RESEARCH HIGHLIGHTS:Four atypical S. Infantis variants differ in the infection dynamics in broilers.Non-motile S. Infantis variants regain motility following host passage.
RESEARCH HIGHLIGHTS: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 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.
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.
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.
In 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 (mASL) 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 require 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 107.6 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.
RESEARCH HIGHLIGHTS: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.
RESEARCH HIGHLIGHTS: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.
Live-attenuated vaccines are widely used for the prevention and control of infectious bursal disease (IBD) in poultry, yet differences in attenuation, replication, and immunogenicity among vaccines remain incompletely characterized. This study comparatively evaluated three live-attenuated IBDV vaccines (Vaccine-A, Vaccine-B and Vaccine-C) using integrated molecular, pathobiological and immunological analyses. Molecular analysis using next-generation sequencing revealed that all vaccines possessed canonical attenuation-associated substitutions (253H, 279N and 284T) in the VP2 hypervariable region, but differed in additional lineage- and virulence-associated residues. Vaccine-A retained several residues characteristic of very virulent IBDV (vvIBDV), including 222A, 242I, 256I, 294I, and 299S, whereas Vaccines-B and -C displayed mixed classical and vvIBDV-associated profiles. Notably, Vaccine-C contained a vvIBDV-derived VP1 polymerase. These molecular differences corresponded to distinct in vivo phenotypes. Vaccine-C showed higher and more persistent vaccine-viral RNA levels in the bursa of Fabricius, with greater lymphoid depletion and lesion severity. In contrast, Vaccine-A exhibited lower residual viral RNA levels and milder pathology. All vaccines induced homologous and cross-neutralizing antibody responses, although response kinetics differed. Vaccine-A elicited earlier cross-neutralizing responses, while Vaccine-C generated higher peak titres at later time points. Cytokine profiling showed stronger pro-inflammatory signals with Vaccine-C and higher early type-I interferon expression with Vaccine-A. Under selective pressure in DT40 cells, Vaccine-A lost vvIBDV-associated residues, Vaccine-B accumulated substitutions including N279D, whereas Vaccine-C exhibited moderate VP2 variability. Overall, these findings suggest that vaccine molecular composition may influence viral replication, tissue pathology and immune responses, with Vaccine-A demonstrating a relatively favourable balance between safety and immunogenicity under the present experimental conditions.
To enable differential detection of virulent and attenuated strains of duck short beak and dwarfism syndrome virus (SBDSV), we developed a fluorescent quantitative PCR (qPCR) assay based on locked nucleic acid (LNA)-TaqMan probes. This assay utilized a pair of universal primers and two strain-specific LNA-TaqMan probes (labelled with FAM and VIC, respectively) targeting the VP1 gene of SBDSV. Validation results showed that the assay specifically detected SBDSV virulent and attenuated strains without cross-reacting with other common waterfowl viruses. It exhibited high sensitivity, with a minimum detection limit of 6.0 × 10⁰ copies/μl, and excellent reproducibility, with intra- and inter-assay coefficients of variation (CVs) all < 3%. Additionally, viral titre estimates from this qPCR assay showed no significant difference from those obtained via the gold-standard 50% tissue culture infectious dose (TCID₅₀) assay (P > 0.05). In conclusion, this LNA-TaqMan qPCR assay provides a specific, sensitive, and reliable tool for the differential diagnosis of SBDSV virulent and attenuated strains in clinical practice, serving as strong technical support for the prevention, control, and eradication of SBDSV.
RESEARCH HIGHLIGHTS:The co-infection rate of infectious tumour disease in chickens is 37% in China.The incidence rate is the highest in Dezhou City.
Highly pathogenic avian influenza (HPAI) H5N8 viruses of clade 2.3.4.4b continue to cause devastating outbreaks worldwide, necessitating effective vaccination strategies. The comparative efficacy of wild-type versus reverse genetics (RG)-based vaccine platforms against emerging H5Nx variants remains poorly characterized, particularly regarding cross-protective immunity mechanisms. We conducted a comprehensive comparative analysis of wild-type and RG-based H5N8 (clade 2.3.4.4b) oil-adjuvanted vaccines in specific-pathogen-free chickens (n = 150, power analysis based on detecting two-fold titre differences with 80% power, α = 0.05). Following single-dose vaccination, we assessed humoral and cellular immune responses, protection against homologous H5N8 (clade 2.3.4.4b), antigenically drifted and heterologous challenges [H5N1 clade 2.3.4.4b and H5N1 clade 2.2.1.2], and viral shedding dynamics using standardized assays. Both vaccines induced robust homologous antibody responses (HI titres: wild-type 7.2 ± 0.15 vs. RG 6.8 ± 0.13 log2, P < 0.05) by 28 days post-vaccination. Challenge studies revealed 100% protection against homologous H5N8 (clade 2.3.4.4b) for both vaccines, while wild-type vaccine demonstrated superior heterologous protection against H5N1 (clade 2.2.1.2): 60% vs. 40% (P = 0.04). Viral load quantification showed >3.0 log10 reduction in respiratory tissues, with strong correlation between HI titres and protection (r = 0.89, P < 0.001). Cellular immunity assessment revealed enhanced Th1-biased responses in wild-type vaccine recipients interferon-gamma/interleukin-4 (IFN-γ/IL-4 ratio: 1.61 vs. 1.49, P = 0.035), potentially explaining superior cross-protection. Wild-type H5N8 vaccines provided enhanced cross-protection compared to RG-based platforms while maintaining equivalent homologous efficacy. HI titres ≥4.5 log2 represent a critical threshold for protection, with optimal protection achieved at ≥6.0 log2. These findings establish evidence-based correlates of protection and inform rational vaccine selection strategies for H5N8 control programs.
Chicken infectious anaemia virus (CIAV), infectious bursal disease virus (IBDV), and Eimeria spp. are major immunosuppressive pathogens in chickens that predispose host to secondary infections, including Clostridium septicum-associated hepatic necrosis. In this case, shotgun metagenomic sequencing was applied to identify C. septicum that could not be isolated by traditional bacterial culture in Samgye chickens. Six 35-day-old Samgye chicken carcasses were submitted for disease diagnosis; histopathological examination, and bacterial and viral isolation/identification were performed. Pooled liver samples were subjected to shotgun metagenomic sequencing to identify microbial composition, virulence factors, and antimicrobial resistance genes. Samgye chickens exhibited dorsal dermatitis, hepatic necrosis, and splenomegaly. Histopathology revealed hepatic necrosis with bacterial colonies and lymphoid depletion. PCR detected CIAV, antigenic variant IBDV, chicken astrovirus, and Eimeria, whereas bacterial culture yielded no growth. Shotgun metagenomic analysis identified C. septicum as the predominant bacterium, and CIAV as the dominant viral pathogen. The α-toxin and the antimicrobial resistance tetA(P) genes were detected in liver samples. This is the first report of concurrent CIAV, antigenic variant IBDV, Eimeria spp., and C. septicum infection from Samgye chickens in South Korea, suggesting that immunosuppressive infections may predispose chickens to C. septicum-associated hepatic necrosis and highlighting the diagnostic utility of shotgun metagenomic sequencing.
RESEARCH HIGHLIGHTS:The prevalence of traumatic skin injuries varies greatly between farms with equal stocking density and the same breeder hybrid.The incidence rate is highest in the first half of the production period.Very few fresh injuries were observed at the end of lay.Flocks with toe-clipped males had numerically fewer injuries.Egg production was suppressed in 55% of hens with unhealed injuries.
Infectious coryza (IC) is a chicken respiratory disease caused by Avibacterium paragallinarum (AP). Recently, multiple naïve-healthy layer (NHL) flocks tested positive for AP by both qPCR and bacterial isolation without clinical signs. Therefore, acquired AP isolates from these NHL flocks were dubbed non-pathogenic AP (npAP). The aim of this study was to investigate the pathogenicity of npAP isolates (Phase I) and their ability to confer protection against IC (Phase II). Seven groups (G1-G7) of 26-week-old laying hens were challenged via intra-sinus injection with seven npAP isolates obtained from five sites out of four states. Positive control group was challenged with Midwestern pathogenic AP (pAP) serovar C1 isolate, while the negative control received saline. No significant differences (P > 0.05) in mean clinical, infraorbital sinus gross and histopathological lesion scores were observed between G1-G7 and the negative control, supporting that these isolates are non-pathogenic, whereas the positive control had significantly higher scores (P < 0.05). Four weeks later (Phase II), G1-G7, as well as the original positive control group, were re-challenged with the same C1 isolate to assess protection. G1-G7 developed clinical signs, infraorbital sinus gross and histopathological lesions comparable to the new positive control. In conclusion, the study results match field observations and confirm the novel npAP isolates are non-pathogenic to chickens. Additionally, npAP isolates do not confer protection against circulating Midwestern pAP serovar C1. Given their prevalence in NHL flocks in the US, npAP should be ruled out before confirming IC diagnosis in flocks without clinical signs.
Runting-stunting syndrome (RSS) is an enteric disease of young chickens with complex aetiopathogenesis. It is often linked to the isolation of multiple virus species and histologically characterized by cystic enteropathy. We investigated the correlation between the detection frequency of avian reovirus, astrovirus, parvovirus, and rotavirus A and the presence of microscopic lesions consistent with cystic enteropathy in 123 broiler chicken submissions to the Alabama State Laboratory System (Auburn, AL, USA). Multi-viral detections were found to be predictive of cystic enteropathy with an increased odds of 4.6 for every additional virus species detection. These results suggest a cumulative effect of enteric viruses on the likelihood of cystic enteropathy. Avian reovirus was frequently associated with cystic enteropathy (59.0% of cases). Next-generation sequencing detected the presence of more than one reovirus strain in seven of the eight tested isolates. Classification into multiple genotypes suggested that genetically diverse avian reoviruses may cause similar clinical signs.
