MASTITIS RESISTANCE TO ENHANCE DAIRY FOOD SAFETY

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0219301
• Project No.: NYC-478838
• Multistate No.: NE-1028
• Project Start Date: Oct 1, 2009
• Project End Date: Sep 30, 2012

Project Director
Bicalho, RO.

Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853

Performing Department
Vet Population Medicine & Diagnostic Science

Non Technical Summary
In the United States, the dairy industry contributes in excess of 65 billion dollars per year to the national economy. Cash receipts from the sale of New York milk during 2004 totaled 1.95 billion dollars (USDA, NASS, NYASS, No. 973-5-05). Dairy production is the largest industry in New York State, which is the 3rd highest producer of milk nationally. The single most costly disease of dairy cattle is bovine mastitis, affecting virtually every dairy farm and approximately 38% of all dairy cows. Recent studies indicate that antibiotic resistance is an important risk factor for a failure to cure after antibiotic therapy. More importantly, antibiotic resistance created with the use of antibiotics for the treatment and prevention of cattle mastitis could be considered a public health concern. In the United States alone, 2 million people per year are diagnosed with hospital acquired infections, of which 90,000 die as a result of their infection. The nature of the antibiotic usage in the food animal industry favors the appearance of antibiotic resistance due to subtherapeutic dosage, mass treatment and long term administration. Bacteriophages are viruses that infect bacteria; they are obligate intracellular parasites and lack their own metabolism (cannot survive outside bacteria). Phages are extremely host specific, able only to infect specific species or even strains of bacteria. The concept of combating pathogens by means of phages is obvious, and was proposed shortly after the discovery of phages approximately 90 years ago. Bacteriophages could be considered the perfect antimicrobial agents; they are highly specific to few bacterial species; they are non-toxic to mammals; and they grow in exponential scale while precisely killing pathogenic bacteria. Unfortunately, the discovery of antibiotics basically eliminated research on phage therapy. Furthermore, the pharmaceutical companies are not attracted by the potential use of phage therapy due to the difficulties associated with patenting the rights of phage therapy use. The objectives of this project are therefore to 1) isolate lytic environmental bacteriophages from dairy farms that are specific and efficacious against the most common mastitis bacterial pathogens, 2) evaluate the in vitro antibacterial efficacy of all bacteriophage isolates, 3) assemble a cocktail of highly effective bacteriophages, and evaluate its efficacy as a treatment of clinical mastitis of dairy cattle. At our laboratory at Cornell University we have already isolated several bacteriophages from environmental samples of commercial dairy farms. The in vitro antimicrobial efficacy of our bacteriophages isolates was assessed and found to be comparable with the following antibiotics: florfenicol, tetracycline, ceftiofur, ampicillin, spectinomycin, and streptomycin. By the end of this project we expect to have developed and tested a novel organic antimicrobial treatment that will be effective against bacterial mastitis of dairy cattle.

Animal Health Component

70%

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3410	1100	30%
311	3410	1101	70%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3410 - Dairy cattle, live animal;

Field Of Science
1101 - Virology; 1100 - Bacteriology;

Keywords

dairy cows

mastitis

bacteriophage therapy

Goals / Objectives
Our long term goals are to harness the antibacterial power of environmental bacteriophages and use it to treat bovine bacterial diseases. Replacing antibiotic with bacteriophage therapy would eliminate the contribution of the food animal industry to the rise of antibiotic resistance. Our specific objectives are: 1. To isolate lytic environmental bacteriophages from dairy farms that are specific and efficacious against the following bacteria: Streptococcus agalactiae, Streptococcus spp., Staphylococcus aureus, Staphylococcus spp., Escherichia coli, Klebsiella spp., and Pseudomonas spp. which are responsible 80% of all culture positive mastitis in New York state. 2. To evaluate the in vitro efficacy of all phage isolates and to assemble a cocktail of highly effective phages. 3. To evaluate the efficacy of the treatment of clinical mastitis with the phage cocktail as compared with positive controls (intramamary antibiotic of choice).

Project Methods
1) To assemble a cocktail of environmental phages and perform in vitro antimicrobial efficacy evaluation the following will be performed: a. Phages will be isolated from environmental samples collected from commercial dairy farms; we will use Streptococcus agalactiae, Streptococcus spp., Staphylococcus aureus, Staphylococcus spp., Escherichia coli, Klebsiella spp., and Pseudomonas spp. isolated from the milk of cows diagnosed with clinical mastitis as target hosts for phage isolation. b. The antimicrobial efficacy of all isolated phages will be tested by assessing the effect of phage inoculation at several different concentrations on the growth curve of the host bacteria. c. We will select the 2 most efficacious phages from each host bacteria for the final phage cocktail. Electron microscopy and genetic characterization will be performed in all 14 selected bacteriophages. d. Evaluation and optimization of different phage lysate purification methods such as cesium chloride density centrifugation and size exclusion chromatography. 2) To evaluate the efficacy of the treatment of clinical mastitis with the phage cocktail as compared with positive controls the following will be performed: a. A prospective, double-blinded clinical trial will be conducted. 160 lactating dairy cows from a large commercial dairy farm diagnosed with clinical mastitis will be randomly assigned to one of two treatments: control (antibiotic of choice) or treatment (intramammary administration of purified phage cocktail). Milk cultures will be repeatedly performed to assess bacteriologic cure.

Progress 10/01/09 to 09/30/12

Outputs
OUTPUTS: Dairy cow mastitis is arguably the most important disease for the dairy industry worldwide, causing economic losses due to reduced milk production, discarded milk, premature culling, and antibiotic usage. Clinical mastitis is also a serious animal welfare issue as it is associated with pain and reduced well-being of the affected animals. Our initial objective was to develop a multivalent bacteriophage cocktail to treat bacterial mastitis caused by the most prevalent gram-positive and gram-negative pathogens. During the last three years we have generated an incredible amount of information, resulting in 10 scientific manuscripts published in several reputable peer-reviewed journals. We have disseminated the information generated by the work funded by this grant mainly by publishing the research results on peer-reviewed journals and by presenting the results in scientific meeting as well as producers meetings such as: Dairy Cattle Reproduction Council, Sacramento, California. 2012 Cornell-China Dairy Institute, Beijing, China. 2012 Alta Genetics Dairy Cattle Management Course. Araras, Uberaba, Belo Horizonte, Brazil. 2012. XXIV Encontro dos Medicos Veterinarios, Goiania, Brazil. 2012. Uruguayan Buiatric Meeting, Paysandu, Uruguay. 2012. World Buiatric Meeting, Lisbon, Portugal. 2012. 4 Dairy Cattle Workshop. Ribeirao Preto, Sao Paulo, Brazil. 2012. Mid-South Ruminant Nutrition Conference, Dallas, Texas. 2012 Cornell Nutrition Conference, Syracuse, New York. 2011. Central Veterinary Conference, San Diego, California. 2011. Conferencia Internacional sobre Ganado Lechero, Guadalajara, Mexico. 2011. The Second Large Herd Dairy Conference, Beijing, China. 2011 XVI International Congress of Bovine Medicine, Avila, Spain. 2011. Western Dairy Management Conference, John Ascuaga's Nugget, Reno, Nevada. 2011. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
After large scale production and purification of our selected bacteriophage cocktail we performed a small safety trial where 6 lactating dairy cows were inoculated with a cocktail containing 10,000,000 phage particles intramammary using a teat cannula. It was observed that the inoculated cows developed a mild inflammatory reaction to the phage cocktail and a significant increase in the somatic cell count of the milk indicated subclinical inflammation of the mammary gland. This inflammatory reaction was recently reported by another group "The effects of phage intramammary infusion on the bovine mammary gland were also studied. In healthy lactating cows, a single infusion of either filter-sterilized broth lysate or a CsCl gradient-purified phage preparation elicited a large increase in the milk somatic cell count." in a report published on the Antimicrobial Agent and Chemotherapy journal. Therefore, we decided that the use of intramammary bacteriophage therapy a large scale trial using client owned dairy cows would be of great risk and therefore we opted to conduct a trial evaluated the effect of intrauterine phage therapy for the prevention of metritis (Machado V. S, M. L. S. Bicalho, R. V. Pereira, L. S. Caixeta, J. H. J. Bittar, G. Oikonomou, R. O. Gilbert, and R. C. Bicalho. 2011. The effect of intrauterine administration of mannose and bacteriophage, and intrauterine presence of Escherichia coli and Arcanobacterium pyogenes on uterine health of dairy cows. J. Dairy Sci. 95:3100-3109.) We also advanced our understanding of bovine mastitis by investigating the microbial diversity of mastitic milk using novel DNA sequencing technologies. This paper was recently published on the PLosOne journal (Oikonomou G., V. S. Machado, C. Santisteban, Y. H. Schukken, and R. C. Bicalho. 2012. Microbial diversity of bovine mastitic milk as described by pyrosequencing of metagenomic 16s rDNA. PLoS ONE 7(10): e47671.doi:10.1371/journal.pone.0047671). One hundred and thirty-six milk samples were collected from cows showing signs of mastitis and used for microbiological culture. Additionally, 20 milk samples were collected from healthy quarters. The mastitis pathogens identified by culture were generally among the most frequent organisms detected by pyrosequencing, and in some cases (Escherichia coli, Klebsiella spp. and Streptococcus uberis mastitis) they were the single most prevalent microorganism. Trueperella pyogenes sequences were the second most prevalent sequences in mastitis cases diagnosed as Trueperella pyogenes by culture, Streptococcus dysgalactiae sequences were the second most prevalent sequences in mastitis cases diagnosed as Streptococcus dysgalactiae by culture, and Staphyloccocus aureus sequences were the third most prevalent in mastitis cases diagnosed as Staphylococcus aureus by culture. In samples that were aerobic culture negative, pyrosequencing identified DNA of bacteria that are known to cause mastitis, DNA of bacteria that are known pathogens but have so far not been associated with mastitis, and DNA of bacteria that are currently not known to be pathogens.

Publications

• Bicalho R. C., T. M. A. Santos, R. O. Gilbert, L. S. Caixeta, L. M. Teixeira, M. L. Bicalho, and V. S. Machado. 2010. Susceptibility of Escherichia coli isolated from uterus of postpartum dairy cows to antibiotic and environmental bacteriophages, PART I: Isolation and lytic activity quantification of bacteriophages. Journal Dairy Sci.93:93-104.
• Santos T. M. A., Bicalho R. C., R. O. Gilbert, L. S. Caixeta, M. L. Bicalho, and V. S. Machado. 2010. Susceptibility of Escherichia coli isolated from uterus of postpartum dairy cows to antibiotic and environmental bacteriophages, PART II: In vitro antimicrobial activity evaluation of a bacteriophage cocktail and several antibiotics. J. Dairy Sci. 93:105-114.
• Santos T. M. A., R. O. Gilbert, L. S. Caixeta, M. L. Bicalho, V. S. Machado, and Bicalho R. C.,. 2010. Antimicrobial resistance and presence of virulence factor genes in Arcanobacterium pyogenes isolated from the uterus of postpartum dairy cows. Vet. Microbiology. 145(1-2): 84 - 89.
• Bicalho R. C., V. S. Machado, M. L. S. Bicalho, A. G. V. Teixeira, L. S. Caixeta, and R. V. V. Pereira. Molecular and epidemiological characterization of bovine Intrauterine Pathogenic Escherichia coli. 2010. Journal Dairy Sci. 93(12):5818-5830.
• Oikonomou G., V. S. Machado, C. Santisteban, Y. H. Schukken, and R. C. Bicalho. 2012. Microbial diversity of bovine mastitic milk as described by pyrosequencing of metagenomic 16s rDNA. PLoS ONE 7(10): e47671. doi:10.1371/journal.pone.0047671.
• Santos T.M.A., Bicalho R.C. 2012. Diversity and Succession of Bacterial Communities in the Uterine Fluid of Postpartum Metritic, Endometritic and Healthy Dairy Cows. PLoS ONE 7(12): e53048. doi:10.1371/journal.pone.0053048.

Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: Our long term goals are to harness the antibacterial power of environmental bacteriophages and use it to treat bovine bacterial diseases. Replacing antibiotic with bacteriophage therapy would eliminate the contribution of the food animal industry to the rise of antibiotic resistance. Our specific objectives are: 1. To isolate lytic environmental bacteriophages from manure lagoons, milk bulk tanks, and other environmental samples that are specific and efficacious against the following bacteria Streptococcus agalactiae, Streptococcus spp., Staphylococcus aureus, Staphylococcus spp., Escherichia coli, Klebsiella spp., and Pseudomonas spp. which are responsible 80% of all culture positive mastitis in New York state. 2. To evaluate the in vitro efficacy of all phage isolates and to assemble a cocktail of highly effective phages. 3. To evaluate the efficacy of the treatment of clinical mastitis with the phage cocktail as compared with positive controls (intramamary antibiotic of choice). Our objective is to develop a multivalent bacteriophage cocktail that will be able to treat indiscriminately cows affected with clinical mastitis. We have so far focus in the isolation and characterization of pages active against E. coli, Klebsiella, and Pseudomonas aeruginosa. For the year of 2011 we have isolated and characterized 50 novel bacteriophages of which we have completely sequenced the genomes of 35 bacteriophages with very promising therapeutic potential. Genome sequencing of lytic bacteriophages is important to rule out the presence of bacterial antibiotic resistant and virulence factor genes that could be present in the phage genome. We have selected the most promising phages and have successfully sequenced their genomes and proved that those phages don't have harmful genes. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Our objective is to develop a multivalent bacteriophage cocktail that will be able to treat indiscriminately cows affected with clinical mastitis. We have so far focus in the isolation and characterization of pages active against E. coli, Klebsiella, and Pseudomonas aeruginosa. For the year of 2011 we have isolated and characterized 50 novel bacteriophages of which we have completely sequenced the genomes of 35 bacteriophages with very promising therapeutic potential. Genome sequencing of lytic bacteriophages is important to rule out the presence of bacterial antibiotic resistant and virulence factor genes that could be present in the phage genome. We have selected the most promising phages and have successfully sequenced their genomes and proved that those phages don't have harmful genes. We have performed very extensive laboratory testing to evaluate the lytic spectrum of over 60 phages against 300 genetically distinct bacterial isolates. The results of extensive testing have allowed us to create a combination of 10 distinct phages to be used in conjunction as a bacteriophage cocktail against E. coli mastitis. We are planning a clinical trial for the beginning of 2012 that will evaluate the use of the phage cocktail in the treatment of clinical gram-negative E. coli mastitis. Recently, our cocktail was tested for safety and efficacy on a clinical trial with a total of 50 calves. A total of 25 calves were orally treated with a cocktail containing 10,000,000 phage particles per calf per day. This trial demonstrated that the use of bacteriophages orally was safe, effectively decreased E. coli counts in the feces, and decreased the incidence of neonatal diarrhea by half. We are now ready to take the final step and evaluate the phage cocktail against gram-negative mastitis.

Publications

• Santos T. M. A. and Bicalho R. C. Complete genome sequence of ECO1230-10, an Escherichia coli bacteriophage with phage therapy potential. 2011. Veterinary Microbiology. 148(2011):267-275.
• Bicalho M. L. S., T. M. A. Santos, D. V. Nydam, V. S. Machado, and R. C. Bicalho. 2011. Evaluation of bacteriophages oral administration in neonatal calves: phage survivability, impact on fecal Escherichia coli and calf health. Livestock Science Journal. Accepted.
• Machado V. S, M. L. S. Bicalho, R. V. Pereira, L. S. Caixeta, J. H. J. Bittar, G. Oikonomou, R. O. Gilbert, and R. C. Bicalho. 2011. The effect of intrauterine administration of mannose and bacteriophage, and intrauterine presence of Escherichia coli and Arcanobacterium pyogenes on uterine health of dairy cows. J. Dairy Sci. Accepted.

Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: Bacteriophages can be considered the perfect antimicrobial agents; they are highly specific to few bacterial species or even strains; they are non-toxic to mammals; they grow in exponential scale while precisely killing targeted bacteria. Several peer reviewed publication evidence the efficacy of phage therapy in controlled animal models with induced disease. However, there is a complete scarcity of peer reviewed literature evaluating the efficacy of phage therapy in the control of natural infections. Our research will be one of a few, or perhaps the only one, that will evaluate the efficacy of phage therapy in the treatment of naturally occurring disease. If we are successful our research will serve as a platform for developing phage therapies for other animal and human bacterial diseases. Our long term goals are to harness the antibacterial power of environmental bacteriophages and use it to treat bovine bacterial diseases. Replacing antibiotic with bacteriophage therapy would eliminate the contribution of the food animal industry to the rise of antibiotic resistance. Our specific objectives are To isolate lytic environmental bacteriophages from manure lagoons, milk bulk tanks, and other environmental samples that are specific and efficacious against the following bacteria Streptococcus agalactiae, Streptococcus spp., Staphylococcus aureus, Staphylococcus spp., Escherichia coli, Klebsiella spp., and Pseudomonas spp. which are responsible 80% of all culture positive mastitis in New York state. To evaluate the in vitro efficacy of all phage isolates and to assemble a cocktail of highly effective phages. To evaluate the efficacy of the treatment of clinical mastitis with the phage cocktail as compared with positive controls (intramamary antibiotic of choice). In the last year (2010) we have made incredible progress towards our goal of assembling and evaluating a multivalent intrammary phage therapy against bacterial mastitis. Thus far we have isolated, characterized, and tested a great collection of Pseudomonas phages; this collection of phages is incredibly effective against a large diversity of multidrug resistant Pseudomonas aeruginosa isolates. Because, of the excellent spectrum of activity that was observed for 4 genetically distinct bacteriophages , 100% coverage against 100 isolates tested, we have decided to stop the isolation of Pseudomonas bacteriophages. We are have made enormous progress in the isolation and characterization of E. coli and Klebsiella bacteriophages. Currently we have an impressive collection of 65 bacteriophages active against one or both of those bacteria. E. coli is an enormous and incredibly diverse species of bacteria and as a consequence, it has been very hard to find a combination of bacteriophages that has acceptable coverage >98%. We believe that we have enough bacteriophages isolated and partially characterized to start spot and microdilution assays which will allow us to build an ideal cocktail. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our objective is to develop a multivalent bacteriophage cocktail that will be able to treat indiscriminately cows affected with clinical mastitis. We have so far focus in the isolation and characterization of pages active against E. coli, Klebsiella, and Pseudomonas aeruginosa. In the last year (2010) we have made incredible progress towards our goal of assembling and evaluating a multivalent intrammary phage therapy against bacterial mastitis. Thus far we have isolated, characterized, and tested a great collection of Pseudomonas phages; this collection of phages is incredibly effective against a large diversity of multidrug resistant Pseudomonas aeruginosa isolates. Because, of the excellent spectrum of activity that was observed for 4 genetically distinct bacteriophages , 100% coverage against 100 isolates tested, we have decided to stop the isolation of Pseudomonas bacteriophages. We are have made enormous progress in the isolation and characterization of E. coli and Klebsiella bacteriophages. Currently we have an impressive collection of 65 bacteriophages active against one or both of those bacteria. E. coli is an enormous and incredibly diverse species of bacteria and as a consequence, it has been very hard to find a combination of bacteriophages that has acceptable coverage >98%. We believe that we have enough bacteriophages isolated and partially characterized to start spot and microdilution assays which will allow us to build an ideal cocktail. Additionally, we have assembled a large collection of Streptococcus agalactiae, Streptococcus spp., and Staphylococcus aureus bacteria which will be used as hosts for the isolation of bacteriophages. These bacteria will be the focus of our work from now until the phage cocktail is finished.

Publications

• Santos T. M. A., Eric C. Ledbetter, L. S. Caixeta, M. L. Bicalho, and R. C. Bicalho. 2011. Isolation and characterization of two bacteriophages with strong in vitro antimicrobial activity against a broad range of multidrug resistant Pseudomonas aeruginosa. AJVR. IN PRESS.
• Santos T. M. A. and Bicalho R. C. Complete genome sequence of ECO1230-10, an Escherichia coli bacteriophage with phage therapy potential. 2010. Veterinary Microbiology. http://dx.doi.org/10.1016/j.vetmic.2010.08.034.