Зборник Матице српске за природне науке / Proc. Nat. Sci, Matica Srpska Novi Sad, 120, 243 248, 2011 UDC 636.371 DOI: 10.2298/ZMSPN1120245L Vesna G. Lalošević, Mirjana N. Jarak, Simonida S. Đ urić, Nemanja S. Obradović Faculty of Agriculture, Trg D. Obradovića 8, Novi Sad, Serbia THE EFFECT OF NEMATOPHAGOUS FUNGUS DUDDINGTONIA FLAGRANS ON THE GASTROINTESTINAL PARASITES IN SHEEP ABSTRACT: Sheep production has serious problems due to the spread of intestinal parasites. These parasites cause loss of appetite, maldigestion, slow growth in body weight and wool, all of which results in economic losses as well. The control measures of infestation with strongyloid parasites in ruminants have until now been based mainly on the organization of grazing and the use of antihelmintics. However, due to the occurrence of resistance, alternative methods of control have been introduced. The use of nematophagous fungus Duddingtonia flagrans, which is capable of decreasing the number of infectious larvae and eggs in feces, has been successful. The aim of this study was to determine whether Duddingtonia flagrans decreases the number of eggs of Trichostrongylus spp in sheep feces. Fecal samples of thirty-four sheep were examined and the parasites were found in twelve sheep, six of which were fed with the fungus, and six of which were used as the control. According to χ 2 test, at the level of certainty of p<0,005, a statistically important difference in the number of eggs was observed between the sheep which were given the fungus and those which were not. KEY WORDS: Duddingtonia flagrans, sheep, gastrointestinal parasite, biological control INTRODUCTION Sheep production has serious problems due to the spread of intestinal parasites. These parasites cause loss of appetite, maldigestion, slow growth in body weight and wool, all of which results in economic losses as well. The control measures of infestation with strongyloid parasites in ruminants have so far been mainly based on the organisation of grazing and the use of antihelmintics. However, due to the occurrence of resistance, alternative methods of control have been introduced. Grazing systems are often impractical and expensive, whereas frequent use of antihelmintics, which has been the main principle of therapy over the last fifty years, develops resistance of parasites to antiparasintics, especially significant in small ruminants, sheep and goats 245
(C o n d e r and J o h n s o n, 1995). Not less important aspect is the increase of consumers demand for animal products without residua of drugs (D i e z T a s c o n et al., 2005; L a l o š e v i ć et al., 2009). For these reasons, increasing number of alternative control methods is being introduced. The most promising one is the use of nematophagous fungus which is capable of reducing pasture contamination and the occurrence of infection (L a r s e n, 2000). In recent years, nematode-destroying, or nematophagous fungi, has become focus of attention of many scientists around the world. They live in different cracks in land and rhizosphere where they feed on different free-living nematodes and dead organic matter. Among the most important and the most frequently investigated fungi are: Duddingtonia flagrans, Monacrosporium spp., Arthrobotrys spp., Pochonia chlamydosporia (C a r v a l h o et al., 2009). A kind of a predatory fungus which attracts the greatest attention is Duddingtonia flagrans (L a r s e n et al., 1997). Modern research shows that D. flagrans spores are capable of passing through the gastrointestinal tract of sheep and lessening the number of infectious larvae in feces, which was proved in the research of pasture contamination during spring grazing season (G o m e z R i n c o n et al., 2006). Duddingtonia flagrans reduces the population of larvae on pastures which later results in a smaller number of parasites in animals up to the level where not only clinical signs but also subclinical effects caused by parasites will be prevented. At the same time, reducing the number of infective larvae on pastures should stimulate the development of naturally acquired immunity in young animals. In the fresh feces, Duddingtonia flagrans forms a three-dimensional net which leads either to egg destruction or larvae destruction (A r a u j o et al., 2009). Besides ruminants, the positive effect of Duddingtonia flagrans was observed in the control of intestinal parasites of pigs (Ascaris suum), and in nematodes of carnivores (Ancylostoma caninum) (L a r s e n et al., 1997, A r a u j o et al., 2009). The aim of the study was to determine the effect of nematophagous fungus Duddingtonia flagrans on the gastrointestinal strongyloids, Trichostrongylus spp. in naturally infected sheep. 246 MATERIALS AND METHODS This research was conducted in the Laboratory for Parasitology, Department of veterinary medicine, Faculty of Agriculture in Novi Sad. Feces samples were collected in the period from July to September 2010 from sheep in Zrenjanin municipality, raised freely on pastures. Before the beginning of the experiment, stool samples were examined and natural infection by Trichostrongylus was found in all animals. Sheep were divided into two groups. The experimental group of six sheep was perorally given 17 ml of water solution of fungus (about 1x10 6 /ml chlamidospora) for five days. The control group of six animals did not receive the fungus suspension. Fecal samples were collected every week in the period of three weeks. The samples were examined by the method of helmint egg concentration with
a saturated solution of saccharose, and the number of eggs per gram of feces was calculated by the method of Stoll. Duddingtonia flagrans (from the collection of Universite catholique de Louvian, Belgium) was multiplied on PDA base (potato dextrose agar) in the Laboratory for Microbiology at the Faculty of Agriculture in Novi Sad. RESULTS AND DISCUSSION With the aim of determining the presence of parasites from the genus Trichostrongylus spp., thirty-four feces samples were examined, and eggs of parasites were found in twelve sheep (35.9%), six of which were fed with the fungus (experimental group), and six of which were used as the control group. One sheep from the control group died during the research. Feces samples were taken from the sheep which had received the nematophagous fungi for three weeks in the amount of 20 grams. A statistically important difference in the number of eggs was observed between the sheep which received the fungus and those which did not. According to a research conducted in Malaysia, already after the second day of the fungus intake, the decrease in larvae development in pastures was over 90%, and when the dose was doubled, the decrease was even 100% (C h a n - d r a w a t h a n i et al., 2003.). A group of Spanish and Brazilian authors discovered that if sheep are fed with spores of fungus Duddingtonia flagrans at the right moment, from the point of view of epizootiology, this can have an important effect on infectious larvae on pastures, which was proved by finding 20% fewer parasites in lambs grazing for three weeks on the same pasture in spring (G o m e z R i n c o n et al., 2006). Apart from the decreased spring contamination of the pasture, the lambs of the treated sheep had smaller total number of parasites and better growth than the lambs from the control group (Table 1). Tab. 1 The number of Trichostrongylus spp. eggs after one, two and three weeks after use of D. flagrans Sheep number. 0 day number 1. week number 2. week number 3. week number 1. experimental 5700 11200 3000 400 2. experimental 5200 3100 1700 600 3. experimental 100 100 200 0 7. experimental 4000 0 200 200 8. experimental 4700 1200 900 100 9. experimental 300 800 0 0 4. control 13700 6300 8600 300 5. control 9200 100 800 0 6. control 1900 died 10. control 500 1100 200 0 11. control 1500 200 1300 800 12. control 500 0 100 0 247
A positive effect of Duddingtonia flagrans was also noticed in the control of intestinal parasites of pigs, which was confirmed by both in vitro experiments and on pastures (L a r s e n et al., 1997). High potential of the fungus to reduce the number of larvae of pulmonary strongyloid Dictyocaulus viviparus was noted in during a lab research, and the already mentioned results against gastrointestinal strongyloids show a wide scope of parasitic worms which can be controlled by this method (L a r s e n et al., 1997, S a r k ū n a s et al., 2000). Fig. 1 Trichostrongylus spp, egg, sheep feces Fig. 2 D. flagrans, chlamidospora It appears that the passing of fungus spores through digestive tract depends on factors related to the nutrition and food flow in the animal, but a good sign is that spores of Duddingtina flagrans can survive in the digestive tract for a significant period of time under difficult conditions (C h a n d r a w a t h a n i et al., 2003). It is important to point out that during the implementation of this measure in the strongyloid control, it should be taken into account that some antihelmintics can have potentially negative or inhibiting influence on some nematophagous fungi. The time between the use and/or the possibility of combining the fungus treatment with medicines should be better explored (L a r s e n et al., 1997). 248 CONCLUSION Our results show that Duddingtonia flagrans significantly reduces the number of eggs of Trichostrongylus spp. in the feces of naturally infected sheep. Since gastrointestinal parasites still represent a serious health problem, especially of small ruminants, antihelmintics are still necessary and prescribed in the control of these infections. Due to the resistance to these drugs, especially of Trichostrongylus spp. in sheep, the implementation of fungus Duddingtonia flagrans in cattle nutrition could be an important way of gastrointestinal and pulmonary strongyloids reduction. By simultaneous decrease
in the number of infected animals, the number of infectious larvae on pasture will decline, and the cycle of parasite development will be disturbed. From the aspect of greater need for safe food for human consumption, the occurrence of residua of drugs will be avoided in the food of animal origin. REFERENCES Araujo, J. V., Braga, F. R., Silva, A. R., Araujo, J. M., Tavela, A. O. (2008): In vitro evaluation of the effect of the nematophagous fungi Duddingtonia flagrans, Monacrosporium sinense, and Pochonia chlamydosporia on Ascaris suum eggs. Vet. Parasitol 102:787-790. Araujo, J. M., De Araujo, J. V., Braga, F. R., Carvalho, R. O., Ferreira, S. R. (2009): Activity of the nematophagous fungi Pochonia chlamydosporia, Duddingtonia flagrans and Monacrosporium thaumasium on egg capsules of Dipylidium caninum. Vet. Parasitol 13, 65-71. Carvalho, R. O., Araujo, J. V., Braga, F. R., Silva, A. R., Tavela, A. O. (2009): Predatory activity of nematophagous fungi on infective larvae of Ancylostoma spp evaluation in vitro and after passing through the gastrointestinal tract of dogs. J Helmintol 83,231-236 Chadrwathani, P., Jamnah, O., Waller, P. J., Larsen, M., Gillespie, A., Zahari, W. M. (2003): Biological control of nematode parasites of small ruminants in Malaysia using the nematophagous fungus Duddingtonia flagrans. Vet. Parasitol 117:173-183. C h a r o n, K. M. (2004): Genes controlling resistance to gastrointestinal nematodes in ruminants. Animal Science Papers and Reports, Vol. 22, no 1., 135-139 Conder, G. A., Johnson, S. S., Nowakowski, D. S., Blake, T. E., Dutton, F. E., Nelson, S. J., Thomas, E. M., Davis, J. P., Thompson, D. P. (1995): Anthelmintic profile of the cyclodepsipeptide PF1022A in vitro and in vivo models. J Antibiot (Tokyo), 48(8):820-3 Diez-Tascon, C., Keane, O. M., Wilson, T., Zadissa, A., Hyndman, D. L., Baird, D. B., McEwan, J. C., Crawford, A. M. (2005): Microarray analysis of selection lines from outbred populations to identify genes involved with nematode parasite resistance in sheep Physiol Genomics. 21(1):59-69 Gómez-Rincón, C., Uriarte, J., Valderrábano, J. (2006): Efficiency of Duddingtonia flagrans against Trichostrongyle infections of sheep on mountain pastures. Vet Parasitol. 10;141(1-2):84-90 L a r s e n, M. (2000): Prospects for controlling animal parasitic nematodes by predacious micro fungi. Parasitology. 120 Suppl:S 121-31 Larsen, M., Nansen, P., Gronvold, J., Wolsturp, J., Henriksen, S. V. (1997): Biological control of gastro-intestinal nematodes-facts, future or fiction? Vet Parasitol 72:479-492 Sarkū nas, M., Larsen, M., Nansen, P., Hansen, J. W. (2000): Biological controlof trichostrongylid infections in calves on pasture in Lithuania using Duddingtonia flagrans, a nematode traping fungus. J Helminthol 74:(4):355-359 249
ЕФЕКАТ НЕМАТОФАГНЕ ГЉИВЕ DUDDINGTONIA FLAGRANS НА ГАСТРОИНТЕСТИНАЛНЕ ПАРАЗИТЕ КОД ОВАЦА Весна Г. Лалошевић, Мирјана Н. Јарак, Симонида С. Ђурић, Немања С. Обрадовић Пољопривредни факултет, Трг Д. Обрадовића 8, Нови Сад, Србија Резиме Производња оваца сусреће се са озбиљним проблемима услед раширености желудачно-цревних паразита. Њихов значај огледа се у економској штети због губитка апетита, малдигестије, слабог прираста телесне тежине и вуне. Кон тро ла инфестације преживара стронгилидама до сада се заснивала углавном на органи зацији напасања и употреби антихелминтика, али, због појаве резистенције, уводе се алтернативне методе контроле. Употреба нематофагне гљиве Duddingtonia flagrans, која је способна да смањи број инфективних ларви и јаја у измету, показала се успешном. Циљ овог рада је да се утврди да ли Duddingtonia flagrans смањује присуство јаја Trichostrongylus spp. у измету оваца. Прегледана су 34 узорка фецеса а паразит је доказан код 12 оваца, од којих је 6 након тога добило гљиву, а 6 је коришћено као контрола. На основу χ 2 теста, на нивоу сигурности од p<0,005 доказана је статистички значајна разлика у броју јаја, у измету оваца које су добијале гљиву у односу на оне које нису. 250