Journal of Avian Medicine and Surgery 28(4):275–279, 2014 Ó 2014 by the Association of Avian Veterinarians

Original Studies

Bacteria Isolated From the Skin of Congo African Grey Parrots (Psittacus erithacus), Budgerigars (Melopsittacus undulatus), and Cockatiels (Nymphicus hollandicus) Stephanie Lamb, DVM, Alicia Sobczynski, MS, Darius Starks, DVM, and Nicholas Sitinas, VMD, Dipl ABVP (Avian) Abstract: Little is known about the normal bacterial flora of the skin of birds. To identify the bacterial organisms that reside on the integument of companion psittacine birds in a normal physiologic state, skin cultures were taken from 75 psittacine birds comprising 25 Congo African grey parrots (Psittacus erithacus), 25 budgerigars (Melopsittacus undulatus), and 25 cockatiels (Nymphicus hollandicus). All birds were adults and in good health with no underlying identifiable diseases, had not been on antibiotics in the preceding 2 months or longer, and had no skin or feather abnormalities. Cultures were taken from the axillary region and incubated on bovine blood agar plates. Positive cultures were identified for 52 out of 75 birds, and a total of 89 bacterial colonies grew. The most frequently identified bacterial organisms belonged to the genus Staphylococcus followed by Corynebacterium. Several other genera of bacteria were also isolated. Of the 89 bacterial colonies, 25 were identified to the species level and 50 to the genus level, and 14 were identified as either a nonfermenter or coliform. Key words: bacteria, skin, psittacine birds, Congo African grey parrot, Psittacus erithacus, budgerigar, Melopsittacus undulatus, cockatiel, Nymphicus hollandicus

birds are assumed to be similar to that of other avian species. Studies of avian species such as raptors and chickens have identified numerous bacterial organisms on the skin.2,3 Culture of the uropygial gland of healthy psittacine birds identified species of Staphylococcus, but failed to identify what other species of bacteria can be present.4 The purpose of this study was to identify bacterial organisms that reside on the skin of companion psittacine birds including Congo African grey parrots (Psittacus erithacus), budgerigars (Melopsittacus undulatus), and cockatiels (Nymphicus hollandicus), housed in an indoor environment in a normal physiologic state.

Introduction Bacterial organisms are ubiquitous in nature and found residing in a variety of habitats. Many are found inhabiting vertebrate organisms in areas such as the integument, mucous membranes, and gastrointestinal systems. Bacteria in these locations are considered normal flora and many are nonpathogenic. They can even be beneficial to the host and participate in local immunity against more destructive or harmful agents.1 Bacterial species that reside on the integument are known for some vertebrate species, including humans and many domestic animals. However, little is known about bacteria residing on the skin of nondomesticated species, such as psittacine birds, in a nondiseased state. The bacterial flora that reside on the skin of psittacine

Materials and Methods The study population comprised 77 companion adult psittacine birds, consisting of 26 Congo African grey parrots, 26 budgerigars, and 25 cockatiels. For inclusion in the study, birds had to be in a normal physiologic state. This was defined as the absence of identifiable disease or

From the South Wilton Veterinary Group, 51 Danbury Rd, Wilton, CT 06897, USA (Lamb, Starks, Sitinas); and the Connecticut Veterinary Medical Diagnostic Laboratory, University of Connecticut, Department of Pathobiology and Veterinary Science, 61 North Eagleville Rd, Unit 3203, Storrs, CT 06269, USA (Sobczynski).

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disorders on physical examination and basic laboratory testing, consisting of a complete blood cell count. The skin and feathers were required to be in normal condition with no evidence of feather damage. Results of the complete blood cell count were required to be within reference intervals for the given species. Additionally, birds that were included in the study were on no medications and had not received antibiotics in the preceding 2 months. All participants came from private indoor home environments in the northeastern United States. Many were housed with other birds, and some were housed individually. All birds were handled by their owners to varying degrees, with some handled daily, whereas others were handled only a few times a month. The diets varied but included pellets, seeds, and fresh vegetables and fruits. Birds were restrained in a towel by a trained technician, and a physical examination was performed. A blood sample was collected from the right jugular vein. The examiner then applied sterile gloves, lifted the feathers from the axillary region to isolate a naturally unfeathered region of skin and, with a sterile culturette swab, swabbed the axilla for 5 seconds. The same procedure was repeated on the contralateral axillary region, with the use of one culturette per bird. Once the sample was collected, the culturette was placed in the transport media, Amies agar gel. The samples were then processed within 48 hours, which was the time period the holding medium viability was guaranteed by the manufacturer. Once transported to the laboratory, the cultures were swabbed onto blood agar plates with bovine blood. Plates were incubated at 378C with 10% CO2. After 24 hours, plates were examined for growth. If no growth or little growth was present, plates were incubated for an additional 24 hours. Samples where growth occurred were then analyzed by the MicroPlate test panel, OmniLog (Biolog Inc, Hayward, CA, USA). This test panel uses 94 biochemical tests, including 71 carbon source utilization assays and 23 chemical-sensitivity assays, to identify a broad range of gramnegative and gram-positive bacteria at the species level. Isolates for OmniLog identification were plated onto either BUG agar blood (Biolog) or chocolate agar (Northeast Laboratories, Waterville, ME, USA) and incubated at 378C for 18–24 hours. Different protocols were then followed according to the manufacturer’s recommendation to identify the bacterial species. Protocol A was used for most of the unknown organisms isolated. Protocol C1 was used for Streptococcus species

isolates. Protocol B was used on organisms that resulted in a false positive under protocol A or C1. Some of the Staphylococcus species were identified with the Staph API kit (bioMerieux, Marcy l’Etoile, France). This test uses miniaturized biochemical tests to identify organisms that belong to the genera Staphylococcus, Micrococcus, and Kocuria. Instructions included in the kit were followed accordingly. Results Of the 77 birds, 75 met the inclusion criteria: 25 Congo African grey parrots, 25 budgerigars, and 25 cockatiels. Two birds were excluded from the study: 1 African grey parrot was omitted because of mild feather chewing, and 1 budgerigar was excluded because of a high white blood cell count. The data from these 2 birds are not included in the results. A culture was classified positive if 1 or more bacterial colonies grew on culture media. A culture was classified as negative if no bacterial colonies grew. Culture results were negative in 23 of 75 (30.6%) birds. Of these negative results, 8 were samples from budgerigars, 5 were from cockatiels, and 10 were from Congo African grey parrots. Culture results were positive in 52 of 75 (69.3%) birds. Of birds with positive culture results, 24 birds had 1 bacterial organism isolated on culture, 21 birds had 2 organisms, 5 birds had 3 organisms, and 2 birds had 4 organisms. A total of 89 bacterial colonies were isolated from all birds (Table 1). The avian species from which these bacteria were isolated is also indicated in Table 1. The most frequently identified bacterial organisms belonged to the genus Staphylococcus followed by Corynebacterium. Nonfermenting bacteria that could not be further identified were the third most commonly isolated colony type. Although the OmniLog and Staph API kit were used, only 25 bacterial colonies could be identified to the species level. Fifty were identified to the genus level, and 14 were simply identified as either a nonfermenter or coliform. Discussion Bacteria exist on the skin of vertebrate species and help contribute to the normal innate immune system.1 In this study we cultured the axillary region of 75 healthy adult companion psittacine birds to isolate and identify the bacterial organisms that can be found under normal physiologic conditions. Although previous studies have identified bacterial organisms on the skin of birds, to our

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Table 1. Bacterial organisms and number of colonies isolated from the axilla of 75 companion psittacine birds by species. All birds were free of identifiable disease and considered in a normal physiologic state. An empty cell indicates no growth for the indicated avian species. Number of bacterial colonies isolated by avian species Bacterial organism

All species

Budgerigars

Cockatiels

African grey parrots

Acinetobacter species Acinetobacter genospecies 6 Acinetobacter schindleri Bacillus species Chryseobacterium species Coliform Corynebacterium species Corynebacterium glutamicum Corynebacterium lipophiloflavum Corynebacterium simulans Curtobacterium species Deinococcus species Enterobacter species Enterococcus feacium Flavimonas oryzihabitans Microbacterium arborescens Microbacterium maritypicum Microbacterium testaceum Micrococcus species Moraxella osloensis Nonfermenter Pantoea species Pantoea agglomerans Pseudomonas lundensis Shingomonas parapaucimobilis Staphylococcus species Staphylococcus aureus Staphylococcus capitis ureolyticus Staphylococcus gallinarum Staphylococcus haemolyticus Staphylococcus hominis subsp hominis Staphylococcus sciuri Staphylococcus xylosus Stenotrophomonas rhizophila Streptococcus species Total

5 1 1 1 1 2 11 1 1 1 3 1 1 1 1 1 2 2 1 2 12 1 1 1 1 21 1 1 1 1 1 1 1 1 4 89

4 1 — — — 1 3 1 — 1 2 — 1 — — — — 1 1 — 6 1 1 — — 3 — — — — — — — 1 1 29

— — — — 1 — 5 — — — — 1 — — 1 1 1 1 — 2 4 — — 1 1 11 1 1 1 1 1 1 1 — — 37

1 — 1 1 — 1 3 — 1 — 1 — — 1 — — 1 — — — 2 — — — — 7 — — — — — — — — 3 23

knowledge, this is the first study identifying the numerous bacteria that can be found on the skin of healthy Congo African grey parrots, budgerigars, and cockatiels in private households. A positive culture, defined as the growth of one or more bacterial colonies, was identified in 69.3% of birds sampled. The most common bacteria isolated were Staphylococcus and Corynebacterium species. Previous reports indicate that bacteria associated with the skin of birds include Staphylococcus, Corynebacterium, and Streptococcus species.5,6 Our results substantiate this claim for Staphylococcus and Corynebacterium species because these were the most common bacterial colonies isolated.

Combined, they accounted for 42 of the 89 (47.2%) bacteria that grew. Streptococcus species, however, were only identified in 4 of 89 (4.5%) colonies. Acinetobacter species, Microbacterium species, and nonfermenters were isolated more frequently than Streptococcus species. In a study performed in free-living goshawks (Accipiter gentilis), culture of the feet of these birds identified numerous species, including Staphylococcus aureus and Staphylococcus epidermis, but also many enteric-associated bacteria such as Escherichia coli and Enterobacter species.2 It can be speculated that some of these organisms came from prey items and were on the feet of birds

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because of contact with the intestinal tract. In our study, we also identified enteric-associated bacteria such as Enterobacter species and Enterococcus feacium. These bacteria accounted for only 2 of 89 (2.2%) colonies in the study. One explanation for the presence of these organisms is that they could have been contaminates that were relocated to the skin during normal grooming behaviors. In this same study of goshawks, potential pathogens were identified such as Pseudomonas and Pasturella species.2 In a separate study in which bacteria were isolated from the feathers of wild eastern bluebirds (Sialis sialis) by molecular analysis, Pseudomonas species was the most common isolate. However, the study results were conflicting because bacterial culture of the same feathers revealed Bacillus species to be the most common isolate.7 One colony of Pseudomonas lundensis was identified in our study and could also potentially act as a pathogen. The individual bird from which this bacterium was isolated appeared healthy and did not show any ill effects from the bacterium. Previous studies have identified the species of Staphylococcus that can be carried on the skin and mucous membranes of birds. In a study of various psittacine species in sanctuary and household environments, researchers identified Staphylococcus species in 49.4% of birds. The most common species isolated were Staphylococcus intermedius, Staphylococcus hominis subsp hominis, and S epidermis.4 In a separate study involving pigeons (Columba livia) and chickens, Staphylococcus xylosus was the most common isolate. The second most common isolate in chickens was Staphylococcus cohnii subsp cohnii, whereas in pigeons it was S intermedius.8 In our study, Staphylococcus species was the most common isolate; however, OmniLog failed to speciate it in 21 of 28 (75%) colonies. In a separate study of 1- to 8-week-old chickens, Staphylococcus species was identified from skin and nasal swabs. Of these, several isolates were randomly sampled to speciate, and results found Staphylococcus sciuri most commonly followed by S epidermis and Staphylococcus saprophyticus.3 Although we did identify S sciuri, S epidermis and S saprophyticus were not found. However, these 2 species could have been present and belonged to the colonies of Staphylococcus that were not speciated. Our study identified numerous other bacterial organisms. Organisms such as Moraxella osloensis, Pantoea species, and Curtobacterium species were detected. Many of these are opportunistic patho-

gens or soil organisms. The significance of these agents in our results is unknown. These organisms could have originated from the environments in which the birds lived or colonized the skin secondary to the owners handling the birds. One study identified that psittacine birds in private home environments were found to have species of Staphylococcus that were more commonly isolated from people. The researchers commented that this was likely secondary to colonization of birds by bacteria from their owners.4 One interesting finding was that 30.6% of birds had negative skin culture results. Bacteriostatic agents exist within the lipids of the epidermis and could potentially be the cause for this finding. The uropygial gland is known to produce alkylsubstituted fatty acids and alcohols that can retard the growth of bacterial and fungal organisms.5,9 Another hypothesis for this finding is that the feathers overlying an area may reduce the contact of skin with environmental organisms and thereby reduce the number of bacteria on the skin. Cultures were only taken from the skin in the axillary region in this study. To test this hypothesis, future studies would need to culture numerous locations on the skin and feathers and compare results. One goal of this study was to obtain a baseline for what could be considered physiologic bacterial skin flora in companion psittacine birds. This is in an effort to help practitioners interpret results of cultures taken from patients. Although primary bacterial infections on the skin of birds seem to be rare, the role of bacteria as secondary pathogens when other disease processes are occurring has been documented.5,10 Ulcerative dermatitis in psittacine birds associated with S aureus bacteria has been reported. In 1 report, a methicillin-resistant S aureus was isolated from the uropygial gland of a Congo African grey parrot.11 In this case, the inciting lesion was thought to be caused by a fall; however, evidence suggested that the bird may have been irritated in the area before the event. It could not be determined if the bacteria isolated in this case was a primary infection or secondary. In a separate report, S aureus was identified in a Nyasa lovebird (Agapornis lilianae) that had been diagnosed with an exudative to ulcerative dermatitis. However, the bacterium was isolated from the liver and not the skin. A skin culture was not performed in this case, although histopathologic results of the integument suggested a bacterial infection.12 In a study of psittacine birds exhibiting feather picking, feather pulp was cultured to determine whether bacterial organisms were present. Of 8

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birds, Staphylococcus species was isolated from only 1 sample, a feather that was associated with mononuclear perifolliculitis. All other feather pulp samples were negative for bacterial growth. Of 4 control birds, only one grew an Enterococcus species. The results of this study concluded that bacterial folliculitis is an unlikely cause for feather picking or mutilation.13 Evaluating these previous studies in companion psittacine birds and comparing those results with results obtained in this study show that the bacteria isolated are similar. This could indicate that, in the cases where disease was present, the normal flora became opportunistic pathogens. The authors of these reports could not state definitively whether the bacterial organisms they identified were likely to be primary or secondary pathogens. Unfortunately, 64 of 89 (72%) colonies could not be identified to the species level. Results of the study would certainly change if these organisms could have been further identified. We believe this is because OmniLog was used as the modality to classify the bacterial species. The organisms that we were unable to speciate are likely not in the OmniLog database. The OmniLog has a database of 1973 organisms, and it primarily focuses on pathogens, whereas the bacteria we isolated were primarily commensal, soil, and environmental organisms. To identify at the species level, OmniLog creates a phenotypic profile of each organism based on the 94 biochemical tests. Not all organisms of the same genus and species have the same exact phenotypic reactions. The phenotypic profile of each organism may have enough slight differences that the OmniLog could not match it in its database. The OmniLog also does not have crucial biochemical tests such as urea and esculin, which could be necessary to differentiate between species. To further speciate the organisms identified, polymerase reaction testing for 16S rRNA could have been performed. The goal of this study was to identify the physiologic bacterial organisms on the skin of healthy adult companion psittacine birds. Species included were Congo African grey parrots, budgerigars, and cockatiels. Positive results were found in 52 of 75 (69.3%) individuals. The most common isolates were Staphylococcus species and Corynebacterium species, which supports previous

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claims about these bacteria being the most common organisms on the skin of psittacine birds. References 1. Janeway CA, Travers P, Walport M, Shlomchik MJ. Innate immunity. In: Janeway CA, Travers P, Walport M, Shlomchik MJ, eds. Immunobiology. 6th ed. New York, NY: Garland Science Publishing; 2005:37–100. 2. Needham JR, Cooper JE, Kenward RE. A survey of the bacterial flora of the feet of free-living goshawks (Accipiter gentilis). Avian Pathol. 1979;8(3):285–288. 3. Kawano J, Shimizu A, Saitoh Y, et al. Isolation of methicillin-resistant coagulase-negative staphylococci from chickens. J Clin Microbiol. 1996;34(9):2072– 2077. 4. Briscoe JA, Morris DO, Rosenthal KL, et al. Evaluation of mucosal and seborrheic sites for staphylococci in two populations of captive psittacines. J Am Vet Med Assoc. 2009;234(7):901–905. 5. Koski MA. Dermatologic disease in psittacine birds: an investigational approach. Semin Avian Exot Pet Med. 2002;11(3):105–124. 6. Fudge AM. Diagnosis and treatment of avian bacterial diseases. Semin Avian Exot Pet Med. 2001;10(1):3–11. 7. Shawkey MD, Mills KL, Dale C, Hill GE. Microbial diversity of wild bird feathers revealed through culture-based and culture-independent techniques. Microb Ecol. 2005;50(1):40–47. 8. Nagase N, Sasaki A, Yamashita K, et al. Isolation and species distribution of staphylococci from animal and human skin. J Vet Med Sci. 2002;64(3):245–250. 9. Shawkey MD, Pillai SR, Hill GE. Chemical warfare? Effects of uropygial oil on feather-degrading bacteria. J Avian Biol. 2003;34(4):345–349. 10. Burgmann PM. Common psittacine dermatologic diseases. Semin Avian Exot Pet Med. 1995;4(4):169– 183. 11. Briscoe JA, Morris DO, Rankin SC, et al. Methicillin-resistant Staphylococcus aureus-associated dermatitis in a Congo African grey parrot (Psittacus erithacus erithacus). J Avian Med Surg. 2008;22(4):336–343. 12. Hermans K, Devriese LA, DeHerdt P, et al. Staphylococcus aureus infections in psittacine birds. Avian Pathol. 2000;29(5):411–415. 13. Rosenthal KL, Morris DO, Mauldin EA, et al. Cytologic, histologic, and microbiologic characterization of the feather pulp and follicles of featherpicking psittacine birds: a preliminary study. J Avian Med Surg. 2004;18(3):137–143.

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Bacteria Isolated From the Skin of Congo African Grey Parrots ( Psittacus erithacus ), Budgerigars ( Melopsittacus undulatus ), and Cockatiels ( Nymphicus hollandicus ).

Little is known about the normal bacterial flora of the skin of birds. To identify the bacterial organisms that reside on the integument of companion ...
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