Determination of spinosad and its metabolites in food and environmental matrices. 2. Liquid chomatography-mass spectrometry

A selective and sensitive method utilizing liquid chromatography-mass spectrometry (LC-MS) has been developed for determining residues of the natural insect control agent spinosad in several crop matrices that are difficult to analyze by HPLC with UV detection. The method determines the active ingredients (spinosyns A and D) and three minor metabolites (spinosyns B and K and N-demethylspinosyn D) in alfalfa hay, wheat hay, wheat straw, sorghum fodder, and corn stover. The analytes are extracted from the samples with an acetonitrile/water solution, and the extracts are purified by solid phase extraction with a C(18) disk and a silica cartridge. All five analytes are determined simultaneously in a single injection using positive atmospheric pressure chemical ionization LC-MS with selected ion monitoring. The average recoveries ranged from 69 to 96% with standard deviations ranging from 4 to 15%. The method has a validated limit of quantitation of 0. 01 microgram/g and a limit of detection of 0.003 microgram/g. The LC-MS method can also provide residue confirmation in addition to quantitation.     

Application potency of engineered G159 mutants on P1 substrate pocket of subtilisin YaB as improved meat tenderizers

A serine protease, subtilisin YaB, produced by alkalophilic Bacillus YaB, shows promises as a potent meat tenderizer, because its substrate specificity is for small amino acids, which are found at high levels in meat connective tissue proteins. Substrate specificity engineering of the substrate binding pockets was used to generate more suitable meat-tenderizing mutants, G124A, G124V, G159A, and G159S, derived from recombinant wild subtilisin YaB and expressed in Bacillus subtilis DB104. The characteristics of these recombinant enzymes were studied to evaluate their usefulness as improved meat tenderizers. The proteolytic activities of recombinant subtilisin YaB, engineered subtilisin YaBs, and commercially available papain, bromelain, collagenase, and elastase were compared using elastin, collagen, casein, and myofibrillar proteins as substrates. Hydrolysis of beef proteins was evaluated using the myofibrillar fragmentation index and collagen solubility. The results demonstrated that recombinant mutant G159A was the most improved meat tenderizer and can be used in the meat pH range of 5.5-6.0 and the temperature range of 10-50 degrees C. Contrary to the result obtained from artificial substrate, mutant enzymes engineered on G124 residues did not exhibit better tenderizing ability when elastin, collagen, or meat was used as substrate, suggesting the necessity of evaluation by real substrate before protein-engineered enzymes are applied commercially.     

Serological Comparison and Molecular Characterization for Verification of Calla lily chlorotic spot virus as a New Tospovirus Species Belonging to Watermelon silver mottle virus Serogroup

ABSTRACT Calla lily chlorotic spot virus (CCSV) isolated from central Taiwan was recently identified as a tospovirus serologically but distantly related to Watermelon silver mottle virus (WSMoV). To clarify the serological relationship between the two viruses, rabbit polyclonal antibody (PAb) to CCSV and mouse monoclonal antibodies (MAbs) to WSMoV NP or CCSV NP were produced in this investigation, using purified nucleocapsid protein (NP) as immunogens. The PAb to CCSV NP reacted stronger with the homologous antigen than with the heterologous antigen, with much lower A(405) readings in indirect enzyme-linked immunosorbent assay (ELISA) and low-intensity banding in immunoblotting. MAbs produced to CCSV NP or WSMoV NP reacted specifically with the homologous antigens but not with the heterologous antigens in both ELISA and immunoblot analyses. The CCSV S RNA was determined to be 3,172 nucleotides in length, with an inverted repeat at the 5' and 3' ends and two open reading frames encoding the NP and a nonstructural (NSs) protein in an ambisense arrangement. A typical 3'-terminal sequence (5'-AUUGCUCU-3') that is shared by all members of the genus Tospovirus also is present in the CCSV S RNA. The CCSV NP and NSs protein share low amino acid identities of 20.1 to 65.1% and 19.9 to 66.1%, respectively, with those of reported tospoviruses. Phylogenetic dendrogram analysis indicates that CCSV is a distinct member in the genus Tospovirus. The results provide evidence that CCSV is a new species in the genus Tospovirus and belongs to WSMoV serogroup.     

Fipronil modulation of GABAA receptor single-channel currents

Fipronil is the first phenylpyrazole insecticide introduced for pest control. Although fipronil is known to inhibit GABA receptors, the detailed mechanism of action remains to be seen. In order to elucidate the mechanism of fipronil interaction with the mammalian GABAA system, single-channel patch clamp experiments were performed using rat dorsal root ganglion neurons. The amplitude of main conductance state (27pS) current was not significantly altered by co-application of 10 microM fipronil and 10 microM GABA. The histograms of open time distribution were fitted to a sum of three exponential functions. After application of 10 microM fipronil, the proportion of the fastest component increased slightly and that of the slowest component decreased slightly. Thus, the mean open time was decreased from 11.4 ms to 7.8 ms by fipronil. The histograms of closed time distribution were fitted to a sum of four exponential functions. Fipronil 10 microM prolonged the slowest time constant resulting in a prolongation of the mean closed time from 29.7 ms to 52.8 ms. Thus, the frequency of channel openings was reduced. Thus, the fipronil suppression of GABA-induced whole-cell currents is caused in part by decreases in the channel open time and the frequency of channel openings.     

Effects of sugar cane extract on pseudorabies virus challenge of pigs

This experiment aimed to evaluate the efficacy of sugar cane extract (SCE) on the modulation of porcine immunity against pseudorabies virus (PrV) infection. Twelve-week-old experimental pigs were fed with SCE (500 mg/kg of body weight per day) for 3 days and challenged with PrV (2 x 10(5) TCID(50)) on the second day. Pigs that were only challenged with PrV and without SCE-treatment served as controls. The leukocyte functional assays were performed on the 7th and 14th day post-PrV challenge. Our results showed a significant enhancement (P<0.05) of natural killer cytotoxicity, lymphocyte proliferation, phagocytic function of monocytes, and interferon-gamma (IFN-gamma) production of CD4(+) and gammadelta T cells in the SCE-treated pigs compared with the controls. In addition, SCE administration reduced the severity of clinical signs and brain lesion in the course of disease in PrV-challenged pigs. SCE-treated pigs showed a 12% growth enhancement compared with untreated controls. SCE administration had an immunostimulating effect on porcine immunity that may subsequently enhance protective activities against PrV infection which may be extensively applied in field for the prevention of infections.     

Animal Biowarfare Research: Historical Perspective and Potential Future Attacks

A biological attack on livestock or poultry could result in the loss of valuable animals, costs related to the containment of outbreaks and the disposal of carcasses, lost trade and other economic effects involving suppliers, transporters, distributors and restaurants; however, it is not possible to secure all livestock, and livestock are much less well guarded than human targets. Thus, the vulnerability of the livestock industry to the introduction of biological agents varies for the following reasons: (i) the majority of lethal and contagious biological agents are environmentally resilient, endemic in foreign countries and harmless to humans, making it easier for terrorists to acquire, handle and deploy these pathogens, (ii) with animals concentrated in fewer production facilities and frequently transported between these facilities, a single pathogen introduction could cause widespread infection and (iii) the extent of human travel around the globe makes it difficult to exclude exotic animal diseases as possible biological agents. Historically, many governments have developed and planned to use biological agents for direct attacks on livestock or poultry. In the past, developed nations have actively developed biological weapons to target animals. The potential spectrum of bioterrorism ranges from isolated acts against individuals by individuals to tactical and strategic military attacks and state‐sponsored international terrorism intended to cause mass casualties in animals, humans or both. This review provides an overview of the past development and use of biological weapons and describes potential future attacks.     

Functional processing of PET fabrics using boehmite/silica/thiazole dye hybrid materials

A series of hybrid materials composed of boehmite/silica/thiazole dyes and prepared via the sol-gel process is synthesized from aluminum isopropoxide (AIP) and tetraethoxysilane using heteroaryl 2-amino-thiazole azo dyes. Heterocyclic 2-amino-thiazole azo dyes undergo a hydrolysis-condensation reaction with an appropriate proportion of AIP under a catalyst, at a constant ratio of vinyltriethoxysilane (VTES) and tetraethoxysilane (TEOS). The structures of these hybrid materials composed of boehmite/silica/thiazole dyes are characterized using Fourier transform infrared (FT-IR) analysis. The surface morphology of polyethylene terephthalate (PET) fabrics is evaluated using scanning electron microscopy (SEM). SEM images show uniform dyeing of the PET fabrics that confirms the reaction of the hybrid materials with the PET fabrics. The water contact angle, washing fastness, color evenness, air permeability, and warmth retention of the PET fabrics dyed with hybrid materials composed of boehmite/silica/thiazole dyes are evaluated. The evaluation results indicate improved warmth retention property and good water repellency.     

First Report on Molecular Detection of Equine Upper Respiratory Infectious Viruses in Republic of Korea

The prevalence of equine respiratory virus infections among a suspected population of race horses was examined using polymerase chain reaction (PCR). One or more of five equine respiratory viruses were detected in the nasal swabs of 45 of 89 horses (50.6%), and the detection rate of equine herpesvirus type 1 (EHV-1), equine herpesvirus type 4 (EHV-4), equine herpesvirus type 5 (EHV-5), equine rhinitis A virus (ERAV) and equine rhinitis B virus (ERBV) were 5.6%, 7.9%, 39.0%, 2.2%, and 6.7%, respectively. Among the 45 infected horses, 7 were co-infected with EHV and/or equine rhinitisvirus (ERV). Equine influenzavirus and equine arteritisvirus were not detected in any samples. Specific antibodies to EHV-1 and/or EHV-4 were detected in 59 of 73 tested sera (80.8%), using a virus neutralization test. This investigation suggests that equine respiratory viruses are endemic at Seoul Race Park and that the impact of viral infections on race horses’ health in Republic of Korea should be evaluated.     

Functional secretion of a type 1 antifreeze protein analogue by optimization of promoter, signal peptide, prosequence, and terminator in Lactococcus lactis

Lactococcus lactis is a food-grade microorganism of major commercial importance. Antifreeze protein is a potent cryogenic protection agent for the cryopreservation of food and pharmaceutical materials. In this study, extracellular expression of a novel recombinant type I antifreeze protein analogue (rAFP) in L. lactis was optimized. An efficient SlpA promoter (P SlpA) was fused to various signal peptides (SPs) and propeptide sequences to examine the extracellular expression levels of rAFP. An efficient signal peptide, SP sacB, fused to prosequence AE, enabled higher extracellular rAFP production; use of the SlpA terminator (Ter SlpA) was a further improvement. The extracellularly expressed rAFP successfully inhibited ice recrystallization and is thus potentially applicable for cryogenic preservation.