Experimental Parasitology 135 (2013) 518–523

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Experimental Parasitology

journal homepage: www.elsevier .com/locate /yexpr

Prevalence and characterization of Cryptosporidium spp. in dairy cattle inNile River delta provinces, Egypt

0014-4894/$ - see front matter Published by Elsevier Inc.http://dx.doi.org/10.1016/j.exppara.2013.09.002

⇑ Corresponding author. Fax: +86 21 6425 0664.E-mail address: yyfeng@ecust.edu.cn (Y. Feng).

Said Amer a,b, Shereif Zidan c, Haileeyesus Adamu a,d, Jianbin Ye a,e, Dawn Roellig a, Lihua Xiao a,Yaoyu Feng e,⇑a Division of Foodborne, Waterborne and Environmental Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention,1600 Clifton Rd, Atlanta, GA, USAb Department of Zoology, Faculty of Science, Kafr El Sheikh University, Kafr El Sheikh 33516, Egyptc Department of Animal Hygiene and Zoonoses, Faculty of Veterinary Medicine, Sadat Branch, Menoufia University, Al Menoufia, Egyptd Addis Ababa University, P.O. Box 42524, Addis Ababa, Ethiopiae State Environmental Protection Key Laboratory of Environmental Risk Assessment and Control on Chemical Process, School of Resources and Environmental Engineering, EastChina University of Science and Technology, Shanghai, People’s Republic of China

h i g h l i g h t s

� Cryptosporidium was found in 13.6% of1974 dairy cattle examined in Egypt.� Four Cryptosporidium species were

commonly found in pre-weanedcalves.� Two IIa and IId subtypes were

dominant C. parvum subtypes.� Calves are likely major reservoirs of

zoonotic cryptosporidiosis.

g r a p h i c a l a b s t r a c t

Al Beheira Farm with Cryptosporidium parvum IIaA15G1R1 Problem

a r t i c l e i n f o

Article history:Received 9 July 2013Received in revised form 19 August 2013Accepted 3 September 2013Available online 13 September 2013

Keywords:CryptosporidiumCryptosporidiosisGenotypeSubtypeCattleZoonosisMolecular epidemiologyEgypt

a b s t r a c t

Molecular characterizations of Cryptosporidium spp. in dairy cattle in industrialized nations have mostlyshown a dominance of Cryptosporidium parvum, especially its IIa subtypes in pre-weaned calves. Fewstudies, however, have been conducted on the distribution of Cryptosporidium species and C. parvum sub-types in various age groups of dairy cattle in developing countries. In this study, we examined the prev-alence and molecular characteristics of Cryptosporidium in dairy cattle in four Nile River delta provinces inEgypt. Modified Ziehl–Neelsen acid-fast microscopy was used to screen for Cryptosporidium oocysts in1974 fecal specimens from animals of different ages on 12 farms. Positive fecal specimens were identifiedfrom all studied farms with an overall prevalence of 13.6%. By age group, the infection rates were 12.5% inpre-weaned calves, 10.4% in post-weaned calves, 22.1% in heifers, and 10.7% in adults. PCR-RFLP and DNAsequence analyses of microscopy-positive fecal specimens revealed the presence of four major Cryptospo-ridium species. In pre-weaned calves, C. parvum was most common (30/69 or 43.5%), but Cryptosporidiumryanae (13/69 or 18.8%), Cryptosporidium bovis (7/69 or 10.2%), and Cryptosporidium andersoni (7/69 or10.2%) were also present at much higher frequencies seen in most industrialized nations. Mixed infec-tions were seen in 12/69 (17.4%) of genotyped specimens. In contrast, C. andersoni was the dominant spe-cies (193/195 or 99.0%) in post-weaned calves and older animals. Subtyping of C. parvum based onsequence analysis of the 60 kDa glycoprotein gene showed the presence of subtypes IIdA20G1 in ninespecimens, IIaA15G1R1 in 27 specimens, and a rare subtype IIaA14G1R1r1b in one specimen. The

S. Amer et al. / Experimental Parasitology 135 (2013) 518–523 519

common occurrence of non-C. parvum species and IId subtypes in pre-weaned calves is a distinct featureof cryptosporidiosis transmission in dairy cattle in Egypt. The finding of the same two dominant IIa andIId C. parvum subtypes recently found in humans in Egypt suggests calves can be potential reservoirs ofzoonotic cryptosporidiosis.

Published by Elsevier Inc.

1. Introduction

Cryptosporidium spp. are ubiquitous, obligatory parasites thatinfect the gastrointestinal epithelium of a wide range of verte-brates, resulting in gastroenteritis manifested as diarrhea of vary-ing severities. The infection is acquired orally, usually by routesof direct contact with infected hosts or ingestion of contaminatedwater or food. In humans, the highest impact is on immune-com-promised individuals such as AIDS patients (Chalmers and Davies,2010). Cryptosporidiosis is especially common in developing coun-tries, creating additional challenges for the poorly supported publichealth infrastructure (Kotloff et al., 2013). Several Cryptosporidiumspecies are commonly found in humans and their distribution dif-fers depending on socioeconomic development and the intensity ofanimal farming. Nevertheless, Cryptosporidium hominis and Cryp-tosporidium parvum in general are responsible for the majority ofhuman Cryptosporidium infections (Xiao, 2010).

Each species of farm animal has been shown to be capable ofbeing infected by multiple species of Cryptosporidium (Xiao et al.,2004; Santin, 2013). Studies in industrialized nations have shownthe presence of four major Cryptosporidium species in dairy cattle,including C. parvum, Cryptosporidium bovis, Cryptosporidium ryanae,and Cryptosporidium andersoni. Each species preferentially infectsdifferent age groups of cattle, with C. parvum almost exclusivelyfound in pre-weaned calves and other species mostly in older ani-mals (Santin et al., 2004, 2008; Fayer et al., 2006, 2007; Santin,2013). Most of the C. parvum infections are caused by IIa subtypesbased on sequence analysis of the 60 kDa glycoprotein (gp60) gene,although some IId subtypes are also occurring in European coun-tries at low frequencies. Intensive farming practices may facilitatethe persistent transmission of C. parvum IIa subtypes on most dairyfarms in industrialized nations (Xiao, 2010; Santin, 2013).

Thus far, relative to the large number of studies in industrial-ized nations, only a few have been conducted to examine the dis-tribution of Cryptosporidium species in dairy cattle in developingcountries, where animals are managed very differently (Ameret al., 2010; Inpankaew et al., 2010; Khan et al., 2010; Imre et al.,2011; Meireles et al., 2011; Muhid et al., 2011; Wang et al.,2011a,b; Kang’ethe et al., 2012; Venu et al., 2012; Silva et al.,2013; Zhang et al., 2013). Most of these studies have shown a com-mon occurrence of C. bovis, C. ryanae, and C. andersoni in pre-weaned calves, in addition to C. parvum (Meireles et al., 2011; Mu-hid et al., 2011; Wang et al., 2011b; Venu et al., 2012; Silva et al.,2013; Zhang et al., 2013). A small number of these studies sub-typed C. parvum (Amer et al., 2010; Imre et al., 2011; Meireleset al., 2011; Muhid et al., 2011; Wang et al., 2011b; Silva et al.,2013), with C. parvum IId subtypes identified as the dominant C.parvum in China, Egypt, and Malaysia (Amer et al., 2010; Muhidet al., 2011; Wang et al., 2011b; Zhang et al., 2013). Thus, the trans-mission of cryptosporidiosis in dairy cattle in developing countriescould be different from industrialized nations at both species andsubtype levels.

Although the vast majority of the population in Egypt lives inthe Nile River delta region, this region occupies only �6% of the to-tal land area (El-Saharty et al., 2005), making it one of the mostdensely populated areas in the world. Because of the high demandfor animal protein, intensive animal farming is common in bothgovernment and private sectors, making zoonoses a major public

health concern. Monitoring of Cryptosporidium spp. in farm animalsin these provinces is important for both veterinary and publichealth. Most previous studies on Cryptosporidium epidemiologyin animals and humans in Egypt used microscopy only (El-Khoderyand Osman, 2008; Abdel-Hafeez et al., 2012). Thus far, only a fewsmall-scale studies have characterized Cryptosporidium in cattle,water buffalo, and humans (Amer et al., 2010, 2013; Abd El Kaderet al., 2012; Helmy et al., 2013). The previous bovine Cryptosporidi-um study was conducted with only 96 dairy calves from the Kafr ElSheikh Province. The present study examines the prevalence anddistribution of Cryptosporidium species and C. parvum subtypes indifferent cattle age groups from four lower delta provinces inEgypt.

2. Materials and methods

2.1. Specimen collection

Specimens for this study were collected during December 2009to November 2011 from 12 dairy farms in Al Menoufia (70 kmnorth of Cairo), Kafr El Sheikh (130 km northwest of Cairo), AlBeheira (160 km northwest of Cairo), and Alexandria (190 kmnorth of Cairo) provinces. Rectal fecal specimens were collectedusing a gloved hand and transferred into a plastic cup labeled withthe animal ear tag number and farm name. Screened animals weredivided into four age groups: pre-weaned calves up to 8 weeks ofage, post-weaned calves 2–6 months of age, heifers 7–18 monthsof age, and adult cattle (Table 1). Fecal specimens were screenedby microscopy for Cryptosporidium oocysts after staining with themodified Ziehl–Neelsen stain (Casemore et al., 1985). Positivespecimens were stored in 2.5% potassium dichromate at 4 �C untilmolecular analysis.

2.2. DNA extraction and PCR amplification

Microscopy-positive fecal specimens were washed twice withdistilled water by centrifugation to remove potassium dichromateprior to DNA isolation. DNA was extracted from the specimensusing the FastDNA SPIN Kit for Soil (BIO 101, Carlsbad, CA). Geno-typing of Cryptosporidium was done by PCR-RFLP analysis of anapproximately 830-bp fragment of the small subunit (SSU) rRNAgene as previously described (Feng et al., 2007). Each specimenwas analyzed at least twice using reagent water as the negativecontrol and DNA of Cryptosporidium baileyi as the positive control.The identity of Cryptosporidium species was confirmed by sequenceanalysis of the secondary PCR products from representative speci-mens. C. parvum was subtyped by sequence analysis of the 60 kDaglycoprotein (gp60) gene (Alves et al., 2003). The establishednomenclature system (Sulaiman et al., 2005) was used in namingsubtypes.

2.3. DNA sequence analysis

Selected secondary PCR products of the SSU rRNA gene and allsecondary PCR products of the gp60 gene were directly sequencedusing Big Dye� Terminator v3.1 Cycle Sequencing Kits (AppliedBiosystems, Foster City, CA) and an ABI 3130 Genetic Analyzer (Ap-plied Biosystems). Sequences were assembled using ChromasPro

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520 S. Amer et al. / Experimental Parasitology 135 (2013) 518–523

(version 1.5) software (http://technelysium.com.au/?pa-ge_id=27). The accuracy of the sequencing reads was confirmedby bidirectional sequencing. The obtained sequences werealigned with each other and reference sequences using ClustalX(http://www.clustal.org/) to determine Cryptosporidium speciesand C. parvum subtypes. Unique nucleotide sequences generatedin this study were deposited in GenBank under accession num-bers AB777172 to AB777196 for SSU rRNA sequences andAB777446 and AB777872 to AB777903 for gp60 sequences.

3. Results

3.1. Prevalence of Cryptosporidium spp.

A total of 1974 animals of four age groups from 12 dairy farmsin four delta provinces were examined microscopically for Cryp-tosporidium oocysts. Cryptosporidium was detected on all farmsexamined. Overall, 74 of 594 (12.5%) pre-weaned calves, 21 of203 (10.4%) post-weaned calves, 94 of 426 (22.1%) heifers, and80 of 751 (10.7%) adults were shedding Cryptosporidium oocysts.

3.2. Distribution of Cryptosporidium species

PCR analysis of the SSU rRNA gene was successful for 69 of 74microscopy-positive specimens from pre-weaned calves and allspecimens from older animals. RFLP analyses of the PCR productsusing restriction enzymes SspI and MboII indicated the occurrenceof single species infection in 57 pre-weaned calves, including C.parvum (30/69 or 43.5%), C. ryanae (13/69 or 18.8%), C. bovis (7/69 or 10.2%), and C. andersoni (7/69 or 10.2%). In addition, 12specimens had concurrent infections: seven (10.2%) having C. rya-nae and C. bovis, four (5.8%) having C. parvum and C. bovis, and one(1.5%) having C. parvum and C. ryanae. Among the four provincesstudied, Kafr El Sheikh had the highest diversity of Cryptosporidi-um species in pre-weaned calves, with all four species being de-tected, including the mixed infections. In contrast, theremaining three provinces (Al Menoufia, Al Beheira, and Alexan-dria) mostly had only C. parvum. Animals older than two monthsof age were mostly infected with C. andersoni, with the exceptionof one post-weaned calf and one adult, which were infected withC. parvum.

3.3. Intra-species variation in the SSU rRNA gene sequences

SSU rRNA sequence analysis of eight specimens of C. andersonifrom pre- and post-weaned calves generated four types of se-quences. The first sequence type was from specimen 37259 andwas identical to GenBank sequences FJ463181, FJ463186, andEU926593 derived from cattle in China. The second sequence typefrom three specimens (35072, 37244, and 37280) was similar tothe previous one but had one thymine deletion at position 704.The third sequence type in two sequences (from specimens37251 and 37318) was identical to sequences JX515549,AB089285, and AB513856 from China, Japan, and Egypt, respec-tively. The fourth type in two sequences (34897 and 37301)was similar to the third type but had one thymine deletion at po-sition 704.

Ten C. parvum specimens generated two types of sequences;one sequence type from three specimens (35090, 35103, and35108) was identical to GenBank sequences JX416362,JQ413434, and AB513880, whereas the second sequence typefrom seven specimens (35089, 350891, 35093, 35097, 35098,35100, and 35106) was similar but had one thymine deletion atposition 678. Similarly, five C. ryanae specimens generated twosequence types, with one sequence type from three specimens

S. Amer et al. / Experimental Parasitology 135 (2013) 518–523 521

(37247, 37257 and 37281) identical to sequences HQ179574 andJX416367 derived from dairy cattle in China. The second typewas seen in two sequences (from 34969 and 37250) and had a sub-stitution of cytosine to thymine at position 71 and an insertion ofthymine at position 476 compared to the former type. These twotypes of sequences had nucleotide substitutions at positions 54and 432 compared to those (such as AB712387) obtained fromthe host-adapted C. ryanae in buffalo from the same study area(Amer et al., 2013). Only one type of SSU rRNA gene sequencewas obtained from two specimens (35047 and 37343) of C. bovis,which had a substitution of cytosine to thymine at position 724compared to sequences JX515546, JQ362488, HQ179575, andGU174540.

3.4. Distribution of C. parvum subtypes

Cryptosporidium DNA from a total of 37 specimens, includingthose from pre-weaned calves with single and mixed infections,one post-weaned calf (35073), and one adult (35088), were suc-cessfully amplified and sequenced at the gp60 locus. Most of theC. parvum specimens belonged to the subtype IIaA15G1R1 (seenin 27 specimens) in the IIa subtype family and IIdA20G1 (seen in9 specimens) in the IId subtype family (Table 2). Most (21/28) ofthe IIa subtype was from one farm (Al Beheira-1) in the Al Beheiraprovince (Table 2).

All sequences of the IIdA20G1 subtype were identical toAB514086, AB514089 and AB712389 obtained from dairy and buf-falo calves from Egypt (Amer et al., 2010, 2013). Likewise, most se-quences of the IIaA15G1R1 subtype were identical to AM937012(Soba and Logar, 2008) and AB712391 and AB712392 (Ameret al., 2013) previously reported in humans in Slovenia and buffalocalves in Egypt. However, the sequence from specimen 35119 hada cytosine to thymine substitution at position 23, while the se-quence from specimen 35124 had an adenine to guanine substitu-tion at position 483.

In addition to IIaA15G1R1 and IIdA20G1, a unique IIa sequencewas obtained from specimen 35125 from Alexandria province,which had 14 copies of TCA, one TCG, and the sequence ACA afterthe trinucleotide repeats but before the R repeat sequence (ACA-TCA). It is not clear whether this was caused by a substitution ofthymine to adenine in the last TCA repeat or a deletion of TCA inthe R2 sequence (ACATCAACATCA). This sequence had two othersubstitutions at positions 251 and 682, resulting in the change ofthe amino acids from proline to leucine and proline to threonine,respectively, compared to other sequences generated in this studyand AY738191, AM937012, and JF727794 in GenBank. Previously,the interruption of TCA repeats by ACA was seen in two GenBanksequences (JX183796 and EU078321), creating some difficultiesin naming these unusual subtypes. We consider the ACA sequencea rare variation of the R repeat (ACATCA), and propose to use a low-er case r to represent it. As the location of ACA is different between35125 and JX183796 or EU078321, sequences JX183796 andEU078321 are named as subtype IIaA14G1R1r1a and the sequencefrom specimen 35125 is named as subtype IIaA14G1R1r1b.

4. Discussion

In the present study, all investigated farms in four provinces ofthe lower delta region of Egypt were positive for Cryptosporidium,indicating that the parasite is widespread in dairy cattle in Egypt.The infection rates of 12.5% in pre-weaned calves and 10.4% inpost-weaned calves are similar to those previously reported in buf-falo (14.5%) and dairy (30.2%) calves in Egypt (El-Khodery and Os-man, 2008; Amer et al., 2010).

Although C. parvum was the more common species in pre-weaned calves in the present study, C. bovis, C. ryanae, and C. ander-soni were seen in high frequency in pre-weaned calves in Kafr ElSheikh. Previously, most studies in industrialized nations haveshown an almost exclusive presence of C. parvum in pre-weaneddairy calves. This was also the case of one small study previouslydone in Kafr El Sheikh (Amer et al., 2010). Several recent studiesin China, India, Malaysia, Australia, Sweden, and Canada, however,have demonstrated the common occurrence of C. bovis and C. rya-nae in these animals in the absence or low occurrence of C. parvum(Feng et al., 2007; Silverlas et al., 2010; Muhid et al., 2011; Wanget al., 2011b; Ng et al., 2011, 2012; Budu-Amoako et al., 2012;Zhang et al., 2013). This difference in the distribution of Cryptospo-ridium species in pre-weaned dairy calves might have been the re-sult of various environmental, host, and management factors. Inagreement with this, recent genotyping studies have shown adominance of C. bovis or C. ryanae and an absence of C. parvum inpre-weaned native calves reared in traditional husbandry systemsin developing countries (Geurden et al., 2006; Ayinmode et al.,2010; Maikai et al., 2011; Feng et al., 2012; Nguyen et al., 2012)and in beef calves reared under the cow-calf grazing system inindustrialized nations (Feltus et al., 2008; Fayer et al., 2010;Murakoshi et al., 2012; Rieux et al., 2013).

With few exceptions (2/188), C. andersoni appears to be the onlyspecies in post-weaned dairy calves, heifers, and adult dairy cattlein the present study. A similar dominance of C. andersoni in theseanimals has also been observed in studies in dairy cattle in China(Liu et al., 2009; Wang et al., 2011a), India (Paul et al., 2009), Mon-golia (Burenbaatar et al., 2008), and some European countries (Ene-mark et al., 2002; Kvac et al., 2006; Robinson et al., 2006;Ondrackova et al., 2009). In the United States and Canada, however,C. bovis and C. ryanae are usually in concurrence with C. andersoniin these animals (Santin et al., 2004, 2008; Fayer et al., 2006, 2007;Budu-Amoako et al., 2012). The very low prevalence of the zoo-notic C. parvum in these animals suggests they are unlikely a majorsource of infection for humans.

Results of gp60 sequence analysis indicate the C. parvum-posi-tive calves in this study were mostly infected with subtypesIIaA15G1R1 and IIdA20G1. The subtype family IIa was seen in allfour studied provinces, but was most common in calves main-tained in individual outdoor pens on one farm in Al Beheira. In con-trast, the subtype family IId was mostly detected in calves from AlMenoufia and Kafr El Sheikh Provinces. The frequency of C. parvumIIa and IId subtype families in cattle is different among geographiclocations, with no clear indication of the factors leading to thedominance of a certain subtype in a particular area. Previous stud-ies reported that IIa subtypes of C. parvum are prevalent in dairycattle in industrialized nations (see a summary in Table 2 of Xiao,2010). Although the subtype IIaA15G1R1 reported in the presentstudy is not among the prevalent IIa subtypes in the world, thereare a few reports of its occurrence in humans in Egypt (Helmyet al., 2013), Kuwait (Sulaiman et al., 2005), Slovenia (Soba and Lo-gar, 2008), and Australia (Waldron et al., 2011). In bovine animals,it was first described in calves on one farm in the Czech Republic(Kvac et al., 2011), and recently in buffalo calves in Egypt (Ameret al., 2013). Results of the present study have shown a commonoccurrence of this subtype in dairy calves in Egypt.

Previously, the subtype family IId was found only at low fre-quencies in cattle and humans in Europe (Alves et al., 2003; Misicand Abe, 2007; Broglia et al., 2008; Quilez et al., 2008; Silverlaset al., 2013) and was largely absent in cattle and humans in otherindustrialized nations such as the United States and Canada (Xiao,2010). However, it is the predominant subtype family in dairycalves in China (Wang et al., 2011b; Zhang et al., 2013), Egypt(Amer et al., 2010), and Malaysia (Muhid et al., 2011) and buffalocalves in Egypt (Amer et al., 2013). In addition, IId is commonly

Table 2Distribution of Cryptosporidium spp. in pre-weaned calves.a

Province C. andersoni C. ryanae C. bovis C. parvum Mixed infectionb

IIa IId C. ryanae + C. bovis C. parvum + C. bovis C. parvum + C. ryanae

Kafr El Sheikh 7 13 7 1 2 7 4 1Al Menoufia, 0 1 0 2 2 0 0 0Al Beheira 0 0 0 21 0 0 0 0Alexandria 0 0 0 2 0 0 0 0

a C. parvum in one post-weaned calf (35073) and one adult cattle (35088) belonged to the IIa subtype family.b The C. parvum in mixed infections with C. bovis or C. ryanae belonged to the IId subtype family.

522 S. Amer et al. / Experimental Parasitology 135 (2013) 518–523

found in children in Mideast countries such as Kuwait, Jordan andIran, and in the nearby Egypt (Sulaiman et al., 2005; Hijjawi et al.,2010; Iqbal et al., 2011; Taghipour et al., 2011; Helmy et al., 2013).It is also common in HIV/AIDS patients in Malaysia (Lim et al.,2011). Reasons for these differences in the distribution of IIa andIId subtype families are not clear.

In summary, both IIa and IId subtype families of C. parvum arecommon in pre-weaned calves in Nile River delta provinces inEgypt, but C. andersoni dominates in older animals. The finding oftwo IIa and IId subtypes as the dominant C. parvum subtypes inboth humans and calves and the close contact of humans with cat-tle suggest zoonotic transmission may be important in cryptospo-ridiosis epidemiology in Egypt and the Middle East. Suchinferences require confirmation from careful case–control studiesand simultaneous sampling and characterization of Cryptosporidi-um spp. in both humans and animals from the same area. Thiswould in turn improve our understanding of the common occur-rence of the IIa and IId subtype families of C. parvum in humansin Mideast countries.

Acknowledgments

This research was supported by the Arab Fund for Economic &Social Development (Zamalat Program), National Natural ScienceFoundation of China (Project Nos. 31229005 and 31110103901),and Centers for Disease Control and Prevention.

The findings and conclusions in this report are those of theauthors and do not necessarily represent the views of the Centersfor Disease Control and Prevention.

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