Early diagnosis of mastitis or garget in milk

Abstract: Samples of milk of humans or cattle are tested to measure the presence of 3-hydroxylacetic acid (3-OHBA), e.g. by means of a known test piece containing nicotinic acid amide adenine dinucleotide, enzymes and nitro blue tetrazolium, and the resultant color compared to a pre-prepared hue diagram. The concentration of 3-OHBA correlates with activity of N-acetyl-beta-D-glucosaminidase which has previously been a test for broken mammary tissue. The values of 3-OHBA in normal mammae and those affected by human mastitis or garget in dairy cattle can be compared, and a difference in the concentration of 3-OHBA in milk of more than 100 µmol indicates disease of the mamma. The test is simple and allows early diagnosis.

Description: This invention relates to the early diagnosis of mastitis or garget by measurement made on the milk of mammals. The concentration of 3-hydroxylactic acid (3-OHBA) in milk, which is less than 100 µmol/l for normal cattle, is well known to increase drastically in the case of ketosis which is the dysbolism of lipid and carbohydrates. Reagents for making an expeditious measurement of the concentration of 3-OHBA are now commercially sold, e.g. by Sanwa Co., Ltd (under the trade name "Lactest"). Various conditions have been studied by the inventors to uncover the association of the ketone bodies (acetone, acetoacetic acid and 3-OHBAs) in such body fluids as urine and blood with the quality of mammalian milk and the health condition of livestock, as already disclosed (see JP-A-62-135771). It has been reported that ketosis conditions are induced not only by diabetes but by various forms of shock, organopathies such as hepatitis, infectious diseases and drug (e.g. salicyclic acid) poisoning as well ("Clinical Diagnosis Management", Vol. 2, p. 549, published by Thunder Co.). Reagents for separation and quantitative determination of a ketone body recently developed by Sanwa Co., Ltd. and other firms are now being used not only for diagnosis of diabetes but also for measuring the degree of activity of mitochondrial cells at the time of liver transplantation, determination of the degree of rejection after transplantation, or for other purposes. However, never until now has there been any report about the association of ketosis with mastitis or garget; in other words, they have been considered to have nothing to do with each other. Nor has anything been reported about the association of a ketone body with mastitis or garget. In general, early detection of mastitis during a period of human lactation is of vital significance for protecting infants and, at the same time, the mothers. Garget, which is a typical disease of dairy cattle, and incurs serious economic losses, is a type of inflammation which attacks the mammary gland tissue and lactiferous duct mainly through bacterial infection, and inhibits the lactigenous function and exacerbates permeation of the milk-secreting cell membrane, thus producing unusual milk in which blood components are incorporated. Dairy cattle produce a ketone body in the liver and digestive tracts after feed intake, which is then used as an energy source after oxidation in the heart, kidney, brain and other organs. In the mammae, the ketone body is used not only as an energy source but also for synthesis of short-chain fatty acids of milk components. A cow with ketosis or having a high concentration of ketones in the blood is found to have a decrease in the activity of peripheral lymphocytes and the number of T-lymphocytes, and this suggests that the cow has a great chance of suffering from garget due to a drop of immune responsibility. In an initial stage of mastitis or garget, the affected mammary gland tissue (cells) is locally inflamed or broken on a small scale, and a part of the blood components is mixed with milk components in the lactiferous duct, aggregated lactiferous duct, lactiferous sinus and other regions, so that all the milk produced is of abnormal composition. Early diagnosis of mastitis or garget to measure to what extent the mammary cells are affected or broken involves measuring the content of chlorine in milk, counting somatic cells in milk, determining NAGase activity and the like. However, a problem with these procedures, which are all awkward to handle, is that they cannot be used for ordinary, expeditious and rapid detection. In view of the above-mentioned problem, it is an object of the present invention to provide for early diagnosis of mastitis or garget, by making it possible to easily learn whether or not the subject mammal suffers from mastitis or garget during lactation or milking. In order to achieve the above-mentioned object and due to the fact that separation and quantitative determination of a ketone body can now be easily carried out the inventors have now discovered the correlation, between 3-OHBA produced in milk and mastitis or garget. Thus, according to one aspect of this invention, there is provided a method of diagnosing disease by measuring the concentration of 3-hydroxylactic acid in milk, a concentration of 3-hydroxylactic acid higher than 100 µmol/l being indicative of mastitis or garget. In the accompanying drawing: Fig. 1 is a graph showing the correlation between NAGase that is an index to mastitis or garget and 3-OHBA. According to the results of experimentation made by the inventors, if the concentration of 3-OHBA in milk is equal to or more than 100 µmol/l, it can then be diagnosed as showing mastitis or garget. A reagent, which can be colored when the concentration of 3-OHBA in milk exceeds 100 µmol/l, may be used for this purpose. Mastitis or garget is an inflammation of the mammary gland tissue or the lactiferous duct, which inhibits normal lactogenesis. This in turn hinders the normal synthesis of acetoacetic acid from 3-OHBA, which would be well achieved if the mammary gland tissue is in good condition. As a result, there is not only an increase in the amount of 3-OHBA transuding in milk, as is the case with ketosis, but also a serious increase in the amount of the transudation of the blood components, which is induced by cytoclasis. Thus, the concentration of 3-OHBA in milk comes to exceed 100 µmol/l. In the case of ketosis in which the concentration of 3-OHBA in the blood is excessively high, there is an increase in the amount and hence concentration of that acid transuding in milk. Thus, the subject, if in good health condition, can be diagnosed as mastitis or garget when the amount of 3-OHBA transuding in the milk is found to increase. While ketosis causes an increase in the concentration of 3-OHBA in milk irrespective of what condition the mammae are in, mastitis or garget allows only the inflamed mamma to show an increase in the concentration of 3-OHBA in the milk. Thus, the subject can be diagnosed as having mastitis or garget when a difference in the concentration of 3-OHBA between the affected and normal mammae is found to be higher than 50 µmol/l. The present invention will now be explained specifically by reference to the following examples. Example 1 This example is provided to explain the correlation between the conventional diagnosis of mastitis or garget by the determination of NAGase activity and the present diagnosis. The NAGase activity (N-acetyl- β -D-glucosaminidase) which is an index to what extent the mammary gland tissue is broken, was assayed by the conventional procedure under the conditions provided by "A Guide to Treating Special Diseases And Wounds In Livestock" published by the National Agricultural Mutual Aid Association. The subject is diagnosed as having mastitis or garget when the NAGase activity exceeds 10 nmol/min/ml, and it is said that the higher the activity, the more serious the degree of inflammation. The concentration of 3-OHBA, on the other hand, was measured with a test piece including a reagent layer impregnated with 25 µg of nicotinic acid amide adenine dinucleotide, 0.5 U of 3-OHBA dehydrogenase, 1.0 U of diaphorase and 25 µg of nitro blue tetrazolium ("Lactest" made by Sanwa Co., Ltd.). Then, the concentration of 3-OHBA was determined by comparing a color developed by the dropwise addition of a milk sample to the reagent layer with a pre-prepared hue diagram. As can be understood from the results set out in Table 1 and illustrated in Fig. 1, it has been found that the concentration of 3-OHBA correlates with the NAGase activity. Correlation between the values of NAGase activity found and the concentration of 3-OHBA in milk Number of case 31 Coefficient of correlation r= 0.864 Regression curve y = 2.12X + 37.95 Example 2 The same test piece as in Ex. 1 was used to determine whether or not cows suffered from garget and a difference in the concentration of 3-OHBA in the milk between the mammae. The results (in µmol/l) are reported in Table 2. t Assay: t value = -9.149, P value = 1.785 x 10⁻⁶ As mentioned above, this invention is efficacious for early detection and treatment of mastitis during a period of human lactation, which is of vital significance for protecting infants and, at the same time, the mothers, and of garget disease in dairy cattle.

Claims: Method for early diagnosis of mastitis or garget which consists of measuring the concentration of 3-hydroxylactic acid in milk, a concentration of 3-hydroxylactic acid higher than 100 µmol/l being indicative of mastitis or garget. Method according to Claim 1, wherein the concentration of 3-hydroxylactic acid is measured in the milk of each of a plurality of the mammae to find a difference in said concentration therebetween. Method according to Claim 1 or 2, in which a reagent reacting with 3-hydroxylactic acid to show a colour at a concentration of 100 µmol/1 or more is used to measure the concentration of 3-hydroxylactic acid in milk.

Inventors: ASAI HIROMOTO, FURUKAWA EIJI, KURONO MASAYASU, SAWAI KIICHI, YAMAMOTO HIDEO, ASAI, HIROMOTO, SANWA KAGAKU KENKYUSHO CO., LTD., FURUKAWA, EIJI, SANWA KAGAKU KENKYUSHO CO., LTD., KURONO, MASAYASU C/O SANWA KAGAKU KENKYUSHO CO.LTD, SAWAI, KIICHI C/O SANWA KAGAKU KENKYUSHO C0.LTD, YAMAMOTO, HIDEO, SANWA KAGAKU KENKYUSHO CO., LTD.
Applicants: SANWA KAGAKU KENKYUSHO CO, SANWA KAGAKU KENKYUSHO CO., LTD.
Application year: 1991
Main classification: G01N33
IPC codes: C12Q1, G01N33, A61B10