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2009, Vol. 4 No. 2, Article 42

 

Susceptibilities of Aerobic Bacteria Associated with Bovine Mastitis to Leaf Extracts of Bauhinia reticulata

Tata Elvis Fon*1, Agba I. Mathias2, Chukwedo A. Anthony4, L. Mgbojikwe5 and Wirkom K. Venasius3


1Senior Medical Laboratory Scientist;2Medical Microbiologist;3Senior Medical Laboratory Scientist;

Igbinedion University-Okada, Benin City, Nigeria.

3Medical Virologist; 4Biochemist;

Federal college of Veterinary and Medical Laboratory technology, National Veterinary Research Institute,
Vom, Plateau State, Nigeria.

 

*Corresponding Author; e-mail address: elfontata@yahoo.com

 


ABSTRACT

The susceptibilities of 16 aerobic bacterial isolates from 76 milk samples collected from 19 cows with udder inflammation to aqueous and methanolic leaf extracts of Bauhinia reticulata was determined by the agar diffusion method. The minimum inhibitory concentration (MIC) of test extracts that gave inhibition were determined using the tube dilution method .The phytochemicals present in the whole leaf powder and the various leaf extracts were also qualitatively assayed using conventional techniques. The aqueous extract of Bauhinia reticulata inhibited Staphylococcus epidermidis, Streptococcus uberis, Pseudomonas aeruginosa, Escherichia coli, Corynebacterium gravis and Bacillus cereus. The methanolic extract inhibited only Escherichia coli. The MIC of the different leaf extracts was found to vary greatly, and ranged from 6.25mg/ml to 25mg/ml.

KEY WORDS

Susceptibility, Bovine, Bacteria, Leaf, Bauhinia

INTRODUCTION

Bovine mastitis is the inflammation of the parenchyma of the mammary glands of cattle (Radostit et al., 1996) associated with microbial infections (Schroeder, 1997) and physiological changes(Spronle, 1995). Mastitis is caused by a group of infective and potentially pathogenic bacteria (Bezek and Hull, 1994), viruses (Wallenberg et al., 2003), mycoplasma (Matrencher, 1995), fungi and algae (Radostit et al., loc.cit.). the most common bacteria causing bovine mastitis include; Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, Streptococcus dysgalactiae and Escherichia coli. In order to minimize the economic losses from bovine mastitis and dissipation of infection resulting from the consumption of contaminated milk and milk products, there is an urgent need to ascertain the current status and involvement of aerobic bacteria in bovine udder inflammation, their role as causative agents of bovine mastitis and their susceptibilities.

MATERIALS AND METHODS

Fresh leaves of Bauhinia reticulata were harvested at the end of October 2006 from within the premises of NVRI, Vom, Nigeria, rinsed properly in sterile distilled water, dried in shadow and then ground to powdered form. The leaf powder was then extracted using methanol and water as solvents with the soxhlet apparatus. The extracts so obtained were weighed and stored in sterile universal bottles at 4 degree in a refrigerator.
Phytochemical Screening: The methanolic extract, aqueous extract and leaf powder of Bauhinia reticulata were used as samples for qualitative phytochemical screening for tannins, resins, alkaloids, saponins, tannins, glycosides and flavonoids following the standard procedures described by Trease and Evans (1989) and Faraz et al, (2003).
Test for resins: To 0.5g of each sample was added 5ml of boiling ethanol. This was filtered through Whatman No.1 filter paper and the filtrate diluted with 4ml of 1% aqueous HCL. The formation of a heavy resinous precipitate indicated the presence of resins (Trease and Evans, 1989).
Test For Alkaloids: 0.5g of each sample was stirred with 5ml of 2N HCL in a steam bath. This was filtered and 1ml of the filtrate tested with a few drops of Dragendorff's reagent and a second 1ml portion treated similarly with Wagners reagent. The formation of a precipitate was an indication of the presence of alkaloids (Faraz et al., 2003).
Test For Saponins: 0.5g of each sample was stirred with water in a test tube. Frothing which persists on warming was taken as preliminary evidence for the presence of saponins (Faraz ,2003).
Test For Tannins: 0.5g of each sample was stirred with 10ml of boiling distilled water. This was filtered and a few milliliters of 6% ferric chloride added to the filtrate. Appearance of deep green coloration indicated the presence of tannins. The second portion of the filtrate was treated with a few milliliters of iodine solution. Appearance of a faint bluish coloration confirmed the presence of tannins (Trease and Evans,1989).
Test For Glycosides: 0.5g of each sample was stirred with 10ml of boiling distilled water. This was filtered and 2ml of the filtrate hydrolized with a few drops of concentrated HCL and the solution rendered alkaline with a few drops of ammonia solution. 5 drops of this solution was added to 2ml of Benedict`s qualitative reagent and boiled. Appearance of reddish brown precipitate showed the presence of glycosides (Trease and Evans, 1989)
Test For Flavonoids: 0.5g of each sample was dissolved in 2ml dilute NaOH solution. A few drops of concentrated H2SO4 was then added. The presence of flavonoids was indicated by the disappearance of color (Trease and Evans, 1989).
Isolation And Identification Of Bacteria: 76 samples (5ml each) of fore milk was aseptically collected from each quarter of the udders of 19 lactating cows into sterile sample bottles labeled with the number of the animal following the method of Schalm et al (1971). Each sample was examined macroscopically for discoloration clots and flakes. Somatic cell counts were done using the manual method. Each sample was then inoculated in duplicate onto 10% sheep blood agar plates and were incubated aerobically at 37 degree (centigrade) for 48 hours and were examined for growth over a period of 24 and 48 hours respectively. Cultures were then identified using conventional techniques (Barrow and Feltham ,1995).
Sensitivity Test: (Garrod et al, 1983) Colonies of fresh cultures were suspended in 20 ml of nutrient broth in different sterile universal bottles and were incubated at 37 degree (centigrade) over night. The concentration of organisms in the broth was determined and was diluted down to 10-6 Macfarland’s standard. One ml of this was used in flooding over nutrient agar plates in the agar diffusion method of the in-vitro antimicrobial sensitivity test. The plates were left on bench for 5mins after which they were dried at 37 degree (centigrade) for 1hour. Four wells, equally distant, were bored round the plate using a sterile cork borer. Various concentrations of the diluted extracts were put inside the wells. Sterile distilled water was put inside the 5th well to serve as negative control while gentamicin was used as positive control in the 4th well. The plates were left on bench for 1 hour,after which there were incubated at 37 degree (centigrade) for 24 hours and were examined for zones of inhibition.
Minimum Inhibitory Concentration (MIC): The broth dilution assay was carried for different dilutions of the extracts using susceptible bacteria as described by Muray et al. (1999).

RESULTS

The phytochemical analysis of the leaf powder and various extracts gave the results as depicted in Table 1. Out of the 76 samples analysed, 18 had somatic cell counts (SCC) greater than 5x105 cells/ml. Sixteen out of these 18 samples gave positive cultures. Hence, 16 samples out of 76 were positive giving a percentage positivity of 21.05%. Staphylococcus aureus was the predominant organism isolated with 5 isolates (6.66 %). Others were Streptococcus uberis and Corynebacterium gravis with 3 isolates (3.95%) each; Staphylococcus epidermidis with 2 Isolates (2.63%); Escherichia coli, Bacillus cereus and Pseudomonas aeruginosa with 1 isolate (1.32%) each. 2 samples gave a mixed culture of Staphylococcus aureus and Corynebacterium gravis. Results obtained from the susceptibility testing of the organisms with the various extracts showed that the aqueous extract inhibited Staphylococcus epidermidis, Streptococcus uberis, Pseudomonas aeruginosa, Escherichia coli, Corynebacterium gravis and Bacillus cereus, while the methanolic extract only inhibited Escherichia coli and Corynebacterium gravis (Table.2). According to results obtained from MIC, the aqueous extract gave an MIC of 6.25mg/ml for Escherichia coli and Staphylococcus epidermidis, 12.5 mg/ml for Streptococcus uberis, Pseudomonas aeruginosa and Corynebacterium gravis, and 25 mg/ml for Bacillus cereus. The methanolic extract gave an MIC of 12.5 mg/ml for Escherichia coli and 25 mg/ml for Corynebacterium gravis (fig.1).

DISCUSSIONS

Normal milk should contain less than 500,000 cells/ml and an elevated somatic cell count is an indication of inflammation in the udder (Schroeder,1997). As such elevated somatic cell counts in the present work was an indication of inflammation. Institute for Animal Health (2003) identifies all the organisms isolated during present trial as causative agents of bovine mastitis and have produced experimental infections in cattle with these organisms The inhibition of Staphylococcus epidermidis, Streptococcus uberis, Pseudomonas aeruginosa, Escherichia coli, Corynebacterium gravis and Bacillus cereus with an MIC range of 6.25 to 25 mg/ml suggest that Bauhinia reticulata stands as a promising alternate source of antibacterial agents for the management of diseases of animals however, further work needs to be done on the toxicity, in-vivo susceptibility, and nutritional value of this plant.

AKNOWLEDGEMENT

The authors wish to acknowledge the management of “Science For Life Foundation Laboratory” for allowing use their laboratory for this research.

REFERENCES

  1. Bezek, D. M and Hall, B. L (1995). Peracute gangrenous mastitis and Chelitis associated with enterotoxin secreting Staphylococci. Canidian Veterinary Journal. 36: 106-107.

  2. Barrow, G. I and Feltham, P. K. A. (1995). Cowan and Steel’s manual for the identification of medical bacteria. 3rd Edn. FAO(1984). Residues of veterinary drugs in food. Report of a joint FAO/WHO expert consultation, Rome. FAO food and nutrition paper. 32nd Edn. P 8-5.

  3. Faraz Mojab, Mohammed Kamalinejad, Naysaneh Ghaderi, Hamid Reza Validipour (2003). Phytochemical screening of some species of Iranian Plants. Iranian Journal of Pharmaceutical research. 77-82.

  4. Institute for Animal Health (2003). Improving milk quality and reducing mastitis. www.iah.bbsrc.ac.uk.

  5. P. R., Baron, E. J., Pfaller, M. A., Tenover. F. C and Yolke, R. H (1999). Manual of clinical microbiology. 7th edn. Washinton: ASM. P 1527-1539

  6. Radostit. O. M, Blood, D.C, and Gray, C. C (1996). Veterinary medicine. Text book of the diseases of cattle, sheep, goats and horses. 8th edn. London Baillere Tindal

  7. Schroeder, J. W (1997). Mastitis control program. Bovine mastitis and milking management. AS-1129.NDSU. Extension service.

  8. Shouky and Magda, S. Sabana (1997). Chemotherapy of bovine mastitis. Egyptian journal of agricultural research. Spronle, R (1995). Outbreak of mastitis in goats. surveillance. 22:16-17.

  9. Trease, G.E and Evans, W.C (1989). Pharmacology 12thn. Bailliere Tindal. 83, 685.

  10. Wallenberg G. J.,Van Der Poal, W. H. M and Vana Oirschot, J. T. (2003).Viral infections and bovine mastitis. A review. Vet microbiology.



Table 1: Phytochemicals analysis of Bauhinia reticulata Leaf Extract


Test Powdered dry leaves Aqueous extracts Methanolic extracts
Resins + - ++
Alkaloids + ++ ++
Saponins + ++ ++
Glycosides ++ ++ -
Flavonoids + ++ ++
Tannins - - -

Key:   + = Present;   ++ = Highly present;   -= Absent


Table 2: Bacterial Isolates

 

Number of cattle selected Milk Samples Samples with SCC Positive samples Percent positive Bacteria Isolated Number of Isolates Percent occurance
19 76 18 16 21.05 Staphylococcus aureus 5 6.56
          Staphylococcus epidermidis 2 2.63
          Streptococcus  uberis 3 3.95
          Escherichia coli 1 1.32
          Pseudomonas aeruginosa 1 1.32
          Corynebacterium gravis 3 3.95
          Bacillus cereus 1 1.32
TOTAL           16 21.05


Table 3: Antibacterial Activity Of Leaf Extracts Of Bauhinia reticulata and diameter of inhibition zones in mm

 

Bacteria Strains tested Aqueous extract (mg/ml) Methanolic extract (mg/ml) Gentamicin (µg/ml)
200 100 50 200 100 50
Staphylococcus aureus 7 0.00 0.00 0.00 0.00 0.00 0.00 15.00
Staphylococcus epidermidis 2 28.00 22.50 17.00 0.00 0.00 0.00 13.00
Streptococcus  uberis 3 18.50 13.30 10.00 0.00 0.00 0.00 16.00
Pseudomonas aeruginosa 1 14.00 12.00 10.00 0.00 0.00 0.00 16.00
Escherichia coli 1 24.00 20.00 17.00 12.00 10.50 10.00 15.00
Corynebacterium gravis 3 13.00 12.00 10.50 13.00 12.00 0.00 18.00
Bacillus cereus 1 20.00 15.00 11.00 0.00 0.00 0.00 10.00

 


Fig 1: Minimum inhibitory concentrations (MIC) of leaf extracts of
Bauhinia reticulata at bacteriostatic level
histogram of MIC

(blue bars: Methanolic extract; Purple bars: Aqueous extract)

Fig 2:
Bauhinia reticulata plant in the month of October
Bauhinia reticulata plant



Fig 3: Inhibition of the growth of
Staphylococcus epidermidis, Streptococcus uberis, Pseudomonas aeruginosa,
Escherichia coli, Corynebacterium gravis
and Bacillus cereus by aqueous leaf extracts of Bauhinia reticulata
Agar plates inhibitory zones


 


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