International Journal of Fisheries and Aquatic Studies 2017; 5(4): 413-417
E-ISSN: 2347-5129
P-ISSN: 2394-0506
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IJFAS 2017; 5(4): 413-417
© 2017 IJFAS
www.fisheriesjournal.com
Received: 01-05-2017
Accepted: 02-06-2017
Ananya Guchhait
MFSc. Student, Department of Aquatic
Animal Health, Faculty of Fishery
Sciences, West Bengal University of
Animal and Fishery Sciences 5,
Budherhat Road, Chakgaria, P.O.
Panchsayar, Kolkata, West Bengal,
India
Koel Bhattacharya Sanyal
Senior Research Fellow, Department of
Aquatic Animal Health, Faculty of
Fishery Sciences, West Bengal
University of Animal and Fishery
Sciences 5, Budherhat Road, Chakgaria,
P.O. Panchsayar, Kolkata, West Bengal,
India
Debapriyo Mukherjee
Senior Research Fellow
Department of Aquatic Animal Health,
Faculty of Fishery Sciences, West
Bengal University of Animal and
Fishery Sciences 5, Budherhat Road,
Chakgaria, P.O. Panchsayar, Kolkata,
West Bengal, India
Prasenjit Mali
Assistant Professor,
Department of Aquatic Animal Health,
Faculty of Fishery Sciences, West
Bengal University of Animal and
Fishery Sciences 5, Budherhat Road,
Chakgaria, P.O. Panchsayar, Kolkata,
West Bengal, India
TJ Abraham
Professor, Department of Aquatic
Animal Health, Faculty of Fishery
Sciences, West Bengal University of
Animal and Fishery Sciences 5,
Budherhat Road, Chakgaria, P.O.
Panchsayar, Kolkata, West Bengal,
India
Gadadhar Dash
Professor, Department of Aquatic
Animal Health, Faculty of Fishery
Sciences, West Bengal University of
Animal and Fishery Sciences 5,
Budherhat Road, Chakgaria, P.O.
Panchsayar, Kolkata, West Bengal,
India
Correspondence
Gadadhar Dash
Professor, Department of Aquatic
Animal Health, Faculty of Fishery
Sciences, West Bengal University of
Animal and Fishery Sciences 5,
Budherhat Road, Chakgaria, P.O.
Panchsayar, Kolkata, West Bengal,
India
Isolation and identification of Myxobolus cerebralis
from brain of Heteropneustes fossilis in the beels of
South 24-Parganas, West Bengal, India
Ananya Guchhait, Koel Bhattacharya Sanyal, Debapriyo Mukherjee,
Prasenjit Mali, TJ Abraham and Gadadhar Dash
Abstract
Myxobolus cerebralis infecting the brain of Heteropneustes fossilis (singhi) was identified
morphologically, histopathologically and by molecular method captured from the beels of Raidighi,
South 24 Parganas, West Bengal, India. Infection of M. cerebralis was uncommon in Heteropneustes
fossilis brain. The infection rate was low to moderate. Microscopically Myxobolus parasites were
detected in brain of Heteropneustes fossilis (singhi). Large plasmodia were localized within brain,
causing severe necrotic changes and vacuolization. Inflammatory infiltrates agglomeration, nuclear
hypertrophy, vacuolization, infiltration of blood cell and haemorrhages were extensively observed in the
brain. Polymerase chain reaction (PCR) with primers specific for the family Myxobolidae was used to
amplify an approximately 1600 base pairs (bp) long fragment of the 18S ribosomal RNA gene.
Keywords: Heteropneustes fossilis, histopathology, morphometry, Myxobolus cerebralis, PCR
1. Introduction
Fisheries have always played a crucial role in food and nutrition all over the world [1].
Aquaculture continues to be the fastest growing animal food producing sector. Health
management in aquaculture is of major concern from production and security point of view.
Diseases are one of the most important constraints on aquaculture production. Several
bacterial, viral, parasitic and fungal diseases have been documented. Among parasitic diseases
myxozoans, monogeneans, digeneans, larval cestodes and ectoparasitic crustaceans are of
great importance [2]. The myxosporea are common parasites of fishes; and some of them are
reported to be serious pathogens associated with epizootics and causing heavy losses to the
aquaculture [1]. One of the richest genera of the Myxosporea is the genus Myxobolus. To date
as many as 500 Myxobolus species infecting fish are known, of which 444 valid species were
recorded by Lom et.al (1992) [3]. Until recently, differentiations of the species were based on a
morphological characterization of spores. The molecular method has been adopted for
confirmation of myxosporean species by using specific sets of primers. The myxozoan
parasite, Myxobolus cerebralis is the causative agent of whirling disease is a widely known
lethal infection of freshwater salmonid fish that has vast economic and ecological impacts [4].
This parasite has a complex life cycle involving two hosts: it first resides in the digestive tract
of Tubifex tubifex, an oligochaete worm, and once expelled it infects salmonid fish [4].
Whirling disease is mostly prevalent in North America, throughout Europe, South Africa,
North-east Asia and New Zealand [5]. Salmonids are not native freshwater fish of India, and are
unlikely to be susceptible to whirling disease. Nevertheless, several reports on Myxobolus spp.
are available from India, especially from West Bengal [5]. The occurrence of Myxobolus
cerebralis in India has been reported by Abidi (2015) [6] from Indian catfish Clarias batrachus.
In this study, we identify the presence of Myxobolus cerebralis in the brain of Heteropneustes
fossilis (singhi) by morphological, histopathological study and by using molecular method.
2. Materials and methods
2.1 Sample Collection
Live Heteropneustes fossilis (singhi) captured from the beels of Raidighi (22.0012° N,
88.4354° E), South 24 Parganas district, West Bengal, India were brought to the laboratory
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International Journal of Fisheries and Aquatic Studies
within 50 min of collection in oxygen filled polythene bags
during early morning. In the laboratory, the length, weight,
external symptoms and general health conditions of the fishes
were recorded immediately. Brains were dissected out, placed
in separate Petri-dishes and thoroughly examined. The
severity of infection was determined by the following scale
proposed by Lightner (1993) [7] with slight modification as
follows:
Table 1
The severity of infection
No signs of parasite
A very few scattered signs of parasitic infection
Low parasitic infection
Low to moderate parasitic infection
Moderate parasitic infection
Severe parasitic infection
Denoted by
0
0.5
1
2
3
4
2.2 Light microscopy
The parasite identification was performed in the laboratory
according to Lom et al. (1989) [8]. For detailed study, fresh
brains were first taken on clean grease free glass slides. Then
it was slightly ruptured and smeared on clean slides with a
few drops of distilled water, covered with cover slips and
sealed with DPX for examination under oil immersion (100X)
lens [Motic BA400]. Permanent mounting of myxosporean
parasites were done by staining with Giemsa solution
(HiMedia, Mumbai). Air dried smears were treated with
acetone free absolute methyl alcohol for about 8 min to fix the
parasites. The stock solution of Giemsa was diluted with
phosphate buffer (pH 7.2) in the ratio of 1:2. The slides were
then placed on a staining rack and covered with Giemsa
working solution for 40 min. The slides were then washed by
distilled water and air dried. The slides containing
myxosporean spores were observed under oil immersion
(100X) lens [Motic BA400]. The morphometric
measurements were done by Motic Image Plus Version2
software.
2.4.2 PCR amplification
The approximately 1600 base pairs (bp) long fragment of the
18S smaller subunit of ribosomal RNA (18S rRNA) was
amplified by PCR using a set of primers (MX5-F, 5´CTGCGGACGGCTCAGTAAATCAGT-3´ and MX3-R,
5´CCAGGACATCTTAGGGCATCACAGA-3´) [10]. The
reaction mixture of 25 μl consisted of 1μl of genomic DNA
(10 to 50 ng), 12.5μl of 2x PCR Taq Mixture (HIMEDIA), 10
p moles each of the two primers and 9.5 μl of molecular
biology grade water (HIMEDIA). Amplification was done by
initial denaturation at 95°C for 5 min, followed by 35 cycles
of denaturation at 95°C for 30 sec, annealing temperature of
primers was 46°C for 30 sec and extension at 72°C for 45 sec.
Final extension was made at 72°C for 5 min.
2.4.3 Agarose gel electrophoresis
The PCR products were analysed on a 1.2% agarose
(HIMEDIA) gels containing 0.5 μg/ml ethidium bromide in
1× tris-acetate- EDTA (TAE) buffer.
3. Results
In the present study Myxobolus cerebralis was detected
microscopically in the brain of H. fossilis. In frontal view
(perpendicular to the sutural plane) the spores were mostly
circular in shape. Mature spores (Fig. 1) from the plasmodia
were round to oval in frontal view, measuring 9.31μm (9.25
to 9.37 μm) in length, 9.24μm (9.19 to 9.28μm) in width. The
polar capsules were pyriform and of equal size, measuring
5.19μm (5.16 to 5.23μm) in length, 3.21μm (3.19 to 3.26μm)
in width. The filament inside the capsule was not very distinct
in this species (Table 2). The severity of infection was low to
moderate.
2.3 Histopathology
The brain samples of H. fossilis were fixed in alcoholic
Bouin’s fixative for48-72 h. After fixation the tissues were
transferred to 70% Ethyl alcohol and kept overnight.
Histopathological analysis was made as described by Roberts
(2001) [9].
2.4 Molecular identification
2.4.1 DNA extraction
The DNA was extracted using DNA, RNA and Protein
purification Kit of Machery-Nagel GmbH & Co. KG,
Germany. After extraction, the DNA was collected and stored
at -20o C for further use.
Fig. 1: Spore of Myxobolus sp. showing polar capsule (PC), spore
(S), collected from brain of Heteropneustes fossilis (400x) (wet
mount).
Table 2: Morphometry of Myxobolus cerebralis spores (on the basis of 20 observations) isolated from the brain of H. fossilis.
Measurement Data given by Hofer, 1903 (µm) Data obtained in the present study (µm) Mean
SD
LS
7.4 - 9.7
9.25-9.37
9.312 0.037077
BS
7 -10
9.19-9.28
9.2435 0.029249
LPC
5-6
5.16-5.23
5.1965 0.020844
BPC
3 - 3.5
3.19-3.26
3.217 0.021788
LS: Length of Spore, BS: Breadth/width of spore, LPC: Length of polar capsule, BPC: Breadth of polar capsule,
SD – standard deviation, CV – coefficient of variation.
By the histopathological analysis we can confirm that infected
by protozoan parasites with binucleated nuclei representing
CV (%)
0.398
0.316
0.401
0.677
Myxobolus cerebralis in the brain of Heteropneustes fossilis
(Fig.2). The brain section also showed inflammatory infiltrate
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International Journal of Fisheries and Aquatic Studies
agglomeration, meningoencephalitis, lesions and enlarged
neural cells (Fig.3). Histopathological analysis of the brain of
H. fossilis, based on histological sections stained with
Hematoxylin-Eosin, allowed us to analyze degenerative
changes and vacuolization in tissue. There was a marked
nuclear hypertrophy, infiltration of blood cells and
haemorrhages were also observed in examined fish brain
(Fig.4 & 5).
Fig 5: The brain cell infected with Myxobolus sp. showing the
presence of infiltration of blood cells (IB), haemorrhage (H),
inflamed neurones (IN), nuclear hypertrophy (NH), vacuolization
(V). H&E, 200 X.
Fig 2: Brain cells of H. fossilis showing the presence of huge
number of Myxobolus spores (M). Cells were necrotised (N),
vacuolization (V) were also present. H&E, 400 X.
By molecular method we can confirm that the primers MX5
and MX3, according to Eszterbauer et al. (2001) [10],
successfully amplified approx. 1600 bp fragments of the 18S
rRNA gene from every sample of the Myxobolus species.
These primers are specific for the family Myxobolidae (Fig.
6).
Fig 3: The brain section of H. fossilis showing inflammatory
infiltrate agglomeration (IIA), meninges indicating
meningoencephalitis (ME), lesions (L) with vacuolization (V),
necrosis (N), enlarged neural cells (EN). H&E, 100 X.
Fig 6: Agarose gel (1.2%) showing amplification of a part of
18S rRNA gene of Myxobolus cerebralis from the brain of H.
fossilis. Lane M: 1kb DNA Ladder (Takara Bio Inc., Japan);
Lane S1: Positive control; Lane S2: Myxobolus cerebralis.
Fig 4: Histopathological changes in the brain of Heteropneustes
fossilis showing inflammatory infiltrates agglomeration (IIA),
nuclear hypertrophy (NH), vacuolization (V) in the tissue and
degenerative changes (D). H&E, 200 X.
4. Discussion
In the present study, mature spores (Fig. 1) from the
plasmodia were round to oval in frontal view, measuring
9.31μm (9.25-9.37 μm) in length, 9.24μm (9.19 to 9.28μm) in
width. The polar capsules were pyriform and of equal size,
measuring 5.19μm (5.16 to 5.23μm) in length, 3.21μm (3.19
to 3.26μm) in width. According to Hofer (1903) [11] in
Myxobolus cerebralis, the spores were mostly broadly oval,
sometimes more elongated, rarely completely circular, and
exceptionally broader than long. The dimensions of spores
were: length 8.7 (7.4 to 9.7) μm, width 8.2 (7 to 10) μm,
thickness 6.3 (6.2 to 7.4) μm. The spore wall could not be
measured accurately but appeared to be about 0.25 μm thick.
The oviform polar capsules measured 5.1 (5 to 6) by 3.2 (3 to
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International Journal of Fisheries and Aquatic Studies
3.5) μm. The comparison of spore morphology shows that the
present species corresponded to M. cerebralis. The spores of
other Myxobolus species, similar to M. cerebralis in shape,
mainly, Myxobolus arcticus, Myxobolus insidiosus but only
the spores of M. kisutchi and the microspores of M. squamalis
were similar in size [12]. However, M. kisutchi was related in a
host of Cohosalman and Chinook salmon and their spores
differed from those of M. cerebralis by appear uniform in
shape, and contain an iodinophilous vacuole. In contrast to M.
cerebralis, the plasmodia of M. arcticus had larger spores
(14.3-16.5x7.6-7.7 µm), with large, elongated polar capsules.
However, plasmodia of M. insidiosus were found in the
muscle of cut throat trout, Chinook salmon and Coho salmon
from the western United State [12].
By the histopathological analysis we can confirm that infected
by protozoan parasites with binucleated nuclei representing
Myxobolus cerebralis in the brain of Heteropneustes fossilis.
We found a correlation between the fish cells and parasitic
spores via the hematoxylin-eosin (HE) histological technique,
which was used to confirm the presence of M. cerebralis in
tissue of fish brain [13]. In the present study the brain of H.
fossilis showed inflammatory infiltrate agglomeration,
lesions, necrosis and enlarged neural cells. Elwell et al.
(2010) [14] showed that cartilage tissue of salmonid fish
infected with M. cerebralis, was visibly eroded and
granulomatous lesions were found in association with the
cartilage destruction (host inflammatory response). This result
is in agreement with the present findings. Uspenskaya 1982
[15]
reported that destruction of cartilage is accomplished both
through extracellular digestion (lysis) of the cartilage matrix
and by phagocytosis of cartilage cells by M. cerebralis [16].
Using histopathological analysis,[17] noted diffuse oedema in
brain tissues, and congestion, degeneration, and focal necrosis
of the cerebral cortex. We also observed a pronounced
quantity of Myxobolus sp. spores dispersed throughout the
brain of H. fossilis. There was a marked nuclear hypertrophy,
infiltration of blood cells and haemorrhages was also
observed in examined fish brain (Fig.4&5). Severe cases of
whirling disease can lead to death in young fish. Early reports
of whirling disease frequently referred to (sometimes heavy)
mortalities associated with the disease [14].
According to Eszterbauer et al. (2001) [10], the 18S rRNA gene
and oligonucleotide primers were used to amplify an
approximately 1600 bp long fragment from the 18S rRNA
gene. The primers were specific for the family Myxobolidae.
This result is in agreement with the present study. The
primers MX5 and MX3 specific for the family Myxobolidae
successfully amplified approx. 1600 bp fragments of the 18S
rRNA gene from every sample of the Myxobolus species (Fig.
6).
5. Conclusion
Existing knowledge on the phylum Myxozoan enigmatic
group of organisms is fragmentary, inadequate and
incomplete in many parts of India, especially in the state of
West Bengal having rich fish biodiversity. There is great need
to identify, describe and satisfactorily classify these parasites
both morphologically and at the molecular level. It is evident
that study on this group is also important in identification of
the pathogenic species which can pose serious threat to
fisheries in state of West Bengal. This knowledge will help in
diagnostics, management and treatment of the diseases caused
by these parasites in freshwater fishes.
6. Acknowledgements
The authors gratefully acknowledge the assistance extended
by the Faculty of Fishery Sciences, West Bengal University of
Animal and Fishery Sciences, West Bengal, India for
providing necessary facilities for undertaking the work.
Special thanks to National Bureau of Fish Genetic Resources
(NBFGR), Lucknow, Uttar Pradesh, India and National
Fisheries Development Board (NFDB), Hyderabad,
Telangana, India for financial support during the study.
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Isolation and identification of Myxobolus cerebralis from brain of Heteropneustes fossilis in the beels of South 24-Parganas, West Bengal, India
International Journal of Fisheries and Aquatic Studies, 2017
Myxobolus cerebralis infecting the brain of Heteropneustes fossilis (singhi) was identified morphologically, histopathologically and by molecular method captured from the beels of Raidighi, South 24 Parganas, West Bengal, India. Infection of M. cerebralis was uncommon in Heteropneustes fossilis brain. The infection rate was low to moderate. Microscopically Myxobolus parasites were detected in brain of Heteropneustes fossilis (singhi). Large plasmodia were localized within brain, causing severe necrotic changes and vacuolization. Inflammatory infiltrates agglomeration, nuclear hypertrophy, vacuolization, infiltration of blood cell and haemorrhages were extensively observed in the brain. Polymerase chain reaction (PCR) with primers specific for the family Myxobolidae was used to amplify an approximately 1600 base pairs (bp) long fragment of the 18S ribosomal RNA gene....Read more
