Compendium July 2001 Small Animal/Exotics 623 EXOTICS ROUNDS Email comments/questions to compendium@medimedia.com or fax 800-556-3288 Use of Enrofloxacin in the Treatment of Piscine Mycobacteriosis Kimberly Lower, DVM University of Georgia Steve Poet, DVM, PhD Best Friend s Animal Hospital Medford, Oregon ABSTRACT: Mycobacteriosis is a common cause of chronic ulcerative skin disease in aquarium fish that can be difficult to treat. This case report describes the use of injectable enrofloxacin (0.23 mg intraperitoneally three times weekly) in the treatment of mycobacterial infection in three aquarium fish (two angelfish and one gourami). The fish were treated for a total of 12 to 14 weeks. One fish initially improved, then died after 12 weeks of therapy from presumptive central nervous system mycobacteriosis. The other two fish were cured of infection after 14 weeks, suggesting that enrofloxacin may be a useful treatment for piscine mycobacteriosis. Mycobacterial infections are common in aquarium fish and can cause various clinical signs, including chronic ulcerative skin or fin lesions, weight loss, ascites, skeletal deformities, and equilibrium abnormalities. Poor tank husbandry and stressors predispose fish to opportunistic infection. Control of mycobacteriosis is best achieved by improved tank management, disinfection, and quarantine and removal of infected fish. Although various medical therapies have been described, mycobacterial infection can be difficult to cure. This case report describes the use of injectable enrofloxacin in the treatment of mycobacteriosis in three aquarium fish. Case Report Background Three fish from a single aquarium were presented with a history of chronic ulcerative skin lesions: Fish 1 Giant golden gourami (Trichogaster trichopterus sumatranus), subadult, unknown gender Fish 2 Angelfish (Pterophyllum scalare), subadult, unknown gender Fish 3 Angelfish, subadult, unknown gender Fish 1 was obtained from a pet store 6 months previously, and skin lesions were noted 4 months before presentation. Fishes 2 and 3 were obtained from the same pet store a year prior to presentation but did not develop skin lesions until 6 to 8 weeks before presentation. Fish 2 developed skin lesions 2 to 3 weeks prior to fish 3. The fish were housed together in a 29-gal aquarium with an over-theside filter (150 gal/hour filtration capacity). Seven other fishes in the tank were unaffected, including one other golden gourami, four rasboras (Rasbora steineri), one zebra danio (Brachydanio rerio), and one cory catfish (Corydoras julii). Aquarium management consisted of monthly 30% water changes, including vacuuming the gravel. A water conditioner was used to remove chlorine and metals at each cleaning. Water temperature was maintained at 75 F (24 C). Ammonia, ph, and nitrites were monitored every 2 to 4 weeks. The ph was maintained at 7.0; ammonia and nitrites were maintained at undetectable levels. The fish were fed a commercial flake ration once daily. All fish had been acting normally and eating well until 3 days before presentation when fish 3 developed a lesion on its mouth and stopped eating. Several over-the-counter medications had been tried without success during the 6 weeks before presentation, including a kanamycin/nitrofurazone mixture (one 150-mg capsule/10 gal of water every other day for three treatments, metronidazole (one 250-mg capsule/10 gal of water every other day for three treatments), and 15% formalin (one drop/gal every other day for three treatments). The formalin treatment seemed to
624 Small Animal/Exotics Compendium July 2001 temporarily slow progression of the lesions when the fish were receiving treatment but had no long-term effects. While medications were used, the charcoal component of the filtration system was removed, and 30% water changes were performed after each treatment regimen. Evaluation and Clinical Findings Physical examination of fish 1 revealed that it was active and in good body condition. Three skin lesions were noted (a 1 0.5 cm erythematous, boggy ulcerated lesion on the left side of the head; a 3 2 mm superficial ulceration on the right lateral abdomen; and a 5 3 mm erythematous ulceration on the dorsal and lateral aspects of the caudal peduncle). Fish 2 was active and in good body condition. Five skin lesions were observed (a 3 2 mm superficial ulceration on the caudolateral right trunk; two similarly sized lesions on the left midabdominal area; a 5 4 mm deep, erythematous ulcerated lesion on the left lateral caudal peduncle that exposed underlying muscle; and a 2 2 mm superficial ulceration on the left dorsal caudal peduncle just caudal to the larger lesion). There were several linear 3- to 4 mm white fuzzy lesions associated with the tail rays, and the pectoral fins were tattered. Fish 3 was lethargic and thin. Two deep pigmented vertically oriented ulcerations (1 4 mm) were present on the right lateral head (Figure 1). A fuzzy white mass (3 1 mm) occupied most of the upper lip. The pectoral fins were tattered. A 3 2 mm indistinct subcutaneous erythematous swelling was noted in the left maxilla and resulted in obstruction of full closure of the mouth. After handling for sampling purposes, fish 3 appeared stressed and tended to float horizontally on the bottom of the container. The tank water was sampled for analysis. Levels of nitrites, nitrates, and ammonia were below detectable limits. The ph was 7.0, which is acceptable for a community tank. Cytology of a skin scraping from an ulcerative lesion (wet mount) revealed clumps of large epithelial cells and mononuclear cells with no visible organisms, suggesting granulomatous inflammation characteristic of chronic mycobacterial, bacterial, or fungal infection. Acid-fast stain of skin scraping revealed several acid-fast rod bacteria typical of mycobacteriosis or nocardiosis. Bacterial culture and sensitivity testing was performed on swabs of lesions, revealing Comamonas acidovorans, Stenotrophomonas maltophilia (neither reported to cause clinical disease), Corynebacterium species (reported to cause neurologic disease in one case 1 ), and Nocardia. Nocardia species occur in fresh water and soil. 2 Clinical infection is uncommon but has been reported to cause ulcerative skin disease and fin and tail rot in wild and experimental fish and resembles mycobacteriosis clinically. 1,2 Some success with chemotherapy using sulfonamides has been reported, but the primary recommendation is to cull the affected fish. 1,2 All bacteria isolated were sensitive to amikacin and enrofloxacin. Cultures taken from the external skin surface of fish are difficult to interpret because many potential pathogenic bacteria are opportunistic and can normally be found in the slime layer of healthy fish. A comparison of isolates from paired cultures taken from both lesions and aquarium water can help differentiate pathogens from environmental contaminants. 3 This was not performed because of cost constraints. Mycobacterial culture from the swab was negative. Skin biopsies for histopathology and culture would provide more direct evidence of a true pathogen. Visualizing intralesional bacteria and obtaining a positive culture on a deep skin sample would decrease the possibility that the bacteria isolated from the swab were not merely contaminants. A biopsy was not performed because of the increase in stress on the fish as well as the potential healing complications of performing a biopsy on an infected wound. The tentative diagnosis was nocardiosis. Mycobacterial infection was still possible because the organism is difficult to culture, resembles nocardiosis clinically, and is a more common disease. Treatment Enrofloxacin (0.1 ml [2.3 mg]) was diluted with 0.9 ml sterile 0.9% sodium chloride to make a 2.3-mg/ml solution in a 1-ml syringe. Each fish was then treated with 0.1 ml of the dilution (0.23 mg) administered intraperitoneally (IP) using a 30-gauge needle inserted just craniolaterally to the anal vent. This was administered three times weekly (suggested dose for enrofloxacin is 10 mg/kg IP or IM every 3 days 1 ). The pharmacokinetics of most therapeutic agents used in ornamental fish have not been studied; therefore, most accepted doses and therapeutic regimens are empiric. 3 A fresh enrofloxacin solution was prepared weekly. Pending the bacterial cultures, the owner was advised to wear gloves when handling the fish because mycobacteriosis (a potentially zoonotic disease) was still a possibility. Injectable therapy was chosen over medicated feed because of dosing and palatability problems, especially since fish 3 was already anorexic. Longterm tank treatments with the antibiotic were not performed because of the difficulty of adequate dosing. Only nifurpirinol and kanamycin have been shown to reach therapeutic levels in fish when absorbed through water. 3 Alternative treatment options included a medicated dip or bath, but either of these would be less effective than injectable or oral therapy. 1 The open ulcerated wounds cause increased stress on the fish to maintain normal body water osmolality and homeostasis. To make the water more isotonic, 0.3% noniodized salt (114 g salt/10 gal water) was added to the
Compendium July 2001 Small Animal/Exotics 625 Figure 1 Fish 3 prior to treatment. Note the deep ulcerative skin lesions. Figure 2 Fish 3 showed clinical improvement of mycobacterial skin lesions after 12 weeks of therapy with injectable enrofloxacin. tank. Salt is also used for prophylaxis and treatment of many freshwater ectoparasites and fungal infections, which could be potential secondary invaders of the skin lesions. 1 To decrease bacterial populations and organic waste load in the environment, water changes were increased to 40% every week. To ensure adequate oxygenation, an air stone was installed to diffuse air through the water continuously. Isolating the fish in a separate tank would reduce the chance of disease transmission to other fish; however, the recommendation was not followed because of cost and space constraints. The fish were rechecked every 2 to 4 weeks over the next 3 months. Although skin lesions on all fish gradually improved, the mouth lesion on fish 3 caused its upper lip to slough 1 week after initial examination, resulting in an irregular, truncated appearance to the upper jaw. The same fish had recurrent problems of waxing and waning anorexia and was noted to have midabdominal distention on day 14 (which had resolved by day 30). Fish 3 also had an episode of distress and acute ascites on day 50 after an antibiotic injection. The ascites persisted for 2 weeks and spontaneously resolved. The other two fish were also noted to have decreased appetites during therapy. When clinical improvement became static on day 45, the concentration of enrofloxacin was increased to 0.15 ml/1 ml saline (3.4 mg/ml) and injections were continued at a dose of 0.1 ml of the 3.4- mg/ml solution. During the treatment period, another fish in the tank (a rasbora) developed an ulcerative skin lesion on the head and died shortly thereafter on day 50. Histopathology of affected tissue from the rasbora revealed granulomatous inflammation and acid-fast organisms consistent with Mycobacterium infection. Cultures of affected tissue were also submitted and grew Staphylococcus and Vibrio alginolyticus. Mycobacterial organisms were not cultured, likely because of their fastidious growth requirements. Although mycobacteria were likely the primary pathogens, secondary invasion and sepsis caused by Vibrio and/or Staphylococcus likely contributed to death. Nocardia was not cultured from the tissue sample, which casts suspicion on the significance of the day 0 swab culture results from the other affected fish. Although other fish in the tank were possibly infected by different organisms, it was more likely that all were infected by Mycobacterium and the prior Nocardia culture reflected surface environmental contamination. Because the fish were improving, treatment with enrofloxacin injections was continued. On day 85, fish 3 developed acute neurologic signs and died. Necropsy revealed granulomatous inflammation and mycobacteria-like organisms present within the maxillary area. Although the lesions were clinically improved (Figure 2), similar inflammation was present within skin and skeletal muscle below the head ulcerations. The liver contained lipid accumulations, possibly secondary to decreased food intake. Hepatic lipidosis and resultant hepatomegaly may have accounted for the previously noted midabdominal swelling. The brain was not visualized in the initial cuts, and the pathologist had accidentally discarded the sample. Therefore, definitive diagnosis of mycobacterial central nervous system infection was not possible. Bacterial culture of head tissue grew Bacillus, Aeromonas hydrophila, and coagulase-negative Staphylococcus. A. hydrophila is a common cause of bacterial skin disease in fish 1 and is ubiquitous in the environment. Aeromonads are also commonly isolated from mucosal surfaces and internal organs of clinically healthy fish. 1,3 Poor management predisposes fish to clinical infection, and aeromonads commonly
626 Small Animal/Exotics Compendium July 2001 invade skin wounds. 1 Clinical lesions of Aeromonas infection include superficial to deep skin lesions and/or bacterial septicemia. On histopathology, the presence of free melanin or lipofuscin from ruptured melanomacrophage centers is characteristic of Aeromonas infection. Septic fish may have renal, splenic, intestinal, liver, or heart necrosis. 1 Definitive diagnosis of Aeromonas infection requires identification of clinically significant numbers of bacteria within tissue, 1 which were not noted in this case; therefore, the bacterial culture likely reflected surface contamination. The Bacillus and Staphylococcus may have been contaminants secondary to handling by the owner or may have been secondary invaders that contributed to mortality. Mycobacteria were not cultured from tissue samples, likely because of the fastidious growth requirements of these organisms. The two surviving fish were treated for 2 more weeks. Although the skin lesions were not completely resolved, the owner was unwilling to continue antibiotic injections; therefore, treatment was discontinued. Improved tank management was continued. Because of the possibility of subclinical carriage of mycobacteriosis in the recovered fish as well as persistence of the organism in the environment, 1 future introduction of new fish into the tank was discouraged. Verbal updates were obtained at 1, 2, and 6 months after discontinuation of treatment, and all skin lesions gradually became fully scaled and appeared normal. An increase in appetite of the treated fish was noted after discontinuation of antibiotic therapy. No new skin lesions developed on previously affected fish or on other fish in the tank. Discussion Ulcerative skin lesions in fish can be caused by bacterial and fungal infections, skin parasites, and even rarely by viral infections (Box 1). 1,2,4 Trauma and barbarism can also cause BOX 1 Causes of Ulcerative Skin Lesions in Fish 1,2,4 Bacterial Infections Aeromonas Enterobacter Flexibacter columnaris Flexibacter psychrophilus Mycobacterium Nocardia Proteus Staphylococcus Streptococcus Vibrio Fungal Infections Atypical water mold infection Ulcerative mycosis Skin Parasites Gyrodactylus Heteropolaria colisarum Hexamita Tetrahymena pyriformis Trichodina Trichodonella Tripartiella Viral Infections (rare) Ulcerative rhabdovirus Angelfish herpesvirus infection skin abrasions that can become secondarily infected. Poor management practices such as overfeeding, overcrowding, or inadequate sanitation can cause stress, thereby predisposing the fish to opportunistic infection. 1,2,4 Although regular water changes were being performed, the density of the fish population in the tank may have mandated more frequent water changes. Ideally, 10% to 20% water changes should be performed weekly to biweekly to reduce concentrations of organic and nitrogenous wastes. 4 Other causes of ulcerative skin disease in other species such as immune-mediated disease or drug eruption have not been reported in fish. The onset of disease in the angelfish after exposure to the affected gourami suggested a transmissible agent. The lack of improvement after treatment with kanamycin or metronidazole may have been due to inadequate dose, a resistant bacterial infection, or an agent not susceptible to antibiotics (parasite or fungus). Inadequate dose was a strong possibility. The metronidazole dose used was 750 mg; however, the appropriate dose for this tank size would be 2755 mg. 1 Similarly, the aquarium was treated with 450 mg of the kanamycin/nitrofurazone mixture (the relative amounts of each drug in the capsules were unspecified on the package), but the suggested kanamycin dose would be 5510 to 11,020 mg for a 29-gal aquarium. 1 The lack of response to formalin rules out most protozoal parasites and fungal infections; however, a secondary infection may have been present accounting for temporary slowing of disease progression. Formalin also has limited antibacterial activity. 1 Mycobacteriosis is common in aquarium fish, most often involving Mycobacterium marinum and Mycobacterium fortuitum; it causes chronic skin ulceration, emaciation, and fin erosion as well as anorexia, muscle wasting, skeletal deformity, ascites, and swimming and/or equilibrium abnormality. 1 3 Mycobacteriosis is transmitted by bacterial shedding from infected skin ulcers, from the intestine, or from ingestion of contaminated food or infected dead fish. 1,2 M. marinum is ubiquitous and has been cultured from streams, beaches, fish tanks, and tap water. 2 It usually is an opportunistic infector of stressed fish housed in suboptimal conditions; once established it is difficult to control because subclinical carriers are possible. 1 Control is best achieved by improvement of water quality, quarantine, removal of infected fish, and disinfection. Treatments have been described with chloramine-b or -T, cyclosporine, doxycycline, minocy-
628 Small Animal/Exotics Compendium July 2001 cline, penicillin, erythromycin, rifampin, streptomycin, sulfonamides, and tetracycline. 1,2 One source 2 cites the drug combination of doxycycline and rifampin as the best treatment choice. Fluoroquinolones are active against mycobacteria and penetrate into tissues and macrophages in which the organisms often reside. 5,6 In human studies, ciprofloxacin, which is a metabolite of enrofloxacin, is one of the more efficacious fluoroquinolones against opportunist mycobacteria, including M. marinum and M. fortuitum. 5,7 9 However, emergence of mycobacterial resistance to fluoroquinolones, especially when used as monotherapy, has been recognized. 5,7 9 Monotherapy is reasonable for minor disease, whereas more extensive disease requires combination therapy. 7 In one human study, monotherapy with ciprofloxacin was successful in the treatment of six of seven patients with cutaneous infection caused by M. fortuitum. However, three patients later relapsed as a result of ciprofloxacin-resistant strains. 9 Monotherapy with enrofloxacin has been used successfully to treat opportunistic mycobacteriosis in cats. 10 Antibiotic therapy for mycobacteriosis has been recommended to be continued for weeks to months. 7,11 Therapy with enrofloxacin specifically is recommended for 3 to 16 weeks, 12 or until 4 to 6 weeks past complete resolution. 13 Definitive diagnosis of mycobacteriosis in the surviving affected angelfish and gourami would have been best achieved by biopsy of lesions for histopathology and culture. This was not performed because the fish were improving with enrofloxacin treatment; therefore, therapy was not altered. Also, more invasive diagnostics would increase stress and skin trauma. The poor response of fish 3 was likely caused by the development of a resistant strain of mycobacteria after prolonged monotherapy with enrofloxacin. Also, poor initial body condition and intermittent periods of anorexia may have contributed to immunosuppression. Mycobacteriosis in fish is potentially zoonotic and can cause localized granulomas or nonhealing skin ulcerations in humans, usually at sites of previous skin injury. 1,2 Lesions become apparent 2 to 3 weeks postexposure. 2 Human epidemics of granulomatous skin lesions have occurred after swimming in infected water, and this mode of transmission appears more common than infections from exposure to infected tropical fish tanks. 2 The high prevalence of mycobacteriosis in pet fish and the apparently low number of infections in fish owners suggest that healthy people may be resistant to infection. M. marinum infections, however, have been reported in HIV-infected individuals after contact with pet fish or aquarium water. Therefore, owners should be advised to wear gloves when handling infected fish or cleaning contaminated aquariums. 1 The residual lesions on the remaining two fish resolved slowly over several months even though antibiotic therapy had been discontinued. The residual lesions may have been caused by persistent mycobacterial infection but alternatively may have been due to scarring as a sequela of the previous severe inflammation and ulceration. The scale beds, which occupy the upper zone of the dermis (stratum spongiosum) 14 may have been damaged in these areas, resulting in the persistent skin and scale defects. Restoration of full epidermal thickness and dermal repair is very slow in fish. Epidermal wound closure is achieved by migration of a single layer of Malpighian cells from neighboring epithelium to fill the defect. Therefore, the surrounding epidermis is markedly thinned for a prolonged period by the loss of cells to the defect. 14 Summary Use of enrofloxacin in the treatment of mycobacteriosis in fish has not previously been described, and this case presentation illustrates the potential effectiveness of enrofloxacin in the therapy of cutaneous (but not disseminated) mycobacteriosis in ornamental fish. Administering intraperitoneal injections to fish every 2 to 3 days is labor-intensive; therefore, patient and owner selection are important. Injection of smaller fish would not only be difficult but impractical. Although complications secondary to the injections (e.g., transient peritonitis, decreased appetite) were observed, in general, the fish seemed to tolerate the procedure fairly well. Alternatives to injectable therapy (fluoroquinolone-medicated food or baths) should be further investigated. References 1. Noga EJ: Fish Disease: Diagnosis and Treatment. St. Louis, Mosby, 1996. 2. Stoskopf M: Fish Medicine. Philadelphia, WB Saunders Co, 1993. 3. Citino S: Basic ornamental fish medicine, in Kirk R, Bonagura JD (eds): Current Veterinary Therapy X: Small Animal Practice. Philadelphia, WB Saunders Co, 1989, 703 721. 4. Moriello KA, Mason IS: Handbook of Small Animal Dermatology. Oxford, England, Elsevier Science Ltd, 1995. 5. Alangaden GJ, Lerner SA: The clinical use of fluoroquinolones for the treatment of mycobacterial diseases. Clin Infect Dis 25(5):1213 1221, 1997. 6. Hooper D, Wolfson J: The fluoroquinolones: Pharmacology, clinical uses, and toxicities in humans. Antimicrob Agents Chemother 28:716 721, 1985. 7. Wallace Jr RJ: Treatment of infections caused by rapidly growing mycobacteria in the era of the newer macrolides. Res Microbiol 147(1 2):30 35, 1996. 8. Cambau E, Jarlier V: Resistance to quinolones in mycobacteria. Res Microbiol 147(1 2):52 59, 1996. 9. Wallace Jr RJ, Bedsole G, Sumter G, et al: Activities of ciprofloxacin and ofloxacin against rapidly growing mycobacteria with demonstration of acquired resistance following single-drug therapy. Antimicrob Agents Chemother 34(1):65 70, 1990. 10. Studdert V, Hughes K: Treatment of opportunistic mycobacterial infections with enrofloxacin in cats. JAVMA 201(9): 1388 1390, 1992. (continues on page 692)
692 Small Animal/Exotics Compendium July 2001 Exotics Rounds (continued from page 628) 11. Jordan H: Canine and feline mycobacterial infection, in Kirk R, Bonagura JD (eds): Current Veterinary Therapy XII: Small Animal Practice. Philadelphia, WB Saunders Co, 1995, pp 320 323. 12. Greene C: Infectious Diseases of the Dog and Cat. Philadelphia, WB Saunders Co, 1998. 13. Scott D, Miller W, Griffen C (eds): Muller & Kirk s Small Animal Dermatology. Philadelphia, WB Saunders Co, 1995. 14. Roberts RJ: Aquatic problems and piscine solutions, in Kwochka K, Willemse T, von Tscharner C (eds): Advances in Veterinary Dermatology, vol 3. Oxford, England, Butterworth Heinemann, 1998, pp 37 45.