Some analyses of exsheathing fluid from infective Haemonchus contortus larvae from Ontario

SOME ANALYSES OF EXSHEATHING FLUID FROM INFECTIVE HAEMONCHI/S CONTORTUS LARVAE FROM ONTARTO J. Department

of Pathology,

OWEN

D.

SLOCOMBL

Ontario Veterinary College, Guelph, Ontario, Canada

University

of Guelph,

( Received 30 Nowmher 1973) J. OWEN D. 1974. Some analysts of exsheathing fluid from infective Haemonchu~ contortits larvae from Ontario. International Jowwa//or Parasitology 4: 397-402. Infective Haemonchus contortus larvae from Ontario were exsheathed, and the exsheathing fluid was prepared, using several AbStraCt-SLOcoMtIE

procedures some of which duplicated those of other researchers. Infective larvae were exsheathed successfully using the rapid (20-min) tetraborate system. Second-stage sheaths were dissected from infective larvae and were incubated with various preparations of exsheathing fluid. Up to 30% of the sheaths incubated with dilute exsheathing fluid for I h had refractile rings. When the fluid was concentrated by dialysis or lyophilization the exsheathing activity was not lost. Heat destroyed the ability of concentrated exsheathing fluid to cause refractile rings in dissected sheaths, but Cu?+, Hg2+ or diaminoethanetetra-acetic acid did not. The enzyme leucine aminopeptidase was not found in con-

centrated exsheathing

fluid.

INDEX

KEY WORDS:

analysis

of exsheathing

f-laemonchus contortrr.v: exsheathment

of infective larvae; exsheathing

fluid;

fluid.

INTRODUCTION

Haemorlchus contortus, a trichostrongyle nematode in the abomasum of sheep, is thought to produce resistance-stimulating antigens during the molting of larval stages (Silverman, 1965). There is considerable interest, therefore, in identifying proteins and antigens elaborated during molting, particularly the exsheathing fluid from infective larvae. However, there is a substantial difference in analyses of exsheathing fluid harvested from infective H. contortus larvae in different parts of the world. Rogers (1965), in Australia, considered the enzyme leucine aminopeptidase (LAP) as the exsheathing enzyme, not only for H. contortus but also for Trichostrongylus colubrifbrmis. a nematode in the small intestine of sheep. Ozerol & Silverman (I 969), in Illinois, found that LAP was not responsible for the cxsheathment of H. contortus. Slocombe & Whitlock (197lb), in New York, also found that LAP was probably not involved in the exsheathment of H. contortus cayugensis. Rogers (1970), and Ozerol & Silver,nan (I 972b) re-affirmed their previous findings respectively, and supported their position with evidence for and against LAP as the exsheathing enzyme. Reasons for the controversy on the content of exsheathing fluid have been dis-

cussed by Whitlock ( I97 I ). Leucine aminopeptidase has been demonstrated histo-chemically in several locations in several nematode species: Ascaris lumbricoides (Lee, 1962; Jenkins & Erasmus. 1971), Nippostrongylw brasiliensis (Lee, 1970), Anisakis sp. (Ruitenberg & Loendersloot, I97 I ), Oesophugostomum radiatum (Douvres & Thompson, 1973), fhoconema decipens (Davey, 1966; Davey & Kan, 1967, 1968). and Xiphinema index (Roggen, Raski & Jones, 1967). Functions attributable to LAP include associations with intestinal processes as in the first four species mentioned, and with molting and ecdysis in the latter two species. Rogers (1970) has insisted that LAP in exsheathing fluid is highly labile and is present in such small quantities that probably his method alone is suitable for its isolation. Certainly, no other researcher has reported using simultaneously, as high a larval concentration, harvested exsheathing fluid after a relatively short incubation period, and maintained through all procedures a pH necessary for the activity of LAP. Rogers’ (1970) procedures were duplicated, therefore, and the results of this work, the ability of infective H. corrtortrts larvae from Ontario to undergo rapid exsheathment, and a partial analysis of the exsheathing fluid harvested, are now reported. 397

398

.I. MATERIALS

AND

OWFN

II.

METHODS

were collected from sheep infected with H. found in Ontario and were prepared for culture (Slocombe, 1969). The cultures were incubated, harvested, cleaned, and stored as described by Slocombe & Whitlock (1970~). When required, infective larvae were used and exsheathed as described below. The exsheathment rate was assessed on 500 larvae as described by Slocombe Rr Whitlock (19706). Exsheathing fluid was harvested and concentrated, and dissected second-stage sheaths were obtained as described below. The assay for activity of exsheathing fluid preparations was the development of refractile rings in the dissected sheaths (observed with the light microscope at a magnification of 100 ; ). Refractile rings appear at 20 pm (and often additionally at 9 pm) from the anterior end of dissected sheaths (Slocombe & Whitlock, 197lh). Feces

cwltortus

Rogers

( 1970) procrdure.~

Rogers’ (1970) “simple and specific” techniques fol using stored larvae, preparing and concentrating exsheathing fluid, testing exsheathing fluid for exsheathing activity and LAP, isolation of second-stage sheaths, and determining refractile ring formation in the sheaths were duplicated. Exsheathing fluid was prepared from 2.5 ’ IO6 I 6% infective larvae, stimulated to exsheath in 0.15 M Tris-hydrochloric acid buffer containing lo-‘M Mg2+ and 100% CO,, and concentrated by dialysis against Carbowax. This concentrated exsheathing fluid was tested for exsheathing activity before and after heating at SO’C for 15 min. or after it was fortified with lo-” M cU’+, Hg?+ or diaminoethanetetra-acetic acid (DAEA), reported as inhibitors of LAP (Smith & Hill, 1960). The concentrated exsheathing fluid was tested for LAP activity spectrophotometrically by determining the rate of hydrolysis of L-leucinamide hydrcchloride at 238 nm (Mitz & Schlueter, 1958) with a Unicam spectrophotometer, SP 800. L-Leucinamide and LAP were obtained from the British Drug Houses Ltd., Poole, England. One unit of LAP hydrolyses I.0 pM of L-Leucinamidejmin at 25’C. In addition to the above techniques. Rogers (1970) used alternate procedures which appeared not to be as as those above and they were “simple” or “specific” not duplicated; exsheathing fluid was not concentrated with Sephadex because dialysis against Carbowax was simpler; LAP was not estimated by release of ammonia from a substrate (Smith & Slonin, 1948). since ammonia is present in the substrate, and is excreted by infective larvae (Slocombe & Whitlock. 197la), and would not be as specific as the method of Mitz & Schlueter (1958).

Slocombe & Whitlock’s (1969) rapid (20~min) method IO6 I 6% larvae in IO ml 0.02 M for ecdysis using I Na,B,O,.IOH,O with 4O:d CO, was duplicated. After incubation, the exsheathing fluid was removed from the larvae by centrifugation at 4 C (Slocombe & Whitlock, 197 I a). Estimates of total protein on this fluid was carried out as described by Lowry et a/. (1951). Protein standards were prepared with bovine albumin, fraction V, from J. T. Baker Chemical Co.. Phillipsburg, New Jerbev. Second-stage sheaths from infective larvae were selectdd by a modification of the method of Ozerol & Silverman (19720) as follows: after the heat-kill of infective larvae, 200,000 larvae in 2 ml of distilled water were placed in a

SLO(.OMBI

l..l.l’.

CUL.

4. 1974

7-ml Pyrex tissue grinder. The pestle was turned thirty times and the crushed larval suspension was flushed out with 7 ml of deionized distilled water and centrifuged at 1390 g for 10 sec. Selected sheaths were retrieved from the supernatant. Sheaths were incubated at 37 C with the various exsheathing fluid preparations either on glass slides (Rogers, 1970), or in depression slides (approximately 0.25 ml of each fluid was placed with sheaths in a depression slide which was covered by a Vaseline-coated coverslip). Exsheathing activity was assessed on fluids (I ) after they were freshly prepared, (2) after a 30-fold concentration by dialysis against Carbowax, (3) after lyophilization and reconstitution with deionized distilled water (100 mg substance/ml), and (4) after the dialired and lyophilized preparations were heated in a water bath at IOO’C for 10 s. Some of the dialyzed preparations were fortified with IO-” M Mg”+ (reported to be essential for LAP activity, Rogers, 1970) and measured for LAP (Mitr & Schiueter, 1958).

RESULTS

The exsheathment rate attained 75% or better. The concentrated exsheathing fluid had a yellow!ish color. Second-stage sheaths incubated with concentrated exsheathing fluid were checked for refractile rings after I and 24 h and the results are shown in Table I. Some sheaths had two refractile rings after incubation for I h. After incubation for 24 h, the cuticle of the sheaths had collapsed and the sheaths appeared crushed and hardly recognizable. The number of sheaths with refractile rings after incubation with concentrated exsheathing fluid, which was either heated at 80 C for I5 min or fortified with IO-” M Cu’+, Hg’+, or DAEA is shown in Table I. Sheaths incubated for 24 h with concentrated exsheathing fluid so fortified were not crushed, although rings were present in the sheaths. No LAP was detected in three preparations of concentrated exsheathing fluid (Fig. I), which nevertheless, caused refractile rings in the dissected sheaths (Table I ).

The exsheathment rate attained 80 per cent or better. Second-stage sheaths incubated with dilute exsheathing fluid, either on a flat glass slide or depression slide, were checked for refractile rings after I and 24 h and the results are shown in Table 2. Some examinations of the sheaths, after they were incubated for 4 h, showed that all sheaths had one refractile ring and many had two. The concentrated exsheathing fluid was colorless. The number of sheaths with refractile rings after incubation with concentrated exsheathing fluid, some of which was heated at 100 C for IO s, is shown in Table 3. Total protein values of exsheathing fluid ranged from 20 to 25 Ltg/ml. L-Leucinamide was not hydrolysed by the exsheathing fluids concentrated by dialysis, although such fluids caused refractile rings in dissected sheaths (Table 3). Exsheathing fluid concentrated by lyophilization was not checked for LAP.

J.I.P. VOL.

TABLE

4. 1974

Exsheathing

fluid from ffaemonchrrs ror~tortus

I--NUMBERS OF SHEATHS WITH REFRACTILE RINGS AFTER INCUBATION WITH TRATED EXSHEATHING FLUID PREPARED BY RoGERs'(I~~~)PROCEDURESIN __

Concentrated

exsheathing

399 VARIOUS PREPARATIONS OF CONC‘ENSEVERAL EXPERIMENTS

fluid ____

Incubalion Untreated

1 I h

I h

_

I

0 0 0

were incubated

-.-

lO-?~-~ith10_L_With

M CLI"+

M Hg?+

1

I

_

Control ,O-’

M DAEA

-

_

_ 0 0 0 0 0 0

5 5 5

7 0 Crushed

24 h I6 days The controls

_

8 0 Crushed 7 0 Crushed

24 h I I1 24 h 7 days

With

Heated 80 C I5 min

I 0 0

6 7 7

6 7 7

I 0 0

6 6 6

I

in 0.15 M Tris-hydrochloric acid buffer containing IO-” M Mg’+ of refractile rings is shown by ! , and the absence by -

__._- --__-_---_-_-

_--__

-___-_-_

____

-_-___-

4 4 6 6 6 6

I

0

6

I

0

6

at pH 8.3. Presence

___-

----_

\ \ .\ ~________

I 20

I

10

__-_

1 40

I 30

---

I SO

____

I GO

__

I 70

I

80

TIMEINMINUTES FIG. I. The action of exsheathing fluid concentrated by dialysis on L-leucinamide when incubated at 38 C and measured continuously using a Unicam Spectrophotometer,

238 nm. The results

shown

are mean

DISCUSSION

Rogers’ (1970) “simple and specific” methods for recovery and concentration of exsheathing fluid, and isolation of LAP were duplicated using infective H.

~77tout7rs larvae

from

Ontario.

No

LAP

was

in this concentrated exsheathing fluid, although it produced refractile rings in dissected sheaths, and caused crushing of the sheaths after incubation for 24 h. When the concentrated fluid was heated at 80 C for 1.5 min the activity was lost.

found

values

of three different

hydrochloride

SP X00. at

runs.

When Cu”+, Hg”+, or DAEA was added refractile rings appeared in the sheaths, but these sheaths did not become crushed. Probably this exsheathing fluid is composed of a mixture of substances some of which may have been inhibited by the additives. Infective H. conrortus larvae from Ontario were exsheathed successfully by the rapid (20-min) tetraborate system which, for several reasons, is a superior method for exsheathment of infective larvae (Slocome & Whitlock, 19706). It provides a suitable pH for the maintenance of LAP, and a yellow pig-

400

D. SLOCOMHC

J. Owt.~ TABLE FRESH

2--NUMBERS DILUTE

OF

SHEATHS

EXSHEATHING

WITH

FLUID

RAPID

REFRACTILL

PREPARED

I.J.P. VOL.

KINGS

AFlLR

BY SLOCOMBE

EXSHEATHMENT

&

INCUBATION WHITLOCK’S

4. 1974

WITH (1971~)

PROCEDURES.

Incubation

time

Medium

Glass slide type

_ Fla1 Depression

lzxsheathing Control Exsheathing

Huid

I

fluid

2 3 Control The controls

were incubated

with 0.02

shown by TABLE PLUID

3-NUMBERS PREPARED

OF SHEATHS WITH BY SLOCOMRE

M Tetraborate.

1, and absence by

REFRACTILE

& WHITLOCK’s

RAPID

87 0 60 75 60 0

7 38 0 0 0 35

Presence of refractile -

RINGS AFItR

(19710)

_

ho 38 46 50 46 35

I4 0 14 25 I4 0

INCUBATION

WITH

FxstgtATiiMEN’r AND

rings is

EXSHEATHING

CONCENTRATION

PROCEDURES.

Exsheathing

Incubation

_

4 days IO days 21 days

Dialysis

Lyophilizalion Unheated

24ihh

fluid concentraled by

43 44

0I

Heated 100 C-IO set

Unheated

I

_

_

00

50 50

13 0 Crushed

0 0 0

The control was incubated with 0.02

50 50 50

Control

Hea!ed 100 CmlOsec

1 ;

_ .5050

1 0 i 50

0 0

50 50

I ;

0 0 0

t ;g 50 50 50

M Tetraborate. Presence of refractile rings is shown by and absence by -

ment did not appear in the exsheathing fluid. The discoloration of exsheathing fluid was found only when infective larvae were stimulated with loo’:;, CO, in Tris-hydrochloric acid buffer, and not when stimulated with 40”/, CO2 in the tetraborate bufler Recently, this rapid method for ecdysis has been found useful in the exsheathment of the nematode, Nematospirokh chbius, in mice (Cypress et al.. 1973). Dilute solutions of exsheathing fluid from H. contortus prepared by the rapid method produced refractile rings in up to 30 per cent of the sheaths after incubation for I h. This activity reflected a very potent fluid since Rogers (1970) found that such solutions would show some action only after 3 h, and Slocombe & Whitlock (197lb) found activity only after I2 h. The levels of protein in dilute solutions (on a limited sample) were similar to those for H. contortus caygensis (Slocombe & Whitlock, 1971~). Contrary to Rogers’ (1965), but similar to Ozerol & Silverman’s (1972~) reports, the activity was not lost after the fluid was concentrated by lyophilization. Ozerol & Silverman (1972b) found that when concentrated fluid was heated (100 C for IO min). rcfractile rings were not found in the sheaths

j,

until 48 h after incubation. Exsheathing fluid from H. conlortus from Ontario was heat-labile (heated at 100 C for 10 s); this agrees with data given by Rogers (1970). When the fluid was concentrated by dialysis and fortified with Mg’+ (required for LAP activity), no LAP was found, although the fluid without Mg’+ produced refractile rings in dissected sheaths. It must be concluded that infective H. contorfus larvae in Ontario do not produce LAP in identifiable amounts, and activity of their exsheathing fluid is independent of LAP. Analyses of ecdysis fluid in Haet~~~~clrus, therefore, have yielded remarkably inconsistent findings. Some of the differences are probably due to techniques, and it would be useful if a single method were used more universally. However, since techniques used in Australia were duplicated in Canada, and since the analyses of exsheathing fluid for the two areas using the same techniques were dissimilar, it might be concluded that the content of exsheathing fluid in at least these two areas is different. This would not be surprising since H. contortus is a polytypic species. Das & Whitlock (1960) have shown morphological differences in populations of H. contorrus in different parts of the world. These morphological differences

I.J.P. VOL. 4. 1974

Exsheathing

fluid from Haemonchus conrorrus

are genetically controlled (Daskalov, 1971 ; Le Jambre, 197 I ). Bionomic differences between populations also exist (Crofton, 1957; Crofton et al., 1965; Conway, 1964), and antigenic differences between two strains have been demonstrated (Slocombe & Whitlock, 19710). Recent findings in Ontario indicate that populations of H. contortus in sheep contain an unusually large percentage of the morphological type “smooth” adult females (Slocombe, 1973), and populations of Haemonchlts in cattle were unlike H. placei or any other recognized HaemorlchLls species (Slocombe, 1974). The genus Huemonchus therefore, contains numerous types, with numerous expressions, and to quote Whitlock (1966). “Skrjabin has pointed out that the trichostrongyles are in their period of greatest evolutionary efflorescence, (and) I suggest the likelihood that the ultimate ecotype system in Huemow C/INS may even rival the Sulmonellu antigenic system in complexity”. AcX/?oll/eJjiements-This work was financed by the National Research Council of Canada and the Ontario Ministry of Agriculture and Food. The skilled assistance of Mrs. Marjorie Hutchison who significantly contributed in the technical procedures is gratefully acknowledged REFERENCES CONWAY D. P. 1964. Some effects of temperature on the development and activity of ~laemonchus contortus. Cornell Veterinarian 54: 266-270. C’R~FT~N H. D. 1957. Nematode parasite populations in sheep on lowland farms-Ill. The seasonal incidence of species. Parasitology 47: 304-3 18. CROFTON H. D., WHITLOCK J. H. & GLAZER R. H. 1965. Ecology and biological plasticity of sheep nematodesenvironmental plasticity in Haemonchus II. Genetic contortus ( Rud I 803). Cornell Veterinarian 55 : 25 I 258. CYPRESS R. H., PRATT E. A. & VAN ZANDT P. 197?. Rapid exsheathment of Nemutospiroides dubius infective larvae. Journal of Parasitology 59: 247-250. DAS K. M. & WHITLOCK J. H. 1960. Subspeciation in Haemonchus contortus (Rudolphi. I803), Nemata, Trichostrongyloidea. Cornell Veterinarian 50: 182-197. DASKALOV P. B. 197 I Haemonchus contorlus genetically determined polymorphism in females. tsperimental Parasitology 29: 351-366. DAVEY K. G. 1966. Neurosecretion and molting in some nematodes. American Zoologist 6: 243-249. DAVFY K. G. & KAN S. P. 1967. Endocrine basis fat ecdysis in a parasitic nematode. h’atlrre 124: 737-738. DAVEY K. G. & KAN S. P. 1968. Mol:ing in a parasitic nematode, Phocanema decipiens. IV. Ecdysis and its control. Canadian Journal of Zoology 46: 893-898. DOUVRES F. W. & THOMPSON D. E. 1973. Histochemical distribution of “Leucine” aminopeptidase in Oesophagostomum radiatum grown in vivo and in virro. Journal o./‘ Parasitology 59: 417-424. JENKINS D. C. & FRASMUS D. A. 1971. The ultrastructure of the intestine of Ascaris suum larvae. Zeitschrifi f& Parasitenkunde 35: 173-187. LCE D. L. 1962. The histochemicnl localization of leucine

401

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402

J. OWEN D. SLOCOMBE

thus contortrt,s ccyrtgensk larvae. Journal oj’Parasitolq~y 57: 7944800. SLOCOMBE J. 0. D. & WHITLOCK J. H. 1971b. Further

analyses of supernatant fluids from exsheathing infective Haemonchrrs contortus cayugetlsis larvae. Jownal r?f’ Parasitology 57: 801-807. SMITH E. I,. & HILL R. L. 1960. Leucine aminopeptidase. In The Enzymes (Eds. BOYER P. D., LARDY H. 61 MYRBAC-K K.). Vol. 4. pp. 37--60. Academic Press, New York.

I.J.P. VOL. 4. 1974

SMITH E. L. & %.ONON N. B. 1948. The specificity of leucine aminopeptidase. Journal of’ Biological Chemivtry 176: 835-841. WHITLOCK J. H. 1966. Biology of a nematode. In Bio/og,v ofParasifes (Ed. SOULSBY J. L.), pp. 185-197. Academic Press, New York. WHITLOCK J. H. 1971. Ecdysis of Haemonchus and hypotheses. C’orm~ll Veterinaricm 61: 349 361,