Sains Malaysiana 48(7)(2019): 1425–1432

http://dx.doi.org/10.17576/jsm-2019-4807-11

Comparison of Molecular Methods for the Detection of Eimeria in Domestic Chickens in Malaysia

(Perbandingan Kaedah Molekul untuk Pengenalpastian Eimeria dalam Ayam Ternakan di Malaysia)

SHU-SAN LOO^1,2,3, LIK-SIN LIM^1,4, NURUL-AIN EFENDI¹, DAMER P. BLAKE⁵, SHIN-ICHIRO KAWAZU⁶ & KIEW-LIAN WAN^1,2,4*

¹School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

²Centre for Biotechnology and Functional Food, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

³Agro-Biotechnology Institute, National Institutes of Biotechnology Malaysia, c/o MARDI Headquarters, 43400 Serdang, Selangor Darul Ehsan, Malaysia

⁴Malaysia Genome Institute, National Institutes of Biotechnology Malaysia, Jalan Bangi, 43000 Kajang, Selangor Darul Ehsan, Malaysia

⁵The Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Hertfordshire, United Kingdom

⁶National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan

Diserahkan: 8 Oktober 2018/Diterima: 30 April 2019

ABSTRACT

Coccidiosis, caused by the Eimeria species, greatly affects the poultry industry. Severity of the disease varies depending on the identity of the infecting parasites, encouraging identification of Eimeria species circulating on a farm as a valuable component of chicken management. Conventional methods of Eimeria species identification are time consuming and can be subjective in nature. Given these limitations, molecular approaches have been developed for specific detection of Eimeria species. In this study, faecal samples were collected from commercial broiler farms and subjected to microscopic examination for Eimeria occurrence. Eimeria species were putatively identified by morphological characterisation and grouped into three categories based on oocyst size. Molecular detection of Eimeria species occurrence in these samples was then performed using two published PCR assays (the individual components of a SCAR-based multiplex PCR, and assays developed for quantitative PCR, termed PCR-SCAR 1 and PCR-SCAR 2 here) and a LAMP assay. Comparison of the results obtained demonstrated that the three molecular methods were capable of detecting all Eimeria species of the reference Houghton strain, but showed varying efficiencies in detecting Malaysian field isolates. PCR-SCAR 2 was found to be the most effective, detecting all seven Eimeria species and indicating the presence of Eimeria parasites in most flocks. Differences in the ability of the molecular methods to detect Eimeria may be a consequence of sequence divergence between isolates from different regions, implying that development of region-specific methods using local Eimeria strains may be required to improve the efficiency of molecular assays for Eimeria detection.

Keywords: Coccidiosis; LAMP; protozoan parasite; SCAR

ABSTRAK

Koksidiosis yang disebabkan oleh spesies Eimeria, memberikan kesan yang besar terhadap industri penternakan ayam. Keparahan penyakit ini bergantung kepada identiti parasit yang menjangkit dan ini menggalakkan usaha pengenalpastian spesies Eimeria yang hadir dalam ladang ayam sebagai komponen yang penting dalam pengurusan ayam. Kaedah konvensional pengenalpastian spesies Eimeria memakan masa dan didapati bersifat subjektif. Disebabkan oleh kekangan kaedah ini, pendekatan molekul telah dibangunkan untuk pengenalpastian spesies Eimeria secara spesifik. Dalam kajian ini, sampel tinja telah dikumpul dari ladang ayam pedaging komersial dan disaring melalui pemerhatian mikroskop untuk mengesan kehadiran Eimeria. Spesies Eimeria telah dikenal pasti secara pencirian morfologi dan dikelompokkan kepada tiga kategori berdasarkan saiz oosista. Pengesanan molekul terhadap kehadiran spesies Eimeria dalam sampel tersebut kemudiannya telah dilakukan dengan menggunakan dua asai PCR yang telah diterbitkan (komponen individu PCR multipleks berasaskan SCAR, dan asai yang dibangunkan untuk PCR kuantitatif yang dikenali sebagai PCR-SCAR 1 dan PCR-SCAR 2) dan asai LAMP. Perbandingan hasil yang diperoleh menunjukkan bahawa ketiga-tiga kaedah molekul ini mampu mengesan kesemua spesies Eimeria daripada strain rujukan Houghton, tetapi menunjukkan kecekapan yang berbeza dalam mengesan pencilan lapangan dari Malaysia. PCR-SCAR 2 didapati paling berkesan dan berupaya mengesan kesemua tujuh spesies Eimeria serta menunjukkan kehadiran parasit Eimeria dalam kebanyakan populasi ayam. Perbezaan dalam keupayaan kaedah molekul untuk mengesan Eimeria mungkin disebabkan oleh perbezaan jujukan antara pencilan dari kawasan yang berbeza, dan ini mencadangkan bahawa pembangunan kaedah berasaskan kawasan yang khusus menggunakan strain Eimeria tempatan adalah diperlukan untuk meningkatkan kecekapan asai molekul untuk pengesanan Eimeria.

Kata kunci: Koksidiosis; LAMP; parasit protozoa; SCAR

RUJUKAN

Al-Natoura, M.Q., Suleimana, M.M. & Abo-Shehadab, M.N. 2002. Flock-level prevalence of Eimeria species among broiler chicks in northern Jordan. Preventive Veterinary Medicine 53: 305-310.

Barkway, C.P., Pocock, R.L., Vrba, V. & Blake, D.P. 2011. Loop-mediated isothermal amplification (LAMP) assays for the species-specific detection of Eimeria that infect chickens. BMC Veterinary Research 7: 67.

Bhaskaran, M.S., Venkatesan, L., Aadimoolam, R., Jayagopal, H.T. & Sriraman, R. 2010. Sequence diversity of internal transcribed spacer-1 (ITS-1) region of Eimeria infecting chicken and its relevance in species identification from Indian field samples. Parasitology Research 106: 513-521.

Blake, D.P. & Tomley, F.M. 2014. Securing poultry production from the ever-present Eimeria challenge. Trends in Parasitology 30(1): 12-19.

Chapman, H.D. 1997. Biochemical, genetic and applied aspects of drug resistance in Eimeria parasites of the fowl. Avian Pathology 28: 221-244.

Chapman, H.D. & Jeffers, T.K. 2014. Vaccination of chickens against coccidiosis ameliorates drug resistance in commercial poultry production. International Journal for Parasitology. Drugs and Drug Resistance 4: 214-217.

Clark, E.L., Macdonald, S.E., Thenmozhi, V., Kundu, K., Garg, R., Kumar, S., Ayoade, S., Fornace, K.M., Jatau, I.D., Moftah, A., Nolan, M.J., Sudhakar, N.R., Adebambo, A.O., Lawal, I.A., Zapata, R.A., Awuni, J.A., Chapman, H.D., Karimuribo, E., Mugasa, C.M., Namangala, B., Rushton, J., Suo, X., Thangaraj, K., Rao, A.S.R.S., Tewari, A.K., Banerjee, P.S., Raj, G.D., Raman, M., Tomley, F.M. & Blake, D.P. 2016. Cryptic Eimeria genotypes are common across the southern but not northern hemisphere. International Journal for Parasitology 46: 537-544.

Fernandez, S., Costa, A.C., Katsuyama, A.M., Madeira, A.M. & Gruber, A. 2003a. A survey of the inter- and intraspecific RAPD markers of Eimeria spp. of the domestic fowl and the development of reliable diagnostic tools. Parasitology Research 89: 437-445.

Fernandez, S., Pagotto, A.H., Furtado, M.M., Katsuyama, A.M., Madeira, A.M. & Gruber, A. 2003b. A multiplex PCR assay for the simultaneous detection and discrimination of the seven Eimeria species that infect domestic fowl. Parasitology 127: 317-325.

Gasser, R.B., Woods, W.G., Wood, J.M., Ashdown, L., Richards, G. & Whithear, K.G. 2001. Automated, fluorescence based approach for the specific diagnosis of chicken coccidiosis. Electrophoresis 22: 3546-3550.

Hadipour, M.M., Olyaie, A., Naderi, M., Azad, F. & Nekouie, O. 2013. Prevalence of Eimeria species in scavenging native chickens of Shiraz, Iran. African Journal of Poultry Farming 1(2): 34-36.

Haug, A., Gjevre, A.G., Thebo, P., Mattsson, J.G. & Kaldhusdal, M. 2008. Coccidial infections in commercial broilers: Epidemiological aspects and comparison of Eimeria species identification by morphometric and polymerase chain reaction techniques. Avian Pathology 37(2): 161-70.

Jatau, I.D., Lawal, I.A., Kwaga, J.K.P., Tomley, F.M., Blake, D.P. & Nok, A.J. 2016. Three operational taxonomic units of Eimeria are common in Nigerian chickens and may undermine effective molecular diagnosis of coccidiosis. BMC Veterinary Research 12: 86.

Karanis, P., Thekisoe, O., Kiouptsi, K., Ongerth, J., Igarashi, I. & Inoue, N. 2007. Development and preliminary evaluation of a loop-mediated isothermal amplification procedure for sensitive detection of Cryptosporidium oocysts in fecal and water samples. Applied and Environmental Microbiology 73(17): 5660-5662.

Kucera, J. 1990. Identification of Eimeria species in Czechoslovakia. Avian Pathology 19: 59-66.

Kumar, S., Garg, R., Moftah, A., Clark, E.L., Macdonald, S.E., Chaudhry, A.S., Sparagano, O., Banerjee, P.S., Kundu, K., Tomley, F.M. & Blake, D.P. 2014. An optimised protocol for molecular identification of Eimeria from chickens. Veterinary Parasitology 199: 24-31.

Lee, B.H., Kim, W.H., Jeong, J., Yoo, J., Kwon, Y.K., Jung, B.Y., Kwon, J.H., Lillehoj, H.S. & Min, W. 2010. Prevalence and cross immunity of Eimeria species on Korean chicken farms. Journal of Veterinary Medical Science 72(8): 985-989.

Lew, A.E., Anderson, G.R., Minchin, C.M., Jenston, P.J. & Jorgensen, W.K. 2003. Inter- and intra-strain variation and PCR detection of the internal transcribed spacer 1 (ITS-1) sequences of Australian isolates of Eimeria species from chickens. Veterinary Parasitology 112: 33-50.

Lien, Y.Y., Sheu, S.C., Liu, H.J., Chen, S.C., Tsai, M.Y., Luo, S.C., Wu, K.C., Liu, S.S. & Su, H.Y. 2007. Cloning and nucleotide sequencing of the second internal transcribed spacer of ribosomal DNA for three species of Eimeria from chickens in Taiwan. Veterinary Journal 173: 186-191.

Long, P.L. & Joyner, L.P. 1984. Problems in identification of species of Eimeria. Journal of Protozoology 31: 535-541.

Long, P.L., Millard, B.J., Joyner, L.P. & Norton, C.C. 1976. A guide to laboratory techniques used in the study and diagnosis of avian coccidiosis. Folia Veterinaria Latina 6: 201-217.

Morgan, J.A.T, Morris, G.M., Wlodek, B.M., Byrnes, R., Jenner, M., Constantinoiu, C.C, Anderson, G.R., Lew-Tabor, A.E., Molly, J.B., Gasser, R.B. & Jorgensen, W.K. 2009. Real-time polymerase chain reaction (PCR) assays for the specific detection and quantification of seven Eimeria species that cause coccidiosis in chickens. Molecular and Cellular Probes 23: 83-89.

Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N. & Hase, T. 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Research 28(12): e63.

Poon, L.L., Wong, B.W., Ma, E.H., Chan, K.H., Chow, L.M., Abeyewikreme, W., Tangpukdee, N., Yuen, K.Y., Guan, Y., Looareesuwan, S. & Peiris, J.S. 2006. Sensitive and inexpensive molecular test for falciparum malaria: Detecting Plasmodium falciparum DNA directly from heat-treated blood by loop-mediated isothermal amplification. Clinical Chemistry 52(2): 303-306.

Schnitzler, B.E., Thebo, P., Mattson, J.G., Tomley, F. & Shirley, M.W. 1998. Development of a diagnostic PCR assay for the detection and discrimination of four pathogenic Eimeria species of the chicken. Avian Pathology 27: 490-497.

Schnitzler, B.E., Thebo, P., Mattson, J.G., Tomley, F., Uggla, A. & Shirley, M.W. 1999. PCR identification of chicken Eimeria. Avian Pathology 27: 490-497.

Shirley, M.W. 1995. Eimeria species and strains of chickens. In Biotechnology - Guidelines on Techniques in Coccidiosis Research, edited by Eckert, J., Braun, R., Shirley, M.W. & Coudert, P. Luxemborg: European Commision. pp. 1-24.

Shirley, M.W., Smith, A.L. & Tomley, F.M. 2005. The biology of avian Eimeria with an emphasis on their control by vaccination. Advances in Parasitology 60: 285-330.

Sotiriadou, I. & Karanis, P. 2008. Evaluation of loop-mediated isothermal amplification for detection of Toxoplasma gondii in water samples and comparative findings by polymerase chain reaction and immunoflourescence test (IFT). Diagnostic Microbiology and Infectious Disease 62(4): 357-365.

Stucki, U., Braun, R. & Roditi, I. 1993. Eimeria tenella: Characterization of 5S ribosomal RNA repeat subunit and its use as a species-specific probe. Experimental Parasitology 76: 68-75.

Su, Y.C., Fei, A.C. & Tsai, F.M. 2003. Differential diagnosis of five avian Eimeria species by polymerase chain reaction using primers derived from internal transcribed spacer 1 (ITS-1) sequence. Veterinary Parasitology 117: 221-227.

Sun, X.M., Pang, W., Jia, T., Yan, W.C., He, G., Hao, L.L., Bentue, M. & Suo, X. 2009. Prevalence of Eimeria species in broilers with subclinical signs from fifty farms. Avian Disease 53: 301-305.

Tsuji, N., Kawazu, S., Ohta, M., Kamio, T., Isobe, T., Shimura, K. & Fujisaki, K. 1997. Discrimination of eight chicken Eimeria species using the two-step polymerase chain reaction. Journal of Parasitology 83: 966-970.

Vrba, V., Blake, D.P. & Poplstein, M. 2010. Quantitative real-time PCR assays for detection and quantification of all seven Eimeria species that infect the chicken. Veterinary Parasitology 174: 183-190.

Williams, R.B., Bushell, A.C., Reperent, J.M., Doy, T.G., Morgan, J.H., Shirley, M.W., Yvore, P., Carr, M.M. & Fremont, Y. 1996. A survey of Eimeria species in commercially-reared chickens in France during 1994. Avian Pathology 25: 113-130.

*Pengarang untuk surat-menyurat; email: klwan@ukm.edu.my