Northern NY Agricultural Development Program 2010 Project Report
Project Leader
Dr Gary Bennett, DVM, QMPS Canton Laboratory Director
Collaborator(s)
Quality Milk Production Services (QMPS) at Cornell University:
Brenda Werner, Ruth Zadoks, Paolo Moroni and Ynte Schukken
Cooperating Producers
Mapleview Farm, St Lawrence County
WH Miner Institute, Clinton County
Bilow Farms, Franklin County
Background
Mastitis caused by gram-negative pathogens is a major concern on many well-managed dairy farms in North America. The main gram-negative pathogens that cause mastitis are Escherichia coli and Klebsiella spp. Cows with Klebsiella spp. Mastitis are more likely to die or to be culled than are cows with other types of mastitis. Klebsiella spp. mastitis causes a considerable and often sustained decrease in milk production, with average losses of 7.6 kg/d shortly after infection and 5 kg/d in subsequent months. Klebsiella spp. Prevention of exposure is the main strategy for control of Klebsiella spp. Mastitis. We have previously shown in our NNY research that antimicrobial treatment of Klebsiella cases is successful under precisely defined treatment conditions. In previous studies we have shown that Klebsiella mastitis is a large problem in NNY dairy farms. In this study our aim was to study the behavior of Klebsiella spp in the environment of the cow and farm and to better understand the genetic differences between clinical mastitis and environmental K. pneumoniae strains.
Methods
We collected a total of 305 samples from 3 dairy farms in Northern New York. Samples included clinical mastitis cases, soil, feed crops, feed, water, rumen content, feces, bedding, and manure from alleyways and holding pens. Klebisella isolates from these samples were identified using rpoB sequencing methods and stored in freezer for future analysis. Suppressive subtractive hybridization was employed on three matched pairs of Clinical mastitis and environmental strains. This method is designed to identify the genetic differences between strains of bacteria.
Results
Klebsiella spp. were detected in 100% of rumen samples, 89% of water samples, and approximately 64% of soil, feces, bedding, alleyway, and holding pen samples. Detection of Klebsiella spp. in feed crops and feed was less common. Genotypic identification of species using rpoB sequence data showed that Klebsiella pneumoniae was the most common species in rumen content, feces, and alleyways, whereas Klebsiella oxytoca,Klebsiella variicola, and Raoultella planticola were the most frequent species among isolates from soil and feed crops. Random amplified polymorphic DNA-based strain typing showed heterogeneity of Klebsiella spp. in rumen content and feces, with a median of 4 strains per 5 isolates.
Observational and bacteriological data support the existence of an oro-fecal transmission cycle, which is primarily maintained through direct contact with fecal contamination or through ingestion of contaminated drinking water.
Fecal shedding of Klebsiella spp. contributes to pathogen loads in the environment, including bedding, alleyways, and holding pens. Hygiene of alleyways and holding pens is an important component of Klebsiella control on dairy farms.
We identified 13 genes uniquely present in the clinical strains. Sequences were tested for homology and screened against a larger pool of 18 clinical mastitis and 11 environmental strains collected from the same farms. Seven sequences were found in both clinical and environmental strains, while 6 were found solely amongst clinical strains. Sequences coding for parB, parA, arsD, and sulfate permease were found with similar frequency among clinical and environmental strains.
Conclusions/Outcomes/Impacts
Klebsiella spp. are commonly present in soil, water troughs, rumen content, feces, bedding, alleyways, and holding pens on dairy farms. Predominant species in soil and plant samples differ from those in the gastrointestinal tract, water, bedding, alleyways, and holding pens, and the presence of Klebsiella spp. in plants appears to result from external contamination rather than endophytic colonization of feed crops. Oro-fecal transmission due to fecal contamination of animals, feed, and water appears more important for maintenance of Klebsiella spp. in the farm ecosystem than does introduction from external sources such as fresh bedding or feed. Fecal contamination of stalls, alleyways, and holding pens may result in exposure of teat ends to Klebsiella. Further studies would be needed to identify methods to reduce oral ingestion and fecal shedding of Klebsiella spp. Meanwhile, hygiene of alleyways and holding pens should be recognized as an important component of prevention of mastitis due to Klebsiella spp.
These results suggest that genes uniquely associated with Klebsiella pneumoniae clinical mastitis strains may contribute to adaptation to the bovine mammary gland and to virulence. These genes would be particularly useful as vaccine or treatment targets.
Outreach
Website: http://ahdc.vet.cornell.edu/sects/QMPS/QMPSNYKlebsiellamastitisbrochure.pdf
Publications for the scientific community:
Sources of Klebsiella and Raoultella species on dairy farms: be careful where you walk. Zadoks RN, Griffiths HM, Munoz MA, Ahlstrom C, Bennett GJ, Thomas E, Schukken YH. J Dairy Sci. 2011 Feb;94(2):1045-51.
Next steps
This study utilized suppressive subtractive hybridization to isolate gene sequences unique to three clinical mastitis strains. In particular, one gene sequence, Ent638_4324 was subsequently found to be present amongst a large set of clinical mastitis strains, and absent amongst a pool of environmental strains not associated with mastitis. Distribution and sequencing and homology analysis of this gene suggests that further investigation and characterization of its contribution to host adaptation and virulence is warranted, particularly with regards to the association of this gene with putative pathogenicity islands.
Additionally, plasmid and antibiotic resistance profiles of clinical Klebsiella strains will provide further insight into Klebsiella pneumoniae persistence in the mammary gland and virulence during an IMI. The unique bacterial strain collection that we have developed from NNY dairy farms, will allow us to study the disease causing factors in more detail.
It is expected that these studies will ultimately lead to the development of vaccines and science based preventative programs for NNY dairy herds
Acknowledgments
We acknowledge the funding from Pfizer, Inc., a pharmaceutical industry located in New York, NY. We also acknowledge the support of the dairy farmers participating in this project. Finally, the QMPS staff in the Canton and Ithaca laboratories are acknowledged for their enthusiastic participation in this project. We thank staff at the W. H. Miner Agricultural Research Institute, Chazy, New York, for their collaboration, questions and support.
For More Information
Dr Gary Bennett, gjb4@cornell.edu