The efficacy of predatory fungi on the control of gastrointestinal parasites in domestic and wild animals—A systematic review

Elsevier Editorial System(tm) for Veterinary Parasitology or its open access mirror Manuscript Draft Manuscript Number: Vetpar-D-20-14091R2 Title: The efficacy of predatory fungi on the control of gastrointestinal parasites in domestic and wild animals - a systematic review Article Type: Review article Keywords: Animals; Biological control; Duddingtonia flagrans; Gastrointestinal parasites; Predatory fungi. Corresponding Author: Professor Luis Manuel Madeira de Carvalho, Ph.D., Habilitation Corresponding Author's Institution: CIISA, Faculty of Veterinary Medicine, University of Lisbon First Author: Miguel Canhao-Dias Order of Authors: Miguel Canhao-Dias; Adolfo Paz Silva; Luis Madeira de Carvalho, DVM, PhD Abstract: Background: Gastrointestinal parasites like nematodes are associated with significant impacts on animal health, causing poor growth rates, diseases and even death. Traditional parasite control includes the use of anthelmintic drugs, albeit being associated with drug resistance and ecotoxicity. In the last decade, biological control of parasites using nematophagous or predatory fungi has been increasingly studied, although systematic evidence of its efficacy is still lacking. The aim of this work was to assess the evidence of efficacy of nematophagous fungi in the control of nematodes and other gastrointestinal parasites in different animal species. Methods: Using the PICO method (Population, Intervention, Comparison and Outcomes), we performed a systematic review on the subject to search for original papers published between January 2006 and October 2019, written in English, and indexed in PubMed/Medline. Medical Subject Headings (MeSH) terms were used in the syntax. Papers were selected for detailed review based on title and abstract. Inclusion and exclusion criteria were applied, and relevant data were collected from the remaining papers. Results: The literature search retrieved 616 papers. Eighty-nine were submitted to a detailed review. In the end, 53 papers were included in the analysis. The studies were very heterogeneous, using different fungi, doses, frequency of administration, duration of treatment, host animals, and target parasites. Considering the 53 papers, 44 studies (83% of the interventions) showed efficacy, with only 9 studies (17%) showing no significant differences when compared to control. Conclusion: With the increasing hazards of drug resistance and ecotoxicity, biological control with predatory fungi stands out as a good tool for future parasite management, whether as a complementary treatment or as an alternative to standard parasite control. Ms. No. Vetpar-D-20-14091R1 The efficacy of predatory fungi on the control of gastrointestinal parasites in domestic and wild animals - a systematic review Dear Professor Michael Philipp Reichel, DrMedVet, PhD, MBA, FRCVS Co-Editor in Chief Veterinary Parasitology, We would like to thank you and the reviewers for the comments to improve the paper. We have performed a proof-reading of the whole manuscript. Please find below the response to the Reviewers’ comments. Reviewer #1: Comment: There are still numerous grammatical errors which have to be corrected. The authors have largely addressed the previous comments of the reviewers, so with some modification, I believe the paper is worthy of publication. Answer: Thank you for these comments. We have revised the manuscript and hope it is now suitable for publication. All the lines mentioned after reviewing, refer to the track changes version. Comments: L 240: L1 of what? This needs to be expanded in this sentence. L 241: This is the first mention of the species name. Answer: These two points have been corrected (Lines 226 to 232). Comment: L 279-280: This sentence should be removed. Answer: The sentence has been removed (Lines 268-269). Comment: L 292: 'ambient' temperature? 'Soil' temperature? Answer: Thank you for noting this. It has been corrected (Line 281). Comment: L 310: I assume this refers to efficacy? Answer: Yes, thank you for pointing this. The sentence has been clarified (Line 297). Revision Note Comment: L 330: 'chicken's organism'? Answer: The sentence has been corrected (Line 315). Comment: L 337: 'translates a better overall welfare'. Not necessarily - this is inference. Also poor grammar. Answer: Thank you for this comment. The sentence has been revised and corrected (Lines 322 and 325). Comment: L 354-358: You need to exclude coccidia from 'helminthophagous' Answer: Thank you for noting this. The paragraph has been corrected (Lines 343 to 348). Reviewer #2: Comment: The authors have addressed the concerns raised by the referees, and it provides a very useful digest of the state of play regarding the potential role of nematophagous fungi for parasite management. Publication is recommended. Answer: Thank you very much. We thank you for the reviewing and guidance through all this process. Best regards, Lisbon, 21st June 2020 Miguel Canhão-Dias Adolfo Paz-Silva Luís Madeira de Carvalho HIGHLIGHTS  Systematic review delivering robust evidence on Biological Control of parasites  New insights on the efficacy of predatory fungi  New fungi products as promising tools for gastrointestinal parasite control *Highlights (for review) 1 The efficacy of predatory fungi on the control of gastrointestinal parasites in 1 domestic and wild animals – a systematic review 2 3 Canhão-Dias M.1, Paz-Silva A.2, Madeira de Carvalho L.M.1 4 5 Affiliations: 6 1 - CIISA – Centre for Interdisciplinary Research in Animal Health, Faculty of 7 Veterinary Medicine, University of Lisbon, Lisbon, Portugal; 8 2 - Control of Parasites Group (COPAR, GI-2120), Department of Animal Pathology, 9 Faculty of Veterinary, University of Santiago de Compostela, Lugo, Spain 10 11 Corresponding author: 12 Luís Madeira de Carvalho 13 CIISA, 14 Faculdade Medicina Veterinária, Universidade de Lisboa, 15 Pólo Universitário do Alto da Ajuda, 16 Avenida da Universidade Técnica, 17 1300-477, Lisboa, 18 Portugal. 19 20 Electronic address: [email protected] 21 22 23 24 25 *Revised Manuscript with changes marked Click here to view linked References 2 Abstract 26 Background: Gastrointestinal parasites like nematodes are associated with significant 27 impacts on animal health, causing poor growth rates, diseases and even death. 28 Traditional parasite control includes the use of anthelmintic drugs, albeit being 29 associated with drug resistance and ecotoxicity. In the last decade, biological control 30 of parasites using nematophagous or predatory fungi has been increasingly studied, 31 although systematic evidence of its efficacy is still lacking. 32 33 The aim of this work was to assess the evidence of efficacy of nematophagous fungi 34 in the control of nematodes and other gastrointestinal parasites in different animal 35 species. 36 37 Methods: Using the PICO method (Population, Intervention, Comparison and 38 Outcomes), We we performed a systematic review on the subject to search, applying 39 the PICO method (Population, Intervention, Comparison and Outcomes), searching 40 for original papers published between January 2006 and October 2019, written in 41 English, and indexed in PubMed/Medline. Medical Subject Headings (MeSH) terms 42 were used in the syntax. Papers were selected for detailed review based on title and 43 abstract. Inclusion and exclusion criteria were applied, and relevant data was were 44 collected from the remaining papers. 45 46 Results: The literature search retrieved 616 papers. Eighty-nine were submitted to a 47 detailed review. In the end, 53 papers were included in the analysis. The studies were 48 very heterogeneous, using different fungi, doses, frequency of administration, 49 duration of treatment, host animals, and target parasites. Considering the 53 papers, 50 3 44 studies (83% of the interventions) showed efficacy, with only 9 studies (17%) 51 showing no significant differences when compared to control. 52 Conclusion: With the increasing hazards of drug resistance and ecotoxicity, biological 53 control with predatory fungi stands out as a good tool for future parasite management, 54 whether as a complementary treatment or as an alternative to standard parasite 55 control. 56 57 Keywords: Animals, Biological control, Duddingtonia flagrans, Gastrointestinal 58 parasites, Predatory fungi. 59 60 61 Introduction 62 Livestock parasites reduce productivity and are a source of economic losses that can 63 reach tens of billions of dollars worldwide (Roeber et al., 2013). Gastrointestinal 64 parasites, namely nematodes, can have a significant impact on animal health and 65 welfare, causing poor growth rates, diseases, and even death (Larsen, 2006). 66 67 The traditional approach to the control of this problem has been the use of 68 anthelmintic drugs, which can present some drawbacks, such as ecotoxicity (Vokřál et 69 al., 2019) and the development of multidrug-resistant parasite populations against 70 most anthelmintic classes (Canever et al., 2013; Peregrine et al., 2014). 71 Simultaneously, the increasing demand for food products produced with reduced 72 chemical use and under eco-friendly standards (Clark et al., 2017; Wee et al., 2012) 73 has further demonstrated the need for alternative types of gastrointestinal parasite 74 control. 75 4 76 This situation has stimulated research in strategies based on natural approaches 77 involving pasture management, rotation of animal species, nutritional optimization, 78 administration of copper oxide wire particles or the utilization of natural antagonists. 79 Nematophagous fungi are microorganisms able to reduce the population of parasites, 80 without damaging the host animals (Buzatti et al., 2015; Hernández et al., 2016). 81 Duddingtonia flagrans has been considered the most promising fungal species for the 82 control of nematode larvae (Larsen, 2006; Sahoo and Khan, 2016). However, it is not 83 the only species with demonstrated efficacy, and fungi such as Arthrobotrys robusta 84 and Monacrosporium thaumasium have also been described as useful tools in the 85 control of parasites (Faedo et al., 1997; Braga and Araújo, 2014). These fungi are able 86 to catch the larvae of some nematodes in the soil through trap formation with their 87 mycelia, and then digesting the parasite (Buzatti et al., 2015). Some species of fungi 88 (Pochonia chlamydosporia, Mucor circinelloides, Purpureocillium lilacinum, 89 Trichoderma spp.) have shown activity against the eggs of helminths and even 90 coccidian oocysts, due to their ability to attach to their eggshells, penetrate and 91 destroy them (Braga and De Araújo, 2014; J. Á. Hernández et al., 2018a). 92 93 Despite the increasing number of studies and publications about the biological control 94 of helminths, there is no systematic review gathering data from the different trials and 95 analyseis on the efficacy of these fungi. In fact, accumulated knowledge arising from 96 research has not yet been translated to daily clinical practice, due to the lack of 97 systematic evidence of efficacy, as well as the scarcity of protocols with standard 98 doses, frequency and duration of treatment, and vehicle of administration. 99 100 11 The use of intraruminal Controlled Release Devices has demonstrated good results in 251 ruminants (Sagüés et al., 2014) and presents itself as a good option for future 252 formulations, especially for free-ranging animals (Waller et al., 2001a). Buzatti et al. 253 (2015) showed that administrations with 3-day intervals in horses are effective in 254 reducing the amount of fecal cyathostomin L3, which is also a good prospect for 255 extensive grazers. 256 257 Nowadays, most studies incorporate the fungi in edible pellets, namely in a sodium 258 alginate matrix, with good results. The fungi retain their nematophagous activity after 259 passing through the animals’ gastrointestinal tract and the pellets allow for an easy 260 dosing and a more even ingestion within the herd; moreover, they are easy to store 261 and preserve (Fitz-Aranda et al., 2015; Hernández et al., 2016). Regarding non-262 herbivores, an effective reduction of L3 can be achieved by mixing mycelia with 263 regular canned food (Carvalho et al., 2009). However, the use of nematophagous 264 fungi in small domestic animals like dogs and cats in a big scale is still a distant 265 reality, due to the lack of a reliable method of administering a standard dose. 266 267 A treatment with nematophagous fungi can be used in combination with other forms 268 of biological control. Hernández et al. (2018) combined D. flagrans and M. 269 circinelloides chlamydospores with a pasture rotation system, showing a reduction of 270 fecal Eggs Per Gram (EPG) of up to 99% in the first six months of fungal 271 administration in horses. Moreover, they registered an Egg Reappearance Period of 16 272 weeks after one ivermectin administration associated with the fungi, in comparison 273 with 6 weeks in the control group using only ivermectin. Similarly, the removal of 274 feces from pasture can have good results in worm control (Paz-Silva et al., 2011). It is 275 Field Code Changed Formatted: French (France) Formatted: French (France) Field Code Changed Formatted: French (France) Formatted: French (France) 12 also suggested that animals with a high parasitic burden must be identified and 276 removed from the herd (Rocha et al., 2007). The concomitant administration of 277 chlamydospores and an energetic supplement was successful in reducing the EPG, 278 parasite load, and pasture contamination in goats, compared to a group receiving only 279 chlamydospores (Gómez-Rincón et al., 2007). 280 281 It has been suggested that abiotic factors like rain, temperature, and sunlight might 282 influence the performance of the nematophagous fungi. Field trials performed with D. 283 flagrans in horses, registered better results in Spring/Summer, than in 284 Autumn/Winter, concerning L3 reduction levels in fecal samples and pasture 285 (Madeira de Carvalho et al., 2007). Indeed, several studies show different fungal 286 efficacies depending on the ambient temperature. Paraud et al. (2006) obtained higher 287 reductions of H. contortus, T. circumcincta and T. colubriformis by D. flagrans at 288 21ºC than at 28ºC. Also, Bilotto et al. (2018) achieved 63.77% and 88.39% L3 289 reduction in pasture in sunny and shaded conditions in summer, respectively, versus 290 1,58% and 3.56% in winter. This shows that the relation between temperature, larval 291 development, and fungal efficacy needs to be understood, in order to design 292 successful parasite-management programs. 293 294 An interesting question lies in the acquisition of useful strains of fungi with 295 parasiticide activity. Due to only two commercial formulations being available 296 (Healey et al., 2018, Braga et al., 2020), with others in progress, most of the 297 information available has been collected by using fungi isolated in different countries. 298 Soto-Barrientos et al. (2011) successfully used three techniques to isolate 299 nematophagous fungi directly from several farms’ soils, an alternative to buying 300 13 commercially formulated fungi. More studies are needed to evaluate the advantages 301 and disadvantages of using locally isolated fungi; some important questions would be: 302 if the ecological impacts on the soil are minimized by using fungi already existing in 303 the same environment; if it is cheaper to isolate than to buy; if it there is better to 304 usegreater efficacy by using a local fungal species/strains, instead of introducing an 305 exotic ones. 306 307 It must be noted that regularly used antiparasitic drugs, namely ivermectin and 308 albendazole, do have an in vitro inhibitory effect over nematophagous fungi, namely 309 on the development of Arthrobotrys oligospora, D. flagrans and P. lilacinum (Vieira 310 et al., 2016). Although not likely to occur due to the buffering effect of the rumen, 311 studies should be performed in order to understand if these effects also occur in vivo. 312 313 From the 53 analyzed articles, only six (11.3%) (Assis et al., 2012, 2013; Dias et al., 314 2013; Hernández et al., 2016, 2018; Palomero et al., 2018) reported data regarding the 315 administration of fungi for 12 or more months. This is elucidative of the lack of 316 studies that measure the long-term impact of nematophagous fungi on a parasite 317 population. All these studies showed a good long-term reduction of EPG, whether by 318 using ovicidal, larvicidal, or a mixture of both types of fungi. One short-term study 319 (Thapa et al., 2018) has shown an unwanted effect when using P. chlamydosporia 320 (ovicidal) against an ascarid parasite of chickens: the reduced parasite burden caused 321 by the exposure to P. chlamydosporia led to a faster development of the existing 322 larvae into adult worms within the chicken’s organismintestine. That, in turn meant a 323 higher EPG and a greater contamination of the environment. This is an extremely 324 important point to have in consideration when designing parasite control programs, 325 14 since it may increase the possibility of environment recontamination. More studies 326 need to be conducted in order to fully understand how beneficial these treatments are 327 in the long-term. 328 329 From 13 studies that measured animal weight changesvariations, six (46.2%) showed 330 animals treated with fungi had significantly higher gains in animals treated with fungi 331 when compared to control groups, which translates a better overall welfare in these 332 animals. The authors have not reported adverse reactions to D. flagrans and M. 333 circinelloides in horses (Hernández et al., 2016), nor against P. chlamydosporia in 334 chickens (Thapa et al., 2018). 335 336 New products using nematophagous fungi are being developed. Bioverm® is a powder 337 containing 105 D. flagrans chlamydospores per gram that has been showingshown 338 over 90% efficacy in the reduction of Strongyloides papillosus and H. contortus 339 larvae in sheep (Braga et al., 2020). Bioworma® is another D. flagrans 340 chlamydospore-based product already commercialized and administered at a 341 concentration of 3x104 chlamydospores per kg of body weight. With a much smaller 342 dose than that regularly used, it is capable of reducing the number of viable larvae of 343 several nematodes in horses, goats, and cows (Healey et al., 2018). Further studies are 344 needed to determine the possibility of driving down costs by reducing the number of 345 chlamydospores used, while maintaining the same parasite reduction efficacy. In this 346 case, the frequency of dosage should also be taken into account. 347 348 Recent studies found that P. chlamydosporia can also be useful against eggs of 349 tapeworms (Araujo et al., 2009; Braga et al., 2011) and trematodes (De et al., 2008; 350 Field Code Changed Formatted: French (France) 15 Dias et al., 2013)., These reports support Braga and Araújo (2014) in arguing that 351 nematophagous fungi can start being called helmintophagous. If the results showing 352 D. flagrans’ efficacy and even against coccidians (Magalhães da Cruz, 2015) are 353 reproduced, they can even be. These reports support Braga and Araújo (2014) in 354 arguing that nematophagous fungi can start being called helmintophagous or, more 355 broadly called, predatory fungi. 356 357 The fungal doses used varied widely, but no significant correlation between low 358 fungal intake and poor outcomes was found with the doses studied. Although several 359 regimens of fungal administration have been tested and overall good results have been 360 achieved, optimal parasite control is expected to be attained when adopting long term 361 schedules, with at least a twice-per-week fungal administration, as showed by Assis et 362 al. (2013), Hernández et al. (2016) and Silva et al. (2009). 363 364 In general, papers with negative or absent results tend to be less published, which can 365 limit the information retrieved by systematic reviews. Despite all the advances in 366 understanding predatory fungi, some questions remain unaddressed. There is scarce 367 information about the fungi’s potential impact on the soil microbiome. The fungi’s 368 attack mechanisms are based on physical and chemical actions simpler than those of 369 anthelmintic drugs. This could be associated with a decreased rate of development of 370 parasite resistance to predatory fungi. Likewise, there is a need to further evaluate the 371 possibility of predatory fungi infecting immunocompromised humans, limiting their 372 potential use. Consequently, this will continue to be a hot topic within the veterinarian 373 community for the coming years. But it will also be a vanguard tool for animal 374 parasite control with great potential in the future, namely because it is an ecological, 375 Formatted: French (France) Field Code Changed Formatted: French (France) Formatted: French (France) Formatted: English (United States) Formatted: Font: Italic Field Code Changed Formatted: English (United States) Formatted: English (United States) Formatted: English (United States) Field Code Changed Formatted: English (United States) Formatted: English (United States) Formatted: English (United States) 16 economical and sustainable approach for doing it in times when less fewer residues in 376 animal tissues and in the environment are a must for animal and human health. 377 378 379 Acknowledgements: 380 MCD and LMMC are supported by Project UID/CVT/00276/202019 (CIISA) funded 381 by Fundação para a Ciência e Tecnologia (FCT). 382 APS is supported by Research Projects RYC-2016-21407 (Ministry of Economy and 383 Competitiveness, Spain) and ED431F 2018/03 (Consellería de Cultura, Educación e 384 Ordenación Universitaria, Xunta de Galicia, Spain). 385 386 387 388 389 390 391 References 392 Aguilar-Marcelino, L., Mendoza-De-Gives, P., Torres-Hernández, G., López-393 Arellano, M.E., Becerril-Pérez, C.M., Orihuela-Trujillo, A., Torres-Acosta, 394 J.F.J., Olmedo-Juárez, A., 2017. 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Trop. 59, 37–52. 686 Tavela, A. de O., Araújo, J.V., Braga, F.R., Silva, A.R., Carvalho, R.O., Araujo, J.M., 687 Ferreira, S.R., Carvalho, G.R., 2011. Biological control of cyathostomin 688 (Nematoda: Cyathostominae) with nematophagous fungus Monacrosporium 689 thaumasium in tropical southeastern Brazil. Vet. Parasitol. 175, 92–96. 690 https://doi.org/10.1016/j.vetpar.2010.09.035 691 Tavela, A. de O., de Araújo, J.V., Braga, F.R., da Silveira, W.F., Dornelas e Silva, 692 V.H., Carretta Júnior, M., Borges, L.A., Araujo, J.M., Benjamin, L. dos A., 693 Carvalho, G.R., de Paula, A.T., 2013. Coadministration of sodium alginate 694 pellets containing the fungi Duddingtonia flagrans and Monacrosporium 695 thaumasium on cyathostomin infective larvae after passing through the 696 gastrointestinal tract of horses. Res. Vet. Sci. 94, 568–572. 697 https://doi.org/10.1016/j.rvsc.2012.11.011 698 Thapa, S., Thamsborg, S.M., Wang, R., Meyling, N. V., Dalgaard, T.S., Petersen, 699 H.H., Mejer, H., 2018. Effect of the nematophagous fungus Pochonia 700 29 chlamydosporia on soil content of ascarid eggs and infection levels in exposed 701 hens. Parasites and Vectors 11, 1–11. https://doi.org/10.1186/s13071-018-2898-1 702 Vieira, Í.S., Oliveira, I.D.C., Campos, A.K., Araújo, J.V. De, 2019. Association and 703 predatory capacity of fungi Pochonia chlamydosporia and Arthrobotrys cladodes 704 in the biological control of parasitic helminths of bovines. Parasitology 146, 705 1347–1351. https://doi.org/10.1017/S003118201900060X 706 Vieira, J.N., Maia Filho, F.S., Ferreira, G.F., Mendes, J.F., Gonçalves, C.L., Villela, 707 M.M., Pereira, D.I.B., Nascente, P.S., 2016. In vitro susceptibility of 708 nematophagous fungi to antiparasitic drugs: interactions and implications for 709 biological control. Brazilian J. Biol. 77, 476–479. https://doi.org/10.1590/1519-710 6984.15715 711 Vilela, V.L.R., Feitosa, T.F., Braga, F.R., Araújo, J.V. de, Souto, D.V. de O., Santos, 712 H.E. da S., Silva, G.L.L. da, Athayde, A.C.R., 2012. Biological control of goat 713 gastrointestinal helminthiasis by Duddingtonia flagrans in a semi-arid region of 714 the northeastern Brazil. Vet. Parasitol. 188, 127–133. 715 https://doi.org/10.1016/j.vetpar.2012.02.018 716 Vokřál, I., Michaela, Š., Radka, P., Jiří, L., Lukáš, P., Dominika, S., Kateřina, L., 717 Barbora, S., Lenka, S., 2019. Ivermectin environmental impact: Excretion profile 718 in sheep and phytotoxic effect in Sinapis alba. Ecotoxicol. Environ. Saf. 169, 719 944–949. https://doi.org/10.1016/j.ecoenv.2018.11.097 720 Waller, P.J., Faedo, M., Ellis, K., 2001a. The potential of nematophagous fungi to 721 control the free-living stages of nematode parasites of sheep: towards the 722 development of a fungal controlled release device. Vet. Parasitol. 102, 299–308. 723 Waller, P.J., Knox, M.R., Faedo, M., 2001b. The potential of nematophagous fungi to 724 control the free-living stages of nematode parasites of sheep: feeding and block 725 Formatted: English (United States) Formatted: Spanish (Spain, International Sort) 30 studies with Duddingtonia flagrans. Vet. Parasitol. 102, 321–330. 726 Waller, P.J., Ljungström, B.-L., Schwan, O., Martin, L.R., Morrison, D.A., Rydzik, 727 A., 2006. Biological control of sheep parasites using Duddingtonia flagrans: 728 Trials on commercial farms in Sweden. Acta Vet. Scand. 47, 23–32. 729 Wee, C.S., Ariff, M.S. Bin, Zakuan, N., Tajudin, M.N., 2012. Key Factors Affecting 730 Consumer Purchase Intention a Study of Safe Vegetable in Ho Chi Minh City , 731 Vietnam. Rev. Integr. Bus. Econ. Res. 3, 378–397. 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 PubMed/Medline n = 616 For detailed review n = 89 Excluded: n= 36 Poor quality design (not Randomized Clinical Trial, no comparator, no treatment doses specified, no outcomes of interest, no measurable outcomes) Excluded by title/abstract: n= 527 - Not in English - Not Original papers (reviews, short communications, letters, editorials) - Published before 2006 Inclusion criteria - Any animal species - Predatory fungi targeting any stage of gastrointestinal parasites 31 751 752 753 Figure 1Flowchart with literature search. 754 755 756 757 758 759 760 761 762 763 764 765 766 Table 1 — PICO method for review question 767 Population Gastrointestinal parasites of any animal species. Intervention Usage of nematophagous or predatory fungi on any stage of gastrointestinal parasites (e.g., eggs, larvae) in in vivo, in vitro and ex vivo settings. Only fungi that predate gastrointestinal parasites of animals were included. Comparison A control group was mandatory. Any relevant comparison was accepted (e.g. placebo, different fungi, different fungus dosage). Included n = 53 32 Outcome Quantified control of gastrointestinal parasites (e.g. reduction of number of infective larvae in feces or pasture, reduction of number of eggs in feces or pasture, reduction of parasite load, reduction of deworming frequency). Only papers that quantified the outcomes with figures were included. 768 769 770 771 772 773 774 775 776 777 778 779 Table 2 – Description of studies’ characteristics 780 Fungi Parasites Administration and Dose Frequency and duration Evaluated Outcomes Ovine N = 15 Duddingtonia flagrans (15); Monacrosporium thaumasium (2); Arthrobotrys robusta (1); Clonostachys rosea (1) Haemonchus contortus (14); Trichostrongylus spp. (8); Teladorsagia spp. (6) and others 2x105 – 1x106 chlamydospores /kg live weight (10); Other forms (fixed dose of mycelia, conidia and chlamydospores) Single administration (2); Daily for 6 days to 5 months (10); Twice/week for 5 to 6 months (3) Eggs per gram (EPG) reduction Fecal L3 reduction Reduction of intestinal adult parasites Reduction of L3 /kg pasture dry matter Reduction in Tracer Sheep parasite load Bovines N = 10 D. flagrans (7); Pochonia Cooperia spp. (8); H. contortus (7); 0,2 – 0,25 g /10 kg live weight (4); Single administration EPG reduction Fecal L3 reduction 33 chlamydosporia (2); Arthrobotrys cladodes (2); M. thaumasium (3); A. robusta (1) Oesophagostomum spp. (7) and others Other forms (fixed dose of mycelia and chlamydospores) (4); Daily for 10 days (1); Every 2 days for 30 days (1); Twice/week for 6 to 18 months (4) Reduction of L3 /kg pasture dry matter Equids N = 8 D. flagrans (7); M. thaumasium (3); A. robusta (1); Mucor circinelloides (2) Cyathostominae (7), Parascaris sp. (1); Strongyloides westeri (1) 1,5x105 – 2x106 chlamydospores /kg live weight (3); Other forms (fixed dose of mycelia and conidia) Single administration (3); Daily for 16 months (1); Twice/week for 21 days to 14 months (4) EPG reduction Fecal L3 reduction Reduction of L3 /kg pasture dry matter Egg Reappearance Period after Ivermectin administration Canids N = 7 D. flagrans (4); Arthrobotrys spp. (6); Monacrosporium spp. (6); P. chlamydosporia (2) Ancylostoma spp. (3); Angiostrongylus vasorum. (2); Toxocara canis (2) Mostly in vitro Single administration (7) Fecal L1 reduction Fecal L3 reduction Others Caprine N = 5 D. flagrans (4); Arthrobotrys spp. (1) Trichostrongylus spp. (5); Haemonchus spp. (4); Teladorsagia spp. (3) and others 5x105 – 1x106 chlamydospores /kg live weight (4); 0,06g mycelia /kg live weight (1) Single administration (1); Daily for 27 days to 5 months (3); Twice/week for 6 months (1) EPG reduction Fecal L3 reduction Reduction of intestinal adult parasites Reduction of L3 /kg pasture dry matter Reduction in Tracer Goat parasite load Swine N = 2 D. flagrans (2) Oesophagostomum spp. (2) Fixed dose of chlamydospores and mycelia (2); 5x105 chlamydospores /kg live weight (1) Single administration (2) Fecal L3 reduction Other N = 6 D. flagrans (5); P. chlamydosporia (2); M. circinelloides (2) Varies with animal species Varies with animal species Single administration (4); Daily for 7 days (1); Every 2 days for 3 years (1) EPG reduction Fecal L3 reduction Reduction of L3 /kg pasture dry matter Others 781 N – Number of studies 782 Between brackets are the number of studies with each characteristic 783 EPG – Eggs per gram of feces 784 L1, L3 – parasite larval stages 785 Formatted: French (France) 1 The efficacy of predatory fungi on the control of gastrointestinal parasites in 1 domestic and wild animals – a systematic review 2 3 Canhão-Dias M.1, Paz-Silva A.2, Madeira de Carvalho L.M.1 4 5 Affiliations: 6 1 - CIISA – Centre for Interdisciplinary Research in Animal Health, Faculty of 7 Veterinary Medicine, University of Lisbon, Lisbon, Portugal; 8 2 - Control of Parasites Group (COPAR, GI-2120), Department of Animal Pathology, 9 Faculty of Veterinary, University of Santiago de Compostela, Lugo, Spain 10 11 Corresponding author: 12 Luís Madeira de Carvalho 13 CIISA, 14 Faculdade Medicina Veterinária, Universidade de Lisboa, 15 Pólo Universitário do Alto da Ajuda, 16 Avenida da Universidade Técnica, 17 1300-477, Lisboa, 18 Portugal. 19 20 Electronic address: [email protected] 21 22 23 24 25 *Revised Manuscript with NO changes marked (clean) Click here to view linked References 2 Abstract 26 Background: Gastrointestinal parasites like nematodes are associated with significant 27 impacts on animal health, causing poor growth rates, diseases and even death. 28 Traditional parasite control includes the use of anthelmintic drugs, albeit being 29 associated with drug resistance and ecotoxicity. In the last decade, biological control 30 of parasites using nematophagous or predatory fungi has been increasingly studied, 31 although systematic evidence of its efficacy is still lacking. The aim of this work was 32 to assess the evidence of efficacy of nematophagous fungi in the control of nematodes 33 and other gastrointestinal parasites in different animal species. 34 35 Methods: Using the PICO method (Population, Intervention, Comparison and 36 Outcomes), we performed a systematic review on the subject to search for original 37 papers published between January 2006 and October 2019, written in English, and 38 indexed in PubMed/Medline. Medical Subject Headings (MeSH) terms were used in 39 the syntax. Papers were selected for detailed review based on title and abstract. 40 Inclusion and exclusion criteria were applied, and relevant data were collected from 41 the remaining papers. 42 43 Results: The literature search retrieved 616 papers. Eighty-nine were submitted to a 44 detailed review. In the end, 53 papers were included in the analysis. The studies were 45 very heterogeneous, using different fungi, doses, frequency of administration, 46 duration of treatment, host animals, and target parasites. Considering the 53 papers, 47 44 studies (83% of the interventions) showed efficacy, with only 9 studies (17%) 48 showing no significant differences when compared to control. 49 3 Conclusion: With the increasing hazards of drug resistance and ecotoxicity, biological 50 control with predatory fungi stands out as a good tool for future parasite management, 51 whether as a complementary treatment or as an alternative to standard parasite 52 control. 53 54 Keywords: Animals, Biological control, Duddingtonia flagrans, Gastrointestinal 55 parasites, Predatory fungi. 56 57 58 Introduction 59 Livestock parasites reduce productivity and are a source of economic losses that can 60 reach tens of billions of dollars worldwide (Roeber et al., 2013). Gastrointestinal 61 parasites, namely nematodes, can have a significant impact on animal health and 62 welfare, causing poor growth rates, diseases, and even death (Larsen, 2006). 63 64 The traditional approach to the control of this problem has been the use of 65 anthelmintic drugs, which can present some drawbacks, such as ecotoxicity (Vokřál et 66 al., 2019) and the development of multidrug-resistant parasite populations against 67 most anthelmintic classes (Canever et al., 2013; Peregrine et al., 2014). 68 Simultaneously, the increasing demand for food products produced with reduced 69 chemical use and under eco-friendly standards (Clark et al., 2017; Wee et al., 2012) 70 has further demonstrated the need for alternative types of gastrointestinal parasite 71 control. 72 73 This situation has stimulated research in strategies based on natural approaches 74 10 shown that D. flagrans can adapt to higher cyathostomin egg-outputs, maintaining 225 great results in the reduction of third-stage larvae (L3). 226 227 Besides the already tested effect of D. flagrans on L3 of gastrointestinal nematodes, 228 there are reports of its predatory activity against Angiostrongylus vasorum first-stage 229 larvae (L1) (Braga et al., 2009b) and Toxocara canis second-stage larvae (L2) (Hiura 230 et al., 2015). Using crude extracts from M. thaumasium, de Freitas Soares et al. 231 (2015) have shown a reduction of A. vasorum L1 in vitro. This is a new approach to 232 parasite control, namely for pets, that deserves further investigation. 233 234 Fitz-Aranda et al. (2015) stated that some of the biggest factors influencing the 235 success of this kind of treatment are the medium in which the fungi are administered, 236 the ingestion of an effective dose, as well as a storage method that does not alter the 237 fungi’s properties. Mixing spores into a nutrient block is an approach already tested, 238 but it has shown the downside of offering a very variable spore intake among animals 239 in the same group, with the ingestion also depending on climate and grazing season 240 (Sagüés et al., 2011). Additionally, the blocks have a relatively high level of moisture 241 comparing to other forms of administration, reducing the product’s shelf life (Larsen, 242 2006). The use of cereal grains combined with chlamydospores has similarly shown 243 to be effective (Facchini Rodrigues et al., 2018; Waller et al., 2001b). 244 245 The use of intraruminal Controlled Release Devices has demonstrated good results in 246 ruminants (Sagüés et al., 2014) and presents itself as a good option for future 247 formulations, especially for free-ranging animals (Waller et al., 2001a). Buzatti et al. 248 (2015) showed that administrations with 3-day intervals in horses are effective in 249 11 reducing the amount of fecal cyathostomin L3, which is also a good prospect for 250 extensive grazers. 251 252 Nowadays, most studies incorporate the fungi in edible pellets, namely in a sodium 253 alginate matrix, with good results. The fungi retain their nematophagous activity after 254 passing through the animals’ gastrointestinal tract and the pellets allow for an easy 255 dosing and more even ingestion within the herd; moreover, they are easy to store and 256 preserve (Fitz-Aranda et al., 2015; Hernández et al., 2016). Regarding non-257 herbivores, an effective reduction of L3 can be achieved by mixing mycelia with 258 regular canned food (Carvalho et al., 2009). However, the use of nematophagous 259 fungi in small domestic animals like dogs and cats in a big scale is still a distant 260 reality, due to the lack of a reliable method of administering a standard dose. 261 262 A treatment with nematophagous fungi can be used in combination with other forms 263 of biological control. Hernández et al. (2018) combined D. flagrans and M. 264 circinelloides chlamydospores with a pasture rotation system, showing a reduction of 265 fecal Eggs Per Gram (EPG) of up to 99% in the first six months of fungal 266 administration in horses. Moreover, they registered an Egg Reappearance Period of 16 267 weeks after one ivermectin administration associated with the fungi, in comparison 268 with 6 weeks in the control group using only ivermectin. It is also suggested that 269 animals with a high parasitic burden must be identified and removed from the herd 270 (Rocha et al., 2007). The concomitant administration of chlamydospores and an 271 energetic supplement was successful in reducing the EPG, parasite load, and pasture 272 contamination in goats, compared to a group receiving only chlamydospores (Gómez-273 Rincón et al., 2007). 274 12 275 It has been suggested that abiotic factors like rain, temperature, and sunlight might 276 influence the performance of the nematophagous fungi. Field trials performed with D. 277 flagrans in horses, registered better results in Spring/Summer, than in 278 Autumn/Winter, concerning L3 reduction levels in fecal samples and pasture 279 (Madeira de Carvalho et al., 2007). Indeed, several studies show different fungal 280 efficacies depending on ambient temperature. Paraud et al. (2006) obtained higher 281 reductions of H. contortus, T. circumcincta and T. colubriformis by D. flagrans at 282 21ºC than at 28ºC. Also, Bilotto et al. (2018) achieved 63.77% and 88.39% L3 283 reduction in pasture in sunny and shaded conditions in summer, respectively, versus 284 1,58% and 3.56% in winter. This shows that the relation between temperature, larval 285 development, and fungal efficacy needs to be understood, in order to design 286 successful parasite-management programs. 287 288 An interesting question lies in the acquisition of useful strains of fungi with 289 parasiticide activity. Due to only two commercial formulations being available 290 (Healey et al., 2018, Braga et al., 2020), with others in progress, most of the 291 information available has been collected by using fungi isolated in different countries. 292 Soto-Barrientos et al. (2011) successfully used three techniques to isolate 293 nematophagous fungi directly from several farms’ soils, an alternative to buying 294 commercially formulated fungi. More studies are needed to evaluate the advantages 295 and disadvantages of using locally isolated fungi; some important questions would be: 296 if the ecological impacts on the soil are minimized by using fungi already existing in 297 the same environment; if it is cheaper to isolate than to buy; if there is greater efficacy 298 by using local fungal species/strains, instead of introducing exotic ones. 299 13 300 It must be noted that regularly used antiparasitic drugs, namely ivermectin and 301 albendazole, do have an in vitro inhibitory effect over nematophagous fungi, namely 302 on the development of Arthrobotrys oligospora, D. flagrans and P. lilacinum (Vieira 303 et al., 2016). Although not likely to occur due to the buffering effect of the rumen, 304 studies should be performed in order to understand if these effects also occur in vivo. 305 306 From the 53 analyzed articles, only six (11.3%) (Assis et al., 2012, 2013; Dias et al., 307 2013; Hernández et al., 2016, 2018; Palomero et al., 2018) reported data regarding the 308 administration of fungi for 12 or more months. This is elucidative of the lack of 309 studies that measure the long-term impact of nematophagous fungi on a parasite 310 population. All these studies showed a good long-term reduction of EPG, whether by 311 using ovicidal, larvicidal, or a mixture of both types of fungi. One short-term study 312 (Thapa et al., 2018) has shown an unwanted effect when using P. chlamydosporia 313 (ovicidal) against an ascarid parasite of chickens: the reduced parasite burden caused 314 by the exposure to P. chlamydosporia led to faster development of the existing larvae 315 into adult worms within the chicken’s intestine. That, in turn meant a higher EPG and 316 a greater contamination of the environment. This is an extremely important point to 317 have in consideration when designing parasite control programs, since it may increase 318 the possibility of environment recontamination. More studies need to be conducted in 319 order to fully understand how beneficial these treatments are in the long-term. 320 321 From 13 studies that measured weight variations, six (46.2%) showed animals treated 322 with fungi had significantly higher gains when compared to control groups. The 323 authors have not reported adverse reactions to D. flagrans and M. circinelloides in 324 14 horses (Hernández et al., 2016), nor against P. chlamydosporia in chickens (Thapa et 325 al., 2018). 326 327 New products using nematophagous fungi are being developed. Bioverm® is a powder 328 containing 105 D. flagrans chlamydospores per gram that has shown over 90% 329 efficacy in the reduction of Strongyloides papillosus and H. contortus larvae in sheep 330 (Braga et al., 2020). Bioworma® is another D. flagrans chlamydospore-based product 331 already commercialized and administered at a concentration of 3x104 chlamydospores 332 per kg of body weight. With a much smaller dose than that regularly used, it is 333 capable of reducing the number of viable larvae of several nematodes in horses, goats, 334 and cows (Healey et al., 2018). Further studies are needed to determine the possibility 335 of driving down costs by reducing the number of chlamydospores used while 336 maintaining the same parasite reduction efficacy. In this case, the frequency of dosage 337 should also be taken into account. 338 339 Recent studies found that P. chlamydosporia can also be useful against eggs of 340 tapeworms (Araujo et al., 2009; Braga et al., 2011) and trematodes (De et al., 2008; 341 Dias et al., 2013). These reports support Braga and Araújo (2014) in arguing that 342 nematophagous fungi can start being called helmintophagous. If the results showing 343 D. flagrans’ efficacy against coccidians (Magalhães da Cruz, 2015) are reproduced, 344 they can even be more broadly called predatory fungi. 345 346 The fungal doses used varied widely, but no significant correlation between low 347 fungal intake and poor outcomes was found with the doses studied. Although several 348 regimens of fungal administration have been tested and overall good results have been 349 15 achieved, optimal parasite control is expected to be attained when adopting long term 350 schedules, with at least a twice-per-week fungal administration, as showed by Assis et 351 al. (2013), Hernández et al. (2016) and Silva et al. (2009). 352 353 In general, papers with negative or absent results tend to be less published, which can 354 limit the information retrieved by systematic reviews. Despite all the advances in 355 understanding predatory fungi, some questions remain unaddressed. There is scarce 356 information about the fungi’s potential impact on the soil microbiome. The fungi’s 357 attack mechanisms are based on physical and chemical actions simpler than those of 358 anthelmintic drugs. This could be associated with a decreased rate of development of 359 parasite resistance to predatory fungi. Likewise, there is a need to further evaluate the 360 possibility of predatory fungi infecting immunocompromised humans, limiting their 361 potential use. Consequently, this will continue to be a hot topic within the veterinarian 362 community for the coming years. But it will also be a vanguard tool for animal 363 parasite control with great potential in the future, namely because it is an ecological, 364 economical and sustainable approach for doing it in times when fewer residues in 365 animal tissues and in the environment are a must for animal and human health. 366 367 368 Acknowledgements: 369 MCD and LMMC are supported by Project UID/CVT/00276/2020 (CIISA) funded by 370 Fundação para a Ciência e Tecnologia (FCT). 371 APS is supported by Research Projects RYC-2016-21407 (Ministry of Economy and 372 Competitiveness, Spain) and ED431F 2018/03 (Consellería de Cultura, Educación e 373 Ordenación Universitaria, Xunta de Galicia, Spain). 374 16 375 376 377 378 379 380 References 381 Aguilar-Marcelino, L., Mendoza-De-Gives, P., Torres-Hernández, G., López-382 Arellano, M.E., Becerril-Pérez, C.M., Orihuela-Trujillo, A., Torres-Acosta, 383 J.F.J., Olmedo-Juárez, A., 2017. 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Res. 3, 378–397. 721 722 723 724 30 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 Figure 1Flowchart with literature search. 743 744 745 746 747 748 749 PubMed/Medline n = 616 For detailed review n = 89 Included n = 53 Excluded: n= 36 Poor quality design (not Randomized Clinical Trial, no comparator, no treatment doses specified, no outcomes of interest, no measurable outcomes) Excluded by title/abstract: n= 527 - Not in English - Not Original papers (reviews, short communications, letters, editorials) - Published before 2006 Inclusion criteria - Any animal species - Predatory fungi targeting any stage of gastrointestinal parasites 31 750 751 752 753 754 755 Table 1 — PICO method for review question 756 Population Gastrointestinal parasites of any animal species. Intervention Usage of nematophagous or predatory fungi on any stage of gastrointestinal parasites (e.g., eggs, larvae) in in vivo, in vitro and ex vivo settings. Only fungi that predate gastrointestinal parasites of animals were included. Comparison A control group was mandatory. Any relevant comparison was accepted (e.g. placebo, different fungi, different fungus dosage). Outcome Quantified control of gastrointestinal parasites (e.g. reduction of number of infective larvae in feces or pasture, reduction of number of eggs in feces or pasture, reduction of parasite load, reduction of deworming frequency). Only papers that quantified the outcomes with figures were included. 757 758 759 760 761 762 32 763 764 765 766 767 768 Table 2 – Description of studies’ characteristics 769 Fungi Parasites Administration and Dose Frequency and duration Evaluated Outcomes Ovine N = 15 Duddingtonia flagrans (15); Monacrosporium thaumasium (2); Arthrobotrys robusta (1); Clonostachys rosea (1) Haemonchus contortus (14); Trichostrongylus spp. (8); Teladorsagia spp. (6) and others 2x105 – 1x106 chlamydospores /kg live weight (10); Other forms (fixed dose of mycelia, conidia and chlamydospores) Single administration (2); Daily for 6 days to 5 months (10); Twice/week for 5 to 6 months (3) Eggs per gram (EPG) reduction Fecal L3 reduction Reduction of intestinal adult parasites Reduction of L3 /kg pasture dry matter Reduction in Tracer Sheep parasite load Bovines N = 10 D. flagrans (7); Pochonia chlamydosporia (2); Arthrobotrys cladodes (2); M. thaumasium (3); A. robusta (1) Cooperia spp. (8); H. contortus (7); Oesophagostomum spp. (7) and others 0,2 – 0,25 g /10 kg live weight (4); Other forms (fixed dose of mycelia and chlamydospores) Single administration (4); Daily for 10 days (1); Every 2 days for 30 days (1); Twice/week for 6 to 18 months (4) EPG reduction Fecal L3 reduction Reduction of L3 /kg pasture dry matter Equids N = 8 D. flagrans (7); M. thaumasium (3); A. robusta (1); Mucor circinelloides (2) Cyathostominae (7), Parascaris sp. (1); Strongyloides westeri (1) 1,5x105 – 2x106 chlamydospores /kg live weight (3); Other forms (fixed dose of mycelia and conidia) Single administration (3); Daily for 16 months (1); Twice/week for 21 days to 14 months (4) EPG reduction Fecal L3 reduction Reduction of L3 /kg pasture dry matter Egg Reappearance Period after Ivermectin administration Canids N = 7 D. flagrans (4); Arthrobotrys spp. (6); Monacrosporium spp. (6); P. chlamydosporia (2) Ancylostoma spp. (3); Angiostrongylus vasorum. (2); Toxocara canis (2) Mostly in vitro Single administration (7) Fecal L1 reduction Fecal L3 reduction Others 33 Caprine N = 5 D. flagrans (4); Arthrobotrys spp. (1) Trichostrongylus spp. (5); Haemonchus spp. (4); Teladorsagia spp. (3) and others 5x105 – 1x106 chlamydospores /kg live weight (4); 0,06g mycelia /kg live weight (1) Single administration (1); Daily for 27 days to 5 months (3); Twice/week for 6 months (1) EPG reduction Fecal L3 reduction Reduction of intestinal adult parasites Reduction of L3 /kg pasture dry matter Reduction in Tracer Goat parasite load Swine N = 2 D. flagrans (2) Oesophagostomum spp. (2) Fixed dose of chlamydospores and mycelia (2); 5x105 chlamydospores /kg live weight (1) Single administration (2) Fecal L3 reduction Other N = 6 D. flagrans (5); P. chlamydosporia (2); M. circinelloides (2) Varies with animal species Varies with animal species Single administration (4); Daily for 7 days (1); Every 2 days for 3 years (1) EPG reduction Fecal L3 reduction Reduction of L3 /kg pasture dry matter Others 770 N – Number of studies 771 Between brackets are the number of studies with each characteristic 772 EPG – Eggs per gram of feces 773 L1, L3 – parasite larval stages 774 Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: *Conflict of Interest