ADAPTATION OF A COMMERCIALLY AVAILABLE WESTERN BLOT KIT FOR THE DETECTION OF ANTIBODY TO ASPERGILLUS IN PENGUINS IN FRANCE AND THE UNITED STATES.

Antemortem serodiagnosis of aspergillosis remains challenging in Sphenisciformes. Protein electrophoresis, serology (antibody, antigen) by ELISA, and gliotoxin detection provide variable diagnostic value. In the present study, a commercially available Western blot (WB) validated for use in humans and dolphins was adapted for use with penguin samples. Using the same method and reagents, samples were analyzed from multiple institutions in the United States and one facility in France. This was inclusive of normal juvenile African penguins (Spheniscus demersus, n = 10) and various species of penguins in the United States with confirmed infection (n = 9) as well as 52 samples from Humboldt penguins (Spheniscus humboldti) in France. Cumulative WB scores (based on reactivity to different antigens) were found to be significantly higher in the group of penguins with confirmed infection (p < 0.0001). Significant differences were also observed between the clinically normal penguins in the two populations, with higher scores in the United States (median score 1.0, 95%CI [0-5], min 0, max 11) compared to France (median score 0,95%CI [0-0], min 0, max 5). The utilization of the WB as a diagnostic tool is inconclusive due to the use of samples from varying institutions, environmental background, age, and stages of infection. However, this tool may provide an overview of antigen reactivity in penguins infected with Aspergillus to help design a more robust serology assay and further understand the humoral immune response during infection.

EFFECT OF SUBSTRATE AND WALKING SURFACES ON CENTRAL METATARSAL FOOT PAD WEIGHT LOADING IN MAGELLANIC PENGUINS (SPHENISCUS MAGELLANICUS) WITH AND WITHOUT PODODERMATITIS: AN EX VIVO STUDY.

Pododermatitis is common in penguins kept under human care. Substrate optimization plays an important role in prevention and treatment; however, there is limited information on biomechanical properties of commonly used substrates on penguin feet. The objectives were to test the ability of different substrates to decrease weight loading on the central metatarsal pad of penguin feet in an ex vivo model using feet with and without bumblefoot harvested from two Magellanic penguin (Spheniscus magellanicus) cadavers. Penguin feet were attached to a digital force gauge mounted onto a stand for compression testing at 2.5 and 5 kg. Forces at the central metatarsal pad were measured in triplicate using small force sensors. Tested substrates included five granular surfaces (sand, wet sand, pea gravel, wet pea gravel, and crushed ice), three compliant surfaces (short-leaf Astroturf, long-leaf Astroturf, and neoprene), and three firm surfaces (tile, rubber drainage mat, and 3M Safety-Walk Wet Area Matting). Data were analyzed using linear mixed models. There were multifaceted effects of applied pressures, substrate surfaces, and pododermatitis on central metatarsal measured pressures. In general, doubling compression forces resulted in higher measured pressures in all firm and compliant surfaces but not in granular surfaces. Firm surfaces were associated with higher recorded plantar pressures at 2.5 kg, but different significance groupings emerged at 5 kg with a high-, medium-, and low-pressure cluster of surfaces. Pododermatitis lesions resulted in significant alterations in statistical significance clustering among substrate surfaces and unique substrate behaviors. The results of this study could help in making recommendations pertaining to foot health for penguin exhibits.

One Health: Urban birds peck up resistant pathogens.

A spatial-genomic analysis reveals that bird species living closer to humans have higher diversity of the pathogen Campylobacter and its antimicrobial resistance genes. This suggests that urbanization could promote pathogen transmission among wild animals and, potentially, humans.

Prevalence and genotypes of Chlamydia psittaci in pet birds of Hong Kong.

Psittacosis, or parrot fever, is a zoonotic disease caused by Chlamydia species associated with birds. One of the causative agents of the disease is Chlamydia psittaci, which is commonly carried by psittacine and other bird species, can be highly pathogenic and virulent to humans. In Hong Kong, a city with high population density, psittacosis is a notifiable disease with over 60% of cases in the last decade resulting in hospitalization. However, the sources of transmission of C. psittaci and its prevalence in pet birds in Hong Kong are currently unknown. To evaluate the risks of psittacosis transmission through pet birds, we tested the presence of C. psittaci and determined its genotypes in samples obtained from 516 captive birds from households, pet shops, and a veterinary hospital in Hong Kong. Results revealed that five samples (0.97%), collected from budgerigars and cockatiels, were C. psittaci-positive, while four (80%) of them were obtained from pet shops. Our phylogenetic analysis revealed that all identified strains belonged to Genotype A and showed high similarity to other sequences of this genotype obtained from various geographical locations and host species, including mammals. Our findings provide evidence for the presence of Chlamydia psittaci and shed light on its sources in captive birds in Hong Kong. They highlight the potential zoonotic risks associated with this pathogen, which can affect both humans and wild birds.

Acanthocephaliasis by Plagiorhynchus sp. (Acanthocephala:Plagiorhynchidae) associated with necrotic enteritis in quetzals (Pharomachrus mocinno) maintained in captivity.

The present report describes a case of acanthocephaliasis associated with necrotic enteritis in quetzals (Pharomachrus mocinno). Three juvenile quetzals exhibited depression, anorexia, ruffled feathers, and sudden death. They were submitted to the laboratory for examination. The birds had hatched and lived in captivity in an aviary in Mexico. Gross lesions observed were hemorrhagic, fibrinoid necrotic enteritis, with acanthocephalans adhered to the intestinal mucosa. The specimens were identified by parasitology, electronic microscopy, and molecular biology as Plagiorhynchus (Prosthorhynchus) sp. Lesions compatible with necrotic enteritis were also confirmed by isolation and genotyping of Clostridium perfringens type E. This is the first report of Plagiorhynchus (Prosthorhynchus) sp. in quetzals in captivity in Mexico. The findings in this manuscript suggest that this parasite could be a predisposing factor for necrotic enteritis as well as a risk component in the breeding programs and survival of this avian species in captivity.

Proximity to humans is associated with antimicrobial-resistant enteric pathogens in wild bird microbiomes.

Humans are radically altering global ecology, and one of the most apparent human-induced effects is urbanization, where high-density human habitats disrupt long-established ecotones. Changes to these transitional areas between organisms, especially enhanced contact among humans and wild animals, provide new opportunities for the spread of zoonotic pathogens. This poses a serious threat to global public health, but little is known about how habitat disruption impacts cross-species pathogen spread. Here, we investigated variation in the zoonotic enteric pathogen Campylobacter jejuni. The ubiquity of C. jejuni in wild bird gut microbiomes makes it an ideal organism for understanding how host behavior and ecology influence pathogen transition and spread. We analyzed 700 C. jejuni isolate genomes from 30 bird species in eight countries using a scalable generalized linear model approach. Comparing multiple behavioral and ecological traits showed that proximity to human habitation promotes lineage diversity and is associated with antimicrobial-resistant (AMR) strains in natural populations. Specifically, wild birds from urban areas harbored up to three times more C. jejuni genotypes and AMR genes. This study provides novel methodology and much-needed quantitative evidence linking urbanization to gene pool spread and zoonoses.

An advance in the understanding the systematics of Echinochasmus Dietz, 1909 and Stephanoprora Odhner, 1902 (Digenea: Echinochasmidae), with the description of a new species of Echinochasmus from the Neotropical region of Mexico.

Echinochasmids are a group of globally distributed digeneans, and the adults are found in the intestines of birds, mammals and reptiles. In the Neotropical region of Mexico, adult specimens were obtained from seven fish-eating bird species in six localities, whereas specimens of Stephanoprora aylacostoma were obtained experimentally in Argentina. Morphologically, the new specimens from the Neotropical region of Mexico were identified as Stephanoprora uruguayense and an undescribed species of Echinochasmus. Sequences for two nuclear (large subunit (28S) and internal transcribed spacer from DNA ribosomal (ITS1-5.8S-ITS2)) molecular markers were generated and analysed together with other sequences downloaded from GenBank. The phylogenies obtained with each molecular marker indicated that Echinochasmus is paraphyletic and agreed with previous phylogenetic studies. The first cluster included the type species (E. coaxatus, which has 24 head-collar spines) plus three congeneric species. The second cluster contained species of Echinochasmus plus Stephanoprora, including the species analysed herein, S. uruguayense, S. aylacostoma (with 22 head-collar spines) and Echinochasmus sp. (with 20 head-collar spines), which formed three independent subclades, allowing us to recognize a lineage that was described morphologically as a new species. Echinochasmus ostrowskiae n. sp. can be distinguished from its congeners by having a head collar with 20 spines in a single row, seven spines on each edge and three angle spines, and a pharynx with an irregular edge and by the body, egg and collar spine sizes. Additionally, new host and locality records for S. uruguayense are presented, expanding its geographical distribution range in the Americas.

Molecular detection, subtyping of Blastocystis sp. in migratory birds from nature reserves in northeastern China.

Migratory birds play an important role in the cross-regional transmission of zoonotic pathogens. Assessing the presence of zoonotic pathogens carried by migratory birds is critical for disease control. However, information about Blastocystis infection in the migratory birds is very limited. Thus, we conducted this study with the aim to explore the occurrence, prevalence and subtyping of Blastocystis in four breeds of migratory birds in northeastern China. From October 2022 to April 2023, a total of 427 fresh fecal samples were obtained from four breeds of migratory birds in five nature reserves in northeastern China, and screened for Blastocystis by PCR amplification. Twenty-one (4.92 %) of the studied samples were confirmed Blastocystis-positive, and two known zoonotic subtypes ST6 and ST7 were founded, with ST7 being the major subtype. Until now, we firstly reported the infection status and subtyping of Blastocystis in the migratory Greater White-Fronted Goose, White Stork, Oriental White Stork and Bean Goose in China. More importantly, these findings present further data on the genetic diversity and transmission routes of Blastocystis and further arouse public health concerns about this organism.

Investigating the virulence-associated genes and antimicrobial resistance of Escherichia fergusonii Isolated from diseased ostrich chicks.

This study investigates the presence of virulence-associated genes and antimicrobial resistance (AMR) in Escherichia fergusonii isolates obtained from ostrich chicks. A total of 287 isolates were recovered from 106 fecal samples from ostrich chicks suffering from diarrhea and subjected to molecular identification and biochemical characterization. E. fergusonii was detected in 10 samples (9.4 %) using two PCR-detection protocols. Notably, the isolates lacked various virulence genes commonly associated with pathogenic E. coli including elt, est, stx, eae, ehly, cdt, iss, iutA, iroN, hlyA, ompT, except for one isolate harboring the astA gene. Antimicrobial susceptibility testing revealed that all isolates were susceptible to ciprofloxacin, while high resistance was observed against amoxicillin clavulanate (AMC), trimethoprim-sulfamethoxazole (SXT), and doxycycline (D). Moreover, eight isolates displayed multidrug resistance (MDR) and four exhibited resistance to 9-11 antimicrobials. The most frequent resistance gene was sul2, which was present in all isolates; the other resistance genes detected consisted of int1 (4/10), int2 (3/10), blaCMY (2/10), and qnrS, blaTEM, blaCMY, blaCTX-M, and flo each were detected only in one E. fergusonii Isolate. Plasmid replicon typing identified the presence of I1 (7/10), N (5/10), and Y (1/10). This study provides valuable insights into the virulence and antimicrobial resistance of E. fergusonii isolates from ostrich chicks, highlighting the complexity of antimicrobial resistance mechanisms exhibited by these bacteria. Further research is essential to understand the transmission dynamics and clinical implications of these findings in veterinary and public health settings.

Detection of pathogenic, heteropathogenic and hybrid Escherichia coli strains in psittacines from zoos and breeders in the state of Ceará, Brazil.

The current study aimed to detect virulence, hetero-pathogenicity, and hybridization genes in Escherichia coli strains, previously isolated from cloacal swabs in commercial breeding psittacines and zoological collections, via multiplex PCR. A total of 68 strains of E. coli, previously isolated from psittacines in zoos and commercial breeding facilities in Ceará, Brazil, were assessed for the presence of the following genes and/or probes: eae, bfpA (EPEC - Enteropathogenic E. coli), CVD432 (EAEC - Enteroaggregative E. coli); LT gene and ST gene (ETEC - Enterotoxigenic E. coli); ipaH (EIEC - Enteroinvasive E. coli); stx1 and stx2 (STEC - Shiga toxin-producing E. coli); iroN, ompT, hlyF, iss, and iutA (APEC - Avian pathogenic E. coli). Of the 68 E. coli strains analyzed, 61 (98.7 %) were positive for the following genes and/or probes: Stx1 (61/98.7 %), ST gene (54/79.4 %), CVD432 (49/72 %), bfpA (44/64.7 %), eae (42/61.8 %), Stx2 (41/60.3 %), ipaH (34/50 %), LT gene (33/48.5 %), iroN (21/30.9 %), hlyF (11/6.2 %), iss (06/8.8 %) and iutA (06/8.8 %). The following diarrheagenic pathotypes were identified: 66 (97 %) from STEC, 49 (72 %) from EAEC, 35 (52 %) from EIEC, 25 (37 %) from ETEC, and one (1.5 %) from EPEC. Regarding hetero-pathogenicity, 50 (74 %) heterogeneous strains were identified. Positivity for APEC was seen in four (6 %) strains, all characterized as pathogenic hybrids. This study describes significant associations of virulence factors in E. coli strains DEC/DEC and DEC/APEC, which were isolated from psittacines and may be potentially harmful to One Health.

First detection of Bagaza virus in Common magpies (Pica pica), Portugal 2023.

Bagaza virus (BAGV) is a mosquito-borne flavivirus of the family Flaviviridae, genus Orthoflavivirus, Ntaya serocomplex. Like other viruses of the Ntaya and Japanese encephalitis serocomplexes, it is maintained in nature in transmission cycles involving viremic wild bird reservoirs and Culex spp. mosquitoes. The susceptibility of red-legged partridge, ring-necked pheasant, Himalayan monal and common wood pigeon is well known. Determining whether other species are susceptible to BAGV infection is fundamental to understanding the dynamics of disease transmission and maintenance. In September 2023, seven Eurasian magpies were found dead in a rural area in the Mértola district (southern Portugal) where a BAGV-positive cachectic red-legged partridge had been found two weeks earlier. BAGV had also been detected in several red-legged partridges in the same area in September 2021. Three of the magpies were tested for Bagaza virus, Usutu virus, West Nile virus, Avian influenza virus and Avian paramyxovirus serotype 1, and were positive for BAGV only. Sequencing data confirmed the specificity of the molecular detection. Our results indicate that BAGV is circulating in southern Portugal and confirm that Eurasian magpie is potential susceptible to BAGV infection. The inclusion of the abundant Eurasian magpie in the list of BAGV hosts raises awareness of the potential role of this species as as an amplifying host.

Detection of Usutu virus in a house martin bug Oeciacus hirundinis (Hemiptera: Cimicidae): implications for virus overwintering in a temperate zone.

The family Cimicidae comprises ectoparasites feeding exclusively on the blood of endothermic animals. Cimicid swallow bugs specifically target swallow birds (Hirundinidae) and their nestlings in infested nests. Bugs of the genus Oeciacus are commonly found in mud nests of swallows and martins, while they rarely visit the homes of humans. Although-unlike other cimicid species-the house martin bug Oeciacus hirundinis has never been reported as a vector of zoonotic pathogens, its possible role in arbovirus circulation in continental Europe is unclear. Samples of O. hirundinis were therefore collected from abandoned house martin (Delichon urbicum) nests in southern Moravia (Czech Republic) during the 2021/2022 winter season and checked for alpha-, flavi- and bunyaviruses by RT-PCR. Of a total of 96 pools consisting of three adult bugs each, one pool tested positive for Usutu virus (USUV)-RNA. Phylogenetic analysis showed that the virus strain was closely related to Italian and some Central European strains and corresponded to USUV lineage 5. The detection of USUV in O. hirundinis during wintertime in the absence of swallows raises the question for a possible role of this avian ectoparasite in virus overwintering in Europe.

Evolutionary drivers of sex-specific parasite prevalence in wild birds.

Males and females often differ in ecology, behaviour and lifestyle, and these differences are expected to lead to sex differences in parasite susceptibility. However, neither the sex differences in parasite prevalence, nor their ecological and evolutionary drivers have been investigated across a broad range of taxa using phylogenetically corrected analyses. Using the most extensive dataset yet that includes 755 prevalence estimates from 151 wild bird species in a meta-analytic framework, here we compare sex differences in blood and gastrointestinal parasites. We show that despite sex differences in parasite infection being frequently reported in the literature, only Haemoproteus infections were more prevalent in females than in males. Notably, only seasonality was strongly associated with the sex-specific parasite prevalence of both Leucocytozoon and Haemoproteus, where birds showed greater female bias in prevalence during breeding periods compared to the non-breeding period. No other ecological or sexual selection variables were associated with sex-specific prevalence of parasite prevalence. We suggest that much of the variation in sex-biased prevalence could be idiosyncratic, and driven by local ecology and behavioural differences of the parasite and the host. Therefore, breeding ecology and sexual selection may only have a modest influence on sex-different parasite prevalence across wild birds.

Evaluation of the stress state based on fecal corticosterone metabolite concentrations in captive penguins in Japan.

Fecal corticosterone metabolite (FCM) concentrations, which can be determined noninvasively, have recently been explored as a stress indicator in birds. In our study, we measured FCM concentrations in penguins under nonmolting or molting conditions, cool or hot season, diseased condition, and incubation period. These measurements were conducted in an aquarium that housed king penguins, gentoo penguins, and African penguins. This study aimed to investigate the validity of fecal matter as a stress indicator. Our findings revealed that FCM concentrations were significantly higher in molting individuals than in nonmolting individuals. Compared with the cool season, FCM concentrations were significantly higher in penguins housed outdoors during the hot season. However, no differences were observed in penguins housed indoors. Diseased individuals and an incubating individual showed notably higher FCM concentrations than healthy individuals. Interestingly, the FCM concentration in king penguin that underwent cataract surgery was extremely high before the surgery. However, 1 month postsurgery, it decreased to a level similar to that of healthy individuals. We observed increased FCM concentrations in penguins considered to be exposed to stressors. Notably, FCM concentration decreased after removing the stress factor. The FCM concentration was found to be consistent with the stress state of penguins, suggesting its usefulness as a stress indicator.

Coccidia of Guinea fowls: Validity of recorded Eimeria spp. (Apicomplexa: Eimeriidae) and first molecular identification of Eimeria grenieri Yvoré & Aycardi, 1967.

Guinea fowls, Numida meleagris (L., 1758), are galliform birds native to sub-Saharan Africa, but introduced in several countries around the world for domestic breeding and/or animal production. This species is considered more resistant to disease by Eimeria spp. than other domestic galliform birds. Here we review the Eimeria spp. known to infect species of Numididae and provide the first molecular identification of an Eimeria sp. from Guinea fowls. There are currently 3 named eimerians from Guinea fowls; Eimeria numidae Pellerdy, 1962; Eimeria grenieri Yvoré and Aycardi, 1967; and Eimeria gorakhpuri Bhatia & Pande, 1967. We reviewed each of these species descriptions and documented their taxonomic shortcomings. From that, we suggest that E. gorakhpuri is a junior synonym of E. numidae. In conclusion, we have morphologically redescribed in detail E. grenieri from N. meleagris from Rio de Janeiro and provided molecular supplementation through sequencing of three non-overlapping loci in cox1 and cox3 genes and fragments of small and large subunit mitochondrial rDNA.

Isospora pichororei n. sp. (Chromista: Apicomplexa: Eimeriidae) from rufous-capped spinetails Synallaxis ruficapilla Vieillot, 1819 (Passeriformes: Furnariidae: Synallaxiinae) in South America.

Spinetails are a suboscine passerines of the genus Synallaxis Vieillot, 1818 which have great interest for ornithology, given the wide diversity of 37 species that are distributed throughout the Neotropical region. Despite this wide diversity and distribution, Synallaxis spp. have never been recorded as hosts of coccidian parasites. In this context, the current study describes a new species of Isospora Schneider, 1881 from rufous-capped spinetails Synallaxis ruficapilla Vieillot, 1819 captured in the Itatiaia National Park, which is a federal conservation unit in Southeastern Brazil. The oocysts of Isospora pichororei Genovez-Oliveira & Berto n. sp. are subspheroidal to ovoidal, measuring on average 25 by 21 µm. Micropyle is present, but discrete. Oocyst residuum absent, but one or two polar granules are present. Sporocysts are ellipsoidal with slightly pointed posterior end, measuring on average 17 by 10 µm. Stieda and sub-Stieda bodies are present. Sporocyst residuum is clustered among the vermiform sporozoites, which have striations, refractile bodies and nucleus. This morphology was different from the other Isospora spp. recorded in the host family Furnariidae. Molecular identification was targeted by the amplification and sequencing of a locus of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. This sequence had the highest similarity of 99.5% with a sequence deposited for Isospora oliveirai Ortúzar-Ferreira & Berto, 2020, which is a coccidian species that parasitizes suboscine tityrids Schiffornis virescens (Lafresnaye, 1838), also in the Itatiaia National Park. Phylogenetic analysis grouped some species in subclades, including I. pichororei with I. oliveirai; however, it was inconclusive in an expectation of parasite-host coevolution. Finally, I. pichororei is established as new to science, being the first description from Synallaxinae and the third description from Furnariidae. Furthermore, this is the first Isospora sp. from the host family Furnariidae to have a molecular supplementation by sequencing a locus of the cox1 gene of the mitochondrial genome.

Haemosporidian intensity and nestlings' life-history along an urban-to-rural gradient.

Urban areas, i.e. dense housing and reduced green spaces, can significantly impact avian health, through altering land use and increasing biotic and abiotic stress. This study assessed the association of urbanization on haemosporidian infections, vectors, immune response, and body condition in Parus major nestlings, across four classes of urbanization along an urban-to-rural gradient in Vienna, Austria. Contrary to our expectations, vector abundance remained consistent across the gradient, while an increase in leukocyte count is positively associated with total parasite intensity. We found that nestlings in more urbanized areas exhibited higher parasite intensity and altered immune response, as evidenced by variations in the heterophil to lymphocyte ratio and leukocyte counts. Culicidae female vectors were associated with nestlings' total parasites, scaled mass index, and industrial units. Nestlings in highly developed areas had higher infection rates than those in forests, suggesting increased exposure to infections. However, there was no clear relationship between total female vectors and total parasites. The level of urbanization negatively affected nestling body condition, with a decrease in fat deposits from forested to highly urbanized areas. Our findings highlight the complex interplay between urbanization, vector-borne parasite transmission, and host immune response, emphasizing the need for comprehensive urban planning to improve wildlife health and guarantee ecosystem functioning. Understanding how urbanization affects bird immunity and parasite infections is critical for adapting urban landscapes for wildlife health and ecosystem integrity.

First detection and a new avian host of the tick Ixodes ventalloi Gil Collado, 1936, in Slovakia.

This study describes the first detection of Ixodes ventalloi in Slovakia. Two engorged females of I. ventalloi were collected from Dunnocks (Prunella modularis) captured in eastern Slovakia. The identification of females was based on morphological and molecular 16S rRNA gene features. Phylogenetic analysis revealed a classification of the females into distinct genogroups. Moreover, comparative morphological analysis highlighted variations between the two females, particularly in the curvature of the auriculae, the shape of coxa I, and the internal spur. These findings suggest the potential for varied phenotypes of I. ventalloi correlated with their genogroups. Nonetheless, I. ventalloi population establishment within Slovakia necessitates further investigation through flagging or drag sampling.