Shih Hsuan Hsiao

Immunogenicity of recombinant GP5 protein of porcine reproductive and respiratory syndrome virus expressed in tobacco plant.

The aim of the study was to evaluate the immunogenicity of the ORF5-encoded major envelop glycoprotein 5 (GP5) of porcine reproductive and respiratory syndrome virus (PRRSV) expressed in tobacco plant as a potential pig oral vaccine in protection against PRRSV infection. Six-week-old PRRSV-free pigs were fed four times orally with 50g of chopped fresh GP5 transgenic tobacco leaves (GP5-T) (GP5 reaching 0.011% of total soluble protein) or wild-type tobacco leaves (W-T) each on days 0, 14, 28, and 42. Samples of serum, saliva, and peripheral blood mononuclear cells (PBMCs) were collected on days -1, 6, 13, 20, 27, 34, 41, and 48 after the initial oral vaccination. A similar vaccination-dependent gradual increase in the responses of serum and saliva anti-PRRSV total IgG and IgA, respectively, and in the levels of PRRSV-specific blastogenic response of PBMCs was seen in GP5-T-treated pigs; all statistically significant elevations occurred after the 2nd vaccination and were revealed after 20 days post-initial oral vaccination (DPIOV). Pigs fed on GP5-T also developed serum neutralizing antibodies to PRRSV at a titer of 1:4-1:8 after the 4th vaccination by 48 DPIOV. No detectable anti-PRRSV antibody responses and PRRSV-specific blastogenic response were seen in W-T-treated pigs. The present study has demonstrated that pigs fed on GP5-T could develop specific mucosal as well as systemic humoral and cellular immune responses against PRRSV. The results also support that transgenic plant as GP5-T can be an effective system for oral delivery of recombinant subunit vaccines in pigs.

The immunogenicity of DNA constructs co-expressing GP5 and M proteins of porcine reproductive and respiratory syndrome virus conjugated by GPGP linker in pigs.

The heterodimer of glycoprotein 5 (GP5) and non-glycosylated matrix protein (M) is the leading target for the development of new generation of vaccines against porcine reproductive and respiratory syndrome virus (PRRSV) infection. It has been demonstrated that DNA vaccine co-expressing GP5 and M proteins as a fusion protein aroused better immunogenicity than that expressing GP5 or M alone, but it was no better than the DNA vaccine co-expressing GP5 and M proteins with two different promoters. Altered natural conformation of the co-expressed GP5 and M fusion protein was considered as the major cause. Glycine-proline-glycine-proline (GPGP) linker can minimize the conformational changes in tertiary structure and provide flexibility of the peptide chain. The objective of this study was to evaluate whether the immunogenicity of DNA constructs co-expressing GP5 and M proteins linked by GPGP could be enhanced in pigs. Three recombinant DNA constructs expressing GP5/M fusion protein without GPGP linker (pcDNA-56), GP5/M fusion protein conjugated by GPGP linker (pcDNA-5L6), and M/GP5 fusion protein conjugated by GPGP linker (pcDNA-6L5) were established. Sixteen PRRSV-free pigs were randomly assigned to four groups and inoculated intramuscularly with 3 consecutive doses of 500 μg of empty vector pcDNA3.1, pcDNA-56, pcDNA-5L6 or pcDNA-6L5 each at a 2-week interval followed by challenge with 5 × 10(5) TCID(50) PRRSV at 3 weeks after the final inoculation. All pcDNA-56-, pcDNA-5L6-, and pcDNA-6L5- but not pcDNA-3.1-inoculated pigs developed neutralizing antibodies (NAs) 3 weeks after the final inoculation and a gradual increase in NA titers after PRRSV challenge, indicating that pigs inoculated with these DNA constructs could establish a sufficient immune memory. The pcDNA-5L6- and pcDNA-6L5-inoculated pigs displayed lower level and shorter period of viremia and lower tissue viral load following PRRSV challenge than did the pcDNA-56-inoculated pigs. The strategy of co-expressing GPGP-linked GP5 and M fusion protein may be a promising approach for future PRRSV vaccine development, possibly via the improvement of natural conformation of the target fusion protein.

Evaluation of the immunogenicity of a transgenic tobacco plant expressing the recombinant fusion protein of GP5 of porcine reproductive and respiratory syndrome virus and B subunit of Escherichia coli heat-labile enterotoxin in pigs.

Escherichia coli heat-labile enterotoxin B subunit (LTB) can be used as an adjuvant for co-administered antigens. Our previous study showed that the expression of neutralizing epitope GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) in transgenic tobacco plant (GP5-T) could induce PRRSV-specific immune responses in pigs. A transgenic tobacco plant co-expressing LTB and PRRSV GP5 as a fusion protein (LTB-GP5-T) was further constructed and its immunogenicity was evaluated. Pigs were given orally three consecutive doses of equal concentration of recombinant GP5 protein expressed in leaves of LTB-GP5-T or GP5-T at a 2-week interval and challenged with PRRSV at 7 weeks post-initial immunization. Pigs receiving LTB-GP5-T or GP5-T developed PRRSV-specific antibody- and cell-mediated immunity and showed significantly lower viremia and tissue viral load and milder lung lesions than wild type tobacco plant (W-T). The LTB-GP5-T-treated group had relatively higher immune responses than the GP5-T-treated group, although the differences were not statistically significant.

Porcine circovirus type 2 (PCV2) induces cell proliferation, fusion, and chemokine expression in swine monocytic cells in vitro

Granulomatous lymphadenitis is one of the pathognomonic lesions in post-weaning multisystemic wasting syndrome (PMWS)-affected pigs. This unique lesion has not been reported in direct association with viral infection in pigs. The objective of the present study was to evaluate whether porcine circovirus type 2 (PCV2) alone is able to induce functional modulation in porcine monocytic cells in vitro to elucidate its possible role in the development of granulomatous inflammation. It was found that the proliferation activity of blood monocytes (Mo) and monocyte-derived macrophages (MDM) was significantly enhanced by PCV2. During monocyte-macrophage differentiation, the PCV2 antigen-containing rate and formation of multinucleated giant cells (MGC) were significantly increased in MDM when compared to those in Mo. The MDM-derived MGC displayed a significantly higher PCV2 antigen-containing rate than did the mono-nucleated MDM. Supernatants from PCV2-inoculated MDM at 24 h post-inoculation induced an increased tendency of chemotactic activity for blood Mo. At the same inoculation time period, levels of mRNA expression of the monocytic chemokines, monocyte chemoattractant protein-1 and macrophage inflammatory protein-1, also significantly increased in PCV2-inoculated MDM. The results suggest that PCV2 alone may induce cell proliferation, fusion, and chemokine expression in swine monocytic cells. Thus, PCV2 itself may play a significant role in the induction of granulomatous inflammation in PMWS-affected pigs.

Immunopathological characterization of porcine circovirus type 2 infection-associated follicular changes in inguinal lymph nodes using high-throughput tissue microarray

The immunopathogenesis of porcine circovirus type 2 (PCV2) infection in conventional pigs is complicated by various environmental factors and individual variation and is difficult to be completely reproduced experimentally. In the present field-based study, a tissue microarray (TMA) consisting of a series of lymphoid follicles having different PCV2-loads was constructed using formalin-fixed and paraffin-embedded superficial inguinal lymph nodes (LNs) from 102 pigs. Using the TMA, a wide range of parameters, including co-infected viral pathogens, immune cell subsets, and cell apoptosis/proliferation activity by immunohistochemical (IHC) staining or in situ hybridization (ISH) were measured, characterized, and compared. The signal location and area extent of each parameter were interpreted by pathologists, semi-quantified by automated image analysis software, and analyzed statistically. The results herein demonstrated a significant negative correlation between PCV2 and CD79a (p<0.001) and a significant positive correlation between PCV2 and lysozyme (p<0.001) or TUNEL (p<0.001) using Pearson correlation analysis. The amount of porcine respiratory and reproductive syndrome virus (PRRSV) and porcine parvovirus antigens did not correlate with the tissue loads of PCV2 nucleic acid. Multiple regression analysis further predicted that PCV2 contributed major effects on CD79a, lysozyme, and TUNEL but PRRSV showed relatively less effects on these parameters. In addition, the total signal intensity of Ki67 (index of cell proliferation activity) did not change significantly among cases with different PCV2 loads; however, as the loading of PCV2 nucleic acid increased, the main contribution of Ki67 signal gradually shifted from B cells in the germinal center to T cells and macrophages in the interfollicular regions. In the present study, the use of TMA to establish a mathematical model with a wider range of statistical analysis can bring us a step forward to understand the immunopathogenesis of PCV2 infection-associated follicular changes in LNs.

Entamoeba invadens Myositis in a Common Water Monitor Lizard (Varanus salvator)

Amoebiasis is one of the most common protozoal diseases of reptiles, but amoebic myositis has not been reported in any animal species. An 11-year-old, male common water monitor lizard (Varanus salvator) was found dead with several subacute ulcerated skin wounds. Gross examination revealed multiple discrete to coalescing, white-yellow to gray, caseous foci scattered in the skeletal muscles and liver. The mucosa of small intestine was thickened, red, and contained many variably sized, dark red ulcers, with depressed and hemorrhagic centers. Histopathologic examination revealed severe necrotizing and granulomatous myositis, hepatitis, and enteritis accompanied by large numbers of intralesional, 10-20-μm diameter, periodic acid-Schiff-positive, amoeboid protozoa. Gene sequence analysis of a 136-bp region of the 18S ribosomal RNA amplified by polymerase chain reaction revealed 98–100% similarity with Entamoeba invadens. Aside from intestinal and hepatic involvement, no other internal organs were affected. The muscular infection by E. invadens likely resulted from a combination of direct invasion of trophozoites via skin wounds and hematogenous spread.

Monocyte-derived dendritic cells enhance cell proliferation and porcine circovirus type 2 replication in concanavalin A-stimulated swine peripheral blood lymphocytes in vitro

Dendritic cells (DCs) are professional antigen presenting cells cooperating with other immune cells for the activation of innate and adaptive immune responses. The objective of the present study was to investigate the replication activity of porcine circovirus type 2 (PCV2) in DCs and/or lymphocytes during their cross talk and its possible mechanism. Two models were set, herein. Swine blood monocyte (Mo)-derived DCs (MoDCs) or peripheral blood lymphocytes (PBLs) were inoculated with PCV2 prior to their co-cultivation. Bacterial lipopolysaccharide (LPS) and concanavalin A (Con A) were used to stimulate MoDCs and PBLs, respectively. During 6 days of cultivation, a high PCV2 antigen-containing rate without detectable intranuclear signals and a slight but significant increase in the copy number of PCV2 genome were detected in PCV2-inoculated MoDCs. The presence of LPS alone or PCV2-free PBLs, however, had no effect on the location of PCV2 antigens or copy number of PCV2 genome in PCV2-inoculated MoDCs. On the contrary, active PCV2 replication occurred in Con A-stimulated PCV2-inoculated PBLs. When compared with blood Mos, MoDCs induced significantly higher cell proliferation and intensified PCV2 replication in Con A-stimulated PCV2-inoculated PBLs, for which direct contact between MoDCs and lymphocytes was required. Among the cytokines secreted by Con A-activated PBLs, interleukin (IL)-2, but not IL-4 or interferon-γ, could induce cell proliferation and PCV2 replication in PCV2-inoculated PBLs. The findings suggest that although MoDCs support only limited PCV2 replication in themselves, their accessory cell function is required for cell proliferation and PCV2 replication in PCV2-infected lymphocytes.

Use of Formalin-Fixed Tissues to Determine Fumonisin B1-Induced Sphingolipid Alterations in Swine

Fumonisin B1 is a mycotoxin that causes lethal pulmonary edema in swine. Sphinganine, sphingosine, and the sphinganine to sphingosine ratio are important biomarkers for fumonisin B1 exposure. Currently, tissues selected for sphinganine and sphingosine analyses are frozen at −80°C until analyses take place. However, for diagnostics and some research projects, formalin is used more routinely as a preservative for long-term storage of tissues. To determine whether formalin-fixed tissues could be used for sphinganine and sphingosine analyses, sphinganine and sphingosine concentrations were quantified in both frozen and formalin-fixed lung, liver, kidney, and heart from fumonisin B1-treated and control pigs. Tissues were evaluated 3 months after freezing and 3, 6, and 12 months after formalin fixation. Sphinganine, sphingosine, and the sphinganine to sphingosine ratio of both frozen and formalin-fixed lung and liver from fumonisin B1-treated pigs were elevated. Formalin-fixed tissues had lower sphinganine and sphingosine concentrations but higher sphinganine to sphingosine ratios than the corresponding frozen tissues. Storage in formalin for up to 12 months did not affect the results. Sphingosine analysis could not be performed in formalin-fixed heart and kidney because of noninterpretable chromatograms. Therefore, formalin-fixed lung and liver can be used to determine fumonisin B1-induced sphinganine and sphingosine alterations in swine, with the sphinganine to sphingosine ratio being the most useful.

Development and evaluation of an indirect in situ polymerase chain reaction for the detection of porcine circovirus type 2 in formalin-fixed and paraffin-embedded tissue specimens

Taking advantage of the high sensitivity of polymerase chain reaction (PCR) and the cell-localizing ability of in situ hybridization (ISH), an indirect in situ PCR (ISPCR) method was developed for detecting the distribution of porcine circovirus type 2 (PCV2) in formalin-fixed and paraffin-embedded inguinal lymph nodes obtained from clinically healthy PCV2-carrier pigs and postweaning multisystemic wasting syndrome (PMWS)-affected pigs. Comparisons of the relative sensitivity of indirect ISPCR with other routinely used diagnostic methods for PCV2 indicated that nested PCR was the most sensitive method followed by indirect ISPCR, conventional PCR, ISH, and immunohistochemical (IHC) staining. Although indirect ISPCR, ISH, and IHC staining all revealed a similar signal distribution pattern of PCV2, using indirect ISPCR allowed specific amplification and detection of previously uneasily detected PCV2 signal than by routine ISH or IHC staining, particularly in those cells within the germinal center in clinically healthy PCV2-carrier pigs. Furthermore, six different PCV2 signal expression patterns in conjunction with the correlated lymphoid lesion stages were classified to describe the tissue morphological changes and viral infection. The result indicates that indirect ISPCR is a more effective, cell-based diagnostic tool with good specificity to detect limited PCV2 infection in formalin-fixed and paraffin-embedded tissue specimens and it would be a useful tool for further exploring the pathogenesis of PCV2 infection.

PHARMACOKINETICS, EFFICACY, AND SAFETY OF VORICONAZOLE AND ITRACONAZOLE IN HEALTHY COTTONMOUTHS (AGKISTRODON PISCIVORUS) AND MASSASAUGA RATTLESNAKES (SISTRURUS CATENATUS) WITH SNAKE FUNGAL DISEASE

Abstract Snake fungal disease (SFD; Ophidiomyces ophiodiicola) is posing a significant threat to several free-ranging populations of pitvipers. Triazole antifungals have been proposed for the treatment of mycoses in reptiles; however, data are lacking about their safety and efficacy in snakes with SFD. Study 1 investigated in vitro susceptibility, and identified that plasma concentrations >250 ng/ml (voriconazole) and >1,000 ng/ml (itraconazole) may be effective in vivo for SFD. In Study 2, the pharmacokinetics after a single subcutaneous voriconazole injection were assessed in apparently healthy free-ranging cottonmouths (Agkistrodon piscivorus). Based on pilot-study results, four snakes were administered a single injection of voriconazole (5 mg/kg). One pilot snake and three full-study snakes died within 12 hr of voriconazole administration. All surviving snakes maintained plasma concentrations >250 ng/ml for 12–24 hr. In Study 3, two Eastern massasaugas (Sistrurus catenatus) and a timber rattlesnake (Crotalus horridus horridus) diagnosed with SFD were treated with voriconazole delivered by subcutaneous osmotic pumps. The timber rattlesnake (12.1–17.5 mg/kg/hr) reached therapeutic concentrations, whereas the massasaugas (1.02–1.6 mg/kg/hr) did not. In Study 4, the pharmacokinetics of a single 10-mg/kg per-cloaca dose of itraconazole (Sporanox®) was evaluated in seven apparently healthy free-ranging cottonmouths. Similarly, the plasma and tissue concentrations did not meet therapeutic concentrations based on in vitro data. The data presented in this report serve as an initial step toward understanding the pharmacokinetics, efficacy, and safety of triazole antifungals in pitviper species with SFD. Further study is needed to determine the appropriate dose and route of administration of triazole antifungals in pitviper species.