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Pseudomonas aeruginosa infection in a group of captive Humboldt penguins (Spheniscus humboldti).

Abstract: Nine Humboldt penguins (Spheniscus humboldti), between 1 and 1.5 years old and kept at Zoo Dresden, developed local and systemic infections with various opportunistic pathogens within a period of 4 months. Affected birds died peracutely without preceding symptoms or showed various clinical signs, including separation from conspecifics, reduced food intake, lethargy, dyspnea, swelling of the salt glands, and ocular discharge. One bird showed central nervous signs, including seizures. Pathologic examination of deceased birds revealed severe necrotizing inflammation of the mucous membranes and deep structures of the glottis, trachea, nasal sinus, and conchae and granulomatous inflammation of the salt glands. Further findings were airsacculitis, pneumonia, hepatitis, conjunctivitis, and myositis. Pseudomonas aeruginosa was the predominant pathogen in 7 cases. Six penguins died or were euthanatized, whereas 3 penguins that received systemic antibiotic treatment with tobramycin (10 mg/kg IM q24h for 10 days) showed rapid clinical improvement. Insufficient turnover rate of the filtration system, biofilm formation on pipe surfaces, and other factors are assumed to have promoted pathogen buildup in the pool water and subsequent infection.

Key words: Pseudomonas aeruginosa, mortality, coliform bacteria, biofilm, opportunistic pathogen, tobramycin, avian, Humboldt penguin, Spheniscus humboldti

Clinical Report

Zoo Dresden has kept Humboldt penguins (Spheniscus humboldti) successfully since the early 1970s. The penguin exhibit at Zoo Dresden comprises an indoor and an outdoor section. Whereas the outdoor part includes a nonchlorinated, 150 000-L, freshwater pool and a pebbled land area with nesting sites, the smaller indoor part comprises a coated-concrete land area and a shallow pool, which is connected to the main pool through a channel. Both areas are always accessible for the birds. A water filtration system composed of sand and activated carbon filters and an ozonator with a turnover rate of 75 000 L/h manages water quality. The pH of the pool is set to 7.7. The penguins are fed thawed Atlantic herring (Clupea harengus) until satiation twice daily, which is supplemented with 1 Fish Eater Tablet (Mazuri Zoo Foods, Claus GmbH, Limburgerhof, Germany) PO q24h per animal.

During a 4-month period in 2014, all 9 penguins kept at that time suffered from severe infections with various opportunistic bacterial pathogens, as described in the following case reports. In 7 cases, almost identical lesions, caused by Pseudomonas aeruginosa, were found within the oral cavity, pharynx, or nasal sinus. Six penguins died or were euthanatized, whereas only 3 penguins were successfully treated.

Case 1

A 1-year-old, male penguin died peracutely, without any preceding clinical signs. Body condition was excellent. Necropsy revealed yellow plaques adhering to the mucous membranes of the conchae and oral cavity as well as a few nodular lesions between 1 to 8 mm in diameter in the cranial air sacs. Histologically, severe necrotic lesions of the conchae and oral mucosa with many intralesional bacterial colonies were found; from which, P aeruginosa and Morganella morganii were cultured in high and moderate amounts, respectively. Chronic, granulomatous, mycotic airsacculitis was seen, and Aspergillus fumigatus was cultured from the lesions. The liver was mildly congested with slightly swollen hepatocytes, showing multifocal, perivascular nonpurulent inflammation.

[FIGURE 1 OMITTED]

Case 2

The second penguin, also a 1-year-old male, showed dyspnea, weakness, and a firm swelling of the right salt gland for 3 days. During this period, the bird's condition deteriorated rapidly. At first presentation on day 3, the bird was found moribund and was euthanatized. Necropsy confirmed marked swelling of the right salt gland in conjunction with multiple, small, white, superficial foci (Fig 1). Histologically, severe pyogranulomatous inflammation with intralesional bacterial colonies was found. Granulomas were surrounded by a mixed population of leukocytes, including multinucleated giant cells. Morphology of intralesional bacteria was consistent with P aeruginosa, which was confirmed by culture. A small, focal mycotic granuloma was found in 1 thoracic air sac. The liver showed multifocal, periportal accumulation of heterophils, lymphocytes, and plasma cells, associated with karyorrhectic necrosis. Mild follicular hyperplasia of the spleen was noted. Body condition was good, with the bird weighing 3.96 kg

Case 3

Initial signs in a 1.3-year-old, male penguin consisted of a slight swelling of the right salt gland, along with serous ocular discharge from the ipsilateral eye and moderate physical weakness. Blood sampling was not done at initial examination to avoid additional restraint. Because the results of bacterial cultures from the previous cases were not yet available, empirical therapy with enrofloxacin (10 mg/kg PO q24h) and terbinafine (50 mg/kg PO q24h) was initiated. Within 2 days of treatment, the clinical condition improved markedly. Ocular discharge was no longer evident, and the swelling of the salt gland as well as the physical weakness were notably reduced. After 7 days, the bird's clinical condition was normal. Medications were stopped after 11 days. Four weeks later, the bird was found dead in the morning without preceding clinical signs. As in case 1, body condition was excellent, and the bird's body weight was 4.2 kg.

At necropsy, a small amount of serous ocular discharge and a few yellow plaques on the mucous membranes in the choanal region were noted. Histologic examination revealed deep focal necrosis of the choana, reaching into the submucosal layer. Large amounts of degenerated leukocytes, fibrin, and bacteria adhered to the lesions. In the submucosa of the conjunctivae, perivascular infiltration with mononuclear leukocytes was present, and the cervical air sac showed a mild infiltration with heterophils and macrophages. No fungal structures were detected microscopically or in culture. The diagnosis was moderate purulent, ulcerative pharyngitis and mild, chronic, catarrhal conjunctivitis as well as subacute fibrinous to purulent airsacculitis of the cervical air sac. Pseudomonas aeruginosa was cultured from ocular, oral, and meningeal swabs, although the meninges did not show signs of inflammation. A number of other bacterial agents were found in multiple organs, including Staphylococcus aureus (liver, kidneys, lungs, meninges), Enterococcus faecalis (lungs, conjunctivae, oral cavity), Aeromonas species (conjunctivae), and Clostridium perfringens (intestines), making a definite etiologie diagnosis impossible. Polymerase chain reaction (PCR) testing of splenic tissue and a conjunctival swab for Chlamydia psittaci were both negative.

Case 4

On initial presentation, this 1.2-year-old, female penguin showed moderate physical weakness, partially closed eyes, and reduced activity and food intake. Body weight was 3.9 kg. Therapy with enrofloxacin (10 mg/kg PO q24h for 21 days), itraconazole (10 mg/kg PO q12h), and a silymarin supplement (56 mg PO q24h for 28 days) was initiated. Within 7 days of treatment, the bird became increasingly strong and active. Food intake improved to normal within 10 days. After 14 days, the penguin was considered to have fully recovered, but medications were continued as prescribed. At 56 days after initial presentation, the penguin suddenly had a seizure for approximately 10 seconds at feeding time, described as "epilepti-form" by the observing keepers. However, it quickly recovered and started eating again. Two days after this incident, a small, circumscribed opacity of the right cornea was noted, which did not stain with fluorescein. Because the bird did not show any further clinical signs, no therapy was initiated. The corneal opacity was still present on day 79 after initial presentation, but at that time, serous, frothy ocular discharge; slight swelling of the salt glands; and reduced overall activity level were noted. A corneal swab was taken; from which, only P aeruginosa was cultured. As the isolate showed good sensitivity for enrofloxacin in vitro, oral medication with enrofloxacin, terbinafine, and silymarin was initiated again on day 83. Additionally, meloxicam (0.5 mg/kg PO q24h) was given to provide analgesia. Increased activity and food intake, decrease of ocular discharge, reduced size of the corneal opacity, and reduced swelling of the salt glands were noted initially. However, clinical signs reappeared on day 93, including paleness of the featherless skin, marked swelling of the salt glands, ocular discharge, and highly reduced activity. When the bird was observed swimming upside down, being disorientated and uncoordinated 97 days after initial presentation, euthanasia was elected. A blood sample taken during anesthesia before euthanasia was submitted for a complete blood cell count (CBC) and serum biochemical analysis (Clinic for Birds, Reptiles, Amphibians and Ornamental Fish, Ludwig-Maximilians-University of Munich, Munich, Germany). Because of insufficient quality of blood slides and degeneration of leukocytes, a CBC could not be obtained. Results of hematologic testing showed the bird was anemic (packed cell volume [PCV], 30%; International Species Information System [ISIS] reference interval, 47.8%), and serum biochemical results showed increased enzyme levels (aspartate aminotransferase, 914 U/L [ISIS reference mean, 209 U/L] and creatine kinase, 3899 U/L [ISIS reference mean, 272 U/L]), hyperkalemia (8.9 mmol/L [mEq/L]; ISIS reference mean, 3.8 mmol/ L [mEq/L]); and hyponatremia (115 mmol/L [mEq/L]; ISIS reference mean, 152 mmol/L [mEq/L]). (1) Serologic testing for Mycobacterium avium was negative for the tested serovars S1-S4. Results of blood samples submitted for blood lead testing were negative, and zinc values were considered physiologic (IDEXX Vet Med Labor GmbH, Ludwigsburg, Germany). The PCR testing for Plasmodium species from a whole blood sample was negative (Genekam Biotechnology AG, Duisburg, Germany).

At necropsy, the penguin was in good body condition and showed extensive subcutaneous fat reserves. Findings consisted of swollen salt glands with slightly granulated consistency and small, white foci. Within the salt glands, multiple small areas of necrosis with central accumulation of degenerated heterophils, peripheral demarcation by multinucleated giant cells, several epithelioid cells, and a mixed population of leukocytes, next to a few accumulations of coccoid bacteria were noted. The main findings, however, were multiple, well-circumscribed, white lesions within the pectoral muscle mass, varying in size up to 3 X 3 X 2 cm. Histologic examination revealed disseminated, interstitial accumulation of heterophils as well as acute muscle cell degeneration. The lungs and liver were hyperemic, and in the small intestine, moderate accumulations of leukocytes were observed within the mucosa. Based on these findings, moderate acute to subacute purulent myositis of the pectoral muscles and moderate purulent to apostematous inflammation of the left salt gland (Fig 2), mild catarrhal enteritis, and acute congestion of the organs were diagnosed. Bacterial culture of the pectoral muscles yielded a moderate growth of C perfringens and a mixture of other bacteria, which were not further characterized. Several other bacterial agents were cultured from various tissues, predominantly Escherichia coli (liver, kidneys, lungs, salt gland), but also E faecalis (liver, kidneys, salt glands), Enterococcus avium (lungs, salt glands), Pseudomonas species (liver, kidneys, lungs, salt glands), and S aureus (salt glands). Although P aeruginosa was found in the lungs and salt glands, it was not the most prevalent pathogen. No fungal pathogens were cultured or found during histologic examination. In contrast to the P aeruginosa isolate cultured from a corneal swab, the isolate cultured at the time of necropsy was resistant to enrofloxacin. As in the previous case, results of PCR testing for C psittaci from splenic tissue and conjunctivae were negative.

[FIGURE 2 OMITTED]

Case 5

A 1.2-year-old, female penguin was found dead without prior symptoms. Body condition was good, with the bird weighing 3.34 kg. Necropsy findings consisted of large amounts of white to yellow fibrin deposits occupying the nasal cavity, and formation of plaques around the glottis. Histologic examination revealed extensive, multifocal accumulations of heterophils within the mucous membranes of the nasal sinuses. Fibrin deposits and cellular debris were present on the surface of both the sinuses and the glottis. The glottis showed necrotic lesions with superficial bacterial colonization and fibrin exudation but also deep heterophilic infiltration with destruction of underlying bony tissues. The lungs, liver, spleen, and brain were hyperemic, and perivascular edema of brain tissue was noted.

[FIGURE 3 OMITTED]

Based on these findings, acute necrotizing inflammation of the larynx with destruction of underlying bony tissue and purulent to fibrinoid sinusitis (Fig 3) were diagnosed. Pseudomonas aeruginosa was isolated in high numbers as the sole bacterial agent from sinus swabs. Toxicologic screening of liver tissue (Institute of Pharmacology, Toxicology, and Pharmacy, Ludwig-Maximilians-University of Munich, Germany) revealed no evidence of toxicosis.

Cases 6-9

Because of the severe infections found in the previous 5 cases, the remaining 4 penguins were examined. Before capture and restraint, the single male and 3 female penguins between 1 and 1.3 years of age seemed clinically healthy and showed satisfactory food intake. However, during initial examination, the male penguin became markedly weak and showed moderate dyspnea, whereas the 3 female birds showed less severe clinical signs, including minor dyspnea and reduced resistance to manual restraint. As in previous cases, the birds were in good to moderate body condition, with body weights between 3.5 and 4 kg. At the time of examination, all penguins showed marked hyperemia of the oral mucosa and glottis with adhering white to yellow plaques or membranes, consistent with pathologic findings in birds submitted for necropsy. In the male penguin, a large, yellow mass within the glottis and cranial aspects of the trachea caused a partial airway obstruction but could not be removed at that time. Two female birds showed a moderate amount of serous, frothy ocular discharge.

Oral, ocular, and cloacal swab samples were submitted for bacterial culture. Additional swabs from oral lesions were stained with Diff-Quik for in-house cytology, and results revealed a single population of short, basophilic, rod-like bacteria in large quantities, consistent with P aeruginosa. Serologic testing for avian bornavirus was performed (Justus-Liebig-University of Giessen, Germany) with no detectable specific antibodies. Real-time PCR testing from combined choanal and cloacal swabs for West Nile virus and hemagglutination inhibition assay for antibodies against paramyxovirus both had negative results (Landesuntersuchungsanstalt Sachsen, Dresden, Germany). Results of PCR testing for Plasmodium species were also negative. An EDTA-anticoagulated blood sample was negative for lead, and serum zinc levels were within the physiologic range.

To facilitate daily treatment, the birds were confined together in the smaller indoor enclosure and provided access to a shallow indoor pool, which was disconnected from the filtration system of the main pool.

Although the results of the antimicrobial sensitivity testing were pending, treatment with marbofloxacin (5 mg/kg IM q24h), itraconazole (10 mg/ kg PO q24h), and meloxicam (0.5 mg/kg IM q24h) was initiated. Ringer's solution (25 mL PO); a commercial solution containing vitamin B-complex, amino acids, electrolytes, and dextrose (Amynin, Merial GmbH, Halbergmoos, Germany; 25 mL PO); and an equine serum protein supplement (Bioserin, WDT, Garbsen, Germany; 10 mL PO) were administered q24h with a stomach tube. All birds received a single dose of 0.3 mL of a liquid vitamin A, [D.sub.3], E, and C supplement PO (Ursovit [AD.sub.3]EC, Serumwerk Bernburg AG, Bernburg, Germany). Despite treatment, the male penguin died on day 1 after initial examination. Similar to case 5, necropsy revealed severe necrotizing laryngitis with destruction of bony tissue and purulent sinusitis. Pseudomonas aeruginosa was cultured from the lesions. Again, results from toxicologic screening of liver tissue were negative.

From all oral and cloacal swabs taken from the 4 penguins during initial examination, P aeruginosa was cultured in high numbers. From cloacal swabs of 2 females, E coli were cultured as well, whereas the other samples revealed no growth of other bacterial or fungal agents. Antimicrobial sensitivity testing of the P aeruginosa isolates showed intermediate sensitivity to enrofloxacin and good sensitivity to amikacin, ceftazidime, colistin, polymyxin B, and tobramycin. Despite treatment with marbofloxacin, a further increase of oral lesions was noted in all penguins until day 3.

Based on these results, practical considerations of treatment, and recommendations in a report of successful treatment of Pseudomonas stomatitis in saker falcons (Falco cherrug), tobramycin was considered the antimicrobial drug of choice. (2,3) To reduce the number of capture and restraint procedures, a dosage of 10 mg/kg IM q24h was chosen, instead of 12-hour intervals as described. (2,3) Administration of tobramycin was initiated on day 3 after examination. Despite doubtful efficacy, marbofloxacin was continued, along with itraconazole, meloxicam, and oral fluid administration. Additionally, silymarin (56 mg PO q24h) and 1 Fish Eater tablet PO q24h were administered. Gentle forced feeding with whole herring was performed from day 3 after initial examination because the birds were reluctant to eat. This was generally tolerated well, and medications were placed in the fish. Administration of fluids was performed via stomach tube. As a supportive measure and to keep bacterial counts at a lower level, sodium chloride was added to the pool water to a concentration of 1.2% at 5-7-day intervals. In between, a continuous washout took place with permanent freshwater flow.

Continuous reduction of oral plaques and hyperemia was noted from day 4 (day 2 of tobramycin administration). The birds quickly became stronger, and dyspnea was not observed after day 7. Direct smear cytology of swab samples from oral lesions taken on day 7 after initial examination revealed a very low quantity of basophilic rods. Oral mucosal swabs taken on the same day showed no growth of P aeruginosa in 2 penguins and only minimal growth in 1 penguin. The former 2 had started to eat fish from the ground or floating in water on day 6, whereas the latter began eating on day 9. To ensure proper treatment, some whole fish containing medications were still force-fed to all birds until day 12.

After 12 days of treatment, all medications were stopped. By that time, visible lesions had completely disappeared and clinical condition was good. An additional 10 days later, 1 of the penguins appeared slightly weak again and showed paleness of the featherless skin and reduced food intake. Because the oral cavity appeared normal and to avoid additional stress, parenteral tobramycin was not considered at this point. However, itraconazole and silymarin at the same dosages as before, along with propentofyllin (5 mg/kg PO q12h) to improve tissue perfusion, were administered for 10 days. The bird quickly and fully recovered within 3 days of treatment. All 3 birds were in a good clinical condition 35 days after initial examination, and no abnormal findings were noted. After 90 days, the penguins were moved back outside into the main exhibit. Results from oral swab samples taken during follow-up examinations after 5 and 9 months were negative on culture for P aeruginosa, and no signs of infection were present. However, P aeruginosa was cultured from a cloacal swab sample of 1 bird taken after 9 months. Because the penguin was clinically healthy and results of radiographs, hematologic testing, and serum biochemical analysis were without abnormal findings, no therapy was initiated. Within 10 months after treatment, no recurrence of clinical signs was noted.

Environmental factors

Sampling of pool water to evaluate microbiologie parameters was started after the first 2 penguins of this case series had died. Coliform, E coli, P aeruginosa, C perfringens, and Enterococcus species counts were determined in water samples taken concurrently from 2 different sites. One sample was taken immediately after filtration passage and ozonization, before water entered the pipes leading back to the main pool. A second sample was taken directly from the main pool at a depth of approximately 0.5 m. Samples were collected with sterilized glass bottles for analysis. At the time of the outbreak, bacterial counts in filtered and ozonized water were low, with a coliform count of 4 per 100 mL, and no E coli, P aeruginosa, C perfringens, or Enterococcus species detected. However, although optical quality of the main pool was good, coliform counts exceeded 24 000 per 100 mL at that time. Counts of E coli (7270 per 100 mL), P aeruginosa (45 per 100 mL), C perfringens (490 per 100 mL), and Enterococcus species (>300 per 100 mL) were also determined.

When the last 4 penguins (cases 6-9) were placed in the indoor exhibit, the pool was emptied. Inspection with a flexible camera revealed a thin layer of soft, amorphous, brown material coating some of the water pipes leading to the pool. Because sampling of the material was not possible, the presence of bacterial biofilms was suspected. Pool surfaces and accessible parts of the pipe system were cleaned, and the pool filled with freshwater. Chlorine was added to a concentration of 5 mg per mL and left to take effect for 2 days; after which, the pool was emptied and refilled once again. For the next 6 months, bacterial counts were measured once every 2 weeks and once every 4 weeks, thereafter. During the first 3 months, with no animals in the exhibit, coliform counts were consistently below 100 per mL and no P aeruginosa or other of the previously cultured bacteria were detectable. After the 3 female penguins had been placed back into the exhibit, a rise of coliform bacteria to 1400/mL was noted, but no further increase was seen for 10 months.

Several measures were implemented to monitor and reduce bacterial numbers. Changes included more frequent high-pressure cleaning and disinfection of surfaces and drains of the indoor exhibit and requirements for keepers to wear dedicated rubber boots when entering the exhibit. Regular water sampling as described above was implemented at 4-week intervals. If bacterial numbers trend upward, additional samples are taken. Complete water changes and chlorination are done at quarterly intervals or in response to increasing bacterial counts. This procedure takes 2-3 days to complete, and during this time, penguins are placed in an indoor pool containing a 1.2% sodium chloride solution. In addition, the birds are examined and weighed, and oral swabs are taken at regular intervals.

Measurements of A fumigatus concentration in the air were conducted by the public health department in October 2013, February 2014, and May 2015 in the indoor and outdoor exhibits. Results were within the reference interval for urban areas (0-30 colony forming units per [m.sup.3]). No obvious sources of A fumigatus could be found in the exhibit.

Discussion

Pseudomonas species have a worldwide distribution and are medium-sized (0.5-1.0 [micro]m X 1.5-5.0 pm) gram-negative rods. They are strict aerobes, oxidative, catalase-positive, and oxidase-positive and are motile because of polar flagella. Pseudomonas aeruginosa is considered a saprophyte, being present in soil and water, but it can also be found on skin, mucous membranes, and in feces. (4) Lipopolysaccharide production, pili, flagella, elastases, proteases, exotoxins, pyocyanin, and extracellular polysaccharides comprise its virulence factors. It is known for biofilm formation in both the environment and in infected hosts and has intrinsic as well as acquired antibiotic resistance. (5) Although isolates of P aeruginosa that are not organized in biofilms show marked antimicrobial resistance already, biofilm cells, because of production of exopolysaccharides, different growth characteristics, and differences in the uptake of nutrients and drugs, prove to be even more resistant. (6) Pseudomonas aeruginosa is considered an opportunistic pathogen, causing local infections but also septicemia, predominantly in immunocompromised individuals of many different species/ In human medicine, it is considered one of the most important causes of nosocomial infections. (7)

As in mammals, Pseudomonas infection in avian patients is usually considered a secondary infection because of reduction in the normal flora, immunosuppression, general weakness, systemic diseases, or injuries to the mucosal membranes. (8) It is a common avian pathogen, causing infections of the oropharynx and respiratory tract, enteritis, peritonitis, bumblefoot, and septicemia. (9-12) In psittacine birds, reports also include panophthalmitis, sinusitis with enophthalmia, ingluviitis, otitis media, and cellulitis. (3,13,14) One report describes unusual cases of Pseudomonas stomatitis after trichomoniasis in saker falcons. (3) In poultry, it is associated with high mortality rates in young chickens. (15-17) In ducklings (Anas platyrhynchos), halotolerant P aeruginosa were cultured from salt glands with granulomatous inflammation. (18)

Infections with P aeruginosa have been reported previously in penguins. (19-20) In a 1977 retrospective study, serositis, pericarditis, airsacculitis, fibrinous pneumonia, and enlarged livers were found in 1 Humboldt penguin, 2 African penguins (Spheniscus demersus), and 2 Magellanic penguins (Spheniscus magellanicus). (21) Similar lesions were found in another retrospective study (22) from 1986. Pseudomonas species may also have a role because pathogens associated with bumblefoot lesions have been found in penguins. (23)

In published reports, antimicrobial drugs with good efficacy against Pseudomonas species include fluoroquinolones, chloramphenicol, gentamicin, streptomycin, tobramycin, oxytetracycline, and erythromycin. (2,6,24) However, biofilm formation has a major role in antimicrobial resistance because antibiotic therapy usually reverses symptoms caused by planktonic cells (ie, not organized in biofilms), whereas it does not necessarily eliminate biofilm bacteria. (6,25) One study showed that planktonic P aeruginosa were sensitive to enrofloxacin, erythromycin, and oxytetracycline, whereas the sessile forms were sensitive to enrofloxacin only. (6) The isolate from case 4, which was cultured from a corneal swab, showed good sensitivity to enrofloxacin in vitro, but treatment did not eliminate infection in vivo because resistant P aeruginosa were cultured from various organs at necropsy. Whether the bacteria had acquired resistance during the course of treatment, or whether there were multiple strains with different sensitivity patterns remains uncertain.

During systemic treatment with tobramycin, the penguins did not show adverse reactions clinically, but neurotoxicity and nephrotoxicity can occur. (2) Whether the transient episode of reduced well-being 10 days after therapy in one bird constitutes a sign of drug toxicity remains elusive. Serial blood sampling to monitor relevant serum parameters and plasma concentrations of tobramycin was not done to avoid additional restraint and stress on the already weakened birds. Furthermore, contaminated puncture wounds with the risk of local infections or systemic spread of environmental P aeruginosa should be minimized. Studies on pharmacokinetics are necessary, however, to determine adequate dosages for Humboldt penguins.

Necropsy of deceased birds and further investigation by various test methods did not reveal predisposing bacterial, viral, or toxic agents that might have led to compromised mucosal or systemic immune function. Concurrent infection with A fumigatus was found in cases 1 and 2 and could have contributed to impaired resistance in these birds. However, increased exposure to A fumigatus could not be demonstrated. Thus, we assumed that excessive bacterial load of the pool water comprised the main factor that compromised immune functions and promoted infections with opportunistic pathogens.

A coliform count of up to 1000 per 100 mL is considered acceptable for penguin pools according to the Association of Zoos and Aquariums Penguin (Spheniscidae) Care Manual, (26) whereas counts measured in the pool were as high as 24 000 per 100 mL during the outbreak. Because bacterial counts had not been measured before, it is unknown how long this condition had been present. Proper disinfection of filtered water was reflected by very low bacterial numbers in samples taken directly after ozonization, thus insufficient water turnover rate appears to be a plausible explanation. According to the Association of Zoos and Aquariums Penguin (Spheniscidae) Care Manual, penguin pools require a turnover rate of 3-5 times the system volume per hour, (26) whereas, at Zoo Dresden, turnover rate was only 0.5 times the pool volume per hour. However, the number of animals and the volume of the pool are not specified. A coliform count of 1400 per 100 mL after cleaning and disinfection still appears high in relation to the number of only 3 animals, but this was considered an acceptable upper limit until definite causes for the elevated bacterial load were found and resolved. The presence of biofilms was assumed because suggestive material was found in water pipes, but it was not proven by culture or on microscopic examination. Porous concrete surfaces of the pool and partially hollow artificial rocks might also represent permanent sources of bacteria and are difficult to clean. However, a definite answer for the elevated bacterial load is lacking.

Feeder fish was considered a possible origin of bacterial pathogens as well. The processing of fish had been improved 1 year before the outbreak, including both thawing and feeding to the penguins. Deep-frozen fish is provided by an established vendor, who delivers to many other zoological institutions. Previously, fish had been thawed in running tap water at room temperature, but this procedure was altered to slow thawing in a cold storage cell at 4[degrees]C to better preserve nutritional value and optimize hygiene. Fish are then rinsed with clean water and fed within 48 hours after thawing is complete. Each fish is checked by the keepers and discarded in case of insufficient quality or signs of decomposition. Currently, fish are hand fed directly to the penguins, and no fish are to remain within the pool after feeding. Only stainless steel buckets are used during feeding and storage and are cleaned and disinfected after each use. However, improper hygiene or bacterial contamination of fish cannot be excluded as a possible origin of infection because bacterial culture of frozen or thawed fish was not conducted during the outbreak.

Traditionally, a "less-is-more" policy regarding the frequency of capture and restraint of penguins has been promoted at our institution because stressful procedures have to be weighed against potential benefits in each case. Thus, few hematologic and serum biochemical values of infected penguins were available. However, the documented cases clearly demonstrate the importance of thorough clinical examination. In future cases, a more straightforward approach will be favored, including early and repeated diagnostic procedures, including blood sampling and bacterial culture, followed by appropriate treatment.

Pseudomonas aeruginosa infection should be considered a differential diagnosis in penguins with nonspecific illness or with clinical signs such as swelling of the salt glands, ocular discharge, oral and choanal inflammation, and central nervous signs. Thus, bacterial culture and sensitivity testing should be included in the diagnostic workup of suggestive cases. Pool water has to be considered a possible source of bacterial pathogens. Therefore, regular water sampling is of particular importance where filtration systems with very little water exchange are present to prevent and control potentially hazardous conditions for the inhabiting animals. The use of tobramycin, in combination with improved hygiene, appeared to be effective against P aeruginosa infection in 3 Humboldt penguins.

Acknowledgments: We thank Astrid Nagel, Birgit Isele-Ruegg, Fritz Fassler, Thomas Wagner, Zoltan Mezoe, and Doris Krumnow for excellent technical assistance and Mirjam Grobbel for bacteriology investigations. We especially thank Baukje Lenting for copyediting the manuscript.

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Dimitri Widmer, DVM, Eva Ziemssen, DVM, Benjamin Schade, DVM, Eva Kappe, DVM, Ferdinand Schmitt, DVM, Hermann Kempf, DVM, and Gudrun Wibbelt, MRCVS, Dipl ECZM (Wildlife Population Health)

From the Zoo Dresden, Tiergartenstrasse 1, D-01219 Dresden, Germany (Widmer, Ziemssen); the Bavarian Animal Health Service, Senator-Gerauer-Strasse 23, D-85586 Poing, Germany (Schade, Kappe, Schmitt); the Tierarztliche Praxis fur Exoten, Neuburger Strasse 30, D-86167 Augsburg, Germany (Kempf); and the Leibniz Institute for Zoo and Wildlife Research, Alfred-Kowalke-Strasse 17, D-10315 Berlin, Germany (Wibbelt).
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Title Annotation:Clinical Report
Author:Widmer, Dimitri; Ziemssen, Eva; Schade, Benjamin; Kappe, Eva; Schmitt, Ferdinand; Kempf, Hermann; Wi
Publication:Journal of Avian Medicine and Surgery
Article Type:Report
Date:Jun 1, 2016
Words:5706
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