We report a variety of pathogenic and intermediate Leptospira,
including two putative new species, found associated with chronic renal colonization of bats in the Peruvian Amazon. Bat-associated leptospires are genetically diverse based on phylogenetic analysis of 16S rDNA sequences. These data are of substantial epidemiologic significance, providing the basis for detailed molecular epidemiologic investigations of bat-associated leptospires as the source of human infection by pathogenic Leptospira
in the Iquitos region, where leptospirosis is highly endemic.30
Although most of the Amazon remains intact,31
the rate of deforestation in the neotropics is alarming32
and results in reducing forested areas, increasing habitat fragmentation, and subjecting natural assemblages to intrusion by humans along with their commensal species (i.e., horses, dogs, cattle, rats). Such ecologic changes promote the emergence of infectious diseases by placing humans into contact with novel reservoirs or infectious agents. Because bats respond to habitat modification, loss, and fragmentation at the level of populations and communities,33
their spatial and temporal dynamics are particularly sensitive to anthropogenic activity.34
The consequences of such altered spatial and temporal dynamics to the ecology of leptospirosis and the likelihood of disease transmission to humans is known poorly. However, anthropogenic activity in previously undisturbed areas could directly or indirectly increase the risk of infection by new serovars.
The role of bats in leptospiral transmission was not assessed here for humans. Evidence of renal infection was found in 20 (3.4%) of 589 bats, a rate lower than that in previous reports from the same area.9
In the previous study, 35% (7 of 20) of the bats were PCR positive as assessed by G1/G2 PCR. However, since only six bat species were evaluated, rates of renal infection might have been biased and artificially high. Unlike a previous report,9
our data derive from animals prospectively collected over an extended period of time at several sites in and around Iquitos. As such, our results likely represent a more accurate estimate of the rate of leptospiral infection of bats in proximity to human populations. In Australia, Smythe and others found that 28% of flying foxes had antibodies to leptospira.13
However, these investigators did not isolate the organism or assess the frequency of renal carriage among these potential mammalian reservoirs.13
Serologic analysis indicates only previous exposure to Leptospira
, but not the presence of renal infection. Antibodies to Leptospira
cannot precisely identify infecting leptospires (particularly a novel leptospire). Therefore, this previous serologic study of flying foxes does not indicate whether these megachiropterans could have been reservoirs for leptospiral transmissions. Because of the limitations of serologic analysis, we did not determine the presence of antibodies to Leptospira
in the present study. It is well known that many leptospiral strains are fastidious in culture or difficult to culture. It is also possible that PCR analysis may miss some infections both for technical reasons (complexity of a DNA extract from kidney) or because we used only the G1/G2 primer set,9
which may fail to detect all leptospiral species.22
Generally, our results should be interpreted as an underestimation of renal colonization of bats in the Peruvian Amazon.
The frequency of renal infection differed among bat species (even among congeners), suggesting that some species may be better suited to carry leptospires than are others. Variation in the frequency of renal carriage among sister taxa has been found in other mammals.35–37
In Hawaii, for instance, Rattus norvegicus
is a more significant carrier and disseminator of leptospires than is either R. rattus
or R. exulans
. In that study, more than 61% of the R. norvegicus
yielded isolates, whereas only 17.8% of R. exulans
were culture positive. In that study, renal carriage was associated strongly with rat population density.36
High population densities facilitate transmission; non-gregarious species that infrequently come into contact with conspecifics should transmit leptospires between themselves infrequently. This is not true for bats in the genus Carollia
, which have low infection rates yet are gregarious.38,39
Environmental factors also could contribute to observed leptospiral transmission and infection rates. In the present study, statistical analyses have shown that the variables habitat, location and species are all inter-related in affecting PCR positivity. It is therefore difficult to determine how each factor influences the rate of leptospiral positivity in bats of Iquitos. Nonetheless, bats collected from mature forests were significantly more likely to carry leptospires than were bats collected in areas associated with human activity. This association could be due in part to differences in species composition with respect to habitat or location.
In Iquitos, we isolated leptospires from the kidneys of three bat species: P. hastatus
, M. crenulatum
, and P. nasutus
. The PCR evidence of infection with identification of the leptospiral species was determined in an additional 17 bats. This report is the first to describe the isolation and identification of leptospires from these bat species. Considering the number of positive PCR reactions, the isolation rate was low. Leptospires are fastidious organisms that are not easily grown in culture media, although some leptospires grow more easily than do others.1,3
Many formulations containing serum have been described that appear to improve growth in primary culture, and standard recommendations are to use more than one type of medium for primary isolation of leptospires from clinical specimens or tissues.1
However, because of logistic concerns, we could only use one type of leptospiral culture medium, and chose to use Ellinghausen-McCullough-Johnson-Harris (EMJH) medium, which does not contain serum. Medium supplemented with serum might have yielded a higher and more accurate rate of isolation.
Despite the low infection rates in bat populations, bats may be an important link in the transmission cycle of leptospirosis in Iquitos. Bats forage in fruit orchards and forest clearings created by human activities, and roost in buildings (under tiles or in attics), water cisterns, culverts, abandoned structures, and bridges.10
Although speculation, inhabitants of rural villages may contact soil or surface waters contaminated with bat urine (e.g., when harvesting wood in forest to make charcoal). In contributing to a sylvatic cycle of leptospiral transmission, ground-dwelling species such as rodents or marsupials that reside or forage under bat roosts could encounter Leptospira
-contaminated urine. Such a consideration leads to the testable hypothesis that leptospires could be maintained by a bat-rodent or bat-marsupial transmission cycle. Molecular and ecologic approaches similar to those in the present study could assess this contention.
Traditional serologic (phenotypic) identification of leptospires is difficult and often does not reflect genetic relatedness.1,3
Molecular approaches based on DNA-DNA hybridization have been described,16,40,41
but are time-consuming and require the analysis of large numbers of bacteria in pure culture.3
Since the late 1980s, rDNA genes universally have used to define phylogenetic relationships among bacteria.42
The application of phylogenetic analysis to the classification of Leptospira
showed that 16S rDNA analysis was consistent with results obtained by DNA-DNA hybridization and distinguished strains at the species level.17,18
More recently, phylogenetic reconstructions also have been used to identify novel leptospiral strains.43–45
The 16S rDNA analysis has the major advantage of not requiring an isolate to enable the analysis.
The application of Bayesian analyses to phylogenetic studies is relatively new, but has generated considerable excitement. Bayesian inferences of phylogeny produce a tree estimate with quantified support for each node. As do maximum likelihood analyses, Bayesian approaches fully capture phylogenetic relationships under a given nucleotide substitution model,46
but require considerably less computation time, otherwise a serious constraint in phylogenetic reconstructions. Other methods, such as neighbor-joining (NJ) and maximum parsimony analysis, although faster, do not fully use all information in a particular dataset.46
Bayesian phylogenetic analysis was used previously to describe the evolutionary relationships among known species of Leptospira
using 16S rDNA lipL32
, and ompL1
Inferred tree topologies were well resolved and consistent with trees generated using NJ and unweighted pair group method with arithmetic mean (UPGMA) tree-generating algorithms,18
indicating that the method could be applied to leptospiral phylogeny.44,47
Our phylogenetic inferences were based on leptospiral 16S rDNA sequences amplified directly from kidney. Tree topology was consistent with published reports.18,47
Three large clusters consistent with groupings based on DNA-DNA hybridization studies were generated. Because of the high degree of similarity between sequences, some terminal taxa were not well resolved, particularly the L. kirschneri
and L. interrogans
strains. Leptospira interrogans
formed a polytomous in-group within the L. kirschneri
cluster. Although other investigators have reported similar branching patterns,47
they were able to resolve all terminal branches (no polytomies). The differences with our results are unclear. In our analyses, the overall sequence similarity of closely related (clustered) terminal taxa may have been higher than in previous studies. Phylogenetic analysis of leptospiral 16S rDNA genes often fails to resolve sequences of more than 97% similarity. Because of less resolving power of 16S rDNA sequence analysis at this level of similarity, Stackebrandt and Goebel suggested confirming conclusions based on 16S rDNA sequence analysis with DNA-DNA hybridization studies.48
Haake and others also concluded that none of the four genes that they evaluated adequately resolved leptospiral species, and suggested that simultaneous analysis of multiple genes would improve resolution.47
Indeed, current strategies explore the possibility of using partitioned datasets: with DNA sequences from 1) more than one gene, 2) a single gene partitioned by codon position (protein coding genes),49
3) stem-loop secondary structure (structural RNA genes),49,50
or 4) mixed partitions including both DNA and protein sequences.51
We used two approaches to investigate the phylogenetic relationship of leptospiral 16S rDNA sequences: weighted parsimony and Bayesian inference. Overall tree topology was similar; however some clades, notably the L. interrogans
and L. kirschneri
strains, were less resolved by parsimony analysis. The disparity of results between the two methods highlights the unique approach that each uses when inferring phylogeny. Maximum parsimony does not adequately account for pleisiomorphy (convergent evolution), but rather assumes that common states in terminal taxa were inherited directly from a common ancestor. Bayesian analyses can implement complex nucleotide substitution models that better account for these invisible changes. It is currently being debated which approach better approximates the true tree. Maximum parsimony may perform better than either maximum likelihood or Bayesian analyses when branch lengths are between 0.15 and 0.35, but the latter generate better results when branch lengths are longer.52
However, with our dataset, Bayesian analysis proved superior: a higher proportion of poorly resolved clades were apparent when using weighted parsimony analysis as compared with Bayesian analysis.
Based on phylogenetic analysis of 16S rDNA sequences, bats in Iquitos maintain a genetically diverse group of leptospires. This is not surprising considering the number of different bat species in the region. Most strains belong to the L. kirschneri (1) or L. interrogans (5) lineage. Both L. kirschneri (serovar Cynopteri) and L. interrogans (serovar Schueffneri) previously have been isolated from bats (Serovar Database; National Veterinary Science Laboratory, Ames, IA). Leptospira interrogans serovar Icterohaemorrhagiae typically is maintained by Rattus spp., which was confirmed by our observations in the urban slum and market area of Belen, Iquitos, where we found that peridomestic rats (R. norvegicus and R. rattus) frequently carry serovar Icterohaemorrhagiae (between 40% and 50%, unpublished data). Of the five L. interrogans clones, one (serovar Icterohaemorrhagiae) was isolated from a bat caught in urban surroundings and was typed as serovar Icterohaemorrhagiae. In addition, four strains showed strong 16S rDNA sequence similarity to L. borg-petersenii, which has been isolated primarily from humans and peridomestic rodents. These results suggest that the transmission of leptospires from peridomestic rodents to bats could occur in Iquitos. The only other L. interrrogans serovar isolated in Peru is serovar Canicola, which is typically associated with dogs. Leptospira interrrogans serovars cause most severe human infections in Iquitos (Cunningham C, unpublished data); thus, bats may contribute to leptospiral transmission to man. To our knowledge, L. borgpetersenii has not been previously detected in bats.
Our data also indicate the possible existence of two new leptospiral species maintained by bats in Iquitos. The first included three clones in bats collected in Varillal, a village near Iquitos, which clustered most closely with L. alexanderi, a strain isolated from humans in China. The second consisted of five clones that clustered closely with L. interrogans and L. kirschneri, but formed a separate monophyletic group. A maintenance host of L. alexanderi has yet to be identified.
The remaining sequence clustered with the intermediate strains, and was almost identical to L. fainei. Leptospira fainei
has been isolated from pigs in Australia53
and humans in Europe.43,44
In Australia, serologic evidence of L. fainei
infection in humans has also been reported.54
The present report is the first demonstration that a wild, as opposed to a domestic, animal has a kidney infection with L. fainei
. An rDNA sequence identical to MMD1100 was detected repeatedly in environmental water samples collected in and around Iquitos (Qanoza CA, unpublished data). Consequently, human exposure to and infection by a leptospiral strain similar to MMD1100 is quite likely.
We identified a number of potential bat reservoirs of leptospires in Iquitos, and highlighted the genetic diversity of bat-associated leptospires, including two undescribed leptospiral species. To understand more fully the role of bats in the maintenance and transmission of leptospires in Iquitos, the relationship of bats with their abiotic environment and with other mammal reservoirs with which they may come into contact needs to be clarified so that mechanisms of transmission and persistence of renal infection can be determined in an ecologic context. Studies to more precisely delineate the transmission of bat-associated leptospires to humans and to peridomestic and domestic animals are needed; this study forms an important starting point for that initiative.