Investigating the Correlations between Single Nucleotide Polymorphisms of Immunity-related Genes and Cell Surface Markers of Immune Cells in Porcine Stocks in Taiwan

Ann Ying-An Chen1, 2, An-Chi Liu1, 2, Mei-Li Wu1, 2 and Hso-Chi Chaung1, 2

1Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan

2Research Center for Animal Biologics, National PingTung University of Science and Technology, Pingtung, Taiwan

Single nucleotide polymorphism (SNP) is a genetic variation controlled gene expressions on transcriptional level. It has been reported that SNPs in immune-relevant genes influence the susceptibility of individuals to pathogens and diseases. The aims of this study are to investigate the distribution of SNP sites in immunity-related genes and its correlations to cell surface markers of immune cells within purebred and crossbred pigs in Taiwan. In this study, 39 SNPs of porcine immune-related genes (IFN-α, IFN-γ, TNF-α, GM-CSF, MCP-1, TLR3, TLR4, TLR7, TLR8 and TLR9) were identified and percentages of positive cells with cell surface markers of CD4, CD8, CD80/86, MHCI, and MHCII were analyzed from blood samples collected from 187 piglets at the age of 8 weeks (Landrace, Yorkshire, Landrace-Yorkshire hybrid, Duroc and Taiwan Black). The results showed that genotype distribution of 28 SNPs were significant associated with breeds, especially between Landrace and Taiwan Black. For instance, frequency of AG genotype of SNP-1 in Taiwan Black and Landrace were 1.6% and 13.4%, respectively. Among them, 18 SNPs significantly had impacts on the expressions of cell surface markers of CD4, CD8, CD80/86, MHCI and MHC II. Interestingly, CD4% (35.42±4.17) in AA genotype of SNP-28 was significantly higher than those in GG genotype (26.65±1.93) in all breeds. An understanding of these SNPs and their correlations with immunity parameters may be beneficial on establishing high pathogen-resistant breeding parameters. This genomic information aids in the discovery of SNPs in genes controlling disease resistance.

Keywords: single nucleotide polymorphism, immune cells, cell surface markers, pig



Establishing a Porcine Immune Synapse System as a Fast Screening System with Response to Immunization with PRRSV Subunit Vaccine

Ann Ying-An Chen1, 2, An-Chi Liu1, 2, Mei-Li Wu1, 2 and Hso-Chi Chaung1, 2

1Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan

2Research Center for Animal Biologics, National PingTung University of Science and Technology, Pingtung, Taiwan

The importance of the interactions of pathogens and the immune system has played an extremely important role in agricultural research for development of vaccinations. Porcine reproductive and respiratory syndrome virus (PRRSV) causes severe economic loss to the swine industry worldwide. The aim of this study is to establish a porcine immune synapse system by co-culturing T subset/antigen-presenting cell as a fast screening system. In this study, blood samples were collected from 12 SPF pigs that were randomly separated into 3 groups for the control group and two different vaccinated immunizing PRRSV subunit vaccines (PRRSV-1 and PRRSV-2).  Different T subsets by fluorescence-activated cell sorting were co-cultured with alveolar macrophage and the profiles of cytokine secretion and mRNA transcription levels of toll-like receptors were examined. The results showed that there were significant interaction differences in IL2, TLR3, TLR8, and TLR9 expressions between vaccinated groups and co-cultured cell systems. For instance, TLR8 expressions in CD4+CD25+ cells of PRRSV-2 (1.005±0.865) were lower than those in same cell system of controls (2.625±1.552). Interesting, the significantly lower IL-10 production were observed in animals vaccinated with PRRSV-2 (1.459± 0.396) as compared with those in control (4.155±1.494) or vaccinated with PRRSV-1 (4.781±1.135). These findings demonstrated that a porcine immune synapse system can be potentially used as screening the micro-environment factors as the adjuvant candidate or effective antigen epitopes. This system will be beneficial in reducing the use of experimental animals when studying the immune system whilst developing disease-resistance biomarkers or immune-modulatory adjuvants.

Keywords: porcine reproductive and respiratory syndrome virus, immune synapse, T subsets, Toll-like receptor, pig


Advanced Development of FMDV Blocking ELISA Based on Site1/Site2 MAbs and VLPs Expressed by SUMO Fusion Protein System

Chieh-Li Liu1, Heng-Wei Lee1, Cheng-Yao Yang2 and Ivan-Chen Cheng1

1Department of Veterinary Medicine, National Taiwan University, Taipei Taiwan

2Agricultural Technology Research Institute, Miaoli Taiwan

Foot-and-mouth virus (FMD) is a non-enveloped virus with an icosahedral capsid containing a positive-sense single-strand RNA. Due to its wide host range and highly contagious activity, FMD is one of the most important diseases in animal husbandry industry in the world. In 1997, a devastating outbreak of FMD in Taiwan caused loses of about US$ 378 million. Until this day, FMD has not been fully eradicated in Taiwan. Hence, establishment of a fast and stable platform for serum monitoring is undoubtedly necessary. Recently, site 1 and site 2 have been regarded as the most immune-dominant neutralization sites. In order to obtain site 2 and site 1 monoclonal antibodies (MAbs) from a previously produced MAbs panel, we established a screening system based on mutated Virus-like-particles (VLPs). Due to its exhibition of authentic antigenicity, VLPs expressed by mammalian cell expression system was chosen as antigen for blocking ELISA (bELISA) instead of recombinant protein. Combining MAbs and VLPs systems, the result of our bELISA for 90 swine serum showed high correlation to SN titer, “r=0.64” in site 1 and “r=0.79” in site 2. Considering cost and application, we intend to express our VLPs with SUMO fusion protein in E.coli, the more productive expression system. With further research, we believe that the bELISA platform we have established could not only be used for FMD researching, but also for serum monitoring.

Keywords: Foot-and-Mouth Disease Virus, Neutralization Site, Monoclonal Antibody, SUMO Fusion Protein, Blocking ELISA



Development of Vector Vaccine Platform Based on Newcastle Disease Virus Backbone by Using Reverse Genetics Technology

Ai-Ping Hsu, Hsuan Chen, Yi-Ta Lu, Yu-Sheng Chou, Jia-Yuan Chang, Yu-Hua Shih and Chun-Hsien Tseng

Animal Health Research Institute, Council of Agriculture, New Taipei City, Taiwan

A novel Newcastle disease (ND) attenuated live vaccine and reverse genetics technology were successfully developed by Animal Health Research Institute. Furthermore, two inventions were incorporated for establishing vector vaccine platform. This platform has been already inserted with antigenic fragment of infectious bronchitis virus (spike protein of IBV) and also of white spot syndrome virus (WSSV). The preliminary assessment revealed chickens immunised by ND vector vaccine harboring the aforementioned antigenic fragments respectively could be induced the protective neutralising immunity against IBV or specific antibodies to WSSV. In order to increase and improve the antigenicity of the vector vaccine platform, the immune-stimulators were designed to be constructed on the vector for immunizing chickens in combination with other bivalent vector vaccine, and the efficacy achievements will be further evaluated.

Keywords: Newcastle disease, vaccine, reverse genetics



Analysis of Banned Veterinary Drugs Residues in Shrimps by Liquid Chromatography Tandem Mass Spectrometry

Geng-Ruei Chang

Department of Veterinary Medicine, National Chiayi University

Safety of seafood is a crucial public health concern for consumers. We applied a validated method to analyze residues of banned veterinary drugs, namely chloramphenicol, malachite green, leucomalachite green, and nitrofuran metabolites namely 5-methylmorpholino-3-amino-2-oxazolidinone (AMOZ), 3-amino-2-oxazolidinone (AOZ), 1-aminohydantoin hydrochloride (AH-HCl), and semicarbazide hydrochloride (SC-HCl). These drugs were simultaneously detected in shrimps through liquid–gas chromatography tandem mass spectrometry. In total, 60 shrimp samples, which included whiteleg, grass, or giant river shrimp, were collected from aquafarms and production areas in Taiwan between January 2011 and December 2012. The detection of 0.4 ng/g of AHD and SC in one whiteleg shrimp, and 0.3 ng/g of AOZ in one whiteleg shrimp, meaning that 3.33% of the samples contained residues of banned veterinary drug. These data can be used for monitoring residues of veterinary drugs in aquatic products and as a reference for food safety. Aquatic samples should be continually surveyed for detecting residues of banned chemicals and ensuring food safety.

Keyword: Banned eterinary drugs, Residues, Shrimp, Liquid chromatography tandem mass spectrometry, Taiwan



Phthalates and Organophosphorus Insecticide Residues in Shrimp Determined by Liquid/Gas Chromatography Tandem Mass Spectrometry and a Health Risk Assessment

Geng-Ruei Chang

Department of Veterinary Medicine, National Chiayi University

Phthalates have endocrine disruption, mutagenicity, and carcinogenic effects. Organophosphorus pesticides are ubiquitous contaminants with high bioaccumulation and persistence levels in the environment. Phthalates and organophosphorus pesticides can both have adverse effects on humans and animals. In this study, we applied a validated method to analyze the phthalate residues in shrimp, including the levels of benzyl butyl phthalate (BBP), di-n-butyl phthalate (DBP), di-2-ethylhexyl phthalate (DEHP), diisodecyl phthalate (DIDP), and diisononyl phthalate (DINP). The Quick, Easy, Cheap, Effective, Rugged, and Safe method was employed to extract shrimp samples and detect 18 insecticides, which were then analyzed using liquid and gas chromatography–tandem mass spectrometry. A total of 46 samples of whiteleg, grass, or giant river shrimp were collected from aquafarms and production areas in Taiwan from June 2015 to December 2016. We detected 0.45–0.70 mg/kg of DEHP in two whiteleg shrimps, 0.50 mg/kg of DEHP in one grass shrimp, 0.02–0.03 mg/kg of chlorpyrifos in two whiteleg shrimps, 0.03 mg/kg of chlorpyrifos in one grass shrimp, and 0.03 mg/kg of trichlorfon in one giant river shrimp, indicating that 6.52% and 8.70% of the samples contained phthalate and insecticide residues, respectively. Daily exposure was calculated and found to not exceed the tolerable daily intake (TDI) for phthalates and acceptable daily intake (ADI) for insecticides. Furthermore, the assessed risk was negligible and considered to be at a safe level, indicating no immediate health risk associated with shrimp consumption. The findings can be used to monitor phthalate and insecticide residues in aquatic organisms and as a reference for food safety. Continual monitoring of the residues in shrimps is critical for further assessment of possible effects on human health.

Keyword: Phthalates, Organophosphorus pesticides, Residues, Shrimp, Mass spectrometry



Persistent Organochlorine Pesticides in Aquatic Environments and Fishes in Taiwan and Their Risk Assessment

Geng-Ruei Chang

Department of Veterinary Medicine, National Chiayi University

Organochlorine pesticides (OCPs) are ubiquitous contaminants with high bioaccumulation and persistence in the environment; they can have adverse effects in humans and animals. This study examined residual concentrations in water, sediments, and fishes as well as the association between the health risks of OCPs and fish consumption in the Taiwanese population. Various water and sediment samples from Taiwanese aquaculture and fish samples from different sources were collected and analyzed through gas chromatography tandem mass spectrometry to determine the concentrations of 20 OCPs, namely aldrin, cis-chlordane, trans-chlordane, dieldrin, endrin, alpha-endosulfan, beta-endosulfan, heptachlor, hexachlorobenzene, alpha-hexachlorocyclohexane, beta-hexachlorocyclohexane, lindane, mirex, pentachlorobenzene, o,p′-dichlorodiphenyltrichloroethane (DDT), p,p′-DDT, and DDT metabolites (o,p'-dichlorodiphenyldichloroethane [DDD], p,p'-DDD, o,p'-dichlorodiphenyldichloroethylene [DDE], and p,p'-DDE). None of the analyzed samples were positive for OCP contamination, suggesting no new input pollution from the land through washing into Taiwanese aquaculture environments. However, OCP residues were detected in fishes caught along the coast, namely skipjack tuna and bigeye barracuda, and in imported fishes, such as codfish and salmon. DDT was the predominant pesticide. The contamination pattern of persistent organic pollutants was as follows: dieldrin > cis-chlordane > hexachlorobenzene, with average concentrations ranging from 0.09 to 2.74 ng/g. The risk was assessed in terms of the estimated daily intake (EDI) for potential adverse indices; the EDI of OCP residues was lower than 1% of the acceptable daily intake established by the Food and Agriculture Organization of the United Nations and World Health Organization. The assessed risk was negligible and considered to be at a safe level, suggesting no association between fish consumption and risks to human health in Taiwan. However, a continuous monitoring program for OCP residues in fishes is necessary to further assess the possible effects on human health.

Keyword: Organochlorine pesticides, Fish, Estimated daily intake, Contamination, Residues