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Very low helminth infection in sheep grazed on pastures fertilised by sewage sludge or cattle slurry
Veterinary Parasitology 131 (2005) 65–70 www.elsevier.com/locate/vetpar
Very low helminth infection in sheep grazed on pastures fertilised by sewage sludge or cattle slurry M.N. Moussavou-Boussougou a, P. Dorny b, J. Cabaret a,* b
a INRA, BASE, 37380 Nouzilly, France Prince Leopold Institute of Tropical Medicine, 155 Nationalestraat, 2000 Antwerpen, Belgium
Received 6 February 2005; received in revised form 26 April 2005; accepted 27 April 2005
Abstract Helminth infection acquired by lambs grazing on pastures fertilised either by urban sewage sludge or cattle slurry were studied in temperate Central Western France. The aim was to assess the risk of larval cestodoses in lambs after sewage application and of digestive tract nematode infection following the slurry application. Twenty-six sheep were allocated on two paddocks of 0.7 ha, one fertilised with sludge and the other with cattle slurry. The delay between application and actual grazing was 6 weeks; grazing on these paddocks extended from mid July to beginning of November 2002. The herbage biomass was slightly increased in the sludge paddock but it did not result in an increase of lamb live weight, compared with the slurry paddock. The lambs did not acquire cysticercosis or any other larval cestodoses in the sewage sludge group and only very limited infections with Cooperia spp. and Nematodirus spp. were observed in the slurry group. It was concluded that in our conditions the helminth risk was extremely low and was not a cause of restriction of the use of these biowastes. # 2005 Elsevier B.V. All rights reserved. Keywords: Sewage sludge; Cattle slurry; Taenia spp.; Cooperia spp.; Nematodirus spp
1. Introduction Among human or animal wastes, sewage sludge, manure and slurry use in agriculture are a real health concern. Sewage sludge has been used for years as a land fertiliser in many countries. It is a possible source of pathogens (Dumontet et al., 2001), of which Taenia spp. is an important component. Sheep is not a recorded host of Taenia saginata (Cabaret et al., * Corresponding author. Tel.: +33 2 47427768; fax: +33 2 47427774. E-mail address: [email protected] (J. Cabaret).
2002), but it may be a putative host as abortive infection was detected in sheep (Geerts et al., 1981). Larval taeniids frequently occur in sheep (Echinococcus granulosus Lezeriuc et al., 2002; Cysticercus tenuicollis Pfister, 1978; Taenia ovis Peris Palau et al., 1987). Among other sources of contamination sewage sludge, when it harbours dog (the definitive host of these cestodes) faeces, may play a role in the transmission of cestodes to sheep. The eggs of the most common adult cestode (Moniezia) in sheep may be found in sewage sludge connected to slaughterhouses (Bergstrom, 1981). The regulations on the use of sewage sludge in agriculture are intended to reduce
0304-4017/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2005.04.029
66
M.N. Moussavou-Boussougou et al. / Veterinary Parasitology 131 (2005) 65–70
the helmintic risk. In France, for sanitised sewage sludge (with less than three helminth eggs in/10 g dry matter), a delay of 3 weeks is required between the sludge application and the actual grazing of pastures. When unsanitised sludge is used this delay extends to 6 weeks according to French law (Arreˆ te´ du 8 janvier, 1998). The use of unsanitized liquid sludge is very common in rural areas and the application on cultivated lands or pastures remains the only economical means to use this type of sludge. It is, however, not known if such a procedure is efficient to reduce risk of infection with cestodes in sheep. Another biowaste that is spread on land is cattle slurry. It represents a much larger volume than sludge (20 times more in dry-matter). Cattle slurry has been described as a source of pathogens, among which are digestive-tract strongyles (Juris et al., 1996). However, the presence of urine in slurry (Helle et al., 1989) and anaerobic conditions (Kates, 1950) in tanks reduce the survival and potential of strongyle eggs to develop into infective larvae (Persson, 1974). Contamination of pastures after slurry fertilisation has been shown in cattle (Downey and Moore, 1977; Nansen et al., 1979, 1981; Moussavou-Boussougou et al., in press) but no information is available for sheep, although several species of nematodes are common to sheep and cattle in condition of mixed grazing, such as Ostertagia ostertagi, Cooperia sp. or Nematodirus sp. (Southcott and Barger, 1975; Cabaret, 1984). No specific regulation is available on the spread of cattle slurry in relation to the risk of digestive-tract nematode transmission; we followed those proposed for sewage sludge (6 weeks before application on pastures and actual grazing). The agronomic interest (increased grass production and sheep live weight) is one of the justifications of the use of these biowastes. Limited helminth infections may be caused by their application on cattle pastures (Moussavou-Boussougou et al., in press) and application on sheep pastures could be possibly a safer alternative when feasible. However, we have to be very careful before proposing such a fertilisation: the use of pig slurry on sheep pasture has resulted in vesicules on lambs liver and that were tentatively attributed to Ascaris suum, an unexpected source of parasite for sheep (Gibson and Lanning, 1981). Therefore, we intended to answer to the following questions: (i) is there any agronomic interest for spreading sewage sludge or slurry on sheep
pastures, (ii) can it be the source of infection with cestodes or digestive tract nematodes in sheep?
2. Materials and methods 2.1. Sewage sludge and slurry The sewage sludge originated from the town of Chaˆ teau-Renault (8000 inhabitants, Centre Western France). Since the sewage treatment plant is not connected to any slaughterhouse, no eggs/larvae of sheep or cattle nematodes or Moniezia were expected. The presence of Toxocara sp. eggs was indicative of the presence of caniids faeces in this sludge. The sludge was stabilized using aerobic digestion; the drymatter was 3.5%. The number of Taeniid eggs was 0.65 per gramme of dry matter, estimated by a modified triple floatation technique with Zinc sulphate (Barbier et al., 1990). Egg viability was 50% based on the methyl tetrazolium coloration test (Madeline et al., 2000, 2003). There were infective T. saginata eggs in the sludge we used, as demonstrated with experimental cattle infection (Moussavou-Boussougou et al., in press). Ten tonnes of sludge were applicated and thus a total of 228,000 Taeniids viable eggs were seeded on one paddock at the beginning of June 2002. Slurry (1% dry-matter) originated from a cattle stable at INRA Nouzilly where the cows and heifers were only slightly infected (five eggs per gramme of faeces); the infection consisted mostly of O. ostertagi. The slurry was stored 3 months in tanks before application in order to reduce the Salmonella risk (Marly, 1994). Thirty eggs per litre were found in the slurry. Eleven tons of slurry were spread on one paddock beginning of June 2002 (corresponding to 330,000 eggs) 6 weeks before turn-out. The methyl tetrazolium coloration was not effective for estimating digestive-tract strongyle eggs viability and faecal cultures were undertaken. The viability of eggs was probably very low as faecal cultures with vermiculite of 1 l (30 eggs equivalent) failed to yield any third stage larvae. 2.2. Pastures The pastures (two paddocks of 0.7 ha) were located at INRA Nouzilly (Centre Western France) under a
M.N. Moussavou-Boussougou et al. / Veterinary Parasitology 131 (2005) 65–70
temperate climate (684 mm yearly rainfall and 11.2 8C average yearly temperature). These pastures was seeded 6 years before this experiment with a mixture of ryegrass (Lolium sp.) and white clover (Trifolium repens), then grazed by sheep (infected with Teladorsagia circumcinta), followed by cattle (in 2001: very slightly infected with Cooperia spp., Nematodirus spp., Trichostrongylus colubriformis and O. ostertagi), and again by sheep in the present experiment. The late turn-out in July was intended to cleaning from previous remainings of digestive-tract strongyle infective larvae (Kerboeuf, 1985) Among the 23 plants found, the most represented were: Poa annua, Festuca pratense, Rumex obtusifolius, Sonchus sp., Taraxacum officinale, T. repens, Bromus hordeaceus. Analyses of herbage biomass (mowing 0.5 m2 on five random different sites in each paddock) and dry-matter as well as herbage height (25 random points done each paddock) were performed on four occasions. 2.3. Sheep and infection measures The Pre´ alpes lambs has been raised indoors and they were put to graze when aged 3 months, on the 17th of July 2003. They remained 111 days on pasture. They were allocated into sludge and slurry groups on the basis of balanced weight and sex (seven males and six females in each group). They were weighed on five occasions during the grazing season. Faecal egg counts were done on day 0 (turn-out), day 27, day 55 and day 83 after grazing of pastures fertilised with slurry or sewage sludge, using McMaster technique with a NaCl floatation solution (d = 1.20; minimal detection of 15 eggs/g). At necropsy (day 134), carcasses were examined in detail for the presence of cysticerci: tongue, heart, diaphragm muscles were examined by cutting them in slices of 1–2 cm thick. The brain and its envelopes were examined for abnormal locations of T. saginata as found in another abnormal host, the reindeer (Kirichek et al., 1984). The absence of other larval cestodoses was also checked on brain, lungs, liver and abdomen cavity. To estimate the digestive tract infections, the abomasum and the small intestine (5 m starting from the pyloric end) were collected. The worms were counted from total washings of each organ; no incubation or peptic digestion was undertaken. Blood samples were taken
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on six occasions (one before turn-out, and then during the grazing season) in order to detect circulating Taenia antigens using a Sandwich-ELISA technique (Dorny et al., 2000), slightly modified as described by (Dorny et al., 2004). The optical density (OD) of each serum sample was compared with a sample of negative sheep serum samples (n = 8) at a probability of P < 0.001 to determine the result of the test (Sokal and Rohlf, 1981). 2.4. Statistical analyses The number of worms at necropsy were analysed with the Kruskal and Wallis non-parametric test, as the distribution was not gaussian. Fisher exact test was used for small groups (less than 5). The Wilcoxon pair test was used to compare the evolution of herbage production between sludge and slurry paddocks. A general linear model was used to compare the live weight of lambs between the sludge and slurry fertilised paddocks. All calculations used a Simstat software (1996).
3. Results 3.1. Cestodes No eggs of Moniezia were shed during the experiment in both groups. The Ag-ELISA remained negative throughout the experiment (OD lower than cut-off value, 0.010) in the sewage sludge group. At necropsy, no larval (Cysticercus or Echinococcus spp.) or adult (Moniezia) cestodes were found. Table 1 Average numbers of digestive-tract nematodes in lambs at the end of grazing season on paddocks fertilised by sewage sludge and slurry Species of nematodes
Sludge a
Cooperia spp. 0 Teladorsagia circumcincta 3.5 Trichostrongylus colubriformis 11.5 Nematodirus spp. 0 Strongyloides papillosus 38.5 Trichuris sp. 2.3
Slurry
Significance
0.3 (0.5) S (2.5)b 0.1 (0.3) S (8.2) 20.2 (19.5) NS 1.8 (1.5) S (31.0) 4.9 (3.6) S (2.3) 11 (6.6) S
S, significant at P < 0.05; NS, non significant. a Mean number of worms. b Standard deviation.
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Table 2 Herbage biomass and mean height of paddocks fertilised by sewage sludge and slurry and grazed by lambs Days after the beginning of grazing
Paddock
Fresh grass biomass (kg/m2)
Dry grass biomass (kg/m2)
Mean height cm (standard deviation)
26
Sludge Slurry
0.143a 0.166
0.038 0.053
3.56 (2.04)b 2.96 (1.54)
7
Sludge Slurry
0.249 0.228
0.082 0.076
3.32 (1.57) 3.12 (1.45)
44
Sludge Slurry
0.165 0.104
0.081 0.052
0.16 (0.47) 0.08 (0.28)
76
Slugde Slurry
0.048 0.065
0.032 0.039
0.04 (0.20) 0 (0)
a b
Average. Standard deviation.
3.2. Digestive-tract nematodes Faecal egg counts remained negative throughout the experiment except for one lamb at day 28 (day 28) at the beginning of the grazing period and one at day 83, in the slurry group. At necropsy only adult worms were recovered. T. colubriformis were recovered from all lambs; percentage of males in adult population was 44 and 50%, in slurry and sludge groups, respectively. Cooperia pectinata, C. oncophora, and C. curticei, Nematodirus chabaudi and N. helvetianus were identified only in the slurry group. The worm counts are presented in Table 1.
biomass, dried grass biomass, grass height, respectively). The GLM model on lamb weight evolution (Table 3) was: weight at time t = weight at turn out + b sex + c group (slurry or sludge). There was no significant effect of sludge or slurry application on pasture on weight gains throughout the grazing season. There was a clear sex effect at the end of the experiment and male and females weighted 39.7 and 37.7 kg, corrected from the initial weight, respectively.
4. Discussion 3.3. Grass production and live weights of lambs Grass production (Table 2) was significantly higher (Wilcoxon-test, P < 0.05) in the sludge group during the whole grazing season (+7, 9 and 12.9% for grass Table 3 Lambs live weight evolution during the grazing season on pastures fertilised by sewage sludge or slurry Days after the beginning of grazing sludge or slurry fertilised paddocks
Sludge (n = 13)
Slurry (n = 13)
0 27 55 83 106
27.4a (5.8)b 31.1 (5.7) 33.9 (6.2) 37.3 (6.5) 38.7 (8.2)
27.4 31.2 34.2 36.6 39.6
a b
Mean (kg). Standard deviation.
(4.8) (4.9) (5.1) (5.8) (6.1)
From an extensive review of literature (Cabaret et al., 2002), sheep was not considered as a good potential host for T. saginata cysticercosis although it could not be fully ruled out. Experimental infections (Geerts et al., 1981; Blazek and Schramlova, 1983) did not result in establishment of cysticerci, even in immunodepressed lambs. Our results show that under field conditions lambs do not acquire T. saginata cysticercosis infection, at least in conditions of low levels of infection. It should be remembered that cattle did not become infected with the same sewage sludge (Moussavou-Boussougou et al., in press) and that infectivity of T. saginata eggs remains low, as corroborated with records on sludge digestion after 15–30 days that corresponds to over 99.5 reduction in infectivity (Morris et al., 1986). The absence of other cestodes (T. hydatigena, T. ovis, E. granulosus) is
M.N. Moussavou-Boussougou et al. / Veterinary Parasitology 131 (2005) 65–70
possibly due to the very limited presence of dog faeces in sludge or to the absence of connection with slaughterhouse wastes (Moniezia) in the sludge used in this experiment. The infections with nematodes are more difficult to interpret. The presence of T. colubriformis in sheep grazed on the sludge and slurry fertilised paddocks can be explained by the grazing of infected cattle on these pastures in the previous years. The percentage of males in the T. colubriformis population was in the range of what is recorded in natural infections (Cabaret, 1984: 39–43%), indicating that the parasite was probably recycled on the pastures by sheep. The presence, even at very low levels of Cooperia spp. and Nematodirus spp., is related to the application of cattle slurry, as it was also found in cattle grazing slurry fertilised paddocks (Moussavou-Boussougou et al., in press). O. ostertagi was the main species found in the cattle housed in the stable from which originated the slurry, but although it could infest sheep in experimental (Borgsteede, 1981) and natural conditions (Southcott and Barger, 1975, Cabaret, 1984), it did not cause infection in lambs in this study. This could be due to poor susceptibility of lambs or low survival under anaerobic conditions in tanks (Furlong and Padilha, 1996) and hence poor contamination of pastures. The presence of T. circumcincta in the sludge group might be related to the grazing of infected sheep 3 years before the present experiment, and the absence in the slurry paddocks would then only be due to chance, since the paddocks had the same sheep grazing history. The use of cattle slurry on lamb paddocks resulted in a very limited infection. The comparative agronomic interest of both biowastes used in this study seems equivalent: the lambs grazed on slurry or sludge paddocks had comparable live weight and the herbage biomass they disposed was also similar. The use of either sewage sludge or cattle slurry, with a 6 weeks delay between application on pasture and turn out of lambs did not present a real helmintic danger. The agronomic advantages are probably modest but the possibility of recycling these biowastes is of real interest.
Acknowledgements The research was funded by ADEME. M.N. Moussavou-Boussougou was financially supported
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by the Gabon Ministry of Public Health. The Chaˆ teauRenault municipality and sewage sludge plant staff are thanked for their cooperation. The following INRA staff helped us: P. Guiard—application of fertilisers, T. Chaumeil—lambs and pasture management, and J. Cortet and C. Sauve´ —parasitological techniques on slurry.
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Gibson, G.M., Lanning, D.G., 1981. Liver damage in lambs. Vet. Rec. 108, 165. Helle, O., Velle, W., Tharaldsen, J., 1989. Effect of ovine urine and some of its components on viability of nematode eggs and larvae in sheep faeces. Vet. Parasitol. 32, 349–354. Juris, P., Vasilkova, Z., Plachy, P., Krupicer, I., Sokol, J., 1996. Coccidial oocysts in cattle slurry on farms in north-eastern Slovakia. Slov. Vet. Casopis 21, 93–95. Kates, K.C., 1950. Survival on pasture of free-living stages of some common gastrointestinal nematodes of sheep. Proc. Helm. Soc. Wash. 17, 39–58. Kerboeuf, D., 1985. Winter survival of Trichostrongyle larvae: a study using tracer lambs. Res. Vet. Sci. 38, 364–367. Kirichek, V.S., Belousov, M.N., Nikitin, A.S., 1984. New data on the epidemiology of taeniasis in the extreme north of the USSR (data of investigations made in the Yamalo-Nenets autonomous region). Med. Parazitol. Bolezni 6, 27–33. Lezeriuc, A., Ionescu, L., Grosu, I., 2002. Parasitic diseases in animals slaughtered at Bacau in 1995-2001. Rev. Rom. Parazitol. 12, 70–72. Madeline, M., Ballandonne, C., Guyon, R., Malas, J.P., Vilagines, P., Cabaret, J., Barbier, D., 2000. Mise en e´vidence de la viabilite´ des œufs de taeniide´ s dans les boues re´ siduaires. J. Eur. Hydrol. 31, 85–90. Madeline, M., Ballandonne, C., Vilagines, P., Malas, J.P., Cabaret, J., Barbier, D., 2003. Boues re´ siduaires: difficulte´ s d’e´valuation du risque parasitaire. J. Eur. Hydrol. 34, 235–244. Marly, J., 1994. Risques sanitaires lie´ s aux effluents d’e´ levage. In: Epandage des boues d’e´ puration sur prairies et cultures fourrage`res. Aspects microbiologiques. ADEME, Paris, p. 92. Morris, D.L., Hughes, D.L., Hewitt, R.C., Norrington, I.J., 1986. Pathogens in sewage sludge: (ii) effects of sludge stabilization
and treatment processes on viability and infectivity of beef tapeworm eggs. Water Pollut. Control 85, 476–481. Moussavou-Boussougou, M.N., Geerts, S., Madeline, M., Ballandonne, C., Barbier, D., Cabaret, J., in press. Sewage sludge or cattle slurry as fertilisers: comparative helminth risk for grazing cattle. Parasitol. Res. Nansen, P., Henriksen, S.A., Jorgensen, R.J., Foldager, J., 1981. Trichostrongylid nematode infections associated with the handling of cattle slurry. A survey of Danish studies. In: Nansen, P., Jorgensen, R.J., Soulsby, E.J.L. (Eds.), Epidemiology and Control of Nematodiasis in cattle. Martinus Nijhoff, la Hague, pp. 497–507. Nansen, P., Midtgaard, N., Jorgensen, M., Sejrsen, K., Jorgensen, R.J., 1979. Trichostrongylid nematode infections in calves grazing slurry manured paddocks. Kgl. Vet. Landbohoejsk. Arsskr. 53–71. Peris Palau, B., Garcia Marin, J.F., Badiola Diez, J.J., 1987. Ovine visceral cysticercosis: main cause of liver condemnation in fat lambs Lesions and incidence. Med. Vet. 4, 289–296. Persson, L., 1974. Studies on the survival of eggs and infective larvae of Ostertagia ostertagi and Cooperia oncophora in liquid cattle manure. Zbl. Vet. Med. B 21, 311–317. Pfister, K., 1978. Cysticerciasis in sheep and its relation to tapeworm infection in the dog. Schweiz. Arch. Tierheilkd. 120, 569–577. Simstat software, 1996. Simstat for Windows. User’s Guide. Normand Peladeau, Provalis Research. Montreal, Canada, 255 pp. Sokal, R.S., Rohlf, J.J., 1981. Biometry: The Principles and Practice of Statistics in Biological Research, second ed. Freeman, New York. Southcott, W.H., Barger, I.A., 1975. Control of nematode parasites by grazing management II Decontamination of sheep and cattle pastures by varying periods of grazing with the alternate host. Int. J. Parasitol. 5, 45–48.
Very low helminth infection in sheep grazed on pastures fertilised by sewage sludge or cattle slurry M.N. Moussavou-Boussougou a, P. Dorny b, J. Cabaret a,* b
a INRA, BASE, 37380 Nouzilly, France Prince Leopold Institute of Tropical Medicine, 155 Nationalestraat, 2000 Antwerpen, Belgium
Received 6 February 2005; received in revised form 26 April 2005; accepted 27 April 2005
Abstract Helminth infection acquired by lambs grazing on pastures fertilised either by urban sewage sludge or cattle slurry were studied in temperate Central Western France. The aim was to assess the risk of larval cestodoses in lambs after sewage application and of digestive tract nematode infection following the slurry application. Twenty-six sheep were allocated on two paddocks of 0.7 ha, one fertilised with sludge and the other with cattle slurry. The delay between application and actual grazing was 6 weeks; grazing on these paddocks extended from mid July to beginning of November 2002. The herbage biomass was slightly increased in the sludge paddock but it did not result in an increase of lamb live weight, compared with the slurry paddock. The lambs did not acquire cysticercosis or any other larval cestodoses in the sewage sludge group and only very limited infections with Cooperia spp. and Nematodirus spp. were observed in the slurry group. It was concluded that in our conditions the helminth risk was extremely low and was not a cause of restriction of the use of these biowastes. # 2005 Elsevier B.V. All rights reserved. Keywords: Sewage sludge; Cattle slurry; Taenia spp.; Cooperia spp.; Nematodirus spp
1. Introduction Among human or animal wastes, sewage sludge, manure and slurry use in agriculture are a real health concern. Sewage sludge has been used for years as a land fertiliser in many countries. It is a possible source of pathogens (Dumontet et al., 2001), of which Taenia spp. is an important component. Sheep is not a recorded host of Taenia saginata (Cabaret et al., * Corresponding author. Tel.: +33 2 47427768; fax: +33 2 47427774. E-mail address: [email protected] (J. Cabaret).
2002), but it may be a putative host as abortive infection was detected in sheep (Geerts et al., 1981). Larval taeniids frequently occur in sheep (Echinococcus granulosus Lezeriuc et al., 2002; Cysticercus tenuicollis Pfister, 1978; Taenia ovis Peris Palau et al., 1987). Among other sources of contamination sewage sludge, when it harbours dog (the definitive host of these cestodes) faeces, may play a role in the transmission of cestodes to sheep. The eggs of the most common adult cestode (Moniezia) in sheep may be found in sewage sludge connected to slaughterhouses (Bergstrom, 1981). The regulations on the use of sewage sludge in agriculture are intended to reduce
0304-4017/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2005.04.029
66
M.N. Moussavou-Boussougou et al. / Veterinary Parasitology 131 (2005) 65–70
the helmintic risk. In France, for sanitised sewage sludge (with less than three helminth eggs in/10 g dry matter), a delay of 3 weeks is required between the sludge application and the actual grazing of pastures. When unsanitised sludge is used this delay extends to 6 weeks according to French law (Arreˆ te´ du 8 janvier, 1998). The use of unsanitized liquid sludge is very common in rural areas and the application on cultivated lands or pastures remains the only economical means to use this type of sludge. It is, however, not known if such a procedure is efficient to reduce risk of infection with cestodes in sheep. Another biowaste that is spread on land is cattle slurry. It represents a much larger volume than sludge (20 times more in dry-matter). Cattle slurry has been described as a source of pathogens, among which are digestive-tract strongyles (Juris et al., 1996). However, the presence of urine in slurry (Helle et al., 1989) and anaerobic conditions (Kates, 1950) in tanks reduce the survival and potential of strongyle eggs to develop into infective larvae (Persson, 1974). Contamination of pastures after slurry fertilisation has been shown in cattle (Downey and Moore, 1977; Nansen et al., 1979, 1981; Moussavou-Boussougou et al., in press) but no information is available for sheep, although several species of nematodes are common to sheep and cattle in condition of mixed grazing, such as Ostertagia ostertagi, Cooperia sp. or Nematodirus sp. (Southcott and Barger, 1975; Cabaret, 1984). No specific regulation is available on the spread of cattle slurry in relation to the risk of digestive-tract nematode transmission; we followed those proposed for sewage sludge (6 weeks before application on pastures and actual grazing). The agronomic interest (increased grass production and sheep live weight) is one of the justifications of the use of these biowastes. Limited helminth infections may be caused by their application on cattle pastures (Moussavou-Boussougou et al., in press) and application on sheep pastures could be possibly a safer alternative when feasible. However, we have to be very careful before proposing such a fertilisation: the use of pig slurry on sheep pasture has resulted in vesicules on lambs liver and that were tentatively attributed to Ascaris suum, an unexpected source of parasite for sheep (Gibson and Lanning, 1981). Therefore, we intended to answer to the following questions: (i) is there any agronomic interest for spreading sewage sludge or slurry on sheep
pastures, (ii) can it be the source of infection with cestodes or digestive tract nematodes in sheep?
2. Materials and methods 2.1. Sewage sludge and slurry The sewage sludge originated from the town of Chaˆ teau-Renault (8000 inhabitants, Centre Western France). Since the sewage treatment plant is not connected to any slaughterhouse, no eggs/larvae of sheep or cattle nematodes or Moniezia were expected. The presence of Toxocara sp. eggs was indicative of the presence of caniids faeces in this sludge. The sludge was stabilized using aerobic digestion; the drymatter was 3.5%. The number of Taeniid eggs was 0.65 per gramme of dry matter, estimated by a modified triple floatation technique with Zinc sulphate (Barbier et al., 1990). Egg viability was 50% based on the methyl tetrazolium coloration test (Madeline et al., 2000, 2003). There were infective T. saginata eggs in the sludge we used, as demonstrated with experimental cattle infection (Moussavou-Boussougou et al., in press). Ten tonnes of sludge were applicated and thus a total of 228,000 Taeniids viable eggs were seeded on one paddock at the beginning of June 2002. Slurry (1% dry-matter) originated from a cattle stable at INRA Nouzilly where the cows and heifers were only slightly infected (five eggs per gramme of faeces); the infection consisted mostly of O. ostertagi. The slurry was stored 3 months in tanks before application in order to reduce the Salmonella risk (Marly, 1994). Thirty eggs per litre were found in the slurry. Eleven tons of slurry were spread on one paddock beginning of June 2002 (corresponding to 330,000 eggs) 6 weeks before turn-out. The methyl tetrazolium coloration was not effective for estimating digestive-tract strongyle eggs viability and faecal cultures were undertaken. The viability of eggs was probably very low as faecal cultures with vermiculite of 1 l (30 eggs equivalent) failed to yield any third stage larvae. 2.2. Pastures The pastures (two paddocks of 0.7 ha) were located at INRA Nouzilly (Centre Western France) under a
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temperate climate (684 mm yearly rainfall and 11.2 8C average yearly temperature). These pastures was seeded 6 years before this experiment with a mixture of ryegrass (Lolium sp.) and white clover (Trifolium repens), then grazed by sheep (infected with Teladorsagia circumcinta), followed by cattle (in 2001: very slightly infected with Cooperia spp., Nematodirus spp., Trichostrongylus colubriformis and O. ostertagi), and again by sheep in the present experiment. The late turn-out in July was intended to cleaning from previous remainings of digestive-tract strongyle infective larvae (Kerboeuf, 1985) Among the 23 plants found, the most represented were: Poa annua, Festuca pratense, Rumex obtusifolius, Sonchus sp., Taraxacum officinale, T. repens, Bromus hordeaceus. Analyses of herbage biomass (mowing 0.5 m2 on five random different sites in each paddock) and dry-matter as well as herbage height (25 random points done each paddock) were performed on four occasions. 2.3. Sheep and infection measures The Pre´ alpes lambs has been raised indoors and they were put to graze when aged 3 months, on the 17th of July 2003. They remained 111 days on pasture. They were allocated into sludge and slurry groups on the basis of balanced weight and sex (seven males and six females in each group). They were weighed on five occasions during the grazing season. Faecal egg counts were done on day 0 (turn-out), day 27, day 55 and day 83 after grazing of pastures fertilised with slurry or sewage sludge, using McMaster technique with a NaCl floatation solution (d = 1.20; minimal detection of 15 eggs/g). At necropsy (day 134), carcasses were examined in detail for the presence of cysticerci: tongue, heart, diaphragm muscles were examined by cutting them in slices of 1–2 cm thick. The brain and its envelopes were examined for abnormal locations of T. saginata as found in another abnormal host, the reindeer (Kirichek et al., 1984). The absence of other larval cestodoses was also checked on brain, lungs, liver and abdomen cavity. To estimate the digestive tract infections, the abomasum and the small intestine (5 m starting from the pyloric end) were collected. The worms were counted from total washings of each organ; no incubation or peptic digestion was undertaken. Blood samples were taken
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on six occasions (one before turn-out, and then during the grazing season) in order to detect circulating Taenia antigens using a Sandwich-ELISA technique (Dorny et al., 2000), slightly modified as described by (Dorny et al., 2004). The optical density (OD) of each serum sample was compared with a sample of negative sheep serum samples (n = 8) at a probability of P < 0.001 to determine the result of the test (Sokal and Rohlf, 1981). 2.4. Statistical analyses The number of worms at necropsy were analysed with the Kruskal and Wallis non-parametric test, as the distribution was not gaussian. Fisher exact test was used for small groups (less than 5). The Wilcoxon pair test was used to compare the evolution of herbage production between sludge and slurry paddocks. A general linear model was used to compare the live weight of lambs between the sludge and slurry fertilised paddocks. All calculations used a Simstat software (1996).
3. Results 3.1. Cestodes No eggs of Moniezia were shed during the experiment in both groups. The Ag-ELISA remained negative throughout the experiment (OD lower than cut-off value, 0.010) in the sewage sludge group. At necropsy, no larval (Cysticercus or Echinococcus spp.) or adult (Moniezia) cestodes were found. Table 1 Average numbers of digestive-tract nematodes in lambs at the end of grazing season on paddocks fertilised by sewage sludge and slurry Species of nematodes
Sludge a
Cooperia spp. 0 Teladorsagia circumcincta 3.5 Trichostrongylus colubriformis 11.5 Nematodirus spp. 0 Strongyloides papillosus 38.5 Trichuris sp. 2.3
Slurry
Significance
0.3 (0.5) S (2.5)b 0.1 (0.3) S (8.2) 20.2 (19.5) NS 1.8 (1.5) S (31.0) 4.9 (3.6) S (2.3) 11 (6.6) S
S, significant at P < 0.05; NS, non significant. a Mean number of worms. b Standard deviation.
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Table 2 Herbage biomass and mean height of paddocks fertilised by sewage sludge and slurry and grazed by lambs Days after the beginning of grazing
Paddock
Fresh grass biomass (kg/m2)
Dry grass biomass (kg/m2)
Mean height cm (standard deviation)
26
Sludge Slurry
0.143a 0.166
0.038 0.053
3.56 (2.04)b 2.96 (1.54)
7
Sludge Slurry
0.249 0.228
0.082 0.076
3.32 (1.57) 3.12 (1.45)
44
Sludge Slurry
0.165 0.104
0.081 0.052
0.16 (0.47) 0.08 (0.28)
76
Slugde Slurry
0.048 0.065
0.032 0.039
0.04 (0.20) 0 (0)
a b
Average. Standard deviation.
3.2. Digestive-tract nematodes Faecal egg counts remained negative throughout the experiment except for one lamb at day 28 (day 28) at the beginning of the grazing period and one at day 83, in the slurry group. At necropsy only adult worms were recovered. T. colubriformis were recovered from all lambs; percentage of males in adult population was 44 and 50%, in slurry and sludge groups, respectively. Cooperia pectinata, C. oncophora, and C. curticei, Nematodirus chabaudi and N. helvetianus were identified only in the slurry group. The worm counts are presented in Table 1.
biomass, dried grass biomass, grass height, respectively). The GLM model on lamb weight evolution (Table 3) was: weight at time t = weight at turn out + b sex + c group (slurry or sludge). There was no significant effect of sludge or slurry application on pasture on weight gains throughout the grazing season. There was a clear sex effect at the end of the experiment and male and females weighted 39.7 and 37.7 kg, corrected from the initial weight, respectively.
4. Discussion 3.3. Grass production and live weights of lambs Grass production (Table 2) was significantly higher (Wilcoxon-test, P < 0.05) in the sludge group during the whole grazing season (+7, 9 and 12.9% for grass Table 3 Lambs live weight evolution during the grazing season on pastures fertilised by sewage sludge or slurry Days after the beginning of grazing sludge or slurry fertilised paddocks
Sludge (n = 13)
Slurry (n = 13)
0 27 55 83 106
27.4a (5.8)b 31.1 (5.7) 33.9 (6.2) 37.3 (6.5) 38.7 (8.2)
27.4 31.2 34.2 36.6 39.6
a b
Mean (kg). Standard deviation.
(4.8) (4.9) (5.1) (5.8) (6.1)
From an extensive review of literature (Cabaret et al., 2002), sheep was not considered as a good potential host for T. saginata cysticercosis although it could not be fully ruled out. Experimental infections (Geerts et al., 1981; Blazek and Schramlova, 1983) did not result in establishment of cysticerci, even in immunodepressed lambs. Our results show that under field conditions lambs do not acquire T. saginata cysticercosis infection, at least in conditions of low levels of infection. It should be remembered that cattle did not become infected with the same sewage sludge (Moussavou-Boussougou et al., in press) and that infectivity of T. saginata eggs remains low, as corroborated with records on sludge digestion after 15–30 days that corresponds to over 99.5 reduction in infectivity (Morris et al., 1986). The absence of other cestodes (T. hydatigena, T. ovis, E. granulosus) is
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possibly due to the very limited presence of dog faeces in sludge or to the absence of connection with slaughterhouse wastes (Moniezia) in the sludge used in this experiment. The infections with nematodes are more difficult to interpret. The presence of T. colubriformis in sheep grazed on the sludge and slurry fertilised paddocks can be explained by the grazing of infected cattle on these pastures in the previous years. The percentage of males in the T. colubriformis population was in the range of what is recorded in natural infections (Cabaret, 1984: 39–43%), indicating that the parasite was probably recycled on the pastures by sheep. The presence, even at very low levels of Cooperia spp. and Nematodirus spp., is related to the application of cattle slurry, as it was also found in cattle grazing slurry fertilised paddocks (Moussavou-Boussougou et al., in press). O. ostertagi was the main species found in the cattle housed in the stable from which originated the slurry, but although it could infest sheep in experimental (Borgsteede, 1981) and natural conditions (Southcott and Barger, 1975, Cabaret, 1984), it did not cause infection in lambs in this study. This could be due to poor susceptibility of lambs or low survival under anaerobic conditions in tanks (Furlong and Padilha, 1996) and hence poor contamination of pastures. The presence of T. circumcincta in the sludge group might be related to the grazing of infected sheep 3 years before the present experiment, and the absence in the slurry paddocks would then only be due to chance, since the paddocks had the same sheep grazing history. The use of cattle slurry on lamb paddocks resulted in a very limited infection. The comparative agronomic interest of both biowastes used in this study seems equivalent: the lambs grazed on slurry or sludge paddocks had comparable live weight and the herbage biomass they disposed was also similar. The use of either sewage sludge or cattle slurry, with a 6 weeks delay between application on pasture and turn out of lambs did not present a real helmintic danger. The agronomic advantages are probably modest but the possibility of recycling these biowastes is of real interest.
Acknowledgements The research was funded by ADEME. M.N. Moussavou-Boussougou was financially supported
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by the Gabon Ministry of Public Health. The Chaˆ teauRenault municipality and sewage sludge plant staff are thanked for their cooperation. The following INRA staff helped us: P. Guiard—application of fertilisers, T. Chaumeil—lambs and pasture management, and J. Cortet and C. Sauve´ —parasitological techniques on slurry.
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