TRADITIONAL AND MOLECULAR STUDY OF CRYPTOSOPORIDIUM SPP. IN DOMESTIC DOGS IN BAGHDAD CITY,IRAQ

1094 TRADITIONAL AND MOLECULAR STUDY OF CRYPTOSOPORIDIUM SPP. IN DOMESTIC DOGS IN BAGHDAD CITY,IRAQ A. A. Faraj* Assist. Prof. *Department of Parasitology, College of Veterinary Medicine, University of Baghdad , Iraq aazhar888@yahoo.com ABSTRACT The aim of this study to investigate the prevalence of Cryptosoridium spp. in domestic dogs in Baghdad city, by using microscopic examination(flotation and staining) and molecular techniques .The results revealed that the rate of Cryptosporidium infection was 15% by microscopic examination and 28.6% using PCR, our study showed that the infection rate was 8.5% in males and 21.42% in females by microscopic examination , while the infection rate by using PCR in males and females was 21.42%, 35.71% respectively. The percentage of infection in the age groups <1 year and >1 year was 20%, 10% respectively by microscopic examination, while by using molecular assay the rate of infection in the age groups <1 year was 40% and 17.14% in >1 year. This is the first study in Iraq to detect Cryptosoridium spp. in domestic dogs using molecular technique.


‫العراقية‬ ‫الزراعية‬ ‫العلوم‬ ‫مجلة‬
the fecal-oral route after the ingestion of contaminated food or water with oocysts (17,3), Diarrhea is the most important clinical signs of disease humans and animals (14). and dogs also act as reservoirs for a large number of parasitic zoonoses, including cryptosporidiosis (20,11). Diagnoses Cryptosporidium of infection in fecal samples of dogs is based by identifying the oocysts using microscopic techniques, identifying the parasite antigen by ELISA, or by pathogen DNA using PCR and the basic interest of last one compared with other assays is that the amplification products can be analyzed by sequencing analysis to define the species of the parasite (13,2). There is little information on the Cryptosporidium spp. present in infected dogs in Iraq. This study was conducted to determine the prevalence and molecular diagnosis Cryptosporidium species in domestic dogs in Baghdad city, Iraq.

MATERIALS AND METHODES Detection of Cryptosporidium oocysts
One hundred and forty fecal samples (10 g) from domestic dogs, of different sexes and ages referred that a small animal private clinics in Baghdad, Iraq. During the period from 1/1/2018 to 31/5/2018, analyzed by flotation methods and modified Ziehl-Neelsen staining (4,6). Finally the samples were saved at 2.5% potassium dichromate (K 2 Cr 2 O 7 ) (12), and stored at (-20 C °) until DNA was extracted. DNA Extraction and PCR DNA extraction procedure was performed using DNA stool kit (ABM, Canada) based on the manufacturer's guideline. By using for 18S ribosomal gene Cryptosoridium . design this primer by the (NCBI)gene-Bank data according to program(Primer3 plus online) (Table1, Figure 1) and negative samples 100 (71.4%). Also, PCR was done and showed 300 bp band which confirmed only 40 samples as Cryptosporidium spp., the specificity was 94.00 and the low sensitivity was( 37.50 ) which means that not useful for con firm the infection also the value of kappa (0.367) reflects a fair agreement between two test (Table 2, Figure 2,3).   Table 3 shows that the infection rate in males was 8.5% and in females 21.42% by microscopic examination , while the infection rate by using PCR in males and females was 21.42%, 35.71% respectively. The association between males and females was not significant

Phylogenetic Analysis
The BLASTIN analysis of the 18S rRNA gene to the our sample that isolated and sequences with the recorded strains that described in the databases using the MEGA 6.06. program. The evolutionary relationship was designed using molecular sequences in sequences in determining cluster due to variation in 18SrRNA monochromatic sequence. It can be stretched between 0.1.when analysis and compare with the NCBI-Gen bank Cryptosporidum parvum isolates that based on offered information with local isolate of Cryptosporidum parvum that recorded under accession number (MH716021.1) (Figure 4). Figure 4: Show the phylogenetic tree inferred the degree of relatedness between 18SrRNA sequence deposited in the international oligonucleotide primers used to amplify and sequence parasite 18S rRNA genes.
Bank sequence database(NCBI) of Cryptosporidium parvum as isolated from infected dogs under accession number (MH716021.1), sample based on a partial sequence of 18S rRNA Gene GenBank Cryptosporidium is a coccidian, intracellular protozoan parasite pathogen, which causes diarrheal illness of animals and humans (18). The present study showed that the rate of infection Cryptosporidium spp. oocysts in dogs fecal was 30% and 57.40% by using staining and molecular methods, respectively. This rate is a high than the results of some previous studies, Bahrami  Results revealed that the microscopic test has a low sensitivity %37. This is attributed to the experience of the examiner to observation of oocysts in the feces specimens , also microscopic examination does not distinguish between different species of parasites.; thus, molecular assay like PCR have been used with high sensitivity, specificity, and rapidly features are capable of differentiating between species and genotypes in different samples, although they are expensive.
Previously, genotyping of Cryptosporidium spp. has been done successfully by PCR method according to (18S rRNA) gene (12, 10 ,19). Our results indicate that the age may affect the rates of infection through the significant high prevalence of Cryptosoridium in the young dogs than in adult dogs using both methods. Similar research were reported in dogs (5,20). This could be attributed to lower resistance in the young compared to older animals. In otherwise, no statistical differences relationship was between the infection of Cryptosporidium and the sex of the dogs. Agree with two studies conducted in China and Brazil recorded similar findings (16,7). This refers that sex is not a major affecting Cryptosporidiosis infection in dogs. In the present study, a first phylogenetic analysis of C. parvum isolates in Iraq from infected domestic dogs, based on the 18S rRNA gene sequence showed that there were relationships between C.parvum local isolate ( MH716021.1) and other global isolates the local isolates were closely related to France isolate under accession number EF158462.1 with nucleotide sequence identity 91%. Furthermore, showed gradual similarities with the other isolates from the high grade to the lower indicated on identity between (91-92%).