Progress 06/01/18 to 01/31/20

Outputs
Target Audience:The target audience of this research endeavor is American Dairy Producers whose herds are afflicted with a high prevalence of bovine leukosis virus. NorthStar, in conjunction with Michigan State University, is currently conducting a field trial comprised of seven herds where we screen cattle through DHI BLV milk ELISA to determine seropositive cattle which we draw blood for BLV qPCR assay to determine proviral load in order to stratify each herd from highest to lowest viral content. At each of the farms we have discussed the possibility of using milk samples to accomplish this, all of which said they would be willing to try. Changes/Problems:The major problem with this project being deemed successful was the finding that BLV microRNAs were unable to reliably be detected in milk from BLV-postive cattle, regardless of which qPCR assay was used. Advance microRNA assays (Thermo Fisher) resulted in the lowest Ct values of any assay, however no positive correlation was found with animal PVL and this protocol was significantly more labor intensive and would not lend to any commercial application. What opportunities for training and professional development has the project provided?This project was intrumental in the development of two young scientists, both enrolled in MSU's School of Veterinary Medicine. From initial validation of automated microRNA extraction approaches to ensure uniform recovery and detection of endogenous mammary microRNAs to incorporation of synthetic spike-in Cel-39 microRNA, our two students evaluated 4 microRNA extraction kits, and 4 microRNA RT-qPCR kits prior to performing the study objectives. These approaches were applied to the MSU dairy which is currently managing a high prevalence of BLV. The MSU dairy submitted routine DHI milk samples for milk component anaylysis, all samples were tested for BLV antibodies and students worked with the herd veternarians to bleed BLV-postive cattle. paired whole blood, plasma, and whole milk samples were stored back for further analysis. Using CentralStar's novel BLV proviral load assay, students performed proviral load testing and compiled a herd report enumerating and prioritizing the management of cattle with advance disease. This is significant because the students gained first-hand experience performing novel molecular diagnostic assays on an entire diary herd, consulted the herd manager, and learned of real life constraints on dairy producers for BLV management.This work was also accepted for two poster presentations at two international scientific conferences (CRWAD, PAG) in 2019-2020. How have the results been disseminated to communities of interest?This research was presented at the annual MSU BLV meeting (November, 2019) to a collective of BLV experts (Bartlett, Norby, Coussens, Taxis, Byrem, Erskine, Williems, Wells, McClure) summarizing our findings and outlining next steps. This meeting was in preparation for CRWAD (Chicago, IL Nov. 2019). No documents have been formally dissemenated to the general public or within academic sectors. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Research Objectives The purpose of this study was to determine whether BLV miRNAs can be detected in milk due to ease of sample access, decrease invasiveness, and decreased cost for sample collection/testing. Increased testing and knowledge of BLV status within herds will enable producers to make more informed decisions about animals in their herd and could lead to decreased rates of BLV nationwide. We hypothesize that BLV miRNAs will be present in milk and indicative of PVL status on BLV positive animals. Objective one of this study was to establish a standardized protocol for miRNA extraction from milk. Objective two of this study was to determine whether BLV miRNAs are present in milk, and further, whether levels of BLV miRNAs are indicative of PVL. Objective I: Isolation of miRNAs from milk Whole milk was collected from dairy cows during routine milking. 600ul of Trizol was added to 200ul of whole milk and mixed thoroughly. Samples were then incubated at room temperature for 5 minutes. 120ul chloroform was added to each sample and shook vigorously for 15 seconds. Samples were then incubated at room temperature for 3 minutes. Samples were centrifuged at 12,000xg at 4 degrees Celsius for 15 minutes, and the aqueous phase was transferred to a King Fisher Flex (KFF) deep well 96-well plate along with a blank well. The MagMax mirVana Total RNA Isolation Kit was used for the remainder of the isolation. Briefly, Proteinase K digestion was completed using solutions included in the kit's protocol. Cel-39 was used as an exogenous control and added to the Proteinase K solution at a concentration of 0.5ul per sample. The plate was vortexed for 5 min at 1050rpm and then incubated at 65 degrees Celsius for 30 min. Lysis Binding Mix and RNA Binding Bead Mix was prepared according to kit instructions. 100ul of Lysis binding mix and 40ul of RNA Binding Bead Mix was added to each sample; the plate was then covered and vortexed for 5 minutes at 1050rpm. 430ul of isopropanol was added to each sample and pipetted up and down to mix. 4 wash plates and 1 DNAse plate was prepared according to KFF protocols. The Bio-fluids protocol on the KFF was used, and processing plates loaded into the correct position when prompted by the instrument; the sample plate was loaded at position 1. 30-35 minutes after the start of the run, the DNAse plate was removed from the instrument. 50ul of rebinding buffer and 100ul of isopropanol was added to each sample well and an elution plate was added to the instrument tray. Samples were stored at -20 degrees Celsius for short-term storage and -80 degrees Celsius for long-term storage. Extensive optimization of our miRNA extraction protocols were performed toward the beginning of these experiments, including a column separation step; we found the King Fisher Flex to be the most successful and consistent method of miRNA extraction. We also tested the addition of lysis binding mix, and found this to improve miRNA yield. We therefore added the lysis binding mix as a permanent step in our protocol. Cel39 was used as an exogenous control for RNA extraction success. 16.5 fmol of Cel39 per reaction was spiked into the aqueous phase post-organic separation samples at the proteinase K digestion step at the beginning of the RNA isolation. Objective II: Detection of BLV miRNAs in milk BLV miRNAs were reliably detected in blood and correlated closely with PVL (i.e. lower Ct values were associated with higher PVL values and higher Ct values were associated with lower PVL values. The correlation between PVL and B4-3p in 75 selected cows (selected based on PVL) was 0.8458. Average Ct values and average PVL values of the high, medium, and low binned cows correlate very strongly (r2= 0.9283). Bin B4-3p Ct Avg Avg PVL High PVL 18.89033 2.24 Medium PVL 23.0423 0.41 Low PVL 25.94215 0.06 Milk samples, collected as part of the Michigan BLV Field trial, were used for miRA extractionusing our validated semi-automated milk extraction protocol. Results for B4-3p and B1-3p indicate thatBLV miRNAs are not indicative of PVL status. Further, Ct values for the BLV miRNAs were either nearly 40 or remained undetected for the entire run, indicating that if they are present, the quantity at which they are present is minute and much greater sensitivity would be required to detect and quantify, however endogenous and spike in Cel-39 miRNAs were robustly detected in these RNA extracts. Target High PVL Low PVL Negative PVL 148a 25.25 + 0.42 24.84 + 0.61 24.88 + 0.50 Cel39 17.05 + 0.36 16.85 + 0.47 16.96 + 0.39 B4-3p 40 + 0 39.72 + 0.88 39.21 + 1.91 B1-3p 38.49 + 1.82 40 + 0 40 + 0 Thirty-Sevenmilk samples and 27 paired plasma samples from MSU was conducted to compare BLV miRNA expression between sample types. MiRNA was extracted using our milk extraction protocol. 148a and Cel39 results indicate that miRNA extraction was successful and outliers (failed extractions) were removed. When comparing detection of miRNAs in blood and milk, 148a again proved itself to be more abundant in milk compared to blood. Interestingly, B5-5p was also found to be more abundant in milk compared to blood, however both Ct values were above 32 cycles, our diagnostic cut off for these assays.B2-5p, B4-3p, and B1-3p were more sensitive in blood and B4-3p and B1-3p were undetectable in milk. Based on this data, BLV miRNAs are indicative of disease and PVL in plasma/blood samples, but not in milk. Several BLV miRNAs (B1-3p, B3-3p, and B4-3p) were undetectable in milk, suggesting that either these miRNAs do not travel to milk or the miRNAs are present in such a small quantity that they cannot be detected by qPCR. Plasma samples reliably and consistently showed a strong correlation between BLV miRNAs and PVL status. However, milk samples did not show the same trend. This suggests that either the two-step RT-qPCR assays primers may not be sensitivec enoughor BLV miRNAs do not proportionally transport to the udder in order toaccurately predict PVL status. Conclusion The purpose of this study was to determine feasibility of using milk samples to detect BLV miRNAs as a new diagnostic tool for BLV status assessment. The BLV profile of the MSU Dairy Cattle Teaching & Research Center revealed a 65% prevalence of BLV within the herd (141 positive and 15 suspect out of 239 cows). We were able to establish an effective and consistent protocol for miRNA extraction from milk (objective one). Using a Trizol separation combined with the King Fisher Flex extraction, we were able to isolate miRNA consistently from milk, which is notoriously a difficult matrix from which to extract. Using this extraction protocol, we confirmed efficacy through an exogenous spike-in control (Cel39) and were able to obtain consistent data. We were also able to establish a reliable endogenous control, miRNA 148a, using this hybrid protocol for miRNA extraction. In fact, our results for milk miRNA extraction were far more consistent than the established blood miRNA extraction protocol. BLV miRNAs correlated with PVL in blood samples. We were able to consistently detect nine different BLV miRNAs and found that these miRNAs correlated closely with PVL status in BLV positive cows. Contrary to blood samples, milk samples did not appear to contain any BLV miRNAs (objective two). We were able to reliably detect our two controls: Cel39 (exogenous) and 148a (endogenous); this indicates that miRNA extractions were successful and reliable. It is possible that BLV miRNAs simply do not reach the milk, or that quantities are not sufficient to reliably predict PVL status. In conclusion, nine different BLV miRNAs correlate very well with PVL in blood. Although miRNAs are not indicative of BLV PVL status in milk samples, there are other potential targets to investigate for this type of diagnostic testing. We established a consistent protocol for miRNA isolation in milk which could have implications for other diagnostic testing in the dairy industry.

Publications

Progress 06/01/18 to 05/31/19

Outputs
Target Audience:Target Audience: The target audience of this research endeavor is American Dairy Producers whose herds are afflicted with a high prevalence of bovine leukosis virus. NorthStar, in conjunction with Michigan State University, is currently conducting a field trial comprised of seven herds where we screen cattle through DHI BLV milk ELISA to determine seropositive cattle which we draw blood for BLV qPCR assay to determine proviral load in order to stratify each herd from highest to lowest viral content. At each of the farms we have discussed the possibility of using milk samples to accomplish this, all of which said they would be willing to try. Efforts: Once high and low proviral load cattle have been identified we collect DHI milk samples from these cattle, in addition to BLV negative cattle for microRNA isolation. Thus far we have compared 3 automated nucleic acid extractors and respective chemistries, Promega's Maxwell RSC 48, Thermo Fisher's King Fisher Flex, and Indical Bioscience's IndyMag 48. Based on throughput, yield and overall pricing we have selected the IndyMag 48 for purchase to continue to optimize microRNA extraction. Preliminary results of the comparison of organic phenol/chloroform to automated magnetic bead isolation has demonstrated that further lysis/fractionation is required to recover maximum amounts of microRNA. Thus, we have also purchased a TissueLyser II (Qiagen) for 2x96 well homogenization prior to ultra-centrifugation accomplished in a refrigerated microcentrifuge, also purchased for this project. With characterized milk samples being collected, and equipment in place, we will continue to optimize high throughput milk microRNA extraction procedures. Changes/Problems:Major challenges include defatting the milk samples of interest without the use of phenol/chloroform and recovery of the microRNAs within the correct fraction of milk. Organic extraction will continue to be our baseline for extractions, while we are working to optimize enzymatic digestion conditions of milk prior to RNA precipitation and purification via either columns or magnetic bead-based approaches. What opportunities for training and professional development has the project provided?This work has attracted two DVM students currently enrolled at Michigan State University College of Veterinary Medicine. They have begun to help with the BLVfield trial which this particular project is monitoring in order to select the previously defined animal disease states: high and low PVL and BLV-negative. We have now collected blood, DNA from blood, and milk samples for these animals. This project has provided opportunities in field experiences (bleeding cattle) as well as commercial lab (diagnostics) and research lab (investigating microRNA isolation and quantification in cow milk).We have also met with a patent attorney and have discussed the steps necessary to patent these processes and analyses. Pending the outcome of these research endeavors, we plan to apply for patents. How have the results been disseminated to communities of interest?No. What do you plan to do during the next reporting period to accomplish the goals?We plan to have Standard Operating Procedures and required equipment in placein order to start evaluating the feasibility, robustness and ruggedness at the commercial scale by the next reporting period.

Impacts
What was accomplished under these goals? As described in the efforts section of this report. We are in the process of determininghigh PVL cattle from seven herdswithin our ongoing field trial and are in the process of collecting DHI milk samples from these cattle for optimization of microRNA isolation via a magnetic bead automated DNA extractor which we have evaluated and are preparing to purchase. We are evaluating microRNA recovery via spiked Cel39 miRNA and endogenous host miRNA RT-qPCR assays. Currently we have identified up to 4 "housekeeping" microRNAs which appear to be reliable endogenous controls for a read out of microRNA recovery from milk samples. These targets will be important as we begin to evaluate relative BLV microRNA expression inmilk from cows which we have determined to be high proviral load (advanced disease), low proviral load (indolent disease), and BLV-negative.

Publications

Progress 06/01/18 to 01/31/19

Outputs
Target Audience:Target Audience: The target audience of this research endeavor is American Dairy Producers whose herds are afflicted with a high prevalence of bovine leukosis virus. NorthStar, in conjunction with Michigan State University, is currently conducting a field trial comprised of seven herds where we screen cattle through DHI BLV milk ELISA to determine seropositive cattle which we draw blood for BLV qPCR assay to determine proviral load in order to stratify each herd from highest to lowest viral content. At each of the farms we have discussed the possibility of using milk samples to accomplish this, all of which said they would be willing to try. Efforts: Once high and low proviral load cattle have been identified we collect DHI milk samples from these cattle, in addition to BLV negative cattle for microRNA isolation. Thus far we have compared 3 automated nucleic acid extractors and respective chemistries, Promega's Maxwell RSC 48, Thermo Fisher's King Fisher Flex, and Indical Bioscience's IndyMag 48. Based on throughput, yield and overall pricing we have selected the IndyMag 48 for purchase to continue to optimize microRNA extraction. Preliminary results of the comparison of organic phenol/chloroform to automated magnetic bead isolation has demonstrated that further lysis/fractionation is required to recover maximum amounts of microRNA. Thus, we have also purchased a TissueLyser II (Qiagen) for 2x96 well homogenization prior to ultra-centrifugation accomplished in a refrigerated microcentrifuge, also purchased for this project. With characterized milk samples being collected, and equipment in place, we will continue to optimize high throughput milk microRNA extraction procedures. Changes/Problems:Major challenges include defatting the milk samples of interest without the use of phenol/chloroform and recovery of the microRNAs within the correct fraction of milk. Organic extraction will continue to be our baseline for extractions, while we are working to optimize enzymatic digestion conditions of milk prior to RNA precipitation and purification via either columns or magnetic bead-based approaches. What opportunities for training and professional development has the project provided?This work has attracted two DVM students currently enrolled at Michigan State University College of Veterinary Medicine. They have begun to help with the BLVfield trial which this particular project is monitoring in order to select the previously defined animal disease states: high and low PVL and BLV-negative. We have now collected blood, DNA from blood, and milk samples for these animals. This project has provided opportunities in field experiences (bleeding cattle) as well as commercial lab (diagnostics) and research lab (investigating microRNA isolation and quantification in cow milk).We have also met with a patent attorney and have discussed the steps necessary to patent these processes and analyses. Pending the outcome of these research endeavors, we plan to apply for patents. How have the results been disseminated to communities of interest?No. What do you plan to do during the next reporting period to accomplish the goals?We plan to have Standard Operating Procedures and required equipment in placein order to start evaluating the feasibility, robustness and ruggedness at the commercial scale by the next reporting period.

Impacts
What was accomplished under these goals? As described in the efforts section of this report. We are in the process of determininghigh PVL cattle from seven herdswithin our ongoing field trial and are in the process of collecting DHI milk samples from these cattle for optimization of microRNA isolation via a magnetic bead automated DNA extractor which we have evaluated and are preparing to purchase. We are evaluating microRNA recovery via spiked Cel39 miRNA and endogenous host miRNA RT-qPCR assays. Currently we have identified up to 4 "housekeeping" microRNAs which appear to be reliable endogenous controls for a read out of microRNA recovery from milk samples. These targets will be important as we begin to evaluate relative BLV microRNA expression inmilk from cows which we have determined to be high proviral load (advanced disease), low proviral load (indolent disease), and BLV-negative.

Publications