MORAXELLA SPECIES DIVERSITY IN INFECTIOUS BOVINE KERATOCONJUNCTIVITIS IN NOTHERN KAZAKHSTAN

Abstract

The purpose of this work was to study bacterial species diversity in Moraxella involved in infectious bovine keratoconjunctivitis (IBK) in Northern Kazakhstan. DNA were isolated and Moraxella spp. cytotoxin A fragments were amplified in 29 samples taken from cattle with clinical signs of IBK. Nucleotide sequence analyses and phylogenetic studies showed that Moraxella bovis DNA was present in ten samples, Moraxella bovoculi/Moraxella ovis DNA was present in eight samples, and that mixed profiles (mixed-infection or contamination) were found in eleven samples. Moraxella bovoculi/Moraxella ovis were isolated from bovine biological material with clinical signs of IBK for the first time in Kazakhstan. Keywords

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UDC 619:616-07,619:579.62
MORAXELLA SPECIES DIVERSITY IN INFECTIOUS BOVINE
KERATOCONJUNCTIVITIS IN NOTHERN KAZAKHSTAN
Kuibagarov M.A.1, Kamalova D.K.1, Shustov A.V.1, Suminov A.A.2, Karibaev T.B.3,
Ryskeldina A.Zh.1, Shevtsov A.B.1
1National Center for Biotechnology
13/5, Korgalzhyn road, Astana, 010000, Kazakhstan
2 “Genesis.KZ” LLP
13/5, Korgalzhyn road, Astana, 010000, Kazakhstan
3National Reference Center for Veterinary Medicine
22/3, 150 let Abaya str., Astana, 010000, Kazakhstan
marat.kuibagarov@gmail.com
ABSTRACT
The purpose of this work was to study bacterial species diversity in Moraxella
involved in infectious bovine keratoconjunctivitis (IBK) in Northern Kazakhstan.
DNA were isolated and Moraxella spp. cytotoxin A fragments were amplified in 29
samples taken from cattle with clinical signs of IBK. Nucleotide sequence analyses
and phylogenetic studies showed that Moraxella bovis DNA was present in ten
samples, Moraxella bovoculi/Moraxella ovis DNA was present in eight samples, and
that mixed profiles (mixed-infection or contamination) were found in eleven samples.
Moraxella bovoculi/Moraxella ovis were isolated from bovine biological material with
clinical signs of IBK for the first time in Kazakhstan.
Keywords: Moraxella in infectious bovine keratoconjunctivitis, bacterial
cytotoxin A analysis, Moraxella bovis in Northern Kazakhstan
__________________________________________________________________________
INTRODUCTION
Infectious bovine keratoconjunctivitis (IBK) or «pink eye» is an acute contagious
disease characterized by lacrimation, hyperemia of conjunctival vessels, photophobia, serous-
purulent discharge, opacity and ulceration of a cornea, deformation of an eyeball in a form of
keratoconus, partial or total loss of vision in an affected animal's eye [1]. IBK is the most
common eye disease of cattle and can affect all breeds of cattle, however, the highest
morbidity is recorded in breeds with no pigmentation in the eye area (mostly Hereford lines)
[2,3,4,5]. On the territory of Kazakhstan, this infection, in addition to Hereford, is most often
registered with the Auliekol and Kazakh white-headed local breeds [6].
A cause of IBK in cattle is thought to be a combination of physical factors and a
biological pathogen. The main causative bacterial agent of IBK which is most often isolated
from sick animals is a bacterium Moraxella bovis [7,8,9]. However, there are also data which
show a possibility of colonization of conjunctiva by Moraxella bovis opportunistically,
without an appearance of typical clinical signs [10]. Among different microorganisms which
can influence the IBK pathogenesis, there should be noted Moraxella bovoculi, Mycoplasma
spp., some viruses which can either aggravate severity of the disease or serve as predisposing
factors for infection [11, 12, 13, 14, 15, 16].
In recent years, IBK in Kazakhstan is often recorded in cattle on farms of various forms
of ownership. However, information about the etiologic agent in the IBK cases varies and is

presented in limited sources. A purpose of this study was to describe specific diversity of
bacterial pathogens which cause IBK.
The research was carried out at the Laboratory of Applied Genetics of the RSE
"National Center for Biotechnology" of the Ministry of Education and Science of the
Republic of Kazakhstan.
MATERIALS AND METHODS
For this study samples of total DNA were isolated from swabs taken from eyes of cattle
with clinical signs of IBK. Collection of samples from the affected eyes of animals was
performed by introducing dry sterile cotton swabs into the conjunctival sac. Swabs were
placed separately in sterile test tubes and delivered to the laboratory in a cooling container
with ice.
To the tubes, 1 ml of 0.5X TE buffer was added, vortexed vigorously for about 1
minute. The liquid was taken into an Eppendorf tube (1.5 ml), centrifuged at 10,000 rpm for 3
min. Portions (100 μl) of 0.5X TE buffer was added to the pellet, resuspended and used for
DNA isolation using the “DNA-sorb-B” kit (InterLabService, Russia). A concentration of
DNA was determined spectrophotometrically using a NanoDrop1000 spectrophotometer.
Analysis of PCR-amplified DNA fragments was carried by electrophoresis in a 1.5%
agarose gel containing ethidium bromide. Electrophoresis was performed in a horizontal
electrophoresis chamber PowerPac, using BioRad Electrophoretic bath current source. A
buffer 1X TAE was used as the electrophoresis buffer. Documenting of the results was done
using a Gel Doc gel documentation system (Bio-Rad) and Quantity One software (Bio-Rad).
Sizes (bp) of the PCR amplificates were determined by comparing their electrophoretic
mobility in the gel with a mobility of marker’s ladder (DNALadder 1kb, Fermentаs).
A selection of PCR-primers was carried out with a use of programs PrimerSelect
(DNASTAR), BioEdit and a web-resource NCBI PrimerBlast. During the primer selection
process, following key parameters were considered: similar annealing temperatures for
forward and reverse primers, length of primers between 18-25 bp, low probability of
formation of secondary structures.
Genotyping was performed by PCR-amplification of a fragment of a cytotoxin A gene
followed by a sequence determination. Purification of the PCR products from primers was
performed by enzymatic method using Exonuclease I (Fermentas) and alkaline phosphatase
(Shrimp Alkaline Phosphatase, Fermentas). The products were sequenced using BigDye
Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer's
instructions. The sequences were produced on an automatic genetic analyzer 3730xl DNA
Analyzer (Applied Biosystems). The nucleotide sequences were analyzed and assembled into
contigs and alignments using SeqMan software (DNAStar). After that, uninformative
sequences were removed (primers, and fragments having a low-quality index). The sequences
obtained were identified using GenBank BLAST algorithm. Phylogenetic trees were
constructed from the sequences deposited in the GenBank database. For phylogenetic studies
a software Mega 5 was used. ClustalW algorithm was used to align the nucleotide sequences,
and phylogenetic trees were constructed using the Neighbor-joining (NJ) method. Verification
of the topology was performed using the "Bootstrap method", number of replications - 1000.
RESULTS
According to published literature, one of the main factors of moraxella pathogenicity is
a cytotoxin A [17, 18, 19, 20, 21, 22]. The cytotoxin A gene is found in all three species of
moraxella associated with a development of keratoconjunctivitis: Moraxella bovis, Moraxella
bovoculi, Moraxella ovis (the genes named as mbxA, mbvA и movA, respectively). Nucleotide
similarity between the mbvA and movA sequences varies from 2 to 5%, and between mbxA

from 77 to 82%. This level of nucleotide variability allows using the cytotoxin A nucleotide
sequences for a selection of groups of primers for use in a species-specific detection of all
three species. To calculate the primers, from the international database GenBank (April 2018),
52 deposited sequences of the Moraxella spp cytotoxin A gene were downloaded. As a result,
a couple of primers (Mor_cуto_F3200 and Mor_cуto_R3675) were designed, which are
specific (PrimerBlast, https://www.ncbi.nlm.nih.gov/tools/primer-blast) to the target gene of
the three species of Moraxella spp.
For this study, from five farms of Akmola region (Central Kazakhstan), 29 ocular swab
samples were taken from diseased cattle (belonging to breeds Kazakh white-headed, Holstein,
Hereford). The cattle presented with varying degrees of clinical signs of IBK: conjunctival
hyperemia, corneal erosion, corneal ulceration, internal membranes prolapsed (keratoconus)
with loss of ocular substance (Fig. 1). Sampling was conducted during May-August 2018.
Fig. 1. Clinical signs of IBK in cattle
PCR on total DNA as the template was performed using the selected primers
Mor_cyto_F3200 and Mor_cуto_R3675, under optimized conditions, which allow a fragment
of a specific length (480 bp) of the cytotoxin A gene to be amplified. As an example, fig. 2
shows the results of amplification of 9 samples.
Lanes: 1-9, DNA samples; M, molecular weight marker (Fermentas, 100-1000 bp, step
100 bp); K-, negative control of an amplification reaction
Fig. 2. PCR results of 9 DNA samples isolated from biological material

Specific PCR products were obtained in 29 samples. Sequencing was carried out for
further species identification. In the sequencing electrophoregrams, for 9 samples
superposition of signals was found at about 15% of the nucleotides, which indicated
contamination of samples with DNA from different but closely related species. Comparison
of these sequences with the reference sequences M. bovis Epp63 strain and M. bovoculi 237
strain made it possible to establish that the mixed signals are because of differing nucleotides
between the species (fig. 3).
Fig. 3. Comparison of sequences from the field samples with the sequences from reference strains
The results allow concluding that in at least 11 samples two moraxella species are
present concominantly: M.bovis and M.bovoculi/M.ovis. Also the analysis of the fragment of
the cytotoxin A gene does not allow for reliable differentiation of M. bovoculi from M.ovis,
since dissimilarity in the nucleotide sequences of the whole gene does not exceed 1.3% (fig.
4).
Fig. 4. Nucleotide similarity of mbvA and movA genes
The nucleotide sequences of the remaining 18 samples showed no signs of contamination
with two or more species and were used to construct a phylogenetic tree (figure 5). The
investigated sequences show grouping into two clades. The first clade includes the reference
sequence of M. bovis, four samples from a farm No.1, two samples from a farm No.4 and four
samples from a farm No.5. The second clade includes the reference sequences from M.
bovoculi and M. ovis, as well as two samples from a farm No.1, four sample from a farm No.2
and by one samples from the farms Nos.5 and 3.

Fig. 5. Phylogenetic tree of the nucleotide sequences of Moraxella spp cytotoxin A gene
In the samples from four farms (out of five locations studied), at least two species of
Moraxella spp. were identified. In the samples from one farm (No.2) only one species was
identified: M. bovoculi/M. ovis (table 1).
Table 1. Species diversity of Moraxella spp.
Farm
Number of samples
Identified species
M. bovis
M. bovoculi /M.ovis
Mixed species
Farm 1
9
4
2
3
Farm2
4
0
4
0
Farm 3
2
0
1
1
Farm 4
4
2
0
2
Farm 5
10
4
1
5
CONCLUSION
There is still no accepted consensus among experts on an etiology of IBK. In connection
with this and an absence of any general strategy for diagnosis and specific prevention of the
disease, we can talk about formation of reservoirs of this infectious disease in Central
Kazakhstan.
There is little information on a species diversity of bacteria isolated from animals with
clinical signs of IBK in the territory of Kazakhstan. In rare publications, data appear on an
CP011158 Moraxella ovis 199
DQ155443 Moraxella ovis strain ATCC 33078
13 ferma3 27 July
12 ferma2 01 August
CP011380 Moraxella bovoculi 57922
CP011374 Moraxella bovoculi 58069
7 ferma1 18 June
DQ155435 Moraxella bovoculi 237
18 ferma5 11 June
9 ferma2 01 August
11 ferma2 01 August
1 ferma1 18 June
10 ferma2 01 August
EF436235.1 Moraxella bovis Epp63
2 ferma1 18 June
3 ferma1 18 June
6 ferma1 18 June
8 ferma1 18 June
14 ferma4 10 May
16 ferma4 10 May
20 ferma5 11 June
24 ferma5 11 June
25 ferma5 11 June
27 ferma5 11 June

isolation of only one species (Moraxella bovis) which was confirmed by bacteriological and
serological methods [6, 25]. For a long time it was believed that from the moraxella genus the
only species taking part in a pathogenesis of IBK is Moraxella bovis. However, based on the
fact that vaccines utilizing antigen from the sole species Moraxella bovis have not always
been effective, search for the other involved species were initiated. Accordingly, Moraxella
bovoculi was isolated and recognized relatively recently. In the summer of 2002, in Northern
California, Angelos J.A. isolated hemolytic gram-negative cocci from calves with signs of
conjunctivitis. After extensive bacteriological, biochemical and molecular-genetic analyzes
the microorganism was classified as a new species Moraxella bovoculi, the nomenclature of
which was published on April 5, 2005 [16, 26].
In recent years, an increasing number of cases of a so-called «Winter Pinkeye» had been
reported, and epizootologic signs of the disease are distinctive in that the disease is registered
off-season, when majority of pathogenic factors are excluded (extreme UV light, flying
insects, wind, plants, dust, etc.) [27, 28]. What exactly causes the winter IBK and what are the
factors that increase its spread, are currently not clear. The causing factor may appear to be a
moraxella of a different species or maybe changes in a quantitative ratio of the known species
play a role.
In a large portion of the examined samples, a presence of sole or mixed species was
determined: M.bovis was detected in 10 samples (34%), M. bovoculi/M.ovis in 8 samples
(27%), and 11 samples contained mixtures of M. bovis and M. bovoculi/M.ovis (37%).
In conclusion, attention should be paid to the occurrence of IBK in the non-typical
seasons such as winter. The obtained data can be used in a development of methods for
genetic diagnosis, prevention and treatment of IBK.
Acknowledgements
This study was funded by the Ministry of Education and Science of Kazakhstan by
BR05236307 STP "Development of new tools and innovative technologies for agriculture and
veterinary medicine" for 2018-2020 years.
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