RU2264171C1 - Method for predicting subclinical mastitis form of microbial etiology in cows - Google Patents

Abstract

FIELD: veterinary science.

SUBSTANCE: the present innovation deals with milk sampling, isolating purified cultures, their identification and detecting in them the capacity to inactivate the factors of natural anti-infectious resistance. In isolated purified cultures of agents one should detect antilactoferrin activity (AlfA) and at the level of pronounced nature of this parameter in, at least, one out of isolated agents both for staphylococci and streptococci AlfA being either above or equal to 2.0 mcg/ml one should predict subclinical mastitis form induced by an isolated agent. The innovation provides high accuracy in quantitative detection of diagnostic signs.

EFFECT: higher efficiency of diagnostics.

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Description

The invention relates to veterinary medicine, namely to veterinary microbiology, and can be used to diagnose subclinical (hidden) forms of mastitis of microbial etiology in cows.

Mastitis is the most common cow disease that causes significant damage to agriculture [Kasyanchuk V.V. Some features of the course of mastitis in cows. // Veterinary medicine. - No. 8. - 1991. - P.40].

The greatest economic and economic problem is subclinical mastitis, which occurs 4-5 times more often than clinically expressed [Zagaevsky I.S. Prevention of diseases of cows with mastitis. Chisinau: "Cartya Moldovenienasca", 1974. - P.9.], And causes great economic damage to livestock due to reduced milk productivity, deterioration in milk quality, premature rejection of animals and treatment costs [V. Goncharov, V. Karpov. ., Yakimchuk I.L. Prevention and treatment of mastitis in animals. M .: Rosselkhozizdat, 1987. - P.3].

The pathogenesis of the disease is complex, and the reasons are not always clear, which causes difficulties in diagnosis and treatment. The leading role in the occurrence of mastitis is the microbial factor [Ivashura A.I. Hygiene of milk production. M .: Rosagropromizdat, 1989. - P.218]. It is known that the spectrum of mastitis pathogens is represented mainly by microorganisms of the genus Staphylococcus and Streptococcus [Demidova L.D. Etiology of mastitis in cows. / Abstracts of the IX International symposium on machine milking of farm animals. - Orenburg, 1997. - S.188-189].

The traditional approach to solving the problem of diagnosing subclinical forms of mastitis is associated with the study of milk using diagnostic tests - tests with dimastin, mastidine, etc. [Goncharov VP, Karpov VA, Yakimchuk I.L. Prevention and treatment of mastitis in animals. M .: Rosselkhozizdat, 1987. - P.36-38].

However, the diagnosis of the subclinical form of mastitis with these methods does not fully assess the picture of the disease and is only the basis for the selection of cows suspected of mastitis for further bacteriological examination of cows [Mutovin V.I. Fighting mastitis of cows. M .: Publishing house of agricultural literature, magazines and posters, 1963. - S. 97].

A known method for the diagnosis of subclinical mastitis of microbial etiology in cows by counting the number of bacterial cells of the main causative agents of the disease in the milk of cows [Serebryakov EV, Vilitok IG Fighting mastitis during machine milking of cows. Kiev: Publishing House "Harvest", 1969. - 53-54; 58-60]. The disadvantage of this method is that it does not take into account the biological properties of mastitis pathogens, while the degree of development of the inflammatory process in the mammary gland depends on the virulence of the pathogen, on the one hand, and the resistance of the cow, on the other hand [Zagayevsky I.S. Prevention of diseases of cows with mastitis. Chisinau: "Cartya Moldovenienasca", 1974. - P.11].

A known method for the diagnosis of chronic and acute course of subclinical mastitis in cows is based on determining biological properties of Staphylococcus aureus and E. coli, including a group of properties aimed at inactivating factors of the natural anti-infection resistance of a macroorganism [Chernova OL, Komarova NK, Matyushina S.B. The role of persistence factors of microorganisms in the chronic course of mastitis. / Abstracts of the IX International symposium on machine milking of farm animals. - Orenburg, 1997. - S.219-220].

However, this method allows you to diagnose only the course of already identified subclinical mastitis caused by these types of pathogens.

Closest to the claimed method for the intended purpose and combination of essential features is a method for the diagnosis of subclinical forms of mastitis of microbial etiology, in particular those caused by S.aureus, S.epidermedis and E.coli, by determining the ability of inactivated factors of natural anti-infection resistance in isolated pure cultures of pathogens. The isolated pure cultures of pathogens determine the antilysocyme and lysozyme activity (ALA and LA, respectively). The presence and level of severity of these signs diagnose the form of mastitis caused by an isolated pathogen [Chernova OL. Features of microflora and lysozyme content in milk in mastitis of cows // Veterinary medicine. - No. 4. - 2001 - S. 32-34].

However, this method has several significant disadvantages:

1) Allows you to diagnose subclinical mastitis initiated by Staphylococcus aureus and Staphylococcus epidermedis, which narrows the scope of the method, since mastitis initiated by streptococci and other types of staphylococci remain beyond the diagnostic uncertainty.

2) The ability to inactivate the factor of natural anti-infectious resistance, namely antilysocyme activity in the isolated pure cultures of pathogens, is determined by the cup method, which is characterized by low sensitivity and significant error in the quantification of diagnostic signs.

The inventive method solves the problem of increasing the efficiency of diagnosis of subclinical mastitis of microbial etiology in cows.

To solve this problem, in the claimed method for the diagnosis of subclinical forms of mastitis of microbial etiology in cows, including milk sampling, isolation of pure cultures, their identification and identification of the ability to inactivate factors of natural anti-infection resistance in isolated pure cultures of pathogens, antilactoferrin activity is determined in the isolated pure cultures of pathogens (ALFA) and with the level of severity of this trait in at least one of the selected pathogens for staphylococci, to and for streptococci ALFA greater than or equal to 2.0 μg / ml, a subclinical form of mastitis caused by an isolated pathogen is diagnosed.

New in the claimed method is that in isolated pure cultures of pathogens determine antilactoferrin activity (ALPA) and with the severity of this trait in at least one of the selected pathogens for staphylococci and streptococci ALFA greater than or equal to 2.0 μg / ml is diagnosed subclinical form of mastitis caused by an isolated pathogen.

The technical result achieved by the invention is that the claimed method improves the diagnosis of subclinical forms of mastitis of microbial etiology in cows due to the possibility of diagnosing subclinical forms of mastitis initiated by different types of staphylococci and streptococci, and the high accuracy of the quantitative determination of diagnostic signs.

In the patent and scientific literature there are no methods for diagnosing diseases of microbial etiology or predicting the course of the disease with determining the ability of microorganisms to inactivate the natural glycoprotein lactoferrin (antilactoferrin activity).

It has now been established that natural glycoprotein lactoferrin has a number of protective properties: bacteriostatic and bactericidal action [Arnold R.R., Russell J.E., Champion W.I. et al. // Immunity. "1982. - Vol.35. - P.792-799], takes part in the processes of local protection of mucous membranes [Dvoretsky L.I., Didkovsky N.A., Charlyev G. Study of the content of lactoferrin in bronchial contents in patients with chronic bronchitis. // Health of Turkmenistan. - 1985. - No. 10. - C.15-18], in the regulation of humoral and cellular immunological reactions, in anti-inflammatory processes, affects the complement system, regulates granulocytopoiesis [Broxmeyer HE, De Sousa M., Smithyman A. // Blood. - 1980. - Vol.55. - P.324], feedback hematopoiesis [Bagby G., Bennett R. // Blood. - 1982. -Vol .60. - P.108-112].

The authors experimentally established the information content of determining the level of severity of antilactoferrin activity in different types of staphylococci and streptococci for the diagnosis of subclinical forms of mastitis in cows. It was found that antilactoferrin activity was determined in all microorganisms isolated from milk of healthy and sick subclinical forms of mastitis in cows, however, the level of expression of this trait was 1.9 times higher in the selected pathogens of subclinical mastitis. The research results are presented in table 1.

A comparative analysis of the informative value of antilactoferrin, antilysocymal and lysozyme activity was carried out; 400 pure cultures of different types of staphylococci and streptococci isolated from milk of 150 cows with a subclinical form of mastitis and 150 healthy animals were studied.

A pure pathogen culture was isolated from the test material (milk). Microorganisms were identified to species using biochemical tests of the company "Lachema" (Czech Republic). In isolated pure cultures, antilactoferrin activity was determined according to a known method [RF patent No. 2156807, IPC 7 C 12 Q 1/02, 1/04, 2000]. In accordance with the prototype was determined in isolated pure cultures of pathogens antilysocyme and lysozyme activity (ALA and LA, respectively). The results are presented in table.2.

As can be seen from Table 2, for different types of staphylococci and streptococci isolated from the milk of healthy animals and patients with a subclinical form of mastitis, 100% antilactoferrin activity is characteristic. Moreover, staphylococci and streptococci isolated from milk of sick cows had a higher level of severity of this trait (from 2.57 to 2.99 μg / ml) compared with healthy cows isolated from milk (from 1.11 to 1.82 μg / ml).

The presence of antilysocyme activity was characterized by 19.0% -58.5% of the causative agents of subclinical mastitis and 11.1% -33.3% of microorganisms isolated from healthy animals. The lysozyme trait was determined in the range from 0% to 50.0%, depending on the type of microorganism.

When studying the average values of ALFA, it was found that ALFA among different types of Staphylococcus isolated from the milk of animals of patients with a subclinical form of mastitis was 2.51 μg / ml, among Streptococcus - 2.73 μg / ml. Alpha in streptococci and staphylococci isolated from the milk of healthy animals is 1.32 and 1.48 mgm / ml, respectively. The average value of antilysocyme activity ranged from 1.0 μg / ml to 3.5 μg / ml and did not significantly differ in the isolated pure culture of pathogens of subclinical mastitis and microorganisms isolated from healthy animals.

Thus, the studies revealed that all types of staphylococci and streptococci isolated from the milk of animals with a subclinical form of mastitis have antilactoferrin activity with a severity level of this trait greater than or equal to 2 μg / ml.

Thus, if the studied pure culture of pathogens isolated from the milk of the examined animal has detected antilactoferrin activity with a severity level of this trait greater than or equal to 2 μg / ml, it is possible to diagnose the subclinical form of mastitis and select the most optimal treatment regimens.

The proposed method for the diagnosis of subclinical forms of mastitis of microbial etiology in cows was tested in BPH them. Kuibyshev of the Orenburg region of the Orenburg region. The effectiveness of the diagnostics carried out in accordance with the claimed method was 95.0%.

The method is as follows.

1. The test material (milk) is collected in sterile tubes and plated on blood agar plates.

2. Crops are incubated in a thermostat at a temperature of 37 ° C for 24-48 hours.

3. The grown colonies are subcultured on mowed agar and studied their morphology when staining Gram smears.

4. A pure culture is identified to a species using biochemical tests of the company "Lachema" (Czech Republic).

5. In all isolated staphylococci and streptococci, antilactoferrin activity is determined by a known method [RF patent No. 2156807, IPC 7 C 12 Q 1/02, 1/04, 2000].

6. If at least one of the selected pathogens detects antilactoferrin activity with a severity level greater than or equal to 2 μg / ml, a subclinical form of mastitis is diagnosed.

Examples of specific performance.

Example 1. A milk sample was taken from a cow in a sterile tube and plated on blood agar plates. Crops were incubated in a thermostat at a temperature of 37 ° C for 24-48 hours. The grown colonies were subcultured on mowed agar and studied their morphology when staining Gram smears. By the combination of morphological (cocci located in clusters) and tinctorial (positive when stained according to Gram) properties, this strain was attributed to representatives of the genus Staphylococcus. A pure culture was then identified to a species using biochemical tests from Lachema (Czech Republic) and identified as Staphylococcus aureus. At the next stage of work, isolated staphylococcus determined antilactoferrin activity.

Based on the results on the presence of the pathogen antilactoferrin activity with a severity level of 2.71 μg / ml, the diagnosis was made - subclinical form of mastitis of microbial etiology.

Further examination of the animal confirmed the correctness of the diagnosis made using the proposed method.

Example 2. Bacteriological examination of milk taken from a clinically healthy animal was carried out according to example 1.

In bacteriological analysis, associations of Staphylococcus aureus with an antilactoferrin activity of 1.34 μg / ml and Streptococcus agalactiae with an antilactoferrin activity of 2.69 μg / ml were isolated from milk. Based on the results, a diagnosis was made - a subclinical form of mastitis of microbial etiology. The diagnosis was confirmed by further examination.

Example 3. During a routine preventive examination, a milk sample was taken from a cow. Clinically, the animal is great. The test with dimastidine is negative. During bacteriological analysis, Staphylococcus epidermidis was isolated from milk with a level of severity of antilactoferrin activity of 2.54 μg / ml. The diagnosis was made - a subclinical form of mastitis of microbial etiology. Further observation of the animal showed the correct diagnosis.

Example 4. During a routine preventive examination, a milk sample was taken from a cow. Clinically, the animal is great. The test with dimastidine is doubtful. In bacteriological analysis, Staphylococcus saprophyticus was isolated from milk with a level of severity of antilactoferrin activity of 1.04 μg / ml. It was concluded that the animal is healthy. Further observation of the animal confirmed the correctness of the conclusion.

The proposed method allows to increase the efficiency of diagnosis of subclinical forms of mastitis of microbial etiology in cows due to the possibility of diagnosing subclinical forms of mastitis, initiated by different types of staphylococci and streptococci, and high accuracy of quantitative determination of diagnostic signs. Timely and efficient identification of animals with a subclinical form of mastitis will reduce the economic damage to livestock.

Table 1
Comparative analysis of the level of severity of antilactoferrin activity in microorganisms isolated from milk of healthy animals and patients with subclinical form of mastitis Types of microorganisms Animal condition ALFA (μg / ml) Lowest value Highest value Mean Staphylococcus Aureus (n = 137) Healthy (n = 18) 1,03 1.87 1,54 Patients (n = 119) 2.08 2.99 2,57 Staphylococcus Epidermidis (n = 43) Healthy (n = 10) 1,1 1.78 1.31 Patients (n = 33) 2.03 2.97 2.78 Staphylococcus Saprophyticus (n = 10) Healthy (n = 2) 1,0 1.22 1,11 Patients (n = 8) 2.03 2,34 2.19 Streptococcus agalactiae (n = 165) Healthy (n = 15) 0.65 1.44 1.17 Patients (n = 150) 2,3 2.79 2.79 Streptococcus Lacttis (n = 14) Healthy (n = 5) 1.33 1.90 1.82 Patients (n = 9) 2.2 2,8 2.45 Streptococcus Uberus (n = 11) Healthy (n = 3) 1,2 1.8 1.70 Patients (n = 8) 2.04 2.88 2.67 Streptococcus pyogenes (n = 20) Healthy (n = 6) 0.9 1,5 1.21 Patients (n = 14) 2.1 3.0 2.91

Claims (1)

A method for diagnosing a subclinical form of mastitis of microbial etiology in cows, including collecting a milk sample, isolating pure cultures, identifying them and determining, in isolated pathogens, the ability to inactivate factors of natural anti-infection resistance, characterized in that antilactoferrin activity (ALPA) is determined in isolated pure cultures of pathogens with the level of severity of this trait in at least one of the selected pathogens, for both staphylococci and streptococci, ALFA is greater than or equal to 2.0 μg / ml, a subclinical form of mastitis caused by an isolated pathogen is diagnosed.