Introduction
Guppy (Poecilia
reticulata; Cyprinodontiformes, Poeciliidae, Poecilia) is the main small
ornamental fish of the freshwater aquaculture industry in China. In recent
years, we have seen an annual increase in guppy production, accompanied by
increase in disease, restricting the development of the ornamental fish
industry. Common guppy diseases have included bacterial diseases caused by Aeromonas
spp. (Chambel 2011) and Vibrio cholerae (Balakrishna
2013); parasitic diseases, such as ichthyophthiriasis, caused by Ichthyophthiriu
smultifiliis (Chen 2002), Gyrodactyliasis spp. (Van-Oosterhout et al.
2007), and Tetrahymena pyriformis (Ponpornpisit et al. 2010); mycosis caused by fungi,
such as Saprolegnia spp. (Qu 2009) and Aspergillus spp. (Haroon et
al. 2014); and
viral nervous necrosis (VNN), caused by nodaviruses (Munday et
al. 2002).
Needle-tail disease has been a high incidence of disease with high mortality in
guppies in recent years. The course of the disease is as follows: first, the
opening angle of the caudal fin becomes smaller, sometimes with rotting tissue,
and finally the fin merges into spicules, which may result in fish death.
However, there are only a few reports of the etiology of needle tail disease from
China or overseas area. Latremouille (2003) reported that fin erosion occurred
when the fins of afflicted fish became degraded for a variety of reasons,
including abrasion from rough surfaces, fin damage from aggressive encounters
between fish, nutritional deficiencies, and bacterial infections (Latremouille
2003). Previous epidemiological investigations on needle tail disease in P.
reticulata showed that guppy needle tail disease can be divided into two
types, one caused by environmental stress and the second, which results in
‘needle tails’ after fin rot, which we refer to here as ‘fester-needle tail
disease’. High ammonia concentration in aquatic environment can cause the
occurrence of needle tail in P. reticulata but does not cause rotting of
the caudal fins (Jin et al. 2019).
In
the current study, the etiology of the fester-needle tail disease was explored
further. We first investigated whether aquatic conditions, fungi, viruses,
parasites, and other pathogens could be causative agents of the disease, and then ruled out those
possibilities and finally we isolated the dominant bacteria. Bacteria isolated
from diseased tissue were identified and their ability to cause fester needle tail
disease was investigated in artificial infection experiments. The results of
the current study support the division of needle tail disease into disease
caused by aquatic conditions and disease caused by bacteria. Such information
will be important for managing and controlling the impact of this disease on
the future development of the guppy aquaculture industry.
Materials and
Methods
Guppies
Healthy and diseased guppies, which had similar age and size (5 months old,
1.3–1.5 cm) were collected from 5 guppy farms in Anshan and Liaoyang cities
in Liaoning. An epidemiological investigation was carried out, and a total of
138 needle tail cases with obvious symptoms of rot were collected. The caudal
fin of the diseased P. reticulate merged into spicules. After the experiment, the fishes
were euthanized refer to the requirements of the Committee of Animal Welfare
and Ethic at Dalian Ocean University.
Ultrastructural observations
The intact healthy and diseased P. reticulate were
fixed with a volume fraction of 2.5% glutaraldehyde and then were sent to the
Shanghai Veterinary Research Institute, Chinese Academy of Sciences for trans
mission electron microscopy studies and ultrastructural observations.
Isolation of
pathogenic microorganisms
The surface of diseased fish was disinfected with 75%
alcohol and then rinsed three times with sterilized normal saline. Each caudal
fin was removed in a sterile environment and placed in a 1.5 mL centrifuge tube
containing sterilized pure water. Each caudal fin was ground to tissue
homogenization by sterile medical tweezers
and vortex mixer (Dragon Lab., China). A total of 100 μL
of homogenate was coated in nutrient agar (Hopebio, China) culture
medium and inverted upside down at 28°C for 24–72 h. Any bacterial growth was
observed and the dominant colonies which had the largest number were selected
for purification by plate streaking according to their morphological
characteristics.
Identification of pathogenic microorganisms
Each purified strain was inoculated in nutrient broth (NB)
liquid medium and cultured in a shaking table at 28°C and 180 rpm for 24 h.
Total bacterial DNA was extracted using a bacterial genomic DNA extraction kit (Tiangen,
China). The extracted DNA was used as a template with the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3')
and 1492R (5'-GGTTACCTTGTTACGACTT-3') (Lane 1991) to amplify the 16S rDNA gene.
The PCR reaction conditions were as follows: denaturation at 94°C for 10 min;
and then 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30
s, extension at 72°C for 1 min 30 s, plus a final extension step at 72°C for 10
min. The PCR products were detected by 1% agarose gel electrophoresis, and then
sent to Sangon Biotech Co. (Shanghai, China) for nucleic acid sequencing. The
sequencing results were analyzed by using BLAST analysis in GenBank
(https://blast.ncbi.nlm.nih.gov/Blast.cgi).
Challenge infection
The dominant bacteria were inoculated in NB liquid medium
and placed in a flask on a constant temperature shaking table at 28°C at 180
rpm for 24 h. The concentration of bacteria was measured by
using a bacterial
chamber. After centrifugation, the supernatant was removed and
the bacterial suspension was serially
diluted to 1×106
CFU mL-1, 1×107 CFU mL-1, 1×108 CFU
mL-1, 1×109 CFU mL-1, and 1×1010
CFU mL-1 with normal saline.
Experimental and control groups were established
with 40 healthy P.
reticulate per group. The control group was soaked in 0.9%
normal saline in a 1 L beaker for 1 h and then transferred to a clean 10 L aquarium.
For each experimental group (one at each bacterial concentration), the fish
were placed in 1 L beakers with either 1×106 CFU mL-1, 1×107 CFU mL-1,
1×108 CFU mL-1, 1×109 CFU mL-1, and
1×1010 CFU mL-1 bacterial suspension. The
1×106 CFU mL-1
bacterial suspension was deemed to be a safe concentration for P. reticulata,
whereas the 1×1010
CFU mL-1 bacterial suspension was deemed to be the most
lethal concentration (being the lower and upper concentration limits). After
soaking for 1 h, the fish were transferred to a clean 10 L aquarium. The fish
were observed continuously for at least 15 days and the morbidity and mortality
rates were recorded. The fish were fed twice a day, with one-third of the water
was changed every 5 days, and were provided with a continuous aeration
supply.
Histopathological analysis
After 15 days of artificial infection, the caudal fins
of each fish with needle tail disease and healthy fish in the control group
were removed by sterile medical tweezers,
placed into 4% paraformaldehyde and fixed at room temperature for 24 h. The
samples were then dehydrated through a graded ethanol series (50–100%), cleaned
in xylene, and embedded in paraffin wax. For staining,
sectioned material was dewaxed in xylene, rehydrated through a graded ethanol
series (0–100%) and stained with hematoxylin and eosin. Permanent preparations
were obtained by dehydrating (through an ethanol series 50–100%), and cleaned
in xylene; neutral resin was used to seal the tissue sections, which were then
examined under a Leica DM4000 microscope (Jin et al. 2020).
Results
Ultrastructure observations of caudal fins of P. reticulate with
needle tail disease
An electron microscope was used to analyze histomorphological
changes between the healthy and diseased tissues. The cells of healthy fish
showed a normal density, were moderate in length, and contained no obvious
vesicles (Fig. 1A,
B). No virus-infected cells or virus inclusion bodies were
found in the samples from the lesions of naturally infected P. reticulata.
The nucleus and cytoplasm of diseased fish were both very dense, with elongated
and narrower cells containing increased numbers of vesicles (Fig. 1C, D).
Bacterial isolation and identification
In total, 138 P. reticulata with needle tail disease were collected. After
isolating the bacteria from the diseased site, 232 strains were identified
based on 16S rDNA molecular biological, physiological, and biochemical
identification. Of the strains identified, Aeromonas spp. accounted for
35.3% and Pseudomonas spp. accounted for 13.7% (Fig. 2). Aeromonas hydrophila accounted for 45.1% of the total Aeromonas isolates,
indicating that it was the most dominant strain isolated from P. reticulata
with needle tail disease.
Artificial regression infection test
After soaking respectively with a 1×106 CFU mL-1,
1×107 CFU mL-1, 1×108 CFU mL-1,
1×109 CFU mL-1, and 1×1010 CFU mL-1
concentration of A. hydrophila suspension, the mortality and
needle tail infection rates of P. reticulata were recorded over 15 days
(Fig. 4), and the
deterioration process was showed in Fig. 3. In the control
group, there were no P. reticulata with needle tail disease or mortalities. However, in all groups
infected with A. hydrophila, both needle tail disease and
mortalities
of fish were recorded, as well as occurrences fin decay. With the increase in
the concentration of A. hydrophila, the needle tail rate and mortality
of P. reticulate
were positively correlated. The mortality rate of the
group with the highest concentration of 1.0×1010CFU mL-1 was
100%, with a needle tail rate of 55%; the mortality rate of the group with the lowest
concentration of 1.0×106CFU mL-1 was 75%, with a needle
tail rate of 30%.
Histopathological changes
In the caudal fins of healthy P. reticulata,
the fiber structure was arranged, and the pigment granules were visible (Fig. 5A);
the muscle fibers were closely packed, with a distinct color hierarchy (Fig. 5C); epithelial
histiocytes were arranged compactly, with clear delineation between cells (Fig.
5E). In contrast, the caudal fins of infected P. reticulata had rotted
and the opening angle of the fins had become smaller. The cells were unevenly
arranged and less compact, and the fiber structure had ruptured (Fig. 5B); the
fins then began to become more needle-like in appearance, with less obvious
cellular boundaries, and pigment granules were transformed and intensively
distributed (Fig.
5D). In the caudal fins of healthy P. reticulata,
the cells were oval, closely packed, with intact nuclei (Fig. 5G).
In the rotting part of the caudal fins of infected P. reticulata, a few bacteria were visible
(red thin arrow in Fig. 5F). Microscopically, the cells could be seen to be
swelling and dissolving, with significant cavitation (red thick arrow), and
fractured cytomembranes, with only a few cells showing any trace of a nucleus.
At the nonulcerated part of the caudal fin of infected P. reticulata, elliptic cells
were reduced, loosely arranged, and vacuoles and karyolysis were observed (red
thin arrow, Fig. 5H).
Discussion
Needle tail
disease does not have a single causative agent, but it is rather a disease
syndrome. Previous research confirmed that high concentrations of ammonia in
the aquatic environment could cause needle tail disease in P. reticulate (Jin
et al. 2019). In the current study, numerous bacterial strains were
isolated from the caudal fins of P. reticulate. Most dominantly strain
was A. hydrophila. Then, Aeromonas veronii and Pseudomonas alcaligenes were 15.5 and 12.5% respectively. However, future studies will
need to test whether the latter two species are also causative agents of needle
tail disease. Thus, needle tail disease might not only a special disease
be caused by the environment or by a particular bacterium, but is rather a syndrome;
a similar situation is seen with Aeromona spunctata f. intestinalis, Aeromonas
sobria, Edwardsiella tarda, and A. hydrophila,
which all cause bacterial enteritis in fish (Zhang et al. 1998; Xuan 2018; Yang et al.
2018), and with Proteus mirabilis (Zhang and Sun 2019), A.
hydrophila, A. sobria, Yersinia ruckeri (Shao 2017),
Vibrio scophthalmi, and Vibrio alginolyticus (Ying et al. 2019),
which can all cause bacterial septicemia in fish. Therefore, we refer to needle
tails as a “syndrome” the disease.
%20397-403_files/image002.png)
Fig. 1: Longitudinal sections of the caudal fin of
healthy Poecilia reticulata (A, B) and of P. reticulata with needle tail disease (C, D).
(A) No damage to the organelles (red
arrow). (B) Cells are completely
oval and contain no virus particles (red arrow). (C) Cell nuclei are pyknotic (red arrow). (D) There are no virus particles in the cells, which have become
narrower (red arrow)
%20397-403_files/image004.jpg)
Fig. 2: Species identification of the 232 strains isolated from Poecilia reticulata with needle tail disease
A. hydrophila is an important pathogen of many aquatic animals, and can cause
different symptoms in different cultured species. The genus Aeromonas not
only can cause diseases of fish and other cold-blooded species, but also can
cause a variety of infectious complications in both immunocompetent and
immunocompromised humans, therefore it is regarded as an important
disease-causing pathogen (Janda and Abbott 2010). Aeromonas has also
been reported as the main pathogen affecting freshwater ornamental fish
(Martínezmurcia et al. 2008). The main pathogenic targets associated
with Aeromonas are surface polysaccharides (capsule, lipopolysaccharide,
and glucan), S-layers, iron-binding systems, exotoxins, and extracellular
enzymes, secretion systems, fimbriae and other nonfilamentous adhesins, motility, and flagella (Tomás 2012). The level of virulence of
different species of Aeromonas is not fixed, and depends on the bacterial
strain, route of infection, and the animal used as a model organism (Yu et
al. 2005). Chambel found that P. reticulata were sensitive to Aeromonas
spp., especially A. hydrophila (Chambel 2011). Aeromonas is a
thermophilic, motile bacterium that exists in fresh-water, sewage, silt, and
soil, and is a primary pathogen that infects fish, amphibians, reptiles, and
mammals (Zhang et al. 2001). The range of disease outcomes caused by A.
hydrophila is broad, including motile septicemia in carp, tilapia, perch,
catfish, and salmon; erythredema in weever fish and carp; and ulcerative
infections in catfish, cod, carp and goby fish, including epiphytic ulcer
syndrome (Holmes et al. 1996). It can also cause poisoning in eels
(Austin et al. 1998). Research showed that carp infected with A.
hydrophila became darker in color with fin ray hyperemia and fin decay (Li et
al. 2007). The caudal fins of hybrid tilapia were also reported to decay as
a result of infection with A. hydrophila (Yang et al. 2009).
Obvious characteristics of A. hydrophila infection in fish include
hemorrhagic septicemia, fin rot, body swelling, and skin ulcers (Jacobs and
Chenia 2007; Abolghait et al. 2010). Such features result mainly from
the production by pathogenic A. hydrophila of hemolysins, enterotoxins,
cytotoxins, and other extracellular enzymes, which result in red blood cell
lysis, tissue cytolysis, and increased tissue permeability (Lu 1992). This
study is the first to report that A. hydrophila infection of P.
reticulata can lead to needle tail disease. However, how the infection
causes the caudal fin to contract into a needle tail remains unclear; besides,
whether the other bacteria which were largely isolated from diseased fins in
the current study, like A. veronii and P. alcaligenes, also cause
guppy needle tail disease, whether by the same mechanism as A. hydrophila,
all will be carried out in further study.
%20397-403_files/image006.png)
Fig. 3: The process of needle tail infection in Poecilia reticulata caused by Aeromonas
hydrophila. (A) Healthy P.
reticulata. (B) Caudal fin ulcer (red arrow). (C) Caudal fin ulcer and a reduced tail (red arrow). (D) Needle tail (red
arrow)
%20397-403_files/image008.jpg)
Fig. 4: The mortality and needle tail
rates in Poecilia reticulata exposed to different concentrations of Aeromonas
hydrophila
The
A. hydrophila that we isolated from naturally diseased guppies were
relatively resistant to antibiotics and presents the trend of parallel
propagation, according to our epidemiological investigation. Therefore,
antibiotics might not be a suitable option for treating P. reticulata needle
tail disease. In recent years, an increasing number of drugs against A.
hydrophila has been developed. It has been reported that scutellaria baicalensis
could affect the membrane permeability of A. hydrophila, causing
intracellular ions and other molecules to exit the cell, destroying the
integrity of the cell membrane, and resulting in its bacteriostatic effect
(Wang et al. 2019). Morin can reduce the pathogenicity of A.
hydrophila by inhibiting aerolysin, an important virulence factors secreted
by this bacterium (Dong et al. 2019). Resveratrol can also affect type I and II gene
expression of the quorum-sensing system in A. hydrophila, influencing
downstream virulence correlation factors (Tan et al. 2019). Adding
dietary fenugreek to fodder could enhance fish immunity and antioxidant
capacities, reducing the visceral damage caused by A. hydrophila (Moustafaet
al. 2020). It has also been reported that the inactivation of the nuclease
(ahn) of A. hydrophila significantly impairs its ability to evade host
immune attack (Ji et al. 2015). Thus, although A. hydrophila has
strong pathogenic potential, its survival and reproduction strategies against
the host immune system have yet to be fully determined (Citterio and Biavasco
2015) and, therefore, its prevention and control still require significant
research attention. We hope other investigators reach more conclusive results
than ours.
Conclusion
An epidemiological
investigation of guppy fester-needle tail disease was carried out for 1 year. In
total, 232 bacterial strains were isolated, and the A. hydrophila was
the most isolated strain. A bacterial challenge experiment with A.
hydrophila revealed it to be a possible causative eagent of fester-needle
tail disease. These results provide a reference for further research and
scientific control of needle tail disease in this economically important
species.
Acknowledgments
%20397-403_files/image010.png)
Fig. 5: Caudalfin
of healthy
Poecilia reticulata
(A, C, E, G) and P. reticulata infected with Aeromonas hydrophila (B,
D, F, H). The shrunken tail fin (B) and needle tail fin of infected P. reticulata (D). (F) Bacteria (red thin arrow) and cellular vacuolation (red
thick arrow) and (H) karyolysis (red
thin arrow) in infected P. reticulata
This work was
funded in part by the Nature Science Foundation Guidance Program of Liaoning
Science and Technology Department (2019-ZD-0733), National Natural Science
Foundation of China (41706177), International Cooperation Project of Liaoning
Education Department (2019GJWYB20) and High-Level Talents Innovation Support
Program of Dalian and support this work.
Author Contributions
YH
performed the data analyses and wrote the manuscript; SJ conducted the
epidemiological investigation and data analyses; SF made the identification of
bacteria; CM dealt with the experiments of transmission electron
microscope; ZJ, DY
and SY performed the data analysis; RL designed and support this work.
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