Molecular Characterization of Paramphistomum cervi in Buffaloes

Naseem Rafiq, Sadaf Niaz, Ismail Zeb, Sultan Ayaz, Itabajara da Silva Vaz Jr., Abid Ali


Background: Paramphistomiasis (Rumen fluke disease) in ruminants is a major health problem, while documented reports on Paramphistomum species are limited in Asian countries. The present study aimed to identify paramphistomoid flukes that infects buffaloes with the goal of characterization of prevalence in Pakistan and its comparison with neighbor countries.

Materials, Methods & Results: In 2018, a total of 178 slaughtered buffaloes aged four to six years were examined and flukes were collected from their infected rumen and reticulum using sterilized forceps. After amplification and sequencing of 18S rRNA partial fragment, the generated sequences were edited (810bp) and aligned with the other sequences of Paramphistomum species retrieved from NCBI. A phylogenetic tree was constructed using maximum likelihood method in MEGA X. The 18S rRNA sequence was found 100% similar with Paramphistomum cervi of China and 98% with Paramphistomum epiclitum and other Paramphistomum species of India. The parasitic Pharamphistomum species was identified molecularly as P. cervi.

Discussion: Molecular studies provide insight into the biology and phylogenetic relationship among various parasites. These studies are reliable in the genetic-based identification and description of several disease causing agents. The 18S rRNA sequence of P. cervi generated in this study was found closely identical to the P. cervi of the neighbor countries (China and India) which may be due to the similar geographical, environmental conditions and transboundary movement of infected hosts. This is the first nature of study which provides the molecular-based evidence of P. cervi existence in Pakistan and revealed the 18S rRNA as novel molecular marker for the identification and further characterization of Paramphistomum species across Pakistan. The submitted sequence of this study will provide a baseline for further molecular characterization and to compare with other Paramphistoma species from different regions of Pakistan.

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Ali Q., Rashid I., Shabbir M.Z., Akbar H., Shahzad K., Ashraf K. & Chaudhry U. 2018. First genetic evidence for the presence of the rumen fluke Paramphistomum epiclitum in Pakistan. Parasitology International. 67(5): 533-537.

Blair D., Campos A., Cummings M.P. & Laclette J.P. 1996. Evolutionary biology of parasitic platyhelminths: the role of molecular phylogenetics. Parasitol Today. 12(2): 66-71.

Chan A.H.E., Chaisiri K., Morand S., Saralamba N. & Thaenkham U. 2020. Evaluation and utility of mitochondrial ribosomal genes for molecular systematics of parasitic nematodes. Parasites Vectors. 13: 1-13.

Chandrasekharan K., Radhakrishnan K. & Jacob V.C. 1982. Efficacy of distodin in the treatment of amphistomiasis in Indian elephants. Kerala Journal of Veterinary Sciences. 13(1): 55-58.

Eduardo S.L. 1937. The taxonomy of the family Paramphistomidae with special reference to the morphology of species occurring in ruminants. Systematic Parasitology. 5: 25-79.

El-Bahy N.M., Bazh E.K. & Elkhtam A. 2017. New approach to molecular characterization of Paramphistomum cervi and Carmyerius gregarius and comparative analyses with selected trematodes. Parasitology Research. 116(5): 1417-1422.

Firdausy L.W., Prahardani R., Wusahaningtyas L., Soedarmanto I., Wahyu M., Nursalim M.T. & Nurcahyo W. 2019. Morphological and molecular identification of Pfenderius heterocaeca (Trematode: Paramphistomoidea) from Sumatran elephant (Elephas maximus sumatranus). Veterinary World. 12(8): 1341-1345.

Foreyt W.J. 2001. Parasites of Cattle, Sheeps and Goats. In: Veterinary parasitology (Reference Manual). 5th edn. Ames: Iowa State Press, pp.69-113.

Godara R., Katoch R., Yadav A. & Rastogi A. 2014. Epidemiology of paramphistomosis in sheep and goats in Jammu, India. Journal of Parasitic Diseases. 38(4): 423-428.

Hernández-Domínguez E.M., Castillo-Ortega L.S., García-Esquivel Y., Mandujano-González V., Díaz-Godínez G. & Álvarez-Cervantes J. 2019. Bioinformatics as a Tool for the Structural and Evolutionary Analysis of Proteins. In Phylogenetics. IntechOpen. DOI: 10.5772/intechopen.89594.

Huson K.M., Oliver N.A. & Robinson M.W. 2017. Paramphistomosis of ruminants: an emerging parasitic disease in Europe. Trends in parasitology. 33(11): 836-844.

Ijaz M., Khan M.S., Avais M., Ashraf K. & Ali M.M. 2008. Infection rate and chemotherapy of various helminths in goats in and around Lahore. Pakistan Veterinary Journal. 28(4): 167-170.

Javed K.U., Akhtar T., Maghbool A. & Anees A. 2006. Epidemiology of Paramphistomiasis in buffaloes different monumental conditions at four districts in Punjab, Pakistan. Iranian Journal of Veterinary Research. 7(3): 68-72.

Kattak A. 2017. Prevalence of gastrointestinal parasite, Paramphistomum in domestic animals (Cows and Buffaloes) of district Swat and Charsadda, KP, Pakistan. Journal of Entomology. 5(3): 907-911.

Khan I., Afshan K., Shah S., Akhtar S., Komal M. & Firasat S. 2019. Morphological and Molecular Identification of Paramphistomum epiclitum from Buffaloes in Pakistan. Acta Parasitologica. 65(3): 225-236.

Khedri J., Radfar M.H., Borji H. & Mirzaei M. 2015. Prevalence and intensity of Paramphistomum spp. in cattle from South-Eastern Iran. Iranian Journal of Parasitology. 10(2): 268-272.

Lotfy W.M., Brant S.V., Ashmawy K.I., Devkota R., Mkoji G.M. & Loker E. S. 2010. A molecular approach for identification of paramphistomes from Africa and Asia. Veterinary Parasitology. 174(3-4): 234-240.

Ma J., He J.J., Liu G.H., Zhou D.H., Liu J.Z., Liu Y. & Zhu X.Q. 2015. Mitochondrial and nuclear ribosomal DNA dataset supports that Paramphistomum leydeni (Trematoda: Digenea) is a distinct rumen fluke species. Parasites & vectors. 8(1): 1-9.

Martinez-Ibeas A.M., Munita M.P., Lawlor K., Sekiya M., Mulcahy G. & Sayers R. 2016. Rumen fluke in Irish sheep: prevalence, risk factors and molecular identification of two Paramphistome species. BMC Veterinary Research. 12(1): 1-11.

Mufti S., Afshan K., Khan I.A., Zafar Y., Raza Rizvi S.S., Nazir F. & Qayyum M. 2014. Genetic Characterization of Fasciola Samples from Bovine Hosts in Pakistan by Sequences of Ribosomal Internal Transcribed Spacer Regions. Pakistan Veterinary Journal. 34(3): 361-366.

Patwardhan A., Ray S. & Roy A. 2014. Molecular markers in phylogenetic studies-a review. Journal of Phylogenetics & Evolutionary Biology. 2(2): 1-9.

Prasad P.K., Tandon V., Biswal D.K., Goswami L.M. & Chatterjee A. 2009. Use of sequence motifs as barcodes and secondary structures of internal transcribed spacer 2 (ITS2, rDNA) for identification of the Indian liver fluke, Fasciola (Trematoda: Fasciolidae). Bioinformation. 3(7): 314-320.

Raza M.A., Murtaza S., Bachaya H.A. & Hussain A. 2009. Prevalence of Paramphistomum cervi in ruminants slaughtered in district Muzaffar Garh. Pakistan Veterinary Journal. 28(1): 34-36.

Rehman A., Jingdong L., Chandio A.A. & Hussain I. 2017. Livestock production and population census in Pakistan: Determining their relationship with agricultural GDP using econometric analysis. Information Processing in Agriculture. 4(2): 168-177.

Rinaldi L., Perugini A.G., Capuano F., Fenizia D., Musella V., Veneziano V. & Cringoli G. 2005. Characterization of the second internal transcribed spacer of ribosomal DNA of Calicophoron daubneyi from various hosts and locations in southern Italy. Veterinary Parasitology. 131(3-4): 247-253.

Sambrook J. & Russell D.W. 2001. Molecular cloning: a laboratory manual. 3rd edn. New York: Cold Spring Harbor Laboratory Press, pp.23-44.

Samn A.A.A.M. 2017. A Molecular and Microscopically Studies of Calicophoron Microbothrium. The Egyptian Journal of Hospital Medicine. 66(1): 28-39.

Shameem H., Devada K. & Lakshmanan B. 2018. Morphological and molecular characterisation of common amphistome species from cattle of South India. Journal of Veterinary Medical Allied Sciences. 2(1): 7-11.

Sindičić M., Martinković F., Strišković T., Špehar M., Štimac I., Bujanić M. & Konjević D. 2017. Molecular identification of the rumen flukes Paramphistomum leydeni and Paramphistomum cervi in a concurrent infection of the red deer Cervus elaphus. Journal of helminthology. 91(5): 637-641.

Stothard J.R., Hughes S. & Rollinson D. 1996. Variation within the ribosomal internal transcribed spacer (ITS) from freshwater snails of the genus Bulinus. Acta Tropica. 61(1): 19-29.

Tandon V., Prasad P.K., Chatterjee A. & Bhutia P.T. 2007. Surface fine topography and PCR-based determination of metacercaria of Paragonimus sp. from edible crabs in Arunachal Pradesh, Northeast India. Parasitology research. 102(1): 21-28.

Titi A., Mekroud A., Chibat H.M., Boucheikhchoukh M., Zein-Eddine R., Djuikwo-Teukeng F.F. & Dreyfuss G. 2014. Ruminal paramphistomosis in cattle from northeastern Algeria: prevalence, parasite burdens and species identification. Parasite. 21: 1-8.

Ullah S., Afshan, K., Arshad, M. & Firasat S. 2019. Genetic Characterization of Gigantocotyle explanatum from Buffaloes in Northwestern Pakistan. Kafkas Universitesi Veteriner Fakultesi Dergisi. 26(2): 225-230. DOI: 10.9775/kvfd.2019.22777.

Yamaguti S. 1952. Parasitic worms mainly from Celebes. Part 5. Trematodes of mammals. Acta Medica Okayama. 8(4): 341-352.

Yamamoto Y. 2002. PCR in diagnosis of infection: detection of bacteria in cerebrospinal fluids. Clinical and Vaccine Immunology. 9(3): 508-514.

Zheng X., Chang Q.C., Zhang Y., Tian S.Q., Lou Y., Duan H. & Zhu X.Q. 2014. Characterization of the complete nuclear ribosomal DNA sequences of Paramphistomum cervi. The Scientific World Journal. 2014: 1-12.


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