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Research Article
 

Molecular Characterization of Bubaline Isolate of Cryptosporidium Species from Egypt



Khaled A. Abdelrahman, Kadria N. Abdel Megeed, Abdel Mohsen M. Hammam, Gazaa H. Morsy, Moshera M.E. Seliem and Dina Aboelsoued
 
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ABSTRACT

Cryptosporidium an apicomplexan parasite has the ability to induce diarrhea in bovines, goats, pigs, dogs and cats worldwide. In this study, buffalo calves fecal samples were examined after staining their smears with Modified Ziehl-Neelsen Stain (MZN). Ileal sections were examined for the detection of pathological changes. Further molecular characterization was done using nested PCR amplification and partial sequence analysis. The detected oocysts were morphologically similar to Cryptosporidium parvum. Light microscopic examination of Cryptosporidium infected ileal Tissue Section (TS) stained with H and E revealed the presence of altered mucosal architecture with congestion of blood vessels, infiltration, sloughing and complete erosion of epithelial cells and shortening, blunting, stunting and atrophy of the intestinal villi. Molecular characterization gave PCR amplicons of 18S SSU rRNA gene products approximately at 823 bp. Sequences proved specified generalized relatedness with 21 species of Cryptosporidium but the nucleotide homogeneity percentage was insufficient to designate species or genotypes. Further bioinformatics analysis showed that resulting Cryptosporidium isolates had the closest match with three isolates. It was implied that the Cryptosporidium isolates is mostly like Cryptosporidium parvum (JX237832.1) previously isolated from buffaloes in Ismailia province.

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Khaled A. Abdelrahman, Kadria N. Abdel Megeed, Abdel Mohsen M. Hammam, Gazaa H. Morsy, Moshera M.E. Seliem and Dina Aboelsoued, 2015. Molecular Characterization of Bubaline Isolate of Cryptosporidium Species from Egypt. Research Journal of Parasitology, 10: 127-141.

DOI: 10.3923/jp.2015.127.141

URL: https://scialert.net/abstract/?doi=jp.2015.127.141
 
Received: September 06, 2015; Accepted: October 17, 2015; Published: October 29, 2015

INTRODUCTION

Cryptosporidiosis is the clinical disease presenting as a gastro enteritis like syndrome in bovines, goats, pigs, dogs and cats (Fayer et al., 2005; Bhat et al., 2013). It causes greenish yellow mucoid or bloody diarrhea, apathy, lack of appetite, mild fever and dehydration in young calves (Abdel Megeed et al., 2015). In Egypt, Cryptosporidium infection was detected in buffalo calves (19.65%) in El Dakahlia Governorate (El-Dessouky and El-Masry, 2005), Middle Egypt (14.19%) (El-Khodery and Osman, 2008), Ismailia (22.5%) (Shoukry et al., 2009), Cairo, Giza, Beni Suef and Qualiobya (52%) (Morsy et al., 2014). Traditionally, the detection of Cryptosporidium oocysts in environmental, water, food, fecal and/or tissue samples had primarily relied on examination by microscopy (O'Donoghue, 1995; Quintero-Betancourt et al., 2003). Oocyst morphology played an important role in Cryptosporidium taxonomy but was inconvenient to clearly differentiate species and genotypes (Fall et al., 2003). Therefore, molecular analyses had been widely used to characterize the genetic structure of Cryptosporidium parasites and assessment of their zoonotic significance (Xiao, 2010). Various PCR-based techniques employing specific primer pairs for the selective amplification of different genetic loci followed by sequencing had been used to characterize and classify Cryptosporidium species or genotypes (Quintero-Betancourt et al., 2002; Xiao et al., 2004). Some key markers included ribosomal RNA genes and spacers, the Cryptosporidium oocyst wall protein (cowp), the 70 kDa heat shock protein (hsp70), the thrombospondin-related adhesive protein (trap) genes and the 60 kDa glycoprotein (gp60) gene (Jex et al., 2008). Cryptosporidium frequently affected and strong relation between C. parvum infection and diarrhea among Egyptian buffalo calves (Warda et al., 2002). Cryptosporidium parvum, C. ryanae and C. bovis were identified as 65.7, 11.8 and 4.1%, respectively, with combinations of C. parvum plus C. ryanae (11.2%), C. parvum plus C. bovis (5.3%) and of C. parvum plus C. andersoni (1.8%) in Egyptian buffaloes from Ismailia province (Helmy et al., 2013). Also, it was found that C. parvum was the dominant species in buffaloes and cattle in Ismailia province (65.7%) (Helmy et al., 2015). While, Amer et al. (2013a) found that the prevailing occurrence of C. ryanae and the subtype family IId of C. parvum and the absence of C. bovis and C. andersoni represent some features of Cryptosporidium transmission in water buffaloes in Egypt. The PCR analysis of the gp60 gene was successful for seven C. parvum positive specimens as well as two specimens that were negative in SSU rRNA PCR. DNA sequence analyses of microscopy-positive fecal specimens revealed the presence of four major Cryptosporidium species. In pre-weaned calves, C. parvum was most common (30/69 or 43.5%) but C. ryanae (13/69 or 18.8%), C. bovis (7/69 or 10.2%) and C. andersoni (7/69 or 10.2%) were also present. Mixed infections were seen in 12/69 (17.4%) of genotyped specimens. In contrast, C. andersoni was the dominant species (193/195 or 99%) in post-weaned calves and older animals (Amer et al., 2013b). In buffaloes of different farms at Kafr El Sheikh province, Egypt, PCR-RFLP analyses of small-subunit rRNA genes from positive specimens revealed the occurrence of C. parvum and C. ryanae. Genotypes distribution showed that C. ryanae was the dominant species (60%) followed by C. parvum (40%) in buffalo calves (Mahfouz et al., 2014). This study was aimed to identify Cryptosporidium species isolated from Egyptian buffalo calves and to study the effect of cryptosporidiosis on intestinal tissues referring to its pathological changes.

MATERIALS AND METHODS

Sample collection: A total number of 571 buffalo calves (age from one day to one year) rectal fecal samples were collected around the year from different Egyptian governorates (Cairo, Giza, Beni Suef, Qualiobya) in a clean labeled container.

Detection of oocysts: Fine fecal smears fixed with methanol spirit and stained with Modified Ziehl-Neelsen Stain (MZN) (Henriksen and Pohlenz, 1981) were examined. The oocysts were measured with the help of stage micrometer conjugated with the light microscope at the eyepiece 10x and the objective 100x. All measurements are in micrometers for about 20-50 oocysts (Fayer and Xiao, 2007).

Histopathological changes: Specimens from different parts of ileum (about 1 cm) were taken from infected buffalo calves for studying histopathological changes. These materials were fixed immediately in 10% formal saline, dehydrated, cleared, embedded in paraffin, sectioned at 4 mm and stained with H and E staining (Drury and Wallington, 1967).

Some infected ileum sections were deparaffinized, hydrated with distilled water and stained with modified-Ziehl-Neelsen (ZN) staining (Sheehan and Hrapchak, 1987). The ileal sections were examined for the detection of pathological changes under microscope.

Genomic DNA extraction: Cryptosporidium oocysts were concentrated by flotation using Sheather’s sugar solution (Current et al., 1983). The floated upper third was washed by centrifugation in distilled water for 3 times and suspended in Phosphate Buffered Saline (PBS) (Current and Reese, 1986). The purified oocysts were stored at -20°C in 2.5% potassium dichromate solutions. DNA was extracted from the washed Cryptosporidium oocysts using QIAamp DNA MiniKit (Qiagen Co., USA) with modifications to the manufacturer’s protocols in which, a total of 200 μL of oocysts solution was suspended in 180 μL of ATL buffer and thoroughly mixed by vortexing. Then, subjected to five extra freezing and thawing cycles in liquid nitrogen and a water bath at 65°C as lysis before extraction protocol.

Nested PCR procedure: Primers were used as described by Xiao et al. (1999), for the primary PCR (expected amplicon size: 1325 bp): 18 SF: 5'-TTC TAG AGC TAA TAC ATG CG-3' (forward) and 18 SR: 5'-CCC ATT TCC TTC GAA ACA GGA-3' (reverse). Each PCR mixture, total volume 100 μL contains 10 μL 10x PCR buffer, 6 mM MgCl2, 200 μM each dNTP, forward and reverse primers at a concentration of 200 nM each, 400 ng μL–1 of non-acetylated BSA, 2.5 U Taq polymerase and 0.5 -3.0 μL DNA template. A total of 35 cycles consisting of; 94°C for 45 sec, 55°C for 45 sec and 72°C for 60 sec make up the PCR program, an initial hot start at 94°C for 3 min and a final extension at 72°C for 7 min were also included. For the nested PCR (expected amplicon size: 819-825 bp, depending on the species) the following primers were used: 18 SNF: 5'-GGA AGG GTT GTA TTT ATT AGA TAA AG-3' (forward) and 18 SNR 5'-CTC ATA AGG TGC TGA AGG AGTA-3' (reverse). The reaction mixture was the same as for the primary step with the following exceptions: no BSA was required, increase primer concentration to a 400 nM and the DNA template volume added was 2 μL of primary PCR product. Cycling conditions were identical to the primary PCR except the annealing temperature was increased to 58°C.

Gel electrophoresis: Following amplification, PCR products were visualized in a 1% agarose gel stained with ethidium bromide by Molecular Imager (Gel DocTM, BIO RAD).

Sequencing of PCR amplicons and BLAST of 18S SSU rRNA gene sequences: All secondary PCR products determined to be Cryptosporidium positive were purified by QIAquick Gel extraction kit (Qiagen Co., USA) and sequenced in both directions in a commercial laboratory (Sigma Scientific Services Co., Egypt). Amplified sequences were compared with reference sequences using Basic Local Alignment Search Tool (BLAST) (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Phylogenetic analysis: The evolutionary history was inferred using the UPGMA method (Sneath and Sokal, 1973). The optimal tree with the sum of branch length equaling 77.60047543 was shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) was shown below the branches (Felsenstein, 1985). The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances. Then, the evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al., 2004) and were in the units of the number of base substitutions per site. All positions containing gaps and missing data eliminated from the dataset (Complete deletion option). There were a total of 560 positions in the final dataset. Phylogenetic analyses were conducted in MEGA4 (Tamura et al., 2007).

RESULTS

Morphology of the detected Cryptosporidium oocysts: The detected Cryptosporidium oocysts in the examined buffalo calf feces stained with Ziehl-Neelsen technique were morphologically similar to C. parvum oocysts that characterized by spherical to ovoid shape with smooth wall and appeared as acid fast (red-pink) on a green background. The measurements of 50 oocysts were varied from 4.4-5.8×4.3-4.9 μm of mean (5.1×4.6 μm) and the shape index was 1.0-1.2 of mean (1.1) (Fig. 1).

Pathological changes in ileal Tissue Sections (TS): Examination of Cryptosporidium-infected ileal TS stained with H and E revealed the presence of altered mucosal architecture, with congestion of blood vessels, infiltration, sloughing and complete erosion of epithelial cells and shortening, blunting (Fig. 2), stunting and atrophy of the intestinal villi (Fig. 3). Basophilic oval or round organism was found on the surface of villi, free or entering the epithelial cells (Fig. 4 and 5). Oval or round structures oocysts were found also in sections stained with MZN staining technique (Fig. 6).

Identification of Cryptosporidium spp. in buffalo calves
Nested PCR amplicons of 18S SSU rRNA gene products: Molecular characterization was done using nested PCR amplification and partial sequence analysis. The PCR amplicons of 18S SSU rRNA gene products of the Cryptosporidium isolates were about 823 bp. These were used for sequencing process (Fig. 7).

BLAST of Egyptian buffalo isolate 18S SSU rRNA gene sequences with GenBank Database: Sequence compared with reference sequences using BLASTn program (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was successful to prove specified generalized relatedness with 21 species of Cryptosporidium but the nucleotide homogeneity percentage were insufficient to designate species or genotypes of Cryptosporidium (Fig. 8).

Fig. 1:Cryptosporidium oocysts in stained fecal smears of buffalo calves (MZN X1000)

Fig. 2:
Ileal tissue section in Cryptosporidium-infected buffalo calf showing severe hemorrhagic enteritis with congestion of blood vessels (double arrow), blunting of villi (blue arrow) and sloughing and complete erosion of epithelial cells (dashed arrow) (H and E X100)

Fig. 3:
Ileal tissue section in Cryptosporidium-infected buffalo calf showing altered mucosal architecture, stunting of villi (green arrows) and Cryptosporidium oocysts (arrow heads) (H and E X400)

Phylogenic tree and evolutionary relationships of Egyptian buffalo isolate with Cryptosporidium species: The evolutionary relationship by bioinformatics analysis and phylogenetic tree construction showed that resulting Cryptosporidium isolates had the closest match with three isolates; Cryptosporidium spp. pig genotype II (isolated from pigs in China, GenBank accession number: HQ844733.1), C. parvum (Egyptian isolate from buffalo in Ismailia province, GenBank accession number: JX237832.1) and C. baileyi (isolated from quails in China, GenBank accession number: EU717805.1) (Fig. 9).

Fig. 4:
Ileal tissue section in Cryptosporidium-infected buffalo calf showing Cryptosporidium oocysts (arrow head) and developmental stages (red arrow) (H and E X1000)

Fig. 5:Ileal tissue section in Cryptosporidium-infected buffalo calf showing Cryptosporidium oocysts (arrow heads) (H and E X1000)

Fig. 6:
Ileal tissue section in Cryptosporidium-infected buffalo calf showing altered mucosal architecture with Cryptosporidium oocysts (arrow heads) (MZN X1000)

Fig. 7:
PCR amplification products of respective Cryptosporidium isolates: Lane 1: 100 bp DNA ladder, lane 2: Negative control, lanes 3-8: 18S rRNA amplification products. PCR amplicons of 18S SSU rRNA gene products of the Cryptosporidium isolates were about 823 bp

Fig. 8: Phylogenetic tree of sequence with GenBank database using BLASTn program utilizing forward strand sequence, *18S rRNA sequences from the respective Cryptosporidium isolates identified

DISCUSSION

Modified Ziehl Neelsen was used in this study for the detection of Cryptosporidium oocysts microscopically. This method has been used in many previous studies; Henriksen and Pohlenz (1981), Fathia (1993), Kvac and Vitovec (2003), El-Sherbini and Mohammed (2006), Diaz-Lee et al. (2011) and Bhat et al. (2012). The MZN staining was the most efficient method in the detection of Cryptosporidium oocysts, so it was recommended as rapid, easy and less costly method for diagnosis of cryptosporidiosis (Abdel-Rady and Sayed, 2008).

In the present study, light microscopic examination of H and E stained Cryptosporidium infected intestinal sections revealed the presence of altered mucosal architecture, with congestion of blood vessels, infiltration, sloughing and complete erosion of epithelial cells and shortening, blunting, stunting and atrophy of the intestinal villi.

Fig. 9:
Phylogenetic tree illustrating the evolutionary relationships of Egyptian buffalo isolate obtained during the present study with 21 Cryptosporidium species recorded in the GenBank database, *18S rRNA sequences from the respective Cryptosporidium isolates identified

Basophilic oval or round organism was found on the surface of villi, free or entering the epithelial cells. These findings agreed with Fathia (1993), Abu El Ezz et al. (2011), Gaafar (2012), Toulah et al. (2012) and Jin et al. (2015).

The Cryptosporidium oocysts detected in buffalo calf fecal smears in this study were morphologically similar to those of C. parvum described in calves in many previous studies (Fall et al., 2003; Fayer et al., 2006; Hassanain et al., 2011; Randhawa et al., 2012b). Identification of the Cryptosporidium species depended upon the oocyst morphology and measurements (Fayer et al., 2000). Fayer et al. (2000) cited that morphometric measurements of oocysts represented the cornerstone of Cryptosporidium taxonomy and was one of the requirements for establishing a new species, however, it was not adequate by itself and multiple parameters as electron microscopy, developmental biology, host specificity, histopathology and/or molecular biology should be used. However, morphological identification only was insufficient to identify species or genotypes of Cryptosporidium (Egyed et al., 2003; Monis and Thompson, 2003).

In the present study, Cryptosporidium identification based on morphology of oocysts had provided generalized prevalence data for the infection but was insufficient alone to identify species or genotypes of Cryptosporidium. Therefore, molecular analysis had been used to characterize the genetic structure of Cryptosporidium oocysts. The PCR could detect up to single oocyst per sample and could ensure specific diagnosis up to species level coupled with 100% diagnostic sensitivity and specificity (Coupe et al., 2005; Shields et al., 2013).

In accordance, during the present study, 823 bp fragments amplified from the 18S SSU rRNA gene could be noted after nested PCR reaction from buffalo’s feces. Previous studies indicated the usefulness of the small subunit (SSU) ribosomal RNA genes as genetic markers for the specific identification of Cryptosporidium having relatively low intraspecific and relatively high interspecific sequence variation (Fayer et al., 2000; Xiao et al., 2004; Jex et al., 2007). Thus, they had been utilized in systematic (phylogenetic) investigations of Cryptosporidium providing the basis for the current classification of members within the genus (Morgan et al., 1999a; Xiao et al., 2004).

The blast of the sequenced PCR products, amplified from the morphologically characterized oocytes in the present study, on GenBank database was successful to prove specified generalized relatedness with 21 species of Cryptosporidium. But the nucleotide homogeneity percentages were insufficient to designate species or genotypes of Cryptosporidium. However, the evolutionary relationship by bioinformatics analysis and phylogenetic tree construction showed that resulting Cryptosporidium isolates had the closest match with three isolates; Cryptosporidium sp. pig genotype II (isolated from pigs in China), C. parvum (Egyptian isolate from buffalo in Ismailia province) and C. baileyi (isolated from quails in China). Despite that these results disagree with McLauchlin et al. (2000) and others (Insulander et al., 2013; Friesema et al., 2012; Wang et al., 2011) who could identify C. parvum genotypes by 18S rRNA gene sequence, they confirmed the previous conclusion of many researchers who found the use of multi-loci analysis had better results with regards to Cryptosporidium genotyping (Abe and Teramato, 2012; Amer et al., 2010). Because their sequences had higher intraspecific variation than the ribosomal RNA gene regions (Morgan et al., 1999b), it was suggested that other Cryptosporidium genes targets should be used for amplification including the Cryptosporidium oocyst wall protein (COWP), 16S rRNA, Hsp70, Actin, β-Tubulin, gp60, microsatellites, minisatellites and extrachromosomal double-stranded RNA (Xiao et al., 2004; Caccio et al., 2005; Coklin et al., 2007). As well as the Internal Transcribed Spacers (ITS) of ribosomal DNA were useful for the detection of genetic variability within species (Chalmers et al., 2005; Schindler et al., 2005). Synchronized analysis of the obtained morphological simultaneous with molecular criteria of Cryptosporidium buffalo’s oocytes in the present study could prove that the isolates were C. parvum. Theoretically, it was known that C. parvum is infectious to many mammalian hosts worldwide (Fayer et al., 2006; Santin and Zarlenga, 2009). Calves were the major recognized reservoirs for C. parvum (Caccio et al., 2000; Warda et al., 2002; Condoleo et al., 2007; Paul et al., 2008; Helmy et al., 2013; Mahfouz et al., 2014) with strong relation between C. parvum infection and diarrhea among Egyptian buffalo calves (Warda et al., 2002). Cryptospordium parvum was mostly dominant in preweaned calves (El-Dessouky and El-Masry, 2005; Santin et al., 2008; Keshavarz et al., 2009; Karanis et al., 2010; Randhawa et al., 2012a). Also, it’s known that C. baileyi infected a broad range of birds and found in the small and large intestine, bursa, respiratory tissues such as the conjunctiva, sinus and trachea. Viable C. baileyi oocysts measured 6.2 by 4.6 μm (5.6-6.3 by 4.5-4.8 μm). Oocysts of C. baileyi were inoculated orally into several animals to determine its host specificity. Mice and goats inoculated with C. baileyi oocysts did not become infected (Current et al., 1986; Xiao et al., 2004). Based on these theories and hence the detected Cryptosporidium oocysts in the examined young buffalo calf feces stained with Ziehl-Neelsen technique were morphologically similar to C. parvum oocysts. It was implied that the Cryptosporidium isolates resulted in this study was mostly approaching to the C. parvum (JX237832.1) isolated previously from buffaloes in Ismailia province. In this study, the Cryptosporidium species with unclear identity were observed. Additional studies, using more genes, with a larger number of isolates from various geographic areas, different husbandry and management systems, covering the item of seasonality should be conducted to identify the species of Cryptosporidium. Little information on gene sequence of isolates from Cryptosporidium species associated with animal hosts in Egypt had been reported so, extensive studies were extremely important including biological aspects associated with molecular techniques (Hassanain et al., 2011). Also, Amer et al. (2010) stated that very little was known about genetic structure of Cryptosporidium spp., in Egypt. Important research gaps remained including lack of subtyping tools for many Cryptosporidium species of public and veterinary health importance, poor understanding of host specificity of Cryptosporidium species and impact of climate change on their transmission (Ryan et al., 2014).

CONCLUSION

In this endeavor the parasitic and pathological studies identified the Cryptosporidium spp. as a causative agent of diarrhea in buffalo calves. While, molecular studies revealed that the similar Cryptosporidium species and C. parvum genotype that was previously isolated in outbreak-associated buffalo calves in Egypt were identified. Sequencing the PCR products obtained from the Egyptian buffalo calf samples may assist in elucidating the Cryptosporidium species C. parvum genotype signature of the amplifiable Cryptosporidium DNA isolated.

REFERENCES
Abdel Megeed, K.N., A.M. Hammam, G.H. Morsy, F.A.M. Khalil, M.M.E. Seliem and D. Aboelsoued, 2015. Control of cryptosporidiosis in buffalo calves using garlic (Allium sativum) and nitazoxanide with special reference to some biochemical parameters. Global Veterinaria, 14: 646-655.
Direct Link  |  

Abdel-Rady, A. and M. Sayed, 2008. Efficiency of hot modified Ziehl-Neelsen staining for detection of Cryptosporidium oocysts. Proceedings of the 4th International Scientific Conference of the Egyptian Society of Environmental Toxicology, November 11-14, 2008, Safaga, Egypt, -.

Abe, N. and I. Teramoto, 2012. Molecular evidence for person-to-person transmission of a novel subtype in Giardia duodenalis assemblage B at the rehabilitation institution for developmentally disabled people. Parasitol. Res., 110: 1025-1028.
CrossRef  |  Direct Link  |  

Abu El Ezz, N.M.T., F.A.M. Khalil and R.M. Shaapan, 2011. Therapeutic effect of onion (Allium cepa) and cinnamon (Cinnamomum zeylanicum) oils on cryptosporidiosis in experimentally infected mice. Global Veterinaria, 7: 179-183.
Direct Link  |  

Amer, S., H. Honma, M. Ikarashi, C. Tada, Y. Fukuda, Y. Suyama and Y. Nakai, 2010. Cryptosporidium genotypes and subtypes in dairy calves in Egypt. Vet. Parasitol., 169: 382-386.
CrossRef  |  Direct Link  |  

Amer, S., S. Zidan, H. Adamu, J. Ye, D. Roellig, L. Xiao and Y. Feng, 2013. Prevalence and characterization of Cryptosporidium spp. in dairy cattle in Nile River delta provinces, Egypt. Exp. Parasitol., 135: 518-523.
CrossRef  |  Direct Link  |  

Amer, S., S. Zidan, Y. Feng, H. Adamu, N. Li and L. Xiao, 2013. Identity and public health potential of Cryptosporidium spp. in water buffalo calves in Egypt. Vet. Parasitol., 191: 123-127.
CrossRef  |  Direct Link  |  

Bhat, S.A., P.D. Juyal and L.D. Singla, 2012. Prevalence of cryptosporidiosis in neonatal buffalo calves in Ludhiana district of Punjab, India. Asian J. Anim. Vet. Adv., 7: 512-520.
CrossRef  |  Direct Link  |  

Bhat, S.A., P.D. Juyal and L.D. Singla, 2013. Bovine cryptosporidiosis: Brief review of its distribution in India. Trends Parasitol. Res., 2: 5-13.
Direct Link  |  

Caccio, S., W. Homan, R. Camilli, G. Traldi, T. Kortbeek and E. Pozio, 2000. A microsatellite marker reveals population heterogeneity within human and animal genotypes of Cryptosporidium parvum. Parasitology, 120: 237-244.
Direct Link  |  

Caccio, S.M., R.C.A. Thompson, J. McLauchlin and H.V. Smith, 2005. Unravelling Cryptosporidium and Giardia epidemiology. Trends Parasitol., 21: 430-437.
CrossRef  |  Direct Link  |  

Chalmers, R.M., C. Ferguson, S. Caccio, R.B. Gasser and Y.G.A. El-Osta et al., 2005. Direct comparison of selected methods for genetic categorisation of Cryptosporidium parvum and Cryptosporidium hominis species. Int. J. Parasitol., 35: 397-410.
CrossRef  |  Direct Link  |  

Coklin, T., J. Farber, L. Parrington and B. Dixon, 2007. Prevalence and molecular characterization of Giardia duodenalis and Cryptosporidium spp. in dairy cattle in Ontario, Canada. Vet. Parasitol., 150: 297-305.
CrossRef  |  PubMed  |  Direct Link  |  

Condoleo, R.U., L. Rinaldi, G. Saralli, M.E. Morgoglione and M. Schioppi et al., 2007. An updating on Cryptosporidium parvum in the water buffalo. Ital. J. Anim. Sci., 6: 917-919.
CrossRef  |  Direct Link  |  

Coupe, S., C. Sarfati, S. Hamane and F. Derouin, 2005. Detection of Cryptosporidium and identification to the species level by nested PCR and restriction fragment length polymorphism. J. Clin. Microbiol., 43: 1017-1023.
CrossRef  |  PubMed  |  Direct Link  |  

Current, W.L. and N.C. Reese, 1986. A comparison of endogenous development of three isolates of Cryptosporidium in suckling mice. J. Protozool., 33: 98-108.
CrossRef  |  Direct Link  |  

Current, W.L., N.C. Reese, J.V. Ernst, W.S. Bailey, M.B. Heyman and W.M. Weinstein, 1983. Human cryptosporidiosis in immunocompetent and immunodeficient persons-studies of an outbreak and experimental transmission. N. Engl. J. Med., 308: 1252-1257.
CrossRef  |  PubMed  |  Direct Link  |  

Current, W.L., S.J. Upton and T.B. Haynes, 1986. The life cycle of Cryptosporidium baileyi n. sp. (Apicomplexa, Cryptosporidiidae) infecting chickens. J. Protozool., 33: 289-296.
CrossRef  |  Direct Link  |  

Diaz-Lee, A., R. Mercado, E.O. Onuoha, L.S. Ozaki and P. Munoz et al., 2011. Cryptosporidium parvum in diarrheic calves detected by microscopy and identified by immunochromatographic and molecular methods. Vet. Parasitol., 176: 139-144.
CrossRef  |  Direct Link  |  

Drury, R.A.B. and E.A. Wallington, 1967. Carleton's Histological Technique. 4th Edn., Oxford University Press, New York, USA., pp: 129-130.

Egyed, Z., T. Sreter, Z. Szell and I. Varga, 2003. Characterization of Cryptosporidium spp.: Recent developments and future needs. Vet. Parasitol., 111: 103-114.
CrossRef  |  Direct Link  |  

El-Dessouky, S.A. and N.M. El-Masry, 2005. Effect of Cryptosporidium parvum infection on the haematology and blood chemistry of buffalo calves with special reference to the prevalence of infection in adult buffaloes. Assiut Vet. Med. J., 51: 108-123.

El-Khodery, S.A. and S.A. Osman, 2008. Cryptosporidiosis in buffalo calves (Bubalus bubalis): Prevalence and potential risk factors. Trop. Anim. Health Prod., 40: 419-426.
CrossRef  |  PubMed  |  Direct Link  |  

El-Sherbini, G.T. and K.A. Mohammad, 2006. Zoonotic cryptosporidiosis in man and animal in farms, Giza Governorate, Egypt. J. Egypt. Soc. Parasitol., 36: 49-58.
PubMed  |  Direct Link  |  

Fall, A., R.C.A Thompson, R.P. Hobbs and U. Morgan-Ryan, 2003. Morphology is not a reliable tool for delineating species within Cryptosporidium. J. Parasitol., 89: 399-402.
CrossRef  |  PubMed  |  Direct Link  |  

Fathia, A.M.K., 1993. Studies on Cryptosporidium infection in calves. M.Sc. Thesis, Faculty of Veterinary Medicine, Cairo University, Cairo, Egypt.

Fayer, R. and L. Xiao, 2007. Cryptosporidium and Cryptosporidiosis. 2nd Edn., CRC Press, Boca Raton, FL., USA., ISBN-13: 9781420052275, Pages: 576.

Fayer, R., M. Santin and L. Xiao, 2005. Cryptosporidium bovis n. sp. (Apicomplexa: Cryptosporidiidae) in cattle (Bos taurus). J. Parasitol., 91: 624-629.
CrossRef  |  Direct Link  |  

Fayer, R., M. Santin, J.M. Trout and E. Greiner, 2006. Prevalence of species and genotypes of Cryptosporidium found in 1-2-year-old dairy cattle in the Eastern United States. Vet. Parasitol., 135: 105-112.
CrossRef  |  Direct Link  |  

Fayer, R., U. Morgan and S.J. Upton, 2000. Epidemiology of Cryptosporidium: Transmission, detection and identification. Int. J. Parasitol., 30: 1305-1322.
CrossRef  |  PubMed  |  Direct Link  |  

Felsenstein, J., 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution, 39: 783-791.
CrossRef  |  Direct Link  |  

Friesema, I.H.M., R.F. de Boer, E. Duizer, L.M. Kortbeek and D.W. Notermans et al., 2012. Etiology of acute gastroenteritis in children requiring hospitalization in the Netherlands. Eur. J. Clin. Microbiol. Infect. Dis., 31: 405-415.
CrossRef  |  Direct Link  |  

Gaafar, M.R., 2012. Efficacy of Allium sativum (garlic) against experimental cryptosporidiosis. Alexandria J. Med., 48: 59-66.
CrossRef  |  Direct Link  |  

Hassanain, M.A., F.A.M. Khalil, K.A. AbdEl-Razik and R.M. Shaapan, 2011. Prevalence and molecular discrimination of Cryptosporidium parvum in calves in Behira provinces, Egypt. Res. J. Parasitol., 6: 101-108.
CrossRef  |  Direct Link  |  

Helmy, Y.A., G. von Samson-Himmelstjerna, K. Nockler and K.H. Zessin, 2015. Frequencies and spatial distributions of Cryptosporidium in livestock animals and children in the Ismailia province of Egypt. Epidemiol. Infect., 143: 1208-1218.
CrossRef  |  Direct Link  |  

Helmy, Y.A., J. Krucken, K. Nockler, G. von Samson-Himmelstjerna and K.H. Zessin, 2013. Molecular epidemiology of Cryptosporidium in livestock animals and humans in the Ismailia province of Egypt. Vet. Parasitol., 193: 15-24.
CrossRef  |  Direct Link  |  

Henriksen, S.A. and J.F. Pohlenz, 1981. Staining of cryptosporidia by a modified Ziehl-Neelsen technique. Acta Vet. Scand., 22: 594-596.
PubMed  |  Direct Link  |  

Insulander, M., C. Silverlas, M. Lebbad, L. Karlsson, J.G. Mattsson and B. Svenungsson, 2013. Molecular epidemiology and clinical manifestations of human cryptosporidiosis in Sweden. Epidemiol. Infect., 141: 1009-1020.
CrossRef  |  Direct Link  |  

Jex, A.R., H.V. Smith, P.T. Monis, B.E. Campbell and R.B. Gasser, 2008. Cryptosporidium-Biotechnological advances in the detection, diagnosis and analysis of genetic variation. Biotechnol. Adv., 26: 304-317.
CrossRef  |  Direct Link  |  

Jex, A.R., U.M. Ryan, J. Ng, B.E. Campbell, L. Xiao, M. Stevens and R.B. Gasser, 2007. Specific and genotypic identification of Cryptosporidium from a broad range of host species by nonisotopic SSCP analysis of nuclear ribosomal DNA. Electrophoresis, 28: 2818-2825.
CrossRef  |  Direct Link  |  

Jin, B., A. Yashpal, N. Deol, A. AbuRashed and H. Saleh, 2015. Cryptosporidiosis in an immunocompetent individual: An unusual case with brief review of the literature. Diagnost. Pathol., Vol. 1. 10.17629/www.diagnosticpathology.eu-2015-1:10

Karanis, P., T. Eiji, L. Palomino, K. Boonrod, J. Plutzer, J. Ongerth and I. Igarashi, 2010. First description of Cryptosporidium bovis in Japan and diagnosis and genotyping of Cryptosporidium spp. in diarrheic pre-weaned calves in Hokkaido. Vet. Parasitol., 169: 387-390.
CrossRef  |  Direct Link  |  

Keshavarz, A., A. Haghighi, A. Athari, B. Kazemi, A. Abadi and E.N. Mojarad, 2009. Prevalence and molecular characterization of bovine Cryptosporidium in Qazvin province, Iran. Vet. Parasitol., 160: 316-318.
CrossRef  |  PubMed  |  Direct Link  |  

Kvac, M. and J. Vitovec, 2003. Prevalence and pathogenicity of Cryptosporidium andersoni in one herd of beef cattle. J. Vet. Med. Ser. B, 50: 451-457.
CrossRef  |  Direct Link  |  

Mahfouz, M.E., N. Mira and S. Amer, 2014. Prevalence and genotyping of Cryptosporidium spp. in farm animals in Egypt. J. Vet. Med. Sci., 76: 1569-1575.
CrossRef  |  Direct Link  |  

McLauchlin, J., C. Amar, S. Pedraza-Diaz and G.L. Nichols, 2000. Molecular epidemiological analysis of Cryptosporidium spp. in the United Kingdom: Results of genotyping Cryptosporidium spp. in 1,705 fecal samples from humans and 105 fecal samples from livestock animals. J. Clin. Microbiol., 38: 3984-3990.
PubMed  |  Direct Link  |  

Monis, P.T. and R.C.A. Thompson, 2003. Cryptosporidium and Giardia-zoonoses: Fact or fiction? Infect. Genet. Evol., 3: 233-244.
CrossRef  |  Direct Link  |  

Morgan, U.M., P. Deplazes, D.A. Forbes, F. Spano and H. Hertzberg et al., 1999. Sequence and PCR-RFLP analysis of the internal transcribed spacers of the rDNA repeat unit in isolates of Cryptosporidium from different hosts. Parasitology, 118: 49-58.
PubMed  |  Direct Link  |  

Morgan, U.M., P.T. Monis, R. Fayer, P. Deplazes and R.C.A. Thompson, 1999. Phylogenetic relationships among isolates of Cryptosporidium: Evidence for several new species. J. Parasitol., 85: 1126-1133.
CrossRef  |  Direct Link  |  

Morsy, G.H., K.N. Abdel Megeed, A.M. Hammam, M.M.E. Seliem, F.A.M. Khalil and D. Aboelsoued, 2014. Prevalence of Cryptosporidium infection in buffalo calves with special reference to urea and creatinine levels. Global Veterinaria, 13: 662-667.
Direct Link  |  

O'Donoghue, P.J., 1995. Cryptosporidium and cryptosporidiosis in man and animals. Int. J. Parasitol., 25: 139-195.
CrossRef  |  PubMed  |  Direct Link  |  

Paul, S., D. Chandra, D. Ray, A.K. Tewari and J.R. Rao et al., 2008. Prevalence and molecular characterization of bovine Cryptosporidium isolates in India. Vet. Parasitol., 153: 143-146.
CrossRef  |  Direct Link  |  

Quintero-Betancourt, W., A.L. Gennaccaro, T.M. Scott and J.B. Rose, 2003. Assessment of methods for detection of infectious Cryptosporidium oocysts and Giardia cysts in reclaimed effluents. Applied Environ. Microbiol., 69: 5380-5388.
CrossRef  |  Direct Link  |  

Quintero-Betancourt, W., E.M. Peele and J.B. Rose, 2002. Cryptosporidium parvum and Cyclospora cayetanensis: A review of laboratory methods for detection of these waterborne parasites. J. Microbiol. Methods, 49: 209-224.
CrossRef  |  Direct Link  |  

Randhawa, S.S., S.S. Randhawa, U.N. Zahid, L.D. Singla and P.D. Juyal, 2012. Drug combination therapy in control of cryptosporidiosis in Ludhiana district of Punjab. J. Parasitic Dis., 36: 269-272.
CrossRef  |  Direct Link  |  

Randhawa, S.S., U.N. Zahid, S.S. Randhawa, P.D. Juyal, L.D. Singla and S.K. Uppal, 2012. Diagnosis and therapeutic management of cryptosporidiosis in cross bred dairy calves. Indian Vet. J., 89: 17-19.

Ryan, U., R. Fayer and L. Xiao, 2014. Cryptosporidium species in humans and animals: Current understanding and research needs. Parasitology, 141: 1667-1685.
CrossRef  |  Direct Link  |  

Santin, M. and D.S. Zarlenga, 2009. A multiplex polymerase chain reaction assay to simultaneously distinguish Cryptosporidium species of veterinary and public health concern in cattle. Vet. Parasitol., 166: 32-37.
CrossRef  |  Direct Link  |  

Santin, M., J.M. Trout and R. Fayer, 2008. A longitudinal study of cryptosporidiosis in dairy cattle from birth to 2 years of age. Vet. Parasitol., 155: 15-23.
CrossRef  |  Direct Link  |  

Schindler, A.R., Y.S.A. EL-Osta, M. Stevens, M.I. Sinclair and R.B. Gasser, 2005. Capillary electrophoretic analysis of fragment length polymorphism in ribosomal markers of Cryptosporidium from humans. Mol. Cell. Probes, 19: 394-399.
CrossRef  |  Direct Link  |  

Sheehan, D.C. and B.B. Hrapchak, 1987. Theory and Practice of Histotechnology. 2nd Edn., Battelle Press, Ohio, USA., ISBN-13: 9780935470390, pp: 235-237.

Shields, J.M., J. Joo, R. Kim and H.R. Murphy, 2013. Assessment of three commercial DNA extraction kits and a laboratory-developed method for detecting Cryptosporidium and Cyclospora in raspberry wash, basil wash and pesto. J. Microbiol. Methods, 92: 51-58.
CrossRef  |  Direct Link  |  

Shoukry, N.M., H.A. Dawoud and F.M. Haridy, 2009. Studies on zoonotic cryptosporidiosis parvum in Ismailia Governorate, Egypt. J. Egypt Soc. Parasitol., 39: 479-488.
PubMed  |  

Sneath, P.H.A. and R.R. Sokal, 1973. Numerical Taxonomy: The Principles and Practice of Numerical Classification. 2nd Edn., WH Freeman and Co., San Francisco, CA., USA., ISBN-13: 9780716706977, Pages: 573.

Tamura, K., J. Dudley, M. Nei and S. Kumar, 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol., 24: 1596-1599.
CrossRef  |  PubMed  |  Direct Link  |  

Tamura, K., M. Nei and S. Kumar, 2004. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc. Natl. Acad. Sci. USA., 101: 11030-11035.
CrossRef  |  Direct Link  |  

Toulah, F.H., A.A. El-Shafei and H.S. Al-Rashidi, 2012. Evaluation of garlic plant and indinavir drug efficacy in the treatment of cryptosporidiosis in experimentally immumosuppressed rats. J. Egypt. Soc. Parasitol., 42: 321-328.
PubMed  |  Direct Link  |  

Wang, R., X. Zhang, H. Zhu, L. Zhang and Y. Feng et al., 2011. Genetic characterizations of Cryptosporidium spp. and Giardia duodenalis in humans in Henan, China. Exp. Parasitol., 127: 42-45.
CrossRef  |  PubMed  |  Direct Link  |  

Warda, M., A. El-Ghaysh, M. Ghoneim, F. Khalil and M. Hilali, 2002. Polymerase chain reaction is a detectable tool to discriminate between Cryptosporidium parvumand other apicomplexan parasites using C. parvum 18s RRNa and Outer Wall Protein (COWP) genes. Proceedings of the 10th Scientific Conference, Faculty of Veterinary Medicine, December 2002, Assiut University, Egypt, pp: 449-457.

Xiao, L., 2010. Molecular epidemiology of cryptosporidiosis: An update. Exp. Parasitol., 124: 80-89.
CrossRef  |  PubMed  |  Direct Link  |  

Xiao, L., R. Fayer, U. Ryan and S.J. Upton, 2004. Cryptosporidium taxonomy: Recent advances and implications for public health. Clin. Microbiol. Rev., 17: 72-97.
CrossRef  |  Direct Link  |  

Xiao, L., U.M. Morgan, J. Limor, A. Escalante and M. Arrowood et al., 1999. Genetic diversity within Cryptosporidium parvum and related Cryptosporidium species. Applied Environ. Microbiol., 65: 3386-3391.
PubMed  |  Direct Link  |  

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