Introduction {#s1}
============

*Cryptosporidium* spp. is an important intestinal protozoa in man and animals ([@R1]). Cryptosporidiosis can cause diarrheal illness in humans, which may be severe in immunocompromised individuals. Transmission usually occurs through the direct fecal-oral route or through oocysts contaminated water or food. However, the potential for zoonotic transmission of *Cryptosporidium* spp*.,* particularly with respect to cattle and other livestock, has generated considerable interest in recent years ([@R2]-[@R4]).

Some livestock, such as cattle, sheep, and goats, harbor *Cryptosporidium* species that are infectious for humans. The use of molecular diagnostic techniques in the characterization of *Cryptosporidium* spp. has led to increased recognition of the diversity of parasites infecting humans and animals and role of animals in the transmission of human cryptosporidiosis ([@R5]).

Studies of cryptosporidiosis in dairy cattle have identified several *Cryptosporidium* species in cattle ([@R6]). Previous studies demonstrated that cattle could be infected with 10 different *Cryptosporidium* species or genotypes at least; nevertheless, *C. parvum, C. andersoni, C. bovis*, and *C. ryanae* were commonly detected in cattle worldwide. All of them were found in the different age groups, especially *C. andersoni* ([@R7],[@R8]); Although dairy cattle have been considered to be a major host for *C. parvum*, only pre-weaned calves are frequently infected with this species ([@R6]), while *C. bovis* and *C. ryanae* are considered predominantly infectious to post-weaned calves ([@R9],[@R10]).

Economic losses due to cryptosporidiosis of neonatal calves depend on the *Cryptosporidium* species/genotypes causing the infection. While *C. parvum* infection is caused diarrhea, growth retardation, dehydration and losses to the dairy industry in terms of increased labour and veterinary costs associated with calf morbidity and, occasionally, mortality ([@R11]), calves are infected with other *Cryptosporidium* species or genotypes exhibit no overt clinical signs ([@R8],[@R12]). Although previous studies demonstrated that some animals were important zoonotic reservoirs of *Cryptosporidium* in humans ([@R13]), and cattle could be infected with 6 out of 15 human pathogenic *Cryptosporidium* species ([@R14]), only *C. parvum* is important for the public health in relation to cattle management.

At least 11 different *C. parvum* families (IIa-IIl) with many subtypes have been described on the basis of sequence analysis of a 60-kDa glycoprotein (GP60) from humans and other mammals including cattle ([@R15]). The objective of this study was to detect and isolate the *Cryptosporidium* spp. from fecal samples of naturally infected pre-wean calves in the Mashhad area, the capital of the Khorasan Razavi Province, Iran and to determine the genetic characterization of these isolates.

Materials and Methods {#s2}
=====================

Study population {#s2a}
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The study was performed in the northeast region of Iran, in Mashhad area, the capital city of Khorasan Razavi Province. The city is located at 36.20° latitude and 59.35° east longitude, in the valley of the Kashaf River near Turkmenistan, between the two mountain ranges of Binalood and Hezar-Masjed. The city benefits from the proximity of the mountains and having very cold winters. Mashhad has more than 500 dairy farms with an estimated 70,000 cows of mostly Holstein/-Friesian breed.

Sample collection and coprodiagnosis {#s2b}
------------------------------------

In the present study, 10 industrial dairy herds were selected. The cow breed in all these farms was Holstein/Friesian, and in all farms, calves were separated from their dams after they received colostrums and were housed in individual pens until weaning at 3 months of age. Approximately 10 g of fecal specimens were collected from the rectum of 300 randomly selected 1--30 days pre-wean calves with or without diarrhea in 10 farms. All the stool samples were previously tested for the presence of oocysts of *Cryptosporidium* spp. using the Ziehl --Neelsen staining technique ([@R24], [@R25], [@R36]). The samples positive in the coprodiagnostic test were stored at 4 °C in 2.5% potassium dichromate ([@R16]).

Oocysts purification {#s2c}
--------------------

The positive samples for *C. parvum*-like oocysts stored in 2.5% potassium dichromate were submitted again to the flotation--concentration method using disposable material and the floated material used in the diagnosis was washed from the slide and the glass coverslip using 1 ml of TE (Tris--EDTA pH 8.0), in a disposable Petri dish, placed in a 1.5 ml microtube, and later submitted to DNA extraction ([@R16]).

DNA extraction from oocysts {#s2d}
---------------------------

The purified oocysts were cleared by centrifugation at 12,000 × g for 5 min, and then ressuspended in 200 ml TE--SDS (Tris--HCl 10 mM, EDTA 1mM, SDS 1%). After that, the oocysts suspension was submitted to seven freeze and thaw cycles in liquid nitrogen and dry bath at 65° C, respectively ([@R17]). DNA was extracted using a DNA isolation kit (MBST, Iran) based on a selective binding of nucleic acids to a silica-based membrane, according to the manufacturer\'s instructions.

Nested-PCR {#s2e}
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Nested polymerase chain reaction (PCR) was used to amplify a fragment (826-- 864 bp) of the SSU rRNA (18S) gene using two sets of oligonucleotide primers as described previously([@R18],[@R19]). PCR products were visualized on 2% agarose gel after ethidium bromide staining.

RFLP using SspI and VspI restriction enzymes {#s2f}
--------------------------------------------

For detection up to species level of *Cryptosporidium*, RFLP analysis using restriction enzymes *SspI* and *VspI* (Fermentas Life Sciences, Lithuania, Vilnius) of nested-PCR products were performed ([@R18]). Products were analyzed by gel electrophoresis in 0.8% agarose containing ethidium bromide (0.5 μg/ml) ([@R17]). The positive *Cryptosporidium* bovine species yields three distinct bands at 449 bp, 254 and 108 bp after the digestion of 2nd PCR products with *SspI*, also the common genotype yields two distinct bands at 104 bp and 628 bp after the digestion of 2nd PCR products with *VspI* ([@R18]).

Sequencing {#s2g}
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All amplicons of Nested-PCR products that have different RFLP patterns were sequenced, and SSU rRNA gene sequence was compared with sequences of *Cryptosporidium spp*. and the obtained sequences were confirmed for their uniqueness by performing BLAST with the NCBI nucleotide database (*<http://www.ncbi.-nih.gov>*).

Statistical analysis {#s2h}
--------------------

The prevalence of Cryptosporidium infection in Pre-weaned dairy calves was compared based on the different age groups, sex and diarrheic or none- diarrheic groups. The Chi-square test was used to analyze the data and differences were considered significant when *P* \< 0.05. Statistical analysis was performed using SPSS software (Ver. 20).

Results {#s3}
=======

*Cryptosporidium* spp. oocysts were microscopically in 28.3% (85/300) of fecal samples. Fecal samples were classified according to the consistency as diarrheic (45/300) and non-diarrheic (255/300). Diarrhea was recorded in 100% (45) of the positive samples. The prevalence of *Cryptosporidium* spp. infection in diarrheic calves was significantly higher than non-diarrheic calves (*P*\< 0.05) ([Table 1](#T1){ref-type="table"}), Also the highest frequency of infection was seen in 8-14 days old group (*P*\< 0.05).

Statistically there was no significant correlation between infection rate and sex factor (*P*\>0.05).

All of the infected samples were also positive by nested PCR ([Fig.1](#F1){ref-type="fig"}). Forty five of nested- PCR positive were selected and identified as *C. parvum* by RLFP using *SspI* and *VspI* ([Fig. 2](#F2){ref-type="fig"}) ([Fig. 3](#F3){ref-type="fig"}). All of the 45 positive samples have shown a similar band on gel electrophoresis. Subsequently, 15 of sequences were obtained; 100% (15/15) were *C. parvum* bovine genotype, Sequence analysis showed 100% homology to the registered in GenBank for *C. parvum* under accession number: *[JX237833.]{.ul} C. parvum* was only isolated from calves aged \<30 days old, with the highest peak at 58.33% detected in 8-14 age group.

Discussion {#s4}
==========

Although many studies dealing with cryptosporidiosis of Pre-weaned calves have been published in Iran, our study represents the second report where the data is supported by molecular techniques. In the present study, the frequency of *Cryptosporidium* spp. infection was 28.3% in the pre-weaned calves. In comparison with studies conducted in other countries, this frequency rate was lower than the infection rates (48--100%) in the pre-weaned dairy calves ([@R9],[@R20]), but, in agreement with results of other studies ([@R21], [@R22]). A few studies were done on the frequency of *Cryptosporidium* spp. infection in calves in Iran. Our frequency was lower than a similar study that was done in calves in Mashhad area, Iran ([@R24]), and higher than another study in Iran. ([@R25]). In the present study the high prevalence of infection was observed in 8-14 days age group of calves and diarrheic. The result was along with the previous study that done this area ([@R24]) and other studies that reported in other countries ([@R6], [@R9], [@R23], [@R26]-[@R28]). All infected samples were examined with nested-PCR and confirmed the microscopic results. In the present study, all positive-PCR samples was detected such a *C. parvum* bovine genotype (100%). In some studies, it has been reported that the zoonotic *C. parvum* is responsible for the majority of *Cryptosporidium* infections in Pre-weaned calves, and only a small percentage of *Cryptosporidium* infections in post-weaned calves and heifers ([@R6], [@R9], [@R29]-[@R31]). Results of some studies have indicated that there are not significant correlations between the calf with age, oocyst excretion and the species/ genotypes of *Cryptosporidium* spp. ([@R26],[@R32]-[@R34]). As well as the man are predominantly infected with the *C. parvum* bovine genotype and *C. hominis* via water, food and person-to-person or animal--human contact ([@R8],[@R35],[@R36]). In the present study, we used a RFLP-PCR with *SspI* and *VspI* restriction enzymes to differentiate of *C. parvum* from *C. hominis.* Our results demonstrated that *C. parvum* is the major cause of infection in the preweaned calves in Mashhad farms. This finding was in agreement with similar studies ([@R6], [@R10], [@R35]). Besides our study indicated that the bovine genotype of *C. parvum* could be the majority of *Cryptosporidium* isolates that is very important as a primary cause of human infections ([@R13], [@R14], [@R35]).

Conclusion {#s5}
==========

In the present study, we used a microscopy and molecular examination to detect and differentiate of *Cryptosporidium* spp. in dairy farms. Our results demonstrated that *C. parvum* is the major cause of infection in preweaned calves in Mashhad farms and it could be a primary cause of human infections.

This study was supported by a grant VPRTFM-21475/3 from The Vice President Research and Technology of Ferdowsi University of Mashhad, Iran.We are very grateful to H Eshrati for his technical assistance.

The authors declare that there is no conflict of interest.

![Ethidium bromide-staining 1% agarose gel with small-subunit rRNA -- based secondary PCR products of calve stool samples. Line B, negative control. Lane M, 100bp 100 bp molecular marker, Line 5, positive control and other lines, DNA from *Cryptosporidium* oocyst in samples 826-834bp](IJPA-8-601f1){#F1}

![Gel electrophoresis of *Cryptosporidium* SSU rRNA. PCR--RFLP products resulting from digestion of the nested-PCR products (secondary PCR product). with *VspI* restriction enzyme. Line A, 100 bp molecular marker. Line B, nested-PCR product. Line 1through 7, *C. parvum*](IJPA-8-601f2){#F2}

![Gel electrophoresis of *Cryptosporidium* SSU rRNA. PCR--RFLP products resulting from digestion of the nested-PCR products (secondary PCR product). with *SspI* Restriction enzyme. Line A, 100 bp molecular marker./ Line 1through 12, *C. parvum*](IJPA-8-601f3){#F3}

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Prevalence of *C. parvum* infections by different risk factors in the pre-weaned calves

  Risk factors        Total   Infection rate   *P* value   
  ------------------- ------- ---------------- ----------- -------------
  Male                145     46               31.72       *P*\>0.05
  Female              155     39               25.16       *P*\>0.05
  Age                                                      
  1-7 days            45      17               37.77       
  8-14 days           48      28               58.33       *P* \< 0.05
  15-21 days          55      17               30.90       
  22-30 days          152     23               15.13       
  Stool Consistency                                        
  diarrheic           45      45               100         *P* \< 0.05
  Non- diarrheic      255     40               15.68       
