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Genotyping of Cryptosporidium Spp. in Domestic Dogs and Humans in Karbala Province Iraq.

Jihad TalibObaid AL-Yasary1,Azhar Ali Faraj 2

Department of Parasitology, College of Veterinary Medicine, University of Baghdad , Iraq1,2 Abstract

In this study, Cryptosporidium sp. was diagnosed in Domestic dogs and humans in Karbala province in Iraq by molecular detection by using a nested polymerase chain reaction (nPCR) by outer primer 18s (9005bp) and inner primer 18s (830bp) were used. our study was beginning of December (2019) to September (2020). A total of 100 fecal samples were collected from adult and young and from both sexes of dogs and 100 stool samples were collected from adult and young and from both sexes humans. The sequence study for 10 samples from the positive samples recorded at the National Center for Biotechnology Information (NCBI) was found 7 species C. parvum under accession number (MT329015.1 - MT329013.1) and 3 samples C. canis under accession number (MT329014.1, MT329016.1, and MT329018.1). The result of the sequence analysis In humans for 10 samples from the positive samples recorded at the (NCBI) was indicate 10 isolate Cryptosporidium parvum. under the accession numbers (MW186198 - MW186207) ).

1-Introduction

Cryptosporidium, a protozoan parasite belonging to the Phylum Apicomplexa and Family Cryptosporiidae, is a common cause of diarrhea in humans, domestic animals and wild vertebrates (Bouzidet al., 2018).. Cryptosporidium genus contains over 30 so far recognized species and numerous genotypes may be infected dogs with C. canis, C. parvum, C. ubiquitum and C. andersoni, about twenty Cryptosporidium spp. have been reported but only a few have been found to infect both humans and animals and these include; Cryptosporidium parvum, Cryptosporidium felis, Cryptosporidium ubiquitum,Cryptosporidium muris, Cryptosporidium meleagridis, Cryptosporidium suisand Cryptosporidium canis. The occurrence of animal Cryptosporidium spp. in humans indicates that humans are constantly at risk of contracting cryptosporidiosis from these reservoir hosts (Bialeket al., 2002). C.parvum was thought to infect all animals. However, it is generally accepted that C. parvum infects primarily ruminants( cattle, sheep and goat) and humansand also have been found occasionally in other mammals such as mice and dogs, although companion animals are most often infected with host-specific Cryptosporidium spp. (Xiao and Fayer, 2008) and (Al- Zubaidei and Kawan, 2020).The infection can be transmitted to human through close contact with dogs (Hunter and Thompson 2005). The close relationship between man and his domestic pets, particularly dogs, makes transmission of zoonotic diseases very easy, especially those that could be acquired through environmental contamination such as cryptosporidiosis. C. parvumis the most frequently

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identified species of Cryptosporidium spp. in dogs and its isolation in human patients from both developed and under-developed countries (Jianet al., 2014). There were many studies in Iraq about Cryptosporidum spp. In humans (Ali et al., 2015) (Maysoonet al ., 2018) Due to no reports between the molecular analysis of the various Cryptosporidium species in human and dogs, the study design for this aim.

2- Material and methods

Collected of 100 hundred fecal samples from domestic dogs and 100 hundred stool samples from humans from different sex and age from several area of Karbala province, during the study period from start of December (2019), to end of September 2020

The kit used for D NA extraction from (Geneaid, Korea) as per the manufacturer’s instruction. The samples were stored at -80˚C until use. To identify Cryptosporidium spp. in the faecal samples, fragments covering 18S rDNA gene were amplified by nested PCR. First,

amplification of the 900 bp Crypto18S1

(FTTCTAGAGCTAATACATGCG.And.Crypto18S1.R.CCCATTTCCTTCGAAACAGGA) region was carried out, and next, for secondary PCR, the 830 bp (Crypto.18S2F GGAAGGGTTGTATTTATTAGATAAAG.AndCrypto18S2R.CTCATAAGGTGCTGAAGGA GTA) fragment was amplified using 3 µl of the first PCR product. Primers, mixture composition and PCR conditions have been described by (18). The secondary PCR products were examined electrophoretically in 1% agarose gels and visualized after staining with Advance DNA stain.

The identified species typing analysis was done by phylogenetic tree analysis in comparison with NCBI-Blast known sequences.

3-Results

The result of present study showed the amplification conditions were optimized for the PCR assay, using specific primers sequences of 18s RNA (900bp. According to nested-PCR examination, samples were subjected to molecular analysis by using small subunit ribosomal RNA gene specific primers(830bp), in order to identify the species of Cryptosporidium.

Prevalence of Cryptosporidium spp. In dogs and humans by using PCR Technique.the infection rates in dogs in DNA samples showed 47% (47/100) positive that among 100 examined the fecal samples that were collected dogs in Karbala city. In humans DNA samples showed the infection rate of Cryptosporidium spp. by using PCR Technique. showed 34%(34/100) were given positive result.

Ten samples from each dogs and humans selected randomly for the sequences to indicated theCryptosporidium species, which had already been identified by nested PCR. The presentstudy indicated Sequences obtained by genotyping were compared with sequences deposited in Gen Bank base found of two Cryptosporidium species in dogs: 7 samples C.

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parvumthat have been deposited in the gene bank under the accession numbers (MT329015.1, MT329017., MT329019.1, MT329020.1, MT329021.1, MT329022.1, and MT329013.1). and3 samples identified Cryptosporidium canis gene (18S) that have been deposited in the gene bank under the accession numbers (MT329014.1, MT329016.1, and MT329018.1). while in humans all samples the identified Cryptosporidium parvum genes (18S) that have been deposited in the gene bank under the accession numbers (MW186198, MW186199, MW186200, MW186201, MW186202, MW186203, MW186204, MW186205, MW186206, MW186207).

4-Discussion

The frequency of infection rates in dogs in DNA samples showed 47% (47/100) positive that among 100 examined the fecal samples of dogs in Karbala city. In Iraq only one previous study was by (Azhar,2020), she found the prevalence of Cryptosporidium spp. In dogs by using PCR technique was 28.6% .Infection rate of Cryptosporidium spp. By using molecular technique Iran 12.3% was done by (Mehdi et al., 2017).The prevalence of Cryptosporidium spp. In dogs in Egypt by Molecular Screening 24% was found by (Rashaet al., 2018), disagree with them.

Also our result different In China was recorded by (Jiayuet al.,2019) and in Japan by (Naoyukiet al., 2019), they found infection rates of Cryptosporidium spp. in dogs by using PCR were 6.9% and 21.0% respectively. The global prevalence of Cryptosporidium spp. in dogs could vary depending on the sensitivity of used diagnostic techniques, geographic locations, and the tested population, In our study, the higher prevalence of Cryptosporidium in dogs compared to the prevalence in previous studies might be attributed to contact with stray dogs, poor feeding such as ingestion of dog feces (dog coprophagia) and water, contaminated soil, poor sanitation with infected animals agree with (Rashaet al., 2018).

In humans DNA samples showed the infection rate of Cryptosporidium spp. by using PCR Technique. showed 34%(34/100) were given positive result. In Iraq the previous study about the infection rate in human by using molecular technique was conducted by (Maysoonet al.,2018), They found infection rate was 47.33%, agree with them. In Neighboring countries such as Iran the prevalence of infection rate by using PCR was 4.94% is done by (Mitraet al.,2015), and in Jordon the frequency of Cryptosporidium infection in humans by using molecular examination was 8.3% recorded by (Hijjawiet al., 2016). Disagree with study was done in Turkey that recorded prevalence of cryptosporidium spp. In humans was 8.93%

(Nadim and Şadiye. 2017). In Egypt (Rashaet al., 2018), find frequency of distribution of Cryptosporidium spp. in 100 stool samples of children was 14% by nested PCR, it is dos not identical to our study.The variation in prevalence of Cryptosporidium spp in humans among different studies may by attributed to samples size, large or small population, hygiene application, level of contamination of food and water, improper disposal of garbage and dog feces, and contact with other livestock (Rashaet al., 2018).

The present Results of molecular study showed Sequences obtained by genotyping were compared with sequences deposited in GenBank base found of two Cryptosporidium species in

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dogs: C. parvum and C. canisGenotyping analysis of the PCR-positive samples from dogs identified C.parvum in Iraq by (Azhar,2020).Our results were in agreement with results recorded by (Piekaraet al.,2020).In Poland he recorded 3 cases of C. canis and 2 cases of C.parvum in dogs were detected.Thesequence analysis of the Cryptosporidium-positive canine samples in Chania by (Shenquanet al., 2020), revealed that presence of C. canisand C.

parvum.While(Jianet al., 2014), he was only recorded C. canis in chain’s dogs. Also in Chain (Jiayuet al.,2019), Whorecorded three species were identified as positive for C. canis, C. muris and the Cryptosporidium rat genotype IV. Genotyping of nested PCR positive dog samples showed the presence of only genotype C. parvum in five samples and the restriction fragments banding patterns of this genotype in Egyptian dogs by (Rashaet al., 2018). In IranMolecular identification of Cryptosporidium species showed that the species in dogs is C. parvum by (Reza et al.,2017). A sequencing analysis in Japans by (Naoyukiet al., 2019), demonstrated that species infected dogs with C. canis. As in current study, C. parvumand C. canis has been identified as the most common Cryptosporidium species in dogs around the world, although a few cases of C. parvum, C. muris, and C. meleagridis infection have also been reported (Jianet al., 2014). The variation of Cryptosporidium spp.In dogs could be due to their close relationship with animals particularly cattle and sheep. Given that dogs with cryptosporidiosis lack clinical symptoms, as they are companion animals for humans. The existence of C. parvum in stool specimens of dogs in this study can probably be due to the close relationship between dogs with sheep and with cattle, and then, these relationships are likely to be a potential risk factor for infecting dogs, agree with (Reza et al.,2017).

DNA sequences of the 18S rRNA gene of ten samples selected randomly from infected human were 99.31% to 99.45% identical to sequences in the GenBank of C. parvum isolates from humans, similar to the results of studies conducted in Iraq by (Maysoonet al.,2018) and (Haider. 2019).record that ( C parvumand C hominis) and (C. parvum ,C. andersoniand C.

hominis) respectivelyisolated from humans samples. Also agree with study conducted in Iran that amplification of small subunit ribosomal RNA gene was performed using a nested polymerase chain reaction protocol and its products were digested using two restriction enzymes for Cryptosporidium species and genotype differentiation and restriction pattern revealed the presence of C.parvum was done by (Mitraet al., 2015). In Jordan (Hijjawiet al., 2016), demonstrated that species infected humans with C. hominis and C. parvum. In Kuwait the DNA sequencing done by (Jamshaidet al., 2011), was used to confirm Cryptosporidium species infect humans genotypes. RFLP analyses of the secondary PCR product and identifiedC.

parvum and C. hominis. In TurkeyGenetic analysis of the Cryptosporidium spp. Infected humans Turkey indicated that C. hominis and C. parvum are species infected humans, that recorded by (Nadim and Şadiye. 2017). Genotyping analysis of the PCR-positive samples from humans. In Egypt genotyping of the nested PCR positive human samples revealed the existence of human genotype C. hominis and genotype C. parvum(Rashaet al.,2018). DNA sequence analysis confirming and identifying the current Cryptosporidium isolates as C. parvum, indicates infect humans in Sudan by (Adam et al., 2019). Current study explained that C. parvum is the main

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Cryptosporidium spp. Infecting humans and animals in Iraq and thought these isolates to be adaptable for both zoonotic and anthroponotic transmissions, by direct or indirect contact. Our findings are supported by previous studies (Mitraet al., 2015), (Chen et al.,2017) and (Adam et al., 2019). Our results investigate that humans could acquire Cryptosporidium infection through anthroponotic and zoonotic transmission. the nested PCR products of (18S) gene demonstrate five C. parvum genotypes from humans samples identical to C. parvum genotypes from dogs samples. These findings indicated that household dogs are potential reservoirs for C. parvum (bovine genotype) with the possibility of direct or indirect zoonotic transmission to humans in close contact with dogs and other domestic livestock agree with (Rashaet al.,2018).

5- Reference

1. Adam, K.Y, Ismail, A.A., Masri, M.A and Gameel, A.A. (2019).First report and molecular characterization of Cryptosporidium spp. in humans and animals in Khartoum state, Sudan, Veterinary World. V. 12(1): 183-189.

2. Al- Zubaidei. H. H, Kawan, M. H.(2020).Prevalence of Cryptosporidiosis in Ostriches from Central and South Parts of Iraq. The Iraqi Journal of Veterinary Medicine, 44 (1):

63-67.

3. Azhar, A. F.(2019).traditional and molecular study of Cryptosoporidium spp. in domestic dogs in baghdadcity,Iraq. Iraqi Journal of Agricultural Sciences –50(4):1094- 1099.

4. Bialek, A.J., Ives, N. J., Gazzard, B. G. and Easterbrook, P. J. ( 2002).The changing pattern of AIDS – defining illness with the introduction of (HAART) in a London clinic.

J Infect. 42: 134 – 139.

5. Bouzid, M., Kintz, E and Hunter, P. R. (2018). Risk factors for Cryptosporidium infection in low and middle income countries: A systematic review and metaanalysis.

PLoS Neglected Trop Dis. 2018;12(6).

6. Chen, S., Atwill, E.R., Zhong, F., Wei, Y and Hou, S. (2017). Prevalence and Risk Factors of Cryptosporidium Infection in Children Hospitalized for Diarrhea in Guangzhou, China. J BacteriolParasitol 8: 308.P, 2 – 8.

7. Haider, H. O. AL. (2019). Prevalence comparative study of Cryptosporidium species in cattle and handlers in Baghdad city, Iraq.Phd thesis in parasitology in veterinary medicine / University of Baghdad

8. Hijjawi, N., Rami. M., Rongchang, Y and Una, R. (2016). Genetic characterization of Cryptosporidium in animal and human isolates from Jordan, Veterinary Parasitology 228 (2016) 116–120

9. Hunter, P.R and Thompson, R.C.(2005).The zoonotic transmission of Giardia and Cryptosporidium.Int J Parasitol.;35(11–12):1181–90.

10. Jamshaid, I, Nabila, K and Parsotam, R. H. (2011).Cryptosporidiosis in Kuwaiti children: association of clinical characteristics with Cryptosporidium species and subtypes.Journal of Medical Microbiology. V 60, P 647–652.

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11. Jian,F., Meng, Q., Xiaoyi, H., Rongjun, W., Sumei, Z, Heping, D and Longxian, Z.

(2014).Occurrence and molecular characterization of Cryptosporidium in dogs in Henan Province, China.BMC Veterinary Research.1746-6148. P.4-4.

12. Jiayu, L, Xiaoyu, D., Kexin, Z., Na, L., Yaqiong, G., Zezhong, Z., Yaoyu, F and Lihua, X. (2019).Genetic characterization of Cryptosporidium spp. and Giardia duodenalis in dogs and cats in Guangdong, China.J. Parasites Vectors ,12:571 /10.1186/s13071-019-3822-.P.9-9

13. Maysoon, A. M; Mohammed, Th. S. Al-Zubaidi; Dalya; B. H; Ilham, A. K and Huda S. J.(2018).Genotyping of Cryptosporidium Spp. Isolated from Human and Cattle in Baghdad Province/ Iraq. Indian Journal of natural science.P,1-12

14. Mitra, S., Ehsan, N. M., Niloofar, T., AbdolSattar., P and Fatemeh, M.

(2015).Prevalence and Genetic Characterization of Cryptosporidium Spp.In Diarrheic Children from GonbadKavoos City, Iran, Iran J Parasitol. 2015 Jul-Sep; 10(3): 441–447.

15. Mehdi, T; Elham, Kord; Rahman, A andFatemeh, A.(2017). Molecular Study of Cryptosporidium spp. in Dogs from Southwest ofIran.Jundishapur Journal of Microbiology, PP.1-6

16. Nadim, Y and Şadiye, K. K.(2017).Cryptosporidiosis in Humans with Reference to the First Case of Cryptosporidium hominis Infection in Turkey. Med Bull Haseki 2017;55:194-8

17. Naoyuki, I., Hazuki, T., Yuko, I., Satoshi, K and Yuya, K. (2019).Molecular Prevalence of Cryptosporidium spp. in Breeding Kennel Dogs. Korean J Parasitol Vol.

57, No. 2: 197-200.

18. Piekara,S. A; Piekarska, J and Gorczykowski, M.(2020).Cryptosporidium spp. in dogs and cats from Poland. Ann Agric Environ Med. doi: 10.26444/ aaem/120467

19. Reza, R., Hossein, M., Morteza, I., Bahador, B and Reza, M. M.(2017). Molecular Identification of Cryptosporidium spp. in Iranian Dogs Using Seminested PCR: A First Report. vector-borne and zoonotic diseases..V,20.P,1-5.

20. Rasha, M.A. G; Abdallah, M.A. M; Ayman, A. S and Amany, M. A.E. (2018).

Molecular Screening and Genotyping of Cryptosporidium Species in Household Dogs and In-Contact Children in Egypt: Risk Factor Analysis and Zoonotic Importance. vector- borne and zoonotic diseases, DOI: 10.1089/vbz.2017.2254. P.P.1-9.

21. Shenquan, L., Xuhui, L., Yongxiang, S., Nanshan, Q., Minna, L., Caiyan, W., Juan, L., Junjing, H., Linzeng,Y., Haiming, C., Wenwan, X., Mingfei, S and Guoqing, L.

(2020). Occurrence and genotypes of Cryptosporidium spp., Giardia duodenalis, and Blastocystis sp. in household, shelter, breeding, and pet market dogs in Guangzhou, southern China.Scientificreports.doi.org/V,10.1038/s41598-020-74299-P,1-11.

22. Xiao, L. and R. Fayer. 2008. Molecular characterization of species and genotypes of Cryptosporidium and Giardia and assessment of zoonotic transmission. Int J Parasitol.

38: 1239-1255

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