Patent Application
20230251217
The present invention relates to a method and a system for testing for mastitis in a domestic animal.
Economic losses in Japan due to mastitis, which is one of infectious diseases suffered by dairy cows, amount to 80 billion yen per year. Since early detection of mastitis can reduce the cost of treatment and inhibit decreased milk production, research and development for early detection of mastitis is underway.
For example, Patent Literature 1 discloses a method for diagnosing mastitis by measuring a somatic cell count in milk. Patent Literature 2 discloses a method for diagnosing mastitis by measuring an electrical conductivity of milk. Patent Literature 3 discloses a method for diagnosing mastitis by decomposing somatic cells in milk to produce cytokine and the like and thus activate somatic cells, and then measuring a somatic cell count. Patent Literature 4 discloses a method for diagnosing mastitis by measuring an electrical conductivity of centrifuged milk.
Methods for diagnosing mastitis by somatic cell count measurement have a problem that devices for measuring a somatic cell count are expensive. Measurement of a somatic cell count by microscopic observation has a problem that the operation is complicated. Methods for diagnosing mastitis by measuring an electrical conductivity of milk are not suitable for early diagnosis of mastitis because the methods observe a change caused by an inflammatory reaction after invasion by a pathogen. Further, since a composition ratio of electrolytes contained in milk varies among quarters of udder of the same individual or among individuals, reproducibility in the measurement of an electrical conductivity of milk is poor. Thus, there has not been developed a technique that can easily detect mastitis in a domestic animal, such as a dairy cow, at an early stage.
It is an object of an aspect of the present invention to provide a test that can easily detect mastitis in a domestic animal, such as a dairy cow, at an early stage.
In order to attain the object, the present invention includes the following aspects.
An aspect of the present invention makes it possible to provide a test that can easily detect mastitis in a domestic animal, such as a dairy cow, at an early stage.
A method (hereinafter, also abbreviated as a “testing method of the present invention”), in accordance with an embodiment of the present invention, for testing for mastitis in a domestic animal includes the step of measuring, by nuclear magnetic resonance (NMR), a spin-spin relaxation time of milk (hereinafter, also referred to as “sample milk”) collected from a quarter of the domestic animal.
Examples of the domestic animal include cattle such as a dairy cow, a goat, a buffalo, a sheep, and a horse. For example, cattle such as a dairy cow have four independent quarters: a right front quarter; a right rear quarter; a left front quarter; and a left rear quarter.
The nuclear magnetic resonance (NMR) used in the testing method of the present invention only needs to be capable of measuring a spin-spin relaxation time (T2) of sample milk, and examples of the NMR include pulsed NMR. From perspectives such as reduction of a size of a measuring device, it is preferable to use pulsed NMR.
The milk can be collected from a quarter of the domestic animal by means ordinarily used by a person skilled in the art, such as a milking machine. By attaching an NMR measurement device to the milking machine, the spin-spin relaxation time (T2) of the milk can be measured easily and non-invasively. In NMR (in particular, pulsed NMR), one measuring time is as short as several tens of seconds. Further, by attaching an NMR measurement device to an automatic milking machine, the spin-spin relaxation time (T2) can be measured continuously at regular intervals. Further, by attaching an NMR measurement device to an automated milking machine, there is no need to sample milk for testing for mastitis, and the burden on a manager of domestic animals can be reduced. The testing method of the present invention therefore allows real-time measurement (testing). Note that it is preferable to have the above configuration in which an NMR measurement device is attached to a milking machine and a portion of the milking machine is used as a flow cell for NMR measurement. However, the present invention is not particularly limited to such a configuration.
The spin-spin relaxation time or a specific surface area of particles contained in the milk, which specific surface area is obtained by converting the spin-spin relaxation time, may be compared with a predetermined standard S1. The comparison allows determining whether or not the quarter from which the milk was collected has mastitis or is likely to have mastitis. Specifically, measured data (the spin-spin relaxation time or the specific surface area) of the sample milk is compared with the predetermined standard S1, and in a case where the measured data of the sample milk is significantly increased or decreased in comparison to the predetermined standard S1, it is possible to determine whether or not the quarter from which the sample milk was collected has mastitis or is likely to have mastitis. When comparing the data of the sample milk with the predetermined standard S1, whether or not there is a significant difference can be determined by a statistical method such as, for example, a t-test, an F-test, a chi-square test, or a Mann-Whitney's U test.
The specific surface area of the particles contained in the milk can be obtained from the following equation (1).
Rav=ΨpSLpp(Rs−Rb)+Rb (1)
In equation (1), Rav is a reciprocal of an average relaxation time. Ψp is a volume fraction (volume concentration) of the particles contained in the milk. L is a thickness (wet thickness) of a liquid layer adsorbed on surfaces of the particles contained in the milk. ρp is a density of the particles contained in the milk. Rs is a reciprocal of a relaxation time of molecules of a liquid adsorbed on the surfaces of the particles contained in the milk. Rb is a reciprocal of a relaxation time of solvent water.
Examples of the predetermined standard S1 include: (1) data of milk collected, from the quarter from which the sample milk was collected, at a time point (e.g., in advance) different from a time point at which the sample milk was collected; (2) data of milk collected from another quarter of the individual from which the sample milk was collected; and (3) data of milk collected from a quarter of an individual different from the individual from which the sample milk was collected. The data (1) through (3) are each a spin-spin relaxation time of the milk or a specific surface area of particles contained in the milk, which specific surface area is obtained by converting the spin-spin relaxation time. The data (2) and (3) may each be data of milk collected simultaneously with the collection of the sample milk, or data of milk collected in advance of the collection of the sample milk. The predetermined standard S1 can be determined such that: a spin-spin relaxation time (specific surface area) of milk collected in advance from a control quarter having no mastitis is measured; and a normal range (e.g., a standard S1 that serves as a threshold) is determined on the basis of the spin-spin relaxation time (specific surface area).
The predetermined standard S1 may be, for example, a collection of time-series data obtained at a plurality of time points with respect to the quarter from which the sample milk was collected. In this case, an evaluation is made of a change over time in the spin-spin relaxation time of milk from the quarter from which the sample milk was collected, or a change over time in the specific surface area of the particles contained in the milk, which specific surface area is obtained by converting the spin-spin relaxation time.
The testing method of the present invention may further include a step of comparing a somatic cell count in the sample milk with a predetermined standard S2. When comparing the somatic cell count of the sample milk with the predetermined standard S2, whether or not there is a significant difference can be determined by a statistical method such as, for example, a t-test, an F-test, a chi-square test, or a Mann-Whitney's U test. In the present specification, “a somatic cell count contained in milk” refers to a total number of blood-derived cells (leukocytes and the like) and mammary gland epithelial cells contained in the milk. The somatic cell count can be measured by a well-known method.
Examples of the predetermined standard S2 include: (4) a somatic cell count in milk collected, from the quarter from which the sample milk was collected, at a timing (e.g., in advance) different from a timing at which the sample milk was collected; (5) a somatic cell count in milk collected from another quarter of the individual from which the sample milk was collected; and (6) a somatic cell count in milk collected from a quarter of an individual different from the individual from which the sample milk was collected. The above data (5) and (6) may each be a somatic cell count in milk collected simultaneously with the collection of the sample milk, or a somatic cell count in milk collected in advance of the collection of the sample milk. The predetermined standard S2 can be determined such that: a somatic cell count in milk collected in advance from a control quarter having no mastitis is measured; and a normal range (e.g., a standard S2 that serves as a threshold) is determined on the basis of the somatic cell count.
The predetermined standard S2 may be, for example, a collection of time-series data obtained at a plurality of time points with respect to the quarter from which the sample milk was collected. In this case, an evaluation is made of a change over time in the somatic cell count in milk from the quarter from which the sample milk was collected, or a change over time in the specific surface area of the particles contained in the milk, which specific surface area is obtained by converting the spin-spin relaxation time.
As indicated in Examples, in a case where mastitis has developed due to infection with Gram-positive bacteria such as Staphylococcus aureus, a specific surface area of particles contained in milk decreases as a somatic cell count increases. This is considered to be because an acidic substance (e.g., lactic acid) produced by the Gram-positive bacteria causes casein micelles, which are dispersed in milk, to aggregate. In contrast, in a case where mastitis (e.g., mastitis due to infection with Gram-negative bacteria such as E. coli, mastitis due to trauma, or the like), which is other than mastitis due to infection with Gram-positive bacteria, has developed, a specific surface area of particles contained in milk increases as a somatic cell count increases. As such, by carrying out somatic cell measurement in addition to NMR measurement, it is possible to presume a cause of mastitis (whether or not the mastitis is caused by Gram-positive bacterial infection).
With reference to
The milking machine 200 can be a well-known milking machine. For example, in a case where four quarters of a dairy cow are tested simultaneously, four milker units may be provided. The flow cell 203 is installed in a predetermined position between the milker unit 201 and a position short of the milk claw 202. This allows a spin-spin relaxation time of milk collected from each quarter to be measured.
It is only necessary that the NMR measurement device 300 can be attached to the flow cell 203 of the milking machine 200. The NMR measurement device 300 can be, for example, a well-known NMR measurement device. From perspectives such as easy attachment to the milking machine 200 and reduction of a size of a measuring device, it is preferable that the NMR measurement device 300 be a pulsed NMR measurement device.
With reference to
The NMR measurement device 300 includes a communication section 301, a measurement section 303, a calculation section 304, and a control section 302 which controls the measurement section 303 and the calculation section 304. The communication section 301 communicates with the management terminal 400. The measurement section 303 measures a spin-spin relaxation time of milk collected from each quarter. On the basis of the spin-spin relaxation time, the calculation section 304 converts a specific surface area of particles contained in the milk.
The management terminal 400 includes a communication section 401, an NMR measured value comparison section 405, a somatic cell count comparison section 406, a determination section 407, an abnormality notification section 408, a storage section 403, a display section 404, and a control section 402. The communication section 401 communicates with the NMR measurement device 300 and the somatic cell measurement device 500. The NMR measured value comparison section 405 compares the spin-spin relaxation time or the specific surface area of the sample milk with the predetermined standard S1. The somatic cell count comparison section 406 compares the somatic cell count in the sample milk with the predetermined standard S2. The determination section 407 determines, on the basis of a result of comparison by the NMR measured value comparison section 405, whether or not the quarter from which the sample milk was collected has mastitis or is likely to have mastitis. In a case where the determination section 407 has determined that the quarter has mastitis or is likely to have mastitis, the abnormality notification section 408 provides notification of such to a user of the management terminal 400. The display section 404 displays the results of measurement by the NMR measurement device 300 and the somatic cell measurement device 500, as well as the notification by the abnormality notification section 408.
The determination section 407 may presume a cause of the mastitis (whether or not the mastitis is due to Gram-positive bacterial infection) on the basis of the result of the somatic cell count comparison section 406 in addition to the result of comparison by the NMR measured value comparison section 405.
The somatic cell measurement device 500 includes: a communication section 501 which communicates with the management terminal 400; a measurement section 503 which measures a somatic cell count in milk collected from each quarter; and a control section 502 which controls the measurement section 503.
The present invention is not limited to the embodiments, but can be altered by a skilled person in the art within the scope of the claims. The present invention also encompasses, in its technical scope, any embodiment derived by combining technical means disclosed in differing embodiments.
The present invention is not limited to the embodiments, but can be altered by a skilled person in the art within the scope of the claims. The present invention also encompasses, in its technical scope, any embodiment derived by combining technical means disclosed in differing embodiments.
The embodiments of the present invention include, for example, the following aspects.
<1> A testing method for testing for mastitis in a domestic animal, said method including the step of: measuring, by nuclear magnetic resonance, NMR, a spin-spin relaxation time of milk (sample milk) collected from a quarter of the domestic animal.
<2> The testing method as set forth in <1>, further including comparing, with a predetermined standard S1, the spin-spin relaxation time or a specific surface area of particles contained in the milk, the specific surface area being obtained by conversion of the spin-spin relaxation time.
<3> The testing method as set forth in <1> or <2>, further including: the step of comparing a somatic cell count in the milk with a predetermined standard S2.
<4> The testing method as set forth in any one of <1> through <3>, wherein the domestic animal is a dairy cow.
<5> A testing system for testing for mastitis in a domestic animal, including: a milking machine including a milker unit and a milk claw; and an NMR measurement device, a flow cell for NMR measurement being provided at a predetermined position between the milker unit and a position short of the milk claw.
<6> The testing system as set forth in <5>, further including a somatic cell measurement device.
<7> The testing system as set forth in <5> or <6>, wherein the domestic animal is a dairy cow.
<8> The testing method as set forth in <2>, wherein whether or not mastitis has developed is presumed by comparison with the predetermined standard S1.
<9> The testing method as set forth in <3>, wherein a cause of mastitis is presumed by comparison with the predetermined standard S2.
<10> Note that the testing methods of <1> through <4> can be interpreted as a method for obtaining data for carrying out testing for mastitis in a domestic animal and a method for analyzing the data.
A dairy cow has four quarters. Each quarter is an independent organ. Accordingly, milk produced by respective quarters differs in concentration of a component and the like, depending on a health condition of each quarter. Further, mastitis develops individually in each quarter. As such, in the following Examples, analysis was conducted with respect to milk collected from each quarter of a dairy cow. In Examples 1 and 2, in a case where a somatic cell count in milk was not less than 20×104/mL, it was determined that a quarter from which the milk was collected had mastitis. In Examples 3 and 4, in a case where a somatic cell count in milk was not less than 50×104/mL, it was determined that a quarter from which the milk was collected had mastitis.
Milk was collected from each of the four udder quarters (right front, right rear, left front, and left rear beasts) of a research dairy cow A kept and managed at the National Institute of Animal Health of the National Agriculture and Food Research Organization. A somatic cell count in the collected milk and a spin-spin relaxation time (T2) of the collected milk were measured. The somatic cell count was measured by a cell counter DCC (manufactured by DeLaval). The collected milk was inverted and mixed, and then sucked into a dedicated cassette and inserted into a main body for measurement. The relaxation time (T2) was measured at 25° C. with a pulsed NMR device (manufactured by Xigo Nanotools, device name: ACORN AREA) with use of the milk which was put into an NMR tube as it was without diluting the milk or adding anything to the milk. A mode in which measurement of T2 was conducted was a Carr-Purcell Meiboom-Gill sequence (CPMG) method.
On the basis of the relaxation time (T2) thus measured, a specific surface area (S) of particles contained in the milk was calculated using the following equation (1):
Rav=ΨpSLpp(Rs−Rb)+Rb (1)
In equation (1), Rav is a reciprocal of an average relaxation time. Ψp is a volume fraction (volume concentration) of the particles contained in the milk. L is a thickness (wet thickness) of a liquid layer adsorbed on surfaces of the particles contained in the milk. ρp is a density of the particles contained in the milk. Rs is a reciprocal of a relaxation time of molecules of a liquid adsorbed on the surfaces of the particles contained in the milk. Rb is a reciprocal of a relaxation time of a solvent water.
As illustrated in
After the collection of milk in Example 1, the research dairy cow A was kept for 2 days, and then milk was collected again from each quarter. Then, a somatic cell count and a relaxation time (T2) were measured and a specific surface area was calculated by the same procedure as in Example 1.
As illustrated in
A somatic cell count and a relaxation time (T2) were measured and a specific surface area was calculated by the same procedure as in Example 1, except that the dairy cow was changed to a research dairy cow B kept and managed at the National Institute of Animal Health of the National Agriculture and Food Research Organization.
As illustrated in
A somatic cell count and a relaxation time (T2) were measured and a specific surface area was calculated by the same procedure as in Example 1, except that the dairy cow was changed to a research dairy cow C kept and managed at the National Institute of Animal Health of the National Agriculture and Food Research Organization.
As illustrated in
To 3 mL of milk collected from a healthy quarter, two different strains of Staphylococcus aureus (SA-VB and SA-VR) were each added in an amount of approximately 30 mg. The Staphylococcus aureus strains used were those preserved at the National Institute of Animal Health of the National Agriculture and Food Research Organization. SA-VR is more toxic than SA-VB. After Staphylococcus aureus was added, the milk was kept at 37° C. At 0, 3, 6, and 9 hours after the addition of Staphylococcus aureus, a relaxation time (T2) of milk was measured and a specific surface area was calculated by the same procedure as in Example 1. A pH of the milk was also measured. Results of the measured pH and the specific surface area calculated on the basis of the measured relaxation time (T2) are illustrated in
In
As illustrated in
To 100 mL of milk collected from a healthy quarter, 1 g of fermented milk containing lactic acid bacteria was added. After the lactic acid bacteria was added, the milk was kept at 37° C. Every hour after the addition of the lactic acid bacteria, a relaxation time (T2) of the milk was measured and a specific surface area was calculated by the same procedure as in Example 1.
In
A somatic cell count and a relaxation time (T2) were measured and a specific surface area was calculated by the same procedure as in Example 1, except that the dairy cow was changed to a research dairy cow D kept and managed at the National Institute of Animal Health of the National Agriculture and Food Research Organization.
In
As illustrated in
A somatic cell count and a relaxation time (T2) were continuously measured and a specific surface area was calculated by the same procedure as in Example 1, with respect to a milk sample collected, directly with use of a catheter, from inside a quarter of each of two dairy cows kept at Rakuno Gakuen University.
In
As illustrated in
A somatic cell count and a relaxation time (T2) were continuously measured and a specific surface area was calculated by the same procedure as in Example 1, with respect to a milk sample collected from each of three dairy cows kept at Hokkaido Agricultural Technical College.
In
As illustrated in
Similarly as in
For quantitative in vitro observation of casein micelle aggregation caused by multiplication of Staphylococcus aureus, Staphylococcus aureus was added to 3% aqueous solutions of skim milk. In respective samples, a bacterial count was adjusted to 1.0×1010 CFU/mL, 5.0×109 CFU/mL, 2.5×109 CFU/mL, and 1.25×109 CFU/mL, respectively. Then, the samples were each cultured at 37° C. Every hour after a start of the culturing, a relaxation time (T2) of milk was measured and a specific surface area was calculated by the same procedure as in Example 1. Results of measurement are shown in
In
From the above results, it was found that, by using NMR to measure a spin-spin relaxation time (T2) of milk collected from each quarter of a domestic animal such as a dairy cow, it is possible to determine whether or not the quarter had mastitis. It was also found that, by also carrying out somatic cell measurement simultaneously, it is possible to presume a type of mastitis (whether or not the mastitis is due to infection with Gram-positive bacteria such as Staphylococcus aureus).
The present invention can be used in testing for mastitis in a domestic animal. Thus, the present invention can be widely used in fields such as food and medical diagnosis.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2019-190477 | Oct 2019 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2020/039008 | 10/16/2020 | WO |
