Qigai He

Isolation, Antimicrobial Resistance, and Virulence Genes of Pasteurella multocida Strains from Swine in China

ABSTRACT A total of 233 isolates of Pasteurella multocida were obtained from 2,912 cases of clinical respiratory disease in pigs in China, giving an isolation rate of 8.0%. Serogroup A P. multocida isolates were isolated from 92 cases (39.5%), and serogroup D isolates were isolated from 128 cases (54.9%); 12 isolates (5.2%) were untypeable. P. multocida was the fourth most frequent pathogenic bacterium recovered from the respiratory tract, after Streptococcus suis, Haemophilus parasuis, and Escherichia coli. All isolates were characterized for their susceptibilities to 20 antibiotics and the presence of 19 genes for virulence factors (VFs). The frequency of antimicrobial resistance among P. multocida isolates from swine in China was higher than that reported among P. multocida isolates from swine in from other countries, and 93.1% of the isolates showed multiple-drug resistance. There was a progressive increase in the rate of multiresistance to more than seven antibiotics, from 16.2% in 2003 to 62.8% in 2007. The resistance profiles suggested that cephalosporins, florfenicol, and fluoroquinolones were the drugs most likely to be active against P. multocida. Use of PCR showed that colonization factors (ptfA, fimA, and hsf-2), iron acquisition factors, sialidases (nanH), and outer membrane proteins occurred in most porcine strains. The VFs pfhA, tadD, toxA, and pmHAS were each present in <50% of strains. The various VFs exhibited distinctive associations with serogroups: concentrated in serogroup A, concentrated in serogroup D, or occurring jointly in serogroups A and D. These findings provide novel insights into the epidemiological characteristics of porcine P. multocida isolates and suggest that the potential threat of such multiresistant bacteria in food-producing animals should not be neglected.

Protection induced by intramuscular immunization with DNA vaccines of pseudorabies in mice, rabbits and piglets.

Glycoprotein gene gB, gC and gD of pseudorabies virus (PrV) strain Ea, which was isolated locally in Wuhan, were cloned from the viral genome DNA and expressed in vitro controlled by the major immediately-early promotor/enhancer of HCMV. In the presented paper, Balb/c mice, rabbits and piglets were vaccinated intramuscularly two times at 2-week interval with those eukaryotic expression plasmid pcDB, pcDC and pcDD, respectively. The animals injected with pcDB, pcDC, pcDD or mix DNA developed anti-PrV antibodies. Neutralizing antibody titers obtained 2-5log(2), 2 weeks after the second vaccination. Cellular immune responses were also detected by lymphoproliferation assay and cytotoxic T lymphocyte (CTL) activity assay in all groups vaccinated with DNA. Immune responses elicited by DNA vaccines provided protections with different degrees against lethal dose PrV challenge. In mice, protections induced by pcDC, pcDD or mix DNA were 100%, similar to that by inactivated vaccine. Protections were more than 50% induced by pcDC, pcDD or mix DNA in rabbits. Protections induced by pcDB were the lowest among DNA immunization in mice or rabbits. However, pcDB could elicit the higher cellular responses in rabbits or piglets. In piglets, body temperatures of animals injected with pcDB, pcDC, pcDD or mix DNA did not change significantly after challenge with 2x10(5) pfu of PrV strain Ea, and the means daily growth post-challenge of those animals were higher than those injected with inactivated vaccine or parental plasmid. Neither DNA vaccines nor inactivated vaccine could prevent or delay virus excretion after challenge. Our experiments in experimental animals and natural hosts suggested the efficiency and potential application of DNA vaccines for pseudorabies in pigs.

Occurrence and investigation of enteric viral infections in pigs with diarrhea in China

Between February 2011 and February 2012, 985 and 324 samples were collected from diarrheal and healthy pigs, respectively, to detect porcine epidemic diarrhea virus (PEDV), porcine kobuvirus (PKoV), porcine bocavirus (PBoV), porcine group A rotavirus (GARV), and transmissible gastroenteritis virus (TGEV). PEDV and PKoV clearly predominated in diarrheal pigs. Compared to healthy pigs, a substantial prevalence of mixed infections was observed in diarrheal pigs (72.3xa0%) (P < 0.001). All of the coinfections were grouped into 13 patterns. The results of quantitative PCR showed PEDV in diarrheal pigs had a slightly higher mean viral load than that in healthy pigs (7.9 × 106 versus 2.0 ×105 copies/g of stool), while similar mean viral loads were observed for PKoV and PBoV. These findings reveal the severity of coinfections in diarrheal disease and suggest that attention should be paid to synthetic administration and vaccination for its prevention and control.

Detection of porcine circovirus type 2, porcine parvovirus and porcine pseudorabies virus from pigs with postweaning multisystemic wasting syndrome by multiplex PCR

Multiplex PCR was established to detect porcine circovirus type 2 (PCV-2), porcine parvovirus (PPV) and porcine pseudorabies virus (PRV) and applied to samples from 137 piglets exhibiting clinical signs of postweaning multisystemic wasting syndrome (PMWS). PCV-2 DNA was detected from all samples. Moreover, 43 samples were positive for PPV but negative for PRV; 11 samples were positive for PRV but negative for PPV; and 35 samples were positive both for PPV and PRV. These results suggests that PCV-2 co-infection with PRV and PPV may play an important role in PMWS. Also, multiplex PCR is an appropriate candidate method for diagnosis of PCV-2, PRV and PPV simultaneously in field cases.

The occurrence of Bordetella bronchiseptica in pigs with clinical respiratory disease

Between January 2003 and September 2008, 652 Bordetella bronchiseptica isolates were cultured from 3506 lung samples collected from pigs with respiratory disease. Over the 6-year period, the average isolation rate was 18.6%, making B. bronchiseptica the fourth most frequently isolated pathogenic bacterium from those lung samples. The isolation rates in different years and provinces ranged from 15.2% to 25.7% and 17.3% to 20.7%, respectively. There were significant influences of sampling month and pig age on bacterial isolation (P<0.05). Streptococcus suis (29.9%), Haemophilus parasuis (26.7%) and Escherichia coli (21.6%) were isolated most frequently in association with B. bronchiseptica. All 12 toxigenic Pasteurella multocida strains co-isolated with B. bronchiseptica from 63 cases of atrophic rhinitis were classified into serogroup D. The results suggest that B. bronchiseptica infection is highly prevalent in pig farms in China, and is often accompanied by co-infection with other bacteria.

Quantum dots-gold(III)-based indirect fluorescence immunoassay for high-throughput screening of APP

Here we report a indirect fluorescence immunoassay for high-throughput screening of APP based on the fluorescence quenching of quantum dots by gold(III), which were dissolved from gold nanoparticles-Rabbit anti-Pig IgG conjugate, and further utilize this system to detect APP in pig serum with high sensitivity and specificity.

Novel pseudorabies virus variant with defects in TK, gE and gI protects growing pigs against lethal challenge

One of the distinct features of the emerging Chinese pseudorabies virus (PRV) variant is its ability to cause severe neurological signs and high mortality in growing pigs in Bartha-K61-vaccinated pig farms. Either single- or multiple-gene-deleted live vaccine candidates have been developed; however, none was evaluated thoroughly in growing pigs. Here, we generated rSMXΔgI/gEΔTK, an attenuated PRV variant with defects in TK, gI and gE genes. The growth kinetics of the attenuated virus was similar to the wild type (wt) strain. It was safe for 1-day-old piglets. Twenty one-day-old weaned pigs were immunized intramuscularly either with 10(6.0) TCID50 of rSMXΔgI/gEΔTK or one dose of commercial Bartha-K61 vaccine, or with DMEM, and were challenged intranasally with 10(7.0) TCID50 wt virus at 28 days post vaccination. rSMXΔgI/gEΔTK elicited higher level neutralization antibody against both PRV variant SMX and Bartha-K61 strain, while Bartha-K61 vaccine elicited lower neutralization activity of antibody against SMX. After challenge, all pigs in rSMXΔgI/gEΔTK group survived without any clinical signs, while unvaccinated group showed 100% mortality, and Bartha-K61 group showed severe respiratory symptoms and 3 out of 5 pigs exhibited severe neurological signs. Pigs in rSMXΔgI/gEΔTK group gained significantly higher body weight and diminished viral excretion titer and period, compared with Bartha-K61 group. Furthermore, the safety and efficacy of rSMXΔgI/gEΔTK was also evaluated in sheep and compared with local vaccine in growing pigs. These data suggest that the attenuated strain rSMXΔgI/gEΔTK is a promising live marker vaccine candidate for PR control in the context of emerging PRV variants.

Inhibition of porcine circovirus type 1 and type 2 production in PK-15 cells by small interfering RNAs targeting the Rep gene

n Abstractn n Porcine circovirus type 1 (PCV1) and type 2 (PCV2) are two genotypes of porcine circovirus. Both of them are presumed to be widespread in the swine population. Currently, there is no specific treatment for their infections. RNA interference (RNAi) is a sequence-specific RNA degradation mechanism mediated by small interfering RNA (siRNA), which represents a possible therapeutic application for the treatment of viral infections. In this study, three siRNA expression plasmids (pS-RepA, pS-RepB and pS-RepC) were generated to target three different coding regions of the Rep protein (Rep) of PCV. These siRNAs were used to inhibit PCV production in a porcine kidney cell line, PK-15 cells. Our results revealed that Rep gene expression was inhibited by pS-RepA, pS-RepB and pS-RepC to different degrees. Moreover, our study also showed that the production of PCV1 and PCV2 was reduced by these siRNAs. pS-RepC, which targets the middle region of Rep gene, proved to be the most efficient siRNA for inhibition of Rep expression and viral production. Taken together, our data suggest that RNAi could be investigated as a potential treatment for PCV infection.n n

Immunogenicity of recombinant protective antigen and efficacy against intranasal challenge with Bordetella bronchiseptica.

Bordetella bronchiseptica is a Gram-negative respiratory pathogen that causes substantial disease in a variety of animals. Filamentous hemagglutinin (FHA) and pertactin are important attachment factors and protective immunogens, which serve as protective antigens in several animal models of infection with B. bronchiseptica. Here, we showed the efficacy of subcutaneous immunization of mice with a recombinant protein rF1P2, which consisted of the important immunodominant protective type I domain (F1) of FHA and the highly immunogenic region II domain (P2) of pertactin. Groups of mice tested, when challenged with different strains of B. bronchiseptica were fully protected, with long-lasting immunity to lethal B.bronchiseptica challenge, whereas mice immunized with Freunds adjuvant alone or PBS were not. In rF1P2-immunized mice, specific antibodies lasted for more than 120 days, and the IgG1/IgG2a ratio remained at a constant level till the end of the study. This suggests that rF1P2-induced a long-lasting balanced humoral immune responses and immunological memory in mice. rF1P2-specific antisera inhibited hemagglutination associated with full-length mature FHA. Furthermore, passive antiserum transfer from immunized animals completely protected naive mice from subsequent B. bronchiseptica challenge. These data may have implications for the development of safe and efficacious subunit vaccines for the prevention of bordetellosis, and may contribute to future acellular whooping cough vaccines.