Evaluation of Antibacterial Effect of Some Sinai Medicinal Plant Extracts on Bacteria Isolated from Bovine Mastitis

Aim: Bovine mastitis is the most economically important disease affecting dairy cattle worldwide from an economic, diagnostic and public-health point of view. The present study aimed to isolate and identify of bacteria causes mastitis in dairy cows and to evaluate the antibacterial activities of some selected medicinal plants extracts comparing antibiotics used in the treatment of mastitis in Egypt. Materials and Methods: A total of 203 milk samples of dairy cows were collected during the period from February to June 2013 at different Governorates in Egypt. The use clinical inspection and California mastitis test examination were provided efficient diagnostic tool for detection of clinical, subclinical mastitis and apparently normal health cattle. The collected milk samples were cultured on Nutrient, Blood agar, Mannitol salt, Edward's and MacConkey agar plates supporting the growth of various types of bacteria for their biochemical studies and isolation. The antimicrobial activity of plants extracts (Jasonia montana and Artemisia herb alba)with different solvent (ethanol, petroleum ether, chloroform and acetone)were studied in vitro against isolated bacteria from mastitis by paper desk diffusion and minimum inhibitory concentration method (MIC). Results: The prevalence of clinical, subclinical mastitis and normal healthy animals were 34.50%, 24.7% and 40.8% respectively. The major pathogens isolated from collected milk samples were Escherichia coli The highest antibacterial activity of J. montana plant extracted with acetone solvent against S. agalactiae, E. coli, S. aureus, Klebsiella spp and coagulase-negative Staphylococci with zone of inhibition values ± standard deviation (SD), ranging from 4.33±0.57 to 25.6±0.60 mm. The MIC values for the extracts ranged from 0.01 to 1.56 mg/ml. when comparing antibacterial activity of A. herb alba plant extracted with acetone solvent on the same bacteria with zone of inhibition values ± SD, ranging from 00±00 to 5.6±0.60 mm. Both extracts from J. montana and A. herb alba plant extracts with petroleum ether, methanol and chloroform solvent were less antibacterial activities than acetone solvent extract. Conclusion: The present study spot highlight on isolation and identification of mastitis pathogens that are fundamental aspects of milk quality, udder health control programs and public health and food safety issues associated with food borne pathogens. J. montana and A. herb alba plants have antibacterial effects more than antibiotics used in the treatment of mastitis. Finally, the medicinal plant extracts can be used to discover bioactive natural product in the form of antibacterial that may be serve the development of new pharmaceutical products. But still …


Introduction
Mastitis, inflammation of the mammary gland, is a costly production disease affecting the dairy cattle industry worldwide [1,2].It causes a fall in milk production, decreased milk quality, economic diagnostic and it's considered of quite vital importance to the public health due to its association with many zoonotic diseases in which the milk act as a vehicle for some infectious agents [3].The most common mastitis pathogens are caused by a wide variety of bacteria, which can be classified as environmental (Escherichia coli, Streptococcus dysgalactiae, Streptococcus uberis, Enterococcus spp.and coagulase-negative Staphylococci [CNS]) or contagious (Staphylococcus aureus and Streptococcus agalactiae) [4].Contagious mastitis pathogens are generally transmitted from cow to cow [5].The infection is transmitted by milk-contaminated fomites at milking, by the Milker's hands, or by the milking machine.Environmental mastitis transmission by contact of the teats with contaminated soil, bedding and water with faecal materials [6,7].Many bacteria strains are resistant to one or more antibiotics used in treatment bovine mastitis [8].The increasingly growing rate of antibiotic resistance to microorganisms necessitates the developed and search of new antimicrobial agents to combat this problem.Medicinal plant-derived compounds have increased widespread interest in the search of alternative antibacterial agents because of the perception that they are safe and have a long history of use in folk medicine for the treatment of infectious diseases [7,8].Natural products of higher plants may possess a new source of antimicrobial agents with possibly novel mechanisms of action [9].Medicinal plants are rich in a wide variety of secondary metabolites such as tannins, alkaloids and flavonoids, which have been found in vitro to have antimicrobial properties [10,11].A number of phyotherapy manuals mentioned that the various natural plants able to treat infectious diseases, fewer side effects and low toxicity [12,13].There are several reports on the antimicrobial activity of different herbal extracts [14].Many herbal plants have been found to cure gastrointestinal disorders, respiratory diseases and cutaneous infections [15].Artemisia herb-alba plant is known as desert wormwood (known in Arabic as shih) [16,17], it has been used in folk medicine to treat arterial hypertension and/or diabetes [18].Herbal tea from this species has been used as analgesic, antibacterial, antispasmodic, and haemostatic agents [19].Jasonia montana belongs to family Asteraceae, growing in the mediterranean and adjacent areas, including the Sinai Peninsula, Egypt and in Arabic known Nohida [20].This herb has a strong aromatic odor and is used in traditional medicine for diarrhea, stomach ache, chest diseases and the different plant extracts as hypoglycemic and antioxidant.But there are no reports of the antibacterial effects on multidrug resistant bacteria.
In Egypt, A. herb alba and J. montana plants are common used in folk medicine as herbal tea for the treatment of renal troubles [20].Many reports in the literature confirmed that J. montana is rich in methoxylated flavonoids that have a cytotoxic activity against carcinogenic cells [21].The present work aims to isolate and identify microorganisms causing bovine mastitis and evaluate the antibacterial activity of selected some Sinai medicinal plant extracts against bacteria isolated from bovine mastitis.

Ethical approval
Approval was taken from the ethical committee of National Research Center, Giza, Egypt for collection of milk samples by describing the protocols of the study.After explanation of the study objective.The study was conducted during the period between February and June 2013.Milk samples were taken by a veterinarian in all examined animals at Beni-Suef, El-Fayoum, Behera and Monofia Governorates in Egypt.

Milk samples
A total of 203 samples (70 clinical and 50 subclinical cases of mastitis and 83 normal healthy)were collected from individuals dairy cows at Beni-Suef, El-Fayoum, Behera and Monofia Governorates in Egypt during the period between February and June 2013.Milk samples were taken by a veterinarian in all examined animals.The bacterial culture medium was prepared in the laboratory as per standard procedures.

California mastitis test (CMT)
The CMT was conducted to diagnose the presence of subclinical mastitis and it was carried out according to procedures given by Quinn et al. [22].A few drops of milk from each quarter of the udder was placed in each of four cups in the CMT paddle and an equal amount of the reagent was added.A gentle circular motion was applied.Positive samples show gel formation within a few seconds.The result was scored based on the gel formation positive and negative, cow was considered mastitic if one or more quarters were CMT positive.

Bacterial isolation and identification
A loop full from fresh and incubated milk was streaked onto plates of nutrient agar, blood agar, mannitol salt agar, Edward's and MacConkey agar media and incubated at 37°C for 24-48 h.Suspected colonies were picked up and examined microscopically in gram stained films before being transferred in semisolid agar to be subjected for further identification of pathogens including gram's staining technique and catalase test to distinguish between Streptococci and Staphylococci.The hemolytic patterns and coagulase reaction with rabbit plasma were used to differentiated between S. aureus and CNS.Also, esculin hydrolysis and Christie-Atkins, Munch-Petersen (CAMP) reaction differentiated between S. agalactiae and other Streptococcus spp.[23].Gram-negative bacteria were identified by sub culturing on differential and selective media and tested to oxidase activity, acid production (glucose, lactose and sucrose fermentation), indole test, Voges-Proskauer test (VP) and hydrogen sulfide production as National Mastitis Council's guidelines [24].Api-20E system (analytical profile index, BioMerieux) used for confirmation the bacterial isolates according to instructions of the manufacture.

Medicinal plants
The aerial parts (steams, leaves and flowers) of A. herba alba and J. montana plants were collected from Saint Catherine, South Sinai, Egypt in January 2014.The plants were performed by a group of genetics and breeding of medicinal and aromatic plants.Department of Genetics and Cytology, Genetic Engineering and Biotechnology Division, National Research Center, Cairo, Egypt.Washed with distilled water, dried in the shade and stored in airtight containers at room temperature in dark until used.

Preparation of plant extracts
The aerial parts (steams, leaves and flowers) samples from plants were subjected to extraction by the method described by AOAC [25].Successive extractions for aerial parts were carried out using four ascending polar solvents: Petroleum ether, chloroform, acetone and ethanol.100 g of aerial parts was placed in a soxhlet apparatus containing petroleum ether and left for 12 h, then recycled for 6 h until full clearance.The mace was left for dryness after the exhaustive extraction with petroleum ether, then repacked in the soxhlet apparatus and successively extracted with chloroform, followed by acetone and ethanol.All the extracts were dried by distilling off the solvents under pressure using a rotary evaporator [25].

Modified disc diffusion method
Antimicrobial plant extracts activity methods were done as the guidelines of National Committee for Clinical Laboratory Standards [26,27].Mueller-Hinton Agar was prepared as the manufacturer's instructions and checked for sterility by incubating the plates overnight at 37°C.Modified discs of 6 mm were prepared using a Whitman filter paper.100 discs were obtained by punching and putting in vials-bottles and sterilizing in an oven at 170°C for 30 min.The discs were impregnating with 20 μl of plant extract (concentration 200 mg/ml).The discs were evaporated at 37°C for 24 h.Prepared discs containing the various extracts were carefully placed on the inoculated plates using a sterilized forceps in each disk according to Wiegand et al. [28].The disc with solvent alone as negative control one and antibiotic discs were the positive controls.The plates were then turned upsidedown and incubated at 37°C for 24 h in an incubator.The results were taken by considering the zone of growth and inhibition of the organisms by the test fractions.Antimicrobial activity was evaluated by measuring the diameter of the inhibition zone (DIZ) around the disc as mean ± standard deviation (SD).

Minimum inhibitory concentration (MIC) of plant extracts using micro dilution method
MIC was determined by micro dilution technique as described by the National Committee for Clinical Laboratories standards [29].The MIC was defined as the lowest concentration of the compound to inhibit the growth of microorganism.The 96-well plates were prepared by dispensing 50 μl of Mueller-Hinton broth for bacteria, into each well.A 50 μl from the stock solution of tested extracts (concentration of 200 mg/ml) was added into the first row of the plate.Then, two-fold, serial dilutions were performed by using a micropipette.The obtained concentration range from 100 to 0.195 mg/ml, and then added 10 μl of inocula to each well except a positive control (inoculums were adjusted to contain approximately 1 × 10 8 CFU/ml.Plant extract with media was used as a positive control and inoculums with media was used as a negative control.Micro titer plates were incubated at 37°C for 24 h.Lack of turbidity was further confirmed by pouring suspension aliquot of 0.1 ml into pre-sterilized petri dishes with nutrient agar medium.The tests were conducted in triplicate.

Statistical analysis
All data were subjected to statistical analysis including the calculation of the mean and SD.Significance between data of control and infected groups was evaluated by the Student's t-test at p<0.05 according to Petrie and Watson [30] using SPSS for windows version 15 (SPSS, Chicago, IL, 2006) computer program.

Results
The results showed that the quarter was considered sub-clinically affected when positively by CMT.A total of 203 milk samples were screened for mastitis by CMT.The prevalence of clinical, subclinical mastitis and normal healthy animals were 34.58%, 24

Antibacterial susceptibility test
The antibiogram profile of different bacterial isolates indicated that the antibiotic enrofloxacin, oxtetracycline and ampecillin showed different activity compared with plant extracts on isolated.There was significant variation in the antibacterial activities of J. montana and A. herb alba plant extracts that have antibacterial effect on Gram-positive isolated bacteria, the highest activity were observed with acetone extracts of J. montana compared to A. herb alba on Gram-positive bacteria S. aureus, Sterptococcus spp., S. agalactiae and CNS with DIZ values in range of 25.6±0.60 and 20.66±0.57,18.67±0.57and 19.66±0.58mm respectively.The petroleum ether, methanol and chloroform extracts of J. montana have shown less antibacterial activity (Table-3).The most effective antibacterial activity was recorded for J. montana which has inhibited S. aureus, Sterptococcus spp., S. agalactiae and CNS (DIZ 25.6±0.57,20.66±0.5 and 19.6±0.56 mm).The second highest antibacterial activity was A. herb alba that recorded a DIZ range between 6.33±0.57and 9.67±0.57mm.The highest antibacterial effect was recorded for S. aureus (9.67±0.58mm).This difference was significant (p<0.05)statistically as in Table -3.Evaluation of antibacterial activity (MIC) was carried out by micro dilution method for methanol, petroleum ether, acetone and chloroform extracts of J. montana and A. herb alba on Gram-positive bacteria S. aureus, Sterptococcus spp.The MIC value of J. montana was found by acetone extracts leaves gave the best antibacterial activity against Gram-positive bacteria.This activity may be attributed to the rich plant contents of active components such as alkaloids and flavonoides.The MIC for A. herb-alba leaves extracts against Gram-positive bacteria particularly was found to be significantly active exhibiting the little potency with all solvents used and this confirms of the need for further studied.Comparing plant extracts with enrofloxacin antibiotic showed antimicrobials against bacteria causing mastitis as in Figures -3a-c.
The results of antibacterial activity of plant extracts were recorded as presence or absence of zones of inhibition around the discs indicated the absence of microbial growth and it as reported as positive and absence of zone as negative antibacterial activity by referring the measurement to a chart, the organisms classified to susceptible (S) as in Figure -4a (1,3,4 and 6) inhibition around the discs or resistant (R) as in (2 and 5) no inhibition zone around the disk.Figure -4b susceptible (S) as in (4 and 6) or resistant (R) as in (5).

Discussion
Mastitis in dairy cows is a serious problem as it is an economically devastating disease causing immense     economic losses in dairy cows and bio health hazard to human worldwide especially in developing countries as in Egypt [31,32].The present study showed clearly that uses of clinical inspection and CMT examination were provided efficient diagnostic tool for detection and differential of clinical and subclinical mastitis and apparently normal health cattle and this observation is in agreement with Gianneechini et al. [33].They found that the screening of clinical and subclinical mastitis in animals by CMT is still the superior diagnostic tool.The high prevalence of subclinical mastitis in dairy cattle may be due poor hygiene and poor management in rural areas which the small or individuals dairy unit owners have no concept of subclinical mastitis, teat dipping, dry cow treatment and usually do not keep adequate herd records, this explain is in agreement with Fadlelmoula et al. [34] On the other hand, this explain is disagreement with El-Attar et al. [35].Isolation of the causative organisms by culturing is considered the most suitable, accurate and reliable method for identification of the causative agent [36,37].Out of these 203 milk samples, a total of 148 different bacterial isolates were recovered, they were identified by biochemical test and found the isolated bacteria belonging to eight species as in Table-2 and Figure-2.These results were in agreement with many authors [38,39].They are considered the culturing of bacterial is a gold standard method.The highest prevalent of E. coli, S. aureus and S. agalactiae may be due to transmission by teat-to-teat or cow-to-cow spread, possibly via by the Milker's hands under the lack of hygiene, these finding were in agreement with Das et al. [38], who considered these microorganisms as major etiological agents of clinical and subclinical mastitis worldwide.The other bacterial species isolated in the present study CNS, S. dysgalactia, Pasteurella spp., Pseudomonas spp., Klebsiella spp.are a minor causes of bovine mastitis [40].Many of these bacteria isolated from bovine mastitis are also the causative agents of human diseases i.e.E. coli, S. aureus and S. agalactiae and higher incidence rate of E. coli may be due to poor hygienic conditions of the environment as E. coli infects the udder via teat canal from the environment.S. aureus is causing food poisoning and frequently found in milk come from dairy cattle with mastitis [7].
Many authors have recorded an increase in the drug resistance bacteria isolated from bovine mastitis [40].Antibiotics sensitivity test is important to suggest suitable antibacterial treatment to prevent antibiotic resistance, potential health risk for humans.The obtained results in Table-3 and Figure -3 and 4 showed clearly that the penicillin, ampicillin and oxtetracycline were found to be less effective against bacteria isolate from bovine mastitis may be due to increased indiscriminate and frequent use of those antibiotic in dairy animals leading to develop of antibiotic resistance bacteria which necessitates develop and search for novel sources as antimicrobial agents.Medicinal plant-derived compounds have increased widespread interest in the search of alternative antibacterial agents.They are safe and have a long history of use in folk medicine for the treatment of infectious diseases [41].According to the World Health Organization, medicinal plants would be the best source to obtain a variety of drugs and active compounds.Therefore, such plants should be investigated in order to understand their properties, safety and efficiency [42].The aim of the current study was to evaluate the antibacterial activity of selected some Sinai medicinal plant extracts against bacteria isolated from bovine mastitis, our results in the results in Table-3 and Figure-4 revealed that the plant extracts by different solvent petroleum ether, methanol, chloroform and acetone exhibited a level of antibacterial activity against Gram-positive bacteria isolated from bovine mastitis.There was significant variation in the antibacterial activities of J. montana and A. herb alba plant extracts that have antibacterial effect against Gram-positive bacteria, the highest activity was observed with acetone extracts of J. montana compared to A. herb alba and this result is in agreement with Sampimona et al. [43].The most effective antibacterial activity was recorded for J. montana showed marked level antibacterial activity against S. aureus, Staphylococci (CNS)and S. dysgalactia followed by A. herb alba were recorded and induce inhibition zone ± SD ranged between 4.33±0.57and 9.67±0.57mm, these results were in agreement with Mothana et al. [44].Evaluation of antibacterial activity by MIC different plant extracts of J. montana and A. herb alba with different solvents (methanolic, petroleum ether, acetone and chloroform)against Gram's-positive bacteria S. aureus, Staphylococci (CNS)and S. dysgalactia.The MIC value of J. montana acetone extract was (1.5625 mg/ml)may be due to attributed to the rich plant contents of active components such as alkaloids and flavonoids.Also, A. herb alba plant extracts gave (50-6.25 mg/ml)against Gram's-positive bacteria particularly found to be significantly active exhibiting the little potency with all solvents used as in Table-3 and Figure-3a-c and 4a and b these results were in agreement with Sampimona et al. and Sumathi et al. [43,45].When comparing antibacterial activities of plant extracts with enrofloxacin antibiotic against bacteria causing bovine mastitis, we found the nearly similar for inhibition patterns were recorded, and this recorded is in agreement with Sumathi et al. [45].Finally, our results proved that the J. montana and A. herb alba have the ability of antibacterial activity against Grampositive bacteria isolated from bovine mastitis.

Conclusion
The present study spot highlight on isolation and identification of mastitis pathogens that are fundamental aspects of milk quality, udder health control programs and public health and food safety issues associated with food borne pathogens.CMT findings represent valuable diagnostic tools in the detection of cows with secretion disorder whose show no clinical signs of disease.Routine milk cultures should be an ongoing part of any mastitis control program.On that perspective, medicinal plants are safe, efficient and a low-cost option for treating bovine mastitis that should also be further explored similar to the synthetic options.Most tested plant extracts showed antibacterial activity against bacteria tested which may reflect the antibacterial activity of plant active ingredients that inhibit bacterial growth.J. montana and A. herb alba acetone extracted were possessed strong antimicrobial activities against Gram-positive bacteria isolated from bovine mastitis.All the solvents (methanolic, petroleum ether and chloroform)used in plants extraction exhibited less antibacterial activities than acetone solvent extract.Our results proved that the J. montana and A. herb alba plant extracts can be used as antimicrobial agent as a natural alternative manner to some of the commonly used antibiotics in human and animals but further researches is still necessary to identify active compounds, effectiveness, toxicity, safety indices and clinical trials in treatment of infectious diseases.

Figure- 1 :
Figure-1: Prevalence of clinical and subclinical mastitis at different Governorates in Egypt.

Figure- 2 :
Figure-2: Percentage of isolated bacteria from bovine mastitis in Egypt.

Figure- 3 :
Figure-3: (a) Revealed that the Jasonia montana plant acetone extract showed the highest antibacterial effect on Staphylococcus aureus, Streptococcus ssp.and coagulase-negative Staphylococci, other extracts have antibacterial effect on the same bacteria but still lower than acetone extract.(b) the Artemisia herb alba had moderate effect on Gram-positive bacteria, (c) antibiotic showed antibacterial effect on Gram-negative and Gram-positive bacteria.c b a

Figure- 4 :
Figure-4: Antimicrobial activity of plant extracts on bacteria isolated from bovine mastitis by disk diffusion method (a) (1,3,4 and 6) inhibition around the discs or resistant (R) as in (2 and 5) no inhibition zone around the desk.(b) susceptible (S) as in (4 and 6) or resistant (R) as in (5).a b .7% and 40.8% respectively.Out of 203 cow's milk samples 171 isolates were obtained from 83 normal healthy, 70 clinical and 50 subclinical cases of mastitis at Benisuef, El-Fayoum, Behera and Monofia Governorates in Egypt.Major bacterial isolates were E. coli (22.16%), S. aureus (20.19%),Streptococcus spp.(13.30%),Pasteurella spp.(2.45%), Klebsiella spp.(1.47%)and Pseudomonas spp.(0.5%) (Table-1 and Figure-1).Gram-positive and catalase test positive distinguished between Streptococci and Staphylococci.The hemolytic patterns and coagulase reaction with rabbit plasma were used to differentiated between S. aureus and CNS.Also, esculin hydrolysis and CAMP reaction differentiated between S. agalactia and another strep.Gram-negative bacteria were identify by sub culturing on differential and selective media and tested to oxidase activity, acid production (glucose, lactose and sucrose fermentation), indole test, VP test and hydrogen sulfide production as National Mastitis Council' s guidelines as in Table-2 and Figure-2.Confirmation test for E. coli with Api-20E0 test.The result of Api-E20 test was revealed the numerical profile [5144552]as confirmed diagnostic test for E. coli isolates.

Table - 1
: Examination of cow's milk samples by CMT and isolated bacteria.

Table - 2
: Biochemical characteristics of isolated bacteria from bovine mastitis.Antimicrobial activity of A. herb alba and J. montana extracted by different chemical solvents on bacteria isolated from bovine mastitis by disk diffusion method (values were calculated as means of triplicates).