Abstract
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.
Highlights
Small ruminants play a major role in the economic status of farmers from developing countries and are considered to be the ‘poor man’s’ financial resources
The primers and probes were modified during assay development by incorporating degenerate bases in order to detect all variants of CaPV genotypes, Peste des petits ruminants virus (PPRV), Pasteurella multocida (PM) and Mycoplasma capricolum subspecies (ssp.) capripneumoniae (Mccp)
Modification of probes was done by adding locked nucleic acids (LNA) internally which allowed the short length of probes, their alignment with the multiple sequences used in the design, and to maintain a high annealing temperature
Summary
Small ruminants play a major role in the economic status of farmers from developing countries and are considered to be the ‘poor man’s’ financial resources. PPRV, a negative-sense, single stranded RNA virus belonging to the morbillivirus group of the Paramoxyviridae family, causes an acute and highly infectious disease of small ruminants leading to high morbidity and mortality. It shares with goat and sheep pox nearly the same endemic regions [5], [7,8]. Mccp is the causative agent of contagious caprine pleuropneumonia (CCPP) which causes significant economic losses to goat production in Africa and Asia It is mainly known in goats, it has been reported in sheep and a few wildlife species [12,13,14]. Because of the similarity in the symptoms caused by these specific pathogens, and their co-localization in nearly the same endemic areas, the proper management of these small ruminant respiratory diseases necessitates the implementation of rapid differential diagnostic tests for accurate identification of the responsible pathogen(s)
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