Source: UNIV OF MINNESOTA submitted to
COW-LEVEL ASSOCIATION BETWEEN SERUM VITAMIN D (25-HYDROXYCHOLECALCIFEROL)CONCENTRATION AND MAP-ANTIBODY SEROPOSITIVITY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0225909
Grant No.
(N/A)
Project No.
MIN-62-020
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
May 1, 2011
Project End Date
Sep 30, 2012
Grant Year
(N/A)
Project Director
Sorge, U. S.
Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108
Performing Department
Veterinary Population Medicine
Non Technical Summary
Before the invention of antibiotics, attempts to treat human tuberculosis included cod liver oil or sending ailing patients to warmer and sunnier climates. Both sunshine (UV-B irradiation) and diet are important sources of vitamin D in both humans and animals. Besides importance for bone metabolism, Vitamin D plays an important part in the modulation of the immune response. For example, deficiency of vitamin D3 or of its co-factor Magnesium has been associated with increased levels of a certain immune response, which is also up-regulated in patients with Crohn's disease. Therefore, it is not unexpected that recent studies found that low serum vitamin D levels were associated with flare-ups in Crohn's disease patients and increased susceptibility to clinical tuberculosis. Similar to Crohn's disease in humans, paratuberculosis is a chronic intestinal inflammation of ruminants caused by the bacterium Mycobacterium avium subsp. paratuberculosis (MAP). Once animals are visibly sick, the disease is called Johne's disease (JD) and is manifested as diarrhea and wasting of the animal. The disease is not curable and can lead to large economic losses on dairy farms. It is interesting to note that, in the US, the percent of dairy operations with ≥5% of cull cows with visible JD varies regionally and more visible JD was observed in areas with lower probabilities of sunshine. Based on this information, we hypothesize that the serum vitamin D levels of cows is also associated with the occurrence of JD. A pilot study at the University of Minnesota with 41 dairy cows found that MAP-seropositive cows tended to have lower serum Vitamin D concentrations than test-negative herd mates. In addition, the vitamin D concentration was associated with the level of antibodies per cow -- the higher the levels, the lower the serum vitamin D concentration. This might indicate that a progression of infection might be associated with reduced vitamin D levels. Whether this is due to impaired absorption of vitamin D through the MAP-infection damaged intestines, lack of sunshine exposure through year-round indoor housing or a higher turnover rate of internally produced Vitamin-D3 for the modulation of an out-of-control immune response or lower vitamin D receptors is currently unknown. The aim of this study is to investigate the association between vitamin D serum levels and MAP antibody and immune response of dairy cows, after accounting for factors that could influence either. We will take blood samples from cows twice and analyze these for vitamin D, antibodies against MAP, and mineral content. In addition, the immune cells and products of a subset of cows will be further analyzed to determine differences to test-negative cows. The results of this study would provide for the first time information about the association between the serum vitamin D concentration and JD sero status. The results of this study could lead to further studies and potential preventive management strategies against JD on farms.
Animal Health Component
40%
Research Effort Categories
Basic
60%
Applied
40%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3023410101010%
3023410102010%
3113410101015%
3113410102015%
3113410109040%
3113410117010%
Goals / Objectives
The three objectives of this study are: 1)Evaluate the cow-level association between serum 25-Hydroxycholecalciferol concentration and MAP seropositivity of dairy cows, adjusting for diet, breed, access to sunlight, cow haircoat color and cow age. 2)Assess the effect of season on the association between serum 25-Hydroxycholecalciferol concentration and MAP seropositivity.3)Assess associations between serum concentration of TNF-α and IL-6 as well vitamin D receptor density of macrophages on MAP seropositivity in dairy cows fed rations with different concentrations of vitamin D. We expect that vitamin D concentration will be negatively associated with JD ELISA-positivity. The results of this study would provide for the first time information about the association between the serum vitamin D concentration and JD serostatus. The results of this study could lead to further studies and potential preventive management strategies against JD on farms. The timeline of this study is as follows: Enrollment of herds (May 2011), first sample collection & analysis (June 2011), second smaple collection and analysis as well as immunological analysis (August 2011), summary of results and article submission to journal (September 2011).
Project Methods
Eight dairy producers in Minnesota and Wisconsin will be asked to participate based on previous Johne's disease (JD) testing by the Minnesota Veterinary Diagnostic Laboratory. Inclusion criteria are: within 150 miles from St Paul, MN, ≥ 5% MAP test-prevalence, DHIA member, milking at least 200 cows, herds housed in freestalls or tiestalls. Exclusion criteria: grass-based housing systems. Ultimately, we will need 80 test-positive and 80 test-negative cows. In June two 9 ml blood samples of all adult cows will be collected in each herd. The JD status of all sampled cows will be determined. All cows with a positive serum sample for MAP antibodies (S/P >0.25) will be enrolled. For each positive cow, a negative herdmate will be enrolled based on age and lactation status as control. In August, the previously identified test-positive cows and their test-negative controls (n=160) will be sampled for a second time. Two blood samples will be collected per cow. On both sample days, cow housing, access to natural light (Y/N), the breed, as well as the 305 d milk production, percent milk fat and milk protein, age, days in milk (DIM) at sample day, sample season, herd and latitude of the farm will be recorded. On the second sampling day, a standardized photograph of the back of each cow will be taken to objectively assess the proportion of white haircoat over the back of the cow. At both times of sample collection, records of the calculated rations currently fed as well as actual feed samples will be obtained from every herd. The feed samples will be analyzed for Vitamin D3 concentration and mineral composition. Feed intake will be estimated for each cow based on her milk production to approximate the daily vitamin D intake. The serum samples of both sampling days of the 160 cow will be analyzed for 25-Hydroxycholecalciferol (25(OH)D) concentration and for their concentration of 15 different minerals (including Magnesium, Calcium, and Phosphorus). Furthermore, the JD ELISA status will be again determined with the second serum sample. To address the third objective, a third blood sample of selected cows will be collected into EDTA tubes. The same day, appropriate methods will be employed to isolate peripheral blood mononuclear cells from the sample and to determine the level of vitamin D receptors (VDR). Using previously frozen samples from the second sampling of those cows, the serum concentration of TNF-α and IL-6 will be determined. The statistical analysis will be conducted with SAS 9.2. Besides descriptive statistics, a logistic mixed model with JD serostatus (positive/negative) as the outcome will be used. Predictors are day of sampling, latitude of herd location, breed, days in milk, 305 d milk production, estimated nutritional vitamin D intake on sample day, and cow haircoat color will be recorded for each cow. Cows will be clustered within herd. The analysis will be repeated with the results from the second sampling. The third objective will be assessed using appropriate descriptive and univariable statistics. Outcome variables will include serum concentrations of VDR, TNF-α, and IL-6. The significance level is set at α = 0.05.

Progress 05/01/11 to 09/30/12

Outputs
OUTPUTS: In the recent years, the importance of Vitamin D deficiency in the etiology of diseases, such as Tuberculosis or Crohns disease, has been highly discussed in the human medical field. This pilot study investigated for the first time, if an association exists between serum vitamin D and Johnes disease of cows (ELISA status). The results of this study have been presented at local, national and international research meetings and have been accepted for publication in a peer-reviewed journal. PARTICIPANTS: All project participants were from the University of Minnesota, College of Veterinary Medicine. Exceptions are noted below. Dr. Ulrike Sorge (PI) not only wrote the study grant, but also trained the students, enrolled herds, collected the data on farm, analyzed the data and was principle author on all publications. Dr. Scott Wells (Co-PI) helped to develop the study idea and provided input throughout the study period and data analysis. Dr. James Linn (Collaborator), Department of Animal Science, provided expertise on ruminant nutrition throughout the study period as well as laboratory space and equipment for the sampling and sample processing of the collected feed samples. Dr. Thomas Molitor (Collaborator) provided expertise and laboratory space and labor for the immunological part of the study. Dr. David Gallaher (Collaborator) provided expertise on vitamin D metabolism throughout the study. Laura Willard, a first year veterinary student, worked as summer student on the project. She collected data on farm, processed the samples in the laboratory so that they could be submitted to respective laboratories for analysis and entered data. Michael Mahero, a public health resident, collected samples on farm and processed the samples in the laboratory so that they could be submitted to respective laboratories for analysis and entered data. TARGET AUDIENCES: This was a pilot study with the aim to establish whether the association between vitamin D and Johnes disease exists. The data is premature for a practical application, e.g. to prevent Johnes disease, but were meant as starting point for future research activities in this field. Therefore, the results of this study have been distributed and presented at scientific meetings to encourage discussion among researchers and future research projects. PROJECT MODIFICATIONS: Due to delay in funding release and enrollment of herds, the sampling frame was slightly changed and objective 2 (effect of seasonality) could therefore not be answered. First, instead of three samplings in April, July (2x), the herds and cows were only targeted twice: July and August. Second, only 5 herds had to be enrolled to obtain the necessary number of cows. Third, fecal samples were collected from the 160 animals to be analyzed for Mycobacterium avium subsp. paratuberculosis using PCR and culture to assess the infection status of the cows in addition to their immunological response (ELISA). This change in sampling was made to prevent criticism that the ELISA status of the cows would only explain exposure, but not infection.

Impacts
Before the invention of antibiotics, attempts to treat human tuberculosis included cod liver oil or sending ailing patients to warmer and sunnier climates. Both sunshine (UV-B irradiation) and diet are important sources of vitamin D in both humans and animals. Besides importance for bone metabolism, Vitamin D plays an important part in the modulation of the immune response. Therefore, it is not unexpected that recent studies found that low serum vitamin D levels were associated with flare-ups in Crohns disease patients and increased susceptibility to clinical tuberculosis. Similar to Crohns disease in humans, Johnes disease is a production limiting, chronic intestinal inflammation of ruminants. It is caused by Mycobacterium avium subsp. paratuberculosis (MAP) and it is not curable. Due to its similarities to Crohns disease, we hypothesized that the serum vitamin D levels of cows is also associated with the occurrence of Johnes disease. The objective of this study were 1) Evaluate the cow-level association between serum 25-Hydroxycholecalciferol concentration and MAP seropositivity of dairy cows, adjusting for diet, breed, access to sunlight, cow haircoat color and cow age. 2) Assess the effect of season on the association between serum 25-Hydroxycholecalciferol concentration and MAP seropositivity. 3) Assess associations between serum concentration of TNF-alpha and IL-6 as well vitamin D receptor density of macrophages on MAP seropositivity in dairy cows fed rations with different concentrations of vitamin D. Five large dairy farms in Minnesota were visited in July and August 2011 for the sample collection. At the first visit, the herds were screened for serum MAP-antibody ELISA positive cows and feed samples and electronic production parameter were obtained. At the second visit, the 160 cows (80 test-positive & 80 test-negative) were bled again to be tested for MAP-antibodies and from one herd, blood samples were collected to address objective 3. In addition, fecal samples were collected to be tested for MAP (PCR and culture), a standardized picture of the cows back was taken and her body condition score assessed. The study found significantly lower serum 25-Hydroxyvitamin D concentrations in ELISA positive cows compared to negative herd mates. Furthermore, the stage of lactation was associated with lower 25-Hydroxyvitamin D. Fresh cows had the lowest serum concentration, which agrees with previous studies investigating the association between serum 25-Hydroxyvitamin D and milk fever. Hair coat color, vitamin D in feed, breed and cow age were not associated with 25-Hyrodroxyvitamin D concentration in the mixed linear regression model. The short time frame of the final study due to funding release and enrollment of herds did not allow for the addressing of objective 2 (seasonality). The investigated immunological parameters did also not show any association with seropositivity or 25-Hydroxyvitamin D status of the cow. This is the first study to show an association between serum 25-Hydroxyvitamin D concentration and serum MAP-antibody ELISA status of cows.

Publications

  • U.S. Sorge, T. Molitor, J. Linn, D. D. Gallaher, and S.W. Wells. Cow-level association between serum 25-Hydroxyvitamin D concentration and MAP-antibody seropositivity: A Pilot Study. Accepted by Journal of Dairy Science. November 1, 2012.
  • U.S. Sorge. Cow-level association between serum 25-Hydroxyvitamin D concentration and MAP-antibody seropositivity: A Pilot Study. Minnesota Dairy Health Conference, Minneapolis, USA, May 24, 2012.
  • U.S. Sorge, T. Molitor, J. Linn, D. D. Gallaher, and S.W. Wells. Cow-level association between serum 25-Hydroxyvitamin D concentration and MAP-antibody seropositivity: A Pilot Study. Poster Presentation at the 11th International Colloquium on Paratuberculosis, Sydney, Australia, February 2-6, 2012.
  • Sorge, U. Johnes disease: New Perspectives to an Old Disease. Presentation at University of Minnesota, College of Veterinary Medicine. St. Paul, USA. December 7, 2011.


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: The sample collection has been completed and the data analysis is ongoing. Preliminary results were presented at the University of Minnesota's College of Veterinary Medicine Research Seminar series in December and a recording of the presentation was made available online. Mycobacterium avium subsp. paratuberculosis culture and data analysis will be completed in 2012. PARTICIPANTS: Dr. Ulrike Sorge (PI): identification and contacting of participating herds, organization of study set-up, training of students (see below), sample collection, and data analysis. This project provided the opportunity to train Michael Mahero (Public Health Resident) and Laura Willard (veterinary student) in the handling of cows, laboratory methods, on-farm sampling procedures and study design. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Due to delays in funding release, the spring season was missed. Therefore, objective 2 was removed from the study. Instead, fecal samples of the cows were collected and analyzed for the presence of Mycobacterium avium subsp. paratuberculosis to determine the infection status of the cows beyond immune response (ELISA) alone.

Impacts
Johne's disease is an economically important disease in dairy cattle and other ruminants. It is a chronic, bacterial, intestinal inflammation and infected animals develop diarrhea, which leads to wasting and ultimately death. There is no cure. Therefore, prevention is important for its control. Vitamin D has been identified as important factor for immune regulations and might be associated with the expression of this disease. This is the first study that investigated the association between vitamin D (25-Hydroxy-and 1,25-DiHydroxy-Vitamin D3) and Johne's disease infection in dairy cattle. Dairy cows from 5 Minnesota herds were screened and 80 MAP-antibody ELISA positive and 80 negative control cows were enrolled. Cows that had antibodies against MAP had significantly lower 25-Hydroxy-Vitamin D3 serum concentrations than test-negative cows. In addition, the stage of lactation was identified as associated factor, which agrees with previously published studies reporting physiological patterns of 25-Hydroxy-Vitamin D3 serum concentration in cows. In the preliminary analysis, the level of vitamin D (vitamin D3 and/or D2) in the nutrition was not associated with neither 25-Hydroxy-vitamin D3 nor 1,25-Dihydroxy-vitamin D3 serum concentration. The analysis of immunological parameters in respect to vitamin D or Johne's status of the animals is currently ongoing. This is the first study that identified that MAP infected cattle have lower vitamin D serum concentrations than MAP-antibody negative cows.

Publications

  • No publications reported this period