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Identification and partial purification of a 26 kilodalton antigen of adult Haemonchus contortus
International
Journalfor
Parenrology,
Pergamon OO20-7519(95)00127-1
Vol. 26. No. 3. pp. 31 I-318. 1996 Australian Society for Parasitology Elsevim Science Ltd Printed in Great Britain CO2LL7519196 $15.00 + 0.00
Identification and Partial Purification of a 26 Kilodalton Antigen ol,Adult Haemonchus contortus M. T. GOMEZ-MUROZ, Departamento
de Patologia
M. CUQUERELLA
and J. M. ALUNDA-
Animal I, Fat. Veterinaria, Universidad Madrid, 28040 Madrid, Spain
(4 November
1994; accepted
7 November
Complutense
de
1995)
Abstract-Gomez-Mmioz M. T., Cuquerella M. & Ahmda J. M. 1996. Identification and partial purification of a 26 kilodalton antigen of adult Haemonchus contortus. International Journal far Parasitology 26: 311-318. Adult H. cantartus soluble extracts were fractionated by means of gel filtration (S-200) and anion exchange chromatography (DEAE-Sephacel). Fractions from both analyses were checked by ELISA and western blotting (WB) with sera from lambs infected with H. contortus, monospecific heterologous sera (anti-TricAostrorrgylus caluhrifiwnis and anti-Teladorsagia circumcinta) and sera from naturally infected sheep with mixed trichostrongylid infections. High cross reactivity was seen between H. cantorfus and heterologous sera, particularly with the anti-T. colubriformis serum, when fractions from gel filtration were checked by ELISA. Individual fractions containing the highest positive/negative and positive/heterologous ratios were pooled and analysed by SDS-PAGE. One of the pools (A4) containing 2 regions around 48-55 and 25-27 kDa were strongly recognized by homologous sera in WB. Similar results were obtained with the first peak eluted in the DEAE-Sephacel chromatography with NaClO.1 M. The pooled fraction A4 from gel filtration was further fractionated by anion exchange chromatography and the peak obtained with the NaCl gradient contained a ea. 26 kDa antigen apparently specific for the diagnosis of H. contortus infections in lambs. Key words: Lambs; H. trichostrongylids; sheep.
contortus;
ELISA;
western
correspondence
should
be addressed.
antigens;
diagnosis;
chromatography;
(detection of genetic material in parasites’ eggs by PCR) (Roos & Grant, 1993; Christensen, Zarlenga & Gasbarre, 1994) could solve some of the problems but are also restricted to patent periods of the infection and have not been assessed economically in sheep production. The detection of parasite antigens would be an ideal method. This approach, however, was unsuccessful when determining circulating H. contortus antigens in infected sheep (Petit, P&y & Luffau, 1981) and the more recent contribution by Ellis et al. (1993) detecting surface antigens of the parasite in the faeces of infected animals fell below expectations since no clear discrimination was seen in spite of the high parasitic burdens found on occasion. Preliminary work carried out with total extracts of the parasite using serology with different immuno-
INTRODUCTION
Haemonchosis constitutes an important constraint in sheep production in both acute and chronic form in many parts of the world. Diagnosis of infection is usually carried out by means of coproscopy, faecal culture and identification of infective larvae. Besides the restriction to patent infections, this method is time consuming, it requires specialized personnel and therefore might not be cost effective. Alternative indirect diagnostic systems would constitute a considerable advantage for clinical and epidemiological work. Recently introduced molecular biology methods *To whom 341-394-3908.
blotting;
Fax:
311
312
M. T. Gomez-Mufioz
logical methods did not yield consistent results (Smith, 1977; Duncan, Smith & Dargie, 1978; Adams & Beh, 1981). Since the lack of sensitivity of the methods assayed was probably more related to the complex nature of the antigens used than to the inability of the sheep to recognize the parasite (Charley-Poulain, Luffau & P&y, 1984) more experimental work was needed. Serological evidence that lambs can recognize H. contortus antigens has been obtained (Haig et al., 1990; Hendrikx, 1990; Cuquerella, G6mez-Mufioz & Alunda, 1991). More recently a potentially useful soluble antigen for diagnosis of H. contortus infections in lambs has been reported (Cuquerella et al., 1993; Cuquerella et al., 1994) and later also a 24 kD component of secretory/excretory products of adult parasites (Schallig et al., 1994). The present isolation
paper describes of a 26 kDa
the identification Ag of adult H.
and partial contortus by
means of gel filtration and anion exchange chromatography. The partially purified Ag was examined for diagnosis of haemonchosis in lambs. MATERIALS AND METHODS Parasitological techniques. Faecal egg output, expressed as eggs per gram of faeces (epg), in the experimentally and naturally infected animals, was determined by coproscopical analysis using a modified McMaster technique. Faecal cultures were done at 26°C (10 days) and recovered L3 were identified (MAFF, 1971). Antigenie material. Adult H. contortus, largely females, were obtained from the abomasa of lambs kept at our department and infected with a pure isolate of the parasite kindly supplied by Merck Sharp and Dohme, Spain. The adult worms were carefully picked off the mucosa and maintained during processing in cold (4°C) PBS and with proteases inhibitors (1 mM Phenylmethylsulphonyl fluoride, PMSF (Merck, Darmstadt); 1 mM Ethylenediaminetetraacetic acid, EDTA (Sigma, St Louis); 50 pg/ml N-Tosyl-LPhenylalanine Chlorometyl Ketone, TPCK and 25 pg/ml Na-p-Tosyl-L-Lysine Chloromethyl Ketone, TLCK (Boehringer, Manheim). Soluble extracts were routinely obtained in a glass-in-glass homogenizer followed by centrifugation at 30,000~ g (Klesius et al., 1984). After recovery of the supernatant fluid, the protein content was estimated by the method of Bradford (Bradford, 1976). The extracts were used immediately or preserved at - 80°C. Serum samples. Eleven serum samples were obtained from worm-free lambs slaughtered at a local abattoir. Homologous sera from infected animals came from 6-month-old lambs (n =-5j 43 days after challenge with 5000 L3 of H. contortus. Heterologous sera were obtained from lambs aged 5-6 months monospecifically infected with 35000 and 40000 L3 of Trichostrongylus colubriformis (n=2) and 25000 of L3 of Teladorsagia circumcinta (n = 2) and bled about 2 mofiths pbst-infection. In addition, 65 sera collected in 8 flocks of the Madrid region, Murcia and Le6n [no. 3: San Sebastian de 10s Reyes (n= 10); no. 4: Torrelaguna (n = 8); no. 5: El Espartal 1 (n = 8); no. 6: El Espartal 2
et al.
(n = 4); no. 7: Murcia (n = 4); no. 8: Le6n (n = 2); no. 9: Talamanca de1 Jarama (n = 9); no. 10: El Molar (n = 20)] from sheep with mixed natural trichostrongylid infections were examined. Average epg value of the sampled flocks was 350 epg although individual counts were highly variable (25- 1680 epg). Faecal cultures from the flocks showed that mixed trichostrongylid infections were due mainly to Te. circumcinta (cu. 50%) and Trichostrongylus (T. axei and T. colubriformis), with low numbers of other genera (Nematodirus, Trichuris). In addition some animals had Dicroceolium dendriticum and Moniezia eggs. H. contortus was present in 2 farms (El Espartal I and El Molar). The sera from the farms were pooled for use in western blotting. Gel @ration and ion exchange chromatography. Gel filtration of H. contortus adult soluble extract (ASE) was
carried out in a 85-cm-column (Pharma%ia,
Sweden).
The
flow
using Sephacryl S-200 rate
was
5.2 ml/h
and
2-ml-fractions were collected. The eluate was read by determining
the absorbance
at 280 nm (A 280).
As molecular
mass markers (Mr), Blue dextran (2000 kDa), P-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa) and Cytochrome C (12.4 kDa) (Sigma, St Louis) were used. Ion exchange chromatography of ASE of H. contortus and the selected’ booled fraction of gel filtration was performed on a DEAE-Sephacel (Sigma, St Louis) 30-cm-column and the soluble extracts were dialyzed and loaded in 50 mM Tris-HCl, pH 7.4. Elution was done with a continuous gradient of NaCl (O-O.4 M) and tested by A 280 determination. ELBA. The assay conditions were as those described previously (Cuquerella et al., 1991) with slight modifications. Briefly, ASE and the fractions obtained were bound to plates (Maxisorp @ Nunc, Denmark) at different concentrations in coating buffer (0.05 M carbonate buffer, pH 9.6) 16 h at 4°C. BSA 5% in PBS was used as blocking solution (1 h at 37°C); sera were used at a l/l00 dilution in PBS-Tween 20 (1 h at 37°C) and the conjugate (alkaline phosphatase-labelled rabbit anti-sheep IgG, Sigma) was used at a l/l000 dilution (1 h at 37°C). Colour was developed with 1 mg/ml I,4 p-nitrophenol phosphate (PNPP) 1 h at 37°C. The optical density was read at 405 nm in a EAR 400 ELISA reader (Invesgen, Madrid). SDS-PAGE. Western blotting and glycoprotein detection. For SDS-PAGE, protein was used at 0.5 mg/ml (ASE) or
25OG120 pg/ml (fractions) mixed with 1% Sodium dodecyl sulphate trations). phoresed running markers Pharmacia gels were transferred blotting pore) for (Bio-Rad).
(SDS) and 5% 2-Mercaptoethanol (final concenAfter boiling for 10 min, samples were electrointo stacking gels containing 5% acrylamide and gels contained 10.5% or 12.5% acrylamide. A4r (94, 67, 43, 30, 20.1, 14.4 kDa) were from (Sweden). After electrophoresis at 150 V, the then fixed and stained with Coomassie Blue or on to Immobilon P (Millipore) for western (WB) or on to Nitrocellulose meqbranes (Milliglycoprotein estimations in a Transblot chamber The WB conditions were as described previously
(Cuquerella et al., 1993). Briefly, after washing 3-times with 10 mM Tris-HC1 containing 0.15 M NaCl (TBS) 0.05% Tween 20 (TBS-T), membranes were blocked
and with
Partial
purification
of a H. contortus
diagnostic
antigen
313
1.4 _’ ‘. ., .yj . ...”
1.2
\ - \
_ _ $;.;; \-
1
0 3
0.8
a
0.6
I
25
1
35 Fraction
45
55
65
number
0.2
c,
o-
0
10
20
I
I
I
I
I
I
40
50
60
70
80
90
Fraction Fig. 1. Elution pooled. Insert:
Elution comparable chromatogram
RESULTS profiles obtained from the ASE were in all 4 trials and a representative is shown in Fig. 1. Eluted peptides
did show
2 main
kDa
and
peaks having 78-42
apparent
kDa. Individual by ELISA with
t
100
number
profile of the gel filtration on Sephacryl S-200 of ASE ratio of the O.D. positive/O.D. negative for homologous (. .) and anti-Teladorsagia (--).
TBS-T and 5% powdered skimmed milk (blocking solution) for 1 h at room temperature. After washing in the same manner the membranes were cut in strips and incubated with 1 : 50 sera diluted in blocking solution for 3 h at 37°C. The conjugate (horseradish peroxidase-labelled donkey anti-sheep IgG, Sigma) was used after a new washing at 1 : 1000 diluted in TBS-T (1 h, 37°C) and colour was developed with 4-chloronaphtol (0.5 mg/ml, 10 min, room temperature). Finally the strips were washed in tap water to stop the colour reaction. Glycosylated proteins were detected with the Digoxigenin glycan detection kit (Boehringer Manheim, Germany) in nitrocellulose membranes.
>200
..
Mr
of
fractions pooled anti-
collected were checked Huemonchus sera (Insert). Highest Positive/Negative (P/N) ratio was observed between fractions nos 36-41, this representing Mr between 150 and 78 kDa. A similar check was done with heterologous sera, resulting in high cross reactivity between H. contortus and T. colubriformis, particularly with peptides over 150 kDa. Given the low recovery of antigenic material, individual fractions were pooled (Al, A2, A3, A4) corresponding to Mr of >200, 150-200, 78-l 50 and 42-78 kDa respectively. The pooled
of H. contortus. Bars: fractions sera (-), anti-Trichostrongylus
fractions were checked by ELISA and analysed by SDS-PAGE and western blotting (WB). ELISA did show an increase in O.D. using pooled fractions A3 and A4 when compared to the values obtained with the ASE (not shown). Aliquots from the fractions were subjected to electroblotting after SDS-PAGE, and probed with sera of lambs infected only with H. contortus. With the exception of the Al fraction which displayed immune reactivity in a region over 94 kDa, A2, A3 and A4 peaks reacted in a similar way with reactivities in the regions of 48-55 kDa and 27-35 kDa, with low stringency washings (not shown). More stringent conditions showed that the A4 fraction (Fig. 2) had two immunodominant regions (48-55, 25527 kDa) recognized by H. contortus infected lambs (lane 2) whereas uninfected control animals sera (lane 1) did not show this recognition pattern; in addition Te. circumcinta infected lambs did not show any noticeable reactivity in the mol.wt range examined (lane 4). However, anti-T. colubriformis sera reacted with a peptide having around 35-36 kDa (lane 3). This heterologous immune recognition could explain the cross reactivity found in the ELISA with this fraction. Parallel experiments were conducted to explore the possibility of using ion exchange chromatography to identify any unique antigens of H. contortus. When applying ASE, dialyzed against 50 mM TrissHCl, to an anion exchange chromatography (DEAE-
314
M. T. Gomez-Mufioz
12
3
enhanced slightly (0.2- and 0.4-times respectively) the sensitivity of the ELISA when compared to the values obtained with total soluble extract of adult H. contortus (not shown); the highest specificity was found with a2. Western blotting analyses carried out with aliquots of these peaks after SDS-PAGE (Fig. 3) showed that fraction a3 mainly displayed reactivity around 55 kDa, of apparently little diagnostic value since it was also recognized by serum. Similar results were anti-T. colubriformis obtained with the a4 peak (not shown). The unbound fraction (al) exhibited reactivity around 25-28 kDa with the homologous serum. Less reactivity was also observed with heterologous reagents, particularly with the anti-Trichostrongylus sera. Homologous sera reacted with the A2 fraction at 25-27 kDa and ea. 55 kDa regions whereas heterologous and control sera apparently did not recognize these peptides; however, anti-Trichostrongylus serum reacted with 22-23 kDa and 67 kDa, peptides not recognized by the homologous sera. The reactivity pattern obtained with this fraction (a2) and the homologous sera was roughly similar to that obtained with the a4 fraction obtained by gel filtration of ASE with S-200. Moredver the ELISA improvement with the A4 fraction was higher, and the quantity of material from gel filtration was greater. Therefore, a4 was subjected to anion exchange chromatography (DEAE-Sephacel) and the elution profile is shown at Fig. 4. A relatively
4
MW
20.1, 14A-
Fig. 2. Western blot analysis of the a4 fraction from gel filtration, probed with negative sera (lane l), homologous sera (lane 2), anti-Trichostrongyl (lane 3) and antiTeladorsagia (lane 4). MW: mol.wt markers in kilodalton (kDa).
Sephacel) column the unbound fraction (al) was followed by 3 peaks eluted between 0.1 and 0.2 M NaCl (not shown). Fractions corresponding to the peaks were pooled and dialyzed overnight (PBS, 4°C). ELISA results with the pooled fractions a3 and a4 NHTO
N
et al.
H
T
0
NHTO
YW 94-
I
67-
Fig. 3. Western blot analysis of the pooled fractions from DEAE-Sephacel chromatography of ASE (al, a2, a3). MW: mol.wt markers in kDa. N: negative sera; H: homologous sera; T: antiTrichostrongylus; 0: anti-Teladorsagia.
Partial purification of a H. contortus diagnostic antigen
315
A41
0.:
z
a 0.1
0 0
10
20
30
40
50
Fraction
60
70
80
90
IO0
110
1200
number
Fig. 4. Elution profile of the a4 fraction from S-200 gel filtration after DEAE-Sephacel chromatography. Bars: pooled fractions. simple pattern was obtained and the 2 fractions with highest A 280 values were pooled (a41). Optical density (OD) at 405 nm in ELISA of positive sera/OD of negative sera (P/N ratio) with this pooled fraction
N
H
T
0
T
0
YW 64. 679
4330-
20.1-
14.4.
6
Fig. 5. Western blot analysis of A41 fractions probed with homologous (anti-Hoemonchus), H; negative sera, N; and heterologous sera (anti-Trichostrongylus, T; anti-Tel&orsagas, 0). MW, mol.wt markers in kDa.
(A41) was 4.04 and OD of positive sera/OD of heterologous sera (P/H ratios) were 2.15 for T. colubriformis and 3.42 for Te. circumcinta-infected animals. These results improved the ELISA findings with ASE and A4. Sera from the monospecifically infected lambs with H. contortus but not those from uninfected control lambs or animals with monospecific heterologous infections (lanes 3-6) (Fig. 5) recognized a peptide having ca. 26 kDa, apparently not glycosylated (not shown). This A41 fraction did not contain the proteins ca. 55 kDa present in the A4 peak from S-200 gel filtration of ASE although a faint reactivity was present at 44 kDa. However, this reactivity disappeared with higher serum dilution or more stringent washings. When A41 was probed with pooled sera from sheep flocks with mixed natural infections with trichostrongylids, WB analysis showed that the 26 kDa antigen present in the fraction was not recognized, under our conditions, by sheep sera from flocks where H. contortus was not present (Fig. 6). Immune recognition was only observed with one of the farms with H. contortus. In addition other reactivities (ca. 44 and 22-23 kDa) were present in some flocks. DISCUSSION Several problems have been found in the use of ELISA methods to detect H. contortus primary
316
M. T. Gomez-Mufioz
et al
MW @4(i
679 43-
I
,
1 i
14.4-.
_ 1
2
-. 9
10
Fig: 6. Western blot analysis of A41 fraction probed with pooled sera of sheep from different flocks wi’th natural mixed trichostrongylid infections (lanes 3-10). Numbers correspond to those given in Materials and Methods. Lane 1: positive control. Lane 2: negative control. MW: mol.wt markers in kDa. infections in lambs, the applications being restricted to a country, breed or age of the animals. One of the possible reasons for this lack of sensitivity and specificity in ELISA could be the extensive antigenic cross reactivity between this parasite and related and unrelated nematodes (Shamansky et al., 1989; Cuquerella et al., 1993; Schallig et al., 1994). In spite of the importance of sheep haemonchosis, information about the characterization of the parasite is relatively scarce. Except for the pioneering work by Neilson (1969) and Ozerol & Silverman (1969, 1970) most research has been related to the cuticle (i.e. Cox et al., ,1989; Shamansky et al., 1989; Gamble et al., 1990), excretory/secretory (E/S) antigens and exsheathing fluid (Ozerol &: Silverman, 1969; Gamble et al., 1990; Schallig et al., 1994). This lack of interest in soluble extracts of the parasite probably relates to the lower immunogenicity suggested (Parkhouse et al., 1987). However, in H. contortus infections in sheep it has been shown either in single point estimations (Hendrikx, 1990; Haig et a/., 1990; Cuquerella et al., 1991) or serial studies (Cuquerella et al., 1993) that some soluble proteins of the parasite are recognized by infected animals, particularly two peptides from adult worms of approximately 25 and 26 kDa mol.wt (Cuquerella et al., 1993). Our results show that a 2-step purification process involving anion exchange chromatography of the 4278 kDa pooled fraction obtained in gel filtration of
soluble extracts of adult H. contortus allows the isolation of a fraction containing a peptide having under reducing and denaturing conditions ca. 26 kDa which is recognized by H. contortus infected lambs but not by negative sera, thus confirming the potential diagnostic value of this region (Cuquerella et al., 1993). An intriguing point is brought up by a 24 kDa antigen reported later by Schallig et al. (1994) in E/S products of adult H. contortus. Although comparable electrophoretic motility does not constitute an absolute criterion for peptide identity, it is likely that the 24 kDa antigen is probably the same peptide reported earlier by us (Cuquerella et al., 1993) and partially purified herein. Actually, immunodetection patterns obtained with E/S components and microdissected oesophagus of H. contortus (Takits et al., 1995) were very close to those obtained by us previously (Cuquerella et al., 1993) although this fact was not acknowledged. Since most infections in sheep by gastrointestinal nematodes under field conditions are mixed, the specificity of reactivity to the partially purified antigen is critical. Besides the extensive antigenic cross reactivity between H. contortus and other nematodes (see above) some heterologous sera react on occasion more strongly than homologous sera with H. contortus antigens (Anderson et al., 1989).‘However, the 26 kDa antigen seems to be specific since it was not recognized by sera of monospecifically infected lambs with T. colubriformis and Te. circumcinta. Moreover,
Partial
purification
of a H. contortus
sheep sera from flocks of the surrounding area with average natural trichostrongylid mixed infections (Trichostrongylus, Teladorsagia) did not show significant reactivity with this antigen, whereas one of the flocks with H. contortus reacted with this antigen. However, the proportion of H. contortus in the mixed infections was low and a more complete study is being done with higher parasitic burdens and different developmental periods (prepatent, patent, postpatent, pretreatment, post-treatment). This antigen has been found to be non-glycosylated and no further characterization of the antigen has been made, so that the original molecular weight of the recognized peptide is not known yet. The yield obtained by using this 2-step process (gel filtration and anion exchange chromatography) was low (the A41 fraction represented as a whole cu. 2% of ASE) and some contaminating peptides were present. Its use for diagnosis or prophylaxis requires some additional research including cloning, chemical synthesis, and monoclonal or polyclonal antibodies which is in progress. Acknowledgements-We deeply thank J. Martin-Checa, 0. Rodriguez and B. Lopez for help provided, and the financial support of CICYT (GAN89/324 and AGF95/648). M. T. Gomez-MuAoz had a predoctoral fellowship from the UCM. Kind gifts of infective larvae by Dr J. Uriarte (ITEA Zaragoza) and Prof. J. L. Duncan (Glasgow, Scotland) (Te. circumcinta and T. colubrzformis) are greatly appreciated. REFERENCES Adams D. B. & Beh K. J. 1981. Immunity acquired by sheep from an experimental infection with Haemonchus contortus. International Journal for Parasitology 11: 381-386. Anderson D. V., Dixon S. C., Graham R. B., Smith W. D. & Tucker E. M. 1989. Ovine monoclonal antibody to Ostertagia circumcinta. Biochemical Society Transactions 17: 736. Bradford M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding. Annales ofBiochemistry 72: 248-254. Charley-Poulain J.. LutTau G. & P&y P. 1984. Serum and abomasal antibody response of sheep to infections with Haemonchus contortus. Veterinary Parasitology 14: 1299141. Christensen C. M., Zarlenga D. S. & Gasbarre L. C. 1994. Ostertagia, Haemonchus. Cooperia and Oesophagostomum: Construction and characterization of genus-specific DNA probes to differentiate important parasites of cattle. E.xperimental Parasitology 78: 933100. Cox G. N., Shamansky L. M. & Boisvenue R. J. 1989. Identification and preliminary characterization of cuticular surface proteins of Haemonchus contortus. Molecular and Biochemical Parasitology 36: 233.-242.
diagnostic
antigen
317
Cuquerella M., Gcmez-Muiioz M. T. & Alunda J. M. 1991. Serum IgG response of Manchego lambs to infection with Haemonchus contortus and preliminary characterization of adult antigens. Veterinary Parasitology 38: 131-143. Cuquerella M., Gomez-Muiioz M. T., Carrera L., de la Fuente C. & Alunda J. M. 1994. Cross antigenicity among ovine trichostrongyloidea. Preliminary report. Veterinary Parasitology 53: 243325 1. Cuquerella M., Gomez-Mufioz M. T., de la Fuente C., Carrera L. & Alunda J. M. 1993. Lamb serum recognition of infective larvae and adult Haemonchus contortus antigens. Veterinary Parasitology 49: 255-264. Duncan J. L., Smith W. D. & Dargie J. D. 1978. Possible relationship of level of mucosal IgA and serum IgG to immune unresponsiveness of lambs to Haemonchus contortus. Veterinary Parasitology 4: 21-27. Ellis T. M., Gregory A., Turnor R., Kalkhoven M. & Wroth R. H. 1993. Detection of Haemonchus contortus surface antigens in faeces from infected sheep. Veterinary Parasitology 51: 85-97. Gamble H. R., Purcell J. P. & Fetterer R. H. 1990. Biochemical characterization of cuticle polipeptides from the infective larvae of Haemonchus contortus. Comparative Biochemistry Physiology 96B: 421-429. Haig D. M., Windon R., Blackie W., Brown D. & Smith W. D. 1989. Parasite-specific T cell responses of sheep following live infection with the gastric nematode Haemonchus contortus. Parasite Immunology 11: 463-477. Hendrikx W. M. L. 1990. De nematode Haemonchus contortus: antigeenkarakter en immuunrespons bij konijnen en schapen. Tijdschrzft Diergeneeskunde 115: 1092-I 101. Klesius P. H., Washburn S. M.. Ciordia H. & Haynes T. B. 1984. Lymphocyte reactivity to Ostertagia ostertagi L3 antigen in type 1 ostertagiasis. American Journal of Veterinarv Research 45: 230-233. MAFF (Ministry of Agriculture, Fisheries and Food). 1971. Manual of Veterinary Parasitological Laborator.y Techniques. HMSO, London. Neilson J. T. M. 1969. Gel Filtration and Disc Electrophoresis of a Somatic Extract and Excretions and Secretions of Haemonchus contortus larvae. Experimental Parasitology 25: 13ll141. Ozerol N. H. & Silverman P. H. 1969. Partial characterization of Haemonchus contortus exsheathing fluid. The Journal of Parasitology 55: 79987. Ozerol N. H. & Silverman P. H. 1970. Further characterization of active metabolites from histotropic larvae of Haemonchus contortus cultured in vitro. The Journal of Parasitology 56: 1199- 1205. Parkhouse R. M. E., Almond N. M.. Cabrera 2. & Harnett W. 1987. Nematode antigens in protection, diagnosis and pathology. Veterinary Immunology and Immunopathology 17: 313-334. Petit A., Pery P. & Luffau G. 1981. Circulating antigens in ovine haemonchosis. Annales de Recherches Veterinaires 12: l-9. Roos M. H. & Grant W. N. 1993. Species-specific PCR for the parasitic nematodes Haenzonchus contortus and Trichostrongylus colubrijormis. International Journal for Parasitology 23: 4 19-42 1.
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Schallig H. D. F. H., van Leeuwen M. A. W. & Hendrikx W. M. L. 1994. Immune responses of Texel sheep to excretory/secretory products of adult Haemonchus contortus. Parasitology 108: 351-351. Shamansky L. M., Pratt D., Boisvenue J. & Cox G. N. 1989. Cuticle collagen genes of Haemonchus contortus and Caenorhabditis &guns are highly conserved. Molecular and Biochemical Parasitology 31: 73-86.
et al.
Smith W. D. 1977. Anti-larval antibodies in the serum and abomasal mucus of sheep hyperinfected with Haemonthus contortus. Research in Veterinary Science 22: 334-338. Takits C., Schallig H. D. F. H., van Leeuwen M. A. W. & Hendrikx W. M. L. 1995. Immune responses of sheep to microdissected parts of Haemonchus contortus. International Journal for Parasitology 25: 857-860.
Journalfor
Parenrology,
Pergamon OO20-7519(95)00127-1
Vol. 26. No. 3. pp. 31 I-318. 1996 Australian Society for Parasitology Elsevim Science Ltd Printed in Great Britain CO2LL7519196 $15.00 + 0.00
Identification and Partial Purification of a 26 Kilodalton Antigen ol,Adult Haemonchus contortus M. T. GOMEZ-MUROZ, Departamento
de Patologia
M. CUQUERELLA
and J. M. ALUNDA-
Animal I, Fat. Veterinaria, Universidad Madrid, 28040 Madrid, Spain
(4 November
1994; accepted
7 November
Complutense
de
1995)
Abstract-Gomez-Mmioz M. T., Cuquerella M. & Ahmda J. M. 1996. Identification and partial purification of a 26 kilodalton antigen of adult Haemonchus contortus. International Journal far Parasitology 26: 311-318. Adult H. cantartus soluble extracts were fractionated by means of gel filtration (S-200) and anion exchange chromatography (DEAE-Sephacel). Fractions from both analyses were checked by ELISA and western blotting (WB) with sera from lambs infected with H. contortus, monospecific heterologous sera (anti-TricAostrorrgylus caluhrifiwnis and anti-Teladorsagia circumcinta) and sera from naturally infected sheep with mixed trichostrongylid infections. High cross reactivity was seen between H. cantorfus and heterologous sera, particularly with the anti-T. colubriformis serum, when fractions from gel filtration were checked by ELISA. Individual fractions containing the highest positive/negative and positive/heterologous ratios were pooled and analysed by SDS-PAGE. One of the pools (A4) containing 2 regions around 48-55 and 25-27 kDa were strongly recognized by homologous sera in WB. Similar results were obtained with the first peak eluted in the DEAE-Sephacel chromatography with NaClO.1 M. The pooled fraction A4 from gel filtration was further fractionated by anion exchange chromatography and the peak obtained with the NaCl gradient contained a ea. 26 kDa antigen apparently specific for the diagnosis of H. contortus infections in lambs. Key words: Lambs; H. trichostrongylids; sheep.
contortus;
ELISA;
western
correspondence
should
be addressed.
antigens;
diagnosis;
chromatography;
(detection of genetic material in parasites’ eggs by PCR) (Roos & Grant, 1993; Christensen, Zarlenga & Gasbarre, 1994) could solve some of the problems but are also restricted to patent periods of the infection and have not been assessed economically in sheep production. The detection of parasite antigens would be an ideal method. This approach, however, was unsuccessful when determining circulating H. contortus antigens in infected sheep (Petit, P&y & Luffau, 1981) and the more recent contribution by Ellis et al. (1993) detecting surface antigens of the parasite in the faeces of infected animals fell below expectations since no clear discrimination was seen in spite of the high parasitic burdens found on occasion. Preliminary work carried out with total extracts of the parasite using serology with different immuno-
INTRODUCTION
Haemonchosis constitutes an important constraint in sheep production in both acute and chronic form in many parts of the world. Diagnosis of infection is usually carried out by means of coproscopy, faecal culture and identification of infective larvae. Besides the restriction to patent infections, this method is time consuming, it requires specialized personnel and therefore might not be cost effective. Alternative indirect diagnostic systems would constitute a considerable advantage for clinical and epidemiological work. Recently introduced molecular biology methods *To whom 341-394-3908.
blotting;
Fax:
311
312
M. T. Gomez-Mufioz
logical methods did not yield consistent results (Smith, 1977; Duncan, Smith & Dargie, 1978; Adams & Beh, 1981). Since the lack of sensitivity of the methods assayed was probably more related to the complex nature of the antigens used than to the inability of the sheep to recognize the parasite (Charley-Poulain, Luffau & P&y, 1984) more experimental work was needed. Serological evidence that lambs can recognize H. contortus antigens has been obtained (Haig et al., 1990; Hendrikx, 1990; Cuquerella, G6mez-Mufioz & Alunda, 1991). More recently a potentially useful soluble antigen for diagnosis of H. contortus infections in lambs has been reported (Cuquerella et al., 1993; Cuquerella et al., 1994) and later also a 24 kD component of secretory/excretory products of adult parasites (Schallig et al., 1994). The present isolation
paper describes of a 26 kDa
the identification Ag of adult H.
and partial contortus by
means of gel filtration and anion exchange chromatography. The partially purified Ag was examined for diagnosis of haemonchosis in lambs. MATERIALS AND METHODS Parasitological techniques. Faecal egg output, expressed as eggs per gram of faeces (epg), in the experimentally and naturally infected animals, was determined by coproscopical analysis using a modified McMaster technique. Faecal cultures were done at 26°C (10 days) and recovered L3 were identified (MAFF, 1971). Antigenie material. Adult H. contortus, largely females, were obtained from the abomasa of lambs kept at our department and infected with a pure isolate of the parasite kindly supplied by Merck Sharp and Dohme, Spain. The adult worms were carefully picked off the mucosa and maintained during processing in cold (4°C) PBS and with proteases inhibitors (1 mM Phenylmethylsulphonyl fluoride, PMSF (Merck, Darmstadt); 1 mM Ethylenediaminetetraacetic acid, EDTA (Sigma, St Louis); 50 pg/ml N-Tosyl-LPhenylalanine Chlorometyl Ketone, TPCK and 25 pg/ml Na-p-Tosyl-L-Lysine Chloromethyl Ketone, TLCK (Boehringer, Manheim). Soluble extracts were routinely obtained in a glass-in-glass homogenizer followed by centrifugation at 30,000~ g (Klesius et al., 1984). After recovery of the supernatant fluid, the protein content was estimated by the method of Bradford (Bradford, 1976). The extracts were used immediately or preserved at - 80°C. Serum samples. Eleven serum samples were obtained from worm-free lambs slaughtered at a local abattoir. Homologous sera from infected animals came from 6-month-old lambs (n =-5j 43 days after challenge with 5000 L3 of H. contortus. Heterologous sera were obtained from lambs aged 5-6 months monospecifically infected with 35000 and 40000 L3 of Trichostrongylus colubriformis (n=2) and 25000 of L3 of Teladorsagia circumcinta (n = 2) and bled about 2 mofiths pbst-infection. In addition, 65 sera collected in 8 flocks of the Madrid region, Murcia and Le6n [no. 3: San Sebastian de 10s Reyes (n= 10); no. 4: Torrelaguna (n = 8); no. 5: El Espartal 1 (n = 8); no. 6: El Espartal 2
et al.
(n = 4); no. 7: Murcia (n = 4); no. 8: Le6n (n = 2); no. 9: Talamanca de1 Jarama (n = 9); no. 10: El Molar (n = 20)] from sheep with mixed natural trichostrongylid infections were examined. Average epg value of the sampled flocks was 350 epg although individual counts were highly variable (25- 1680 epg). Faecal cultures from the flocks showed that mixed trichostrongylid infections were due mainly to Te. circumcinta (cu. 50%) and Trichostrongylus (T. axei and T. colubriformis), with low numbers of other genera (Nematodirus, Trichuris). In addition some animals had Dicroceolium dendriticum and Moniezia eggs. H. contortus was present in 2 farms (El Espartal I and El Molar). The sera from the farms were pooled for use in western blotting. Gel @ration and ion exchange chromatography. Gel filtration of H. contortus adult soluble extract (ASE) was
carried out in a 85-cm-column (Pharma%ia,
Sweden).
The
flow
using Sephacryl S-200 rate
was
5.2 ml/h
and
2-ml-fractions were collected. The eluate was read by determining
the absorbance
at 280 nm (A 280).
As molecular
mass markers (Mr), Blue dextran (2000 kDa), P-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa) and Cytochrome C (12.4 kDa) (Sigma, St Louis) were used. Ion exchange chromatography of ASE of H. contortus and the selected’ booled fraction of gel filtration was performed on a DEAE-Sephacel (Sigma, St Louis) 30-cm-column and the soluble extracts were dialyzed and loaded in 50 mM Tris-HCl, pH 7.4. Elution was done with a continuous gradient of NaCl (O-O.4 M) and tested by A 280 determination. ELBA. The assay conditions were as those described previously (Cuquerella et al., 1991) with slight modifications. Briefly, ASE and the fractions obtained were bound to plates (Maxisorp @ Nunc, Denmark) at different concentrations in coating buffer (0.05 M carbonate buffer, pH 9.6) 16 h at 4°C. BSA 5% in PBS was used as blocking solution (1 h at 37°C); sera were used at a l/l00 dilution in PBS-Tween 20 (1 h at 37°C) and the conjugate (alkaline phosphatase-labelled rabbit anti-sheep IgG, Sigma) was used at a l/l000 dilution (1 h at 37°C). Colour was developed with 1 mg/ml I,4 p-nitrophenol phosphate (PNPP) 1 h at 37°C. The optical density was read at 405 nm in a EAR 400 ELISA reader (Invesgen, Madrid). SDS-PAGE. Western blotting and glycoprotein detection. For SDS-PAGE, protein was used at 0.5 mg/ml (ASE) or
25OG120 pg/ml (fractions) mixed with 1% Sodium dodecyl sulphate trations). phoresed running markers Pharmacia gels were transferred blotting pore) for (Bio-Rad).
(SDS) and 5% 2-Mercaptoethanol (final concenAfter boiling for 10 min, samples were electrointo stacking gels containing 5% acrylamide and gels contained 10.5% or 12.5% acrylamide. A4r (94, 67, 43, 30, 20.1, 14.4 kDa) were from (Sweden). After electrophoresis at 150 V, the then fixed and stained with Coomassie Blue or on to Immobilon P (Millipore) for western (WB) or on to Nitrocellulose meqbranes (Milliglycoprotein estimations in a Transblot chamber The WB conditions were as described previously
(Cuquerella et al., 1993). Briefly, after washing 3-times with 10 mM Tris-HC1 containing 0.15 M NaCl (TBS) 0.05% Tween 20 (TBS-T), membranes were blocked
and with
Partial
purification
of a H. contortus
diagnostic
antigen
313
1.4 _’ ‘. ., .yj . ...”
1.2
\ - \
_ _ $;.;; \-
1
0 3
0.8
a
0.6
I
25
1
35 Fraction
45
55
65
number
0.2
c,
o-
0
10
20
I
I
I
I
I
I
40
50
60
70
80
90
Fraction Fig. 1. Elution pooled. Insert:
Elution comparable chromatogram
RESULTS profiles obtained from the ASE were in all 4 trials and a representative is shown in Fig. 1. Eluted peptides
did show
2 main
kDa
and
peaks having 78-42
apparent
kDa. Individual by ELISA with
t
100
number
profile of the gel filtration on Sephacryl S-200 of ASE ratio of the O.D. positive/O.D. negative for homologous (. .) and anti-Teladorsagia (--).
TBS-T and 5% powdered skimmed milk (blocking solution) for 1 h at room temperature. After washing in the same manner the membranes were cut in strips and incubated with 1 : 50 sera diluted in blocking solution for 3 h at 37°C. The conjugate (horseradish peroxidase-labelled donkey anti-sheep IgG, Sigma) was used after a new washing at 1 : 1000 diluted in TBS-T (1 h, 37°C) and colour was developed with 4-chloronaphtol (0.5 mg/ml, 10 min, room temperature). Finally the strips were washed in tap water to stop the colour reaction. Glycosylated proteins were detected with the Digoxigenin glycan detection kit (Boehringer Manheim, Germany) in nitrocellulose membranes.
>200
..
Mr
of
fractions pooled anti-
collected were checked Huemonchus sera (Insert). Highest Positive/Negative (P/N) ratio was observed between fractions nos 36-41, this representing Mr between 150 and 78 kDa. A similar check was done with heterologous sera, resulting in high cross reactivity between H. contortus and T. colubriformis, particularly with peptides over 150 kDa. Given the low recovery of antigenic material, individual fractions were pooled (Al, A2, A3, A4) corresponding to Mr of >200, 150-200, 78-l 50 and 42-78 kDa respectively. The pooled
of H. contortus. Bars: fractions sera (-), anti-Trichostrongylus
fractions were checked by ELISA and analysed by SDS-PAGE and western blotting (WB). ELISA did show an increase in O.D. using pooled fractions A3 and A4 when compared to the values obtained with the ASE (not shown). Aliquots from the fractions were subjected to electroblotting after SDS-PAGE, and probed with sera of lambs infected only with H. contortus. With the exception of the Al fraction which displayed immune reactivity in a region over 94 kDa, A2, A3 and A4 peaks reacted in a similar way with reactivities in the regions of 48-55 kDa and 27-35 kDa, with low stringency washings (not shown). More stringent conditions showed that the A4 fraction (Fig. 2) had two immunodominant regions (48-55, 25527 kDa) recognized by H. contortus infected lambs (lane 2) whereas uninfected control animals sera (lane 1) did not show this recognition pattern; in addition Te. circumcinta infected lambs did not show any noticeable reactivity in the mol.wt range examined (lane 4). However, anti-T. colubriformis sera reacted with a peptide having around 35-36 kDa (lane 3). This heterologous immune recognition could explain the cross reactivity found in the ELISA with this fraction. Parallel experiments were conducted to explore the possibility of using ion exchange chromatography to identify any unique antigens of H. contortus. When applying ASE, dialyzed against 50 mM TrissHCl, to an anion exchange chromatography (DEAE-
314
M. T. Gomez-Mufioz
12
3
enhanced slightly (0.2- and 0.4-times respectively) the sensitivity of the ELISA when compared to the values obtained with total soluble extract of adult H. contortus (not shown); the highest specificity was found with a2. Western blotting analyses carried out with aliquots of these peaks after SDS-PAGE (Fig. 3) showed that fraction a3 mainly displayed reactivity around 55 kDa, of apparently little diagnostic value since it was also recognized by serum. Similar results were anti-T. colubriformis obtained with the a4 peak (not shown). The unbound fraction (al) exhibited reactivity around 25-28 kDa with the homologous serum. Less reactivity was also observed with heterologous reagents, particularly with the anti-Trichostrongylus sera. Homologous sera reacted with the A2 fraction at 25-27 kDa and ea. 55 kDa regions whereas heterologous and control sera apparently did not recognize these peptides; however, anti-Trichostrongylus serum reacted with 22-23 kDa and 67 kDa, peptides not recognized by the homologous sera. The reactivity pattern obtained with this fraction (a2) and the homologous sera was roughly similar to that obtained with the a4 fraction obtained by gel filtration of ASE with S-200. Moredver the ELISA improvement with the A4 fraction was higher, and the quantity of material from gel filtration was greater. Therefore, a4 was subjected to anion exchange chromatography (DEAE-Sephacel) and the elution profile is shown at Fig. 4. A relatively
4
MW
20.1, 14A-
Fig. 2. Western blot analysis of the a4 fraction from gel filtration, probed with negative sera (lane l), homologous sera (lane 2), anti-Trichostrongyl (lane 3) and antiTeladorsagia (lane 4). MW: mol.wt markers in kilodalton (kDa).
Sephacel) column the unbound fraction (al) was followed by 3 peaks eluted between 0.1 and 0.2 M NaCl (not shown). Fractions corresponding to the peaks were pooled and dialyzed overnight (PBS, 4°C). ELISA results with the pooled fractions a3 and a4 NHTO
N
et al.
H
T
0
NHTO
YW 94-
I
67-
Fig. 3. Western blot analysis of the pooled fractions from DEAE-Sephacel chromatography of ASE (al, a2, a3). MW: mol.wt markers in kDa. N: negative sera; H: homologous sera; T: antiTrichostrongylus; 0: anti-Teladorsagia.
Partial purification of a H. contortus diagnostic antigen
315
A41
0.:
z
a 0.1
0 0
10
20
30
40
50
Fraction
60
70
80
90
IO0
110
1200
number
Fig. 4. Elution profile of the a4 fraction from S-200 gel filtration after DEAE-Sephacel chromatography. Bars: pooled fractions. simple pattern was obtained and the 2 fractions with highest A 280 values were pooled (a41). Optical density (OD) at 405 nm in ELISA of positive sera/OD of negative sera (P/N ratio) with this pooled fraction
N
H
T
0
T
0
YW 64. 679
4330-
20.1-
14.4.
6
Fig. 5. Western blot analysis of A41 fractions probed with homologous (anti-Hoemonchus), H; negative sera, N; and heterologous sera (anti-Trichostrongylus, T; anti-Tel&orsagas, 0). MW, mol.wt markers in kDa.
(A41) was 4.04 and OD of positive sera/OD of heterologous sera (P/H ratios) were 2.15 for T. colubriformis and 3.42 for Te. circumcinta-infected animals. These results improved the ELISA findings with ASE and A4. Sera from the monospecifically infected lambs with H. contortus but not those from uninfected control lambs or animals with monospecific heterologous infections (lanes 3-6) (Fig. 5) recognized a peptide having ca. 26 kDa, apparently not glycosylated (not shown). This A41 fraction did not contain the proteins ca. 55 kDa present in the A4 peak from S-200 gel filtration of ASE although a faint reactivity was present at 44 kDa. However, this reactivity disappeared with higher serum dilution or more stringent washings. When A41 was probed with pooled sera from sheep flocks with mixed natural infections with trichostrongylids, WB analysis showed that the 26 kDa antigen present in the fraction was not recognized, under our conditions, by sheep sera from flocks where H. contortus was not present (Fig. 6). Immune recognition was only observed with one of the farms with H. contortus. In addition other reactivities (ca. 44 and 22-23 kDa) were present in some flocks. DISCUSSION Several problems have been found in the use of ELISA methods to detect H. contortus primary
316
M. T. Gomez-Mufioz
et al
MW @4(i
679 43-
I
,
1 i
14.4-.
_ 1
2
-. 9
10
Fig: 6. Western blot analysis of A41 fraction probed with pooled sera of sheep from different flocks wi’th natural mixed trichostrongylid infections (lanes 3-10). Numbers correspond to those given in Materials and Methods. Lane 1: positive control. Lane 2: negative control. MW: mol.wt markers in kDa. infections in lambs, the applications being restricted to a country, breed or age of the animals. One of the possible reasons for this lack of sensitivity and specificity in ELISA could be the extensive antigenic cross reactivity between this parasite and related and unrelated nematodes (Shamansky et al., 1989; Cuquerella et al., 1993; Schallig et al., 1994). In spite of the importance of sheep haemonchosis, information about the characterization of the parasite is relatively scarce. Except for the pioneering work by Neilson (1969) and Ozerol & Silverman (1969, 1970) most research has been related to the cuticle (i.e. Cox et al., ,1989; Shamansky et al., 1989; Gamble et al., 1990), excretory/secretory (E/S) antigens and exsheathing fluid (Ozerol &: Silverman, 1969; Gamble et al., 1990; Schallig et al., 1994). This lack of interest in soluble extracts of the parasite probably relates to the lower immunogenicity suggested (Parkhouse et al., 1987). However, in H. contortus infections in sheep it has been shown either in single point estimations (Hendrikx, 1990; Haig et a/., 1990; Cuquerella et al., 1991) or serial studies (Cuquerella et al., 1993) that some soluble proteins of the parasite are recognized by infected animals, particularly two peptides from adult worms of approximately 25 and 26 kDa mol.wt (Cuquerella et al., 1993). Our results show that a 2-step purification process involving anion exchange chromatography of the 4278 kDa pooled fraction obtained in gel filtration of
soluble extracts of adult H. contortus allows the isolation of a fraction containing a peptide having under reducing and denaturing conditions ca. 26 kDa which is recognized by H. contortus infected lambs but not by negative sera, thus confirming the potential diagnostic value of this region (Cuquerella et al., 1993). An intriguing point is brought up by a 24 kDa antigen reported later by Schallig et al. (1994) in E/S products of adult H. contortus. Although comparable electrophoretic motility does not constitute an absolute criterion for peptide identity, it is likely that the 24 kDa antigen is probably the same peptide reported earlier by us (Cuquerella et al., 1993) and partially purified herein. Actually, immunodetection patterns obtained with E/S components and microdissected oesophagus of H. contortus (Takits et al., 1995) were very close to those obtained by us previously (Cuquerella et al., 1993) although this fact was not acknowledged. Since most infections in sheep by gastrointestinal nematodes under field conditions are mixed, the specificity of reactivity to the partially purified antigen is critical. Besides the extensive antigenic cross reactivity between H. contortus and other nematodes (see above) some heterologous sera react on occasion more strongly than homologous sera with H. contortus antigens (Anderson et al., 1989).‘However, the 26 kDa antigen seems to be specific since it was not recognized by sera of monospecifically infected lambs with T. colubriformis and Te. circumcinta. Moreover,
Partial
purification
of a H. contortus
sheep sera from flocks of the surrounding area with average natural trichostrongylid mixed infections (Trichostrongylus, Teladorsagia) did not show significant reactivity with this antigen, whereas one of the flocks with H. contortus reacted with this antigen. However, the proportion of H. contortus in the mixed infections was low and a more complete study is being done with higher parasitic burdens and different developmental periods (prepatent, patent, postpatent, pretreatment, post-treatment). This antigen has been found to be non-glycosylated and no further characterization of the antigen has been made, so that the original molecular weight of the recognized peptide is not known yet. The yield obtained by using this 2-step process (gel filtration and anion exchange chromatography) was low (the A41 fraction represented as a whole cu. 2% of ASE) and some contaminating peptides were present. Its use for diagnosis or prophylaxis requires some additional research including cloning, chemical synthesis, and monoclonal or polyclonal antibodies which is in progress. Acknowledgements-We deeply thank J. Martin-Checa, 0. Rodriguez and B. Lopez for help provided, and the financial support of CICYT (GAN89/324 and AGF95/648). M. T. Gomez-MuAoz had a predoctoral fellowship from the UCM. Kind gifts of infective larvae by Dr J. Uriarte (ITEA Zaragoza) and Prof. J. L. Duncan (Glasgow, Scotland) (Te. circumcinta and T. colubrzformis) are greatly appreciated. REFERENCES Adams D. B. & Beh K. J. 1981. Immunity acquired by sheep from an experimental infection with Haemonchus contortus. International Journal for Parasitology 11: 381-386. Anderson D. V., Dixon S. C., Graham R. B., Smith W. D. & Tucker E. M. 1989. Ovine monoclonal antibody to Ostertagia circumcinta. Biochemical Society Transactions 17: 736. Bradford M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding. Annales ofBiochemistry 72: 248-254. Charley-Poulain J.. LutTau G. & P&y P. 1984. Serum and abomasal antibody response of sheep to infections with Haemonchus contortus. Veterinary Parasitology 14: 1299141. Christensen C. M., Zarlenga D. S. & Gasbarre L. C. 1994. Ostertagia, Haemonchus. Cooperia and Oesophagostomum: Construction and characterization of genus-specific DNA probes to differentiate important parasites of cattle. E.xperimental Parasitology 78: 933100. Cox G. N., Shamansky L. M. & Boisvenue R. J. 1989. Identification and preliminary characterization of cuticular surface proteins of Haemonchus contortus. Molecular and Biochemical Parasitology 36: 233.-242.
diagnostic
antigen
317
Cuquerella M., Gcmez-Muiioz M. T. & Alunda J. M. 1991. Serum IgG response of Manchego lambs to infection with Haemonchus contortus and preliminary characterization of adult antigens. Veterinary Parasitology 38: 131-143. Cuquerella M., Gomez-Muiioz M. T., Carrera L., de la Fuente C. & Alunda J. M. 1994. Cross antigenicity among ovine trichostrongyloidea. Preliminary report. Veterinary Parasitology 53: 243325 1. Cuquerella M., Gomez-Mufioz M. T., de la Fuente C., Carrera L. & Alunda J. M. 1993. Lamb serum recognition of infective larvae and adult Haemonchus contortus antigens. Veterinary Parasitology 49: 255-264. Duncan J. L., Smith W. D. & Dargie J. D. 1978. Possible relationship of level of mucosal IgA and serum IgG to immune unresponsiveness of lambs to Haemonchus contortus. Veterinary Parasitology 4: 21-27. Ellis T. M., Gregory A., Turnor R., Kalkhoven M. & Wroth R. H. 1993. Detection of Haemonchus contortus surface antigens in faeces from infected sheep. Veterinary Parasitology 51: 85-97. Gamble H. R., Purcell J. P. & Fetterer R. H. 1990. Biochemical characterization of cuticle polipeptides from the infective larvae of Haemonchus contortus. Comparative Biochemistry Physiology 96B: 421-429. Haig D. M., Windon R., Blackie W., Brown D. & Smith W. D. 1989. Parasite-specific T cell responses of sheep following live infection with the gastric nematode Haemonchus contortus. Parasite Immunology 11: 463-477. Hendrikx W. M. L. 1990. De nematode Haemonchus contortus: antigeenkarakter en immuunrespons bij konijnen en schapen. Tijdschrzft Diergeneeskunde 115: 1092-I 101. Klesius P. H., Washburn S. M.. Ciordia H. & Haynes T. B. 1984. Lymphocyte reactivity to Ostertagia ostertagi L3 antigen in type 1 ostertagiasis. American Journal of Veterinarv Research 45: 230-233. MAFF (Ministry of Agriculture, Fisheries and Food). 1971. Manual of Veterinary Parasitological Laborator.y Techniques. HMSO, London. Neilson J. T. M. 1969. Gel Filtration and Disc Electrophoresis of a Somatic Extract and Excretions and Secretions of Haemonchus contortus larvae. Experimental Parasitology 25: 13ll141. Ozerol N. H. & Silverman P. H. 1969. Partial characterization of Haemonchus contortus exsheathing fluid. The Journal of Parasitology 55: 79987. Ozerol N. H. & Silverman P. H. 1970. Further characterization of active metabolites from histotropic larvae of Haemonchus contortus cultured in vitro. The Journal of Parasitology 56: 1199- 1205. Parkhouse R. M. E., Almond N. M.. Cabrera 2. & Harnett W. 1987. Nematode antigens in protection, diagnosis and pathology. Veterinary Immunology and Immunopathology 17: 313-334. Petit A., Pery P. & Luffau G. 1981. Circulating antigens in ovine haemonchosis. Annales de Recherches Veterinaires 12: l-9. Roos M. H. & Grant W. N. 1993. Species-specific PCR for the parasitic nematodes Haenzonchus contortus and Trichostrongylus colubrijormis. International Journal for Parasitology 23: 4 19-42 1.
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Schallig H. D. F. H., van Leeuwen M. A. W. & Hendrikx W. M. L. 1994. Immune responses of Texel sheep to excretory/secretory products of adult Haemonchus contortus. Parasitology 108: 351-351. Shamansky L. M., Pratt D., Boisvenue J. & Cox G. N. 1989. Cuticle collagen genes of Haemonchus contortus and Caenorhabditis &guns are highly conserved. Molecular and Biochemical Parasitology 31: 73-86.
et al.
Smith W. D. 1977. Anti-larval antibodies in the serum and abomasal mucus of sheep hyperinfected with Haemonthus contortus. Research in Veterinary Science 22: 334-338. Takits C., Schallig H. D. F. H., van Leeuwen M. A. W. & Hendrikx W. M. L. 1995. Immune responses of sheep to microdissected parts of Haemonchus contortus. International Journal for Parasitology 25: 857-860.