Hayat et al., The J. Anim. Plant Sci. 29(6):2019 PCR-BASED SCREENING OF PLASMODIUM SPECIES IN MOSQUITO VECTORS OF FAISALABAD DISTRICT, PUNJAB, PAKISTAN K. Hayat 1 , M.S. Sajid *1,2 , Z. Iqbal 1 , M. Saqib 3 , F.A. Raza 4 , W. Akram 5 , H. Abbas 6 1 Department of Parasitology, University of Agriculture, Faisalabad, Pakistan 2 Center for Advanced Studies in Agriculture and Food Security (CAS-AFS) University of Agriculture, Faisalabad, Pakistan 3 Department of Clinical Medicine and Surgery, Faculty of Veterinary Science, University of Agriculture, Faisalabad, Pakistan 4 Pakistan Health Research Council, Research Center, Fatima Jinnah Medical University, Lahore, Pakistan 5 KBCMA College of Veterinary & Animal Sciences (CVAS), Narowal, Sub-campus of University of Veterinary & Animal Sciences, Lahore, Pakistan 6 Department of Entomology, University of Agriculture, Faisalabad, Pakistan Corresponding Author’s Email: drsohailuaf@hotmail.com ABSTRACT Plasmodium (P), mosquito-borne unicellular parasite, is responsible for “malaria”. Pakistan remains at risk of malaria and almost 1.6 million cases of malaria are reported every year. The present study was planned to screen the mosquito vectors for Plasmodium sp. in Faisalabad district, Punjab, Pakistan using nested PCR. For this purpose, convenient sampling of adult mosquitoes was done from different places including: animal populated areas, lavatories, water storage tanks, livestock farms and road-side ditches in 70% ethanol. DNA extraction was done after stereomicroscopic identification of the specimens. Species identification of P. falciparum, P. vivax, P. ovale and P. malariae was done through universal forward and species-specific reverse primers in the nested PCR. Prevalence of Culex mosquitoes was higher as compared to Anopheles. Plasmodium falciparum and P. vivax were found higher as compared to other species of Plasmodium. The overall prevalence of Plasmodium sp. in mosquito vectors was 46% (14 out of 30 pools for Plasmodium sp.). Results were analyzed through chi-square analyses. Present study may explore the vectorial capacity of mosquitoes which can be an indicator of Plasmodium sp. distribution in an area for large scale metagenomics. Key words: PCR, Mosquito, Plasmodium, Faisalabad, Molecular epidemiology. INTRODUCTION Plasmodium (Haemosporida: Plasmodiidae) is responsible for the occurrence of “malaria”. Various species of Plasmodium, which have been reported to cause malaria, include P. falciparum, P. hylobati, P. cynomolgi, P. malariae, P. vivax, P. inui, P. jefferyic, P. knowlesi, P. fieldi, P. simiovale, P. silvaticum, P. youngi, P. eylesi, P. coatneyi, and P. pitheci (White 2008; Beignonet al. 2014; Indra et al. 2014; Maenoet al. 2015). In most parts of the world (Asia, Africa and America), it is considered as an endemic disease. Every year, more than 200 million cases and a million of those ending as death have been reported resulting in economic losses in the form of macroeconomics (like depletion of foreign investments and human capital) and microeconomics (like direct cost invested in treatment and prevention and indirect cost due to sickness) leading to increased poverty level and interruption in the economic development (Kenneth et al. 2004; Anonymous, 2014, 2017). According to a survey, about 216 million cases and 0.445 million deaths due to malaria were reported in 2016, globally (Anonymous, 2017). Endemic reports of malaria have been reported from different countries of the Indian subcontinent like Pakistan, India, Bangladesh and Sri Lanka. According to World Health Organization (WHO), Pakistan contributes 31% of estimated malaria cases in 2016 and falls among the countries which showed increase in case incidence from 2010 to 2016 (Anonymous, 2017). The prevalence of different species of mosquitoes depends upon the environmental conditions of the area under study, for example from rural areas; Anopheles (An.) culicifacies has been reported (Pervez and Shah 1989; Rasheed et al. 2013) while An. stephensi is mostly reported in urban areas (Regmiet al. 2016). Similarly, An. fluviaitlis and An. pulcherrimus have been found in the mountainous areas of Punjab and Khyber Pakhtunkhwa (KPK) provinces of Pakistan (Suleman et al. 1993). There are different diagnostic techniques (conventional and molecular) available for the detection of malaria. The gold standard and the older one conventional method used for the diagnostic purpose is the blood smear. It is cost effective and applicable on large scale (Hanscheid 1999). The disadvantages of conventional diagnosis are (a) time consuming and (b)