ࡱ> '`PbjbjVwVw 844~H(*2 2 2 8j F!,*rH,~!!"!!!"."" GGGGGGG$IhLjH2+""2+2+H!!,H9992+b !!G92+G99:>,T ?!r! 2 66> GBH0rH>.pL7xpL ? ?LpLU?"6%9&D'A"""HHB9^"""rH2+2+2+2+***D****** Running head D. AGAMARUM INFECTIONS IN SPINY-TAILED LIZARDS Title FINDINGS OF DEVRIESEA AGAMARUM ASSOCIATED INFECTIONS IN SPINY-TAILED LIZARDS (UROMASTYX SP.) IN CROATIA Authors Maja Lukac, D.V.M., Danijela Horvatek-Tomic, D.V.M., Ph.D., and Estella Prukner-Radovcic, D.V.M., Ph.D. From the: Department of Avian Diseases with Clinic, Faculty of Veterinary Medicine, University of Zagreb 10000, Croatia. Corresponding author Maja Lukac, Department of Avian Diseases with Clinic, Faculty of Veterinary Medicine, Heinzelova 55, Zagreb 10000, Croatia Tel.: +3851 2390 281; e-mail: maja.lukac@vef.hr Abstract: Actinobacteria are common agents causing skin diseases in captive desert lizards, including the recently described Devriesea agamarum. To date, infections caused by D. agamarum, their signs and treatment have been described only by the research group from Belgium that isolated the species in 2008. This paper presents the signs indicating the possibility of a D. agamarum associated infection, such as scaly changes around the mouth in a juvenile lizard (Uromastyx ocelatta), and dermatitis in the form of skin scaling around the mouth and cloaca and over the dorsal part of the body in a group of four spiny-tailed lizards (Uromastyx geyri). In two animals, a swelling of the front limbs with the loss of some toes was also present, as a sign not previously described in relation to D. agamarum infections. Bacteriological analysis of dermal lesion samples confirmed the presence of D. agamarum in all subjects. The treatment with ceftazidime was carried out and the signs of dermatitis resolved, followed by negative bacteriological findings. This is the first report describing the diagnostics, detailed clinical picture with newly described signs, and treatment of lizards with D. agamarum associated skin lesions out of Belgium. The results also confirm the effectiveness of the systemic administration of 3rd generation cephalosporin antibiotics in combination with local chlorhexidine in the treatment of D. agamarum infections. Key words: Dermatitis, Devriesea agamarum, desert lizards, Uromastyx sp. INTRODUCTION Bacterial skin infections in captive desert lizards most commonly occur secondary to traumatic injuries or burns, or may be caused by inadequate housing conditions including excessive humidity, a dirty substrate, malnutrition or too low temperature which may negatively affect the immune system.4,11 In desert lizards, these infections are manifested in the form of chronic dermatitis, cheilitis or occasionally septicemia, often associated with the presence of coryneform bacteria either as causative agents or as complicating factors.5,14 These infections are most commonly attributed to bacteria from the genus Dermabacter; however, a new species called Devriesea agamarum was recently isolated, predominantly from skin lesions of spiny-tailed lizards (Uromastyx sp.).12,13,15 D. agamarum is part of the normal microflora of the oral cavity in healthy bearded dragons and these lizards can be asymptomatic carriers for spiny-tailed lizards, causing chronic dermatitis, cheilitis and occasionally septicemia in the latter species8. The bacterium seems to be facultatively pathogenic, causing a dermal disease when the integrity of lizards skin is breached.8 The mortality in spiny-tailed lizards is low and the disease is most commonly restricted to skin problems, though the mortality rate can reach up to 100% in other agamid lizards.8,13,15 Currently, the available information about the signs, diagnostics and treatment of D. agamarum associated infections in lizards were provided by the Belgian research group that first isolated this pathogen.6,8-10,13 This paper describes the diagnostic methods and treatment of D. agamarum associated infections in a group of spiny-tailed lizards (Uromastyx sp.), including some cases with clinical signs not previously described in association with D. agamarum infections. CASE SERIES Case Report 1 A juvenile spiny-tailed lizard (Uromastyx ocelatta), several months of age, imported from France two weeks before the visit to the clinic, presented with signs of scaly changes around the mouth. The changes were mild in the nature and restricted to the lips. No other visible signs of disease or behavioral changes were present. Skin scrapings from the affected areas were obtained using a sharp sterile curette and sent for microbiological analysis. Case Report 2 A group of four spiny-tailed lizards (Uromastyx geyri) presented for the clinical examination. According to the case history obtained from the owner, the animals were wild caught and transferred to Croatia six years before the clinical visit. The animals were housed in a terrarium 170 x 60 x 60 cm in size with 6 compartments and with a substrate of ordinary sand of 0-4 mm granulation. The daily temperature was approximately 30oC, with a hot spot providing a temperature of 40oC. The night temperature was 22-25oC. The animals were fed with dandelion, clover leaves and flowers, plantain, Chinese cabbage, carrot, pollen, oat cereals and a fine seed of leguminous plants. The animals hibernated for approximately three months during the winter period. During this period, the heating lamp was switched off though a UVB lamp remained on and the temperature was ambient. The photoperiod was always 13 hours of light and 11 hours of darkness. In 1 to 2 months following the last hibernation, the dermatitis signs occurred in all animals in the form of more or less expressed skin scaling around the mouth (Figure 1) and cloaca, and over the dorsal part of the body (Figure 2). The scaling was the same in appearance in all animals though its intensity varied from site to site and from animal to animal. In two of four animals a swelling of the front limbs with the loss of toes was also present (Figure 3). Skin scrapings from affected areas were obtained from all animals using a sharp sterile curette and sent for microbiological analysis. Microbiological diagnostics The skin scrapings samples taken from the dermal changes described above were plated using a microbiological loop as described by Brown on (i) Columbia agar supplemented with 5% sheep blood (BBL, Becton Dickinson and Company, Sparks, Maryland 21152, USA) and incubated under microaerophilic condition, (ii) on neutral agar (Difco Nutrient Agar, Becton, Dickinson and Company, Sparks, MD 21152, USA) and (iii) on brilliant green agar (brilliant green agar -modified, Oxoid Ltd., Basingstoke, Hampshire RG24 8PW, England) and incubated under aerobic conditions.1 All plates were incubated for 24 hours at 37oC and examined after 24 and 48 h. After 24 hours of incubation small, smooth, mucoid colonies were formed, surrounded by a moderately expressed zone of alpha-haemolysis on the Columbia agar supplemented with 5% sheep blood. The typical bacterial colonies were randomly selected and examined microscopically to confirm their morphology. Catalase and oxidase reactions were conducted as described by Brown, and Gram staining was carried out.1 Bacteria isolated on blood agar plates from all examined samples were described as D. agamarum based on growth performance, positive catalase and oxidase reaction and Gram staining that revealed short positive rods. There was no bacterial growth on other agar plates. For identification of fungi, including Nannizziopsis vriesii, skin scrapings were tested at the Department of Pathology, Bacteriology and Avian Diseases of the Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium, by culturing the samples on Sabouraud dextrose agar (Oxoid GmbH, D-46467, Wesel, Germany) for 4 days of incubation at 25C as described by Hellebuyck et al.7 No fungal growth was detected. The antimicrobial susceptibility of the isolated D. agamarum strains were detected by using the disk diffusion test on Mueller-Hinton II agar (Mueller Hinton II Agar, Becton Dickinson and Company, Sparks, Maryland 21152, USA), according to the Clinical and Laboratory Standards Institute (CLSI) recommendations.3 To detect the most effective treatment, the sensitivity was determined for enrofloxacin (Bayer HealthCare, Animal Healthcare Division, Shawnee Mission, Kansas 66201, USA), ceftazidime, erythromycin, gentamicin, penicillin, and tetracycline (Bio-Rad Laboratories, Hercules, California 94547, USA). Zones of inhibition were described according to the recommendation of the British Society for Antimicrobial Chemotherapy (BSAC) recommendation for coryneform bacteria belonging to the same subclass Actinobacteridae as D. agamarum, since there are no susceptibility references for disk diffusion for Devriesea species.2 Treatment and microbiological/clinical outcomes The highest in vitro sensitivity was observed to ceftazidime, erythromycin and tetracycline and the highest resistance to enrofloxacin, penicillin and gentamicin. According to these results, a course of treatment was recommended to the owners of infected animals. In Case 1, the owner did not accept the treatment due to the high price of the medication and the further development of the disease and its outcome are unknown. The four animals from Case 2 were placed on systemic ceftazidime (Mirocef , Pliva, Zagreb 10000, Croatia) therapy at a dose of 10 mg/kg IM every third day for 15 days (An Martel, personal communication). After 15-day systemic ceftazidime treatment and a local rinsing of the lesions with a 0.1% chlorhexidine solution (Plivasept glukonat, Pliva, Croatia), the scaly changes were resolved in three of the animals and remained barely visible in the fourth. The swellings of distal parts of the extremities remained unchanged and surgical treatment of those changes was recommended. Microbiological analysis of scrapings taken at the end of treatment did not reveal the presence of D. agamarum or other bacteria in any sample. The owner was instructed to completely disinfect the terrarium and equipment with 1% chlorhexidine and to keep the animals environment dry. DISCUSSION Signs were presented indicating the possibility of D. agamarum associated infection, i.e. scaly changes around the mouth in a juvenile lizard (Uromastyx ocelatta), and dermatitis in the form of skin scaling around the mouth and cloaca and over the dorsal part of the body in a group of four spiny-tailed lizards (Uromastyx geyri), were presented. Bacteriological analysis of the dermal changes collected from infected animals confirmed the presence of D. agamarum in all cases examined at the clinic. D. agamarum was recently designated to a novel genus and species of coryneform bacteria.13 Some other bacterial species like Pasteurella and Actinobacillus are very similar to Devriesea and no single phenotypic feature reliably distinguishes between them. To reconfirm our findings, the same skin scrapings were also sent to the Department of Pathology, Bacteriology and Avian Diseases of the Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium, where the presence of D. agamarum was confirmed in all cases described above. Also, the presence of fungus Nannizziopsis vriesii in those skin scraping samples was excluded by the Belgian laboratory. D. agamarum belongs to a group of bacteria involved in reptile diseases causing chronic skin disease and/or septicemia in lizards.8,14 The disease in the collection of lizards is a chronic problem, which, if left untreated, persists for several years and compromises captive maintenance.6 The disease can be transmitted via direct or indirect contact, and the entire collection of animals can be infected within several months.14,15 Since the number of pet reptiles including lizards is rapidly increasing in Croatia and in other parts of the world, the risk of bringing various infective agents including D. agamarum into existing collections is also increasing. According to the results published by Hellebuyck et al., dermatitis associated with D. agamarum infections seems to develop more frequently in desert lizards species in captivity, and these authors isolated D. agamarum from all cases of cheilitis combined with dermatitis in spiny-tailed lizards (Uromastyx spp.).8 The clinical signs described by Pasmans et al. and Hellebuyck et al. were also noticeable in our cases, and lead to the suspicion of D. agamarum infection, in two lizards distinct swelling of distal parts of the front limbs accompanied with a skin dryness and loss of toes were also presented.8,14 Although the exact pathogenesis of these signs was not elucidated, the presence of fungi and bacteria other than Devriesea was excluded by skin sraping cultures. These signs have not previously been described in the context of D. agamarum infections in dab lizards. Regarding treatment, Hellebuyck et al. reported that ceftiofur at a dose of 5 mg/kg BW in 24-hour intervals is the most effective, resolving the infection with this bacterium in an average interval of 18 days.10 In our case, systemic ceftazidime at a dose of 10 mg/kg BW every third day in combination with local chlorhexidine was used, 5 doses in total, and proved to be an effective alternative to ceftiofur. The signs of dermatitis were resolved after the treatment, followed by negative bacteriological findings. CONCLUSION D. agamarum, a recently described Gram positive pathogen, was recognized as one of primary etiological agents in skin diseases of reptiles in captivity, particularly in spiny-tailed lizards. These infections spread quickly and may affect the entire animal collection in a short period of time. Since the popularity of reptiles as pet animals is continuously increasing, the educated veterinarians able to diagnose and treat reptilian diseases including bacterial infections, among them D. agamarum infections, are needed. One of the prerequisites for this is reliable recognition of the clinical symptomatology as part of the diagnostic process. Since it was shown that spiny-tailed lizards are prone to D. agamarum infections, while bearded dragons can be asymptomatic carriers of this infective agent, it is also very important to educate owners not to keep different reptile species together in order to preserve the health of the animal population and to prevent the spread of disease. To our knowledge, this paper is the first report describing the diagnostics, detailed clinical picture and description of novel signs, and treatment of lizards with D. agamarum associated skin lesions out of Belgium. Results also confirm the effectiveness of systemic administration of the 3rd generation cephalosporin antibiotics in the treatment of D. agamarum infections. Acknowledgment: The authors would like to thank Dr. An Martel, Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium, for providing confirmation of bacterial isolation and useful advice regarding the treatment of infected animals. LITERATURE CITED Brown, A. E. 2005. Bensons Microbiological Applications: Laboratory Manual in General Microbiology. McGraw-Hill Companies Inc., New York. Pp. 121-159. British Society for Antimicrobial Chemotherapy. 2011. BSAC Methods for Antimicrobial Susceptibility Testing. Version 10.2. Clinical and Laboratory Standards Institute. 2012. Performance standards for Antimicrobial Disk Susceptibility Tests: Approved Standard-Eleventh Edition. M02-A11. Harkewicz K. A. 2000. Dermatology of reptiles: A clinical approach to diagnosis and treatment. Vet Clin North Am Exotic Anim Pract. 4: 441-461. Harkewicz K. A. 2002. Dermatologic problems in reptiles. Semin Avian Exotic Pet Med. 11: 151-161. Devloo R., A. Martel, T. Hellebuyck, F. Haesebrouck, and F. Pasmans. 2011. Bearded dragons (Pogona vitticeps) asymptomatically infected with Devriesea agamarum are a source of persistent clinical infection in captive colonies of dab lizards (Uromastxy sp.). Vet. Microbiol. 150: 297-301. Hellebuyck, T., K. Baert, F. Pasmans, L. Van Waeyenberghe, L. Beernaert, K. Chiers, P. De Backer, F. Haesebrouck, and A. Martel. 2010. Cutaneous hyalohyphomycosis in a girdled lizard (Cordylus giganteus) caused by the Chrysosporium anamorph of Nannizziopsis vriesii and successful treatment with voriconazole. Vet Dermatol. 21: 429-433. Hellebuyck, T., A. Martel, K. Chiers, F. Haesebrouck, and F. Pasmans. 2009. Devriesea agamarum causes dermatitis in bearded dragons (Pogona vitticeps). Vet Microbiol. 134: 267-271. Hellebuyck T., F. Pasmans, M. Blooi, F. Haesebrouck, and A. Martel. 2011. Prolonged environmental persistence requires efficient disinfection procedures to control Devriesea agamarum-associated disease in lizards. Lett. Appl. Microbiol. 52: 28-32. Hellebuyck T., F. Pasmans, F. Haesebrouck, and A. Martel. 2009. Designing a successful antimicrobial treatment against Devriesea agamarum infections in lizards. Vet. Microbiol. 139: 189-192. Hoppmann E., and H. Wilson Barron. 2007. Dermatology in reptiles. J Exotic Pet Med. 16: 210-224. Koplos, P., M. Garner, T. Besser, R. Nordhausen, and R. Monaco. 2000. Cheilitis in lizards of the genus Uromastyx associated with a filamentous Gram positive bacterium. Proceedings of the Association of Reptilian and Amphibian Veterinarians. Pp. 7375. Martel A., F. Pasmans, T. Hellebuyck, F. Haesebrouck, and P. Vandamme. 2008. Devriesea agamarum gen. nov., sp. nov., a novel actinobacterium associated with dermatitis and septicaemia in agamid lizards. Int. J. Syst. Evol. Microbiol. 58: 2206-2209. Pasmans F., S. Blahak, A. Martel, and N. Pantchev. 2008. Introducing reptiles into a captive collection: the role of the veterinarian. Vet. J. 175: 53-68. Pasmans, F., A. Martel, M. van Heerden, L. Devriese, A. Decostere, and F. Haesebrouck. 2004. Dermatitis and septicaemia in a captive population of Agama impalearis caused by unknown Actinobacteria. In: Seybold, J., and F. Mutschmann (eds.). Proceedings of the 7th International Symposium of Pathology and Medicine of Reptiles and Amphibians, Chimaira, Frankfurt, Germany. P. 303  HYPERLINK "http://www.nhbs.com/frankfurt_contributions_to_natural_history_tefno_124938.html"  Figure captions Figure 1. Uromastyx geyri, typical skin scaling around the mouth (arrow) Figure 2. Uromastyx geyri, skin scaling over the over the dorsal part of the body (arrow) Figure 3. 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