Morphological and molecular characterisation of common amphistome species from cattle of South India. Shameem H 1* , Devada K 1 , Lakshmanan B 1 , Joseph S 1 , Sabu L 2 , Usha AP 3 1 Department of Veterinary Parasitology, College of Veterinary and Animal Sciences, Mannuthy, Kerala, India 2 Department of Veterinary Microbiology, College of Veterinary and Animal Sciences, Pookode, Kerala, India 3 Centre for Pig production and Research, Mannuthy, Kerala Veterinary and Animal Sciences University, Pookode, Kerala, India Abstract Bovine amphistomosis is a highly neglected snail borne trematode disease causing great economic loss to dairy farmers. The aim of the present study was morphological identification of different amphistome species and molecular characterization of the most common amphistomes prevalent in the study area. Among the ten different species of amphistomes identified in the present study four common species namely, F. cobboldi, F. elongatus, G. crumenifer and Paramphistomum spp. were genetically characterized by amplifying second internal transcribed spacer sequence flanking 5.8S and 28S partial ribosomal gene sequences (ITS-2+) which produced amplicons of 494 bp, 503 bp, 514 bp and 494 bp respectively. The nucleotide sequences of four species of amphistomes F. cobboldi, F. elongatus, G. crumenifer and Paramphistomum spp. obtained in the present study is the first report from Kerala (India) which provides the primary sequence data of the rDNA ITS-2+ region of the amphistome flukes. Nucleotide sequence analysis of the four species revealed eight nucleotide differences between the sequences of pouched amphistomes (F. cobboldi, F. elongatus and G. crumenifer) and unpouched (Paramphistome spp.). Nucleotide sequence analysis and in silico restriction map analysis of four amphistomes suggest the possibility of ITS-2+ region as a useful genetic marker for species identification. Keywords: Amphistomes, Cattle, PCR, ITS2. Accepted on 24 February, 2018 Introduction Amphistomosis is a common ruminant trematode disease causing high morbidity and mortality in cattle, goats, sheep and water buffaloes. The disease is characterized by gastro- enteritis, reduced milk production and lowered feed conversion resulting in economic loss. The disease has widespread geographical distribution in tropical and subtropical areas and has recently emerged as an important cause of productivity loss [1]. Paramphistomosis is caused by trematodes belonging to different genera and species of the family Paramphistomidae. Even though these species are difficult to identify due to their morphological similarities they may differ in certain aspects like susceptible hosts, pathogenicity and geographical distribution. More than forty species of amphistomes have been recorded, among which four species viz., Gastrothylax crumenifer, Fischoederius cobboldi, F. elongatus and Paramphistomum are regarded as common amphistomes of domestic ruminants [2]. Paramphistomes are ubiquitous and abundant within hosts but the importance of these worms has been underestimated widely. Since amphistome species are morphologically similar alternative approaches using molecular biology techniques are useful for species identification [3,4]. The second internal transcribed spacer (ITS2) region of ribosomal DNA (rDNA) is particularly shown to be a useful genetic marker in species identification and for phylogenetic analyses [3,5-7]. Hussain et al. [7] characterized three different genus of amphistomes based on rDNA ITS2+ region whereas Ghatani et al. [8] derived molecular phylogeny of five gastrothylacid species using the ITS2 sequence and secondary structure analyses. Little attention has been received in the use of molecular tools to identify the species prevalent in South India. Molecular characterization of the prevalent species of our area will help to explore the regional variation and to contribute a molecular phylogenetic framework for paramphistomes in Kerala. Apart from morphological identification the present study thus aimed to characterize the ITS2+region of four predominant amphistome species viz., Gastrothylax crumenifer, Fischoederius cobboldi, F. elongatus and Paramphistomum spp. infecting ruminants in South India. Research Article http://www.alliedacademies.org/veterinary-medicine-and-allied-science/ J Vet Med Allied Sci 2018 Volume 2 Issue 1 7 ISSN: 2591-7978