43 Introduction Cryptosporidium is an intracellular protozoan parasite of a phylum of Apicomplexa. Ernest Edward Tyzzer first identified and reported the parasite, found frequently in the gut of the laboratory mice (Tyzzer 1907). This parasite causes acute and self‑limiting gastroenteritis in humans. The parasite is detected in healthy individuals whereas persistent and fatal infection can be observed in immunocompromised individuals. It is estimated that millions of cases of disease occur annually in developing and developed countries (Putignani and Menichella 2010). Moreover, this parasite has been reported as one of the most common pathogens in human intestine (Mendonca et al. 2007, Paul et al. 2009). The genus Cryptosporidium includes a variety of species found in many domestic animals and human (Dillingham et  al. 2002, Fayer and Ungar 1986, Kvac and Vitovec 2003, Mendonca et al. 2007, O'Donoghue 1995). This protozoan often detected in calves, lambs, piglets, horses, puppies and kittens, chicken and turkeys, with diarrhea. This study aimed to establish Cryptosporidium prevalence and species identification in dairy cattle farms located in Mashhad, Northeast of Iran. Materials and methods Sampling In this study, a cross‑sectional study with single random sampling was done. A total of 800 stool samples were collected from healthy and diarrheal (from one month before the beginning of testing sampling) Holstein cattle in the city of Mashhad, within different age groups (less than 6 months, between 6‑18 months and more than 18 months) from 2011 to 2015. Individuals with diarrhea among those showing diarrhea for a month before sampling. Direct microscopic detection The specimens were stained by modified Ziehl‑Neelsen method and observed with a microscope (100X). Samples were then stored in a freezer at ‑ 20 °C. DNA extraction Genomic DNA was extracted with two different procedures, a standard phenol‑chloroform procedure [H.E. McKiernan, P.B. Danielson, in Razi Vaccine & Serum Research Institute, Agricultural Research, Education and Extension Organization, Mashhad, Islamic Republic of Iran *Corresponding author at: Razi Vaccine & Serum Research Institute, Agricultural Research, Education and Extension Organization, Mashhad, Islamic Republic of Iran. E‑mail: a.sadr@rvsri.ac.ir. Keywords Cryptosporidium andersoni, Cattle, PCR‑RFLP, Mashhad, Iran. Summary Cryptosporidium is an intracellular and extracytoplasmic protozoan that belongs to the phylum Apicomplexa. In this observational study, fecal samples were randomly collected from 800 dairy cattle, in 10 industrial dairy farms in Mashhad, Iran (from 2011 to 2015 years). The presence of Cryptosporidium oocysts was determined by modified cold Ziehl‑Neelsen’s staining. Results of microscopy observation showed that 23 samples (2.87%) were positive for Cryptosporidium oocyst. Twenty two samples were confirmed by PCR. The identification of Cryptosporidium andersoni was determined by restriction digestions of PCR products, using SspI, VspI, and DdeI enzymes. Differences between healthy and diarrheic groups as well as between age groups were not observed. Alireza Sadrebazzaz Molecular identification of Cryptosporidium andersoni in healthy and in cattle with diarrhea of Mashhad, Northeast of Iran Veterinaria Italiana 2020, 56 (1), 43‑46. doi: 10.12834/VetIt.1771.9343.3 Accepted: 30.07.2019 | Available on line: 24.04.2020 44 Cryptosporidium andersoni in cattle of Iran Sadrebazzaz Veterinaria Italiana 2020, 56 (1), 43‑46. doi: 10.12834/VetIt.1771.9343.3 differences (P < 0.05) by Statistix for windows, Trial version 9.0 (Analytical software, Tallahassee, FL 32317, USA). Results No differences have been observed between the two extraction methods in terms of final DNA concentration. Results of microscopic tests 23/800  samples were positive for Cryptosporidium oocysts. Analysis according to health status and age is reported in Table I and II. PCR‑RFLP data Out of 23 samples tested positive by ZN, 22 were correctly amplified by the adopted nested PCR. The PCR products were then digested with the SspI and demonstrated similar products (385 bp and 448 bp); with the VspI enzyme showing two bands of 731 bp and 102  bp, and with the DdeI enzyme showing three bands of 470 bp, 186 bp and 156 bp. All the 22  samples were identified positive for Cryptosporidium andersoni (2.75%). Statystical analysis No significant difference was found between age groups, and between healthy and diarrheal cows, using Chi‑square test (overall x2  =  0.07, p‑value = 0.7847, d.f. = 1). It can be said that Cryptosporidium andersoni causes the same infection in all age groups (overall x2 = 1.27, p‑value = 0.5292, d.f. = 2) . Molecular Diagnostics (Third Edition), 2017] and a Nucleospin® Tissue Kit (MN), following manufacturer's instructions, from 23 positive samples by modified Ziehl‑Neelsen method. PCR‑restriction fragment length polymorfism (RFLP) Cryptosporidium oocysts were identified at species level using a nested PCR of 18S rRNA gene followed by RFLP (Xiao et al. 1999). Primary PCR DNA from positive samples was amplified using the protocol described by Xiao and colleagues (Xiao et al. 1999). The PCR master mix was prepared by using the AccuPower® PCR PreMix kit (Bioneer, Korea). The PCR master mix reaction was prepared according to kit instructions in 40 µl total volume by adding 1 µl of purified genomic DNA and 1 µl of 10 pmol/µl of F1 and 1 µl of 10 pmol/µl of R1 and briefly mixed. The following thermal profile was used: denaturation at 95 °C for 3 minutes, followed by 35 cycles of denaturation at 94  °C for 45  seconds, annealing at 55 °C for 45 seconds, extension at 72 °C for 1 minute, and then a final extension at 72 °C for 7 minutes. Then, PCR products were loaded on 1.5% agarose gel stained with SYBR safe, and electrophoresed. The bands were visualized by UV and photographed using the gel Doc system. Secondary PCR By using published primers (Xiao et al. 1999) the following thermal profile was used: initial denaturation at 94  °C for 3 minutes, followed by 35  cycles of denaturation 94 °C for 45 seconds, annealing at 58 °C for 45 seconds, extension at 72 °C for 1 minute, and then a final extension at 72  °C for 7 minutes. RFLP A total volume of 10 μl of the secondary PCR product was digested for 2 h at 37  °C with 10 U of each enzyme, SspI, DdeI and VspI (Fermentas) in 32 μl following manufacturer’s instruction. The digested products were separated on a 2% agarose gel electrophoresis using SYBR safe staining and photographed by UV transilluminator. Statistical analysis The chi‑square test was used to evaluate significant Table I. Prevalence of Cryptosporidium spp. infection in cattle by age in Mashhad, Northeast of Iran. Age group (months) N. of cattle Cryptosporidium spp. positive Prevalence (%) ≤ 6 220 6 2.72 6-18 200 8 4 ≥ 18 380 9 2.36 Total 800 23 2.87 Table II. Prevalence of Cryptosporidium spp. infection in healthy and diarrheic cattle in Mashhad, Northeast of Iran. Group N. of cattle Cryptosporidium spp. positive Prevalence (%) Healthy 500 15 3 Diarrheic 300 8 2.66 Total 800 23 2.87 Veterinaria Italiana 2020, 56 (1), xxx-xxx. doi: 10.12834/VetIt.xxxxx 45 Sadrebazzaz Cryptosporidium andersoni in cattle of Iran be used as suggested methods in laboratories for veterinary and medical use. The rate of Cryptosporidium infections in this study was consistent with the contamination rate found in China, 5.12%, in Sweden 1.8%, in Wales 6.9% and in Japan 1.5%, in India 12.85%, Brazil 5.88%, Portugal (4.5% in adult cattle), Western Czech Republic (4.1%) (Fiuza et  al. 2011, Koyama et  al. 2005, Kvac and Vitovec 2003, Mendonca et al. 2007, Ondrackova et  al. 2009, Paul et  al. 2009, Robinson et al. 2006, Silverlas et al. 2010, Wang et al. 2011). The consistency of the results of the above‑mentioned studies with our data suggests that Cryptosporidium andersoni has a relatively high rate in animal contamination in different countries. In Iran, similar contamination rate was found in different cities, such as Isfahan, Tehran, Tabriz, Kerman and Ahvaz (Fotouhi Ardakani 2008, Nouri et al. 1995, Nouri 2003). According to other studies and considering the role of Cryptosporidium andersoni in cattle industry prevention methods should be considered. Acknowledgment The authors thank Dr. Gereon Schares (Friedrich‑Loeffler‑Institut, Federal Research Institute for Animal Health, Institute of Epidemiology, 17493 Greifswald‑Insel Riems, Germany) for valuable comments on the manuscript. We also acknowledge and thank Dr Lihua Xiao (Division of Foodborne, Waterborne and Environmental Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA, United States) for valuable and practical comments. This study was financially supported by the Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Mashhad, Iran. Nucleotide sequence accession number One of the sequences used in this study has been deposited in the GenBank database under the accession no. MH395839. Discussion Molecular techniques for the detection and differentiation of Cryptosporidium oocysts, along with conventional methods such as condensation and staining of stool specimens have significantly increased our understanding of the parasite dispersion and epidemiology. Selection of molecular targets for species identification of the parasite should be performed appropriately, as the parasite genome plays an important role in the interpretation of the information obtained by the PCR method and real‑time PCR (Burnet et  al. 2013, Morgan et  al. 1995). Molecular tools for Cryptosporidium identification at species level can provide valuable information about the detection of the protozoa in different hosts, and help to recognize the epidemiology of Cryptosporidiosis (Blears et  al. 2000, Fayer and Xiao 2008, Fiuza et  al. 2011, Ondrackova et al. 2009, Xiao et al. 2001). 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