Prevalence of West Nile virus in domesticated mammals (cattle, goat, horse and pig) in selected areas of Peninsular Malaysia

West Nile virus (WNV) is the most widespread cause of arboviral encephalitis in the world. It is a re-emerging zoonotic virus that has the potential of significantly impacting public health and animal welfare. In nature, the virus is maintained in a cycle between wild birds that served as amplifiers and Culex mosquitos which are the vectors. Non-avian species are dead-end hosts that can become infected with WNV. The reservoirs and vectors of WNV are abundant in Malaysia and are often found in close to livestock farms, yet limited information is available on WNV status in domesticated mammals in Malaysia. This study was carried out to determine the WNV seroprevalence in domesticated mammals, including cattle, goats, horses and pigs in selected areas of Peninsular Malaysia and to determine the association between WNV infection and host factors (objective 1); to detect WNV infection through reverse transcriptase- Polymerase chain reaction (RT-PCR) in cattle, goats, horses and pigs and the association between WNV infection and host factors (objective 2); and to carry out phylogenetic analysis on the WNV isolates from this study to determine their genetic relatedness to other strains (objective 3). A total of 283 animals (n=283; of which were 80 pigs, 91 horses, 29 goats and 83 cattle) were sampled in this study, which included 283 serum samples and 203 nasopharyngeal swab samples. The serum samples were screened for WNV IgG using a competitive ELISA (c- ELISA) kit (ID VET, France), and for Japanese encephalitis virus (JEV) IgG using double-antibody sandwich ELISA kit (Sunred, China). Total RNA extracted from nasopharyngeal swabs were tested using reverse-transcriptase polymerase chain reaction (RT-PCR) test targeting conserved gene of WNV capsid and premembrane. The samples that produced bands following RT-PCR test were purified and subjected to partial DNA sequencing. Using MEGA 7, the sequences were aligned with the MUSCLE method. Phylogenetic analysis was done with BEAST2 with the Bayesian MCMC method, and statistical analyses were done using Chi-square (Fisher’s exact test) and logistic regression in IBM SPSS 28. In total, 140 samples were positive for WNV IgG. Among the different species, the highest seroprevalence was in pigs with 62.5% [(50/80); 95% CI (0.5155 – 0.7231)], followed by 53.85% [(49/91); 95% CI (0.4366 – 0.6373)] in horse, 48.3% [(14/29); 95% CI (0.3139 – 0.4828)] in goats and 32.53% [(27/83); 95% CI (0.2339 – 0.4322)] in cattle. WNV seroprevalence was associated with the species, age of the animal and location of sampling. Among the species, pigs were more likely to be WNV seropositive. In the cattle, location of sample collection was associated with WNV seropositivity, whereas in the goat, age was associated with WNV seropositivity. Location was the only factor in the horse that was associated with WNV seropositivity. Meanwhile, both age and location were associated with WNV seropositivity in the pigs. Serological results indicate past exposure to WNV in all the species in this study. For the molecular analysis, 7.7% [(7/91) at 95% CI (0.0353 to 0.1528)] of the horses are positive for WNV RNA, comprising of four males and three females. The horses were from Cheras (n=4) and Putrajaya (n=3). There were no significant differences between sex and location with the WNV molecular positivity (p > 0.05). RT-PCR test positivity indicating a recent and ongoing infection in the horse at the time of sample collection. From the phylogenetic analysis of the partial sequence of the isolates, they grouped closely with lineage 2 WNV isolates, with close similarity (greater that 98%) with South African strain, and with recently reported Malaysian isolates of WNV. These findings show that domesticated mammals in Malaysia were exposed to WNV infection of which highlights the need for increased vigilance and surveillance for WNV to prevent future outbreak.

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