Four atypical S. Infantis variants differ in the infection dynamics in broilers.Non-motile S. Infantis variants regain motility following host passage.

The H9N2 subtype of avian influenza A virus has been endemic in poultry populations across Asia and the Middle East, posing ongoing economic and public health concerns. Since their initial detection in Pakistan in the 1990s, H9N2 viruses have caused repeated outbreaks in commercial poultry, leading to substantial economic losses and raising concerns about zoonotic transmission. To characterize the recent genetic evolution and zoonotic potential of circulating strains, five H9N2 isolates were obtained from poultry in Pakistan between January and March 2023. Phylogenetic analysis of the haemagglutinin gene revealed that all five isolates belong to the G5.3.2, formerly known as B2, sub-lineage of the G1 Eurasian lineage, in accordance with the revised global classification system for H9 viruses. Comparative genomic analysis confirmed that all eight gene segments were closely related to previously reported Pakistani strains, with no evidence of recent reassortment, suggesting localized persistence and ongoing genetic drift. Notably, several mammalian-adaptive mutations were identified in internal gene segments of the isolates, suggesting a potential risk of cross-species transmission. In addition, all five isolates exhibited dual receptor-binding characteristics, recognizing both α2,3-linked (avian-type) and α2,6-linked (human-type) sialic acid glycans, with a stronger affinity for α2,6-linked glycans. These findings underscore the need for continuous surveillance and risk assessment of H9N2 viruses circulating in Pakistan.

First serological survey on rotavirus A (RVA) in domestic and wild pigeons.RVA widely distributed (∼80%) in domestic and feral pigeons but not in wild common wood pigeons.Almost all adult domestic pigeons (98.1%) possess anti-RVA antibodies.Antibody titres in vaccinated juvenile pigeons are similar to reconvalescents and adults.

The immunosuppressive effects of the infectious bursal disease virus (IBDV), together with its ongoing genome evolution, still represent a major challenge for poultry farmers. This study characterized a novel reassortant IBDV strain, named UK2020, which was isolated in broiler farms from the United Kingdom in 2020 and has now spread across Europe. The phylogenetic analysis revealed that UK2020 belongs to the genotype A3B1, with segment A derived from a very virulent strain (vvIBDV) and segment B from a classical attenuated virus. The pathogenicity studies conducted in both specific-pathogen-free and commercial broiler chickens, with and without maternally-derived antibodies (MDAs), demonstrated that UK2020 caused subclinical infections. These infections resulted in splenomegaly, moderate inflammation of the bursa with histopathological changes, such as lymphoid depletion, inflammatory cell infiltration and plical oedema, and a significant humoral response, despite the absence of clinical signs. The efficacy of a commercial IBD immune complex (Icx) vaccine was also evaluated. Vaccinated birds showed significantly higher antibody titres and reduced lymphoid organ damage compared to mock-vaccinated controls. These results highlight the need for continuous field surveillance to monitor the circulation and impact of reassortant IBDV strains under the current control programmes. The complete sequencing of segments A and B is essential for accurate detection and classification of such strains. The use of Icx vaccines may contribute to mitigating bursal and splenic lesions associated with A3B1 infections like UK2020, especially in flocks with variable MDA levels.

ABSTRACTThe study evaluated the efficacy of dietary benzoic acid (BA) and oregano essential oil (OEO) as alternatives to a commercial coccidiostatic drug in broiler chickens challenged with Eimeria spp. A total of 252 one-day-old broiler chicks (Cobb-500™) were randomly assigned to six treatments, each with six replicates (n = 42 per treatment): (i) negative control (NC); (ii) positive control, coccidia-challenged and untreated (PC); (iii) supplemented with salinomycin at 60 mg/kg of feed and challenged (Sal); (iv) BA at 500 mg/kg of feed and challenged (BA); (v) OEO at 500 mg/kg of feed and challenged (OEO); and (vi) BA at 500 mg/kg plus OEO at 500 mg/kg of feed and challenged (B&O). The study demonstrated that supplementation with BA and OEO, either individually or in combination, improved intestinal villus morphology, particularly in the jejunum, and mitigated the adverse effects of Eimeria infection. The combined supplementation of BA and OEO reduced infectivity and faecal scores to levels comparable to those observed with salinomycin treatment. Moreover, the combined treatment ameliorated gut structural damage and restored growth performance to near that of the non-infected control group by 21 days post-infection (DPI). The study demonstrated that combined supplementation with benzoic acid (BA) and oregano essential oil (OEO) reduced Eimeria infectivity and improved faecal scores comparable to salinomycin, while enhancing intestinal integrity, and jejunal morphology. These effects, accompanied by increased expression of Zonula occludens-1 and Claudin-1, support the potential of BA and OEO as natural anticoccidial alternatives in broiler chickens.

SummaryIn the present study, the virulence of two Mycoplasma synoviae (MS) field isolates was evaluated in specific pathogen-free (SPF) and modern-day commercial broilers. Both MS isolates belonged to Multilocus Sequence Typing (MLST) clonal complex (STCplx) 56, the most prevalent STCplx isolated from the respiratory tract of Dutch commercial poultry in the last two decades. Additionally in this study a well-defined histologic air sac lesion (ASL) score was combined with an in situ hybridization (ISH) test to further study the cellular reaction in the air sacs and tracheal tissue. Birds were challenged intratracheally with MS at 5 days of age. The synergistic effect of a mild infectious bronchitis virus (IBV) H120 vaccine given 5 days prior to MS challenge was also evaluated in the commercial broilers. The clinical impact of the MS strains was comparable to earlier studies with older isolates, as were the synergistic effects of IBV. The present study demonstrated a difference in effect on growth, macroscopic air sac lesions (m ASL) and histologic (h)ASL between the two broiler types. A relevant new finding with economic impact for the poultry industry was that ascites was observed related to both isolates, however the mechanisms underlying MS-associated ascites requires further investigation. The hASL, combined with the MS ISH assay, revealed differences in cellular reaction in the air sacs compared to the trachea, explaining the lower MS detection rate in the air sacs in the chronic phase of MS infection. This also emphasizes the risk of misdiagnosing MS-related airsacculitis in broilers.

Enterococcus cecorum lesions include bacterial chondronecrosis with osteomyelitis (BCO) of the femur and free thoracic vertebrae (FTV). Mechanical stress can be applied using wire-obstacle models and has been suggested as a predisposing factor for BCO. Here, the effect of a wire-obstacle model was assessed following inoculation with E. cecorum.Two groups SPF broilers were orally inoculated at day of hatch with 10[7.6] colony-forming units of E. cecorum in peptone physiological saline (PPS) per bird. In one group, a wire-obstacle was placed between the feed and water. This obstacle had a 66% slope, a height of 30 cm and was covered with iron mesh. Additionally, two control groups, were inoculated with PPS, with an obstacle placed in one group. At 3, 7, 10, 14, 21, 28, 35 and 42 days post-inoculation, twenty birds from the E. cecorum-inoculated groups and eight birds from the PPS-inoculated groups were euthanized and macroscopically examined. Samples from spleen, femur and FTV were subjected to bacteriological examination.No significant differences in the number of birds with typical lesions or positive re-isolations were observed between the E. cecorum-inoculated groups with and without obstacle. Typical lesions were observed in 58 of the 320 inoculated birds, with pericarditis being the most common lesion (46 birds). Femoral BCO was observed in 7 birds. E. cecorum was re-isolated in 45 of the 320 inoculated birds and in none of the controls. A wire-obstacle model does not predispose for BCO in an E. cecorum model using SPF broilers.

AbstractAnatid herpesvirus 1 (AnHV-1) is still a significant limiting factor in the waterfowl industry and there is a clear demand for field-applicable serodiagnostic assays that are robust, sensitive and reproducible. In this report, we designed and conducted a comprehensive comparative evaluation of whole virus or recombinant glycoprotein C (gC), glycoprotein D (gD) alone or a mixture of gC-gD ELISA, to determine which one had the better diagnostic performance. The recombinant proteins were produced, purified, and optimized for use in indirect ELISA, and subsequent validation was carried out using a set of 120 field serum samples. Receiver operating characteristic (ROC) curve analysis demonstrated that the combined gC-gD ELISA exhibited an almost perfect diagnostic performance (AUC = 0.9925), and high sensitivity (93%) and specificity (95%) surpassed those of two antigen-based assays and whole virus assay. Although both gC ELISA (high sensitivity 95% but moderate specificity 80%) and gD ELISA (outstanding sensitivity 99%, but lower overall accuracy: AUC = 0.9150) performed well, the sensitivity of the whole virus ELISA was lower (87%) with marked variation. In particular, intra-assay precision analysis showed a significant decrease in coefficient of variation for the gC-gD ELISA (∼4%) compared with the gD (∼15%), gC (∼20%) and whole virus (∼40%) formats, indicating the highest reproducibility of the gC-gD ELISA. Taken together these results demonstrate that the gC-gD ELISA provides a novel and robust tool for high-throughput AnHV-1 serosurveillance which represents a major breakthrough in terms of economy, when compared to traditional whole virus based ELISAs for large field applications.

Five vaccination-challenge trials were performed with a live attenuated vaccine which includes an infectious bronchitis virus (IBV) Massachusetts strain (H120, GI-1), an IBV Q1 strain (BNF 28/86, GI-16), and a Newcastle disease virus (NDV) cloned strain. The efficacy of the vaccine was tested in maternally-derived antibody-positive (MDA+) commercial layers against challenge with IBV M41, 793B, D388(QX) or Q1 challenge virus. The efficacy of the vaccine was also tested in SPF layers against challenge with IBV M41, D388(QX) or Variant 2 challenge virus. The vaccine was applied at day of hatch, and all challenges were performed at 3 weeks of age. At 5 days post-challenge, the ciliary activity of tracheal explants was examined to determine the level of protection. The level of protection induced by the vaccine in commercial layers against IBV M41, 793B, D388(QX) and Q1 challenge was 91%, 94%, 73% and 84%, respectively. The level of protection in the SPF layers against IBV M41, D388(QX) and Var2 challenge was 89%, 69%, and 96%, respectively. In summary, vaccination with a live attenuated vaccine which includes an IBV Massachusetts strain (H120, GI-1) and an IBV Q1 strain (BNF 28/86, GI-16) provides a substantial level of protection against the homologous and most heterologous challenge viruses, which are representatives of the IBV strains circulating worldwide.

In this study, a novel nucleic acid detection method based on multienzyme isothermal rapid amplification (MIRA) combined with the Pyrococcus furiosus Argonaute (PfAgo) cleavage system (MIRA-PfAgo) was developed for the detection of Tembusu virus (TMUV), an important pathogen threatening the waterfowl industry. The method targets the highly conserved NS5 gene of TMUV. Target nucleic acids are rapidly enriched by MIRA under isothermal conditions at 39°C, followed by PfAgo-mediated cleavage guided by sequence-specific guide DNA (gDNA), enabling visual detection through fluorescence signal output. Systematic optimization identified 0.5 μM gDNA, 1 μM PfAgo, and 5 mM Mn[2+] as the optimal reaction conditions. The assay exhibited excellent specificity for TMUV, with no cross-reactivity observed with common waterfowl pathogens, including H9 subtype avian influenza virus (AIV-H9), duck enteritis virus (DEV), goose parvovirus (GPV), Muscovy duck reovirus (MDRV), Newcastle disease virus (NDV), and duck circovirus (DuCV). The sensitivity was 10[0] copies/μl. In the analysis of 21 clinical samples, the results of the MIRA-PfAgo assay were fully consistent with those obtained by RT-PCR. This method does not require complex thermal cycling equipment and combines high sensitivity, strong specificity, and operational simplicity, providing a reliable and practical technical tool for rapid on-site monitoring of TMUV.RESEARCH HIGHLIGHTSThis is the first study to utilize the MIRA-PfAgo detection assay for the detection of TMUV.The results are observable by the unaided eye.The developed assay potentially offers an alternative means for detection of TMUV, requiring minimal equipment.

Infectious bursal disease (IBD) is a highly contagious and immunosuppressive viral disease of poultry, causing severe economic losses worldwide. Although, intermediate plus live strain vaccines are widely used for IBD control, these vaccines may induce immunosuppression and metabolic stress, predisposing birds to secondary bacterial infections such as avian pathogenic Escherichia coli. Azolla pinnata, a nutrient rich aquatic fern possessing antioxidant and hepatoprotective properties, has emerged as a promising feed supplement in poultry nutrition. The present study was designed to evaluate the ameliorative potential of Azolla pinnata in broiler chickens challenged with Escherichia coli (E. coli) and vaccinated with an infectious bursal disease virus live strain vaccine on biochemical, oxidative parameters and immunological response. Broiler chicks were vaccinated 9[th] day of age and fed diets with or without Azolla pinnata supplementation at the rate of 5% in the feed in dried form followed by experimentally challenged with E. coli. Serum biochemical parameters, including total protein, albumin, globulin, liver enzyme activities were assessed to evaluate hepatic status. Oxidative parameters like lipid peroxidation, glutathione peroxidase and humoral immune response evaluated to access the immune status of birds. Dietary supplementation of Azolla pinnata resulted in improved biochemical, oxidative profiles and humoral immune response in vaccinated and E. coli challenged birds achieved mainly through its rich phytochemical constituents and antioxidant properties which indicating reduced hepatic stress. The findings suggested that Azolla pinnata supplementation may help to maintain homeostasis with live IBD vaccination and E. coli challenged broiler chickens.

Molecular characterization of MDV strains from British small chicken flocks showed great diversity.Sequencing showed a variety of Meq protein isoforms: standard, long, very long, and very very long.Phylogenetic analysis identified MDV strains similar to those from diverse geographical regions, varying in predicted virulence.

Vaccinated broilers were protected against mortality from early H9N2-vNDV-VII.1.1 co-infections.Vaccination of broilers significantly decreased but did not stop shedding of either virus.H9N2 infection prior to vNDV infection in nonvaccinated broilers induced faster mortality.

Infectious bursal disease (IBD), caused by infectious bursal disease virus (IBDV), poses a major threat to the global poultry industry. In this study, we identified and characterized a novel field IBDV strain NN040124, which exhibits both reassortment and recombination features. Genotyping identified this field strain as A3B1a, with segment A derived from a very virulent IBDV (vvIBDV) strain (A3) and segment B from a classical-like attenuated vaccine (attIBDV) strains (vv-A/att-B IBDV)). Crucially, recombination analysis revealed that segment B is a backbone originated from vaccine strain B87, with the N-terminal part replaced by the homologous region from a vvIBDV strain (Harbin-1). This replacement is implicated in the restored virulence of the strain, as demonstrated by a challenge experiment in 4-week-old commercial Three-Yellow chickens, which resulted in 40% mortality, typical clinical signs, and severe bursal lesions. These findings confirm the emergence of a novel dually reassortant and recombinant IBDV in Southern China, identify the N-terminal region of segment B as a potential virulence determinant in a naturally occurring field strain, and highlight the potential risks of live vaccine use. Our findings underscore the importance of continuous genetic and pathogenic surveillance of IBDV to inform effective prevention and control strategies.