In recent years, plasma-activated solutions (PASs) have made good progress in the disinfection of medical devices, tooth whitening, and fruit preservation. In this study, we investigated the inactivation efficacy of Newcastle disease virus by PASs. Water, 0.9% NaCl, and 0.3% H2O2 were excited by plasma to obtain the corresponding solutions PAS(H2O), PAS(NaCl), and PAS(H2O2). The complete inactivation of virus after PAS treatment for 30 min was confirmed by the embryo lethality assay (ELA) and hemagglutination (HA) test. Scanning electron microscopy (SEM) results showed that the morphology of the viral particle changed under PAS treatments. The total protein concentration of virus decreased as measured by a Bradford protein assay due to PAS treatment. The nucleic acid integrity assay demonstrated that viral RNA degraded into smaller fragments. Moreover, the physicochemical properties of PASs, including the oxidation-reduction potential (ORP), electrical conductivity, and H2O2 concentration, and electron spin resonance spectra analysis indicated that reactive oxygen and nitrogen species play a major role in the virus inactivation. Therefore, the application of PASs, as an environmentally friendly method, would be a promising alternative strategy in poultry industries.IMPORTANCE Newcastle disease (ND), as an infectious viral disease of avian species, caused significant economic losses to domestic animal and poultry industries. The traditional chemical sanitizers, such as chlorine-based products, are associated with risks of by-product formation with carcinogenic effects and environmental pollution. On the basis of this, plasma-activated water as a green disinfection product is a promising alternative for applications in stock farming and sterilization in hospitals and public places. In this study, we explored the inactivation efficacy of different plasma-activated solutions (PASs) against ND virus (NDV) and the possible underlying mechanisms. Our results demonstrated that reactive oxygen and nitrogen species detected in PASs, including short-lived OH˙ and NO˙ and long-lived H2O2, changed the morphology, destroyed the RNA structure, and degraded the protein of the virus, consequently resulting in virus inactivation. These lay a foundation for the application of PASs to resolve the issues of public health and environmental sanitation.

Clostridium perfringens encodes at least two different quorum sensing (QS) systems, the Agr-like and LuxS, and recent studies have highlighted their importance in the regulation of toxin production and virulence. The role of QS in the pathogenesis of necrotic enteritis (NE) in poultry and the regulation of NetB, the key toxin involved, has not yet been investigated. We have generated isogenic agrB-null and complemented strains from parent strain CP1 and demonstrated that the virulence of the agrB-null mutant was strongly attenuated in a chicken NE model system and restored by complementation. The production of NetB, a key NE-associated toxin, was dramatically reduced in the agrB mutant at both the transcriptional and protein levels, though not in a luxS mutant. Transwell assays confirmed that the Agrlike QS system controls NetB production through a diffusible signal. Global gene expression analysis of the agrB mutant identified additional genes modulated by Agrlike QS, including operons related to phospholipid metabolism and adherence, which may also play a role in NE pathogenesis. This study provides the first evidence that the Agr-like QS system is critical for NE pathogenesis and identifies a number of Agr-regulated genes, most notably netB, that are potentially involved in mediating its effects. The Agr-like QS system thus may serve as a target for developing novel interventions to prevent NE in chickens.KEYWORDS NetB, Clostridium perfringens, Agr-like quorum sensing, LuxS, VirS/VirR, necrotic enteritis, poultry, quorum sensing C lostridium perfringens is a Gram-positive, spore-forming, anaerobic bacterium that is widely distributed in soil, feces, and foods as well as the normal intestinal microbiota of both humans and animals (1). C. perfringens is responsible for a number of human and animal diseases owing to an arsenal of at least 16 different extracellular toxins, including food poisoning, gas gangrene in humans, enterotoxemia of sheep and goats, lamb dysentery, and necrotic enteritis (NE) in poultry (2). Strains are classified into five toxinotypes (A to E) based on the production of four major typing toxins (alpha-, beta-, epsilon-, and iota-toxin) (3, 4).Certain type A strains can produce NE, an enteric disease of poultry that, in 2015, was estimated to cost the worldwide poultry industry nearly $6 billion in losses (5). The disease occurs when C. perfringens proliferates to high numbers in the intestinal tract and produces extracellular toxins, resulting in characteristic necrotic lesions and often high rates of mortality (6, 7). In addition to producing alpha-toxin (CPA), which is carried

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BackgroundNecrotic enteritis (NE) is an economically important disease of poultry caused by certain Clostridium perfringens type A strains. The NetB toxin plays a critical role in the pathogenesis of NE. We previously demonstrated that netB is located within a 42 kb plasmid-encoded pathogenicity locus (NELoc-1), which also encodes 36 additional genes. Although NetB clearly plays a role in pathogenesis, the involvement of the other NELoc-1 genes has not yet been established. The current study was to provide experimental evidence to confirm the involvement of these genes in NE pathogenesis.ResultsThe present study has characterized a virulent C. perfringens strain (CP1) that has spontaneously lost the NELoc-1-encoding plasmid, pCP1netB. When assessed for cytotoxicity on Leghorn Male Hepatoma (LMH) cells, the culture supernatant of the pCP1netB-deficient CP1 variant (CP1ΔpCP1netB) demonstrated significantly reduced cytotoxicity compared to the wild-type. In addition, CP1ΔpCP1netB was unable to cause intestinal lesions in chickens in a NE disease model. When netB alone was introduced into CP1ΔpCP1netB, in vitro cytotoxicity was restored to the wild-type level; however, it did not completely restore virulence when used to challenge broiler chickens [mean lesion score of 0.71 compared to 3.23 in the wild type control group (n = 14)].ConclusionsThe results of this study suggest that other genes present in NELoc-1, in addition to netB, are required for full virulence in the chicken challenge model.

Campylobacter jejuni is the leading bacterial cause of human enteritis in developed countries. Chicken is the major animal reservoir of C. jejuni and a powerful infection model for human campylobacteriosis. No commercial vaccine against C. jejuni is available to date. The high affinity iron acquisition mediated through enterobactin (Ent), a small siderophore, plays a critical role in the colonization of C. jejuni in the intestine. Recently, an innovative Ent conjugate vaccine has been demonstrated to induce high-level of Ent-specific antibodies in rabbits; the Ent-specific antibodies displayed potent binding ability to Ent and inhibited Ent-dependent growth of C. jejuni. In this study, using specific-pathogen-free (SPF) chickens, we performed three trials to evaluate the immunogenicity of the Ent conjugate vaccine and its efficacy to control C. jejuni colonization in the intestine. The purified Ent was conjugated to the carrier keyhole limpet hemocyanin (KLH). Intramuscular immunization of chickens with the Ent–KLH conjugate for up to three times did not affect the body weight gain, the development of major immune organs and the gut microbiota. In the first two trials, immunizations of chickens with different regimens (two or three times of vaccination) consistently induced strong Ent-specific immune response when compared to control group. Consistent with the high-level of systemic anti-Ent IgG, C. jejuni colonization was significantly reduced by 3–4 log10 units in the cecum in two independent vaccination trials. The third trial demonstrated that single Ent–KLH vaccination is sufficient to elicit high level of systemic Ent-specific antibodies, which could persist for up to eight weeks in chickens. Taken together, the Ent–KLH conjugate vaccine could induce high-level of Ent-specific antibodies in chickens and confer host protection against C. jejuni colonization, which provides a novel strategy for Campylobacter control in poultry and humans.

A multiplex (m)PCR and a PCR followed by restriction fragment length polymorphism (RFLP) analysis of Avibacterium paragallinarum have been proposed as alternatives to conventional serotyping by the Page scheme. We evaluated both methods, and also sequenced the PCR-RFLP target fragment to reexamine the capacity of molecular serotyping. Eleven reference strains and 27 field isolates were used. Many reference strains and isolates were misidentified as Page serogroup B. The sequence analysis revealed 6 profiles based on the matching rates of the target sequence with the 3 reverse primers of the mPCR. The reference strains and field isolates in profiles 1 and 4 were correctly identified as serogroup A or C by the mPCR. The strains and/or isolates in profiles 2, 3, 5, and 6 could be misidentified as serogroup B or as nontypeable by the mPCR. The homology comparison of the sequences showed that the target sequence of the mPCR, called region 2, was not Page serogroup specific, although some Kume serovars, such as A-1 and C-2, were correctly serotyped. In addition, there was a 9 nucleotide deletion in the sequences of profiles 1, 3, and 5, but not of profiles 2, 4, and 6. Overall, we confirmed that the mPCR and PCR-RFLP molecular assays are not suitable for identifying the serogroups of A. paragallinarum isolates. With further study, analysis of region 2 sequences may have potential as a means of recognizing the Kume serovars of A. paragallinarum isolates.

Vaccination has been regarded as the most effective way to reduce death and morbidity caused by infectious diseases in the livestock industry. In this study, plasma activated water (PAW) was introduced to prepare the inactivated Newcastle disease vaccine. Humoral immune response was tested by hemagglutination inhibition (HI) assay and enzyme-linked immunosorbent assay (ELISA). In addition, cell-mediated immune response was evaluated by lymphocyte proliferation assay and flow cytometry. The results demonstrated that the vaccine prepared by PAW at appropriate volume ratio could induce similar antibody titers in specific pathogen-free (SPF) chickens compared with the formaldehyde-inactivated vaccine. The challenge experiment further confirmed that the vaccine prepared by PAW conferred solid protection against virulent NDV. Moreover, it was found that the vaccine could promote the proliferation of lymphocytes and stimulate cell-mediated immunity of SPF chickens. Furthermore, analysis of electron spin resonance (ESR) spectroscopy and physicochemical properties of PAW suggested reactive oxygen and nitrogen species (RONS) played an essential role in the virus inactivation. Therefore, this study indicated that NDV treated by PAW in an appropriate ratio retained immunogenicity on the premise of virus inactivation. PAW as a promising strategy could be used to prepare inactivated vaccine for Newcastle disease.

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