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An inactivated bovine virus diarrhoea virus (BVDV) type 1 vaccine affords clinical protection against BVDV type 2
Vaccine 19 (2001) 3261 – 3268 www.elsevier.com/locate/vaccine
An inactivated bovine virus diarrhoea virus (BVDV) type 1 vaccine affords clinical protection against BVDV type 2 B. Makoschey 1,*, M.G.J. Janssen 1, M.P. Vrijenhoek 2,1, J.H.M. Korsten 3,2, P.v.d. Marel 1 1
Department of Virological R&D, Inter6et International B.V., P.O. Box 31, 5830 AA Boxmeer, The Netherlands 2 Department of Pathology, Inter6et International B.V., P.O. Box 31, 5830 AA Boxmeer, The Netherlands 3 Department of Toxicology and Drug Disposition, N.V. Organon, P.O. Box 20, 5340 BH Oss, The Netherlands Received 18 July 2000; received in revised form 1 November 2000; accepted 12 December 2000
Abstract This study was designed to answer to two distinct questions. Firstly, is it possible to reproduce clinical signs of acute bovine virus diarrhoea virus (BVDV) type 2 infection including signs of haemorrhagic disease under experimental conditions in cattle at 20 weeks of age? Secondly, what is the extent of the protection afforded by vaccination with an inactivated BVDV type 1 vaccine against BVDV type 2 infection? Calves were vaccinated at 12 and 16 weeks of age with a commercially available inactivated BVDV type 1 vaccine (Bovilis® BVD). At 20 weeks they were challenge infected with BVDV type 2 virus together with unvaccinated control calves. The unvaccinated animals developed typical signs of respiratory disease, diarrhoea with erosions and haemorrhages along the whole length gastro-intestinal tract, and depletion of lymphocytes in lymphatic organs. These signs were either absent or markedly less severe in the vaccinated animals. The beneficial effects of vaccination were most striking in the haematological parameters thrombocytopenia and leukopenia. It can be concluded that vaccination with Bovilis® BVD affords cross-protection against clinical effects of a challenge-infection with heterologous type 2 BVDV. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Vaccine; Bovine; Diarrhoea
1. Introduction Bovine virus diarrhoea virus (BVDV) is a major viral pathogen in cattle and causes considerable economic losses throughout the world [1]. The clinical manifestations vary in both type and degree. Traditionally, these are classified into three categories, (1) acute virus infection of an immunocompetent animal, (2) foetal infection and (3) mucosal disease. Categories 2 and 3 refer to the potential transplacental spread of the virus during viraemia. Depending on the stage of gestation at the time of transplacental infection, the outcome may be embryonic death, abortion, stillbirth or the birth of immunotolerant calves persistently infected with BVDV
* Corresponding author. Tel.: +31-485-587600; fax: + 31-485587339. 1 Tel. : + 31-485-587600, fax: + 31-485-587339. 2 Tel.:+ 31-412-666112, fax: + 31-486-469131.
[2,3]. These calves may appear healthy, but are a permanent source of BVDV in the herd. They are also at risk of developing fatal mucosal disease if superinfected with a cytopathic biotype of the virus [4]. The classical bovine virus diarrhoea, which gave the virus its name is only one of the possible manifestations of acute BVDV infections of immunocompetent animals. BVDV also infects white blood cells [5] and causes depletion of circulating B- and T-lymphocytes [6]. This results in an unspecific immunosuppression that favours infections with other viral or bacterial pathogens. Furthermore, there is epidemiological [7] and experimental [8] evidence that BVDV is directly associated with respiratory disease. New forms of acute BVDV infection have been reported in North America and Canada [9–12]: thrombocytopenia resulting in the so-called haemorrhagic syndrome. The different BVDV strains vary considerably in their virulence [13]. There is also considerable
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antigenic and genetic variation in the BVDV strains that have been characterised so far. This lead to the classification of BVDV into two types (1 and 2) [14]. Most cases of the haemorragic form of BVDV infection have been attributed to BVDV type 2 viruses. The attenuated and inactivated BVDV vaccines that have been developed to date have been reviewed by van Oirschot and colleagues [15]. The broad antigenic diversity of BVDV strains places high demands on these vaccines. The extent of cross-protection afforded by a vaccine has to be determined experimentally and is difficult to predict. The aim of the present study was to investigate the protection provided by a new inactivated BVDV type 1 vaccine against severe challenge with a BVDV type 2 strain that induces thrombocytopenia.
2.3. Clinical signs After challenge, clinical observations were performed daily for 2 weeks. Special attention was paid to behaviour, faecal consistency, sneezing, coughing, ocular and nasal discharge, hyperaemia or lesions of the nasal and oral mucosae, and abnormal breathing. The clinical parameters were recorded daily for each calf and scored 0 when normal, 1 when mild, 2 when moderately and 3 when markedly abnormal. The mean daily clinical scores per group were calculated. Rectal temperature was measured as a separate parameter. This was done daily for 2 weeks and the mean temperature per group and per day was calculated.
2.4. Haematology 2. Materials and Methods
2.1. Viruses and cells Virus titration, virus isolation and serum neutralisation tests were performed on Bovine Embryo Lung (BEL) cells. The cells were cultured in a combination of Glasgow’s and Eagles modified minimal essential medium (MEM) supplemented with 5% foetal calf serum and a cocktail of antibiotics neomycin (50 mg/ ml), polymyxin B (50 mg/ml), pimafucin (2.5 mg/ml) and tylosin (10 mg/ml). The cells were incubated at 37°C in humidified atmosphere with 5% CO2. The challenge virus BVDV-2 Giessen-1 was isolated originally from the blood of a naturally infected calf [16] (kindly provided by H.-J. Thiel, Gießen, Germany). The inoculum consisted of a pair of non-cytopathic and cytopathic viruses. This virus mixture was passaged five times in cell culture and finally amplified on calf kidney cells. The inoculum titre was 7.7 log10TCID50/ml non-cytopathic BVDV and 5.4 log10TCID50/ml cytopathic BVDV. The cell and virus stocks were sterile.
2.2. Experimental design Twelve Holstein calves obtained from dams seronegative for BVDV were used for this experiment. One group of four calves was vaccinated with a commercial batch of Bovilis®BVD (Intervet) administered by intramuscular injection at 12 and 16 weeks of age. Four weeks after the second vaccination, the vaccinates were challenged along with five age matched unvaccinated controls. Challenge virus was administered intravenously into the jugular vein (2 ml) and intranasally (2 ml, 1 ml per nostril). A group of three untreated calves of the same age were kept as a negative control.
The haematological examinations focussed on the changes in thrombocyte and leukocyte counts. The former due to the numerous reports of BVDV induced thrombocytopenia, and the latter as an indicator for the immunosuppressive effects of BVDV. Blood samples were taken at 2–3 days interval beginning 5 days before challenge until 3 weeks after challenge. Thrombocyte and leukocyte counts were determined using an automated cell counter (Cell-Dyn 3500 haematology analyser with veterinary software, Abbott) (according to their size, complexity and depolarisation capacity).
2.5. Determination of 6irus excretion with nasal discharge Nasal swab samples were taken using swabsticks that contain a sterile cotton plug starting at the day of challenge. The swabs were submerged in sterile phosphate buffered saline medium for approximately 1 h. The medium was transferred into sterile tubes and stored frozen at − 70°C until tested. Virus titrations were performed using the end point dilution method according to standard procedures. Four replicates of a series of 10-fold dilutions were made in microtitre plates on monolayers of BEL cells. Titres were determined using immunofluorescence testing and calculated according to the formula of Ka¨rber [17].
2.6. Virus isolation from peripheral blood cells and plasma samples Whole blood samples were taken three times a week, for the determination of cell-bound viraemia. The buffy coat was recovered after centrifugation of the sample, and treated with erythrocyte lysis buffer. Then centrifugation was repeated. The cell pellet was re-washed in the buffer and then stored frozen until tested. For virus isolation, aliquots of 107 cells were co-cultured in 4-fold
B. Makoschey et al. / Vaccine 19 (2001) 3261–3268
on monolayers of BEL cells and virus infection was judged by immunofluorescent staining. The cell free virus titre in the plasma samples was determined by titration using the end point dilution method, in 4-fold, on monolayers of BEL cells. BVDV infection was judged by immunofluorescence testing and calculated according to the formula of Ka¨rber [17].
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3. Results
3.1. Virus neutralisation
Serum samples were collected from the calves immediately before each vaccination and at the time of autopsy. They were stored frozen at − 20°C until tested. A standard virus neutralisation assay was performed against the homologous vaccine virus strain and the heterologous challenge virus strain. The test was performed on monolayers of BEL cells and neutralisation titres were recorded as the highest dilution of serum that inhibited BVDV virus growth, judged by immunofluorescent staining.
At 4 weeks after the first vaccination, all four vaccinates had detectable levels of neutralising antibody versus the vaccine virus (Table 1). By the time of challenge, i.e. at 4 weeks after the second vaccination, the homologous titres had increased to 13 log2. The heterologous titres against the BVDV type 2 challenge virus ranged from 5–10 log2. The animals of both unvaccinated groups were free from detectable BVDV neutralising antibodies at the time of challenge. At the time of autopsy (post challenge-infection days 9, 10, 13 or 22) all five unvaccinated animals had seroconverted for BVDV, and the homologous and heterologous titres of the vaccinated animals had clearly increased to titres of 11–13 log2. The three negative challenge control calves remained negative for BVDV neutralising antibodies until the end of the trial.
2.8. Pathological examination
3.2. Clinical findings
On days 9, 10 and 13, one animal from both challenge infected groups was killed. The remaining animals from these two groups were killed on day 22. Tissue samples were collected from the oesophagus, abomasum, ileum, colon, Peyer’s patches, mesenteric lymph nodes, pancreas, tonsils, thymus, spleen and bone marrow. Formalin fixed tissue samples for histological examination were processed using hematoxylin-eosin stain and the slides were examined microscopically.
Four out of the five unvaccinated animals developed diarrhoea 6 or 7 days post challenge-infection. This was mild in two animals and moderate to severe in the two other calves. In contrast, the consistency of the faeces of three out of the four vaccinated calves remained normal throughout the observation period. After challenge the vaccinated animals exhibited some respiratory signs such as coughing, serous ocular and nasal discharge, and hyperventilation. Coughing and nasal dis-
2.7. Determination of 6irus neutralising antibodies
Table 1 Titres of neutralising antibodies at different time points [log2] Animal number (date of necropsy)
1st Vaccinationb
2nd Vaccination
At challenge
Virus strain
Vaccine
Vaccine
Vaccine
Vaccinated 265 (9a) 1174 (22) 2025 (10) 6077 (13)
53 53 53 53
5 7 6 5
At necropsy Challenge
13 13 13 13
6 10 8 8
Vaccine
13 13 13 13
Challenge
12 13 13 13
Un6accinated 1176 (22) 6036 (13) 6075 (10) 7184 (22) 9152 (9)
n.t.c n.t. n.t. n.t. n.t.
n.t. n.t. n.t. n.t. n.t.
53 53 53 53 53
53 53 53 53 53
n.t n.t. n.t. n.t. n.t.
11 11 7 13 5
Controls 1077 (-) 2121 (-) 7185 (-)
n.t. n.t. n.t.
n.t. n.t. n.t.
53 53 53
53 53 53
n.t. n.t. n.t.
53 53 53
a
Days after challenge infection. The two vaccinations were performed at 12 and 16 weeks of age, the animals were challenge virus infected at 20 weeks. c Not tested. b
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an established scoring system. There was a clear peak around 7 days after challenge in both challenged groups. The average daily scores per group were lower in the vaccinated animals (Fig. 1a). Two days after challenge, four unvaccinated animals became febrile with rectal temperatures of 39.6–41.2°C, whereas the vaccinated animals had only slightly elevated rectal temperatures (39.4° to 39.9°C). A second, more pronounced increase in rectal temperature was measured on days 6 and 7 after challenge when the rectal temperature of the unvaccinated animals ranged from 40.9°–41.5°C on day 6 and from 40.4–41.5°C on day 7. On day 6 post-challenge, the maximum rectal temperature of the vaccinated animals was 41.0°C. The next day, all of the calves in this group had rectal temperatures within the reference range. The time courses of mean rectal temperature per group are depicted in Fig. 1b.
3.3. Haematology
Fig. 1. Mean daily clinical scores (a) and daily rectal temperatures (b) after challenge infection of calves with a cp/ncp pair of a BVDV type II strain. Four calves were vaccinated twice intramuscularly, 4 weeks apart with an inactivated BVDV type I vaccine ( ). One group of calves (n = 5) was not vaccinated (") and one group of calves (n =3) served as uninfected control ( ×).
charge were also reported sporadically in the uninfected control calves. The clinical signs were validated using
In the unvaccinated calves there was a dramatic drop in the thrombocyte counts post-challenge, with the minimal value at 10 days post-challenge (Fig. 2). Blood samples taken from these calves 15 and 17 days postchallenge contained markedly elevated numbers of thrombocytes. Thrombocyte counts did not decline to values within the normal reference range until 3 weeks post-challenge. As expected, the thrombocyte counts of the uninfected controls stayed within the normal range throughout the whole observation period. The animals that were vaccinated prior to challenge-infection showed only a slight decrease in thrombocyte counts 3 days post challenge-infection and the numbers of circulating thrombocytes normalised by day 13 post challenge-infection. Similar patterns were observed for the leukocyte counts (Fig. 3). Similarly, the unvaccinated animals had a dramatic drop in leukocyte counts that
Fig. 2. Individual thrombocyte counts after challenge infection of calves with a cp/ncp pair of a BVDV type II strain. Four calves were vaccinated twice intramuscularly, 4 weeks apart with an inactivated BVDV type I vaccine (a). One group of calves was not vaccinated (b) and one group of calves served as uninfected control (c).
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Fig. 3. Individual white blood cell counts after challenge infection of calves with a cp/ncp pair of a BVDV type II strain. Four calves were vaccinated twice intramuscularly, 4 weeks apart with an inactivated BVDV type I vaccine (a). One group of calves was not vaccinated (b) and one group of calves served as uninfected control (c).
peaked 6 days post-challenge and normalised not before 2 weeks post-challenge. The leukocyte counts of the vaccinated animals also dropped after infection, but they rose again at 6 or 8 days after infection. The leukocyte counts of the uninfected controls oscillated around the reference value.
3.4. Virus excretion with nasal discharge BVDV was re-isolated from nasals swab samples at low titres only and only from three of the unvaccinated animals. This was detected at days 6 and 7 post challenge-infection. All swabs taken from vaccinated or uninfected animals were negative (Table 2)
during autopsy. These were more numerous and more severe in the unvaccinated animals. Three of the unvaccinated animals appeared to have pneumonia and the blood of two of the unvaccinated animals (6075, 9152) was watery and did not clot completely during autopsy. Histopathological examination revealed lesions that can be considered typical of acute BVDV infection and these were more apparent in the unvaccinated animals. The lesions consisted of hyperaemia and haemorrhages as well as erosions and ulcerations of the intestinal mucosa, associated with crypt necrosis, formation of micro-abscessses, lymphocytic depletion in both central and peripheral lymphatic tissues (Fig. 4) and megakaryocytosis. This is likely to be related to the thrombocytopenia.
3.5. Viraemia Except for one vaccinated animal, cell bound BVDV was detected in all challenge-infected animals on at least one day. The semi-quantitative detection method revealed that the peak of cell bound viraemia was 6 days after challenge-infection when all unvaccinated animals were clearly positive for virus isolation (Table 3). On post challenge day 8 still three (out of five) unvaccinated animals were positive in this test. In contrast, only one vaccinated animal each was tested positive on days 6 and 8 after challenge. The number of positive wells out of four wells tested was clearly lower in the vaccinated animals indicating a lower virus load. Infectious BVDV was detected in plasma samples of all infected animals for at least 1 day and at most on 3 consecutive days. The titres were quite low, with a maximum of 40 TCID50/ml (Table 3).
4. Discussion
Animal
6 dpia
7 dpi
3.6. Pathology
1176 6036 9152
3.2 2.9 Negative
1.4 Negative 1.4
Erosions and petechial haemorrhages throughout the gastro-intestinal tract were the predominant findings
BVDV type 2 infection causes a syndrome in young calves that is characterised by thrombocytopenia both in the field [9] and experimentally [10,11,18,19]. Due to the young age of the animals used in these models, they are unsuitable for vaccination — challenge trials. A challenge model that produces clinical disease including severe thrombocytopenia in calves at 20 weeks of age has been developed in the present study. Unpublished results of an earlier study indicated that the pathogenTable 2 BVDV (non cytopathic) infectivity titres in positive nasal swab samples [log10TCID50/ml] of unvaccinated animals
a
Days post infection.
B. Makoschey et al. / Vaccine 19 (2001) 3261–3268
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Table 3 Detection of BVDV in peripheral blood cells and plasma 0 dpi
1 dpi
3 dpi
6 dpi
Sample
Cells
Plasma
Cells
Plasma
Vaccinated 265 1174 2025 6077
– – – –
– – – –
– – +a +
– – – –
– – + +
Un6accinated 1176 – 6036 – 6075 – 7184 – 9152 –
– – – – –
– – + – –
– – – – –
– +++ ++ + –
Controls 1077 2121 7185
n.t.c n.t. n.t.
– cont.d –
n.t. n.t. n.t.
– – –
– – –
Cells
Plasma
1.4b 1.2 – 1.5
8 dpi
Cells
Plasma
10 dpi
Cells
Plasma
Cells
Plasma
– – – –
– – – –
– – – –
++ – – –
– 1.3 1.2 1.3
– – + –
– 1.3 1.2 – 1.4
+++ ++ ++++ +++ ++++
1.2 – – – 1.2
– +++ ++ – ++
1.2 – 1.6 – 1.2
– – – – –
– – – 1.2 –
n.t. n.t. n.t.
– – –
n.t. n.t. n.t.
– – –
n.t. n.t. n.t.
– – –
n.t. n.t. n.t.
a
+ to ++++ indicates the number of positive wells (out of four wells tested) each incubated with 107 cells. Titres are expressed as log10TCID50 per ml. c Not tested. d Contaminated. b
icity of the challenge strain was related to the cp virus of the cp/ncp pair. The rather high challenge dose in this study was used in order to inoculate a sufficient amount of cp virus to achieve pathogenicity. The typical biphasic febrile response after BVDV infection [10,19] was also reproduced in this challenge model. Furthermore, four out of five unvaccinated animals developed diarrhoea, the classical clinical sign of BVDV infection. There were also signs of respiratory disease. These signs were also present — to a much lower extent — in the uninfected control animals. Therefore, it is difficult to decide whether they were caused or only exacerbated by BVDV infection. Potgieter and colleagues [20] reported that BVDV alone caused mild pneumonic lesions whereas sequential inoculation of BVDV and Pasteurella hemolytica resulted in marked respiratory disease. The most striking effect of the BVDV infection in the present system was the thrombocytopenia followed by a dramatic increase in thrombocyte counts several days later. This was also reflected in the pronounced megakaryocytosis that was seen in the bone marrow of the calves and can be interpreted as an over-compensation. The calves did not display the haemorrhages that had been described for the herd, from which the challenge virus was originally isolated. It has been observed by others, that viruses isolated from severe outbreaks may only induce mild illness in experimentally infected animals [21]. This may be due to the absence of the pathogens or other co-factors that exacerbated BVDV infection in the field or to the attenuation of the viruses
during cell culture propagation. The unvaccinated animals infected in the present trial developed severe leukopenia. BVDV induced leukopenia has been studied in more detail by Bolin and colleagues [6]. This study indicated that the T-lymphocyte population was the most affected, with substantial decreases in the percentage and absolute numbers of cells. One can easily imagine, that the effects of BVDV impair normal immune defences and, thereby, potentiate the activities of other microorganisms. The present model reproduced clinical BVDV in terms of diarrhoea, respiratory signs, thrombopaenia and leukopenia in calves at 20 weeks of age. This challenge model was then used to determine the efficacy of an inactivated BVDV type 1 vaccine in protection against a BVDV type 2 strain. There is some epidemiological evidence that proper vaccination with BVDV type 1 vaccines protect against clinical disease due to BVDV type 2 infection [22]. The efficacy of live BVDV type 1 vaccines against type 2 challenge has been demonstrated by Dean and colleagues [23], but the results of Potgieter and colleagues [24] questioned the efficacy of killed virus vaccines at least in heterologous protection. In the present study, the vaccine reduced the different kinds of clinical signs. Also the haematological changes were reduced compared with the unvaccinated animals. It has to be stressed, that the leukocyte counts decreased but recovered very soon, indicating that the immune system was less affected by the BVDV infection than in the unvaccinated animals. This is a very beneficial effect of the vaccination. Another item
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to consider is the reduction of challenge virus excretion with nasal discharges. Under field conditions, the amount of excreted virus has important consequences for transmission of virus and the size of an outbreak. In this study, challenge virus could be re-isolated from nasal discharge only at low titre from three (out of five) unvaccinated animals only on 2 days. Therefore, it is difficult to draw conclusions about the effect of the vaccine on the vertical transmission of the challenge virus. In the vaccinated animals, the cell bound viraemia after challenge was reduced both in extent and duration as compared with the unvaccinated animals. In contrast, there was no clear effect of the vaccine with regard to the cell free viraemia. The latter is considered to be critical for vertical transmission of BVDV. In an earlier vaccination-challenge study with Bovilis® BVD animals were protected against cell-free viraemia after challenge with a pool of 12 ncp BVDV strains and Border disease virus strains (data not shown). In the latter study, the challenge was performed intranasally but not intravenously, which might explain the different results in the two studies.
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The mechanisms that underlie protection against BVDV infection are not completely understood. Bolin and Ridpath [25] suggest that the level of neutralising antibodies is critical for protection, with a titre of ]28 giving considerable protection. The animals vaccinated with the type 1 vaccine in the present trial had neutralising antibody titres of 26 – 210 against the heterologous type 2 challenge virus. Therefore, the protection seen in the present study may well be based on antibody mediated neutralisation. Typically, titers of neutralising antibodies decline by a factor of 12–16 during 1 year after vaccination with Bovilis® BVD (data not shown). It can be assumed that the extent of protection would also become less in the course of time after vaccination. It has been shown that inactivated vaccines protect cattle against foetal infection [26] and respiratory signs[27] after homologous challenge. Heterologous protection by live vaccines [23] has been reported earlier. The present study is the first, which to our knowledge demonstrated the efficacy of an inactivated BVDV type 1 vaccine against clinical signs including thrombocytopenia after infection with a BVDV type 2 strain.
Fig. 4. Lymphoid follicles from ileal Peyers’ patches 9 days after infection (a, b) and the thymus 10 days after infection (c, d). The lymphoid follicles and the thymic cortex of unvaccinated animals are severely depleted for lymphocytes (b, d) whereas those of the vaccinates are normal (a, c). Objective magnification, 4 × .
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Acknowledgements The authors thank H.-J. Thiel for kindly providing the challenge virus and J. Jansen for technical assistance. The comments of L. Horspool were highly appreciated.
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An inactivated bovine virus diarrhoea virus (BVDV) type 1 vaccine affords clinical protection against BVDV type 2 B. Makoschey 1,*, M.G.J. Janssen 1, M.P. Vrijenhoek 2,1, J.H.M. Korsten 3,2, P.v.d. Marel 1 1
Department of Virological R&D, Inter6et International B.V., P.O. Box 31, 5830 AA Boxmeer, The Netherlands 2 Department of Pathology, Inter6et International B.V., P.O. Box 31, 5830 AA Boxmeer, The Netherlands 3 Department of Toxicology and Drug Disposition, N.V. Organon, P.O. Box 20, 5340 BH Oss, The Netherlands Received 18 July 2000; received in revised form 1 November 2000; accepted 12 December 2000
Abstract This study was designed to answer to two distinct questions. Firstly, is it possible to reproduce clinical signs of acute bovine virus diarrhoea virus (BVDV) type 2 infection including signs of haemorrhagic disease under experimental conditions in cattle at 20 weeks of age? Secondly, what is the extent of the protection afforded by vaccination with an inactivated BVDV type 1 vaccine against BVDV type 2 infection? Calves were vaccinated at 12 and 16 weeks of age with a commercially available inactivated BVDV type 1 vaccine (Bovilis® BVD). At 20 weeks they were challenge infected with BVDV type 2 virus together with unvaccinated control calves. The unvaccinated animals developed typical signs of respiratory disease, diarrhoea with erosions and haemorrhages along the whole length gastro-intestinal tract, and depletion of lymphocytes in lymphatic organs. These signs were either absent or markedly less severe in the vaccinated animals. The beneficial effects of vaccination were most striking in the haematological parameters thrombocytopenia and leukopenia. It can be concluded that vaccination with Bovilis® BVD affords cross-protection against clinical effects of a challenge-infection with heterologous type 2 BVDV. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Vaccine; Bovine; Diarrhoea
1. Introduction Bovine virus diarrhoea virus (BVDV) is a major viral pathogen in cattle and causes considerable economic losses throughout the world [1]. The clinical manifestations vary in both type and degree. Traditionally, these are classified into three categories, (1) acute virus infection of an immunocompetent animal, (2) foetal infection and (3) mucosal disease. Categories 2 and 3 refer to the potential transplacental spread of the virus during viraemia. Depending on the stage of gestation at the time of transplacental infection, the outcome may be embryonic death, abortion, stillbirth or the birth of immunotolerant calves persistently infected with BVDV
* Corresponding author. Tel.: +31-485-587600; fax: + 31-485587339. 1 Tel. : + 31-485-587600, fax: + 31-485-587339. 2 Tel.:+ 31-412-666112, fax: + 31-486-469131.
[2,3]. These calves may appear healthy, but are a permanent source of BVDV in the herd. They are also at risk of developing fatal mucosal disease if superinfected with a cytopathic biotype of the virus [4]. The classical bovine virus diarrhoea, which gave the virus its name is only one of the possible manifestations of acute BVDV infections of immunocompetent animals. BVDV also infects white blood cells [5] and causes depletion of circulating B- and T-lymphocytes [6]. This results in an unspecific immunosuppression that favours infections with other viral or bacterial pathogens. Furthermore, there is epidemiological [7] and experimental [8] evidence that BVDV is directly associated with respiratory disease. New forms of acute BVDV infection have been reported in North America and Canada [9–12]: thrombocytopenia resulting in the so-called haemorrhagic syndrome. The different BVDV strains vary considerably in their virulence [13]. There is also considerable
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antigenic and genetic variation in the BVDV strains that have been characterised so far. This lead to the classification of BVDV into two types (1 and 2) [14]. Most cases of the haemorragic form of BVDV infection have been attributed to BVDV type 2 viruses. The attenuated and inactivated BVDV vaccines that have been developed to date have been reviewed by van Oirschot and colleagues [15]. The broad antigenic diversity of BVDV strains places high demands on these vaccines. The extent of cross-protection afforded by a vaccine has to be determined experimentally and is difficult to predict. The aim of the present study was to investigate the protection provided by a new inactivated BVDV type 1 vaccine against severe challenge with a BVDV type 2 strain that induces thrombocytopenia.
2.3. Clinical signs After challenge, clinical observations were performed daily for 2 weeks. Special attention was paid to behaviour, faecal consistency, sneezing, coughing, ocular and nasal discharge, hyperaemia or lesions of the nasal and oral mucosae, and abnormal breathing. The clinical parameters were recorded daily for each calf and scored 0 when normal, 1 when mild, 2 when moderately and 3 when markedly abnormal. The mean daily clinical scores per group were calculated. Rectal temperature was measured as a separate parameter. This was done daily for 2 weeks and the mean temperature per group and per day was calculated.
2.4. Haematology 2. Materials and Methods
2.1. Viruses and cells Virus titration, virus isolation and serum neutralisation tests were performed on Bovine Embryo Lung (BEL) cells. The cells were cultured in a combination of Glasgow’s and Eagles modified minimal essential medium (MEM) supplemented with 5% foetal calf serum and a cocktail of antibiotics neomycin (50 mg/ ml), polymyxin B (50 mg/ml), pimafucin (2.5 mg/ml) and tylosin (10 mg/ml). The cells were incubated at 37°C in humidified atmosphere with 5% CO2. The challenge virus BVDV-2 Giessen-1 was isolated originally from the blood of a naturally infected calf [16] (kindly provided by H.-J. Thiel, Gießen, Germany). The inoculum consisted of a pair of non-cytopathic and cytopathic viruses. This virus mixture was passaged five times in cell culture and finally amplified on calf kidney cells. The inoculum titre was 7.7 log10TCID50/ml non-cytopathic BVDV and 5.4 log10TCID50/ml cytopathic BVDV. The cell and virus stocks were sterile.
2.2. Experimental design Twelve Holstein calves obtained from dams seronegative for BVDV were used for this experiment. One group of four calves was vaccinated with a commercial batch of Bovilis®BVD (Intervet) administered by intramuscular injection at 12 and 16 weeks of age. Four weeks after the second vaccination, the vaccinates were challenged along with five age matched unvaccinated controls. Challenge virus was administered intravenously into the jugular vein (2 ml) and intranasally (2 ml, 1 ml per nostril). A group of three untreated calves of the same age were kept as a negative control.
The haematological examinations focussed on the changes in thrombocyte and leukocyte counts. The former due to the numerous reports of BVDV induced thrombocytopenia, and the latter as an indicator for the immunosuppressive effects of BVDV. Blood samples were taken at 2–3 days interval beginning 5 days before challenge until 3 weeks after challenge. Thrombocyte and leukocyte counts were determined using an automated cell counter (Cell-Dyn 3500 haematology analyser with veterinary software, Abbott) (according to their size, complexity and depolarisation capacity).
2.5. Determination of 6irus excretion with nasal discharge Nasal swab samples were taken using swabsticks that contain a sterile cotton plug starting at the day of challenge. The swabs were submerged in sterile phosphate buffered saline medium for approximately 1 h. The medium was transferred into sterile tubes and stored frozen at − 70°C until tested. Virus titrations were performed using the end point dilution method according to standard procedures. Four replicates of a series of 10-fold dilutions were made in microtitre plates on monolayers of BEL cells. Titres were determined using immunofluorescence testing and calculated according to the formula of Ka¨rber [17].
2.6. Virus isolation from peripheral blood cells and plasma samples Whole blood samples were taken three times a week, for the determination of cell-bound viraemia. The buffy coat was recovered after centrifugation of the sample, and treated with erythrocyte lysis buffer. Then centrifugation was repeated. The cell pellet was re-washed in the buffer and then stored frozen until tested. For virus isolation, aliquots of 107 cells were co-cultured in 4-fold
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on monolayers of BEL cells and virus infection was judged by immunofluorescent staining. The cell free virus titre in the plasma samples was determined by titration using the end point dilution method, in 4-fold, on monolayers of BEL cells. BVDV infection was judged by immunofluorescence testing and calculated according to the formula of Ka¨rber [17].
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3. Results
3.1. Virus neutralisation
Serum samples were collected from the calves immediately before each vaccination and at the time of autopsy. They were stored frozen at − 20°C until tested. A standard virus neutralisation assay was performed against the homologous vaccine virus strain and the heterologous challenge virus strain. The test was performed on monolayers of BEL cells and neutralisation titres were recorded as the highest dilution of serum that inhibited BVDV virus growth, judged by immunofluorescent staining.
At 4 weeks after the first vaccination, all four vaccinates had detectable levels of neutralising antibody versus the vaccine virus (Table 1). By the time of challenge, i.e. at 4 weeks after the second vaccination, the homologous titres had increased to 13 log2. The heterologous titres against the BVDV type 2 challenge virus ranged from 5–10 log2. The animals of both unvaccinated groups were free from detectable BVDV neutralising antibodies at the time of challenge. At the time of autopsy (post challenge-infection days 9, 10, 13 or 22) all five unvaccinated animals had seroconverted for BVDV, and the homologous and heterologous titres of the vaccinated animals had clearly increased to titres of 11–13 log2. The three negative challenge control calves remained negative for BVDV neutralising antibodies until the end of the trial.
2.8. Pathological examination
3.2. Clinical findings
On days 9, 10 and 13, one animal from both challenge infected groups was killed. The remaining animals from these two groups were killed on day 22. Tissue samples were collected from the oesophagus, abomasum, ileum, colon, Peyer’s patches, mesenteric lymph nodes, pancreas, tonsils, thymus, spleen and bone marrow. Formalin fixed tissue samples for histological examination were processed using hematoxylin-eosin stain and the slides were examined microscopically.
Four out of the five unvaccinated animals developed diarrhoea 6 or 7 days post challenge-infection. This was mild in two animals and moderate to severe in the two other calves. In contrast, the consistency of the faeces of three out of the four vaccinated calves remained normal throughout the observation period. After challenge the vaccinated animals exhibited some respiratory signs such as coughing, serous ocular and nasal discharge, and hyperventilation. Coughing and nasal dis-
2.7. Determination of 6irus neutralising antibodies
Table 1 Titres of neutralising antibodies at different time points [log2] Animal number (date of necropsy)
1st Vaccinationb
2nd Vaccination
At challenge
Virus strain
Vaccine
Vaccine
Vaccine
Vaccinated 265 (9a) 1174 (22) 2025 (10) 6077 (13)
53 53 53 53
5 7 6 5
At necropsy Challenge
13 13 13 13
6 10 8 8
Vaccine
13 13 13 13
Challenge
12 13 13 13
Un6accinated 1176 (22) 6036 (13) 6075 (10) 7184 (22) 9152 (9)
n.t.c n.t. n.t. n.t. n.t.
n.t. n.t. n.t. n.t. n.t.
53 53 53 53 53
53 53 53 53 53
n.t n.t. n.t. n.t. n.t.
11 11 7 13 5
Controls 1077 (-) 2121 (-) 7185 (-)
n.t. n.t. n.t.
n.t. n.t. n.t.
53 53 53
53 53 53
n.t. n.t. n.t.
53 53 53
a
Days after challenge infection. The two vaccinations were performed at 12 and 16 weeks of age, the animals were challenge virus infected at 20 weeks. c Not tested. b
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an established scoring system. There was a clear peak around 7 days after challenge in both challenged groups. The average daily scores per group were lower in the vaccinated animals (Fig. 1a). Two days after challenge, four unvaccinated animals became febrile with rectal temperatures of 39.6–41.2°C, whereas the vaccinated animals had only slightly elevated rectal temperatures (39.4° to 39.9°C). A second, more pronounced increase in rectal temperature was measured on days 6 and 7 after challenge when the rectal temperature of the unvaccinated animals ranged from 40.9°–41.5°C on day 6 and from 40.4–41.5°C on day 7. On day 6 post-challenge, the maximum rectal temperature of the vaccinated animals was 41.0°C. The next day, all of the calves in this group had rectal temperatures within the reference range. The time courses of mean rectal temperature per group are depicted in Fig. 1b.
3.3. Haematology
Fig. 1. Mean daily clinical scores (a) and daily rectal temperatures (b) after challenge infection of calves with a cp/ncp pair of a BVDV type II strain. Four calves were vaccinated twice intramuscularly, 4 weeks apart with an inactivated BVDV type I vaccine ( ). One group of calves (n = 5) was not vaccinated (") and one group of calves (n =3) served as uninfected control ( ×).
charge were also reported sporadically in the uninfected control calves. The clinical signs were validated using
In the unvaccinated calves there was a dramatic drop in the thrombocyte counts post-challenge, with the minimal value at 10 days post-challenge (Fig. 2). Blood samples taken from these calves 15 and 17 days postchallenge contained markedly elevated numbers of thrombocytes. Thrombocyte counts did not decline to values within the normal reference range until 3 weeks post-challenge. As expected, the thrombocyte counts of the uninfected controls stayed within the normal range throughout the whole observation period. The animals that were vaccinated prior to challenge-infection showed only a slight decrease in thrombocyte counts 3 days post challenge-infection and the numbers of circulating thrombocytes normalised by day 13 post challenge-infection. Similar patterns were observed for the leukocyte counts (Fig. 3). Similarly, the unvaccinated animals had a dramatic drop in leukocyte counts that
Fig. 2. Individual thrombocyte counts after challenge infection of calves with a cp/ncp pair of a BVDV type II strain. Four calves were vaccinated twice intramuscularly, 4 weeks apart with an inactivated BVDV type I vaccine (a). One group of calves was not vaccinated (b) and one group of calves served as uninfected control (c).
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Fig. 3. Individual white blood cell counts after challenge infection of calves with a cp/ncp pair of a BVDV type II strain. Four calves were vaccinated twice intramuscularly, 4 weeks apart with an inactivated BVDV type I vaccine (a). One group of calves was not vaccinated (b) and one group of calves served as uninfected control (c).
peaked 6 days post-challenge and normalised not before 2 weeks post-challenge. The leukocyte counts of the vaccinated animals also dropped after infection, but they rose again at 6 or 8 days after infection. The leukocyte counts of the uninfected controls oscillated around the reference value.
3.4. Virus excretion with nasal discharge BVDV was re-isolated from nasals swab samples at low titres only and only from three of the unvaccinated animals. This was detected at days 6 and 7 post challenge-infection. All swabs taken from vaccinated or uninfected animals were negative (Table 2)
during autopsy. These were more numerous and more severe in the unvaccinated animals. Three of the unvaccinated animals appeared to have pneumonia and the blood of two of the unvaccinated animals (6075, 9152) was watery and did not clot completely during autopsy. Histopathological examination revealed lesions that can be considered typical of acute BVDV infection and these were more apparent in the unvaccinated animals. The lesions consisted of hyperaemia and haemorrhages as well as erosions and ulcerations of the intestinal mucosa, associated with crypt necrosis, formation of micro-abscessses, lymphocytic depletion in both central and peripheral lymphatic tissues (Fig. 4) and megakaryocytosis. This is likely to be related to the thrombocytopenia.
3.5. Viraemia Except for one vaccinated animal, cell bound BVDV was detected in all challenge-infected animals on at least one day. The semi-quantitative detection method revealed that the peak of cell bound viraemia was 6 days after challenge-infection when all unvaccinated animals were clearly positive for virus isolation (Table 3). On post challenge day 8 still three (out of five) unvaccinated animals were positive in this test. In contrast, only one vaccinated animal each was tested positive on days 6 and 8 after challenge. The number of positive wells out of four wells tested was clearly lower in the vaccinated animals indicating a lower virus load. Infectious BVDV was detected in plasma samples of all infected animals for at least 1 day and at most on 3 consecutive days. The titres were quite low, with a maximum of 40 TCID50/ml (Table 3).
4. Discussion
Animal
6 dpia
7 dpi
3.6. Pathology
1176 6036 9152
3.2 2.9 Negative
1.4 Negative 1.4
Erosions and petechial haemorrhages throughout the gastro-intestinal tract were the predominant findings
BVDV type 2 infection causes a syndrome in young calves that is characterised by thrombocytopenia both in the field [9] and experimentally [10,11,18,19]. Due to the young age of the animals used in these models, they are unsuitable for vaccination — challenge trials. A challenge model that produces clinical disease including severe thrombocytopenia in calves at 20 weeks of age has been developed in the present study. Unpublished results of an earlier study indicated that the pathogenTable 2 BVDV (non cytopathic) infectivity titres in positive nasal swab samples [log10TCID50/ml] of unvaccinated animals
a
Days post infection.
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Table 3 Detection of BVDV in peripheral blood cells and plasma 0 dpi
1 dpi
3 dpi
6 dpi
Sample
Cells
Plasma
Cells
Plasma
Vaccinated 265 1174 2025 6077
– – – –
– – – –
– – +a +
– – – –
– – + +
Un6accinated 1176 – 6036 – 6075 – 7184 – 9152 –
– – – – –
– – + – –
– – – – –
– +++ ++ + –
Controls 1077 2121 7185
n.t.c n.t. n.t.
– cont.d –
n.t. n.t. n.t.
– – –
– – –
Cells
Plasma
1.4b 1.2 – 1.5
8 dpi
Cells
Plasma
10 dpi
Cells
Plasma
Cells
Plasma
– – – –
– – – –
– – – –
++ – – –
– 1.3 1.2 1.3
– – + –
– 1.3 1.2 – 1.4
+++ ++ ++++ +++ ++++
1.2 – – – 1.2
– +++ ++ – ++
1.2 – 1.6 – 1.2
– – – – –
– – – 1.2 –
n.t. n.t. n.t.
– – –
n.t. n.t. n.t.
– – –
n.t. n.t. n.t.
– – –
n.t. n.t. n.t.
a
+ to ++++ indicates the number of positive wells (out of four wells tested) each incubated with 107 cells. Titres are expressed as log10TCID50 per ml. c Not tested. d Contaminated. b
icity of the challenge strain was related to the cp virus of the cp/ncp pair. The rather high challenge dose in this study was used in order to inoculate a sufficient amount of cp virus to achieve pathogenicity. The typical biphasic febrile response after BVDV infection [10,19] was also reproduced in this challenge model. Furthermore, four out of five unvaccinated animals developed diarrhoea, the classical clinical sign of BVDV infection. There were also signs of respiratory disease. These signs were also present — to a much lower extent — in the uninfected control animals. Therefore, it is difficult to decide whether they were caused or only exacerbated by BVDV infection. Potgieter and colleagues [20] reported that BVDV alone caused mild pneumonic lesions whereas sequential inoculation of BVDV and Pasteurella hemolytica resulted in marked respiratory disease. The most striking effect of the BVDV infection in the present system was the thrombocytopenia followed by a dramatic increase in thrombocyte counts several days later. This was also reflected in the pronounced megakaryocytosis that was seen in the bone marrow of the calves and can be interpreted as an over-compensation. The calves did not display the haemorrhages that had been described for the herd, from which the challenge virus was originally isolated. It has been observed by others, that viruses isolated from severe outbreaks may only induce mild illness in experimentally infected animals [21]. This may be due to the absence of the pathogens or other co-factors that exacerbated BVDV infection in the field or to the attenuation of the viruses
during cell culture propagation. The unvaccinated animals infected in the present trial developed severe leukopenia. BVDV induced leukopenia has been studied in more detail by Bolin and colleagues [6]. This study indicated that the T-lymphocyte population was the most affected, with substantial decreases in the percentage and absolute numbers of cells. One can easily imagine, that the effects of BVDV impair normal immune defences and, thereby, potentiate the activities of other microorganisms. The present model reproduced clinical BVDV in terms of diarrhoea, respiratory signs, thrombopaenia and leukopenia in calves at 20 weeks of age. This challenge model was then used to determine the efficacy of an inactivated BVDV type 1 vaccine in protection against a BVDV type 2 strain. There is some epidemiological evidence that proper vaccination with BVDV type 1 vaccines protect against clinical disease due to BVDV type 2 infection [22]. The efficacy of live BVDV type 1 vaccines against type 2 challenge has been demonstrated by Dean and colleagues [23], but the results of Potgieter and colleagues [24] questioned the efficacy of killed virus vaccines at least in heterologous protection. In the present study, the vaccine reduced the different kinds of clinical signs. Also the haematological changes were reduced compared with the unvaccinated animals. It has to be stressed, that the leukocyte counts decreased but recovered very soon, indicating that the immune system was less affected by the BVDV infection than in the unvaccinated animals. This is a very beneficial effect of the vaccination. Another item
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to consider is the reduction of challenge virus excretion with nasal discharges. Under field conditions, the amount of excreted virus has important consequences for transmission of virus and the size of an outbreak. In this study, challenge virus could be re-isolated from nasal discharge only at low titre from three (out of five) unvaccinated animals only on 2 days. Therefore, it is difficult to draw conclusions about the effect of the vaccine on the vertical transmission of the challenge virus. In the vaccinated animals, the cell bound viraemia after challenge was reduced both in extent and duration as compared with the unvaccinated animals. In contrast, there was no clear effect of the vaccine with regard to the cell free viraemia. The latter is considered to be critical for vertical transmission of BVDV. In an earlier vaccination-challenge study with Bovilis® BVD animals were protected against cell-free viraemia after challenge with a pool of 12 ncp BVDV strains and Border disease virus strains (data not shown). In the latter study, the challenge was performed intranasally but not intravenously, which might explain the different results in the two studies.
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The mechanisms that underlie protection against BVDV infection are not completely understood. Bolin and Ridpath [25] suggest that the level of neutralising antibodies is critical for protection, with a titre of ]28 giving considerable protection. The animals vaccinated with the type 1 vaccine in the present trial had neutralising antibody titres of 26 – 210 against the heterologous type 2 challenge virus. Therefore, the protection seen in the present study may well be based on antibody mediated neutralisation. Typically, titers of neutralising antibodies decline by a factor of 12–16 during 1 year after vaccination with Bovilis® BVD (data not shown). It can be assumed that the extent of protection would also become less in the course of time after vaccination. It has been shown that inactivated vaccines protect cattle against foetal infection [26] and respiratory signs[27] after homologous challenge. Heterologous protection by live vaccines [23] has been reported earlier. The present study is the first, which to our knowledge demonstrated the efficacy of an inactivated BVDV type 1 vaccine against clinical signs including thrombocytopenia after infection with a BVDV type 2 strain.
Fig. 4. Lymphoid follicles from ileal Peyers’ patches 9 days after infection (a, b) and the thymus 10 days after infection (c, d). The lymphoid follicles and the thymic cortex of unvaccinated animals are severely depleted for lymphocytes (b, d) whereas those of the vaccinates are normal (a, c). Objective magnification, 4 × .
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Acknowledgements The authors thank H.-J. Thiel for kindly providing the challenge virus and J. Jansen for technical assistance. The comments of L. Horspool were highly appreciated.
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