Capillary Zone Electrophoresis in Humboldt Penguins (Spheniscus humboldti).

Electrophoresis is a useful diagnostic tool for detecting inflammation, including inflammation associated with infectious diseases (eg, aspergillosis in penguins). To our knowledge, reference intervals are not available for plasma proteins via electrophoresis in Humboldt penguins (Spheniscus humboldti). Therefore, preliminary reference intervals for blood plasma proteins measured by capillary zone electrophoresis were calculated for Humboldt penguins from a single zoological collection, and possible differences between the sexes and the ages of the birds were evaluated. Lithium heparinized plasma samples from 39 Humboldt penguins were analyzed. The following sex- and age-independent reference intervals were calculated: total protein 33.8-70.4 g/L, prealbumin 1.9-4.9 g/L, albumin 12.9-31.1 g/L, albumin: globulin ratio 0.7-1.7, α-globulins 4.5-11.6 g/L, ß-globulins 5.6-20.6 g/L, and γ-globulins 2.6-8.4 g/L. Male penguins had a significantly (P = 0.047) higher albumin: globulin ratio and lower percentage of ß-globulins (P = 0.015) in comparison with female penguins. Prealbumin (g/L) significantly (P = 0.021) decreased with increased age of the penguins. These results showed some differences between the sexes and ages of the penguins, which should be considered when interpreting the results. Further studies are needed to determine whether differences in other age groups or seasons exist, and also to evaluate which infectious diseases affect plasma proteins and how the reference values calculated here may deviate in ill penguins.

PHARMACOKINETICS OF PRIMAQUINE PHOSPHATE AFTER A SINGLE ORAL ADMINISTRATION TO AFRICAN PENGUINS (SPHENISCUS DEMERSUS).

Primaquine is an 8-aminoquinolone drug commonly used for the chemoprophylaxis and treatment of avian malarial infections in managed penguin populations worldwide. Little is known about its pharmacokinetic properties in avian species. The objective of this study was to describe the disposition of primaquine phosphate after a single oral dose in 15 healthy African penguins (Spheniscus demersus). A single tablet containing 26.3 mg of primaquine phosphate (equivalent to 15 mg primaquine base) was administered orally to each bird in a herring fish. Blood samples were collected prior to drug administration and at predetermined timepoints through 144 hr postadministration. Plasma was analyzed for drug concentration by high-performance liquid chromatography with ultraviolet detection. Mean maximum plasma concentration of primaquine phosphate was 277 ± 96 ng/ml at approximately 3.1 hr following oral administration. The mean disappearance half-life was 3.6 ± 1.6 hr. Plasma concentrations were below detectable limits in all but one penguin by 36 hr. A single oral administration of 26.3 mg of primaquine phosphate in African penguins resulted in a pharmacokinetic profile comparable to those attained in human studies. These results suggest that a dosing interval similar to human regimens may be of potential use in the prevention and treatment of avian malaria in penguins. Additional clinical studies are needed to determine the efficacy and safety of this regimen.

Evolved increases in hemoglobin-oxygen affinity and the Bohr effect coincided with the aquatic specialization of penguins.

Dive capacities of air-breathing vertebrates are dictated by onboard O2 stores, suggesting that physiologic specialization of diving birds such as penguins may have involved adaptive changes in convective O2 transport. It has been hypothesized that increased hemoglobin (Hb)-O2 affinity improves pulmonary O2 extraction and enhances the capacity for breath-hold diving. To investigate evolved changes in Hb function associated with the aquatic specialization of penguins, we integrated comparative measurements of whole-blood and purified native Hb with protein engineering experiments based on site-directed mutagenesis. We reconstructed and resurrected ancestral Hb representing the common ancestor of penguins and the more ancient ancestor shared by penguins and their closest nondiving relatives (order Procellariiformes, which includes albatrosses, shearwaters, petrels, and storm petrels). These two ancestors bracket the phylogenetic interval in which penguin-specific changes in Hb function would have evolved. The experiments revealed that penguins evolved a derived increase in Hb-O2 affinity and a greatly augmented Bohr effect (i.e., reduced Hb-O2 affinity at low pH). Although an increased Hb-O2 affinity reduces the gradient for O2 diffusion from systemic capillaries to metabolizing cells, this can be compensated by a concomitant enhancement of the Bohr effect, thereby promoting O2 unloading in acidified tissues. We suggest that the evolved increase in Hb-O2 affinity in combination with the augmented Bohr effect maximizes both O2 extraction from the lungs and O2 unloading from the blood, allowing penguins to fully utilize their onboard O2 stores and maximize underwater foraging time.

Trade-off between aerobic performance and egg production in migratory macaroni penguins.

When successive stages of an organism's life-history overlap, conflicts and trade-offs can emerge due to competition among physiological pathways. For example, long periods of sustained locomotion in migrating birds are supported by the androgenic up-regulation of aerobic factors, such as new red blood cell production and hematocrit. However, towards the end of migration, many female birds begin up-regulating 17ß-estradiol (E2) to support vitellogenesis and egg production, but E2 secretion is known to have suppressive effects on red blood cell production (anti-erythropoiesis). We explored potential trade-offs between factors related to aerobic performance (hematocrit, reticulocyte index) and the expression of factors related to E2-mediated vitellogenesis (i.e. yolk precursor production) in female macaroni penguins (Eudyptes chrysolophus), a species in which the physiologies controlling egg production and migratory activity run simultaneously (e.g. females experience a migratory conflict). We collected blood samples from penguins immediately upon their return to the colony, prior to egg laying. Hematocrit was elevated when the penguins returned to the colony (50.05% ± 3.40 SD), which is similar to pre-laying values observed in other migratory bird species. Furthermore, mean reticulocyte levels were elevated (34.87% ± 2.34), which is the highest level yet recorded in birds. Similarly, both plasma vitellogenin and yolk-targeted very low density lipoprotein levels were upregulated (2.30 ± 0.06 µg Zn ml-1, and 9.70 ± 0.19 mmol l-1, respectively), indicating that penguins were reproductively active and producing eggs during migration and upon arrival on land. As predicted, a negative relationship between hematocrit and plasma vitellogenin was found, but we found no evidence to suggest that birds were experiencing reproductive anemia. Alternatively, we attribute the negative relationship to a hemodilution effect of yolk precursor secretion into circulation. It appears that female macaroni penguins are able to preserve hematocrit levels and new red blood cell production when migratory activity overlaps with reproductive processes.

Effects of nest type and sex on blood saccharide profiles in Humboldt penguins (Spheniscus humboldti): Implications for habitat conservation.

Reproductive success of endangered Humboldt penguin (Spheniscus humboldti) colonies in Peru has been associated with nesting habitat type, presumably due to differences in environmental exposure and activity patterns that may affect energy demands and metabolism. Gas chromatography and mass spectrometry were used to determine serum concentrations of 19 saccharides from 30 Humboldt penguins nesting at Punta San Juan, Peru in order to evaluate differences in metabolic state between penguins nesting in a sheltered burrow or crevice (n = 17) and those in exposed surface nests (n = 13). Univariate and multivariate statistical analyses identified serum saccharides (arabinose, maltose, glucose-6-phosphate, and levoglucosenone in particular) that were nest-dimorphic with substantial differences between surface- and sheltered-nesting penguins. Four sugars (arabinose, xylose, fructose-6-phosphate, and sucrose) had ≥ 2-fold difference in concentration between nest types. Seven saccharides were in the top five subsets generated by discriminant analysis; four of these are simple sugars (D-glucopyranose, α ⇄ ß; D-glucose; D-maltose; and D-mannose) and three are derivatives (glucose 6-phosphate, levoglucosenone, and N-acetylglucosamine). D-ribose had the highest information values (generated from weight-of-evidence values) followed by glucose 6-phosphate, levoglucosenone, and D-galactose. Sex was not a significant predictor of saccharide concentration. Levoglucosenone, which is a metabolite of the environmental contaminant levoglucosan, was significantly higher in surface-nesting penguins, reflecting a higher rate of exposure in non-sheltered penguins. Differences in the saccharide profiles of surface- and sheltered-nesting Humboldt penguins likely reflect increased metabolic requirements of surface-nesters at Punta San Juan. Conservation of appropriate sheltered-nesting habitat for penguins is essential for sustained reproductive success and colony health.

Circulating nutrient concentrations in free-ranging Humboldt penguins (Spheniscus humboldti) in Punta San Juan, Peru.

Free-ranging Humboldt penguin (HP, Spheniscus humboldti) populations are under pressure from resource competition with industrial fisheries, habitat loss, and El Niño Southern Oscillation events. Foraging patterns for this top marine predator change during periods of aberrant oceanographic conditions and scarce fish stock numbers. These radical dietary fluctuations can lead to poor fertility, early embryonic death, poor hatchability, suppressed immune function, high chick mortality, and illness. To understand the variability of nutrient status in reproductive seasons, we measured select circulating nutrient concentrations (fat-soluble vitamins A, D, E, and carotenoids, fatty acids, amino acids, minerals, and electrolytes) of 105 HP at Punta San Juan, Peru during the first reproductive seasons of 2007 and 2008. We determined significant differences in nutrient status between sexes, years of sampling, and reproductive stages. Males (4.5 ± 0.38 kg) weighed more than females (4.0 ± 0.29 kg) and exhibited higher concentrations of vitamin A (0.71 ± 0.11 vs. 0.61 ± 0.12 µg/ml) and docosahexaenoic acid (6.70 ± 1.61 vs. 5.65 ± 1.59%). Males also displayed lower concentrations of ß-carotene (0.01 ± 0.01 vs. 0.012 ± 0.001 µg/ml) and phosphorus (3.43 ± 0.83 vs. 4.40 ± 1.66 mg/dl). Comparison between the 2 years showed most circulating amino acid concentrations were higher in 2007. Significant differences in circulating amino acids and vitamins were also noted between different reproductive stages. These results demonstrate concentrations of nutrients can vary due to the physiological state of the animal, as well as the overall dynamics of their marine ecosystem habitat.

HEMATOLOGY AND PLASMA CHEMISTRY VALUES IN BEACHED MAGELLANIC PENGUIN (SPHENISCUS MAGELLANICUS) IN NORTHERN ARGENTINA DURING THE NONBREEDING SEASON.

Seabirds have been widely used for monitoring the health of the oceans in diverse marine regions. Among low-cost survey strategies, systematic surveys of seabirds beached on coasts have been developed since the 20th century. However, these studies do not always address blood aspects. The assessment of the health status of birds based on the analysis of hematological and plasma chemistry is crucial to evaluate the overall health status profile of live organisms. Here, the authors study the variability of blood parameters by sex, age class, and year of beached Magellanic Penguin during the nonreproductive period in northern Argentina. Of 44 penguins, 77% were categorized as younger juveniles and the rest as older juveniles, and were captured and studied in coastal areas of Buenos Aires Province during the summers of 2017 and 2018. The mean body weight of beached penguins was affected by the age class of the individuals; most of the younger juveniles showed poor condition in terms of body mass (1,761 ± 235 g). No significant differences were observed in body weight between years and sex. Still, there were significant differences between years for alkaline phosphatase (ALP) and creatine phosphokinase (CPK) values. Twelve of the 20 blood parameters analyzed differ significantly with the age class of the beached penguins; younger juveniles were in a state of inanition. Our results may serve as a necessary first step in improving the conservation status of the Magellanic Penguin in nonbreeding grounds of Argentina, and call for a better knowledge of the health status of the species along its annual cycle.

Diet-tissue discrimination factors (δ15 N and δ13 C values) for blood components in Magellanic (Spheniscus magellanicus) and southern rockhopper (Eudyptes chrysocome) penguins.

RATIONALE: Analysis of the stable isotope ratios of carbon and nitrogen (δ13 C and δ15 N values) is increasingly being used to gain insight into predator trophic ecology, which requires accurate diet-tissue discrimination factors (DTDFs), or the isotopic difference between prey and predator. Accurate DTDFs must be calculated from predators consuming an isotopically constant diet over time in controlled feeding experiments, but these studies have received little attention to date, especially among seabird species. METHODS: In this study, aquarium-housed Magellanic (Spheniscus magellanicus) and southern rockhopper (Eudyptes chrysocome) penguins were fed a single-prey source diet (capelin Mallotus villosus) for eight weeks. Stable isotope ratios (δ13 C and δ15 N values) of penguin blood (cellular component and plasma) and capelin were measured using mass spectrometry and then used to calculate DTDFs for both components of penguin blood by comparison with prey values. RESULTS: The DTDFs for plasma were -0.63 ± 0.49 (mean ± SD) and -0.27 ± 0.22 for δ13 C values, and 2.60 ± 0.50 and 2.78 ± 0.22 for δ15 N values for Magellanic and southern rockhopper penguins, respectively, while the DTDFs for the cellular component were 1.22 ± 0.03 and 1.26 ± 0.03 for δ13 C values, and 2.54 ± 0.07 and 2.43 ± 0.17 for δ15 N values. CONCLUSIONS: We compare our DTDFs with published values from blood components of penguins and discuss the effects that lipid extraction, sample storage, and diet have on the DTDFs of penguin blood components. This study provides accurate DTDFs of blood components for two seabird species of conservation concern, and is one of the first to provide plasma DTDFs for penguins, which are underrepresented in the seabird literature.

Effects of feeding on plasma concentrations of vitamin A in captive African penguins (Spheniscus demersus).

Vitamin A comprises vitamin A1 and vitamin A2; vitamin A1 is retinol and its fatty-acid esters and vitamin A2 is 3,4-didehydroretinol and its fatty-acid esters. Although vitamin A1 is generally recognized as the major vitamin A, vitamin A2 is found in some birds and mammals that eat fish containing vitamin A2. Plasma concentration of retinyl esters, but not retinol, is known to increase postprandially in humans. The objectives of this study were to confirm the presence of vitamin A2 in fish fed to penguins, and in penguin plasma, and the postprandial changes in vitamin A concentration in penguin plasma. Blood was collected from six male African penguins (Spheniscus demersus) before and after feeding on jack mackerels (Trachurus japonicus) along with a vitamin premix containing vitamin A1. Vitamin A1 concentration in fish was much higher than the requirement, and was 5-fold higher than the vitamin A2 concentration. Vitamin A2 was present in plasma but its concentration was at least 100-fold below that of plasma retinol, suggesting that vitamin A2 is much less bioavailable than vitamin A1 in penguins. Plasma retinol and retinyl palmitate concentrations were found to be stable after the meal. Plasma retinol concentration is suggested to be homeostatically controlled in penguins against the rapid flow of vitamin A1 after meal. The absorbed vitamin A1 is thought to be transported to the liver via the portal vein for storage in penguins, resulting in stable retinyl palmitate concentration in plasma after meal.

Survival rates of oil-rehabilitated and non-rehabilitated little penguins after the C/V Rena oil spill, New Zealand.

We monitored the post-release survival of little penguins (Eudyptula minor) rehabilitated after the 2011 C/V Rena oil spill in New Zealand to assess the effectiveness of the rehabilitation process. Surveys were conducted over a 23-month period after the spill to assess whether survival differed between rehabilitated and control penguins. Survival probabilities from mark-recapture analyses were lower for both oiled and control penguins in the first six months of the study (monthly probability 0.92) but increased and remained high thereafter (monthly probabilities 0.97-1.0). Importantly, survival did not differ significantly between oiled and control birds throughout the study. Post-release survival of rehabilitated birds was not influenced by the degree of oiling, body mass (at admission or release), blood parameters (admission packed cell volume, total protein or blood glucose) or the duration of captivity. Rehabilitation therefore appeared to successfully reverse the negative effects of oiling on the post-release survival of treated penguins.

Clinical biochemistry of rescued Magellanic Penguins (Spheniscus magellanicus): serum or heparinized plasma? / Bioquímica clínica de pinguins-de-magalhães (Spheniscus magellanicus) de resgate: soro ou plasma heparinizado?

IIn the last few years, an increasing number of debilitated Magellanic penguins (Spheniscus magellanicus) has been rescued and taken to rehabilitation centers on Brazil's southern coast to be clinically treated and evaluated for re-introduction. This work aims to compare the viability of heparinized plasma with the viability of serum for biochemistry analyses under rehabilitation conditions. Blood sampled from 31 physically healthy rescued penguins was processed into serum/plasma-paired samples and analyzed for 12 biochemical parameters: alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), cholesterol (CHOL), creatine kinase (CK), gamma-glutamyl transpeptidase (GGT), glucose, (GLU) lactate dehydrogenase (LDH), total proteins (TP), triglycerides (TG), urea (UR), and uric acid (UA).The results showed that six paired samples presented visual signs of hemolysis (visual hemolytic score≥1), four of which occurred exclusively in the serum counterpart. Significant differences (P≤ 0.5) between sample types were found for CHOL (3%), GLU (6%) and TG (52%). Only TG was considered clinically relevant (>10%). All mean/median results fell within the available reference intervals by the Association of Zoos and Aquariums (Penguin, 2014). In conclusion, we verified that heparinized plasma is a viable sample for the clinical biochemistry of rescued Magellanic penguins as it yields compatible results with serum, while providing practical benefits. The adoption of this practice favors a faster bird recovery, by minimizing blood sampling volume, and optimizes material resources, allowing use of the same collector tube as for hematology.(AU)

Nos últimos anos, um número crescente de pinguins-de-magalhães (Spheniscus magellanicus) debilitados vem sendo resgatado e encaminhado aos centros de reabilitação do litoral sul do Brasil para cuidados clínicos e posterior avaliação de reintrodução. Este trabalho teve como objetivo comparar a viabilidade do plasma heparinizado com a do soro para análises bioquímicas, em condições de reabilitação. Amostras de sangue de 31 pinguins de resgate fisicamente saudáveis foram processadas em amostras pareadas de soro e plasma heparinizado, e 12 parâmetros bioquímicos foram analisados: alanina aminotransferase (ALT), fosfatase alcalina (ALP), aspartato aminotransferase (AST), colesterol (CHOL), creatina quinase (CK), gamaglutamil transpeptidase (GGT), glicose (GLU), lactato desidrogenase (LDH), proteínas totais (TP), triglicérides (TG), ureia (UR) e ácido úrico (UA). Os resultados mostraram que seis amostras pareadas apresentaram sinais visuais de hemólise (escore hemolítico visual≥1), das quais quatro ocorreram exclusivamente no soro. Observaram-se diferenças significativas (P≤0,5) entre os tipos de amostra em CHOL (3%), GLU (6%) e TG (52%), sendo apenas TG considerado clinicamente relevante (>10%). Todos os resultados de médias e medianas situaram-se dentro dos intervalos de referência disponíveis fornecidos pela Associação de Zoológicos e Aquários (AZA). Como conclusão, verificou-se que o plasma heparinizado é uma amostra viável para a bioquímica clínica de pinguins-de-magalhães de resgate, produzindo resultados compatíveis com os do soro. Além disso, a adoção dessa prática favorece uma recuperação mais rápida dos animais, ao diminuir o volume de sangue amostrado, e otimiza os recursos materiais, ao permitir o aproveitamento do mesmo tubo de colheita de hematologia.(AU)

Effect of molting on the concentration of plasma 25-hydroxyvitamin D in captive African penguins (Spheniscus demersus).

Feed intake and body weight are drastically altered in penguins during peri-molting period, and molting is known to affect the nutritional status of vitamin A and E. Although vitamin D status is not known in penguins during peri-molting period, vitamin D intake is supposed to be remarkably altered. The objective of the present study was to clarify the alterations in plasma 25-hydroxyvitamin D (25(OH)D) concentration, the most reliable biomarker for assessing vitamin D status, and vitamin D intake during peri-molting period. Blood samples were collected from seven adult male African penguins (Spheniscus demersus) in the control period, pre-molting period, early-molting period, and late-molting period. The dietary content of vitamin D and calcium (Ca) were higher than that of the estimated requirements. Feed intake increased in the pre-molting period and drastically decreased during the molting periods. Body weight increased in the pre-molting period, followed by the loss of weight towards the end of the experiment. Although vitamin D and Ca intakes decreased during the molting periods, plasma 25(OH)D concentration increased during the molting periods and the increase in plasma Ca concentration was also observed in the late-molting period. These results suggest that the reduction in body fat induced by reducing feed intake stimulated the release of vitamin D from body fat, which increased plasma 25(OH)D and Ca concentrations in molting penguins. Penguins are unlikely to suffer from typical hypervitaminosis D even during molting and vitamin D toxicity is not a realistic problem in penguins because of the short duration of molting.

Analysis of Toxic and Essential Elements in the Blood and Feathers of Humboldt Penguins ( Spheniscus humboldti) at Punta San Juan, Peru.

Whole blood, serum, and feather samples from 29 Humboldt Penguins ( Spheniscus humboldti) at the Punta San Juan Marine Protected Area, Peru, were analyzed for 55 toxic and essential elements by using inductively coupled plasma mass spectrometry. Mercury (Hg) was analyzed by cold vapor atomic fluorescence. Maximum Hg concentrations in serum (0.0056 mg/g), whole blood (0.297 mg/kg), and feathers (1.8 mg/kg dry weight) were at levels generally not considered to cause health impairment. Of the elements analyzed, only eight (aluminum, calcium, iron, Hg, potassium, magnesium, sodium, and zinc) were detected in serum. These elements, plus selenium and titanium, were also quantifiable in whole blood. Feather analysis detected quantifiable values for the elements found in serum, plus arsenic, boron, barium, copper, manganese, and titanium. Results indicate this important breeding population of endangered penguins did not appear to be exposed to environmental elemental contaminants at levels detrimental to health and reproductive success. However, identification of measurable concentrations of toxic elements at low levels underscores the need for continued environmental monitoring, particularly in the face of expanding regional human populations and industrial growth. These results provide important reference data for temporospatial monitoring of this and other penguin populations.

Application of different pharmacokinetic models to describe and predict pharmacokinetics of voriconazole in magellanic penguins following oral administration.

Aspergillosis is a condition causing serious morbidity and mortality in captive penguins and other bird species. It can be treated with antifungal drugs, such as voriconazole. However, the pharmacokinetics of voriconazole are variable between different animal and bird species. Therefore, the pharmacokinetics of voriconazole were investigated in this study in Magellanic penguins. Pharmacokinetic models were constructed and applied to predict the pharmacokinetics of voriconazole during long-term treatment in Magellanic penguins, since the voriconazole treatment duration in chronic aspergillosis cases can last up to several months. Plasma voriconazole concentration-time data from adult Magellanic penguins (Spheniscus magellanicus; n = 15) following a single oral (PO) dose of either 2.5 mg/kg or 5 mg/kg in a herring in three separate study periods 7-12 months apart were collected. Mean plasma voriconazole concentrations were above the targeted MIC for Aspergillus fumigatus for 2 hr following a single 2.5 mg/kg voriconazole dose while the plasma concentrations exceeded the MIC for least 24 hr following a 5 mg/kg dose. Nonlinear mixed-effects modeling was used to fit two pharmacokinetic models, one with first-order and another with saturable elimination, to the single-dose data. Fits were good for both, as long as dose was included as a covariate for the first-order model so that clearance was lower and the half-life longer for animals receiving the 5 mg/kg dose. Although the single-dose data suggested saturated elimination at higher concentrations, the model with saturable elimination did not predict plasma voriconazole concentrations well for a clinical aspergillosis case receiving long-term treatment, possibly because of induction of metabolizing enzymes with chronic exposure. Pharmacokinetic models should accurately predict plasma drug concentrations for different dosage regimens in order to be applicable in the field. Future studies should focus on determining clearance at steady-state to be able to refine the pharmacokinetic models presented here and improve model performance for long-term oral voriconazole administration in Magellanic penguins.

Occurrence of tissue cyst forming coccidia in Magellanic penguins (Spheniscus magellanicus) rescued on the coast of Brazil.

The main motivation for this study was to determine the occurrence of Toxoplasma gondii, a cosmopolitan widespread zoonotic parasite distribution that can infect a wide variety of mammals and birds, in Magellanic penguins (Spheniscus magellanicus) in Brazil. In recent decades there has been a significant increase in the number of penguins originating from Argentinian and Chilean Patagonia, where these birds are born, that arrive on the Brazilian coast, where many of them are stranded and rescued. Tissue samples were collected from 330 individuals surveyed from 2012-2015 at the Institute for Marine Animal Research and Rehabilitation (IPRAM) located in Cariacica, state of Espirito Santo, Brazil. Serum were collected from 145 animals surveyed in 2015 for the detection of anti-T. gondii antibodies using the Modified Agglutination Test (MAT ≥20) and 18 birds were positive, with titers of 20 (7 birds), 40 (9 birds) and 80 (2 birds). Mouse bioassay for the isolation of T. gondii was performed using tissues from 54 penguins that were also surveyed in 2015, but no isolates were obtained. DNA from tissue samples of 330 individuals was PCR amplified and sequenced to detect tissue cyst forming coccidians by using pan sarcocystids-directed primers (based on 18S rDNA). These samples were from animals surveyed in 2015 and from frozen stocked tissues from animals surveyed in the years 2012 and 2013. The positives were PCR amplified and sequenced with genus Sarcocystis-specific primers (based on internal transcribed spacer 1, RNA polymerase beta subunit coding gene, and cytochrome B coding gene) and with Sarcocystis falcatula/Sarcocystis neurona- specific primers (based on surface antigens SAG2, SAG3 and SAG4). Sixteen (3.0%) of pectoral muscle samples were positive by all the seven molecular markers and all the samples were identical to each other. Organisms close related to Sarcocystis falcatula were confirmed in all cases. This is the first report on molecular detection of infection by S. falcatula-related organisms and the first report of seropositivity for T. gondii in free-living Magellanic penguins in Brazil. Felids and didephid opossums are definitive hosts of T. gondii and S. falcatula, respectively. Where the penguins acquire the infective forms of the parasites shed by the terrestrial mammals remains to be elucidated.

APPLICATION OF 3-HYDROXYBUTYRATE MEASUREMENT AND PLASMA PROTEIN ELECTROPHORESIS IN THE DIAGNOSIS OF ASPERGILLOSIS IN AFRICAN PENGUINS ( SPHENISCUS DEMERSUS).

New alternative laboratory means are needed to improve the options for antemortem diagnosis of avian aspergillosis. In this study, 3-hydroxybutyrate was measured in plasma samples collected from a cohort of African penguins ( Spheniscus demersus) maintained under human care. Results were interpreted in combination with those of protein electrophoresis and compared with anti- Aspergillus antibody and galactomannan antigen detection. Overall, 3-hydroxybutyrate levels were found significantly increased in Aspergillus-diseased cases versus the control penguin group ( P = 0.002). Mean absolute concentration of ß-globulins was increased >20% in samples from infected birds, and α2-globublins were also found to be significantly increased versus clinically normal controls ( P < 0.001 and P = 0.001 respectively). Of note, the α2-globulins were also significantly increased versus penguins with inflammatory (non-aspergillosis) diseases ( P = 0.001). The specificity of 3-hydroxybutyrate, ß-globulins, and α2-globulins for aspergillosis was 78.6%, 79.6%, and 92.2%, respectively. Using these measures in tandem resulted in high specificity (>90%) and negative predictive value (≥80%). In contrast, anti- Aspergillus antibody and galactomannan antigen did not distinguish between infected cases and controls ( P > 0.05). This study demonstrates that basic testing in tandem with the new biomarker 3-hydroxybutyrate may provide reliable evidence for the diagnosis of aspergillosis in penguins.

PLASMA CONCENTRATIONS OF VITAMIN A1, B1, D3, AND E IN HUMBOLDT PENGUINS ( SPHENISCUS HUMBOLDTI) BEFORE AND AFTER DIETARY VITAMIN SUPPLEMENTATION OF THEIR FISH DIET.

In a practical feeding trial at Ouwehand Zoo, plasma concentrations of vitamin A1, calcidiol (D3), α-tocopherol (E), and B1 in 17 Humboldt penguins ( Spheniscus humboldti) were measured before and after supplementation to gain insight into the effect of supplementing these vitamins in animals being fed thawed frozen-fish diets. None of the penguins received vitamin supplements for at least 6 mo before the supplementation trial, which was conducted prior to their normal nesting and molting period. During the trial period, eight penguins received daily vitamin A1, D3, tocopheryl acetate, and B1 supplementation placed in their fish immediately prior to feeding and nine control penguins received no supplementation. Concentrations of vitamins A1, D3, α-tocopherol, and B1 were also measured in the thawed ready-to-feed fish. Concentrations of vitamins B1 and α-tocopherol were below the Association of Zoos and Aquariums (AZA) recommendations for penguin diets, while concentrations of vitamins A1 and D3 were far above AZA recommendations. At the start of the study and after 70 days of supplementation, plasma concentrations were determined for these vitamins. Vitamin B1 concentrations in plasma increased significantly ( P < 0.05) between Day 0 (mean 39.9 µg/L) and day 70 (mean 160.5 µg/L) in the supplemented group. Plasma vitamin D3 and α-tocopherol did not show a significant change. Vitamin A1 levels in the supplemented group decreased significantly from 1.65 mg/L on day 0 to 1.4 mg/L on day 70. In the control group no significant changes were observed. The results of the study support the necessity of supplementing vitamin B1 in penguins fed thawed frozen fish. Depletion of vitamin A and E concentrations in frozen food fish over time support recommendations to regularly measure vitamin concentrations in different batches of frozen fish.

UV-light and dietary vitamin D and their effects on ionized calcium and 25-OH-D plasma concentrations in captive gentoo penguins (Pygoscelis papua).

In this study, the effect of ultraviolet (UV) light and dietary vitamin D on calcium metabolism in permanently indoor-housed gentoo penguins (Pygoscelis papua) was investigated. The study consisted of three periods, each completed with blood samples to analyse plasma concentrations of 25-OH-D, 1,25-(OH)2 -D, ionized (iCa) and total calcium (tCa). During the first study period (D), animals were housed under routine conditions without UV-light and fed a diet of different fish species, supplemented with 1,000 IU vitamin D per animal and day. The following study period (Baseline) of 28-day duration consisted of the same diet without any vitamin D supplementation and without UV-light. During the study period (UVB) artificial UV-light was added for 3 weeks. The vitamin D content of fish was measured by high-performance liquid chromatography. It varied between fish species and between facilities, ranging from no measurable content in capelin (Mallotus villosus) to 7,340 IU vitamin D/kg original matter (OM) in herring (Clupea spp). The average dietary vitamin D content was 311 IU/kg OM at facility 1 and 6,325 IU/kg OM at facility 2, resulting in a vitamin D intake per animal and day without supplementation of 130 IU (25.5 IU/kg body weight BW) and 2,454 IU (438.2 IU/kg BW) respectively. The supplementation of vitamin D elevated significantly the plasma concentrations of 25-OH-D by an intraindividual difference of 15 (range -2 to 59) nmol/L and tCa by 0.1 (0.0-0.3) mmol/L only at facility 2. The exposure to UV-light raised the blood concentrations of tCa at facility 2 by 0.15 (0.1-0.2) mmol/L, and of iCa and tCa for females at facility 1 by 0.23 (0.13-0.41) mmol/L and 1.8 (1.1-2.5) mmol/L respectively. No significant influence of the study periods (D) and (UVB) was found for the concentrations of 1,25-(OH)2 -D at both facilities.

Influence of biological factors on hematological and serum biochemistry values in captive Adélie, Chinstrap, Gentoo, and Macaroni penguins in Pingtung, Taiwan.

Hematology and serum biochemistry reference values are essential for health evaluation and disease diagnosis in penguins. However, there are currently no published physiological values for captive Adélie (Pygoscelis adeliae) and Chinstrap penguins (P. antarcticus), nor for wild or captive Macaroni penguins (Eudyptes chrysolophus). The present study is the first investigation regarding hematology and serum biochemistry reference values for captive Adélie, Gentoo (P. papua), Chinstrap, and Macaroni penguins in Asia. Fixed effect models for repeated measure were applied to determine the influence of penguin species, age, gender, and age-gender interaction on each blood parameter. Hematology and serum biochemical data from 122 apparently healthy penguins (24 Adélie, 38 Chinstrap, 46 Gentoo, and 14 Macaroni) were collected between 2009 and 2014. The effects of penguin species were observed for most blood parameters, except total bilirubin, creatine kinase (CK), creatinine, and potassium ion (K+ ). Values of mean corpuscular volume, mean corpuscular hemoglobin (MCH), heterophil, ratio of heterophils to lymphocytes (H/L), alanine aminotransferase (ALT), and chloride ion (Cl- ) had significant positive correlation with age, while significant negative correlation with age was observed in total red blood cells (RBCs), lymphocytes, thrombocytes, alkaline phosphatase (ALP), CK, lactate dehydrogenase (LDH), and plasma iron. Compared to male penguins, females had lower mean corpuscular hemoglobin concentration (MCHC) and blood urea nitrogen (BUN) but higher calcium ion (Ca2+ ) values. As for age-gender interaction, significant positive correlation was shown in MCHC and K+ , and the reverse was true in H/L ratio.

ASSESSMENT OF POLYCHLORINATED BIPHENYLS, ORGANOCHLORINE PESTICIDES, AND POLYBROMINATED DIPHENYL ETHERS IN THE BLOOD OF HUMBOLDT PENGUINS ( SPHENISCUS HUMBOLDTI) FROM THE PUNTA SAN JUAN MARINE PROTECTED AREA, PERU.

Persistent organic pollutants were assessed in Humboldt Penguins ( Spheniscus humboldti) from the Punta San Juan Marine Protected Area, Peru, in the austral winter of 2009. Plasma samples from 29 penguins were evaluated for 31 polychlorinated biphenyl (PCB) congeners and 11 organochlorine pesticides (OCPs) by using gas chromatography coupled to an ion trap mass spectrometer and for 15 polybrominated diphenyl ether (PBDE) congeners by using gas chromatography coupled with high-resolution mass spectrometry. The detection rate for PCBs in the samples was 69%, with congeners 105, 118, 180, and 153 most commonly detected. The maximum ΣPCB concentration was 25 ng/g. The detection rate for DDT, DDD, and/or DDE was higher than for other OCP residues (90%; maximum concentration=10 ng/g). The detection rate for PBDEs was 86%, but most concentrations were low (maximum ΣPBDE concentration=3.81 ng/g). This crucial breeding population of S. humboldti was not exposed to contaminants at levels detrimental to health and reproductive success; however, the identified concentrations of legacy and recently emerged toxicants underscore the need for temporal monitoring and diligence to protect this endangered species in the face of regional human population and industrial growth. These results also provide key reference values for spatial comparisons throughout the range of this species.