Phagocytic Plasma Cells in Teleost Fish Provide Insights into the Origin and Evolution of B Cells in Vertebrates.

Teleost IgM+ B cells can phagocytose, like mammalian B1 cells, and secrete Ag-specific IgM, like mammalian B2 cells. Therefore, teleost IgM+ B cells may have the functions of both mammalian B1 and B2 cells. To support this view, we initially found that grass carp (Ctenopharyngodon idella) IgM+ plasma cells (PCs) exhibit robust phagocytic ability, akin to IgM+ naive B cells. Subsequently, we sorted grass carp IgM+ PCs into two subpopulations: nonphagocytic (Pha-IgM+ PCs) and phagocytic IgM+ PCs (Pha+IgM+ PCs), both of which demonstrated the capacity to secrete natural IgM with LPS and peptidoglycan binding capacity. Remarkably, following immunization of grass carp with an Ag, we observed that both Pha-IgM+ PCs and Pha+IgM+ PCs could secrete Ag-specific IgM. Furthermore, in vitro concatenated phagocytosis experiments in which Pha-IgM+ PCs from an initial phagocytosis experiment were sorted and exposed again to beads confirmed that these cells also have phagocytic capabilities, thereby suggesting that all teleost IgM+ B cells have phagocytic potential. Additionally, we found that grass carp IgM+ PCs display classical phenotypic features of macrophages, providing support for the hypothesis that vertebrate B cells evolved from ancient phagocytes. These findings together reveal that teleost B cells are a primitive B cell type with functions reminiscent of both mammalian B1 and B2 cells, providing insights into the origin and evolution of B cells in vertebrates.

Identification and functional analyses of CD4-1+ cells in grass carp (Ctenopharyngodon idella).

In mammals, CD4 is found to be expressed on T cells and innate immune cells, however, teleost cells bearing CD4 have not been well identified and characterized. In this study, we identified two different CD4-1+ cell subsets in grass carp (Ctenopharyngodon idella): CD4-1+ lymphocytes (Lym) and CD4-1+ myeloid cells (Mye), both of which had the highest proportions in the head kidney. The mRNA expression analysis showed that CD4-1, CD4-2, TCRß, CD3γ/δ, and LCK1 are highly expressed in CD4-1+ Lym and also expressed in CD4-1+ Mye. Furthermore, we found that CD4-1+ Lym have a Lym morphology and highly express T-cell cytokines, suggesting that they are CD4+ T cells equivalent to mammalian Th cells. On the other hand, CD4-1+ Mye were found to have a morphology of macrophage and highly express macrophage marker gene MCSFR, indicating that they are macrophages. In addition, functional analysis revealed that CD4-1+ Mye possess phagocytic ability and great antigen-processing ability. Taken together, our study sheds further light on the composition and function of CD4+ cells in teleost fish.

Antimicrobial Protein LECT2-b Helps Maintain Gut Microbiota Homeostasis via Selectively Targeting Certain Pathogenic Bacteria.

Antimicrobial peptides/proteins (AMPs) constitute a critical component of gut immunity in animals, protecting the gut from pathogenic bacteria. However, the interactions between AMPs and gut microbiota remain elusive. In this study, we show that leukocyte-derived chemotaxin-2 (LECT2)-b, a recently discovered AMP, helps maintain gut homeostasis in grass carp (Ctenopharyngodon idella), one of the major farmed fish species globally, by directly regulating the gut microbiota. Knockdown of LECT2-b resulted in dysregulation of the gut microbiota. Specifically, LECT2-b deficiency led to the dominance of Proteobacteria, consisting of proinflammatory bacterial species, over Firmicutes, which includes anti-inflammatory bacteria. In addition, the opportunistic pathogenic bacteria genus Aeromonas became the dominant genus replacing the probiotic bacteria Lactobacillus and Bacillus. Further analysis revealed that this effect was due to the direct and selective inhibition of certain pathogenic bacterial species by LECT2-b. Moreover, LECT2-b knockdown promoted biofilm formation by gut microbiota, resulting in tissue damage and inflammation. Importantly, LECT2-b treatment alleviated the negative effects induced by LECT2-b knockdown. These findings highlight the crucial role of LECT2-b in maintaining the gut microbiota homeostasis and mucosal health. Overall, our study provides important data for understanding the roles of AMPs in the regulation of gut homeostasis in animals.

Vector-Borne Pathogens with Veterinary and Public Health Significance in Melophagus ovinus (Sheep Ked) from the Qinghai-Tibet Plateau.

Melophagus ovinus (sheep ked) is a hematophagous ectoparasite that mainly parasitizes sheep. In addition to causing inflammation, wool loss, and skin damage to the animal hosts, M. ovinus also serves as a vector for a variety of pathogens and is highly likely to participate in the life and transmission cycle of pathogenic organisms. Herein, we investigated the presence and molecular characterization of vector-borne pathogens in M. ovinus from Qinghai-Tibet Plateau, China. A total of 92 M. ovinus pools collected from the Qinghai province of China were screened for the presence of selected vector-borne pathogens. The overall positive rate of A. ovis, A. bovis, A. phagocytophilum, and T. ovis in M. ovinus was 39.1%, 17.4%, 9.8%, and 89.1%, respectively. All of the samples were negative for Border disease virus (BDV), other Anaplasma species, Babesia spp., Rickettsia spp., and Borrelia spp. Co-infection of different Anaplasma species and T. ovis occurred in 51.2% of all samples with T. ovis. The positive rates of A. ovis, A. bovis, and A. phagocytophilum in different regions and altitudes of the sampling sites were significantly different. Sequence and phylogenetic analysis of target genes confirmed their identity with corresponding pathogens. Our results elucidate the occurrence and molecular characterization of Anaplasma spp. and Theileria spp. in M. ovinus, which could act as potential zoonotic reservoirs. To the best of our knowledge, this is the first report of the detection of A. bovis and A. phagocytophilum DNA in M. ovinus. This study gives the first extensive molecular survey of vector-borne pathogens with veterinary and public health significance in M. ovinus from the Qinghai-Tibet Plateau, China.

Identification and characterisation of tumour necrosis factor receptor (TNFR) associated factor 3 from Nile tilapia, Oreochromis niloticus.

In this study, we cloned the complementary (c)DNA sequences of tumour necrosis factor receptor (TNFR)-associated factor 3 (traf3) in Nile tilapia, Oreochromis niloticus. The expression patterns of the traf3 gene were investigated and preliminary functional analyses were performed. In healthy fish, traf3 transcript was broadly expressed in all examined tissues, with the highest expression level in the blood and the lowest in the liver. The traf3 gene reached its highest expression at 8 days post-fertilisation (dpf) during embryonic development. Moreover, we found that expression of traf3 was clearly altered following stimulation with Streptococcus agalactiae in vivo and that traf3 could be induced by lipopolysaccharides (LPS), Poly I: C and S. agalactiae WC1535 in Nile tilapia macrophages. Overexpression in 293T cells showed that Traf3 protein was mainly distributed in the cytoplasm and could significantly increase nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. Taken together, these results implied that traf3 could play important roles in the immune response to pathogen invasion.

Establishment and optimization of a liquid bead array for the simultaneous detection of ten insect-borne pathogens.

BACKGROUND: Insect-borne diseases could induce severe symptoms in human and clinical signs in animals, such as febrility, erythra, arthralgia and hemorrhagic fever, and cause significant economic losses and pose public health threat all over the world. The significant advantages of Luminex xMAP technology are high-throughput, high parallel and automation. This study aimed to establish a liquid bead array based on Luminex xMAP technology that was able to simultaneously detect multiple insect-borne pathogens. METHODS: Specific probes and primers to detect the nucleic acid of 10 insect-borne pathogens were designed. Probes were coupled with fluorescent carboxylated microspheres. The parameters of the system were optimized, including ratio of forward/reverse primers (1:2), hybridization temperature (50 °C) and duration (30 min) and quantity of PCR product (2 µl). The sensitivity and specificity of the system were also evaluated. Moreover mixed nucleic acid of 10 insect-borne pathogens, including Bluetongue virus, Epizootic hemorrhagic disease virus of deer, Coxiella burnetii, African swine fever virus, West Nile fever virus, Borrelia burgdorferi, vesicular stomatitis virus, Rift Valley fever virus, Ebola virus and Schmalenberg's disease virus, and 3000 clinical samples were tested for practicability. RESULTS: The optimized detection system showed high sensitivity, specificity and reproducibility. Each probe showed specific fluorescence signal intensity without any cross-hybridization for the other insect-borne pathogens tested, which included dengue virus, tick-borne encephalitis virus, Japanese encephalitis virus, Xinjiang hemorrhagic fever virus, spotted fever group rickettsiae, ehrlichiae and chikungunya virus. The limit of detection was 10 copies of target gene. Insect-borne pathogens were successfully detected among the 3000 clinical samples, and the results were consistent with those obtained using gold-standard assays or commercial nucleic acid detection kits. CONCLUSIONS: This optimized liquid array detection system was high-throughput and highly specific and sensitive in screening of the insect-borne pathogens. It was promising in detection of these pathogens for molecular epidemiological studies.

[Prokaryotic expression, protein purification and reactivity analysis of the VP1 and its C terminus of a South African isolate of FMDV serotype SAT2].

AIM: To induce the expression of structure protein VP1 and its C terminus of foot-and-mouth disease virus (FMDV) serotype SAT2 in E.coli and analyze their reactivities with FMDV positive antiserum. METHODS: The plasmid pGEM-SAT2P1 carrying the VP1 coding region of FMDV serotype SAT2 isolated from South African was used as template for RT-PCR to get the coding fragment of VP1 and its C terminus. The fragments were then cloned into expression vector pET-30a(+) to get recombinant plasmids pET-SAT2VP1 and pET-SAT2VP1C. The recombinant plasmids were transformed into E.coli BL21(DE3)pLysS and induced by IPTG to express VP1 and its C terminus protein. The expressed VP1 and its C terminus were then purified by Ni-NTA His Bind Resin affinity chromatography and analyzed by Western blot. New Zealand rabbits were immunized to prepare polyclonal antibodies against VP1 and VP1C. The antisera were obtained and polyclonal antibody was characterized by ELISA. RESULTS: SDS-PAGE demonstrated that VP1and its C terminus expressed in the E.coli transformants had a molecular weight of 33000 and 19000 and contained in the inclusion body. Purified VP1 and its C terminus was obtained by Ni-NTA His Bind Resin affinity chromatography and a single clear band appeared in the SDS-PAGE gel. Western blot analysis showed that the purified VP1 and VP1 C terminus could react with bovine antiserum against the same serotype FMDV without cross-reactivity with the negative bovine serum. CONCLUSION: Rabbit polyclonal antibodies against VP1 and VP1C were successfully prepared, the titers of which were above 1:12800 and had obvious specificity.

[Expression of phosphoprotein P2 of Cysticercus cellulosae in Pichia pastoris and its application].

Cysticercosis is caused by the metacestode form of Taenia solium-Cysticercus cellulosae and it causes great economic losses and threatens the people's health. There are some problems on how to control cysticercosis, in order to resolve the key problem that the native antigen to diagnose and prevent cysticercosis is very limited and is not satisfied, Pichia pastoris Expression System was used to express recombinant P2 protein. The interested P2 gene was got by digesting the pGEM - P2 vector using restriction endonuclease, then it was inserted into the secretory pPIC9K Pichia pastoris expression vector and transformed into E. coli. Positive recombinant plasmids were selected sequenced and named pPIC9K-P2 and it was linearized by Sal I and Bgl II, then the linear DNA transfored into Pichia pastoris GS115 by electroporation. The recombinant expression vector pPIC9K - P2 integrated into GS115 via homologous recombination between the transforming DNA and regions of homology within the genome. The transformants were screened for multicopy recombinants using G418 and were distinguished for Mut phenotypes by MD and MM. Two different phenotypes were generated-HIS+ MUT+ (Methanol utilization plus) and HIS+ MUT(S) (Methanol utilization slow). PCR analysis of the multicopy recombinants indicated that the P2 gene was integrated within the genome of pichia Pastoris. The multicopy recombinants were named GS115/pPIC9K - P2HIS+ MUT+ and GS115/pPIC9K-P2HIS+ MUT(S), both HIS+ MUT+ and HIS+ MUT(S) were induced with methanol. The results of SDS-PAGE and Western blot demonstrated that the culture supernatant of the induced Pichia pastoris contained P2 protein which was accumulated up to 33 % of total proteins in the culture supernant and its molecular weight is 12.6kD. The results of the clinical study indicated that the expression P2 protein could be used to diagnose human cysticercosis and swine cysticercosis as diagnosis antigen.

[Study on the expression of E2 gene of classical swine fever virus in Pichia pastoris and the immunological activity of its expression product].

E2 gene of classical swine fever virus (CSFV) was cloned into secretory pPIC9K Pichia pastoris expression vector. After being linearized by digestion, the vector was transformed into Pichia pastoris by electroporation to integrate with the genome, the transformants with high copies were screened by G418 and were induced to express with methonal. The results of SDS-PAGE and Western blot demonstrated that the supernatant of the induced P. pastoris culture contained protein E2. The results of the study on the immunological activity indicated that the protein E2 expressed in P. pastoris can elicit animal bodies to produce antibodies against protein E2.

[Cloning of the major antigen region of E2 gene of hog cholera virus and expression in Escherichia coli].

The major antigen region of E2 gene of Hog Cholera Prevalent Strain (Guangxi Yuling Strain) and Chinese Hog Cholera Lapinised Virus (C-strain) derived from hog and rabbit spleen tissue, was amplified by reverse transcription polymerase chain reaction(RT-PCR) and the nested Polymerase Chain Reaction (nPCR). After the amplified fragments were cloned into the expression vector pPROEX-HTb, the recombinant plasmids pPROEX-GXYL and pPROEX-C were obtained. The insert position, the size and the reading frame were right by PCR, restriction digestion and the sequence analysis. SDS-PAGE indicated that both of the reciepient germs transducted and induced by the recombinant plasmids pPROEX-GXYL and pPROEX-C could express the major antigen region of E2 gene. Western-blot indicated that the expressed antigen protein could be recognized by the positive serum of CSFV.