ABSTRACT Automated behavioral monitoring may offer opportunities to assess health in broiler chickens. This study aimed to (i) evaluate behavioral changes following challenge with Eimeria spp. and infectious bronchitis virus (IBV) combined with avian pathogenic Escherichia coli (APEC), and (ii) characterize patterns across age and time of the day. Across four rounds, 132 Ross 308 broilers were assigned to three groups (control, Eimeria, and IBV+APEC), with four replicate pens of 11 birds each. Challenges were applied on day 14 (Eimeria), day 21 (IBV), and day 24 (APEC). A total of 12 behaviors were quantified from 10-min video recordings collected every 2 h between 07:00 and 21:00 from days 14 to 41, divided into four periods: Period 1 (d14–20), Period 2 (d21–27), Period 3 (d28–34), and Period 4 (d35–41). Behavior was primarily driven by age. In control birds, locomotion and exploratory/foraging decreased progressively, while sleeping increased; inactivity remained stable. Time-of-day analysis revealed diurnal patterns with higher afternoon/evening activity and lower sleeping in these periods compared with morning. Relative to controls, Eimeria-infected birds showed variable drinking and sustained increased locomotion and exploratory behavior (periods 1–4), representing a non-classical response to intestinal challenge. IBV+APEC-challenged birds exhibited modest changes: decreased inactivity (period 2) and increased panting (period 4), with largely unchanged activity patterns. Disease challenge altered diurnal patterns, with disease-specific effects on circadian-dependent behaviors. These findings highlight the importance of age-specific baselines and the potential of time-of-day-dependent behavioral analyses and circadian markers for detecting disease-related deviations and enabling early disease detection through automated behavioral monitoring.

ABSTRACT The immune responses elicited in chickens by wild-bird-origin serotype 1 avian paramyxoviruses (APMV-1) Mallard/US(OH)/04-411/2004 (virus A) and Northern pintail/US(OH)/87-486/1987 (virus B) were characterized and compared with those elicited by the Newcastle disease virus (NDV) LaSota strain. Specific-pathogen-free chickens were inoculated via the ocular route with each virus. Viral RNA was detected in lachrymal fluid 7 days after inoculation in all groups. By day 14 viral RNA persisted in only a few chickens inoculated with the wild-bird-origin viruses. Both wild-bird-origin APMVs induced systemic antibody responses similar to those elicited by the LaSota strain, with virus B eliciting earlier seroconversion. By day 21 after inoculation, all vaccinated groups exhibited similar antibody levels and antibody avidity. IgA in lachrymal fluid achieved the highest levels on day 14 post-inoculation in all groups, with LaSota eliciting the highest mucosal IgA response. Flow cytometry of lymphocytes isolated from the Harderian gland (HG) showed B- (CD3-Bu-1+) and T- (CD3+CD8+ and CD3+CD4+) cell expansion that was evident 8 days after inoculation in all virus-inoculated chicken groups. Consistently, tracheal histomorphometry showed enhanced lymphocytic infiltration 8 days after inoculation in all inoculated groups compared to uninoculated controls. These results suggest that wild-bird-origin APMV-1s can induce robust systemic and mucosal immune responses upon inoculation into chickens. RESEARCH HIGHLIGHTS Wild-bird APMV-1s elicited antibody responses at levels similar to LaSota strain. APMV-1s induced CD8+-, CD4+-, and Bu-1+-cell expansion in the Harderian gland.

ABSTRACT Infectious bursal disease virus (IBDV) is a highly contagious, economically relevant immunosuppressive pathogen of chickens. Despite belonging to a single serotype, virulent IBDVs display a remarkable heterogeneity in genetic and functional features. Traditionally, strains are categorized into classical, variant and very virulent viruses, but many atypical IBDVs have been recently identified. A primary example is the emergence of North-Western European (NW EU) reassortants (genotype A3B1), first identified in 2017, which rapidly became predominant in Western Europe. A notable exception was Italy, where circulating IBDVs belonged to a separate A3B1 clade related to Russian and Middle Eastern strains, but the present study describes the entry of NW EU reassortants into this country as well. During a 2-year-long survey (March 2023-January 2025), 256 bursal pools were collected from broiler farms and subjected to molecular analyses: 97 samples tested positive for field strains (38%), 43 for vaccine strains (17%), and 116 were negative (45%). Notably, 46 field strains were NW EU reassortants (47%), whereas 50 belonged to the Italian Russian-Middle Eastern (ITA/RUS/ME) clade (52%). The remaining strain showed VP2 features consistent with the ITA/RUS/ME clade but clustered with classical attenuated IBDVs at VP1 level, either due to reassortment or coinfection between field and vaccine viruses. Following their first detection, NW EU reassortants reached all major poultry producing regions and seemingly became prevalent over the ITA/RUS/ME clade. Such a swift epidemiological shift emphasizes the threat posed by atypical IBDVs and the subsequent need for enhanced surveillance and control measures. RESEARCH HIGHLIGHTS North-Western European reassortants (A3B1) were found in Italy for the first time. Local A3B1 clade, related to Russian and Middle Eastern strains, also persisted. Infectious pressure was seemingly stable despite this epidemiological shift.

ABSTRACT Although poultry meat production supports the livelihood and provides food security for billions of people worldwide, it is facing substantial challenges. The emergence of broiler breast myopathies (white striping, woody breast, spaghetti meat) at large scale is one of the most significant economic and welfare challenges that menace poultry production sustainability and for which there is currently no effective prevention, due to its unknown aetiologies. Here, by inviting and gathering several experts with diverse, but complementary disciplines, the objective of the present review is to highlight the current progress and knowledge on these myopathies. Five sections are presented, describing in detail the history and geographic occurrence of these breast myopathies, their macroscopic morphologies and microscopic characteristics, their putative aetiologies and causes as well as their underlying molecular mechanisms, and potential strategies and solutions. The is review summarizes both descriptive and functional mechanistic studies, highlights the complexity of these myopathies and the kinship between broiler genome, nutrition, and management, and outlines some of the promising molecular signatures. It aims to offer new fundamental frameworks for future investigations.

ABSTRACT Gastrointestinal nematode infections, particularly Ascaridia galli (A. galli), pose challenges to the poultry industry especially in free-range chickens. These nematodes employ sophisticated immunomodulation strategies to evade host immune defences, allowing them to establish chronic infections. Excretory-secretory (ES) products, including extracellular vesicles (EVs), play a pivotal role in host–parasite communication. Thus, this study aimed to investigate the immunomodulatory properties of nematode-derived ES/EVs, and evaluate the impact of ivermectin (IVM) on these interactions in vitro. Adult A. galli worms were collected from the jejunum of infected chickens and cultured in vitro with or without IVM treatment. ES products were collected, and EVs were enriched using size-exclusion chromatography. T-cell proliferation and macrophage activation assays were performed to assess the immunomodulatory effects of ES/EVs. A. galli-derived ES products suppressed T-cell activation (CD25+CD57+ cells) and reduced blast transformation in both CD4+ and CD8+ T-cells (P < 0.05). Furthermore, ES promoted TLR4-induced macrophage NO production, changed macrophage-activated surface phenotype, and inhibited macrophage phagocytosis in high concentration but promoted it in low concentration (P < 0.05). Additionally, A. galli-derived EVs were detected in culture supernatants, and this EV secretion was decreased with the IVM treatment (P < 0.05). Fourier-transform infrared spectroscopy confirmed IVM treatment altered EV cargo composition. Finally, the EVs influenced cytokine profiles of activated macrophages, decreasing pro-inflammatory cytokine gene expression (IL-1β and IL-8, P < 0.05) which may mirror immune evasion in vivo. In summary, this study highlights immunomodulatory mechanisms of A. galli ES/EVs and provides fundamental information for developing diagnostic and therapeutic strategies targeting nematode ES products including EVs. RESEARCH HIGHLIGHTS Excretory-secretory (ES) products, including extracellular vesicles (EVs), demonstrated immunomodulatory properties. A. galli ES suppressed T-cell activation, proliferation, and macrophage phagocytosis. A. galli ES, including EVs, may help in evading the host immune system.

ABSTRACT Over the last decade, the emergence of aviadenovirus infections, namely inclusion body hepatitis (IBH) and gizzard erosion and ulceration syndrome (GEUS) has occurred in Morocco with significant losses to the poultry industry. In this work, 255 suspected flock cases of IBH and GEUS were assessed among various poultry production types in the country from 2012–2024. Macroscopic pathological changes in affected chickens included an enlarged, discoloured liver or a distended gizzard with haemorrhagic content and erosion/ulceration on the koilin layer. Microscopic findings revealed severe necrotic hepatitis, consistent with IBH, and/or necrotizing ventriculitis, which is characteristic of GEUS, with intranuclear viral inclusion bodies (INIB) in the hepatocytes or epithelial cells of the gizzard, which are attributable to fowl adenovirus (FAdV). The real-time PCR results revealed that 71% (182/255) of the suspected FAdV cases tested positive. The prevalence varied by poultry production type, with broiler farms being the most affected at 79% (144/194), followed by layer farms at 15% (28/42), breeder farms at 5% (9/13), and turkey farms at 1% (1/6). Sequencing of the isolates identified FAdV serotype 11 (species D) and serotype 8b (species E) in 22 and six cases of IBH, respectively. Additionally, FAdV serotype 1 (species A) and serotypes 8a and 8b (species E) were detected in nine and two cases of GEUS, respectively. These findings underscore the significant impact of fowl adenoviruses on the Moroccan poultry industry and highlight the need for continuous surveillance and targeted control interventions. RESEARCH HIGHLIGHTS Increase in FAdV infections (IBH and GEUS) in Morocco over past decade. FAdV-positive cases were mainly found in broiler farms (79%). Identification of FAdV-11 and 8b serotypes for IBH, and FAdV-1, 8a and 8b for GEUS.

ABSTRACT Captive breeding of endangered species, such as the African Houbara bustard (Chlamydotis undulata) is an essential part of conservation programmes. However, several factors may negatively impact the health and reproductive performance of captive animals, including genital infections. Between 2010 and 2023, a total of 439 cases of salpingo-peritonitis were diagnosed in a flock of African Houbara breeders belonging to a Moroccan conservation project, relying exclusively on artificial insemination and artificial incubation of eggs (annual prevalence from 0.07% to 1.64%). To improve the overall knowledge of reproductive pathology in this avian species, a representative sample of 14 cases registered in 2022 were characterized, combining gross pathology, histopathology, microbiology and molecular biology. Overall, 4/14 bustards died spontaneously and exhibited septicaemic changes, while 10/14 euthanized birds presented signs consistent with chronic salpingo-peritonitis and oviductal impaction. The latter was further confirmed at histopathology and, in eight bustards, was associated with mucosal changes resembling cystic endometrial hyperplasia, a condition commonly reported in mammals and potentially predisposing to secondary bacterial infections. Escherichia coli was isolated from the reproductive tract of 12/14 birds. Molecular characterization of nine selected isolates revealed the presence of several virulence factors associated with avian pathogenic E. coli. The variety of virulence, as well as antimicrobial sensitivity profiles identified, combined with the pathological presentation, suggested the involvement of multiple bacterial strains. Overall, in addition to the presence of specific virulence genes, management and host-related factors should also be considered as important determinants of the pathogenic potential of E. coli in captive African Houbaras. RESEARCH HIGHLIGHTS First comprehensive description of salpingo-peritonitis in African Houbaras. First report of cystic oviductal hyperplasia in African Houbaras. Escherichia coli isolated from the reproductive tract of the majority of birds. Several virulence and antimicrobial sensitivity profiles identified.

ABSTRACT The highly pathogenic avian influenza virus (HPAIV) A(H5N1) has caused the most extensive and severe epizootic event affecting both poultry and wild birds globally. This study investigated the impact of HPAIV on the breeding population of the Black-headed Gull Chroicocephalus ridibundus, the most abundant gull species in Poland. During the 2023 outbreak, this species was reported as the most frequently infected in the country. A higher-than-natural adult mortality rate (greater than 1.5% of the breeding individuals) was observed in 114 surveyed colonies across all regions of Poland. Laboratory tests confirmed the presence of HPAIV in all 17 colonies sampled, with average adult mortality estimated at 26.1%, and ranging from 1.7% to 77.8%. The estimated mortality rate across all surveyed colonies was 22.2%. Extrapolations across the entire Polish breeding population (at least 115,000 pairs according to the national census) indicated that approximately 51,000 adult Black-headed Gulls might have perished due to HPAIV in 2023. The number of adults found dead was positively correlated with colony size (r = 0.733, P < 0.001). The deaths were associated with a single HPAIV genotype (BB) across all confirmed cases. Understanding the spread and severity of HPAIV in colonially breeding waterbirds, such as gulls, is essential for assessing the full extent of the threats this virus poses to wild bird populations.

ABSTRACT In the last decade, the emergence of variant strains of avian orthoreovirus (ARV) has caused an enormous economic impact on the poultry industry across China and other countries. This study aimed to evaluate the molecular evolution of the ARV lineages detected in Chinese commercial broiler farms. Firstly, ARV isolation and identification of commercial broiler arthritis cases from different provinces in China from 2016 to 2021 were conducted. A total of 51 pure ARV isolates were obtained. Sequencing results showed that there were five genotypes of the strains isolated in this study, of which genotype 1 ARV predominated, accounting for 56.9% (29/51). The whole gene sequences of 19 ARV representative isolates were successfully obtained. The genetic evolution analysis of 10 genome segments of 19 ARV isolates showed that the σC-encoding gene had evolved into six different lineages, while the other genome segments only differentiated into two to four different lineages. The results of recombination analysis showed that recombination events were present in the L3, M1 and S1 genome segments. Analysis of the variation of the key factor σC protein showed that the nucleotide and amino acid homologies of the σC were low among the different genotypes. Three-dimensional structural visualization analysis showed that all the structural changes of σC protein were concentrated in the spherical domain at the C-terminal, which is associated with host receptor binding.

ABSTRACT Mycoplasma gallisepticum (Mg) and Mycoplasma synoviae (Ms) are regarded as the most important avian mycoplasma species for today’s chicken and turkey farming industry from clinical and economical perspectives. Control strategies for Mg and Ms have become more efficient due to investments in mycoplasma research over the last 70 years. These investments have contributed to the further implementation of serological and molecular testing, the development of vaccines, and the improvement of antimicrobial treatment strategies. However, the increasing spotlight on welfare, the pressure on prudent use of antimicrobials, and the expected global increase in poultry production, are going to have an impact on the future control of avian mycoplasmas in commercial poultry. In this paper a group of avian mycoplasma experts discuss the future challenges in mycoplasma control considering the background of these expected changes and the relevance for future avian mycoplasma research.