Enteric Clostridial Colonization in Psittacine Birds

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550 Small Animal/Exotics Compendium July 2002 EXOTICS ROUNDS Comments? Questions? Email: compendium@medimedia.com Web: VetLearn.

1 550 Small Animal/Exotics Compendium July 2002 EXOTICS ROUNDS Comments? Questions? Web: VetLearn.com Fax: Enteric Clostridial Colonization in Psittacine Birds G. Heather Wilson, DVM, DABVP (Avian) a Deidre K. Fontenot, DVM b Cheryl B. Greenacre, DVM, DABVP (Avian) c Branson W. Ritchie, DVM, DABVP (Avian) a a University of Georgia b Disney s Animal Kingdom Lake Buena Vista, FL c University of Tennessee ABSTRACT: Bacteria with morphologic characteristics suggestive of Clostridium species were demonstrated by culture or microscopic examination (Gram s stain) of feces in two groups of psittacine birds. The most common clinical change in affected birds was passage of a fetid, loose stool. Although only a few species of Clostridium were cultured, the variable characteristics of the bacteria detected in excrement suggest that multiple Clostridium-like species may colonize the intestinal tract of psittaciforms. The role that each of these Clostridium-like organisms plays in disease has not been determined, but their detection in birds with clinical signs of enteritis warrants attention. Case 1 Background A clostridial-associated epornitic was documented in a psittacine aviary with approximately 263 chicks and 320 adults representing 70 different species. All of the adults were maintained in large, suspended, welded-wire enclosures in six separate buildings that were partially roofed and enclosed with wire. Adults were fed a commercial formulated diet in addition to fresh produce and some seed or nuts during the breeding season. Hygiene and preventive medical care were comparatively excellent. The aviary s preventive health program required full necropsy and histologic examination of all dead birds, including dead-in-shell chicks. Numerous free-range chickens had access around, but not within, the aviary. The nursery was located in two adjoining rooms in the client s house, approximately 20 miles from the aviary. These rooms had been successfully used as a nursery for approximately 5 years. All chicks in the nursery were routinely monitored with fecal Gram s staining. Evaluation and Clinical Findings Clinical changes associated with enteritis were first noted in the chicks. The most consistent signs were a fetid smell to the excrement and failure to gain weight despite a normal feeding response and cropemptying time. Other associated signs included diarrhea or soft stool, dehydration, stunting (Figure 1), Figure 1 A conure chick (Aratinga species) with clostridial enteritis and classic signs of stunted growth. abnormal plumage development, depression, inappetence, regurgitation, and crop stasis. Several morphologically different gram-positive, spore-forming rods of varying sizes (0.3 to to 10 µm) suggestive of Clostridium species were demonstrated microscopically in approximately 23% (60 of 263) of the chicks Exotics Rounds is designed to disseminate information on emerging disease in exotic animals. Readers interested in submitting papers for Exotics Rounds can contact Dr. Michelle Hawkins (A Pet Care Clinic, th Ave. W, Mountlake Terrace, WA 98043; ; fax ; hawkvet@attbi.com) or Dr. Lisa Tell (Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, Tupper Hall, Room 2108, Davis, CA 95616; ; fax ; latell@usdavis.edu).

Compendium July 2002 Enteric Clostridial Colonization 551 Figure 2 Clostridium-like organisms detected on fecal Gram s stain may have differing spore characteristics suggestive of infection with

2 Compendium July 2002 Enteric Clostridial Colonization 551 Figure 2 Clostridium-like organisms detected on fecal Gram s stain may have differing spore characteristics suggestive of infection with multiple species. (Oil immersion; original magnification 100.) from one breeding season. Approximately 20% (12 of 60) of those chicks had subclinical signs (including no detectable odor to their droppings) despite the microscopic detection of Clostridium-like bacteria. Crop and fecal cytology using Gram s stain or other stains and, in some cases, aerobic and anaerobic culture or complete blood counts were used to rule out other potential causes of enteritis in the affected chicks. Clostridium sordelli and Clostridium perfringens were isolated from the feces of one severely affected Moluccan cockatoo (Cacatua moluccensis) chick. The National Veterinary Services Laboratory in Ames, Iowa, classified the isolate of C. perfringens as toxin type A. C. perfringens was the most frequently isolated (8 of 10 isolates) anaerobe from the excrement of affected birds. Treatment In an attempt to limit the spread of Clostridium-like organisms in the nursery, all chicks testing positive for the organisms were treated regardless of clinical signs. Chicks were treated with clindamycin (Antirobe Aquadrops, Upjohn) 50 mg/kg PO bid for 5 to 7 days. Most chicks with clinical signs responded to treatment within 24 hours. Treatment in these chicks was considered successful when clinical signs improved and the quantity of Clostridium-like organisms in the feces was reduced to microscopically undetectable levels. Posttreatment weight gain in some affected chicks was remarkable. None of the treated chicks died from a documented clostri- dial infection; however, Clostridium tertium was isolated from one dead-in-shell chick. The breeding flock was considered to be one potential source of chick exposure to Clostridium species. Adults from the breeding collection were screened with fecal Gram s stain. Four morphologically different gram-positive, spore-forming rods of varying sizes (0.3 to to 10 µm) suggestive of Clostridium species were demonstrated microscopically in approximately 17% (54 of 320) of the adult population (Figure 2). Contaminated hand-feeding formula was considered to be another potential source of infection for the chicks in the nursery. The hand-feeding formula was a commercial brand that used egg as a protein source. Samples from two new, properly stored, recently opened bags of formula were cultured in aerobic and anaerobic conditions. C. perfringens, Enterococcus species, and Klebsiella pneumoniae were recovered from one bag, and Bacillus species and Clostridium subterminale were recovered from the other. Case 2 Background A large gram-positive, spore-forming rod morphologically suggestive of Clostridium species was detected using fecal Gram s stain as part of a preshipment examination of a 20- year-old female eclectus parrot (Eclectus roratus) maintained in a zoologic collection. Questioning of the keeper staff revealed that this bird recently had voluminous, malodorous stool. Clostridium could not be recovered from the excrement using anaerobic techniques. The bird was treated with clindamycin (100 mg/kg/day PO for 7 days). Posttreatment fecal cytology demonstrated the absence of gram-positive, sporeforming rods. Evaluation and Clinical Findings One month later, the female s cagemate was reported to have undigested food in its stool and was found dead the following morning. Microscopic evaluation of the feces and hepatic impression smears using Gram s stain revealed numerous gram-positive, spore-forming rods. However, anaerobic culture of the feces and liver was negative. Gross pathologic findings included thin body condition, diffuse intestinal gas, and hemorrhage in the intestines. Histopathology revealed hemorrhagic enteritis, hepatitis, and nephritis (all with intralesional bacilli); severe, diffuse, thoracoabdominal air sac edema; and pulmonary anthracosis. Physical examinations and fecal cytology were routinely performed biannually on all psittacines in the collection. However, clinical findings in these two eclectus parrots prompted immediate fecal evaluation of all psittacine birds in the propagation facility and on exhibit. The gross characteristic of the excrement from each of these birds was noted and a sample collected. Excrement was further evaluated by direct saline examination, fecal flotation with zinc sulfate, and microscope using Gram s stain and a modified acid-fast stain. Loose or discolored stool was noted in 11.6% (5 of 43) of the fecal samples. All five of the abnormal stool samples were positive for gram-positive, spore-forming rods. In addition,

552 Small Animal/Exotics Compendium July 2002 Figure 3 Fecal Gram s stain with two morphologically distinct, gram-positive, spore-forming rods suggestive of Clostridium species.

(Oil immersion; original magnification 100.) Clostridium-like organisms were detected in four birds with normalappearing droppings.

3 552 Small Animal/Exotics Compendium July 2002 Figure 3 Fecal Gram s stain with two morphologically distinct, gram-positive, spore-forming rods suggestive of Clostridium species. (Oil immersion; original magnification 100.) Figure 4 Fecal Gram s stain from a psittacine demonstrates normal flora (100% gram-positive, non spore-forming rods and cocci). (Oil immersion; original magnification 100.) Clostridium-like organisms were detected in four birds with normalappearing droppings. The five clinically affected birds were in the same propagation facility that housed the index eclectus parrots. Two pairs of golden conures, positive for the organisms but with subclinical signs, were maintained on exhibit at the opposite end of the zoologic park. Clostridial organisms were not recovered from the feces of any birds (in which organisms had been detected by microscope) by using anaerobic microbiologic culture techniques. Microscopically, spores were noted to be terminal in some organisms and central in others, suggesting that at least two different species of Clostridium were present (Figure 3). Treatment All five birds with clinical signs in the propagation facility were treated via gavage with clindamycin (100 mg/kg/day PO for 7 days). Posttreatment fecal Gram s stains were negative for Clostridium-like organisms (Figure 4). An attempt was made to identify potential risk factors for organism transmission. The diet consisted of 75% (by volume) commercial parrot pellets and 25% fruit/vegetable mixture. Food was changed twice daily. All birds were fed in elevated food pans away from defecation sites. Gram s stains of food (both fresh and collected at the end of the day) were negative for Clostridium-like organisms. However, food contamination was suspected because of the high number of waterfowl and raptors in the collection that were cared for by the same keepers. Strict hygiene, including footbaths and handwashing between exhibits, was enforced, and birds in psittacine exhibits were fed and their cages cleaned before daily care of the waterfowl and raptor exhibits. No further problems were noted after these husbandry changes were implemented. Discussion The cases reported here suggest that clostridial-associated problems may be more common than realized, and potential infections may be missed if clinicians are using culture to the exclusion of fecal cytology to evaluate patients with signs of gastrointestinal (GI) disease. While we are unaware of any experimental studies that would satisfy Koch s postulates with respect to clostridial-associated disease in psittacine birds, the cases in this report at least suggest that the presence of organisms with morphologic characteristics suggestive of Clostridium species are a marker for certain clinical signs consistent with enteritis that are commonly responsive to treatment using antibiotics with an anaerobic spectrum. Bacillus species may also appear microscopically as gram-positive, spore-forming rods but are considered normal flora in the GI tract of psittacine birds and are unlikely to cause the clinical signs described. Culture may help differentiate between Bacillus and Clostridium species. Clostridium species are considered autochthonous flora in carnivorous and ground-feeding birds (e.g., falconiforms, phasianiforms, anseriforms). In these species, clostridial overgrowth sufficient to cause disease can occur. 1,2 Clostridium species are not considered autochthonous flora in psittaciforms and passeriforms, and clostridial enteritis in these species has been less frequently documented than in ground-feeding birds The chronic problems described in the first aviary in this report suggest that there was a persistent source of exposure. The breeding adults were considered a potential source because some chicks were briefly parent raised before being introduced to the nursery. A single, definitive source of exposure was never identified, but clostridial spores are extremely resistant to inactivation and can remain infectious for years. 13 It was undetermined what role, if any, the low level of Clostridium species recovered from the hand-feeding formula played in the epornitic. In most birds described in this

4 Compendium July 2002 Enteric Clostridial Colonization 553 report, Clostridium-like organisms were detected using fecal Gram s stain. Culture was frequently ineffective for recovering these organisms, even when large quantities of clostridial bacteria could be demonstrated by fecal Gram s stain and anaerobic techniques were used. Some clostridia (e.g., C. perfringens) are facultative anaerobes that can occasionally be cultured in aerobic conditions. Other clostridia are strict anaerobes that are difficult to culture unless clinical specimens are collected and maintained in absolute anaerobic conditions. Confirmation that a suspected organism is a Clostridium species may be accomplished by performing anaerobic cultures on fresh (recently evacuated) feces or a fresh cloacal swab. Samples collected for culture should be immediately placed in beefbroth media or semisolid agar culturettes for transport to a laboratory. A nucleic-acid sequencing technique for identifying C. perfringens in psittacine birds was recently described. 14 Microscopically, Clostridium species appear as gram-positive, spore-forming rods, but their morphology and spore characteristics vary widely by species. The spore can be eccentric, subterminal or terminal, and invisible (in some species [e.g., C. perfringens] because the developing spore does not displace the sporangiophore). In other Clostridium species, the spores can be identified as a swollen area that, depending on the stage of development, may remain unstained (clear) or can stain slightly darker with Gram s stain than the vegetative portion of the organism. C. perfringens is a ubiquitous organism that is 1.0 µm wide and 4 to 8 µm long. 1 This organism produces potent exotoxins that have been associated with disease in a variety of birds. 15 The clinical course of clostridial infections may vary, depending on the site of infection, the species of Clostridium, and the species of bird. Although exotoxins are important in most clostridial diseases, the potency of the toxins produced and the invasive ability of the clostridia vary. 16 In most cases, clostridial organisms are considered opportunistic pathogens. It has been proposed that clostridial enteritis is more common in animals with reduced GI motility; low levels of dietary fiber; high levels of dietary protein; medication-induced changes in normal GI flora; or concomitant bacterial, viral, or parasitic infection. 1 3 Clostridium species are gas producers and have been documented in nectivorous birds, particularly lories, fed high-sugar diets. 4 7 In one outbreak involving a group of non-loridae psittacines, seven birds died over a 30-day period. The source of the Clostridium was traced to contaminated peanuts, and further deaths were prevented by removing the contaminated food and treating the birds with erythromycin. 8 In another reported case, Clostridium species were identified by fecal Gram s stain in a Catalina macaw (Ara species) that passed voluminous, tan, malodorous droppings. (Normal, freshly evacuated psittacine bird feces should have no detectable odor.) The bird was otherwise considered normal and responded to clindamycin (50 mg/kg PO bid). 9 Antibiotics that are frequently recommended for treating clostridial infections include chloramphenicol, metronidazole, fluoroquinolones, potentiated penicillins, and clindamycin. Some reports suggest that 70% of C. perfringens are resistant to clindamycin. 1 However, nearly all of the birds in this report responded to treatment with clindamycin. These differences may reflect a variance between in vitro and in vivo sensitivity of Clostridium cultured from psittacine birds. Alternatively, Clostridium could have been a concomitant colonizer in birds with enteritis caused by other organisms that were susceptible to clindamycin. While the pharmacokinetics of clindamycin have not been studied extensively in any veterinary species, the elimination half-life in dogs is reportedly 3 to 5 hours. 17 Therefore, in this report, birds that were more easily medicated received a dosage of 50 mg/kg bid. The published dosage suggested for use in pigeons is 100 mg/kg/day. 18 Further studies on the pharmacokinetics of this drug in psittacine birds are needed, but both of these dosages appeared to be effective with no clinically obvious side effects. References 1. Pisani J, Speer B, Howerth EW, Clubb S: Clostridial infections in psittacine birds. J Avian Med Surg 12(3): , Gerlach H: Bacteria, in Ritchie BW, Harrison GJ, Harrison LR, et al (eds): Avian Medicine: Principles and Application. Lake Worth, FL, Wingers Publishing, 1994, pp Wilson GH, Greenacre CB, Howerth EW, et al: Ascaridosis in a group of psittacine birds. J Avian Med Surg 13(1):32 39, Gill J: Parrot diseases and nutrition. Caged Bird Med and Surg Symp: , O'Tool D, Mills K, Ellis R, et al: Clostridial enteritis in red lories (Eos bornea). J Vet Diag Invest 5: , McOrist S, Reece RL: Clostridial enteritis in free-living lorikeets (Trichoglossus spp.). Avian Pathol 21: , Ferrell ST, Tell L: Clostridium tertium infection in a rainbow lorikeet (Trichoglossus haematodus haematodus) with enteritis. J Avian Med Surg 15(3): , Dhillon AS: An outbreak of enteritis in a psittacine flock. Proc Assoc Avian Vet : , Kolmstetter C, Carpenter JW, Ernst S: What s your diagnosis? J Avian Med Surg 9(3): , Gould WJ: Common digestive tract disorders in pet birds. Vet Med 90(1):40 52, Rupiper DJ: Hemorrhagic enteritis in a group of great-billed parrots (Tanygnathus megalorhynchos). J Avian Med Surg (4)7: , Hess L, Bartick T, Hoefer H: Clostrid

5 554 Small Animal/Exotics Compendium July 2002 ium tertium infection in a moluccan cockatoo (Cacatua moluccensis) with megacolon. J Avian Med Surg 12(1):30 35, Oderdonk AB, Allen SD: Clostridium, in Murray PR (ed): Manual of Clinical Microbiology. Washington, DC, ASM Press, 1995, pp Garner M, Cui J, Murray S, Lung N: Fatal acute clostridial enteritis and detection of the causative agent. Proc Assoc Avian Vet : , Cato EP, George WL, Finegold SM: Clostridium, in Sneath PHA (ed): Bergey s Manual of Systemic Bacteriology, ed 8. Baltimore, Williams & Wilkins, 1986, pp Quinn PJ, Carter ME, Markay BK, Carter GR: Clinical Veterinary Microbiology. London, Mosby, Plumb DC: Veterinary Drug Handbook. White Bear Lake, MN, Pharmavet Publishing, 1991, p Carpenter JW, Mashima TY, Rupiper DJ: Exotic Animal Formulary. Manhattan, KS, Greystone Publications, 1996, p 98.

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