Zhijun Xiao

Porcine reproductive and respiratory syndrome virus and bacterial endotoxin act in synergy to amplify the inflammatory response of infected macrophages

In 2006 China experienced outbreaks of a severe form of porcine reproductive and respiratory syndrome (PRRS) characterized by high fever, morbidity and mortality in swine irrespective of age. It is thought that secondary bacterial infections may contribute to the generation of this severe form of the disease. To determine the mechanisms by which a highly pathogenic PRRSV strain causes high fever we used an in vitro model to investigate the production of the pro-inflammatory cytokines IL-1β and TNF-α by macrophages in response to inoculation with PRRSV with or without LPS. Firstly we demonstrated, through an animal inoculation trial, that the isolate HN07-1 was a highly pathogenic strain and sequencing showed that the virus had the same genomic characteristics as previously described isolates. Porcine alveolar macrophage (PAM) cultures infected with PRRSV strains showed increased cytokine secretion and this was greater in the more virulent strain. Addition of LPS further increased cytokine secretion and again the effect was greater with the more virulent strain. Incubation of PAMs with PRRSV strain HN07-1 resulted in a significant increase in surface CD14 expression. This may explain the synergistic action between PRRSV and LPS in the induction of inflammatory cytokine secretion seen in the PAMs and so offer an explanation for the high fever that is characteristic of infections by the highly pathogenic PRRSV.

Development of a One-Step Strip Test for the Diagnosis of Chicken Infectious Bursal Disease

Abstract A rapid diagnostic strip for chicken infectious bursal disease (IBD) was developed based on membrane chromatography using high-affinity monoclonal antibodies directed to chicken infectious bursal disease virus (IBDV). The diagnostic strip has high specificity for detection of chicken IBDV antigen and recognizes a variety of the virus isolates, including virulent and attenuated strains, with no cross-reactivity to other viruses, such as Newcastle disease virus, Mareks disease virus, infectious bronchitis virus, infectious laryngotracheitis virus, and egg-drop-syndrome virus. The results showed that its specificity was highly consistent with the agar-gel precipitation test (AGP). The diagnostic strip detected as low as 800 median egg lethal dose (ELD50) viruses in the IBDV BC6/85-infected sample, which was comparable with AC-ELISA (400 ELD50) and 32 times more sensitive than the AGP test (2.56 × 104 ELD50). In experimental infection, IBDV was detected in the bursa as early as 36 hr postinfection with the diagnostic strip before the clinical signs and gross lesions appeared. It takes only 1–2 min to do a strip test to detect chicken IBDV antigen after the specimen is grounded in a whirl pack with finger massage.

Development of rapid immunoassays for the detection of ractopamine in swine urine

The monoclonal antibodies (mAbs) against ractopamine (Rac) were prepared and their properties identified by indirect competitive enzyme-linked immunoabsorbant assay (ELISA). The IC50 of mAbs was 2.7 ng ml−1 towards Rac or 9.3 ng ml−1 towards Rac-glucuronides and no cross-reactivity (CR) towards other competitors except dobutamine (CR: 3.76%). Based on the mAbs, the Rac-kit (kit) and Rac-strip (strip) were developed to detect Rac residues in swine urine. The strip and kit assay could be performed within 5–10 min and 2 h, respectively, allowing the analysis of urine samples without the need for sample clean-up. The detection limits were 1 ng ml−1 for kit and 3 ng ml−1 with the unaided eye, and 0.2 ng ml−1 with the Strip Reader for strip. The correlation coefficients (R 2) were 0.988 for kit in the range 0–128.0 ng ml−1, and 0.987 for strip in the range 0–10.8 ng ml−1. Comparing the gas chromatography-mass spectrometry (GC-MS) with the kit or strip in swine urine spiked with Rac standards, the differences ranged from 1.4% to 4.5% for kit and 1.0% to 4.7% for strip. However, the differences were greater than 54% for the kit and 55% for the strip test for the analysis of urine from swine treated with Rac. The results obtained from GC-MS using hydrolysed urine samples were generally in good agreement with those obtained from strip or kit using non-hydrolysed urine samples.

Surface IgM on DT40 cells may be a component of the putative receptor complex responsible for the binding of infectious bursal disease virus.

To investigate the host–pathogen interactions between infectious bursal disease virus (IBDV) and target B-lymphocytic cells, a cDNA T7 phage display library from the chicken bursa of Fabricius was constructed and screened for virus binding. Surface immunoglobulin M (sIgM) was isolated as a putative candidate binding site and its interactions with IBDV were further investigated using a chicken bursal lymphoma-derived cell line DT40. The results showed that the λ light chain of sIgM specifically interacted with IBDV in a virulence-independent manner in vitro, and most of the binding of IBDV to DT40 cells was inhibited by sIgM-specific monoclonal antibodies. Further, the infectivity of IBDV in vitro was reduced by sIgM-specific monoclonal antibodies. Our data provided evidence that sIgM may participate as one of the putative membrane binding sites responsible for IBDV infection.

Development of immunoassays for the detection of sulfamethazine in swine urine

The use of sulfonamides, such as sulfamethazine (SM2), in pig production is recognized as a public health risk as it inevitably results in sulfamethazine residues in pork. This study is aimed at establishing rapid, simple, reliable methods, with both sensitivity and specificity, for detecting sulfamethazine residues. For this purpose, monoclonal antibodies against sulfamethazine were prepared and characterized. No cross-reaction of the monoclonal antibodies was identified with other sulfonamides or analytes. Based on the competitive immunoassay principle, an indirect competitive ELISA kit (SM2 kit) and a rapid detection strip for detecting sulfamethazine residues were developed using monoclonal antibodies and the colloidal gold technique. The indirect competitive ELISA kit and the strip assay could be performed within 2 h and 5–10 min, respectively. The results showed that the detection limits were 1 ng ml−1 for the indirect competitive ELISA kit and 8 ng ml−1 with the unaided eye and 1 ng ml−1 with the strip reader for the rapid strip assay. Comparing the HPLC method with the SM2-kit or the strip in pig urine spiked with standards of SM2, the difference was <4.6% for SM2-kit and 4.3% for the strip. The two methods are suitable for the rapid screening of sulfamethazine residues in swine urine. Experimental data revealed that the two methods, especially the strip, proved to be sensitive, specific, rapid and easy to use for the quantitative, semi-quantitative or qualitative detection of SM2 residues in swine urine.