Fast diagnosis and quantification for porcine circovirus type 2 (PCV-2) using real-time polymerase chain reaction.

BACKGROUND/PURPOSE: The postweaning multisystemic wasting syndrome, caused by the porcine circovirus type 2 (PCV-2), is a major disease that poses a significant threat to the global swine industry. The purpose of this study was to establish a real-time polymerase chain reaction (PCR) method for the quantification of PCV-2 and to enable the rapid differentiation of porcine circoviruses type 1 and 2 (PCV-1 and PCV-2). Such a method would significantly speed up the process of clinical diagnosis, and could also be used to study the pathogenic mechanisms of diseases associated with PCV-2. METHODS: Multiplex real-time PCR, together with LightCycler PCR data analysis software, was used for the quantification of PCV-2, and for the rapid differentiation of PCV-1 and PCV-2. A 263-bp DNA fragment was amplified from the 3' end of the open reading frame-2 of PCV-2 by nested PCR, and its DNA sequence was verified as having 100% identity with a PCV-2 standard (NCBI accession number: AF055394). The 263-bp DNA fragment was cloned into the pGEM-T easy vector, and the recombinant plasmid was serially diluted and quantified using real-time PCR. A standard curve was then constructed for quantification of the PCV-2 levels in field samples. The differentiation of PCV-1 and PCV-2 was carried out by analyzing the melting temperatures of the genotype-specific PCR products. RESULTS: To quantify the PCV-2 levels in field samples, a standard curve (1 x 10(2) -1 x 10(9) copies/microL) was constructed. PCV-2 concentrations as low as 1 x 10(2) copies/microL could be detected in specimens taken from the lymph nodes or infected tissues in samples of PCV-2-infected pigs. The diagnosis of PCV-1 and PCV-2 infections and the quantification of the viral load in the field samples could be completed within 45 minutes after extracting the viral DNA using a commercial extraction kit. CONCLUSION: This study demonstrate that real-time PCR is a clinically feasible method for the accurate quantification of PCV-2, and for the rapid differentiation of PCV-1 and PCV-2.

Characterisation of antimicrobial resistance patterns and class 1 integrons among Escherichia coli and Salmonella enterica serovar Choleraesuis strains isolated from humans and swine in Taiwan.

Escherichia coli isolates from humans (n=110) and swine (n=61) and Salmonella enterica serovar Choleraesuis isolates (n=95) from swine in southern Taiwan were characterised for antimicrobial resistance patterns and class 1 integrons. All E. coli isolates and S. Choleraesuis isolates were multidrug resistant and demonstrated high resistance to beta-lactams, aminoglycosides, tetracycline, sulfonamides, spectinomycin, chloramphenicol and nalidixic acid. By polymerase chain reaction and DNA sequencing, 104 (61%) E. coli isolates and 31 (33%) S. Choleraesuis isolates were found to carry class 1 integrons. The gene cassette array dfrA12-orfF-aadA2 was the most prevalent (24%) among the human and swine E. coli isolates, whilst the gene cassette array dfrA12-orfF-aadA2-sul1 was the most prevalent (24%) among S. Choleraesuis strains. For E. coil isolates, all class 1 integrons were located on conjugated plasmids. Meanwhile, human and swine E. coli isolates carrying identical gene cassettes were genetically unrelated. Our results revealed that multidrug resistance and class 1 integrons were widely present in E. coli and S. Choleraesuis isolates obtained in Taiwan and that class 1 integrons might play an important role in contributing to the horizontal transfer of antimicrobial resistance.

Protective immunity against Toxoplasma gondii in mice induced by a chimeric protein rSAG1/2.

The aim of this study was to test the protective immunity against Toxoplasma gondii in mice induced by a chimeric protein rSAG1/2. The PCR-amplified SAG2 fragment (558 bp) digested with the restriction enzyme XhoI was inserted into the XhoI site of plasmid pGEX-6p-1, termed pGexSAG2. The PCR-amplified SAG1 fragment (1,008 bp) digested with restriction enzymes EcoRI and XhoI was cloned into the EcoRI/ XhoI sites of a separate plasmid pGEX-6p-1, termed pGexSAG1. The SAG2 fragment (HindIII/HindIII) excised from pGexSAG2 was inserted into the HindIII site of pGexSAG1 and a chimeric vector constructed, pGexSAG1/2. The fusion proteins GST-SAG1/2, GST-SAG1 and GST-SAG2 were expressed in BL21 Star (DE3) Escherichia coli and purified by GSTrap FF columns. After removing the GST tag, the recombinant proteins rSAG1/2, rSAG1 and rSAG2 were independently collected and injected into different groups of mice to evaluate their protective capability. The highest proliferation of splenocytes stimulated with tachyzoite sonicate antigen (TsoAg) was observed in BALB/c mice which had received two intraperitoneal injections of rSAG1/2. Maximum production of IFN-gamma was also found in the culture supernatants of TsoAg-stimulated splenocytes from rSAG1/2-immunized mice. Finally, 73% (11/15) of mice immunized with rSAG1/2 survived at least 28 days after a lethal challenge of 1 x 10(3) live tachyzoites which killed all non-immunized mice within 10 days. Moderate survival rates were observed in mice immunized with either rSAG1 (60%) or rSAG2 (53%). These results show that the chimeric protein rSAG1/2 can elicit a Th1-associated protection against T. gondii infections in mice.

Protective immunity against Toxoplasma gondii in mice induced by the SAG2 internal image of anti-idiotype antibody.

A monoclonal antibody TGCM5 (isotype G, subclass 1, kappa light chain) against SAG2, a major surface antigen of Toxoplasma gondii tachyzoites, was produced in quantity and its Fab-containing idiotype (Id) was injected into rabbits for production of rabbit anti-Id sera. The rabbit anti-Id IgG (aId-IgG) with a SAG2 internal image was obtained by purification with a protein A column. Significant lymphocyte proliferation induced by tachyzoite lysate antigen was observed in BALB/c mice that received two injections of aId-IgG, but not in non-immunized control mice. Large amounts of gamma interferon were detected in supernatants of splenocyte cultures prepared from mice immunized with aId-IgG. Of the mice immunized with aId-IgG, 75-87.5% survived at least 28 days after a lethal challenge of 1 x 10(3) live tachyzoites, which killed all non-immunized control mice within 10 days. The results showed that the SAG2 internal image presented by aId-IgG indeed elicited a protection against the infection of T. gondii in mice.

Specific polymerase chain reaction for differential diagnosis of Dirofilaria immitis and Dipetalonema reconditum using primers derived from internal transcribed spacer region 2 (ITS2).

Both Dirofilaria immiti and Dipetalonema reconditum may be found in blood of infected dogs but it is not easy to distinguish D. immitis from D. reconditum in morphology. We cloned and sequenced the contiguous internal transcribed spacer (ITS) region, ITS1-5.8S-ITS2, of these two different parasites and published on GenBank as AF217800 for D. immiti and AF217801 for D. reconditum in this study. We designed two pairs of specific primers derived from ITS2 being used for polymerase chain reaction (PCR). The amplicons of ITS2 from D. immiti and D. reconditum are 302 and 348bp, respectively. Moreover, the limitation for amplifying ITS2 gene using this PCR demonstrated that 1 x 10(-2) microfilaria of each species of parasite smashed or even with mixed samples could be detected and the PCR products were predicted as the same as that described above. Thus, D. immiti and D. reconditum could be differentially diagnosed by this specific PCR. Seventeen clinical cases were evaluated and all of them were correctly identified. In this study, ITS1-5.8S-ITS2 of D. immiti or D. reconditum were the first time sequenced and analyzed. No significant similarity of ITS1 and ITS2 between D. immiti and D. reconditum could be observed.