Identification and function analysis of BCL2 in immune response of Pteria penguin.

B-cell lymphoma/leukemia-2 (BCL2), an anti-apoptotic factor in the mitochondrial regulatory pathway of apoptosis, is critically important in immune defenses. In this study, a novel BCL2 gene was characterized from Pteria penguin (P. penguin). The PpBCL2 was 1482 bp long, containing an open reading frame (ORF) of 588 bp encoding 195 amino acids. Four highly conserved BCL-2 homology (BH) domains were found in PpBCL2. Amino acid alignment and phylogenetic tree showed that PpBCL2 had the highest similarity with BCL2 of Crassostrea gigas at 65.24 %. Tissue expression analysis showed that PpBCL2 had high constitutive expression in gill, digestive diverticulum and mantle, and was significantly increased 72 h of Vibrio parahaemolyticus (V. parahaemolyticus) challenge in these immune tissues. Furthermore, PpBCL2 silencing significantly inhibited antimicrobial activity of hemolymph supernatant by 1.4-fold, and significantly reduced the survival rate by 51.7 % at 72 h post infection in P. penguin. These data indicated that PpBCL2 played an important role in immune response of P. penguin against V. parahaemolyticus infection.

Evidence of Pathogen-Induced Immunogenetic Selection across the Large Geographic Range of a Wild Seabird.

Over evolutionary time, pathogen challenge shapes the immune phenotype of the host to better respond to an incipient threat. The extent and direction of this selection pressure depend on the local pathogen composition, which is in turn determined by biotic and abiotic features of the environment. However, little is known about adaptation to local pathogen threats in wild animals. The Gentoo penguin (Pygoscelis papua) is a species complex that lends itself to the study of immune adaptation because of its circumpolar distribution over a large latitudinal range, with little or no admixture between different clades. In this study, we examine the diversity in a key family of innate immune genes-the Toll-like receptors (TLRs)-across the range of the Gentoo penguin. The three TLRs that we investigated present varying levels of diversity, with TLR4 and TLR5 greatly exceeding the diversity of TLR7. We present evidence of positive selection in TLR4 and TLR5, which points to pathogen-driven adaptation to the local pathogen milieu. Finally, we demonstrate that two positively selected cosegregating sites in TLR5 are sufficient to alter the responsiveness of the receptor to its bacterial ligand, flagellin. Taken together, these results suggest that Gentoo penguins have experienced distinct pathogen-driven selection pressures in different environments, which may be important given the role of the Gentoo penguin as a sentinel species in some of the world's most rapidly changing environments.

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.

Diversity in the Toll-Like Receptor Genes of the African Penguin (Spheniscus demersus).

The African penguin, Spheniscus demersus, is listed as Endangered by the IUCN Red List of Threatened Species due to the drastic reduction in population numbers over the last 20 years. To date, the only studies on immunogenetic variation in penguins have been conducted on the major histocompatibility complex (MHC) genes. It was shown in humans that up to half of the genetic variability in immune responses to pathogens are located in non-MHC genes. Toll-like receptors (TLRs) are now increasingly being studied in a variety of taxa as a broader approach to determine functional genetic diversity. In this study, we confirm low genetic diversity in the innate immune region of African penguins similar to that observed in New Zealand robin that has undergone several severe population bottlenecks. Single nucleotide polymorphism (SNP) diversity across TLRs varied between ex situ and in situ penguins with the number of non-synonymous alterations in ex situ populations (n = 14) being reduced in comparison to in situ populations (n = 16). Maintaining adaptive diversity is of vital importance in the assurance populations as these animals may potentially be used in the future for re-introductions. Therefore, this study provides essential data on immune gene diversity in penguins and will assist in providing an additional monitoring tool for African penguin in the wild, as well as to monitor diversity in ex situ populations and to ensure that diversity found in the in situ populations are captured in the assurance populations.

Serological monitoring of antibodies for an early diagnosis ofaspergillosis in captive penguins / Monitoramento sorológico com detecção de anticorpos para diagnóstico precoce da aspergilose em pinguins em cativeiro

This study aimed to evaluate the efficacy of detection of anti-Aspergillus fumigatus antibodies in captive penguins by double radial agar gel immunodiffusion (AGID) for the aspergillosis diagnosis. We included 134 Magellanic penguins (Spheniscus magellanicus) in rehabilitation at the Center for Recovery of Marine Animals (CRAM / FURG). All of them were monitored by AGID weekly until its final destination (death or release), totalizing 660 serum samples studied. All animals were clinically accompanied and post-mortem examinations was performed in penguins that died during the studied period. A total of 28% (37/134) of the penguins died, 89.2% (33/37) due to aspergillosis, 11% (4/37) by other causes and 97 were released. From the 33 animals with proven aspergillosis, 21 presented anti- A. fumigatus antibodies by AGID, being the average interval between death and positive AGID 16.4 days. Twelve animals with negative serology died of aspergillosis. The sensitivity and specificity rates were 63.6% and 95% respectively, and the positive and negative predictive values were 80.7% and 88.9% respectively. These data demonstrate that the serological monitoring for detection of antibodies by AGID can be an important tool for the diagnosis of aspergillosis in penguins.

Este estudo teve como objetivo avaliar a eficácia da detecção de anticorpos anti- Aspergillus fumigatus em pinguins em cativeiro por imunodifusão radial dupla em gel de ágar (IDGA) para diagnóstico da aspergilose. Foram incluídos 134 pingüins de Magalhães (Spheniscus magellanicus) em reabilitação no Centro de Recuperação de Animais Marinhos (CRAM/FURG), que foram monitoradas por IDGA, semanalmente, até o seu destino final (morte ou de liberação), totalizando 660 amostras de soro estudadas. Todos os animais foram acompanhados clinicamente e exames post mortem foram realizados em pingüins que vieram a óbito durante o período de estudo. Um total de 28% (37/134) dos pinguins foram a óbito, 89,2% (33/37) de aspergilose, 11% (4/37) de outras causas, e 97 foram liberados. A partir dos 33 animais com aspergilose comprovada, 21 apresentaram anticorpos anti- A. fumigatus por IDGA, sendo o intervalo médio entre a morte e IDGA positivas 16,4 dias. Doze animais com sorologia negativa vieram a óbito por aspergilose. As taxas de sensibilidade e especificidade foram de 63,6% e 95%, respectivamente, e os valores preditivos positivos e negativos foram de 80,7% e 88,9 %, respectivamente. Estes dados demonstram que o monitoramento sorológico para detecção de anticorpos por IDGA pode ser uma ferramenta importante no diagnóstico de aspergilose em pinguins.

Analysis of hematologic and serum chemistry values of Spheniscus magellanicus with molecular detection of avian malarial parasites (Plasmodium spp) / Análise dos valores hematológicos e bioquímicos de Spheniscus magellanicus com detecção molecular de parasitos maláricos aviários (Plasmodium spp)

Magellanic penguins (Spheniscus magellanicus) routinely migrate from their breeding colonies to Southern Brazil often contracting diseases during this migration, notably avian malaria, which has been already reported in Brazil and throughout the world. Detection of Plasmodium spp. in blood smears is the routine diagnostic method of avian malaria, however it has a low sensitivity rate when compared to molecular methods...

O pinguim-de-Magalhães (Spheniscus magellanicus) migra das suas colônias reprodutivas até o extremo sul do Brasil. Esses pinguins frequentemente são acometidos por doenças, notavelmente a malária aviária, que é relatada no Brasil e no mundo. A detecção de Plasmodium spp. no esfregaço sanguíneo é o método de rotina mas apresenta baixa sensibilidade quando comparado aos métodos moleculares...

Leucocyte profiles and corticosterone in chicks of southern rockhopper penguins.

The immune system is essential for health and survival of vertebrates, yet still little is known about the ontogeny of the immune system in wild birds. The southern rockhopper penguin (Eudyptes chrysocome chrysocome) is a semi-altricial seabird with a long developmental period and reversed hatching asynchrony, favouring the survival of B-chicks. We compared leucocyte counts and baseline corticosterone levels of southern rockhopper penguin chicks under different preconditions such as sex and origin from an A- or B-egg from 4 to 51 days of age. We conducted an experiment to compare leucocyte profiles and baseline corticosterone levels in A- and B-chicks in single-egg clutches as well as in B-chicks from normal two egg-clutches (one A- and one B-egg). None of these treatments influenced leucocyte counts or corticosterone levels, indicating a similar investment in the immune system. Our main finding was an increase of leucocytes/10,000 erythrocytes with age, which was mainly caused by an increase in lymphocyte numbers. This suggests differential investment into acquired immunity at this stage of development. As the granulocyte/lymphocyte (G/L) ratio did not change with age or body condition, G/L ratios seem not to reflect stress caused by poor provisioning of penguin chicks. This was also reinforced by the decrease of plasma corticosterone levels with age. Body condition was negatively correlated with monocyte numbers, suggesting a poorer health status of penguin chicks in poorer body condition. Yet, there was no link between body condition and other leucocyte parameters, indicating that chicks in a good body condition did not additionally invest into their immune system.

Trans-species polymorphism of the Mhc class II DRB-like gene in banded penguins (genus Spheniscus).

The Major Histocompatibility Complex (Mhc) class II DRB locus of vertebrates is highly polymorphic and some alleles may be shared between closely related species as a result of balancing selection in association with resistance to parasites. In this study, we developed a new set of PCR primers to amplify, clone, and sequence overlapping portions of the Mhc class II DRB-like gene from the 5'UTR end to intron 3, including exons 1, 2, and 3 and introns 1 and 2 in four species (20 Humboldt, six African, five Magellanic, and three Galapagos penguins) of penguin from the genus Spheniscus (Sphe). Analysis of gene sequence variation by the neighbor-joining method of 21 Sphe sequences and 20 previously published sequences from four other penguin species revealed overlapping clades within the Sphe species, but species-specific clades for the other penguin species. The overlap of the DRB-like gene sequence variants between the four Sphe species suggests that, despite their allopatric distribution, the Sphe species are closely related and that some shared DRB1 alleles may have undergone a trans-species inheritance because of balancing selection and/or recent rapid speciation. The new primers and PCR assays that we have developed for the identification of the DRB1 DNA and protein sequence variations appear to be useful for the characterization of the molecular evolution of the gene in closely related Penguin species and might be helpful for the assessment of the genetic health and the management of the conservation and captivity of these endangered species.

West Nile virus seroconversion in penguins after vaccination with a killed virus vaccine or a DNA vaccine.

To investigate the serologic response of penguins to West Nile virus (WNV) vaccines, four species of exclusively indoor-housed penguins, negative for WNV by serology, were evaluated: Humboldt (Spheniscus humboldti), Magellanic (Spheniscus magellanicus), Gentoo (Pygoscelis papua), and Rockhopper (Eudyptes chrysoscome) penguins. Birds were inoculated with either a killed virus vaccine or a plasmid-mediated DNA WNV vaccine, and postinoculation serology was evaluated. Both vaccines induced seroconversion in all four species, and no adverse reactions were noted. Postvaccination serology results varied across species and vaccine types. However, in all four species, the killed virus vaccine resulted in a greater seroconversion rate than the DNA vaccine and in a significantly shorter time period. Additionally, the duration of the seropositive titer was significantly longer in those birds vaccinated with the killed virus vaccine compared with those vaccinated with the DNA vaccine. A subset of unvaccinated penguins serving as negative controls remained negative throughout the duration of the study despite the presence of WNV in the geographic locations of the study, suggesting that indoor housing may minimize exposure to the virus and may be an additional means of preventing WNV infection in penguins.

Low MHC variation in the endangered Galápagos penguin (Spheniscus mendiculus).

The major histocompatibility complex (MHC) is one of the most polymorphic regions of the genome, likely due to balancing selection acting to maintain alleles over time. Lack of MHC variability has been attributed to factors such as genetic drift in small populations and relaxed selection pressure. The Galápagos penguin (Spheniscus mendiculus), endemic to the Galápagos Islands, is the only penguin that occurs on the equator. It relies upon cold, nutrient-rich upwellings and experiences severe population declines when ocean temperatures rise during El Niño events. These bottlenecks, occurring in an already small population, have likely resulted in reduced genetic diversity in this species. In this study, we used MHC class II exon 2 sequence data from a DRB1-like gene to characterize the amount of genetic variation at the MHC in 30 Galápagos penguins, as well as one Magellanic penguin (S. magellanicus) and two king penguins (Aptenodytes patagonicus), and compared it to that in five other penguin species for which published data exist. We found that the Galápagos penguin had the lowest MHC diversity (as measured by number of polymorphic sites and average divergence among alleles) of the eight penguin species studied. A phylogenetic analysis showed that Galápagos penguin MHC sequences are most closely related to Humboldt penguin (Spheniscus humboldti) sequences, its putative sister species based on other loci. An excess of non-synonymous mutations and a pattern of trans-specific evolution in the neighbor-joining tree suggest that selection is acting on the penguin MHC.