Final Report for LNC12-343
This project aims to develop intra-mammary, non-antibiotic alternatives to treating mastitis in dairy cows raised under organic and conventional production methods. In 2013, we determined the minimum inhibitory concentrations (MICs) of Manuka honey for E. coli, Staphylococcus and Streptococcus isolated from cows with clinical mastitis. The MICs obtained with Manuka honey were very likely too high to be able to achieve needed concentrations of honey in the quarters of cows with clinical mastitis. Hence, we redirected our efforts to involve essential oils from plants that have been shown to have very good antibacterial properties. The tested essential oils had very good antibacterial properties against pathogens isolated form mammary glands of cows with clinical mastitis. Based on literature/web searches that provided information that essential oils in in vitro and in vivo are very tissue irritant and caused up to a 100-fold increase in somatic cell counts and bloody milk. Hence, we terminated attempts to assess the safety of intra-mammary infusions of essential oils.
Mastitis is the most common health condition requiring management on both conventional and organic dairy farms (APHIS, 2008; Ruegg, 2009; Werner et al., 2010). The cost of each case of mastitis on a dairy averages $155.08 per each episode (Cha et al., 2011) and is estimated to cost the entire US Dairy industry $1.7 – 2 billion each year (Jones and Bailey, 2009). Vaccinations to reduce the occurrence and/or severity of mastitis, adequate hygiene throughout milking procedures and sanitation in cow housing are effective control methods common to both organic and conventional dairy farms (Fluckey et al., 2009;Ruegg, 2009;USDA, 2008c). Although conventional dairies rely on antibiotics to prevent new cases of mastitis during the cow’s non-lactating (dry) period and to treat clinical cases of mastitis during lactation (AHPIS, 2008; Werner et al., 2010), antibiotics are not allowed for use in organic dairies under the US National Organic Program (USDA, 2008c;Ruegg, 2009; Werner et al., 2010). Both organic and conventional dairy producers would benefit from the development of new non-antimicrobial alternatives to bacterial infections in livestock. These reasons are based on the intense scrutiny of antimicrobial drug use in animal agriculture because of antimicrobial resistance issues, a decrease in market incentives for developing new antimicrobial drugs, and the growing antibiotic-free segment of the dairy industry.
Alternative mastitis treatments are of importance to improve the sustainability of organic dairy farmers and conventional dairy farmers who are committed to reducing their use of antimicrobial drugs. Although several guidelines for judicious and prudent use of antimicrobial drugs are available to conventional dairy farmers, there are few documented safe and efficacious alternative treatment options. Additionally, if there is risk to human health from use of antimicrobial drugs and antimicrobial resistance, the environment and society as a whole will benefit from the availability of non-antimicrobial alternatives for treatment of mastitis.
The objectives of this project are to:
To determine the minimum inhibitory concentration (MIC) of Manuka honey and guava leaf extract to bacteria commonly associated with bovine mastitis.
To establish in-animal safety of candidate formulations and in-milk inhibition metabolites residue depletion
To determine cure rate of mild to moderate clinical mastitis in dairy cows using candidate formulations
Work towards completing Objective 1 was performed during 2012-2015. The first objective of this NCR-SARE project was to assess the in-vitro effect of Manuka Honey and Guava Extract to inhibit the growth of common mastitis pathogens isolated from dairy cows with clinical mastitis. To accomplish that goal, we proposed to determine the minimum inhibitory concentrations (MIC) of Manuka Honey and Guava Extract for bacteria causing clinical mastitis in dairy cows. We were not able to identify a source of Guava extract that was consistent in quality and quantity. The MICs obtained for Manuka honey ~6-8% (weight/volume) are likely so high that we would not be able to obtain high enough concentrations of the honey in the udder of dairy cows to reach the MIC needed.
To compensate for the loss of Guava extract and Manuka honey as viable options for treatment of mastitis, we identified several essential oils that appeared to have much lower MICs than Manuka honey. These oils include: oregano oil, two constituents of oregano oil; carvacrol and thymol, and trans-cinnamaldehyde.
In 2015, we finished work to determined the MICs to the following essential oils: oregano oil, carvacrol, thymol and trans-cinnamaldehyde.
Based on literature/web searches that provided information that essential oils in in vitro and in vivo are very tissue irritant and caused up to a 100-fold increase in somatic cell counts and bloody milk. Hence, we had to abandon the safety studies of mammary infusion of essential oils into the quarters of lactating cows.
Minimum inhibitory concentrations of Manuka honey and essential oils were determined and compared to MICs for antibiotics using methodology as described in CLSI Guidance M31-A3. In short, Manuka honey, essential oils with an emulsifier and antibiotics were added to Mueller-Hinton (MH) agar at predetermined concentrations. For all studies we used the following QC strains: Staphylococcus aureus (ATCC29213), Escherichia coli (25922) and Streptococcus pneumonia (49619)
MICs were determined using a micro agar-dilution method with a Cathra replicator. This allowed use to place 16 strains of the same bacterial strain in duplicate on an agar plate. In short, bacterial cultures were grown in tryptic soy broth to a concentration of ½ MacFarland standard and the broth cultures were placed on agar plates using the Cathra replicator. After incubation for 18-24 hours at 37C, the agar plates were examined for growth by two people.
The concentrations for Manuka honey 13+ were: 1, 2, 4, 6, 8, 10, 12%. Additionally, we used a Mock honey consistent of 33.5g D-glucose, 7.5g maltose, and 1.5g sucrose into 17ml of sterilized distilled water at the same concentrations as for Manuka honey. The Mock honey had the same approximate osmolality as the Manuka honey. We also tested the mastitis pathogen isolates used with Manuka honey against an antibiotic (Cephalothin as 1st gen. cephalosporin) at the following concentration 4µg/ml to 128 µg/ml. Cephalothin is commonly used to treat clinical mastitis (Cephapirin; ToDAY, Cefa-Lak) and has the resistance breakpoint of ≥32 µg/ml
The concentrations used for the essential oils ranged from 0.0075(vol/vol)% to 1(v/v)%.
The isolates tested for essential oils were also assessed for their MIC to ampicillin (4µg/ml to 128 µg/ml ) and ceftiofur (4µg/ml to 128 µg/ml )
For determination of MICs to Manuka honey, we used 46 Clinical mastitis strains from MSU mastitis lab: 6 S. aureus, 15 Streptococcus spp., 18 E. coli, 6 Klebsiella and 1 CNS.
For determination of MICs to essential oils and antibiotics, we used 88 clinical mastitis isolates against essential oils: Escherichia coli n = 33, Staphylococcus spp. n = 27, Streptococcus spp. n = 28. For essential oils studies, each isolate was included in duplicate on duplicate plates (4 repetitions)
The MICs obtained for Manuka honey ~6-8% (weight/volume) are likely so high that we would not be able to obtain high enough concentrations of the honey in the udder of dairy cows to reach the MIC needed.
To compensate for the loss of Guava extract and Manuka honey as viable options for treatment of mastitis, we initiated a search for other practical options. We identified several essential oils that appeared to have much lower MICs than Manuka honey. These oils include: oregano oil, two constituents of oregano oil; carvacrol and thymol, and trans-cinnamaldehyde.
Manuka honey could still be pursued as a potential emollient for intra-mammary solutions containing mainly essential oils.
We determined the MICs for Manuka honey to 18 E. coli and 15 Streptococcus isolates, respectively. We also determined the MICs of these isolates to the antimicrobial drug cephalothin and mock honey (a solution of different sugars).
For E. coli isolates, the MIC was 6%, 8% and 12% for 11, 6 and 1 isolates, respectively. The MIC was 6% for all Streptococcus isolates. Two isolates with a Manuka MIC of 6 or 8% were resistant to the antibiotic Cephalothin.
The project did not progress entirely as expected, because we realized that we would very likely not be able to achieve high enough concentrations of Manuka honey in the udder of a lactating cow in order to treat an infected quarter(s). Likewise, we were not able to find a high quality Guava product. However, we identified viable alternatives in the form of essential oils (liquid containing volatile aroma compounds from plants).
Assessing MICs of essential oils in mastitis pathogens.
The Michigan State University, College of Veterinary Medicine mastitis lab collected clinical mastitis isolates from Michigan farms in 2014. The National Masstitis Council Protocol and standards for isolation and classification were used for isolation and identification.
We collected over 200 isolates, but we screened the following 88 clinical mastitis isolates for MICs to essential oils and antimicrobial drugs: Escherichia Coli n = 33, Staphylococcus spp. n = 27, and Streptococcus spp. n = 28
For the 33 E. coli isolates, the MICs ranged from 2 to >64 for ampicillin, ≤0.0626 to 16 for ceftiofur, 0.0125 to >1 for oregano, 0.0625 to 1 for carvacrol, 0.0625 to >1 for thymol, and 0.03125 to 0.0625 for trans-cinnamaldehyde, respectively.
For the 27 Stapylococcus isolates, the MICs ranged from ≤0.0625 to >64 for ampicillin, ≤0.0626 to 16 for ceftiofur, 0.015625 to >1 for oregano, 0.015625 to >1 for carvacrol, 0.015625 to >1 for thymol, and ≤0.003906 to 0.0625 for trans-cinnamaldehyde, respectively.
For the 28 Streptococcus isolates, the MICs ranged from ≤0.0625 to 0.25 for ampicillin, MICs to ceftiofur was not done for streprococcal isolates, 0.5 to >1 for oregano, 0.015625 to 0.0625 for carvacrol, 0.03125 to 0.0625 for thymol, and 0.015625 to 0.0625 for trans-cinnamaldehyde, respectively.
For the E. coli isolates, the MIC50 and MIC 90 for the essential oils were 0.5 and >1 for oregano, 0.125 and 0.125 for carvacrol, 0.125 and 1 for thymol and 0.03125 and 0.03125 for trans-cinnamaldehyde, respectively.
For the Stapylococcus isolates, the MIC50 and MIC 90 for the essential oils were 0.5 and >1 for oregano, 0.125 and 0. 5 for carvacrol, 0.0625 and 0.25 for thymol and 0.03125 and 0.0625 for trans-cinnamaldehyde, respectively.
For the Streptococcus isolates, the MIC50 and MIC 90 for the essential oils were 1 and >1 for oregano, 0.03125 and 0.0625 for carvacrol, 0.03125 and 0.03125 for thymol and 0.0625 and 0.0625 for trans-cinnamaldehyde, respectively.
As expected the median MICs for essential oils were much lower – almost by a factor 100 – than for Manuka honey. Hence, essential oils may be better targets for alternatives to antimicrobial drugs for treatment of mastitis in cattle.
Our determination of MICs of various essential oils, strongly suggests that essential oils have antibacterial properties against pathogens isolated from clinical cases of mastitis. If the tissue irritant properties of essential oils can be mitigated, they may become a powerful tool in fighting mastitis in dairy cattle in the future.
Essential oils have excellent antibacterial properties with Trans-cinemaldehyde having the lowest MICs. However, because of being very tissue irritant, they will likely not be useful without chemical modifications for application in the udder or on mucosal surfaces. Although the MICs are low for essential oils, they will need to be applied at high concentration to a quarter because they get greatly diluted in the milk in the quarter.
Assuming the concentration of essential oils needed in the udder to reach at least the MICs determined previously, we determine the amount of antimicrobial drugs to inject into a mammary quarter will have to be a 50%-100% concentration of the essential oils. Such concentrations may be too irritating to the mammary epithelium. We initiated a web and literature search for safety and tissue reactivity/irritation of essential oils. Most essential oils are very tissue irritant. In a yearly report from a USDA, NIFA grant entitled: “Investigating the potential of natural antimicrobials for controlling bovine mastitis”, it was reported that infusion of trans-cinnamaldehyde at concentrations of 10%, 20, and 30% increased signs of discomfort (tail flicking and kicking) in the cows as well as a 100-fold increase in somatic cells counts and blood in the milk. Based on the findings reported in the grant report we terminated the 2nd objective of testing safety of the essential oils when injected into mammary glands at concentration we calculated to be needed to achieve concentrations above the mastitis organisms’ MIC. The next steps for us would be determine if essential oils can be manipulated to reduce their tissue irritant properties, however because of the switch in focus from Manuka honey to essential oils we did not have time to initiate this research within the time frame of the grant.
Since Manuka honey and essential oils likely are not viable options for treatment of clinical mastitis, we were unable to calculate potential economic impacts of the procedure.
No farmer adoption to report.
Educational & Outreach Activities
Antimicrobial activity of essential oils against mastitis isolates Scott Henderson, Lisa Halbert, Ron Erskine, Paul Bartlett, and Bo Norby (2014). Phi Zeta day, Michigan State University, East Lansing
Non-antibiotic treatment options for mastitis: NCR SARE funded research at Michigan State University, Bo Norby and Lisa Halbert. Ohio Ecological Food and Farm Association OEFFA, February 16, 2014
Areas needing additional study
There is a world-wide call for improving antibiotic stewardship in human and animal medicine. One option to reduce antibiotic use would be alternatives to antibiotics. Such substitutes to antibiotics may include alternatives that primarily aim at preventing diseases by improving immunity and better vaccines. However, alternatives are also need to treat clinical bacterial infections that previously have been treated with antibiotics. Perhaps essential oils and other natural compounds may be modified chemically in a manner so they are not so tissue irritant, and they may become viable alternatives to intra-mammary antibiotics in the future. Additionally, essential oils may become a viable option as antibacterial compounds to teat dips and external teat sealants or controlling pathogens in the environemnt.