RU2748979C1 - Method for diagnosis of calf malnutrition (bos taurus) based on analysing expression of pro-inflammatory cytokine gene of interleukin il1α - Google Patents

Abstract

FIELD: biotechnology; veterinary science.SUBSTANCE: present invention relates to biotechnology and veterinary science, can be used for diagnosing calf hypotrophy. Method comprises extracting total RNA from collected calf blood, then cDNA is obtained. That is followed by PCR with cDNA, interleukin 1α gene expression is calculated in relation to housekeeping gene expression by indicator 2-ΔΔct, where ΔΔCt = ΔCt (calf with suspected hypotrophy) - ΔCt (healthy calf); ΔCt = Ct (interleukin 1α gene) - Ct (β-actin gene). Intercalating colouring agent used in PCR is SYBR Green.EFFECT: invention enables assessing the risk of developing hypotrophy in calves within 4–5 hours after blood sampling.1 cl, 3 dwg, 1 tbl, 1 ex

Description

The present invention relates to veterinary medicine and can be used to diagnose malnutrition in calves (Bos taurus).

Among the diseases of non-infectious etiology, hypotrophy has a special role, since it is a widespread disease of calves, piglets, lambs and other animal species associated with impaired development and growth in the antenatal period. Economic losses from this pathology consist of the death of young animals, growth retardation, loss of breeding qualities, deterioration in the quality of animal meat and a decrease in the return on feed (Anokhin B.M., Causes of diseases of young animals, diagnosis, control measures. - M .: MEINF, 2002. - 191 p; Samokhin V.T.Optimization of the metabolic status of mothers cows - the basis for the prevention of neonatal diseases of calves / V.T.Samokhin // Actual problems of diseases of young animals in modern conditions: mater, international conference, Voronezh, September 23-25, 2002 - Voronezh: VGAU, 2002. - S. 37-38.). Hypotrophic calves are distinguished by morphological and functional underdevelopment of various organs and their systems. Such animals have a weight 1.5-2 times lower than their peers. In such calves, in addition to a decrease in the concentration of hemoglobin and erythrocytes, blood plasma proteins, disorders of water-electrolyte metabolism, disorders of neuroendocrine regulation, the body's immunobiological reactivity and its resistance to infectious diseases are sharply reduced. With hypotrophy, the protective function of the skin and mucous membranes, the barrier role of the lymph nodes, the activity of the system of phagocytic micro- and macrophages, substances that have a bactericidal and antiviral effect - properdin, lysozyme, complement, interferon are reduced (Boyarentsev L.E. Immunomodulatory activity of ligaverine in calf hypotrophy / L.E.Boyarantsev // Veterinary. - 2002. - No. 9. - P. 41-44). Hypotrophy is a pathology of the fetus, manifested by a violation (inhibition, inhibition) of its development and arising as a pathophysiological reaction to insufficient supply of the fetus with oxygen, nutrients and biological active substances or in violation of their digestibility. Hypotrophy reflects the concept of "physiological immaturity" of newborns. Hypochromic anemia in calves is a pathological condition characterized by hypochromia of erythrocytes, their low content, the predominance of microcytes, anemicity of varying degrees of visible mucous membranes, anorexia. Anemia is proposed to be considered as a pathology of transsyndromic comorbid hypotrophy, pathogenetically associated and interrelated. The comorbid profile of morbidity with multisystem multiple organ failure is one of the most dangerous risks of violation of the harmonious modeling of the organism of young farm animals, the likelihood of which increases with a decrease in the adaptability of technologies. Savrasov / / Topical issues of veterinary biology. 2018. No. 1 (37). S. 7-10. As a result, hypotrophic calves easily develop diseases of the respiratory system and gastrointestinal tract, even caused by opportunistic microorganisms. With this pathology, in terms of its physical parameters, the fetus does not correspond to the size appropriate for a given gestational age. Delay in fetal development is associated with the impact on the maternal organism of a variety of stress factors of exogenous and endogenous origin. Early diagnosis of this disease in calves is difficult due to the wide range of features that characterize this condition. It is necessary to search for a marker that would reflect the general condition of calves with malnutrition.

A known method for early diagnosis of intrauterine growth retardation of the embryo and fetus in cows (RU 2539015 C1). In the method, in the early stages of gestation, ultrasound scanning of both the embryo and the fetus in cows is carried out, on the basis of which the fetometric parameters of the embryo and fetus are determined. If the embryo length is within 12-16 mm and the body diameter is 7-9 mm at 38-40 days of gestation, the syndrome of delayed embryo development in cows is diagnosed. In case of detection on days 60-65 of the length of the fetus in the range of 25-45 mm and the diameter of the body - 12-16 mm, the syndrome of delayed fetal development in cows is diagnosed. The method allows at the early stages of gestation to diagnose the syndrome of intrauterine growth retardation of the embryo and fetus in cows to prevent intrauterine embryo death, antenatal fetal malnutrition, intra- and neonatal diseases in the fetus and newborn calves, birth and postpartum pathology in mothers cows.

A known method for predicting the risk of malnutrition in children born at the time of very early preterm labor, to the corrected age of 6 months (patent RU 2698564 C1). The method determines the period of restoration of body weight at birth (in days). Consider the presence or absence of severe preeclampsia during pregnancy in a woman. Calculate the prognostic index X according to the original calculation formula. The method is not applicable for predicting malnutrition in calves.

A known method for the diagnosis of malnutrition in calves on the basis of clinical and hematological data and indicators of articles of the body (Hypotrophy of calves: Diagnostics. Prevention. Treatment. Savrasov DA LAP LAMBERT Academic Publishing. Pages 77-91), taken as a prototype. The method determines the physical properties of the blood, the morphological properties of the blood, the biochemical parameters of the blood, the habit of the animal.

The disadvantage of this method is the need to use a wide range of methods for identifying hypotrophy, which significantly increase both the time and complexity of the analysis, and the cost of the analysis.

The technical result of the proposed invention is to assess the risk of malnutrition in calves within 4-5 hours after taking blood.

To achieve the technical result in the method for diagnosing calf malnutrition, according to the invention, total RNA is isolated from the selected calf blood, sequentially reverse transcription is performed to obtain cDNA, real-time PCR from the obtained cDNA, the expression of the interleukin 1α gene is calculated in relation to the expression of the household gene according to indicator 2 ^-ΔΔct , where ΔΔCt = ΔCt (calf with suspected malnutrition) - ΔCt (healthy calf); ΔCt = Ct (interleukin 1α gene) - Ct (β-actin gene), and in the case of a obtained value of 2 ^-ΔΔct ≥ 1.7, malnutrition is diagnosed in a calf with a 90% probability. Primers SEQ ID NO: 1 and SEQ ID NO: 2 are used to amplify a region of the IL1α gene, primers SEQ ID NO: 3, SEQ ID NO: 4 are used to amplify the housekeeping gene (β-actin), and as an intercalating dye in PCR use SYBR Green.

At the All-Russian Scientific Research Veterinary Institute of Pathology, Pharmacology and Therapy, studies were carried out to study the expression of cytokine genes in calves with malnutrition. In the course of the research, it was revealed that 90% of calves with malnutrition (9 out of 10 calves) on days 2-3 had an increase of at least 1.7 times in the expression of the pro-inflammatory cytokine interleukin 1α (IL1α) gene in relation to the expression of this gene in healthy calves ... Thus, this feature can be used as a diagnostic one in assessing the risks of malnutrition.

In the presented method, hypotrophy is determined by analyzing the expression of the proinflammatory cytokine IL1α gene. The method is carried out as follows.

In a calf, from the selected blood immediately (within 30 minutes), total RNA is isolated by any available method (if it is impossible to isolate RNA from the blood immediately, then the blood must be stored at -20 ° C for no more than two weeks). Then, reverse transcription is carried out to obtain cDNA. Then, real-time PCR is carried out with the obtained cDNA, while primers SEQ ID NO: 1 and SEQ ID NO: 2 are used to amplify the IL1α gene region. Primers are used to amplify the housekeeping gene (β-actin): SEQ ID NO: 3, SEQ ID NO: 4. SYBR Green was used as an intercalating dye in PCR. PCR is carried out according to the following protocol: primary denaturation at 95 ° C - 3 minutes; 38 cycles, including denaturation at 95 ° C for 30 seconds, annealing of primers at 58 ° C for 30 seconds, chain elongation at 72 ° C for 30 seconds, signal registration. The calculation of the expression of the interleukin 1α gene in relation to the expression of the housekeeping gene is based on the calculation of the index 2 ^-ΔΔct , where ΔΔCt = ΔCt (calf with suspected malnutrition) - ΔCt (healthy calf); ΔCt = Ct (interleukin 1α gene) - Ct (β-actin gene). To make a diagnosis, it is necessary to have cDNA from the blood of healthy calves 2-3 days old, which can be obtained earlier and stored for a year at -20 ° C. If, when calculating by the formula 2 ^-ΔΔct , a value of ≥ 1.7 was obtained, then the calf with a probability of 80% has malnutrition.

Example.

Blood was obtained from the blood of a calf aged 3 days, from which RNA was immediately isolated using a commercially available kit "RNA-EXTRAN" (Syntol, Moscow) according to the attached instructions. After RNA isolation, reverse transcription was performed using the MMLV RT kit (Evrogen, Russia) according to the attached instructions. Real-time quantitative polymerase chain reaction was performed on a CFX96 Real-Time PCR Detection System (BioRad, USA) using a qPCRmix-HS SYBR + LowROX mixture (Evrogen, Russia) according to the following protocol: primary denaturation 95 ° C - 3 minutes; 38 cycles, including denaturation at 95 ° C for 30 seconds, annealing of primers at 58 ° C for 30 seconds, chain elongation at 72 ° C for 30 seconds, signal registration. Mixed in a test tube: 16 μl of sterile water, 5 μl of qPCRmix-HS SYBR + LowROX (Evrogen, Russia), 1 μl of primer SEQ ID NO: 1 at a concentration of 5 μM, 1 μl of primer SEQ ID NO: 2 at a concentration of 5 μM , 2 μl of the obtained cDNA. As a control, we used cDNA from a healthy calf 3 days old. During PCR, the following Ct values were obtained:

Healthy calf: IL1α - 31.28, actin - 25.40;

Calf with suspected malnutrition: IL1α - 29.01, actin - 24.84.

Further, the expression level of the IL1α gene was assessed:

ΔCt (healthy calf) = 31.28 - 25.40 = 5.88; ΔCt (calf with suspected malnutrition) = 29.01 - 24.84 = 4.17;

ΔΔCt = 4.17 - 5.88 = -1.71;

2 ^-ΔΔct = 2 ^{- (- 1.71)} = 3.27

Thus, the expression of the IL1α gene in the blood of a calf with suspected malnutrition was 3.27 times higher than in the blood of a healthy calf, which indicates that a calf suspected of malnutrition is 90% likely to have this pathological condition, since the expression level the IL1α gene was more than 1.7 times higher than the expression of this gene in a healthy calf.

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Sequence listing

<110> State Scientific Institution All-Russian Research Veterinary Institute of Pathology, Pharmacology and Therapy

<120> Primers for the study of the level of expression of the gene IL1α

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<213> Artificial sequence

<400> 2

cagcagcagc aaactgagac 20

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<212> DNA

<213> Artificial sequence

<400> 3

ctcttccagc cttccttcct 20

<210> 4

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<212> DNA

<213> Artificial sequence

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agcactgtgt tggcgtacag 20

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Claims (2)

1. A method for diagnosing calf malnutrition, characterized in that total RNA is isolated from the selected calf blood, sequentially reverse transcription is carried out to obtain cDNA, real-time PCR from the obtained cDNA, the expression of the interleukin 1α gene is calculated in relation to the expression of the housekeeping gene according to indicator 2 ^-ΔΔct , where ΔΔCt = ΔCt (calf with suspected malnutrition) - ΔCt (healthy calf); ΔCt = Ct (interleukin 1α gene) - Ct (β-actin gene), and in the case of a obtained value of 2 ^-ΔΔct ≥ 1.7, malnutrition is diagnosed in a calf with a 90% probability. 2. The method according to claim 1, characterized in that primers SEQ ID NO: 1 and SEQ ID NO: 2 are used to amplify the IL1α gene region, primers SEQ ID NO: 3, SEQ ID NO: 4, and SYBR Green was used as an intercalating dye in PCR.

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