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Antibodies to major pasture borne helminth infections in bulk-tank milk samples from organic and nearby conventional dairy herds in south-central Sweden
Veterinary Parasitology 171 (2010) 293–299
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Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Antibodies to major pasture borne helminth infections in bulk-tank milk samples from organic and nearby conventional dairy herds in south-central Sweden Johan Höglund a,∗ , Frida Dahlström b , Annie Engström a , Anna Hessle b , Eva-Britt Jakubek a , Thomas Schnieder c , Christina Strube c , Sofia Sollenberg a a
Department of Biomedicine and Veterinary Public Health, Section for Parasitology (SWEPAR), Swedish University of Agricultural Sciences (SLU), Ulls väg 2B 7036, SE 750 07 Uppsala, Sweden b Department of Animal Environment and Health, Swedish University of Agricultural Sciences (SLU), Sweden c Institute for Parasitology, University of Veterinary Medicine Hannover, Germany
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Article history: Received 5 February 2010 Received in revised form 23 March 2010 Accepted 1 April 2010 Keywords: Dairy cattle Parasite control Grazing management Organic farming ELISA Herd health Ostertagia Dictyocaulus Fasciola
a b s t r a c t The objective of this randomised pairwise survey was to compare the regional distribution of antibody levels against the three most important helminth infections in organic and conventional dairy herds in Sweden. Bulk-tank milk from 105 organic farms and 105 neighbouring conventional dairy farms with access to pasture in south-central Sweden were collected in September 2008. Samples were also collected from 8 organic and 8 conventional herds located in a much more restricted area, on the same as well as 3 additional occasions during the grazing season, to reveal evidence for seasonal patterns against cattle stomach worm (Ostertagia ostertagi). Antibody levels to the stomach worm (O. ostertagi), liver fluke (Fasciola hepatica) and lungworm (Dictyocaulus viviparus) were then determined by detection of specific antibodies using three different enzyme-linked immunosorbent assays (ELISAs). According to the Svanovir® Ostertagia ELISA, the mean optical density ratio (ODR) was significantly higher in the milk from organic compared to conventional herds, i.e. 0.82 (95% CL = 0.78–0.86) versus 0.66 (0.61–0.71). However, no significant differences were observed in the samples collected at different time points from the same 16 herds (F3,39 = 1.18, P = 0.32). Antibodies to D. viviparus infection were diagnosed with an ELISA based on recombinant major sperm protein (MSP), and seropositivity was found in 21 (18%) of the 113 organic herds and 11 (9%) of the 113 conventional herds. The seroprevalence of D. viviparus was somewhat higher in the organic herds (Chi-square = 3.65, P = 0.056), but with the positive conventional herds were located in the vicinity of infected organic herds. Of the 16 herds that were sampled on repeated occasions, as many as 10 (63%), were seropositive on at least one sampling occasion. Many of these turned positive towards the end of the grazing season. Only one herd was positive in all 4 samples and 3 were positive only at turnout. Considering F. hepatica there was no difference in seroprevalence between organic and conventional herds according to the Institute Pourquier® ELISA. In general, liver fluke infection was low and it was only diagnosed in 8 (7%) organic and 7 (6%) conventional herds. © 2010 Elsevier B.V. All rights reserved.
1. Introduction ∗ Corresponding author. Tel.: +46 18 67 14 56; fax: +46 18 67 43 04. E-mail address: [email protected] (J. Höglund). 0304-4017/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2010.04.002
Cattle are economically the most important livestock in Sweden where milk and beef originate from approx-
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imately 1.6 million cattle, including 355,000 dairy cows, which accounts for 64% of the economic value in the domestic animal production (Anon., 2009). Most dairy cattle (93%) in Sweden are raised in conventional herds (Anon., 2009). At the same time there is a political goal to increase the Swedish organic production of agricultural commodities to 20%. As a result the proportion of Swedish livestock production run organically is steadily increasing. A major difference between organic and conventional cattle production is that the prophylactic use of anthelmintics is prohibited in organic herds (Anon., 2008), although anthelmintics are not always used even in conventionally reared cattle. There are also regulations about prolonged minimum lengths of the grazing periods in organic production (Anon., 2008). However, irrespective of whether Swedish dairy producers are organic or conventional, according to the national animal welfare legislation act cattle older than 6 months, bulls excluded, must have outdoor access during grazing season for at least 2–4 months depending on region (DFS, 2007:5). Thus all dairy cattle in Sweden are exposed for at least several months to a wide range of helminth parasites with larval stages that are picked up with the grass grazed on pasture. Among many different species of pasture borne nematode parasites that can infect cattle, there are only a few that cause major problems, notably the stomach worm Ostertagia ostertagi and lungworm Dictyocaulus viviparus. Both of these parasites are widely distributed in Sweden, particularly in first season grazing (FSG) animals, where they may act as the major causes of impaired or reduced productivity of both beef and milk even where infection levels are sub-clinical. For example, the weight-gain penalties in unprotected set stocked FSG animals were on an average in the range of 20–65 kg from turn-out to housing, compared to simultaneously grazed calves that were fully protected from parasites by the use of effective anthelmintics (Dimander et al., 2000, 2003; Larsson et al., 2007). Although, it is particularly the FSG that are at risk of developing disease and sub-clinical production losses, pasture borne nematodes are also important infections potentially limiting production in adult dairy cattle (for reviews see Ploeger, 2002; Charlier et al., 2009b). The liver fluke Fasciola hepatica is another important helminth parasite, but baseline data on its distribution and importance in dairy in Sweden is currently missing. In contrast to the nematodes, this trematode parasite is indirectly transmitted, mainly through the snail Galba (Lymnaea) truncatula with a preference for wet areas (Torgerson and Claxton, 1999). It is often anticipated that organic livestock production leads to an increased risk of being exposed to pasture borne parasite infection, but comparative information on the level of parasitism in organic versus conventional dairy herds is limited (Kijlstra and Eijck, 2006). According to an earlier Swedish study, the levels of gastrointestinal nematode infections in FSG animals were not dramatically increased in 15 organic herds monitored over a 2-year period (Höglund et al., 2001). This was so despite a restricted use of anthelmintic drugs in combination with increased access to outdoor areas, including a prolonged
period of grazing. The loss of an effect could partly be explained by the fact that alternative preventive antiparasitic measures were adopted more frequently by organic than conventional dairy farmers (Svensson et al., 2000). However, it is nearly a decade since these studies were conducted, and some of the farms that participated had been managed in an organic fashion only for a few years when the studies were started. There is still limited scientific evidence as to whether the movement towards increased organic production in Sweden has had any significant effects on the severity of parasite infection in grazing livestock. Especially for adult dairy cattle, information on the level of parasitism and its constraints on organic farming is restricted. The purpose of the present study was to compare the antibody levels towards major pasture borne helminth parasites in organic and nearby conventional dairy herds, and thereby to examine if the level of parasitism has been changed in response to this managerial shift. To this end we have for the first time in Sweden tested a novel surveillance technique based on the examination of antibody levels to all major cattle helminths in bulk-tank milk samples (Bennema et al., 2009). 2. Material and methods 2.1. Milk samples A research system was established consisting of farmpairs, where each pair included one organic farm matched with one conventional. Overall a total of 210 bulk-tank milk samples were collected in September 2008 by a technician at the Eurofins laboratory in Jönköping, Sweden, who randomly selected milk from each of 105 organic farms, representing 22% of all organic herds in Sweden (n = 470), and from 105 matching nearby conventional herds. All samples, except for 16 herds monitored more closely, were taken from herds situated in a restricted district that delivered their milk to Arla-Foods (Fig. 1). This is a dairy intensive area situated in south-central Sweden covering about 65% of the total milk production in Sweden. Furthermore, 8 organic and 8 matching nearby conventional herds were selected, located in an even more restricted area between the large lakes shown in Fig. 1. On these farms, additional milk samples were also taken throughout the grazing season to study the time course of antibody responses to O. ostertagi. These samples were first taken at the time for turn-out and then again 6, 15 and 20 weeks post-turn-out. All milk samples were collected in special tubes pre-treated with bronopol as a preservative. Upon arrival at our laboratory, fat was removed from the milk by centrifugation (16,000 × g for 5 min) and samples were stored at −20 ◦ C before being analysed. 2.2. Antibodies in milk Three different enzyme-linked immunosorbent assays (ELISAs) were used to determine the antibody levels in the collected bulk-tank milk samples. Two tests are commercially available and were used and interpreted according to the manufacturers’ instructions. First, specific antibod-
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Fig. 1. Map of Sweden showing the geographical distribution of all 226 bulk-tank milk samples that were analysed for antibodies against the three most important pasture borne helminth parasite infections of cattle in Sweden. The 16 farms that were sampled on up to four occasions were all located between the big lakes. Pyramids denote organic and circles conventional farms.
ies against the stomach worm O. ostertagi were detected using the SVANOVIR® Ostertagia-Ab ELISA kit (SVANOVA Biotech, Uppsala, Sweden) using a crude adult worm capture antigen. This test was recently standardised and validated for milk samples in 3 European laboratories, the SWEPAR laboratory included (Charlier et al., 2009a). The optical density (OD) of the sample were expressed as a ratio (ODR) calculated according to the formula ODR = (sample − OD − NC)/(PC − NC), where NC and PC are the ODs of a negative and positive test control samples included on each plate. ODR values above 0.8 units are associated with a high exposure to the parasite and with decreased milk production (Charlier et al., 2007b; Forbes et al., 2008).
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Secondly, antibodies to the liver fluke F. hepatica was detected with the Institut Pourquier fasciolosis kit (Idexx Laboratories, Montpellier, France) based on a purified f2 antigen prepared from crude parasite extracts as capture antigen. This liver fluke ELISA was validated under Australian conditions using >1500 samples from artificial and natural infections from both endemic and non-endemic areas, and has both high specificity (95–98% depending on samples) and sensitivity (99%; Hutchinson and Macarthur, 2003). The F. hepatica results were expressed as a ratio between the sample OD and a PC included on each plate. Cut-off values above 30% give adequate sensitivity, whereas ODs between 100% and 200% show a moderate and >200% strong F. hepatica infection levels. Lungworm infection was analysed with an indirect in-house antibody-ELISA developed by the Institute for Parasitology of the University of Veterinary Medicine in Hannover. It is based on a recombinant major sperm fusion protein used as capture antigen. The detailed procedure for serum is described by von Holtum et al. (2008). Validation for use with milk was presented by Fiedor (2009), who showed that specific IgG1 can be detected by 30(±5) days post-infection and for up to 2–6 months post-infection in experimentally infected cows. 2.3. Data analysis Raw data were collated in Excel spreadsheets, and later imported and analysed and/or graphically displayed in JMPTM version 6.00 (SAS Institute Inc., Cary, NC, USA) or GraphPad Prism® version 4.0c (San Diego, CA, USA) for MacIntosh. Herd mean antibody levels were compared with a two-tailed Mann–Whitney’s U-test, whereas herd prevalences with a likelihood-ratio Chi-square test, as well as odds ratio calculations. Differences in the time course of the Ostertagia ODR were tested in the fit-model platform of JMP using a random-effects split-plot repeated measures analysis of variance (ANOVA) design with 1 between-
Fig. 2. The geographical location of bulk-tank milk samples from farms that had antibodies with an optical density ratio against Ostertagia ostertagi of ≥0.8.
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Fig. 3. Box-plots of the ODR values against Ostertagia ostertagi as determined with the SVANOVIR® ELISA in bulk-tank milk from conventional and organic dairy herds in Sweden. Groups means are significantly different (P < 0.0001) according to a two-tailed Mann–Whitney’s U-test.
subject factor (management: i.e. organic or conventional) and 2 within-subject factors (herd identity and sampling date). We set significance levels of P less than 0.05 for all statistical comparisons. Information on the geographical location of the farms was retrieved from a database, and their geographical positions were plotted using ArcGIS version 9.9.1 (ESRI® Inc., Redlands, CA, USA). 3. Results
Fig. 5. The geographical location of bulk-tank milk samples from farms that were seropositive for Dictyocaulus viviparus.
P = 0.0027), but not different among samples collected at different time points during the grazing season (F3,39 = 1.18, P = 0.32), which indicates a non-seasonal pattern. Furthermore, there was no significant interaction for management by sample (F3,39 = 0.89, P = 0.49), but there was for herd identity nested by management (F15,39 = 2.34, P = 0.017) with the largest ODRs observed in organic herds.
3.1. O. ostertagi 3.2. D. viviparus The antibody levels based on the results from the SVANOVIR® ELISA are shown in Fig. 2, and also displayed as box-plots in Fig. 3. It was shown that the average ODR in milk from the organic herds was 0.82 (95% CL = 0.78–0.86), which is significantly (P < 0.001) higher than the mean value observed in the milk from the conventional herds, with a mean ODR of 0.66 (0.61–0.71). Of the 210 samples 88 (42%) had an ODR ≥0.8, of which 55 (52%) and 33 (32%) of the samples were from organic and conventional milk, respectively. The same pattern was observed for the ODRs in the subset of milk samples collected from the same herds but at different occasions during the grazing season (Fig. 4). According to the split-plot design ANOVA, ODRs in organic milk were lower than in conventional herds (F1,15 = 12.42,
The results of the lungworm ELISA are shown in Fig. 5. Elevated antibodies were found in 21 (18%) of the 113 organic herds and 11 (9%) of the 113 conventional herds. They tended to be higher in the organic herds (Chisquare = 3.65, P = 0.055), and the few positive conventional herds were located in the same geographical area as an infected organic herd. The corresponding odds ratio for an organic herd to be infected compared to a conventional herd was 2.1 (±95% CL: 0.96–4.57). Of the 16 herds that were sampled on up to 4 occasions during the grazing season, 10 (63%) were seropositive on at least one sampling occasion. Only in one herd were all 4 samples positive, whereas the majority of the infected herds turned positive towards the end of the grazing season. 3.3. F. hepatica The results from the liver fluke ELISA are shown in Fig. 6. Overall, 7.1% (15 of 210) milk samples analysed were found to be positive. Of these positive samples, 8 (7.6%) were from organic herds whereas 7 (6.7%) came from conventional herds. Accordingly, there was not a significant difference between the numbers of positive samples. A low to moderate seroprevalence was observed in 7 of the herds, whereas 4 samples from each type of management showed strong infections. 4. Discussion
Fig. 4. Time course of antibody levels to Ostertagia ostertagi in bulk-tank milk samples from organic and conventional herds as determined with the SVANOVIR® ELISA.
It is natural that intensive rearing of livestock kept on pasture is associated with an increased risk of exposure
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Fig. 6. The geographical location of bulk-tank milk samples from farms that were seropositive for the liver fluke Fasciola hepatica.
to a range of pasture borne pathogens. Concerning parasites of grazing livestock, it has long been anticipated that the situation will be particularly severe for animals raised on organic farms, because the prophylactic use of chemotherapy (anthelmintics) is prohibited and the animals are usually kept on pasture for prolonged periods of time (Waller, 2006). Despite the added regulations in organic production, the differences in rearing conditions between conventional and organic dairy cattle in Sweden are relatively minor. This includes the use of anthelmintics, which is also set to a minimum especially in adult stock also in conventional dairy herds in Sweden. In spite of this, significantly elevated antibody levels to O. ostertagi and a tendency to increased risk for D. viviparus infections were demonstrated in the organic compared to the conventional herds investigated. In this study the average ODR value was 0.75 from bulk-tank milk samples collected in one of the most dairy intensive regions in southern Sweden. This is higher compared with the ODR values in some other northern European countries including Denmark, investigated in the autumn of 2005 and 2006 (Forbes et al., 2008). However, in the study Forbes et al. (2008) permanently housed herds were also included. All herds in our study had access to pasture and ODR values above 0.8, indicating the presence of economically important O. ostertagi infection levels, were found in 39% (n = 88) of the 226 milk samples collected. Of these samples, 24% (n = 55) were from organic herds, whereas 15% (n = 33) came from conventional herds. Although the proportion of samples with a high ODR was significantly lower in conventional herds, the present results somewhat contrast with earlier observations collected in a more extensive European survey that involved nearly 529 conventional Swedish herds examined in 2006 (Bennema et al., submitted for publication). When we compare the data from the conventional Swedish dairy herds in these previous surveys, it is evident that there are not only major regional differences but apparently also fluctuations between years. Irrespective of the explanation for the variations observed, exposure to pasture borne para-
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sites reflects a dynamic situation that is influenced both by climatic and management factors (Bennema et al., 2009). The present study also shows that the longitudinal data on ODRs to O. ostertagi in bulk-tank milk from the 16 different herds sampled on several occasions was not significantly different during the grazing season according to the split-plot design ANOVA. This is in line with previous results from Canada (Sanchez et al., 2002), but contrasts the situation on two dairy farms in Normandy in France, where a clear seasonal pattern was observed with a gradual increase in the ODR during the grazing season and the highest values observed from June until October (Charlier et al., 2007a). However, a major difference between the present and previous seasonal bulk-tank milk surveys is that in the current study we only analysed samples collected during the grazing season, whereas the French and Canadian herds were sampled all year round. Nevertheless, the seasonal ODR patterns seem to differ between the geographical areas, which probably reflects differences in the epidemiology of O. ostertagi in slightly different temperate climates. For example, a large number of overwintered infective larvae is the key factor in the epidemiology of bovine gastrointestinal nematode infections in the Scandinavian countries including both Norway (Helle and Tharaldsen, 1976) and Sweden (Dimander et al., 2003), whereas high pasture infectivity seems to occur from midseason and onwards in most European countries (Charlier et al., 2007a). The lack of a seasonal pattern for ODR in Sweden, however, is of great practical importance, because in this situation it means that milk analysed soon after turn-out can be used to predict whether antiparasitic measures are necessary later in the same grazing season, which is beneficial for practical prognostic control. The seroprevalence of F. hepatica was only 7% in the present study, which is a much lower level than for O. ostertagi and D. viviparus. In the case of the liver fluke, surveys based on bulk-tank milk samples are available from the UK (Salimi-Bejestani et al., 2005) and Belgium (Bennema et al., 2009) with herd prevalences ranging from 37% to 86%. Liver fluke infection is thus a much more limited problem in Swedish dairy herds. This was so irrespective of whether the farms were managed according to the rules of organic or conventional production. The few positive herds were mainly located in damp areas along the coastline or were from farms located near the two big lakes in southcentral Sweden (Fig. 6). Although the seroprevalence was somewhat lower than expected, the locations of the positive herds were as expected, since livers condemned by F. hepatica are more commonly reported at slaughter when the abattoirs receive cattle raised in the same places. The low seroprevalence of F. hepatica can be a consequence of a non-optimal sampling time for this parasite. Nevertheless, dairy herds in Sweden seem to have a relatively low prevalence of F. hepatica in comparison with other European countries, and its effects on production is therefore probably very restricted. Previously published information on the seroepidemiology in bulk-tank milk to Dictyocaulus is restricted to a single study from Flanders in Belgium, in which the herd prevalence was around 20% (Bennema et al., 2009). This agrees with the levels observed in the organic herds tested
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here but is approximately two times higher than in the conventional herds. Although the prevalence of lungworms in general is lower in younger stock, this result contrasts with previous findings on this parasite in Sweden, based on comparisons of serum samples from first season grazing dairy calves collected from organic and conventional farms both with and without anthelmintic treatment, and where no major differences in seroprevalence in relation to management could be detected (Höglund et al., 2004). There are typical limitations of any antibody-ELISA tests, such as the inability to detect newly acquired infections and to distinguish between previous and ongoing infections, as well as the existence of potential cross-reactions. It has been reported that both the D. viviparus (von Holtum et al., 2008) and F. hepatica tests (Hutchinson and Macarthur, 2003) have high sensitivity and specificity. In contrast, the O. ostertagi ELISA is based on a crude antigen made from adult worm homogenates. Thus it cross-reacts to a range of internal parasites, in particular the closely related intestinal nematode Cooperia oncophora, but also with F. hepatica (Bennema et al., 2009). This does not necessarily constitute a problem if the objective of the survey is to measure the overall exposure to pasture borne parasite infections rather than to diagnose different species of parasites. However, as pointed out by Bennema et al. (2009), it is more of a problem when the purpose is to identify hot spots of a particular parasite infection and locations where regional parasite control programmes should be applied. At the same time, it needs to be realised that classical parasitological faecal examination is certainly not a feasible and realistic alternative, as with these techniques we will be limited to the detection of patent infection or ongoing egg/larval production. 5. Conclusion This randomised pairwise study demonstrates that ODR values for the parasitic nematode O. ostertagi were significantly higher in bulk-tank milk collected in organic compared to conventional dairy herds. It also shows that the ODR in response to this parasite was stable throughout the grazing season. We also detected a tendency to higher seroprevalence of the bovine lungworm D. viviparus in samples that originated from organic herds. On the other hand, there was no difference between organic and conventional herds in the seroprevalence of the liver fluke F. hepatica. To sum up, this study further confirms that various milk ELISAs can be explored to monitor important helminth infections in dairy cows, and that this is a non-invasive and inexpensive method that easily can be used map to parasite hot spots. Acknowledgements Funding was provided by FORMAS (220-2007-1616) and the 6th framework program of the EU (Parasol, Project FOOD-CT-2005-022851). Jacob Lagerstedt and Tobias Rydlinge are thanked for their support with the GIS analyses.
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Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Antibodies to major pasture borne helminth infections in bulk-tank milk samples from organic and nearby conventional dairy herds in south-central Sweden Johan Höglund a,∗ , Frida Dahlström b , Annie Engström a , Anna Hessle b , Eva-Britt Jakubek a , Thomas Schnieder c , Christina Strube c , Sofia Sollenberg a a
Department of Biomedicine and Veterinary Public Health, Section for Parasitology (SWEPAR), Swedish University of Agricultural Sciences (SLU), Ulls väg 2B 7036, SE 750 07 Uppsala, Sweden b Department of Animal Environment and Health, Swedish University of Agricultural Sciences (SLU), Sweden c Institute for Parasitology, University of Veterinary Medicine Hannover, Germany
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Article history: Received 5 February 2010 Received in revised form 23 March 2010 Accepted 1 April 2010 Keywords: Dairy cattle Parasite control Grazing management Organic farming ELISA Herd health Ostertagia Dictyocaulus Fasciola
a b s t r a c t The objective of this randomised pairwise survey was to compare the regional distribution of antibody levels against the three most important helminth infections in organic and conventional dairy herds in Sweden. Bulk-tank milk from 105 organic farms and 105 neighbouring conventional dairy farms with access to pasture in south-central Sweden were collected in September 2008. Samples were also collected from 8 organic and 8 conventional herds located in a much more restricted area, on the same as well as 3 additional occasions during the grazing season, to reveal evidence for seasonal patterns against cattle stomach worm (Ostertagia ostertagi). Antibody levels to the stomach worm (O. ostertagi), liver fluke (Fasciola hepatica) and lungworm (Dictyocaulus viviparus) were then determined by detection of specific antibodies using three different enzyme-linked immunosorbent assays (ELISAs). According to the Svanovir® Ostertagia ELISA, the mean optical density ratio (ODR) was significantly higher in the milk from organic compared to conventional herds, i.e. 0.82 (95% CL = 0.78–0.86) versus 0.66 (0.61–0.71). However, no significant differences were observed in the samples collected at different time points from the same 16 herds (F3,39 = 1.18, P = 0.32). Antibodies to D. viviparus infection were diagnosed with an ELISA based on recombinant major sperm protein (MSP), and seropositivity was found in 21 (18%) of the 113 organic herds and 11 (9%) of the 113 conventional herds. The seroprevalence of D. viviparus was somewhat higher in the organic herds (Chi-square = 3.65, P = 0.056), but with the positive conventional herds were located in the vicinity of infected organic herds. Of the 16 herds that were sampled on repeated occasions, as many as 10 (63%), were seropositive on at least one sampling occasion. Many of these turned positive towards the end of the grazing season. Only one herd was positive in all 4 samples and 3 were positive only at turnout. Considering F. hepatica there was no difference in seroprevalence between organic and conventional herds according to the Institute Pourquier® ELISA. In general, liver fluke infection was low and it was only diagnosed in 8 (7%) organic and 7 (6%) conventional herds. © 2010 Elsevier B.V. All rights reserved.
1. Introduction ∗ Corresponding author. Tel.: +46 18 67 14 56; fax: +46 18 67 43 04. E-mail address: [email protected] (J. Höglund). 0304-4017/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2010.04.002
Cattle are economically the most important livestock in Sweden where milk and beef originate from approx-
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imately 1.6 million cattle, including 355,000 dairy cows, which accounts for 64% of the economic value in the domestic animal production (Anon., 2009). Most dairy cattle (93%) in Sweden are raised in conventional herds (Anon., 2009). At the same time there is a political goal to increase the Swedish organic production of agricultural commodities to 20%. As a result the proportion of Swedish livestock production run organically is steadily increasing. A major difference between organic and conventional cattle production is that the prophylactic use of anthelmintics is prohibited in organic herds (Anon., 2008), although anthelmintics are not always used even in conventionally reared cattle. There are also regulations about prolonged minimum lengths of the grazing periods in organic production (Anon., 2008). However, irrespective of whether Swedish dairy producers are organic or conventional, according to the national animal welfare legislation act cattle older than 6 months, bulls excluded, must have outdoor access during grazing season for at least 2–4 months depending on region (DFS, 2007:5). Thus all dairy cattle in Sweden are exposed for at least several months to a wide range of helminth parasites with larval stages that are picked up with the grass grazed on pasture. Among many different species of pasture borne nematode parasites that can infect cattle, there are only a few that cause major problems, notably the stomach worm Ostertagia ostertagi and lungworm Dictyocaulus viviparus. Both of these parasites are widely distributed in Sweden, particularly in first season grazing (FSG) animals, where they may act as the major causes of impaired or reduced productivity of both beef and milk even where infection levels are sub-clinical. For example, the weight-gain penalties in unprotected set stocked FSG animals were on an average in the range of 20–65 kg from turn-out to housing, compared to simultaneously grazed calves that were fully protected from parasites by the use of effective anthelmintics (Dimander et al., 2000, 2003; Larsson et al., 2007). Although, it is particularly the FSG that are at risk of developing disease and sub-clinical production losses, pasture borne nematodes are also important infections potentially limiting production in adult dairy cattle (for reviews see Ploeger, 2002; Charlier et al., 2009b). The liver fluke Fasciola hepatica is another important helminth parasite, but baseline data on its distribution and importance in dairy in Sweden is currently missing. In contrast to the nematodes, this trematode parasite is indirectly transmitted, mainly through the snail Galba (Lymnaea) truncatula with a preference for wet areas (Torgerson and Claxton, 1999). It is often anticipated that organic livestock production leads to an increased risk of being exposed to pasture borne parasite infection, but comparative information on the level of parasitism in organic versus conventional dairy herds is limited (Kijlstra and Eijck, 2006). According to an earlier Swedish study, the levels of gastrointestinal nematode infections in FSG animals were not dramatically increased in 15 organic herds monitored over a 2-year period (Höglund et al., 2001). This was so despite a restricted use of anthelmintic drugs in combination with increased access to outdoor areas, including a prolonged
period of grazing. The loss of an effect could partly be explained by the fact that alternative preventive antiparasitic measures were adopted more frequently by organic than conventional dairy farmers (Svensson et al., 2000). However, it is nearly a decade since these studies were conducted, and some of the farms that participated had been managed in an organic fashion only for a few years when the studies were started. There is still limited scientific evidence as to whether the movement towards increased organic production in Sweden has had any significant effects on the severity of parasite infection in grazing livestock. Especially for adult dairy cattle, information on the level of parasitism and its constraints on organic farming is restricted. The purpose of the present study was to compare the antibody levels towards major pasture borne helminth parasites in organic and nearby conventional dairy herds, and thereby to examine if the level of parasitism has been changed in response to this managerial shift. To this end we have for the first time in Sweden tested a novel surveillance technique based on the examination of antibody levels to all major cattle helminths in bulk-tank milk samples (Bennema et al., 2009). 2. Material and methods 2.1. Milk samples A research system was established consisting of farmpairs, where each pair included one organic farm matched with one conventional. Overall a total of 210 bulk-tank milk samples were collected in September 2008 by a technician at the Eurofins laboratory in Jönköping, Sweden, who randomly selected milk from each of 105 organic farms, representing 22% of all organic herds in Sweden (n = 470), and from 105 matching nearby conventional herds. All samples, except for 16 herds monitored more closely, were taken from herds situated in a restricted district that delivered their milk to Arla-Foods (Fig. 1). This is a dairy intensive area situated in south-central Sweden covering about 65% of the total milk production in Sweden. Furthermore, 8 organic and 8 matching nearby conventional herds were selected, located in an even more restricted area between the large lakes shown in Fig. 1. On these farms, additional milk samples were also taken throughout the grazing season to study the time course of antibody responses to O. ostertagi. These samples were first taken at the time for turn-out and then again 6, 15 and 20 weeks post-turn-out. All milk samples were collected in special tubes pre-treated with bronopol as a preservative. Upon arrival at our laboratory, fat was removed from the milk by centrifugation (16,000 × g for 5 min) and samples were stored at −20 ◦ C before being analysed. 2.2. Antibodies in milk Three different enzyme-linked immunosorbent assays (ELISAs) were used to determine the antibody levels in the collected bulk-tank milk samples. Two tests are commercially available and were used and interpreted according to the manufacturers’ instructions. First, specific antibod-
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Fig. 1. Map of Sweden showing the geographical distribution of all 226 bulk-tank milk samples that were analysed for antibodies against the three most important pasture borne helminth parasite infections of cattle in Sweden. The 16 farms that were sampled on up to four occasions were all located between the big lakes. Pyramids denote organic and circles conventional farms.
ies against the stomach worm O. ostertagi were detected using the SVANOVIR® Ostertagia-Ab ELISA kit (SVANOVA Biotech, Uppsala, Sweden) using a crude adult worm capture antigen. This test was recently standardised and validated for milk samples in 3 European laboratories, the SWEPAR laboratory included (Charlier et al., 2009a). The optical density (OD) of the sample were expressed as a ratio (ODR) calculated according to the formula ODR = (sample − OD − NC)/(PC − NC), where NC and PC are the ODs of a negative and positive test control samples included on each plate. ODR values above 0.8 units are associated with a high exposure to the parasite and with decreased milk production (Charlier et al., 2007b; Forbes et al., 2008).
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Secondly, antibodies to the liver fluke F. hepatica was detected with the Institut Pourquier fasciolosis kit (Idexx Laboratories, Montpellier, France) based on a purified f2 antigen prepared from crude parasite extracts as capture antigen. This liver fluke ELISA was validated under Australian conditions using >1500 samples from artificial and natural infections from both endemic and non-endemic areas, and has both high specificity (95–98% depending on samples) and sensitivity (99%; Hutchinson and Macarthur, 2003). The F. hepatica results were expressed as a ratio between the sample OD and a PC included on each plate. Cut-off values above 30% give adequate sensitivity, whereas ODs between 100% and 200% show a moderate and >200% strong F. hepatica infection levels. Lungworm infection was analysed with an indirect in-house antibody-ELISA developed by the Institute for Parasitology of the University of Veterinary Medicine in Hannover. It is based on a recombinant major sperm fusion protein used as capture antigen. The detailed procedure for serum is described by von Holtum et al. (2008). Validation for use with milk was presented by Fiedor (2009), who showed that specific IgG1 can be detected by 30(±5) days post-infection and for up to 2–6 months post-infection in experimentally infected cows. 2.3. Data analysis Raw data were collated in Excel spreadsheets, and later imported and analysed and/or graphically displayed in JMPTM version 6.00 (SAS Institute Inc., Cary, NC, USA) or GraphPad Prism® version 4.0c (San Diego, CA, USA) for MacIntosh. Herd mean antibody levels were compared with a two-tailed Mann–Whitney’s U-test, whereas herd prevalences with a likelihood-ratio Chi-square test, as well as odds ratio calculations. Differences in the time course of the Ostertagia ODR were tested in the fit-model platform of JMP using a random-effects split-plot repeated measures analysis of variance (ANOVA) design with 1 between-
Fig. 2. The geographical location of bulk-tank milk samples from farms that had antibodies with an optical density ratio against Ostertagia ostertagi of ≥0.8.
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Fig. 3. Box-plots of the ODR values against Ostertagia ostertagi as determined with the SVANOVIR® ELISA in bulk-tank milk from conventional and organic dairy herds in Sweden. Groups means are significantly different (P < 0.0001) according to a two-tailed Mann–Whitney’s U-test.
subject factor (management: i.e. organic or conventional) and 2 within-subject factors (herd identity and sampling date). We set significance levels of P less than 0.05 for all statistical comparisons. Information on the geographical location of the farms was retrieved from a database, and their geographical positions were plotted using ArcGIS version 9.9.1 (ESRI® Inc., Redlands, CA, USA). 3. Results
Fig. 5. The geographical location of bulk-tank milk samples from farms that were seropositive for Dictyocaulus viviparus.
P = 0.0027), but not different among samples collected at different time points during the grazing season (F3,39 = 1.18, P = 0.32), which indicates a non-seasonal pattern. Furthermore, there was no significant interaction for management by sample (F3,39 = 0.89, P = 0.49), but there was for herd identity nested by management (F15,39 = 2.34, P = 0.017) with the largest ODRs observed in organic herds.
3.1. O. ostertagi 3.2. D. viviparus The antibody levels based on the results from the SVANOVIR® ELISA are shown in Fig. 2, and also displayed as box-plots in Fig. 3. It was shown that the average ODR in milk from the organic herds was 0.82 (95% CL = 0.78–0.86), which is significantly (P < 0.001) higher than the mean value observed in the milk from the conventional herds, with a mean ODR of 0.66 (0.61–0.71). Of the 210 samples 88 (42%) had an ODR ≥0.8, of which 55 (52%) and 33 (32%) of the samples were from organic and conventional milk, respectively. The same pattern was observed for the ODRs in the subset of milk samples collected from the same herds but at different occasions during the grazing season (Fig. 4). According to the split-plot design ANOVA, ODRs in organic milk were lower than in conventional herds (F1,15 = 12.42,
The results of the lungworm ELISA are shown in Fig. 5. Elevated antibodies were found in 21 (18%) of the 113 organic herds and 11 (9%) of the 113 conventional herds. They tended to be higher in the organic herds (Chisquare = 3.65, P = 0.055), and the few positive conventional herds were located in the same geographical area as an infected organic herd. The corresponding odds ratio for an organic herd to be infected compared to a conventional herd was 2.1 (±95% CL: 0.96–4.57). Of the 16 herds that were sampled on up to 4 occasions during the grazing season, 10 (63%) were seropositive on at least one sampling occasion. Only in one herd were all 4 samples positive, whereas the majority of the infected herds turned positive towards the end of the grazing season. 3.3. F. hepatica The results from the liver fluke ELISA are shown in Fig. 6. Overall, 7.1% (15 of 210) milk samples analysed were found to be positive. Of these positive samples, 8 (7.6%) were from organic herds whereas 7 (6.7%) came from conventional herds. Accordingly, there was not a significant difference between the numbers of positive samples. A low to moderate seroprevalence was observed in 7 of the herds, whereas 4 samples from each type of management showed strong infections. 4. Discussion
Fig. 4. Time course of antibody levels to Ostertagia ostertagi in bulk-tank milk samples from organic and conventional herds as determined with the SVANOVIR® ELISA.
It is natural that intensive rearing of livestock kept on pasture is associated with an increased risk of exposure
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Fig. 6. The geographical location of bulk-tank milk samples from farms that were seropositive for the liver fluke Fasciola hepatica.
to a range of pasture borne pathogens. Concerning parasites of grazing livestock, it has long been anticipated that the situation will be particularly severe for animals raised on organic farms, because the prophylactic use of chemotherapy (anthelmintics) is prohibited and the animals are usually kept on pasture for prolonged periods of time (Waller, 2006). Despite the added regulations in organic production, the differences in rearing conditions between conventional and organic dairy cattle in Sweden are relatively minor. This includes the use of anthelmintics, which is also set to a minimum especially in adult stock also in conventional dairy herds in Sweden. In spite of this, significantly elevated antibody levels to O. ostertagi and a tendency to increased risk for D. viviparus infections were demonstrated in the organic compared to the conventional herds investigated. In this study the average ODR value was 0.75 from bulk-tank milk samples collected in one of the most dairy intensive regions in southern Sweden. This is higher compared with the ODR values in some other northern European countries including Denmark, investigated in the autumn of 2005 and 2006 (Forbes et al., 2008). However, in the study Forbes et al. (2008) permanently housed herds were also included. All herds in our study had access to pasture and ODR values above 0.8, indicating the presence of economically important O. ostertagi infection levels, were found in 39% (n = 88) of the 226 milk samples collected. Of these samples, 24% (n = 55) were from organic herds, whereas 15% (n = 33) came from conventional herds. Although the proportion of samples with a high ODR was significantly lower in conventional herds, the present results somewhat contrast with earlier observations collected in a more extensive European survey that involved nearly 529 conventional Swedish herds examined in 2006 (Bennema et al., submitted for publication). When we compare the data from the conventional Swedish dairy herds in these previous surveys, it is evident that there are not only major regional differences but apparently also fluctuations between years. Irrespective of the explanation for the variations observed, exposure to pasture borne para-
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sites reflects a dynamic situation that is influenced both by climatic and management factors (Bennema et al., 2009). The present study also shows that the longitudinal data on ODRs to O. ostertagi in bulk-tank milk from the 16 different herds sampled on several occasions was not significantly different during the grazing season according to the split-plot design ANOVA. This is in line with previous results from Canada (Sanchez et al., 2002), but contrasts the situation on two dairy farms in Normandy in France, where a clear seasonal pattern was observed with a gradual increase in the ODR during the grazing season and the highest values observed from June until October (Charlier et al., 2007a). However, a major difference between the present and previous seasonal bulk-tank milk surveys is that in the current study we only analysed samples collected during the grazing season, whereas the French and Canadian herds were sampled all year round. Nevertheless, the seasonal ODR patterns seem to differ between the geographical areas, which probably reflects differences in the epidemiology of O. ostertagi in slightly different temperate climates. For example, a large number of overwintered infective larvae is the key factor in the epidemiology of bovine gastrointestinal nematode infections in the Scandinavian countries including both Norway (Helle and Tharaldsen, 1976) and Sweden (Dimander et al., 2003), whereas high pasture infectivity seems to occur from midseason and onwards in most European countries (Charlier et al., 2007a). The lack of a seasonal pattern for ODR in Sweden, however, is of great practical importance, because in this situation it means that milk analysed soon after turn-out can be used to predict whether antiparasitic measures are necessary later in the same grazing season, which is beneficial for practical prognostic control. The seroprevalence of F. hepatica was only 7% in the present study, which is a much lower level than for O. ostertagi and D. viviparus. In the case of the liver fluke, surveys based on bulk-tank milk samples are available from the UK (Salimi-Bejestani et al., 2005) and Belgium (Bennema et al., 2009) with herd prevalences ranging from 37% to 86%. Liver fluke infection is thus a much more limited problem in Swedish dairy herds. This was so irrespective of whether the farms were managed according to the rules of organic or conventional production. The few positive herds were mainly located in damp areas along the coastline or were from farms located near the two big lakes in southcentral Sweden (Fig. 6). Although the seroprevalence was somewhat lower than expected, the locations of the positive herds were as expected, since livers condemned by F. hepatica are more commonly reported at slaughter when the abattoirs receive cattle raised in the same places. The low seroprevalence of F. hepatica can be a consequence of a non-optimal sampling time for this parasite. Nevertheless, dairy herds in Sweden seem to have a relatively low prevalence of F. hepatica in comparison with other European countries, and its effects on production is therefore probably very restricted. Previously published information on the seroepidemiology in bulk-tank milk to Dictyocaulus is restricted to a single study from Flanders in Belgium, in which the herd prevalence was around 20% (Bennema et al., 2009). This agrees with the levels observed in the organic herds tested
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here but is approximately two times higher than in the conventional herds. Although the prevalence of lungworms in general is lower in younger stock, this result contrasts with previous findings on this parasite in Sweden, based on comparisons of serum samples from first season grazing dairy calves collected from organic and conventional farms both with and without anthelmintic treatment, and where no major differences in seroprevalence in relation to management could be detected (Höglund et al., 2004). There are typical limitations of any antibody-ELISA tests, such as the inability to detect newly acquired infections and to distinguish between previous and ongoing infections, as well as the existence of potential cross-reactions. It has been reported that both the D. viviparus (von Holtum et al., 2008) and F. hepatica tests (Hutchinson and Macarthur, 2003) have high sensitivity and specificity. In contrast, the O. ostertagi ELISA is based on a crude antigen made from adult worm homogenates. Thus it cross-reacts to a range of internal parasites, in particular the closely related intestinal nematode Cooperia oncophora, but also with F. hepatica (Bennema et al., 2009). This does not necessarily constitute a problem if the objective of the survey is to measure the overall exposure to pasture borne parasite infections rather than to diagnose different species of parasites. However, as pointed out by Bennema et al. (2009), it is more of a problem when the purpose is to identify hot spots of a particular parasite infection and locations where regional parasite control programmes should be applied. At the same time, it needs to be realised that classical parasitological faecal examination is certainly not a feasible and realistic alternative, as with these techniques we will be limited to the detection of patent infection or ongoing egg/larval production. 5. Conclusion This randomised pairwise study demonstrates that ODR values for the parasitic nematode O. ostertagi were significantly higher in bulk-tank milk collected in organic compared to conventional dairy herds. It also shows that the ODR in response to this parasite was stable throughout the grazing season. We also detected a tendency to higher seroprevalence of the bovine lungworm D. viviparus in samples that originated from organic herds. On the other hand, there was no difference between organic and conventional herds in the seroprevalence of the liver fluke F. hepatica. To sum up, this study further confirms that various milk ELISAs can be explored to monitor important helminth infections in dairy cows, and that this is a non-invasive and inexpensive method that easily can be used map to parasite hot spots. Acknowledgements Funding was provided by FORMAS (220-2007-1616) and the 6th framework program of the EU (Parasol, Project FOOD-CT-2005-022851). Jacob Lagerstedt and Tobias Rydlinge are thanked for their support with the GIS analyses.
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