Rubin and. Anderson, 1968; Rubin tv w/., 1971; Hurley, Day and Mitchell, 1980). TliLMiiost impressive result to dale, however, was reported by Mitchell and ...
Aosl. J. Exp. Biol. Med. Sci., 63 (Pi. 4t 423-430 (I985t
©VACCINATION AGAINST NEMATOSPIROIDES DUBIUS IN MICH USING ADULT WORM EXTRACTS AS ANTIGENS by FERNANDO MONROY G.. PAUL J. BRINDLEY AND COLIN DOBSON (From the Departmeni of Parasiiology, University of Queensland, St. Lucia 4067. Ausiralia.) (Accepted for publication .March 25, 1985.)
!^uminar>. Various preparaiions of somatk- Nematiitpiraides duhius antigens in (-rcuiid\ compteie adju\ani and assayed in yiuckcnhusli inia- I'or clfiuauy as vaccine-. against hdmiilo^ous infL-ciittn. Snluhlc and paiiiculaic fractions were pwn by iwo dillercni Klines. Onlv minimal proieeiion was induced by vaetinaiion wiili ciilici soluble or pariiciilaie parasiie liaciioiis used indepcndenily when compaied with ihe adjuvani conirol. t-ewer aiul smaller worms Acrt: recovered from, and signifieanily fewer parasiie eggs were voided b>, mice which had been (reaicd previously wilh boih soluble sottialic proiein (IS Mg V dubius per mouse) by ihe inirapcriloncai route and particiitaie aniigcn by subcutaneous injection. These mice showed higher aniibody litres in comparison with other mice treated wiih either fraction alone. Further, ircaimeni with boih antigen IraL-lions togetlu-r appeared lo cceri a synergislic cITeci in cornpurison with either fraction administered alone.
INTRODUCTION The Nematospiroides dubius mouse system has proved useful for ihe characterization and application of worm vaccines. Van Zandt (1971) reported a 30"/o reduction in challenge infections in mice vaccinated wiih soluble cxiracis of adult N. c/»/;/i/.^ emulsified in Freund's complete adjiivani (FCA). Slightly better levels of protection (SC/o) were obtained with vaccines prepared from soluble extracts of infective N. dubius larvae emulsified in FCA (Cypess. 1970) and in a double emulsion adjuvant (Goven atid De Buysscher, 1980). Also, other workers have shown that larvae or adult N. dubius implanted ectopically confer some protection against challenge infection (Lueker. Rubin and Anderson, 1968; Rubin tv w/., 1971; Hurley, Day and Mitchell, 1980). TliLMiiost impressive result to dale, however, was reported by Mitchell and Muno/ (1983) who induced >95^o protection in genetically susceptible C57BI/6 mice against N. dubius by vaccination with .soluble extracts of adults and pertussigen as adjuvant. This effect was dependent on the dose and route of vaccination. Particulate antigen from adult worms, plus pertussigen. gave little or no protection, whereas a tnixturc of aduh soluble and particulate antigens and pertussigen induced up to 90*^^0 protection. Ahhreviutioiis used in this paper: epg, eggs voided per gram of faeces; efd, parasite egg voided per fetnalc per day; QIl. Quackcnbtish strain; SC. sLibcutaneiuis(h); IP. intrapcriioncal(ly); SE. standard error of the mean (x); ^ "Student's" f-lest \ahie for comparison of means; /'. sunisiical probability; I t ' A , l-reund\ complete adjuvant; ht.lSA, en/vme linked imnuinosnrbctit assay; PliS. pliospluiti'-l-iufleietl saline.
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FERNANDO MONROY G., PAUL J. BRINDLEY \M) COLIN DOBSON
This Study was done to examine the fate of challenge infection with N. dubius in mice vaccinated with adult worm extracts in FCA, with particular reference to the nature, quantity, timing and route of antigen presentation required to stimulale protection.
M A T F R I A L S A N D METHOtJS Mici' and parasite Outbred 6-weeks-otd Quackenbush (QB) mice were obtained from the Cenrral Anima! Breeding House, University of Queensland. Nemaiospirokles diihius was maintained in QB mice. Infeciive N. tltihiiis larvae were washed from 7-day-old fuecal cultures, conuemraied by sedimcniaiion and stored at 4" in a shallow layer of waier in lealcd boilles and ajrcd for 14 days before use. Larval counts wore made for len 0'05 ml aliquots from a stock suspension which vvas adjiisied in volume lo give 100 larvae in aboui 0'2 ml of water. Mice were inoculated with UH) larvae by sioniach gavage through a blunted 18 G needle.
Panisifological methods All mice were killed under ether anaesthesia by cervical dislocation after ex-.anguination hy cardiac puncture using a 26 G needle fitted to a 1 ml tuberculin syringe, rhc blood was alloued lo clot al 4 ' for 18 h and the serum was then collected and stored at -20-". I h e peritoneal caviiy was opened and the gut stripped from Iho mesenteries and transferred into saline in a Petri dish. The small intestine was teased open under a stereo microscope. The worms were removed with forceps, counted, sexed, fixed in hot 7O"?o ethanol and measured from camera lucida drawings using a map measurer c^libraied in millimetres. Fifty worms of each sex from each treatment group were measured. Counts of parasite eggs \oided per gram ol' faeces (epg) were made on laei;es from individual mice using [he McMaster technique (Roberts and O'Sullivan, 1950) from day I? to day 20 alter infection. The fecundity of female N. dubius was calculated as eggs per letnaie per day (efd) lestimated from the regression Y (faeces, g) = 0035 + 0 00017 x (mouse body wt,g) and the number ol" female worms recovered 21 days after infection (Brindley and Dobson, 1982)|, A ntigeris Adult V. (liihius worms collected from 2l-dav-o!d infections in QB mice were thoroughly washed in ()• 15 M NaCI and homogenized in a glass tissue grinder in 2 ml of phasphate-bulfered saline (PBS). pH 7 2.0 01 M in an ice bath. The homogenate was centrifuged at 10 OOOrev./min for 30 min at 4 to separate the soluble (A) and particutate (B) fractions, fraction A was clarified by passage through a 0-2 ^m pore size membrane and the protein concentration determined by the Lowry ei al (I95H method and adjusted to 20 mg ml ' with PBS. l-raction B was prepared by homogeni/ation of 600 adult worms and the pellet after centrilugationasabove wasmadeup to2 ml with PBS. This w;is then diluted 1 in 10 for use as a vaccine,
Adjuvanr Equal volumes of both parasite fractions were thoroughly mixed individually witlj FCA . injected boiti with 0-1 ml IP and SC with an adjuvant-PBS (()'(I5 nil : 0-05 ml} mixture. The remaining group served as a noii-vaccinatcd control. These mice wt'rc inoculated vMth 100 ,V diihiu.s larvae by stomaeh gavage 14 days alter vaceinaticm. Analyses of variance and Student's ^test•. were done where appropriate. Values nf /•
