Induction of protective immunity to Dictyocaulus viviparus in calves while under treatment with endectocides

Veterinary Parasitology 88 (2000) 219–228

Induction of protective immunity to Dictyocaulus viviparus in calves while under treatment with endectocides S.M. Taylor ∗ , J. Kenny, H.W. Edgar, T.R. Mallon, A. Canavan Veterinary Sciences Division, Department of Agriculture for Northern Ireland, Stormont, Belfast BT4 3SD, UK Received 27 July 1999; accepted 18 October 1999

Abstract Three groups of five parasite-naive calves were used. The treatments were: (a) Group 1 calves were weighed on Day 0 and injected with doramectin at 200 ␮g/kg. From Day 1 to 19 they were dosed orally with 2000 infective larvae of Dictyocaulus viviparus. On Day 28 they were again injected with doramectin, and infected with D. viviparus larvae from Days 33 to 41. They were then left untreated until Day 81 when they were infected with 20 infective larvae of D. viviparus per kg body weight. They were killed on Day 110 and lungworms were counted; (b) Group 2 calves were immunised with oral lungworm vaccine on Days 0 and 28, and infected and slaughtered as Group 1 on Days 81 and 110, respectively; (c) Group 3 calves acted as infection controls. Blood samples were taken at Days 0, 21, 49, 77 and 110 for antibody tests to D. viviparus. At autopsy there were no significant differences between the number of lungworms from Groups 1 and 2 (Means 17.4 and 31.3, respectively); Group 1 had significantly less value than Group 3 (Mean 228) (p < 0.05). Increased antibody titres to the larval sheath of the infective larvae were observed from Groups 1 and 2, showing that the larvae in Group 1 had penetrated the intestine before being killed by the circulating anthelmintic. This experiment shows that if calves are exposed to infective larvae while under systemic endectocide cover, an immune reaction is stimulated. ©2000 Elsevier Science B.V. All rights reserved. Keywords: Cattle-Nematoda; Control methods — Nematoda; Dictyocaulus viviparus; Immunity; Doramectin; Endectocides

∗ Corresponding author. Present address: 8 Grey Point, Helens Bay, Bangor, Co Down, Northern Ireland, BT19 1LE, UK. Tel.: +44-1247-853331; fax: +44-1247-853523. E-mail address: stuart [email protected] (S.M. Taylor).

0304-4017/00/$ – see front matter ©2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 9 9 ) 0 0 2 1 6 - 2

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1. Introduction It has been recognised for many years that cattle infected with Dictyocaulus viviparus and subsequently treated with anthelmintic, both before the infection becomes patent and after patency, will develop some protective immunity to further reinfection (Downey, 1980; Oakley, 1982). After the introduction of endectocides with persistent activity, it was noted that provided infection with D. viviparus took place during the first grazing season when the endectocide regimes were applied, that cattle in their second year were functionally immune to reinfection (Taylor et al., 1988, 1997; Jacobs et al., 1989). Similar results were also seen with some types of intraruminal slow-release boluses (Jacobs et al., 1989; Schnieder et al., 1992; Downey et al., 1993), although with the ivermectin intraruminal bolus, Schnieder et al. (1996) noted that immunity in second year cattle was less than previously reported with other boluses or endectocide applications. It has been generally assumed that the immunity was stimulated by infection at times when there were no nematocidal plasma concentrations of anthelmintic, and that the subsequent anthelmintic treatment killed prepatent or adult worms and released parasite antigen to prime the immune reaction to subsequent reinfection. No experiments have been reported which examined the relative importance of larvae or adults in the process. In studies of immune reactions to antigens from various stages of D. viviparus, Gilleard et al. (1995) and McKeand et al. (1996) reported that the most highly immunogenic larval tissue was the sheath surface of the third stage larva, which is the retained cuticle of the second stage. Prior to these reports, Soliman (1953) had reported that this sheath was not shed prior to parasite penetration, but was retained until after penetration. If this process does occur in lungworm infections, it forms the basis of an explanation of why immunity to infection can be stimulated even in the presence in plasma of nematocidal concentrations of persistent endectocides that stop further larval development. The experiment described in this paper reports an examination of this hypothesis.

2. Materials and methods 2.1. Experimental design Three groups of five parasite-naive calves of 6 months of age and one calf which had been frequently infected with D. viviparus and acted as a source of positive control plasma were used. They were divided into four treatments: (a) Group 1 were carefully weighed on Day 0 and then injected with the endectocide doramectin (Dectomax, Pfizer Ltd.) at the recommended dose rate of 200 mcg/kg. On each day from Day 1 to Day 19 they were orally dosed with 2000 infective larvae (L3) of D. viviparus. On Day 28 they were again treated with doramectin, and from Day 33 to Day 41 each animal was again dosed with 2000 L3 daily. They were then left until Day 81, 53 days after the second treatment, when they were weighed and given a challenge infection of 20 L3/kg of D. viviparus. They were killed on Day 110 and lungworm counts made using the modification of Oakley (1980) of the Inderbitzen method. (b) Group 2 calves were immunised with irradiated lungworm

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larvae (Huskvac, Intervet Ltd.) administered orally on Days 0 and 28, and were given the challenge infection on the same day as Group1 (Day 81), slaughtered on Day 110 and lungworm counts made as for Group 1: (c) Group 3 acted as controls for the challenge infection, and were infected on Day 81 and killed on Day 110 as per Groups 1 and 2; (d) the one calf of Group 4 was used as a source of positive control plasma for ELISA and immunofluorescence assays. 2.2. Observations Blood samples were taken from all the calves every 21 days starting at Day 0 and on Day 110 prior to slaughter, and faeces samples were taken per rectum weekly from Day 21. The blood samples were used for ELISA to D. viviparus adult somatic antigen, adult excretory/secretory (e/s) antigen, and developing larvae(L4) e/s antigen, for immunofluorescence tests to the L3 larval sheath, and for quantifying plasma concentrations of doramectin in Group 1. The faeces samples were examined for lungworm larvae by the adaptation of the Baermann method of Eysker (1997), in which 30 g of faeces were examined to increase the sensitivity of detection. 2.3. ELISA antigen preparation 2.3.1. Excretory/secretory antigens Lungworm adults and/or larvae were recovered from the lungs of donor calves. For adult antigen, animals were killed between Days 28 and 35 after infection; for L4, calves were slaughtered 11 days after infection. In all cases worms were recovered from the lungs by a modification of the Inderbitzen perfusion technique (Oakley, 1980). The recovered worms or larvae were rinsed in RPMI 1640 tissue culture fluid (GIBCOBRL Life Technologies Ltd., Paisley PA4 9RF, UK) containing antibiotics (100 ␮g/ml NA-penicillin and 0.25 mg/ml streptomycin) and a fungistat (Nystatin, Sigma N3503, Sigma Aldridge, Poole, Dorset, UK). After rinsing, the worms were transferred into roller tubes containing fresh RPMI with antibiotics and Nystatin, and incubated on a roller at 37◦ C for 24 h. The supernatant fluid was decanted and the worms transferred to similarly prepared fresh tubes, and then incubated for 48 h. After 48 h the fluid from these tubes was decanted and passed through a sterilising filter (0.22 ␮). The volume of fluid was then reduced using a molecular exclusion filter (YM10-Amicon Inc., Beverley, MA 01915, USA), the fluid retained above the filter being used as antigen. It was then dialysed against 0.01M phosphate buffered saline (PBS) overnight before being recovered and stored in aliquots at −70◦ C. 2.3.2. Somatic antigens After recovery as for e/s antigens, worms were placed in a Teflon pestle grinder in a ground glass tube (Cole Palmer, Vernon Hills, Illinois, USA) containing a small volume of PBS that included antibiotics as for e/s antigens plus 0.05 ␮g/ml N-tosyl-l-phenyl alanine chloromethylketone (TPCK) and 0.025 ␮g/ml of Na-p-tosyl-l-lysine chloromethylketone (TLCK) at 4◦ C. The worms were ground up before transfer to a sonicator (MSE Soniprep 150, Sanyo, Japan). Further PBS was added to the suspension and it was stirred overnight

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at 4◦ C after which it was centrifuged for 20 min at 10 000 × g at 4◦ C. The supernatant fluid was passed through a sterilising filter and stored at −70◦ C. 2.4. ELISA methods Tests were carried out on 96-well flat-bottomed microtitre plates (Immulon, Dynatech Laboratories, Chantilly, VA, USA). 100 ␮l of all the antigens (protein estimation 30 ␮g/ml) in each well were incubated at 4◦ C for 16 h. The plates were washed three times in a microplate washer (Titertek Microplate Washer M96V) with 0.01M PBS at pH 7.2. 100 ␮l of 5% bovine serum albumin were added to each well and incubated at 37◦ C for 1 h. The plates were then washed again in the washer with 0.01M PBS plus 0.05% Tween 20. 100 ␮l of test serum diluted with 0.1M PBS at pH 7.2, 2% NaCl, 0.05% Tween 80 and 1% bovine serum albumin, was then added to each well, and the plates incubated at 37◦ C for 1 h in a shaking incubator (Inshake-Luminar Technology). The plates were then washed five times with washing fluid (0.01M PBS, pH 7.2 plus 0.05% Tween 20). 100 ␮l of conjugate (RAB/IgG (whole molecule) HRPO) (Sigma A5295) diluted with 1/2560 with serum and conjugate diluent (0.1M PBS pH 7.2, 2.0% NaCl, 0/05% Tween 80, 1% bovine serum albumin) was added to the wells and the plates incubated for 1 h at 37◦ C. The plates were then washed five times in 0.01M PBS at pH 7.2, after which 100 ␮l of substrate (3,30 5,50 –tetramethyl-benzidine in 0.1 citrate/phosphate buffer (Chemicon ES001 TMB/E) was added and plates incubated for 10 min at room temperature. The reactions were stopped with 50 ␮l of 2.5M H2 SO4 , and the absorbance read on a plate reader (Titertek Multiscan) at 450 nm. The ELISA values were calculated using the formula (V − N/(P − N)) × 100, where V is the test serum value, N is the known negative serum value and P is the known positive serum value. 2.5. Plasma doramectin concentration analysis 2 ml of the test plasma was diluted with acetonitrile : water (2 ml, 1 : 1, v/v) and applied to a Bakerbond C18 solid phase extraction cartridge (J.T. Baker Inc., Phillipsburg, NJ, USA) that had been pre-washed with 4 ml acetonitrile followed by 4 ml of distilled water. After application of the sample, the cartridge was washed with a further aliquot of distilled water (7 ml), and allowed to dry for 5 min under vacuum. Doramectin was eluted twice with methanol (2 ml), and the methanol evaporated to dryness under nitrogen at 60◦ C. The residue was derivatised with 1-methylimidazole and trifluoracetic acid anhydride as described previously for ivermectin (De Montigny et al., 1990). The extracts were analysed by reversed-phase HPLC using a Luna 5 ␮m C18 column (250 mm × 4.6 mm) (Phenomenex, Macclesfield Cheshire, England) with methanol as the mobile phase at a flow rate of 1.8 ml/min. Fluorescence detection was employed with an excitation wavelength of 365 nm, and an emission wavelength of 470 nm. Results were calculated by comparison of sample peak areas of dilute standards derivatised simultaneously with the samples. The method was validated at 10 and 1 ng/ml with recoveries between 80 and 105%, and coefficients of variation of 2.7 and 8.3%. The limit of detection, based on a signal/noise ratio of 3, was <0.5 ng/ml.

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2.6. Immunofluorescence assays on D. viviparus larval L3 sheath The L3 used in the test were supplied by Intervet Ltd., Milton Road Cambridge, England. 250 larvae were placed in 1 ml of PBS in glass agglutination tubes. The larvae were washed three times in PBS for 5 min. After each washing the supernatant was drawn off by Pasteur pipette attached to a suction pump. A stop was placed on the pipette to prevent any larvae from being siphoned out. Each tube was then centrifuged and the washing, fluid removed. The test sera had been diluted in PBS at dilutions of 1/40, 1/80, 1/160, and 1/320. 1 ml of the sera under test was added to each tube and incubated overnight at 4◦ C. In the following morning the tubes were allowed to return to ambient room temperature (@20◦ C), before centrifugation to sediment the larvae. After the supernatant was removed the larvae were again washed three times in 1 ml PBS. After the last wash 1 ml of RAB/FITC IgG (Nordic Immunological Laboratories BV 5000AA Tilburg, The Netherlands) at a dilution of 1/100 was added to the larvae, and incubated at 37◦ C for 1 h. The larvae were then again sedimented by centrifugation and washed three times in PBS. After the final wash the larvae were transferred to a microscope slide, a coverslip (22 mm × 40 mm No.1) placed above and the slides were examined using a fluorescent microscope with incident light fluorescence and a 10× objective lens. A positive result was recorded when more than 50% of the larvae displayed specific cuticle fluorescence at the relevant serum dilution. 2.7. Statistical methods The D. viviparus counts from each group were compared non–parametrically by Kruskal Wallis ANOVA. The ELISA values at each sampling date and the Area Under Curve (AUC) of the graphs of the group mean ELISA values were compared after logarithmic transformation. The same method was used for the immunofluorescent assays of the larval sheaths. 3. Results 3.1. Clinical observations With the exception of one calf in the vaccinated Group 2 that died of bacterial pneumonia before the challenge infection, all the calves showed normal health until after the challenge infection. After that some mild coughing and slightly increased respiratory rates were observed in all groups, but at no time was the disease considered to be severe. Lungworm larvae were not found in the faeces of the calves in Groups 1 and 2 during the time from Day 0 until Day 106. On Day 106 they were observed in one calf in Group 2, and in all calves in Group 3. None were observed in faeces samples from Group 1 at any time during the experiment. 3.2. Total lungworm counts after slaughter on Day 110 As can be seen in Table 1 the counts from Groups 1 and 2 were much lower than that of Group 3; the difference between Group 1 and Group 3 was statistically significant (p < 0.05),

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Table 1 Lungworm counts after slaughter Group 2

Group 3

Lungworm count Lungworm count Lungworm count Lungworm count Lungworm count

Group 1 42 45 0 0 0

2 6 13 104 dieda

319 330 82 195 214

Arithmetic mean Geometric mean

17.4 4.5

31.3 11.3

228 204.8

a

Died due to bacterial pneumonia before challenge infection.

Table 2 Plasma concentrations of doramectin (ng/ml) Animal number

Day 0

Day 21

Day 49

Day 81

1 2 3 4 5

<1a <1 <1 <1 <1

3.0 1.7 2.8 3.4 5.7

3.7 2.7 9.4 5.4 7.5

<1 <1 <1 <1 <1

<1

3.3

5.7

<1

Arithmetic mean a

<1 = below detectable concentrations.

but the difference between Group 2 and Group 3 did not reach significance. There was no statistically significant difference between the counts of Groups 1 and 2. 3.3. Plasma doramectin concentrations in Group 1 calves The plasma analyses indicated that inhibitory concentrations of doramectin were still present 2 and 8 days respectively after the last administration of both the first and second lungworm infections, but that none was detectable at the time of the challenge infection (Table 2). The number of worms recovered after the challenge infection therefore should not have been affected by the presence of residual anthelmintic, unless the worm is affected by concentrations below the level of detection. 3.4. ELISA values (a) Adult somatic antigen (Fig. 1). From Day 0 until Day 81 there were no significant differences between the group mean ELISA. At the time of slaughter on Day 110 the group mean value of Group 1 was significantly lower (p < 0.05) than that of Group 3. There were no significant differences between the other groups. (b) L5 e/s antigen (Fig. 2). There were no significant differences between the reactions of the groups, all rising after infection. (c) L4 e/s antigen. (Fig. 3) From Fig. 3 it appears that Groups 1 and 2 did not react after infection to the same extent as Group 3. However due to large variations in the readings there were no statistically significant differences between the group mean values.

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Fig. 1. ELISA values with adult somatic antigen.

Fig. 2. ELISA values with L5 E/S antigen.

Fig. 3. ELISA values with L4 E/S antigen.

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Fig. 4. Reciprocal immunofluorescence titres to L3 larval sheath.

3.5. Immunofluorescence assays to larval sheath The titres of both Groups 1 and 2 increased during the infection period for Group 1 and the vaccination of Group 2, from Day 49 until Day 110 their mean titres were significantly greater (p < 0.001) than that of the uninfected controls, which remained low (Fig. 4). The AUC of the graphs of their mean values were similarly significantly larger (p < 0.001). 4. Discussion One of the major considerations in the design of this experiment was the persistence of the activity of doramectin against D. viviparus. The result of Weatherley et al. (1993), that doramectin has 100% efficacy at 21 days was taken as the maximum period for administration of L3 after doramectin injection, to ensure that no larvae would be able to continue development for long after penetration of the intestine. The first series of 2000 L3 per day were therefore given to Group 1 from Day +1 to Day +19 to stay within that limit, and the second series from Day +33 to Day +41, after the second treatment on Day +28. The second consideration was to ensure that nematocidal plasma concentrations of doramectin were not present at the time of the challenge infection on Day +81. The longest reported persistent efficacy after treatment with doramectin is that of Burden and Ellis (1997), who found 88.2% protection at Day +42. The same authors cited 96.4% efficacy at 35 days, and Stromberg et al. (1999) more recently cited 97.5% at 28 days. Toutain et al. (1997) in a pharmacokinetic analysis, have also shown that plasma concentrations were unlikely to be present for longer than 42 days. For these reasons the interval between the last treatment and the challenge infection of 53 days was thought to be adequate to ensure that no residual plasma doramectin would be present to interfere with the development of the larvae in the challenge infection. As an additional safeguard, the plasma concentrations of doramectin were analysed in samples taken at the time of the challenge infection on Day +81, and were all below the limit of detection of 0.5 ng/ml. Unless the larvae are susceptible to concentrations below that level, their development after the challenge infection should not have been affected by the presence of doramectin.

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When the lungworm counts were completed, two features of the results were of special interest. Firstly in Group 1 three calves had no worms at all and the remaining two had 42 and 45 respectively. Two possibilities exist: either the three calves had a very high level of immunity or enough residual doramectin had been present to inhibit development. The latter explanation is unlikely given that none was detectable at the day of challenge, and previous experiments cited (Weatherley et al., 1993; Burden and Ellis, 1997; Stromberg et al., 1999) had shown that persistent efficacy was less than 100% at 28 days after treatment. In addition, after 53 days some worm development would have been expected even if very low concentrations of doramectin were still present. The second feature was the relatively high number of worms found in one of the vaccinated Group 2 calves, which illustrated that this calf would have been a source of considerable larval output in a grazing situation. The ELISA results were not unexpected. In previous experiments many authors have found that reactions to adult somatic antigen frequently reflect the number of adult worms recovered, and the same was observed in this study. The highest reaction after infection was in Group 3, which had the largest number of adult worms, with Groups 1 and 2 having lower reactions (Fig. 1). The reactions to L4 e/s antigen indicated a significantly larger post challenge increase in the calves of Group 3 in contrast to the other groups (Fig. 3), mirroring the number of larvae that developed to the L4 stage in each. There were no large differences between the groups in the reactions to adult (L5) e/s antigens (Fig. 2). The most interesting immunological reactions were those to the larval sheath (Fig. 4). Both Groups 1 and 2 showed increasing reactions during their respective treatment and vaccination periods, and a further increase after challenge infection. There was, as would be expected, no reaction in Group 3 before infection and only a small increase after infection. The results in Group 1 showed that reaction to larval sheath antigens was taking place despite the high plasma concentrations of doramectin at the time, and in Group 2 to the larval sheath of the irradiated larvae. This experiment has shown that in situations of heavy lungworm infection and simultaneous systemic doramectin treatment, significant immunity can be stimulated without further development of the larvae. This must have taken place after the early death of the larvae and systemic antigenic release, both from the larval sheath and somatic antigens. However, since immunity to lungworm involves protection against larval establishment as well as inhibition of further adult development, the protection in this study may extend only to the former, and may wane during succeeding months if no further stimulus is received (Michel et al., 1965). The degree of immunity stimulated may reflect the large number of L3 administered (56 000), and in normal field infections when the larval intake is much lower, the reaction would be expected to be less. The same reactions may apply to other injected or topically applied endectocides, but may not to continuous-release intraruminal boluses where the anthelmintic concentration in the ruminal and reticular fluid may reduce the viability of the larvae and inhibit the penetration of the larvae into the intestine.

Acknowledgements The authors are grateful to Intervet Ltd. for the gift of infective larvae of D. viviparus, and to Pfizer Ltd. for the gift of a doramectin standard for analysis of the plasma doramectin con-

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