RU2729106C1 - Method for non-invasive monitoring of the upper respiratory tract in calves - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention represents a method for monitoring the condition of upper airways in calves, involving selection of sterile cotton tampons or cotton gauze tampons of left and right nasal holes, placing them in sterile test tubes and transferring to device "electronic nose" based on five piezoelectric sensors, with base oscillation frequency of 10–15 MHz, electrodes of which there are films of acetone solutions of polyethylene glycol succinate (PEGS), bromocresol blue (BCB), triton X-100 (TX-100), chloroform suspension of multilayer carbon nanotubes (MCNT) and an ethanol suspension of hydroxyapatite (HA) with coating weight after removing solvent 4–10 mcg, recording sensor signals in the "electronic nose" program for 180 s, wherein 80 s of the array load are carried out by holding a cell with an open inlet above tampon and 120 with unloading without a sample in turns for each sample, recording the area of the circular signal diagram of the sensors in the software and applying the criterion as the evaluation value during monitoring, wherein if relative differences of areas for left and right samples from one animal exceed 20 %, this means that on the side giving large responses, an inflammatory process begins / proceeds, reduced areas of circular diagrams of sensor signals when conducting therapeutic actions for averaged sample is associated with positive dynamics (recovery), and increase by more than 20 % is associated with development of disease.

EFFECT: invention allows to considerably simplify and increase efficiency of monitoring and control of efficiency of treatment of inflammatory process of upper respiratory tract, time costs for clinical and laboratory investigations.

1 cl, 3 ex

Description

The invention relates to veterinary medicine and can be used for express diagnostics of the inflammatory process of the upper respiratory tract in calves in order to monitor the condition of animals and control the effectiveness of treatment by the composition of the easily volatile fraction of nasal mucus.

For the intravital diagnosis of the inflammatory process in the respiratory organs in calves, a complex of clinical and laboratory research methods is currently used [Shakhov A.G. Respiratory diseases of calves / A.G. Shakhov, A.I. Anufriev, S.M. Suleimanov, A.I. Zolotarev, S.I. Pershina // Complex ecologically safe system of veterinary protection of animal health. - M .: FGNU "Rosinformagrotech", 2000. - S. 163-186]. The diagnosis of respiratory diseases in calves is carried out in a comprehensive manner based on the analysis of epizootological data, clinical symptoms, pathological changes and the results of laboratory (virological, bacteriological, serological, mycological, etc.) studies. In this case, they carry out:

- selection and transfer of biological material to a specialized laboratory or research institution;

- preparation of biological material for research;

- isolation of pathogens, antigens and retrospective diagnostics;

- if necessary, morphological and biochemical blood tests and histological examination of the material.

Clinical methods are aimed at detecting changes in the physiological parameters of the body: body temperature, heart rate and respiratory movements, respiratory minute volume and tidal volume (breathing depth), wheezing, shortness of breath, coughing, nasal discharge. Laboratory studies (hematological, immunological, biochemical) blood parameters - leukograms, erythrocyte sedimentation rate, immunoglobulin classes, concentration of C-reactive protein, interleukins, defensins, cortisol content, antioxidants, peroxidation products, etc. - are aimed at identifying the inflammatory process in the body of calves , however, they are all nonspecific and change during inflammation of any localization (bronchopulmonary inflammation, omphalitis, enteritis, hepatitis, etc.). The final diagnosis of the infectious nature of respiratory pathology in calves is made on the basis of the results of comprehensive laboratory studies of nasal and tracheal lavages, sputum and bronchoalveolar lavage using virological, bacteriological and molecular genetic research methods.

The disadvantages of this method are: invasiveness (trauma) of obtaining biological material (blood, nasal and tracheal lavage, broncho-alveolar lavage), significant material and time costs (up to 72 hours) for conducting research, the possibility of obtaining false negative results, since inflammation of the upper respiratory tract (bronchitis, tracheobronchitis) in calves often has a blurred clinical picture and is asymptomatic in the early stages of development, the need for special laboratory equipment and highly qualified personnel.

For the diagnosis of the inflammatory process in the respiratory organs in calves informative is the study of exhaled air condensate (EBC) - an organ-specific biological material obtained non-invasively [Zolotarev A.I. Early diagnosis of bronchitis in newborn calves / A.I. Zolotarev, A.E. Chernitsky, M.I. Retskiy, L.I. Efanova // Veterinary Medicine. - 2013. - No. 3. - S.43-47].

As a prototype, the selected method of non-invasive express diagnostics of the inflammatory process in the respiratory organs in calves by the composition of the equilibrium gas phase above the samples of exhaled air condensate. For this purpose, the condensate of exhaled air is collected and the equilibrium gas phase above it is analyzed using a detecting device of the "electronic nose" type based on an array of three piezosensors with a base vibration frequency of 10-15 MHz, on the electrodes of which films are applied from acetone solutions of bromocresol blue (BCS ), Triton X-100 (TX-100), and a chloroform suspension of multilayer carbon nanotubes (MCNTs) with a coating mass after removal of the solvent of 4-10 μg. Registration in the "electronic nose" program of the maximum signals of the array of piezosensors in 60 s and the calculation of the inflammation index (PV) according to the formula:

where ΔF _BKC is the greatest change in the oscillation frequency of a piezosensor with a BKS film,

ΔF _TX-100 - the greatest change in the oscillation frequency of a piezosensor with a TX-100 film,

ΔF _MWCNT - the greatest change in the oscillation frequency of a piezosensor with a film of MWCNT, diagnostics of an inflammatory process in the respiratory organs of calves with a PV value greater than 1.2 ± 0.1. The use of the invention will make it possible to quickly diagnose inflammatory processes in the respiratory organs of calves [US Pat. RU 2564877 Method of non-invasive express-diagnostics of the inflammatory process in the respiratory organs of calves / Chernitskiy AE, Kuchmenko TA, Shuba AA, Retskiy MI]. The disadvantage of this method is the need to collect the condensate of exhaled air for at least 30 minutes, a significant increase in the time of obtaining information if it is necessary to re-measure, which limits the number of animals studied and reduces the effectiveness of monitoring in the farm. In addition, the procedure for collecting the condensate of the exhaled air requires efforts from the veterinarian, due to the need for long-term fixation of the animal, and is inconvenient when keeping young animals in groups.

The technical objective of the invention is to develop a method for monitoring the state of the upper respiratory tract in calves, including newborns, which makes it possible to significantly simplify and increase the performance of monitoring the state of the upper respiratory tract and control the effectiveness of treatment of the inflammatory process of the upper respiratory tract, reduce the economic and time costs of clinical and laboratory research, analysis of their results, form a database and draw up an individual map of the state of the animal based on the results.

To solve the technical problem of the invention, a method is proposed for monitoring the state of the upper respiratory tract in calves, including the selection of sterile cotton or cotton-gauze swabs from the left and right nasal openings, placing them in sterile test tubes and transferring them to the device "electronic nose" based on five piezosensors, with a basic vibration frequency of 10-15 MHz, on the electrodes of which films are deposited from acetone solutions of polyethylene glycol succinate (PEGS), bromcresol blue (BCS), Triton X-100 (TX-100), chloroform suspension of multilayer carbon nanotubes (MWCNT) and ethanol suspension of hydroxyapatite (GA) with a coating mass after removal of the solvent of 4-10 μg, registration of sensor signals in the "electronic nose" program for 180 s, while carrying out 80 s of the array load by holding the cell with an open inlet over the tampon and 120 s of unloading without a sample in turn for of each sample, record in the software the area of the signal circle sors and use this criterion as an evaluation criterion for monitoring, while if the relative differences in the areas for the left and right samples from one animal exceed 20%, this means that from the side that gives great responses, an inflammatory process begins / goes on, a decrease in the areas of circular the diagrams of sensor signals during treatment for the averaged sample is associated with positive dynamics (recovery), and an increase of more than 20% is associated with the development of the disease.

The method for monitoring the state of the upper respiratory tract in calves is carried out as follows.

To select swabs of nasal mucus, sterile cotton or cotton-gauze tampons on a holder are used, placed in test tubes, and a detection device of the "electronic nose" type [Kuchmenko, T.A. Innovative solutions in analytical control: textbook / T.A. Kuchmenko. - Voronezh: Voronezh State Technological Academy, 2009, ISBN 978-5-89448-596-6, p. 26-62] based on an array of 5 piezosensors with a base vibration frequency of 10-15 MHz, on the electrodes of which films are applied from acetone solutions of polyethylene glycol succinate (PEGS), bromocresol blue (BCS), Triton X-100 (TX-100), chloroform suspension multilayer carbon nanotubes (MCNTs) and ethanol suspension of hydroxyapatite (HA) with a coating mass after solvent removal of 4-10 μg (a change in the coating mass on the electrodes of piezoelectric sensors, the nature of sorbents, algorithms for processing analytical information worsens the metrological characteristics of the method, contributes to an increase in the proportion of erroneous conclusions). The prepared detecting device is connected to a computer and controlled by a program that registers in real time the signals of the piezosensor array in the form of chronofrequency patterns. When examining calves using a detecting device of the "electronic nose" type for the presence of an inflammatory process of the upper respiratory tract, an open detection cell is used and the following measurement mode is used: the cell is held over the tampon for 80 s (load) and for 120 s without a sample (unload) ... The area of the circular diagram of the sensor signals for the entire measurement time is recorded in the software and this criterion is used as an evaluation criterion for monitoring and diagnosing the condition.

At the same time, if the relative differences in the areas of the figures of the circular diagrams of the sensor signals for samples from the left and right nasal openings for one animal exceed 20%, this means that from the side giving large responses and, accordingly, the area, an inflammatory process begins / goes on, a decrease areas of circular diagrams of sensor signals during therapeutic measures for an average sample is associated with positive dynamics (recovery), and an increase of more than 20% is associated with the development of the disease.

The method for monitoring the condition of the upper respiratory tract in calves is illustrated by examples.

Example 1.

Calf with inv. No. 2676 at the age of 9 days. At the initial examination healthy from the respiratory system, body temperature 39.0 ºC, spontaneous and induced cough, wheezing and nasal discharge, clinical assessment on the WI scale 3 points [McGuirk SM Disease management of dairy calves and heifers / SM McGuirk // Vet ... Clin. North Am. Food Anim. Pract. - 2008. - Vol. 24. - Issue 1. - P. 139-153]. In a laboratory study of blood, hematological signs of inflammation are absent (leukocyte count 6.6 × 10 ⁹ / L, neutrophils 3.7 × 10 ⁹ / L, nuclear neutrophilic shift index 0.08), the concentration of haptoglobin in the blood serum is increased - 4.49 g / L, during the PCR study of tracheal lavage, the adenovirus genome was isolated.

To take nasal mucus swabs from the calf, sterile cotton swabs on a holder were used, and the mucus taken alternately from the left and right was placed in a test tube (averaged sample). A detecting device of the "electronic nose" type was preliminarily prepared for analysis based on an array of 5 piezosensors with a basic vibration frequency of 10-15 MHz, with films of polyethylene glycol succinate (PEGS), bromocresol blue (BCS), Triton X-100 (TX-100), phases from multilayer carbon nanotubes (MCNT) and hydroxyapatite (HA). During the examination, an open detection cell was used and the following measurement mode was used: the cell was held over the tampon for 80 s (load) and for 120 s without a sample (unload). The area of the circular diagram of the sensor signals was recorded in the software for the entire time of measurement and this criterion was used as an evaluation criterion for monitoring and diagnosing the condition. The area of the pie chart figure at the first control point was 9900 ± 100 (measurement error) Hz ² .

At the second examination at the age of 18 days, the calf was diagnosed with early manifestations of tracheobronchitis (induced cough on palpation of the last tracheal ring and after 30 seconds of apnea), body temperature within the normal range of 38.6 ºC, spontaneous cough, wheezing and nasal discharge are absent, clinical score on the WI scale 4 points. Hematological (leukocyte count in the blood 12.0 × 10 ⁹ / l, neutrophils 6.4 × 10 ⁹ / l, nuclear neutrophilic shift index 0.06) and biochemical (concentration of haptoglobin in the blood serum 4.92 g / l) signs of inflammation confirm the clinical diagnosis. PCR study of tracheal lavage isolated the adenovirus genome. At this control point, the mucus was taken again (averaged sample) and analyzed using the "electronic nose" device. The area of the pie chart for the sample was 23 300 Hz ² .

The relative differences in the areas of the figures of the circular diagrams of the sensor signals for the samples at the first and second control points is significantly more than 20%, which is associated with the development of the inflammatory process.

From example 1 it can be seen that when the calf was re-examined after 9 days, a deterioration was observed, indicating the development of tracheobronchitis of a viral (adenovirus) etiology, which is adequately reflected by the results of the analysis of easily volatile compounds of nasal mucus (averaged samples).

Example 2.

Calf with inv. No. 2671 at the age of 19 days. At the initial examination: bilateral serous nasal discharge, hyperemia of the mucous membrane of the nasal cavity, spontaneous cough with sputum discharge, mixed shortness of breath, increased sensitivity of the trachea to palpation, with auscultation of the chest - moist large bubbling rales, body temperature 40.1 ºC, clinical assessment on a scale WI 8 points. In a laboratory study of blood, hematological (leukocytosis 11.0 × 10 ⁹ / l and a shift in the leukocyte formula to the left) and biochemical (haptoglobin content in the blood serum 4.37 g / l) signs of inflammation. PCR study of tracheal lavage isolated the adenovirus genome, bacteriological study - E. coli serovariant O142 and Ent. faecalis. Based on the results of clinical and laboratory studies, the patient was diagnosed with rhinitis and tracheobronchitis of viral-bacterial etiology. Etiotropic and symptomatic treatment was started. To take nasal mucus swabs from the calf, sterile cotton swabs on a holder were used, and the mucus taken alternately from the left and right was placed in a test tube (averaged sample). Analyzed as described in example 1. The area of the circular diagram of the sensor signals for this sample was 15100 Hz ² .

When the animal was re-examined after 9 days, the animal had a normal body temperature of 38.3 ºC, nasal discharge, spontaneous and induced cough after 30 seconds of apnea, and wheezing was absent, the clinical assessment on the WI scale was 2 points, of the residual phenomena, cough was detected with palpation of the last tracheal ring. Despite obvious clinical improvements, the calf retains hematological (leukocytosis 12.0 × 10 ⁹ / L and a shift of the leukocyte formula to the left) and biochemical (serum haptoglobin content 5.19 g / L) signs of inflammation.

At this control point, the mucus was taken again (averaged sample) and analyzed using the "electronic nose" device. The area of the circle diagram of the sensor signals for the sample was 11 185 Hz ² .

The relative differences in the areas of the figures of the circular diagrams of the sensor signals for the samples at the first and second control points is 26%, which is associated with an improvement in the animal's condition.

From example 2, it can be seen that when re-examining after 9 days of etiotropic and symptomatic therapy, the animal showed an improvement in the condition, indicating a positive trend.

Example 3.

Calf with inv. No. 2673 at the age of 14 days. At the initial examination: spontaneous cough with sputum release, mixed dyspnea, increased sensitivity of the trachea to palpation, during auscultation of the chest, moist large-bubble rales are heard, nasal discharge is absent, body temperature is increased to 40.4 ºC, clinical assessment on the WI scale is 6 points. In a laboratory study of blood, the content of leukocytes is normal 5.8 × 10 ⁹ / l, neutrophils - 2.0 × 10 ⁹ / l, the nuclear neutrophilic shift index is 0.06, the concentration of haptoglobin in the blood serum is increased 4.41 g / l. PCR study of tracheal lavage isolated the adenovirus genome, bacteriological study - E. coli serovariant O142 and Ent. faecium. Based on the results of clinical and laboratory studies, tracheobronchitis of viral-bacterial etiology was diagnosed. Etiotropic and symptomatic treatment was started. To take nasal mucus swabs from the calf, sterile cotton swabs on a holder were used, and the mucus taken alternately from the left and right was placed in a test tube (averaged sample). Analyzed as described in example 1. The area of the circular diagram of the sensor signals for this sample was 6850 Hz ² .

When the animal was re-examined 9 days later, the animal's body temperature (38.1 ºC) and respiratory rate (26 per minute) were normal, no nasal discharge, the clinical WI score decreased to 3 points, but spontaneous cough and wet rales remained on auscultation chest. Despite clinical improvements, negative dynamics of laboratory parameters was found (the content of leukocytes in the blood increased from 5.8 × 10 ⁹ / L to 8.4 × 10 ⁹ / L, neutrophils - from 2.0 × 10 ⁹ / L to 4.5 × 10 ⁹ / l, the concentration of haptoglobin in the blood serum increased from 4.41 g / l to 5.03 g / l). The prognosis of the disease is doubtful.

At this control point, the mucus was taken again (averaged sample) and analyzed using the "electronic nose" device. The area of the circular diagram of the sensor signals for the sample was 6800 Hz ² .

The relative difference in the areas of the figures of the circular diagrams of sensor signals for samples at the first and second control points does not exceed 1% (less than 20%), which is associated with the stability of the animal's state.

Example 3 shows that the calf's condition after 9 days of treatment did not change significantly.

The presented examples show that the proposed method for monitoring the state of the upper respiratory tract in calves by the composition of easily volatile compounds of nasal mucus is characterized by high sensitivity, simplicity and reliability, it can significantly simplify and increase the productivity of monitoring and control the effectiveness of treatment of the inflammatory process of the upper respiratory tract, reduce economic and time spent on clinical and laboratory studies, analysis of their results, to form a database and draw up an individual map of the animal's condition based on the results.

Claims (1)

A method for monitoring the state of the upper respiratory tract in calves, including the selection of sterile cotton or cotton-gauze swabs from the left and right nasal openings, placing them in sterile test tubes and transferring them to the device "electronic nose" based on five piezosensors, with a basic vibration frequency of 10 15 MHz, on the electrodes of which films of acetone solutions of polyethylene glycol succinate (PEGS), bromcresol blue (BCS), Triton X-100 (TX-100), chloroform suspension of multilayer carbon nanotubes (MWCNT) and ethanol suspension of hydroxyapatite (HA) with a mass of coating after removing the solvent 4-10 μg, registration of the sensor signals in the "electronic nose" program for 180 s, while carrying out 80 s of the array load by holding the cell with an open inlet over the swab and 120 s of unloading without a sample alternately for each sample, register in the software ensuring the area of the circular diagram of the sensor signals and apply this criterion as an estimate during monitoring, in this case, if the relative differences in the areas for the left and right samples for one animal exceed 20%, this means that an inflammatory process begins / goes on from the side that gives large responses, a decrease in the areas of the circular diagrams of sensor signals during treatment the average sample is associated with positive dynamics (recovery), and an increase of more than 20% is associated with the development of the disease.