collection of specimens and examination : samples of this study were 
 collected during june 2010november 2011 from 5 private farms designated a , b , c , d and e , 
 which were situated at different localities in kafr el sheikh ( 130 km north of cairo ) , egypt 
 ( table 1table 1.infection rates of cryptosporidium in different age groups of 
 buffalo , cattle and sheep in kafr el sheikh province , egypt , based on acid fast 
 staining - microscopyfarm namefarm animalsneonatesheifersadultssample sizeno . positiveinfection rate ( % ) farm acattle15442.6012521.6037020.54farm bcattle1591610.101231310.60318216.60farm ccattle137118.0751824.02363314.0farm dbuffalo5711.808900.011600.0farm ebuffalo6346.405000.09111.10farm csheep4524.40 - --7511.30 ) . in cattle and buffalo farms , the neonates were kept in separate pens with 
 milk feeding of suckling calves performed manually , allowing no direct contact of calves 
 with their dams . based on animals age , the examined cattle and buffalo were classified into 
 3 groups : less than 3 months of age ( calves ) , 424 months ( heifers ) and older than 2 years 
 ( adults ) . a total of 2,163 fecal samples were collected from 570 calves including 450 cattle 
 and 120 buffalo , 462 heifers comprising 323 cattle and 139 buffalo and 1,131 adults with 924 
 cattle and 207 buffalo . additionally , 120 sheep samples ( 45 neonates and 75 adults ) were 
 sampled . fecal specimens were collected directly from the rectum of each animal with a 
 gloved hand and transferred into plastic cups labeled with the ear tag number and farm name . the fecal samples were screened for cryptosporidium oocysts by microscopy 
 using the modified ziehl - neelsen stain . positive 
 specimens were stored in 2.50% potassium dichromate at 4.0c for molecular biologic 
 analyses . dna extraction and cryptosporidium genotyping and subtyping : 
 microscopy - positive specimens were washed off potassium dichromate with distilled water by 
 centrifugation . dna was extracted using the qiamp dna stool mini kit ( qiagen , hilden , 
 germany ) , according to the manufacturer s protocol . genotyping of 
 cryptosporidium was done by pcr - rflp analysis of ssu rrna gene as 
 described by feng et al . . because 60 kda glycoprotein ( gp60 ) gene is highly polymorphic , it is favorably used as a 
 good tool for genotyping and subtyping of many cryptosporidium species 
 including c. parvum , c. hominies,  etc . in the present 
 study , samples from c. parvum were subtyped by nested pcr - sequence analysis 
 of gp60 as described previously . dna sequencing , sequence analysis and phylogenetic analysis : secondary pcr 
 products of the representative samples of each rflp pattern were sequenced directly using 
 big dye terminator v3.1 cycle sequencing kit ( applied biosystems , foster city , ca , u.s.a . ) 
 and read on an abi 3130 genetic analyzer ( applied biosystems ) . sequences were assembled 
 using the chromaspro software ( version 1.5 ) 
 ( http://www.technelysium.com.au/chromaspro.html ) . the obtained sequences were aligned with reference sequences in 
 database using clustalx ( ftp://ftp-igbmc.u-trasbg.fr/pub/clustalx/ ) to determine the 
 identity of the examined species . unique nucleotide sequences generated in this study were 
 deposited in genbank under accession numbers ab922117ab922125 . the neighbor - joining ( nj ) 
 method as implemented in the mega5 ( http://www.megasoftware.net/ ) was used in a phylogenetic 
 analysis of ssu rrna sequences , utilizing eimeria tenella ( af026388 ) as an 
 out - group . cryptosporidium infection : cryptosporidium oocysts were 
 detected in all examined farms in spite of species or age of the subjected animals ( table 1 ) . in water buffalo , 
 cryptosporidium oocysts were detected microscopically in 5 out of 120 
 ( 4.17% ) fecal samples from calves and in one out of 207 of adults ( 0.48% ) , while no oocysts 
 were detected in the 139 examined specimens from heifers . nonetheless , the infection rate in 
 cattle was higher than that detected in buffalo animals , addressing the effect of animal 
 species on the cryptosporidium infection rate , especially in case of older 
 animals . a total of 31 out of 450 ( 6.90% ) fecal samples from calves , 33 out of 323 ( 10.20% ) 
 from heifers and 56 out of 924 ( 6.10% ) from adult cattle proved to have 
 cryptosporidium oocysts . microscopic examination of sheep samples 
 revealed the occurrence of cryptosporidium oocysts in 2 out of 45 lambs 
 ( 4.40% ) and only one sample out of 75 adult sheep ( 1.30% ) . cryptosporidium species : rflp analysis of pcr products of the ssu rrna 
 gene indicated the presence of c. ryanae in 4 specimens ( 66.67% , 3 calves 
 and one adult ) and c. parvum in 2 specimens ( 33.33% ) of buffalo samples . in 
 addition , results revealed that 3 cryptosporidium species were detected in 
 cattle calves with varying prevalence ; c. parvum ( 23/31 ; 74.23% ) , 
 c. ryanae ( 5/31 ; 16.10% ) and c. bovis ( 3/31 ; 9.70% ) , 
 indicating the dominance of c. parvum . on the other hand , in heifers and 
 adults , c. andersoni was the overwhelming species accompanied with a minor 
 fraction of c. parvum ( 2/89 ; 2.30% ) . in sheep , c. xiaoi 
 was the only detected species in the 3 positively infected animals . based on the rflp 
 patterns , no mixed infections in one animal were detected in the present study . dna sequencing of representative samples confirmed the identification of the 
 cryptosporidium species . blasting the obtained sequences with those in 
 database and the deduced phylogenetic analysis ( fig . 1.phylogenetic relationship of cryptosporidium spp . based on sequences 
 of the partial ssu rrna gene . evolutionary relationships of 43 taxa were inferred 
 using the neighbor - joining method   with the 
 coccidian eimeria tenella ( af026388 ) as an out - group . egyptian 
 isolates are bolded . ) demonstrated that out of all the isolated samples , the sequences of c. 
 parvum were 100% identical to each other in spite of the difference in host 
 species and age of the animals . moreover , these sequences were identical to reference 
 sequences of c. parvum ( ab777182 ) from egypt and showed one base pair 
 difference from the reference sequences reported from egypt ( ab513881 ) , iran ( kf830257 ) and 
 brazil ( jn247404 ) . meanwhile , specimens of c. andersoni generated one 
 sequence that was identical to those from china ( kf271477 ) , japan ( ab089285 ) and egypt 
 ( ab513856 ) . phylogenetic relationship of cryptosporidium spp . based on sequences 
 of the partial ssu rrna gene . evolutionary relationships of 43 taxa were inferred 
 using the neighbor - joining method   with the 
 coccidian eimeria tenella ( af026388 ) as an out - group . the first was derived 
 from two isolates of buffalo and was identical to reference sequences reported from buffalo 
 in brazil , egypt and india ( jx559848 , ab712388 and jq002562 , respectively ) . the second 
 sequence type was generated from 3 specimens of cattle and was identical to that derived 
 from dairy calves in china ( hq179574 ) as well as from cattle in egypt ( ab777174 ) , while it 
 has one base pair difference ( 99.0% homology ) to sequences jx515551 isolated from dairy calf 
 and kf128757 isolated from yak in china . one type of sequence was obtained from c. bovis that was identical to the 
 reference reported from egypt ( ab777172 ) and 99.0% similarity , with one base pair 
 difference , compared to the sequences described from yak in china ( kf128742 ) , from cattle in 
 japan ( ab746197 ) and from humans in australia ( jq362488 ) . c. xiaoi from sheep generated one sequence type that was identical to 
 sequences from sheep in u.s.a . and china ( fj896044 and kf907825 ) . in addition , 99.0% 
 similarity , one base pair difference , was noticed when compared to sequences from goat in 
 spain and sheep in romania ( gu553016 and jx258864 , respectively ) . c. parvum subtypes : in the current study , sequence analysis of c. 
 parvum isolates on the gp60 gene locus revealed no difference among subtypes 
 isolated from buffalo or cattle and generated two types of sequences . the first includes 3 
 sequences and belongs to the subtype iid ( iida20g1 subtype ) . this sequence type was 
 identical to sequences obtained from dairy cattle and buffalo in egypt ( ab514086 , ab514089 
 and ab712389 ) and 99.0% with sequences with those reported from children in kuwait and 
 sweden ( ay738186 and jq028866 ) . these sequences were identical to those reported from humans in kuwait 
 , slovenia   and australia   as well as from 
 dairy calves in the czech republic , dairy and 
 buffalo calves from egypt [ 4 , 5 ] and dairy calves and humans from egypt . the coccidian protozoa cryptosporidium infects a wide range of livestock , 
 resulting in significant economic loss and public health concern . in the present study , the 
 infection rate varies according to the age and species of the host . cryptosporidium oocysts were identified microscopically in 4.17% fecal 
 samples of water buffalo calves and in 0.48% of adults , while no oocysts were detected in 
 specimens from heifers . the infection rate in calves reported herein is much lower than that 
 reported by amer et al . ( 9.50% ) 
 and el - khodery and osman   ( 14.10% ) , for water 
 buffalo from egypt and bhat et al . the differences in the 
 reported infection rates may be attributed to differences in the age and breed of animals , 
 season of specimen collection , environmental settings as well as management and husbandry 
 regimens . results from older animals indicated the negativity of all heifers samples and 
 very low infection rate ( 0.48% ) in adults . these results are comparable to those reported by 
 abou - eisha   and amer et al .   for adult water buffalo in egypt and for buffalo in cuba , 
 who reported no oocysts detection in adult water buffalo . although it is difficult to 
 explain the low incidence of cryptosporidium in adult buffalo , it might , 
 partially , be attributed to the sensitivity of microscopic investigation or more likely to 
 the resistance of adult buffalo either as species or owing to previous infections . in cattle , the infection rates ( overall 7.07% ) were higher than that detected in buffalos , 
 addressing the effect of animal species on the cryptosporidium infection 
 rate , especially in case of older animals . the prevalence was almost similar in both calves 
 and adults ( ~6.90% ) which relatively increased in case of heifers ( 10.20% ) . these results 
 are comparable to those previously reported in dairy cattle in egypt ; 30.20%   and 13.60% . 
 in the present study , high differences were detected in the infection rates between 
 different farms ; 2.60% versus 10.10% in calves , 1.60% versus 24.0% in heifers and 0.54% 
 versus 14.0% in adults , in low and high rate infected farms , respectively . these differences 
 reflect the effect of farm related factors on the incidence of 
 cryptosporidium . the management regimen in the farm a imposes very 
 restricting roles on the in and out of the animals , and the suckling calves up to the ~6 
 weeks of age are reared in ground separated pens with very limited opportunity of calves to 
 contact with the discharged fecal matters . because farms b and c belong to the same body , 
 the move of animals between the two farms allows introducing new members to the herds . the 
 calves are reared in ground pens , which in turn afford high possibility to contact with the 
 fecal matter . microscopic examination of sheep samples revealed the occurrence of 
 cryptosporidium oocysts in 4.40% of lambs and 1.30% of adult sheep . similar results were reported in sheep from brazil   and egypt . however , this infection 
 rate is remarkably lower than that reported in sheep and lambs in norway ( 1524.0% ) , romania 
 ( 13.70% ) , brazil ( 25.0% ) and australia ( 16.90% ) [ 23 , 
 34 , 37 , 
 53 ] , addressing the habitat on the incidence of the 
 disease . cryptosporidium species : rflp and sequence analyses of ssu rrna gene 
 fragment ( table 2table 2.distribution of cryptosporidium spp . in different age groups of 
 buffalo , cattle and sheep in kafr el sheikh province , egypt , based on pcr - rflp 
 patterns of ssu rrna gene fragmentfarm nameneonatesheifersadultsc . b113200001310020farm c91100001810032farm d100000000000farm e130000000100farm c2 specimens of c. xiaoione specimen of c. xiaoi ) indicated the presence of c. ryanae ( 66.67% ) and c. 
 parvum ( 33.33% ) in buffalo samples . similar results were reported in water 
 buffalo from egypt . on the other hand , caccio 
 et al .   and gomez - couso 
 et al . reported only 
 c. parvum in water buffalo calves in spain and italy , respectively . feng 
 et al .   reported only 
 c. ryanae in buffalo calves from nepal . these discrepancies may be 
 attributed to farm , breed and/or climatic factors . c. parvum was the dominant prevailed species in cattle calves ( 74.23% ) 
 versus c. ryanae ( 16.10% ) and c. bovis ( 9.70% ) , whereas , 
 in heifers and adults , c. andersoni was the prevailing along with a small 
 fraction of with c. parvum ( 2.30% ) . it is evident that the distribution of 
 cryptosporidium species in pre - weaned calves differs from a country to 
 another and from a study to others , reflecting the profound effect of the environment and 
 husbandry regimen on the infection rate . an exclusive presence of c. 
 parvum , or with a minor fraction of other cryptosporidia , in pre - weaned dairy 
 calves was previously reported in many countries [ 3 , 
 44 ] , contrasting the predominance of c. 
 bovis and c. ryanae in some other countries [ 17 , 29 ] . in the 
 present study , c. andersoni appears to be the major species existing in 
 heifers and adult cattle ( 87/89 ; 97.80% ) . a similar dominance was reported in a number of 
 asian countries , such as china [ 28 , 49 ] , as well as european countries . in the united states and canada , c. andersoni 
 occurs in adult cattle beside different proportions of c. bovis and 
 c. ryanae[8 , 15 ] . although c. andersoni represents no public health 
 concerns , it can cause impairment of weight gain in growing animals and milk production in 
 dairy cows and thus reduced feed efficiency . in concordance with results reported herein , c. xiaoi was the dominant 
 cryptosporidium species in ewes and lambs in china   and in sheep in australia . in contrast , imre et al . reported the dominance of the zoonotic c. parvum in newborn 
 lambs in romania , while c. cervine was the major genotype in adult sheep in 
 china . , especially c. xiaoi , 
 from ewes to lambs was addressed for cycling of the infection . in spite of the limitation of sample size , the obtained results 
 indicated that cryptosporidium in sheep has no communal health concerns . generally , the high identity of the generated sequences of similar type may be due to the 
 semi - conservative nature of the ssu rrna gene locus   common origin of the infection and/or the possibility that the infection was 
 due to epidemic clone ( the genetic fitness of this cryptosporidium strain ) 
 [ 2 , 7 ] . moreover , geographic differences in the distribution of specific alleles may exist . c. parvum subtypes : occurrence of iid and iia subtypes highlights the 
 public health risk posed by cryptosporidium parvum recovered in the present 
 study . the incidence of c. parvum subtype families differs widely according 
 to geographic locations and hosts . subtype iid was reported as the predominant subtype 
 family of c. parvum in cattle in egypt , malaysia and china [ 3 , 29 , 50 ] , sheep and goats in spain   and human in kuwait , jordan , iran and egypt [ 21 , 22 , 42 , 43 ] in addition to hiv / aids 
 patients in malaysia . however , it was detected 
 at low frequencies in cattle in europe and is largely absent in cattle and humans in u.s.a . 
 and canada . meanwhile , iia is the most prevalent 
 subtype family of c. parvum in calves worldwide , especially in 
 industrialized countries [ 19 , 25 ] , implicating the cattle as the main source of human iia 
 infections . in conclusion , cryptosporidium oocysts were detected in all of the 
 examined farms in spite of species or age of animals , which signify the widespread of the 
 infection in egypt . in cattle , the infection rates were higher than that detected in buffalo 
 or sheep , revealing the effect of animal species on cryptosporidium 
 infection rate , especially in case of older animals . the common occurrence of the c. 
 parvum iia and iid subtype families in both buffalo and dairy calves in egypt 
 highlights the potential role of cryptosporidium zoonotic in these 
 animals . the study was approved by the institutional committee of the post graduate studies 
 and research at kafr el sheikh university , egypt .
abstractin this study , we examined the 
 prevalence and molecular characteristics of cryptosporidium in buffalo , 
 dairy cattle and sheep in different farms at kafr el sheikh province , egypt . 
 rectal fecal 
 samples , including 466 samples from buffalo , 1697 from cattle and 120 from sheep , were 
 collected from different ages and screened by modified ziehl - neelsen acid - fast microscopy 
 for detection of cryptosporidium oocysts . 
 all studied farms were 
 positives with an overall prevalence of 1.29% in buffalo ( 4.17% in claves versus 0.48% in 
 adults ) , 7.07% in cattle ( 6.90% in calves versus 10.20% and 6.10% in heifers and adults , 
 respectively ) and 2.50% in sheep ( 4.40% in lambs versus 1.30% in adults ) . 
 pcr - rflp 
 analyses of small - subunit rrna genes from positive specimens revealed the occurrence of 
 c. parvum and c. ryanae in buffalo ; c. 
 parvum , c. ryanae , c. bovis and c. 
 andersoni in cattle and only c. xiaoi in sheep . 
 genotypes 
 distribution showed that c. ryanae was the dominant species ( 60.0% ) 
 followed by c. parvum ( 40.0% ) in buffalo calves . 
 meanwhile , in cattle 
 calves , c. parvum was the commonest species ( 74.23% ) followed by 
 c. ryanae ( 16.10% ) and c. bovis ( 9.70% ) . 
 subtyping of 
 c. parvum based on sequence analysis of the polymorphic 60 kda 
 glycoprotein gene locus showed the presence of subtypes iida20g1 and iiaa15g1r1 in both 
 buffalo and cattle calves , addressing the potential role of calves in zoonotic 
 cryptosporidiosis in egypt .