Cytotoxicity effect and transcriptome analysis of PCV3-infected cells revealed potential viral pathogenic mechanisms.

Porcine circovirus type 3 (PCV3) has become an important pathogen in the global swine industry and poses a threat to pig health, but its pathogenic mechanism remains unknown. In this study, we constructed an innovative, linear infectious clone of PCV3 for rescuing the virus, and explored the transcriptome of infected cells to gain insights into its pathogenic mechanisms. Subsequently, an in vivo experiment was conducted to evaluate the pathogenicity of the rescued virus in pig. PCV3 nucleic acid was distributed across various organs, indicating systemic circulation via the bloodstream and viremia. Immunohistochemical staining also revealed a significant presence of PCV3 antigens in the spleen, lungs, and lymph nodes, indicating that PCV3 had tropism for these organs. Transcriptome analysis of infected ST cells revealed differential expression of genes associated with apoptosis, immune responses, and cellular metabolism. Notably, upregulation of genes related to the hypoxia-inducible factor-1 pathway, glycolysis, and the AGE/RAGE pathway suggests activation of inflammatory responses, ultimately leading to onset of disease. These findings have expanded our understanding of PCV3 pathogenesis, and the interplay between PCV3 and host factors.

A prospective CSFV-PCV2 bivalent vaccine effectively protects against classical swine fever virus and porcine circovirus type 2 dual challenge and prevents horizontal transmission.

Classical swine fever virus (CSFV) infection leading to CSF outbreaks is among the most devastating swine diseases in the pig industry. Porcine circovirus type 2 (PCV2) infection, resulting in porcine circovirus-associated disease (PCVAD), is also a highly contagious disease affecting pig health worldwide. To prevent and control disease occurrence, multiple-vaccine immunization is necessary in contaminated areas or countries. In this study, a novel CSFV-PCV2 bivalent vaccine was constructed and demonstrated to be capable of eliciting humoral and cellular immune responses against CSFV and PCV2, respectively. Moreover, a CSFV-PCV2 dual-challenge trial was conducted on specific-pathogen-free (SPF) pigs to evaluate vaccine efficacy. All of the vaccinated pigs survived and showed no clinical signs of infection throughout the experimental period. In contrast, placebo-vaccinated pigs exhibited severe clinical signs of infection and steeply increased viremia levels of CSFV and PCV2 after virus challenge. Additionally, neither clinical signs nor viral detections were noted in the sentinel pigs when cohabitated with vaccinated-challenged pigs at three days post-inoculation of CSFV, indicating that the CSFV-PCV2 bivalent vaccine completely prevents horizontal transmission of CSFV. Furthermore, conventional pigs were utilized to evaluate the application of the CSFV-PCV2 bivalent vaccine in field farms. An adequate CSFV antibody response and a significant decrease in PCV2 viral load in the peripheral lymph nodes were observed in immunized conventional pigs, suggesting its potential for clinical application. Overall, this study demonstrated that the CSFV-PCV2 bivalent vaccine effectively elicited protective immune responses and the ability to prevent horizontal transmission, which could be a prospective strategy for controlling both CSF and PCVAD in commercial herds.

Detection and phylogenetic analysis of porcine circovirus type 3 in Taiwan.

Porcine circovirus type 3 (PCV3) is a newly emerging porcine circovirus that infects pig populations worldwide. In this study, we investigated the prevalence of PCV3 in Taiwan and analyzed the phylogenetic relationships between the Taiwanese PCV3 strains and those from other countries. A total of 463 clinical specimens from sick pigs were collected in 2016-2019 and analyzed for PCV3 by PCR. The positivity rate for PCV3 was 10.6% in 2016, increasing markedly to 34.78% in 2019. A phylogenetic analysis based on full-length genomic sequences of PCV3 divided the PCV3 strains into three clades, with the Taiwanese strains in clade 1.

Characterization of the monoclonal antibody specific to the ORF72 protein of koi herpesvirus and cellular distribution analysis of the viral protein.

Koi herpesvirus (KHV) is an emerging pathogen of koi and common carp that causes a severe disease and mass mortality of infected fish. The KHV ORF72 protein is an important capsid protein that has been suggested to be a candidate for the development of diagnostic reagents and KHV vaccines. The purpose of this study was to clone and express the KHV ORF72 gene for further preparation of a specific monoclonal antibody (mAb) and to analyse cellular distribution of the viral protein. The mAb 3E1 could specifically recognize the expressed ORF72 protein of transfected cells by indirect immunofluorescence, and the antigenic site recognized by the mAb 3E1 was mapped to the region of N-terminal 124 residues of KHV ORF72. This mAb was further demonstrated to specifically detect the KHV-infected fish tissue by immunohistochemistry, thereby suggesting its high diagnostic potential. In addition, the cellular distribution analysis of the KHV ORF72 protein revealed that the region of amino acid residues 125-247 was related to mitochondrial localization and proliferation. Furthermore, a putative nuclear export signal (NES) of ORF72 at the residues 201-212 was confirmed on the basis of its function associated with NES activity.

Exploring the surface epitope and nuclear localization analysis of porcine circovirus type 3 capsid protein.

Porcine circovirus 3 (PCV3) is a newly emerging virus associated with porcine dermatitis and nephropathy syndrome (PDNS) and reproductive disorders, impacting global pig populations. Porcine circoviruses contain two major open reading frames (ORFs), and the ORF2 encodes the viral capsid protein (Cap). Cap is the most antigenic structural protein and an ideal candidate for the development of vaccines and diagnostic reagents. This study generated a monoclonal antibody (MAb) specific to PCV3 Cap, MAb CCC160, for diagnosis and pathogenesis studies of this novel virus. The MAb specifically recognized PCV3-infected swine lymph node tissue in an immunohistochemical analysis confirming its clinical diagnostic potential. In addition, a novel linear B-cell epitope recognized by MAb CCC160 was identified at the amino acid region 120-134 of Cap. Nuclear localization analysis of PCV3 Cap revealed a potential nuclear localization signal (NLS) in the middle region (aa 131-143) in addition to the dominant N-terminal NLS that is already known. A cell viability assay further demonstrated that the cytotoxicity of PCV3 Cap is correlated with its nuclear localization, indicating a crucial role of Cap in the pathogenic mechanism of PCV3. A full-length construct of PCV3 Cap was successfully expressed using a baculovirus expression system and purified recombinant proteins self-assembled into virus-like particles (VLPs). The protein constitution of the VLPs was confirmed by MAb CCC160 recognition, indicating the correct conformation and specificity of VLP and exhibiting the linear epitope aa 120-134 on the VLP surface. These results provide insights for developing diagnostic tools and potential VLP vaccines for PCV3, revealing its pathogenesis and antigenic properties.

Characterization of porcine circovirus type 2 (PCV2) capsid particle assembly and its application to virus-like particle vaccine development.

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases in pigs. The sole structural capsid protein of PCV2, Cap, consists of major antigenic domains, but little is known about the assembly of capsid particles. The purpose of this study is to produce a large amount of Cap protein using Escherichia coli expression system for further studying the essential sequences contributing to formation of particles. By using codon optimization of rare arginine codons near the 5'-end of the cap gene for E. coli, a full-length Cap without any fusion tag recombinant protein (Cap1-233) was expressed and proceeded to form virus-like particles (VLPs) in normal Cap appearance that resembled the authentic PCV2 capsid. The N-terminal deletion mutant (Cap51-233) deleted the nuclear localization signal (NLS) domain, while the internal deletion mutant (CapΔ51-103) deleted a likely dimerization domain that failed to form VLPs. The unique Cys108 substitution mutant (CapC/S) exhibited most irregular aggregates, and only few VLPs were formed. These results suggest that the N-terminal region within the residues 1 to 103 possessing the NLS and dimerization domains are essential for self-assembly of stable Cap VLPs, and the unique Cys108 plays an important role in the integrity of VLPs. The immunogenicity of PCV2 VLPs was further evaluated by immunization of pigs followed by challenge infection. The Cap1-233-immunized pigs demonstrated specific antibody immune responses and are prevented from PCV2 challenge, thus implying its potential use for a VLP-based PCV2 vaccine.

Immunostimulation of sugar cane extract on neutrophils to Salmonella typhimurium infection in mice.

The aim of this study was to evaluate the immunomodulatory effects of sugar cane extract (SCE) on the biological activities of neutrophils in mice. Six-week-old BALB/c mice were fed 1250 mg/kg of SCE once. The generation, migration and biological functions of neutrophils and the survival rates of the mice in response to Salmonella typhimurium infection were evaluated. The results show that the numbers of both bone marrow cells and neutrophils were significantly increased in response to SCE administration (p < 0.05) compared with controls. The migration, phagocytosis and H2O2 generation of neutrophils were all significantly enhanced in SCE-treated mice (p < 0.05). After challenge with S. typhimurium (lethal dose, 50% (LD50), SCE-treated mice had a 19.2% higher survival rate and milder hepatic lesions than the controls. Additionally, fewer invasive bacteria were recovered from the spleens of SCE-treated mice. In conclusion, our results suggest that SCE has a positive regulatory effect on the biological function of mouse neutrophils that may increase host resistance against bacterial infections.

Mechanisms underlying Actinobacillus pleuropneumoniae exotoxin ApxI induced expression of IL-1ß, IL-8 and TNF-α in porcine alveolar macrophages.

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) causes fibrino-hemorrhagic necrotizing pleuropneumonia in pigs. Production of proinflammatory mediators in the lungs is an important feature of A. pleuropneumoniae infection. However, bacterial components other than lipopolysaccharide involved in this process remain unidentified. The goals of this study were to determine the role of A. pleuropneumoniae exotoxin ApxI in cytokine induction and to delineate the underlying mechanisms. Using real-time quantitative PCR analysis, we found native ApxI stimulated porcine alveolar macrophages (PAMs) to transcribe mRNAs of IL-1ß, IL-8 and TNF-α in a concentration- and time-dependent manner. Heat-inactivation or pre-incubation of ApxI with a neutralizing antiserum attenuated ApxI bioactivity to induce cytokine gene expression. The secretion of IL-1ß, IL-8 and TNF-α protein from PAMs stimulated with ApxI was also confirmed by quantitative ELISA. In delineating the underlying signaling pathways contributing to cytokine expression, we observed mitogen-activated protein kinases (MAPKs) p38 and cJun NH2-terminal kinase (JNK) were activated upon ApxI stimulation. Administration of an inhibitor specific to p38 or JNK resulted in varying degrees of attenuation on ApxI-induced cytokine expression, suggesting the differential regulatory roles of p38 and JNK in IL-1ß, IL-8 and TNF-α production. Further, pre-incubation of PAMs with a CD18-blocking antibody prior to ApxI stimulation significantly reduced the activation of p38 and JNK, and subsequent expression of IL-1ß, IL-8 or TNF-α gene, indicating a pivotal role of ß2 integrins in the ApxI-mediated effect. Collectively, this study demonstrated ApxI induces gene expression of IL-1ß, IL-8 and TNF-α in PAMs that involves ß2 integrins and downstream MAPKs.

Characterization of the monoclonal antibody against classical swine fever virus glycoprotein E(rns) and its application to an indirect sandwich ELISA.

Classical swine fever virus (CSFV) E(rns) is an envelope glycoprotein possessing RNase activity. The E(rns)-based enzyme-linked immunosorbent assay (ELISA) has been considered a discriminating diagnostic test for differentiating infected from vaccinated animals. The purpose of this study was to produce a specific monoclonal antibody (MAb) to E(rns) for further developing an indirect sandwich ELISA. The MAb CW813 was shown to specifically recognize both the monomer and dimer forms of Pichia pastoris yeast-expressed E(rns) (yE(rns)). The antigenic site recognized by MAb CW813 was mapped to the region of amino acid residues 101-160 of E(rns) where it was neither a neutralizing epitope nor essential to RNase activity. Furthermore, MAb CW813 was utilized as a capture antibody to develop a yE(rns)-based indirect sandwich ELISA for detecting swine antibody to E(rns). The assay demonstrated a high sensitivity and specificity that may provide an alternative method for developing a diagnostic kit with easy manipulation and low cost.

Evaluation of classical swine fever E2 (CSF-E2) subunit vaccine efficacy in the prevention of virus transmission and impact of maternal derived antibody interference in field farm applications.

BACKGROUND: Classical swine fever (CSF) is one of the most devastating pig diseases that affect the swine industry worldwide. Besides stamping out policy for eradication, immunization with vaccines of live attenuated CSF or the CSF-E2 subunit is an efficacious measure of disease control. However, after decades of efforts, it is still hard to eliminate CSF from endemically affected regions and reemerging areas. Most of previous studies demonstrated the efficacy of different CSF vaccines in laboratories under high containment conditions, which may not represent the practical performance in field farms. The inadequate vaccine efficacy induced by unrestrained factors may lead to chronic or persistent CSF infection in animals that develop a major source for virus shedding among pig populations. In this study, a vaccination-challenge-cohabitation trial on specific-pathogen-free (SPF) pigs and long-term monitoring of conventional sows and their offspring were used to evaluate the efficacy and the impact of maternally derived antibody (MDA) interference on CSF vaccines in farm applications. RESULTS: The trials demonstrated higher neutralizing antibody (NA) titers with no clinical symptoms and significant pathological changes in the CSF-E2 subunit vaccine immunized group after CSFV challenge. Additionally, none of the sentinel pigs were infected during cohabitation indicating that the CSF-E2 subunit vaccine could provoke adequately acquired immunity to prevent horizontal transmission. In field farm applications, sows immunized with CSF-E2 subunit vaccine revealed an average of higher and consistent antibody level with significant reduction of CSF viral RNA detection via saliva monitoring in contrast to those of live attenuated CSF vaccine immunized sows possessing diverse antibody titer distributions and higher viral loads. Furthermore, early application of the CSF-E2 subunit vaccine in 3-week-old piglets illustrated no MDA interference on primary immunization and could elicit consistent and long-lasting adequate antibody response suggesting the flexibility of CSF-E2 subunit vaccine on vaccination program determination. CONCLUSIONS: The CSF-E2 subunit vaccine demonstrated significant efficacy and no MDA interference for immunization in both pregnant sows and piglets. These advantages provide a novel approach to avoid possible virus shedding in sow population and MDA interference in piglets for control of CSF in field farm applications.

Expression and immunological studies of classical swine fever virus glycoprotein E2 in the bi-cistronic baculovirus/larvae expression system.

To develop an economical, easy technique for producing recombinant E2 glycoprotein (rE2) of classical swine fever virus (CSFV) as a candidate immunogen, a bi-cistronic baculovirus/larvae expression vector was constructed using p10 promoter, an internal ribosome entry site, and the gfp gene. Trichoplusia ni larvae were successfully infected with the occluded recombinant baculovirus via feed, and the characteristics of rE2 were confirmed by immunoblot and glycosylation stain. rE2 at a concentration of 0.6-0.8 mg/ml without degradation was obtained from hemolymphs of infected larvae that emitted high levels of green fluorescence. Immunization assays indicated that mice and piglets immunized with rE2-containing hemolymph elicited high titers of anti-CSFV E2 antibodies with virus-neutralizing activity. This is the first study to indicate that baculovirus/T. ni larvae-expressed rE2 can be served as a vaccine candidate. This system provides an economical alternative for the production of vaccine components in the veterinary industry.

Evaluation of a type 2 modified live porcine reproductive and respiratory syndrome vaccine against heterologous challenge of a lineage 3 highly virulent isolate in pigs.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most common diseases in the global swine industry. PRRSV is characterized by rapid mutation rates and extensive genetic divergences. It is divided into two genotypes, which are composed of several distinct sub-lineages. The purpose of the present study was to evaluate the cross-protective efficacy of Fostera PRRS MLV, an attenuated lineage 8 strain, against the heterologous challenge of a lineage 3 isolate. Eighteen pigs were randomly divided into mock, MLV and unvaccinated (UnV) groups. The pigs in the MLV group were administered Fostera PRRS vaccine at 3 weeks of age and both the MLV and UnV groups were inoculated with a virulent PRRSV isolate at 7 weeks. Clinically, the MLV group showed a shorter duration and a lower magnitude of respiratory distress than the UnV group. The average days of fever in the MLV group was 3.0 ± 0.5, which was significantly lower than the 6.2 ± 0.5 days of the UnV group (P < 0.001). The average daily weight gains of the mock, MLV and UnV groups were 781 ± 31, 550 ± 44 and 405 ± 26 g/day, respectively, during the post-challenge phase. The pathological examinations revealed that the severity of interstitial pneumonia in the MLV group was milder compared to the UnV group. Furthermore, PRRSV viremia titers in the MLV pigs were consistently lower (101-101.5 genomic copies) than those of the UnV pigs from 4 to 14 DPC. In conclusion, vaccination with Fostera PRRS MLV confers partial cross-protection against heterologous challenge of a virulent lineage 3 PRRSV isolate.

The N-terminus of porcine circovirus type 2 replication protein is required for nuclear localization and ori binding activities.

Porcine circovirus type 2 possesses a circular, single-stranded DNA genome that requires the replication protein (Rep) for virus replication. To characterize the DNA binding potential and the significant region that confers the nuclear localization of the Rep protein, the defined coding regions of rep gene were cloned and expressed. All of the recombinant proteins except for the N-terminal 110 residues deletion mutant could bind to the double-stranded minimal binding site of replication origin (ori). In addition, the N-terminal deletion mutant lacking 110 residues exhibited mainly cytoplasmic staining in the transfected cells in contrast to the others, which localized dominantly in the nucleus, suggesting that this N-terminal domain is essential for nuclear localization. Furthermore, a series of green fluorescence proteins (GFP) containing potential nuclear localization signal (NLS) sequences were tested for their cellular distribution. The ability of the utmost 20 residues of the N-terminal region to target the GFP to the nucleus confirmed its role as a functional NLS.

Actinobacillus pleuropneumoniae serotype 10 derived ApxI induces apoptosis in porcine alveolar macrophages.

Actinobacillus pleuropneumoniae (AP) is the causative agent of swine pleuropneumonia, a fibrinous, exudative, hemorrhagic, necrotizing pleuropneumonia affecting all ages of pigs. Actinobacillus pleuropneumoniae exotoxins (Apx) are one of the major virulence factors of AP. Due to the complex nature of Apx toxins produced by AP, little is known regarding the interactions of individual species of Apx toxin with target cells. The objective of this study was to examine whether AP serotype 10-derived exotoxin, ApxI, caused apoptosis in porcine alveolar macrophages (PAMs) and to delineate the underlying signaling pathways. Isolated PAMs were stimulated with different concentrations of native ApxI and monitored for apoptosis using Hoechst staining, TUNEL, and DNA laddering assays. The ApxI-stimulated PAMs exhibited typical morphological features of apoptosis, including condensation of chromatin, formation of apoptotic bodies and DNA laddering. ApxI-induced apoptosis in a concentration- and time-dependent manner. Furthermore, to delineate the signaling events involved in ApxI-induced apoptosis, it was observed that caspase 3 was activated in ApxI-stimulated PAMs. Ablation of caspase 3 activity via specific inhibitors protected PAMs from apoptosis by ApxI. This study is the first to demonstrate that native ApxI causes apoptosis in PAMs at low concentrations and that these apoptotic events are mediated via a caspase 3-dependent pathway. These findings suggest a role of ApxI in AP infection as it might impair the host defense system through the induction of apoptosis in PAMs.

Effect of immunostimulation by detoxified E. coli lipopolysaccharide combined with inactivated Propionibacterium granulosum cells on porcine immunity.

The aim of this experiment was to evaluate the immunomodulating activities of inactivated Propionibacterium granulosum cell walls and E. coli lipopolysaccharide (PG/LPS) on porcine immunity. Piglets were intramuscularly administered PG/LPS (1 ml/10 kg body weight) once or twice. The function of natural killer cells, lymphocytes and neutrophils and the adjuvant effect on antibody induction by attenuated classical swine fever virus (CSFV) and inactivated Mycoplasma hyopneumoniae vaccination were evaluated. The results showed that the cytotoxicity of natural killer cells and proliferation of lymphocytes in response to mitogen stimulation were significantly enhanced (P<0.05) in those pigs receiving PG/LPS injection compared with the controls. However, there was no significant effect on the phagocytic activity of neutrophils (P>0.05). PG/LPS also displayed adjuvant effects with CSFV and Mycoplasma hyopneumoniae vaccines. Moreover, pigs receiving two injections of PG/LPS showed a 20.8% growth enhancement compared with untreated pigs. Thus, PG/LPS caused positive immunoregulation of porcine innate immune system effectors, non-specific activation of lymphocytes and antibody production.

N-terminus of Classical swine fever virus strain TD96 glycoprotein Erns contains a potential heparin-binding domain.

Classical swine fever virus (CSFV) envelope glycoprotein Erns has been shown to bind to cell surface sulphated-heparin-like glycosaminoglycans (GAGs), which participate in cell attachment of the virus. In this study, the CSFV Erns gene was codon optimized for expression in the yeast Pichia pastoris. A C-terminally truncated Erns recombinant protein lacking the previously identified heparin-binding domain (HBD) bound to heparin column, suggesting the presence of another HBD in CSFV Erns. Sequence analyses of the CSFV Erns coding region revealed a common potential N-terminal HBD at residues 301－311. Site-directed mutagenesis of the basic amino acids at K303 and K306 significantly reduced the heparin-binding affinity of the protein. Further mutations of both T310 and H311 had little effect. Thus, a novel potential heparin-binding site near the N-terminus of CSFV strain TD96 Erns has been detected, and the two basic amino acids K303 and K306 are crucial for binding activity to heparin matrix and cell-surface GAGs.

The impact of porcine circovirus associated diseases on live attenuated classical swine fever vaccine in field farm applications.

Porcine circovirus associated diseases (PCVADs) are among the most important diseases affecting the worldwide swine industry. Vaccination against porcine circovirus type 2 (PCV2) infection has been utilized for disease control and effectively reduces clinical signs of PCVADs. To evaluate the efficacy of the PCV2 vaccine in field farms, we conducted a trial using conventional pigs immunized with the subunit PCV2 vaccine followed by PCV2 challenge. Immunized pigs demonstrated lower serum viral loads, less viral antigen staining in lymph nodes, and higher average daily weight gain, confirming the protective efficacy of the vaccine. However, low levels of PCV2 infection were still detected in vaccinated pigs after challenge, suggesting that the PCV2 vaccine was unable to eradicate the virus, which could lead to asymptomatic PCV2 subclinical infection (PCV2-SI) in pig farms. Additionally, PCV2 infection is a risk factor for impaired pig immune response development during the weaning to growth stages, which is a crucial period to receive vaccines against classical swine fever (CSF). Therefore, the impact of PCV2-SI or PCV2-systemic disease (PCV2-SD) on live attenuated CSF vaccine was investigated. After PCV2 challenge, there was no difference in levels of classical swine fever virus (CSFV) neutralizing antibodies (NA) between pigs with PCV2-SD and PCV2-SI, suggesting that the efficacy of CSF vaccine was compromised. Moreover, results of long-term monitoring of CSFV NA titers in PCV2-SI pigs with minimized interference by maternally-derived antibodies suggested that serum PCV2 viral loads greater than 102 copies/mL may compromise the efficacy of CSF vaccine. Overall, a conventional pig model was established to demonstrate the impaired efficacy of the subunit PCV2 vaccine and its impact on the CSF vaccine in vaccination-challenge trials. Additionally, the impaired efficacy of the PCV2 vaccine resulted in increased PCV2-SI, eventually leading to compromised the live attenuated CSF vaccine induced NA response in field farm applications.

Expression of the porcine circovirus type 2 capsid protein subunits and application to an indirect ELISA.

Porcine circovirus type 2 (PCV2) is considered to be associated with post-weaning multisystemic wasting syndrome (PMWS), which is a newly emerged economically important swine disease. The entire coding region of open reading frame 2 (ORF2), encoding the viral capsid protein (Cap), of PCV2 was cloned and sequenced from the clinical specimen obtained from PMWS-affected piglets. Six recombinant subunits, A-F, spanning the defined regions of Cap were produced by E. coli expression system and used as antigens for testing their reactivities with swine sera in the indirect ELISA. The recombinant Cap subunit-based ELISA was evaluated by examining a panel of 12 PCV2-negative and 26 PCV2-positive sera. When the positive/negative cut-off value was set at the mean value of negative sera plus 3 standard deviations, all subunits-based ELISA demonstrated 100% specificities. The N-terminal subunits, A and B, revealed poor reactivity with positive swine sera, whereas, greater immunoreactivity was observed for the C-terminal subunits of which subunits C and D demonstrated good sensitivities of 96.2% and 84.6%, respectively. The recombinant Cap subunits possessing defined antigenicity are easy to produce and the subunit-based ELISA was developed with a high specificity and sensitivity that may provide a useful method for routine serodiagnosis of PCV2 infection.

Spontaneous rhabdomyosarcoma in a young Sprague-Dawley rat.

An eight-week-old male Sprague-Dawley (SD) rat showed signs of emaciation, and masses were found in the subcutis around the cervical and thoracic regions. At necropsy, a multilobular mass, 2.2 x 1.8 x 2.0 cm in size, had grown from the left neck into the thoracic cavity. On a cutting surface, masses were firm and whitish to tan, with necrotic and hemorrhagic plaques. Microscopically, masses were composed of multiple nodules of tumor cells that were incompletely encapsulated with fibrous connective tissue. The tumor cells exhibited varied forms, from spindle to globoid shapes with minimal to abundant eosinophilic cytoplasm, and appeared as large, multinucleated cells; many of the tumor cells were vacuolated. Histochemistry results revealed that tumor cells exhibited some cross-striation in the cytoplasm using PTAH staining. There were some multinuclear tumor cells with vacuoles located around the nuclei, and these vacuoles showed reddish staining by the periodic acid Schiff (PAS) method. Immunohistochemical staining also expressed intense granular cytoplasmic staining for desmin and myoglobin, with highly positive staining for PCNA, whereas alpha-smooth muscle actin (alpha-SMA) was negative. Based on the pathology results, a spontaneous rhabdomyosarcoma with pleomorphic type was diagnosed in a young SD rat.

The emergence in Taiwan of fluoroquinolone resistance in Salmonella enterica serotype choleraesuis.

BACKGROUND: Salmonella enterica serotype choleraesuis is a cause of serious systemic infections. Because fluoroquinolones are the drug of choice for the treatment of severe salmonella infections, the emergence and dissemination of fluoroquinolone-resistant S. enterica serotype choleraesuis have clinical consequences. METHODS: In Taiwan, a hospital-based surveillance system has been in place since 1987 to monitor the incidence of S. enterica serotype choleraesuis infections and the antimicrobial susceptibility of the isolates. We investigated the rapid emergence of fluoroquinolone resistance in this serotype in 2000 and 2001. Pigs in Taiwan were evaluated as a potential source of the resistant salmonella. RESULTS: A total of 501 clinical isolates of S. enterica serotype choleraesuis were recovered in our hospital from 1987 through 2000. The proportion of total salmonella isolates made up by S. enterica serotype choleraesuis decreased from an average of 8.4 percent before 1995 to 2.7 percent in 1996 through 1998. During 1999 and 2000, this proportion increased significantly, to an average of 5.0 percent. Ciprofloxacin resistance in S. enterica serotype choleraesuis has been observed since 2000. In the third quarter of 2001, 60 percent of isolates were resistant to ciprofloxacin. Molecular typing indicated that the primary source of S. enterica serotype choleraesuis isolates was herds of swine. All the resistant isolates from humans and swine had mutations that led to the substitution of phenylalanine for serine at position 83 and asparagine for aspartic acid at position 87 in the gene for DNA gyrase A. CONCLUSIONS: This investigation in Taiwan indicates that fluoroquinolone-resistant S. enterica serotype choleraesuis can spread from swine to humans. The use of fluoroquinolones in food animals should be prohibited.