Attenuation of Mycoplasma hyopneumoniae Strain ES-2 and Comparative Genomic Analysis of ES-2 and Its Attenuated Form ES-2L

Mycoplasma hyopneumoniae causes swine respiratory disease worldwide. Due to the difficulty of isolating and cultivating M. hyopneumoniae, very few attenuated strains have been successfully isolated, which hampers the development of attenuated vaccines. In order to produce an attenuated M. hyopneumoniae strain, we used the highly virulent M. hyopneumoniae strain ES-2, which was serially passaged in vitro 200 times to produce the attenuated strain ES-2L, and its virulence was evidenced to be low in an animal experiment. In order to elucidate the mechanisms underlying virulence attenuation, we performed whole-genome sequencing of both strains and conducted comparative genomic analyses of strain ES-2 and its attenuated form ES-2L. Strain ES-2L showed three large fragment deletion regions including a total of 18 deleted genes, compared with strain ES-2. Analysis of single-nucleotide polymorphisms (SNPs) and indels indicated that 22 dels were located in 19 predicted coding sequences. In addition to these indels, 348 single-nucleotide variations (SNVs) were identified between strains ES-2L and ES-2. These SNVs mapped to 99 genes where they appeared to induce amino acid substitutions and translation stops. The deleted genes and SNVs may be associated with decreased virulence of strain ES-2L. Our work provides a foundation for further examining virulence factors of M. hyopneumoniae and for the development of attenuated vaccines

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Epidemiological and genetic characteristics of swine pseudorabies virus in mainland China between 2012 and 2017

The outbreak of pseudorabies (PR) in many Bartha-K61 vaccinated farms in China in late 2011 has seriously damaged the pig industry of one of the largest producers of pork products in the world. To understand the epidemiological characteristics of the pseudorabies virus (PRV) strains currently prevalent in China, a total of 16,256 samples collected from pig farms suspected of PRV infection in 27 Provinces of China between 2012 and 2017 were evaluated for detection of PRV. Since the extensive use of gE-deleted PRV vaccine in China, the PRV-gE was applied for determining wild-type virus infection by PCR. Of the 16,256 samples detected, approximately 1,345 samples were positive for the detection of PRV-gE, yielding an average positive rate of 8.27%. The positive rates of PRV detection from 2012 to 2017 were 11.92% (153/1284), 12.19% (225/1846), 6.70% (169/2523), 11.10% (269/2424), 5.57% (147/2640), and 6.90% (382/5539), respectively. To understand the genetic characteristics of the PRV strains currently circulating, 25 PRV strains isolated from those PRV-gE positive samples were selected for further investigation. Phylogenetic analysis based on gB, gC, and gE showed that PRV strains prevalent in China had a remarkably distinct evolutionary relationship with PRVs from other countries, which might explain the observation that Bartha-K61 vaccine was unable to provide full protection against emergent strains. Sequence alignments identified many amino acid changes within the gB, gC, and gE proteins of the PRVs circulating in China after the outbreak compared to those from other countries or those prevalent in China before the outbreak; those changes also might affect the protective efficacy of previously used vaccines in China, as well as being associated in part with the increased virulence of the current PRV epidemic strains in China

New Variants of Porcine Epidemic Diarrhea Virus, China, 2011

In 2011, porcine epidemic diarrhea virus (PEDV) infection rates rose substantially in vaccinated swine herds. To determine the distribution profile of PEDV outbreak strains, we sequenced the full-length spike gene from samples from 9 farms where animals exhibited severe diarrhea and mortality rates were high. Three new PEDV variants were identified

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

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

Subcutaneous Vaccination with Attenuated Salmonella enterica Serovar Choleraesuis C500 Expressing Recombinant Filamentous Hemagglutinin and Pertactin Antigens Protects Mice against Fatal Infections with both S. enterica Serovar Choleraesuis and Bordetella bronchiseptica▿

Salmonella enterica serovar Choleraesuis strain C500 is a live, attenuated vaccine that has been used in China for over 40 years to prevent piglet paratyphoid. We compared the protective efficacies of subcutaneous (s.c.) and oral vaccination of BALB/c mice with C500 expressing the recombinant filamentous hemagglutinin type I domain and pertactin region 2 domain antigen (rF1P2) of Bordetella bronchiseptica. Protective efficacy against both S. enterica serovar Choleraesuis infection in an oral fatal challenge model and B. bronchiseptica infection in a model of fatal acute pneumonia was evaluated. Both the s.c. and oral vaccines conferred complete protection against fatal infection with the virulent parent S. enterica serovar Choleraesuis strain (C78-1). All 20 mice vaccinated s.c. survived intranasal challenge with four times the 50% lethal dose of virulent B. bronchiseptica (HH0809) compared with 4 of 20 vector-treated controls and 1 of 18 phosphate-buffered saline-treated controls that survived, but no significant protection against HH0809 was observed in orally vaccinated animals. Both the s.c. and oral vaccines elicited rF1P2-specific serum immunoglobulin G (IgG) and IgA antibodies. However, lung homogenates from s.c. vaccinated animals had detectably high levels of rF1P2-specific IgG and IgA; a much lower level of rF1P2-specific IgG was detected in samples from orally vaccinated mice, and the latter showed no evidence of local IgA. Furthermore, a more abundant and longer persistence of vaccine organisms was observed in the lungs of mice immunized s.c. than in those of mice immunized orally. Our results suggest that s.c. rather than oral vaccination is more efficacious in protecting mice from fatal challenge with B. bronchiseptica

Clonal analysis and virulent traits of pathogenic extraintestinal <it>Escherichia coli</it> isolates from swine in China

<p>Abstract</p> <p>Background</p> <p>Extraintestinal pathogenic <it>Escherichia coli</it> (ExPEC) can cause a variety of infections outside the gastrointestinal tract in humans and animals. Infections due to swine ExPECs have been occurring with increasing frequency in China. These ExPECs may now be considered a new food-borne pathogen that causes cross-infections between humans and pigs. Knowledge of the clonal structure and virulence genes is needed as a framework to improve the understanding of phylogenetic traits of porcine ExPECs.</p> <p>Results</p> <p>Multilocus sequence typing (MLST) data showed that the isolates investigated in this study could be placed into four main clonal complexes, designated as CC10, CC1687, CC88 and CC58. Strains within CC10 were classified as phylogroup A, and these accounted for most of our porcine ExPEC isolates. Isolates in the CC1687 clonal complex, formed by new sequence types (STs), was classified as phylogroup D, with CC88 isolates considered as B2 and CC58 isolates as B1. Porcine ExPECs in these four clonal complexes demonstrated significantly different virulence gene patterns. A few porcine ExPECs were indentified in phylogroup B2, the phylogroup in which human ExPECs mainly exist. However some STs in the four clonal groups of porcine ExPECs were reported to cause extraintestinal infections in human, based on data in the MLST database.</p> <p>Conclusion</p> <p>Porcine ExPECs have different virulence gene patterns for different clonal complexes. However, these strains are mostly fell in phylogenentic phylogroup A, B1 and D, which is different from human ExPECs that concentrate in phylogroup B2. Our findings provide a better understanding relating to the clonal structure of ExPECs in diseased pigs and indicate a need to re-evaluate their contribution to human ExPEC diseases.</p

Isolation of a T7-Like Lytic <i>Pasteurella</i> Bacteriophage vB_PmuP_PHB01 and Its Potential Use in Therapy against <i>Pasteurella multocida</i> Infections

A lytic bacteriophage PHB01 specific for Pasteurella multocida type D was isolated from the sewage water collected from a pig farm. This phage had the typical morphology of the family Podoviridae, order Caudovirales, presenting an isometric polyhedral head and a short noncontractile tail. PHB01 was able to infect most of the non-toxigenic P. multocida type D strains tested, but not toxigenic type D strains and those belonging to other capsular types. Phage PHB01, the first lytic phage specific for P. multocida type D sequenced thus far, presents a 37,287-bp double-stranded DNA genome with a 223-bp terminal redundancy. The PHB01 genome showed the highest homology with that of PHB02, a lytic phage specific for P. multocida type A. Phylogenetic analysis showed that PHB01 and PHB02 were composed of a genus that was close to the T7-virus genus. In vivo tests using mouse models showed that the administration of PHB01 was safe to the mice and had a good effect on treating the mice infected with different P. multocida type D strains including virulent strain HN05. These findings suggest that PHB01 has a potential use in therapy against infections caused by P. multocida type D

Comparative Genome Analysis of a Pathogenic Erysipelothrix rhusiopathiae Isolate WH13013 from Pig Reveals Potential Genes Involve in Bacterial Adaptions and Pathogenesis

Erysipelothrix rhusiopathiae is a common pathogen responsible for pig erysipelas. However, the molecular basis for the pathogenesis of E. rhusiopathiae remains to be elucidated. In this study, the complete genome sequence of the E. rhusiopathiae strain WH13013, a pathogenic isolate from a diseased pig, was generated using a combined strategy of PacBio RSII and Illumina sequencing technologies. The strategy finally generated a single circular chromosome of approximately 1.78 Mb in size for the complete genome of WH13013, with an average GC content of 36.49%. The genome of WH13013 encoded 1633 predicted proteins, 55 tRNAs, as well as 15 rRNAs. It contained four genomic islands and several resistance-associated genes were identified within these islands. Phylogenetic analysis revealed that WH13013 was close to many other sequenced E. rhusiopathiae virulent strains. The comprehensive comparative analysis of eight E. rhusiopathiae virulent strains, including WH13013, identified a total of 1184 core genes. A large proportion (approximately 75.31%) of these core genes participated in nutrition and energy uptake and metabolism as well as the other bioactivities that are necessary for bacterial survival and adaption. The core genes also contained those encoding proteins participating in the biosynthesis and/or the components of the proposed virulence factors of E. rhusiopathiae, including the capsule (cpsA, cpsB, cpsC), neuraminidase (nanH), hyaluronidase (hylA, hylB, hylC), and surface proteins (spaA, rspA, rspB). The obtaining of the complete genome sequence of this virulent strain, WH13013, and this comprehensive comparative genome analysis will help in further studies of the genetic basis of the pathogenesis of E. rhusiopathiae