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The effect of treatment with eprinomectin on lungworms at early patency on the development of immunity in young cattle
Veterinary Parasitology 114 (2003) 205–214
The effect of treatment with eprinomectin on lungworms at early patency on the development of immunity in young cattle Johan Höglund a,∗ , Charina Gånheim b , Stefan Alenius b a
b
Department of Parasitology (SWEPAR), National Veterinary Institute and Swedish University of Agricultural Sciences, 751 89 Uppsala, Sweden Department of Ruminant Medicine and Veterinary Epidemiology, Swedish University of Agricultural Sciences, 750 07 Uppsala, Sweden Received 23 October 2002; received in revised form 19 March 2003; accepted 28 March 2003
Abstract An experiment was carried out to study the effect of topical application of eprinomectin at early patency on the build up of infection and development of protection against Dictyocaulus viviparus in young cattle. Three groups of six calves were used and parasitological and blood variables were monitored at weekly intervals throughout the trial. At the start of the experiment calves in groups A and B were experimentally inoculated with 100 D. viviparus infective third-stage larvae (L3) for five consecutive days, whereas calves in group C served as uninfected controls. The calves in group A were each treated with eprinomectin (0.5 mg/kg bodyweight) in a pour-on formulation at early patency at day 24 post the first inoculation, whereas the calves in groups B and C were left untreated. Seven weeks following anthelmintic treatment all groups were challenged with 1500 L3. Another 4 weeks later the animals were sacrificed and established worms in the lungs were counted. Moderate transient signs of lungworm disease occurred both in groups A and B. However, group B calves were found to be about 8 times more resistant than those in group A, whereas the naive infection controls in Group C was found to be about 35 times more susceptible to infection. Also the ELISA values showed that the course of infection was different between experimental groups. The eosinophil counts prior to and at the time of slaughter indicate that immunity was involved in the protection and the response was correlated with previous exposure and worm load. Weight gains differed significantly, but only between groups A and C and between groups B and C that on an average were approximately 13 kg heavier at the termination of the experiment. It was concluded that eprinomectin was effective against established adult lungworms. However, the untreated calves (group B) developed a more marked
∗ Corresponding author. Tel.: +46-18-674156; fax: +46-18-309162. E-mail address: [email protected] (J. Höglund).
0304-4017/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0304-4017(03)00155-9
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resistance to lungworms compared to those that were subjected to anthelmintic treatment at early patency (group A). On the other hand, the cumulative number of excreted larvae was on an average 43 times higher in group B as compared to group A. Consequently, infected calves that remain out on pasture should be treated. This will restrain transmission of the parasite despite the fact that immunity is deteriorated. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Cattle; Dictyocaulus viviparus; Immunity; Eosinophils; Protection; Eprinomectin
1. Introduction Dictyocaulus viviparus is an extremely pathogenic nematode of cattle in temperate regions with mild summers and high rainfall. It causes husk, or bovine parasitic bronchitis, which is a serious lung disease that in general is observed from August and onwards in Sweden. The costs of a moderate outbreak in a herd with 100 dairy cows in the UK has been estimated to be in the magnitude of ∼30 000–35 000 Euro (Woolley, 1997). D. viviparus has a direct life cycle with infective third stage larvae (L3) that are ingested with herbage (Eysker, 1994). In contrast to gastrointestinal nematodes of cattle, D. viviparus travel from the small intestine to the predilection sites in the lungs via the lymphatic system and pulmonary circulation. This induces a very powerful immune response which normally results in a high level of protection against reinfection (Gilleard et al., 1995). In recent years there is evidence for an increased prevalence of D. viviparus in Sweden. A regional survey among organic cattle producers showed that lungworm infected animals were found in approximately 80% of the herds examined at the time of housing in late autumn (Höglund et al., 2001). Accordingly lungworm infections have attracted an increased attention in Sweden. As an outcome of this it has become increasingly more evident that also many conventional dairy and beef producers are experiencing problems with this infection. Thus it seems that the situation in Sweden is in many respects similar to that found elsewhere in northwestern Europe (Ploeger et al., 2000; Schnieder et al., 1993). An irradiated larval lungworm vaccine has never been available on the Swedish market. Although early season prophylactic measures against nematode parasites, based on the application of anthelmintics in combination with grazing management are advocated to Swedish farmers, these measures have never been specifically directed against lungworms. Instead, when clinical signs of dictyocaulosis are diagnosed, farmers are advised to treat all animals in the affected grazing group, including those individuals harbouring sub-clinical infections. To what extent this strategy interferes with the development of acquired resistance to re-infection is unclear. The aim of the study was to evaluate the influence of eprinomectin treatment on the level of protection against lungworm in young cattle following reinfection to moderate number of infective larvae. We were particularly interested to investigate to what extent treatment of calves with sub-clinical D. viviparus infection at early patency interfered with development of protection to reinfection some weeks later.
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2. Materials and methods 2.1. Animals and parasite strain Eighteen Swedish Red and White Breed Cattle (SRB) male calves were purchased at an age of about 2 months from three commercial farms. These herds were certified to be free from bovine virus diarrhoea (BVDV). Upon arrival to the Department of Ruminant Medicine and Veterinary Epidemiology at the Swedish University of Agricultural Sciences (SLU) in January 2002, the calves were allocated to three experimental groups (A, B and C) based on their initial weights. The calves were then acclimatised in three identical pens (3–3.7 m) for another 4 weeks before the experiment was started. Throughout the experiment the calves were inspected daily by clinicians. They were fed hay and water ad libitum as well as about 1.2 kg supplement per day. The D. viviparus larvae used in this experiment were obtained from Intervet (The Netherlands). These were administered to two donor calves, which acted as a source of larvae once infections reached patency. These calves were housed similar to the experimental calves, and the larvae used for the experimental inoculations were less than 3 weeks old. 2.2. Experimental design At day 1 of the experiment, all calves in groups A and B were inoculated with 100 infective D. viviparus third stage larvae (L3) for five consecutive days, whereas those in group C remained uninfected. At day 24 following the first inoculation, when patency first had been observed, the calves in group A were treated with eprinomectin (Eprinex Pour-On Vet.® , Merial) applied topically at a dose rate 0.5 mg/kg bodyweight according to the manufacturers instructions. The animals in groups B and C were left untreated. Seven weeks following treatment of group A, all animals in experimental groups A, B and C were challenged with 1500 L3. Following another period of 4 weeks post-challenge (p.c.), the experimental animals were sacrificed and worms established in the lungs were recovered and counted. 2.3. Observations Throughout the experimental period individual blood and faecal samples were collected at weekly intervals for determination of specific serum antibodies, eosinophils and excreted larvae. Enumeration of larvae was based on the numbers in 50 g of fresh faeces extracted overnight at room temperature in Baermann funnels with a diameter of 30 cm. Total and differential leucocyte counts were performed with Cell-Dyn 3500 using software for veterinary specimens (Abbott Diagnostic Division, Abbott Park, IL, USA). Serum and EDTA samples were taken from the jugular vein with vacutainer tubes (Becton-Dickinson, USA). A specific antibody response reflecting patent D. viviparus infection were measured according to the procedures described in a commercially available indirect enzyme-linked immunosorbent assay kit (ELISA) (Ceditest, IDO-DLO, The Netherlands). To account for inter-assay variation control sera was added to fixed positions on all plates, whereas the sera to be tested was added randomly in duplicates. The OD values of the test sera
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were then converted to percent seropositivity by dividing with the mean OD values obtained for the negative control sera diluted 1:50 on the sample plate. The animals were weighed upon arrival and thereafter every fortnight. Clinical signs such as coughing and listlessness as well as rectal temperature were recorded on a daily basis by clinicians. Number of established worms were established at necropsy after perfusion of the lungs according to procedures described by Andrews and James (1994) and Borgsteede et al. (1998). 2.4. Statistical evaluation Differences between experimental groups were assessed using a repeated measures model and one-way analysis of variance (ANOVA) with Tukey Cramer post hoc test. Groups A–C and time served as independent variables and number of adult worms, L3s, antibody levels, eosinophils and weight gain were dependent response variables. Data were handled in Excel© (Microsoft) and statistical analyses were performed in StatViewTM (SAS) software with P ≤ 0.05 as the significance level. Where necessary the data were logarithmically transformed. 3. Results 3.1. Clinical observations Most calves in groups A and B developed moderate transient signs of respiratory disease approximately 14 days after the first inoculation, whereas those in the negative control group C were clinically healthy until they experienced the challenge infection. In three and four animals of groups A and B, respectively, fever increased and these individuals were treated with benzylpenicillinprokain (Ethacilin Vet.® , Intervet), at a dose rate of 20 mg/kg bodyweight, on five consecutive days. One animal in the B group was sacrificed 21 days post-infection (p.i.) for humane reasons. At autopsy no adult lungworms were found, however, pneumonia of viral origin was observed. 3.2. Larvae in faeces Mean faecal larval counts are shown in Fig. 1a. Larvae were detected in all calves in groups A and B from day 24 p.i. The eprinomectin treatment of group A at day 24 p.i., subsequently eliminated larval excretion. Faecal larval counts were highest in group B and the values increased rapidly and reached its maxima at day 31 p.i. when larval numbers per gram of faeces varied between 8 and 27. Thereafter larval numbers dropped gradually and no larvae were observed at day 52 p.i. At day 22 p.c. the excretion of larvae increased in most animals. One week later mean larval numbers varied between 16, 19 and 154 in groups A, B and C, respectively. The differences observed in larval numbers were significant during the course of the infection (P < 0.0001). However, cumulative number of larvae varied only between experimental groups A and B (P < 0.0001), A and C (P < 0.0001), but not between B and C (P = 0.71).
Fig. 1. (a) Mean faecal counts of D. viviparus first stage larvae, (b) ELISA values indicating the development of IgG1 antibodies to D. viviparus adult antigens expressed as mean seropositivity in relation to control sera, (c) mean eosinophil counts (number of cells × 109 /l). Calves in groups A and B were inoculated with 5 × 100 D. viviparus infective third-stage larvae (L3), whereas calves in group C served as uninfected controls. Group A were treated with eprinomectin (0.5 mg/kg bodyweight) at day 24 post the first inoculation, whereas groups B and C were left untreated. Seven weeks following anthelmintic treatment all groups were challenged with 1500 L3.
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3.3. ELISA results Fig. 1b demonstrates that parasite-specific IgG1 levels were slightly elevated day 24 p.i. both in groups A and B. In group B antibodies continued to increase and maximum levels were observed at day 52 p.i., whereas in group A the levels started to decrease approximately 14 days after anthelmintic treatment. The antibody responses in groups A and B against the challenge infection occurred earlier compared to after the primary infection. In group A elevated levels were observed already 7 days p.c. In group B antibody levels continued to decrease from very high levels also after challenge, however, they were increased again 21 days p.c. The antibody responses were significantly (P < 0.0001) different between experimental groups. 3.4. Number of eosinophils The mean eosinophil counts are shown in Fig. 1c. Both in groups A and B eosinophils increased steadily from the baseline levels between days 10 and 17 p.i. However, the response was significantly (P = 0.0025) higher in group B, where the number of eosinophils reached a maximum of 7.9 × 109 cells/l and were elevated from day 17 p.i. Then they remained elevated until day 59 p.i. when they started to decrease. Also in group A, a maximum of 7.8 × 109 cells/l was observed at day 18 p.i., but in this group the numbers started to decrease already from day 31 p.i. After challenge the eosinophils were increased again and in both groups elevated levels were observed 14 days p.c. As with the antibodies the response of the eosinophils occurred somewhat earlier after challenge as compared with after the primary infection.
Fig. 2. Mean number of established D. viviparus ± S.D. in the lung at slaughter 28 days after challenge with 1500 L3. Group A: primary infected (5 × 100 L3) between days 0–4 and treated with eprinomectin (0.5 mg/kg bodyweight) at day 24, group B: only primary infected, and group C: challenge control. Seven weeks following anthelmintic treatment all groups were challenged with 1500 L3.
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Fig. 3. Mean cumulative weight gains (kg). Calves in groups A and B were inoculated with 5 × 100 D. viviparus infective third-stage larvae (L3), whereas calves in group C served as uninfected controls. Groups A and B: primary infected with 5 × 100, group C: challenge control.
3.5. Lungworm counts Worm counts at necropsy are shown in Fig. 2. There was a significant (P < 0.0001) difference in the number of adult lungworms that were recovered from each of the experimental groups. Post hoc testing showed that the post mortem counts within group A was significantly (P < 0.0001) higher compared to group B that in turn was significantly (P < 0.0001) lower than in group C. 3.6. Weight gains Weight gains are shown in Fig. 3. There was a significant difference in weight performance between calves in the different experimental groups (P = 0.019), but only between the calves in the uninfected challenge control group C that grew significantly better than those in groups A and B. Calves in group C had an average weight gain of 1.16 kg per day, whereas the animals in groups A and B gained 0.96 and 0.94 kg, respectively.
4. Discussion In previous studies protection against D. viviparus in calves has been defined as the proportion of ingested infective larvae that survives to maturity after challenge (Oakley, 1981; Ploeger and Eysker, 2002). In this study we showed that a good level of protection could be stimulated in calves that had been treated with a topical formulation of eprinomectin at early patency. This is in agreement with earlier observations that have shown that D. viviparus infected cattle developed some degree of protection to reinfection when drenched either with levamisole, or fenbendazole around the onset of patency of the parasite (Downey, 1980). We also observed that a relatively low dose level of larvae, 5×100 per animal induced
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protection. This is in accordance with Ploeger and Eysker (2002), who found an inoculation dose of 500 larvae to be sufficient to stimulate the development of a reasonable protection against a challenge infection 35 days after primary infection. In this trial we also demonstrated that the level of protection was significantly improved in animals that were allowed to recover naturally from infection when compared to the levels in the treated infection control group (group B). Thus this result confirms the conclusion that development of protection against D. viviparus in cattle is a gradual process, as it seemed to be beneficial when the immune system was stimulated by all stages of the parasite. It has been demonstrated that a strong protective immunity against D. viviparus can be induced without further development of larvae before the larval stages reach maturity in the lungs (Taylor et al., 2000). On the contrary, it has been established that high levels of protection was induced when the migratory phase of the life cycle was avoided. In a vaccination trial (Bain and Urquhart, 1988) showed that passage of irradiated larvae through the intestine and mesenteric lymph nodes was not necessary for the stimulation of a high degree of immunity. Significant levels of protection have been observed in guinea pigs as an infection model, that were immunised with protective adult ES-antigens that seemed to be involved in some form of antibody mediated mechanism (McKeand et al., 1995). On the other hand, when the protective effect of these antigens was tested in calves, conflicting results were obtained (Matthews et al., 2001). As mentioned earlier, the results of the present experiment indicated that the level of protection against a constant challenge was enhanced in the untreated group and probably as a result of the combined immune responses directed both against the migrating juvenile worms and established adults in the lungs. However, treatment of the animals in group A was applied 24 days after the first trickle infection and immediately when patency first was observed. Consequently, with the design of the experiment it was not possible to completely rule out that the differences in challenge protection between calves in experimental groups A and B was a reflection of the number of adult worms that previously had been established in the lungs and the time the animals had been exposed to these worms. Although it has been demonstrated in several field experiments that varying degrees of protection were induced in first season grazers subjected to different suppressive anthelmintic regimes (Borgsteede et al., 1998; Jacobs et al., 1989; Taylor et al., 1997, 1990), the results from the present experiment clearly indicated that a significantly stronger protection was induced in calves that were allowed to recover naturally from the infection as compared to if they were treated at early patency. It has been documented that topical application of eprinomectin at the 0.5 and 0.2 mg/kg dosage levels remove more than, or equal to 99% of a wide range of species of adult nematode parasites in cattle (Shoop et al., 1996). In this trail we observed that larval excretion dropped to zero in all of the treated animals 1 week after application of the drug. The activity of eprinomectin against D. viviparus at the lower dose level was thus confirmed. The drop in larval excretion was accompanied by a decrease in the antibody levels and in the number of peripheral eosinophils, which indicates that all adult lungworms were removed. The persistent efficacy of eprinomectin at the lower dosage level has been shown to last for a minimum of 28 up to 35 days after treatment (Cramer et al., 2000). In this study we gave the challenge infection 7 weeks after treatment. Thus it cannot be argued that the difference between the primary trickle infected groups was due to residual activity of the drug.
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Notable is that the growth was similar in both of the primary infected groups, but significantly reduced as compared to the uninfected challenge control group. Accordingly the damage induced by the parasite was caused already in the initial phase of the infection before the animals were treated with anthelmintic. It also seemed like the treated animals were unable to compensate for this initial loss in weight gains. Furthermore, all animals in the trickle-infected groups were susceptible to challenge 11 weeks after the primary infection. Consequently, the immunity induced by the primary infection was not able to completely protect the animals against challenge. Those in the eprinomectin group were also about eight times more susceptible to re-infection than the group that recovered from the infection. These observations are interesting both from a practical and epidemiological point of view. Applied in a possible field situation it can be argued that both groups are likely to be involved in the propagation of the infection and it cannot be expected that the performance should be improved in case of the animals are treated at early patency. On the other hand, although the protection is enhanced if the animals are allowed to recover naturally from the infection, these animals will continue to excrete larvae that will contaminate the pastures. The cumulative number of larvae that were excreted by the animals in the untreated group was 43 times higher as compared to the animals in the group that were allowed to recover naturally from the infection. Based on the results of this experiment it can be concluded that topical eprinomectin treatment of lungworm-infected cattle at early patency interfered with the development of protection against reinfection. This is in conflict with the current advice given to Swedish farmers. However, in a field situation animals are exposed to infective larvae also after treatment has been applied. There is evidence that challenge infection in this situation will result in the development of some degree of protection (Borgsteede et al., 1998). Another factor that needs to be taken into consideration is the continued contamination of the pastures with larvae where animals are kept out on pasture, but remain untreated. As larval excretion was eliminated following the application of eprinomectin, this measure will clearly restrain transmission and thereby prevent, or reduce, the risk of re-infection to co-grazing animals. However, one possibility to avoid this scenario would be to stable infected, but clinically healthy animals for a period of some weeks in order to allow them to build up immunity. This will obviously induce a significantly stronger protection against re-infection compared to animals that are treated. Acknowledgements We are grateful to Miss M. Moberg and MSc A. Engström for technical assistance. Dr. A. Vermeulen, Intervet, is thanked for originally providing us with larvae. Thanks also to the animal attendants at Department of Ruminant Medicine for taking care of the animals. Formas (22.9/2001-1991) provided funding support to JH for this trial that was approved by the Swedish Animal Ethics Committee (permission C4/2). References Andrews, S.J., James, F.M., 1994. Further evaluation of a perfusion technique for the recovery of Dictyocaulus viviparus from bovine lungs. J. Helminthol. 68, 81–82.
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Bain, R.K., Urquhart, G.M., 1988. Parenteral vaccination of calves against the cattle lungworm Dictyocaulus viviparus. Res. Vet. Sci. 45, 270–271. Borgsteede, F.H., van der Linden, J.N., Cornelissen, J.B., Gaasenbeek, C.P., Ascher, F., 1998. Effect of three sustained-release devices on parasitic bronchitis in first year calves. Vet. Rec. 142, 696–699. Cramer, L.G., Pitt, S.R., Rehbein, S., Gogolewski, R.P., Kunkle, B.N., Langhoff, W.K., Bond, K.G., Maciel, A.E., 2000. Persistent efficacy of topical eprinomectin against nematode parasites in cattle. Parasitol. Res. 86, 944–946. Downey, N.E., 1980. Effect of treatment with levamisole or fenbendazole on primary experimental Dictyocaulus viviparus infection and on resistance in calves. Vet. Rec. 107, 271–275. Eysker, M., 1994. Dictyocaulosis in cattle. Compend. Contin. Educ. Pract. Vet. 16, 669–675. Gilleard, J.S., Duncan, J.L., Tait, A., 1995. An immunodominant antigen on the Dictyocaulus viviparus L3 sheath surface coat and a related molecule in other strongylid nematodes. Parasitology 111, 193–200. Höglund, J., Svensson, C., Hessle, A., 2001. A field survey on the status of internal parasites in calves on organic dairy farms in southwestern Sweden. Vet. Parasitol. 99, 113–128. Jacobs, D.E., Fox, M.T., Pilkington, J.G., Ross, D.B., Ryan, W.G., 1989. Chemoprophylaxis and immunity to parasitic bronchitis in cattle—a field experiment comparing topical ivermectin and an oxfendazole intraruminal device. J. Vet. Pharmacol. Ther. 12, 444–450. Matthews, J.B., Davidson, A.J., Freeman, K.L., French, N.P., 2001. Immunisation of cattle with recombinant acetylcholinesterase from Dictyocaulus viviparus and with adult worm ES products. Int. J. Parasitol. 31, 307– 317. McKeand, J.B., Knox, D.P., Duncan, J.L., Kennedy, M.W., 1995. Protective immunisation of guinea pigs against Dictyocaulus viviparus using excretory/secretory products of adult parasites. Int. J. Parasitol. 25, 95–104. Oakley, G.A., 1981. Protection developed against reinfection by Dictyocaulus viviparus following anthelmintic treatment of a one-day-old primary infection in cattle. Vet. Rec. 109, 485–487. Ploeger, H.W., Eysker, M., 2002. Protection against and establishment of Dictyocaulus viviparus following primary infection at different dose levels. Vet. Parasitol. 106, 213–223. Ploeger, H.W., Borgsteede, F.H., Sol, J., Mirck, M.H., Huyben, M.W., Kooyman, F.N., Eysker, M., 2000. Cross-sectional serological survey on gastrointestinal and lung nematode infections in first and second-year replacement stock in The Netherlands: relation with management practices and use of anthelmintics. Vet. Parasitol. 90, 285–304. Schnieder, T., Bellmer, A., Tenter, A.M., 1993. Seroepidemiological study on Dictyocaulus viviparus infections in first year grazing cattle in northern Germany. Vet. Parasitol. 47, 289–300. Shoop, W.L., Egerton, J.R., Eary, C.H., Haines, H.W., Michael, B.F., Mrozik, H., Eskola, P., Fisher, M.H., Slayton, L., Ostlind, D.A., Skelly, B.J., Fulton, R.K., Barth, D., Costa, S., Gregory, L.M., Campbell, W.C., Seward, R.L., Turner, M.J., 1996. Eprinomectin: a novel avermectin for use as a topical endectocide for cattle. Int. J. Parasitol. 26, 1237–1242. Taylor, S.M., Mallon, T.R., Green, W.P., 1990. Comparison of the efficacy of dermal formulations of ivermectin and levamisole for the treatment and prevention of Dictyocaulus viviparus infection in cattle. Vet. Rec. 126, 357–359. Taylor, S.M., Kenny, J., Edgar, H.W., Whyte, M., 1997. Protection against Dictyocaulus viviparus in second year cattle after first year treatment with doramectin or an ivermectin bolus. Vet. Rec. 141, 593–597. Taylor, S.M., Kenny, J., Edgar, H.W., Mallon, T.R., Canavan, A., 2000. Induction of protective immunity to Dictyocaulus viviparus in calves while under treatment with endectocides. Vet. Parasitol. 88, 219–228. Woolley, H., 1997. The economic impact of husk in dairy cattle. Cattle Pract. 5, 315–317.
The effect of treatment with eprinomectin on lungworms at early patency on the development of immunity in young cattle Johan Höglund a,∗ , Charina Gånheim b , Stefan Alenius b a
b
Department of Parasitology (SWEPAR), National Veterinary Institute and Swedish University of Agricultural Sciences, 751 89 Uppsala, Sweden Department of Ruminant Medicine and Veterinary Epidemiology, Swedish University of Agricultural Sciences, 750 07 Uppsala, Sweden Received 23 October 2002; received in revised form 19 March 2003; accepted 28 March 2003
Abstract An experiment was carried out to study the effect of topical application of eprinomectin at early patency on the build up of infection and development of protection against Dictyocaulus viviparus in young cattle. Three groups of six calves were used and parasitological and blood variables were monitored at weekly intervals throughout the trial. At the start of the experiment calves in groups A and B were experimentally inoculated with 100 D. viviparus infective third-stage larvae (L3) for five consecutive days, whereas calves in group C served as uninfected controls. The calves in group A were each treated with eprinomectin (0.5 mg/kg bodyweight) in a pour-on formulation at early patency at day 24 post the first inoculation, whereas the calves in groups B and C were left untreated. Seven weeks following anthelmintic treatment all groups were challenged with 1500 L3. Another 4 weeks later the animals were sacrificed and established worms in the lungs were counted. Moderate transient signs of lungworm disease occurred both in groups A and B. However, group B calves were found to be about 8 times more resistant than those in group A, whereas the naive infection controls in Group C was found to be about 35 times more susceptible to infection. Also the ELISA values showed that the course of infection was different between experimental groups. The eosinophil counts prior to and at the time of slaughter indicate that immunity was involved in the protection and the response was correlated with previous exposure and worm load. Weight gains differed significantly, but only between groups A and C and between groups B and C that on an average were approximately 13 kg heavier at the termination of the experiment. It was concluded that eprinomectin was effective against established adult lungworms. However, the untreated calves (group B) developed a more marked
∗ Corresponding author. Tel.: +46-18-674156; fax: +46-18-309162. E-mail address: [email protected] (J. Höglund).
0304-4017/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0304-4017(03)00155-9
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resistance to lungworms compared to those that were subjected to anthelmintic treatment at early patency (group A). On the other hand, the cumulative number of excreted larvae was on an average 43 times higher in group B as compared to group A. Consequently, infected calves that remain out on pasture should be treated. This will restrain transmission of the parasite despite the fact that immunity is deteriorated. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Cattle; Dictyocaulus viviparus; Immunity; Eosinophils; Protection; Eprinomectin
1. Introduction Dictyocaulus viviparus is an extremely pathogenic nematode of cattle in temperate regions with mild summers and high rainfall. It causes husk, or bovine parasitic bronchitis, which is a serious lung disease that in general is observed from August and onwards in Sweden. The costs of a moderate outbreak in a herd with 100 dairy cows in the UK has been estimated to be in the magnitude of ∼30 000–35 000 Euro (Woolley, 1997). D. viviparus has a direct life cycle with infective third stage larvae (L3) that are ingested with herbage (Eysker, 1994). In contrast to gastrointestinal nematodes of cattle, D. viviparus travel from the small intestine to the predilection sites in the lungs via the lymphatic system and pulmonary circulation. This induces a very powerful immune response which normally results in a high level of protection against reinfection (Gilleard et al., 1995). In recent years there is evidence for an increased prevalence of D. viviparus in Sweden. A regional survey among organic cattle producers showed that lungworm infected animals were found in approximately 80% of the herds examined at the time of housing in late autumn (Höglund et al., 2001). Accordingly lungworm infections have attracted an increased attention in Sweden. As an outcome of this it has become increasingly more evident that also many conventional dairy and beef producers are experiencing problems with this infection. Thus it seems that the situation in Sweden is in many respects similar to that found elsewhere in northwestern Europe (Ploeger et al., 2000; Schnieder et al., 1993). An irradiated larval lungworm vaccine has never been available on the Swedish market. Although early season prophylactic measures against nematode parasites, based on the application of anthelmintics in combination with grazing management are advocated to Swedish farmers, these measures have never been specifically directed against lungworms. Instead, when clinical signs of dictyocaulosis are diagnosed, farmers are advised to treat all animals in the affected grazing group, including those individuals harbouring sub-clinical infections. To what extent this strategy interferes with the development of acquired resistance to re-infection is unclear. The aim of the study was to evaluate the influence of eprinomectin treatment on the level of protection against lungworm in young cattle following reinfection to moderate number of infective larvae. We were particularly interested to investigate to what extent treatment of calves with sub-clinical D. viviparus infection at early patency interfered with development of protection to reinfection some weeks later.
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2. Materials and methods 2.1. Animals and parasite strain Eighteen Swedish Red and White Breed Cattle (SRB) male calves were purchased at an age of about 2 months from three commercial farms. These herds were certified to be free from bovine virus diarrhoea (BVDV). Upon arrival to the Department of Ruminant Medicine and Veterinary Epidemiology at the Swedish University of Agricultural Sciences (SLU) in January 2002, the calves were allocated to three experimental groups (A, B and C) based on their initial weights. The calves were then acclimatised in three identical pens (3–3.7 m) for another 4 weeks before the experiment was started. Throughout the experiment the calves were inspected daily by clinicians. They were fed hay and water ad libitum as well as about 1.2 kg supplement per day. The D. viviparus larvae used in this experiment were obtained from Intervet (The Netherlands). These were administered to two donor calves, which acted as a source of larvae once infections reached patency. These calves were housed similar to the experimental calves, and the larvae used for the experimental inoculations were less than 3 weeks old. 2.2. Experimental design At day 1 of the experiment, all calves in groups A and B were inoculated with 100 infective D. viviparus third stage larvae (L3) for five consecutive days, whereas those in group C remained uninfected. At day 24 following the first inoculation, when patency first had been observed, the calves in group A were treated with eprinomectin (Eprinex Pour-On Vet.® , Merial) applied topically at a dose rate 0.5 mg/kg bodyweight according to the manufacturers instructions. The animals in groups B and C were left untreated. Seven weeks following treatment of group A, all animals in experimental groups A, B and C were challenged with 1500 L3. Following another period of 4 weeks post-challenge (p.c.), the experimental animals were sacrificed and worms established in the lungs were recovered and counted. 2.3. Observations Throughout the experimental period individual blood and faecal samples were collected at weekly intervals for determination of specific serum antibodies, eosinophils and excreted larvae. Enumeration of larvae was based on the numbers in 50 g of fresh faeces extracted overnight at room temperature in Baermann funnels with a diameter of 30 cm. Total and differential leucocyte counts were performed with Cell-Dyn 3500 using software for veterinary specimens (Abbott Diagnostic Division, Abbott Park, IL, USA). Serum and EDTA samples were taken from the jugular vein with vacutainer tubes (Becton-Dickinson, USA). A specific antibody response reflecting patent D. viviparus infection were measured according to the procedures described in a commercially available indirect enzyme-linked immunosorbent assay kit (ELISA) (Ceditest, IDO-DLO, The Netherlands). To account for inter-assay variation control sera was added to fixed positions on all plates, whereas the sera to be tested was added randomly in duplicates. The OD values of the test sera
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were then converted to percent seropositivity by dividing with the mean OD values obtained for the negative control sera diluted 1:50 on the sample plate. The animals were weighed upon arrival and thereafter every fortnight. Clinical signs such as coughing and listlessness as well as rectal temperature were recorded on a daily basis by clinicians. Number of established worms were established at necropsy after perfusion of the lungs according to procedures described by Andrews and James (1994) and Borgsteede et al. (1998). 2.4. Statistical evaluation Differences between experimental groups were assessed using a repeated measures model and one-way analysis of variance (ANOVA) with Tukey Cramer post hoc test. Groups A–C and time served as independent variables and number of adult worms, L3s, antibody levels, eosinophils and weight gain were dependent response variables. Data were handled in Excel© (Microsoft) and statistical analyses were performed in StatViewTM (SAS) software with P ≤ 0.05 as the significance level. Where necessary the data were logarithmically transformed. 3. Results 3.1. Clinical observations Most calves in groups A and B developed moderate transient signs of respiratory disease approximately 14 days after the first inoculation, whereas those in the negative control group C were clinically healthy until they experienced the challenge infection. In three and four animals of groups A and B, respectively, fever increased and these individuals were treated with benzylpenicillinprokain (Ethacilin Vet.® , Intervet), at a dose rate of 20 mg/kg bodyweight, on five consecutive days. One animal in the B group was sacrificed 21 days post-infection (p.i.) for humane reasons. At autopsy no adult lungworms were found, however, pneumonia of viral origin was observed. 3.2. Larvae in faeces Mean faecal larval counts are shown in Fig. 1a. Larvae were detected in all calves in groups A and B from day 24 p.i. The eprinomectin treatment of group A at day 24 p.i., subsequently eliminated larval excretion. Faecal larval counts were highest in group B and the values increased rapidly and reached its maxima at day 31 p.i. when larval numbers per gram of faeces varied between 8 and 27. Thereafter larval numbers dropped gradually and no larvae were observed at day 52 p.i. At day 22 p.c. the excretion of larvae increased in most animals. One week later mean larval numbers varied between 16, 19 and 154 in groups A, B and C, respectively. The differences observed in larval numbers were significant during the course of the infection (P < 0.0001). However, cumulative number of larvae varied only between experimental groups A and B (P < 0.0001), A and C (P < 0.0001), but not between B and C (P = 0.71).
Fig. 1. (a) Mean faecal counts of D. viviparus first stage larvae, (b) ELISA values indicating the development of IgG1 antibodies to D. viviparus adult antigens expressed as mean seropositivity in relation to control sera, (c) mean eosinophil counts (number of cells × 109 /l). Calves in groups A and B were inoculated with 5 × 100 D. viviparus infective third-stage larvae (L3), whereas calves in group C served as uninfected controls. Group A were treated with eprinomectin (0.5 mg/kg bodyweight) at day 24 post the first inoculation, whereas groups B and C were left untreated. Seven weeks following anthelmintic treatment all groups were challenged with 1500 L3.
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3.3. ELISA results Fig. 1b demonstrates that parasite-specific IgG1 levels were slightly elevated day 24 p.i. both in groups A and B. In group B antibodies continued to increase and maximum levels were observed at day 52 p.i., whereas in group A the levels started to decrease approximately 14 days after anthelmintic treatment. The antibody responses in groups A and B against the challenge infection occurred earlier compared to after the primary infection. In group A elevated levels were observed already 7 days p.c. In group B antibody levels continued to decrease from very high levels also after challenge, however, they were increased again 21 days p.c. The antibody responses were significantly (P < 0.0001) different between experimental groups. 3.4. Number of eosinophils The mean eosinophil counts are shown in Fig. 1c. Both in groups A and B eosinophils increased steadily from the baseline levels between days 10 and 17 p.i. However, the response was significantly (P = 0.0025) higher in group B, where the number of eosinophils reached a maximum of 7.9 × 109 cells/l and were elevated from day 17 p.i. Then they remained elevated until day 59 p.i. when they started to decrease. Also in group A, a maximum of 7.8 × 109 cells/l was observed at day 18 p.i., but in this group the numbers started to decrease already from day 31 p.i. After challenge the eosinophils were increased again and in both groups elevated levels were observed 14 days p.c. As with the antibodies the response of the eosinophils occurred somewhat earlier after challenge as compared with after the primary infection.
Fig. 2. Mean number of established D. viviparus ± S.D. in the lung at slaughter 28 days after challenge with 1500 L3. Group A: primary infected (5 × 100 L3) between days 0–4 and treated with eprinomectin (0.5 mg/kg bodyweight) at day 24, group B: only primary infected, and group C: challenge control. Seven weeks following anthelmintic treatment all groups were challenged with 1500 L3.
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Fig. 3. Mean cumulative weight gains (kg). Calves in groups A and B were inoculated with 5 × 100 D. viviparus infective third-stage larvae (L3), whereas calves in group C served as uninfected controls. Groups A and B: primary infected with 5 × 100, group C: challenge control.
3.5. Lungworm counts Worm counts at necropsy are shown in Fig. 2. There was a significant (P < 0.0001) difference in the number of adult lungworms that were recovered from each of the experimental groups. Post hoc testing showed that the post mortem counts within group A was significantly (P < 0.0001) higher compared to group B that in turn was significantly (P < 0.0001) lower than in group C. 3.6. Weight gains Weight gains are shown in Fig. 3. There was a significant difference in weight performance between calves in the different experimental groups (P = 0.019), but only between the calves in the uninfected challenge control group C that grew significantly better than those in groups A and B. Calves in group C had an average weight gain of 1.16 kg per day, whereas the animals in groups A and B gained 0.96 and 0.94 kg, respectively.
4. Discussion In previous studies protection against D. viviparus in calves has been defined as the proportion of ingested infective larvae that survives to maturity after challenge (Oakley, 1981; Ploeger and Eysker, 2002). In this study we showed that a good level of protection could be stimulated in calves that had been treated with a topical formulation of eprinomectin at early patency. This is in agreement with earlier observations that have shown that D. viviparus infected cattle developed some degree of protection to reinfection when drenched either with levamisole, or fenbendazole around the onset of patency of the parasite (Downey, 1980). We also observed that a relatively low dose level of larvae, 5×100 per animal induced
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protection. This is in accordance with Ploeger and Eysker (2002), who found an inoculation dose of 500 larvae to be sufficient to stimulate the development of a reasonable protection against a challenge infection 35 days after primary infection. In this trial we also demonstrated that the level of protection was significantly improved in animals that were allowed to recover naturally from infection when compared to the levels in the treated infection control group (group B). Thus this result confirms the conclusion that development of protection against D. viviparus in cattle is a gradual process, as it seemed to be beneficial when the immune system was stimulated by all stages of the parasite. It has been demonstrated that a strong protective immunity against D. viviparus can be induced without further development of larvae before the larval stages reach maturity in the lungs (Taylor et al., 2000). On the contrary, it has been established that high levels of protection was induced when the migratory phase of the life cycle was avoided. In a vaccination trial (Bain and Urquhart, 1988) showed that passage of irradiated larvae through the intestine and mesenteric lymph nodes was not necessary for the stimulation of a high degree of immunity. Significant levels of protection have been observed in guinea pigs as an infection model, that were immunised with protective adult ES-antigens that seemed to be involved in some form of antibody mediated mechanism (McKeand et al., 1995). On the other hand, when the protective effect of these antigens was tested in calves, conflicting results were obtained (Matthews et al., 2001). As mentioned earlier, the results of the present experiment indicated that the level of protection against a constant challenge was enhanced in the untreated group and probably as a result of the combined immune responses directed both against the migrating juvenile worms and established adults in the lungs. However, treatment of the animals in group A was applied 24 days after the first trickle infection and immediately when patency first was observed. Consequently, with the design of the experiment it was not possible to completely rule out that the differences in challenge protection between calves in experimental groups A and B was a reflection of the number of adult worms that previously had been established in the lungs and the time the animals had been exposed to these worms. Although it has been demonstrated in several field experiments that varying degrees of protection were induced in first season grazers subjected to different suppressive anthelmintic regimes (Borgsteede et al., 1998; Jacobs et al., 1989; Taylor et al., 1997, 1990), the results from the present experiment clearly indicated that a significantly stronger protection was induced in calves that were allowed to recover naturally from the infection as compared to if they were treated at early patency. It has been documented that topical application of eprinomectin at the 0.5 and 0.2 mg/kg dosage levels remove more than, or equal to 99% of a wide range of species of adult nematode parasites in cattle (Shoop et al., 1996). In this trail we observed that larval excretion dropped to zero in all of the treated animals 1 week after application of the drug. The activity of eprinomectin against D. viviparus at the lower dose level was thus confirmed. The drop in larval excretion was accompanied by a decrease in the antibody levels and in the number of peripheral eosinophils, which indicates that all adult lungworms were removed. The persistent efficacy of eprinomectin at the lower dosage level has been shown to last for a minimum of 28 up to 35 days after treatment (Cramer et al., 2000). In this study we gave the challenge infection 7 weeks after treatment. Thus it cannot be argued that the difference between the primary trickle infected groups was due to residual activity of the drug.
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Notable is that the growth was similar in both of the primary infected groups, but significantly reduced as compared to the uninfected challenge control group. Accordingly the damage induced by the parasite was caused already in the initial phase of the infection before the animals were treated with anthelmintic. It also seemed like the treated animals were unable to compensate for this initial loss in weight gains. Furthermore, all animals in the trickle-infected groups were susceptible to challenge 11 weeks after the primary infection. Consequently, the immunity induced by the primary infection was not able to completely protect the animals against challenge. Those in the eprinomectin group were also about eight times more susceptible to re-infection than the group that recovered from the infection. These observations are interesting both from a practical and epidemiological point of view. Applied in a possible field situation it can be argued that both groups are likely to be involved in the propagation of the infection and it cannot be expected that the performance should be improved in case of the animals are treated at early patency. On the other hand, although the protection is enhanced if the animals are allowed to recover naturally from the infection, these animals will continue to excrete larvae that will contaminate the pastures. The cumulative number of larvae that were excreted by the animals in the untreated group was 43 times higher as compared to the animals in the group that were allowed to recover naturally from the infection. Based on the results of this experiment it can be concluded that topical eprinomectin treatment of lungworm-infected cattle at early patency interfered with the development of protection against reinfection. This is in conflict with the current advice given to Swedish farmers. However, in a field situation animals are exposed to infective larvae also after treatment has been applied. There is evidence that challenge infection in this situation will result in the development of some degree of protection (Borgsteede et al., 1998). Another factor that needs to be taken into consideration is the continued contamination of the pastures with larvae where animals are kept out on pasture, but remain untreated. As larval excretion was eliminated following the application of eprinomectin, this measure will clearly restrain transmission and thereby prevent, or reduce, the risk of re-infection to co-grazing animals. However, one possibility to avoid this scenario would be to stable infected, but clinically healthy animals for a period of some weeks in order to allow them to build up immunity. This will obviously induce a significantly stronger protection against re-infection compared to animals that are treated. Acknowledgements We are grateful to Miss M. Moberg and MSc A. Engström for technical assistance. Dr. A. Vermeulen, Intervet, is thanked for originally providing us with larvae. Thanks also to the animal attendants at Department of Ruminant Medicine for taking care of the animals. Formas (22.9/2001-1991) provided funding support to JH for this trial that was approved by the Swedish Animal Ethics Committee (permission C4/2). References Andrews, S.J., James, F.M., 1994. Further evaluation of a perfusion technique for the recovery of Dictyocaulus viviparus from bovine lungs. J. Helminthol. 68, 81–82.
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