A nosocomial Pseudomonas aeruginosa ST3495 isolated from a wild Burmese python (Python bivittatus) with suppurative pneumonia and bacteremia in Hainan, China.

Pseudomonas aeruginosa is a common infectious agent associated with respiratory diseases in boas and pythons, however, the histopathology, resistance and virulence are yet described for this species. In this study, we investigated a dying Burmese python rescued from tropical rainforest in Hainan. Clinical signs were open-mouthed breathing, abnormal shedding and anorexia. Abundant yellow mucopurulent secretions were observed in highly ectatic segmental bronchi by postmortem. Histopathological lesions included systemic pneumonia, enteritis, nephritis and carditis. P. aeruginosa was the only species isolated from heart blood, kidney, trachea and lung. The phenotype analysis demonstrated that the isolates had strong biofilm, and were sensitive to amikacin, spectinomycin, ciprofloxacin, norfloxacin and polymyxin B, moreover, the LD50 of the most virulent isolate was 2.22×105 cfu/mL in a zebrafish model. Molecular epidemiological analysis revealed that the isolates belonged to sequence type 3495, the common gene patterns were toxA + exoSYT + phzIM + plcHN in virulence and catB + blaTEM + ant (3'')-I+ tetA in resistance. This study highlights that P. aeruginosa should be worth more attention in wildlife conservation and raise the public awareness for the cross infection and cross spread between animals and human.

Complete genome sequence analysis of Edwardsiella tarda SC002 from hatchlings of Siamese crocodile.

Edwardsiella tarda is a Gram-negative, facultative anaerobic rod-shaped bacterium and the causative agent of the systemic disease "Edwardsiellosis". It is commonly prevalent in aquatic organisms with subsequent economic loss and hence has attracted increasing attention from researchers. In this study, we investigated the complete genome sequence of a highly virulent isolate Edwardsiella tarda SC002 isolated from hatchlings of the Siamese crocodile. The genome of SC002 consisted of one circular chromosome of length 3,662,469 bp with a 57.29% G+C content and four novel plasmids. A total of 3,734 protein-coding genes, 12 genomic islands (GIs), 7 prophages, 48 interspersed repeat sequences, 248 tandem repeat sequences, a CRISPR component with a total length of 175 bp, and 171 ncRNAs (tRNA = 106, sRNA = 37, and rRNA = 28) were predicted. In addition, the coding genes of assembled genome were successfully annotated against eight general databases (NR = 3,618/3,734, COG = 2,947/3,734, KEGG = 3,485/3,734, SWISS-PROT = 2,787/3,734, GO = 2,648/3,734, Pfam = 2,648/3,734, CAZy = 130/3,734, and TCDB = 637/3,734) and four pathogenicity-related databases (ARDB = 11/3,734, CARD = 142/3,734, PHI = 538/3,734, and VFDB = 315/3,734). Pan-genome and comparative genome analyses of the complete sequenced genomes confirmed their evolutionary relationships. The present study confirmed that E. tarda SC002 is a potential pathogen bearing a bulk amount of antibiotic resistance, virulence, and pathogenic genes and its open pan-genome may enhance its host range in the future.

Novel Streptococcus uberis sequence types causing bovine subclinical mastitis in Hainan, China.

AIM: To determine the molecular epidemiology, genotypes and phenotypes of the major species of Streptococcus associated with bovine subclinical mastitis in Hainan, China. METHODS AND RESULTS: In total, 150 subclinical mastitis milk samples were collected from two large dairy farms in Hainan. On the basis of biochemical tests and 16S rDNA sequencing, 39 samples were Streptococcus positive and the most frequently isolated species was Streptococcus uberis (n = 29, 74.4%). According to multilocus sequence typing (MLST), and assays of biofilm formation, antimicrobial susceptibility, resistance and virulence genes, the S. uberis isolates were clustered into nine new sequence types (STs; ST986-ST994) but were not merged into a clonal group (except for ST991 [CC143]). All isolates produced biofilm, but most weakly. The dominant virulence pattern was hasABC + sua + gapC + oppF + pauA + mtuA + cfu (27/29, 91.1%), based on the 11 virulence genes tested. The majority of isolates (88.46%) carried at least one resistance gene, and more than half (58.62%) were multidrug-resistant. The main resistance genes were linB (65.5%), ermB (37.9%) and tetS (34.5%), among the six antibiotic resistance genes and 11 antimicrobials tested. CONCLUSION: Environmental S. uberis is important in bovine subclinical mastitis in Hainan. SIGNIFICANCE AND IMPACT OF THE STUDY: Streptococcus uberis isolates in Hainan, China, show distinct MLST, virulence and antibiotic resistance characteristics.

Reduced glutathione does not further reduce contrast-induced nephropathy in elderly patients with diabetes receiving percutaneous coronary intervention.

OBJECTIVE: To investigate the preventive effect of hydration combined with reduced glutathione on contrast-induced nephropathy (CIN) after coronary intervention therapy in elderly Chinese patients with diabetes. METHODS: Patients with diabetes aged ≥65 years, who received percutaneous coronary intervention (PCI) between 1 August 2016 and 31 December 2018, were enrolled and randomized into two groups: patients treated with hydration combined with reduced glutathione (treatment group) and patients who received hydration alone (controls). Serum creatinine and creatinine clearance levels were measured in all patients before PCI and then daily for 3 days after PCI. Occurrence of CIN (the primary endpoint) was defined as serum creatinine value 25% or 44.2 mmol/l (0.5 mg/dl) above baseline at 72 h after an exposure to contrast medium. RESULTS: A total of 396 patients were included (treatment group, n = 204; and controls, n = 192). The CIN occurrence rate in the treatment and control group was 5.88% and 6.77%, respectively, with no statistically significant between-group difference. CONCLUSION: In elderly patients with diabetes receiving PCI, the risk of CIN was not effectively lowered by hydration combined with reduced glutathione.

A double-locus scarless genome editing system in Escherichia coli.

OBJECTIVE: To develop a convenient double-locus scarless genome editing system in Escherichia coli, based on the type II Streptococcus pyogenes CRISPR/Cas9 and λ Red recombination cassette. RESULTS: A two-plasmid genome editing system was constructed. The large-sized plasmid harbors the cas9 and λ Red recombination genes (gam, bet, and exo), while the small-molecular plasmid can simultaneously express two different gRNAs (targeting genome RNAs). The recombination efficiency was tested by targeting the galK, lacZ, and dbpA genes in E. coli with ssDNA or dsDNA. Resulting concurrent double-locus recombination efficiencies were 88 ± 5.5% (point mutation), 39.7 ± 4.3% (deletion/insertion), and 57.8 ± 3.4%-58.5 ± 4.1% (mixed point and deletion/insertion mutation), depending on 30 (ssDNA) or 40 bp (dsDNA) homologous side arms employed. In addition, the curing efficiency of the guide plasmid expressing gRNAs for negative selection was higher (96 ± 3% in 4 h) than the help plasmid carrying cas9 and λ Red (92 ± 2% in 9 h). CONCLUSIONS: The new editing system is convenient and efficient for simultaneous double-locus recombination in the genome and should be favorable for high-throughput multiplex genome editing in synthetic biology and metabolic engineering.

Identification of a Novel Ichthyic Parvovirus in Marine Species in Hainan Island, China.

Parvoviruses are a diverse group of viruses that are capable of infecting a wide range of animals. In this study, we report the discovery of a novel parvovirus, tilapia parvovirus HMU-HKU, in the fecal samples of crocodiles and intestines of tilapia in Hainan Province, China. The novel parvovirus was firstly identified from crocodiles fed with tilapia using next-generation sequencing (NGS). Screening studies revealed that the prevalence of the novel parvovirus in crocodile feces samples fed on tilapia (75-86%) was apparently higher than that in crocodiles fed with chicken (4%). Further studies revealed that the prevalence of the novel parvovirus in tilapia feces samples collected at four areas in Hainan Province was between 40 and 90%. Four stains of the novel parvovirus were identified in this study based on sequence analyses of NS1 and all the four strains were found in tilapia in contrast only two of them were detected in crocodile feces. The nearly full-length genome sequence of the tilapia parvovirus HMU-HKU-1 was determined and showed less than 45.50 and 40.38% amino acid identity with other members of Parvoviridae in NS1 and VP1 genes, respectively. Phylogenetic analysis based on the complete helicase domain amino acid sequences showed that the tilapia parvovirus HMU-HKU-1 formed a relatively independent branch in the newly proposed genus Chaphamaparvovirus in the subfamily Hamaparvovirinae according to the ICTV's most recent taxonomic criteria for Parvoviridae classification. Tilapia parvovirus HMU-HKU-1 likely represented a new species within the new genus Chaphamaparvovirus. The identification of tilapia parvovirus HMU-HKU provides further insight into the viral and genetic diversity of parvoviruses and its infections in tilapia populations need to be evaluated in terms of pathogenicity and production losses in tilapia farming.

Effects of palytoxins extracted from Ostreopsis ovata on the oxidative stress and immune responses in Pacific white shrimp Litopenaeus vannamei.

Palytoxins (PLTXs) are a group of complex and poisonous marine natural products that are toxic to marine life and even human beings. In the present study, the oxidative stress and immune response in the hepatopancreas and gills of Litopenaeus vannamei were assessed for 72 h after injection with PLTX extracts. Chemical and physiological parameters, e.g., the respiratory burst (O2-), activities of antioxidant enzymes, oxidative damage to lipids, carbonylation of proteins, and immune gene mRNA expression levels, were analysed. The results showed that the PLTX extract was not fatal to the shrimp but could reduce their mobility. The O2- levels in the gills gradually increased after exposure to PLTX extracts and were significantly higher than those in the control from 6 to 72 h. The malondialdehyde content, lipid peroxidation, protein carbonyl levels, and total antioxidant capacity in the gills all peaked at 12 h. At the same time, the gills were loosely connected, there was a clear disintegration of the epithelial tissue, and the stratum corneum disappeared after 12 h. In addition, compared to those in the control group, the PLTX extract treatment increased the O2- content, malondialdehyde content, lipid peroxidation, and protein carbonyl levels from 12 to 72 h, 24-48 h, 12-24 h, and 12-72 h after injection in the hepatopancreas of the shrimp, respectively. Both the Crustin and Toll gene expression levels significantly increased in the hepatopancreas compared to those in the control 6-72 h after injection of the toxin. In parallel, the expression levels of the manganese superoxide dismutase gene gradually decreased from 6 to 48 h and returned to normal levels after 72 h. Interestingly, the total antioxidant capacity also significantly increased compared to that in the control from 6 to 72 h. Our results indicate that although PLTX extracts cause lipid peroxidation and carbonylation of proteins in hepatopancreatic cells, leading to their damage, they did not cause a decrease in the total antioxidant capacity of the hepatopancreas.

Streptomyces tirandamycinicus sp. nov., a Novel Marine Sponge-Derived Actinobacterium With Antibacterial Potential Against Streptococcus agalactiae.

A novel actinobacterium, strain HNM0039T, was isolated from a marine sponge sample collected at the coast of Wenchang, Hainan, China and its polyphasic taxonomy was studied. The isolate had morphological and chemical characteristics consistent with the genus Streptomyces. Based on the 16S rRNA gene sequence analysis, strain HNM0039T was closely related to Streptomyces wuyuanensis CGMCC 4.7042T (99.38%) and Streptomyces spongiicola HNM0071T (99.05%). The organism formed a well-delineated subclade with S. wuyuanensis CGMCC 4.7042T and S. spongiicola HNM0071T in the Streptomyces 16S rRNA gene tree. Multi-locus sequence analysis (MLSA) based on five house-keeping gene alleles (atpD, gyrB, rpoB, recA, trpB) further confirmed their relationship. DNA-DNA relatedness between strain HNM0039T and its closest type strains, namely S. wuyuanensis CGMCC 4.7042T and S. spongiicola HNM0071T, were 46.5 and 45.1%, respectively. The average nucleotide identity (ANI) between strain HNM0039T and its two neighbor strains were 89.65 and 91.44%, respectively. The complete genome size of strain HNM0039T was 7.2 Mbp, comprising 6226 predicted genes with DNA G+C content of 72.46 mol%. Thirty-one putative secondary metabolite biosynthetic gene clusters were also predicted in the genome of strain HNM0039T. Among them, the tirandamycin biosynthetic gene cluster has been characterized completely. The crude extract of strain HNM0039T exhibited potent antibacterial activity against Streptococcus agalactiae in Nile tilapia. And tirandamycins A and B were further identified as the active components with MIC values of 2.52 and 2.55 µg/ml, respectively. Based on genotypic and phenotypic characteristics, it is concluded that strain HNM0039T represents a novel species of the genus Streptomyces whose name was proposed as Streptomyces tirandamycinicus sp. nov. The type strain is HNM0039T (= CCTCC AA 2018045T = KCTC 49236T).

Acute myocardial infarction in pregnancy: spasm caused by hyperthyroidism?

We present a 26-year-old woman with ST-segment elevation myocardial infarction in the 14th week of pregnancy. Coronary angiography revealed no abnormalities in the coronary arteries. She had no history of coronary risk factors such as smoking, diabetes mellitus, hypertension, or dyslipidemia. Although we do not have direct evidence of coronary spasm in this patient, several factors suggest that coronary spasm is the most likely cause of myocardial infarction. We suspect that hyperthyroidism may have played an important role in coronary spasm in this patient. Early use of coronary angiography is helpful to identify the types of coronary artery lesions.

Adding ReputationRank to member promotion using skyline operator in social networks.

BACKGROUND: To identify potential stars in social networks, the idea of combining member promotion with skyline operator attracts people's attention. Some algorithms have been proposed to deal with this problem so far, such as skyline boundary algorithms in unequal-weighted social networks. METHODS: We propose an improved member promotion algorithm by presenting ReputationRank based on eigenvectors as well as Influence and Activeness and introduce the concept of skyline distance. Furthermore, we perform skyline operator over non-skyline set and choose the infra-skyline as our candidate set. The added ReputationRank helps a lot to describe the importance of a member while the skyline distance assists us to obtain the necessary condition for not being dominated so that some meaningless plans can be pruned. RESULTS: Experiments on the DBLP and WikiVote datasets verify the effectiveness and efficiency of our proposed algorithm. CONCLUSIONS: Treating the infra-skyline set as candidate set reduces the number of candidates. The pruning strategies based on dominance and promotion cost decrease the searching space.

Soluble expression of biologically active methionine sulfoxide reductase B1 (PaMsrB1) from Carica papaya in Escherichia coli and isolation of its protein targets.

Plant methionine sulfoxide reductase B1 (MsrB1) protects the photosynthetic apparatus from oxidative damage by scavenging reactive oxygen species to repair Met-oxidized proteins in response to abiotic stresses and biotic attack. Papaya MsrB1 (PaMsrB1) was identified previously to interact with papaya ringspot virus NIa-Pro, and this interaction inhibits the import of PaMsrB1 into the chloroplast. Further functional characterization of PaMsrB1 requires the production of a biologically active purified recombinant protein. In this report, PaMsrB1 as a fusion protein containing an N-terminal maltose-binding protein (MBP) was expressed in Escherichia coli Rosetta (DE3) cells and purified. Production of soluble fusion protein was greater when the cells were cultured at 16 °C than at 37 °C. The Factor Xa protease digested MBP-PaMsrB1 fusion protein and subsequently purified recombinant PaMsrB1 specifically reduced the R-diastereomer of methionine sulfoxide (MetSO) and Dabsyl-MetSO to Met in the presence of dithiothreitol. Eight chloroplast-localized and five non-chloroplast-localized candidate proteins that interact with PaMsrB1 were isolated by affinity chromatography and liquid chromatography coupled to tandem mass spectrometry. The results provide a platform to further understand the anti-oxidative defense mechanism of PaMsrB1.

The function of PlcR in Bacillus anthracis vaccine strain A16R.

Bacillus anthracis, B. thuringiensis and B. cereus are members of the B. cereus group. They share high genetic similarity. Whereas plcR (Phospholipase C regulator) usually encodes a functional pleiotropic activator protein in B. cereus and B. thuringiensis isolates, a characteristic nonsense mutation is found in all B. anthracis strains investigated, making the gene dysfunctional. To study the function of PlcR in B. anthracis, we used the B. cereus CMCC63301 genome as a template and constructed a recombinant expression plasmid pBE2A-plcR, and introduced it into the B. anthracis vaccine strain A16R, and then analyzed the activity of the hemolysin and sphingomyelinase. The results showed that transformation of B. anthracis with plasmid pBE2A-plcR carrying the native B. cereus plcR gene active the expression of sphingomyelinase gene, but did not activate expression of hemolysin genes of B. anthracis A16R.

The extent of error-prone replication restart by homologous recombination is controlled by Exo1 and checkpoint proteins.

Genetic instability, a hallmark of cancer, can occur when the replication machinery encounters a barrier. The intra-S-phase checkpoint maintains stalled replication forks in a replication-competent configuration by phosphorylating replisome components and DNA repair proteins to prevent forks from catastrophically collapsing. Here, we report a novel function of the core Schizosaccharomyces pombe checkpoint sensor kinase, Rad3 (an ATR orthologue), that is independent of Chk1 and Cds1 (a CHK2 orthologue); Rad3(ATR) regulates the association of recombination factors with collapsed forks, thus limiting their genetic instability. We further reveal antagonistic roles for Rad3(ATR) and the 9-1-1 clamp - Rad3(ATR) restrains MRN- and Exo1-dependent resection, whereas the 9-1-1 complex promotes Exo1 activity. Interestingly, the MRN complex, but not its nuclease activity, promotes resection and the subsequent association of recombination factors at collapsed forks. The biological significance of this regulation is revealed by the observation that Rad3(ATR) prevents Exo1-dependent genome instability upstream of a collapsed fork without affecting the efficiency of recombination-mediated replication restart. We propose that the interplay between Rad3(ATR) and the 9-1-1 clamp functions to fine-tune the balance between the need for the recovery of replication through recombination and the risk of increased genome instability.

Molecular phylogenies and evolutionary behavior of AhR (aryl hydrocarbon receptor) pathway genes in aquatic animals: implications for the toxicology mechanism of some persistent organic pollutants (POPs).

Phylogenetic analysis of AhR pathway genes and their evolutionary rate variations were studied on aquatic animals. The gene sequences for the proteins involved in this pathway were obtained from four major phylogenetic groups, including bivalvia, amphibian, teleostei and mammalia. These genes were distributed under four major steps of toxicology regulation: formation of cytosolic complex, translocation of AhR, heterodimerization of AhR and induction of CYP1A. The NJ, MP, and ML algorithm were used on protein coding DNA sequences to deduce the evolutionary relationship for the respective AhR pathway gene among different aquatic animals. The rate of non-synonymous nucleotide substitutions per non-synonymous site (d(N)) and synonymous nucleotide substitutions per synonymous site (d(S)) were calculated for different clade of the respective phylogenetic tree for each AhR pathway gene. The phylogenetic analysis suggests that evolutionary pattern of AhR pathway genes in aquatic animals is characterized mainly through gene duplication events or alterative splicing. The d(N) values indicate that all AhR pathway genes are well conserved in aquatic animals, except for CYP1A gene. Furthermore, compare with other aquatic animals, the d(N) value indicates that AhR pathway genes of fish are less conserved, and these genes likely go through an adaptive evolution within aquatic animals.

Identification of loss of function mutations in human genes encoding RIG-I and MDA5: implications for resistance to type I diabetes.

Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are essential for detecting viral RNA and triggering antiviral responses, including production of type I interferon. We analyzed the phenotype of non-synonymous mutants of human RIG-I and MDA5 reported in databases by functional complementation in cell cultures. Of seven missense mutations of RIG-I, S183I, which occurs within the second caspase recruitment domain repeat, inactivated this domain and conferred a dominant inhibitory function. Of 10 mutants of MDA5, two exhibited loss of function. A nonsense mutation, E627*, resulted in deletion of the C-terminal region and double-stranded RNA (dsRNA) binding activity. Another loss of function mutation, I923V, which occurs within the C-terminal domain, did not affect dsRNA binding activity, suggesting a novel and essential role for this residue in the signaling. Remarkably, these mutations are implicated in resistance to type I diabetes. However, the A946T mutation of MDA5, which has been implicated in type I diabetes by previous genetic analyses, affected neither dsRNA binding nor IFN gene activation. These results provide new insights into the structure-function relationship of RIG-I-like receptors as well as into human RIG-I-like receptor polymorphisms, antiviral innate immunity, and autoimmune diseases.

Construction of a novel Shigella live-vector strain co-expressing CS3 and LTB/STm of enterotoxigenic E.coli.

AIM: To construct and evaluate a polyvalent recombinant vaccine strain Shigella flexneri 2a T32 against enterotoxigenic E.coli (ETEC). METHODS: By using a host-plasmid balanced lethal system based on asd gene, a polyvalent recombinant strain was constructed to highly express CS3 and regularly express fusion enterotoxin of LTB subunit and mutant ST (LTB/STm) in a vaccine strain Shigella flexneri 2a T32 with specific deletion of asd gene. Fimbria CS3 was observed by immunofluorescence and electron microscopy assay. The security of LTB/STm was examined by ileal loop assay and suckling mouse assay. To evaluate this new candidate vaccine, it was compared with a previous vaccine strain in plasmid and protein level, growth assay and immunogenicity in Balb/c mice. RESULTS: The newly constructed vaccine expressed CS3 and grew better than the previously constructed vaccine except for the lower expression of LTB/STm. Serum IgG and mucosal IgA against CS3, LTB, ST, and host lipopolysaccharide (LPS) were produced after immunization of Balb/c mice by oral route with the new strain. The titers were not significantly different from the Balb/c mice with the previous strain. CONCLUSION: This novel candidate diarrheal vaccine can effectively induce serum and mucosal antibody responses against ETEC and Shigella.

[Biodiversity of tree species, their populations' spatial distribution pattern and interspecific association in mixed deciduous broadleaved forest in Changbai Mountains].

Based on the data collected from the sampling plot with an area of 1 hm2 at the Experimental and Management Bureau of Jiaohe in Jilin Province, this paper analyzed and researched the community structure, biological diversity, spatial distribution pattern and interspecific association between tree species populations in a mixed deciduous broadleaved forest. The results indicated that there was no obvious dominant order among tree species populations, and the indices of tree species diversity such as Shannon-Wiener index (H'), Pielou evenness index (E) and Simpson ecological dominant index (D) were all low. Shrub and herb species diversity indices were also not high. The spatial distribution pattern of five dominant tree species all presented aggregative. Based on 2 x 2 contigency table, the interspecific associations of 15 tree species in the community were examined by means of chi2-test, together with the association coefficient (AC). The results showed that there were 14 species pairs in 15 species showing significantly negative association. The proportion of species pairs showing negative association was much more than that of showing positive association. It could be deduced that the community was instable and at the stage of autogenic succession from soft deciduous broadleaved forest to hard deciduous broadleaved forest, and then, the Korean pine broadleaved forest.

[Co-expression of CFA/I and CS6 of Enterotoxigenic Escherichia coli ( ETEC ) in Shigella flexneri 2a T32 Derivative Strain FWL01].

Among the known colonization factors of enterotoxigenic Escherichia coli (ETEC), CFA/I and CS6 (the common antigen in the CFA/IV fimbrial antigens ) are two of the most prevalent fimbriae found in clinical isolates but are never expressed by the same wild-type strains. In this study, CFA/I and CS6 of ETEC were co-expressed in Shigella flexneri 2a T32 derivative strain FWL01 by using a host-plasmid lethal balancing system based on asd gene. The results indicate that the recombinant plasmid carrying CFA/I and CS6 could be stably integrated in FWL01. Expression of the two antigens did not interfere the host growth. The results of immunofluorescence analysis showed that CFA/I and CS6 were localized on the surface of the strain FWL01. In Balb/c mice orally immunized with the recombinant strain, the immune responses against CFA/I and CS6 were observed. Those observations show the feasibility of a multivalent vaccine expressing different fimbrial antigens in attenuated Shigella flexneri.

[Simultaneous expression of CS3 colonization factor antigen and LT-B/ST fusion enterotoxin antigen of enterotoxigenic Escherichia coli by attenuated Shigella flexneri 2a].

Enterotoxigenic Escherichia coli (ETEC) causes watery dehydrating diarrhea in infants in developing countries, and is the most common cause of travelers diarrhea. It has been known that the colonazition factor antigens (CFAs) and enterotoxins are important virulence factors of ETEC, and these two kinds of proteins should be included in any effective vaccine against ETEC. In this study, a host-plasmid lethal balancing system was constructed based on asd gene in an avirulent strain of S.flexneri to express CS3 antigens and the fusion LT-B/ST enterotoxins of Escherichia coli. Both of these antigens were expressed steadily in the S. flexneri vector without any antibiotic markers. Antibodies against CS3, LT, ST and LPS of Shigella were detected in sera of mice that were immunized with recombinant bacteria either oragastrically (o.g.) or intranasally (i.n.). SIgA against CS3 and enterotoxins were detected simultaneously in feces of mice. This work is helpful for constructing multivalent recombinant vaccine for prevention of bacterial diarrhea.