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Congenital infection of pigs with ruminant-type pestiviruses
J. Comp. Path. 1994Vol. 111, 151-163
Congenital Infection of Pigs with Ruminant-type Pestiviruses D.J. Paton and S. H. Done Central Veterinary Laboratory (Weybridge), New Haw, Addlestone, Surrey KT15 3NB, UB"
Summary Congenital infections of pigs were induced with two ruminant-type pestiviruses isolated from pigs. One of the viruses was bovine viral diarrhoea virus-like and the other border disease virus-like. Both produced symptoms similar to those observed with low virulence strains of classical swine fever virus. A striking effect of persistent virus infection in post-natal life was stunting in viraemic animals. It was also shown that a congenitally infected pig shed virus for 2.5 years and in sufficient quantity to infect other pigs, even by indirect contact. Unlike ruminants, congenitally infected pigs sometimes had persistent viraemia but eventually eliminated the virus. Clearance of virus from the blood was related to the appearance of neutralizing antibodies. However, clearance from the tissues sometimes took as much as 5 months longer than from the blood.
Introduction Classical swine fever virus (CSFV), bovine viral diarrhoea virus (BVDV) and ovine border disease virus (BDV) are serologically related members of the genus Pestivirus within the family Flaviviridae. In Britain, CSFV has been eradicated, whereas B V D V and BDV, termed "ruminant-type" pestiviruses, are widespread. The occasional porcine infections produced by these viruses do not constitute a significant disease problem, but distinguishing such infections from those caused by CSFV can be difficult both on the farm and in the laboratory. Natural infection of pigs with ruminant-type pestiviruses has been demonstrated by serological surveys (Snowdon and French, 1968) and by occasional virus isolations (Fernelius et al., 1973). Such infections are usually subclinical but have sometimes been associated with reproductive or neonatal illness (Terpstra and Wensvoort, 1988). They have generally been attributed either to the use of pestivirus-contaminated vaccines (Vannier et al., 1988; Wensvoort and Terpstra, 1988) or to contact between pigs and ruminants (Terpstra and Wensvoort, 1992). T w o ruminant-type pestiviruses were isolated from British pigs in 1987 and 1991, in the course of investigations of suspected cases of swine fever. The virus obtained in 1987 (strain "87/6") was shown to be BDV-like by genetic (Roehe et al., 1992) and antigenic (Edwards and Sands, 1990; Paton et al., 1994) 0021-9975/94/060151+ 13 $08.00/0
9 1994AcademicPressLimited
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D.J. Paton and S. H. Done
analysis. It also b e h a v e d like an ovine pestivirus in e x p e r i m e n t a l l y infected p r e g n a n t sows and sheep, crossing the p l a c e n t a b u t causing no discernible illness in adults (Roehe, 1991). T h e "91 / 1" strain o f virus, which was isolated from a farm on which b o t h cattle and pigs were kept, was shown to be B V D V like a n d a p p e a r e d also to cause illness only as a result o f congenital porcine infection ( P a t o n et al., 1992). T h e present study was designed to e x a m i n e the consequences o f in-utero infection with these viruses.
Materials and Methods
Viruses An 87/6 virus challenge inoculum was prepared from the tonsil of a pig affected in the original outbreak, by three serial passages in PK15 cells. A 91/1 virus challenge inoculum consisted of serum from a persistently infected steer thought to have been the index case in the original outbreak.
Infection and Monitoring of Experimental Animals Pigs obtained from the herd of specific pathogen-free animals at the Central Veterinary Laboratory were all initially seronegative to pestiviruses. Sows were served naturally and pregnancy was confirmed 3 weeks later by vaginal biopsy. The route and timing of challenge inoculation for each of sows 1-6 are shown in Table 1. At intervals after challenge, blood samples were collected to monitor seroconversion, and a blood sample collected 83-90 days after service was used to diagnose pregnancy by oestrone sulphate assay. To induce farrowing at a convenient time, pregnant sows were given an injection of 184 gg ofcloprostenol sodium (Planate, Pitman-Moore Ltd) 113 days after servme. At birth, the piglets were weighed and pre-colostral blood samples were collected from the umbilical cord. Subsequent progress of piglets was monitored at intervals by clinical observation, weighing and collection of blood for virological and serological examination. Weight differences between litter mates born viraemic and non-viraemic were analysed statistically by the t-test. Pigs were weaned at 5 weeks of age and usually retained until approximately one year old. Post-mortem samples, including, tonsil, submandibular lymph node, lung, liver, spleen, kidney, ileum, mesentenc lymph node and ovary or testis, were collected from most pigs after death or slaughter. Strain 87/6 Study. Four pregnant sows (nos 1-4) were inoculated with the 87/6 virus, two intranasally and two by intra-uterine injection via a laparotomy (Table 1). Intranasally inoculated sows (nos 1 and 2) each received 3 ml of culture supernate containing 105STCID~0 of virus. In sows 3 and 4 each conceptus received a 0"5 ml volume containing 102'~'CID50. Strain 91/1 study. Two sows (nos 5 and 6) were inoculated intranasally on day 27 of pregnancy with 2 ml of serum containing 105STCID50 of the 91/1 virus. Transmission study. Pig-to-pig transmission of strain 87/6 virus from a congenitally infected boar that developed a persistent viraemia was investigated by means of two seronegative 10-week-old pigs, used as sentinels. The sentinels and the persistently infected boar were moved in rotation between three strawed pens, as follows. Each day the boar was moved into a clean pen, and the sentinels were moved into the boar's vacated pen, which had not been cleaned out. After a week, the sentinels were moved to separate accommodation, and after a further 3 and 6 weeks they were bled and tested for the presence of 87/6 virus neutralizing antibodies.
Congenital
Pestivirus
Infections
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in Pigs
Table 1 Summary of sow inoculations and consequences
Observation
N u m b e r of piglets belonging to sow no.
1 (87/6;44;i.n.)
N u m b e r of piglets Whole litter Mummies Stillbirths Other abnormality Precolostral status of liveborns V+/Ab+ V+/AbV-/Ab+ V -/Ab Not tested Piglet progress Survival > 1 wk Post-weaning illnessw Stunting Post-natal viraemia Post-natal seroconversion Persistent infection ( > 6 months)
--------
------
2 (87/6;37;i.n.)
13 -1* 2"~ 1 3 4 4
7 --
--
3 (87/6;35;i,u.)
4 (87/6;42;i.u.)
12 ---.
6 (91/1;27;i.n.)
-----
11 --
--
5 -6
4 2 1* 1++ .
9
5 (91/1;27;i,n;)
.
-3
1 ----
11 1 6 6 10 2
-------
1 JJ
.
---
------
6 3 4 4 2 1
Brackets show: (infecting strain; day of pregnancy on which sow inoculated; route of inoculation). i.n., Intranasal; i.u., intrauterine, *Virus positive; ~'haemorrhages and oedema; ++meningocoele; IIoedema of ear pinnae; w leading to death or euthanasia. V + / A b + , V + / A b - , V - / A b + , V - / A b - : see text.
Virus Detection and Typing V i r u s e s w e r e i s o l a t e d i n P K 1 5 cell c u l t u r e s k n o w n to b e f r e e o f p e s t i v i r u s e s a n d pestivirus inhibitors. Virus-infected cultures were identified by an immunoperoxidase staining method and epitopic differences between isolates were compared with a panel of 13gp53-specific BVDV monoclonal antibodies ( P a t o n et al., 1 9 9 2 ) . T i s s u e homogenates and nasal swabs received two passages before staining, whereas sera received only one. Some blood samples from neonatal piglets were tested for pestivirus antigen by ELISA, since this may detect virus that cannot be cultured because of prior contact with colostral antibody. The method, which detects a pestivirus protein in h e p a r i n i z e d b l o o d , w a s a s d e s c r i b e d b y D r e w et al. ( 1 9 9 3 ) .
Serology Serum-virus neutralization tests (VNT) were carried out according to standard protocols (Edwards, 1990). The virus used was either the 87/6 virus or a porcine isolate derived from the same farm as the 91/1 virus, whichever accorded with the challenge s t r a i n . T h e t i t r e s w e r e e x p r e s s e d a s t h e d i l u t i o n (log~0) n e u t r a l i z i n g 5 0 % o f v i r u s r e p l i c a t e s , a n d a t i t r e o f < 1-0 w a s c o n s i d e r e d n e g a t i v e . I n a m o d i f i e d t e s t , t o c o m p a r e the neutralization of two viruses by a single serum, ten-fold virus dilutions were incubated with doubling serum dilutions.
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D.J. Paton and S. H. Done
Two CSFV-specific competition ELISAs based on different pairs of monoclonal antibodies were used to measure antibody responses in pigs that developed very high titres of neutralizing antibody. One was the assay of Wensvoort et al. (1988) and the other was an equivalent test designed at our laboratory (Paton, unpublished).
Histopathology Post-mortem samples for histopathological examination were fixed in buffered formalin, embedded in paraffin wax, and stained with haematoxylin and eosin (HE).
Results
Monitoring of Sows and Piglets The main findings are summarized in Table 1.
Strain 87/6 study. None of the sows (nos 1 4) showed any signs of illness, but all "seroconverted" to 87/6 virus. Sow 1. This animal was found to be non-pregnant by oestrone sulphate assay at 85 days after service and failed to farrow. Sow 2. Five of the piglets born to this sow died in the first three days of life, all with macroscopical haemorrhages and oedema. Three of these animals were born viraemic and seronegative ( V + / A b - ) , one was born viraemic and seropositive ( V + / A b + ) , and one was born non-viraemic and seropositive ( V - / A b + ) . The other members of the litter were born V - / A b - ( f o u r ) or V - / A b + (four) and remained completely healthy. Piglets born seropositive had antibody titres in the range of 2"2-2"6. Sow 3. This animal had a litter of 12 apparently normal piglets, one of which was overlain at one day old. Nine of the piglets were V + / A b - at birth, and the other three were not tested. After ingesting colostrum, all of the piglets became V - / A b + (antibody titres of 1-5-2.4 at 3 weeks of age), but when colostral antibodies waned at between 8 and 20 weeks of age, six piglets became V+/Ab-, and five actively seroconverted without a detectable post-natal viraemia. These pigs developed high titres of neutralizing antibody (maximum titres 4"2-4"8) and all gave strongly positive results in both CSFV-specific ELISAs. Five of the six pigs that developed post-natal viraemia subsequently seroconverted and became non-viraemic at between 20 and 25 weeks of age (maximum antibody titres 3"7-4.6). It was nevertheless possible to isolate virus from the tissues of three of these five pigs that were killed up to 8 months after seroconversion. T w o of these animals, killed 5 months after seroconversion, yielded virus from a wide range of (but not all) tissues, whereas the third, killed 8 months after seroconversion, yielded virus only from the testis. The other two pigs, killed 10 and 12 months after seroconversion, were completely virus negative. Viraemic animals were observed to be stunted, in comparison with their non-viraemic litter mates (Fig. 1), and seroconversion seemed to be related to
Congenital Pestivirus Infections in Pigs
Fig. 1.
155
Stunted and normal pig. Stunted pig was persistently viraemic with 87/6 virus; normal pig was a non-viraemic litter mate.
an improvement in growth (Fig. 2). A single pig remained viraemic until its death at 2"5 years of age. Serum virus titres for this animal ranged from 1053/ml at 5 months of age to 1025/ml 15 months later. Virus was also recovered from nasal swabs collected when the pig was 16 and 23 months old. The animal was undersized and had several intermittent periods of nonspecific illness and anorexia over a 12-month period. Shortly before being killed on humane grounds it developed a severe dyspnoea. Sow 4. Only four piglets were born, two being mummified and one stillborn with a meningocoele. The fourth pig appeared normal but died within 24 h. Virus was isolated from the tissues of the stillborn pig, and the liveborn pig was V+/Ab-.
Strain 91/1 study. Neither of the sows (nos 5 and 6) showed any signs of illness, although both seroconverted to 91/1 virus. Sow 5. This animal was negative in the pregnancy test 90 days after service and failed to farrow. Sow 6. This sow farrowed 11 piglets that were normal at birth, except one which had oedematous ear pinnae. Thereafter, the progress of the litter was similar to that observed for sow 3. Six piglets born V - / A b were all seropositive at 3 weeks old and thereafter their antibody titres declined slowly. In most cases, despite regular bleeding and no indication of active seroconversion, residual antibody (titres of 1"0-1"8) could still be detected at the end of the experiment, when the pigs were 10-11 months old. Five piglets born V + / A b - fell into two groups. Three became non-viraemic at 3 weeks old, with low or undetectable antibody titres. By 7 weeks of age, they were again V + / A b - ,
156
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although in one pig virus was only detectable by ELISA and not by virus isolation. The other two pigs developed high antibody titres (maximum titres of >4.6 and 4-0 in VNT, and positive in CSFV-specific ELISAs), associated with clearance of virus from serum. T h e y differed in that one seroconverted soon after birth and the other at about 20 weeks of age. When these animals were killed at 10 months old, virus could be isolated from tissues (testis, ileum, spleen, tonsil) of the one that had showed delayed seroconversion, but not from the one that had seroconverted earlier. The pigs born with viraemia grew poorly as compared with their litter mates, except for the animal that seroconverted just after birth, and body weights at 21 and 52 days were significantly lower for the "viraemic-at-birth" group (95% confidence by ttest; 99% confidence if early seroconverting pig excluded). The three pigs that did not seroconvert died or were killed due to illness at between 83 and 196 days of age. All appeared stunted compared with litter mates. The first pig died suddenly, whereas the next two became listless and lethargic over a 2-3 day period and did not respond to antibiotic therapy.
Transmission study. Three weeks after their last indirect contact with the persistently infected boar, only one sentinel pig from the transmission study had seroconverted. When next bled, three weeks later, both were seropositive. Pathological Observations Litter of sow 2. Five pigs that died in the first three days of life were examined. A
Congenital Pestivirus Infections in Pigs
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stillborn and two liveborn piglets showed pinhead-sized haemorrhages and anasarca at birth, but the other two were apparently normal until 1-3 days old. The five dead piglets showed widespread internal oedema and ascites. The spleen, liver and lymph nodes were congested and enlarged and fine haemorrhages were detected in lymph nodes, bladder and kidneys. Litter of sow 3. Most necropsies were conducted on animals killed at 11-14 months of age. Gross pathology was unremarkable. Histopathology carried out on one animal revealed a mild hyperplasia of the Peyer's patches and the spleen. The persistently viraemic animal killed at 2"5 years old showed a marked lymphoid depletion, lymph nodes being hard to find (Fig. 3) and Peyer's patches not evident. There were omental adhesions to the spleen, which was itself severely atrophied and nodular (Fig. 4). Histopathological examination confirmed lymphoid depletion, revealed an absence of germinal centres, and demonstrated focal haemorrhagic lymphadenitis. Litter of sow 4. The liveborn and stillborn piglets showed mild subcutaneous oedema, swollen liver and spleen, and fine petechiation of the surface of the kidneys. The stillborn animal had a large meningocoele protruding from the dorsal cranium. Litter of sow 6. The first pig to die had a pale and watery carcass with enlarged and haemorrhagic lymph nodes. Histopathological examination confirmed haemorrhage within lymph nodes, liver and kidney (Fig. 5). Widespread deposition of haemosiderin and increased splenic haemopoiesis indicated that this haemorrhage had been of a chronic nature. Other findings included oedema and lymphocyte depletion within lymph nodes, villous atrophy of the small intestine, Peyer's patch hyperplasia, and abscessation of tonsillar crypts. The second pig to die had a pale, watery and oedematous carcass, the oedema being most pronounced in the submandibular region. All lymph nodes were enlarged and haemorrhagic and further haemorrhages were found on the thymus, epicardium and cerebellum. Histopathological findings were similar to those for the first pig with, in addition, evidence of myocarditis. The last pig to die because of illness was also normal in appearance. Submandibular, mesenteric and iliac nodes were enlarged, with a firm rubbery texture and a pinkishred discolouration. Histopathological examination revealed haemorrhage and oedema of the submandibular lymph node.
Comparative Neutralization and Monoclonal Antibody Typing
Strain 91/1 virus, isolated from the testis of an 11-month-old pig 5 months after it had seroconverted, was compared with virus obtained from a precolostral serum sample taken from the same animal. Serum collected from the same pig at the time of the second virus isolation neutralized both viruses equally effectively in vitro (Fig. 6), and no epitopic differences were detected in the major envelope protein of the virus by monoclonal antibody typing.
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D.J. Paton and S. H. Done
Fig. 3.
Lack of visible mesenteric lymph nodes in a 2"5-year-old, persistently infected pig at death
Fig. 4.
Spleen of a 2.5-year-old, persistently infected pig at death.
Fig. 5.
Kidney from newborn congenitally infected piglet, showing haemorrhages. HE. x300.
159
Congenital Pest/virus Infections in Pigs
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C o m p a r a t i v e n e u t r a l i z a t i o n of two 91/1 viruses isolated from the same congenitally infected pig before and after seroconversion. Virus 1 (-t~-) was isolated from pre-colostral serum a n d virus 2 (-~j from p o s t - m o r t e m tissues collected 320 days later. Both viruses were tested against a p o s t - m o r t e m serum sample.
Discussion
In the two field outbreaks from which the 87/6 and 91/1 viruses were isolated, disease was only seen in pigs less than 3 months old, and in the second outbreak affected piglets were all litter mates, some of which were shown to have been persistently viraemic (Paton et al., 1992). This suggested that the illness observed was a delayed consequence of congenital infection. In other reports, disease in pigs due to ruminant-type pestiviruses has generally affected young animals, pregnant sows, or both (Terpstra and Wensvoort, 1988; Vannier et al., 1988; Wensvoort and Terpstra, 1988). In the present study, no illness was seen in any of the post-natally infected pigs.
160
D.J. Paton and S. H. Done
The stage of gestation at which infection with pestiviruses occurs is known to affect the outcome (Roeder et al., 1986; van Oirschot and Terpstra, 1989). Infection in early pregnancy, before fetal immunocompetence develops, favours persistent infection and subsequent disease in early post-natal life. In the study with strain 87/6, different challenge times and routes of inoculation were used. To ensure that some viraemic piglets were born, two litters were inoculated through the wall of the uterus by means of laparotomy. Direct fetal inoculation with 87/6 virus at 35 days of gestation produced large numbers of viraemic piglets. In the study with strain 91/1, on the assumption that the development of maternal viraemia and transmission of virus would take approximately 8 days, an intranasal challenge was given on the 27th day of gestation. Two sows, one infected with the 91/1 virus and one with the 87/6 virus, failed to farrow, having been inoculated intranasally at 27 and 44 days of gestation respectively and having been confirmed by vaginal biopsy as pregnant 3 weeks after service. It was not proved that the inoculations had a role in the pregnancy terminations, but ruminant-type pestiviruses have been implicated previously as a cause of infertility in pigs (Vannier et al., 1988). Furthermore, a third sow had a litter of only four pigs, despite the presence of 14 conceptuses when 87/6 virus was inoculated through the uterus 42 days after service. Abnormalities detected at birth in a number of different litters included mummification, haemorrhages, oedema and meningocoele. All of these lesions have been observed in pigs congenitally infected with CSFV (Trautwein, 1988; van Oirschot and Terpstra, 1989). In some pigs illness was delayed and appeared at between one day and 2"5 years later. One pig died when 2 days old, having been born V - / A b + , and another was overlain, but these apart, all of the morbidity and mortality occurred in viraemic animals. Pigs that died soon after birth tended to show few premonitory signs but marked haemorrhages and oedema at death. Those that died or were killed later often had a period of poorly defined ill-thrift and showed few striking lesions at necropsy. The most consistent feature was the presence of enlarged, rubbery lymph nodes with a pink-red discoloration. Unlike cattle, pigs apparently do not develop a mucosal disease-like syndrome associated with superinfection by cytopathogenic biotypes of pestivirus. However, late onset disease is a well recognized feature of pre-natal porcine infection with low virulence strains of both CSFV (van Oirschot and Terpstra, 1977) and BVDV (Terpstra and Wensvoort, 1988). The trigger for such onset, as in the present cases, is not known and no cytopathogenic viruses were identified from pigs in this study. Possibly some other change in virus virulence took place, or the disease was immunologicatly mediated. Most viraemic pigs were readily recognized because of their poor growth as compared with that of healthy litter mates. Stunting is a well known consequence of congenital BVDV infection in ruminants (Roeder et al., 1986) and has also been described in pigs (Leforban et al., 1990). The mechanisms that underlie such growth retardation are not known, but are possibly related to generalized viral interference with cell division or to specific disturbance of
Congenital Pestivirus Infections in Pigs
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particular cell functions, such as hormone production (Sawyer et al., 1991). Whatever the mechanisms, it was noticed that pigs that seroconverted when still growing subsequently resumed normal growth and were not significantly lighter than healthy litter mates at 10 months of age. This suggests that growth retardation is the result of a continuing process and not of an irreversible lesion acquired in early development. Most of the pigs born with viraemia either died, were killed due to illness, or seroconverted and became non-viraemic. Only one pig remained a long-term virus shedder, and this was an animal that had been infected in utero with the 87/6 virus. Once colostral antibody was cleared from this animal, virus was isolated from serum and nasal swabs on every occasion on which samples were taken, and the virus titre in the serum was similar to that reported for persistently viraemic ruminants (Drew and Edwards, personal communication). This pig was also able to infect others, even by indirect contact, suggesting that ruminant-type pestiviruses may be maintained in pigs without a requirement for a continuous ruminant contact. The delayed seroconversion in the second sentinel pig suggested that it might have beeen infected from its transiently infected pen-mate rather than from the persistently infected pig. The causes of the terminal illness and of the severe immunological depletion in the persistently infected pig are unknown. A general depletion of the lymphoid system has been described in pigs suffering from chronic classical swine fever (van Oirschot, 1988). M a n y of the pigs born with viraemia seroconverted in later life. This was associated with an immediate clearance of virus from serum, but a delayed clearance from the tissues. It also seemed to be associated with an improvement in growth rate and survival. Delayed recovery from congenital infection with pestiviruses is generally not seen in ruminants but has been reported in a persistently infected lamb (Nettleton et al., 1992). This is possibly an indication of a difference in the speed of maturation of the immune system of young ruminants and pigs. A chronic form of CSFV infection has been described in pigs, in which antibodies appeared but failed to eliminate virus (Mengeling and Cheville, 1968). However, unlike the results reported here, recovery was incomplete and the pigs ultimately died. T e m p o r a r y persistence of virus, with occasional continued persistence in the tissues, in the presence of neutralizing antibodies has been described for congenital rubella in man (Rawls, 1974). In the present study, the appearance of serum antibodies was related to the clearance of virus from serum but not tissues. Furthermore, neither comparative neutralization nor monoclonal antibody typing revealed any evidence that virus persistence was due to phenotypic changes that resulted in an acquired resistance to neutralization by antibody. These findings suggest that neutralizing antibodies play a very limited role in the clearance of BVDV from ceils. This is consistent with in vitro studies by Curia and McClurkin (1981), who showed that antibody was ineffective in clearing BVDV from persistently infected cell cultures. It is also in agreement with the known incapacity of colostrum-derived antibodies to clear virus permanently from persistently infected newborn animals. Sera from some of the pigs that had recovered from congenital infection
162
D.J. Paton and S. H. Done
with either B V D V or BDV gave false positive results when assessed with ELISAs designed to be CSFV-specific. The high levels of antibody probably caused steric inhibition to the binding of the CSFV-specific monoclonal antibodies employed in the tests. Although naturally occurring cases of this type are probably very rare, they are precisely the ones likely to be confused clinically with CSFV infection. This emphasizes the importance of confirming ELISA-positive results by comparative neutralization tests, especially when new outbreaks of disease are being investigated.
Acknowledgments
We are indebted to many staff at the Central Veterinary Laboratory for assistance in maintaining the experimental animals and for providing technical laboratory assistance.
References
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Paton, D.J., Simpson, V. and Done, S.H. (1992). Infection of pigs and cattle with bovine viral diarrhoea virus on a farm in England. Veterinary Record, 131,185 188. Paton, D.J., Sands, J.J. and Edwards, S. (1994). Border disease virus: delineation by monoclonal antibodies. Archives of Virology, in press. Rawls, W. E, (1974). Viral persistence in congenital rubella. Progress in Medical Virology, 18, 273-288. Roeder, P. L., Jeffrey, M. and Cranwell, M. P. (1986). Pestivirus fetopathogenicity in cattle: changing sequelae with fetal maturation. Veterinary Record, 118, 44-48. Roche, P. M. (1991). Studies on the comparative virology of pestiviruses. PhD Thesis, Surrey University. Roche, P. M., Woodward, M.J. and Edwards, S. (1992). Characterization of p20 gene sequences from a border disease-like pestivirus isolated from pigs. Veterinary Microbiology, 33, 23 t 238. Sawyer, M.M., Schore, C.E. and Osburn, B.I. (1991). Border disease of sheep--aspects for diagnostic and epidemiological consideration. Archives of Virology, [Suppl 3], 97-100. Snowdon, W. A. and French, E. L. (1968). The bovine mucosal disease-swine fever virus complex in pigs. Australian Veterinary Journal, 44, 179-184. Terpstra, C. and Wensvoort, G. (1988). Natural infections of pigs with bovine viral diarrhoea virus associated with signs resembling swine fever. Research in Veterinary Science, 45, 137-142. Terpstra, C. and Wensvoort, G. (1992). Bovine viral diarrhoea virus infections in pigs. Tijdschrift Diergeneeskundig, 116, 943-948. Trautwein, G. (1988). Pathology and pathogenesis of the disease. In: Classical Swine Fever and Related Infections, B. Liess, Ed. Martinus Nijhoff Publishing, Boston, pp. 27 54. Vannier, P., Leforban, Y., Carnero, R. and Cariolet, R. (1988). Contamination of a live virus vaccine against pseudorabies (Aujeszky's disease) by an ovine pestivirus pathogenic for the pig. Annales de Recherches Vitlrinaires, 19, 283-290. Wensvoort, G., Bloemraad, M. and Terpstra, C. (1988). An enzyme immunoassay employing monoclonal antibodies and detecting specifically antibodies to classical swine fever virus. Veterinary Microbiology, 17, 129-140. Wensvoort, G. and Terpstra, C. (1988). Bovine viral diarrhoea virus infections in piglets born to sows vaccinated against swine fever with contaminated vaccine. Research in Veterinary Science, 45, 143-148.
Received, January 4 th, 1994] Accepted, March 25th, 1994J
Congenital Infection of Pigs with Ruminant-type Pestiviruses D.J. Paton and S. H. Done Central Veterinary Laboratory (Weybridge), New Haw, Addlestone, Surrey KT15 3NB, UB"
Summary Congenital infections of pigs were induced with two ruminant-type pestiviruses isolated from pigs. One of the viruses was bovine viral diarrhoea virus-like and the other border disease virus-like. Both produced symptoms similar to those observed with low virulence strains of classical swine fever virus. A striking effect of persistent virus infection in post-natal life was stunting in viraemic animals. It was also shown that a congenitally infected pig shed virus for 2.5 years and in sufficient quantity to infect other pigs, even by indirect contact. Unlike ruminants, congenitally infected pigs sometimes had persistent viraemia but eventually eliminated the virus. Clearance of virus from the blood was related to the appearance of neutralizing antibodies. However, clearance from the tissues sometimes took as much as 5 months longer than from the blood.
Introduction Classical swine fever virus (CSFV), bovine viral diarrhoea virus (BVDV) and ovine border disease virus (BDV) are serologically related members of the genus Pestivirus within the family Flaviviridae. In Britain, CSFV has been eradicated, whereas B V D V and BDV, termed "ruminant-type" pestiviruses, are widespread. The occasional porcine infections produced by these viruses do not constitute a significant disease problem, but distinguishing such infections from those caused by CSFV can be difficult both on the farm and in the laboratory. Natural infection of pigs with ruminant-type pestiviruses has been demonstrated by serological surveys (Snowdon and French, 1968) and by occasional virus isolations (Fernelius et al., 1973). Such infections are usually subclinical but have sometimes been associated with reproductive or neonatal illness (Terpstra and Wensvoort, 1988). They have generally been attributed either to the use of pestivirus-contaminated vaccines (Vannier et al., 1988; Wensvoort and Terpstra, 1988) or to contact between pigs and ruminants (Terpstra and Wensvoort, 1992). T w o ruminant-type pestiviruses were isolated from British pigs in 1987 and 1991, in the course of investigations of suspected cases of swine fever. The virus obtained in 1987 (strain "87/6") was shown to be BDV-like by genetic (Roehe et al., 1992) and antigenic (Edwards and Sands, 1990; Paton et al., 1994) 0021-9975/94/060151+ 13 $08.00/0
9 1994AcademicPressLimited
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analysis. It also b e h a v e d like an ovine pestivirus in e x p e r i m e n t a l l y infected p r e g n a n t sows and sheep, crossing the p l a c e n t a b u t causing no discernible illness in adults (Roehe, 1991). T h e "91 / 1" strain o f virus, which was isolated from a farm on which b o t h cattle and pigs were kept, was shown to be B V D V like a n d a p p e a r e d also to cause illness only as a result o f congenital porcine infection ( P a t o n et al., 1992). T h e present study was designed to e x a m i n e the consequences o f in-utero infection with these viruses.
Materials and Methods
Viruses An 87/6 virus challenge inoculum was prepared from the tonsil of a pig affected in the original outbreak, by three serial passages in PK15 cells. A 91/1 virus challenge inoculum consisted of serum from a persistently infected steer thought to have been the index case in the original outbreak.
Infection and Monitoring of Experimental Animals Pigs obtained from the herd of specific pathogen-free animals at the Central Veterinary Laboratory were all initially seronegative to pestiviruses. Sows were served naturally and pregnancy was confirmed 3 weeks later by vaginal biopsy. The route and timing of challenge inoculation for each of sows 1-6 are shown in Table 1. At intervals after challenge, blood samples were collected to monitor seroconversion, and a blood sample collected 83-90 days after service was used to diagnose pregnancy by oestrone sulphate assay. To induce farrowing at a convenient time, pregnant sows were given an injection of 184 gg ofcloprostenol sodium (Planate, Pitman-Moore Ltd) 113 days after servme. At birth, the piglets were weighed and pre-colostral blood samples were collected from the umbilical cord. Subsequent progress of piglets was monitored at intervals by clinical observation, weighing and collection of blood for virological and serological examination. Weight differences between litter mates born viraemic and non-viraemic were analysed statistically by the t-test. Pigs were weaned at 5 weeks of age and usually retained until approximately one year old. Post-mortem samples, including, tonsil, submandibular lymph node, lung, liver, spleen, kidney, ileum, mesentenc lymph node and ovary or testis, were collected from most pigs after death or slaughter. Strain 87/6 Study. Four pregnant sows (nos 1-4) were inoculated with the 87/6 virus, two intranasally and two by intra-uterine injection via a laparotomy (Table 1). Intranasally inoculated sows (nos 1 and 2) each received 3 ml of culture supernate containing 105STCID~0 of virus. In sows 3 and 4 each conceptus received a 0"5 ml volume containing 102'~'CID50. Strain 91/1 study. Two sows (nos 5 and 6) were inoculated intranasally on day 27 of pregnancy with 2 ml of serum containing 105STCID50 of the 91/1 virus. Transmission study. Pig-to-pig transmission of strain 87/6 virus from a congenitally infected boar that developed a persistent viraemia was investigated by means of two seronegative 10-week-old pigs, used as sentinels. The sentinels and the persistently infected boar were moved in rotation between three strawed pens, as follows. Each day the boar was moved into a clean pen, and the sentinels were moved into the boar's vacated pen, which had not been cleaned out. After a week, the sentinels were moved to separate accommodation, and after a further 3 and 6 weeks they were bled and tested for the presence of 87/6 virus neutralizing antibodies.
Congenital
Pestivirus
Infections
153
in Pigs
Table 1 Summary of sow inoculations and consequences
Observation
N u m b e r of piglets belonging to sow no.
1 (87/6;44;i.n.)
N u m b e r of piglets Whole litter Mummies Stillbirths Other abnormality Precolostral status of liveborns V+/Ab+ V+/AbV-/Ab+ V -/Ab Not tested Piglet progress Survival > 1 wk Post-weaning illnessw Stunting Post-natal viraemia Post-natal seroconversion Persistent infection ( > 6 months)
--------
------
2 (87/6;37;i.n.)
13 -1* 2"~ 1 3 4 4
7 --
--
3 (87/6;35;i,u.)
4 (87/6;42;i.u.)
12 ---.
6 (91/1;27;i.n.)
-----
11 --
--
5 -6
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9
5 (91/1;27;i,n;)
.
-3
1 ----
11 1 6 6 10 2
-------
1 JJ
.
---
------
6 3 4 4 2 1
Brackets show: (infecting strain; day of pregnancy on which sow inoculated; route of inoculation). i.n., Intranasal; i.u., intrauterine, *Virus positive; ~'haemorrhages and oedema; ++meningocoele; IIoedema of ear pinnae; w leading to death or euthanasia. V + / A b + , V + / A b - , V - / A b + , V - / A b - : see text.
Virus Detection and Typing V i r u s e s w e r e i s o l a t e d i n P K 1 5 cell c u l t u r e s k n o w n to b e f r e e o f p e s t i v i r u s e s a n d pestivirus inhibitors. Virus-infected cultures were identified by an immunoperoxidase staining method and epitopic differences between isolates were compared with a panel of 13gp53-specific BVDV monoclonal antibodies ( P a t o n et al., 1 9 9 2 ) . T i s s u e homogenates and nasal swabs received two passages before staining, whereas sera received only one. Some blood samples from neonatal piglets were tested for pestivirus antigen by ELISA, since this may detect virus that cannot be cultured because of prior contact with colostral antibody. The method, which detects a pestivirus protein in h e p a r i n i z e d b l o o d , w a s a s d e s c r i b e d b y D r e w et al. ( 1 9 9 3 ) .
Serology Serum-virus neutralization tests (VNT) were carried out according to standard protocols (Edwards, 1990). The virus used was either the 87/6 virus or a porcine isolate derived from the same farm as the 91/1 virus, whichever accorded with the challenge s t r a i n . T h e t i t r e s w e r e e x p r e s s e d a s t h e d i l u t i o n (log~0) n e u t r a l i z i n g 5 0 % o f v i r u s r e p l i c a t e s , a n d a t i t r e o f < 1-0 w a s c o n s i d e r e d n e g a t i v e . I n a m o d i f i e d t e s t , t o c o m p a r e the neutralization of two viruses by a single serum, ten-fold virus dilutions were incubated with doubling serum dilutions.
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D.J. Paton and S. H. Done
Two CSFV-specific competition ELISAs based on different pairs of monoclonal antibodies were used to measure antibody responses in pigs that developed very high titres of neutralizing antibody. One was the assay of Wensvoort et al. (1988) and the other was an equivalent test designed at our laboratory (Paton, unpublished).
Histopathology Post-mortem samples for histopathological examination were fixed in buffered formalin, embedded in paraffin wax, and stained with haematoxylin and eosin (HE).
Results
Monitoring of Sows and Piglets The main findings are summarized in Table 1.
Strain 87/6 study. None of the sows (nos 1 4) showed any signs of illness, but all "seroconverted" to 87/6 virus. Sow 1. This animal was found to be non-pregnant by oestrone sulphate assay at 85 days after service and failed to farrow. Sow 2. Five of the piglets born to this sow died in the first three days of life, all with macroscopical haemorrhages and oedema. Three of these animals were born viraemic and seronegative ( V + / A b - ) , one was born viraemic and seropositive ( V + / A b + ) , and one was born non-viraemic and seropositive ( V - / A b + ) . The other members of the litter were born V - / A b - ( f o u r ) or V - / A b + (four) and remained completely healthy. Piglets born seropositive had antibody titres in the range of 2"2-2"6. Sow 3. This animal had a litter of 12 apparently normal piglets, one of which was overlain at one day old. Nine of the piglets were V + / A b - at birth, and the other three were not tested. After ingesting colostrum, all of the piglets became V - / A b + (antibody titres of 1-5-2.4 at 3 weeks of age), but when colostral antibodies waned at between 8 and 20 weeks of age, six piglets became V+/Ab-, and five actively seroconverted without a detectable post-natal viraemia. These pigs developed high titres of neutralizing antibody (maximum titres 4"2-4"8) and all gave strongly positive results in both CSFV-specific ELISAs. Five of the six pigs that developed post-natal viraemia subsequently seroconverted and became non-viraemic at between 20 and 25 weeks of age (maximum antibody titres 3"7-4.6). It was nevertheless possible to isolate virus from the tissues of three of these five pigs that were killed up to 8 months after seroconversion. T w o of these animals, killed 5 months after seroconversion, yielded virus from a wide range of (but not all) tissues, whereas the third, killed 8 months after seroconversion, yielded virus only from the testis. The other two pigs, killed 10 and 12 months after seroconversion, were completely virus negative. Viraemic animals were observed to be stunted, in comparison with their non-viraemic litter mates (Fig. 1), and seroconversion seemed to be related to
Congenital Pestivirus Infections in Pigs
Fig. 1.
155
Stunted and normal pig. Stunted pig was persistently viraemic with 87/6 virus; normal pig was a non-viraemic litter mate.
an improvement in growth (Fig. 2). A single pig remained viraemic until its death at 2"5 years of age. Serum virus titres for this animal ranged from 1053/ml at 5 months of age to 1025/ml 15 months later. Virus was also recovered from nasal swabs collected when the pig was 16 and 23 months old. The animal was undersized and had several intermittent periods of nonspecific illness and anorexia over a 12-month period. Shortly before being killed on humane grounds it developed a severe dyspnoea. Sow 4. Only four piglets were born, two being mummified and one stillborn with a meningocoele. The fourth pig appeared normal but died within 24 h. Virus was isolated from the tissues of the stillborn pig, and the liveborn pig was V+/Ab-.
Strain 91/1 study. Neither of the sows (nos 5 and 6) showed any signs of illness, although both seroconverted to 91/1 virus. Sow 5. This animal was negative in the pregnancy test 90 days after service and failed to farrow. Sow 6. This sow farrowed 11 piglets that were normal at birth, except one which had oedematous ear pinnae. Thereafter, the progress of the litter was similar to that observed for sow 3. Six piglets born V - / A b were all seropositive at 3 weeks old and thereafter their antibody titres declined slowly. In most cases, despite regular bleeding and no indication of active seroconversion, residual antibody (titres of 1"0-1"8) could still be detected at the end of the experiment, when the pigs were 10-11 months old. Five piglets born V + / A b - fell into two groups. Three became non-viraemic at 3 weeks old, with low or undetectable antibody titres. By 7 weeks of age, they were again V + / A b - ,
156
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although in one pig virus was only detectable by ELISA and not by virus isolation. The other two pigs developed high antibody titres (maximum titres of >4.6 and 4-0 in VNT, and positive in CSFV-specific ELISAs), associated with clearance of virus from serum. T h e y differed in that one seroconverted soon after birth and the other at about 20 weeks of age. When these animals were killed at 10 months old, virus could be isolated from tissues (testis, ileum, spleen, tonsil) of the one that had showed delayed seroconversion, but not from the one that had seroconverted earlier. The pigs born with viraemia grew poorly as compared with their litter mates, except for the animal that seroconverted just after birth, and body weights at 21 and 52 days were significantly lower for the "viraemic-at-birth" group (95% confidence by ttest; 99% confidence if early seroconverting pig excluded). The three pigs that did not seroconvert died or were killed due to illness at between 83 and 196 days of age. All appeared stunted compared with litter mates. The first pig died suddenly, whereas the next two became listless and lethargic over a 2-3 day period and did not respond to antibiotic therapy.
Transmission study. Three weeks after their last indirect contact with the persistently infected boar, only one sentinel pig from the transmission study had seroconverted. When next bled, three weeks later, both were seropositive. Pathological Observations Litter of sow 2. Five pigs that died in the first three days of life were examined. A
Congenital Pestivirus Infections in Pigs
157
stillborn and two liveborn piglets showed pinhead-sized haemorrhages and anasarca at birth, but the other two were apparently normal until 1-3 days old. The five dead piglets showed widespread internal oedema and ascites. The spleen, liver and lymph nodes were congested and enlarged and fine haemorrhages were detected in lymph nodes, bladder and kidneys. Litter of sow 3. Most necropsies were conducted on animals killed at 11-14 months of age. Gross pathology was unremarkable. Histopathology carried out on one animal revealed a mild hyperplasia of the Peyer's patches and the spleen. The persistently viraemic animal killed at 2"5 years old showed a marked lymphoid depletion, lymph nodes being hard to find (Fig. 3) and Peyer's patches not evident. There were omental adhesions to the spleen, which was itself severely atrophied and nodular (Fig. 4). Histopathological examination confirmed lymphoid depletion, revealed an absence of germinal centres, and demonstrated focal haemorrhagic lymphadenitis. Litter of sow 4. The liveborn and stillborn piglets showed mild subcutaneous oedema, swollen liver and spleen, and fine petechiation of the surface of the kidneys. The stillborn animal had a large meningocoele protruding from the dorsal cranium. Litter of sow 6. The first pig to die had a pale and watery carcass with enlarged and haemorrhagic lymph nodes. Histopathological examination confirmed haemorrhage within lymph nodes, liver and kidney (Fig. 5). Widespread deposition of haemosiderin and increased splenic haemopoiesis indicated that this haemorrhage had been of a chronic nature. Other findings included oedema and lymphocyte depletion within lymph nodes, villous atrophy of the small intestine, Peyer's patch hyperplasia, and abscessation of tonsillar crypts. The second pig to die had a pale, watery and oedematous carcass, the oedema being most pronounced in the submandibular region. All lymph nodes were enlarged and haemorrhagic and further haemorrhages were found on the thymus, epicardium and cerebellum. Histopathological findings were similar to those for the first pig with, in addition, evidence of myocarditis. The last pig to die because of illness was also normal in appearance. Submandibular, mesenteric and iliac nodes were enlarged, with a firm rubbery texture and a pinkishred discolouration. Histopathological examination revealed haemorrhage and oedema of the submandibular lymph node.
Comparative Neutralization and Monoclonal Antibody Typing
Strain 91/1 virus, isolated from the testis of an 11-month-old pig 5 months after it had seroconverted, was compared with virus obtained from a precolostral serum sample taken from the same animal. Serum collected from the same pig at the time of the second virus isolation neutralized both viruses equally effectively in vitro (Fig. 6), and no epitopic differences were detected in the major envelope protein of the virus by monoclonal antibody typing.
158
D.J. Paton and S. H. Done
Fig. 3.
Lack of visible mesenteric lymph nodes in a 2"5-year-old, persistently infected pig at death
Fig. 4.
Spleen of a 2.5-year-old, persistently infected pig at death.
Fig. 5.
Kidney from newborn congenitally infected piglet, showing haemorrhages. HE. x300.
159
Congenital Pest/virus Infections in Pigs
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Discussion
In the two field outbreaks from which the 87/6 and 91/1 viruses were isolated, disease was only seen in pigs less than 3 months old, and in the second outbreak affected piglets were all litter mates, some of which were shown to have been persistently viraemic (Paton et al., 1992). This suggested that the illness observed was a delayed consequence of congenital infection. In other reports, disease in pigs due to ruminant-type pestiviruses has generally affected young animals, pregnant sows, or both (Terpstra and Wensvoort, 1988; Vannier et al., 1988; Wensvoort and Terpstra, 1988). In the present study, no illness was seen in any of the post-natally infected pigs.
160
D.J. Paton and S. H. Done
The stage of gestation at which infection with pestiviruses occurs is known to affect the outcome (Roeder et al., 1986; van Oirschot and Terpstra, 1989). Infection in early pregnancy, before fetal immunocompetence develops, favours persistent infection and subsequent disease in early post-natal life. In the study with strain 87/6, different challenge times and routes of inoculation were used. To ensure that some viraemic piglets were born, two litters were inoculated through the wall of the uterus by means of laparotomy. Direct fetal inoculation with 87/6 virus at 35 days of gestation produced large numbers of viraemic piglets. In the study with strain 91/1, on the assumption that the development of maternal viraemia and transmission of virus would take approximately 8 days, an intranasal challenge was given on the 27th day of gestation. Two sows, one infected with the 91/1 virus and one with the 87/6 virus, failed to farrow, having been inoculated intranasally at 27 and 44 days of gestation respectively and having been confirmed by vaginal biopsy as pregnant 3 weeks after service. It was not proved that the inoculations had a role in the pregnancy terminations, but ruminant-type pestiviruses have been implicated previously as a cause of infertility in pigs (Vannier et al., 1988). Furthermore, a third sow had a litter of only four pigs, despite the presence of 14 conceptuses when 87/6 virus was inoculated through the uterus 42 days after service. Abnormalities detected at birth in a number of different litters included mummification, haemorrhages, oedema and meningocoele. All of these lesions have been observed in pigs congenitally infected with CSFV (Trautwein, 1988; van Oirschot and Terpstra, 1989). In some pigs illness was delayed and appeared at between one day and 2"5 years later. One pig died when 2 days old, having been born V - / A b + , and another was overlain, but these apart, all of the morbidity and mortality occurred in viraemic animals. Pigs that died soon after birth tended to show few premonitory signs but marked haemorrhages and oedema at death. Those that died or were killed later often had a period of poorly defined ill-thrift and showed few striking lesions at necropsy. The most consistent feature was the presence of enlarged, rubbery lymph nodes with a pink-red discoloration. Unlike cattle, pigs apparently do not develop a mucosal disease-like syndrome associated with superinfection by cytopathogenic biotypes of pestivirus. However, late onset disease is a well recognized feature of pre-natal porcine infection with low virulence strains of both CSFV (van Oirschot and Terpstra, 1977) and BVDV (Terpstra and Wensvoort, 1988). The trigger for such onset, as in the present cases, is not known and no cytopathogenic viruses were identified from pigs in this study. Possibly some other change in virus virulence took place, or the disease was immunologicatly mediated. Most viraemic pigs were readily recognized because of their poor growth as compared with that of healthy litter mates. Stunting is a well known consequence of congenital BVDV infection in ruminants (Roeder et al., 1986) and has also been described in pigs (Leforban et al., 1990). The mechanisms that underlie such growth retardation are not known, but are possibly related to generalized viral interference with cell division or to specific disturbance of
Congenital Pestivirus Infections in Pigs
161
particular cell functions, such as hormone production (Sawyer et al., 1991). Whatever the mechanisms, it was noticed that pigs that seroconverted when still growing subsequently resumed normal growth and were not significantly lighter than healthy litter mates at 10 months of age. This suggests that growth retardation is the result of a continuing process and not of an irreversible lesion acquired in early development. Most of the pigs born with viraemia either died, were killed due to illness, or seroconverted and became non-viraemic. Only one pig remained a long-term virus shedder, and this was an animal that had been infected in utero with the 87/6 virus. Once colostral antibody was cleared from this animal, virus was isolated from serum and nasal swabs on every occasion on which samples were taken, and the virus titre in the serum was similar to that reported for persistently viraemic ruminants (Drew and Edwards, personal communication). This pig was also able to infect others, even by indirect contact, suggesting that ruminant-type pestiviruses may be maintained in pigs without a requirement for a continuous ruminant contact. The delayed seroconversion in the second sentinel pig suggested that it might have beeen infected from its transiently infected pen-mate rather than from the persistently infected pig. The causes of the terminal illness and of the severe immunological depletion in the persistently infected pig are unknown. A general depletion of the lymphoid system has been described in pigs suffering from chronic classical swine fever (van Oirschot, 1988). M a n y of the pigs born with viraemia seroconverted in later life. This was associated with an immediate clearance of virus from serum, but a delayed clearance from the tissues. It also seemed to be associated with an improvement in growth rate and survival. Delayed recovery from congenital infection with pestiviruses is generally not seen in ruminants but has been reported in a persistently infected lamb (Nettleton et al., 1992). This is possibly an indication of a difference in the speed of maturation of the immune system of young ruminants and pigs. A chronic form of CSFV infection has been described in pigs, in which antibodies appeared but failed to eliminate virus (Mengeling and Cheville, 1968). However, unlike the results reported here, recovery was incomplete and the pigs ultimately died. T e m p o r a r y persistence of virus, with occasional continued persistence in the tissues, in the presence of neutralizing antibodies has been described for congenital rubella in man (Rawls, 1974). In the present study, the appearance of serum antibodies was related to the clearance of virus from serum but not tissues. Furthermore, neither comparative neutralization nor monoclonal antibody typing revealed any evidence that virus persistence was due to phenotypic changes that resulted in an acquired resistance to neutralization by antibody. These findings suggest that neutralizing antibodies play a very limited role in the clearance of BVDV from ceils. This is consistent with in vitro studies by Curia and McClurkin (1981), who showed that antibody was ineffective in clearing BVDV from persistently infected cell cultures. It is also in agreement with the known incapacity of colostrum-derived antibodies to clear virus permanently from persistently infected newborn animals. Sera from some of the pigs that had recovered from congenital infection
162
D.J. Paton and S. H. Done
with either B V D V or BDV gave false positive results when assessed with ELISAs designed to be CSFV-specific. The high levels of antibody probably caused steric inhibition to the binding of the CSFV-specific monoclonal antibodies employed in the tests. Although naturally occurring cases of this type are probably very rare, they are precisely the ones likely to be confused clinically with CSFV infection. This emphasizes the importance of confirming ELISA-positive results by comparative neutralization tests, especially when new outbreaks of disease are being investigated.
Acknowledgments
We are indebted to many staff at the Central Veterinary Laboratory for assistance in maintaining the experimental animals and for providing technical laboratory assistance.
References
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Received, January 4 th, 1994] Accepted, March 25th, 1994J