A multiplex digital PCR assay for detection and quantitation of porcine circovirus type 2 and type 3.

Porcine circovirus (PCV) has become a major pathogen, causing major economic losses in the global pig industry, and PCV type 2 (PCV2) and 3 (PCV3) are distributed worldwide. We designed specific primer and probe sequences targeting PCV2 Cap and PCV3 Rap and developed a multiplex crystal digital PCR (cdPCR) method after optimizing the primer concentration, probe concentration, and annealing temperature. The multiplex cdPCR assay permits precise and differential detection of PCV2 and PCV3, with a limit of detection of 1.39 × 101 and 1.27 × 101 copies/reaction, respectively, and no cross-reaction with other porcine viruses was observed. The intra-assay and interassay coefficients of variation (CVs) were less than 8.75%, indicating good repeatability and reproducibility. To evaluate the practical value of this assay, 40 tissue samples and 70 feed samples were tested for both PCV2 and PCV3 by cdPCR and quantitative PCR (qPCR). Using multiplex cdPCR, the rates of PCV2 infection, PCV3 infection, and coinfection were 28.45%, 1.72%, and 12.93%, respectively, and using multiplex qPCR, they were 25.00%, 0.86%, and 4.31%, respectively This highly specific and sensitive multiplex cdPCR thus allows accurate simultaneous detection of PCV2 and PCV3, and it is particularly well suited for applications that require the detection of small amounts of input nucleic acid or samples with intensive processing and complex matrices.

Lactic acid bacteria reduce bacterial diarrhea in rabbits via enhancing immune function and restoring intestinal microbiota homeostasis.

BACKGROUND: Numerous previous reports have demonstrated the efficacy of Lactic acid bacteria (LAB) in promoting growth and preventing disease in animals. In this study, Enterococcus faecium ZJUIDS-R1 and Ligilactobaciiius animalis ZJUIDS-R2 were isolated from the feces of healthy rabbits, and both strains showed good probiotic properties in vitro. Two strains (108CFU/ml/kg/day) were fed to weaned rabbits for 21 days, after which specific bacterial infection was induced to investigate the effects of the strains on bacterial diarrhea in the rabbits. RESULTS: Our data showed that Enterococcus faecium ZJUIDS-R1 and Ligilactobaciiius animalis ZJUIDS-R2 interventions reduced the incidence of diarrhea and systemic inflammatory response, alleviated intestinal damage and increased antibody levels in animals. In addition, Enterococcus faecium ZJUIDS-R1 restored the flora abundance of Ruminococcaceae1. Ligilactobaciiius animalis ZJUIDS-R2 up-regulated the flora abundance of Adlercreutzia and Candidatus Saccharimonas. Both down-regulated the flora abundance of Shuttleworthia and Barnesiella to restore intestinal flora balance, thereby increasing intestinal short-chain fatty acid content. CONCLUSIONS: These findings suggest that Enterococcus faecium ZJUIDS-R1 and Ligilactobaciiius animalis ZJUIDS-R2 were able to improve intestinal immunity, produce organic acids and regulate the balance of intestinal flora to enhance disease resistance and alleviate diarrhea-related diseases in weanling rabbits.

Dual Gene Detection of H5N1 Avian Influenza Virus Based on Dual RT-RPA.

The H5N1 avian influenza virus seriously affects the health of poultry and humans. Once infected, the mortality rate is very high. Therefore, accurate and timely detection of the H5N1 avian influenza virus is beneficial for controlling its spread. This article establishes a dual gene detection method based on dual RPA for simultaneously detecting the HA and M2 genes of H5N1 avian influenza virus, for the detection of H5N1 avian influenza virus. Design specific primers for the conserved regions of the HA and M2 genes. The sensitivity of the dual RT-RPA detection method for HA and M2 genes is 1 × 10-7 ng/µL. The optimal primer ratio is 1:1, the optimal reaction temperature is 40 °C, and the optimal reaction time is 20 min. Dual RT-RPA was used to detect 72 samples, and compared with RT-qPCR detection, the Kappa value was 1 (p value < 0.05), and the clinical sample detection sensitivity and specificity were both 100%. The dual RT-RPA method is used for the first time to simultaneously detect two genes of the H5N1 avian influenza virus. As an accurate and convenient diagnostic tool, it can be used to diagnose the H5N1 avian influenza virus.

Enhancing Cross-Lingual Entity Alignment in Knowledge Graphs through Structure Similarity Rearrangement.

Cross-lingual entity alignment in knowledge graphs is a crucial task in knowledge fusion. This task involves learning low-dimensional embeddings for nodes in different knowledge graphs and identifying equivalent entities across them by measuring the distances between their representation vectors. Existing alignment models use neural network modules and the nearest neighbors algorithm to find suitable entity pairs. However, these models often ignore the importance of local structural features of entities during the alignment stage, which may lead to reduced matching accuracy. Specifically, nodes that are poorly represented may not benefit from their surrounding context. In this article, we propose a novel alignment model called SSR, which leverages the node embedding algorithm in graphs to select candidate entities and then rearranges them by local structural similarity in the source and target knowledge graphs. Our approach improves the performance of existing approaches and is compatible with them. We demonstrate the effectiveness of our approach on the DBP15k dataset, showing that it outperforms existing methods while requiring less time.

Verification of Bacteroidales 16S rRNA markers as a complementary tool for detecting swine fecal pollution in the Yangtze Delta.

To correctly assess and properly manage the public health risks associated with exposure to contaminated water, it is necessary to identify the source of fecal pollution in a watershed. In this study, we evaluated the efficacy of our two previously developed real time-quantitative PCR (qPCR) assays for the detection of swine-associated Bacteroidales genetic markers (gene 1-38, gene 3-53) in the Yangtze Delta watershed of southeastern China. The results indicated that the gene 1-38 and 3-53 markers exhibited high accuracy (92.5%, 91.7% conditional probability, respectively) in detecting Bacteroidales spp. in water samples. According to binary logistic regression (BLR), these two swine-associated markers were well correlated (P < 0.05) with fecal indicators (Escherichia coli and Enterococci spp.) and zoonotic pathogens (E. coli O157: H7, Salmonella spp. and Campylobacter spp.) in water samples. In contrast, concentrations of conventional fecal indicator bacteria (FIB) were not correlated with zoonotic pathogens, suggesting that they are noneffective at detecting fecal pollution events. Collectively, the results obtained in this study demonstrated that a swine-targeted qPCR assay based on two Bacteroidales genes markers (gene 1-38, gene 3-53) could be a useful tool in determining the swine-associated impacts of fecal contamination in a watershed.

[Molecular beacon based PNA-FISH method combined with fluorescence scanning for rapid detection of Listeria monocytogenes].

Objective: To simplify the PNA-FISH (Peptide nucleic acid-fluorescence in situ hybridization) test, molecular beacon based PNA probe combined with fluorescence scanning detection technology was applied to replace the original microscope observation to detect Listeria monocytogenes Methods: The 5' end and 3' end of the L. monocytogenes specific PNA probes were labeled with the fluorescent group and the quenching group respectively, to form a molecular beacon based PNA probe. Results: When PNA probe used for fluorescence scanning and N1 treatment as the control, the false positive rate was 11.4%, and the false negative rate was 0; when N2 treatment as the control, the false positive rate decreased to 4.3%, but the false negative rate rose to 18.6%. When beacon based PNA probe used for fluorescence scanning, taken N1 treatment as blank control, the false positive rate was 8.6%, and the false negative rate was 1.4%; taken N2 treatment as blank control, the false positive rate was 5.7%, and the false negative rate was 1.4%. Compared with PNA probe, molecular beacon based PNA probe can effectively reduce false positives and false negatives. The success rates of hybridization of the two PNA probes were 83.3% and 95.2% respectively; and the rates of the two beacon based PNA probes were 91.7% and 90.5% respectively, which indicated that labeling the both ends of the PNA probe dose not decrease the hybridization rate with the target bacteria. Conclusions: The combination of liquid phase PNA-FISH and fluorescence scanning method, can significantly improve the detection efficiency.

MALDI-TOF nucleic acid mass spectrometry for simultaneously detection of fourteen porcine viruses and its application.

BACKGROUND: Mixed infections of multiple viruses significantly contribute to the prevalence of swine diseases, adversely affecting global livestock production and the economy. However, effectively monitoring multiple viruses and detecting mixed infection samples remains challenging. This study describes a method that combines single-base extension PCR with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to detect important porcine viruses. RESULTS: Our approach accurately and simultaneously identified 14 porcine viruses, including porcine circovirus types 1-3, porcine bocaviruses groups 1-3, African swine fever virus, pseudorabies virus, porcine parvovirus, torque teno sus virus, swine influenza virus, porcine reproductive and respiratory syndrome virus, classical swine fever virus, and foot-and-mouth disease virus. The low limit of detection for multiplex identification ranges from 13.54 to 1.59 copies/µL. Inter- and intra-assay stability was found to be ≥98.3 %. In a comprehensive analysis of 114 samples, the assay exhibited overall agreement with qPCR results of 97.9 %. CONCLUSIONS: The developed MALDI-TOF NAMS assay exhibits high sensitivity, specificity, and reliability in detecting and distinguishing a wide spectrum of porcine viruses in complex matrix samples. This underscores its potential as an efficient diagnostic tool for porcine-derived virus surveillance and swine disease control.

Simultaneous detection of three nitrofuran antibiotics by the lateral flow immunoassay based on europium nanoparticles in aquatic products.

Nitrofuran (NF) antibiotics have been banned worldwide in aquaculture due to their potential carcinogenicity and mutagenicity. Because of the short half-life of NF antibiotics, an easy and sensitive multiple lateral flow immunoassay (mLFIA) based on europium nanoparticles (EuNPs) has been successfully established to simultaneously and quantitatively detect 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), 3-amino-2-oxazolidinone (AOZ) and sodium nifurstylenate (NFS) in aquatic products. The EuNP-mLFIA assay was accomplished within 10 min. The limits of detection (LODs) for AOZ, AMOZ and NFS were 0.013, 0.019 and 0.023 ng/mL, respectively. The average recoveries of AOZ, AMOZ and NFS were 98.0-104.4%, 96.0-102.6% and 98.0-102.8%, respectively. It showed satisfactory consistency, and the feasibility was validated by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Briefly, this method will become a powerful tool for monitoring multiple NF antibiotics and provide promising applications in the field of food safety and environmental testing.

Dietary dicarbonyl compounds exacerbated immune dysfunction and hepatic oxidative stress under high-fat diets in vivo.

High-fat diets (HFDs) predispose to obesity and liver dysfunctions, and α-dicarbonyl compounds (α-DCs) present in highly processed foods are also implicated in relevant pathological processes. However, the synergistic harmful effects of α-DCs co-administered with HFDs remain to be elucidated. In this study, 6-week-old C57BL/6 mice were fed with a HFD co-administered with 0.5% methylglyoxal (MGO)/glyoxal (GO) in water for 8 weeks, and multi-omics approaches were employed to investigate the underlying toxicity mechanisms. The results demonstrated that the MGO intervention with a HFD led to an increased body weight and blood glucose level, accompanied by the biological accumulation of α-DCs and carboxymethyl-lysine, as well as elevated serum levels of inflammatory markers including IL-1ß, IL-6, and MIP-1α. Notably, hepatic lesions were observed in the MGO group under HFD conditions, concomitant with elevated levels of malondialdehyde. Transcriptomic analysis revealed enrichment of pathways and differentially expressed genes (DEGs) associated with inflammation and oxidative stress in the liver. Furthermore, α-DC intervention exacerbated gut microbial dysbiosis in the context of a HFD, and through Spearman correlation analysis, the dominant genera such as Fusobacterium and Bacteroides in the MGO group and Colidextribacter and Parabacteroides in the GO group were significantly correlated with a set of DEGs involved in inflammatory and oxidative stress pathways in the liver. This study provides novel insights into the healthy implications of dietary ultra-processed food products in the context of obesity-associated disorders.

Porcine circovirus type 2 induces autophagy via the AMPK/ERK/TSC2/mTOR signaling pathway in PK-15 cells.

Porcine circovirus type 2 (PCV2) uses autophagy machinery to enhance its replication in PK-15 cells. However, the underlying mechanisms are unknown. By the use of specific inhibitors, RNA interference, and coimmunoprecipitation, we show that PCV2 induces autophagy in PK-15 cells through a pathway involving the kinases AMP-activated protein kinase (AMPK) and extracellular signal-regulated kinase 1/2 (ERK1/2), the tumor suppressor protein TSC2, and the mammalian target of rapamycin (mTOR). AMPK and ERK1/2 positively regulate autophagy through negative control of the mTOR pathway by phosphorylating TSC2 in PCV2-infected PK-15 cells. Thus, PCV2 might induce autophagy via the AMPK/ERK/TSC2/mTOR signaling pathway in the host cells, representing a pivotal mechanism for PCV2 pathogenesis.

In vivo characterization of chimeric PCV DNA clones containing heterogeneous capsid protein nuclear localization signals (NLS).

BACKGROUND: PCV ORF2 capsid protein was predicted to contribute to the control of replication via an interaction between the Cap and Rep proteins in the nucleoplasm. We previously showed that the nuclear localization signal (NLS) on the capsid protein plays an accessory role in the replication of PCV in vitro. To further evaluate the in vivo characteristics of NLS-chimeric PCV DNA clones, BALB/C mice were inoculated intranasally and intraperitoneally with the DNA clones. RESULTS: As expected, no gross lesions were detected during the study of the inoculated animals. The chimeric PCV12-, PCV1-NLS2- and PCV2-NLS1-inoculated animals had significantly fewer and less severe histopathological lesions in lymphoid tissues than the PCV2-inoculated animals (P < 0.05). PCV12 induced a specific antibody response against PCV2 ORF2 comparable to that induced by wild-type PCV2 but demonstrated a shorter period of viremia and much lower level of virus loads in sera than those in PCV2-inoculated mice. Remarkably, the PCV2-NLS1 and PCV1-NLS2 chimeras replicated in inoculated mice and induced specific antibody responses but failed to produce viral antigens in the lymph nodes or a detectable viremia. CONCLUSIONS: The chimeric PCV2-NLS1 and PCV1-NLS2 demonstrated a lower replication level as compared with wild type of PCV2 or PCV1 in vivo, suggesting that ORF2 NLSs played an accessory role in PCV replication. The chimeric PCV12 is a good candidate for vaccination against PCV2 infection.

Triplex-Loop-Mediated Isothermal Amplification Combined with a Lateral Flow Immunoassay for the Simultaneous Detection of Three Pathogens of Porcine Viral Diarrhea Syndrome in Swine.

Porcine epidemic diarrhea virus (PEDV), porcine bocavirus (PBoV), and porcine rotavirus (PoRV) are associated with porcine viral diarrhea. In this study, triplex loop-mediated isothermal amplification (LAMP) combined with a lateral flow dipstick (LFD) was established for the simultaneous detection of PEDV, PoRV, and PBoV. The PEDV-gp6, PoRV-vp6, and PBoV-vp1 genes were selected to design LAMP primers. The amplification could be carried out at 64 °C using a miniature metal bath within 30 min. The triplex LAMP-LFD assay exhibited no cross-reactions with other porcine pathogens. The limits of detection (LODs) of PEDV, PoRV, and PBoV were 2.40 × 101 copies/µL, 2.89 × 101 copies/µL, and 2.52 × 101 copies/µL, respectively. The consistency between rt-qPCR and the triplex LAMP-LFD was over 99% in field samples testing. In general, the triplex LAMP-LFD assay was suitable for the rapid and simultaneous detection of the three viruses in the field.

Functional exchangeability of the nuclear localization signal (NLS) of capsid protein between PCV1 and PCV2 in vitro: Implications for the role of NLS in viral replication.

BACKGROUND: Porcine circovirus type 2 (PCV2) is believed to be the primary causative agent of postweaning multisystemic wasting syndrome (PMWS). It is supposed that capsid protein of PCV may contribute to replication control via interaction between Cap and Rep in the nucleoplasm. In this study, we described the construction and in vitro characterization of NLS-exchanged PCV DNA clones based on a PMWS-associated PCV2b isolate from China to determine the role of ORF2 NLS in PCV replication. RESULTS: The PCV1, PCV2, PCV2-NLS1 and PCV1-NLS2 DNA clone were generated by ligating a copy of respective genome in tandem with a partial duplication. The PCV2-NLS1 and PCV1-NLS2 DNA clone contained a chimeric genome in which the ORF2 NLS was exchanged. The four DNA clones were all confirmed to be infectious in vitro when transfected into PK-15 cells, as PCV capsid protein were expressed in approximately 10-20% of the transfected cells. The in vitro growth characteristics of the DNA clones were then determined and compared. All the recovered progeny viruses gave rise to increasing infectious titers during passages and were genetically stable by genomic sequencing. The chimeric PCV1-NLS2 and PCV2-NLS1 viruses had the final titers of about 104.2 and 103.8 TCID50/ml, which were significantly lower than that of PCV1 and PCV2 (105.6 and 105.0 TCID50/ml, respectively). When the ORF2 NLS exchanged, the mutant PCV2 (PCV2-NLS1) still replicated less efficiently and showed lower infectious titer than did PCV1 mutant (PCV1-NLS2), which was consistent with the distinction between wild type PCV1 and PCV2. CONCLUSIONS: Recovery of the chimeiric PCV1-NLS2 and PCV2-NLS1 progeny viruses indicate that the nuclear localization signal sequence of capsid protein are functionally exchangeable between PCV1 and PCV2 with respect to the role of nuclear importing and propagation. The findings also reveal that ORF2 NLS play an accessory role in the replication of PCV. However, we found that ORF2 NLS was not responsible for the distinction of in vitro growth characteristic between PCV1 and PCV2. Further studies are required to determine the in vivo viral replication and pathogenicity of the NLS chimeric DNA clones.

Immune responses of Fenneropenaeus chinensis against white spot syndrome virus after oral delivery of VP28 using Bacillus subtilis as vehicles.

The protective efficacy of oral administration of VP28 using Bacillus subtilis as vehicles (rVP28-bs) in shrimp, Fenneropenaeus chinensis, upon challenge with white spot syndrome virus (WSSV) was investigated. The calculated relative percent survival (RPS) value of rVP28-bs fed shrimp was 83.3% when challenged on the 14th day post-administration, which is significantly higher (p < 0.001) than that of the group administered recombinant Escherichia coli over-expressing rVP28 (rVP28-e21). After immunization, activities of phenoloxidase (PO), superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) in hemolymph were analyzed. It was found that the supplementation of rVP28-bs into shrimp food pellets resulted in the most pronounced increase of iNOS activity (p < 0.001), but had the least influence on activities of PO and SOD. Besides, in the shrimp orally administered with rVP28-bs, the caspase-3 activity was one-fifth that of the control, though the signs of apoptosis (chromatin margination, nuclear fragmentation and apoptotic bodies) could not be observed by transmission electron microscope (TEM). These results suggest that by oral delivery of rVP28-bs, shrimp showed significant resistance to WSSV and an effect on the innate immune system of shrimp. The remarkably enhanced level of iNOS after rVP28-bs administration might be responsible for antiviral defense in shrimp.

Porcine reproductive and respiratory syndrome viruses predominant in southeastern China from 2004 to 2007 were from a common source and underwent further divergence.

The full-length glycoprotein 5 (GP5) gene and a partial nonstructural protein 2 (NSP2) gene fragment of 46 porcine reproductive and respiratory syndrome viruses (PRRSV) from pig farms in southeastern China between 2004 and 2007 were sequenced for phylogenetic analysis. All of the PRRSV isolates in this study were of the North American type, and the majority of them were clustered in subgroup II and had 84.1-89.1% amino acid sequence identity to those of subgroup I including the North American strain VR-2332. Three variable regions containing epitopes A and B in the N-terminal region were identified and found to be under positive selection. Several additional mutations, which were also located in the variable regions, were seen in isolates from the years 2006 and 2007 in subgroup II, as compared with those of earlier years (2004-2005) in the same group. Further analysis revealed that the majority of the subgroup II PRRSV isolates prevalent in the region since 2004 had thirteen mutation sites that distinguished them from subgroup I strains, indicating a possible introduction of a certain strain from the same source in the region or elsewhere well before 2004. A 29-aa deletion in the NSP2 fragment was found in PRRSV isolates as early as in 2005, one year earlier than the virulent PRRSV with the same deletion became dominant in China. Taken together, this study shows that subgroup II PRRSV strains with a partial deletion of nsp2 are currently prevailing in southeastern China.

Genetic diversity of the envelope glycoprotein E2 of classical swine fever virus: recent isolates branched away from historical and vaccine strains.

The envelope glycoprotein E2 of classical swine fever virus (CSFV) plays multiple roles in the viral life cycle, interaction with host and pathogenesis. We sequenced the E2 gene of 34 CSFV isolates from southeastern China for analysis of genetic diversity in this particular region. Phylogenetic analysis revealed that genotype 2.1b viruses became predominant in southeastern China with 33 isolates clustered in 2.1b and only 1 isolate belonged to 2.2. Pairwise comparisons demonstrated isolates in this study showed a homology of 78.4-82.6% to Chinese C-strain in the 190nt of the E2 fragment. The minimum similarity within the 2.1b isolates was 91.1%. Variability of the full length E2 amino acid sequences of 45 CSFV isolates representative of three main groups of CSFV including 19 from southeastern China during 2004--2007 and 26 from other parts of China and other countries was analyzed by the differences between non-synonymous (dN) and synonymous (dS) rates and the entropy values. Two variable and three conserved regions as well as some specific positions under positive selection were defined. Our results suggest that recent isolates from southeastern China have shifted away from historical CSFV isolates including the vaccine strains probably as a result of their adaptive abilities to the selection forces within the host.

Mapping of the nuclear localization signals in open reading frame 2 protein from porcine circovirus type 1.

Porcine circovirus type 1 (PCV1) contains two major open reading frames encoding the replication-associated proteins and the major structural capsid (Cap) protein. PCV1 Cap has an N-terminus carrying several potential monopartite or bipartite nuclear localization signals (NLS). The contribution of these partially overlapping motifs to nuclear importing was identified by expression of mutated PCV1 Cap versions fused to enhanced green fluorescent protein (EGFP). The C-terminus truncated PCV1 Cap-EGFP was localized in nuclei of PK-15 cells similar to the wild-type PCV1 Cap-EGFP, whereas truncation of the N-terminus rendered the fusion protein distributed into cytoplasm, indicating that the nuclear import of PCV1 Cap was efficiently mediated by its N-terminal region. Substitutions of basic residues in stretches 9RRRR12 or the right part of 25RRPYLAHPAFRNRYRWRRK43 resulted in a diffused distribution of the fusion protein in both nuclei and cytoplasm, indicating that the two NLSs were responsible for restricted nuclear targeting of PCV1 Cap.

A survey on porcine circovirus type 2 infection and phylogenetic analysis of its ORF2 gene in Hangzhou, Zhejiang Province, China.

Porcine circovirus type 2 (PCV2) is closely related to the postweaning multisystemic wasting syndrome (PMWS). In this study, the pig serum and tissue samples collected from different regions of Hangzhou District in Zhejiang Province of China between 2003 and 2005 were analyzed by enzyme-linked immunosorbent assay (ELISA) for PCV2 antibody and by polymerase chain reaction (PCR) for ORF2 gene. The results show that out of 1250 randomly collected serum samples, 500 sera (40%) were seropositive for PCV2. PCR results demonstrate that Hangzhou PCV2 with more than 50% Chinese PCV2 strains and French PCV2 formed Cluster A. Only one PCV2 from Hangzhou belonged to Cluster B with some other Chinese PCV2 and Netherlands's isolates. Cluster C consisted of PCV2 isolates from China, US, Canada, UK and Germany. The results indicate that the PCV2 infection was widespread in Hangzhou.

Characterization and potential use of truncated PCV2 capsid protein and its polyclonal antibody for diagnosis of PCV2 infections.

As porcine circovirus 2 (PCV2) ORF2 encodes the major structural protein (capsid) that is closely related to the pathogenesis, the capsid (Cap) protein could be used as a target antigen for serological analysis. The immunoactivities of the truncated capsid proteins containing immunogenic epitopes of PCV2 (Cap2s) or PCV1 (Cap1s) expressed in Escherichia coli were described, as well as the characteristic of their polyclonal antibodies in diagnosis of PCV2 infection. Western blot analysis revealed that both Cap2s and Cap1s gave strong signals on nitrocellulose membranes to their corresponding polyclonal antibody. Furthermore, either PCV2-positive sera from PMWS cases or PCV1-positive swine sera could only recognize Cap2s or Capls, respectively. There was also no cross-reactivity between the two polyclonal antibodies when reacted with natural Cap proteins of viral particles on cells by immunofluorescence assay (IFA). Thus, an ELISA was then developed using PCV2 Cap as coating antigen to evaluate the sero-prevalence of PCV2 infection in pigs. The PCV2-positive rate ranged from 48.28% to 100% among different herds (n = 13) with an average of 80.69% (209/259). These results indicate that Cap2s was type-specific and could be used as a discriminative antigen for monitoring PCV2 antibody in serum. The polyclonal antibodies were also useful for differential identification of PCV1 and PCV2 infection by immunohistochemistry.

The S-layer proteins of Lactobacillus crispatus strain ZJ001 is responsible for competitive exclusion against Escherichia coli O157:H7 and Salmonella typhimurium.

Lactobacillus crispatus ZJ001, isolated from pig intestines and identified by sequencing analysis based on partial 16S rRNA gene, was examined in vitro for probiotic activity exerted by the surface layer proteins (S-layer). The characteristics of L. crispatus ZJ001 were compared to Lactobacillus acidophilus ATCC 4356 from the same genus which also produces the S-layer proteins. The strain ZJ001 was resistant to acidic condition and bile salt. Its antagonistic properties such as adhesion, inhibition of the pathogen growth and competitive exclusion against Escherichia coli O157:H7 and Salmonella typhimurium were apparently advantageous over L. acidophilus ATCC 4356. SDS-PAGE analysis of cell surface proteins revealed the presence of S-layer proteins, approximately at 42 kDa in L. crispatus ZJ001. Removal of the S-layer proteins reduced autoaggregation and adhesion to HeLa cells. The functional role of the S-layer proteins in adhesion was also confirmed by the antibody-mediated inhibition assay using the polyclonal antibody against the S-layer protein. The S-layer proteins from L. crispatus ZJ001 inhibited adhesion of S. typhimurium and E. coli O157:H7 to HeLa cells. These results suggest that L. crispatus ZJ001 possesses probiotic properties and the S-layer proteins are involved in the adhesion and competitive exclusion of pathogens to HeLa cells.