Low molecular weight Cooperia oncophora antigens. Potential to discriminate between susceptible and resistant calves after infection

Low M&m&w Weight Coope~iu PO 1 to Discriminate between Resistant Calves after h&e&h P. M. VAN DIEMEN,* H. W. PLOEGER,* M. G. B. NIEUWLAND,* F. W. RIETVELD,” M. EYSKER,? F. N. J. KOOYMAN,? A. KLOOSTERMAN* and H. K. PARMENTIER”: *Department

of Animal Husbandry, Animal Health and Reproduction, Agricultura/ Unit~ersrt~. Wageningen, The Netherlands SDepartment qf Parasitology, Veterinary Science, Universit,v qf Utrecht, Utrecht, The Netherlands ( Receiced

I8 June 1996: accepted

6 Januurj,

19971

Abstract-Diemen P. M. van, Ploeger H. W., Nieuwlaod M. G. B., Rietveid F. W., Eysker M., Kooyumn F. N. J., Khstezmon A. & Parmentier H. K. 1997. Low molectdar weight Coqericr oncqhnw Potential to te between swcepthie aod resistant calves after inhtion. ItierJmtmd fur Purusitofogy 27: 9874593. The remgdoo of low molecoiar weight oral (primmy} imfectkwi witf8 100 000 3rd-stage Cooperip 0~coQirrra hrvae was stah4I in uhzs, of6or7es~~were~~basedondinraenteggexcre4ktll~~~f CQopk mdar rdndng condiths, followed by Western C&OS t0 c. @SQ2Qphetw II@bt be refated with aotisody respOneeS(4bfayS (14-16 LDa and 27 kDa). The 14-16-kDa pro?eic~ indivbInaf sem fmm au cafves.TfIe intensity of stainhg was negativety correGzw%& eggcotmtsonDay21p.i.eitkerdidnotoron@weakiy wbeher *& 14-16 kDa (or recombinant 14.2kDa) It bps to Be e a tool for kmnoated resistance to ne8 wiaetber the 27-kDa fragment can beip fhther maravel Cooperia. 6 1997 Australian Society for Parasitology. Published by Elsevier Science Ltd. Key words: Cooperiu oncophoru; cattle; gastrointestinal proteins; Western blotting;humoralimmunity.

INTRODUCTION

Calves infected with the nematode Cooperia oncophora mount an antibody responseto a rangeof low to moderateweight adult worm protein fragments(de Graaf er al., 1993;Parmentieret nl., 1995;Nieuwland et al., 1995;van Diemen er al., 1996).The antigen recognition profile and parametersof resistancesuch as faecaleggexcretion, however, vary widely among fTo whom partment of

correspondence

should

be addressed

at: De-

Animal Husbandry,Agricultural University, POBox 338,6700AH Wageningen, TheNetherlands. Fax: + 3 1 3 17 485006; WAU.NL.

E-mail:

[email protected].

nematodes:

egg excretion

pattern;

recombinant

infected calves, despite the use of a highly standardized infection model (similar age and infection level). Genetic differencesin resistanceof calves to Cooperiainfections do exist, although only a minor role is suggestedfor geneticfactors (Albers, 1981).It hasbeenobservedrepeatedlythat calvesshow& high faecaleggcountsgenerallyhave lower antibody titers againstcrude adult antigen than calvesshowinglow faecal egg counts, both after primary infection and after trickle infection (Albers, 1981;Kloosterman & Ploeger, unpublished observations). The antibody responsesare mainly directed againstlow molecular weight antigens(Parmentieret al., 1995,de Graaf et al., 1993).Further, someof theseantigen fragments, suchas a I&16-kDa! complex, are recognizedby all

587

P. M. van Diemen et ~1

588

calves but their quantitative response to this varies (Nieuwland et ul., 199.5; van Diemen et al., 1996). The staining intensity was found to correlate with infection dose (Nieuwland et al., 1995). Other adult parasite fragments are not recognized by sera from all infected calves, e.g., a 27-kDa component (Nieuwland et al., 1995; van Diemen et al., 1996). The function and relevance of these antigen fragments regarding immune-mediated resistance to infection is not yet clear. The above mentioned observations indicate that a closer examination of the relationship between the specificity and responsiveness of antibodies from Cooperia-infected calves to various low molecular weight antigens and parasitological parameters of resistance may be worthwhile with respect to revealing relevant factors determining immune-mediated resistance. A group of 174 calves resident at the university farm offered a good opportunity to conduct a preliminary study on the relationship between EPG and responsiveness to the above-mentioned protein fragments. Thus, the aim of the present study was to investigate the antigen recognition of groups of calves characterized by specific egg excretion patterns after a single primary infection with 100 000 3rd-stage infective Cooperiu larvae. MATERIALS

AND

METHODS

Calves. Three-month-old female calves (Dutch HolsteinFriesian, n= 174). born on the university farm “De A. P. Minderhoudhoeve” in 1993 and 1994, were infected orally with a single dose of 100 000 C. oncophora 3rd~stage larvae (L,). Faeces were collected weekly from the rectum for estimation of the number of eggs per gram of faeces (EPG) according to the method described by Van den Brink (1971). Blood was collected from the jugular vein just before infection (Day 0) and weekly thereafter from Day 14 until Day 42 post infection (p.i.). Sera were stored at -20°C until use. The larvae had been collected from faecal cultures from calves infected with C. oncophora according to standard procedures (Borgsteede & Hendriks, 1973). For the present study, sera of 20 calves were selected from the 174 available, based on their EPG pattern (see below). Faecal egg excretion categories. Selection of the 20 calves in this study was based on a previous study in which different EPG excretion patterns were identified through cluster analysis of data (Ploeger, unpublished data). In those experiments, calves had undergone similar oral primary infection with 100000 L,, and were necropsied on day 42 p.i. Three clear egg excretion patterns (cluster 1 to cluster 3, Fig. 1A) were selected out of 5 partly overlapping patterns which were identified. The post-mortem findings from the respective clusters are given in Table 1. For each of the 3 clusters, 6 or 7 calves were selected, with an egg output pattern matching the cluster patterns (Fig. 1A). The mean faecal egg counts of the selected groups of calves are shown in Fig. 1B. C. oncophora antigens. Adult C. oncophora worms. obtained from apparently susceptible calves showing a high worm burden after primary infection, were homogenized in 0.05 M Tris-HCl, 1 mM phenylmethylsulphonyl fluoride and

1mM EDTA (pH 8.6). After 2 h incubation on ice, the extracts were centrifuged at 25 000 g at 4°C for 30 min. The supernatants were dialysed using a 0.45pm dialysis tube (Schleicher & Schuell, Dassel. Germany), and stored at - 20’ C until use. The protein concentration, as determined with a Perkin-Elmer Lambda 1 u.v./vis spectrophotometer (Oak Brook, IL, U.S.A.) according to Bradford (1976). was 5.85mgmI-‘. A genus-specific 14.2-kDa recombinant C. oncophora protein (protein concentration 1.8 pg ~1~ ‘) was also used. Details on this recombinant protein will be published elsewhere (J. Poot, F. N. J. Kooyman, M. Eysker, H. W. D. H. Schallig, A. W. C. A. Cornelissen, unpublished results, 1995-I 996). Sodium dodecyl phoresis (SDS-PAGE).

sulphate-polyacrvlamide Adult Cooperia

gel

electro-

antigen fragments were separated on molecular weight using 12.5% SDSPAGE gels under reducing (5% P-mercaptoethanol) conditions using a Midget Electrophoresis unit (Pharmacia, Uppsala, Sweden). The recombinant protein was run under non-reducing conditions. Some gels were stained directly with 0.25% Coomassie Blue solution (R 250, LKB, Villeneuve-la-Garenne, France) to visualize the proteins. Western blot. Western blots with sera from all individual calves, were carried out with a Multiphor II Nova Blot electrophoresis transfer unit (Pharmacia). Proteins from the SDS-PAGE gels were blotted for 1 h at 0.8 mA cm-’ onto 0.45~pm nitrocellulose (NC) membranes (Bio-Rad, Richmond, CA, U.S.A.) using a continuous buffer system. Immunostaining was performed on individual lanes using a Miniblotter 16 (Immunetics, Cambridge, MA, U.S.A.). The NC membranes were blocked for 1 h in phosphate buffered saline (PBS. pH 7.2) containing 0.05% Tween-20 and 1% Bovine Serum Albumin (BSA, Sigma, St. Louis, MO, U.S.A.). Subsequently the NC membranes were washed with PBS-Tween (3 x 10 min) and incubated for 1 h with the individual sera, 1:10 diluted in PBS-Tween containing 0.5% horse serum to prevent non-specific binding. After washing (PBS-Tween, 3 x 10 min), the membranes were incubated for 1 h with 1:250 diluted rabbit anti-bovine immunoglobulin G (heavy and light chains) coupled to horseradish peroxidase (RAB-IgGn+,/PO, Sigma), followed by another wash-step. Binding of the bovine antibodies to proteins was visualized by incubation of the membranes for no more than 1h with the chromogen. 4-chloro-1-naphthol (Sigma). The reaction was stopped by rinsing with water. Western blot staining was evaluated with an Ultroscan XL (Pharmacia). Molecular weights (mol. wts) of the individual bands on the blots were calculated as compared to standard prestained mol. wt markers (Bio-Rad). The staining intensity of individual bands on a blot is reflected as an absorption value (laserbeam 6=633nm). Statistical analysis. When appropriate, comparisons were made between the 3 groups of calves as characterized by their faecal egg excretion pattern. Analyses involved the use of ANOVA to detect differences in group means. Further, correlations between EPG on Day 21 and Dav 42 and staining intensity of the 1416-kDa and 27-kDa bands were analysed by the method of Pearson’s partial correlation, both within groups and over groups. RESULTS analysis of crude adult worm extract Polyacrylamide gel electrophoresis of adult C. oncophora antigen under reduced conditions revealed SDS-PAGE

Recognition of low weight C. oncophoro antigens

A 5000

[---

2000

;/-

-.-._--.I

.- .__.

- __..

-~

-

1000 Ii 28

21

35

49

42

Day p.i. A

8000

Cluster

l

1

Cluster

2

n

Cluster

3

-----

y---

0

7

14

21

28

Day -A-

Group

1

-*-

Group

35

42

49

p.i. 2

-a-

Group3

Fig. 1. (A) The course of the arithmetic mean faecal egg counts as identified through cluster analysis of data from calves infected with a single dose of IO0 000 L, Cooperiu oncophoru in other experiments. and (H) the mean faecal egg counts of the selected calves by egg excretion pattern groups, after infection with 100000 L, C. oncoplrorn. a,b. Means with different superscripts differ significantly (PtO.OO1).

P. M. van Diemen

590 Table

l--Post

mortem

findings

(day 42 p.i.) from the respective clusters. analysis of data from other experiments Cluster

Total No. of worms (+S.D.) Sexratio (X 6 of total) (+S.D.) Male worm length (mm) (+S.D.) Female worm length (mm) (fS.D.)

1

8900 (2095) 42.5 (3.0) 8.92 (0.27) 12.05 (0.29)

Cluster

2

103.3

43.1

(14.6)

(22.9)

identified

through

F-value

3

30840 (15820) 46.2 (2.7) 8.92 (0.34) 11.87 (0.36)

26.3 (12.0) 8.48 (0.62) 11.05 (1.37)

(5S.D.)

Clusters

Cluster

11100 (10770)

No. eggsin uteroifemale ‘***P < 0.001; ns, not significant

et al.

79.8

cluster

P value”

5.93

***

6.28

***

1.02

ns

1.72

ns

6.23

***

(23.7)

P > 0.05.

numerousfragments. In Fig. 2 the antigenic com- ular Cooperia proteins (Nieuwland et al., 1995;van position of the crude C. oncophora antigenand of the Diemenet al., 1996). recombinant14.2-kDaprotein aregiven after staining The stainingintensity of individual bandson a blot the proteins on the gelwith Coomassie Blue. was reflected as an absorption value. The mean absorptionvaluesare given in Table 2. Group 1 and Group 2 calvesshoweda clear stainingof the 14-16Western blot analysis of antibody reactivity kDa complex. The stainingintensity of this complex Incubation of blots containingadult worm proteins by seraof Group 3 calveswassignificantly(P < 0.01) with sera obtained from the individual animals lower. Serafrom 5 out of 6 Group 1 calvesshoweda resultedin the identification of severalfragmentsof good recognition of the 27-kDa band, whereasfrom Cooperia adultsthat were bound by the immunesera the Group 2 calves,5 had an absorptionvalue under (Fig. 3). Forty-two daysp.i., serafrom infectedcalves 0.1 to this fragment.In Group 3, individual serafrom bound a protein complex located at approximately 3 calvesrecognizedthe 14-16-kDa protein complex the 14-16-kDa level, and 1 at 27kDa. Lessintense and the 27-kDa fragmentin a weak manner.One calf stainingwasobservedat 31.6kDa and at variousother had a low responseto the 14-16-kDa complex and a bands of higher molecular weight. Day 0 (non- good recognition of the 27-kDa band (calf 2828). immune) sera and sera from calves mono-infected In Table 3 the Pearson’s partial correlation with other nematodes,suchasDictyocaulus viviparus coefficientsbetweenEPG at Day 21 and Day 42 p.i. and Ostertagia ostertagi, did not bind the low molec- on the onehand, and the antibody recognitionof the 14-16-kDaprotein complex,the 27-kDa bandand the 14.2-kDarecombinantprotein on the other hand, are MARKER given.Correlationsweresimilarbetweenwithin-group and overall analyses.Responsesof calves to the recombinant14.2-kDaprotein werecomparablewith the responses to the 14-16-kDa complexof the crude Table 2-Staining intensity (mean va1uefS.E.M.) after Western blot analysis from 3 groups of calves, 42 days after infection

absorption of antibodies with 100 000

Cooperia L,

Fig. 2. Antigenic composition of (A) crude C. oncophora protein (73 pg/lane) and (B) 14.2 kDa recombinant protein (0.5pg/lane), as evaluated on 12.5% SDS-PAGE gel and visualized by Coomassie Blue staining. The crude protein was run under reduced @-mercaptoethanol) conditions.

Group

n

1416

1 2 3

6 7 6

0.531a’

“Results gen. ‘Results ‘Means, nificantly

kDa” (0.067)

27 kDa” 0.143

14.2 kDab

(0.01)

0.127ab

(0.03)

0.468a (0.031) 0.084 (0.01) 0.184a (0.04) 0.249b (0.033) 0.101(0.02) 0.049b (0.01)

from

blotting

using crude

adult

C.

oncophora anti-

from blotting using the recombinant protein. within a column. with different letters differ

(P < 0.05).

sig-

Recognition of low weight C. oncophora

antigens

El

Fig. 3. lmmunoblots of adult C. oncophoru antigen (73Oj~g/gel) after incubation with (A) sera obtained on Day 42 pi. {IO calves) and standard positive serum, and (B) sera obtained on Day 42 pi. (10 calves), and standard positive serum, followed by incubation with RAB/PO. The 12.5% gel was run under reduced conditions. Group 1 calves: 2670, 2694, 2707. 2708. 2743, 2766. 2769; Group 2 calves: 2656, 2661, 2662, 2684, 2706, 2738, 2757: Group 3 calves: 2667, 2703. 2741. 2804 2827, 2828.

P. M. van Diemen ef al

592

Table 3-Pearson’s partial correlation coefficient between protein bands (crude or recombinant) recognized by Day 42 sera and egg excretion counts (EPG) on Days 21 and 42 from calves after oral infection with 100 000 Cooperia L, EPG Day 21 EPG Day 42 14.2 kDa 14-16 kDa 27 kDa 14.2 kDa (recomb.)

-0.31 -0.53* 0.12

-0.70*** -0.24 -0.58**

0.82***

d.f. = 19. *P < 0.05; **P < 0.01; ***P < 0.001. antigen, only less intense (Table 2). In all Groups, the 2 parameters were positively correlated (r > 0.75, P < 0.01). Significant negative correlations were

found betweenthe stainingintensity of both the 14 1dkDa (crude) protein complex and the 14.2-kDa recombinantprotein, and EPG levelson Day 42 p.i. (Table 3). The stainingintensity of the 27-kDa band was significantly negatively correlated with EPG on Day 21 pi. (Table 3). DISCUSSION

In this study, binding of individual sera from 3 groupsof calvesto adult Cooperia antigenfragments was compared.The calveswere selectedon their egg excretion pattern as an indication of resistance/susceptibilityto primary infection with 100000 infective C. oncophora larvae. The clusteranalyseshad revealedthat parasiteestablishment and expulsionare 2 underlying mechanismsof variation betweenEPG curves (Ploeger, unpublished).Basedon the cluster analysesresultsand their own EPG pattern, Group 1 calveswereclassifiedasresistant,having a low establishmentof worms. The Group 2 and 3 calveswere classifiedas susceptibleto establishmentof worms as indicatedby the initially high EPG on day 21. Further, Group 2 calveswere also classifiedas responsiveto an already establishedworm burden in contrast with Group 3 calves.In comparisonwith Group 3 calves, on day 42 p.i. Group 2 calves had a lower worm burdenand a reducedsexratio, aswell asa substantial reduction in EPG betweendays 21 and 28p.i. These observationsindicate that Group 2 calves showed worm expulsion.Additionally, the data on the number of eggs in utero per female worm, and again the reduction in EPG in the 4th week p.i., indicate that particularly Group 2 calves also had respondedto the reproductive capacity of the worm population, thereby reducingfecundity. Other than beingresistant to establishmentof infection, Group 1 calvesdid not appearto respondto the low numbersof surviving worms,asjudged by, for example.a normal sexratio and a high number of eggs in utero per femaleworm. This apparentunresponsiveness of Group 1 calvesto

an establishedadult worm population may simply be due to the relatively low numbersof worms present. It seemsthat Group 1 and Group 2 calves,in spiteof their different EPG patterns had little problem with the Cooperiu infection. Group 3, however,represented calvesthat wereunableto respondto Cooperiu infection, and might need the help of anthelmintics to prevent health problemsand growth retardation, as well as to prevent the high egg output and resulting pasture contamination. If any relationship between the recognition of low weight C. oncophora antigens and EPG pattern exists,it shouldshowin thesegroups of selectedcalves. The presentresultsconfirm the potential value of the 14-l6-kDa protein fragment of adult Cooperia for serodiagnosticpurposesas proposedpreviously (Nieuwland et al., 1995;van Diemenet al., 1996).All calvesinvestigatedso far recognizedthis complex of protein fragmentsto, at least,someextent after infection. The recognition of the 14-16-kDaprotein complex alone, however, may not be sufficient to distinguishbetweenanimalsresistantor susceptibleto Cooperia infections. Yet, when combinedwith recognition of the 27-kDa fragment, it may be possible to differentiate between“types” of calvesaswasindicatedby the bindingcharacteristicsof serafrom calves selected on their egg excretion pattern. The current data suggest that resistance of calves to C. oncophoru,

as illustrated by Group 1 calves,may be relatedto a systemicantibody responseagainstat least2 protein fragments(14-16kDa and 27kDa) of adult Cooperiu antigen. The “totally unresponsive”-classified Group 3 had an evident lower responseto thesefragments (Table 2). The present results and the above-mentioned characterizationof the clustersmakeit tempting to speculatethat humoral responsiveness to the 27-kDa fragmentis associated with protection against establishment of Cooperia spp.in the gut, assupported by the negativecorrelation of 27-kDa recognitionand EPG on Day 21 pi. Theseresultsindicate the need for further studiesusingthe presentlyinvestigatedand possiblyother adult antigen fragments.Such studies shouldinclude for instance:repeatingthe study with other and preferably more calves; investigating the temporal kinetics of the antibody responsesto unravel, for instance,the recognition patterns at the time that the EPG of Group 2 calvesdecreases dramatically; investigating (accompanying) cellular responses;and extending the infection regimewith secondary challengeinfections. The recombinant 14.2kDa may provide a tool for immunodiagnostics,becauseof its high positive correlation with the recognition of the l+16-kDa (crude) protein complex, its unlimited production potential,

supposedlybetter standardization, and possibly a

Recognition of low weight C. oncophoru antigens

;i):

quantitation of microgram quantities of protein utilizing higher specificity.A disadvantageof the recombinant the principle of proteinAye binding. A/ro/yirc,a/ Bio14.2kDa may be a lower sensitivity, as indicated by chemistry 72: 248-254. the difference in the correlation coefficient between De Graaf D. C.. Berghen P., Hilderson H.. de ( ‘ock H the recombinant and crude fragmentsand EPG on & Vercruysse J. 1993. Identification and purification of Cooperia oncophorcr-specific antigens to improve seroDay 42 p.i. The presentstudy suggeststhat devellogical diagnosis. fnrrmatic~nu~.iournalf~r Parasi~c&~,v~~ 23: opment of a recombinant27-kDa protein may prove 141m144. worthwhile for further studieson immune-mediated Nieuwland M. G. B.. Ploeger H. W.. Kloosterman A. & resistanceto Cooperia. Parmentier H. K. 1995. Systemic antibody respconsesof AcknowledgementsThe authors acknowledge the staff of the university farm “De A. P. Minderhoudhoeve” for their assistance in this study.

REFERENCES

Albers G. A. A. 1981. Genetic Resistance to Experimental Cooperia oncophora Irzfections in Calves. Communications Agricultural University Wageningen No. 811. H. Veenman & zonen B.V., Wageningen. Borgsteede F. H. M. & Hendriks J. 1973. Fen kwantitatieve methode voor het kweken en verzamelen van infectieuze larven van maagdarmwormen. Tijdschrift voor Diergeneeskunde

98: 280-286.

Bradford M. M. 1976. A rapid and sensitive method for the

calves to low molecular weight Cooperia oncophr~rtr antigens. Veterinary Parasitology 59: 23 l-239. Parmentier H. K.. Ploeger H. W.. Nieuwland M. G. 8,. Souren P. J. E., van Pinxteren L. A. H., Rietveld F W .. de Vries Reilingh G. & Kloosterman A. 1995. Low molecular weight Cooperia oncophora antigens: character&non and humoral immune responses in calves mono-infected with 100 000 infective larvae. Veterinary Parmifo/frg,~ 59: 2 I9 230. Van den Brink R. 197 I Een eenvoudige McMaster methode voor het tellen van Trichostrongyiiden eieren in runderfaeces. T@!schrijt war Diergeneeskunde 5: 26 I- 269 Van Diemen P. M.. Ploeger H. W., Nieuwland M. G. H., Rietveld F. W. & Parmentier H. K. 1996. Recogtution of low molecular weight Cooperia oncophora antigens after primary and trickle-infection of calves with third-stage infective iarvae. Intw~afiotud Jourml lix Paru.sitt~li~~~~ 26: 1305m1310.