A parasitological survey of zoonotic cestodes carried by house rats in Aswan, Egypt, reveals cryptic diversity at the molecular level

Abstract Background and Aim: Some rat cestodes are zoonotic and are capable of parasitizing humans and animals, raising serious concerns regarding human and veterinary health. The study aimed to determine the prevalence and risk factors for cestodes in Egyptian house rats and to characterize the cestodes molecularly. Materials and Methods: The current survey examined 115 house rats (Rattus rattus) in two cities (Edfu and Aswan) in Egypt’s Aswan Governorate for cestode infection using integrated molecular approaches (polymerase chain reaction, sequencing, and phylogenetic analysis) and morphological/morphometrical approaches. Results: The cestodes identified in this study exhibited the typical morphological characteristics of Hymenolepis diminuta (Rudolphi, 1819), Hymenolepis nana (Siebold, 1852) (from rat intestine), and Hydatigera taeniaeformis (from rat liver). The species prevalence rates from these three studies were reported to be 8.7%, 10.4%, and 20.9%, respectively. The ribosomal DNA (ITS1, 18S, and complete ITS) sequences revealed that the hymenolepid sequences were highly distinct but were related to other sequences in the GenBank database, with some sequences showing high similarities to those of H. nana and H. diminuta. In addition, the H. taeniaeformis sequences (ITS2 and mitochondrial cytochrome c oxidase subunit 1 [mtCOX1]) obtained in this study were highly similar to some Taenia taeniaeformis GenBank sequences. The constructed phylogram revealed that the hymenolepidid tapeworms examined in this study were classified into four major branches (the majority of which were hybrids of the two species) and belonged to the genus Hymenolepis. In addition, the phylogram of H. taeniaeformis assigned this species to T. taeniaeformis. Conclusion: When typical hymenolepid morphology is combined with molecular and phylogenetic divergence, it may indicate the existence of possible cryptic species. In addition, on the basis of the phylogenetic analysis, genetic diversity within T. taeniaeformis may exist as determined by comparing the metacestode mtCOX1 sequences. The current study presents the prevalence values of zoonotic cestodes and contributes to the body of knowledge, including identification keys and the use of molecular tools for species confirmation.


Introduction
Around 25% of the world's population may be infected with transmissible parasitic infections, which are typically prevalent in tropical and subtropical regions. Such infections lead to reduced worker productivity and the depletion of economic resources [1]. Rodent-borne helminthiases are neglected diseases that disproportionately affect (but are not limited to) residents of low-and middle-income countries. These cause a variety of health problems in humans, including malnutrition, increased prevalence of malaria and HIV/AIDS, decreased vaccine effectiveness, and increased incidence of allergies [2].
The house rat, Rattus rattus, is one of the world's most widespread mammals [3]. It is regarded as a serious pest in urban and rural areas due to the economic damage it causes and the fact that it harbors and spreads zoonotic pathogens such as viruses, bacteria, protozoa, and helminths [4][5][6].
Rodents can serve as intermediate hosts for cestode parasites and can transmit them to other animals such as cats, in the case of the parasite Hydatigera taeniaeformis (Taenia taeniaeformis), or as reservoir hosts, in the case of Hymenolepis spp. [7]. Hymenolepiasis is a zoonotic parasitic infection spread by the cestodes Hymenolepis nana and Hymenolepis diminuta. Of these, H. nana is by far the most prevalent because it is frequently transmitted directly to children through contaminated hands, dust, food, and water. Human infections with H. diminuta are uncommon and typically result from accidental ingestion of small arthropod intermediate hosts. The family Hymenolepididae contains over 920 species of tapeworms that infect birds and mammals [8].
H. taeniaeformis is the larval stage of the adult tapeworm T. taeniaeformis (alternative names: Available at www.veterinaryworld.org/Vol.14/August-2021/24.pdf Cysticercus fasciolaris, Hydatigera fasciolaris, Strobilocercus fasciolaris, or T. crassicolis), which is found worldwide and primarily infects the small intestines of felines and canines following the ingestion of infected rodent livers [9]. H. taeniaeformis is a potential zoonotic parasite. Although this cestode can infect humans, this is a rare occurrence and has only been reported in individuals from Argentina, the Czech Republic, Denmark, Taiwan, and Sri Lanka [10][11][12].
Molecular biology techniques such as polymerase chain reaction (PCR) and sequencing can easily identify parasites [13,14]. However, the taxonomic and systematic statuses of Hymenolepididae and Taeniidae, as well as their family and generic phylogenetic relationships, remain unknown [15]. At present, the use of nuclear ribosomal DNA (rDNA) and mitochondrial cytochrome c oxidase subunit 1 (mtCOX1) as markers makes these methods powerful tools for resolving exceptional taxonomic challenges and separating closely related genera and species [16].
Recognizing cestode distribution and population genetics is critical for controlling them since it influences their systematics, epidemiology, genesis, and ecology [17]. The house rat likewise plays a critical role in understanding the epidemiology of diseases transmitted by cestode hosts. Inadequate urban infrastructure and sanitation significantly affect rat abundance and increase the rate of rat-human contact. Thus, residents of underprivileged urban areas are more susceptible to rat-borne infections, with drug users, the immunocompromised, and the homeless being the most disadvantaged [4,18,19]. However, few studies on rodent polyparasitism have been conducted in Egypt, particularly in the Upper Egypt region. There is currently no information on rodent tapeworms in Egypt's Aswan Governorate, an ancient region of international tourist importance that serves as the country's southernmost region and gateway to Africa.
This study aimed to conduct a prevalence survey in two locations (Edfu and Aswan) in the Aswan Governorate to ascertain the presence of cestode parasites in house rats and the effect of house rats on cestode parasite transmission. We additionally performed molecular and phylogenetic analyses on nuclear and mitochondrial loci to validate our identifications, examine the rates of genetic variation and differentiation, and augment the cestode genetic dataset.

Ethical approval
The current study was approved by Aswan University's Research and Graduate Studies Council. It follows a standard operating procedure that has been approved by Aswan University's Animal Use and Care Committee.

Study period and area
The study was conducted from January 2016 to December 2019 in the residential communities of Aswan and Edfu in Egypt's Governorate of Aswan. This covers an area of 62,726 square kilometers. Edfu, Aswan's northernmost city, is located on the border with Luxor, while Aswan, Egypt's southernmost city, and is located on the Sudanese border ( Figure-1) [20]. The two neighborhoods are located in a rural/urban area with a middle socioeconomic status and are defined by paved streets, a variety of small businesses, middle-class housing, and vacant lots. In these neighborhoods, pets (i.e., dogs and cats), weeds, shrubs, and trees are common.

Animals and parasites
Wire traps were baited with baked cheese and distributed throughout the yard near areas where rodent activity was observed. Fifty traps were set daily for 3 days at each trapping site, comprising approximately 2 km of land. A total of 115 house rats (45 in Edfu and 70 in Aswan) were captured and identified in the field using a morphological rodent identification key [21] and immediately transported to the laboratory. This study used only house rats, and any other species that were captured were released. Rats were then anesthetized and killed in a small container of ether. The rats were dissected immediately after killing, and their internal organs were immersed in physiological saline and examined for tapeworm infections. Any tapeworms found were extracted from the infected rats, washed with saline, and examined under a light stereomicroscope for initial identification. Individual tapeworm fragments were stored at −20°C in 70% ethanol for molecular experiments. The remainder of each tapeworm was fixed in neutral buffered formalin for further morphological confirmation.

Examination of morphology
Each rat's isolated tapeworm species were counted to determine the prevalence and intensity of infection. Mature eggs of Hymenolepididae were obtained from terminal gravid proglottids in saline solution. The fixed cestode parasites were then sequentially fixed, stained with alum carmine, dehydrated in ascending grades of alcohol, cleared in xylene, and finally mounted in Dibutylphthalate Polystyrene Xylene. The tapeworms were then morphologically and morphometrically examined using a light microscope (Olympus CX41, Japan) and photographed using a digital camera (Olympus DP74) attached to the microscope (Olympus BX43). Drawings were created using a drawing tube, and measurements in millimeters were taken using a light microscope. The tapeworms were then identified using morphological taxonomic Key [22].

Molecular analysis
Genomic DNA (gDNA) extraction gDNA was isolated from individual ethanol-preserved tapeworms using a slightly adapted phenol/ chloroform protocol [23]. In brief, the parasite materials were digested with proteinase K in ALT buffer overnight at 56°C (Dneasy Kit, Qiagen) and then precipitated with 5.2 M ammonium acetate. The samples were diluted in 20-50 μL of dH 2 O according to pellet size, and concentrations of gDNA samples were determined spectrophotometrically using a Nanodrop spectrophotometer (Implen NP80, Germany).

Primer design and PCR
The following markers and primers were used: A. Hymenolepis spp.

Sequencing
The PCR products were purified according to the manufacturer's protocol using the DNA Clean and Concentrator Kit (Zymo Research, USA) or QIAquick ® PCR Purification Kit (Qiagen, Hilden, Germany). Purified PCR products were sequenced in both directions (forward and reverse) using the same primers as in the initial PCR using a dideoxy termination method (Macrogen Inc., Korea) and an Applied BioSystems sequencer automated DNA sequencing system (Model 3730XL) (Applied BioSystems, USA).

Sequence and phylogenetic analysis
The NCBI Blast program was used to conduct homology searches on each sequence (http://www. ncbi.nlm.nih.gov/). Each forward sequence was then compared to its reverse complement and manually adjusted and assembled using the CAP3 program. Following that, the generated sequences were aligned to each other and to the most homologous sequences in the database using the multiple sequence alignment program CLUSTALW. Phylogenetic trees were constructed using the online tool Phylogeny.fr, aligned using MUSCLE (v3.8.31), and optimized for maximum accuracy (MUSCLE with default settings). After alignment, ambiguous regions (i.e., containing gaps and/or misaligned) were removed using Gblocks (v0.91b). The phylogenetic tree was then reconstructed using the PhyML software's maximum likelihood approach (v3.1/3.0 aLRT). Finally, TreeDyn (v198.3) was used to graphically represent and edit the phylogenetic tree [27].

Morphological identification
The morphological characters depicted in Figures H. nana was identified as 10 small cestodes found in the rat's small intestinal lumen, and these were creamy white and measured 35 mm (ranging from 27.5 to 42.5 mm) in total body length. The body consisted of a small scolex with an armed rostellum and a multistrobila that was wider than its length. H. diminuta was identified as eight slightly longer cestodes found in the rat's small intestinal lumen, and these were creamy white and measured approximately 330 mm (ranging from 220 to 450 mm) in total body length. The body consisted of a small scolex and a series of long multistrobila with greater length    The strobila consisted of short segments that terminated in a bladder, resembling a small tapeworm but lacking reproductive organs. All of these morphological characteristics were consistent with the larval stage of T. taeniaeformis (Figure-4).

Prevalence and distribution
In our survey, we identified 115 rats as R. rattus based on morphological characteristics. Of these, 42 rats (36.52%) were found to be infected with cestodes. Two species of hymenolepidid cestodes were identified in the rat intestines, namely, H. nana and H. diminuta, which were found in 10.43% and 8.70%, respectively, of the total number of rats. We additionally detected the taeniid H. taeniaeformis cyst stage in 20.87% of the examined rat livers (Table-1).
Table-2 shows the distribution of cestode infection in house rats for two sites in the Aswan Governorate. All three detected species were discovered at both of the sites investigated. H. nana and H. diminuta were both prevalent in Edfu City, accounting for more than 57% and 35% of infected rats, respectively, and H. taeniaeformis was more prevalent in Aswan than in Edfu. Aside from a few cases where two species coinfected a single rat, the rats were mostly infected by a single species. No rat was found to be coinfected by all three species (Table-3).

Molecular analysis
The ITS1, complete ITS, and 18S amplicons obtained from the gDNA samples of H. diminuta and H. nana were 900, 1500, and 1050 bp, respectively (Figure-5a). Three H. diminuta sequences were generated and deposited into GenBank for validation under the accession numbers MF143798 (ITS1), MF067416 (complete ITS), and MT448710 (18S). Blast analyses of the three sequences revealed that they were distinct from all other sequences in the GenBank databases. The ITS1 and 18S sequences bore no resemblance to other H. diminuta sequences. However, the complete ITS sequence had a high identity (92%) to two H. diminuta sequences (accession numbers MK787167 and MK787168) in the GenBank databases with 48% query coverage. Interestingly, both the newly submitted ITS1 and the complete ITS sequences demonstrated 100% identity with 54% coverage due to the fact that ITS1 is a fragment of the Only one of the designed primer pairs (CesS2F/ CesS2R) successfully amplified the H. taeniaeformis ITS2 rDNA marker. Amplicons of 250 bp (ITS2) and 200 bp (mitochondrial COX1) were generated (Figure-5b). Two sequences were obtained and submitted to GenBank for validation under the accession numbers MT454664 (ITS2) and MT512661 (COX1). A blast search of the ITS2 sequence revealed 98.84% identity to T. taeniaeformis (GenBank accession number FJ939133). Additional blast analyses of the COX1 sequence revealed a high degree of identity (99.53%) to eight T. taeniaeformis sequences (GenBank accession Available at www.veterinaryworld.org/Vol.14/August-2021/24.pdf  Figure-6). A constructed phylogram revealed that the hymenolepidid tapeworms examined in this study comprised four major branches, mostly mixed with both species, with no effect on their geographical distributions. These were found to be embedded within the genus Hymenolepis with considerable divergence, particularly in H. diminuta.
An additional phylogenetic tree was constructed by comparing the metacestodes mtCOX1 sequences to the most closely related T. taeniaeformis sequences obtained from GenBank (Figure-7).

Discussion
This study is the first to use integrated molecular and morphological approaches to characterize cestodes from house rats (R. rattus) in Aswan, Egypt. The cestodes detected in this study were classified as belonging to H. nana, H. diminuta, and H. taeniaeformis of the order Cyclophyllidea. These findings corroborate Owen's assertion [28] that rat cestodes are all Cyclophyllideans and belong to the Hymenolepididae and Taeniidae families in particular. Our measurements of H. diminuta and H. nana morphology independently verify Younis' description [29] and Hughes' key [30] to Hymenolepis species.
We identified two hymenolepid species from rat small intestines in the Aswan Governorate. We found that H. diminuta had a similar prevalence in our study (8.70%) as it did in a similar study (11.67%) in which these species were observed in rats of Egypt's Assiut Governorate [29]. However, our observed H. diminuta prevalence was lower than that of [31], who reported H. diminuta infection prevalence in 15.3% of R. rattus. In addition, H. nana had a prevalence of 10.4% in our study, which was similar to the prevalence rate of 13.33% reported in the Assiut Governorate [29]. We detected infection with H. taeniaeformis at a prevalence of 20.9%, which is consistent with the previous study in Rattus spp. in the Assiut Governorate [29], but higher than the prevalence of 7.8% reported in the same host in Yucatan Mexico [9] and of 7.4% reported in Rattus spp. in Iran [32]. On the other hand, the present study detected a lower prevalence of H. taeniaeformis in rats than did similar studies in Argentina (39.5%) [33], Korea (33.8%) [34], and Grenada, West Indies (67.6%) [35]. Although the prevalence of helminth infection in rats varied between studies, these variations may be attributed to the differences in climatic conditions, environmental hygiene, previous control interference, socioeconomic status of the occupants, intermediate host abundance, and host vulnerability to parasite infection [2,36].
The morphological characteristics of the present H. taeniaeformis specimens are consistent with previous work in Egypt [29] and in India [37], who detected and described metacestode infection in R. rattus. We detected pseudosegmentation along the strobila and a small terminal bladder, correlating with [9,38], who reported H. taeniaeformis to be the only metacestode in which the scolex is not invaginated at the bladder but rather attached to it through strobilae.
Our molecular analysis revealed that the H. nana sequences were distinct but related to other sequences from the same species in the GenBank database, whereas ITS1 and complete ITS showed 98.2% and 94.3% similarity, respectively, to H. nana sequences obtained from Australia [39]. In addition, the 18S sequence was 97.3% identical to H. nana isolated from a human in London [40]. Although our complete ITS sequence of H. diminuta showed 92% similarity to H. diminuta obtained from a rodent in Iraq [41], the ITS1 and 18S sequences showed no similarity to other hymenolepid reference sequences in the GenBank database.
Our phylogenetic analyses assigned H. diminuta and H. nana to four hybrid branches, indicating the presence of possible cryptic species. These findings corroborate those of Nkouawa et al. [42], who reported that identifying and diagnosing hymenolepidid tapeworms is challenging, most likely due to the presence of cryptic species.
The metacestode larval stage sequences obtained in this study were highly similar to T. taeniaeformis sequences in GenBank, particularly the ITS2 sequence, which was found to be highly similar (98.9%) to the ITS2 sequence of T. taeniaeformis obtained from R. rattus in India [37]. In addition, the mtCOX1 sequence was found to be highly similar (99.53%) to the COX1 sequence of T. taeniaeformis collected from cats in Georgia, USA [43]. Given the phylogenetic tree's relative branch lengths and the nodal support that connects them, it is possible that genetic diversity exists within T. taeniaeformis. This finding corroborates the findings of Lavikainen et al., [44] Jia et al. [45], who discovered significant sequence variations in isolate gene markers of T. taeniaeformis that are predictive of cryptic species.
The current study confirms that rodents pose a parasitic threat to both human [46,47] and veterinary [48] health, and corroborates Dyab's report [49] that some rodent-borne parasites were detected in Aswan schoolchildren, including 3% H. nana infection.

Conclusion
This study sheds light on the potential public health and veterinary consequences of house rats in Egypt's Aswan Governorate. Three zoonotic cestodes were identified and characterized: H. diminuta, H. nana, and H. taeniaeformis, all of which exhibit molecular characteristics consistent with the presence of cryptic species. This study contributes to the narrowing of the knowledge gap on rat-borne helminthiases by providing additional references to identification keys and the use of molecular tools for species confirmation. However, additional research on the dynamics and socio-environmental risk factors of human-rat-helminth infection remains necessary.

Authors' Contributions
AEY: Supervisor who contributed to the technique and practical aspects of molecular biology, as well as data analysis and interpretation. AIS: Head supervisor who developed the idea and the study design and contributed to the morphological section's explanation. This study is a part of IRME's Master's thesis, for which he conducted the majority of the practical experiments. NMKS: Assistant supervisor who assisted with the methodology and practical aspects of morphological identification. All authors drafted and revised the manuscript then read and approved the final manuscript.