GTPVs based on their geographical origin using the alignment-free method. This approach can help determine the source of GTPV responsible for an outbreak in disease-free countries and detect the introduction of new strains in disease-endemic countries. Finally, we investigated the expression of thirteen genes involved in the innate immune response in sheep peripheral blood mononuclear cells (PBMC) and infected in vitro, by quantitative real-time PCR. The results showed that both WT and LAV infections in sheep upregulated the expression of RIG-1, TNFα, IL-15, and IL-10. The expression of RIG-1 was significantly higher in WT than LAV. These findings advance our knowledge on SPPV induction of a protective immune response and will further help to develop safer and more potent vaccines against SPP and GTP.by Tesfaye Rufael ChibssaDissertation Universität für Bodenkultur Wien 2019Zusammenfassungen in deutscher Sprach
Molecular and biological characterization of the Capripoxviruses and the development of molecular diagnostocs assays
No full textSheeppox (SPP), Goatpox (GTP) und Lumpy Skin Disease (LSD) sind drei wichtige Pockenviruserkrankungen bei Schafen, Ziegen und Rindern. Eine Reihe von Lebendinpfstoffen steht als Präventivmaßnahme zur Verfügung. Um zwischen dem Lebendimpfstoffstamm gegen SPPV (LAV-SPPV) und dem wild-typ SSPV (WT-SSPV) unterscheiden zu können, wurde zunächst eine 84 Nukleotid lange Deletion zwischen dem Ligasegen und dem Vriolavirus B22r homologen Gen, die in LAV-SPPV identifiziert wurde heramngezogen. Die von uns dafür enwickelte gelbasierte PCR-Methode konnte in weiterer Folge jedoch nicht zwischen WT-SPPV und WT-GTPV, beziehungsweise WT-LSDV. Daher wurde eine hochauflösende „High Resolution Melting“-Methode (HRM) entwickelt, die eine genaue Differenzierung der einzelnen Stämme ermöglichte. Beide Untersuchingsmethoden sind wichtige und wertvolle Werkzeuge für epidemologische Untersuchungen während der Impfkampagnen. Wir untersuchten LSDVs aus Ostafrika, die zwischen 2007 und 2012 isoliert wurden. Wir entdeckten, dass LSDV Embu/B338/2011, ein Isolat aus Kenia, eine Mischung aus dem WT-LSDV und dem dazugehörigen Lebendimpfstoffstamm darstellt. Diese Erkenntnis zeigt, wie wichtig ein ständiges Monitoring der im Umlauf befindlichen Virusstämme ist.Um mehr Einblick in die Epidemologie zu erlangen wurden molkularbiologische Untersuchungen anhand des GPCR-Gens von GTPV durchgeführt. „Alignment“- und „Alignment free“ basierte Methodenerlauben die Segregation von GTPV zu verfolgen, und so die Quellen neuer Ausbrüche zu identifizieren, wie auch voraus zu sehen.Bezüglich besseren Verständnisses der immunologischen Wechselwirkung zwischen Virus und Wirt wurden 13 Gene, die maßgeblich an der angeborenen Immunantwort beteiligt sind, in „Peripheral blood mononuclear cells“ (PBMC) aus Schafen mittels quantitativer PCR analysiert. Es zeigte sich, dass vier der analysierten Gene (RIG-1, TNFA, IL-15, IL-10) sowohl in WT-SPPV als auch in LAV-SPPV deutlich hochreguliert waren und ein Gen (RIG-1) in WT-SPPV im Vergleich zu LAV-SPPV nochmals deutlich erhöht war. Diese und weitere Erkenntnisse über protektive Immunantwort werden dazu beitragen effektivere und sichere Impfstoffe gegen SPPV und GTPV zu entwickeln.Sheeppox (SPP), goatpox (GTP) and lumpy skin disease (LSD) are three important pox diseases of sheep, goat, and cattle caused by SPP virus (SPPV) and GTP virus (GTPV), and LSD virus (LSDV), respectively. Live attenuated vaccines (LAV) are available for the control of these viruses. However, the efficacy and safety are not always satisfactory; therefore, studies are necessary to understand the immunology of SPPV infections, and tools are required to distinguish vaccinated from infected animals, characterize virus variants and trace the source of infections. To differentiate LAV SPPV from WT SPPV, we firstly identified an 84-nucleotide deletion between the DNA ligase gene and the variola virus B22R homolog gene of LAV SPPV and developed a gel-based PCR method. Furthermore, we designed a high-resolution melting (HRM) method exploiting specific markers in the CaPV B22R gene, which was successful in differentiating LAV SPPV from WT SPPV, GTPV, and LSDV. These two assays are a valuable tool for epidemiological investigations during vaccination programs. Secondly, we investigated LSDVs from East Africa collected between 2007 and 2012. We discovered that LSDV Embu/B338/2011, a field isolate from Kenya, presented mixed characteristics of the LSDV vaccine and WT isolates, highlighting the need for constant monitoring of LSDV genetic variability. Thirdly, we studied the GPCR gene of GTPVs using an alignment-based and an alignment-free method. This work enabled us to follow the segregation of GTPVs based on their geographical origin using the alignment-free method. This approach can help determine the source of GTPV responsible for an outbreak in disease-free countries and detect the introduction of new strains in disease-endemic countries. Finally, we investigated the expression of thirteen genes involved in the innate immune response in sheep peripheral blood mononuclear cells (PBMC) and infected in vitro, by quantitative real-time PCR. The results showed that both WT and LAV infections in sheep upregulated the expression of RIG-1, TNFα, IL-15, and IL-10. The expression of RIG-1 was significantly higher in WT than LAV. These findings advance our knowledge on SPPV induction of a protective immune response and will further help to develop safer and more potent vaccines against SPP and GTP.by Tesfaye Rufael ChibssaDissertation Universität für Bodenkultur Wien 2019Zusammenfassungen in deutscher Sprach
CRISPR-Cas12/Cas13: Bibliometric analysis and systematic review of its application in infectious disease detection
No full textBackground: Infectious diseases impose a significant burden on the global public health and economy, resulting in an estimated 15 million deaths out of 57 million annually worldwide. This study examines the current state of CRISPR-Cas12/Cas13 research, focusing on its applications in infectious disease detection and its evolutionary trajectory. Methods: A bibliometric analysis and systematic review were conducted by retrieving CRISPR-Cas12/Cas13-related articles published between January 1, 2015 to December 31, 2022, from the Web of Science database. The research protocol was registered with International Platform of Registered Systematic Review and Meta-analysis Protocols (INPLASY202380062). Results: Our search identified 1987 articles, of which, 1856 were included in the bibliometric analysis and 445 were used in qualitative analysis. The study reveals a substantial increase in scientific production on CRISPR-Cas12/Cas13, with an annual growth rate of 104.5%. The United States leads in the number of published articles. The systematic review identified 580 different diagnostic assays targeting 170 pathogens, with SARS-CoV-2 dominating with 158 assays. Recombinase polymerase amplification (RPA)/reverse transcription-RPA (RT-RPA) emerged as the predominant amplification method, while lateral flow assay was the most common readout method. Approximately 72% of the diagnostic assays developed are suitable for point-of-care testing. Conclusion: The rapid increase in research on CRISPR-Cas12/Cas13 between 2015 and 2022 suggests promising potential for advancements in infectious disease diagnosis. Given the numerous advantages of CRISPR-Cas technology for disease detection over other methods, and the dedicated efforts of scientists from around the world, it is reasonable to anticipate that CRISPR-Cas technology may emerge as a formidable alternative, offering the possibility of expedited point-of-care testing in the not-too-distant future
Molecular Characterization of Lumpy Skin Disease Virus Isolates from Outbreak Cases in Cattle from Sawena District of Bale Zone, Oromia, Ethiopia
No full textLumpy skin disease (LSD) is a viral disease caused by LSD virus and is one of the most economically significant transboundary and emerging diseases of cattle. LSD causes considerable economic losses due to emaciation, damage to hides, infertility, and loss of milk production. In Ethiopia, the disease is distributed almost in all regions and is regarded as one of the most economically important livestock diseases in the country. An outbreak investigation of the disease was monitored from October 2016 to April 2017 in southern pastoral areas of Bale Zone, Oromia, Ethiopia. In December 2016, LSD outbreak occurred in Sawena district of Bale Zone, from which necessary biopsy samples were collected from actively infected animals for the purpose of virus isolation, and characterization using different molecular techniques at National Animal Health and Diagnostic Investigation Center (NAHDIC) of Sebeta, Ethiopia. In addition, clinical examination of infected and in-contact animals was carried out together with a questionnaire survey. Based on the clinical manifestations, LSD was recorded in 18% (94/522) of examined cattle, whereas biopsy samples from 20 clinically positive animals were collected for further laboratory process. The morbidity rate was higher in animals less than two years 28.97% (31/107) than other ages and showed a statistically significant difference with P<0.05. Female animals showed higher morbidity rate of 20.59% (76/369) than male animals (11.76%) (18/153) with a significant difference at P≤0.003. Mortality rate and case fatality were also significantly higher in young animals than other age groups. Viruses were isolated from both skin biopsies and nasal swabs on Vero cell line. From both skin biopsies and nasal swabs, the virus DNA was identified by amplifying the 172 bp DNA fragment using real-time and conventional PCR. Providing adequate diagnostic facilities, establishing strategic policies for effective control and eradication and awareness creations for communities for early identification or reporting were recommendations made to minimize economic losses of the disease
Isolation and Molecular Characterization of Peste des Petits Ruminants Virus from Outbreaks in Southern Ethiopia, 2020
No full textPeste des petits ruminants (PPR) is one of the most important transboundary diseases of small ruminants. In this study, nasal and oral swabs (n = 24) were collected from sheep (n = 7) and goats (n = 17) with clinical signs in southern Ethiopia in March 2020. PPR virus was isolated on Vero dog cells expressing the signaling lymphocyte activation molecule (VDS) and screened using RT-qPCR. Positive samples were confirmed by conventional RT-PCR followed by sequencing of a partial nucleoprotein (N) gene segment. Results revealed that 54% (n = 13/24) of the tested samples were PPRV-positive Phylogenetic analysis revealed that the viruses belonged to lineage IV and lineage II. The lineage IV viruses were similar, although not identical, to other lineage IV viruses previously reported in Ethiopia and other East African countries while the lineage II viruses have been reported for the first time in Ethiopia showed a high nucleotide identity (99.06%) with the vaccine (Nigeria 75/1) that is currently used in Ethiopia for the prevention of PPR. Further investigations are therefore recommended in order to fully understand the true nature of the lineage II PPRVs in Ethiopia
Innate Immune Responses to Wildtype and Attenuated Sheeppox Virus Mediated Through RIG-1 Sensing in PBMC In-Vitro
No full textInternational audiencetSheeppox (SPP) is a highly contagious disease of small ruminants caused by sheeppox virus (SPPV) and predominantly occurs in Asia and Africa with significant economic losses. SPPV is genetically and immunologically closely related to goatpox virus (GTPV) and lumpy skin disease virus (LSDV), which infect goats and cattle respectively. SPPV live attenuated vaccines (LAVs) are used for vaccination against SPP and goatpox (GTP). Mechanisms related to innate immunity elicited by SPPV are unknown. Although adaptive immunity is responsible for long-term immunity, it is the innate responses that prevent viral invasion and replication before LAVs generate specific long-term protection. We analyzed the relative expression of thirteen selected genes that included pattern recognition receptors (PRRs), Nuclear factor-kappa beta p65 (NF-kappa beta), and cytokines to understand better the interaction between SPPV and its host. The transcripts of targeted genes in sheep PBMC incubated with either wild type (WT) or LAV SPPV were analyzed using quantitative PCR. Among PRRs, we observed a significantly higher expression of RIG-1 in PBMC incubated with both WT and LAV, with the former producing the highest expression level. However, there was high inter-individual variability in cytokine transcripts levels among different donors, with the expression of TNF alpha, IL-15, and IL-10 all significantly higher in both PBMC infected with either WT or LAV compared to control PBMC. Correlation studies revealed a strong significant correlation between RIG-1 and IL-10, between TLR4, TNF alpha, and NF-kappa beta, between IL-18 and IL-15, and between NF-kappa beta and IL-10. There was also a significant negative correlation between RIG-1 and IFN gamma, between TLR3 and IL-1 beta, and between TLR4 and IL-15 (P< 0.05). This study identified RIG-1 as an important PRR in the signaling pathway of innate immune activation during SPPV infection, possibly through intermediate viral dsRNA. The role of immunomodulatory molecules produced by SPPV capable of inhibiting downstream signaling activation following RIG-1 upregulation is discussed. These findings advance our knowledge of the induction of immune responses by SPPV and will help develop safer and more potent vaccines against SPP and GTP
Differentiation of sheep poxvirus vaccines from field isolates and other Capripoxvirus species
No full textBackground The genus Capripoxvirus (CaPV) within the family Poxviridae com-prises three closely related viruses, sheep poxvirus (SPPV), goat poxvirus (GTPV) and lumpy skin disease virus (LSDV) causing sheep pox (SPP), goat pox (GTP) and lumpy skin disease (LSD) in small ru-minants and cattle respectively. LSD has emerged in Europe in 2015 and first incursions of SPP in the European Union were reported in Bulgaria and Greece in 2013. Live attenuated SPPV vaccines are widely used in many countries to control SPP and GTP. With the in-creasing number of reports on SPP in previously vaccinated sheep herds, it is imperative to develop new diagnostic tools for differen-tiation of SPPV field strains from attenuated vaccine strains. Objective This work aimed at identifying appropriate diagnostic targets to de-velop assays for the rapid and accurate differentiation SPPV vaccine strains from SPPV field isolates and other CaPVs. Methodology To identify a suitable molecular target for the development of these assays, the full genomes of several SPPV vaccines strains and SPPV field isolates were compared. A unique 84-base pair nucleotide dele-tion located between the DNA ligase and the B22R gene was exploit-ed to develop a gel-based PCR, and a region containing a 48bp de-letion within the B22R gene of SPPV vaccines strains only, as well as species-specific nucleotide difference between SPPV field isolates, GTPV and LSDV, was targeted to develop a HRM assay. Results The gel-based assay was readily able to differentiate SPPV vaccines from field isolates. However, this method alone could not differen-tiate SPPV field isolates from GTPV and LSDV. In contrast, the HRM based method allowed the differentiation of SPPV vaccines from field isolates and further enabled the genotyping of capripoxviruses isolates. Out of 61 samples tested, we identified 4 SPPV vaccines, 14 SPPV field isolates, 11 GTPVs and 32 LSDVs. The two assays were both sensitive and specific and in agreement with the sequencing data of the tested samples. Conclusion The assays described herein are reliable and rapid methods for the differentiation of SPPV Vaccines from SPPV field isolates. While the gel based assay needs to be combined with capripoxvirus spe-cies-specific assays, the HRM assay stands alone as a tool to differen-tiate SPPV vaccines from field isolates and simultaneously genotype SPPVs, GTPVs and LSDVs. The methods are suited for routine use during outbreak investigations in both capripoxvirus enzootic and disease-free countries
Development and evaluation of a real-time PCR panel for the detection of 20 immune markers in cattle and sheep
No full textPhenotypic, molecular detection, and antibiogram analysis of Pseudomonas species from Oreochromis niloticus.L 1758 (Nile Tilapia) from aquaculture pond, Ethiopia
No full textAbstract Background Pseudomonas species, including P. aeruginosa, P. putida, and P. fluorescens, are zoonotic bacterial pathogens responsible for significant disease and mortality in both farmed and wild fish worldwide. In Ethiopia, these bacteria have been identified in Sebeta fish ponds and Rift Valley lakes, yet there is limited data on their molecular and phenotypic characteristics in local aquaculture systems. To address this gap, a cross-sectional study was conducted from November 2022 to May 2023 in selected aquaculture ponds in Ethiopia. Results A total of 637 samples were aseptically collected from the muscle, liver, spleen, and kidney of fish using purposive sampling. Pseudomonas base agar selective medium morphological characteristic and biochemical tests were used to isolate and identify pseudomonas species. Pseudomonas species were isolated from 81 samples, representing 12.7% of the total. Among these isolates, 85.6% displayed virulence traits, including β- hemolysis on 5% sheep blood agar. Additionally, 75 isolates (92.59%) was confirmed using conventional PCR with Pseudomonas-specific primers. Of the PCR-positive samples, 8 (10.66%) were identified as P. aeruginosa, 28 (37.63%) as P. putida, and 39 (52%) as P. fluorescens from Nile Tilapia (O. niloticus). Antibiotic susceptibility testing on ten representative isolates revealed that all strains were sensitive to Ciprofloxacin, Gentamicin, and Ceftriaxone but resistant to Amoxicillin and Penicillin. Conclusions The findings indicate that Pseudomonas species carrying virulence genes, exhibiting β- hemolytic activity, and showing resistance to commonly used antibiotics in both human and veterinary medicine are present in aquaculture. The detection of this pathogen in 75 fish samples raises concerns about potential outbreaks and zoonotic transmission. Therefore, further research on the molecular epidemiology of the disease is necessary to understand inter-host transmission and antibiotic resistance patterns
Use of an Alignment-Free Method for the Geographical Discrimination of GTPVs Based on the GPCR Sequences
No full textInternational audienceThis article is an oGoatpox virus (GTPV) belongs to the genus Capripoxvirus, together with sheeppox virus (SPPV) and lumpy skin disease virus (LSDV). GTPV primarily affects sheep, goats and some wild ruminants. Although GTPV is only present in Africa and Asia, the recent spread of LSDV in Europe and Asia shows capripoxviruses could escape their traditional geographical regions to cause severe outbreaks in new areas. Therefore, it is crucial to develop effective source tracing of capripoxvirus infections. Earlier, conventional phylogenetic methods, based on limited samples, identified three different nucleotide sequence profiles in the G-protein-coupled chemokine receptor (GPCR) gene of GTPVs. However, this method did not differentiate GTPV strains by their geographical origins. We have sequenced the GPCR gene of additional GTPVs and analyzed them with publicly available sequences, using conventional alignment-based methods and an alignment-free approach exploiting k-mer frequencies. Using the alignment-free method, we can now classify GTPVs based on their geographical origin: African GTPVs and Asian GTPVs, which further split into Western and Central Asian (WCA) GTPVs and Eastern and Southern Asian (ESA) GTPVs. This approach will help determine the source of introduction in GTPV emergence in disease-free regions and detect the importation of additional strains in disease-endemic area
