METHOD FOR THE DETECTION OF MASTITIS AND MILK QUALITY AND MASTITIS SENSOR

Information

  • Patent Application
  • 20120115184
  • Publication Number
    20120115184
  • Date Filed
    June 09, 2009
    15 years ago
  • Date Published
    May 10, 2012
    12 years ago
Abstract
A mastitis sensor and a method for detection of mastitis and determination of milk quality in real time (on-line). Methods and apparatuses for the rapid non-invasive determination of the concentration of dissolved molecular oxygen in milked milk are disclosed. Mastitis sensors are disclosed that include a fiberoptic, amperometric or potentiometric device for the determination of oxygen concentration, a device for data acquisition and processing, mastitis indicator and a device that generates a signal for the automatic on-line elimination of substandard milk of infected animals to prevent the pollution of bigger quantities of milk.
Description
TECHNICAL FIELD OF THE INVENTION

The present invention relates to the fields of veterinary and milk production, particularly to the determination of raw milk quality. The invention is useful for rapid detection of mastitis and other inflammatory processes in real time, but also for the quick determination of the quality of raw milk and on-line separation of substandard milk to avoid the pollution of dairy production.


BACKGROUND OF THE INVENTION

Mastitis is the most common infection of dairy cattle and it causes economic losses, being a major problem in the whole world.


The diagnosis of mastitis is currently based on different non-invasive methods:

    • detection of somatic cell count (SCC) in milk spectrometrically (patent application US2008000426; Grabek et al., 2008) or viscosimetrically, where anionic surfactant is added to the milk, causing the formation of gel of the proteins in somatic cells, which viscosity is measured and calibrated against the somatic cell concentration (U.S. Pat. No. 2,935,384; Schalm, O., Noorlander, D., 1960);
    • detection of lactate in milk (patent EP1192460, Agresearch, 2003)—bacteria causes the elevated concentration of lactate in the udder (anaerobic environment);
    • measuring the conductivity of milk; this method is relatively nonspecific, as milk conductivity is influenced by other factors than bacteria and normal biological variation in conductivity has nothing to do with mastitis. (Kamphuis C. Making sense of sensor data: detecting clinical mastitis in automatic milking systems. Dissertation. Faculty of Veterinary Medicine, Utrecht University, the Netherlands, 2010);
    • monitoring the ratios of various ions in milk. As the level of mastitic infection progresses, the concentration of sodium ions increases and potassium ions decreases (international patent application WO/2006/127921, Westfall, G., 2006);
    • detection of MAA. In response to an infection, mammalian immune system produces acute phase proteins, e.g. Milk Amyloid A (MAA) protein is produced in cow's udder (“PHASE”™ Milk Amyloid A (MAA) Assay Cat. No.: TP-807, Tridelta Development Limited (Ireland), www.trideltaltd.com);
    • microbiological tests for the detection of mastitis-causing bacteria, e.g. RAPIDEC Staph tests for the detection of S. aureus (analysis time 24 h) (Boerlin, P. et al., J Clin Microbial., 2003, 41(2):767-771);
    • spectrophotometric methods based on the application of chemical reagents (patents CN100460866, Ox. Biolog. Tech. Co. Ltd, 2009 and U.S. Pat. No. 6,979,550 Rivas et al., 2005), which produce a coloured product with the detectable compound;
    • detection method based on infrared thermography (international patent application WO0057164, Emerge Interactive Inc., 2000);
    • fresh milk is centrifuged in special pipettes and pathogens are detected by the number of cells in different sedimentation layers (Garcia-Cordero, J. L., Barrett L. M., O'Kennedy, R & Ricco, A. J. Microfluidic Sedimentation Cytometer for Milk Quality and Bovine Mastitis Monitoring. Biomedical Microdevices, 12:1051-1059, 2010).
    • the chemiluminescence assay is used to measure the ability of phagocytes to emit light after bacterial invasion (Takahashi, H. “Cytokine Therapy for Staphylococcus Mastitis in Dairy Cows” Science & Technonews Tsukuba, 1999, 50:55-56).


Another disclosed approach is the method of mastitis detection, which is based on the determination of lactate in milk and comparing the lactate level with the lactate levels of healthy animals' milk (U.S. Pat. No. 7,033,836, Pastoral Agric. Res. Inst. Nz Ltd., 2006).


However, the above methods have several disadvantages:

    • Relatively low lifetime of the sensor. For example, the lactate sensor needs frequent renewal, as its recognition system is based on enzymes. The (optical) oxygen sensor, applied in the present invention, can be operated for years;
    • Lactate concentration in milk depends on many different factors—feeding, milking frequency, lactation phase etc.;
    • Most of the abovementioned methods are not applicable on-line in real time course and it is not possible to eliminate substandard milk in the course of milking;
    • Some methods require addition of different compounds to the milk;
    • Some methods require costly equipment and highly-qualified personnel.


In scientific studies, the dissolved oxygen concentration in udder (before milking) has been studied with the purpose of studying whether the dissolved oxygen content in udder of normal cows and those of mastitis were sufficient to support normal neutrophil, function to eliminate S. aureus. Neutrophils kill bacteria by 2 methods: oxidative and non-oxidative. When neutrophils are stimulated to phagocytose, there will be an increase in oxygen consumption and the production of oxygen radicals (e.g., superoxide), resulting from the activation of NADPH oxidase, which forms an electron transport chain converting molecular O2 to superoxide. It was found, that mastitis led to a dramatic drop in O2 concentration and the antimicrobial activity of neutrophils in udder was depressed. Normal cows have the levels of dissolved O2 in milk similar to those in venous blood; the levels of dissolved O2 in mastitic cows are less than 10% of control values (Mayer S J, Waterman A E, Keen P M, Craven N, Bourne J. “Oxygen concentration in milk of healthy and mastitic cows and implications of low oxygen tension for the killing of Staphylococcus aureus by bovine neutrophils.” Journal of Dairy Research 1988; 55(4): 513-9).


There are no methods known in which the determination of dissolved O2 in milk have been used for the detection of mastitis.





BRIEF DESCRIPTION OF THE DRAWINGS

For the present invention to be clearly understood and readily practiced, the present invention will be described in conjunction with the following FIGURES, wherein like reference characters designate the same or similar elements, which figures are incorporated into and constitute a part of the specification, wherein:



FIG. 1 displays a histogram of oxygen concentration data in freshly milked milk (385 measurements) and the approximation of these data to the normal distribution (continuous line). ◯—results close to the mean value (377 measurements); —outliers (8 measurements).





DISCLOSURE OF THE INVENTION

The present invention proposes a quick and reliable method for the detection of mastitis and the determination of the quality of milk in real time and a mastitis sensor. The method for the detection of mastitis and the determination of the quality of milk is based on the noninvasive measurement of dissolved molecular oxygen in milk. In this instance, the oxygen concentration is substantially bigger or smaller from the normal concentration of dissolved oxygen in milk (difference is more than 3 standard deviation σ values from the mean value of the typical concentration or other given threshold), mastitis or other inflammatory processes in the organism of the animal and the substandard milk are detected on-line. In milking systems, based on the application of vacuum devices, the measurement of oxygen is carried out as quickly as possible, but not later than 60 seconds after the beginning of the milking process to avoid the mass transfer of oxygen from air. The oxygen concentration is measured in milk from one or several teats.


The mastitis sensor comprises a fiberoptic, amperometric or potentiometric device for the determination of oxygen concentration; a device for data acquisition and processing; mastitis indicator and a device, generating a signal for the automatic on-line elimination of substandard milk. The mastitis sensor is used for the application of the method, described in the present invention for the detection of mastitis and the determination of the quality of milk.


Proposed in the present invention methods and mastitis sensor have several advantages in comparison with earlier solutions, as they allow:

    • to detect quickly the potential mastitis sources in subclinical phase in milking animals, like cows, goat, sheep etc.;
    • to detect mastitis and other inflammatory processes and determine the quality of milk on-line;
    • to remove the infected animals' substandard milk before milk collecting tank;
    • the application of mastitis sensor is very easy and does not require special skills.


Maintenance costs of the proposed method and device are low due to the long lifetime of the sensors and fact, that there is no regular need for waste materials or renewal of the system components. Detection of infected animals in real time enables the reduction of production costs, as the substandard milk can be eliminated early in the milking process and the pollution of bigger amounts of milk prevented. There is no need for time-consuming and expensive analytical procedures. Application of the proposed device does not require special training of the personnel. The detection of animals with sub-clinical mastitis in the early phase of the infection allows starting early treatment of the animals with more effective results. So the application of the method and device gives economic effect in reducing both the steady and running costs of production, but also in the improvement of animal welfare. The following examples illustrate the application of the invention, although the invention is not limited with the following examples, but can be applied according to the claims.


DESCRIPTION OF EMBODIMENTS

Method for the detection of mastitis and determination of milk quality was used in a farm, where we measured the concentration of dissolved oxygen with a Clark-type sensor in the milk of 385 cows. Milk probes of 12 ml were taken from the milking device and oxygen was measured in the probes as quickly as possible, but not later than 60 seconds after taking the probes from the device to prevent the mass transfer of oxygen from the surrounding air into milk.


We analyzed the obtained results and calculated the normalized mean value and the standard deviation σ of oxygen concentration. The results are shown on FIG. 1 and Table 1.









TABLE 1







The normalized mean concentration of oxygen in milk of


different cows.















cO2

cO2

cO2

cO2



normal-

normal-

normal-

normal-


No.
ized
No.
ized
No.
ized
No.
ized





 1.
0.8410041
 98.
1.018828
195.
1.023013
292.
0.9767441


 2.
0.7175732
 99.
1.108787
196.
0.9853556
293.
1.023256


 3.
0.8556485
100.
1.087866
197.
1.002092
294.
0.9790697


 4.
1.010460
101.
1.010460
198.
1.002092
295.
1.006977


 5.
1.056485
102.
1.104602
199.
1.012552
296.
1.074419


 6.
0.9707112
103.
1.135983
200.
1.129707
297.
1.065116


 7.
0.8807531
104.
0.9958159
201.
0.9707112
298.
0.9186046


 8.
0.9205021
105.
1.129707
202.
1.012552
299.
1.097674


 9.
0.9748953
106.
1.064854
203.
1.020921
300.
1.134884


10.
0.9225941
107.
1.041841
204.
0.916318
301.
0.8930232


11.
0.878661
108.
1.008368
205.
0.9016736
302.
1.172093


12.
0.9435146
109.
1.043933
206.
0.4651163
303.
1.074419


13.
0.962343
110.
0.9958159
207.
1.144186
304.
1.090698


14.
0.7740586
111.
1.309623
208.
1.090698
305.
1.044186


15.
0.9832635
112.
1.138075
209.
0.8860465
306.
1.569767


16.
0.9790794
113.
1.215481
210.
1.046512
307.
1.083721


17.
0.9058577
114.
1.284519
211.
0.9186046
308.
1.081395


18.
0.9414226
115.
1.012552
212.
0.9186046
309.
1.006977


19.
0.9205021
116.
1.152720
213.
1.104651
310.
1.058140


20.
0.9832635
117.
1.223849
214.
0.8116279
311.
1.453488


21.
1.016736
118.
1.148535
215.
0.7953488
312.
1.034884


22.
0.9205021
119.
1.046025
216.
1.046512
313.
1.093023


23.
0.962343
120.
1.096234
217.
0.9186046
314.
1.109302


24.
0.9518828
121.
1.037657
218.
0.9813952
315.
1.067442


25.
0.9351463
122.
1.085774
219.
0.9604651
316.
1.141860


26.
0.8849372
123.
1.046025
220.
1.093023
317.
1.044186


27.
0.8033472
124.
1.014644
221.
1.255814
318.
1.069767


28.
0.8828451
125.
1.031381
222.
1.069767
319.
1.295349


29.
0.9853556
126.
1.062761
223.
1.088372
320.
1.018605


30.
0.792887
127.
1.152720
224.
0.9279069
321.
1.067442


31.
0.8619246
128.
1.184100
225.
1.155814
322.
1.062791


32.
0.9811715
129.
1.056485
226.
1.093023
323.
1.097674


33.
0.956067
130.
1.002092
227.
0.9720929
324.
1.104651


34.
0.962343
131.
1.014644
228.
1.123256
325.
1.076744


35.
0.9100418
132.
1.169456
229.
1.041860
326.
1.006977


36.
0.956067
133.
1.052301
230.
0.9209302
327.
1.369767


37.
1.106694
134.
0.8472803
231.
0.8999999
328.
0.9139535


38.
0.9811715
135.
1.033473
232.
0.9069767
329.
1.051163


39.
1.087866
136.
0.9456066
233.
1.074419
330.
1.039535


40.
0.9079498
137.
0.9832635
234.
1.139535
331.
1.034884


41.
0.8995816
138.
1.115063
235.
1.051163
332.
0.9860464


42.
0.9309623
139.
1.046025
236.
0.8651162
333.
1.055814


43.
1.027197
140.
1.303347
237.
1.004651
334.
1.032558


44.
0.9895397
141.
1.054393
238.
0.8906976
335.
1.158139


45.
0.9916317
142.
1.052301
239.
0.9953489
336.
1.074419


46.
0.9414226
143.
1.110879
240.
1.079070
337.
1.013953


47.
0.9539748
144.
0.9937238
241.
0.9418605
338.
0.9860464


48.
1.018828
145.
1.056485
242.
0.9558139
339.
0.9697674


49.
0.9351463
146.
0.9539748
243.
1.018605
340.
1.041860


50.
0.9916317
147.
1.004184
244.
0.9860464
341.
0.9581395


51.
0.9539748
148.
0.9853556
245.
0.8930232
342.
1.034884


52.
0.8807531
149.
0.9539748
246.
0.844186
343.
1.006977


53.
0.9581589
150.
0.9790794
247.
0.8697674
344.
0.9767441


54.
1.039749
151.
1.004184
248.
0.8511628
345.
0.9976743


55.
0.9456066
152.
0.9351463
249.
0.9488372
346.
0.9325582


56.
1.006276
153.
1.025105
250.
1.002326
347.
1.041860


57.
1.083682
154.
1.025105
251.
0.9255813
348.
1.025581


58.
1.016736
155.
0.9769874
252.
0.8139535
349.
0.9348837


59.
1.023013
156.
0.9665271
253.
0.8162791
350.
1.023256


60.
1.127615
157.
1.066946
254.
0.988372
351.
1.102325


61.
0.9686192
158.
1.138075
255.
0.9279069
352.
1.027907


62.
1.050209
159.
0.9895397
256.
1.123256
353.
0.7604651


63.
0.9748953
160.
0.9665271
257.
1.125581
354.
0.9418605


64.
0.916318
161.
0.960251
258.
0.988372
355.
1.016279


65.
1.004184
162.
1.048117
259.
1.000000
356.
0.9232558


66.
0.8284519
163.
1.077406
260.
0.9372093
357.
0.9395348


67.
0.9539748
164.
0.9874476
261.
0.9093023
358.
0.9697674


68.
0.834728
165.
0.9790794
262.
0.9418605
359.
1.006977


69.
0.9644352
166.
0.8640167
263.
1.006977
360.
0.9953489


70.
1.073222
167.
0.9539748
264.
0.8860465
361.
0.9813952


71.
1.058577
168.
1.006276
265.
0.9139535
362.
1.004651


72.
0.9832635
169.
0.9539748
266.
0.9744186
363.
0.8372092


73.
0.916318
170.
1.094142
267.
0.8790697
364.
0.9279069


74.
0.9288703
171.
1.004184
268.
1.065116
365.
0.9325582


75.
0.8744769
172.
0.9958159
269.
1.023256
366.
0.8744186


76.
1.048117
173.
0.9539748
270.
1.004651
367.
0.9418605


77.
1.025105
174.
1.232218
271.
1.011628
368.
0.9767441


78.
0.9832635
175.
0.9414226
272.
1.102325
369.
1.037209


79.
0.8870292
176.
0.9267781
273.
1.093023
370.
0.7418604


80.
0.9832635
177.
0.9267781
274.
0.8558139
371.
0.8790697


81.
0.9937238
178.
0.9100418
275.
1.209302
372.
0.8418604


82.
1.066946
179.
1.035565
276.
1.102325
373.
0.8860465


83.
1.274059
180.
0.9769874
277.
1.106977
374.
0.8023255


84.
0.9728034
181.
1.052301
278.
1.162791
375.
0.9860464


85.
1.002092
182.
0.9686192
279.
0.9069767
376.
0.944186


86.
0.956067
183.
0.9790794
280.
1.055814
377.
0.9232558


87.
0.956067
184.
1.194561
281.
1.132558
378.
0.944186


88.
0.9979079
185.
1.100418
282.
1.072093
379.
0.9465116


89.
0.9309623
186.
1.104602
283.
1.109302
380.
0.8279069


90.
1.012552
187.
0.9958159
284.
1.030232
381.
0.9395348


91.
1.000000
188.
1.066946
285.
1.060465
382.
0.9697674


92.
0.9497907
189.
1.079498
286.
1.018605
383.
0.8604651


93.
0.9895397
190.
0.9686192
287.
1.065116
384.
0.8465116


94.
0.960251
191.
0.9267781
288.
1.218605
385.
0.9069767


95.
1.000000
192.
0.9351463
289.
1.025581




96.
1.033473
193.
0.8451883
290.
1.076744




97.
0.960251
194.
0.9769874
291.
1.086046









According to FIG. 1, in 377 cases (97.9%) the obtained results were within span, which in the case of normal distribution should include 99% of results (area between 2 arrows on the FIGURE). The results were out of this span in 8 cases (12.1%).


Cows, whose milk oxygen levels were different from the established threshold (mean cO2 value±3σ), were taken under special observation. From this group, 50% of cows were diagnosed clinical mastitis during the observation period.


The concentration of dissolved oxygen was measured with Clark-type oxygen sensor also in the milk from different tits of the infected with mastitis cows, milked manually. In milk probes of 12 ml the oxygen concentration was measured right after milking within 60 seconds. The results of the measurements of oxygen in milk from infected udder quarters of mastitic animals were notably different from the results in milk from healthy animals. In most probes of the milk from infected udder quarters of mastitic animals, the oxygen concentration was considerably lower in comparison with milk of healthy animals (approximately 2 times lower), milked in similar conditions. There were also probes from infected udder quarters, in which oxygen concentration was considerably higher than in milk from healthy animals, taken in similar conditions. In conclusion the measured oxygen concentrations in all milk probes from infected udder quarters of mastitic animals were drastically different from the mean value of oxygen concentration in milk from healthy animals.


The above-described procedure of measuring oxygen concentration in milk was also used for the determination of milk quality. In case, the measured oxygen concentration in milk probes from infected udder quarters was drastically different from the mean value of oxygen concentration in milk from healthy animals (difference more than 3 σ values), substandard milk was identified and this milk was separated on-line before reaching the milk tubes and directed to waste.


The mastitis sensor consisted of an oxygen sensor, a device for the digitalization of the sensor analogue output signal, an automatic data acquisition and processing system and a mastitis indicator, where the results were compared with the normalized mean value of oxygen concentration and in case of establishing significant difference (over 3 σ) in the results, a signal lamp lightened on the panel of the indicator. The mastitis sensor enables to generate a signal, which starts the system of on-line separation of substandard milk from quality milk if necessary. The mastitis sensor is placed in milking tubes or in small collecting tanks in milking system, calibrated according to the temperature of the testing place and the concentration of dissolved oxygen in milk is measured in real time.


In case the measured oxygen concentration in milk is considerably different from the mean value of oxygen concentration (normally the concentration of oxygen in milk is 65 to 75% of the oxygen saturation concentration at 38.6° C. or 4.30 to 4.95 mg/l accordingly; the oxygen saturation concentration at 38.6° C. is 6.60 mg/l) and the difference with the mean value is more than 3 σ values, the animal is likely to have subclinical or clinical mastitis; in case the difference is 2-3 σ values, additional examination of the animal is recommended and in case the difference is smaller, the animal is healthy. The oxygen concentration in milk from infected udder quarters of animals suffering from mastitis, is 2-3 times lower than normal (23-49% of oxygen saturation concentration at 38.6° C.) or on the other extreme equals to the oxygen saturation concentration (100%).


Results, obtained with the mastitis sensor, are displayed on the screen of the device in the form of a continuous or discrete colour scale (e.g. difference over 3 σ generates a red, difference between 2 to 3 σ generates a yellow and difference under 2 σ values generates a green indicator colour) or as a numerical output.

Claims
  • 1. A method for the detection of mastitis in animals, comprising the following steps: a) an animal is milked and the concentration of dissolved molecular oxygen in milk is measured non-invasively;b) the concentration of dissolved molecular oxygen in milk is compared with typical concentration of dissolved molecular oxygen found in uninfected milk; andc) in case the oxygen concentration in milk is significantly different from the typical oxygen concentration in uninfected milk (difference is more than 3 standard deviation σ values from the mean value of the typical concentration or other given threshold), mastitis is detected in the animal in real time course.
  • 2. The method according to claim 1, wherein the concentration of dissolved oxygen in milk is measured with a fiberoptic, amperometric or potentiometric device.
  • 3. The method according to claim 1, wherein an animal is milked with a vacuum milking system and the concentration of dissolved molecular oxygen in milk is determined in the milking system with no access of external air.
  • 4. The method according to claim 1, wherein an animal is milked manually and the concentration of dissolved molecular oxygen in milk is determined not later than 60 seconds after the beginning of milking.
  • 5. The method according to claim 1, wherein the concentration of dissolved molecular oxygen in milk is determined in one or more udder quarters.
  • 6. The method according to claim 1, wherein the typical concentration of dissolved molecular oxygen in milk is determined as the mean dissolved oxygen concentration of the farm or the herd, or as the mean of the measured dissolved oxygen concentrations of an animal and the allowed deviation from the typical value of the concentration of the dissolved molecular oxygen is established on the basis of the mean value of the dissolved molecular oxygen of the farm or herd.
  • 7. A method for the determination of milk quality in real time, wherein the milk of the animal, in whose organism mastitis has been detected according to claim 1, is determined as substandard.
  • 8. The method according to claim 1, wherein the animal is a cow, a goat or a sheep.
  • 9. Mastitis sensor for the application of the method according to claim 1, comprising a fiberoptic, amperometric or potentiometric device for the determination of oxygen concentration, a device for data acquisition and processing, mastitis indicator and a device, generating a signal for the automatic on-line elimination of substandard milk.
CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase application pursuant to 35 U.S.C. §371 of International Application No. PCT/EE2009/000009, filed Jun. 9, 2009.

PCT Information
Filing Document Filing Date Country Kind 371c Date
PCT/EE09/00009 6/9/2009 WO 00 1/23/2012