Insight into the Epidemiology of Leptospirosis: A Review of Leptospira Isolations from “Unconventional” Hosts

Simple Summary The isolation of Leptospira is the most important test to assess infection in animal species. Several animals play a role as maintenance-host for specific serovars and in the last 30 years the incidence of leptospirosis has constantly increased in well-known reservoirs as well as in “unconventional” hosts. The emergence and the identification of Leptospira infection in such “unconventional” hosts could be related to several factors including problematic or inaccurate sampling modes during the Leptospira isolation, newly identified Leptospira strains, underestimated leptospirosis cases and climatic changes causing modifications of ecological niches. The aim of this review was to report the Leptospira isolations of the last 60 years from animals that could be considered “unconventional” hosts. Thus, the identification of “unconventional” hosts is crucial because they almost surely play an important role in the epidemiology of Leptospira infection. Abstract Leptospirosis is a re-emerging worldwide zoonotic disease. Even though the primary serological test for diagnosis and surveying is the microscopic agglutination test (MAT), isolation remains the gold-standard test to detect Leptospira infections. The leptospirosis transmission is linked to maintenance and accidental hosts. In the epidemiology of Leptospira some serovar are strictly related to specific maintenance hosts; however, in recent years, the bacterium was isolated from an even wider spectrum of species. The aim of this review is to report the isolation of Leptospira strains in animals which could be recognized as “unconventional” hosts, analyzing studies from 1960 to 2020 that highlighted the Leptospira isolation. This scientific literature aimed to provide evidence of infection in several animal species including of the Carnivora, Didelphimorphia, Rodentia, Cetacea, Cingulata, Afrosoricida, Chiroptera and Primate orders, as well as in Reptilia and Amphibia classes. In conclusion, the spreading of Leptospira is attention-worthy because the infection could occur in all the animal species ranging in a specific area. Further screening and isolations are needed to collect all necessary data to gain a complete understanding of leptospirosis epidemiology and its modifications.


Introduction
Leptospirosis is a neglected and re-emerging zoonoses caused by a Gram-negative bacterium belonging to the Spirochaetaes phylum, Leptospiraceae family, Leptospira genus [1,2]. These microorganisms appear spiral-shaped, with a diameter of 0.1 µm and a length of 6-20 µm and a pointed end that is typically folded into a characteristic hook shape [1]. Leptospira is highly mobile and performs rotational movements around the central axis, translation, undulation, and flexion thanks to two periplasmic axial flagella located under the cell membrane [3]. Although Leptospira is a microaerophile, it develops well even in conditions of complete aerobiosis. The optimum temperature for its growth is between 28 • C and 30 • C, although it also grows at 37 • C. The ideal pH range is between 7.2 and 7.4 [2].
Traditional classification divided the genus Leptospira in two species: L. interrogans, pathogenic strains, and L. biflexa, saprophytic strains. On the basis of antigenic agglutina-form the primary isolation of fastidious pathogenic strains directly from infected host tissues, especially for strains belonging to L. borgpetersenii species [15]. Leptospirosis is a worldwide diffused disease, occurring in tropical, subtropical, and temperate zones [4,16]. The spread of the disease is favored by a large variety of both wild and domestic animals which can be natural reservoirs of Leptospira [1,2,10,[17][18][19][20]. Several specimens are asymptomatic Leptospira renal carriers, contributing to maintain the infection in the environment by constantly shedding bacteria with urine and developing symptoms only after a long period of incubation [2,21]. The incidental contact with Leptospira infected urine by non-adapted animals, the so-called accidental hosts, could cause infections that evolve in the above-mentioned clinical diseases. For this reason, the epidemiology of leptospirosis in a particular ecosystem is related to the close relationship between specific Leptospira serovars and specific maintenance hosts [17,21,22]. For example, rodents are reservoirs of Icterohaemorrhagiae and Ballum serogroups [23,24], swine of Pomona and Tarassovi serogroups [25][26][27][28][29], horses of Bratislava serogroups [30,31], bovines and ovine of Sejroe serogroups [32,33]. In the last 30 years, the incidence of leptospirosis has constantly increased in well-known reservoirs as well as in never-before detected animal species [34][35][36]. The Leptospira incidence in these atypical hosts has become even higher, reaching several animal species from different classes and orders [37][38][39], including a wide spectrum of avian species (seagulls, doves, ibis, and owl) [40,41], reptiles [40,42] and fishes [43]. The emergence and the identification of Leptospira infection in such "unconventional" hosts could be related to: (i) problematic or inaccurate sampling methods to isolate Leptospira [12]; (ii) newly identified Leptospira strains [44]; (iii) underestimated leptospirosis cases, characterized by a downward trend [36]; (iv) climatic changes modifying ecological niches [16,45]; (v) presence of domestic animals raised in semi-extensive or extensive farms, promoting contact with wild species [16,46]; (vi) Leptospira strain antimicrobial resistances [47][48][49].
The epidemiology of Leptospira and leptospirosis is strictly related to the presence of susceptible hosts, both maintenance and accidental. Since isolation of viable bacteria represents the highest level of diagnosis and the best way to prove the relation between a pathogen and a host, the aim of this review was to report the Leptospira isolations of the last 60 years from animals that could be considered "unconventional" hosts. Animal species that are well recognized Leptospira hosts were not taken in consideration. Although in many cases, the data available in the literature are not sufficient to classify these "unconventional" hosts as incidental or maintenance, further knowledge of them could give new insights into the epidemiology of leptospirosis.

Leptospira Isolation on "Unconventional" Host
From 1960 to 2020, 34 papers were published about the isolation of Leptospira from species not recognized as leptospirosis reservoir. As showed in Figure 1, the published works increase constantly year after year, reporting Leptospira isolation from several animal species. Leptospira spp. isolated from animals belonging to the Carnivora order are the most numerous, followed by Didelphimorphia and Rodentia. However, isolation was obtained from Cetacea, Cingulata, Afrosoricida, Chiroptera and Primate, as well as in Reptilia and Amphibia classes. The geographical distribution of Leptospira isolation ( Figure 2) is more abundant in South America, especially in Brazil and Argentina, due to the high animal species variability present in this geographic area. Moreover, other isolations were performed in the North American West Coast, Italy, Netherlands, Japan, and Madagascar.

Carnivora
Among the Carnivora order, Leptospira was isolated from pinnipeds, racoons, and Gray fox (Table 1). Within Carnivora, pinnipeds were the most investigated animals, in which high number of isolations were reported. Within California sea lion (Zalophus californianus) and fur seal (Callorhinus ursinus) colonies Leptospira isolations were obtained from four leptospirosis outbreaks. From all of these, isolations from blood, kidney and urine, L. interrogans serogroup Pomona serovar Pomona were obtained [50][51][52][53][54]. The serovar Pomona is considered the most important cause of diseases in California sea lions, as widely reported from serological surveys and post-mortem lesions, reaching the 100% of colony population [50,51,[55][56][57][58]. Moreover, serovar Pomona seems to be the reason of high incidence of abortion in this animal species, as suggested by the isolation obtained

Carnivora
Among the Carnivora order, Leptospira was isolated from pinnipeds, racoons, and Gray fox (Table 1). Within Carnivora, pinnipeds were the most investigated animals, in which high number of isolations were reported. Within California sea lion (Zalophus californianus) and fur seal (Callorhinus ursinus) colonies Leptospira isolations were obtained from four leptospirosis outbreaks. From all of these, isolations from blood, kidney and urine, L. interrogans serogroup Pomona serovar Pomona were obtained [50][51][52][53][54]. The serovar Pomona is considered the most important cause of diseases in California sea lions, as widely reported from serological surveys and post-mortem lesions, reaching the 100% of colony population [50,51,[55][56][57][58]. Moreover, serovar Pomona seems to be the reason of high incidence of abortion in this animal species, as suggested by the isolation obtained

Carnivora
Among the Carnivora order, Leptospira was isolated from pinnipeds, racoons, and Gray fox (Table 1). Within Carnivora, pinnipeds were the most investigated animals, in which high number of isolations were reported. Within California sea lion (Zalophus californianus) and fur seal (Callorhinus ursinus) colonies Leptospira isolations were obtained from four leptospirosis outbreaks. From all of these, isolations from blood, kidney and urine, L. interrogans serogroup Pomona serovar Pomona were obtained [50][51][52][53][54]. The serovar Pomona is considered the most important cause of diseases in California sea lions, as widely reported from serological surveys and post-mortem lesions, reaching the 100% of colony population [50,51,[55][56][57][58]. Moreover, serovar Pomona seems to be the reason of high incidence of abortion in this animal species, as suggested by the isolation obtained from its placenta [53].
Considering fur seal, on 29 specimens collected in Alaska, two isolations were obtained from a new-born puppy liver and another one from the urine of an adult male. Both isolations, as for California sea lions, belong to serovar Pomona [53], as demonstrated by MAT surveys on dead animals with hemorrhagic lesions [57]. Moreover, two groups from Phocidae family tested positive for Leptospira in the American West Coast, serovar Pomona was isolated from the kidney of a Northern elephant seal (Mirounga angustirostris) [59]. However, serological positivity, other than for serovar Pomona, was reported for Grippotyphosa and Bratislava, at high titers [60,61]. Additionally, from a harbor seal (Phoca vitulina) sampled in Netherlands, L. interrogans serogroup Icterohaemorrhagiae serovar Icterohaemorrhagiae was isolated [62]. The Leptospira infection was also detected in these animals through serological and molecular surveys of both free-ranging and captive specimens with even higher positivity, to Icterohaemorrhagiae, Grippotyphosa and Bratislava [60][61][62][63][64][65][66]. From two kidneys of racoons (Procyon lotor) L. interrogans serogroup Icterohaemorrhagiae and L. interrogans serogroup Hebdomadis serovar Hebdomadis were isolated in Japan [67]. This is the first isolation from this animal species, although anti-Leptospira antibodies were detected in racoon sera in North America [68][69][70][71]; furthermore, it was demonstrated that, in Canada, trappers contracted leptospirosis from contact with these carnivores [71]. Two isolations were obtained from the Canidae family from South America Gray fox (Lycalopex griseus) and Maikong (Cerdocyon thous). Serovar Icterohaemorrhagiae was isolated from one out of five south America Gray fox specimens collected in Argentina [72]. True foxes, belonging to Vulpes genus, are well recognized as Leptospira reservoir, in particular Red fox (Vulpes vulpes), [73][74][75], but no isolation was performed among them. Leptospira infection was detected only by serological studies that highlighted antibodies against serovars Icterohaemorrhagiae, Canicola, Hebdomadis, Hardjo and Grippotyphosa [76,77]. A Leptospira surveillance was never conducted on maikong (Cerdocyon thous) until 2015, when in Brazil a strain belonging to Pomona serogroup was isolated from these animals, while Bratislava, Shermani and Whitcombi was also detected by MAT [78].

Cetacea
Among species belonging to the Cetacea order, only two isolations have occurred so far (Table 1). One study describes the characterization of Leptospira strain Manara, as collected from a Southern right whale (Eubalaena australis) stranded in Patagonia [79]. Moreover, this strain turned out to be halophilic, surviving in a salt environment, at different concentrations, for a few days. The other isolation of serovar Pomona was obtained from the kidney of a common bottlenose dolphin (Tursiops truncates) collected in Sardinia (Italy) [80]. These are the only isolations from whale and dolphin species, among several investigated species around the world [40,[81][82][83][84][85].

Cingulata
Very few studies were conducted on armadillo's species and only in Brazil. As reported in Table 1, serogroup Autumnalis, Cynopteri, Hebdomadis and Pomona were isolated from kidneys of nine-banded armadillo (Dasypus novemcinctus) [88,95]. Also, a strain belonging to serogroup Pomona was isolated from the urine of a six-banded armadillo (Euphractus sexcinctus) [78]. Other serological investigations on armadillo specimens indicate the presence of antibodies to serogroups Autumnalis, Cynopteri, and Pomona at titer reaching 1:1600 [96,97], suggesting that this animal could be susceptible to leptospirosis as an incidental host.

Rodentia
Detailed and recent reviews of literature about leptospirosis in rodent species have been widely treated elsewhere hence they will not be discussed in this article. Indeed, rodents are considered to be one of the most important reservoirs of Leptospira [2]. Infections have already been demonstrated in different urban, wild, and imported rodent species, such as Apodemus, Delomys, Mus, Necromys, Oryzomys, Rattus, Thaptomys, Trinomys and Myocastor coypus [98,99]. However, few data are present in the literature about the role of squirrels and the largest rodents, capybara, and porcupine, in the leptospirosis epidemiology. Among squirrels (Table 1), Ballum and Grippotyphosa serovars were isolated from fox squirrels (Sciurus niger) [100,101], while Grippotyphosa serogroup from Southern flying squirrel (Glaucomys volans) [102], serogroup Javanica from Pallas's squirrel (Callosciurus flavimanus) [103] and serovar Icterohaemorrhagiae and Canicola from redbellied tree squirrel (Callosciurus erythraeus) [104]. All of these studies highlighted the role of squirrels as renal carriers, although their role in epidemiology remains unknown. The largest rodents in the world, capybara (Hydrochoerus hydrochaeris), seem to be incidental hosts of Leptospira, as demonstrated by the isolation of serovar Icterohaemorrhagiae and serovar Bananal in Brazil [105,106]. Moreover, other findings suggest that incidental contact with the pathogens indicates asymptomatic infection due to L. santarosai identified through molecular amplification [106], and by the experimental infection with a strain belonging to serovar Pomona [107]. Among porcupines, isolations of serovar Pomona were performed only from crested porcupine (Hystrix cristata) in Italy, and from North American porcupine (Erethizon dorsatum) in Canada [108,109]. The role of this animal species remains unknown because it could be an incidental host or a reservoir of Leptospira. However, crested porcupine specimens resulted positive for serogroup Icterohaemorrhagiae (also a titer ≤1:1600), and for Australis and Pomona (at low titers) during a serological survey [110]. Also, anti-Leptospira antibodies to serovars Javanica, Hurstbridge, Ballum, Celledoni and Hardjoprajitno were detected [111]. Finally, molecular analysis of the urine of orange-spined hairy dwarf porcupine (Sphiggurus villosus) reported a renal Leptospira infection, despite the MAT negative results [112], suggesting a role as reservoir.

Afrosoricida
The only investigation on the Afrosoricida order highlighted the isolation of L. mayottensis from a tailless tenrec (Tenrec ecaudatus) ( Table 1) sampled in Mayotte Islands [113]. This valuable piece of information was the nearly perfect identity scored form the tenrec strains and they obtained from ill human patients. The L. mayottensis typically circulates in the Mayotte islands, reaching not only humans but also domestic animals [7,50,114]. Probably, tenrec was an incidental host, but a role as reservoir is not to be excluded

Primates
Within Leptospira epidemiology, non-human primates could provide important data due to their close lineage with human beings. Although some investigations were carried out on animals kept in captivity, other studies performed on free-ranging monkeys indicated an interesting prevalence of Leptospira infection [135]. The infections occurring in several primate species were mainly caused by strain of L. interrogans serogroup Icterohaemorrhagiae and L. borgpetersenii serogroup Ballum, serologically detected [136][137][138][139][140][141][142][143][144]. The serological results were confirmed with Leptospira isolation in only two cases (Table 1). Among prosimian, blood samples collected from a specimen of ring-tailed lemur (Lemur catta) kept in a Portuguese zoological park scored positive to Leptospira infection and serovar Copenhageni was isolated [116]. Moreover, serogroup Icterohaemorrhagiae have been isolated from the blood of captive specimens of squirrel monkey (Saimiri sciures) in French Guyana and of white-faced capuchin monkey (Cebus capuchinus and Cebus apella) in Colombia and in Portugal and of a common marmoset (Callithrix jacchus) kept in a Portuguese zoological park [116][117][118]. The isolation from squirrel monkey was performed after an outbreak in a colony bred in captivity, where some animals showed jaundice and hemorrhagic syndrome, death, and abortion [117]. All symptoms were referable to leptospirosis and were confirmed by serology and isolation. In the case of white-faced capuchin monkey, too, the isolation was performed after the incoming of leptospirosis symptoms in a colony kept in a recovery center, in which specimens had died despite antibiotic treatments [118].

Reptilia
Among Leptospira hosts, reptiles seem to be relevant as well. In these animals, leptospirosis could occur after the ingestion of rodents, typical maintenance-Leptospira-host, or due to contact with humid and contaminated environment [42,145]. Moreover, implication and pathogenesis remain unclear in these animals. Presently, only two isolations were reported in two snake species (Table 1). Serovar Andamana strains were isolated in Brazil from a kidney of a Prado's lancehead snake (Bothrops pradoi) [121]. Furthermore, a strain belonging to serogroup Ballum was found in hognosed snake (Heterodon platghrinus) [120]. Serogroup Ballum was previously detected in one more snake species [119]. In several snake species, different Leptospira serogroups were detected using serological assay [146,147], including Andamana and Ballum [119]. This suggests that snakes are incidental hosts, susceptible to environmental Leptospira contaminations due to different landscapes and living fauna.

Amphibia
As for reptiles, little information is available about leptospirosis in toads and frogs. In the Barbados islands, the marine toad (Bufo maninus) and the whistling frog (Eleutherodactylus johnstonei) were investigated, with very similar results (Table 1). L. interrogans serogroup Autumnalis serovar Bim were isolated from three out of four marine toads and two out of three whistling frogs [121,122]. The other specimens were both infected by L. interrogans serogroup Australis serovar Bajan [121,122]. Moreover, serological data for toads are available, showing Australis, Autumnalis and Panama infection [122], while no data are present for frogs. No piece of data clarifies if amphibians develop severe and lethal infection, but as well as reptiles, the infections are strictly related to Leptospira infection in a specific geographical area. The infection could penetrate in amphibians through water, as these animals can absorb water through their skin, store it in their urinary bladder and reabsorb it during dehydration stress. This mechanism seems to be the most probable cause of leptospirosis in toads and frogs [42].

Conclusions
Leptospirosis is probably the most prevalent, underestimated, and re-emerging zoonotic disease; animals, both wild and domestic, represent one of its most important transmission sources. This review highlighted several worldwide Leptospira isolations from a wide range of a "new" potential reservoir, ranging from rodent, opossum and carnivora species to bats, armadillos, cetaceans, reptiles, and amphibians. The amount of isolation has constantly increased during the last 50 years, suggesting that all free-ranging animals could be an incidental Leptospira host, including marine mammals. Probably, Leptospira infection is endemic in many countries with no surveillance or diagnostic facilities, especially for animals, such as in several African countries where leptospirosis has rarely or never been reported. These findings are undoubtedly of high importance for human public health, due to the risk of human infection through interaction with the reservoir or incidental hosts or contact with biological materials, including blood, urine, tissue, and excretions. Such risks could affect not only already confirmed worker categories (i.e., veterinarians, trappers, abattoir workers, farm workers, hunters, animal shelter workers and scientists and technologists handling animals in laboratories or during field work) but also marine mammal workers, fishmen, researchers, wildlife rehabilitators, trainers, and zoological park workers.
A better knowledge of the epidemiology of this infectious disease is essential to facilitate the creation of efficient prevention and control programs using a One Health approach. Constant monitoring is needed to control the evolution of the dynamics of leptospirosis epidemiology, mainly focused on new animal species that could contribute to its spreading, in order to better clarify their role as a reservoir or incidental hosts.

Conflicts of Interest:
The authors declare no conflict of interest.