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.     

High-amylose corn exhibits better antioxidant activity than typical and waxy genotypes

The consumption of fruits, vegetables, and whole grains rich in antioxidative phytochemicals is associated with a reduced risk of chronic diseases such as cancer, coronary heart disease, diabetes, Alzheimer's disease, cataract, and aged-related functional decline. For example, phenolic acids are among the main antioxidative phytochemicals in grains that have been shown to be beneficial to human health. Corn (Zea mays L.) is a major staple food in several parts of the world; thus, the antioxidant activity of several corn types was evaluated. The 2,2-Diphenyl-1-picryhydrazyl free radical (DPPH*) scavenging activity, total phenolic content (TPC), antioxidant capacity of lipid-soluble substances (ACL), oxygen radical absorbance capacity (ORAC), and phenolic acid compositions of typical and mutant genotypes (typical-1, waxy, typical-2, and high-amylose) were investigated. The DPPH* scavenging activity at 60 min was 34.39-44.51% in methanol extracts and 60.41-67.26% in HCl/methanol (1/99, v/v) extracts of corn. The DPPH* scavenging activity of alkaline hydrolysates of corn ranged from 48.63 to 64.85%. The TPC ranged from 0.67 to 1.02 g and from 0.91 to 2.15 g of ferulic acid equiv/kg of corn in methanol and HCl/methanol extracts, respectively. The TPC of alkaline hydrolysates ranged from 2.74 to 6.27 g of ferulic acid equiv/kg of corn. The ACL values were 0.41-0.80 and 0.84-1.59 g of Trolox equiv/kg of corn in methanol and HCl/methanol extracts, respectively. The ORAC values were 10.57-12.47 and 18.76-24.92 g of Trolox equiv/kg of corn in methanol and HCl/methanol extracts, respectively. ORAC values of alkaline hydrolysates ranged from 42.85 to 68.31 g of Trolox equiv/kg of corn. The composition of phenolic acids in alkaline hydrolysates of corn was p-hydroxybenzoic acid (5.08-10.6 mg/kg), vanillic acid (3.25-14.71 mg/kg), caffeic acid (2.32-25.73 mg/kg), syringic acid (12.37-24.48 mg/kg), p-coumaric acid (97.87-211.03 mg/kg), ferulic acid (1552.48-2969.10 mg/kg), and o-coumaric acid (126.53-575.87 mg/kg). Levels of DPPH* scavenging activity, TPC, ACL, and ORAC in HCl/methanol extracts were obviously higher than those present in methanol extracts. There was no significant loss of antioxidant capacity when corn was dried at relatively high temperatures (65 and 93 degrees C) postharvest as compared to drying at ambient temperatures (27 degrees C). Alkaline hydrolysates showed very high TPC, ACL, and ORAC values when compared to methanol and HCl/methanol extracts. High-amylose corn had a better antioxidant capacity than did typical (nonmutant) corn genotypes.     

Comparison of the phosphorus and mineral concentrations in bran and abraded kernel fractions of a normal barley (Hordeum vulgare) cultivar versus four low phytic acid isolines

Phytic acid consists of 65-80% of the total phosphorus (P) in cereal grains. Its salts are concentrated in the germ and aleurone layers, which are typically removed during milling. We hypothesize that concentrations of different types of P and minerals in milled products will be greatly altered in low phytic acid (lpa) barleys. Seeds of cv. Harrington (control) and four lpa isolines-lpa1-1, lpa2-1, lpa3-1, and M955-were abraded by a laboratory method into five surface layer and four remaining kernel fractions. Results show that phytic acid in the four lpa lines ranged from 75% to 5% of the control. The decrease in phytic acid P concentration was matched almost equally by an increase in inorganic P, so that the rest of P (the sum of all P-containing compounds other than phytic acid P and inorganic P) and total P levels remained relatively unchanged among the five genotypes. These trends were also observed for the processed fractions. The major mineral elements in barley seeds were P, K, Mg, S, and Ca, while minor ones were Fe, Zn, Mn, Cu, and Ba. All types of P and other minerals measured were generally concentrated in the outer layers of the grain. Although there were substantial differences in mineral contents of bran fractions among genotypes, the level of phytic acid P had little effect on mineral contents in whole or abraded kernels. One major exception was Fe, which had the highest level in all tissues of M955 genotype. The above findings were all confirmed by analyzing another set of barley samples grown in a different environment. Thus, in general, breeding lpa barleys does not lead to reduced mineral contents in whole grains or elevated mineral levels in milled products.     

Simple, rapid, and inexpensive cleanup method for quantitation of aflatoxins in important agricultural products by HPLC

A chemical cleanup procedure for low-level quantitative determination of aflatoxins in major economically important agricultural commodities using HPLC has been developed. Aflatoxins were extracted from a ground sample with MeOH/H2O (80:20, v/v), and after a cleanup step on a minicolumn packed with Florisil, aflatoxins were quantified by HPLC equipped with a C18 column, a photochemical reactor, and a fluorescence detector. Water/MeOH (63:37, v/v) served as the mobile phase. Recoveries of aflatoxins B1, B2, G1, and G2 from peanuts spiked at 5, 1.7, 5, and 1.7 ng/g were 89.5+/-2.2, 94.7+/-2.5, 90.4+/-1.0, and 98.2+/-1.1, respectively (mean+/-SD, %, n=3). Similar recoveries, precision, and accuracy were achieved for corn, brown and white rice, cottonseed, almonds, Brazil nuts, pistachios, walnuts, and hazelnuts. The quantitation limits for aflatoxins in peanuts were 50 pg/g for aflatoxin B1 and 17 pg/g for aflatoxin B2. The minimal cost of the minicolumn allows for substantial savings compared with available commercial aflatoxin cleanup devices.     

Molecular characteristics of an immobilization antigen gene of the fish-parasitic protozoan Ichthyophthirius multifiliis strain ARS-6

Ichthyophthirius multifiliis (Ich), a ciliated protozoan parasite of fish, expresses surface antigens (i-antigens), which react with host antibodies that render them immobile. The nucleotide sequence of an i-antigen gene of I. multifiliis strain ARS-6 was deduced. The predicted protein of 47 493 Da is comprised of 460 amino acids (aa's) arranged into five imperfect repeats with periodic cysteine residues with the structure: CX₍₁₉₎₂₀CX₂CX₁₆₋₂₇CX₂CX₂₀₍₂₁₎CX₃. The N-terminal aa's typify a signal peptide motif while a stretch of C-terminal aa's resemble a glycosyl–phosphatidyl–inositol (GPI)-anchor addition site. The degree of deduced i-antigen aa sequence identity of strain ARS-6 (GenBank accession # ACH87654 and # ACH95659) with other I. multifiliis i-antigen sequences present in GenBank ranges from 99% to 36% identity with 52 kDa i-antigens of I. multifiliis strain G5 (accession #s AAK94941 and AAK01661 respectively). Immunoblot analysis of i-antigens following exposure of I. multifiliis theronts to catfish anti- I. multifiliis immune serum did not show any appreciable alteration in i-antigen expression. The mechanism that regulates i-antigen expression in I. multifiliis remains a puzzling question.     

Pan‐continental invasion of Pseudorasbora parva: towards a better understanding of freshwater fish invasions

In recent years, policy‐makers have sought the development of appropriate tools to prevent and manage introductions of invasive species. However, these tools are not well suited for introductions of non‐target species that are unknowingly released alongside intentionally‐introduced species. The most compelling example of such invasion is arguably the topmouth gudgeon Pseudorasbora parva, a small cyprinid species originating from East Asia. A combination of sociological, economical and biological factors has fuelled their rapid invasion since the 1960s; 32 countries (from Central Asia to North Africa) have been invaded in less than 50 years. Based on a combination of monitoring surveys (2535 populations sampled) and literature reviews, this paper aims to quantify and characterise important invasion parameters, such as pathways of introduction, time between introduction and detection, lag phase and plasticity of life history traits. Every decade, five new countries have reported P. parva introduction, mainly resulting from the movement of Chinese carps for fish farming. The mean detection period after first introduction was 4 years, a duration insufficient to prevent their pan‐continental invasion. High phenotypic plasticity in fitness related traits such as growth, early maturity, fecundity, reproductive behaviour and the ability to cope with novel pathogens has predisposed P. parva to being a strong invader. The Pseudorasbora parva invasion has provided quantitative data for the development of 1) early warning systems across different spatial scales; 2) rapid eradication programmes prior to natural spread in open systems and 3) sound risk assessments with emphasis on plasticity of life history traits.     

Induction of triploidy in spotted sand bass (Paralabrax maculatofasciatus Steindachner, 1868) by cold shock

Conditions for the induction of triploidy with cold shock of fertilized eggs of the spotted sand bass Paralabrax maculatofasciatus (Steindachner) were investigated. Different temperatures (12, 8 and 4 °C), timing of cold shock application (5, 10 and 15 min after fertilization) and duration of the shock (5, 10, 15 and 20 min) were tested. Triploidy was determined using flow cytometry at 12 h after larvae hatched. Triploids were produced only when the cold shock treatment was applied 5 min after fertilization. No significant difference was observed in the percentage of triploidy between temperature and the shock duration. At 8 and 4 °C, 100% triploidy was obtained at different durations of cold shock. Survival was significantly lower at 12 or 4 °C than at 8 °C. No significant difference was observed for shock duration at the temperature of 8 or 12 °C; however, at 4 °C, survival was significantly lower at longer durations. We recommend induction of triploidy by applying cold shock at 8 °C for a duration of 15–20 min starting at 5 min after fertilization, in the spotted sand bass.     

Effect of the VP3 gene of chicken anemia virus on canine mammary tumor cells

OBJECTIVE: To investigate the antitumor effect of the chicken anemia virus (CAV) VP3 gene in canine mammary tumor (CMT) cells. SAMPLE POPULATIONS: Established primary canine cell lines that originated from epithelial cells of resected CMTs and nonneoplastic mammary gland epithelial (MGE) cells. PROCEDURES: Expression vectors and lentiviral vectors encoding the VP3 gene from a Taiwan-Ilan isolate of CAV were used to deliver the VP3 gene into CMT cells and nonneoplastic MGE cells. Ectopic gene expression and the pro-apoptotic effect of the VP3 gene on CMT and nonneoplastic MGE cells by either transfection or viral infection were evaluated via immunofluorescence microscopy, western blot analysis, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling analysis. RESULTS: Overexpression of the enhanced green fluorescent protein-VP3 fusion protein was detected predominantly in the nuclei of CMT cells. In contrast, the VP3 protein was localized to the cytoplasm of nonneoplastic MGE cells. Among the fusion protein-expressing CMT cells, most underwent characteristic changes of apoptosis, whereas apoptosis was not detected in fusion protein-expressing, nonneoplastic MGE cells. Induction of apoptosis by VP3 gene overexpression in CMT cells was associated with the caspase-9-, but not the caspase-8-, mediated apoptosis pathway. CONCLUSIONS AND CLINICAL RELEVANCE: These data indicate that the VP3 gene of the CAV induces apoptosis in malignant CMT cells, but not in nonneoplastic canine MGE cells. On the basis of such tumor cell-specific killing, the VP3 gene may be a promising agent for the treatment of malignant mammary gland tumors in dogs.