<p> 1</p><p>Running head</p><p>D. AGAMARUM INFECTIONS IN SPINY-TAILED LIZARDS </p><p>Title </p><p>5FINDINGS OF DEVRIESEA AGAMARUM ASSOCIATED INFECTIONS IN SPINY-</p><p>TAILED LIZARDS (UROMASTYX SP.) IN CROATIA</p><p>Authors</p><p>Maja Lukac, D.V.M., Danijela Horvatek-Tomic, D.V.M., Ph.D., and Estella Prukner-</p><p>10Radovcic, D.V.M., Ph.D.</p><p>From the: Department of Avian Diseases with Clinic, Faculty of Veterinary Medicine, </p><p>University of Zagreb 10000, Croatia.</p><p>15Corresponding author </p><p>Maja Lukac, Department of Avian Diseases with Clinic, Faculty of Veterinary Medicine, </p><p>Heinzelova 55, Zagreb 10000, Croatia</p><p>Tel.: +3851 2390 281; e-mail: [email protected]</p><p>20 2</p><p>Abstract: Actinobacteria are common agents causing skin diseases in captive desert </p><p> lizards, including the recently described Devriesea agamarum. To date, infections caused by </p><p>D. agamarum, their signs and treatment have been described only by the research group from </p><p>Belgium that isolated the species in 2008. This paper presents the signs indicating the </p><p>5possibility of a D. agamarum associated infection, such as scaly changes around the mouth in </p><p> a juvenile lizard (Uromastyx ocelatta), and dermatitis in the form of skin scaling around the </p><p> mouth and cloaca and over the dorsal part of the body in a group of four spiny-tailed lizards </p><p>(Uromastyx geyri). In two animals, a swelling of the front limbs with the loss of some toes </p><p> was also present, as a sign not previously described in relation to D. agamarum infections. </p><p>10Bacteriological analysis of dermal lesion samples confirmed the presence of D. agamarum in </p><p> all subjects. The treatment with ceftazidime was carried out and the signs of dermatitis </p><p> resolved, followed by negative bacteriological findings. This is the first report describing the </p><p> diagnostics, detailed clinical picture with newly described signs, and treatment of lizards with </p><p>D. agamarum associated skin lesions out of Belgium. The results also confirm the </p><p>15effectiveness of the systemic administration of 3rd generation cephalosporin antibiotics in </p><p> combination with local chlorhexidine in the treatment of D. agamarum infections. </p><p>Key words: Dermatitis, Devriesea agamarum, desert lizards, Uromastyx sp. 3</p><p>INTRODUCTION</p><p>Bacterial skin infections in captive desert lizards most commonly occur secondary to </p><p> traumatic injuries or burns, or may be caused by inadequate housing conditions including </p><p>5excessive humidity, a dirty substrate, malnutrition or too low temperature which may </p><p> negatively affect the immune system.4,11 In desert lizards, these infections are manifested in </p><p> the form of chronic dermatitis, cheilitis or occasionally septicemia, often associated with the </p><p> presence of coryneform bacteria either as causative agents or as complicating factors.5,14 </p><p>These infections are most commonly attributed to bacteria from the genus Dermabacter; </p><p>10however, a new species called Devriesea agamarum was recently isolated, predominantly </p><p> from skin lesions of spiny-tailed lizards (Uromastyx sp.).12,13,15 D. agamarum is part of the </p><p> normal microflora of the oral cavity in healthy bearded dragons and these lizards can be </p><p> asymptomatic carriers for spiny-tailed lizards, causing chronic dermatitis, cheilitis and </p><p> occasionally septicemia in the latter species8. The bacterium seems to be facultatively </p><p>15pathogenic, causing a dermal disease when the integrity of lizard’s skin is breached.8 The </p><p> mortality in spiny-tailed lizards is low and the disease is most commonly restricted to skin </p><p> problems, though the mortality rate can reach up to 100% in other agamid lizards.8,13,15 </p><p>Currently, the available information about the signs, diagnostics and treatment of D. </p><p> agamarum associated infections in lizards were provided by the Belgian research group that </p><p>20first isolated this pathogen.6,8-10,13 </p><p>This paper describes the diagnostic methods and treatment of D. agamarum associated </p><p> infections in a group of spiny-tailed lizards (Uromastyx sp.), including some cases with </p><p> clinical signs not previously described in association with D. agamarum infections.</p><p>25 CASE SERIES 4</p><p>Case Report 1</p><p>A juvenile spiny-tailed lizard (Uromastyx ocelatta), several months of age, imported from </p><p>5France two weeks before the visit to the clinic, presented with signs of scaly changes around </p><p> the mouth. The changes were mild in the nature and restricted to the lips. No other visible </p><p> signs of disease or behavioral changes were present. Skin scrapings from the affected areas </p><p> were obtained using a sharp sterile curette and sent for microbiological analysis. </p><p>10Case Report 2</p><p>A group of four spiny-tailed lizards (Uromastyx geyri) presented for the clinical </p><p> examination. According to the case history obtained from the owner, the animals were wild </p><p> caught and transferred to Croatia six years before the clinical visit. The animals were housed </p><p>15in a terrarium 170 x 60 x 60 cm in size with 6 compartments and with a substrate of ordinary </p><p> sand of 0-4 mm granulation. The daily temperature was approximately 30oC, with a hot spot </p><p> providing a temperature of 40oC. The night temperature was 22-25oC. The animals were fed </p><p> with dandelion, clover leaves and flowers, plantain, Chinese cabbage, carrot, pollen, oat </p><p> cereals and a fine seed of leguminous plants. The animals hibernated for approximately three </p><p>20months during the winter period. During this period, the heating lamp was switched off </p><p> though a UVB lamp remained on and the temperature was ambient. The photoperiod was </p><p> always 13 hours of light and 11 hours of darkness. In 1 to 2 months following the last </p><p> hibernation, the dermatitis signs occurred in all animals in the form of more or less expressed </p><p> skin scaling around the mouth (Figure 1) and cloaca, and over the dorsal part of the body </p><p>25(Figure 2). The scaling was the same in appearance in all animals though its intensity varied 5</p><p> from site to site and from animal to animal. In two of four animals a swelling of the front </p><p> limbs with the loss of toes was also present (Figure 3). Skin scrapings from affected areas </p><p> were obtained from all animals using a sharp sterile curette and sent for microbiological </p><p> analysis. </p><p>5</p><p>Microbiological diagnostics</p><p>The skin scrapings samples taken from the dermal changes described above were plated </p><p> using a microbiological loop as described by Brown on (i) Columbia agar supplemented with </p><p>105% sheep blood (BBL, Becton Dickinson and Company, Sparks, Maryland 21152, USA) and </p><p> incubated under microaerophilic condition, (ii) on neutral agar (Difco Nutrient Agar, Becton, </p><p>Dickinson and Company, Sparks, MD 21152, USA) and (iii) on brilliant green agar (brilliant </p><p> green agar -modified, Oxoid Ltd., Basingstoke, Hampshire RG24 8PW, England) and </p><p> incubated under aerobic conditions.1 All plates were incubated for 24 hours at 37oC and </p><p>15examined after 24 and 48 h. After 24 hours of incubation small, smooth, mucoid colonies </p><p> were formed, surrounded by a moderately expressed zone of alpha-haemolysis on the </p><p>Columbia agar supplemented with 5% sheep blood. The typical bacterial colonies were </p><p> randomly selected and examined microscopically to confirm their morphology. Catalase and </p><p> oxidase reactions were conducted as described by Brown, and Gram staining was carried out.1</p><p>20Bacteria isolated on blood agar plates from all examined samples were described as D. </p><p> agamarum based on growth performance, positive catalase and oxidase reaction and Gram </p><p> staining that revealed short positive rods. There was no bacterial growth on other agar plates. </p><p>For identification of fungi, including Nannizziopsis vriesii, skin scrapings were tested at the </p><p>Department of Pathology, Bacteriology and Avian Diseases of the Faculty of Veterinary </p><p>25Medicine, Ghent University, Merelbeke, Belgium, by culturing the samples on Sabouraud 6</p><p> dextrose agar (Oxoid GmbH, D-46467, Wesel, Germany) for 4 days of incubation at 25°C as </p><p> described by Hellebuyck et al.7 No fungal growth was detected.</p><p>The antimicrobial susceptibility of the isolated D. agamarum strains were detected by </p><p>5using the disk diffusion test on Mueller-Hinton II agar (Mueller Hinton II Agar, Becton </p><p>Dickinson and Company, Sparks, Maryland 21152, USA), according to the Clinical and </p><p>Laboratory Standards Institute (CLSI) recommendations.3 To detect the most effective </p><p> treatment, the sensitivity was determined for enrofloxacin (Bayer HealthCare, Animal </p><p>Healthcare Division, Shawnee Mission, Kansas 66201, USA), ceftazidime, erythromycin, </p><p>10gentamicin, penicillin, and tetracycline (Bio-Rad Laboratories, Hercules, California 94547, </p><p>USA). Zones of inhibition were described according to the recommendation of the British </p><p>Society for Antimicrobial Chemotherapy (BSAC) recommendation for coryneform bacteria </p><p> belonging to the same subclass Actinobacteridae as D. agamarum, since there are no </p><p> susceptibility references for disk diffusion for Devriesea species.2</p><p>15</p><p>Treatment and microbiological/clinical outcomes </p><p>The highest in vitro sensitivity was observed to ceftazidime, erythromycin and tetracycline</p><p> and the highest resistance to enrofloxacin, penicillin and gentamicin. According to these </p><p>20results, a course of treatment was recommended to the owners of infected animals. In Case 1, </p><p> the owner did not accept the treatment due to the high price of the medication and the further </p><p> development of the disease and its outcome are unknown. The four animals from Case 2 were</p><p> placed on systemic ceftazidime (Mirocef ®, Pliva, Zagreb 10000, Croatia) therapy at a dose </p><p> of 10 mg/kg IM every third day for 15 days (An Martel, personal communication). 7</p><p>After 15-day systemic ceftazidime treatment and a local rinsing of the lesions with a 0.1% </p><p> chlorhexidine solution (Plivasept glukonat, Pliva, Croatia), the scaly changes were resolved in</p><p> three of the animals and remained barely visible in the fourth. The swellings of distal parts of </p><p> the extremities remained unchanged and surgical treatment of those changes was </p><p>5recommended. </p><p>Microbiological analysis of scrapings taken at the end of treatment did not reveal the presence</p><p> of D. agamarum or other bacteria in any sample.</p><p>The owner was instructed to completely disinfect the terrarium and equipment with 1% </p><p> chlorhexidine and to keep the animal’s environment dry. </p><p>10</p><p>DISCUSSION</p><p>Signs were presented indicating the possibility of D. agamarum associated infection, i.e. </p><p> scaly changes around the mouth in a juvenile lizard (Uromastyx ocelatta), and dermatitis in </p><p>15the form of skin scaling around the mouth and cloaca and over the dorsal part of the body in a </p><p> group of four spiny-tailed lizards (Uromastyx geyri), were presented. Bacteriological analysis </p><p> of the dermal changes collected from infected animals confirmed the presence of D. </p><p> agamarum in all cases examined at the clinic. </p><p>D. agamarum was recently designated to a novel genus and species of coryneform </p><p>20bacteria.13 Some other bacterial species like Pasteurella and Actinobacillus are very similar to </p><p>Devriesea and no single phenotypic feature reliably distinguishes between them. To reconfirm</p><p> our findings, the same skin scrapings were also sent to the Department of Pathology, </p><p>Bacteriology and Avian Diseases of the Faculty of Veterinary Medicine, Ghent University, </p><p>Merelbeke, Belgium, where the presence of D. agamarum was confirmed in all cases 8</p><p> described above. Also, the presence of fungus Nannizziopsis vriesii in those skin scraping </p><p> samples was excluded by the Belgian laboratory. </p><p>D. agamarum belongs to a group of bacteria involved in reptile diseases causing chronic </p><p> skin disease and/or septicemia in lizards.8,14 The disease in the collection of lizards is a </p><p>5chronic problem, which, if left untreated, persists for several years and compromises captive </p><p> maintenance.6 The disease can be transmitted via direct or indirect contact, and the entire </p><p> collection of animals can be infected within several months.14,15 Since the number of pet </p><p> reptiles including lizards is rapidly increasing in Croatia and in other parts of the world, the </p><p> risk of bringing various infective agents including D. agamarum into existing collections is </p><p>10also increasing. According to the results published by Hellebuyck et al., dermatitis associated </p><p> with D. agamarum infections seems to develop more frequently in desert lizards species in </p><p> captivity, and these authors isolated D. agamarum from all cases of cheilitis combined with </p><p> dermatitis in spiny-tailed lizards (Uromastyx spp.).8 </p><p>The clinical signs described by Pasmans et al. and Hellebuyck et al. were also noticeable in</p><p>15our cases, and lead to the suspicion of D. agamarum infection, in two lizards distinct swelling </p><p> of distal parts of the front limbs accompanied with a skin dryness and loss of toes were also </p><p> presented.8,14 Although the exact pathogenesis of these signs was not elucidated, the presence </p><p> of fungi and bacteria other than Devriesea was excluded by skin sraping cultures. These signs </p><p> have not previously been described in the context of D. agamarum infections in dab lizards. </p><p>20 Regarding treatment, Hellebuyck et al. reported that ceftiofur at a dose of 5 mg/kg BW in </p><p>24-hour intervals is the most effective, resolving the infection with this bacterium in an </p><p> average interval of 18 days.10 In our case, systemic ceftazidime at a dose of 10 mg/kg BW </p><p> every third day in combination with local chlorhexidine was used, 5 doses in total, and proved</p><p> to be an effective alternative to ceftiofur. The signs of dermatitis were resolved after the </p><p>25treatment, followed by negative bacteriological findings. 9</p><p>CONCLUSION</p><p>D. agamarum, a recently described Gram positive pathogen, was recognized as one of primary</p><p>5etiological agents in skin diseases of reptiles in captivity, particularly in spiny-tailed lizards. </p><p>These infections spread quickly and may affect the entire animal collection in a short period </p><p> of time. Since the popularity of reptiles as pet animals is continuously increasing, the </p><p> educated veterinarians able to diagnose and treat reptilian diseases including bacterial </p><p> infections, among them D. agamarum infections, are needed. One of the prerequisites for this </p><p>10is reliable recognition of the clinical symptomatology as part of the diagnostic process. Since </p><p> it was shown that spiny-tailed lizards are prone to D. agamarum infections, while bearded </p><p> dragons can be asymptomatic carriers of this infective agent, it is also very important to </p><p> educate owners not to keep different reptile species together in order to preserve the health of </p><p> the animal population and to prevent the spread of disease. To our knowledge, this paper is </p><p>15the first report describing the diagnostics, detailed clinical picture and description of novel </p><p> signs, and treatment of lizards with D. agamarum associated skin lesions out of Belgium. </p><p>Results also confirm the effectiveness of systemic administration of the 3rd generation </p><p> cephalosporin antibiotics in the treatment of D. agamarum infections. </p><p>20</p><p>Acknowledgment: The authors would like to thank Dr. An Martel, Department of </p><p>Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent </p><p>University, Merelbeke, Belgium, for providing confirmation of bacterial isolation and useful </p><p> advice regarding the treatment of infected animals.</p><p>25 10</p><p>LITERATURE CITED</p><p>1. Brown, A. E. 2005. Benson’s Microbiological Applications: Laboratory Manual in </p><p>General Microbiology. McGraw-Hill Companies Inc., New York. Pp. 121-159.</p><p>5 2. 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Beernaert, K. </p><p>Chiers, P. De Backer, F. Haesebrouck, and A. Martel. 2010. Cutaneous hyalohyphomycosis </p><p> in a girdled lizard (Cordylus giganteus) caused by the Chrysosporium anamorph of </p><p>20Nannizziopsis vriesii and successful treatment with voriconazole. Vet Dermatol. 21: 429-</p><p>433.</p><p>8. Hellebuyck, T., A. Martel, K. Chiers, F. Haesebrouck, and F. Pasmans. 2009. </p><p>Devriesea agamarum causes dermatitis in bearded dragons (Pogona vitticeps). Vet Microbiol. </p><p>134: 267-271. 11</p><p>9. Hellebuyck T., F. Pasmans, M. Blooi, F. Haesebrouck, and A. Martel. 2011. </p><p>Prolonged environmental persistence requires efficient disinfection procedures to control </p><p>Devriesea agamarum-associated disease in lizards. Lett. Appl. Microbiol. 52: 28-32.</p><p>10. Hellebuyck T., F. Pasmans, F. Haesebrouck, and A. Martel. 2009. Designing a </p><p>5successful antimicrobial treatment against Devriesea agamarum infections in lizards. Vet. </p><p>Microbiol. 139: 189-192.</p><p>11. Hoppmann E., and H. Wilson Barron. 2007. Dermatology in reptiles. J Exotic </p><p>Pet Med. 16: 210-224.</p><p>12. Koplos, P., M. Garner, T. Besser, R. Nordhausen, and R. Monaco. 2000. </p><p>10Cheilitis in lizards of the genus Uromastyx associated with a filamentous Gram positive </p><p> bacterium. Proceedings of the Association of Reptilian and Amphibian Veterinarians. Pp. </p><p>73–75.</p><p>13. Martel A., F. Pasmans, T. Hellebuyck, F. Haesebrouck, and P. Vandamme. </p><p>2008. Devriesea agamarum gen. nov., sp. nov., a novel actinobacterium associated with </p><p>15dermatitis and septicaemia in agamid lizards. Int. J. Syst. Evol. Microbiol. 58: 2206-2209.</p><p>14. Pasmans F., S. Blahak, A. Martel, and N. Pantchev. 2008. Introducing reptiles </p><p> into a captive collection: the role of the veterinarian. Vet. J. 175: 53-68.</p><p>15. Pasmans, F., A. Martel, M. van Heerden, L. Devriese, A. Decostere, and F. </p><p>Haesebrouck. 2004. Dermatitis and septicaemia in a captive population of Agama impalearis </p><p>20caused by unknown Actinobacteria. In: Seybold, J., and F. Mutschmann (eds.). Proceedings </p><p> of the 7th International Symposium of Pathology and Medicine of Reptiles and Amphibians, </p><p>Chimaira, Frankfurt, Germany. P. 303 12</p><p>Figure captions 5</p><p>Figure 1. Uromastyx geyri, typical skin scaling around the mouth (arrow)</p><p>Figure 2. Uromastyx geyri, skin scaling over the over the dorsal part of the body (arrow) 10 Figure 3. Uromastyx geyri, a swelling of the front limb with the loss of some toes</p><p>15</p><p>20</p>