- Email: [email protected]
Comparative study of rotavirus strains of bovine and rabbit origin
Comp. lmmun. Microbiol. lnfect. Dis. Vol. 7. No. 3/4, pp. 171 178, 1984 Printed in Great Britain
COMPARATIVE
STUDY
BOVINE
AND
OF
0147-9571/8453.00+0.00 Pergamon Press Ltd
ROTAVIRUS
RABBIT
STRAINS
OF
ORIGIN
G. CASTRUCCI 1, F. FRIGERI l, M. FERRARI 2, V. CILLI t, V. ALDROVANDI 3, F. CALEFVl3 and R. GATTI 4 qstituto di Malattie Infettive, Profilassi e Polizia Veterinaria dell'Universita' di Perugia, Perugia, 2Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia, Brescia, 3Centro Provinciale Svezzamento Vitelli, Tripoli, MN, and 4Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia, Sezione di Mantova, Mantova, Italy Abstract--Newborn calves were susceptible to infection with a rotavirus strain isolated from rabbits and, conversely, rabbits 4~5 weeks of age became ill and some died when inoculated with the rotavirus strain of bovine origin. However, the latter isolate appeared to be more virulent than the rabbit isolate in either calves or rabbits. Moreover, in reciprocal cross protection tests, carried out in calves and in rabbits, the rabbit rotavirus antiserum afforded only weak protection to challenge infection with either the homologous or the heterologous virus. By contrast, the protective level of the bovine rotavirus antiserum was relatively high. Key words: Comparative study, rotavirus, bovine, rabbits
ETUDE COMPARATIVE DES SOUCHES DE ROTAVIRUS PROVENANT DE LAPINS ET DE BOVINS R6sum~--Des veaux nouveau-n6s se sont av+r6s sensibles 5. l'infection par une souche de rotavirus isol~e de lapins. Par ailleurs, des lapins, agbs de 4 5. 5 semaines, on bt6 rendus malades et certains sont morts par suite de l'inoculation d'une souche de rotavirus d'origine bovine. Cene derni6re souche semblait cependant plus virulente 5. la fois chez les veaux et chez les lapins. Des exp6riences portant sur la protection crois6e instaur6e par les antis6rums contre ces souches ont 6t6 men6es chez des veaux et chez des lapins. Elles ont d6montr6 qu'un antis6rum contre le virus provenant de lapins ne conf6rait qu'une protection limit6e contre les souches homologue et h&6rologue. Un plus haut degr6 de protection +tait cependant induit par l'antis6rum contre le rotavirus bovin. Mots-clefs: Etude comparative, rotavirus, bovins, lapins
INTRODUCTION From several comprehensive studies on rotaviruses [1-3], there are convincing indications that these viruses infect a variety of animal species in which they have been associated as the cause of diarrhea in the young. It has also been shown that rotavirus from one species can infect members of certain other species. Thus, propagation of bovine rotavirus in cats, dogs [4] and pigs [5] has been reported. Moreover, experimental infection of pigs [6-8], calves [9] and lambs [10], was obtained with human rotavirus. In view of the importance that inter-species transmission may have in the epidemiology of rotavirus infections it appeared worthwhile to extend such a comparative study as much as possible. The purpose of this report was to obtain data regarding the possibility that rotavirus strains from bovine and rabbits, respectively, can infect the heterologous host. Also, an attempt was made to induce a passive immunity in calves and in rabbits against the heterologous virus. 171
172
G. CASTRUCC1 et al.
MATERIALS AND METHODS Virus
Two strains of rotavirus, one of which, 81/36F, was from cattle [11] and the other, 82/311F, from rabbits [12], were used for the present study. Both viruses were at the 7th passage level in MA-104 cell cultures and their titers were 108.5o median tissue culture infectious doses (TCIDs0) per 0.2 ml. Animals Calves. Twenty-nine conventionally reared Friesian calves 2 3 days of age were used. All calves were from rotavirus unvaccinated cows, and were obtained from dairy farms where no history of enteric conditions had been recently reported. The calves had ingested colostrum from their dams at least once before they were transferred to the laboratory animal facilities. Thereafter, they were fed a milk substitute twice daily. For the experimental infection trial 13 calves were divided into three groups of 5, 6 and 2 animals, respectively. The calves of the first group were infected with the bovine rotavirus strain, whereas those of the second group received the rabbit rotavirus. Each of the remaining two calves was given Eagle's minimum essential medium (MEM) and served as control. The inoculum was given orally in a volume of 30 ml per calf. Infected and control calves were kept in separate accommodations and observed for 30 days. During this period each calf was inspected for the appearance of clinical signs of disease. Fecal swabs for virus isolation were taken at the time of infection (time 0), and again on post infection day (PID) 2, 4, 6, 8 and 10. In addition, blood samples for serology were obtained from the calves at time 0 and at PID 30. In order to enhance the antibody level, the calves which survived the infection were re-exposed to the virus on P I D 30. Each calf received the same virus as that given originally in a dose of 10ml by the oral route, and of 2 m l by subcutaneous inoculation. For oral administration, undiluted virus culture was used, whereas in the case of the subcutaneous injection the virus was mixed with an equal volume of Freund's incomplete adjuvant. Eighteen days later the calves were bled, the serum was separated, and pools were made according to the groups of calves. Thus, two pools were obtained which were referred to as bovine rotavirus and rabbit rotavirus antiserum, respectively. The bovine rotavirus antiserum had a titer of 1:128, while that of rabbit rotavirus antiserum was 1:180. These serums were used to conduct passive immunity tests. For cross protection tests, 16 calves were segregated into three groups of 6, 6 and 4 animals, respectively. Each calf of one group of 6 animals received 250ml of bovine rotavirus antiserum which was given orally on two consecutive days in a volume of 125 ml per day. The calves of the other group of 6 were given rabbit rotavirus antiserum according to the above procedure. The remaining 4 calves, each was given M E M and served as virus controls. Two hours after the first inoculation of the antiserum or M E M , the calves were challenge exposed according to the following method: each of the two challenge viruses (bovine rotavirus and rabbit rotavirus) was given orally in a volume of 30 ml to 3 calves of each of the two groups of immune serum treated animals, and to 2 calves of the control group. Fecal swabs for virus isolation and blood samples for serology were taken according to the procedure for the experimental infection trial. The calves were kept under observation for 30 days. Particular attention was paid to the character of the feces.
Rotaviruses from bovine and rabbits
173
Rabbits. Twenty-four New Zealand White rabbits, 4-5 weeks of age, were used in the experiment. The experimental infection trial was carried out in 12 rabbits, of which 5 received rabbit rotavirus, 5 the bovine rotavirus and 2, which served as control, were given MEM. The volume of inoculum was 2 ml, given by gastric tube. The three groups of rabbits were separated from one another just prior to inoculation and kept in three separate cages for the duration of the experiment (30 days). Stools were collected from each cage prior to inoculation (time 0) and on PID 2, 4, 6, 8 and 10. Rabbits were bled before inoculation and on PID 30. As in the case of the calves, the rabbits which survived in each group were used for the production of immune serum to be used in the cross immunity tests. The same procedure as described for the calves was followed, the only exception being the volume of inoculum. In the case of the rabbits it was 2 ml of undiluted virus culture given by the oral route, and 1 ml of a mixture, consisting of equal amounts of virus culture and Freund's incomplete adjuvant given by subcutaneous injection. Both serums which were obtained from rabbits had a titer of 1:180 when used in the cross protection tests. Cross protection tests were carried out in 12 rabbits in the same manner as for the analogous tests in the calves. Thus, 4 rabbits received rabbit rotavirus immune serum and 4 rabbits were given immune serum to the bovine rotavirus. The remaining 4 rabbits were given MEM. The inoculum was given by gastric tube in a volume of 10 ml per rabbit several times over a period of two consecutive days. Two hours after the first inoculation of antiserum or M E M , the rabbits were exposed to the two challenge viruses (rabbit rotavirus and bovine rotavirus). Each virus was inoculated by gastric tube in a volume of 2 ml to 2 rabbits in each of the two groups of immunized rabbits, and of the control group. Stools for virus isolation, and blood samples for serology, were collected at the intervals mentioned above. Virus isolation Attempts to recover the virus from rectal swabbings obtained from calves were made in MA-104 cell cultures as described elsewhere [11]. Recovery of virus from the rabbit stools was done in the same way as in the case of the calves, the only exception being that the stools were previously prepared as a 10~o suspension in MEM. When virus was isolated, its identity was determined by neutralization tests carried out with rabbit antiserum specific for 81/36F bovine rotavirus [11] or for 82/311F rabbit rotavirus [12].
Serologic tests The serums obtained from calves and rabbits were tested against both the homologous and the heterologous rotavirus strains. The tests were carried out in microtiter plates as previously reported [13]. RESULTS
Experimental infection Calves. The results are depicted in Table 1. All calves became depressed and anorexic after an incubation period of 2-5 days. The diarrhea first appeared (PID 2.6) in the calves that were inoculated with bovine rotavirus, and about 3 days later (PID 5.6) in the calves infected with the rabbit rotavirus. In all cases the feces were fluid, yellowish, and they quite
174
G . CASTRUCCI et al. Table 1. Response of calves to oral inoculation of rotavirus of either bovine or rabbit origin Diarrhea
lnoculum: rotavirus strain Bovine: 81/36F Rabbit: 82/311F Controls : MEM
No. of calves studied
Duration* days
Live-weight* gain: g per day
Survival rate ratio
Affected calves, No.
Onset* PID
5
5
2.6
8.0
50
2/5t
6
6
5.6
3.1
80
6/6
2
0
NA
NA
120
2/2
Virus recovery from fecal swabs, PID/ No. of calves 2/5, 8/4, 2/2, 8/6,
4/5, 6/3, 10/4 4/5, 6/4, 10/5.
Negative
*Average values, tDied on PID 8, 11 and 13, respectively. PID post infection day. MEM culture medium. NA not applicable.
often contained mucous and blood. All calves were afebrile. The diarrhea lasted from 3.1 (rabbit rotavirus infected group) to 8.0 (bovine rotavirus infected group) days. Three calves of those that had been infected with the bovine rotavirus died on PID 8, 11 and 13, respectively. All had lesions limited to the small intestines where a general thickening of the mucosa, particularly evident in the ileum, was found. All calves which received the rabbit rotavirus survived the infection. N o significant differences were observed in virus shedding between the two groups of infected animals. The control calves remained healthy and virus was not isolated from their feces. The serological response of the calves is shown in Table 2. Low antibody titers were found at the time of infection. On PID 30 the titers had increased by apporximately 3 to 4, two-fold dilution steps in calves infected with the rabbit rotavirus or with the bovine rotavirus, respectively. There were no significant differences in titers to the homologous or the heterologous virus. The antibody titers in the control calves did not vary significantly during the experiment. Rabbits. From Table 3 it is seen that the rabbits gave a similar response to inoculation of the two rotaviruses. The diarrhea appeared in 3 of the 5 inoculated with the rabbit rotavirus, and in 2 of 5 of those which were infected with bovine rotavirus. However, all were depressed, anorexic, and underwent a significant loss in condition. One rabbit in the group inoculated with rabbit rotavirus and 2 among those infected with bovine rotavirus died. At autopsy the intestines were found to be markedly congested and distended. Virus was consistently isolated from the pooled feces of each group. N o signs of diseases was observed in the two control rabbits, and their feces were negative for virus.
Table 2. Serological response of calves experimentally infected with rotavirus of bovine or rabbit origin Neutralizing antibody* to rotavirus strain: Inoculum: rotavirus strain Bovine: 81/36F Rabbit: 82/311F Controls : MEM
No. of calves studied
Day 0
PID 30
Day 0
PID 30
5
1:5
1:647
1:10
1:64t
6
1:20
1:130
1:18
1:130
2
1:24
1:16
1:12
1:8
Bovine 81/36F
Rabbit 82/311F
* Average values, tValues referred to the 2 calves which survived. Day 0 the day of infection. PID post infection day. MEM culture medium.
Rotaviruses from bovine and rabbits
175
Table 3. Response of rabbits to oral inoculation of rotavirus of either bovine or rabbit origin Diarrhea Inoculum: Rotavirus strain
Duration* days
No. of rabbits showing stunting
Survival rate ratio
Viral recovery from fecal pools on PID:
4.6
4.3
5
4/53"
2, 4, 6, 8
2
7.0
2.0
5
3/5~
2, 4, 6, 8
0
NA
NA
0
2/2
Negative
No. of rabbits studied
Affected rabbits, No.
Onset* PID
5
3
5
2
Rabbit: 82/311F Bovine: 81/36F Controls:
MEM
* Average values, tDied on PID 5, or :~on PID 8 and 28, respectively. PID post infection day. MEM culture medium. NA not applicable.
Table 4. Serological response of rabbits experimentally infected with rotavirus of bovine or rabbit origin Neutralizing antibody* to rotavirus strain: Inoculum: rotavirus strain Rabbit: 82/311F Bovine: 81/36F
Rabbit 82/311 F
Bovine 81/36F
No. of rabbits studied
Day 0
PID 30
Day 0
PID 30
5
1:3
1: 120t
1:2
1 : 114t
5
1:2
1:128:~
1;2
1:138~:
2
< 1:4
1:4
1:2
1:4
Controls:
MEM
* Average values, t Values referred to the four or Sthe three rabbits which survived. Day 0 day of infection. PID post infection day. MEM culture medium.
The behavior of the neutralizing antibody (Table 4) in the rabbits was generally similar to that reported above for calves. However, the increased antibody titers on PID 30 (about 6 steps) in the rabbits was higher than that registered for the calves. Cross protection tests Calves. The results are shown in Table 5. The immune serum to the bovine rotavirus completely protected the calves against challenge infection with the homologous virus in that the animals remained clinically normal. The same antiserum was also partially Table 5. Cross protection tests in calves with rotavirus of bovine and rabbit origin Diarrhea Immune serum to rotavirus strain Bovine: 81/36F
Rabbit: 82/311F
Challenge rotavirus strain Bovine: 81/36F Rabbit: 82/311F Rabbit: 82/311 F Bovine: 81/36F
No. of calves studied
Affected calves, No.
Onset* PID
Duration* days
Virus recovery from fecal swabs, PID/No. of calves
3
0
NA
NA
4/1,6/1
3
2
5.5
2.5
2/1,4/1, 6/2
3
2
4.5
5.5
2/1,4/3, 6/2
3
3
5.0
5.6
2/1,4/2, 6/1,8/2
2
2
4.5
8.0
2/2, 4/2, 6/2, 8/2
2
2
3.5
9.0
2/2, 4/2, 6/2, 8/2, 10/1
Controls:
MEM
Bovine: 81/36F Rabbit: 82/311F
* Average values. PID post infection day. NA not applicable. MEM culture medium.
176
G. CASTRUCCI et al.
Table 6. Cross protection tests in rabbits with rotavirus of bovine and rabbit origin Diarrhea Immune serum to rotavirus strain Rabbit: 82/311F
Bovine: 81/36F
Controls: MEM
Challenge rotavirus strain
Survival rate ratio
Virus recovery from fecal pools on PID:
No. of rabbits studied
Affected rabbits, No.
Onset* PID
Duration* days
Rabbit: 82/311F Bovine: 81/36F Bovine: 81/36F Rabbit: 82/31 l F
2
0
NA
NA
2/2
Negative
2
2
9,0
7.0
0/2¢
4, 6, 8, 10
2
1
10.0
4.0
2,'2
4, 6
2
1
10.0
6.0
I/2 +
2, 4, 6
Rabbit: 82/311F Bovine: 81/36F
2
2
7.0
15.0
I/2§
2, 4, 6, 8, 10
2
2
8.0
10.0
1/211
2,4, 6, 8, 10
* Average values. PID post infection day. NA not applicable. MEM culture medium. Death of the rabbits occurred on PID: I1 and 167, 16 ++, 29 § and 14 ]].
protective for the calves which were challenge exposed to the heterologous virus, i.e., the rabbit rotavirus. In this group of calves, one did not show any signs of disease, while the other two had a mild diarrhea of short duration. The rabbit rotavirus immune serum appeared to be, in general, less protective than the bovine rotavirus antiserum, in that two of the three calves that received the homologous virus, and all three which were given the heterologous virus, had diarrhea, although its duration was shorter compared with that of the control calves. Virus was recovered only twice from one calf in the group immunized with the bovine rotavirus antiserum. In the other groups virus was isolated several times, especially in those calves which had rabbit rotavirus antiserum and were challenge infected with the bovine rotavirus. The control calves reacted with clinical signs of disease, i.e. depression, stunting, and diarrhea, without there being any significant difference with regard to the virus inoculated. Virus was reisolated from the fecal swabbings from PID 2 through 8 (bovine rotavirus) or 10 (rabbit rotavirus). All calves had antibody titers at time 0 ranging from 1:8 to 1:32. At PID 30 the titers had increased slightly to 1:64. Rabbits. From Table 6 it is apparent that the immune serum to the rabbit rotavirus completely protected the rabbits which were challenge infected with the homologous virus, whereas it was without any effect in rabbits which were exposed to challenge infection with the bovine virus. The latter animals had diarrhea and died on PID 11 and 16, respectively. The intestines of these two rabbits were generally congested and edematous. Of the rabbits immunized with bovine rotavirus antiserum, and then challenge exposed to the homologous virus, one out of two had mild diarrhea, but both survived. One of the two rabbits that were challenged with the rabbit rotavirus was fully protected, whereas the other underwent diarrhea and died on PID 16. The intestines of this rabbit were congested and markedly distended. Virus was not recovered from the rabbits immunized with rabbit rotavirus antiserum and infected with the rabbit rotavirus, whereas in the case of rabbits which had the same antiserum, but were challenge exposed to the bovine rotavirus, the feces were positive for virus from PID 4 to 10. The virus was also isolated from rabbits immunized with bovine rotavirus antiserum, i.e. on two days (PID 4, 6) in
Rotaviruses from bovine and rabbits
177
the rabbits exposed to the bovine rotavirus and three times (PID 2, 4, 6) in those exposed to the rabbit rotavirus. In each group the rabbits that served as control of the challenge viruses had profuse diarrhea, and one rabbit in each group died. The serologic situation was the same as described for the calves, and no differences were detected among the groups: the titer of the antibody rose from an average value of 1:4 at time 0 to a level of 1:32 on PID 30.
DISCUSSION The results of this study disclose that a rotavirus, isolated from calves, can be propagated in rabbits in which it induces the classic signs of rotaviral infection. Conversely, a rotavirus of rabbit origin is able to cause a clinically manifested infection in calves. This conclusion is based on the finding that the five rabbits inoculated with the bovine rotavirus became stunted, two had a severe diarrhea, two died, and the virus was reisolated from their feces. On the other hand, of the 6 calves that were exposed to the rabbit rotavirus, all had diarrhea, they suffered loss of condition, and excreted virus consistently in the feces. However, it is of interest that the 81/36F strain of bovine rotavirus seems to be more virulent than the rabbit isolate. In this experiment the lowest live-weight gain per day (50 g) was registered by the calves inoculated with the bovine rotavirus, whereas higher values (80 g) were gained by the calves which received the rabbit rotavirus. The average daily gain for the control calves was 120 g. Moreover, the bovine isolate caused the death of 3 calves out of 5, whereas all calves inoculated with the rabbit isolate survived the infection. This finding appears to be in agreement with previous observations which demonstrated that the same strain of bovine rotavirus (81/36F) used in this experiment was the most virulent when compared with two other bovine isolates [13]. The results of the cross protection tests show that the bovine rotavirus seems to be more immunogenic than the rabbit isolate. This is seen in calves where the immune serum to the bovine isolate was fully protective in all animals that were challenge exposed to the homologous virus, whereas the immune serum to the rabbit isolate failed to protect 1 calf out of 3 infected with the rabbit rotavirus. This result seems to reinforce the hypothesis that the 81/36F bovine rotavirus strain might be considered to be a predominant antigenic type [13]. The antibody produced in calves and in rabbits neutralized the homologous and the heterologous virus alike. This is not surprising since the bovine and the rabbit rotavirus strains used in this experiment were found to be closely related when compared by serum neutralization tests [12]. This study and others [6-10] demonstrate the ability of rotaviruses to be transmitted and to produce illness in heterologous species. Unfortunately, there is insufficient information to evaluate the possibility of inter-species infection under natural conditions. However, in view of the many successful experiments, it seems reasonable that inter-species spread of rotaviruses would be possible under the field conditions also. If this could be proven, it might be interesting to investigate the possible existence of just o n e rotavirus which is potentially able to infect regardless animal species. The genetic diversity that is known to exist among the rotaviruses isolated from the same animal species [14, 15] and the difficulty of finding the real electrophoretype considered C.LM.I.D. 7/3-4~C
178
G. CASTRUCC1et al.
t o b e specific f o r o n e a n i m a l species, p r o v i d e s g o o d e v i d e n c e i n s u p p o r t o f t h e h y p o t h e s i s proposed above. Acknowledgements--This work was supported by the Italian National Research Council, Progetto Finalizzato Controllo Malattie da Infezione, 1984 funds, and by the Italian Department of Education, research funds (40 and 60~) 1984. We are grateful to Mr. A. Picchietti, Director of "Centro Provinciale Svezzamento Vitelli", Tripoli San Giorgio, Mantova, and to his highly qualified staff for their important contribution to conduct this experiment.
REFERENCES 1. McNulty M. S. Rotaviruses. J. gen. ViroL 40, 1-18 (1978). 2. Flewett T. H. and Woode G. N. The rotaviruses. Archs ViroL 57, 1-23 (1978). 3. Cilli V. and Castrucci G. Viral diarrhea of young animals: a review. Comp. Immun. Microbiol. infect. Dis. 4, 229-242 (1981). 4. Schwers A., Hoyois Ph., Chappuis G., Dagenais L. and Pastoret P. P. Propagation of bovine rotavirus by cats and dogs. Ann. Rech. vet. 13, 303-308 (1982). 5. Woode G. N., Bridger J. C., Jones J. M., Flewett T. H., Bryden A. S., Davies H. A. and White G. B. B. Morphological and antigenic relationships between viruses (rotaviruses) from acute gastroenteritis of children, calves, piglets, mice and foals. Infect. Immun. 14, 804-810 (1976). 6. Bridger J. C., Woode G. N., Jones J. M., Flewett T. H., Bryden A. S. and Davies H. A., Transmission of human rotaviruses to gnotobiotic piglets. J. reed. Microbiol. 8, 565-569 (1975). 7. Middleton P. J., Petrie M. and Szymansky M. T. Propagation of infantile gastroenteritis virus (orbi group) in conventional and germ free piglets. Infect. lmmun. 12, 1276-1280 (1975). 8. Torres-Medina A., Wyatt R. G., Mebus C. A., Underdahl N. R. and Kapikian A. Z. Diarrhea caused in gnotobiotic piglets by the reovirus-like agent of human infantile gastroenteritis. J. Infect. Dis. 133, 22-27 (1976). 9. Mebus C. A., Wyatt R. G., Sharpes R. L., Sereno M. M., Kalica A. R., Kapikian A. Z. and Twiehaus M. J. Diarrhea in gnotobiotic calves caused by reovirus-like agent of human infantile gastroenteritis. Infect. Immun. 14, 471-474 (1976). 10. Snodgrass D. R., Madeley C. R., Wells P. W. and Angus K. W. Human rotavirus in lambs: infection and passive protection. Infect. Immun. 16, 263-274 (1977). 11. Castrucci G., Ferrari M., Frigeri F., Cilli V., Donelli G., Angelillo G. and Bruggi M. A study of cytopathic rotavirus strains isolated from calves with acute enteritis. Comp. Immun. Microbiol. Infect. Dis. 6, 253-264 (1983). 12. Castrucci G., Ferrari M., Frigeri F., Cilli V., Perucca L. and Donelli G. Isolation and characterization of cytopathic strains of rotavirus from rabbits. Archs Virol. (In press) (1984). 13. Castrucci G., Ferrari M., Frigeri F., Cilli V., Caleffi F., Aldrovandi V., Rampichini L. and Tangucci F. Experimental infection and cross protection tests in calves with cytopathic strains of bovine rotavirus. Comp. lmmun. Microbiol. Infect. Dis. 6, 321-332 (1983). 14. Oejeh C. K., Snodgrass D. R. and Herring A. J. Evidence for serotypic variation among bovine rotaviruses. Archs Virol. 79, 161 171 (1984). 15. Rodger S. M. and Holmes I. H. Comparison of the genomes of Simian, bovine, and human rotaviruses by gel electrophoresis and detection of genomic variation among bovine isolates. J. Virol. 30, 839-846 (1979).
COMPARATIVE
STUDY
BOVINE
AND
OF
0147-9571/8453.00+0.00 Pergamon Press Ltd
ROTAVIRUS
RABBIT
STRAINS
OF
ORIGIN
G. CASTRUCCI 1, F. FRIGERI l, M. FERRARI 2, V. CILLI t, V. ALDROVANDI 3, F. CALEFVl3 and R. GATTI 4 qstituto di Malattie Infettive, Profilassi e Polizia Veterinaria dell'Universita' di Perugia, Perugia, 2Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia, Brescia, 3Centro Provinciale Svezzamento Vitelli, Tripoli, MN, and 4Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia, Sezione di Mantova, Mantova, Italy Abstract--Newborn calves were susceptible to infection with a rotavirus strain isolated from rabbits and, conversely, rabbits 4~5 weeks of age became ill and some died when inoculated with the rotavirus strain of bovine origin. However, the latter isolate appeared to be more virulent than the rabbit isolate in either calves or rabbits. Moreover, in reciprocal cross protection tests, carried out in calves and in rabbits, the rabbit rotavirus antiserum afforded only weak protection to challenge infection with either the homologous or the heterologous virus. By contrast, the protective level of the bovine rotavirus antiserum was relatively high. Key words: Comparative study, rotavirus, bovine, rabbits
ETUDE COMPARATIVE DES SOUCHES DE ROTAVIRUS PROVENANT DE LAPINS ET DE BOVINS R6sum~--Des veaux nouveau-n6s se sont av+r6s sensibles 5. l'infection par une souche de rotavirus isol~e de lapins. Par ailleurs, des lapins, agbs de 4 5. 5 semaines, on bt6 rendus malades et certains sont morts par suite de l'inoculation d'une souche de rotavirus d'origine bovine. Cene derni6re souche semblait cependant plus virulente 5. la fois chez les veaux et chez les lapins. Des exp6riences portant sur la protection crois6e instaur6e par les antis6rums contre ces souches ont 6t6 men6es chez des veaux et chez des lapins. Elles ont d6montr6 qu'un antis6rum contre le virus provenant de lapins ne conf6rait qu'une protection limit6e contre les souches homologue et h&6rologue. Un plus haut degr6 de protection +tait cependant induit par l'antis6rum contre le rotavirus bovin. Mots-clefs: Etude comparative, rotavirus, bovins, lapins
INTRODUCTION From several comprehensive studies on rotaviruses [1-3], there are convincing indications that these viruses infect a variety of animal species in which they have been associated as the cause of diarrhea in the young. It has also been shown that rotavirus from one species can infect members of certain other species. Thus, propagation of bovine rotavirus in cats, dogs [4] and pigs [5] has been reported. Moreover, experimental infection of pigs [6-8], calves [9] and lambs [10], was obtained with human rotavirus. In view of the importance that inter-species transmission may have in the epidemiology of rotavirus infections it appeared worthwhile to extend such a comparative study as much as possible. The purpose of this report was to obtain data regarding the possibility that rotavirus strains from bovine and rabbits, respectively, can infect the heterologous host. Also, an attempt was made to induce a passive immunity in calves and in rabbits against the heterologous virus. 171
172
G. CASTRUCC1 et al.
MATERIALS AND METHODS Virus
Two strains of rotavirus, one of which, 81/36F, was from cattle [11] and the other, 82/311F, from rabbits [12], were used for the present study. Both viruses were at the 7th passage level in MA-104 cell cultures and their titers were 108.5o median tissue culture infectious doses (TCIDs0) per 0.2 ml. Animals Calves. Twenty-nine conventionally reared Friesian calves 2 3 days of age were used. All calves were from rotavirus unvaccinated cows, and were obtained from dairy farms where no history of enteric conditions had been recently reported. The calves had ingested colostrum from their dams at least once before they were transferred to the laboratory animal facilities. Thereafter, they were fed a milk substitute twice daily. For the experimental infection trial 13 calves were divided into three groups of 5, 6 and 2 animals, respectively. The calves of the first group were infected with the bovine rotavirus strain, whereas those of the second group received the rabbit rotavirus. Each of the remaining two calves was given Eagle's minimum essential medium (MEM) and served as control. The inoculum was given orally in a volume of 30 ml per calf. Infected and control calves were kept in separate accommodations and observed for 30 days. During this period each calf was inspected for the appearance of clinical signs of disease. Fecal swabs for virus isolation were taken at the time of infection (time 0), and again on post infection day (PID) 2, 4, 6, 8 and 10. In addition, blood samples for serology were obtained from the calves at time 0 and at PID 30. In order to enhance the antibody level, the calves which survived the infection were re-exposed to the virus on P I D 30. Each calf received the same virus as that given originally in a dose of 10ml by the oral route, and of 2 m l by subcutaneous inoculation. For oral administration, undiluted virus culture was used, whereas in the case of the subcutaneous injection the virus was mixed with an equal volume of Freund's incomplete adjuvant. Eighteen days later the calves were bled, the serum was separated, and pools were made according to the groups of calves. Thus, two pools were obtained which were referred to as bovine rotavirus and rabbit rotavirus antiserum, respectively. The bovine rotavirus antiserum had a titer of 1:128, while that of rabbit rotavirus antiserum was 1:180. These serums were used to conduct passive immunity tests. For cross protection tests, 16 calves were segregated into three groups of 6, 6 and 4 animals, respectively. Each calf of one group of 6 animals received 250ml of bovine rotavirus antiserum which was given orally on two consecutive days in a volume of 125 ml per day. The calves of the other group of 6 were given rabbit rotavirus antiserum according to the above procedure. The remaining 4 calves, each was given M E M and served as virus controls. Two hours after the first inoculation of the antiserum or M E M , the calves were challenge exposed according to the following method: each of the two challenge viruses (bovine rotavirus and rabbit rotavirus) was given orally in a volume of 30 ml to 3 calves of each of the two groups of immune serum treated animals, and to 2 calves of the control group. Fecal swabs for virus isolation and blood samples for serology were taken according to the procedure for the experimental infection trial. The calves were kept under observation for 30 days. Particular attention was paid to the character of the feces.
Rotaviruses from bovine and rabbits
173
Rabbits. Twenty-four New Zealand White rabbits, 4-5 weeks of age, were used in the experiment. The experimental infection trial was carried out in 12 rabbits, of which 5 received rabbit rotavirus, 5 the bovine rotavirus and 2, which served as control, were given MEM. The volume of inoculum was 2 ml, given by gastric tube. The three groups of rabbits were separated from one another just prior to inoculation and kept in three separate cages for the duration of the experiment (30 days). Stools were collected from each cage prior to inoculation (time 0) and on PID 2, 4, 6, 8 and 10. Rabbits were bled before inoculation and on PID 30. As in the case of the calves, the rabbits which survived in each group were used for the production of immune serum to be used in the cross immunity tests. The same procedure as described for the calves was followed, the only exception being the volume of inoculum. In the case of the rabbits it was 2 ml of undiluted virus culture given by the oral route, and 1 ml of a mixture, consisting of equal amounts of virus culture and Freund's incomplete adjuvant given by subcutaneous injection. Both serums which were obtained from rabbits had a titer of 1:180 when used in the cross protection tests. Cross protection tests were carried out in 12 rabbits in the same manner as for the analogous tests in the calves. Thus, 4 rabbits received rabbit rotavirus immune serum and 4 rabbits were given immune serum to the bovine rotavirus. The remaining 4 rabbits were given MEM. The inoculum was given by gastric tube in a volume of 10 ml per rabbit several times over a period of two consecutive days. Two hours after the first inoculation of antiserum or M E M , the rabbits were exposed to the two challenge viruses (rabbit rotavirus and bovine rotavirus). Each virus was inoculated by gastric tube in a volume of 2 ml to 2 rabbits in each of the two groups of immunized rabbits, and of the control group. Stools for virus isolation, and blood samples for serology, were collected at the intervals mentioned above. Virus isolation Attempts to recover the virus from rectal swabbings obtained from calves were made in MA-104 cell cultures as described elsewhere [11]. Recovery of virus from the rabbit stools was done in the same way as in the case of the calves, the only exception being that the stools were previously prepared as a 10~o suspension in MEM. When virus was isolated, its identity was determined by neutralization tests carried out with rabbit antiserum specific for 81/36F bovine rotavirus [11] or for 82/311F rabbit rotavirus [12].
Serologic tests The serums obtained from calves and rabbits were tested against both the homologous and the heterologous rotavirus strains. The tests were carried out in microtiter plates as previously reported [13]. RESULTS
Experimental infection Calves. The results are depicted in Table 1. All calves became depressed and anorexic after an incubation period of 2-5 days. The diarrhea first appeared (PID 2.6) in the calves that were inoculated with bovine rotavirus, and about 3 days later (PID 5.6) in the calves infected with the rabbit rotavirus. In all cases the feces were fluid, yellowish, and they quite
174
G . CASTRUCCI et al. Table 1. Response of calves to oral inoculation of rotavirus of either bovine or rabbit origin Diarrhea
lnoculum: rotavirus strain Bovine: 81/36F Rabbit: 82/311F Controls : MEM
No. of calves studied
Duration* days
Live-weight* gain: g per day
Survival rate ratio
Affected calves, No.
Onset* PID
5
5
2.6
8.0
50
2/5t
6
6
5.6
3.1
80
6/6
2
0
NA
NA
120
2/2
Virus recovery from fecal swabs, PID/ No. of calves 2/5, 8/4, 2/2, 8/6,
4/5, 6/3, 10/4 4/5, 6/4, 10/5.
Negative
*Average values, tDied on PID 8, 11 and 13, respectively. PID post infection day. MEM culture medium. NA not applicable.
often contained mucous and blood. All calves were afebrile. The diarrhea lasted from 3.1 (rabbit rotavirus infected group) to 8.0 (bovine rotavirus infected group) days. Three calves of those that had been infected with the bovine rotavirus died on PID 8, 11 and 13, respectively. All had lesions limited to the small intestines where a general thickening of the mucosa, particularly evident in the ileum, was found. All calves which received the rabbit rotavirus survived the infection. N o significant differences were observed in virus shedding between the two groups of infected animals. The control calves remained healthy and virus was not isolated from their feces. The serological response of the calves is shown in Table 2. Low antibody titers were found at the time of infection. On PID 30 the titers had increased by apporximately 3 to 4, two-fold dilution steps in calves infected with the rabbit rotavirus or with the bovine rotavirus, respectively. There were no significant differences in titers to the homologous or the heterologous virus. The antibody titers in the control calves did not vary significantly during the experiment. Rabbits. From Table 3 it is seen that the rabbits gave a similar response to inoculation of the two rotaviruses. The diarrhea appeared in 3 of the 5 inoculated with the rabbit rotavirus, and in 2 of 5 of those which were infected with bovine rotavirus. However, all were depressed, anorexic, and underwent a significant loss in condition. One rabbit in the group inoculated with rabbit rotavirus and 2 among those infected with bovine rotavirus died. At autopsy the intestines were found to be markedly congested and distended. Virus was consistently isolated from the pooled feces of each group. N o signs of diseases was observed in the two control rabbits, and their feces were negative for virus.
Table 2. Serological response of calves experimentally infected with rotavirus of bovine or rabbit origin Neutralizing antibody* to rotavirus strain: Inoculum: rotavirus strain Bovine: 81/36F Rabbit: 82/311F Controls : MEM
No. of calves studied
Day 0
PID 30
Day 0
PID 30
5
1:5
1:647
1:10
1:64t
6
1:20
1:130
1:18
1:130
2
1:24
1:16
1:12
1:8
Bovine 81/36F
Rabbit 82/311F
* Average values, tValues referred to the 2 calves which survived. Day 0 the day of infection. PID post infection day. MEM culture medium.
Rotaviruses from bovine and rabbits
175
Table 3. Response of rabbits to oral inoculation of rotavirus of either bovine or rabbit origin Diarrhea Inoculum: Rotavirus strain
Duration* days
No. of rabbits showing stunting
Survival rate ratio
Viral recovery from fecal pools on PID:
4.6
4.3
5
4/53"
2, 4, 6, 8
2
7.0
2.0
5
3/5~
2, 4, 6, 8
0
NA
NA
0
2/2
Negative
No. of rabbits studied
Affected rabbits, No.
Onset* PID
5
3
5
2
Rabbit: 82/311F Bovine: 81/36F Controls:
MEM
* Average values, tDied on PID 5, or :~on PID 8 and 28, respectively. PID post infection day. MEM culture medium. NA not applicable.
Table 4. Serological response of rabbits experimentally infected with rotavirus of bovine or rabbit origin Neutralizing antibody* to rotavirus strain: Inoculum: rotavirus strain Rabbit: 82/311F Bovine: 81/36F
Rabbit 82/311 F
Bovine 81/36F
No. of rabbits studied
Day 0
PID 30
Day 0
PID 30
5
1:3
1: 120t
1:2
1 : 114t
5
1:2
1:128:~
1;2
1:138~:
2
< 1:4
1:4
1:2
1:4
Controls:
MEM
* Average values, t Values referred to the four or Sthe three rabbits which survived. Day 0 day of infection. PID post infection day. MEM culture medium.
The behavior of the neutralizing antibody (Table 4) in the rabbits was generally similar to that reported above for calves. However, the increased antibody titers on PID 30 (about 6 steps) in the rabbits was higher than that registered for the calves. Cross protection tests Calves. The results are shown in Table 5. The immune serum to the bovine rotavirus completely protected the calves against challenge infection with the homologous virus in that the animals remained clinically normal. The same antiserum was also partially Table 5. Cross protection tests in calves with rotavirus of bovine and rabbit origin Diarrhea Immune serum to rotavirus strain Bovine: 81/36F
Rabbit: 82/311F
Challenge rotavirus strain Bovine: 81/36F Rabbit: 82/311F Rabbit: 82/311 F Bovine: 81/36F
No. of calves studied
Affected calves, No.
Onset* PID
Duration* days
Virus recovery from fecal swabs, PID/No. of calves
3
0
NA
NA
4/1,6/1
3
2
5.5
2.5
2/1,4/1, 6/2
3
2
4.5
5.5
2/1,4/3, 6/2
3
3
5.0
5.6
2/1,4/2, 6/1,8/2
2
2
4.5
8.0
2/2, 4/2, 6/2, 8/2
2
2
3.5
9.0
2/2, 4/2, 6/2, 8/2, 10/1
Controls:
MEM
Bovine: 81/36F Rabbit: 82/311F
* Average values. PID post infection day. NA not applicable. MEM culture medium.
176
G. CASTRUCCI et al.
Table 6. Cross protection tests in rabbits with rotavirus of bovine and rabbit origin Diarrhea Immune serum to rotavirus strain Rabbit: 82/311F
Bovine: 81/36F
Controls: MEM
Challenge rotavirus strain
Survival rate ratio
Virus recovery from fecal pools on PID:
No. of rabbits studied
Affected rabbits, No.
Onset* PID
Duration* days
Rabbit: 82/311F Bovine: 81/36F Bovine: 81/36F Rabbit: 82/31 l F
2
0
NA
NA
2/2
Negative
2
2
9,0
7.0
0/2¢
4, 6, 8, 10
2
1
10.0
4.0
2,'2
4, 6
2
1
10.0
6.0
I/2 +
2, 4, 6
Rabbit: 82/311F Bovine: 81/36F
2
2
7.0
15.0
I/2§
2, 4, 6, 8, 10
2
2
8.0
10.0
1/211
2,4, 6, 8, 10
* Average values. PID post infection day. NA not applicable. MEM culture medium. Death of the rabbits occurred on PID: I1 and 167, 16 ++, 29 § and 14 ]].
protective for the calves which were challenge exposed to the heterologous virus, i.e., the rabbit rotavirus. In this group of calves, one did not show any signs of disease, while the other two had a mild diarrhea of short duration. The rabbit rotavirus immune serum appeared to be, in general, less protective than the bovine rotavirus antiserum, in that two of the three calves that received the homologous virus, and all three which were given the heterologous virus, had diarrhea, although its duration was shorter compared with that of the control calves. Virus was recovered only twice from one calf in the group immunized with the bovine rotavirus antiserum. In the other groups virus was isolated several times, especially in those calves which had rabbit rotavirus antiserum and were challenge infected with the bovine rotavirus. The control calves reacted with clinical signs of disease, i.e. depression, stunting, and diarrhea, without there being any significant difference with regard to the virus inoculated. Virus was reisolated from the fecal swabbings from PID 2 through 8 (bovine rotavirus) or 10 (rabbit rotavirus). All calves had antibody titers at time 0 ranging from 1:8 to 1:32. At PID 30 the titers had increased slightly to 1:64. Rabbits. From Table 6 it is apparent that the immune serum to the rabbit rotavirus completely protected the rabbits which were challenge infected with the homologous virus, whereas it was without any effect in rabbits which were exposed to challenge infection with the bovine virus. The latter animals had diarrhea and died on PID 11 and 16, respectively. The intestines of these two rabbits were generally congested and edematous. Of the rabbits immunized with bovine rotavirus antiserum, and then challenge exposed to the homologous virus, one out of two had mild diarrhea, but both survived. One of the two rabbits that were challenged with the rabbit rotavirus was fully protected, whereas the other underwent diarrhea and died on PID 16. The intestines of this rabbit were congested and markedly distended. Virus was not recovered from the rabbits immunized with rabbit rotavirus antiserum and infected with the rabbit rotavirus, whereas in the case of rabbits which had the same antiserum, but were challenge exposed to the bovine rotavirus, the feces were positive for virus from PID 4 to 10. The virus was also isolated from rabbits immunized with bovine rotavirus antiserum, i.e. on two days (PID 4, 6) in
Rotaviruses from bovine and rabbits
177
the rabbits exposed to the bovine rotavirus and three times (PID 2, 4, 6) in those exposed to the rabbit rotavirus. In each group the rabbits that served as control of the challenge viruses had profuse diarrhea, and one rabbit in each group died. The serologic situation was the same as described for the calves, and no differences were detected among the groups: the titer of the antibody rose from an average value of 1:4 at time 0 to a level of 1:32 on PID 30.
DISCUSSION The results of this study disclose that a rotavirus, isolated from calves, can be propagated in rabbits in which it induces the classic signs of rotaviral infection. Conversely, a rotavirus of rabbit origin is able to cause a clinically manifested infection in calves. This conclusion is based on the finding that the five rabbits inoculated with the bovine rotavirus became stunted, two had a severe diarrhea, two died, and the virus was reisolated from their feces. On the other hand, of the 6 calves that were exposed to the rabbit rotavirus, all had diarrhea, they suffered loss of condition, and excreted virus consistently in the feces. However, it is of interest that the 81/36F strain of bovine rotavirus seems to be more virulent than the rabbit isolate. In this experiment the lowest live-weight gain per day (50 g) was registered by the calves inoculated with the bovine rotavirus, whereas higher values (80 g) were gained by the calves which received the rabbit rotavirus. The average daily gain for the control calves was 120 g. Moreover, the bovine isolate caused the death of 3 calves out of 5, whereas all calves inoculated with the rabbit isolate survived the infection. This finding appears to be in agreement with previous observations which demonstrated that the same strain of bovine rotavirus (81/36F) used in this experiment was the most virulent when compared with two other bovine isolates [13]. The results of the cross protection tests show that the bovine rotavirus seems to be more immunogenic than the rabbit isolate. This is seen in calves where the immune serum to the bovine isolate was fully protective in all animals that were challenge exposed to the homologous virus, whereas the immune serum to the rabbit isolate failed to protect 1 calf out of 3 infected with the rabbit rotavirus. This result seems to reinforce the hypothesis that the 81/36F bovine rotavirus strain might be considered to be a predominant antigenic type [13]. The antibody produced in calves and in rabbits neutralized the homologous and the heterologous virus alike. This is not surprising since the bovine and the rabbit rotavirus strains used in this experiment were found to be closely related when compared by serum neutralization tests [12]. This study and others [6-10] demonstrate the ability of rotaviruses to be transmitted and to produce illness in heterologous species. Unfortunately, there is insufficient information to evaluate the possibility of inter-species infection under natural conditions. However, in view of the many successful experiments, it seems reasonable that inter-species spread of rotaviruses would be possible under the field conditions also. If this could be proven, it might be interesting to investigate the possible existence of just o n e rotavirus which is potentially able to infect regardless animal species. The genetic diversity that is known to exist among the rotaviruses isolated from the same animal species [14, 15] and the difficulty of finding the real electrophoretype considered C.LM.I.D. 7/3-4~C
178
G. CASTRUCC1et al.
t o b e specific f o r o n e a n i m a l species, p r o v i d e s g o o d e v i d e n c e i n s u p p o r t o f t h e h y p o t h e s i s proposed above. Acknowledgements--This work was supported by the Italian National Research Council, Progetto Finalizzato Controllo Malattie da Infezione, 1984 funds, and by the Italian Department of Education, research funds (40 and 60~) 1984. We are grateful to Mr. A. Picchietti, Director of "Centro Provinciale Svezzamento Vitelli", Tripoli San Giorgio, Mantova, and to his highly qualified staff for their important contribution to conduct this experiment.
REFERENCES 1. McNulty M. S. Rotaviruses. J. gen. ViroL 40, 1-18 (1978). 2. Flewett T. H. and Woode G. N. The rotaviruses. Archs ViroL 57, 1-23 (1978). 3. Cilli V. and Castrucci G. Viral diarrhea of young animals: a review. Comp. Immun. Microbiol. infect. Dis. 4, 229-242 (1981). 4. Schwers A., Hoyois Ph., Chappuis G., Dagenais L. and Pastoret P. P. Propagation of bovine rotavirus by cats and dogs. Ann. Rech. vet. 13, 303-308 (1982). 5. Woode G. N., Bridger J. C., Jones J. M., Flewett T. H., Bryden A. S., Davies H. A. and White G. B. B. Morphological and antigenic relationships between viruses (rotaviruses) from acute gastroenteritis of children, calves, piglets, mice and foals. Infect. Immun. 14, 804-810 (1976). 6. Bridger J. C., Woode G. N., Jones J. M., Flewett T. H., Bryden A. S. and Davies H. A., Transmission of human rotaviruses to gnotobiotic piglets. J. reed. Microbiol. 8, 565-569 (1975). 7. Middleton P. J., Petrie M. and Szymansky M. T. Propagation of infantile gastroenteritis virus (orbi group) in conventional and germ free piglets. Infect. lmmun. 12, 1276-1280 (1975). 8. Torres-Medina A., Wyatt R. G., Mebus C. A., Underdahl N. R. and Kapikian A. Z. Diarrhea caused in gnotobiotic piglets by the reovirus-like agent of human infantile gastroenteritis. J. Infect. Dis. 133, 22-27 (1976). 9. Mebus C. A., Wyatt R. G., Sharpes R. L., Sereno M. M., Kalica A. R., Kapikian A. Z. and Twiehaus M. J. Diarrhea in gnotobiotic calves caused by reovirus-like agent of human infantile gastroenteritis. Infect. Immun. 14, 471-474 (1976). 10. Snodgrass D. R., Madeley C. R., Wells P. W. and Angus K. W. Human rotavirus in lambs: infection and passive protection. Infect. Immun. 16, 263-274 (1977). 11. Castrucci G., Ferrari M., Frigeri F., Cilli V., Donelli G., Angelillo G. and Bruggi M. A study of cytopathic rotavirus strains isolated from calves with acute enteritis. Comp. Immun. Microbiol. Infect. Dis. 6, 253-264 (1983). 12. Castrucci G., Ferrari M., Frigeri F., Cilli V., Perucca L. and Donelli G. Isolation and characterization of cytopathic strains of rotavirus from rabbits. Archs Virol. (In press) (1984). 13. Castrucci G., Ferrari M., Frigeri F., Cilli V., Caleffi F., Aldrovandi V., Rampichini L. and Tangucci F. Experimental infection and cross protection tests in calves with cytopathic strains of bovine rotavirus. Comp. lmmun. Microbiol. Infect. Dis. 6, 321-332 (1983). 14. Oejeh C. K., Snodgrass D. R. and Herring A. J. Evidence for serotypic variation among bovine rotaviruses. Archs Virol. 79, 161 171 (1984). 15. Rodger S. M. and Holmes I. H. Comparison of the genomes of Simian, bovine, and human rotaviruses by gel electrophoresis and detection of genomic variation among bovine isolates. J. Virol. 30, 839-846 (1979).