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Feeding Lactobacilli as Probiotic and Proportion of Escherichia Coli in the Intestine of Calves

S. M. Amanullah
Bangladesh Livestock Research Institution, Savar, Dhaka-1341, Bangladesh

M. S. Alam
Bangladesh Livestock Research Institution, Savar, Dhaka-1341, Bangladesh

R. N. Subarna
Bangladesh Livestock Research Institution, Savar, Dhaka-1341, Bangladesh

R. Bateen
Department of Animal Science, Faculty of Animal Husbandry, Bangladesh Agricultural University, Mymensingh, Bangladesh

K. S. Huque
Bangladesh Livestock Research Institution, Savar, Dhaka-1341, Bangladesh

A. Sultana
Bangladesh Livestock Research Institution, Savar, Dhaka-1341, Bangladesh

Abstract/ Executive Summary:

A study was conducted to determine the ability of oral Lactobacillus bacteria as probiotic to increase the Lactobacilli and decrease the Escherichia coli (E. coli) population in the intestine of calves. Bacteria were isolated from yoghurt (Dahi) with selective de Man, Rogosa and Sharpe (MRS) agar media and identified as Lactobacillus, followed by mass production of bacterial cells and freeze drying. Four one-day-old calves were divided into two groups. One group (n=2) was fed freeze-dried bacterial cells and remaining group (n=2) was a control. After 60 days, one calf from each group was slaughtered to enumerate Lactobacillus and E. coli bacteria on intestinal wall. The number of colony-forming units (cfu) of Lactobacillus was significantly (p>0.01) higher in the intestinal wall of Lactobacillus-fed calf than in the control. On the other hand the number of E. coli was significantly (p>0.01) lower in Lactobacillus-fed calf.

Keywords: Feeding, Lactobacilli as probiotic and proportion, Escherichia coli, Intestine of calves

Location: Bangladesh Livestock Research Institution, Savar, Dhaka

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Calf

Objectives:

To investigate the ability of Lactobacilli isolated from yoghurt (Dahi) in removing E. coli from intestine in newborn calves.

Methodology:

This study was conducted in three steps: a) Isolation and identification of bacteria Isolation of Lactobacillus bacteria: Selective MRS agar media was used. The samples were inoculated through a pour-plate method. After inoculation, plates were incubated at 37^oC for 24-48 hours. Phenotypic characterization: Gram’s stain: Gram’s stain was used as described by Collins and Lyne (1980). Catalase test: To check the production of enzyme catalase, a drop of 3% hydrogen peroxide was placed on a clean microscope slide. A visible amount of bacterial growth was added aseptically with the help of an inoculating loop. Both were mixed and observed for gas bubble production. Identification tests: The strains showing Gram (+) and Catalase (-) were identified by the following tests: Sugar fermentation: One percent solutions of lactose, sucrose, glucose, maltose and mannitol were prepared by dissolving one gram of sugar in 10 ml of distilled water and sterilized by passing through 0.45 μm filter. Nutrient broth was prepared by dissolving 0.8g of Nutrient broth powder in 100ml distilled water, one ml of phenol red was added, and it was autoclaved at 121^oC for 15 minutes and cooled at room temperature. Five ml of broth and 100 μl of sugars were taken into sterilized test tubes, labelled and placed at room temperature for 24 hours to check for contamination. After 24 hours, the purified colonies were inoculated into test tubes and incubated at 37oC for 48 hours. If the colour changed from red to yellow, the test was positive. Sodium chloride (NaCl) utilization test: NaCl solution was prepared at 4% and 6.5%. Colonies were inoculated in MRS broth containing NaCl in test tubes. Test tubes were incubated at 37^oC for 48-72 hours. b) Mass production of bacterial cells and probiotic powder The isolated bacteria were transferred to MRS broth for multiplication. After 48 hours of incubation culture cells were harvested by centrifugation at 6000 rpm for 30 minutes. The cell suspension was washed once with sterile saline, and then inoculated in sterile 10% skim milk solution. Finally, it was freeze-dried to get probiotic Lactobacilli powder. c) Intestinal colonization of Lactobacilli Animals and their management: Four male calves aged one-day were distributed in two groups of two calves in each group and kept in an open shed with other calves in the farm. All calves were allowed to feed twice a day through suckling. Feeding of probiotic: Prepared probiotic powder was given orally with water @ 0.5g/day/calf before evening suckling. All drenching bottles were sterilized with boiling water for half an hour before and after drinking. Slaughter of calves and Sample collection: At 60 days, one calf from each group was selected at random for slaughtering. After removal of skin, the abdomen was washed with sterile water; intestine was removed and placed onto a sterile surgical cloth. A 10 to 15-cm segment from three different portions of each of small and large intestines was cut and placed into a sterile stainless steel tray. Enumeration of Lactobacillus from the mucus of small intestine (modified from Fuller et al., 1981) : Each segment of the intestine was cut longitudinally, the contents removed and washed gently with sterile water. The mucus was collected by scraping gently with a glass slide from previously marked one square cm area of the wall. Mucus was weighed and kept in test tube. Distilled water was added to this mucus at a ratio of nine ml of water to one ml of mucus. It was then vortexed, and centrifuged for 15 minutes. The supernatant was serially diluted ten-fold and plated on MRS and Eosin Methylene Blue (EMB) agar plates and incubated at 37^o C for 72 hours to identify the number of Lactobacillus and E. coli cfu.

Source of Information: The Bangladesh Veterinarian (2009) 26(1) : 17 – 22

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

It can be concluded that the probiotic Lactobacilli isolated from yoghurt (Dahi) are able to increase the proportion of Lactobacillus and decrease the proportion of E. coli in the intestine of newborn calves, which may open an opportunity for the dairy farmers to use an alternative to antibiotics to improve calf health.

Knowledge Management: Journal