Prevalence of dog's Echinococcus granulosus with study of the Genetic Variability of Antigen B2

Prevalence of dog's Echinococcus granulosus with study of the Genetic Variability of Antigen B2 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Prevalence of dog's Echinococcus granulosus with study of the Genetic Variability of Antigen B2 Noor Jarad, Saba Klaif, Noaman A'aiz This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7736067/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Echinococcosis is caused by infection with tapeworms, represented by Echinococcus granulosus. The antigen B is a main protein produced by the metacestode of this parasite and is a member of a family of genes that help evade the host's immune system. There are many different types of E. granulosus (G1-G10), each with its own unique set of biological traits.in this study we investigate of sequencing and polymorphism of AGb2 of Echinococcosis in final host of dog. Two hundred and seventy dog ​​feces samples were examined microscopically to detect the parasite eggs, and then DNA extraction was done for each positive sample, where conventional PCR technique used specific AgB2 gene primer and gene sequencing to detect E. granulosus strains. The results of the microscopic examination indicated that 24 (8.8%) of the cases were positive (13 males and 11 females), including 8 puppies and 16 adults. That 17 out of 24 succeeded in amplifying the DNA and two sequences were submitted and gotten accession number OP272485.1, OP272486.1 and aligned with group of AGB2 groups. The results showed AgB2 play functional genes exclusively in dog as final host of E. granulosus , suggesting inter-strain variation of this gene. Echinococcus granulosus AgB2 dog Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Echinococcus granulosus , also known as the "dog small tapeworm," affect humans and domestic animals ( 1 ). Transmission of E. granulosus occurs primarily within a domestic cycle, with dogs serving as the definitive host and ungulates acting as the intermediary host ( 2 ). Definitive hosts typically show no symptoms of infection, though pruritus may emerge if gravid proglotiids gain access to the anal glands ( 3 ). The onset of symptoms in the intermediate host is determined by the size and location of the cyst within the infected organ, the impact of the cyst on the organ and adjacent tissues, and any complications brought on by the cyst's rupture or a secondary infection ( 4 ). Most cases are discovered in adults because the parasite grows so slowly ( 5 ). Depending on the conditions, taeniid eggs can remain infectious for up to a year after being laid if stored between 4 and 15 degrees Celsius (39 and 59 degrees Fahrenheit) ( 6 ). There is a higher incidence of infection during the rainy season because canine feces are more likely to be spread over grasses by the rain. Other contributing variables include humidity and temperature ( 7 ). Antigen B (AgB) is a lipoprotein produced by E. granulosus larvae. It is synthesized by the protoscolices tegumental cells and, in smaller quantities in both the laminated and the germinal layers of the brood capsules ( 8 ). AgB is a thermostable lipoprotein that separates into three bands at 8, 16, and 24 kDa when run through SDS-PAGE ( 9 ). It is widely used in the immunodiagnosis of cystic Echinococcus (CE) is considered one of the common proteins in hydatid cyst fluid (HCF) ( 10 ). In patients with progressive CE, it plays a role in immune evasion with reason of its capacity to suppress elastase enzyme activity and neutrophil cells recruitment and to induce a T-helper 2 (Th2) cell responses associated with immunopathology ( 11 ). AgB varies in composition and grade depending on the host species and geographical location. AgB proteins are capable to antigenic variation, which increase the impact of their practical application, as indicated by Rosenzvit et al. ( 10 ). Because of this, it is imperative to always consider the possibility of conducting a serologic evaluation of prepared AgB in various E. granulosus hosts at endemic regions or nations using local facilities ( 12 ). Five different gene regions together make up the AgB gene family: AgB1, AgB2, AgB3, and AgB4 ( 16 ). In conclusive hosts, diagnosis can be made through coprology, necropsy, purging, and serology ( 17 ). Egg detection and differentiation from dog to canine, or from dog to feces, is a crucial diagnostic challenge ( 18 ). The eggs of Echinococcu s and other Taeniidae species look so identical those conventional carpological techniques of feces as concentration methods cannot tell them apart ( 19 ). There has been widespread use of necropsy and purgation with arecoline compounds in dogs, but these procedures can be dangerous, take a long time, and should only be performed by experts. Other methods exist that use polymerase chain reaction to identify infectious copro-DNA molecules ( 20 ). Echinococosis have been identified using numerous methods, including restriction fragment length polymorphism (RFLP PCR) and Real time PCR ( 21 ). Eggs were analyzed with a multiplex-PCR for the presence of Echinococcus ( 22 ). This study was conceived due to a lack of epidemiological data of this parasite in the province of Al-Diwaniyah and lack of sequence variation studies involving Echinooccus granulosus in dogs. Materials and Methods Ethical approval Ethical approval for conducting this research was obtained with the approval of the Scientific Research Ethics Committee at the College of Veterinary Medicine / University of Al-Qadisiyah, pursuant to approval letter numbered 1890 on 28/8/2023. Samples collection The current study included the detection of infection with the parasite E. granulosus in a number of stray dogs in Al-Diwaniyah region / Iraq, 180 km south of Baghdad. Fecal samples of 270 dogs were collected, from 143 males and 127 females, of which 106 were puppies and 164 adults. Stool samples were collected in a dry plastic bags and transport with cold conditions to the laboratory of parasitology in College of Veterinary Medicine / university of Al-Qadisiyah for to a perform the required examinations. Microscopical study The floatation method was adopted using Shether solution, where 2-4 grams of each fecal sample were mixed with 10 ml of distilled water until emulsion was obtained. The mixture was filtered through two layers of gauze. Then the precipitate was placed in a test tube and centrifuged at 3,000 rpm for 3 min. The supernatant was poured off and Sheather's sugar solution was added to the precipitate. The tube was filled close to the tube nozzle and the mixture was mixed well with a wooden stick. Then it was transferred to the centrifuge at 3,000 rpm for 2 min. The tube was placed vertically in the tubes rack and drops of sugar solution were added until the tip. The cover slip was placed on it to touch the sugar solution and left for 10 min. Then it was gently lifted and placed on the glass slide and examined under the microscope at 10x and 40x magnifications to scan the E. granulosus eggs (34). DNA extraction In order to obtain genomic DNA from feces samples, Stool DNA extraction Kit (Bioneer, South Korea) was used, according to the manufacturer information. The purity of extracted DNAs was double-checked with a Nano drop spectrometer, and then gDNAs were frozen at -20 degrees Celsius until use in a PCR technique. PCR study Primers targeting the AgB2 gene in Echinococcus granulosus were used in a polymerase chain reaction (PCR) test. The primers were developed using the NCBI Gene-Bank database and the web application Primer 3 plus, based on the sequence of Echinococcus granulosus species (GenBank: AY569356.1). (Bioneer company, Korea). The 400bp of the Antigen B (AGB) Antigen gene in Echinococcus granulosus . was amplified using the primers F5′GGATCCTTCGTGGCCGTCGTTCAAGC3 and-R-primer (5′TCGACAA ATCA TGTGTC CCGACGCA). (23). The PCR reaction construction was prepared in a total volume of 20 µl by adding 5 µl of extracted DNA, 1µl (10 picomoles) of each forward and reverse primers were added to the PCR master mix tube which (AccuPower® PCR PreMix, Bioneer, Korea) , then 13 µl of deionized PCR water were added to obtain the total volume. The construction was briefly mixed by exispin vortex. A thermocycler (Mygene Bioneer. Korea) was used to carry out the reaction, with the initial denaturation temperature set to 95 °C for 5 minutes, followed by 30 cycles, each one with denaturation at 95 °C for 30 seconds, annealing at 58 °C for 30 seconds, extension at 72 °C for 1 minute, and final extension at 72 °C for 5 minutes. Electrophoresis in 1% agarose gel, staining with ethidium bromide, and visualization under UV light visible the PCR results. Gene sequencing and phylogenetic study The AgB2 genome was sequenced, and a phylogenetic tree was constructed to determine the various strains of E. granulosus . DNA sequencing was done on the AgB2 gene PCR product (400 bp) by the Bioneer Company /SouthKorea using the AgB2 forward primer (AB DNA sequencing system). Phylogenetic analysis was carried out using Neighbor-Joining and NCBI-Blast Alignment recognition for tree construction (Mega X). Results Microscopical results The infection rate with suspected Echinococcus parasite were detected in 24 (8.8%) out of 270 examined fecal samples. Eggs appeared surrounded by two envelops separated by septa, the egg envelope looks thick and radially striated with a six-hooked oncosphere, Figure (1). The results of the current study indicated that the infection rate in male and female dogs was relatively close and with no significant difference (P>0.05), (Table 1). Table (1): Infection rate of suspected E. granulosus according to the sex The highest prevalence rate of infection with E. granulosus (9.75%) was in adults in compare to the puppies infection rate (7.54%) with significant differences (P<0.05) between these two age groups, (Table 2). Table (2): Infection rate of suspected E.granulosus according to age. P value X 2 Percentage (%) No. of infected No. of examined Age group 0.020 5.333 7.54 8 106 Puppies 9.75 16 164 Adult 8.8 24 270 Total PCR results Out of 24 microscopic positive samples, 17 samples revealed positive results when successful to amplifying their extracted DNA to 400bp through polymerase chain reaction technique to confirm the infection with E. granulosus (Figure 2). The DNA sequencing has been done for two clones to gotten accession number OP272485.1 andOP272486.1.Phylogenetic tree revealed that the two strains alignment with AY314986.1 with identity 99.86% which related to the E.granulosus that have AgB2 groups Ag B 2 were present as functional genes exclusively dog as final host of E.granulosus Gene sequencing and phylogenetic results It was observed through phylogenetic analysis that there is a genetic difference in the EgAgB family genes. Evolutionary genetic analysis revealed genetic variation in the EgAgB family. This was achieved after studying the evolutionary tree of the amino acid sequences involved in the construction of EgAgB proteins, using MEGA X methods to reveal the genetic relationship between these proteins found in species belonging to the genus Taenia. The results revealed a very similar genetic evolutionary pattern. Analysis of phylogenetic tree revealed highly bootstrap values for deeper nodes (Fig. 3), as anticipated due to the similarities among sequences of different subfamilies, and different other species. Multiple sequence alignment showed the variation of amino acid between groups of AgB family as shown in figure (4, 5). The two strains OP272485.1 and OP272486.1 show mutation with reference strain AY314986.1. at position 54 (C-T) and 75 (A-G) for first strain OP272485.1 ,while OP272486.1 show one mutation at 54 (C-T) with reference strain as in figure(5) and table (6). Both OP272485.1 and OP272486.1 share the cytosine C→T thiamin mutation at position 54 . And OP272485.1 has an additional A→G mutation at position 75 The mutations appear to be synonymous mutations or located in non-coding/5′ regions . Table (6) Main mutation and substitution of two strain and position with type of mutation . Discussion In Iraq, there were few reports of canine echinococcosis; however, this study was designing to obtain some aspects related to this disease (24). The current research focused on rate of spread of Echinococcus granulosus in dog. The current research focused on microscopically examination and parasite suspected eggs detection, molecular examination for infection confirmation and parasite strain detection and prevalence study in related to some risky factors in Al-Diwaniyah province. Through the current results confirmed by the PCR examination, which consider the end in diagnosis of disease, it was noted that the prevalence of the parasite E. granulosus in dogs in Al- Diwaniyah province was 6.29%. The male dogs had a higher prevalence (9.09%) than the females (8.66%) and in the adults were higher (9.75%) than in puppies (7.54%).The relatively high rate of infection may be attributed to slaughtering outside slaughterhouses without veterinary supervision, where considered one of the important sources of echinicoccosis among dogs (25). The movement of infected dogs between different regions may also increase the presence of infection in non-endemic areas. In the present study, the molecular diagnosis of E. granulosus by AGB2 was important in diagnosis of disease and focused of type of AgB2 more prevalence than other type of this antigen. The parasite larvae secrete a type of lipoprotein known as antigen B into the hydatid fluid. Previous studies have identified eight individual units of this antigen (antigen B1 - antigen B8), (26). It is the most reliable immunological antigen in diagnosing the genus Echinococcus granulosus. Its weight is 230 KD, and polar and neutral lipids constitute 50% of its mass, including fatty acids (FA) and sterols, which cannot synthesize by Echinococcus . This has led to emphasize its hypothetical role in parasite lipid metabolism, taking up host lipids as building blocks for parasite metabolic demands (27). Moreover, this hypothesis is supported by the fact that AgB belongs to a cestode-specific family of proteins exhibiting ability to bind hydrophobic ligands (HLBP for hydrophobic ligand binding protein) (33). This family arose through independent gene expansion events, resulting in genetically distinct monophyletic species and subspecies. Interestingly, all HLBP members are immunodominant. However, genetic diversity was observed among these Agb2 isolates from other populations using sequencing and phylogenetic tree analysis. Genetic distances were not correlated with geographic distances, indicating local divergent evolution rather than isolation by distance. In conventional PCR, 17 of the 24 positive cases were diagnosed. The high acceptance rate among researchers in Iraqi Kurdistan, who focused on stray dogs (28).The genomic and phylogenetic tree confirmed that EgAgB2 local strain was 99.68%,99.63% identical to world AgB2 with AY314986.1. GU166200.1 respectively from Brazil and Australia respectively. EgAgB is a polymorphic gene, likely dependent on host type for adaptation, as E. granulosus strains are often specific to their intermediate hosts (29). Some studies have indicated that E. granulosus AgB has a high degree of genetic diversity. Differences in the gene sequences of E. granulosus isolates encoding the five AgB subunits may affect the diagnostic performance of the proteins of these subunits. Therefore, understanding the similarities in AgB genes may contribute to the development and application of AgB-based diagnostic assays (30). Molecular analysis of E. granulosus isolates revealed differences in the EgAgB nucleotide sequences, resulting in variants in the amino acid sequence. (29) Given the positive diagnostic performance of the EgAgB2 subunit, we sought to identify potential polymorphisms in the gene encoding this antigen in different E. granulosus isolates. In our search, we discovered that all EgAgB82 sequences from dogs were closely related to AgB5 (accession number GU166215.1), isolated from the kangaroo host of E. granulosus from Australia with 99.43% identity . While AgB3 and AgB2 (accession numbers GU166214.1 and GU166204.1) from E. granulosus from China were isolated from dogs with AgB, 3, and 2 . Overall, this study has provided important information about the composition, abundance, and assembly of AgB2 subunits, OP272485.1 and OP272486.1 two strain show polymorphism at position 54 (C-T) and 75 (A-G) for first strain OP272485.1 ,while OP272486.1 show one mutation at 54 (C-T) with reference strain (Accession no.: AY569356.1)greatly expanding our understanding of AgB. Conclusion This study focused on polymorphism of two strain We were able to structurally characterize the oligomers formed by different AgB subunits and correlate them with those formed by AgB subunits from E. granulosus using reconstituted subunits.this polymorphism makes genetic diversity and immunological of epitopes that make of change of main humoral immunity Although further research is needed to fully understand the structure of full genome of AgB, our findings will help understand the roles of AgB in host-parasite interactions during Echinococcus infection (31, 32). 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06:47:31","extension":"xml","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":85417,"visible":true,"origin":"","legend":"","description":"","filename":"1b11198c14e643e790d6d57cff6d0b161structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7736067/v1/0e5af3d0a42d4176d58458dc.xml"},{"id":93556878,"identity":"b8bc0bae-1d55-4656-a5c1-a8db472ccff7","added_by":"auto","created_at":"2025-10-15 06:47:31","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":95697,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7736067/v1/80e0bba781ac00972a2f229d.html"},{"id":93556858,"identity":"afaab2f2-6e2e-42c6-aeed-58e6abc8d51d","added_by":"auto","created_at":"2025-10-15 06:47:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":419533,"visible":true,"origin":"","legend":"\u003cp\u003emicroscopic examination of egg of \u003cem\u003eE.granulosus\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7736067/v1/059ab8e7205eebc71c8adf3d.png"},{"id":93558451,"identity":"749e8f67-a8b1-49f1-9af3-9bb66ed1b665","added_by":"auto","created_at":"2025-10-15 07:03:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":290617,"visible":true,"origin":"","legend":"\u003cp\u003eFigure (1): Electrophoreses image of 1% agarose gel with the PCR products of AgB2 gene from of \u003cem\u003eE.granulosus\u003c/em\u003e .M marker is the ladder (100-2000), Lanes (1 to 6) are positive samples at 400bp.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7736067/v1/0b350e9620b373dd175fb3e3.png"},{"id":93556861,"identity":"bfca3502-94ca-4a5c-bc5f-c066b1442252","added_by":"auto","created_at":"2025-10-15 06:47:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":663754,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure (4) Phylogenetic tree of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE.granulosus aligned with \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eworld strains.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7736067/v1/3843f19f930b4c2697b9d5a2.png"},{"id":93556864,"identity":"3f0dea08-268a-4fed-b5cd-e4525629ba15","added_by":"auto","created_at":"2025-10-15 06:47:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1838447,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure (5) Main mutation of OP272485.1 and OP272486.1 with reference AY314986.1.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7736067/v1/66ab33460cf86f824d8f0ea1.png"},{"id":93924476,"identity":"ad4fcf4d-5633-4b4c-a0c7-4b6b8ad226b2","added_by":"auto","created_at":"2025-10-20 10:17:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4340164,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7736067/v1/d2c8ffa2-ecdd-4f0b-aa02-de5fe0b9292e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Prevalence of dog's Echinococcus granulosus with study of the Genetic Variability of Antigen B2","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eEchinococcus granulosus\u003c/em\u003e, also known as the \"dog small tapeworm,\" affect humans and domestic animals (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Transmission of \u003cem\u003eE. granulosus\u003c/em\u003e occurs primarily within a domestic cycle, with dogs serving as the definitive host and ungulates acting as the intermediary host (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Definitive hosts typically show no symptoms of infection, though pruritus may emerge if gravid proglotiids gain access to the anal glands (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The onset of symptoms in the intermediate host is determined by the size and location of the cyst within the infected organ, the impact of the cyst on the organ and adjacent tissues, and any complications brought on by the cyst's rupture or a secondary infection (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Most cases are discovered in adults because the parasite grows so slowly (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Depending on the conditions, taeniid eggs can remain infectious for up to a year after being laid if stored between 4 and 15 degrees Celsius (39 and 59 degrees Fahrenheit) (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). There is a higher incidence of infection during the rainy season because canine feces are more likely to be spread over grasses by the rain. Other contributing variables include humidity and temperature (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAntigen B (AgB) is a lipoprotein produced by \u003cem\u003eE. granulosus\u003c/em\u003e larvae. It is synthesized by the protoscolices tegumental cells and, in smaller quantities in both the laminated and the germinal layers of the brood capsules (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). AgB is a thermostable lipoprotein that separates into three bands at 8, 16, and 24 kDa when run through SDS-PAGE (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). It is widely used in the immunodiagnosis of cystic Echinococcus (CE) is considered one of the common proteins in hydatid cyst fluid (HCF) (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). In patients with progressive CE, it plays a role in immune evasion with reason of its capacity to suppress elastase enzyme activity and neutrophil cells recruitment and to induce a T-helper 2 (Th2) cell responses associated with immunopathology (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). AgB varies in composition and grade depending on the host species and geographical location. AgB proteins are capable to antigenic variation, which increase the impact of their practical application, as indicated by Rosenzvit \u003cem\u003eet al.\u003c/em\u003e (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Because of this, it is imperative to always consider the possibility of conducting a serologic evaluation of prepared AgB in various \u003cem\u003eE. granulosus\u003c/em\u003e hosts at endemic regions or nations using local facilities (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Five different gene regions together make up the AgB gene family: AgB1, AgB2, AgB3, and AgB4 (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn conclusive hosts, diagnosis can be made through coprology, necropsy, purging, and serology (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Egg detection and differentiation from dog to canine, or from dog to feces, is a crucial diagnostic challenge (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). The eggs of \u003cem\u003eEchinococcu\u003c/em\u003es and other Taeniidae species look so identical those conventional carpological techniques of feces as concentration methods cannot tell them apart (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). There has been widespread use of necropsy and purgation with arecoline compounds in dogs, but these procedures can be dangerous, take a long time, and should only be performed by experts. Other methods exist that use polymerase chain reaction to identify infectious copro-DNA molecules (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Echinococosis have been identified using numerous methods, including restriction fragment length polymorphism (RFLP PCR) and Real time PCR (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Eggs were analyzed with a multiplex-PCR for the presence of \u003cem\u003eEchinococcus\u003c/em\u003e (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThis study was conceived due to a lack of epidemiological data of this parasite in the province of Al-Diwaniyah and lack of sequence variation studies involving \u003cem\u003eEchinooccus granulosus\u003c/em\u003e in dogs.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval for conducting this research was obtained with the approval of the Scientific Research Ethics Committee at the College of Veterinary Medicine / University of Al-Qadisiyah, pursuant to approval letter numbered 1890 on 28/8/2023.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSamples collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe current study included the detection of infection with the parasite \u003cem\u003eE. granulosus\u003c/em\u003e in a number of stray dogs in Al-Diwaniyah region / Iraq, 180 km south of Baghdad. Fecal samples of 270 dogs were collected, from 143 males and 127 females, of which 106 were puppies and 164 adults. Stool samples were collected in a dry plastic bags and transport with cold conditions to the laboratory of parasitology in College of Veterinary Medicine / university of Al-Qadisiyah for to a perform the required examinations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicroscopical study \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe floatation method was adopted using Shether solution, where 2-4 grams of each fecal sample were mixed with 10 ml of distilled water until emulsion was obtained. The mixture was filtered through two layers of gauze. Then the precipitate was placed in a test tube and centrifuged at 3,000 rpm for 3 min. The supernatant was poured off and Sheather\u0026apos;s sugar solution was added to the precipitate. The tube was filled close to the tube nozzle and the mixture was mixed well with a wooden stick. Then it was transferred to the centrifuge at 3,000 rpm for 2 min. The tube was placed vertically in the tubes rack and drops of sugar solution were added until the tip. The cover slip was placed on it to touch the sugar solution and left for 10 min. Then it was gently lifted and placed on the glass slide and examined under the microscope at 10x and 40x magnifications to scan the \u003cem\u003eE. granulosus\u003c/em\u003e eggs (34).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to obtain genomic DNA from feces samples, Stool DNA extraction Kit (Bioneer, South Korea) was used, according to the manufacturer information. The purity of extracted DNAs was double-checked with a Nano drop spectrometer, and then gDNAs were frozen at -20 degrees Celsius until use in a PCR technique.\u003c/p\u003e\n\u003cp\u003ePCR study\u003c/p\u003e\n\u003cp\u003ePrimers targeting the AgB2 gene in \u003cem\u003eEchinococcus granulosus\u003c/em\u003e were used in a polymerase chain reaction (PCR) test. The primers were developed using the NCBI Gene-Bank database and the web application Primer 3 plus, based on the sequence of \u003cem\u003eEchinococcus granulosus\u003c/em\u003e species (GenBank: AY569356.1). (Bioneer company, Korea). The 400bp of the Antigen B (AGB) Antigen gene in Echinococcus \u003cem\u003egranulosus\u003c/em\u003e. was amplified using the primers F5\u0026prime;GGATCCTTCGTGGCCGTCGTTCAAGC3 and-R-primer (5\u0026prime;TCGACAA ATCA TGTGTC CCGACGCA). (23). The PCR reaction construction was prepared in a total volume of 20 \u0026micro;l by adding 5 \u0026micro;l of extracted DNA, 1\u0026micro;l (10 picomoles) of each forward and reverse primers were added to the PCR master mix tube which (AccuPower\u0026reg; PCR PreMix, Bioneer, Korea) , then 13 \u0026micro;l of deionized PCR water were added to obtain the total volume. The construction was briefly mixed by exispin vortex. A thermocycler (Mygene Bioneer. Korea) was used to carry out the reaction, with the initial denaturation temperature set to 95 \u0026deg;C for 5 minutes, followed by 30 cycles, each one with denaturation at 95 \u0026deg;C for 30 seconds, annealing at 58 \u0026deg;C for 30 seconds, extension at 72 \u0026deg;C for 1 minute, and final extension at 72 \u0026deg;C for 5 minutes. Electrophoresis in 1% agarose gel, staining with ethidium bromide, and visualization under UV light visible the PCR results. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene sequencing and phylogenetic study \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe AgB2 genome was sequenced, and a phylogenetic tree was constructed to determine the various strains of \u003cem\u003eE. granulosus\u003c/em\u003e. DNA sequencing was done on the AgB2 gene PCR product (400 bp) by the Bioneer Company /SouthKorea using the AgB2 forward primer (AB DNA sequencing system). Phylogenetic analysis was carried out using Neighbor-Joining and NCBI-Blast Alignment recognition for tree construction (Mega X).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eMicroscopical results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe infection rate with suspected Echinococcus parasite were detected in 24 (8.8%) out of 270 examined fecal samples. Eggs appeared surrounded by two envelops separated by septa, the egg envelope looks thick and radially striated with a six-hooked oncosphere, Figure (1).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The results of the current study indicated that the infection rate in male and female dogs was relatively close and with no significant difference (P\u0026gt;0.05), \u0026nbsp; \u0026nbsp; \u0026nbsp;(Table 1).\u003c/p\u003e\n\u003cp\u003eTable (1): Infection rate of suspected \u003cem\u003eE. granulosus\u003c/em\u003e according to the sex\u003c/p\u003e\n\u003cp\u003e\u003cimg 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v6T0GzdupDQCN/KVNF6fr/ztZP/YtjhODhw40Aw6OU7u3r2b0llHXk8Myv51o+Puks2bN6fxlStX0hi0ThBcaPDs3LkzHaBVyncDWLx4cePIkSNpes2aNc2KUfVoVT7nzp0rphqpDCgLDYbHjx+ni0E4ceJEusiVXbp0qbF///40zUWRiwHBYqfvH3VxwV2/fn0ac0NDXpSx3A8//JDu2ssXaS62nB8E4rnvv/++GYh88cUXjc8++yxNz6VO92/Hjh3NZTZu3JiGEPUAr4+Pj6dpDML+davjIGPPnj1pfOHChTTG9evXG19//XUx98HZs2c76kKhVSSWOXz4cEord7+o/3788cdmQBHyOwRezy92mjtRDlRUVDp5uWh+vX79elqA/sknnxRT03GnuWTJkmLufZny3k7fP+r+97//pcArkA/lYAHc+MTdPC1A0apHHp4/fz5NR5dCiPMl8jku4nOp0/3Lz21aLrjxQJ5OMBWtNBiE/etWx0HG9u3bi6n3gUArBBTHjh1r7Nu3r3Hz5s2URjBShQiNLhS6WQheCDrofok0W0n6jxN19+7dxdx0HLicsJzAPg8wf6hYqFRpJi3fwWn+cNdInZYHCq1UBRDdvH/U5RfSVmIZLqQEGnQtgBYhrh0E4QQhTOf1FV0Iy5cvT60g89Xd2Mn+ge2LOrfc9RHpecCCQdi/bnQcZCCeqSAQICBYu3Ztms/x7AUBAi0c0cxDBF8WgQoBSbRYPHr0KLWYkMYDp0+fPk3p6h8i5lbRLycGJy3PZFAZeoGbH7QiUQ5UJFXNrJofnDd0d1AuXAAIBLvpVpzt+xcy8i4utlxPqKvizp/gI28poJU2go8ITAYV+8C2clwQNORIJ7jiuZ48mBim/UNXQcbWrVvT+Pbt2+nZjHXr1qX5MgIEoq1oyZhJtFowHDp0KLWYME20b3dJf3GgEhlTwTGACq8cDXPgR4Co+fPrr78WUxoUEQAygD7yMgIHmswDdVn0nXfy/lFHXtClFN68edNRsBWtA3RFddIaNF9dvr3sX6ttjcC0ynztX7e6CjJopYgHQPM+xxwtHAQhmzZtSi0TraxevTqNL168mMa8j2c5CFCYjlYMptUfETXnlRxRctwRBE5g7gxatXhobvAAod/yGTycHwTp3GVWNYvTHRkPdNIaWD6X2r1/1EVeREspd+OtunADN0LxzUXqq7ylNX/QNsc3U6rS69br/rUKJrgxLNfRmK/969rUxSZh8t27d/8apoKK9BrDVMGmgenJycl/jh8/3nwthv/+97/N6X379jWnp+7KmtMMrHsqwGjO8zmk8Z74zEhzqB7Io1amKrBm3rZajvSpICNNTx3gzWWXL1+e0jQ7rfIdVeVDWeRpExMTKV1zK8qjjPKIsikbHx9PQzh58mRz2TjHZnr/KJppP/NjnbwKeT7mdRLTufz8yV+LNIZ8vXXgM1rpZP+oZ2OZfB/yY4chl6fXvX+9Km+zv10yxOhOKopPA4hzyvIZPpZbf4x6PnqcVCvnS1fdJZIkSZ0yyJAkSbUwyJAkSbUwyJAkSbUwyJAkSbUwyJAkSbUwyJAkSbUwyJAkSbUwyJAkSbUwyJAkSbUwyJAkSbUwyJAkSbUwyJAkSbUwyJAkSbUwyJAkSbUwyJAkSbUwyJAkSbVY9M+UNLFoUUqQJKkfisvLSOKaOcr716tyvkwLMt69e5cSNRw++ugjD/IBZiU0nCy3/hj1fPQ4qVbOF7tLJElSLQwyJElSLQwyRtjly5dT01Xu/v37KY1hxYoVRep7kR7DwYMHi1dUh6ryCaTzuiQNM4OMEcUFateuXcXcBydOnEj9ZdFnFoEEy0c6w8TERGPjxo3pNfVfq/JBq8BDkoaNQcaI2rlzZ+PevXvF3Ac3btwopt4HHC9evEjTa9asSeNw9+7dtA7Vo1X5gCBv+fLlxZwkDS+DjAVu2bJl08Yhgg9JknplkLGA0Vrx/fffF3Mf8NzGN998U8xJktQbg4wFikCCZy7KLRi4dOnSv7pPJEnqlkHGAkRXCM9jzPTMRVXwIUlSNwwyFqAvv/yy+QAo33KgVSMwPTY2VsxJktQ7g4wRRbCwYcOGNB1fiSSN6efPn6cxw9GjRxvr169Pr4Oukh07dhRzqktV+QT+fwllxFdc/V8ZkoaZv10yxPztksHGOWX5DB/LrT9GPR89TqqV88WWDEmSVAuDDEmSVAuDDEmSVAuDDEmSVAuDDEmSVAuDDEnqUHwNnIGvGrfDMixb/iryqVOnUvqWLVuKFGk0GWRIUof43yb8ei5f0SOAIFioEsHI/v3707L5f9flfb/88ktKz38VWRpFBhmS1IXFixen8Uz/ej+CkSNHjhQp7x08eDD9x91nz54VKdJoM8iQpA4dOHAgBQq4devWv4II0LoxPj6efsmY1oxYnt8MOn/+fJomnUEadQYZktShc+fOpTEBQqvWiDt37qRWDoIQ/j08gQXPZDx+/DgFH/z6MV0lTPtMhkadQYYkdYhggd/7OXnyZAo0aJ0oI/jYvXt3CjQYaP14/fp1GpiP5zNYj90mGnUGGZLUAQIKfrSOHxSkm4RAI7pCcjzY+ebNm2LugyVLllQGJdIoM8iQpC7wzZFQ9fAnz2LQShHoLuGXjWnBuHnzZvP9/OIx3z6RRplBhiR1gIBiYmIifXOErhK+hhrPaPBsRTxfQTDBN0ji4U6+ZRLBCNPxflQ9OCqNEn/qfYj5U++DjXPK8hk+llt/jHo+epxUK+eLLRmSJKkWBhmSJKkWBhmSJKkWBhmSJKkWBhmSJKkWBhmSJKkWbYOMbdu2pa9KVg2HDx8ulqpHfM7Zs2eLFEmSNCzaBhnXrl1rHD9+PE3v27cv/S8NhsnJyZRWp4sXLxZTkiRp2PTcXTI2NtY4ffp0MSdJkjRdT0EGXSg5ujOia+Pq1auNV69eTevqiOmHDx82u1/yrhbeE8vEclXy9cY2sGz+XpaRJEnzr6sg48KFC+lCnv+SIBf5Y8eOpR/+Ydi7d29q5YguFro8omtlfHw8/a/+zZs3p3UF3kNaLHflypU0Lvvqq6+aXTZsA8EJy0Ya65AkSYOhqyAjLub5Lw9GQEAAwYC8JYIAJBAErFu3rph73zIB1nnmzJnGypUr0/zLly/TOMc6SY9Ah+kHDx40vv7665S2atWq9PwIAY4kSZp/PXWXcDEvoxWDYIEhDyQ6QdcJrRSdPEyaP3zKMyF8FtMEPnaXSJI0OHp+8DN8/vnnaRwtGjyD0eqZiioEBbRE0Mrx5MmTIvXfInCJbhbeR3DCQLcJgc/SpUtnXIckSZo7bX/qnQcsb9++Xcw10oX86dOnxdx7XOjj4s+zGFu3bm12fYDWh3id90d3CIEFwQEtEMiX41kOntUItHK8ffu22SUT2xEPkPK+qm0bZeSbPzU8uPwp6OFkufXHqOejx0m1cr60DTI0uAwyBpuV0HCy3Ppj1PPR46RaOV9m3V0iSZJUxSBDkiTVwiBDkiTVwiBDkiTVwiBDkiTVwiBDkiTVwiBDkiTVwiBDkiTVwiBDkiTVwiBDkiTVwiBDkiTVwiBDkiTVwiBDkiTVwiBDkiTVwiBDkiTVwiBDkiTVwiBDkiTVYtE/U9LEokUpQZKkfiguLyOJa+Yo71+vyvkyLcgww4aLZTbYLJ/hZLn1x6jno8dJtXK+2F0iSZJqYZAhSZJq0TbIOHXqVGr+qBp4bbZevHiR1qX+OnjwYGU5VZUnZYAoC4bLly+nNNWjVfnkVqxY0SwbDYb79+83y43yaaWT8pUWgrZBxqtXrxrPnz9PfSwHDhxonDx5Mk3fu3evWKI1Tsh2F6vly5cXU+oX8nzjxo3Ncvrhhx9SWWDJkiUpPYbx8fHGsmXL0muURZT10aNHvcDVZKbyCVyYKAsNlg0bNqQyo+wIMqoCCMr31q1baRnKkPL1XNJC1TbIOHfuXPMilFu/fn3jyJEjxVy1EydOFFOtWZH2386dO9MAyolA4s2bN2l+zZo1aQwqvi+++CJNUzHmAcf+/fsbP/74Y5pWf81UPvCCNNgWL16cxlX1YohWjpmWkRaCvjyTkTfBb9myJaUxvnnzZmPXrl3NNE68WK5dC4f665NPPknjvNL7/fffG5999lmavnv37rTXaPHwYjd3onxAcNcugNf8oDWXrhDQWlFVTgSQ1H3UcQy0/hpsaKGadZBBMy9dKjQNMoCT8MaNG+kObWJiIk1zshFksAxpv/76a1pWc4M75jLKLU8fGxsrpjTXohw4T3bv3p2mNXho2QU3Ss+ePUvTVWih5QaL4N2AUQvZrIOMS5cupf7lwElIhF9GdE+wQQDCyae5EQFfma0Ug6FcPlyUqgJCDQaCQJ5XonWCQKPVeURrFDdU1IXRkistRH3pLnn9+nUx1Vo8lU1AQkuG6kc3Vh4A5h4/fjztjpnl7ty5U8y9L9N4XkP1KJcPF7Dz5883uxTBw7ika/4RUHCDRBBI6wSBRnSd5PKHQWntYLAMtVDNOsigkuTp6cDFi4cGy2jx4KSMB95Ur6jUIr/LlSHdVfkdc/Qjx50ZZbpjx440rf6rKh+mo9sxuh5pdvecGSz5N4FaPWtRbs3Nn7mRFpSpyizJJitN3VGlZRjGx8eL1PemgofmawcOHChS/0nTpPHee/fuNZeJdcXrkabukG9V8nyNgTIKUxeuaeUU8jKamJgoUtUr8rFKu/IJpFNWmlvkeyucF1FmeZ1FnZjXi0zHclVluxDMlI+jYNT3r1flfPG3S4aYZTbYLJ/hZLn1x6jno8dJtXK+9OWZDEmSpDKDDEmSVAuDDEmSVAuDDEmSVAuDDEmSVAuDDEmSVAuDDEmSVAuDDEmSVAuDDEmSVAuDDEmSVAuDDEmSVAuDDEmSVAuDDEmSVAuDDEmSVAuDDEmSVAuDDEmSVAuDDEmSVItF/0xJE4sWpQRJkqReFWFF0gwyJEmS+qfR+H8/naLsJl6GUwAAAABJRU5ErkJggg==\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe highest prevalence rate of infection with \u003cem\u003eE. granulosus\u003c/em\u003e (9.75%) was in adults in compare to the puppies infection rate (7.54%) with significant differences (P\u0026lt;0.05) between these two age groups, (Table 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable (2): Infection rate of suspected \u003cem\u003eE.granulosus\u003c/em\u003e according to age.\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable dir=\"rtl\" border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp dir=\"LTR\"\u003eP value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp dir=\"LTR\"\u003eX\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp dir=\"LTR\"\u003ePercentage (%)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp dir=\"LTR\"\u003eNo. of infected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp dir=\"LTR\"\u003eNo. of examined\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp dir=\"LTR\"\u003eAge group\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\"\u003e\n \u003cp dir=\"LTR\"\u003e0.020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\"\u003e\n \u003cp dir=\"LTR\"\u003e5.333\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp dir=\"LTR\"\u003e7.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp dir=\"LTR\"\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp dir=\"LTR\"\u003e106\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;Puppies\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp dir=\"LTR\"\u003e9.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp dir=\"LTR\"\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;164\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;Adult\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp dir=\"LTR\"\u003e8.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp dir=\"LTR\"\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp dir=\"LTR\"\u003e270\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp dir=\"LTR\"\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePCR results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOut of 24 microscopic positive samples, 17 samples revealed positive results when successful to amplifying their extracted DNA to 400bp through polymerase chain reaction technique to confirm the infection with \u003cem\u003eE. granulosus\u003c/em\u003e (Figure 2). \u0026nbsp; The DNA sequencing has been done for two clones to gotten accession number OP272485.1 andOP272486.1.Phylogenetic tree revealed that the two strains \u0026nbsp;alignment with AY314986.1 with identity 99.86% which related to the \u003cem\u003eE.granulosus\u003c/em\u003e\u0026nbsp; that have \u003cem\u003eAgB2\u003c/em\u003e groups Ag\u003cem\u003eB\u003c/em\u003e2 were present as functional genes exclusively dog as final host of \u003cem\u003eE.granulosus\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eGene sequencing and phylogenetic results\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt was observed through phylogenetic analysis that there is a genetic difference in the EgAgB family genes. Evolutionary genetic analysis revealed genetic variation in the EgAgB family. This was achieved after studying the evolutionary tree of the amino acid sequences involved in the construction of EgAgB proteins, using MEGA X methods to reveal the genetic relationship between these proteins found in species belonging to the genus Taenia. The results revealed a very similar genetic evolutionary pattern. Analysis of phylogenetic tree revealed highly bootstrap values for deeper nodes (Fig. 3), as anticipated due to the similarities among sequences of different subfamilies, and different \u0026nbsp;other \u0026nbsp; species. Multiple sequence alignment showed the variation of amino acid between groups of AgB family as shown in figure (4, 5). \u0026nbsp; \u0026nbsp;\u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The two strains OP272485.1 \u0026nbsp;and \u0026nbsp; OP272486.1 \u0026nbsp;show mutation with reference strain \u0026nbsp;AY314986.1. at position 54 (C-T) \u0026nbsp; and 75 (A-G) for first strain OP272485.1 ,while \u0026nbsp; OP272486.1 \u0026nbsp; show one mutation at 54 (C-T) with reference strain \u0026nbsp;as in figure(5) and table (6).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBoth OP272485.1 and OP272486.1 share the \u0026nbsp; cytosine \u003cstrong\u003eC→T \u0026nbsp;thiamin mutation at position 54\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eAnd \u0026nbsp;OP272485.1 has an \u003cstrong\u003eadditional A→G mutation at position 75\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cstrong\u003emutations\u0026nbsp;\u003c/strong\u003eappear to be \u003cstrong\u003esynonymous mutations\u003c/strong\u003e or located in \u003cstrong\u003enon-coding/5′ regions\u003c/strong\u003e.\u003c/p\u003e\u003cp\u003eTable (6) Main mutation and substitution of two strain and position with type of mutation .\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn Iraq, there were few reports of canine echinococcosis; however, this study was designing to obtain some aspects related to this disease (24).\u003c/p\u003e\n\u003cp\u003eThe current research focused on rate of spread of \u003cem\u003eEchinococcus granulosus\u003c/em\u003e in dog. The current research focused on microscopically examination and parasite suspected eggs detection, molecular examination for infection confirmation and parasite strain detection and prevalence study in related to some risky factors in Al-Diwaniyah province. Through the current results confirmed by the PCR examination, which consider the end in diagnosis of disease, it was noted that the prevalence of the parasite \u003cem\u003eE. granulosus\u003c/em\u003e in dogs in Al- Diwaniyah province was 6.29%. The male dogs had a higher prevalence (9.09%) than the females (8.66%) and in the adults were higher (9.75%) than in puppies (7.54%).The relatively high rate of infection may be attributed to slaughtering outside slaughterhouses without veterinary supervision, where considered one of the important sources of echinicoccosis among dogs (25).\u003c/p\u003e\n\u003cp\u003eThe movement of infected dogs between different regions may also increase the presence of infection in non-endemic areas. In the present study, the molecular diagnosis \u003cem\u003eof E. granulosus\u003c/em\u003e by AGB2 was important in diagnosis of disease and focused of type of AgB2 more prevalence than other type of this antigen. The parasite larvae secrete a type of lipoprotein known as antigen B into the hydatid fluid. Previous studies have identified eight individual units of this antigen (antigen B1 - antigen B8), (26).\u003c/p\u003e\n\u003cp\u003eIt is the most reliable immunological antigen in diagnosing the genus Echinococcus granulosus. Its weight is 230 KD, and polar and neutral lipids constitute 50% of its mass, including fatty acids (FA) and sterols, which cannot synthesize by \u003cem\u003eEchinococcus\u003c/em\u003e. This has led to emphasize its hypothetical role in parasite lipid metabolism, taking up host lipids as building blocks for parasite metabolic demands (27).\u003c/p\u003e\n\u003cp\u003eMoreover, this hypothesis is supported by the fact that AgB belongs to a cestode-specific family of proteins exhibiting ability to bind hydrophobic ligands (HLBP for hydrophobic ligand binding protein) (33). This family arose through independent gene expansion events, resulting in genetically distinct monophyletic species and subspecies. Interestingly, all HLBP members are immunodominant. However, genetic diversity was observed among these Agb2 isolates from other populations using sequencing and phylogenetic tree analysis. Genetic distances were not correlated with geographic distances, indicating local divergent evolution rather than isolation by distance. In conventional PCR, 17 of the 24 positive cases were diagnosed. The high acceptance rate among researchers in Iraqi Kurdistan, who focused on stray dogs (28).The genomic and phylogenetic tree confirmed that EgAgB2 local strain was 99.68%,99.63% identical to world AgB2 with AY314986.1. GU166200.1 respectively from Brazil and Australia respectively.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e EgAgB \u003c/em\u003eis a polymorphic gene, likely dependent on host type for adaptation, as \u003cem\u003eE. granulosus\u003c/em\u003e strains are often specific to their intermediate hosts (29). Some studies have indicated that \u003cem\u003eE. granulosus\u003c/em\u003e AgB has a high degree of genetic diversity. Differences in the gene sequences of \u003cem\u003eE. granulosus\u003c/em\u003e isolates encoding the five AgB subunits may affect the diagnostic performance of the proteins of these subunits. Therefore, understanding the similarities in AgB genes may contribute to the development and application of AgB-based diagnostic assays (30). Molecular analysis of \u003cem\u003eE. granulosus\u003c/em\u003e isolates revealed differences in the EgAgB nucleotide sequences, resulting in variants in the amino acid sequence. (29) Given the positive diagnostic performance of the EgAgB2 subunit, we sought to identify potential polymorphisms in the gene encoding this antigen in different \u003cem\u003eE. granulosus\u003c/em\u003e isolates. In our search, we discovered that all EgAgB82 sequences from dogs were closely related to AgB5 (accession number GU166215.1), isolated from the kangaroo host of \u003cem\u003eE. granulosus\u003c/em\u003e from Australia with 99.43% identity\u003cspan dir=\"RTL\"\u003e.\u003c/span\u003eWhile AgB3 and AgB2 (accession numbers GU166214.1 and GU166204.1) from \u003cem\u003eE. granulosus\u003c/em\u003e from China were isolated from dogs with AgB, 3, and 2\u003cspan dir=\"RTL\"\u003e.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eOverall, this study has provided important information about the composition, abundance, and assembly of AgB2 subunits, OP272485.1 and OP272486.1 two strain show polymorphism at position 54 (C-T) and 75 (A-G) for first strain OP272485.1 ,while OP272486.1 show one mutation at 54 (C-T) with reference strain (Accession no.: AY569356.1)greatly expanding our understanding of AgB. \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study focused on polymorphism of two strain We were able to structurally characterize the oligomers formed by different AgB subunits and correlate them with those formed by AgB subunits from \u003cem\u003eE. granulosus\u003c/em\u003e using reconstituted subunits.this polymorphism makes genetic diversity and immunological of epitopes that make of change of main humoral immunity Although further research is needed to fully understand the structure of full genome of AgB, our findings will help understand the roles of AgB in host-parasite interactions during Echinococcus infection (31, 32).\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge all the efforts of the veterinarians at the Al-Diwaniyah city abattoir for kindly helping in collecting the blood samples. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe declare that we have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eTorgerson PR, Deplazes P. 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Diagnostic parasitology for Veterinary technique. Mosby Elsevier. 2006; 3rd. pp: 320-331\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Echinococcus, granulosus, AgB2, dog","lastPublishedDoi":"10.21203/rs.3.rs-7736067/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7736067/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEchinococcosis is caused by infection with tapeworms, represented by \u003cem\u003eEchinococcus granulosus.\u003c/em\u003e The antigen B is a main protein produced by the metacestode of this parasite and is a member of a family of genes that help evade the host's immune system. There are many different types of \u003cem\u003eE. granulosus\u003c/em\u003e (G1-G10), each with its own unique set of biological traits.in this study we investigate of sequencing and polymorphism of AGb2 of Echinococcosis in final host of dog. Two hundred and seventy dog ​​feces samples were examined microscopically to detect the parasite eggs, and then DNA extraction was done for each positive sample, where conventional PCR technique used specific AgB2 gene primer and gene sequencing to detect \u003cem\u003eE. granulosus\u003c/em\u003e strains. The results of the microscopic examination indicated that 24 (8.8%) of the cases were positive (13 males and 11 females), including 8 puppies and 16 adults. That 17 out of 24 succeeded in amplifying the DNA and two sequences were submitted and gotten accession number OP272485.1, OP272486.1 and aligned with group of AGB2 groups. The results showed AgB2 play functional genes exclusively in dog as final host of \u003cem\u003eE. granulosus\u003c/em\u003e, suggesting inter-strain variation of this gene.\u003c/p\u003e","manuscriptTitle":"Prevalence of dog's Echinococcus granulosus with study of the Genetic Variability of Antigen B2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-15 06:47:26","doi":"10.21203/rs.3.rs-7736067/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d87b09f5-88bb-405d-bc95-b7521a88c6d0","owner":[],"postedDate":"October 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-20T10:08:59+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-15 06:47:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7736067","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7736067","identity":"rs-7736067","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}