The invention concerns the test set for identification of agents causing mastitis-inflammation of udder tissue, and cow herd sorting method in relation to mastitis causing agent.

State of the Art

Currently it is known a wide range of diagnostic methods for mastitis causing agents identification.

The basic method is counting of somatic cells present in untreated milk. The number of somatic cells is an important mark, which indicates mastitis. For somatic cells counting it is used for example direct cell counting under microscope, California mastitis test, and fluoro- opto- electronic cell counting. The main disadvantages of these methods are the facts that they are only approximate and indicate only if the udder could be inflamed or not. Because it is not possible to identify the concrete pathogen, after high number of somatic cells detection, there are used broad- spectrum antibiotics, which are expensive and in case of non-bacterial agent useless. The another disadvantage is depreciation of milk by the antibiotics.

In the patent application CZ PV2001 -4305, there is described a physical method of udder inflammatory diagnosing. This method uses visible and/ or nearly infrared rays radiation into urine, untreated milk, or into mammary gland, detection of rays and the assessment. The main disadvantage of this method is the fact that it is only approximate and facilitates only to recognize if inflammation is present or not. Next disadvantage is high price of the equipment.

From the current technology, there are known another methods of mastitis diagnosing. For example diagnostics using detecting of albumin in milk described in patent document CS191082. In this method is the 1- 3% gel boiled with physiological solution and cooled to 50- 60°C. After that, there is incorporated albumin precipitating serum. Agar is poured on Petri plates and after solidification there are made holes, in which is poured milk serum. After the specimens are absorbed, the Plates are left at room temperature for the period from 17 to 34 hours. Diameters of circles made by the milk serum are directly related to the amount of albumin in the specimen. This is compared to the standard amount of albumin in milk. Disadvantage of this method is the fact that it is complicated, time consuming and it is possible to perform it only in a laboratory. It is expensive and its results are only approximate.

There is known wide range of methods using chromogenic substrates to identify mastitis causing agent. These methods facilitate accurate identification of mastitis causing agent Yet, design of the methods makes them expensive and time consuming and the identification is not as rapid and easy.

From the situation in current technology it is clear that the main disadvantages of current methods are the absence of rapid and accurate test facilitating mastitis causing agent identification.

Our goal was a test set and its use, which facilitates rapid mastitis causing agent identification, which leads to rapid problem solving, for example narrow- spectrum antibiotics prescription.

Principle of the Invention

The disadvantages of the methods mentioned above removes and the solution offers the test set, especially the test set for the mastitis causing agent identification, which contains diagnostic substance. The test set consist of at least two sectors, which contain various diagnostic substances with chromogenic components for various mastitis causing agents identification.

In the advantageous version the test set consists of at least three sectors with diagnostic substances with various colour reactions to identify mastitis causing agent.

Benefit of the test set is facilitating rapid and easy mastitis causing agent identification and, with respect to results, quick sorting of cows with different treatment needs. The next benefit is that he set can be used not only in laboratory, but, due to chromogenic component, straight in the field.

Next benefit is that the test set contains a sector for Staphylococci identification, a sector for Enterococci identification and sector for Gram- negative bacteria and yeast identification. This composition is useful considering the most common mastitis causing pathogens in cows.

Advantageous is when every sector contains diagnostic substances for identification of expected mastitis causing agents. This facilitates accurate results and the mastitis causing agents are identified at the genus or species level. The advantage is rapidness of identification, which provides the opportunity to begin with a targeted treatment. The advantage is that it is possible to identify especially highly infectious agents.

Another possibility is searching for pathogens spreading cows before lactation stopping at the end of pregnancy. It facilitates the opportunity to use targeted treatment in the period (called "dry period") when the cow does not produce milk. It is profitable for practical reasons that the certain sectors are separated from each other and are marked according to contained substances. It makes using in the field easier.

The disadvantages of the methods mentioned above removes and the solution offers the cows herd sorting method, especially sorting according to mastitis causing agent.

Every particular specimen of milk is spread on agar in sectors. Afterwards the test set is placed into hatchery, where it stays for fixed period at fixed temperature. After that the reactions of chromogenic substances are assessed. It is beneficial to separate the cows into three groups: group of healthy individuals, group where antibiotics are not needed and the group where antibiotics are needed.

The big advantage is a rapidness of the process. Currently, the first results of testing of cows herd where high number of somatic cells appeared, are known after four days. Our method provides accurate results in 24 hours already. This provides an opportunity to separate diseased individuals and prevent further infection spreading.

Furthermore, it is useful when the group with antibiotic treatment is separated into certain groups according to mastitis causing pathogen and the type of antibiotic needed.

Profitable is that the diseased individuals can be treated with narrow-spectrum antibiotics and the risk of antibiotic-resistant pathogens development is lower than by global broad-spectrum antibiotics use. Special antibiotics (antibiotic with restricted indication) are prescribed only in individuals, where there is no other possibility.

The method using the test offers a cost saving and it enhances future treatment success rate. The test set and the cow herd sorting method facilitates rapid and cheap mastitis causing agent identification in particular individuals. The another advantage is rapid mastitis causing agent identification leading to narrow- antibiotic treatment. The fact that there are treated only diseased individuals brings the cost saving and the antibiotic use decrease. This leads to reduction of antibiotics in milk and reduction of antibiotics residues in human food chain. Prescribing of broad- spectrum antibiotics and antibiotic with restricted indication only if indicated is a basic means of antibiotic resistant bacteria strains development prevention.

Overview of the Figures

The invention is described in detail in the Fig. 1 , where the test set with three sectors is depicted.

Examples of the Performance of the Invention

The test set 4 (Fig. 1 ) for mastitis causing agent identification, which contains diagnostic substances for mastitis causing agent identification, consists of three separated sectors 1,2,3 where are present various test substances with chromogenic components for mastitis causing agent identification.

Every sector1,2,3 contains chromogenic substances with chromogenic components with various colour reactions for various mastitis causing agent identification.

Sector 1 contains the following diagnostic substances with chromogenic components for the group of Staphylococci identification:

- coagulase-negative Staphylococci:

- diagnostic substance for identification of Staphylococcus chromogenes with a white colour reaction,

- diagnostic substance for identification of Staphylococcus haemolyticus with a light green colour reaction,

- diagnostic substance for identification of Staphylococcus sciuri with a dark green colour reaction,

- coagulase-positive Staphylococci:

- diagnostic substance for identification of Staphylococcus aureus with dark ping colour reaction.

Sector 2 contains the following diagnostic substances with chromogenic components for the group of Enterococci identification:

- diagnostic substance for identification of Streptococcus agalactiae with a light blue colour reaction, - diagnostic substance for identification of Streptococcus uberis with a dark blue colour reaction,

-diagnostic substance for identification of Streptococcus dysgalactiae with blue- green colour reaction,

- diagnostic substance for identification of Enterococcus faecalis with dark violet colour reaction.

Sector 3 contains the following diagnostic substances with chromogenic components for Gram- negative bacteria and yeasts identification:

- diagnostic substance for identification of Eschericha coli with a red colour reaction,

- diagnostic substance for identification of Klebsiella species with a violet colour reaction,

- diagnostic substance for identification of Proteus vulgaris with a light grey colour reaction,

- diagnostic substance for identification of Candida species with a white colour reaction.

Every medium in each sector1, 2, 3 contains nutrient components.

Each sector 1_, 2, 3 is marked according to contained components for identifying of mastitis causing agents group.

After collecting samples, every specimen is spread onto media in sectors 1, 2, 3 and the test set is placed in a hatchery, where it is incubated for a period of 24 hours.

After this time the pathogens can be identified according to reactions of particular chromogenic components.

According to the results cows can be sorted into three groups: The group of healthy individuals without need for treatment, the group of individuals with need for non- antibiotics treatment, and the group of individuals where antibiotics are needed. The last group is sorted according to mastitis causing agent and each individual is cured by narrow- spectrum antibiotic.

Industrial Application

The test is purposed for identification of mastitis causing agents. The test set is great of utility for easy and rapid separating of diseased cows and the targeted treatment, which leads to decrease of somatic cells in milk and the milk quality increase.lt is useful to use test set in pregnancy before cows stop producing milk. The cows are tested and consequently the diseased individuals are cured during the period when the cows does not produce milk. It is profitable to use test set again after treatment to prove its effectiveness.

List of Reference Marks

Sector I Sector II Sector III Test set