Blood parasites of passerines in the Brazilian Pampas and their implications for a potential population supplementation program for the endangered Yellow Cardinal (Gubernatrix cristata).

Espinilho savanna ("seasonal steppe savanna") is a unique vegetation formation of the Pampas biome that is found near the tri-border of Brazil, Uruguay, and Argentina. The Yellow Cardinal (Gubernatrix cristata) is a flagship species of this ecosystem, but it is classified as "critically endangered" in Brazil due to habitat loss and poaching for the illegal trade. Population supplementation through the release of individuals that were captive-bred or apprehended by authorities from the illegal trade has been considered as a conservation strategy for this species; however, the risk of pathogen introduction is a critical concern. We used microscopy and molecular methods to investigate the occurrence of blood parasites in wild passerines (n = 64, including three Yellow Cardinals) at Espinilho State Park, Rio Grande do Sul, Brazil, and in captive Yellow Cardinals (n = 30) at three facilities in Brazil. Haemosporidian parasites were detected in the blood smears of 10.9% of the wild passerines, comprising the morphospecies Haemoproteus erythrogravidus in Rufous-collared Sparrow (Zonotrichia capensis), H. quiscalus in Grayish Baywing (Agelaioides badius), and H. tyranni in Great Kiskadee (Pitangus sulphuratus); these are the southernmost records for these morphospecies and their first record for the Pampas biome. No haemosporidian parasites were detected in the blood smears of the Yellow Cardinals, wild or captive. Microfilariae were detected in the blood smears of 14.1% of the wild passerines, including all wild Yellow Cardinals, and in 43.3% of captive Yellow Cardinals. Trypanosoma sp. was detected in the blood smear of one captive Yellow Cardinal. Nested PCR and gene sequencing of the cyt-b gene of Haemoproteus/Plasmodium was used to test a subset of wild passerines and captive Yellow Cardinals, allowing for the molecular barcoding of H. quiscalus lineage AGEBAD04 and H. tyranni lineage PITSUL01; additionally, DNA identical to that of lineage PITSUL01 was detected in the blood of one captive Yellow Cardinal. This study provides valuable data to support the conservation management of the Yellow Cardinal and other threatened passerines from the Pampas and highlights the need for further studies on the epidemiology and pathology of filarioid worms and trypanosomes in passerines from this biome.

Improved subtyping affords better discrimination of Trichomonas gallinae strains and suggests hybrid lineages.

Trichomonas gallinae is a protozoan pathogen that causes avian trichomonosis typically associated with columbids (canker) and birds of prey (frounce) that predate on them, and has recently emerged as an important cause of passerine disease. An archived panel of DNA from North American (USA) birds used initially to establish the ITS ribotypes was reanalysed using Iron hydrogenase (FeHyd) gene sequences to provide an alphanumeric subtyping scheme with improved resolution for strain discrimination. Thirteen novel subtypes of T. gallinae using FeHyd gene as the subtyping locus are described. Although the phylogenetic topologies derived from each single marker are complementary, they are not entirely congruent. This may reflect the complex genetic histories of the isolates analysed which appear to contain two major lineages and several that are hybrid. This new analysis consolidates much of the phylogenetic signal generated from the ITS ribotype and provides additional resolution for discrimination of T. gallinae strains. The single copy FeHyd gene provides higher resolution genotyping than ITS ribotype alone. It should be used where possible as an additional, single-marker subtyping tool for cultured isolates.

An outbreak of trichomonosis in European greenfinches Chloris chloris and European goldfinches Carduelis carduelis wintering in Northern France.

Avian trichomonosis is a common and widespread disease, traditionally affecting columbids and raptors, and recently emerging among finch populations mainly in Europe. Across Europe, finch trichomonosis is caused by a single clonal strain of Trichomonas gallinae and negatively impacts finch populations. Here, we report an outbreak of finch trichomonosis in the wintering populations of Chloris chloris (European greenfinch) and Carduelis carduelis (European goldfinch) from the Boulonnais, in northern France. The outbreak was detected and monitored by bird ringers during their wintering bird ringing protocols. A total of 105 records from 12 sites were collected during the first quarter of 2017, with 46 and 59 concerning dead and diseased birds, respectively. Fourteen carcasses from two locations were necropsied and screened for multiple pathogens; the only causative agent identified was T. gallinae. Genetic characterization was performed by four markers (small subunit ribosomal RNA, hydrogenosomal iron-hydrogenase, and RNA polymerase II subunit 1 genes, and the internal transcribed spacers (ITS) region) and confirmed the T. gallinae strain to be A1, which affects the finch populations of Europe. This was also confirmed by an ITS-based phylogenetic analysis which further illustrated the diversity of the Trichomonas infecting birds. Preliminary data on the survival and dispersion of infected birds were obtained from ring-returns of diseased individuals. The anthropogenic spread of diseases through bird feeding practices is highlighted and some suggestions to prevent pathogen transmission via backyard supplementary feeders for garden birds are given.

Comparative morphometric evaluation of hepatic hemosiderosis in wild Magellanic penguins (Spheniscus magellanicus) infected with different Plasmodium spp. subgenera / Avaliação morfométrica comparativa da hemossiderose hepática em pinguins-de-Magalhães (Spheniscus magellanicus) de vida livre infectados por diferentes subgêneros de Plasmodium spp

Abstract Avian malaria is one of the most important diseases of captive penguins. We employed morphometric techniques to evaluate hepatic hemosiderosis in rehabilitating wild Magellanic penguins (Spheniscus magellanicus) that were negative (n = 9) or naturally infected by different subgenera of Plasmodium spp. (n = 24), according with: Plasmodium subgenera (Haemamoeba, Huffia, Other lineages, and Unidentified lineages), severity of Plasmodium histopathological lesions, and concurrent diseases, age class (juvenile or adult plumage), sex (male, female or not determined), body score (emaciated, thin, good, excellent, not available), molt, presence or absence of oil contamination upon admission, iron supplementation, and rehabilitation center. The percentage of the area occupied by hemosiderin was called 'Index of Hepatic Hemosiderosis (IHH)'. Plasmodium-positive females presented significantly higher IHH values (17.53 ± 12.95%) than males (7.20 ± 4.25%; p = 0.041). We observed higher levels of congestion (p = 0.0182) and pneumonia (p = 0.0250) severity between Unidentified lineages vs. Huffia. We believe that the hepatic hemosiderosis observed in this study was multifactorial, the result of pathological processes caused by malaria, molting, hemoglobin and myoglobin catabolism during migration, anemia, concomitant diseases, and iron supplementation, all possibly potentiated by decreased liver mass. Further studies are needed to clarify the mechanisms of these hypotheses.

Resumo Malária aviária é uma das mais relevantes doenças em pinguins cativos. Foram aplicadas técnicas morfométricas para avaliar a hemossiderose hepática em pinguins-de-Magalhães (Spheniscus magellanicus ) de vida livre em reabilitação negativos (n = 9) e naturalmente infectados por diferentes subgêneros de Plasmodium spp. (n = 24), quanto a: subgênero de Plasmodium (Haemamoeba , Huffia, Outras Linhagens, e Linhagens não identificadas), severidade das lesões histopatológicas causadas por Plasmodium e doenças concomitantes, faixa etária (plumagem juvenil ou adulta), sexo (macho, fêmea, indeterminado), condição corporal (emaciado, magro, bom, excelente, indisponível), muda, presença/ausência de óleo a admissão, suplementação de ferro, e centro de reabilitação. A porcentagem da área ocupada por hemossiderina foi denominada "Índice de Hemossiderose Hepática (IHH)". Fêmeas Plasmodium -positivas apresentaram IHH significativamente mais elevado que machos, respectivamente, 17,53 ± 12,95% e 7,20 ± 4,25% (p = 0,041). Níveis mais elevados de congestão (p = 0,0182) e pneumonia (p = 0,0250) foram observados entre Linhagens não identificadas vs. Huffia. Possivelmente, a hemossiderose hepática observada nesse estudo seja multifatorial, resultado de processos patológicos causados por malária, muda, catabolismo de hemoglobina e mioglobina durante a migração, anemia, doenças concomitantes e suplementação de ferro, potencialmente intensificados por massa hepática reduzida. Estudos complementares são necessários para esclarecer os mecanismos de tais hipóteses.

Comparative morphometric evaluation of hepatic hemosiderosis in wild Magellanic penguins (Spheniscus magellanicus) infected with different Plasmodium spp. subgenera.

Avian malaria is one of the most important diseases of captive penguins. We employed morphometric techniques to evaluate hepatic hemosiderosis in rehabilitating wild Magellanic penguins (Spheniscus magellanicus) that were negative (n = 9) or naturally infected by different subgenera of Plasmodium spp. (n = 24), according with: Plasmodium subgenera (Haemamoeba, Huffia, Other lineages, and Unidentified lineages), severity of Plasmodium histopathological lesions, and concurrent diseases, age class (juvenile or adult plumage), sex (male, female or not determined), body score (emaciated, thin, good, excellent, not available), molt, presence or absence of oil contamination upon admission, iron supplementation, and rehabilitation center. The percentage of the area occupied by hemosiderin was called 'Index of Hepatic Hemosiderosis (IHH)'. Plasmodium-positive females presented significantly higher IHH values (17.53 ± 12.95%) than males (7.20 ± 4.25%; p = 0.041). We observed higher levels of congestion (p = 0.0182) and pneumonia (p = 0.0250) severity between Unidentified lineages vs. Huffia. We believe that the hepatic hemosiderosis observed in this study was multifactorial, the result of pathological processes caused by malaria, molting, hemoglobin and myoglobin catabolism during migration, anemia, concomitant diseases, and iron supplementation, all possibly potentiated by decreased liver mass. Further studies are needed to clarify the mechanisms of these hypotheses.

In vitro nematicidal effect of Chenopodium ambrosioides and Castela tortuosa n-hexane extracts against Haemonchus contortus (Nematoda) and their anthelmintic effect in gerbils.

The in vitro nematicidal effect of Chenopodium ambrosioides and Castela tortuosa n-hexane extracts (E-Cham and E-Cato, respectively) on Haemonchus contortus infective larvae (L3) and the anthelmintic effect of these extracts against the pre-adult stage of the parasite in gerbils were evaluated using both individual and combined extracts. The in vitro confrontation between larvae and extracts was performed in 24-well micro-titration plates. The results were considered 24 and 72 h post confrontation. The in vivo nematicidal effect was examined using gerbils as a study model. The extracts from the two assessed plants were obtained through maceration using n-hexane as an organic agent. Gerbils artificially infected with H. contortus L3 were treated intraperitoneally with the corresponding extract either individually or in combination. The results showed that the highest individual lethal in vitro effect (96.3%) was obtained with the E-Cham extract at 72 h post confrontation at 40 mg/ml, followed by E-Cato (78.9%) at 20 mg/ml after 72 h. The highest combined effect (98.7%) was obtained after 72 h at 40 mg/ml. The in vivo assay showed that the individual administration of the E-Cato and E-Cham extracts reduced the parasitic burden in gerbils by 27.1% and 45.8%, respectively. Furthermore, the anthelmintic efficacy increased to 57.3% when both extracts were administered in combination. The results of the present study show an important combined nematicidal effect of the two plant extracts assessed against L3 in gerbils.

INVESTIGATION OF THE PRESENCE OF ATOXOPLASMA SPP. IN BLUE-CROWNED LAUGHINGTHRUSH (DRYONASTES COURTOISI) ADULTS AND NEONATES.

Between 1996 and 2013, 71 blue-crowned laughingthrush (Dryonastes courtoisi) chicks, a small passerine bird endemic to China, were born at Mulhouse Zoo in northeast France. None of them survived past 1 yr, and 82% died between 0 and 6 days old of an unidentified cause and despite an attempt to establish an artificial breeding protocol. Atoxoplasma spp., causing a disease known as systemic isosporosis, is a coccidian parasite that can infect several species of birds. Mulhouse's adult birds were suspected to be infected with Atoxoplasma spp. and to transmit this parasite to their offspring. A treatment with toltrazuril (Baycox® 2.5%) was implemented in the four adult birds. Coprologic examinations were performed before, during, and after the treatment to quantify the parasite load in feces. Polymerase chain reaction (PCR) assays were used to test blood samples from the adult and liver, lung, gizzard, and kidney samples from 10 chicks to detect Atoxoplasma spp. Five of the 10 chicks had some tissue samples positive for Atoxoplasma spp. in at least one of the three repeats of the atoxoplasmosis PCR. An average of 181 Isospora spp. oocysts per gram of feces were found in the group of adults before treatment. This number was reduced to zero 1 wk after the beginning of the toltrazuril treatment. The PCR results suggest a transovarian transmission of Atoxoplasma spp., but further investigation is needed for confirmation. The treatment with toltrazuril appears to allow a significant reduction of the parasite excretion.

Treatment of atoxoplasmosis in the Blue-crowned Laughing Thrush (Dryonastes courtoisi).

Passerines are frequently parasitized by coccidia, especially species of the genus Isospora, with extra-intestinal stages that can be highly pathogenic causing serious clinical damage in young birds. Whilst there is still no effective treatment to completely clear isosporoid coccidia with extra-intestinal stages from a host species, our results showed that prolonged treatment with toltrazuril (BAYER AG, Leverkusen, Germany) can decrease the oocysts in faeces and thus reduce the extra-intestinal phase of the infection. The toltrazuril treatment is therefore probably indirectly effective against the systemic form of atoxoplasmosis.

Anthelmintic efficacy of pumpkin seed (Cucurbita pepo Linnaeus, 1753) on ostrich gastrointestinal nematodes in a semiarid region of Paraíba State, Brazil.

The aim of this study was to verify the in vivo effectiveness of pumpkin seed (Curcubita pepo Linnaeus, 1753) in naturally infected ostriches in the Cariri zone, semiarid region of Paraíba State, Brazil. Forty-eight ostriches were used, African Black breed, of 14 to 36 months old, naturally infected by gastrointestinal nematodes. These animals were divided into four groups of 12 ostriches. Group 1 consists of animals treated with 0.5 g/kg live weight (l. w.) of pumpkin seed meal; group 2 received 1 g/kg l. w. of pumpkin seed meal; group 3 was treated with Albendazole 5 %, at the dosage of 1 mL/10 kg l. w.; and Group 4 was the control group and do not received treatment. Groups 1 and 2 received the treatment for three consecutive days, orally, at intervals of 7 days, totaling nine administrations. The Albendazole 5 % was administered one time, at the beginning of the experiment, according to the manufacturer's recommendations. The groups treated with pumpkin seed showed a significant decrease in egg counts per gram of feces (EPG), wherein group 2 (1 g/kg l. w.) was the most effective. The control and drug groups showed no reduction in EPG. The results of the present study demonstrate that the administration of pumpkin seed was effective in controlling gastrointestinal helminths in naturally infected ostriches.

Effects of a neem seed extract (MiteStop®) on mallophages (featherlings) of chicken: in vivo and in vitro studies.

Mallophages of birds (featherlings) are mostly very tiny and can even as adults better be recognized by their movements than by their elongate body shape when using just the naked eye. Since some species (e.g., the "shaft louse" Menopon gallinae, the elongate feather louse Lipeurus caponis, or Columbicola sp.) may pierce the pulp of feathers or the skin by their biting or scratching mandibles and thus lick the excreted blood, they may be extremely dangerous especially to young birds, even if they only feed by nibbling along the feather surface and/or eat epidermal debris. The present paper reports on the successful treatment of different races of fowls being severely infested with both above cited species. This in vivo treatment was done either by a short dipping of the whole fowl into the 1:33 dilution (with tap water) of a neem seed extract (MiteStop®) or by spraying them with the freshly diluted product. It was seen that the dead mallophages dropped down from the feathers as soon as they were dry again. As a precaution, a second treatment was done by some owners 1 week after the first one in order to eliminate all stages, which eventually might have hatched from untouched nits during the time interval between the two treatments. When controlling the treated fowls 4 weeks after the treatment, in no case (treated once or twice), living motile stages were diagnosed indicating the high efficacy of this nontoxic neem seed extract. When treating in vitro cutoff feathers contaminated with L. caponis, it was seen under the stereomicroscope, that the mallophages tried to run away from the 1:33 water-diluted active compound indicating that there is also a repellent effect. Treated L. caponis stopped leg movements within 3 min and died on their feathers within 1-20 min. Then, the last slight trembling movements of their legs and convulsions of their intestine stopped finally.

Influence of petroleum hydrocarbons on the endoparasitic helminths of the common eider, Somateria mollissima, from Newfoundland.

A comparison of commonly occurring metazoan parasites in the digestive tract was made between common eiders, Somateria mollissima, that were contaminated with oil and reference birds confiscated from illegal hunting. There was a greater number of commonly occurring parasites and their abundance in reference than in oiled eiders. Except for an acanthocephalan, Polymorphus botulus, which was embedded in the wall of the intestinal tract, most of the other taxa of parasites, including trematodes, cestodes and nematodes, were probably voided from the birds following ingestion of oil. Reference eiders harboured fewer species and a substantially lower mean abundance of parasites than those studied in Newfoundland and Labrador more than four decades ago; this may be a signal of a changing prey base or an increase in prey availability as winter ice cover continues to decline.

Effects of carotenoids, immune activation and immune suppression on the intensity of chronic coccidiosis in greenfinches.

Allocation trade-offs of carotenoids between their use in the immune system and production of integumentary colouration have been suggested as a proximate mechanism maintaining honesty of signal traits. We tested how dietary carotenoid supplementation, immune activation and immune suppression affect intensity of coccidian infection in captive greenfinches Carduelis chloris, a passerine with carotenoid-based plumage. Immune activation with phytohaemagglutinin (PHA) decreased body mass among birds not supplemented with lutein, while among the carotenoid-fed birds, PHA had no effect on mass dynamics. Immune suppression with dexamethasone (DEX) induced loss of body mass and reduced the swelling response to PHA. DEX and PHA increased the concentration of circulating heterophils. Lutein supplementation increased plasma carotenoid levels but had no effect on the swelling response induced by PHA. PHA and DEX treatments did not affect plasma carotenoids. Immune stimulation by PHA suppressed the infection, but only among carotenoid-supplemented birds. Priming of the immune system can thus aid in suppressing chronic infection but only when sufficient amount of carotenoids is available. Our experiment shows the importance of carotenoids in immune response, but also the complicated nature of this impact, which could be the reason for inconsistent results in studies investigating the immunomodulatory effects of carotenoids. The findings about involvement of carotenoids in modulation of an immune response against coccidiosis suggest that carotenoid-based ornaments may honestly signal individuals' ability to manage chronic infections.

Effect of Lysimachia ramosa (Primulaceae) on helminth parasites: motility, mortality and scanning electron microscopic observations on surface topography.

The alcoholic extract of Lysimachia ramosa Wall (Primulaceae) was tested in vitro against helminth parasites, Fasciolopsis buski and Ascaris suum, from porcine hosts and Raillietina echinobothrida from domestic fowl. The live adult parasites, collected from a freshly autopsied host, were exposed to different concentrations (5-50mg) of the test plant extract in physiological phosphate-buffered saline (PBS) having 0.1% dimethyl sulphoxide (DMSO) at 37+/-1 degrees C. The treated parasites revealed complete inactivation and flaccid paralysis that was followed by death at varying periods of time. A dose-dependent loss of motility and mortality was observed in all the treated parasites. Scanning electron microscopic observations revealed conspicuous deformity of the surface architecture in all the parasites exposed to the test plant extract. The general tegument in F. buski showed shrinkage and loss of scale-like spines; proglottides all along the strobilar length in R. echinobothrida appeared shrunken and deformed and the cuticular surface of A. suum appeared disorganised, having lost transverse striations. The botanicals of the test plant seem to be effective against all the three types of helminth parasites.

Interspecific differences in Aethia spp. auklet odorants and evidence for chemical defense against ectoparasites.

The true auklets (Genus Aethia) are small planktivorous seabirds of the Bering Sea and North Pacific. Two species, the crested and whiskered auklets produce volatile citrus-like odorants. We here show that the whiskered auklet odorant is composed predominantly of two odd-numbered aldehydes (heptanal and nonanal) with no detectable unsaturated aldehydes. By comparison the crested auklet odorant is dominated by even-numbered aldehydes, both saturated and monounsaturated, ranging in size from 6 to 12 carbons. This is evidence of species-specific acquisition or biosynthetic pathways. We clarify the chemistry of the crested auklet odorant. We cite evidence that the C-12:1 aldehyde in crested auklets is actually two isomers, (Z)-4-dodecenal and (Z)-6-dodecenal. We also report on experimental evidence that aldehyde constituents kill and repel ectoparasites. Efficacy of the aldehydes may increase when they are combined in a mixture. The repellency of the mixture increases with chemical concentration. This suggests that individuals with higher chemical production are likely to repel ectoparasites more effectively.