https://doi.org/10.52973/rcfcv-e33280
Received: 07/06/2023 Accepted: 04/07/2023 Published: 28/07/2023
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Revista Científica, FCV-LUZ / Vol. XXXIII, rcfcv-e33280, 1 – 5
ABSTRACT
The present study aimed to detect Yersinia ruckeri bacteria in sh
samples collected from the rainbow trout (Oncorhynchus mykiss,
Walbaum, 1792) farms in the South Eastern Anatolia Region of
Turkey through bacteriological culturing and polymerase chain
reaction. Separate visits were made to trout farming facilities in
Diyarbakır, Adıyaman, Şanlıurfa, and Batman in January and February
2021, and 30sh with an average live weight of 200–250 g were
collected from each facility. A total of 1,200 samples were taken
from 40 trout farms. Liver, spleen, kidney, and tissue specimens
were collected from rainbow trout. Yersinia ruckeri was isolated
from the kidney, liver and muscle tissue of sh on McConkey Agar
for bacteriological examinations. Isolated strains were identied
by MALDI–TOF and PCR. It was detected the bacteria in the liver,
kidney, and muscle tissue samples of sh from 23 out of 40 farms. In
conclusion, these bacteria were detected in local farms. These farms
must implement vaccination, minimize stress factors affecting the
sh, and avoid overstocking. The best methods to protect against
infection include avoiding overstocking, low oxygen levels, and
underqualied labor force.
Key words: MALDI–TOF; Oncorhynchus mykiss; PCR; Yersinia ruckeri
RESUMEN
El presente estudio tuvo como objetivo detectar la bacteria Yersinia
ruckeri en muestras de pescado recolectadas de las granjas de
trucha arco iris (Oncorhynchus mykiss, Walbaum, 1792) en la región
de Anatolia del sudeste de Turquía a través del cultivo bacteriológico y
la reacción en cadena de la polimerasa. Además, también tenía como
objetivo determinar la susceptibilidad antibiótica de la bacteria. Se
realizaron visitas separadas a las instalaciones de cultivo de truchas
en Diyarbakır, Adıyaman, Şanlıurfa y Batman en enero y febrero de
2021, y se recolectaron 30 peces con un peso vivo promedio de 200
a 250 g en cada instalación. Se tomaron un total de 1.200 muestras
de 40 criaderos de truchas. Se recolectaron especímenes de hígado,
bazo, riñón y tejido de trucha arcoíris. Se aisló Yersinia ruckeri del
riñón, el hígado y el tejido de pescado en Agar McConkey para realizar
exámenes bacteriológicos. Las cepas aisladas fueron identicadas
por MALDI–TOF y PCR. Se detectó el agente bacteriano en muestras
de hígado, riñón y músculo tejido de peces en 23 de 40 granjas. En
conclusión, estas bacterias fueron detectadas en granjas locales.
Estas granjas deben implementar la vacunación, minimizar los
factores de estrés que afectan a los peces y evitar el exceso de la
sobre población. Los mejores métodos para protegerse contra la
infección incluyen evitar la alta densidad los bajos niveles de oxígeno
y la mano de obra poco calicada.
Palabras clave: MALDI–TOF; Oncorhynchus mykiss; PCR; Yersinia
ruckeri
Detection of Yersinia ruckeri in naturally infected rainbow trout
(Oncorhynchus mykiss Walbaum, 1792) from trout farms in Anatolia, Turkey
Detección de Yersinia ruckeri en truchas arcoíris (Oncorhynchus mykiss Walbaum, 1792) infectadas
naturalmente en criaderos de truchas en Anatolia, Turquía
Filiz Özcan
1
* , Neval Berrin Arserim
2
, Muzaffer Aydın Ketani
3
, Kasım Aydin
4
, Nurdan Karacan–Sever
5
, Nida Özcan
6
, Burçak Aslan–Çelik
7
1
Dicle University,Veterinary Faculty, Fisheries and Fisheries Diseases Department,Diyarbakır, Turkey.
2
Dicle University, Veterinary Faculty, Department of Microbiology, Diyarbakır, Turkey.
3
Dicle University, Veterinary Faculty, Department of Histology and Embryology, Diyarbakır, Turkey.
4
Metropolitan Municipality of Diyarbakır, Diyarbakır, Turkey.
5
Dicle University, Veterinary Faculty, Department of Microbiology, Diyarbakır, Turkey.
6
Dicle University Medical Faculty, Department of Medical Microbiology, Diyarbakır, Turkey.
7
Siirt University,Veterinary Faculty, Department of Parasitology, Siirt, Turkey.
*Corresponding Author: felizozcan@gmail.com
FIGURE 2. A: Colony morphology of Yersinia ruckeri. B: Spectrum images of MALDI–
TOF Yersinia ruckeri
A
B
Yersinia ruckeri in Rainbow Trout from Turkish Trout Farms / Özcan et al. __________________________________________________________
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INTRODUCTION
Currently, one of the greatest challenges faced by the developing
World is nutrition from a balanced, healthy, and adequate diet. A
major cause for this challenge is the population boom [1]. Feeding the
rapidly increasing World population is contingent upon increasing the
agricultural output and ensuring its equitable distribution. Considering
the prevalent rise in population and aggravated nutritional problems,
these challenges are expected to persist in the future as well [2].
Therefore, the demand for seafood, a rich source of protein, increases
by the day. It is necessary to ensure the continuity of production by
protecting the scarce resources to benet from the shery stocks in
an economic and sustainable way [3]. Accordingly, intensive efforts
are in place to progressively increase the current shery output
through aquaculture production [4]. The shery output in Turkey was
785,811 tons in 2020, including the aquaculture production of 421,411
tons where shing accounted for 364,400 tons [5]. Aquaculture
production increased by 12.9% in 2020. Trout was the most important
fish species with an output of 127,905 tons [5] Rainbow trout
(Oncorhynchus mykiss) is one of the most widespread cold water sh
species farmed throughout the World. Production of rainbow trout
in Turkey accounts for approximately 50% of the total aquaculture
production [6] Infectious diseases and associated economic losses
are the leading factors that have an adverse effect on aquaculture.
Karabulut and Kurtoğlu [7], suggested that feed costs and diseases
were the primary development associated with rainbow trout farming.
The disease–associated annual economic losses increased with the
rapid expansion of aquaculture and increased sh production levels,
accurating for billions of dollars Worldwide [8]. Bacteria, fungi, viruses,
and parasites were reported as the cause of the most prominent diseases
jeopardizing the aquaculture industry [9] There are a number of agents
associated with diseases in aquatic organisms.
Microorganisms typically occurring in the normal microora of the
aquatic environment and sh can be transmitted by broodstock sh to
the roes as well as to sh during each stage of production. Vaccines,
immunostimulants, prebiotics and probiotics have been shown to
confer some protection against Y. ruckeri [10]. But the immune system
is developed only in trout sh with ≥2 g of body weight; therefore,
sucient protection cannot be provided to smaller sh by means of
vaccination. Accordingly, intensive antimicrobial use is inevitable for
controlling the diseases [11].
Studies have demonstrated that aquatic and sh microora have
similar characteristics and that many bacteria that induce diseases
in sh are present in aquatic resources. In particular, the causative
factors associated with Y. ruckeri, which induces the systemic disease
enteric red mouth disease, also known as yersiniosis in trout [12],
ranked as the top disease–causing factor in rainbow trout farms.
Yersiniosis was rst detected in the Hagerman Valley in Idaho
State, United States, during the early 1950s; it has now proliferated
in other Countries located in distant geographical Regions across
Europe, South America, West Africa, and Australia, affecting trout
farms almost all over the World [1]. Yersiniosis is associated with
fry mortality in trout production. Mortality due to yersiniosis was
reported in the rainbow trout species that generally populate fresh
water and farmed in cages in the Black Sea region of Turkey [13]. In
aquaculture, the acute epizootic infected formations are associated
with a mortality rate of up to 70% [14], for yersiniosis, which occurs
as a result of stress in sh farming and follows an acute or chronic
course. The disease presents with symptoms such as darkening of
the skin, hemorrhage at the inner and outer mouth, operculum, outer
surface of the body and base of the ns, abdominal bloating, uid
accumulation, and unilateral or bilateral exophthalmos. Furthermore,
lysis around anus, ns, and skin and hemorrhages in the orbit and iris
may also occur. Hemorrhage in the internal organs and a yellowish
uid accumulated inside the abdominal cavity and stomach may be
observed on postmortem examination [15].
MATERIAL AND METHODS
Trout farming facilities in Diyarbakır, Adıyaman, Şanlıurfa, and
Batman Provinces were contacted and visited separately during
January and February, 2021. All cages and ponds were individually
examined and 30 sh showing signs of disease with an average live
weight of 200–250 g were collected from each facility. All collected
sh were insensitive to feed, immobile, oating on the water surface,
and showing signs of disease such as color darkening. Overall,
40 trout farms were visited, and 1,200 samples were collected,
which were transferred to Dicle University, Faculty of Veterinary
Medicine, Department of Fisheries and Diseases Laboratory, on
ice. There, the samples were rst macroscopically examined and
then dissected to examine the internal organs. The liver, kidneys,
and muscle tissues were collected from the dissected samples and
inoculated to bacteriological culture media to detect the presence of
bacteria. Blood agar (BA) and McConkey agar (McA) plates were used
for bacterial isolation by incubation for 24 h at 28°C (NÜVE–NC40M,
Turkey) The dominant uniform bacterial colonies were puried by
streaking onto the BA and McA plates three times (FIG. 1). Then, the
causative agents were identied using MALDI–TOF mass spectrometry
by MALDI Biotyper (Bruker, Daltonics, Germany) based on Gram staining
and culture growth proles (FIG. 1).
A B
C D
FIGURE 2. Postmortem examination revealing bleeding in the internal organs; at the base of the ns; and swelling and uid accumulation in the abdomen (A); the
outer surface of the body (B); bleeding in the operculum (C); yellowish uid accumulated inside the abdominal cavity and stomach (D)
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The disease usually has an acute course and is associated with
high mortality rates. Therefore, there was a need for accurate and
rapid diagnostic methods to combat it. PCR is the most widely used
method for this purpose as it provides fast and accurate results [16].
Nyztech Y. ruckeri Real Time PCR Kit (Catalogue number: MD0317)
was used for identication.
RESULTS AND DISCUSSION
The present study investigated 1,200 samples and Y. ruckeri
was detected in the liver, kidneys, and tissues of the sh collected
from 23 out of the 40 farms. The trout farms in the SouthEastern
Anatolia Region were contaminated by Y. ruckeri at a rate of 0 –100%
and different levels of resistance to antimicrobial agents were
observed. Y. ruckeri is responsible for causing yersiniosis, one of
the most important bacterial diseases in both freshwater and marine
salmonids. Outbreak of this disease can lead to heavy economic losses
[17]. The course of the disease is characterized by acute, subacute,
and chronic forms. Although the disease spontaneously occurs
due to sudden changes in water temperatures, lysis was reported
at the base of the ns, around the anus, and inside the operculum
[18]. Hemorrhage can be observed on the skin and at other parts of
the body. Moreover, erythema and petechial hemorrhages may also
occur within the internal organs, peritoneum, body fat, gonads, and
mesenterium. Intestinal organs are typically erythemal and lled with
bloody mucus. Additionally, the spleen and kidneys are swollen and
the liver is pale. Furthermore, petechiae in the muscles have been
reported [1]. Most of these abovementioned symptoms were also
observed in the present study (FIG. 2).
Symptoms mostly observed in the study; bleeding in the operculum,
at the base of the ns; and swelling and uid accumulation in the
abdomen the outer surface of the body.
Consistent with the results of the current study, Kumar et al.
[18] reported exophthalmos, intestinal fluid accumulation, and
darkening skin color in their study on salmon in 2015. In another study,
Mohammed Saeed [19] reported clinical symptoms of diculty in
swimming; swimming closer to the water surface or near the ground;
hemorrhage in the gills, around the eyes, and in the mouth and oral
cavity; dark pigmentation; and petechial hemorrhages on the inner
surface; these are consistent with the clinical symptoms observed in
the current study. Most of the samples in this study had hemorrhages
on the skin and various parts of the body and lysis in the operculum
and ns.Y. ruckeri was detected in the tissue, liver, and kidney samples
of the sh. Yersiniosis is a disease characterized by the occurrence of
hemorrhagic zones in various tissues and organs, and general signs
of hemorrhagic septicemia are manifested in course of the disease
(FIG. 2). Yersiniosis due to Y. ruckeri is a disease that may induce high
mortality rates and heavy economic losses in rainbow trout farming
facilities [20]. This may be because adequate quarantine measures
were not in place in the farms with the infected sh. Furthermore,
the health specications issued during sh transportation lacked
adequate controls in terms of the carrier sh might have accounted for
the spread of the infection throughout the entire region. In addition,
the condition of sh scoops and negligence during feeding suggested
that the pools might not have been disinfected. The occurrence of the
disease was associated with organic pollution of the environment,
heat, dense stocking, direct contact, and oxygen deciency, all of
which contributed in the stress factor [21].
Yersinia ruckeri in Rainbow Trout from Turkish Trout Farms / Özcan et al. __________________________________________________________
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In this study, Y. ruckeri was isolated at a higher rate in the sh
farms with excessively turbid water, higher water temperature, and
overstocking. Tinsley et al. [22] reported that although the bacterial
agents could also be isolated in the normal microora of water, the
incidence of disease increased in the presence of stress–inducing
factors, including sudden seasonal changes in the water temperature,
hygienic negligence, overpopulated production areas, and lack of
clean barriers. Generally, bacterial infection occurs when sh are
under physiological stress and hygiene conditions are below adequate
levels. Avoiding overstocking, low oxygen, and underqualied labor
force were reported as the best ways to avoid infection [23]. A number
of similar reports indicated that Y. ruckeri had a wider host range and
geographic distribution and can cause both epizootic and zoonotic
diseases [17]. Therefore, further studies must be conducted in sh
farms. Moreover, it was understood that regular vaccination was in
place in certain farms.
CONCLUSIONS
Pathogens were detected in 23 farms. This is the rst research to
study yersiniosis in the SouthEastern Anatolia Region; the obtained
results contribute to scientic knowledge regarding this disease.
Although adequate care is essential for the treatment and control of
bacterial diseases in aquaculture, vaccination and immunostimulant
drugs can also be used. In general, diseases can be prevented and
controlled by good administration and management practices and
vaccination. Considering that clinical yerniosis is dened as a stress–
related disease, the severity of the disease should be mitigated
by reducing stress, controlling water quality, maintaining proper
nutrition, and implementing good health practices across the regions
where the disease is already prevalent. Appropriate care and feeding
conditions, improved water quality, reduced stock intensity, and the
removal of dead sh can reduce disease emergence and result in
faster treatment results.
ACKNOWLEDGEMENTS
This work was supported by the Ministry of Industry and Technology
Southeastern Anatolia Project Regional Development Administration
(Grant number 2019–E2536).
Conict of interest
The authors have no declaration of competing interests.
BIBLIOGRAPHIC REFERENCES
[1] Ummey S, Khan S, Vayakumar PPN, Ramya A. Enteric Red Mouth
disease and its causative bacterium, Yersinia ruckeri, in Indian
Major Carps from culture ponds in Andhra Pradesh, India. Aquacult.
Fisheries. [Internet]. 2021; 6:289–299. doi: https://doi.org/kj3x
[2] Wang D, Sun S, Li S, Lu T, Shi D. Transcriptome proling of
immune response to Yersinia ruckeri in spleen of rainbow trout
(Oncorhynchus mykiss) l. BMC Genomics. [Internet]. 2021; 22:292.
doi: https://doi.org/w9b2
[3] Cao G, Yu Z, Zheng L, Lei X, Chen D. Isolation and identication
of Yersinia ruckeri from hybrid sturgeon and pathological lesions
of its infection. J. South China Agricult. Univ. [Internet]. 2018;
39(4): 13–19. doi: https://doi.org/kj3z
[4] Mohammed–Saeed G. Causualty agent (yersiniosis) detection in
reared rainbow trout (Oncorhynchus mykiss) in Kohgiluyeh– VA–
boyerahmad province in Iran. Oceanogr. Fisheries Open AccessJ.
[Internet]. 2017; 4(2): 555–633. doi: https://doi.org/kj32
[5] Türkish Statistical Institute (TUİK). 2020; 60 p.
[6] Türkish Statistical Institute (TUİK). 2021; 63 p.
[7] Karabulut HA, Kurtoglu IZ. The Effect of Red Pepper to Add into
Commercial Trout Feed on Growth Performance and Meat Quality
of Rainbow Trout (Oncorhynchus mykiss). Fresenius Environm.
Bull. 2017; 26: 7473–7477.
[8] Wang D, Sun S, Li S, Lu T, Shi D. Transcriptome proling of
immune response to Yersinia ruckeri in spleen of rainbow trout
(Oncorhynchus mykiss) l. BMC Genomics. [Internet]. 2021; 22:292.
doi: https://doi.org/w9b2
[9] Vega–Heredia S, Mendoza–Cano F, Sanchez–Paz A. The infectious
hypodermal and haematopoietic necrosis virus: A brief review of
what we do and do not know. Transb. Emerging. Dis. [Internet].
2012; 59:95–105. doi: https://doi.org/ftkjr5
[10] Adel M, Dawood MA, Shafiei S, Sakhaie F, Shekarabi SPH.
Dietary Polygonum minus extract ameliorated the growth
performance, humoral immune parameters, immune–related
gene expression and resistance against Yersinia ruckeri in rainbow
trout (Oncorhynchus mykiss). Aquacult. [Internet]. 2020; 519:
734–738. doi: https://doi.org/gmn4hw
[11] Küçükköşker B. Üryani eriği (Prunus domestica) ekstraktinin
gökkuşaği alabaliğinda (Oncorhynchus mykiss) kizilağiz hastaliğina
(Yersinia ruckeri) karşi bağişiklik yaniti üzerine etkilerinin
belirlenmesi. [Master's Thesis]. Turkey: Kastamonu Üniversitesi;
2000. 53 p.
[12] Altinok I, Capkin E, Boran H. Comparison of molecular and
biochemical heterogeneity of Yersinia ruckeri strains isolated
from Turkey and the USA. Aquacult. [Internet]. 2016; 450: 80–88.
doi: https://doi.org/kj7v
[13] Karataş S, Candan A, Demircan D. Enteric redmouth disease in
cultured rainbowtrout (Oncorhynchus mykiss) on the black sea coast
of Turkey. Israeli J. Aquacult. Bamidgeh. 2004; 56 (3): 226–231.
[14] Timur G, Timur M. Balık Hastalıkları . 5th. ed. İstanbul Üniversitesi.
Su Ürünleri Fakültesi. 2003; 538 p.
[15] Sibinga N.A, Marquis H. Tissue–specic differences in detection
of Yersinia ruckeri carrier status in rainbow trout (Oncorhynchus
mykiss). J. Fish Dis. [Internet]. 2021; 44:2013–2021 doi: https://
doi.org/kj7x
[16] Gibello A, Blanco MM, Morena MA, Cutuli MT, Domenench A,
Dominguez L, Fernandez–Garayzabal JF. Development of a PCR
assay for detection of Yersinia ruckeri in tissues of inoculated and
naturally infected trout. Appl. Environm. Microbiol. [Internet].
1999; 65:346–350. doi: https://doi.org/kj7z
[17] Kumar G, Menanteau–Ledouble S, Saleh M, El–Matbouli M.
Yersinia ruckeri, the causative agent of enteric redmouth disease
in sh. Vet. Res. 2015; 46(1):103.
______________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIII, rcfcv-e33280, 1 – 5
5 of 5
[18] Abdel–Latif HMR, Khalil RH, Saad TT, El–Bably RY. Identication
and molecular characterization of Yersinia ruckeri isolated from
mass mortalities of cultured Nile tilapia at Kafr El– Sheikh
Governorate. Global J. Fisheries Aquacult. Res. 2014; 1: 17– 01
[19] Mohammed–Saeed G. Causualty agent (yersiniosis) detection
in reared rainbow trout (Oncorhynchus mykiss) in Kohgiluyeh–
VA–boyerahmad province in Iran. Oceanogr. Fisheries Open
Access J. 2017; 4: 555–633.
[20] Pajdak–Czaus J, Platt–Samoraj A, Szweda W, Siwicki AK,
Terechmajewska E. Yersinia ruckeri. A threat not only to rainbow
trout. Aquacult. Res. 2019; 50: 3083–3096.
[21] Zorriehzahra M, Adel M, Torabi –Delshad S. Enteric redmouth
disease: Past, present and future: A review. Iranian J. Fisheries
Sci. 2017; 17: 1135–1156.
[22] Tinsley JW, Lyndon AR, Austin B. Antigenic and cross–protection
studies of biotype 1 and biotype 2 isolates of Yersinia ruckeri
in rainbow trout, Oncorhynchus mykiss (Walbaum). J. Appl.
Microbiol. 2011; 111: 8–16.
[23] Liu T, Wang KY, Wang J, Chen DF, Huang XL, Ouyang P, Geng
YI, He Y, Zhou YI, Min J. Genome sequence of the sh pathogen
Yersinia ruckeri SC09 provides insights into niche adaptation and
pathogenic mechanism. Intern. J. Molec. Sci. 2016; 17(4): 557.
