Northern NY Agricultural Development Program 2008-2009 Project Report
Quality Milk Promotion Services (QMPS), Cornell University: Gary J. Bennett, Ruth N. Zadoks and Ynte H. Schukken
Heidi Sharkey, QMPS Canton
Marcos Munoz, Brad Rauch and Natalia Belomestnykh, QMPS Ithaca
Murcrest Farm, Jefferson County
Mapleview Farm, St. Lawrence County
WH Miner Institute, Clinton County
Bilow Farm, Franklin County
We have previously shown that Klebsiella spp and E. coli (both coliform bacteria) are very important causes of mastitis in Northern New York (NNY). In our previous work we have shown that important sources of infection include the cow’s environment and the water and bedding in the barn. We have performed a treatment trial and shown that extended therapy with a third generation cephalosporin treatment resulted in a significant improvement compared to non-treated controls. Both Klebsiella and E. coli bacteria are abundant in the environment of the modern dairy cow. Although we do observe many different strains of these bacteria in the cow environment, only a relative small number of strains are causing mastitis on farms. In most farms with a Klebsiella problem we observe a dominant strain of the Klebsiella bacteria in the mastitis samples from the farm. This suggests that some strains of Klebsiella are more likely to cause mastitis compared to other more environmentally and less bovine host-adapted strains. The objective of the research in this year is to identify phenotypic (visual) and genetic difference between Klebsiella bacteria from NNY that are found in clinical mastitis cases versus the environment of the dairy cow.
A collection of 33 K. pneumoniae isolates from CM and 37 K. pneumoniae isolates from the on-farm environment from 4 purposely selected New York dairy farms were included in the study. The samples of milk from Clinical Mastitis and environmental samples were collected from May 2005 through October 2007.
Environmental K. pneumoniae isolates were randomly selected from a larger collection of environmental isolates from the farms. Clinical mastitis K. pneumoniae isolates were purposely selected from precisely described mastitis cases. K. pneumoniae isolates’ ability to utilize 3 iron acquisition systems was detected by assessing the presence of DNA coding regions of siderophore and siderophore-receptors.
Dry Cow Secretum (DCS) from fully involuted infection-free mammary glands was obtained from 3 dairy cows at 20, 24 and 34 d of the dry period, respectively. Pooled NIBS was obtained from blood samples from 20 selected healthy dairy cows. Bacterial growth was evaluated using 10 µL of each bacterial culture containing 100 cfu that was placed in 290 µL of DCS. This DCS-bacterial culture mix was incubated at 37 ºC. Fifty µL of each DCS-bacterial duplicate culture was harvested at 0, 4, and 12 h of incubation and plated on agar plates. These agar plates were incubated for 24 h at 37 ºC and colonies were counted.
We identified that Klebsiella bacteria that cause clinical mastitis have vastly different characteristics compared to bacteria that were cultured from the environment (Figure 1). Apparently, bacteria that are ‘successful’ in causing intramammary infections form a sub-populations of all Klebsiella bacteria. The initial results from our current work on Klebsiella isolates from NNY farms are shown in Figure 1. Here we show that many of the mastitis bacteria are either able to grow in dry cow secretum in non-immune bovine serum or in both. In sharp contrast to this, virtually all environmental Klebsiella isolates do not grow in either dry cow secretum or bovine serum and are easily killed by the immune substances in these body fluids.
In Table 1, the siderophore profiles of mastitis and environmental Klebsiella isolates are shown. Siderophores are important tools for the bacteria to use iron from the environment where it lives. This is particularly important in dry cow secretum as a high lactoferrin concentration results in a very low availability of iron for the bacteria. Consequently, siderophores are important pathogenicity characteristics of Klebsiella bacteria.
Table 1. Siderophore profiles found in Klebsiella pneumoniae isolates from both environmental and mastitis origin classified as resistant or sensitive to dry cow secretion (DCS) and non-immune bovine serum (NIBS)*.
No siderophore Enterobactin Enterobactin and Fisher’s exact
DCS sensitive 6 (11.5) 46 (88.5) 0 (0) 0.035
DCS resistant 0 (0) 16 (89.9) 2 (11.1)
NIBS sensitive 6 (12.8) 41 (87.2) 0 (0) 0.029
NIBS resistant 0 (0) 21 (91.3) 2 (8.7)
*Values in parenthesis are percentages of isolates within rows.
**Differences in distribution of siderophore profiles between DCS-sensitive and DCS-resistant groups and between NIBS-sensitive and NIBS-resistant groups respectively.
Interestingly, when we plot the isolates by the time that the clinical mastitis occurred during lactation, the cases that occurred in the first month after calving are significantly more associated with resistance to dry cow secretum. This further emphasizes the importance of infection in the dry period as an important high risk period for Klebsiella intramammary infections (figure 2).
These findings are important for two reasons:
1) the results indicate that mastitis Klebsiella bacteria have special characteristics that make them more successful in causing bovine mastitis. We are currently trying to identify the genes associated with this survival success in dry cow secretum and bovine serum. These genes would be potential vaccine targets; and
2) the results indicate that particularly dry cow secretum is likely killing many Klebsiella isolates, but allowing a sub group of Klebsiella bacteria to grow. For this sub group of cow-adapted bacteria the dry period is suspected to be a very important time of first infection. Klebsiella bacteria may enter the mammary gland in the dry period and grow in dry cow secretum, cause a persistent infection and then later in lactation cause high somatic cell counts and clinical mastitis.
We plan to publish the results of our studies in Eastern Dairy Business, the Journal of Dairy Science and several meetings throughout the US and worldwide. Results will also be presented in materials that can be downloaded from our website. The results have already been used in the 2009-2010 cycle of the Winter Dairy Management program. The case studies developed through this Klebsiella research program were used for teaching in the NNY Dairy Institute mastitis modules.
We now suspect that many of the Klebsiella infections originate in the dry period. The key to prevention of both long term infections and clinical cases lies in prevention of infection in the first place. For our future studies we hypothesize that klebsiella and other coliform infections originate in the dry period, and that preventative programs in the dry period will result in a reduced incidence of infections. In our future research we plan to use samples collected on farms in NNY and molecular typing of the collected isolates we will be able to test whether the dry period is the key to a long term resolution of Klebsiella spp. and E. coli mastitis.
In addition to NNYADP funding, support for thisKlebsiella project has been obtained from several sources. Formula funds provided funding for essay set up and actual testing. Pfizer inc. provided funds for our treatment trial and subsequent educational programs using data from farms in NNY.