Toxocara vitulorum: maternal transfer of antibodies from buffalo cows (Bubalis bubalis) to calves and levels of infection with T vitulorum in the calves

Research in Veterinary Science 1994, 57, 81-87

Toxocara vitulorum: maternal transfer of antibodies from buffalo cows (Bubalis bubalis) to calves and levels of infection with T vitulorum in the calves R. P. V. J. RAJAPAKSE, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Peradeniya, Sri Lanka, S. LLOYD, Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, S. T. FERNANDO, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Peradeniya, Sri Lanka

The levels of antibody to the excretory/secretory antigens of the infective larvae and adults of Toxocara vitulorum were measured by gel precipitation and ELISAS in the serum and colostrum of 12 buffalo cows naturally infected with T vitulorum and in the serum of their calves. The antibody levels were compared with the extent of T vitulorum infection as judged by faecal egg counts in the calves. The patterns of bands of the larval antigens and gel precipitating antibodies in cow serum taken one month before calving, in cow colostrum and in calf serum were very similar. Nine cows and their calves had gel precipitating antibodies but the remaining three cows and their calves did not. The ELISA detected anti-larval antibodies in the colostrum of all 12 cows and calves. With the exception of one calf there was a strong correlation (r=0-902) between the antibody titre in cow colostrum and the titre of passively acquired antibody in calf serum. The titres of these passively acquired antibodies declined to their lowest levels in calves 12 to 25 days of age; the antibody concentrations then began to increase up to day 42 and remained stable for the remainder of the experiment (105 days). The titres of antibodies to the antigens of the adult worms, examined in four cows and their calves, were lower than the titres to the larval antigens; the calves absorbed this anti-adult antibody from the colostrum and the antibody levels reached a plateau between days 12 and 30 and remained stable for the rest of the experiment. The peak titres of antibodies to the larval antigens in the cows' colostrum and in the serum of their

calves (both passively acquired and actively produced) showed a significant inverse correlation with the m a x i m u m faecal egg count of T vitulorum in the calves (r=-0.728 to -0.817).

TOXOCARA vitulorum infection is transferred vertically from buffalo cows to their calves. The infective larvae in eggs ingested by the cow migrate into the tissues where it is assumed they become dormant (Warren 1971). Larval activation, growth and migration to the mammary gland are reported to begin one to eight days before parturition and the majority of the larvae are passed in the milk in the first five to six days of lactation (Roberts 1990a, Roberts et al 1990). The ingestion of milk by the suckled calf leads to adult T vitulorum infection in the calf (Warren 1971). Fernando et al (1987) reported gel-precipitating antibodies against an extract of embryonated T vitulorum eggs in the serum and colostrum of some pregnant buffaloes and in the serum of their suckled calves. Antibodies to other gastrointestinal nematodes have been detected in the colostrum of ruminants (Filmer and McClure 1951, Kloosterman et al 1980) although there was little evidence of a role for these maternal antibodies in protection (Connan 1968, Kloosterrnan et al 1980). Femando et al (1989a) did not find any antibody-mediated effects on the development of T vitulorum infections or their pathogenic effects in calves, and deaths due to heavy burdens of T vitulorum occurred both in calves with gel-precipitating antibodies and in calves without them. Kelley and Nayak (1965), however, did demonstrate a colostral transfer of protection against Ascaris suum in pigs. 81

82

R. P. V. J. Rajapakse, S. Lloyd, S. T. Fernando

The excretory/secretory (ES) antigens of T canis and other helminths have greater antigenic specificity than extracts of the parasites (Ogilvie and de Savigny 1982). In this study Es antigens were therefore used to monitor the levels of antibody to T vitulorum in the serum and colostrum of pregnant buffalo cows and in the serum of their calves in relation to the levels of infection of the calves with T vitulorum. Materials and methods Buffaloes

Twelve pregnant buffaloes were purchased from an area where T vitulorum is endemic and where all calves are born infected with T vitulorum (Roberts 1989). The cows were moved to the Experimental Farm, housed in a concrete yard at night and allowed to graze and wallow by day. After calving, each cow and its calf were penned together and fed cut grass for one week so that the calf could suck only its dam for this period. Thereafter, the cow and calf grazed together during the day but the calf was housed separately at night. Serum, milk and faecal samples

Blood was collected from the cows by jugular venepuncture about one month before parturition, the serum separated and stored at -20°C. Immediately after parturition a colostrum sample was collected and colostrum or milk samples were collected before the calf sucked every morning for 10 days. Colostrum or milk whey was used in the serological tests. The samples were centrifuged at 3000 g for 30 minutes at 4°C and the solidified fat removed. Casein was precipitated with 1 per cent rennin at 37°C for one hour and the whey recovered by centrifugation. Each calf was bled immediately after birth before it sucked, and was allowed to suck colostrum within one hour. The calves were bled on days 2 and 4, then every four days until day 28, and then weekly until 105 days. Rectal faecal samples were collected from the calves twice weekly from day 4 to day 41 and weekly thereafter. Antigens Larval ES antigen. Toxocara eggs were collected from the faeces of buffalo calves housed at the

Experimental Farm. The eggs were embryonated in 0.1 N sulphuric acid, washed, decorticated in hypochlorite, and the larvae hatched and cultured as described by Rajapakse et al (1992). The supernatant medium was harvested weekly, centrifuged at 3500 g for 30 minutes and concentrated 100fold in dialysis tubing against polyethylene glycol (Aquacide III, Calbiochem). Adult ES antigen. Live, adult T vitulorum were collected from buffalo calves presented for post mortem examination, washed repeatedly in phosphate buffered saline (PBS) and 100 male and female worms were then suspended in RPMI 1640 containing 10X antibiotic/antimycotic (Gibco) at 37°C for one hour (Rajapakse et al 1992). The worms were then incubated in 500 ml RPMI 1640 containing 1 per cent glucose, 10 mM HEPES, 0.85 per cent sodium bicarbonate and 1 x antibiotic/ antimycotic (penicillin G [200 units ml-1], streptomycin sulphate [100 gg m1-1] and amphotericin B [2.5 gg ml-1]) (Rajapakse et al 1992) for 48 hours at 37°C and in an atmosphere containing 5 per cent carbon dioxide. The cultures were examined microscopically for contamination, and no culture contaminated with bacteria/protozoa was harvested. The supernatant was treated as described for the larval ES. Faecal egg counts

Faecal samples were examined by the McMaster technique and the egg counts recorded as eggs g-t of faeces (epg). Precipitating antibodies

Double immunodiffusion in 1 per cent agarose in veronal buffer containing 8 per cent sodium chloride (Fernando et al 1989a) was used to detect the serum precipating antibodies against larval ES. The precipitation reaction was judged subjectively as -, +, ++ or +++ by the number of bands and by the intensity of the reaction. ELISA

The indirect ELISA was carried out as described by Voller et al (1979). All volumes were 200 gl per well. All the washes (four on each occasion) and dilutions were made in PBS/1 per cent Tween 20 containing, for adult ES, 1 per cent fetal calf

83

Toxocara vitulorum in buffalo cows and calves serum (FCS) (Flow Laboratories). The larval and adult ES were used to coat PVC plates (Titertec, Flow Laboratories) at 23 and 125 gg m1-1, respectively (determined by previous chequer-board titration), in carbonate/bicarbonate buffer, pH 9-6, overnight at 4°C. The plates were washed, doubling serial dilutions of test sera were added and the plates were incubated at room temperature for two hours. After washing, horseradish peroxidaseconjugated, rabbit anti-bovine IgG (heavy and light chain) (Cappel Laboratories) at a 1:500 dilution (determined by chequer-board titration) was added and the plates incubated for four hours. Substrate (3, 3, 5, 5 tetramethyl benzidine) and hydrogen peroxide were used to develop the reaction which was stopped after 30 minutes with 50 ~tl of sulphuric acid and read at 450 nm in a semiautomatic ELISA reader (Uniscan). The titre was recorded as that serum dilution which yielded a reading that was eight times greater than the reading at the same dilution of the serum controls which consisted of FCS and serum samples from Ayrshire cattle in Cambridge, free of T vitulorum infection.

with one to three bands visible in nine of the 12 cows (Table 1). The responses in maternal serum and colostrum and in the serum of the corresponding calves (two calf patterns are depicted in Fig 1) were very similar with the exception of cow 608

0

0 24

24

f~

24,

28

70~ |ES ' ~63

70

~12t

,~

2u

35

E~ '42 ' 56

77

24

28

'0" .....

49

35 42 ili

9

84 91

Results

Antibody response to larval ES Response in cows. Serum taken one month before

calving, and the first colostrum sample at day 0, had gel-precipitating antibodies against larval ES

FIG 1: Precipitin bands formed between T vitulorum larval excretory/secretory antigens and serum of calves 606 (left) and 607 (right) collected at birth to 98 days

TABLE 1 : Serum and colostrum antibody responses to larval excretory/secretory antigens in 12 buffalo cows naturally infected with T vitulorum, the passively acquired antibody responses in their calves, and the maximum T vitulorum faecal egg counts in the calves Cow Precipitin reaction* Cow

Serum1-

600 601 602 603 604 605 606 607 608 609 610 611

+++ ++ ++ +++ +++ +++ (+) +++ +++ -

Serum from two-day-old calf

Reciprocal ELISA titre in Colostrum:l: colostrum ++ ++ ++ +++ 3+(+) ++(+) ++(+) +++ +++ -

3200 200 200 6400 50 50 6400 6400 3200 3200 6400 50

Precipitin reaction ++ ++ ++(+) +++ +++ +++ + +++ ++ -

Reciprocal ELISA titre 800 50 50 800 25 25 1600 800 25 800 1600 25

* -, +, ++, +++ denotes 0, 1,2, 3 precipitation bands; (+) denotes weak reaction + Serum sample one month before parturition $ Colostrum sample on day of parturition

T vitulorum maximum epg 88,500 108,300 99,200 61,000 146,500 132,200 78,800 59,600 148,300 39,100 43,100 133,400

R. P. V. J. Rajapakse, S. Lloyd, S. T. Fernando

84

which had a stronger response in its colostrum than in either its own or its calf's serum. The number of bands in the colostrum/milk reactions decreased to zero after between one and six days in individual animals. By the ELISAtechnique, the colostrum of all 12 cows had antibodies to larval ES (Table 1). The titres were highest in the cows which had the strongest gel-precipitating antibodies and lowest in those with none. Colostral titres were high on the day of parturition and then declined rapidly over the first four to 10 days of lactation (Fig 2).

Passively acquired response in calves. The pattern of the gel-precipitating antibodies to larval Es in the serum of the calves is presented in Table 1 and those from calves 606 and 607 are shown in Fig 1. There were no reactions in the sera of the calves at No609

birth but nine of the 12 calves had reactions on day two; the reactions reached a maximum on day four and then disappeared by days 12 to 28. The three calves which showed no passively acquired antibodies were born to cows which themselves had no gel-precipitating antibodies (Table 1). The titres in the serum of eight of the nine positive calves were comparable with those in the colostrum of their dams except for calf 608 whose serum showed only a very faint reaction compared with its dam's colostrum. The passively acquired antibody titres detected by ELISA are shown in Fig 2. None of the calves had detectable serum antibodies on their day of birth, but antibodies were present in all 12 calves, sera on day two, reached a maximum on days two to four, and in some of the calves this high titre remained until day eight. The maximum antibody

No.610

~No607

150

6400 1600

I00 50

I00

1

0 NO 600

!I

150 I00 50

I

~ x

0

cnr~ 0 ua .m o9

8~

No602

N0.605

No.601

150

6400

m L~ U

u_

16oo

100

U

50

100'

% 101

0

m

No.61 I

NO608

No604

150

6400 U 0 U

1600

1oo

50

I00 IO~ I0 20

40

60

80

I00

10 20

40

60

80

I00

0

10 20

40

60

80

100

FIG 2: Antibody to larval excretory/secretory antigens of T vitulorum detected by ELISA in the colostrum/milk of buffalo cows and in the serum of their calves, and the T vitulorum faecal egg counts in the calves. Graphs arranged from lowest peak (top left) to highest peak

(bottom right) epg in the calves' faeces

85

Toxocara vitulorum in buffalo cows and calves

800I0"603

No.600

!

,, ,oo,i V-

_

:I IL ~

2

~ 1

!

,

,

i

|

NO.601

800 N0.602

d

u ~ u

100

I

ILl FF

10

....

0

1

i

10

20

I

,

i

40

60

80

, [~ . . . . .

1O0

0

!

1

I

I

I

10

20

40

60

80

! 1O0

DAYS AFTER BIRTH OF CALVES Fig 3: Antibody to adult excretory/secretory antigens of T vitulorum detected by ELISA in the colostrum/milk of buffalo cows, and in the serum of their calves

titre in calf serum was lower than that detected in the respective dam's colostrum, but the antibody titres in the calves' serum were highly correlated with the dams' colostral antibody titre (r=0.861) except again for calf 608. Excluding this calf, coefficient of correlation of 0.902 was achieved. In six calves with low peak titres (<1:50), passively acquired antibody was no longer detected by days 12 to 24. In the remaining six calves, which had peak titres >1:800, the antibody declined to a minimum on days 20 to 35 but never became undetectable. Active antibody response in calves. The antibody titres, both by gel-precipitation and ELISA, began to increase on days 24 to 42 in individual calves (Figs 1 and 2); the maximum titres occurred from about day 70 in 11 of the 12 calves. Calf 608 which had no detectable second peak of gel-precipitating antibodies reached an ELISAtitre of only 1:25 at the time of its death at 71 days of age. Antibody response to adult ES

The ELISAantibody titres to adult ES were two to three dilutions lower than to larval ES in the same cows and calves. The reciprocal titres to adult Es on day 0 were 400, 50, 50 and 800 in the four cows, respectively (Fig 3).

The pattern of anti-adult ES antibodies in the calves was different from that against larval ES. In the two calves which ingested the colostrum with higher titres, the serum titre peaked on day two at 1:200 and then decreased to 1:25 to 1:50 until day 70 (Fig 3). In the two calves which ingested the 1:50 titre colostrum, the titre reached only 1:10 on day two, but increased to 1:25 by day 10 and remained between 1:25 and 1:50 until day 70. After day 70 the titres fell slightly and remained at 1:25 for the remainder of the experiment. Parasitological data

The faecal egg counts of T vituIorum in the individual calves are shown in Fig 2 and the peak values are given in Table 1. T vitulorum eggs were detected for the first time when the calves were 16 to 25 days of age; the epg of the faeces then increased to a maximum of between 39,100 to 148,300 between 40 to 60 days of age, and then declined rapidly before 70 days in most of the calves. When the peak epg of the calves' faeces were compared with the peak passively acquired antibody titres or the peak actively acquired antibody titres, there were significant inverse correlations of (r=-0-728 including calf 608; r=-0-817 excluding 608, both significant at the 1 per cent level).

86

R. P. V. J. Rajapakse, S. Lloyd, S. T. Fernando

Calf 608 died when it was 71 days old and had petechiae in the small intestine and 185 adult T vitulorum. The eggs of Strongyloides species appeared in the faeces of all the calves, beginning when they were seven to 40 days old, but they were not counted.

Discussion Antibodies to the ES of infective larvae and adults of T vitulorum occurred in the serum and colostrum of all the buffalo cows and their calves. The calves were infected with a Strongyloides species in addition to T vitulorum and the specificity of the antibody detected may therefore be questioned. Nevertheless, when T vitulorum, T canis and Strongyloides species larvae were incubated in the buffalo colostmm for 48 hours, IgG antibodies were detected, by strong indirect immunofluorescence with anti-bovine IgG, on the surface and in oral precipitates of T vitulorum; there was some cross reaction because slight immunofluorescence was detected on T canis but no immunofluorescence was detected on Strongyloides species larvae (Rajapakse 1993). Furthermore, T canis larval ES antigens have shown relatively good genus and species specificity (Ogilvie and de Savigny 1982, Glickman et al 1985) and the same may be true of T vitulorum larval ES antigens. Nevertheless, the specificity of the antibodies detected has not been extensively tested. The presence of antibody to larval ES in the serum of buffalo cows can be related to larval infection; the infection in the cows can be confirmed indirectly because calves only acquire infection with adult T vitulorum from milk (Warren 1971, Roberts 1990a). Since the cow and its calf were penned with each other for the first week, which is the main period for the transmission of larvae from colostrum/milk (Roberts et al 1990), essentially all the adult T vitulorum acquired by each calf would have originated from its own dam. The anti-larval ES antibodies in the colostrum would have been concentrated from. those identified in the serum. The immunoglobulin isotype was not determined but it is mainly IgG 1 serum antibodies that are concentrated into ruminant colostrum (Newby and Bourne 1977); antibodies to an extract of T vitulorum larvae were found mainly in the IgG 1 subclass (Fernando et al 1989b), and specific IgG antibodies to T vitulorum in cow serum declined at about the time of parturition (Amerasinghe et al 1994).

Cattle calves absorb colostral immunoglobulins unselectively (Penhale et al 1973). The high correlation between anti-T vitulorum antibody titres in the cow colostrum and the calf serum indicates that most of the buffalo calves were similarly unselective. The exception was calf 608 but, as up to 25 per cent of cattle calves fail to absorb colostral immunoglobulin (Penhale et al 1973) this animal may fall into this category. The passively acquired antibodies to larval ES in the calves declined over three weeks, but the titres began to increase again when the calves were one month older. This increase in titre was probably an active response to a newly acquired egg infection. T vitulorum-infected animals are common in the area and the eggs of T vitulorum are ubiquitous and long-lived in wallow water, in soil, on pasture and on the udder and teats of cows (Roberts 1989) and the calves would have ingested these eggs from a young age. Furthermore, Fernando et al (1989a) have shown that the experimental infection of buffalo with eggs of T vitulorum induced an increase in antibody titre to a somatic larval antigen. However, at the time of these rising antibody titres, the calves carried adult T vitulorum and, since cross-reactions between the antigens from different stages of a helminth's life cycle are common, this increase in the titres of the calves over one month old could have been due to these adult parasites. Nevertheless, some specificity in the responses might have been present, because the anti-adult ES antibody titres declined from 1:50 to 1:25 at about 70 days (Roberts 1990b) after the decline in the faecal egg counts and the loss of the adults by most of the calves, whereas the anti-larval antibody titres remained high. High antibody titres in both the cows and the calves were correlated significantly with low faecal egg counts and therefore possibly with protection against infection. A number of explanations can be put forward to account for these correlations, because the antibodies could act against the parasite in either the cow or the calf. First, the high inverse correlation between the passively acquired calf antibody titre and low faecal egg counts might indicate a colostral transfer to the calf of protection against T vitulorum. Secondly, the inverse correlation between the antibody titre in the cow's colostrum and the calf's faecal egg count could represent anti-larval, antibody-mediated immunity in the cow itself, which might limit

Toxocara vitulorum in buffalo cows and calves the number of larvae in the tissues of the cow and/or the number activated and transferred to the calf. Certainly, the numbers of larvae detected in the colostrum and milk of buffalo cows were similar to the numbers of adults found in the intestines of buffalo calves (Roberts et al 1990). Finally, the correlations described could reflect a geneticallymediated individual variation, inherited from cow to calf, in both the resistance and susceptibility to T vitulorum infection and the ability to produce antibody. For example, Windon and Dineen (1981) and Wagland et al (1984) have demonstrated the inheritance from ewe to lamb of resistance to T r i c h o s t r o n g y l u s colubriformis, with the manifestation of resistance being highly correlated with the serum antibody titre. Acknowledgements The authors thank the Overseas Development Administration, United Kingdom, the Natural Resources Energy and Science Authority, Sri Lanka (NARESA), the Swedish Agricultural Research Cooperation with Developing Countries, Sweden (SAREC), and the University of Peradeniya Grants RG/47/90/Vt and RG/8/C91/Vt for generous financial support. R. P. V. J. R. received a NARESA scholarship and a Technical Cooperation Training Award from the British Council. References AMERASINGHE, P. H., VASANTHATHILAKE, V. W. S. M., LLOYD, S. & FERNANDO, S. T. (1994) Peripartufient reduction in antibody to Toxocara vitulorum and in mitogen-induced lymphocyte proliferation in buffalo. Tropical Animal Health and Production (In press) CONNAN, R. M. (1968) An attempt to demonstrate passive resistance in iambs to three species of gastrointestinal nematodes as a result of suckling the ewe. Research in Veterinary Science 9, 591-593 FERNANDO, S. T., GUNAWARDANA, V .K., MASOODI, M. A. & RAJAPAKSE, R. P. V. J. (1987) Serum and colostral precipitin reactions in buffalo cows and colostral transmission of antibodies to the calves. Buffalo Journal 2, 195-203 FERNANDO, S. T., SAMARASINGHE, B. T. & GUNAWARDANA, V. K. (1989a) Immunological response of buffaloes infected with Toxocara vitulorum. Buffalo Journal 2, 205-218 FERNANDO, S. T., SAMARASINGHE, B. T. & GUNAWARDANA, V. K. (1989b) Immunoglobulin classes of antibodies in the sera of

87

buffaloesinfectedwithToxocara vitulorum.Journal of the National Science Council, Sri Lanka 17, 213-228 FILMER, D. B. & McCLURE, T. J. (1951) Absorption of anti-nematode antibodies from ewe's colostmm by the new-born lambs. Nature 168, 170 GLICKMAN, L. T., GRIEVE, R. B., LAURIA, S. S. & JONES, D. L. (1985) Serodiagnosis of ocular toxocariaisis: a comparison of two antigens. Journal of Comparative Pathology 38, 103-107 KELLEY, G. W. & NAYAK, D. P. (1965) Passive immunity to Ascaris suum transferred in colostmm from sows to their offspring. American Journal of Veterinary Research 26, 948-950 KLOOSTERMAN, A., BENEDICTUS, J. & AGHINA, H. (1980) Colostral transfer of antinematode antibodies in cattle and its significance to protection. Veterinary Parasitology 7, 133-142 NEWBY, T. & BOURNE, J. (1977) The nature of the local immune system of the bovine mammary gland. Journal of Immunology 118, 461-465. OGILVIE, B. M. & DE SAVIGNY, D. (1982) Immune response to nematodes. In Immunology of Parasitic Infections. Eds S. Cohen, and K.S. Warren. Oxford, BlackwelI Scientific Publications. pp 715-757 PENHALE, W. J., LOGAN, E. F., SELMAN, I. E., FISHER, E. W. & McEWAN, A. D. (1973) Observations on the absorption of colostral irrtmunoglobulins by the neonatal calf and their significance in colibacillosis. Annales de la Recherche Veterinaire 4, 223-233 RAJAPAKSE, R. P. V. J. (1993) Immunological response of buffalo cows and calves to Toxocara vitulorum infection. PhD thesis, University of Peradeniya, Sfi Lmlka RAJAPAKSE, R. P. V. J, VASANTHATHILAKE, V. W. S. M., LLOYD, S. & FERNANDO, S. T. (1992) Collection of eggs and hatching and culture of second-stage larvae of Toxocara vitulorum in vitro. Journal of Parasitology 78, 1090-1092 ROBERTS, J. A. (1989) The extra-parasitic life cycle of Toxocara vimlorum In the village environment of Sri Lanka. Veterinary Research Communications 13, 377-388 ROBERTS, J. A. (1990a) The life cycle of Toxocara vitulorum in Asian buffalo (Bubalis bubalis). International Journal for Parasitology 20, 833-840 ROBERTS, J. A. (1990b) The egg production of Toxocara vitulorum in Asian buffalo (Bubalis bubalis). Veterinary Parasitology 37, 113-120 ROBERTS, J. A., FERNANDO, S. T. & SIVANATHAN, S. (1990) Toxocara vitulorum in the milk of buffalo (Bubalis bubalis) cows. Research in Veterinary Science 49, 289-291 WAGLAND, B. M., STEEL, J. W., WINDON, R. G. & DINEEN, J. K. (1984) The response of lambs to vaccination and challenge with Trichostrongylus colubriformis: effect of plane of nutrition on, and the inter-relationship between immunological responsiveness and resistance. International Journal for Parasitology 14, 39-44 WARREN, E. G. (1971) Observations on the migration and development of Toxocara vitulorum in natural and experimental hosts. International Journal for Parasitology 1, 85-91 WINDON, R. G. & DINEEN, J. K. (1981) Effect of selection of both sire and dam on the response of lambs to vaccination with irradiated Trichostrongylus colubriformis larvae. International Journal for Parasitology 11, 11-18 VOLLER, A., BIDWELL, D. E. & BARTLETT A. (1979) Enzyme immunoassay in diagnostic medicine. Theory and practice. Bulletin of the Worm Health Organization 53, 55-63 Received August 7, 1992 Accepted January 7, 1994