Pathogenicity In Chickens Research Articles

The Antimicrobial Effect of Thymol and Carvacrol in Combination with Organic Acids Against Foodborne Pathogens in Chicken and Beef Meat Fillets

Bioactive compounds and organic acids are applied to a wide range of foods against different types of foodborne pathogens. In the present study, carvacrol and thymol (1000 mg/L) were applied in wine-based marinades, alone or in combination with them and in combination with tartaric acid, malic acid, ascorbic acid, citric acid, and acetic acid (in concentration 0.1% w/v), in chicken and beef fillets and their antimicrobial activity, antioxidant capacity, and pH were estimated during refrigerated storage. Likewise, their antimicrobial activity was recorded against Enterobacteriaceae, total mesophilic bacteria, yeasts/molds, and lactic acid bacteria. The outcome demonstrated that both meats kept under similar storage conditions (4 °C/9 days) exhibited lower microbial growth, particularly with Enterobacteriaceae, when treated with wine-based carvacrol—thymol marinades and may extend their shelf-life. This antimicrobial action was more pronounced in the beef samples. The total phenolic content (TPC) and the antioxidant activity of the applied marinades were determined using the Folin−Ciocalteau method and ABTS and DPPH radical scavenging activity methods, respectively. The results revealed that marinades with thymol and/or carvacrol in combination with acetic or ascorbic acid had greater TPC and antioxidant activity. The pH values of the respective marinades applied to both chicken and beef fillets exhibited an upturn during storage. Consequently, these marinades, even at low concentrations, could be used as natural preservatives in meat products.

The evolution of hemagglutinin-158 and neuraminidase-88 glycosylation sites modulates antigenicity and pathogenicity of clade 2.3.2.1 H5N1 avian influenza viruses

Clade 2.3.2.1 of the H5N1 avian influenza virus (AIV) evolved into several subclades. However, the effect of glycosylation on the biological characteristics of hemagglutinin (HA) and/or neuraminidase (NA) from H5N1 AIVs remains unclear. Here, we determined that the global prevalence of clade 2.3.2.1 H5N1 AIVs with deglycosylated residue 158 on HA (HA158-) and glycosylated residue 88 on NA (NA88+) were predominant via multiple sequence analysis. The deglycosylation of residue on NA 88 (NA88-) was observed in clade 2.3.2.1a (new) and clade 2.3.2.1e H5N1 AIVs. Interestingly, NA88- was coupled with the acquisition of 158 glycosylation sites on HA (HA158+) in clade 2.3.2.1e H5N1 AIVs from China, and clade 2.3.2.1a (new) H5N1 AIVs exhibiting the HA158-NA88- pattern were predominant in Bangladesh. Meanwhile, the temporal distribution of strain HA158+ NA88- was highly consistent with the implementation of Re-6 vaccine in China. The recombinant H5N1 AIVs constructed using a reverse genetic system showed that the acquisition of the HA158 glycosylation site facilitated viral evasion form Re-6 antisera, and the virus lacking glycosylation sites at HA158 and NA88 resulted in reduced NA activity, replication in mammalian cells, and pathogenicity in both chickens and mice compared to that of the viruses with alternative glycosylation patterns. Therefore, the acquisition of HA158+ in clade 2.3.2.1e H5N1 AIVs enables evasion of Re-6 vaccination pressure, and the virulence of clade 2.3.2.1 H5N1 AIVs is modulated by the absence of glycosylation sites at HA158 and NA88. Our finding highlighting the importance of epidemiological surveillance and timely updating vaccines of H5 AIVs.

The Role of Dual Mutations G347E and E349D of the Pigeon Paramyxovirus Type 1 Hemagglutinin-Neuraminidase Protein In Vitro and In Vivo.

Pigeon Newcastle disease (ND) is the most common viral infectious disease in the pigeon industry, caused by pigeon paramyxovirus type 1 (PPMV-1), a variant of chicken-origin Newcastle disease virus (NDV). Previous studies have identified significant amino acid differences between PPMV-1 and chicken-origin NDV at positions 347 and 349 in the hemagglutinin-neuraminidase (HN) protein, with PPMV-1 predominantly exhibiting glycine (G) at position 347 and glutamic acid (E) at position 349, while most chicken-origin NDVs show E at position 347 and aspartic acid (D) at position 349. However, the impact of these amino acid substitutions remains unclear. In this study, we generated a recombinant virus, NT-10-G347E/E349D, by introducing the G347E and E349D dual mutations into a PPMV-1 strain NT-10 using reverse genetics. The biological characteristics of NT-10 and NT-10-G347E/E349D were compared both in vitro and in vivo. In vitro, the G347E and E349D dual mutations reduce NT-10's replication and neuraminidase activity in pigeon embryo fibroblast (PEF) cells while enhancing both in chicken embryo fibroblast (CEF) cells. Additionally, these mutations decrease NT-10's binding affinity to the α-2,6 sialic acid receptor while significantly increasing its affinity for the α-2,3 receptor. In vivo, NT-10-G347E/E349D exhibited reduced pathogenicity in pigeons but increased pathogenicity in chickens compared to the parental NT-10 strain. The mutations also reduced the pigeon-to-pigeon transmission of NT-10 but enhanced its transmission from pigeons to chickens. Notably, significant antigenic differences were observed between NT-10 and NT-10-G347E/E349D, as an inactivated vaccine based on NT-10 provided full protection against NT-10 challenge in immunized pigeons but only 67% mortality protection against NT-10-G347E/E349D. Overall, these findings underscore the critical role of amino acids at positions 347 and 349 in PPMV-1 infection, pathogenicity, and transmission, providing a theoretical foundation for the scientific prevention and control of PPMV-1.

16S rRNA gene sequencing for bacterial identification and infectious disease diagnosis

16S rRNA gene sequence is the most common housekeeping genetic marker to study bacterial phylogeny and taxonomy. Therefore, 16S rRNA gene sequencing has the potential to identify novel bacteria and diagnose bacteria. This study compared 16S rRNA gene sequencing with conventional PCR for bacterial identification and disease diagnosis. The bacterial community in healthy and diseased hosts was analyzed by 16S rRNA gene sequencing. 16S rRNA gene sequencing is more sensitive than conventional PCR in detecting bacteria. Moreover, 16S rRNA gene sequencing is adequate to identify novel bacteria. 16S rRNA gene sequencing demonstrated that most pathogenic bacteria persist in diseased or healthy hosts in different abundance. Pathogenic bacteria, such as well-known chicken pathogen Avibacterium paragallinarum, Ornithobacterium rhinotracheale, and Gallibacterium anatis, were identified as indicator species of diseased samples. Alpha diversity analysis showed that the healthy group species is significantly higher than in the diseased groups. Beta diversity analysis also demonstrated differences between healthy and infected groups. The study concluded that 16S rRNA gene sequencing is a more sensitive method for detecting pathogens, and microbiota analysis can distinguish between healthy and diseased samples. Eventually, 16S rRNA gene sequencing has represented the potential in human and animal clinical diagnosis and novel bacterial identification.

Hemagglutinin and neuraminidase of a non-pathogenic H7N7 avian influenza virus coevolved during the acquisition of intranasal pathogenicity in chickens.

Polybasic amino acid residues at the hemagglutinin (HA) cleavage site are insufficient to induce the highly pathogenic phenotype of avian influenza viruses in chickens. In our previous study, an H7N7 avian influenza virus named "Vac2sub-P0", which is nonpathogenic despite carrying polybasic amino acids at the HA cleavage site, was passaged in chick air sacs, and a virus with high intravenous pathogenicity, Vac2sub-P3, was obtained. Intranasal infection with Vac2sub-P3 resulted in limited lethality in chickens; therefore, in this study, this virus was further passaged in chicken lungs, and the resultant virus, Vac2sub-P3L4, acquired high intranasal pathogenicity. Experimental infection of chickens with recombinant viruses demonstrated that mutations in HA and neuraminidase (NA) found in consecutive passages were responsible for the increased pathogenicity. The HA and NA functions of Vac2sub-P3L4 were compared with those of the parental virus in vitro; the virus growth at 40 °C was faster, the binding affinity to a sialic acid receptor was lower, and the rate of release by NA from the cell surface was lower, suggesting that these changes enabled the virus to replicate efficiently in chickens with high intranasal pathogenicity. This study demonstrates that viruses that are highly pathogenic when administered intranasally require additional adaptations for increased pathogenicity to be highly lethal to intranasally infected chickens.

Pathogenicity of Duck Adenovirus Type 3 in Chickens.

Duck adenovirus Type 3 (DAdV-3) severely affects the health of ducks; however, its pathogenicity in chickens remains unknown. The objectives of this study were to evaluate the pathogenicity and major pathological changes caused by DAdV-3 in chickens. Viral DNA was extracted from the liver of the Muscovy duck, and the fiber-2 and hexon fragments of DAdV-3 were amplified through polymerase chain reaction (PCR). The evolutionary tree revealed that the isolated virus belonged to DAdV-3, and it was named HE-AN-2022. The mortality rate of chicks that received inoculation with DAdV-3 subcutaneously via the neck was 100%, while the mortality rate for eye-nose drop inoculation was correlated with the numbers of infection, with 26.7% of chicks dying as a result of exposure to multiple infections. The main symptoms exhibited prior to death were hepatitis-hydropericardium syndrome (HHS), ulceration of the glandular stomach, and a swollen bursa with petechial hemorrhages. A histopathological examination revealed swelling, necrosis, lymphocyte infiltration, and basophilic inclusion bodies in multiple organs. Meanwhile, the results of quantitative real-time PCR (qPCR) demonstrated that DAdV-3 could affect most of the organs in chickens, with the gizzard, glandular stomach, bursa, spleen, and liver being the most susceptible to infection. The surviving chicks had extremely high antibody levels. After the chickens were infected with DAdV-3 derived from Muscovy ducks, no amino acid mutation was observed in the major mutation regions of the virus, which were ORF19B, ORF66, and ORF67. On the basis of our findings, we concluded that DAdV-3 infection is possible in chickens, and that it causes classic HHS with ulceration of the glandular stomach and a swollen bursa with petechial hemorrhages, leading to high mortality in chickens. The major variation domains did not change in Muscovy ducks or in chickens after infection. This is the first study to report DAdV-3 in chickens, providing a new basis for preventing and controlling this virus.

FAdV-4 can cause more noticeable clinical symptoms compared to FAdV-8b after infecting specific pathogen free chickens

Chickens infected with FAdV-4 and FAdV-8b both exhibit hepatic lesions characterized by hemorrhagic necrosis and intranuclear inclusion body formation. However, only FAdV-4 induces pericardial effusion and acute mortality in chickens. To investigate the similarities and differences in the pathogenicity of HPS and IBH, this study intends to compare the infectivity and pathogenicity of FAdV-4 and FAdV-8b, two serotypes of fowl adenovirus isolated in our laboratory. The two viruses were respectively inoculated subcutaneously into SPF chicks at the neck. The clinical manifestations and pathological changes in these infected groups of chickens differed to some extent. Chickens infected with FAdV-4 exhibit evident depression and acute mortality, with a mortality rate of 60%; while those infected with FAdV-8b only display mild depression. Postmortem examination reveals serosanguinous effusion in the pericardial sac, spot-like hemorrhage, and focal necrosis in the liver of chickens infected with FAdV-4. Additionally, various degrees of edema are observed in organs such as the lungs, spleen, kidneys, and pancreas. In contrast, chickens infected with FAdV-8b exhibit spot-like hemorrhage and focal necrosis in the liver but do not display pericardial effusion or widespread organ edema. Histopathological examination demonstrates that both FAdV-4 and FAdV-8b can induce inflammatory reactions of varying degrees in the kidneys, pancreas and duodenum of chickens, while reducing the necrosis of bursa of Fabricius, thymus, and spleen lymphocytes. Our data preliminarily reveal that both FAdV-4 and FAdV-8b can induce strong pathogenicity in chickens.

Co-infection of H9N2 subtype avian influenza virus and QX genotype live attenuated infectious bronchitis virus increase the pathogenicity in SPF chickens

Avian influenza virus (AIV) infection and vaccination against live attenuated infectious bronchitis virus (aIBV) are frequent in poultry worldwide. Here, we evaluated the clinical effect of H9N2 subtype AIV and QX genotype aIBV co-infection in specific-pathogen-free (SPF) white leghorn chickens and explored the potential mechanisms underlying the observed effects using by 4D-FastDIA-based proteomics. The results showed that co-infection of H9N2 AIV and QX aIBV increased mortality and suppressed the growth of SPF chickens. In particular, severe lesions in the kidneys and slight respiratory signs similar to the symptoms of virulent QX IBV infection were observed in some co-infected chickens, with no such clinical signs observed in single-infected chickens. The replication of H9N2 AIV was significantly enhanced in both the trachea and kidneys, whereas there was only a slight effect on the replication of the QX aIBV. Proteomics analysis showed that the IL-17 signaling pathway was one of the unique pathways enriched in co-infected chickens compared to single infected-chickens. A series of metabolism and immune response-related pathways linked with co-infection were also significantly enriched. Moreover, co-infection of the two pathogens resulted in the enrichment of the negative regulation of telomerase activity. Collectively, our study supports the synergistic effect of the two pathogens, and pointed out that aIBV vaccines might increased IBV-associated lesions due to pathogenic co-infections. Exacerbation of the pathogenicity and mortality in H9N2 AIV and QX aIBV co-infected chickens possibly occurred because of an increase in H9N2 AIV replication, the regulation of telomerase activity, and the disturbance of cell metabolism and the immune system.

Molecular epidemiology of infectious bronchitis virus in eastern and southern China during 2021–2023

As a highly infectious and contagious pathogen in chickens, infectious bronchitis virus (IBV) is currently grouped into nine genotypes (GI to GIX). However, the classification of serotypes of IBV is still not clear. In this study, 270 field strains of IBV were isolated from dead or diseased chicken flocks in eastern and southern China during Jan. 2021-Apr. 2023. These isolated IBV strains could be classified into two genotypes, GI (including five lineages GI-1, GI-13, GI-19, GI-22 and GI-28) and GVI based on the complete S1 sequence. Further analysis showed that the GI-19, GI-13, GI-22, GI-28 and GVI were the dominant genotypes with the proportions of 61.48%, 8.89%, 8.89%, 7.78% and 8.89% respectively, and the homology of S1 protein of these isolates ranged from 86.85% to 100% in GI-19, 92.22% to 100% in GI-13, 83.1% to 100% in GI-22, 94.81% to 100% in GI-28 and 90.0% to 99.8% in GVI, respectively. Moreover, cross-neutralization test with sera revealed that these isolates in GI-19 lineage could be classified into at least three serotypes according to the antigenic relationship. In addition, structure assay using PyMOL indicated that one mutation such as S120 in receptor binding site (RBD) of GI-19 might alter the antigenicity and conformation of S protein of IBV. Overall, our data demonstrate that not only multiple genotypes, but also multiple serotypes in a single genotype or lineage have been co-circulated in eastern and southern China, providing novel insights into the molecular evolution of the antigenicity of IBV and highlighting the significance of the selection of the dominant isolate for vaccine development in IBV endemic region.

Evolution of H6N6 viruses in China between 2014 and 2019 involves multiple reassortment events

ABSTRACT H6N6 avian influenza viruses (AIVs) have been widely detected in wild birds, poultry, and even mammals. Recently, H6N6 viruses were reported to be involved in the generation of H5 and H7 subtype viruses. To investigate the emergence, evolutionary pattern, and potential for an epidemic of H6N6 viruses, the complete genomes of 198 H6N6 viruses were analyzed, including 168 H6N6 viruses deposited in the NCBI and GISAID databases from inception to January 2019 and 30 isolates collected from China between November 2014 and January 2019. Using phylogenetic analysis, the 198 strains of H6N6 viruses were identified as 98 genotypes. Molecular clock analysis indicated that the evolution of H6N6 viruses in China was constant and not interrupted by selective pressure. Notably, the laboratory isolates reassorted with six subtype viruses: H6N2, H5N6, H7N9, H5N2, H4N2, and H6N8, resulting in nine novel H6N6 reassortment events. These results suggested that H6N6 viruses can act as an intermediary in the evolution of H5N6, H6N6, and H7N9 viruses. Animal experiments demonstrated that the 10 representative H6N6 viruses showed low pathogenicity in chickens and were capable of infecting mice without prior adaptation. Our findings suggest that H6N6 viruses play an important role in the evolution of AIVs, and it is necessary to continuously monitor and evaluate the potential epidemic of the H6N6 subtype viruses.

Synergy of Subgroup J Avian Leukosis Virus and Chicken Infectious Anemia Virus Enhances the Pathogenicity in Chickens.

Subgroup J avian leukemia virus (ALV-J) and chicken infectious anemia virus (CIAV) are widely acknowledged as significant immunosuppressive pathogens that commonly co-infect chickens, causing substantial economic losses in the poultry industry. However, whether co-infection of ALV-J and CIAV have synergistic pathogenicity remains uncertain. To explore their synergistic pathogenesis, we established a co-infection model of ALV-J and CIAV in HD11 cells and specific-pathogen-free (SPF) chickens. We discovered that ALV-J and CIAV can synergistically promote the secretion of IL-6, IL-10, IFN-α, and IFN-γ and apoptosis in HD11 cells. In vivo, compared to the ALV-J and CIAV mono-infected group, the mortality increased significantly by 27% (20 to 47%) and 14% (33 to 47%) in the co-infected group, respectively. We also discovered that ALV-J and CIAV synergistically inhibited weight gain and exhibited more severe organ damage in co-infected chickens. Furthermore, we found that CIAV can promote the replication of ALV-J in HD11 cells and significantly enhance ALV-J viral load in blood and tissues of co-infected chickens, but ALV-J cannot promote the replication of CIAV. Moreover, by measuring the immune organ indexes and proportions of blood CD3+CD4+ and CD3+CD8+ lymphocytes, more serious instances of immunosuppression were observed in ALV-J and CIAV co-infected chickens than in mono-infected chickens. Taken together, our findings demonstrate that ALV-J and CIAV synergistically enhance pathogenicity and immunosuppression.

Role of ArcA in the regulation of antibiotic sensitivity in avian pathogenic Escherichia coli

Avian pathogenic Escherichia coli (APEC) is one of the common extraintestinal infectious disease pathogens in chickens, geese, and other birds, inducing serious impediments to the development of the poultry industry. Hence, investigating how bacteria regulate themselves amidst different challenging conditions is immense essential in prevention and treatment for bacterial pathogen infections. The ArcA regulatory factor has been reported to regulate oxygen availability in strains, but its role in regulation of antibiotics resistance in APEC is unclear. This study delved into understanding how ArcA regulates antibiotic resistance in APEC. An E. coli APEC40 arcA knockout strain was constructed, and the regulatory mechanism of arcA on APEC antibiotic susceptibility was identified by drug sensitivity test, colony counting assay, real-time quantitative PCR, β-galactosidase assays and electrophoretic mobility shift assay (EMSA). The results showed that ArcA directly binds to the promoter region of the outer membrane protein OmpC/OmpW and regulates bacterial susceptibility to kanamycin and penicillin G. At the same time, the double knockout of ompW and ompW/arcA resulted in an increase in resistance to kanamycin compared to the deletion of the arcA gene. This outcome provided experimental proof suggesting that the outer membrane protein OmpW could serve as a crucial pathway for the ingress of kanamycin into cells. These results confirmed the important regulatory role of ArcA transcription factors under APEC antibiotic stress.

Analysis of H5N8 influenza virus infection in chicken with mApple reporter genes in vivo and in vitro

H5N8 highly pathogenic avian influenza virus (HPAIV) has caused huge losses to the global poultry industry and critically threatens public health. Chickens are the important host for the transmission. However, the distribution of H5N8 avian influenza virus (AIV) in chicken and the infected cell types are limitedly studied. Therefore, in this study, we detected viral replication and infection by generating recombinant H5N8 AIV expressing an easily tracked mApple fluorescent reporter. The results showed that recombinant viruses passaged four times in chicken embryos successfully expressed mApple proteins detected by fluorescence microscopy and WB, which verified that the constructed recombinant viruses were stable. Compared to parental virus, although recombinant virus attenuated for replication in MDCK cells, it can still replicate effectively, and form visible plaques. Importantly, the experiments on infection of chicken PBMCs in vitro showed a strong correlation between mApple positivity rate and NP positivity rate (r = 0.7594, P =0.0176), demonstrating that mApple reporter could be used as an indicator to accurately reflect AIV infection. Then we infected monocytes/macrophages in PBMCs in vitro and detected the mApple positive percentage was 55.1%-80.4%, which confirmed the chicken primary monocytic/macrophages are important target cells for avian influenza virus infection. In chicken, compared with parental virus, the recombinant virus-infected chickens had lower viral titers in oropharyngeal cloacal and organs, but it can cause significant pathogenicity in chicken and the mortality rate was approximately 66%. In addition, the results of bioluminescent imaging showed that the fluorescence in the lungs was strongest at 5 days post-infection (DPI). Finally, we discovered the mApple positive expression in chicken lung immune cells (CD45+ cells), especially some T cells (CD4 and CD8 T cells) also carrying mApple, which indicates that the H5N8 AIV showed a tropism for immune cells including chicken T cells causing potentially aggressive against cellular immunity. We have provided a simple visualization for further exploration of H5N8 AIV infected chicken immune cells, which contributes to further understanding pathogenic mechanism of H5N8 AIV infection in chicken.

Genome sequences of haemagglutinin cleavage site predict the pathogenicity phenotype of avian influenza virus: statistically validated data for facilitating rapid declarations and reducing reliance on in vivo testing

ABSTRACT Based on the pathogenicity in chickens, most H1–H16 avian influenza viruses (AIV) cause mild diseases, whereas some of the H5 and H7 AI viruses cause severe, systemic disease. The number of basic amino acids in the haemagglutinin (HA) cleavage site of AIV plays a critical role in pathogenicity. As we gain a greater understanding of the molecular mechanisms of pathogenicity, genome sequencing of the HA0 cleavage site has assumed a greater role in assessment of the potential pathogenicity of H5 and H7 viruses. We validated the use of HA cleavage site motif analysis by comparing molecular pathotyping data against experimental in vivo (intravenous pathogenicity index [IVPI] and lethality) data for determination of both low pathogenicity and high pathogenicity AI virus declaration with the goal of expediting pathotype confirmation and further reducing the reliance on in vivo testing. Our data provide statistical support to the continued use of molecular determination of pathotype for AI viruses based on the HA cleavage site sequence in the absence of an in vivo study determination. This approach not only expedites the declaration process of highly pathogenic AIV (HPAIV) but also reduces the need for experimental in vivo testing of H5 and H7 viruses.

Paper-based electrochemical immunosensor for highly sensitive detection of chicken anemia virus

Chicken anemia virus (CAV) is one of the primary causes of morbidity and mortality in young chickens. Given the importance of timely detection for maintaining livestock quality, there is a pressing need for rapid and field-deployable diagnostic tools. This study introduces a highly sensitive paper-based electrochemical immunosensor (PEI) for the detection of the 60 amino acid N-terminally truncated viral protein 1 (Δ60VP1), a derivative of the CAV capsid (VP1). A custom antibody was produced for precise immunoassay detection, with results obtainable within 30 min using Square Wave Voltammetry (SWV). The underlying mechanism involves an immunocomplex in the sample zone that hinders the electron transfer of redox species, thereby reducing the current signal in proportion to the Δ60VP1 concentration. Under optimal conditions, the detection linearity for Δ60VP1 ranged from 80 to 2500 ng/mL, with a limit of detection (LoD) of 25 ng/mL. This device was then successfully applied to detect VP1 in 29 chicken serum samples, achieving 91.6% sensitivity and 94.1% selectivity. In conclusion, the PEI device presents a promising solution for rapid, sensitive, and disposable detection of chicken pathogens, potentially revolutionizing productivity and quality assurance in chicken farming.

The coinfection of ALVs causes severe pathogenicity in Three-Yellow chickens

The coinfection of ALVs (ALV-J plus ALV-A or/and ALV-B) has played an important role in the incidence of tumors recently found in China in local breeds of yellow chickens. The study aims to obtain a better knowledge of the function and relevance of ALV coinfection in the clinical disease of avian leukosis, as well as its unique effect on the pathogenicity in Three-yellow chickens. One-day-old Three-yellow chicks (one day old) were infected with ALV-A, ALV-B, and ALV-J mono-infections, as well as ALV-A + J, ALV-B + J, and ALV-A + B + J coinfections, via intraperitoneal injection, and the chicks were then grown in isolators until they were 15 weeks old. The parameters, including the suppression of body weight gain, immune organ weight, viremia, histopathological changes and tumor incidence, were observed and compared with those of the uninfected control birds. The results demonstrated that coinfection with ALVs could induce more serious suppression of body weight gain (P < 0.05), damage to immune organs (P < 0.05) and higher tumor incidences than monoinfection, with triple infection producing the highest pathogenicity. The emergence of visible tumors and viremia occurred faster in the coinfected birds than in the monoinfected birds. These findings demonstrated that ALV coinfection resulted in considerably severe pathogenic and immunosuppressive consequences.

Prokaryotic and eukaryotic dual-expression plasmid-mediated delivery of Campylobacter jejuni antigens by live-attenuated Salmonella: A strategy for concurrent Th1 and Th2 immune activation and protection in chickens

Salmonella and Campylobacter are food-borne pathogens that significantly affect poultry production and are transmitted to humans. Long-term protection against these pathogens in chicken relies on a balanced Th1 and Th2 response. C. jejuni antigens were screened and a fusion antigen, including CadF + FlaA adhesin and flagellin antigenic fragments was developed and safely delivered by low-endotoxicity S. Typhimurium through pJHL270, a dual-expression plasmid featuring prokaryotic (Ptrc) and eukaryotic (CMV) promoters. Antigen expression in Salmonella and host cells was confirmed by western blotting and IFA. The vaccine construct JOL2999, triggered significant increases in IgY, IgA antibodies, CD4+ and CD8+ T cells, indicating humoral, mucosal, and cell-mediated responses against both pathogens. Elevations in pro-inflammatory cytokines TNFα, INF-γ, IL-2, and IL-4 and MHC I and II cell populations further suggest simultaneous Th1 and Th2 immune activation. Reduced pathogen load and histopathological inflammatory signs in vital organs upon challenge confirmed the protective efficacy in chickens.

Isolation, characterization, evaluation of pathogenicity, and immunomodulation through interferon production of duck adenovirus type-3 (DAdV-3)

Duck adenovirus type-3 (DAdV-3) is a poorly characterized duck virus. A comprehensive analysis of the DAdV-3 pathogenicity and host immune response could be a valuable addition. Herein, DAdV-3 was isolated from Muscovy duck and virus-specific genes were confirmed by polymerase chain reaction (PCR). The obtained gene fragments were sequenced and compared with the reference sequence. Results confirmed that the clinically isolated virus was DAdV-3, named as HF-AN-2020. To evaluate DAdV-3 host immune response, the expression levels of MDA5, STING, IRF7, MAVS, and NF-κB, and inflammatory cytokines (IFN-β, IFN-γ, and IL-1β) were determined by quantitative reverse transcriptase PCR (qRT-PCR). The expression levels of IFN-β and IFN-γ were 32.6- and 28.6-fold, respectively, higher (P < 0.01) than the control group. It was found that the upregulation of STING and NF-κB pathways was directly involved in the regulation of inflammatory cytokines (IFN-β, IFN-γ, and IL-1β). Furthermore, the gene regulation pathways consecutively upregulated the expression levels of MDA5, STING, IRF7, MAVS, and NF-κB up to 31.6, 10.5, 31.4, 2.2, and 2.6-fold, respectively, higher (P < 0.01) than the control group. The TCID50 of DAdV-3 for Muscovy duck and chicken was 10-3.24/0.1 mL with 0% mortality, indicating low pathogenicity in both Muscovy ducks and chickens, but DAdV-3 can induce higher expression of interferons. Genome analysis showed mutations in 4 amino acids located in ORF19B (Ser to Thr), ORF66 (Leu to Phe, Ile to Leu), and ORF67 (Gly to stop codon). This study provides essential and basic information for further research on the mechanism of the cellular immune responses against adenoviruses.

Osteomielite vertebral causada por Enterococcus faecalis em frangos de corte no sul do Brasil

ABSTRACT: Enterococcal spondylitis affects poultry and causes progressive lameness. This study reports what seems to be the first case of vertebral osteomyelitis caused by Enterococcus in broiler chickens in southern Brazil. We also conducted an experimental infection to evaluate microorganismal characteristics and pathogenicity in broiler chickens. We performed bacterial isolation, identification, and histopathology. The isolates were tested for their growth and survival capacity at different temperatures, pH values, and antimicrobial resistance profiles. The experiment infection was conducted with broiler breeders (n=9). Group 1 = negative control, Group 2 = challenged orally, Group 3 = challenged via air sac. The autopsy was performed on the 50th day of life (DOL). The report showed spondylitis and fusion of thoracic vertebra, accompanied by spinal cord compression, and femoral head necrosis. We used the isolates (n=17) to test their growth at 10°C and 45°C, survival capacity for up to 60° for 30 min, and growth under pH levels from four to 12. Higher resistance was observed against macrolides and quinolones. On experimental infections, all animals expressed signs of lameness and “sitting on the hocks”. Enterococcus faecalis is the causal agent of enterococcal spondylitis in broilers in southern Brazil, which is an underreported and emerging pathological condition that requires attention.

Molecular characterization of chicken astrovirus and pathogenicity of a novel isolate in China.

As an enteric virus, chicken astrovirus has been related to various kinds of diseases in chickens, including white chick syndrome, runting-stunting syndrome, severe kidney disease, urate deposits and visceral gout, generating economic losses in the poultry industry globally. The complete ORF2 gene of 31 CAstV isolates in six provinces of China during 2020-2022 was characterized and analyzed with the purpose of better understanding the molecular epidemiology and genetic diversity of CAstV field isolates. Phylogenetic analysis which was based on the complete ORF2 (capsid) amino acid sequence of 31 CAstV isolates and 57 reference strains indicated that 2 isolates belonged to subgroup Ai, 10 isolates belonged to subgroup Bi, 3 isolates belonged to subgroup Bii, 5 isolates belonged to subgroup Biii, 7 isolates belonged to subgroup Biv, 3 isolates belonged to subgroup Bv, and one isolate (JS202103) belonged to a new B subgroup. In addition, the novel CAstV strain JS202103 was successfully isolated in vitro, and its whole genome shared 76.9-94.3% identity with the 29 CAstV reference strains. JS202103 caused hatchability reduction, dead embryos, kidney disease and visceral gout in chicken embryos. Moreover, this is the also the initial study focusing on diverse CAstV strains including subgroups Biii, Biv, and Bv circulate in China. The current work contributes to improving our understanding of CAstV isolates in China, and it will also provide references for developing efficient measures to control this virus.