Leptospirosis is a disease caused by several species of Leptospira, which are spirochetes belonging to the order Spirochaetales of the family Leptospiraceae, and include more than 250 pathogenic serovars. Under favorable conditions and in the presence of suitable hosts, these organisms can survive for weeks or months. They are common in convalescent animals, and asymptomatic carriers contribute to the maintenance of leptospirosis hotspots1.
Globalization and associated processes, such as urban demographic growth, forest clearing for the advancement of agriculture and livestock, and industrialization, have reduced the boundaries between populations of humans and domestic animals and those of wild animals, facilitating the spread of infectious diseases2.
Marsupials in the family Didelphidae have distinct anatomical and physiological characteristics and extensive behavioral diversity. They inhabit forests, marshes, and semi-arid areas, at high or low altitudes, as well as aquatic environments; additionally, they are able to survive in fluctuating environments. They may become synanthropic or live in areas of fragmented vegetation3.
Ecological disturbances cause marsupials to invade urban areas in search of food and shelter. Accordingly, they are common in cities, with substantial litter, which favors the spread of zoonoses, such as leptospirosis4. Therefore, in the present study, anti-Leptospira antibodies were examined in free-living marsupials in the municipalities of Peixe-Boi (01º11’31 “S, 47º18’44” W), Castanhal (07º20’53 “S, 50º23’45” W), and Viseu (01º11’48 “S, 46º08’24” W) within the metropolitan mesoregion of Belém and Northeast Pará, Brazil.
A total of 19 marsupials (Table 1) were collected using Sherman, Pitfall, and Tomahawk bait traps. Traps were separated by 10m and remained open for at least ten consecutive nights, with daily morning inspections. The captured animals were anesthetized with a combination of xylazine hydrochloride (0.1mg/kg) and ketamine hydrochloride (5mg/kg) injected intramuscularly. Blood was collected by venipuncture at the base of the tail or by intracardiac puncture using sterile needles (0.45mm × 13mm), placed in tubes without an anticoagulant, and sent to the Laboratory of Zoonoses and Public Health (Federal University of Pará). Then, the serum was removed, transferred to 1.5-ml microtubes, and stored at -22°C until the serological test.
TABLE 1: Distribution of captured marsupial species.
| Species | Number |
|---|---|
| Didelphis marsupialis | 10 |
| Caluromys sp. | 1 |
| Micoureus cf. demerarae | 1 |
| Philander opossum | 1 |
| Marmosa murina | 5 |
| Metachirus cf. nudicaudatus | 1 |
| Total | 19 |
Antibodies were detected using the microscopic agglutination test (MAT) with antigens from 31 serovars of Leptospira spp Serovars Australis, Bratislava, Autmnalis, Butembo, Castellonis, Bataviae, Canicola, Whitcombi, Cynopteri, Djasiman, Sentot, Gryppotyphosa, Hebdomadis, Copenhageni, Icterohaemorrhagie, Javanica, Panamá, Pomona, Pyrogenes, Hardjo, Wolffi, Shermani, Tarassovi, Andamana, Patoc, Guaricura (isolated at USP-São Paulo) Nupezo, Hardjo miniswajizak, Hardjo prajitno, Hardjo CTG, and Hardjo bovis were used as references.
Serum exhibiting an agglutination reaction equal to or greater than 50% on the 1:50 dilution were considered positive. The cutoff point was a dilution of 1:1005. The most probable serovar was defined as the serovar with the highest titer and frequency. Samples that presented equal titers for two or more serovars were excluded from the analysis and considered only as reagents for Leptospira spp6.
Of the 19 samples collected, 36.8% (7/19) were positive for Leptospira spp according to the MAT. Among these, five samples were from the municipality of Peixe-Boi, and two were from the municipality of Viseu. Co-agglutination, i.e., reaction to more than one serovar, was observed in two samples; therefore, these were not considered in the analysis of the most frequent serovars. The most frequent serovars were Icterohaemorrhagiae, representing 60% (3/5) of positive samples, followed by Panama and Nupezo, each at 20% (1/5). The titers ranged from 100 to 400, and 100 and 200 were the most frequent titers (Table 2).
TABLE 2: Characterization of serovars in marsupial samples according to locality, frequency, and antibody titers.
| Localities | Species | Serovars | |||
|---|---|---|---|---|---|
| Icterohaemorragiae | Panama | Nupezo | |||
| titers | |||||
| Peixe-Boi | Didelphis marsupialis | 100 | – | – | |
| Peixe-Boi | Didelphis marsupialis | 200 | – | – | |
| Peixe-Boi | Caluromys sp. | 100 | – | – | |
| Peixe-Boi | Didelphis marsupialis | – | – | 400 | |
| Viseu | Marmosa murina | – | 200 | – | |
| Total | 5 | 3 | 1 | 1 | |
To our knowledge, this is the first report of the Icterohaemorrhagiae serovar in marsupials in the Brazilian Amazon. The serovar is considered pathogenic for various wild and domestic animals, as well as humans. We cannot confirm that the marsupials serve as a source of Leptospira infection, since the immune response in these animals is unknown. However, environmental disturbances can increase the proximity of these animals to urban areas and may promote the spread of pathogenic Leptospira to humans and domestic animals7.
In this study, one sample was reactive to the Panama serovar, with a titer of 200. Similarly, Paixão8 detected this serovar in free-living rodents at a conservation center of wild fauna in Ilha Solteira, SP. As rodents move freely in the forest in search of food, they acquire Leptospira and transmit it indirectly to animals of different species. This may explain why we found the Panama serovar in marsupials, since they live in the same habitat as several rodent species.
The Nupezo serovar (isolated from canine urine), belonging to the Canicola serogroup, was also identified in this study. Considering that marsupials are synanthropic and forage near human residences, it is possible that these wild animals had contact with domestic animals or environments containing the agent. A previous study of zoo animals involved 22 serovars and identified two samples that were reactive to the Patoc serovar9. However, we tested for 31 serovars in this study. The variation in the number and type of tested serovars may explain the differences in results. By including more serovars, we were able to obtain a more comprehensive evaluation of Leptospira circulation in the region.
In a study of domestic and wild animals at the university campus of Faculdade de Ciências Agrárias e Veterinárias(FCAV), Universidade Estadual Paulista Julio de Mesquita Filho (UNESP), Jaboticabal10, the detection rate (11/25; 44%) was higher than that in the present study. They examined 25 opossums (Didelphis albiventris) housed with horses, pigs, and dairy cattle and observed a higher frequency of reactive horses, pigs, and cattle during the period in which there was a high prevalence of reactive opossums, reinforcing the hypothesis that marsupials may be important reservoirs of Leptospira spp.
Lower detection rates have been observed for opossums (Didelphis virginiana) collected in peridomestic areas of Yucatán, Mexico11, with a seropositivity of 4.9% (4/81) and a high prevalence of Pomona and Wolffi serovars. However, only 10 strains were considered, which may explain the low detection rate. Furthermore, a lack of knowledge of the circulating serovars in Mexico and Brazil may result in underestimates of seropositivity.
In a study of a farm area in the southern area of Lake Maracaibo, Mérida, Venezuela12, serological tests were performed on small mammals (37 rodents and 2 marsupials) caught in pastures. No seroreactivity for Leptospiraspp was detected, despite a high seropositivity rate in cattle in the area. Although we did not examine the seroprevalence in domestic animals in the study areas, the potential that they serve as a source of infection for wild animals cannot be ruled out.
We conclude that anti-Leptospira spp antibodies are currently circulating in free-living marsupials in the municipalities of Peixe-Boi and Viseu, Pará, suggesting that animals in these areas may be important sources of Leptospira infection.
ETHICAL CONSIDERATIONS
The manipulation of wild animals followed the recommendations of the National Council for Control of Animal Experimentation (CONCEA), with authorization from the Ethics Committee on the Use of Animals of the Evandro Chagas Institute under protocol number 028/2014. Authorization for the collection of biological samples was obtained from the Biodiversity Information and Authorization System (SISBIO), under protocol number 37174-1.
