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 oC. 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.