METHOD FOR TESTING OF SAMPLE DAY MILK FOR PATHOGENS BACKGROUND OF THE INVENTION

CROSS REFERENCE TO RELATED APPLICATION(S)

[0001] The present application relates and claims priority to U.S. Provisional Application No. 63/316537, filed March 4, 2022, the entire contents/disclosure of which is hereby incorporated by reference.

1. FIELD OF THE INVENTION

[0002] The present invention relates to milk testing methods and, more specifically, to a method for testing milk for pathogens.

2. DESCRIPTION OF THE RELATED ART

[0003] Dairy operations frequently test milk for a wide variety of reasons and applications. Milk testing requires accurate sample preparation and testing to prevent inconclusive results, misidentification and/or misdiagnosis issues, and other problems, all of which are expensive and time-consuming. Automating milk sample preparation and testing can prevent or mitigate against these expensive and time-consuming issues. However, these approaches usually involve specialized aseptic collection to avoid pathogens that may be present on the udder, teat, or a liquid on outside of teat but not in the milk. As a result, sample of milk obtained from normal dairy herd information (DHI) type collection processes cannot be used for such testing. Additionally, traditional milk sample analysis for pathogens can take twenty-four hours or longer to receive a diagnosis or other test results, a delay which is both time-consuming and expensive. Conventional collection processes. Accordingly, there is a need in the art for a testing approach that can process large numbers of samples of milk and that will provide accurate results even if the milk is collected using standard, nonaseptic collection processes. BRIEF SUMMARY OF THE INVENTION

[0004] The present invention provides an approach for milk testing that can be performed quickly and with high throughput. More specifically, the present invention includes the steps of collecting sample day milk and immediately (if desired) analyzing the samples in a multi-well system using specific reagents and polymerase chain reaction (PCR) amplification approaches. The present invention can be used on samples collected through normal dairy herd information (DHI) type collection process (i.e., not aseptic) as any pathogens present in the milk will be detected without false positives due to pathogens that might have been present on the udder, teat, or liquid on outside of teat. In addition, although the DHI collection has a preservative in it that kills any pathogens, the present invention remains capable of detecting the presence of such pathogen based on the presence of any remaining fragments of DNA.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S) [0005] The present invention will be more fully understood and appreciated by reading the following Detailed Description in conjunction with the accompanying drawings, in which:

[0006] FIG. l is a flowchart of a method of detecting pathogens in collection day milk samples according to the present invention.

[0007] FIG. 2 is a first graph of the results of a STREP assay according to the present invention.

[0008] FIG. 3 is a second graph of the results of a STREP assay according to the present invention.

[0009] FIG. 4 is a chart of the results for DHIA Complete-16 for the 10 highest SCC Samples in Comparison to Acu-Reagents STREP and MYPRO. DETAILED DESCRIPTION OF THE INVENTION

[0010] Referring to the figures, wherein like numerals refer to like parts throughout, there is seen in FIG. 1 a method 10 according to the present invention for milk testing that involves collecting sample day samples of milk 12, such as by conventional dairy herd information (DHI) type collection non-aseptic processes. Next, the samples are lysed 14 and organism specific reagents for the detection of any genetic material, such as DNA, or pathogenic organisms 16 are added. For example, oligonucleotides probes along with primers and fluorophores may be used. The samples are then plated in a multi-well system 18 and thermocycled for PCR amplification in the presence of standard PCR reagents 20. The presence of any target organisms may then be determined based on changes in fluorescence 22. Method 10 thus employs polymerase chain reaction (PCR) amplification using specific reagents according to the present invention to determine the presence of any deoxyribonucleic acid (DNA) indicating the presence of organisms associated with dairy herd infections. Analysis using method 10 is reliable even if the samples were collected using dairy herd information (DHI) type collection process that are non-aseptic and include the antimicrobials, such as bronopol, as method 10 is robust enough to identify the presence of contaminating organisms even after treatment with antimicrobials.

[0011] In a first study, samples were selected from cows whose PSCC <200,000 and SCC >400,000 in order to sort the new infections from the chronic infections. This sorting yielded 103 samples. Of the 103 samples, 51 cows had not been previously sampled but currently had a SCC >400,000 on the current test. 103 samples with requisite negative controls and positive controls (known bacterial concentrations of 10 ² , 10 ³ , 10 ⁴ , and 10 ⁵ genome copies) seemed appropriate for this pilot. After the samples were sorted based on SCC criteria, the sample cups were wiped down with 70% IPA and processed through the Acu-Lysis Reagent. Once processing was completed, the 103 processed samples were placed into the 3.5 °C refrigerator for overnight storage. The samples were then tested to determine if DNA was present from Staph aureus, Coagulase Negative Staphs, and/or Mycoplasma bovis. Of the 103 samples, the following were found: 0 Staph aureus, 2 Coagulase Negative Staphs; 0 Mycoplasma bovis.

[0012] Following the completion of the STAPH and MYCOB runs, the STREP reagent was tested to determine if it might be useful for these samples. A subset of 10 samples were selected based on the sample being among the 10 highest SCC (383,800 < SCC < 1,000,000). 3/10 of the samples gave strong positives for Strep uberis and Strep species. 3/10 of the samples demonstrated late amplification and were considered close but negative (Retest). 4/10 of the samples demonstrated no amplification and are negative. Table 1 below shows the results:

Table 1

[0013] Table 2 below shows a summary of all results from the sample day.

Table 2

[0014] The results show that sample day milk can be used for analysis with Acu-PCR. Contaminated samples have very late amplification with levels of DNA present that are well below the Level of Quantification. These results also show that milk samples can be analyzed in a 96 well system to quickly (<3 hrs.) get Acu-PCR results. Test day samples with bronopol can be used in a 96 well plate with appropriate positive and negative controls in the Acu-PCR system.

[0015] In a second study, the cow selection criteria were changed to include cows with HIGHEST Test Day SCC (down to 400,000) - 218 cows. PCR tests included the STREP Reagent (Strep uberis and Strep spp( as well as the STAPH Reagent (Staph aureus and Staph spp.) and 1 :4 Dilutions of milk were evaluated to see if the approach was still sensitive enough to detect DNA. FIGS. 1 and 2 show the results of the second study, with Table 3 below showing the STREP and STAPH Results for all 218 Feb. Samples.

Table 3 [0016] Table 4 below shows the Pooling Results with the STREP Reagent.

Table 4

[0017] FIG. 3 shows the Results for DHIA Complete- 16 for the 10 highest SCC

Samples in Comparison to Acu-Reagents STREP and MYPRO. The use of Acumen Reagents in a 96 well Bio-Rad plate can provide a valuable screening test using DHI Samples. Bronopol does not affect the results. Low level contamination in DHI Samples does not affect results. In fact, 1 :4 Dilutions of milk were still sensitive enough to detect DNA (dependent on amount of DNA coming from target organisms).

[0018] Knowledge of prevalent mastitis-causing organisms and prevalence level of pathogens in the herd can help with the reagent selection/pooling decision. Prevalence of >10% or unknown warrants testing each cow in the group vs. pooling. If problem pathogens are known, it is possible to focus on the use of appropriate Acu-Reagents. If problem pathogens are unknown, initial testing with MYPRO and SASUB is indicated. [0019] Materials and Methods

[0020] Sample Storage

[0021] Non-aseptic milk samples preserved in 16% Bronopol and obtained during regular Dairy Herd Improvement Association collection. Sample Days are received and stored at 3.5 ° Celsius. The samples are then sorted based on previously determined Somatic Cell Count thresholds to increase the likelihood that they would yield a positive result. [0022] Sample Processing with Acu-Lysis Reagent

[0023] Once sorted, the samples are then placed into a Biosafety Cabinet. Each sample is mixed using a vortex and ImL of preserved raw milk and is transferred to the Acu- Lysis Reagent Tube A. This Tube A is then inverted to mix and placed back into the tube rack until all the samples had been transferred to their respective Tube As and inverted to mix. Following the mixing steps, all Tube As are placed into dry heat baths at a temperature of 100° C for 20 minutes. Following the conclusion of the 20-minute heat step, the samples are removed from the heat block and allowed to cool for one or more minutes. Following the cooling period, the Tube As are placed into a centrifuge and spun for approximately 10 seconds. This step is repeated until all the Tube As had been centrifuged. The Tube As are then placed into the Biosafety Cabinet and 200uL from each tube A is transferred to a corresponding Acu-Lysis Reagent Tube B. Following the transfer, Tube B is inverted to mix. At this point the sample processing is complete and the Tube Bs are placed into the 3.5°Celsius fridge and refrigerated overnight.

[0024] PCR Reagent Loading Procedure Unknown Samples

[0025] Single use lyophilized or premixed Acu-Reagent contained in a .2 mL high profile optically clear PCR tube are loaded with lOOuL of the Tube B solution from each sample. The Tube is then flicked to mix and placed into a PCR Tube Rack. Either one of the following two options may then be used. [0026] First, following the loading of 12 consecutive PCR Tubes and aligning them in a single row of the PCR Tube Rack, the caps are opened for each individual PCR tube and a 12-channel pipette is used to transfer approximately lOOuL from each PCR Assay Tube to the corresponding well in a 96-Well Hardshell BioRad Plate, assigned by random number generation in an Excel Spreadsheet.

[0027] Second, the pre-populated liquid or lyophilized Acu-Reagent 96-well plate is loaded with lOOuL of sample into each well as assigned by random number generation in an Excel Spreadsheet and pipetted to mix.

[0028] The Acu-Reagents suitable for use with the method described above are the MYPRO Reagent, the MYCOB Reagent, the SASUB Reagent, the STAPH Reagent, and the STREP Reagent. The Acu-Reagents are all ready-to-use lyophilized one channel (simplex) or two channel (duplex) PCR reagents packaged in 0.2ml plastic tubes with 10 microliters of PCR master mix, primers, probes (oligonucleotides), fluorophores for in vitro detection of target DNA sequences in the associated organisms. For example, MYPRO is a duplex reagent that detects oligonucleotides from Mycoplasma species and Prototheca species. MYCOB is a simplex reagent that detects oligonucleotides from Mycoplasma bovis, SASUB is a duplex reagent that detects oligonucleotides from Staph aureus and Strep uberis, STAPH is a duplex reagent that detects oligonucleotides from Staph species and Staph aureus, STREP is a duplex reagent that detects oligonucleotides from Strep species and Strep uberis. It should be recognized by those of skill in the art that other reagents may be used including those to detect oligonucleotides from specific organisms as desired.

[0029] PCR Reagent Loading Procedure Positive Controls/ Standards

[0030] Single use lyophilized or premixed Acu-Reagents contained in a .2 mL high profile optically clear PCR tube are loaded with 99uL of molecular grade Hyclone Water. The tube is then flicked to mix and placed into a PCR tube rack. luL of genomic DNA is then added to each positive control with triplicates of each of the following concentrations: 10 ⁵ Genome Copies/uL, 10 ⁴ Genome Copies/uL, 10 ³ Genome Copies/uL, and 10 ² Genome Copies/uL. For example, the STAPH Reagent would be loaded with Staph aureus gDNA purchased from ATCC and used as the standard genomic material. For another example, the STREP Reagent would be loaded with Strep uberis gDNA purchased from ATCC and used as the standard genomic material. Either of the two following approaches may then be used. [0031] First methodology - Following the loading of 12 consecutive PCR tubes and aligning them in a single row of the PCR Tube Rack, the caps are opened for each individual PCR tube and a 12-channel pipette is used to transfer approximately lOOuL from each PCR assay tube to the corresponding well in a 96-Well Hardshell BioRad Plate, as assigned by a random number generator in an Excel spreadsheet.

[0032] Second methodology - The pre-populated liquid or lyophilized Acu-Reagent 96-well plate is loaded with 99uL of molecular grade Hyclone Water into each well and pipetted to mix. luL of genomic DNA is then added to each positive control, with triplicates of each of the following concentrations: 10 ⁵ Genome Copies/uL, 10 ⁴ Genome Copies/uL, 10 ³ Genome Copies/uL, and 10 ² Genome Copies/uL. For example, the STAPH Reagent, would be loaded with Staph aureus gDNA purchased from ATCC and used as the standard genomic material. Another example, the STREP Reagent, would be loaded with Strep uberis gDNA purchased from ATCC and used as the standard genomic material.

[0033] The Acu-Reagents suitable for use with the method described above are the MYPRO Reagent, the MYCOB Reagent, the SASUB Reagent, the STAPH Reagent, and the STREP Reagent. The Acu-Reagents are all ready-to-use lyophilized one channel (simplex) or two channel (duplex) PCR reagents packaged in 0.2ml plastic tubes with 10 microliters of PCR master mix, primers, probes (oligonucleotides), fluorophores for in vitro detection of target DNA sequences in the associated organisms. For example, MYPRO is a duplex reagent that detects oligonucleotides from Mycoplasma species and Prototheca species. MYCOB is a simplex reagent that detects oligonucleotides from Mycoplasma bovis, SASUB is a duplex reagent that detects oligonucleotides from Staph aureus and Strep uberis, STAPH is a duplex reagent that detects oligonucleotides from Staph species and Staph aureus, STREP is a duplex reagent that detects oligonucleotides from Strep species and Strep uberis. It should be recognized by those of skill in the art that other reagents may be used including those to detect oligonucleotides from specific organisms as desired. It should be recognized by those of skill in the art that other reagents may be used including those to detect oligonucleotides from specific organisms as desired.

[0034] Thermocycler Setup: Once all samples are loaded into the 96-Well Plate(s), the plate is sealed using a BioRad Microseal® 'C PCR Plate Sealing Film. The plate is then placed into a BIORAD CFX96 Cl 000 Thermocycler and a run is made using User-Defined Parameters. The cycling protocol selected is the previously established proprietary cycling temperatures and cycle numbers. The plate file is created using the BioRad Manager Version 3.1. This plate is set to detect fluorescent amplification for the FAM Channel and Texas Red Channel which corresponds to the fluorescent emission generated by the STAPH and STREP Reagents. Each sample name is then added to the plate using the SpreadsheetZExcel Importer function of the BioRad Software. The samples identified as standards (gDNA Positives) are assigned as such in the BioRad software as well as assigned the log starting concentration of genome copies and selected as replicates. Negative controls are also identified as such using the BioRad Software with all other samples identified as “Unknown.” The plate file is then saved for future use in experiments with a similar setup. The “Run” button is then clicked and the thermocycler begins the heating and cooling protocol as well as simultaneously recording amplification/fluorescence for the identified channels. [0035] Result Interpretation: Following the completion of the run, the BioRad software populates a standard curve with the known positives and the unknowns plotted against log concentration and Cq values. The comparison of the unknowns to the standard curve generated allows us to set a Cq threshold of 35.5 to separate positive samples (below 35.5) from those that had environmental, and carry-over contaminants (above 35.5).

[0036] Significance: This ability to separate contaminants from positives with DHI

Samples is a novel approach as it eliminates the time-consuming effort of obtaining individual aseptic samples from individual cows and expedites the results turnaround time when paired with the Acu-Reagents.