Genetic Identification of Parasitic Giardia enterica in Three Wild Rodent Species from a Zoological Institution: First Host Records in Brazilian Porcupine (Coendou prehensilis) and Naked Mole Rat (Heterocephalus glaber), and Detection in Crested Porcupine (Hystrix cristata)

Abstract

Flagellates of the genus Giardia are intestinal parasites with a broad host range. Several Giardia duodenalis variants (assemblages) recently elevated to species rank—G. duodenalis (assemblage A1), G. intestinalis (A2) and Giardia enterica (B) are human pathogens. Giardia enterica has been reported in some hystricomorph rodents such as wild crested porcupines (Hystrix cristata), but no data were previously available from Brazilian porcupines (Coendou prehensilis) and naked mole rats (Heterocephalus glaber). The aim of this study is to genetically identify the Giardia isolates from these three rodent species, all housed in a zoological institution. Fecal samples were processed using the Bailenger concentration method, and DNA was extracted from the sediments using commercial kits. Partial PCR amplification and sequencing of the glutamate dehydrogenase, beta-giardin, and triose-phosphate isomerase genes revealed that all isolates belonged to G. enterica, showing 99–100% identity with sequences available in GenBank. Prevalences could not be reliably estimated due to small group sizes and the resulting proportions may be biased. To our knowledge, this is the first report identifying Giardia (G. enterica) in C. prehensilis and H. glaber, thus expanding the known host range of this parasite species and reinforcing the importance of surveillance in captive wild hosts.

1. Introduction

Rodents (order Rodentia) represent the most diverse mammal group, comprising nearly 2600 described species [1] accounting for more than 40% of currently living mammal species [2]. A defining feature of the group is a pair of open-rooted, chisel-shaped incisor teeth in each jaw, adapted for gnawing and requiring continuous wear to maintain functionality. The systematic of the order remains under revision; the suborders Sciuromorpha and Hystricomorpha are widely accepted, while a third suborder, Supramyomorpha, includes three infraorders (Myomorphi, Castorimorphi, and Anomaluromorphi) [1] which were previously classified as distinct suborders (Myomorpha, Castorimorpha and Anomaluromorpha) [2].

The Hystricomorpha, characterized by a hystricomorphous zygomasseteric system (in which the medial masseter muscle extends anteriorly through a greatly enlarged infraorbital foramen to insert on the lateral surface of the rostrum) [3] includes 17 families [1]. Among these are the Old World and New World porcupines and the African mole rats. A distinctive feature of porcupines is the presence of quills (modified hairs coated with thick keratin plates) located on the dorsal region and serving as a defense mechanism against predators. Old World porcupines (family Hystricidae), such as the crested porcupine (Hystrix cristata) and the Indian crested porcupine (Hystrix indica) are large rodents (reaching up to 27 kg in the crested porcupine). They are fossorial and of nocturnal habits. Primarily herbivorous, they can also consume insects and small vertebrates. They are frequently found in anthropogenic agricultural systems and are known to damage crops, thus being regarded as pests by farmers [4,5]. Consequently, they may play a role in the transmission of parasites to and from domestic animals and humans. In contrast, New World porcupines (family Erethizontidae) are smaller (the Brazilian porcupine, Coendou prehensilis, weighs approximately 4 kg), arboreal, and adapted to feeding in trees. In the wild, they have limited contact with humans; however, certain species, such as the Brazilian porcupine, are easily maintained in captivity in zoological gardens, increasing their potential contact with humans. With respect to the mole rats, this term refers to two families: Heterocephalidae (which includes a single genus and species, the naked mole rat Heterocephalus graber) and Bathyergidae. They are small, mouse-sized African rodents that inhabit complex underground tunnel systems and feed on tuberous plants. Their relevance to human interaction has increased in recent years due to their emergence as important animal models in biomedical research due to their eusocial behavior, unique ecophysiological adaptations, and exceptional longevity (up to 30 years in captivity) accompanied by sustained good health most of their lifespan [6,7].

Zoological institutions maintain a wide range of wild species under human supervision and care, facilitating closer contact between animals, zoo personnel, and visitors. These conditions can favor cross-species transmission of pathogens, particularly for parasites with direct life cycles, such as Giardia [8]. Monitoring gastrointestinal parasites in captive wildlife is therefore essential, not only for ensuring animal health but also for assessing zoonotic risks and preventing potential outbreaks [9]. This is especially relevant in zoological institutions, where the possibility of reverse zoonosis [9,10] may affect the health of captive populations and potentially impact wild populations if infected animals are released for reintroduction in the wild. Despite the importance of these issues, genetic characterization of Giardia isolates from captive zoo animals remains limited, particularly among lesser-studied rodent groups.

In routine parasitological monitoring of animals in zoological institutions in Madrid, Spain, Giardia cysts have been found in crested porcupines [11] and, more recently, in Brazilian porcupines and naked mole rats. The genus Giardia comprises diplomonadid flagellate protozoan parasites that infect a wide range of terrestrial vertebrates, including species parasitizing amphibians (Giardia agilis), reptiles (Giardia varani), birds (Giardia ardeae, Giardia psittaci) and mammals (Giardia duodenalis sensu lato, Giardia muris, Giardia microti, Giardia peramelis, and Giardia cricetidarum) [12]. These parasites have a direct life cycle, with eight-flagellated, binucleated trophozoites inhabiting the small intestine of the host, and four-nucleated cysts as the infective stage.

The taxonomy of G. duodenalis sensu lato has long been a subject of considerable debate, with three different names (G. duodenalis, Giardia intestinalis, and Giardia lamblia) being used in the scientific literature: G. lamblia has traditionally been favored in medical literature, whereas G. duodenalis has been more prevalent in non-medical contexts, with G. intestinalis generally regarded as a synonym in most cases [13]. The application of molecular tools has revealed extensive genetic variability within the species, leading to the identification of genetically distinct variants (assemblages) with varying host specificities, and ultimately to the proposal of several distinct species [14]: G. duodenalis sensu stricto (=G. duodenalis assemblage A1), G. intestinalis (=G. duodenalis A2), Giardia enterica (=G. duodenalis B), Giardia canis (=G. duodenalis C), Giardia lupus (=G. duodenalis D), Giardia bovis (=G. duodenalis E), Giardia cati (=G. duodenalis F), and Giardia simoni (=G. duodenalis G). Two additional assemblages (A3 and H) have been suggested as new species (Giardia cervus and Giardia pinnipedis, respectively), though they have not yet been formally proposed [14]. In citing identifications from previous studies, we will adopt this taxonomic framework and refer to these species names, rather than by assemblage letter designations. Giardia duodenalis, G. intestinalis, and G. enterica are all recognized as etiological agents of human giardiasis, a common gastrointestinal disease characterized by symptoms such as diarrhea, vomiting, and abdominal pain. Giardiosis is among the most prevalent gastrointestinal human infections worldwide, affecting an estimated 180 million people annually [15], and is responsible for numerous outbreaks in both low- and high-income countries [16].

In rodents, G. muris, G. microti, G. simoni, and G. cricetidarum have been identified through genetic analyses in species belonging to the Supramyomorpha-Myomorphi clade, primarily within murids and cricetids. However, these species have not been reported in members of Supramyomorpha-Castorimorphi, Hystricomorpha, or Sciuromorpha. In contrast, G. duodenalis sensu lato has been detected across all these clades, with a predominance in Hystricomorpha and Sciuromorpha (

Table 1. Species of Giardia identified in rodent hosts by using molecular markers.

Rodent Order-Suborder	Family	Common Name	Rodent Genus	Giardia Species *	References
Sciuromorpha	Sciuridae	Squirrels	Callosciurus	G. enterica	[19]
			Spermophilus	G. enterica	[20]
		Chipmunks	Eutamias	G. duodenalis, G. simoni	[21]
		Marmots	Marmota	G. duodenalis, G. enterica, G. bovis	[20]
		Prairie dogs	Cynomys	G. duodenalis, G. intestinalis, G. enterica	[22]
Hystricomorpha	Caviidae	Guinea pigs	Cavia	G. enterica	[23]
		Patagonian cavy	Dolichotis
	Chinchillidae	Chinchillas	Chinchilla	G. duodenalis, G. intestinalis, G. enterica, G. canis, G. lupus, G. bovis	[24,25,26,27,28]
	Echimyidae	Nutrias	Myocastor	G. duodenalis, G. enterica	[29]
		Hutias	Capromys	G. enterica	[26]
	Hystricidae	Old World porcupines	Hystrix	G. enterica	[17,18,30]
Supramyomorpha
Myomorphi	Cricetidae	Hamsters	Mesocricetus	G. cricetidarum	[31]
		Dwart hamsters	Phodopus	G. muris, G, cricetidarum, G. duodenalis s.l.	[31]
		Voles	Arvicola	G. microti	[32]
			Clethrionomys	G. microti, G. duodenalis, G. intestinalis	[33]
			Eothenomys	G. microti	[31]
			Microtus	G. microti, G. intestinalis	[32,33,34,35]
			Myodes	G. microti, G. duodenalis/G. intestinalis, G. enterica	[33]
		Muskrats	Ondrata	G. microti, G. enterica, G. canis	[36,37]
		Deer mice	Peromyscus	G. microti	[38]
	Muridae	Mice	Apodemus	G. microti, G. duodenalis s.l.	[32,33,35]
			Mus	G. microti, G. duodenalis s.l., G. simoni	[23,32,39,40]
		Rats	Niniventer	G. muris	[35]
			Rattus	G. microti, G. enterica, G. simoni	[23,31,35,39,40]
	Spalacidae	Bamboo rats	Rhizomys	G. enterica	[41]
Castorimorphi	Castoridae	Beavers	Castor	G. duodenalis, G. enterica	[36,42]

* The identifications as G. duodenalis assemblages made by some authors have been renamed in the table as the corresponding species G. duodenalis (assemblage A1), G. intestinalis (A2), G. enterica (B), G. canis (C), G. lupus (D) or G. simoni (G). following Wielinga et al. (2023) [14]. When the assemblage is not indicated, the identification is noted as sensu lato (s.l.).

). For the family Hystricidae (Old World porcupines), genetic data are available only from a few wild individuals in Europe [17,18], while no molecular data are currently available for isolates from Erethizontidae (New World porcupines) or mole rats. The aim of the present study is to genetically identify Giardia isolates from these hosts. To our knowledge, this is the first time that Giardia has been genetically identified to the species level in the Brazilian porcupine and the African naked mole rats. These findings contribute to the epidemiological understanding of Giardia infections in rodents and help assess the potential role of these hosts as reservoirs for zoonotic transmission.

2. Materials and Methods

2.1. Sample Collection and Microscopical Analysis

The present study is based on Giardia-positive fecal samples collected in 2024 from crested porcupines, Brazilian porcupines, and naked mole rats housed at Faunia, a zoological park located in Madrid (Spain). These are the samples now analyzed to achieve the genetic identification of Giardia isolates in these host species. All animals were kept in isolated enclosures designed to mimic their natural habitats, with controlled lightning and humidity conditions. In all three species, animals were separated from visitors by transparent glass panels, preventing any direct visitor-animal contact.

Sample collection was performed by zookeepers early in the morning during routine cleaning and environmental maintenance procedures; no direct handling of the animals occurred during the collection of samples used in this study. Care was taken to avoid contamination with soil or plant debris. For the porcupine species, animal groups were formed by two individuals and fecal samples in each sampling event were obtained from both of them. In the case of the naked mole rats, which were housed as a colony of 11 individuals, pooled fecal boluses were collected from the “toilet chamber” of the colony. Samples were stored in clean, new plastic containers and transported to the laboratory within 1–3 h. after collection for immediate processing. Fecal concentrates were prepared using the sodium acetate-ether stool concentration technique [43]. Briefly, a small amount of each sample (2–3 g for the porcupine samples, 10–15 fecal pellets for the naked mole rats) was homogenized in 30 mL of acetate-acetic acid buffer (1.5% sodium acetate and 0.36% acetic acid in distilled water), then filtered through a metal sieve. The filtrate was mixed 1:1 with diethyl ether, vortexed for 30 s, and centrifuged at 1500 rpm for 2 min. The resulting aqueous sediment was examined microscopically on temporary slides, either unstained or stained with Lugol’s iodine. Cysts were photographed and measured using an Olympus DP20 camera mounted on an Olympus BX51 microscope (Olympus, Tokyo, Japan).

2.2. DNA Extraction, Gene Selection and Amplification

One microscopically positive sample from each host species was further processed for genetic analysis. DNA was extracted using the Speedtools tissue DNA extraction kit (Biotools B&M Labs S.A., Madrid, Spain) following the manufacturer’s instructions. Extracted DNA was either used immediately for PCR amplification or stored at −20 °C until used. Following Capewell et al. (2011) [44], partial fragments of four genes commonly used for Giardia genotyping were amplified by nested or semi-nested PCR and subsequently sequenced: small subunit rRNA (SSU-rRNA), beta-giardin (bg), triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh). All PCR amplifications were performed by using the PureTaq^TM Ready-To-Go^TM PCR beads (Merck KGaA, Darmstadt, Germany) in a final volume of 25 µL, containing 2 µL of each primer solution and 5 µL of template DNA (either total DNA extracted from the sample or the first-round PCR product). Reactions were carried out in an Eppendorf Master Cycler Gradient thermocycler (Eppedorf AG, Hamburg, Germany).

For the SSU-rRNA gene, the amplicon obtained from an initial PCR using forward primer Gia2029 (5′-AAGTGTGGTGCAGACGGACTC-3′) and reverse primer Gia2150c (5′-CTGCTGCCGTCCTTGGATGT-3′) was used as a template for a second (nested) PCR with forward primer RH11 (5′-CATCCGGTCGATCCTGCC-3′) and reverse primer RH4 (5′-AGTCGAACCCTGATTCTCCGCCAGG-3′), generating a 292 bp fragment [45]. The thermocycler was programmed as follows: initial denaturation at 96 °C for 4 min, followed by 35 cycles of 96 °C for 45 s denaturation), 55 °C (first PCR) or 59 °C (nested PCR) for 30 s (annealing), and 72 °C for 45 s (extension), with a final extension at 72 °C for 4 min [45].

Amplification of a 511 bp fragment of the bg gene was achieved using a primary PCR with forward primer G7 (5′-AAGCCCGACGACCTCACCCGCAGTGC-3′) and reverse primer G759 (5′-GAGGCCGCCCTGGATCTTCGAGACGAC-3′) [46], followed by a secondary PCR with forward primer 5′-GAACGAGATCGAGGTCCG-3′ and reverse primer 5′-CTCGACGAGCTTCGTGTT-3′ [47]. The thermocycling conditions were: initial denaturation at 95 °C for 15 min, 35 cycles of 95 °C for 30 s, 65 °C (primary PCR) or 55 °C (secondary PCR) for 30 s, and 72 °C for 1 min, with a final extension of 72 °C for 7 min [47].

For the tpi fragment (530 bp fragment), a primary amplification was performed using forward primer AL3543 (5′-AAATTATGCCTGCTCGTCG-3′) and reverse primer AL3546 (5′-CAAACCTTTTCCGCAAACC-3′), followed by a second amplification with forward primer AL3544 (5′-CCCTTCATCGGTGGTAACTT-3′) and reverse primer AL3545 (5′-GTGGCCACCACTCCCTGTCC-3′) [36]. PCR conditions for both rounds included an initial denaturation at 94 °C for 5 min, followed by 35 cycles of 94 °C for 45 s, 50 °C for 45 s, and 72 °C for 60 s, with a final extension of 72 °C for 10 min [36].

Amplification of a fragment of 432 bp of the gdh gene was conducted via an initial PCR with forward primer GDHeF (5′-TCAACGTYAAYCGYGGYTTCCGT-3′) and reverse primer GDHiR (5′-GTTRTCCTTGCACATCTCC-3′), followed by a semi-nested PCR using forward primer GDHiF (5′-CAGTACAACTCYGCTCTCGG-3′) and reverse primer GDHiR [48]. Thermocycling conditions for both PCR amplifications were: initial denaturation at 94 °C for 2 min, followed by 55 cycles of 94 °C for 30 s, 56 °C for 20 s, and 72 °C for 45 s, with a final extension of 72 °C for 7 min [48].

2.3. Sequence Analysis and Comparisons

In all cases, final PCR products obtained after the second (nested or semi-nested) amplification were visualized on 1% agarose gels stained with Pronasafe (Condalab, Torrejón de Ardoz, Spain) using a UV transilluminator (NuGenius Syngene, Cambridge, UK). Amplicons were purified using the QIAquick^®® PCR Purification Kit (Qiagen, Hilden, Germany) and sequenced using an AbiPrism 3730XL DNA Analyzer (Applied Biosystems, now ThermoFisher Scientific, Waltham, MA, USA). Chromatograms were analyzed with ChromasPro ver. 2.1.10 (Technelysium Pty Ltd., South Brisbane, Australia) and compared against sequences in GenBank using the blastn algorithm available on the Nacional Center for Biotechnology Information website (https://blast.ncbi.nlm.nih.gov/Blast.cgi; last accessed on 25 May 2025).

Phylogenetic trees were constructed separately for each gene to enable comparative analyses between the sequences obtained in this study and those previously published from Giardia isolates in rodents. For this purpose, Giardia sequences from rodent hosts, as well as reference sequences for G. duodenalis, G. intestinalis, and G. enterica [14], including subtypes BIII and BIV of G. enterica [49], were retrieved from GenBank and aligned using the Muscle algorithm [50] implemented in MEGA-X [51]. For each gene, alignments were trimmed to match the length of the second-round amplicons; sequences covering less than 50% of alignment positions were excluded from the analysis. The best-fitting nucleotide substitution model was selected for each gene based on both the Akaike information criterion and the Bayesian information criterion, as calculated in MEGA-X. Phylogenetic trees were generated using the Neighbor-Joining method; boostrap resampling was performed 1000 times to assess branch support.

3. Results

3.1. Microscopical Findings

During 2024, several fecal samples tested positive for Giardia cysts: 3 out of 13 samples (23.1%) from crested porcupines, 3 out of 14 (21.4%) from Brazilian porcupines, and 1 out of 3 (33.3%) from naked mole rats. In both porcupine species, only one animal from each group (not always the same individual each time) was found to be infected at any given sampling event. For naked mole rats, the number of infected individuals could not be determined, as samples were collected as pooled feces and individual testing was not possible. In all cases, fecal consistency was normal and no clinical signs were observed in the animals.

The cysts exhibited typical morphological characteristics (Figure 1). They were observed in large numbers in the concentrated fecal sediments from crested porcupines, whereas they were scarce in the fecal concentrates from the Brazilian porcupine and the naked mole rat.

3.2. Genetic Analyses and Comparisons

PCR amplicons of the expected size were successfully obtained for all four genetic markers. However, the sequences corresponding to the SSU-rRNA fragments were illegible due to the presence of numerous double and triple peaks in the chromatograms. In contrast, the sequences obtained for the other markers were clear and unambiguous. Only in the case of the gdh marker was a consistent nucleotide ambiguity (Y = C/T) observed across all isolates, located at the same position as the ambiguity (R = G/A) in the GDHiR primer. The sequences obtained in this study have been deposited in the GenBank/EMBL/DDBJ databases under accession numbers PV391923-PV391925 (partial bg gene), PV391926-PV391928 (partial tpi gene) and PV391929-PV391931 (partial gdh gene).

A comparison of the sequences obtained from the different host species revealed that they were not identical, with some nucleotide differences observed across all three markers (bg, tpi, and gdh) (

Table 2. Condensed alignment of beta-giardin sequences obtained from Giardia isolates of Brazilian porcupine, crested porcupine, and naked mole rat (highlighted in bold), compared with the reference Giardia enterica sequence (ACGJ01002392). Nucleotide positions are numbered according to the alignment shown in Supplementary File S1.

	Position
Sequence	97	232	301
ACGJ01002392 G. enterica (strain GS/M)	R	T	C
AHHH01000111 G. enterica (strain GS)	G	T	C
PV391923 (Brazilian porcupine)	G	C	T
PV391924 (crested porcupine)	G	C	T
PV391925 (naked mole rat)	G	C	C

,

Table 3. Condensed alignment of triose-phosphate isomerase sequences obtained from Giardia isolates of Brazilian porcupine, crested porcupine, and naked mole rat (highlighted in bold), compared with the reference Giardia enterica sequences (ACGJ01002000 and AHHH01000009). Nucleotide positions are numbered according to the alignment shown in Supplementary File S2.

	Position
Sequence	24	30	76	153	161	195	256	265	282	387	519
ACGJ01002000 G. enterica (strain GS/M)	A	T	T	T	G	A	C	A	A	A	T
AHHH01000009 G. enterica (strain GS)	A	T	T	T	G	A	C	A	A	A	T
PV391926 (Brazilian porcupine)	A	T	C	T	G	A	C	A	A	G	A
PV391927 (crested porcupine)	G	C	C	C	A	G	T	G	G	G	A
PV391928 (naked mole rat)	A	T	T	T	G	A	C	A	A	A	A

and

Table 4. Condensed alignment of glutamate dehydrogenase sequences obtained from Giardia isolates of Brazilian porcupine, crested porcupine, and naked mole rat (highlighted in bold), compared with the reference Giardia enterica sequences (ACGJ01002929 and AHHH01000018). Nucleotide positions are numbered according to the alignment shown in Supplementary File S3.

	Position
Sequence	120	309	333	405	429
ACGJ01002929 G. enterica (strain GS/M)	C	C	C	C	T
AHHH01000018 G. enterica (strain GS)	C	C	C	C	T
PV391929 (Brazilian porcupine)	T	C	C	T	Y
PV391930 (crested porcupine)	C	A	C	C	Y
PV391931 (naked mole rat)	C	C	T	C	Y

, Supplementary Files S1–S3). Comparisons with sequences available in GenBank confirmed that all isolates corresponded to G. enterica for each of the three markers. For the tpi gene, the sequences obtained were identical to previously deposited sequences. However, for the bg and gdh genes, novel sequence variants were identified (

Table 5. Comparisons of beta-giardin, triose phosphate isomerase, and glutamate dehydrogenase sequences obtained from Giardia isolates of Brazilian porcupine, crested porcupine, and naked mole rat (highlighted in bold) with reference sequences of Giardia duodenalis, Giardia intestinalis and Giardia enterica (highlighted in bold), and well as sequences available in GenBank using the blastn algorithm.

Beta-giardin Gene	G. duodenalis	G. intestinalis	G. enterica	G. enterica	Other Highly Similar
	AACB03000002	AHGT01000121	ACGJ01002392	AHHH01000111	Giardia Sequences
PV391923 (Brazilian porcupine)	93.94% (479/511)	93.43% (478/511)	99.61% (509/511)	99.61% (509/511) *	100.00% (511/511) AB618785 (human isolate)
					100.00% (511/511) FJ009209 (anteater isolate)
PV391924 (crested porcupine)	93.74% (479/511)	93.43% (478/511)	99.61% (509/511)	99.61% (509/511) *	100.00% (511/511) AB618785 (human isolate)
					100.00% (511/511) FJ009209 (from anteater)
PV391925 (naked mole rat)	93.93% (480/511)	93.73% (479/511)	99.80% (510/511)	99.80% (510/511) *	100.00% (511/511) KU504703 (human isolate)
					100.00% (511/511) LC865371 (human isolate)
Triose-phosphate isomerase Gene	AACB03000001	AHGT01000004	ACGJ01002000	AHHH01000009
PV391926 (Brazilian porcupine)	80.00% (424/530)	80.38% (426/530)	99.43% (527/530)	99.43% (527/530)	99.43% (527/530) LC865535 (human isolate)
					99.43% (527/530) EU637591 (barbary macaque isolate)
PV391927 (crested porcupine)	80,57% (427/530)	80.94% (429/530)	97.92% (519/530)	97.92% (519/530)	99.25% (526/530) KM190834 (human isolate)
					98.68% (523/530) MH310971 (human isolate)
PV391928 (naked mole rat)	79.62% (422/530)	80.94% (429/530)	99.81% (529/530)	99.81% (529/530)	99.81% (529/530) KM190822 (beaver isolate)
					99.81% (529/530) HG970113 (human isolate)
Glutamate dehydrogenase Gene	AACB03000002	AHGT01000014	ACGJ010022929	AHHH01000018
PV391929 (Brazilian porcupine)	90,28% (390/432) **	89,81% (388/432) **	99.31% (429/432) **	99.31% (429/432) **	99.77% (431/432) HM134209 (Alouatta fusca isolate)
					99.77% (431/432) HM134211 (Alouatta fusca isolate)
PV391930 (crested porcupine)	90,28% (390/432) **	89,81% (388/432) **	99.31% (429/432) **	99.31% (429/432) **	99.54% (430/432) HM134215 (ostrich isolate)
					99.54% (430/432) KM190702 (beaver isolate)
PV391931 (naked mole rat)	90,51% (391/432) **	90,05% (389/432) **	99.54% (430/432) **	99.54% (430/432) **	99.77% (430/431) LC430563 (human isolate)
					99,54% (429/431) EF5076821 (human isolate)

* The ambiguity R (G/A) at position 97 of sequence AHHH01000111 is not considered a difference, as it is compatible with the nucleotides present at the corresponding position in the compared sequences. ** The ambiguity Y (C/T) at position 429 of sequences PV391929-PV391931 is not considered a difference, as it is compatible with the nucleotides present at the corresponding position in the reference sequences.

; Supplementary Files S1–S3). In phylogenetic analyses, the sequences clustered consistently within the G. enterica clade. A condensed tree based on the bg gene is shown in Figure 2 as representative of the results obtained for each gene; full phylogenetic trees for the bg, tpi, and gdh markers are provided in Supplementary Files S4–S6.

4. Discussion

The present results confirm that the crested porcupine can serve as a natural host for G. enterica, and provide the first genetic evidence of G. enterica infecting the Brazilian porcupine and the naked mole. Although this parasite is known to cause giardiasis in humans, no apparent clinical signs were observed in any of the infected rodents sampled in this study.

Zoo animals are particularly susceptible to infections by parasites with direct life cycles due to stress and the increased likelihood of encountering infective stages in confined environments. Additionally, there is potential for reverse zoonosis or “spill-back” transmission from humans (such as caretakers and visitors) to animals [9,10]. In a previous study [11], Giardia was detected in crested porcupines at a low frequency (8.5%) over a ten-year period, which increases to 13% when the current findings are included. The observed prevalence in the present study (50% in each positive sampling event) may be biased due to the small number of individuals sampled (n = 2), but it is comparable to prevalence in wild animals (38–100%) [30,52]. A similar limitation applies to the Brazilian porcupines, where only two individuals were sampled. Despite this, the prevalence observed (21.4% of samples testing positive over the year, and 50% of positive animals in the positive sampling events) is in line with previous findings in wild New World porcupines: in one study, one out of two Coendou villosus individuals tested positive for Giardia cysts [53], and in another, two out of ten individuals were infected [54]. In naked mole rats, determining prevalence would require individual handling of colony members, which was not conducted in the present study. The only previous report of Giardia in wild naked mole rats dates back several decades [55], describing a small number of trophozoites in the intestine and cysts in feces. To our knowledge, no other reports exist for Giardia infection in captive colonies of this species, nor in wild Cryptomys mole rats [56] or in captive Fukomys damarensis (Damaraland mole rats) [57].

The reasons for the failure to obtain readable sequences of the SSU-rRNA gene remain unclear. Nested or semi-nested PCR approaches are commonly employed to enhance the sensitivity and specificity of amplification, particularly when working with fecal DNA, which may contain low concentrations of target DNA, and PCR inhibitors [44]. In each case, the second-round PCR (nested or semi-nested) used a new set of internal primers and the product from the first round as a template, thereby minimizing the likelihood of non-specific amplification [36,45,47,48]. Given that high-quality sequences were obtained for the other markers (which are single-copy genes), it is unlikely that the SSU-rRNA gene amplification failure was due to low DNA concentration, poor DNA quality, or the presence of PCR inhibitors. It is therefore more plausible that the issue was related to amplicon purification or suboptimal sequencing conditions. Since the bg, gdh, and tpi markers yielded clear and informative sequences for genotyping, additional attempts to sequence the SSU-rRNA gene were not pursued, particularly in light of its lower discriminatory power for differentiating assemblages or species within the G. duodenalis complex [44]. The results obtained from the other gene markers are consistent with previous studies that identified G. enterica (G. duodenalis assemblage B) in Old World porcupines using SSU-rRNA, tpi, and bg gene sequences [17,18,30,58]. Notably, an infection by G. intestinalis (G. duodenalis assemblage A2) was also reported in wild crested porcupines in Italy [30], although the authors did not publish sequence data, preventing direct comparison.

Importantly, a comparative analysis of the sequences obtained from the three host species revealed that each was infected by a different G. enterica variant/genotype. In the case of the crested porcupine, the sequences differed from those previously reported in wild animals from Italy [17,18]. In natural environments, crested porcupines are exposed to a broad range of sympatric wildlife, including other reservoirs, and seasonal variation in habitat use and diet, all of which may influence both the risk of exposure and the selective pressures acting on circulating parasite strains. In contrast, zoo environments are characterized by increased host density, greater contact with humans (caretakers, visitors), artificial diets, reduced environmental microbial diversity, and potential cross-species transmission from other captive animals or contaminated sources. Such conditions may favor the establishment or persistence of different Giardia species and subspecific variants genotypes, or may facilitate the introduction and maintenance of variants not typically encountered in natural habitats. The present findings support this hypothesis and underscore the potential for captive environments to shape or concentrate specific parasite variants/genotypes of zoonotic relevance.

No genetic data are currently available for comparison in the case of the Brazilian porcupine and the naked mole rat. In Brazilian porcupines, previous reports have referred to the parasite as Giardia sp. [53,59] or as G. intestinalis [54], based solely on morphological identification. In naked mole rats, the parasite was identified as Giardia cuniculi [55], a junior synonym of G. duodenalis sensu lato. Both of these identifications in Brazilian porcupines [54] and naked mole rats [55] are compatible with the present identification as G. enterica.

Although the number of animals in the groups analyzed in this study is small, the objective was not to assess infection intensity or prevalence but rather to achieve molecular identification of Giardia isolates from zoo-housed rodent species that had not previously been genetically characterized. Accordingly, no attempts were made to quantify cysts in fecal samples, and as previously discussed, reported prevalence values may be biased. Notably, in both porcupine species, each group tested positive for Giardia on several occasions over time (although not always in the same individual), suggesting persistent infection within the group. Although only one positive sample per species was subjected to molecular analysis, and the possibility of additional genotypes circulating over time cannot be excluded, the sequence obtained from the crested porcupine matches those previously identified in wild individuals [17,18,30]. In contrast, the sequences from the isolates of Brazilian porcupine and naked mole rats represent the first available genetic data for Giardia in these host species. These findings provide a valuable reference for future comparative and epidemiological studies involving both captive and wild populations.

It is noteworthy that the sequences obtained from the three rodent species were not identical, indicating that different transmission events likely occurred; however, the specific routes of transmission remain unknown. The earliest detection of Giardia cysts in crested porcupine at this zoo was in 2020 [11], and in 2023 for the Brazilian porcupine [60]. All samples from naked mole rats had consistently tested negative until the current findings. It could be hypothesized that crested porcupines were initially infected and that the parasite was subsequently transmitted to other species through mechanical carriers (e.g., insects or fomites such as zookeepers’ footwear), through infected local microfauna (e.g., small rodents), or potentially via infected zookeepers. However, given that distinct sequence variants were identified in each host species, it is more plausible that independent transmission events involving different G. enterica lineages occurred.

Transmission of the parasite from these animals to humans can be considered unlikely in the current zoo setting, as the animals are housed in isolated enclosures with minimal human contact. Only trained zookeepers enter the enclosures, and they do not directly interact with the animals except during veterinary check-ups. Enclosure cleaning is performed according to established protocols, and fecal material is disposed of under controlled conditions. Nevertheless, transmission risks cannot be completely excluded, and zoo animal-human transmission has been documented in other institutions [61]. In the present case, further investigation is needed within this zoological facility to clarify the potential routes of introduction and transmission of the parasite. Additionally, studies in porcupines and naked mole rats from other zoological institutions and wild populations are needed to better assess their potential role as zoonotic reservoirs of Giardia spp.

While further research and future discoveries may reveal additional Giardia genotypes or species in previously unreported hosts, there appears to be an association between G. muris, G. microti, and G. cricetidarum and rodents of the suborder Myomorphi. In contrast, within other rodent taxa, the predominant (and in some cases, the only) species identified belong to the G. duodenalis sensu lato complex, which is also capable of infecting myomorphic rodents. To date, no Giardia species have been reported in rodents of the suborder Anomaluromorphi (springhares, scaly-tailed flying squirrels, and Zenkerella).

5. Conclusions

This study provides the first genetic identification of G. enterica in the Brazilian porcupine and the naked mole rat, and confirms the presence of this parasite in captive individuals of the crested porcupine. Although all isolates were identified as G. enterica through three independent genetic markers (tpi, gdh, and bg), the sequences differed among host species and from sequences available in GenBank, suggesting independent infection events and previously undescribed variants. These findings support the hypothesis that captive environments may influence the circulation and selection of specific Giardia genotypes, which may differ from those found in wild populations.

No clinical signs of giardiasis were observed in infected individuals, and in both porcupine species, repeated positive results over time suggest persistent colonization. Given the zoonotic potential of G. enterica, ongoing surveillance in both captive and wild rodent populations is warranted, particularly to clarify transmission dynamics and evaluate the role of these hosts as reservoirs. The genetic data generated in this study provide a valuable reference for future epidemiological, comparative, and taxonomic research on Giardia in porcupines and mole rats.