Rapid diagnostic methods for classifying avian leukosis subgroups in the field were needed for routine, large-scale screening.  As a first step in method development, we inserted the avian leukosis virus subgroup A (ALV-A) env gene into plasmid pcDNA3.1/Zeo (+) and used this construct to transfect DF-1 cells.  Zeocin-resistant cells were obtained after 2 weeks of zeocin selection.  Then, the cells were analyzed using PCR, immunofluorescence, and Western blot for expression of the envA-encoded envelope protein after 30 serial passages.  The DF-1/A cell line was completely resistant to 10⁴ TCID₅₀/0.1 mL (50% tissue culture infective dose) ALV-A and was partially resistant to 10⁵ TCID₅₀/0.1 mL ALV-A viral particles.  By comparing the DF-1/A and DF-1 cell lines, an ALV-A isolate was identified using a gag-specific ELISA for capsid protein p27.  Thus, we established a DF-1/A cell line that was resistant to ALV-A infection.  This cell line will be useful as a diagnostic tool.

Alternative splicing is a cellular mechanism in eukaryotes that results in considerable diversity of gene products. It plays an important role in several diseases and cellular signal regulation. Heat stress is a major factor that induces immunosuppression in pigs. Little is known about the correlation between alternative splicing and heat stress in pigs. Therefore, this study aimed to clone, sequence and quantify the alternative splicing variant of toll-like receptor 4 (TLR4) in Bama miniature pigs (Sus scrofa domestica) following exposure to heat stress. The results showed that the second exon of TLR4 was spliced and 167 bp shorter in the alternative splicing variant, and the protein was putatively identified as a type of truncated membrane protein consisting of extramembrane, transmembrane and intramembrane regions lacking a signal peptide. Further, it was not a nonclassical secretory protein. Five potential reference genes were screened for their potential as reliable standards to quantify the expression of TLR4 alternative spliced variants by real-time quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). The stability of these reference genes was ranked using the geNorm and NormFinder programs, and ribosomal protein L4 (RPL4) and TATA box-binding protein (TBP) were found to be the two genes showing the most stable expression in the in vitro cultured peripheral blood mononuclear cells (PBMCs) during heat shock. The mRNA level of the TLR4 gene (both classical and spliced) in stressed pigs increased significantly (P<0.05). Further, the expression levels of the alternative spliced variant of TLR4 (TLR4-ASV) showed a 2-3 folds increase in heat-stressed PBMCs as compared to control pigs. The results of the present study suggested that heat shock might modulate the host immune response by regulating the expressions of TLR4 and its alternative splicing variant.

To characterize the β-lactam resistance in veterinary clinical isolates of Haemophilus parasuis, 115 isolates were examined for the β-lactam resistance, the possession of β-lactamase, and the presence of β-lactamase genes. The genetic relationship among isolates was evaluated by pulsed-field gel electrophoresis (PFGE). Overall, the commonly detected resistance phenotypes were resistant to ampicillin (26.09%), penicillin (22.61%), amoxicillin (21.74%), cefazolin (14.78%), cefaclor (12.17%), and cefotaxime (6.96%). These strains showed high minimal inhibitory concentration (MICs) to oxacillin. 20.87% strains produced β-lactamase, and 4.35% strains showed extended-spectrum b-lactamase (ESBL) phenotype. Moreover, 19 strains harboured bla genes including TEM-1 (n=5), TEM-116 (n=10), and ROB-1 (n=5). Significantly, one strain possessed both TEM-1 and ROB-1, and displayed resistance to cefotaxime (MIC=8 mg L-1). The epidemiological analysis of PFGE revealed high genetic diversity among bla-positive isolates. This work shows that TEM- and ROB-type β-lactamases are prevalent in H. parasuis isolates in China.