- Email: [email protected]
Ability of the fungus Duddingtonia flagrans to adapt to the cyathostomin egg-output by spreading chlamydospores
Veterinary Parasitology 179 (2011) 277–282
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Ability of the fungus Duddingtonia flagrans to adapt to the cyathostomin egg-output by spreading chlamydospores ˜ a , J.A. Sánchez a , R. Francisco a , A. Paz-Silva a,∗ , I. Francisco a , R.O. Valero-Coss b , F.J. Cortinas a a b M. Arias , J.L. Suárez , M.E. López-Arellano , R. Sánchez-Andrade a , P. Mendoza de Gives b , Equine Diseases Study Group (Epidemiology, Parasitology and Zoonoses) a
Animal Pathology Department, Faculty of Veterinary, University of Santiago de Compostela, 27002-Lugo, Spain Área de Helmintología, Centro Nacional de Investigación Disciplinaria en Parasitología Veterinaria, Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, Paseo Cuaunahuac 8534, Jiutepec, Morelos-62550, Mexico
b
a r t i c l e
i n f o
Article history: Received 8 September 2010 Received in revised form 9 February 2011 Accepted 17 February 2011 Keywords: Nematodes Duddingtonia flagrans Biological control Grazing horses Nematophagous fungi
a b s t r a c t The analysis of the capability of the nematode trapping-fungus Duddingtonia flagrans to adapt to the cyathostomin egg-output in horses was evaluated. Fecal samples from 196 pasturing autochthonous Pura Raza Galega horses were collected from the rectum and then divided according to the egg-output into three groups: ≤300, 310–800 and >800 eggs per gram feces. Four doses of chlamydospores (0.1, 0.2, 0.4 and 0.8 × 106 /100 g feces) were directly spread onto fecal pats on the ground, remaining one without treatment as control. Fecal pats confirmed the presence of gastrointestinal nematode larvae belonging to strongylid cyathostomins (Cyathostomum and Gyalocephalus spp). An overall 94% (95% CI 91, 97) percentage of reduction was obtained, and an increase in the activity of the trappingfungi simultaneously to the rising in the number of cyathostomin eggs and larvae in the coprocultures was detected. A significantly highest reduction of the cyathostomin L3 in the coprocultures with more than 800 EPG was found, which indicates that Df trapping activity is larvae nematode density-dependant. The present research showed the high biological activity of D. flagrans against nematode larvae can adjust to the cyathostomin egg-output, and underlines its efficacy as a practical method for the control of these parasites in grazing horses. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Some livestock managing involve animals under pasturing conditions, but the possibility for infection by gastrointestinal nematode parasites is notably enhanced under this regime (Francisco et al., 2009). Larvae belonging to several genera (Strongylus, Triodontophorus, Gyalocephalus, Trichonema, Ostertagia, Trichostrongylus, Haemonchus, Chabertia or Oesophagostomum) exit the egg
∗ Corresponding author. Tel.: +34 982285900x22126; fax: +34 982252195. E-mail address: [email protected] (A. Paz-Silva). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.02.014
and develop in the environment to attain the infective stage (L3), which are ingested when the animals feed the grass. These parasitic infections reduce the benefits and the inputs afforded (poor gain weight, low reproductive indexes), being chemotherapy the most employed procedure for their control. Duddingtonia flagrans is a nematophagous fungus which develops traps to capture nematodes present in the soil (Mendoza de Gives et al., 2006). Successful results have been achieved to reduce gastrointestinal nematode infections in horses, calves and sheep under northern temperate, tropical or subtropical climates (Gómez-Rincón et al., 2006; Braga et al., 2009; Campos et al., 2009; Maciel et al., 2010; Silva et al., 2010). This fungus is capable to produce a
278
A. Paz-Silva et al. / Veterinary Parasitology 179 (2011) 277–282
large quantity of chlamydospores able to resist the passage through the gastrointestinal tract of ruminants and monogastrics, hence different investigations regarding the oral administration of aqueous suspensions have been carried out (Baudena et al., 2000). This practice requires the correct immobilization of the animals, thus the use of multinutritional pellets containing spores of the nematode-trapping fungi D. flagrans and Monacrosporium thaumasium as vehicles has been checked in sheep (Casillas-Aguilar et al., 2008) and horses (Tavela Ade et al., 2011). In the last decades an increasing tendency to the abandonment of productive lands has been observed in many European countries. Under the requirement for keeping these properties free of shrubs and bushes and thus for reducing the risk for fire, the presence of 1–2 animals (mainly horses and then donkeys, sheep and goats) is becoming usual for making advantage of <0.5 ha pastures. There is a lack of information about the efficacy of the addition of the chlamydospores directly onto the pasture. The possible influence of the numbers of eggs passed by feces on the efficacy of D. flagrans spores to form trapping devices in feces remains also untested. In the current work, the utility of the direct administration of spores on the pasture was evaluated, and their ability to adapt to different cyathostomin egg-output numbers also. An annual coprological survey was conducted to get a useful knowledge about the risk periods for infection in horses.
By taking into account previous investigations concerning the cut-off egg-output values to establish the anthelmintic treatments the horses were grouped in the basis of the counts of strongyle eggs per grams (EPG): G-1 (≤300 EPG), G-2 (310–800), G-3 (>800 EPG) (Nielsen et al., 2006; Uhlinger, 2007).
2. Materials and methods
2.3.2. D. flagrans chlamydospore production The Mexican strain of D. flagrans FTHO-8 (CENIDPAVET, INIFAP-MEXICO) was used and chlamydospores were produced, harvested and managed according to Llerandi-Juárez and Mendoza de Gives (1998).
2.1. Area of study The current research was developed in SW Europe (Galicia, Spain) (42◦ 20 –43◦ 45 N, 6◦ 49 –8◦ 00 W), an agricultural area where cattle livestock represents the main farm activity. 2.2. Annual variations of nematode egg-output and on climatic parameters To determine the kinetics of nematode egg-output and the highest risk periods for infection, a herd of 25 indigenous Pura Raza Galega (PRG) horses in a farm in Lugo (NW Spain) was monthly sampled throughout 2008. Data corresponding to the maximal temperature, minimal temperature, rainfall, and relative humidity were monthly obtained from 2 automated meteorological stations to establish the climatic pattern. All the management of the animals has been carried out in accordance with the EC Directive 86/609/EEC for animal experiments. 2.3. Design of the study of the efficacy of D. flagrans According to the results from the previous experience, fecal samples were collected between June and October 2009 from 196 indigenous Pura Raza Galega (PRG) grazing horses in several farms located at different sites interspersed within this area, and the distance among the farms is 20–100 km approx.
2.3.1. Coprological techniques Fecal samples were individually collected from the rectum of the horses and analyzed by the coprological flotation method. Five grams of each fecal sample were processed (by duplicate) by using the copromicroscopical flotation technique (MAFF, 1986), with a sensitivity of 10 eggs per gram of feces. The counts of nematode eggs were expressed as counts of EPG. The microscope analysis of fecal samples was performed and additional blind samples were run to diminish possible technique errors. To gain more information about the different genera/specie of the strongyles affecting the horses, fecal samples were cultured for 10–15 days at 22–24 ◦ C to allow the development of eggs to infective larvae. Once larvae showed the typical morphological features of L3, they were collected by means of the Baermann procedure (Osterman Lind et al., 1999; Kuzmina et al., 2006) and taxonomically identified according to Lichtenfels et al. (1998). The counts were expressed as numbers of larvae/gram (LPG).
2.3.3. Efficacy of D. flagrans The efficacy of D. flagrans against nematode larvae was measured by comparing the number of L3 recovered from fecal pats with (LPGDR) and without D. flagrans chlamydospores (LPG). For every horse, three pats were prepared by leaving 50 g feces/each in plastic trays (50 cm × 50 cm) placed in the pastures fed by the horses. The spores of D. flagrans were diluted in an aqueous solution and added to two pats, and the other remained as control. Four doses of 0.1 (D1), 0.2 (D2), 0.4 (D3) and 0.8 × 106 (D4) spores/100 g feces were employed. Three replicates were considered in all cases. The efficacy in the larval reduction by the fungal action was determined as follows: Larval reduction (LR %) =
control mean L3 − fungus mean L3 control mean L3
× 100
Twelve days after the addition of the chlamydospores, the pats were collected and analyzed by using the flotation and the Baermann techniques. 2.4. Statistical analysis Considering that EPG and LPG counts are not normally distributed, these data were presented as the quartiles 1,
A. Paz-Silva et al. / Veterinary Parasitology 179 (2011) 277–282
279
Fig. 1. Variations on the cyathostomin egg-output and on the climatic parameters in the area of study (NW Spain).
2 and 3, and analyzed by using the 2 test. The differences among the groups were assessed by the Mann–Whitney U test. The values of reduction were expressed as percentages and the 95% confidence interval (CI). The Spearman’s rho test was employed to determine the correlation among the different parameters. Statistic significance was considered when P < 0.05. All tests were performed by the statistical package SPSS, version 15 (SPSS Inc.). 3. Results 3.1. Experiment I: annual variations of nematode egg-output and on climatic parameters Only cyathostomins (Cyathostomum and Gyalocephalus spp) were observed in the coprocultures. The lowest numbers of cyathostomin eggs in feces were observed in February–April, according to the highest rainfall record and reduced temperature values (Fig. 1). The greatest egg-output occurred from June to October, with the lowest values of rainfall and average temperatures above 15 ◦ C. Significant differences regarding the month in the numbers of eggs observed in feces were found (2 = 200.815, P = 0.001). 3.2. Experiment II: coprological results Table 1 summarizes the results of the coprological analysis. A statistical difference both in the EPG counts and in the mean of larvae per gram (LPG) in relation to the group of equines was observed (P < 0.05). The percentage of larvae developed in the control coprocultures ranged from 42% (95% CI 29, 55) in G-1 to 46% (35, 57) in G-3, but significant differences were not observed. The Spearman’s rho
test showed a significant positive correlation between the EPG and the LPG (Table 3). 3.3. Effect of the nematode egg-output on the efficacy of D. flagrans A ninety-four percent (91, 97) overall reduction of the L3 cyathostomin counts in the fecal pats with spores was obtained (Table 2). Low numbers of larvae were recovered in the coprocultures with D. flagrans (LPGDR) by using the four doses of spores, and no correlation to the EPG was observed (Table 3). Significant differences in the percentage of larval reduction (LR) according both to the egg-output (2 = 14.432, P = 0.001) and to the LPG (2 = 26.360, P = 0.001) were recorded. By means of the Mann–Whitney U test, the differences among the group G-1 and the others were established. The LR values rose together with the cyathostomin-egg burden, being the highest values in the fecal pats containing more than 800 EPG (G-3). A significant LR-correlation with the EPG and with the LPG was observed (Table 3). Significantly elevated LR percentages (>95%) were observed by using the highest dosages of spores (D3 and D4) (2 = 10.591, P = 0.005), and these differences were established among D1 and D2, and D3 and D4 by means of the Mann–Whitney U test. A chlamydospore dosedependent efficacy was recorded by a positive significant correlation between the dosage of chlamydospores and the percentage of reduction (Table 3). Nevertheless, the D. flagrans dose and the amount of the larval recovered (LPGDR) were negatively correlated. The simultaneous analysis of the effect of the eggoutput and the dose of spores indicated the lowest percentages of reduction in the cyathostomin 3rd stages when applying the minimal dose (0.1 × 106 ) to fecal pats with ≤300 EPG (Table 2), whereas the best results by
280
A. Paz-Silva et al. / Veterinary Parasitology 179 (2011) 277–282
Table 1 Values of cyathostomin egg-output (EPG), larvae in the control fecal pats (LPG) and larvae in the fecal pats with D. flagrans chlamydospores (LPGDR). Results are expressed as counts per gram of feces. The descriptive parameters were the quartiles 1, 2 and 3. G-1: fecal samples with ≤300 EPG; G-2: 310–800 EPG; G-3: >800 EPG. Egg-output
G-1 (n = 59)
G-2 (n = 52)
G-3 (n = 85)
Fecal pats EPG
Control (LPG)
Chlamydospores added (LPGDR)
Q1 Q2 Q3 Q1 Q2 Q3 Q1 Q2 Q3
230 280 290 500 690 767 1400 1640 1900
60 90 150 116 207 411 575 732 900
0 4 21 2 5 13 2 10 20
Statistics
2 = 169.511 P = 0.001
2 = 144.039 P = 0.001
2 = 41.371 P = 0.001
Table 2 Analysis of the efficacy of D. flagrans chlamydospores regarding the dosage assayed and the EPG counts. LR: percentage of reduction of L3 cyathostomins. G-1: fecal samples with ≤300 EPG; G-2: 310–800 EPG; G-3: >800 EPG. Group
Chlamydospores (106 /100 g feces)
G-1
G-2
G-3
Total
LR (%)
(95% CI)
LR (%)
(95% CI)
LR (%)
(95% CI)
LR (%)
(95% CI)
0.1 0.2 0.4 0.8
84 81 91 99
(69, 98) (53, 100) (79, 100) (92, 100)
92 95 97 99
(79, 100) (80, 100) (89, 100) (93, 100)
97 95 98 99
(91, 100) (86, 100) (93, 100) (96, 100)
91 92 95 99
(83, 98) (84, 100) (90, 100) (96, 100)
Total
89
(81, 97)
96
(90, 100)
97
(94, 100)
94
(91, 97)
Table 3 Analysis of the correlations among the different parameters established by using the Speraman’s rho test. CC: Spearman’s coefficient of correlation; EPG: counts of cyathostomin eggs per gram of feces; LPG: counts of L3 cyathostomins in the control fecal pats per gram of feces; LPGDR: counts of L3 cyathostomins in the fecal pats with D. flagrans chlamydospores. EPG Spearman’s rho Chlamydospores dosage
LR
LPGDR
CC P
0.065 0.361
0.365 0.001
−0.396 0.001
LPG
CC P
0.906 0.001
0.290 0.001
0.183 0.010
LPGDR
CC P
0.135 0.059
−0.828 0.001
LR
CC P
0.278 0.001
administering a 0.8x106 dose of D. flagrans spores to the coprocultures containing higher than 800 EPG (G-3) were achieved. 4. Discussion It is well known that the ingestion of pastures contaminated with L3 infective stages of gastrointestinal nematodes provokes the infection of the animals, so the prevention of these infections requires the action on the environment to reduce their presence (Pook et al., 2002). In the current research, cyathostomin specimens were only identified in the feces of the horses, and the observation of the greatest values of egg-output from June to October indicated the highest risk periods for infection in the
grazing horses were during spring turnout and late summer (Elsener and Villeneuve, 2009). By taking into account these results, a second experiment to establish the larvicidal activity of D. flagrans against parasite nematodes was developed between June and October 2009. In the present work, the efficacy of the D. flagrans on the reduction of the infective L3 cyathostomids ranged from 91% with a dosage of 0.1 × 106 chlamydospores to 99% by administering 0.8 × 106 spores. Several pasture hygiene procedures such as regular removal of feces have previously been pointed as an effective worm control approach resulting in reduced strongyle infection risk in horses (Herd and Coles, 1995). Nevertheless, removal of feces at least once or twice weekly is required to achieve an effect on worm burdens, depending
A. Paz-Silva et al. / Veterinary Parasitology 179 (2011) 277–282
on weather conditions, which reduces severely their practical use. Furthermore, it has recently reported the failure of this measure to reduce the strongyle fecal egg-counts in horses (von Samson-Himmelstjerna et al., 2009). Considering the cut-off values previous to establish the anthelmintic treatments (Nielsen et al., 2006; Uhlinger, 2007), in the present investigation horses were grouped into low (≤300 EPG), moderate (310–800) and high shedding levels (>800 EPG). The ability of D. flagrans to adapt to the nematode burden was demonstrated by the negative significant correlation between the percentage of larval reduction and the counts of larvae. The rise in the LR values together with the increment in the cyathostominegg-output, and the finding of highest LR values in the fecal pats containing more than 800 EPG support this record. Most of the measures for controlling the infection by nematodes are based on highly efficient chemotherapy, however the emergence of resistance in different countries has been highlighted (Traversa et al., 2007; Molento et al., 2008; Elsener and Villeneuve, 2009). Searching for biological procedures to decrease the use of anthelmintics against gastrointestinal nematode has involved great efforts (Baudena et al., 2000), with the main aim focused on preventive measures to remove free-living infective stages from pasture. This becomes especially important in areas with oceanic climate, as the present and others in the west coasts at the middle latitudes (40–60◦ N) of all the world continents and in southeastern Australia (Kottek et al., 2006), due to the occurrence of mild temperatures and frequent rainfall throughout the year can favor the survival of parasitic infective stages. Several investigations have shown the usefulness of D. flagrans to capture and destroy nematodes (Baudena et al., 2000), and thus these fungi should be used for removing the gastrointestinal nematode larvae from pastures were horses feed. In a recent report, the usefulness of an enzymatic extract of D. flagrans (isolate AC001) as an alternative for biological control of cyathostomin larvae has been proposed (Braga et al., 2010). Our data demonstrated that the addition of four doses (0.1, 0.2, 0.4 and 0.8 × 106 ) of D. flagrans chlamydospores directly onto fecal samples sited on the ground provides the successful elimination of almost all the larval infective stages of cyathostomins, preventing in consequence the infection of pasturing animals. The ability of the trapping fungus to adapt to the parasite burden has been also tested. Further studies are in progress to assess the extent of this effect and the frequency for administrating the chlamydospores.
Acknowledgements We are grateful to Mrs. B. Valcárcel for her contribution to the language corrections. This work was partly supported by the Autochthonous Government (Xunta de Galicia 07MDS021261PR & 10MDS261023PR), and conducted in agreement with the Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, (Jiutepec, Morelos, México), where Dr. A. Paz-Silva and Dr. I. Francisco have developed a scientific stay.
281
We thanked the participation of the PRG horse associations PURAGA (Muras, Lugo, Galicia), Cabalo de Pura ˜ Raza Galega (Boqueixón, A Coruna) “Granxa do Souto” ˜ (Ortigueira, A Coruna), and Grupoportichol (Muras, Lugo).
References Baudena, M.A., Chapman, M.R., Larsen, M., Klei, T.R., 2000. Efficacy of the nematophagous fungus Duddingtonia flagrans in reducing equine cyathostome larvae on pasture in south Louisiana. Vet. Parasitol. 89, 219–230. Braga, F.R., Araújo, J.V., Silva, A.R., Araújo, J.M., Carvalho, R.O., Tavela, A.O., Campos, A.K., Carvalho, G.R., 2009. Biological control of horse cyathostomin (Nematoda: Cyathostominae) using the nematophagous fungus Duddingtonia flagrans in tropical southeastern Brazil. Vet. Parasitol. 163, 335–340. Braga, F.R., Araújo, J.V., Soares, F.E., Araújo, J.M., Genier, H.L., Silva, A.R., Carvalho, R.O., Queiroz, J.H., Ferreira, S.R., 2010. Optimizing protease production from an isolate of the nematophagous fungus Duddingtonia flagrans using response surface methodology and its larvicidal activity on horse cyathostomins. J. Helminthol., doi:10.1017/S0022149X10000416. Campos, A.K., Araújo, J.V., Guimarães, M.P., Dias, A.S., 2009. Resistance of different fungal structures of Duddingtonia flagrans to the digestive process and predatory ability on larvae of Haemonchus contortus and Strongyloides papillosus in goat feces. Parasitol. Res. 105, 913–919. Casillas-Aguilar, J.A., Mendoza de Gives, P., López-Arellano, M.E., Hernández, L., 2008. Evaluation of multinutritional pellets containing Duddingtonia flagrans chlamydospore for the control of ovine haemonchosis. Animal biodiversity and emerging diseases. Ann. N. Y. Acad. Sci. 1149, 161–163. Elsener, J., Villeneuve, A., 2009. Comparative long-term efficacy of ivermectin and moxidectin over winter in Canadian horses treated at removal from pastures for winter housing. Can. Vet. J. 50, 486–490. ˜ Francisco, I., Arias, M., Cortinas, F.J., Francisco, R., Mochales, E., Sánchez, J.A., Suárez, J.L., Morrondo, P., Uriarte, J., Sánchez-Andrade, R., Díez˜ Banos, P., Paz-Silva, A., 2009. Silvopastoralism and autochthonous equine livestock. Analysis of the infection by endoparasites. Vet. Parasitol. 164, 357–362. Gómez-Rincón, C., Uriarte, J., Valderrábano, J., 2006. Efficiency of Duddingtonia flagrans against Trichostrongyle infections of sheep on mountain pastures. Vet. Parasitol. 141, 84–90. Herd, R.P., Coles, G.C., 1995. Slowing the spread of anthelmintic resistant nematodes of horses in the United Kingdom. Vet. Rec. 136, 481–485. Kottek, M., Grieser, J., Beck, C., Rudolf, B., Rubel, F., 2006. World map of the Köppen–Geiger climate classification updated. Meteorol. Z. 15, 259–263. Kuzmina, T.A., Kuzmin, Y.I., Kharchenko, V.A., 2006. Field study on the survival, migration and overwintering of infective larvae of horse strongyles on pasture in central Ukraine. Vet. Parasitol. 141, 264–272. Lichtenfels, J.R., Kharchenko, V.A., Krecek, R.C., Gibbons, L.M., 1998. An annotated checklist by genus and species of 93 species level names for 51 recognized species of small strongyles (Nematoda: Strongyloidea: Cyathostominae) of horses, asses and zebras of the world. Vet. Parasitol. 79, 65–79. Llerandi-Juárez, R.D., Mendoza de Gives, P., 1998. Resistance of chlamydospores of nematophagous fungi to the digestive processes of sheep. J. Helminthol. 72, 209–213. Maciel, A.S., Freitas, L.G., Campos, A.K., Lopes, E., Araújo, J.V., 2010. The biological control of Ancylostoma spp. dog infective larvae by Duddingtonia flagrans in a soil microcosm. Vet. Parasitol. 173, 262–270. MAFF, 1986. Manual of Veterinary Parasitological Laboratory Techniques. Ministry of Agriculture, Fisheries and Food, HMSO, London, UK. Mendoza de Gives, P., Zapata Nieto, C., Hernández, E.L., Arellano, M.E., ˜ R.G., 2006. Biological control of gastroinRodríguez, D.H., Garduno, testinal parasitic nematodes using Duddingtonia flagrans in sheep under natural conditions in Mexico. Ann. N. Y. Acad. Sci. 1081, 355–359. Molento, M.B., Antunes, J., Bentes, R.N., Coles, G.C., 2008. Anthelmintic resistant nematodes in Brazilian horses. Vet. Rec. 162, 384–385. Nielsen, M.K., Monrad, J., Olsen, S.N., 2006. Prescription-only anthelmintics—a questionnaire survey of strategies for surveillance and control of equine strongyles in Denmark. Vet. Parasitol. 135, 47–55. Osterman Lind, E., Höglund, J., Ljungström, B.L., Nilsson, O., Uggla, A., 1999. A field survey on the distribution of strongyle infections of horses in Sweden and factors affecting faecal egg counts. Eq. Vet. J. 31, 68–72.
282
A. Paz-Silva et al. / Veterinary Parasitology 179 (2011) 277–282
Pook, J.E., Power, M.L., Sangster, N.C., Hodgson, J.L., Hodgson, D.R., 2002. Evaluation of tests for anthelmintic resistance in cyathostomes. Vet. Parasitol. 106, 331–343. Silva, B.F., Mauad, J.R.C., Braga, F.R., Amarante, A.F.T., Araújo, J.V., Campos, A.K., 2010. Efficacy of Duddingtonia flagrans and Arthrobotrys robusta in controlling sheep parasitic gastroenteritis. Parasitol. Res. 106, 1346–1350. Tavela Ade, O., Araújo, J.V., Braga, F.R., Silva, A.R., Carvalho, R.O., Araújo, J.M., Ferreira, S.R., Carvalho, G.R., 2011. Biological control of cyathostomin (Nematoda: Cyathostominae) with nematophagous fungus Monacrosporium thaumasium in tropical southeastern Brazil. Vet. Parasitol. 175, 92–96.
Traversa, D., Klei, T.R., Iorio, R., Paoletti, B., Lia, R.P., Otranto, D., Sparagano, O.A., Giangaspero, A., 2007. Occurrence of anthelmintic resistant equine cyathostome populations in central and southern Italy. Prev. Vet. Med. 82, 314–320. Uhlinger, C.A., 2007. Evidence-based parasitology in horses. Vet. Clin. North Am. Equine Pract. 23, 509–517. von Samson-Himmelstjerna, G., Traversa, D., Demeler, J., Rohn, K., Milillo, P., Schurmann, S., Lia, R., Perrucci, S., di Regalbono, A.F., Beraldo, P., Barnes, H., Cobb, R., Boeckh, A., 2009. Effects of worm control practices examined by a combined faecal egg count and questionnaire survey on horse farms in Germany, Italy and the UK. Parasit. Vectors Suppl. 2, S3.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Ability of the fungus Duddingtonia flagrans to adapt to the cyathostomin egg-output by spreading chlamydospores ˜ a , J.A. Sánchez a , R. Francisco a , A. Paz-Silva a,∗ , I. Francisco a , R.O. Valero-Coss b , F.J. Cortinas a a b M. Arias , J.L. Suárez , M.E. López-Arellano , R. Sánchez-Andrade a , P. Mendoza de Gives b , Equine Diseases Study Group (Epidemiology, Parasitology and Zoonoses) a
Animal Pathology Department, Faculty of Veterinary, University of Santiago de Compostela, 27002-Lugo, Spain Área de Helmintología, Centro Nacional de Investigación Disciplinaria en Parasitología Veterinaria, Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, Paseo Cuaunahuac 8534, Jiutepec, Morelos-62550, Mexico
b
a r t i c l e
i n f o
Article history: Received 8 September 2010 Received in revised form 9 February 2011 Accepted 17 February 2011 Keywords: Nematodes Duddingtonia flagrans Biological control Grazing horses Nematophagous fungi
a b s t r a c t The analysis of the capability of the nematode trapping-fungus Duddingtonia flagrans to adapt to the cyathostomin egg-output in horses was evaluated. Fecal samples from 196 pasturing autochthonous Pura Raza Galega horses were collected from the rectum and then divided according to the egg-output into three groups: ≤300, 310–800 and >800 eggs per gram feces. Four doses of chlamydospores (0.1, 0.2, 0.4 and 0.8 × 106 /100 g feces) were directly spread onto fecal pats on the ground, remaining one without treatment as control. Fecal pats confirmed the presence of gastrointestinal nematode larvae belonging to strongylid cyathostomins (Cyathostomum and Gyalocephalus spp). An overall 94% (95% CI 91, 97) percentage of reduction was obtained, and an increase in the activity of the trappingfungi simultaneously to the rising in the number of cyathostomin eggs and larvae in the coprocultures was detected. A significantly highest reduction of the cyathostomin L3 in the coprocultures with more than 800 EPG was found, which indicates that Df trapping activity is larvae nematode density-dependant. The present research showed the high biological activity of D. flagrans against nematode larvae can adjust to the cyathostomin egg-output, and underlines its efficacy as a practical method for the control of these parasites in grazing horses. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Some livestock managing involve animals under pasturing conditions, but the possibility for infection by gastrointestinal nematode parasites is notably enhanced under this regime (Francisco et al., 2009). Larvae belonging to several genera (Strongylus, Triodontophorus, Gyalocephalus, Trichonema, Ostertagia, Trichostrongylus, Haemonchus, Chabertia or Oesophagostomum) exit the egg
∗ Corresponding author. Tel.: +34 982285900x22126; fax: +34 982252195. E-mail address: [email protected] (A. Paz-Silva). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.02.014
and develop in the environment to attain the infective stage (L3), which are ingested when the animals feed the grass. These parasitic infections reduce the benefits and the inputs afforded (poor gain weight, low reproductive indexes), being chemotherapy the most employed procedure for their control. Duddingtonia flagrans is a nematophagous fungus which develops traps to capture nematodes present in the soil (Mendoza de Gives et al., 2006). Successful results have been achieved to reduce gastrointestinal nematode infections in horses, calves and sheep under northern temperate, tropical or subtropical climates (Gómez-Rincón et al., 2006; Braga et al., 2009; Campos et al., 2009; Maciel et al., 2010; Silva et al., 2010). This fungus is capable to produce a
278
A. Paz-Silva et al. / Veterinary Parasitology 179 (2011) 277–282
large quantity of chlamydospores able to resist the passage through the gastrointestinal tract of ruminants and monogastrics, hence different investigations regarding the oral administration of aqueous suspensions have been carried out (Baudena et al., 2000). This practice requires the correct immobilization of the animals, thus the use of multinutritional pellets containing spores of the nematode-trapping fungi D. flagrans and Monacrosporium thaumasium as vehicles has been checked in sheep (Casillas-Aguilar et al., 2008) and horses (Tavela Ade et al., 2011). In the last decades an increasing tendency to the abandonment of productive lands has been observed in many European countries. Under the requirement for keeping these properties free of shrubs and bushes and thus for reducing the risk for fire, the presence of 1–2 animals (mainly horses and then donkeys, sheep and goats) is becoming usual for making advantage of <0.5 ha pastures. There is a lack of information about the efficacy of the addition of the chlamydospores directly onto the pasture. The possible influence of the numbers of eggs passed by feces on the efficacy of D. flagrans spores to form trapping devices in feces remains also untested. In the current work, the utility of the direct administration of spores on the pasture was evaluated, and their ability to adapt to different cyathostomin egg-output numbers also. An annual coprological survey was conducted to get a useful knowledge about the risk periods for infection in horses.
By taking into account previous investigations concerning the cut-off egg-output values to establish the anthelmintic treatments the horses were grouped in the basis of the counts of strongyle eggs per grams (EPG): G-1 (≤300 EPG), G-2 (310–800), G-3 (>800 EPG) (Nielsen et al., 2006; Uhlinger, 2007).
2. Materials and methods
2.3.2. D. flagrans chlamydospore production The Mexican strain of D. flagrans FTHO-8 (CENIDPAVET, INIFAP-MEXICO) was used and chlamydospores were produced, harvested and managed according to Llerandi-Juárez and Mendoza de Gives (1998).
2.1. Area of study The current research was developed in SW Europe (Galicia, Spain) (42◦ 20 –43◦ 45 N, 6◦ 49 –8◦ 00 W), an agricultural area where cattle livestock represents the main farm activity. 2.2. Annual variations of nematode egg-output and on climatic parameters To determine the kinetics of nematode egg-output and the highest risk periods for infection, a herd of 25 indigenous Pura Raza Galega (PRG) horses in a farm in Lugo (NW Spain) was monthly sampled throughout 2008. Data corresponding to the maximal temperature, minimal temperature, rainfall, and relative humidity were monthly obtained from 2 automated meteorological stations to establish the climatic pattern. All the management of the animals has been carried out in accordance with the EC Directive 86/609/EEC for animal experiments. 2.3. Design of the study of the efficacy of D. flagrans According to the results from the previous experience, fecal samples were collected between June and October 2009 from 196 indigenous Pura Raza Galega (PRG) grazing horses in several farms located at different sites interspersed within this area, and the distance among the farms is 20–100 km approx.
2.3.1. Coprological techniques Fecal samples were individually collected from the rectum of the horses and analyzed by the coprological flotation method. Five grams of each fecal sample were processed (by duplicate) by using the copromicroscopical flotation technique (MAFF, 1986), with a sensitivity of 10 eggs per gram of feces. The counts of nematode eggs were expressed as counts of EPG. The microscope analysis of fecal samples was performed and additional blind samples were run to diminish possible technique errors. To gain more information about the different genera/specie of the strongyles affecting the horses, fecal samples were cultured for 10–15 days at 22–24 ◦ C to allow the development of eggs to infective larvae. Once larvae showed the typical morphological features of L3, they were collected by means of the Baermann procedure (Osterman Lind et al., 1999; Kuzmina et al., 2006) and taxonomically identified according to Lichtenfels et al. (1998). The counts were expressed as numbers of larvae/gram (LPG).
2.3.3. Efficacy of D. flagrans The efficacy of D. flagrans against nematode larvae was measured by comparing the number of L3 recovered from fecal pats with (LPGDR) and without D. flagrans chlamydospores (LPG). For every horse, three pats were prepared by leaving 50 g feces/each in plastic trays (50 cm × 50 cm) placed in the pastures fed by the horses. The spores of D. flagrans were diluted in an aqueous solution and added to two pats, and the other remained as control. Four doses of 0.1 (D1), 0.2 (D2), 0.4 (D3) and 0.8 × 106 (D4) spores/100 g feces were employed. Three replicates were considered in all cases. The efficacy in the larval reduction by the fungal action was determined as follows: Larval reduction (LR %) =
control mean L3 − fungus mean L3 control mean L3
× 100
Twelve days after the addition of the chlamydospores, the pats were collected and analyzed by using the flotation and the Baermann techniques. 2.4. Statistical analysis Considering that EPG and LPG counts are not normally distributed, these data were presented as the quartiles 1,
A. Paz-Silva et al. / Veterinary Parasitology 179 (2011) 277–282
279
Fig. 1. Variations on the cyathostomin egg-output and on the climatic parameters in the area of study (NW Spain).
2 and 3, and analyzed by using the 2 test. The differences among the groups were assessed by the Mann–Whitney U test. The values of reduction were expressed as percentages and the 95% confidence interval (CI). The Spearman’s rho test was employed to determine the correlation among the different parameters. Statistic significance was considered when P < 0.05. All tests were performed by the statistical package SPSS, version 15 (SPSS Inc.). 3. Results 3.1. Experiment I: annual variations of nematode egg-output and on climatic parameters Only cyathostomins (Cyathostomum and Gyalocephalus spp) were observed in the coprocultures. The lowest numbers of cyathostomin eggs in feces were observed in February–April, according to the highest rainfall record and reduced temperature values (Fig. 1). The greatest egg-output occurred from June to October, with the lowest values of rainfall and average temperatures above 15 ◦ C. Significant differences regarding the month in the numbers of eggs observed in feces were found (2 = 200.815, P = 0.001). 3.2. Experiment II: coprological results Table 1 summarizes the results of the coprological analysis. A statistical difference both in the EPG counts and in the mean of larvae per gram (LPG) in relation to the group of equines was observed (P < 0.05). The percentage of larvae developed in the control coprocultures ranged from 42% (95% CI 29, 55) in G-1 to 46% (35, 57) in G-3, but significant differences were not observed. The Spearman’s rho
test showed a significant positive correlation between the EPG and the LPG (Table 3). 3.3. Effect of the nematode egg-output on the efficacy of D. flagrans A ninety-four percent (91, 97) overall reduction of the L3 cyathostomin counts in the fecal pats with spores was obtained (Table 2). Low numbers of larvae were recovered in the coprocultures with D. flagrans (LPGDR) by using the four doses of spores, and no correlation to the EPG was observed (Table 3). Significant differences in the percentage of larval reduction (LR) according both to the egg-output (2 = 14.432, P = 0.001) and to the LPG (2 = 26.360, P = 0.001) were recorded. By means of the Mann–Whitney U test, the differences among the group G-1 and the others were established. The LR values rose together with the cyathostomin-egg burden, being the highest values in the fecal pats containing more than 800 EPG (G-3). A significant LR-correlation with the EPG and with the LPG was observed (Table 3). Significantly elevated LR percentages (>95%) were observed by using the highest dosages of spores (D3 and D4) (2 = 10.591, P = 0.005), and these differences were established among D1 and D2, and D3 and D4 by means of the Mann–Whitney U test. A chlamydospore dosedependent efficacy was recorded by a positive significant correlation between the dosage of chlamydospores and the percentage of reduction (Table 3). Nevertheless, the D. flagrans dose and the amount of the larval recovered (LPGDR) were negatively correlated. The simultaneous analysis of the effect of the eggoutput and the dose of spores indicated the lowest percentages of reduction in the cyathostomin 3rd stages when applying the minimal dose (0.1 × 106 ) to fecal pats with ≤300 EPG (Table 2), whereas the best results by
280
A. Paz-Silva et al. / Veterinary Parasitology 179 (2011) 277–282
Table 1 Values of cyathostomin egg-output (EPG), larvae in the control fecal pats (LPG) and larvae in the fecal pats with D. flagrans chlamydospores (LPGDR). Results are expressed as counts per gram of feces. The descriptive parameters were the quartiles 1, 2 and 3. G-1: fecal samples with ≤300 EPG; G-2: 310–800 EPG; G-3: >800 EPG. Egg-output
G-1 (n = 59)
G-2 (n = 52)
G-3 (n = 85)
Fecal pats EPG
Control (LPG)
Chlamydospores added (LPGDR)
Q1 Q2 Q3 Q1 Q2 Q3 Q1 Q2 Q3
230 280 290 500 690 767 1400 1640 1900
60 90 150 116 207 411 575 732 900
0 4 21 2 5 13 2 10 20
Statistics
2 = 169.511 P = 0.001
2 = 144.039 P = 0.001
2 = 41.371 P = 0.001
Table 2 Analysis of the efficacy of D. flagrans chlamydospores regarding the dosage assayed and the EPG counts. LR: percentage of reduction of L3 cyathostomins. G-1: fecal samples with ≤300 EPG; G-2: 310–800 EPG; G-3: >800 EPG. Group
Chlamydospores (106 /100 g feces)
G-1
G-2
G-3
Total
LR (%)
(95% CI)
LR (%)
(95% CI)
LR (%)
(95% CI)
LR (%)
(95% CI)
0.1 0.2 0.4 0.8
84 81 91 99
(69, 98) (53, 100) (79, 100) (92, 100)
92 95 97 99
(79, 100) (80, 100) (89, 100) (93, 100)
97 95 98 99
(91, 100) (86, 100) (93, 100) (96, 100)
91 92 95 99
(83, 98) (84, 100) (90, 100) (96, 100)
Total
89
(81, 97)
96
(90, 100)
97
(94, 100)
94
(91, 97)
Table 3 Analysis of the correlations among the different parameters established by using the Speraman’s rho test. CC: Spearman’s coefficient of correlation; EPG: counts of cyathostomin eggs per gram of feces; LPG: counts of L3 cyathostomins in the control fecal pats per gram of feces; LPGDR: counts of L3 cyathostomins in the fecal pats with D. flagrans chlamydospores. EPG Spearman’s rho Chlamydospores dosage
LR
LPGDR
CC P
0.065 0.361
0.365 0.001
−0.396 0.001
LPG
CC P
0.906 0.001
0.290 0.001
0.183 0.010
LPGDR
CC P
0.135 0.059
−0.828 0.001
LR
CC P
0.278 0.001
administering a 0.8x106 dose of D. flagrans spores to the coprocultures containing higher than 800 EPG (G-3) were achieved. 4. Discussion It is well known that the ingestion of pastures contaminated with L3 infective stages of gastrointestinal nematodes provokes the infection of the animals, so the prevention of these infections requires the action on the environment to reduce their presence (Pook et al., 2002). In the current research, cyathostomin specimens were only identified in the feces of the horses, and the observation of the greatest values of egg-output from June to October indicated the highest risk periods for infection in the
grazing horses were during spring turnout and late summer (Elsener and Villeneuve, 2009). By taking into account these results, a second experiment to establish the larvicidal activity of D. flagrans against parasite nematodes was developed between June and October 2009. In the present work, the efficacy of the D. flagrans on the reduction of the infective L3 cyathostomids ranged from 91% with a dosage of 0.1 × 106 chlamydospores to 99% by administering 0.8 × 106 spores. Several pasture hygiene procedures such as regular removal of feces have previously been pointed as an effective worm control approach resulting in reduced strongyle infection risk in horses (Herd and Coles, 1995). Nevertheless, removal of feces at least once or twice weekly is required to achieve an effect on worm burdens, depending
A. Paz-Silva et al. / Veterinary Parasitology 179 (2011) 277–282
on weather conditions, which reduces severely their practical use. Furthermore, it has recently reported the failure of this measure to reduce the strongyle fecal egg-counts in horses (von Samson-Himmelstjerna et al., 2009). Considering the cut-off values previous to establish the anthelmintic treatments (Nielsen et al., 2006; Uhlinger, 2007), in the present investigation horses were grouped into low (≤300 EPG), moderate (310–800) and high shedding levels (>800 EPG). The ability of D. flagrans to adapt to the nematode burden was demonstrated by the negative significant correlation between the percentage of larval reduction and the counts of larvae. The rise in the LR values together with the increment in the cyathostominegg-output, and the finding of highest LR values in the fecal pats containing more than 800 EPG support this record. Most of the measures for controlling the infection by nematodes are based on highly efficient chemotherapy, however the emergence of resistance in different countries has been highlighted (Traversa et al., 2007; Molento et al., 2008; Elsener and Villeneuve, 2009). Searching for biological procedures to decrease the use of anthelmintics against gastrointestinal nematode has involved great efforts (Baudena et al., 2000), with the main aim focused on preventive measures to remove free-living infective stages from pasture. This becomes especially important in areas with oceanic climate, as the present and others in the west coasts at the middle latitudes (40–60◦ N) of all the world continents and in southeastern Australia (Kottek et al., 2006), due to the occurrence of mild temperatures and frequent rainfall throughout the year can favor the survival of parasitic infective stages. Several investigations have shown the usefulness of D. flagrans to capture and destroy nematodes (Baudena et al., 2000), and thus these fungi should be used for removing the gastrointestinal nematode larvae from pastures were horses feed. In a recent report, the usefulness of an enzymatic extract of D. flagrans (isolate AC001) as an alternative for biological control of cyathostomin larvae has been proposed (Braga et al., 2010). Our data demonstrated that the addition of four doses (0.1, 0.2, 0.4 and 0.8 × 106 ) of D. flagrans chlamydospores directly onto fecal samples sited on the ground provides the successful elimination of almost all the larval infective stages of cyathostomins, preventing in consequence the infection of pasturing animals. The ability of the trapping fungus to adapt to the parasite burden has been also tested. Further studies are in progress to assess the extent of this effect and the frequency for administrating the chlamydospores.
Acknowledgements We are grateful to Mrs. B. Valcárcel for her contribution to the language corrections. This work was partly supported by the Autochthonous Government (Xunta de Galicia 07MDS021261PR & 10MDS261023PR), and conducted in agreement with the Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, (Jiutepec, Morelos, México), where Dr. A. Paz-Silva and Dr. I. Francisco have developed a scientific stay.
281
We thanked the participation of the PRG horse associations PURAGA (Muras, Lugo, Galicia), Cabalo de Pura ˜ Raza Galega (Boqueixón, A Coruna) “Granxa do Souto” ˜ (Ortigueira, A Coruna), and Grupoportichol (Muras, Lugo).
References Baudena, M.A., Chapman, M.R., Larsen, M., Klei, T.R., 2000. Efficacy of the nematophagous fungus Duddingtonia flagrans in reducing equine cyathostome larvae on pasture in south Louisiana. Vet. Parasitol. 89, 219–230. Braga, F.R., Araújo, J.V., Silva, A.R., Araújo, J.M., Carvalho, R.O., Tavela, A.O., Campos, A.K., Carvalho, G.R., 2009. Biological control of horse cyathostomin (Nematoda: Cyathostominae) using the nematophagous fungus Duddingtonia flagrans in tropical southeastern Brazil. Vet. Parasitol. 163, 335–340. Braga, F.R., Araújo, J.V., Soares, F.E., Araújo, J.M., Genier, H.L., Silva, A.R., Carvalho, R.O., Queiroz, J.H., Ferreira, S.R., 2010. Optimizing protease production from an isolate of the nematophagous fungus Duddingtonia flagrans using response surface methodology and its larvicidal activity on horse cyathostomins. J. Helminthol., doi:10.1017/S0022149X10000416. Campos, A.K., Araújo, J.V., Guimarães, M.P., Dias, A.S., 2009. Resistance of different fungal structures of Duddingtonia flagrans to the digestive process and predatory ability on larvae of Haemonchus contortus and Strongyloides papillosus in goat feces. Parasitol. Res. 105, 913–919. Casillas-Aguilar, J.A., Mendoza de Gives, P., López-Arellano, M.E., Hernández, L., 2008. Evaluation of multinutritional pellets containing Duddingtonia flagrans chlamydospore for the control of ovine haemonchosis. Animal biodiversity and emerging diseases. Ann. N. Y. Acad. Sci. 1149, 161–163. Elsener, J., Villeneuve, A., 2009. Comparative long-term efficacy of ivermectin and moxidectin over winter in Canadian horses treated at removal from pastures for winter housing. Can. Vet. J. 50, 486–490. ˜ Francisco, I., Arias, M., Cortinas, F.J., Francisco, R., Mochales, E., Sánchez, J.A., Suárez, J.L., Morrondo, P., Uriarte, J., Sánchez-Andrade, R., Díez˜ Banos, P., Paz-Silva, A., 2009. Silvopastoralism and autochthonous equine livestock. Analysis of the infection by endoparasites. Vet. Parasitol. 164, 357–362. Gómez-Rincón, C., Uriarte, J., Valderrábano, J., 2006. Efficiency of Duddingtonia flagrans against Trichostrongyle infections of sheep on mountain pastures. Vet. Parasitol. 141, 84–90. Herd, R.P., Coles, G.C., 1995. Slowing the spread of anthelmintic resistant nematodes of horses in the United Kingdom. Vet. Rec. 136, 481–485. Kottek, M., Grieser, J., Beck, C., Rudolf, B., Rubel, F., 2006. World map of the Köppen–Geiger climate classification updated. Meteorol. Z. 15, 259–263. Kuzmina, T.A., Kuzmin, Y.I., Kharchenko, V.A., 2006. Field study on the survival, migration and overwintering of infective larvae of horse strongyles on pasture in central Ukraine. Vet. Parasitol. 141, 264–272. Lichtenfels, J.R., Kharchenko, V.A., Krecek, R.C., Gibbons, L.M., 1998. An annotated checklist by genus and species of 93 species level names for 51 recognized species of small strongyles (Nematoda: Strongyloidea: Cyathostominae) of horses, asses and zebras of the world. Vet. Parasitol. 79, 65–79. Llerandi-Juárez, R.D., Mendoza de Gives, P., 1998. Resistance of chlamydospores of nematophagous fungi to the digestive processes of sheep. J. Helminthol. 72, 209–213. Maciel, A.S., Freitas, L.G., Campos, A.K., Lopes, E., Araújo, J.V., 2010. The biological control of Ancylostoma spp. dog infective larvae by Duddingtonia flagrans in a soil microcosm. Vet. Parasitol. 173, 262–270. MAFF, 1986. Manual of Veterinary Parasitological Laboratory Techniques. Ministry of Agriculture, Fisheries and Food, HMSO, London, UK. Mendoza de Gives, P., Zapata Nieto, C., Hernández, E.L., Arellano, M.E., ˜ R.G., 2006. Biological control of gastroinRodríguez, D.H., Garduno, testinal parasitic nematodes using Duddingtonia flagrans in sheep under natural conditions in Mexico. Ann. N. Y. Acad. Sci. 1081, 355–359. Molento, M.B., Antunes, J., Bentes, R.N., Coles, G.C., 2008. Anthelmintic resistant nematodes in Brazilian horses. Vet. Rec. 162, 384–385. Nielsen, M.K., Monrad, J., Olsen, S.N., 2006. Prescription-only anthelmintics—a questionnaire survey of strategies for surveillance and control of equine strongyles in Denmark. Vet. Parasitol. 135, 47–55. Osterman Lind, E., Höglund, J., Ljungström, B.L., Nilsson, O., Uggla, A., 1999. A field survey on the distribution of strongyle infections of horses in Sweden and factors affecting faecal egg counts. Eq. Vet. J. 31, 68–72.
282
A. Paz-Silva et al. / Veterinary Parasitology 179 (2011) 277–282
Pook, J.E., Power, M.L., Sangster, N.C., Hodgson, J.L., Hodgson, D.R., 2002. Evaluation of tests for anthelmintic resistance in cyathostomes. Vet. Parasitol. 106, 331–343. Silva, B.F., Mauad, J.R.C., Braga, F.R., Amarante, A.F.T., Araújo, J.V., Campos, A.K., 2010. Efficacy of Duddingtonia flagrans and Arthrobotrys robusta in controlling sheep parasitic gastroenteritis. Parasitol. Res. 106, 1346–1350. Tavela Ade, O., Araújo, J.V., Braga, F.R., Silva, A.R., Carvalho, R.O., Araújo, J.M., Ferreira, S.R., Carvalho, G.R., 2011. Biological control of cyathostomin (Nematoda: Cyathostominae) with nematophagous fungus Monacrosporium thaumasium in tropical southeastern Brazil. Vet. Parasitol. 175, 92–96.
Traversa, D., Klei, T.R., Iorio, R., Paoletti, B., Lia, R.P., Otranto, D., Sparagano, O.A., Giangaspero, A., 2007. Occurrence of anthelmintic resistant equine cyathostome populations in central and southern Italy. Prev. Vet. Med. 82, 314–320. Uhlinger, C.A., 2007. Evidence-based parasitology in horses. Vet. Clin. North Am. Equine Pract. 23, 509–517. von Samson-Himmelstjerna, G., Traversa, D., Demeler, J., Rohn, K., Milillo, P., Schurmann, S., Lia, R., Perrucci, S., di Regalbono, A.F., Beraldo, P., Barnes, H., Cobb, R., Boeckh, A., 2009. Effects of worm control practices examined by a combined faecal egg count and questionnaire survey on horse farms in Germany, Italy and the UK. Parasit. Vectors Suppl. 2, S3.