One-step multiplex RT-qPCR assay for the detection of Peste des petits ruminants virus, Capripoxvirus, Pasteurella multocida and Mycoplasma capricolum subspecies (ssp.) capripneumoniae

Respiratory infections, although showing common clinical symptoms like pneumonia, are caused by bacterial, viral or parasitic agents. These are often reported in sheep and goats populations and cause huge economic losses to the animal owners in developing countries. Detection of these diseases is routinely done using ELISA or microbiological methods which are being reinforced or replaced by molecular based detection methods including multiplex assays, where detection of different pathogens is carried out in a single reaction. In the present study, a one-step multiplex RT-qPCR assay was developed for simultaneous detection of Capripoxvirus (CaPV), Peste de petits ruminants virus (PPRV), Pasteurella multocida (PM) and Mycoplasma capricolum ssp. capripneumonia (Mccp) in pathological samples collected from small ruminants with respiratory disease symptoms. The test performed efficiently without any cross-amplification. The multiplex PCR efficiency was 98.31%, 95.48%, 102.77% and 91.46% whereas the singleplex efficiency was 93.43%, 98.82%, 102.55% and 92.0% for CaPV, PPRV, PM and Mccp, respectively. The correlation coefficient was greater than 0.99 for all the targets in both multiplex and singleplex. Based on cycle threshold values, intra and inter assay variability, ranged between the limits of 2%–4%, except for lower concentrations of Mccp. The detection limits at 95% confidence interval (CI) were 12, 163, 13 and 23 copies/reaction for CaPV, PPRV, PM and Mccp, respectively. The multiplex assay was able to detect CaPVs from all genotypes, PPRV from the four lineages, PM and Mccp without amplifying the other subspecies of mycoplasmas. The discriminating power of the assay was proven by accurate detection of the targeted pathogen (s) by screening 58 viral and bacterial isolates representing all four targeted pathogens. Furthermore, by screening 81 pathological samples collected from small ruminants showing respiratory disease symptoms, CaPV was detected in 17 samples, PPRV in 45, and PM in six samples. In addition, three samples showed a co-infection of PPRV and PM. Overall, the one-step multiplex RT-qPCR assay developed will be a valuable tool for rapid detection of individual and co-infections of the targeted pathogens with high specificity and sensitivity. (Résumé d'auteur

A rare case of aerococcus urinae infective endocarditis

Introduction: Aerococcus urinae is a rare cause of infective endocarditis. Aerococcus is a gram positive cocci that is easily misidentified as Staphylococci or Streptococci. The true incidence rate of this pathogen is likely underestimated. Recent advances in laboratory diagnostic methods with matrix-associated laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) have lead to increased recognition of this pathogen in the clinical microbiology lab, and awareness as a cause of infective endocarditis in the infectious disease community. Case reports: Aerococcus usually affects males with underlying urinary tract conditions. Herein, we report a case of prosthetic aortic valve endocarditis caused by Aerococcus urinae. Discussion: Our patient was considered high risk for cardiac surgery and was treated successfully with intravenous antibiotics alone for six weeks. Conclusion: Infective endocarditis should be considered in all cases of Aerococcus bacteremia and appropriate diagnostic evaluations pursued. Abbreviations: AV: Aortic valve; IE: Infective endocarditi

Molecular Characterization of the 2020 Outbreak of Lumpy Skin Disease in Nepal

Lumpy skin disease (LSD) is a transboundary viral disease of cattle and buffaloes transmitted by blood-feeding vectors and causes high morbidity and low-to-moderate mortality. Since the first observation of LSD in Zambia in 1929, it has spread in cattle populations across African countries, the Middle East, Europe, and Asia. Following the recent outbreaks of LSD in South Asian countries such as India and Bangladesh, the disease was first reported in cattle farms in Nepal in June 2020. This study investigated the Nepalese LSD outbreak and confirmed that the disease spread rapidly to three neighboring districts in a month, infecting 1300 animals. Both cattle and buffaloes showed common clinical signs of LSD, with the exception that the buffaloes presented small nodular lesions without centered ulcerations. The collected samples were first tested for the presence of LSDV by real-time PCR. We further applied molecular tools, RPO30, GPCR, EEV glycoprotein gene, and B22R, for additional characterization of the LSDV isolates circulating in Nepal. Using a PCR-based Snapback assay, we confirmed that samples collected from cattle and buffaloes were positive of LSDV. Furthermore, sequence analysis (phylogenetic and multiple sequence alignments) of four selected LSDV genes revealed that the Nepal LSDVs resemble the Bangladesh and Indian isolates and the historic isolates from Kenya. We also highlight the importance of a unique B22R gene region harboring single-nucleotide insertions in LSDV Neethling and LSDV KSGPO-240 vaccine strains, enabling us to differentiate them from the Nepalese isolates and other fields isolates. This study demonstrates the importance of disease surveillance and the need to determine the source of the disease introduction, the extent of spread, modes of transmission, and the necessary control measures

Viral Co-Infections of Warthogs in Namibia with African Swine Fever Virus and Porcine Parvovirus 1

Understanding virus circulation in wild animals, particularly those that have contact with domestic animals, is crucial for disease management and control. In Africa, warthogs are known to be asymptomatic carriers of porcine pathogens; a recent study in Namibia has shown them to be positive for Porcine circovirus-2 (PCV-2). In this study, the same samples used for the PCV-2 investigation in Namibia were further screened for the presence of African swine fever virus (ASFV) and porcine parvovirus 1 (PPV1) by PCR. Of the 42 animals tested, 2 (4.8%) and 13 (31%) were positive for AFSV and PPV1, respectively. The two AFSV were also co-infected with PPV1. Combing the results of this study with the results of the previous PCV-2 investigation, four warthogs were shown to be co-infected with both PPV1 and PCV-2. Sequence and phylogenetic analysis revealed that the AFSV belonged to genotype (Ib) but were from different serogroups. Unexpectedly, the ASFVs from the warthogs were genetically distinct to those observed in an outbreak in the same region of Namibia that occurred less than fifteen months prior to the sampling of the warthogs. In fact, a stronger genetic relationship was observed between the warthog viruses and historical Namibian and South African ASFVs identified in 1980, 2004 and 2008. For the PPV1s, the closest relative to the Namibian PPV1 were viruses identified in wild boar in Romania in 2011. This study confirms that warthogs are carriers of porcine pathogens and the data should encourage further studies on larger populations of wild and domestic swine to more fully understand the epidemiology and transmission of viral pathogens from these species

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First genetic characterization of Peste des Petits Ruminants from Niger: On the advancing front of the Asian virus lineage

International audiencePeste des Petits Ruminants (PPR) is a serious transboundary infectious disease of small ruminants. The causal agent, PPR virus (PPRV), can be separated into four genetically distinct lineages using phylogenetic analysis. In recent decades, lineage IV of PPRV has dramatically extended its geographic distribution from Asia to the Middle East and to Africa, where it has progressively replaced other PPRV lineages. Lineages I and II are historically distributed in West Africa. Currently, lineage II appears to dominate the region, whereas the last recorded occurrence of lineage I dates back to 1994. Recent studies reported the presence of lineage IV in Nigeria, suggesting that this lineage is expanding in West Africa. In Niger, a close neighbour of Nigeria, PPRV has never been genetically characterized, despite reports of PPR incidence. In this study, pathological samples collected from sick goats were collected in 2013 during a suspected PPR outbreak in southern Niger close to the Nigerian border were compared to samples collected in a previous investigation in October 2001 in south-western Niger. These strains were characterized by sequencing and phylogenetic analysis to identify their genetic lineage. Our results show that in 2001, lineages I and II were cocirculating in south-western Niger, whereas the strain that caused the outbreak in 2013 belonged to lineage IV and is closely related to strains identified in Nigeria. These results confirm the progression of lineage IV in West Africa. The process of PPRV lineage replacement and its implications for the epidemiology and the control of the disease in this region are unclear and should be the subject of further studies in the field

Low Dose Gamma Irradiation of Trypanosoma evansi Parasites Identifies Molecular Changes That Occur to Repair Radiation Damage and Gene Transcripts That May Be Involved in Establishing Disease in Mice Post-Irradiation

International audienceThe protozoan parasite Trypanosoma evansi is responsible for causing surra in a variety of mammalian hosts and is spread by many vectors over a wide geographical area making it an ideal target for irradiation as a tool to study the initial events that occur during infection. Parasites irradiated at the representative doses 100Gy, 140Gy, and 200Gy were used to inoculate BALB/c mice revealing that parasites irradiated at 200Gy were unable to establish disease in all mice. Cytokine analysis of mice inoculated with 200Gy of irradiated parasites showed significantly lower levels of interleukins when compared to mice inoculated with non-irradiated and 100Gy irradiated parasites. Irradiation also differentially affected the abundance of gene transcripts in a dose-dependent trend measured at 6- and 20-hours post-irradiation with 234, 325, and 484 gene transcripts affected 6 hours post-irradiation for 100Gy-, 140Gy- and 200Gy-irradiated parasites, respectively. At 20 hours post-irradiation, 422, 381, and 457 gene transcripts were affected by irradiation at 100Gy, 140Gy, and 200Gy, respectively. A gene ontology (GO) term analysis was carried out for the three representative doses at 6 hours and 20 hours post-irradiation revealing different processes occurring at 20 hours when compared to 6 hours for 100Gy irradiation. The top ten most significant processes had a negative Z score. These processes fall in significance at 140Gy and even further at 200Gy, revealing that they were least likely to occur at 200Gy, and thus may have been responsible for infection in mice by 100Gy and 140Gy irradiated parasites. When looking at 100Gy irradiated parasites 20 hours post-irradiation processes with a positive Z score, we identified genes that were involved in multiple processes and compared their fold change values at 6 hours and 20 hours. We present these genes as possibly necessary for repair from irradiation damage at 6 hours and suggestive of being involved in the establishment of disease in mice at 20 hours post-irradiation. A potential strategy using this information to develop a whole parasite vaccine is also postulated