Systematic antibody responses of calves to low molecular weight Cooperia oncophora antigens

ELSEVIER

Veterinary Parasitology59 (1995) 231-239

Systemic antibody responses of calves to low molecular weight Cooperia oncophoraantigens M.G.B. Nieuwland, H.W. Ploeger, A. Kloosterman, H.K. Parmentier * Departmentof Animal Husbandry, Agricultural University, PO Box 338, 6700AH, Wageningen,Netherlands Accepted 30 September1994

Abstract

The systemic antibody responses to adult Cooperia oncophora antigen were studied using sera obtained from calves during a 6-week period following a single oral infection with either 20 000 or 100 000 third-stage C. oncophora larvae. Dose dependent increasing titres of IgG binding complete adult Cooperia antigen were found in the sera of Cooperia-infected calves. SDS-gel electrophoresis under reducing conditions, followed by Western blotting, revealed that the increase of IgG binding Cooperia antigens could be attributed mainly to specific binding of IgG to a complex of 12-15 kDa protein fragments of Cooperia adult antigen. This protein may represent a Cooperia oncophora-specific component that can be used for serodiagnosis.

Keywords: Cooperiaoncophora;Cattle-Nematoda;Proteins;Westernblotting; Humoralimmunity

1. Introduction

Cooperia oncophora is a ubiquitous intestinal nematode in young grazing cattle in the temperate regions. Infection with C. oncophora in calves is accompanied by a systemic antibody response, that can be serodiagnosed using complete C. oncophora-antigen preparations (Keus et al., 1981). The role of the increasing systemic antibody titres during infection is not completely understood, but antibody titres correlated with various parasitological parameters of resistance (Kloosterman et al., 1991). Variation in immune responses of calves to gastro-intestinal nematodes depends on infection dose (Albers, 1981; Klesius, 1988), age (Herlich, 1979; Armour, 1989; Kloosterman et al., 1991), sex (Leigh* Correspondingauthor. 0304-4017/95/$09.50 © 1995 ElsevierScience B.V. All fights reserved SSD10304 -4017 ( 94 ) 00754-3

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ton et al., 1989) and genetic background ( Kloosterman et al., 1978; Albers, 1981; Leighton et al., 1989). Furthermore, antibody titres were also shown to reflect differences in the level of exposure to infection among groups of calves (Ploeger et al., 1994). In the present study, sera from three groups of calves of similar age and sex, that had been infected with different doses of third-stage C. oncophora larvae, were used to study dose-dependence of the systemic humoral immune response, and the identity of Cooperia protein fragments to which the antibody responses are directed.

2. Materials and methods

2.1. Calves Twenty Holstein-Friesian and Dutch-Friesian bull calves were purchased at 1 week of age. They were fed with a milk replacer until weaning at 10 weeks of age, with hay and water ad libitum from the date of purchase, and thereafter their feed was supplemented with concentrates at a maximum of 2 kg per day. The animals were individually stalled on a floor of rubber mats and straw. At 2 months of age the calves were vaccinated against infectious bovine rhinotracheitis (IBR), parainfluenza (PI-3) and bovine respiratory syncytial virus (BRSV). At the age of 3 months the animals were weighed and divided into three groups. Half-sibs were evenly distributed among groups. Seven calves were orally infected with a single dose of 100 000 C. oncophora third-stage larvae, and seven other calves were infected with 20 000 larvae. Six calves were not infected. Blood was collected from the jugular vein twice weekly for 6 weeks. Faeces were collected from the rectum twice weekly for estimation of egg counts according to the method described by Van den Brink (1971). The infected animals were necropsied 6 weeks post-infection (pi). 2.2. Infective larvae Third-stage (L3) larvae were collected from faecal cultures from C. oncophora monoinfected calves according to standard procedures. The strain of C. oncophora used in this experiment was isolated in 1967 from naturally infected calves. Since then, it has been maintained in our laboratory by one or two calf passages per year. The C. oncophora isolate contains approximately 30% C. surnabada. 2.3. C. oncophora antigens Adult worms had been obtained in previous experiments from calves with large worm burdens. They were homogenised in 0.05 M Tris-HCl, 1 mM phenylmethylsulphonyl fluoride, 1 mM EDTA (pH 8.6). After 2 h incubation on ice, the extracts were centrifuged at 25 000 X g at 4°C for 30 min. The supernatants were dialysed using a 45 gm filter (Schleicher & Schuell, Dassel, Germany), and stored at - 20°C until use. Protein contents were determined with a Perkin Elmer Lambda 1 UV/VIS spectrophotometer (Oak Brook, IL, USA) according to Bradford (1976).

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2.4. Immunological reagents Rabbit anti-bovine immunoglobulin coupled to horseradish peroxidase (PO) (RAB/ IgGu+L/PO) was purchased from Sigma (St. Louis, MO, USA).

2.5. Antibody titres Titres of serum antibodies with specificity for adult C. oncophora antigen were measured with the enzyme linked immunosorbent assay (ELISA) on micro-titre plates coated with saline extract of homogenized adult worms of C. oncophora, according to the method described by Keus et al. (1981). Titres were expressed as the log2 values of the highest dilution giving a positive reaction. Positivity was derived from the extinction values of a standard positive serum present on every microtitre plate. Sera were doubly diluted from 1/20 to 1/2560.

2.6. Sodium dodecyl sulfate polyacrylamide gel electrophoresis Antigen preparations of adult worms were separated on their molecular weight using 12.5% SDS-PAGE gels under reduced (/3-mercaptoethanol) conditions using a Midget electrophoresis unit (Pharmacia, Uppsala, Sweden). Bands were visualised by silver nitrate staining.

2.7. Western blots Western blots were carded out with a Multhiphor II Nova blot electrophoresis transfer unit (Pharmacia). Proteins from the SDS-PAGE gels were blotted for 1 h at 0.8 mA cm -2 on 0.45/xm nitrocellulose membranes (Bio-Rad, Richmond, CA, USA) using a continuous buffer system. Immunostaining was performed on individual lanes using a Miniblotter 16 (Immunetics, Cambridge, MA, USA). To determine the identity of Cooperia antigen fragments that were bound by the sera, blots were blocked for 1 h in phosphate buffered saline (pH 7.3) containing 0.05% Tween20 (PBS-T) and 1% bovine serum albumin, and subsequently for 1 h in PBS-Tween containing 0.5% horse serum to prevent non-specific binding. After washing, the blots were incubated for 1 h with 1:20 diluted sera obtained at weekly intervals from individual C. oncophora-infected calves ("immune sera") and the non-infected control calves. After washing in PBS-T, the blots were incubated overnight with 1:200 diluted RAB/IgGH+L/ PO. Binding of the bovine antibodies to proteins was visualized by incubation for no more than 1 h with the chromogen 4-chloro-1-naphthol (Sigma). The reaction was stopped with distilled water.

2.8. Evaluation of Western blot staining Calculation of molecular weights (Mw) as compared to standard prestained Mw markers (Bio-Rad), and semi-quantitative evaluation of the staining intensity of individual bands on the blots were performed with an Ultroscan XL (Pharmacia).

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2.9. Statistical evaluation

The differences in systemic antibody titres between groups were tested by one-way analysis of variance (ANOVA) (Statistical Analysis Systems Institute Inc., 1985). The staining intensity of the bands was semiquantified after classifying the bands in 5 kDa categories from the 0-5 kDa class to the 96-100 kDa class, Differences between infection levels were tested with Bonferroni's pair-wise comparisons of means.

3. Results 3.1. Faecal egg counts

Fig. 1 shows the patterns of faecal egg excretion in the two infected groups. The noninfected control calves did not excrete eggs at any time. 3.2. Systemic antibody titres

Increasing titres of IgG binding to Cooperia antigen were found in sera from calves infected with either 20 000 or 100 000 larvae (Fig. 2). The mean titres of the infected groups were highest at the end of the observation period (Day 42 pi). From Day 28 pi onwards, the mean titre of calves infected with 100 000 larvae differed significantly from the control group. The mean titre of the animals infected with 20 000 larvae differed significantly from the control group from Day 35 on. At that date all three groups differed significantly from each other. This remained the case on Day 39. On Day 41 pi the two infected groups no longer differed significantly from each other. 4000

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Fig. 1. The course of the arithmetic mean faecal egg counts ± SE by calves infected with 20 000 or 100 000 infective third-stage C. oncophora larvae.

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Fig. 2. Mean + SE of systemic antibody titres to complete adult C. oncophora antigen of non-infected control calves, or calves infected orally with either 20 000 or 100 000 Cooperia third-stage larvae. Means with different superscripts differ significantly (P < 0.05 ).

3.3. Western blot analysis of antibody reactivity Polyacrylamide gel electrophoresis of adult C. oncophora antigen under reduced conditions revealed numerous fragments (Fig. 3). Incubation of blots containing adult worm proteins with sera obtained from individual animals from all three groups during the observation period resulted in the identification of several fragments of Cooperia adults that were kDa 10 8

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Fig. 3. Silver-stained SDS gel showing the protein fragments of adult C. oncophora (lane 2). Lane 1 represents molecular weight markers. The 12.5 % gel was electrophorised under reduced conditions.

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kDa

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Fig. 4. A representative immunoblot of adult C. oncophora antigen (60 pg per lane) after incubation with sera obtained at weekly intervals up to 6 weeks pi from a non-infected calf (lanes 2-8, Weeks O-6) and a calf infected with 100 000 Cooperia third-stage larvae (lanes 9-15, Weeks O-6), followed by incubation with RAB/PO. Lane 1 represents prestained molecular weight markers, lane 16 was incubated with RAB/PO alone. The 12.5% gel was run under reduced conditions.

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Fig. 5. Staining intensity (mean absorption value f SE) after Western blot analysis with protein fragments between I1 and 15 kDa of antibodies from non-infected calves, or calves infected orally with either 20 000 or 100 000 Cooperia third-stage larvae. Means with different superscripts differ significantly (P < 0.05).

bound by immune serum (Fig. 4). A particularly strong reactivity of sera from infected calves was found for bands which were located at approximately 12, 13 and 15 kDa. This 12-15 kDa complex corresponded with a strong silver staining of a similar region in the SDS gel. Intense staining was also observed regularly at approximately the 27 kDa level. Analysis of variance revealed that only the “class” of II-15 kDa was consistently and

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increasingly stained by immune sera obtained on Day 28 up to Day 41 pi, but not by sera obtained at any time from control calves. Absorption values of sera binding by Western blotting the 11-15 kDa class is shown in Fig. 5. There was a significant difference for the 11-15 kDa class between the group infected with 100 000 larvae and the non-infected group on Days 35 and 42 pi, and a significant difference between both infection levels on Day 35 pi ( P < 0 . 0 5 ) . The staining intensity of the 20 000 group was intermediate between the control group and the 100 000 group. There was no significant difference at any time between the 20 000 group and the control group.

4. Discussion

Cooperia oncophora infections in calves are accompanied by increasing systemic antibody titers to adult Cooperia antigen. Little is known of the contribution of the humoral response in the immune-mediated resistance to Cooperia, or the Cooperia antigenic fragments which initiate a (protective) immune response. It has been proposed, however, that the increase of systemic Cooperia antigen binding IgG antibodies in infected calves is related to various (parasitological) parameters of resistance (Kloosterman et al., 1991 ). SDS-PAGE analysis of Cooperia adults revealed numerous peptide fragments, only a few of which were bound by sera from Cooperia-infected animals. Furthermore, not all peptide fragments were consistently recognised in time by sera obtained from individual calves. However, in sera from calves infected with either 20 000 or 100 000 Cooperia larvae, a significant and increasing binding activity was found to a (complex of) protein fragment(s) that under reducing conditions had a molecular weight between 12 and 15 kDa. These results are consistent with those from De Graaf et al. (1993), who reported that only 14.2 and 14.9 kDa bands were specifically recognised by sera from C. oncophora trickle-infected calves, while other bands were shared with other nematodes. In this respect, it could be that the inconsistent and variable staining of the other protein bands such as the 27 kDa depended on the presence in the sera of antibodies directed to other (cross-reacting) microorganisms. The intensity of staining of the 12-15 kDa fragment in immunoblots corresponded with the increasing antibody titres to whole Cooperia antigen as determined with ELISA. It is therefore proposed that a fragment or a complex of fragments in the 1215 kDa region represents the active component in the complete Cooperia antigen used in ELISA (Keus et al., 1981). It remains to be established whether this protein represents a somatic antigen or an excretory / secretory peptide. Specific low molecular peptide fragments of other nematodes have been reported previously. These include fragments identified by Western blotting of C. oncophora (De Graaf et al., 1993), Dictyocaulus viviparus (De Leeuw and Cornelissen, 1991; Schnieder, 1992) and O. ostertagi (Mansour et al., 1990), or SDS-PAGE patterns (Baker and Gershwin, 1993). Since the 12-15 kDa fragments of C. oncophora were not bound by immune sera from calves mono-infected with several other nematodes, such as D. viviparus and O. ostertagi (Parmentier et al., 1995), it may thus represent a Cooperia-specific fragment that could have potential value for serodiagnosis of Cooperia infections in calves. The relevance of the humoral immune response from calves infected once to the antigenic proteins of Cooperia adult worms has yet to be determined. Variation in immune responses

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of calves to gastro-intestinal nematodes was reported to depend on infection dose (Albers, 1981; Klesius, 1988), age (Herlich, 1979; Armour, 1989; Kloosterman et al., 1991), sex (Leighton et al., 1989) and genetic background (Kloosterman et al., 1978; Albers, 1981; Leighton et al., 1989). In this study, animals of similar age and sex were used. Variations in antibody titres and parasitological parameters were found within both the 20 000 and 100 000 larvae infected groups. Between groups, the level of antibody titres reflected the infection dose level and faecal egg excretion patterns, which was also found by Ploeger et al. (1994). A systemic IgG response to Cooperia antigen was found in sera from both infected groups of calves but not in the control animals. No significant differences in ELISA titres or immunoblot-"titres" were found between infected groups and the non-infected control group until Day 28 pi. ELISA titres differed significantly from Day 32 to Day 39 pi, but were subsequently similar for both infected groups. Immunoblot-"titres" differed between both infected groups on Day 35 pi only; on Day 42 they were similar. The difference in results may be attributed to a difference in sensitivity between both assays, and the fact that in ELISA, titres represent reaction with complete worm antigen, whereas in immunoblot the binding o f antibodies is directed to separate fragments. Further studies on the specificity and responsiveness of antibodies (and T cells) of calves to Cooperia antigen fragments in relation to variation in (parasitological) parameters of resistance, such as egg production or the number and length of worms, and further characterization of the antigens recognized by infected calves, such as the 12-15 kDa fragment, may reveal the importance of these antigen fragments in immune-mediated resistance.

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