Improvement of Laboratory Diagnosis for Detection and Identification
of Bovine Clostridiosis
NATALIA A. BEZBORODOVA*, EVGENIA N. SHILOVA,
VERONIKA V. KOZHUKHOVSKAYA, VLADLENA D. ZUBAREVA, OLGA V. SOKOLOVA,
NIKOLAI A. MARTYNOV
Federal State Budgetary Scientific Institution “Ural Federal Agrarian Scientific Research Centre, Ural
Branch of Russian Academy of Sciences”
Ekaterinburg
RUSSIAN FEDERATION
*Corresponding Author
Abstract: - Objective: Clostridiosis is a toxic infectious disease; the pathogenicity factor of causative agents is
the secreted toxins. A characteristic feature of clostridiosis pathogens is their polytropism. They affect both
humans and agricultural, domestic, and wild animals. Our research aimed to monitor Clostridium perfringens
and Clostridium difficile spread among agricultural organizations of the Ural region.
Materials and Methods: 137 biological samples were obtained from cattle with symptoms of clostridial
infection. For PCR species and toxinotype identification commercial kits and previously described protocols
were used. Results verification was conducted using MALDI-TOF MS.
Results: Out of 137 samples of selected material Clostridium was detected in 40.6% of samples: Cl. difficile in
35.8%, Cl. perfringens in 25.3%, Cl. difficile+Cl. perfringens in 16.4%. Cl. difficile and Cl. perfringens were
found in 30.5% of fecal samples, in pathological material from dead calves and cows – 8.7%, in milk samples –
1.4%.
Conclusion: Laboratory methods made it possible to verify the diagnosis: infectious anaerobic enterotoxemia
of calves in one case, necrotic enteritis in 3 animals, and intestinal toxic infection caused by Cl. perfringens
type A in 2 cows and 5 calves. The diagnostics of toxinotypes of Cl. perfringens have made it possible to
conduct toxin-specific vaccination against clostridial infection in farms.
Key-Words: - cattle, Clostridium difficile, Clostridium perfringens, PCR, toxinotypes.
Received: August 2, 2022. Revised: October 6, 2023. Accepted: October 21, 2023. Published: November 1, 2023.
1 Introduction
Bovine clostridiosis in cattle is one of the major
problems in veterinary medicine, animal
husbandry, and food safety. This pathogen causes
infectious diseases related to zooanthroponosis
diseases, [1], [2], [3], [4], [5].
Anaerobic spore-forming bacteria of the genus
Clostridium cause infections, which occur
ubiquitary. Specific features of this group of
diseases are stationarity and high mortality.
Generally, the disease manifests itself in sporadic
form, sometimes as an epidemic outbreak. The
main reservoir of these anaerobes is soil, as well as
the intestines of animals and humans, [1], [6].
Under certain conditions clostridia, which normally
inhabits the gastrointestinal tract, can gain
pathogenicity properties. Metabolic disorders,
stress factors, injuries, and misuse of antibacterial
drugs contribute to the spreading of toxigenic
anaerobes, [7], [8], [9].
Active exotoxins are the main factors of
clostridiosis pathogenesis. The most common
zoonotic pathogens of clostridial infections include
Clostridium perfringens, [6], [10]. There are five
toxinotypes of this pathogen: A, B, C, D, and E,
each of which causes a disease with specific
clinical signs, [11]. All toxinotypes cause anaerobic
enterotoxemia in young animals, [8], [10].
Toxinotype A causes malignant edema, and gas
gangrene in cows with injuries. Cl. perfringens
toxinotype A causes less malignant diseases than
other toxinotypes. Mortality of animals with
toxinotype A-caused infection does not exceed
25%, [2], [11], [12], [13]. Toxinotype B provokes
enterotoxemia in calves with hemorrhages in all
vital organs. Cl. perfringens toxinotype C causes
necrotizing enteritis in animals. This toxin causes
WSEAS TRANSACTIONS on BIOLOGY and BIOMEDICINE
DOI: 10.37394/23208.2023.20.31
Natalia A. Bezborodova, Evgenia N. Shilova,
Veronika V. Kozhukhovskaya, Vladlena D. Zubareva,
Olga V. Sokolova, Nikolai A. Martynov
E-ISSN: 2224-2902
305
Volume 20, 2023
intestinal necrosis, bleeding, septic shock, and
eventually death, [14], [15]. Cl. perfringens type D
is another pathogen that causes enterotoxemia in
young ruminants with specific pathological
findings also known as pulpy kidney, [16], [17].
Toxinotype D causes dysentery and nephrolithiasis
in sheep and lambs, [18]. In calves it causes
enterotoxemia with neurological signs, without
extensive intestinal lesions. Toxinotype E is the
cause of necrotic hemorrhagic enteritis in calves,
[19].
Cl. difficile is a gram-positive toxin-producing
bacterium. Being an intestinal pathogen it is
capable of releasing enterotoxin A, cytotoxin B,
and binary toxin CDT. Toxins A and B are encoded
by separate genes (tcdA, tcdB), while the binary
CDT toxin is encoded by two genes (cdtA and
cdtB), [10], [20]. Toxin A causes pronounced
enterotoxic manifestation and pro-inflammatory
activity of interleukins in intestinal epithelial cells.
Toxin B triggers receptor-mediated endocytosis,
[21]. Clostridia that produce these toxins have
increased adhesion to the intestinal epithelium, [4],
[22]. Toxin-negative isolates of Cl. difficile are
often detected in the feces of animals and humans
with intestinal dysbiosis. The acquisition of
toxigenic properties in initially toxin-negative
isolates of Cl. difficile can occur through horizontal
gene transfer, [4], [23].
The species composition of the genus
Clostridium, which provokes diseases in livestock
farms, has not been studied enough, especially in
the Russian Federation. There are reports of bovine
clostridiosis being caused by seven species of the
genus Clostridium with a predominance of Cl.
perfringens, Cl. septicum, Cl. sporogenes, [24],
[25]. It is necessary to know causative species to
improve the effectiveness of preventive measures
against clostridial infections in cattle. Currently,
several methods of laboratory diagnostics of
Clostridium spp. are used: culture, enzyme
immunoassay, immunochromatographic analysis,
and polymerase chain reaction, [26], [27]. Only one
commercial PCR kit for the detection of Cl.
perfringens is available in the Russian Federation.
Yet, several commercial kits allow simultaneous
detection of toxinotypes: BactoReal® Kit
Clostridium perfringens (Austria), and
RIDA®GENE Clostridium difficile (Germany).
The development of new extended assays that
allow genotyping of clostridial pathogens is an
important task for the development of an effective
strategy for the elimination and prevention of these
dangerous, often incurable diseases, [4], [11].
The purpose of this study was to improve
laboratory diagnosis for the detection and
identification of clostridium infections in cattle. To
do that monitoring for the spread of Cl. difficile and
Cl. perfringens toxinotypes on farms of the Ural
region was conducted.
2 Materials and Methods
2.1 Ethical Approval
The institutional ethics committee of the Federal
State Budgetary Scientific Institution “Ural Federal
Agrarian Scientific Research Centre, Ural Branch
of Russian Academy of Sciences” approved this
study with protocol number: 515.
2.2 Sample Collection, Isolation and
Identification
Overall, in 2023, 137 samples of biological
material from cows and calves (Bos taurus) from
21 agricultural organizations of the Ural region
were studied. Collected biomaterial included: feces,
milk, and swabs from the wounded hooves.
Samples of pathological material from dead calves
and cows included: the heart, liver, kidneys, spleen,
lungs, rumen, abomasum, and reticulum.
The Diatom DNA Prep 200 kit (IsoGen LLC,
Moscow) was used to extract DNA from the
biomaterial. The HiPure Stool DNA Kit
(Guangzhou Magen Biotechnology Co., Ltd
(Magen), China) was used to obtain high-quality
DNA from feces. Assay kits "RealBest-Vet DNA
Cl.difficile/Cl.perfringens" were used to detect
clostridial infection in the biomaterial. RealBest-
Vet DNA Cl.difficile tcdA/tcdB/CDT kit (JSC
Vector-Best, Moscow) was used for typing Cl.
difficile. Amplification with real-time detection was
performed using QuantStudio 5 (USA). MALDI-
TOF mass spectrometry verification of the results
was conducted on a VITEK MS analyzer
(bioMerieux SA, France) in the laboratory of
Quality Med LLC (Ekaterinburg).
2.3 Molecular Identification of etx, iap, plc,
cpe, and cpb Genes by PCR
Toxinotypes Cl. perfringens was determined by
PCR based on the presence of the etx, iap, plc, cpe
and cpb genes. Genotyping was carried out
according to the protocol proposed by Julian I.
Rood, [11], Primers’ sequence are shown in Table
1. 10 µl reaction mix included: SibEnzyme SE-
Buffer (60 mM Tris-HCl (pH 8.6), 25 mM KCl, 10
mM 2-mercaptoethanol, 0.1% Trion X-100), 0.18
WSEAS TRANSACTIONS on BIOLOGY and BIOMEDICINE
DOI: 10.37394/23208.2023.20.31
Natalia A. Bezborodova, Evgenia N. Shilova,
Veronika V. Kozhukhovskaya, Vladlena D. Zubareva,
Olga V. Sokolova, Nikolai A. Martynov
E-ISSN: 2224-2902
306
Volume 20, 2023
mM of each dNTP , 1.16 mM MgCl2, 0.06 units of
Taq-polymerase (LLC SibEnzyme, Russia); 15-40
ng of DNA, 0.35 µM of each primer (LLC DNA-
Synthesis, Russia).
Table 1. Primers’ sequence for etx, iap, plc, cpe and cpb genes detection
Gene
Primers’ sequence (5’-3’)
Amplicon
etx
ETX_F-CCACTTACTTGTCCTACTAAC
656 b.p.
ETX_R-GCGGTGATATCCATCTATTC
iap
IAP_F-GGAAAAGAAAATTATAGTGATTGG
461 b.p.
IAP_R-CCTGCATAACCTGGAATGGC
plc
PLC_F-GGACCAGCAGTTGTAGATA
324 b.p.
PLC_R-CCTCTGATACATCGTGTAAG
cpe
CPE_F-GGAGATGGTTGGATATTAGG
233 b.p.
CPE_R-GGACCAGCAGTTGTAGATA
cpb
CPB_F-GCGAATATGCTGAATCATCTA
196 b.p.
CPB_R-GCAGGAACATTAGTATATCTTC
Amplification protocol: preliminary
denaturation at 95 °С – 5 minutes; further 35
cycles: denaturation at 94°C – 30 seconds, primer
annealing at 60°C – 30 seconds, elongation at 72°C
– 30 seconds, final elongation at 72°C – 10
minutes. The result of amplification was evaluated
by electrophoresis in 3% agarose gel. Clostridium
perfringens ATCC 13124 type A (BD Microtrol,
USA) was used as a positive control.
Statistical data processing was carried out using
Microsoft Office Excel 2019.
3 Results
PCR results showed that Clostridium DNA was
found in 40.6% out of 137 samples. Cl. difficile
was detected in 48 samples (35.8%). Cl.
perfringens was found in 34 samples (25.3%). The
simultaneous presence of Cl. difficile and Cl.
perfringens was observed in 16.4% of samples. Cl.
difficile and Cl. perfringens were found both in
feces (30.5%) and in pathological material from
dead calves and cows (8.7%), as well as in milk
samples (1.4%) (Figure 1).
Fig. 1: Clostridium is detected from biological
material from cows.Electrophoresis was used for
the detection of the etx, iap, plc, cpe, and cpb genes
in Cl. perfringens (Figure 2).
Fig. 2: Results of amplicon electrophoresis for
genotyping Cl. perfringens toxinotype A.
Designations: plc gene (324 b.p.); etx, iap, cpe, cpb
are Cl. perfringens toxin genes; M is a size
standard with a step of 100 b.p.
Cl. perfringens with the plc gene is responsible
for the production of ɑ-toxin. It was detected by
PCR in 6 fecal samples from cows and calves that
had signs of diarrhea. Also, plc, etx, cpe genes were
detected in 3 fecal samples from calves. These
genes are responsible for the production of ɑ, ε-
toxins and enterotoxin, respectively. Those isolates
belong to toxinotype D. Cl. perfringens toxinotype
С (plc, cpb) was detected in some fecal samples.
Cl. perfringens toxinotype A was detected in
parenchymal organs of the dead calf.
Cl. difficile was toxigenic in 56.2% of isolates.
The genes of binary toxin (CDT) were found most
often – in 70.0% of isolates, genes of toxin B – in
51.8% and toxin A – in 48.1% of isolates. Several
Cl. difficile toxinotypes were detected
simultaneously: toxins A + B + CDT in 18.5%
isolates, A + CDT in 14.8% isolates, and few
isolates had A + B and B + CDT combination.
Also, non-toxigenic Cl. difficile was found in
43.7% of samples.
WSEAS TRANSACTIONS on BIOLOGY and BIOMEDICINE
DOI: 10.37394/23208.2023.20.31
Natalia A. Bezborodova, Evgenia N. Shilova,
Veronika V. Kozhukhovskaya, Vladlena D. Zubareva,
Olga V. Sokolova, Nikolai A. Martynov
E-ISSN: 2224-2902
307
Volume 20, 2023
Overall, toxigenic Cl. difficile (68.7% isolates)
was detected in calves and cows with diarrhea,
which could indicate the presence of an acute
intestinal infection in animals. It is known that Cl.
difficile is the leading cause of antibiotic-associated
diarrhea. The genes (TcdA, TcdB, CDI) responsible
for the production of toxins are the main virulence
factors, [28]. Toxins secreted by Cl. difficile
damage the intestinal epithelium in young animals,
which leads to inflammation, tissue damage,
production of pro-inflammatory cytokines in the
macroorganism, and the development of the
disease, [29].
Cl. difficile was present in 20.8% samples of
pathological material from cows and calves. At the
same time, Cl. difficile was always present in
samples with several types of microorganisms
(Escherichia coli, Salmonella enterica, Cl.
perfringens), which may also indicate a septic
process (Figure 3, and Figure 4).
Fig. 3: Sample preparation of pathological material
of calves with suspected clostridial infection (1 –
kidneys, 2 – liver).
Fig. 4: Sample preparation of pathological material
from cows (1 – lung, 2 – spleen, 3 – lymphatic
nodes), with suspected mixed bacterial infection (E.
coli, S. enterica, Cl. perfringens, Cl. difficile).
Cl. difficile (31.2%), and Cl. perfringens
(12.5%) were found with the simultaneous presence
of Staphylococcus spp., E. coli, Streptococcus
agalactiae and Staphylococcus aureus in 16 milk
samples from cows with signs of mastitis. In a few
samples, toxinotypes of Cl. difficile A, B, CDT
were detected and the rest of the anaerobes were
toxin-negative.
2
1
1
2
3
WSEAS TRANSACTIONS on BIOLOGY and BIOMEDICINE
DOI: 10.37394/23208.2023.20.31
Natalia A. Bezborodova, Evgenia N. Shilova,
Veronika V. Kozhukhovskaya, Vladlena D. Zubareva,
Olga V. Sokolova, Nikolai A. Martynov
E-ISSN: 2224-2902
308
Volume 20, 2023
4 Discussion
The species composition of bacteria of the genus
Clostridium, which causes pathology in cattle in the
livestock farms of the Russian Federation, has not
been studied enough. Determination of toxins
produced by a particular strain or isolate (Cl.
perfringens, Cl. difficile) is important for predicting
the course of the disease. In addition, the typing of
bacteria of the Clostridium genus is an urgent task
for a deeper understanding of the epizootic process
and should be taken into account when developing
vaccine-preventive measures in agricultural
enterprises, [30].
The most common pathogenic agent affecting
humans and animals is Cl. perfringens. Many years
of experience of cultivating anaerobes has shown
that isolation of Clostridium by microbiological
methods is very difficult, since they require strict
anaerobic conditions. Moreover, samples are often
contaminated with a mixed bacterial flora, which
complicates the diagnosis. The standard Cl.
perfringens toxinotype identification by toxins
neutralization with specific sera is very laborious,
expensive, and time-consuming. Before
biochemical identification, it is important to have a
pure culture of the strain, which is an arduous
process, [31]. PCR has expanded the possibilities of
studying clostridial infections in animals with its
exceptional sensitivity, specificity, and fast
laboratory analysis, [31]. An important advantage
of the PCR method is bypassing the stage of
cultivation. Real-time PCR studies have opened up
a wide range of possibilities in the field of
quantitative analysis of bacterial nucleic acids.
Currently, only one commercial PCR test system is
available in the Russian Federation, which detects
the DNA of Cl. perfringens without determining
the genes responsible for the production of toxins.
Therefore, the development of a test system for
the identification of various types of Cl.
perfringens by molecular genetic methods is very
promising. However, in the course of work on the
test system, a number of problems arose when
working with such biological material as animal
feces. Those samples contain many foreign
microorganisms and substances that inhibit the
PCR reaction (calcium salts and phosphates, bile
salts, bilirubins, polysaccharides, undigested plant
fibers, mucus, and insoluble products of the
gastrointestinal tract), and therefore obtaining pure
and high-quality DNA is difficult, [32]. It is also
worth noting that some genes encoding toxins are
localized on plasmids, which are not permanent
inclusions in the cell, and may be lost in the course
of obtaining a pure culture, leading to false
negative results. Therefore, to obtain high-quality
DNA for our work, we used a specialized HiPure
Stool DNA Kit (Guangzhou Magen Biotechnology
Co., Ltd (Magen), China) designed to isolate
nucleic acids from fecal samples. Current test
system requires electrophoretic visualization of
obtained results and has its drawbacks. They
include high probability of contamination,
increased requirements for the PCR laboratory, and
work with ethidium bromide. To overcome the
abovementioned drawbacks we will improve this
test system by developing real-time PCR detection
of Cl. perfringens toxins. Novel test systems will
be created to identify significant bacteria of the
Clostridium genus (Cl. perfringens, Cl. difficile, Cl.
chauvoei, Cl. histolyticum, Cl. sordellii, Cl.
septicum, Cl. novyi, Cl. tetani, Cl. botulinum) and
detect antibiotic resistance genes. Using the
developed test systems, an analysis of the species
diversity of clinical isolates of Clostridium
circulating in dairy farms will be carried out.
Preventive measures for the clostridial infections in
dairy herds will be improved taking into account
conducted studies. The obtained data will be used
to develop appropriate schemes of vaccination and
effective therapy against this dangerous anaerobic
infection in agricultural organizations.
5 Conclusion
Out of 137 samples of obtained biological material
Clostridium was detected in 40.6% of samples: Cl.
difficile in 35.8%, Cl. perfringens in 25.3%, Cl.
difficile+Cl. perfringens in 16.4%. Cl. difficile and
Cl. perfringens were found in 30.5% of fecal
samples, in pathological material from dead calves
and cows – 8.7%, in milk samples – 1.4%.
The complex diagnostics of individual cases of
clostridiosis in cattle in agricultural organizations
of the Ural region was conducted. Epizootic data,
clinical signs, and characteristic pathological
changes were taken into account. Utilized
laboratory methods made it possible to verify
infectious anaerobic enterotoxemia in one case,
necrotic enteritis in 3 animals, and intestinal
toxicoinfection caused by Cl.perfringens type A in
2 cows and 5 calves.
Conducted PCR studies showed the presence of
various toxinotypes of Cl. difficile and Cl.
perfringens. In some samples, a mixed infection
was identified, which included aerobic and
anaerobic bacteria. The diagnostics of toxinotypes
of Cl. perfringens have made it possible to conduct
toxin-specific vaccination against clostridial
infection in farms. For example, farms were
WSEAS TRANSACTIONS on BIOLOGY and BIOMEDICINE
DOI: 10.37394/23208.2023.20.31
Natalia A. Bezborodova, Evgenia N. Shilova,
Veronika V. Kozhukhovskaya, Vladlena D. Zubareva,
Olga V. Sokolova, Nikolai A. Martynov
E-ISSN: 2224-2902
309
Volume 20, 2023
provided with recommendations for changing the
type of vaccine according to diagnostic results.
List of Abbreviations:
PCR, Polymerase chain reaction; MALDI-TOF
MS, Matrix-assisted laser desorption ionization
time-of-flight mass spectrometry; CDT, Cytolethal
distending toxin; DNA, Deoxyribonucleic acid.
Acknowledgments:
The study was carried out within the framework of
project No. 23-26-00053 “Development of test
systems for molecular genetic diagnostics of
Clostridia with identification of toxinotypes and
antibiotic resistance genes” with the financial
support of the Russian Science Foundation.
References:
[1] Rahman M.T., Sobur M.A., Islam M.S., Ievy
S., Hossain M.J., El Zowalaty M.E., Rahman
A.T., Ashour H.M. Zoonotic diseases:
etiology, impact, and control.
Microorganisms, Vol. 8 No. 9, 2020, 1405;
https://doi.org/10.3390/microorganisms80914
05.
[2] Geier R.R., Rehberger T.G., Smith A.H.
Comparative genomics of Clostridium
perfringens reveals patterns of host-associated
phylogenetic clades and virulence factors.
Front Microbiol, Vol. 12, 2021, 649953.
http://doi.org/10.3389/fmicb.2021.649953
[3] Xiu L., Liu Y., Wu W., Chen S., Zhong Z.,
Wang H. Prevalence and multilocus sequence
typing of Clostridium perfringens isolated
from 4 duck farms in Shandong province,
China. Poult Sci, Vol. 99, No. 10, 2020, pp.
5105–5117;
http://doi.org/10.1016/j.psj.2020.06.046
[4] Fourie J.C.J., Bezuidenhout C.C., Sanko T.J.,
Mienie C., Adeleke R. Inside environmental
Clostridium perfringens genomes: antibiotic
resistance genes, virulence factors and
genomic features. J Water Health, Vol. 18, No
4, 2020, pp. 477–493.
http://doi.org/10.2166/wh.202 0.029.
[5] Hoelzer K., Bielke L., Blake D.P., Cox E.,
Cutting S.M., Devriendt B., Erlacher-Vindel
E., Goossens E., Karaca K., Lemiere S.,
Metzner M., Raicek M., Collell Surinach M.,
Wong N.M,. Gay C., Van Immerseel F.,
Vaccines as alternatives to antibiotics for food
producing animals. Part 1: challenges and
needs. Vet Res, Vol. 49, No 1, 2018, pp. 64;
http://doi.org/10.1186/s13567-018-0560-8
[6] Lobzin Yu.V., Kvetnaya A.S., Skripchenko
N.V., Zhelezova L.I., Current notions about
etiopathogenic and genetics specific features
of Сlostridium perfringens toxins. Journal of
microbiology, epidemiology and
immunobiology; Vol. 98, 2021, pp. 91–103.
http://doi.org/10.36233/0372-9311-37
[7] Hu W.S., Woo D.U., Kang Y.J., Koo O.K.,
Biofilm and spore formation of Clostridium
perfringens and its resistance to disinfectant
and oxidative stress. Antibiotics (Basel), Vol.
10, No 4, 2021, pp. 396; http://doi.org/10
.3390/antibiotics10040396.
[8] Gohari I.M., Navarro M.A., Li J., Shrestha A.,
Uzal F., McClane B.A., Pathogenicity and
virulence of Clostridium perfringens.
Virulence Vol. 12, No 1, 2021, pp. 723–753.
http://doi.org/10.1080/21505594.2021.188677
7.
[9] Simpson K.M., Callan R.J., Van Metre D.C.,
Clostridial abomasitis and enteritis in
ruminants. Vet Clin North Am Food Anim
Pract, Vol. 34 No 1, 2018, pp. 155–184;
http://doi.org/10.1016/j.cvfa.2017.10.010.
[10] Uzal F.A., Navarro M.A., Li J., Freedman
J.C., Shrestha A., McClane B.A., Comparative
pathogenesis of enteric clostridial infections in
humans and animals. Anaerobe, Vol. 53,
2018, pp. 11–20. http://doi.org/10.1016/j.ana
erobe.2018. 06.002.
[11] Rood J.I., Adams V., Lacey J., Lyras D,
McClane BA, Melville SB, Moore RJ, Popoff
MR, Sarker MR, Songer JG, Uzal FA, Van
Immerseel F. Expansion of the Clostridium
perfringens toxin-based typing scheme.
Anaerobe Vol. 53, 2018, pp. 5–10;
http://doi.org/10.1016/j.anaerobe.2018.04.011.
[12] Verherstraeten S., Goossens E., Valgaeren B.,
Pardon B., Timbermont L., Haesebrouck F.,
Ducatelle R., Deprez P., Van Immerseel F.,
Non-toxic perfringolysin O and α-toxin
derivatives as potential vaccine candidates
against bovine necrohaemorrhagic enteritis.
Vet. J., Vol. 217, 2016, pp. 89–94;
https://doi.org/10.1016/j.tvjl.2016.09.008.
[13] Kapustin A.V., Laishevtcev A.I., Ivanov E.V.,
Danilyuk A.V., Species diversity of Clostridia
causing malignant edema in cattle. IOP
Conference Series: Earth and Environmental
Science Vol. 548 (7), 2020, 072041;
http://doi.org/10.1088/1755-1315/548/7/072
041.
WSEAS TRANSACTIONS on BIOLOGY and BIOMEDICINE
DOI: 10.37394/23208.2023.20.31
Natalia A. Bezborodova, Evgenia N. Shilova,
Veronika V. Kozhukhovskaya, Vladlena D. Zubareva,
Olga V. Sokolova, Nikolai A. Martynov
E-ISSN: 2224-2902
310
Volume 20, 2023
[14] Posthaus H., Kittl S., Tarek B., Bruggisser J.,
Clostridium perfringens type C necrotic
enteritis in pigs: diagnosis, pathogenesis, and
prevention. J Vet Diagn Invest, Vol. 32, No 2,
2020, pp. 203–212;
https://doi.org/10.1177/1040638719900180.
[15] Wagley S., Bokori-Brown M., Morcrette H.,
Malaspina A., D'Arcy C., Gnanapavan S.,
Lewis N., Popoff M.R., Raciborska D.,
Nicholas R., Turner B., Titball R.W.,
Evidence of Clostridium perfringens epsilon
toxin associated with multiple sclerosis. Mult
Scler, Vol. 25, No 5, 2019, pp. 653–660;
http://doi.org/10.1177/1352458518767327.
[16] Hussain R., Guangbin Z., Abbas R.Z.,
Siddique AB, Mohiuddin M, Khan I, Rehman
TU, Khan A. Clostridium perfringens types A
and D involved in peracute deaths in goats
kept in Cholistan ecosystem during winter
season. Front Vet Sci, Vol. 9, 2022, 849856;
http://doi.org/10.3389/fvets.2022.849856.
[17] Moustafa S., Zakaria I., Moustafa A.,
AboSakaya R., Selim A., Bacteriological and
serological investigation of Clostridium
perfringens in lambs. Sci Rep, Vol. 12 No 1,
2022, pp.19715
http://doi.org/10.1038/s41598-022-21918-6
[18] Goossens E., Valgaeren B.R., Pardon B.,
Haesebrouck F., Ducatelle R., Deprez P.R.,
Van Immerseel F., Rethinking the role of
alpha toxin in Clostridium perfringens-
associated enteric diseases: a review on
bovine necro-haemorrhagic enteritis. Vet Res,
Vol. 48, No 1, 2017, pp. 9.
http://doi.org/10.1186/s13567-017-0413-x.
[19] Sholeh M., Krutova M., Forouzesh M.,
Mironov S., Sadeghifard N., Molaeipour L.,
Maleki A., Kouhsari E., Antimicrobial
resistance in Clostridioides (Clostridium)
difficile derived from humans: a systematic
review and meta-analysis. Antimicrob Resist
Infect Control, Vol. 9, No 1, 2020, pp.158.
http://doi.org/10.1186/s13756-020-00815-5.
[20] Kachrimanidou M., Tzika E., Filioussis G.
Clostridioides (Clostridium) Difficile in food-
producing animals, horses and household pets:
a comprehensive review. Microorganisms,
Vol. 7, No 12,n 2019, pp. 667.
http://doi.org/10.3390/microorganisms712066
7.
[21] Venhorst J., van der Vossen J., Agamennone
V. Battling enteropathogenic clostridia: phage
therapy for Clostridioides difficile and
Clostridium perfringens. Front Microbiol,
Vol. 13, 2022, 891790. http://doi.org/10.3389
/fmicb.2022.891790
[22] Knight D.R., Riley T.V., Genomic delineation
of zoonotic origins of Clostridium difficile.
Front Public Health, Vol. 7, 2019, pp. 164.
http://doi.org/10.3389/fpubh.2019.00164
[23] Glotova T., Koteneva S.V., Nefedchenko A.,
Glotov A. Etiological agents causing
reproduction pathology in cows in big dairy
farms. Veterinary medicine journal 3–8, 2023.
https://doi.org/10.30896/0042-
4846.2023.26.2.03-08.
[24] Danilyuk A.V., Kapustin A.V. The prevalence
and species diversity of clostridia, the
causative agents of anaerobic infections in
cattle. Trudi VIEV, Vol. 81, 2019, pp. 19–26.
http://doi.org/10.30917/ATT-PRINT-2019-10
[25] Zaragoza N.E., Orellana C.A., Moonen G.A.,
Moutafis G., Marcellin E. Vaccine production
to protect animals against pathogenic
Clostridia. Toxins, Vol. 11, No 9, 2019, 525;
https://doi.org/10.3390/toxins11090525.
[26] Lu W., Sun H., Xu Z.-M., Du Z., Si L., Yuan
S., Jin J., Jin C.-H. Diagnostic and therapeutic
strategy for Clostridium perfringens infection
in postpartum dairy cows: a report of 14 cases,
Journal of Applied Animal Research, Vol. 50,
No 1, 2022, pp. 350-354;
https://doi.org/10.1080/09712119.2022.20783
29.
[27] Carter G.P., Chakravorty A., Pham Nguyen
T.A., Mileto S., Schreiber F., Li L., Howarth
P., Clare S., Cunningham B., Sambol S.P.,
Cheknis A., Figueroa I., Johnson S., Gerding
D., Rood J.I., Dougan G., Lawley T.D., Lyras
D. Defining the roles of TcdA and TcdB in
localized gastrointestinal disease, systemic
organ damage, and the host response during
Clostridium difficile infections. mBio, Vol. 6,
No 3, 2015, e00551;
http://doi.org/10.1128/mBio.00551-15.
[28] Chandrasekaran R., Lacy D.B. The role of
toxins in Clostridium difficile infection. FEMS
Microbiol Rev, Vol. 41, No 6, 2017, pp. 723–
750. http://doi.org/10.1093/femsre/fux048.
[29] Amini M., Kheirkhah B., Shamsaddini Bafti
M., Rokhbakhsh Zamin F. Molecular
identification and sequence analysis of
Clostridium perfringens virulence genes
isolated from sheep and goats. Journal of the
Hellenic Veterinary Medical Society, Vol. 74
No 1, 2023, pp. 5345–5354;
https://doi.org/10.12681/jhvms.29434.
[30] Ahmed H.A., El Bayomi R.M., Hamed R.I.,
Mohsen R.A., El-Gohary F.A., Hefny A.A.,
WSEAS TRANSACTIONS on BIOLOGY and BIOMEDICINE
DOI: 10.37394/23208.2023.20.31
Natalia A. Bezborodova, Evgenia N. Shilova,
Veronika V. Kozhukhovskaya, Vladlena D. Zubareva,
Olga V. Sokolova, Nikolai A. Martynov
E-ISSN: 2224-2902
311
Volume 20, 2023
Elkhawaga E., Tolba H.M.N. Genetic
Relatedness, antibiotic resistance, and effect
of silver nanoparticle on biofilm formation
by Clostridium perfringens isolated from
chickens, pigeons, camels, and human
consumers, Veterinary sciences, Vol. 9, No 3,
2022, pp. 109.
https://doi.org/10.3390/vetsci9030109.
[31] Blau K., Gallert C. Prevalence, antimicrobial
resistance and toxin-encoding genes of
Clostridioides difficile from environmental
sources contaminated by feces. Antibiotics,
Vol. 12, No 1, 2023, pp. 162.
https://doi.org/10.3390/antibiotics12010162.
Contribution of Individual Authors to the
Creation of a Scientific Article (Ghostwriting
Policy)
- N.A. Bezborodova designed the study, conducted
PCR, interpreted the data and drafted the
manuscript.
- E. N. Shilova contributed in study design and
critical checking of manuscript.
- V.V. Kozhukhovskaya was involved in DNA
extraction and conducting of PCR.
- V.D. Zubareva contributed in preparing and
critical checking of this manuscript.
- O.V. Sokolova contributed in sample collection
and critical checking of this manuscript.
- N.A. Martynov helped with PCR protocol
optimization.
Sources of Funding for Research Presented in a
Scientific Article or Scientific Article Itself
No funding was received for conducting this study.
Conflict of Interest
The authors have no conflicts of interest to declare.
Creative Commons Attribution License 4.0
(Attribution 4.0 International, CC BY 4.0)
This article is published under the terms of the
Creative Commons Attribution License 4.0
https://creativecommons.org/licenses/by/4.0/deed.e
n_US
WSEAS TRANSACTIONS on BIOLOGY and BIOMEDICINE
DOI: 10.37394/23208.2023.20.31
Natalia A. Bezborodova, Evgenia N. Shilova,
Veronika V. Kozhukhovskaya, Vladlena D. Zubareva,
Olga V. Sokolova, Nikolai A. Martynov
E-ISSN: 2224-2902
312
Volume 20, 2023
