AP09259105 «Study of Coxiella burnetii distribution and genotypes in South Kazakhstan»

Coxiellaburnetii is the etiologic agent of Q fever. Preliminary data obtained by us showed high seroprevalence of antibodies to this pathogen in the population of the southern region of Kazakhstan, however, there is no surveillance of Q fever in Kazakhstan. The main goal of the project is to study the distribution of C. burnetii in the southern region of Kazakhstan and genotyping of the pathogen. Diagnosis of Q fever is impossible without studying the prevalence of the pathogen causing it, its genotypes, which will allow to justify the surveillance strategy, reduce the risk of spread of infection, and develop test systems based on the characteristics of local strains. The work will be carried out by a group of employees headed by Dr. A.M. Dmitrovsky, whose scientific interests lie in the field of zoonotic natural focal vector-borne infections, PCR-diagnostics and genotyping.


Q fever (Coxiellosis) is a zoonotic infection characterized by multiple sources and transmission factors. The disease is caused by Coxiella (C.) burnetii, a strictly intracellular Gram-negative bacterium that affects farm animals and humans. The circulation of C. burnetii in nature involves many species of mammals, birds, and ticks.

The greatest frequency of human infection is directly or indirectly from farm animals. Aspiration and contact routes of transmission are predominant, and alimentary transmission is less important. Clinically, Q fever often presents under the guise of other infections (influenza, acute respiratory viral infections, brucellosis, etc.). The most characteristic features are acute onset, high fever, fever lasting about a week or more, chills, sweating, headache, joint and muscle pain, hepatolienal syndrome, possible development of interstitial pneumonia, complications in the form of thrombophlebitis, pancreatitis. The first press reports about Q fever in Kazakhstan (and in the USSR) were made by E.N.Bartoshevich in 1946, when she described short-term febrile illnesses occurring in rural areas among inhabitants of the southern region of Kazakhstan, later they were serologically identified as coxiellosis. In 1953-1954, Q fever was confirmed in Uzbekistan, Tajikistan and Kyrgyzstan. Subsequently, in the works of H.Zhumatov, A.M. Kurochkin, A.F. Petrov, and others, a description of the widespread spread of this infection in Kazakhstan was given. At the same time, in our opinion, the southern region of Kazakhstan is of the greatest interest due to the developed livestock breeding and a large number of private farms.

Because the symptoms of Q fever are nonspecific, the incidence of Q fever in both humans and animals cannot be estimated in most countries. However, current epidemiological studies indicate that Q fever should be considered a public health problem in many countries, such as France, the United Kingdom, Italy, Spain, Germany, Israel, Greece, etc., as well as in many other countries where Q fever is common but unrecognized due to lack of epidemiological surveillance of the disease. For example, in the USA, Q fever has been registered only since 1999, which led to a 250% increase in the number of human cases between 2000 and 2004 due to the monitoring of this infection [15].In Kazakhstan, Q fever has not been monitored since the 1980s, and there is no epidemiologic surveillance of this infection. Thus, there is currently no information on the prevalence of this infection among farm animals and humans in Kazakhstan.

Nevertheless, since 1995, among residents of the southern region of Kazakhstan there has been an increase in the number of cases of fevers of unclear etiology, with most cases diagnosed on the basis of clinical and epidemiologic data without laboratory confirmation. This group may include a significant proportion of Q fever. Also, a significant number of brucellosis cases are registered in the region, given that the clinical picture and epidemiologic factors of these two infections are often indistinguishable, and Q fever may also be present in this group. Taking into account the livestock breeding developed in the southern region, as well as the registration of diseases with clinical manifestations similar to Q fever in the southern oblasts without their etiological interpretation, the aim of this project is to clarify the prevalence of C. burnetii in the southern region of Kazakhstan, their main reservoirs, genotyping of C. burnetii, as well as improving laboratory diagnosis and epidemiological surveillance of Q fever.

One of the main objectives of the study will be the genetic characterization of C. burnetii strains circulating in the southern region of Kazakhstan. As far as we know, studies of this kind have not been conducted in the Republic before.

The aim

to study the distribution of C.burnetii in the southern region of Kazakhstan, to carry out genotyping of the detected pathogen, to improve laboratory and etiologic diagnostics of Q fever and to develop a strategy of epidemiologic surveillance of Q fever in the Republic of Kazakhstan.

Expected Results

As part of this task, primers for genotyping C. burnetii in biological material will be synthesized. Based on the study of clinical and epidemiologic manifestations of Q fever in the population in the region, a standard definition of a case of Q fever will be developed. Fulfillment of this task will make it possible to introduce a diagnostic algorithm for coxiellosis, determining on the basis of what criteria it is necessary to suspect a case of Q fever, which requires the transmission of an emergency notification and laboratory examination for this infection, what criteria confirm the probability of diagnosis and require the implementation of measures in the focus, and what laboratory tests confirm it.

Project manager

A.M. Dmitrovsky, Doctor of Medical Sciences, specialty infectious diseases, professor, head of the laboratory, infectious diseases doctor. Hirsch Index: 2. ORCID: https://orcid.org/0000-0003-4714-3079. ScopusID: 57217423246.WoS ID: AAZ-2816-2020.

Executive team members

Perfilieva Y.V., Ph.D. (biology). H-index: 3. ORCID: 0000-0001-6803-0773. Scopus ID: 56823500600. WoSID: AAF-9666-2020.

S.M. Mamadaliev, Doctor of Veterinary Sciences, Professor, virologist, specialist in especially dangerous animal diseases. H-index: 3. ORCID: 0000-0002-7767-0251. Scopus ID: 37000092700. WoSID: N-8389-2017. 

Ostapchuk E.O., Ph.D. (Biology). H-index: 3. ORCID: 0000-0002-3771-423X. Scopus ID: 56823472400. WoSID: D-1254-2015.

Bisenbay A.O., PhD-doctoral student (biotechnology). ORCID: 0000-0002-7109-2534. ScopusID: 57217425178.

Berdygulova J.A., PhD-doctoral student (biology), specialist in virology and molecular biology. H-index: 4. ORCID: https://orcid.org/0000-0003-0379-2472. Scopus ID: 23977664200. WoS ID: I-2943-2018.

Berezovsky D.V., Master’s degree (medicine), doctor hygienist-epidemiologist. H-index: 1. ORCID: https://orcid.org/0000-0002-2830-1994. Scopus ID: 57212528777. WoS ID: AAY-6245-2020.

Kuatbekova S.A., Master’s degree, veterinary specialist .ORCID:https://orcid.org/0000-0001-5569-1847. WoS ID:ABA-3202-2020.

Publications of the project manager and members of the research team on the topic of the project

  1. PerfilyevaY.V., ShapiyevaZ.Zh., OstapchukY.O., Berdygulova Z.A., Bissenbay A.O., Kulemin M.V., Ismagulova G.A., Maltseva E.R., SkibaY.A., Sayakova Z.Z., Mamadaliyev S.M., DmitrovskiyA.M. Tick-borne pathogens and their vectors in Kazakhstan – areview. Ticks and Tick-borne diseases. 2020;11(5):101498. https://doi.org/10.1016/j.ttbdis.2020.101498.IF 2.749; Q2; Cite score 5.2; SJR 1.182; percentile 95. 
  2. Abdiyeva K., Turebekov N., Dmitrovsky A., et al. Seroepidemiological and molecular investigations of infections with Crimean–Congo haemorrhagic fever virus in Kazakhstan. Int.J.Inf.Dis. 2019; 78:121-127. IF 3.202; Q2.Cite score 5.3; SJR 1.44; percentile 79.
  3. Turebekov N., Abdiyeva K., Yegemberdiyeva  R., Dmitrovsky A., et al. Prevalence of Rickettsia species in ticks including identification of unknown species in two regions in Kazakhstan. Parasites and Vectors. 2019; 12:197. IF 2.824; Q1. Citescore 5.3; SJR 1.406; percentile87.
  4. OstapchukY.O.,ZhigailovA.V., PerfilyevaY.V.,ShumilinaA.G., YeraliyevaL.T., NizkorodovaA.S., KuznetsovaT.V., IskakovaF.A., BerdygulovaZ.A., NeupokoyevaA.S., MamadaliyevS.M., DmitrovskiyA.M.Two case reports of neuro invasive West Nile Virus infection in the Almaty region, Kazakhstan. ID Cases. 2020; 21:e00872. SJR 0.294.
  5. Perfilyeva Yu.V., Nizkorodova A.S., Berdygulova Zh.A., Ostapchuk Ye.A.,Naizabayeva D.A., Neupokoyeva A.S., Kuznetsova T.V., Shishkina T.S., Abuova G.N., Yegemberdiyeva R.A., Bissenbay A.O., Maltseva E.R., Mamadaliyev S.A., Dmitrovsky A.M. Detection of IgG against Rickettsia typhi: A population-based study in Southern Kazakhstan. Infektološkiglasnik. 2019; 39(4) .https://doi.org/10.37797/ig.39.4.2. SJR 0.104.
  6. Bissenbay A.O.,Zhigailov A.V., Maltseva E.R., Egemberdieva R.A., Skiba Y.A., Mamadaliyev S.M. Borreliosis: a Hidden Threat for Kazakhstan. Eurasian Journal of Applied Biotechnology. 2019; 2:5-27. DOI: 10.11134/btp.2.2019.2. ККСОН РК.
  7. Bissenbay A.O., Zhigailov A.V., Neupokoyeva A.S., Naizabaeva D.A., Skiba Y.A., Dmitrovsky A.M., Shapiyeva Zh.Zh., Mamadaliyev S.M. West Nile fever virus: biology, epidemiology, molecular genetic characteristics and research priorities. Eurasian Journal of Applied Biotechnology. 2019; 2:28-40. DOI: 10.11134/btp.2.2019.3. ККСОН РК.
  8. Sansyzbay A.R., Erofeeva M.K., Khairullin B.M., Sandybayev N.T., KydyrbayevZh.K. ;Mamadaliyev S.M., et al. An Inactivated, Adjuvanted Whole Virion Clade 2.2 H5N1 (A/Chicken/Astana/6/05) Influenza Vaccine Is Safe and Immunogenic in a Single Dose in Humans. Clinical and Vaccine Immunology. 2013;20(8):1314-1319. https://doi.org/10.1128/CVI.00096-13. IF 3.233; Q2.
  9. Mamadaliyev S.M.,Sandybayev N.T., et al. Basic results of development of a production technology and control of a pandemic influenza A/H5N1 vaccine. Influenza and Other Respiratory Viruses. 2011;5:350-353. IF 3.288; Q2.
  10. Mamadaliyev S.M., Sandybayev N.T., et al. Development of production technology and pre-clinical testing of a pandemic influenza A/H1N1 vaccine. Influenza and Other Respiratory Viruses. 2011; 5:354-357. IF 3.288; Q2.
  11. Kiseleva I.V., Voeten J.T.M., Teley L.C.P., Larionova N.V., Dubrovina I.A., Berdygulova Z.A., et al. Genome Composition Analysis of Reassortant Influenza Viruses Used in Seasonal and Pandemic Live Attenuated Influenza Vaccine. 2011; 26(4):174-185. IF 0.25; Q4.
  12. Berdygulova Z.,Westblade L.F., Florens L., et al. Temporal regulation of gene expression of the Thermus thermophilus bacteriophage. J.Mol.Biol. 2011; 405(1):125-142. IF 4.76; Q1.
  13. Berdygulova Z., Esyunina D., Miropolskaya N., et al. A novel phage-encoded transcription antiterminator acts by suppressing bacterial RNA polymerase pausing. Nucleic Acids Research. 2012; 40(9):4052-4063. IF 11.501; Q1.

Achieved Results

2021 year

Collection and morphological identification of ticks of the southern region of Kazakhstan in the epidseason 2021 was carried out. In Turkestan region 1331 ticks were collected and their species diversity was analyzed. In total 6 species of ticks belonging to 4 genera were morphologically identified: Argaspersicus, Hyalommaasiaticum, H. scupense, H. anatolicum, H. marginatum, Dermacentormarginatus, Haemaphysalissulcata. Argaspersicus (48.1%; 640/1331; 95% CI: 45.4-50.7%) and Ha. sulcata (30.10%; 400/1331; 95% CI: 27.7-32.6%) were the most common tick species collected.

Blood samples from cattle in the southern region of Kazakhstan were analyzed. Serum/plasma from blood samples from 247 domestic sheep (Ovis aries), 25 goats (Capra hircus) and 10 cows (Bostaurus) were obtained.

Blood samples were collected from residents of the southern region of Kazakhstan. In the epidemiologic season of 2021, 100 blood samples were collected from residents of Taraz, Zhambyl region. From them serum/plasma samples were obtained. Samples were collected in hospitals and polyclinics of the city. A total of 92 serum samples were collected for ELISA. The mean age of the subjects was 33.7±8.7 years (range 23-50 years), of whom 42 were women (56.1%) and 45 were men (43.9%), for 5 donors the sex was unknown.

Serologic and PCR analysis of collected samples was performed. Cross-sectional serologic examination of 282 farm animals was carried out. Total seropositivity to C.burnetii antigens I and II among farm animals was shown to be 27.7% (29.0%; 78/282; 95% CI: 22.8-33.2%), with the highest number of seropositive animals detected in Turkestan oblast (32.2%: 46/143; 95% CI: 25.1-40.2%) compared to Zhambyl oblast (23.0%; 32/139; 95% CI: 16.8-30.7%). Of the 78 seropositive animals, 66 belonged to sheep and 12 to goats. No positive samples were detected among cattle, which may have been due to the small sample size (n=10). – ELISA analysis with sera from residents of the southern region of Kazakhstan was performed using a validated kit “Immunoenzyme test system for detection of IgG class antibodies to coxiella-Bernet antigens” (ELISA-anti-Cu-G) (Pasteur Research Institute of Epidemiology and Microbiology). Of 92 samples analyzed, 3 were positive for IgG to C. burnetii. These samples were obtained from 20 ticks collected in the Almaty region and 237 single ticks and 10 pools of ticks (2-3 ticks) collected in Turkestan region during the epidemiological season of 2021, nucleic acids were isolated and DNA was analyzed by classical PCR using two pairs of primers. The expected PCR products (738 bp and 775 bp, respectively) were not detected in any of the analyzed samples.

Epidemiologic analysis and GIS mapping of the obtained data were carried out. The serologic analysis revealed that in Turkestan and Zhambyl regions of Kazakhstan a significant proportion of livestock (27.7%) has antibodies to C. burnetii, which indicates a fairly wide prevalence of coxiellosis among farm animals in the surveyed area. At the same time, the most epizootically active foci were found in Baidibek, Sairam and Kazygurt districts of Turkestan oblast and in T. Ryskulov district of Zhambyl oblast. Identification of regional peculiarities of coxiellosis epidemiology showed that the risk of seropositivity was two and a half times higher in Caprahircus, suggesting that this animal species is more vulnerable to C. burnetii infection. Nevertheless, the larger sheep population in the region and the observed seroprevalence of 26.7% among Ovis aries emphasize the active participation of this species in the circulation of the pathogen in the study area. Serologic analysis of serum samples obtained from residents of Taraz-Zhambyl oblast showed that there is a small layer of the population with antibodies to C. burnetii (3.3%), which indicates the legitimate possibility of human infection with C. burnetii in the study area.

2022 year

Collection and morphological identification of ticks from the southern region of Kazakhstan was carried out. Homogenization of collected ticks and isolation of DNA preparations were carried out.

– Collection of ticks from vegetative vegetation, cattle and small horned cattle and big gerbil in 2 districts of Turkestan region (Otyrar and Shardara) was carried out. 185 ticks were collected, morphologically identified 5 species belonging to 3 genera: Hyalomma (H.) asiaticum, H. scupense, H. anatolicum, Dermacentor (D) niveus, Haemaphysalis (Ha.) erinacei.

– In the epidemiologic season of 2022, ticks were collected by the “flag” method from vegetative vegetation in the foothills of the Zailiyskiy Alatau and from bitten people and animals. 66 ticks were collected, their morphological classification was carried out. 4 species of ticks were identified: Ixodes persulcatus (41 ticks), D. marginatus (5 ticks), Haemaphysalis punctata (12 ticks), Rhipicephalus spp (8 ticks).

– The isolation of nucleic acid preparations from 96 homogenized ticks collected in 2021 in Turkestan region by selective precipitation method using the kit “RIBO-sorb” (“AmpliSens”) was carried out.

2023 year

– In order to identify C. burnetii and subsequent DNA sequencing, ticks were collected from Almaty and Zhambyl regions in 2023. Their morphological identification was performed according to the identification tables using a stereoscopic microscope Micromed MS-2-ZOOM var.2A. Homogenization of 136 single iscodon mites with their subsequent pooling (n=34) and aliquoting was carried out. Total DNA preparations from 34 pools of ticks were isolated by selective precipitation using the RIBO-Prep kit (AmpliSens) and PCR-analyzed using the validated commercial test system “AmpliSens® Coxiella burnetii-FL”. Four positive pools were identified. DNA preparations were isolated from homogenates of single ticks, pools of which gave PCR-positive results. PCR analysis of DNA samples obtained from single ticks showed that the total infection rate of collected ticks with C. burnetii in the investigated areas was 2.9% (4/136). All information was entered into the database in Excell format.

– Amplification was performed on three loci of icd (expected product 738 bp), 16S rRNA (expected product 1459 bp) and com1 genes (amplification was performed using two primer pairs “CoxBurnetii_com1_F and “CoxBurnetii_com1_R”, “CoxBurn_Com-1ab-F” and “CoxBurn_Com-4ab-R”, expected products 775 and 757 bp, In addition, nested PCR was performed using the primer pair “CoxBurn_Com-1ab-F” and “CoxBurnetii_com1_R” and the mass of the expected amplification product 259 bp) for 13 DNA samples isolated from single ticks collected in 2022 and showing the presence of C. burnetii DNA, and 3 DNA samples isolated from the blood of PCR-positive animals. Electrophoretic analysis of the obtained PCR reaction products with their subsequent visualization in transmitted ultraviolet light on GelDox XR+ gel-documentation system (Bio-Rad, USA) was performed.

– In addition, for genetic characterization of the detected C. burnetii strains, primers for the transposon repeat region of C. burnetii “Trans 1” and “Trans 2” were synthesized on a DNA synthesizer and purified by high-performance liquid chromatography.

– An algorithm for diagnosing Q fever in patients with determination of IgG antibody titers to phase I (chronic coxiellosis) and phase II (acute coxiellosis) C. burnetii was developed.

– Serum and plasma samples were collected from patients from Turkestan region (endemic for coxiellosis) with clinical signs of chronic coxiellosis (prolonged pneumonia, endocarditis) (n=14), their aliquoting with subsequent entry into the database.

– Serological screening of the population of Zhambyl oblast (n=316) and Turkestan oblast (n=14) for the presence of antibodies to C.burnetii using a validated kit “Immunoenzyme test system for detection of IgG class antibodies to coxiella burnetii antigens” (Pasteur Research Institute of Epidemiology and Microbiology). To detect chronic and acute forms of Q fever, all positive samples were further tested for the presence of antibodies to C. burnetii phase I and II using validated anti-Coxiella burnetii (Q-Fever) Phase 1 IgG Human ELISA and anti-Coxiella burnetii (Q-Fever) Phase 2 IgG Human ELISA (Abcam) kits with subsequent determination of antibody titers in serum dilutions from 1:100 to 1:1600. Interpretation of the obtained results was performed. DNA isolation from serum samples from residents with detected chronic coxiellosis (n=4) and their subsequent PCR analysis were performed.

– Collection of medical history from a patient with detected acute coxiellosis (collection of objective data, epidemiologic anamnesis, results of laboratory and diagnostic tests, prescribed medications). For epidemiologic investigation of the detected case of acute coxiellosis, blood samples were collected from persons living together with the patient, as well as collection of milk from livestock in the patient’s backyard. ELISA testing of the collected samples for the presence of IgG class antibodies to C. burnetii antigens was performed using a validated kit “ID Screen® Q Fever Indirect Multi-species” (IDVet) according to the manufacturer’s instructions.

– Based on the literature data, primers targeting the groEL molecular chaperone gene were selected for genetic characterization of the detected C. burnetii strains: “Cox-GrF1” (5′-ttttgaaaayatgggcgckcaaatggt), “Cox-GrR2” (5′-(cgrtcrccaaarccaggtgc), “Cox-GrR2” 5′-(cgrtcrccaaarccaggtgc), “Cox-GrF2” (5′-gaagtggcttcgcrtacwtcagacg), “Cox-GrR1” (5′-ccaaarccaggtgtgctttyac).

– Amplification using primers “Trans 1” and “Trans 2” was performed for DNA samples isolated in 2022 from 21 single ticks showing the presence of C. burnetii DNA and from the blood of 3 PCR-positive animals, followed by electrophoretic analysis. No amplifications of appropriate size were detected.

– Amplification was performed at four loci of icd (expected product 738 bp), 16S rRNA (expected product 1459 bp), com1 (expected product 775 bp) genes for 4 DNA samples isolated from single ticks collected in 2023 that showed the presence of C. burnetii DNA according to the results of a commercial kit. Electrophoretic analysis of the obtained PCR reaction products with their subsequent visualization in transmitted UV light on GelDox XR+ gel-documentation system (Bio-Rad, USA) was performed. A single amplificate of 738 bp was obtained, purified from agarose gel, and sequenced at both ends on a 24-capillary ABI 3500XL genetic analyzer (Life Technologies).

– Developed:

1) Definition of a presumptive case of acute febrile or primary generalized form of Q fever (57 – 68%);

2) Definition of a presumptive case of secondary focal hepatitic form of Q fever (23 – 37%);

3) Identification of a presumptive case of secondary focal carditic form of Q fever (4-10%);

4) Identification of a presumptive case of secondary focal meningo-encephalitic form of Q fever (up to 2%);

5) Probable case of Q fever;

6) Confirmed case of Q fever.

– Published 1 (one) article in a peer-reviewed scientific publication with CiteScore percentile in Scopus database not less than 80 (eighty): 1) Perfilyeva Y.V., Berdygulova Z.A., Mashzhan A. S. Ostapchuk Y.O., Zhigailov A.V., Ostapchuk Y.O., Naizabayeva D.A., Cherusheva A. S., Bissenbay A.O., Kuatbekova S., Abdolla N., Maltseva E.R., Nizkorodova A.S., Kulemin M. V., Shapiyeva Z.Z., Sayakova Z. Z., Perfilyeva A. V., Maltseva E. R., Skiba Y.A., Mamadaliyev S.M., Dmitrovskiy A.M. Molecular and seroepidemiological investigation of Сoxiella burnetii and spotted fever group rickettsiae in the southern region of Kazakhstan // Ticks Tick Borne Dis. – 2023. – N.14:102240. doi: 10.1016/j.ttbdis.2023.102240 (Impact Factor-3.7 WOS, Cite Score, Scopus – 91%, 15/73 Insect Science).