Veterinary Immunology and Immunopathology 160 (2014) 288–292

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Short communication

Immunogenicity of an inactivated Chinese bovine viral diarrhea virus 1a (BVDV 1a) vaccine cross protects from BVDV 1b infection in young calves Wei Wang a,b , Xinchuan Shi b , Yongwang Wu b , Xiaoxin Li c , Ye Ji b , Qingsen Meng b , Shucheng Zhang c , Hua Wu b,∗ a b c

Institute of Special Economic Animal and Plant Science, CAAS, No. 4899, Juye Street, Changchun 130122, China Sinovet (Beijing) Biotechnology Co., Ltd., No. 5 Kaituo Street, Haidian District, Beijing 100085, China VMRD, APAC, Zoetis, Unit 1400, 14th Floor, Sunflower Tower, No. 37 Maizidian Street, Chaoyang District, Beijing 100125, China

a r t i c l e

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Article history: Received 7 January 2014 Received in revised form 1 April 2014 Accepted 22 April 2014 Keywords: Bovine viral diarrhea virus type 1a Inactivated vaccine Bovine viral diarrhea virus type 1b Cross protection Challenge study China

a b s t r a c t Bovine viral diarrhea virus (BVDV) 1a and 1b strains are the predominant subgenotypes in China. Because of the genetic and antigenic variability among different BVDV strains, a vaccine effective in one region may fail to protect against infections caused by different virus strains in another region. No BVDV vaccine developed with the predominant strains in China are available. In this study, the immunogenicity of an inactivated Chinese BVDV 1a NM01 vaccine strain was evaluated by challenging with a Chinese BVDV 1b JL strain. Ten 2–4-month-old calves were intramuscularly vaccinated with a single dose of the vaccine strain and boosted with same dose three weeks after the first vaccination, with five mock immunized calves serving as a control group. The average titer of neutralization antibody to BVDV 1a and BVDV 1b of immunized calves reached 1:410 and 1:96, respectively, at 21 days post the second vaccination. Twenty-one days post the second vaccination, all calves were challenged with strain JL. The clinical signs, such as the temperature and leukopenia of the immunized calves and viral shedding, were significantly less than the mock immunized calves after challenging with the virulent BVDV 1b strain, indicating that the BVDV 1a vaccine strain elicited efficacious protection against the endemic BVDV 1b strain in China. To the best of our knowledge, this is the first report of an inactivated BVDV vaccine which demonstrated effective cross-protection against BVDV type 1b infection in China. © 2014 Published by Elsevier B.V.

1. Introduction Bovine viral diarrhea virus (BVDV), a member of the pestivirus genus of the flaviviridae, is a major pathogen causing bovine respiratory disease complex (BRDC) of ruminants worldwide, resulting in severe economic losses (Alkan

∗ Corresponding author. Tel.: +86 1062980176. E-mail address: [email protected] (H. Wu). http://dx.doi.org/10.1016/j.vetimm.2014.04.007 0165-2427/© 2014 Published by Elsevier B.V.

et al., 2000). Two genotypes, BVDV1 and BVDV2, are identified based on comparison of the conserved genes of 5 UTR, Npro or E2. Each genotype is further divided into different genetic subgroups, and at least 15 genetic subgroups of BVDV1 and four genetic subgroups of BVDV2 are identified (Xue et al., 2010a), as well as two biotypes cytopathic (cp) and noncytopathic (ncp) according to their cytopathic effects. BVDV related-diseases range from subclinical to clinical diseases, depending on the virulence of different virus strains, and are complicated with fever, leukopenia,

W. Wang et al. / Veterinary Immunology and Immunopathology 160 (2014) 288–292

diarrhea, lacrimation, nasal discharge, persistent infections (PI), abortions, hemorrhagic and systemic diseases such as mucosal disease (Laureyns et al., 2011). Although measures, including identification and removal of PI cattle and biosecurity procedures, are taken to control and prevent BVDV exposure to a herd (Borsberry, 2012), vaccination is still a basic and important strategy to control and prevent BVDV exposure (Xue et al., 2011). Inactivated BVDV vaccines used worldwide to protect against BVDV are generally safer in pregnant cattle; thus, it was advocated that inactivated instead of live BVDV vaccines be used during cattle pregnancy in some vaccination programs (Beer et al., 2000). It is very important to consider that the presence of multiple genotypes and antigenic diversity of BVDV isolates may affect protective efficacy of BVDV vaccines (Xue et al., 2010b). A vaccination may fail to protect against virus infection due to the genetic and antigenic variability among different strains (Ridpath et al., 2010). Previous studies revealed that the cross protection of a BVDV vaccine from different strains with different genotypes or subgroups is significantly different (Fairbanks et al., 2003; Makoschey et al., 2001; Xue et al., 2009, 2010b). Although the crossprotection between the BVDV 1a Singer strain and the BVDV 1b NY1 and T1186a strains in modified live virus (MLV) vaccines has been confirmed (Ho et al., 2013), the cross-protection of the inactivated BVDV 1a vaccine against BVDV 1b infection, especially among the different regional strains, still remains unknown. BVDV 1a and BVDV 1b are the predominant subgenotypes in China (Wang et al., 2014; Xue et al., 2010a). Unfortunately, no commercial vaccine or immunogenicity data of the BVDV isolates has been available in China. In this study, we developed a new inactivated vaccine candidate containing Chinese strain NM01 of cpBVDV 1a. Young calves were immunized with the vaccine strain through intramuscular injection, and the efficacy of the vaccine was evaluated by challenging with the predominant ncpBVDV 1b JL strain in China.

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then emulsified. Antigen in the vaccine was batched at a minimum immunization level of 107.0 TCID50 /dose. 2.3. Animal immunizations and challenging Ten 2–4-month-old calves were intramuscularly immunized with a single 2 ml dose of the inactivated vaccine and calves in the control group (n = 5) were mock immunized with 2 ml of PBS diluent. The immunization, including the mock-immunization, was repeated once three weeks later. Blood samples were collected on day 0 of vaccination and 7, 14, 21, 28, 37 and 42 days post-immunization. The sera were used to test neutralization antibody against BVDV by the virus neutralization (VN) method. Twenty-one days post the second vaccination, all calves were intranasally challenged with 3 ml of aerosolized BVDV 1b JL strain virus, a non-cytopathic (ncp) strain isolated from Jilin province of China, into each nostril using a Devilbiss Atomizer (Devilbiss, Somerset, PA, USA). Each animal received approximately 107.5 TCID50 (50% tissue culture infectious dose) of the challenge virus. 2.4. Clinical assessment The calves were observed for clinical signs, including depression, cough, asthma, excessive lacrimation, and rectal temperature. Rectal temperature was taken at the same time each morning at days −2, −1 and 0 prior to the challenging and at days 1 through 14 after the challenging. 2.5. Sample collection Blood samples were collected from each calf using EDTA-coated tubes from day 2 pre-challenge through day 8 post-challenge and white blood cell (WBC) counts were conducted by a Vetscan HM5 veterinary hematology system (Abaxis, USA). Deep nasal swab specimens were collected at 1 day prior to challenge through 8 days postchallenge and used for virus isolation.

2. Materials and methods

2.6. Virus neutralizing antibody analysis

2.1. Experimental animals

The neutralization antibody titers to BVDV were measured using a standard microplate VN procedure with BVDV 1a strain NM01 or BVDV 1b strain JL as the neutralizing viruses in the assay. Briefly, 56 ◦ C-heat inactivated serum samples were two-fold serially diluted in 96-well plates. Approximately 200 TCID50 of the VN challenge virus was added to each diluted serum sample and incubated at 37 ◦ C for 1 h. The serum-virus mixture was then inoculated onto MDBK cells. At 4–5 days post the incubation, the plates were evaluated for CPE or immunofluorescence assay. The neutralizing antibody titer each sample was calculated using the Spearman-Kärber method.

Fifteen 2–4-month-old healthy calves free of persistent infection of non-cytopathic BVDV were purchased from a calf farm in Inner Mongolia Autonomous Region, China. All calves were seronegative to BVDV antibody. All animal experiments were approved by the ethics committee of the Chinese Academy of Agricultural Science.

2.2. Vaccine and administration BVDV1 NM01 strain, used as the vaccine strain, was isolated from Inner Mongolia Autonomous Region in China. The virus was infected into MDBK cells and harvested, frozen and thawed three times. Viruses were inactivated with binary ethyleneimine (BEI; Sigma, USA), mixed with MontanideTM ISA 206 VG (Seppic, France) adjuvant and

2.7. Virus isolation Viruses were isolated from nasal swabs and blood leukocytes on MDBK cell monolayers in 24-well tissue culture plates for two passages and finally were placed in

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Table 1 Leukopenia viremia and temperature of vaccinated and control animal groups on day of challenge. Group

Animal #

Leukopenia (%)a

Viraemiab

Highest temperaturec

Total days with temperature >39.7 ◦ Cd

Vaccinated

W72 W78 W79 W77 W68 W61 W63 W75 W74 W66

19.9 6.8 8.5 10 14 23.5 24.7 34.8 13.9 7.8

0/0 0/0 0/0 2/0 0/0 0/0 0/0 2/2 0/0 0/0

39.5 39.5 39.2 39.5 39.6 39.2 39.5 40.2 39.6 39.3

0 0 0 0 0 0 0 1 0 0

Control

821 833 798 135 783

51.2 44.0 44.0 54.2 33.3

7/3 6/2 5/3 6/3 5/3

41.0 40.7 41.3 41.7 41.3

2 3 2 6 3

a b c d

Maximum percent reduction in cell count (3–8 dpc), compared to an average of two pre-inoculation values. Number of days with positive virus isolation from blood/nasal swab 1–8 dpc. The highest rectal temperature during 1–14 dpc. Total days of >39.7 ◦ C during 1–14 dpc.

96-well tissue culture plates for an indirect immunofluorescence assay (Corapi et al., 1990). 2.8. Gross pathology and histopathology Three control calves and two vaccinated calves were randomly euthanized at day 14 post-challenge. Tissues, including tonsil, mandibular lymph node, mesenteric lymph node, spleen, thymus, trachea, lung, liver, kidney, and heart, were observed and collected. All tissue samples were fixed in 10% neutral buffered formalin for histology analysis. 2.9. Data analysis The efficacy of the inactivated vaccine candidate was evaluated based on primary clinical signs, including rectal temperature, WBC counts and virus shedding using Fisher Exact test of SPSS Version 20 (IBM, China). The significance of the variability among the groups was determined by one-way analysis of variance using GraphPad Prism (version 4.0, GraphPad Software, Inc. USA) software. Statistical significance was set at p < 0.05. 3. Results and discussion

antibody (Ridpath et al., 2010). The vaccinated calves were completely protected from the challenge at BVDV 1a antibody titer of 1:256 or greater, but were partially (1/2) protected at a titer of 1:128. These results are in accordance with previous studies in which the level of neutralizing antibodies is suggested to be critical for protection and a neutralization titer at 1:256 is sufficient for preventing clinical signs and viremia virus shedding (Beer et al., 2000; Bolin and Ridpath, 1995; Fulton and Burge, 2000). 3.2. Clinical signs after inoculation of challenge virus After challenged with BVDV 1b JL, all control calves showed disease symptoms associated with BVDV infection, including depression, nasal discharge, excessive lacrimation, and coughing. All control calves showed high rectal temperatures, reaching peak rectal temperatures at days 7–8 post-challenge. In contrast, none of the vaccinated calves (0/10) developed clinical signs after the challenge (Table 1). The vaccinated group had significantly (p < 0.05) lower temperatures at 6–8 days post-challenge than the control group (Fig. 1A). These results were similar to a previous study in which all control calves had elevated rectal temperatures, with peak temperatures at days 7–8 postchallenge (Xue et al., 2010b).

3.1. Neutralization antibody 3.3. Leukopenia All calves were free of VN antibodies to both BVDV 1a and BVDV 1b at the time of vaccination. Following vaccination with the inactivated vaccine, the elicited average titers of VN antibodies reached 1:410 against BVDV 1a and 1:96 against BVDV 1b. These titers were significantly (p < 0.001) higher in the vaccinated calves than in the control calves. The BVDV 1a vaccine induced VN antibody which recognized BVDV 1b. The discrepancy between the titers induced by BVDV 1a and BVDV 1b is similar to that of a previous study, in which BVDV 1a induced a significantly higher titer of anti-BVDV 1a antibody than that of anti-BVDV 1b

Results of WBC counts demonstrated a decrease of WBCs beginning at 2 days post-challenge in all control calves, and the lowest number of WBCs occurred at 5–6 days post-challenge. The extents of WBC count decrease among the control calves are shown in Table 1. The average WBC counts decreased by more than 40% in the control group over two days compared with the vaccinated group, and WBC counts in the vaccinate group were significantly higher (p < 0.05) than in the control group at 5 to 7 days post-challenge (Fig. 1B). The WBC reduction in the control

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group is similar to that caused by challenge with the BVDV NY-1 strain (Ridpath et al., 2007). 3.4. Virus shedding

Fig. 1. Vaccination with BVDV 1a and challenge with BVDV 1b. (A) Elevated rectal temperatures. (B) Decrease in white blood cell counts. Statistical values are *p < 0.05 and ***p < 0.001.

All calves were negative for virus isolation prior to challenge. In the vaccinated group, one of ten and two of ten calves had virus isolations from nasal swabs and the blood leukocytes, respectively. The control group was all positive from nasal swabs or blood leukocytes. However, the frequency of virus isolation from blood leukocytes was higher than that from nasal swabs in both the vaccinated and control groups (Table 1). Statistical analysis of virus isolation, in terms of positive or negative and the total number of days positive indicates that the shed viruses from the vaccinated group were significantly (p < 0.01) less than those from the control group. Additionally, the vaccinated calves were virus positive for significantly (p < 0.01) fewer days than the control calves. We found that virus was more frequently isolated from WBCs than from the nasal swab in the mock immunized

Fig. 2. Histopathologic changes of intestine, spleen and mesenteric lymph nodes. (A) The infected calves in the control group developed significant inflammatory bowel disease; inflammation occurred in the mucosa, submucosa, muscularis and the serosa. (B) No obvious lesions were observed in the intestinal lamina or the other intestinal layers. (C) Lymphocytosis was observed in the mesenteric lymph nodes. (D) Lymphoid nodules increased lymphoproliferation (E) Varying degrees of hyperplasia were seen in spleen lymphocytes. (F) No significant damage was observed in vaccinated cattle.

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calves. This phenomenon is similar to a previous study where BVDV was isolated from WBC and nasals, but the frequency of virus isolation in WBCs was greater than in the nasal swab (Beer et al., 2000). 3.5. Gross pathology and histopathology analysis The control calves inoculated with ncp BVDV 1b JL had gross and histologic lesions, while the vaccinated calves had no visible lesions. Gross pathological findings of the control group included hemorrhages in the spleen and mesenteric lymph nodes. Histopathologic changes included thymic and spleen atrophy, enteritis, and the lymph node was lymphadenopathy and hemorrhagic with lymphoid depletion of the Peyer’s patches in all the infected calves. The infected calves in the control group developed significant inflammatory bowel disease, inflammation in the mucosa, submucosa, muscularis and the serosa, a small amount of necrosis in mesenteric lymph nodes, spleen lymphocyte, and mild lung necrosis in the trachea and bronchial mucosa, but no obvious lesions in the liver, kidney or submandibular gland (Fig. 2). These results are similar to those of previous studies in which the control calves manifest typical histopathology and symptoms of respiratory disease, including the depletion of lymphocytes in lymphatic organs (Makoschey et al., 2001). In conclusion, we demonstrated, for the first time, that immunization of calves with inactivated BVDV 1a strain effectively cross-protects against endemic BVDV type 1b strain infection, suggesting that the inactivated BVDV 1a strain may be an effective vaccine candidate to protect against both BVDV 1a and BVDV 1b infections in China. Conflict of interest statement The authors have agreed that there is no conflict of interest between them. Acknowledgements The authors thank all of the technicians who were involved in the animal care and sample collection. References Alkan, F., Ozkul, A., Bilge-Dagalp, S., Yesilbag, K., Oguzoglu, T.C., Akca, Y., Burgu, I., 2000. Virological and serological studies on the role of

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Immunogenicity of an inactivated Chinese bovine viral diarrhea virus 1a (BVDV 1a) vaccine cross protects from BVDV 1b infection in young calves.

Bovine viral diarrhea virus (BVDV) 1a and 1b strains are the predominant subgenotypes in China. Because of the genetic and antigenic variability among...
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