Outbreaks of Erysipelothrix rhusiopathiae infection are well documented in poultry but only sporadically reported in gamebirds. We report two erysipelas outbreaks causing 7% and 9% mortality in separate flocks of ring-necked pheasants (Phasianus colchicus) in New York State in 2024. Gross examination of three pheasants from each flock (n = 6) revealed splenomegaly and renomegaly in birds from one flock and Syngamus trachea in two birds from the other flock. Bacteriological culture of samples of lungs, livers, and spleens yielded E. rhusiopathiae from birds in both flocks based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) of bacterial isolates. Serotyping of the E. rhusiopathiae isolates revealed serovar 5 from one flock and serovar 6 from the other flock. Histological examination revealed vasculitis and bacilli within the cytoplasm of endothelial cells and macrophages, as well as intravascular bacterial thrombi and emboli in multiple tissues. Other histologic findings included splenic necrosis, myocarditis, interstitial pneumonia, and hepatitis. Immunohistochemistry of tissue samples with a rabbit polyclonal antiserum raised against serotypes 1a, 1b, and 2 identified E. rhusiopathiae serotype 6 but not serotype 5. This report highlights multimodal diagnostic approaches to confirming erysipelas outbreaks in gamebirds using histopathology and immunohistochemistry combined with bacteriological culture and isolate identification by MALDI-TOF MS and serotyping.

Laridae birds, such as gulls, are known reservoirs of H13 and H16 low pathogenicity avian influenza virus subtypes. However, during the recent outbreaks linked to the reemergence of high pathogenicity avian influenza virus (HPAIV) H5N1 clade 2.3.4.4b of the Goose/Guangdong lineage, European populations of Laridae birds suffered significant losses. HPAI cases were reported not only along the coastlines but also in inland areas, particularly in France and Central Europe. During a diagnostic investigation of a group of Laridae birds, part of a HPAIV outbreak reported in the South of France in 2023, larval stages of Ornithodoros maritimus, a nidicolous soft tick parasitizing seabirds, were recovered from a slender-billed gull (Chroicocephalus genei). Affected birds exhibited gross and histopathological lesions consistent with systemic HPAIV infection. Immunohistochemistry revealed marked neurotropism, oculotropism, and multicentric epitheliotropism. Viral isolation and sequencing analysis confirmed the presence of HPAIV H5N1 clade 2.3.4.4b in both the gull and ectoparasites, showing from 99.64% to 100% nucleotide identity across five of eight RNA segments. While additional research is needed to properly assess the vector competence of O. maritimus for HPAIV, ticks may represent an interesting noninvasive surveillance tool for these viruses. This is the first time a HPAIV has been successfully isolated from tick larvae. These findings represent a first step toward understanding the potential role played by ticks in the spread of avian influenza viruses within marine bird colonies and among other ecosystems, considering the occurrence of specific behavioral traits, such as kleptoparasitim and the position of gulls at the interface between wild and domestic species.

The endangered crested ibis (Nipponia nippon), once reduced to just seven individuals in the wild worldwide in 1981, has rebounded to more than 10,000 through intensive conservation. Yet the species remains highly vulnerable to acute environmental disturbance, with stress-induced sudden death (SISD) posing persistent challenges for captive management and reintroduction programs. We investigated three SISD cases, integrating behavioral context with necropsy and histopathology in all cases, and computed tomography/magnetic resonance imaging neuroimaging and metagenomic pathogen screening in a subset. Major organs showed no diagnostic lesions, with only mild, nonspecific digestive tract changes. Neuroimaging identified localized subdural hemorrhage without diffuse parenchymal damage. Metagenomic screening detected no high-pathogenicity avian viruses, fungi, or parasites and only trace environmental bacteria. Using published datasets, we also compared baseline corticosterone (CORT) from feathers and blood across five avian species; crested ibis occupied the lower end of baseline CORT distributions, consistent with reduced endocrine buffering capacity. These observations suggest that peracute death may occur in association with acute disturbance even in the absence of overt infectious or major degenerative lesions, with panic flight and collision trauma likely contributing to the terminal events. Framing SISD as a failure of physiological tolerance thresholds highlights actionable management: minimizing startle stimuli, reducing predator cues and human disturbance during sensitive periods, carefully staging transfers to new enclosures, managing group dynamics to limit social transmission of stress, designing for safe flight, and early-warning monitoring. Taken as case-based evidence, this work may help refine captive husbandry and reintroduction protocols aimed at improving survival and long-term population resilience.

Mongolia lies at the intersection of major migratory flyways, the East Asian-Australasian Flyway (EAAF) and the Central Asian Flyway (CAF), serving as a pivotal region for avian influenza virus (AIV) transmission among wild bird populations across Eurasia. Continued surveillance in this region remains important for understanding AIV evolution and dissemination across migratory flyways. In this study, active surveillance was conducted in wild bird habitats in Mongolia during the summer of 2025 to investigate the genetic diversity of circulating AIVs. A total of 1656 fresh fecal samples were collected, resulting in the isolation of 13 viruses, including H3N8, H4N6, H10N8, and H12N5 subtypes. Whole-genome sequencing and phylogenetic analyses revealed high genetic diversity and extensive reassortment, with gene segments closely related to viruses circulating in East Asia, Southeast Asia, Central Asia, South Asia, Europe, and North Africa. Some isolates contained polymerase or nucleoprotein genes clustering within European lineages, whereas most retained Asian genomic backgrounds. These findings support the role of wild birds in Mongolia in facilitating the evolution and spread of AIVs across Eurasia, highlighting the need for continued genomic surveillance in this region.

Despite the widespread use of live attenuated and recombinant vaccines against infectious laryngotracheitis, the disease continues to pose substantial economic challenges to poultry production. This study aims to assess the protective efficacy of DNA plasmid pool formulation (PLgBDI) expressing infectious laryngotracheitis virus glycoproteins gB, gD, and gI designed to preferentially drive cell-mediated immune responses. The plasmid formulation was administered to specific-pathogen-free chickens via eye drop at 3 and 17 days of age (doa) by itself, and to chickens previously immunized in ovo with a commercial rHVT-ND-ILT recombinant vaccine that expresses gD and gI ILTV antigens. In contrast to in ovo vaccination with the recombinant rHVT-ND-ILT vaccine, eye drop immunizations with the PLgBDI formulation did not induce seroconversion. Postchallenge, the PLgBDI, rHVT-ND-ILT, and rHVT-ND-ILT+PLgBDI vaccinated groups of chickens exhibited survival rates of 91%, 80%, and 78%, compared to 65% for the NVx/Ch group and 100% for the group vaccinated with the live attenuated chicken embryo origin (CEO) vaccine via eye drop at 9 doa. Although not statistically different to the NVx/Ch group, the PLgBDI and rHVT-ND-ILT groups presented a trend toward reduction in clinical signs that was not observed for the rHVT-ND-ILT+PLgBDI group. Neither the PLgBDI, rHVT-ND-ILT, nor rHVT-ND-ILT+PLgBDI vaccinated groups showed a decrease in challenge virus replication. Overall, this study indicated that eye drop immunization with DNA plasmid pool formulation induced partial protection comparable to that induced by rHVT-ND-ILT, while it deterred protection when administered to previously in ovo rHVT-ND-ILT vaccinated chickens.

Avian leukosis (AL), a tumorigenic disease induced by avian leukosis virus (ALV) that leads to tumors, immunosuppression, and reduced productivity, has caused enormous economic losses in the poultry industry since its emergence. While the disease has been virtually eradicated in Western commercial flocks, it still poses a persistent threat in Chinese indigenous chicken breeds, causing significant economic losses. Currently, detection and eradication are the best ways to eliminate avian leukosis. This study compared the performance of quantitative reverse transcription PCR (RT-qPCR), conventional RT-PCR, and ELISA for ALV detection. Results showed that RT-qPCR had the highest sensitivity, with a detection limit of 10[-2] TCID50/ 100 µl, significantly outperforming RT-PCR (10[0] TCID50/100 µl) and ELISA (10[3] TCID50/100 µl). Evaluation of 84 clinical samples revealed that RT-qPCR achieved 100% accuracy, aligning perfectly with the reference positive rate (50%, 42/84); the positive rate recorded by conventional RT-PCR was only 42.86% (36/84), with a positive concordance rate of 85.7%, whereas ELISA identified only 33.33% (28/84) of samples as positive, yielding a concordance rate of only 66.7%. Our findings underscore that the high false-negative rate of ELISA-based screening poses substantial economic risks and may undermine ALV eradication efforts. Given its superior analytical sensitivity, RT-qPCR is poised to become the cornerstone of future ALV surveillance. However, it should be implemented as a complementary strategy-rather than a total replacement for traditional methods-to establish a more robust and integrated diagnostic framework.

Environmental testing of poultry farms after an outbreak of infectious viral disease such as infectious bronchitis or avian influenza is essential for verifying viral clearance and supporting the safe resumption of operations. To find optimal sampling locations inside and outside of chicken farms, a laboratory study simulating a respiratory virus-infected poultry house with wire layer cages was conducted using a commercial live attenuated infectious bronchitis virus vaccine as a surrogate virus administered to 21-day-old layer chickens. Oropharyngeal swab samples of chickens, air, and four locations within the poultry house, including manure conveyor belt, floor surface, environmental dust, and on-farm boots, were evaluated on the day before and 1, 3, 5, and 8 days after spray vaccination. Tools that were used outside the poultry house, such as off-farm shoes, farm work clothes, protective face masks, cotton gloves, egg transport vehicles, and egg transport pallets, were also evaluated. Due to inherent differences in environmental conditions and facility structures across farms, environmental sampling was performed following a standardized protocol designed in this study to ensure consistency across all collection sites. Virus loads at each sampling site were determined by quantitative real-time reverse transcription PCR and expressed as 50% egg infectious dose (EID50) equivalents. In addition, a subset of samples was further subjected to virus re-isolation through egg inoculation. The highest viral RNA loads inside and outside of the farm were observed from the manure conveyer belts and off-farm shoes, respectively. Samples taken from floor surfaces and on-farm boots also consistently showed high amounts of viral RNA. While infectious viruses were successfully re-isolated from several internal samples, no infectious virus was detected in samples collected outside the farm, suggesting that no viral spillover occurred under the experimental conditions. Our findings identified high-risk virus accumulation sites within poultry farms and highlight the utility of optimized, standardized environmental sampling protocols in enhancing detection sensitivity and supporting effective risk-based surveillance.

Pigeon paramyxovirus-1 (PPMV-1) is a genetically and antigenically distinct variant of avian paramyxovirus-1 that is endemic in Columbiformes (pigeons and doves) globally. This endemicity poses an ongoing risk of Newcastle disease (ND) incursion in poultry because PPMV-1 typically possesses a virulent fusion gene cleavage site. Indeed, numerous ND outbreaks have been attributed to the spillover of PPMV-1 from Columbiformes into poultry. However, the mechanisms of this cross-species transmission and virulence in different species are poorly defined. Here, we examine the infection dynamics, pathogenicity, and transmission potential of a contemporary genotype XXI.1.1 PPMV-1 isolated from a racing pigeon (Columba livia) in Great Britain (January 2021), focusing on the potential transmission pathways into poultry. Importantly, conventionally reared pigeons were used to better reflect field conditions and understand the implications of prior exposure on infection. Following infection, directly inoculated pigeons exhibited low-level viral shedding in the absence of overt clinical disease. Critically, onward transmission to poultry occurred, with subsequent viral shedding from chickens in the absence of clinical disease or seroconversion. These findings suggest that subclinical PPMV-1 infection of poultry may occur following exposure to infected pigeons, raising questions about the role and necessity of canonic virulence factors in mediating infection across different avian hosts.

Surveillance programs for low pathogenic avian influenza virus frequently rely on serological tests to determine whether poultry flocks have been infected with the virus. Poultry sera that are positive by type A influenza screening tests are frequently tested at reference laboratories by an hemagglutination inhibition (HI) assay as a subtype-specific confirmatory test. Because of the potential to become highly pathogenic, H5 and H7 are the most common targets. An alternative to HI assay is the microneutralization (MN) assay, which is widely used for humans, wildlife, and mammalian research animals because of its sensitivity. Subtype-specific assays are also used to monitor response to vaccines. Here the observable endpoint of the HI assay was compared with the MN assay using two methods to read the MN assay results, hemagglutination assay (MN-HA), and observation of cytopathic effects (MN-CPEs). Rates of positivity for individual samples by HI assay, MN-HA, and MN-CPEs were 66.7% (32/48), 89.6% (43/48), and 87.5% (42/ 48), respectively. Agreement was low with a Cohen's kappa statistic (κ) of 0.377 for HI vs. MN-HA and 0.333 for HI vs. MN-CPEs, where discrepancies were almost exclusively where samples were negative by HI assay and positive by MN-HA or MN-CPEs (κ = 0.897 for MN-HA vs. MN-CPEs). Titer values were also significantly higher with both MN-HA and MN-CPEs than with the HI assay (P < 0.0001 and P = 0.150, respectively). Although not significant, there was a trend for MN-HA to produce higher titers and detect more positive samples than MN-CPEs.

High pathogenicity avian influenza viruses (HPAIVs) continue to pose a major threat to poultry and wildlife through repeated introductions by migratory birds, and active surveillance has, therefore, been implemented in South Korea as an early warning measure. Here, we analyzed virologic and serologic surveillance data collected from captured wild birds between 2019 and 2025 to characterize the circulation dynamics of avian influenza virus (AIV). Clear seasonal patterns were observed, with AIV detection and H5 seroprevalence occurring predominantly during the wintering season. Increased HPAIV detection and H5 seroprevalence in wild birds during the 2020-21 season coincided with increased high pathogenicity avian influenza outbreaks in poultry, and spatial analyses revealed consistent hot spot regions along major migratory flyways. At the species level, mallards (Anas platyrhynchos) and spot-billed ducks (Anas zonorhyncha), which were captured most frequently, as well as pintails (Anas acuta), despite lower capture frequency, exhibited high H5 seroprevalence. Mandarin ducks (Aix galericulata) were repeatedly identified as the earliest HPAIV-positive species each winter and exhibited diverse group-level infection patterns, underscoring a role as sentinel hosts. Overall, these findings demonstrate pronounced seasonality, species-specific differences, and flyway-associated clustering of AIV circulation in wild birds, highlighting the value of targeted active surveillance for early warning of HPAIV incursions into poultry.

Viral subpopulations in Arkansas Delmarva Poultry Industry (ArkDPI)-derived IBV vaccines are rapidly selected in inoculated chickens, resulting in tracheal damage and virus persistence. These subpopulations with distinct spike (S) amino acid sequences were previously eliminated from an ArkDPI-derived vaccine strain during serial passage in chicken embryonic kidney (CEK) cells, and a new viral population with an S protein harboring three amino acid differences relative to the original vaccine major population became predominant. Recombinant S protein representing the CEK-adapted vaccine strain showed severely reduced or no detectable binding to CEK cells. The two amino acid substitutions in the S1 subunit and the single amino acid substitution in the S2 subunit independently reduced binding. Structural models predicted that each of the amino acid substitutions involved an amino acid located at the interface between monomers in the trimer, suggesting that the conformation and/or stability of alternative conformations of the trimer might be affected. Recombinant S protein representing the CEK-adapted virus also exhibited reduced or nondetectable binding to most relevant chicken tissues in vitro. We compared replication after ocular inoculation in 1-day-old chickens between our CEK-adapted ArkDPI virus and a commercial ArkDPI-derived vaccine. Viral replication in individual chickens was monitored by relative levels of viral RNA in tears 3, 5, and 8 days postvaccination (DPV) and in choanal and tracheal swabs 5 and 8 DPV, determined by reverse transcription quantitative polymerase chain reaction. At 3 DPV, tears of chickens inoculated with 10[5] 50% embryo infectious doses (EID50) of CEK-adapted vaccine and 10[4] EID50 commercial ArkDPI vaccine had similar viral loads, while those of chickens given 10[4] EID50 CEK-adapted vaccine were two orders of magnitude lower. Viral loads in tears of both chicken groups given the CEK-adapted vaccine remained at levels similar to 3 DPV through days 5 and 8 DPV, while those in chickens given the commercial vaccine increased three orders of magnitude during the same period. Statistically significant differences among groups were also observed in viral loads in choanal and tracheal swabs at both 5 and 8 DPV, though less pronounced. The sharp increase in viral load in tears of chickens given the commercial vaccine was likely due to efficient replication of vaccine subpopulations selected in chickens, which are lacking in the CEK-adapted vaccine. Selection of vaccine subpopulations in chickens given the commercial vaccine, but not in chickens given the CEK-adapted vaccine, was verified by S1 gene sequencing of the IBV genomes in samples from individual chickens in all vaccinated groups. Protection following ocular challenge at 19 DPV with virulent Ark IBV, as assessed by reductions in viral loads in tears, correlated with vaccine viral loads at 5 and 8 DPV, with the greatest correlation (r = 0.52) with vaccine viral load in choanal swabs at 8 DPV.

Backyard chickens have become more popular in the United States in recent years. Due to the private nature of owning backyard chickens, information on these flocks is difficult to obtain. Given the large scale of the commercial poultry industry in the United States, and particularly in Pennsylvania, understanding disease risks that backyard chickens face is vital for public health and the health of commercial flocks. Respiratory pathogens are of particular concern for backyard chickens because they are common and can spread rapidly among birds and cause severe disease. Mycoplasma synoviae (MS), Mycoplasma gallisepticum (MG), Avibacterium paragallinarum, and infectious bronchitis virus (IBV) are among the most common respiratory pathogens detected in backyard flocks. This retrospective cross-sectional study analyzed 835 backyard chicken necropsy submissions to the Pennsylvania Animal Diagnostic Laboratory System from September 1, 2019, to August 31, 2024, for the presence of these four pathogens. Avibacterium paragallinarum was found to be the most common pathogen detected, with 229 positive submissions (229/589, 39%). MS, MG, and IBV had 148 (148/502, 29%), 71 (71/631, 11%), and 71 (71/467, 15%) PCR-positive submissions, respectively. A total of 381 submissions were tested for all four pathogens of interest, and 245 of these submissions (64%) were positive for one or more pathogens. Seasonality was significant for detection of A. paragallinarum and IBV, with the greatest number of detections occurring in the summer and fall. Detections were predominantly in birds from southeastern Pennsylvania, coinciding with the most poultry-dense region of the state.

Footpad dermatitis (FPD) is a major welfare and economic concern in broiler production and is closely linked to litter conditions and bird performance. Although wet litter is a well-recognized risk factor, the contribution of early litter microbiota to paw quality remains poorly understood. In this study, we evaluated footpad lesion severity in Cobb 500 broilers at harvest (49 days) and examined litter microbiologic and physicochemical parameters throughout the grow-out period to identify factors associated with differences in paw quality. Birds were raised under four treatments: a negative control; a positive control challenged with Salmonella enterica serovar Infantis; a feed-applied Bacillus velezensis probiotic (Calsporint) with Salmonella Infantis challenge; and a litter-applied B. velezensis probiotic (proLitterbiotic) with Salmonella Infantis challenge. Footpad lesion scores differed modestly among treatments, with the positive control and birds receiving proLitterbiotic exhibiting a higher mean lesion severity score at harvest compared with the Calsporin and negative control birds. Litter moisture, water activity, pH, and metal composition varied by treatment at select time points but did not consistently align with lesion severity. Female birds in the positive control and proLitterbiotic treatments had higher mean body weights at 42 days. The 16S rRNA gene sequencing revealed that litter bacterial communities were strongly structured by time and showed treatment-associated differences immediately after placement. Early litter bacterial communities reflected contributions from the broiler house environment, starter feed, and the meconium with distinct separation between treatments receiving litter-applied probiotics and those receiving feed-applied probiotics. Although litter microbial communities converged later in the grow-out, early differences coincided with subsequent variation in paw quality. These findings indicate that poorer paw quality was associated with a combination of early changes in litter microbial communities and litter physicochemical differences, rather than a single dominant factor.

This case report details listeriosis in a chicken from a public botanic garden and the ancillary testing required to confirm Listeria monocytogenes. A 6-mo-old female Barred Plymouth Rock chicken, found deceased within its flock at a public botanic garden, was submitted to The Pennsylvania State University Animal Diagnostic Laboratory for postmortem examination. The clinical history included bloody feces, weight loss, and pallor prior to death. On postmortem examination, the chicken was emaciated with vent fecal staining. Gross lesions suggested bacterial septicemia, including necrotic fibrinous hepatitis, peritonitis, myocarditis, and pancreatitis. The heart was rounded, with pericardial effusion and a focal, dry, white bulging area around the left ventricle. Ulceration of the gizzard mucosa, severe, focal, unilateral cecal dilation, and ulceration with hemorrhage and necrosis were supportive of gastroenteritis and typhlitis. Differential diagnoses included lymphoid neoplasia, chronic coccidial or bacterial enteritis or typhlitis, bacterial septicemia, and mycobacteriosis. On histology, major findings included granulomatous myocarditis and endocarditis, as well as transmural coccidial enteritis and typhlitis. Preliminary results from ancillary testing confirmed severe coccidiosis and bacterial myocarditis. Although matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry identified nonpathogenic Listeria innocua, the severity of the heart lesions and the hemolysis observed on culture suggested a pathogenic species. Whole-genome sequencing confirmed the presence of L. monocytogenes, rather than the L. innocua incorrectly identified by MALDI-TOF. This case highlights the presentation of listeriosis in a chicken, the importance of confirmatory testing in the face of equivocal results, and potential disease risks in public spaces shared with avian species.

Phasianus chaphamaparvovirus 1 is a newly recognized pathogen associated with high mortality in young pheasant chicks with significant losses for pheasant breeders. Here we present an outbreak of necrotizing hepatitis with high mortality in a flock of young pheasant chicks in Bosnia and Herzegovina associated with Phasianus chaphamaparvovirus 1 and coccidiosis coinfection. Severe necrotizing hepatitis with characteristic intranuclear inclusion bodies in affected hepatocytes was observed on microscopic examination. In addition, multifocal areas of intestinal mucosal necrosis with numerous developing stages (meronts) of coccidia were present. Phasianus chaphamaparvovirus 1 was detected with qPCR from the livers of diseased birds. Our report represents the first data on the presence of this new virus in pheasant flocks in the Balkan region and suggests that the virus is widespread across Europe. It further supports the inclusion of Chaphamaparvovirus as a differential diagnosis of pheasant mortality outbreaks both in breeding facilities and in the wild.