JPS60500144A - How to measure mastitis in cows, etc. - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] How to measure mastitis in cows, etc. The present invention relates to a method for quantifying mastitis in cattle, etc. This invention relates to a method for measuring the trypsin inactivation effect of milk. Above trypsin inactivation The main reason for the increase in effectiveness is α in milk in the case of mastitis.

- Because antitrypsin flows from the blood to the milk chamber.

The most important method of measuring and detecting bovine mastitis currently available is based on bacteriology. The method is based on measuring the number of cells in a milk sample.

This method has the following drawbacks.

- Equipment for counting and measuring cells is expensive, labor-intensive to use, and It takes a lot of labor to maintain it. Under these circumstances, the existing equipment Large laboratories in dairy factories [e.g. Valio J in Helsinki] - Used only for storing milk samples for counting and measuring cell counts. It is technically inconvenient to maintain it and requires special equipment.

Depending on when and how a sample is taken, the number of cells measured in a milk sample may vary. Affected by causes outside of adenitis.

In addition to the above-mentioned reasons, especially to date, there has been a It was not possible to conduct tests with certainty on a cell-by-cell basis. Measuring latent mastitis It was very uncertain. Apart from this, for example in Finland, all livestock It is estimated that up to 40% of women suffer from acute or latent mastitis. child This also results in considerable economic losses.

The method of the present invention includes: a) With the standard amount of trypsin, N-henzoylalkyne-p-nitroanilide? The colored compound p-nitroaniline is produced, but in this colored compound α,-antitriply The idea that trypsin acts as a color inhibitor by interfering with the activity of trypsin. or b) based on the clarifying action of the milk's own casein. It is characterized by measuring the trypsin inhibitor content.

The method of measuring mastitis of the present invention has been compared with the prior art method to track the disease. It is possible. In the method of the invention, a new method of sample pretreatment is also introduced. It is very simple compared to conventional technology.

The permeability of the cell wall of the tube changes. As a result, blood proteins (especially (lower molecular weight) flows into milk. Therefore, in relation to mastitis, e.g. Blood albumin and α1-antitrypsin, i.e., these compounds have low molecular weight in the blood. It was known that a member of the protein family is found in milk. Regarding mastitis The presence of antitrypsin in associated milk is described, for example, in the report below. .

T, Honkanen-Buzalski (T, Honkanen-Buzal) ski), T, Katila and M.

By Sandholm (M, 5andho 1m) °°Therapeutic and experimental bovine milk Milk antitryp-sin act during adenitis ivity durin, g clinical an4 experiment tal bov-ine mastitis)” Acta Vet, 5c and, 1981 22.

360-368.

6 Honkanen-Buzarski and M. Sandholm, “Mastitis Milk and Breast Cancer” Trypsin inhibitor in milk; trypsin inhibitor capacity, bovine serum albumin and Correlation between somatic cell content ypsininhibitor capacity, bovjne serum 　al-bumin and somaticcell　conteuts)” , J., Dairy Res, 1981, 4, 8.

213-223; M., Sanodholm, T., Honkanen-Buzarski and Earl, Kangasni. Written by R.Kangasniemi, “’As an indicator for mastitis in cows. Milk trypsin inhibitor ability, a novel principle that can be automated (, Milk trypsin in 1nhi-bitor capacity as an 1ndicat or of bovine mastitis.

a novel principle which can be autom ated)” (manuscript).The method of the present invention has been developed to be suitable for automation. Uno is based on this knowledge.

Thus, the appearance of blood albumin and α1-antitrypsin in the milk pile In principle, it indicates mastitis, and its concentration is correlated with the severity of mastitis. Two.

In practice, measuring blood albumin from milk is labor-intensive and especially Although it requires chemical methods, the measurement of α1-antitrypsin is a protease inhibitor in milk. It is very simple because it can be carried out enzymatically by measuring the ability of harmful factors.

Therefore, it is possible to extract antitoxification agents from milk samples accurately on the basis of their protease inhibitor potency. Based on the measurement of lipsin, mastitis in cattle is measured as described below. A method was developed.

While developing this method, we realized that it was not suitable for processing very large numbers of samples. Particular attention was paid to the fact that it had to be accurate.

In the simplest method, protease inhibitor activity is determined by adding trypsin to the sample. Mix and measure the proteolytic activity of excess trypsin Can be done. Proteolytic activity is a) Low molecular weight organic substances, such as BAPNA (N-benzyl alcohol) nitroanilide) or b) Degradation of proteins such as casein as a standard [e.g. By using the month of Easy, can be measured.

The present invention will be described in more detail below with reference to drawings of a carp served as an embodiment of the present invention. I will clarify. In the drawings, FIG. 1 shows a so-called microcontroller used in the method of the present invention. It is a top view of the crotiter disk (m1crotiter aisc), and it is a $2 figure. is a graph showing the measurement results obtained by the method of the present invention. The great invention can be implemented in two different ways.

Method A: Sample pretreatment: 1 ml of milk sample was pretreated with 0.1 molar acetic acid at pH 5.0. Clear using rennin 0.1mM diluted to 1150.000 using buffer. do. Clarification is carried out by incubating the milk and rennin mixture at 37°C for 30 minutes, followed by sanitation. This is done by centrifuging or filtering the pull.

This clarified milk sample, 0.1mM, was used to measure trypsin inhibitor content. However, the measurement is performed as follows.

Dispense the clarified milk sample into the well of the microtiter disk. To this person Trypsin standard solution (2,5 ILg/m solution dissolved in 1 mH hydrochloric acid) 0. L m days and 1 mg = i of N-benzoylarginine-p-nitroanilide ( Add 0.1 ml of substrate solution containing 1 mg of BAPNA into the well.

One milk sample is processed in each well of the microtiter disk. Furthermore , in the microtiter disk to determine the background value, an empty column, i.e. A so-called ``blank column'' is created, which also represents the average quality of the milk. Standard dilutions (1, 1/2° 1/4...1/64) of the milk obtained, and further A 100% lipsin control is also created.

The amount of trypsin inhibitor in milk is proportional to the degree of mastitis; It was observed that the degree of

The milk mixed in the milking shed under these circumstances (this represents the average health of the cow). The idea of using the standard diluent system is to standardize the results for each sample. The fact that it is given by comparison with a semi-dilute liquid system, that is, the individual in question by indicating whether the person is healthier or less healthy compared to the average. This means that the result is given. This makes standardization of this method quite convenient. are doing.

After adding the reagents, place the microtiter disc or FP cuvette block (label). LabsystemsOy, Helsinki, Finland) was incubated for 3.5 hours, and then the amount of yellow color produced in the depressions was measured using Titer Techma. Titertek Multiskan f lab OY), Helsinki, Finland) or FP-901 Anara Measured using Iser (Labo Systems Oy, Helsinki, Finland) . Both of these devices operate using the so-called vertical measurement principle; The location is described in the report and patent below; Suova Niemi, O, (Suova・niemi) i+, Inpipette Min. Performance and Property of the analyzer system sof the Finnpipette Analyzer System) ”, Bulletin of the 2nd Finnish National Conference on Biophysics and Physical Technology, Kairen-t. o, A, L, , RiihiiMki, E, and Tarkka, P, , Eds, pp. 183-187 (1976); Suobaniemi, 0. Author: “Method for Improving the Speed and Measurement Accuracy of Chemical Analysis” d for Improving the Rate and Meas -a remer+t Accuracy of Chemical Analysis s)”, U.S. Patent No. 4,144,030.

Suopaniemi, 0. ．． Written by J Mrnefelt °Discrete Multi-channel analysis system el Analysis System)”, International Labo-ratory, April 1982 and American Labo ratory June 1982, and Suohe Niemi, 0. Canadian Patent No. 1.031 , No. 193, French Patent No. 7,442,147, British Patent No. 1,542 ．． 526, U.S. Pat. No. 1.392,792, U.S. Patent No. 4,144,030, U.S. Pat. No. 4,147,752.

FIG. 1 shows the arrangement of samples on a microtiter disk. Number 1 is a serial number Represents a sample given in numbers 80 to 80. Standard dilution system (1/1.I/ 2.1/4...l/64) is represented by the number 2. Number 3 is trypsin 1<3 Number 4 represents the blank row, representing the 0% control (no milk sample). milk sa Measurement of trypsin inhibitor in samples is based on the following biochemical reaction. Ru.

(Colored compounds, 405 (Adsorption at 6m) In other words, the trypsin added to the cavity of the microtiter tape is BA. PNA (substrate) is decomposed to produce colored compounds. Antitrypsin from milk This reaction is inhibited by inactivating trypsin.

The more trypsin inhibitors contained in milk, the more active trypsin is. effectively inhibited and therefore color development is further inhibited.

The presence of trypsin inhibitors in milk is therefore inversely proportional to the intensity of the color measured.

As previously known, the intensity of trypsin inhibitors in milk is proportional to the degree of mastitis. For example, based on the test arrangement above, the color development on a microtiter disk is The more effectively the milk sample inhibits growth, the more severe the associated mastitis. It can be said that

This situation is shown graphically in Figure 2. In the diagram, curve 5 is from “healthy” milk. Curve 6 shows the results from mixed milk at the milking parlor, Curve 7 shows the results from dilution to 1/40. Curve 8 is from the milk with mastitis. The results are shown below.

While developing this method, only one dilution was made within the range of 1/4 to 1/10; Compare the results obtained with the standard curve obtained for milk mixed in the milking parlor (graph Rough comparison), conditions were set to be sufficient for each milk sample. .

Under the test conditions chosen, the differences between unhealthy and healthy cows are most clearly shown. This is the dilution.

Milk t+ mixed by the milking plant (in this case milk from about 5000 cows) ) is better than milk obtained from healthy cows, based on the measurement results (see graph). It should be noted that there are many anti-1 lipids involved. This also means that the current Approximately 40% of cows suffer from mastitis, some of which are latent. Currently, there is no way to systematically observe cows for each animal or each udder. This can be easily understood if you keep this in mind.

Method B: Another method is based on casein degradation. Ru. Add cheese rennin to the milk sample at the tip of the disc as in method A. When calcium is added, calcium paracaseinate is produced in the presence of calcium, which is Make the sample your own station. Adding trypsin as in method A results in casein degradation. As a result, the sample becomes transparent. After incubation for 1 hour, the degree of clarity is determined. Method A and Similarly, standardize. Thus, for methods A and B, under the selected conditions 80 milk samples can be processed on one microtiter disc Ru. Approximately 100 holes can be processed by one person in one working day using this method. You can get those results.

In some situations, such as connecting to a computer programmed to obtain It becomes even easier.

The first rather extensive study using the method described here involved milk samples from 1029 cows. This was done by performing pull measurements.

The accuracy and reproducibility of measurements by the method described herein was approximately 6% from the average. this Values come from differences between microtiter discs and daily variations. It also includes errors caused by

The results obtained in this study were compared to two other methods of measuring mastitis (albumin in milk). measurement and cell counting method).

11　°− Antitrypsin assay, based on other methods (e.g. albumin assay or When compared with the anti-trypsin assay (cell counting method), the anti-trypsin assay has even more significant advantages. You can get sex. In other words, the pretreatment of the sample is easy. or by freezing the sample by adding Rium azide (a bacterial inhibitory compound) This is because it can be preserved and stored. For antitrypsin measurement This operation (unlike other methods) does not interfere with the measurement.

The ease of pretreatment means that it is possible to process a very large number of samples. This is very important because it will not happen. This also means that animals can be treated individually and in their breasts. It also makes it possible to observe each time.

Fig, 1 international search report

Claims (1)

[Claims] 1. The factor is trypsin inhibitor present in milk associated with mastitis in cows etc. In the method of measuring trypsin inhibitor as a) Standard amounts of trypsin replace the colored compound P-nitroaniline with N-benzoylalcohol. It is produced from ginine-p-cotroanilide, but in the colored compound, antitrypsin is It utilizes a coloring reaction that acts as a coloring inhibitor by inhibiting the activity of lipsin. to use or b) Bovine, characterized in that the measurement is based on the clarification effect of casein in the milk itself. Quantitative measurement method of mastitis such as. 2. The coloring reaction or the clarification effect of casein was measured optically using an optical density measuring device. It is characterized by measuring as a change in density and measuring the amount of antitrypsin as optical density. The method according to claim 1, characterized in that 3. Perform measurements using a so-called microtiter disk or FP cuvette block. A measuring device (titrate) operating on the principle of so-called vertical measurement that multiscan and FP-901 analyzer) and connected to the measuring device. a suitable microcomputer pre-programmed to process the results. The method according to claim 1 or 2, characterized in that the method is carried out using a computer. Method.