Comp. lmmun. Microbiol. infect. Dis. Vol. 15, No. 4, pp. 261-270, 1992 Printed in Great Britain. All rights reserved
0147-9571/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press Ltd
EFFECTS IN CALVES OF MIXED INFECTIONS WITH BOVINE VIRAL DIARRHEA VIRUS A N D SEVERAL OTHER BOVINE VIRUSES G. CASTRUCCI, I* M. FERRARI, 2 V. TRALDI 3 and E. TARTAGLIONE 4 ~lstituto di Malattie lnfettive, Profilassi e Polizia Veterinaria, Laboratorio Universitario di Virologia "V. Cilli", Universita di Perugia, 2c/o Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia, Brescia, 3Centro Provinciale Svezzamento Vitelli, Tripoli S. Giorgio, Mantova and 4Unit~i Sanitaria Locale, Venafro, lsernia, Italy Abstract--The objective of this study was to verify whether a mixed infection in calves with bovine viral diarrhea virus (BVDV) and other bovine viruses, such as bovid herpesvirus-4 (BHV-4), parainfluenza-3 (PI-3) and infectious bovine rhinotracheitis (IBR) virus, would influence the pathogenesis of the BVDV infection sufficiently to result in the typical form o f mucosal disease being produced. Accordingly, two experiments were undertaken. In one experiment calves were first infected with BVDV and subsequently with BHV-4 and IBR virus, respectively. The second experiment consisted in a simultaneous infection o f calves with BVDV and PI-3 virus or BVDV and IBR virus. From the first experiment it seems that BVDV infection can be reactivated in calves by BHV-4 and IBR virus. Evidence of this is that BVDV, at least the cytopathic (CP) strain, was recovered from calves following superinfection. Moreover, following such superinfection the calves showed signs which could most likely be ascribed to the pathogenetic activity of BVDV. Superinfection, especially by IBR virus, created a more severe clinical response in calves that were initially infected with CP BVDV, than in those previously given the non-cytopathic (NCP) biotype of the virus. Simultaneous infection with PI-3 virus did not seem to modify to any significant extent the pathogenesis o f the experimentally induced BVDV infection whereas a severe clinical response was observed in calves when simultaneous infection was made with BVDV and IBR virus. Key word~: Bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, bovid herpesvirus-4, parainfluenza-3 virus, mixed infections, calves. R6sum6--L'objectif de cette 6rude 6tait de v6rifier si une infection mixte chez des veaux provoqu6e par le virus de la diarrh6e bovine virale (BVDV) et d'autres virus bovins, tels que rherpesvirus-4 du bovin (BHV-4), le virus parainfluenza-3 (PI-3) et de la rhinotrach6ite bovine infectieuse (IBR), influenceraient assez la pathog6nie de rinfection par BVDV pour en arriver fi produire la forme typique de la maladie des muqueuses. Deux exp6riences ont 6t6 entreprises en ce sens. Dans l'une, les veaux ont tout d'abord 6t6 soumis fi l'infection par BVDV, puis, respectivement, par BHV-4 et le virus IBR. La deuxi6me experience consistait en une infection simultan6e des veaux au moyen de BVDV et du virus PI-3 ou de BVDV et du virus IBR. D'apr6s la premiere exp6rience, il semble que rinfection par BVDV puisse 6ire r6activ6e chez les veaux par BHV-4 et le virus IBR. Une preuve e n e s t que BVDV, ou tout au moins la souche cytopathique (CP), a 6t6 retrouv6e chez les veaux suite ~i la superinfection. En outre, ,,i la suite de chaque surinfection, les veaux ont montr6 des signes pouvant tr6s vraisemblablement ~tre imput6s ~i I'activit6 pathog6nique de BVDV. La superinfection, particuli6rement par le virus IBR, a provoqu6 une r6ponse clinique plus grave chez les veaux quit avaient initialement 6t~ infect6s par CP BVDV que chez ceux qui avaient regu le biotype non cytopathique (NCP) du virus. L'infection simultan6e par le virus PI-3 ne semble pas avoir modifi6 de fagon significative la pathog6nie de rinfection par BVDV exp~rimentalement provoqu~e, alors qu'une r~ponse clinique grave a 6t6 observ6e chez les veaux lors de I'infection simultange par BVDV et le virus IBR. Mots clefs: Virus de la diarrh6e bovine virale, virus de la rhinotrachgite bovine infectieuse, herpesvirus-4 du bovin, virus parainfluenza-3, infection mixte, veaux.
*Author for correspondence: Istituto di Malattie Infettive, Facoltfi di Medicina Veterinaria, Via S. Costanzo 4, 06100 Perugia, Italy. 261
262
G. CASTRUCC1 et al.
INTRODUCTION The biology of bovine viral diarrhea virus (BVDV) is complex, so that multiple and diverse clinical manifestations may occur in cattle infected with the virus. It is generally known that the most common forms of infection are subclinical or mild [I]. However, the virus is also responsible for a sporadic form of disease, known as "mucosal disease", which generally involves cattle ranging in age from 6 months to 2yr. Mucosal disease is characterized by severe clinical signs, low morbidity and a fatality rate approaching 100%. According to several studies on the pathogenesis of BVDV infection, mucosal disease occurs in cattle that are immunotolerant and viraemic with non-cytopathic (NCP) virus when they become superinfected with a cytopathic (CP) strain of BVDV which is antigenically closely related to the persistently infecting NCP strain [2--4]. However, in other studies [5, 6] the CP strains failed to induce mucosal disease in persistently infected cattlc and it was suggested that only some combinations of biotypes of the virus may cause mucosal disease. Considering that bovine viral diarrhea-mucosal disease has to be regarded as a multifactorial syndrome [7], several factors, many of which are still unknown, could be involved in altering the pathogenesis of the disease. Among the factors which appear worthy of some consideration is the possibility that a mixed infection could play an important role in the pathogenesis of BVDV infection. It is possible that certain infectious agents could provide the required conditions for BVDV to produce typical mucosal disease in cattle. The purpose of the present study therefore, was to determine whether the clinical response to BVDV infection might be altered by the intervention of other viral infections affecting cattle, such as those induced by infectious bovine rhinotracheitis (IBR), parainfluenza-3 (PI-3) viruses, and bovid herpesvirus-4 (BHV-4).
MATERIALS AND METHODS (?ell cultures
Secondary cell cultures from bovine embryo kidney (BEK) were prepared by standard methods and were grown in minimum essential medium (MEM) with Earle's salts supplemented with 5% (v/v) bovine fetal serum and containing antibiotics (penicillin, 1000 U/ml, streptomycin, 500 #g/ml and amphotericin B, 2 pg/ml. Viruses
BVDV. Two strains of the virus have been used, i.e. the NCP New York-I strain (kindly supplied by Dr Osburn, University of California, Davis, Calif., U.S.A.) and the CP TVM2 strain [8]. The NCP virus had an undetermined number of passages in tissue culture and its titer, as found by immunofluorescence in BEK cells, was 105~ median tissue culture infectious doses (TCID~o) per 1 ml. The CP strain was used at the 6th BEK cell cultures passage and contained 1065 TCIDso/ml. IBR virus. The field isolate 90/180 TN (Castrucci, unpublished data) was selected for the study. The virus was at its 2nd passage level in BEK cell cultures with a titer of 10s5 TCIDso/ml. PI-3. This virus was represented by the field isolate 90/1 TN (Castrucci, unpublished data), at the 4th BEK cell cultures passage and had a titer of 10s.s TCIDs0/ml.
M i x e d i n f e c t i o n s in calves with b o v i n e viral d i a r r h e a virus a n d o t h e r b o v i n e viruses
263
BHV-4. The virulent strain 81/16 TV [9] at the 4th passage in BEK cell cultures and a titer of 106.5 TCIDso/ml, was used. Experimental design: inoculation of calves Sixteen Friesian calves, 30-40 days of age, were used throughout the experiment. They were without detectable serum neutralizing antibodies to the viruses under study. As shown in Table I, the calves were allotted to 5 groups of 2 each. An additional control group of 6 calves was added at the appropriate time during the experiment. The calves of the first two groups were inoculated sequentially as follows: first with NCP (group I) or CP (group 2) BVDV, then, 40 days later, calves of both groups received BHV-4. Finally, 32 days after the inoculation of the second virus, all calves were given IBR virus. The calves of groups 3, 4 and 5, were simultaneously infected with NCP BVDV (group 3) or CP BVDV (group 4) and IBR virus or, in the case of group 5, with NCP BVDV (i calf) or CP BVDV (I calf) together with PI-3 virus. Of the control calves of group 6, two were given BHV-4, 2 were inoculated with IBR virus and 2 with PI-3 virus at the time these viruses were given to the experimental calves. The CP and NCP BVDV, were inoculated intravenously (i.v.), each calf receiving 5 ml of virus. The other 3 viruses (IBR, PI-3 and BHV-4) were given by nasal spray in a volume of 5 ml per calf. The calves were observed for 30 days after administration of each virus and their temperatures were taken daily. Daily white blood cells (WBC) counts were also made. Nasal swabbings and blood samples were obtained at predetermined intervals post-inoculation. Samples of serum were also collected from each calf for serology.
Evaluation of clinical response As the purpose of the present study was to investigate the possibility that a mixed infection would eventually lead to a severe clinical form of mucosai disease, an inventory of the main clinical signs of the disease was made. The list incuded the following signs: depression, fever, leukopenia, cough, nasal discharge, lacrimation, dyspnoea, hypersalivation, diarrhea and mouth lesions. The grade of severity of the clinical response was indicated as follows: - , no clinical signs; + , mild reaction with at least two main clinical Table I. Experimental design: inoculation of calves Calves
Simultaneous infection
Sequential infections Group No.
No. of animals
I 2 3 4
2 2 2 2 I I
5
.......... Ist Virus
] t
2nd Virus*
3rd Virus*
Viruses
NCP BVDV CP BVDV
BHV-4 BHV-4
IBR IBR
NA
NA
NA
NA NA NCP BVDV + IBR CP BVDV t- IBR NCP BVDV + PI-3 CP BVDV ~ PI-3
NA NA NA
BHV-4 NA NA
NA NA NA
Controls f
2 2 2
NA IBR PI3
*40 days after the Ist infection. *32 days after the 2nd infection. BVDV = bovine viral diarrhea virus. NCP = non cytopathic or CP = cytopathic biotype of BVDV. BHV-4 = bovid herpesvirus-4. IBR = infectious bovine rhinotracheitis virus. Pl-3 = parainfluenza-3 virus. NA = not applicable.
264
G. CASTRUCCI et al.
signs; + + , reaction with more than two clinical signs of moderate intensity; + + + , several clinical signs of moderate to severe intensity, + + + + , severe clinical reaction.
Viral isolation The nasal swabbings were expressed into 2 ml of MEM. The blood was collected in tubes containing a chelating agent (EDTA-Vacutainer tubes, Becton-Dickson), and buffy coat obtained by centrifugation at 600g for 30 min. The fluid from each swab was homogenized in a glass grinder, centrifuged at 850g for 10min and the supernatant fraction was inoculated on to monolayers of BEK cells grown in wells in plastic plates. The buffy coat samples were frozen and thawed once and then inoculated on to BEK cell cultures as indicated previously. Samples were considered to be negative if cytopathic effects (CPE) failed to appear or immunofluorescence (NCP BVDV) was negative after two serial subpassages of the inoculum had been made. Any virus recovered from the samples was identified by serum neutralization tests. In the case of calves subjected to a mixed infection when their samples were positive for a virus, the virus was neutralized with the appropriate specific antiserum, in order to detect any other virus which might also be present in the sample.
Immunofluorescence (IF) The IF procedure was used when the detection of NCP BVDV in the cultured samples obtained from the inoculated calves was involved. A reference antiserum to BVDV conjugated with fluorescein-isothiocianate was used as previously described [10].
Serum neutralization Serial dilutions of each serum were mixed with 100 TCIDso of the appropriate virus (the CP strain of BVDV, IBR, PI-3 or BHV-4) in 96-well microtiter plates, using one well for each serum-virus dilution. The plates were held for 90 min at room temperature (22'C) and then 20,000 BEK cells, in 0.05 ml vol were added to each well. Neutralization titers were expressed as the highest dilution inhibiting cytopathology. RESULTS
(1) Response of calves to sequential infections: BVDV, BHV-4 and IBR virus (a) BVDV infection. As shown in Table 2, when calves were subjected to BVDV infection, they reacted as previously observed with the strains used in this experiment [10]. Thus, the two calves inoculated with the NCP strain of BVDV (Nos 45 and 47), did not show any clinical signs of the disease with the exception of a slight leukopenia on post-infection day (PID) 2 which lasted for 5-7 days. On the other hand, as expected, the calves Nos 49 and 50, which were given the CP TVM2 strain of BVDV had a moderate ( + + ) response, characterized by fever, leukopenia, nasal discharge and diarrhea. The two calves recovered from the experimental infection in about 10 days. Virus was recovered (Table 3) from buffy coat and the nasal swabbings of all four claves, from PID 2 to 7, the only exception being the calf No. 47, inoculated with NCP BVDV, which was negative on PID 2. Neutralizing antibodies were present at PID 30 at titers between 1:32 and 1: 128. Co) 1st Superinfection: BHV-4. When the four calves which had experienced BVDV infection were superinfected with BHV-4, a variation in response was observed depending
M i x e d i n f e c t i o n s in c a l v e s with b o v i n e viral d i a r r h e a v i r u s a n d o t h e r b o v i n e v i r u s e s
265
Table 2. The evaluation of the grade of severity of the clinical response in calves exposed to sequential infections with bovine viral diarrhea virus (BVDV), bovid herpesvirus-4 (BHV-4) and infectious bovine rhinotracheitis (IBR) virus Clinical response:l: Calves Group No.
BVDV infection
Superinfection with
No.
NCP
CP
BHV-4*
IBRt
I I 2 2
45 47 49 50
-NA NA
NA NA *+ ++
+ + ++ ++
+ + + + + + +t-++ ++++
Controls 6 6 6
51 52 55
NA NA NA
NA NA NA
+ + NA
NA NA + +
*BHV-4 was inoculated to calves 40 days after BVDV infection. $1BR virus was inoculated to calves 32 days after BHV-4 infection. ~Clinical response: absence of signs - ; mild +; moderate + +; moderate to severe + + +; severe + + + + . NCP = non cytopathic or CP = cytopathic biotype of BVDV. NA = not applicable.
on the biotype of the BVDV (NCP or CP) given earlier. As shown in Table 2, the calves first inoculated with NCP BVDV had a mild clinical response ( + ) of the kind generally observed with BHV-4 infection [1 I]. Thus, the calves had nasal discharge and were slightly depressed for about 1 week after superinfection. However, they showed few signs which could be related to BVDV infection, with only a slight leukopenia and a moderate diarrhea observed on PID 5-6, both of which lasted 2-3 days. The clinical response to BHV-4 was more evident in the calves previously given CP BVDV and was considered to be of moderate intensity ( + +). In this case, the animals reacted with all signs usually reported for calves experimentally infected with the virulent strain 85/16 TV of BHV-4, such as fever, depression and nasal discharge, but in addition they showed also the typical signs of BVDV infection. The latter were represented by a marked leukopenia and diarrhea. The leukopenia started on PID 2 and lasted 10-14 days with the lowest WBC count (1900-2000) being registered on PID 7-9. Diarrhea consisted of watery feces which occasionally appeared blood-stained, on PID 7-8 and lasted, in both calves, for 4 days. The two calves which served as control to the BHV-4 infection (Nos 51 and 52), had a mild clinical response ( + ) , similar to the calves of Group 1. BHV-4 was recovered (Table 3) from the nasal swabs of all the inoculated calves, but only on PID 2 from those calves which were previously given BVDV, whereas it was consistently recovered from the controls until PID 12. Following BHV-4 infection NCP BVDV was never recovered from the inoculated calves, whereas the CP BVDV was isolated on PID 7 and 9 from the buffy coats obtained from the two calves previously infected with CP BVDV. No significant rise in the antibody titers to BVDV was observed after superinfection with BHV-4. The immunologic reaction of calves to BHV-4, in terms of neutralizing antibody production, as usually [1 I] was very poor, the average titer being 1:4. (c) 2nd Superinfection: IBR virus. The superinfection with IBR virus induced a moderate to severe reaction ( + + + , as indicated in Table 2) in the two calves which were first inoculated with NCP BVDV (Nos 45 and 47). The calves developed signs that could be considered as typical of either BVDV or IBR virus infections, i.e. fever, nasal discharge
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266
Table 3. Virus recovery in calves exposed to sequential infections with bovine viral diarrhea virus (BVDV), bovid herpesvirus-4 (BHV-4) and infectious bovine rhinotracbeitis virus {IBR) Virus recovery* After BVDV infection
Calves Group No,
BVDV LE NS
After BHV-4 infection
No.
First infection
BVDV LE NS
I I 2 2
45 47 49 51)
N ( ' P BVDV N C P BVDV CP BVDV CP BVDV
2-7~" 7 2 7 2 7
2 7 7 2-7 2-7
7, 9 7. 9
Controls 6 6 6
51 52 55
BHV-4 BHV-4 IBR virus
NA NA NA
NA NA NA
NA NA NA
BHV-4 I,E NS
BVDV LE NS
----
2 2 2 2
10 -10
2 12 2-12 NA
NA NA NA
. . . .
NA NA NA
After IBR virus infection
-NA
BHV-4 LE NS
--
IBR LE m
.-
.--
NA NA NA
NA NA NA
NA NA NA
NA NA 2.12
NS 3.6 3.6 3,6 3.6 NA NA 212
*Virus recovery was made in bovine embryo kidney cell cultures and samples (LE = buffy coat; NS = nasal swabbings) were obtained from calves on post infection days: 2.3, 6. 7 . 9 and 12. N C P = non cytopathic or CP = cytopathic biotype of BVDV. NA = not applicable. - - Virus was not recovered. +The numbers indicate the post infection day when the virus was recovered.
and salivation. The two calves which were previously infected with CP BVDV (Nos 49 and 50), when superinfected with IBR virus showed a wider variety of clinical signs, compared to the calves previously given NCP BVDV. Here the clinical response was more severe ( + + + + , as indicated in Table 2) and signs of both IBR virus and BVDV infection were seen. Both the affected calves had high fever, marked leukopenia, heavy nasal discharge, profuse salivation, watery diarrhea, coughing, dyspnoea and viscous oculo-conjunctival discharge. The calf which served as control of IBR virus infectivity (No. 55), had a typical clinical response as recognized for experimentally induced IBR. Thus, the calf developed a febrile reaction and respiratory symptoms, such as nasal discharge, dyspnoea and cough, but recovered in about 10 days. The IBR virus was recovered (Table 3) from nasal swabbings of calves on PID 3 and 6, when all samples were negative for BVDV. However, BVDV was recovered from the buffy coat of one calf previously infected with NCP BVDV (No. 45) and from one of the two pre-infected with CP BVDV (No. 49). No IBR virus was isolated at this time of infection (PID 10). IBR virus was isolated from the nasal swabbings and from the blood of the control from PID 2 until 12. After IBR superinfection, no significant modification was observed in the antibody titers to either BVDV or BHV-4, as determined by neutralization tests carried out on the serum samples collected 30 days after superinfection. At this time, neutralizing antibody to IBR virus was present at titers ranging from i : i 6 to 1:64. (2) Response o f cah, es to simultaneous injection: B VD V 4- I B R virus or B VD V 4- P I - 3 virus (a) B VD V 4- I B R virus. When the calves were subjected to simultaneous infection with BVDV and IBR virus, they developed a severe clinical response ( + + + + , in Table 4) without any difference being observed between calves that were given the CP or the NCP strain of BVDV. All calves showed clinical signs similar to those described for calves Nos 49 and 50 (Table 2), subjected to sequential infections with CP BVDV, BHV-4 and IBR virus. Thus, they reacted with high fever, mucoid nasal discharge, copious salivation, intense diarrhea and a marked leukopenia from which they recovered very slowly, so that after three weeks from the time of onset of the clinical illness, their general condition was
Mixed infections in calves with bovine viral diarrhea virus and other bovine viruses
267
Table 4. The evaluation o f the grade o f severity o f the clinical response in calves exposed to simultaneous infection with bovine viral diarrhea virus (BVDV) and infectious bovine rhinotracheitis (1BR) virus or BVDV and parainttuenza-3 (PI-3) virus Calves Group No.
Clinical response*
No.
N C P BVDV + I B R virus
CP BVDV + I B R virus
N C P BVDV + P I - 3 virus
CP BVDV + P I - 3 virus
IBR virus only
PI-3 virus only
3 3 4 4 5 5
34 53 31 54 29 28
+ + + + + + ~- + NA NA NA NA
NA NA ++ 4 + + + + + NA NA
NA NA NA NA -~ NA
NA NA NA NA NA #- +
NA NA NA NA NA NA
NA NA NA NA NA NA
Controls 6 6 6
30 27 56
NA NA NA
NA NA NA
NA NA NA
NA NA NA
+ -'r NA NA
NA -
*Clinical response: absence o f signs - ; mild + ; moderate + + ; moderate to severe + + + ; severe + + + + . N C P = non cytopathic or CP = cytopathic biotype o f BVDV. N A = not applicable.
still very poor. The calf which was given IBR virus only (No. 30), had a milder clinical response, characterized by fever, nasal discharge, slight salivation and coughing. Neither ieukopenia nor diarrhea was observed in this calf. The only virus recovered (Table 5) from the first samples taken on PID 3 from the calves was IBR virus, which was isolated from all calves either from buffy coats or nasal swabbings. BVDV was first isolated on PID 7 from either buffy coats or nasal swabbings from both calves which were given the N C P BVDV and from one calf inoculated with the CP biotype of BVDV. The last isolation of IBR was obtained from nasal swabbings and buffy coats at PID 7, when it was recovered from the two calves which were coinfected with CP BVDV and from one calf coinfected with the N C P biotype of BVDV. BVDV was recovered also on P I D 24 from the buffy coat of all inoculated calves. The calves produced neutralizing antibody to either BVDV and IBR virus. The antibody titers to BVDV were in the range 1:32 to 1 : 128, whereas the maximum titer found for IBR virus was 1: 16. The control calf, inoculated with IBR virus only, did not produce antibody to BVDV, but 30 days after infection it had a titer to IBR virus of 1:8. (b) B V D V + P I - 3 virus. Simultaneous infection with BVDV and PI-3 virus induced in calves a clinical response which was considered mild ( + ) in the case of the calf coinfected with N C P BVDV (calf No. 29) or of moderate intensity ( + + ) in the other calf which was coinfected with the CP BVDV (calf No. 28). Both calves were afebrile but became leukopenic and had a nasal discharge and hypersalivation. Although the two calves showed the same signs, these were more pronounced and lasted longer in the calf which was coinfected with the CP biotype of BVDV. No clinical signs were observed in the calves that were inoculated with PI-3 virus only (control calves, Nos 27 and 56). BVDV was recovered (Table 5) from the buffy coats of the calves on PID 7 ( N C P BVDV) or 9 (CP BVDV). PI-3 virus was reisolated from the nasal swabbings of the two calves coinfected with BVDV on PID 3 till PID 12. The virus was also recovered from the nasal swabbings obtained from the control calves on PIDs 3, 6 and 12. At PID 30 neutralizing antibody to PI-3 virus was found at titers of I : 8 and 1: 16 in the two calves coinfected with CP BVDV or N C P BVDV. The two calves produced neutralizing antibody also to BVDV at a titer of 1:64. The serum of the control calves neutralized PI-3 virus at an average titer of 1:128, but failed to neutralize BVDV at the lowest dilution tested of 1:4. C I M I D 15,4 -C
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Table 5. Virus recovery in calves exposed to simultaneous infection with bovine viral diarrhea virus (BVDV) and infectious bovine rbinotracheitis (IBR) virus or BVDV and parainfluenza-3 (Pl-3) virus Virus recovery after simultaneous infection* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Infected calves Group No.
BVDV and Pl-3 virus
B V D V and IBR virus
No,
N C P BVDV LE NS
CP BVDV LE NS
IBR virus LE NS
N C P BVDV LE NS
C P BVDV LE NS
PI-3 ~irus LE NS
NA
NA
N C P B V D V + IBR virus 3 3
34 53
7, 24~" 7. 24
7 7
NA NA
NA NA
3, 7 3
3, 7 3
NA NA
NA NA
7, 24 24
7
3, 7 3, 7
3. 7 3, 7
"1
C P B V D V + IBR virus 4 4
31 54
NA
N C P B V D V t- Pl-3 virus 7 5 29 t C P B V D V + Pl-3 virus 5
NA
NA
NA
....
3 12
NA
28
NA
NA
9
3 12
Controls IBR virus 6
3O
NA
NA
27 56
NA NA
NA NA
3 12
3 12
NA
NA
NA NA
NA NA
NA
PI-3 virus 6 6
NA NA
-.
3. 6. 12 3, 6. 12
*Virus recovery was made in bovine embryo kidney cell cultures and samples (I.E = buffy coat; N S = nasal swabbings) were obtained on post infection days: 3, 6. 7, 12 and 24. N C P = non cytopathic or C P = cytopathic biotype of BVDV. eThe numbers indicate the post infection day when the virus was recovered. - Virus was not recovered. N A = not applicable.
DISCUSSION The aim of this study was to verify whether the intervention of other bovine viruses would influence the pathogenesis of B V D V infection to a point where a mixed virus infection would eventually cause the B V D V to produce typical mucosal disease in cattle. Accordingly. two different methods have been followed. One method consisted in infecting calves, first with B V D V and subsequently with BHV-4 and IBR virus, respectively. The second method was the simultaneous infection with BVDV and Pl-3 virus or BVDV and IBR virus. In the sequential infection trial (first method), the superinfecting viruses (BHV-4 and IBR), did not modify, to a significant extent, the response to the reactivated BVDV infection in calves that were initially infected with N C P strain o f BVDV. As previously seen [10], the N C P N e w York-I strain o f B V D V did not cause any signs of disease with the exception of a slight leukopenia. When the same calves were superinfected 40 days later with BHV-4 and subsequently with IBR virus, they showed the characteristic signs associated with infection by these two viruses. Whereas, as far as the BVDV infection is concerned, the response of calves was actually the same observed with BVDV before superinfection. This could mean that BVDV was reactivated in calves by the superinfecting viruses, but they failed to create the conditions necessary to modify the pathogenic ability of the N C P BVDV which, as usually, caused only leukopenia and a slight diarrhea.
Mixed infections in calves with bovine viral diarrhea virus and other bovine viruses
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In the case of calves that were first infected with the CP TVM2 strain of BVDV, both superinfecting viruses were able to reactivate BVDV infection, so that BVDV was recovered from the calves. However, only IBR virus induced in calves a situation where the reactivated BVDV contributed to a severe clinical response, whereas only slight clinical manifestations due to BVDV were seen following superinfection by BHV-4. The BHV-4 itself produced the usual mild respiratory signs [1 1] and the reactivated BVDV produced the typical clinical response which is commonly observed with each strain, CP or NCP, of BVDV used [10]. Simultaneous infection with BVDV and PI-3 virus, produced in calves a clinical response which was very similar to that observed in the case of calves first inoculated with BVDV and subsequently superinfected with BHV-4. So, as in the case of BHV-4, the two strains of BVDV did not undergo any significant modification in their pathogenic response in the presence of PI°3 coinfecting virus. A different situation was observed when the simultaneous infection was made with BVDV and IBR virus. The two viruses together induced in calves a severe clinical response characterized by a wide variety of signs which included mucoid nasal discharge, profuse salivation, pronounced diarrhea and marked leukopenia. The reaction of the control calf, inoculated with only IBR virus, was much milder. The work reported here indicates that BVDV infection can be reactivated in calves by other bovine viruses, such as BHV-4 and IBR virus, since BVDV was consistently recovered from calves following superinfection. Moreover, following each superinfection, the calves showed symptoms, such as leukopenia and diarrhea which most likely can be attributed to the reactivated BVDV. It is noteworthy that the superinfection, especially that induced by IBR virus, promoted a more severe clinical response in calves which were previously infected with CP BVDV, than in those which were previously given the NCP biotype of the virus. On the other hand, an interesting finding which was made with the simultaneous infection trial was that when BVDV and IBR virus were given together to calves, the biotype of BVDV involved, CP or NCP, did not seem to be as important as it proved to be when calves were infected with BVDV alone or in combination with PI-3 virus or BHV-4. This was evident in the case of the N C P strain which, when combined with IBR virus, induced in calves clinical signs of BVDV infection similar to those resulting from infection with the CP strain. These results are consistent with the observation that a mixed infection by IBR virus and N C P BVDV, was responsible for a severe disease in a group of calves which showed signs of classical IBR infection together with lameness and marked alimentary tract involvement [12]. It has also been shown that BVDV may enhance dissemination in calves of IBR virus so that the virus could be recovered from many tissues unrelated to the respiratory tract [13]. To conclude: our findings do not allow us to state that a mixed infection represents a "key factor" in creating the conditions which cause BVDV to produce typical mucosal disease. However, the more severe clinical response that resulted when calves were superinfected or coinfected with IBR virus, as well as, previously published data [12, 13], support the idea that particular attention should be given to this aspect in studying the pathogenesis of BVDV infection.
Acknowledgements--This study was financially supported by the Ministry of Public Health, Department of Veterinary Services, the Ministry for the University (funds 40 and 60%) and by the National Research Council.
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I. Ames T. R. The causative agent of BVD: its epidemiology and pathogenesis. Vet. Med. 81,629 633 (1986). 2. Bolin S. R., McClurkin A. W., Cutlip R. C. and Coria M. F. Severe disease induced in cattle persistently infected with noncytopathic bovine viral diarrhea virus. Am. J. vet. Res. 46, 573-576 (1985). 3. Brownlie J., Clarke M. C. and Howard C. J. Experimental production of fatal mucosal disease in cattle. Vet. Rec. 114, 535--536 (1984). 4. McClurkin A. W., Bolin S. R. and Coria M. F. Isolation of cytopathic and noncytopathic bovine viral diarrhea virus from the spleen of cattle acutely and chronically affected with bovine viral diarrhea. J. Am. vet. med. Assoc. 186, 568--569 (1985). 5. 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). 6. Bolin S. R., McClurkin A. W., Cutlip R. C. and Coria M. F. Response of cattle persistently infected with noncytopathic bovine viral diarrhea virus to vaccination for bovine viral diarrhea and to subsequent challenge exposure with cytopathic bovine viral diarrhea virus. Am. J. vet. Res. 46, 2467 2470 (1985). 7. Harkness J. W. The control of bovine viral diarrhea virus infection. Ann. Rech. Vbt. 18, 167 174 (1987). 8. Castrucci G., Cilli V. and Gagliardi G. Bovine virus diarrhea in Italy. I. Isolation and characterization of the virus. Archs Ges. Virusforsch. 24, 48-64 (1968). 9. Castrucci G., Frigeri F., Cilli V., Donelli G., Ferrari M., Chicchini U. and Bordoni E. A study of a herpesvirus isolated from dairy cattle with a history of reproductive disorders. Comp. Imrnun. Microhiol. infect. Dis. 9, 13-21 (1986). 10. Castrucci G., Frigeri F., Osburn B. 1., Ferrari M.. Sawyer M. M. and Aldrovandi V. A study ot some pathogenetic aspects of bovine viral diarrhea virus infection. Comp. lmmun. Microbiol. its/bet. Dis. 13, 41 49 (1990). 1 I. Castrucci G., Frigeri F., Ferrari M., Cilli V., Aldrovandi V.. Rampichini L. and Gatti R. A study of the pathogenesis of Bovid herpesvirus-4 in calves. J. vet. Med. B. 34, 473479 (1987). 12. Greig A., Gibson I. R., Nettleton P. F. and Herring J. A. Disease outbreak in calves caused by a mixed infection with infectious bovine rhinotracheitis virus and bovine virus diarrhea virus. Vet. Rec. 108, 480 (1981). 13. Potgieter I,. N. D., McCracken M. D., ltopkins F. M. and Walker R. D. Effect of bovine viral diarrhea virus infection on the distribution of infectious bovine rhinotracheitis virus in calves. Am. J. ret. Re~. 45, 687-690 (1984).
0147-9571/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press Ltd
EFFECTS IN CALVES OF MIXED INFECTIONS WITH BOVINE VIRAL DIARRHEA VIRUS A N D SEVERAL OTHER BOVINE VIRUSES G. CASTRUCCI, I* M. FERRARI, 2 V. TRALDI 3 and E. TARTAGLIONE 4 ~lstituto di Malattie lnfettive, Profilassi e Polizia Veterinaria, Laboratorio Universitario di Virologia "V. Cilli", Universita di Perugia, 2c/o Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia, Brescia, 3Centro Provinciale Svezzamento Vitelli, Tripoli S. Giorgio, Mantova and 4Unit~i Sanitaria Locale, Venafro, lsernia, Italy Abstract--The objective of this study was to verify whether a mixed infection in calves with bovine viral diarrhea virus (BVDV) and other bovine viruses, such as bovid herpesvirus-4 (BHV-4), parainfluenza-3 (PI-3) and infectious bovine rhinotracheitis (IBR) virus, would influence the pathogenesis of the BVDV infection sufficiently to result in the typical form o f mucosal disease being produced. Accordingly, two experiments were undertaken. In one experiment calves were first infected with BVDV and subsequently with BHV-4 and IBR virus, respectively. The second experiment consisted in a simultaneous infection o f calves with BVDV and PI-3 virus or BVDV and IBR virus. From the first experiment it seems that BVDV infection can be reactivated in calves by BHV-4 and IBR virus. Evidence of this is that BVDV, at least the cytopathic (CP) strain, was recovered from calves following superinfection. Moreover, following such superinfection the calves showed signs which could most likely be ascribed to the pathogenetic activity of BVDV. Superinfection, especially by IBR virus, created a more severe clinical response in calves that were initially infected with CP BVDV, than in those previously given the non-cytopathic (NCP) biotype of the virus. Simultaneous infection with PI-3 virus did not seem to modify to any significant extent the pathogenesis o f the experimentally induced BVDV infection whereas a severe clinical response was observed in calves when simultaneous infection was made with BVDV and IBR virus. Key word~: Bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, bovid herpesvirus-4, parainfluenza-3 virus, mixed infections, calves. R6sum6--L'objectif de cette 6rude 6tait de v6rifier si une infection mixte chez des veaux provoqu6e par le virus de la diarrh6e bovine virale (BVDV) et d'autres virus bovins, tels que rherpesvirus-4 du bovin (BHV-4), le virus parainfluenza-3 (PI-3) et de la rhinotrach6ite bovine infectieuse (IBR), influenceraient assez la pathog6nie de rinfection par BVDV pour en arriver fi produire la forme typique de la maladie des muqueuses. Deux exp6riences ont 6t6 entreprises en ce sens. Dans l'une, les veaux ont tout d'abord 6t6 soumis fi l'infection par BVDV, puis, respectivement, par BHV-4 et le virus IBR. La deuxi6me experience consistait en une infection simultan6e des veaux au moyen de BVDV et du virus PI-3 ou de BVDV et du virus IBR. D'apr6s la premiere exp6rience, il semble que rinfection par BVDV puisse 6ire r6activ6e chez les veaux par BHV-4 et le virus IBR. Une preuve e n e s t que BVDV, ou tout au moins la souche cytopathique (CP), a 6t6 retrouv6e chez les veaux suite ~i la superinfection. En outre, ,,i la suite de chaque surinfection, les veaux ont montr6 des signes pouvant tr6s vraisemblablement ~tre imput6s ~i I'activit6 pathog6nique de BVDV. La superinfection, particuli6rement par le virus IBR, a provoqu6 une r6ponse clinique plus grave chez les veaux quit avaient initialement 6t~ infect6s par CP BVDV que chez ceux qui avaient regu le biotype non cytopathique (NCP) du virus. L'infection simultan6e par le virus PI-3 ne semble pas avoir modifi6 de fagon significative la pathog6nie de rinfection par BVDV exp~rimentalement provoqu~e, alors qu'une r~ponse clinique grave a 6t6 observ6e chez les veaux lors de I'infection simultange par BVDV et le virus IBR. Mots clefs: Virus de la diarrh6e bovine virale, virus de la rhinotrachgite bovine infectieuse, herpesvirus-4 du bovin, virus parainfluenza-3, infection mixte, veaux.
*Author for correspondence: Istituto di Malattie Infettive, Facoltfi di Medicina Veterinaria, Via S. Costanzo 4, 06100 Perugia, Italy. 261
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INTRODUCTION The biology of bovine viral diarrhea virus (BVDV) is complex, so that multiple and diverse clinical manifestations may occur in cattle infected with the virus. It is generally known that the most common forms of infection are subclinical or mild [I]. However, the virus is also responsible for a sporadic form of disease, known as "mucosal disease", which generally involves cattle ranging in age from 6 months to 2yr. Mucosal disease is characterized by severe clinical signs, low morbidity and a fatality rate approaching 100%. According to several studies on the pathogenesis of BVDV infection, mucosal disease occurs in cattle that are immunotolerant and viraemic with non-cytopathic (NCP) virus when they become superinfected with a cytopathic (CP) strain of BVDV which is antigenically closely related to the persistently infecting NCP strain [2--4]. However, in other studies [5, 6] the CP strains failed to induce mucosal disease in persistently infected cattlc and it was suggested that only some combinations of biotypes of the virus may cause mucosal disease. Considering that bovine viral diarrhea-mucosal disease has to be regarded as a multifactorial syndrome [7], several factors, many of which are still unknown, could be involved in altering the pathogenesis of the disease. Among the factors which appear worthy of some consideration is the possibility that a mixed infection could play an important role in the pathogenesis of BVDV infection. It is possible that certain infectious agents could provide the required conditions for BVDV to produce typical mucosal disease in cattle. The purpose of the present study therefore, was to determine whether the clinical response to BVDV infection might be altered by the intervention of other viral infections affecting cattle, such as those induced by infectious bovine rhinotracheitis (IBR), parainfluenza-3 (PI-3) viruses, and bovid herpesvirus-4 (BHV-4).
MATERIALS AND METHODS (?ell cultures
Secondary cell cultures from bovine embryo kidney (BEK) were prepared by standard methods and were grown in minimum essential medium (MEM) with Earle's salts supplemented with 5% (v/v) bovine fetal serum and containing antibiotics (penicillin, 1000 U/ml, streptomycin, 500 #g/ml and amphotericin B, 2 pg/ml. Viruses
BVDV. Two strains of the virus have been used, i.e. the NCP New York-I strain (kindly supplied by Dr Osburn, University of California, Davis, Calif., U.S.A.) and the CP TVM2 strain [8]. The NCP virus had an undetermined number of passages in tissue culture and its titer, as found by immunofluorescence in BEK cells, was 105~ median tissue culture infectious doses (TCID~o) per 1 ml. The CP strain was used at the 6th BEK cell cultures passage and contained 1065 TCIDso/ml. IBR virus. The field isolate 90/180 TN (Castrucci, unpublished data) was selected for the study. The virus was at its 2nd passage level in BEK cell cultures with a titer of 10s5 TCIDso/ml. PI-3. This virus was represented by the field isolate 90/1 TN (Castrucci, unpublished data), at the 4th BEK cell cultures passage and had a titer of 10s.s TCIDs0/ml.
M i x e d i n f e c t i o n s in calves with b o v i n e viral d i a r r h e a virus a n d o t h e r b o v i n e viruses
263
BHV-4. The virulent strain 81/16 TV [9] at the 4th passage in BEK cell cultures and a titer of 106.5 TCIDso/ml, was used. Experimental design: inoculation of calves Sixteen Friesian calves, 30-40 days of age, were used throughout the experiment. They were without detectable serum neutralizing antibodies to the viruses under study. As shown in Table I, the calves were allotted to 5 groups of 2 each. An additional control group of 6 calves was added at the appropriate time during the experiment. The calves of the first two groups were inoculated sequentially as follows: first with NCP (group I) or CP (group 2) BVDV, then, 40 days later, calves of both groups received BHV-4. Finally, 32 days after the inoculation of the second virus, all calves were given IBR virus. The calves of groups 3, 4 and 5, were simultaneously infected with NCP BVDV (group 3) or CP BVDV (group 4) and IBR virus or, in the case of group 5, with NCP BVDV (i calf) or CP BVDV (I calf) together with PI-3 virus. Of the control calves of group 6, two were given BHV-4, 2 were inoculated with IBR virus and 2 with PI-3 virus at the time these viruses were given to the experimental calves. The CP and NCP BVDV, were inoculated intravenously (i.v.), each calf receiving 5 ml of virus. The other 3 viruses (IBR, PI-3 and BHV-4) were given by nasal spray in a volume of 5 ml per calf. The calves were observed for 30 days after administration of each virus and their temperatures were taken daily. Daily white blood cells (WBC) counts were also made. Nasal swabbings and blood samples were obtained at predetermined intervals post-inoculation. Samples of serum were also collected from each calf for serology.
Evaluation of clinical response As the purpose of the present study was to investigate the possibility that a mixed infection would eventually lead to a severe clinical form of mucosai disease, an inventory of the main clinical signs of the disease was made. The list incuded the following signs: depression, fever, leukopenia, cough, nasal discharge, lacrimation, dyspnoea, hypersalivation, diarrhea and mouth lesions. The grade of severity of the clinical response was indicated as follows: - , no clinical signs; + , mild reaction with at least two main clinical Table I. Experimental design: inoculation of calves Calves
Simultaneous infection
Sequential infections Group No.
No. of animals
I 2 3 4
2 2 2 2 I I
5
.......... Ist Virus
] t
2nd Virus*
3rd Virus*
Viruses
NCP BVDV CP BVDV
BHV-4 BHV-4
IBR IBR
NA
NA
NA
NA NA NCP BVDV + IBR CP BVDV t- IBR NCP BVDV + PI-3 CP BVDV ~ PI-3
NA NA NA
BHV-4 NA NA
NA NA NA
Controls f
2 2 2
NA IBR PI3
*40 days after the Ist infection. *32 days after the 2nd infection. BVDV = bovine viral diarrhea virus. NCP = non cytopathic or CP = cytopathic biotype of BVDV. BHV-4 = bovid herpesvirus-4. IBR = infectious bovine rhinotracheitis virus. Pl-3 = parainfluenza-3 virus. NA = not applicable.
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signs; + + , reaction with more than two clinical signs of moderate intensity; + + + , several clinical signs of moderate to severe intensity, + + + + , severe clinical reaction.
Viral isolation The nasal swabbings were expressed into 2 ml of MEM. The blood was collected in tubes containing a chelating agent (EDTA-Vacutainer tubes, Becton-Dickson), and buffy coat obtained by centrifugation at 600g for 30 min. The fluid from each swab was homogenized in a glass grinder, centrifuged at 850g for 10min and the supernatant fraction was inoculated on to monolayers of BEK cells grown in wells in plastic plates. The buffy coat samples were frozen and thawed once and then inoculated on to BEK cell cultures as indicated previously. Samples were considered to be negative if cytopathic effects (CPE) failed to appear or immunofluorescence (NCP BVDV) was negative after two serial subpassages of the inoculum had been made. Any virus recovered from the samples was identified by serum neutralization tests. In the case of calves subjected to a mixed infection when their samples were positive for a virus, the virus was neutralized with the appropriate specific antiserum, in order to detect any other virus which might also be present in the sample.
Immunofluorescence (IF) The IF procedure was used when the detection of NCP BVDV in the cultured samples obtained from the inoculated calves was involved. A reference antiserum to BVDV conjugated with fluorescein-isothiocianate was used as previously described [10].
Serum neutralization Serial dilutions of each serum were mixed with 100 TCIDso of the appropriate virus (the CP strain of BVDV, IBR, PI-3 or BHV-4) in 96-well microtiter plates, using one well for each serum-virus dilution. The plates were held for 90 min at room temperature (22'C) and then 20,000 BEK cells, in 0.05 ml vol were added to each well. Neutralization titers were expressed as the highest dilution inhibiting cytopathology. RESULTS
(1) Response of calves to sequential infections: BVDV, BHV-4 and IBR virus (a) BVDV infection. As shown in Table 2, when calves were subjected to BVDV infection, they reacted as previously observed with the strains used in this experiment [10]. Thus, the two calves inoculated with the NCP strain of BVDV (Nos 45 and 47), did not show any clinical signs of the disease with the exception of a slight leukopenia on post-infection day (PID) 2 which lasted for 5-7 days. On the other hand, as expected, the calves Nos 49 and 50, which were given the CP TVM2 strain of BVDV had a moderate ( + + ) response, characterized by fever, leukopenia, nasal discharge and diarrhea. The two calves recovered from the experimental infection in about 10 days. Virus was recovered (Table 3) from buffy coat and the nasal swabbings of all four claves, from PID 2 to 7, the only exception being the calf No. 47, inoculated with NCP BVDV, which was negative on PID 2. Neutralizing antibodies were present at PID 30 at titers between 1:32 and 1: 128. Co) 1st Superinfection: BHV-4. When the four calves which had experienced BVDV infection were superinfected with BHV-4, a variation in response was observed depending
M i x e d i n f e c t i o n s in c a l v e s with b o v i n e viral d i a r r h e a v i r u s a n d o t h e r b o v i n e v i r u s e s
265
Table 2. The evaluation of the grade of severity of the clinical response in calves exposed to sequential infections with bovine viral diarrhea virus (BVDV), bovid herpesvirus-4 (BHV-4) and infectious bovine rhinotracheitis (IBR) virus Clinical response:l: Calves Group No.
BVDV infection
Superinfection with
No.
NCP
CP
BHV-4*
IBRt
I I 2 2
45 47 49 50
-NA NA
NA NA *+ ++
+ + ++ ++
+ + + + + + +t-++ ++++
Controls 6 6 6
51 52 55
NA NA NA
NA NA NA
+ + NA
NA NA + +
*BHV-4 was inoculated to calves 40 days after BVDV infection. $1BR virus was inoculated to calves 32 days after BHV-4 infection. ~Clinical response: absence of signs - ; mild +; moderate + +; moderate to severe + + +; severe + + + + . NCP = non cytopathic or CP = cytopathic biotype of BVDV. NA = not applicable.
on the biotype of the BVDV (NCP or CP) given earlier. As shown in Table 2, the calves first inoculated with NCP BVDV had a mild clinical response ( + ) of the kind generally observed with BHV-4 infection [1 I]. Thus, the calves had nasal discharge and were slightly depressed for about 1 week after superinfection. However, they showed few signs which could be related to BVDV infection, with only a slight leukopenia and a moderate diarrhea observed on PID 5-6, both of which lasted 2-3 days. The clinical response to BHV-4 was more evident in the calves previously given CP BVDV and was considered to be of moderate intensity ( + +). In this case, the animals reacted with all signs usually reported for calves experimentally infected with the virulent strain 85/16 TV of BHV-4, such as fever, depression and nasal discharge, but in addition they showed also the typical signs of BVDV infection. The latter were represented by a marked leukopenia and diarrhea. The leukopenia started on PID 2 and lasted 10-14 days with the lowest WBC count (1900-2000) being registered on PID 7-9. Diarrhea consisted of watery feces which occasionally appeared blood-stained, on PID 7-8 and lasted, in both calves, for 4 days. The two calves which served as control to the BHV-4 infection (Nos 51 and 52), had a mild clinical response ( + ) , similar to the calves of Group 1. BHV-4 was recovered (Table 3) from the nasal swabs of all the inoculated calves, but only on PID 2 from those calves which were previously given BVDV, whereas it was consistently recovered from the controls until PID 12. Following BHV-4 infection NCP BVDV was never recovered from the inoculated calves, whereas the CP BVDV was isolated on PID 7 and 9 from the buffy coats obtained from the two calves previously infected with CP BVDV. No significant rise in the antibody titers to BVDV was observed after superinfection with BHV-4. The immunologic reaction of calves to BHV-4, in terms of neutralizing antibody production, as usually [1 I] was very poor, the average titer being 1:4. (c) 2nd Superinfection: IBR virus. The superinfection with IBR virus induced a moderate to severe reaction ( + + + , as indicated in Table 2) in the two calves which were first inoculated with NCP BVDV (Nos 45 and 47). The calves developed signs that could be considered as typical of either BVDV or IBR virus infections, i.e. fever, nasal discharge
G . CASTRUCCX et al.
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Table 3. Virus recovery in calves exposed to sequential infections with bovine viral diarrhea virus (BVDV), bovid herpesvirus-4 (BHV-4) and infectious bovine rhinotracbeitis virus {IBR) Virus recovery* After BVDV infection
Calves Group No,
BVDV LE NS
After BHV-4 infection
No.
First infection
BVDV LE NS
I I 2 2
45 47 49 51)
N ( ' P BVDV N C P BVDV CP BVDV CP BVDV
2-7~" 7 2 7 2 7
2 7 7 2-7 2-7
7, 9 7. 9
Controls 6 6 6
51 52 55
BHV-4 BHV-4 IBR virus
NA NA NA
NA NA NA
NA NA NA
BHV-4 I,E NS
BVDV LE NS
----
2 2 2 2
10 -10
2 12 2-12 NA
NA NA NA
. . . .
NA NA NA
After IBR virus infection
-NA
BHV-4 LE NS
--
IBR LE m
.-
.--
NA NA NA
NA NA NA
NA NA NA
NA NA 2.12
NS 3.6 3.6 3,6 3.6 NA NA 212
*Virus recovery was made in bovine embryo kidney cell cultures and samples (LE = buffy coat; NS = nasal swabbings) were obtained from calves on post infection days: 2.3, 6. 7 . 9 and 12. N C P = non cytopathic or CP = cytopathic biotype of BVDV. NA = not applicable. - - Virus was not recovered. +The numbers indicate the post infection day when the virus was recovered.
and salivation. The two calves which were previously infected with CP BVDV (Nos 49 and 50), when superinfected with IBR virus showed a wider variety of clinical signs, compared to the calves previously given NCP BVDV. Here the clinical response was more severe ( + + + + , as indicated in Table 2) and signs of both IBR virus and BVDV infection were seen. Both the affected calves had high fever, marked leukopenia, heavy nasal discharge, profuse salivation, watery diarrhea, coughing, dyspnoea and viscous oculo-conjunctival discharge. The calf which served as control of IBR virus infectivity (No. 55), had a typical clinical response as recognized for experimentally induced IBR. Thus, the calf developed a febrile reaction and respiratory symptoms, such as nasal discharge, dyspnoea and cough, but recovered in about 10 days. The IBR virus was recovered (Table 3) from nasal swabbings of calves on PID 3 and 6, when all samples were negative for BVDV. However, BVDV was recovered from the buffy coat of one calf previously infected with NCP BVDV (No. 45) and from one of the two pre-infected with CP BVDV (No. 49). No IBR virus was isolated at this time of infection (PID 10). IBR virus was isolated from the nasal swabbings and from the blood of the control from PID 2 until 12. After IBR superinfection, no significant modification was observed in the antibody titers to either BVDV or BHV-4, as determined by neutralization tests carried out on the serum samples collected 30 days after superinfection. At this time, neutralizing antibody to IBR virus was present at titers ranging from i : i 6 to 1:64. (2) Response o f cah, es to simultaneous injection: B VD V 4- I B R virus or B VD V 4- P I - 3 virus (a) B VD V 4- I B R virus. When the calves were subjected to simultaneous infection with BVDV and IBR virus, they developed a severe clinical response ( + + + + , in Table 4) without any difference being observed between calves that were given the CP or the NCP strain of BVDV. All calves showed clinical signs similar to those described for calves Nos 49 and 50 (Table 2), subjected to sequential infections with CP BVDV, BHV-4 and IBR virus. Thus, they reacted with high fever, mucoid nasal discharge, copious salivation, intense diarrhea and a marked leukopenia from which they recovered very slowly, so that after three weeks from the time of onset of the clinical illness, their general condition was
Mixed infections in calves with bovine viral diarrhea virus and other bovine viruses
267
Table 4. The evaluation o f the grade o f severity o f the clinical response in calves exposed to simultaneous infection with bovine viral diarrhea virus (BVDV) and infectious bovine rhinotracheitis (1BR) virus or BVDV and parainttuenza-3 (PI-3) virus Calves Group No.
Clinical response*
No.
N C P BVDV + I B R virus
CP BVDV + I B R virus
N C P BVDV + P I - 3 virus
CP BVDV + P I - 3 virus
IBR virus only
PI-3 virus only
3 3 4 4 5 5
34 53 31 54 29 28
+ + + + + + ~- + NA NA NA NA
NA NA ++ 4 + + + + + NA NA
NA NA NA NA -~ NA
NA NA NA NA NA #- +
NA NA NA NA NA NA
NA NA NA NA NA NA
Controls 6 6 6
30 27 56
NA NA NA
NA NA NA
NA NA NA
NA NA NA
+ -'r NA NA
NA -
*Clinical response: absence o f signs - ; mild + ; moderate + + ; moderate to severe + + + ; severe + + + + . N C P = non cytopathic or CP = cytopathic biotype o f BVDV. N A = not applicable.
still very poor. The calf which was given IBR virus only (No. 30), had a milder clinical response, characterized by fever, nasal discharge, slight salivation and coughing. Neither ieukopenia nor diarrhea was observed in this calf. The only virus recovered (Table 5) from the first samples taken on PID 3 from the calves was IBR virus, which was isolated from all calves either from buffy coats or nasal swabbings. BVDV was first isolated on PID 7 from either buffy coats or nasal swabbings from both calves which were given the N C P BVDV and from one calf inoculated with the CP biotype of BVDV. The last isolation of IBR was obtained from nasal swabbings and buffy coats at PID 7, when it was recovered from the two calves which were coinfected with CP BVDV and from one calf coinfected with the N C P biotype of BVDV. BVDV was recovered also on P I D 24 from the buffy coat of all inoculated calves. The calves produced neutralizing antibody to either BVDV and IBR virus. The antibody titers to BVDV were in the range 1:32 to 1 : 128, whereas the maximum titer found for IBR virus was 1: 16. The control calf, inoculated with IBR virus only, did not produce antibody to BVDV, but 30 days after infection it had a titer to IBR virus of 1:8. (b) B V D V + P I - 3 virus. Simultaneous infection with BVDV and PI-3 virus induced in calves a clinical response which was considered mild ( + ) in the case of the calf coinfected with N C P BVDV (calf No. 29) or of moderate intensity ( + + ) in the other calf which was coinfected with the CP BVDV (calf No. 28). Both calves were afebrile but became leukopenic and had a nasal discharge and hypersalivation. Although the two calves showed the same signs, these were more pronounced and lasted longer in the calf which was coinfected with the CP biotype of BVDV. No clinical signs were observed in the calves that were inoculated with PI-3 virus only (control calves, Nos 27 and 56). BVDV was recovered (Table 5) from the buffy coats of the calves on PID 7 ( N C P BVDV) or 9 (CP BVDV). PI-3 virus was reisolated from the nasal swabbings of the two calves coinfected with BVDV on PID 3 till PID 12. The virus was also recovered from the nasal swabbings obtained from the control calves on PIDs 3, 6 and 12. At PID 30 neutralizing antibody to PI-3 virus was found at titers of I : 8 and 1: 16 in the two calves coinfected with CP BVDV or N C P BVDV. The two calves produced neutralizing antibody also to BVDV at a titer of 1:64. The serum of the control calves neutralized PI-3 virus at an average titer of 1:128, but failed to neutralize BVDV at the lowest dilution tested of 1:4. C I M I D 15,4 -C
G . CASTRtJCCI et al.
268
Table 5. Virus recovery in calves exposed to simultaneous infection with bovine viral diarrhea virus (BVDV) and infectious bovine rbinotracheitis (IBR) virus or BVDV and parainfluenza-3 (Pl-3) virus Virus recovery after simultaneous infection* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Infected calves Group No.
BVDV and Pl-3 virus
B V D V and IBR virus
No,
N C P BVDV LE NS
CP BVDV LE NS
IBR virus LE NS
N C P BVDV LE NS
C P BVDV LE NS
PI-3 ~irus LE NS
NA
NA
N C P B V D V + IBR virus 3 3
34 53
7, 24~" 7. 24
7 7
NA NA
NA NA
3, 7 3
3, 7 3
NA NA
NA NA
7, 24 24
7
3, 7 3, 7
3. 7 3, 7
"1
C P B V D V + IBR virus 4 4
31 54
NA
N C P B V D V t- Pl-3 virus 7 5 29 t C P B V D V + Pl-3 virus 5
NA
NA
NA
....
3 12
NA
28
NA
NA
9
3 12
Controls IBR virus 6
3O
NA
NA
27 56
NA NA
NA NA
3 12
3 12
NA
NA
NA NA
NA NA
NA
PI-3 virus 6 6
NA NA
-.
3. 6. 12 3, 6. 12
*Virus recovery was made in bovine embryo kidney cell cultures and samples (I.E = buffy coat; N S = nasal swabbings) were obtained on post infection days: 3, 6. 7, 12 and 24. N C P = non cytopathic or C P = cytopathic biotype of BVDV. eThe numbers indicate the post infection day when the virus was recovered. - Virus was not recovered. N A = not applicable.
DISCUSSION The aim of this study was to verify whether the intervention of other bovine viruses would influence the pathogenesis of B V D V infection to a point where a mixed virus infection would eventually cause the B V D V to produce typical mucosal disease in cattle. Accordingly. two different methods have been followed. One method consisted in infecting calves, first with B V D V and subsequently with BHV-4 and IBR virus, respectively. The second method was the simultaneous infection with BVDV and Pl-3 virus or BVDV and IBR virus. In the sequential infection trial (first method), the superinfecting viruses (BHV-4 and IBR), did not modify, to a significant extent, the response to the reactivated BVDV infection in calves that were initially infected with N C P strain o f BVDV. As previously seen [10], the N C P N e w York-I strain o f B V D V did not cause any signs of disease with the exception of a slight leukopenia. When the same calves were superinfected 40 days later with BHV-4 and subsequently with IBR virus, they showed the characteristic signs associated with infection by these two viruses. Whereas, as far as the BVDV infection is concerned, the response of calves was actually the same observed with BVDV before superinfection. This could mean that BVDV was reactivated in calves by the superinfecting viruses, but they failed to create the conditions necessary to modify the pathogenic ability of the N C P BVDV which, as usually, caused only leukopenia and a slight diarrhea.
Mixed infections in calves with bovine viral diarrhea virus and other bovine viruses
269
In the case of calves that were first infected with the CP TVM2 strain of BVDV, both superinfecting viruses were able to reactivate BVDV infection, so that BVDV was recovered from the calves. However, only IBR virus induced in calves a situation where the reactivated BVDV contributed to a severe clinical response, whereas only slight clinical manifestations due to BVDV were seen following superinfection by BHV-4. The BHV-4 itself produced the usual mild respiratory signs [1 1] and the reactivated BVDV produced the typical clinical response which is commonly observed with each strain, CP or NCP, of BVDV used [10]. Simultaneous infection with BVDV and PI-3 virus, produced in calves a clinical response which was very similar to that observed in the case of calves first inoculated with BVDV and subsequently superinfected with BHV-4. So, as in the case of BHV-4, the two strains of BVDV did not undergo any significant modification in their pathogenic response in the presence of PI°3 coinfecting virus. A different situation was observed when the simultaneous infection was made with BVDV and IBR virus. The two viruses together induced in calves a severe clinical response characterized by a wide variety of signs which included mucoid nasal discharge, profuse salivation, pronounced diarrhea and marked leukopenia. The reaction of the control calf, inoculated with only IBR virus, was much milder. The work reported here indicates that BVDV infection can be reactivated in calves by other bovine viruses, such as BHV-4 and IBR virus, since BVDV was consistently recovered from calves following superinfection. Moreover, following each superinfection, the calves showed symptoms, such as leukopenia and diarrhea which most likely can be attributed to the reactivated BVDV. It is noteworthy that the superinfection, especially that induced by IBR virus, promoted a more severe clinical response in calves which were previously infected with CP BVDV, than in those which were previously given the NCP biotype of the virus. On the other hand, an interesting finding which was made with the simultaneous infection trial was that when BVDV and IBR virus were given together to calves, the biotype of BVDV involved, CP or NCP, did not seem to be as important as it proved to be when calves were infected with BVDV alone or in combination with PI-3 virus or BHV-4. This was evident in the case of the N C P strain which, when combined with IBR virus, induced in calves clinical signs of BVDV infection similar to those resulting from infection with the CP strain. These results are consistent with the observation that a mixed infection by IBR virus and N C P BVDV, was responsible for a severe disease in a group of calves which showed signs of classical IBR infection together with lameness and marked alimentary tract involvement [12]. It has also been shown that BVDV may enhance dissemination in calves of IBR virus so that the virus could be recovered from many tissues unrelated to the respiratory tract [13]. To conclude: our findings do not allow us to state that a mixed infection represents a "key factor" in creating the conditions which cause BVDV to produce typical mucosal disease. However, the more severe clinical response that resulted when calves were superinfected or coinfected with IBR virus, as well as, previously published data [12, 13], support the idea that particular attention should be given to this aspect in studying the pathogenesis of BVDV infection.
Acknowledgements--This study was financially supported by the Ministry of Public Health, Department of Veterinary Services, the Ministry for the University (funds 40 and 60%) and by the National Research Council.
270
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