Assessing the Public Health and Zoonotic Impacts of Giardia duodenalis Assemblages in Domestic Animals of Southwestern Iran

Assessing the Public Health and Zoonotic Impacts of Giardia duodenalis Assemblages in Domestic Animals of Southwestern Iran | 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 Assessing the Public Health and Zoonotic Impacts of Giardia duodenalis Assemblages in Domestic Animals of Southwestern Iran Ali Asghari, Mohammad Hossein Motazedian, Qasem Asgari, Laya Shamsi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4326318/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Aug, 2024 Read the published version in Journal of Parasitic Diseases → Version 1 posted 5 You are reading this latest preprint version Abstract Domestic animals play a vital role in the spread and transmission of various zoonotic agents to humans. Giardia duodenalis , an important enteric zoonotic parasite, is widespread globally. This research aimed to assess the presence and genetic diversity of G. duodenalis assemblages in domestic animals in Shiraz, southern Iran. 245 fresh fecal samples were collected from 87 dogs, 63 cats, and 95 cattle in Shiraz, the capital city of Fars province, between July 2021 and August 2022. None of the animals showed gastrointestinal signs such as diarrhea, and all fecal samples had normal consistency. Upon parasite observation using light microscopy, a DNA fragment of G. duodenalis tpi gene was amplified using nested-PCR. Using direct wet mount and Wheatley’s trichrome staining examination, 9 out of 245 samples (3.7%; 1 from dogs and 8 from cattle) tested positive for G. duodenalis . Molecular methods confirmed 1.1% (1/87) of dogs and 8.4% (8/95) of cattle to be positive. Giardiasis was not detected in cats. Phylogenetic analysis revealed that Giardia isolates infecting dogs and cattle in Shiraz belonged to three genotypes/assemblages: A, B (zoonotic), and E (non-zoonotic). In cattle, assemblages E (75%; 6/8), A (12.5%; 1/8), and B (12.5%; 1/8) were identified, while dogs were infected with assemblage A (100%; 1/1). In Shiraz, southwestern Iran, domestic animals like cattle and dogs could potentially serve as reservoirs for zoonotic infections caused by G. duodenalis . Giardia duodenalis Prevalence Domestic animals Cattle Dog Cat Figures Figure 1 Full Text Giardiasis, a parasitic infection caused by Giardia duodenalis (also known as Giardia lamblia or Giardia intestinalis ), is a global zoonotic disease [1]. The infection can either be asymptomatic or present as acute diarrhea and malabsorption syndrome [2]. The zoonotic nature of giardiasis has been emphasized due to numerous water-borne outbreaks, which serve as the primary source of infection. In some areas where the disease is endemic, domestic and pet animals are closely associated with the direct transmission of the parasite to humans [3, 4]. The life cycle of Giardia involves two main stages: the trophozoite (non-infective) and the cyst (infective). When contaminated food or water is ingested, the resistant cysts of Giardia enter the small intestine. Once there, the cysts release trophozoites that attach themselves to the epithelial cells lining the intestine. Trophozoites multiply through binary fission and may move downstream towards the colon, where they can be released into the environment as infectious cysts. The latter form remains infectious for several months in cool, damp places, posing a significant health risk to animals and humans alike [1, 5–7]. Giardia species exhibit considerable heterogeneity and are referred to as genetic groups or assemblages. Eight assemblages have been identified for the parasite, including A and B (humans, livestock, companion animals), C and D (domestic/wild canids), E (domestic ruminants and pigs), F (cats), G (rodents), and H (seals and gulls) [8, 9]. Molecular characterization of Giardia isolates requires a fundamental understanding of the gene fragments that may be involved. Various genes that have sufficient polymorphism, such as the small subunit ribosomal RNA gene ( SSU RNA ) and the elongation factor 1 gene ( ef1a ), have been employed as markers for distinguishing between species. For the typing of Giardia assemblages, the most commonly used approach is multilocus genotyping (MLG). This method involves amplifying highly-conserved target genes, including glutamate dehydrogenase ( gdh ), triose phosphate isomerase ( tpi ), and beta giardin ( bg ), through polymerase chain reaction (PCR). Subsequently, downstream experiments such as restriction fragment length polymorphism (RFLP) or sequencing of PCR products are conducted [1, 10, 11]. Domestic animals play a crucial role in the food chain for the human population. Therefore, they are raised and kept close to humans, especially in rural areas [9, 12, 13]. The proximity between animals and humans can potentially lead to an increased risk of transmitting zoonotic agents like G. duodenalis . Hence, in order to enhance our understanding of the epidemiological aspects of giardiasis in both humans and animals in Iran, it is necessary to conduct further studies on the prevalence and genotyping of Giardia . The present study focused on identifying the Giardia assemblages present in fecal samples from dogs, cats, and cattle in Shiraz, located in southwestern Iran. The present study was confirmed by the Ethics Committee of the Shiraz University of Medical Sciences, Fars Province, Iran (Approval No: IR.SUMS.REC.1400.460). Between July 2021 and August 2022, a total of 245 fresh stool samples were gathered from cattle (n = 95), dogs (n = 87), and cats (n = 63) in Shiraz, after receiving permission from their owners. Of each collected specimen, single aliquots were prepared and stored without preservatives at -20 o C. No clinical symptoms, such as diarrhea, were observed in the examined animals, and their stool consistency was normal. Various demographic parameters, including sex, age, breed, and living conditions, were recorded for canids, while sex and age were documented for cattle. The parasite's morphology, including size, shape, and internal structures, was examined using light microscopy (Olympus BX41TF, Tokyo, Japan) at 400× magnification. To purify the parasitic DNA from about 200 mg of fecal samples, the AccuPrep Stool DNA extraction kit (Bioneer Corporation, Seoul, South Korea) was utilized. The manufacturer's protocol was modified as follows: samples were mixed with 600 μL digestion buffer (100 mM NaCl, 10 mM Tris-HCl pH 8.0, and 25 mM EDTA), glass pearls (0.45-0.52 mM in diameter) were added and vortexing was done for 10 min. Five cycles of freeze/thawing in liquid nitrogen and boiling water were also applied for cyst wall disruption. Subsequently, 20 μL of proteinase K enzyme (final concentration: 200 μg/mL) and 40 μL of 2% sodium dodecyl sulfate (SDS) were added to each sample and placed in a water bath at 60°C overnight. Finally, the recommended protocol by the manufacturer was exerted. The concentration of purified DNA was measured by NanoDrop (Thermo Scientific 2000C, Wilmington, USA) and the extracted DNAs were stored at −20°C. The primer pair used to amplify a 530 bp fragment of the tpi gene was derived from the study by Sulaiman et al., which included AL3543 and AL3546 (amplifying a 605 bp sequence) and AL3544 and AL3545 (for second-round amplification) [14]. All PCR reactions in a final volume of 25 μL included 1 μL (10 pm) of each primer, 12.5 μL of 1 × Taq DNA Polymerase Master Mix RED (Ampliqon, Odense, Denmark), 7.5 μL of DW, and 3 μL of extracted DNA for primary PCR or 1 μL of the first-round PCR product for secondary PCR. The first-round PCR conditions were as follows: initial denaturation step (95°C for 5 min), followed by 35 cycles of denaturation (95°C for 45 s), annealing (50°C for 45 s), and extension (72°C for 60 s). A final extension step was performed at 72°C for 4 min. The second-round PCR conditions remained unchanged, except for the annealing temperature, which was adjusted to 54°C. G. duodenalis assemblage A DNA served as the positive control, while the negative control consisted of the same positive control materials, with the exception that distilled water was used instead of DNA. Lastly, the products of the second PCR were observed through electrophoresis on a 1% agarose gel stained with safe stain (SinaClon, Tehran, Iran). The PCR products were purified from agarose gel for DNA sequencing, and both strands were sequenced by MACROGEN (Korea). The results were presented as chromatograms, which were subsequently edited and aligned using MEGA version X. The Neighbor-Joining parameter model was utilized to build phylogenetic trees with the utmost probability logarithm value. The credibility of clusters was assessed by employing a bootstrap of 1000 iterations. The tree was rooted by incorporating the sequence of Giardia muris (AF069565) as an out-group. Collected data were analyzed by SPSS software (version 20, IBM Inc., USA). To determine the correlation between G. duodenalis infection and reported variables in cattle, the Chi-square test was employed. P<0.05 was considered statistically significant. Microscopy of the direct wet mount and Wheatley’s trichrome stained fecal samples demonstrated the presence of G. duodenalis protozoan in 9 [3.7% (1 from dogs and 8 from cattle)] stool samples. Of note, cat samples were negative for Giardia infection. The molecular prevalence matched the microscopic results, with a reported prevalence of 1.1% (1/87) in dogs and 8.4% (8/95) in cattle. Based on the reported demographic parameters, there was no statistically significant difference between these variables in cattle (age, sex) with the prevalence of giardiasis (Table 1). The sequence identity between the 9 sequenced isolates and the reference G. duodenalis sequences in GenBank was 98-100%. Phylogenetic analysis using the neighbor-joining method revealed three assemblages: 2 zoonotic (A and B) and one non-zoonotic (E). The majority of assemblages were found in cattle, with assemblage E comprising 75% (6/8), assemblage A including 12.5% (1/8), and assemblage B also composing 12.5% (1/8). Additionally, one assemblage A was detected in dogs, accounting for 100% (1/1) of the samples (Fig. 1). All 9 sequenced Giardia isolates from domestic animals (dogs and cattle) in this study were shown in the Fig. 1, under isolate numbers of A1 (OP312619, assemblage A/Cattle), A2 (OP312621, assemblage A/ dogs), B1 (OP312620, assemblage B/ cattle), and CT1-CT6 (OP312613-OP312618, assemblage E/cattle). The parasitic zoonotic agent, G. duodenalis , infects a wide range of domestic and wildlife animals as well as humans [15]. The transmission of the parasite from animals to humans is a significant public health threat that requires further investigation [16]. Zoonotic assemblages A and B have previously been found in various domestic animals, such as cattle, dogs, and cats [17, 18]. Therefore, this study aims to evaluate the prevalence, distribution of assemblages, and zoonotic significance of G. duodenalis infection in dogs, cats, and cattle in Shiraz, southwestern Iran. In this study, 245 stool samples were collected and analyzed using microscopy and molecular methods. The results revealed a relatively low infection rate (3.7%). Among the sampled animals, cattle showed a higher parasitic rate (8.4%) compared to dogs (1.1%). Interestingly, cats tested negative for Giardia infection. These variations in infection rates could be attributed to factors such as host species, sampled areas, animal breeding, detection methods, and sample size. A proper comparison of the difference in prevalence among these hosts could not be made due to the unequal number of samples and sampling areas. In Iran, there is a lack of information regarding the prevalence of G. duodenalis and the distribution of assemblages among animal hosts. Based on available epidemiological data, the reported prevalence of the parasite has been estimated to be between 4.2-9.3% [19, 20] in cattle, 0.7-4% [21–23] in dogs, and 1.3-11.6% [22–25] in cats. These estimates are somewhat consistent with the calculated prevalence in the current study. Furthermore, the previous meta-analyses have reported that the prevalence of Giardia infection in cattle [26], dogs and cats [17] have been estimated at 22% (95% CI, 17-28%), 15.2% (95% CI 13.8-16.7%), and 12% (95% CI 9.2-15.3%), respectively. Our own findings align with this, indicating that the infection rate in cattle is relatively high compared to the other two hosts. However, it's worth noting that discrepancies in factors such as the number of studies examined, the sensitivity of diagnostic methods, and the sampled areas can contribute to differences in the final prevalence. No significant association was found between demographic variables in cattle and the prevalence of Giardia . Of note, the small sample size and the limited number of positive Giardia cases, particularly in dogs (only one sample), prevent us from drawing statistical conclusions. However, the infection was more common in younger animals than in older ones, which is consistent with previous studies [18, 21, 25]. Additionally, animals kept outdoors had lower parasite levels compared to those kept indoors. Waterborne transmission of protozoan parasites, such as Cryptosporidium and Giardia , poses a significant problem for water facilities and can potentially lead to human infections [16, 27, 28]. However, there is still limited knowledge regarding the possible non-human sources of water contamination, and the extent to which zoonotic infections contribute should be precisely determined. In many European countries, animals like beavers, muskrats, companion animals (such as dogs and cats), and livestock have been identified as potential sources of Giardia zoonotic infections [29–31]. In Iran, studies have mostly concentrated on companion animals, rodents, and livestock to determine the prevalence, distribution of assemblages, and likelihood of zoonotic transmission [8, 9, 20–22, 25]. Among the reported Giardia assemblages, assemblages A (sub-assemblages AI and AII) and B (sub-assemblages BII, BIII, and BIV), especially AI, have been proven to be highly transmissible between human and animal populations. Additionally, several animal assemblages (C-F) have been found in human infections in various countries, highlighting the zoonotic transmission of Giardia species. However, their impact on human health is still a subject of debate [10, 11]. Humans and various classes of animals, including cattle, dogs, and cats, are parasitized by Giardia assemblages A and B. However, other genotypes/assemblages show host-specificity. For example, assemblage E mostly infects cattle, while assemblages C and D infect dogs, and assemblage F infects cats [12, 32]. However, our study did not report any positive cases of Giardia in cats or assemblages D and C in dogs. In accordance with our findings, previous studies conducted in Iran have also identified assemblages E and B in cattle, C, D, and A in dogs, and assemblages F and A in cats [20–23, 25, 33]. Although our study only found Giardia infections in cattle and dogs, with zoonotic assemblages present, all three animal hosts (cattle, dog, cattle) can play a significant role in transmitting Giardia zoonotic infections to humans. It is important to note, however, that the presence of zoonotic assemblages in these animal hosts does not necessarily indicate the circulation of these assemblages between humans and animals. It should be noted that the current study's sample size is relatively small. To obtain more precise results, broader and more comprehensive studies are necessary. In addition, fresh smears have low sensitivity in detecting G. duodenalis in stool samples, even when performed by experts. Nowadays, other more sensitive techniques are typically used for diagnosing G. duodenalis , such as fluorescence, for instance. Furthermore, in most epidemiological studies, the correct determination of Giardia infecting genotypes/assemblages in different hosts is achieved using the multilocus genotyping method based on various genes. This method is considered appropriate in this field. However, in the present study, we solely relied on the tpi gene (single locus) to assess the prevalence and genotyping of Giardia . This approach may be prone to some potential errors in detecting various assemblages, and therefore, it is suggested to interpret the results of the genotyping section in the current study with caution. Overall, these issues are included among the limitations of the present study. Although the number of positive cases of G. duodenalis in the samples examined from various animals (cattle, dog, and cat) in this study was insufficient for drawing epidemiological conclusions. However, it was shown that dogs and cattle could be potential reservoirs of Giardia zoonotic infections in Shiraz, southwestern Iran. Declarations Acknowledgements and Funding The current study was a part of the Ph.D. thesis of Ali Asghari, financed by the Vice-Chancellor for Research of Shiraz University of Medical Sciences (Grant No: 21882). Hereby, we would like to express our gratitude and appreciation for the comprehensive support of this center. Compliance with Ethical Standards Conflict of Interest The authors declare that they have no conflict of interest. Author Contributions A.A and MH.M conceived and designed the study. A.A, MH.M, and Q.A had a role in collecting samples and methodology. A.A performed the molecular analysis. A.A and L.S wrote the manuscript. 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Parasit Vectors 13:1–11. https://doi.org/10.1186/s13071-020-04248-2. Table 1 Table 1. Potential host factors associated with Giardia infection in cattle Potential host factors No. examined animals (%) No. positive (%) P value Age (years) ≥1 ≤1 Gender Male Female 43 52 38 57 4 (9.3) 4 (7.7) 3 (7.9) 5 (8.8) >0.05 >0.05 Cite Share Download PDF Status: Published Journal Publication published 27 Aug, 2024 Read the published version in Journal of Parasitic Diseases → Version 1 posted Reviewers agreed at journal 28 Apr, 2024 Reviewers invited by journal 27 Apr, 2024 Editor invited by journal 27 Apr, 2024 Editor assigned by journal 26 Apr, 2024 First submitted to journal 25 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4326318","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":296059870,"identity":"a10eff21-c906-421b-a755-20275b43d400","order_by":0,"name":"Ali Asghari","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYHACNiCWsOOHMEjQkizZQKIWBsYNB4jVwt9//NqDn3ssmI1vJD978KGCQZ5f7AB+LRI3csoNe55J8JndSDM3nHGGwXDm7AQC1tzgSZPgOSDBbHYjwUyat40hweA2AS3y58+kSf45IMG4eUb6N+K0GBxIPyYNtIVxg0QOkbYY3shhk5Y5IJEsceZNmeSMMxKE/SJ3/vgzyTcH6uz429O3SXyosJHnlyaghYGBxwBCC4BVShBSDgLsDyA0/wFiVI+CUTAKRsFIBADvL0EqjqTKrgAAAABJRU5ErkJggg==","orcid":"","institution":"Qazvin University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Ali","middleName":"","lastName":"Asghari","suffix":""},{"id":296059871,"identity":"e33682d9-908e-450c-860e-511751b62d0f","order_by":1,"name":"Mohammad Hossein Motazedian","email":"","orcid":"","institution":"Shiraz University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"Hossein","lastName":"Motazedian","suffix":""},{"id":296059872,"identity":"9b5e97ba-20c8-42ff-a76b-6eaef4e9dda9","order_by":2,"name":"Qasem Asgari","email":"","orcid":"","institution":"Shiraz University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Qasem","middleName":"","lastName":"Asgari","suffix":""},{"id":296059873,"identity":"92997226-cc2a-4dfe-af95-e50fa0d3c8d7","order_by":3,"name":"Laya Shamsi","email":"","orcid":"","institution":"Urmia University","correspondingAuthor":false,"prefix":"","firstName":"Laya","middleName":"","lastName":"Shamsi","suffix":""}],"badges":[],"createdAt":"2024-04-25 23:25:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4326318/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4326318/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12639-024-01727-6","type":"published","date":"2024-08-27T15:56:51+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":55781784,"identity":"9befc6f0-af7b-43c3-a2e3-022227fe1ed7","added_by":"auto","created_at":"2024-05-03 05:02:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":98020,"visible":true,"origin":"","legend":"\u003cp\u003eThe phylogenetic tree reconstructed of \u003cem\u003eGiardia duodenalis\u003c/em\u003e using the Neighbor-Joining method, showing the \u003cem\u003eGiardia\u003c/em\u003eisolates infecting the cattle and dogs of the present study belonging to the assemblages of A, B, and E. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4326318/v1/f5499b4032bbd78eb172ae68.png"},{"id":63821572,"identity":"b996e9e2-392e-469e-aab5-938c8c5a89ff","added_by":"auto","created_at":"2024-09-02 16:14:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":364713,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4326318/v1/bca4e14b-cb88-4a2d-b1d9-cd332b0ff510.pdf"}],"financialInterests":"","formattedTitle":"Assessing the Public Health and Zoonotic Impacts of Giardia duodenalis Assemblages in Domestic Animals of Southwestern Iran","fulltext":[{"header":"Full Text","content":"\u003cp\u003eGiardiasis, a parasitic infection caused by \u003cem\u003eGiardia duodenalis\u003c/em\u003e (also known as \u003cem\u003eGiardia lamblia\u003c/em\u003e or \u003cem\u003eGiardia intestinalis\u003c/em\u003e), is a global zoonotic disease\u0026nbsp;[1]. The infection can either be asymptomatic or present as acute diarrhea and malabsorption syndrome\u0026nbsp;[2]. The zoonotic nature of giardiasis has been emphasized due to numerous water-borne outbreaks, which serve as the primary source of infection. In some areas where the disease is endemic, domestic and pet animals are closely associated with the direct transmission of the parasite to humans\u0026nbsp;[3, 4]. \u0026nbsp;The life cycle of \u003cem\u003eGiardia\u003c/em\u003e involves two main stages: the trophozoite (non-infective) and the cyst (infective). When contaminated food or water is ingested, the resistant cysts of \u003cem\u003eGiardia\u003c/em\u003e enter the small intestine. Once there, the cysts release trophozoites that attach themselves to the epithelial cells lining the intestine. Trophozoites multiply through binary fission and may move downstream towards the colon, where they can be released into the environment as infectious cysts. The latter form remains infectious for several months in cool, damp places, posing a significant health risk to animals and humans alike\u0026nbsp;[1, 5\u0026ndash;7]. \u003cem\u003eGiardia\u003c/em\u003e species exhibit considerable heterogeneity and are referred to as genetic groups or assemblages. Eight assemblages have been identified for the parasite, including A and B (humans, livestock, companion animals), C and D (domestic/wild canids), E (domestic ruminants and pigs), F (cats), G (rodents), and H (seals and gulls)\u0026nbsp;[8, 9]. Molecular characterization of \u003cem\u003eGiardia\u003c/em\u003e isolates requires a fundamental understanding of the gene fragments that may be involved. Various genes that have sufficient polymorphism, such as the small subunit ribosomal RNA gene (\u003cem\u003eSSU RNA\u003c/em\u003e) and the elongation factor 1 gene (\u003cem\u003eef1a\u003c/em\u003e), have been employed as markers for distinguishing between species. For the typing of \u003cem\u003eGiardia\u003c/em\u003e assemblages, the most commonly used approach is multilocus genotyping (MLG). This method involves amplifying highly-conserved target genes, including glutamate dehydrogenase (\u003cem\u003egdh\u003c/em\u003e), triose phosphate isomerase (\u003cem\u003etpi\u003c/em\u003e), and beta giardin (\u003cem\u003ebg\u003c/em\u003e), through polymerase chain reaction (PCR). Subsequently, downstream experiments such as restriction fragment length polymorphism (RFLP) or sequencing of PCR products are conducted\u0026nbsp;[1, 10, 11]. Domestic animals play a crucial role in the food chain for the human population. Therefore, they are raised and kept close to humans, especially in rural areas\u0026nbsp;[9, 12, 13]. The proximity between animals and humans can potentially lead to an increased risk of transmitting zoonotic agents like \u003cem\u003eG. duodenalis\u003c/em\u003e. Hence, in order to enhance our understanding of the epidemiological aspects of giardiasis in both humans and animals in Iran, it is necessary to conduct further studies on the prevalence and genotyping of \u003cem\u003eGiardia\u003c/em\u003e. The present study focused on identifying the \u003cem\u003eGiardia\u0026nbsp;\u003c/em\u003eassemblages present in fecal samples from dogs, cats, and cattle in Shiraz, located in southwestern Iran.\u003c/p\u003e\n\u003cp\u003eThe present study was confirmed by the Ethics Committee of the Shiraz University of Medical Sciences, Fars Province, Iran (Approval No: IR.SUMS.REC.1400.460).\u0026nbsp;Between July 2021 and August 2022, a total of 245 fresh stool samples were gathered from cattle (n = 95), dogs (n = 87), and cats (n = 63) in Shiraz, after receiving permission from their owners. Of each collected specimen, single aliquots were prepared and stored without preservatives at -20 \u003csup\u003eo\u003c/sup\u003eC. No clinical symptoms, such as diarrhea, were observed in the examined animals, and their stool consistency was normal. Various demographic parameters, including sex, age, breed, and living conditions, were recorded for canids, while sex and age were documented for cattle. The parasite\u0026apos;s morphology, including size, shape, and internal structures, was examined using light microscopy (Olympus BX41TF, Tokyo, Japan) at 400\u0026times; magnification. To purify the parasitic DNA from about 200 mg of fecal samples, the AccuPrep Stool DNA extraction kit (Bioneer Corporation, Seoul, South Korea) was utilized. The manufacturer\u0026apos;s protocol was modified as follows: samples were mixed with 600 \u0026mu;L digestion buffer (100 mM NaCl, 10 mM Tris-HCl pH 8.0, and 25 mM EDTA), glass pearls (0.45-0.52 mM in diameter) were added and vortexing was done for 10 min. Five cycles of freeze/thawing in liquid nitrogen and boiling water were also applied for cyst wall disruption. Subsequently, 20 \u0026mu;L of proteinase K enzyme (final concentration: 200 \u0026mu;g/mL) and 40 \u0026mu;L of 2% sodium dodecyl sulfate (SDS) were added to each sample and placed in a water bath at 60\u0026deg;C overnight. Finally, the recommended protocol by the manufacturer was exerted. The concentration of purified DNA was measured by NanoDrop (Thermo Scientific 2000C, Wilmington, USA) and the extracted DNAs were stored at \u0026minus;20\u0026deg;C. The primer pair used to amplify a 530 bp fragment of the \u003cem\u003etpi\u003c/em\u003e gene was derived from the study by Sulaiman et al., which included AL3543 and AL3546 (amplifying a 605 bp sequence) and AL3544 and AL3545 (for second-round amplification)\u0026nbsp;[14]. All PCR reactions\u0026nbsp;in a final volume of 25\u0026nbsp;\u0026mu;L included 1 \u0026mu;L (10 pm) of each primer, 12.5 \u0026mu;L of 1 \u0026times; Taq DNA Polymerase Master Mix RED (Ampliqon, Odense, Denmark), 7.5 \u0026mu;L of DW, and 3 \u0026mu;L of extracted DNA for primary PCR or 1 \u0026mu;L of the first-round PCR product for secondary PCR. The first-round PCR conditions were as follows: initial denaturation step (95\u0026deg;C for 5 min), followed by 35 cycles of denaturation (95\u0026deg;C for 45 s), annealing (50\u0026deg;C for 45 s), and extension (72\u0026deg;C for 60 s). A final extension step was performed at 72\u0026deg;C for 4 min. The second-round PCR conditions remained unchanged, except for the annealing temperature, which was adjusted to 54\u0026deg;C. \u003cem\u003eG. duodenalis\u003c/em\u003e assemblage A DNA served as the positive control, while the negative control consisted of the same positive control materials, with the exception that distilled water was used instead of DNA. Lastly, the products of the second PCR were observed through electrophoresis on a 1% agarose gel stained with safe stain (SinaClon, Tehran, Iran). The PCR products were purified from agarose gel for DNA sequencing, and both strands were sequenced by MACROGEN (Korea). The results were presented as chromatograms, which were subsequently edited and aligned using MEGA version X. The Neighbor-Joining parameter model was utilized to build phylogenetic trees with the utmost probability logarithm value. The credibility of clusters was assessed by employing a bootstrap of 1000 iterations. The tree was rooted by incorporating the sequence of \u003cem\u003eGiardia muris\u003c/em\u003e (AF069565) as an out-group. Collected data were analyzed by SPSS software (version 20, IBM Inc., USA). To determine the correlation between \u003cem\u003eG. duodenalis\u003c/em\u003e infection and reported variables in cattle, the Chi-square test was employed.\u0026nbsp;P\u0026lt;0.05 was considered statistically significant.\u003c/p\u003e\n\u003cp\u003eMicroscopy of the direct wet mount and Wheatley\u0026rsquo;s trichrome stained fecal samples demonstrated the presence of \u003cem\u003eG. duodenalis\u003c/em\u003e protozoan in 9 [3.7% (1 from dogs and 8 from cattle)] stool samples. Of note, cat samples were negative for \u003cem\u003eGiardia\u003c/em\u003e infection. The molecular prevalence matched the microscopic results, with a reported prevalence of 1.1% (1/87) in dogs and 8.4% (8/95) in cattle. Based on the reported demographic parameters, there was no statistically significant difference between these variables in cattle (age, sex) with the prevalence of giardiasis (Table 1). The sequence identity between the 9 sequenced isolates and the reference \u003cem\u003eG. duodenalis\u003c/em\u003e sequences in GenBank was 98-100%. Phylogenetic analysis using the neighbor-joining method revealed three assemblages: 2 zoonotic (A and B) and one non-zoonotic (E). The majority of assemblages were found in cattle, with assemblage E comprising 75% (6/8), assemblage A including 12.5% (1/8), and assemblage B also composing 12.5% (1/8). Additionally, one assemblage A was detected in dogs, accounting for 100% (1/1) of the samples (Fig. 1). All 9 sequenced \u003cem\u003eGiardia\u003c/em\u003e isolates from domestic animals (dogs and cattle) in this study were shown in the Fig. 1, under isolate numbers of A1 (OP312619, assemblage A/Cattle), A2 (OP312621, assemblage A/ dogs), B1 (OP312620, assemblage B/ cattle), and CT1-CT6 (OP312613-OP312618, assemblage E/cattle).\u003c/p\u003e\n\u003cp\u003eThe parasitic zoonotic agent, \u003cem\u003eG. duodenalis\u003c/em\u003e, infects a wide range of domestic and wildlife animals as well as humans\u0026nbsp;[15]. The transmission of the parasite from animals to humans is a significant public health threat that requires further investigation\u0026nbsp;[16]. Zoonotic assemblages A and B have previously been found in various domestic animals, such as cattle, dogs, and cats \u0026nbsp;[17, 18]. Therefore, this study aims to evaluate the prevalence, distribution of assemblages, and zoonotic significance of \u003cem\u003eG. duodenalis\u003c/em\u003e infection in dogs, cats, and cattle in Shiraz, southwestern Iran. In this study, 245 stool samples were collected and analyzed using microscopy and molecular methods. The results revealed a relatively low infection rate (3.7%). Among the sampled animals, cattle showed a higher parasitic rate (8.4%) compared to dogs (1.1%). Interestingly, cats tested negative for \u003cem\u003eGiardia\u003c/em\u003e infection. These variations in infection rates could be attributed to factors such as host species, sampled areas, animal breeding, detection methods, and sample size. A proper comparison of the difference in prevalence among these hosts could not be made due to the unequal number of samples and sampling areas. In Iran, there is a lack of information regarding the prevalence of \u003cem\u003eG. duodenalis\u003c/em\u003e and the distribution of assemblages among animal hosts. Based on available epidemiological data, the reported prevalence of the parasite has been estimated to be between 4.2-9.3%\u0026nbsp;[19, 20]\u0026nbsp;in cattle, 0.7-4%\u0026nbsp;[21\u0026ndash;23]\u0026nbsp;in dogs, and 1.3-11.6%\u0026nbsp;[22\u0026ndash;25]\u0026nbsp;in cats. These estimates are somewhat consistent with the calculated prevalence in the current study. Furthermore, the previous meta-analyses have reported that the prevalence of \u003cem\u003eGiardia\u003c/em\u003e infection in cattle\u0026nbsp;[26], dogs and cats\u0026nbsp;[17]\u0026nbsp;have been estimated at 22% (95% CI, 17-28%), 15.2% (95% CI 13.8-16.7%), and 12% (95% CI 9.2-15.3%), respectively. Our own findings align with this, indicating that the infection rate in cattle is relatively high compared to the other two hosts. However, it\u0026apos;s worth noting that discrepancies in factors such as the number of studies examined, the sensitivity of diagnostic methods, and the sampled areas can contribute to differences in the final prevalence. \u0026nbsp;No significant association was found between demographic variables in cattle and the prevalence of \u003cem\u003eGiardia\u003c/em\u003e. Of note, the small sample size and the limited number of positive \u003cem\u003eGiardia\u003c/em\u003e cases, particularly in dogs (only one sample), prevent us from drawing statistical conclusions. However, the infection was more common in younger animals than in older ones, which is consistent with previous studies\u0026nbsp;[18, 21, 25]. Additionally, animals kept outdoors had lower parasite levels compared to those kept indoors. Waterborne transmission of protozoan parasites, such as \u003cem\u003eCryptosporidium\u003c/em\u003e and \u003cem\u003eGiardia\u003c/em\u003e, poses a significant problem for water facilities and can potentially lead to human infections\u0026nbsp;[16, 27, 28]. However, there is still limited knowledge regarding the possible non-human sources of water contamination, and the extent to which zoonotic infections contribute should be precisely determined. In many European countries, animals like beavers, muskrats, companion animals (such as dogs and cats), and livestock have been identified as potential sources of \u003cem\u003eGiardia\u0026nbsp;\u003c/em\u003ezoonotic infections\u0026nbsp;[29\u0026ndash;31]. In Iran, studies have mostly concentrated on companion animals, rodents, and livestock to determine the prevalence, distribution of assemblages, and likelihood of zoonotic transmission\u0026nbsp;[8, 9, 20\u0026ndash;22, 25]. Among the reported \u003cem\u003eGiardia\u003c/em\u003e assemblages, assemblages A (sub-assemblages AI and AII) and B (sub-assemblages BII, BIII, and BIV), especially AI, have been proven to be highly transmissible between human and animal populations. Additionally, several animal assemblages (C-F) have been found in human infections in various countries, highlighting the zoonotic transmission of \u003cem\u003eGiardia\u003c/em\u003e species. However, their impact on human health is still a subject of debate\u0026nbsp;[10, 11]. Humans and various classes of animals, including cattle, dogs, and cats, are parasitized by \u003cem\u003eGiardia\u003c/em\u003e assemblages A and B. However, other genotypes/assemblages show host-specificity. For example, assemblage E mostly infects cattle, while assemblages C and D infect dogs, and assemblage F infects cats\u0026nbsp;[12, 32]. However, our study did not report any positive cases of \u003cem\u003eGiardia\u003c/em\u003e in cats or assemblages D and C in dogs. In accordance with our findings, previous studies conducted in Iran have also identified assemblages E and B in cattle, C, D, and A in dogs, and assemblages F and A in cats\u0026nbsp;[20\u0026ndash;23, 25, 33]. Although our study only found \u003cem\u003eGiardia\u0026nbsp;\u003c/em\u003einfections in cattle and dogs, with zoonotic assemblages present, all three animal hosts (cattle, dog, cattle) can play a significant role in transmitting \u003cem\u003eGiardia\u003c/em\u003e zoonotic infections to humans. It is important to note, however, that the presence of zoonotic assemblages in these animal hosts does not necessarily indicate the circulation of these assemblages between humans and animals. It should be noted that the current study\u0026apos;s sample size is relatively small. To obtain more precise results, broader and more comprehensive studies are necessary. In addition, fresh smears have low sensitivity in detecting \u003cem\u003eG. duodenalis\u003c/em\u003e in stool samples, even when performed by experts. Nowadays, other more sensitive techniques are typically used for diagnosing \u003cem\u003eG. duodenalis\u003c/em\u003e, such as fluorescence, for instance. Furthermore, in most epidemiological studies, the correct determination of \u003cem\u003eGiardia\u003c/em\u003e infecting genotypes/assemblages in different hosts is achieved using the multilocus genotyping method based on various genes. This method is considered appropriate in this field. However, in the present study, we solely relied on the \u003cem\u003etpi\u003c/em\u003e gene (single locus) to assess the prevalence and genotyping of \u003cem\u003eGiardia\u003c/em\u003e. This approach may be prone to some potential errors in detecting various assemblages, and therefore, it is suggested to interpret the results of the genotyping section in the current study with caution. Overall, these issues are included among the limitations of the present study.\u003c/p\u003e\n\u003cp\u003eAlthough the number of positive cases of \u003cem\u003eG. duodenalis\u003c/em\u003e in the samples examined from various animals (cattle, dog, and cat) in this study was insufficient for drawing epidemiological conclusions. However, it was shown that dogs and cattle could be potential reservoirs of \u003cem\u003eGiardia\u003c/em\u003e zoonotic infections in Shiraz, southwestern Iran.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements and Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe current study was a part of the Ph.D. thesis of Ali Asghari, financed by the Vice-Chancellor for Research of Shiraz University of Medical Sciences (Grant No: 21882). Hereby, we would like to express our gratitude and appreciation for the comprehensive support of this center.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.A and MH.M conceived and designed the study. A.A, MH.M, and Q.A had a role in collecting samples and methodology. A.A performed the molecular analysis. A.A and L.S wrote the manuscript. A.A critically revised the manuscript. All the authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available in the online version.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWu Y, Yao L, Chen H, Zhang W, Jiang Y, Yang F, Liu A, Shen Y (2022) Giardia duodenalis in patients with diarrhea and various animals in northeastern China: prevalence and multilocus genetic characterization. Parasit Vectors 15:1\u0026ndash;11. https://doi.org/10.1186/s13071-022-05269-9.\u003c/li\u003e\n\u003cli\u003eXie S-C, Zou Y, Chen D, Jiang M-M, Yuan X-D, Li Z, Zou F-C, Yang J-F, Sheng J-L, Zhu X-Q (2018) Occurrence and multilocus genotyping of Giardia duodenalis in Yunnan Black Goats in China. Biomed Res Int 2018. https://doi.org/10.1155/2018/4601737.\u003c/li\u003e\n\u003cli\u003eChen L, Zhao J, Li N, Guo Y, Feng Y, Feng Y, Xiao L (2019) Genotypes and public health potential of Enterocytozoon bieneusi and Giardia duodenalis in crab-eating macaques. Parasit Vectors 12:1\u0026ndash;11. https://doi.org/10.1186/s13071-019-3511-y.\u003c/li\u003e\n\u003cli\u003eAgresti A, Berrilli F, Maestrini M, Guadano Procesi I, Loretti E, Vonci N, Perrucci S (2021) Prevalence, Risk Factors and Genotypes of Giardia duodenalis in Sheltered Dogs in Tuscany (Central Italy). Pathogens 11:12. https://doi.org/10.3390/pathogens11010012.\u003c/li\u003e\n\u003cli\u003eBelkessa S, Ait-Salem E, Laatamna A, Houali K, S\u0026ouml;nksen UW, Hakem A, Bouchene Z, Ghalmi F, Stensvold CR (2021) Prevalence and clinical manifestations of Giardia intestinalis and other intestinal parasites in children and adults in Algeria. Am J Trop Med Hyg 104:910. https://doi.org/10.4269/ajtmh.20-0187.\u003c/li\u003e\n\u003cli\u003eLevecke B, Geldhof P, Claerebout E, Dorny P, Vercammen F, Cacci\u0026ograve; SM, Vercruysse J, Geurden T (2009) Molecular characterisation of Giardia duodenalis in captive non-human primates reveals mixed assemblage A and B infections and novel polymorphisms. Int J Parasitol 39:1595\u0026ndash;1601. https://doi.org/10.1016/j.ijpara.2009.05.013.\u003c/li\u003e\n\u003cli\u003eReboredo-Fernandez A, Ares-Mazas E, Caccio SM, Gomez-Couso H (2015) Occurrence of Giardia and Cryptosporidium in wild birds in Galicia (Northwest Spain). Parasitology 142:917\u0026ndash;925. https://doi.org/10.1017/S0031182015000049.\u003c/li\u003e\n\u003cli\u003eAsghari A, Motazedian MH, Asgari Q, Shamsi L, Sarkari B, Shahabi S, Mohammadi-Ghalehbin B (2022) Occurrence, genetic characterization, and zoonotic importance of Giardia duodenalis in various species of rodents (Mus musculus, Rattus norvegicus, and Rattus rattus). Comp Immunol Microbiol Infect Dis 101812. https://doi.org/10.1016/j.cimid.2022.101812.\u003c/li\u003e\n\u003cli\u003eAsghari A, Mahdavi F, Shamsi L, Motazedian MH, Asgari Q, Shahabi S, Mohammadi-Ghalehbin B, Sadrebazzaz A (2022) Prevalence and molecular characterization of Giardia duodenalis in small ruminants of Shiraz, southwestern Iran: A zoonotic concern. Comp Immunol Microbiol Infect Dis 86:101819. https://doi.org/10.1016/j.cimid.2022.101819.\u003c/li\u003e\n\u003cli\u003eMahdavi F, Sadrebazzaz A, Chahardehi AM, Badali R, Omidian M, Hassanipour S, Asghari A (2022) Global epidemiology of Giardia duodenalis infection in cancer patients: a systematic review and meta-analysis. Int Health 14:5\u0026ndash;17. https://doi.org/10.1093/inthealth/ihab026.\u003c/li\u003e\n\u003cli\u003eMahdavi F, Shams M, Sadrebazzaz A, Shamsi L, Omidian M, Asghari A, Hassanipour S, Salemi AM (2021) Global prevalence and associated risk factors of diarrheagenic Giardia duodenalis in HIV/AIDS patients: A systematic review and meta-analysis. Microb Pathog 160:105202. https://doi.org/10.1016/j.micpath.2021.105202.\u003c/li\u003e\n\u003cli\u003eZhao Z-Y, Li M-H, Lyu C, Meng X-Z, Qin Y-F, Yang X-B, Ma N, Zhao Q, Zhang Y, Jiang J (2022) Prevalence of Giardia duodenalis Among Dogs in China from 2001 to 2021: A Systematic Review and Meta-Analysis. Foodborne Pathog Dis. https://doi.org/10.1089/fpd.2021.0073.\u003c/li\u003e\n\u003cli\u003eLi J, Dan X, Zhu K, Li N, Guo Y, Zheng Z, Feng Y, Xiao L (2019) Genetic characterization of Cryptosporidium spp. and Giardia duodenalis in dogs and cats in Guangdong, China. Parasit Vectors 12:1\u0026ndash;9. https://doi.org/10.1186/s13071-019-3310-5.\u003c/li\u003e\n\u003cli\u003eSulaiman IM, Fayer R, Bern C, Gilman RH, Trout JM, Schantz PM, Das P, Lal AA, Xiao L (2003) Triosephosphate isomerase gene characterization and potential zoonotic transmission of Giardia duodenalis. Emerg Infect Dis 9:1444\u003c/li\u003e\n\u003cli\u003eWang H, Zhang Y, Wu Y, Li J, Qi M, Li T, Wang J, Wang R, Zhang S, Jian F (2018) Occurrence, molecular characterization, and assessment of zoonotic risk of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in Pigs in Henan, Central China. J Eukaryot Microbiol 65:893\u0026ndash;901\u003c/li\u003e\n\u003cli\u003eSahraoui L, Thomas M, Chevillot A, Mammeri M, Polack B, Vall\u0026eacute;e I, Follet J, Ain-Baaziz H, Adjou KT (2019) Molecular characterization of zoonotic Cryptosporidium spp. and Giardia duodenalis pathogens in Algerian sheep. Vet Parasitol Reg Stud Reports 16:100280\u003c/li\u003e\n\u003cli\u003eBouzid M, Halai K, Jeffreys D, Hunter PR (2015) The prevalence of Giardia infection in dogs and cats, a systematic review and meta-analysis of prevalence studies from stool samples. Vet Parasitol 207:181\u0026ndash;202\u003c/li\u003e\n\u003cli\u003eSouza SLP, Gennari SM, Richtzenhain LJ, Pena HFJ, Funada MR, Cortez A, Gregori F, Soares RM (2007) Molecular identification of Giardia duodenalis isolates from humans, dogs, cats and cattle from the state of Sao Paulo, Brazil, by sequence analysis of fragments of glutamate dehydrogenase (gdh) coding gene. Vet Parasitol 149:258\u0026ndash;264\u003c/li\u003e\n\u003cli\u003eKiani-Salmi N, Fattahi-Bafghi A, Astani A, Sazmand A, Zahedi A, Firoozi Z, Ebrahimi B, Dehghani-Tafti A, Ryan U, Akrami-Mohajeri F (2019) Molecular typing of Giardia duodenalis in cattle, sheep and goats in an arid area of central Iran. Infect Genet Evol 75:104021\u003c/li\u003e\n\u003cli\u003eMalekifard F, Ahmadpour M (2018) Molecular detection and identification of Giardia duodenalis in cattle of Urmia, northwest of Iran. In: Veterinary Research Forum. Faculty of Veterinary Medicine, Urmia University, Urmia, Iran, p 81\u003c/li\u003e\n\u003cli\u003eShoorijeh SJ, Sadjjadi SM, Asheri A, Eraghi K (2008) Giardia spp. and Sarcocystis spp. status in pet dogs of Shiraz, Southern part of Iran. Trop Biomed 25:154\u0026ndash;159\u003c/li\u003e\n\u003cli\u003eHomayouni MM, Razavi SM, Asadpour M (2019) Prevalence and molecular characterization of Cryptosporidium spp. and Giardia intestinalisin household dogs and catsfrom Shiraz, Southwestern Iran. Vet Ital 55:311\u0026ndash;318\u003c/li\u003e\n\u003cli\u003eMOSALANEZHAD B, Avizeh R, RAZI JMH, Alborzi AR (2010) ANTIGENIC DETECTION OF GIARDIA DUODENAL IS IN COMPANION DOGS OF AHVAZ AREA, SOUTH-WEST OF IRAN.\u003c/li\u003e\n\u003cli\u003eKhademvatan S, Abdizadeh R, Rahim F, Hashemitabar M, Ghasemi M, Tavalla M (2014) Stray cats gastrointestinal parasites and its association with public health in Ahvaz City, South Western of Iran. Jundishapur J Microbiol 7. https://doi.org/10.5812/jjm.11079.\u003c/li\u003e\n\u003cli\u003eZarebavani M, Pezeshki A, Jamshidi S, Rezaeian M (2006) Study of Giardia infection in cats. Iran J Public Health 35:77\u0026ndash;80\u003c/li\u003e\n\u003cli\u003eTaghipour A, Sharbatkhori M, Tohidi F, Ghanbari MR, Karanis P, Olfatifar M, Majidiani H, Khazaei S, Bahadory S, Javanmard E (2022) Global prevalence of Giardia duodenalis in cattle: A systematic review and meta-analysis. Prev Vet Med 105632. https://doi.org/10.1016/j.prevetmed.2022.105632.\u003c/li\u003e\n\u003cli\u003eTan TK, Low VL, Ng WH, Ibrahim J, Wang D, Tan CH, Chellappan S, Lim YAL (2019) Occurrence of zoonotic Cryptosporidium and Giardia duodenalis species/genotypes in urban rodents. Parasitol Int 69:110\u0026ndash;113. https://doi.org/10.1016/j.parint.2018.12.007.\u003c/li\u003e\n\u003cli\u003eGal\u0026aacute;n-Puchades MT, Trelis M, S\u0026aacute;ez-Dur\u0026aacute;n S, Cifre S, Gos\u0026aacute;lvez C, Sanxis-Furi\u0026oacute; J, Pascual J, Bueno-Mar\u0026iacute; R, Franco S, Peracho V (2021) One health approach to zoonotic parasites: Molecular detection of intestinal protozoans in an urban population of Norway rats, Rattus norvegicus, in Barcelona, Spain. Pathogens 10:311. https://doi.org/10.3390/pathogens10030311.\u003c/li\u003e\n\u003cli\u003eFayer R, Sant\u0026iacute;n M, Trout JM, DeStefano S, Koenen K, Kaur T (2006) Prevalence of Microsporidia, Cryptosporidium spp., and Giardia spp. in beavers (Castor canadensis) in Massachusetts. J Zoo Wildl Med 37:492\u0026ndash;497. https://doi.org/10.1638/06-013.1.\u003c/li\u003e\n\u003cli\u003eMeerburg BG, Singleton GR, Kijlstra A (2009) Rodent-borne diseases and their risks for public health. Crit Rev Microbiol 35:221\u0026ndash;270. https://doi.org/10.1080/10408410902989837.\u003c/li\u003e\n\u003cli\u003ePerec-Matysiak A, Bunkowska-Gawlik K, Zalesny G, Hildebrand J (2015) Small rodents as reservoirs of Cryptosporidium spp. and Giardia spp. in south-western Poland. Ann Agric Environ Med 22.\u003c/li\u003e\n\u003cli\u003eMa X, Wang Y, Zhang H-J, Wu H-X, Zhao G-H (2018) First report of Giardia duodenalis infection in bamboo rats. Parasit Vectors 11:1\u0026ndash;6. https://doi.org/10.1186/s13071-018-3111-2.\u003c/li\u003e\n\u003cli\u003eCostache C, Kalm\u0026aacute;r Z, Colosi HA, Baciu AM, Opriş RV, Gy\u0026ouml;rke A, Colosi IA (2020) First multilocus sequence typing (MLST) of Giardia duodenalis isolates from humans in Romania. Parasit Vectors 13:1\u0026ndash;11. https://doi.org/10.1186/s13071-020-04248-2.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003ePotential host factors associated with \u003cem\u003eGiardia\u003c/em\u003e infection in cattle\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.874439461883405%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePotential host factors\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.457399103139014%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. examined animals (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.869955156950674%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. positive (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.798206278026905%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.874439461883405%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge (years)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026ge;1\u003c/p\u003e\n \u003cp\u003e\u0026le;1\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eGender\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.457399103139014%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e43\u003c/p\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003cp\u003e57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.869955156950674%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4 (9.3)\u003c/p\u003e\n \u003cp\u003e4 (7.7)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e3 (7.9)\u003c/p\u003e\n \u003cp\u003e5 (8.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.798206278026905%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026gt;0.05\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026gt;0.05\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-parasitic-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jopd","sideBox":"Learn more about [Journal of Parasitic Diseases](https://www.springer.com/journal/12639)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jopd/default.aspx","title":"Journal of Parasitic Diseases","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Giardia duodenalis, Prevalence, Domestic animals, Cattle, Dog, Cat","lastPublishedDoi":"10.21203/rs.3.rs-4326318/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4326318/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDomestic animals play a vital role in the spread and transmission of various zoonotic agents to humans. \u003cem\u003eGiardia duodenalis\u003c/em\u003e, an important enteric zoonotic parasite, is widespread globally. This research aimed to assess the presence and genetic diversity of \u003cem\u003eG. duodenalis\u003c/em\u003e assemblages in domestic animals in Shiraz, southern Iran. 245 fresh fecal samples were collected from 87 dogs, 63 cats, and 95 cattle in Shiraz, the capital city of Fars province, between July 2021 and August 2022. None of the animals showed gastrointestinal signs such as diarrhea, and all fecal samples had normal consistency. Upon parasite observation using light microscopy, a DNA fragment of \u003cem\u003eG. duodenalis tpi\u003c/em\u003e gene was amplified using nested-PCR. Using direct wet mount and Wheatley\u0026rsquo;s trichrome staining examination, 9 out of 245 samples (3.7%; 1 from dogs and 8 from cattle) tested positive for \u003cem\u003eG. duodenalis\u003c/em\u003e. Molecular methods confirmed 1.1% (1/87) of dogs and 8.4% (8/95) of cattle to be positive. Giardiasis was not detected in cats. Phylogenetic analysis revealed that \u003cem\u003eGiardia\u003c/em\u003e isolates infecting dogs and cattle in Shiraz belonged to three genotypes/assemblages: A, B (zoonotic), and E (non-zoonotic). In cattle, assemblages E (75%; 6/8), A (12.5%; 1/8), and B (12.5%; 1/8) were identified, while dogs were infected with assemblage A (100%; 1/1). In Shiraz, southwestern Iran, domestic animals like cattle and dogs could potentially serve as reservoirs for zoonotic infections caused by \u003cem\u003eG. duodenalis\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Assessing the Public Health and Zoonotic Impacts of Giardia duodenalis Assemblages in Domestic Animals of Southwestern Iran","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-03 05:02:16","doi":"10.21203/rs.3.rs-4326318/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-04-28T08:52:17+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-27T06:23:29+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Journal of Parasitic Diseases","date":"2024-04-27T06:07:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-26T08:28:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Parasitic Diseases","date":"2024-04-25T19:24:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-parasitic-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jopd","sideBox":"Learn more about [Journal of Parasitic Diseases](https://www.springer.com/journal/12639)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jopd/default.aspx","title":"Journal of Parasitic Diseases","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"2a32a23a-37e0-4532-861d-1de7fd10850a","owner":[],"postedDate":"May 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-02T16:08:45+00:00","versionOfRecord":{"articleIdentity":"rs-4326318","link":"https://doi.org/10.1007/s12639-024-01727-6","journal":{"identity":"journal-of-parasitic-diseases","isVorOnly":false,"title":"Journal of Parasitic Diseases"},"publishedOn":"2024-08-27 15:56:51","publishedOnDateReadable":"August 27th, 2024"},"versionCreatedAt":"2024-05-03 05:02:16","video":"","vorDoi":"10.1007/s12639-024-01727-6","vorDoiUrl":"https://doi.org/10.1007/s12639-024-01727-6","workflowStages":[]},"version":"v1","identity":"rs-4326318","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4326318","identity":"rs-4326318","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}