The Stimulus From the Animal

8/2/2019 The Stimulus From the Animal

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The Physiology of Infective Processes of Nematode Parasite; the Stimulus from the AnimalHostAuthor(s): W. P. RogersSource: Proceedings of the Royal Society of London. Series B, Biological Sciences, Vol. 152, No.948 (Jun. 14, 1960), pp. 367-386Published by: The Royal SocietyStable URL: http://www.jstor.org/stable/75342 .

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lost so that the parasite would be dependent on the host to replace it (figure 1).For instance, the infective stage, lacking part of this system for restarting develop-ment, would remain in a 'resting' condition until, in the right host, the missingcomponents were provided.

The host might function by providing: (I) a stimulus which causes the infectivestage to produce the internal secretions; or (II) by providing the substances whichreplace the missing internal secretions; or (III) by producing substances whichcause exsheathment or hatching by their direct action on the sheath or egg shell.The strongest argument against (III) stems from the fact that the hosts do notnormally produce the chitinases or the particular proteases needed to break down

infective eggorlarva

( Ihlost . .- > internal I secretion(II)

(III)hatching orexsheathingfluid(chitinase,esterase,protease)

FIGURE 1. Host-parasite relationships in the process of infection. The host may start infectionby providing an environment (I) which stimulates the infective agent to produce,directly or indirectly, hatching or exsheathing fluids which contain certain enzymes.There is no evidence, as yet, to show that the host may function as in (II) or (III).

the outer coverings of most eggs or infective larvae. It is possible that a host couldproduce substances which play a highly specific role in the physiology of a parasiteas suggested in (II). Indeed the unsaturated lactone which serves as the 'hatchingfactor' for Heterodera rostochiensis (Calam, Todd & Waring I949; Ellenby &Gilbert I957) might act in this way. There is, however, no evidence to supportthis view. The suggestion (I) that the host stimulates the production of a fluidwhich completes the process of moulting (exsheathment) in infective larvae oftrichostrongyle parasites of sheep was made by Rogers & Somerville (I957) whoused the rumen fluid of the host to stimulate the larvae to produce exsheathingfluid. Rogers (I958) gave more details of the chemistry of the stimulus whichinduced infective eggs of Ascaris lumbricoides to produce a 'hatching fluid'which contained the enzymes which attacked the egg shell.In this paper the components of the stimulus which cause the exsheathment ofinfective larvae of Trichostrongylus axei, T. colubriformis, Haemonchus contortus,and the hatching of infective eggs of Ascaris lumbricoides (pig strain), Ascaridia galliand Toxocara mystax are given. Trichostrongylus axei and Haemonchus contortuscommonly exsheath in the rumen of the host though the adults live in theabomasum. With Trichostrongylus colubriformis exsheathment takes place in theabomasum, and the adults live in the small intestine. The eggs of Ascaris lumbri-coides, Ascaridia galli and Toxocara mystax hatch in the small intestine of the pig,fowl and cat, respectively, where the adults also live.

368 W. P. Rogers


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Infective processes of nematode parasitesMost of the experiments which are described here were carried out to examinethe three chief components of the stimulus. To make the interpretation of resultseasier the less important factors which enhanced activity were seldom added.Thus the activity reported for many experiments was less than that which could

have been obtained under ideal conditions in vitro.When infection occurs, the processes of stimulating the infective stages toproduce exsheathing and hatching fluids, and the enzymic breakdown of thesheath or egg shell, take place in the one medium. Nevertheless, it is necessarythat these two processes should be studied separately, otherwise it would not beknown if a given condition was affecting the stimulus or the enzymes. In this work,it was convenient to carry out most of the experiments so that both processes tookplace in the one medium. From the results so obtained more critical experimentswere carried out in which the second process, during which the enzymes in theexsheathing and hatching fluids attacked the sheaths and egg shells, took place inbuffer or water. In this way it was found that the major components of thestimulus normally had little effect on the second process.

MATERIALS AND METHODSLarvae were obtained from cultures of faeces from sheep infected with Tricho-

strongylus axei, T. colubriformis or Haemonchus contortus. Eggs were dissectedfrom the posterior 2 cm of the uteri of Ascaris lumbricoides. Ascaridia galli andToxocara mystax were incubated for 24 h in saline and the eggs laid during thisperiod were collected.The eggs of Ascaris lumbricoides were washed for 2 h in 0-5 N-sodium hydroxideat room temperature (Fairbairn 1955); the eggs of the other species were washedfor 10 min. Thereafter they were incubated at 28 ?C either in 01 N-sulphuric acidor in 1 % formalin. The eggs were either shaken continuously throughout theincubation period or else they were kept in layers less than 2 mm deep.Larvae were stored in water at 5 ?C. They were about 2 to 3 weeks old whenused. To estimate the degree of exsheathment larvae were mixed with Lugol'siodine and examined with a microscope. A larva which had emerged even partlyfrom the sheath was regarded as exsheathed. At least 100 larvae were counted foreach experiment; those which obviously had been dead for some time were notcounted.

After incubation for 20 days at 28 ?C eggs were stored in 0-lN-sulphuric acidor in water at 5 ?Cuntil they were needed. It was best to use eggs of A. lumbricoideswithin 10 days otherwise the results were sometimes variable. The eggs of theother species could be stored for 30 days before use.The proportion of eggs which had hatched in an experiment was measured bycounting at least 100 of the eggs or larvae with a microscope. If the larva was freefrom the egg, or even if the embryo had emerged sufficiently to break the innermembrane of the egg, it was considered to be hatched. Unembryonated eggs werenot counted. It was necessary to mix the eggs and larvae to be counted with athick solution of methyl cellulose before placing the coverslip in position. Otherwisethey were distributed unevenly.

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Bicarbonate-carbon dioxide buffers (Umbreit, Burris & Stauffer 1957) werechecked with a glass electrode. In early experiments gas mixtures were passedover copper turnings at 400 ?Cbefore use. The removal of traces of oxygen in thisway did not affect the results and in later experiments gas was used directly as itcame from cylinders.Solutions of cysteine and ascorbic acid were brought to the appropriate pHwith sodium hydroxide. Sodium dithionite (British Drug Houses) was dissolvedin oxygen-free water under mixtures of nitrogen and carbon dioxide immediatelybefore use. The solutions of sodium dithionite were added, under nitrogen ornitrogen and carbon dioxide, to the rest of the system after it had been gassed toremove oxygen. In this way the formation of acid in the solutions was kept lowand the buffers were not disturbed appreciably.The sum of the concentrations of undissociated carbonic acid and dissolvedgaseous carbon dioxide in the bicarbonate-carbon dioxide buffers was calculatedwith the formula

[H2C03][+ 1 ( + [1 )1 Pco255where [12CO31 = the molar concentration of undissociated carbonic acid plus

gaseous dissolved carbon dioxide;K- = Henry's law constant;Pco,

= the partial pressure of carbon dioxide; in the gas phase in mmof mercury;and K, and K2 = the first and second dissociation constants, respectively.The effect of ionic strength on the dissociation constants was neglected. Thevalue of pK, at 37 ?Cwas taken as 6-317 (Shedlovsky & Maclnnes I935) and thecorrection at maximum ionic strength (pH 7.3, Pco, 285 mm of mercury, 0-05 M-sodium chloride, 004 M-sodium dithionite) would have been less than 0-03(Umbreit et al. I957). The consequent change in K, would have corrected an errorof less than 4%.

The solubility of carbon dioxide in dilute hydrochloric acid was taken fromgraphs of Van Slyke, Sendroy, Hastings & Neill (1928). For calculating the totalcarbon dioxide in solution it was assumed that the carbonic acid was undissociatedwhen the pH was below 3.The effects of varying the period the larvae were in the stimulating mediumwere examined. After exposure to the medium at 37? C, chilled water or 1 x 10-3ir-magnesium chloride in 001 m-phosphate buffer at pH 7 2 was added and the larvaewere concentrated by centrifuging. This process was repeated three times afterwhich larvae were incubated for a further period in water or buffer. Exsheathmentwas measured after both periods of incubation. Similar experiments were carriedout with eggs. After exposure to the stimulus, however, the eggs were washed inwarm saline and then incubated.

Unless otherwise stated experiments were carried out at 37 ?C and the periodof incubation was 3 h.

W. P. Rogers70


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Infective processes of nematode parasites 371RESULTS

Some results from preliminary experiments are shown in table 1. From theresults it appeared that carbon dioxide was a necessary part of the stimulus.Sodium bicarbonate alone was not sufficient but some activity could be obtainedwithout sodium bicarbonate if carbon dioxide were present. It seems then, thatthe undissociated carbonic acid or the dissolved gaseous carbon dioxide ratherthan carbonate or bicarbonate ions was a component of the stimulus. It has notbeen possible to separate the actions of these un-ionized components nor havetheir separate concentrations been calculated. For convenience, therefore, theundissociated carbonic acid referred to in the rest of this paper will be regarded asincluding the dissolved gaseous carbon dioxide.

TABLE 1. THE GENERAL EFFECT OF GASES, PH, ANDREDUCING AGENTS ON LARVAE AND EGGS

exsheathment of larvae

composition of mediumgas phase pHair 6-0N2 6-05% C2-N2 c. 605% C02-N2 c. 6-05% CO2--N2 6-05 % C02-N2 6-0

5% C02-N2 805% C02-N2 8-0N2 c. 8-3N2 c. 8-3

(%)reducing Haemonchusagent contortus- 0

+ 0- 1+ 0- 3+ 14- 3+ 5- 0+ 0

Tricho-strongylusaxei

004655422103

hatching of eggs (%)Toxocara Ascarismystax lumbricoides

0 00 01 13 15 616 3840 762 640 00 0

The reducing agent was 0O02M-sodium dithionite except for Toxocara mystax for which 0O02M-cysteinewas used

Activity was low when the nitrogen in the gas phase was replaced by air oroxygen. Reducing agents under nitrogen without carbon dioxide had littleactivity. Their main action seemed to be the enhancement of activity due to theundissociated carbonic acid. The pH was important because it affected the con-centration of undissociated carbonic acid; but it seemed to have other actions also.Sodium chloride, wetting agents and blood serum, which are not shown in table 1,affected activity slightly.

(1) The action of undissociated carbonic acid(a) Experiments with larvae

The exsheathment of larvae of Haemonchus contortus, Trichostrongylus axeiand T. colubriformis was examined in sodium bicarbonate-carbon dioxide bufferswith and without 0-02M-sodium dithionite. The gas mixtures consisted of nitrogen

'buffer'phosphatephosphatephosphatephosphatebicarbonatebicarbonatebicarbonatebicarbonatebicarbonatebicarbonate


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372 W. P. Rogerscontaining 1, 2-5, 5, 10, 20 and 40 % carbon dioxide (vol./vol.) and the concentra-tions of sodium bicarbonate were chosen to give pH 6*0, 6.9, 7-3, and 8*0 for allconcentrations of carbon dioxide except 40 % for which pH 7 3 was the highest used.

80- -?

c ~60 / /

rS f I

40-6, 40

)20 .

0 2 46[H2CO3]x 10M

FIGURE 2. The effect of different concentrations of undissociated carbonic acid on the ex-sheathment of larvae of Trichostrongylus axei. Bicarbonate-carbon dioxide buffersunder nitrogen containing carbon dioxide at different partial pressures were used.Sodium dithionite, 0.02M, was present in experiments which gave the results for theupper curves; 0.05M-sodium chloride was present in all experiments. El, I, pH 7.3;0, 0, pH 6-0.

o60-

1 40-

20-

l{ . i I I ,....0 2 4 6[H2CO3]x 103M

FIGURE 3. The effect of different concentrations of undissociated carbonic acid on the ex-sheathment of larvae of Haemonchus contortus. Bicarbonate-carbon dioxide buffersunder nitrogen containing carbon dioxide at different partial pressures were used.Sodium dithionite, 0.02M, was present in experiments which gave the results for theupper curves; 0-05N-sodium chloride was present in all experiments. l, S, pH 7.3;0, e, pH 6.0.


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Infective processes of nematode parasites 373The results from one set of experiments with T. axei and Haemonchus contortusat pH 6*0and 7-3 are shown in figures 2 and 3. Some variation was obtained withdifferent lots of larvae; in particular, older larvae were more sensitive to un-dissociated carbonic acid. However, the general relationships shown in figures 2and 3 were consistent.Never more than 18 % of Trichostrongylus colubriformis exsheathed under theseconditions (table 2). Activity was greatest at the lowest pH. For this reason, andbecause T. colubriformis normally exsheaths in the abomasum, experiments were

TABLE 2. THE EXSHEATHMENT OF LARVAE OF TRICHOSTRONGYLU SCOLUBRIFORMIScomposition of the medium

sodiumchloride, cysteine, exsheathed

pH gas phase 0-1M 0-02M (%)1.6 air - - 0(HC1)1.6 air + - 4(HC1)1'6 N2 + - 1(HC1)1-6 N2 + + 7(HC1)1-6 20%C02-N2 + - 71(HCI)1*6 20%C02-N2 - - 21(HC1)1.6 20%CO2-N2 + + 62(HC1)6'0 20 % CO2-N + - 12(NaHCO3)6.0 20%CO2-N2 + + 18(NaHCO,)

conducted in 0-025N-hydrochloric as shown in table 2. Exsheathment was en-hanced by carbon dioxide in the gas phase so the effect of varying the concentra-tion of undissociated carbonic acid was examined by using mixtures of nitrogenand carbon dioxide (figure 4). This experiment was only repeated once; greatestactivity, 91 %, was again obtained at 4-8 x 10-3vr.(b) Experiments with infective eggs

The hatching of eggs was examined in sodium bicarbonate under nitrogen ormixtures of nitrogen and carbon dioxide within the range pH 6-0 to 8.3 with andwithout reducing agents. Some of the results are shown in figure 5 and tables3 and 4. With Ascaris lumbricoides the optimum concentration of undissociatedcarbonic acid was about 0-25 to 0-5 x 10-3 at pH 7-3 and about 1 to 2 x 10-3M atpH 6-0. At high concentrations no eggs hatched. At a given concentration ofundissociated carbonic acid and pH the addition of reducing agents alwaysincreased activity. The eggs of the other two species, which were examined in two


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experiments only, needed somewhat lower concentrations of undissociated carbonicacid.The results of these and other experiments with the eggs and larvae showedcertain features about the action of the undissociated carbonic acid.

40-

? 20I

1 I J l l l l l f I0 2 4 6 8 10[H2CO3] x 103M

FIGURE 4. The effect of different concentrations of undissociated carbonic acid on exsheath-ment of larvae of Trichostrongylus colubriformis. The medium consisted of 0-025Nhydrochloric acid, 0 05M-sodium chloride under mixtures of nitrogen and carbon dioxide.

I ti40-I

XeAg I / \o I / \, 20+- I\g \

I.1

0 20 2 4 6

[Hl2CO3]x 103MFIGURE 5. The hatching of eggs of Ascaris lumbricoides at different concentrations of undis-sociated carbonic acid and hydrogen ions. The medium was bicarbonate-carbon dioxidebuffer at pH 7-3 (broken line) and pH 6.0 (continuous line) containing 0.02M-sodiumdithionite under different mixtures of nitrogen and carbon dioxide.

(i) For larvae of Haemonchus contortus and Trichostrongylus axei activity of thestimulus increased with increasing concentration of the undissociated carbonicacid at least up to 6-2 x 10-3M. And inhibition did not occur at high concentrations;high activity was even obtained under 100 % carbon dioxide at pH 6-0.

374 W. P. Rogers


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Infective processes of nematode parasites(ii) With the other species high concentrations of undissociated carbonic aciddecreased the activity of the stimulus so it would appear that only a small pro-

portion of the larvae or eggs would infect a host if the concentration was aboveabout 4 x 10-3M in solutions below pH 7-3.(iii) Under conditions that were otherwise similar, the exsheathment of Haemon-chus contortusrequired much higher concentrations of undissociated carbonic acidthan was necessary for the infective agents of the other species.

TABLE 3. THE EFFECT OF 0-02M-CYSTEINE ON THE HATCHING OF EGGS OF TOXOCARAMYSTAX AT DIFFERENT CONCENTRATIONS OF UNDISSOCIATED CARBONIC ACID

% hatched in 3 hcarbonic ---acid without withpH x 104M cysteine cysteine6.0 1.6 5 166.3 1.1 8 196.9 0.5 8 288.0 0.05 40 62

TABLE 4. THE EFFECT OF SODIUM DITHIONITE ON THE HATCHING OF EGGS OFASCARIS LUMBRICOIDES AT DIFFERENT CONCENTRATIONS OF UNDISSOCIATEDCARBONIC ACID % of eggs hatched

concentration of undissociated carbonic acid, x 104Msodium at pH 6-0 at pH 8-0dithionite --x 102M 7-9 159 159 024 0-47

0 2 16 < 2 5 241 15 31 - 24 372 38 45 < 2 43 404 47 58 - 54 508 48 45 - 54 53

(2) The action of reducing agents(a) Experiments with larvae

When undissociated carbonic acid was present, reducing agents generally in-creased the proportion of larvae of Trichostrongylus axei and Haemonchus contortuswhich exsheathed (figures 2, 3). The optimum concentration of sodium dithionitefor exsheathment of larvae of Trichostrongylus axei in a medium containing0 16 x 10-3 or 1-6 x 10-3M-undissociated carbonic acid and 0-05M-sodium chlorideat pH 6-0 was 0-04M. Only one experiment was carried out with each of ascorbicacid and cysteine; the optimum concentrations were 0.02 and 0-04M, respectively.With Haemonchus contortus the optimum concentrations of the three reducingagents always fell between 0-02 and 0-06M.The relative increase in the activity of the stimulus due to sodium dithionitewas always greater at the lower concentrations of undissociated carbonic acid(table 5, figures 2, 3). This was generally true of ascorbic acid and cysteine also(table 5).

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W. P. RogersThe activity of the reducing agents at pH 6-0 for stimulating exsheathment oflarvae of Trichostrongylus axei was sodium dithionite > cysteine > ascorbic acid;i.e. activity increased as the oxidation-reduction potential fell. Similar resultswere obtained with larvae of Haemonchus contortusthough sometimes the activity

of cysteine and sodium dithionite was about the same. The change in the relativeactivity of the reducing agents when the pH was raised from 6-0 to 7-3 was inaccordance with the fall in the oxidation-reduction potential which would occurunder such circumstances (table 5).As exsheathment of Trichostrongylus colubriformis could not be obtained exceptat relatively high hydrogen ion concentrations, 0-02 M-cysteine was tested insteadof sodium dithionite (see, for example, table 2). It had no effect.TABLE 5. THE RELATIVE ACTIVITY OF REDUCING AGENTS (0-04M) FOR THEEXSHEATHMENT OF LARVAE OF TRICHOSTRONGYLUS AXEI AND HAIEMONCHUS

CONTORTUSrelative

activity:carbonic sodiumacid, ascorbic sodium dithionite/

species pH x 104M control acid cysteine dithionite controlT. axei 6.0 7.9 9 9 25 100 11

6.0 15.9 28 62 72 100 3.57.3 1.1 1 25 130 100 1007.3 2.2 10 12 120 100 10

H. contortus 6.0 7.9 7 60 110 100 146.0 15.9 64 77 94 100 1.66*0 31.8 74 88 97 100 1.37.3 1.1 26 100 107 100 3.87.3 2.2 55 124 89 100 1.87.3 4.4 63 122 81 100 1.6

(b) The hatching of eggsReducing agents increased the proportion of eggs of Ascaris lumbricoides whichhatched in bicarbonate-carbon dioxide buffers. The optimum concentrations andthe relative activity of the reducing agents were similar to those obtained with

Trichostrongylus axei and Haemonchus contortus (figure 6) but the relative activityof cysteine and sodium dithionite was not affected by changes in pH (figure 8).The effect of adding sodium dithionite at concentrations between 0.01 and 0-08Mwas always relatively less at higher concentrations of undissociated carbonic acidboth at pH 6-0 and 8*0 (table 4). The addition of reducing agents also increasedthe proportion of eggs of Toxocara mystax (table 3) and Ascaridia galli whichhatched.These and other results showed that the reducing agents had similar actions oneggs and larvae. Except in experiments with Trichostrongylus colubriformis theaddition of a reducing agent always increased activity at a given concentration ofundissociated carbonic acid. But the effect of the reducing agent was alwaysdecreased at a given pH when the concentration of the carbonic acid was increased.

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Infective processesof nematodeparasitesIncreases in the concentration of sodium dithionite had little effect on exsheathmentor hatching unless carbon dioxide was present in the gas phase.As the three reducing agents are different in structure their action on the eggsand larvae cannot be attributed to any of their individual properties. It seemslikely, therefore, that it was the oxidation-reduction potential of the system whichwas important in the stimulus.

60-

40- /

bD .^/ ._ _X20

0 1 2 3time (h)

FIGURE6. The effect of reducing agents on the hatching of eggs of Ascaris lumbricoides. Themedium consisted of bicarbonate-carbon dioxide buffer at pH 7-3 with a gas phase ofnitrogen containing 5% carbon dioxide. The reducing agents were OOl1M-sodiumdi-thionite (upper curve), OOlM-cysteine (middle curve) and 0Ol M-ascorbic acid (lowercurve).(3) The effect of pH

(a) Experiments with larvaeExsheathment of Trichostrongylus axei and Haemonchus contortusin bicarbonate-carbon dioxide buffers under a given concentration of carbon dioxide was greatestat the lower end of the range pH 6-0 to 8-0 (figure 9). This was because increasesin the concentration of undissociated carbonic acid always lead to increased ex-sheathment with these species. But the pH also had an effect on exsheathment

independent of its effect on the concentration of the carbonic acid; at a givenconcentration of undissociated carbonic acid increases in pH increased activityboth with and without a reducing agent (figures 2, 3).The exsheathment of larvae of Trichostrongylus axei and Haemonchus contortuswas also examined in solutions of 0-025N-hydrochloric acid at different concentra-tions of undissociated carbonic acid and with and without cysteine. Larvae ofH. contortus did not exsheath. The highest proportion of Trichostrongylus axeiwhich exsheathed, 11 %, was obtained under nitrogen-40 % carbon dioxide withcysteine present.Within the usual range of bicarbonate-carbon dioxide buffers, pH 6-0 to 8-0,exsheathing of T. colubriformiswas low. Only when the hydrogen ion concentration

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W. P. Rogersexceeded 10-3M was appreciable activity obtained. When the concentration ofundissociated carbonic acid was about 5 x 10-3M, optimum activity was obtainedin the range pH 1-5 to 2-5 (figure 7).

40-

20-0 20-/ \

1 2 3pH

FIGURE 7. The effect of pH on the exsheathment of Trichostrongylus colubriformis. Themedium consisted of dilute hydrochloric acid containing 0.lM-sodium chloride undernitrogen containing 20% carbon dioxide so that the concentration of undissociatedcarbonic acid was about 4-80 to 4-85 x 10-3M (upper curve). The results shown in thelower curve were obtained when there was no carbon dioxide in the gas phase.

80-

b\

T? /^ \

0 \

0 J . . . .6 7 8pH

FIGURE 8. The effect of pH on the hatching of eggs of Ascaris lumbricoides with 0.02M-sodiumdithionite (upper curve) and 0.02M-cysteine (lower curve). The medium consisted ofbicarbonate under nitrogen (pH 8.3) or bicarbonate-carbon dioxide buffers undernitrogen containing 5 % carbon dioxide. Sodium chloride was added in amounts rangingfrom 0-025M at pH 8-3 and pH 8-0 to 0-1M at pH 6-0.

(b) The hatching of eggsThe hatching of eggs of Ascaris lumbricoides increased as the pH was raisedfrom 6-0 to 8-0 in bicarbonate-carbon dioxide buffers containing ascorbic acid,

cysteine or sodium dithionite under nitrogen-5 % carbon dioxide. At pH 8*3,

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Infective processes of nematode parasiteswhen nitrogen alone formed the gas phase, activity was greatly decreased (figure 8).This was probably due to the low concentration of undissociated carbonic acidrather than the low hydrogen ion concentration. The stimulus for the hatchingof eggs of A. lumbricoides,Toxocara mystax and Ascaridia galli at a givenconcentration of undissociated carbon dioxide was always greatest at the higher pH(figure 5).The results obtained with eggs and larvae indicated that hydrogen ions formedan important part of the stimulus. The pH influenced the action of the stimulusby affecting: (a) the concentration of undissociated carbonic acid in the medium,

80

6 7pH

FIGURE 9. The effect of pH on the exsheathment of Trichostrongylus axei. Experiments werecarried out in bicarbonate-carbon dioxide buffers containing 0.02m-sodium dithioniteand under nitrogen containing 5 % carbon dioxide. In one set of experiments (continuousline) a constant amount of sodium chloride, 0.05m, was added; in the other (broken line)the concentration of sodium chloride was decreased in steps from 0.1M at pH 6.0 to0025M at p60-80.

(b) the larvae and eggs so that a given concentration of the carbonic acid wasmore effective at a higher pH, and (c), the oxidation-reduction potential ofthe reducing agents.(4) Miscellaneous actorsin thestimulus

Though bicarbonate or carbonate ions had little direct effect on larvae or eggs,it seemed possible that the different concentrations of sodium bicarbonate inbuffers may have had an indirect effect by changing the osmotic pressure. Thiswas examined by adding sodium chloride to the bicarbonate-carbon dioxidebuffers containing a reducing agent, to give a concentration of-1M at pH 6.0

PH

falling in steps to a concentration of T025Mt pH 8 xei.iff erence sn osmoticpressure due to the varyingmounts of sodiumicarbonate were thus largelyremoved. These experiments,onducted with Haemonchus contortus and Tricho-str ongylusxei, gave results similar to those obtained w he nconstant amount ofsodiumhloride,.05m, was resentfigure 9). The relationship between pH and

0w025M at pE 8B0.

(9) Miscelloneous fctors in the stimulus

buIfers may have had an indirect effect by chaingig the osmotic pressure. Thiswas examined by adding sodium chloride to the bicarbonate-carbon dioxidebufers containing a reducing agent, to give a concentration of 0-IM At pHi 6@0falling in steps to a concentration of0o025M at pH 8m0. Differences in osmoticpressure due to the varying amounts of sodium bicarbonate were thus largelyremoved. These experiments, conducted with Hcaemonc7hu,sontortus and Tricho-strongylus axei, gave results similar to those obtained when a constant amount ofsodium chloride, 0 05M, was present (figure 9). The relationship between pH and

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the hatching of eggs of Ascaris lumbricoideswas also little affected when appropriateamounts of sodium chloride were added to avoid large changes in osmotic pressureof the buffers (figure 8).When the exsheathment of larvae was compared under similar conditions, butwith and without 01 M-sodium chloride, it was found that activity was generallyincreased by 5 to 15 %. The effect of sodium chloride was greatest in experimentswith Trichostrongylus colubriformis in 0-025 N-hydrochloric acid. At a concentra-tion of 0-05M, the salt sometimes increased exsheathment by 20 %. With al threespecies of larvae 0-4M-sodium chloride inhibited exsheathment.Sodium chloride in concentrations up to 0-1Mincreased the hatching of eggs ofAscaris lumbricoides by 15 to 30 % in bicarbonate-carbon dioxide buffer at pH 7.3(0*013M-sodium bicarbonate) containing 0-02M-sodium dithionate. At 0-2M,sodium chloride was less effective. But even when the concentration was 0-3Mthe proportion of eggs which hatched was often greater than 30 %, In the twoexperiments which were carried out, 0-1 and 0.2M-potassium chloride increasedthe hatching of eggs between 5 and 15 %, as did 0-1M-ammonium chloride. Thehatching of eggs decreased slightly when 0*2M-ammonium chloride or 0-4M-sucrose was added.

Concentrations of sodium taurocholate up to 0.05M in bicarbonate-carbondioxide buffers containing reducing agents, increased the exsheathing of Tricho-strongylus axei and Haemonchus contortus by 5 to 15 %. Similarly, horse serum,10 %, and 'Tween 80', 0-01 to 0-001 %, increased the hatching of eggs of Ascarislumbricoides, but octyl alcohol and a commercial 'anti-foaming' agent were stronginhibitors.

(5) The time relations of the stimulusIf larvae of Trichostrongylus axei were incubated in bicarbonate-carbon dioxidebuffer at pH 6-9 containing 0-04M-sodium dithionite under 100 % carbon dioxidefor 15 min and then washed and incubated in water or phosphate buffer containingmagnesium chloride the subsequent exsheathment of larvae reached 75 % after

2| h. About the same proportion of larvae exsheathed if they were incubatedcontinuously in the stimulating medium (figure 10). The proportion of larvae ofTrichostrongylus axei 'triggered' for exsheathment decreased as the time ofexposure to the stimulus was reduced below 12 min. With larvae of Haemonchuscontortus exposure to the stimulus for about 30 min was necessary to stimulatethe proportion of larvae which would exsheath if they were exposed to the stimuluscontinuously for 3 h.

Eggs of Ascaris lumbricoides were incubated in bicarbonate-carbon dioxidebuffer at pH 8-0 containing 0-04M-sodium dithionite under nitrogen-5 % carbondioxide. After 1, 2 and 3 h samples were taken and the proportions of eggs whichhad hatched were measured. The hatching of these eggs when they were subse-quently incubated in saline was measured at intervals up to 19 h after the beginningof the experiment (figure 11). This experiment was varied by using differentstimulating media and by using a 'balanced salt' medium (Fenwick 1939a), knownto favour the survival of the hatched larvae, instead of saline. As before, the eggscontinued to hatch, but at a reduced rate.

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Infective processes of nematode parasites(6) The stimulus in vitro and in vivo

For comparing the response of infective agents to the stimulus in vivo and invitro the eggs of Ascaris lumbricoides were used (table 6). The capacity of differentlots of eggs to hatch in the small intestine of the mouse closely paralleled the80-

3h h 3h 3h

0vq~

. 4(ca

I ,31 45minmir

15min

FIGURE10. The time relations of the stimulus for the exsheathment of larvae of Tricho-strongylus axei. The proportion of larvae which exsheathed when they were incubatedin bicarbonate-carbon dioxide buffer at pH 6*9 containing 0.02M-sodium dithionite and0-05M-sodium chloride under 100 % carbon dioxide is shown by the lengths of the blackareas. The exsheathment which took place subsequently in 0001 M-magnesium chloridein 001M-phosphate buffer at pH 7 2 is shown by the lengths of the open areas.

61

oX-t 41a)C)

CoI

? 21

0

0I- !

i ._0i

I~-.-

0-

/ \I I I I I I 1, jI I0 4 1' 16 20time (h)FIGURE 11. The time relations for the hatching of eggs of Ascaris lumbricoides.The proportionof eggs which hatched in bicarbonate-carbon dioxide buffer containing 0.04M-sodiumdithionite under nitrogen-5 % carbon dioxide is indicated by the arrows. The subsequenthatching of the eggs occurred when they were incubated in saline.

25-2

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__

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W. P. Rogershatching of eggs in vitro. Thus the eggs of lot B 10 which hatched only in smallnumbers in vitro also failed to hatch in vivo.TABLE 6. THE HATCHING OF EGGS OF ASCARIS LUMBRICOIDES IN VIVO AND IN VIT?RO

% of eggs hatchedincubation in vitro in vivo

time r A - r --(h) B8 BiO Al A2 B8 B10 Al A22 24 7 - 29 93 31 5 73 76 26 8 48 624 58 12 - - 67 6 - -

Four different lots of eggs were examined. A 1 and A2 were tested about a week afterembryonation was complete; eggs in lots B 8 and B 10 were much older.

DISCUSSION(1) The nature of the stimulus in vivo

Larvae have been exsheathed in vitro in a variety of media. Lapage (1935 a, b)removed the sheaths of third-stage larvae of trichostrongyle parasites with hypo-chlorites, sodium sulphide, or organic compounds containing sulphur. Morephysiological media were used by Crofton (1947) who exsheathed larvae ofTrichostrongylus retortaeformis in acid solutions containing pepsin but incubationhad to be continued for 60 h before the majority of the larvae exsheathed. Poynter(1954a, b) found that larvae of Trichostrongylus axei exsheathed slowly in vitro inthe duodenal contents of the horse; when coliform organisms from horses werepresent exsheathment was rapid. Rogers & Sommerville (1957) showed thatcultures of Escherichia coli, when they had reached low oxidation-reductionpotentials, caused exsheathment of Trichostrongylus axei. The concentration ofundissociated carbonic acid in these cultures was probably high; this wouldexplain the activity which was obtained.Infective eggs have been caused to hatch by detergents, hypochlorite and theaction of abrasives or pressure (Jaskoski I952; Fenwick I939b; Pitts 1948).More physiological media have been used (Pick 1948) but long periods of incubationwere needed.

It is probable that the processes of exsheathment and hatching described in thispaper are similar to the processes in vivo. Thus the hatching of eggs in vitro paralleledthe hatching of eggs in vivo (table 6). Moreover, the capacity to hatch in vitroand in vivo developed at the same time (Rogers 1958). And when eggs wereincubated in the fungicide 'Shirlan' (salicyl anilide) the delay in development ofthe infective stage measured by tests in vitro gave the same results as tests in vivo.The eggs of Ascaris lumbricoides did not appear to hatch in vitro until after thefirst moult had occurred, as also was shown to be the case in vivo by Alicata(I934)-

Morphological changes during exsheathment of larvae in vitro followed thesame stages as those which occur in vivo (Veglia 1916; Lapage I935 b; Rogers &Sommerville 1960). The changes in the egg shell of A. lumbricoides during hatching

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Infective processes of nematode parasitesin vitro showed the same two forms that were seen in eggs which hatched in thesmall intestine of the mouse (Rogers I958).

(2) Characteristic properties of gut fluidsIt seems reasonable that undissociated carbonic acid, low oxidation-reduction

potentials and low oxygen pressures should be the important components of theenvironment which start infection in the alimentary canals of animals. Theseproperties distinguish regions in the alimentary canal from most other habitats,and ensure that development from the infective stage would not start until theinfective agent was eaten by the host. The factors which enhance the activity of thestimulus, such as sodium taurocholate and salts, also occur at appropriate con-centrations in gut fluids.

The instability of rumen fluid for inducing exsheathment of larvae of Tricho-strongylus axei and Haemonchus contortus in vitro (Rogers & Sommerville i960)can now be explained because all three components of the stimulus would changewhen the fluid was exposed to air. Moreover, the need for high concentrations ofundissociated carbonic acid for the exsheathment of H. contortus explains whyreducing agents partly restored the activity of rumen fluid for the exsheathmentof Trichostrongylus axei but failed for Haemonchus contortus.

(3) The stimulus for infection in different parts of the alimentary canalThe high concentration of undissociated carbonic acid and the low oxidation-reduction potential needed for the exsheathment of Haemonchus contortus suggeststhat the rumen is one of the few organs where infection with this species could bestarted. Indeed the rumen of the sheep, and presumably of other ruminants also,is a habitat which must be almost unique. Thus the rumen contents of six of the

sheep examined by Turner & Hodgetts (I955) had a pH range of 6-6 to 7.3 andthe carbon dioxide in the gas phase varied from about 65 to 39 % so that the un-dissociated carbonic acid may have reached concentrations from 3 x 10-3 to7 x 10-3M. Oxidation-reduction potentials in rumen fluid may range from -210to -260 mV (Dewey, Lee & Marston 1958). Rumen fluid would thus be suitablefor causing the exsheathment of larvae of Trichostrongylus axei and Hlaemonchuscontortus. The high pH and concentration of undissociated carbonic acid in therumen fluid would inhibit exsheathment of Trichostrongylus colubriformis and thehigh concentration of undissociated carbonic acid alone would prevent the hatchingof the eggs of Ascaris lumbricoides, Ascaridia galli and Toxocara mystax. Thisconclusion has been verified experimentally with eggs of Ascaris lumbricoides.Similarly Sommerville (I957) showed that Trichostrongylus colubriformis did notexsheath in the rumen.

The pH of fluid in the abomasum ranges from 1-05 to 3-6 in sheep (SpectorI956). The concentration of undissociated carbonic acid may be high because thePco2 in the mucous membrane of the cat and rabbit lies between 40 and 60 mmof mercury (Campbell 1933). In the bulk of the stomach contents where exsheath-ment would occur the po2 would be very low (Rogers I949) but the oxidation-reduction potential, + 150 mV in the rat (Bergeim, Kleinberg & Kirch I945) is

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probably somewhat higher than in many other parts of the gut. Conditions ofthis sort in vitro especially favoured the exsheathment of larvae of T. colubriformis;and though a small proportion of T. axei also exsheathed, very few larvae ofHaemonchus contortus were affected. No eggs would be expected to hatch underthese conditions.These results agree very largely with those obtained from studies on life cyclesof the parasites. Thus the eggs of Ascaris lumbricoides, Ascaridia galli and Toxocaramystax hatch in the small intestine of the host-not in the stomach. Tricho-strongylus colubriformis exsheathed very rapidly when placed in a Cellophane sacin a fistula in the abomasum of the sheep. Under the same conditions exsheath-ment of T. axei and Haemonchus contortus was much slower. And when testedin vivo by conventional methods H. contortus did not exsheath at all; Tricho-strongylus axei was not tested in vivo (Sommerville I957).

The pattern of the components which stimulates the hatching of eggs would befound in the fluids of the small intestine in vivo. Thus the Po2would be low exceptclose to the mucosa (von Brand I946; Rogers I949). The Eh would also be low;it is about -100 mV for the small intestine and -200 mV for the caecum andcolon of the rat (Jahn i933; Bergeim et al. I945). In the intestinal juices of thedog the pH in the jejunum ranged from 6-1 to 7-3 (Robinson, Luckey & Mills 1943)and the corresponding concentrations of undissociated carbonic acid would havebeen about 1 to 2 x 10-3M at pH 6-8. Under conditions like these the eggs ofAscaris lumbricoides, Ascaridia galli and Toxocara mystax, which would be un-affected in the anterior parts of the gut, would be expected to hatch.

(4) The stimulus of infection in relation to host specificityIt is difficult to discuss the relationship between the nature of the stimulus for

starting infection and specificity because little is known about the range of hostsof many parasites. Also specificity may often be determined partly by thebehaviour of the infective stage and the host, and not wholly by the ability of theparasite to survive within a particular host. Thus behaviour may determinespecificity between broad limits. The capacity of the host to provide a pattern ofcomponents necessary to start development of the infective stage within the hostmight narrow these limits. Finally, the development of the parasite to differentstages within a host would depend upon a number of factors concerning the anatomyand physiology of the host.The lack of a high degree of specificity in the stimuli which start infection withdifferent species of parasites is thus not surprising. Moreover, specificity of manynematode parasites is not high (Chandler 1932). Thus Trichostrongylus axei andT. colubriformis have a wide range of hosts (see, for example, Roth 194I; Lie KianI946; Bearup & Bolliger 1949; Drudge, Leland, Wyant & Elam I955; Tromba &Douvres I957). On the other hand, Haemonchus contortus is seldom found exceptin ruminants. Is this because the stimulus for infection with H. contortus rarelyoccurs outside the rumen

Eggs of Ascaris lumbricoides and Toxocara mystax hatch in the small intestineof a variety of hosts. This would be expected because the stimuli needed in vitro

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Infective processes of nematode parasitesare not exacting. The range of hosts in which Ascaridia galli will hatch has notbeen examined extensively.

(5) The process of infection as an adaptation to parasitismIn parasitic nematodes the infective stage forms a 'bridge' by which the parasitepasses from one environment to another. The suspension of development in this

stage, and the requirement that a stimulus from the host is necessary to restart it,is an adaptation to parasitism. It ensures that protective structures such as thesheath or egg shell are not discarded until an environment which might supportfuture development is reached.The hatching of eggs of Trichostrongylus retortaeformis, which occurs outsidethe host, is retarded by concentrations of salts as low as 0-05M (Wilson I958).But higher concentrations did not seriously inhibit the hatching of eggs of Ascarislumbricoides. This may be regarded as an adaptation to parasitism because it allowsthe hatching of infective eggs in vivo under conditions where eggs like those ofTrichostrongylus retortaeformis would be inhibited.

(6) How does the stimulus act?The egg shell of Ascaris lumbricoides is impermeable to some ions; cyanide andazide ions do not affect development or respiration whereas hydrogen cyanideand hydrazoic acids inhibit (Resnitschenko I927, I928; Passey & Fairbairn I955).

During respiration oxygen passes into the egg and carbon dioxide is produced.Labelled carbon dioxide (14CO2) asses in through the shell in some form or other(Passey & Fairbairn 1957). The results which suggest that undissociated carbonicacid or dissolved gaseous carbon dioxide rather than bicarbonate or carbonateions is a component of the stimulus thus seem reasonable. It seems that undis-sociated carbonic acid or dissolved gaseous carbon dioxide is the principle com-ponent of the stimulus and that other factors, chiefly the Eh and pH, simplyenhance or decrease its effect. The action of pH on the relative activity of thedifferent reducing agents (table 5) was probably due to changes in the values of Eowhich would fall as the pH: was raised from 6-0 to 7-3.

Dissolved gaseous carbon dioxide affects the rate of cell division and differentia-tion in some organisms. It also controls sexual differentiation in Hydra (LoomisI957). It is not known how carbon dioxide causes these changes.The importance of the stimulus from the host for starting the development ofthe infective stage of nematode parasites generally is difficult to assess. But itseems possible that when infection takes place in the gut of the host a processsimilar to that described here may operate even for parasites like Trichinellaspiralis which have no free-living stages. On the other hand the culture of Haemon-chus contortus throughout its life cycle in media composed of embryo- and liver-extract, casein hydrolysate and serum (Silverman 1959) suggests that undercertain conditions a stimulus which is necessary for infection in vivo is not required.

I wish to thank Mrs M. Ross for technical assistance. My thanks are also due toDr M. Creeth and Dr P. Dunlop of the Department of Physical Chemistry, Uni-versity of Adelaide for advice. Mr R. I. Sommerville of C.S.I.R.O., McMaster

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386 W. P. RogersLaboratory, Sydney, with whom I had several helpful discussions, supplied thelarvae. Part of this work was carried out during the tenure of a visiting professor-ship at McGill University and I wish to thank Professor T. W. M. Cameron andDr D. Fairbairn of the Institute of Parasitology, McGill University, who arrangedmy visit and gave helpful advice.Grants in aid of this work from the George Aitken Pastoral Research Trust,Melbourne, and the Rockefeller Foundation are gratefully acknowledged.

REFERENCESAlicata, J. E. 1934 Proc. Helm. Soc. Wash. 1, 12.Bearup, A. J. & Bolliger, A. I949 Aust. J. Sci. 12, 75.Bergeim, O., Kleinberg, J. & Kirch, E. R. I945 J. Bact. 49, 453.Calam, C. T., Todd, A. R. & Waring, W. S. 1949 Biochem. J. 45, 520.Campbell, A. I933 Quart. J. Exp. Physiol. 22, 159.Chandler, A. C. 1932 J. Parasit. 18, 135.Crofton, H. D. I947 Parasitology, 38, 101.Dewey, D. W., Lee, H. J. & Marston, H. R. I958 Nature, Lond. 181, 1367.Drudge, J. H1.,Leland, S. E., Wyant, Z. N. & Elam, G. W. 1955 J. Parasit. 41, 505.Ellenby, C. & Gilbert, A. B. I957 Nature, Lond. 180, 1105.Fairbairn, D. I955 Canad. J. Biochem. Physiol. 33, 122.Fenwick, D. W. I939a J. Helminth. 17, 211.Fenwick, D. W. I939b J. Helminth. 17, 69.Jahn, T. L. I933 J. Parasit. 20, 129.Jaskoski, B. J. 1952 Exp. Parasit. 1, 291.Lapage, G. 1935a Parasitology, 27, 186.Lapage, G. I935 b Univ. Cambridge,Inst. Animal Path., Rept. Director, 4th Rep., p. 208.Lie Kian Joe I946 Naturk. Tijdschp. Ned-Ind. 102, 41.Loomis, W. F. 1957 Science, 126, 735.Passey, BR.F. & Fairbairn, D. 1955 Canad. J. Biochem. Physiol. 33, 1033.Passey, R. F. & Fairbairn, D. I957 Canad. J. Biochem. Physiol. 35, 511.Pick, F. 1948 Bull. Soc. Path. exot. 41, 208.Pitts, T. D. 1948 Proc. Soc. Exp. Biol., N.Y., 69, 348.Poynter, D. 1954a Nature, Lond. 173, 781.Poynter, D. i954b Nature, Lond. 177, 481.Resnitschenko, M. S. 1927 Biochem. Z. 191, 345.Resnitschenko, M. S. I928 Biochem. Z. 201, 110.Robinson, C. S., Luckey, H. & Mills, H. 1943 J. Biol. Chem. 147, 175.Rogers, W. P. 1949 Aust. J. Sci. Res. B2, 157.Rogers, W. P. I958 Nature, Lond. 181, 1410.Rogers, W. P. & Sommerville, R. I. I957 Nature, Lond. 179, 619.Rogers, W. P. & Sommerville, R. I. I960 Parasitology. (In the Press.)Roth, H. 194I J. Parasit. 27, 363.Shedlovsky, T. & Maclnnes, D. A. I935 J. Amer. Chem. Soc. 57, 1705.Silverman, P. H. 1959 Nature, Lond. 183, 197.Sommerville, R. I. 1957 Exp. Parasit. 6, 18.Spector, W. S. I956 Handbook of biological data. Springfield: Carpenter Lithio and PrintingCompany.Tromba, F. G. & Douvres, F. W. 1957 J. Parasit. 43, Suppl. 12.Turner, A. W. & Hodgetts, V. E. 1955 Aust. J. Agric. Res. 6, 115.Umbreit, W. W., Burris, R. H. & Stauffer, J. F. 1957 Manometric techniques. Minneapolis:Burgess Publishing Company.Van Slyke, D. D., Sendroy, J., Hastings, A. B. & Neill, J. M. I928 J. Biol. Chem. 78, 765.Veglia, F. 1916 Rept. Director Vet. Education Research, Onderstepoort,3rd and 4th Rep.,1915-16, p. 349.Von Brand, T. I946 Anaerobiasis in invertebrates. Normandy, Missouri: Biodynamica.Wilson, P. A. G. I958 J. Exp. Biol. 35, 584.

The Stimulus From the Animal

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