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The development of a potency test for Leptospira hardjo vaccines: a comparison of protection in calves and serology in guinea-pigs
Journal of Biological Standardization
(1986) 14, 337-344
The development of a potency test for Leptospira hardjo vaccines: a comparison of protection in calves and serology in guinea-pigs*
R. D. Goddard,? 1. G. Hopkins’f and Denise H. Thornton?
The protective properties of two commercial Leptospira hard/o vaccines in calves were compared with the serological responses induced in guinea-pigs with a view to establishing a potency cesr based on serology. Groups of calves were given graded doses of the two vaccines and after eighr weeks were challenged with virulent L. hardjo. Infection was monitored by microscopy and culture of urine and, eight weeks after challenge, by culture of the kidneys post mortem. Groups of guinea-pigs also were tested by the microscopic agglurlnation test (MAT). A clear dose response was observed and the response was related to the degree of protection acheived in the calves. The MAT titre in guinea-pigs indicative of an effective vaccine was calculated for the minimum dose of each vaccine that gave full protection In calves. A close correlation was observed. Two further batches of each vaccine were tested in guinea-pigs on two occasions with reproducible results. A potency test based on the MAT response of guinea-pigs LS proposed.
INTRODUCTION Infection of cattle with Leptospira intewogans serovar hardjo (L. hardjo) has increased in importance during recent years and in consequence vaccines to protect against this infection have become commercially available. These have proved effective, ‘Z but there is as yet no satisfactory method by which batches of vaccine can be readily and accurately tested for potency. This is due to the relatively low pathogenicity of * Received for publication 23 March 1986. i- Central Veterinary Laboratory, New Haw, Weybridge, 0092-l
157/86/040337+07
$O?.OOlO
Surrey, KT! 5 3NB, UK
@ 1986 The Internarronal
Assoctation
of Biological
Srandardlzarlon
337
R. D. GODDARD
E7’AL
L. hardjo3,* Both farm and laboratory animals can be infected and yet show no obvious clinical signs other than effects on lactation or pregnancy.“35 This makes traditional protection tests extremely difficult as infection can be gauged reliably only by recovery of the organism. This is usually achieved by culture from the kidneys post rr~tem. The protection test in cattle is not economic and the animal of choice for experimental work with L. bardjo is the hamster. The test in this species requires considerable time, Iv6 the interval from vaccination to challenge ranging from 14-2 1 days and the interval from challenge to sacrifice and culture ranging from 14-28 days. The culture of leptospires on primary isolation often takes several weeks and cultures are not usually pronounced negative and discarded until 12 weeks have elapsed. A test may therefore take between 7 and 19 weeks. In addition, a challenge culture has to be maintained in a virulent state, bearing in mind that L. hardjo is a human pathogen, and the culture media used for isolation have to be tested for their ability to support the growth of the organism from minimal inocula. Strains capable of causing major clinical signs or death in hamsters have been isolated’.* but, even with these, culture IS still desirable for the confirmation of infection. The work reported here was carried out to ascertain whether protection in vaccinated cattle could be related to the serological responses induced by the same vaccines in guinea-pigs. If this were the case then the potency test could be performed solely by serology in a laboratory animal. MATERIALS
AND
METHODS
Vaccines The two vaccines commercially available in the UK were used. Both were inactivated, adjuvanted vaccines and both had a cattle field dose of 2 ml, to be administered subcutaneously. Under field conditions the first dose would be followed by a booster dose after not less than 4 weeks and annual boosters thereafter. In this experiment only the initial dose was given. Dilutions of the vaccine were prepared in sterile saline so that three graded doses could be administered to the animals. Challenge culture The L8 strain of L. hardjo,’ passaged once through a calf and thereafter held in liquid nitrogen, was used. It was grown at 30°C in Difco EMJH medium for three weeks by which time a count of 3 X IO8 organisms/ml was achieved. Each calf was given 2 ml intravenously. Culture The medium used for the isolation of leptospires was Difco EMJH medium with the addition ofO* 1% Noble agar, 1% rabbit serum and 400 pg/ml of 5-fluorouracil. Ir had been previously established that the challenge strain both from cultures and from urine would grow readily in this medium. To examine blood or urine two or three drops of a sample were inoculated into 8 ml volumes of the medium immediately after the sample was taken. On reaching the laboratory 0.5 ml from the first bottle was inoculated to a second, and 0.5 ml from this to a third. To culture the organism from the kidneys small pieces were cut from different areas of the kidney, mashed in a stomacher with an equal quantity of EMJH medium, and two to three drops of the resultant mash were 338
POTENCY
OF LEPTOSPIRA
HARDJO VACCINES
cultured as for blood or urine. All bottles were incubated at 30°C and examined weekly, both macroscopically and by dark ground microscopy. Most positive cultures were detected in four to six weeks. Remaining bottles were retained for four months before being discarded as negative. Microscopic agglutination
test (MAT)
Serum samples were collected weekly, heated at 56°C for 30min and stored at -20°C. The MAT was carried out in Nunc 96 well Microwell plates. One hundred microlitres of serum was placed in the first well of each row and 50 ~1 of saline in the remainder. Using an Eppendorf multichannel micropipette, 50 ~1 was then transferred from each well to the next with thorough mixing and,, finally, 50 ~1 was discarded from the last wells, leaving in each well 50 ~1 of a twofold dilution series starting from undiluted serum. Then SO~1 of antigen was added to each well and and the plates were incubated at 30°C for two hours. The antigen used was a five day, live culture of L. hardjo in Difco EMJH medium grown at 30°C and diluted to contain 1 X 10” organisms per millilitre after counting in a Thoma counting chamber. After mcubation, lop.1 drops were transferred to glass microscope slides using a four channel micropipette and agglutination was looked for using dark ground microscopy ac a magnification of X 100. The titre was taken as the highest dilution producing at least 50% agglutination and expressed as the log* of its reciprocal. Calves
Thirty-live Channel Island bull calves were used. These came from farms from which previously purchased calves had been shown to be free from antibodies to L. hardjo. In addition the calves were bled on arrival and only those with a negative MAT to L. hnvdjo at 2’ were retained. Calves were brought in at two to seven days old and kept in individual pens until all health problems, mainly scouring, were resolved. They were then housed in groups of five in loose boxes. At the time of vaccination the average age was 2 1 days with a range of 12-28 days. The calves were divided into seven groups of five such that the average age in each group was the same. On the day prior to vaccination all of the calves were bled and the sera tested by the MAT to ensure that all were still negative to L. havdjo at the start of the experiment. Calves in group 1 received no vaccine and acted as controls. Calves in the remainmg groups received graded doses of 1, ‘/‘ICYor %oc, of the field dose of either vaccine A or B, each calf receiving 2 ml subcutaneously. The calves were bled every seventh day until the end of the experiment. Eight weeks after vaccination, when it was considered that the MAT titres had reached a maximum, all of the calves were challenged mtravenously with 2 ml of the L. hardjo L8 culture. Blood samples were taken for culture one, four and seven days after challenge. Urine was cultured weekly from the twelfth day after challenge. Urine was collected into 2 02 bottles containing 10 ml of form01 saline. The samples were centrifuged at 4000 K for 30 min, the sediment was resuspended in 1 ml of saline and examined by dark ground microscopy at a magnification of X 250. The experiment was terminated after a further eight weeks and the kidneys of each calf were removed pmt vzortem and cultured 339
R. D. GODDARD
ET AL.
Gzrintu-pigs Seventy female albino guinea-pigs, weighing between 250 and 3OOg, were used. These were of the Dunkin Hartley strain, classified as Category 3 according to the Medical Research Council accreditation scheme. They were divided into seven groups of ten. Group 1 received no vaccine and served as a negative control. The animals in the other groups were given graded doses of l/5, %o or Y500 of the field dose, each receiving 0*4ml by the intramuscular route. Blood samples were taken by cardiac puncture one, two, three and four weeks after vaccination and the sera were separated for testing by the MAT. After completion of the initial experiment further work was undertaken with guinea-pigs to determine whether the response to L. hardjo vaccines was a reproducible effect. Two more batches of vaccine A and two of vaccine B were tested in guinea-pigs using the same format as described above but with bleeding only once at seven days after inoculation. After a one month interval the same four batches were tested again in the same way, blood samples again being taken at seven days for testing by the MAT. RESULTS Calves Fifteen calves became infected. These were all five of the control group and all five of the group receiving %oo of the field dose of vaccine B, three of the group receiving %o of the field dose of vaccine B and two of the group receiving %oo of the held dose ofvaccine A. All but one of these had a positive blood culture after one day and four gave positive cultures after four days. At seven days all blood cultures were negative. Leptospires were found in the urine of 13 calves by dark ground microscopy and were cultured from the urine of these same 1.3calves, the earliest positive being attained at 15 days after challenge. Kidney cultures were positive from 15 calves. At no time did any calf show visible signs of illness. Although both vaccines proved to be protective, vaccine A could be diluted ten times more than vaccine B before losing its protective efficacy. It could also be diluted tenfold and still induce the same MA titre as vaccine B (Fig. 1)
0
12345678 Time (weeks)
Fig. 1. vaccination. vaccine B. 340
Calves: mean microscopic agglutination test (MAT) titres for four dose groups after 0, Full dose vaccine A; A, ‘/IO dose vaccine A; n , YIMI dose vaccine A; 0. full dose
POTENCY
OF LEPTOSPIRA
HARDJO
VACCINES
Vaccine B produced a serological response only in its undiluted form, the initial appearance of a titre averaging 32 days (range 28-42) and the highest titre (with a mean of 23.“, range 21-25) being reached after an average of4 1 days (range 35-49). With the exception of one calf at the %oo of the field dose level, vaccine A produced serological responses in all animals at all three dilutions, the mean titres being 2”” (range 25-28) for the full dose, 2” (range 2’-25) for the 9’10dose and 2’.’ (range O-2’) for the L/IOUdose. The average time from vaccination to the first detectable response was 15 days (range 14-21), 24 days (range 14-35) and 22 days (range 2 l-42), respectively. The average time to reach maximum response was 45 days (range 2X-56), 39 days (range 21-56) and 27 days (range 21-42), respectively. The control group remained negative until challenged. The three vaccinated calves with the highest titres of 2”, 2’ and 28 showed no alteration in titre when challenged. All others had an increase in titre although the titre after challenge bore no relation to that before challenge. The calves that later proved to be uninfected increased to a mean of 28.25 (range 24-21 ‘) while those that proved to be infected increased to a mean of 2 I*.” (range 2”-2 l’). Of the 15 infected calves only one showed a MAT response prior to challenge. This calf was in the vaccine A %oo of the field dose group and it maintained a titre of 2’ for three weeks. Of the 20 uninfected calves, only two failed to produce a MAT response prior to challenge. These were both in the vaccine B I/IO of the field dose group. Two further calves, one in the vaccine B full dose group and one in the vaccine A ‘+‘I()of the field dose group had titres of only 2’ prior to challenge but remained uninfected. Guinea-pigs Vaccine A could be diluted ten-fold and still induce a similar response to vaccine B, (Fig. 2) and the response to both vaccines was in proportion to the dose (Fig. J). All animals reached their maximum MAT titre by the time of the first bleeding at seven days, with the exception of five animals in the high dose groups (three in the 1%dose
(b)
7: ;
6-
5
5-
d P
4
g
3-
_
2-
v
1234012345 Time [weeks)
Fig. 2 Guinea-pigs: mean microscopic agglutination test (MAT) titres for three dose groups (a) after vaccination with vaccine A and (b) vaccine B. A, % dose; n %o dose; l > %IIO dose.
341
R. D. GODDARD
E’I AL
0
-I
-2
-3
log,, dose
Fig. 3. Dose-response lines at maximum tlrre III rhe microscopic agglutmation guinea-pigs, vaccine A; m, guinea-pigs, vaccine R; 0, calves, vaccine A
rest (MAT).
A,
vaccine A group and two in the So dose vaccine A group) which had peak levels at the second bleeding. The %oo dose of vaccine B induced a response in only four of the ten animals at levels of 2’ and 2’. The control group remained negative throughout. When the further groups of guinea-pigs were tested a similar pattern appeared (Table 1). Although there was generally a falling away in response at the ‘/SOOlevel only one of the sets of figures showed a significant deviation from linearity (vaccine B 1, second test, P < 0.5). A clear dose-response relationship was apparent throughout and reproducibility between tests on the same vaccine batch was good; of the 12 pairs of TABLE 1. Mean microscopic agglutination test (MAT) titres (10g~) oi sera from groups of ten guinea-pigs with regression analysis on vaccine dose (log ,(J omitting the %oo dilution Mean MAT titresi (log?) for dose of vaccine: ----___ Vac55 !‘5ll ‘/so0 tine Tesr Al
Regression analysis omitting
YWOdilution$
Slope F SE
Mean
95% CL#
95?FTL[l
I 2
7.9 7.2”
6.5 5*Lx”*
1.7 3.4
1.40 k 0.356 2.00 t 0.462
8.88 8.60
7.91 2 9.85 7.14 + 9.86
7.7 -- lO*O 7.2 ~~ 104
1
8.2 X.0
7.1 6.4X
4.6 3.8”
1.27 * 0.232 1.64 31 0.210
937
2
9.18
8.80 k 9.95 8.66 + 9.71
8.6 8.5
Bl
1 2
7-o 6.9
4.7 5.1
1.1 14
2.20 5 0.327 2-00 k 0.415
8-54 8.50
7.65 + 9.4i 7.28 zk 9.71
7.5 7.1 -
B2
1 2
0.5 6.9
4.2
1.4 1.2
230 f. 0.260 3.00 k 0.362
8.1 I 9.00
7.40 k 8.82
3.9
8.01
7.3 ~ 8.9 7.9 ~ 10.1
A2
t Significantly different from mean for Test I’ * = P< $ Estimate oi log,, titres given by near vaccine. Q Cl = Confidence limits. 11TL = Tolerance limits.
342
0.05;
k 9.98
*** = P < 0~001.
lo. 1 9.8 9.6 9.9
POTENCY
OF LEPTOSPZRA HARDjO
VACCINES
mean titres calculated only four showed significant differences. The slope for vaccine A was less steep than for vaccine B, but not significantly so. Vaccine A gave similar results to those obtained before while the two batches of vaccine B both induced a response greater than that observed in the first test although in both tests it still appeared to be several times less potent than vaccine A. Since the lowest dose of vaccine ('/500) appeared in many cases to be approaching a limiting dilution, the results were subjected to statistical analysis omitting these figures (Table 1). DISCUSSfON It is generally accepted when testing sera for evidence of leptospiral infection that only Although valid titres of Yioo (26’“44) or more should be considered significant. when looking for evidence of infection this premise is not so when following the effects of a vaccine and it possibly accounts for the opinion that I,. havdjo vaccines often fail to induce a serological response. As seen from this work, titres as low as 7’ can be significant when an animal has been previously negative and the ritre can be attributed to a known input of antigen, in this case the vaccines. It would perhaps be unwise to accept these very low titres in isolation even ifa vaccine had been given, but when they are part of a rising titre or a response to a vaccine which gives higher ritres with higher doses there can be little doubt that they are sqnificant. An important point to make when testing at such low levels is that high titre sera often give negative MAT results at low dilutions (below about 2”) and this should be borne in mind when testing, the full range ofdilutions being examined before a serum is pronounced negative. It was often found when prrformlng the MAT on very low titre serd that there was complete inactivation of the leptospires but no agglutin~tlon. This made lc ncc-essary to use a higher magnification to assess the degree of-movement and provided very erraticresults. This effect was eliminated and the normal apglutinatlon partcrn resrored by heating the sera at 56°C for 10 mins. A comparison ofheated ,~tl unhcarc~! \t’ra showed that those sera with low titres gave signlticantly higher values (P < 0.00 I), or pave agglutination where there had previously been doubt, while the hlpher titres etcher remained the same or varied up or down by only one twofold step. Since the important part of the serology was the response to the vaccines involving low titre scril from both the calves and the guinea-pigs it was decided to perform all MATS o:t heated sera. In order to correlate protection in CJ~VCS with a srrolo,q~cal response In ,~u~nea-pigs, a c-lear relationship must be shown IO exlsr between these two tft&crz ot the vaccine. Protection of‘ 100% was achieved by vaccine A with ~1“IO dust ;Ind val.clne B with a full dose. By interpolation (vaccxne A) and cxtrapolat!on (vaccine U) from i’rp. 3, these doses in guinea-pigs would have given titres of 2”’ and 1’ ‘, respectivrly. It would therefore appear that a vaccine that Induced a titre of at leasr 2‘ ’ when lnoiulated Into pumea-pigs could expected to protect d calf. Since borh ~,LCCIIICSarl- t(l !Y given as a primary vaccination with a booster vaccination after at least one rnonrtl t;jllowed by annual boosters the protection afforded by the single dose used in this cxper:ment IS the minimum likely to be achieved in the field, allowing a margin of error in both the choice of tltre in the guinea-pig and the possibility of a grown animal giving a lesser response than a calf. The examination of furrher batches of both vaccines on two separate occasions Indicated that the serological response induced in guinea-pigs was a reproducible effect
R. D. GODDARD
ET AL
and was reasonably constant for a given vaccine formulation. By the criteria set out above, all four further vaccine batches would have been calf protective. If the two dose r/s and %o of a field dose were to be used in groups of six levels of vaccine, guinea-pigs it is estimated that there is a chance of only one in 20 that a vaccine of adequate potency would give a mean titre of less than 2’ when extrapolation to full dose level was made. Since it is hoped that no vaccine as borderline in potency as this would be produced, these figures have been used to reduce the number of animals required for a test. The possibility that other strains of guinea-pigs might react differently to L. ham’) vaccines would make it desirable to have some form of standard preparation as a reference. However, the vaccines currently in production have a limited shelf-life with long term stability unknown. A reference preparation made to conform with these vaccines would thus be of limited use and is a problem requiring further attention. The following is thus proposed as a potency test for L. hardjo vaccines for cattle. Two groups of six guinea-pigs (250-300g in weight) are to be given, intramuscularly, respectively, of a dose of the vaccine under test. One week after 1% and %o, vaccination the animals are bled and the serum is heated at 56°C for 10 min and subjected to the MAT using live L. havdjo antigen as described in this paper. To pass the potency test the guinea-pigs must show a dose related response to the dilutions and extrapolation of this must indicate that a full dose would have elicited a MAT response of at least 2T. It is suggested that the system is verified under particular test conditions using a full test of three dose groups of ten animals each before the abbreviated test described here is adopted as a routine. Acknowledgements We wish to thank Miss C. N. Hebert for invaluable work on the statistical analysis
REFERENCES 1.
Flint SH, Liardet DM. A trivalent leptospiral vaccine with emphasis on a Leptospzra intcrvogans serovar hardjo component to prevent leptospiruria. NZ Vet J 1980; 28: 263-266. 2. Mackintosh CG, Marshal RB, Boughton ES. The use ofa hardjolpomona vaccine to prevent leptospiruria in cattle exposed to natural challenge with L. har~/~. NZ Vet J 1980; 28: 174-177. 3. Hathaway SC, Marshall RB. Experimental infection of sheep with Leptnspira intevwgans. serovars hardjn and balcanica. NZ Vet J 1979; 27: 197. 4. Hoare RJ, Claxton PD. Observations on Leptospiva ha$jo infection in New South Wales. Aust Vet J 1972; 48: 228-232. 5. Higgins RJ, Harbourne JF, Little TWA, Stevens AE. Mastitis and abortion in dairy cattle associated with Leptospira of the serotype hardjo. Vet Ret 1980; 107: 307-3 10. 6. Eardella MA, Kellog JL, Baldwin CD, Nervig RM. Potency testing of the hard’o component of Leptospira bactirins. Ann Proc AM Ass Vet Lab Diag 1977; 20: 107-122. 7. Kingscore B. Isolation of a hamster-lethal strain of LeptoJpiva ba~dp. Can Vet J 1980; 2 1: 266-267. 8. Woods SB, Maley AD, Frerichs GN, Bailey J. Isolation of a hamster-lethal strain of LeptoJpira interqans serotype bardjo. Vet Ret 1981; 112: 4.37438.
344
(1986) 14, 337-344
The development of a potency test for Leptospira hardjo vaccines: a comparison of protection in calves and serology in guinea-pigs*
R. D. Goddard,? 1. G. Hopkins’f and Denise H. Thornton?
The protective properties of two commercial Leptospira hard/o vaccines in calves were compared with the serological responses induced in guinea-pigs with a view to establishing a potency cesr based on serology. Groups of calves were given graded doses of the two vaccines and after eighr weeks were challenged with virulent L. hardjo. Infection was monitored by microscopy and culture of urine and, eight weeks after challenge, by culture of the kidneys post mortem. Groups of guinea-pigs also were tested by the microscopic agglurlnation test (MAT). A clear dose response was observed and the response was related to the degree of protection acheived in the calves. The MAT titre in guinea-pigs indicative of an effective vaccine was calculated for the minimum dose of each vaccine that gave full protection In calves. A close correlation was observed. Two further batches of each vaccine were tested in guinea-pigs on two occasions with reproducible results. A potency test based on the MAT response of guinea-pigs LS proposed.
INTRODUCTION Infection of cattle with Leptospira intewogans serovar hardjo (L. hardjo) has increased in importance during recent years and in consequence vaccines to protect against this infection have become commercially available. These have proved effective, ‘Z but there is as yet no satisfactory method by which batches of vaccine can be readily and accurately tested for potency. This is due to the relatively low pathogenicity of * Received for publication 23 March 1986. i- Central Veterinary Laboratory, New Haw, Weybridge, 0092-l
157/86/040337+07
$O?.OOlO
Surrey, KT! 5 3NB, UK
@ 1986 The Internarronal
Assoctation
of Biological
Srandardlzarlon
337
R. D. GODDARD
E7’AL
L. hardjo3,* Both farm and laboratory animals can be infected and yet show no obvious clinical signs other than effects on lactation or pregnancy.“35 This makes traditional protection tests extremely difficult as infection can be gauged reliably only by recovery of the organism. This is usually achieved by culture from the kidneys post rr~tem. The protection test in cattle is not economic and the animal of choice for experimental work with L. bardjo is the hamster. The test in this species requires considerable time, Iv6 the interval from vaccination to challenge ranging from 14-2 1 days and the interval from challenge to sacrifice and culture ranging from 14-28 days. The culture of leptospires on primary isolation often takes several weeks and cultures are not usually pronounced negative and discarded until 12 weeks have elapsed. A test may therefore take between 7 and 19 weeks. In addition, a challenge culture has to be maintained in a virulent state, bearing in mind that L. hardjo is a human pathogen, and the culture media used for isolation have to be tested for their ability to support the growth of the organism from minimal inocula. Strains capable of causing major clinical signs or death in hamsters have been isolated’.* but, even with these, culture IS still desirable for the confirmation of infection. The work reported here was carried out to ascertain whether protection in vaccinated cattle could be related to the serological responses induced by the same vaccines in guinea-pigs. If this were the case then the potency test could be performed solely by serology in a laboratory animal. MATERIALS
AND
METHODS
Vaccines The two vaccines commercially available in the UK were used. Both were inactivated, adjuvanted vaccines and both had a cattle field dose of 2 ml, to be administered subcutaneously. Under field conditions the first dose would be followed by a booster dose after not less than 4 weeks and annual boosters thereafter. In this experiment only the initial dose was given. Dilutions of the vaccine were prepared in sterile saline so that three graded doses could be administered to the animals. Challenge culture The L8 strain of L. hardjo,’ passaged once through a calf and thereafter held in liquid nitrogen, was used. It was grown at 30°C in Difco EMJH medium for three weeks by which time a count of 3 X IO8 organisms/ml was achieved. Each calf was given 2 ml intravenously. Culture The medium used for the isolation of leptospires was Difco EMJH medium with the addition ofO* 1% Noble agar, 1% rabbit serum and 400 pg/ml of 5-fluorouracil. Ir had been previously established that the challenge strain both from cultures and from urine would grow readily in this medium. To examine blood or urine two or three drops of a sample were inoculated into 8 ml volumes of the medium immediately after the sample was taken. On reaching the laboratory 0.5 ml from the first bottle was inoculated to a second, and 0.5 ml from this to a third. To culture the organism from the kidneys small pieces were cut from different areas of the kidney, mashed in a stomacher with an equal quantity of EMJH medium, and two to three drops of the resultant mash were 338
POTENCY
OF LEPTOSPIRA
HARDJO VACCINES
cultured as for blood or urine. All bottles were incubated at 30°C and examined weekly, both macroscopically and by dark ground microscopy. Most positive cultures were detected in four to six weeks. Remaining bottles were retained for four months before being discarded as negative. Microscopic agglutination
test (MAT)
Serum samples were collected weekly, heated at 56°C for 30min and stored at -20°C. The MAT was carried out in Nunc 96 well Microwell plates. One hundred microlitres of serum was placed in the first well of each row and 50 ~1 of saline in the remainder. Using an Eppendorf multichannel micropipette, 50 ~1 was then transferred from each well to the next with thorough mixing and,, finally, 50 ~1 was discarded from the last wells, leaving in each well 50 ~1 of a twofold dilution series starting from undiluted serum. Then SO~1 of antigen was added to each well and and the plates were incubated at 30°C for two hours. The antigen used was a five day, live culture of L. hardjo in Difco EMJH medium grown at 30°C and diluted to contain 1 X 10” organisms per millilitre after counting in a Thoma counting chamber. After mcubation, lop.1 drops were transferred to glass microscope slides using a four channel micropipette and agglutination was looked for using dark ground microscopy ac a magnification of X 100. The titre was taken as the highest dilution producing at least 50% agglutination and expressed as the log* of its reciprocal. Calves
Thirty-live Channel Island bull calves were used. These came from farms from which previously purchased calves had been shown to be free from antibodies to L. hardjo. In addition the calves were bled on arrival and only those with a negative MAT to L. hnvdjo at 2’ were retained. Calves were brought in at two to seven days old and kept in individual pens until all health problems, mainly scouring, were resolved. They were then housed in groups of five in loose boxes. At the time of vaccination the average age was 2 1 days with a range of 12-28 days. The calves were divided into seven groups of five such that the average age in each group was the same. On the day prior to vaccination all of the calves were bled and the sera tested by the MAT to ensure that all were still negative to L. havdjo at the start of the experiment. Calves in group 1 received no vaccine and acted as controls. Calves in the remainmg groups received graded doses of 1, ‘/‘ICYor %oc, of the field dose of either vaccine A or B, each calf receiving 2 ml subcutaneously. The calves were bled every seventh day until the end of the experiment. Eight weeks after vaccination, when it was considered that the MAT titres had reached a maximum, all of the calves were challenged mtravenously with 2 ml of the L. hardjo L8 culture. Blood samples were taken for culture one, four and seven days after challenge. Urine was cultured weekly from the twelfth day after challenge. Urine was collected into 2 02 bottles containing 10 ml of form01 saline. The samples were centrifuged at 4000 K for 30 min, the sediment was resuspended in 1 ml of saline and examined by dark ground microscopy at a magnification of X 250. The experiment was terminated after a further eight weeks and the kidneys of each calf were removed pmt vzortem and cultured 339
R. D. GODDARD
ET AL.
Gzrintu-pigs Seventy female albino guinea-pigs, weighing between 250 and 3OOg, were used. These were of the Dunkin Hartley strain, classified as Category 3 according to the Medical Research Council accreditation scheme. They were divided into seven groups of ten. Group 1 received no vaccine and served as a negative control. The animals in the other groups were given graded doses of l/5, %o or Y500 of the field dose, each receiving 0*4ml by the intramuscular route. Blood samples were taken by cardiac puncture one, two, three and four weeks after vaccination and the sera were separated for testing by the MAT. After completion of the initial experiment further work was undertaken with guinea-pigs to determine whether the response to L. hardjo vaccines was a reproducible effect. Two more batches of vaccine A and two of vaccine B were tested in guinea-pigs using the same format as described above but with bleeding only once at seven days after inoculation. After a one month interval the same four batches were tested again in the same way, blood samples again being taken at seven days for testing by the MAT. RESULTS Calves Fifteen calves became infected. These were all five of the control group and all five of the group receiving %oo of the field dose of vaccine B, three of the group receiving %o of the field dose of vaccine B and two of the group receiving %oo of the held dose ofvaccine A. All but one of these had a positive blood culture after one day and four gave positive cultures after four days. At seven days all blood cultures were negative. Leptospires were found in the urine of 13 calves by dark ground microscopy and were cultured from the urine of these same 1.3calves, the earliest positive being attained at 15 days after challenge. Kidney cultures were positive from 15 calves. At no time did any calf show visible signs of illness. Although both vaccines proved to be protective, vaccine A could be diluted ten times more than vaccine B before losing its protective efficacy. It could also be diluted tenfold and still induce the same MA titre as vaccine B (Fig. 1)
0
12345678 Time (weeks)
Fig. 1. vaccination. vaccine B. 340
Calves: mean microscopic agglutination test (MAT) titres for four dose groups after 0, Full dose vaccine A; A, ‘/IO dose vaccine A; n , YIMI dose vaccine A; 0. full dose
POTENCY
OF LEPTOSPIRA
HARDJO
VACCINES
Vaccine B produced a serological response only in its undiluted form, the initial appearance of a titre averaging 32 days (range 28-42) and the highest titre (with a mean of 23.“, range 21-25) being reached after an average of4 1 days (range 35-49). With the exception of one calf at the %oo of the field dose level, vaccine A produced serological responses in all animals at all three dilutions, the mean titres being 2”” (range 25-28) for the full dose, 2” (range 2’-25) for the 9’10dose and 2’.’ (range O-2’) for the L/IOUdose. The average time from vaccination to the first detectable response was 15 days (range 14-21), 24 days (range 14-35) and 22 days (range 2 l-42), respectively. The average time to reach maximum response was 45 days (range 2X-56), 39 days (range 21-56) and 27 days (range 21-42), respectively. The control group remained negative until challenged. The three vaccinated calves with the highest titres of 2”, 2’ and 28 showed no alteration in titre when challenged. All others had an increase in titre although the titre after challenge bore no relation to that before challenge. The calves that later proved to be uninfected increased to a mean of 28.25 (range 24-21 ‘) while those that proved to be infected increased to a mean of 2 I*.” (range 2”-2 l’). Of the 15 infected calves only one showed a MAT response prior to challenge. This calf was in the vaccine A %oo of the field dose group and it maintained a titre of 2’ for three weeks. Of the 20 uninfected calves, only two failed to produce a MAT response prior to challenge. These were both in the vaccine B I/IO of the field dose group. Two further calves, one in the vaccine B full dose group and one in the vaccine A ‘+‘I()of the field dose group had titres of only 2’ prior to challenge but remained uninfected. Guinea-pigs Vaccine A could be diluted ten-fold and still induce a similar response to vaccine B, (Fig. 2) and the response to both vaccines was in proportion to the dose (Fig. J). All animals reached their maximum MAT titre by the time of the first bleeding at seven days, with the exception of five animals in the high dose groups (three in the 1%dose
(b)
7: ;
6-
5
5-
d P
4
g
3-
_
2-
v
1234012345 Time [weeks)
Fig. 2 Guinea-pigs: mean microscopic agglutination test (MAT) titres for three dose groups (a) after vaccination with vaccine A and (b) vaccine B. A, % dose; n %o dose; l > %IIO dose.
341
R. D. GODDARD
E’I AL
0
-I
-2
-3
log,, dose
Fig. 3. Dose-response lines at maximum tlrre III rhe microscopic agglutmation guinea-pigs, vaccine A; m, guinea-pigs, vaccine R; 0, calves, vaccine A
rest (MAT).
A,
vaccine A group and two in the So dose vaccine A group) which had peak levels at the second bleeding. The %oo dose of vaccine B induced a response in only four of the ten animals at levels of 2’ and 2’. The control group remained negative throughout. When the further groups of guinea-pigs were tested a similar pattern appeared (Table 1). Although there was generally a falling away in response at the ‘/SOOlevel only one of the sets of figures showed a significant deviation from linearity (vaccine B 1, second test, P < 0.5). A clear dose-response relationship was apparent throughout and reproducibility between tests on the same vaccine batch was good; of the 12 pairs of TABLE 1. Mean microscopic agglutination test (MAT) titres (10g~) oi sera from groups of ten guinea-pigs with regression analysis on vaccine dose (log ,(J omitting the %oo dilution Mean MAT titresi (log?) for dose of vaccine: ----___ Vac55 !‘5ll ‘/so0 tine Tesr Al
Regression analysis omitting
YWOdilution$
Slope F SE
Mean
95% CL#
95?FTL[l
I 2
7.9 7.2”
6.5 5*Lx”*
1.7 3.4
1.40 k 0.356 2.00 t 0.462
8.88 8.60
7.91 2 9.85 7.14 + 9.86
7.7 -- lO*O 7.2 ~~ 104
1
8.2 X.0
7.1 6.4X
4.6 3.8”
1.27 * 0.232 1.64 31 0.210
937
2
9.18
8.80 k 9.95 8.66 + 9.71
8.6 8.5
Bl
1 2
7-o 6.9
4.7 5.1
1.1 14
2.20 5 0.327 2-00 k 0.415
8-54 8.50
7.65 + 9.4i 7.28 zk 9.71
7.5 7.1 -
B2
1 2
0.5 6.9
4.2
1.4 1.2
230 f. 0.260 3.00 k 0.362
8.1 I 9.00
7.40 k 8.82
3.9
8.01
7.3 ~ 8.9 7.9 ~ 10.1
A2
t Significantly different from mean for Test I’ * = P< $ Estimate oi log,, titres given by near vaccine. Q Cl = Confidence limits. 11TL = Tolerance limits.
342
0.05;
k 9.98
*** = P < 0~001.
lo. 1 9.8 9.6 9.9
POTENCY
OF LEPTOSPZRA HARDjO
VACCINES
mean titres calculated only four showed significant differences. The slope for vaccine A was less steep than for vaccine B, but not significantly so. Vaccine A gave similar results to those obtained before while the two batches of vaccine B both induced a response greater than that observed in the first test although in both tests it still appeared to be several times less potent than vaccine A. Since the lowest dose of vaccine ('/500) appeared in many cases to be approaching a limiting dilution, the results were subjected to statistical analysis omitting these figures (Table 1). DISCUSSfON It is generally accepted when testing sera for evidence of leptospiral infection that only Although valid titres of Yioo (26’“44) or more should be considered significant. when looking for evidence of infection this premise is not so when following the effects of a vaccine and it possibly accounts for the opinion that I,. havdjo vaccines often fail to induce a serological response. As seen from this work, titres as low as 7’ can be significant when an animal has been previously negative and the ritre can be attributed to a known input of antigen, in this case the vaccines. It would perhaps be unwise to accept these very low titres in isolation even ifa vaccine had been given, but when they are part of a rising titre or a response to a vaccine which gives higher ritres with higher doses there can be little doubt that they are sqnificant. An important point to make when testing at such low levels is that high titre sera often give negative MAT results at low dilutions (below about 2”) and this should be borne in mind when testing, the full range ofdilutions being examined before a serum is pronounced negative. It was often found when prrformlng the MAT on very low titre serd that there was complete inactivation of the leptospires but no agglutin~tlon. This made lc ncc-essary to use a higher magnification to assess the degree of-movement and provided very erraticresults. This effect was eliminated and the normal apglutinatlon partcrn resrored by heating the sera at 56°C for 10 mins. A comparison ofheated ,~tl unhcarc~! \t’ra showed that those sera with low titres gave signlticantly higher values (P < 0.00 I), or pave agglutination where there had previously been doubt, while the hlpher titres etcher remained the same or varied up or down by only one twofold step. Since the important part of the serology was the response to the vaccines involving low titre scril from both the calves and the guinea-pigs it was decided to perform all MATS o:t heated sera. In order to correlate protection in CJ~VCS with a srrolo,q~cal response In ,~u~nea-pigs, a c-lear relationship must be shown IO exlsr between these two tft&crz ot the vaccine. Protection of‘ 100% was achieved by vaccine A with ~1“IO dust ;Ind val.clne B with a full dose. By interpolation (vaccxne A) and cxtrapolat!on (vaccine U) from i’rp. 3, these doses in guinea-pigs would have given titres of 2”’ and 1’ ‘, respectivrly. It would therefore appear that a vaccine that Induced a titre of at leasr 2‘ ’ when lnoiulated Into pumea-pigs could expected to protect d calf. Since borh ~,LCCIIICSarl- t(l !Y given as a primary vaccination with a booster vaccination after at least one rnonrtl t;jllowed by annual boosters the protection afforded by the single dose used in this cxper:ment IS the minimum likely to be achieved in the field, allowing a margin of error in both the choice of tltre in the guinea-pig and the possibility of a grown animal giving a lesser response than a calf. The examination of furrher batches of both vaccines on two separate occasions Indicated that the serological response induced in guinea-pigs was a reproducible effect
R. D. GODDARD
ET AL
and was reasonably constant for a given vaccine formulation. By the criteria set out above, all four further vaccine batches would have been calf protective. If the two dose r/s and %o of a field dose were to be used in groups of six levels of vaccine, guinea-pigs it is estimated that there is a chance of only one in 20 that a vaccine of adequate potency would give a mean titre of less than 2’ when extrapolation to full dose level was made. Since it is hoped that no vaccine as borderline in potency as this would be produced, these figures have been used to reduce the number of animals required for a test. The possibility that other strains of guinea-pigs might react differently to L. ham’) vaccines would make it desirable to have some form of standard preparation as a reference. However, the vaccines currently in production have a limited shelf-life with long term stability unknown. A reference preparation made to conform with these vaccines would thus be of limited use and is a problem requiring further attention. The following is thus proposed as a potency test for L. hardjo vaccines for cattle. Two groups of six guinea-pigs (250-300g in weight) are to be given, intramuscularly, respectively, of a dose of the vaccine under test. One week after 1% and %o, vaccination the animals are bled and the serum is heated at 56°C for 10 min and subjected to the MAT using live L. havdjo antigen as described in this paper. To pass the potency test the guinea-pigs must show a dose related response to the dilutions and extrapolation of this must indicate that a full dose would have elicited a MAT response of at least 2T. It is suggested that the system is verified under particular test conditions using a full test of three dose groups of ten animals each before the abbreviated test described here is adopted as a routine. Acknowledgements We wish to thank Miss C. N. Hebert for invaluable work on the statistical analysis
REFERENCES 1.
Flint SH, Liardet DM. A trivalent leptospiral vaccine with emphasis on a Leptospzra intcrvogans serovar hardjo component to prevent leptospiruria. NZ Vet J 1980; 28: 263-266. 2. Mackintosh CG, Marshal RB, Boughton ES. The use ofa hardjolpomona vaccine to prevent leptospiruria in cattle exposed to natural challenge with L. har~/~. NZ Vet J 1980; 28: 174-177. 3. Hathaway SC, Marshall RB. Experimental infection of sheep with Leptnspira intevwgans. serovars hardjn and balcanica. NZ Vet J 1979; 27: 197. 4. Hoare RJ, Claxton PD. Observations on Leptospiva ha$jo infection in New South Wales. Aust Vet J 1972; 48: 228-232. 5. Higgins RJ, Harbourne JF, Little TWA, Stevens AE. Mastitis and abortion in dairy cattle associated with Leptospira of the serotype hardjo. Vet Ret 1980; 107: 307-3 10. 6. Eardella MA, Kellog JL, Baldwin CD, Nervig RM. Potency testing of the hard’o component of Leptospira bactirins. Ann Proc AM Ass Vet Lab Diag 1977; 20: 107-122. 7. Kingscore B. Isolation of a hamster-lethal strain of LeptoJpiva ba~dp. Can Vet J 1980; 2 1: 266-267. 8. Woods SB, Maley AD, Frerichs GN, Bailey J. Isolation of a hamster-lethal strain of LeptoJpira interqans serotype bardjo. Vet Ret 1981; 112: 4.37438.
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