V i S A M D M a A b c d a A R R A K P C D F U B j ( ( m ( p ( h 0 Preventive Veterinary Medicine 134 (2016) 1–5 Contents lists available at ScienceDirect Preventive Veterinary Medicine j o ur na l ho me page: www.elsev ier .com/ locate /prevetmed alidation of a new technique to detect Cryptosporidium spp. oocysts n bovine feces andra Valéria Inácioa,∗, Jancarlo Ferreira Gomesb, Bruno César Miranda Oliveiraa, lexandre Xavier Falcãoc, Celso Tetsuo Nagase Suzukic, Bianca Martins dos Santosd, onally Conceiç ão Costa de Aquinoa, Rafaela Silva de Paula Ribeiroa, anilla Mendes de Assunç ãoa, Pamella Almeida Freire Casemiroa, arcelo Vasconcelos Meirelesa, Katia Denise Saraiva Bresciania UNESP − Universidade Estadual Paulista Júlio de Mesquita Filho, Departamento de Apoio, Produç ão e Saúde Animal, Faculdade de Medicina Veterinária de raç atuba, Araç atuba, São Paulo, Brazil UNICAMP, Universidade Estadual de Campinas, Institutos de Biologia e Computaç ão, Campinas, São Paulo, Brazil UNICAMP, Universidade Estadual de Campinas, Instituto de Computaç ão, Campinas, São Paulo, Brazil UNICAMP, Universidade Estadual de Campinas, Instituto de Biologia, Campinas, São Paulo, Brazil r t i c l e i n f o rticle history: eceived 4 February 2016 eceived in revised form 15 August 2016 ccepted 23 September 2016 eywords: arasites occidiosis iagnostic a b s t r a c t Due to its important zoonotic potential, cryptosporidiosis arouses strong interest in the scientific commu- nity, because, it was initially considered a rare and opportunistic disease. The parasitological diagnosis of the causative agent of this disease, the protozoan Cryptosporidium spp., requires the use of specific techniques of concentration and permanent staining, which are laborious and costly, and are difficult to use in routine laboratory tests. In view of the above, we conducted the feasibility, development, evalua- tion and intralaboratory validation of a new parasitological technique for analysis in optical microscopy of Cryptosporidium spp. oocysts, called TF-Test Coccidia, using fecal samples from calves from the city of Araç atuba, São Paulo. To confirm the aforementioned parasite and prove the diagnostic efficiency of the new technique, we used two established methodologies in the scientific literature: parasite concen- tration by centrifugal sedimentation and negative staining with malachite green (CSN-Malachite) and Nested-PCR. We observed good effectiveness of the TF-Test Coccidia technique, being statistically equiva- lent to CSN-Malachite. Thus, we verified the effectiveness of the TF-Test Coccidia parasitological technique for the detection of Cryptosporidium spp. oocysts and observed good concentration and morphology of the parasite, with a low amount of debris in the fecal smear. © 2016 Elsevier B.V. All rights reserved. ∗ Corresponding author at: Departamento de Apoio, Produç ão e Saúde Animal, aculdade de Medicina Veterinária de Araç atuba, Universidade Estadual Paulista − NESP, Rua Clóvis Pestana, 793, Jardim D. Amélia, CEP 16050-680, Araç atuba, SP, rasil. E-mail addresses: sandra byol@yahoo.com.br (S.V. Inácio), gomes@ic.unicamp.br (J.F. Gomes), bruno.9988@hotmail.com B.C.M. Oliveira), afalcao@ic.unicamp.br (A.X. Falcão), celso.suzuki@gmail.com C.T.N. Suzuki), biancamsantos@yahoo.com.br (B.M. dos Santos), onallyaquino@yahoo.com.br (M.C.C. de Aquino), rafaela.ribeiro123@hotmail.com R.S. de Paula Ribeiro), danilla.assuncao@hotmail.com (D.M. de Assunç ão), amellacasemiro@hotmail.com (P.A.F. Casemiro), marcelo@fmva.unesp.br M.V. Meireles), bresciani@fmva.unesp.br (K.D.S. Bresciani). ttp://dx.doi.org/10.1016/j.prevetmed.2016.09.020 167-5877/© 2016 Elsevier B.V. All rights reserved. 1. Introduction Being an important zoonotic pathogen, Cryptosporidium spp. infects mammals, birds, reptiles, amphibians and fish (Fayer, 2010; Xiao, 2010). This parasite causes diarrhea in calves (Vargas et al., 2014), with growth retardation, mortality and consequent eco- nomic loss (Olson et al., 2004; Santín et al., 2008). In cattle, the most common species are Cryptosporidium parvum (Santín et al., 2008), Cryptosporidium ryanae, Cryptosporidium andersoni (Xiao, 2010) and Cryptosporidium bovis (Feng et al., 2007; Fayer et al., 2008; Rieux et al., 2013). Laboratory diagnosis of Cryptosporidium oocysts in feces can be performed by means of parasite concentration techniques fol- lowed by the use of specific and permanent stains as Ziehl-Neelsen (Henriksen and Pohlenz, 1981), Kinyoun (Lennette et al., 1985), dx.doi.org/10.1016/j.prevetmed.2016.09.020 http://www.sciencedirect.com/science/journal/01675877 http://www.elsevier.com/locate/prevetmed http://crossmark.crossref.org/dialog/?doi=10.1016/j.prevetmed.2016.09.020&domain=pdf mailto:sandra_byol@yahoo.com.br mailto:jgomes@ic.unicamp.br mailto:bruno.9988@hotmail.com mailto:afalcao@ic.unicamp.br mailto:celso.suzuki@gmail.com mailto:biancamsantos@yahoo.com.br mailto:monallyaquino@yahoo.com.br mailto:rafaela.ribeiro123@hotmail.com mailto:danilla.assuncao@hotmail.com mailto:pamellacasemiro@hotmail.com mailto:marcelo@fmva.unesp.br mailto:bresciani@fmva.unesp.br dx.doi.org/10.1016/j.prevetmed.2016.09.020 2 eterin n m 2 p d i o s d ( T l 2 b ( I t g i e p t s 2 F p 2 s ( c w t w s c u c 2 t t ( s 3 3 3 a i F M S.V. Inácio et al. / Preventive V egative malachite green staining (Elliot et al., 1999), safranin ethylene blue (Garcia, 2007, 2009) and trichrome (Garcia, 2007, 009). Other techniques can also be used for this diagnostic pur- ose, such as phase contrast microscopy (Teixeira et al., 2011), irect fluorescent antibody test (CDC, 2015) and enzyme-linked mmunosorbent assay (CDC, 2015). Nowadays, the laboratory diagnosis of Cryptosporidium spp. ocysts requires the use of specific concentration and permanent taining techniques, which are labor-intensive and costly, and have ifficulty in its implementation in the routine of a clinical laboratory Gomes et al., 2004; Garcia, 2007; Carvalho et al., 2012, 2016). The precursor technique of this study, called TF-Test (Three Fecal est), has been used with practicality for years for fecal parasito- ogical diagnosis in humans, with excellent results (Gomes et al., 004; Carvalho et al., 2012, 2016). More recently, this technique has een extended to research in animal area by studies involving sheep Lumina et al., 2006) and canine (Coelho et al., 2013, 2015) species. n these works, new diagnostic procedures were validated with gas- rointestinal helminths and protozoa, not taking into account the enus Cryptosporidium. In view of the above, continuing the line of research regard- ng the above technique, we analysed the feasibility, development, valuation and intralaboratory validation of a new and practical arasitological technique for analysis in optical microscopy of Cryp- osporidium spp. oocysts, called TF-Test Coccidia (Fig. 3), using fecal amples from calves from the city of Araç atuba, São Paulo. . Material and methods This study was approved by the Animal Ethics Committee of the aculdade de Odontologia do Campus de Araç atuba − UNESP, with rotocol number 2013-00847. .1. Harvest and storage of fecal samples Considering the irregular shedding of oocysts of Cryptosporidium pp. (Garcia, 2007, 2009; CDC, 2015), fecal samples were harvested collected) on three alternate days, directly from the rectum of each alf. This material was divided into three parts: the first part 50 mL as stored under refrigeration for use in the parasite concentra- ion technique by centrifugal sedimentation and negative staining ith malachite green, named in our study as CSN-Malachite; the econd part (5.4 g) was kept in the collecting tubes of TF-Test Coc- idia; and the third 200 milligrams part was kept frozen at −20 ◦C ntil the time of genomic DNA extraction and nested polymerase hain reaction (nested PCR). .2. Processing of fecal samples For the confirmation of oocysts and statistical comparison of echniques, besides the new TF-Test Coccidia technique, we used wo established techniques in the scientific literature: nested PCR confirmation of oocysts) and CSN-Malachite (statistical compari- on), respectively. . Experimental design .1. Protocol standardization of the new technique .1.1. Study description This intralaboratory study was conducted with the support of farm of dairy cattle breeding in the city of Araç atuba, São Paulo, dentified with positive samples, during the period of March 2014. or this step, fecal samples (n = 60) were processed only by the CSN- alachite technique. As a result, we obtained five positive samples ary Medicine 134 (2016) 1–5 for Cryptosporidium spp. oocysts, which were later divided into 100 aliquots for the development and standardization of the operating protocol of the TF-Test Coccidia technique. A total of 60 Holstein calves, 11 males and 49 females, aged from six to 480 days, were involved in this study. 3.2. New technique validation 3.2.1. Study description This intralaboratory research was conducted with fecal samples in 10 dairy cattle breeding farms in the city of Araç atuba, São Paulo, during the period from March 2014 to October 2015. For this step, we used the CSN-Malachite technique to perform the screening of samples to assess the occurrence of Cryptosporidium spp. oocysts in the fecal content of cattle. After this screening, we applied the stan- dard protocol of the new TF-Test Coccidia diagnostic technique. The diagnostic of fecal samples were confirmed by nested PCR. Lastly, a total of 68 calves of Holstein and Girolanda races, 15 males and 53 females, aged between one and 540 days, were examined to assess the presence of the parasite, resulting in 34 positive samples and 34 negative samples for intralaboratory evaluation. These ani- mals were classified according to their age in two groups: ≤ 30 days and ≥ 31 days. 3.3. Centrifugal sedimentation with negative malachite green stain modified technique (CSN-Malachite) For this procedure, we obtained a pool of three samples for each animal. This material was processed in the laboratory in accor- dance with the literature of centrifugal sedimentation (Meloni and Thompson, 1996) and staining with malachite green (Elliot et al., 1999). 3.3.1. New TF-Test coccidia technique This technique consisted of the following steps: The three collection tubes (Fig. 3) of TF-Test Coccidia (contain- ing formalin) containing 5.4 g of the collected feces were placed in an appropriate shelf. In this material, 25 �L of surfactant organic solvent (colorless neutral detergent) and 3 mL of ethyl acetate pro analysis (Formula C4H8O2) were added to each tube. Then, the respective tubes with their caps were closed and shaken vigorously for 30 s with a vortex mixer. After that, the caps of the tubes were removed and connected to the other two parts, called set of filters and centrifuge tube. The union of all these parts was denominated in this study “set processor”. Subsequently, this set was centrifuged for two minutes at 500 x G (gravitational force). After this centrifu- gation, the centrifuge tube was disengaged from the set of filters and collection tubes. Next, the supernatant liquid was decanted from the centrifuge tube, so leaving 500 �L of sediment in the con- ical bottom of the centrifugation tube. In this sediment, 250 �L of treated water were added and mixed, providing the formation of a fecal suspension. This suspension was redeemed using an auto- matic pipette, and 150 mL of the fecal suspension were transferred to a TF-Test Coccidia collection tube. Then a drop of colorless neu- tral detergent was added to this material and the whole suspension was manually mixed. After that, 3 mL of neutral formalin solution were added to this material and all material was mixed vigorously for 30 s with a vortex mixer. In sequence, 3 mL of ethyl acetate pro analysis were added, and all material was shaken again for another 30 s on the vortex mixer. Then, the tube was centrifuged at 333 x G for one minute. After this centrifugation, the supernatant was decanted using a pipette, until a small aliquot of suspension on the bottom of the demarcated tube was left, and 25 �L of this material were transferred to a microscope slide. Then, on the slide, 25 �L of the dye developed and standardized in the study (25 �L of mod- ified D’Antoni’s iodine solution and 50 �L of modified Masson’s eterinary Medicine 134 (2016) 1–5 3 t s m 3 w t D n t m t h k l 3 C 3 n e f K a 3 t p i 1 m 5 G 5 A T a R w p T p T f 4 e 3 s t t l 4 n Fig. 1. Image of Cryptosporidium spp. oocysts in a sample of calf feces, with the use of a dry objective lens with 60 x magnification. S.V. Inácio et al. / Preventive V richrome) were added. Finally, a fecal smear was made on the lide and a coverslip was superimposed for examination by optical icroscopy. .3.2. Staining and microscopy The operational protocol of the new technique allowed, even ith samples fixed with neutral formalin solution, the prepara- ion of fecal smear using temporary staining composed of modified ’Antoni’s iodine solution and modified Masson’s trichrome. This ew dye allowed the reading of a microscope slide with dry objec- ive lens of 60 x and 100 x magnification in a conventional optical icroscope, without the need for immersion oil. It is worth men- ioning that these objective lenses for use without immersion oil ave good image quality and were recently introduced in the mar- et by Olympus industry, with the following descriptions: objective ens 60 x − UPlan FLN and objective lens 100 x − Plan FLN. .4. Nested PCR targeting the 18S ribosomal RNA gene of ryptosporidium spp .4.1. Fecal DNA extraction The DNA was extracted from 200 mg of 68 fecal samples “in atura”, stored at −20 ◦C until genomic DNA extraction; for the xtraction of samples it was performed heating to 99 ◦C for 60 min or breaking the wall of the oocyst, using QIAamp® DNA Stool Mini it (Qiagen®). The DNA was eluted with 50 �L AE buffer and stored t −20 ◦C. .4.2. Nested PCR Nested PCR was performed for amplification of a fragment of he 18S ribosomal RNA gene (Xiao et al., 2000). Cryptosporidium arvum DNA was used as positive control and ultrapure water was ncluded as a negative control, followed by electrophoresis from a .5% agarose gel. For amplification of the 18S subunit ribosomal RNA gene frag- ents it was used nested PCR with primers from Sigma-Aldrich® ‘TTC AGC TAG TAA ATG TAC CG 3′ and 5 ‘CCC ATT ACA GGA TCC AA TTC 3 ‘for the primary reaction, with 1325 base pairs (bp) and ′-AGG GTT GGA GTA TTT ATT AGA AAT GA 3′ and 5 ‘AAT GGA AG GAG ACC ACA TCC 3′ for the secondary reaction (bp 826–840). o perform the following reaction conditions, there was prepared solution with a final volume of 25 �L from JumpStart TaqTM EADYMIXTM reagent from Sigma-Aldrich®, for primary reaction as used: JumpStart TaqTM READYMIXTM 12.5 �L, primer 1 0.5 �L, rimer 2 0.5 �L, H2O 8.25 �L, BSA 0.75 �L and DNA target 2.5 �L. o use secondary reaction: JumpStart TaqTM READYMIXTM 12.5 �L, rimer 3 0.5 �L, primer 4 0.5 �L, H2O 9.0 �L and DNA target 2.5 �L. he samples were subjected to DNA initial denaturation at 94 ◦C or 3 min followed by 34 cycles each consisting of denaturation for 5 s at 94 ◦C, 45 s annealing at 55 ◦C and 60 s extension at 72 ◦C with xtension end at 72 ◦C for 7 min (Xiao et al., 2000). .5. Statistical analysis The variables (sex, breed and age) were analysed by the chi- quare test (�2) or Fisher’s exact test for the association between he variables. The Kappa statistic was used to measure the agreement between he mentioned techniques. We used the SAS software (SAS, 2015) for the statistical calcu- ations, with 5% significance level. . Results In the intralaboratory study of the new TF-Test Coccidia tech- ique, we observed good sharpness and morphology of oocysts of Fig. 2. Image of Cryptosporidium spp. oocyst in a sample of calf feces, with the use of a dry objective lens with 100 x magnification. Cryptosporidium spp. (Figs. 1 and 2). The same could not be demon- strated in most slides processed by the CSN-Malachite technique, in accordance with literature reports (Coelho et al., 2015). Furthermore, the new technique presented fecal smear slides with low quantity of debris, good concentration of parasite on a microscope slide (Fig. 1) and demonstrated the preparation of fecal smears with the use of a new temporary stain. Unlike conventional staining, the dye of the new technique allowed to highlight the sporozoites of oocysts with a purple hue (Fig. 2), and the fecal debris with a green color. The concentration adjustment between an iodine solution and Masson allowed this new composition dye work with same accu- racy in fecal samples collected in neutral formalin solution, unlike literature indications (Garcia, 2007, 2009). In this intralaboratory study, the same examiner detected 34 positive samples in 68 calves examined by both CSN-Malachite and 4 S.V. Inácio et al. / Preventive Veterinary Medicine 134 (2016) 1–5 n (A) T t fi a n b t b ( 5 o t f c p c t a m s o t r h C c t t o T t l Fig. 3. Illustration of the set of centrifugatio F-Test Coccidia techniques, with full statistical correlation between hese results (Kappa = 1.000). However, with nested-PCR we con- rmed 24 positive and 10 false negative samples. Regarding the negative samples (n = 34), there are statistic greement between the CSN Malachite and TF −Test Coccidia and ested PCR techniques. By the chi-square test (�2), a higher frequency of infection y Cryptosporidium spp. was observed in younger calves in rela- ion to other age groups (P<0.0001), and with respect to sex and reed, there was no significant difference in parasitic infection rate P>0.05). . Discussion Through the data shown in this intralaboratory study, we bserved good efficacy in oocyst detection of the protozoan Cryp- osporidium spp. by using the TF-Test Coccidia technique, which avors the use of this new technique in laboratory routine. For this research, we prefer to take fecal samples of newborn alves (Fayer and Santín, 2009; Silverlås et al., 2010) because they resent higher frequency of cryptosporidial infection, which was onfirmed by our results that showed a significant difference for his age group compared to animals of other ages. The new technique clearly showed fecal smears with good par- sitic concentration, and largely free of debris (Figs. 1 and 2). In any slides it was possible to view a concentration of more than even oocysts per field, with 60 x magnification objective lens with- ut immersion (Fig. 1). This should favor the detection of oocysts of his parasite in microscope reading practiced in laboratory routine, educing fatigue and diagnostic interpretation error practiced by umans. The use of the new dye preserved the morphological integrity of ryptosporidium spp. oocysts, always dyeing the sporozoites in lilac olor (Fig. 2) and almost all of the impurities in green. This condi- ion should facilitate the microscopy reading, significantly reducing he diagnostic interpretation error practiced by humans, which can ccur with a false positive or false negative result. Unlike literature recommendation (Garcia, 2007, 2009), the TF- est Coccidia technique allowed the preparation of slides with emporary coloring and microscopy reading with dry objective ens. This eliminated the limitations provided by specific tech- and collectors tubes (B) of TF-Test Coccidia. niques using permanent dyes, for example, those derived from carbol fuchsin, which despite demonstrating excellent diagnostic performance, are rarely used in routine laboratory because they are labor-intensive and costly. For the CSN-Malachite and TF-test Coccidia techniques, the results were concordant for the presence of the protozoan Cryp- tosporidium spp., that is, the same animals showed positive (n = 34) and negative (n = 34) samples. PCR was used only for confirma- tion of fecal samples, to indicate that TF-Test Coccidia detected the oocysts in the samples correctly. The same examiner performed the processing of stool samples with the negative staining with malachite green (CSN-Malachite) and TF-Test Coccidia techniques, which were later confirmed by Nested PCR. The samples were processed randomly in each tech- nique, without a sort order for the reading of the fecal samples, and thus the examiner was not suggestible when interpreting the data. The proper selection of an extraction and purification method is crucial to ensure reliable results (Paulos et al., 2016), and is widely known that molecules found in fecal samples, or in the composition of buffers used for DNA extraction, can inhibit the enzymes used in amplification (Monteiro et al., 1997; Schrader et al., 2012; Paulos et al., 2016). Therefore, the detection efficiency of the molecular test may have been influenced by the possible presence of inhibitory agents in the material (Ward and Wang, 2001), or the vegetable intake by the animals (Monteiro et al., 2001). There are differences in the performance of kits to extract DNA from oocysts depend- ing on the pathogen, and the intensity of the infection (Paulos et al., 2016). The justification for the lower sensitivity of PCR is because the stool samples were used “in natura”, without purifica- tion and in a small amount (200 mg) when compared with TF-Test Coccidia, which was processed with 5.4 g of feces. It is notewor- thy that, despite the comments above, the use of Nested-PCR in this study was extremely important, especially for the molecular confirmation of the parasite Cryptosporidium spp. TF-Test Coccidia was considered an efficient technique and could be applied in the veterinary medicine field for the fecal diagnosis of Cryptosporidium spp. oocysts. The preliminary results of this study allow us to move forward to the next research step: the interlaboratory validation of the TF-Test Coccidia technique. With this, we can begin with the study of com- puterized image analysis of Cryptosporidium spp. oocysts, which eterin s p 6 p o p A P S 4 t r R C C C C C E F F F F G G and sources of Cryptosporidium oocysts in storm waters with a small-subunit rRNA-based diagnostic and genotyping tool. Appl. Environ. Microbiol. 66, S.V. Inácio et al. / Preventive V hould result in advances in this type of diagnosis in public and rivate laboratory routines, as well as government programs. . Conclusion In this study, we verified the effectiveness of the TF-Test Coccidia arasitological technique for the detection of Cryptosporidium spp. ocysts and observed a good concentration and morphology of the arasite, with a low amount of debris in the fecal smear. cknowledgements We thank the Agência de Coordenaç ão de Aperfeiç oamento de essoal de Nível Superior − CAPES, for granting Phd scholarship, ão Paulo Research Foundation (FAPESP) for grant PIPE #99/06228- and ImmunoCamp Science and Technology for research support, hrough investments in human resources and consumption mate- ial. eferences enter for disease control and prevention (CDC), 2015. Cryptosporidium: Diagnosis & Detection, Retrieved from http://www.cdc.gov/parasites/crypto/ diagnosis.html (accessed 01.08.16.). arvalho, G.L.X., Moreira, L.E., Pena, J.L., Marinho, C.C., Bahia, M.T., Machado-Coelho, G.L.L., 2012. 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dx.doi.org/10.1016/S0732-8893(01)00276-0 dx.doi.org/10.1016/S0732-8893(01)00276-0 dx.doi.org/10.1016/S0732-8893(01)00276-0 dx.doi.org/10.1016/S0732-8893(01)00276-0 dx.doi.org/10.1016/S0732-8893(01)00276-0 dx.doi.org/10.1016/S0732-8893(01)00276-0 dx.doi.org/10.1016/S0732-8893(01)00276-0 dx.doi.org/10.1128/AEM.66.12.5492-5498.2000 dx.doi.org/10.1128/AEM.66.12.5492-5498.2000 dx.doi.org/10.1128/AEM.66.12.5492-5498.2000 dx.doi.org/10.1128/AEM.66.12.5492-5498.2000 dx.doi.org/10.1128/AEM.66.12.5492-5498.2000 dx.doi.org/10.1128/AEM.66.12.5492-5498.2000 dx.doi.org/10.1128/AEM.66.12.5492-5498.2000 dx.doi.org/10.1128/AEM.66.12.5492-5498.2000 dx.doi.org/10.1128/AEM.66.12.5492-5498.2000 dx.doi.org/10.1128/AEM.66.12.5492-5498.2000 dx.doi.org/10.1128/AEM.66.12.5492-5498.2000 dx.doi.org/10.1128/AEM.66.12.5492-5498.2000 dx.doi.org/10.1016/j.exppara.2009.03.018 dx.doi.org/10.1016/j.exppara.2009.03.018 dx.doi.org/10.1016/j.exppara.2009.03.018 dx.doi.org/10.1016/j.exppara.2009.03.018 dx.doi.org/10.1016/j.exppara.2009.03.018 dx.doi.org/10.1016/j.exppara.2009.03.018 dx.doi.org/10.1016/j.exppara.2009.03.018 dx.doi.org/10.1016/j.exppara.2009.03.018 dx.doi.org/10.1016/j.exppara.2009.03.018 dx.doi.org/10.1016/j.exppara.2009.03.018 dx.doi.org/10.1016/j.exppara.2009.03.018 Validation of a new technique to detect Cryptosporidium spp. oocysts in bovine feces 1 Introduction 2 Material and methods 2.1 Harvest and storage of fecal samples 2.2 Processing of fecal samples 3 Experimental design 3.1 Protocol standardization of the new technique 3.1.1 Study description 3.2 New technique validation 3.2.1 Study description 3.3 Centrifugal sedimentation with negative malachite green stain modified technique (CSN-Malachite) 3.3.1 New TF-Test coccidia technique 3.3.2 Staining and microscopy 3.4 Nested PCR targeting the 18S ribosomal RNA gene of Cryptosporidium spp 3.4.1 Fecal DNA extraction 3.4.2 Nested PCR 3.5 Statistical analysis 4 Results 5 Discussion 6 Conclusion Acknowledgements References