IMPROVEMENT OF BRUCELLOSIS DIAGNOSTICS MANUAL
ABNUREL TRIAD REACTION
A. A. ALIYEV N. G. GULİYEV E. H. HASANOV
H. G. KHALİLOV F. K. GANJALİYEV O.M.SADYGLY
Brucellosis is a zooanthroponosis disease, i.e. transmitting from an animal to a human, having a history of more than 1 century. According to statistical data, approximately more than 200-400 people are diagnosed to brucellosis with serological methods based on detection of present specific antibodies and involved to treatment every year in the Republic. More than 2000 cattle and small ruminants are diagnosed to brucellosis with those methods. Unlike humans, as sick animals are not an effective treatment method from an economic point of view, they are subject to mandatory slaughtering in units of special regime, thus, this means that more than 50% of total value of an animal is damage to animal owners. Positive results of serological analyses (Rose Bengal Test (RBT), Agglutination Reaction (AR), Complement Fixaton Test (CFT), Enzyme-Linked ImmunoSorbent Assay (ELISA), Milk Ring Reaction (MRR) or reactions based on detection of other specific antibodies) of brucellosis does no meet triad condition suggested by german microbiologist Robert Cox to prove the disease. There are some features characterizing infectious diseases; features such as development of diseases over times, having an agent, formation of specific antibodies, immunity against it, presenting with characteristic clinic signs and establishing the disease model during a biological test, etc. However, the above mentioned is not seen in the animals diagnosed to brucellosis with serological tests. It appeared from the carried out practical studies that specific antibodies form against single inactivated brucellosis antigen on 4-5 days, attenuated brucellosis vaccines on 15-21 days, virulent brucellas on day 30 in 25% inoculated guinea pigs and rabbits. The results of available serological reactions were negative, while the results of bacteriological tests were positive up to 135 days in the rest 75% inoculated guinea pigs and rabbits. Thus, while the virulence of brucellas increases, the formation period of specific antibodies increases as well. Therefore, as detection of existing specific antibodies of animals spreading brucellas is not possible with serological tests, secretions and materials of those animals cause animals and humans to be infected. Agents of brucellosis consist of somatic antigen and hyaluronidase enzyme. Its enzyme provides invasiveness to Brucella. The reason for formation of specific antibodies is somatic antigen of brucella. Antigen does not have potential to multiply in separate or to cause disease.
Enzymes fall apart at temperatures higher than 43° C. It is confirmed during verification of the obtained results by the committee that even if boiled for 3 hours, somatic antigen of brucellosis does not lose its potential of forming specific antibody. Meat of animals with brucellosis is conserved, dairy products are pasteurized. When somatic brucellosis antigen gets into human body with the meat cooked again and pasteurized milk, it causes formation of specific antibodies within 7-10 days, it results in saying that the person has brucellosis due to Wright, Heddeleson serological analyses, involvement of humans to treatment with bacteriocidal chemicals against brucellosis and impairment of functions of organs that are essential for life. It appeared during the studied we conducted that specific antibodies melt compeletely (lysis) single brucellosis antigen added in a test tube at diagnostic titre indicators, the result of biological test with that precipitation is negative with RBT, RPR. As single brucellosis antigen is not lysed in a test tube at titres after a diagnsotic titre, it leads to formation of specific antibodies during biological test. As this process happens in vivo, it results in clearance of that body from brucellas consequently. As a result of this, those individuals cannot infect others and are called “biological alley”! Taking into consideration that animals are sent to mandatory slaughtering, humans are involved in treatment with additional anti-bactericidal chemicals due to specific antibodies and organisms that spread virulent microbes are not detected with available serological tests, we recommend the methods based on detection of specific antibodies for determination of immune system background only, but “ABNUREL” Triad reaction (comprehensive evaluation of bacteriological, serological Ring Precipitation Reaction (RPR) and biological test) named conditionally by the authors in diagnostics of brucellosis.
As animal bodies are at different immune status, it is difficult to diagnose with available serological methods at different periods of the disease. Serological methods based on detection of specific antibodies do not completely detect organisms spreading brucellas, it requires looking for more effevtive methods for massive analysis based on detection of brucellas. Not defining the problem precisely put a barrier for improvement of diagnostics and treatment of the disease, more effective treatment ways of the disease have not been developed so far because of this!
Application of the results we obtained in appropriate fields will stimulate specific prophylactics and facilitation of treatment of the disease, this will cause economic development, provide food safety, eliminate existing problems and is of great importace for elimination of the disease. Addressing to solve this problem for the the first time in the world, as well in the Republic is important in this respect.
2. STUDY MATERIALS AND METHODS
Study works were conducted in the laboratory studying Brucellosis and Tuberculosis in Azerbaijan Scientific-Research Veterinary Institute between 1994-2009, afterwards in the laboratory of animals’ infectious diseases, bacteriological kitchen, scientific production and application department according to the latest diagnostic and prophylactic instuctions. Serological methods used currently in diagnostics of brucellosis are based on the principle of detection of specific antibodies at serum. Agglutination reaction is run at portions of 1:25, 1:50 in sheep and goats, 1:50, 1:100 in cattle during massive analysis of animals. 0,1 ml analysed serum is taken for analysis at initial dilution, diluted in physiologic solution in shown titres and 0.5 ml single brucellosis antigen diluted at 1:10 physiologic solution is added on it. There are 10 billion brucella bodies in 1 ml single brucellosis antigen. If 1 ml 1:10 dilution is prepared, 1 billion microbe bodies remain in 1 ml, when 0.5 ml from this is added over the serum, each test tube consists of 500 million-brucellosis body. If 1:100 and 1:50 at least 2++ agglutination is observed in cattle and sheep and goats respectively, the reaction is considered to be positive, and an animal to have brucellosis. If 1:50, 1:100 dilutions of 0,1 ml analysed serum lyse 500 million brucellosis body in an electrolytic medium “in vitro” (in a test tube) precipitating after 18-24 hours, serum contains at least 45% of an animal having 20-litre blood, and that means approximately 9-litre serum for approximately 1 animal. It is possible to see clearly with mathematical calculations that how many brucellas it can neutralise “in vivo”. In this case, can those organisms be considered to be dangerous, the infection source or sick? Evidently, not! Because the conflicting sources and obtained results confirm the above mentioned once more.
Besides this, 45 rabbits were immunized intramuscularly to the thigh site with single brucellosis antigen on day 1 with 1 ml, on day 6 with 2 ml, on day 14 with 2,5 ml, titre of serum was checked with AR on day 21, the lowest titre was 1:160, the highest one was 1:1280. Then the precipitation of test tubes of both titres was immunized to rabbits from 1:5 to 1:1280, the results were as the following after 5 days: when precipitation of serum that the last titre is 1:160 with AR diluted up to 1:5, 1:10, 1:20 after 18-24 hours immunized to rabbits, specific antibodies did not form, they formed against precipitation of further titres. Specific titres did not form against 1:5, 1:10, 1:20, 1:40, 1:80 dilutions of the serum that the last titre is 1:1280 with AR, but specific antibodies formed against precipitations higher than 1:160. Experiments were repeated 3 times, the same results were obtained. It was informed in 2009 report, scientific council of Azerbaijani Scientific-Research Veterinary Institute accepted the report. In the next experiment, 10 rabbits were each immunized in a subdermally, intraperitonally from under abdominal site on amount of 1 ml with 19 strains that are productions of 1973. At the same time 10 rabbits were immunized at 1 ml volume in the same way with single brucellosis antigen that is production of 1989. Non-expired Rev-1, strain-82 and single brucellosis antigen were taken in control. With a 7-day interval, they were immunized with increasing dose again 3 times. After each immunization, 1.5-2 ml blood was taken from ear vein of rabbits, the serum was isolated. The results were positive with RBT. 1:10 suspension (1 kg mass, 10 ml 0.85% physiologic solution) was prepared from internal organs of paralel rabbits, their blood being drained, 1 ml from each sample was injected to thigh muscle of 2 rabbits, blood was taken from ear vein after 5 days and the serum was isolated. The results of reaction with RBT were negative in all animals. In another experiment, blood sample was taken with a 10 gr disposable syringe from 59 breed cattle in compliance with sterillity, cultured new AF2 and broth and agar nutrient media with meat peptone, liver, glucose, glicerin (MPLGG) on amount of 1 ml at place. The rest blood samples were taken for serological analysis, checked comparatively with comprehensive available and new analysis methods in the laboratory, as a result, 8 animals were positive with RBT, AR, 2 animals were positive with RBT. The results of bacteriological analysis were negative in all samples. Besides, 1:50, 1:100 precipitations of positive 8 sera with AR after 20 hours were run with a biological test in 40 rabbits, the results of precipitations in a diagnostic titre were negative after 5 days, but positive at higher titres. 2 positive ones with RBT were at 1:40 titre with AR. Precipitations after 20 hours were injected to thigh muscle of 2 rabbits on amount of 1 ml at 1:50, 1:100 titres each. The result was positive with RBT. 1:50, 1:100 precipitations of standard single brucellosis antigen of hyperimmune brucellosis serum after 18 hours in control were injected to thigh muscle of 2 rabbits each in the same way, in the result, as standard antigen was lysed in vitro (in a test tube) due to the effect of specific antibodies at diagnostic titres on day 5, specific antibodies did not emerge. The results of positive sera with single brucellosis antigen were not the same with Ring Precipitation Reaction, delay was observed, reaction was positive in minute 16 in control (cross agglutination). In the 3rd experiment, single brucellosis antigen was taken in 6 test tubes, each containing 4 ml conditionally, boiled in water 30, 60, 90, 120, 150, 180-minute intervals, injected in enteral, subdermal, intraperitonal and intramuscular way to 2 rabbits from each sample. After 5 days, the results were checked with RBT, the result was positive. Even after being kept for 3 hours at a boiling temperature, the antigen kept the capability to form specific antibody. If formation of specific antibodies happens within 5 days, formation of specific antibodies on the 7-10th days in enteral immunization was confirmed in Azerbaijan Scientific-Research Veterinary Institute by committee in 2008. In the next experiment, single brucellosis antigen was injected to 7 rabbits or thigh muscle of 3 rams on amount of 1.5 ml, with a 7-day interval on amount of 2 ml on day 7 with increasing dose, 2.5 ml on day 14, the serum of blood was subject to serological analysis every 10 days. During each serological analysis, one of rabbits was subjected to blood draining, the serum was isolated, samples were cultured to available and new broth nutrient media from internall organs and bone marrow under a steril condition, controlled beginning from day 1 till day 3, pH of the nutrient medium was checked and it was neutral. Rev-1 brucellosis vaccine of Jordania and Kyrgyzstan production was cultured in control. After 1-3 days, pH in broth was 8-10, cultured to agar, 2-day agar was washed with physiological solution of 0.5% phenol, yhe prepared suspension was inactivated in water bath for 1 hour at 70° C, then being kept in the fridge for 16 hours, monospecific hyperimmune brucellosis serum was checked with Ring Precipitation Reaction (RPR). In control, the antigen prepared form Rev-1, single brucellosis antigen, positive, negative or the analysed animal’s serum were taken, the results were compared, evaluated in a diagnostic way. In Ring Precipitation Reaction (RPR) test experiment, 0,3 ml 1:10 prepared suspension, standard positive serum over 0,3 ml, positive, negaive and the analysed serum with standard single brucellosis antigen in the same way in control were kept for 4 minutes at water bath at 55°, then the test tubes were processed in cold water for 4 minutes. The process is kept with hot-cold regime with 4-minute interval. In control, the result of the reaction was suspended when standard single brucellosis antigen was run with positive serum and the results were evaluated in a diagnostic way being compared. According to the results obtained over antigen, specific antibodies form beginning from 4-5 days in blood of rabbits. The different feature of Rev-1 antigen from single brucellosis antigen is that result with HPR delayed within 1-3 hours, not in 16-30 minutes. Suspension prepared from organs was injected on amount of 1 ml to thigh or pectoral muscles of 2 rabbits having 2 kg of live weight or 1-month broiler chicks. The result of serum was negative with RBT and RPR on day 5 with biological test, as there were Rev-1 antigen and single brucellosis antigen in control, the result was positive with both reactions. However, specific antibodies against Rev-1 antigen were positive afte 11-14 days. Only liver suspension from suspensions prepared from organs was positive with RPR, but the result of the biological test was negative. These experiments were conducted with Strain-82, Rev-1, Strain-19 in paralel, the results of serum analysed with RBT, AR, CFT and RPR were positive beginning from 7-14 days. However, reaction with RPR delayed up to 1 hour. The results of suspensions prepared from organs were the same with the results of standard single brucellosis antigen. Analysis of literature data and conducted experiments gives basis to us to conclude the followings.
3. THE OBTAINED MAJOR RESULTS
1. It was clear to us from the experiments that specific antibodies form against single brucellosis antigen on the 4-5th days, against antigen prepared from REV-1 vaccine after 11-14 days. The different feature of antigen prepared from vaccine strain from single brucellosis antigen; causes delay of reaction within 1-3 hours with RPR, not in 16-30 minutes, this is considered to be the best technique in comparison of vaccinated animals from those infected with virulent microbe and cross agglutination.
2. The results of bacteriological, biological, Polimerase Chain Reaction (PCR) and “ABNUREL” Triad are positive, but negative with available serological tests (RBT, AR, CFT, MRR, ELISA, etc.) in acute infection phase of brucellosis. Positive results of available serological tests show negative with bacteriological, biological, PCR and “ABNUREL” Triad reaction. Specific growth of brucella is observed in 72 hours in the new method, the result of biological test is compared 5 days after under control and evaluated. Being local production, reaction components are easy, effective from economic point of view and for massive analysis. Healthy animal is provided with expertise in 72 hours, but sick animal after 8 days. The advantage of this test is that it can compare animals with brucellosis having acquired steril immunity, vaccinated or immunosuppressed and organisms providing cross agglutination, it prevents subjecting those animals in a mixed form to mandatory slaughtering.
3. Blood is taken with disposable sterile 1, 3, 5 and 10 gr syringes in the new method, cultured to broth directly at place. Work regime of bacteriological, biological methods and nutrient media and RPR is improved for the first time. As presence of diagnostic titre features is “in vitro” bacteriolysis, it is a feature of natural recovery from brucellosis. Specific antibodies against precipitation (in vitro) in a test tube in diagnostic titre after 18 hours of AR do not form during the biological test, but specific antibodies have formed in further titres.
4. Serum of an animal identified as sick or rabbits hyperimmunized with single brucellosis antigen at 1:1280 was inoculated with lethal dose in an experiment way, resulted in clinical recovery during interperitonal immunization (of lethal rabbit) with a 1-day interval, 6.5 ml 2 times at acute infection phase of brucellosis. However, serological reactions were positive, specific growth was not observed as a result while culturing from spleen, liver, lymph node and tubular bone marrow of that rabbit to available and new liquid, solid nutrient media after 10 days, biological tests were conducted on 2 rabbits from each sample with 1:10 suspensions prepared from organs, the result was negative after 5 days with RBT, single brucellosis antigen and antigen prepared from REV-1 vaccine were injected during the same period in control, blood serum was positive at RBT and AR 1:80 titre against single brucellosis antigen on the 4-5th days, against antigen prepared from REV-1 after 11-14 days. Pathogenicity of REV-1 vaccine increases for rabbits and rats during culturing to nutrient media 5-6 times, 1:2560 titre specific hyperimmune brucellosis serum against antigen prepared from vaccine resulted in clearance of brucellas in the body of the rat, but rats died of activation of secondary infections. This requires complex administration of analgetics, antibiotics and immunomodulators in paralel with hyperimmune serum in future.
5. It is not correct to identify the obtained results on brucellosis antigen with pathogen brucellas, because brucella antigen has long-term resistance in external environment, it does not lose the capability to form specific antibody even after being boiled for 3 hours! This feature of brucellosis antigen must be taken into consideration while diagnosing for brucellosis!
6. Naturally acquired sterile immunity and immunological reactions after vaccination can be positive in different degrees, without having clinical signs of the disease, it does not provide basis for diagnosing to brucellosis if serological reactions are positive, isolation of hemoculture, positiveness of biological test confirm diagnosis.
7. In order to check comparatively with available and new methods, blood and milk samples taken from 25 sick and 20 healthy animals provided by Guba Zonal Veterinary Laboratory (expertise #291) were submitted to the Republic Veterinary Laboratory to check effectiveness of new diagnostic method with available methods in comparison according to order #13, dated 21.04.2014 of the State Veterinary Service under the Ministry of Agriculture of the Republic of Azerbaijan, the samples were tested with bacteriological, PCR, ELISA and the new method. According to the obtained results, as a result of PCR verification of 25 sick animals being positive with serological methods, only 5 of 25 animals were identified as sick. According to ELISA results, 2 of IgG 24 sick animals were negative, the rest was positive, as well 9 from healthy ones were positive with IgG. Comparable results of studies with avaialble and new methods confirmed 25 animals not to be sick with bacteriological and new methods, PCR confirmed 80% of animals, 20 animals to be sick, ELISA – 22 animals from 25 sick animals were positive, 9 animals from 20 healthy animals were positive. Moderate temperature was 38.8 C at healthy and six animals. Characteristic clinical signs of the disease were not observed during visual analysis.
4. GENERAL PROVISIONS
4.1. Brucellosis is diagnosed at animals with epizootological status of the farm, characteristic clinic signs of the disease, anamnesis and complex evaluation of “ABNUREL” Triad reaction.
4.2. While diagnosing brucellosis, measures that can prevent human infection and environment contamination should be adhered in accordance with guidelines and instuctions in force while taking milk, blood samples and pathological materials.
4.3. Diagnosis of brucellosis is considered to be correct if brucella is isolated as pure culture, behaves like standard brucellosis antigen in control and experiment test tubes. That means, clearly visible negative result with negativ serum, clearly visible ring with monospecific positive (hyperimmune) serum should form (precipitation) at the same time at both control test tubes and should precipitate to the bottom of the test tube. The result of that suspension with biological test should be positive. (“ABNUREL” Triad reaction). Positive results can be analysed in accordance with existing instruction accepted with ELISA for IgM and of PCR in paralel.
4.4. The animals identified as sick should be isolated immediately, processed in slaughtering units of specific regime, other animals are involved in treatment and service objects are decontaminated with the last disinfection regime we suggested currently, areas inside the building, its surrounding are disinfected.
5. TAKING AND SENDING SAMPLES FOR ANALYSIS
5.1. Sample for laboratory analysis is taken from each animal in separate with a disposable syringe adhering sterility, cultured to broth nutrient media at place and sent to the laboratory on that day being packed.
5.2. Milk, blood, urine, animal secretions and materials, bone marrow, lymph node and so on from slaughtered sick animal can be taken for analysis to brucellosis.
5.3. Attached sheet is added to milk, blood and pathological materials sent to the laboratory. Anamnesis data are recorded in the sheet about if characteristic relevant clinic signs are present to brucellosis in the animal, short epizootic status of the farm due to brucellosis, suspicious signs are present similar to other diseases at the same.
APPLICATION TO OTHER FIELDS
1. The developed methodical instruction can be used in diagnostics of all types of animals susceptible to brucellosis, including diagnostics of humans, as it is a zooanthroponosis disease.
2. Herd can die without specific antibodies being formed at serum at acute, semi-acute periods of infectious diseases, therefore, it is not possible to diagnose at incubation period of the disease with available serological tests, when the disease progresses to prodromal period, the damage increases more. Regarding this, “ABNUREL” Triad reaction can also be applied in diagnostics of other infectious diseases with acute progression, however, some specific features of agents must be taken into consideration!
1. A. A. Aliyev N. G. Guliyev, H. M. Isgandarov, N. F. Atakishiyev – There is a contradiction between brucellosis antigen and pathogen brucellas. Azerbaijan agrarian scientific journal, 2007, # 8 - 9, p.75. In Azerbaijani.
2. A. A. Aliyev, N. G. Guliyev, F. R. Gulaliyeva Agrarian scientific journal, #3, 2011, “Comparison of bodies with brucellosis and immunity”, p.122. In Azerbaijani.
3. A. A. Aliyev, F. R. Gulaliyeva Agrarian scientific journal, #3, 2011, “Studying specific growth duration of brucellas in artificial nutrient media” p.125. In Azerbaijani.
4. A. A. Aliyev, N. G. Guliyev, F. R. Gulaliyeva – The “SCIENCE” newspaper, body of National Academy of Sciences or Higher Attestation Commission, 17 November 2011, 29-30 (1007-1008), About application of “ABNUREL” Triad reaction in diagnostics of brucellosis, p.9. In Azerbaijani.
5. A. A. Aliyev, N. G. Guliyev, E. H. Hasanov, F. R. Gulaliyeva, monthly international scientific-thereotical Scientific Work journal, on 02 (28) 2011 edition, “About improvement of diagnostics of brucellosis of farm animals” p. 35 – 40. In Azerbaijani.
6. A. A. Aliyev, N. G. Guliyev, E. H. Hasanov The “SCIENCE” newspaper, body of National Academy of Sciences or Higher Attestation Commission, 21 June 2004, “About improvement of diagnostics of brucellosis” p.14. In Azerbaijani.
7. A. A. Aliyev, N. G. Guliyev, E. H. Hasanov, M. A. Garayeva – Specific response of organism against Brucellosis is immune response. Azerbaijan international scientific-theoretical journal, #6-7, p.24-30. In Azerbaijani.
8. A. A. Aliyev, N. G. Guliyev, E. H. Hasanov, M. A. Garayeva – Presence of specific antibodies during brucellosis is a recovery feature. Azerbaijan international scientific-theoretical journal, #8-9, p.34-38. In Azerbaijani.
9. A. A. Aliyev - Bacteriolysis – as a presence of diagnostic titre features of specific antibodies during brucellosis p.118-122, Contemporary achievements of Azerbaijani Medicine, #1, 2012. p.224. Quarterly scientific-practical journal. In Azerbaijani.
10. R. F. Akhundov, H. A. Rahimova, A. A. Aliyev, F. R. Gulaliyeva – Studying effectiveness of new nutrient medium for growth of microorganisms, 154-155, Contemporary achievements of Azerbaijani Medicine, p.224. Quarterly scientific-practical journal. #1, 2012. In Azerbaijani.
11. Contemporary achievements of Azerbaijani Medicine, #4, 2012. About improvement of diagnostics of brucellosis. p.224. Quarterly scientific-practical journal. In Azerbaijani.
Chief scientist of the laboratory studying Animals Infectious diseases of Azerbaijan Scientific-Research Veterinary Institute, Doctor of Veterinary Sciences, Professor of Veterinary Microbiology, Epizootology, Virology and Immunology – Gara Ali Sariyev
Manual –being discussed widely in the Department of Infection of Veterinary Medicine Faculty of Agrarian University, (protocol #5, dated 29 December 2005) in the Department of Microbiology and Immunology of the Medicine University, (protocol #9, dated 30 June 2006, the review of A. Gurbanov, associate professor, candidate of medical sciences, dated 2006) All-Russian Scientific-Research Institute of Experimental Veterinary named after Y.R.Kovalenka, the idea and issues put forward have been approved. (Review of Moscow scientists, 1-6 June 2009) The obtained results have been checked and confirmed by committee in Azerbaijan Scientific-Research Veterinary Institute. (act dated 24.12.2008) The obtained major results have been discussed widely, reports have been approved in Scientific Council of Azerbaijan Scientific-Research Veterinary Institute and scientific articles have been published. (2009-2010 annual reports) Scientists of Kazan Veterinary Academy named after N.E.Bauman, chief of epizootology department, doctor of veterinary sciences, Professor R.Kh. Ravilov and head of microbiology and virology department, doctor of veterinary sciences, professor A.K.Galiulin have approved and recommended to be applied in veterinary practice. (the review dated 06.12.2011) Have been discussed in Medical Scientific Council of the Ministry of Health of the Azerbaijan Republic, relevant recommendations have been provided by specialists. (their reviews have been added to the letter # 17/19-4653, dated 06.06.2013) Comparative effectiveness of the new method with the existing method was checked by committee according to order #13 of the State Veterinary Service on 21.04.2014. (The results have been confirmed by the chief of the Khachmaz Veterinary Office and the director of the Republic Veterinary Laboratory). Comparative results shown in those acts were discussed widely in the Department of Microbiology and Immunology of the Medicine University on 01.07.2014, approved and their publication was recommended.