The flow of calcium ions (Ca2+) is involved in numerous vital activities of Toxoplasma gondii. Calreticulin is a type of Ca2+-binding protein in the endoplasmic reticulum (ER), that is involved in Ca2+ signaling pathway regulation, Ca2+ storage, and protein folding. In this work, the calreticulin (CALR), a protein predicted to possess a conserved domain of calreticulin in T. gondii, was characterized. The CALR localized in the ER. Using reverse genetics, we discovered that CALR is not necessary for the lytic cycle, including invasion and replication. However, depletion of CALR affected microneme secretion triggered by A23187, which is a Ca2+ ionophore used to increase cytoplasmic Ca2+ concentration. Furthermore, we discovered that CALR influences Ca2+ release. Transcriptomic comparison between Δcalr and Δku80 parasites showed that 226 genes in the Δcalr parasites were significantly down-regulated (p<0.05). The cellular biological functions of the down-regulated genes were mainly involved in calmodulin-dependent protein kinase pathways. Furthermore, in the absence of CALR, tachyzoites were still able to cause acute infection in mice. These results imply that by influencing ER Ca2+ release content, CALR may further impair the ionophore-induced secretion of the parasite. However, this protein is not required for the completion of the parasite's lytic cycle or for the acute virulence of the parasite.

While canonical Wnt signaling is well recognized for its crucial regulatory functions in cell fate decisions, the role of non-canonical Wnt signaling in adult stem cells remains elusive and contradictory. Here, we identified Mcam , a potential member of the non-canonical Wnt signaling, as an important negative regulator of mammary gland epithelial cells (MECs) by genome-scale CRISPR-Cas9 knockout (GeCKO) library screening. Loss of Mcam increases the clonogenicity and regenerative capacity of MECs, and promotes the proliferation, differentiation, and ductal morphogenesis of mammary epithelial in knockout mice. Mechanically, Mcam knockout recruits and polarizes macrophages through the Il4-Stat6 axis, thereby promoting secretion of the non-canonical Wnt ligand Wnt5a and its binding to the non-canonical Wnt signaling receptor Ryk to induce the above phenotypes. These findings reveal Mcam roles in mammary gland development by orchestrating communications between MECs and macrophages via a Wnt5a/Ryk axis, providing evidences for non-canonical Wnt signaling in mammary development.

Botryosphaeria dothidea infects hundreds of woody plants and causes a severe economic loss to apple production. In this study, we characterized BdLM1, a protein from B. dothidea that contains one LysM domain. BdLM1 expression was dramatically induced at 6 h post-inoculation in wounded apple fruit, strongly increased at 7 d post-inoculation (dpi), and peaked at 20 dpi in intact shoots. The knockout mutants of BdLM1 had significantly reduced virulence on intact apple shoots (20%), wounded apple shoots (40%), and wounded apple fruit (40%). BdLM1 suppressed programmed cell death caused by the mouse protein BAX through Agrobacterium-mediated transient expression in Nicotiana benthamiana, reduced H2O2 accumulation and callose deposition, downregulated resistance gene expression, and promoted Phytophthora nicotianae infection in N. benthamiana. Moreover, BdLM1 inhibited the active oxygen burst induced by chitin and flg22, bound chitin, and protected fungal hyphae against degradation by hydrolytic enzymes. Taken together, our results indicate that BdLM1 is an essential LysM effector required for the full virulence of B. dothidea and that it inhibits plant immunity. Moreover, BdLM1 could inhibit chitin-triggered plant immunity through a dual role, i.e., binding chitin and protecting fungal hyphae against chitinase hydrolysis.

Background Uridyl peptide compounds are renowned as a subclass of nucleoside antibiotics for their highly specific antibacterial activity against Gram-negative bacteria and the unique target of action. We previously activated the biosynthetic gene cluster of a uridyl peptide antibiotic, mureidomycin, in Streptomyces roseosporus NRRL 15998 by introducing an exogenous positive regulator gene ssaA , and the generated strain was designated as Sr-hA. This study aims to further explore mureidomycin analogs from Sr-hA as well as the collaborative roles of two wide-spread genes, SSGG-02980 and SSGG-03002 encoding putative nuclease/phosphatase and oxidoreductase respectively, in mureidomycin diversification. Results In order to understand how SSGG-02980 and SSGG-03002 contribute to mureidomycin biosynthesis, the gene disruption mutants and complementary strains were constructed. Mass spectrometry analyses revealed that two series of pairwise mureidomycin analogs were synthesized in Sr-hA with a two-dalton difference in molecular weight for each pair. By disruption of SSGG-03002 , only mureidomycins with lower molecular weight (MRDs, 1 – 6 ) could be specifically accumulated in the mutant (∆03002-hA), whereas the other series of products with molecular weight plus 2 Da (rMRDs, 1ʹ – 6ʹ ) became dominant in SSGG-02980 disruption mutant (∆02980-hA). Further comprehensive NMR analyses were performed to elucidate the structures, and three MRDs ( 3 , 4 , 5 ) with unsaturated double bond at C5-C6 of uracil group were characterized from ∆03002-hA. In contrast, the paired rMRDs analogs ( 3ʹ , 4ʹ , 5ʹ ) from ∆SSGG-02980 corresponding to 3 , 4 and 5 were shown to contain a single bond at this position. The results verified that SSGG-03002 participates in the reduction of uracil ring, whereas SSGG-02980 antagonizes the effect of SSGG-03002, which has been rarely recognized for a phosphatase. Conclusions Overall, this study revealed the key roles of two wide-spread families of enzymes in Streptomyces . Of them, oxidoreductase, SSGG-03002, is involved in dihydro-mureidomycin biosynthesis of S. roseosporus , whereas nuclease/phosphatase, SSGG-02980, has an adverse effect on SSGG-03002 . This kind of unusual regulation model between nuclease/phosphatase and oxidoreductase is unprecedented, providing new insights into the biosynthesis of mureidomycins in Streptomyces . The findings would be of significance for structural diversification of more uridyl peptide antibiotics against Gram-negative bacteria.

Sarcocystis spp., Neospora caninum and Toxoplasma gondii are globally ubiquitous pathogens, and domestic sheep are considered to be one of the intermediate hosts. 83 myocardial samples of sheep were collected from 12 retail stores in Beijing, China. Sarcocystis spp., N. caninum and T. gondii were identified by PCR amplification of the 18S rRNA gene, Nc-5 gene and 529bp DNA fragment with a prevalence of 86.7% (95% CI: 77.5–93.2) and 43.4% (95% CI: 32.5–54.7) for Sarcocystis spp. and N. caninum infections, respectively, and no T. gondii was detected. The co-infection prevalence of Sarcocystis and N. caninum was 38.6% (95% CI: 28.1–49.9). Two Sarcocystis species were subtyped by analyzing 18SrRNA sequences and were identified as Sarcocystis tenella and Sarcocystis arieticanis. The prevalence of S. tenella and S. arieticanis infections was 84.3% (95% CI: 74.7–91.4) and 56.6% (95% CI: 45.3–67.5), respectively. This study shows that sheep have a high risk of infection with Sarcocystis and N. caninum, suggests that effective prevention measures are needed to avoid the spread of these parasites in sheep. Toxoplasmosis in sheep poses a threat to human and animal health and requires monitoring and preventing continuously.

CRISPR/Cas9 technology has been widely used for gene editing in organisms. Gene deletion of the ku80/ku70 complex can improve the efficiency of gene replacement in Arabidopsis thaliana, Cryptococcus neoformans, and Toxoplasma gondii, which remained elusive in Neospora caninum. Here, we knock out the ku80 gene in Nc1 strain by using CRISPR/Cas9, detect the growth rate and virulence of NcΔku80. Then we compare the efficiency of gene replacements between NcΔku80 and Nc1 strains by transfected with the same HA-tagged plasmids, and the percentage of HA-tagged parasites was investigated by IFA. The results showed that gene targeting efficiency was increased in the NcΔku80 strain via double crossover at several genetic loci, but its growth rate and virulence were unaffected. In conclusion, the NcΔku80 strain can be used as an effective strain for rapid gene editing of N. caninum.

Cancers appear to escape surveillance by the immune system at least in part because they fail to induce a protective immune response. Therapeutic vaccines based on specific tumour antigens and tumour cells modified ex vivo by genetic techniques are but two strategies being used to circumvent this problem. In this report, we describe a simple, yet effective alternative in which tumour-specific responses are induced by in situ administration of a well-characterized liposomal formulation of the cytokine interleukin 2 (IL-2). Using the non-immunogenic B16 melanoma model, intratumoural injections of liposomal IL-2 L(IL2), were shown to induce a long-lived immune response specific for the injected tumour. In conjunction with subsequent removal of the primary tumours by surgery, the injections increased mean survival to 57 days from a control value of 32 days and partially protected surviving mice against re-challenge with B16. L(IL2) induced an early infiltration of inflammatory cells within the tumours which was followed several days later by an influx of CD3+ T cells. The cellular influx and a coincident decrease in tumour growth were noted in both injected tumours and a second non-injected tumour on the same animal, thereby demonstrating the systemic nature of the immune response. Intratumoural injections of soluble IL-2 at the same dose failed to induce B16-specific cellular immunity or to prolong survival of the mice. Thus, liposomal formulation of the cytokine was fundamental to successful induction of immunity by this in situ vaccination regimen.

Maternally derived IgG antibodies to protective Eimeria antigens have great potential to control chicken coccidiosis and multivalent vaccines are more practical to resist against co-infection with several species of Eimeria under natural conditions. In this study, five good protective antigens of Eimeria species were combined into two combinations based on previous studies, namely C1(EtROPK-Eten5-A, EtGAM22, Ea3-1E and EmGAM56) and C2(EtM2AP and EtGAM22, Ea3-1E and EmGAM56). Then, five antigens were expressed in the Escherichia coli system and purified to inoculate breeding hens. After three times immunization, the specific antibodies could sustain for 11 and 10 weeks in hens' plasma and egg yolk, respectively. Moreover, maternally derived antibodies against recombinant proteins could retain for 14 days in hatchlings' serum. Then, protective efficacies of specific antibodies on hatchlings against mixed infection of E. tenella, E. acervulina and E. maxima were evaluated. The results showed that the hatchlings of the immunized hens had a higher survival rate on day 7 of hatching. Moreover, body weight gains within the hatchlings of immunized hens were higher than those of unvaccinated hens on 7 days (C1: p = 0.0744; C2: p = 0.4020) and 14 days (p < 0.0001). Moreover, hatchlings from vaccinated hens showed significantly alleviated lesion scores in the small intestine and duodenum at day 7 (p < 0.01) and day 14 (C1: p < 0.05). Particularly, the number of oocyst excretion from hatchlings of immunized hens was significantly reduced at day 7 (p < 0.0001) and day 14 (p < 0.0001). Our findings suggest that the maternal immunization with multivalent recombinant vaccines has the potential to be transmission blocking vaccines against mixed infection of Eimeria.

Neospora caninum, an intracellular protozoan, has attracted much attention because of the etiology of reproductive disorders in cattle. Although numerous relevant studies have been published, the overall prevalence of Neospora caninum infection among cattle in mainland China is still unknown, and associated risk factors need to be evaluated to establish preventive measures. We systematically searched four databases for epidemiologic studies that investigated the prevalence and associated risk factors of this parasite among cattle in mainland China. A total of 33,945 cattle from 51 studies were tested. The pooled prevalence of Neospora caninum among cattle in mainland China was 13.69% (95% CI: 10.86%-17.12%) through the random-effects model, which showed high heterogeneity, I-2 = 98%. The analysis showed that Neospora infection-positive cows were 2.42 times more likely to abort than infection-negative cows (OR 2.42; 95% CI: 2.07-2.84). Our analysis indicated that Neospora-infection is widely distributed among cattle in China, which reinforces the importance of adequate prevention and control strategies to reduce economic losses for the cattle industry.

Toxoplasma gondii is an obligate intracellular apicomplexan parasite causing lethal diseases in immunocompromised patients. UBL-UBA shuttle proteins (DDI1, RAD23, and DSK2) are important components of the ubiquitin-proteasome system. By degrading ubiquitinated proteins, UBL-UBA shuttle proteins regulate many cellular processes. However, the specific processes regulated by UBL-UBA shuttle proteins remain elusive. Here, we revealed that the deletion of shuttle proteins results in a selective accumulation of ubiquitinated proteins in the nucleus and aberrant DNA replication. ROP18 was mistargeted and accumulated in the shuttle protein mutant strain, resulting in the recruitment of immunity-related GTPases to the parasitophorous vacuole membrane (PVM). Furthermore, the mistargeting of ROP18 and the recruitment of Irgb6 to the PVM were also observed in the DDI1 mutant strain. DDI1 is a nonclassical UBL-UBA shuttle protein homologous to the HIV-1 protease. Molecular docking showed that DDI1 was a potential target of HIV-1 protease inhibitors. However, these inhibitors blocked the growth of T gondii in vitro but not in vivo. In conclusion, the Toxoplasma UBL-UBA shuttle protein regulates several important cellular processes and the mistargeting of ROP18 may be a representative of the abnormal homeostasis caused by shuttle protein mutation.

Toxoplasma gondii (T. gondii) is an important human and veterinary pathogen causing life-threatening disease in immunocompromised patients. The UBL-UBA shuttle protein family are important components of the ubiquitin–proteasome system. Here, we identified a novel UBL-UBA shuttle protein DSK2b that is charactered by an N-terminal ubiquitin-like domain (UBL) and a C-terminal ubiquitin-associated domain (UBA). DSK2b was localized in the cytoplasm and nucleus. The deletion of dsk2b did not affect the degradation of ubiquitinated proteins, parasite growth in vitro or virulence in mice. The double-gene knockout of dsk2b and its paralogs dsk2a (ΔΔdsk2adsk2b) results in a significant accumulation of ubiquitinated proteins and the asynchronous division of T. gondii. The growth of ΔΔdsk2adsk2b was significantly inhibited in vitro, while virulence in mice was not attenuated. In addition, autophagy occurred in the ΔΔdsk2adsk2b, which was speculated to degrade the accumulated ubiquitinated proteins in the parasites. Overall, DSK2b is a novel UBL-UBA shuttle protein contributing to the degradation of ubiquitinated proteins and is important for the synchronous cell division of T. gondii.

Glutaredoxins (GRXs), important components of the intracellular thiol redox system, are involved in multiple cellular processes. In a previous study, we identified five GRXs in the apicomplexan parasite, Neospora caninum. In the present study, we confirmed that the GRXs S14 and C5 are located in the apicoplast, which suggests unique functions for these proteins. Although single-gene deficiency did not affect the growth of parasites, a double knockout (Δgrx S14Δgrx C5) significantly reduced their reproductive capacity. However, there were no significant changes in redox indices (GSH/GSSG ratio, reactive oxygen species and hydroxyl radical levels) in double-knockout parasites, indicating that grx S14 and grx C5 are not essential for maintaining the redox balance in parasite cells. Key amino acid mutations confirmed that the Cys203 of grx S14 and Cys253/256 of grx C5 are important for parasite growth. Based on comparative proteomics, 79 proteins were significantly downregulated in double-knockout parasites, including proteins mainly involved in the electron transport chain, the tricarboxylic acid cycle and protein translation. Collectively, GRX S14 and GRX C5 coordinate the growth of parasites. However, considering their special localization, the unique functions of GRX S14 and GRX C5 need to be further studied.

Background: The CRISPR/Cas9 technology based on homologous recombination has been widely used for gene editing in organism. Gene deletion of ku80 complex can increase the efficiency of homologous recombination(HR), or gene editing by CRISPR/Cas9 in Arabidopsis thaliana and Toxoplasma gondii , which has remained elusive in Neospora caninum . Methods: Here, we knocked out the ku80 in Nc1 strain, named Δku80 , using CRISPR/Cas9. In vitro, phenotype assays, including plaque formation, invasion, replication and egress were carried out to determination of the growth of Δku80 . In vivo, we respectively injected BALB/c mice with different doses of NcKU80 and Nc1, and measured the survival curves and parasites burdens of mice infected. To test the efficiency of HR, Δku80 and Nc1 strains were transfected with the same HA-tagged plasmids, and the percentage of parasites with HA-tagged was investigated. Results: It is showed that the growth and virulence of Δku80 kept unaffected, while the efficiency of gene targeting via double-crossover at several genetic loci was increased 2 to 3 fold in Δku80 . Conclusions: It is concluded that Δku80 can be used as an effective strain for rapid gene editing in N. caninum .

Coccidiosis, caused by parasites of the genus Eimeria, is one of the most widespread and economically detrimental diseases in the global poultry industry. Because the merozoite stage of Eimeria tenella is immunologically vulnerable, motile, and functionally important for the parasites, the proteins expressed in these stages are considered to be potentially immunoprotective antigens, especially the secreted antigens and surface antigens. Here, we detected a previously unidentified MIC2-associated protein (Et-M2AP) from E. tenella and determined its localization. An immunofluorescence assay revealed that Et-M2AP was distributed in the apical part of second generation merozoites and sporozoites. In addition, an expression profile analysis revealed that the transcriptional level of Et-M2AP is significantly higher in the merozoite stage. To assess the potential of Et-M2AP protein as a coccidiosis vaccine, we expressed recombinant Et-M2AP (rEt-M2AP) and compared the immune protective efficacy of rEt-M2AP with 3 surface antigens that are highly expressed by merozoites (rEt-SAG23, rEt-SAG16, and rEt-SAG2 proteins). The immune protective efficacy of these vaccine candidates was assessed based on survival rate, lesion score, BW gain, relative BW gain, and oocyst output. The results show that the survival rate was 90%, which are significantly higher than those in the challenge control group. The BW gain rate was 42% (P < 0.001) in rEt-M2AP–immunized chickens, which are significantly higher than those in the challenge control group and rEt-SAG23, rEt-SAG16, and rEt-SAG2 proteins–immunized chickens. In addition, chickens immunized with rEt-M2AP (88% oocyst output decrease rate, P < 0.001) had the least oocyst output, compared with those immunized with rEt-SAG16 (59.2% oocyst output decrease rate, P < 0.001), rEt-SAG23 (22% oocyst output decrease rate), and rEt-SAG2 (1.36% oocyst output decrease rate). These results demonstrate that rEt-M2AP provided effective protection against challenge with E. tenella, suggesting that rEt-M2AP is a promising candidate antigen gene for development as a coccidiosis vaccine.

Toxoplasma gondii is an important zoonotic protozoan of the phylum Apicomplexa that can infect nearly all warm-blooded animals. The parasite can exist as the interconvertible tachyzoite or bradyzoite forms, leading to acute or latent infection, respectively. No drug has been reported to penetrate the cyst wall and reduce bradyzoite survival and proliferation till now. The transcriptional level of metacaspases 2 (TgMCA2) in T. gondii is significantly upregulated during the formation of bradyzoites in the Pru strain, indicating that it may play an important role in the formation of bradyzoites. To further explore the function of TgMCA2, we constructed a TgMCA2 gene-knockout variant of the Pru strain (Δ mca2 ). Comparative analysis revealed that the proliferative capacity of Pru Δ mca2 increased, while the invasion and egressing properties were not affected by the knockout. Further data shows that the tachyzoites of Δ mca2 failed to induce differentiation and form bradyzoites in vitro, and the transcriptional levels of some of the bradyzoite-specific genes (such as BAG1, LDH2, and SAG4A) in Δ mca2 were significantly lower compared with that in the Pru strain at the bradyzoite stage. In vivo, no cysts were detected in Δ mca2 -infected mice. Further determination of parasite burden in Δ mca2 - and Pru-infected mice brain tissue at the genetic level showed that the gene load was significantly lower than that in Pru. In summary, we confirmed that TgMCA2 contributes to the formation of bradyzoites, and could provide an important foundation for the development of attenuated vaccines for the prevention of T. gondii infection.

Neospora caninum is an obligate intracellular protozoan parasite that infects a wide range of mammalian species and causes spontaneous abortion in cattle. N. caninum is exposed to oxidative stress during its life cycle. Oxidoreductase is crucial for parasite response to the environmental stresses. Glutaredoxins (Grxs) are small oxidoreductases of the thioredoxin family proteins that catalyze thiol-disulfide exchange reactions by utilizing electrons from the tripeptide glutathione (γGlu-Cys-Gly; GSH). Grxs are key elements in redox signaling and cell signal transduction. However, Grxs are an unexplored set of oxidoreductases in N. caninum. Here, we identified two cytoplasm located glutaredoxin domain-containing proteins (NcGrx1 and NcGrx3) in N. caninum. To better understand the functions of these Grx proteins, we generated NcGrx1 and NcGrx3 deficiency and overexpression strains. The deletion or overexpression of NcGrx3 had no significant effect on the growth of N. caninum in vitro and in vivo. NcGrx1 knockout parasites displayed a significant growth defect, which was due to the influence on invasion and egress abilities. Moreover, NcGrx1 deficiency decreased the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) (GSH/GSSG ratio), caused a significant accumulation of hydroxyl radical in parasites, and an increase in apoptotic cells under oxidative stress (H2O2) condition. To determine the cause of growth defects in ΔNcGrx1, we examined the transcription levels of various invasion-egress related genes as measured by qPCR. We found a significant decrease in MIC1, MIC4, and MIC6 genes. Further investigation found that the secretion of MIC1, MIC4, and MIC6 proteins was significantly affected. Collectively, Ncgrx1 is important for microneme protein-mediated parasite growth, and maybe a potential intervention target for the N. caninum.

Eimeria tenella (E. tenella) is a highly pathogenic and prevalent species of Eimeria that infects chickens, and it causes a considerable disease burden worldwide. The secreted proteins and surface antigens of E. tenella at the sporozoite stage play an essential role in the host–parasite interaction, which involves attachment and invasion, and these interactions are considered vaccine candidates based on the strategy of cutting off the invasion pathway to interrupt infection. We selected two highly expressed surface antigens (SAGs; Et-SAG13 and Et-SAG) and two highly expressed secreted antigens (rhoptry kinases Eten5-A, Et-ROPK-Eten5-A and dense granule 12, Et-GRA12) at the sporozoite stage. Et-ROPK-Eten5-A and Et-GRA12 were two unexplored proteins. Et-ROPK-Eten5-A was an E. tenella-specific rhoptry (ROP) protein and distributed in the apical pole of sporozoites and merozoites. Et-GRA12 was scattered in granular form at the sporozoite stage. To evaluate the potential of rEt-ROPK-Eten5-A, rEt-GRA12, rEt-SAG13 and rEt-SAG proteins as a coccidiosis vaccine, the protective efficacy was examined based on survival rate, lesion score, body weight gain, relative body weight gain and oocyst output. The survival rate was significantly improved in rEt-ROPK-Eten5-A (100%) and rEt-GRA12 (100%) immune chickens compared to the challenged control group (40%). The average body weight gains of rEt-ROPK-Eten5-A, rEt-GRA12, rEt-SAG13 and rEt-SAG immunized chickens were significantly higher than those of unimmunized chickens. The mean lesion score and oocyst output of the rEt-ROPK-Eten5-A immunized chickens were significantly reduced compared to unimmunized challenged chickens. These results suggest that the rEt-ROPK-Eten5-A protein effectively triggered protection against E. tenella in chickens and provides a useful foundation for future work developing anticoccidial vaccines.

The α-galactosidase gene (galC) was cloned from Aspergillus oryzae YZ1 and expressed in Pichia pastoris. The galC (2319 bp) containing two introns encoded a protein of 726 amino acids. The activity of the α-galactosidase (GalC) increased 1-fold after coding sequence optimization. Purified GalC exhibited a single protein band (100 kDa) in SDS-PAGE. The optimum pH and temperature of GalC were pH 4.66 and 50 °C, respectively. Like many GH36 family α-galactosidases, GalC displayed its activities towards raffinose and stachyose. The Km values for pNPG, raffinose and stachyose were 2.16, 4.63 and 8.54 mM, respectively. The GalC retained about 90% activity within the pH range 3.0-8.0. The activity of GalC was inhibited by Cu2+, while Ca2+ increased the enzyme activity. Different concentrations of glucose, mannose, galactose, xylose and sucrose slightly affected the activity of GalC. The GalC displayed strong resistance to trypsin, α-chymotrypsin, and proteinase K. Under simulated gastric conditions, GalC maintained most of its native activity after pepsin treatment for 3 h. The GalC could also effectively degrade raffinose and stachyose in soymilk. The GalC with high hydrolysis efficiency towards raffinose family oligosaccharides (RFOs) and strong resistance to proteases is considered to have great potential in food and feed industries.

Insects can produce various antimicrobial peptides (AMPs) upon immune stimulation. One class of AMPs are characterized by their high proline content in certain fragments. They are generally called proline-rich antimicrobial peptides (PrAMPs). We previously reported the characterization of Spodoptera litura lebocin-1 (SlLeb-1), a PrAMP proprotein. Preliminary studies with synthetic polypeptides showed that among the four deductive active fragments, the C-terminal fragment SlLeb-1 (124-158) showed strong antibacterial activities. Here, we further characterized the antibacterial and antifungal activities of 124-158 and its four subfragments: 124-155, 124-149, 127-158, and 135-158. Only 124-158 and 127-158 could agglutinate bacteria, while 124-158 and four subfragments all could agglutinate Beauveria bassiana spores. Confocal microscopy showed that fluorescent peptides were located on the microbial surface. Fragment 135-158 lost activity completely against Escherichia coli and Staphylococcus aureus, and partially against Bacillus subtilis. Only 124-149 showed low activity against Serratia marcescens. Negative staining, transmission, and scanning electron microscopy of 124-158 treated bacteria showed different morphologies. Flow cytometry analysis of S. aureus showed that 124-158 and four subfragments changed bacterial subpopulations and caused an increase of DNA content. These results indicate that active fragments of SlLeb-1 may have diverse antimicrobial effects against different microbes. This study may provide an insight into the development of novel antimicrobial agents.

Infectious coryza (IC), an acute respiratory disease of chickens, is caused by Avibacterium paragallinarum. Here, the current epidemiological status of IC was investigated in China over 5 yr (2013 to 2018). A total of 28 Av. paragallinarum field isolates were identified by PCR tests and by sequence analysis of the hemagglutinin gene. The pathogenicities of 4 field isolates, the efficacy of 2 commercial inactivated oil-emulsion IC vaccines and vaccines containing different Av. paragallinarum isolates were also evaluated. The PCRs revealed a high rate (51.5%) of sample positivity for Av. paragallinarum during 2013 to 2018. Phylogenetic analysis showed that most field strains fell into the same cluster and had a farther genetic relationship with the early isolates from China. Pathogenicity testing revealed that the Chinese Av. paragallinarum isolates were able to induce the typical clinical signs of IC; hence, they were clearly pathogenic to chickens. Vaccine efficacy tests revealed that the 2 commercial inactivated oil-emulsion IC vaccines we tested had low protection rates against 2 selected Av. paragallinarum isolates after a single immunization, whereas the inactivated vaccine containing the Av. paragallinarum BJ26 isolate generated a relatively high protection rate against the field isolates compared with other three tested vaccines. The results indicate that IC is currently prevalent in China, and that commercial vaccines have not counteracted its presence in this country.