There is a need to identify the presence of lesions in fish skin as soon as they erupt. Fish skin lesions are either macroscopic (can be visualized by the naked eye) or microscopic (difficult to detect with the naked eye). Skin wounds resulting in loss of the epithelium (superficial or deep ulcers) are serious as they may interfere with osmoregulation and open portals for opportunistic pathogens. Herein, we report on the use of a fluorescein dye for the detection of skin ulcers that cannot be seen by the naked eye. Due to their importance in aquaculture endeavors in Egypt, this study focused on two indigenous species, the Nile tilapia (Oreochromis niloticus) and the scale-less African sharptooth catfish (Clarias gariepinus). Fluorescein dye was tested for safety to fish without interfering with microbiological analysis. Parallel to the use of the flourescein dye, the detected ulcers were examined for the presence of bacteria or tissue alterations. Further, we experimentally induced the formation of skin ulcers in O. niloticus physically or by injecting Aeromons hydrophila, and then assessed the utility of fluorescein dye in detecting the induced skin lesions.
Trang 1ORIGINAL ARTICLE
Determining the safety and suitability of fluorescein dye for characterization of skin ulcerations in cultured
Nile tilapia (Oreochromis niloticus) and African
sharptooth catfish (Clarias gariepinus)
a
Department of Fish Diseases and Management, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt
b
Department of Pathology, Faulty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt
Received 29 December 2009; revised 7 February 2010; accepted 19 April 2010
Available online 17 September 2010
KEYWORDS
Fluorescein dye;
Abrasions;
Ulcers;
Nile tilapia;
Catfish;
Detection
Abstract There is a need to identify the presence of lesions in fish skin as soon as they erupt Fish skin lesions are either macroscopic (can be visualized by the naked eye) or microscopic (difficult to detect with the naked eye) Skin wounds resulting in loss of the epithelium (superficial or deep ulcers) are serious as they may interfere with osmoregulation and open portals for opportunistic pathogens Herein, we report on the use of a fluorescein dye for the detection of skin ulcers that cannot be seen by the naked eye Due to their importance in aquaculture endeavors in Egypt, this study focused on two indigenous species, the Nile tilapia (Oreochromis niloticus) and the scale-less African sharptooth catfish (Clarias gariepinus) Fluorescein dye was tested for safety to fish without interfering with microbiological analysis Parallel to the use of the flourescein dye, the detected ulcers were examined for the presence of bacteria or tissue alterations Further, we experimentally induced the formation of skin ulcers in O niloticus physically or by injecting Aeromons hydrophila, and then assessed the utility of fluorescein dye in detecting the induced skin lesions Results obtained in this study demonstrated that fluorescein dye application is harmless to Nile tilapia at
* Corresponding author Tel.: +202 33800575; fax: 202 35725240.
E-mail addresses: mai_ibrahim12@yahoo.com , ibrahemmai20@yahoo.
com (M.D Ibrahem).
2090-1232 ª 2010 Cairo University Production and hosting by
Elsevier B.V All rights reserved.
Peer review under responsibility of Cairo University.
doi: 10.1016/j.jare.2010.04.002
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Cairo University Journal of Advanced Research
Trang 2concentrations up to 0.5 mg fluorescein/ml water for up to 15 min Indeed, a low dose of fluorescein (0.10 mg/ml for 5 min) could identify very minute skin abrasions We highly recommend the use of fluorescein dye for the evaluation of skin health in farmed fish species and the visualization of min-ute skin abrasions
ª 2010 Cairo University Production and hosting by Elsevier B.V All rights reserved.
Introduction
Loss of skin and underlying tissue is considered one of the
most common lesions affecting fish worldwide and is
consid-ered to be a reflection of the surrounding environment There
are a number of causes for skin ulceration including pathogens
[1]and chemical and physical factors[2] Skin loss, even if
min-ute, can bring serious complications such as osmoregulatory
failure, swimming imbalance, respiratory distress dehydration
[1]and can predispose affected fish to opportunistic bacteria
and fungi Skin ulcers vary in both depth and size from
non-visible microscopical erosions to grossly non-visible ulcers[3]
Fluoresceinating sodium salt (which will be referred to as
‘‘fluorescein’’) is a non-toxic dye that produces an intense
green fluorescence colour when dissolved in water It has been
safely used to detect ophthalmic lesions including ulcers and
degeneration of the cornea in humans[4–7] Fluorescein
nei-ther penetrates intact epithelium nor forms a firm bond with
vital tissue However, when there is a break in the skin
epithe-lium, fluorescein can rapidly penetrate[8], thereby staining
ex-posed underlying skin layers[9] The present study builds upon
the study of Noga and Udomkusonsri[10]and examines their
findings for the utility, safety and efficiency of the fluorescein
dye as a tool for the detection of natural and experimentally
induced skin ulcers in native fish species, and compares the
re-sults of histopathology and the use of fluorescein dye for early
detection of skin lesions
Material and methods
Field studies
Naturally collected fish
African sharptooth catfish (Clarias gariepinus) of 50–60 mm
total length [TL] and Nile tilapia (Orecochromis niloticus) of
120–150 mm TL (10 fish/species) were obtained from a
semi-intensive aquaculture facility and transported alive in
well-aer-ated containers to the laboratory of the Department of Fish
Disease and Management (FDML), Faculty of Veterinary
Medicine, Cairo University Upon arrival, the fish were
imme-diately screened for the presence of exposed skin areas using
the fluorescein dye
Experimental studies
Fish
For monitoring the safety and efficiency of fluorescein to
de-tect the skin ulcers, experimental studies were performed on
O niloticus Nile tilapia (20 ± 0.2 g) (240 fish) were carefully
collected alive from a semi-intensive fish farm to avoid any
in-jury and transported alive in tanks to FDML Fish were
accli-matised to laboratory conditions for two weeks prior to the
experiment They were maintained in 40 l tanks containing
static dechlorinated water[11] Acclimatisation was performed
in temperature-controlled aquaria at 23 ± 1C The fish were fed twice a day with a balanced commercial fish pelleted diet (Zoocontrol Company, Cairo, Egypt) that contained 30% pro-tein[12] Water quality in all aquaria was monitored regularly for ammonia, and pH, kept within the acceptable limits during the course of the study[13] All fish were subjected to clinical and bacteriological examination to prove that they were free from Aeromons hydrophila infection
Safety of fluorescein dye to fish Eighty of the pre-acclimatised Nile tilapia used for the safety test were transferred to glass aquaria (30· 40 · 80 cm3
), each containing 40 l of 23C freshwater (10 fish/ aquarium) The fish were first placed in a high concentration of dye, up to 0.5 mg fluorescein/ml for 15 min, and then observed for seven days Control fish (10 fish/aquarium) were similarly treated All tests were performed in duplicate
In vitro antimicrobial assay (agar spot assay)
A reference culture pathogenic, A hydrophila (ATCC 7966 strain), was cultured in Trypticase Soya broth (Biomerieux, France) for 18 h at 25C Soft agar (composed of Trypticase Soya broth + 0.7% bacteriological agar) containing 5% of overnight culture of A hydrophila in Trypticase Soya broth was prepared Spots were made by pipetting 10 ll of fluores-cein dye dilutions (from 0.1 to 0.7 mg fluoresfluores-cein/ml, each con-centration on a separate plate)[14] The plates were incubated for 24 h Inhibition was recorded by measuring the clearing zone around the spot All tests were performed in duplicate
[14] Fish anesthesia Tricaine methane sulfonate (MS222) obtained from Argent Chemical Laboratories, Redmond, WA, was used to anesthe-tise fish in a dose of 50 mg/l MS222 with 100 mg/l sodium bicarbonate as a buffering reagent to adjust water alkalinity
to exceed 50 mg/l as CaCO3as recommended by[15] Potency of the fluorescein dye to fish
Naturally affected fish
The fish (both African sharptooth catfish and Nile tilapia) were placed (one at a time) in a solution of 0.10 mg fluorescein (Fluor, 10% fluorescein sodium injection, 100 mg/ml, Sigma, USA) per ml of water for 5 min The fish were then removed from the fluorescein solution and immediately rinsed by plac-ing them in clean water for 1–2 min The fish were then euthan-atized with MS222 and immediately examined for skin damage under an ultra violet source from a UV Trans-illuminator (Spectoline Bi-O-Vision Model TVD-1000R/F, USA) in com-plete darkness Photographs were taken using digital camera
Trang 3with automatic adjustment (Fujinon ptical zoom lens camera;
Model No F 7U2540; China) Fluorescein-positive areas from
Nile tilapia were fixed for histopathological examination[16]
Experimentally ulcerated Nile tilapia
The pre-acclimatised fish were transferred to identical aquaria
containing 40 l of water (10 fish/aquarium, two replications of
the treatment) Water temperature was adjusted to 23C Fish
were collected one at a time from each aquarium and
anesthe-tised with MS222 A few scales were hand-removed from each
fish; then fluorescein dye was used for the detection of the
in-duced ulcers via the regime described above
Experimentally infected Nile tilapia
A culture suspension of A hydrophila (ATCC 7966) was
pre-pared by spreading onto Tryptic soya agar for 24 h at 25C;
4–5 colonies were suspended in sterile saline 0.85% and matched
to contain 108bacteria ml 1using McFarland standard tubes
The test fish were held in replicate aquaria containing 40 l of
thermostatically controlled water at 23C (10 fish/aquarium,
two replications of the treatment); fish were infected
intraperito-neally with 0.2 ml of the culture suspension The infected fish
were examined at 8, 12, 18, 24, 48 and 72 h post infection by
fluo-rescein dye for ulceration via the regime described above
Bacteriological investigations
Smears from skin ulcers, kidney and liver of naturally examined
Nile tilapia and African sharptooth catfish and experimentally
infected Nile tilapia were spread onto Trypticase Soya Agar
(TSA) (Oxoid) and blood agar plates Culture plates were
incu-bated at 25C for 24 h and then inspected for bacterial growth
Further morphological and biochemical identification of the
re-trieved isolates was done according to Whitman[17]
Histopathology
For histopathological examination, skin lesions from naturally
ulcerated Nile tilapia were fixed in 10% neutral buffered
forma-lin, and then processed in paraffin embedding method sections
of 5 lm were stained with hematoxylin and eosin (H&E)[16]
Results and discussion
Safety of fluorescein dye to fish
In the first group, fish were subjected to a single application
of a high concentration of fluorescein dye (0.5 mg
fluores-cein/ml water for 15 min) and then monitored for seven days The dye proved safe as no adverse clinical or behav-ioural abnormalities appeared in Nile tilapia post exposure The present results coincide with those of Noga and Udom-kusonsri [10] who used the same stain with rainbow trout (Oncorhynchus mykiss), channel catfish (Ictalurus punctatus), goldfish (Carassius auratus) and hybrid striped bass (Morone saxatilis male X M chrysops female), and found it non toxic with no evidence of health hazards to the examined fish Pouliquen et al [18] found that exposure of turbot (Scoph-thalmus maximus) to 700 mg fluorescein/l for 96 h was not lethal As it will eventually find its way to the gills and gas-trointestinal tract, the potential effects of fluorescein dye on fish were discussed O’goshi and Serup[19]systematically re-viewed literature on fluorescein and concluded that it was useful in cutaneous research; the intradermal injection of fluorescein is being used increasingly to investigate skin con-ditions in vivo with non-invasive devices such as con focal scanning laser microscopy Sodium fluorescein was used intravenously for decades for the examination of the vascu-lature of the ocular fundus (fluorescein angiography) and
as eye drops for diagnosis of corneal erosions, and proved its safety Watson and Rosen [20] stated that the injection
of fluorescein intravenously for fundal angiography in hu-mans is associated with a high incidence of minor adverse ef-fects with a very low incidence of serious (life threatening) reactions There are no reports of oral fluorescein causing
a serious reaction, and minor adverse effects are uncommon Furthermore, under the diagnostic procedures used for iden-tifying ulcers in fish, it is unlikely that any significant con-centration of fluorescein would be taken up by humans In addition, in a farming setting, only a sample of the popula-tion may be tested at one time
In vitro antimicrobial assay (agar spot assay) This test was intended to evaluate the possible risk of fluores-cein dye in masking the diagnosis of skin injury due to bacte-ria The results showed that there was no difference between the growth of A hydrophila in the culture plates and when ex-posed to fluorescein dye Such a result indicates that fluores-cein had no bactericidal effects in our studied bacterium Thus, the dye does not appear to mask the presence of A hydrophiladuring diagnosis of skin ulceration The agar spot assay was used by a number of researchers to study the
in vitro activity of bacteria against other bacteria or biological agents[21,22] In the present study, it was essential to evaluate the risk of fluorescein masking the bacteria in ulcerated skin Fluorescein is a water soluble and diffusible agent [4,5] and can readily diffuse into the agar; if it has a bactericidal effect
it would be expected to inhibit the growth of A hydrophila, resulting in a clear inhibition zone
Table 1 Summary of field studies on fish obtained from an aquaculture facility, showing the number of gross ulcers, number of fluorescein-positive ulcers and the number of microscopic ulcers
No of
fish
No of gross lesions
No of fluorescein-positive lesions
No of microscopic ulcers
No of fish
No of gross lesions
No of fluorescein-positive lesions
No of microscopic ulcers
Trang 4Potency of fluorescein dye to fish
A bright, apple-green fluorescent colour appeared after
fluorescein treatment, indicating ulcerated areas in naturally
collected scaled (Nile tilapia) and scale-less fish (African
sharp-tooth catfish), seeTable 1 Ulcerated spots were mainly
ob-served on the ventral aspect of the head region (isthmus and
operculum((Fig 1) as well as on the lateral aspect (body
re-gion) of African catfish (Fig 2) Meanwhile, the ulcerated
areas appeared mainly at the dorsal aspect of the abdomen
in Nile tilapia (Figs 3 and 4)
A dose of 0.10 mg fluorescein/ml for 5 min was sufficient to identify even minute undetected skin ulcers The optimum fluorescein exposure concentration was different from that of Noga and Udomkusonsri [10] who used a concentration of 0.2 mg/ml for 3 min sufficient to stain the ulcers and recorded
it They stated that a lower concentration of fluorescein resulted in weaker staining of the ulcers This difference in the effectiveness of the concentrations may be attributable to the different fish species and the time of exposure, although our tested dose lies on the same safety margins that were tested
by Noga and Udomkusonsri[10] Bacteriological investigations
The importance of detecting skin damage early in bacterial infections is of major concern [21] In the present study,
A hydrophila and Streptococcus feacalis were isolated from naturally collected Nile tilapia skin ulcers with a prevalence
of 20% In African sharptooth catfish, no bacterial growth that was negative was obtained from the ulcerated skin sam-ples, (Table 2) This can be attributed to several causes of ul-cers including the aggressive behaviour of African catfish However, these ulcers can be a portal of entry for opportunis-tic bacteria Being able to detect and then culture from the ear-liest invisible lesions, will definitely improve the ability to identify important pathogens, hence bacterial diagnosis The importance of detecting early skin damage in bacterial infec-tions is exemplified by the studies of Elliott and Shotts [23]
who found that Aeromonas salmonicida, the primary bacterial pathogen of atypical furunculosis in goldfish, was only present
in the earliest stages of the disease
Histopathology Histopathological examination of lesions detected by fluores-cein from naturally affected Nile tilapia revealed ulceration
in the skin as shown by superficial to deep desquamation of the epidermal epithelium In some cases, vacuolar degenera-tion with focal erosion was evident in the cells of the stratum spinosum (Fig 5) Remarkable vacuolation with mononuclear cell infiltration was evident (Fig 6) In severely affected cases,
a complete loss of the epidermis with ulcer formation was seen where the underlying dermis was exposed to the exterior (Fig 7)
The results of the histopathological examination assessed the efficiency of the fluorescein dye in detecting various stages
of skin ulcers of the naturally affected Nile tilapia Such
Fig 1 Lateral view of African catfish; the eroded/ulcerated areas
appeared as apple green coloured areas at the lateral aspect of the
abdomen (body region)
Fig 2 Head of African catfish (isthmus and operculum) showing
apple green coloured areas indicating eroded/ulcerated areas
Fig 3 Lateral view of Nile tilapia; the eroded/ulcerated areas
appear as apple green coloured areas at the dorsal aspect of the
abdomen (body region)
Fig 4 Lateral view of Nile tilapia; the eroded/ulcerated areas appear as apple green coloured areas at the dorsal aspect of the abdomen (body region)
Trang 5findings proved that the stain is safe for use as there were no
obvious tissue reactions
Experimental infection on Nile tilapia with A hydrophila
Experimental infection of Nile tilapia using A hydrophila was carried out to assess the ability of fluorescein dye to detect early skin ulcers induced by bacterial infection Exposure of the infected fish to the dye (starting from 8 h post infection) re-sulted in detection of the minute skin ulcers and beyond the site of injection at 12 h post infection Re-isolation of A hydro-philafrom the lesions was successful at 12 h post infection Aeromons hydrophila is a ubiquitous, opportunistic bacte-rial pathogen that produces ulcerative dermatitis under stress conditions and inflicts severe losses, manifested by both high mortality and deterioration of product quality from global fisheries and fish culture[24–27].The anticipation of A hydro-phila infection as early as 12 h will definitely lower the eco-nomic losses and thus amplify the net gain from the farm
A sealed package of 250 g of fluorescein stain powder costs
130 Egyptian pounds, and can produce up to 2500 litres of the diagnostic stain Therefore, in addition to its reliability as diag-nostic procedure, it is considered to be an inexpensive and cost effective early disease detection method
Acknowledgments
We thank Dr Omar El-Tookhy, Department of Surgery, Fac-ulty of Veterinary Medicine, Cairo University, for supplying
Fig 5 Skin of naturally affected Nile tilapia showing
vacuola-tion with superficial ulceravacuola-tion (H&E stain,·100)
Fig 6 Skin of naturally affected Nile tilapia showing edema and
inflammatory cell infiltration (H&E stain,·100)
Fig 7 Skin of naturally affected Nile tilapia showing a complete loss of the epidermis and ulcer formation where the underlying dermis was exposed to the exterior (H&E stain,·100)
Table 2 Summary of field studies on fish, showing the number and percent of the bacterial isolates from fluorescein-positive ulcers
Fish Fluorescein-positive lesions Bacterial isolates Fish Fluorescein-positive ulcers Bacterial isolates
Trang 6the fluorescein dye We also thank Dr Rawhia Doghaim and
Dr Osama El-Shazly, Department of Pathology, Faculty of
Veterinary Medicine, Cairo University, for revising and
evalu-ating the gross figures and histopathology results in this study
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