Profile and efficacy of whole-plant corn silage juice against Escherichia coli and Salmonella sp. isolated from feses of diarrheic calves

Abstract

Pre-ruminant phase of calf is a crucial stage on raising dairy calves. High morbidity and mortality rates commonly found on pre-ruminant calves are triggered by diarrhea occurrence. Coliform bacteria mainly Escherichia coli and Salmonella are gram negative bacteria and are normal inhabitant of the intestinal tract of many animal species which are usually at low number. However, under diarrheic calves the fecal numbers of coliform exceed lactic acid bacteria (LAB). This research was conducted to study the juice profile of whole corn ensiled for seventy days and its antibacterial activity against Escherichia coli and Salmonella sp. isolated from diarrheic calves. This research encompasses three stages. The first stage of this study was to produce the whole-plant corn silage juice. Whole-plant corn with half milk line of kernel maturity was harvested and chopped with 1-2 cm length of cut. The chopped corn was then mixed thoroughly and filled into 0.35 mm double-lined plastic bags and was vacuum-packed and stored at 25-28 oC storage temperature. After seventy day preservation the plastic bag was pressed using hydraulic pressure to obtain the juice. The whole-plant corn silage juice was then evaluated for acid fermentation products, lactic acid bacteria (LAB) numbers and pH level. The second stage of this study was isolation of Eschericihia coli and Salmonella sp. from fecal samples of five diarrheic calves with fecal score 3-4. Eschericihia coli isolation was performed by inoculating the fecal samples on Levine’s-Eosin Methylene Blue (L-EMB) Agar. The isolated colonies were assayed for Indol, Methyl-Red, Voges Proskaeur, Citrate (IMViC), gram staining and blood haemolysis test. In addition Salmonella sp. isolation was performed by inoculating the fecal samples on Salmonella Shigella Agar. The isolated colonies were inoculated on Tripple Sugar Iron (TSI) Agar and assayed for IMViC, gram staining and blood haemolysis test. The third stage of this study was antibacterial activity determination of the whole-plant corn silage juice against Escherichia coli and Salmonella sp. using agar well diffusion assay. In addition the commercial antibiotic VITA Tetra-Chlor® solution at 50 μg/ml final concentration was used as control treatment. Escherichia coli and Salmonella sp. isolates used as test organisms were inoculated on Mueller Hinton Agar at 1-2 x 108 CFU/ml suspension. Diameter of inhibition zone was measured after overnight pre-incubation at 4 oC and followed with overnight incubation at 37 oC. The whole-plant corn silage juice was dominated by lactic and acetic acids which the concentration of lactic and acetic was 7.71 ± 0.73 mg/ml and 1.48 ± 0.45 mg/ml, respectively. Moreover LAB numbers of the whole-plant corn silage juice were 10.32 ± 9.84 log10 CFU/ml and the pH level was 3.87 ± 0.26. Escherichia coli numbers of fecal samples of five diarrheic calves were more than 3x106 CFU/gram. Based on isolation procedures it was obtained thirteen isolates of Escherichia coli and nine isolates of Salmonella sp.. The whole-plant corn silage juice showed antibacterial activity against all of the test organisms. The diameter mean of inhibition zone of the whole-plant corn silage juice was 3.0±1.1 mm against Escherichia coli and 4.7±1.7 mm against Salmonella sp.. The antibacterial activity of the whole-plant corn silage juice in this study was greater than 50 μg/ml VITA Tetra-Chlor® solution against Salmonella sp. (p<0.05). The antibacterial activity of the whole-plant corn silage juice against Escherichia coli, however, was lower than 50 μg/ml VITA Tetra-Chlor® solution (p<0.05). The results showed that the juice obtained from well preserved whole-plant corn silage contains acid fermentation products dominated by lactic and acetic acids. Moreover the juice possesses antimicrobial agents which are capable of inhibiting the growth of Escherichia coli and Salmonella sp.