Abstract
Introduction:
Leptospirosis is a zoonotic disease caused by the bacterium Leptospira spp., which is divided into saprophytic and pathogenic. Transmission in zoos can occur due to infected urine in poorly sanitized environments, rodents, and animal accumulation. Aim: This study investigated the presence of antibodies and DNA of Leptospira spp. in nonhuman primates and wild felids from a zoo in western Paraná, Brazil.
Materials and Methods:
Samples were collected through chemical restraint and venipuncture. Antibody testing was performed by microscopic agglutination test and polymerase chain reaction (PCR).
Results:
Of the 24 samples analyzed (17 capuchin monkeys and 7 felines), 12.5% presented antibodies for the Butembo serovar, all in primates. No feline showed reaction or DNA amplification.
Conclusion:
The results highlight the importance of studies on leptospirosis in wild animals in captivity, considering factors such as biome and management of the zoo, which houses rescued species that cannot be reintroduced into the wild.
Introduction
Leptospirosis is a zoonotic, cosmopolitan disease with an acute to chronic course and endemic in several regions and locations. It is caused by a highly mobile, aerobic spirochete bacterium of the genus Leptospira. The bacterium is divided into saprophytic serovars, which are present in the environment, and pathogenic serovars, which lodge in the host’s renal tubules and are excreted in the urine. It can affect different species of domestic and wild animals (Santos et al., 2023).
Upon entering its host, Leptospira spp. migrates to the kidneys and, from there, actively and intermittently eliminates itself through urine, even if there are no clinical signs (Aliaga‐Samanez et al., 2022).
In zoos, the occurrence of this disease may occur due to the absence and/or deficiency of hygiene, presence of rodents, overcrowding of animals in the enclosures, lack of sanitary structure for disposal of animal waste, presence of food remains around the enclosures, and presence of garbage in the vicinity of the zoo, among other situations that favor the presence of rodents, which are the main reservoirs of this disease (Aliaga‐Samanez et al., 2022; Hardgrove et al., 2021).
Transmission is influenced by characteristics of the infectious agent, host susceptibility, demographic agglomeration, and interaction between species. Rodents of the species Mus musculus, Rattus rattus, and Rattus novergicus are involved in the epidemiology of the disease by eliminating the microorganism through urine. Infection can occur through direct exposure (contact with infected urine) or indirect exposure (contact with the bacteria through food, water, and the environment) (Bierque et al., 2020).
Primates, wild felines, and marsupials play an important role as reservoirs of the bacteria, as well as for the persistence of foci of infection. However, rodents are still considered the main reservoirs of the disease, as they have alkaline urine and low antibody levels. Thus, being able to actively eliminate the bacteria from the environment, enabling the contamination of water, soil, and food (Aliaga‐Samanez et al., 2022; Bierque et al., 2020; Santos et al., 2023).
Zoos play an important role in the conservation of wild animal species, which are protected from smuggling, predatory hunting, illegal breeding, accidents, abandonment, or exploitation in a variety of ways. It acts as a place of environmental education, refuge, and recovery environment for such animals. On the other hand, when they stop living in their natural habitat and start living in urban environments, these animals become more exposed to different reservoirs, becoming susceptible to various pathogens (Artigas and Fischer, 2019; Piredda et al., 2021).
Wild animals kept in captivity, whether in a zoo or by a registered keeper, can be an important link in the transmission of different zoonotic pathogens, such as Leptospira spp., to humans and other animal species present there. For this reason, the objective of this study was to investigate the presence of antibodies and DNA of Leptospira spp. in nonhuman primates and wild felines from a municipal Zoo in the western region of the state of Paraná.
Materials and Methods
Study location and population
This research project was carried out in a municipal zoo, in a city in the western region of the state of Paraná, Brazil.
Ethical aspects
This study was submitted and approved by the Ethics Committee for Research Involving Animal Experimentation of UNIPAR, under protocol 39559/2022; and by the Chico Mendes Institute for Biodiversity Conservation (ICMBio), under Number: 82499-1. All collections were conducted during the year 2023.
Animal species and sampling
Twenty-four animals were used, including seven wild felines and 17 nonhuman primates.
The feline species studied were four cougars (Puma concolor), one jaguar (Panthera onca), and two ocelots (Leopardus pardalius). The nonhuman primates researched were all of the Capuchin Monkey species (Sapajus apella). Animals that had been at the zoo for more than a year were selected, and biological samples were collected when routine physical examinations of the animals were necessary, as per standard procedure at the respective zoo. At this time, the project team and the zoo team worked together.
Capture and containment of animals
All capture and handling procedures for these animals were carried out by properly trained veterinarians and biologists belonging to the zoo team and according to the species, using drugs already used in the zoo’s routine according to the standard protocol kindly provided by the respective team: Feline containment protocol: 7 µg/kg dexmedetomidine hydrochloride; 2 mg/kg ketamine hydrochloride; 0.15 mg/kg midazolam maleate; 0.2 mg/kg methadone hydrochloride; Frequency: Single dose; Duration: Not applicable
The containment of the large felines was carried out in the handling area through the association of the aforementioned drugs, in their respective dosages, mixed in a single dart, which was fired into the animal’s muscles using a blowgun.
In the case of small felines (Leopardus pardalius), capture was carried out in the enclosure using a punch. After capture, a trained employee immobilized the animal using leather gloves so that the veterinarian in charge could apply the drugs listed according to each animal’s weight intramuscularly using a sterile needle and syringe. Primate restraint protocol: 2 µg/kg dexmedetomidine hydrochloride; 2 mg/kg ketamine hydrochloride; 0.15 mg/kg midazolam maleate; 0.1 mg/kg methadone hydrochloride; Frequency: Single dose; Duration: Not applicable
The capture of Capuchin Monkeys (Sapajus apella) was carried out in the enclosure using a net. After capture, a trained employee immobilized the animal using leather gloves so that the veterinarian in charge could apply the drugs according to each animal’s weight, intramuscularly, using a sterile needle and syringe.
Collection of biological samples
Blood collections were performed by the zoo’s responsible veterinarian, accompanied by the team of this research project, and were performed, preferably, through the cephalic, jugular, or caudal vein, respecting antisepsis measures with the use of 70% alcohol.
For each collection, a sterile needle, catheter, or syringe of the appropriate size for each animal species was used. The collected samples were stored in isothermal boxes and sent to the Preventive Veterinary Medicine and Public Health Laboratory of the Postgraduate Program in Animal Science with Emphasis on Bioactive Products at Universidade Paranaense. After collecting the biological samples, the animals were returned to their place of origin.
Diagnostic methods
Serological
For the detection of anti-Leptospira spp. antibodies, serum samples were subjected to a microscopic agglutination test (MAT). Fourteen reference serovars were used: Bratislava, Butembo, Castellonis, Canicola, Grippotyphosa, Icterohaemorrhagiae, Copenhageni, Pomona, Pyrogenes, Hardjo, Wolffi, Hardjobovis, and Tarassovi, maintained at 28°C for 5 to 10 days in modified EMJH (Ellinghausen-McCullough-Johnson-Harris) medium (DIFCO®-USA). Serum samples that presented at least 50% of agglutinated Leptospiras at a dilution of 1:100 were considered reactive and then diluted at a ratio of two to one to determine the maximum positive dilution.
Molecular
DNA extraction from blood samples was performed using the PureLink Genomic DNA Mini Kit (Invitrogen—USA).
The DNAs were subjected to the PCR technique, using the Platinum PCR SuperMix Kit (Invitrogen—USA), following the manufacturer’s instructions, adding the specific primers for the partial sec Y gene (F 5′-ATGCCGATCATTTTTGCTTC-3′ and R 5′-CCGTCCCTTAATTTTAGACTTCTTC-3′) with the aim of identifying and confirming the genetic species (Piredda et al., 2021). The final product of PCR amplification was subjected to electrophoresis in a 2% agarose gel containing ethidium bromide (0.05 μg/μL), and visualization was performed on a transilluminator with ultraviolet light, and the molecular weight was estimated according to the comparison with the 100 bp molecular marker. In all reactions, positive and negative controls using autoclaved ultrapure water were included.
Results
For the research of anti-Leptospira spp. antibodies in the 24 samples of nonhuman primates and wild felines, a prevalence of 12.5% was detected; that is, three reactive samples, and all for the antibody against the Butembo serovar. Of the three samples, all from primates, two (8.33%) had a titer of 100, and one (4.16%) had a titer of 200. Regarding the research of anti-Leptospira spp. antibodies in the seven feline samples, none were reactive.
There was no DNA amplification for the DNA research of Leptospira spp. in the 24 samples between nonhuman primates and wild felines.
Discussion
Paraná is the state that presented the highest rate of deforestation of the Atlantic Forest between 1995 and 2000, with this plant formation being the habitat of many animal species belonging to the wild fauna. Paraná is also considered an area for capturing, passing through, and exporting wild animals [Instituto Paranaense de Desenvolvimento Econômico e Social (IPARDES), Rodrigues et al., 2023].
The Paraná Environmental Institute seized 20,275 wild animals between 1980 and 2002. Most of the animals seized were destined for illegal trade, and there was a greater seizure rate in the regions located in the western region of the state. The number of fauna seizures is correlated with the ease of land access, which facilitates the transport or trafficking of animals between the states of São Paulo and Mato Grosso, as well as easy access to neighboring countries such as Argentina and Paraguay, making inspection actions difficult, since in terms of vegetation, these areas are largely occupied by agricultural and livestock areas (Mendes França et al., 2021).
In the present study, none of the wild felines tested using the MAT presented reactive results; however, in the primate samples, three (17.64%) were considered reactive with titers ranging from 100 to 200, with the antibody against serovar Butembo being the most prevalent. This serovar has already been reported by other researchers in a study of free-living wild animals in Brazil, including in the State of Paraná (Lenharo et al., 2012; Vieira et al., 2013). Regarding the detected titration, it is believed that it must correspond to the immunological profile of the animals since they must have been exposed to the etiological agent at some point in their lives, characterized by the presence of antibodies.
The serological results of the present study are inferior to the studies carried out in the State of Bahia, where Pinna et al. (2012), in the city of Salvador, detected in 44 primates (Callithrix jacchus, Cebus, and Callithrix pennicilata), 56.8% of reagent samples in the MAT also with a titration of 100; however, the most prevalent serovars were Icterohaemorrhagie and Canicola. In another state, Rio Grande do Sul, Grumann (2015), in the city of Passo Fundo in Rio Grande do Sul, evaluated 21 nonhuman primates (Sapajus apella and Alouatta guariba) also using the MAT method, having detected 90.47% of reactive samples, with titers ranging from 100 to 400, and the most commonly found serovars were Sejroe, Panamá, and Andamana.
It should be noted that the detection of Leptospira spp. DNA by PCR in blood samples presents significant limitations related to the timing of sample collection and the stage of the disease. For example, during the leptospiremic phase, the bacteria circulate in the bloodstream, which allows for their identification through molecular methods. However, after this period, the pathogens tend to migrate to tissues and be excreted in the urine, substantially reducing the sensitivity of PCR in blood samples (Aymée et al., 2025; Fonseca et al., 2006).
Another important detail that may interfere with the result of the present study is that wild felines can act as refractory hosts of Leptospira spp., meaning they can be infected by the bacteria without showing obvious clinical signs of the disease. This suggests that these animals possess some immune or biological mechanism that limits the progression of the infection. Although refractory, these felines can excrete the bacteria in their urine, contributing to the spread of the pathogen in the environment, which poses a risk to other species, including humans. The ability of these animals to harbor and disseminate Leptospira without suffering from the typical effects of leptospirosis has important implications for understanding the ecology of the disease, especially in natural ecosystems, where transmission dynamics may be more complex and difficult to control (Mazzotta et al., 2023). It is worth noting that another factor that may have influenced the results is the pest control implemented at the site, from the feeding of these animals to their management.
The serological results of this study may have been influenced by the difference in the prevalence of animal leptospirosis in the respective study sites, in addition to reflecting the study period, which may have provided greater or lesser probabilities of infection occurring in the animals (Benitez et al., 2021). Another fact that may have also contributed to the low prevalence of this study was that the time when blood collections were carried out from the animals was carried out in July 2023, considered a dry season in the region. This is in line with the work of Azevedo et al. (2004), where it is reported that the choice of time of year for collecting biological material can also influence the positivity of animals since the dry season can reduce the environmental conditions favorable for the survival of the etiological agent in the environment and consequently the spread of the disease.
Another environmental factor that may have contributed to this low prevalence in this research is that the western region of the State of Paraná has deep, clayey soils with a dark red color that arefriable and very porous, thus being highly draining (Larach et al., 1984), which may have influenced the nonsurvival of the microorganism, since humidity is a necessary condition for the survival of the etiological agent.
According to information obtained by Larach et al. (1984), through soil studies in the western region of Paraná, it is observed that this is not an endemic area for leptospirosis, given that due to its geographic location, soil composition, and water disposition of rivers and tributaries, the probability of flooding is low; thus, it does not provide the ideal scenario for the dissemination of the etiological agent by the different hosts.
All animals present at the respective zoo come from rescues and seizures collected by environmental agencies such as the Water and Land Institute (IAT), Environmental Police, the Brazilian Institute of the Environment and Renewable Natural Resources (IBAMA), and the Wildlife Support Center (CAFS). These animals sent to the zoo can no longer be reintroduced into the wild. Upon arrival, they are housed in the “extra sector,” a designated area within the zoo for quarantine before being placed in their enclosures. Until the samples for this study were collected, the newly arrived animals had not undergone any examinations. Due to this situation, it is believed that these animals that reacted in MAT may have been exposed to Leptospira spp. at some point in their lives. According to the MAT antibody titration, this exposure very possibly occurred in the environment prior to their rescue.
The primate enclosure has no connection with other species in the zoo, as well as the drainage of their waste (urine and feces); the urine, as well as the water used to clean and sanitize the enclosures, flows into a containment box and finally returns to the sewer. The feces are collected daily and sent to the local compost bin. Likewise, the enclosures for big cats do not have connections or considerable proximity to other enclosures in the zoo. The management of the drainage and collection of organic materials occurs in the same way as in the primate enclosure, but this management is carried out by another employee, who is solely responsible for the feline sector, which contributes to the good sanitary practices of the zoo.
Environmental and hygiene management are essential to maintain the health of animals in captivity; therefore, the results obtained demonstrate the good operating and management status of the animals residing in the aforementioned zoo.
This study presents highly relevant findings within the One Health context, demonstrating that the animals in this study, residing in the zoo, receive proper care in terms of hygiene, health, infrastructure, and management. This is evident in the zoo’s operational practices, including the assignment of a dedicated employee to each sector, ensuring limited exposure between different areas of the facility.
The primate and feline enclosures receive treated water from the Basic Sanitation Company of the State of Paraná (SANEPAR); and all discarded water passes through a sediment containment box, thus returning to the sewer. The feces are collected and sent to the zoo’s compost bin, and it is observed that the different facilities/enclosures are maintained within the expected basic care so that no presence of reservoirs could allow the spread of different pathogenic agents among wild animals.
Conclusions
It is concluded that the serological results indicate the prevalence of anti-Leptospira spp. antibodies among primates in the western region of Paraná and that the animals were exposed to the etiological agent at some point in their lives. This highlights the importance of regional studies of captive wild animals to understand leptospirosis and its associated factors, such as biome, environmental characteristics, and management practices. These studies help identify risks and improve the management of rescued animals living in zoos.
Footnotes
Acknowledgment
The authors thank the zoo staff for their ongoing collaboration.
Authors’ Contributions
Conceptualization, A.C.S.A., I.C.S., and D.D.G.; Methodology, A.C.S.A., D.D.G., X.X., and R.N.R.; Formal analysis, F.G.C.B.; Collection of biological samples, A.C.S.A., B.P.B.S., E.M.S.J., R.N.R., G.C.C.S., and R.P.J.; Laboratory analysis, F.G.C.B., L.N.B., and L.G.P.G.; Writing—review and editing, S.G.D.C., I.C.S., Z.C.G., and D.D.G.; Supervision, L.N.B.; Project administration, D.D.G.; Funding acquisition, D.D.G.
Author Disclosure Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Funding Information
This research was funded by UNIPAR, CAPES for granting the PROSUP school fee and CNPq (CP 09/2023—Research Productivity Grants) Grand Number 310953/2023-5.