RU2808377C1 - Method of early diagnostics of postpartum endometritis in dairy cows - Google Patents

Abstract

FIELD: medicine; veterinary medicine; veterinary obstetrics.

SUBSTANCE: invention can be used for early diagnostics of postpartum endometritis in dairy cows. 5–10 ml of cervical-vaginal mucus from a cow is taken, placed in a sterile tube which is transferred to an “electronic nose-diagnostic” device with an array of “Bio” sensors for analyzing biosamples, the device is warmed up and the stability of the sensors is checked for 5–10 minutes according to the passport, selecting at least 0.5 ml of mucus with a cotton swab for a liquid sample and 0.1 g for a thick sample, transferring the stick to a Petri dish and measuring volatile compounds with sensors for 60–80 s, interrupting the measurement and calculating 6 parameters of the qualitative composition of volatiles sample connections by dividing the maximum sensor signals when measuring dƒ_i /dƒ_j in pairs and recording the number of positive hits within the boundaries labeled “inflammation”. Positive hits are the calculated value for the samples falling within the range of values of the “inflammation” parameters: dƒ₁ /dƒ₅ =[0.84–0.96]; dƒ₂ /dƒ₃ =[0.60–0.79]; dƒ₃ /dƒ₅ =[0.60–0.75]; dƒ₃ /dƒ₈ =[0.24–0.30]; dƒ₆/dƒ₈ =[0.10–0.13]; dƒ₇ /dƒ₈ =[0.080–0.13], where the indices correspond to the number of sensors in the array, and dƒ₁–dƒ₈ are responses of all sensors, Hz. When the number of positive hits is within the following boundaries: from 0 to 2 parameters in “inflammation”, the normal status is diagnosed; from 3 to 4 parameters of “inflammation”, the initial/mild stage of endometritis is diagnosed. With more than 4 positive parameters of “inflammation” endometritis is diagnosed.

EFFECT: method provides the possibility of express, simple and objective early diagnostics of postpartum endometritis in cows with the establishment of a risk group for the development of endometritis through the analysis of mucus using gas sensors in an “electronic nose-diagnostic” device.

1 cl, 2 dwg, 2 tbl, 3 ex

Description

The invention relates to the field of veterinary medicine, in particular to veterinary obstetrics.

Postpartum endometritis is an inflammation of the predominantly uterine mucosa. Depending on the nature of the inflammation, postpartum endometritis is divided into catarrhal, purulent-catarrhal and fibrinous. It is believed that the main form of manifestation of postpartum endometritis in cows is purulent-catarrhal endometritis. Clinical signs of the disease appear 6-7 or 8-10 days after birth. In the first case, endometritis develops, as a rule, against the background of retained placenta or acute subinvolution of the uterus. From the genital organs of an animal, when lying down, straining or massaging, a large amount of purulent-mucous, purulent or serous-purulent exudate of a liquid consistency, gray-brown or yellow-brown in color is released through the rectum. Postpartum endometritis is recorded in 20-57% of calving cows [Nezhdanov A.G., Misailov V.D., Shakhov A.G. Diseases of the reproductive organs in cows and problems of their diagnosis, therapy and prevention // Current problems of diseases of the reproductive organs and mammary gland in animals. Mater. International, scientific and practical conference dedicated to the 35th anniversary of VNIVIPiT. Voronezh, 2005. - P. 8-11].

The absence in production conditions of qualified and timely diagnosis of diseases of the reproductive organs of cows and, accordingly, their adequate treatment gives rise to the widespread spread of these pathologies among the breeding stock of farms, and, as a consequence, an increase in the share of diseases of the reproductive organs in the overall morbidity of livestock I.V. Gordeeva, 2005).

There is a known method for cytological diagnosis of postpartum pathology in cows. The essence of the method is a microscopic examination of epithelial cells (superficial, parabasal, basal). With a predominant number of superficial cells (49-60%), a normal course of the postpartum period is diagnosed, with a predominance of parabasal epithelial cells, its complicated course is diagnosed [RU 2304773 C2, G01N 33/48, A61B 10/00, A01K 67/02, 08/20/2007 Bulletin . No. 23].

The disadvantage of this method is its use at a later date, the high cost of reagents, it requires a certain amount of time, highly qualified specialists and is not technologically advanced enough in production conditions.

There is a known method for predicting postpartum diseases in cows by measuring blood pressure in the area of the caudal artery using a medical tonometer twice on days 220-275 of pregnancy. If the average systolic blood pressure is detected more than 120 mmHg. and diastolic more than 80 mm Hg predict the development of complications in the postpartum period [RU 2380903 C1, A01K 67/02, 02/10/2010 Bull. No. 4].

The disadvantage of this method is the labor intensity in its implementation associated with fixation of animals, as well as low accuracy - 72.7%.

There is a known method for predicting postpartum diseases in cows by determining 15 days before calving in the blood the content of positive neutrophils in the stimulated NBT test and the concentration of placental alkaline phosphatase, followed by calculating the coefficient determined by the ratio of the content of positive neutrophils and the concentration of placental alkaline phosphatase [RU 2740531 C1, G01N 33/48, 01/15/2021, Bulletin. No. 2].

The disadvantage of this method is the lack of qualified personnel and laboratory equipment in production conditions, as well as the time required.

The closest technical solution, taken as a prototype, is a method for predicting postpartum endometritis in cows based on the presence of a mucus plug 24-48 hours after birth [RU 2019135 C1, A61B 10/00, publ. 09/15/1994].

However, this method is not diagnostically informative enough. In the presence of a mucus plug, inflammatory diseases develop in a significant proportion of fresh cows.

The purpose of the invention is to develop an express, informative method for early diagnosis and prediction of postpartum endometritis in cows by analyzing cervical-vaginal mucus using gas sensors in an “electronic nose-diagnostic” device.

To solve a technical problem, a method for early diagnosis of the development of endometritis in cows is proposed, including collecting 5-10 ml of cervical-vaginal mucus from a cow, placing it in a sterile tube, transferring it to an “electronic nose-diagnostic” device with an array of “Bio” sensors for analyzing biosamples , warming it up and checking the stability of the sensors for 5-10 minutes. according to the passport, selecting at least 0.5 ml of mucus with a cotton swab for a liquid sample and 0.1 g for a thick sample, transferring the stick to a Petri dish and measuring volatile compounds with sensors for 60-80 seconds, interrupting the measurement and calculating 6 parameters of the qualitative composition of volatiles sample connections by dividing the maximum sensor signals when measuring in pairs dƒ_i/dƒ_j and fixing the number of positive hits within the boundaries labeled “inflammation”, while positive ones are hits of the calculated value for the sample in the intervals of the “inflammation” parameter values: dƒ₁/dƒ₅=[0.84-0.96]; dƒ₂/dƒ₃=[0.60-0.79]; dƒ₃/dƒ₅=[0.60-0.75]; dƒ₃/dƒ₈=[0.24-0.30]; dƒ₆/dƒ₈=[0.10-0.13]; dƒ₇/dƒ₈=[0.080-0.13], where the indices correspond to the number of sensors in the array, and dƒ₁– dƒ₈– responses of all sensors, Hz; and when the number of positive hits within the boundaries: from 0 to 2 parameters in “inflammation”, the norm is diagnosed; from 3 to 4 parameters of “inflammation” diagnose the initial/mild stage of endometritis; more than 4 positive parameters of “inflammation” diagnose endometritis.

The invention makes it possible to determine risk groups for developing endometritis in cows by analyzing mucus using gas sensors in an “electronic nose-diagnostic” device.

To explain the method, photos of selected mucus samples in test tubes with cotton swabs are shown (Fig. 1) and a photo of the process of measuring volatile molecules over the swab with an “electronic nose-diagnostic” device (Fig. 2)

A method for early diagnosis of the development of endometritis in cows is carried out as follows.

Sampling of cervical-vaginal mucus from a cow of at least 5-10 ml is carried out in a sterile polymer tube with filler (Fig. 1). The test tube is closed and transferred to the device, which can be located anywhere on the farm, except where animals are kept. To study the material, test tubes (a swab probe with a cotton tip) and a detecting device of the “electronic nose-diagnostic” type [A mobile device based on an array of sensors for the analysis of small-volume biosamples are used. RU 2764964 C1, G01N 27/12, 01/24/2022 Bulletin. No. 3]. The device is equipped with an array of sensors with the trade name “Bio©” (Sensorika-New Technologies LLC. Russia), it is heated for 5-10 minutes and the stability of the sensors is checked according to the passport (Kuchmenko, T.A. Portable electronic nose system for fast gynecological-conditions diagnosis in consulting room: A case study / TA Kuchmenko et. al // Sensors and Actuators: B. Chemical Chemical 358 (2022) 131538: (1-12) Take at least 0.5 ml of liquid mucus with a cotton swab and 0.1 g thick, transfer the stick with mucus to a Petri dish and measure volatile compounds with sensors for 60-80 s (Fig. 1, 2). After the specified time has elapsed, interrupt it and transfer the device to the stand. In this case, in the software device, the responses of all sensors are recorded (dƒ _1-8 , Hz), six parameters are calculated that reflect the qualitative composition of the volatile compounds of the sample. To do this, the maximum signals of the sensors are divided in pairs dƒ _i / dƒ _j , where i and j are the numbers of sensors in the array (they are constant and regulated by the manufacturer). The obtained values are compared with reference limits, which were obtained when training the device using samples from healthy, sick, and treated animals. These boundaries are most closely related to the chemicals that mucus contains during inflammation and the development of endometritis, as well as with varying degrees of severity, and register a group of animals diagnosed with inflammation. The number of positive hits within the boundaries labeled “inflammation” is recorded. Next, a decision is made on the state of the animal’s reproductive health in accordance with the number of positive hits of the measured and recommended values in the “inflammation” group. The animal is considered healthy (normal) if 0-2 parameters for the measured sample are positive; the animal has an initial/mild stage of endometritis, if 3 to 4 parameters of inflammation are positive; an animal has endometritis if more than 4 positive parameters coincide with the boundaries of inflammation. Positive results are when the calculated value for the sample falls within the range of values of the “inflammation” parameters: dƒ ₁ /dƒ ₅ =[0.84-0.96]; dƒ ₂ /dƒ ₃ = [0.60-0.79]; dƒ ₃ /dƒ ₅ = [0.60-0.75]; dƒ ₃ /dƒ ₈ =[0.24-0.30]; dƒ ₆ /dƒ ₈ = [0.10-0.13]; dƒ ₇ /dƒ ₈ = [0.080-0.13].

The method is demonstrated by the following examples.

Example 1. During a clinical and obstetric examination of healthy cows (a veterinarian’s conclusion based on research results - see Table 1, column 10) under accession numbers 4918, 4648, 1.1354, 151.1.9, 1, 14181, O1, 15228, 2172 mucus samples with a volume of at least 5-10 ml were collected. The samples were placed in sterile polymer tubes with filler (Fig. 1). The tubes were closed and transferred to the “electronic nose-diagnostic” device. The device is equipped with an array of sensors with the trade name “Bio©”; it was previously prepared for measurement by heating it for 5-10 minutes. and checking the stability of the sensors according to the data sheet. We took at least 0.5 ml of liquid mucus and 0.1 g of thick mucus with a cotton swab, transferred the stick with mucus to a Petri dish and measured volatile compounds with sensors for 60-80 seconds (Fig. 1, 2). After the specified time had expired, it was interrupted and the device was transferred to the stand, while the sensors were restored within 120-140 seconds. The samples were measured one at a time. At the same time, the responses of all sensors in the array were recorded in the device software (dƒ _1-8 , Hz). Six parameters were calculated that reflect the qualitative composition of volatile compounds in the sample. To do this, we divided the maximum sensor signals in pairs dƒ _i /dƒ _j , where i and j are the numbers of sensors in the array (they are constant and regulated by the manufacturer). The obtained values (Table 1) were compared with the reference limits that were obtained earlier when training the device using samples from healthy, sick, and animals after treatment. These boundaries are most closely related to the chemicals that mucus contains during inflammation and the development of endometritis, as well as with varying degrees of inflammation and separate the group of animals diagnosed with inflammation from healthy ones. The number of positive hits within the boundaries labeled “inflammation” was recorded (Table 1, column 11). Next, a decision was made on the clinical and obstetric status of the animal in accordance with the number of positive hits of the measured and recommended values in the “inflammation” group. The animal is considered healthy (normal) if 0-2 parameters for the measured sample are positive. Positive results are when the calculated value for the sample falls within the range of values of the “inflammation” parameters: dƒ ₁ /dƒ ₅ =[0.84-0.96]; dƒ ₂ /dƒ ₃ = [0.60-0.79]; dƒ ₃ /dƒ ₅ =[0.60-0.75]; dƒ ₃ /dƒ ₈ =[0.24-0.30]; dƒ ₆ /dƒ ₈ = [0.10-0.13]; dƒ ₇ /dƒ ₈ = [0.080-0.13].

Of the 9 samples, 8 test results coincide with the conclusion of the veterinary specialist. Sample 15119 at the time of measurement belonged to the group of healthy animals, but after 2 weeks signs of the development of postpartum endometritis were recorded (Table 1, verification sample). This means that at the time of the study the animal’s condition was already different from the norm.

The method is feasible.

Example 2.

During a clinical examination of healthy cows (conclusion of a veterinary specialist based on research results - see Table 1, column 10) under inventory numbers 4098, 183500, 18222, 9978, 3420, 522265, 522190, 8926, 64, 2238, 4768 by a veterinarian mucus samples with a volume of at least 5-10 ml were taken. Samples were placed in sterile tubes (Fig. 1). The tubes were closed and transferred to the “electronic nose-diagnostic” device. The device is equipped with an array of sensors with the trade name “Bio©”; it was previously prepared for measurement by heating it for 5-10 minutes. and checking the stability of the sensors according to the data sheet. We took at least 0.5 ml of liquid mucus and 0.1 g of thick mucus with a cotton swab, transferred the stick with mucus to a Petri dish and measured volatile compounds with sensors for 60-80 seconds (Fig. 2). After the specified time had expired, it was interrupted and the device was transferred to the stand, while the sensors were restored within 120-140 seconds. The samples were measured one at a time. At the same time, the responses of all sensors in the array were recorded in the device software (dƒ _1-8 , Hz). Six parameters were calculated that reflect the qualitative composition of volatile compounds in the sample. To do this, we divided the maximum sensor signals in pairs dƒ _i /dƒ _j , where i and j are the numbers of sensors in the array (they are constant and regulated by the manufacturer). The obtained values (Table 1) were compared with the reference limits that were obtained earlier when training the device using samples from healthy, sick, and treated animals. These boundaries are most closely related to the chemicals that mucus contains during inflammation and the development of endometritis, as well as with varying degrees of inflammation and separate the group of animals diagnosed with inflammation from healthy ones. The number of positive hits within the boundaries labeled “inflammation” was recorded (Table 1, column 11). Next, a decision was made on the state of the animal’s gynecological health in accordance with the number of positive hits of the measured and recommended values in the “inflammation” group. The animal is considered healthy (normal) if 0-2 parameters for the measured sample are positive. Positive results are when the calculated value for the sample falls within the range of values of the “inflammation” parameters: dƒ ₁ /dƒ ₅ =[0.84-0.96]; dƒ ₂ /dƒ ₃ = [0.60-0.79]; dƒ ₃ /dƒ ₅ =[0.60-0.75]; dƒ ₃ /dƒ ₈ =[0.24-0.30]; dƒ ₆ /dƒ ₈ = [0.10-0.13]; dƒ ₇ /dƒ ₈ = [0.080-0.13].

The assessment of the condition of all samples coincided with the specialist’s conclusion.

The method is feasible.

Example 3.

Testing the method on a separate sample blindly, with subsequent monitoring of the condition by a veterinary specialist, showed the reliability and correctness of the results (Table 1 - test sample), as well as greater unambiguity in assessing the condition of the animals.

From the presented examples it is clear that the proposed method for the early diagnosis of postpartum endometritis in cows based on the composition of easily volatile mucus compounds is highly sensitive, simple and reliable, can significantly simplify and increase control over the effectiveness of treatment of the inflammatory process, reduce economic and time costs for clinical and laboratory studies, analysis of their results, forming a database and drawing up an individual map of the animal’s condition based on the results, assessing therapeutic effectiveness and establishing the onset of the inflammatory process at an earlier stage.

Reducing the detection time of volatile components over mucus samples to less than 60 seconds leads to false-negative results; An increase in the measurement time of more than 80 seconds leads to an unjustified increase in the time of analysis and system recovery after measurement. A decrease in the volume/weight of a mucus sample leads to an increase in the frequency of a false-negative prognosis about the animal’s condition and a transition from the “inflammation” group to the normal group.

From 20 samples of cervical-vaginal mucus (Table 2), when measuring the qualitative composition of volatile compounds, the following results were obtained:

true positive result (IP) - 11 samples;

false positive result (LP) - 0 samples;

true negative result (TN) - 8 samples;

false negative result (LF) - 1 sample.

The accuracy of the method was: (IP+IO):N×100%=(11+7):20×100=90.0%.

The specificity indicator of the method was: AI:(IO+LP)×100%=7:(7+1)×100=87.5%.

Claims (1)

A method for early diagnosis of postpartum endometritis in dairy cows, including collecting 5-10 ml of cervical-vaginal mucus from the cow, placing it in a sterile tube, transferring it to an “electronic nose-diagnostic” device with an array of “Bio” sensors for analyzing biosamples, warming it up and checking the stability of the sensors for 5-10 minutes according to the passport, selecting at least 0.5 ml of mucus with a cotton swab for a liquid sample and 0.1 g for a thick sample, transferring the stick to a Petri dish and measuring volatile compounds with sensors for 60-80 s, interrupting the measurement and calculating 6 parameters of the qualitative composition of the volatile compounds of the sample by dividing the maximum sensor signals when measuring in pairs dƒ _i / dƒ _j and recording the number of positive hits within the boundaries labeled “inflammation”, while positive values are hits of the calculated value for the sample in the intervals of parameter values “inflammation”: dƒ ₁ /dƒ ₅ = [0.84-0.96]; dƒ ₂ /dƒ ₃ = [0.60-0.79]; dƒ ₃ /dƒ ₅ = [0.60-0.75]; dƒ ₃ /dƒ ₈ =[0.24-0.30]; dƒ ₆ /dƒ ₈ = [0.10-0.13]; dƒ ₇ /dƒ ₈ =[0.080-0.13], where the indices correspond to the number of sensors in the array, and dƒ ₁ - dƒ ₈ – responses of all sensors, Hz; and when the number of positive hits within the boundaries: from 0 to 2 parameters in “inflammation”, the norm is diagnosed; from 3 to 4 parameters of “inflammation” diagnose the initial/mild stage of endometritis; more than 4 positive parameters of “inflammation” diagnose endometritis.