Epidemiological and genetic characteristics of porcine reproduction and respiratory syndrome virus 2 in mainland China, 2017-2018.

Porcine reproductive and respiratory syndrome virus 2 (PRRSV2) is a major threat to the global pig industry, particularly in China, the world's largest pig-rearing and pork-production country. Continuously monitoring the epidemiological and genetic characteristics of PRRSV epidemic strains is beneficial for prevention and control of infection. Previously, we reported the epidemiological and genetic characteristics of PRRSV2 in China from 2012 to 2016. Here, the epidemiological and genetic characteristics of PRRSV2 in China from 2017 to 2018 are reported. During these two years, we collected different types of porcine samples from 2428 pig farms in 27 provinces in China. Of the 7980 samples collected, 2080 (26.07%) were positive for PRRSV2 ORF5 by RT-PCR. The positive rate of PRRSV detection between different regions of China ranged from 8.12% to 29.33%, and from 7.96% to 55.50% between different months. Phylogenetic analysis based on the ORF5 gene revealed that the PRRSV2 strains currently circulating in China belong to five clades, and most of the PRRSVs detected are highly pathogenic PRRSVs (HP-PRRSVs; clade IV) and PRRSV NADC30-like strains (clade I). Sequence analysis revealed multiple amino acid mutation types, including amino acid changes and deletions in both the GP5 and Nsp2 proteins. The presence of these mutations may have an effect on the evolution of the virus by altering the viral titer and/or affecting the antibody response against the virus.

O-serogroups, virulence genes, antimicrobial susceptibility, and MLST genotypes of Shiga toxin-producing Escherichia coli from swine and cattle in Central China.

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) is a leading cause of worldwide food-borne and waterborne infections. Despite an increase in the number of STEC outbreaks, there is a lack of data on prevalence of STEC at the farm level, distribution of serogroups, and virulence factors. RESULTS: In the present study, a total of 91 (6.16%) STEC strains were isolated from 1477 samples including pig intestines, pig feces, cattle feces, milk, and water from dairy farms. The isolation rates of STEC strains from pig intestines, pig feces, and cattle feces were 7.41% (32/432), 4.38% (21/480), and 9.57% (38/397), respectively. No STEC was isolated from the fresh milk and water samples. By O-serotyping methods, a total of 30 types of O-antigens were determined, and the main types were O100, O97, O91, O149, O26, O92, O102, O157, and O34. Detection of selected virulence genes (stx1, stx2, eae, ehxA, saa) revealed that over 94.51% (86/91) of the isolates carried more than two types of virulence associated genes, and approximately 71.43% (65/91) of the isolates carried both stx1 and stx2, simultaneously. Antimicrobial susceptibility tests showed that most of the STEC isolates were susceptible to ofloxacin and norfloxacin, but showed resistance to tetracycline, kanamycin, trimethoprim-sulfamethoxazole, streptomycin, amoxicillin, and ampicillin. MLST determined 13 categories of sequence types (STs), and ST297 (31.87%; 29/91) was the most dominant clone. This clone displayed a close relationship to virulent strains STEC ST678 (O104: H4). The prevalence of ST297 clones should receive more attentions. CONCLUSIONS: Our preliminary data revealed that a heterogeneous group of STEC is present, but the non-O157 serogroups and some ST clones such as ST297 should receive more attentions.

A capsule/lipopolysaccharide/MLST genotype D/L6/ST11 of Pasteurella multocida is likely to be strongly associated with swine respiratory disease in China.

Pasteurella multocida is a leading cause of respiratory disease in pigs worldwide. In this study, we determined the genetic characteristics of 115 P. multocida isolates from the lungs of pigs with respiratory disease in China in 2015 using capsular typing, lipopolysaccharide (LPS) genotyping, and virulence genotyping based on the detection of virulence-associated genes. The results showed that the isolates belonged to three capsular types: A (49.6%), D (46.1%), and nontypable (4.3%); and two LPS genotypes: L3 (22.6%) and L6 (77.4%). When combining the capsular types with the LPS genotypes, a genotype group D: L6 (46.1%) was the most prevalent among the strains. Among the 23 virulence-associated genes detected in this study, a small number of them displayed a certain level of "genotype-preference". We found that pfhA, hgbA, and hgbB had a close association with P. multocida LPS genotypes, while tadD was more associated with P. multocida capsular types. In addition, multilocus sequence typing (MLST) on 40 P. multocida isolates identified four sequence types: ST3, ST10, ST11, and ST16, and the distribution of ST11 was significantly higher than the other MLST genotypes. Interestingly, all of the ST11 isolates detected in this study were genotype D: L6 strains and they were 100% positive for hgbB. Our data suggest that a capsule/LPS/MLST genotype D/L6/ST11 is likely to be strongly associated with respiratory clinical manifestation of the disease in pigs.

Genome analysis and in vivo virulence of porcine extraintestinal pathogenic Escherichia coli strain PCN033.

BACKGROUND: Strains of extraintestinal pathogenic Escherichia coli (ExPEC) can invade and colonize extraintestinal sites and cause a wide range of infections. Genomic analysis of ExPEC has mainly focused on isolates of human and avian origins, with porcine ExPEC isolates yet to be sequenced. To better understand the genomic attributes underlying the pathogenicity of porcine ExPEC, we isolated two E. coli strains PCN033 and PCN061 from pigs, assessed their in vivo virulence, and completed and compared their genomes. RESULTS: Animal experiments demonstrated that strain PCN033, but not PCN061, was pathogenic in a pig model. The chromosome of PCN033 was 384 kb larger than that of PCN061. Among the PCN033-specific sequences, genes encoding adhesins, unique lipopolysaccharide, unique capsular polysaccharide, iron acquisition and transport systems, and metabolism were identified. Additionally, a large plasmid PCN033p3 harboring many typical ExPEC virulence factors was identified in PCN033. Based on the genetic variation between PCN033 and PCN061, corresponding phenotypic differences in flagellum-dependent swarming motility and metabolism were verified. Furthermore, the comparative genomic analyses showed that the PCN033 genome shared many similarities with genomic sequences of human ExPEC strains. Additionally, comparison of PCN033 genome with other nine characteristic E. coli genomes revealed 425 PCN033-special coding sequences. Genes of this subset included those encoding type I restriction-modification (R-M) system, type VI secretion system (T6SS) and membrane-associated proteins. CONCLUSIONS: The genetic and phenotypic differences between PCN033 and PCN061 could partially explain their differences in virulence, and also provide insight towards the molecular mechanisms of porcine ExPEC infections. Additionally, the similarities between the genomes of PCN033 and human ExPEC strains suggest that some connections between porcine and human ExPEC strains exist. The first completed genomic sequence for porcine ExPEC and the genomic differences identified by comparative analyses provide a baseline understanding of porcine ExPEC genetics and lay the foundation for their further study.

Development of a multiplex reverse transcription-quantitative PCR (qPCR) method for detecting common causative agents of swine viral diarrhea in China.

BACKGROUND: Diarrheal diseases caused by viral agents have led to a great morbidity, mortality, and economic loss in global pig industry. Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and group A porcine rotavirus (RVA) are main causative agents of swine viral diarrhea with similar clinical signs on Chinese farms and their co-infection is also common. However, it is still lack of a convenient method to detect these four agents. METHODS: A TaqMan multiplex qPCR method was developed to detect PEDV, TGEV, PDCoV, and RVA, simultaneously. This method was then applied to investigate 7,342 swine fecal samples or rectal swabs, as well as 1,246 swine intestinal samples collected from 2075 farms in China in 2022. RESULTS: Minimum detection limits of this method were 3 copies/µL for PEDV, 4 copies/µL for TGEV, 8 copies/µL for RVA, and 8 copies/µL for PDCoV, suggesting a good sensitivity. No signals were observed by using this method detecting other viral agents commonly prevalent in pigs, which is suggestive of a good specificity. Application of this method on investigating clinical samples demonstrated a relatively high positive rate for PEDV (22.21%, 1907/8588) and RVA (44.00%, 3779/8588). In addition, co-infection between PEDV and RVA was observed on 360 investigated farms, accounting for 17.35% (360/2075) of the farms where co-infection events were screened. CONCLUSIONS: A TaqMan multiplex qPCR method targeting PEDV, TGEV, PDCoV, and RVA was developed in this study. This method demonstrated a good specificity and sensitivity on investigating these four common viruses responsible for viral diarrhea on Chinese pig farms, which represents a convenient method for the monitoring and differential diagnosis of swine viral diarrhea.

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 (72.3 %) (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 × 10(6) versus 2.0 ×10(5) 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.

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.

Clonal analysis and virulent traits of pathogenic extraintestinal Escherichia coli isolates from swine in China.

BACKGROUND: Extraintestinal pathogenic Escherichia coli (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. RESULTS: 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. CONCLUSION: 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.

A triplex real-time PCR method to detect African swine fever virus gene-deleted and wild type strains.

Currently there is still no effective vaccines and drugs available for African swine fever virus (ASFV), a life-threatening virus to domestic pigs and wild boars. Therefore, accurate diagnosis is important for the prevention and control of the virus. In this study, we developed a triplex real-time PCR method to detect and differentiate ASFV gene-deleted and wild type strains based on three viral genes B646L, MGF_360-14L gene, and CD2v. Standard curves plotted showed that there was a strong linear correlation (R 2 > 0.99) between Ct values and the corresponding copy numbers of synthesized standard plasmids. The detection limits of the method for B646L, MGF_360-14L, and CD2v were 78.9, 47.0, and 82.1 copies/µl, respectively. Detection results of different types of swine viruses showed that the method only gave amplification curves to ASFV. Finally, we found the triplex real-time PCR method developed in this study displayed better results on detecting the laboratory sample mocks, and it could be used as a supplemental method to detect ASFV genotype I strains. These findings suggest that the triplex real-time PCR method developed in this study have good specificity and sensitivity. This triplex real-time PCR method might also represent an effective tool for the detection of ASFV gene-deleted and wild type strains.

Propidium Monoazide Combined With RT-qPCR Detects Infectivity of Porcine Epidemic Diarrhea Virus.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) allows sensitive detection of viral particles and viruses in epidemic samples but it cannot discriminate noninfectious viruses from infectious ones. Propidium monoazide (PMA) coupled with quantitative polymerase chain reaction (qPCR) was assessed to detect infectious viruses. Currently, there is no established test method to detect the infection of the porcine epidemic diarrhea virus (PEDV). In this study, propidium monoazide coupled with qPCR detects infectivity of PEDV. We optimized the method from the selection of primers, the working concentration of PMA, and the inactivation method using heat or ultraviolet (UV). The viruses which were treated with PMA before qPCR were inactivated using heat or UV. However, the addition of PMA alone did not affect the detection of live viruses, which indicates that a viral capsid break may be essential for PMA to bind to the genome. A repetition of the method on naked PEDV RNA suggests that it can be used to detect potentially infectious PEDV. The results indicated that an optimal plan of PMA could be extremely useful for evaluating infectious and noninfectious viruses.

Detection of suid herpesvirus 1 infectivity in pigs by propidium monoazide-qPCR.

At present, there is no effective experimental method for detecting whether the suid herpesvirus 1 (SHV-1) detected in pigs is infectious. Although the technique of quantitative polymerase chain reaction (qPCR) has significantly improved the detection rate and accuracy of the disease, it does not differentiate between infective and non-infective status of the virus. Propidium monoazide (PMA) is a dye that can be combined with DNA molecules. The decomposition of PMA produces an azene compound covalently crosslinked with DNA molecules, thereby inhibiting PCR amplification of DNA. In this study, the combination of PMA and qPCR was used to determine the infectivity of SHV-1. We optimized the method from the selection of primers, the working concentration of PMA, and the method of inactivation using UV or heat inactivation. We found that when specific primer 1 was used and a PMA working concentration was 50-100 µM, heat inactivation was able to distinguish whether SHV-1 was infectious or not. We also showed that UV prevented the virus from replicating, it did not destroy the capsid of the virus, and therefore, PMA cannot enter the virus and bind to the nucleic acid of the virus. Consequently, there is no way to identify the infectivity of the virus using UV inactivation. The study showed that the method was stable and the detection rate reached 96%. In conclusion, this method exhibited strong specificity and high sensitivity and can identify the infectivity of SHV-1. This method has practical significance for clinical virus isolation and the effects of disinfection of farms.

Genome sequence of a porcine extraintestinal pathogenic Escherichia coli strain.

Extraintestinal pathogenic Escherichia coli (ExPEC) is an important pathogen which can infect humans and animals and cause many diseases outside the intestine. Here, we report the first draft genome sequence of a porcine ExPEC strain, PCN033, isolated from a pig with meningitis.

Antimicrobial resistances of extraintestinal pathogenic Escherichia coli isolates from swine in China.

Antibiograms and relevant genotypes of porcine extraintestinal pathogenic Escherichia coli (ExPEC) isolates (n = 315) recovered between 2004 and 2007 in China were assessed. Among the 14 antimicrobials tested, the most prevalent resistance was to ampicillin, trimethoprim, sulfadimidine, tetracycline, neomycin, streptomycin, kanamycin, ciprofloxacin and ofloxacin (ranging from 81.9 to 100%). Forty-six multiresistant patterns were found. For each antimicrobial agent, ampicillin resistance was primarily mediated by bla(TEM,) streptomycin resistance by strA and strB, kanamycin/neomycin resistance by aphA1, gentamicin resistance by aac(3)-IV, quinolones resistance by mutations in gyrA, tetracycline resistance by tet(A), tet(B) and tet(G), trimethoprim resistance by dfrA7, dfrA12 and dfrA13, and sulfadimidine resistance by sul1 and sul2. Both bla(TEM-1) and bla(CTX-M-14) were found in two ESBLs-producing isolates. Strains that harbored several genes that conferred resistance to the same antimicrobial agent were often significantly more multiresistant than others. Class 1 integrons were identified in 86 (27.3%) ExPEC isolates, which harbored dfrA14, aadA2, aadA22, dfrA17, aadA5, dfrA17-aadA2, dfrA1-aadA1, dfrA12-aadA2, dfrA17-aadA5 gene cassettes in five major different variable regions, conferring resistance to trimethoprim and aminoglycosides. These results provide novel insights into the epidemiological characteristics of porcine ExPEC strains in China, and suggest the need for the prudent use of antimicrobial agents in food animals.

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.

Epidemiological Analysis From 2018 to 2020 in China and Prevention Strategy of Porcine Circovirus Type 2.

Porcine circovirus type 2 (PCV2) is one of the smallest known animal viruses and is the main pathogen of PCV-associated diseases (PCVAD). Epidemiological surveillance results have shown that the PCV2 infection rate is on the rise in China, thus, PCV2 disease prevention and control has become a huge challenge for the Chinese swine industry. We collected clinical samples from multiple different provinces in China from 2018 to 2020 and found that the positive rate of PCV2 was 53% (3619/6872), identity between the cloned 62 ORF2 genes was 84.4-100% and identity between the cloned 62 ORF2 sequences and reference sequence was 72.9-99.8%. Genetic evolution analysis found that PCV2d accounted for 79% (49/62 samples), PCV2a for 12.9% (8/62 samples), PCV2b for 8% (5/62 samples), and PCV2c and PCV2e genotypes were not found. However, most commercial PCV2 subunit vaccines are based on the PCV2a genotype, and there are very few vaccines based on PCV2b or PCV2d. Therefore, the homologous and heterologous protection ability of PCV2b and PCV2d Cap proteins based on the baculovirus against the PCV2b and PCV2d infections was evaluated, which is expected to design and develop excellent PCV2 protein vaccine candidates. This study found that both PCV2b and PCV2d Cap proteins can increase the level of humoral immunity and cellular immune response in mice. Importantly, both PCV2b and PCV2d cap proteins can provide homologous and heterologous protection against the PCV2b and PCV2d viruses. Overall, this study provides a reference for the prevention and control of PCVAD in mainland China and the development of PCV2 vaccines.

Isolation, Antimicrobial Resistance Phenotypes, and Virulence Genes of Bordetella bronchiseptica From Pigs in China, 2018-2020.

Bordetella bronchiseptica is a leading cause of respiratory diseases in pigs. However, epidemiological data of B. bronchiseptica in pigs particularly in China, the largest pig rearing country in the world is still limited. We isolated 181 B. bronchiseptica strains from 4259 lung samples of dead pigs with respiratory diseases in 14 provinces in China from 2018 to 2020. The average isolation rate of this 3-year period was 4.25% (181/4259). Antimicrobial susceptibility testing performed by disc diffusion method revealed that most of the B. bronchiseptica isolates in this study were resistant to ampicillin (83.98%), while a proportion of isolates were resistant to cefotaxime (30.39%%), chloramphenicol (12.71%), gentamicin (11.60%), florfenicol (11.60%), tetracycline (8.84%), amoxicillin (8.29%), tobramycin (6.63%), ceftriaxone (4.97%), and cefepime (0.55%). There were no isolates with resistant phenotypes to imipenem, meropenem, polymyxin B, ciprofloxacin, enrofloxacin, and amikacin. In addition, ~13.18% of the isolates showed phenotypes of multidrug resistance. Detection of antimicrobial resistance genes (ARGs) by PCR showed that 16.57% of the B. bronchiseptica isolates in this study was positive to aac(3)-IV, while 3.87%, 2.21%, 1.10%, 0.55%, 0.55%, and 0.55% of the isolates were positive to aac6'-Ib, rmtA, bla TEM, bla SHV, oqxB, and tetA, respectively. Detection of virulence factors encoding genes (VFGs) by conventional PCR showed that over 90% of the pig B. bronchiseptica isolates in this study were positive to the five VFGs examined (fhaB, 97.24%; prn, 91.16%; cyaA, 98.34%; dnt, 98.34%; betA, 92.82%). These results demonstrate B. bronchiseptica as an important pathogen associated with pig respiratory disorders in China. The present work contributes to the current understanding of the prevalence, antimicrobial resistance and virulence genes of B. bronchiseptica in pigs.

Whole-Genome Sequence Analysis of Pseudorabies Virus Clinical Isolates from Pigs in China between 2012 and 2017 in China.

Pseudorabies virus (PRV) is an economically significant swine infectious agent. A PRV outbreak took place in China in 2011 with novel virulent variants. Although the association of viral genomic variability with pathogenicity is not fully confirmed, the knowledge concerning PRV genomic diversity and evolution is still limited. Here, we sequenced 54 genomes of novel PRV variants isolated in China from 2012 to 2017. Phylogenetic analysis revealed that China strains and US/Europe strains were classified into two separate genotypes. PRV strains isolated from 2012 to 2017 in China are highly related to each other and genetically close to classic China strains such as Ea, Fa, and SC. RDP analysis revealed 23 recombination events within novel PRV variants, indicating that recombination contributes significantly to the viral evolution. The selection pressure analysis indicated that most ORFs were under evolutionary constraint, and 19 amino acid residue sites in 15 ORFs were identified under positive selection. Additionally, 37 unique mutations were identified in 19 ORFs, which distinguish the novel variants from classic strains. Overall, our study suggested that novel PRV variants might evolve from classical PRV strains through point mutation and recombination mechanisms.

Pasteurella multocida capsular: lipopolysaccharide types D:L6 and A:L3 remain to be the main epidemic genotypes of pigs in China.

Pasteurella multocida is a leading cause of respiratory disorders in pigs. This study was designed to understand the genotypical and antimicrobial resistant characteristics of P. multocida from pigs in China. To achieve this, we briefly investigated 158 P. multocida isolates from pigs with respiratory disorders in China between 2019 and 2020. Genotyping through multiplex PCR assays assigned these 158 isolates into capsular genotypes A (60.13%, 95/158), D (35.44%, 56/158), F (4.43%, 7/158), and/or lipopolysaccharide (LPS) genotypes L3 (28.48%, 45/158) and L6 (66.46%, 105/158). In addition, eight isolates (5.06%, 8/158) were found to be nontypable using the LPS genotyping method. When combining the capsular genotypes and the LPS genotypes, D: L6 (34.81%, 55/158) and A: L6 (31.65%, 50/158) were the predominant genotypes, followed by A: L3 (24.05%, 38/158). PCR detection of virulence factor-encoding genes showed that over 80% of the isolates were positive for exbB, tonB, exbD, ompH, ptfA, fimA, sodA, sodC, fur, ompA, oma87, plpB, hsf-2, nanH and hgbB, suggesting the presence of these genes were broad characteristics of P. multocida. We also found approximately 63.92% (101/158), 51.27% (81/158), 8.86% (14/158), 7.59% (12/158), 3.16% (5/158), 0.63% (1/158), and 0.63% (1/158) of the isolates grew well in media with the presence of colistin (4 µg/mL), tetracycline (16 µg/mL), tigecycline (1 µg/mL), ampicillin (32 µg/mL), chloramphenicol (32 µg/mL), cefepime (16 µg/mL), and ciprofloxacin (1 µg/mL), respectively. This study contributes to the understanding of genotypes and antimicrobial resistance profile of P. multocida currently circulation in pigs of China. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s44149-021-00031-7.

[Prokaryotic expression and immunization of glutamyl tRNA synthetase of Streptococcus suis].

OBJECTIVE: Glutamyl tRNA Synthetase (Gts) is a protease which catalyzes esterification between tRNA and glutamine. The immunogenicity of Gts was evaluated through immunization and challenge experiment. METHODS AND RESULTS: We cloned gts from the genome of SC19, and inserted it into prokaryotic expression plasmid pET28a-gts. The recombinant vector was transformed into E. coli BL21. Induced by IPTG, one 58 kD protein, was expressed and purified by using Ni - NTA column (Novagen). The purity of rGtS was 93.3% and the concentration of purified protein was 433 microg/mL. We proved the immunogenicity of recombinant protein rGtS by western blot analysis. We immunized Balb/c mice with rGtS and Freund's adjuvant, and after two boost vaccinations, all mice were challenged with 4 times LD50 amount of SC19 (1.2 x 10(9) CFU). The survive rate of vaccination group is 50% (4/8), significantly higher than blank vector control group (1/8). CONCLUSION: These results proved that GtS has certain immunogenicity and can offer partial protection against high dose challenge. Therefore Gts could be a potential candidate of subunit vaccine against Streptococcus suis.

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.