A preliminary study of the biological control of strongyles affecting equids in a zoological park

Accepted Manuscript A preliminary study of the biological control of strongyles affecting equids in a zoological park Mariasol Arias, Cristiana Cazapal-Monteiro, Esther Valderrábano, Silvia Miguélez, José Luis Rois, María Eugenia López-Arellano, Luis Madeira de Carvalho, Pedro Mendoza de Gives, Rita Sánchez-Andrade, Adolfo Paz-Silva PII: S0737-0806(13)00336-5 DOI: 10.1016/j.jevs.2013.04.013 Reference: YJEVS 1571 To appear in: Journal of Equine Veterinary Science Received Date: 24 October 2012 Revised Date: 6 December 2012 Accepted Date: 17 April 2013 Please cite this article as: Arias M, Cazapal-Monteiro C, Valderrábano E, Miguélez S, Rois JL, LópezArellano ME, Madeira de Carvalho L, Mendoza de Gives P, Sánchez-Andrade R, Paz-Silva A, A preliminary study of the biological control of strongyles affecting equids in a zoological park, Journal of Equine Veterinary Science (2013), doi: 10.1016/j.jevs.2013.04.013. 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MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 1 A PRELIMINARY STUDY OF THE BIOLOGICAL CONTROL OF STRONGYLES AFFECTING EQUIDS IN A ZOOLOGICAL PARK Mariasol Arias1, Cristiana Cazapal-Monteiro1, Esther Valderrábano2, Silvia Miguélez1, José Luis Rois2, María Eugenia López-Arellano3, Luis Madeira de Carvalho4, Pedro Mendoza de Gives3, Rita Sánchez-Andrade1, Adolfo Paz-Silva1,♦ ♦♦ ♦. 1Equine Diseases Study Group (COPAR), Parasitology Diseases, Animal Pathology Department, Veterinary Faculty, Santiago de Compostela University, 27002-Lugo (Spain); 2Marcelle Natureza Zoological Park, Outeiro de Rei, 27122-Lugo (Spain); 3National Disciplinary Centre of Veterinary Parasitology Research, INIFAP, Cuernavaca (México); 4CIISA/FMV/UTL, Pólo Universitário do Alto, Avenida da Universidade Técnica, 1300, Lisboa (Portugal). ♦ Corresponding author: Adolfo Paz-Silva, DVM, PhD, DipEVPC. Epidemiology and Zoonoses, Veterinary Faculty, Campus Universitario s/n, 27002-Lugo (Spain). Phone: 34982822126. E-mail: [email protected]. MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 2 SUMMARY The main goal in this research was to determine the beneficial effect of incorporating biological procedures to the parasite control programs on equids from Zoological Parks. Two trials were developed on Equus quagga, E. asinus and E. africanus asinus. The first (September 2010 to August 2011) consisted of chemotherapy (ivermectin + praziquantel) only, and the second (September 2011 to September 2012) in the administration of chemotherapy and chlamydospores of the nematophagous fungus Arthrobotrys (Duddingtonia) flagrans. The effect of these measures was evaluated by the estimation of the reduction in the faecal egg-counts (FECR). In the first trial, 100% FECR values were achieved fifteen days after treatment in all the animals. The Egg Reappearance Period (ERP) was 2-3 months for the equids, and all of them were passing strongyle eggs in the faeces 2-4 months after their deworming. In the second experiment, the FECR values were 100% in the three species. An ERP of 3 months in the European donkeys, 4 months in the Africans and 6 in the zebras was recorded. All the equids were positive to the coprological flotation test 4-8 months after the anthelmintic administration. This preliminary study demonstrates the incorporation of chlamydospores of nematophagous fungus as A. flagrans appears highly promising to reduce the infective stages of the strongyles affecting captive animals, but the experimental design precludes true determination of whether the treatment is fully efficacious. Keywords: equids, zoological park, strongyles, biological control, Arthrobotrys flagrans MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 3 1. INTRODUCTION The role of zoological parks has changed significantly in the last few decades. The former objective consisting of displaying wild animals for visitor entertainment has been replaced by the conservation of endangered species, education, and research [1]. This change has required large modifications to zoological parks to ensure animals have an appropriate habitat, thus making breeding possible in many cases. Besides the transformation of the exhibits into more adequate enclosures, attention is also given to enrichment, motivating animals to search for food and explore their environment, and improved veterinary care. As a consequence, preservation of several endangered or vulnerable species (African wild ass (Equus africanus asinus), Dromedary (Camelus dromedarius), plains zebra (Equus quagga), and eland (Tragelaphus oryx)) has been successful [2]. Animals in zoological parks may be more likely to become infected by parasites if they are housed on land previously occupied by domestic animals [3, 4]. Grazing animals are at an elevated risk of parasitic infection because of the presence of oocysts, eggs, cysts or larvae in the soil and/or herbage, which can be ingested simultaneously when feeding on pasture [5, 6]. Helminths (cestodes, trematodes and nematodes) are commonly reported in grazing horses, especially strongylid nematodes [7, 8]. Their generalized life cycle begins with the release of unembryonated eggs by adult worms located in the gastrointestinal tract, which are passed in the faeces. After their embryonation in the faecal pat, eggs hatch to first-stage larvae (L1), then moult into second-stage larvae (L2) and finally to third-stage larvae (L3), the infective phase [9]. Third stage larvae leave the faecal pats and move to adjacent plants, which are subsequently ingested by the animals on pasture. Strongyles in the equids are responsible for significant inflammation, unexplained weight loss, diarrhea, poor hair coat, and in extreme cases death. MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 4 Control of parasites in horses is most commonly based on anthelmintic treatment, but the absence of useful measures against the free-living stages in the environment makes it difficult to clear the infection. This becomes very important if pasture rotation can not be observed, due to a horse infected with strongyles can be passing millions of eggs/ day in the dung. Arthrobotrys (formerly Duddingtonia) flagrans is a predatory fungus very frequently found in natural and agricultural soils [10]. This fungus can live either saprotrophytically or predatorily (in the presence of larval nematodes). A. flagrans is characterized for producing thick-walled chlamydospores in abundance, which are eliminated intact in the faeces of herbivores after passing through their gastrointestinal tract [11]. As a consequence, the fungus establishes in the environment and develops an extensive hyphal system, together with traps at intervals along the hyphae, with the purpose to capture larval nematodes for obtaining N and C. The objective of this study was to investigate the effect of incorporating biological procedures to the parasite control programs for equids at a zoological park. In the first trial, an anthelmintic mixture was administered to the animals, whereas the second trial utilized anthelmintic treatment plus A. flagrans chlamydospores. 2. MATERIAL & METHODS 2.1. Marcelle Natureza Zoological Park Marcelle Natureza is a 20 Ha zoological park located in NW Spain (Outeiro de Rei, Lugo) (43° 4' 14.71" N, 7° 37' 53.50" W). Collection animals live in fenced, semi-free ranging exhibits of various sizes. The animals are routinely dewormed in spring and autumn by adding granulated anthelmintic preparations to the concentrate (feedstuff) portion of the diet, on the basis of coprological determinations previously performed. Deworming is also provided when diarrhoea is observed, mainly from late spring to early autumn. MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 5 Removal of faecal material is performed daily in the paddocks, paying special attention to avoid or at least minimize their presence during the visiting hours. 2.2. Equids Four equid species are maintained in the zoological park, including Equus quagga (plains zebra), E. caballus (Falabella miniature horse), E. asinus (European donkey) and E. africanus asinus (African wild ass). The Falabella miniature horse was not included in this study, as it is singly housed. There are 3 adult zebras (3-10 yr; 1 stallion and 2 mares) housed in a 4038 m2 meadow. The European donkeys (3-9 yr; 3 stallions and 3 mares) are housed in a 2015 m2 pasture. The African wild assesses (3-6 yr; 1 stallion and 5 mares) are maintained in a 1000 m2 parcel where sand is the main soil component throughout the year. For this reason, these animals are given herbage cut from areas both inside and outside of the park. All equids have access to pasture and are supplemented with pellets each two days. Water is available ad libitum. 2.3. Arthrobotrys flagrans (Af) Chlamydospores were produced, harvested and managed in Petri dishes (9 cm diameter) containing wheat meal agar [12]. The medium was comprised of 20 g agar, 25 g wheat flour and 1 L distilled water. 2.4. Experimental design Between September 2010 and September 2012, two trials were carried out. Faecal samples were collected directly from the soil of the paddocks where the zebras, European and African assesses were housed. MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 6 Excluding any type of immobilization of the animals due to their avoidance of stressful situations, faecal samples were collected from the soil. An equal number of stools to the animals in each enclosure were obtained. Collection of faeces was done early in the morning (0800-0900), prior to the daily cleaning of the paddocks by the animal keepers. A concise explanation about the need for taking the samples in different places of the paddocks was given to the keepers. This experimental design makes difficult true determination of the treatment successfulness, because of the impossibility for having control animals under the same conditions than those receiving chlamydospores with the feedstuff. a) Anthelmintic only (September 2010 to August 2011) In September 2010 and March 2011, after faecal analysis, the equids were dewormed by the application of ivermectin + praziquantel (Equimax®, Virbac, Spain) at a dosage of 1.07 g gel / 100 kg body weight. Body weights were estimated by visual examination. b) Anthelmintic + biological control (September 2011 to September 2012) Anthelmintic therapy, as described previously, was administered to the equids in September 2011. A. flagrans chlamydospores were then administered bi-weekly to the equids. By considering previous investigations [13], the total quantity of spores required for each individual was 2 x 106 Af chlamydospores kg bw-1. This was delivered orally by carefully dissolving the spores into 50 mL of water and then mixing with the feedstuff immediately prior to feeding the equids. 2.5. Faecal analyses MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 7 Faecal samples were applied in duplicate by using copromicroscopic techniques [14]. Five grams of faeces were analyzed by flotation to determine the presence of protozoan oocysts and eggs of cestodes and nematodes. The same quantity of faeces was processed by sedimentation to ascertain the existence of trematode infections. A McMaster technique with a sensitivity of 10 eggs/g faeces (EPG) was employed for the egg counting [14]. For determining the presence of lungworm infection, 10 g faeces were analyzed by means of the Baermann method. Coprocultures were performed to determine the genera of strongyles affecting the equids [15]. Briefly, 20 g pools of the faeces were prepared from samples collected monthly from each enclosure. These were cultured at 22-24ºC for 15 days, and then larvae were recovered by using the Baermann technique, and their identification done according to morphological keys [16, 17]. 2.6. Efficacy of deworming procedures The success of the two trials was determined by faecal analysis. Eggs per gram (EPG) values were determined on the day of treatment, 15 days after treatment, and then once per month. The Faecal Egg Count Reduction (FECR) was calculated by using the following formula [15]: FECR (%) = [1 – (FECpost-treatment / FECpretreatment)] x 100 2.7. Climatic pattern For gaining information about possible climatic variations between the two years of study, the values of maximal temperature, minimal temperature and relative humidity were monthly obtained from an automated meteorological station near (10 Km) to the Park. MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 8 2.8. Statistical analyses The values of FECR were expressed as percentages (Table 1). ANOVA was utilized to examine total differences among the different equids. Differences were considered significant when P< 0.05. 3. RESULTS 3.1. Climatic pattern As presented in Figure 2, a similar climatic pattern was established in the two years of study, and significant differences in the values of maximal temperature, minimal temperature and relative humidity were not observed. 3.2. Parasitological examinations At the beginning of the study, strongyle eggs were observed in the faeces of the equids. No protozoa, trematodes, cestodes or lungworms were found in any of the examined stool samples. Specimens belonging to the genera Cyathostomum sensu lato, Trichostrongylus, Gyalocephalus and Poteriostomum were identified in the coprocultures. 3.2. Administration of anthelmintics only (September 2010 to August 2011) As shown in Figure 3, egg-output values higher than 500 EPG were recorded in September 2010 for all the equids. The FECR showed a 100% efficacy 14 days after the administration of anthelmintics to the equids. The Egg Reappearance Period (ERP) was 2 months for the European donkeys, and 3 months for the African asses and the zebras (Table 1). All the equids were positive to strongyle egg-excretion 3 months after their deworming. MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 1 [14] Francisco I, Sánchez JA, Cortiñas FJ, Francisco R, Mochales E, Arias M, Mula P, Suárez JL, Morrondo P, Díez-Baños P, Sánchez-Andrade R, Paz-Silva A. Clinical trial of efficacy of ivermectin pour-on against gastrointestinal parasitic nematodes in silvopasturing horses. Equine Vet J 2009;41:713-715. [15] Francisco I, Sánchez JA, Cortiñas FJ, Francisco R, Suárez J, Cazapal C, Suárez JL, Arias MS, Morrondo P, Sánchez-Andrade R, Paz-Silva A. Efficacy of ivermectin pour-on against nematodes infecting foals on pasture: coprological and biochemical analysis. J Equine Vet Sci 2011;31:530-535. [16] Lichtenfels JR, Kharchenko VA, Dvojnos GM. Illustrated identification keys to strongylid parasites (Strongylidae: Nematoda) of horses, zebras and asses (Equidae). Vet Parasitol 2008;156:4-161. [17] Cernea M, Madeira de Carvalho LM, Cozma V. Atlas of Diagnosis of Equine Strongyloidosis. London: Academic Press; 2008. [18] Rehbein S, Holste JE, Doucet MY, Fenger C, Paul AJ, Reinemeyer CR, Smith LL, Yoon S, Marley SE. Field efficacy of ivermectin plus praziquantel oral paste against naturally acquired gastrointestinal nematodes and cestodes of horses in North America and Europe. Vet. Ther. 2003;4:220-227. [19] Uhlinger CA. Evidence-based parasitology in horses. Vet Clin N Am Equine Pract 2007;23:509-517. [20] Eysker M, van Doorn DC, Lems SN, Weteling A, Ploeger HW. Frequent deworming in horses; it usually does not do any good, but it often harms. Tijdschr Diergeneeskd 2006;131:524-530. [21] Garretson PD, Hammond EE, Craig TM, Holman PJ. Anthelmintic resistant Haemonchus contortus in a giraffe (Giraffa camelopardalis) in Florida. J Zoo Wildl Med 2009;40:131-139. MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 1 [22] Floate KD. Endectocide use in cattle and fecal residues: environmental effects in Canada. Can J Vet Res 2006;70:1-10. [23] Larsen ML, Ritz C, Petersen SL, Nielsen MK. Determination of ivermectin efficacy against cyathostomins and Parascaris equorum on horse farms using selective therapy. Vet J 2011;188:44-47. [24] Chandrawathani P, Jamnah O, Waller PJ, Höglund J, Larsen M, Zahari WM. Nematophagous fungi as a biological control agent for nematode parasites of small ruminants in Malaysia: a special emphasis on Duddingtonia flagrans. Vet Res 2002;33:685-696. [25] Braga FR, Araújo JV, Silva AR, Araújo JM, Carvalho RO, Tavela AO, Campos AK, Carvalho GR. Biological control of horse cyathostomin (Nematoda: Cyathostominae) using the nematophagous fungus Duddingtonia flagrans in tropical southeastern Brazil. Vet Parasitol 2009;163:335-340. [26] Arias M, Suárez J, Cortiñas FJ, Francisco I, Suárez JL, Romasanta A, Cazapal-Monteiro C, Sánchez-Andrade R, Paz-Silva A. Restoration of fungal biota in the soil is essential to prevent infection by endoparasites in grazing animals. In: Paz-Silva A & Arias MS, editors. Fungi: Types, environmental impact and role in disease, 1st edn, Hauppauge (NY): Nova Science Publishers; 2011, p. 341-358. [27] Gómez-Rincón C, Valderrábano J, Uriarte J. Nematophagous fungi as control agents of gastrointestinal nematodes in small ruminants. In: Paz-Silva A & Arias MS, editors. Fungi: Types, environmental impact and role in disease, 1st edn, Hauppauge (NY): Nova Science Publishers; 2011, p. 359-375. 11. CAPTION FIGURES Figure 1: Values of climatic parameters from September 2010 to September 2012 in the Marcelle Zoological Park (Outeiro de Rei, NW Spain). MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 1 Figure 2: Dynamics of strongyle egg-output in equids at the Marcelle Natureza Zoological Park (Outeiro de Rei, NW Spain). IVM: ivermectin; PZQ: praziquantel.   : Zebra (Equus quagga);   : African Wild Ass (E. africanus asinus);   : European Donkey (E. asinus). Values are means plus 2 SD. Figure 3: Dynamics of strongyle egg-output in equids at the Marcelle Natureza Zoological Park (Outeiro de Rei, NW Spain) receiving a bi-weekly dosage of 2 x 106 Arthrobotrys flagrans chlamydospores kg bw-1.   : Zebra (Equus quagga);   : African Wild Ass (E. africanus asinus);   : European Donkey (E. asinus). Values are means plus 2 SD. Table 1: Strongyle Faecal Egg Count Reduction (FECR) and Positive Equids Reduction (PER) from equids housed at the Marcelle Natureza Zoological Park (Outeiro de Rei, NW Spain). MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 1 12. CONTRIBUTORS Mariasol Arias, Cristiana Cazapal-Monteiro, Esther Valderrábano, Silvia Miguélez, José Luis Rois, María Eugenia López-Arellano, Luis Madeira de Carvalho, Pedro Mendoza de Gives, Rita Sánchez-Andrade, Adolfo Paz-Silva. 1) Conception and design of the study Mariasol Arias, María Eugenia López-Arellano, Luis Madeira de Carvalho, Pedro Mendoza de Gives, Rita Sánchez-Andrade, Adolfo Paz-Silva. 2) Acquisition of data Mariasol Arias, Cristiana Cazapal-Monteiro, Esther Valderrábano, Silvia Miguélez, José Luis Rois. 3) Analysis and interpretation of data Mariasol Arias, María Eugenia López-Arellano, Luis Madeira de Carvalho, Pedro Mendoza de Gives, Rita Sánchez-Andrade, Adolfo Paz-Silva. 3) Redaction and revision of the article Mariasol Arias, Cristiana Cazapal-Monteiro, Esther Valderrábano, Silvia Miguélez, José Luis Rois, María Eugenia López-Arellano, Luis Madeira de Carvalho, Pedro Mendoza de Gives, Rita Sánchez-Andrade, Adolfo Paz-Silva. 4) Final approval of the version to be submitted MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 1 Mariasol Arias, Cristiana Cazapal-Monteiro, Esther Valderrábano, Silvia Miguélez, José Luis Rois, María Eugenia López-Arellano, Luis Madeira de Carvalho, Pedro Mendoza de Gives, Rita Sánchez-Andrade, Adolfo Paz-Silva. MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT African donkeys (n = 6) European donkeys (n = 6) Zebras (n = 3) Month Strongyle Control FECR PER FECR PER FECR PER Sep 10 Anthelm. Oct - 100 100 100 100 100 100 Nov - 100 100 94 33 100 100 Dec - 26 0 56 0 46 0 Jan 11 - 11 0 60 0 11 0 Feb - 0 0 33 0 0 0 Mar Anthelm. 15 0 0 0 23 0 Apr - 100 100 100 100 100 100 May - 91 33 71 0 100 100 Jun - 4 0 20 0 88 33 Jul - 0 0 0 0 53 0 Aug - 0 0 0 0 6 0 Sep Anthelm.+ Af 23 0 24 0 0 0 Oct Af 100 100 100 100 100 100 Nov Af 100 100 100 100 100 100 Dec Af 100 100 96 50 100 100 Jan 12 Af 70 0 91 33 100 100 Feb Af 10 0 94 0 100 100 Mar Af 53 0 85 0 73 33 Apr Af 52 0 84 0 65 33 May Af 74 0 93 0 79 0 Jun Af 91 33 86 0 72 0 Jul Af 82 33 87 0 87 0 Aug Af 87 0 88 0 53 0 Sep Af 81 0 95 0 69 33