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

Received: 8 October 2018/Accepted: 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 spesiesEimeria, memberikan kesan yang besar terhadap industri penternakan ayam. Keparahan penyakit ini bergantung kepada identiti parasit yang menjangkit dan ini menggalakkan usaha pengenalpastian spesiesEimeria yang hadir dalam ladang ayam sebagai komponen yang penting dalam pengurusan ayam. Kaedah konvensional pengenalpastian spesiesEimeria memakan masa dan didapati bersifat subjektif. Disebabkan oleh kekangan kaedah ini, pendekatan molekul telah dibangunkan untuk pengenalpastian spesiesEimeria secara spesifik. Dalam kajian ini, sampel tinja telah dikumpul dari ladang ayam pedaging komersial dan disaring melalui pemerhatian mikroskop untuk mengesan kehadiranEimeria. SpesiesEimeria telah dikenal pasti secara pencirian morfologi dan dikelompokkan kepada tiga kategori berdasarkan saiz oosista. Pengesanan molekul terhadap kehadiran spesiesEimeria 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 spesiesEimeria 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 parasitEimeria 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

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*Corresponding author; email: klwan@ukm.edu.my