Influence of serial passage on the infectivity and immunogenicity of Nematospiroides dubius in mice

Influence of serial passage on the infectivity and immunogenicity of Nematospiroides dubius in mice

Internutionol Journul /or Prrru~ito!o.qy. 1977. Vo!. 7. pp. 463-466. Per,pamon Press. Printed irr Great Briloin INFLUENCE OF SERIAL PASSAGE ...

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Vo!. 7. pp. 463-466.


Press. Printed

irr Great



of Parasitology,

University of Queensland, (Received

4 March

St. Lucia, Brisbane, Qld. 4067, Australia 1977)

Abstract-DossoN C. and OWEN M. E. 1977. Influence of serial passage on the infectivity and immunogenicity of Nen afospiroides dubills in mice. Ifiternatiocal Journal for Parasitology 7: 463-466. The infectivity of Nematospiroides dubius was increased for Quackenbush (Q) mice by ten serial passages through this host. At the same time GH mice became more refractory to infection with successive Q generations of the parasite. Both strains of mice rejected the most highly selected parasites more readily than parasites from the earlier generations. These responses were shown to be immunological in nature by infections in hypothymic Balb/c CBA nu/nu and nu/+ mice and to be dose dependent. The selection of N. dubius for increased infectivity in Q mice enhanced its immunogenicity in this and other mouse strains possibly because of increased genetic homogenicity in the selected populations. N. dubius selected for Q mice showed a degree of immunological adaptation to Q but not to GH mice. INDEX KEY WORDS: Nemarospiroides genetics: infectivity; immunogenicity.

INTRODUCTION ?Vematospiroides duhius is a common parasite of microtine and to a lesser extent murine rodents. In general however mice are highly susceptible to infection with N. dubius but variations in the levels of infection attained in different host genotypes have frequently been reported (Spurlock, 1943; Liu, 1965, Hepler & Lueker, 1974). Because of this it appeared possible to select parasite subpopulations which have different degrees of infectivity in various mouse strains. The present study investigates the influence of serial passage of N. dub& through outbred Quackenbush mice on the infectivity and survival of successive generations in this and other mouse strains.





Elrperimental infections. All the mice used were obtained from the Central Animal Breeding House, University of Queensland. The Quackenbush (Q) mice were from an outbred colony. The GH were inbred and together with the Balb/c CBA nujrzu and nu/+ mice were kept specific pathogen free until infected. The recessive gene nude (nlc) is associated with hairlessness and congenital aplasia of the thymus in mice (Pantelouris, 1968). All the infected mice were kept in the Department of Parasitology under the same environmental conditions and were fed rodent cubes (Queensland Institute of Medical Research formula) and water on demand. Nematospiroides dubius was obtained from Dr. D. C. Jenkins, Department of Microbiology, University of Adelaide. Stock infections were maintained in 7-week-old Q mice and for the purposes of these experiments passaged using larvae culture from eggs voided 2-3 weeks after infection. The infective larvae were

CjH, Balb/c CBA II~L/~IImice;

obtained from cultures prepared by streaking washed faecal slurries on to two layers of filter paper in Petri dishes which were then completely moistened and kept at 23°C in a dark humidity incubator. The cultures were aired each day and the larvae collected in water after 7 days, cleaned and concentrated by sedimentation. Larvae were stored at 4°C in a shallow layer of water in sealed bottles for 2 weeks before being used to infect mice. Each mouse was infected at 7 weeks of age with 100 N. dubius larvae suspended in about 0.2 ml water using a 1 ml tuberulin syringe with a blunt 18 gauge needle. Larval suspensions were adjusted to this dilution after counting larvae in ten 0.05 ml aliquots from a stock suspension. The dilutions of larvae was assessed again in the same way after adjusting the larval concentration to 100/0.2 ml. The error in sampling was “3% on counts done on the larval suspension before and at the completion of infection. Infections were terminated after II, 21 and 28 days. Thg mice were killed by cervical dislocation and the gastrointestinal tract stripped from its mesenteries and placed in 6.15 M-saline in a Petri dish. The gut was opened along its whole length and individual worms counted and removed with forceps under a disecting microscope. Experimental design. Ten successive generations of N. dubius were raised by infecting groups of 10 male and 10 female 7-week-old Q mice with 100 infective larvae at intervals of 5 weeks. The parasite progeny from each generation were also used to infect groups of at least ten age matched (7 weeks) Q and GH strain mice. These animals were killed after 11, 21 and 28 days and the total number of worms counted in each mouse. These data were used to show the influence of serial passage on the survivial of N. dubius by calculating linear regressions on worm burdens against parasite generation number. Further infections using 100,110,120 and 150 larvae from the eleventh generation through Q mice (Qll) were set up in groups of at least IO, 7-week-old Q mice to assess the influence of larval dose on the survival of the parasite



464 after 28 days infection.

The final experiments were done to show the infectivity of Q11 N. duhius in 7-week-old Q mice compared with that in age matched hypothymic SPF Balb/c CBA nu,Inu and SPF Balb/c CBA nu] + mice. Each mouse was infected with 150 larvae. All the data was treated mathematically to obtain linear regression lines, correlation coefficients and where appropriate ‘I’ test comparisons between experimental groups.



7. 1977

(t 28 1 P
RESULTS The infectivity of N. duhius for Q mice increased after ten serial passages through this host strain (r=0.246 and 0.345 PcO.01) but decreased in CsH mice (r= -0.354 and -0.419 P
DlSCUSSlOlV The infectivity of nematode parasites can be increased by serial passage through selected hosts (Herlich, Douvres & Isenstein, 1956; Haley, 1962). Moreover parasite variants have been described for nematode species which have arisen through isolaC,H



60 .I doys


r =0.246








21 days



r= 0.305


0 Infection






28 days InfectIon r=-0.492



o fi

“= 96

FIG. 1. The effect of repeated passage through Quackenbush mice on the survival of Nenmtospiruidcs ~l&l~.~ in Quackenbush and CJH mice infected with 100 larvae for 11, 21 and 28 days. l male. ’ female mice.


7. 1977

Nematospiroidesduhirrsin mice

8 0

FIN. 2. Effect of larval dose on the survival of Nematospiroides dub&s passaged eleven times through Quackenbash mice 28 days after infection in this host. l male, 0 female mice. tion (Le Jambre & Whitlock, 1973), after chemotherapy (Le Jambre, Southcott & Dash, 1976) or after adaptation to an immunological response (Ogilvie & Jones, 1971). The present results show that the infectivity of Nematospiroides dubius for Q mice can be increased by selection following serial passage through this strain of host. The increase was small (6%) but significant, and was commensurate with the high initial infectivity of N. dubius for the mouse and the small number of generations involved, it represented 38% of the possible increase. Moreover the change in infectivity was specific for Q mice because inbred SPF C&i mice became progressively more refractory to infection, showing greater variation in worm recoveries than Q mice, with successive Q generations of N. duhius. These results equate with the influence of sampling and environmental selection pressures reducing TABLE



genetic variation between parasites and increasing their fitness for Q mice. it is unlikely that the immuile response of the host influenced the seiection process because the eggs used to culture each generation of infective larvae were voided between 14 and 21 days after infection before the onset of rejection of adult N. da&us. This may have importance because the ability of Q and CJH mice to reject each successive generation of N. dubius 28 days after infection increased dramstically. The loss of significant numbers of adult N. dubius at this time was shown to have immunological basis by infecting hypothymic SPF Balb/c CBA cross nu/nu mice. The recessive gene nude (nu) is associated with hairlessness and congenital aplasia of the thymus in mice (Pante~ouris, 1968). Mice homozygous for n~~~~ show profound impairment of cell-mediated immune responses against various antigens (Rygaard, 1959) and do not exert immune responses which will eliminate a number of gastrointestinal parasite species (Ruitenberg & Steernberg, 1974). Information from various sources indicate that immunity to gastrointestinal nematodes in rodents is elicited through a thymus dependent mechanism (see Larsh & Weatherly, 1975). Therefore the failure of nujnu mice to reject Ql I N, dubius after 28 days infection while their nlr/--I-- sibling counterparts did so demonstrated the immunological basis for this reaction. Thus the selection of N. ~~~~j~~ for increased infectivity in Q mice enhanced its immunogenicity in this and other mouse strains which possibly relates to the genetic homogeneity of the selected populations. This suggests that the greater rejection of selected N. dubius in Q and CjH mice may depend on elevated immune responses to increased concentrations of structurally uniform immunogens from these parasites. Increasing the dose of Ql 1 N. dubius given to Q mice enhanced the ability of these mice to reject the parasite 28 days after infection which supports this hypothesis. It is also possible however that selected parasites are not able to control the effects on themselves of the immune response they elicit. The N. du~iu.~/mouse mode1 is not unique in demonstrating this phenomenon. Tr~c~ostru~gy/u~~ co/lfbri~rmi~ selected for infectivity is guinea pigs are rejected more readily by this host than by sheep

Nematospiroidesdubius RECOVERED WITH


Mouse strain -~_-


Balb/c CBA ilU/ -t

male female

n 10 10

BaIbiCBA nu/nu Quackenbush

male female male

10 10 IO

female IO *X. mean: SE. standard error.



Numbers of worms recovered at 21 days n x S.E. 132+.4.5 10 11514.2 IO 11913.3 13812.4 10 13612.9 138 i-I.5 134+2.8 10 139-2.2 134;3,5 10 135+3+j 132--2.4 120+3.9 10 131: 3.2

II days S.E. x* S.E. Y _~____ __I-..134++9 I 275-3.5





intervals after infection 28 days n x S.E. x S.E. x Y.t. _..__.___-._. 123 -!::3.6 IO 103 T4.9 952 3.7 IO 86 4~4.2 13611 1.9 4 135 54-9 135 i 2,9 4 134 i-3.8 127-z 3.1 IO 10813.7 IO4i 2.6 10 [email protected] 3.4 -



in which the parasite is found naturally (Herlich et nl., 1956). A comparison of data from early studies on ~~~~us~~~~gy~~{~brasi~~e~s~s in rats (Africa, I93 I ; Chandler, 1932) with results from more recent studies (see Qgilvie & Jones, 1971) provides circumstantial evidence that selection has increased the immunogenicity and reduced the longevity of this parasite in rats. Graham & Porter (1934) very early reported that a 3-year-old strain of N. hrasiiiensis from Texas passaged through laboratory rats produced fewer eggs per worm than either of two strains isolated from wild rats in Baltimore, Maryland. Moreover Haley & Parker (1961) showed that the longevity of N, hrasiliensis passaged through Sprague-Dawley rats for many years was strongly dose dependent, fewer worms surviving for a shorter time from large compared with small doses of larvae during primary infections in this host strain. These results are similar to those reported here. The host-parasite relationship has been considered to evolve immunologically towards a state of tolerance either by a reduction of antigenic stimulation by the parasite (Sprent, 1962; Damian, 1962; Dineen, 1963) or by specific unresponsiveness of the host in spite of the presence of parasite antigens (Sprent, 1969). The present data indicate that parasites become more infective for a given host at the risk of enhancing the immune response against them; this appears contrary to the initial premis of Sprent (1962). Nevertheless the rejection of N. ~~~~~~.~ by C3F-l was greater than that in Q mice which implies that a degree of adaptation towards antigen complementariness has occurred in the parasite to the Q mouse strain. There are obvious survival advantages to the parasite to adapt towards increased infectivity for their host despite the enhanced immune responses they may elicit because they still may counteract this by masking themselves with host components or by depressing the immune response they engender (Brown, Crandall & Crandall, 1976).





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