An investigation on the diversity of bacterial organisms in hepatopanueas ,spleen and kidney of healthy hybridized crussian carp (Carassius auratus gibelio) was carried out in six large‐scale breeding farms in Zhejiang province during June —July and September—October in 2013 .From a total of 12 bat‐ches ,seven opportunistic bacterial species capable of affecting humans ,animals and fishes were identified . A eromonas spp .predominated both in June—July and September—October batches and aerA +act+alt‐act‐was the major virulence‐gene pattern ,indicating that the Aeromonas isolates exhibited potential pathogenic inclination .On the contrary ,Acinetobacter lwof fii ,Edwardsiella hoshinae ,Plesiomonas shigelloides , Shewanella putref aciens , Stenotrophomonas maltophilia and Escherichia coli showed varying isolation rates between June—July and September—October batches .Bacterial species composition and high carry‐ing‐rate of pathogenic A eromonas in hybridized prussian carp posed significant risks to aquaculture industry and public health .It is necessary to develop the surveillance system of potential bacterial pathogens ,espe‐cially A eromonas spp .,in hybridized crussian carp ,and to clarify the pathogenic mechanism to establish comprehensive prevention and control schemes .

The genus Listeria consists of six closely related species and forms three phylogenetic groups: L. monocytogenes- L. innocua, L. ivanovii-L. seeligeri-L. welshimeri, and L. grayi. In this report, we attempted to examine the evolutionary relationship in the L. monocytogenes-L. innocua group by probing the nucleotide sequences of 23S rRNA and 16S rRNA, and the gene clusters lmo0029-lmo0042, ascBdapE, rplS-infC, and prs-ldh in L. monocytogenes serovars 1/2a, 4a, and 4b, and L. innocua. Additionally, we assessed the status of L. monocytogenes-specific inlA and inlB genes and 10 L. innocua-specific genes in these species/serovars, together with phenotypic characterization by using in vivo and in vitro procedures. The results indicate that L. monocytogenes serovar 4a strains are genetically similar to L. innocua in the lmo0035-lmo0042, ascB-dapE, and rplS-infC regions and also possess L. innocua-specific genes lin0372 and lin1073. Furthermore, both L. monocytogenes serovar 4a and L. innocua exhibit impaired intercellular spread ability and negligible pathogenicity in mouse model. On the other hand, despite resembling L. monocytogenes serovars 1/2a and 4b in having a nearly identical virulence gene cluster, and inlA and inlB genes, these serovar 4a strains differ from serovars 1/2a and 4b by harboring notably altered actA and plcB genes, displaying strong phospholipase activity and subdued in vivo and in vitro virulence. Thus, by possessing many genes common to L. monocytogenes serovars 1/2a and 4b, and sharing many similar gene deletions with L. innocua, L. monocytogenes serovar 4a represents a possible evolutionary intermediate between L. monocytogenes serovars 1/2a and 4b and L. innocua.

Three pairs of polymerase chain reaction(PCR) primers were designed for the gdh、mrp and cps2J genes,and the triplex-PCR assay was developed to identify Streptococcus suis serotype 2(SS-2) both in purified cultures and pork samples.688 bp,532 bp and 459 bp DNA products were amplified from Streptococcus suis type 2 under following conditions in 30 μL reaction systems:the concentration ratio of primer pair for gdh,mrp and cps2J is 1∶1∶1.67;and the cycle detailed circumstance are 3 min of pre-incubation at 94℃,30 cycles of 1 min at 94℃,40 s at 55℃,50 s at 72℃,followed by 5 min at 72℃.The lowest detection limit of the triplex-PCR for purification cultures and artificially polluted pork samples are 450 CFU and 4 500 CFU,respectively.Results indicated that the established triplex-PCR assay could be used to detect SS-2 in purified cultures and pork samples,and had potentially clinical and epidemiological applications.

A pair of primers was designed according to the sequence of LMO1847 of Listeria monocytogenes strain EGD from GenBank.The gene fragment of LMO1847 without signal peptide sequence was obtained by PCR from genomic DNA of L.monocytogenes,and cloned into prokaryotic expression plasmid pET-30a.The recombinant plasmid was transferred into E.coli BL21 and expressed successfully.The purified recombinant protein was used for polyclonal antibody production in rabbits.ELISA analysis showed that the polyclonal antibody of LMO1847 was reactive to whole listeial cells and the polyclonal antibody against the whole cells of L.monocytogenes was reactive to the recombinant LMO1847 protein.Dot-ELISA and the whole bacteria coated ELISA showed that LMO1847 existed on listerial cell surface with good immune reactivity.The expression level of LMO1847 remained stable under short period(<3 h)of elevated temperature at 45 ℃,5.5% NaCl or acidic(pH 5.5)stress conditions,but the reaction disappeared under acidic stress at pH 3.0.Expression level of LMO1847 by L.monocytogenes in these conditions was decreased depending on the concentration of salt and temperature variation,and the reaction ability wais steady except grown in 4 ℃.

1 发病情况 南通市某肉鸭场于2003年7月份出孵雏鸭5 000余只,采用池边网床饲养.雏鸭16日龄开始发病,并出现死亡.随后2 d内死亡1 300多只.用链霉素、恩诺沙星给鸭饮水,但效果不佳,病情得不到控制.