Differences in Pathogenicity and Vaccine Resistance Discovered between Two Epidemic Strains of Marek's Disease Virus in China

Abstract

Despite highly effective vaccines, Marek's disease (MD) causes great economic loss to the poultry industry annually, largely due to the continuous emergence of new MD virus (MDV) strains. To explore the pathogenic characteristics of newly emerged MDV strains, we selected two strains (AH/1807 and DH/18) with clinically different pathotypes. We studied each strain's infection process and pathogenicity and observed differences in immunosuppression and vaccine resistance. Specific pathogen-free chickens, unvaccinated or vaccinated with CVI988, were challenged with AH/1807 or DH/18. Both infections induced MD damage; however, differences were observed in terms of mortality (AH/1807: 77.8%, DH/18: 50%) and tumor rates (AH/1807: 50%, DH/18: 33.3%). The immune protection indices of the vaccine also differed (AH/1807: 94.1, DH/18: 61.1). Additionally, while both strains caused interferon-β and interferon-γ expression to decline, DH/18 infection caused stronger immunosuppression than AH/1807. This inhibition persisted even after vaccination, leading to increased replication of DH/18 that ultimately broke through vaccine immune protection. These results indicate that both strains have different characteristics, and that strains such as DH/18, which cause weaker pathogenic damage but can break through vaccine immune protection, require further attention. Our findings increase the understanding of the differences between epidemic strains and factors underlying MD vaccination failure in China.

Keywords: IFN-β; IFN-γ; Marek’s disease virus; immunosuppression; pathogenicity; vaccination.

Figure 1

Detection of Marek’s disease virus (MDV) by polymerase chain reaction (PCR). DNA of MDV-infected duck embryo fibroblasts (DEFs) was used as the template for PCR amplification. (a) PCR amplification of the Marek’s EcoRI-Q (meq) gene of MDV. The PCR products of AH/1807 and DH/18 were 1,403 base pairs (bp) long; (b) PCR amplification of the 132-bp repeat (bpr) of MDV. The PCR product of AH/1807 132-bpr was 448 bp long with a copy number of 3, while the PCR product length for DH/18 was 316 bp with a copy number of 2. (M) DL 2000 DNA Marker; CVI988: Culture of CVI988 vaccine stain infected duck cells; GA: Culture of MDV strain GA-infected duck cells; MOCK: Culture of uninfected duck cells.

Figure 2

Hematoxylin and eosin-stained histological lesions of collected tissues. Control, AH/1807-, and DH/18-challenged groups were compared for each tissue. (a) Liver, DH/18 invasive growth of tumor cells, pyknosis of liver nuclei, and excessive necrosis. AH/1807, a large number of tumor cells infiltrating and proliferating, with frequent pathological mitotic phases. Liver cells are necrotic in large numbers, and normal tissues are rarely visible. Tumor cells are mainly lymphoblastic. Scale bar: 50 µm. (b) Thymus, DH/18 atrophy, significant decrease in cortical lymphocytes, proliferation of macrophages, and proliferation of adipose tissue around the thymus. AH/1807 thymus atrophy, massive necrosis, and reduction in cortical lymphocytes, and proliferation of macrophages. Scale bar: 200 µm. (c) Spleen, DH/18 local necrosis of the parenchyma with a large number of tumor cells infiltrating, and the tumor cells are mainly lymphoblastic. AH/1807, local necrosis of the parenchyma with a large number of tumor cells infiltrating, most of which are mitotic, and the tumor cells are mainly lymphoblastic. Scale bar: 50 µm. (d) Bursa, DH/18 follicle atrophy, multiple necrosis, massive necrosis, reduction in lymphocytes, and mild interstitial hyperplasia. AH/1807 fold atrophy, significant necrosis of follicular lymphocytes, and interstitial hyperplasia. Scale bar: 200 µm.

Figure 3

Chicken survival curves for each treatment group. The survival patterns in the AH/1807-challenged and DH/18-challenged groups exhibited effects of infection with two strains. It exhibited significant differences (p < 0.05) by log-rank (Mantel–Cox) test.

Figure 4

Ratios of immune organ weight to body weight in chickens of each treatment group. The body weights and immune organ indices of MDV strains at 4, 7-, 14-, 21-, and 28-days post challenge were analyzed. (a) Body weight of chickens in each group. (b) Ratio of spleen weight to body weight in chickens of each group. (c) Ratio of thymus weight to body weight in chickens of each group. (d) Ratio of bursa weight to body weight in chickens of each group. Two-way ANOVA was performed for significance analysis. The data are shown as mean with standard deviations (SDs). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 5

Body weight and ratio of immune organ weight to body weight in control and challenged groups at 72-days post challenge. (a) Body weight. (b) Ratio of spleen weight to body weight. (c) Ratio of thymus weight to body weight. (d) Ratio of bursa weight to body weight. T tests were performed for significance analysis. The data are shown as means with standard deviations (SDs). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 6

Normalized viral loads in the spleens of chickens from various treatment groups. (a) The normalized viral loads from the spleens of three chickens randomly selected from each group at 4-, 7-, 14-, 21-, and 28-days post challenge (dpc). (b) The normalized viral loads in the spleens of all surviving chickens in each group at 72 dpc. Normalized viral loads were calculated as the logarithm of the MDV copy number per million cells. T tests were performed for significance analysis. The data are shown as means with standard deviations (SDs). * p < 0.05, **** p < 0.0001.

Figure 7

The mRNA expression of IFN-β and IFN-γ in the spleen. Three chickens were randomly selected at each time point (4-, 7-, 14-, 21-, and 28-days post challenge), and the relative expression of interferons (IFNs) was detected using quantitative real-time polymerase chain reaction. Bars above the horizontal line were upregulated and those below were downregulated. Comparing the expression levels of (a) IFN-β and (b) IFN-γ between AH/1807 and DH/18 groups. Comparing the expression levels of (c) IFN-β and (d) IFN-γ between CVI988-AH/1807 and CVI988-DH/18 groups. Two-way ANOVA was performed for significance analysis. The data are shown as means with standard deviations (SDs). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 8

Phylogenetic tree based on meq gene complete amino acid sequences of AH/1807 and DH/18 that marked with a colored dot. Other strains were retrieved from GenBank.