AU775568B2 - Mastitis detection - Google Patents

Description

WO 01/35728 PCT/NZ00/00232 MASTITIS DETECTION TECHNICAL BACKGROUND The present invention relates to mastitis detection and to related apparatus, methods and procedures.

The present invention recognises that an advantage is obtained for a farmer where there is the prospect of on-line monitoring of the somatic cell count indicative of mastitis infection in individual animals. Such monitoring either allows a treatment regime to be instituted for the particular animal or animals and/or for the isolation of or recriminations, if appropriate, or in respect of any such affected milk (whether at the milking parlour, farm, factory or elsewhere).

BACKGROUND ART Procedures exist whereby a non isolated flow of milk might directly be tested for mastitis. Such procedures have tended to find favour but at the cost of reduced accuracy over isolated milk testing procedures.

Journal ofDairy Research (1998) 65 187-198 "Changes in Electrical Conductivity and Somatic Cell Count Between Milk Fractions From Quarters Subclinically Infected with Particular Mastitis Pathogens" M W Woolford et al. discloses that intramammary infection (which seriously reduces milk yields) is frequently associated with increases in electrical conductivity in milk owing to increased levels of sodium and chloride ions in the milk. The monitoring of electrical conductivity can therefore be a means of automatically tracking udder health. Yet Woolfordet al states that factors such as temperature, fat concentration, milk solids, bacterial type, and milk fraction have variously been found to influence the measure of electrical conductivity. It is stated that such factors are important since the increase in electrical conductivity induced by intramammary infection is typically in the range of 15% to 50% only whereas the increase in somatic cell count (SCC) is usually at least 1000% increase.

Moreover Woolford et al states there are substantial physiological variations in the normal electrical conductivity levels that preclude comparisons of absolute milk electrical conductivity levels amongst cows for the purposes of identifying infection.

Examples of procedures and related apparatus that have been developed reliant on electrical conductivity to test milk flows include EP0748156 of Gasgoine-Melotte B.V. and PCT/SE97/00671 (published as W097/40374) of Alfa Laval Agri AB.

The full content of such Woolford el al publication and specifications EP0748156 and W097/40374 are hereby here included by way of reference.

WO 01/35728 PCT/NZ00/00232 -2- Gasgoine-Melotte B Vin their EPO 748156 comment on two sampling procedures so that still milk can be tested for conductance. They refer inter alia to EP-B-137367.

The Woolford et al publication in addition makes reference to somatic cell count (SCC) as a diagnostic criteria by indicating that, for example, greater than 500,000 cells/ml might be indicative of infection whilst less than 500,000 cells/ml may be indicative of no infection.

Table 1 (from Woolford et al) Diagnostic criteria for electrical conductivity, within-cow electrical conductivity ratio and somatic cell count used for prediction of the infection status of individual quarters.

Required diagnostic criteria Predicted quarters status Conductivity Somatic cell count, cells/ml Infected ECR> 1.15 500000 or EC 7000 p.S Uninfected ECR 1.5 <500000 where EC is electrical conductivity and ECR is electrical conductivity ratio Woolford et al indicates that reliance on somatic cell count (SCC) using a criteria based on 500,000 cells/ml provided greater sensitivity than did the electrical conductivity measures.

Test procedures for mastitis for manually isolated milk are known where the milk that is tested is not subsequently returned to the main volume of the milk.

One type of such isolated milk testing regime is a cow-side gel forming mastitis test procedure. This is typified by the Rapid Mastitis Test (RMT) [or California Mastitis Test See "Journal ofAmerican Veterinary Medical Association, Vol 130, March 1, 1957 No. 5 "Experiments and Observations Leading to Development of the California Mastitis Test" of O. W. Schalm and D. O. Noorlander.

The CMT procedure utilises an anionic surfactant a detergent such as, for example, sodium lauryl sulphate commonly marketed at TEEPOLTM). The CMT procedure results in a precipitate or gel indicative of the degree of infection the SCC).

Australian Journal of Dairy Technology 23, 129 (1968) E A Kernohan has shown that accuracy of somatic cell count (SCC) reliant upon the CMT procedure is dependant on the relative amounts of milk and reagent utilised. For best results preferably a near one to one WO 01/35728 PCT/NZOO/00232 -3volume ratio of a suitable reagent to milk is used in equal volumes for best results [for example, 2% w/v sodium laurel sulphate (commonly marketed as TEEPOL T M in water used in equal volume with milk].

Historically the CMT procedure of Schalm and Noorlander was graded on a score outlined below.

This score corresponds to SCC (Milchwissenschaft 19, 65-69 (1964) Halbquantitative Ausarbeitung des Schalmtestes fiir wissenschaftliche Zwecke of Keirmeier and Keis). The measurement technique used an "eyeball" technique. Therefore some variation exists in interpretation: negative, remains liquid, trace, slight precipitate which tends to disappear with more movement, >116 000 cells/ml weak positive, precipitate but not gel formation, >315 000 cells/ml distinct positive, thickness immediately with some gel formation, >600 000 cells/ml strong positive, distinct gel formation adherence to bottom of paddle and during swirling peaks forms >1 000 000 cells/ml The Journal ofMilk and Food Technology 27,271-275 (1964) "The Wisconsin Mastitis Test An Indirect Estimation of Leucocytes in Milk" of Thompson and Postle discloses the Wisconsin Mastitis Test (WMT). In the WMT the milk and detergent were mixed in a tube.

The tube was then inverted and a small hole in the top (3/64 inch diameter) allows the watery part to drain out. The height of the residual was then measured after at least a 1 min inversion This is still in use in some small laboratories today with an active SCC range from 100 000 to 1.2 million cclls/ml.

This test was then investigated in the mid 1970s (Milchwissenschaft 27 1972 "A simple semi-automatic viscosimeter for the estimation of somatic cells in milk", Whittlestone et al., and Milne et al., 1976) to try and automate the testing for use in the laboratory.

It was found that some ways of measuring the viscosity of the gel destroyed the gel.

Ultimately a rolling ball viscometer where the time for a ball to roll through the gel was timed.

This eliminated errors due to gel destruction. The active SCC ranges were 250 000 2 million cells/ml (Whittlestone et al.) and 100 000 1.3 million cells/mi (Milne et al.) respectively. This was also undertaken in Germany (Kiermier and Keis 1964).

-4- See also the publications: The Australian Journal of Diary Technology, 21, 138-139, (1966) "An Automatic Viscometer for the Measurement of the California Mastitis Reaction" Whittlestone et al.; Journal of Milk Food Technology 33, 35-354 "A Viscometric Method for the Estimation of Milk Cell Count" Whittlestone et al.; Milchwissenschaft 27, 84-86 (1972) "A Simple Semi-automatic Viscometer for the estimation of somatic cells in Milk" Whittlestone et al.; and New Zealand Journal of Diary Science and Technology, 11, 21-23 (1976) Milne et al.

Each discusses procedures for measuring the viscosity of the gels of such prior art detergent/milk SCC procedures with a view to determining a viable measurement regime.

0 15 Procedures disclosed include orifice or capillary viscometers (i.e.

0 moving gel) as well as falling ball or rolling ball viscometers stationary gel).

Milne et al. standardised a Whittlestone et al. type rolling ball viscometer to a tilt angle of 250 in preference to less accurate falling ball viscometers as even very gentle shear forces were found to cause a significant 20 decrease in viscosity. Milne et al. found about a 3.5 sec tilt time resulted at that 250 tilt angle when they used a ball of 4.7 mm diameter in a tube of 5.5 mm I.D. in a gel 0 made using 2% w/v TEEPOL 610TM (Shell Chemical Company) in water solution o 0 i as the reagent in volume ratios of 10 mL of the reagent to 5 mL of milk.

DISCLOSURE OF THE INVENTION In a first aspect the present invention consists in a method of testing a milk flow for mastitis, said method comprising: diverting a sample from said milk flow; and automatically testing said diverted sample flow by measuring an attribute of any somatic cells in at least part of the milk of that sample; wherein said measuring is by reference to a gelling characteristic of the milk under the action of an appropriate predetermined anionic detergent, and said measuring is by reliance upon the viscosity of at least part of the milk of that sample when in a predetermined admixture for a predetermined time with said anionic detergent.

The invention also provides a method of determining whether or not there is a somatic cell count change between milkings in milk of an animal by applying the method of the above-described aspect to separate milkings, and comparing data resulting from said testings to determine any somatic cell count

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e o change. goS S lO •coo Preferably the attribute is of the DNA of the somatic cells.

Preferably the milk flow is from a single animal and an identifier of that animal and an indicator of viscosity and thus of the somatic cell count is coupled for l 20 comparative purposes.

Sr l In another aspect the present invention provides a method of testing a milk flow for mastitis, said method comprising: diverting a sample flow from a said milk flow; providing an inflow of an anionic detergent and an inflow of said milk sample in predetermined proportions into a dwell zone from whence the detergent/milk admixture as a gelling and/or gelled product can outflow only at a -6lesser rate than said inflow(s); feeding the outflow admixture product of said dwell zone to a viscosity testing zone; and testing in said viscosity testing zone the outflow admixture product for viscosity and generating an output signal indicative of such viscosity.

Preferably the method comprises clearing the viscosity testing zone or (ii) both the viscosity testing zone and said dwell zone of said admixture without feedback into the milk flow from which the sample flow was diverted.

Preferably the inflows are not mixed prior to said dwell zone.

"Preferably the milk flow is the milk from at least one teat ofa known animal, such animal being identified by an output signal from an animal identifying sensor.

Preferably a receiver receives the output signal indicative of viscosity •i and associates such output signal with the animal by reference to either an input signal or a said animal identifying sensor output signal.

20 Preferably the data received by said receiver is stored for comparative purposes with subsequent data received at subsequent milkings from the same animal.

Preferably the method comprises drawing said sample flow off a milk flow during milking, said drawing off being substantially identical for each milking having regard to any one or more of: commencement of milking generated milk flow, -7- (ii) duration or volume of draw off, and (iii) elapsed milk flow.

In one particular embodiment, the testing in said viscosity testing zone of the outflow admixture product for viscosity is whilst the at least partly gelled product is stationary.

Preferably a rolling ball test for viscosity is utilised which generates said output signal indicative of such viscosity, such output signal being an elapsed time or a function of an elapsed time.

go Preferably the dwell zone and said viscosity testing zone are a common chamber of apparatus into which said inflows are provided.

oooo In yet another aspect the present invention provides a method of testing a milk flow for mastitis, said method comprising (and in any workable order) diverting a sample from a said milk flow from an animal; providing an animal identifier input or signal capable of identifying said animal to data acquisition, analysis and storage means; providing an inflow of an anionic detergent and an inflow of said milk sample in predetermined proportions into a dwell zone to allow at least partial gelling of the mixture; and providing an outflow of the at least partially gelled mixture of said dwell zone to and/or through and/or using a viscosity testing zone and (immediately or subsequently) generating an output signal to said data acquisition, analysis and storage means indicative of the viscosity of a predetermined part of the mixture that has been subject to a predetermined gel forming dwell time post mixing (whether -8prior to, during or post said inflow(s)).

Preferably the method further comprises clearing the viscosity testing zone or (ii) both the viscosity testing zone and said dwell zone of said mixture without feedback into the milk flow from which the sample was diverted.

Preferably the inflows are not mixed prior to said dwell zone.

Preferably the method includes storing the data received by said acquisition, analysis and storage means for comparative purposes with subsequent data received at subsequent milkings from the same animal.

o• Preferably the method includes drawing the sample flow offa milk flow 0*40 during milking, the drawing off being substantially identical for each milking 0*• 15 having regard to any one or more of: o00000 commencement of milking generated milk flow, (ii) duration or volume of draw off, and (iii) elapsed milk flow.

0~ 20 Preferably the testing in said viscosity testing zone of the outflow o admixture product for viscosity is whilst the at least partly gelled product is stationary.

Preferably method includes using a rolling ball test for viscosity that generates said output signal indicative of such viscosity, such output signal being an elapsed time or a function of an elapsed time.

-9- Preferably method includes vacuum recocking the ball for a subsequent viscosity test along with the emptying of the already tested admixture product from said viscosity testing zone or said viscosity testing zone and said dwell zone.

Preferably the predetermined proportions are in the volume ratios of 5:1 to 1:5, and more preferably the predetermined proportions are in the range of from 2:1 to 1:2 inclusive. Still more preferably the predetermined proportions are substantially 1:1.

Preferably the anionic detergent is a Gardinol Type Detergent as i defined in the Merk Index. More preferably the anionic detergent is an aqueous solution of about 2% w/v sodium laurel sulphate.

:ooo In still another aspect, the present invention provides an apparatus for testing a milk flow for mastitis, comprising: means for diverting a sample from said milk flow; and ~means for automatically testing said diverted sample flow by measuring *an attribute of any somatic cells in at least part of the milk of that sample; wherein said measuring is by reference to a gelling characteristic of the 20 milk under the action of an appropriate predetermined anionic detergent, and said measuring is by reliance upon the viscosity of at least part of the milk of that sample when in a predetermined admixture for a predetermined time with said anionic detergent.

Preferably the apparatus is such that an identical testing regime is followed for each sample taken from the milk flow such that for an individual animal or as between individual animals, or both, there is a comparative basis.

Preferably the apparatus further comprises data acquisition, analysis and storage means and means to provide an animal identifier input or signal capable of identifying an animal being milked to said data acquisition, analysis and storage means and said means to generate a signal or representation or record itself provides an output signal to said data acquisition, analysis and storage means indicative of the viscosity of the milk sample gel in the apparatus for tying to the animal identified.

Preferably the apparatus includes a rolling ball type viscosity tester capable of generating an output signal reliant on a elapsed time or a function of :elapsed time.

The invention also provides an apparatus for determining whether or not 15 there is a somatic cell count change between milkings in milk of an animal, comprising the above-described apparatus of the previous aspect, and further comprising means for comparing data resulting from said respective milkings to determine any somatic cell count change.

S 20 Preferably the apparatus further comprises means for generating a gel S°from the milk sample by means of an appropriate surfactant, means for testing the gel thus generated for viscosity or some function of viscosity, and means for causing or allowing the gel to clear from the apparatus.

In a further aspect the present invention provides a method of testing an animal for mastitis by on-line testing of milk from such an individual animal as it is milked, comprising: -11automatically diverting a sample from said milk flow; and automatically measuring said diverted sample flow by measuring an attribute of any somatic cells in at least part of the milk of that sample; wherein said automated measuring is by reference to a gelling characteristic of the milk under the action of an appropriate predetermined anionic detergent, and said automated measuring is by reliance upon the viscosity of at least part of the milk of that sample when in a predetermined admixture for a predetermined time with said anionic detergent.

In a further aspect the present invention provides an apparatus for testing the milk of an animal for somatic cells (and thus for testing an animal being S• milked for mastitis), suitable for-or for association with-a milk line ofa milking machine, the apparatus comprising: *means defining a liquid chamber having an ability to receive a first 15 liquid inlet feed to be an inflow of a sample flow of milk from the milk line and a second liquid inlet feed to be an inflow of an anionic detergent for causing a viscosity increase as a result of at least partial gelling of the resultant milk/detergent fluid; means to provide a measure of and to generate a data output indicative S• 20 of the viscosity or a function of viscosity of at least part of the resultant milk/detergent fluid calibratable to the somatic cell count of the milk inflow; and means for clearing said milk/detergent fluid from the apparatus without contamination of milk in the milk line after said means to provide a measure of and to generate a data output has at least taken a reading indicative of viscosity or a function of viscosity, in order to allow a subsequent milk flow sample to be likewise tested.

-12- In another aspect the present invention provides an apparatus for testing the milk of an animal for somatic cells (and thus for testing an animal being milked for mastitis), suitable for-or for association with-a milk line of a milking machine, the apparatus comprising: means defining a first chamber having an ability to receive a first liquid inlet feed to be an inflow of a sample flow of filing from the milk line and a second liquid inlet feed to be an inflow of an anionic detergent of a kind that will cause a viscosity increase as a result of at least partial gelling of the resultant milk/detergent fluid; means defining a second chamber to receive at least part of the resultant milk/detergent fluid from said first chamber; means to provide a measure indicative of viscosity or a function of viscosity of the resultant milk/detergent fluid in said second chamber calibratable to oooo the somatic cell count of the said sample flow of milk; and S 15 means to generate a data output indicative of said measure.

Preferably the apparatus comprises means adapted to in use reproducibly control the first and second inlet feeds.

20 Preferably there is provision in said means to provide a measure whereby said milk/detergent fluid can be cleared from the apparatus without contamination of milk in the milk line in order to allow a subsequent milk flow sample to be likewise tested.

Preferably the means to provide a second chamber is or includes the barrel of a rolling ball viscometer. Preferably the means to provide a measure are spaced sensors of a rolling ball viscometer.

-13- Preferably the means defining a first chamber and said means defining a second chamber are interconnected such that a chamber is defined into which there is adapted to be a greater infeed of fluids than outfeed therefrom thereby in use ensuring, for at least part of the milk/detergent fluid, a reproducible gelling time at which the fluid is measured by said means to provide a measure.

In yet another aspect the present invention provides a method of testing an animal substantially as hereinbefore described with reference to any one of the accompanying drawings.

o.

:In still a further aspect the invention consists in somatic cell count data and/or comparisons thereof generated using a method or apparatus of the present invention.

In still a further aspect the invention consists in a milking shed having provision for a testing regime in accordance with any of the earlier embodiments

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:stated.

20 Reference herein to diversion of or the taking of a sample of milk includes (where the context might allow) milk from one or more teats of the same animal or bulk milk, i.e. milk from a number of animals.

SCC data can be used for comparative purposes of for an absolute decision greater or less than 500,000 cells/mL) for an individual teat, for an animal or for a grouping of animals.

-14- DETAILED DESCRIPTION OF THE INVENTION In order that the present invention may be more clearly ascertained, preferred embodiments will now be described, by way of example, with reference to the accompanying drawing in which: Figures 1A and 1B show for the same herd (hereafter referred to as "DRC" herd) the time changes in somatic cell count (SCC) measured in thousands of cells per millilitre with the peaks showing the actual somatic cell count; Figure 1A shows for a July to April period the upper 50% of the DRC herd ranked in order of average SCC, the peaks being against a scale ranging from 0 to 5 million cells/mL, whilst correspondingly Figure 1B shows the remainder of the herd likewise ranked for the same period but over a range of somatic cell count depicted on an axis running to 300,000 cells/mL only, thus showing the significant variation per animal over the period and the extent and localised nature of infection in a herd, Figure 2 is a diagrammatic view of apparatus which shows a method 15 whereby a diverted flow can be dispatched into a container and thence from a

TM

machine controlled valve down a tube or the like a Gilmont Viscometer available from Cole-Parmer International, USA) *o *oo WO 01/35728 PCT/'NZ0/00232 with the viscosity of the gel formed in the container by appropriate means (not shown) being determined by the time of passage between two zones, (eg; measuring the gel flow commencement interface and gel flow completion interface using a light emitter and detector).

Figure 3 shows the effect of sodium laurel sulphate concentration in respect of

TEEPOL

T (14% w/v) of Shell Chemical Company using stock solution of bovine blood leucocytes, Figure 4 shows the effect of angle on a rolling ball type viscometer when compared with a Gilmont type falling fall viscometer, ie; which allows the ball to fall vertically (with respect to various aqueous concentrations of glycerine), Figure 5 shows the effect of ball size on the time the ball descends at various angles using 50% glycerine in water as the fluid, the slope of the resultant lines if plotted through the data points being approximately equal, Figure 6 shows the effect of changing ratio of milk to anionic detergent in water reagent (the milk being held to 1), Figure 7 shows the effect of SCC on viscosity using a 1:1 milk ratio, Figure 8 shows the effect of SCC on viscosity using a 2:1 milk ratio, Figure 9 shows a simplified plot of time for a rolling ball fall against SCC thereby showing a dynamic range of preference within which any testing regime should preferably operate, such dynamic range being described hereafter, Figure 10 shows a cross section of a rolling ball tube of rolling ball type viscometer of a kind in respect of which results for a quarter inch and five sixteenth inch balls are plotted in Figure Figure 11 is a plot (While and Raltray, 1965) showing for a particular cow and its four teats the changes in SCC during an after milking, the rise in cell count during milking as they have indicated often being a factor or 10 or more and with any decline hi cell count between milkings arising from dilution effects in the lower cisternal region of the animal and which decline may be subject to daily fluctuations in yield, Figure 12 is a prototype set up of apparatus to which the drawing sequence 12a to 12g refers, Figures 12A through 12G show in respect of a milk tube line or the like apparatus in accordance with the present invention showing diagrammatically various stages of a mastitis testing regime, Figure 13 is a block diagram showing preferred flow parameters and options, and WO 01/35728 PCT/NZ00/00232 -16- Figure 14 is a block flow diagram of electronic systems useful in apparatus and methods of the present invention.

Our invention provides a system on-line preferably in the milking parlour (eg; whether conventional or robotic) preferably using a gel type test. The viscosity of the gel can be measured by standard viscosity measurements (Cole Parmer 1999) time of air bubble travel, time of draining from container, or other].

In Figure 2 a container 1 leads to a drainage tube 3 controlled by a valve 2. In the container 1 there can be a merging or other mixing of a milk sample and sufficient non-anionic detergent to allow a standard gel representative of somatic cell count to be generated. A light emitter 4 or 6 in conjunction with a light detector 5 and 7 respectively determines the commencement of gel flow down the tube 3 and its termination. Such light sensor system if desired can be used in the apparatus of Figures 12 to 12g.

Figure 3 This figure was obtained by spinning down blood obtained from the Ruakura research abattoir, with heparin to stop blood clots. It was spun down so that the leukocytes could be extracted without red blood cells. This was then diluted with 0.15M NaCI to produce three solutions. The first solution was between 500 000-1 000 000 cells/ml. The second and third were /z and 1/5, of the first respectively.

Within 12 hours of blood being harvested it was tested. This was done by adding 4ml of reagent to 4ml cell solution in a 12ml centrifuge tube. When they were mixed a timer started, and the tube inverted once. When the timer reached 20 seconds the solution was poured into the Gilmont viscometer. Before the first sample the viscometer was rinsed once with water.

Then the stainless steel ball was added and the cap screwed on. The time taken for the ball to pass between the marks on the tube (10 Ocm apart) was timed. The viscometer was then inverted and the ball timed between the points again. This was then averaged.

The gel was then tipped out from the viscometer and it was rinsed once with water and the cycle started again.

Figure 4 this figure was obtained by using viscosity standards made from glycerine mixed with water. These standards were 25%, 50% and 75% glycerine in water. These standards were run through the Gilmont viscometer. This result was compared to the viscometer made in the laboratory. This was a 8.4mm diameter ID tube with a 7.9mm stainless ball. One result was taken with the viscometer at 90 degrees vertical) and the other at degrees from the horizontal.

Figure 5 this figure was obtained using the viscometer in the laboratory, using the WO 01/35728 PCT/NZ00/00232 -17glycerine standards. The angle was varied from the horizontal with two different diameter balls.

The 6.4mm and 7.9mm balls were stainless and carbon steel respectively. See Figure Figure 10 shows a cross section of the tube. The tube T, has an internal diameter D, and the ball B, has a diameter D 2 The difference between the two diameters determines the speed through the liquid. See Figure 5 for example. As the ball diameters increases it slows the movement down. However if the ball diameter becomes too large the non-homogonous nature of the gel stops the ball from rolling even though it could roll though a standard gel solution.

glycerine mixed with water).

Figure 6 this figure was taken with raw milk from the DRC herd within four hours of harvesting. 14% TeepolTM was used as the reagent. It was mixed with the same milk sample at different ratios using the same procedure as Figure 3. It was measured in the Gilmont viscometer in the same way as Figure 3.

Figure 7 this figure was created by mixing regent, (14% TeepolTM), 1:1 with milk before two hours had elapsed since harvest. The tests were done between 15-25 degrees centigrade.

Both the mixing and measuring procedure was the same as Figure 3.

Figure 8 this is exactly same as Figure 7 except the ratio reagent:milk was 1:2.

Figure 12A through 12G depict diagrammatically a preferred system in accordance with the present invention (eg; as shown in Figure 12) with the explanations as to the system modules and electronic systems being shown by way of flow diagrams Figures 13 and 14 respectively. In Figures 12A through 12G are shown the following milk tube or line 8 showing the pulsing movement of milk there along, a reagent reservoir milk line flow diversion valves V,, a sample reservoir reagent valve V 3 milk sample flow control valve V,, a flow control pump P, a dwell zone 9 with inflow greater than outflow, cell solenoid E to control a ball 10 retention pin to hold the ball until released in its upper cot condition as shown in Figure 12B, sensor locations 11 and 12, an air filter 13, a vacuum valve V 4 and WO 01/35728 PCT/NZ00232 -18- S a waste reservoir W.

The operation of the apparatus can best be described by reference to the cycle description in Table 2.

Description of Cycle: Table 2 Step in Cycle Description Components Used Figure 12A Evacuation and reset a.1.1 Turn on valve to evacuate the chamber and move ball V 4 On up a.1.2 Move pin up to allow ball to pass E (electro magnet) On a.1.3 Turn on valve and pump to remove any previous V, and P On sample a.2 Time delay a.3.1 Pin moved down E Off a.3.2 Sample valve and pump stopped V, and P Off a.3.3 Time delay a.3.4 Vacuum switched off V, Off Figure 12B Sampling milk sample b.1 Valve open V, On b.2 Time delay b.3 Valve dosed V, Off Figure 12C Mixing and sample into tube c.1.1 Sample valve open V, On c.1.2 Reagent valve open

V

3 On (may alternate with V,) c.1.3 Pump on P On c.1.4 Time delay c.2.1 Pump Off P Off c.2.2 Sample valve dosed V, Off c.2.3 Reagent valve closed V 3 Off Figure 12D Settling time d.1 Time for ball to pass sensors Figure 12E Ball release and measure e.1 Pin moved up E On e.2 Time ball to pass to predetermined places in measured Figure 12F Ball reaches bottom Figure 12G Reset and evacuation g.1.1 Turn on valve to empty the chamber and move ball up V 4 On g.1.2 Move pin up to allow ball to pass E (electro magnet) On g.1.3 Turn on valve and pump to remove any previous V 2 and P On sample g.2 Time delay g.3.1 Pin moved down E Off WO 01/35728 PCTINZ00/00232 Step In Cycle Description Components Used g.3.2 Sample valve and pump stopped V, and P Off g.3.3 Time delay g.3.4 Vacuum switched off V, Off The aforementioned cycle in respect of Figures 12A through 12G shows one method whereby apparatus could be worked. Another arrangement could be typified by the following tabulated procedure in Table 3.

Block Diagram of Rolling Ball Sensor (Table 3).

Way to hold ball at top system. Include Pin actuated by electromagnet or electromagnet holding ball directly Method of getting sample into tube. This could include pumps, gravity Tube with ball in it.

Tube diameter could be Svariable, and ball diameter viable.

Method to get ball up, this could include air flow/vacuum evacuation, gravity or electric field.

Method to evacuate the tube, this could include gravity, pumping or vacuum.

System for measuring ball, this could include light beam(s) being blocked, electromagnetic detection.

WO 01/35728 PCT/NZOO/00232 Still a further arrangement could be that depicted by Table 4.

Table 4 Method of getting sample into measurement system, this could include pumps, gravity Measurement system.

Method to evaluate and dean measurement system. Could include gravity, pumping or vacuum.

The apparatus is preferably automated online but can be. if desired, provided with a command feature whereby a farmer can run the testing regime for a particular cow or stall or during particular milkings.

Placement in Herringbone, rotary and robot In an herringbone milking system the SCC measuring device preferably will be placed such that it will analyse milk flowing between the claw and the milk line. It will be attached to the milk line and have the milk from the dropper tubes flow through it.

In a rotary it preferably will be again placed such that it will analyse milk flowing between the claw and the milk line. It will be attached to the rotary platform or milk line.

In a robotic milker preferably there will be one device per quarter or one device for the composite milk. It will be placed between the cups and milk collection vessel. Or after the collection vessel to test the composite milk.

Calibration equations Figure 9 shows an example of a calibration curve; this is obtained by measuring a sample with a known amount of SCC and recording the change in time These results are then transformed using some linear transformation technique. The results are then plotted with At versus SCC.

Over the dynamic range the At is related to the SCC by the following equation where b 0 WO 01/35728 PCTINZ00/00232 -21and b, are the coefficients of linear regression. These are calculated from least squares analysis of the data.

At= bo bSCC The SCC can then be determined by solving for SCC, which yields the following equation. Therefore by measuring the At, it is possible to determine the SCC.

SCC-

bi From the foregoing therefore it is believed that standard surfactant amounts coupled with standard milk sample amounts in substantially similar ambient conditions will provide sufficient consistency to allow worthwhile data to be generated from the detectors and to be processed to provide effective information to a dairy farmer.

Changes over milking, and over day to day Woolford et al 1998 shows that SCC can be used to determine mastitis,. If a threshold of 500000 cells/ml (see Table 1) is used the disease status can be determined. If the first milk is used (fore milk) the SCC only gives 5% false positives. If main milk is used then less false positives are obtained but more infections are missed. If the last milk (strippings) are used 9% of cows are false positives but more are detected (see tables 4 5 in Woolford et al).

As shown in attached Figure 11 the SCC changes over a milking. White and Rattray (1965) reported changes in cell count levels during and after milking, the rise in cell count during milking often being by a factor often or more.

Figure 1 1 shows individual quarter variations in cell count over a 70 minute period prior to, during, and after milking.

Therefore it is important to analyse the milk at a predetermined time after the start of milking every day. Schalm et al 1957 also stated that the SCC rose between the start of milking (foremilk) and the end (strippings).

Woolford et al (1998) also showed the measurement of milk every day is important to determine the health of the cow due to high day-to-day variation. In this respect see Figures 1A and 1B which are Figures 2 3 from Woolford et al.

-22- The present invention envisages preferably any desired same time consistency during milking, for example, tied to any one or more of: commencement of milking generated milk flow, (ii) duration or volume of draw off, and (iii) elapsed milk flow.

Modifications within the scope of the invention may be readily effected by those skilled in the art. It is to be understood, therefore, that this invention is not limited to the particular embodiments described by way of example hereinabove.

In the claims that follow and in the preceding description of the invention, except where the context requires otherwise owing to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

15 Further, any reference herein to prior art is not intended to imply that such prior f art forms or formed a part of the common general knowledge.

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Claims (38)

1. A method of testing a milk flow for mastitis, said method comprising: diverting a sample from said milk flow; and automatically testing said diverted sample flow by measuring an attribute of any somatic cells in at least part of the milk of that sample; wherein said measuring is by reference to a gelling characteristic of the milk under the action of an appropriate predetermined anionic detergent, and said measuring is by reliance upon the viscosity of at least part of the milk of that sample when in a predetermined admixture for a predetermined time with said anionic detergent.

2. A method of determining whether or not there is a somatic cell count change between milkings in milk of an animal by applying the method as claimed in claim 1 to separate milkings, and comparing data resulting from said testings to determine any 1 somatic cell count change.

3. A method claimed in either claim 1 or claim 2, wherein said attribute is of the DNA of the somatic cells. .ooooi

4. A method as claimed in any one of the preceding claims, wherein said milk flow is from a single animal and an identifier of that animal and an indicator of viscosity and thus of the somatic cell count is coupled for comparative purposes. A method of testing a milk flow for mastitis, said method comprising: diverting a sample flow from a said milk flow; Sproviding an inflow of an anionic detergent and an inflow of said milk sample in predetermined proportions into a dwell zone from whence the detergent/milk admixture as a gelling and/or gelled product can outflow only at a lesser rate than said inflow or inflows; feeding the outflow admixture product of said dwell zone to a viscosity testing zone; and testing in said viscosity testing zone the outflow admixture product for viscosity and generating an output signal indicative of such viscosity. -24-

6. A method as claimed in claim 5, comprising clearing the viscosity testing zone or (ii) both the viscosity testing zone and said dwell zone of said admixture without feedback into the milk flow from which the sample flow was diverted.

7. A method as claimed in either claim 5 or 6, wherein said inflows are not mixed prior to said dwell zone.

8. A method as claimed in any one of claims 5 to 7, wherein said milk flow is the milk from at least one teat of a known animal, such animal being identified by an output signal from an animal identifying sensor.

9. A method as claimed in either claim 7 or 8, wherein a receiver receives the output signal indicative of viscosity and associates such output signal with the animal by S: 15 reference to either an input signal or a said animal identifying sensor output signal.

10. A method as claimed in claim 9, wherein the data received by said receiver is stored for comparative purposes with subsequent data received at subsequent milkings ooooo S•from the same animal. o:oo

11. A method as claimed in any one of claims 7 to 10, comprising drawing said sample flow off a milk flow during milking, said drawing off being substantially identical for each milking having regard to any one or more of: commencement of milking generated milk flow, (ii) duration or volume of draw off, and (iii) elapsed milk flow.

12. A method as claimed in any one of claims 5 to 11, wherein the testing in said viscosity testing zone of the outflow admixture product for viscosity is whilst the at least partly gelled product is stationary.

13. A method as claimed in claim 12, wherein a rolling ball test for viscosity is utilised which generates said output signal indicative of such viscosity, such output signal being an elapsed time or a function of an elapsed time.

14. A method as claimed in claim 13, wherein said dwell zone and said viscosity testing zone are a common chamber of apparatus into which said inflows are provided. A method of testing a milk flow for mastitis, said method comprising: diverting a sample from a said milk flow from an animal; providing an animal identifier input or signal capable of identifying said animal to data acquisition, analysis and storage means; providing an inflow of an anionic detergent and an inflow of said milk sample in predetermined proportions into a dwell zone to allow at least partial gelling of the mixture; and providing an outflow of the at least partially gelled mixture of said dwell zone to and/or through and/or using a viscosity testing zone and generating an output signal to 15 said data acquisition, analysis and storage means indicative of the viscosity of a predetermined part of the mixture that has been subject to a predetermined gel forming dwell time post mixing. 0 00•

16. A method as claimed in claim 15, further comprising clearing the viscosity testing zone or (ii) both the viscosity testing zone and said dwell zone of said mixture without feedback into the milk flow from which the sample was diverted.

17. A method as claimed in either claim 15 or 16, wherein said inflows are not mixed 2 prior to said dwell zone. S•18. A method as claimed in any one of claims 15 to 17, including storing the data received by said acquisition, analysis and storage means for comparative purposes with subsequent data received at subsequent milkings from the same animal.

19. A method as claimed in any one of claims 15 to 18, comprising drawing said sample flow off a milk flow during milking, said drawing off being substantially identical for each milking having regard to any one or more of: commencement of milking generated milk flow, 26 (ii) duration or volume of draw off, and (iii) elapsed milk flow. A method as claimed in any one of claims 15 to 19, wherein the testing in said viscosity testing zone of the outflow admixture product for viscosity is whilst the at least partly gelled product is stationary.

21. A method as claimed in claim 20, including using a rolling ball test for viscosity that generates said output signal indicative of such viscosity, such output signal being an elapsed time or a function of an elapsed time.

22. A method as claimed in claim 21, including vacuum recocking the ball for a subsequent viscosity test along with the emptying of the already tested admixture product from said viscosity testing zone or said viscosity testing zone and said dwell zone.

23. A method as claimed in any one of claims 5 to 22, wherein said predetermined 0*• proportions are in the volume ratios of5:1 to

24. A method as claimed in claim 23 wherein said predetermined proportions are in the range of from 2:1 to 1:2 inclusive.

25. A method as claimed in claim 24 wherein said predetermined proportions are o substantially 1:1. S• o

26. A method as claimed in any one of the preceding claims, wherein said anionic detergent is a Gardinol Type Detergent as defined in the Merk Index.

27. A method as claimed in claim 26, wherein said anionic detergent is an aqueous solution of about 2% w/v sodium laurel sulphate.

28. An apparatus for testing a milk flow for mastitis, comprising: means for diverting a sample from said milk flow; and 27 means for automatically testing said diverted sample flow by measuring an attribute of any somatic cells in at least part of the milk of that sample; wherein said measuring is by reference to a gelling characteristic of the milk under the action of an appropriate predetermined anionic detergent, and said measuring is by reliance upon the viscosity of at least part of the milk of that sample when in a predetermined admixture for a predetermined time with said anionic detergent.

29. An apparatus as claimed in claim 28, wherein said apparatus is such that an identical testing regime is followed for each sample taken from the milk flow such that for an individual animal or as between individual animals, or both, there is a comparative basis. An apparatus as claimed in either claim 28 or 29, further comprising data acquisition, analysis and storage means and means to provide an animal identifier input or signal capable of identifying an animal being milked to said data acquisition, analysis and storage means and said means to generate a signal or representation or record itself provides an output signal to said data acquisition, analysis and storage means indicative *00* Oof the viscosity of the milk sample gel in the apparatus for tying to the animal *eve*: identified. An apparatus as claimed in any one of claims 28 to 30, wherein said apparatus includes a rolling ball type viscosity tester capable of generating an output signal reliant on a elapsed time or a function of elapsed time. 25 32. An apparatus for determining whether or not there is a somatic cell count change S° between milkings in milk of an animal, comprising the apparatus as claimed in claim 28, and further comprising means for comparing data resulting from said respective milkings to determine any somatic cell count change.

33. An apparatus as claimed in claim 32, further comprising means for generating a gel from the milk sample by means of an appropriate surfactant, means for testing the gel thus generated for viscosity or some function of viscosity, and means for causing or allowing the gel to clear from the apparatus. -28-

34. A method of testing an animal for mastitis by on-line testing of milk from such an individual animal as it is milked, comprising: automatically diverting a sample from said milk flow; and automatically measuring said diverted sample flow by measuring an attribute of any somatic cells in at least part of the milk of that sample; wherein said automated measuring is by reference to a gelling characteristic of the milk under the action of an appropriate predetermined anionic detergent, and said automated measuring is by reliance upon the viscosity of at least part of the milk of that sample when in a predetermined admixture for a predetermined time with said anionic detergent. An apparatus for testing the milk of an animal for somatic cells, said apparatus comprising: means defining a liquid chamber having an ability to receive a first liquid inlet "feed to be an inflow ofa sample flow of milk from a milk line and a second liquid inlet •:feed to be an inflow of an anionic detergent for causing a viscosity increase as a result of at least partial gelling of the resultant milk/detergent fluid; means to provide a measure of and to generate a data output indicative of the 20 viscosity or a function of viscosity of at least part of the resultant milk/detergent fluid calibratable to the somatic cell count of the milk inflow; and means for clearing said milk/detergent fluid from the apparatus without contamination of milk in the milk line after said means to provide a measure of and to generate a data output has at least taken a reading indicative of viscosity or a function of 25 viscosity, in order to allow a subsequent milk flow sample to be likewise tested.

36. An apparatus for testing milk of an animal for somatic cells, said apparatus comprising: means defining a first chamber having an ability to receive a first liquid inlet feed to be an inflow of a sample flow of filing from a milk line and a second liquid inlet feed to be an inflow of an anionic detergent of a kind that will cause a viscosity increase as a result of at least partial gelling of the resultant milk/detergent fluid; means defining a second chamber to receive at least part of the resultant II -29- milk/detergent fluid from said first chamber; means to provide a measure indicative of viscosity or a function of viscosity of the resultant milk/detergent fluid in said second chamber calibratable to the somatic cell count of the said sample flow of milk; and means to generate a data output indicative of said measure.

37. An apparatus as claimed in either claim 35 or 36, comprising means adapted to in use reproducibly control the first and second inlet feeds.

38. An apparatus as claimed in any one of claims 35 to 37, wherein there is provision in said means to provide a measure whereby said milk/detergent fluid can be cleared from the apparatus without contamination of milk in the milk line in order to allow a subsequent milk flow sample to be likewise tested.

39. An apparatus as claimed in any one of claims 35 to 38, wherein said means to provide a second chamber is or includes the barrel of a rolling ball viscometer. An apparatus as claimed in claim 39, wherein said means to provide a measure are spaced sensors ofa rolling ball viscometer.

41. An apparatus as claimed in any one of claims 35 to 38, wherein said means defining a first chamber and said means defining a second chamber are interconnected such that a chamber is defined into which there is adapted to be a greater infeed of fluids than outfeed therefrom thereby in use ensuring, for at least part of the milk/detergent fluid, a reproducible gelling time at which the fluid is measured by said means to provide a measure.

42. A method of testing an animal substantially as hereinbefore described with reference to any one of the accompanying drawings.

43. A method of testing animals substantially as hereinbefore described with reference to any one of the accompanying drawings. I

44. A method of testing a milk flow substantially as hereinbefore described with reference to any one of the accompanying drawings. A method of testing milk substantially as hereinbefore described with reference to any one of the accompanying drawings.

46. An apparatus substantially as hereinbefore described with reference to any one of the accompanying drawings.

47. Somatic cell count data and/or comparisons thereof generated using a method as claimed in any one of claims 1 to 27, 34 and 42 to

48. Somatic cell count data and/or comparisons thereof generated using an apparatus as claimed in any one of claims 28 to 33, 35 to 41 and 46. Dated this 15th day of June 2004 SENSORTEC LIMITED By their Patent Attorneys GRIFFITH HACK 2 0 Fellows Institute of Patent and Trade Mark Attorneys of Australia