Comp. lmmun. Microbiol. infect. Dis. Vol. 15, No. 3, pp. 163-169, 1992 Printed in Great Britain. All rights reserved
0147-9571/92 $5.00+0.00 Copyright © 1992 Pergamon Press Ltd
A N E X P E R I M E N T A L C O N T R I B U T I O N TO THE S T U D Y OF THE P A T H O G E N E S I S OF BOVINE VIRAL DIARRHEA VIRUS INFECTION G. CASTRUCCI, I* B. I. OSBURN, 2 M. FERRARI 3 and V. TRALD14 qstituto di Malattie Infettive, Facoltfi di Medicina Veterinaria, Via S. Costanzo 4, 06100 Perugia, Italy, 2Department of Veterinary Pathology, University of California, Davis, CA 95616, U.S.A., 3Istituto Zooprofillatico Sperimentale della Lombardia e della Emilia, via A. Bianchi 7, 25100 Brescia, Italy and 4Centro Provinciale Svezzamento Vitelli, Tripoli S. Giorgio, Mantova, Italy Abstract--This presentation summarizes the results of a study on the pathogenesis of bovine viral diarrhea (BVDV) infection. The cytopathic (CP) strain TVM-2 of BVDV induced in calves an overt clinical disease which is usually recorded as the acute primary BVDV infection observed under natural conditions. In contrast the non-cytopathic (NCP) strain New York- 1 of BVDV did not cause any significant signs of disease. However, when the calves were immunosuppressed by treatment with dexamethasone (DMS) the biotype of BVDV involved did not seem to be as important as it appeared to be in an immunologically normal animal. This was shown in this study by the NCP BVDV which caused a fatal disease in calves treated with DMS. A mixed infection given to calves by injecting them with both CP and NCP BVDV, did not result in any particularly serious disease. So, the potential immunosuppressive activity of BVDV itself for the host has not been proven under the experimental procedures used in this experiment. Finally, a modified-live CP BVDV vaccine was unable to cause clinical disease when injected into calves that had been infected previously with strain New York-I of BVDV. Key words: Bovine viral diarrhea virus, infection, pathogenesis, calves.
UNE CONTRIBUTION EXPERIMENTALE A L ' E T U D E D E LA P A T H O G E N I E D E L ' I N F E C T I O N D U E A U V I R U S D E LA D I A R R H E E BOVINE VIRALE R r s u m ~ C e t t e prrsentation synthrtise les resultats d'une 6tude sur la pathogrnie de l'infection de la diarrhre bovine virale (BVDV). La souche cytopathique (CP) TVM-2 de BVDV a provoqu6 chez les veaux une maladie clinique manifeste qui est grnrralement enregistrre comme l'infection primaire aigu6 de BVDV observre dans les conditions naturelles. Par contre, la souche non cytopathique (NCP) New York-I de BVDV n'a provoqu6 aucun signe significatif de maladie. Toutefois, lorsque les veaux ont 6t6 soumis un traitement d'immunosuppression par dexam&hasone (DMS), le biotype de BVDV impliqu6 n'a pas sembl6 6tre aussi important qu'il parait l'&re chez un animal immunologiquement normal. Cela a 6t~ d~montr6 dans cette 6tude par la souche NCP de BVDV, qui a provoqu6 une maladie mortelle chez les veaux traitrs au DMS. Une infection mixte, provoqure en injectant au veaux tant la souche CP que la souche NCP de BVDV, n'a donn~ aucun maladie particulidrement grave. Ansi, l'activit6 immunosuppressive potentielle de BVDV lui-mrme pour l'hrte n'a pas 6t6 prouvre au cours des procrdures utilis~es pour cette exprrience. Enfin, l'inoculation, chez des veaux prralablement infectrs par la souche New York-I de BVDV, d'un vaccin vivant attrnu6 de la souche CP de BVDV n'a pu provoquer aucun maladie clinique. Mots-clefs: Virus de la diarrh~e bovine virale, infection, pathogrnie, veaux. *Author for correspondence. 163
164
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INTRODUCTION In the last few years studies on the pathogenesis of bovine viral diarrhea virus (BVDV) infection have been concentrated mostly on the different roles of the two biotypes of the virus, one of which is non-cytopathogenic (NCP) and the other cytopathogenic (CP). Apparently, the time of infection and interplay between these biotypes are the most outstanding factors which eventually lead BVDV infections to produce clinical disease in cattle ranging from mild to fatal [1]. It was demonstrated that persistently viraemic calves, superinfected by CP BVDV, succumbed to mucosal disease [2, 3]. According to a number of experimental data it seems that immunosuppression plays a paramount role in the pathogenesis [4]. Considering that BVDV infection may cause one of the most economically important viral diseases of cattle, it seemed worthwhile to investigate some aspects of the pathogenesis of BVDV which apparently came out after the above mentioned observations. Accordingly, a study was planned to verify the possible involvement in the pathogenesis of BVDV infection of the following factors: (1) The biotype of the virus, i.e. CP or NCP and the influence of an immunosuppressive state of the host. (2) Simultaneous infection in the same host by both biotypes of the virus. (3) The effects of vaccination with a modified-live (ML) BVDV vaccine in cattle latently infected with a NCP strain of BVDV. Two previous papers [5, 6] together with the present paper give the findings made.
EXPERIMENTAL PROCEDURES--RESULTS Tissue culture
Primary or secondary cultures of bovine embryo kidney (BEK) cells were used throughout the study. They were grown in minimum essential medium (MEM) with Earle's balanced salt solution and L-glutamine, enriched with 10% fetal bovine serum and containing antibiotics (penicillin, 1000 U/ml, streptomycin, 500 p g/ml and amphotericin B, 2/~g/ml). The fetal bovine serum was replaced by horse serum at a concentration of 5% whenever the cultures were to be used for virus cultivation or for the recovery of the virus from the experimental animals. Virus
The NCP strain New York-1 (kindly supplied by Dr B. I. Osburn, University of California, Davis) and the CP strain TVM-2 [7] of BVDV, grown in BEK cells, were used. The New York-1 strain had an undetermined number of passages in tissue cultures and a titer, as detected by immunofluorescence (IF) in BEK cells, of l055 median tissue culture infectious doses (TCIDs0) per 1 ml. The TVM-2 strain was at its 6th passage level in BEK cell cultures and had a titer of 1065/ml. Viral isolation
Pharyngeal swabbings and buffy coat obtained from the blood were tested in the BEK cell cultures for the presence of the virus according to the procedures described previously [5]. Samples were considered to be negative for the virus if cytopathic effects (CPE) failed
P a t h o g e n e s i s o f b o v i n e viral d i a r r h e a virus infection
165
to appear or IF was negative after 2 serial subpassages of the inoculum had been made. The IF procedure [5] was used for the detection of virus in BEK cell cultures inoculated with the specimens collected from calves that had been exposed to NCP BVDV.
Serum neutralization Serum samples were serially diluted two-fold in MEM in 96-well cell culture microtitration plate. An equal volume (0.025 ml) of MEM containing 200 TCIDs0 of CP BVDV/0.025 ml was added to the wells. After incubation at 22°C for 1 hr, 20,000 BEK cells, in a volume of 0.05 ml, were added to each well: Plates were incubated at 37°C for 3 days, and each well was scored for the presence of CPE [5]. Experimental design (1) The biotype of BVD V and the immunosuppressive state of the host. This experiment was designed to answer the question, whether the clinical and virological findings made in calves might be influenced by the biotype of the virus responsible for the infection. The tests were carried out in 20 Friesian calves, 30--40 days of age, allotted into two groups of I0. In each group, 8 calves were inoculated with CP or NCP strains of BVDV, whereas the remaining 2 in either groups were given dexamethasone (DMS) in addition to the appropriate biotype of BVDV. The NCP and the CP BVDV strains were inoculated intravenously (i.v.), each calf receiving 5 ml of virus culture. The DMS was given to the calves i.v. for 4 consecutive days and each calf was injected with 0.1 mg/kg of body weight per day, the first injection being made a few hours before virus inoculation. Calves of each group were housed in isolation facilities. Calves were observed for 30 days for the presence of clinical signs. Daily white blood cell (WBC) counts were also made. Pharyngeal swabbings and blood samples were obtained for virus isolation at pre-determined intervals post-inoculation. Serum was collected from each calf on the day of infection (post-infection day 0) and on post-infection day (PID) 30. The calves inoculated with CP TVM-2 strain on BVDV (Table 1) all showed overt clinical disease, however, the clinical response was generally more severe in the case of Table 1. Clinical and virological findings in calves treated or not treated with dexamethasone (DMS) and exposed to experimental infection with cytopathic (CP) TVM-2 strain of bovine viral diarrhea virus (BVDV) Clinical signs
Virus recovery on PID
Calf No.
DMS treatment
Fever ~>40°C*
Nasal discharge*
Diarrhea*
Leukopenia*
PSt
LEt
141 132 133 134 135 136 193 194 191 192,
NT NT NT NT NT NT NT NT T T
3/4 2/2 3/7 3/5 5/6 2/7 3/6 5/3 5/10 4/4
3/7 2/7 2/4 2/10 3/7 2/6 3/6 5/3 6/9 6/1
4/1 4/1 2/5 6/1 3/5 2/6 7/5 7/7 5/13 5/3
2/6 2/5 2/7 2/6 2/10 3/10 3/6 3/5 10/4 6/2
2-11 2-11 2-11 2/11 2/11 2/11 2/6 6 2 2,6~
2 2, 6, 17 2, 17 11, 17 4, 11, 17 NI 2 2 2, 6 2,6~
*Onset, post infection day (PID)/duration days. tSamples (PS = pharyngeal swabbings; LE = buffy coat) were collected on PIDs 2 , 4 , 6 , 8 , 1 1 , 1 7 . 1Calf No. 192 died on PID 7. §Virus was also isolated from the lung and the spleen of this calf. NT = not treated; T = treated; NI = virus was not isolated.
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G. CASTRUCCl et al.
animals which were treated with DMS. The 8 calves which were given the virus only reacted with fever, nasal discharge, diarrhea and leukopenia and recovered completely in about 2 weeks. The two calves (Nos 191, 192) which in addition to the virus, were also given DMS, developed classical mucosal disease of which the most consistent sign was diarrhea. One calf (No. 192) died on PID 7, i.e. 3 days following the last injection of DMS. At necropsy the mesenteric lymph nodes appeared enlarged and the mucosa of the small intestine was slightly congested. Virus was consistently recovered from the pharyngeal swabbings of all calves, whereas virus recovery from the buffy coat was less regular. At PID 30 neutralizing antibody to BVDV was found at titers ranging from 1:32 to 1:128. In the case of those calves exposed to the NCP New York-1 strain of BVDV (Table 2), the 8 animals which were given the virus only did not show any clinical signs of disease, even if the virus was recovered from their pharyngeal swabbings and buffy coats. In contrast, the two calves which were simultaneously treated with DMS underwent a severe clinical disease. They had high fever (41~2°C), copious nasal discharge, very marked leukopenia and diarrhea. One calf (No. 150) died on PID 11 and the other (No. 149) on PID 17. The symptoms appeared on PIDs 5 6 and lasted until they died. The most serious sign was diarrhea. Initially, the fluid feces were yellowish; later they became brownish in colour and were mixed with large fragments of necrotic material. The two calves were markedly dehydrated when they died. At necropsy the mucosa of the upper digestive tract appeared congested, whereas superficial erosions were found in the mucosa of the small intestine. The mesenteric lymph nodes were hemorrhagic and enlarged. (2) Simultaneous infection in the same host by both biotypes of BVDV. Two Friesian calves of the same age as those of the previous group ( 3 0 4 0 days), were both injected simultaneously with CP and NCP BVDV. Assuming that BVDV is an immunosuppressive agent itself, the expectation from this test was that a simultaneous infection with the 2 biotypes of the virus would result in the classically fatal expression of mucosal disease. However, the clinical response of the 2 calves was analogous to that observed in the 8 calves that were inoculated with the CP TVM-2 strain of BVDV (Table 1) in that they reacted with fever, leukopenia and diarrhea. The symptoms appeared on PIDs 5-7 and lasted about 1 week. It seems, therefore, that the mixed infection did not result in any
Table 2. Clinical and virological findings in calves treated or not treated with dexamethasone (DMS) and exposed to experimental infection with noncytopathic (NCP) New York-1 strain of bovine viral diarrhea virus (BVDV) Clinical signs Calf No.
DMS treatment
Fever ~>40"C*
Nasal discharge*
Diarrhea*
Virus recovery on PIDt PSi.
LE~
162
NT')
Leukopenia*
2,6
6
163 164 165 166 167 151 152 149§ 150¶1
NT NT t NT NT NT NT NT T T
2,26 2, 6 2, 6 2, 6 2 11 2 11 2, 4, 11 2, 4, 8
22 6 2 2 2,6,8,11 2,6,8,11 2, 6, 8, I I 2, 6, 8
No clinical signs have been observed
5/10 6/5
6/8 6/2
6/10 6/5
6/8 6/5
*Onset, post infection day (PID)/duration days. "l'Virus detection was made by immunofluorescence. :~Samples (PS = pharyngeal swabbings; LE = buffy coat) were collected on PIDs 2, 4, 6, 8, 11. §Died on PID 17. ¶lDied on PID 11.
P a t h o g e n e s i s o f b o v i n e viral d i a r r h e a v i r u s infection
167
particularly serious pathological situation like the one which is usually observed with the true mucosal disease: evidently neither BVDV biotype was sufficiently immunosuppressive for the host. (3) The effects of vaccination with M L CP B V D V vaccine in cattle latently infected with a N C P strain of B V D V [6]. One of the most common hypotheses on the pathogenesis of BVDV infection is that fatal mucosal disease could occur in animals persistently infected with a NCP strain of BVDV when they are superinfected with a homologous CP biotype of the virus [2, 3]. These findings raise the question whether cattle, which are persistently infected, would develop mucosal disease when vaccinated with a modified, live cytopathic BVDV vaccine. Accordingly, the purpose of this experiment was to verify the effects of vaccination in calves which were previously infected with NCP BVDV. Six Friesian calves, approx. 3 month old, which were determined to be free of neutralizing antibody to BVDV [6], were used. Latent infection was established in four calves by i.v. inoculation of NCP New York-I strain of BVDV, each calf receiving 10.0 ml of virus culture. Thirty days later the four latently infected calves and two additional uninfected calves (vaccine control), were each inoculated intramuscularly (i.m.) with 2.0 ml of a commercial vaccine containing a CP strain of BVDV 005.5 TCIDs0/ml ), and in addition infectious bovine rhinotracheitis (IBR) (1065 TCIDs0/ml ) and parainfluenza-3 (PI-3) (1055 TCIDs0/ml ) viruses. The animals were observed for 73 days from the start of the experiment. Their temperatures were taken daily for at least 25 days following the inoculation with the NCP strain of BVDV, as well as after vaccination. WBC counts were also made daily. Pharyngeal swabbings and blood samples were obtained for virus isolation and also samples of serum were collected from each calf for serology. As shown in Table 3, with the exception of a slight leukopenia, the four calves exposed to NCP BVDV did not develop any signs of disease during the 30 days between infection and the subsequent vaccination. However, the virus was consistently detected by IF in the BEK cell cultures inoculated with the pharyngeal swabbings and the buffy coats of each calf and this indicated that the infection with NCP virus was established. Moreover, all the calves at PID 30 had neutralizing antibody to BVDV at titers ranging from 1 : 8 to 1 : 64, depending on the calf.
Table 3. The response of calves to experimental infection with noncytopathic (NCP) strain of bovine viral diarrhea virus (BVDV) and to subsequent vaccination with a modified-live (ML) cytopathic (CP) BVDV vaccine Inoculation of NCP BVDV
Vaccination with ML CP BVDV
Virus isolation on PID* Calf No. 3 4 5 6
"] I
Vaccine controls 11 12
Clinical response Leukopenia only which appeared on PID 3-5 and lasted 2~. days NA NA
PSI"
LEt
4, 4, 4, 4,
4, 4, 4, 4,
16 16 16 16
NA NA
16 16 16 16
NA NA
Virus isolation on PVD Clinical response
PSt
LEt
None None Slight leukopenia Slight leukopenia
0, 6, 14 0, 6 0, 6, 14 0, 6
0, 6, 14 0, 6, 14 0, 6 0, 6, 14
Slight leukopenia Slight leukopenia
6, 14 6
6 6, 14
*Virus detection was made by immunofluorescence. tSamples (PS = pharyngeal swabbings; LE = buffy coat) were collected on post infection day (PID) 4 and 16 or on post vaccination day (PVD) 0 (the day of vaccination) and on PVDs 6 and 14. When PVDs are underlined it means that CP BVDV virus was isolated. NA = not applicable.
168
G. CASTRUCCI et al.
After vaccination with a vaccine containing a CP strain of BVDV (Table 3), the calves previously infected with NCP BVDV, did not show any significant signs of disease within the 43 days during which the calves were kept under observation. Only two calves (Nos 5 and 6) became leukopenic on post vaccination day (PVD) 3; however, their WBC counts were back to the normal range within 2-3 days. A similar picture was observed in the controls which were leukopenic for 3-4 days. BVDV was isolated from pharyngeal swabbings and buffy coats of all calves. The four calves which had been infected 30 days before vaccination, proved to be still infected on the day they were vaccinated (PVD 0 in Table 3). BVDV was subsequently detected by IF in the specimens obtained from the calves on PVDs 6 and 14. From the control calves CP BVDV of vaccine origin, was isolated. IBR virus, one of the components of the vaccine, was occasionally isolated from pharyngeal swabbings and from the buffy coats of all calves. PI-3, the other viral component of the vaccine was never isolated, nor was the viral antigen detected by hemadsorption tests in any of the cultures inoculated with pharyngeal swabbings or the blood samples obtained from the calves.
CONCLUSIONS One of the objectives of this work was to verify whether the biotype of the virus, i.e. CP or NCP, could influence the clinical response of calves to BVDV infection. Based on the results [5], the CP TVM-2 strain of BVDV induced in calves clinical signs of disease which included depression, fever, leukopenia, respiratory symptoms and diarrhea. Thus, the CP virus reproduced in calves the clinical picture which is usually reported for the acute primary BVDV infection observed under natural conditions. In contrast, the NCP New York-1 strain did not cause in calves any significant signs of disease, even if virus was recovered from the specimens obtained from the inoculated calves. These findings support the evidence for a potential diversity among BVDV isolates in their pathogenetic pathway toward the host. The possibility of the existence of a close association between BVDV strains and the clinical development of the disease has been already suggested by others [8]. However, when the animal is immunosuppressed, the biotype of BVDV involved does not seem to be as important as it might be in an immunologically normal animal. This was proved in the present study by the NCP strain which caused a fatal disease in the DMS treated calves while it was unable to give rise to overt disease in calves that were not immunosuppressed. Apparently, the immunosuppression could really be considered one of the."key factors" regulating the pathogenetic mechanism of bovine virus diarrhea/ mucosal disease [4]. On the other hand, the potential immunosuppressive activity of BVDV itself for the host, has not been proven under the experimental conditions adopted in these tests. Evidence of this is that the dual infection, i.e. the CP and NCP BVDV did not result in more serious diseases than produced by the CP virus alone [5, 9]. In the present experiment vaccination, with a modified-live CP BVDV vaccine, of calves previously infected with a NCP strain of BVDV did not cause mucosal disease. In a similar study others [3] failed to induce mucosal disease in persistently infected cattle by vaccination with M L CP BVDV. Failure to reproduce experimental mucosal disease by superinfection with CP BVDV was also reported [10]. It has been suggested that only CP BVDV with an antigenic structure identical to that of the endogenous NCP BVDV would be capable of inducing the disease [11]. This was recently confirmed when others succeeded
Pathogenesis of bovine viral diarrhea virus infection
169
in r e p r o d u c i n g acute, severe disease u s i n g pairs o f closely r e l a t e d N C P a n d C P B V D V [12]. H o w e v e r , the close h o m o l o g y b e t w e e n the e n d o g e n o u s a n d the s u p e r i n f e c t i n g viruses, w h i c h was the case o f the t w o s t r a i n s ( C P T V M - 2 a n d N C P N e w Y o r k - l ) used in this e x p e r i m e n t , a p p a r e n t l y is n o t the o n l y " k e y " for r e p r o d u c i n g the classical m u c o s a l disease in cattle c o n s i d e r i n g the n e g a t i v e results o f the tests r e p o r t e d herein. B o v i n e virus d i a r r h e a / m u c o s a l disease has to be r e g a r d e d i n c r e a s i n g l y as a real m u l t i f a c t o r i a l s y n d r o m e w h e r e several aspects o f the p a t h o g e n e s i s n e e d to be elucidated. P a r t i c u l a r a t t e n t i o n s h o u l d be d e d i c a t e d to the s t u d y o f the m u l t i f a c e t e d biologic p r o p e r t i e s o f the different b i o t y p e s o f the virus w h i c h e v e n t u a l l y will ease the w a y for a b e t t e r u n d e r s t a n d i n g o f the p a t h o g e n e s i s o f the disease. Acknowledgements--This work was financially supported by the Department of Public Health, Directory of
Veterinary Services, the Ministry of the University and Research (funds 40 and 60%) and by National Research Council (Bilateral Project, Italy/U.S.A.). The scientific advice of Dr W. B. Martin, Visiting Researcher, is greatly appreciated. REFERENCES 1. McClurkin A. W., Bolin S. R. and Coria M. F. Isolation of cytopathic and noncytopathic bovine viral diarrhea virus from spleen of cattle acutely and chronically affected with bovine viral diarrhea. J. Am. Vet. Med. Assoc. 186, 568-569 (1985). 2. Brownlie J., Clarke M. C. and Howard C. J. Experimental production of fatal mucosal disease in cattle. Vet. Rec. 114, 535 536 (1984). 3. Bolin S. R., McClurkin A. W., Cutlip R. C. and Coria M. F. Severe clinical disease induced in cattle persistently infected with noncytopathic viral diarrhea virus by superinfection with cytopathic bovine viral diarrhea virus. Am. J. Vet. Res. 46, 573-576 0985). 4. Potgieter L. N. D. Immunosuppression in cattle as a result of bovine viral diarrhea virus infection. Agri-practice--Viral/Immun. 9, 7-14 (1988). 5. Castrucci G., Frigeri F., Osburn B. l., Ferrari M., Sawyer M. M. and Aldrovandi V. A study of some pathogenetic aspects of bovine viral diarhea virus infection. Comp. Immun. MicrobioL infect. Dis. 13, 41-49 (1990). 6. Castrucci G., Frigeri F., Ferrari M. and Traldi V. The vaccination and challenge with bovine viral diarrhea virus (BVDV) of calves previously infected with a non-cytopathic BVDV. Comp. Immun. MicrobioL infect. Dis. 14, 31-38 (1991). 7. Castrucci G., Cilli V. and Gagliardi G. Bovine virus diarrhea in Italy. I. Isolation and characterization of the virus. Archs ges. Virusforsch. 24, 48~4 (1968). 8. Harkness J. W. The control of bovine viral diarrhea virus infection. Ann. Rech. V~t. 18, 167 174 (1987). 9. Avellini G., Castrucci G., Morettini B. and Cilli V. Bovine virus diarrhea in Italy. II. Reproduction of the disease. Archs ges. Virusforsch. 2,4, 65-75 (1968). 10. Harkness J. W., Roeder P. L. and Wood L. Mucosal disease in cattle. Vet. Rec. 115, 186 (1984). 11. Howard C. F., Brownlie J. and Clarke H. C. Comparison by the neutralisation assay of pairs of non-cytopathogenetic and cytopathogenetic strains of bovine virus diarrhoea virus isolated from cases of mucosal disease. Vet. MicrobioL 13, 361 369 (1987). 12. Moennig V., Frey H. R., Liebler E., Pohlenz J. and Liess B. Reproduction of mucosal disease with cytopathogenic bovine viral diarrhea virus selected in vitro. Vet. Rec. 127, 200~203 (1990).
0147-9571/92 $5.00+0.00 Copyright © 1992 Pergamon Press Ltd
A N E X P E R I M E N T A L C O N T R I B U T I O N TO THE S T U D Y OF THE P A T H O G E N E S I S OF BOVINE VIRAL DIARRHEA VIRUS INFECTION G. CASTRUCCI, I* B. I. OSBURN, 2 M. FERRARI 3 and V. TRALD14 qstituto di Malattie Infettive, Facoltfi di Medicina Veterinaria, Via S. Costanzo 4, 06100 Perugia, Italy, 2Department of Veterinary Pathology, University of California, Davis, CA 95616, U.S.A., 3Istituto Zooprofillatico Sperimentale della Lombardia e della Emilia, via A. Bianchi 7, 25100 Brescia, Italy and 4Centro Provinciale Svezzamento Vitelli, Tripoli S. Giorgio, Mantova, Italy Abstract--This presentation summarizes the results of a study on the pathogenesis of bovine viral diarrhea (BVDV) infection. The cytopathic (CP) strain TVM-2 of BVDV induced in calves an overt clinical disease which is usually recorded as the acute primary BVDV infection observed under natural conditions. In contrast the non-cytopathic (NCP) strain New York- 1 of BVDV did not cause any significant signs of disease. However, when the calves were immunosuppressed by treatment with dexamethasone (DMS) the biotype of BVDV involved did not seem to be as important as it appeared to be in an immunologically normal animal. This was shown in this study by the NCP BVDV which caused a fatal disease in calves treated with DMS. A mixed infection given to calves by injecting them with both CP and NCP BVDV, did not result in any particularly serious disease. So, the potential immunosuppressive activity of BVDV itself for the host has not been proven under the experimental procedures used in this experiment. Finally, a modified-live CP BVDV vaccine was unable to cause clinical disease when injected into calves that had been infected previously with strain New York-I of BVDV. Key words: Bovine viral diarrhea virus, infection, pathogenesis, calves.
UNE CONTRIBUTION EXPERIMENTALE A L ' E T U D E D E LA P A T H O G E N I E D E L ' I N F E C T I O N D U E A U V I R U S D E LA D I A R R H E E BOVINE VIRALE R r s u m ~ C e t t e prrsentation synthrtise les resultats d'une 6tude sur la pathogrnie de l'infection de la diarrhre bovine virale (BVDV). La souche cytopathique (CP) TVM-2 de BVDV a provoqu6 chez les veaux une maladie clinique manifeste qui est grnrralement enregistrre comme l'infection primaire aigu6 de BVDV observre dans les conditions naturelles. Par contre, la souche non cytopathique (NCP) New York-I de BVDV n'a provoqu6 aucun signe significatif de maladie. Toutefois, lorsque les veaux ont 6t6 soumis un traitement d'immunosuppression par dexam&hasone (DMS), le biotype de BVDV impliqu6 n'a pas sembl6 6tre aussi important qu'il parait l'&re chez un animal immunologiquement normal. Cela a 6t~ d~montr6 dans cette 6tude par la souche NCP de BVDV, qui a provoqu6 une maladie mortelle chez les veaux traitrs au DMS. Une infection mixte, provoqure en injectant au veaux tant la souche CP que la souche NCP de BVDV, n'a donn~ aucun maladie particulidrement grave. Ansi, l'activit6 immunosuppressive potentielle de BVDV lui-mrme pour l'hrte n'a pas 6t6 prouvre au cours des procrdures utilis~es pour cette exprrience. Enfin, l'inoculation, chez des veaux prralablement infectrs par la souche New York-I de BVDV, d'un vaccin vivant attrnu6 de la souche CP de BVDV n'a pu provoquer aucun maladie clinique. Mots-clefs: Virus de la diarrh~e bovine virale, infection, pathogrnie, veaux. *Author for correspondence. 163
164
G. CASTRUCCI et al.
INTRODUCTION In the last few years studies on the pathogenesis of bovine viral diarrhea virus (BVDV) infection have been concentrated mostly on the different roles of the two biotypes of the virus, one of which is non-cytopathogenic (NCP) and the other cytopathogenic (CP). Apparently, the time of infection and interplay between these biotypes are the most outstanding factors which eventually lead BVDV infections to produce clinical disease in cattle ranging from mild to fatal [1]. It was demonstrated that persistently viraemic calves, superinfected by CP BVDV, succumbed to mucosal disease [2, 3]. According to a number of experimental data it seems that immunosuppression plays a paramount role in the pathogenesis [4]. Considering that BVDV infection may cause one of the most economically important viral diseases of cattle, it seemed worthwhile to investigate some aspects of the pathogenesis of BVDV which apparently came out after the above mentioned observations. Accordingly, a study was planned to verify the possible involvement in the pathogenesis of BVDV infection of the following factors: (1) The biotype of the virus, i.e. CP or NCP and the influence of an immunosuppressive state of the host. (2) Simultaneous infection in the same host by both biotypes of the virus. (3) The effects of vaccination with a modified-live (ML) BVDV vaccine in cattle latently infected with a NCP strain of BVDV. Two previous papers [5, 6] together with the present paper give the findings made.
EXPERIMENTAL PROCEDURES--RESULTS Tissue culture
Primary or secondary cultures of bovine embryo kidney (BEK) cells were used throughout the study. They were grown in minimum essential medium (MEM) with Earle's balanced salt solution and L-glutamine, enriched with 10% fetal bovine serum and containing antibiotics (penicillin, 1000 U/ml, streptomycin, 500 p g/ml and amphotericin B, 2/~g/ml). The fetal bovine serum was replaced by horse serum at a concentration of 5% whenever the cultures were to be used for virus cultivation or for the recovery of the virus from the experimental animals. Virus
The NCP strain New York-1 (kindly supplied by Dr B. I. Osburn, University of California, Davis) and the CP strain TVM-2 [7] of BVDV, grown in BEK cells, were used. The New York-1 strain had an undetermined number of passages in tissue cultures and a titer, as detected by immunofluorescence (IF) in BEK cells, of l055 median tissue culture infectious doses (TCIDs0) per 1 ml. The TVM-2 strain was at its 6th passage level in BEK cell cultures and had a titer of 1065/ml. Viral isolation
Pharyngeal swabbings and buffy coat obtained from the blood were tested in the BEK cell cultures for the presence of the virus according to the procedures described previously [5]. Samples were considered to be negative for the virus if cytopathic effects (CPE) failed
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165
to appear or IF was negative after 2 serial subpassages of the inoculum had been made. The IF procedure [5] was used for the detection of virus in BEK cell cultures inoculated with the specimens collected from calves that had been exposed to NCP BVDV.
Serum neutralization Serum samples were serially diluted two-fold in MEM in 96-well cell culture microtitration plate. An equal volume (0.025 ml) of MEM containing 200 TCIDs0 of CP BVDV/0.025 ml was added to the wells. After incubation at 22°C for 1 hr, 20,000 BEK cells, in a volume of 0.05 ml, were added to each well: Plates were incubated at 37°C for 3 days, and each well was scored for the presence of CPE [5]. Experimental design (1) The biotype of BVD V and the immunosuppressive state of the host. This experiment was designed to answer the question, whether the clinical and virological findings made in calves might be influenced by the biotype of the virus responsible for the infection. The tests were carried out in 20 Friesian calves, 30--40 days of age, allotted into two groups of I0. In each group, 8 calves were inoculated with CP or NCP strains of BVDV, whereas the remaining 2 in either groups were given dexamethasone (DMS) in addition to the appropriate biotype of BVDV. The NCP and the CP BVDV strains were inoculated intravenously (i.v.), each calf receiving 5 ml of virus culture. The DMS was given to the calves i.v. for 4 consecutive days and each calf was injected with 0.1 mg/kg of body weight per day, the first injection being made a few hours before virus inoculation. Calves of each group were housed in isolation facilities. Calves were observed for 30 days for the presence of clinical signs. Daily white blood cell (WBC) counts were also made. Pharyngeal swabbings and blood samples were obtained for virus isolation at pre-determined intervals post-inoculation. Serum was collected from each calf on the day of infection (post-infection day 0) and on post-infection day (PID) 30. The calves inoculated with CP TVM-2 strain on BVDV (Table 1) all showed overt clinical disease, however, the clinical response was generally more severe in the case of Table 1. Clinical and virological findings in calves treated or not treated with dexamethasone (DMS) and exposed to experimental infection with cytopathic (CP) TVM-2 strain of bovine viral diarrhea virus (BVDV) Clinical signs
Virus recovery on PID
Calf No.
DMS treatment
Fever ~>40°C*
Nasal discharge*
Diarrhea*
Leukopenia*
PSt
LEt
141 132 133 134 135 136 193 194 191 192,
NT NT NT NT NT NT NT NT T T
3/4 2/2 3/7 3/5 5/6 2/7 3/6 5/3 5/10 4/4
3/7 2/7 2/4 2/10 3/7 2/6 3/6 5/3 6/9 6/1
4/1 4/1 2/5 6/1 3/5 2/6 7/5 7/7 5/13 5/3
2/6 2/5 2/7 2/6 2/10 3/10 3/6 3/5 10/4 6/2
2-11 2-11 2-11 2/11 2/11 2/11 2/6 6 2 2,6~
2 2, 6, 17 2, 17 11, 17 4, 11, 17 NI 2 2 2, 6 2,6~
*Onset, post infection day (PID)/duration days. tSamples (PS = pharyngeal swabbings; LE = buffy coat) were collected on PIDs 2 , 4 , 6 , 8 , 1 1 , 1 7 . 1Calf No. 192 died on PID 7. §Virus was also isolated from the lung and the spleen of this calf. NT = not treated; T = treated; NI = virus was not isolated.
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animals which were treated with DMS. The 8 calves which were given the virus only reacted with fever, nasal discharge, diarrhea and leukopenia and recovered completely in about 2 weeks. The two calves (Nos 191, 192) which in addition to the virus, were also given DMS, developed classical mucosal disease of which the most consistent sign was diarrhea. One calf (No. 192) died on PID 7, i.e. 3 days following the last injection of DMS. At necropsy the mesenteric lymph nodes appeared enlarged and the mucosa of the small intestine was slightly congested. Virus was consistently recovered from the pharyngeal swabbings of all calves, whereas virus recovery from the buffy coat was less regular. At PID 30 neutralizing antibody to BVDV was found at titers ranging from 1:32 to 1:128. In the case of those calves exposed to the NCP New York-1 strain of BVDV (Table 2), the 8 animals which were given the virus only did not show any clinical signs of disease, even if the virus was recovered from their pharyngeal swabbings and buffy coats. In contrast, the two calves which were simultaneously treated with DMS underwent a severe clinical disease. They had high fever (41~2°C), copious nasal discharge, very marked leukopenia and diarrhea. One calf (No. 150) died on PID 11 and the other (No. 149) on PID 17. The symptoms appeared on PIDs 5 6 and lasted until they died. The most serious sign was diarrhea. Initially, the fluid feces were yellowish; later they became brownish in colour and were mixed with large fragments of necrotic material. The two calves were markedly dehydrated when they died. At necropsy the mucosa of the upper digestive tract appeared congested, whereas superficial erosions were found in the mucosa of the small intestine. The mesenteric lymph nodes were hemorrhagic and enlarged. (2) Simultaneous infection in the same host by both biotypes of BVDV. Two Friesian calves of the same age as those of the previous group ( 3 0 4 0 days), were both injected simultaneously with CP and NCP BVDV. Assuming that BVDV is an immunosuppressive agent itself, the expectation from this test was that a simultaneous infection with the 2 biotypes of the virus would result in the classically fatal expression of mucosal disease. However, the clinical response of the 2 calves was analogous to that observed in the 8 calves that were inoculated with the CP TVM-2 strain of BVDV (Table 1) in that they reacted with fever, leukopenia and diarrhea. The symptoms appeared on PIDs 5-7 and lasted about 1 week. It seems, therefore, that the mixed infection did not result in any
Table 2. Clinical and virological findings in calves treated or not treated with dexamethasone (DMS) and exposed to experimental infection with noncytopathic (NCP) New York-1 strain of bovine viral diarrhea virus (BVDV) Clinical signs Calf No.
DMS treatment
Fever ~>40"C*
Nasal discharge*
Diarrhea*
Virus recovery on PIDt PSi.
LE~
162
NT')
Leukopenia*
2,6
6
163 164 165 166 167 151 152 149§ 150¶1
NT NT t NT NT NT NT NT T T
2,26 2, 6 2, 6 2, 6 2 11 2 11 2, 4, 11 2, 4, 8
22 6 2 2 2,6,8,11 2,6,8,11 2, 6, 8, I I 2, 6, 8
No clinical signs have been observed
5/10 6/5
6/8 6/2
6/10 6/5
6/8 6/5
*Onset, post infection day (PID)/duration days. "l'Virus detection was made by immunofluorescence. :~Samples (PS = pharyngeal swabbings; LE = buffy coat) were collected on PIDs 2, 4, 6, 8, 11. §Died on PID 17. ¶lDied on PID 11.
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167
particularly serious pathological situation like the one which is usually observed with the true mucosal disease: evidently neither BVDV biotype was sufficiently immunosuppressive for the host. (3) The effects of vaccination with M L CP B V D V vaccine in cattle latently infected with a N C P strain of B V D V [6]. One of the most common hypotheses on the pathogenesis of BVDV infection is that fatal mucosal disease could occur in animals persistently infected with a NCP strain of BVDV when they are superinfected with a homologous CP biotype of the virus [2, 3]. These findings raise the question whether cattle, which are persistently infected, would develop mucosal disease when vaccinated with a modified, live cytopathic BVDV vaccine. Accordingly, the purpose of this experiment was to verify the effects of vaccination in calves which were previously infected with NCP BVDV. Six Friesian calves, approx. 3 month old, which were determined to be free of neutralizing antibody to BVDV [6], were used. Latent infection was established in four calves by i.v. inoculation of NCP New York-I strain of BVDV, each calf receiving 10.0 ml of virus culture. Thirty days later the four latently infected calves and two additional uninfected calves (vaccine control), were each inoculated intramuscularly (i.m.) with 2.0 ml of a commercial vaccine containing a CP strain of BVDV 005.5 TCIDs0/ml ), and in addition infectious bovine rhinotracheitis (IBR) (1065 TCIDs0/ml ) and parainfluenza-3 (PI-3) (1055 TCIDs0/ml ) viruses. The animals were observed for 73 days from the start of the experiment. Their temperatures were taken daily for at least 25 days following the inoculation with the NCP strain of BVDV, as well as after vaccination. WBC counts were also made daily. Pharyngeal swabbings and blood samples were obtained for virus isolation and also samples of serum were collected from each calf for serology. As shown in Table 3, with the exception of a slight leukopenia, the four calves exposed to NCP BVDV did not develop any signs of disease during the 30 days between infection and the subsequent vaccination. However, the virus was consistently detected by IF in the BEK cell cultures inoculated with the pharyngeal swabbings and the buffy coats of each calf and this indicated that the infection with NCP virus was established. Moreover, all the calves at PID 30 had neutralizing antibody to BVDV at titers ranging from 1 : 8 to 1 : 64, depending on the calf.
Table 3. The response of calves to experimental infection with noncytopathic (NCP) strain of bovine viral diarrhea virus (BVDV) and to subsequent vaccination with a modified-live (ML) cytopathic (CP) BVDV vaccine Inoculation of NCP BVDV
Vaccination with ML CP BVDV
Virus isolation on PID* Calf No. 3 4 5 6
"] I
Vaccine controls 11 12
Clinical response Leukopenia only which appeared on PID 3-5 and lasted 2~. days NA NA
PSI"
LEt
4, 4, 4, 4,
4, 4, 4, 4,
16 16 16 16
NA NA
16 16 16 16
NA NA
Virus isolation on PVD Clinical response
PSt
LEt
None None Slight leukopenia Slight leukopenia
0, 6, 14 0, 6 0, 6, 14 0, 6
0, 6, 14 0, 6, 14 0, 6 0, 6, 14
Slight leukopenia Slight leukopenia
6, 14 6
6 6, 14
*Virus detection was made by immunofluorescence. tSamples (PS = pharyngeal swabbings; LE = buffy coat) were collected on post infection day (PID) 4 and 16 or on post vaccination day (PVD) 0 (the day of vaccination) and on PVDs 6 and 14. When PVDs are underlined it means that CP BVDV virus was isolated. NA = not applicable.
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G. CASTRUCCI et al.
After vaccination with a vaccine containing a CP strain of BVDV (Table 3), the calves previously infected with NCP BVDV, did not show any significant signs of disease within the 43 days during which the calves were kept under observation. Only two calves (Nos 5 and 6) became leukopenic on post vaccination day (PVD) 3; however, their WBC counts were back to the normal range within 2-3 days. A similar picture was observed in the controls which were leukopenic for 3-4 days. BVDV was isolated from pharyngeal swabbings and buffy coats of all calves. The four calves which had been infected 30 days before vaccination, proved to be still infected on the day they were vaccinated (PVD 0 in Table 3). BVDV was subsequently detected by IF in the specimens obtained from the calves on PVDs 6 and 14. From the control calves CP BVDV of vaccine origin, was isolated. IBR virus, one of the components of the vaccine, was occasionally isolated from pharyngeal swabbings and from the buffy coats of all calves. PI-3, the other viral component of the vaccine was never isolated, nor was the viral antigen detected by hemadsorption tests in any of the cultures inoculated with pharyngeal swabbings or the blood samples obtained from the calves.
CONCLUSIONS One of the objectives of this work was to verify whether the biotype of the virus, i.e. CP or NCP, could influence the clinical response of calves to BVDV infection. Based on the results [5], the CP TVM-2 strain of BVDV induced in calves clinical signs of disease which included depression, fever, leukopenia, respiratory symptoms and diarrhea. Thus, the CP virus reproduced in calves the clinical picture which is usually reported for the acute primary BVDV infection observed under natural conditions. In contrast, the NCP New York-1 strain did not cause in calves any significant signs of disease, even if virus was recovered from the specimens obtained from the inoculated calves. These findings support the evidence for a potential diversity among BVDV isolates in their pathogenetic pathway toward the host. The possibility of the existence of a close association between BVDV strains and the clinical development of the disease has been already suggested by others [8]. However, when the animal is immunosuppressed, the biotype of BVDV involved does not seem to be as important as it might be in an immunologically normal animal. This was proved in the present study by the NCP strain which caused a fatal disease in the DMS treated calves while it was unable to give rise to overt disease in calves that were not immunosuppressed. Apparently, the immunosuppression could really be considered one of the."key factors" regulating the pathogenetic mechanism of bovine virus diarrhea/ mucosal disease [4]. On the other hand, the potential immunosuppressive activity of BVDV itself for the host, has not been proven under the experimental conditions adopted in these tests. Evidence of this is that the dual infection, i.e. the CP and NCP BVDV did not result in more serious diseases than produced by the CP virus alone [5, 9]. In the present experiment vaccination, with a modified-live CP BVDV vaccine, of calves previously infected with a NCP strain of BVDV did not cause mucosal disease. In a similar study others [3] failed to induce mucosal disease in persistently infected cattle by vaccination with M L CP BVDV. Failure to reproduce experimental mucosal disease by superinfection with CP BVDV was also reported [10]. It has been suggested that only CP BVDV with an antigenic structure identical to that of the endogenous NCP BVDV would be capable of inducing the disease [11]. This was recently confirmed when others succeeded
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169
in r e p r o d u c i n g acute, severe disease u s i n g pairs o f closely r e l a t e d N C P a n d C P B V D V [12]. H o w e v e r , the close h o m o l o g y b e t w e e n the e n d o g e n o u s a n d the s u p e r i n f e c t i n g viruses, w h i c h was the case o f the t w o s t r a i n s ( C P T V M - 2 a n d N C P N e w Y o r k - l ) used in this e x p e r i m e n t , a p p a r e n t l y is n o t the o n l y " k e y " for r e p r o d u c i n g the classical m u c o s a l disease in cattle c o n s i d e r i n g the n e g a t i v e results o f the tests r e p o r t e d herein. B o v i n e virus d i a r r h e a / m u c o s a l disease has to be r e g a r d e d i n c r e a s i n g l y as a real m u l t i f a c t o r i a l s y n d r o m e w h e r e several aspects o f the p a t h o g e n e s i s n e e d to be elucidated. P a r t i c u l a r a t t e n t i o n s h o u l d be d e d i c a t e d to the s t u d y o f the m u l t i f a c e t e d biologic p r o p e r t i e s o f the different b i o t y p e s o f the virus w h i c h e v e n t u a l l y will ease the w a y for a b e t t e r u n d e r s t a n d i n g o f the p a t h o g e n e s i s o f the disease. Acknowledgements--This work was financially supported by the Department of Public Health, Directory of
Veterinary Services, the Ministry of the University and Research (funds 40 and 60%) and by National Research Council (Bilateral Project, Italy/U.S.A.). The scientific advice of Dr W. B. Martin, Visiting Researcher, is greatly appreciated. REFERENCES 1. McClurkin A. W., Bolin S. R. and Coria M. F. Isolation of cytopathic and noncytopathic bovine viral diarrhea virus from spleen of cattle acutely and chronically affected with bovine viral diarrhea. J. Am. Vet. Med. Assoc. 186, 568-569 (1985). 2. Brownlie J., Clarke M. C. and Howard C. J. Experimental production of fatal mucosal disease in cattle. Vet. Rec. 114, 535 536 (1984). 3. Bolin S. R., McClurkin A. W., Cutlip R. C. and Coria M. F. Severe clinical disease induced in cattle persistently infected with noncytopathic viral diarrhea virus by superinfection with cytopathic bovine viral diarrhea virus. Am. J. Vet. Res. 46, 573-576 0985). 4. Potgieter L. N. D. Immunosuppression in cattle as a result of bovine viral diarrhea virus infection. Agri-practice--Viral/Immun. 9, 7-14 (1988). 5. Castrucci G., Frigeri F., Osburn B. l., Ferrari M., Sawyer M. M. and Aldrovandi V. A study of some pathogenetic aspects of bovine viral diarhea virus infection. Comp. Immun. MicrobioL infect. Dis. 13, 41-49 (1990). 6. Castrucci G., Frigeri F., Ferrari M. and Traldi V. The vaccination and challenge with bovine viral diarrhea virus (BVDV) of calves previously infected with a non-cytopathic BVDV. Comp. Immun. MicrobioL infect. Dis. 14, 31-38 (1991). 7. Castrucci G., Cilli V. and Gagliardi G. Bovine virus diarrhea in Italy. I. Isolation and characterization of the virus. Archs ges. Virusforsch. 24, 48~4 (1968). 8. Harkness J. W. The control of bovine viral diarrhea virus infection. Ann. Rech. V~t. 18, 167 174 (1987). 9. Avellini G., Castrucci G., Morettini B. and Cilli V. Bovine virus diarrhea in Italy. II. Reproduction of the disease. Archs ges. Virusforsch. 2,4, 65-75 (1968). 10. Harkness J. W., Roeder P. L. and Wood L. Mucosal disease in cattle. Vet. Rec. 115, 186 (1984). 11. Howard C. F., Brownlie J. and Clarke H. C. Comparison by the neutralisation assay of pairs of non-cytopathogenetic and cytopathogenetic strains of bovine virus diarrhoea virus isolated from cases of mucosal disease. Vet. MicrobioL 13, 361 369 (1987). 12. Moennig V., Frey H. R., Liebler E., Pohlenz J. and Liess B. Reproduction of mucosal disease with cytopathogenic bovine viral diarrhea virus selected in vitro. Vet. Rec. 127, 200~203 (1990).
