Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
1999-08-04
2001-10-02
Beisner, William H. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S287300, C435S303300
Reexamination Certificate
active
06297045
ABSTRACT:
FIELD OF THE INVENTION AND RELATED ART STATEMENT
The present invention relates to a mastitis diagnosing apparatus, and particularly it relates to a mastitis diagnosing apparatus that is capable of easily measuring the state of progression of mastitis based on a chemiluminescence method (CL activity measurement).
The incidence of mastitis among dairy cows in Japan hovers between 20-25% of all dairy cow diseases, and it has the highest mortality rate. Mastitis is the disease to which dairy cows are most susceptible, and it is also a very difficult disease to cure. Appropriate methods are being sought for dealing with mastitis, through early discovery of the diseased condition and optimum treatment based on the course (state of progression) of the mastitis.
Various conventional methods are known for diagnosis of mastitis.
FIG. 9
is a diagram showing the relationship between progression of mastitis and effectiveness of the major diagnostic methods. As shown in
FIG. 9
, mastitis usually takes a course from bacterial invasion→mastitis signs→mild mastitis→moderate mastitis→severe mastitis. Common diagnostic methods for mastitis include somatic cell counting, CMT (California Mastitis Test), conductometry, etc., among which somatic cell counting is effective for diagnosis from bacterial invasion up to moderate mastitis, and CMT and conductometry are effective for diagnosis from mild mastitis up to severe mastitis.
Somatic cell counting involves measurement of the number of cells in milk, including lactic gland epithelial cells, neutrophils, acidophils and other phagocytes, and lymphocytes, monocytes, plasma cells and the like, and it can successfully detect during the period from initial infection up to moderate mastitis.
CMT is a method of estimating the number of somatic cells by utilizing the difference in the extent of aggregation reaction depending on the number of somatic cells, when a surfactant is added to the milk. Since a BTB reagent is also included for pH measurement, it is used as an evaluation index for mastitis by utilizing the fact that increased vascular permeability and accelerated conflict between leukocytes and bacteria during mastitis results in increased salts such as sodium chloride and potassium chloride in the milk, creating a higher alkalinity, and causing a color change from yellow→green→blue. The advantages of this measurement are that it can be easily performed by anyone, it can generally distinguish between the presence or absence of mastitis, and it is an extremely low-cost method.
Conductometry takes advantage of the fact that mastitic milk has greater vascular permeability and contains more electrolyte components such as sodium, chlorine and other plasma components due to inflammation reaction in the mammary tissue, thus allowing electricity to flow more readily than with normal milk, and its advantage is that an objective value can be obtained by quantifying the conductivity.
Milk cell counters exist for somatic cell counting, but their problems are high cost and the excessive labor required for smear measurement using optical microscopes. For the colostrum in particular, it has been difficult to the smear and fix the milk. Another problem is that as mastitis progresses, numerous leukocytes adhere to the bacteria producing bulb-like forms which make measurement impossible.
The drawbacks of CMT are that diagnosis is difficult until the reaction has occurred, involving the conflict between leukocytes and the bacteria, or after promotion of vascular permeability, and that diagnosis depends on subjective human judgment, so that this method can only serve as an approximate diagnosis method. Diagnosis has been particularly rough in cases where the milk somatic cell count is 300,000/ml or less.
Conductometry has had disadvantages in that it depends on changes occurring by inflammation reaction after the bacteria invade and conflict with the leukocytes, and therefore it is unsuitable for diagnosis in the initial stages of mastitis, while it has poor reproducibility due to substantial differences in electrolyte components and concentrations in different teats or different cows even with normal milk, such that diagnosis is risky by this diagnostic method alone.
Thus, one of the present inventors, Hideyuki Takahashi, focusing on the fact that phagocytic leukocytes such as neutrophils which constitute the larger part of somatic cells in milk mount an excellent initial reaction (mobilization) during infection, has already proposed a method for diagnosis of mastitis (chemiluminescence method) by measurement of phagocytic leukocyte function in milk in terms of CL activity (Takahashi, Hideyuki, “Manual for High-Quality Milk Production by Environmental Stress Reduction”, ed. Hokkaido National Agricultural Experimental Station, March, 1997).
This chemiluminescence method (CL activity measurement) will now be explained. Milk contains somatic cells such as lactic gland epithelial cells, neutrophils, acidophils, lymphocytes, monocytes, plasma cells, etc. These cells are virtually absent in milk during times of health, but once the mamma suffers invasion of bacteria, leukocytes with phagocytic bactericidal activity (phagocytic leukocytes=neutrophils, acidophils, etc.) accumulate in the milk in an attempt to halt bacterial proliferation. Among these, it is the neutrophils that mobilize to the greatest extent after bacterial invasion, working to halt proliferation of the bacteria by overwhelming superiority of numbers. The neutrophils kill bacteria mainly with active oxygen, and also emit a trace intensity of light (photons) in proportion to the amount of active oxygen released.
Chemiluminescence is a method whereby that light (of photons) is detected, amplified and quantified, and it is characterized in that the phagocytic leukocytes with bactericidal activity that accumulate in response to bacterial invasion can be detected with high sensitivity during a long period from the initial bacterial invasion through to severe mastitis.
However, no apparatus (mastitis diagnosing apparatus) has yet been provided for convenient and reliable diagnosis of mastitis based on the aforementioned chemiluminescence method (CL activity measurement).
OBJECT AND SUMMARY OF THE INVENTION
It is an object of the present invention to provide a mastitis diagnosing apparatus capable of performing convenient and reliable diagnosis of the state of progression from invasion of bacteria into the mamma up to severe mastitis, based on chemiluminescence (CL activity measurement).
The mastitis diagnosing apparatus of the invention is characterized by comprising a memory for pre-storage of a corresponding relationship between the number of photons emitted when phagocytic leukocytes phagocytose bacteria in milk and the state of progression of mastitis due to the bacteria; photon counting means which counts within a prescribed period of time the number of photons emitted when phagocytic leukocytes in milk phagocytose bacteria, by addition of a culture solution, emission enhancer and phagocyte stimulator to the milk itself; diagnosing means which refers to the memory and, based on the number of photons counted by the photon counting means, diagnoses the state of progression of mastitis corresponding to the number of photons; and output means which allows output of the state of progression of mastitis diagnosed by the diagnosing means.
The mastitis diagnosing apparatus according to the invention is further characterized by comprising a memory for pre-storage of a corresponding relationship between the number of photons emitted when phagocytic leukocytes phagocytose bacteria in milk and the state of progression of mastitis due to the bacteria; reagent injection means which injects an emission enhancer and phagocyte stimulator into a set test tube in which the milk itself and a culture solution are mixed; stirring means which stirs with a prescribed period of stirring the test tube into which the emission enhancer and phagocyte stimulator have been injected from the reagent i
Matue Takahisa
Shimizu Masakatu
Takahashi Hideyuki
Beisner William H.
Japan as represented by National Institute of Animal Health, Min
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