Calodium hepaticum (syn. Capillaria hepatica) infection in synanthropic small mammals (rodents and shrews) in Northeastern India: Zoonotic and One Health implications

Abstract

Rapid urbanization has increased overlap between human and synanthropic rodent habitats. This intensifies human–animal–environment interactions and facilitates the emergence and re-emergence of zoonotic diseases, highlighting the importance of the “One Health” concept. Calodium hepaticum (syn. Capillaria hepatica) is a neglected zoonotic parasite where rodents serve as the principal reservoirs. Human infections are often asymptomatic, but they can also cause liver failure and death in severe cases, with definitive diagnosis requiring liver biopsy. This study was conducted in Meghalaya, Northeastern India, where 173 rodents and shrews representing seven species were captured from synanthropic environments, with B. bengalensis comprising the largest proportion of captured animals (49.13%), followed by B. indica (17.34%), S. murinus (14.45%), M. musculus (8.67%), R. rattus (4.63%), M. booduga (4.05%), and R. norvegicus (1.73%). Liver samples were examined using gross pathology, histopathology, parasitological, and molecular methods, including 18S rRNA gene sequencing and phylogenetic analysis. Histopathological examination revealed non-embryonated barrel-shaped eggs in hepatic tissues, while molecular analysis confirmed the identity of C. hepaticum. Based on molecular detection, the overall prevalence of C. hepaticum was 36.99% (64/173; 95% CI: 30.16–44.44). Species-level prevalence was highest in Bandicota indica (80%; 95% CI: 62.69–90.49), followed by Mus booduga (57.14%; 95% CI: 25.05–84.18), B. bengalensis (31.76%; 95% CI: 22.84–42.27), and M. musculus (26.67%; 95% CI: 10.90–51.95), with lower rates in S. murinus (16%; 95% CI: 6.40–34.65) and R. rattus (12.50%; 95% CI: 2.24–47.09). Given the high prevalence observed, appropriate rodent control and carcass disposal should be encouraged to minimize the potential risk of environmental contamination, as eggs may be released during decomposition, predation, or cannibalism and subsequently embryonate in soil, becoming infective to humans or other susceptible hosts.

Graphical abstract

Highlights

• •High prevalence of Calodium hepaticum in synanthropic small mammals in Meghalaya
• •Molecular prevalence was 40.54% in rodents and 16% in shrews.
• •Highest prevalence was in B. indica (80%), M. booduga (57.14%), and B. bengalensis (31.76%).
• •Findings indicate contamination, zoonotic risk, and carcass role in transmission.

1. Introduction

Environmental health is a key part of the One Health framework. Small mammals living in synanthropic environments such as towns, livestock farms, and crop fields are an important environmental component and can significantly impact public health by acting as reservoirs of zoonotic pathogens [1], [2]. Rodentia represents one of the most species-diverse groups within Mammalia worldwide, comprising 2552 known species and representing approximately 39.3% of all mammalian species [3], [4]. Apart from their beneficial ecological roles, such as soil aeration and insect control, some rodent species are of considerable public health importance [4]. It is estimated that nearly 10% of the global rodent species serves as carriers or reservoirs of pathogens of zoonotic importance [4], [5], [6]. Rodents readily adapt to human-altered environments and continue to inhabit newly urbanized areas, whereas many other wildlife species are displaced during urban expansion [4], [7]. Rodent-borne zoonotic diseases continue to pose significant global health concerns, as synanthropic rodents constitute an important component of urban and peri-urban wildlife communities and are involved in the maintenance and transmission of diverse zoonotic pathogens, including those responsible for leptospirosis, hantavirus infections, plague, and salmonellosis [8], [9], [10]. Furthermore, environmental and anthropogenic factors, including climate change and habitat alteration, can influence rodent population density, distribution, species composition, and vector abundance, thereby increasing opportunities for pathogen transmission [11], [12], [13]. Rodent-associated pathogens can be transmitted to humans through direct contact, arthropod vectors, or contamination of crops, stored food, and water sources with excretions and secretions from infected rodents, thereby compromising food and water safety and increasing the risk of disease outbreaks [2], [4], [8], [14], [15]. This zoonotic potential is further illustrated by studies of synanthropic Rattus norvegicus in New York City detected a diverse range of zoonotic pathogens, including atypical enteropathogenic Escherichia coli, Clostridium difficile, Salmonella enterica, Bartonella spp., Streptobacillus moniliformis, Leptospira interrogans, and Seoul hantavirus [10].

More than 200 species of parasites are recognized as zoonotic, posing a substantial burden on global public health [16], [17]. A number of zoonotic parasites for which rodents serve as reservoir or intermediate hosts have been well documented, including helminths such as Calodium hepaticum and Trichinella spiralis, as well as protozoan parasites such as Toxoplasma gondii and Leishmania spp., which are associated with significant human diseases [18]. In addition, rodent ectoparasites such as Xenopsylla cheopis and Ixodes spp. play important roles in the transmission of severe diseases, including plague and Lyme disease, respectively [18].

Calodium hepaticum (syn. Capillaria hepatica) is a cosmopolitan nematode with zoonotic potential infecting murid rodents, marsupials, carnivores, primates, and other mammals, including humans [19]. Muroid rodents, particularly Murinae and Arvicolinae, act as the primary reservoir hosts; however, the parasite has been reported from more than 70 non-murid species due to its low host specificity [19], [20], [21]. Unlike intestinal nematodes such as Ascaris lumbricoides and Trichuris trichiura, whose eggs are shed in the faeces of living hosts, C. hepaticum eggs remain within the liver and are released into the environment only following host decomposition or through predation/cannibalism, with ingested eggs subsequently passed in faeces [19], [22]. In the environment, eggs embryonate over 5–8 weeks and remain infective for up to one year [23]. After ingestion by a suitable host, embryonated eggs hatch in the intestine, releasing first-stage (L1) larvae that migrate through the portal circulation to the liver, where they mature and complete the life cycle in approximately 28 days [23], [24]. Non-embryonated eggs passed in faeces are non-infective and represent pseudoparasitism [19], [23]. The distribution of this parasite has been reported across Asia, Australia, Africa, Europe, and North and South America, with documented presence in more than 50 countries [25].

Although human infections have been reported sporadically, underreporting is likely due to the absence of characteristic clinical signs, limited availability of diagnostic tools, and the inherent difficulty in diagnosis, with liver tissue biopsy followed by histological evaluation remaining the definitive diagnostic method [26]. Humans typically acquire infection through ingestion of food or drinking water contaminated by infective embryonated eggs of the parasite, with paediatric population considered at higher risk [25], [27]. Human hepatic capillariasis may present with non-specific clinical features such as fever, marked eosinophilic leukocytosis, and hepatic fibrosis. In some cases, infections remain asymptomatic, while others may present as visceral larva migrans. Severe infections can lead to significant hepatic dysfunction and may occasionally result in fatal outcomes [25], [27]. C. hepaticum infection may cause respiratory symptoms notably cough and chest pain due to ectopic larval migration [28]. Owing to its diverse and non-specific clinical features, hepatic capillariasis is commonly misdiagnosed as other hepatic conditions, such as viral hepatitis, bacterial (pyogenic) liver abscess, or hepatic tumours [28].

Despite the importance of this pathogen, India, where zoonotic diseases are common due to extensive human-animal interactions, has reported only a few human cases of capillariasis and infections in dogs [29], [30], [31]. Regarding rodent studies, only a few have been conducted to date in northern and southern India, each involving limited sample sizes and a restricted range of rodent species [25], [32]. Therefore, the present study was designed and undertaken in Northeastern India, a region predominantly inhabited by tribal populations with heavy dependence on agricultural activities, where a large number of rodents and shrews were trapped and examined for C. hepaticum. The objectives were to determine the prevalence of infection, characterize the associated gross and histopathological changes, and confirm the identity of the parasite using parasitological, molecular, and phylogenetic approaches.

2. Materials and methods

2.1. Study area

The present study was conducted in two districts of Meghalaya (Fig. 1), a remote and predominantly hilly state in northeastern India: Ri Bhoi (25.8747° N, 91.8837° E) and East Khasi Hills (25.3805° N, 91.78905° E) (Fig. 1). Meghalaya shares an international border with Bangladesh and an interstate boundary with Assam. The state is predominantly inhabited by three major indigenous tribal communities, the Khasi, Jaintia, and Garo, which collectively constitute more than 90% of its population. Agriculture, animal husbandry, and fisheries are important livelihood activities among the local population [33]. The two study districts exhibit substantial climatic and ecological heterogeneity. Average annual rainfall is approximately 2410 mm at Umiam in Ri Bhoi, whereas Mawsynram in East Khasi Hills receives more than 11,800 mm annually, reflecting the marked rainfall gradient between the two study areas. The region also experiences considerable seasonal variation in temperature and relative humidity. During 2018–2022, population densities were 151.04 persons km⁻² in Ri Bhoi and 373.25 persons km⁻² in East Khasi Hills, compared with 171.46 persons km⁻² for Meghalaya and 431.11 persons km⁻² for India [34]. Shifting cultivation and monocropping remain important components of the traditional agricultural system in the study area. During the same period, forest cover accounted for 35.57% and 34.94% of the geographical area of Ri Bhoi and East Khasi Hills, respectively, while their corresponding ecological sustainability indices were 0.091 and 0.069 [34].

Fig. 1.

Map of Meghalaya, India, depicting the study sampling sites in Ri Bhoi and East Khasi Hills districts and the trapped small mammal species.

2.2. Sample size estimation

The sample size for estimating the prevalence of C. hepaticum was calculated using the standard formula for prevalence studies [35]: n = Z²P(1 − P)/d², where n is the required sample size, Z is the standard normal deviate corresponding to the desired confidence level (1.96 for 95% confidence), P is the expected prevalence, and d is the desired absolute precision. Assuming an expected prevalence of 17% (P = 0.17) in rodents [36], with a 95% confidence level and 6% absolute precision (d = 0.06), the minimum required sample size was calculated to be 151 small mammals. Trapping continued beyond attainment of the minimum required sample size, resulting in a final sample of 173 eligible small mammals, all of which were included in the study.

2.3. Trapping of small mammals and collection of samples

Small-mammal trapping was conducted between July 2020 and June 2025 as part of an ongoing, convenience-based surveillance program across multiple sites for the epidemiological investigation and surveillance of multiple pathogens [2], [37]. Trapping was not organized into predefined seasonal sampling periods or a fixed temporal schedule but was conducted intermittently and opportunistically throughout the study period based on logistical feasibility and the availability of suitable trapping locations. Sampling sites included residential areas, livestock and fish farms, livestock feed-storage facilities, agricultural fields, wet animal markets, restaurants, and forest-fringe habitats. Rodents were captured using foldable Sherman traps and indigenous live-capture trap (Supplementary Fig. S1). Traps were primarily baited with dried fish, while peanuts, biscuits, and sweet potatoes were used as alternative baits. A total of 10 traps were placed approximately 10 m apart, with small and large traps alternated to effectively sample mice, rats, and bandicoots. The traps were set in the evening and retrieved the following morning, with the timing of placement and retrieval adjusted according to climatic conditions and season. Captured animals were transported to the animal house facility, where species identification was carried out by trained personnel, including entomologists experienced in rodent identification. Following capture, the body weight of each animal was measured using a digital weighing scale, and sex was determined based on external genital characteristics. Relevant data, including body weight, sex, and sample identification number, were recorded. The animals were then humanely euthanized using chloroform anaesthesia followed by cervical dislocation, in accordance with the IAEC-approved study protocol and whole liver samples were subsequently collected for detailed analysis of C. hepatica. All procedures involving animal trapping, handling, euthanasia, and sample collection were conducted in accordance with institutional ethical guidelines and approved by the Institutional Animal Ethics Committee (IAEC; Approval No. IAEC01, dated 11 June 2020) of the Indian Council of Agricultural Research (ICAR) Research Complex for North Eastern Hill (NEH) Region, Meghalaya, registered under the Committee for the Control and Supervision of Experiments on Animals (CCSEA), Government of India.

Supplementary Fig. S1.

Traps used for the capture of small mammals during the study.

2.4. Gross examination and histopathology for detection of parasites and eggs

The collected livers were thoroughly examined across all lobes for abnormalities such as white tracts, patches, cysts, and streaks. For parasitological examination, since C. hepaticum eggs are unevenly distributed within the liver and are often embedded in both granulomatous and fibrotic tissues, representative tissue samples were collected from all lobes, including both superficial and deeper parenchymal regions. For histopathological examination, liver tissues were fixed in 10% neutral buffered formalin, dehydrated through ascending grades of alcohol, cleared in xylene, and embedded in paraffin wax. Sections of 4–5 μm thickness were cut using a rotary microtome, mounted on glass slides, and stained with haematoxylin and eosin (H&E) as previously described [38]. The stained sections were examined under an Olympus CX23-TR LED trinocular microscope (Olympus Corporation, Tokyo, Japan) for histopathological alterations and the presence of C. hepaticum eggs and adult worms. By compression (squash) wet mount preparation, thin slices of liver tissue were prepared using a scalpel and gently crushed between two grease-free sterile glass slides. A coverslip was then applied, and the preparation was examined under 100× and 400× magnification using a microscope for the detection of C. hepaticum eggs.

2.5. DNA extraction and PCR analysis

Liver tissue samples were homogenized in 1× PBS prior to DNA extraction. Genomic DNA was extracted using the QIAamp DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. DNA concentration and purity were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and the extracted DNA was stored for downstream molecular analysis. Polymerase chain reaction (PCR) was performed targeting a 204 bp fragment of the 18S rRNA gene of C. hepaticum, as previously described [39], using the primer pair 233f (5′-CGGTTCGCTGTTCAGTTGTT-3′) and 436r (5′-TGCTGCCTTCCTTGGATGTA-3′). Each reaction was prepared using 2× DreamTaq Master Mix (Thermo Fisher Scientific, Waltham, MA, USA), with primers synthesized by Integrated DNA Technologies (IDT, USA). Thermal cycling consisted of an initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 52 °C for 30 s, and extension at 72 °C for 60 s, with a final extension at 72 °C for 7 min. In the absence of a positive control, the earliest PCR-positive samples were sequenced to confirm their identity and were subsequently used as positive controls for rescreening and further PCR assays. Nuclease-free water was used as the no-template control (NTC) in all PCR assays. PCR amplification was carried out in a Mastercycler Nexus GX2 thermal cycler (Eppendorf, Germany). Amplified products were separated on 2.0% agarose gel, stained with ethidium bromide, and visualized under UV transillumination using a gel documentation system (Vilber Lourmat, Marne-la-Vallée, France).

2.6. Sequencing and phylogenetic analysis

Representative PCR-positive samples were selected for confirmation through Sanger sequencing and phylogenetic analysis. Bi-directional Sanger sequencing of the PCR amplicons was outsourced to M/s HiMedia Laboratories Pvt. Ltd. The obtained sequence reads were quality-checked, cleaned, and assembled using BioEdit v7.7.1 (updated upto 5/10/2021; available at https://thalljiscience.github.io/), MEGA v12.0.9 [40], and EMBOSS CONS [41] (available at https://www.ebi.ac.uk/jdispatcher/msa/emboss_cons). The consensus sequences were further verified using NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and submitted to the NCBI GenBank database via BankIt to obtain accession numbers. Percent identity, query coverage, and E-values were used for taxonomic confirmation. For phylogenetic analysis, multiple sequence alignment was performed using the MUSCLE algorithm, and a maximum likelihood phylogenetic tree was constructed in MEGA v12.0.9. The Jukes–Cantor substitution model was selected as the best-fit model using a model selection tool, and phylogenetic tree robustness was assessed using 1000 bootstrap replicates [42], [43], [44]. The 18S rRNA region of Strongyloides ratti was used as the outgroup.

2.7. Statistical analysis

Statistical analyses were performed using R software (version 4.4.1) [45]. The packages used included “epiDisplay” v.3.5.0.2 [46], “vcd” v.1.4–13 [47], and “logistf” v.1.26.0 [48]. The association between variables such as sex, animal category, and species with positivity for C. hepaticum was statistically evaluated using Fisher's exact test and the likelihood ratio chi-squared test for univariable analysis, depending on whether the respective prerequisite conditions were satisfied. Odds ratios along with the 95% confidence intervals were calculated using the respective reference categories. Firth's penalized logistic regression was performed in situations where sparse data were present. A p-value <0.05 was considered statistically significant.

3. Results

3.1. Characteristics of sampled small mammals and gross hepatic lesions

The trapped small mammals (n = 173) comprised seven species, dominated by B. bengalensis (49.13%; 85/173), followed by B. indica (17.34%; 30/173), S. murinus (14.45%; 25/173), M. musculus (8.67%; 15/173), R. rattus (4.62%; 8/173), M. booduga (4.05%; 7/173), and R. norvegicus (1.73%; 3/173) (Table 1). Of the 173 small mammals examined, 106 were males and 67 were females. The median body weights (interquartile range) of rats, mice, bandicoots, and shrews were 85.00 g (37.00–116.32 g), 16.05 g (12.37–16.05 g), 270.10 g (118.00–420.00 g), and 48.65 g (37.95–58.18 g), respectively.

Table 1.

Distribution of small mammals included in the study according to sex, category, and species.

Factor	Attribute	No. of animals	Percentage of total population
Sex	Male	106	61.27
	Female	67	38.73
Category	Bandicoot	115	66.47
	Mouse	22	12.72
	Rat	11	6.36
	Shrew	25	14.45
Species	Bandicota bengalensis	85	49.13
	Bandicota indica	30	17.34
	Suncus murinus	25	14.45
	Mus musculus	15	8.67
	Rattus rattus	8	4.63
	Mus booduga	7	4.05
	Rattus norvegicus	3	1.73

Gross hepatic lesions were observed in the livers of 34 of the 173 animals examined (19.65%). The typical hepatic lesions of C. hepaticum infection were characterized by irregular, tortuous linear to coalescing yellowish-white discolorations on the liver surface (Fig. 2). These lesions appeared as slightly raised or flat tan to yellow streaks, striae, or patchy areas distributed over both dorsal and ventral surfaces of all liver lobes. Livers were often shrunken or, in severe cases, showed hepatomegaly, with lesions extending into the hepatic parenchyma on incision. The affected areas exhibited a sinuous, irregular, and sometimes cheese-like appearance, with lesions ranging from small focal spots to extensive involvement of more than half of the liver tissue (Fig. 2).

Fig. 2.

Gross hepatic lesions of C. hepaticum infection in B. bengalensis, with more than 75% of the liver affected, showing irregular, tortuous, coalescing yellowish-white streaks and patchy areas on the liver surface (yellow arrows). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.2. Histological and parasitological analysis

On histopathological examination of infected liver samples, characteristic lesions of C. hepaticum infection were observed, including multifocal clusters of large, non-embryonated, elliptical to barrel-shaped eggs with polar prominences and a rough, pitted shell, predominantly located in the subcapsular and hepatic parenchyma. These egg clusters were surrounded by a thin fibrovascular stroma with associated pressure atrophy of adjacent hepatocytes (Fig. 3A-C). Higher magnification revealed prominent granulomatous inflammation with eosinophilic infiltration, coagulative necrosis, and areas containing degenerating parasitic cuticle (Fig. 3A, D). Multiple cross, oblique, and longitudinal sections of nematode larvae and adult worms were also observed within the hepatic parenchyma and immediately beneath the liver capsule (Fig. 3E-F). Larval stages showed developing intestinal and gonadal structures, while adult worms exhibited characteristic morphological features including a coelomate body with cuticle and stichosome surrounding the oesophagus. Female worms contained uterine structures with ova, whereas male worms showed a well-defined intestinal tract (Fig. 3E-F). Severe pathological changes included eosinophilic coagulative necrosis, haemorrhage, and fibroplasia at sites of parasite localization, accompanied by dense granulomatous inflammation composed of eosinophils, macrophages, lymphocytes, and neutrophils (Fig. 3D-F). In addition, degenerating parasitic structures and migratory tracts containing parasite remnants were observed within affected hepatic tissue (Fig. 3F). Eggs recovered from rat liver were examined under light microscopy at 100× and 400× magnifications. The eggs were unembryonated, ellipsoidal to barrel-shaped, with a double-layered, striated shell and a bi-operculated structure bearing shallow polar plugs (Fig. 4). The polar plugs were situated within the eggshell outline and did not project externally. Eggs were frequently observed in aggregates, often associated with female worms and embedded within a thin fibrous tissue matrix.

Fig. 3.

Histopathological lesions in Calodium hepaticum-infected liver. A–D show multifocal clusters of parasitic eggs with granulomatous inflammation, eosinophilic necrosis, and fibrovascular stroma; eggs are barrel-shaped, bioperculated, and striated. E–F show live and degenerating larvae within hepatic parenchyma and beneath the capsule, associated with necrosis and fibroplasia (H&E: ×100× & 400×).

Fig. 4.

Microscopic view (400×) of C.hepaticum eggs recovered from hepatic tissue (B.bengalensis).

3.3. Prevalence based on molecular analysis

Genomic confirmation using a sensitive and specific PCR assay targeting the 18S rRNA gene of C. hepaticum showed a prevalence of 36.99% (95% CI: 30.16–44.44; 64/173) in the trapped small mammals. Overall, 64 liver samples tested positive for C. hepaticum by PCR out of 173 liver samples examined. Gross lesions were observed in 53.12% (34/64, 95% CI: 41.07–64.82%) of PCR-confirmed infected small mammals, whereas 46.88% (45/64, 95% CI: 35.18–58.93%) of PCR-positive animals did not exhibit any visible gross lesions. Species-wise PCR analysis targeting the 18S rRNA gene of C. hepaticum revealed variable prevalence among small mammals. The highest prevalence was observed in B. indica (24/30; 80%; 95% CI: 62.69–90.49), followed by M. booduga (4/7; 57.14%; 95% CI: 25.05–84.18), B. bengalensis (27/85; 31.76%; 95% CI: 22.84–42.27), M. musculus (4/15; 26.67%; 95% CI: 10.90–51.95). Lower prevalence rates were recorded in S. murinus (4/25; 16%; 95% CI: 6.40–34.65) and R. rattus (1/8; 12.50%; 95% CI: 2.24–47.09), while no infection was detected among the three R. norvegicus examined. The prevalence was 40.54% (60/148) in rodents and 16.00% (4/25) in shrews. Among rodents, the prevalence rate was 44.35% (51/115) for bandicoots, 36.36% (8/22) for mice and 9.09% (1/11) for rats. The confirmed samples showed the expected PCR amplicon of 204 bp (Fig. 5). Variation in sample size among species reflects differences in field abundance and trapping success; therefore, prevalence estimates should be interpreted together with their corresponding confidence intervals.

Fig. 5.

PCR amplification of a 204 bp fragment of the partial 18S rRNA gene of C. hepaticum. Lane M: 100 bp DNA ladder; Lanes 1–10: positive samples from infected liver of rats, mice, bandicoots, and shrews; Lane NTC: no-template control.

3.4. Phylogenetic analysis

The sequences were assembled and compared with reference sequences using BLAST and subsequently submitted to the NCBI GenBank database for accession number allocation (Fig. 6). The sequences generated in the present study showed 100% identity with previously reported C. hepaticum 18S rRNA sequences in GenBank, while maximum-likelihood phylogenetic analysis showed clustering with reference sequences from South India, Indonesia, and Zambia, confirming species-level identification (Fig. 6). No sequence variation was detected among the study isolates; therefore, the 18S rRNA fragment was not informative for assessing intraspecific diversity or population structure.

Fig. 6.

Maximum Likelihood phylogenetic analysis of the partial 18S rRNA gene of C. hepaticum with 1000 bootstrap replicates. Red triangles denote sequences generated in the present study and deposited in GenBank. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.5. Statistical analysis of factors influencing C. hepaticum positivity

Univariable analysis was performed to assess the association of sex, animal category (rats, mice, bandicoots, and shrews), and species with C. hepaticum positivity (Table 2). No significant association was observed between sex and C. hepaticum positivity (Likelihood ratio χ² = 0.51, p = 0.48, df = 1). The odds of positivity were lower in males (34.91%; 37/106) than in females (40.30%; 27/67) (OR = 0.79; 95% CI: 0.41–1.09); however, the difference was not statistically significant (p = 0.48). A significant association was observed between animal category and positivity for C. hepaticum (Fisher's exact test p < 0.01). Animal category represented the broader groups of bandicoots, mice, rats, and shrews, with individual species pooled within their respective categories. Compared with bandicoots (reference category), rats had significantly lower odds of infection (OR = 0.13; 95% CI: 0.0067–0.6873; p = 0.05), and shrews also showed significantly lower odds (OR = 0.24; 95% CI: 0.0666–0.6757; p < 0.01). Although mice exhibited lower odds of positivity (OR = 0.72; 95% CI: 0.55–1.15), the association was not statistically significant (p = 0.49). At the individual species level, a significant association with positivity was also observed (p < 0.01). Compared with B. bengalensis (reference species), B. indica exhibited significantly higher odds of positivity (OR = 8.02; 95% CI: 3.17–22.89; p < 0.01). Other species, including S. murinus, M. musculus, R. rattus, M. booduga, and R. norvegicus, did not show statistically significant associations with positivity (Likelihood ratio χ² = 36.73, p < 0.01, df = 6).

Table 2.

Univariable analysis of sex, animal category, and species associated with C. hepaticum positivity.

Factor	Attribute	Positivity (%)	Confidence interval	Odds ratio	2.5%	97.5%	p value
Sex							0.48
	Male	37/106 (34.91)	26.50–44.36	0.79	0.41	1.09	0.47
	Female	27/67 (40.30)	29.39–52.26	Reference	–	–	–
Category							<0.01*
	Bandicoot	51/115 (44.35)	35.60–53.46	Reference	–	–	–
	Mouse	8/22 (36.36)	19.73–57.05	0.72	0.55	1.15	0.49
	Rat	1/11 (9.09)	1.62–37.74	0.13	0.01	0.69	0.05
	Shrew	4/25 (16.00)	6.40–34.65	0.24	0.07	0.68	<0.01*
Species							<0.01*
	Bandicota bengalensis	27/85 (31.76)	22.84–42.27	Reference	–	–	–
	Bandicota indica	24/30 (80.00)	62.69–90.49	8.02	3.17	22.89	<0.01*
	Suncus murinus	4/25 (16.00)	6.40–34.65	0.45	0.13	1.26	0.13
	Mus musculus	4/15 (26.67)	10.90–51.95	0.83	0.23	2.58	0.76
	Rattus rattus	1/8 (12.50)	2.24–47.09	0.43	0.04	2.09	0.32
	Mus booduga	4/7 (57.14)	25.05–84.18	2.74	0.62	13.01	0.18
	Rattus norvegicus	0/3 (0.00)	0.00–56.15	0.30	0.01	3.29	0.37

4. Discussion

Calodium hepaticum infection is an important yet neglected zoonotic parasitic disease. Because its clinical manifestations are non-specific and diagnosis is challenging, its true incidence is likely to be significantly underestimated [28]. Our study, the first epidemiological investigation from Northeastern India conducted in synanthropic environments of tribal agricultural communities, demonstrated the presence of C. hepaticum, a neglected zoonotic nematode, in multiple small mammal species. The study demonstrated the presence of C. hepaticum in multiple small mammal species, indicating that, in addition to rats, other small mammals may contribute to the maintenance of the parasite. Such low host specificity has been reported previously, with C. hepaticum detected in the livers of nearly 180 mammalian species, including rabbits, cats, and dogs [21], [28]. This broad host range is of zoonotic importance because infection in a wide range of mammalian hosts may increase the potential for environmental contamination with parasite eggs, thereby facilitating transmission to other susceptible animals and humans [21], [28].

In the present study, species-wise variation in C. hepaticum prevalence should be interpreted cautiously because reliable population data for these small mammal species are unavailable for the study area. Furthermore, differences in prevalence among species may have been influenced by variation in species abundance, capture frequency, and sample size. Sampling was conducted across diverse synanthropic settings within tribal agricultural communities, where close human-animal-environment interactions may facilitate exposure to C. hepaticum. As C. hepaticum transmission is associated with reservoir abundance and activity, the 37% prevalence observed suggests potential human exposure. However, human infections may remain undetected due to mild or asymptomatic presentations and diagnostic challenges [28], [49].

In agreement with our study, which recorded an overall prevalence of 37% (64/173), a previous study from Hong Kong reported a comparable prevalence of 36.7% (81/221) among four rat species (R. norvegicus, R. tanezumi, R. andamanensis, and Niviventer huang) [23]. In that study, gross hepatic lesions were absent in 17% of infected rats, whereas in our study a substantially higher proportion (46.88%) of infected animals showed no visible gross lesions [23]. A more recent study from Chennai, South India, reported a prevalence of 21.81% (12/55) based on gross lesions and 38.18% (21/55) by PCR in urban Rattus rattus populations, with 43% of PCR-confirmed animals showing no macroscopic lesions [25]. The absence of gross macroscopic lesions in infected animals may reflect variation in infection stage, parasite burden, lesion distribution, or host response. Thus, macroscopic examination alone may underestimate infection, highlighting the importance of confirmatory diagnostic methods [19], [23]. As false-positive and false-negative results may occur when relying solely on macroscopic examination, confirmatory methods such as PCR, artificial digestion of liver tissue followed by microscopic identification, or histopathological examination are important for more accurate detection and estimation of C. hepaticum infection [19], [23], [25], [50].

A study from Punjab, North India, reported a higher prevalence of 55% in Rattus rattus, B. bengalensis, and M. musculus based on gross and histopathological examinations [32]. In contrast, another study from the same region documented a lower prevalence of 22.22% in B. bengalensis using similar diagnostic approaches [51]. Previous studies have reported a wide range of C. hepaticum prevalence in small mammals globally, ranging from 4.5% to 52%, with intermediate values of 10.68%, 34.5%, and 44.9% reported from different geographical regions [19], [52], [53], [54], [55]. In our study, the shrew species S. murinus (Asian house shrew) showed a prevalence of 16% (4/25). Similarly, a previous study from Slovakia reported C. hepaticum prevalence in two shrew species, Sorex araneus (common shrew; 15%) and Neomys fodiens (Eurasian water shrew; 33.3%) [19].

Sequencing of the 18S rRNA gene confirmed the molecular identity of C. hepaticum in the examined small mammals, with all study sequences showing 100% identity irrespective of host species or sampling site. Phylogenetic analysis supported species-level identification by clustering the study sequences with available C. hepaticum reference sequences from South India, Indonesia, and Zambia. However, no sequence variation was detected among the study isolates, indicating that the conserved 18S rRNA fragment provides insufficient resolution for assessing intraspecific diversity or population structure. Future studies using more variable markers, such as mitochondrial cox1, are warranted to investigate the genetic diversity and population structure of C. hepaticum [56].

The high prevalence of hepatic calodiosis in small mammals may pose a potential zoonotic risk to public health, as illustrated by the parasite's transmission cycle, including its hosts and infective and diagnostic stages (Fig. 7). Accidental ingestion of these eggs from environment by humans and other mammals, including livestock, pets, and wildlife, can lead to infection, thereby sustaining transmission across multiple hosts and promoting the parasite's persistence in the environment [28], [57]. True infection occurs following ingestion of embryonated eggs, whereas ingestion of unembryonated eggs from infected tissues results in spurious infection, with eggs passing through the gastrointestinal tract and subsequently becoming infective after embryonation in the environment [28], [57]. C. hepaticum infection is considered more common in children, individuals living under poor sanitary conditions, and those with greater environmental exposure [28]. In rodent-dense areas, cannibalism may further promote parasite transmission across generations and contribute to its persistence in rodent populations [28]. Therefore, understanding the prevalence of hepatic calodiosis among different rodent species and geographical regions is important for developing effective prevention and control strategies.

Fig. 7.

Schematic representation of the transmission cycle of Calodium hepaticum (Source for clipart images: Mind the graph https://mindthegraph.com/). The black lines indicate the probable routes of true infection, whereas the red dashed lines indicate the spurious passage of the parasite through different hosts. Detection of parasite eggs in the faeces of animals following spurious passage represents pseudoparasitism. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

The present study has several limitations. Small-mammal trapping was convenience-based and conducted intermittently across sites and years, which may limit the temporal and spatial representativeness of the observed positivity. The study primarily addresses the animal-health component of the One Health framework while providing ecological relevance through the investigation of synanthropic hosts. However, environmental contamination and human exposure were not directly assessed and warrant further investigation through integrated animal, environmental, and human surveillance. Furthermore, species-specific estimates for M. booduga, R. rattus, and R. norvegicus should be interpreted cautiously due to their small sample sizes. The absence of detected infection in R. norvegicus (0/3) should be interpreted cautiously due to the small sample size, reflecting differences in field abundance and trapping success.

5. Conclusion

This is the first study on the prevalence of C. hepaticum in synanthropic small mammal populations (rodents and shrews) in Northeastern India, a predominantly tribal-inhabited region. Our findings highlight the potential zoonotic risk in the study area, particularly for local communities engaged in agriculture and allied activities that may increase contact with synanthropic small mammals and their environments. Children may also be particularly vulnerable due to behaviours that increase their exposure to potentially contaminated environments. Rodent control measures should incorporate appropriate carcass disposal to minimize the potential release and environmental dispersal of C. hepaticum eggs and reduce associated public health risks. Furthermore, large-scale epidemiological investigations using a One Health approach are warranted, particularly in the context of rapid urbanization and increasing human-synanthropic animal interactions. Such efforts are essential to better understand and address the potential risk of this neglected parasitic zoonosis in vulnerable populations.

The following are the supplementary data related to this article.

Animal subject

This study was conducted in accordance with the following guidelines for animal welfare and/or reporting: The Committee for Control and Supervision of Experiments on Animals (CCSEA) is a statutory Committee of Department of Animal Husbandry and Dairying (DAHD), Ministry of Fisheries, Animal Husbandry and Dairying (MoFAH&D), Government of India.

This study was approved by the Institutional Animal Ethics Committee (IAEC), ICAR Research Complex for NEH Region, Meghalaya.

(Approval No. IAEC01 dated 11 June 2020)

Declaration of generative AI and AI-assisted technologies in manuscript preparation

During the preparation of this manuscript, the authors used ChatGPT to assist in improving language clarity and enhancing readability. The authors critically reviewed, edited, and verified all content, and take full responsibility for the final version of the manuscript.

CRediT authorship contribution statement

Arockiasamy Arun Prince Milton: Writing – original draft, Methodology, Investigation, Funding acquisition, Conceptualization. Aleimo G. Momin: Investigation. M. Saminathan: Investigation, Formal analysis. K. Srinivas: Visualization, Investigation, Formal analysis. D.M. Firake: Investigation, Formal analysis. P.N. Gandhale: Writing – review & editing, Supervision, Funding acquisition. Nagappa Karabasanavar: Supervision, Funding acquisition, Formal analysis. Purna Bahadur Chetri: Investigation. Azhahianambi Palavesam: Writing – review & editing, Methodology. Sandeep Ghatak: Writing – review & editing, Formal analysis. Samir Das: Writing – review & editing, Visualization, Methodology.

Ethical approval

Ethical approval for this study was obtained from the Institutional Animal Ethics Committee (IAEC) of the ICAR Research Complex for NEH Region, Meghalaya, registered under the Committee for the Control and Supervision of Experiments on Animals (CCSEA), Government of India (IAEC01 dated 11 June 2020).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Data availability

No data was used for the research described in the article.