Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
Vet Population Medicine & Diagnostic Science
Non Technical Summary
Mastitis is a highly prevalent disease in dairy cows and the number one cause of economic losses to the dairy industry worldwide. Economic losses are a consequence of reduced milk production, discarded milk, lower conception rates, premature culling, and treatment costs (Grohn et al., 2005, Bar et al., 2008, Hertl et al., 2010). The well-documented reduction in milk production resulting from mastitis is estimated at approximately 15% of the milk production potential of the affected cow but highly dependent on the type of pathogen (Seegers et al., 2003, Bar et al., 2007, Schukken et al., 2009). Clinical mastitis is also a serious animal welfare issue as it is associated with pain, reduced well-being, and behavioral changes (Medrano-Galarza et al., 2012).Current guidelines do not recommend the use of intramammary antibiotics for cows diagnosed with Gram-negative mastitis, and the treatment of coliform organisms with intramammary antibiotics is typically considered to be of only limited value (Erskine et al., 2003, Suojala et al., 2013). In a recent randomized controlled field trial, our research team showed no improvement on clinical and bacterial cures and microbiome dynamics regarding treatment of mild and moderate coliform mastitis with a third generation cephalosporin in comparison to a no-treatment control (Ganda et al., 2016).Treatment of Klebsiella spp. mastitis is a complicated issue due to increasing resistance toward antibiotics, production of endotoxins that induce septic shock (Kim et al., 2002), bacterium capsular polysaccharide (CPS) that cause immuno-paralysis by inhibiting phagocytosis (Podschun and Ullmann, 1998), and resistance to complement-mediated phagocytosis (Merino et al., 1992). Therefore, immunization appears to provide a powerful tool to control Klebsiella spp intramammary infections.Therefore, our overarching objective is to develop strategies to control the incidence of Klebsiella mastitis. Our specific aim to evaluate the effect of two available vaccines, a commercial vaccine and a novel subunit vaccine, on the prevention of K. pneumonia mastitis in a large clinical trial.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
The multifactorial etiology of mastitis presents a major challenge for disease prevention and treatment of affected animals. Coliform organisms are also among the highest incidence organisms at farms with otherwise excellent udder health management. Implementation of programs for mastitis control has reduced the prevalence of important contagious pathogens, and currently approximately 40% of all clinical mastitis cases are associated with opportunistic Gram-negative bacteria such as Klebsiella spp., Escherichia coli, Pseudomonas spp., and Pasteurella spp. (Erskine et al., 1988, Olde Riekerink et al., 2008, Botrel et al., 2010). Among the major mastitis pathogens, coliform organisms such as Klebsiella spp. and E. coli are causing the most severe clinical cases with greater milk loss, and an average cost of at least U$ 221.03 per case (Cha et al., 2011).Studies have shown differences in the pathogenicity of Klebsiella spp. as a mastitis pathogen and other Gram negatives (Rajala-Schultz and Grohn, 2001, Bannerman, 2009). Klebsiella spp. causes longer intramammary infections than E. coli (Todhunter et al., 1991), more severe clinical episodes than Serratia spp and E. coli (Erskine et al., 2002, Roberson et al., 2004), and greater milk production loss and risk of culling than IMI's caused by E. coli (Grohn et al., 2004, Grohn et al., 2005).Current guidelines do not recommend the use of intramammary antibiotics for cows diagnosed with Gram-negative mastitis, and the treatment of coliform organisms with intramammary antibiotics is typically considered to be of only limited value (Erskine et al., 2003, Suojala et al., 2013). In a recent randomized controlled field trial, our research team showed no improvement on clinical and bacterial cures and microbiome dynamics regarding treatment of mild and moderate coliform mastitis with a third generation cephalosporin in comparison to a no-treatment control (Ganda et al., 2016).Treatment of Klebsiella spp. mastitis is a complicated issue due to increasing resistance toward antibiotics, production of endotoxins that induce septic shock (Kim et al., 2002), bacterium capsular polysaccharide (CPS) that cause immuno-paralysis by inhibiting phagocytosis (Podschun and Ullmann, 1998), and resistance to complement-mediated phagocytosis (Merino et al., 1992). Therefore, immunization appears to provide a powerful tool to control Klebsiella spp intramammary infections.Therefore, our overarching objective is to develop strategies to control the incidence of Klebsiella mastitis. Our specific aim to evaluate the effect of two available vaccines, a commercial vaccine and a novel subunit vaccine, on the prevention of K. pneumonia mastitis in a large clinical trial.
Project Methods
Clinical trialProduction and purification of recombinant yidRFermentation of E. coli BL21 containing the yidR plasmid: Culture medium and fermentation parameters: Fermentation will be performed using a Bioflo® & Celligen® 310 Fermenter/Bioreactor unit (New Brunswick Scientific, NJ, USA) using a fed-batch fermentation method. Bioengineered E. coli BL21, containing the yidR plasmid, will be cultured in chemically defined medium according with Li and Sha (2015). The composition of the initial fermentation medium will be; 450 mL 10× phosphate/citric acid buffer (133 g/L KH2PO4,40 g/L (NH4)2HPO4, 17 g/L citric acid), 4.05 L deionized (DI) water. The vessel containing the initial medium will be autoclaved for 90 min. After the vessel is removed from the autoclave the medium will cooled to room temperature and the following ingredients will be added; 45 mL of 240 g/L MgSO4, 1.02 mL of 20 g/L thiamine, 45 mL of 100× trace element solution, and 66 mL of 70% glucose solution. The concentrated trace element solution will be prepared, and filter sterilized as follows; 10 g/L iron (III) citrate, 0.25 g/L CoCl2·6H2O, 1.5 g/L MnCl2·4H2O, 0.15 g/L CuCl2·6H2O, 0.3 g/L H3BO3, 0.25 g/L Na2MoO4·2H2O, 1.3 g/L zinc acetate·2H2O, 0.84 g/L EDTA. Finally, kanamycin will be added to the initial medium to a final concentration of 30µg/ml. A concentrated feeding medium will be prepared in a 2L glass bottle; 197.1mL of 240 g/L MgSO4, 7.47 mL of 20 g/L thiamine solution, 67.5 mL of 100× trace element solution, and 70% glucose solution will be added to a final volume of 2 L.The fermenter system is equipped with calibrated probes for pH, temperature, foam level, and dissolved oxygen (DO). A solution of 30% ammonium hydroxide will be automatically pumped (Pump 1) into the vessel to maintain the pH at 6.8. A 20% antifoam solution (Antifoam B Emulsion, aqueous-silicone emulsion, Sigma Life Science) will be automatically pumped (Pump 2) into the vessel as needed. Pump 3 will be assigned to the feeding medium. The feeding strategy will maximized the bacterial growth without accumulation of glucose in the medium and maintaining DO levels around 25%. To maintain DO levels around 25%, a calibrated DO probe will be used, medical grade Oxygen sparging and agitation speed (100-1200 rpm) varied as needed.Protein purification:The soluble fraction of the supernatant will be used for purification of brIL-8 using a custom packed 60 ml column of TALON® Metal Affinity Resin (Tanaka Bio USA Inc., CA, USA). Automated multi-dimensional purifications of the his-tagged yidR protein will be performed using the ÄKTA-pure instrument (GE Healthcare Life Sciences) controlled by UNICORN 7 software.Clinical Trial DesignThis study will be conducted to evaluate the efficacy of a commercially available bacterin vaccine (KlebVaxTM; USDA registered and commercially available in the USA), a recombinant subunit vaccine containing the protein yidR, against placebo treated cows. A total of 3,000 cows will be blocked by parity group (nulliparous, primiparous, and multiparous). Within each block, cows will be assigned randomly to one of 3 treatment groups at around 240 days of gestation: placebo (1,000 cows), KlebVaxTM (1,000 cows), and yidr (1,000 cows). Cows will receive two vaccinations: 240 ± 3 days of pregnancy and 270 ± 3 days of pregnancy. Control cows will receive a placebo and all research personnel will be blinded to vaccination treatment. Vaccine and placebo preparations will be stored in identical containers and labeled "A", "B", and "C". Their identity will not be known to the persons administering the treatment, examining the cows, or collecting clinical data, or to farm personnel. This code will be broken at the end of the experiment; after all data have been analyzed. The total sample size of 3,000 animals will allow us to detect a reduction 40% of mastitis caused by KP considering a type-1 error of 0.025 (Dunnet multiple comparison adjusted), a study power of 0.8, two-sided statistical test, and a baseline incidence of 10% of KP mastitis (current KP incidence at the study farm).The following six outcome variables will be analyzed statistically: clinical mastitis incidence, sub-clinical mastitis incidence, Klebsiella spp. mastitis incidence, severity of clinical Klebsiella spp. mastitis cases, percentage of dead/culled animals by the end of follow-up period, and average daily milk production. Table 1 provides information regarding the treatment groups.Table 1 Treatment groupsTreatment group# of animalsVaccineIdentificationDoseRouteAdministration schedule11,000Sterile saline5 mLSQDays 240 ±3, and 270 ±3 DCC21,000KlebVaxTM5 mLSQDays 240 ±3, and 270 ±3 DCC31,000YIDR1mg/dose5 mLSQDays 240 ±3, and 270 ±3 DCCVaccinationAdministration of the Biological ProductsEach animal in the study will be vaccinated two times: the first vaccination will be on 240 ±3 and the second vaccination on 270 ±3 DCC. According to randomized grouping, the animals will receive 5.0 mL of the respective vaccine by the SQ route each time.Post Vaccination ProceduresBlood SamplesBlood samples of 10-15 mL will be collected from all heifers at 30 and 60 DIM. Blood will be collected in a SST.To evaluate the effect of vaccination on antigen-specific IgG response, ELISA will be used. Blood samples will be collected from all study cows at 240 ± 3 days of gestation, 270 ± 3 days of gestation, and 30 ± 3 DIM from coccygeal vein/artery using a Vacutainer tube without anticoagulant and a 20 gauge x 2.54 cm Vacutainer needle (Becton, Dickinson and Company, Franklin Lakes, NJ). After collection, all blood samples will be transported to the laboratory on ice and spun in a centrifuge at 2,000 x g for 15 min at 4oC, and the serum will be harvested and frozen at - 80o C.Post Mastitis Diagnosis ProceduresMastitis management on farmThe farm currently culture submit milk samples from clinical mastitis cases to the Cornell QMPS laboratory, results are automatically downloaded into the farms DC-305 software daily. All intramammary treatment is dependent upon culture results; the farm only administers intramammary treatment to cows with Streptococcus spp. mastitis. Streptococcus spp. cows get an identification red leg band to indicate that they have a milk withhold and are treated for 5 consecutive days using intramammary infusion of ceftiofur (Spectramast LC, Zoetis). If a cow is culture negative or any other species besides Streptococcus, Staphylococcus aureus, and Mycoplasma bovis, she is monitored for health and given a green leg band to ensure farm personnel keep an eye on her, once milk is normal she will return to her pen with her green leg band. If a cow with a green leg band comes up clinical within 14 days of her last culture, she is sent back to the hospital pen to recover further; if she is beyond 14 days from her last culture she will be cultured again. All on farm cultures, supportive therapy (fluids, systemic antibiotic, etc.), days in the hospital and health events are recorded into DC305.Statistical analysisAnalysis of data collected will be performed by the study investigator's group. To assess the effect of treatment group on the probability of the outcome variables, a mixed logistic regression model will be fitted to the data for each one of the disease outcome variables. Data will be analyzed by multivariate mixed logistic regression. Additionally, the effect of treatment group on milk production, linear scores, and levels of antibody to vaccines antigens will be assessed by repeated measures ANOVA.