Influence of serum donor and recipient mouse genotype on the passive transfer of protective immunity with serum against Nematospiroides dubius

Influence of serum donor and recipient mouse genotype on the passive transfer of protective immunity with serum against Nematospiroides dubius

0020-7519/82/060567-06$03.OU/O Pergomon Press L td, C‘ 1982 Aus~ro/un Sonefy for P,rmito/ogy INFLUENCE OF SERUM DONOR AND RECIPIENT MOUSE GENOTYPE ON...

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0020-7519/82/060567-06$03.OU/O Pergomon Press L td, C‘ 1982 Aus~ro/un Sonefy for P,rmito/ogy

INFLUENCE OF SERUM DONOR AND RECIPIENT MOUSE GENOTYPE ON THE PASSIVE TRANSFER OF PROTECTIVE IMMUNITY WITH SERUM AGAINST NEMATOSPIROIDES DUBIUS COLIN DOBSON, PAUL J. BRINDLEY and PETRUS SITEPU Department

of Parasitology,

University

of Queensland,

St. Lucia,

4067, Qld, Australia

(Received 23 February 1982) Abstract-DoBsoN

C.,

BRINDLEY P. J. and

SITEPU P. 1982. Influence

of serum donor and recipient with serum against Nematospiroides dub&. International Journal for Parasitology 12: 567-572. Different strains of serum donor mice showed variations in innate immunity to primary infections with Nemafospiroides dubius. Different levels of anti-N. dubius antibodies were detected in sera from these mouse strains; there was no correlation between antibody titre and numbers of worms recovered. Serum from donor wild and six laboratory strains of mice protected female Quack&bush (Q) recipients against N. dubius infections; donor mouse strain influenced the degree of protection conferred and donor serum antibody titre related to the degree of stunting of worm growth in recipient mice. Five laboratory strains of mice developed different levels of protective immunity following multiple experimental infections with N. dubius. Antibody titres in these mice were strongly correlated with the percentage protection observed after l-4 infections: Q and CBA mice produced more anti-N. dubius antibody and were better protected than DBA/2, BALB/c and C3H mice. However BALB/c, C3H and CBA mice attained similar anti-N. dubius antibody titres after a single infection with N. dubius but serum from BALB/c gave better protection when transferred to female Q recipients than that from the other two strains. This suggested qualitative differences in the protective antibodies in sera between mouse strains. Five mouse strains were passively immunized with a uniform dose of serum from female Q donors: DBA/2 female recipients showed the least, BALB/c and C3H females were intermediate, and Q and CBA female mice attained the greatest level of passive protection against N. dubius. A close positive correlation existed between the degree of actively acquired and the level of passively acquired protection between the five strains of mice. mouse

genotype

on the passive

transfer

of protective

Passive serum immunity; Nematospiroides dubius; mouse CBA; C3H; DBA/Z; BALB/c mice; parasite growth; fecundity;

INDEX KEY WORDS: Quackenbush; multicolour;

INTRODUCTION IMMUNITY

TO

Nematospiroides

immunity

MATERIALS

dubius in mice can be

genotype; sex-ratio.

AND METHODS

Hosts and parasite. Wild

(W) mice were caught in suburban Brisbane gardens and bred in the laboratory. Outbred Quackenbush (Q) and inbred C3H, CBA, BALB/c and DBA/2 mice were obtained from the University of Queensland Central Animal Breeding House. Outbred Multicoloured (MC) mice were obtained from the Queensland Institute for Medical Research. All the mice were 7-8 weeks old when infected and were maintained under uniform conditions and fed pelleted food and water on demand. Nematospiroides dubius was maintained in Q mice. All the parasites used were from the 26th and 27th generation in this host. Infective N. dubius larvae were obtained from 7 day fresh faecal cultures, and used to infect mice following the procedures outlined in Dobson (1982). Collection of serum. Groups of 40 outbred female donor Q mice were given one, and seven infections with 100 N. dubius larvae. The mice given a single infection were exsanguinated 28 days after infection by cardiac puncture and killed by cervical dislocation; mice infected seven times were bled and killed 21 days after the seventh infection. Serum from female Q donors was previously shown to be

passively transferred with serum from immune mice. The level of protection attained following serum transfer related to the quality and quantity of immune serum injected and it influenced the survival, growth and fecundity of the parasite. Moreover the host environment regulated the manner in which the passively transferred serum influenced N. dubius so that immunotherapy was more efficacious in female than male mice (Dobson & Owen, 1978; Dobson, 1982; Dobson & Cayzer, 1982). It has also been shown that various laboratory strains of mice differ in their ability to develop protective immunity to N. dubius (see Wakelin, 1978 and Mitchell, 1979). The present investigation shows relationships between serum donor and recipient strains of mice in their ability to confer and develop protective immunity against N. dubius following passive immune serum transfer. 567

568

COLIN

DOBSON, PAUL J. BRINDLEYand PETRUS SITEPU

highly protective when passively transferred to female Q recipients (Dobson, 1982). Female W mice and female inbred DBA/Z, BALB/c, C3H and CBA outbred laboratory strains were infected with 100 N. dubius larvae, bled by cardiac puncture under ether anaesthesia and killed 28 days later. The blood for each strain and group within strains was pooled, the serum collected and stored at - 20°C. Serum from noninfected age-matched Q donors was collected at the same time and stored frozen. Serum was sterilized by filtration through 0.2 pm pore size membranes (Nuclepore, Pleasanton, California) before injection into mice. Saline for injection was sterilized in an autoclave. The intestine from each donor mouse was searched for parasites. Parasitological techniques. Mice were killed, searched for parasites and the worms treated according to the methods in Dobson (1982). Faecal egg counts were done by a modified McMaster method and expressed as eggs g-’ faeces (epg) and eggs per female per day (efd) (Brindley & 1981). Worm lengths were obtained from Dobson, drawings of worms using a map measurer calibrated in millimetres. Serology. Passive haemagglutination tests were done to assay mouse serum anti-N. dubius antibodies using the method outlined in Dobson (1982). Experimental design. Groups of five or six female Q mice were injected intraperitoneally (ip) with 3 ml sterile serum from wild and 2 outbred and 4 inbred laboratory strains of female mice which had been actively immunized against N. dubius. Groups of control mice were injected with 3 ml normal mouse serum or saline, one group of mice was given a sham injection and another group was retained without treatment. All the mice were infected 3 h after treatment with 100 N. dubius larvae and exsanguinated and killed 21 days after infection when the parasites were recovered, counted, sexed and measured. Faecal egg counts were made 14 and 15 days after infection. The experiment was designed to test variations in the protective potency of serum from mice of different genotype in Q female recipient mice infected with N. dubius. Three millilitres of sterile serum from female Q donors, which had been infected concurrently 7 times with N. dubius, was injected ip into 8-10 female Q, CBA, C3H, DBA/Z and BALB/c mice. Groups of treatment and no treatment control female mice in each strain were set up as before. All these mice were infected 3 h later with 150 N. dubius larvae. The parasitological procedures used in the previous experiment were followed. The experiment was done to show variations in the levels of protection attained following passive transfer of immune serum in recipient mice of different genotype following infection with N. dubius. Groups of Q, DBA/2, BALB/c, CBA and C3H female mice were infected 4 times with 100 N. dubius larvae, after terminating the previous infection with anthelminitic (162 mg kg-‘, ‘Nilverm’, I.C.I., Australia); reinfections were done at 24 day intervals and 3 days after anthelminitic treatment. Infected mice were bled for serology and killed 21 days after infection and the gut searched for parasites. The purpose of these infections was to show the antibody responses and the

degree of protection actively conferred following reinfection in each of 5 strains of mice. The experiments were repeated and the data pooled. Mathematics. Multitreatment experiments were analysed in an analysis of variance. Individual comparisons, where appropriate, were made using Student’s ‘t’ test (Snedecor & Cochran, 1968).

I.J.P. VOL. 12. 1982

RESULTS

Infections in serum donor mice There was no relationship between the numbers of worms recovered and the anti-N. dubius antibody titres 21 days after a primary infection in wild mice, and 2 outbred and 4 inbred strains of female serum donor mice. There were, however, differences between the numbers of worms recovered between the different mouse strains (Fig. lA, Table 1). Female Q donor mice given 7 infections with N. dubius did not harbour any worms and their pooled serum gave an anti-N. dubius antibody titre of log,

60 -

I

0

I

I

I

I

I

I

‘234567

I

8

log, antibody

I

I

9

ic

,I

I/

titre

FIG. 1. Relationship between worm burden and serum antiN. dubius antibody titres in mice infected with 100 larvae and killed 28 days after a single (A) and 21 days after multiple infections (B). TABLE 1-Nematospiroides dubius RECOVERED, AND SERUM ANTI-N. dub&s ANTIBODYTITRESFROMGROUPS OF 40 FEMALE MICE FROM 7 STRAINS 28 DAYS AFTER A PRIMARY INFECTION WITH

Mouse strain Wild DBA/Z BALB/c CBA C3H Multicolour Quackenbush

100

LARVAE

Worm number R SE 67 64 71 70 75 85 80

3 2 2 2 1 4 3

Log, serum antibody titre

R SE 9 3 5 6 5 5 3

0.4 0.4 0.3 0.2 0.4 0.4 0.2

Multiple infections in different strains of mice Protective immunity increased in 4 inbred and 2 outbred strains of mice over four infections; Q and CBA mice were better protected than DBA/Z, BALB/c and C3H mice (Table 2). There was a correlation between mouse strains in the degree of

Passive serum immunity to Nematospiroides dubius

I.J.P. VOL. 12. 1982 TABLE

I-Nemalospiroides dubius RECOVERED AND SERUMANTI-N. dubius ANTIBODY AFTER 1-4 INFECTIONS WITH looLARVAE AFTER ANTHELMINTIC TERMINATION

TITRES

FROM

FEMALE

MICE

OF FIVE STRAINS

PREVIOUS INFECTION

OFTHE

Log, Mice antibody titre X SE n

recovered after multiple infections Third Second Worm Log, Mice Log, Mice Worm number antibody number antibody titre titre X SE X SE n R SE X SE n

2 5 4 6 3

68 60 78 52 25

Parasites

Mouse

strain Mice

DBA/2 BALB/c C3H CBA Q

569

First Worm number

n

X

SE

51 59 53 20 21

12 12 81 75 80

2 2 2 3 3

TABLE FROM

0.5 0.5 2.0 0.6 I.1

19 10 27 37 29

3--Nematospiroides

dubius

3 7 2 4 4

FROM

DAYS

0.8 0.5 0.5 0.2 0.2

FEMALE

r: Accumulated Controls Wild Multicolour Quackenbush BALB/c DBA/Z C3H CBA

AFTER

INFECTIONS

Number Male-female sex-ratio R SE P SE

21

91

4

5 6 5 5 6 6 6

49 60 61 49 60 71 1-I

12 2 5 4 6 4 2

53 51 54 33 19

QUACKENBUSH

WITH

9 6 4 8 3

6 5 6 8 7

RECIPIENTS AND

1.1 0.9 O-3 0.3 0.3

OF

CBA

12 12 55 34 42

IMMUNE

58 42 62 22 8

Male R SE

Female P SE

Fecundity X 10m3 efd epg X SE X SE

0.1

20.7

0.5

32.1

5.0

5.3

0.7

0.58 0.81 0.78 0.92 0.66 0.73 0.88

5.1 6.7 7.1 6.6 6.9 6.6 6.3

0.05 0.2 0.2 0.3 0.5 0.2 0.1

15.5 18.6 18.3 18.2 17.1 16.8 17.4

0.2 0.6 0.3 0.6 0.8 1.1 0.5

7.0 3.3 29.0 7.2 18.4 2.3 16.6 3.7 21.1 4.4 32.9 6.4 30.2 3.8

1.5 4.9 3.7 3.9 3.5 5.4 4.5

0.2 1.2 0.3 0.6 0.3 0.7 0.4

Passive transfer qf serum from donor mice of differen t strain There were no differences between the numbers of worms, their lengths, sex-ratios and fecundities recovered from normal mouse serum, saline and sham injection treatment and no treatment control mice. These data were accumulated to give single control values (Table 3). Fewer and shorter worms which showed reduced male-female sex-ratios and fecundities were recovered from immune mouse serum recipients than from control female Q mice (F 49.1; 42.8 female, 20.2 male worms; 12.2; 4.9, df 1:58, P O.Ol< O+)Ol respectively). Donor mouse strain significantly influenced the degree of protection observed in

1,o 0.8 0.5 0.4 0.6

SERUM

7.3

protection observed, as a proportion of the primary infection, and the serum antibody titre 21 days after infection (r 0.8, o’f 18, P < 0.001). This correlation was most apparent for Q and CBA mice (r 0.98, 0.92 df 2, P < 0.02). In general there was a negative correlation between the numbers of worms recovered and the anti-N. dubius antibody titre in the serum from the donor and the experimentally infected mice (r - 0.73, df 26, P < 0.001) (Fig. 1B).

6 5 I 8 8

STRAINSOFMICE

0.95 0.06 0.09 0.03 0.09 0.13 0.04 0.08 0.09

7 11 4 5 3

100 LARVAE

Parasite Length mm

Treatment Mice

15 20 21 39 18

BALB/c,DBA/Z,C3H

WILD,QUACKENBUSH,MULTICOLOURED,

21

4 5 6 7 I

Fourth Worm Log, number antibody titre SE X SE x

immune mouse serum recipients measured in terms of reduced worm numbers, lengths and fecundities but not in terms of the sex-ratio of the parasites recovered (F 13.4; 2.9 female, 6.1 male worms; 4.3 1; 2.0; df 6:38; P 0~05-0.01 & N.S. respectively). Immune serum from wild and BALB/c donors protected female Q mice, measured as reduced worm burdens, to a greater extent than serum from Q, DBA/2 and MC mouse strains; serum from C3H and CBA donors was the least protective of all those tested (Table 3). There was a positive correlation between the percentage loss of worms and the mean percentage change in the sex-ratio, worm length and fecundity in the immunized compared with the control mice but the relationship was not statistically significant (r 0.55, df 5, P N.S.). There was no correlation between the anti-N. dubius antibody titre of the donated serum and its ability to protect mice against N. dubius infections except that the worm length decreased as the antibody titre of the immunizing serum increased (r 0.91, df 5, P < 0.01) and that serum from wild mice, with the highest anti-N. dubius antibody titre was the most protective among the sera tested, measured as reduced worm burdens, lengths, fecundities and sexratios.

570

1.1.~. VOL. 12. 1982

C~LIN D~BSON, PAUL J. RRINDLEVand PETRUSSKEW

TABLE 4-Nematospiroides dubius FROMFEMALEDBA/Z, CBA, BALB/c, C3H AND QUACKENBUSH(Q) RECIPIENTS OF IMMUNESERUMFROMFEMALEQUACKENBUSHDONORMICE 21 DAYS AFTERINFECTIONWITH 150 LARVAE -____-...__ Mouse

Treatment

Parasite Length mm Male Female R SE X SE

Mice

strain Numbers x SE

n

-______ DBA/2

Control Immune CBA Control Immune BALB/‘c Control Immune C3H Control Immune C? Control Immune ~______.___

--___ 33 8 33 IO 25 8 24 8 30 10 ___-.

96 9-I 105 68 107 93 112 95 108 50

5 6 4 4 5 4 4 5 3 4

Sex-ratio R SE

---....

0.93 0.92 O-87 0.93 0.88 0.87 0.90 0.98 0.99 0.72

__-.....-

0.05 0.05 0.05 0.06 0.05 O-04 0.06 0.07 0.05 0.09

Passive fransfer of serum to recipients of different strain Treatment of mice with saline or normal mouse serum did not influence and number, sex-ratio, lengths or fecundities of IV. dubius compared with similar data from infections in untreated controls within mouse strains. These data among the controls were pooled and expressed as single values within mouse strains (Table 4). There were significant differences between host strains in the numbers and lengths of worms recovered (F 4.4, df 4:130, P < O-01; 6.13 male and 2.6 female worms, df 4:6, P < O-01, < 0.05 respectively) but not in the sexratios and fecundities of the parasites from the trea~ent and no-treatment control mice. There was a positive relationship between the body size of the host strain in the control groups and the number of N. dubius recovered such that small mice like DBA/2 (21g) had fewer and shorter worms than large Q mice (36 g). The passive transfer of immune female Q strain serum to mice of five strains significantly reduced the number, lengths and fecundities of worms recovered compared with the homologous strain control mice (F91.5; 58.5 and 75.3 for male and female worms; 19.0; & 1:174, P < 0.01). The strain of the immune serum recipient mice significantly influenced the degree of passive protection achieved for each of the parasite parameters (F 4.6; 6.0 male 3.7 female worms; 42.9; df 4: 174, P < O-01 respectively) (Table 4). Passive immune serum transfer was highly protective in two mouse strains (Q, 54; CBA, 35%) intermediate in two (C3H, 15; BALB/c, 13%) but not protective in one (DBA/2, -1%). There appeared to be a direct relationship between peicentage protection in terms of worm number and the debilitative effect of the transferred serum on worm length, sex-ratio and fecundity between mouse

strains although the correlation (r 0.7, df 3, P N.S.). There

was a significant

was not significant

correlation

between

the

7.4 6.6 8.2 7.5 8.1 6.4 7-7 7.4 8.1 6.6

0.1 0.3 0.1 0.3 0.2 o-4 0.2 0.2 0.2 0.1

18.8 16.1 20.6 17.5 19% 13.7 19.6 18.3 20.1 15.1

0.7 1.2 0.4 0.3 0.7 0,9 0.6 0.6 0.8 0.7

Egg output efd

epg

K 24.2 28.5 26.1 15.8 56.4 31-8 39.0 27.0 20.4 6.5

SE X .----2.2 2.8 2.5 1.2 5.2 2.8 4.0 3.5 2.0 1.1

1.1 0.9 0.9 0.8 1.5 0.9 1.3 0.9 2.1 1.0

~__

SE 0.09 0.09 0.06 0.07 0.10 0.05 0.10 0.06 0.15 0.10

percentage protection achieved after two, three and four infections and the degree of passive protection afforded by female Q immune serum in the five strains of mice (r 0.92, 0.99 and 0.95, df 3, P < O-001 respectively) (Fig. 2). DISCUSSION

in innate immunity were shown among seven strains of female mice, after primary infection with Nematospiroides dubius when used as serum donors, in experimental infections or as controls in the passive serum transfer experiments. This, in part, Differences

Posslve protectIon in recipients,

%

FIG. 2. Relationship between the percentage protection actively achieved after 2 (e), 3(O) and 4 (0) infections with 100 N. dubius larvae compared with percentage protection against infections with 150 larvae after passive immunization with serum in five strains of mice.

I.J.P. VOL. 12. 1982

Passive serum immunity

to .Nemarospiroides

mice related to mouse size, such that smaller harboured fewer worms and additionally to the influence of mouse genotype restricting larval establishment (Brindley & Dobson, 1981). The levels of antibody detected in different mouse strains did not correlate with the size of the primary worm burdens. Wild mice, which harboured the fewest worms, had the highest anti-N. dubius serum antibody titres and there was a spectrum of antibody titres among the different strains which suggested that some strains of mice produced antibody against N. dubius better than others. Serum from wild mice and six laboratory strains of mice protected female Q recipient mice against N. dub&s when passively transferred 3 h before infection and reduced parasite survival, length, fecundity and mate-female sex-ratio compared with the treatment and no-treatment controls, but not always to the same extent. The data suggested that there was a relationship between the titre of antibody transferred and the degree of passive protective immunity similar to that reported by Dobson (1982) but a significant positive correlation was only shown between donor anti-N. dubius antibody titre and percentage reduction in parasite length. Nevertheless, a strong positive relationship was evident between anti-N. dubius antibody titre and active protective immunity in terms of number of worms recovered after single and multiple experimental infections in wild and six laboratory strains of serum donor mice. N. dubius reacted differently in response to passively transferred sera from each mouse strain in female Q recipients assessed as a reduction in numbers, lengths, fecundities and sex-ratio of the parasites. This may reflect the mode of action of the transferred antibodies against N. dubius. The antibodies may act directly to inhibit larval establishment, growth and fecundity (Dobson & Cayzer, 1982) or indirectly to reduce the survival and biological efficiency of the larvae and adults by activating the afferent arm of the immune response (Dobson, 1982). experimental multiple infections with N. dubius showed that some among five mouse strains were better protected than others against challenge infections despite the production of significant antiN. dub&s antibody titres: Q and CBA mice produced more anti-N. dubius antibody and were better protected than DBA/2, BALB/c and C3H mice. However BALB/c, C3H and CBA mice attained similar anti-N. dubius antibody titres after a single infection, but the primary infection serum from BALB/c mice gave better protection when transferred to female Q recipients, than that from the other two strains. Thus qualitative as well as quantitative differences in the serum antibodies may have contributed to the differences between strains in the protective potencies of their sera. This conclusion was supported by the observation that the anti-N. &b&s antibody titre in five mouse strains with

dubius

571

different levels of protective immunity after four infections only differed by two log, serial dilutions. Furthermore Brindley & Dobson (1982) found that antisera from mice, selected as liable to primary infection with N. dub&s, unlike that from refractory mice, failed to passively immunize female recipients against N. dubius despite a high anti-N. dubius antibody titre and attributed this failure to the poor protective qualities of the antibodies produced. Strain variation in antigen recognition as well as antibody synthesis could also account for the differences in active and passive protective immunity reported here between genetically different mice infected with N. dubius. When five strains of mice were passively immunized with a uniform dose of immune serum from female Q donors the degree of protection conferred was influenced by strain such that DBA/2 female recipients developed the least, BALB/c and C3H were intermediate, and Q and CBA mice attained the greatest level of protection. Moreover a close positive correlation was demonstrated between the degree of actively acquired protection in each mouse strain after each of two through four experimental infections and the level of passively acquired protection conferred in each strain by immune serum. Thus passive immunization estimated the potential of each strain to mount a protective immune response to N. dubjus infection and because the Q donor immune serum had a known high protective potency in Q recipients, this suggested that the afferent arm of the immune response was deficient in some strains of mice against N. dub&s. Cross (1960) pointed out that rodents which were naturally refractory to primary N. dubius infections developed intense inflammatory reactions and that reduction of these responses, following treatment with cortisone, allowed the parasite to complete its life cycle in these hosts. The importance of efficient reticuloendothelial function had also been implicated in the protection of mice against N. d~bius by Liu (1966) and Cypess, Lucia, Zidian & Rivera-Ortiz (1977). Spurlock (1943) found splenomegally in mice following infections with N. dub&s which suggests that the spleen is involved in immunity against this parasite, particularly since Cypess (1970) found that spleen cells from infected mice would passively immunize recipient mice against N. dubius. More recently Chaicumpa & Jenkin (1978) showed that macrophages, activated by a trypsin-labile substance thought to be specific antibody adhered to and damaged larval N. dubius in vitro and in artificially induced intraperitoneal infections in mice. Splenomegally may thus result from a mobilization and proliferation of macrophages and other reticuloendothelial cells involved in protective immunity against N. dubius in the gut. It is interesting that DBA/Z mice, which failed to exhibit, to an appreciable extent, either

572

COLIN DOBSON. PAUL J. BRINDLEYand PETRUS SITEP~J

active or passive protective immunity in terms of worm survival, produced significant titres of anti-N. &&us antibody and that the worms recovered from actively and passively immunized DBAi2 mice were stunted and showed reduced fecundity. It is possible that these results illustrate the direct effect of antibodies on the parasite in the absence of an antibody mediated cellular response against N. dubius. Protective immunity against N. dubius in mice appeared to result from the direct effects of antibodies reducing larval infectivity and reducing the biological efficiency of surviving worms and further, from the indirect effects of antibody mediated cellular reaction killing the developing larvae in the tissues of the gut and inducing expulsion of some of those parasites which survived to return to the gut lumen as adults. Strain variations that exist among mouse strains in their liability to infection and their ability to develop protective immunity to Nematospiroides dubius infections were clearly demonstrated by the results of the present experiments. Moreover the variations outlined were similar to those reported by other workers for certain of these mouse strains (see Spurlock, 1943; Liu, 1966; Cypess & Zidian, 1975; Behnke & Wakelin, 1977; Prowse, Mitchell, Ey & Jenkin, 1979). For example, C3H mice were highly susceptible to infection with ff. dub&s and were moderately protected against a challenge infection while BALBlc mice were less susceptible to primary infection and developed a greater degree of protective immunity than C3H mice. These results compare with those of Liu (1966), Cypess & Zidian (1975) and Behnke & Wakelin (1977), but they contrast with the work of Prowsc et al. (1979). In this latter case the differences in immunity achieved within the strains of mice may relate to the manner and route of immunization (Behnke & Wakelin, 1977) and variations in the strains of N. dubius used (Hepler & Leuker, 1975; Dobson & Owen, 1977). Acknowledgemenls-This

work was supported by a grant

from the Australian Research Grants Committee and a Commonwealth Postgraduate Award held by P. J. B. and a Colombo Plan Award held by P. S.

REFERENCES BEHNKE J. M. & WAKELIN D. 1977. Nematospiroides dubius: stimulation of acquired immunity in inbred strains of mice. Journal of Helminthology 51: 167-175. BRINDLEY P. J. & DOBSON C. 1981. Genetic control of liability to infection with Nematospiroides dubius in mice: selection of refractory and liable populations of mice. Parasitology 83: 5 I-65. BRINDLEY P. J. & DOBS~N C. 1982. Multiple infections with Nema~ospirojdes dubius in mice selected for liabi-

I.J.P. VOL. 12. 1982

iity to a single infection. Australian Journal of Experimental Biology and Mpdjcaf Research 60: 319-327. CHAICU~~PA R. & JENKIN C. K. 1978. Studies in vitro on the reaction of peritoneal exudate cells from mice immune to infection with Nemafospiro~des dubius with the infective third stage larvae of this parasite. Australian Journal of Experimental Biology and Medical Science 53: 61-68. CROSS J. H. 1960. The natural resistance of the white rat to Nematospiroides dubius and the effect of cortisone on this resistance. Journal of Parasitology 46: 175-185. CYPESS R. 1970. Demonstration of immunity to Nematospiroides dubius in recipient mice given spleen cells. Journal of Parasitology 56: 199-200. CYPESS R. H., LUCIA H. L., ZIDIAN J. L. & RIVERAORTIZ C. I. 1977. He~~gmosomoides pofygyrus: temporal, spatial and morphological population characteristics in LAFt/J mice. experimental ParasitoIogy 42: 34-43. CVZWS R. H. & BDIAN J. L. 1975. Heiigmasomoides polygyrus ~Nemafospiroides dub&s): the development of self-cure and/or protection in several strains of mice. Journal of Parasitology 61: 819-824. DOBSON C. 1982. Passive transfer of immunity with serum in mice infected with Nematospiroides dubius: influence of quality and quantity of immune serum. International Journal for Parasitology 12: 207-213. DOBSON C. & CAYZER C. J. R. 1982. Passive transfer of immunity with serum in mice infected with Nernafospiroides dub&s: in vitro effect of immune serum on larval infectivity. International Journal for Parasitology 12: 413-421. DOBSON C. & OWEN M. E. 1977. influence of seriai passage on the infectivity and immunogenicity of Nematospirojdes dubius in mice. ~nternafiona~ Journal for Parasitology 7: 463-466. DOBSON C. & OWEN M. E. 1978. Effect of host sex on passive immunity in mice infected with Nematospiroides dubius. International Journal for Parasitology 8: 359364. HEPLER D. 1. & LEUKER D. C. 1974. Enhancement of virulence and immunogenicity of Nematospiroides dubius. Journal of Parasitology 60: 1057-1058. Lru S-K. 1966. Genetic influence on resistance of mice to Nematospiroides dub&s. Experimental Parasitology 18: 311-319. MITCHELL G. F. 1979. Responses to infection with metazoan and protozoan parasites in mice. Advances in ~~7~7z~~o/o~y 28: 45 t-51 1, PROWSE S. J., MITCHELL G. F., EY P. L. & JENKIN C. R. 1979. The development of resistance in different inbred strains of mice to infection with Nemafospiroidesdubjus. Parasite Immunology I: 277-288. SNEDECOR G. W. & COCHRAN W. G. 1968. Statistical Methods. 6th Ed. Ames, The Iowa State University Press. SPURLOCK G. M. 1943. Observations on the host-parasite relations between laboratory mice and Nematospiroides dubius Baylis. Journal of Parasitology 29: 303-311. WAKELIN D. 1978. Genetic control of susceptibility and resistance to parasitic infection. Advances in Parasitology 16: 219-308.