EFFECTIVE NATURAL THERAPY USING SOME COMMON WILD HERBS AGAINST STAPHYLOCCAL MASTITIS IN CATTLES.

Presented by: Miss Reetibala L. Bhagadkar Guided By: Prof. D. A. Chouhan

D.B. Science College, Gondia.

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Department of Microbiology

BACKGROUND :

Drug resistance is a common phenomenon seen in pathogenic microorganisms. Excessive and unregulated use of antibiotics in therapeutics is a one of the important cause. Traditional uses of plant remedies are familiar to most peoples. The way in which information about herbal use has been passed between generations has been generally empirical and unquestioning. Mastitis is considered to be the most costly disease of dairy animals worldwide. The control of mastitis becomes difficult in prevailing circumstances. The major constraints include small herd size, low level of farmers education and lack of milk quality premium incentive etc. In the absence of any mastitis control program and presence of high antibiotic resistance, searching for new cost effective treatment line is essential.

INTRODUCTION:
MASTITIS

Mastitis , the inflammation of mammary glands, is a major production limiting disease of dairy animals all over the globe. It not only reduces milk quantity but also impairs quality thus adversely affecting milk production economics (DeGraves and Fetrow, 1993; Sordillo et al., 1997) Infectious agents: Although, a wide variety of microorganisms are associated with infectious mastitis, yet the bacteria are predominantly important. Among bacteria, the most frequent mastitogens include Staphylococcus aureus (S. aureus), Streptococcus agalactiae (Str. agalactiae), Escherichia coli (E. coli), Corynebacterium pyogenes (C. pyogenes), Streptococcus dysgalactiae (Str. dysgalactiae) and Streptococcus uberis (Str. uberis) etc. OUT OF WHICH S.aureus is predominant.

INTRODUCTION:
Mastitis

cont

About two decades ago, worldwide losses due to mastitis have been estimated as 35 billion dollars annually (Ratafia, 1987). In USA, economic losses estimated to be $200 per cow per year due to decreased production, discarded milk, treatment cost, veterinary fees, labor costs and increased replacement costs (Smith and Hogan, 2001). Mastitis is one of the major causes of economic loss in dairy cattle. Staphylococcus aureus is the main pathogenic species causing the subclinical form of mastitis. This type of udder infection impairs alveolar function, reduces milk yield and has a deleterious effect on milk composition, including increased milk somatic cell count (SCC) (Gudding et al., 1984; Nickerson, 1989).

INTRODUCTION
Mastitis

Cont.

Despite significant advances in mastitis control over the past 5 decades S. aureus is still an important cause of udder infection in dairy herds (Berkema et al., 1998; Dingwell et al., 2003; Fox and Gay, 1993; Sargeant et al., 1998; Wilson et al., 1997). Being contagious in nature, the pathogen may be easily transmitted within herds (Fox and Gay, 1993; Lam et al., 1996). Intrammamary infection (IMI) with S. aureus resulted in reduced milk production and deterioration of milk quality both in terms of milk constituents and increase somatic cell count (SCC).

INTRODUCTION
HERBAL MEDICINES

Cont.

As one would expect herbal medicine is widely used for the treatment of animals The use of herbal treatments for mastitis is limited by convention, regulation and lack of qualified advisers. However there appears to be strong interest from a small but significant part of the dairy industry for reliable advice and products. The world wide trend continues to be one of increasing importance of herbal medicine. Total worldwide sales are hard to measure but in 2001 were estimated at $16-20 billion (Journal of Nutrition. 2001;131:1120S-1123S) More reliable figures are available from the US where sales grew from $2 to $4.4 billion over the period 1994 to 2005. (Ferrier et al. Nutrition Business Journal 2006).

OBJECTIVE OF STUDY
There are a large number of complementary medicines available to treat mastitis and many opinions about which is the most effective.
Primary objective of study is to evaluate occurrence of drug resistance among the clinical isolates of Staphylococcus aureus from mastitic cattles. This study was extended for evaluation of antibacterial potential of some commonly occurring wild herbs against these antibiotic resitistant pathogens.

OBJECTIVE OF STUDY
Fine objectives were

Collection of clinical specimen from mastitic cattles Isolation and identification of Staphylococcus aureus. Studies on occurrence of Antibiotic resistance among Clinical isolate (Staphylococcus aureus). Screening of antibacterial potential of wild herb extracts against clinical isolates (Staphylococcus aureus). Phytochemical study of effective wild herb extract. Determination of MIC

METHODOLOGY

Collection and Identification of Plant materials Phyto-chemical analysis Isolation and identification of S. aureus from mastitic cattles: Antibiotic susceptibility testing. Extract preparations: Aqueous and organic Evaluation of Antibacterial activity of plant extracts: Well diffusion method. Determination of minimum inhibitory concentration (MIC): Determination of effect of exposure time.

METHODOLOGY

cont..

Isolation of S. aureus from mastitic cattles: A loop full of each clinical specimen was inoculated Baired Parker agar by four way streaking method. The inoculated plants were incubated at 370C for 24 to 48 hours acerbically. After completion of incubation, macroscopic, microscopic, examination of colonies on plant was carried out, and Black colored colonies from BPA selected. Selected colonies were sub-cultured on appropriate solid culture media for purification. They were later subculture on nutrient agar slants and stored at 40C for further analysis. Identification of clinical isolates: Identification of isolates carried out on basis of morphological, cultural and biochemical characteristic. Each isolate subjected to morphological examination like Gram staining, motility test (hanging drop method), cultural characteristic on appropriate media, biochemical test like sugar fermentation test, IMViC test, Coagulase test, Nitrate reduction test, Urease test, Catalase test etc.
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METHODOLOGY

cont..

Antibiotic susceptibility testing: All isolates were subjected to analysis for susceptibility or resistance towards available antibiotics of choice. The chosen antibiotics were Amoxicillin, Ciprofloxacin, Gentamycin, Chloramphenicol, Ceftriazone, Cotiramaxazole, Penicillin, Tetracycline, Carbenicillin, and Kanamycin. Antibiotic susceptibility of different isolates was tested using Kirby Bauer disc diffusion method. The size of the zone of inhibition was interpreted by referring zone diameter interpretive standards of the NCCLS/M 100, S12 performance standard for antimicrobial susceptibility testing.
Measurement of multiple antibiotic resistant (MAR index) MAR index of each isolates was calculated as recommended by Krumpreman No. of antibiotic to which test isolates Showed resistance MAR index of isolates = ----------------------------------------------------------No. of antibiotic used in studies
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METHODOLOGY

cont..

Extract preparations Aqueous extract: To obtain the aqueous extracts, dried and finely powdered leaves wild herbs were weighed about 30 grams each and homogenized using 150ml of water. They were added to Soxhlet apparatus and the boiling point of water was set up at 100C. The water evaporates continuously and was recycled, thereby extracting the compounds present in the samples. They were continuously extracted until the solution looses the color. Organic Solvent extract: To obtain the solvent extracts, dried and finely powdered leaves of wild herbs were weighed about 30 grams each and homogenized using 150ml of 70% organic solvent. They were added Soxhlet apparatus set up at boiling point of solvent. The solvent was recycled, thereby extracting the compounds present in the samples. They were continuously extracted until the solvent loses its color. The extract was then transferred to a sterile Petri dish and kept for evaporation of acetone at room temperature. Residues of extracts were collected and stored in the refrigerator
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METHODOLOGY

cont..

Phytochemical analyses Each test sample was subjected to phytochemical analysis to detect presence of tannins, alkalosiss and Flavanoids. Tannins: 70 mg of pulverized plant maternal was dissolved in one ml of distilled water and was filtered. 2ml of the filtrate was added to 2ml of FeCl3 , blue black color indicates presence of tannins. Alcaloids: 70 mg of plant material was dissolved in 1ml methanol and was filtered. 2ml filtrate was added to 1% HCl and was heated .One ml of filtrate added to 6 drops of Mayors reagent resulted in a cream colour patch which indicates presence of alkaloid. Flavonoids: 70 mg of plant material dissolved in 10 ml ethanol and was filtered. 2ml filtrate was added to Conc. HCl and add Magnesium ribbon .Pink tomato red color indicated the presence of Flavanoids. (Parke at al.2005) 13

METHODOLOGY

cont..

Antibacterial activity of plant extracts: Well diffusion method Antibacterial activities of the aqueous and organic extracts of leaves of different wild herbs were tested using well diffusion method. 100g of the sterile aqueous and organic extracts of each plant were added to each well. The diameter of inhibition zones were measured in mm and the results are recorded. Determination of minimum inhibitory concentration (MIC): The determination of minimum inhibitory concentration of the ethyl acetate extracts of Tridex procumbens leaf was carried out using the broth dilution method described by Lajubutu et al. Different concentrations of the extract were prepared to give a final concentration in the range of 0.05 to 0.50. mg/ml. The lowest concentration inhibiting growth was regarded as the minimum inhibitory concentration of the extracts.
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METHODOLOGY

cont..

Effect of exposure time on (MIC) : The determination of minimum inhibitory concentration of the ethyl acetate extract of Tridex procumbens leaf was carried out using the broth dilution method described by Lajubutu et al. Different concentrations of the extract were prepared to give a final concentration in the range of 0.1 to 0.50 mg/ml. Different exposure time is allotted in study is 30 minutes, 60 minutes and 120 minutes. The lowest concentration inhibiting growth was regarded as the minimum inhibitory concentration of the extracts for respective exposure time.

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RESULTS AND DISSCUSSION

Isolation and identification of S. aureus from mastitic cattles: 10 clinical specimens were tested for presence of S aureus. All 10 samples were founded to be positive for bacterial pathogens. Total 20 S. aureus strains were isolated from these clinical specimens and identified on the basis of morphological, Biochemical and cultural characteristics.

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RESULTS AND DISSCUSSION

Antibiogram of clinical isolates:
All 20 strain tested were multiple antibiotic resistant. The antibiotics used during study were Ampicillin (10 mcg), Amikacin (10 mcg), Amoxicillin (10mcg) Co-trimaxozole (25 mcg), Ceftizidime (30 mcg), Gentamycin (10 mcg), Imipenem (10 mcg), Kanamycin (30 mcg), Nalidixic acid (30 mcg), Methicillin (5 mcg), Vancomycin (10 mcg) and Penicillin (10 mcg). All Staphylococcus aureus strain isolate found to be resistance to Penicillin, Amoxicillin, Methicillin, Ampicillin and Ceftizidime. 15% strains were found to be resistant to Vancomycin & Nalidixic acid, 20% strain resistance co-trimaxozole, 30% strain resistant to Kanamycin, Gentamycin and Amikacin. Most of the strain was showing MDR (Multiple Drug resistance). S13, S4 strain sowing MDR index 0.75. Most of the strain showed MDR index greater than 0.6.
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RESULTS AND DISSCUSSION

Antibiogram of clinical isolates:
Sr. No. 1 2 3 4 5 6 7 8 9 10 11 12 No. of isolates Tested Resistan Sensitive t Amoxicillin 20 20 00 Kanamycin 20 00 17 Amikacin 20 00 19 Ceftizidime 20 20 00 Ampicillin 20 20 00 Imipenem 20 05 13 Gentamycin 20 00 19 Nalidixic acid 20 10 10 Cotrimaxazole 20 03 16 Methicillin 20 17 00 Vancomycin 20 10 10 Penicillin 20 20 00 Antibiotics Intermediat e 00 03 01 00 00 02 01 00 01 03 00 00 R 100 00 00 100 100 25 00 50 15 85 50 100 % S 00 85 95 00 00 65 95 50 80 00 50 00 I 00 15 05 00 00 10 05 00 05 15 00 00

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RESULTS AND DISSCUSSION

Antibiogram of clinical isolates:

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RESULTS AND DISSCUSSION

Phytochemical analyses of wild herbs: Sr. No 1. 2 3 Plant Material Alkaloids Flavanoids Tannins

Tridex +++ procumbens L +++ Solanum xanthocarpum +++ Wild neem

++++
++++ ++++

++
--++

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RESULTS AND DISSCUSSION

Antibiogram of clinical isolates: According to world health organization report in 2000. Staphylococcus aureus is multiple drug resistance in the many region of the world. reported prevalence rate indicates the wild variation exist regionally and from herd to herd. reported more than 50% staphylococcus isolates resistance the high penicillin resistance. The wild use of intra-mammary preparation containing combination and browd spectrum antibiotics.(Ebrahimi A et al 2007 ) Ebrahimi A et al 2007 reported that 78.5% isolate were resistance to Amikacin, 50% resistance to Penicillin, 50% resistance to tetracycline, 42.8% resistance to ampicillin, while 100% susceptible to Ciprofloxacin, Carbenicillin and Gentamycin.
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RESULTS AND DISSCUSSION

Antibiogram of clinical isolates: According to Deluyker et al 2005 reported Erythromycin and Methicillin in in-effective against Staphylococcus aureus. According to Deluyker et al 2005 broad range of antibiotics such as Tetracycline Penicillin and Pirlimycin shows only 15 13% cure rate in mastitis cause by Staphylococcus aurous. Reason for this low success rate include 1. Poor penetration of antibiotics into areas of scarring and inflammation 2. Inactivation of antibiotics by milk and serum components 3. Intracellular or metabolically inactive organisms bacterial L-forms resistance to antibiotics and improper treatment procedures.

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RESULTS AND DISSCUSSION

Antibacterial activity of selected herbs: In this study different extract (Aqueous, methanolic, Ethanolic and ethyl acetate) of different wild herbs were screened for their antibacterial potential. Antibacterial activities of different extracts were summarized in Table 5.7, 5.8, 5.9, 5.10. Results obtained suggested that ethyl acetate extract of Tridex pcocumbens leaf was more effective than Aqueous and methanolic extract against all clinical isolates tested. It is also suggested that methanol extract of Mentha arvensis leaf was more effective than its Aqueous and ethyl acetate extract in inhibiting Staphylococcus aureus. In comparison Tridex procumbens leaf extract in ethyl acetate solvent was more effective among all wild herb tested for screening there antiStaphylococcus potential. Sharma B. and Kumar P., 2009 Studied antimicrobial potential of Tridex procumbens and reported that Tridex procumbens has broad spectrum of antimicrobial activity due to free and bound Flavanoids. Antibacterial potential difference extract of some wild herbs against multi drug resistance Staphylococcus aureus from mastitis cattles was evaluated by Kirby Bauer well diffusion method. 23

RESULTS AND DISSCUSSION

Antibacterial activity of selected herbs: Result of the presence study showed that out of 16 different extract tested ethyl acetate extract of Tridex procumbens leaf inhibiting effectively growth of Staphylococcus aureus isolates from mastitic cattles. Bioactive potential of this extract is due to presence of Flavanoids. Several works Ilic et al 2004 also reported bioactive potential of Flavanoids. Samy and Ignalimupthu 1999, Taddy et al 2000 and Sanchiz et al 2005 demonstrated presence of Flavanoids in Tridex procumbens. Tridex juices are effective against Bacillus subtilis, Staphylococcus aureus, E. coli, Pseudomonas areuginosa and is known for is wound healing properties. (Dhar et al 2003).

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RESULTS AND DISSCUSSION

Antibacterial activity of selected herbs:
Table 5.8Antimicrobial activity of Solanum xanthocarpum aganist S. aureus:
Sr. No Strain Inhibition zone (mm) Gentamyci EE ME EaE n 16 00 12 13 20 00 00 11 16 00 12 13 18 00 11 00 24 00 11 00 23 00 12 11 20 00 00 13 23 00 00 11 20 00 15 11 13 00 00 00 23 00 12 15 23 00 00 11 AE 00 00 00 00 00 00 00 00 00 00 00 00 EE 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Activity index ME EaE 0.75 0.00 0.76 0.61 0.45 0.52 0.00 0.00 0.75 0.00 0.52 0.00 0.81 0.55 0.81 0.00 0.00 0.47 0.61 0.47 0.55 0.00 0.65 0.47 AE 00 00 00 00 00 00 00 00 00 00 00 00

1 2 3 4 5 6 7 8 9 10 11 12

Sa1 Sa2 Sa4 Sa8 Sa10 Sa11 Sa13 Sa16 Sa17 Sa18 Sa19 Sa20

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RESULTS AND DISSCUSSION

Antibacterial activity of selected herbs:
Table 5.7 Antimicrobial activity of Tridex procumbens against S. aureus: Sr. No Strain Inhibition zone (mm) Gentamyci EE ME EaE n 16 16 16 21 20 00 00 22 16 16 16 24 18 15 15 20 24 13 13 22 23 00 00 20 20 00 00 20 23 00 00 18 20 16 16 23 13 11 11 23 23 11 11 21 23 11 11 19 Activity index ME EaE 1.00 0.00 1.00 0.83 0.54 0.55 0.00 0.00 0.80 0.84 0.47 0.47 1.31 1.10 1.50 1.11 0.91 0.86 1.00 0.78 1.15 1.76 0.91 0.80

AE 00 00 00 00 00 00 00 00 00 00 00 00

EE 1.00 0.00 1.00 0.83 0.54 0.55 0.00 0.00 0.80 0.84 0.47 0.47

AE 00 00 00 00 00 00 00 00 00 00 00 00
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1 2 3 4 5 6 7 8 9 10 11 12

Sa1 Sa2 Sa4 Sa8 Sa10 Sa11 Sa13 Sa16 Sa17 Sa18 Sa19 Sa20

RESULTS AND DISSCUSSION

Antibacterial activity of selected herbs:
Table 5.9 Antimicrobial activity of Mantha arvensis against S. aureus:
Sr. No Strain Inhibition zone (mm) Gentamyc EE ME EaE in 16 00 12 11 20 00 00 14 16 00 00 16 18 00 11 13 24 00 11 14 23 00 00 16 20 00 13 12 23 00 00 13 20 00 00 00 13 00 00 00 23 00 12 13 23 00 00 15 Activity index ME EaE 0.75 0.00 0.00 0.61 0.45 0.00 0.65 0.00 0.00 0.00 0.52 0.00 0.68 0.70 0.10 0.72 0.58 0.69 0.60 0.56 0.00 0.00 0.56 0.60

AE 00 00 00 00 00 00 00 00 00 00 00 00

EE 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

AE 00 00 00 00 00 00 00 00 00 00 00 00
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1 2 3 4 5 6 7 8 9 10 11 12

Sa1 Sa2 Sa4 Sa8 Sa10 Sa11 Sa13 Sa16 Sa17 Sa18 Sa19 Sa20

RESULTS AND DISSCUSSION

Antibacterial activity of selected herbs:
Table 5.10 Antimicrobial activity Andrographis peniculata (wild neem) aganist S. aureus:
Sr. No 1 2 3 4 5 6 7 8 9 10 11 12 Strain Inhibition zone (mm) Gentamyc EE ME EaE in 16 00 00 00 20 00 00 00 16 00 00 00 18 00 00 00 24 00 00 00 23 00 00 00 20 00 00 13 23 00 00 00 20 00 00 00 13 00 00 00 23 00 00 00 23 00 00 00 Activity index ME EaE 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.65 0.00 0.00 0.00 0.00 0.00

AE 00 00 00 00 00 00 00 00 00 00 00 00

EE 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

AE 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
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Sa1 Sa2 Sa4 Sa8 Sa10 Sa11 Sa13 Sa16 Sa17 Sa18 Sa19 Sa20

RESULTS AND DISSCUSSION

Determination of MIC of effective extract of herbs: When ethyl acetate extract of Tridex procumbens leaf subjected to determination of minimum inhibitory concentration. It was found that minimum inhibition concentrate this extract against tested clinical isolates was 0.10mg/ml against Staphylococcus aureus (S4) strain. MIC and MBC values for mast resistance strain of Staphylococcus aureus were evaluated for ethyl acetate extract of Tridex procumbens which showing highest activity in agar well diffusion method. In the presence investigation MIC value range from 0.10 0.22 mg/ml was recorded against multi drug resistance Staphylococcus aureus (S4) strain. Amount of extract isolated from plant parts and total activity was calculated and recorded in table 5.12.

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RESULTS AND DISSCUSSION

Determination of MIC of effective extract of herbs:
Table 5.12 Quantity and total activity, MIC and MBC of Tridex procumbens ethyl acetate extract against S. aureus

Sr. No

Strain

1. 2 3 4 5 6

Sa2 Sa4 Sa10 Sa13 Sa17 Sa18

Amount of extract mg/gm dried plant part 3.5 3.5 3.5 3.5 3.5 3.5

MIC mg/ml 0.19 0.10 0.19 0.22 0.15 0.15

MBC mg/ml 0.38 0.20 0.38 0.44 0.30 0.30

Total activity

0.05 0.02 0.05 0.06 0.04 0.04

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RESULTS AND DISSCUSSION

Total activity of effective extract of herbs: Total activity indicates volume at which extract can with diluted steel having ability to kill microorganisms. Lean et al 1999 and Palombo and Sample 2001 reported that Tridex pcocumbens have broad spectrum of antibacterial activity. In current study inhibition zone, activity index, MIC, MBC and total activity have been evaluated. Extract under study not only inhibit growth of selected bacterial strain but inhibition zone develop was more or less permanent as compare to antibiotics used during study. In the light of this fact microorganisms are become resistance against antibiotic used. Present investigation is of a great significance as far as future drugs are considered and selected plant by used by pharmaceutical industry for preparing plant west antimicrobial drugs.

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RESULTS AND DISSCUSSION

Studies on effect of exposure time on MIC values of Ethyl acetate extract of Tridex pcocumbens: When clinical strain S4 strain of Staphylococcus aureus subjected to action of different concentration of ethyl acetate extract of Tridex pcocumbens leaf and also allows the exposure for different time duration like 30min 60 min and 120 min. With increasing the exposure time there was decrease in MIC valve was reported. For 30 min exposure time MIC of extract was found to be 0.05 mg, for 60 min exposure time MIC of extract was found to be 0.04 mg and for 120 min exposure time MIC valve is 0.02 mg/ml.

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RESULTS AND DISSCUSSION

Studies on effect of exposure time on MIC values of Ethyl acetate extract of Tridex pcocumbens: Table 5.13 Quantity and total activity, MIC and MBC of Tridex procumbens ethyl acetate extract against S. aureus Sr. No Exposure time (min) 30 60 120 MIC mg/ml 0.05 0.04 0.02 MBC mg/ml 0.10 0.07 0.04

1. 2 3

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Significance of study
The

present results revealed that the extract of Tridax procumbens L. was effective against mastitis causing S. aureus. Presence of chemical compounds viz. alkaloids, tannins, flavanoids of Tridax procumbens L. may inhibit the bacterial growth. Traditionally, Tridax procumbens L. was employed using/mixing with water for treating the bacterial and other infections. Naturally, the biologicallly active compounds whose activity can be enhanced in the presence of ethyl acetate could have been produced number of active compound responsible for antibacterial activity. The present study provides the scientific information about the leaf extract of Tridax procumbens L. and supports the usage of this plant for curing mastitis in cattle by traditional healing and antibacterial properties. Further, phytochemical separation studies of this plant and in VIVO antibacterial study of Tridex extract is in under progress.
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CONCLUSION
In the absence of any mastitis control program and presence of high antibiotic resistance, searching for new cost effective treatment line is essential. The use of herbal treatments for mastitis is appears to be strong interest from a small but significant part of the dairy industry for reliable advice and products. Ethno-veterinary information about plants used in the prevention and control of bovine mastitis. The results of this study have shown that the leaf extract of Tridax procumbens L. have great potential as antibacterial agents in the treatment of mastitis caused by multidrug resistant S. aureus. Further, detailed investigation of the active compounds of the plant for the exact mechanism of action will contribute greatly to the development new pharmaceuticals.
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