Detection of zoonotic pathogens in invasive black rats (Rattus rattus) inside and outside a coastal protected area in southern Peru

Abstract

Introduction

This study investigates the occurrence of Leptospira spp., Toxoplasma gondii, and respiratory viruses in invasive black rats (Rattus rattus) from Punta San Juan (PSJ), a coastal protected area in southern Peru. Rodents can harbor zoonotic pathogens at the wildlife–human interface, posing ecological and public health risks.

Methods

Fifty-three rats were trapped inside and outside PSJ in June 2025 from two contrasting zones: inside PSJ (n = 29), corresponding to the protected coastal habitat within the reserve, and outside PSJ (n = 24), including adjacent urban and human-impacted coastal areas. Serum, blood, and tissues were analyzed for T. gondii using Western blot and quantitative PCR. Leptospira spp. detection was performed by real-time PCR targeting the lipL32 gene in blood samples. Respiratory viruses, including influenza A and B, were screened using the Illumina Respiratory Virus Panel through next-generation sequencing (NGS) technology in respiratory tract and lung swabs.

Results

One adult female (1/49, 2.0%) captured inside PSJ was seropositive for T. gondii, but all PCR tests were negative in blood and tissues. Leptospira spp. DNA was detected in 18/53 rats (34.0%), with higher frequency outside the reserve (54.2%) than inside (17.2%) (p = 0.008). No amplifications were obtained for respiratory viruses.

Discussion

The higher Leptospira frequency outside PSJ suggests that human-associated environments increase infection risk in the study area. In contrast, evidence for other zoonotic pathogens was limited, with only a single serological detection of T. gondii and no respiratory viruses identified. Although these findings are restricted to a local context, they highlight Leptospira spp. as the primary zoonotic pathogen detected and support the need for broader surveillance to better assess the epidemiological role of invasive rodents in coastal protected areas.

1. Introduction

Invasive rodents have had catastrophic effects on island and coastal ecosystems worldwide, particularly through their predation on seabirds and eggs, leading to population declines and local extinctions (1). Beyond their direct ecological impact, rodents also pose significant health risks by harboring zoonotic pathogens capable of infecting wildlife, domestic animals, and humans (2). Including species inhabiting coastal and marine ecosystems (3, 4).

Punta San Juan (PSJ), located in the Ica region of Peru, is part of the National Reserve System of Guano Islands, Islets, and Capes (Reserva Nacional Sistema de Islas, Islotes y Puntas Guaneras, RNIPG). This reserve system has hosted guano harvests which involve camps of over 100 workers that live for months at these sites to harvest or extract seabird guano that is then distributed as fertilizer. The protected area harbors remarkable biodiversity, including three residential marine mammal species: the South American sea lion (Otaria byronia), the South American fur seal (Arctocephalus australis), and the marine otter (Lontra felina). PSJ is also home to guano-producing seabirds the Guanay cormorant (Phalacrocorax bougainvilllii), the Peruvian booby (Sula variegata) and the Peruvian pelican (Pelecanus thagus). It is also recognized as an important breeding colony for Humboldt penguins (Spheniscus humboldti), a globally threatened species (5). A perimeter 1.2 km long and 2.5 m high wall surrounding the reserve has been crucial in maintaining biodiversity by limiting human and domestic animal access. This physical barrier was built to protect the area from external pressures such as urban expansion from the nearby city (2 km) of San Juan de Marcona.

However, the presence of invasive rodents in Punta San Juan poses a serious threat to wildlife health, ecological balance, and public health. Black rats (Rattus rattus), which have colonized the area, negatively affect the ecosystem by preying on seabird eggs and chicks (6–8). Rattus rattus is a highly adaptable and opportunistic rodent species that thrives in coastal and insular environments, particularly in areas with seabird colonies and human influence. Its omnivorous diet, and close association with anthropogenic habitats facilitate frequent contact with wildlife, domestic animals, and contaminated environments, making it a relevant species for the circulation and maintenance of zoonotic pathogens at the terrestrial–marine interface (2, 9, 10).

Toxoplasma gondii is a protozoan parasite that causes toxoplasmosis in warm-blooded vertebrates and exhibits a complex life cycle involving both definitive and intermediate hosts (11). Felids are the definitive hosts, with domestic cats (Felis catus) being the primary host species responsible for the global spread of the parasite (12, 13). Wildlife may become infected through ingestion of infected prey, or by contact with contaminated water, soil, or fecal matter from infected felids (14, 15). In intermediate hosts such as rodents, T. gondii can establish in multiple tissues, particularly in the brain and heart, forming cysts that can persist throughout the host’s lifetime (16). In wildlife populations, toxoplasmosis can cause sublethal effects such as reproductive disorders (17) or contribute to population weakening, increasing vulnerability to stochastic environmental events (18). Aquatic animals are particularly susceptible to T. gondii exposure, as toxoplasmosis is a waterborne disease and oocysts can be transported through freshwater and marine runoff (11, 19).

Similarly, the genus Leptospira, is a globally distributed spiroqueta that can cause leptospirosis in humans and a wide range of animal species (20). These bacteria infect mammals, birds, and reptiles, with both domestic and wild rodents, carnivores, and ruminants serving as important reservoirs (21). Pathogenic Leptospira are shed in the urine of infected hosts, contaminating soil and water, and are efficiently transmitted in aquatic or humid environments (22). Exposure to Leptospira has been reported in numerous wildlife species, particularly in aquatic animals, as leptospirosis is primarily a waterborne disease (23, 24). The risk of transmission is especially high in areas with poor sanitation, agricultural activity, and urban development, where contact between humans, domestic animals, and wildlife is more frequent (25).

The outbreak of highly pathogenic avian influenza (HPAI) A(H5N1) in November 2022 in Peru, which affected both marine and terrestrial species, including seabirds, marine mammals, and poultry nationwide, resulted in significant mortality among sea lions and seabirds (26). Punta San Juan and its surrounding areas were not spared from this epizootic event, as both avian and marine mammal populations were affected (27–29). Experimental and field evidence further indicates that wild and synanthropic rodents, such as R. rattus and R. norvegicus, can harbor and replicate avian-origin H5N1 viruses in respiratory tissues, suggesting that they may act as replication-competent hosts and contribute to viral maintenance in human-dominated environments (30–34). The proximity of Punta San Juan to urban areas, where domestic animals such as cats, dogs, and livestock are present, represents a significant risk factor for the introduction and transmission of these and other zoonotic pathogens.

Although rodents are widely recognized as hosts of zoonotic pathogens, information from coastal protected areas in southern Peru is virtually absent. In particular, no studies have evaluated pathogen occurrence in R. rattus populations inhabiting the interface between a marine protected area and nearby urban environments. Therefore, the present study aims to evaluate the occurrence and detection frequency of selected zoonotic pathogens, including respiratory virus such as influenza A, T. gondii, and Leptospira spp. in black rats (Rattus rattus) captured across two contrasting areas: within Punta San Juan (protected area) and in adjacent urban and coastal zones.

2. Materials and methods

2.1. Study area and design

The study was conducted in and around Punta San Juan (PSJ), located in the Ica region of southern Peru (15°22′S, 75°11′W). A cross-sectional comparative study design was adopted to evaluate Rattus rattus populations across two zones with contrasting ecological and anthropogenic characteristics: (i) inside Punta San Juan (PSJ): comprises the coastal habitat within the perimeter wall of the reserve; (ii) outside Punta San Juan (urban and coastal zone): This area includes sites located more than 0.5 km beyond the perimeter wall, encompassing zones of intense human activity, such as tourist beaches, a marble quarry, and informal waste disposal sites. It also includes the city of San Juan de Marcona, characterized by elevated levels of anthropogenic pressure and frequent interactions among humans, domestic animals (dogs, cats, poultry, and livestock), and wildlife.

2.2. Ethical statement

This study was conducted in accordance with relevant national legislation and institutional guidelines for the use of animals in research. Fieldwork and sample collection were authorized under research permits RJ N° 000008-2024-SERNANP/RNIPG-SGD and RJ N° 000008-2025-SERNANP/RNIPG-SGD issued by the National Service of Natural Protected Areas (SERNANP), and RD N° D00033-2025-MIDAGRI-SERFOR-DGGSPFFS-DGSPFS issued by the National Forest and Wildlife Service (SERFOR), Peru. All animal handling and sampling procedures were approved by the Institutional Animal Care and Use Committee of Universidad Peruana Cayetano Heredia (CIEA-UPCH: CONSTANCIA-CIEA-038-08-24; CONSTANCIA-CIEA-E-035-12-24; CONSTANCIA-CIEA-R-013-02-25).

2.3. Sample collection and processing

Black rats were captured inside and outside Punta San Juan during June 2025 using Tomahawk live traps baited with oatmeal, peanut butter and vanilla extract. Within the reserve, four Sherman traps were also used to increase capture efficiency. Trapping effort totaled 234 trap-nights, with 67 active traps inside the reserve (117 trap-nights) and 78 outside (117 trap-nights). Capture success was 23.9% (28/117) inside and 20.5% (24/117) outside the reserve, yielding 53 Rattus rattus in total, including two individuals captured in a single Tomahawk trap. Traps were checked early each morning, and captured animals were transported to a field laboratory for examination.

Animals were euthanized using a CO₂ chamber, in accordance with American Veterinary Medical Association (35) guidelines. Each individual was then sexed, aged (juvenile/adult), and measured for body weight and morphometric variables (total length, body, tail, hind foot, and ear). All procedures were carried out in accordance with biosafety and field handling recommendations outlined by the PREDICT Consortium (36).

Blood was collected by cardiac puncture using sterile syringes and divided into serum, whole blood with EDTA, and coagulated fractions. Blood samples were centrifuged at 3,500 × g for 10 min to separate serum for serological testing. Heart and brain tissues were collected for molecular analysis and preserved in 70% ethanol. Respiratory tract and lung swabs were obtained by making a small incision in the trachea and placed in Universal Transport Medium (UTM).

All samples requiring freezing, including serum, whole blood with EDTA, coagulated fractions and UTM swabs, were stored at −20 °C in the field between 4 to 16 days, transported on dry ice to Lima, and subsequently kept at −80 °C until processing, a maximum of 4 months after. Heart and brain tissues preserved in ethanol were maintained at room temperature during both storage and transport to the laboratory.

Different biological samples were collected to target each investigated pathogen based on their known tissue tropism and recommended diagnostic approaches. Serum samples were used for serological detection of T. gondii exposure (37, 38). Whole blood (EDTA) was collected for molecular detection of Leptospira spp., as leptospires circulate in blood during infection (39, 40). Kidney tissue and urine samples, which are required to assess renal colonization and shedding of Leptospira spp., were not analyzed in this study. Therefore, molecular detection was limited to blood samples and aimed at identifying active or recent infection rather than chronic carriage or shedding status. Blood clots, brain, and heart tissues were collected for molecular confirmation of T. gondii, given the parasite’s affinity for neural and muscular tissues (41). Respiratory tract and lung swabs were obtained for molecular screening of influenza A and other respiratory viruses, as these pathogens primarily replicate in the respiratory epithelium (30, 33).

2.4. Pathogen detection

For molecular detection of influenza A and other respiratory viruses, RNA was extracted from respiratory tract and lung swabs using the QIAamp Viral RNA Mini Kit (Qiagen, Germany) following the manufacturer’s instructions. Extracted RNA samples were analyzed using the Illumina Respiratory Virus Oligo Panel (Illumina, San Diego, CA, United States), which allows targeted enrichment and detection of more than 40 respiratory viruses, including influenza A and B, coronaviruses, adenoviruses, parainfluenza viruses, and metapneumovirus (42). This approach allowed broad, unbiased screening of respiratory viruses using next-generation sequencing (NGS).

Serological detection of T. gondii was carried out using Western blot analysis on serum samples, following the procedures described by Saavedra and Ortega (38) and Flores et al. (37). For molecular confirmation, DNA was extracted from blood clot, brain, and heart tissues using a High Pure PCR Template Preparation Kit (Roche Diagnostics Corp., Indianapolis, IN) according to the manufacturer’s instructions. Quantitative PCR (qPCR) targeting the REP529 repetitive element of T. gondii was subsequently performed on these samples, following the protocol of Gutiérrez-Loli et al. (41).

For detection of Leptospira spp., DNA was extracted from whole blood samples using the High Pure PCR Template Preparation Kit (Roche Applied Science, Mannheim, Germany) according to the manufacturer’s instructions. Pathogenic Leptospira spp. were detected by real-time PCR targeting the lipL32 gene, following the protocols described by Stoddard et al. (43) and Silva-Caso et al. (40).

2.5. Statistical analyses

Detection frequency for each pathogen was calculated as the proportion of positive individuals among the sampled rats, with 95% confidence intervals (CI). A principal component analysis (PCA) was first performed on standardized morphometric variables (body weight, total length, tail length, ear, and hind foot) to generate a body size index (PC1) representing overall body size. Body size was included as a proxy for age and cumulative exposure, as larger and older rodents are more likely to have experienced repeated contact with contaminated environments and pathogens, potentially increasing their probability of infection (44, 45). Associations between infection status and categorical variables (sex, age, and sampling zone) were evaluated using Fisher’s exact tests, while comparisons involving the body size index (PC1) were assessed using the Mann–Whitney U test. A multivariable logistic regression model was then fitted to identify predictors of infection, including sampling zone (inside vs. outside the reserve), sex, age, and body size index. Odds ratios (OR) and 95% confidence intervals were estimated, and statistical significance was set at p < 0.05. All statistical analyses were conducted using R software (version 4.1.1 (46);) with the packages epiR (47), FactoMineR (48), and stats (46).

3. Results

A total of 53 Rattus rattus individuals were analyzed, with different biological samples tested according to the diagnostic target of each pathogen (serum for T. gondii serology; blood for molecular detection of Leptospira spp.; respiratory tract and lung swabs for respiratory viruses).

No amplifications were obtained for respiratory viruses in any respiratory tract or lung swab. Serological testing for T. gondii was performed on 49 serum samples, as insufficient blood was obtained from four individuals. One adult female (1/49, 2.0, 95% CI, 0.1–10.7%), captured inside the reserve, tested positive for T. gondii IgG antibodies. Molecular analysis of this individual’s blood clot, heart, and brain tissues yielded negative results by qPCR. Subsequently, brain samples from an additional 24 individuals were tested by qPCR targeting the REP529 gene, and all were negative for T. gondii DNA.

Leptospira spp. DNA was detected in 18 of 53 rats’ blood samples (34.0%; 95% CI: 21.2–48.8%) (Table 1; Figure 1). The proportion of positives was significantly higher outside the reserve (13/24; 54.2, 95% CI: 33.2–73.7%) than inside (5/29, 17.2, 95% CI: 7.6–34.5%), according to Fisher’s exact test (p = 0.008). No significant differences in infection status of Leptospira spp. were observed between sexes, age classes, or body size index (PC1) values. In the multivariable logistic regression model, sampling zone (inside vs. outside the reserve) was the only significant predictor of infection by Leptospira spp. (OR = 7.3, 95% CI: 1.9–28.1, p = 0.004).

Table 1.

Molecular detection of Leptospira spp. by qPCR in invasive black rats (Rattus rattus) from Punta San Juan (PSJ) and surrounding coastal areas, southern Peru.

Variable	Positive/total (%)	95% CI or mean ± SDa	p-value
Sex			0.565 b
Male	6/21 (28.6)	11.3–52.2
Female	12/32 (37.5)	21.1–56.3
Age class			0.83 b
Adult	11/31 (35.5)	19.2–54.6
Juvenile	7/22 (31.8)	14.7–54.9
Zone			0.008 b
Inside PSJ	5/29 (17.2)	7.6–34.5
Outside PSJ	13/24 (54.2)	33.2–73.7
Body size index (PCA1)			0.324 c
Positive		0.290 ± 1.748
Negative		−0.149 ± 1.989
Overall	18/53 (34.0)	21.2–48.8

^aCI, Confidence interval; SD, Standard deviation; ^bFisher’s exact tests; ^cMann–Whitney U test.

Figure 1.

Study area of Punta San Juan (PSJ) and surrounding coastal zones in southern Peru. Red heat maps show the sampling effort in the entire study area. Circles with numbers show the PCR-positive detections for Leptospira spp. in black rats (Rattus rattus).

4. Discussion

This study provides new insights into the occurrence of Leptospira spp. and T. gondii in invasive Rattus rattus populations from Punta San Juan (PSJ) and the surrounding coastal areas in southern Peru. Previous health assessments of marine fauna in PSJ reported no serological evidence of exposure to T. gondii or Leptospira spp. in South American fur seals (Arctocephalus australis) (49). However, these assessments were conducted several years ago, and the epidemiological context may have changed substantially. Recent studies in Chile have reported seropositivity to both pathogens in pinnipeds, particularly in individuals that inhabit areas with greater human activity or domestic animal presence (50, 51). Both Leptospira and T. gondii can cause morbidity and mortality in marine mammals, including pinnipeds (52, 53), and infected rodents near reproductive colonies of fur seals and/or sea lions could represent a potential source of pathogen spillover (54, 55). Moreover, pinnipeds themselves may act as carriers and play an important role in the maintenance and transmission of Leptospira spp. within coastal ecosystems (56). Chronic shedding of Leptospira interrogans in California sea lions (Zalophus californianus) has been shown to sustain bacterial persistence between outbreaks, enabling long-term maintenance of infection within populations despite seasonal variation in transmission (57).

Surveys of T. gondii and Leptospira spp. exposure have not yet been conducted in seabird populations of PSJ, including the Humboldt penguin (Spheniscus humboldti), Guanay cormorant (Leucocarbo bougainvillii), and Peruvian pelican (Pelecanus thagus) (58, 59). Further investigation into these species is warranted given their ecological overlap with terrestrial mammals, the presence of invasive rodents, and their potential exposure to contaminated environments. In other coastal regions, serological evidence of T. gondii infection has been reported in seabirds (60–62), and fatal toxoplasmosis has been documented in penguins (63, 64). In contrast, information on Leptospira infection in birds remains scarce. Although exposure to Leptospira spp. has been reported in two Magellanic penguins (Spheniscus magellanicus) and other wild birds in Chile (50), other studies from Chile and Brazil have failed to detect exposure in other seabird species (60, 61). Thus, the epidemiological role of Peruvian seabirds in Leptospira transmission, their potential contribution to pathogen circulation, and the clinical significance of infection remain unknown.

The detection of T. gondii in a single rodent inside the reserve and the higher molecular detection frequency of Leptospira spp. infection in black rats captured outside PSJ suggests distinct ecological and epidemiological dynamics of pathogens associated with habitat type and human influence. These findings highlight the potential involvement of Rattus rattus in pathogen circulation at the interface between wildlife, domestic animals, and humans in coastal ecosystems (20). Because Leptospira detection was based on PCR amplification of bacterial DNA in blood, these results reflect active or recent infection (39). Therefore, the present findings demonstrate ongoing circulation of pathogenic Leptospira among black rats in the study area, but do not allow inference on chronic carriage, shedding dynamics, or long-term reservoir competence.

Leptospira detection was reported at the genus level, as the real-time PCR protocol used targets the lipL32 gene, which is conserved among pathogenic Leptospira species (40, 43). While molecular typing or serovar identification would provide higher epidemiological resolution, such analyses were beyond the scope of this study and require additional molecular markers, isolation, or sequencing approaches. Future studies incorporating genotyping or multilocus sequence typing would substantially enhance understanding of transmission pathways and public health relevance.

The presence of Leptospira-positive black rats in areas surrounding the reserve indicates the possible introduction of pathogenic strains from nearby urban zones, where synanthropic rats thrive and may facilitate pathogen spillover into protected habitats (65). Leptospira can persist for extended periods in humid environments and be transmitted through urine-contaminated soil or water, posing infection risks for both wildlife and humans (22). Consequently, its detection on beaches adjacent to the reserve represents a direct occupational hazard for local fishers and kelp collectors, who often work barefoot or in close contact with seawater, kelp, caves, sand and gravel on the beach. Moreover, as marine mammals and seabirds frequently congregate in these areas, cross-species transmission events cannot be ruled out, underscoring the need for coordinated pathogen monitoring under a One Health framework.

The absence of viral RNA in the respiratory samples analyzed does not necessarily exclude rodent participation in viral maintenance or transmission but rather provides an essential baseline for future surveillance. Considering the increasing diversity of respiratory and zoonotic viruses recently identified in wild rodent populations worldwide (66, 67), continued metagenomic screening in coastal ecosystems of South America is warranted to better elucidate the viral communities circulating in invasive species and their potential for interspecies transmission to inform and promote eradication or removal of these reservoirs from sensitive wild areas.

This study has several limitations that should be considered when interpreting the results. The sample size was relatively small, which may have limited the ability to detect statistical associations. An additional limitation of this study is the absence of serological testing for Leptospira spp., which precludes assessment of past exposure and population-level prevalence. Furthermore, leptospiral persistence and shedding are primarily associated with renal colonization; therefore, future studies should include molecular and histopathological analyses of kidney tissue, and ideally urine samples, to confirm renal carriage and assess chronic infection status and reservoir potential in invasive rodent populations (39, 68, 69). In the urban area outside the reserve, trapping success was constrained by low public acceptance of rodent capture and the abundance of alternative food sources reduced bait efficiency. The presence of free-roaming domestic cats in the city may also have influenced rodent activity and spatial distribution (70).

Beyond the influence of urbanization, environmental conditions likely play a critical role in Leptospira persistence and transmission. Accumulation of garbage, organic waste, and informal dumpsites in the desert and on polluted beaches around PSJ creates microhabitats that favor rodent proliferation and bacterial survival in moist substrates (70). Seasonal and longitudinal sampling would also help reveal temporal fluctuations in Leptospira circulation and identify environmental drivers of infection. Expanding surveillance to include domestic animals, livestock, and local human populations would further clarify the ecological dynamics of leptospirosis in the area (25). Integrating these approaches under a One Health perspective will be essential for identifying transmission pathways and developing effective mitigation strategies.

Overall, these findings emphasize the importance of sustained surveillance and rodent management at the human–wildlife interface in coastal protected areas. Continuous monitoring of invasive species, combined with wildlife and environmental pathogen surveillance, will support early detection of emerging diseases and mitigate health risks to both biodiversity and local communities.

Funding Statement

The author(s) declared that financial support was received for this work and/or its publication. This work was funded by CONCYTEC through the PROCIENCIA program under the call “Proyectos Especiales: Proyectos de Incorporación de Investigadores Posdoctorales en Instituciones Peruanas 2024”, according to contract PE501092495-2024.

Data availability statement

The data presented in this study are included in the article and its supplementary material. Further inquiries can be directed to the corresponding author.

Ethics statement

The animal study was approved by Institutional Animal Care and Use Committee of Universidad Peruana Cayetano Heredia (CIEA-UPCH: CONSTANCIA-CIEA-038-08-24; CONSTANCIA-CIEA-E-035-12-24; CONSTANCIA-CIEA-R-013-02-25). The study was conducted in accordance with the local legislation and institutional requirements.

Author contributions

CC-M: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. WS-C: Investigation, Methodology, Validation, Writing – review & editing. JV-M: Investigation, Methodology, Supervision, Validation, Writing – review & editing. YT-C: Investigation, Methodology, Validation, Writing – review & editing. JC: Investigation, Methodology, Validation, Writing – review & editing. EM-M: Investigation, Methodology, Validation, Writing – review & editing. MC: Investigation, Methodology, Supervision, Validation, Writing – review & editing. AM-V: Investigation, Methodology, Validation, Writing – review & editing. PT: Investigation, Methodology, Supervision, Validation, Writing – review & editing. SC-A: Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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