J. Vet. Med. B 39, 337-344 (1992) Q 1992 Paul Parey Scientific Publishers, Berlin and Hamburg ISSN 093 1- 1793
Animal Pathology Dpt. I. Veterinary Faculty UCM, Avda. Puerta de Hierro sln, 28040 - Madrid
Field Trials of an Inactivated Virus Vaccine Against Porcine Parvovirus J. M. CASTRO', M. DEL POZ02 and I. SIMARROl Address of authors: ' Animal Pathology Dpt. I. Faculty of Veterinary, UCM, Avda. Puerta de Hierro s/n, 28040 - Madrid (Espafia) * Porcine Pathology Laboratory, Proinserga, Segovia (Espafia)
With 5 tables (Received for publication Juli 12, 1991)
Summary Serological response and reproductive performance were estimated in field trials of an inactivated virus vaccine against porcine parvovirus. Experiments were carried out in 10 selected pig breeding herds. A total of 277 seronegative gilts were used. Two hundred and twenty animals were vaccinated twice before mating, fourteen days apart and revaccinated after farrowing. Blood samples were obtained from both vaccinated and non-vaccinated (57 animal) control gilts, one week after the 2nd dose of vaccination, at farrowing time and one week after revaccination. Although there were considerable variations among the herds, the number of returns to oestrus in all herds was higher in vaccinated gilts (11.81 YO) than in the controls (10.52%). This difference, however, was not statistically significant. The reproductive performance results revealed the absence of an increase in the total born, as pooled values, in vaccinated gilts compared to controls. However, when these results are interpreted in relation to serological data, many control gilts were already seropositive before mating, or remained seronegative at farrowing. According to our results, the duration of immunity with this vaccine is apparently short, as there is a clear decrease in the titres between the 1st and the 2nd sampling times (2.35 k 0.14 and 1.97? 0.08, respectively).
Introduction Porcine parvovirus (PPV) is an infectious agent most common in farms involved in pig production. The virus has been recognized worldwide as the main infectious cause of reproductive failure in swine (7). The main features of these dysfunctions are embryonaric death and foetal mummification, return to oestrus, weak newborn piglets and small litter sizes, especially in gilts (8). In view of these features, PPV is regarded as responsible for a considerable part of the economic losses in the affected herds. In order to control PPV, both modified live and inactivated vaccines with either oil or aluminum hydroxide adjuvants have been developed (4, 5 , 6, 9, 10, 13, 15, 19, 21) and tested in pregnant sows. Most of these vaccines proved effective as they produced immunity in pregnant sows and their litters when challenged with a live virus. However, important differences have been observed depending on the antibodies titre induced with the adjuvants used, and on the duration of these antibodies (2, 4, 21). US.Copyright Clearance Center Code Statement:
0931 - 1793/92/3905 -0337$02.50/0
338
CASTRO, DEL Pozo and SIMARRO
T h e present paper describes the results of a field trial in 10 herds carried o u t with an inactivated vaccine against PPV with aluminum hydroxide adjuvant. In each herd, the serological responses and reproductive performance of vaccinated gilts were compared to those of contemporary non-vaccinated controls.
Material and Methods General. Ten pig breeding herds were selected to participate in the field trial. Each herd had a history of reproductive failure typical of PPV. Parvovirus had been identified, in most of the herds, by virological or serological tests (3). All the herds were housed and managed under similar conditions. The service area was in pens with different types of floor, either a partial slat floor or straw floor without slat. All gilts were mated according to the normal herd management and carried out on natural conditions. Experimental vaccine. The vaccine was prepared and inactivated according to MENGELING et al. (9). Briefly, the vaccine consisted of the PPV- NADL-2 strain grown on porcine embryonic kidney cells on their passage 7. The virus seed, with a titer of at least 1 :256 hemagglutination units, was added at the ratio of 1 ml to 400 ml of media (1 : 1,000 to 1 : 10,000). The virus was inactivated by adding 2.0ml of 0.1 binary ethylenimine (BEI)/100 ml virus. After an incubation period, surplus BE1 was neutralized by adding 1.0 M sodium thiosulfate at a rate of 0.5ml/100ml inactivated PPV. Sterile aluminum hydroxide (10 %) was added to a final concentration of 1 % and sterile saponin (2 YO)to a final concentration of 0.5 mg/ml. Once the product was blended, its p H was adjusted to 6.3-6.5 by the addition of 5N NaOH. Finally, a fungistatic agent (2.5 mg/ml) was added. The final product was standardized so that it would contain at least 1 : 1,024 hemagglutination units/2 ml (vaccination dose). Blood sampling and Vaccination. Seronegative gilts only (titres < 1/10 by hemagglutination inhibition test) were used. The trial comprised a total of 277 gilts, of which 220 (79.42%) were vaccinated twice fourteen days apart at least two weeks before mating and the remaining 57 (20.57%) were used as non-vaccinated controls. Seven days after the second vaccination, blood samples were drawn from both vaccinated and non-vaccinated gilts (1st sample). When these gilts farrowed, a further blood sample was taken (2nd sample). Vaccinated sows were re-vaccinated with a third dose at weaning. A third blood sample was drawn from vaccinated and non-vaccinated sows, at least seven days after revaccination and before the new mating (3rd sample). Farmers were asked to record any abnormal localized or generalized reactions after vaccination. Serological test and interpretation. Serum samples were tested for levels of porcine parvovirus antibody by the hemagglutination inhibition test (HI) (3). Although all the samples were fully titrated and a very wide range of titres was found, it was decided after a preliminary analysis of the data to simplify the results. The sera were grouped into three categories according to the titre: reciprocal HI titres < 1/10, considered as negative, titers ranging between 1/10 and 1/640, and titers > 11640, considered to have arisen from active immunizations in vaccinated gilts or active infection in control gilts. Reproduction records. Production data were recorded in every herd, including the number of gilts mating, returns to oestrus, and gilts sent to slaughter. The total number of births, including liveborn, stillborn and mummified, was also recorded. Data analysis. Statistical analyses were performed by applying ANOVA and Turkey's test.
Results No post-vaccination reactions were seen in a n y of the animals. Table 1 shows the number of vaccinated and non-vaccinated groups which were served, farrowed o r dropped out the trial for any reason. The number of gilts pregnant at the first mating, and those returning to a new oestrus, one or several times, are summarized in Table 2. Although the number of returns to oestrus was higher in vaccinated gilts (11.81 %) than in the controls (10.5 Yo), this difference was not statistically significant, and was caused mainly by an increase in the number of returns, either normal o r abnormal, in vaccinated gilts from herd 1. In this herd, 13 out of 36 vaccinated gilts (36.11 "/.) returned to oestrus compared to 1 o u t of 8 (12.50%) in the control gilts (data not shown). Details of the litters, including the number of liveborn, stillborn, mummified and total litter size produced by vaccinated and control gilts in the 10 herds in the trial are shown in
339
Field Trials of an Inactivated Virus Vaccine Table 1. Fate of gilts participating in the vaccine trial (all herd)
Number of gilts farrowed (Yo of gilts in trial) Gilts dropped out - Slaughter after service - Non-pregnant - Surplus of repeats - Deaths - Vaginal Discharge Number of gilts served (% of gilts) Gilts dropped out - Lameness - N o heat before service Number of gilts in trial
Vaccinated gilts
Controls
Total
220 (79.42)
57 (20.57)
277
3 1 4 1 1 230 (79.58)
59 (20.41)
289
2 5 237 (79.53)
1 1 61 (20.46)
298
1 1
Table 3. Although there were considerable variations among the herds, none of the differences between controls and vaccinated gilts within herds, or overall, were significant. Table 4 shows the serological titres categorized into the three classes for the vaccinated and control gilts at their Ist, 2nd, and 3rd samplings. Combined results for all the combined herds show that out of the 220 vaccinated gilts with two doses of the vaccine, 19 (8.6 Yo) were seronegative at the 1st sampling; 153 (69.5 %) had titres ranging between 1 : 10 and 1 : 640; and 48 (21.9 %) had titres over 1 :640. At farrowing (2nd sampling), 54 (24.5 Yo) were seronegative; 115 (52.2 %) had titres between 1 : 10 and 1 : 640; and 51 (23.3 Yo) had titres over 1 : 640. Titres obtained after the third dose of vaccine (after the first farrowing, 3rd sampling) show that 16 (1 7.3 %) gilts were seronegative; 101 (45.9 Yo) had titres between 1 : 10 and 1 : 640; and 103 (46.8 Yo) showed titres over 1 : 640. Of the 19 gilts vaccinated with two dose and were seronegative at the 1st sampling, 14 stayed seronegative and 5 had seroconverted at the 2nd sampling. Of the 153 vaccinated gilts with titres between 1 : 10 and 1 : 640,21 had seroconverted, and 39 returned to seronegative in the 2nd sampling. After the third vaccine doses, 13 of the 54 seronegative gilts at the 2nd sampling continued to be seronegative; 3 which had shown a titre between 1 : 10 and 1 : 640 returned to seronegativity, adding a total of 16 seronegative sows. If the titres are expressed as the mean of the log10 of the highest serum dilution which inhibits four hemagglutination units of PPV, the values are 2.35 k 0.14, 1.97 & 0.08, and 2.77 k 0.13, for the Ist, 2nd and 3rd sampling, respectively.
Table 2. Detail of services and repeat services in gilts which farrowed in the trial
Served once Served twice Served three times Total number of services Mean number services per farrowing Repeat services at intervals < 18 days Repeat services at normal intervals (18-24) Repeat services at intervals longer than 24 days Repeat services at intervals longer than 48 days
Vaccinated (per cent)
Control (per cent)
190 26 (11.81) 4 (1.80) 254 1.15 3 (8.82) 12 (35.29) 15 (44.11) 4 (11.76)
50 6 (10.52) 1 (1.75) 65 1.14 0 4 (50.00) 1 (12.50) 3 (37.50)
CASTRO, DEL Pozo and SIMARRO
340
Table 3. Details of litters produced by the control and vaccinated gilts Vaccinated Mean number Stillborn %
Herd
Number of Litters
Mean number Liveborn
1 2 3 4 5 6 7 8 9 10 All herd
36 15 31 26 31 15 20 12 21 13 220
8.33 9.73 8.67 8.42 9.12 8.00 9.25 7.58 10.40 9.07 8.85
Herd
Number
Mean
Litters
Mean number Liveborn
8 4 10 4 9 3 7 2 7 3 57
8.62 10.25 9.30 8.50 7.88 8.66 9.28 9.50 12.14 8.66 9.27
0.00- 0.00 0.25- 2.38 0.3 - 3.12 0.00- 0.00 0.34- 4.05 1.34-13.33 0.00- 0.00 0.00- 0.00 0.86- 6.59 0.67- 7.14 0.37- 3.66
0.22-2.59 0.33-3.31 0.68-7.24 0.15-1.71 0.88-8.70 0.46-5.51 0.75-7.50 0.66-8.08 0.694.00 0.23-2.47 0.50-5.31
Mean Mean number total born Mummified YO 0.02-0.32 0.13-1.32 0.12-1.37 0.004.00 0.12-1.29 0 .oo-0 .00 0.00-0.00 0.00-0.00 0.09-0.85 0.00-0.00 0.05-0.51
8.55 10.06 9.35 8.57 10.00 8.46 10.00 8.25 11.09 9.30 9.36
Control of
1 2 3 4 5 6 7 8 9 10
All herd
number Stillborn %
Mean Mean number total born Mummified % 0.004.00 0 .oo-0 .00 0.1 -1.04
0.00-0.00 0.00-0.00 0.664.66 0.00-0.00 0.00-0.00 0.28-2.19 0.00-0.00 0.104.98
8.62 10.50 9.60 8.50 8.22 10.00 9.28 9.50 13.00 9.33 9.65
O u t of the 57 control gilts (Tab. 4), 32 (56.1 YO)were seronegative in the 1st sampling, while the remaining 25 (43.9 %) had titres equal to or over 1 : 10. When analysing these 32 seronegative control gilts individually, 19 remained seronegative, while the remaining 13 seroconverted. Thus, these 19 gilts, were considered as the only true controls. Productive data of the control gilts are shown in Table 5 . A higher percentage of mummified foetuses and repeat matings was seen in seroconverted controls (non-statistical significance) than in non-seroconverted. Table 4 also reveals some major differences between the herds, especially in the proportions of gilts with negative HI titres at the 1st sampling and in the proportions of controls moving into the category indicative of active infection at the 2nd sampling. Herd 5, with no seroconversion in the control gilts, was at one end of the spectrum in this respect, and herds I , 4, and 7 with seroconversions were at the other. The variations were probably due to the relative levels of the activity of “wild” parvovirus in the herds. However, the seroconversion observations (Tab. 4) did not correspond to the productive performance results (Tab. 3).
34 1
Field Trials of an Inactivated Virus Vaccine
Table 4. Reciprocal serum hemagglutination inhibition titres of vaccinated and control gilts for the first, second and third samples Titres of vaccinated gilts Second sample
First sample Herd 1 2 3 4 5 6 7 8 9 10 Total of herds
Number of samples 36 15 31 26 31 15 20 12 21 13 220
640
640
(YO)
7(12.28) 3 (23.07) l(5.26) 3 (12.00)
342
CASTRO, DEL Pozo and SIMARRO
Discussion Other researchers have carried out field trials with inactivated vaccines against PPV (2, 14, 17, 19), but only WRATHALL(19) has used such a large number of sows with contemporary controls. However, WRATHALL (19) initiated the field trials in animals with a varied range of antibody titres. In the present study, we only used seronegative gilts ( < 1/10). O n the other hand, serological data were not always shown, which would have provided information about the possible presence of wild parvovirus in herds, the rate of viral spread, and sero-conversions among gilts. Furthermore, there is contradicting information about the efficacy of vaccines against PPV, and it is difficult to establish whether the efficacy measured is in terms of protection and depends on the H I serological titre degree obtained. Otherwise, it is important to emphasize that there are clear differences between vaccines adjuvanted with oil or with aluminum hydroxide. The former provide high, uniform and long duration titres (4, 17, 21) while the titres of the latter are the opposite (2, 4, 10, 14, 16). In addition to these differences, maternal antibodies to PPV in the range of 1 :40 to 1 : 80 may block the immune response to the vaccine adjuvanted with aluminum hydroxide (12). It seems that this effect is null, o r at least very small, in oiladjuvanted vaccines (20). The reproduction results shown in Table 3 surprisingly reveal the absence of an increase in the total number of piglets born, as pooled values, in vaccinated gilts compared with controls. However, when these results are interpreted relative to serological data (Tab. 4) it is not so surprising, given that many control gilts were already seropositives at the 1st sampling, or they remained seronegative at farrowing. By analyzing individual herds, we can see that there was no evidence of wild parvovirus challenging control gilts in Herd 5. According to WRATHALL (19), our results could be used as an evidence against the need to implement widespread vaccination, especially because of the expense of the vaccine and by the shift in the balance of the “wild” virus in herds. The lack of correlation between the serological data from herds I , 4 and 7 and the productive performance sharply contrast with those obtained by WRATHALL(19) in spite of seroconversion observed in the above mentioned herds, which represent eight out of a total of 19 control gilts. When the serological results of gilts was analyzed (Tab. 4), 8.6 % of the gilts did not develop antibodies against PPV after two vaccine doses. These results agree with those reported by other authors studying similar vaccines (2, 4, 14, 16). In the present study the mean titres reached with two doses of the vaccine were log10 2.35 f 0.14 HI. These titres were similar to the vaules reported by SORENSEN and ASKAA (14) and BROWNet al. (2). However, our values were much lower than those obtained by EDWARDS et al. (4), VANNIER et al. (17), WRATHALL et al. (21), who have worked with oiladjuvanted vaccines. There may be several reasons which explain the different behavior of these vaccines. First, variations in the amount of antigenic mass used, provided that a certain amount of virus might have been added in the foetal material used in the vaccine preparation (4, 21). Second, the viruses included in vaccines such as the one used by BROWNet al. (2), o r in this research, may have been less immunogenic as a result of changes induced in their antigenic properties, when these viruses are selected through cell cultures. In laboratory tests with other viruses, it has been found that those antigens can cause an important proportion in the total antigenic contents of the infected material (18). Finally, the adjuvant used may also determine a different response; ANDERSON et al. (1) reported that oil-adjuvanted vaccines may cause a higher and longer immunity in pigs. According to our results, the duration of immunity with this vaccine is apparently short, as there is a clear decrease in the titres between the 1st (8.6 % seronegative gilts, mean titre log10 2.35 -t 0.14) and the 2nd sampling (24.5 % seronegative gilts, mean titre log10 1.97 k 0.8). This agrees with results reported by others (2, 10, 14) who described a significant decrease of HI titres 8-10 weeks following the first vaccination. Results published by BROWNet al. (2) show that 75 % of the vaccinated gilts were seronegative 112 days after vaccination. O n the contrary, reports by WRATHALL et al. (21), EDWARDS et al.
Field Trials of an Inactivated Virus Vaccine
343
(4), and VANNIER et al. (17) show that the duration of the HI titres after vaccinating with an oil emulsion preparation was at least between 7 and 12 months, or longer. Vaccinated gilts seropositive at the 1st sampling whose titres decreased markedly or even to seronegativity or those which seroconverted at 2nd sampling by wild parvovirus infection, did not have visible dysfunctions during pregnancy. Nevertheless, in the present work we did not measure whether there was transplacental infection or not. However, the remarkable increase in the titres at farrowing of some of the vaccinated gilts showed that there had been virus replication. Virus replication in vaccinated gilts with no transplacental foetal infection has been noted before, suggesting that PPV replication in such gilts only occurred in exposure sites (9). In spite of the fact that the virus may replicate in these animals, the animals showed no viremia whatsoever (13). This may explain why transplacental infection does not take place, provided that viremia appears to be a pre-requirement for transplacental infection by PPV (8). Since our experiment was stopped after the first farrowing we could not confirm the potential value of vaccination as reported by WRATHALL (19).
References 1. ANDERSON, E.C., R.C. MASTERS,and G . N . MOWAT,1971: Immune response of pigs to inactivated Foot and Mouth diseases vaccines. Res. Vet. Sci. 12, 342-350. T. T., M. D. WHITACRE, and 0.WAYNE ROBISON, 1987: Use of an inativated vaccine for 2. BROWN, prevention of parvovirus-induced reproductive failure in gilts. J. A. V. M. A. 190 (2), 179-181. J. M., M. DEL Pozo, E. RODRIGUEZ y A. DE LAS HERAS,1986: Prevalencia de anticuerpos 3. CASTRO, frente a parvovirus porcino en granjas de producci6n en ciclo cerrado. Med. Vet. 3 (7), 401 -405. K.R., M.A. EMMERSON, P.R. LUFF,D.E. WELLS,J. C. MUSKETT, A. E. WRATHALL, 4. EDWARDS, 1986: Efficacy of porcine parvovirus C. RICHARDSON, B. N . J. PARKER,and D. H. THORTON, vaccines. Vet. Rec. 119, 203-205. 5. FUJISAKI, Y., T. ICHIHARA,N. SASAKI, F. SHIMIZU, Y. MURAKAMI, T. SUEIMORI,and J. SASAHARA, 1978 b: Field trials on inactivated porcine parvovirus vaccine for prevention of viral stillbirth among swine. Bull. Natl. Inst. Anim. Healt 77, 12-14. 6. J o o , H . S., and R. H. JOHNSON,1977: Serological response in pigs vaccinated with inactivated porcine parvovirus. Aust. Vet. J. 53, 550-552. 7. MENGELING, W. L., 1986: Porcine parvovirus infection. In: A. D. LEMAN,B. STRAW,R. D. GLOCK,W. L. MENGELING, R. H. C. PENNY,and E. SCHOLL,Edit. Diseases of swine, p. 411-424, Iowa State University Press. AMES. IOWA U.S.A. 8. MENGELING, W. L., and R. C. CUTLIP,1976: Reproductive disease experimentally induced by exposing pregnant gils to porcine parvovirus. Am J. Vet. Res. 37, 1393-1400. 1979: Efficacy of an 9. MENGELING, W. L., T.T. BROWN,P. S. PAUL, and E. E. GUTEKUNST, inactivated virus vaccine for prevention of porcine parvovirus induced reproductive failure. Am. J. Vet. Res. 40, 204-207. 10. MENGELING, W. L., D. E. GUTEKUNST, E. C. PIRTLE,and P. S. PAUL,1981: Immunogenicity of bivalent vaccine for reproductive failure of swine induced by pseudorabies virus and porcine parvovirus. Am J. Vet. Res. 42, 600-603. 11. OuLDRtDGE, E. J., M.J. FRANCIS, and L. BLACK,1982: Antibody response of pigs to foot and mouth disease oil emulsion vaccine: The antibody classes involved. Res Vet. Sci. 32, 327-331. 12. PAUL,P.S., and W.L. MENGELING, 1986: Vaccination of swine with an inactivated porcine parvovirus vaccine in the presence of passive immunity. J. A. V. M. A. 188 (4), 410-413. and T. T. BROWN,1980: Effect of vaccinal and passive immunity 13. PAUL,P. S., W. L. MENGELING, on experimental infection of pigs with porcine parvovirus. Am. J. Vet. Res. 41, 1368-1371. K. J., and J. ASKAA,1981: Vaccination against porcine parvovirus infection. Acta Vet. 14. SORENSEN, Scand. 22, 171-179. H., and F. FUJISAKI,1976: Immunizing effects of inactivated porcine parvovirus vaccine 15. SUZUKI, on piglets. Bull. Natl. Inst. Anim. Health. 72, 17-23. and R. SALINE,1986: Evaluation of commercial 16. TRACKER, B. J., L. BATISTA,R. L. GONZALEZ, porcine parvovirus vaccines: post-vaccination serological responses, hemagglutinating activity and long term serostatus of vaccinated gilts in endemically infected herd. Proceeding of the Pig Veterinary Society, p. 87. 17. VANNIER,P., A.BRuN, G.CHAPPUIS,and G.REYNAUD, 1986: Study of the efficacy of an inactivated virus vaccine against porcine parvovirus. Ann. Rech. Vet. 17, (4), 425-432.
344
CASTRO, DEL P o z o and SIMARRO
18. WALLACE, B. L., J. K. MCMILLEN, and J. D. TODD,1983: Canine parvovirus serum neutralizing
antibody assay: Assessment of factors responsible for disparity of results between tests. Cornell. Vet. 73, 52-57. A. E., 1988: Field trials of an inactivated oil-emulsion porcine parvovirus vaccine in 19. WRATHALL, British pig herds. Vet. Rec. 122, 411-418. A. E., S. F. CARTWRIGHT, D. E. WELLS,and P. C. JONES,1987: Maternally-derived 20. WRATHALL, antibodies to porcine parvovirus and their effect on active antibody production after vaccination with an inactivated oil-emulsion vaccine. Vet. Rec. 120, 475-478. 21. WRATHALL, A. E., D. E. WELLS,S. F. CARTWRIGHT, and G. N. FRERICHS, 1984: An inactivated, oil-emulsion vaccine for the prevention of porcine parvovirus induced reproductive failure. Res. Vet. Sci. 36, 136-143.
Animal Pathology Dpt. I. Veterinary Faculty UCM, Avda. Puerta de Hierro sln, 28040 - Madrid
Field Trials of an Inactivated Virus Vaccine Against Porcine Parvovirus J. M. CASTRO', M. DEL POZ02 and I. SIMARROl Address of authors: ' Animal Pathology Dpt. I. Faculty of Veterinary, UCM, Avda. Puerta de Hierro s/n, 28040 - Madrid (Espafia) * Porcine Pathology Laboratory, Proinserga, Segovia (Espafia)
With 5 tables (Received for publication Juli 12, 1991)
Summary Serological response and reproductive performance were estimated in field trials of an inactivated virus vaccine against porcine parvovirus. Experiments were carried out in 10 selected pig breeding herds. A total of 277 seronegative gilts were used. Two hundred and twenty animals were vaccinated twice before mating, fourteen days apart and revaccinated after farrowing. Blood samples were obtained from both vaccinated and non-vaccinated (57 animal) control gilts, one week after the 2nd dose of vaccination, at farrowing time and one week after revaccination. Although there were considerable variations among the herds, the number of returns to oestrus in all herds was higher in vaccinated gilts (11.81 YO) than in the controls (10.52%). This difference, however, was not statistically significant. The reproductive performance results revealed the absence of an increase in the total born, as pooled values, in vaccinated gilts compared to controls. However, when these results are interpreted in relation to serological data, many control gilts were already seropositive before mating, or remained seronegative at farrowing. According to our results, the duration of immunity with this vaccine is apparently short, as there is a clear decrease in the titres between the 1st and the 2nd sampling times (2.35 k 0.14 and 1.97? 0.08, respectively).
Introduction Porcine parvovirus (PPV) is an infectious agent most common in farms involved in pig production. The virus has been recognized worldwide as the main infectious cause of reproductive failure in swine (7). The main features of these dysfunctions are embryonaric death and foetal mummification, return to oestrus, weak newborn piglets and small litter sizes, especially in gilts (8). In view of these features, PPV is regarded as responsible for a considerable part of the economic losses in the affected herds. In order to control PPV, both modified live and inactivated vaccines with either oil or aluminum hydroxide adjuvants have been developed (4, 5 , 6, 9, 10, 13, 15, 19, 21) and tested in pregnant sows. Most of these vaccines proved effective as they produced immunity in pregnant sows and their litters when challenged with a live virus. However, important differences have been observed depending on the antibodies titre induced with the adjuvants used, and on the duration of these antibodies (2, 4, 21). US.Copyright Clearance Center Code Statement:
0931 - 1793/92/3905 -0337$02.50/0
338
CASTRO, DEL Pozo and SIMARRO
T h e present paper describes the results of a field trial in 10 herds carried o u t with an inactivated vaccine against PPV with aluminum hydroxide adjuvant. In each herd, the serological responses and reproductive performance of vaccinated gilts were compared to those of contemporary non-vaccinated controls.
Material and Methods General. Ten pig breeding herds were selected to participate in the field trial. Each herd had a history of reproductive failure typical of PPV. Parvovirus had been identified, in most of the herds, by virological or serological tests (3). All the herds were housed and managed under similar conditions. The service area was in pens with different types of floor, either a partial slat floor or straw floor without slat. All gilts were mated according to the normal herd management and carried out on natural conditions. Experimental vaccine. The vaccine was prepared and inactivated according to MENGELING et al. (9). Briefly, the vaccine consisted of the PPV- NADL-2 strain grown on porcine embryonic kidney cells on their passage 7. The virus seed, with a titer of at least 1 :256 hemagglutination units, was added at the ratio of 1 ml to 400 ml of media (1 : 1,000 to 1 : 10,000). The virus was inactivated by adding 2.0ml of 0.1 binary ethylenimine (BEI)/100 ml virus. After an incubation period, surplus BE1 was neutralized by adding 1.0 M sodium thiosulfate at a rate of 0.5ml/100ml inactivated PPV. Sterile aluminum hydroxide (10 %) was added to a final concentration of 1 % and sterile saponin (2 YO)to a final concentration of 0.5 mg/ml. Once the product was blended, its p H was adjusted to 6.3-6.5 by the addition of 5N NaOH. Finally, a fungistatic agent (2.5 mg/ml) was added. The final product was standardized so that it would contain at least 1 : 1,024 hemagglutination units/2 ml (vaccination dose). Blood sampling and Vaccination. Seronegative gilts only (titres < 1/10 by hemagglutination inhibition test) were used. The trial comprised a total of 277 gilts, of which 220 (79.42%) were vaccinated twice fourteen days apart at least two weeks before mating and the remaining 57 (20.57%) were used as non-vaccinated controls. Seven days after the second vaccination, blood samples were drawn from both vaccinated and non-vaccinated gilts (1st sample). When these gilts farrowed, a further blood sample was taken (2nd sample). Vaccinated sows were re-vaccinated with a third dose at weaning. A third blood sample was drawn from vaccinated and non-vaccinated sows, at least seven days after revaccination and before the new mating (3rd sample). Farmers were asked to record any abnormal localized or generalized reactions after vaccination. Serological test and interpretation. Serum samples were tested for levels of porcine parvovirus antibody by the hemagglutination inhibition test (HI) (3). Although all the samples were fully titrated and a very wide range of titres was found, it was decided after a preliminary analysis of the data to simplify the results. The sera were grouped into three categories according to the titre: reciprocal HI titres < 1/10, considered as negative, titers ranging between 1/10 and 1/640, and titers > 11640, considered to have arisen from active immunizations in vaccinated gilts or active infection in control gilts. Reproduction records. Production data were recorded in every herd, including the number of gilts mating, returns to oestrus, and gilts sent to slaughter. The total number of births, including liveborn, stillborn and mummified, was also recorded. Data analysis. Statistical analyses were performed by applying ANOVA and Turkey's test.
Results No post-vaccination reactions were seen in a n y of the animals. Table 1 shows the number of vaccinated and non-vaccinated groups which were served, farrowed o r dropped out the trial for any reason. The number of gilts pregnant at the first mating, and those returning to a new oestrus, one or several times, are summarized in Table 2. Although the number of returns to oestrus was higher in vaccinated gilts (11.81 %) than in the controls (10.5 Yo), this difference was not statistically significant, and was caused mainly by an increase in the number of returns, either normal o r abnormal, in vaccinated gilts from herd 1. In this herd, 13 out of 36 vaccinated gilts (36.11 "/.) returned to oestrus compared to 1 o u t of 8 (12.50%) in the control gilts (data not shown). Details of the litters, including the number of liveborn, stillborn, mummified and total litter size produced by vaccinated and control gilts in the 10 herds in the trial are shown in
339
Field Trials of an Inactivated Virus Vaccine Table 1. Fate of gilts participating in the vaccine trial (all herd)
Number of gilts farrowed (Yo of gilts in trial) Gilts dropped out - Slaughter after service - Non-pregnant - Surplus of repeats - Deaths - Vaginal Discharge Number of gilts served (% of gilts) Gilts dropped out - Lameness - N o heat before service Number of gilts in trial
Vaccinated gilts
Controls
Total
220 (79.42)
57 (20.57)
277
3 1 4 1 1 230 (79.58)
59 (20.41)
289
2 5 237 (79.53)
1 1 61 (20.46)
298
1 1
Table 3. Although there were considerable variations among the herds, none of the differences between controls and vaccinated gilts within herds, or overall, were significant. Table 4 shows the serological titres categorized into the three classes for the vaccinated and control gilts at their Ist, 2nd, and 3rd samplings. Combined results for all the combined herds show that out of the 220 vaccinated gilts with two doses of the vaccine, 19 (8.6 Yo) were seronegative at the 1st sampling; 153 (69.5 %) had titres ranging between 1 : 10 and 1 : 640; and 48 (21.9 %) had titres over 1 :640. At farrowing (2nd sampling), 54 (24.5 Yo) were seronegative; 115 (52.2 %) had titres between 1 : 10 and 1 : 640; and 51 (23.3 Yo) had titres over 1 : 640. Titres obtained after the third dose of vaccine (after the first farrowing, 3rd sampling) show that 16 (1 7.3 %) gilts were seronegative; 101 (45.9 Yo) had titres between 1 : 10 and 1 : 640; and 103 (46.8 Yo) showed titres over 1 : 640. Of the 19 gilts vaccinated with two dose and were seronegative at the 1st sampling, 14 stayed seronegative and 5 had seroconverted at the 2nd sampling. Of the 153 vaccinated gilts with titres between 1 : 10 and 1 : 640,21 had seroconverted, and 39 returned to seronegative in the 2nd sampling. After the third vaccine doses, 13 of the 54 seronegative gilts at the 2nd sampling continued to be seronegative; 3 which had shown a titre between 1 : 10 and 1 : 640 returned to seronegativity, adding a total of 16 seronegative sows. If the titres are expressed as the mean of the log10 of the highest serum dilution which inhibits four hemagglutination units of PPV, the values are 2.35 k 0.14, 1.97 & 0.08, and 2.77 k 0.13, for the Ist, 2nd and 3rd sampling, respectively.
Table 2. Detail of services and repeat services in gilts which farrowed in the trial
Served once Served twice Served three times Total number of services Mean number services per farrowing Repeat services at intervals < 18 days Repeat services at normal intervals (18-24) Repeat services at intervals longer than 24 days Repeat services at intervals longer than 48 days
Vaccinated (per cent)
Control (per cent)
190 26 (11.81) 4 (1.80) 254 1.15 3 (8.82) 12 (35.29) 15 (44.11) 4 (11.76)
50 6 (10.52) 1 (1.75) 65 1.14 0 4 (50.00) 1 (12.50) 3 (37.50)
CASTRO, DEL Pozo and SIMARRO
340
Table 3. Details of litters produced by the control and vaccinated gilts Vaccinated Mean number Stillborn %
Herd
Number of Litters
Mean number Liveborn
1 2 3 4 5 6 7 8 9 10 All herd
36 15 31 26 31 15 20 12 21 13 220
8.33 9.73 8.67 8.42 9.12 8.00 9.25 7.58 10.40 9.07 8.85
Herd
Number
Mean
Litters
Mean number Liveborn
8 4 10 4 9 3 7 2 7 3 57
8.62 10.25 9.30 8.50 7.88 8.66 9.28 9.50 12.14 8.66 9.27
0.00- 0.00 0.25- 2.38 0.3 - 3.12 0.00- 0.00 0.34- 4.05 1.34-13.33 0.00- 0.00 0.00- 0.00 0.86- 6.59 0.67- 7.14 0.37- 3.66
0.22-2.59 0.33-3.31 0.68-7.24 0.15-1.71 0.88-8.70 0.46-5.51 0.75-7.50 0.66-8.08 0.694.00 0.23-2.47 0.50-5.31
Mean Mean number total born Mummified YO 0.02-0.32 0.13-1.32 0.12-1.37 0.004.00 0.12-1.29 0 .oo-0 .00 0.00-0.00 0.00-0.00 0.09-0.85 0.00-0.00 0.05-0.51
8.55 10.06 9.35 8.57 10.00 8.46 10.00 8.25 11.09 9.30 9.36
Control of
1 2 3 4 5 6 7 8 9 10
All herd
number Stillborn %
Mean Mean number total born Mummified % 0.004.00 0 .oo-0 .00 0.1 -1.04
0.00-0.00 0.00-0.00 0.664.66 0.00-0.00 0.00-0.00 0.28-2.19 0.00-0.00 0.104.98
8.62 10.50 9.60 8.50 8.22 10.00 9.28 9.50 13.00 9.33 9.65
O u t of the 57 control gilts (Tab. 4), 32 (56.1 YO)were seronegative in the 1st sampling, while the remaining 25 (43.9 %) had titres equal to or over 1 : 10. When analysing these 32 seronegative control gilts individually, 19 remained seronegative, while the remaining 13 seroconverted. Thus, these 19 gilts, were considered as the only true controls. Productive data of the control gilts are shown in Table 5 . A higher percentage of mummified foetuses and repeat matings was seen in seroconverted controls (non-statistical significance) than in non-seroconverted. Table 4 also reveals some major differences between the herds, especially in the proportions of gilts with negative HI titres at the 1st sampling and in the proportions of controls moving into the category indicative of active infection at the 2nd sampling. Herd 5, with no seroconversion in the control gilts, was at one end of the spectrum in this respect, and herds I , 4, and 7 with seroconversions were at the other. The variations were probably due to the relative levels of the activity of “wild” parvovirus in the herds. However, the seroconversion observations (Tab. 4) did not correspond to the productive performance results (Tab. 3).
34 1
Field Trials of an Inactivated Virus Vaccine
Table 4. Reciprocal serum hemagglutination inhibition titres of vaccinated and control gilts for the first, second and third samples Titres of vaccinated gilts Second sample
First sample Herd 1 2 3 4 5 6 7 8 9 10 Total of herds
Number of samples 36 15 31 26 31 15 20 12 21 13 220
640
640
(YO)
7(12.28) 3 (23.07) l(5.26) 3 (12.00)
342
CASTRO, DEL Pozo and SIMARRO
Discussion Other researchers have carried out field trials with inactivated vaccines against PPV (2, 14, 17, 19), but only WRATHALL(19) has used such a large number of sows with contemporary controls. However, WRATHALL (19) initiated the field trials in animals with a varied range of antibody titres. In the present study, we only used seronegative gilts ( < 1/10). O n the other hand, serological data were not always shown, which would have provided information about the possible presence of wild parvovirus in herds, the rate of viral spread, and sero-conversions among gilts. Furthermore, there is contradicting information about the efficacy of vaccines against PPV, and it is difficult to establish whether the efficacy measured is in terms of protection and depends on the H I serological titre degree obtained. Otherwise, it is important to emphasize that there are clear differences between vaccines adjuvanted with oil or with aluminum hydroxide. The former provide high, uniform and long duration titres (4, 17, 21) while the titres of the latter are the opposite (2, 4, 10, 14, 16). In addition to these differences, maternal antibodies to PPV in the range of 1 :40 to 1 : 80 may block the immune response to the vaccine adjuvanted with aluminum hydroxide (12). It seems that this effect is null, o r at least very small, in oiladjuvanted vaccines (20). The reproduction results shown in Table 3 surprisingly reveal the absence of an increase in the total number of piglets born, as pooled values, in vaccinated gilts compared with controls. However, when these results are interpreted relative to serological data (Tab. 4) it is not so surprising, given that many control gilts were already seropositives at the 1st sampling, or they remained seronegative at farrowing. By analyzing individual herds, we can see that there was no evidence of wild parvovirus challenging control gilts in Herd 5. According to WRATHALL (19), our results could be used as an evidence against the need to implement widespread vaccination, especially because of the expense of the vaccine and by the shift in the balance of the “wild” virus in herds. The lack of correlation between the serological data from herds I , 4 and 7 and the productive performance sharply contrast with those obtained by WRATHALL(19) in spite of seroconversion observed in the above mentioned herds, which represent eight out of a total of 19 control gilts. When the serological results of gilts was analyzed (Tab. 4), 8.6 % of the gilts did not develop antibodies against PPV after two vaccine doses. These results agree with those reported by other authors studying similar vaccines (2, 4, 14, 16). In the present study the mean titres reached with two doses of the vaccine were log10 2.35 f 0.14 HI. These titres were similar to the vaules reported by SORENSEN and ASKAA (14) and BROWNet al. (2). However, our values were much lower than those obtained by EDWARDS et al. (4), VANNIER et al. (17), WRATHALL et al. (21), who have worked with oiladjuvanted vaccines. There may be several reasons which explain the different behavior of these vaccines. First, variations in the amount of antigenic mass used, provided that a certain amount of virus might have been added in the foetal material used in the vaccine preparation (4, 21). Second, the viruses included in vaccines such as the one used by BROWNet al. (2), o r in this research, may have been less immunogenic as a result of changes induced in their antigenic properties, when these viruses are selected through cell cultures. In laboratory tests with other viruses, it has been found that those antigens can cause an important proportion in the total antigenic contents of the infected material (18). Finally, the adjuvant used may also determine a different response; ANDERSON et al. (1) reported that oil-adjuvanted vaccines may cause a higher and longer immunity in pigs. According to our results, the duration of immunity with this vaccine is apparently short, as there is a clear decrease in the titres between the 1st (8.6 % seronegative gilts, mean titre log10 2.35 -t 0.14) and the 2nd sampling (24.5 % seronegative gilts, mean titre log10 1.97 k 0.8). This agrees with results reported by others (2, 10, 14) who described a significant decrease of HI titres 8-10 weeks following the first vaccination. Results published by BROWNet al. (2) show that 75 % of the vaccinated gilts were seronegative 112 days after vaccination. O n the contrary, reports by WRATHALL et al. (21), EDWARDS et al.
Field Trials of an Inactivated Virus Vaccine
343
(4), and VANNIER et al. (17) show that the duration of the HI titres after vaccinating with an oil emulsion preparation was at least between 7 and 12 months, or longer. Vaccinated gilts seropositive at the 1st sampling whose titres decreased markedly or even to seronegativity or those which seroconverted at 2nd sampling by wild parvovirus infection, did not have visible dysfunctions during pregnancy. Nevertheless, in the present work we did not measure whether there was transplacental infection or not. However, the remarkable increase in the titres at farrowing of some of the vaccinated gilts showed that there had been virus replication. Virus replication in vaccinated gilts with no transplacental foetal infection has been noted before, suggesting that PPV replication in such gilts only occurred in exposure sites (9). In spite of the fact that the virus may replicate in these animals, the animals showed no viremia whatsoever (13). This may explain why transplacental infection does not take place, provided that viremia appears to be a pre-requirement for transplacental infection by PPV (8). Since our experiment was stopped after the first farrowing we could not confirm the potential value of vaccination as reported by WRATHALL (19).
References 1. ANDERSON, E.C., R.C. MASTERS,and G . N . MOWAT,1971: Immune response of pigs to inactivated Foot and Mouth diseases vaccines. Res. Vet. Sci. 12, 342-350. T. T., M. D. WHITACRE, and 0.WAYNE ROBISON, 1987: Use of an inativated vaccine for 2. BROWN, prevention of parvovirus-induced reproductive failure in gilts. J. A. V. M. A. 190 (2), 179-181. J. M., M. DEL Pozo, E. RODRIGUEZ y A. DE LAS HERAS,1986: Prevalencia de anticuerpos 3. CASTRO, frente a parvovirus porcino en granjas de producci6n en ciclo cerrado. Med. Vet. 3 (7), 401 -405. K.R., M.A. EMMERSON, P.R. LUFF,D.E. WELLS,J. C. MUSKETT, A. E. WRATHALL, 4. EDWARDS, 1986: Efficacy of porcine parvovirus C. RICHARDSON, B. N . J. PARKER,and D. H. THORTON, vaccines. Vet. Rec. 119, 203-205. 5. FUJISAKI, Y., T. ICHIHARA,N. SASAKI, F. SHIMIZU, Y. MURAKAMI, T. SUEIMORI,and J. SASAHARA, 1978 b: Field trials on inactivated porcine parvovirus vaccine for prevention of viral stillbirth among swine. Bull. Natl. Inst. Anim. Healt 77, 12-14. 6. J o o , H . S., and R. H. JOHNSON,1977: Serological response in pigs vaccinated with inactivated porcine parvovirus. Aust. Vet. J. 53, 550-552. 7. MENGELING, W. L., 1986: Porcine parvovirus infection. In: A. D. LEMAN,B. STRAW,R. D. GLOCK,W. L. MENGELING, R. H. C. PENNY,and E. SCHOLL,Edit. Diseases of swine, p. 411-424, Iowa State University Press. AMES. IOWA U.S.A. 8. MENGELING, W. L., and R. C. CUTLIP,1976: Reproductive disease experimentally induced by exposing pregnant gils to porcine parvovirus. Am J. Vet. Res. 37, 1393-1400. 1979: Efficacy of an 9. MENGELING, W. L., T.T. BROWN,P. S. PAUL, and E. E. GUTEKUNST, inactivated virus vaccine for prevention of porcine parvovirus induced reproductive failure. Am. J. Vet. Res. 40, 204-207. 10. MENGELING, W. L., D. E. GUTEKUNST, E. C. PIRTLE,and P. S. PAUL,1981: Immunogenicity of bivalent vaccine for reproductive failure of swine induced by pseudorabies virus and porcine parvovirus. Am J. Vet. Res. 42, 600-603. 11. OuLDRtDGE, E. J., M.J. FRANCIS, and L. BLACK,1982: Antibody response of pigs to foot and mouth disease oil emulsion vaccine: The antibody classes involved. Res Vet. Sci. 32, 327-331. 12. PAUL,P.S., and W.L. MENGELING, 1986: Vaccination of swine with an inactivated porcine parvovirus vaccine in the presence of passive immunity. J. A. V. M. A. 188 (4), 410-413. and T. T. BROWN,1980: Effect of vaccinal and passive immunity 13. PAUL,P. S., W. L. MENGELING, on experimental infection of pigs with porcine parvovirus. Am. J. Vet. Res. 41, 1368-1371. K. J., and J. ASKAA,1981: Vaccination against porcine parvovirus infection. Acta Vet. 14. SORENSEN, Scand. 22, 171-179. H., and F. FUJISAKI,1976: Immunizing effects of inactivated porcine parvovirus vaccine 15. SUZUKI, on piglets. Bull. Natl. Inst. Anim. Health. 72, 17-23. and R. SALINE,1986: Evaluation of commercial 16. TRACKER, B. J., L. BATISTA,R. L. GONZALEZ, porcine parvovirus vaccines: post-vaccination serological responses, hemagglutinating activity and long term serostatus of vaccinated gilts in endemically infected herd. Proceeding of the Pig Veterinary Society, p. 87. 17. VANNIER,P., A.BRuN, G.CHAPPUIS,and G.REYNAUD, 1986: Study of the efficacy of an inactivated virus vaccine against porcine parvovirus. Ann. Rech. Vet. 17, (4), 425-432.
344
CASTRO, DEL P o z o and SIMARRO
18. WALLACE, B. L., J. K. MCMILLEN, and J. D. TODD,1983: Canine parvovirus serum neutralizing
antibody assay: Assessment of factors responsible for disparity of results between tests. Cornell. Vet. 73, 52-57. A. E., 1988: Field trials of an inactivated oil-emulsion porcine parvovirus vaccine in 19. WRATHALL, British pig herds. Vet. Rec. 122, 411-418. A. E., S. F. CARTWRIGHT, D. E. WELLS,and P. C. JONES,1987: Maternally-derived 20. WRATHALL, antibodies to porcine parvovirus and their effect on active antibody production after vaccination with an inactivated oil-emulsion vaccine. Vet. Rec. 120, 475-478. 21. WRATHALL, A. E., D. E. WELLS,S. F. CARTWRIGHT, and G. N. FRERICHS, 1984: An inactivated, oil-emulsion vaccine for the prevention of porcine parvovirus induced reproductive failure. Res. Vet. Sci. 36, 136-143.
