Discussion
This study stands as the most comprehensive investigation ofLeptospira spp. infection in domestic and wild animals from Fernando de Noronha archipelago, also providing the description of the first leptospiral strain ever isolated from an insular setting in Brazil. Overall, most animals reacted against serogroup Icterohaemorrhagiae, corresponding to almost 80% of all positive samples that the most likely infecting serogroup could be determined by MAT. The presence of anti-Leptospira antibodies in animal species from Fernando de Noronha have already been described in 2018 by Moraiset al . (2018), who also found Icterohaemorrhagiae as the main serogroup infecting cattle and rodents in the island. Although prevalence of renal carriage has not been accessed in the present study, the identification of Icterohaemorrhagiae as the most prevalent serogroup in practically all species strongly suggests that rodents play a major role in leptospiral transmission in Fernando de Noronha. Rodents are recognized as the most important reservoir of leptospires worldwide, and renal carriage of different pathogenic serovars causing disease in humans and other animals have been frequently reported (Boey et al., 2019).
Invasive rodents in Fernando de Noronha are subject of major concern, once they can rapidly reproduce and adapt to human activity (Benavidez et al., 2019). Furthermore, invasion of rodents induced devastating effects on local biodiversity, leading to extinction of native wildlife (Russell et al., 2018). These ecosystem disruptions are frequently associated to diminished diversity of species within ecological communities, potentially leading to increased incidence of leptospirosis in insular environments (Derne et al., 2011). Rats, especially black rats, are widely distributed in Fernando de Noronha and density is currently estimated at 3,700 individuals/km2 in the main island (Russell et al., 2018). In our study, both R. rattusand R. norvergicus presented agglutinins exclusively against Icterohaemorrhagiae serogroup. Icterohaemorrhagiae have also been identified as the most prevalent serogroup among rodent populations from numerous insular locations (Boey et al., 2019) and strains belonging to this serogroup are considered the main pathogen causing human leptospirosis in the Reunion Islands (Guernier et al., 2016) and French Polynesia (Guernier et al., 2017).
Interestingly, even in face of the high exposure to Icterohaemorrhagiae, the characterization of the leptospiral strain recovered from a black rat revealed infection caused by L. interrogans serogroup Pyrogenes. Pyrogenes strains have been isolated from humans in New Caledonia (Thibeaux et al., 2017), Philippines (Villanueva et al., 2014), Puerto Rico (Valverde et al., 2013) and Mayotte islands (Bourhy et al., 2010). In the Brazilian mainland, two strains belonging to the Pyrogenes serogroup were recovered from a grey opossum (Phylander opossum ) and water rats (Nectomys squamipes ) in previous attempts to isolate leptospires from wildlife (Santa Rosa et al., 1975, 1980).
Despite no novel STs was revealed through the MLST analysis, further whole genome sequencing should be performed in future studies for more accurate phylogenetic inference and for a more comprehensive understanding on the pathogenicity of the isolated strain, including key aspects related to host-serovar adaptation. Our results suggest that Fernando de Noronha might present a mixed pattern of leptospiral diversity, where serogroups probably brought along in more recent introduction of rodents coexist with older lineages, as proposed by Guernier et al . (2018) and Dietrich et al. (2018) in Western Indian Ocean islands. Nevertheless, further studies focusing on more in-depth analysis of the leptospiral strains circulating locally should be conducted to fully elucidate the epidemiology of leptospirosis in Fernando de Noronha.
From the 18 seroreactive bovines, 14 had the highest titers against Icterohaemorrhagiae serogroup. These results are markedly different from those found in Brazilian mainland and most parts of the world, where seroreactivity is predominantly associated with serogroup Sejroe, particularly serovar Hardjo (da Silva Pinto et al., 2016). Acute and incidental infection is rare and has been associated with serovars Pomona, Grippotyphosa and Icterohaemorrhagiae (da Silva Pinto et al., 2016). Nine out of the 14 bovines seroreactive for Icterohaemorrhagiae had titers ranging from 400 to 1.600, thus indicating recent exposure to the pathogen. Similarly to bovines, most seroreactive sheep and goats showed the highest titers against Icterohaemorrhagiae, which also diverges from serological data found in continental Brazilian territory, where leptospirosis in small ruminants is also historically associated with serogroup Sejroe, particularly serovar Hardjo (Martins and Lilenbaum, 2014). Horses had the highest seroprevalence found and presented the highest titers mostly against Icterohaemorrhagiae. Although serological surveys in Brazil have confirmed infection caused by serogroup Australis, including horses from Northeast region (Morais et al., 2019), Icterohaemorrhagiae infection has been consistently demonstrated in numerous other studies, currently categorizing this serogroup as the most prevalent among horses in Brazil (Pinto et al., 2017).
Only a small proportion of cats were positive by MAT, and most individuals had titers against serogroup Icterohaemorrhagiae. Curiously, none of the 200 cats previously surveyed in Fernando de Noronha by Morais et al. , (2018) presented titers against Leptospira . The epidemiology of feline leptospirosis is yet to be determined, and serovars infecting cats vary greatly across different environmental conditions. Overall, seroreactivity is low, and Pomona has been frequently attributed to leptospiral infection in cats, however infection by serovars from Autumnalis and Ballum serogroups can also occur (Murillo et al., 2020). Under insular conditions, an even wider range of serovars can be found in cats, such as Bataviae in Malaysia (Alashraf et al., 2020), Semaranga in Thailand (Sprißler et al., 2019) and Cynopteri in Saint Kitts (Pratt et al., 2017). Cats have long been believed to control rodent populations, and low proportion of seroreactive cats in Fernando de Noronha requires further investigation, especially considering the high density of rodents found in the island. Still, these findings may indicate naturally acquired immune response to leptospiral infection. Alternatively, most of the cats sampled were found around the inhabited areas and under supervised care by local inhabitants, and although not specifically kept as pets, cats were predominantly subsidized by human feeding, which might attenuate predatory behavior and consequentially lead to less exposure to pathogenic Leptospira .
It is noteworthy that dogs had the most variable serological response in this study. Although Icterohaemorrhagiae was indeed the predominant serogroup found in the canine population, dogs with the highest titers (≥400) may have possibly presented infection by different serovars such as Whitcombi, Autumnalis and Australis, with the former being the serogroup with the highest titration among all seroreactive animals in the study. These findings are consistent with former studies that also found highly diverse serological response in dogs living on insular settings (Pratt et al., 2017). Such repertoire might be influenced by immunization, which tends to promote paradoxical reactions found in MAT, leading to titration against multiple serovars unrelated to infection. Nevertheless, the dogs included in the study were mostly supervised pets with unrestricted movement and no history of recent immunization, and serovar diversity infecting dogs may reflect intense environmental exposure to leptospires or contact with livestock and peridomestic wildlife reservoirs. Further studies should be addressed to clarify the actual role of dogs in the epidemiology of leptospirosis in Fernando de Noronha, and serological findings may at least indicate dogs as important sentinels for human infection in the island.
Few birds (Gallus domesticus and Bubulcus ibis ) had antibody titers detectable by MAT. The only three seroreactive cattle egrets presented titers against Icterohaemorrhagiae serogroup. Icterohaemorrhagiae could also be detected in one out of the four chickens positive by MAT. Little is known about leptospirosis in birds, but previous reports have also shown low seropositivity in domestic and wildlife birds (Brasil et al., 2013; Acosta et al., 2019). The prevalence found in tegu lizards was relatively high, with four out of the 13 samples reacting against serogroups Icterohaemorrhagiae and Autumnalis, and although the small sample size, similar high prevalence is usually reported in Brazil (Biscola et al., 2011; Paz et al., 2019; Miranda et al., 2020). Still, the role of reptiles as reservoirs of leptospirosis and their risks for public health is debatable (Fornazari, 2017). Few studies have tackled infection and pathogenesis in avian and reptile species, and serological findings in the present study indicate exposure to environmental leptospiral strains circulating locally.
Overall, the serological pattern found in all species highlight the importance of rodents for leptospiral transmission in the island. Rodent control is currently limited to poison bait stations around areas with more infrastructure, which are checked and re-stocked at regular intervals. No rat surveillance in the in the bay area is taken, and invasion from shipped cargo is neglected. Therefore, there is an urgent need for the implementation of a systematic rodent control program. Likewise, the poor sanitary infrastructure conditions found in the island also poses as risk of Leptospira spp. transmission not only to the local human population but also for tourists. The improvement of the sanitary conditions in the archipelago is however a logistical challenge to local managers, since the long distances from the continent and high freight costs make any civil construction intervention very difficult to implement. Although no permanent water stream is found in the archipelago, most of the small farms are located around one large water reservoir, which was used for human consumption in the past. This water reservoir is probably not only associated with the transmission of Leptospira spp. between farm animals, but also wild animals. Cattle egrets, for example, were found at the airport strip, which is located between this water reservoir and the archipelago garbage dump.