311 1Department of Parasitology and Mycology, School of Medicine, AJA University of Medical Sciences, Tehran, Islamic Republic of Iran. 2Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran. *Corresponding author at: Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran. E-mail: m.asadpour65@gmail.com. Parole chiave Cryptosporidium, G. intestinalis, Cani, Gatti, Genotipizzazione, Beta-giardin, SSU rRNA. Riassunto Nel presente studio 615 campioni fecali provenienti da cliniche veterinarie sono stati esaminati per la presenza di Cryptosporidium e Giardia. Sono state inoltre effettuate la genotipizzazione molecolare dei due microganismi mediante PCR e l’analisi di sequenza. Complessivamente, sono state rilevate oocisti di Cryptosporidium e Giardia rispettivamente nello 0,6% (2/315) e nell'1,9% (6/315) dei cani e nello 0,7% (2/300) e 1,3% (4/300) dei gatti. I dati molecolari hanno dimostrato la presenza di C.  canis (n = 2) nei cani e C. felis (n = 2) nei gatti; inoltre nei cani sono stati evidenziati G.  intestinalis assemblage  D (n  =  2), C (n = 3) e A, sub-assemblage AII (n = 1); G. intestinalis assemblage F (n = 3) e assemblage A, sub-assemblage AI (n = 1) nei gatti. La più alta prevalenza di Giardia è stata osservata nei cani di età inferiore a un anno (6/315) e in quelli con diarrea (p < 0,05). Caratterizzazione molecolare e potenziale zoonotico di Cryptosporidium spp. e Giardia intestinalis in cani e gatti domestici di Shiraz, sud-ovest dell'Iran Keywords Cryptosporidium, G. intestinalis, Dogs, Cats, Genotyping, Beta-giardin, SSU rRNA. Summary In the present study, a total of 615 fecal samples from veterinary clinics were screened by microscopy for the presence of Cryptosporidium and Giardia oocysts. Molecular genotyping of Cryptosporidium and Giardia were carried out using PCR and sequence analysis. Overall, Cryptosporidium and Giardia oocysts were detected in the 0.6% (2/315) and 1.9% (6/315) of dogs and in the 0.7% (2/300) and 1.3% (4/300) of cats, respectively. Sequencing revealed the presence of C. canis (n = 2) in dogs and C. felis (n = 2) in cats. Moreover, G.  intestinalis assemblage D (n = 2), C (n = 3) and A, sub-assemblage AII (n = 1) were identified in dogs; G.  intestinalis assemblage F (n = 3) and assemblage A, sub-assemblage AI (n = 1) were identified in cats. The highest prevalence of Giardia was observed in dogs younger than one year (6/315), and in those with diarrhea (p < 0.05). Data of the study suggest that dogs and cats play a minor role in the zoonotic transmission of cryptosporidiosis and giardiasis in Southwestern Iran. Mohamad Mohsen Homayouni1, Seyed Mostafa Razavi2, Minoo Shaddel1 and Mohammad Asadpour1* Prevalence and molecular characterization of Cryptosporidium spp. and Giardia intestinalis in household dogs and cats from Shiraz, Southwestern Iran Veterinaria Italiana 2019, 55 (4), 311-318. doi: 10.12834/VetIt.1710.9049.3 Accepted: 14.01.2018 | Available on line: 31.12.2019 312 Molecular characterization of Cryptosporidium and Giardia in Iran Homayouni et al. Veterinaria Italiana 2019, 55 (4), 311-318. doi: 10.12834/VetIt.1710.9049.3 IIaA15G2R1) and C.  hominis are the most common agents of cryptosporidiosis (Berahmat et  al. 2017). G. intestinalis AII, BIII, and BIV are the most common assemblages identified in humans in Iran (Hooshyar et  al. 2017, Kasaei et  al. 2018). The frequency of G.  intestinalis and Cryptosporidium in Iranian dogs and cats has been investigated mostly by microscopic methods. In a study conducted in dogs, in central Iran, Cryptosporidium oocysts were found in the 2.1% (3/140) in Isfahan (Ranjbar et al. 2017), in the 6% (4/77) in Mashhad (Beiromvand et al. 2013), and in the 0.4% (2/450) in Zanjan (Kohansal et  al. 2017). Cryptosporidium was also detected in the 18% (9/50) of cats in Tehran (Mirzaghavami et  al. 2016). Based on microscopic data, prevalence rates of G.  intestinalis in dogs and cats range from 0.6% (1/147) to 1.6% (6/450) in dogs (Jafari Shoorijeh et  al. 2008, Kohansal et  al. 2017), and 10.7%, (15/140) to 18.9% (7/37) in cats (Bahrami et al. 2011, Khademvatan et  al. 2014). Data upon molecular epidemiology and genetic diversity of G. intestinalis and Cryptosporidium in Iranian dogs and cats are scarce and poorly understood. Accordingly, the aim of this study was to evaluate the prevalence and molecular diversity of Cryptosporidium and G.  intestinalis in household dogs and cats from Shiraz, Southwestern Iran. Material and methods Samples collection The study was carried out from July 2017 to March  2018. Fecal samples were collected from 615   household dogs (n  =  315) and cats (n  =  300) referred to three veterinary clinics in Shiraz, the capital of Fars province, Iran. The majority of dogs (304/315) and cats (202/300) were asymptomatic. Metadata such as gender, breed, age, keeping conditions (indoor/outdoor), fecal consistency (diarrheic/non-diarrheic), and diet were recorded. A safe diet was defined when clean food (e.g. canned and/or packed and cooked food) and water were provided to the animal. Microscopic examination and sucrose flotation All stool samples were screened for Cryptosporidium oocysts using modified acid-fast staining method. Furthermore, wet smears with saline and Lugol’s iodine were prepared for all fecal samples for detection of G.  intestinalis oocysts. As for purification of Giardia and Cryptosporidium oocysts, sucrose gradient flotation technique was performed as previously described (Lagapa et  al. 2009, Asadpour et  al. 2018). Then, the recovered Introduction Foodborne zoonotic pathogens are a serious public health issue and result in significant global economic losses. Giardia and Cryptosporidium, genera of common protozoan parasites that infect domestic and wild animals and humans, generally cause diarrhea. As for Cryptosporidium, the most common species that causes human infection are the Cryptosporidium parvum and Cryptosporidium hominis. They are divided into many species/ genotype and subtypes using various molecular methods. These molecular tools are necessary for epidemiological purposes and understanding of the transmission of infection to humans and animals. Domestic dogs and cats are frequently infected by C. canis and C. felis (Itoh et al. 2014, Sotiriadou et al. 2013, Berahmat et al. 2017, Xiao 2010). Furthermore, C.  parvum and C. muris are frequently reported in domestic dogs and cats, respectively (Pavlasek and Ryan 2007, Santín et  al. 2006). Giardia is extremely common and is responsible for ~ 280 million human cases of diarrhoea every year (total giardiasis acquired by all transmission routes) and infects >  40 animal species (Horlock-Roberts et  al. 2017). Currently eight species of Giardia are accepted as valid, including the recently described Giardia cricetidarum in hamsters and Giardia peramelis in bandicoots (Hillman et al. 2016, Lyu et al.2018). Giardia intestinalis infects humans and is a species complex consisting of eight assemblages (A-H) (Ryan and Cacciò 2013). Assemblages A and B are the predominant assemblages in humans, but assemblages C, D, E and F have also been identified (Cacciò et  al. 2017). Within Assemblage A, sub-assemblages AI, AII and AIII have been identified and of these AI and AII are commonly reported in humans and animals with sub- assemblage AIII reported in wild ruminants (Feng and Xiao 2011). Assemblages C-H, are generally the host-specific Giardia assemblages. Assemblages C and D are widespread in dogs and assemblage F is the prevalent assemblage in cats (Yang et  al. 2015). Some researchers suggest that dogs and cats may play a role in zoonotic transmission of cryptosporidiosis and giardiasis (Berrilli et  al. 2012, Pallant et  al. 2015), while others reject this hypothesis (De Lucio et  al. 2017, Rehbein et  al. 2019). In Iran, like in other developing countries, cryptosporidiosis and giardiasis are a public health concern with socio-economic impact. Prevalence rates of G.  intestinalis and Cryptosporidium have been reported in Iran. G.  intestinalis was found in the 2.7% (12/450) of children in Behbahan (Kasaei et al. 2018) while Taghipour and colleagues (Taghipour et  al. 2011) reported the 2.4% of Cryptosporidium prevalence in humans with diarrhea in Tehran. C.  parvum (particularly subtype 313 Homayouni et al. Molecular characterization of Cryptosporidium and Giardia in Iran Veterinaria Italiana 2019, 55 (4), 311-318. doi: 10.12834/VetIt.1710.9049.3 and genotyping of Giardia. A 292-bp fragment of the DNA sequence coding for the SSU rRNA was amplified using RH4 (5’- AGTCGAACCCTGATTCTCCGCCAGG-3’) and RH11 (5’-CATCCGGTCGATCCTGCC-3’) (Hopkins et  al. 1997). PCR amplification was performed with the following conditions: 25 µl of master mix, supplemented with 2µl (20 pmol) of each forward and reverse primer, 8 μl of DNA (~ 40 ng) and filled to 50 µl with distilled water. PCR program was set as follows: after a primary denaturation step at 96  °C for 10 min, 35 cycles were performed consisting of denaturation at 95 °C for 30 s, annealing at 65 °C for 35 s, extension at 72 °C for 60 s, and a final extension at 72  °C for 7 min. Moreover, a 753-bp fragment of the Beta-giardin (bg) locus was amplified using G7 (5’-AAGCCCGACGACCTCACCCGCAGTGC-3’) and G759 (5’-GAGGCCGCCCTGGATCTTCGAGACG AC-3’) primers as described previously (Caccio et al. 2002). PCR was performed in a reaction mixture containing 25 µl of master mix, supplemented with 2 µl (20 pmol) of each primer, 8 μl of DNA (~ 40 ng) as template and filled to 50  µl with distilled water. PCR program consisted of an initial denaturation at 96 °C for 5 min, followed by 30 cycles consisting of denaturation at 94 °C for 35 s, annealing at 58 °C for 40 s, extension at 72 °C for 60 s, and a final extension at 72 °C for 7 min. For positive and negative controls, consisting of a G. intestinalis assemblage A (isolated from humans) and sterile water were included in all amplifications, respectively. Analysis of PCR products was conducted by 1.5% agarose gel electrophoresis (Fermentas, USA) and visualization using a UV transilluminator. Purification of PCR products and sequencing analysis PCR products were purified by gel excision (Vivantis Technologies, Selangor, Malaysia). The recovered products were sequenced by Sanger dideoxy sequencing technology. Obtained sequences data were aligned by CLC Main Workbench 6.0 software (CLC bio, Denmark) and Clustal W MEGA 6 software. Neighbor-joining method and bootstrap analysis over 1,500 replicates were used for reconstructing phylogenetic trees (Tamura et al. 2013). Statistical analysis Statistical analysis was performed using SPSS version 21 software (SPSS Inc. Chicago, IL, USA). Pearson’s chi-squared (χ2) for independence and Fisher’s exact two-sided tests were conducted to evaluate association between infections and host factors including gender, breed, age, fecal consistency, keeping conditions, and diet. p  <  0.05 was considered significant. oocysts were washed 3 times (1,500 rpm for 15 min) with phosphate buffer saline (PBS) (1  M, pH  =  7.4), kept in 2.5% potassium dichromate and stored at 4 ºC until further use. DNA extraction Total DNA was extracted using Stool Genomic DNA Extraction commercial kit (Bioneer, cat. no. K-3036, Daejeon, Korea) based on the manufacturer’s procedure with some modifications as described earlier (Asadpour et al. 2018). Briefly, the recovered oocyst were washed three times with tap water (2,000 rpm, 12 min), the supernatant was removed, oocyst were supplemented with 400 μl of lysis buffer and 40 μl of proteinase K (Bioneer, cat. no. KB-0111, South Korea), mixed gently, and incubated at 65 °C overnight. Then, the supernatant was transferred to a fresh tube and centrifugated (6,000  rpm, 5  min). The remaining steps were accomplished according to the kit procedure. Extracted DNA (~  150  µl) was kept at - 20 °C until further use. PCR for Cryptosporidium Cryptosporidium -positive samples were genotyped by nested-PCR amplification of a 830-bp fragment of the DNA sequence coding for the SSU rRNA as described previously (Xiao et  al. 2006). CryptF1 (5’-TTCTAGAGCTAATACATGCG-3’) and CryptR1 (5’-CCCATTTCCTTCGAAACAGGA-3’) in the first PCR, and CryptF2 (5’-GGAAGGGTTGTATTTATTAGATA-3’) and CryptR2 (5’- CTCATAAGGTGCTGAAGGAGTA-3’) for the second PCR reaction were used. In the present study we used a ready to use master mix (Taq DNA Polymerase Master Mix RED, Ampliqon, Denmark). Each 50  µl PCR tube contained 25  µl of master mix, 2  µl (20  pmol) of each forward and reverse primer (Bioneer, Daejeon, South Korea), 8 μl of DNA (~ 40 ng) was extracted as template, filled to 50 μl with distilled water. For amplification, a Bio-Rad thermocycler machine (Bio-Rad, CA, USA) was used with an initial denaturation at 95  °C for 4  min, followed by 30 cycles, consisting of denaturation at 94  °C for 40  s, annealing at 55 °C (for primary PCR) and 58 °C (for secondary PCR) for 45 s, extension at 72 °C for 60 s. A final extension at 72 °C for 7 min was included at the end of the amplification cycles. For positive and negative controls, a C.  parvum isolate (https://www.ncbi.nlm.nih.gov/nuccore/KY410237) and sterile water were included in each reaction, respectively. In order to confirm the genotype, all secondary PCR-products were sequenced. PCR for Giardia Two target genes were used for molecular detection 314 Molecular characterization of Cryptosporidium and Giardia in Iran Homayouni et al. Veterinaria Italiana 2019, 55 (4), 311-318. doi: 10.12834/VetIt.1710.9049.3 (Tables I and II). In this study, all examined cats belonged to the same breed (Persian short hair). Molecular results Genotyping of Cryptosporidium isolates Sequencing analysis revealed the presence of C.  canis (n  =  2) in dogs and C.  felis (n  =  2) in cats, respectively. Nucleotide sequences were deposited in GenBank under accession numbers MG888049.1- MG888051.1 and MG889862.1, as shown in Table III. Figure 1 shows the phylogenetic relatedness of the samples of this study. Phylogenetic analysis showed that C. canis and the two C. felis isolates grouped in specific clusters. Results Prevalence rates of Cryptosporidium and G. intestinalis As shown in Table I and II, Cryptosporidium and G.  intestinalis were detected in the 0.6% (2 of 315) and 1.9% (6 of 315) of dogs; and in the 0.7% (2 of 300) and 1.3% (4 of 300) of cats, respectively. Statistical analysis revealed a significant higher prevalence of Giardia in dogs younger than one year (6/315) (p < 0.05). Besides, a significant correlation resulted between G.  intestinalis infection and diarrhea in dogs (p  =  0.001). No association was evidenced between gender, breed or diet with the presence of G. intestinalis and Cryptosporidium in dogs (p > 0.05) Table I. Potential host factors associated with Cryptosporidium and Giardia infections in household dogs. Potential host factors No. of screened dogs (%) (n = 315) Positive for Cryptosporidium (n = 2) (%) P value # Positive for Giardia (n = 6) (%) P value# Age (years) ≥ 1 143 (45.4) 2 (100) 0 (0.00) ≤ 1 172 (54.6) 0 (0.00) 0.503 6 (100) 0.034* Gender Male 157 (49.8) 2 (100) 0.248 3 (50) Female 158 (50.2) 0 (0.00) 3 (50) 0.655 Breed Terrier 63 (20.0) 1 (16.66) 2 (33.33) Great Dane 10 (3.2) 0 (0.00) 0 (0.00) Husky 53 (16.8) 0 (0.00) 1 (16.66) German Shepherd 62 (19.7) 1 (16.66) 0 (0.00) Dobermann 85 (27.0) 0 (0.00) 0.801 2 (33.33) 0.277 Shih Tzu 34 (10.8) 0 (0.00) 0 (0.00) Pomeranian 8 (2.5) 0 (0.00) 1 (16.66) Fecal consistency Diarrheic 11(3.49) 2 (100) 0.069 6 (100) < 0.001* Non-diarrheic 304 (96.50) 0 (0.00) 0 (0.00) Keeping condition Indoor 65 (20.63) 1 (50) 1 (16.66) Outdoor 250 (79.36) 1 (50) 0.371 5 (83.33) 0.105 Unsafe Diet Yes 145 (46.03) 2 (100) 0.801 6 (100) 0.127 No 170 (53.96) 0 (0.00) 0 (0.00) # Statistical significance, p < 0.05. * The association was evaluated using Fisher exact test. Table II. Potential host factors associated with Cryptosporidium and Giardia infections in household cats. Potential host factors No. of screened cats (%) (n = 300)a Positive for Cryptosporidium (n = 2) (%) P value b Positive for Giardia (n = 4) (%) P value b Age (years) ≥ 1 172 (57.3) 1 (50.0) 0.672 4 (100.0) 0.139 ≤ 1 128 (42.7) 1 (50.0) 0.0 (0.00) Gender Male 174 (58.0) 1 (50.0) 0.664 2 (50.0) 0.142 Female 126 (42.0) 1 (50.0) 2 (50.0) Fecal consistency Diarrheic 98 (32.7) 2 (100.0) 2 (50.0) Non-diarrheic 202 (67.3) 0.0 (0.00) 0.106 2 (50.0) 0.599 Keeping condition Indoor 55 (18.3) 0.00 (0.00) 2 (50.0) Outdoor 245 (81.7) 2 (100.0) 0.666 2 (50.0) 0.155 Unsafe Diet Yes 258 (86.0) 2 (100.0) 0.843 4 (100.0) 0.099 No 42 (14.0) 0.0 (0.00) 0.0 (0.00) a All screened cats were Persian short hire breed. Statistical significance, p < 0.05. b The association was evaluated using Fisher exact test. 315 Homayouni et al. Molecular characterization of Cryptosporidium and Giardia in Iran Veterinaria Italiana 2019, 55 (4), 311-318. doi: 10.12834/VetIt.1710.9049.3 Discussion In this study, a total of 615 stool samples were collected from household dogs and cats and screened for detection of Cryptosporidium and Giardia (oo) cysts. Moreover, PCR methods using different targets genes were used to determine the species/genotype/assemblage of Cryptosporidium and G.  intestinalis detected in this study. The prevalence rates of Cryptosporidium in household dogs and cats in this study was lower compared to those reported previously (Mirzaghavami et al. 2016, Genotyping of Giardia isolates DNA sequence analysis revealed the presence of G.  intestinalis assemblages C (3/6), D (2/6) and sub-assemblage AII (1/6) in dogs. G.  intestinalis assemblage F (3/4), and sub-assemblage AI (1/4) were identified in cats. Nucleotide sequence data were deposited in GenBank (Table IV). Phylogeny is depicted in Figure 2. Table III. Cryptosporidium positive samples identified in household dogs and cats by nested-PCR of the SSUrRNA coding gene. Sample name Host* Species GenBankAccession No. 24 Cat C. felis MG888051.1 111 Dog C. canis MG888050.1 216 Dog C. canis MG888049.1 218 Cat C. felis MG889862.1 *Cryptosporidium were detected in 2 dogs and 2 cats. 100 C. bovis (MG516770.1: cattle - Australia) C. bovis (KT922232.1: calf - Ethiopia) C. bovis (KP793007.1: cattle - China) C. felis (MG889862.1: cat - Iran) C. felis (MG888051.1: cat - Iran) C. parvum (DQ656352.1: human - Iran) C. parvum (KY410237.1: calf - Iran) C. parvum (KF830259.1: calf - Iran) C. suis (KJ790232.1: swine - China) C. suis (KJ790239.1: swine - China) C. ubiquitum (MG516761.1: sheep - Australia) C. ubiquitum (KX259133.1: deer - China) C. canis (MG888050.1: dog - Iran) C. canis (MG888049.1: dog - Iran) C. baileyi (EU827305.1: chicken - China) C. baileyi (GU377276.1: ostrich - China) C. baileyi (GU377272.1: ostrich - China) C. muris (KJ469984.1: horse - Poland) Eimeria bovis (KU052226.1: Bos taurus - Turkey) 93 51 63 62 50 77 75 95 90 0.05 Figure 1. Neighbor-joining (NJ) tree based on the SSU rRNA coding sequences. Black dots represent the Cryptosporidium species detected in household dogs and cats of this study (Shiraz, Southwestern Iran). Table IV. Giardia intestinalis identified in dogs and cats by PCR of the 16srRNA gene. Sample name Host* Assemblage GenBankAccession No. 1 Dog D MG851692.1 2 Cat F MG832845.1 3 Cat A MG832842.1 4 Cat F MG832844.1 11 Cat F MG832843.1 36 Dog C MG851690.1 113 Dog D MG851691.1 192 Dog C MG851695.1 226 Dog C MG851693.1 306 Dog A MG851694.1 *Giardia was detected in 6 dogs and 4 cats. 99 99 G. intestinalis (Dog isolate 226) G. intestinalis (Dog isolate 192) G. intestinalis (Dog isolate 36) G. intestinalis (Dog isolate 113) G. intestinalis (Dog isolate 1) G. intestinalis (Cat isolate 4) G. intestinalis (Cat isolate 11) G. intestinalis (Cat isolate 2) G. intestinalis (Cat isolate 3) G. intestinalis (Dog isolate 306) Assemblage A G. muris (EF455599.1: Rattus norvegicus - Sweden) G. intestinalis (KR051227.1: meerkat - China) G. intestinalis (KU156663.1: dog - China) G. intestinalis (KJ888976.1: Macaca mulatta - China) G. intestinalis (KP899898.1: Homo sapiens - Ethiopia) G. intestinalis (KJ888974.1: monkey - China) G. intestinalis (KT698974.1: cattle- China) G. intestinalis (KJ188080.1: lemur - China) G. intestinalis (KJ188080.1: cattle - Bangladesh) G. intestinalis (KT698972.1: cattle - China) G. intestinalis (GQ919292.1: �sh - Australia) 53 63 100 94 97 99 58 100 57 62 57 74 98 Assemblage D Assemblage E Assemblage F Assemblage B Assemblage C Figure 2. Neighbor-joining (NJ) tree based on b-giardin coding sequences of G. intestinalis assemblages. Black dots represent G. intestinalis assemblages from dogs and cats of this study (Shiraz, Southwestern Iran). 316 Molecular characterization of Cryptosporidium and Giardia in Iran Homayouni et al. Veterinaria Italiana 2019, 55 (4), 311-318. doi: 10.12834/VetIt.1710.9049.3 in zoonotic transmission of these parasites, at least in the areas under investigation. Conclusions The overall prevalence of Cryptosporidium and Giardia in household dogs and cats was low. Further studies using multiple target genes are recommended in different geographical areas of Iran to provide a better understanding of the epidemiology of these two parasites. Acknowledgements We appreciate AJA University of Medical Sciences for financial support (Grant No. 15/86051). The authors also would like to thank Mr Shahriyari, Mr Bahrami, and Mr Nabavi for their participation in sampling. We are also thankful to Lucy J Robertson (Department of Food Safety and Infection Biology, Norwegian School of Veterinary Science) for her kind advise on molecular techniques. Ranjbar et  al. 2017, Beiromvand et  al. 2013). This study indicates C.  canis and C.  felis as the prevalent species detected in dogs and cats, respectively, in this area of Iran. This finding is in line with other studies (Ranjbar et al. 2017, Neves et al. 2014, Pallant et  al. 2015, Yang et  al. 2015). The overall prevalence of G. intestinalis in dogs (1.9%) was higher than that of previous studies (0/6%) (Shoorijeh et  al. 2008). G.  intestinalis was detected in the 1.3% of sampled cats. These rates were also lower compared to previous studies (Bahrami et al. 2011, Khademvatan et al. 2017). Molecular data revealed that household dogs were mostly infected with G.  intestinalis host-specific assemblages C and D. It is well established that G. intestinalis assemblages C and D are the most prevalent assemblages in dogs (Ryan and Cacciò 2013, Sotiriadou et  al. 2013, Uehlinger et al. 2013) while several studies, including this one, indicates also assemblage F capable of infecting cats (Cacciò et al. 2005, Santín et al. 2006, Yang et al. 2015). Overall, data obtained in the present study showed that household dogs and cats likely play a minor role 317 Homayouni et al. Molecular characterization of Cryptosporidium and Giardia in Iran Veterinaria Italiana 2019, 55 (4), 311-318. doi: 10.12834/VetIt.1710.9049.3 Asadpour M., Namazi F., Razavi S.M. & Nazifi S. 2018. Comparative efficacy of curcumin and paromomycin against Cryptosporidium parvum infection in a BALB/c model. Vet Parasitol, 250, 7-14. Bahrami A., Doosti, A., Nahravanian H., Noorian A.M. & Asbchin S.A. 2011. Epidemiological survey of gastro-intestinal parasites in stray dogs and cats. Aust J Basic Appl Sci, 5, 1944-1948. Beiromvand M., Akhlaghi L., Massom S.H.F., Meamar A.R., Motevalian A., Oormazdi H. & Razmjou E. 2013. Prevalence of zoonotic intestinal parasites in domestic and stray dogs in a rural area of Iran. Prev Vet Med, 109, 162-167. Berahmat R., Spotin A., Ahmadpour E., Mahami-Oskouei M., Rezamand A., Aminisani N., Ghojazadeh M., Ghoyounchi R. & Mikaeili-Galeh T. 2017. Human cryptosporidiosis in Iran: a systematic review and meta-analysis. Parasitol Res, 116, 1111-1128. Berrilli F., D’Alfonso R., Giangaspero A., Marangi M., Brandonisio O., Kaboré Y., Glé C., Cianfanelli C., Lauro R. & Di Cave D. 2012. Giardia duodenalis genotypes and Cryptosporidium species in humans and domestic animals in Côte d’Ivoire: occurrence and evidence for environmental contamination. Trans R Soc Trop Med Hyg, 106, 191-195. Bowman D.D. & Lucio-Forster A. 2010. Cryptosporidiosis and giardiasis in dogs and cats: veterinary and public health importance. Exp Parasitol, 124, 121-127. Cacciò S.M., De Giacomo M. & Pozio E. 2002. Sequence analysis of the β-giardin gene and development of a polymerase chain reaction-restriction fragment length polymorphism assay to genotype Giardia duodenalis cyst from human faecal samples. Int J Parasitol, 32, 1023-1030. Cacciò S. & Lalle M. 2015. Giardia (L. Xiao, U. Ryan & Y. Feng, eds). Biology of Foodborne Parasites, CRC Press, USA, 175-193. Cacciò S.M. & Ryan U. 2008. Molecular epidemiology of giardiasis. Mol Biochem Parasitol, 160, 75-80. Cacciò S.M., Thompson R.A., McLauchlin J. & Smith H.V. 2005. Unravelling Cryptosporidium and Giardia epidemiology. Trends Parasitol, 21, 430-437. De Lucio A., Bailo, B., Aguilera, M., Cardona, G.A., Fernández-Crespo, J.C. & Carmena, D. 2017. No molecular epidemiological evidence supporting household transmission of zoonotic Giardia duodenalis and Cryptosporidium spp. from pet dogs and cats in the province of Álava, Northern Spain. Acta Trop, 170, 48-56. Feng Y. & Xiao L. 2011. Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev, 24, 110-140. Hillman A., Ash A., Elliot A., Lymbery A., Perez C. & Thompson R.C.A. 2016. Confirmation of a unique species of Giardia parasitic in the quenda (Isoodon obesulus). Int J Parasitol Parasites Wildl, 5, 110-115. Hooshyar H., Ghafarinasab S., Arbabi M., Delavari M. & References Rasti S. 2017. Genetic variation of Giardia lamblia isolates from food-handlers in Kashan, Central Iran. Iran J Parasitol, 12, 83. Hopkins R.M., Meloni B.P., Groth D.M., Wetherall J.D., Reynoldson J.A. & Thompson R.A. 1997. Ribosomal RNA sequencing reveals differences between the genotypes of Giardia isolates recovered from humans and dogs living in the same locality. J Parasitol, 83, 44-51. Itoh N., Muraoka N., Kawamata J., Aoki M. & Itagaki T. 2006. Prevalence of Giardia intestinalis infection in household cats of Tohoku district in Japan. J Vet Med Sci, 68, 161-163. Itoh N., Oohashi Y., Ichikawa-Seki M., Itagaki T., Ito Y., Saeki H., Kanai K., Chikazawa S., Hori Y. & Hoshi F. 2014. Molecular detection and characterization of Cryptosporidium species in household dogs, pet shop puppies, and dogs kept in a school of veterinary nursing in Japan. Vet Parasitol, 200, 284-288. Kasaei R., Carmena, D., Jelowdar, A. & Beiromvand, M. 2018. Molecular genotyping of Giardia duodenalis in children from Behbahan, southwestern Iran. Parasitol Res, 117, 1425-1431. Karim M.R., Zhang S., Jian F., Li J., Zhou C., Zhang L., Sun M., Yang G., Zou F. & Dong H. 2014. Multilocus typing of Cryptosporidium spp. and Giardia duodenalis from non-human primates in China. Int J Parasitol, 44, 1039-1047. Khademvatan S., Rahman Abdizadeh F.R., Hashemitabar M., Ghasemi M. & Tavalla M. 2014. Stray cats gastrointestinal parasites and its association with public health in Ahvaz City, South Western of Iran. Jundishapur J Microbiol, 7, 8. Kohansal M.H., Fazaeli A., Nourian A., Haniloo A. & Kamali K. 2017. Dogs’ gastrointestinal parasites and their association with public health in Iran. J Vet Res, 61, 189-195. Lagapa J.T.G., Oku Y., Kaneko M., Ganzorig S., Ono T., Nonaka N., Kobayashi F. & Kamiya M. 2009. Monitoring of environmental contamination by Echinococcus multilocularis in an urban fringe forest park in Hokkaido, Japan. Environ Health Prev Med, 14, 299. Lyu Z., Shao J., Xue M., Ye Q., Chen B., Qin Y. & Wen H. 2018. A new species of Giardia Künstler, 1882 (Sarcomastigophora: Hexamitidae) in hamsters. Parasit Vectors, 11, 202. Lucio-Forster A., Griffiths J.K., Cama V.A., Xiao L. & Bowman D.D. 2010. Minimal zoonotic risk of cryptosporidiosis from pet dogs and cats. Trends Parasitol, 26, 174-179. Mirzaghavami M., Sadraei J. & Forouzandeh M. 2016. Detection of Cryptosporidium spp. in free ranging animals of Tehran, Iran. J Parasit Dis, 40, 1528-1531. Neves D., Lobo L., Simões P.B. & Cardoso L. 2014. Frequency of intestinal parasites in pet dogs from an urban area (Greater Oporto, northern Portugal). Vet Parasitol, 200, 295-298. Pallant L., Barutzki D., Schaper R. & Thompson R.A. 2015. The epidemiology of infections with Giardia species 318 Molecular characterization of Cryptosporidium and Giardia in Iran Homayouni et al. Veterinaria Italiana 2019, 55 (4), 311-318. doi: 10.12834/VetIt.1710.9049.3 Molecular identification of Giardia and Cryptosporidium from dogs and cats. Parasite, 20, 8. Taghipour N., Nazemalhosseini-Mojarad E., Haghighi A., Rostami-Nejad M., Romani S., Keshavarz A., Alebouyeh M. & Zali M. 2011. Molecular epidemiology of cryptosporidiosis in Iranian children, Tehran, Iran. Iran J Parasitol, 6, 41. Tamura K., Stecher G., Peterson D., Filipski A. & Kumar S. 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol, 30, 2725-2729. Uehlinger F.D., Greenwood S.J., McClure J.T., Conboy G., O’Handley R. & Barkema H.W. 2013. Zoonotic potential of Giardia duodenalis and Cryptosporidium spp. and prevalence of intestinal parasites in young dogs from different populations on Prince Edward Island, Canada. Vet Parasitol, 196, 509-514. Xiao L. 2010. Molecular epidemiology of cryptosporidiosis: an update. Exp Parasitol, 124, 80-89. Xiao L., Alderisio K. & Singh A. 2006. Development and standardization of a Cryptosporidium genotyping tool for water samples. J Am Water Works Assoc, 5-9. Yang R., Ying J.L.J., Monis P. & Ryan U. 2015. Molecular characterisation of Cryptosporidium and Giardia in cats (Felis catus) in Western Australia. Exp Parasitol, 155, 13-18. and genotypes in well cared for dogs and cats in Germany. Parasites & vectors, 8, 2. Pavlasek I. & Ryan U. 2007. The first finding of a natural infection of Cryptosporidium muris in a cat. Vet Parasitol, 144, 349-352. Ranjbar R., Mirhendi H., Izadi M., Behrouz B. & Mohammadi Manesh R. 2018. Molecular identification of Cryptosporidium spp. in Iranian dogs using seminested PCR: a first report. Vector Borne Zoonotic Dis, 18, 96-100. Rehbein S., Klotz C., Ignatius R., Müller E., Aebischer A. & Kohn B. 2019. Giardia duodenalis in small animals and their owners in Germany: a pilot study. Zoonoses Public Health, 66, 117-124. Ryan U. & Cacciò S.M. 2013. Zoonotic potential of Giardia. Int J Parasitol, 43, 943-956. Santín M., Trout J.M., Vecino J.A.C., Dubey J. & Fayer R. 2006. Cryptosporidium, Giardia and Enterocytozoon bieneusi in cats from Bogota (Colombia) and genotyping of isolates. Vet Parasitol, 141, 334-339. Shoorijeh S.J., Sadjjadi S.M., Asheri A. & Eraghi K. 2008. Giardia spp. and Sarcocystis spp. status in pet dogs of Shiraz, Southern part of Iran. Trop Biomed, 25, 154-159. Sotiriadou I., Pantchev N., Gassmann D. & Karanis P. 2013.