He Qigai

Loop‐mediated isothermal amplification targeting the apxIVA gene for detection of Actinobacillus pleuropneumoniae

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method performed under isothermal conditions with high specificity and efficiency. We developed a diagnostic method based on LAMP for detection of Actinobacillus pleuropneumoniae. Using six specific primers targeting the apxIVA gene, the LAMP assay rapidly amplified the target gene within 30 min, requiring only a laboratory water bath for the reaction to occur. The resulting amplificon was visualized by adding SYBR Green I to the mixture. The results obtained from testing 15 A. pleuropneumoniae reference strains and other seven bacterial species strains showed that the LAMP was as specific as and 10 times more sensitive than nested PCR. Sixty-five tonsil samples were collected from 65 healthy pigs. All the samples were negative for A. pleuropneumoniae by immunomagnetic separation-based (IMS) bacterial isolation, nested PCR and LAMP, respectively. Meanwhile, 115 tonsil samples were also collected from 115 pigs with apparent respiratory problems. Twenty-two were positive by IMS bacterial isolation. All the samples that were positive by IMS bacterial isolation were also positive by nested PCR and LAMP. The LAMP assay demonstrated exceptionally higher sensitivity than nested PCR by picking up 14 additional positive cases (chi(2) test, P<0.0001); we concluded that LAMP was a highly sensitive and reliable method for detection of A. pleuropneumoniae infection.

Development and evaluation of loop-mediated isothermal amplification for rapid detection of Haemophilus parasuis

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method performed under isothermal conditions and has a high specificity and efficiency. We developed a LAMP assay targeting the 16S rRNA gene for rapid detection of Haemophilus parasuis. The results obtained from testing 31 H. parasuis strains and 28 other bacterial species strains showed that LAMP was as specific as, and more sensitive than, nested PCR. Fifty-five lung samples were collected from 55 healthy pigs. All the samples were negative for H. parasuis by bacterial isolation, nested PCR and LAMP, respectively. In addition, 122 lung samples were collected from 122 pigs with apparent respiratory problems. Sixty-five were positive by bacterial isolation. All the samples that were positive by bacterial isolation were also positive by nested PCR and LAMP. The LAMP assay demonstrated higher sensitivity than nested PCR, picking up 16 additional cases. The LAMP assay also gave a same result compared with the nested PCR when the two assays were used, respectively, to detect H. parasuis from samples obtained from experimentally infected pigs. We concluded that LAMP is a highly sensitive and reliable method for detection of H. parasuis infection.

The development and application of the latex agglutination test to detect serum antibodies against Japanese encephalitis virus

The attenuated SA14-14-2 strain of Japanese encephalitis virus (JEV) was cultured in BHK-21 cells. The viral supernatant was purified and concentrated with PEG (MW 20 000). A suitable concentration of JEV antigen was used to sensitize latex to prepare the latex antigen. The specificity, sensitivity and stability of the antigen were assessed. A latex agglutination test (LAT) was developed for rapidly detecting antibody against JEV infection. The LAT and haemagglutination inhibition (HI) assay were compared by simultaneously testing 35 porcine serum samples from five farms. Ninety per cent (20/23) of the samples were seropositive by both assays. No significant difference was found between the two methods (p > 0.05). Furthermore, when 1613 porcine sera from 120 farms were tested by LAT, the number of positive sera was 652, while that of negative sera was 961, ranging from 20% to 50% positive throughout the year. These results indicate that LAT is an appropriate candidate method for epidemiological surveys for and diagnosis of Japanese encephalitis.

Development of a latex agglutination test using the major epitope domain of glycoprotein E of pseudorabies virus expressed in E. coli to differentiate between immune responses in pigs naturally infected or vaccinated with pseudorabies virus.

A 0.8 kb DNA fragment encoding the major epitope domain of glycoprotein E (gE) of pseudorabies virus (PRV) was inserted downstream of the T7 promoter of an expression vector, pET-28b, to yield the recombinant plasmid pETgE804. After induction by isopropy1-β-D-thiogalactopyranoside (IPTG), a high level expression of fusion protein was obtained. SDS-PAGE and western immunoblotting analysis showed that the fusion protein was 38 kDa and could bind with antisera against PRV. The protein existed mainly in the form of the inclusion body. After being denatured and renatured, the protein was used to prepare the latex antigen. The concentration of antigen, temperature and time for sensitization were optimized. The latex agglutination test (LAT) was able to differentiate sera of PRV-infected pigs from those of gE-deletion vaccine-immunized pigs. The diagnostic specificity and sensitivity of the developed gE latex agglutination test (gE-LAT) were also evaluated by using sets of sera. The diagnostic specificity and diagnostic sensitivity of the gE-LAT were 96.77% and 95.76%, respectively. For comparison between gE-LAT and a commercial blocking enzyme-linked immunosorbent assays (ELISA), 260 serum samples were tested. The coincidence frequency of both assays was 96.94% (252/260). No significant difference was found between the two methods (p>0.05). For comparison between the abilities of gE-LAT and gE-ELISA to detect sera with low titres of gE-specific antibody, 66 sera from 22 pigs were tested. The data indicate that the gE-LAT is of similar sensitivity to gE-ELISA. These results indicate that gE-LAT using recombinant gE might be very useful as a routine screening method for the differential diagnosis of PRV infection.

Microarray-Based Detection and Differentiation of Virulent and Attenuated Pseudorabies Virus

DNA microchip used in this study was formed from miniature arrays of pseudorabies virus (PrV) gene-specific probes immobilized on a glass surface. Hybridization using DNA microchip (microarrays) was used for differentiation between virulent and attenuated PrV. The presence of four gene segments (gB, gD, gE+, and gE-) encoding conservative glycoprotein B (gB), D (gD), and E (gE) of PrV was monitored using multiplex PCR. The amplicons were labeled with Cy5 or Cy3 dyes followed by hybridization to the gene-specific capture probes on the microchip. The presence of gD and gB, gE+ gene fragments was shown in virulent (gE+ genotype) and attenuated PrV (gE- genotype), whereas gE- gene (deleted domain in gE gene) was demonstrated only in virulent, not in attenuated, virus. No cross-hybridization was observed when fluorescence labeled-PCR products of PrV were hybridized using capture probes of related viruses, such as porcine respiratory and reproductive syndrome virus (PRRSV), porcine parvovirus (PPV), Japanese encephalitis virus (JEV), and porcine circovirus type 2 (PCV-2). The assay was 10 times sensitive than gD gene-specific PCR. Overall, the results of this study suggested that the microarray might be very useful for detection and differentiation of virulent PrV from attenuated one.

Cloning and Expression of Actinobacillus pleuropneumoniae Gene Coding for TbpA and Development of an Indirect TbpA-ELISA

This study presents the cloning and expression of gene encoding transferrin-binding protein A from Actinobacillus pleuropneumoniae in Escherichia coli expression system and the development of an indirect TbpA-ELISA. The gene coding TbpA was amplified from the A. pleuropneumoniae serotype 2 genome using polymerase chain reaction and cloned to pET-28b expression vector under the control of strong, inducible T7 promoter. The recombinant plasmid was expressed in E. coli BL21 (DE3). The expressed fusion protein was analyzed using SDS-PAGE and Western blotting. The diagnostic potential of recombinant TbpA (rTbpA) was evaluated through an antibody-detection indirect ELISA based on the purified rTbpA. The TbpA antibodies were detectable in mice on day 7 after vaccination with purified rTbpA protein or infection with A. pleuropneumoniae serotype 10 with the TbpA-based ELISA. In addition, the TbpA-ELISA was able to detect 12 serotyping rabbit antisera postinoculation (PI) with A. pleuropneumoniae 12 serotypes experimentally. The comparable result was obtained by detecting the 117 clinical serum samples using, respectively, the TbpA-ELISA and indirect hemagglutination test (IHA) based on multiplex antigen. The result indicates that the TbpA-ELISA was the more sensitive method compared with the Mix-IHA method because of its consistent presence in A. pleuropneumoniae serotypes. In conclusion, the conserved TbpA of A. pleuropneumoniae can be used for the development of a cross-serotype diagnostic method for the detection of antibodies against A. pleuropneumoniae.

Development and preliminary application of gE enzyme-linked immunosorbent assay for detection of the antibody to gE protein of Pseudorabies virus in pigs

Abstract To differentiate pigs infected with Pseudorabies virus (PrV) from pigs vaccinated with gE-PrV, aglycoprotein E enzyme-linked immunosorbent assay (gE-ELISA) based on recombinantglycoprotein E (gE) (which was expressed by Escherichia coli, purified, denatured andrenatured) was developed. By testing 115 serum samples, the diagnostic specificity andsensitivity of the developed gE-ELISA were evaluated to be 94.5% and 96.7%, respectively. Fiveserum samples were tested with plates from five lots, and the results had a coefficient ofvariation of less than 10%, showing good reproducibility of gE-ELISA. This gE-ELISA wascompared with a commercial blocking ELISA by testing 356 serum samples. The agreement rateof the two assays was 92.13% (328/356). These results suggested that the gE-ELISA developedin our laboratory could be used in differentiating PrV-infected and gE-PrV-vaccinated pigs. Keywords: Pseudorabies virus; glycoprotein E; enzyme-linked immunosorbent assay (ELISA) Introduction Aujeszky’s disease is one of the most serious infectiousdiseases of several domestic and wild animals. It is causedby Pseudorabies virus (PrV). Swine are its natural hostand reservoir. Once piglets and other animals are infectedby PrV, the mortality is generally 100%. Reproductionrates will be low if boars and sows are infected by PrV.Pseudorabies can easily spread between pigs. It causeshigh economic losses in the pig industry (Mettenleiter,2000). Control of pseudorabies depends mainly onvaccination, as there are no effective therapeutic drugs.In many developed countries, pseudorabies has beencontrolled or eradicated by using marker vaccines anddifferentiation diagnostic assays (Stegemam, 1997). But inChina, pseudorabies still breaks out frequently and badlyaffects the Chinese pig industry. Therefore, studying themarker vaccine and differentiation diagnostic assays, andputting eradication campaigns into practice, are essentialin China.The genome of PrV codes for more than 100 proteins,and there are about 50 proteins on the mature virus. Atleast 11 glycoproteins (gB, gC, gD, gE, gG, gH, gI, gK, gL,gM and gN) were found in the membrane of PrV (Hampl,1984). The gE gene is a non-essential glycoprotein genefor PrV replication, but is associated with its virulence.Thus deletion of the gE gene does not influencereplication, but decreases the virulence of the virus.