Safety and Efficacy Evaluation of Recombinant Marek’s Disease Virus with REV-LTR

Figures

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  Figure 1. In vitro growth rate of recombinant Marek’s disease virus (rMDV) and Rispens. Rispens and rMDV were separately inoculated onto chicken embryo fibroblast cultures and harvested at each time point. The virus titer from each culture was determined by a plaque assay.

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  Figure 2. Gross lesions, histopathological analysis and weight gains, and tissue weight in the safety test. (A) At day 60 after immunization, the tissues of all chickens were collected to observe their gross lesions. (B) At day 60 after immunization, the samples of tissues were fixed in 4% formalin for 24 h and dehydrated, embedded, sliced, and stained with hematoxylin-eosin (HE) for histopathological analysis. (C) At each time point, all chickens were weighed to analyze their weight gains, and at day 60 the tissues of each group were collected to analyze the organ weights (a: body weight; b: tissue weight; NS: no significant difference).

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  Figure 3. rMDV detection by PCR in the persistence and detoxification test. At each time point, DNA was extracted from the blood, feathers, swabs, feed, feces, litter, water, and tissues of the immunized chickens, and PCR was carried out to detect rMDV (a): target sequence 1730bp could be detected in blood and tissues at 3 days after immunization; (b): target sequence 1730bp could be detected in blood and tissues at 7 days after immunization; (c): 21 days; (d): 42 days; (e): 60 days; M: Marker 1: blood; 2: feather; 3: liver; 4: spleen; 5: kidney; 6: lung; 7: tracheal swab; 8: rectal swab; 9: feed; 10: feces; 11: litter feces; 12: water; 13: positive; 14: negative).

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  Figure 4. PCR detection in horizontal test. DNA was extracted from the blood, feathers, swabs, and tissues of immunized and cohabiting chickens, and PCR was carried out to determine the horizontal transmission capacity of rMDV (a: blood; b: feather; c: tracheal swab; d: liver; e: spleen; f: kidney; g: lung; h: rectal swab; M: marker; 1: rMDV-immunized chicken; 2: rMDV cohabiting chicken; 3: rMDV-positive; 4: Rispens-immunized chicken; 5: Rispens cohabiting chicken; 6: Rispens-positive).

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  Figure 5. PCR detection and histopathological analysis in the passage test. (A) DNA was extracted from the blood of the first four generations, and PCR was carried out using primers 1 + 4 to detect rMDV; the target gene was 1730 bp (a: first generation; b: second generation; c: third generation; d: fourth generation; 1, 2, 3, 4, 5, 6: chickens of each generation). (B) DNA was extracted from the feathers of the fifth generation, and PCR was carried out using primers 1 + 2, 1 + 3, 1 + 4 to detect rMDV (a: rMDV detection by primers 1 + 2, and the target gene was 827 bp; b: rMDV detection by primers 1 + 3, and the target gene was 1448 bp; c: rMDV detection by primers 1 + 4, and the target gene was 1730 bp). (C) On the 60th day, visceral tissues of the fifth generation were collected for histological examination (a: heart; b: liver; c: spleen; d: lung; e: kidney; f: bursa).

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  Figure 6. Clinical signs, survival curves, and gross lesions in the comparative experiment. (A) Clinical signs were observed after the Md5 challenge in each group ((a), (b): GC group; (c): G3 group; (d): G6 group). (B) The survival curves of each group were generated after the Md5 challenge. (C) Gross lesions of the survived chickens of the control ((a): enlarged kidneys; (b): testicular swelling; (c): splenomegaly) and G4 groups ((d): a diffused tumor in the liver).