A detailed analysis of 16S rRNA gene sequencing and conventional PCR-based testing for the diagnosis of bacterial pathogens and discovery of novel bacteria.

This study represents the first analysis of the bacterial community in chickens affected by swollen head syndrome, utilizing 16S rRNA gene sequencing. Samples were obtained from clinical laying chickens and were examined for the presence of Avibacterium paragallinarum (APG) and Ornithobacterium rhinotracheale (ORT) using conventional polymerase chain reaction (PCR). From the samples, five APG-positive (APG) and APG-negative (N-APG) samples were chosen, along with five specific pathogen-free chickens, for 16S rRNA gene sequencing. Results showed that APG and ORT were widely detected in the chicken samples with swollen head syndrome (SHS, 9/10), while APG was detected in all five specific pathogen-free (SPF) samples. In contrast, conventional PCR sensitivity was found to be inadequate for diagnosis, with only 35.7% (5/14) and 11.1% (1/9) sensitivity for APG and ORT, respectively, based on 16S rRNA gene sequencing data. Furthermore, 16S rRNA gene sequencing was able to quantify the bacteria in the samples, revealing that the relative abundance of APG in the APG group ranged from 2.7 to 81.3%, while the relative abundance of APG in the N-APG group ranged from 0.1 to 21.0%. Notably, a low level of APG was also detected in all 5 SPF samples. The study also identified a significant number of animal and human common bacterial pathogens, including but not limited to Gallibacterium anatis, Riemerella columbina, Enterococcus cecorum, Mycoplasma synoviae, Helicobacter hepaticus, and Staphylococcus lentus. In conclusion, 16S rRNA gene sequencing is a valuable tool for bacterial pathogen diagnosis and the discovery of novel bacterial pathogens, while conventional PCR is not reliable for diagnosis.

The emergence of new antigen branches of H9N2 avian influenza virus in China due to antigenic drift on hemagglutinin through antibody escape at immunodominant sites.

Vaccination is a crucial prevention and control measure against H9N2 avian influenza viruses (AIVs) that threaten poultry production and public health. However, H9N2 AIVs in China undergo continuous antigenic drift of hemagglutinin (HA) under antibody pressure, leading to the emergence of immune escape variants. In this study, we investigated the molecular basis of the current widespread antigenic drift of H9N2 AIVs. Specifically, the most prevalent h9.4.2.5-lineage in China was divided into two antigenic branches based on monoclonal antibody (mAb) hemagglutination inhibition (HI) profiling analysis, and 12 antibody escape residues were identified as molecular markers of these two branches. The 12 escape residues were mapped to antigenic sites A, B, and E (H3 was used as the reference). Among these, eight residues primarily increased 3`SLN preference and contributed to antigenicity drift, and four of the eight residues at sites A and B were positively selected. Moreover, the analysis of H9N2 strains over time and space has revealed the emergence of a new antigenic branch in China since 2015, which has replaced the previous branch. However, the old antigenic branch recirculated to several regions after 2018. Collectively, this study provides a theoretical basis for understanding the molecular mechanisms of antigenic drift and for developing vaccine candidates that contest with the current antigenicity of H9N2 AIVs.

[Development of a double-antibody sandwich ELISA targeting the receptor binding domain of TcdB toxin of ST11 type Clostridium difficile of porcine origin].

Clostridium difficile is an important zoonotic intestinal pathogen, which is widely present in humans and a variety of animals. The ST11 type C. difficile is one of the most widespread and harmful subtypes in the world. As a large country in pig farming, China lacks efficient methods for detecting C. difficile of porcine origin, leaving hidden dangers for the prevention and control of C. difficile. The aim of this study was to develop a specific and sensitive double-antibody sandwich ELISA for the epidemiological investigation of ST11 type C. difficile of porcine origin. Firstly, a 97 kDa receptor binding domain (RBD) was expressed in a prokaryotic host and purified. A hybridoma cell line AE2D3 capable of stably secreting monoclonal antibody targeting the RBD was screened, and the antibody subtype was determined to be IgG2b (κ). Secondly, a double antibody sandwich ELISA method was developed, where the monoclonal antibody targeting the RBD was used as a detection antibody, and the rabbit polyclonal antibody was used as a capture antibody. The chessboard method was used to determine the matching concentration of the capture antibody and the detection antibody, the antigen coating conditions, the blocking conditions, the incubation conditions for detection antibody and samples to be tested, as well as the reaction conditions of HRP-conjugated and reaction conditions of TMB chromogenic solution. The negative cutoff OD450 was 0.152, and no cross-reaction with 13 strains of non-ST11 type C. difficile was found. The minimum detection concentration of RBD was 8.83 ng/mL. This specific and sensitive double-antibody sandwich ELISA provides a reliable serological detection method for epidemiological investigation of the ST11 type C. difficile in pig industry.

Generation of an avian influenza DIVA vaccine with a H3-peptide replacement located at HA2 against both highly and low pathogenic H7N9 virus.

A differentiating infected from vaccinated animals (DIVA) vaccine is an ideal strategy for viral eradication in poultry. Here, according to the emerging highly pathogenic H7N9 avian influenza virus (AIV), a DIVA vaccine strain, named rGD4HALo-mH3-TX, was successfully developed, based on a substituted 12 peptide of H3 virus located at HA2. In order to meet with the safety requirement of vaccine production, the multi-basic amino acid located at the HA cleavage site was modified. Meanwhile, six inner viral genes from a H9N2 AIV TX strainwere introduced for increasing viral production. The rGD4HALo-mH3-TX strain displayed a similar reproductive ability with rGD4 and low pathogenicity in chickens, suggesting a good productivity and safety. In immuned chickens, rGD4HALo-mH3-TX induced a similar antibody level with rGD4 and provided 100% clinical protection and 90% shedding protection against highly pathogenic virus challenge. rGD4HALo-mH3-TX strain also produced a good cross-protection against low pathogenic AIV JD/17. Moreover, serological DIVA characteristics were evaluated by a successfully established competitive inhibition ELISA based on a 3G10 monoclonal antibody, and the result showed a strong reactivity with antisera of chickens vaccinated with H7 subtype strains but not rGD4HALo-mH3-TX. Collectedly, rGD4HALo-mH3-TX is a promising DIVA vaccine candidate against both high and low pathogenic H7N9 subtype AIV.

Truncation or Deglycosylation of the Neuraminidase Stalk Enhances the Pathogenicity of the H5N1 Subtype Avian Influenza Virus in Mallard Ducks.

H5N1 subtype avian influenza virus (AIV) with a deletion of 20 amino acids at residues 49-68 in the stalk region of neuraminidase (NA) became a major epidemic virus. To determine the effect of truncation or deglycosylation of the NA stalk on virulence, we used site-directed mutagenesis to insert 20 amino acids in the short-stalk virus A/mallard/Huadong/S/2005 (SY) to recover the long-stalk virus (rSNA+). A series of short-stalk or deglycosylated-stalk viruses were also constructed basing on the long-stalk virus, and then the characteristics and pathogenicity of the resulting viruses were evaluated. The results showed that most of the short-stalk or deglycosylated-stalk viruses had smaller plaques, and increased thermal and low-pH stability, and a decreased neuraminidase activity when compared with the virus rSNA+. In a mallard ducks challenge study, most of the short-stalk or deglycosylated-stalk viruses showed increased pathological lesions and virus titers in the organ tissues and increased virus shedding in the oropharynx and cloaca when compared with the rSNA+ virus, while most of the short-stalk viruses, especially rSNA-20, showed higher pathogenicity than the deglycosylated-stalk virus. In addition, the short-stalk viruses showed a significantly upregulated expression of the immune-related factors in the lungs of the infected mallard ducks, including IFN-α, Mx1, and IL-8. The results suggested that NA stalk truncation or deglycosylation increases the pathogenicity of H5N1 subtype AIV in mallard ducks, which will provide a pre-warning for prevention and control of H5N1 subtype avian influenza in the waterfowl.

A Live Attenuated H9N2 Avian Influenza Vaccine Prevents the Viral Reassortment by Exchanging the HA and NS1 Packaging Signals.

The H9N2 avian influenza virus is not only an important zoonotic pathogen, it can also easily recombine with other subtypes to generate novel reassortments, such as the H7N9 virus. Although H9N2 live attenuated vaccines can provide good multiple immunities, including humoral, cellular, and mucosal immunity, the risk of reassortment between the vaccine strain and wild-type virus is still a concern. Here, we successfully rescued an H9N2 live attenuated strain [rTX-NS1-128 (mut)] that can interdict reassortment, which was developed by exchanging the mutual packaging signals of HA and truncated NS1 genes and confirmed by RT-PCR and sequencing. The dynamic growth results showed that rTX-NS1-128 (mut) replication ability in chick embryos was not significantly affected by our construction strategy compared to the parent virus rTX strain. Moreover, rTX-NS1-128 (mut) had good genetic stability after 15 generations and possessed low pathogenicity and no contact transmission characteristics in chickens. Furthermore, chickens were intranasally immunized by rTX-NS1-128 (mut) with a single dose, and the results showed that the hemagglutination inhibition (HI) titers peaked at 3 weeks after vaccination and lasted at least until 11 weeks. The cellular immunity (IL-6 and IL-12) and mucosal immunity (IgA and IgG) in the nasal and trachea samples were significantly increased compared to inactivated rTX. Recombinant virus provided a good cross-protection against homologous TX strain (100%) and heterologous F98 strain (80%) challenge. Collectively, these data indicated that rTX-NS1-128(mut) lost the ability for independent reassortment of HA and NS1-128 and will be expected to be used as a potential live attenuated vaccine against H9N2 subtype avian influenza.

Escherichia coli-Derived Outer Membrane Vesicles Deliver Galactose-1-Phosphate Uridyltransferase and Yield Partial Protection against Actinobacillus pleuropneumoniae in Mice.

In our previous studies, we have identified several in vivo-induced antigens and evaluated their potential as subunit vaccine candidates in a murine model, in which the recombinant protein GalT showed the most potent immunogenicity and immunoprotective efficacy against Actinobacillus pleuropneumoniae. To exploit a more efficient way of delivering GalT proteins, in this study, we employed the widely studied E. coli outer membrane vesicles (OMVs) as a platform to deliver GalT protein and performed the vaccine trial using the recombinant GalT-OMVs in the murine model. Results revealed that GalT-OMVs could elicit a highly-specific, IgG antibody titer that was comparable with the adjuvant GalT group. Significantly higher lymphocyte proliferation and cytokines secretion levels were observed in the GalT-OMVs group. 87.5% and 50% of mice were protected from a lethal dose challenge using A. pleuropneumoniae in active or passive immunization, respectively. Histopathologic and immunohistochemical analyses showed remarkably reduced pathological changes and infiltration of neutrophils in the lungs of mice immunized with GalT-OMVs after the challenge. Taken together, these findings confirm that OMVs can be used as a platform to deliver GalT protein and enhance its immunogenicity to induce both humoral and cellular immune responses in mice.

Galactose-1-phosphate uridyltransferase (GalT), an in vivo-induced antigen of Actinobacillus pleuropneumoniae serovar 5b strain L20, provided immunoprotection against serovar 1 strain MS71.

GALT is an important antigen of Actinobacillus pleuropneumoniae (APP), which was shown to provide partial protection against APP infection in a previous study in our lab. The main purpose of the present study is to investigate GALT induced cross-protection between different APP serotypes and elucidate key mechanisms of the immune response to GALT antigenic stimulation. Bioinformatic analysis demonstrated that galT is a highly conserved gene in APP, widely distributed across multiple pathogenic strains. Homologies between any two strains ranges from 78.9% to 100% regarding the galT locus. Indirect enzyme-linked immunosorbent assay (ELISA) confirmed that GALT specific antibodies could not be induced by inactivated APP L20 or MS71 whole cell bacterin preparations. A recombinant fusion GALT protein derived from APP L20, however has proven to be an effective cross-protective antigen against APP sevorar 1 MS71 (50%, 4/8) and APP sevorar 5b L20 (75%, 6/8). Histopathological examinations have confirmed that recombinant GALT vaccinated animals showed less severe pathological signs in lung tissues than negative controls after APP challenge. Immunohistochemical (IHC) analysis indicated that the infiltration of neutrophils in the negative group is significantly increased compared with that in the normal control (P<0.001) and that in surviving animals is decreased compared to the negative group. Anti-GALT antibodies were shown to mediate phagocytosis of neutrophils. After interaction with anti-GALT antibodies, survival rate of APP challenged vaccinated animals was significantly reduced (P<0.001). This study demonstrated that GALT is an effective cross-protective antigen, which could be used as a potential vaccine candidate against multiple APP serotypes.