Description:
Rabies is an acute zoonotic disease viral etiology, which affects warm-blooded animals and humans. The disease is characterized by progressive damage to the central nervous system and 100% mortality.
Causes:
Rabies is viral infection. The neurotropic virus belongs to the Lysovirus family. They have a bullet shape. The virus has single-stranded RNA.
There are 2 types of rabies virus: a street (wild) virus circulating naturally among animals and a fixed virus - used to obtain an anti-rabies vaccine.
Replication (multiplication) of the rabies virus in nerve cells accompanied by the formation of Babes-Negri bodies. The rabies virus is unstable in the external environment and dies after 2 minutes of boiling.
The reservoir is carnivorous animals - foxes, raccoon dogs, cats, dogs. Bats, which release the virus into the external environment. The highest probability of infection is observed in the spring and summer.
Rabies is widespread throughout the globe, but is not found in Antarctica, Australia, island countries, as well as in Sweden, Spain, Norway and some other countries.
Pathogenesis:
The entry gate for the virus is damage to the skin and mucous membranes. From the site of introduction, the rabies virus spreads through the perineural spaces, reaches the central nervous system, where it is fixed and begins to replicate in neurons, the medulla oblongata, the hippocampus, and the nodes of the base of the brain, and is localized in the spinal cord. This ensures an increase in reflex excitability, and the associated development of paralysis. Respiratory muscles associated with damage to the vagus, glossopharyngeal and hypoglossal cranial nerves are observed. Irritation of the sympathetic nervous system causes increased salivation and sweating. Defeat vagus nerve causes disruption to work cardiovascular system. The virus is “screened out” from the central nervous system into various organs, primarily the salivary glands, adrenal glands, kidneys, skeletal muscles, skin, heart, as a result of which dystrophic changes in the myocardium and hemorrhages in the mucous and serous membranes occur.
Symptoms:
The incubation period lasts from 10 to 90 days. Prodromal phenomena last from 1 to 3 days. The first signs are at the site of the bite - swelling, redness of the wound, scar, itching, neurological pain along the way nerve pathways located close to the bite site. I'm worried about feeling unwell, increased sensitivity to auditory and visual stimuli, sleep disturbance with nightmares. The patient experiences unreasonable fear.
After 2-3 days, the peak period of the disease begins. Respiratory and swallowing disorders also appear. During the paralytic period of the disease, there is a decrease in all symptoms, mental calm, cessation of hydrophobia and aerophobia, and the ability to drink and eat appears. This phase is also called "sinister calm."
Further paralysis of the upper and lower limbs, cranial nerves. Development most often follows the type of Landry's ascending paralysis. The function of the pelvic organs is impaired. Body temperature rises to 42C. Death occurs from paralysis of the cardiac or respiratory center. The total duration of the disease is from 3 to 7 days.
Diagnostics:
Epidemiological anamnesis plays a very important role in the diagnosis of rabies. During the conversation, the patient or relatives can report recent contact with animals.
Material for post-mortem diagnostics – skin and brain biopsies.
In brain sections, eosnophilic Babes-Negri bodies can be detected; their size is up to 10 microns. These bodies represent an accumulation of viral nucleocapsids.
The method of immunofluorescence and biological testing is used.
A modern method for diagnosing the disease is the detection of viral antigen in prints taken from the membrane of the eye.
Treatment:
For treatment the following is prescribed:
Rabies is a disease with 100% mortality, and there is no pathogenetic treatment.
Possible appointment only symptomatic treatment– painkillers, anticonvulsants, hypnotics. At the stage of development of respiratory disorders, it is indicated.
Only a few are known in medicine clinical cases cure for rabies when manifestations of the disease have already begun.
Thus, in the USA in 2005, Gina Gis, a patient with rabies, was put into an artificial coma while receiving immunostimulation. After spending a week in a coma, the girl was discharged from the hospital with recovery. This method treatment was called the Milwaukee Protocol, but it does not give effective results in all cases.
Prevention:
Developed primary and secondary preventive measures regarding rabies.
With the aim of primary prevention All domestic and farm animals are vaccinated. It is necessary to timely detect natural foci of infection and destroy them.
Preventive immunization is indicated for persons whose profession is related to increased risk rabies infection, for example, hunters, veterinarians, foresters, etc.
If an animal bite does occur, the animal should be taken to a veterinarian for examination. Such an animal is subject to a mandatory 10-day quarantine. If within the specified period the animal remains alive and has not changed its behavior, then it is probably healthy and there is no risk of getting sick. Otherwise, emergency rabies prevention must be started immediately.
Methods of nonspecific prevention include aseptic wound treatment. The bite site is treated with running water and soap; deep injuries must be washed with a catheter. The wound should be managed open method, without stitches.
Specific prevention of rabies involves the administration of a combination of immunoglobulin and rabies vaccine.
The most effective method is passive immunization with rabies immunoglobulin or rabies serum followed by active immunization (vaccination). Passive and active immunization are carried out simultaneously, introducing drugs into different places.
Indications for the administration of rabies vaccination.
Vaccinal prophylaxis should be started immediately in the following situations:
- for all bites and other damage to the skin, salivation of the skin and mucous membranes inflicted on animals with symptoms of rabies, suspected rabies or unknown animals;
- if the wound was caused by an object contaminated with the brain or saliva of a rabid animal;
- if clothing was damaged by the animal’s teeth during a bite;
- the bite was carried out through thin or knitted fabric;
- in case of bites, salivation and application to healthy animals at the time of contact, if within 10 days thereafter it became ill, died or disappeared;
- the bite was caused by wild rodents;
- in case of obvious drooling or damage to the skin by a person diagnosed with rabies.
Vaccinations are not carried out if:
- the bite occurred through very thick or multi-layered clothing;
- damage caused by non-predatory birds;
- the bite was caused by domestic mice or rats in non-epidemic areas for rabies; - accidental consumption of thermally processed meat and milk of rabid animals;
- if the animal did not become ill during the observation period;
- the bite occurred earlier than 10 days before the animal was supposedly infected;
- with mild salivation and shallow bites that were inflicted by animals without clinical manifestations of rabies, with favorable data (low probability of a rabies case for the given area, isolated keeping of the animal, the bite was provoked by the victim, the animal is vaccinated against rabies). But even in this case, the animal is monitored for a period of 10 days in order to begin vaccinations if the animal exhibits symptoms of rabies, as well as death or disappearance;
- in case of provoked salivation of intact skin by an unknown domestic animal in rabies-free areas;
- in cases of contact with a person with rabies, in the absence of obvious salivation of the mucous membranes or damage to the skin.
For the purpose of active immunization, the vaccine is administered intramuscularly, 1 ml 5 times according to the following scheme: on the day of infection, then on the 3rd, 7th, 14th and 28th day. When vaccinated according to this scheme, it is possible to achieve satisfactory immunity. According to the recommendations of the World Health Organization, another 6th injection should be performed 90 days after the first.
May appear at the injection site side effects- pain, swelling and hardening of soft tissues. Sometimes these reactions manifest themselves in a more pronounced form. In addition, there may be an increase in temperature to 38 C or higher, arthritis, and enlarged lymph nodes. May bother you headache, general weakness, and allergic reactions.
Vaccinations against rabies can be carried out both on an outpatient basis and in an inpatient setting. Hospitalization is recommended for persons with severe bites and residents of rural areas; persons receiving repeated vaccinations; persons suffering from diseases of the nervous system, concomitant allergies; pregnant women, as well as persons vaccinated with other drugs during the previous two months.
Particular attention should be paid to vaccination while taking cytostatics and corticosteroids. In this case, it is necessary to conduct a study of the level of antibodies in order to decide on additional vaccination.
During active immunization, the patient's condition must be monitored. If there are complaints of deterioration of the condition, hospitalization is indicated, and vaccinations are temporarily suspended. The patient should be consulted by related specialists - a therapist and a neurologist. Further vaccination is decided by a commission examination consisting of a neurologist, radiologist and therapist.
During the vaccination course and for 6 months after it, you should not drink alcoholic beverages. This is necessary to ensure the proper level of post-vaccination immunity and prevent post-vaccination reactions.
The use of other vaccines simultaneously with rabies is not allowed; only when necessary can it be carried out emergency prevention. People with rabies are not vaccinated.
Rabies(Rabies) is an infectious disease dangerous to animals and humans. This disease occurs in all countries of the world. Characterized by 100% lethal outcome.
Pathogen– An RNA virus belonging to the Rhabdovirus family. This virus has an affinity for nervous tissue. It is found in the highest titers in the brain of sick animals. In addition, viruses are also found in the spinal cord, salivary and lacrimal glands.
There are:
- a “wild” virus that circulates in natural conditions and is highly pathogenic for humans and animals.
- And “fixed”, obtained in laboratory conditions, is not pathogenic for animals and humans when administered extraneurally.
The rabies virus is not resistant to high temperatures: at 60C o is inactivated after 10 minutes, at 100C o - almost instantly. But a frozen brain can remain viable for up to several months. It also persists for several months in rotting tissue. Resistant to iodine solutions. But it is quickly inactivated by disinfectant solutions based on formaldehyde, chloramine and alkali.
Epizootological data.
All warm-blooded animals are susceptible to the rabies virus. The most sensitive animals are wild dogs (such as foxes, arctic foxes, wolves, raccoon dogs, jackals), mustelids, and cat rodents. Birds are less susceptible.
In the wild, the reservoirs of the rabies virus are predatory animals and the bats, there are stray dogs in the city.
The source of infection is sick animals that shed the virus in their saliva. The presence of the virus in saliva is noted already 8-10 days before signs of the disease appear. Infection occurs through a bite, when saliva gets on mucous membranes or damaged areas of the skin.
Pathogenesis .
After entering the body, the virus begins to multiply at the site of introduction. And after 24 hours it begins to penetrate the nervous tissue. In the nerves it moves towards the spinal cord and brain at a rate of 7 cm/day. Having reached the brain, the virus begins to multiply vigorously, then the virus travels along the nerves to all organs and, first of all, to the salivary glands and eyes. Incubation period (from the moment of infection to the appearance of clinical signs) averages 14 – 60 days, sometimes it can reach 6-12 months.
Clinical signs.
There are 3 types of disease progression.
- Classic three-stage course or violent form. Initially, a change in the animal’s behavior is noted (stage 1), it becomes very affectionate or, on the contrary, timid and uncommunicative. This stage can last from several hours to 4 days. Then comes the next stage - excitement (stage 2), which lasts 1-4 days. Animals become aggressive, prone to wandering and attacking animals and people. Anorexia, anxiety, and drooling develop. And 3-4 days before death, a paralytic, or depressive, stage develops, characterized by progressive paralysis.
- Paralytic, or silent, form. Characterized by the development of paralysis without a previous stage of excitation. With this form, drooling and sagging are noted. lower jaw, difficulty swallowing, anorexia. Then paralysis of the muscles of the trunk and limbs develops. The death of the animal occurs after 3-4 days.
- "Atypical rabies." This is a chronic subclinical (without the development of symptoms) course of the disease, which can last up to 3 months.
Diagnosis.
A preliminary diagnosis is made based on medical history and clinical symptoms. If rabies is suspected after the death of an animal, the animal's corpse is sent to the laboratory. If this is not possible, use the head or brain. Strict precautions must be taken when taking material!
Laboratory diagnostics rabies includes several research methods.
- Detection of Babes-Negri bodies. To make a diagnosis of rabies in the laboratory, histological examination brain of a deceased animal. The presence of rabies nodules in the structures of the bottom of the fourth ventricle of the brain and the presence of Babes-Negri bodies (cytoplasmic eosinophilic inclusions consisting of rabies virus particles) in the brain are noted. They can be found in the hippocampus, cortical and stem structures of the brain, in the cerebellum and dorsal ganglia of the spinal cord and the corneal epithelium of the eye. However, if Babes Negri bodies are not detected, rabies cannot be ruled out.
- Antigen detection using fluorescent antibodies (MFA) or immunofluorescence analysis (ELISA). In addition to these reactions, a new polymerase diagnostic method is currently being developed. chain reaction(PCR).
- Biological test with infection of newborn mice with a virus from a suspension of the brain and salivary glands.
According to the legislation of the Russian Federation, in cases of bites of people by dogs or other animals (except for those clearly suffering from rabies), the victims must immediately contact medical institutions. And animals that have bitten them must be taken to the nearest state veterinary institution for examination by specialists and quarantine for 10 days.
To prevent the spread of rabies, annual mandatory vaccination of dogs and cats with rabies vaccines is provided.
Currently, to import animals into a number of countries, a document confirming the number of valid antibodies to the rabies virus is required. For effective protection against rabies, the minimum level of virus-neutralizing antibodies in the blood must be at least 0.5 IU/ml. This type of study is not diagnostic and is intended only to determine the strength of immunity against rabies.
In Moscow, you can donate blood to determine the titer of antibodies to the rabies virus at the Molecular Diagnostics Center, located on Zvenigorodskoye Shosse, 5. The CDC has received international accreditation to conduct this test and issues International Certificates valid for the entire life of the animal, subject to compliance with the revaccination deadlines.
Determination of antibody titers is carried out no earlier than 30 days after vaccination. Therefore, if you plan to travel with your pets to the EU or other countries, you must take care in advance of vaccination and donating blood for this test (results are issued in 7-14 days).
Laboratory assistant at a veterinary laboratory
"BioVetLab" Lazareva N.V.
A. In the nucleus of brain neurons
B. In the cytoplasm of myocytes
*WITH. In the cytoplasm of neurons
D. In the nucleus of spinal cord neurons
E. In the cerebrospinal fluid
How are brain smears processed in the laboratory diagnosis of rabies?
A. Complement and hemolytic serum
*IN. Antirabies gamma globulin bound to a fluorescein molecule.
C. Antibodies of the IgM class
D. Erythrocyte diagnostics with fixed rabies lipoproteins
E. Fluorescein solution
B. Humoral lifelong
*WITH. Post-infectious immunity is not detected
D. Antitoxic long lasting
E. Acquired active
In what case, when a person is infected with rabies, will the use of anti-rabies immunoglobulin be more effective compared to the anti-rabies vaccine?
A. When bitten by wild animals
B. For bites stray dogs
C. If you experience difficulty breathing
*D. For bites to the head
E. When the patient becomes agitated
What is advisable to use to increase the effectiveness of vaccination in people infected with rabies?
*A. Anti-rabies immunoglobulin
B. Antibiotics
C. Vitamins of group D
D. Immunomodulators
E. Hemadsorbents
For how long is rabies vaccination effective for sick people?
A*. Within 14 days from the moment of the bite
B. Within 1 month after the bite
C. In the first minutes after the bite
D. Within 2 months after the bite
E. Within 3 months after the bite
Which method is the fastest early method for intravital diagnosis of rabies?
*A. Immunofluorescence of skin biopsies
B. Study of Bebes Negri bodies in brain biopsies
C. Detection of anti-rabies antibodies in the blood by neutralization reaction
D. Study of Bebesh-Negri bodies in biopsies of the horn of Ammon
E. Isolation of the virus in saliva by infecting a cell culture
120. Name the most important structure for the reproduction of the rabies virus?
A. DNA-dependent RNA polymerase
*B. RNA-dependent RNA polymerase
C. RNA-dependent DNA polymerase
D. Topoisomerase
E. DNA polymerase
Who first described specific inclusions in the brain cells of animals that died from rabies?
A. A. Negri
*B. V. Babesh
C. L. Pasteur
D. K. Celsus
E. V. Babes and A. Negri
Considering the resistance of the rabies virus, can first aid be provided to a person bitten by a dog by treating the wound?
A. A solution of 0.1% acetic acid
B Hydrogen peroxide solution
C. Rabies vaccine
D. Anti-rabies immunoglobulin
*E. Soapy solution
Which of the following families does the rabies virus belong to?
A. Orthomyxoviruses
B. Picornaviruses
C. Flaviviruses
*D. Rhabdoviruses
E. Picoviruses
What is used to treat patients with clinical manifestations rabies?
*A. No treatment
B. Rabies immunoglobulin
C. Anti-rabies serum
D. Rabies vaccine
E. Treat the wound with a 5% formaldehyde solution
How is anti-rabies immunoglobulin obtained?
A. From rabbits infected with the rabies virus
B. When immunizing rabbits with street rabies virus
C. Isolated from the blood of people with rabies using electrophoresis
*D. When hyperimmunizing horses with a fixed rabies virus
E. Isolated from the blood of people vaccinated with street rabies virus
What is the main way the rabies virus spreads in the body?
A. Hematogenous
B. In circulating blood cells
*C. Neurogenic
D. Lymphogenic
E. By motor neurons
How can you prevent the development of complications after rabies vaccinations?
*A. Administration of rabies immunoglobulin
B. Use of antiallergic drugs
C. Use of immunostimulants
D. Administration of human gamma globulin
E. Use of eubiotics
What type of immunity for rabies is formed in a person who has not been vaccinated against this virus?
A. Cellular short-lived
B. Humoral lifelong
C. Post-infectious immunity is not detected
*D. Antitoxic long lasting
E. Acquired active
Blood was taken from a patient with suspected hepatitis B for testing. What antigen must be detected in the blood serum to confirm this disease?
V. Nvs and Nwe
GRANDMOTHER'S TAURUS[named after the Romanian scientist V. Babes, 1887, and the Italian scientist A. Negri, 1903], specific cytoplasmic inclusions found in the neurons of the central nervous system in animals and people who died from rabies. They are oxyphilic polymorphic formations of various shapes, ranging in size from 0.251 to 27 microns. Detection of bodies in the brain of dead animals is one of the methods for laboratory diagnosis of rabies.
- - neoplasms in nerve cells of the brain affected by the rabies virus. Located in the cytoplasm of the cell...
Dictionary of microbiology
- - Grandma's body, specific cytoplasmic inclusions found in neurons of the central nervous system in animals and people who died from rabies...
- - Babesha Negri bodies, specific cytoplasmic inclusions found in neurons of the central nervous system in animals and people who died from rabies...
Veterinary encyclopedic dictionary
- - ...
Ethnographic terms
- - NEGRI Ada - Italian poetess. R. in the family of a farm laborer in the year of the unification of Italy...
Literary encyclopedia
- - rigidity abdominal wall with pain on palpation in the area of the projection of the splenic artery; may be observed with an abdominal aortic aneurysm...
- - see Babesha-Negri calf...
Large medical dictionary
- - cytoplasmic eosinophilic inclusions found in hippocampal cells during rabies...
Large medical dictionary
- - cytoplasmic inclusions in microbial cells, appearing in the form of large granules, especially when special methods coloring...
Large medical dictionary
- - see Babesha-...
Large medical dictionary
- - Italian writer and politician; originally from Lombardy; was a professor of public law in Padua...
encyclopedic Dictionary Brockhaus and Euphron
- - Italian poetess, originally from Lodi; in childhood, as can be seen from her poems, she lived in great need; From the age of 18 she was a public teacher...
Encyclopedic Dictionary of Brockhaus and Euphron
- - Ada, Italian poetess. She came from a working-class family and taught at a public school. In N.’s poems, for the first time in Italian poetry, pictures of the labor of factory workers appeared, and motives of rebellion were heard...
- - Negri Ada, Italian poet. She came from a working-class family and taught at a public school. In N.’s poems, for the first time in Italian poetry, pictures of the labor of factory workers appeared, and motives of rebellion were heard...
Great Soviet Encyclopedia
- - Babes-Bolyai University of Kluj - Romania, founded in 1872 as a Hungarian university; since 1919 Romanian. In 1991 approx. 6 thousand students...
Large encyclopedic dictionary
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From the book Newspaper Tomorrow 971 (28 2012) author Zavtra NewspaperRabies (B) is an acute disease of warm-blooded animals, characterized by damage to the central nervous system. Domestic and wild animals of all species, as well as humans, are susceptible.
The disease is recorded in various regions of the globe. There have been no cases of the disease spreading in Australia, Great Britain, or Japan. The disease almost always ends in death. There are actually documented cases of recovery from rabies in humans and dogs after experimental infection.
Rabies virus (RBV) belongs to the family Rhabdoviridae, genus Lyssavirus. It has now been established that VB has 4 serotypes, which is apparently due to differences in composition membrane proteins. All variants of VD are immunobiologically related, but differ in virulence. WB has GA and GAD properties. Between infectious and GA activity there is linear dependence. Animals immunized against rabies produce VNA, CSA, antiGA and lytic (destroying cells infected with the virus in the presence of complement) antibodies.
The diagnosis is made on the basis of epidemiological data, symptoms of the disease, pathological changes (they are of less importance) and, mainly, the results laboratory research.
Laboratory diagnostics consists of studying the brain of animals in order to detect viral hypertension in IF, RDP, detection of Babes-Negri bodies and a bioassay on white mice.
IN Russian Federation Currently, the production of diagnostic kits for B in the IF and RDP has been organized at VNITIBP and KazNIVI.
Virus isolation. Fresh corpses of small animals are sent to the laboratory for research; from large animals - the head or brain. In some cases, it is possible to preserve the brain in 50% glycerol. The corpse or head must be carefully packed in a plastic bag, the brain in a jar with a ground glass or rubber stopper, filled with paraffin. The material is packaged in moisture-proof containers. For virological research Only non-canned brain is suitable. It must be remembered that dissection of a corpse, brain extraction and other operations with pathological material should be carried out under sterile conditions and strict adherence to personal preventive measures: the animal’s head is firmly fixed, hands are protected with 2 pairs of gloves (surgical and anatomical), goggles are worn to protect the eyes , and a 6-layer gauze bandage on the nose and mouth.
Laboratory research of the material rabies tests are carried out out of turn; the results are immediately reported to the farm doctor and the chief doctor of the district (city).
Virus indication and identification. The procedure for conducting research: from each part of the brain on the left and right sides (Ammon's horn, cerebellum, cerebral cortex and medulla oblongata), 4 fingerprint smears are prepared for IF and detection of Babes-Negri bodies; RDP is placed with brain tissue; if the results are negative, a biotest is performed.
Detection of specific inclusion bodies. Imprint strokes are stained using Sellers, Muromtsev or other methods. After staining, the preparations are viewed under a light microscope with an immersion system. A positive result is considered to be the presence of specific Babesch-Negri bodies (when stained according to Sellers - clearly defined oval or oblong granular formations of pink-red color in the protoplasm, when stained according to Muromtsev - light purple with dark blue inclusions of Babesch-Negri bodies, more often they are located outside the nerve cells.
Most characteristic feature Babesch - Negri bodies - their internal structure, which allows them to be absolutely accurately differentiated. Small grains are visible inside - basophilic granules of dark blue, even black color, 0.2-0.5 microns in size.
The diagnostic value of detecting inclusion bodies to prove infection with VD is generally accepted. However, healthy animals, especially cats and white mice, have formations whose presence can cause diagnostic difficulties. IN in some cases in the cat’s brain, it is possible to confidently differentiate such inclusions from Babes-Negri bodies, and here it is recommended to use identification methods, especially IF. Likewise, in dogs that died as a result of poisoning by snake venom or defeat electric shock, you can find inclusion bodies resembling Babes-Negri bodies. Babesh-Negri bodies are detected only in 65-85% of rabies cases, so their absence is not a negative answer, and the material is examined in other tests (IF, RDP, bioassay).
IF. One of the main tests for diagnosing B. When performed in a highly qualified manner, a 99-100% agreement with the bioassay method is obtained. Typically, in diagnostic practice, the direct IF method is used, which is carried out using anti-rabies fluorescent Ig. The preparations are fixed in cooled (8-10°C) acetone for at least 4 hours. Brain smears of healthy white mice are used as a negative control. The results are taken into account visually in a fluorescence microscope based on an assessment of the luminescence intensity of the AG-AT complex. Ag VB is detected in the form of bright yellow-green or green granules of various shapes and sizes in cells (usually outside cells). The diagnosis is considered established if a sufficient number (at least 10) of typical granules with a bright green glow or many smallest points, There should not be such a glow in the control.
To prove the specificity of the luminescence of the AG-AT complex, the method of suppressing IF is used, which consists in the ability of rabic AG associated with non-fluorescent AT to not combine again with fluorescent specific AT. To do this, 5% anti-rabies non-fluorescent Ig is applied to fixed preparations prepared from the brain under study, kept for 30 minutes at 37°C in a humid chamber, washed with saline, and then stained with fluorescent anti-rabies Ig using the generally accepted direct method. There should be no fluorescence in preparations treated in this way.
The IF method makes it possible to detect VD in the cells of the cornea of the eye and make a preliminary diagnosis intravitally: during the animal’s illness, as well as 1-2 days or more before its clinical manifestation. The method can be used to study animals suspected of disease B, as well as clinically. healthy dogs and cats that have bitten people and animals. To do this, prepare prints from the cornea, observing all the rules of personal safety, open the animal’s palpebral fissure with a large and index fingers and slightly bulging eyeball press with the surface of the glass slide, retreating 0.5 cm from the end. It is necessary to ensure that the animal does not blink with the third eyelid, since epithelial cells are removed from the glass and a poor-quality smear is obtained. At least 2 preparations containing 2 prints are made from each eye. For control, corneal imprints from healthy animals are prepared in the same way. They can be prepared at home. The prints are dried in air, fixed in acetone for 4 hours at 4°C, packaged and sent to the laboratory. The preparations are stained as for IF, according to the generally accepted method.
In preparations obtained from sick animals or at the end of the incubation period of the disease, brightly luminous granules of various shapes are observed in the cytoplasm of many epithelial cells different sizes- from dust-like points to 2 microns or more. In order to obtain reliable results In each preparation, 50-100 cells are examined, and in total from an animal - at least 200-400 cells. The results of microscopy are considered positive if 11% or more cells with characteristic foci of luminescence are found in the imprints of the animal’s cornea. It must be borne in mind that in preparations from healthy animals (control), due to autofluorescence, single cells with foci of similar shape and luminescence may be encountered.
It is important to note that IF makes it possible to speed up the response when making a final diagnosis using a bioassay, since the diagnosis of B can be established only on the 4-8th day after infecting mice with the test material, and incubation period Diseases in mice can reach 20 days. IF can detect VD in the tissues of the submandibular salivary gland. Smear preparations are prepared from the submandibular salivary glands, taking material from at least 6 different parts of the gland, since the distribution of the virus in it is uneven. Often, to get a print, you have to apply strong pressure, because due to the abundance of mucin, little material remains on the glass.
The possibility of identifying VB in the skin using the IF method has been shown. For this purpose, samples are taken from the scalp, as well as follicles of sensory and tactile hair of the muzzle or lateral sensory papillae (on the dog’s cheek). Samples are stored at -20 or -70°C. Cryosections are made from them and treated with fluorescent globulin. The results of IF analysis obtained from the identification of VB in the skin highly correlate with the data obtained from studying the brain of the same animal. A close correlation was shown between the detection of virus AG in brain and lip tissue samples by the IF method.
RDP. Used to detect WB AG in the unpreserved brain of animals that have died from street rabies, or pups used in a bioassay. RDPs are placed using the micromethod on glass slides using 1-1.5% agar gel according to the generally accepted method. The highest percentage of positive results is detected when using the following stencil: A - ammon's horn ( Right side); B - cerebral cortex (right side); C - cerebellum (right side);
D - medulla oblongata (right side); + (plus) - positive control; - (minus) - negative control; 1, 2, 3, 4 - wells with specific immunoglobulin dilution 1:2, 1:4, 1:8, 1:16, respectively
A homogeneous paste-like mass is prepared from each part of the brain using tweezers, which is placed in the corresponding wells. The entire brain is examined from mice. AG is prepared from parts of the brain on the left side in a similar way (a total of 4 slides with agar gel are required for each examination). The reaction is taken into account after 6, 24, 48 hours. If there are 1 or 2-3 lines of precipitation between the wells containing AG and immunoglobulin, the reaction is considered positive.
RDP is simple to perform and specific, but the percentage of detection of viral Ag in the test material is 45-70. When examining the brain of mice obtained with a positive bioassay, RDP detects up to 100% of cases. The absence of Babes-Negri bodies, specific fluorescence and negative RDP in the studied material does not provide grounds to exclude the presence of a virus. In this case, the final diagnosis is made based on the results of a bioassay on white mice followed by identification of the virus.
Bioassay. It is generally accepted that a bioassay is a more effective method than detecting Babes-Negri bodies, IF, etc. However, in some cases it turned out to be negative, despite confirmation of diagnosis B by detecting inclusion bodies and IF. The percentage of negative results for the bioassay ranged from 1.3 to 12.
Information about the varying effectiveness of a bioassay can be explained by a number of factors: the choice of experimental animal, the number of animals in the experiment, the method of infection, the method and period of storage of the material before entering the laboratory. The phenomenon of interference of infectious particles with inactive particles may also play a role if insufficiently diluted material is used for inoculation.
In the brain and salivary glands foxes and skunks that died from rabies, a substance was found that inhibits the infectivity of the virus, which does not allow diagnosing the disease in these animals by intracerebral infection of mice. The presence of an inhibitory substance in the test material does not prevent the detection of viral Ag by the IF method; for skunks and foxes this is the most sensitive diagnostic method.
Of animals of all kinds (rabbits, Guinea pigs, adult white mice and hamsters) used for bioassays, many prefer suckling mice because they are more sensitive to different strains of VB and are less dangerous at work. Syrian hamsters are as sensitive as mice, but they are less accessible.
RSK. Detection of specific antigens in RSCs when diagnosing rabies is used less frequently than other methods. An AG is prepared from the brain sent for research. To do this, brain tissue (parts of the thalamus and brain stem are especially rich in antigen that fixes complement) is ground in veronal buffer in a ratio of 1:10 and left at room temperature for 1 hour, after which the suspension is inactivated at 56°C for 5 hours. This treatment kills the virus and removes the anticomplementarity of the brain tissue without damaging the specific antigen. The suspension is centrifuged for 15 minutes at 3500 min-1 from the supernatant liquid, which is the material for testing for the presence of AG, 2-fold increasing dilutions from 1:2 to 1:64 are prepared and used for testing in the RSC.
ELISA. In ELISA, specific staining of AG in the brain cells of animals that died from rabies is detected both in freshly taken samples stored in glycerol, as well as in samples stored without glycerol at 20°C for 8-18 hours. This test suitable for routine diagnosis of rabies in animals, for the detection of VB Ag in tissue paraffin sections when fixing preparations with 10% formalin solution with pH 5.3 and subsequent processing of preparations embedded in paraffin with pepsin.
Unlike RN in mice and in cell culture, ELISA can detect AT in animals within a few hours, ELISA is the most promising laboratory method detection of AT and indication of the virus itself. An express method for diagnosing rabies is the capture technique and the IF method for detecting AG EVs. It has been proven that the method for detecting WB AG in paraffinized sections using the peroxidase-antiperoxidase method is significantly superior to the ELISA method. In 1987, a rapid rabies enzyme immunodiagnostic (RREID) kit was created, suitable for epidemiological and laboratory studies.
Variant identification using mAbs. Using mAbs to VB glycoproteins, AG variants were selected, among which phenotypically thermolabile (avirulent) variants were identified. When using 2 groups of mAbs, it was shown that wild strains differ from pcs. Fixe (Pasteur), CVS, Flury HEP, ERA and “duck” strains for AG determinants. The study of 7 strains isolated from sick people using mAb allowed us to identify certain differences from the vaccine strain in relation to antigen determinants.
It is generally accepted that AT differences between wild strains can be detected using mAbs against the nucleocapsid AT N, which react with cytoplasmic inclusions of virus-infected cells; against glycoprotein (G AG), which react with the membranes of infected cells, lyse these cells in the presence of complement and neutralize the virus. Due to the possible AG variability of the surface glycoprotein of VB from different geographical zones, a ribonucleoprotein characterized by a conserved AG structure was used as a protective AG. Thus, not only G protein, but also WB RNP have protective activity.
Serodiagnosis and retrospective diagnosis. These methods are unusual for rabies, as they are used only for the purpose of testing post-vaccination immunity. To detect and titrate post-vaccination AT, pH is used, which is determined using the generally accepted method. A fixed WB is used as an AG. pH on BHK-21 cells was more sensitive than indirect IF in detecting AT in the sera of vaccinated foxes. In addition, RTGA and ELISA have been proposed.
RTGA. It has not yet found widespread use in diagnostic practice due to the presence of nonspecific inhibitors in blood serum, to which VB is highly sensitive, and most importantly, GA AG, which have not been sufficiently purified, have low sensitivity. For the preparation of GA AG WB, it is proposed to use pcs. Moscow, grown in VNK-21 cell culture, after its treatment with saponin, followed by purification and concentration. GA is separated from other virion components by ultracentrifugation. The resulting preparation had a purity degree of 99.92%, had high GA activity (1:128), which was well preserved for 1 month at pH 5-9.
Before performing RTGA, moderate 2-fold trypsinization of goose erythrocytes should be carried out to sensitize them. When using a 0.25% suspension of trypsinized erythrocytes (preferably 10 7 cells in 1 ml), the sensitivity of RTGA increases 4 times. To dilute AG and serums, borate-salt solution (pH 9) with the addition of 0.4% bovine serum albumin is used. A suspension of erythrocytes is prepared in a saline solution with an acidic pH, so that after combining with a mixture of virus and serum, the pH of which is 9, the final pH would be established within 6.2. After adding the red blood cell suspension to the wells, the panel is shaken, sealed with a transparent film and placed on ice. The results of RTGA are taken into account after 40-50 minutes, and with erythrocytes from 2-day-old chickens or rhesus monkeys - after 1-1.5 hours.
A radioimmunoassay has been developed based on the ability of AT to bind to 125-labeled 1-AG VB. Labeled IgG isolated from anti-rabies hyperimmune serum can be used to detect WB antigens by solid-phase RIA. The best results were obtained when using a phosphate-saline solution (pH 6.0) with an ionic strength of 0.01 M and labeled IgG with an activity of 200-250 thousand pulses/min.
Solid-phase competitive RIA is applicable for the detection of anti-rabies antibodies in serum and hybridoma supernatants.
Differential diagnosis. It is necessary to exclude Aujeszky's disease, in which sick animals are non-aggressive and there is no perversion of appetite. In dogs, the nervous form of plague is excluded. Suspicion of rabies may arise from equine infectious encephalomyelitis. A set of laboratory tests makes it possible to make an accurate diagnosis of rabies. Proposed new method differentiation of various strains of VD, based on restriction enzyme digestion of PCR amplification products of the N gene segment.
