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Original Paper Comparative Analyses of Coproscopical Techniques To Annals of Parasitology 2020, 66(3), 397–406 Copyright© 2020 Polish Parasitological Society doi: 10.17420/ap6603.279 Original paper Comparative analyses of coproscopical techniques to diagnose enteroparasites in a group of captive Indian peafowl (Pavo cristatus ) Bárbara r odrIguEs 1, Paula Andrea Borges s ALgAdo 1, Irys hany Lima goNzALEz 1, Adelini Q uAdrINI 1, M árcia Moreira h oLCMAN 2, Patr ícia Locosque r AMos 1, Carolina romeiro fernandes C hAgAs 1 1S ão Paulo Zoological Park Foundation, Av. Miguel Stéfano 4241, S ão Paulo, SP, CEP 04301-905, Brazil 2Division of Vector Control, Superintendence for Endemic Disease Control, R. Paula Sousa, 166, S ão Paulo, SP, CEP 01027-000, S ão Paulo, Brazil Corresponding Author: Carolina R.F. CHAGAS ; e-mail: [email protected] ABsTrACT. Captive animals commonly have infections by direct life cycle parasites, since they are easily transmitted between individuals. However, diagnosing these infections in the laboratory is challenging due to the wide variety of parasite, their life stages and to the variety of available diagnose techniques, being difficult to choose the best one. The present study sampled a group of captive Indian peafowl ( Pavo cristatus ) from S ão Paulo Zoological Park Foundation, São Paulo, Brazil, to test and compare different coproscopical techniques commonly applied in veterinarian clinical analysis laboratories: direct smear, concentrations by sodium chlorite, sucrose, zinc sulphate, faecal sedimentation and formalin-ether followed by modified Ziehl-Neelsen staining. Sensitivity, specificity, predictive values (positive and negative) and Cohen’s kappa index were calculated. In total 108 samples were processed and parasites found were: non- sporulated coccidian oocysts (91.7%), Capillarinae eggs (89.8%), unidentified nematode larvae (75%), Ascarididae eggs (63%), unidentified nematode adults (60.2%), unidentified nematode eggs (42.6%), strongylid-like eggs (42.6%), Cryptosporidium spp. (28.7%), flagellated (15.7%) and ciliated (10.2%) protozoans, trematode eggs (0.9%), Acanthocephala eggs (0.9%), Adeleidae oocysts (0.9%) and Cruzia sp. eggs (0.9%). Sensitivity and specificity varied considerably between parasite groups. Cohen’s Kappa index reinforces the recommendation of applying more than one technique to diagnose enteroparasites infections. Keywords: Capillarinae, coccidian, nematode, Ascarididae, parasites, Pavo cristatus Introduction which most common symptoms include diarrhoea, anaemia, weight loss, growing problems in nestlings Captive animals might have a high prevalence of and juveniles and even death [8,9] . Many parasitic infections, mainly because they live in enteroparasites were described to infect wild birds restricted spaces and usually in high densities, (free-living and captive), the most frequently which facilitates the transmission of parasites. In reported are: Isospora spp., Atoxoplasma spp., captivity, the presence of parasites with direct life Eimeria spp., Cryptosporidium spp., Sarcocystis cycles, such as nematodes, protozoans and spp., Giardia sp., Trichomonas gallinae , Histo mo - coccidian, have been extensively reported and nas meleagris , Toxoplasma gondii , Balantidium seems to be more common than parasites with spp., Blastocystis spp., Entamoeba spp., Ascaridia indirect life cycles, such as trematodes, cestodes and spp., Heterakis spp., Capillaria spp., Barusca pilla - acanthocephalans [1–7] . ria spp., Philophthalmus gralli and Taenia sp. Parasitic infections may be asymptomatic, with [1,2,8,10 –16 ]. no clinical symptoms, or have a symptomatic form, Diagnose of parasitic infections is essential to 398 B. r odriGuES et al. guarantee the health of captive wild animals. with specific gravity 1.18 g/ml and sucrose solution Choosing the best technique to be used is an with specific gravity 1.27 g/ml [19–21]; faecal important part of diagnose process, since there are sedimentation [17,22,23], and formalin-ether many different methods with different sensibility, sedimentation followed by modified Ziehl-Neelsen specificity and indications [17] . According to the staining technique to identify the presence of literature, it is recommended to use at least two Cryptosporidium -like oocysts [19,24,25]. Parasites different techniques when performing a parasitolo - were identified according to Foreyt [11], Greiner [9] gical diagnose, especially for entero parasites due to and Henriksen et al. [26]. Preparations were the wide variety of parasite species and their life analysed in their totality under 100× magnification. cycle [18] . Some techniques are broadly used, such Slides stained with modified Ziehl-Neelsen as direct smear, flotation protocols (using different technique were also analysed in their totality and solutions) and faecal sedimentation [19] . under high magnification (1000×) in oil immersion. In order to choose the best diagnose technique All samples were screened using a Zeiss PrimoStar for coproscopical diagnosis, we studied a captive light microscope (Carl Zeiss MicroImaging GmbH, population of Indian peafowl Pavo cristatus that Jena, Germany). lives in S ão Paulo Zoological Park Foundation statistical analysis. The flotation technique (FPZSP), S ão Paulo, Brazil. The aim of this study using NaCl solution was considered as gold was (i) to identify parasite diversity infecting a standard because it is used in the routine captive population of Indian peafowl and (ii) to coproscopical exams of the Clinical Analysis compare different coproscopical techniques of Laboratory at FPZSP. The results were compared parasite diagnose: direct smear, concentration among techniques and separated by the type of techniques with sodium chloride, sucrose and zinc parasite found (protozoans, coccidian, nematodes, sulphate solutions, faecal sedimentation and cestodes, trematodes and acanthocephalans), not formalin-ether concentration followed by modified taking into account the parasite stage that was Ziehl-Neelsen staining. found, such as larvae, eggs and/or adults. For parasites diagnosed in all applied techniques, Materials and Methods sensitivity, specificity, positive predictive values (PPV) and negative predictive values (NPV) were studied population. This study was conducted calculated, along with their respective 95% in the Clinical Analysis Laboratory at Applied confidence interval (95% CI). Cohen’s Kappa index Research Department of S ão Paulo Zoological Park (ĸ) was calculated to evaluate the concordance Foundation (FPZSP) ( 23°39′S, 46°37′W ). The between applied techniques and the gold standard samples used in this study were collected from the method, being considered as follow: ĸ<0, no enclosure where 28 Indian peafowl ( Pavo cristatus) agreement; ĸ=0 –0.20, poor agreement; ĸ=0.21 – were kept in a semi-captivity environment, with a 0.40, fair agreement; ĸ=0.41 –0.60, moderate big wooded area where they can freely circulate. agreement; ĸ=0.61 –0.80, substantial agreement; They were kept with water ad libitum , dry food for ĸ=0.81 –1.00, almost perfect agreement [27,28]. birds (produced at FPZSP), dry corn, dry oat and fresh chicory and catalonia. results sample collection and parasitological analysis. Fresh faeces were randomly chosen and collected in diversity of parasites a way to prevent soil contamination. Samples were In total, one hundred and eight samples were collected between October 2017 and March 2018, analysed during the present study and all of them twice a week in alternated weeks. After being were positive for at least one parasite (Table 1). The collected, they were stored in clean containers with highe r prevalence was reported for non-sporulated screw caps and kept at room temperature during coccidian oocysts (Fig. 1B), followed by Capillarinae transport to the laboratory, which did not take more eggs (Fig. 1D,E). Different life stages of unidentified than 3 0 minutes. Sample processing started as soon nematodes were found, such as eggs (Fig. 1F), larvae as samples arrived at the laboratory. The techniques and adults. The presence of Ascarididae (Fig 1J) and used were: direct smear [17,19]; faecal flotation strongylid-like (Fig. 1I) eggs were also detected in with sodium chloride (NaCl) solution with specific the present study. Among samples that were positive gravity of 1.20 g/ml, zinc sulphate (ZnSO 4) solution for Capillarinae eggs, it was possible to observe two Comparative analyses 399 Table 1. Diversity and prevalence of infections in captive Indian peafowl ( Pavo cristatus ), S ão Paulo Zoo/Brazil, October 2017 and March 2018 Sedimentation Ether- Parasite No+ (%) Direct (%) NaCl (%) ZnSO (%) Sucrose (%) 4 (%) formalin (%) Non-sporulated coccidian 99 (91.7) 30 (30.3) 80 (80.8) 68 (68.7) 79 (79.8) 40 (40.4) – oocysts Capillarinae eggs 97 (89.8) 32 (33.0) 74 (76.3) 58 (59.8) 86 (91.8) 40 (41.2) – Unidentified nematode 81 (75.0) 42 (51.9) 16 (19.8) 72 (88.9) 54 (66.7) 41 (50.6) – larvae Ascarididae eggs 68 (63.0) 16 (23.5) 39 (57.4) 44 (64.7) 55 (80.9) 34 (50.0) – Unidentified nematode 65 (60.2) 26 (40.0) 6 (9.2) 53 (35.4) 36 (55.4) 25 (38.5) – adults Unidentified nematode 46 (42.6) 7 (15.2) 23 (50.0) 25 (54.4) 29 (64.4) 13 (28.3) – eggs Strongylids -like eggs 42 (38.9) 1 (2.4) 17 (40.5) 21 (50.0) 28 (66.7) 6 (14.3) – Cryptosporidium spp. 31 (28.7) – – – – – 31 (100) Trophozoites of flagelated 17 (15.7) 17 (100) – – – – – protozoans Trophozoites of ciliated 11 (10.2) 5 (45.5) – – – 8 (72.7) – protozoans Non-sporulated coccidian 1 (0.9) – 1 (100) 1 (100) 1 (100) – – oocysts ( Adeleidae) Cruzia sp. eggs 1(0.9)
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