Genotyping of cystic echinococcosis isolates from clinical samples of human and domestic animals

Cystic hydatid disease is a cosmopolitan important disease in both human and animals. Many strains were investigated in this parasite. The aim of study was to characterize genotype variations of Echinococcus granulosus isolates collected from human and domestic animals in Al-Qadisiyah province/ Iraq based on sequencing of nad1 mitochondrial gene. Eighty hydatid cysts of human (12), sheep (15), cattle (36), and camels (17) were collected from hospital and slaughter house of the province, during October 2014 to June 2015; microscopic examination was made for cysts fluid to determine the fertility. DNAs extraction was done for each sample in addition to purify and concentrate of extracted DNA samples was performed to determine nad1 (400bp) gene used conventional PCR method. Phylogenetic analysis was performed using NCBI-Blast Alignment identification and Unweighted Pair Group Method with Arithmetic Mean. Twenty five (10 from human and 5 from each studied animals) samples were chosen due to their fertility and high DNA purity, in which three strains (genotypes) were investigated including sheep strain (G1) 40%, buffalo strain (G3) 48% and camel strain (G6) 12%, where human samples related to G1(20%) and G3(80%); sheep samples related to G1(80%) and G3(20%); cattle samples related to G1(60%), G3 (20%) and G6 (20%); camels samples related to G1(20%), G3(40%) and G6(40%). The dominant strain is a buffalo strain (G3); both of buffalo strain (G3) and sheep strain (G1) represented the actual source of human infection. There is no host specificity of detected genotypes.


Introduction
Echinococcus granulosus is one of the most important zoonotic parasites that cause hydatid cysts in human and domestic animals, which called "dog small tapeworm"; it lives in the small intestine of dogs mainly (1).The adult worm required two hosts to complete its life cycle which are intermediate host like human and domestic animals and definitive host like canids (2).
Cystic echinococcosis disease is caused by the larval stage (hydatid cyst) of E. granulosus, the hydatid cyst develops after ingestion of eggs contain oncosphere embryo that shown by King and Fairley (3); the cyst characterized as unilocular filled with fluid surrounded by a two layers of hydatid cyst wall, nucleated inner germinal layer, where protoscolices grow, and a cellular outer laminated layer; these layers are surrounded by fibrous capsules of host (4,5).
Oku (6) and Al-Mutaywiti (7) referred to that the adult worm settles down in the mucosal layer of small intestine of definitive host; general life cycle of Echinococcus spp.occur through passing of gravid segment or free eggs by adult parasite with feces of definitive host, where the intermediate host ingested the eggs with contaminated food lead to develop of hydatid cyst containing protoscolices (fertile cysts).The cycle is completing if the definitive host eats the infected part of intermediate host then protoscolices grow to adult cestode in definitive host small intestine (8).Clinical signs of hydatid disease may occur after a highly variable incubation period of several months to years; hepatic cyst may cause abdominal pain and hepatomegaly while pulmonary cyst may cause chronic cough, dyspnea and expectoration (5,9,10).Romig (11) reported that hydatid cysts was cosmopolitan distribution.It remains public health threatened in endemic areas such as Mediterranean countries, North and East Africa, Western and Central Asia, China, South America and Australia.Diagnosis of the hydatid cyst in the infected animals do not explained, but most dependent detections during carcass inspection and at post mortem examination (12,13).
To date more sensitive molecular techniques are used for determination species and strains of E. granulosus (14).There are ten distinct genotypes (G1-G10) have been recorded in the world based on nucleotide sequence analysis of the mitochondrial cytochrome C oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) genes.
McManus and Thampson (18) recorded the most common geographic distribution around the world is sheep strain (G1 genotype); it is also dominant in the Mediterranean area.
Due to there is inadequate study in Iraq related to genotyping diversity and sequence variations of E. granulosus isolates from human and animals hydatid cysts, this study was designed and it`s aims were: characterize genotype variations of E. granulosus isolates from human and animal's hydatid cysts based on nad1 gene and determine the relationship between strains in relative countries.

Materials and methods
Eighty hydatid cysts of human (12), sheep (15), cattle (36) and camels (17) were collected from hospital and slaughter house of Al-Qadisiyah province during October 2014 to June 2015.Microscopic examination was made for cysts fluid to determine the cyst fertility through investigation of protoscolices which were rinsed three times with phosphate buffer saline (PBS).
DNA extraction was done for each sample by Genomic DNA extraction kit (Geneaid, USA), according to the company instruction; the purity and concentration of extracted DNA samples were analyzed by Nanodrop.The extracted DNAs were stored at -20 ○ C until used for PCR.
The mitochondrial nad1 gene was used for PCR amplification the primers were designed by (19) which provided by Bioneer Company, Korea.
PCR amplification was prepared by added of 20 µl including: 5 µl of DNA template, 1.5 µl (10 pmol) of each (forward) 5'-TAAAGAAAGAACATAATGAAAATG-3' and reverse 5'-CCATAATCAAATGGCGTACGAT-3' primers, 12 µl PCR water to the PCR tube of AccuPower PCR PreMix Kit(Bioneer, Korea) which contain other PCR reaction requirements (Taq DNA polymerase, dNTPs, Tris-HCl pH: 9.0, KCl, MgCl2, stabilizer, and tracking dye), were used to amplify a 400bp fragment of nad1 gene under the following conditions: initial denaturation at 94 ○ C for 5 min, followed by 30 cycles of denaturation at 94 ○ C for 30 second; annealing at 50 ○ C for 45 second; elongation at 72 ○ C for 30 second, then the final extension at 72 ○ C for 5 minute and holding at 4 ○ C. The PCR product of mitochondrial nad1 gene (400bp) was analyzed by agarose gel electrophoresis.
The PCR products were purified from agarose gel by using commercial purification kit (EZ EZ-10 Spin Column DNA Gel Extraction Kit, Biobasic, Canada) and sent to Bioneer Company in Korea for DNA sequencing by AB DNA sequencing system.
Phylogenetic analyses were performed based on NCBI-Blast Alignment identification and unweight pair Group Method with Arithmetic Mean Tree (UPGMA tree).

Results
Twenty five (10 from human and 5 from each studied animals) samples has been chosen due to their fertility and high purity DNA.
Among all analyzed samples the G3 strain recorded the highest percentage (48%) followed by the G1 strain (40%) with the lowest rate was appeared in G6 (12%).

Discussion
Genotyping of E. granulosus is the initial step in detection of parasite and controlling its virulence then minimize the infection by genotype determination.The results showed there are three common genotypes existed in Iraq depending on nad1 gene sequencer analysis, which are: sheep strain (G1); buffalo strain (G3) and camel strain (G6).Two strains were existed in human isolates which are: sheep strain (G1) and buffalo strain (G3); and the most predominant genotype that infects Iraqi people was G3 (80%).
The results mismatched the previous studies in Iraq such as (20) who detected G1 in human and animals where the result showed 100% matching sheep strain (G1), also (21) detected G1 strain in all human isolates in Turkey.Anyway, present result agreed with (16) in Italy; (22) in Romania and (19,23) in Iran.This may be attributed to the study encircled by a determinant such as the reality of high risk of surgery in such infections; many people prefer to visit more developed hospitals and might necessity to involvement a wide provinces in Iraq for accurate genotypes diagnoses.Two strains were existed in sheep isolates which are: sheep strain (G1) and buffalo strain (G3) depending on nad1 gene; G1 was the predominant (80%) genotype that infects sheep in Iraq.The result matches most of previous studies in Iraq such as (20,24) that sequenced nad1 gene and (25) who detected G1, G3 and G7 genotypes in Turkey.The reason may attribute to sheep obviously sensitive to sheep strain (G1) of E. granulosus; shortest life span of parasite depending on sheep slaughtering age in compare to other animals and the hydatid cysts in this intermediate host being predominantly fertile so the sheep is essential source of echinococcosis in dogs (26).
Three strains were existed in cattle and camels isolates which are: sheep strain (G1); buffalo strain (G3) and camel strain (G6) depending on nad1 gene.The G1 was the predominant (60%) genotype in cattle; the result matched (20,24) who detected G1 in all cattle isolates in Iraq whereas (27) detected G3 in all Indian livestock.G3 and G6 are the predominant genotypes in camel's isolates that which mismatched to the study of (28) in Tunisia who detected most of camels infected with sheep strain (G1) whereas ( 29) detected G1, G3, and G6 in camels isolates in Iran.
Phylogenetic analysis of Iraqi isolates showed that hydatid cysts were produced by G3, G1 and G6 respectively.This study indicated that commonly circulating genotype which cause hydatid cystic disease in Iraq was buffalo strain (G3) in general, but the result mismatched to (24) who referred to that G1 is the common genotype in Iraqi sheep, cattle and camels.
The commonest of G3 strain infectivity may be due to its highly fertility that aid in infects not typical host rather than other strains as proved by (27); additionally Al-Qadisiyah is agricultural province and existence of buffalo's breeders and settle down in such province then enhancement of buffalo strain to transmission easily to human and animals; likewise G6 could not be predominant strain mostly attributed to the fact of little use of camel's meat, thus providing little or no access for dog to camel carcass.
The result of phylogenetic analysis that based on nad1 gene revealed there is close relationship between strains that infects human, sheep, cattle and camels; so each strain do not specific to infects one host without another.
Results of this study indicated that G1 genotype detected in ten (40%) isolates and could be infective for human, sheep, cattle and camels that agreed with (25,(30)(31)(32).Bowles (33) explained when cattle were infected with sheep strain (G1) it be considered as accidental host and the cyst mostly infertile so they do not agreed with the present study.G3 strain represented of most of isolates (twelve isolates 48%).Studies on G3 strain revealed this genotype is most predominant strain in human, sheep, cattle and camels as reported by (34)(35)(36); but Grosso (37) disagreed with the current study and explained that G3 has no susceptibility among human.Pednekar (27); Sharbatkhori (29); Rostami (38) investigated that G3 genotype was detected in sheep, cattle and camels and it is the predominant strain in Iranian cattle and camels, while Capuano (39) detected G1 in Italy in most of buffalo's isolates, so G3 rarely infected its typical host.
G6 strain composed of few isolates (only three isolates 12%); the study agreed with the previous investigation in some points; such study of Fasihi-Harandi (40) that used PCR-RFLP method on the internal transcribed spacer (Its1) region and reviewed that camels' strains have a crosstransmitted between human, sheep, cattle and camels.Sadjjadi (1); McManus and Thompson (18) and McManus (41) also detected that G6 related to infection of studied hosts with hydatidosis; while (34) identified G6 in human isolates; furthermore (42,43) recorded G6 in sheep and cattle samples while recorded in camels also in addition to sheep and cattle by (44).In Egypt, all human, sheep, buffalo and camels isolates indicated to presence of G6 strain (45,46).The mitochondrial genes have more power than nuclear genes in reconstruction of the phylogenetic relationship among closely related species due to their rapid sequence evolution (44).Demonstrated of G3 was not exclusively infect its typical host (buffalo), but it can be considerably ingested by human and other animals.Higher frequency of strains with G3 genotype compared with other reports is of great concern that suggested human, cattle and camels as a new appropriate host for G3 genotype.G1, G3 and G6 have a possibility to transmission between livestock and human (47).
Relationship of detected genotypes in Iraq with others of relative countries indicated that the phylogenetic analysis of nad1 gene revealed that some of sheep, cattle and camels isolates related to Egyptian, Iranian and Moroccan isolates whereas other local isolates considered as unique isolates due to it is out of tree.Sheep strain is the worldwide predominant genotype among extent of intermediate hosts (48).In Ilam province, Iran, G1 have been detected by isolation of DNA from protoscolices of human, sheep and cattle hydatid cysts (49).G1 is the predominant genotype in Turkey; it is essential agent in human and animal hydatidosis as reported by (50); majority of sheep and cattle were infected with G1 strains in different Turkish regions (30).In Greece, G1 have been detected in sheep isolates (51).
Sadjjadi (1); Bardonnet (52) and Azab (53) detected in Africa and Middle East the sheep strain in human and animals, additionally common circulating genotype in Tunisia and Libya was G1 which be identified in camels also as investigated by (28,54).Zhong (55) collected of 45 hydatid cysts belongs to human and sheep in China and detected G1 in all samples when he used cytb gene; in contrast to Grosso (37) who could be find G1 and G6 as the predominant strains that infects human.
Buffalo strain (G3) detected in human and domestic animals in Iran whereas G1 was the common infective strain in them as reported by (23); while Sharbatkhouri (29) identified buffalo strains as a predominant in dromedaries that matched the present investigation.
G6 have been detected in human and animal hydatidosis in Iran (1,29,40) and in Sub-Saharan Africa where as it is proven as predominant strain in Egypt (45,46).
The concourse of these results with Iran and other lands may due to reality of these countries are neighboring to Iraq and shares borders overlapping naturally in addition to these countries specialized in breeding different species of animals to each other; therefore the pathogenic strains in human and animals are the same in these countries and this is analogous to that proven by in addition to there are no studies in other surrounding countries involving genotyping and phylogenetic analysis (21,56).

Figure 1 :
Figure1: Phylogenetic tree analysis based on nad1 gene partial sequence that used for E. granulosus genotyping detection of human isolates.The phylogenetic tree was constructed using Unweighted Pair Group method with Arithmetic Mean (UPGMA tree) in (MEGA 6.0 version).

Figure 2 :
Figure 2: Phylogenetic tree analysis based on nad1 gene partial sequence that used for E. granulosus genotyping detection of sheep isolates.The phylogenetic tree was constructed using Unweighted Pair Group method with Arithmetic Mean (UPGMA tree) in (MEGA 6.0 version).

Figure 3 :
Figure3: Phylogenetic tree analysis based on nad1 genes partial sequence that used for E. granulosus genotyping detection of cattle isolates.The phylogenetic tree was constructed using Unweighted Pair Group method with Arithmetic Mean (UPGMA tree) in (MEGA 6.0 version).

Figure 4 :
Figure4: Phylogenetic tree analysis based on nad1 genes partial sequence that used for E. granulosus genotyping detection of camel isolates.The phylogenetic tree was constructed using Unweighted Pair Group method with Arithmetic Mean (UPGMA tree) in (MEGA 6.0 version).

Figure ( 5 )
Figure (5): Phylogenetic tree analysis based on nad1 gene partial sequence that used for E. granulosus host relationship study.The phylogenetic tree was constructed using Unweighted Pair Group method with Arithmetic Mean (UPGMA tree) in (MEGA 6.0 version).E.g = E. granulosus, H = human, S = sheep, C = cattle, CM = camel, 1-5 samples numbers.

Figure ( 6 )
Figure(6): Phylogenetic tree analysis based on nad1 genes partial sequence that used for E. granulosus genotyping detection relationship to other relative countries.The phylogenetic tree was constructed using Unweighted Pair Group method with Arithmetic Mean (UPGMA tree) in (MEGA 6.0 version).E.g = E. granulosus, H = human, S = sheep, C = cattle, CM = camel, 1-5 samples numbers.

Table 1 :
The genotypes of E. granulosus in human and animals (sheep, cattle and camels) using partial sequence of nad1 gene according to phylogenetic tree analysis and NCBI-BLAST alignment tool H: E. granulosus Human, EG.S: E. granulosus Sheep, EG.C: E. granulosus Cattle, EG.CM: E. granulosus Camel