Infectious bovine keratoconjunctivitis (IBK, pink-eye) is considered to be the most important eye disease worldwide with high economic losses. Moraxella bovis is the only pathogen reproducing IBK-like eye lesions in various experimental models. In this regard, most often, developed and used for the prevention of vaccines against IBK contain inactivated strains of Moraxella spp or fragments thereof. Field trials of such vaccines more often show their low effectiveness, which may indicate the involvement of different groups of bacteria in pathogenesis, and that attention to only one potential pathogen may be incorrect.

Relevance

Infectious keratoconjunctivitis is a highly contagious infectious disease characterized by conjunctivitis and ulcerative keratitis, affecting many species of ruminants worldwide, including wild animals. Bovine keratoconjunctivitis (pink-eye) is considered the most important eye disease worldwide, with high economic losses due to reduced weight gain, milk production, infertility of heifers and increased treatment costs.

Infectious keratoconjunctivitis, despite the fact that it does not lead to death, causes great economic damage. Farmers’ costs are associated with lost weight gain, culling of breeding animals, and treatment costs. The annual damage from IBK in the United States reaches 150 million US dollars. In Kazakhstan, the IBK is registered in many areas. In the East Kazakhstan region, more than 50% of young animals get sick every year. The most effective way to combat this is to prevent it with vaccines. Registered vaccines show low efficacy in field trials, which is directly associated with differences in microbial diversity and its genetic characteristics. The project aims to explore the white spots in this issue.

Aim

To study changes in the ocular microbiota in calves with varying degrees of severity of infectious keratoconjunctivitis and to conduct whole genome sequencing of bacterial species whose numbers increase during the infectious process.

Expected results

• Ocular microbiome data will be obtained from 300 calves with infectious keratoconjunctivitis of varying severity and a control group.
• A possible correlation between species diversity, the number of bacteria and the nature of the development of infectious keratoconjunctivitis will be studied.
• Whole genome data of at least 150 bacterial strains of bacterial species potentially associated with IBK will be obtained.
• Virulence and drug resistance factors in isolated strains will be determined.

The dissemination of the results of the work among potential users, the community of scientists and the general public will be carried out by publishing the results in scientific articles with open access. Whole genome bacterial data and raw microbiome sequencing data will be published in open databases (NCBI).

Data on the microbiome and genetic diversity of bacteria in cattle with IBK may be of interest to scientists conducting research in the field of diagnosis and specific prevention of eye diseases. The study has a long-term economic effect, as the data will improve the prevention and treatment of IBK, which will subsequently have a positive impact on the profitability of cattle breeding.

The results can be used by biotechnologists in the planning of new vaccines for the prevention of keratoconjunctivitis in cattle and by practicing veterinary specialists in the preparation of treatment regimens.

Project manager

Shevtsov Alexander Borisovich, Candidate of Biological Sciences, author of 49 articles published in journals indexed in Scopus.

https://orcid.org/0000-0002-0307-1053

https://www.scopus.com/authid/detail.uri?authorId=57201604158

Members of the research group

Kuibagarov Marat Amangeldyevich, Candidate of Veterinary Sciences. Within the framework of the project, participates in microbiological research, species identification of bacteria, DNA isolation, and analysis of results.

https://www.scopus.com/authid/detail.uri?authorId=57220278412

https://orcid.org/0000-0001-7428-7620

Amirgazin Asylulan Orazgalievich. Within the framework of the project, participates in DNA isolation, genome-wide sequencing, microbiome analysis, and analysis of results.

https://orcid.org/0000-0001-9418-7758

https://www.scopus.com/authid/detail.uri?authorId=57218628470

Abdigulov Bolat Baurzhanuly, Master of Natural Sciences, participates in DNA isolation, whole genome sequencing, microbiome analysis, and analysis of results within the framework of the project.

https://orcid.org/0009-0004-4550-252X

https://www.scopus.com/authid/detail.uri?authorId=58653782400

Yerzhanova Nurdina Serikzy. Within the framework of the project, participates in microbiological research, species identification of bacteria, DNA isolation and analysis of results.

https://orcid.org/0000-0003-0612-455X

https://www.scopus.com/authid/detail.uri?authorId=57972056800&origin=recordpage

Publications and protection documents on the project topic

1. Shevtsov A., Cloeckaert A., Berdimuratova K., Shevtsova E., Shustov A. V., Amirgazin A., Karibayev T., Kamalova  D., Zygmunt M. S., Ramanculov Y. Vergnaud G.. Brucella abortus in Kazakhstan, population structure and comparison with worldwide genetic diversity. Frontiers in Microbiology, (2023) 14, 1106994. https://doi.org/10.3389/fmicb.2023.1106994
2. Kuibagarov, M., Abdullina, E., Ryskeldina, A., Abdigulov, B., Amirgazin, A., Shevtsov, A., & Angelos, J. A. (2023). Association of different microbes and pathogenic factors in cases of infectious bovine keratoconjunctivitis in cattle from Eastern Kazakhstan. Veterinary world, 16(9), 1833–1839. https://doi.org/10.14202/vetworld.2023.1833-1839
3. Shevtsov A., Aushakhmetova Z., Amirgazin A., Khegay O., Kamalova D., Sanakulova B., Abdaliyev A., Bayesheva D., Seidullayeva A., Ramankulov Y., Shustov A., Vergnaud G. (2022). Whole genome sequence analysis of Neisseria meningitidis strains circulating in Kazakhstan, 2017–2018. Plos one, 17(12), e0279536. https://doi.org/10.1371/journal.pone.0279536  
4. Kuibagarov M., Zhylkibayev A., Kamalova D., Ryskeldina A., Yerzhanova N., Ramankulov Y., Shevtsov A., Angelos J. Genetic diversity of Pilin from Kazakh isolates of Moraxella bovoculi. /Adv. Anim. Vet. Sci. – 2022. – Vol. 10. – Issue 9. – P.2148-2159. http://dx.doi.org/10.17582/journal.aavs/2022/10.11.2376.2383
5. Shevtsov A., Lukhnova L., Izbanova U., Vernadet J.-P., Kuibagarov M., Amirgazin A., Ramankulov Y., Vergnaud G. Bacillus anthracis Phylogeography: New Clues From Kazakhstan, Central Asia. Frontiers in microbiology 12 (2021). https://doi.org/10.3389/fmicb.2021.778225
6. Shevtsov V., Kairzhanova A., Shevtsov A., Shustov A., Kalendar R., Abdrakhmanov S., Lukhnova L., Izbanova U., Ramankulov Y., Vergnaud G. Genetic diversity of Francisella tularensis subsp. holarctica in Kazakhstan. PLoS Neglected Tropical Diseases 15.5 (2021): e0009419. https://doi.org/10.1371/journal.pntd.0009419
7. Nurmakhanov T., Sansyzbaev Y., Atshabar B., Berlin V., Kobzhasarov D., Yeskhojayev O., Vilkova A., Ayazbayev T., Andryuchshenko A., Bidashko F., Hay J., Shvetsov A. Phylogenetic characteristics of West Nile virus isolated from Culex modestus mosquitoes in West Kazakhstan. Frontiers in public health 8 (2021): 1089. https://doi.org/10.3389/fpubh.2020.575187
8. Shevtsova E., Vergnaud G., Shevtsov A., Shustov A., Berdimuratova K., Mukanov K., Syzdykov M., Kuznetsov A., Lukhnova L., Izbanova U., Filipenko M., Ramankulov Y. Genetic diversity of Brucella melitensis in Kazakhstan in relation to world-wide diversity. Frontiers in Microbiology (2019): 1897. https://doi.org/10.3389/fmicb.2019.01897
9. Marat Kuibagarov, Riza Makhamed, Assylbek Zhylkibayev, Maxat Berdikulov, Sarsenbay Abdrakhmanov, Mazhit Kozhabayev, Ilyas Akhmetollayev, Kasim Mukanov, Anara Ryskeldina, Yerlan Ramankulov, Alexandr Shustov, Christian Bauer, Alexandr Shevtsov. Theileria and Babesia infection in cattle – first molecular survey in Kazakhstan. Ticks and Tick-borne Diseases. 2023., Vol.4, Issue 1. 102078,ISSN 1877-959X, https://doi.org/10.1016/j.ttbdis.2022.102078
10. Kuibagarov, M., Amirgazin, A., Vergnaud, G., Shustov, A., Ryskeldina, A., Ramankulov, Y., & Shevtsov, A. (2020). Draft Genome Sequence of Moraxella bovoculi Strain KZ-1, Isolated from Cattle in North Kazakhstan. Microbiology resource announcements, 9(30), e00670-20. https://doi.org/10.1128/MRA.00670-20
11. Yessembekova G.N., Shuang X., Abenov A., Karibaev T., Shevtsov A. Amirgazin A., Mukhanbetkaliyev Y.Y., Lei S., Zhi-gao B. Abdrakhmanov S.K. (2023). Molecular epidemiological study of animal rabies in Kazakhstan. Journal of Integrative Agriculture, 22(4), https://doi.org/10.1016/j.jia.2022.11.011
12. Amirgazin A, Shevtsov A, Karibayev T, Berdikulov M, Kozhakhmetova T, Syzdykova L, Ramankulov Y, Shustov AV. Highly pathogenic avian influenza virus of the A/H5N8 subtype, clade 2.3.4.4b, caused outbreaks in Kazakhstan in 2020. PeerJ. 2022 Mar 2;10:e13038. PMID: 35256921; PMCID: PMC8898005. DOI: 10.7717/peerj.13038
13. Kuibagarov, M., Kairzhanova, A., Vergnaud, G., Amirgazin, A., Lukhnova, L., Izbanova, U., Shevtsov, A. (2020). Draft Genome Sequence of the Strain Francisella tularensis subsp. mediasiatica 240, Isolated in Kazakhstan. Microbiology Resource Announcements, 9(35), e00766-20. https://doi.org/10.1128/mra.00766-20

Achieved results

2024

Whole genome DNA sequencing of 85 collection strains of M. bovis and M. bovoculi isolated from animals with infectious keratoconjunctivitis (IBK) from different farms in the period 2018-2022 was performed.

Biological samples were collected from calves with IBK of varying severity and a control group without IBK in the amount of 100 samples. A 30-day monitoring of the clinical condition of the animals was carried out with registration of the response to therapy and the presence/absence of IBK in the control group.

Bacterial cultures were isolated and identified from collected swabs of lacrimal swabs from calves. A total of 75 bacterial cultures were isolated including: Acinetobacter, Dietzia, Agrococcus, Bacillus, Bavariicoccus, Corynebacterium, Micrococcus, Moraxella, Staphylococcus and Streptococcus. DNA was isolated from swabs and isolated bacterial cultures.

The analysis of the eye microbiota in calves with IBK of varying severity and the control group without IBK, selected in the amount of 100 samples by sequencing regions V3 and V4 of 16S rRNA gene, was carried out. The data of bacteriological diversity in lacrimal fluid swabs from calves with varying degrees of severity of IBK were obtained.