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Vaccination against the nematode Trichostrongylus colubriformis—I. Vaccination of guinea-pigs with worm homogenates and soluble products released during in vitro maintenance
VACCINATION AGAINST THE NEMATODE T~~C~~S~R~~G YLUS C~~~~~IF~R~~S-~. VACCINATION OF GUINEA-PIGS WITH WORM HOMOGENATES AND SOLUBLE PRODUCTS RELEASED DlJRING IN VITRO MAINTENANCE T. L. W. ROTHWELL and R. J. LOVE C.S.I.R.O.,
Division of Animal
Health, McMaster
Laboratory,
(Received 8 August 1973 : amended
Glebe, N.S.W. 2037, Australia
I October 1973)
Abstract-RorwwErLT. L. W. and LOVE R. .I. 1974. Vaccination against the nematode T’ichoutrongy/@s co/~tb$&mis--1. Vaccination of guinea-pigs with worm homogenates and soluble products released during in v&o nlaintenance. I~ic~t~ati~~aIJoi~r~?al~~ Pa~as~to/u~~ 4: 293-299. Vaccination of guineapigs with homogenates of fourth larval stage and adult Trichostrongylus colubviformis and soluble products released by fourth stage larvae during itl vitro maintenance, induced a high level of protection against challenge infection with the parasite. INDEX KEY WORDS: vaccination.
Antigens; guinea-pigs; immunity; nematode; Trkhosrrorrgyhts colrtbrifornris;
INTRODUCTION DESPITE the demonstration of acquired immunity against many parasites and advances in the under-
standing of the immune responses parasites generate in their hosts, protection of man and animals against parasitic disease by vaccination (used here as “administration of antigenic material to stimulate active immunity with the aim of protecting against infection”) has proved an elusive goal (Stall, 1961 : World Health Organisation, 196.5; Terry, 1968; Silverman, 1970). In the case of nematodes, limited success has been achieved using live vaccines (Jarrett, Jennings, McIntyre, Mulligan & Urquhart, 1958; Mulligan, Gordon, Stewart & Wagland, 1961; Sokolic, Jovanovic, Cuperlovic & Movsesijan, 1965; Miller, 1965), but vaccination with non-living nematode material has been generally unsuccessful. The intestinal nematode Tr~~~ostr~t~~~[its cohbri.f&nks is an important pathogen of sheep, which in addition reaches patency in guinea pigs. Silverman, Poynter & Podger (1962) and Connan (1965) have reported that guinea pigs can be vaccinated against T. colubrifkwmis. Silverman et al. (1962) gave two intraperitoneal injections of crude homogenates of infective larvae, fourth stage larvae and adult T. ~(l~ub~~~~rj~l~.~. The methods used in the preparation of these homogenates were not given in detail, but it appears that the fourth larval stage preparation contained non-exsheathed third stage larvae, sheaths and metabolic products. The dosage of the fourth larval stage preparation administered is expressed
as equivalents of the number of larvae which had undergone ecdysis to the fourth larval stage, but the dosages of a third larval stage and adult preparations were not revealed. Connan (1965) published only a brief report of his experiments, referring to “a marked degree of protection” in guinea-pigs “vaccinated with metabolic solutions”. His attempts to vaccinate guinea-pigs with somatic antigen preparations, however, failed. Silverman et al. (1962) and Connan’s (1965) results suggest that this host-parasite system might be well suited for studying the nematode antigens responsible for host protective immunity. Insufficient detail, however, is provided in these publications to confidently use the system for this purpose. Therefore, we have re-examined vaccination of guinea-pigs against T. cohbr$wnis with worm homogenates and metabolites prior to attempting to identify the antigens responsible for host imnlunity. In the experiments described, the ability of a single injection of worm homogenate to stimulate protective immunity was determined. Most attention was given to the fourth larval stage of the parasite because large numbers can easily be collected after a wait of only eight days. In addition, the subventral glands of the excretory system, which are a possible source of protective antigens, are relatively well developed in fourth-stage larvae (Rothwell, Ogilvie & Love, 1973), making them especially suitable for attempts to isolate these antigens. Infective larvae are also readily available, but because they did not
stimulate solid protective immunity, they were not examined in deta;l. However, for completeness, the results of these studies and limited observations made on adult worms are also presented. Worm metaboljtes, because of their freedom from somatic material and possible value in planned antigen purification studies, were also examined for their ability ta protect guinea-pigs against infection with T. colubriformis. This was done by determining whether implantation of millipore chambers containing viable worms, or injection of metabolites released by worms i~r vitro, protected guinea-pigs against infection. METHODS Experimental
AND MATERIALS
unimds
Outbred guinea-pigs, body weight 300-550 g of both sexes and from a variety of sources, were used in these experiments. tn individual experiments, animals of about the same size, and from the same source and generally of only one sex were used, When it was necessary to use animals of both sexes. equal numbers were allocated to each experimental group. Sheep used were between 6- and l&months-old and had been raised worm-free from birth. T. cn(ubri~rrrri.s-infective larvae were obtained from cultures of the faeces of sheep harbouring pure experimental infections. Fourth stage 7’. roluhriformis larvae were collected from the anterior small intestine of sheep eight days after infection with 100,000 infective larvae. Adult worms were collected 28 days after infection with I S.ooOlarvae. immediately after slaughter, the anterior part of the small intestine was removed, opened tongitudinally, food particles rinsed off in @SS”/o w/v sodium chloride at 38°C and the intestine placed on a Baermann apparatus in saline at the same temperature. After from I.5 to 2 h, the worms were pipetted off any underlyjng debris and rinsed in warm saline. Third stage larvae were exsheathed by exposure to 0.05% sodium hypochlorite as described by Robinson {1967). Recipient guinea-pigs were anaesthetized for administration of infective larvae, which were injected through a metal stomach tube. Preparation qf’worm homogenates. After collection, the parasites were blotted with fine filter paper, then hornagenized with @I M pH 7-O phosphate buffer using an ice-cooled Potter-Elvehjem tissue homogenizer. Cu/~~~~~~~i$ s&a& p~i~~~~F~ rdeased rfrtrit?i: in vitro maintenamx. In the first experiment with these materials. fourth-stage larvae were collected as described above and approximately O-4 g wet wt transferred to 50 ml phosphate buffered saline pW 7.0 at 38°C in a conical flask. They were then maintained under nitrogen, while gently agitated in a water bath For 3 h. ln the second experiment, the larvae were allowed to settle in a centrifuge tube and approximately 5 ml cultured, without agitation, under air in a conical flask in 20 ml of 0.85% w/v sodium chloride at 38°C for 6 h. In both experiments, the maintenance fluid was pipetted off the worms, lightly centrifuged and then filtered through a 0.45 urn pore size Millipore filter. Xn the second experiment, the maintenance fluid was concentrated to approximately one-fifth
of its original volume with a membrane ultra-filtration apparatus (Amicon Diaflo, membrane UM-2). The second culture technique was adopted because it
simpler, and because in other experiments fourth stage larvae maintained under both conditions have been is
found equally capabte of estabtishing themsefves in the smati intestine of guinea-pigs into which they were surgically transplanted. Milhpore chambers. Millipore chambers (13 mm dia., membrane pore size 0.45 pm were prepared and freshly collected fourth-stage larvae suspended in saline or saline alone injected into them, They were then placed in the peritoneal cavity of recipient animals through a mid-line incision and removed through the same incision after 72 h or seven days. Protein determinariom. The protein content of the worm homogenates and culture fluids was determined by the micro-Kjeldahl method. Vaccinatiorr of guinea-pigs. Vaccination was done on the day the worms were collected. in some experiments, homogenates or maintenance fluids were emulsified with an equal volume (O-15 ml) of incomplete or comptere Freund’s adjuvant (Difco Laboratories, Michigan, U.S.A.). Injections were given in one or more sites subcutaneously on the back. Control guinea-pigs received similar injectians, without homogenate or metabulites. Worm cmm/s+ Prior to slaughter for worm counts, guinea-pigs were starved for 24-36 h. They were then killed by a blow on the head and their intestinal tract, from pylorus to rectum, removed. The small intestine was freed from the mesentery, cut into short lengths, and digested overnight at 38‘C in a mixture of I % w/v pepsin and 0.17 N hydrochloric acid, after which it was shaken vigorously and formalin added to give a concentration of IO’%. The large intestine was opened into 10% formalin. Before counting, the small intestine digest was washed through a 75pm aperture sieve and the large intestine contents through a f78 wrn aperture sieve, Des&u qf e.uperiments. In all experiments, guinea-pigs were given a single immunizing or control injection, challenged with 2000 T. roluhri/irrmi.~ infective larvae 21 days later and killed for worm counts after a further I4 days. The efficacy of vaccination was then determined by comparing worm counts in control and vaccinated groups. Stafbfifaf ana/ysis c~~rrctrn~ (‘(INNI tiara. Prior to %naiySis, logarithmic transformation of worm count data was performed. This transformation is variance-stabilizing and normalizes such data. The data are therefore presented in the results as means of log worm counts and as geometric means fantilog of mean worm count). RESULTS Vaccinotiorr with imreasing
doses of worm honmgnrut~~
In order to study the ability of increasing doses of worm homogenate to protect guinea-pigs against T. co~~br~~~r~?~s infection, groups of IO animals were injected with 10, 100 or loo0 pg of fourth Iarvaf stage homogenate protein in Freund”s complete adjuvant and then challenged with K roltrbri,formis larvae. The results of this experiment are summarized in Table 1, These results show that vaccination with both 100 1.18and 1000 pg. but not IO gig of homo-
I.J.P. VOL. 4. 1974
Vaccination
against
TABLE I-WORM COUNTS IN GUINEA PIGS VACCINATED WITH INCREASING AMOUNTS OF T. rolubriformis FOURTH LARVAL STAGE HOMOGENATE IN COMPLETE FREUND'S ADJUVANT THEN CHALLENGED WITH T. cohrbriformis Mean log worm count
Group Controls (adjuvant alone) IO ug homogenate protein 100 ug homogenate protein 1000 ug homogenate protein
The previous experiment showed that vaccination of guinea-pigs with fourth larval stage homogenate in complete Freund’s adjuvant stimulated significant immunity against infection with T. cohbriformis. In order to determine whether adjuvants enhanced the immunogenicity of the homogenate, groups of seven guinea-pigs were injected with IO, 100 or 1000 ug of homogenate protein in buffer, complete or incomplete Freund’s adjuvant. These animals, and controls given buffer or the adjuvants alone, were then challenged with T. colubriformis larvae. The results of this experiment are summarized in Tables 2a and 2b and their analysis of variance in Table 2c. This analysis shows that worm homogenates again stimulated significant immunity to infection, and that vaccination with homogenate in buffer was as effective as vaccination with homogenate in either adjuvant.
1203 775 183 91
Pooled estimate of standard error (log worm counts) 0.15. * Means bearing the same superscript are not significantly different at the 5% level by Duncan’s (1955) “new multiple range” test. Means bearing different superscripts are significantly different at the I y. level. genate
protein
protective 1000
)tg
stimulated
immunity. and
100
In )tg
statistically addition
were
significant
they
effective
show
to
a
‘Y5
The efecf of’ adjuvants 011 the abi/itJj of worm homogenates to protect against itlfectiotl
Geometric mean
3,08”* 2.89” 2,2@ I ,96b
T. co/uh~~b~~;s
that
similar
degree. TABLE 2-WORM COUNTSINGUINEA-PIGSVACCINATEDWITH~NCREASING AMOUNTSOF T, colubrifhnis FOURTH LARVAL STAGE HOMOGENATE IN BUFFER,INCOMPLETE OR cOMPLETE FREUND'S ADJUVANTTHFN CHALLENGED WITH T.colubrifirmis
(a)
Doses of homogenate Controls 10 ug homogenate protein 100 ug homogenate protein 1000 ug homogenate protein
Mean
log worm
No adjuvant
Incomplete Freund’s adjuvant
Complete Freund’s adjuvant
2.79 2.38 1.26 I.80
2.98 2.12 2.28 2.17
2.67 2.28 2.1 I
I.54
2.81 2.26 I.88 I.84
2.06
2.39
2.15
2.20
Complete Freund’s adjuvant
Means
~~ ..______
Means
(b) Geometric
Group
No adjuvant ___~_
Controls IO ug homogenate protein 100 ug homogenate protein 1000 ug homogenate protein Means Pooled estimates of standard all homogenate doses 0.13.
Dose level Adjuvants Interaction Residual
mean worm
613 239 18 64
964 132 190 149
463 I91 129 35
650 182 76 69
II4
245
141
158
of variance
Degrees of freedom 3 2 6 72
Means
counts
Incomplete Freund’s adjuvant ~~~~
error (log worm counts).
(c) Analysis
Source
counts
Each group
of log worm
Mean square 4.27 0.82 0.76 0.50
0.27;
all adjuvants
counts
F
P
8.59 I .64 I.52
OTIOI 0.20 0.18
0.15;
I.J.P. VOL.
T. L. W. Rtt-II~WI-LLand R. J. Lovk
296 TABLE
3-WORM
INFECTIVE
COUN Is
THIRII
IN
STAGE LARVAE,
GUINEA-PIGS FOURTH
VACC’lNATtD
STAGE LARVAE
WITH
0~
r.
ADULT
Co/lthriJi,mli.S
WORMS
Experiment A (100 pig protein per animal) Group Controls Homogenate of third-stage larvae Homogenate of fourth-stage larvae Homogenate of adult worms
IIOMCIGENAlt
THFN CHALLENGED
Mean log
Standard
Geometric
error
mean
2.9P 2.79’ 2.67’ 2.60h
0,095 0.112 0,095 0,095
870 622 465 398
FROM
T. rohrhrifi,rmi.v
WITH
Experiment B protein per ICOgbody weight) Mean log Standard Geometric mean worm count error
( IOOpg
worm counl
I’RFPARtD
4. 1974
0.14 0.16 0, I4 0. I4
2,22”* I .99” I ,39” I .44”
164 93 25 28
* Within each dxperiment. means bearing the same superscript are not sj~nificantly different at the 5”/, level by Duncan’s (1955) “new muftiple range” test. Means bearing different superscripts are significantly different at the 5”,/, level, at least. TABLE
~--WORM
‘THIRD
LARVAL
COUNTS
IN C;UINEA-PIGS
STAGE tiOMOGENATE
VACCINATED
PROTEIN WITH
WITH
PER 100 g BODY
100 pg
WEIGHT.
T. chhrijkwzi.~
THEN
CHALLENGED
T. ~~/ll~rjf~~rrn~.~
Mean log* Group Controls Homogenate of infective third-stage larvae Homogenate of exsheathed third-stage larvae
uorm count
Standard error
Geometric mean
2.68 2.25 2.18
0.20 0.23 O-20
4x1 177 I52
* Mean log worm counts are not significantly different at the ST, level by Duncan.5 f 1955) “new multiple range” test. Analysis of the pooled data for each dosage level of homogenate by Duncan’s (1955) “new multiplerange” test showed that the worm counts in all the vaccinated groups were signi~cantly less (at least at the 5 7: level) than those in the control groups. Vaccination with hon~ogeriates prepareci,f~oni stages qf’ the life cycle
di#Prerrt
The ability of the homogenates prepared from different stages of the Life-cycle to stimulate protective immunity against T. c#/~br~f~r~??i.~ was studied in three experiments. In the first of these experiments, groups of seven guinea-pigs were vaccinated with homogenates prepared from infective larvae, fourth-stage larvae
or adult worms. In each case, the homogenate was injected without adjuvant and each animal was given 100 pg of homogenate protein. The vaccinated animals and seven non-vaccinated controls were then challenged with T. colubr-jfomri.s. The worm counts from this experiment are summarized in Table 3 (experiment A). They show that vaccination with homogenates prepared from adult worms, but not from third or fourth stage larvae, protected guineapigs against infection with T. c~/i~br~f~r/?~;.s. Previous results (summarized in Table 2), however, showed that 100 pg fourth larval stage homogenate protein protected guinea-pigs against infection. The experiment was therefore repeated, injecting in this case 100 pg of homogenate protein per 100 g body wt. Worm counts from this experi-
ment are summarized in Table 3 (experiment B). These worm counts were found to be a great deal lower than generally found after challenge with 2000 T. eo/~~r~f~r~?i~ larvae. Nevertheless homogenates prepared from fourth-stage larvae and adult worms, but not from third stage larvae protected against infection with T. colubr(fkwi.s. In both the preceding experiments, worm counts in guinea-pigs vaccinated with homogenates of third stage larvae were considerably lower than those in the controls. In a third experiment a group of guineapigs was vaccinated with 100 pg of third larval stage homogenate protein per 100 g body weight. In this experiment, an additional group was vaccinated with the same dose of exsheathed third stage larvae. The worm counts from this experiment are summarized in Table 4. Once again, the worm counts in the vaccinated guinea-pigs were considerably lower than the controk but the difference was not quite significant at the 5’:,; level. Protectiou
by worm nwtabolites
of T. cohrbr~f~rniis metabolites to protect guinea-pigs against infection with T. colorbr~f~r~?i~~ was studied by implanting millipore The
ability
chambers containing fourth stage larvae into the peritoneal cavity, or by injecting soluble products released by T. colubr~formis fourth stage larvae in Ljitro, then challenging treated guinea-pigs with T. rolubr[fbrnlis.
In one
experiment,
groups
of five guinea-pigs
I.J.1’. VOL. 4. 1974
‘97
were implanted with either a millipore chamber containing approximately 250 live 7. colubr~fbrmis fourth stage larvae or physiological saline. After seven days the chambers were removed, and the animals challenged with T. co/ubr(fbrmis after a further I4 days. The worm counts from this experiment are given in Table 5 and show that guinea-pigs implanted with larvae enclosed in a Millipore chamber were solidly immune to challenge infection. TARL~
~--WORM
MILLIPORE FOURTH
MOUNTS
CHAMBERS STAGE
IN GUINEA-PIGS
CONTAINING
LARVAE
THEN
VIABLE
CHALLENGED
IMPLANTED
TABLE
Controls Implanted with Millipore chambers containing larvae
3.23
I688
IMPLANTED
Mean log worm count 2
8fjd9
I ,74”
Geometric mean 723 55 6
0.78’
Pooled estimate of standard error (log worm counts) : 0.30. * Means bearing different superscripts are significantly different at the 5 % level at least, by Duncan’s (1955) “new multiple range” test.
mean
0.42
GUINEA-PIGS
Controls Implanted with millipore chamber containing dead larvae Implanted with millipore chamber containing live larvae
T. rolubriformis WITH T. C&4-
Geometric
IN
CONTAINING T. colubrij2wmi.s CHALLENGED WITH THEN
Group
WITH
Mean log worm count
COUNTS
T. colubr~~rmis
hrij~wmis
Group
~-WORM
WITH MILLIPORE CHAMBERS FOURTH STAGE LARVAE
3
protein
stimulated
significant
immunity
against
challenge.
The larvae in the Millipore chambers removed during the experiment were dead, and immunization might have been achieved by the leakage of somatic material from the chambers. Another experiment was therefore done in which groups of five guineapigs were implanted with millipore chambers containing physiological saline, foul-th stage larvae killed by freezing in dry-ice alcohol, or viable larvae. In this experiment, the chambers were removed after 72 h, by which time the viable larvae had died. The guinea-pigs were challenged 21 days after removal of the chambers. The worm counts from this experiment are summarized in Table 6. This experiment shows that implantation of millipore chambers containing both live and dead larvae protected guinea-pigs against challenge infection, although live larvae were more effective. Vaccination with soluble products released by fourth stage larvae hl vitro was attempted in two experiments. In the first experiment, groups of IO guinea-pigs were injected with IO pg of maintenance fluid protein in complete Freund’s adjuvant or with adjuvant alone, thenchallenged with T. colubv$wmis. The worm counts from this experiment are summarized in Table 7 (experiment A), and they show that vaccination with IO 1.18 maintenance fluid TABLE
l--WORM
C‘OLINTS
FOURTH
IN
GUINEA-PIGS
STAGE LARVAE
VACCINATED
In the second experiment, groups of seven guineapigs were injected with 100 pg of maintenance fluid protein without adjuvant per 100 g body weight and with controls, challenged with T. colubr(fhmis. The worm counts from this experiment are summarized in Table 7 (experiment B) and they confirm that vaccination with soluble products collected during iti vitro maintenance protects guinea-pigs against infection with the parasite.
DISCUSSION The aim of the experiments described in this paper was to determine the suitability of T. cohtbr~fbrmi.~ infections in guinea-pigs for study of nematode protective antigens. The experimental results, which broadly confirm those of Silverman et al. (I 962) and Connan (l965), show that a high level of immunity can be stimulated by the administration of a single subcutaneous injection of approximately 100 pg of worm protein. The model is therefore admirably suited to studying these antigens. The ready stimulation of a high level of immunity to infection by vaccination with non-living worm material is not consistent with the almost traditional view that these materials are incapable of stimulating WITH
SOLUBLE
in vitro THEN CHALLENGED
PRODUCTS
WITH
Experiment A (IO pg prolein in complete Freund’s adjuvant) Mean log Standard error worm count
Group Controls Vaccinated Significance
of difference
between groups
3.08 2.83 p - 0.06
0.09 0.09
Geometric mean I202 672
RELEASED
BY
T. colubrij~rmi.v
T. colubrij?wmis
( IO0
Experiment B pg protein per IO0 g body weight. No adjuvant)
Mean log Standard worm count error 2.97 I.89 p :m OGIIO6
0.16 0.17
Geometric mean 933 78
298
I-. L. W. Rox-f(wtLL
significant protective immunity (Stall, 1961; World Health Organization, 1965; Terry, 1968; Silverman, 1970). However, T. colubr~fiwmis is not a natural parasite of guinea-pigs, and it could be argued that the host-parasite relationship is already biased towards the host and that protective immunity is consequently much more readily stimulated than in natural host-parasite systems. However, even if this is true, it cannot detract from the suitability of the system for study of the parasite’s antigens. The experimental results show that fourth stage larvae, adult worms and soluble products collected during itz vitro maintenance of fourth stage larvae contain protective antigen(s). Mean worm counts in animals vaccinated with homogenates of third stage larvae were lower than those in the control groups in all the experiments described, but in none was the difference statistically significant (at the 5 0; level). Silverman et al. (1962) reported evidence of protection against T. ~o/~br~~orn?;~ infection in guinea-pigs vaccinated with infective larvae, with a homogenized mixture of infective larvae, exsheathed third stage larvae, fourth stage larvae and metabolites and with adult worms. Analysis of their data by the methods used in the present paper, however, showed statistically significant protection (at the 5 “/, level) only in guinea-pigs vaccinated with adult worm homogenate. Connan (I 965) reported that his attempts to vaccinate guinea-pigs with somatic preparations failed, but no information on the identity of these preparations was given. The experiments utilizing the implantation of fourth stage larvae in millipore chambers were designed to provide evidence for the stimulation of protective immunity by worm metabolites. However, dead worms in Millipore chambers were almost as effective as live worms in protecting against challenge, and so attempts were made to collect the metabolites released by fourth stage larvae during itr vitro culture. The soluble products collected in the maintenance fluid might have contained somatic material released from damaged larvae, but as the larvae appeared viable after culture and few damaged larvae were found, the protective antigen(s) in the soluble products were probably worm metabolites. The finding that incomplete or complete Freund’s adjuvants did not enhance the immunizing ability of the worm homogenates might be explained by the possession of similar adjuvant properties by worm somatic materials. However, the soluble products administered without adjuvant in the final experiment stimulated solid protective immunity, and it appears that these adjuvants do not enhance the immunizing ability of the protective antigen(s). Attempts to study the protective antigens in 7: co/ubr~f&mis using this host-parasite system are currently in progress. Attention is being given to two antigens known to be present in fourth larval stage T. colttbr(fbrrGs and which have been impli-
l.J.I’. VOL.
and R. J. Lovr:
4. 1974
cated in other host-parasite systems, the enzyme acetylcholinesterase (Edwards, Burt & Ogilvie, 1971 ; Jones & Ogilvie, 1972; Rothwell P/ a/., 1973) and worm allergen (Andrews, 1962; Rothwell, 1969). Ar~no~~/~~~emenir-We wish to thank Dr. D. A. Grifiths for statistical analysis of some of the data, and Miss Valma lsberg and Messrs. P. Marshall and J. Weave1 for their able technical
assistance.
REFERENCES ANDREWSJ. M. 1962. Parasitism and allergy. Tfze Jortnml of Para.~;tolo~y 48: 3-l 2. CONNANR. 1965. Immunity in the guinea pia- to Trick+ stronylus
colubriformis--a
study
of
the
antigen
associated with immunity. P~ra.siro/o~y 55: IOP. DUNCAND. B. 1955. Multiple range and multiple F tests. Biometrics 11: l-42. EDWARDSA. J., BURTJ. S. & OC~ILVIE BRIDGETM. 1971. The effect of immunity upon some enzymes of the parasitic nematode, ~ipp[~stro~~yl~/~~ brasjiien.s;.s, Parasitology
62:
339-347.
JARRETTW. F. H., JENNINEX F. W.. MC~NTYKEW. I. M., MULLIGANW. & URQUHARTC. M. 1958. Irradiated helminth larvae in vaccination. Proceedings ofthe Royal Society qf‘ Medicine 51: 743-744. JONESVALERIEE. & OCILVIE BRm(;Er M. 1972. Protective ~mmLtnity to ~ippf~.~tro~~Jl/u‘~ bro.~i/;e~.si.~ in the rat. III. Modulation of worm a~etylcholinesterase by antibodies. itnrnl~~o/o~y 22: 119.--129. MILLER T. A. 1965. Effect of route of administration of vaccine and challenge on the immunogenic efficiency of double vaccination with irradiated Ancylcwtoma caninum larvae. The Journal of’ Parasitology 51: 200206. MULLIGAN W., GORDON H. Mr-L., STEWART D. F. & WAGLAND B. M. 1961. The use of irradiated larvae as immunizing agents in Haemonchrrs contortus and Trichostrongylus co/ubrifi,rmis infections of sheep. Australian
Jounlal
of
Agricultural
Resrarrh
12:
1175-l 187. ROBINSON D. L. H. 1967. in vifro studies on Haemonchrrs contortus. Symposium of’ the British Society .fiw Parasitology 5: 61-69. ROTHWELL. T. L. W. 1969. Mechanisms of immunity to parasites. Ph.D. Thesis, University of Sydney. ROTHWELL T. L. W., OGILVIE BKIDCET M. & LOVE R. J.
1973. Acetylcholinesterase secretion by parasitic Trichostrot1~~~lu.s spp. ltfternational nematodes-II. Journal ,/iw Parasitology 3 : 599-608 SILVERMAN P. H. 1970. Vaccination: progress problems. In ln?~rr~/lity to Parasitic Animals. Y. 2
and
(Eds. JACKSONG. J., HERMANR.&L SINGERI.)pp. 1165-l 185. Appleton-Century-Crofts, N.Y. SILVERMAN P. H., PovNTER D. & PODGER K. R. 1962. Studies on larval antigens derived by cultivation of some parasitic nematodes in simple media: protection tests in laboratory animals. 7”lle Journal of Parasitology 48: 562-571. SOKOLIC A., JOVANOVIC. M., CUPERLOVIC K.& MOVSESIJAN M. 1965. Vaccination against Dirtyocactlus Filaria with irradiated larvae. British Veterinary Jowual 121: 212.-222. STOLL N. R. 1961, The worms: can we vaccinate against them? Americun Journal o/’ Tropical Medicine rmd Hygiene 10: 293-303.
I.J.P. VOL. 4. 1974
Vaccination
against
TERRY R. J. 1968. The application of immunology to helminth diseases. Symposiwn nfthe BritishSocietyfor Pnrcisitolqyy 6 : 9 I -I 03.
T. colrrbriformi.5 WORLD HEALTH ORGANISATION Series No. 315. Immunology 64 PP.
299 1965. Technical Report and Parasitic Diseases.
Division of Animal
Health, McMaster
Laboratory,
(Received 8 August 1973 : amended
Glebe, N.S.W. 2037, Australia
I October 1973)
Abstract-RorwwErLT. L. W. and LOVE R. .I. 1974. Vaccination against the nematode T’ichoutrongy/@s co/~tb$&mis--1. Vaccination of guinea-pigs with worm homogenates and soluble products released during in v&o nlaintenance. I~ic~t~ati~~aIJoi~r~?al~~ Pa~as~to/u~~ 4: 293-299. Vaccination of guineapigs with homogenates of fourth larval stage and adult Trichostrongylus colubviformis and soluble products released by fourth stage larvae during itl vitro maintenance, induced a high level of protection against challenge infection with the parasite. INDEX KEY WORDS: vaccination.
Antigens; guinea-pigs; immunity; nematode; Trkhosrrorrgyhts colrtbrifornris;
INTRODUCTION DESPITE the demonstration of acquired immunity against many parasites and advances in the under-
standing of the immune responses parasites generate in their hosts, protection of man and animals against parasitic disease by vaccination (used here as “administration of antigenic material to stimulate active immunity with the aim of protecting against infection”) has proved an elusive goal (Stall, 1961 : World Health Organisation, 196.5; Terry, 1968; Silverman, 1970). In the case of nematodes, limited success has been achieved using live vaccines (Jarrett, Jennings, McIntyre, Mulligan & Urquhart, 1958; Mulligan, Gordon, Stewart & Wagland, 1961; Sokolic, Jovanovic, Cuperlovic & Movsesijan, 1965; Miller, 1965), but vaccination with non-living nematode material has been generally unsuccessful. The intestinal nematode Tr~~~ostr~t~~~[its cohbri.f&nks is an important pathogen of sheep, which in addition reaches patency in guinea pigs. Silverman, Poynter & Podger (1962) and Connan (1965) have reported that guinea pigs can be vaccinated against T. colubrifkwmis. Silverman et al. (1962) gave two intraperitoneal injections of crude homogenates of infective larvae, fourth stage larvae and adult T. ~(l~ub~~~~rj~l~.~. The methods used in the preparation of these homogenates were not given in detail, but it appears that the fourth larval stage preparation contained non-exsheathed third stage larvae, sheaths and metabolic products. The dosage of the fourth larval stage preparation administered is expressed
as equivalents of the number of larvae which had undergone ecdysis to the fourth larval stage, but the dosages of a third larval stage and adult preparations were not revealed. Connan (1965) published only a brief report of his experiments, referring to “a marked degree of protection” in guinea-pigs “vaccinated with metabolic solutions”. His attempts to vaccinate guinea-pigs with somatic antigen preparations, however, failed. Silverman et al. (1962) and Connan’s (1965) results suggest that this host-parasite system might be well suited for studying the nematode antigens responsible for host protective immunity. Insufficient detail, however, is provided in these publications to confidently use the system for this purpose. Therefore, we have re-examined vaccination of guinea-pigs against T. cohbr$wnis with worm homogenates and metabolites prior to attempting to identify the antigens responsible for host imnlunity. In the experiments described, the ability of a single injection of worm homogenate to stimulate protective immunity was determined. Most attention was given to the fourth larval stage of the parasite because large numbers can easily be collected after a wait of only eight days. In addition, the subventral glands of the excretory system, which are a possible source of protective antigens, are relatively well developed in fourth-stage larvae (Rothwell, Ogilvie & Love, 1973), making them especially suitable for attempts to isolate these antigens. Infective larvae are also readily available, but because they did not
stimulate solid protective immunity, they were not examined in deta;l. However, for completeness, the results of these studies and limited observations made on adult worms are also presented. Worm metaboljtes, because of their freedom from somatic material and possible value in planned antigen purification studies, were also examined for their ability ta protect guinea-pigs against infection with T. colubriformis. This was done by determining whether implantation of millipore chambers containing viable worms, or injection of metabolites released by worms i~r vitro, protected guinea-pigs against infection. METHODS Experimental
AND MATERIALS
unimds
Outbred guinea-pigs, body weight 300-550 g of both sexes and from a variety of sources, were used in these experiments. tn individual experiments, animals of about the same size, and from the same source and generally of only one sex were used, When it was necessary to use animals of both sexes. equal numbers were allocated to each experimental group. Sheep used were between 6- and l&months-old and had been raised worm-free from birth. T. cn(ubri~rrrri.s-infective larvae were obtained from cultures of the faeces of sheep harbouring pure experimental infections. Fourth stage 7’. roluhriformis larvae were collected from the anterior small intestine of sheep eight days after infection with 100,000 infective larvae. Adult worms were collected 28 days after infection with I S.ooOlarvae. immediately after slaughter, the anterior part of the small intestine was removed, opened tongitudinally, food particles rinsed off in @SS”/o w/v sodium chloride at 38°C and the intestine placed on a Baermann apparatus in saline at the same temperature. After from I.5 to 2 h, the worms were pipetted off any underlyjng debris and rinsed in warm saline. Third stage larvae were exsheathed by exposure to 0.05% sodium hypochlorite as described by Robinson {1967). Recipient guinea-pigs were anaesthetized for administration of infective larvae, which were injected through a metal stomach tube. Preparation qf’worm homogenates. After collection, the parasites were blotted with fine filter paper, then hornagenized with @I M pH 7-O phosphate buffer using an ice-cooled Potter-Elvehjem tissue homogenizer. Cu/~~~~~~~i$ s&a& p~i~~~~F~ rdeased rfrtrit?i: in vitro maintenamx. In the first experiment with these materials. fourth-stage larvae were collected as described above and approximately O-4 g wet wt transferred to 50 ml phosphate buffered saline pW 7.0 at 38°C in a conical flask. They were then maintained under nitrogen, while gently agitated in a water bath For 3 h. ln the second experiment, the larvae were allowed to settle in a centrifuge tube and approximately 5 ml cultured, without agitation, under air in a conical flask in 20 ml of 0.85% w/v sodium chloride at 38°C for 6 h. In both experiments, the maintenance fluid was pipetted off the worms, lightly centrifuged and then filtered through a 0.45 urn pore size Millipore filter. Xn the second experiment, the maintenance fluid was concentrated to approximately one-fifth
of its original volume with a membrane ultra-filtration apparatus (Amicon Diaflo, membrane UM-2). The second culture technique was adopted because it
simpler, and because in other experiments fourth stage larvae maintained under both conditions have been is
found equally capabte of estabtishing themsefves in the smati intestine of guinea-pigs into which they were surgically transplanted. Milhpore chambers. Millipore chambers (13 mm dia., membrane pore size 0.45 pm were prepared and freshly collected fourth-stage larvae suspended in saline or saline alone injected into them, They were then placed in the peritoneal cavity of recipient animals through a mid-line incision and removed through the same incision after 72 h or seven days. Protein determinariom. The protein content of the worm homogenates and culture fluids was determined by the micro-Kjeldahl method. Vaccinatiorr of guinea-pigs. Vaccination was done on the day the worms were collected. in some experiments, homogenates or maintenance fluids were emulsified with an equal volume (O-15 ml) of incomplete or comptere Freund’s adjuvant (Difco Laboratories, Michigan, U.S.A.). Injections were given in one or more sites subcutaneously on the back. Control guinea-pigs received similar injectians, without homogenate or metabulites. Worm cmm/s+ Prior to slaughter for worm counts, guinea-pigs were starved for 24-36 h. They were then killed by a blow on the head and their intestinal tract, from pylorus to rectum, removed. The small intestine was freed from the mesentery, cut into short lengths, and digested overnight at 38‘C in a mixture of I % w/v pepsin and 0.17 N hydrochloric acid, after which it was shaken vigorously and formalin added to give a concentration of IO’%. The large intestine was opened into 10% formalin. Before counting, the small intestine digest was washed through a 75pm aperture sieve and the large intestine contents through a f78 wrn aperture sieve, Des&u qf e.uperiments. In all experiments, guinea-pigs were given a single immunizing or control injection, challenged with 2000 T. roluhri/irrmi.~ infective larvae 21 days later and killed for worm counts after a further I4 days. The efficacy of vaccination was then determined by comparing worm counts in control and vaccinated groups. Stafbfifaf ana/ysis c~~rrctrn~ (‘(INNI tiara. Prior to %naiySis, logarithmic transformation of worm count data was performed. This transformation is variance-stabilizing and normalizes such data. The data are therefore presented in the results as means of log worm counts and as geometric means fantilog of mean worm count). RESULTS Vaccinotiorr with imreasing
doses of worm honmgnrut~~
In order to study the ability of increasing doses of worm homogenate to protect guinea-pigs against T. co~~br~~~r~?~s infection, groups of IO animals were injected with 10, 100 or loo0 pg of fourth Iarvaf stage homogenate protein in Freund”s complete adjuvant and then challenged with K roltrbri,formis larvae. The results of this experiment are summarized in Table 1, These results show that vaccination with both 100 1.18and 1000 pg. but not IO gig of homo-
I.J.P. VOL. 4. 1974
Vaccination
against
TABLE I-WORM COUNTS IN GUINEA PIGS VACCINATED WITH INCREASING AMOUNTS OF T. rolubriformis FOURTH LARVAL STAGE HOMOGENATE IN COMPLETE FREUND'S ADJUVANT THEN CHALLENGED WITH T. cohrbriformis Mean log worm count
Group Controls (adjuvant alone) IO ug homogenate protein 100 ug homogenate protein 1000 ug homogenate protein
The previous experiment showed that vaccination of guinea-pigs with fourth larval stage homogenate in complete Freund’s adjuvant stimulated significant immunity against infection with T. cohbriformis. In order to determine whether adjuvants enhanced the immunogenicity of the homogenate, groups of seven guinea-pigs were injected with IO, 100 or 1000 ug of homogenate protein in buffer, complete or incomplete Freund’s adjuvant. These animals, and controls given buffer or the adjuvants alone, were then challenged with T. colubriformis larvae. The results of this experiment are summarized in Tables 2a and 2b and their analysis of variance in Table 2c. This analysis shows that worm homogenates again stimulated significant immunity to infection, and that vaccination with homogenate in buffer was as effective as vaccination with homogenate in either adjuvant.
1203 775 183 91
Pooled estimate of standard error (log worm counts) 0.15. * Means bearing the same superscript are not significantly different at the 5% level by Duncan’s (1955) “new multiple range” test. Means bearing different superscripts are significantly different at the I y. level. genate
protein
protective 1000
)tg
stimulated
immunity. and
100
In )tg
statistically addition
were
significant
they
effective
show
to
a
‘Y5
The efecf of’ adjuvants 011 the abi/itJj of worm homogenates to protect against itlfectiotl
Geometric mean
3,08”* 2.89” 2,2@ I ,96b
T. co/uh~~b~~;s
that
similar
degree. TABLE 2-WORM COUNTSINGUINEA-PIGSVACCINATEDWITH~NCREASING AMOUNTSOF T, colubrifhnis FOURTH LARVAL STAGE HOMOGENATE IN BUFFER,INCOMPLETE OR cOMPLETE FREUND'S ADJUVANTTHFN CHALLENGED WITH T.colubrifirmis
(a)
Doses of homogenate Controls 10 ug homogenate protein 100 ug homogenate protein 1000 ug homogenate protein
Mean
log worm
No adjuvant
Incomplete Freund’s adjuvant
Complete Freund’s adjuvant
2.79 2.38 1.26 I.80
2.98 2.12 2.28 2.17
2.67 2.28 2.1 I
I.54
2.81 2.26 I.88 I.84
2.06
2.39
2.15
2.20
Complete Freund’s adjuvant
Means
~~ ..______
Means
(b) Geometric
Group
No adjuvant ___~_
Controls IO ug homogenate protein 100 ug homogenate protein 1000 ug homogenate protein Means Pooled estimates of standard all homogenate doses 0.13.
Dose level Adjuvants Interaction Residual
mean worm
613 239 18 64
964 132 190 149
463 I91 129 35
650 182 76 69
II4
245
141
158
of variance
Degrees of freedom 3 2 6 72
Means
counts
Incomplete Freund’s adjuvant ~~~~
error (log worm counts).
(c) Analysis
Source
counts
Each group
of log worm
Mean square 4.27 0.82 0.76 0.50
0.27;
all adjuvants
counts
F
P
8.59 I .64 I.52
OTIOI 0.20 0.18
0.15;
I.J.P. VOL.
T. L. W. Rtt-II~WI-LLand R. J. Lovk
296 TABLE
3-WORM
INFECTIVE
COUN Is
THIRII
IN
STAGE LARVAE,
GUINEA-PIGS FOURTH
VACC’lNATtD
STAGE LARVAE
WITH
0~
r.
ADULT
Co/lthriJi,mli.S
WORMS
Experiment A (100 pig protein per animal) Group Controls Homogenate of third-stage larvae Homogenate of fourth-stage larvae Homogenate of adult worms
IIOMCIGENAlt
THFN CHALLENGED
Mean log
Standard
Geometric
error
mean
2.9P 2.79’ 2.67’ 2.60h
0,095 0.112 0,095 0,095
870 622 465 398
FROM
T. rohrhrifi,rmi.v
WITH
Experiment B protein per ICOgbody weight) Mean log Standard Geometric mean worm count error
( IOOpg
worm counl
I’RFPARtD
4. 1974
0.14 0.16 0, I4 0. I4
2,22”* I .99” I ,39” I .44”
164 93 25 28
* Within each dxperiment. means bearing the same superscript are not sj~nificantly different at the 5”/, level by Duncan’s (1955) “new muftiple range” test. Means bearing different superscripts are significantly different at the 5”,/, level, at least. TABLE
~--WORM
‘THIRD
LARVAL
COUNTS
IN C;UINEA-PIGS
STAGE tiOMOGENATE
VACCINATED
PROTEIN WITH
WITH
PER 100 g BODY
100 pg
WEIGHT.
T. chhrijkwzi.~
THEN
CHALLENGED
T. ~~/ll~rjf~~rrn~.~
Mean log* Group Controls Homogenate of infective third-stage larvae Homogenate of exsheathed third-stage larvae
uorm count
Standard error
Geometric mean
2.68 2.25 2.18
0.20 0.23 O-20
4x1 177 I52
* Mean log worm counts are not significantly different at the ST, level by Duncan.5 f 1955) “new multiple range” test. Analysis of the pooled data for each dosage level of homogenate by Duncan’s (1955) “new multiplerange” test showed that the worm counts in all the vaccinated groups were signi~cantly less (at least at the 5 7: level) than those in the control groups. Vaccination with hon~ogeriates prepareci,f~oni stages qf’ the life cycle
di#Prerrt
The ability of the homogenates prepared from different stages of the Life-cycle to stimulate protective immunity against T. c#/~br~f~r~??i.~ was studied in three experiments. In the first of these experiments, groups of seven guinea-pigs were vaccinated with homogenates prepared from infective larvae, fourth-stage larvae
or adult worms. In each case, the homogenate was injected without adjuvant and each animal was given 100 pg of homogenate protein. The vaccinated animals and seven non-vaccinated controls were then challenged with T. colubr-jfomri.s. The worm counts from this experiment are summarized in Table 3 (experiment A). They show that vaccination with homogenates prepared from adult worms, but not from third or fourth stage larvae, protected guineapigs against infection with T. c~/i~br~f~r/?~;.s. Previous results (summarized in Table 2), however, showed that 100 pg fourth larval stage homogenate protein protected guinea-pigs against infection. The experiment was therefore repeated, injecting in this case 100 pg of homogenate protein per 100 g body wt. Worm counts from this experi-
ment are summarized in Table 3 (experiment B). These worm counts were found to be a great deal lower than generally found after challenge with 2000 T. eo/~~r~f~r~?i~ larvae. Nevertheless homogenates prepared from fourth-stage larvae and adult worms, but not from third stage larvae protected against infection with T. colubr(fkwi.s. In both the preceding experiments, worm counts in guinea-pigs vaccinated with homogenates of third stage larvae were considerably lower than those in the controls. In a third experiment a group of guineapigs was vaccinated with 100 pg of third larval stage homogenate protein per 100 g body weight. In this experiment, an additional group was vaccinated with the same dose of exsheathed third stage larvae. The worm counts from this experiment are summarized in Table 4. Once again, the worm counts in the vaccinated guinea-pigs were considerably lower than the controk but the difference was not quite significant at the 5’:,; level. Protectiou
by worm nwtabolites
of T. cohrbr~f~rniis metabolites to protect guinea-pigs against infection with T. colorbr~f~r~?i~~ was studied by implanting millipore The
ability
chambers containing fourth stage larvae into the peritoneal cavity, or by injecting soluble products released by T. colubr~formis fourth stage larvae in Ljitro, then challenging treated guinea-pigs with T. rolubr[fbrnlis.
In one
experiment,
groups
of five guinea-pigs
I.J.1’. VOL. 4. 1974
‘97
were implanted with either a millipore chamber containing approximately 250 live 7. colubr~fbrmis fourth stage larvae or physiological saline. After seven days the chambers were removed, and the animals challenged with T. co/ubr(fbrmis after a further I4 days. The worm counts from this experiment are given in Table 5 and show that guinea-pigs implanted with larvae enclosed in a Millipore chamber were solidly immune to challenge infection. TARL~
~--WORM
MILLIPORE FOURTH
MOUNTS
CHAMBERS STAGE
IN GUINEA-PIGS
CONTAINING
LARVAE
THEN
VIABLE
CHALLENGED
IMPLANTED
TABLE
Controls Implanted with Millipore chambers containing larvae
3.23
I688
IMPLANTED
Mean log worm count 2
8fjd9
I ,74”
Geometric mean 723 55 6
0.78’
Pooled estimate of standard error (log worm counts) : 0.30. * Means bearing different superscripts are significantly different at the 5 % level at least, by Duncan’s (1955) “new multiple range” test.
mean
0.42
GUINEA-PIGS
Controls Implanted with millipore chamber containing dead larvae Implanted with millipore chamber containing live larvae
T. rolubriformis WITH T. C&4-
Geometric
IN
CONTAINING T. colubrij2wmi.s CHALLENGED WITH THEN
Group
WITH
Mean log worm count
COUNTS
T. colubr~~rmis
hrij~wmis
Group
~-WORM
WITH MILLIPORE CHAMBERS FOURTH STAGE LARVAE
3
protein
stimulated
significant
immunity
against
challenge.
The larvae in the Millipore chambers removed during the experiment were dead, and immunization might have been achieved by the leakage of somatic material from the chambers. Another experiment was therefore done in which groups of five guineapigs were implanted with millipore chambers containing physiological saline, foul-th stage larvae killed by freezing in dry-ice alcohol, or viable larvae. In this experiment, the chambers were removed after 72 h, by which time the viable larvae had died. The guinea-pigs were challenged 21 days after removal of the chambers. The worm counts from this experiment are summarized in Table 6. This experiment shows that implantation of millipore chambers containing both live and dead larvae protected guinea-pigs against challenge infection, although live larvae were more effective. Vaccination with soluble products released by fourth stage larvae hl vitro was attempted in two experiments. In the first experiment, groups of IO guinea-pigs were injected with IO pg of maintenance fluid protein in complete Freund’s adjuvant or with adjuvant alone, thenchallenged with T. colubv$wmis. The worm counts from this experiment are summarized in Table 7 (experiment A), and they show that vaccination with IO 1.18 maintenance fluid TABLE
l--WORM
C‘OLINTS
FOURTH
IN
GUINEA-PIGS
STAGE LARVAE
VACCINATED
In the second experiment, groups of seven guineapigs were injected with 100 pg of maintenance fluid protein without adjuvant per 100 g body weight and with controls, challenged with T. colubr(fhmis. The worm counts from this experiment are summarized in Table 7 (experiment B) and they confirm that vaccination with soluble products collected during iti vitro maintenance protects guinea-pigs against infection with the parasite.
DISCUSSION The aim of the experiments described in this paper was to determine the suitability of T. cohtbr~fbrmi.~ infections in guinea-pigs for study of nematode protective antigens. The experimental results, which broadly confirm those of Silverman et al. (I 962) and Connan (l965), show that a high level of immunity can be stimulated by the administration of a single subcutaneous injection of approximately 100 pg of worm protein. The model is therefore admirably suited to studying these antigens. The ready stimulation of a high level of immunity to infection by vaccination with non-living worm material is not consistent with the almost traditional view that these materials are incapable of stimulating WITH
SOLUBLE
in vitro THEN CHALLENGED
PRODUCTS
WITH
Experiment A (IO pg prolein in complete Freund’s adjuvant) Mean log Standard error worm count
Group Controls Vaccinated Significance
of difference
between groups
3.08 2.83 p - 0.06
0.09 0.09
Geometric mean I202 672
RELEASED
BY
T. colubrij~rmi.v
T. colubrij?wmis
( IO0
Experiment B pg protein per IO0 g body weight. No adjuvant)
Mean log Standard worm count error 2.97 I.89 p :m OGIIO6
0.16 0.17
Geometric mean 933 78
298
I-. L. W. Rox-f(wtLL
significant protective immunity (Stall, 1961; World Health Organization, 1965; Terry, 1968; Silverman, 1970). However, T. colubr~fiwmis is not a natural parasite of guinea-pigs, and it could be argued that the host-parasite relationship is already biased towards the host and that protective immunity is consequently much more readily stimulated than in natural host-parasite systems. However, even if this is true, it cannot detract from the suitability of the system for study of the parasite’s antigens. The experimental results show that fourth stage larvae, adult worms and soluble products collected during itz vitro maintenance of fourth stage larvae contain protective antigen(s). Mean worm counts in animals vaccinated with homogenates of third stage larvae were lower than those in the control groups in all the experiments described, but in none was the difference statistically significant (at the 5 0; level). Silverman et al. (1962) reported evidence of protection against T. ~o/~br~~orn?;~ infection in guinea-pigs vaccinated with infective larvae, with a homogenized mixture of infective larvae, exsheathed third stage larvae, fourth stage larvae and metabolites and with adult worms. Analysis of their data by the methods used in the present paper, however, showed statistically significant protection (at the 5 “/, level) only in guinea-pigs vaccinated with adult worm homogenate. Connan (I 965) reported that his attempts to vaccinate guinea-pigs with somatic preparations failed, but no information on the identity of these preparations was given. The experiments utilizing the implantation of fourth stage larvae in millipore chambers were designed to provide evidence for the stimulation of protective immunity by worm metabolites. However, dead worms in Millipore chambers were almost as effective as live worms in protecting against challenge, and so attempts were made to collect the metabolites released by fourth stage larvae during itr vitro culture. The soluble products collected in the maintenance fluid might have contained somatic material released from damaged larvae, but as the larvae appeared viable after culture and few damaged larvae were found, the protective antigen(s) in the soluble products were probably worm metabolites. The finding that incomplete or complete Freund’s adjuvants did not enhance the immunizing ability of the worm homogenates might be explained by the possession of similar adjuvant properties by worm somatic materials. However, the soluble products administered without adjuvant in the final experiment stimulated solid protective immunity, and it appears that these adjuvants do not enhance the immunizing ability of the protective antigen(s). Attempts to study the protective antigens in 7: co/ubr~f&mis using this host-parasite system are currently in progress. Attention is being given to two antigens known to be present in fourth larval stage T. colttbr(fbrrGs and which have been impli-
l.J.I’. VOL.
and R. J. Lovr:
4. 1974
cated in other host-parasite systems, the enzyme acetylcholinesterase (Edwards, Burt & Ogilvie, 1971 ; Jones & Ogilvie, 1972; Rothwell P/ a/., 1973) and worm allergen (Andrews, 1962; Rothwell, 1969). Ar~no~~/~~~emenir-We wish to thank Dr. D. A. Grifiths for statistical analysis of some of the data, and Miss Valma lsberg and Messrs. P. Marshall and J. Weave1 for their able technical
assistance.
REFERENCES ANDREWSJ. M. 1962. Parasitism and allergy. Tfze Jortnml of Para.~;tolo~y 48: 3-l 2. CONNANR. 1965. Immunity in the guinea pia- to Trick+ stronylus
colubriformis--a
study
of
the
antigen
associated with immunity. P~ra.siro/o~y 55: IOP. DUNCAND. B. 1955. Multiple range and multiple F tests. Biometrics 11: l-42. EDWARDSA. J., BURTJ. S. & OC~ILVIE BRIDGETM. 1971. The effect of immunity upon some enzymes of the parasitic nematode, ~ipp[~stro~~yl~/~~ brasjiien.s;.s, Parasitology
62:
339-347.
JARRETTW. F. H., JENNINEX F. W.. MC~NTYKEW. I. M., MULLIGANW. & URQUHARTC. M. 1958. Irradiated helminth larvae in vaccination. Proceedings ofthe Royal Society qf‘ Medicine 51: 743-744. JONESVALERIEE. & OCILVIE BRm(;Er M. 1972. Protective ~mmLtnity to ~ippf~.~tro~~Jl/u‘~ bro.~i/;e~.si.~ in the rat. III. Modulation of worm a~etylcholinesterase by antibodies. itnrnl~~o/o~y 22: 119.--129. MILLER T. A. 1965. Effect of route of administration of vaccine and challenge on the immunogenic efficiency of double vaccination with irradiated Ancylcwtoma caninum larvae. The Journal of’ Parasitology 51: 200206. MULLIGAN W., GORDON H. Mr-L., STEWART D. F. & WAGLAND B. M. 1961. The use of irradiated larvae as immunizing agents in Haemonchrrs contortus and Trichostrongylus co/ubrifi,rmis infections of sheep. Australian
Jounlal
of
Agricultural
Resrarrh
12:
1175-l 187. ROBINSON D. L. H. 1967. in vifro studies on Haemonchrrs contortus. Symposium of’ the British Society .fiw Parasitology 5: 61-69. ROTHWELL. T. L. W. 1969. Mechanisms of immunity to parasites. Ph.D. Thesis, University of Sydney. ROTHWELL T. L. W., OGILVIE BKIDCET M. & LOVE R. J.
1973. Acetylcholinesterase secretion by parasitic Trichostrot1~~~lu.s spp. ltfternational nematodes-II. Journal ,/iw Parasitology 3 : 599-608 SILVERMAN P. H. 1970. Vaccination: progress problems. In ln?~rr~/lity to Parasitic Animals. Y. 2
and
(Eds. JACKSONG. J., HERMANR.&L SINGERI.)pp. 1165-l 185. Appleton-Century-Crofts, N.Y. SILVERMAN P. H., PovNTER D. & PODGER K. R. 1962. Studies on larval antigens derived by cultivation of some parasitic nematodes in simple media: protection tests in laboratory animals. 7”lle Journal of Parasitology 48: 562-571. SOKOLIC A., JOVANOVIC. M., CUPERLOVIC K.& MOVSESIJAN M. 1965. Vaccination against Dirtyocactlus Filaria with irradiated larvae. British Veterinary Jowual 121: 212.-222. STOLL N. R. 1961, The worms: can we vaccinate against them? Americun Journal o/’ Tropical Medicine rmd Hygiene 10: 293-303.
I.J.P. VOL. 4. 1974
Vaccination
against
TERRY R. J. 1968. The application of immunology to helminth diseases. Symposiwn nfthe BritishSocietyfor Pnrcisitolqyy 6 : 9 I -I 03.
T. colrrbriformi.5 WORLD HEALTH ORGANISATION Series No. 315. Immunology 64 PP.
299 1965. Technical Report and Parasitic Diseases.