Xiaomin Zhao

Evaluation of a chimeric multi-epitope-based DNA vaccine against subgroup J avian leukosis virus in chickens.

The prokaryotic expressed recombinant chimeric multi-epitope protein X (rCMEPX) had been evaluated with good immunogenicity and protective efficacy against subgroup J avian leukosis virus (ALV-J) in our previous study. In the present research, we cloned the chimeric multi-epitope gene X into the eukaryotic expression vector pVAX1 to evaluate its potency as a DNA vaccine. The purified recombinant gp85 protein and rCMEPX were used as positive controls and a DNA prime-protein boost strategy was also studied. Six experimental groups of 7-day-old chickens (20 per group) were immunized intramuscularly three times at 2weeks interval with PBS, gp85, rCMEPX, pVAX1, pVAX-X and pVAX-X+rCMEPX respectively. The antibody titers and cellular immune responses were assayed after immunization. The efficacy of immunoprotection against the challenge of ALV-J NX0101 strain was also examined. The results showed that the DNA vaccine could elicit both neutralizing antibodies and cellular responses. Immune-challenge experiments showed good protection efficacy against ALV-J infection. Particularly, the regimen involving one priming pVAX-X and twice recombinant rCMEPX boosting, induced the highest antibody titers in all immunized groups. Our results suggest that the constructed chimeric multi-epitope DNA has potential for a candidate vaccine against ALV-J when used in proper prime-boost combinations. The data presented here may provide an alternative strategy for vaccine design in chicken ALV-J prevention.

Distribution of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) in different stages of gestation sows: HP-PRRSV distribution in gestation sows

Highly pathogenic PRRSV (HP-PRRSV) emerged in China in 2006 and caused severe reproductive losses, particularly in late-term sows. To determine whether these reproductive failures were related to the susceptibility of late-term sows to HP-PRRV, 60- and 90-days of gestation sows were infected with HP-PRRSV isolate TA-12 (GenBank accession HQ417620). A monoclonal antibody specific to the C-terminal of the nucleocapsid protein was used to evaluate viral distribution by IHC. This showed that HP-PRRSV had a similar distribution in both sets of sows. However, HP-PRRSV infection led to dramatically decreased serum levels of luteinizing hormone (LH) and 17-β-estradiol (E2) in late-term sows, while only E2 was decreased in the 60-day sows. These results indicate that HP-PRRSV-induced reproductive failure is more likely due to reproductive hormone level imbalances rather than tissue tropism differences.

Comparison of genomes of Brucella melitensis M28 and the B. melitensis M5-90 derivative vaccine strain highlights the translation elongation factor Tu gene tuf2 as an attenuation-related gene.

ABSTRACT Brucella melitensis causes brucellosis, a disease affecting sheep, cattle, and sometimes humans. Attenuated B. melitensis strain M5-90, derived from virulent strain M28, is widely used as a live vaccine in ruminants in China. Genetic differences between the strains may cast light on the mechanism of attenuation. We recently reported the complete genomic sequences of M28 and M5-90. Genome organization is highly conserved between these isolates, and also with virulent strains 16 M and ATCC 23457. Analysis revealed 23 open reading frames (ORFs) with consistent differences between M5-90 and the virulent strains. Notably, the tuf2 gene encoding translation elongation factor EF-Tu from M5-90 contained 50 single nucleotide polymorphisms (SNPs) and 9 gaps (indels) compared to tuf2 of M28 or of the other virulent strains. There were no changes in tuf1. To evaluate the potential role of EF-Tu in pathogenesis, tuf1 and tuf2 mutants of M28 and an M5-90 strain harboring wild-type tuf2 were constructed, and their virulence/attenuation was evaluated in vivo. We report that the tuf2 gene plays an important role in the attenuation of M5-90 virulence.

Prevalence of Trichomonas spp. in domestic pigeons in Shandong Province, China, and genotyping by restriction fragment length polymorphism.

Oropharyngeal swabs (nu2009=u2009609) were collected randomly from 80,000 domestic pigeons (Columba livia domestica) on five pigeon farms and at one pigeon slaughterhouse in Shandong Province, China, from September 2012 to July 2013. Trichomonas spp. were detected in 206/609 (33.8%) samples. The prevalence was 14.9-31.1%, depending on different levels of sanitation and management, and was 4.8% in nestling pigeons, 13.6% in breeding pigeons and 35.2% in adolescent pigeons. Trichomonas gallinae genotypes A and B, and Trichomonas tenax-like isolates were identified by PCR-restriction fragment length polymorphism (RFLP) analysis and sequencing of the 5.8S rDNA-internal transcribed spacer (ITS) regions. RFLP analysis with the restriction enzyme BsiEI generated different RFLP band patterns between T.u2009gallinae and T.tenax-like isolates. When BsiEI RFLP analysis was combined with HaeIII RFLP analysis, all infection types of T.u2009gallinae and T.tenax-like isolates could be identified.

Synergy of subgroup J avian leukosis virus and Eimeria tenella to increase pathogenesis in specific-pathogen-free chickens

To investigate the effects of co-infections of subgroup J avian leukosis virus (ALV-J) and Eimeria tenella on the pathogenesis in specific-pathogen-free (SPF) white leghorn chickens, groups of chickens were infected with ALV-J strain NX0101 at one day of age or with E. tenella at 14 days of age or both. The control group was left uninfected and was mock-inoculated with phosphate buffer saline (PBS). Mortality rates, body weights, cecal lesions, and viremia of infected chickens in each group were evaluated. Immune status was evaluated by measuring several parameters: immune organ weight/body weight index, specific humoral responses to inactivated NDV vaccine and to inoculated E. tenella, proportions of blood CD3+CD4+ and CD3+CD8α+ lymphocytes and transcriptional levels of cytokines in blood and cecal tonsils. The results show that co-infections of ALV-J and E. tenella induced a higher mortality rate and a lower body weight in SPF chickens compared to single-pathogen infection. In co-infected chickens, ALV-J accelerated the disease symptoms induced by E. tenella, and the E. tenella extended the ALV-J viremia. Thymus atrophy, decrease in the humoral response levels to pathogens and the NDV vaccine, modifications in the blood lymphocyte sub-populations and transcriptional cytokine disorders were found in co-infected chickens compared to chickens infected with one pathogen alone and to controls. We underline a synergy between ALV-J and E. tenella that results in increasing pathogenesis in SPF chickens.

Evaluation of infection status in Chinese swine with porcine reproductive and respiratory syndrome virus by nested RT-PCR targeting nsp2 gene

One of the biggest obstacles in containing porcine reproductive and respiratory syndrome virus (PRRSV) results from its genetic diversity due to the high mutation rate. The nsp2 gene of PRRSV is the most hypervariable region of the genome. Since the emergence of highly pathogenic (HP)-PRRSV, many of PRRSV strains with a mutated nsp2 gene have been reported. To decipher the epidemiology of the PRRSV and identify the epidemic strains, a nested RT-PCR able to differentiate the nsp2 gene from different PRRSV strains was developed and used to test 550 clinical samples. The amplified products of 301-bp, 211-bp and 154-bp were corresponding to low pathogenic PRRSV (LP-PRRSV) infection without deletion in nsp2 gene, HP-PRRSV infection with 90-bp deletion in nsp2 gene and a variant PRRSV strain with 147-bp deletion in nsp2 gene, respectively. Of the 550 clinical samples, 192 including 108 serum samples and 84 tissue samples were tested PRRSV RNA positive. Of the 192 positive samples, 107 were infected with a single strain and 85 were infected with two strains. 84 out of 85 samples harboring two PRRSV strains of HP-PRRSV and PRRSV variant strain were detected. 97 out of 107 samples with single strain were detected with HP-PRRSV infection. The data indicated that HP-PRRSV containing a 90-bp deletion in the nsp2 gene remained the predominant strain in China.

Intranasal inoculation of sows with highly pathogenic porcine reproductive and respiratory syndrome virus at mid-gestation causes transplacental infection of fetuses

Transplacental infection plays a critical role in the reproductive failure induced by porcine reproductive and respiratory syndrome virus (PRRSV), yet exposure of sows and gilts to classical PRRSV generally leads to reproductive failure after 85 days of gestation. We report, for the first time, that the susceptibility of fetuses to highly pathogenic PRRSV (HP-PRRSV) is similar at 60 days and 90 days of gestation. This difference from classical PRRSV may contribute to its high pathogenicity. A field study of the HP-PRRSV vaccine in pregnant sows at mid-gestation should be considered.

Improving the immunogenicity and protective efficacy of the EtMIC2 protein against Eimeria tenella infection through random mutagenesis

In recent years, directed evolution has emerged as an efficient tool to develop and identify novel protein variants. Eimeria tenella microneme-2 (EtMIC2) is a promising vaccine candidate for use against E. tenella infection; however, it only yields partial protection. The present study aimed to improve the immunogenicity and protective efficacy of EtMIC2 through random mutagenesis. Mutagenesis gene libraries of EtMIC2 were generated using error-prone polymerase chain reaction (epPCR), and the corresponding variant proteins were displayed on the yeast cell surface. Variant EtMIC2 proteins with high immunogenicity were screened through fluorescence-activated cell sorting (FACS) based on the affinity between polyclonal antibodies and antigens. Seven effective variant proteins were screened out and heterogeneously expressed in Escherichia coli as subunit vaccines. The protective efficacy of the variant proteins against E. tenella infections was then evaluated in chicken. Two variant proteins (1130 and 2119) displayed higher immunogenicity and protective efficacy than the wild-type EtMIC2 protein against E. tenella infections, increasing body weight gains and significantly decreasing lesion scores and fecal oocyst shedding, and increasing sIgA antibody production and lymphocyte proliferation. These variants displayed potential for use in the development of subunit vaccines for coccidiosis in chickens. The present results also indicate that directed evolution technology is useful for improving the immunogenicity and protective efficacy of parasite antigens.

Effect of Dual Infection with Eimeria tenella and Subgroup J Avian Leukosis Virus on the Cecal Microbiome in Specific-Pathogen-Free Chicks

Understanding gut microflora alterations associated with gut parasites and other pathogens that drive these alterations may help to promote the understanding of intestinal flora’s role in multiple-infected individuals. This study examined the effects of dual infection with Eimeria tenella and subgroup J avian leukosis virus (ALV-J) on the chick cecal microbiome. Specific-pathogen-free (SPF) chicks were infected with either ALV-J strain NX0101 at 1u2009day of age or E. tenella at 14u2009days of age, another group was infected with both pathogens. Cecal contents from chicks were extracted at the 21u2009days of age and examined using 16S rRNA genes illumina sequencing. A genus-level opportunistic pathogen enrichment and a decrease in possible resident probiotics were observed in response to all infection groups. Of note, E. tenella mainly induced a sharp decrease in the richness and diversity of cecal microflora from infected chicks because of the serious E. tenella-induced damage to intestinal tissues. ALV-J infection led to structural changes and increased the richness and diversity of the cecal microflora. As for E. tenella and ALV-J dual infected chicks, a marked enrichment of opportunistic pathogens in addition to some other bacteria that may play a role involving cecal microbiota carbohydrate transport and metabolic functions were also found compared to single pathogen-infected chicks. Overall, this study provides valuable insights into the SPF chick cecal microbial community, the modulations of this community in response to different pathogenic infections of single or dual infections, and the interactions between different pathogens and hosts from the perspective of intestinal microflora.