Đang tải... (xem toàn văn)
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.
Journal of Advanced Research (2010) 1, 361–366 Cairo University Journal of Advanced Research ORIGINAL 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) Mai D Ibrahem a b a,* , Salah Mesalhy b Department of Fish Diseases and Management, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt Department of Pathology, Faulty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt Received 29 December 2009; revised 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 Production and hosting by Elsevier 362 M.D Ibrahem and S Mesalhy concentrations up to 0.5 mg fluorescein/ml water for up to 15 Indeed, a low dose of fluorescein (0.10 mg/ml for 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 minute 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 considered 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 minute, 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 nonvisible microscopical erosions to grossly 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 neither penetrates intact epithelium nor forms a firm bond with vital tissue However, when there is a break in the skin epithelium, fluorescein can rapidly penetrate [8], thereby staining exposed 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 results of histopathology and the use of fluorescein dye for early detection of skin lesions static dechlorinated water [11] Acclimatisation was performed in temperature-controlled aquaria at 23 ± °C The fish were fed twice a day with a balanced commercial fish pelleted diet (Zoocontrol Company, Cairo, Egypt) that contained 30% protein [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 23 °C 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) Field studies A reference culture pathogenic, A hydrophila (ATCC 7966 strain), was cultured in Trypticase Soya broth (Biomerieux, France) for 18 h at 25 °C 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 fluorescein dye dilutions (from 0.1 to 0.7 mg fluorescein/ml, each concentration 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] Naturally collected fish Fish anesthesia 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 semiintensive aquaculture facility and transported alive in well-aerated containers to the laboratory of the Department of Fish Disease and Management (FDML), Faculty of Veterinary Medicine, Cairo University Upon arrival, the fish were immediately screened for the presence of exposed skin areas using the fluorescein dye Tricaine methane sulfonate (MS222) obtained from Argent Chemical Laboratories, Redmond, WA, was used to anesthetise 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 CaCO3 as recommended by [15] Experimental studies The fish (both African sharptooth catfish and Nile tilapia) were placed (one at a time) in a solution of 0.10 mg fluorescein (FluorÒ, 10% fluorescein sodium injection, 100 mg/ml, Sigma, USA) per ml of water for The fish were then removed from the fluorescein solution and immediately rinsed by placing them in clean water for 1–2 The fish were then euthanatized 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 complete darkness Photographs were taken using digital camera Material and methods Fish For monitoring the safety and efficiency of fluorescein to detect 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 injury and transported alive in tanks to FDML Fish were acclimatised to laboratory conditions for two weeks prior to the experiment They were maintained in 40 l tanks containing Potency of the fluorescein dye to fish Naturally affected fish Diagnosis of ulcerations using fluorescein dye 363 with 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 23 °C Fish were collected one at a time from each aquarium and anesthetised with MS222 A few scales were hand-removed from each fish; then fluorescein dye was used for the detection of the induced ulcers via the regime described above Experimentally infected Nile tilapia A culture suspension of A hydrophila (ATCC 7966) was prepared by spreading onto Tryptic soya agar for 24 h at 25 °C; 4–5 colonies were suspended in sterile saline 0.85% and matched to contain 108 bacteria mlÀ1 using McFarland standard tubes The test fish were held in replicate aquaria containing 40 l of thermostatically controlled water at 23 °C (10 fish/aquarium, two replications of the treatment); fish were infected intraperitoneally 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 fluorescein 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 incubated at 25 °C for 24 h and then inspected for bacterial growth Further morphological and biochemical identification of the retrieved isolates was done according to Whitman [17] Histopathology For histopathological examination, skin lesions from naturally ulcerated Nile tilapia were fixed in 10% neutral buffered formalin, and then processed in paraffin embedding method sections of 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 behavioural abnormalities appeared in Nile tilapia post exposure The present results coincide with those of Noga and Udomkusonsri [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 (Scophthalmus maximus) to 700 mg fluorescein/l for 96 h was not lethal As it will eventually find its way to the gills and gastrointestinal tract, the potential effects of fluorescein dye on fish were discussed O’goshi and Serup [19] systematically reviewed literature on fluorescein and concluded that it was useful in cutaneous research; the intradermal injection of fluorescein is being used increasingly to investigate skin conditions in vivo with non-invasive devices such as focal scanning laser microscopy Sodium fluorescein was used intravenously for decades for the examination of the vasculature 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 humans is associated with a high incidence of minor adverse effects 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 identifying ulcers in fish, it is unlikely that any significant concentration of fluorescein would be taken up by humans In addition, in a farming setting, only a sample of the population may be tested at one time In vitro antimicrobial assay (agar spot assay) This test was intended to evaluate the possible risk of fluorescein dye in masking the diagnosis of skin injury due to bacteria The results showed that there was no difference between the growth of A hydrophila in the culture plates and when exposed to fluorescein dye Such a result indicates that fluorescein had no bactericidal effects in our studied bacterium Thus, the dye does not appear to mask the presence of A hydrophila during 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 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 Nile tilapia (Oreochromis niloticus) African catfish (Clarias gariepinus) No of fish No of gross lesions No of fluoresceinpositive lesions No of microscopic ulcers No of fish No of gross lesions No of fluoresceinpositive lesions No of microscopic ulcers 10 11 11 10 5 364 M.D Ibrahem and S Mesalhy Fig 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 Lateral view of African catfish; the eroded/ulcerated areas appeared as apple green coloured areas at the lateral aspect of the abdomen (body region) A dose of 0.10 mg fluorescein/ml for 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 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 Fig Head of African catfish (isthmus and operculum) showing apple green coloured areas indicating eroded/ulcerated areas Fig Lateral view of Nile tilapia; the eroded/ulcerated areas appear as apple green coloured areas at the dorsal aspect of the abdomen (body region) Potency 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 sharptooth catfish), see Table Ulcerated spots were mainly observed on the ventral aspect of the head region (isthmus and operculum((Fig 1) as well as on the lateral aspect (body region) of African catfish (Fig 2) Meanwhile, the ulcerated areas appeared mainly at the dorsal aspect of the abdomen in Nile tilapia (Figs and 4) 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 samples, (Table 2) This can be attributed to several causes of ulcers including the aggressive behaviour of African catfish However, these ulcers can be a portal of entry for opportunistic bacteria Being able to detect and then culture from the earliest invisible lesions, will definitely improve the ability to identify important pathogens, hence bacterial diagnosis The importance of detecting early skin damage in bacterial infections 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 fluorescein 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 degeneration 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 Diagnosis of ulcerations using fluorescein dye Table 365 Summary of field studies on fish, showing the number and percent of the bacterial isolates from fluorescein-positive ulcers Nile tilapia (Oreochromis niloticus) African catfish (Clarias gariepinus) Fish Fluorescein-positive lesions Bacterial isolates Fish Fluorescein-positive ulcers Bacterial isolates A hydrophila % Strept faecalis % 10 20 Fig Skin of naturally affected Nile tilapia showing vacuolation with superficial ulceration (H&E stain, ·100) A hydrophila % Strept faecalis % 20 10 11 0 0 Fig 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) 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 h post infection) resulted in detection of the minute skin ulcers and beyond the site of injection at 12 h post infection Re-isolation of A hydrophila from the lesions was successful at 12 h post infection Aeromons hydrophila is a ubiquitous, opportunistic bacterial 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 hydrophila infection as early as 12 h will definitely lower the economic 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 diagnostic procedure, it is considered to be an inexpensive and cost effective early disease detection method Fig Skin of naturally affected Nile tilapia showing edema and inflammatory cell infiltration (H&E stain, ·100) Acknowledgments findings proved that the stain is safe for use as there were no obvious tissue reactions We thank Dr Omar El-Tookhy, Department of Surgery, Faculty of Veterinary Medicine, Cairo University, for supplying 366 the 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 evaluating the gross figures and histopathology results in this study References [1] Noga EJ Skin ulcers in fish: Pfiesteria and other etiologies Toxicol Pathol 2000;28(6):807–23 [2] Au DW The application of histo-cytopathological biomarkers in marine pollution monitoring: a review Mar Pollut Bull 2004;48(9-10):817–34 [3] Noga EJ, Botts S, Yang MS, Avtalion R Acute stress causes skin ulceration in striped bass and hybrid bass (Morone) Vet Pathol 1998;35(2):102–7 [4] Berkow JW, Orth DH, Kelley JS Fluorescein angiography: technique and interpretation San Francisco: American Academy of Ophthalmology; 1991 [5] Pavlopoulos GP, Giannakos GI, Theodosiadis PG, Moschos MM, Iliakis EK, Theodosiadis GP Rubeola keratitis: a photographic study of corneal lesions Cornea 2008;27(4):411–6 [6] De Almeida Torres RJ, Muccioli C, Barbante Casella AM, Regonha E, Luchini A, Weiss W, et al Angiography: safety · economy [Angiografia: Seguranc¸a · economia] Arq Bras Oftalmol 2006;69(6):837–43 [7] Chan WM, Lai TYY, Lai RYK, Tang EWH, Liu DTL, Lam DSC Safety enhanced photodynamic therapy for chronic central serous chorioretinopathy: one-year results of a prospective study Retina 2008;28(1):85–93 [8] Bartlett JD, Ghormley NR, Jaanus SD, Rowsey JJ, Zimmerman TJ Ophthalmic dyes Ophthalmic drug facts Facts and comparisons St Louis, MO: Wolters Kluwer Co.; 1996 [9] Ferguson H Systemic pathology of fish: a text and atlas of comparative tissue responses in diseases of teleosts Arnes, IA: Iowa State University Press; 1989 [10] Noga EJ, Udomkusonsri P Fluorescein: a rapid, sensitive, nonlethal method for detecting skin ulceration in fish Vet Pathol 2002;39(6):726–31 [11] Best JH, Pflugmacher S, Wiegand C, Eddy FB, Metcalf JS, Codd GA Effects of enteric bacterial and cyanobacterial lipopolysaccharides and of microcystin-LR, on glutathione Stransferase activities in zebra fish (Danio rerio) Aquat Toxicol 2002;60(3–4):223–31 [12] Jauncey K, Ross B A guide to tilapia feeds and feeding Stirling, Scotland: Institute of Aquaculture, University of Stirling; 1982 M.D Ibrahem and S Mesalhy [13] Clesceri LS, Greenberg AE, Trussell RR Standard methods for the examination of water and wastewater 17th ed Washington, D.C., USA: American Public Health Association (APHA), American Water Works Association (AWWA); 1989 [14] Aly SM, Abdel-Galil AY, Abdel-Aziz GA, Mohamed MF Studies on Bacillus subtilis and Lactobacillus acidophilus, as potential probiotics, on the immune response and resistance of Tilapia nilotica (Oreochromis niloticus) to challenge infections Fish Shellfish Immunol 2008;25(1–2):128–36 [15] Davis MW, Stephenson J, Noga EJ The effect of tricaine on use of the fluorescein test for detecting skin and corneal ulcers in fish J Aquat Anim Health 2008;20(2):86–95 [16] Bancroft JD, Stevens A Theory and practice of histological techniques 4th ed Hong Kong: Churchill Livingstone; 1996 [17] Whitman KA Finfish and shellfish bacteriology manual: techniques and procedures USA: Blackwell Publishing Company; 2004 [18] Pouliquen H, Algoet M, Buchet V, Le Bris H Acute toxicity of fluorescein to turbot (Scophthalmus maximus) Vet Hum Toxicol 1995;37(6):527–9 [19] O’goshi K, Serup J Safety of sodium fluorescein for in vivo study of skin Skin Res Technol 2006;12(3):155–61 [20] Watson AP, Rosen ES Oral fluorescein angiography: reassessment of its relative safety and evaluation of optimum conditions with use of capsules Br J Ophthalmol 1990;74(8):458–61 [21] Jacobsen CN, Rosenfeldt Nielsen V, Hayford AE, Moller PL, Michaelsen KF, Paerregaard A, et al Screening of probiotic activities of forty-seven strains of Lactobacillus spp by in vitro techniques and evaluation of the colonization ability of five selected strains in humans Appl Environ Microbiol 1999;65(11):4949–56 [22] Luthi Peng Q, Dileme FB, Puhan Z Effect of glucose on glycerol bioconversion by Lactobacillus reuteri Appl Microbiol Biotechnol 2002;59(2–3):289–96 [23] Elliott DG, Shotts Jr EB Aetiology of an ulcerative disease in goldfish Carassius auratus (L.): microbiological examination of diseased fish from seven locations J Fish Dis 1980;3:133–43 [24] Cipriano RC Aeromonas hydrophila and motile aeromonad septicemias of fish Fish Dis Leaflet 2001;68:1–24 [25] Groff JM, LaPatra SE Infectious diseases impacting the commercial culture of salmonids J Appl Aquacult 2000;10(4):17–90 [26] Karunasagar I, Karunasagar I, Otta SK Disease problems affecting fish in tropical environments J Appl Aquacult 2003;13(3–4):231–49 [27] Harikrishnan R, Balasundaram C In vitro and in vivo studies of the use of some medicinal herbals against the pathogen Aeromonas hydrophila in goldfish J Aquat Anim Health 2008;20(3):165–76 ... thereby staining exposed 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 results of histopathology and the use of fluorescein dye for early... recommend the use of fluorescein dye for the evaluation of skin health in farmed fish species and the visualization of minute skin abrasions ª 2010 Cairo University Production and hosting by Elsevier