Development and preliminary testing of a probe-based duplex real-time PCR assay for the detection of African swine fever virus.

An outbreak of African swine fever (ASF) in China in 2018 caused substantial economic losses to the swine industry. To accurately diagnose clinical infection with ASF virus (ASFV), we developed a TaqMan probe-based duplex real-time PCR that simultaneously detected two discontinuous genes in the virus genome, thereby preventing the inaccurate results obtained with only one reaction. Two sets of ASFV gene-specific primers, along with two fluorescent TaqMan probes were designed to target conserved regions of the B646L and B438L genes. This method had high sensitivity and specificity, with a limit of detection of 10 copies/µL, and it did not cross-react with the genomes of other viral pathogens that affect pigs (i.e., CSFV, PRRSV, PEDV, PRV, PPV and PCV2). Overall, 180 clinical samples from ASFV-infected pig farms were used to compare this method with a commercial kit, which yielded excellent consistency (98.3%). This new diagnostic method should greatly improve the efficiency of ASFV surveillance and reduce economic losses, providing benefits for both animal and public health.

Imbalance of intestinal immune function in piglets infected by porcine circovirus type 2 during the fetal period.

Porcine circovirus type 2- (PCV2-) associated reproductive disorders and enteritis have commonly been observed on PCV2-contaminated pig farms in recent years. In order to investigate disorders of intestinal immunity in piglets infected by PCV2 during the fetal period, 9 PCV2b-infected piglets and 6 non-infected piglets at one day of age were selected and euthanised prior to suckling. Samples of mesenteric lymph nodes (MLNs) and duodena were collected to investigate factors related to intestinal immunity and to detect lymphocytic apoptosis. The results indicated that there were no significant changes in the levels of IL-2, IL-10 and transforming growth factor-ß (TGF-ß) in the PCV2b-infected piglets but IFN-γ levels were significantly lower (P < 0.01) and IL-4 levels were significantly higher (P < 0.05) in infected piglets than in the controls. Furthermore, lymphocytic apoptosis increased in PCV2b-infected piglets and CD4+ to CD8+ ratios were lower in these piglets than in the controls. These findings suggest vertical transmission of PCV2b to fetuses, leading to an imbalance of intestinal immune function in piglets.

Transmission of porcine circovirus type 2b (PCV2b) in Kunming mice.

To investigate porcine circovirus type 2b (PCV2b) transmission by contact and vertical infection in Kunming mice (an outbred mouse stock deriving from Swiss albino mice with a high ratio of gene heterozygosis), four mice in cage 6 were inoculated with PCV2b and 25 mice without any treatment were placed into cages 1 to 5 (five mice in each cage). Seven days after being infected, the PCV2-binoculated mice were co-mingled with non-inoculated mice from cages 1 to 5 successively at 7, 14, 21, 28 and 35 days post infection (dpi), respectively, for 3 days. In addition, eleven pregnant mice were injected with PCV2b. Samples were collected from non-inoculated mice and three newborn mice from each litter for PCV2b detection by polymerase chain reaction (PCR) and immunohistochemistry (IHC). The PCR results showed that PCV2b transmission rate among mice in cages 1, 2, 3, 4 and 5 was 0/5, 2/5, 5/5, 5/5 and 1/5, respectively. PCV2b antigen signals generally appeared in most organs of the non-inoculated mice in which viruses were detected by PCR. PCV2b DNA was also detected in newborn mice of PCV2b-infected litters, and viral antigen signals were observed in their organs as well. PCV2b was transmitted in Kunming mice by contact, and it also caused vertical infection through the placenta.

Viral distribution and lesions in Kunming mice experimentally infected with porcine circovirus type 2b.

The viral distribution and lesions in Kunming mice experimentally infected with porcine circovirus type 2b (PCV-2b) were investigated. Seventy special pathogen free mice were divided into 2 groups with 35 mice in each group. The test group (TG) was infected with PCV-2b, the control group (CG) was inoculated with sterile cell cultures. Five mice in each group were sacrificed at 3, 7, 14, 21, 28, 35 and 42 dpi (day post infection), respectively. Necropsies were performed on all mice and tissues were collected for testing by histopathology, immunohistochemistry, transmission electron microscope (TEM) and polymerase chain reaction (PCR). Apoptosis and necrosis in lymphoid organs were observed in virus-infected mice, and became severe from 14 to 28 dpi. The proportion of PCV-2b antigen-positive cells was moderate in lung, heart, thymus, liver or kidney, and low in brain from TG. In spleen and cervical lymph node, the proportions of PCV-2b antigen-positive cells were low to high from 7 to 28 dpi, and moderate from 35 to 42 dpi. PCV-2b DNA was detected in all tissues examined in TG from 7 to 42 dpi. Viral inclusion bodies presented in the cytoplasm of lymphocytes, macrophages, hepatocytes, podocytes, neurocytes, spermatids and uterine epithelial cells in TG. In CG, no viruses and viral lesions were detected. PCV-2b could replicate in mice, and PCV-2b associated lesions in mice were similar to those observed in pigs. The present results indicate that it is possible to use Kunming mouse as an animal model for PMWS research.

[Cloning and analysis of phage Fab antibodies of mouse male specific antigen].

To clone mouse phage antibodies against H-Y antigen from a phage antibody library, three cycles of affinity enrichment of the mouse phage antibody library with male spleen cells and two cycles of nonspecific absorption with female spleen cells were performed. The presence of mouse Fab on the phage surface was determined by ELISA and sequence analysis. 9 of 15 strains can bind to male spleen cells with the specific activity. Recombination rate of the phage antibody library clones is 60%. Sequence analysis of the PCR products of plasmid DNA of E5 clones show VH and Vkappa had common characteristics shared by other known variable region of antibodies. The mouse phage Fab antibody could be used for identifying H-Y antigen, and for the development of sex determination of early embryos in mammals.