STALL SIDE METHOD FOR THE DETECTION OF BACTERIA IN DAIRY CATTLE

Abstract

The present invention relates to several methods to detect gram positive mastitis pathogens in a small sample of bovine milk by luminescence using a combination of specific reagents giving a “cow side” “in-stall” indication of the presence or absence of gram positive mastitis pathogens within a short period of time.

Claims

1-22. (canceled)

23. A method for determining amounts of bacteria in bovine milk comprising the steps of: i) Contacting the bovine milk with a material which selectively attracts bacteria ii) Separating the bacteria from the material and placing the bacteria into solution iii) Treatment of the bacterial solution with a bacterial releasing agent capable of lysing bacterial cells to give a released ATP second solution iv) Treatment of the second solution with a Luciferin/Luciferase reagent to give a third solution and v) Quantitation of bacteria in the third solution by luminescence;

24. The method according to claim 23 wherein the material is selected from the group consisting of antibody coated surfaces, lectin coated surfaces, lytic enzyme binding domains coated surfaces, glass wool membranes and treated glass surfaces or any charged or uncharged surface.

25. The method of claim 23 wherein the bovine milk is obtained from a surface using a cloth, gauze, swab, wipe, non woven fiber or sponge.

26. The method according to claim 23 wherein the bacterial releasing agent is a bacteriophage lytic enzyme (endolysin) or modified lytic enzyme (genetic or chimeric), quaternary amines, ionic and or non-ionic surfactants.

27. The method according to claim 26 wherein the ionic surfactant is selected from the group consisting of anionic surfactants cationic surfactants and zwitterion surfactants.

28. The method according to claim 27 wherein the anionic surfactant is selected from the group consisting of alkyl sulfates, alkyl ether sulfates, docusates, sulfonate fluorosurfactants, alkyl benzene sulfonates, alkyl aryl ether phosphates, alkyl ether phosphates, alkyl carboxylates, and carboxylate fluorosurfactants, more preferably selected from the group consisting of ammonium lauryl sulfate, sodium dodecyl sulfate (SDS), sodium deoxycholate, sodium-n-dodecylbenzenesulfonate, sodium lauryl ether sulfate (SLES), sodium myreth sulfate, dioctyl sodium sulfosuccinate, perfluorooctanesulfonate (PFOS), perfluorobutanesulfonate, sodium stearate, sodium lauroyl sarcosinate, perfluorononanoate, and perfluorooctanate (PFOA or PFO).

29. The method according to claim 27 wherein the cationic surfactant is selected from the group consisting of cetyl trimethylammonium bromide (CTAB), cetyl trimethylammonium chloride (CTAC), cetylpyridinium chloride (CPC), Polyethoxylated tallow amine (POEA), benzalkonium chloride (BAC), benzthonium chloride (BZT), 5-bromo-5-nitro-1,3-dioxane, dimethyldioctadecylammonium chloride, laureltrimethylammonium bromide (DTAB), benzyldimethyldodecylammonium bromide (BDDABr), dioctadecyldimethylammonium bromide (DODAB).

30. The method according to claim 27 wherein the ionic surfactant is selected from DTAB, CTAB and BDDABr.

31. The method according to claim 27 wherein the zwitterion surfactant is sulfobetaine-3-10.

32. The method according to claim 26 wherein the bacteriophage lytic endolysin is selected from lysostaphin, LysK, lambdaSa2, OSH3b, and KSN383, lysA, lysA2, LysgaY, truncated lambda Sa2 and plyC.

33. The method according to claim 23 wherein the Luciferin/Luciferase reagent is chosen from the group consisting of Hygiena ATP Biomass Kit #CCK4, Promega Bright Glo system and any formulations which contain naturally occurring or genetically recombinant Luciferase.

34. The method according to claim 23 wherein the bacteria is gram positive bacteria.

35. The method according to claim 34 wherein the gram positive bacteria is selected from Staphylococcus spp., Streptococcus spp., Propionibacterium spp., Enterococcus spp., Bacillus spp., Corynebacterium spp., Nocardia spp., Clostridium spp., Actinobacteria spp., Lactococcus spp. and Listeria spp.

36. The method according to claim 23 wherein the bacteria is selected from the group consisting of streptococcus agalactiae, streptococcus spp., staphylococcus aureus and staphylococcus spp.

37-55. (canceled)

Description

DETAILED DESCRIPTION

EXAMPLES

[0076] The following examples are intended to illustrate the present invention without limitations.

Example 1

Determination of Usefulness of Luciferin/Luciferase Reagent in Measuring ATP Concentrations in Raw Bovine Milk Sample

[0077] Raw Bovine milk samples are known to have endogenous ATP which potentially could interfere with a bioluminescent assay. Raw Bovine milk samples were prepared containing various amounts of ATP standard from Sigma Chemical (#A2383.) to produce raw Bovine milk samples with concentrations of 10.sup.−6M to 10.sup.−10M. In each test, 50 uL of Promega luciferin-luciferase reagent (containing 25 mM HEPES buffer (pH 7.5), 40 pg luciferase, 100 pM luciferin, and 10 mM MgSO4). were added to 50 uL of Bovine milk sample and light output was determine using Hygiena Ensure System. All measurements were performed by first obtaining signal output of raw Bovine milk sample devoid of additional ATP and final ATP readings for each sample described above were corrected for the blank. In all cases the ATP levels were detected as expected for the ATP concentrations noted above. For comparison, standard solutions of ATP prepared in buffer were tested against their blank, and results similar to the raw Bovine milk study were obtained. This indicates that raw Bovine milk samples do not appear to hinder the Luciferin-Luciferase reaction, and are useful for the determination of bacteria in raw milk.

Example 2

Determination of Optimum Method to Filter Bovine Milk Sample

[0078] Some assays it may be necessary to pretreat the milk sample to remove fats and other endogenous materials that may be present in raw Bovine milk. In this procedure, raw Bovine milk (with endogenous bacteria) was filtered via gravity flow for 2 min using 2 different commercially available filter papers (after evaluation of numerous filter media). The filter media of choose are: Ahlstrom 222 (A222) and Ahlstrom 142 (A142). The filter papers where supplied by Ahlstrom Corporation. Samples (100 uL) prior to and after filtering on Ahlstrom 222 (A222) and Ahlstrom 142 (A142)) were tested for ATP with 50 uL of Promega luciferin-luciferase reagent (containing 25 mM HEPES buffer (pH 7.5), 40 pg luciferase, 100 pM luciferin, and 10 mM MgSO4), as well as for colony forming units (CFUs) via serial dilution on rich media tryptic soy agar (TSA) plates. The endogenous ATP appears to have largely (70%) bound to the A222 filter, while nearly 95% of the bacteria appear to have passed through. The A142 filter does not appear useful in this process since it does not allow the bacteria to pass through. There was no testing for somatic cells in the filtrate of the A222 filter, such that the reduction in ATP after filtration might reflect the capturing of the somatic cells on the filter and the resultant loss of intracellular somatic cells stores of ATP. All determinations of ATP concentration were performed using a Hygiena Ensure luminometer.

Example 3

Determination of Methods to Rupture Somatic Cells Present in Raw Bovine Milk Samples

[0079] A number of surfactants (detergents) were evaluated for their ability to rupture the somatic cells present in raw Bovine milk samples. Among the reagents tested were Triton X100 (Sigma Chemical) and Neonol AF9-10 (Nonoxynol-9) (Elarum Petrochemicals). Determinations using both surfactants were performed on raw Bovine milk samples in which somatic cell counts were predetermined. It was determined that the Neonol-9-10 was superior to the Triton X100 in its ability to rupture somatic cells in under 90 seconds. The number of somatic cell ruptured was determined by quantifying the ATP released in 50 uL samples of treated raw Bovine milk (as a function of time) using 50 uL of Promega luciferin-luciferase reagent (containing 25 mM HEPES buffer (pH 7.5), 40 pg luciferase, 100 pM luciferin, and 10 mM MgSO4). Bioluminescent measurements were performed using a Hygiena Ensure luminometer and all readings were blank corrected.

Example 4

Determination of Method to Eliminate Endogenous ATP Present in Raw Bovine Milk Sample

[0080] To demonstrating the ability to eliminate the endogenous ATP from raw Bovine milk, we selected Apyrase, an ATPase enzyme, from Sigma Chemical (A6535, ATPase ≥200 units/mg protein). All assays were performed using 50 uL of raw Bovine milk and 50 uL of Promega luciferin-luciferase reagent (containing 25 mM HEPES buffer (pH 7.5), 40 pg luciferase, 100 pM luciferin, and 10mM MgSO4). Bioluminescent measurements were determined using the Hygiena Ensure System. The test was performed in both 100% and 50% raw Bovine milk. Results indicate that Apyrase works well in both 100% and 50% raw Bovine milk samples. At a concentration of 284 mUnits the apyrase is able to deplete the endogenous ATP in 50 μL of raw milk in less than 30 seconds. The diminution of ATP was so fast with higher concentrations of Apyrase (diluting the enzyme) resulting in the inability to take meaningful reading, since all of the ATP was gone within 10 seconds. There are numerous commercially available ATP-degrading enzymes that can be tested for this purpose, but in our system, the Apyrase enzyme appears more than sufficient.

Example 5

Determination of Method to Evaluate “Eliminating Reagents” for Excess Apyrase Enzyme Present in Raw Bovine Milk Sample after Treatment to Eliminate Endogenous ATP (Example #4)

[0081] It is important to eliminate any excess Apyrase that may remain in the raw Bovine milk sample after treatment with the Apyrase enzyme that was used to eliminate endogenous ATP in Example #4. We examined a number of anionic and cationic surfactants (detergents) to determine those that are most effective at inactivate Apyrase. Among the reagents evaluated were: dimethyldioctadecylammonium chloride, laureltrimethylammonium bromide (DTAB), benzyldimethyldodecylammonium bromide (BDDABr), and cetyl trimethylammonium bromide (CTAB). The experiment was performed by adding between 0.02% and 1% of the selected surfactant to 50 ul of raw Bovine milk that had treated previously been treated with 284 mU of Apyrase as detailed in example #4. The levels of ATP were confirmed by adding 50 uL of Promega luciferin-luciferase reagent (containing 25 mM HEPES buffer (pH 7.5), 40 pg luciferase, 100 pM luciferin, and 10 mM MgSO4). As expected the readings determined on the Hygiena Ensure Luminometer were too low to measure, since all of the ATP had already been eliminated by Apyrase treatment in Example #4. After the initially readings were determined as described above, 10 uL of a 10.sup.−8M solution of ATP standard (Sigma Chemical) was added to the samples above and the bioluminescent signal was determined on a Hygiena Ensure Luminometer. As expected a signal was now detected, since all of the excess Apyrase added earlier had been eliminated by the surfactants being evaluated. The best results were seen when the detergents benzyldimethyldodecylammonium bromide (BDDABr) at 0.05% concentration and, laureltrimethylammonium bromide (DTAB) at 0.5% and 1.0% concentrations were added to the samples.

Example 6

Demonstrate the Ability of Both Streptococcal and Staphylococcal Phage Lytic Enzyme (Endolysins or Peptidoglycan Hydrolases) to Effectively Rupture Gram Positive Bacteria in a Raw Bovine Milk Sample

[0082] As the goal of this invention is to target and detect only gram positive organisms active in mastitis such as: S. aureus, Coagulase negative staphylococci (CoNS) and S. uberi, it is important to identify lytic enzymes that can effectively rupture the cell walls of these pathogens in the presence of raw Bovine milk. Lysostaphin (Lyso) and Streptococcal phage endolysin (PlyC), as well many other phage lytic enzymes, some which were developed by Donovan, have been shown to have high activity against the major Gram positive mastitis pathogens. The action against specific gram-positive organisms of these enzymes has been verified in PBS buffered solutions, but their ability to rupture bacteria had to be verified to in raw milk. The first candidates tested in for their activity in Bovine milk were Lysostaphin (Lyso) which attacks S. aureus, and Streptococcal phage lytic enzyme (PlyC) which attacks Streptococcus uberis. In all reactions, a concentration a of 0.05% of the phage lytic enzyme was used to evaluate the time required to rupture the cell wall of gram-positive bacteria in raw Bovine milk, as well as in a Phosphate-buffered saline (PBS) solution. It was determined that that in one hour, PlyC can eradicate up to 6 logs of S. uberis in PBS buffer, and either mastitic milk or healthy milk. The degree of lysing was determined by bioluminescence produced by the ATP released from the ruptured cells as described earlier. We then lowered the bacterial load to 2 logs of S. uberis in PBS buffer, and either mastitic milk or healthy milk, and it was determined that the PlyC can eradicate this bacterial load in 3 minutes or less. Similar experiments were performed with Lysostaphin (Lyso), which attacks S. aureus, and we determined we that can eradicate up to 6 logs of S. aureus in PBS buffer, and either mastitic milk or healthy milk in under 25 minutes. We then lowered the bacterial load to 2 logs of S. aureus in PBS buffer and either mastitic milk or healthy milk, and it was determined that the Lyso can eradicate this bacterial load in 2 minutes or less.