Kajian Fenotip dan Genotip Verocytotoxigenic Escherichia coli (VTEC) pada ternak di Yogyakarta

DRASTINI, Yatri, Promotor Prof.drh. Setyawan Budiharta, MPH.,Ph.D

2007 | Disertasi | S3 Ilmu Pertanian (Sain Veteriner)

Ternak merupakan reservoir VTEC, yang dapat menyebabkan berbagai penyakit, bahkan kematian pada manusia. Penelitian ini bertujuan untuk identifikasi, dan karakterisasi VTEC sapi perah, sapi potong, babi dan kambing/domba di Daerah Istimewa Yogyakarta (DIY) didasarkan atas fenotip dan genotip. Sampel yang digunakan dalam penelitian ini sebanyak tujuh ratus tiga puluh berupa tinja 142 ekor sapi perah, 211 sapi potong, 193 babi, dan 184 kambing/domba dari empat kabupaten dan satu kota di Yogyakarta, dikumpulkan secara rambang tahapan ganda dan klaster. Dua isolat standar yakni galur O157:H7 (EDL933) dan O91:H- digunakan sebagai pembanding positif. Escherichia coli diisolasi melalui pengayaan dan penanaman pada media standar untuk bakteri tersebut. Galur VTEC dideteksi melalui reaksi rantai polymerase (polymerase chain reaction, PCR) dengan primer oligonukleotida spesifik verotoksin 1 (vt1) dan verotoksin 2 (vt2) dari isolat yang tidak menfermentasi sorbitol. Galur O157 ditentukan dalam reaksi aglutinasi lateks O157. Fenotip VTEC dievaluasi berdasarkan aktivitas toksin pada sel Vero, dan hasilnya dianalisis dengan one way analysis of variance (anova). Genotip VTEC ditentukan dengan analisis DNA VTEC dengan metode rRNA gene restriction fragment patterns dengan probe DNA yang menyandi RNA ribosomal (ribotiping). Kemiripan antar isolat dianalisis dengan simple matching coeficient (SSM) dan algorhythm unweighted pair-group method using arithmetic averages (UPGMA) dalam program Multivariate Statistical Package (MVSP) 3.1 dan disajikan dalam bentuk dendrogram. Analisis data berdasarkan perbedaan jenis vt, spesies hewan dan geografi asal ternak, dan diujibandingkan dengan data isolat standar. Isolat VTEC yang teridentifikasi sejumlah 264, yang terbanyak dari babi (84%, 162/193), diikuti kambing/domba (78%, 143/184), sapi perah (54%, 76/142), dan sapi potong (44%, 93/211). Wilayah Yogyakarta yang terdapat VTEC paling banyak adalah kabupaten Gunungkidul (80%, 51/64), selanjutnya Kulonprogo (66%, 61/93), Sleman (56%, 81/146), Kota Yogyakarta (51%, 36/71) dan Bantul (47%, 35/74). Isolat VTEC dari babi yang terbanyak berasal dari Kulonprogo, sedang VTEC dari Gunungkidul yang terbanyak berasal dari sapi perah. Verocytotoxin E. coli terbagi dalam galur VTEC O157 dan nir O157. Tujuh (1,5%) VTEC O157 didapat dari babi (5), domba (1) dan sapi perah (1). Isolat O157 memiliki kedua vt1 dan vt2 . Karakter toksisitas O157 pada sel Vero menunjukkan bahwa toksisitas O157 lebih rendah daripada O157:H7, tetapi lebih tinggi daripada toksisitas nirO157. Toksisitas tertinggi adalah O157 dari sapi perah, kemudian babi dan domba. Seperti halnya O157:H7 dari manusia, VTEC O157 dari ternak menyandang kedua vt1 dan vt2,. Pada gambaran dendrogram, O157 dan O157:H7 terletak dalam klaster yang berbeda pada tingkat kemiripan 54% (EcoRI) atau 20% (SalI). Sebanyak 257 (57%) VTEC nir O157 diisolasi dari babi, kambing/domba (masing-masing 65%), sapi perah, dan sapi potong (masingmasing 42%). Berbeda dengan O157:H7 dari manusia, mayoritas VTEC nirO157 mempunyai vt1 serta toksisitas lebih rendah, dan terletak dalam klaster yang berbeda pada tingkat kemiripan 77,6% (EcoRI) dan 59,9% (SalI). Toksisitas antar spesies ternak, jenis VT dan kabupaten asal ternak tidak berbeda signifikan (P>0,05). Hasil analisis keseluruhan menunjukan bahwa babi, kambing/domba, sapi perah dan sapi potong merupakan reservoir VTEC di wilayah DIY. Berdasarkan sifat fenotip, verositotoksisitas VTEC tidak berbeda signifikan antar spesies ternak, jenis vt maupun tempat ternak berasal. Genotip VTEC yang identik terdapat pada kambing di desa yang sama di Gunungkidul, dan antar sapi perah dengan sapi potong di beda kabupaten yaitu Gunungkidul dan kota Yogyakarta. Spesies ternak yang sama dan terletak dalam satu kandang cenderung memiliki VTEC. Penelitian ini menunjukkan juga bahwa jenis vt dari O157 identik dengan O157:H7 manusia, tetapi profil ribotipenya berbeda.

Livestock is considered as reservoir of VTEC, organism that may cause different diseases and even death in human. This study was to identify and characterize phenotypically and genotypically VTEC of livestock in Yogyakarta Special Province (DIY). A total of 730 fecal samples was taken from dairy cow (142), beef cattle (211), pigs (193), and goat/sheep (184) in the 5 districts of DIY. The samples were taken using multistage and cluster strategy to detect the presence of VTEC.Standard strains (O157:H7 EDL933 and O91:H-) were used as control organisms. Escherichia coli was isolated through the enrichment and culture using standard media for the bacteria. The VTEC strains was detected by means of polymerase chain reaction (PCR) using oligonucleotides specific for vt1 and vt2. Strains of O157 was verified by O157 latex agglutination test. The activity of the toxin (200 mg/mL). was measured based on its toxic lethal dose 50 against Vero cell, and the results were subjected to one-way analysis of variance. The stages of ribotyping included the digestion of sample DNA with EcoRI and SalI separately, transfer of DNA to nylon membrane in blotting method, labelling of probe (DNA probe consisting of rRNA) with nonradioactive label (Dig DNA Labeling and Detection Kit), and hibridization. Similarity of isolates was analyzed by simple matching coefficient (SSM) and unweighted pair group method using arithmetic averages (UPGMA) in Multivariate Statistical Package (MVSP) 3.1 programme and was showed in dendrogram. The total of VTEC isolated from livestock in Yogyakarta was 264. The majority of VTEC isolates was obtained from pigs (84%, 162/193), followed by goat/sheep (78%, 143/184), dairy cows (54%, 76/142) and beef cattle (44%, 93/211). Gunungkidul was a district having the most VTEC (80%, 51/64), followed by Kulonprogo (66%, 61/93), Sleman (56%, 81/146), Kota Yogyakarta (51%, 36/71) and Bantul (47%, 35/74). But, the most pigs were from Kulonprogo, while the most VTEC from Gunungkidul were isolated from dairy cattle Verocytotoxigenic E.coli was grouped into VTEC O157 and VTEC nonO157. Seven (1.5%) VTEC O157 isolates were from pigs (5), sheep (1) and dairy cow (1). All of them have both vt1 or vt2 .. Characters of O157 on Vero cells showed that toxicity of O157 was lower than that of nonO157. The highest toxicity was O157 from dairy cattle, then pigs and sheep. All of O157 consisted of both vt1 and vt2 the same as O157:H7 from human.The dendrogram showed that O157 and O157:H7 were in different cluster at 54% (EcoRI) and 20% (SalI). All of 257 (57%) VTEC nonO157 were isolated from pigs and goat/sheep (65% each), followed by dairy cows and beef cattle (42% each). Toxicity among species, VT and geography was not significantly different (P>0.05). Unlike O157:H7, majority of nonO157 VTEC had vt1 of low toxicity, and was in different cluster 77.6% and 59.9% with EcoRI and SalI, respectively. Toxicity of nonO157 among species, kinds of vt and district of original geography was not different significantly (P>0,05). The results showed that pigs, goat/sheep, dairy cattle and beef cattle were reservoirs in Yogyakarta. Phenotypically, the verocytotoxicity among isolates was not significantly different among species, kinds of vt, and geographical location. Identical genotype of VTEC was found distributed among goats in the same village, and between dairy and beef cattle of Gunungkidul and Yogyakarta city. The genotype of VTEC of the same livestock species in the same barn tended to be identical. This investigation also revealed that the vt’s from O157 isolated from livestock were identical with those of human O157:H7, while the ribotypes were different from each other.

Kata Kunci : Ternak,Penyakit,Fenotip dan Genotip VTEC,VTEC, Vero cells, verotoxin, phenotype, genotype