Avian Influenza Research Articles

An influenza mRNA vaccine protects ferrets from lethal infection with highly pathogenic avian influenza A(H5N1) virus.

The global spread of the highly pathogenic avian influenza (HPAI) A(H5N1) virus poses a serious pandemic threat, necessitating the swift development of effective vaccines. The success of messenger RNA (mRNA) vaccine technology in the COVID-19 pandemic, marked by its rapid development and scalability, demonstrates its potential for addressing other infectious threats, such as HPAI A(H5N1). We therefore evaluated mRNA vaccine candidates targeting panzootic influenza A(H5) clade 2.3.4.4b viruses, which have been shown to infect a range of mammalian species, including most recently being detected in dairy cattle. Ferrets were immunized with mRNA vaccines encoding either hemagglutinin alone or hemagglutinin and neuraminidase, derived from a 2.3.4.4b prototype vaccine virus recommended by the World Health Organization. Kinetics of the immune responses, as well as protection against a lethal challenge dose of A(H5N1) virus, were assessed. Two doses of mRNA vaccination elicited robust neutralizing antibody titers against a 2022 avian isolate and a 2024 human isolate. Further, mRNA vaccination conferred protection from lethal challenge, whereas all unvaccinated ferrets succumbed to infection. It also reduced viral titers in the upper and lower respiratory tracts of infected ferrets. These results underscore the effectiveness of mRNA vaccines against HPAI A(H5N1), showcasing their potential as a vaccine platform for future influenza pandemics.

Revealing novel CD8+ T-cell epitopes from the H5N1 avian influenza virus in HBW/B1 haplotype ducks

The duck CD8+ T-cell response effectively defends against H5N1 highly pathogenic avian influenza virus (HPAIV) infection, but the recognized peptide is rarely identified. Here, we found that the ratio of CD8+ T cells and the expression of IFN-γ and cytotoxicity-associated genes, including granzyme A/K, perforin and IL2, at 7 days post-infection in peripheral blood mononuclear cells (PBMCs) from B1 haplotype ducks significantly increased in the context of defending against H5N1 AIV infection in vivo. Moreover, similar results were observed in cultured and sorted H5N1 AIV-stimulated duck CD8+ T cells in vitro. Next, we selected 109 epitopes as candidate epitopes on the basis of the MHC-I restriction binding peptide prediction website database and further identified twelve CD8+ T-cell epitopes that significantly increased IFN-γ gene expression after stimulating B1 haplotype duck memory PBMCs. In particular, NP338−346, NP473−481, M2−10, PB1540−548 and PA80−88 were highly conserved in H5N1, H5N6, H5N8, H7N9, and H9N2 AIVs. These findings provide directions for the development of universal T-cell epitope vaccines for AIV in ducks.

Recombinant avian metapneumovirus subtype C expressing HA protein of H9N2 avian influenza virus are stable and induce protection

To prevent H9N2 avian influenza virus (AIV) and Avian metapneumonovirus/C (aMPV/C) infections, we constructed recombinant aMPV/C viruses expressing the HA protein of H9N2 AIV. In addition, EGFP was inserted into the intermediate non-coding region of P-M protein in the aMPV/C genome using a reverse genetic system. The conditions for rescuing the recombinant virus were enhanced followed by insertion of the H9N2 AIV HA gene into the same location in the aMPV/C. The constructed recombinant virus raMPV/C-HA expressed the H9N2 AIV HA protein and showed good stability. Immunization of chicks with raMPV/C-HA increased the generation of neutralizing antibodies against aMPV and H9N2 AIV for 21 days. In the late challenge experiment, raMPV/C-HA effectively inhibited the replication of the virus in vivo, decreased the incidence of infection and conferred protection effects.

Avian flu suspected on South Africa’s remote Marion Island

Avian flu suspected on South Africa’s remote Marion Island

Recurring incursions and dissemination of novel Eurasian-origin H5Nx avian influenza viruses in Atlantic Canada

Abstract Wild birds are important hosts of influenza A viruses (IAVs) and play an important role in their ecology. The emergence of the A/goose/Guangdong/1/1996 H5N1 (Gs/GD) lineage marked a shift in IAV ecology, leading to recurrent outbreaks and mortality in wild birds from 2002 onwards. This lineage has evolved and diversified over time, with a recent important derivative being the 2.3.4.4b sub-lineage, which has caused significant mortality events in wild bird populations. An H5N1 clade 2.3.4.4b virus was transmitted into North America from Eurasia in 2021, with the first detection being in Newfoundland and Labrador in Atlantic Canada, and this virus and its reassortants then spread broadly throughout North America and beyond. Following the first 2021 detection, there have been three additional known incursions of Eurasian-origin strains into Atlantic Canada, a second H5N1 strain in 2022 and two H5N5 strains in 2023. In this study we document a fifth incursion in Atlantic Canada that occurred in 2023 by another H5N5 strain. This strain spread throughout Atlantic Canada and into Quebec, infecting numerous species of wild birds and mammals. Genomic analysis revealed mammalian-adaptive mutations in some of the detected viruses (PB2-E627K and PB2-D701N) and mutations in the hemagglutinin (HA) and neuraminidase (NA) genes that are associated with enhanced viral fitness and avian transmission capabilities. Our findings indicate that this virus is continuing to circulate in wildlife, and confirms Atlantic Canada is an important North American entry point for Eurasian IAVs. Continued surveillance and genomic analysis of IAVs detected in the region is crucial to monitor the evolution of these viruses and assess potential risks to wildlife and public health.

Detection of antibodies against H5 subtype highly pathogenic avian influenza viruses in multiple raccoons in Tokachi District, Hokkaido, Japan, from 2022 to 2023.

In recent years, infection cases of H5 subtype highly pathogenic avian influenza viruses (HPAIVs) in wild mammals have increased globally. To obtain recent epidemiological information regarding influenza A virus (IAV) infection in raccoons (Procyon lotor), the prevalence of anti-IAV antibodies in sera was analyzed among raccoons captured in Tokachi District, Hokkaido, Japan, from 2019 to 2023. Screening of serum samples using enzyme-linked immunosorbent assay and agar gel precipitation test detected anti-IAV antibodies in 5 of 114 (4.4%) raccoons. All positive sera were from raccoons captured from 2022 to 2023. The hemagglutination inhibition test revealed that all five serum samples contained anti-H5 subtype HPAIV antibodies, and one also contained anti-H1 subtype antibodies. The neuraminidase inhibition test revealed that all five sera contained anti-N1 subtype antibodies, and one also contained anti-N8 subtype antibodies. In the virus neutralization test, these five sera showed stronger neutralization activity against the H5 subtype clade 2.3.4.4b HPAIV strain recently circulating worldwide compared to the old H5 HPAIV strain isolated in Japan in 2007. Findings suggested that raccoons could be involved in the circulation of H5 HPAIVs in nature.

Development of a Large-Volume Concentration Method to Recover Infectious Avian Influenza Virus from the Aquatic Environment

Since late 2021, outbreaks of highly pathogenic avian influenza virus have caused a record number of mortalities in wild birds, domestic poultry, and mammals in North America. Wetlands are plausible environmental reservoirs of avian influenza virus; however, the transmission and persistence of the virus in the aquatic environment are poorly understood. To explore environmental contamination with the avian influenza virus, a large-volume concentration method for detecting infectious avian influenza virus in waterbodies was developed. A variety of filtering, elution, and concentration methods were explored, in addition to testing filtering speeds using artificially amended 20 L water matrices (deionized water with sterile dust, autoclaved wetland water, and wetland water). The optimal protocol was dead-end ultrafiltration coupled with salt solution elution and centrifugation concentration. Using this method, infectious virus was recovered at 1 × 10−1 50% egg infectious dose per milliliter (EID50/mL), whereas viral RNA was detected inconsistently down to 1 × 100 EID50/mL. This method will aid in furthering our understanding of the avian influenza virus in the environment and may be applicable to the environmental detection of other enveloped viruses.

Marked neurotropism and potential adaptation of H5N1 clade 2.3.4.4.b virus in naturally infected domestic cats

ABSTRACT In April 2024, ten cats died in a rural South Dakota (SD) residence, showing respiratory and neurological symptoms. Necropsy and laboratory testing of two cats confirmed H5N1 clade 2.3.4.4b infection. The viral genome sequences are closely related to recent SD cattle H5N1 sequences. Cat H5N1 genomes had unique mutations, including T143A in haemagglutinin, known to affect infectivity and immune evasion, and two novel mutations in PA protein (F314L, L342Q) that may affect polymerase activity and virulence, suggesting potential virus adaptation. Dead cats showed systemic infection with lesions and viral antigens in multiple organs. Higher viral RNA and antigen in the brain indicated pronounced neurotropism. Lectin-histochemistry revealed widespread co-expression of sialic acid α-2,6 and α-2,3 receptors, suggesting cats could serve as mixing vessels for reassortment of avian and mammalian influenza viruses. No differences in clade 2.2 or 2.3.4.4b H5 pseudoviruses binding to cat lung/brain tissues indicated the neurotropism is unlikely mediated by receptor binding affinity.

H5 subtype avian influenza virus induces Golgi apparatus stress response via TFE3 pathway to promote virus replication.

During infection, avian influenza virus (AIV) triggers endoplasmic reticulum (ER) stress, a well-established phenomenon in previous research. The Golgi apparatus, situated downstream of the ER and crucial for protein trafficking, may be impacted by AIV infection. However, it remains unclear whether this induces Golgi apparatus stress (GAS) and its implications for AIV replication. We investigated the morphological changes in the Golgi apparatus and identified GAS response pathways following infection with the H5 subtype AIV strain A/Mallard/Huadong/S/2005. The results showed that AIV infection induced significant swelling and fragmentation of the Golgi apparatus in A549 cells, indicating the presence of GAS. Among the analyzed GAS response pathways, TFE3 was significantly activated during AIV infection, while HSP47 was activated early in the infection process, and CREB3-ARF4 remained inactive. The blockade of the TFE3 pathway effectively inhibited AIV replication in A549 cells and attenuated AIV virulence in mice. Additionally, activation of the TFE3 pathway promoted endosome acidification and upregulated transcription levels of glycosylation enzymes, facilitating AIV replication. These findings highlight the crucial role of the TFE3 pathway in mediating GAS response during AIV infection, shedding light on its significance in viral replication.

Reassortment of newly emergent clade 2.3.4.4b A(H5N1) highly pathogenic avian influenza A viruses in Bangladesh

ABSTRACT Avian influenza active surveillance was conducted in Bangladesh from January 2022 to November 2023 in live-poultry markets (LPMs) and Tanguar Haor wetlands. The predominant viruses circulating in LPMs were low pathogenic avian influenza (LPAI) A(H9N2) and clade 2.3.2.1a highly pathogenic avian influenza (HPAI) A(H5N1) viruses. Non-H9N2 LPAIs were found at Tanguar Haor and at a lower prevalence in LPMs. Starting from June 2023, we detected novel genotypes of clade 2.3.4.4b A(H5N1) viruses from ducks in LPMs. The HA, NA, and M genes of these viruses are related to those of 2020 European clade 2.3.4.4b H5N1 viruses such as A/Eurasian Wigeon/Netherlands/1/2020 (Netherlands/1). However, analyses of the other five gene segments’ sequences identified three distinct genotypes (BD-G2, BD-G3, and BD-G4). BD-G2 viruses were closely related to the clade 2.3.4.4b H5N1 viruses that have been detected in Japan and nearby regions since November 2022. BD-G3 viruses were reassortants, with gene segments from other Eurasian LPAI viruses. BD-G4 viruses were similar to BD-G2 viruses, but their NS gene was accrued from contemporary Bangladeshi clade 2.3.2.1a A(H5N1) viruses. The ability of any of the clade 2.3.4.4b viruses to displace the long-entrenched 2.3.2.1a A(H5N1) viruses in Bangladesh is unknown.

An epizootic of highly pathogenic avian influenza virus H7N3 in a Mexican ecological reserve.

In this case study, we describe an outbreak of highly pathogenic avian influenza (HPAI) virus subtype H7N3 in an ecological reserve in Chiapas, Mexico, affecting captive and wild birds. The virus was detected mainly in plain chachalacas displaying respiratory and gastrointestinal clinical signs and death within 24 hours. Mortality in white-fronted parrots and a clay-colored thrush was also recorded. We describe control strategies implemented to prevent virus dissemination and active surveillance within the risk area. Phylogenetic analysis revealed that the HPAI H7N3 virus detected in affected birds shared a close genetic relationship with Mexican H7N3 isolates from 2012.

Natural infection of common cranes (Grus grus) with highly pathogenic avian influenza H5N1 in Serbia

IntroductionThe late autumn epizootic of the highly pathogenic avian influenza virus (HPAIV) subtype H5N1 in Serbia in 2023 caused massive mortality in the migratory population of common cranes (Grus Grus). This is the first time HPAIV has been identified in the common crane in Serbia, leading to mass mortality of this bird species.MethodsTo understand the pathological impact of HPAIV in cranes, we evaluated the pathological changes in the tissues of common cranes. Additionally, we report genomic characterization of HPAI/H5N1. In total, 14 juvenile common crane carcasses were examined.ResultsInfected birds primarily exhibited neurologic signs, including ataxia and incoordination. Grossly, necrotizing pancreatitis was the most common finding, while microscopic lesions included necrosis, inflammation and hemorrhages in the lungs, spleen, brain, liver and kidneys. Based on RT-PCR, all birds were infected with the HPAI H5N1 virus, as viral RNA was detected in all 14 selected tissues. Genetic analysis revealed that our H5N1 isolate could be grouped with highly pathogenic avian influenza clade 2.3.4.4b, subgroup DA, and is very closely related to the H5N1 strains isolated from the common crane and turkey from Croatia, the common crane from Italy and the Ural owl from Slovakia.DiscussionOur findings showed that common cranes are highly susceptible to natural infection with the HPAI H5N1 virus of clade 2.3.4.4b and may serve as bio-sentinels for the presence of the HPAI virus in wildlife.

Molecular characterisation of novel reassortants of the G57 genotype of low-pathogenic avian influenza H9N2 virus isolated from poultry farms in Malaysia.

In late 2017, Malaysia reported repeated outbreaks of low-pathogenic avian influenza virus (LPAI) H9N2 infections in commercial poultry flocks. Two H9N2 viruses, A/chicken/Malaysia/Negeri Sembilan/UPM994/2018 and A/chicken/Malaysia/Johore/UPM2033/2019, which were isolated from breeder and layer flocks in Peninsular Malaysia, were characterised in this study. Phylogenetic analysis revealed that both viruses were multiple-genotype reassortant strains with genes originating from Y280-like (HA gene), F/98-like (NS, NP and PA), G1-like (M and PB2), and Korean-like (PB1) lineages, indicating that they belong to a novel genotype that is divergent from the G57 lineage of Chinese origin. Both isolates were predicted to have a dibasic cleavage site (333-PSRSSRGLF-341) in the HA gene cleavage locations. Thus, the novel Malaysian H9N2 strain is a Y280-like virus resembling H9N2 isolates from Indonesia, Taiwan, Japan, and Cambodia. This virus is of the G57 lineage but has a novel genotype of the PB1 gene originating from a Korean-lineage H9N2 virus, which has not been detected before in the region.

Confirmation of highly pathogenic avian influenza H5N1 in skuas, Antarctica 2024

From December 2023 to March 2024, a surveillance program aiming to detect Highly Pathogenic Avian Influenza (HPAI) H5N1 was conducted on Antarctica territories, specifically at Fildes Peninsula (King George Island, Maritime Antarctic), and James Ross Island. At Fildes Peninsula, samples from marine birds and mammals were collected from four accessible sampling locations with significant animal colonies: Ardley Island, hosting a large concentration of Gentoo penguins (Pygoscelis papua); Ardley Cove, where small groups of likely non-breeding Chinstrap penguins (Pygoscelis antarcticus) were present; seal haul-out sites of Southern elephant (Mirounga leonina) and Weddell (Leptonycotes wedellii); and, a nesting site of Southern giant petrels (Macronectes giganteus). Additionally, six samples were collected from five dead skuas near the Lachman lakes on James Ross Island (63.7989S, 57.8105W) on March 3, 2024. Despite collecting a total of 943 samples from Fildes Peninsula, all results tested negative for HPAI, and no animals displayed clinical signs or behaviors consistent with HPAI infection. However, all skua samples from James Ross Island tested positive for HPAI H5N1 clade 2.3.4.4 by specific real-time RT-PCR reactions, confirming the first recorded HPAI-related mortality event in Antarctica (south of 60°S), specifically in skuas. Further research is necessary to genetically characterize the virus and better understand the role of skuas in viral dissemination in Antarctica.

Analysis of the Monophyletic Lineage of Avian Influenza H5N1 Which Circulated in Venezuelan Birds During the 2022–2023 Outbreak

Avian influenza subtype H5N1 has caused outbreaks worldwide since 1996, with the emergence of the Guandong lineage in China. The current clade 2.3.4.4b has evolved from this lineage, with increased virulence and mass mortality events in birds and mammals. The objective of this study was the analysis of 17 viral genomes of H5N1 avian influenza isolated in Venezuela during the 2022–2023 outbreak. The eight viral genomic segments were amplified using universal primers and sequenced via next-generation sequencing. The sequences were analyzed to confirm the H5 hemagglutinin clade, identify possible genetic reassortments, and perform a phylogenetic and docking analysis of the viral isolates. The viruses found in Venezuela belonged, as expected, to clade 2.3.4.4b and formed a monophyletic clade with North American influenza viruses, with no evidence of further reassortment. The introduction of the virus in South America is associated with bird migration through the Atlantic (Venezuela), Atlantic/Mississippi (Choco, Colombia), and Pacific migratory flyways, with the emergence of several viral lineages. Several mutations were found in all segments of the genome, although none of the key mutations was involved in mammalian adaptation. Moreover, in silico structural analysis suggests, as expected, that the viral hemagglutinin maintained a predilection for avian α2,3-linked sialic acid. The unprecedented pathogenic outbreak of avian influenza disease in South America was associated with the circulation of three different lineages, which maintain a lower affinity for the mammalian receptor.

Avian influenza: A new threat to wild bird conservation?

<p>Avian influenza, also known as bird flu, significantly threatens wild bird populations and global biodiversity. As wild birds are natural reservoirs for various strains of the influenza virus, they have a crucial role in the epidemiology of the disease, which has profound implications for both wildlife conservation and public health. The emergence and dispersion of highly pathogenic avian influenza strains, particularly H5N1, have resulted in large-scale mortality events in wild bird populations, disrupting ecosystems and threatening endangered species. The conservation of wild birds in the context of avian flu involves several critical actions, including surveillance, rapid response to outbreaks, habitat management, and minimizing human-wildlife interactions that facilitate virus transmission. Studying avian influenza’s impact on wild bird populations is crucial due to its dual importance in wildlife conservation and public health. Wild birds, as natural reservoirs of the virus, play a central role in its spread, with highly pathogenic strains like H5N1 causing devastating mortality events that disrupt ecosystems and endanger species. Effective management, including monitoring, rapid outbreak response, and habitat protection, is essential to mitigate these effects. Collaboration among experts is vital to protect biodiversity, sustain ecological balance, and reduce risks to human health, ensuring the long-term survival of wild bird populations.<strong></strong></p>

Avian influenza: A new threat to wild bird conservation?

<p>Avian influenza, also known as bird flu, significantly threatens wild bird populations and global biodiversity. As wild birds are natural reservoirs for various strains of the influenza virus, they have a crucial role in the epidemiology of the disease, which has profound implications for both wildlife conservation and public health. The emergence and dispersion of highly pathogenic avian influenza strains, particularly H5N1, have resulted in large-scale mortality events in wild bird populations, disrupting ecosystems and threatening endangered species. The conservation of wild birds in the context of avian flu involves several critical actions, including surveillance, rapid response to outbreaks, habitat management, and minimizing human-wildlife interactions that facilitate virus transmission. Studying avian influenza’s impact on wild bird populations is crucial due to its dual importance in wildlife conservation and public health. Wild birds, as natural reservoirs of the virus, play a central role in its spread, with highly pathogenic strains like H5N1 causing devastating mortality events that disrupt ecosystems and endanger species. Effective management, including monitoring, rapid outbreak response, and habitat protection, is essential to mitigate these effects. Collaboration among experts is vital to protect biodiversity, sustain ecological balance, and reduce risks to human health, ensuring the long-term survival of wild bird populations.<strong></strong></p>

Highly pathogenic avian influenza: considerations for healthcare settings.

In this manuscript, we discuss a systematic approach that healthcare facilities can adopt to prepare to identify, confirm, and safely manage highly pathogenic avian influenza in the healthcare setting.

Genetic diversity of highly pathogenic avian influenza H5N6 and H5N8 viruses in poultry markets in Guangdong, China, 2020-2022.

H5 highly pathogenic avian influenza (HPAI) viruses of the A/Goose/Guangdong/1/96 (Gs/Gd) lineage continue to evolve and cause outbreaks in domestic poultry and wild birds, with sporadic spillover infections in mammals. The global spread of clade 2.3.4.4b viruses via migratory birds since 2020 has facilitated the introduction of novel reassortants to China, where avian influenza of various subtypes have been epizootic or enzootic among domestic birds. To determine the impact of clade 2.3.4.4b re-introduction on local HPAI dynamics, we analyzed the genetic diversity of H5N6 and H5N8 detected from monthly poultry market surveillance in Guangdong, China, between 2020 and 2022. Our findings reveal that H5N6 viruses clustered in clades 2.3.4.4b and 2.3.4.4h, while H5N8 viruses were exclusively clustered in clade 2.3.4.4b. After 2020, the re-introduced clade 2.3.4.4b viruses replaced the clade 2.3.4.4h viruses detected in 2020. The N6 genes were divided into two clusters, distinguished by an 11 amino acid deletion in the stalk region, while the N8 genes clustered with clade 2.3.4.4 H5N8 viruses circulating among wild birds. Genomic analysis identified 10 transient genotypes. H5N6, which was more prevalently detected, was also clustered into more genotypes than H5N8. Specifically, H5N6 isolates contained genes derived from HPAI H5Nx viruses and low pathogenic avian influenza in China, while the H5N8 isolates contained genes derived from HPAI A(H5N8) 2.3.4.4b and A(H5N1) 2.3.2.1c. No positive selection on amino acid residues associated with mammalian adaptation was found. Our results suggest expanded genetic diversity of H5Nx viruses in China since 2021 with increasing challenges for pandemic preparedness.IMPORTANCESince 2016/2017, clade 2.3.4.4b H5Nx viruses have spread via migratory birds to all continents except Oceania. Here, we evaluated the impact of the re-introduction of clade of 2.3.4.4b on highly pathogenic avian influenza (HPAI) virus genetic diversity in China. Twenty-two H5N6 and H5N8 HPAI isolated from monthly surveillance in two poultry markets in Guangdong between 2020 and 2022 were characterized. Our findings showed that clade 2.3.4.4h, detected in 2020, was replaced by clade 2.3.4.4b in 2021-2022. H5N6 (n = 18) were clustered into more genotypes than H5N8 (n = 4), suggesting that H5N6 may possess better replication fitness in poultry. Conversely, the H5N8 genotypes are largely derived from the clade 2.3.4.4b wild bird isolates. As clade 2.3.4.4b continues to spread via migratory birds, it is anticipated that the genetic diversity of H5N6 viruses circulating in China may continue to expand in the coming years. Continuous efforts in surveillance, genetic analysis, and risk assessment are therefore crucial for pandemic preparedness.

Moving to Venice and settling there: a synthesis on the biology of Sandwich Tern (<i>Thalasseus sandvicensis</i>) in the largest Mediterranean wetland during the 1995-2023 years

In 1995, the first colony of Thalasseus sandvicensis settled in a saltmarsh islet in the Lagoon of Venice, NE Italy. The number of nesting pairs rose from 202 (1995) pairs to 3503 (2023), with the arrival of immigrants peaking in 2014 (+887 pairs) and 2023 (+1884). From 1995 to 2023, the number of breeding pairs showed a moderate increase (p<0.01), with a yearly rate of +5.7%. Two kinds of colony sites were used: i) salt marsh islets, where birds nested mostly on windrows. These sites were usually in well-secluded area, with low levels of human disturbance; ii) man-made sites such as dredge islands, with nests placed on the bare ground. These man-made sites, in one case located at about 200 m from the town of Venice, were used only since 2014 and now support a large fraction of the breeding population. Overall, 73 colonies were found (361±423 pairs, range: 5-2762; median: 203). Each year there were one to six colonies; 21 colony sites were used at least once. On average, a site was used for 3.5±5.4 years (range 1-25), but the first site was used 25 years out of 29. The turnover rate was overall 41.4%. Over the study period, the biggest cause of clutch loss was flooding during extreme high tides, which are becoming more and more frequent and responsible for more than 90% of losses until 2022. In 2023, an H5N1 avian influenza outbreak annihilated the productivity of the whole nesting season. Clutch size was 1.7±0.5 (n=1338) without differences between saltmarsh and dredge islands: 1.64±0.5 vs 1.81±0.45 eggs per clutch. At the colonies, the association between T. sandvicensis and Larus ridibundus was strong and moderate with Sterna hirundo; instead, a negative and moderate correlation was found between T. sandvicensis and Sternula albifrons. The persistence of the largest Italian population appears threatened by several factors, among which the multiple effects of climate change are the most dangerous.