Humoral immune response of water buffalo monitored with three different antigens of Toxocara vitulorum

Veterinary Parasitology 122 (2004) 67–78

Humoral immune response of water buffalo monitored with three different antigens of Toxocara vitulorum E.M. de Souza a , W.A. Starke-Buzetti a,∗ , F.P. Ferreira a , M.F. Neves a , R.Z. Machado b a

Departmento de Biologia e Zootecnia, FEIS/UNESP, Ilha Solteira 15385-000, São Paulo, Brazil b Departamento de Patologia Veterinária, FCAV/UNESP, São Paulo, Brazil Received 1 February 2003; received in revised form 1 March 2004; accepted 17 March 2004

Abstract Humoral immune response of water buffalo naturally infected with Toxocara vitulorum was monitored using three different antigens of this parasite in serum and colostrum of buffalo cows and calves. Soluble extract (Ex) and excretory/secretory (ES) larval antigens and perienteric fluid antigen (Pe) of adult T. vitulorum were used to measure the antibody levels by an indirect ELISA. Serum of 7–12 buffalo cows for the first 365 days and colostrum of the same number of buffalo cows for the first 60 days of parturition, and serum of 8–10 buffalo calves for the first 365 days after birth were assayed. The ELISA detected antibodies against all three T. vitulorum antigens in the colostrum and serum of 100% of buffalo cows and calves examined. The highest antibody levels against Ex, ES and Pe antigens were detected in the buffalo cow sera during the perinatal period and were maintained at high levels through 300 days after parturition. On the other hand, colostrum antibody concentrations of all three antigens were highest on the first day post-parturition, but decreased sharply during the first 15 days. Concomitantly to the monitoring of immune response, the parasitic status of the calves was also evaluated. In calves, antibodies passively acquired were at the highest concentrations 24 h after birth and remained at high levels until 45 days coincidentally with the peak of T. vitulorum infection. The rejection of the worms by the calves occurred simultaneously with the decline of antibody levels, which reached their lowest levels between 76 and 150 days. Thereafter, probably because of the presence of adults/larvae stimulation, the calves acquired active immunity and the antibodies started to increase slightly in the serum and plateaued between the days 211 and 365. All three antigens were detected by the serum antibodies of buffalo calves; however, the concentration of anti-Pe antibody was higher than anti-EX and anti-ES, particularly after 90 days of age. By conclusion, the buffalo cows develop immunity and keep high levels of antibodies against ∗ Corresponding author. Tel.: +55-18-3743-1152; fax: +55-18-3743-1186. E-mail address: [email protected] (W.A. Starke-Buzetti).

0304-4017/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2004.03.013

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T. vitulorum-Ex, ES and Pe antigens and these antibodies are transferred to their calves through the colostrum. This passively acquired immunity does not protect the calves against the acquisition of the infection, but these antibodies, passively or actively acquired, may have an important role during worm rejection by the calves and prevention of intestinal reinfection. © 2004 Elsevier B.V. All rights reserved. Keywords: Toxocara vitulorum; Water buffalo; ELISA

1. Introduction Toxocara vitulorum is a parasite of the small intestine of ruminants, particularly buffalo calves of 1–3 months of age. It is responsible for high morbidity and mortality rates (Das and Singh, 1955; Patnaik and Pande, 1963; Starke et al., 1983; Gupta et al., 1976) resulting in serious economic losses (Enyenihi, 1969). This parasite is acquired by calves when they suckle colostrum/milk contaminated with infective larvae from infected cows (Chauhan et al., 1974; Mia et al., 1975; Roberts et al., 1990; Starke et al., 1992). It is common to find buffalo calves highly infected between 15 and 90 days of age with the peak egg output occurring 31–45 days post-infection (Starke et al., 1983; Roberts, 1990a). The life cycle of the hosts is synchronized with the life cycle of T. vitulorum to the benefit of the parasite resulting in infection to offspring during pregnancy. The infective larvae in eggs ingested by the buffalo cow migrate into the tissues after which the dam can effectively act as an intermediate host for the parasite (Mia et al., 1975). In pregnant host, larvae grow in the liver and lung 1–8 days before parturition and migrate to the mammary gland around the time of parturition (Roberts, 1990b), then pass into the milk during the first 26 days after parturition (Roberts et al., 1990; Starke et al., 1992). While the adult parasites are relatively easy to remove from the intestines by antihelminthic, the larvae are difficult to kill, particularly larvae that can be hypobiotic in the musculature and the brain (Abo-Shehada and Herbert, 1984). Barriga and Omar (1992) suggested vaccination of the dams to kill the larvae in tissues before they are transferred to the calves could control this parasite. Antibodies against larval excretory/secretory (ES) (Rajapakse et al., 1994a) and larval soluble extract (Ex) (Starke-Buzetti et al., 2001) of T. vitulorum have been detected in serum of buffalo cows and calves naturally infected with T. vitulorum, indicating that T. vitulorum infection can stimulate the immune system of the buffalo. The immunization of mice with ES antigen of larvae and perienteric fluid (Pe) antigen of adults of T. vitulorum has induced protection against larval migration in their tissues (Amerasinghe et al., 1992). Therefore, Rajapakse et al. (1994b) confirmed the antibody-mediated protection against T. vitulorum larvae through the ability of buffalo serum or colostrum to inhibit migration of T. vitulorum larvae in mice. Based on the hypothesis that T. vitulorum infection stimulates the immune system of the buffaloes, the objective of the present study was to monitor the levels of T. vitulorum anti-ES, anti-Ex and anti-Pe antibodies in the serum and colostrum of buffalo cows during the pregnancy and lactation periods and also in the serum of the calves. In addition, the levels of antibodies were compared with the parasitic status of the buffalo calves naturally infected with T. vitulorum during their first year.

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2. Materials and methods 2.1. Buffalo housing Water buffaloes naturally infected with T. vitulorum were kept for about 12 months on a 12 ha pasture of Brachiaria decumbens grass with a pond as the source of water. The cows were not milked and the calves were grazed together in this area with the dams for a period of 12 months. 2.2. Fecal, serum and colostrum/milk samples from buffalo calves and cows Rectal fecal samples were collected from the buffalo calves (n = 10–31) according to the following schedule: weekly (from birth to 90 days) and fortnightly (from day 91 until attaining two consecutive weeks with an absence of T. vitulorum eggs). Fecal examinations were performed according to Whitlock (1948) and results expressed as eggs per gram of feces (EPG). The sera of buffalo calves (n = 8–10) were sampled at 1 day of age before and after suckling the colostrum and then sequentially as follows: weekly (from the birth to 60 days), fortnightly (from 61 to 180 days) and monthly (from 180 to 365 days of age). Simultaneously, the serum samples of buffalo cows were collected weekly from parturition to 60 days post-parturition, fortnightly from 61 to 180 days and then monthly from 181 to 365 days. Colostrum/milk of buffalo cows were sampled on the day of parturition and weekly through 60 days post-parturition. The samples were centrifuged at 4 ◦ C in a refrigerated centrifuge at 460 × g for 15 min. After removal of solidified fat, the samples were left in an incubator at 37 ◦ C for 1 h for casein precipitation with 1% rennin (Chymosin bovine, Sigma® , R-4879). Then the colostrum/milk serum was separated by centrifugation for 15 min at 460 × g at 4 ◦ C. Serum and colostrum/milk samples were separated, aliquoted and stored at −70 ◦ C. 2.3. T. vitulorum antigen preparation T. vitulorum adults were recovered by expulsion of this parasite through the feces of naturally infected water buffalo calves by administration of 100 mg/kg of piperazine. Mature females were dissected and the uteri and eggs removed. The eggs were incubated in PBS solution (phosphate-buffered saline, 0.1 M; 7.5 pH) with several drops (5–10 drops/ml of egg suspension) of commercial sodium hypochlorite solution (1% available chlorine) in Petri dishes for 20–45 days at room temperature. The dishes with the egg suspension were gently stirred for daily aeration while the development of eggs was daily observed with an optical microscopic until the infective third-stage larvae (L3). After that, the egg suspension was transferred to tubes (15 ml) and washed in distilled water by centrifugation. Sediment from eggs was collected then combined with an equal volume of sodium hypochlorite solution (14% available chlorine) and incubated for 20 min at room temperature until the eggs were completely decorticated. PBS was added to this solution to increase the volume to 15 ml

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after which the mixture was centrifuged 10 times at 460 × g for 2 min or until chlorine odor could not be detected (Crowcroft and Gillespie, 1991). The decoated eggs were suspended in PBS and placed in a water bath at 37 ◦ C for 1 h while air was bubbled with a Pasteur pipette through the suspension until the L3 were hatched. Almost a 100% of larvae were hatched and mobile in only 5 min after the air bubbling method. 2.3.1. Larval soluble extract Ex antigen was obtained from infective L3, as described previously by Starke-Buzetti et al. (2001), by ultrasonic homogenization in ice, using three pulsating cycles of 1 min/ cycle, in an ultrasonic processor (Vir Sonic 1001, Virtis). To the larvae suspension, a mixture of water-soluble protease inhibitors solution with specificity for the inhibition of serine, cysteine, aspartic and metalloproteases (Protease Inhibitor Coktail General Use, Sigma® P-2714 containing AEBSF, E-64, bestatin, leupeptin, aprotinin and sodium EDTA) was added and the larval suspension was left resting for overnight at 4 ◦ C. Later the resulting suspension was primarily centrifuged at 690 × g for 10 min and then at 3700 × g for 30 min in a refrigerated centrifuge at 10 ◦ C. The supernatant was filtered through a membrane (Gelman Sciences, membrane filter, pore size 0.22 ␮m), dehydrated in an vacuum centrifuge (Vacufuge Concentrator 5301, Eppendorf) and stored at −70 ◦ C. 2.3.2. Excretory–secretory antigen The larval suspension was recovered by centrifugation and the sediment was suspended in RPMI-1640 culture medium (Sigma® , R-4130-L-Glutamine, 25 mM HEPES) containing 100× antibiotic/antimycotic (Sigma® A-5955). The 10,000 larvae/ml in RPMI-1640 containing antibiotic/antimycotic (100×) solution (5000 UI penicillin, 5 mg streptomycin and 10 ␮g amphotericin B) plus 1% glucose and 0.85% NaHCO3 were placed in a flat glass tissue culture in a 5% CO2 incubator according to Rajapakse et al. (1992). At least two times a week a sample of 100 ␮l of culture medium with larvae was collected, placed on slide and observed under light microscope in order to see the viability of the larvae. Every week the culture medium without any larvae was removed and centrifuged at 460 × g for 5 min and the supernatant was filtered in a membrane filter of 0.2 ␮m pore size to which protease inhibitor was added. This medium was then dialyzed in 25,000 Spectra/Por DispoDialysers (Spectrum® ) for 24 h at 4 ◦ C in 2 l of PBS stirred constantly at 10 rpm. The dialyzed material was filtered in a membrane filter with a 0.2 ␮m pore size and then dehydrated in a vacuum centrifuge and stored at −70 ◦ C. 2.3.3. Perienteric fluid antigen (Pe) Pe antigen was collected of adult male and female parasites according to Amerasinghe et al. (1992). The posterior end of each parasite was punctured with a hypodermic needle and the perienteric fluid was drained and collected. The perienteric liquid was centrifuged at 460 ×g for 5 min and the supernatant was filtered in a membrane filter with a 0.2 ␮m pore size after which protease inhibitor was added. Aliquots of 500 ␮l were stored at −70 ◦ C until used.

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2.4. Protein concentration Protein concentration of each antigen was measured using a Protein Assay Kit (Sigma® P-5656) using Lowry’s reagent. The concentration of ES was 500 ␮g/ml, Ex was 950 ␮g/ml and Pe was 2700 ␮g/ml. 2.5. Indirect ELISA procedure IgG serum and colostrum levels to T. vitulorum antigens (ES, Ex and Pe antigens) were analyzed by an indirect method according to Starke-Buzetti et al. (2001). In brief, 100 ␮l of antigens diluted in carbonate/bicarbonate buffer (0.05 M, pH 9.6) was added in each well of an ELISA microplate, sealed and incubated overnight at 4 ◦ C in a humid chamber. Antigenic protein concentration was determined by block standardization as 40 ␮g/ml for ES, and 10 ␮g/ml for both Ex and Pe antigens of T. vitulorum. In the intervals of reaction phases, microplates were washed three times for 3 min each using PBS-Tween 20 (0.05% Tween-20:Polyoxyethylene-Sorbitan Monolaurate, Sigma® P-7949), pH 7.4. After coating the plates with antigens, plates were blocked for 1 h at 37 ◦ C with 5% powered milk diluted in 0.05 M carbonate/bicarbonate buffer at pH 9.6. After three washings (3 min of each washing), 100 ␮l of diluted buffalo sera or colostrum (1/100) in PBS-Tween-20 plus 5% normal rabbit serum were added in duplicate to the plate and incubated in a humid chamber at 37 ◦ C for 60 min. A 100 ␮l aliquot of alkaline phosphatase conjugate rabbit anti-bovine IgG (Sigma® A-7914) at 1:5000 dilution in PBS was added to each well and incubated at 37 ◦ C for 60 min in a humid chamber. After washings, 100 ␮l of substrate (p-nitrophenyl phosphate, Sigma® 104) at 1 mg/ml in diethanolamine buffer, pH 9.8 was added and incubated for 30 min at room temperature. The reaction was stopped by the addition of 30 ␮l of 3.2 M sodium hydroxide and the plates were read at 405 nm wavelength using an EL-800 Universal Microplate Reader (BIO-TEK® Instruments) running the KCjunior® software (series S:164290). To control inter-plate variation, positive and negative controls were included in each plate. Control assays were run without antigen, buffalo serum/colostrum, conjugate or substrate. The calibration of the ELISA system was done according to Starke-Buzetti et al. (2001) using positive reference serum collected from 1-day post-parturition buffalo cows and negative reference serum from buffalo calves at birth and before suckling the colostrum. The enzymatic activity of each serum/colostrum sample was determined by calculating the sample for the positive rate (S/P) at each dilution, considering positive and negative sera as references using the following equation: S MSA − MAN = P MAP − MAN where MSA is the mean sample absorbance of calf or cow sera or cow colostrum, MAN is the mean absorbance of negative reference sera and MAP is the mean absorbance of positive reference sera. Sample/positive (S/P) values were grouped into ELISA levels (EL), which ranged from 0 (lowest level) to 9 (highest level). The subsequent levels were determined in increments of

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35% as described by Machado et al. (1997) and adapted by Starke-Buzetti et al. (2001) to the Toxocara system. The cut off point was determined multiplying the minimal SP value (EL = 0) by the coefficient 2.5 according to Machado et al. (1997). The cut off point was determined to be EL equal or below level 4.

3. Results 3.1. Humoral immune response 3.1.1. Buffalo cows Fig. 1 shows data of anti-Ex, anti-ES and anti-Pe antibody reactivity against T. vitulorum infection in buffalo cow sera. The ELISA antibody reactivity was high and similar for all three antigens with only minor oscillation over the year. During this year, the lowest S/P value was 0.8605 ± 0.0808 (average ± standard deviation) and the highest was 1.1984 ± 0.1825 with ELISA levels ranging from 6 to 8. The anti-ES level was slightly higher than the other two, but only after 120 days post-parturition. Fig. 2, on the other hand, shows data of antibody reactivity found in colostrum/milk of 7–8 buffalo cows; all cows examined had antibodies against T. vitulorum-ES, Ex and Pe antigens. The concentration of the antibodies was highest on the day of parturition which had S/P values ranging from 0.893±0.106 to 1.211±0.133 and ELISA levels ranging from

1.6

Sample/Positive (S/P)

1.4 1.2 1 0.8 0.6

Anti-Pe

0.4

Anti-Ex

0.2

Anti-ES

335-365

304-334

273-303

242-272

211-241

180-210

166-180

151-165

136-150

121-135

106-120

91-105

76-90

61-75

46-60

31-45

16-30

8-15

2-7

1

0

Post-parturition (days) Fig. 1. Values for S/P (sample/positive) of T. vitulorum anti-Pe, anti-Ex and anti-ES antibodies in sera of buffalo cows (n = 7–12) during the first 365 days post-parturition. Standard deviation of the average is also represented.

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73 6000

1.4

5000

1

4000

Anti-Pe 0.8

Anti-Ex 3000

Anti-ES 0.6

EPG

Sample/Positive (S/P)

1.2

EPG 2000

0.4

1000

136-150

121-135

106-120

Post-parturition (days)

91-105

76-90

61-75

46-60

31-45

16-30

8-15

1

0

2-7

0.2

0

Fig. 2. Values for S/P (sample/positive) of T. vitulorum anti-Pe, anti-Ex and anti-ES antibodies in colostrum of buffalo cows (n = 7–12) during the first 60 days post-parturition compared with the parasitic status of the calves (n = 10–31) represented by the EPG counts. Standard deviation of the average is also represented.

7 to 9, which then declined rapidly after the seventh day and reached very low concentration and ELISA levels equal to 0 on days 8–15 for Ex and ES antigens and on days 24–30 for Pe antigens. 3.1.2. Buffalo calves Antibody levels of buffalo calves naturally infected with T. vitulorum during their first year of life was assayed by ELISA using T. vitulorum-ES, Ex and Pe antigens with the results shown in Fig. 3. At birth but before suckling the colostrum from all calves had no detectable antibodies in their sera; but 1 day after suckling, the antibody concentration was highest for all three antigens (S/P values and ELISA levels ranging from 0.798 ± 0.191 to 0.907 ± 0.143 and 6–7, respectively), indicating that the antibodies were passively acquired through the colostrum. During approximately the first 45 days after the birth, the antibody levels maintained high, particularly against T. vitulorum-Ex and Pe antigens, but thereafter the concentration of antibodies of all three antigens started to decline reaching the lowest levels on days 76–90 for Pe antigen and days 136–150 for ES and Ex antigens. After reaching the lowest levels, the concentration of antibodies again increased, probably due acquisition of active immunity. These antibody levels had remained stable until the calves reached 365 days old. The lowest S/P value and ELISA level observed were 0.195 ± 0.065 and 2, respectively, whereas the highest were 0.921 ± 0.104 and 7, respectively. The reactivity of anti-Ex and anti-Pe antibodies were more intense than that of anti-ES in calves, particularly after 106 days (Fig. 3).

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1.2

Anti-Ex

1

5000

Anti-ES EPG

0.8

4000

0.6

3000

0.4

2000

0.2

1000

335-365

273-303

211-241

166-180

136-150

106-120

76-90

46-60

16-30

2-7

0 1*

0

EPG

Sample/Positive (S/P)

6000

Anti-Pe

Ages ofbuffalo calves (days) Fig. 3. Values for S/P (sample/positive) of T. vitulorum anti-Pe, anti-Ex and anti-ES antibodies in sera of buffalo calves (n = 7–10) during the period from 1 to 365 post-birth compared with the parasitic status of the calves (n = 10–31) represented by the EPG counts. Standard deviation of the average is also represented.

3.2. Antibody-mediated immune response versus parasitological status of the buffalo calves The pre-patent period for buffalo calves naturally infected with T. vitulorum ranged from 15 to 36 days and the maximum peak output of eggs occurred when the calves were 44.3 ± 6.6 days of age with the EPG ranged between 1250 and 26,300. The patent period was 57.5 ± 20.8 days and the EPG decline occurred between days 106 and 120. Antibodies passively acquired by calves through the dams’ colostrum remained at high concentrations until 45 days coincidental to the peak of EPG maximum counts for T. vitulorum eggs. After the peak, worm expulsion began and simultaneously the antibody levels started to decline, reaching their lowest levels when the calves were between 76 and 150 days. Thereafter, when the animal ages were greater than 120 days and the EPG counts were zero, the antibody levels started increasing slightly and plateaued between days 211 and 365 (Fig. 3).

4. Discussion Three different antigens of T. vitulorum were used to monitor the humoral immune response of buffalo cows and calves naturally infected with T. vitulorum. This ELISA method initially used by Machado et al. (1997) for diagnosis of bovine babesiosis was

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standardized for serological detection of T. vitulorum antibodies of naturally infected water buffaloes by Starke-Buzetti et al. (2001). In the present work, serum and colostrum/milk samples were analyzed during a period of 365 days including the lactation and pregnancy periods. The results revealed 100% of the buffalo cows with high levels of antibodies in their serum and colostrum against all three antigens: two larva antigens (Ex and ES) and one of adult worms (Pe). Larval ES antigen antibodies were present in slightly higher concentrations after the lactation period (106 days post-parturition), but antibodies against the other two antigens remained high throughout the year, suggesting the presence of resident migrating larvae in the tissues and possible acquisition of new infection by oral ingestion of infective eggs that might continuously stimulate the immune system of the cows. In agreement with the development of the antibody-mediated immune response in cows against T. vitulorum infection, Rajapakse et al. (1994a) showed positive gel-precipitating bands of reaction of anti-ES in the serum of 66.6% of buffalo cows 30 days before calving. Furthermore, Starke-Buzetti et al. (2001) detected also high level of anti-Ex antibody during the perinatal period (30 days prior and 30 days post-parturition). Besides the high concentrations of antibodies in buffalo cow sera against T. vitulorum infection during the lactation and pregnancy periods, some larvae were not killed in tissues and migration to the mammary gland was not totally prevented. Situations considered stressful like parturition and lactation may depress the immune response. Suppression of mitogen-induced lymphocyte and antibody titter decrease were reported by Amerasinghe et al. (1994) in buffalo cows at parturition time and for 2–3 months into lactation. The decline in antibody titter was suggested to be due to translocation of maternal serum IgG into colostrum for passive transfer of immunity to the calf (Rajapakse et al., 1994a). In cattle, according to Brandon et al. (1971), IgG1 serum concentrations decrease by up to 50% 2–3 weeks prior to parturition due selective secretion of IgG1 across the mammary epithelium into the mammary secretion, but with a return to normal serum levels by 4 weeks after parturition. However, a decrease in antibody concentration indicating immunosuppression was not detected in the serum of the buffalo cows in the present experiment. Moreover, IgG antibodies were found in the colostrum of the buffalo cows reacting strongly against T. vitulorum antigens. All eight cows (100%) had antibodies to ES, Ex and Pe antigens in their colostrum. The antibody levels were the highest on the day of parturition, but then declined rapidly after the seventh day and reached very low concentrations on days 8–15 for Ex and ES antigens and on days 24–30 for Pe antigen. Similarly, Rajapakse et al. (1994a) detected high titter of anti-T. vitulorum antibodies against larval ES antigen by ELISA in the colostrum of buffalo cows on the parturition day and over the first 10 days of lactation. The immunoglobulin isotype was not determined in the present work; however, it is known that IgG1 is the most abundant serum immunoglobulin that is concentrated in ruminant colostrum (Newby and Bourne, 1977). In both serum and colostrum of buffaloes, the highest levels of activity against T. vitulorum were present in the IgG fraction, particularly IgG1 (Fernando et al., 1989; Rajapakse et al., 1994b). The buffalo calves were naturally infected with T. vitulorum which started to shed T. vitulorum eggs through the feces on day 15 and with peak output of eggs between 35 and 57 days. The precocity of this infection may be explained by acquisition of infective larvae by the colostrum (Chauhan et al., 1974; Mia et al., 1975; Roberts et al., 1990; Starke et al., 1992). On the other hand, while the buffalo calves received T. vitulorum infective larvae they

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also received specific T. vitulorum IgG antibodies through the colostrum. High serum levels of passively acquired antibodies were detected in all buffalo calves on the first day after ingesting the colostrum to 46 and 60 days after birth; however, even receiving the antibodies against T. vitulorum, the calves developed patent infection. The concentration of antibodies in the serum of the calves was high until the peak of T. vitulorum EPG counts and then, coincident with declining antibody levels, the EPG counts also declined, which occurred after 45 days of age. The rapid decline of fecal EPG counts were simultaneous to the expulsion of adult worms in the feces of buffalo calves. After 120 days post-birth, eggs of T. vitulorum were no longer seen in the feces of the animals, suggesting a process of self-cure and protection against intestinal reinfection. The acquisition of passive immunity by calves from the colostrum was confirmed in the present work by the absence of anti-T. vitulorum IgG against larval antigens (ES and Ex) and adult antigen (Pe) in the sera of calves before suckling the colostrum and by the presence of high levels of these antibodies 24 h after such ingestion. After reaching the lowest levels between days 76 and 150, the antibody levels in the serum started to increase slightly and remained at a plateau level on days 211 to 365, possibly reflecting acquisition of active immunity by the calves. Anti-Pe antibodies started increasing earlier (on days 76–90) than anti-ES or Ex (on days 136–150). The calves had high levels of anti-ES during the first 60 days which then declined below the cut off point (EL 4) and then increased after 210 days, but never above the cut off point. The antibodies to Ex and Pe, on the other hand, never had values less than the cut off point. Because Pe antigen was obtained from adult worms may be the reason of higher anti-Pe level, particularly in calves. Intestinal adult or juvenile worms may release antigenic molecules in the lumen of the intestine; and these molecules may stimulate the mucosal immune system. The experimental animals shared the same pasture with other T. vitulorum-infected animals and, according to Roberts (1989), the eggs of T. vitulorum are ubiquitous and long-lived in shallow water, in soil, pasture or udder of the cows favoring a continuous infestation of the pasture. But, the absence of eggs in feces of the calves during the period (120–365 days of age) after expulsion of the adult worms suggests that intestinal reinfection did not occur. Although the negative EPG, the calves could feed infective eggs from the environment and the migratory larvae could be trapped in the tissues instead of return back to the intestines as adult worms. It could stimulate the immune response and explain the presence of T. vitulorum antibodies in the serum of calves after adult worm rejection. In conclusion, the buffalo cows develop immunity and keep high levels of antibodies against T. vitulorum-Ex, ES and Pe antigens and these antibodies are transferred to their calves through the colostrum. This passively acquired immunity does not protect the calves against the acquisition of the infection, but these antibodies, passively or actively acquired, may have an important role during worm rejection by the calves and prevention of intestinal reinfection.

Acknowledgements The authors gratefully acknowledge the contribution to this review by Dr. S.G. Kayes from the Department of Cell Biology and Neuroscience, University of South Alabama, Mobile, AL, USA.

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