Dyah Ayu Widiasih

Frequency and risk-factors analysis of Escherichia coli O157:H7 in Bali-cattle

Cattle are known as the main reservoir of zoonotic agents verocytotoxin-producing Escherichia coli. These bacteria are usually isolated from calves with diarrhea and/or mucus and blood. Tolerance of these agents to the environmental conditions will strengthen of their transmission among livestock. A total of 238 cattle fecal samples from four sub-districts in Badung, Bali were used in this study. Epidemiological data observed include cattle age, sex, cattle rearing system, the source of drinking water, weather, altitude, and type of cage floor, the cleanliness of cage floor, the slope of cage floor, and the level of cattle cleanliness. The study was initiated by culturing of samples onto eosin methylene blue agar, then Gram stained, and tested for indole, methyl-red, voges proskauer, and citrate, Potential E.coli isolates were then cultured onto sorbitol MacConkey agar, and further tested using O157 latex agglutination test and H7 antisera. Molecular identification was performed by analysis of the 16S rRNA gene, and epidemiological data was analyzed using STATA 12.0 software. The results showed, the prevalence of E. coli O157:H7 in cattle at Badung regency was 6.30% (15/238) covering four sub districts i.e. Petang, Abiansemal, Mengwi, and Kuta which their prevalence was 8.62%(5/58), 10%(6/60), 3.33%(2/60), and 3.33(2/60)%, respectively. The analysis of 16S rRNA gene confirmed of isolates as an E. coli O157:H7 strain with 99% similarities. Furthermore, the risk factors analysis showed that the slope of the cage floor has a highly significant effect (P<0.05) to the distribution of infection. Consequently, implementing this factor must be concerned in order to decrease of infection.

Detection of Trenbolone Acetate in Beef Muscle and Liver Using High-Performance Liquid Chromatography Method

The use of trenbolone acetate (TBA) as growth promotor was legal in some regions, and it has to be considered since there are no rules yet in Indonesia committed to TBA application in beef. As a step for preconditioning the policy of TBA used as growth promotor in beef, the validated method of hormone detection is needed to be established. This research was aimed to validate the high-performance liquid chromatography (HPLC) method for TBA detection as a tool to control the hormone residue in meat. The imported beef muscles and liver were obtained from a supermarket in Yogyakarta. All samples (n = 10) were kept in the freezer (−18 °C) before analysis. The HPLC method is developed from sample extraction, cleanup with solid-phase extraction (SPE) cartridge, and the hormone concentration analysis using standard curve of TBA. The method has validated and gave the values of LOD (limit of detection): 1 × 10−5 μg · L−1 (muscle) and 1 × 10−4 μg · L−1 (liver). The LOQ (limit of quantification): 1 × 10−4 μg · L−1 (muscle) and 1 × 10−3 μg · L−1 (liver) and R2 = 0.99 (muscle and liver). The analysis results were three positive TBA in muscles (0.730 μg · L−1, 0.074 μg · L−1 and 0.116 μg · L−1) and two positive in livers (0.080 μg · L−1 and 0.130 μg · L−1). All TBA levels in tissues were not exceeding the maximum residue limits of TBA according to the Codex Alimentarius Commissions. This HPLC analysis procedure could be done as a detection method for TBA in beef tissues.

Serological and Molecular Biological Diagnosis for Leptospirosis

In this study, serological and molecular biological approaches were implicated to be used in the diagnosis of leptospirosis. We developed specific antigens of leptospira by using leptospiral outer membrane, LipL32, and leptospiral immunoglobulin-like protein A (LigA). By using these recombinant antigens, ELISA for IgG and IgM tests for sera from leptospirosis cases found in Sri Lanka were performed. These cases were already diagnosed by the microscopic agglutination test (MAT) and PCR. Serological approaches revealed the preparation of specific antigens to be used in ELISA system and the sensitivity of ELISA system compared with MAT. The chi-square test (χ 2 ) defined that IgG-LipL32, IgG-LigA, and IgM-ELISA in comparison with MAT were sensitive as 5.439 (significance p ≤ 0.01), 9.188 (significance p ≤ 0.001), and 5.915 (significance p ≤ 0.01), respectively. Molecular studies approaches revealed the sensitivity of PCR to be diagnostic tool in leptospirosis. PCR can be considered as effective diagnostic tools to diagnose leptospirosis. From the results of experiments for comparing sensitivity, several contradictions were found. Too much amount of DNA might be inhibiting specific PCR. Anyway, both PCR methods targeting flaB gene and WHO recommendations were followed to detect genomic DNA of leptospira effectively.

Regulatory elements of stx2 gene and the expression level of Shiga-like toxin 2 in Escherichia coli O157:H7

BACKGROUND/PURPOSE Shiga-like toxin (Stx) is an important factor in the pathogenesis of Escherichia coli O157:H7 infection and is responsible for some severe complications. Stx2 is usually associated with hemolytic uremic syndrome in humans. Its expression is regulated by elements located upstream of the stx2 gene, including stx2-promoter sequence, ribosome binding site, and the antiterminator q gene. The present study aimed to find the correlation between regulatory elements and the expression level of Stx2 in two local isolates of E. coli O157:H7. METHODS Two local E. coli O157:H7 strains SM-25(1) and KL-48(2), originating from human and cattle feces, respectively, and an E. coli reference strain, ATCC 43894, were investigated. The complete stx2 gene covering the sequences of promoter, ribosome binding site, and open reading frame and q gene of each strain was analyzed. The magnitude of Stx2 production was detected with a reverse passive latex agglutination method and Stx mediated cellular damage was determined with the Vero cell assay. RESULTS A comparison of the complete stx2 gene contained stx2-promoter, ribosome binding site, and q genes of two local strains KL-48(2) and SM25(1), and the E. coli ATCC 43894 showed that the amino acid sequences were identical. Both local isolates were Stx negative in the reverse passive latex agglutination test and nontoxic in the Vero cell assay. CONCLUSION The expression level of Shiga-like toxin of the two local isolates of E. coli O157:H7 did not only depend on the regulatory elements of the stx2 gene.