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)

Schistosomes and Microfilarial Parasites in Magellanic Penguins.

The Magellanic Penguin ( Spheniscus magellanicus) is native to Argentina, Chile, and the Falkland/Malvinas Islands, and is a regular winter migrant in Uruguayan and Brazilian coastal waters. The species is known to be susceptible to a variety of gastrointestinal nematodes, cestodes, trematodes, and acanthocephalans, as well as renal trematodes and pulmonary nematodes. Schistosomes (Platyhelminthes, Trematoda, Schistosomatidae) and microfilariae (Nematoda, Secernentea, Onchocercidae) were histologically identified in Magellanic Penguins ( Spheniscus magellanicus) that died while under care at rehabilitation centers in southern Brazil. Phylogenetic analysis of the COI gene, ITS-1 region, 5.8S rRNA gene, ITS-2 region, and 28S rRNA gene sequences of the schistosome revealed that it is closely related to, but distinct from, a schistosome reported from the African Penguin ( Spheniscus demersus). The schistosomes from Magellanic and African Penguins were grouped with Gigantobilharzia huronensis, Gigantobilharzia melanoidis, and Dendritobilharzia pulvurenta; however, the lack of a clearly monophyletic origin precludes determining their genus. The incidental discovery of novel parasites during a study that did not specifically aim to investigate the occurrence of helminths underscores the value of histopathological examination as an exploratory diagnostic approach.

Persistent organic pollutants in juvenile Magellanic Penguins (Spheniscus magellanicus) in South America.

Magellanic penguins, Spheniscus magellanicus, are the most abundant penguins living in temperate regions of South America and are good indicators of environmental pollution in the region. Persistent organic pollutants (POPs) were detected in the liver of Magellanic penguins found debilitated or dead on the beaches of Brazil (states of Rio de Janeiro, Sao Paulo, Santa Catarina and Rio Grande do Sul) between 2008 and 2012 as well as in Uruguay and Chile in 2011. Polychlorinated biphenyls (PCBs) were more prevalent than organochlorine pesticides (DDTs ∼ HCB ∼ Drins) and polybrominated diphenyl ethers (PBDEs). Among PCBs, penta-, hexa- and hepta-chlorinated congeners were predominant. Concentrations of POPs were similar between the Pacific and Atlantic penguin populations, except for PCBs, which were relatively higher in the Pacific population. During the study years (2008-2012), large variations were found in organochlorine pesticides and PCBs tended to decline. Overall, the southern portion of South America has low concentrations of POPs, with either a constant trend or evidence of decline.

Lacaziosis-like disease in Tursiops truncatus from Brazil: a histopathological and immunohistochemical approach

ABSTRACT: Cetacean lacaziosis-like disease or lobomycosis-like disease (LLD) is a chronic skin condition caused by a non-cultivable yeast of the order Onygenales, which also includes Lacazia loboi, as well as Paracoccidioides brasiliensis and P. lutzii, respectively responsible for lacaziosis and paracoccidioidomycosis in humans. Complete identification and phylogenetic classification of the LLD etiological agent still needs to be elucidated, but preliminary phylogenetic analyses have shown a closer relationship of the LLD agent to Paracoccidioides spp. than to L. loboi. Cases of LLD in South American cetaceans based on photographic identification have been reported; however, to date, only 3 histologically confirmed cases of LLD have been described. We evaluated multiple tissue samples from 4 Tursiops truncatus stranded in the states of Santa Catarina (n = 3) and Rio Grande do Sul (n = 1), southern Brazil. Macroscopically, all animals presented lesions consistent with LLD. Hematoxylin-eosin, periodic acid-Schiff, Grocott's methenamine silver, and Mayer's mucicarmin stains were used for histological evaluation. Microscopically, numerous refractile yeasts (4-9 µm in diameter) were observed in skin samples (4/4), and for the first time in dolphins, also in a skeletal muscle abscess (1/4). Immunohistochemistry using anti-P. brasiliensis glycoprotein gp43 as a primary antibody, which is known to cross-react with L. loboi and the LLD agent, was performed and results were positive in all 4 cases. We describe 3 new cases of LLD in cetaceans based on histopathology and immunohistochemistry. This is the first report of LLD in the muscle of cetaceans.

Assessment of adenovirus, hepatitis A virus and rotavirus presence in environmental samples in Florianopolis, South Brazil.

AIMS: To assess the presence of human adenovirus (HAdV), hepatitis A (HAV) virus and rotavirus A (RV-A) in environmental samples from the Southern region of Brazil and to provide viral contamination data for further epidemiological studies and governmental actions. METHODS AND RESULTS: Water samples from various sources (seawater, lagoon brackish water, urban wastewater, drinking water sources-with and without chlorination and water derived from a polluted creek) and oysters of two growing areas were analysed by enzymatic amplification (nested PCR and RT-PCR), quantification of HAdV genome (qPCR) and viral viability assay by integrated cell culture-PCR (ICC-PCR). From June 2007 to May 2008 in a total of 84 water samples, 54 (64·2%) were positive for HAdV, 16 (19%) for RV-A and 7 (8·3%) for HAV. Viability assays showed nonpositive samples for HAV; though, infectious viruses were confirmed for RV-A (12·5%) and HAdV (88·8%). Oyster samples by PCR were positive for HAdV (87·5%) and RV-A (8·3%), but none for HAV. Quantitative PCR in oysters showed means loads in genomic copies (gc) of 9·1 × 10(4) gc g(-1) (oyster farm south) and 1·5 × 10(5) gc g(-1) (oyster farm north) and in waters ranging from 2·16 × 10(6) (lagoon water) to 1·33 × 10(7) gc l(-1) (untreated drinking water). CONCLUSIONS: This study has shown a widespread distribution of the analysed viruses in this particular region with high loads of HAdV in the environment which suggests the relevance of evaluating these viruses as positive indicators of viral contamination of water. SIGNIFICANCE AND IMPACT OF THE STUDY: The environmental approach in this study provides data concerning the prevalence, viability and quantification of enteric viruses in environmental waters and oysters in the South region of Brazil and has indicated that their presence might pose a risk to population in contact with the environmental samples searched.

Assessment of norovirus contamination in environmental samples from Florianópolis City, Southern Brazil.

AIMS: To assess norovirus (NoV) contamination in aquatic ecosystems in the city of Florianópolis, in Southern Brazil, to provide epidemiological data that can support actions for environmental contamination control. METHODS AND RESULTS: An adsorption-elution method, followed by ultrafiltration, was performed to concentrate the viruses. NoV were detected using semi-nested PCR and quantified by real-time PCR. From June 2007 to May 2008, NoV were detected in 23% (22/94) of the samples analysed, including seawater, drinking water, superficial water (creek and brackish lagoon) and treated sewage. The mean viral loads for genogroups (G)I and GII in treated sewage samples were 297 and 440 genomic copies (gc) l(-1) , respectively, whereas creek water samples contained 2603 and 1361 gc l(-1) , respectively. Six samples were sequenced: two samples were GII.4, two were GII.2 and two were GI.3. CONCLUSIONS: NoV were detected in all water types analysed, demonstrating the widespread contamination of this geographical area with several cocirculating strains belonging to GI and GII. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the environmental spread of NoV in environmental waters and highlights the potential hazard for human health following the consumption of or contact with these waters, which could result in waterborne or foodborne acute gastroenteritis.

Evaluation of HA negatively charged membranes in the recovery of human adenoviruses and hepatitis A virus in different water matrices.

Human adenoviruses (HAdV) and hepatitis A virus (HAV) are shed in the faeces and consequently may be present in environmental waters, resulting in an increase in pathogen concentration that can affect water quality and human health. The aim of this study was to evaluate an adsorption-elution method which utilizes negatively charged membrane HA to determine the efficient recovery of HAdV and HAV from different water matrices and to combine this procedure with a qualitative molecular method (nested RT-PCR and nested PCR). The best efficiency recovery was achieved in distilled water and treated wastewater effluent (100%) for both viruses and in recreational lagoon water for HAV (100%). The efficiency recovery was 10% for HAdV and HAV in seawater and 10% for HAdV in lagoon water. The viral detection limit by nested PCR for HAV in water samples ranged between 20-0.2 FFU/mL and 250 and 25 TCID50/mL for HAdV. In conclusion, these results suggest that the HA negatively charged membranes vary their efficiency for recovery of viral concentration depending upon the types of both enteric viruses and water matrices.