Progress 02/01/12 to 01/31/17
Outputs
Target Audience:Dairy producers and managers, Veterinarians, Extension and outreach personnel, Nutritionists, Scientists, Consumer groups, Veterinary students and residents Changes/Problems:As described above for the project assessing the effect of Manuka honey and guava as potential treatments of mastitis, we terminated the project to identify antibiotic alternatives to treat mastitis because the selected compounds were too tissue irritant for infusion in the udder of cows. What opportunities for training and professional development has the project provided?One PostDoc, one technician, one PhD student, five veterinary students, and one undergraduate student were trained indeveloping study designs, basic microbiological methods as for exampleplating feces on agar to enumerate suceptible as well as resistant colifrom bacteria. Aditionally everyone were trained in the logistics of conducting research with farm animals and animal care, data management and data analysis. How have the results been disseminated to communities of interest?Results were disseminated at several localand national scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1. One of our previously reported findings has been that there is a smaller proportion of dry cows in which we can detect ceftiofur-resistant coliforms as compared to cows in other stages of their lactation cycle. We were curious about this finding because the farm at which we conducted the longitudinal study uses a ceftiofur product for intra-mammary dry-cow treatment, and we would expect that this might affect levels of ceftiofur-resistant coliforms in the feces of dry cows. Although, not a part of the original objective, we decided to investigate if dry-cow treatment with a ceftiofur product would affect the levels of ceftiofur-resistant coliform bacteria in cows treated intra-mammary with a ceftiofur product. Because this farm does blanket treats all cows at dry-off, we did not have a control group consisting of cows not dry-treated and moved to the dry-cow pen with the dry-cow treated cows. However, we followed a group of cows that we to be dried off the following week, but who stayed in the pen the dry cows were in be they were dried off and moved to the dry-cow pen. The total coliform counts decreased by about 1 log concentration and ceftiofur-resistant coliform counts in dry cows increased by approximately half a log. These findings are similar to findings when cows are injected parentally with ceftiofur products. However, the effect was not as large. No changes were seen in the group of cows that were not dried off. Using a multiple singleplex qPCR platform that allows us to quantitate over 100 resistance genes, we found that the gene coding for ceftiofur resistance (blaCMY-2) increased from less than 0.01% to over 0.1% in relative abundance as compared to cows that were not treated. Although, this finding cannot be definitively attributed to the effect of intra-mammary infusion of a ceftiofur product, it suggests that intra-mammary infusion of ceftiofur in dry cows, results in some systemic absorption of ceftiofur which in term affects the intestinal microbiome and resistome. Analysis of CFUs per gram of feces is complicated because the data are not normal distributed and a large proportion of samples have zero counts Several samples with zero counts are likely not truly zero but are between zero and the detection limit of ~240 CFUs/g feces. Hence, we are investigating using count models and potentially imputing CFUs for samples with <240 CFUs. The analyses are further complicated by the longitudinal/repeated sampling design. Final analyses have to be completed Objective 2, Investigating probiotics as an intervention to reduce antimicrobial resistance in pre-weaned dairy calves: We developed a probiotic solution consisting of 10 E. coli isolates from pre-weaned calves. The 10 E. coli isolates were tested for resistance using National Antimicrobial Resistance Monitoring System (NARMS) antimicrobial susceptibility Sensititre® plate to ensure that those strains were susceptible to the common antimicrobials used in veterinary medicine for gram-negative infections. Susceptible bacteria were screened for virulence genes (eaeA, Stx1, Stx2, STa, STb, and LT) at Michigan State University's Diagnostic Center for Population and Animal Health (DCPAH) to ensure that the isolates were non-pathogenic strains of Escherichia coli. In a preliminary safety and efficacy trial, four calves were randomly assigned to receive the probiotic of E. colis and four calves served as controls and were fed the carrier of whole milk. Two calves fed the probiotic developed clinical signs consistent with abomasal stasis and abomasitis and they were treated with antibiotics and excluded from the study. Results suggest that there might be an approximate 1 log10 reduction in E. coli resistant to ceftiofur in the group fed the probiotic (power=~20%). However, no differences were observed for resistance to ampicillin and tetracycline and further studies assessing the safety of the product are needed. The fisrt study of an E. coli probiotic was repeated in 2016, but we only used only 7 of the 10 E. coli strains. Fourteen calves were housed at the MSU Beef Cattle Teacher and Research Center in individual hutches. As opposed to the 2014 study when calves received the probiotic E. coli culture from birth to the end of the study, this summer feeding of the probiotic culture started at the age of about 3 weeks. Calves fed the probiotic did not have lower counts of intestinal ceftiofur-resistant coliforms as compared to the calves only fed the carrier. However, there were, as seen in most studies, a decline in total coliforms and resistant coliforms over time. One explanation for observing an effect of the probiotic in the 2014 study and no effect in the 2016 study may be that probiotic have to be fed immediately after birth to be able to outcompete resistant bacteria. In other words, to be able to outcompete resistant coliforms, susceptible coliforms have to be fed from birth as with other established competitive exclusion cultures for pathogens. Work towards completing Objective 3 was performed during 2015/2016. To compensate for the loss of Guava extract and Manuka honey as viable options for treatment of mastitis, we initiated a search for other viable 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. 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 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. Assuming the concentration of essential oils needed in the udder needed to be 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. Most essential oils are very tissue irritant, and needed intra-mammary 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. 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 here we terminated the 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.
Publications
|
Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Dairy producers and managers, Veterinarians, Extension and outreach personnel, Nutritionists, Scientists, Consumer groups Changes/Problems:Most essential oils are very tissue irritant, and needed intra-mammary 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. 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 here we terminated the 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. Assuming the concentration of essential oils needed in the udder needed to be 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 largely exceed what can be infused into a quarter of a cow according to the information we unveiled. What opportunities for training and professional development has the project provided?OUTPUTS: A veterinary students presented results at national meetings. PARTICIPANTS: Bo Norby is the PI of the project and he committed approximately 4 month to the project. His role was to oversee the overall management of the project and project development and design. He also participated in sample collection and animal care and data analysis. Erik Corbett is a graduate student who worked 1 months the project. He participated in study design and was in charge data analysis. Daniel Stern is a veterinary student who worked approximately 3 months on the project. He participated in calf management, sample collection, sample analysis and data analysis for the study of the effect of an E. coli probiotic in dairy calves. Christa Finley is a veterinary student who worked approximately 3 months on the project. He participated in calf management, sample collection, sample analysis and data analysis for the study of the effect of an E. coli probiotic in dairy calves. Alecca Como is an undergraduate student who worked 3 months on the project. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?The umbrella project is about to expire, but data analyses for Objective 1 will continue regardless.
Impacts
What was accomplished under these goals?
Objective 1. One of our previously reported findings has been that there is a smaller proportion of dry cows in which we can detect ceftiofur-resistant coliforms as compared to cows in other stages of their lactation cycle. We were curious about this finding because the farm at which we conducted the longitudinal study uses a ceftiofur product for intra-mammary dry-cow treatment, and we would expect that this might affect levels of ceftiofur-resistant coliforms in the feces of dry cows. Although, not a part of the original objective, we decided to investigate if dry-cow treatment with a ceftiofur product would affect the levels of ceftiofur-resistant coliform bacteria in cows treated intra-mammary with a ceftiofur product. Because this farm does blanket treats all cows at dry-off, we did not have a control group consisting of cows not dry-treated and moved to the dry-cow pen with the dry-cow treated cows. However, we followed a group of cows that we to be dried off the following week, but who stayed in the pen the dry cows were in be they were dried off and moved to the dry-cow pen. The total coliform counts decreased by about 1 log concentration and ceftiofur-resistant coliform counts in dry cows increased by approximately half a log. These findings are similar to findings when cows are injected parentally with ceftiofur products. However, the effect was not as large. No changes were seen in the group of cows that were not dried off. Using a multiple singleplex qPCR platform that allows us to quantitate over 100 resistance genes, we found that the gene coding for ceftiofur resistance (blaCMY-2) increased from less than 0.01% to over 0.1% in relative abundance as compared to cows that were not treated. Although, this finding cannot be definitively attributed to the effect of intra-mammary infusion of a ceftiofur product, it suggests that intra-mammary infusion of ceftiofur in dry cows, results in some systemic absorption of ceftiofur which in term affects the intestinal microbiome and resistome. Analysis of CFUs per gram of feces is complicated because the data are not normal distributed and a large proportion of samples have zero counts Several samples with zero counts are likely not truly zero but are between zero and the detection limit of ~240 CFUs/g feces. Hence, we are investigating using count models and potentially imputing CFUs for samples with <240 CFUs. The analyses are further complicated by the longitudinal/repeated sampling design. Understanding the variation in susceptibility patterns of fecal coliforms in dairy cows will assist in the design of potential interventions to reduce levels and patterns of antimicrobial resistance in cull cows entering the food chain. Objective 2, Investigating probiotics as an intervention to reduce antimicrobial resistance in pre-weaned dairy calves: During the summer of 2014 we developed a probiotic solution consisting of 10 E. coli isolates from pre-weaned calves. The 10 E. coli isolates were tested for resistance using National Antimicrobial Resistance Monitoring System (NARMS) antimicrobial susceptibility Sensititre® plate to ensure that those strains were susceptible to the common antimicrobials used in veterinary medicine for gram-negative infections. Susceptible bacteria were screened for virulence genes (eaeA, Stx1, Stx2, STa, STb, and LT) at Michigan State University's Diagnostic Center for Population and Animal Health (DCPAH) to ensure that the isolates were non-pathogenic strains of Escherichia coli. In a preliminary safety and efficacy trial, four calves were randomly assigned to receive the probiotic of E. colis and four calves served as controls and were fed the carrier of whole milk. Two calves fed the probiotic developed clinical signs consistent with abomasal stasis and abomasitis and they were treated with antibiotics and excluded from the study. Results suggest that there might be an approximate 1 log10 reduction in E. coli resistant to ceftiofur in the group fed the probiotic (power=~20%). However, no differences were observed for resistance to ampicillin and tetracycline and further studies assessing the safety of the product are needed. During the summer of 2016, we repeated the study from 2014 but used only 7 of the 10 E. coli strains. Fourteen calves were housed at the MSU Beef Cattle Teacher and Research Center in individual hutches. As opposed to the 2014 study when calves received the probiotic E. coli culture from birth to the end of the study, this summer feeding of the probiotic culture started at the age of about 3 weeks. Calves fed the probiotic did not have lower counts of intestinal ceftiofur-resistant coliforms as compared to the calves only fed the carrier. However, there were, as seen in most studies, a decline in total coliforms and resistant coliforms over time. One explanation for observing an effect of the probiotic in the 2014 study and no effect in the 2016 study may be that probiotic have to be fed immediately after birth to be able to outcompete resistant bacteria. In other words, to be able to outcompete resistant coliforms, susceptible coliforms have to be fed from birth as with other established competitive exclusion cultures for pathogens. Work towards completing Objective 3 was performed during 2015/2016. To compensate for the loss of Guava extract and Manuka honey as viable options for treatment of mastitis, we initiated a search for other viable 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. 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 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. Assuming the concentration of essential oils needed in the udder needed to be 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. Most essential oils are very tissue irritant, and needed intra-mammary 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. 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 here we terminated the 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.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Corbett, E.M.; Norby, B.; Halbert, L.W.; Henderson, S.T.; Grooms, D.L.; Manning, S.D.; Kaneene, J.B. (2015) Effect of feeding a direct-fed microbial on total and antimicrobial-resistant fecal coliform counts in preweaned dairy calves. Am.J.Vet.Res. 86:748-835
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Bactericidal activity of Human Beta Defensin-3 on susceptible and resistant enteric E. coli from cattle. C.A. Pieprzyk, S.T. Henderson, B. Norby Phi Zeta day, Michigan State University, East Lansing, Michigan, October 2, 2015
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Use of susceptible and avirulent E. coli to reduce antimicrobial-resistant coliforms in milk-fed calves, Daniel Stern and Bo Norby (2016) Merial-NIH National Veterinary Scholars Symposium. The Ohio State University, Columbus Ohio, July 29-Aug 1, 2016
|
Progress 10/01/14 to 09/30/15
Outputs
Target Audience:Dairy producers and managers, Veterinarians, Extension and outreach personnel, Nutritionists, Scientists, Consumer groups Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?OUTPUTS: A graduate student and two veterinary students presented results local and national meetings. PARTICIPANTS: Bo Norby is the PI of the project and he committed approximately 4 month to the project. His role was to oversee the overall management of the project and project development and design. He also participated in sample collection and animal care and data analysis. Erik Corbett is a graduate student who worked 1 month on the project. He participated in study design and was in charge of study conduct, including sample collection, sample processing, sample analysis and data analysis. Scott Henderson is a veterinary student and laboratory technician who worked approximately 3 months on the project. He tested the MICs of mastitis pathogens to essential oils. Cedar Pieprzyk is a veterinary student who worked on the project for 3 months. She developed the bioassay to test the bacterial killing effect of antimicrobial peptides on E. coli isolates. Stephen Carney is an undergraduate student who worked 1 month on the project. How have the results been disseminated to communities of interest?Results from this project have been presented at local research and extension meetings. What do you plan to do during the next reporting period to accomplish the goals?Repeat the study using an E. coli probiotic to lower the abundance of resistant coliforms in dairy calves. Develop a safety model for essential oils as treatment for clinical mastitis in dairy cows.
Impacts
What was accomplished under these goals?
Objective 1, On-going analysis will further determine if quantities of total coliforms and resistant coliforms are associated with stage of production, diet/location, treatment history, or seasonality in dairy cows. Analysis of CFUs per gram of feces is complicated because the data are not normal distributed and a large proportion of samples have zero counts. Several samples with zero counts are likely not truly zero but are between zero and the detection limit of ~240 CFUs/g feces. Hence, we are investigating using count models and potentially imputing CFUs for samples with <240 CFUs. The models are exceedingly complicated because we need to account for repeated sampling (~26) per cow over a year, and we are continuing working on these model. Understanding the variation in susceptibility patterns of fecal coliforms in dairy cows will assist in the design of potential interventions to reduce levels and patterns of antimicrobial resistance in cull cows entering the food chain. We have already discovered that cows that are in the dry period, have a lower risk of having ceftiofur-resistant coliforms detected in their feces as compared to cows that are in other stages of production. Objective 2, Investigating probiotics as an intervention to reduce antimicrobial resistance in pre-weaned dairy calves: We conducted a second study during the summer of 2013, to further investigate the effect of DFM2 on resistant coliforms in dairy calves. In this study, three treatment groups were used: 1) calves treated with DFM2 at birth and twice daily with 1g of DFM2, 2) calves treated with DFM2 at birth and twice daily with 0.5g of DFM2,and 3) control calves that were not fed DFM2. This study lasted 28 days and fecal samples were collected daily for the first 3 days and every other day thereafter. Phenotypic microbiological and statistical analyses have been completed. A manuscript has been submitted and is in review. Results suggest that the absolute numbers of total coliforms did not significantly differ between treatment groups, however for ceftiofur-resistant coliforms, there was a significant reduction in control calves as compared to groups fed the DFM at different concentrations. During the summer of 2014 we developed a probiotic solution consisting of 10 E. coli isolates from pre-weaned calves. The 10 E. coli isolates were tested for resistance using National Antimicrobial Resistance Monitoring System (NARMS) antimicrobial susceptibility Sensititre® plate to ensure that those strains were susceptible to the common antimicrobials used in veterinary medicine for gram-negative infections. Susceptible bacteria were screened for virulence genes (eaeA, Stx1, Stx2, STa, STb, and LT) at Michigan State University's Diagnostic Center for Population and Animal Health (DCPAH) to ensure that the isolates were non-pathogenic strains of Escherichia coli. In a preliminary safety and efficacy trial, four calves were randomly assigned to receive the probiotic of E. colis and four calves served as controls and were fed the carrier of whole milk. Two calves fed the probiotic developed clinical signs consistent with abomasal stasis and abomasitis and they were treated with antibiotics and excluded from the study. Results suggest that there might be an approximate 1 log10 reduction in E. coli resistant to ceftiofur in the group fed the probiotic (power=~20%). However, no differences were observed for resistance to ampicillin and tetracycline and further studies assessing the safety of the product are needed. In 2015, we submitted a grant to the Michigan Alliance for Animal Agriculture, to further test the E. coli probiotic in a larger set of calves. During the summer of 2015, we decided to investigate our finding from 2012 where calves fed a probiotic had higher ceftiofur-resistant coliform counts as compared to controls not fed the probiotic. From the human literature it is known that some probiotics, including an E. coli probiotic, increase the intestinal epithelium's release of antimicrobial peptides. Antimicrobial peptides are peptides with antibacterial properties release by the animal host. We therefore decided to test if antibiotic-resistant E. coli were more likely to be resistant to antimicrobial peptides than mainly susceptible E. coli. In other words, we wanted to investigate if antibiotic resistance in E. coli was associated with resistance to antimicrobial peptides. If so, this could explain why feeding probiotics to calves increased abundance of resistance. Work towards completing Objective 3 was performed during 2013. The first objective of this 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 initiated a search for other viable 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 may still be pursued as a potential emollient for intra-mammary solutions containing mainly essential oils. In future work we will focus on these essential oils as targets for non-antibiotic treatments of mastitis. During the current reporting period, we determined the minimum inhibitory concentrations of oregano oil, carvacrol and thymol, and trans-cinnamaldehyde and also compared them to MICs for commonly used antibiotics. The MICs of oregano oil, carvacrol and thymol, and trans-cinnamaldehyde as well as ceftiofur and ampicillin were tested in 33. E. coli, 27 Staphylococcus, and 28 streptococcus isolates. MICs were lowest for the antibiotics as compared to essential oils. However, for essential oils, MICs were lowest for trans-cinnamaldehyde followed by carvacrol, thymol and oregano oils.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Corbett, E.M.; Norby, B.; Halbert, L.W.; Henderson, S.T.; Grooms, D.L.; Manning, S.D.; Kaneene, J.B. (2015) Effect of feeding a direct-fed microbial on total and antimicrobial-resistant fecal coliform counts in preweaned dairy calves. Am.J.Vet.Res. 86:748-835
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Antimicrobial susceptibility, nonpathogenic Escherichia coli used as a probiotic to decrease resistant coliforms in dairy calves (2014) K.C. VanFleet, Erik Corbett , Scott Henderson, and B. Norby. Phi Zeta day, Michigan State University, East Lansing, Michigan.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
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
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Bactericidal activity of Human Beta Defensin-3 on susceptible and resistant enteric E. coli from cattle. C.A. Pieprzyk, S.T. Henderson, B. Norby. Merial-NIH Research Symposium, U.C. Davis, CA, August 1st, 2015
|
Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Dairy producers and managers, Veterinarians, Extension and outreach personnel, Nutritionists, Scientists, Consumer groups Changes/Problems: As described above for the project assessing the effect of Manuka honey and guava as potential treatments of mastitis, we changed the focus to essential oils as the MIC of Manuka honey was too high for practical purposes. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest? A graduate student and two veterinary students presented results local and national meetings. What do you plan to do during the next reporting period to accomplish the goals? For objective 1, we plan to finish data analyses and submit a manuscript for publication. For objective 2, we will publish a manuscript objective 3, we plan to complete in vitro MIC analyses of clinical mastitis isolates, submit a manuscript for publication, and initiate the in-animal safety trial.
Impacts
What was accomplished under these goals?
Objective 1, On-going analysis will further determine if quantities of total coliforms and resistant coliforms are associated with stage of production, diet/location, treatment history, or seasonality in dairy cows. Analysis of CFUs per gram of feces is complicated because the data are not normal distributed and a large proportion of samples have zero counts Several samples with zero counts are likely not truly zero but are between zero and the detection limit of ~240 CFUs/g feces. Hence, we are investigating using count models and potentially imputing CFUs for samples with <240 CFUs. Understanding the variation in susceptibility patterns of fecal coliforms in dairy cows will assist in the design of potential interventions to reduce levels and patterns of antimicrobial resistance in cull cows entering the food chain. Objective 2, Investigating probiotics as an intervention to reduce antimicrobial resistance in pre-weaned dairy calves: We conducted a second study during the summer of 2013, to further investigate the effect of DFM2 on resistant coliforms in dairy calves. In this study, three treatment groups were used: 1) calves treated with DFM2 at birth and twice daily with 1g of DFM2, 2) calves treated with DFM2 at birth and twice daily with 0.5g of DFM2,and 3) control calves that were not fed DFM2. This study lasted 28 days and fecal samples were collected daily for the first 3 days and every other day thereafter. Phenotypic microbiological and statistical analyses have been completed. A manuscript has been submitted and is in review. Results suggest that the absolute numbers of total coliforms did not significantly differ between treatment groups, however for ceftiofur-resistant coliforms, there was a significant reduction in control calves as compared to groups fed the DFM at different concentrations. During the summer of 2014 we developed a probiotic solution consisting of 10 E. coli isolates from pre-weaned calves. The 10 E. coli isolates were tested for resistance using National Antimicrobial Resistance Monitoring System (NARMS) antimicrobial susceptibility Sensititre® plate to ensure that those strains were susceptible to the common antimicrobials used in veterinary medicine for gram-negative infections. Susceptible bacteria were screened for virulence genes (eaeA, Stx1, Stx2, STa, STb, and LT) at Michigan State University’s Diagnostic Center for Population and Animal Health (DCPAH) to ensure that the isolates were non-pathogenic strains of Escherichia coli. In a preliminary safety and efficacy trial, four calves were randomly assigned to receive the probiotic of E. colis and four calves served as controls and were fed the carrier of whole milk. Two calves fed the probiotic developed clinical signs consistent with abomasal stasis and abomasitis and they were treated with antibiotics and excluded from the study. Results suggest that there might be an approximate 1 log10 reduction in E. coli resistant to ceftiofur in the group fed the probiotic (power=~20%). However, no differences were observed for resistance to ampicillin and tetracycline and further studies assessing the safety of the product are needed. Work towards completing Objective 3 was performed during 2013. The first objective of this 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 initiated a search for other viable 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 may still be pursued as a potential emollient for intra-mammary solutions containing mainly essential oils. In future work we will focus on these essential oils as targets for non-antibiotic treatments of mastitis.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
The Effect of Feeding a Direct Fed Microbial on Antimicrobial Resistance in Fecal Coliforms from Dairy Calves. Erik Corbett, Bo. Norby, Lisa .W. Halbert, John B. Kaneene, Dan L. Grooms (2013). Conference for Research Workers in Animal Diseases (CRWAD), December 8-10, Chicago, Illinois.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Antimicrobial susceptibility, nonpathogenic Escherichia coli used as aprobiotic to decrease resistant coliforms in dairy calves (2014) K.C. VanFleet, Erik Corbett , Scott Henderson, and B. Norby. Phi Zeta day, Michigan State University, East Lansing, Michigan.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
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
|
Progress 12/01/12 to 09/30/13
Outputs
Target Audience: Dairy producers and managers, Veterinarians, Extension and outreach personnel, Nutritionists, Scientists, Consumer groups. Changes/Problems: We have no changes to report, but we conducted a second trial in dairy calves investigating further the effect of direct-fed microbials on resistance in coliform intestinal bacteria. We are behind schedule for objective 3 due to investigations into methods to sterilize Manuka honey. What opportunities for training and professional development has the project provided? OUTPUTS: Research results have been presented at local and national professional meetings. PARTICIPANTS: Bo Norby is the PI of the project and he committed approximately 4 month to the project. His role was to oversee the overall management of the project and project development and design. He also participated in sample collection and animal care and data analysis. Lisa Halbert is a research associate who worked approximately 8 months on the project. Her role was to develop and oversee management of the project. She also participated in sample collection and animal care. Erik Corbett is a graduate student who worked 10 months the project. He participated in study design and was in charge of study conduct, including sample collection, sample processing, sample analysis and data analysis. Scott Henderson is a laboratory technician who worked approximately 1 months on the project. Ashley Kwasniewski is a veterinary student who worked on the project for 3 months. She participated in sample collection and processing, as well as microbial analyses and data entering. Wanda Gonzales is a veterinary student from Tuskegee who worked on the project for 3 months, and who participated in sample collection, microbiological analyses and data entry. She also conducted her own study on samples from the calf project (2013) identifying total CFUs of enterococci and enterococci resistant to vancomycin, tetracycline, ampicillin and ciprofloxacin. Stephen Carney is an undergraduate student who worked 12 months on the project. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? For objective 1, we plan to finish data analyses and submit a manuscript for publication. For objective 2, we plan to complete data analyses, submit two manuscripts, and initiate community microbial analyses using a 16S or metagenomic approach. For objective 3, we plan to complete in vitro MIC analyses of clinical mastitis isolates, submit a manuscript for publication, and initiate the in-animal safety trial.
Impacts
What was accomplished under these goals?
Objective 1, Temporal changes in antimicrobial resistance in dairy cattle: The sampling phase and microbiological analyses of the study has been completed. Enrolled cows were sampled every other week for up to 66 weeks, and absolute and relative numbers of total coliform colony forming units (CFUs) and CFUs of coliform bacteria resistant to ampicillin, ceftiofur and tetracycline have been completed. Analyzing the dichotomous outcome of detection of resistance to the tested antimicrobials and comparing dry cows to cows in other stages of production, dry cows had decreased odds of resistance being detected to one or more antibiotics (OR=2.6, p<0.001) , to two or more antibiotics (OR=2.7, p<0.001) as well as detection of coliforms resistant to ampicillin (2.8, P<0.001) and tetracycline (OR 2.6, p=0.001). Detection of resistance to ceftiofur was marginally significant (OR=1.7, P=0.06). Results from our preliminary analyses suggest that the stage of production may have an effect on shedding of coliforms and resistant coliforms. Further analysis will assess if quantities of total coliforms and resistant coliforms are associated with stage of production, diet/location, treatment history, or seasonality in dairy cows. Understanding the variation in susceptibility patterns of fecal coliforms in dairy cows will assist in the design of potential interventions to reduce levels and patterns of antimicrobial resistance in cull cows entering the food chain. Objective 2, Investigating probiotics as an intervention to reduce antimicrobial resistance in pre-weaned dairy calves: Our preliminary data from the study conducted in 2012 suggest that feeding of a DFM (DFM2) containing Bifidobacterium animalis ssp. Lactis, Lactobacillus acidophilus, Bacillus licheniformis, Bacillus subtilis and Lactobacillus lactis at certain time-points significantly reduced the total absolute counts of total coliform bacteria, and absolute numbers of coliforms resistant to ampicillin, ceftiofur and tetracycline by an approximate 2 log10 concentration. We conducted a second study during the summer of 2013, to further investigate the effect of DFM2 on resistant coliforms in dairy calves. In this study, three treatment groups were used: 1) calves treated with DFM2 at birth and twice daily with 1g of DFM2, 2) calves treated with DFM2 at birth and twice daily with 0.5g of DFM2,and 3) control calves that were not fed DFM2. This study lasted 28 days and fecal samples were collected daily for the first 3 days and every other day thereafter. Phenotypic microbiological analyses have been completed. Preliminary results suggest that the absolute numbers of total coliforms did not significantly differ between treatment groups, however for resistant coliforms, it preliminarily appears as though control calves had lower CFUs as compared to groups fed the DFM at different concentrations . Objective 3, Investigate Manuka honey and oregano as an alternative non-antibiotic treatment for mastitis in dairy cows. We have determined a method to “sterilize” Manuka honey, and we are currently collecting clinical mastitis isolates provided by the MSU, CVM Mastitis Lab for further MIC testing. Additionally, we have decided to add oregano extract as a potential antimicrobial for use in the study. Preliminary MIC results show that Manuka honey’s MIC with our quality control strains was 5% to 7%, which were lower concentrations than what we experienced using a mock honey solution.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2012
Citation:
Temporal Changes in Antimicrobial Resistance in Fecal Coliforms Isolated from Dairy Cows. (2012) E.M. Corbett, B. Norby, L.W. Halbert, J.B. Kaneene, D.L. Grooms. Conference for Research Workers in Animal Diseases (CRWAD), December 2-4, Chicago, Illinois.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2013
Citation:
Evaluation of Direct Fed Microbials Effect on (Phenotypic) Antimicrobial Resistance from Enterococci Strains Isolated from Dairy Calves (2013). W. M. Gonzalez-Quintana, B. Norby, L.W. Halbert, E. Corbett, S.T. Henderson, A. Kwasniewski, S.V. Carney. Summer Student Presentations Michigan State University July 24-26, East Lansing, Michigan.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
The Effects of Feeding a Direct Fed Microbial on Antimicrobial Resistance in Fecal Coliforms from Dairy Calves. (2013) E.M Corbett, B. Norby, L.W. Halbert, J.B. Kaneene, D.L. Grooms. Phi Zeta day, Michigan State University, East Lansing, Michigan.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2013
Citation:
The Effect of Feeding a Direct Fed Microbial on Antimicrobial Resistance in Fecal Coliforms from Dairy Calves. E.M. Corbett, B. Norby, L.W. Halbert, J.B. Kaneene, D.L. Grooms (2013)
Conference for Research Workers in Animal Diseases (CRWAD), December 8-10, Chicago, Illinois.
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Progress 12/01/11 to 11/30/12
Outputs
OUTPUTS: Objective 1, Temporal changes in antimicrobial resistance in dairy cattle: 50 dairy cows, 10 in each of 5 different production stages were enrolled in this study in the month of November 2011. Fecal samples were collected from the cows every 2 weeks for 12 months. Production data, health events and movements between barns were recorded. Total coliform counts, and counts of coliforms resistant to ceftiofur, ampicillin, tetracycline and ciprofloxacin in 1g of feces have been determined for the first 19 of 26 sampling dates. Microbiological and statistical analyses have not been completed for this objective. Preliminary descriptive statistics for total coliform counts and coliforms resistant to ceftiofur, ampicillin, and tetracycline have been performed. Additionally, analytical statistics have been conducted for detection of resistance in a sample (dichotomous; yes/no) as compared to the production group the cows was in at the time of sampling. Mixed effect count models and zero-inflated count models are being developed in order to most appropriately analyze our coliform count data. Objective 2, Investigating probiotics as an intervention to reduce antimicrobial resistance in pre-weaned dairy calves: In a pilot randomized controlled clinical trial, we investigated the effect(s) of feeding direct-fed microbials (DFMs) to pre-weaned dairy calves on the counts of total and antimicrobial-resistant fecal coliforms and coliforms resistant to ceftiofur, ampicillin, tetracycline and ciprofloxacin. A total of 42 (6 batches x 7 calves) calves were enrolled in the study. The seven treatment groups consisted of: 1) DFM 1 + Naxcel treatment for three days (days 4, 5, and 6), 2) DFM 1, 3) control (no DFM) + Naxcel treatment for three days, 4) DFM 2 + Naxcel treatment for three days, 5) DFM 2, 6 and 7) control groups (no DFM). To control for the potential effect of ambient temperature and other variables, calves were enrolled into the study in batches of seven. When seven calves were assembled, treatments were assigned at random to the calves. All the calves were fed non-medicated milk-replacer twice daily according to the participating dairy's standard protocol, and they had free access to calf starter grain and fresh water. DFMs were dissolved in 2% milk and administered per os by a catheter tip syringe. Feces was collected by digital stimulation form the rectum of calves every third day after a batch was assembled until day 27 and every 6th day thereafter until weaning at 8 weeks of age. Microbiological and statistical analyses have not been completed for this objective. Preliminary descriptive and analytical statistics for total coliform counts and coliforms resistant to ceftiofur, ampicillin, and tetracycline have been performed. Objective 3, Investigate Manuka honey as an alternative non-antibiotic treatment for mastitis in dairy cows: External funding for this project has been obtained from USDA NCR SARE. We have commenced testing of a batch of Manuka honey for bacterial contamination. One graduate student, two veterinary students (summer students), two undergraduate students and a laboratory technician were tutored as part of these project. PARTICIPANTS: Bo Norby is the PI of the project and he committed approximately 4 month to the project. His role was to oversee the overall management of the project and project development and design. He also participated in sample collection and animal care and data analysis. Lisa Halbert is a research associate who worked approximately 4 months on the project. Her role was to develop and oversee overall management of the project. She also participated in sample collection and animal care. Erik Corbett is a graduate student who worked full time on the project. He participated in study design and was in charge of study conduct, including sample collection, sample processing, sample analysis and data analysis. Jessica Reis is a veterinary student who worked on the project for 3 months. She participated in sample collection and processing, as well as microbial analyses and data entering. Ashley Kwasniewski is a veterinary student who worked on the project for 3 months. She participated in sample collection and processing, as well as microbial analyses and data entering. Kate Elbourne is an undergraduate student who worked approximately 3 months on the project. She helped with all laboratory procedures, including sample processing and analysis. Stephen Carney is an undergraduate student who worked approximately 3 months on the project. She helped with all laboratory procedures, including sample processing and analysis. TARGET AUDIENCES: Dairy producers and managers, Veterinarians, Extension and outreach personnel, Nutritionists, Scientists, Consumer groups PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Objective 1, Temporal changes in antimicrobial resistance in dairy cattle: Analyzing the dichotomous outcome of detection of resistance to the tested antimicrobials and comparing dry cows to cows in other stages of production, dry cows had a decreased chance of resistance being detected to one or more antibiotics, as well as detection of resistance to ampicillin and tetracycline (p<0.05). Detection of resistance to ceftiofur was not statistically significant at p<0.05. Results from our preliminary analyses suggest that the stage of production may have an effect on shedding of coliforms and resistant coliforms. Further analysis will assess if stage of production, diet, treatment history, or seasonality affect the levels and patterns of resistance in dairy cows. Understanding the variation in susceptibility patterns of fecal coliforms in dairy cows will assist in the design of potential interventions to reduce levels and patterns of antimicrobial resistance in cull cows entering the food chain. Objective 2, Investigating probiotics as an intervention to reduce antimicrobial resistance in pre-weaned dairy calves: Our preliminary data suggest that feeding of a DFM (DFM2) containing Bifidobacterium animalis ssp. Lactis, Lactobacillus acidophilus, Bacillus licheniformis, Bacillus subtilis and Lactobacillus lactis at certain time-points significantly reduced the total absolute counts of total coliform bacteria, and absolute numbers of coliforms resistant to ampicillin, ceftiofur and tetracycline by more than 2 log10 concentrations. Resistance to ciprofloxacin was not detected. In our models for total coliform counts and coliforms resistant to ceftiofur, ampicillin, and tetracycline, there was a significantly lower counts in absolute numbers of total coliforms (an approximately 2 log10 reduction), and coliforms resistant to ceftiofur, ampicillin and tetracycline (1 to 2 log10 reduction) on day 10 for calves fed DFM 2 as compared to calves fed DFM 1 and the control calves. There was no significant difference between calves fed DFM 1 and the control calves on day 10. Objective 3, Investigate Manuka honey as an alternative non-antibiotic treatment for mastitis in dairy cows: Few Gram positive and Gram negative bacterial strains have been isolated from the Manuka honey. Further microbial analyses of the bacterial isolates will be conducted, and methods for sterilization of the honey will be explored. Three presentations were given to primarily veterinarians as part of outreach activities: Norby, B. (2012) "The Fuss about Food Animal Antibiotic Use, Resistance and Drug Residues: Fact or Folly." Presentation at the Michigan Veterinary Conference, Lansing, January 27, 2012. Halbert, L. (2012) "Regulatory and Residue Insights." Presentation to CVM/MSU Practice-Based Ambulatory Veterinarians, East Lansing, November 1, 2012. Corbett, E.; Norby, B.; Halbert, L.W.; Kaneene, J.B.; Grooms, D.L. (2012) Temporal changes in antimicrobial resistance of fecal coliforms isolated from dairy cows. Presentation to CVM/MSU Practice-Based Ambulatory Veterinarians, East Lansing, November 1, 2012.
Publications
- Corbett, E.; Norby, B.; Halbert, L.W.; Kaneene, J.B.; Grooms, D.L. (2012) Temporal changes in antimicrobial resistance of fecal coliforms isolated from dairy cows. (Abstract proceedings) American Association of Bovine Practitioners - Annual Meeting, Montreal, QC, September 21, 2012
- Corbett, E.; Norby, B.; Halbert, L.W.; Kaneene, J.B.; Grooms, D.L. (2012) Temporal patterns of antimicrobial resistance in dairy cows and factors that affect them. (Abstract proceedings) Phi Zeta Research Day in the College of Veterinary Medicine, Michigan State University, East Lansing, October 5, 2012
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