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

Linearity of multiplex and singleplex assays.

<p>The calibration curves were generated by amplification of 10-fold dilutions of plasmid DNA standards for CaPV, PM, Mccp and RNA transcripts of PPRV using one-step multiplex RT-qPCR and singleplex RT-qPCR for respective target. Each dilution was run in triplicate at three different intervals. The mean Cq values are plotted against the log of concentration of the target gene copies/reaction. The PCR efficiency (<b><i>E</i></b>) for each target was calculated using the slope of each calibration curve with the formula <b><i>E</i></b> = 10^−1/slope−1 was greater than 90% and the correlation coefficient (R²) was greater than 0.99.</p

Primers and probes used for the amplification and detection of CaPV, PPRV, PM and Mccp.

<p>The different fluorescent labels on the 5’ end (FAM/HEX/TEXAS RED/CY5) and compatible black hole quencher on the 3’ end of the probes (BHQ 1/2/3) are displayed (LNAs are indicated by ‘+’ prior to the base). The targeted nucleotide sequence details are also provided.</p

Inter- and Intra-assay variability of the one-step multiplex RT-qPCR assay.

<p>The variability was calculated based on the threshold values for the amplification of three different concentrations of controls—higher (10⁷), medium (10⁵) and lower concentrations (10³) of each targeted pathogen run at three different intervals.</p

Amplification patterns obtained using the One-step multiplex RT-PCR.

<p><b>(A) Simultaneous detection of CaPV, PPRV, PM and Mccp in a single tube:</b> Amplification curves of three separate plasmids harbouring three selected gene fragments and of RNA transcripts of PPRV are shown. The amplification curves are shown for CaPV (Cyan), PM (Blue), Mccp (Red) and PPRV (Green). The curves were generated by the amplification of a pooled control sample at a final concentration of 10⁵ copies /μL of PPRV, PM and Mccp controls and 10⁶ copies /μL of CaPV control. <b>(B) Simultaneous detection of mixed infection while screening the swab samples of infected sheep at Burkina Faso</b>. Two amplification curves of FAM-Blue and HEX-Green colours indicate the clear presence of dual infection of PM and PPRV respectively. The absence of TEXAS RED-Red and CY5-Cyan coloured peaks or background indicate the absence of Mccp and CaPV infections, respectively.</p

Summary of viral/bacterial isolates and field samples screened by one-step multiplex RT-qPCR.

<p>The positivity was further confirmed by the respective confirmatory tests.</p

First Report of Lumpy Skin Disease in Myanmar and Molecular Analysis of the Field Virus Isolates

Lumpy skin disease virus (LSDV) causes lumpy skin disease in cattle and buffaloes, which is associated with significant animal production and economic losses. Since the 2000s, LSDV has spread from Africa to several countries in the Middle East; Europe; and Asia; including, more recently, several south-east Asian countries. In November 2020, Myanmar reported its first LSD outbreak. This study reports on the first incursion of LSD in Myanmar and the molecular analysis of the LSDV detected. Staff from the Livestock Breeding and Veterinary Department (LBVD) of the Ministry of Agriculture, Livestock, and Irrigation collected samples from cattle with suspected LSD infection. The Food and Agriculture Organization (FAO) of the United Nations&rsquo; Emergency Centre for Transboundary Animal Diseases (ECTAD) and the Joint International Atomic Energy Agency (IAEA)/FAO program&rsquo;s Animal Health and Production laboratory provided LSDV diagnostic support to two regional veterinary diagnostic laboratories in Myanmar. Samples from 13 cattle tested positive by real-time PCR. Selected samples underwent sequence analysis in IAEA laboratories. The results show that the Myanmar LSDV sequences clustered with LSDV isolates from Bangladesh and India, LSDV Kenya, and LSDV NI-2490. Further characterization showed that the Myanmar LSDV is 100% identical to isolates from Bangladesh and India, implying a common source of introduction. These findings inform diagnosis and development of control strategies