DETECTION AND TREATMENT OPTIONS FOR MASTITIS TREATMENT AND PREVENTION OF MASTITIS IN CAPRINES AND SUBSEQUENT DRUG RESIDUES AND DRUG RESISTANCES IN CAPRINE MILK

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: EVANS-ALLEN
• Reporting Frequency: Annual
• Accession No.: 1009423
• Project No.: GEOX-3224
• Project Start Date: Mar 22, 2016
• Project End Date: Mar 5, 2021

Project Director
McCommon, GE.

Recipient Organization
FORT VALLEY STATE UNIVERSITY
1005 STATE UNIVERSITY DRIVE
FORT VALLEY,GA 31030

Performing Department
Veterinary Science 1912

Non Technical Summary
CURRENT ISSUE/PROBLEM: The demand for goat and dairy products has grown in the United States (US) and continues to increase through the second decade of the 21st Century. This is due to the nutritive advantages that include provision of higher protein and lower cholesterol levels as well as ease of digestibility in milk, chevre (cheese) and ice cream. Goat milk also has an increased demand in health and beauty aids including lotions, soaps and creams. "Mastitis", a disease caused by bacterial introduction into the mammary system, presents as an inflammatory response with subsequent increase in the leukocyte count (somatic cell count)and bacterial counts within milk, thusreducing the amount of marketable milk as well as market value. Because of commercial availability, many producers "self-medicate" animals, which leads to antibiotic resistance as well as antibiotic residue, which further contributes to a lack of commercial production.BASIC METHODS/APPROACHES: Probiotics have shown promising effects in prevention and treatment of mastitis in humans. Goat milk also has shown promise as a carrier for probiotic bacteria. Three areas will be studied within a series of experiments: 1)The incidence of current antibiotic resistance in dairy goats, 2) The duration of antibiotic residue in goats undergoing treatment for mastitis and 3) the use of probiotic bacteria as a preventative as well as treatment of mastitis. Studies will include: evaluation of antibiotic resistance in dairy goats treated for mastitis, evaluation of antibiotic residue in dairy goats treated for mastitis and the use of probiotics for intra-mammary infusion treatment of clinical and sub-clinical mastitis in dairy goats as compared to an oral probiotic.The animals to be used will be identified from within the Dairy Goat Herd at FVSU. Identification and use will be based on a) availability of animals from other researchers, b) animals that are diagnosed with mastitis, c) animals that have undergone antibiotic therapy to treat cases of mastitis. A plan will be developed and presented to the Animal Care and Use Committee prior to the start of the project regarding use of animals in study. Once approved, animals will be assessed to see if have sub-clinical or clinicalmastitis, then assigned to resarch groups in order to receive oral probiotics or probiotic intra-mammary infusion. Other goats that have already undergone antibitic treatment willhave milk tested for antibioic residue or to assess antibiotic resistance.Results of research will be shared with producers during University field days, community outreach presentations (Agricultural Exposition @ Moultrie, etc.), publications (both producer-friendly and refereed journals) as well as presentations at state, national and international scientific meetings. These presentations and publications will be presented by agricultural reserchers, veterinarians, veterinary technicians and graduate students associated with the project.ULTIMATE PROJECT GOALS: The goals of this project is to a) determine if there is antibiotic residue past the manufacturers guidelines in goat milk (post treatment), b) determine is some goats are resistant to antibiotics and if so, what classes of antibiotics, c) are probiotics an acceptable form of novel treatment for sub-clinical or clinical mastitis in dairy goats and if so in what form - oral ingestion or intra-mammary infusion technique. It is hoped that the studies will provide farmers and producers an alternative to costly antibiotic therapy in treating mastitis in any form as well as to reinforce to farmers/producers the dangers of overuse of antibiotics. Societal benefits will include a healthier form of goat milk and a more robust supply of the product on the open market to be used for nutritive purposes, health and beauty aids, etc. This is turn will be beneficial to the farmer/producer as there is a higher demand for their product, one that they are prepared to meet.

Animal Health Component

100%

Research Effort Categories

Basic

30%

Applied

70%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3999	1060	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3999 - Animal research, general;

Field Of Science
1060 - Biology (whole systems);

Keywords

antibiotic resistance

probiotics

antibiotic residue

caprine milk

mastitis

food safety

goat milk

Goals / Objectives
GOAL # 1: To determine the inhibitory effect of various probiotics (Lactobacillus plantarum v. commercially available probiotics) on the growth of Staphylococcus aureus isolated from lactating dairy goats. Once the probiotic exhibiting the highest inhibitory effect is determined, this product will be fed to goats and the somatic cell count will be evaluated to determine if probiotics are beneficial in decreasing subclinical mastitis in goatsObjectives:1. To decrease somatic cell count in milk due to mastitis by 40% (post-treatment) using oral probiotics.2. To decrease somatic cell count in milk due to mastitis by 40% (post-treatment) using intramammary infusion of probiotics.3. To effectively treat sub-clinical mastitis in dairy goats yielding a 70% cure rate through use of oral probiotics.4. To effectively treat clinical mastitis in dairy goats yielding a 70% cure rate through use of oral probiotics.5. To effectively treat sub-clinical mastitis in dairy goats yielding a 70% cure rate through use ofintramammaryprobiotics.6.To effectively treat clinical mastitis in dairy goats yielding a 70% cure rate through use ofintramammaryprobiotics.GOAL # 2To identify the presence of antibiotic resistance and antibiotic residue in dairy goat milk.Objectives:1. Establish a percentage of goats displaying resistance and residueto Penicillin G past manufacturer guidelines.2. Establish a percenage of goats displaying resistance and residueto Amoxicillin and Ampicillin past manufacturer guidelines.3. Establish a percentage of goats displaying resistance and residueto Ceftiofer and Cephapircin past manufacturer guidelines .4. Establish a percentage of goats displaying resistance and residueto the Tetracycline class of antibiotics past manufacturer guidelines.5. Establish a percentage of goats displaying resistance and residueto the Aflatoxin M-1 class of antibiotics past manufacturer guidelines.6. Establish a percentage of goats displaying resistance and residueto the Gentamycin class of antibiotics past manufacturer guidelines.7. Establish a percentage of goats displaying resistance and residueto the Sulfamethazine class of antibiotics past manufacturer guidelines.GOAL # 3: To compare the efficacy of a commercially available probiotic versus a compounded intra-mammary infusion on both subclinical and clinical mastitis.1. To determine the percentage of goats with favorable outcome through treatment of sub-clinical mastitis with commercilly available probiotic usage.2. To determine the percentage of goats with favoable outcome through treatment of clinical mastitis with commercially available probiotic usage.3. To determine the percentage of goats with favorable outcomes through treatment of sub-clinical mastitis with compounded intra-mammary infusion.4. To determine the percentage of goats with favorable outcomes through treatment of clinical mastitis with compouned intra-mammary infusion.GOAL # 4: Assist small and economically challenged farmers by increasing the safety of their milk product.1. Decrease the amount of sub-clinical and clinical mastitis on farms through the use of probiotics.2. Increase the amount of viable milk products that are available as a result of less mastitis in dairy herds due to use of probiotics.

Project Methods
METHODS: Objective 1 and 3 Experiment to be conducted on 40, 1-2 year old pasture-raised dairy does to include Nubian, Alpine, Toggenburg and Saanen breeds. Year 1- animals used by other researchers will remain at current locations; those purchased will be housed at the Department of Veterinary Science (year 2-5 of study). Pre-study, all goats will be weighed, vaccinated, dewormed and bled for Complete Blood Count (CBC). During study a CBC will be performed monthly assessing health status of animals for entire trial. Pre-treatment, somatic cell count of each goat will be determined via commercial reference laboratory.Dairy goats with documented mastitis will be separated into a subclinical group and a clinical group. Goats with legally recognized normal somatic cell counts of 1,000,000mL-1 pre-project constitute control group (Paape, M.J. 2007). Within each group, goats will then be subdivided into sub-groups and randomly assigned to 1 of 2 treatment groups commercial probiotic fed vs. compounded probiotic mammary infusion). Within each group, "N" is based on number diagnosed with each form of mastitis.All three groups (subclinical mastitis, clinical mastitis, and control) will be maintained on a diet of a commercial pellet (Wrangler 12%), Bermuda grass hay, and ad lib access to mineral block and water throughout the study. Housing of 7-10 goats per pen with milking continued per the University farm protocol. The two sub-groups receiving the probiotic will be fed Dairyman's Choice® (7g/head/day plus 10 cc of Dairyman's Choice paste on Day 0) for 28 days, while the two sub-groups treated with intra-mammary infusion receive a probiotic (provided by a compounding pharmacy) once every 7 days after thoroughly cleaning the mammary glands. Upon completion of each treatment, somatic cell counts will be determined using IDEXX Reference Laboratory, Westbrook, Maine. Decreases in somatic cell counts will be evaluated and results statistically analyzed. Milk production prior to treatments and after treatments will also be documented and evaluated for significance in noted increase.Objective 2:Experiment will be conducted on the same 1-2 year old pasture-raised does (caprine) as used in Objective 1 and 3 and include two groups: Experimental Group (N=20) and Control Group (N=20) that are designated by simple random sampling (Perrin, 2015). Grouped animals will be maintained on separate pastures with ad libatum access to fresh water, mineral block and pasture. Pre-study, all goats will be weighed, vaccinated, dewormed and bled for CBC. During study, CBC will be performed monthly assessing health status of all animals for entire trial. Milk samples will be taken and cultured using mannitol salt agar. The primary pathogen will be isolated and identified (suspected to be Staphylococcus aureus). Pure cultures of the dominant pathogen will undergo four separate treatments determining the antagonistic activity, if any, of probiotics against the dominant pathogen. The four treatment groups include: Lactobacillus plantarum, Bacillus subitilus, Bacillus licheniformis, and Saccharomyces cerevisiae. These are the individual biologic ingredients of the commercially available product, Dairyman's Choice® as well as Lactobacillus plantarum, shown to be effective against Bovine Mastitis Staphylococcus aureus. Once pure cultures are obtained the organism will then be tested for growth against each probiotic organism using the Spot-on-lawn method (Tambekar, Bhutada, 2016).The zone of inhibition will be measured determining organism effectiveness in growth inhibition of S. aureus. The same treatments will be applied to Staphylococcus aureus (ATCC25923) serving as a control. Significance of the results will be determined using statistical analysis.Objective 4:: A two-part experiment will be conducted on the same identified goat herd as utilized in Objectives 1,2 and 3 including 1-2 year old, pasture-raised does being treated with traditional antibiotic therapy for mastitis. During year one, 40 goats will be identified within the dairy goat population at FVSU for use depending on lactation status and infection with mastitis. During year 2-5, 40 goats will be purchased and kept at the Department of Veterinary Science on the main campus at FVSU. Pre-study, all goats will be weighed, vaccinated, dewormed and a CBC completed to assess overall health status. The animals will be maintained on pasture with ad libatum access to fresh water and mineral block. During study, CBC will be performed monthly to assess health status of animals throughout the trial. Currently IDEXX has five SNAP tests available to test for a variety of antibiotic residue in dairy animals (See Table 1).The first part of the experiment involves milk samples collected from lactating does in the University dairy herd. Utilizing the appropriate SNAP testing methodology, milk will be collected from 40 does and tested for presence of antibiotic residue of tetracycline utilizing the Rapid One-Step Assay lateral flow technology (Charm MRL-BLTET; Charm Sciences, Inc. Lawrence, MA) and recorded as confirmatory data of presence of treatment-related antibiotics. The Charm MRL-BLTET test measures tetracycline residue in raw goat milk (Beltran, 2013). Once animal has completed the antibiotic regimen, a second round of testing utilizing the same SNAP methodology will be completed at the end of the manufacturers stated withdrawal time to ascertain continued presence of antibiotics in milk samples. If positive, samples will be carried out at one week intervals until results are negative. Significance of results will be determined using statistical analysis.The second part of the study will involve studying possibility of antibiotic resistance to mastitis-causing bacteria. Does from the above-mentioned test group will be examined via California Mastitis Test to determine presence of mastitis after antibiotic regimen has been completed. Positive animals (after completion of antibiotic therapy) will be logged into a database based on antibiotic resistance displayed. A milk culture will be made in order to run in-vitro drug sensitivity testing determining antibiotic resistance using the Kirby-Bauer method as described by Sharmar et al (2012). According to Rajeev, et al (2010), the Kirby-Bauer method is appropriate for in vitro drug sensitivity testing of amoxicillin, penicillin, streptomycin, cloxicillin and neomycin in goat milk. Old and new generation antibiotics will be utilized in testing in-vitro efficiency including amoxicillin, penicillin, gentamicin, tetracycline, and sulfamethazine. The antibiotic discs representing a) the antibiotic just used and b) other types of antibiotics that may be utilized in animals that are displaying resistance will be placed on the surface of agar plates previously seeded with mastitis positive milk from animals displaying antibiotic resistance. Plates will be incubated at 37oC for 18-24 hours before examining for the development of zones of inhibition (Rajeev, 2010). The diameter of the zone of inhibition will be measured in mm and compared with values listed in manufacturers standardized chart on the basis of (R) resistance, (MS) mild sensitivity, (MoS) moderately sensitive or (HS) highly sensitive to the antimicrobial action of each disc. Those that are completely resistant will be further examined to determine if mastitis resistance can be characterized as one of the following: Prototheca mastitis (grows on blood agar) and Cryptococcus neoformans and Candida albicans (yeast) or "other". All data will be analyzed statistically and reported in scientific formatTABLE 1: SNAP TEST & ANTIOBIOTIC DETECTEDSNAP Beta-LactumPenicillin GAmoxicillinAmpicillinCeftioferCephapircinSNAP TetracyclineTetracyclineChlortetracyclineOxetetracyclineSNAP Aflatoxin M-1Aflatoxin M1SNAP GentamycinGentamycinSNAP SulfamethazineSulfamethazine (SMTZ)

Progress 03/22/16 to 03/05/21

Outputs
Target Audience:The target audience consists of small ruminant farmers and those that consume small ruminant products along with veterinary and scientific professionals that assist and advise them. Changes/Problems:The COVID 19 pandemic has been problematic both for research and distributing our data. We have collectedpromising information and will endeavour to refine our knowledge in this area for small ruminant producers and agricultural professionals. What opportunities for training and professional development has the project provided?Trained graduate students on microbiological techniques and clinical pathology. Graduate students have been trained in small ruminanthusbandry. Students are using critical thinking and are learning advanced laboratory techniques. Through numerous presentations at public gatherings such as the annual American Dairy Goat Convention, the results have been disseminated to the public. How have the results been disseminated to communities of interest?Multiple scientifis posters, abstracts, and presentations have been presented. They were limited this year due to the pandemic. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Using of commercial strains of probiotics for treatment of Staphylococcus aureus in Mastitis Goats.Goat mastitis is the most important infectious disease on dairy goat farms. Conventional antibiotic therapy often yieldunsatisfactory results and alternative treatments are continually under investigation. In the previous work Lactobacillus acidophilusCL1285, Bifidobacterium bifidum, and Lactobacillus plantarum commercial strains showed the highest inhibition activityagainst growth of S. aureus isolates in vitro. Their effects were mediated both by direct cell competitive exclusion as well asproduction of acids or bacteriocin-like inhibitors. Very few clinical data has been found on the interactions between probioticsand S. aureus isolates, but the few identified clinical cases pointed to the feasibility of elimination or reduction of S. aureuscolonization with probiotics use.During the period of May to July 2020, twenty (20) dairy goats with mastitis from the veterinary science department were enrolledin the clinical trial to exam and compare the effectivenessof three probiotics which showed the highest inhibition activity against growth of S.aureus isolates in vitro.Goats selected where divided into 4 groups with each group having 5 does. All goats were diagnosed and confirmed to havemastitis (seepage 25 of report dated04/27/2021,United States Department of AgricultureProgress Report Accession No. 1009423 Project No. GEOX-3224).California mastitis test and Staphylococcus aureus isolates where cultured, isolated and identified from milk.Group 1 was comprised of goats which were treated with antibiotics (control), group 2 were infused with Lactobacillus acidophilusCL1285, group 3 were infused with Bifidobacterium bifidum and group 4 were infused with Lactobacillus plantarum.Group 1 were infused with pirlimycin intramammary infusion once daily for 5 days and the other groups were infused with 0.5ml of 106 CFU of the respective probiotics once daily for 5 days. Twenty-four (24) hours after the first dose of antibiotic or probiotic infusion,milk was collected for culture, isolation and identificationofS. aureus and respective probiotics used. This process was conductedfor10 days and bacteria CFU were recorded.It was noted that the CFU in group 1 were markedly reduced by day 3 and no bacteria was isolated by day 5.Whilegroups that received probiotics the S. aureus CFU declined by days 5 and no bacteria was isolated by days 8 or 9. Theprobiotics continued to be recovered each day though the CFU had markedly reduced by day 10.Very few clinical data has been found on the interactions between probiotics and S. aureus isolates, but our clinical trial pointsto the feasibility of elimination or reduction of S. aureus colonization with probiotics use. In conclusion, due to the small sample size used in this study, additional studies are needed to ascertain whetherprobiotics can truely eliminate S.aureus from mastitisinfected udders of dairy goats.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: 2017: Georgia Public Health Association Conference: POSTER: Probiotics as a Novel Treatment of Mastitis in Dairy Goats to Combat a Nutritional Concern, V. Singh, Mofya, S, Samples, OM, Knight, M, McCommon, G Research Day - FVSU POSTER: Effect of Probiotic Treatment on Raw Milk Quality Tests in Mastitic Dairy Goats, Singh, V, Ocloo, K, Samples OM, McCommon G, Mofya S, Knight M ORAL: Detection of Antibiotic-resistant Staphylococcus aureus Isolated From Mastitis Dairy Goats Treated with Cephpirin Sodium at Fort Valley State University, Ocloo O, Mofya, S, Samples OM, Knight M, McCommon G ORAL: Exploring Effects of Probiotic (Lactobaccillus sp.) on Mastitis Pathogens (Staphylococcus aureus) Isolated From Caprines, Ocloo O, Mofya,S, Samples OM, Knight M, McCommon G ORAL: Mastitis in Goats  A National Concern, Singh, V, Mofya,S, Samples OM, Knight M, McCommon G ARD Meeting POSTER: Detection of Antibiotic Resistant Mastitis in Dairy Goats, Ocloo O, Samples OM, Gillespie RA, McCommon G, Mofya S, Knight M POSTER: Treatment of Antibiotic Resistant Mastitis in Dairy Goats with Probiotic sp. Lactobacillus, Ocloo O, Samples OM, Gillespie RA, McCommon G, Mofya S, Knight M POSTER: Detection of Antibiotic Resistant Mastitis in Dairy Goats, Wimes J, Ocloo O, Samples OM, Gillespie RA, McCommon G, Mofya S, Knight M POSTER: Detection of Antibiotic Resistant Staphlococcus aureus Isolated From Mastitic Dairy Goats Treated with Cephapirin Sodium, Ocloo O, Samples OM, Gillespie RA, McCommon G, Mofya S, Knight M 2018: National Goat Conference POSTER: Novel Use of Probiotics in Treatment of Antibiotic-resistant Mastitis-Causing Disease in Dairy Goats, Singh, V, Ocloo O, Samples OM, Mofya S, Knight M, McCommon G POSTER: Preliminary Assessment of Antibiotic Resistant Mastitis in Dairy Goats and Identification of Causative Agents, Singh, V, Samples OM, Mofya S, Knight M, McCommon G Georgia Environmental Health Conference POSTER: A Comparative of Treatment of Mastitis in Humans and Caprine Species, Kalu K, Samples OM, Mofya S, McCommon G 2019: State of Georgia Public Health UGA Conference POSTER: A Comparative of Treatment of Mastitis in Humans and Caprine Species, Kalu K, Samples OM, Mofya S, McCommon G 2020: ARTICLE National Association of Veterinary Technicians: Mastitis in Dairy Goats, Samples, OM, Kalu, Kingsley Students Supported by Grant: Vishal Singh Anurag Singh Kofi Otis Ocloo Jeremy Wimes

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:The target audience consists of small ruminant farmers and those that consume small ruminant products along with veterinary and scientific professionals that assist and advise them. Changes/Problems:The Covid 19 pandemic has been problematic both for research and distributing our data. We need an extension to accomplish all of our goals. What opportunities for training and professional development has the project provided?Trained graduate students on microbiological techniques and clinical pathology. Graduate students have been trained in small food animal husbandry. Students are using critical thinking and are learning advanced laboratory techniques. How have the results been disseminated to communities of interest?Multipe scientific poasters, abstracts and presentations have been presented. They were limited this year due to the pandemic. What do you plan to do during the next reporting period to accomplish the goals?Our next step is to increase the sample size as well as recruit goats with mastitis from different farms throughout the state of Georgia which are reared under different management. A large sample size and different management bsystems may help us to gather information and results which may be helpful to determine if probiotics can be used as alternative treatments and prevention of mastitis in dairy goats.

Impacts
What was accomplished under these goals? Using of commercial strains of probiotics for treatment of Staphylococcus aureus in Mastitis Goats. Goat mastitis is the most important infectious disease on dairy goat farms. Conventional antibiotic therapy is often unsatisfactory and alternative treatments are continually under investigation. In the previous work Lactobacillus acidophilus CL1285, Bifidobacterium bifidum, and Lactobacillus plantarum commercial strains showed the highest inhibition activity against growth of S. aureus isolates in vitro. Their effects were mediated both by direct cell competitive exclusion as well as production of acids or bacteriocin-like inhibitors. Very few clinical data has been found on the interactions between probiotics and S. aureus isolates, but the few identified clinical cases pointed to the feasibility of elimination or reduction of S. aureus colonization with probiotics use. During the period of May to July 2020, twenty dairy goats with mastitis from the veterinary science department where enrolled in the clinical trial to establish how the use of three probiotics which showed the highest inhibition activity against growth of S. aureus isolates in vitro. The goats where divided into 4 groups with each group having 5 goats. All the goats were diagnosed with mastitis using California mastitis test and Staphylococcus aureus isolates where cultured, isolated and identified from milk. Group 1 comprised goats which were treated with antibiotics( control), group 2 were infused with Lactobacillus acidophilus CL1285, group 3 were infused with Bifidobacterium bifidum and group 4 were infused with Lactobacillus plantarum. Group 1 were infused with pirlimycin intramammary infusion once daily for 5 days and the other groups were infused with 0.5 ml of 106 CFU of the respective probiotics once daily for 5 days. 24 hours after the first dose of antibiotic or probiotic infusion milk was collected for culture, isolation and identified for S. aureus and respective probiotics used. This process was done for 10 days and bacteria CFU were recorded. It was noted that the CFU in group 1 were markedly reduced by day 3 and no bacteria was isolated by day 5, while for the groups that received probiotics the S. aureus CFU declined by days 5 and no bacteria was isolated by days 8 or 9. The probiotics continued to be recovered each day through the CFU had markedly reduced by day 10. Very few clinical data has been found on the interactions between probiotics and S. aureus isolates, but our clinical trial points to the feasibility of elimination or reduction of S. aureus colonization with probiotics use. However, due to the small sample size in this trial we cannot definitively concluded that probiotics eliminate S.aureus from the infected udder.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: 2017: Georgia Public Health Association Conference: POSTER: Probiotics as a Novel Treatment of Mastitis in Dairy Goats to Combat a Nutritional Concern, V. Singh, Mofya, S, Samples, OM, Knight, M, McCommon, G Research Day - FVSU POSTER: Effect of Probiotic Treatment on Raw Milk Quality Tests in Mastitic Dairy Goats, Singh, V, Ocloo, K, Samples OM, McCommon G, Mofya S, Knight M ORAL: Detection of Antibiotic-resistant Staphylococcus aureus Isolated From Mastitis Dairy Goats Treated with Cephpirin Sodium at Fort Valley State University, Ocloo O, Mofya, S, Samples OM, Knight M, McCommon G ORAL: Exploring Effects of Probiotic (Lactobaccillus sp.) on Mastitis Pathogens (Staphylococcus aureus) Isolated From Caprines, Ocloo O, Mofya,S, Samples OM, Knight M, McCommon G ORAL: Mastitis in Goats  A National Concern, Singh, V, Mofya,S, Samples OM, Knight M, McCommon G ARD Meeting POSTER: Detection of Antibiotic Resistant Mastitis in Dairy Goats, Ocloo O, Samples OM, Gillespie RA, McCommon G, Mofya S, Knight M POSTER: Treatment of Antibiotic Resistant Mastitis in Dairy Goats with Probiotic sp. Lactobacillus, Ocloo O, Samples OM, Gillespie RA, McCommon G, Mofya S, Knight M POSTER: Detection of Antibiotic Resistant Mastitis in Dairy Goats, Wimes J, Ocloo O, Samples OM, Gillespie RA, McCommon G, Mofya S, Knight M POSTER: Detection of Antibiotic Resistant Staphlococcus aureus Isolated From Mastitic Dairy Goats Treated with Cephapirin Sodium, Ocloo O, Samples OM, Gillespie RA, McCommon G, Mofya S, Knight M 2018: National Goat Conference POSTER: Novel Use of Probiotics in Treatment of Antibiotic-resistant Mastitis-Causing Disease in Dairy Goats, Singh, V, Ocloo O, Samples OM, Mofya S, Knight M, McCommon G POSTER: Preliminary Assessment of Antibiotic Resistant Mastitis in Dairy Goats and Identification of Causative Agents, Singh, V, Samples OM, Mofya S, Knight M, McCommon G Georgia Environmental Health Conference POSTER: A Comparative of Treatment of Mastitis in Humans and Caprine Species, Kalu K, Samples OM, Mofya S, McCommon G 2019: State of Georgia Public Health UGA Conference POSTER: A Comparative of Treatment of Mastitis in Humans and Caprine Species, Kalu K, Samples OM, Mofya S, McCommon G 2020: ARTICLE IN REVIEW: National Association of Veterinary Technicians: Mastitis in Dairy Goats, Samples, OM, Kalu, Kingsley

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:The target audience consists of small ruminant farmers and those that consume small ruminantproducts along with veterinary and scientific professionals that assist and advise them. Changes/Problems:The Covid 19 pandemic has been problematic both for research and distributing our data. We need an extension to accomplish all of our goals. What opportunities for training and professional development has the project provided?Faculty and staff continue to improve microbiological techniques and use as examples for undergraduate students education. How have the results been disseminated to communities of interest?2019: State of Georgia Public Health UGA Conference POSTER: A Comparative of Treatment of Mastitis in Humans and Caprine Species, Kalu K, Samples OM, Mofya S, McCommon G 2020: ARTICLE IN REVIEW: National Association of Veterinary Technicians: Mastitis in Dairy Goats, Samples, OM, Kalu, Kingsley What do you plan to do during the next reporting period to accomplish the goals?The next step is to try to determine the efficacy of probiotics in vivo in animal infectious models.

Impacts
What was accomplished under these goals? Sixty-two Staphylococcus aureus isolates where cultured, isolated and identified from milk from goats which had mastitis. The milk samples were cultured on mannitol salt agar, gram stained, catalase and coagulase tested. Among the 86 milk samples tested, 62 were identified as Staphylococcus aureus after subjecting them to specific bacteriological tests. Antibiotic sensitivity tests were then performed onisolates using 12 different antibiotics in order to determine their antimicrobial resistance profiles. Once the antimicrobial resistance profiles were determined the isolates were then subject to in vitro experiments using five stains of probiotics: Lactobacillus acidophilus CL1285, Lactobacillus casei LBC80R, Bifidobacterium bifidum, and Lactobacillus plantarum commercial strains. Lactobacillus acidophilus CL1285, Bifidobacterium bifidum, and Lactobacillus plantarum commercial strains showed the highest inhibition activity against growth of S. aureus isolates in vitro. Their effects were mediated both by direct cell competitive exclusion as well as production of acids or bacteriocin-like inhibitors. Very few clinical data were found on the interactions between probiotics and S. aureus isolates, but the few identified clinical cases pointed to the feasibility of elimination or reduction of S. aureus colonization with probiotics use. The next step is to try todetermine the efficacy of probiotics in vivo in animal infectious models.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: 2019 Poster: State of Georgia Public Health UGA Conference: A Comparative of Treatment of Mastitis in Humans and Caprine Species, Kalu K, Samples OM, Mofya S, McCommon, GArticle:Caprine Mastitis as a Nutritional Concern: Samples, O., McCommon, G.NAVTA 2018 National Goat Conference Poster: Novel Use of Probiotics in Treatment of Antibiotic-resistant Mastitis-Causing Disease in Dairy Goats, Singh, V., Ocloo, O., Samples, O., Mofya, S., Knight, M., McCommon, G. Poster: Prelimanary Assessment of Antibiotic-resistant Mastitis in Dairy Goatsand Identification of Causative Agents:Singh, V.,Samples, O., Mofya, S., Knight, M., McCommon, G. Georgia Environmental Health Conference Poster: A Comparative of Treatment of Mastitis in Humans and Caprine Species, Kalu, K., Samples, O., Mofya, S. McCommon, G.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:The target audience consists of small ruminant farmers and those that consume their products along with veterinary, Cooperative Extension and scientific professionals that assist and advcise them. Multiple presentations and abstacts have been presented. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate students were trained in laboratory procedures and dairy goathusbandry practices.. Graduate students used research results to fulfill M.S. degree thesis requirements. As previously stated in previous reports the graduate students became very familiar with laboratory procedures. How have the results been disseminated to communities of interest?Multiple scientific posters and abstracts have been submitted. Articles have been published. Graduate student thesis' results have been presented asinvited speakers at the National Dairy Goat Association annual meeting. What do you plan to do during the next reporting period to accomplish the goals?1. All bacterial cultured will be preserved for year round testing. 2. The bacteria will continue to be tested with common antibiotics. 3. Bacteria will continue to be tested with various probiotics searching for which probiotic is most effective.

Impacts
What was accomplished under these goals? 1. Multiple graduate students presented their research in multiple environments. 2. Diary goats were sampled andsamples were placed on multiple agarplates. The microbes were exposed to multiple antibiotics and their susceptibility was recorded. 3. Bacteria were isolated and probiotic compounds were tested andinformation recorded.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Article:Caprine Mastitis as a Nutritional Concern: Samples, O., McCommon, G.NAVTA 2018 National Goat Conference Poster: Novel Use of Probiotics in Treatment of Antibiotic-resistant Mastitis-Causing Disease in Dairy Goats, Singh, V., Ocloo, O., Samples, O., Mofya, S., Knight, M., McCommon, G. Poster: Prelimanary Assessment of Antibiotic-resistant Mastitis in Dairy Goatsand Identification of Causative Agents:Singh, V.,Samples, O., Mofya, S., Knight, M., McCommon, G. Georgia Environmental Health Conference Poster: A Comparative of Treatment of Mastitis in Humans and Caprine Species, Kalu, K., Samples, O., Mofya, S. McCommon, G.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:The target audience consists of small ruminant farmers and those that consume their products along with veterinary and scientific professionals that assist and advise them. Multiple presentations and abstracts have been presented this past year with more scheduled this fall. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided numerous opportunities for students to increase their laboratory and microbiological skill sets through training in the following areas: Collect fresh milk samples from goats via manual expression Appropriately preserve & transport milk samples to lab for testing Test milk using California Mastitis Testing methodology and determine results Record and report data Trained students to recognize leukocyte types in prepared blood smears. Trained graduate students on microbiological plating techniques and preservation to identify bacterial germs & species of collected bacteria from milk. Graduate students have been trained in small ruminant husbandry. Multiple abstracts and posters have been presented. Scheduled to speak atNational Dairy Goat Association annual meeting in October 2017 How have the results been disseminated to communities of interest?Multiple scientific posters and abstracts have been presented. Scheduled to speak atNational Dairy Goat Association annual meeting in October 2017 What do you plan to do during the next reporting period to accomplish the goals?1. All bacteria cultured from last summer 2016 and 2017 are now been tested for their biochemical characteristics and identified at species level. 2. The identified species will be tested for antibiotic resistance against 12 antibiotics commonly used in the treatment of mastitis in food animal and humans. 3. The identified species with be tested against several probiotics in vitro. The probiotic(s) that will be very effective in the lab will then be used in the clinical trial in summer of 2018 as alternative therapy for mastitis treatment in goats.

Impacts
What was accomplished under these goals? Requisitioned supplies for project, set up laboratory environment Identified and purchasedcaprine subjects (Goats) for use in pilot project. Breeding females with males. Identifying graduate students to work on project in fulfillment of Thesis research projects Trained 4 graduate students to do the following: Collect fresh milk samples from 39 goats in milking parlor Appropriately preserve & transport milk samples for microbiological testing Testing milk using California Mastitis Testing methodology Compiles and report data collected from testing Performed testing approximately 7 times overseveral months to assess & track sub-clinical and clinical mastitis in identified goats. Trained students to recognize leukocyte types in prepared blood smears for future research. Trained graduate students on microbiological plating techniques and preservation of bacterial cultures in order to identify bacterial genus & species (of collected bacteria from milk) Graduate students have been trained in husbandry of small ruminents. All samples that tested positive for mastitis were cultured and preserved for further biochemical tests Milk samples from treated goats were also cultured to check for antibiotic resistance before, during and after treatments. Pastures were fenced in. Goats were purchased, sorted and treated for a variety of ailments. ?Graduate students became familiar with common goat illnesses, biosecurity, and evaluating goats Multiple abstracts and posters have been presented

Publications

Progress 03/22/16 to 09/30/16

Outputs
Target Audience:The target audience consists of small ruminant dairy farmers and those that consume their products along with veterinary and scientific professionals that assist and advise them. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided numerous opportunities for students to increase their laboratory and microbiological skill sets through training in the following areas: Collect fresh milk samples from goats via manual expression Appropriately preserve & transport milk samples to lab for testing Test milk using California Mastitis Testing methodology and determine results Record and report data Trained students to recognize leukocyte types in prepared blood smears. Trained graduate students on microbiological plating techniques and preservation to identify bacterial germs & species of collected bacteria from milk. How have the results been disseminated to communities of interest?The results of pilot testing (conducted over the summer) were reported to the PI (Dr. George McCommon). These results show a preliminary need to continue testing while administering novel treatments as stated in original grant proposal. What do you plan to do during the next reporting period to accomplish the goals?Based on pilot study the following will be accomplished during next the reporting period. Differentiate groups of goats for testing. (i.e. subclinical; clinical mastitis) Begin administrating novel treatment options to infected animals. Continue testing milk via California Mastitis Test. Continue identifying microbes. Continue data collection.

Impacts
What was accomplished under these goals? Requisitioned supplies for project, set up laboratory environment Identifying caprine subjects (Goats) for use in pilot project Identifying graduate students to work on project in fulfillment of Thesis research projects Trained 4 graduate students to do the following: Collect fresh milk samples from 39 goats in milking parlor Appropriately preserve & transport milk samples for microbiological testing Testing milk using California Mastitis Testing methodology Compiles and report data collected from testing Performed testing approximately 7 times over 2 months to assess & track sub-clinical and clinical mastitis in identified goats. Trained students to recognize leukocyte types in prepared blood smears for future research. Trained graduate students on microbiological plating techniques and preservation of bacterial cultures in order to identify bacterial genus & species (of collected bacteria from milk) Bacterial samples have been preserved to allow continued testing during the non-milking season. Samples will be tested to determine antibiotic susceptability and for residues.

Publications