Various Profiles of tet Genes Addition to tet(X) in Riemerella anatipestifer Isolates From Ducks in China.

To investigate tetracycline resistance and resistant genotype in Riemerella anatipestifer, the tetracycline susceptibility of 212 R. anatipestifer isolates from China between 2011 and 2017 was tested. The results showed that 192 of 212 (90.6%) R. anatipestifer isolates exhibited resistance to tetracycline (the MICs ranged from 4 to 256 µg/ml). The results of PCR detection showed that, 170 of 212 (80.2%) R. anatipestifer isolates possessed the tet(X) gene. Other genes, including tet(A), tet(M), tet(Q), tet(O), tet(B), and tet(O/W/32/O), were found at frequencies of 20.8, 4.7, 1.4, 0.9, 0.9, and 0.5%, respectively. However, tet(C), tet(E), tet(G), tet(K), and tet(W) were not detected in any isolate. In these tet gene positive strains, 31 (14.6%), 2 (0.9%), 5 (2.4%), 1 (0.5%), 3 (1.4%) were detected containing tet(A)/tet(X), tet(M)/tet(O), tet(M)/tet(X), tet(O)/tet(X), and tet(Q)/tet(X) simultaneously, respectively. One isolates, R131, unexpectedly contained three tet genes, i.e., tet(M), tet(O), and tet(X). Sequence analysis of the tet gene ORFs cloned from R. anatipestifer isolates confirmed that tet(A), tet(B), tet(M), tet(O), tet(Q) and an unusual mosaic tet gene tet(O/W/32/O) were present in R. anatipestifer. The MIC results of R. anatipestifer ATCC 11845 transconjugants carrying tet(A), tet(B), tet(M), tet(O), tet(O/W/32/O), tet(Q), and tet(X) genes exhibited tetracycline resistance with MIC values ranging from 4 to 64 µg/ml. Additionally, the tet(X) gene could transfer into susceptible strain via natural transformation (transformation frequencies of ~10-6). In conclusion, the tet(A), tet(B), tet(M), tet(O), tet(O/W/32/O), tet(Q), and tet(X) genes were found and conferred tetracycline resistance in R. anatipestifer isolates. Moreover, the tet(X) is the main mechanism of tetracycline resistance in R. anatipestifer isolates. To our knowledge, this is the first report of tet(A), tet(B), tet(M), tet(O), tet(Q), and mosaic gene tet(O/W/32/O) in R. anatipestifer.

A novel resistance gene, lnu(H), conferring resistance to lincosamides in Riemerella anatipestifer CH-2.

The Gram-negative bacterium Riemerella anatipestifer CH-2 is resistant to lincosamides, having a lincomycin (LCM) minimum inhibitory concentration (MIC) of 128 µg/mL. The G148_1775 gene of R. anatipestifer CH-2, designated lnu(H), encodes a 260-amino acid protein with ≤41% identity to other reported lincosamide nucleotidylyltransferases. Escherichia coli RosettaTM (DE3) containing the pBAD24-lnu(H) plasmid showed four- and two-fold increases in the MICs of LCM and clindamycin (CLI), respectively. A kinetic assay of the purified Lnu(H) enzyme for LCM and CLI showed that the protein could inactive lincosamides. Mass spectrometry analysis demonstrated that the Lnu(H) enzyme catalysed adenylylation of lincosamides. In addition, an lnu(H) gene deletion strain exhibited 512- and 32-fold decreases in LCM and CLI MICs, respectively. The wild-type level of lincosamide resistance could be restored by complementation with a shuttle plasmid carrying the lnu(H) gene. The transformant R. anatipestifer ATCC 11845 [lnu(H)] acquired by natural transformation also exhibited high-level lincosamide resistance. Moreover, among 175 R. anatipestifer field isolates, 56 (32.0%) were positive for the lnu(H) gene by PCR. In conclusion, Lnu(H) is a novel lincosamide nucleotidylyltransferase that inactivates LCM and CLI by nucleotidylylation, thus conferring high-level lincosamide resistance to R. anatipestifer CH-2.

Contribution of RaeB, a Putative RND-Type Transporter to Aminoglycoside and Detergent Resistance in Riemerella anatipestifer.

Riemerella anatipestifer is an important pathogenic bacterium that infects ducks. It exhibits resistance to multiple classes of antibiotics. Multidrug efflux pumps play a major role as a mechanism of antimicrobial resistance in Gram-negative pathogens and they are poorly understood in R. anatipestifer. In this study, a gene encoding the B739_0873 protein in R. anatipestifer CH-1, which belongs to the resistance-nodulation-cell division (RND) efflux pump family, was identified. With respect to the substrate specificity of B739_0873, the antibiotic susceptibility testing showed that the B739_0873 knockout strain was more sensitive to aminoglycosides and detergents than the wild-type strain. The transcription of B739_0873 was up-regulated when R. anatipestifer CH-1 was exposed to sub-inhibitory levels of these substrates. From the gentamicin accumulation assay, we concluded that B739_0873 was coupled to the proton motive force to pump out gentamicin. Furthermore, site-directed mutagenesis demonstrated that Asp 400, Asp 401, Lys 929, Arg 959, and Thr 966 were the crucial function sites of B739_0873 in terms of its ability to extrude aminoglycosides and detergents. Finally, we provided evidence that B739_0873 is co-transcribed with B739_0872, and that both B739_0872 and B739_0873 are required for aminoglycoside and detergent resistance. In view of these results, we designate B739_0873 as RaeB (Riemerella anatipestifer efflux).

Type B Chloramphenicol Acetyltransferases Are Responsible for Chloramphenicol Resistance in Riemerella anatipestifer, China.

Riemerella anatipestifer causes serositis and septicaemia in domestic ducks, geese, and turkeys. Traditionally, the antibiotics were used to treat this disease. Currently, our understanding of R. anatipestifer susceptibility to chloramphenicol and the underlying resistance mechanism is limited. In this study, the cat gene was identified in 69/192 (36%) R. anatipestifer isolated from different regions in China, including R. anatipestifer CH-2 that has been sequenced in previous study. Sequence analysis suggested that there are two copies of cat gene in this strain. Only both two copies of the cat mutant strain showed a significant decrease in resistance to chloramphenicol, exhibiting 4 µg/ml in the minimum inhibitory concentration for this antibiotic, but not for the single cat gene deletion strains. Functional analysis of the cat gene via expression in Escherichia coli BL21 (DE3) cells and in vitro site-directed mutagenesis indicated that His79 is the main catalytic residue of CAT in R. anatipestifer. These results suggested that chloramphenicol resistance of R. anatipestifer CH-2 is mediated by the cat genes. Finally, homology analysis of types A and B CATs indicate that R. anatipestifer comprises type B3 CATs.

Complete Genome Sequence of Mycobacterium avium, Isolated from Commercial Domestic Pekin Ducks (Anas platyrhynchos domestica), Determined Using PacBio Single-Molecule Real-Time Technology.

Mycobacterium avium is an important pathogenic bacterium in birds and has never, to our knowledge, reported to be isolated from domestic ducks. We present here the complete genome sequence of a virulent strain of Mycobacterium avium, isolated from domestic Pekin ducks for the first time, which was determined by PacBio single-molecule real-time technology.

Outbreak of Avian Tuberculosis in Commercial Domestic Pekin Ducks ( Anas platyrhynchos domestica).

Avian tuberculosis is a contagious disease affecting various domestic and wild bird species, and is caused by Mycobacterium avium . It is reported extremely rarely in commercial poultry flocks and has not been reported in commercial domestic ducks to date, with domestic ducks reported to be moderately resistant to M. avium infection. Here, we report the outbreak of avian tuberculosis in commercial Pekin duck ( Anas platyrhynchos domestica) flocks. Postmortem and histopathologic findings included nodules presenting in the visceral organs of ducks, and granulomas with central caseous necrosis surrounded by infiltrating lymphocytes. The M. avium pathogen was isolated and further identified by Ziehl-Neelsen staining and PCR based on insert sequence IS901 and the 16S rRNA gene. We highlight that avian tuberculosis not only has economic significance for the duck industry, but also presents a potential zoonotic hazard to humans.

Comparative genomic analysis identifies structural features of CRISPR-Cas systems in Riemerella anatipestifer.

BACKGROUND: Riemerella anatipestifer infection is a contagious disease that has resulted in major economic losses in the duck industry worldwide. This study attempted to characterize CRISPR-Cas systems in the disease-causing agent, Riemerella anatipestifer (R. anatipestifer). The CRISPR-Cas system provides adaptive immunity against foreign genetic elements in prokaryotes and CRISPR-cas loci extensively exist in the genomes of archaea and bacteria. However, the structure characteristics of R. anatipestifer CRISPR-Cas systems remains to be elucidated due to the limited availability of genomic data. RESULTS: To identify the structure and components associated with CRISPR-Cas systems in R. anatipestifer, we performed comparative genomic analysis of CRISPR-Cas systems in 25 R. anatipestifer strains using high-throughput sequencing. The results showed that most of the R. anatipestifer strains (20/25) that were analyzed have two CRISPR loci (CRISPR1 and CRISPR2). CRISPR1 was shown to be flanked on one side by cas genes, while CRISPR2 was designated as an orphan. The other analyzed strains harbored only one locus, either CRISPR1 or CRISPR2. The length and content of consensus direct repeat sequences, as well as the length of spacer sequences associated with the two loci, differed from each other. Only three cas genes (cas1, cas2 and cas9) were located upstream of CRISPR1. CRISPR1 was also shown to be flanked by a 107 bp-long putative leader sequence and a 16 nt-long anti-repeat sequence. Combined with analysis of spacer organization similarity and phylogenetic tree of the R. anatipestifer strains, CRISPR arrays can be divided into different subgroups. The diversity of spacer organization was observed in the same subgroup. In general, spacer organization in CRISPR1 was more divergent than that in CRISPR2. Additionally, only 8 % of spacers (13/153) were homologous with phage or plasmid sequences. The cas operon flanking CRISPR1 was observed to be relatively conserved based on multiple sequence alignments of Cas amino acid sequences. The phylogenetic analysis associated with Cas9 showed Cas9 sequence from R. anatipestifer was closely related to that of Bacteroides fragilis and formed part of the subtype II-C subcluster. CONCLUSIONS: Our data revealed for the first time the structural features of R. anatipestifer CRISPR-Cas systems. The illumination of structural features of CRISPR-Cas system may assist in studying the specific mechanism associated with CRISPR-mediated adaptive immunity and other biological functions in R. anatipestifer.

Genome Sequence of Riemerella anatipestifer Strain RCAD0122, a Multidrug-Resistant Isolate from Ducks.

Riemerella anatipestifer is an important pathogenic bacterium in waterfowl and other avian species. We present here the genome sequence of R. anatipestifer RCAD0122, a multidrug-resistant strain isolated from infected ducks. The isolate contains at least nine types of antibiotic resistance-associated genes.

Quantitative real-time PCR study of the expression and regulation of the tetracycline resistance gene in Riemerella anatipestifer.

Riemerella anatipestifer (RA) is one of the most important pathogens of 1- to 8-wk-old ducklings that severely affects the development of the duck industry in China. Every year, antibiotic medicines including tetracycline and doxycycline are used in the duck industry. Few reports compare the expression of multidrug-resistant genes in RA before and after addition of chemical drugs. With this in mind, the direct effects of gradient concentration of tetracyclines on the expression of tetracycline resistance genes (TETr) in RA at the cDNA level were studied by using a quantitative real-time PCR method. The expression of TETr, tetA, tetC, and tetM was investigated in ATCC11845 and in 30 RA isolated from different samples. Using a range of doxycycline concentrations up to 50% of the minimum inhibitory concentration (MIC), the optimal induction concentration of 0.0625 µg/mL was selected. Under the optimal inducible expression, concentrations of TETr, tetC, and tetM cDNA were detected in all isolates, and the highest mRNA expression level of TETr genes was shown. Additionally, the expression levels of 3 TETr genes in RA14 (tetA and tetC) and RA17 (tetM and tetC) were compared. Both tetC and tetA found in isolate RA14 was found to express both tetC and tetA, and tetC cDNA was detected in isolate RA17 at all doxycycline concentrations tested, whereas tetM cDNA was not detected at any concentration. We can conclude that resistance pump is the main mechanism of tetracycline antibiotic resistance, and under the action of drug resistance pump tetC, the expression of tetM was not activated in RA17. These data suggest that the mRNA expression level of TETr genes was correlated with the MIC values, indicating that the degree of drug resistance is determined by the expression levels of TETr genes. Also, the induction of TETr is the major tetracycline resistance mechanism in RA, especially the resistance pump. However, lower concentrations of doxycycline induced higher TETr expression, and higher concentrations inhibited TETr expression. Maybe that is the reason for selection mutation to make tolerated bacteria survive.

Transcription phase, protein characteristics of DEV UL45 and prokaryotic expression, antibody preparation of the UL45 des-transmembrane domain.

BACKGROUND: Some UL45 gene function of Herpesvirus was reported. While there was no any report of the duck enteritis virus (DEV) UL45 protein as yet. RESULTS: The UL45 gene and des-transmembrane domain of UL45 (named UL45Δ gene, 295-675bp of UL45) of DEV were amplified by PCR and subcloned into the prokaryotic expression vector pET-32a(+). The constructed recombinant plasmids were transformed into the host strain BL21(DE3) PLysS and induced by IPTG. SDS-PAGE analysis showed the UL45 gene couldn't express while UL45Δ gene was highly expressed. His Purify Kit or salting-out could purify the protein effectively. Using the purified protein to immunize New-Zealand rabbits and produce polyclonal antibody. The agar diffusion reaction showed the titer of antibody was 1:32. Western blot analysis indicated the purified rabbit anti-UL45Δ IgG had a high level of specificity and the UL45 gene was a part of DEV genome. The transcription phase study of UL45 gene showed that expression of UL45 mRNA was at a low level from 0 to 18 h post-infection (pi), then accumulated quickly at 24 h pi and peaked at 42 h pi. It can be detected till 72 h pi. Besides, western blot analysis of purified virion and different viral ingredients showed that the UL45 protein resided in the purified virion and the viral envelope. CONCLUSIONS: The rabbit anti-UL45Δ IgG was produced successfully and it can serve as a good tool for penetrating studies of the function of DEV UL45 protein. The transcription phase and protein characteristics analysis indicated that DEV UL45 gene was a late gene and UL45 protein may be a viral envelope protein.

Characterization of the duck plague virus UL35 gene.

OBJECTIVE: Previous study has demonstrated that the duck plague virus (DPV) UL35 gene can be expressed as a recombinant fusion protein, and the prepared antiserum has a high reactivity and specificity against the purified recombinant protein. In the present study, to elucidate the properties and functions of its encoding protein, the UL35 gene product (VP26) was identified by using the prepared rabbit polyclonal antiserum. METHODS: Real-time PCR, Western blot and immunofluorescence analysis were used to determine the transcription and expression kinetics and subcellular localization of DPV VP26 in DPV-infected cells. RESULTS: A protein of approximately 13 kDa that reacted with the antiserum was detected in immunoblot of DPV-infected cellular lysates. Real-time PCR and Western blot analysis of DPV-infected cells showed that VP26 was produced predominantly at the late stage of infection, its production was highly dependent on viral DNA synthesis, and the UL35 gene was regulated as a late viral gene, suggesting that the gene should be categorized as gamma2 class. Additionally, analysis of the association of DPV VP26 with purified virions revealed that VP26 was a component of extracellular mature DPV virions. Subcellular localization demonstrated that VP26 firstly localized in cytoplasm, then it transferred to the nucleus and aggregated in the punctate region of the nucleus in DPV-infected cells. CONCLUSION: Taken together, these results will provide a foundation for further functional analysis of the DPV UL35 gene.

[Cloning, prokaryotic expression and subcellular localization in the infected host cells of the duck plague virus DPV UL35 gene].

Based on the duck plague virus (DPV) UL35 gene sequence that our laboratory obtained (GenBank accession number EF643558), a pair of primers was designed using Oligo6.0 and primer5.0, then the UL35 gene was amplified from DPV CHv strain genomic DNA and cloned into the pMD18-T to construct a clone plasmid pMD18-T-UL35. After identification of the pMD18-T-UL35 by PCR amplification and restriction digestion, the fragment of the UL35 gene was subcloned into the prokaryotic expression vector pET-32a(+). The resultant recombinant plasmid pET-32a(+)-UL35 was then transformed into E. coli BL21 (DE3) strain and optimally-expressed under the induction of 1.0 mmol/L IPTG at 34 degrees C for 5 hours. SDS-PAGE analysis showed the recombinant protein (VP26) had a molecular weight of about 33KDa and accounted for 32.3% of total bacterial protein by gel scanning. The protein was then purified by Ni(2+)-affinity chromatography and used to immunize rabbit for producing the VP26 anti-serum and its antibody titer was up to 1:32 detected by agar diffusion reaction. After the IgG of the polyclonal antibodies was purified by High-Q anion-exchange chromatography, Western blot analysis indicated that the IgG had specific reaction with the VP26. Moreover, the subcellular localization detection was observed using immunofluorescence technique. The results showed that the specific fluorescences appeared relatively few in nucleus in 2 to 8 hours and increased gradually in 12 to 36 hours and eventually reached to the maximum, which aggregated in the spot region of the nucleus after the duck embryo fibroblast (DEF) were infected by DPV. However, there were only a small amount of specific fluorescences in the cytoplasm in 12 hours and increased with the extension of infection time in 24 to 48 hours. The specific fluorescences finally reached to the maximum in the cytoplasm in 72 hours. The results provided significant data for furthering the study on the function of DPV UL35 gene.

Development of a fluorescent quantitative real-time polymerase chain reaction assay for the detection of Goose parvovirus in vivo.

BACKGROUND: Goose parvovirus (GPV) is a Dependovirus associated with latent infection and mortality in geese. Currently, it severely affects geese production worldwide. The objective of this study was to develop a fluorescent quantitative real-time polymerase chain reaction (PCR) (FQ-PCR) assay for fast and accurate quantification of GPV DNA in infected goslings, which can aid in the understanding of the regular distribution pattern and the nosogenesis of GPV in vivo. RESULTS: The detection limit of the assay was 2.8 x 10(1) standard DNA copies, with a sensitivity of 3 logs higher than that of the conventional gel-based PCR assay targeting the same gene. The real-time PCR was reproducible, as shown by satisfactory low intraassay and interassay coefficients of variation. CONCLUSION: The high sensitivity, specificity, simplicity, and reproducibility of the GPV fluorogenic PCR assay, combined with a high throughput, make this method suitable for a broad spectrum of GPV etiology-related applications.

Characterization of synonymous codon usage bias in the duck plague virus UL35 gene.

OBJECTIVE: The aim was to identify the codon usage bias between the newly identified duck plague virus (DPV) UL35 gene (GenBank accession No. EF643558) and the UL35-like genes of 27 other reference herpesviruses. METHODS: A comparative analysis of the codon usage bias of the 28 herpesviruses was performed by using the CodonW 1.4 program and CUSP (create a codon usage table) program of EMBOSS (The European Molecular Biology Open Software Suite). RESULTS: The results showed obvious differences of the synonymous codon usage bias in the 28 herpesviruses indicated by the Codon Adaptation Index, effective number of codons (ENc), and the value of G + C content at the 3rd codon position. The codon usage pattern of the DPV UL35 gene was phylogenetically conserved and similar to that of the UL35-like genes of the avian alpha-herpesvirus, with a strong bias towards the codons with A and T at the 3rd codon position. A cluster analysis of codon usage pattern of the DPV UL35 gene with other reference herpesviruses demonstrated that the codon usage bias of the UL35 genes of the 28 herpesviruses had a very close relation with their gene function. The ENc-plot revealed that the genetic heterogeneity in the DPV UL35 gene and the 27 reference herpesviruses were constrained by G + C content, while gene length exerted relatively weaker influences. In addition, comparisons of the codon preferences in the UL35 gene of DPV with those of Escherichia coli, yeast and humans revealed that there were 33 codons showing distinct usage differences between the DPV and yeast, and 38 between the DPV and humans, but only 31 between the DPV and E. coli. Therefore, the E. coli system may be more suitable for the expression of the DPV UL35 gene. CONCLUSION: Together, these results may improve our understanding of the evolution, pathogenesis and functional studies of DPV, as well as contribute significantly to the area of herpesvirus research and possibly studies with other viruses.

Identification and characterization of the duck enteritis virus UL51 gene.

Compared to the UL51 gene of other alphaherpesviruses, the duck enteritis virus (DEV) UL51 gene contains ten conserved motifs and has a close evolutionary relationship with members of the genus Mardivirus. The DEV UL51 gene product was identified using a rabbit polyclonal antiserum raised against a 6-His-UL51 fusion protein expressed in Escherichia coli as a 34-kDa protein. Western blotting and RT-(real time) PCR analysis of DEV-infected cells showed that the protein was produced at the late stage of infection and that its production was highly dependent on viral DNA synthesis, suggesting that the gene should be classified as gamma2 class. Analysis of extracellular virions revealed that the protein was a component of extracellular mature DEV virions. Indirect immunofluorescence studies localized most of the protein to the juxtanuclear region. These results will provide a basis for further functional analysis of the gene.

His6-tagged UL35 protein of duck plague virus: expression, purification, and production of polyclonal antibody.

OBJECTIVE: Duck plague virus (DPV), the causative agent of duck plague (DP), is an alphaherpesvirus that causes an acute, febrile, contagious, and septic disease of waterfowl. UL35 protein (VP26) is a major capsid protein encoded by the UL35 gene, which is located in the unique long (UL) region of the DPV genome. To investigate the specific roles of VP26, the UL35 gene was amplified from the DPV DNA by polymerase chain reaction (PCR) and subcloned into pET-32a(+). METHODS: The resultant prokaryotic expression vector, pET-32a(+)/UL35, includes an amino-terminal His6 as a fusion partner. Escherichia coli BL21 (DE3) competent cells were transformed with the construct and protein expression was subsequently induced by the addition of isopropyl-beta-D-thiogalactopyranoside to the culture medium. Protein lysates were submitted to SDS-PAGE to evaluate recombinant protein expression. RESULTS: The band that corresponded to the predicted protein size (33 kDa) was observed on the SDS-PAGE gel. The recombinant His6-tagged VP26 fusion protein was expressed at a high level in an insoluble form (inclusion bodies) and constituted about 24% of the total cellular protein. Then, the fusion protein was purified to near homogeneity using single-step immobilized metal affinity chromatography on a nickel-nitrilotriacetic acid affinity resin, yielding about 620 mg per liter culture. After purification, New Zealand white rabbits were immunized with purified His6-tagged VP26 in order to raise polyclonal antibody against this recombinant protein. Using the resultant sera, Western blot analysis showed that the recombinant protein was recognized by the polyclonal antibody. CONCLUSION: Thus, the polyclonal antibody prepared here may serve as a valuable tool to study the functional involvement of VP26 in the DPV life cycle.

New strategies for electrophoresis analysis of enterobacterial repetitive intergenic consensus PCR in animal intestinal microflora.

Enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) is a molecular biological technology that can be used to study microbial community diversity and dynamics. In many reports, investigations of microbial diversity from environmental samples were based on the agarose gel electrophoresis (AGE) patterns of ERIC-PCR amplified products. This is not a sound practice, since bands with identical positions can contain different sequences; thus, this practice could possibly exaggerate the similarities or diversities among samples. To mitigate this issue, we employed a denaturing gradient gel electrophoresis (DGGE) strategy to explore DNA bands with the same size, between ERIC-PCR profiles of samples. DPS software was used with Shannon-Wiener diversity index (H') to analyze ERIC-PCR fingerprint profiles. H' revealed that the microbial community diversity at DGGE was higher than with AGE. The results of this study suggest that the ERIC-PCR assays with DGGE can provide a better assessment of electrophoresis pattern with regards to the structure of an intestinal microbial community.

Antibiotic susceptibility of Riemerella anatipestifer field isolates.

Kirby-Bauer tests were used to analyze the antibiotic resistance of 224 isolates of Riemerella anatipestifer isolated between 1998 and 2005. Among the 36 antibiotics tested, 50% of the analyzed isolates were resistant to ampicillin, ceftazidime, aztreonam, cefazolin, cefepime, cefuroxime, oxacillin, penicillin G, rifampin, and trimethoprim/sulfamethoxazole. Higher levels of resistance were detected for aztreonam, cefepime, oxacillin, penicillin G, ceftazidime, and trimethoprim/sulfamethoxazole (87.8%, 64.3%, 88.6%, 86.9%, 75.9%, and 79.2% resistance, respectively). The lowest resistance rates were observed for amikacin (9.5%), cefoperazone (7.2%), imipenem (3.2%), and neomycin (9.5%). Four isolates were found to be resistant to 29 different antimicrobials. Riemerella anatipestifer drug resistance profiles changed over time, and the only consistent patterns observed were the resistance of R. anatipestifer to cefoperazone, piperacillin, spectinomycin, and aztreonam. In addition to determining the antibiotic-resistance profiles of R. anatipestifer isolates, we also examine the therapeutic efficacy of these antibiotics against lethal R. anatipestifer infection in ducks in vivo. According to these data, we have extrapolated an antibiotic treatment approach for veterinarians attending flocks of ducks. These data suggest that disk-diffusion analyses can be extrapolated to predict in vivo efficacy, thereby facilitating the identification of effective antibacterial treatments and potentially diminishing the irresponsible use of antibiotics.

[Replication of duck plague virus in artificially infected ducks detected by in situ hybridization].

Replication of duck plague virus(DPV) in artificially infected ducks were detected by in situ hybridization (ISH) which employed a 37bp oligonucleotide as probe designed according to DPV DNA sequence in GenBank. The results indicated that DPV DNA was detected in liver, intestine and bursa Fabricius at 4 h, in spleen and esophagus at 6h, in thymus at 12h post infection; DPV DNA in lung and kidney was detected only in dead ducks and no positive signal was detected in muscle, heart, cerebrum and pancreas. DPV DNA was distributed in cell nucleus and cytoplasm. Hepatocytes, sinus endodermal cells and Kuffer's cells were the mainly infected cell types in liver. DPV DNA was mainly detected in epithelium of villi, in lamina propria of intestinal villi of duodenum, in stratum spinosum of esophagus, and in epithelium, cortex, medulla of bursa Fabricius. The positive signals were mainly detected in medulla of thymus, lymphocytes and macrophages of spleen. The research suggests that ISH is a direct and specific method in detecting DPV DNA in paraffin sections and it's also a good method for virus diagnosis and DNA location of DPV.