Domestic cats as overlooked reservoirs for zoonotic parasites: New records and molecular confirmation of Echinochasmus spp., Opisthorchis felineus, Metagonimus romanicus and Physaloptera praeputialis

Abstract

Domestic cats can act as important carrier for numerous zoonotic parasites. This study aimed to investigate the prevalence, diversity, and risk factors associated with digestive and respiratory endoparasites in 338 domestic cats originating from nine Romanian counties between 2017 and 2025. Copro-microscopic examinations using flotation, sedimentation, and larval concentration techniques, complemented by molecular confirmation for unusual findings, revealed an overall parasitic prevalence of 44.1%, comprising 17 species, of which 10 had zoonotic potential. Toxocara cati (26.6%), Ancylostoma spp. (13.3%), Aelurostrongylus abstrusus (9.5%), and Cystoisospora spp. (6.2%) were the most frequently identified parasites. Outdoor access and mouser lifestyle were significantly associated with increased infection risk, whereas other host-related factors showed limited influence. Three potentially zoonotic trematodes, Echinochasmus spp., Opisthorchis felineus, and Metagonimus romanicus were identified in cats from Tulcea County. Physaloptera praeputialis was identified in a cat from Alba County, representing the first confirmed case in Romania and the first molecular confirmation in Europe.

Continuous epidemiological surveillance, improved diagnostic strategies, and public education are essential.

Highlights

• •Ten out of 17 parasites identified in infected cats (58.82%) have zoonotic potential.
• •Toxocara cati, Ancylostoma spp., A. abstrusus, and Cystoisospora spp. predominated.
• •First Romanian and European molecular report of Physaloptera praeputialis in cats.
• •Three zoonotic trematodes were identified in a city adjacent to the Danube Delta.
• •Outdoor access and mouser behavior significantly increased infection risk.

1. Introduction

Parasites of domestic cats (Felis catus), can pose significant threats to both animal and human health [1]. Most digestive and respiratory parasites are frequently transmitted via the oral-fecal route, while some can cause zoonotic infections and pose substantial epidemiological concerns [2]. The spread of parasitic forms (eggs, cysts, or oocysts) is directly correlated with their release in large numbers through feces by infected animals into the environment, and control measures primarily involve systematic anthelmintic treatments. In infected animals, clinical signs range from asymptomatic to severe digestive or respiratory distress, depending on many factors, including age, diet, and environmental conditions, as well as parasite burden and deworming status [3]. Heavy infections by endoparasites can lead to anemia, diarrhea, dehydration, severe weight loss, respiratory distress, or even death [[1], [3]].

Zoonotic food-borne helminths (FBH) refer to a wide range of nematodes, cestodes, and trematodes transmitted through ingestion of infected intermediate or paratenic hosts, contaminated food, or other raw animal products [[4], [5]]. Among these, food-borne nematodes, Toxocara cati, Ancylostoma spp., and Physaloptera spp. infect cats and occasionally humans, causing severe clinical manifestations or even life-threatening symptoms [[1], [6], [7]]. Food-borne trematodes, also known as fish-borne parasites, such as Opisthorchis spp., Echinochasmus spp., and Metagonimus spp., are of particular concern, as they may be accidentally transmitted to fish consumers, in which they can evolve asymptomatically or cause severe disease manifested by fever, diarrhea, abdominal pain, headache, and other symptoms, including acute pancreatitis and liver abscesses [5]. In addition, cestodes like Dipylidium caninum and Echinococcus multilocularis can infect both animals and humans, highlighting the zoonotic potential of feline helminths [1].

Previously, few studies on domestic feline parasites were conducted in Romania and have reported prevalences ranging from 25.2% to 34.3% for digestive endoparasites [[8], [9],10] and between 2.9% and 31.5% for respiratory nematodes [11].

Nationwide epidemiological studies are needed to monitor parasitic infections in domestic cats, determine standard deworming protocols, and develop public awareness campaigns to reduce the risk of zoonotic transmission. The variations in the prevalence of many parasitic diseases are correlated to the climatic, geographical, and socio-economic factors, all of which are in continuous change [12]. Such changes emphasize the need for ongoing studies of endoparasites infecting cats to understand their distribution and prevalence better, and to adapt control protocols to current needs.

Considering all these, the present study aimed to determine the prevalence and diversity of endoparasites (respiratory and digestive) of cats from various geographical areas in Romania, and to assess the associated risk factors.

2. Materials and methods

2.1. Study population

A total of 338 domestic cats were included in this study, originating from Alba (n = 7), Arad (n = 8), Argeș (n = 50), Bihor (n = 14), Botoșani (n = 100), Cluj (n = 85), Hunedoara (n = 7), Maramureș (n = 51), and Tulcea (n = 16) counties from Romania. The cats were enrolled through collaborations with private owners, animal shelters, veterinary medicine students, and caretakers, in order to capture a wide diversity of management conditions and exposure risks. The investigated cats were either kept as pets for companionship, housed in shelters, or kept for their role as “mousers” in rural or peri-urban settings. The “mouser” group comprised cats primarily used for rodent control, with unrestricted outdoor access and increased exposure to prey species, as reported by owners or caretakers.

Both housed and outdoor animals were included, as well as cats with unrestricted outdoor access, to ensure samples from cats of different lifestyles and environmental exposures. The animals were examined between 2017 and 2025 and enrolled with the informed consent of their owners or caretakers. Cats were selected regardless of health status, allowing the inclusion of both clinically healthy animals and cats showing clinical signs potentially compatible with parasitic infections. For each animal, information about the locality, age, sex, neutering status, breed, service, environment, lifestyle, food, deworming, and the presence or absence of clinical manifestations was recorded using a standard data collection form. All collected data were entered into a Microsoft Excel spreadsheet, which served as the primary database for further statistical analysis. Due to the broad age range of the study population, the animals were categorized as follows: young (0–12 months) and adult (>12 months).

2.2. Fecal sample collection and processing

A single fecal sample was collected from each cat included in the study (n = 338). Sample collection was performed either by direct observation of spontaneous defecation followed by immediately collecting the feces from the environment, or directly from the litter box when possible. Only freshly voided feces were collected for ensuring diagnosis accuracy. Each sample was placed in clean, sealed, and individually labeled plastic container to prevent cross-contamination. Samples were stored and transported in thermal boxes with ice packs to preserve parasitic structures and prevent egg or larval degradation. Upon arrival at the laboratory, fecal samples were stored in the refrigerator for up to 48 h to maintain sample integrity.

Each collected sample was macroscopically examined to detect the possible presence of adult parasites or proglottids, and fecal consistency (normal, pasty, liquid, liquid-bloody) was noted as an indicator (indirect) of gastrointestinal affections. For coproscopic analysis, the total amount of feces collected from each cat was not weighed. Each sample was homogenized directly in the plastic container using a single-use wood stick and divided into three approximately equal portions (visual estimation), with one aliquot assigned to each diagnostic method. Coproscopic examinations were performed using three diagnostic techniques: Willis flotation with saturated salt solution (specific gravity 1.18), sedimentation, and the Baermann larval concentration technique [9]. These methods were employed to maximize the detection of a broad range of intestinal and tissue parasites, including nematodes, cestodes, and protozoa.

Identification of the endoparasites was based on morphological features and the size of the detected parasitic forms, as shown by Mircean et al. [9]. When surprising or rare parasitic forms for Romania were detected, the eggs were concentrated from the feces using sedimentation and flotation techniques and further processed for DNA isolation and molecular confirmation.

2.3. Molecular analysis

Genomic DNA was isolated from the concentrated eggs using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Germany) from 200 μl fecal pellets, according to the manufacturer's instructions.

For trematode egg samples, PCR amplification of a ∼ 780 bp fragment of the 18S rDNA gene was performed using the C for/A rev primer pair [13], with a modified thermal profile as described by Hołówka et al. [14].

For Physaloptera praeputialis, as there was a single sequence available in GenBank (18S rDNA, Accession Number MW410927), the primers used were the ones described among the sequence's source modifiers: PHYSA_F 5’-GCGAACGGCTCATTATAA-3′, and PHYSA_R 5’-AATTTCACCTCTCAGCA-3′. The PCR amplification was performed in a gradient, with annealing temperatures ranging between 45 and 55 °C. It consisted of an initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 45 s, annealing [45–55 °C] for 45 s, extension at 72 °C for 45 s, and a final extension at 72 °C for 5 min. Following gel electrophoresis and visual inspection of the attained bands, the optimal annealing temperature was established to be 46 °C. Additionally, the amplification of a ∼ 670 bp fragment of the mitochondrial cytochrome c oxidase I gene (COI) using the COIintF/ COIintR primer pair, as described in the literature [15], was also performed.

All PCR products were bidirectionally sequenced by an external service (Macrogen Europe, NL), and the resulting chromatograms were assembled and compared to other sequences available in the GenBank® database using the Basic Local Alignment Search Tool (BLAST).

2.4. Statistical analysis

Frequency, prevalence, and their 95% confidence interval (CI) were calculated for each identified parasite and overall. Risk factors were analyzed first by univariate analysis using the chi-square test, followed by logistic regression analysis if the p-value in the univariate analysis was ≤0.05. The analyzed risk factors were: age (young ≤1 year, and adults >1 year), environment (urban, and rural), lifestyle (housed, outdoor, housed/outdoor), service (pet, shelter, and mouser), deworming status (yes, and no), digestive signs (yes, and no), and respiratory signs (yes, and no). Few or no parasite-specific positive cases in the housed category led to sparse data and quasi-complete separation, resulting in inflated odds ratios with wide confidence intervals. Epi Info™ version 3.5.1 (CDC, USA) was used for statistics. The distribution map was elaborated using ArcMap 10.6.1 software.

3. Results

Overall, 44.1% (149/338; 95% CI: 38.7–49.6%) of the investigated cats harbored at least one endoparasite, with 17 different species identified (Table 1). Ten (58.82%) out of 17 identified species (from the positive cats) have zoonotic potential, and were identified in 37.57% (127/338, 95% CI: 32.58–42.85) of examined cats. The lifestyle and service seemed to influence the overall prevalence of endoparasites, with significantly higher prevalences in outdoor and mouser cats compared to housed, pet, and shelter cats (Supplementary material 1). No other significant differences were noted.

Table 1.

The frequency, prevalence, and 95% confidence interval (95% CI) of individual parasites by microscopy.

Parasite species	Frequency (n = 338)	Prevalence (%)	95% CI
Giardia duodenalis*	3	0.9	0.2–2.8
Toxoplasma gondii*/Hammondia hammondi	2	0.6	0.1–2.4
Cystoisospora (total)	21	6.2	4.0–9.5
Cystoisospora felis	20	5.9	3.7–9.1
Cystoisospora rivolta	2	0.6	0.1–2.4
Echinochasmus spp.*	5	1.5	0.5–3.6
Opisthorchis felineus*	3	0.9	0.2–2.8
Metagonimus romanicus§	1	0.3	0.0–1.9
Taenia taeniaeformis	5	1.5	0.5–3.6
Dypilidium caninum*	2	0.6	0.1–2.4
Toxocara cati*	90	26.6	22.1–31.7
Toxascaris leonina	8	2.4	1.1–4.8
Ancylostoma spp.*	45	13.3	10.0–17.5
Physaloptera praeputialis*	1	0.3	0.0–1.9
Eucoleus aerophilus*	10	3.0	1.5–5.5
Eucoleus boehmi	1	0.3	0.0–1.9
Aelurostrongylus abstrusus	32	9.5	6.7–13.2
Troglostrongylus brevior	6	1.8	0.7–4.0
Single infection	91	26.9	22.3–32.0
Mixed infections (2–5 parasites)	58	17.2	13.5–21.5
Total parasite infection	149	44.1	38.7–49.6

Legend: *Parasitic species that can cause confirmed zoonoses; ^§Parasitic species that can cause potential zoonoses.

The most frequent endoparasite identified was T. cati (26.6%), followed by Ancylostoma spp. (13.3%), Aelurostrongylus abstrusus (9.5%), Cystoisospora spp. (6.2%), Eucoleus aerophilus (3%), T. leonina (2.4%), Troglostrongylus brevior (1.2%), Echinochasmus spp. (1.5%) (Fig. 1A), Taenia spp. (1.5%), Giardia duodenalis (0.9%), T. gondii/Hammondia spp. (0.6%), O. felineus (0.9%) (Fig. 1B), P. praeputialis (0.3%) (Fig. 1C, E), M. romanicus (0.3%) (Fig. 1D), E. boehmi (0.3%) (Table 1). Ninety-one cats (26.9%, 95% CI: 22.3–32.0) harbored single infections, whereas 58 (17.2%, 95% CI: 13.5–21.5) showed mixed infections. Nematodes constituted the predominant group of parasites detected. In young cats, co-infections involving nematodes and Cystoisospora spp. were most frequently observed.

Fig. 1.

The morphological aspects of the identified Trematoda eggs and the gastric spirurid. A.Echinochasmus spp. eggs. Note the oval shape, tan colour, measuring 98–121 μm. A small operculum is visible at one pole. B. Opistorchis felineus eggs. Note the oval form, dark yellow/brownish parasitic forms, measuring 28–30 μm, operculated at one pole and showing a knot at the opposite one. C. Physaloptera praeputialis embryonated eggs obtained by flotation. Note the elliptical form, measuring between 42 and 50 μm, containing a larva. D. Metagonimus romanicus embryonated eggs, measuring 33–36 μm. Note the oval, yellow-colored eggs, and the operculum at one pole. Eggs contain a miracidium. E. Physaloptera preaeputialis adults recovered from the cat's vomit (photo sent by the owner). Specific cuticle at the posterior end that continues over the worm's body, forming a protective sheath from which the name of the species “praeputialis” was given. This aspect is not visible in other Physaloptera spp. Note the general aspect of a female with dark yellow cement material around the vulvar opening. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Following logistic regression analysis, statistically significant associations were identified for age category with C. felis, T. cati, Ancylostoma spp., and E. aerophilus (Table 2, Supplementary material 1). Regarding environmental factors, significance was observed only for C. felis. Lifestyle showed a significant effect on overall parasite occurrence. When analyzed by service type, significant associations were observed for both total parasite burden and mixed infections (Supplementary material 1). Deworming history was significantly associated with T. cati infection, mixed infections, and the total prevalence (Table 2, (Supplementary material 2).

Table 2.

Multivariable logistic regression analysis of risk factors associated with parasitic infections in cats (OR, 95% CI).

Variable	Category	C. felis	T. cati	Ancylostoma spp.	E. aerophilus	A. abstrusus	Mixed inf.	Total
Age category	Adults	ref.	ref.	2.2 (1.1–4.7)	8.5 (1.1–68.0)	NS	NS	NS
	Young	7.0 (2.0–24.4)	2.9 (1.7–5.2)	ref.	ref.	NS	NS	NS
	P	0.002	0.0002	0.04	0.05	–	–	–
Environment	Urban	ref.	NS	ref.	NS	NS	ref.	NS
	Rural	3.2 (1.0–10.1)	NS	1.7 (0.8–3.5)	NS	NS	1.3 (0.6–2.8)	NS
	P	0.05	–	0.14	–	–	0.46	–
Lifestyle	Housed	ref.	ref.	NS	ref.	ref.	ref.	ref.
	Mixed	4.8E5 (0.0–1.0E12)	1.5 (0.4–6.3)	NS	2.1E5 (0.0- > 1.0E12)	1.0E6 (0.0- > 1.0E12)	6.9 (0.7–72.8)	7.8 (2.1–28.4)
	P	0.97	0.57	–	0.98	0.98	0.11	0.002
	Outdoor	1.1E6 (0.0- > 1.0E12)	1.0 (0.2–4.8)	NS	9.6E5 (0.0- > 1.0E12)	1.8E6 (0.0- > 1.0E12)	5.6 (0.5–64.1)	6.7 (1.7–26.5)
	P	0.97	0.99	–	0.97	0.97	0.17	0.006
Service	Shelter	NS	ref.	ref.	NS	NS	ref.	ref.
	Pet	NS	2.9 (0.9–9.5)	1.5 (0.3–7.7)	NS	NS	8.3 (1.4–49.4)	5.1 (2.0–13.3)
	P	–	0.08	0.65	–	–	0.02	0.0008
	Mousers	NS	4.5 (1.8–11.0)	5.9 (1.7–20.8)	NS	NS	10.6 (2.2–51.5)	3.3 (1.6–6.9)
	P	–	0.001	0.006	–	–	0.004	0.001
Deworming	N/A	NS	ref.	NS	NS	NS	ref.	ref.
	No	NS	0.7 (0.3–1.6)	NS	NS	NS	0.5 (0.2–1.5)	0.7 (0.3–1.5)
	P	–	0.35	–	–	–	0.24	0.30
	Yes	NS	0.2 (0.1–0.5)	NS	NS	NS	0.3 (0.1–0.8)	0.4 (0.2–0.7)
	P	–	0.00001	–	–	–	0.01	0.001
Digestive signs	No	ref.	ref.	NS	NS	NS	ref.	ref.
	Yes	12.3 (4.2–36.1)	3.9 (1.8–8.3)	NS	NS	NS	4.8 (2.2–10.7)	3.0 (1.3–6.6)
	P	0.00001	0.0006	–	–	–	0.0001	0.008
Respiratory signs	No	NS	ref.	ref.	NS	ref.	ref.	ref.
	Yes	NS	3.2 (1.0–11.0)	0.4 (0.1–3.3)	NS	13.9 (4.4–44.0)	7.1 (2.1–23.7)	3.5 (0.9–13.5)
	P	–	0.06	0.40	–	0.00001	0.002	0.07

Fig. 2 (map) shows the origin of the cats included, as well as the location of the positive cat to P. praeputialis and those positive for trematodes.

Fig. 2.

Map showing Romania's shape and the bordering countries. Grey triangles represent the locations of investigated cats. The red circle represents the location of the positive cat for P. praeputialis. Blue circle represents the location of positive cats for trematodes. One symbol represents the origin of multiple cats. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Sequence analysis revealed 100% nucleotide identity with other GenBank entries in the case of M. romanicus (with PP378514, isolated from a golden hamster in Hungary), and O. felineus (with MF077357, isolated from a cat in Russia). In case of Echinochasmus sp., the sequence had a similarity of 97.39% with E. milvi (LT904765, isolated in Russia), and between 97.39 and 98.4% with E. japonicus (LT904764, isolated in Russia; PQ824438, isolated from a stone moroko in China; OR509030, isolated from a human in Vietnam).

For P. praeputialis, the 18S rDNA sequence showed the highest similarity (98.75%) with an isolate from a cat in India (MW410927), followed by P. sibirica (98.51%), isolated from hog badgers in China (OQ846900-OQ846902). The COI sequence was closest to a Physaloptera sp. (89.16%) recovered from a bobcat in the USA (PV369934), followed by P. calusa (86.31%), isolated from a hedgehog in China (NC_086779). The sequences will be deposited in GenBank.

4. Discussion

Besides showing that more than 50% of the identified parasites are zoonotic, the present study provides comprehensive data on digestive and respiratory parasites identified in 44.1% of examined cats across various groups. This overall prevalence is slightly higher but still comparable to previous studies conducted in Romania [[9], [11]]. The high prevalence could be attributed to the lack of deworming [9]; however, it is surprising that the prevalence is slightly higher than it was 15 years ago, when routine deworming in companion animals and particularly in cats was not routinely done, and owners were less informed about the zoonotic risks.

Overall, no significant age-related differences in overall prevalence were observed between the two age groups, which showed nearly identical values (44.6% vs. 43.6%). This contrasts with the age-related patterns previously reported in Romanian cats [[8], [9]]. In general, a trend toward slightly lower prevalences in older cats is reported, showing that parasitic exposure over the years might have contributed to a cumulative immune development or could be related to the more frequent or repeated dewormings in older, owned cats [3]. Nevertheless, younger cats were 2.9 and 7 times more likely to be infected with T. cati and C. felis, respectively, than adults. In contrast, adult cats were 2.2 and 8.5 times more likely to be infected with Ancylostoma spp. and E. aerophilus, respectively, than younger cats.

The lifestyle (housed/outdoor) seems to be one of the two factors associated with endoparasites, with cats with outdoor access being 6.74–7.8 times more likely to be at infection risk than those kept housed. Extremely wide confidence intervals of the odds ratios for C. felis, E. aerophilus, and A. abstrusus reflect sparse parasite-specific events rather than strong biological effects. This finding was not surprising, as it is now generally accepted and consistent with numerous other epidemiological studies linking outdoor exposure to greater environmental contamination and possible contact with intermediate or paratenic hosts [[8], [9]]. The significantly higher prevalence among mousers (OR: 3.34) further supports the risks of hunting behavior in parasite transmission [7], particularly for species such as T. cati, and Ancylostoma spp.

As previously shown [9], T. cati was the most prevalent endoparasite (26.6%), followed by hookworms (13.3%) and A. abstrusus (9.5%). The predominance of zoonotic T. cati is consistent with the global literature, which consistently reports this species as the most prevalent feline endoparasite due to its direct life cycle and wide range of transmission routes [[8], [12]]. Hookworm infections were mainly influenced by environmental and behavioral factors, being more frequent in rural, mousers, and outdoor cats, where soil contamination favors larval persistence. Considering the poor sensitivity of coproscopy in the detection of cestode infections, mainly by D. caninum and Taenia spp. [16], the real prevalence was most likely underestimated in this study, with only 2 cats confirmed.

Among protozoans, Cystoisospora spp. infections confirmed that coccidiosis remains common in younger and free-roaming cats, while T. gondii/H. hammondii was identified in only 2 cats.

The most common respiratory nematode, A. abstrusus, was associated with outdoor lifestyle and respiratory clinical signs, consistent with its life cycle involving gastropod intermediate hosts, but identified in higher prevalences than in previous studies [9].

Additionally, compared with the available data from 2010 [9], the present study provides a temporal updated situation of parasite occurrence in domestic cats and extends the geographical range by including samples from South and Eastern Romania, like Argeș (south), Botoșani (north-eastern), and Tulcea (south-eastern), the latter being a city adjacent to the Danube Delta and known for its major fluvial-maritime port. Such an expanded area resulted in the novel identification of trematode species not previously reported, as well as the identification of a gastric spirurid, representing new, significant records.

The identification of Echinochasmus spp. in 5 cats from Tulcea is a novelty and of particular importance due to rare reports in Europe and the first report in a cat from Romania. Although a sequence was obtained, due to the scarcity of GenBank entries, a high similarity to Asian isolates was noted, but the molecular identification down to the species level was not possible. However, based on the morphological aspects and the size of the eggs, as well as the molecular data (which is highly suggestive of the genus Echinochasmus), and the previous identification of E. perfoliatus in dogs from Romania in 1902 (mentioned in Hu et al. [17]), one could state that E. perfoliatus cannot be excluded in these cases. Echinochasmus definitive hosts include wild carnivores such as wolves and red foxes, domestic dogs and cats, domestic pigs, fish-eating birds, and humans [17].

The identification of O. felineus in 3 domestic cats, including two with co-infection with Echinochasmus spp. and one with M. romanicus from Tulcea County, provides further evidence of the persistence of opisthorchiid flukes in the Danube Delta basin. Opisthorchis felineus is transmitted by ingestion of raw or insufficiently cooked freshwater fish containing metacercariae, after development in Bithynia spp. snails and cyprinid fish [18]. The occurrence of O. felineus in domestic cats strongly suggests local circulation of the parasite's larval stages in aquatic ecosystems of Tulcea County, most likely transmitted via the Danube River. A very recent case from Austria, near the Danube as well, was published [19]. This finding correlates with the old evidence of opisthorchiid infections in 0.66% humans, 3.3% dogs, and 13% pigs (Ungureanu et al., [20]) as well as in cats and Bithynia snails from the same region, Danube Delta, Romania (Erhardt et al., 1962[5]). In the latest study from 1967, 35.9% of examined pigs from Tulcea were confirmed infected by examination of the carcasses, with all harboring trematodes in the pancreas and none in the liver (Igrițan [21]). No other cases have been reported since then in humans, nor in animals. In humans, opisthorchiasis can lead to acute febrile illness, cholangitis, cholelithiasis, and, in chronic infections, even cholangiocarcinoma [[5], [18]].

Originally described as Loossia romanica from a domestic dog in Somova, Tulcea County, Romania, M. romanicus [22,23] was not reported since then in definitive hosts in Romania. Similar to Echinochasmus spp., it is an intestinal trematode with a wide distribution among the Danube cities, parasitizing piscivorous birds and fish-eating mammals. More recently, data from intermediate and paratenic hosts include reports in the bordering countries (Hungary, Serbia, Bulgaria and Ukraine) [[24], [25]]. The identification of M. romanicus in a cat from Romania supports its persistence in the Danube River over the decades and highlights the importance of continuous parasitic surveillance.

All three trematode species are associated with complex life cycles involving aquatic or semi-aquatic environments and the presence of specific intermediate and paratenic hosts, which are characteristic of Tulcea County in the Danube Delta [18]. These findings raise concern about the potential emergence or re-emergence of zoonotic trematodes in Romania, as no recent reports have been published for several decades for O. felineus. The reappearance or the lack of reports of O. felineus could reflect changes in local ecosystems, such as the introduction or proliferation of suitable snail and fish hosts, increased movement of animals, or could be due to the lack of studies on this subject. Metagonimus romanicus has not yet been reported in humans, possibly due to the traditional lack of raw-fish consumption in the geographic areas where this species circulates, rather than a true lack of zoonotic capacity. Moreover, of 16 cats examined from Tulcea, 37.5% (n = 6) harbored at least one trematode. Such a high prevalence highlights the need for improved surveillance and awareness of feline trematodes with public health implications.

Another very important finding of this study was the identification of P. praeputialis in one cat from Alba County. Although the prevalence was low, this gastric nematode has considerable epidemiological importance due to its indirect life cycle, being transmitted through ingestion of infected intermediate hosts, such as beetles, or paratenic hosts, including reptiles and small mammals, and its zoonotic potential. Its occurrence in a cat from Romania is highly important from an epidemiological point of view, as in cats, only one previous report from Europe was published, in a cat from Greece (mentioned in Gupta and Gupta, [26]). The non-nodular cat stomach worm, P. praeputialis was first described from a domestic cat in Brazil (Linstow [27]), and it is currently reported mainly from North to South America, but also in South Asia and South Africa [26]. The infection is clinically associated with vomitus and melena [28], vomitus being reported by the owners of the present cat as well, but no blood in the feces was observed. Additionally, the infection can be fatal in wild captive felines, as shown in UK, in a zoo tiger more than 100 years ago [29]. The real prevalence of P. praeputialis in Romania as well as in Europe can be underestimated due to the difficulties in diagnosing it using standard coproscopic techniques [26].

It should be specified that the authors are aware that only a single fecal sample was collected from each cat, which may have led to an underestimation of the true prevalence of parasitic infections, as the elimination of most parasitic forms/elements is intermittent, and repeated sampling over several days should have been done to improve diagnostic sensitivity. Additionally, the real prevalence could have been underestimated due to the low sensitivity of coproscopical techniques for some of the identified parasitic forms, like Giardia spp. and cestodes [[16], [30]]. Moreover, the long study period (2017–2025) might have introduced temporal heterogeneity due to potential changes in deworming practices, diagnostics, or even environmental conditions that could influence prevalence estimates and risk factor analysis.These results provide new insights into the parasitic fauna of Romanian cats and highlight the potential reemergence of zoonotic food-borne trematodes in Romania. There is a clear need for continuous epidemiological monitoring, public awareness, and the integration of veterinary and human health data under the One Health approach.

5. Conclusions

This study revealed a high prevalence and diversity of parasitic zoonoses in domestic cats from Romania, particularly among animals originating from rural areas or those with unrestricted outdoor access. The identification of three zoonotic trematodes in cats from Tulcea is of major epidemiological relevance, suggesting that these fish-borne trematodes may represent re-emerging zoonotic threats in the region after decades without reported cases.

This paper reports for the first time the presence of P. praeputialis in a domestic cat from Romania and offers the first molecular confirmation of this nematode species in Europe.

The occurrence of zoonotic helminths in cats reinforces the importance of continuous monitoring in domestic animals, raising awareness in pet owners to reduce environmental contamination and limit the transmission to humans. Under the One Health framework, collaboration between veterinarians, veterinary clinics, animal hospitals, faculties of veterinary medicine, and human doctors is crucial for prevention, control, and early detection of emerging or re-emerging zoonotic diseases.

Ethics approval for animal samples

The methodology and the use of data were approved by the bioethical committee of the University of Agricultural Sciences Veterinary Medicine of Cluj-Napoca (Permit nr.: 210 from 12.03.2020).

CRediT authorship contribution statement

Georgiana Deak: Writing – original draft, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation. Adriana Gyӧrke: Writing – review & editing, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. George-Iulian Enacrachi: Methodology. Angela Monica Ionică: Writing – review & editing, Writing – original draft, Methodology, Formal analysis. Viorica Mircean: Writing – review & editing, Validation, Supervision, Investigation, Data curation, Conceptualization.

Declaration of generative AI and AI-assisted technologies in the writing process

Before publication, the authors used https://app.grammarly.com/ to correct any grammatical errors, punctuation, and typing mistakes.

Funding

The molecular biology work done by Georgiana Deak was supported by a grant of the Ministry of Research, Innovation, and Digitization from Romania, CNCS-UEFISCDI, project number PN-IV-P2-‐2.1-TE-2023-0054, within PNCDI IV.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

Supplementary material

Data availability

Data will be made available on request.