Biological control of soil transmitted helminths (STHs) in a zoological park by using saprophytic fungi

1 Biological control of soil transmitted helminths (STHs) in a zoological park 1 by using saprophytic fungi 2 J.A. Hernández1, C.F. Cazapal-Monteiro1, F.L. Arroyo1, M.I. Silva1, A.M. Palomero1, A. Paz3 Silva1, R. Sánchez-Andrade1, M.S. Arias1,*. Control of Parasites Group (COPAR, GI-2120). 4 5 1Department of Animal Pathology, Faculty of Veterinary, University of Santiago de 6 Compostela, 27002-Lugo (Spain). 7 8 9 * Corresponding author. 10 E-mail address: mar[email protected] (M.S. Arias) 11 12 13 2 Abstract 1 Toxascaris leonina and Trichuris sp. are soil-transmitted helminths (STHs) infecting domestic 2 and wild mammals. The antagonistic effect of the saprophytic filamentous fungi Mucor 3 circinelloides and Verticillium sp. was examined on eggs of T. leonina passed in the feces of 4 captive lynxes (Lynx lynx) kept in a zoological park. The activity of M. circinelloides and 5 Trichoderma atrobrunneum was tested on eggs of Trichuris sp. shed by captive dromedaries 6 (Camelus dromedarius). The parasiticide activity was assessed by measuring the ovistatic 7 (delayed development) and ovicidal (non-viability) effects on eggs placed in Petri plates, and 8 by spraying spores directly onto fecal samples. 9 An ovicidal type 3 effect (hyphae adhere to the eggshells, penetrate and destroy the inner 10 embryo) was observed in the Petri plates with M. circinelloides, Verticillium sp. and T. 11 atrobrunneum. Development of eggs of T. leonina and Trichuris sp. in the feces was delayed 12 in the presence of all fungi, and one third remained at the stage of zygote. A significant 13 reduction of T. leonina viable eggs was recorded in the feces sprayed spores of M. 14 circinelloides (58%) or Verticillium sp. (67%). Fifty percent of the eggs of Trichuris sp. 15 became into non-viable by 30 days after the exposure to either M. circinelloides or T. 16 atrobrunneum. It is concluded that distribution of the filamentous fungi M. circinelloides, 17 Verticillium sp. and T. atrobrunneum constitutes a novel approach to conduct the biological 18 control of the STHs T. leonina and Trichuris sp. affecting wild animals captive in a zoological 19 park. 20 21 Keywords: soil-transmitted helmiths, egg-parasitic fungi, prevention, Lynx lynx, Camelus 22 dromedaries, zoo 23 24 3 1. Introduction 1 Soil-transmitted helminths (STHs) are parasites involving roundworms (ascarids), 2 hookworms and whipworms which can affect animals and humans. Infection occurs through 3 the accidental ingestion of infective eggs in contaminated soil or food (Vandemark et al., 4 2010; Elsheika, 2011), which develop in the feces from unembryonated eggs shed by adult 5 female worms localized in the intestine (Mateus et al., 2014; Hoopes et al., 2015). 6 Embryonation happens in the feces or ground after several weeks, resulting in that a second 7 stage larva (L2) originates inside the eggs of ascarids, which becomes into infective (Hendrix, 8 2014), whereas in the case of whipworms the eggs are infective when a L1 larva develops 9 inside (Felsmann et al., 2017). The role of rodents as paratenic hosts has been indicated in the 10 transmission of Toxocara and Toxascaris (Okulewicz et al., 2012), and thus it has been 11 pointed that the cycles of these nematodes could be considered as non-strictly monoxenous 12 (Reperant et al., 2007). 13 Some ascarid species have zoonotic potential, as Toxocara canis, T. cati, Baylisascaris 14 procyonis, Ascaris suum or Toxascaris leonina, frequently detected in domestic (cats, dogs, 15 pigs) and wild mammals (foxes, wolves, lynxes, raccoons) (Okulewicz and Buńkowska, 16 2009; Dado et al., 2012; Carver et al., 2012; Beiromvand et al., 2013; Neves et al., 2014; 17 Figueiredo et al., 2016). The zoonotic role of Trichuris spp. has been also reported (Gałęcki et 18 al., 2015; Felsmann et al., 2017; Gawor and Borecka, 2017). 19 As indicated for other STHs, control of ascarids regularly comprises the administration 20 of anthelmintics together with adequate hygiene (Alemu et al., 2011). Despite successful 21 deworming is frequently administered among captive wild carnivores in zoo gardens (lions, 22 lynxes, foxes, wolves), they become infected again by helminths because their maintenance in 23 the same parcels enhances that ground is permanently contaminated by eggs passed in the 24 4 feces (Fagiolini et al., 2010; Maesano et al., 2014). In the same way, frequent infection by 1 Trichuris sp. in dromedaries has been also described (Gurler et al., 2010; Eo et al., 2014). 2 The eggs of ascarids and whipworms are very resistant to chemical and climatic factors, thus 3 can remain infective in the soil for several years (Gavin et al., 2005; Overgaauw and van 4 Knapen, 2013; Gałęcki et al., 2015; Gawor and Borecka, 2017). Nevertheless, there is 5 insufficient information regarding measures to reduce the presence of infective stages in the 6 ground. Prior investigations reported that certain saprophytic soil fungi perform an 7 antagonistic activity on eggs of T. canis and A. suum, based on their ability to adhere to the 8 eggshell, penetrate and destroy the embryo inside (Carvalho et al., 2010; Cortiñas et al., 9 2015). In this way, significant reductions on egg-viability have been recorded both in the soil 10 and in feces of parasitized animals by using Pochonia chlamydosporia, Trichoderma sp. and 11 Mucor circinelloides, saprophytic filamentous fungi harmless for humans, animals and plants 12 (Maciel et al., 2012; Arias et al., 2013a; Cazapal-Monteiro et al., 2015). 13 Few investigations have been carried out concerning biological control of parasites 14 among captive wild animals kept in zoo gardens, involving some ovicide fungi (Mucor 15 circinelloides, Paecilomyces lilacinus and Verticillium sp.) against Baylisascaris procyonis 16 (Cazapal-Monteiro et al., 2015) or a trapping nematophagous fungus (Duddingtonia flagrans) 17 against strongyles (Terry, 2009; Arias et al., 2013b). The usefulness of the fungi M. 18 circinelloides and Verticillium sp. to conduct biological control of T. leonina eggs excreted in 19 the feces of lynxes captive in a zoological park has been assayed in the present study. The 20 activity of M. circinelloides and Trichoderma atrobrunneum was tested on eggs of Trichuris 21 spp. shed in the feces of confined dromedaries. 22 23 2. Material and methods 24 2.1. “Marcelle Natureza” Zoological Park 25 5 The present investigation was conducted in the “Marcelle Natureza” Zoological Park 1 (43º4’14.71’’ N, 7º37’53.50’’ W; Outeiro de Rei, Lugo, NW Spain), where a number of 150 2 animal species are maintained, most of them in fenced parcels. 3 Three adult Eurasian lynxes (Lynx lynx; one male and two females) are housed in a 0.5 4 ha plot with vegetation and trees. Food is provided to lynxes in feeders placed in individual 5 cages of concrete soil and iron walls, and each one takes it from the same cage. 6 Four adult dromedaries (Camelus dromedarius; one male and three females) are 7 maintained in a 0.6 ha sandy area with palm trees, feeders and drinkers. There is also a large 8 shelter for the dromedaries can keep under adverse climatic conditions. All the cages and 9 plots in the zoo are cleaned daily with water, and feces removed prior to the visitors arrive. 10 11 2.2. Control of parasites 12 Two parasiticide treatments are yearly administered in March and in September, 13 unless additional treatment is considered needed in the basis of the coprological tests. 14 Fecal analyses are performed monthly in the COPAR Lab (Control of Parasites, Faculty of 15 Veterinary, University of Santiago de Compostela, Spain), located at a distance of 12 Km 16 from the zoological park. The apical region of the feces is taken directly from the ground of 17 the paddocks and then examined by means of flotation technique with saturated sodium 18 chloride solution (ρ= 1.20 g/cm3), sedimentation and migration tests (Arias et al., 2013b). 19 Deworming of lynxes consists of giving, for three consecutive days, anthelmintic 20 granules (10 mg Fenbendazole/Kg body weight (bw); Panacur® Granules 22.2%, Intervet 21 GesmbH, Vienna, Austria) previously mixed with the food. Consequently, lynxes remain 22 briefly caged every day until all the premixed food is eaten to ensure that each individual 23 ingests the prescribed dosage. Despite T. leonina eggs are not observed by 15 days post24 treatment and successful of the deworming is concluded, eggs appear again by 2–3 months 25 6 after treatment and counts increase until lynxes are dewormed again (unpublished data). It has 1 been observed that lynxes defecate mainly in two zones of the parcel, under a tree. 2 Dromedaries are provided an oral dosage of 10 mg Fenbendazole/Kg bw (Panacur 3 suspension 10%; MSD Animal Health, Madrid, Spain) during five consecutive days. 4 5 2.3. Fungal specimens 6 The CECT 208724 strain of Mucor circinelloides, CECT strain of Trichoderma 7 atrobrunneum and a wild-type strain of Verticillium sp. were utilized in the present research. 8 These specimens were isolated from feces of domestic and wild animals (Hernández et al., 9 2017), identified according to their morphology (de Hoog, 2000) and cultured individually in 10 a submerged medium for the production of spores of different fungal species (COPFr) 11 (Cazapal-Monteiro et al., 2015). 12 13 2.4. Collection of feces 14 Fresh fecal samples were collected directly from the soil in the two parcels, early in 15 the morning. The samples were kept at 4 ºC and brought to the Lab. Eggs of T. leonina were 16 detected in all the samples from the lynxes, and eggs of Trichuris sp. in those of dromedaries. 17 18 2.5. Purification of eggs of helminths 19 For each of the animal species, a total of 16 samples of feces (200 g approximately) 20 taken from the ground were pooled and mixed with 2 L distilled water (Cazapal-Monteiro et 21 al., 2015). This solution was filtered consecutively through wire sieves of 300, 150 and 40 µm 22 in a decreasing order. The sediment obtained between the two last wire sieves was collected 23 and resuspended in distilled water, then transferred to acetate tubes and centrifuged at 2500 24 rpm for 5 min. The supernatant without eggs was removed by aspiration and distilled water 25 7 added for sediment resuspension. These steps were performed until the supernatant was 1 wholly transparent, then acetate tubes were filled with saturated NaCl solution and 2 centrifuged at 2500 rpm for 5 min. The exceeding supernatant with eggs (around 2 mL) was 3 collected by repeated pipetting, washed with distilled water and centrifuged again at 2500 rpm 4 for 5 min. After discarding the supernatant without eggs, the sediment was cleaned with 5 distilled water and centrifuged three times to eliminate NaCl residues. Finally, 50 µl aliquots 6 were placed on glass slides with glass covers and observed under an optical microscope 7 (Leica DM2500; 20-40x) to estimate the numbers of eggs per mL. The aqueous solutions 8 were adjusted to 400 eggs of T. leonina or Trichuris sp./mL and kept at 4 ºC. 9 10 2.6. Experimental design 11 The fungal activity was assayed in Petri plates to establish the antagonistic effect on 12 the eggs of the helminths, and in fecal pats to ascertain the activity of the filamentous fungi on 13 eggs in fecal samples. 14 15 2.6.1. Plate assays 16 Thirty Petri plates with agar-water medium (2%) were added 200 eggs of T. leonina in 17 one edge, and 0.5 mL of liquid culture containing about 2 x·106 spores of each fungus (n = 10 18 plates M. circinelloides, and n = 10 Verticillium sp.) on the opposite side. Ten plates serving 19 as controls were also placed 200 eggs in one edge, plus 0.5 mL distilled water on the opposite 20 side. 21 Similarly, 30 Petri plates with agar-water medium (2%) were placed 200 eggs of 22 Trichuris sp. Ten plates were also provided 0.5 mL of liquid culture containing about 2·x 106 23 spores of M. circinelloides and other 10 dishes 2·x 106 spores of T. atrobrunneum. Other 10 24 plates serving as controls were placed eggs of Trichuris sp. and 0.5 mL distilled water. 25 8 Petri dishes were maintained at room temperature (15-20 ºC) and darkness for 22 days. 1 During this period, plates were examined daily under an optical microscope at 10x and 40x 2 objectives (Leica DM2500) until a minimum of 120 eggs were visualized in each. The fungal 3 effect was defined as type 1 when eggshells remained unaltered with hyphae adhered; type 2 4 if disrupted eggshells without hyphal penetration were observed, and type 3 in the presence of 5 eggshell injured, infiltration of fungal hyphae and embryo destruction (Lýsek and Krajcí, 6 1987; Cazapal-Monteiro et al., 2015). 7 8 2.6.2. Fecal pats assays 9 The feces collected from the soil of the lynxes parcel where pooled and divided into 10 two groups. Five grams of feces were placed in 38 polypropylene translucent boxes (15 × 6 × 11 15 cm) (1.3 L volume) with a cover (Cazapal-Monteiro et al. 2015); twelve boxes (group 12 TlMc) were added 3 mL of liquid medium with 2 x 106 spores of M. circinelloides, other 12 13 boxes (group TlVe) received 3 mL of medium containing 2 x 106 spores of Verticillium sp.; 14 fourteen boxes were added 3 mL distilled water as controls (group TlC). 15 Similarly to that described before, after pooling the fresh samples of feces of 16 dromedary, a quantity of 5 g were placed in 30 boxes which were divided into three lots of 10 17 each. Group TcMc received 3 mL of medium with 2 x 106 spores of M. circinelloides, group 18 TcTa was added 2 x 106 spores of T. atrobrunneum, and group TcC was added 3 mL distilled 19 water as controls. 20 All the boxes were maintained outdoors in a wooded grass, and feces examined after 21 10, 20 and 30 days through the flotation test and sucrose saturated solution ( = 1.25 g/cm3). 22 Aliquots of 50 µL placed between a glass slide and a glass coverslip were examined at 20x 23 and 40x under a light microscope (Leica DM2500), until a minimum of 150 eggs were 24 visualized in each. 25 9 1 2.7. Ovicide and ovistatic effects 2 The effect of the soil fungi on the eggs of T. leonina and Trichuris sp. was ascertained 3 by measuring their viability and development rate. Eggs were considered viable when 4 showing no apparent damage and/or unaltered eggshells with attached hyphae (type 1 effect) 5 (Cazapal-Monteiro et al., 2015), and the percentage of reduction estimated according to the 6 formula: 7 8 %Viability reduction= [1 – (mean viable eggsday0 / mean viable eggsday of assay)] × 100 9 10 With the aim to assess the ovistatic effect, the development of the eggs of T. leonina 11 and Trichuris sp. was measured. Therefore, viable eggs without cellular division (zygote) 12 were classified as non-developed, whereas eggs were considered as developed if contained a 13 morula, blastula, gastrula or larva inside. 14 15 2.8. Statistical analysis 16 Normality of data collected in the current investigation was assessed by performing the 17 Kolmogorov-Smirnov test. Because Z values resulted lower than 0.05, it was concluded that 18 data do not have a normal distribution. Besides this, the Levene's test demonstrated the 19 variances were not homogeneous (P < 0.05). Thus, non-parametric tests (Kruskal-Wallis and 20 Mann-Whitney U) were performed at a significance level of P < 0.05 (Thrusfield, 2007). 21 All the tests were carried out by using the statistical package SPSS, version 20 (IBM SPSS 22 Inc., Chicago, IL, USA). 23 24 3. Results 25 16 absence of side effects regarding respiration, digestion, reproduction or even the skin, among 1 domestic and wild captive animals provided spores of M. circinelloides and/or D. flagrans 2 (Arias et al., 2013a; Hernández et al., 2016). 3 Biological control of STHs appears very useful in zoological parks, because of data in 4 the present investigation showed the capability of three innocuous saprophytic fungi, M. 5 circinelloides, T. atrobrunneum and Verticillium sp., to reduce the presence of infective stages 6 of T. leonina and Trichuris sp. by more than half, as well as to delay the development to the 7 infective stage by more than one third, in feces of captive lynxes and dromedaries. One 8 interesting question relies on the appropriate way to ensure that fungal spores are properly 9 spread in the feces (Despommier, 2003). Due to fungal spores in the current study have been 10 obtained in a submerged culture, direct spraying on feces appears very useful especially when 11 animals are maintained in small/medium size parcels. 12 13 5. Conclusions 14 Proper preventive strategies are needed to support the control of STHs in animal 15 species captive in zoological parks. Results obtained in the current research led us to conclude 16 the usefulness of hand spraying spores of filamentous fungi as M. circinelloides, T. 17 atrobrunneum or Verticillium sp. directly on feces of infected animals to reduce the viability 18 of their eggs and/or their development to the infective stages. Moreover, spores should be also 19 sprayed on the ground after the routinely collection of feces from the plots, minimizing thus 20 the risk of infection by soil transmitted helminths. 21 22 6. Conflict of interest 23 The final article has been approved by all authors, whose assert the absence of any 24 financial or personal interests that could improperly influence the present paper. 25 17 1 7. Acknowledgments 2 This research was partly supported by the Research Project CTM2015-65954-R 3 (Spanish Ministry of Economy and Competitiveness; FEDER). María Sol Arias Vázquez is 4 recipient of a Ramón y Cajal (Spanish Ministry of Economy and Competitiveness) contract 5 and Cristiana F. Cazapal-Monteiro is recipient of a postdoctoral research fellowship (Xunta 6 de Galicia, Spain). These funding sources had no involvement in study design, collection, 7 analysis and interpretation of data, writing of the report and in the decision to submit the 8 article for publication. 9 All authors have approved the final article. 10 11 References 12 Alemu, A., Atnafu, A., Addis, Z., Shiferaw, Y., Teklu, T., Mathewos, B., Birhan, W., 13 Gebretsadik, S., Gelaw, B., 2011. 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Percentages of non-viable eggs of Toxascaris leonina in the feces of lynxes captive in a zoological park. TlC: control (untreated) group; TlMc: feces hand sprayed spores of Mucor circinelloides; TlVe: feces hand sprayed spores of Verticillium sp. Bars represent the 95% Confidence Interval for mean. Statistical analysis: a means statistical differences between TlMc and TlC; b indicates significant differences between TlVe and TlC. Fig. 2. Non-viable egg of T. leonina due to hyphae of the saprophytic fungus Mucor circinelloides attached to the shell, penetrated and destroyed the inner embryo. Fig. 3. Non-viable egg of T. leonina in feces of captive lynxes hand sprayed spores of Verticillium sp. Fig. 4. Percentages of non-viable eggs of Trichuris sp. in the feces of dromedaries captive in a zoological park. TcC: control (untreated) group; TcMc: feces hand sprayed spores of Mucor circinelloides; TcVe: feces hand sprayed spores of Verticillium sp. Bars represent the 95% Confidence Interval for mean. Statistical analysis: a means statistical differences between TlMc and TlC; b indicates significant differences between TlVe and TlC. Fig. 5. Hyphae of Mucor circinelloides were capable to penetrate eggs of Trichuris sp. by removing one of the polar plugs. Fig. 6. Non-viable egg of Trichuris sp. in feces of captive lynxes hand sprayed spores of Trichoderma atrobrunneum. Fig. 7. Egg of T. leonina in feces of captive lynxes hand sprayed spores of Verticillium sp., showing two different sized blastomeres. Fig. 8. Vacuolization was observed in eggs of Trichuris sp. exposed to Mucor circinelloides. Table Captions Table 1. Percentages of the different stages of development of eggs of Toxascaris leonina in the feces of lynxes captive in a zoological park. TcC: control (untreated) group; TcMc: feces hand sprayed spores of Mucor circinelloides; TcVe: feces hand sprayed spores of Verticillium sp. Table 2. Percentages of the different stages of development of eggs of Trichuris sp. in the feces of dromedaries captive in a zoological park. TcC: control (untreated) group; TcMc: feces hand sprayed spores of Mucor circinelloides; TcVe: feces hand sprayed spores of Verticillium sp.