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Two different formulations containing a blend of spores of the filamentous fungi Mucor circinelloides and Duddingtonia flagrans have been assayed against trichostrongylids in wapitis captive in a grassy parcel from a zoological park and dewormed with fenbendazole. One approach (sprayed-on pellets) consisted of providing them, during 10 months, nutritional pellets sprayed the spores just before to be ingested, and the other (formulated pellets) involved the administration of pellets industrially manufactured with the spores for an identical period. Data collected on wapitis treated with fenbendazole without receiving spores during a 10-months interval were considered as controls. The effect of these strategies was evaluated through the analysis of feces collected directly from the soil, by estimating the reduction of the counts of eggs of trichostrongylids per gram of feces (EPG). The possibility of side effects in the digestive tract, respiratory apparatus or on the skin was assessed. The coprological analyses showed that, despite the successfulness of fenbendazole, the values of trichostrongylids egg-output increased four months later above 300 EPG. Through the administration of pellets sprayed spores, a significant EPG reduction of 69% (30–100) was recorded, and 71% (36–100) by giving the wapitis pellets industrially enriched with the spores. No unfavorable effects were observed among the wapitis taking the spores. It is concluded that ingestion of a blend of spores of M. circinelloides and D. flagrans every two days affords a beneficial long-term effect on controlling the infection by trichostrongylids in wild captive animals.

Soil-transmitted helminths (STHs) are parasites transmitted through contact with soil contaminated with their infective eggs/larvae. People are infected by exposure to human-specific species or animal species (zoonotic agents). Fecal samples containing eggs of Ascaris suum or Lemurostrongylus sp. were sprayed with spores of the soil saprophytic filamentous fungi Clonostachys rosea (CR) and Trichoderma atrobrunneum (TA). The antagonistic effect was assessed by estimating the viability of eggs and their developmental rate. Compared to the controls (unexposed to fungi), the viability of the eggs of A. suum was halved in CR and decreased by two thirds in TA, while the viability of the eggs of Lemurostrongylus sp. was reduced by one quarter and one third in CR and TA treatments, respectively. The Soil Contamination Index (SCI), defined as the viable eggs that attained the infective stage, reached the highest percentages for A. suum in the controls after four weeks (66%), with 21% in CL and 11% in TA. For Lemurostrongylus sp., the values were 80%, 49%, and 41% for control, CR and TA treatments, respectively. We concluded that spreading spores of C. rosea or T. atrobrunneum directly onto the feces of animal species represents a sustainable approach under a One Health context to potentially reduce the risk of zoonotic STHs in humans This research was funded by the Research Projects CTM2015-65954-R and RYC-2016-21407 (Ministerio de Economía y Competitividad, Spain; FEDER), and ED431F 2018/03 (Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia, Spain). María Sol Arias Vázquez is recipient of a Ramón y Cajal (Ministerio de Economía y Competitividad, Spain) contract SI

The integrated control of strongyles was assayed for a period of three years in wild equids (zebras, European donkeys, and African wild asses) captive in a zoo and infected by strongyles. During three years control of parasites consisted of deworming with ivermectin + praziquantel; equids also received every two days commercial nutritional pellets containing a blend of 104 - 105 spores of the fungi Mucor circinelloides + Duddingtonia flagrans per kg meal. Coprological analyses were done monthly to establish the counts of eggs of strongyles per gram of feces (EPG). The reductions in the fecal egg counts (FECR) and in the positive horses (PHR) were calculated fifteen days after deworming; the egg reappearance period (ERP) and the time elapsed from the previous deworming (TPD) were also recorded. Four anthelmintic treatments were administered during the assay, three times throughout the first 2 yrs, and another treatment during the last one. FECR values of 96-100% and 75-100% for the PHR were recorded. The ERP oscillated between eight and twenty-eight weeks, and the TPD ranged from four to eighteen months, increasing to the end of the trial. No side effects were observed in any of the equids. It is concluded that integrated control of strongyles among equids captive in a zoo can be developed by anthelmintic deworming together with the administration of pellets manufactured with spores of parasiticide fungi every two days. This trial was partly supported by the Research Project CTM2015-65954-R (Ministerio de Economía y Competitividad, Spain; FEDER). Dr. María Sol Arias Vázquez is recipient of a Ramón y Cajal contract (Spanish Ministry of Economy and Competitiveness), and Dr. C. F. Cazapal-Monteiro is beneficiary of a postdoctoral research fellowship (Xunta de Galicia, Spain) SI

An ATP diphosphohydrolase was identified in the plasma membranes isolated from promastigote forms of Leishmania amazonensis. Both ATP and ADP were hydrolysed at similar rates by the enzyme. Other nucleotides such as UTP, GTP and CTP were also degraded, revealing a broad substrate specificity. Adding ATP and ADP simultaneously, the amount of hydrolysis achieved was compatible with the presence of a single enzyme. ATPase activity was not affected by addition of vanadate, ouabain, thapsigargin, dicyclohexylcarbodiimide, oligomycin and bafilomycin A, thus excluding involvement of P-, F- and V-type ATPases. The effects of pH in the range 6·5–8·5 were examined using ATP or p-NPP as substrate. At pH 7·4, the phosphatase activity decreased, and did not show a significant contribution to ATP hydrolysis. In addition, the enzyme was not inhibited by levamisole and ammonium molybdate, excluding alkaline phosphatase and nucleotidase activities, respectively. Sodium azide (5–10 mM) caused inhibition of the ATP and ADP hydrolysis in a dose-dependent manner. Calcium was the best activating metal ion for both ATPase and ADPase activities. Ultrastructural cytochemical microscopy showed ATP diphosphohydrolase on the surface and flagellar pocket of the parasite. We have proposed that L. amazonensis ATP diphosphohydrolase may participate in the salvage pathway of nucleosides.