Spheniscus Humboldti Research Articles

Microplastics in Seabird Feces from Coastal Areas of Central Chile.

Pollution from plastic waste thrown into the ocean affects all levels of the food chain. Marine species of birds are affected by plastic particles of different sizes, especially the mesoplastics (1 to 10 mm) found in their digestive tract, which mainly cause obstructions. In the case of microplastics (1.000 µm to 1 mm), their presence in the digestive tract of these species has been widely reported. We studied fecal samples of the Dominican gull (Larus dominicanus) (n = 14), Magellanic penguins (Spheniscus magellanicus) (n = 8), and Humboldt penguin (Spheniscus humboldti) (n = 1) obtained from the Wildlife Rehabilitation Center of the Biobío region, Chile. Microfibers of various colors were present in the feces of Dominican gulls and Magellanic penguins, corresponding mainly in composition to polypropylene (PP) (83%) and rayon (77%). These results demonstrate that microplastic particles occur in the coastal environments of central Chile and suggest that they are probably circulating in the food chain.

A comprehensive investigation of negative visitor behaviour in the zoo setting and captive animals' behavioural response

Negative visitor behaviour in zoos such as banging, shouting and feeding animals are unwanted, but under-studied, visitor actions. It is not known how prevalent negative behaviour is, which species or enclosure type receives the most negative behaviour or how these behaviours affect zoo-housed animals. In this study, a comprehensive assessment of negative visitor behaviour, using an innovative methodology, was conducted at 25 different enclosures at Fota Wildlife Park, Ireland. Additionally, animal activity level and out of sight behaviour was observed. Descriptive statistics and general linear models were used to investigate which variables affected behaviour. Banging was the most common negative behaviour, while Humboldt penguins, lion-tailed macaques and Sumatran tigers were the most harassed species. Negative actions increased as visitor number increased and at traditional-style viewing areas. Active animal behaviour and out of sight animals were effected as negative visitor behaviours increased, but there appeared to be a tolerance threshold before a behavioural response was observed. By understanding negative behaviours, zoos can strive to reduce them and promote positive animal welfare.

DETECTION AND PREVALENCE OF SPHENISCID ALPHA-HERPESVIRUS-1 (SpAHV-1) IN A SAMPLE OF HUMBOLDT PENGUINS (SPHENISCUS HUMBOLDTI) AT PUNTA SAN JUAN, PERU.

Infections with herpesvirus have contributed to respiratory, enteric, and neurological disease reports in avian species worldwide. Herpesviruses have been detected in penguin species before but have not been studied extensively. To better understand the impact of these viruses in free-living populations, an initial retrospective survey was performed on a wild population of Humboldt penguins (Spheniscus humboldti) in the Punta San Juan Marine Protected Area, Peru (15°22'S, 75°12'W) using tracheal swabs collected from 28 penguins in 2016 and 34 penguins in 2018. DNA extracted from these swabs was analyzed using a consensus herpesviral PCR assay targeting the DNA polymerase gene, and positive samples were sequenced. A single sample from 2016 was positive for spheniscid alpha-herpesvirus-1 (SpAHV-1), establishing an overall sample prevalence of 1.6% (95% CI: 0-8.6%). The positive animal was an adult male that did not show any clinical signs of herpesviral infection and was otherwise healthy based on physical exam and laboratory findings. This is the first detection of a herpesvirus in penguins at Punta San Juan, Peru, and the first step toward characterizing the implications of SpAHV-1 for Humboldt penguins. This investigation highlights the importance of continual disease surveillance in wild populations over time to monitor for changes that may impact long-term population viability.

Pulmonary Aspergillosis in Humboldt Penguins-Susceptibility Patterns and Molecular Epidemiology of Clinical and Environmental Aspergillus fumigatus Isolates from a Belgian Zoo, 2017-2022.

Aspergillus fumigatus is the main causative agent of avian aspergillosis and results in significant health problems in birds, especially those living in captivity. The fungal contamination by A. fumigatus in the environment of Humboldt penguins (Spheniscus humboldti), located in a Belgian zoo, was assessed through the analysis of air, water, sand and nest samples during four non-consecutive days in 2021-2022. From these samples, potential azole-resistant A. fumigatus (ARAF) isolates were detected using a selective culture medium. A total of 28 veterinary isolates obtained after necropsy of Humboldt penguins and other avian species from the zoo were also included. All veterinary and suspected ARAF isolates from the environment were characterized for their azole-resistance profile by broth microdilution. Isolates displaying phenotypic resistance against at least one medical azole were systematically screened for mutations in the cyp51A gene. A total of 14 (13.6%) ARAF isolates were identified from the environment (n = 8) and from Humboldt penguins (n = 6). The TR34/L98H mutation was observed in all resistant environmental strains, and in two resistant veterinary strains. To the best of our knowledge, this is the first description of this mutation in A. fumigatus isolates from Humboldt penguins. During the period 2017-2022, pulmonary aspergillosis was confirmed in 51 necropsied penguins, which reflects a death rate due to aspergillosis of 68.0%, mostly affecting adults. Microsatellite polymorphism analysis revealed a high level of diversity among environmental and veterinary A. fumigatus isolates. However, a cluster was observed between one veterinary isolate and six environmental strains, all resistant to medical azoles. In conclusion, the environment of the Humboldt penguins is a potential contamination source of ARAF, making their management even more complex.

Suspected Mefloquine Toxicity in a Colony of Humboldt Penguins (Spheniscus humboldti).

Avian malaria is an important cause of mortality in captive penguins housed in outdoor exhibits. Mefloquine was used as a prophylaxis to treat a colony of 19 Humboldt penguins (Spheniscus humboldti) for avian malaria. A target dose of 30 mg/kg was obtained from anecdotal literature for sphenisciforms that was not based on pharmacokinetic or toxicity studies. For this reason, preliminary plasma concentrations of mefloquine were acquired after the first dose in some penguins to ensure that plasma concentrations reached human malaria prophylactic concentrations. Afterward, each penguin in the entire colony received mefloquine (26-31 mg/kg [125 mg in toto] PO q7d). Regurgitation was frequently observed starting after the fourth weekly administration. Plasma concentrations of mefloquine after the seventh dose showed elevated concentrations, and the treatment was immediately terminated. Eight penguins died during and after the treatment period. The first fatality occurred after the fifth weekly administration, and 7 birds died within 7-52 days after the seventh weekly administration. Three penguins were found dead without previous symptoms. The other five presented with marked lethargy, dyspnea, poor appetite, and vomiting, and all died despite medical care. The remaining 11 penguins of the colony survived without any supportive care; 5 did not exhibit any clinical disease signs, while the other 6 showed a mild apathy and decreased appetite. Mefloquine toxicity was highly suspected on the basis of clinical signs, the elevated mefloquine plasma concentrations, and no other underlying pathologic disease conditions identified through postmortem examinations. Nonspecific lesions, including pulmonary congestion and edema and hepatic perivascular hematopoiesis, were noted in the birds that died. Additionally, 1 case presented with myocarditis, and mycobacteria were observed within granulomas in the respiratory tract of 2 penguins. Caution is advised, and further studies are encouraged before administering mefloquine to penguins.

Comparison of Plasma Trace Elements Between 2 Captive Humboldt Penguin (Spheniscus humboldti) Populations.

Environmental pollutants and their effect on wildlife health play an important role in the conservation of endangered species and can be clinically relevant in captive animals too. Data on relevant concentrations of trace elements in captive birds with no known exposures are rare. For this study, silver, arsenic, gold, barium, cadmium, cobalt, chromium, copper, iron, mercury, magnesium, manganese, molybdenum, thallium, selenium, and zinc were measured by inductively coupled plasma mass spectrometry in lithium heparinized plasma samples from 18 Humboldt penguins at 2 zoological collections in Germany. The results showed that the plasma concentrations of silver, arsenic, gold, cadmium, cobalt, chromium, mercury, and selenium differed significantly (P ≤ 0.05) between the 2 penguin collections. The results indicate that the location of the birds has a strong influence on the plasma trace element concentrations. Well water used in the enclosures was suspected to be associated with these significant differences. Trace elemental concentrations in feed (eg, marine fish) and contamination from enclosure construction materials may also play a role. This study could provide a basis for further comparative, biomonitoring, toxicity, and reference interval studies.

Effectiveness of Marine Wildlife Rescue and Rehabilitation Centers in Chile in relation to strandings of sea turtles, penguins, sea lions, and sea otters

Marine Wildlife Rescue and Rehabilitation Centers (MWRRCs) are crucial for the rehabilitation of endangered marine species, such as penguins, sea turtles, sea lions, and/or sea otters. In Chile, rescue of marine fauna is coordinated by the National Fisheries and Aquaculture Service (SERNAPESCA). This organization is responsible for assisting stranded animals and transporting them to 6 of the 13 MWRRCs that exist throughout the 6,435 km coastline of the country. Stranding events were analyzed for two species of pinnipeds (Otaria flavescens and Arctocephalus australis), two species of penguins (Spheniscus humboldti and Spheniscus magellanicus), two species of chelonians (Chelonia mydas and Lepidochelys olivacea), and one species of sea otter (Lontra felina) found stranded at the coast of Chile during 2009-2019 period. Success in post-rehabilitation release of individuals was also examined. A total of 2,818 stranding events were recorded with a total of 3,198 stranded animals, corresponding to O. flavescens (52.9%), S. humboldti (20.4%), S. magellanicus (17.9%) and L. olivacea (4.3%). Of the 3,198 stranded animals, 721 specimens were referred to MWRRC, and only 136 were released post-rehabilitation. This shows an18.8% success rate in the release of marine fauna species post-rehabilitated in rescue centers after their stranding. It is necessary to improve coordination between SERNAPESCA and MWRRCs in Chile to improve the release rate and to increase the number of MWRRCs to recover endangered marine species that strand along the country’s coast.

Behavioural patterns in zoo-housed Humboldt penguins (Spheniscus humboldti) revealed using long-term keeper-collected data: Validation of approaches and improved husbandry

Given the increasing obligation to elevate animal welfare beyond minimum expectations, zoos need robust mechanisms to monitor physical activity and species-appropriate behaviours. This is not without challenge as animal behaviour can vary seasonally or be influenced by external factors such as weather and visitors. In theory, keepers who work with animals year-round are ideally situated to collect behavioural data. However, time pressure often means any data collection is minimal, ad-hoc, or skewed towards particular times of day. Here we compare infrequent low-intensity ad-hoc data on activity and behaviour of zoo-housed Humboldt penguins (Spheniscus humboldti) collected by keepers with simultaneous high-intensity systematic data collected by a researcher. Once out-of-sight records (more frequent in researcher data) were excluded from both datasets, we found excellent agreement between keeper and researcher data. After this validation step, we collected extensive multi-year keeper data (diurnal hourly scan samples; 262 days across a 5-year study period) to assess potential influence of time of day, time of year, visitors, and weather on penguin behaviour. There were pronounced seasonal patterns: aquatic active behaviours were highest in autumn (38.9% in October) compared to early spring and late summer (February = 16.7%; August = 19.4%). Time of day was also important: terrestrial inactive behaviours increased throughout the day and this pattern was significantly more pronounced in summer than winter. Aquatic active behaviours were more frequent during warmer/sunnier conditions compared to cooler/wetter conditions, while the reverse was true for terrestrial inactive behaviours. Birds were much less active on days when the site was closed to visitors. Overall, we demonstrate that where keeper-collected data are validated and found to be robust and representative, there are real opportunities to study long-term behavioural patterns that can quickly translate to tangible husbandry benefits and improved animal welfare.

Sex identification of Humboldt penguins using a morphometric analysis

Primorsky Aquarium keeps a population of 32 Humboldt penguins. The employees of Ornithology Department are faced with a problem of sex identification of 18 immature/young penguins. Male and female Humboldt penguins have not significant differences in feather coloration, so reliable sex identification by direct observation is not possible. The most reliable ultrasound and genetic methods require special equipment, reagents and qualified specialists as well as provoke stress that is dangerous for the health of the birds. Sex identification by morphometric parameters for captivity birds is more preferable, since this procedure is non-invasive, does not require special equipment and specialists. We have performed a statistical analysis of measurements of the beak length in 3 variations, of the dorsal part of flipper length, and weight of females and males of the studied group. For the first time, we have added to the analysis the length of the beak from the nostril, which was not previously taken into account. It has been found that the use of 4 traits simultaneously in the analysis does not demonstrate a significant correlation with the sex of the bird, and therefore is not suitable for the sex identification of penguins. The most consistent features for correlation analysis were the beak length and the flipper length. The maximum grouping of birds by sex within the study group was observed while comparing these morphometric characteristics.

Does the evolutionarily stable strategy apply to captive Humboldt penguins? Study on S. humboldti lateralized behavior

So far, little has been done on the motor and perceptual asymmetry in the genus Spheniscus, which on the contrary, has been the target of much research in the wild due to its vulnerable ecological condition. The present study aimed to investigate the presence of lateralization on different asymmetry dimensions in Humboldt penguins by studying fourteen subjects housed in a captive colony in Italy. For each individual, the preferred foot to start walking and climbing on a weighing scale, the preferred side to reach the uropygium, and the preferred eye to scrutinize visitors outside the exhibit were recorded. Asymmetry was found at the individual level for the preferred foot to climb onto the scale (three left and five right out of 14) and the preferred side to reach the preen gland (two left and five right out of 14). Only thirteen penguins were seen interacting with people outside the exhibit using their left (four) or right (three) eye while swimming in the pool. No lateralization could be documented at the population level. The lack of lateralization at the population level could be attributable to the small size of the colony studied, hampering speculations on the social life of S. humboldti. Since the requirement for coordinated behaviors at the population level occurs when social interactions become common and recurrent in the natural behavior of a species, and because only one among the items studied was devoted to the study of social behavior, it will be necessary to broaden the research to other captive S. humboldti populations to expand our results. Data obtained from a larger number of animals could unveil a little piece of the puzzle that will allow evaluating lateralization at the population level in this species and ascertain if the Evolutionarily Stable Strategy could also be applied to S. humboldti.

Comparing Data Collection Tools for Zoo Management Decision-Making: A Case Study Examining Behavioral Measures of Humboldt Penguin Bond Strength.

Systematic data collection has become increasingly important in zoos as it facilitates evidence-based decision-making. Here, we describe the results of a two-year study on exhibit use and pair-bonding in a colony of Humboldt penguins. We used two different data collection apps to evaluate their effectiveness and suitability for evaluating pair-bond strength. Data were collected using instantaneous scan sampling and all-occurrence sampling 2-3 times per week for 2 years for a total of nearly 240 h of observation (19 h with one system and 219 h with the other system). The activity patterns (in particular, time spent in the water) differed amongst penguins and between the two data collection tools. Patterns of courtship-related behaviors varied tremendously across individuals. The longer pairs had been bonded, the more time they spent in close proximity. We highlight two important considerations for institutions aiming to collect such systematic data. First, it is critical to interpret all findings in context by incorporating husbandry details and keeper insights to highlight explanations that may not be readily apparent from the data. Second, one must explore all aspects of any data collection system before committing to its use-system setup, ease of data collection, format and accessibility of exported data. Not doing so may negate the value of systematic data collection by limiting the use and interpretability of the data.

Assessing the influence of Humboldt penguin (Spheniscus humboldti) by excrements on the levels of trace and rare earth elements in the soil

Rare earth elements (REE) and some transition metals (e.g. Nb) are a group of chemicals that have recently been widely used in industrial processes due to the increasing demand for new technologies. As a result, these chemicals are increasingly being released into the environment, which could mean that these pollutants could modify marine and terrestrial ecosystems. Seabirds, such as penguins, can biotransport pollutants and nutrients from the sea to land through excreta. However, there is no information about the role of the Humboldt penguin (Spheniscus humboldti) in bio-transporting emerging contaminants such as REE. This study aimed to assess any possible contribution of Humboldt penguins to the geochemical composition of some terrestrial areas. Excreta samples were collected from Chañaral Island, one of the most important sites in Chile for the conservation of Humboldt penguins. The results showed that this species tends to contribute to soil enrichment with REE (Ce, La, Nd, and Pr) and Nb through excreta, as well as with carbon. More studies are needed to see the potential impacts on the soil.

An integrative insight into the synsacral canal of fossil and extant Antarctic penguins.

The lumbosacral-canal system in birds most likely operates as a sense organ involved in the control of balanced walking and perching, but our knowledge of it is superficial. Penguins constitute interesting objects for the study of this system due to their upright walking, but only the Humboldt penguin, Spheniscus humboldti, and some incomplete fossil penguin synsacra have been studied in this respect. Here, we give an integrative comparative insight into the synsacral canal of extant Emperor penguin, Aptenodytes forsteri, Adelie penguin, Pygoscelis adeliae, and Eocene giant Anthropornis and/or Palaeeudyptes Antarctic penguins, using computed tomography imaging and associated data-extraction methodologies, complemented by analytical approaches ranging from geometric morphometrics to modularity, curvature, and wavelet analyses. We document that the variability in the number of synsacro-lumbar vertebrae is evolutionarily conserved, and all studied synsacra possess osteological correlates of the lumbosacral-canal system. We also found that Eocene and extant Antarctic penguins were separable on the basis of the main direction of the shape-related (size-independent) variability within said system, and A. forsteri was unique in the entire studied set in terms of the relative cranial shift of this compound structure. Moreover, we suggest that the evolutionary processes, shaping both the terrestrial posture and gait, were responsible, in extant penguins, for the increased simplicity and stability of the synsacral canal cross-sectional periodic patterns, as well as pave the way for the lumbosacral-canal system modularity characterized by reduced atomization/complexity.

Long and winding road: Training progress and trainability variation across a psychoacoustic experiment in penguins

Operant conditioning has been used in a variety of animal species both in captivity (laboratories, zoological institutions) and in the wild. Such training may enrich the environment of animals or facilitate their manipulation but it can also be used to answer scientific questions (so far primarily regarding sense modalities). However, the trainability of animals may vary because of cognitive differences between species or because of individual and external factors within species. The assumed difficulty of training seabirds presumably explains, why they have been mostly overlooked in studies based on operant conditioning, such as psycho-acoustic studies. Here, as part of a broad project examining the hearing capacity of penguins, we trained four captive Humboldt penguins (Spheniscus humboldti) for psychoacoustic trials, following well-established operant conditioning methods. In addition to describing the different steps of the training of penguins, we examined how the trainability of each individual (concentration, response to call and stationing quality) was affected by different life stages (moult, pairing interest) and environmental conditions (daylength, temperature, humidity, visitors). After 22 months and ca. 700 trials, all four penguins were ready for psychoacoustic measurements, which in turn extended over 22 additional months. However, the trainability of penguins was not stable over these 44 months, as their concentration decreased over time (presumably because of habituation) while the quality of their call response and stationing increased for most of them (presumably reflecting learning progress). Moreover, all trainability parameters were strongly reduced during moult while the effects of pairing interest and environmental conditions were more variable between individuals. Our study demonstrates that, despite the length and the instability of training as well as some interindividual differences in trainability, it is possible to train captive penguins for scientific purposes. As such, we hope that our study opens the path for further studies based on the training of animals, for which training is assumed to be challenging but for which scientific data are urgently needed because of their vulnerability in the face of environmental changes.

P467 Aspergillosis in Humboldt penguins — susceptibility patterns of clinical and environmental isolates from a Belgian zoo, 2018-2022

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM ObjectivesAvian aspergillosis causes a heavy burden on birds in captivity, such as Humboldt penguins. In recent years the colony of a Belgian zoo has experienced very high mortality rates and the zoo has already taken several measures to lower the burden. This study was set up to see if the penguins acquire A. fumigatus via the environment and if so, if additional measures can be taken to limit the incidence.MethodsA total of 29 clinical strains collected from 2018 to 2022 were included in the study. From April 2021 until January 2022, four samplings have been performed, accounting for every season. A combination of sand, water, nest swabs, and air was analyzed for the presence of (azole-resistant) A. fumigatus. In brief, air samples were collected at fixed locations in the penguin enclosure by impacting 1000 L on an agar plate [malt + chloramphenicol (MC) and MC + tebuconazole (MC + T)]. A total of 100 mL water was collected and 50 mL water was filtered through a 0.22 μm filter and placed on an MC plate, and repeated with the other 50 ml on MC + T. A total of 9 ml 0.1% Tween 20 + 0.85% NaCl was added to 1 g of sand and vortexed for 1 min. Both MC and MC + T plates were incubated with 100 μL of this suspension. All plates were incubated at 48°C ± 1°C for 48 h. The phenotypical resistance pattern of all clinical isolates was determined using the EUCAST method.ResultsThe phenotypical resistance pattern showed resistance in 7 isolates (24%) with 5 of them showing resistance to posaconazole, one was resistant against voriconazole, isavuconazole, and posaconazole, and 1 showing pan-resistance. No A. fumigatus colonies could be detected from water samples, nor from the sand. One A. fumigatus isolate was retrieved from the nest swabs. In total 64 A. fumigatus colonies were isolated from air samples collected on MC + T medium. All have been subjected to EUCAST microbroth dilution determination and 4 resistant isolates could be detected (all had a MIC value for posaconazole of 0.5 μg/ml and one strain showed additional resistance against isavuconazole with a MIC of 4 μg/ml. Cyp51a sequencing of all resistant strains is ongoing and will give more insight in the molecular mechanisms involved to investigate the potential link with the environment.ConclusionThis study showed high resistance rates in the clinical isolates. Four resistant isolates were found in environmental air samples. Sequencing of the cyp51A gene will give more information on a possible relation between the resistance mechanisms found in the clinical and the environmental isolates. More research should be done to investigate the origin of the resistant isolates in the environment.

P468 Aspergillosis in a colony of Humboldt penguins (Spheniscus humboldti) under managed care: a clinical and environmental investigation in a French zoological park

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM ObjectivesAspergillosis is an opportunistic fungal infection due to Aspergillus spp., and primarily A. fumigatus. The disease is pervasive in avian populations, especially those under managed care. The incidence and the management of avian aspergillosis can be critically impacted by exposure to high levels of spores via environmental contamination and by strains resistant to azole drugs. The present study aimed at assessing the impact of environmental contamination on the clinical incidence of aspergillosis in penguins managed under human care. ZooParc de Beauval was chosen for the investigations, because this zoological facility hosts a large colony of Humboldt penguin (Spheniscus humboldti) and it is geographically located in the rural countryside of the Loir-et-Cher district, France, surrounded by numerous large crop fieldsMaterial and methodsThe fungal contamination in the environment was assessed through three serial sessions of surface sampling in bird nests: all isolates were counted, identified by DNA sequencing, and then systematically screened for resistance mutations and MICs (minimal inhibitory concentrations) elevation for the A. fumigatus strains. In addition, the clinical incidence of aspergillosis was evaluated in the penguin population over a 3-year period. A microsatellite-based analysis tracked A. fumigatus isolates circulation.ResultsEnvironmental investigations highlighted a substantial increase in the fungal load during the Autumn season (>12-fold vs. the other timepoints) and a large overrepresentation of species belonging to the Aspergillus section Fumigati (ranging from 22.7 to 94.6%) (Fig. 1). Only one cryptic species (A. nishimurae) and one isolate exhibiting resistance mutation (G138S in the cyp51A gene; MIC itraconazole >4 μg/ml) were detected. The overall incidence of aspergillosis was measured at ∼3.4% case-years and was observed mostly in juvenile penguins (Fig. 1). The analysis of microsatellite polymorphism revealed a high level of genetic diversity among A. fumigatus isolates, except for one strain that was largely over-represented during the Autumn sampling session. A limited number of isolate genotypes was collected from clinical cases and from the environment.ConclusionsFungal environmental contamination and incidence of aspergillosis in penguins appeared variable depending on seasons, but the rural location of the penguin habitat did not seem to influence the emergence of resistant strains.

Long-Term Follow-up of Osteoarthritis in Three Humboldt Penguins Treated with Acupuncture

Humboldt penguins, (Spheniscus humbolti) are medium-sized penguins from the rocky shores of Chile and Peru. Their lifespan in the wild is 20-25 years and in captivity 20-30 years. Three geriatric penguins at the Santa Barbara Zoological Park displayed various degrees of lameness and decreased swim time in their pool. Radiographs revealed osteoarthritis in numerous joints. Conventional treatments with various non-steroidal anti-inflammatory drugs (NSAIDs) were minimally effective at improving their mobility. The penguins were part of a zoo rookery and were not used to being handled. The traditional Chinese veterinary medicine (TCVM) examinations were kept brief and treatments done under the supervision of the zoo veterinary staff. The three penguins received a TCVM Pattern diagnosis of Kidney Qi Deficiency with local Stagnation in various joints. Dry needle acupuncture treatments utilizing minimal restraint were administered monthly for a year. Between acupuncture treatments, conventional medication was also administered. During the year of treatment, the geriatric penguins’ quality of life showed some improvement. One of the penguin’s mobility improved enough to again swim in the pool and catch fish during the feeding sessions. The other two penguins demonstrated some mild improvement with their mobility on land and swam more frequently in the pool. These improvements allowed them to remain part of their colony.

Molecular and epidemiological surveillance of Plasmodium spp. during a mortality event affecting Humboldt penguins (Spheniscus humboldti) at a zoo in the UK.

In 2017, a mortality event affected Humboldt penguins at Chester Zoo (UK), which coincided with the diagnosis of avian malaria (AM) in some birds. AM is found worldwide wherever a competent mosquito vector is present, but the disease is particularly severe in penguins and other species that originate from non-endemic regions. To better understand the role of AM and manage its threat to penguin collections, Plasmodium was surveyed through PCR at Chester Zoo in mosquitoes, penguins, and dead free-living wild birds during and around the mortality event. Additional sequences were obtained from penguin fatalities from four other UK zoological collections. All sequences were integrated into phylogenetic analyses to determine parasite species and lineages. In total, 753/6459 positive mosquitoes were recorded (11.7% prevalence), reaching a weekly peak of 30% prevalence in mid-summer. Among penguin fatalities at Chester Zoo, several penguins presented signs and lesions compatible with AM; nevertheless, exoerythrocytic meronts were identified in only one case and Plasmodium spp. was identified in 5/22 birds. Phylogenetic analysis revealed at least five parasite cytb lineages of three Plasmodium species (P. matutinum, P. relictum and P. vaughani) circulating in mosquitoes at Chester Zoo; however, infections in free-living wild birds and penguins were only from P. matutinum. Plasmodium matutinum was confirmed as the cause of death of one penguin and was highly suspected to be the cause of death of another three. The lineage LINN1 was associated with 4/5 penguin infections. AM had a key role in the penguin multicausal mortality event. Understanding the risk of AM to penguin collections at Chester Zoo and elsewhere requires long-term surveillance to examine the association between Plasmodium infection and penguin mortality and the variability in parasite virulence. Surveillance of Plasmodium spp. in mosquitoes and local birds provides information about the parasite's transmission cycle locally, and could warn about infection risks to species of interest, which is essential for efficient disease control and prevention.

Aspergillosis in a colony of Humboldt penguins (Spheniscus humboldti) under managed care: a clinical and environmental investigation in a French zoological park.

AbstractAspergillosis is pervasive in bird populations, especially those under human care. Its management can be critically impacted by exposure to high levels of conidia and by resistance to azole drugs. The fungal contamination in the environment of a Humboldt penguin (Spheniscus humboldti) group, housed in a French zoological park next to numerous large crop fields, was assessed through three serial sessions of surface sampling in nests, in 2018–20: all isolates were counted and characterized by sequencing. When identified as Aspergillus fumigatus, they were systematically screened for resistance mutations in the cyp51A gene and tested for minimal inhibitory concentrations (MICs) determination. At the same time, the clinical incidence of aspergillosis was evaluated in the penguin population by the means of systematic necropsy and mycological investigations. A microsatellite-based analysis tracked the circulation of A. fumigatus strains. Environmental investigations highlighted the substantial increase of the fungal load during the summer season (>12-fold vs. the other timepoints) and a large overrepresentation of species belonging to the Aspergillus section Fumigati, ranging from 22.7 to 94.6% relative prevalence. Only one cryptic species was detected (A. nishimurae), and one isolate exhibited G138S resistance mutation with elevated MICs. The overall incidence of aspergillosis was measured at ∼3.4% case-years, and mostly in juveniles. The analysis of microsatellite polymorphism revealed a high level of genetic diversity among A. fumigatus clinical isolates. In contrast, one environmental strain appeared largely overrepresented during the summer sampling session. In all, the rural location of the zoo did not influence the emergence of resistant strains.Lay summary

Assessment of the Safety and Efficacy of an Oral Probiotic-Based Vaccine Against Aspergillus Infection in Captive-Bred Humboldt Penguins (Spheniscus humboldti).

Aspergillosis is a fungal infection caused mainly by Aspergillus fumigatus that often results in respiratory disease in birds. Aspergillosis is a major cause of morbidity and mortality in captive-bred penguin species. Currently, there is no registered vaccine to prevent aspergillosis. Recent research demonstrated that oral administration of gram-negative bacteria expressing high levels of galactose-α-1,3-galactose (α-Gal) modulates anti-α-Gal immunity and protects turkeys from clinical aspergillosis caused by experimental A. fumigatus infection. The role of anti-α-Gal immunity in penguins has not been studied. Here, we tested the distribution of α-1,3-galactosyltransferase (α1,3GT) genes in the fecal microbiome of Humboldt penguins (Spheniscus humboldti). The occurrence of natural anti-α-Gal antibodies (Abs) in sera and eggs of healthy Humboldt penguins was also assessed. A trial was then conducted to test whether oral administration of Escherichia coli Nissle, expressing high α-Gal levels, modulates anti-α-Gal immunity in a colony of Humboldt penguins. Animals in the vaccination and placebo groups were evaluated before the trial and followed for one year for aspergillosis detection using a diagnostic panel including computed tomography scans, capillary zone electrophoresis, 3-hydroxybutyrate levels, and anti-A. fumigatus Abs. Anti-α-Gal Abs were detected in sera (IgM and IgY) and eggs (IgY) of healthy penguins. Microbiota analysis and functional predictions revealed the presence of α1,3GT genes in the microbiota of Humboldt penguins and other penguin species. A strong decrease in anti-α-Gal IgM levels was observed in all animals in the placebo group three months after vaccination protocol. This decrease was not observed in E. coli Nissle-treated penguins. After the vaccination protocol, we found a positive correlation between anti-E. coli IgY and anti-α-Gal IgY in the E. coli Nissle group, suggesting a correlation between the presence of the bacteria and these Abs. During the study period, three penguins exhibited respiratory signs consistent with aspergillosis. Two were from the placebo group whose symptoms resolved with specific treatments, while a single vaccinated individual developed fatal respiratory aspergillosis eight months after the trial. We conclude that E. coli Nissle represents a safe potential probiotic with a protective effect against aspergillosis in Humboldt penguins that deserves to be further explored for therapeutic uses in these animals.