Assessment of probiotic Bacillus velezensis supplementation to reduce Campylobacter jejuni colonization in chickens.

Campylobacter jejuni continues to be a major public health issue worldwide. Poultry are recognized as the main reservoir for this foodborne pathogen. Implementing measures to decrease C. jejuni colonization on farms has been regarded as the most effective strategy to control the incidence of campylobacteriosis. The probiotics supplementation has been regarded as an attractive approach against C. jejuni in chickens. Here the inhibitory effects of one probiotic B. velezensis isolate CAU277 against C. jejuni was evaluated in vitro and in vivo. The in vitro antimicrobial activity showed that the supernatant of B. velezensis exhibited the most pronounced inhibitory effects on Campylobacter strains compared to other bacterial species. When co-cultured with B. velezensis, the growth of C. jejuni reduced significantly from 7.46 log10 CFU/mL (24 h) to 1.02 log10 CFU/mL (48 h). Further, the antimicrobial activity of B. velezensis against C. jejuni remained stable under a broad range of temperature, pH, and protease treatments. The in vivo experiments demonstrated that oral administration of B. velezensis significantly reduced the colonization of C. jejuni by 2.0 log10 CFU/g of feces in chicken cecum at 15 d postinoculation. In addition, the supplementary of B. velezensis significantly increased microbial species richness and diversity in chicken ileum, especially enhanced the bacterial population of Alistipes and Christensenellaceae, and decreased the existence of Lachnoclostridium. Our study presents that B. velezensis possesses antimicrobial activities against C. jejuni and promotes microbiota diversity in chicken intestines. These findings indicate a potential to develop an effective probiotic additive to control C. jejuni infection in chicken.

Transcriptional Differential Analysis of Nitazoxanide-Mediated Anticanine Parvovirus Effect in F81 Cells.

Canine parvovirus (CPV) is a single-stranded DNA virus that can cause typical hemorrhagic enteritis, and it is one of the common canine lethal viruses. In previous studies, we screened the Food and Drug Administration (FDA)'s drug library and identified nitazoxanide (NTZ), which has anti-CPV capabilities. To investigate the potential antiviral mechanisms, we first reconfirmed the inhibitory effect of NTZ on the CPV by inoculating with different doses and treating for different lengths of time. Then, the differences in the transcription levels between the 0.1%-DMSO-treated virus group and the NTZ-treated virus group were detected using RNA-seq, and a total of 758 differential expression genes (DEGs) were finally identified. Further Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the DEGs revealed that these genes are involved in a variety of biological processes and/or signaling pathways, such as cell cycle, mitosis and cell proliferation and differentiation. A protein-protein interaction (PPI) analysis further identified hub genes associated with cell cycle and division among the DEGs. In addition, the expression levels of some of the enriched genes were detected, which were consistent with the high-throughput sequencing results. Moreover, when the cell cycle was regulated with cell cycle checkpoint kinase 1 (Chk1) inhibitor MK-8776 or Prexasertib HCl, both inhibitors inhibited the CPV. In summary, the transcriptome differential analysis results presented in this paper lay the foundation for further research on the molecular mechanism and potential targets of NTZ anti-CPV.

Chaperonin TRiC/CCT subunit CCT7 is involved in the replication of canine parvovirus in F81 cells.

Canine parvovirus (CPV) is one of the most common lethal viruses in canines. The virus disease is prevalent throughout the year, with high morbidity and mortality rate, causing serious harm to dogs and the dog industry. Previously, yeast two hybrid method was used to screen the protein chaperonin containing TCP-1 (CCT7) that interacts with VP2. However, the mechanism of interactions between CCT7 and VP2 on CPV replication remains unclear. In this study, we first verified the interaction between CCT7 and viral VP2 proteins using yeast one-to-one experiment and co-immunoprecipitation (CoIP) experiment. Laser confocal microscopy observation showed that CCT7 and VP2 were able to co-localize and were mostly localized in the cytoplasm. In addition, the study of VP2 truncated mutant found that the interaction region of VP2 with CCT7 was located between amino acids 231 and 320. Cycloheximide (CHX) chase experiments showed that CCT7 can improve the stability of VP2 protein. After further regulation of CCT7 expression in F81 cells, it was found that the expression level of VP2 protein was significantly reduced after knocking down CCT7 expression by RNA interference (RNAi) or HSF1A inhibitor, and increased after overexpressing host CCT7. The study reveals the role of VP2 interacting protein CCT7 in the replication process of CPV, which could provide a potential target for the prevention and control of CPV.

Exposure to Sublethal Ciprofloxacin Induces Resistance to Ciprofloxacin and Cross-Antibiotics, and Reduction of Fitness, Biofilm Formation, and Apx Toxin Secretion in Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae, the etiological agent of porcine pleuropneumonia, is increasingly resistant to antibiotics. However, little is known about the mechanisms of antibiotic resistance in this pathogen. In this study, we experimentally evolved the reference strain of both A. pleuropneumoniae serovar 1 and serovar 7, the most prevalent serovars worldwide, to quinolone resistance by sequential exposure to subinhibitory concentrations of ciprofloxacin. The adaptive ciprofloxacin-resistant mutants of A. pleuropneumoniae serovar 1 and serovar 7 had a minimum inhibitory concentration (MIC) increment from 0.004 to 1 or 2 µg/mL, respectively. Adaptation to ciprofloxacin was shown to confer quinolone resistance with a 32- to 512-fold increase (serovars 1 and 7, respectively) as well as cross-resistance to ampicillin with an increased MIC by 16,384- and 64-fold (serovars 1 and 7, respectively). The genetic analysis of quinolone resistance-determining region mutations showed that substitutions occurred in gyrA (S83A) and parC (D84N) of serovar 1, and gyrA (D87N) of serovar 7. The ciprofloxacin-resistant mutants showed significantly reduced bacterial fitness. The mutants also showed changes in efflux ability and biofilm formation. Notably, the transcription and secretion levels of Apx toxins were dramatically reduced in ciprofloxacin-resistant mutants compared with their wild-type strains. Altogether, these results demonstrated marked phenotypic changes in ciprofloxacin-resistant mutants of A. pleuropneumoniae. The results stress the need for further studies on the impact of both the genotypic and phenotypic characteristics of A. pleuropneumoniae following exposure to subinhibitory concentrations of antibiotics.

Ct value-based real time PCR serotyping of Glaesserella parasuis.

Glaesserella parasuis is the causative agent of Glässer's disease in swine. Serotyping plays an essential role in prevalence investigations and in the development of vaccination strategies for the prevention of this disease. Molecular serotyping based on variation within the capsule loci of the 15 serovars is more accurate and efficient than traditional serological serotyping. To reduce the running time and facilitate ease of data interpretation, we developed a simple and rapid cycle threshold (Ct) value-based real time PCR (qPCR) method for the identification and serotyping of G. parasuis. The qPCR method distinguished between all 15 serovar reference strains of G. parasuis with efficiency values ranging between 85.5 % and 110.4 % and, R2 values > 0.98. The qPCR serotyping was evaluated using 83 clinical isolates with 43 of the isolates having been previously assigned to a serovar by the gel immuno-diffusion (GID) assay and 40 non-typeable isolates. The qPCR results of 41/43 (95.3 %) isolates were concordant with the GID assay except two isolates of serovar 12 were assigned to serovar 5. In addition, the qPCR serotyping assigned a serovar to each of the 40 non-typeable isolates. Of the 83 isolates tested to assign a serovar, a concordance rate of 98.8 % (82/83) was determined between the qPCR and the previously reported multiplex PCR of Howell et al. (2015) (including those that were either serovars 5 or 12). Despite the inability to differentiate between serovars 5 and 12, the Ct value-based qPCR serotyping represents an attractive alternative to current molecular serotyping method for G. parasuis and could be used for both epidemiological monitoring and the guidance of vaccination programs.

Immunization of Chickens with the Enterobactin Conjugate Vaccine Reduced Campylobacter jejuni Colonization in the Intestine.

Campylobacter jejuni is the leading bacterial cause of human enteritis in developed countries. Chicken is the major animal reservoir of C. jejuni and a powerful infection model for human campylobacteriosis. No commercial vaccine against C. jejuni is available to date. The high affinity iron acquisition mediated through enterobactin (Ent), a small siderophore, plays a critical role in the colonization of C. jejuni in the intestine. Recently, an innovative Ent conjugate vaccine has been demonstrated to induce high-level of Ent-specific antibodies in rabbits; the Ent-specific antibodies displayed potent binding ability to Ent and inhibited Ent-dependent growth of C. jejuni. In this study, using specific-pathogen-free (SPF) chickens, we performed three trials to evaluate the immunogenicity of the Ent conjugate vaccine and its efficacy to control C. jejuni colonization in the intestine. The purified Ent was conjugated to the carrier keyhole limpet hemocyanin (KLH). Intramuscular immunization of chickens with the Ent-KLH conjugate for up to three times did not affect the body weight gain, the development of major immune organs and the gut microbiota. In the first two trials, immunizations of chickens with different regimens (two or three times of vaccination) consistently induced strong Ent-specific immune response when compared to control group. Consistent with the high-level of systemic anti-Ent IgG, C. jejuni colonization was significantly reduced by 3-4 log10 units in the cecum in two independent vaccination trials. The third trial demonstrated that single Ent-KLH vaccination is sufficient to elicit high level of systemic Ent-specific antibodies, which could persist for up to eight weeks in chickens. Taken together, the Ent-KLH conjugate vaccine could induce high-level of Ent-specific antibodies in chickens and confer host protection against C. jejuni colonization, which provides a novel strategy for Campylobacter control in poultry and humans.

Oral Immunization of Chickens with Lactococcus lactis Expressing cjaA Temporarily Reduces Campylobacter jejuni Colonization.

Campylobacter jejuni is the leading cause of human foodborne enteritis worldwide. Poultry products are regarded as the main source of human campylobacteriosis. Strategies are being developed to reduce colonization of poultry by Campylobacter. The membrane transport protein CjaA was reported to stimulate mucosal immune responses, which can reduce the C. jejuni load in chickens. In this study, oral immunization of broilers with food-grade Lactococcus lactis NZ3900/pNZ8149 carrying the C. jejuni cjaA gene was examined for the ability to reduce colonization of broilers by Campylobacter. The Usp45 signal peptide and the Escherichia coli heat-labile enterotoxin B subunit (LTB) gene fragments were inserted into the upstream and downstream of the cjaA gene for secretory expression and immune enhancement, respectively. The cjaA gene and the fusion cjaA-ltb gene were both expressed in recombinant L. lactis, and the single cjaA gene was secretory expressed in the recombinant strain. Oral administration of two recombinant L. lactis strains expressing the cjaA gene and the fusion cjaA-ltb gene both stimulated specific anti-CjaA serum IgY responses significantly. While the average intestinal sIgA responses in these groups were higher compared with the control groups, they were not significantly different. Chicken challenge experiments showed that the colonization levels of C. jejuni in the groups provided oral immunization with two recombinant L. lactis-delivered CjaA strains were significantly lower than that of the control group at 5 d postinoculation, but there was no significant difference in C. jejuni colonization among all groups at 9 d. These results indicated that recombinant L. lactis with secretory expression of CjaA is a promising live vector vaccine against C. jejuni colonization of chickens. The immunization regimen requires further optimization to ideally stimulate detectable levels of intestinal sIgA to enhance the level of inhibition of C. jejuni colonization.

Inhibitory Effects of Antiviral Drug Candidates on Canine Parvovirus in F81 cells.

Canine parvovirus (CPV) is a common etiological agent of acute enteritis, which occurs globally in domestic and wild carnivores. Despite the widespread use of inactivated or live attenuated vaccines, the emergence of antigenic variants and the influence of maternal antibodies have raised some concerns regarding the efficacy of commercial vaccines. While no specific antiviral therapy for CPV infection exists, the only treatment option for the infection is supportive therapy based on symptoms. Thus, there is an urgent medical need to develop antiviral therapeutic options to reduce the burden of CPV-related disease. In this study, a cytopathic effect (CPE)-based high-throughput screening assay was used to screen CPV inhibitors from a Food and Drug Administration (FDA)-approved drug library. After two rounds of screening, seven out of 1430 screened drugs were found to have >50% CPE inhibition. Three drugs-Nitazoxanide, Closantel Sodium, and Closantel-with higher anti-CPV effects were further evaluated in F81 cells by absolute PCR quantification and indirect immunofluorescence assay (IFA). The inhibitory effects of all three drugs were dose-dependent. Time of addition assay indicated that the drugs inhibited the early processes of the CPV replication cycle, and the inhibition effects were relatively high within 2 h postinfection. Western blot assay also showed that the three drugs had broad-spectrum antiviral activity against different subspecies of three CPV variants. In addition, antiapoptotic effects were observed within 12 h in Nitazoxanide-treated F81 cells regardless of CPV infection, while Closantel Sodium- or Closantel-treated cells had no pro- or antiapoptotic effects. In conclusion, Nitazoxanide, Closantel Sodium, and Closantel can effectively inhibit different subspecies of CPV. Since the safety profiles of FDA-approved drugs have already been extensively studied, these three drugs can potentially become specific and effective anti-CPV drugs.

Inactivation Efficacy of Nonthermal Plasma-Activated Solutions against Newcastle Disease Virus.

In recent years, plasma-activated solutions (PASs) have made good progress in the disinfection of medical devices, tooth whitening, and fruit preservation. In this study, we investigated the inactivation efficacy of Newcastle disease virus by PASs. Water, 0.9% NaCl, and 0.3% H2O2 were excited by plasma to obtain the corresponding solutions PAS(H2O), PAS(NaCl), and PAS(H2O2). The complete inactivation of virus after PAS treatment for 30 min was confirmed by the embryo lethality assay (ELA) and hemagglutination (HA) test. Scanning electron microscopy (SEM) results showed that the morphology of the viral particle changed under PAS treatments. The total protein concentration of virus decreased as measured by a Bradford protein assay due to PAS treatment. The nucleic acid integrity assay demonstrated that viral RNA degraded into smaller fragments. Moreover, the physicochemical properties of PASs, including the oxidation-reduction potential (ORP), electrical conductivity, and H2O2 concentration, and electron spin resonance spectra analysis indicated that reactive oxygen and nitrogen species play a major role in the virus inactivation. Therefore, the application of PASs, as an environmentally friendly method, would be a promising alternative strategy in poultry industries.IMPORTANCE Newcastle disease (ND), as an infectious viral disease of avian species, caused significant economic losses to domestic animal and poultry industries. The traditional chemical sanitizers, such as chlorine-based products, are associated with risks of by-product formation with carcinogenic effects and environmental pollution. On the basis of this, plasma-activated water as a green disinfection product is a promising alternative for applications in stock farming and sterilization in hospitals and public places. In this study, we explored the inactivation efficacy of different plasma-activated solutions (PASs) against ND virus (NDV) and the possible underlying mechanisms. Our results demonstrated that reactive oxygen and nitrogen species detected in PASs, including short-lived OH˙ and NO˙ and long-lived H2O2, changed the morphology, destroyed the RNA structure, and degraded the protein of the virus, consequently resulting in virus inactivation. These lay a foundation for the application of PASs to resolve the issues of public health and environmental sanitation.

Necrotic enteritis locus 1 diguanylate cyclase and phosphodiesterase (cyclic-di-GMP) gene mutation attenuates virulence in an avian necrotic enteritis isolate of Clostridium perfringens.

Necrotic enteritis (NE) caused by netB-positive strains of Clostridium perfringens is an important disease of intensively-reared broiler chickens. It is widely controlled by antibiotic use, but this practice that has come under increasing scrutiny and alternative approaches are required. As part of the search for alternative approaches over the last decade, advances have been made in understanding its pathogenesis but much remains to be understood and applied to the control of NE. The objective of this work was to assess the effect on virulence of mutation of the cyclic-di-GMP signaling genes present on the large pathogenicity locus (NELoc-1) in the tcp-encoding conjugative virulence plasmid, pNetB. For this purpose, the diguanylate cyclase (dgc) and phosphodiesterase (pde) genes were individually insertionally inactivated and the two mutants were subsequently complemented with their respective genes. Southern blotting showed that a single gene insertion was present. Mutation of either gene resulted in almost total attenuation of the mutants to cause NE in experimentally-infected broiler chickens, which was fully restored in each case by complementation of the respective mutated gene. Production of NetB-associated cytotoxicity for Leghorn male hepatoma (LMH) cells was unaffected in mutants. We conclude that the cyclic-di-GMP signaling system is important in controlling virulence in a NE C. perfringens strain and might be a target for control of the disease.

The Agr-Like Quorum Sensing System Is Required for Pathogenesis of Necrotic Enteritis Caused by Clostridium perfringens in Poultry.

Clostridium perfringens encodes at least two different quorum sensing (QS) systems, the Agr-like and LuxS, and recent studies have highlighted their importance in the regulation of toxin production and virulence. The role of QS in the pathogenesis of necrotic enteritis (NE) in poultry and the regulation of NetB, the key toxin involved, has not yet been investigated. We have generated isogenic agrB-null and complemented strains from parent strain CP1 and demonstrated that the virulence of the agrB-null mutant was strongly attenuated in a chicken NE model system and restored by complementation. The production of NetB, a key NE-associated toxin, was dramatically reduced in the agrB mutant at both the transcriptional and protein levels, though not in a luxS mutant. Transwell assays confirmed that the Agr-like QS system controls NetB production through a diffusible signal. Global gene expression analysis of the agrB mutant identified additional genes modulated by Agr-like QS, including operons related to phospholipid metabolism and adherence, which may also play a role in NE pathogenesis. This study provides the first evidence that the Agr-like QS system is critical for NE pathogenesis and identifies a number of Agr-regulated genes, most notably netB, that are potentially involved in mediating its effects. The Agr-like QS system thus may serve as a target for developing novel interventions to prevent NE in chickens.

Influence of pCP1NetB ancillary genes on the virulence of Clostridium perfringens poultry necrotic enteritis strain CP1.

BACKGROUND: Necrotic enteritis (NE) is an economically important disease of poultry caused by certain Clostridium perfringens type A strains. The NetB toxin plays a critical role in the pathogenesis of NE. We previously demonstrated that netB is located within a 42 kb plasmid-encoded pathogenicity locus (NELoc-1), which also encodes 36 additional genes. Although NetB clearly plays a role in pathogenesis, the involvement of the other NELoc-1 genes has not yet been established. The current study was to provide experimental evidence to confirm the involvement of these genes in NE pathogenesis. RESULTS: The present study has characterized a virulent C. perfringens strain (CP1) that has spontaneously lost the NELoc-1-encoding plasmid, pCP1netB. When assessed for cytotoxicity on Leghorn Male Hepatoma (LMH) cells, the culture supernatant of the pCP1netB-deficient CP1 variant (CP1ΔpCP1netB) demonstrated significantly reduced cytotoxicity compared to the wild-type. In addition, CP1ΔpCP1netB was unable to cause intestinal lesions in chickens in a NE disease model. When netB alone was introduced into CP1ΔpCP1netB, in vitro cytotoxicity was restored to the wild-type level; however, it did not completely restore virulence when used to challenge broiler chickens [mean lesion score of 0.71 compared to 3.23 in the wild type control group (n = 14)]. CONCLUSIONS: The results of this study suggest that other genes present in NELoc-1, in addition to netB, are required for full virulence in the chicken challenge model.

Evaluation of a proposed molecular methodology for the serotyping of Avibacterium paragallinarum.

A multiplex (m)PCR and a PCR followed by restriction fragment length polymorphism (RFLP) analysis of Avibacterium paragallinarum have been proposed as alternatives to conventional serotyping by the Page scheme. We evaluated both methods, and also sequenced the PCR-RFLP target fragment to reexamine the capacity of molecular serotyping. Eleven reference strains and 27 field isolates were used. Many reference strains and isolates were misidentified as Page serogroup B. The sequence analysis revealed 6 profiles based on the matching rates of the target sequence with the 3 reverse primers of the mPCR. The reference strains and field isolates in profiles 1 and 4 were correctly identified as serogroup A or C by the mPCR. The strains and/or isolates in profiles 2, 3, 5, and 6 could be misidentified as serogroup B or as nontypeable by the mPCR. The homology comparison of the sequences showed that the target sequence of the mPCR, called region 2, was not Page serogroup specific, although some Kume serovars, such as A-1 and C-2, were correctly serotyped. In addition, there was a 9 nucleotide deletion in the sequences of profiles 1, 3, and 5, but not of profiles 2, 4, and 6. Overall, we confirmed that the mPCR and PCR-RFLP molecular assays are not suitable for identifying the serogroups of A. paragallinarum isolates. With further study, analysis of region 2 sequences may have potential as a means of recognizing the Kume serovars of A. paragallinarum isolates.

[Immunological modulation of chickens by recombinant Lactococcus lactis expressing ferric enterobactin receptor CfrA of Campylobacter jejuni].

Objective: The purpose of this study is to reduce the colonization level of Campylobacter jejuni in chicken intestine by oral immunization of recombinant Lactococcus lactis expressing the ferric enterobactin receptor CfrA of C. jejuni. Methods: The whole cfrA gene and its N-terminal fragments were amplified by PCR, inserted into the expression vector pNZ8149 and transformed into L. lactis NZ3900. Based on the expression of CfrA in recombinant L. lactis by Western blot, the expression level was optimized by screening nisin concentration, induction temperature and time. Then the recombinant L. lactis strains were used to orally immunize specific-pathogen-free chickens. After oral immunization, the duration of recombinant L. lactis in chickens was determined by PCR, and the antibody levels of anti-CfrA serum IgG and intestinal mucosal sIgA were measured by ELISA. Finally, the immunized chickens were orally inoculated with C. jejuni to evaluate the inhibitory effect of recombinant L. lactis on colonization of C. jejuni. Results: Western blot results determined that the whole cfrA gene and its N-terminal fragments were both expressed in recombinant L. lactis in soluble forms whereas no secreted CfrA protein was detected outside bacterial cells. The optimal conditions for inducing the expression were grown at 37℃ for 1 h with nisin concentration of 25 ng/mL. Detection of chicken cloacal swabs showed that the duration of oral L. lactis was less than 10 days in chicken. The immunized groups produced higher antibody titers of anti-CfrA specific serum IgG and mucosal sIgA than the control groups. Moreover, the colonization rate of C. jejuni in the immunized groups was significantly lower than that in the control groups. Conclusion: Oral immunization of chickens with recombinant L. lactis expressing CfrA inhibited the colonization of C. jejuni. Our findings can be useful to develop oral vaccines with recombinant Lactobacillus for control of C. jejuni infection in chickens.

Identification and Genome Characterization of the First Sicinivirus Isolate from Chickens in Mainland China by Using Viral Metagenomics.

Unlike traditional virus isolation and sequencing approaches, sequence-independent amplification based viral metagenomics technique allows one to discover unexpected or novel viruses efficiently while bypassing culturing step. Here we report the discovery of the first Sicinivirus isolate (designated as strain JSY) of picornaviruses from commercial layer chickens in mainland China by using a viral metagenomics technique. This Sicinivirus isolate, which contains a whole genome of 9,797 nucleotides (nt) excluding the poly(A) tail, possesses one of the largest picornavirus genome so far reported, but only shares 88.83% and 82.78% of amino acid sequence identity to that of ChPV1 100C (KF979332) and Sicinivirus 1 strain UCC001 (NC_023861), respectively. The complete 939 nt 5'UTR of the isolate strain contains at least twelve stem-loop domains (A-L), representing the highest set of loops reported within Sicinivirus genus. The conserved 'barbell-like' structure was also present in the 272 nt 3'UTR of the isolate as that in the 3' UTR of Sicinivirus 1 strain UCC001. The 8,586 nt large open reading frame encodes a 2,862 amino acids polyprotein precursor. Moreover, Sicinivirus infection might be widely present in commercial chicken farms in Yancheng region of the Jiangsu Province as evidenced by all the tested stool samples from three different farms being positive (17/17) for Sicinivirus detection. This is the first report on identification of Sicinivirus in commercial layer chickens with a severe clinical disease in mainland China, however, further studies are needed to evaluate the pathogenic potential of this picornavirus in chickens.

Identification of putative virulence-associated genes of Haemophilus parasuis through suppression subtractive hybridization.

Haemophilus parasuis is the causative agent of Glässer's disease. Up to now 15 serovars of H. parasuis have been identified, with significant differences existing in virulence between serovars. In this study, suppression subtractive hybridization (SSH) was used to identify the genetic difference between Nagasaki (H. parasuis serovar 5 reference strain, highly virulent) and SW114 (H. parasuis serovar 3 reference strain, non-virulent). A total of 191 clones were obtained from the SSH library. Using dot hybridization and PCR, 15 clones were identified containing fragments that were present in the Nagasaki genome while absent in the SW114 genome. Among these 15 fragments, three fragments (ssh1, ssh13, ssh15) encode cell surface-associated components; three fragments (ssh2, ssh5, ssh9) are associated with metabolism and stress response; one fragment (ssh8) is involved in assembly of fimbria and one fragment (ssh6) is a phage phi-105 ORF25-like protein. The remaining seven fragments are hypothetical proteins or unknown. Based on PCR analysis of the 15 serovar reference strains, eight fragments (ssh1, ssh2, ssh3, ssh6, ssh8, ssh10, ssh11 and ssh12) were found in three to five of most virulent serovars (1, 5, 10, 12, 13 and 14), zero to two in three moderately virulent serovars (2, 4 and 15), but absent in the low virulent serovar (8) and non-virulent serovars (3, 6, 7, 9 and 11). In vivo transcription fragments ssh1, ssh2, ssh8 and ssh12 were identified in total RNA samples extracted from experimental infected pig lung by RT-PCR. This study has provided some evidence of genetic differences between H. parasuis strains of different virulence.