Leptospira interrogans in the Galapagos sea lions (Zalophus wollebaeki)

Abstract

Background

Leptospira are endemic and cause disease among California sea lions (Zalophus californianus), but the epidemiology of Leptospira in Galapagos sea lions (Z. wollebaeki) is almost completely unknown. Understanding the presence and circulation of this zoonotic pathogen is essential not only for the conservation of Galapagos sea lions, but also for assessing potential health risks to humans and other animals within the Galapagos archipelago. This study fills knowledge gaps by investigating Leptospira in this endemic host species to provide valuable insights into the dynamics of the pathogen in an isolated island ecosystem that is completely different from others in which Leptospira have been documented.

Results

In 2016, serological analyses detected 24 positive samples in the “El Malecón” rookery (n = 43) and 18 positive samples in the “Punta Pitt” rookery (n = 33), confirming exposure to pathogenic Leptospira species. In 2017, we identified 15 (n = 29) and 13 (n = 30) positive samples, respectively. Molecular analyses further detected leptospiral DNA in 70.8% (n = 24) of sand with urine samples and 45.8% (n = 24) of kidney samples. Amplicon sequencing of the secY gene fragment yielded 10 consensus sequences, confirming the circulation of Leptospira interrogans among Galapagos sea lions.

Conclusions

Our findings confirm that Galapagos sea lions are exposed to pathogenic Leptospira species and, for the first time, identify L. interrogans as the circulating species over two consecutive years. By expanding sampling across multiple rookeries, we provide a clearer picture of pathogen exposure in this endemic population. These results improve our understanding of Leptospira dynamics in marine mammals on remote islands, where management efforts aim to reduce disease risks from human activity, livestock, and synanthropic species.

Background

Over the past 40 years, the Galapagos sea lion (GSL, Zalophus wollebaeki) has experienced a population decline of around 50% [1], leading to its categorization as an endangered species by the International Union for Conservation of Nature (IUCN) [2]. The main causes of this decline are the effects of environmental variability in the region [3]. However, there are other factors such as introduced species, habitat degradation and overfishing that have been identified as likely stressors for the species [4]. As GSL inhabits a region of highly variable marine productivity amid a tropical environment, it is exposed to frequent periods of low productivity (i.e., El Niño–Southern Oscillation ENSO) that generates food stress, and increases mortality rates [5]. Additionally, the close contact with human settlements exposes GSL to infections from pathogens originating in humans and domestic animals, such as Leptospira, canine distemper virus, Mycoplasma, dirofilariasis, among others [6–9]. Leptospira are bacterial spirochetes that cause leptospirosis, a disease with a broad range of hosts that has been identified as a primary reason for the population reduction of the California sea lion (CSL, Z. californianus) [10, 11], the closest relative of GSL.

Leptospirosis in CSL, likely caused by Leptospira interrogans serovar Pomona, was first recorded in 1970 and associated with the deaths of hundreds of animals [12–15]. Since then, leptospirosis has become endemic, with periodic outbreaks observed every 3 to 5 years [13], resulting in high mortality among sea lions of the Gulf of California [12, 16–18]. Leptospira colonizes the kidneys of infected sea lions, and can cause renal failure and abortions [19, 20]. Infections in sea lions can manifest as severe clinical disease or silent, potentially chronic infections, providing a mechanism for maintenance and spread by excreting Leptospira in urine for months, or even years [20]. Despite the available information, there is still a lack of comprehensive understanding regarding the epidemiology of leptospirosis in CSL, and the precise role these animals play in the complex North American coastal ecosystem in the persistence and circulation of this zoonotic disease remains elusive [13, 20].

To date, two studies have been published on Leptospira in GSL [21, 22]. The presence of antibodies against various Leptospira serovars was reported using the microscopic agglutination test (MAT) on blood samples from apparently healthy GSL pups sampled during 2002 and 2003. 56% of samples obtained from 9 islands in the archipelago tested positive for at least one serovar with titers from 1:20 to 1:320. The most prevalent serovars were Patoc, Australis, and Hebdomadis [22]. The only study that incorporated molecular detection using PCR reported the presence of Leptospira DNA in 5 out of 7 tissue samples from deceased GSL collected in 2010 on San Cristobal Island. The DNA sequences obtained from 3 of these samples were indicative of pathogenic Leptospira species [21]. While these studies provide evidence that GSL are exposed to pathogenic species of Leptospira, the identity of the circulating species and the disease dynamics in the population are still unknown.

In this study, we report the results of Leptospira serology in GSL over a period of two consecutive years. We increased sampling efforts compared to previous studies by including samples from individuals in different rookeries. Furthermore, DNA analysis enabled us to identify the species of pathogenic Leptospira circulating among GSL. These data contribute to our understanding of Leptospira circulation in sea lions in the relatively simple setting of remote islands without the complex interactions inherent in the mainland communities of Z. californianus. Moreover, the GSL populations are actively managed to reduce interactions with locals, tourists, and synanthropic species specifically to reduce the risk of exposure to imported diseases.

Results

Assessing Leptospira seroprevalence by MAT

Serum samples collected during the breeding season of 2016 showed agglutination with Leptospira serovars in 55.8% (24/43) and 54.5% (18/33) samples of the “El Malecón” and “Punta Pitt” rookeries, respectively. One year later, during the breeding season of 2017 serum samples showed agglutination with Leptospira serovars in 51.7% (15/29) and 43.3% (13/30) respectively (Table 1). Agglutination titers for each sample are shown in Table 1, no significant differences were found between the proportion of seropotive samples in these sites in 2016 (Chi-square = 2.3346e-30, p-value = 1, CI: -0.22560–0.25097) and 2017 (X² = 0.14784, p-value = 0.7006, CI: -0.20401- 0.37182). A total of 20 samples showed agglutination for > 1 serovar and are thus labeled as cross-reactions (Table 1).

Table 1.

MAT results for blood samples from GSL living on San Cristobal Island

Year	Rookery	Serovar	Positives	Titers	Year	Rookery	Serovar	Positives	Titers
2016	El Malecón	CR* PAT TAR HEB BAT GRIP DJA SEJ CAN	11 2 3 3 1 1 1 1 1	----- 1:20 1:20 1:80 1:40 1:40 1:20 1:40 1:20	2017	El Malecón	GRIP SEJ HEB AUS CAN HAR PYR PAT SAX	2 2 3 2 2 1 1 1 1	1:20 1:20 1:40 1:40 1:80 1:20 1:40 1:20 1:20
	Punta Pitt	CR* POM AUS HEB CAN HAR GRIP TAR SEJ	5 3 4 1 1 1 1 1 1	------ 1:80 1:80 1:20 1:20 1:20 1:20 1:20 1:20		Punta Pitt	CR* HEB CAN AUS GRIP PYR POM	4 2 2 2 1 1 1	------ 1:80 1:40 1:20 1:40 1:80 1:40

Legend: *Cross-reactions. Leptospira serovars: Icterohaemorrhagiae (ICT), Canicola (CAN), Pomona (POM), Hardjo (HAR), Grippotyphosa (GRIP), Bratislava (BRAT), Autumnalis (AUT), Hebdomadis (HEB), Bataviae (BAT), Cynopteri (CYN), Australis (AUS), Saxkoebing (SAX), Patoc (PAT), Celledoni (CELL), Djasiman (DJA), Shermani (SHE), Tarassovi (TAR), Sejroe (SEJ), Pyrogenes (PYR) and Javanica (JAV)

Detection and identification of pathogenic Leptospira spp. From GSL

Leptospiral DNA was detected in 70.8% (17/24) and 45.8% (11/24) of the sand and kidney samples, respectively. Identification of Leptospira spp. by amplicon sequencing of the secY gene fragment, was successful for 2 sand samples and 8 kidney samples. We obtained 10 consensus sequences, all showed 100% similarity with Leptospira interrogans (Table 2). When sequences were phylogenetically compared with other secY sequences from pathogenic Leptospira species extracted from GenBank, they were positioned within the L. interrogans clade (Fig. 1). Based on the alignment of the secY fragment, one SNP was identified in position 3, 739, 025 when compared to Leptospira interrogans serovar Canicola strain 782 (Accesion number: CP043884.1) in samples L2, PC2, PC1, SL102, and PM11.

Table 2.

Molecular identification of Leptospira in kidney and sand with urine samples from “El Malecón” rookery

Sample Code	Sample Barcode*	Sample Type	Sample Beach	Identified Species	BLASTn percentage	Accession number
PC2	24	Kidney	Punta Carola	L. interrogans	100	SRX21597789
PC1	28	Kidney	Punta Carola	L. interrogans	100	SRX21597798
L2	20	Sand with urine	Lobería	L. interrogans	100	SRX21597799
C2	22	Sand with urine	Punta Carola	L. interrogans	100	SRX21597801
NA2	35	Kidney	Base Naval	L. interrogans	100	SRX21597802
PM11	38	Kidney	Playa Mann	L. interrogans	100	SRX21597803
NA24	41	Kidney	Base Naval	L. interrogans	100	SRX21597791
SL102	42	Kidney	Lobería	L. interrogans	100	SRX21597793
NA05	43	Kidney	Base Naval	L. interrogans	100	SRX21597794
PO2	44	Kidney	Playa de Oro	L. interrogans	100	SRX21597796

Legend: *Sample barcode at (NCBI Number)

Fig. 1.

Phylogenetic tree for Leptospira species assignment based on the secY gene. Legend: GenBank accession numbers for each Leptospira reference used are indicated. Leptospira biflexa MN862542.1 served as the outgroup for rooting. Numbers at the nodes represent bootstrap values

Discussion

This work confirms the presence of pathogenic Leptospira in GSL, contributing to a limited but growing body of knowledge on its epidemiology in the archipelago. Despite the absence of reported leptospirosis outbreaks in GSL populations, early evidence of exposure to the pathogen (using the Microscopic Agglutination Test) was first in 57.6% of individuals sampled in 2002–2003 [22]. Ten years later, pathogenic Leptospira DNA was first detected in pup tissues and placentas from GSL living in San Cristobal Island [21]. Although no serological data was associated with any pathology, all these findings highlighted the need for long term surveillance and set the background for this study.

Our data from 2016 to 2017 and 2021 are consistent with previous exposure rates, suggesting continued presence of the pathogen in the population [21, 22]. While during our study, we did not detect leptospirosis, the presence of DNA belonging to L. interrogans in urine, and kidneys and 50% seropositivity confirm the hypothesis of persistent exposure in GSL. However, without data from annual monitoring, it is difficult to infer temporal trends in transmission, exposure dynamics, and of impact on GSL health.

One major limitation, not only in the Galapagos but globally, is the lack of locally isolated Leptospira strains for use in serological analyses. With over 200 known L. interrogans serovars, the absence of isolates reduces the accuracy of MAT results due to cross-reactivity, and make it difficult to confidently assign circulating Leptospira to specific serovars. This was a challenge in both previous studies and in our current work, and contributes to the uncertainty regarding specific serovars assigned to the GSL.

Ecologically, Galapagos pinnipeds play an important role in the archipelago due to their movements between marine and terrestrial ecosystems during their feeding and breeding activities [23, 24]. As a species on remote islands, interactions with other potential Leptospira hosts can be expected to be much more limited compared to their Z. californianus counterparts, making them ideal model to study simplified transmission system. Understanding Leptospira in such a close and simple system may offer valuable insights for more complex ecosystems.

Nevertheless, several important knowledge gaps remain. The origin, frequency, persistence, and outbreak potential of the circulating Leptospira still unknown. It is unclear whether the infection pattern of pathogenic Leptospira reflects acute signs, chronic asymptomatic carriage, or some intermediate manifestation similar to CSLs, with characteristics of both accidental and maintenance hosts [13]. The low anti-leptospira antibody titers observed in this study may be due to a lack of an isolate with a similar serotype in the MAT panel or past exposure but not necessarily active infection. We therefore cannot determine when exposure occurred or confidently identify the serovar(s) present. Without additional monitoring we cannot determine the impact of Leptospira on GSL. However, the identification of L. interrogans and confirmation of ongoing exposure allow us to draw concerning parallels to the leptospirosis epidemiology in CSLs that have been characterized by continued outbreaks and high mortality [13].

The detection of L. interrogans DNA and evidence of pathogen exposure at the “El Malecón” rockery deserve particular attention, as this site is located on a human-impacted island. The rockery is highly exposed to interactions with dogs, cats, and humans, factors that are associated with increased exposure to multiple pathogens compared to colonies with minimal human influence [25]. While some Leptospira serovars show host specificity, cross-species transmission is common. This highlights the need for surveillance and mitigation strategies that consider interactions among wildlife, humans, livestock, and synanthropic species in the Galapagos. Our findings reinforce the importance of a One Health approach. Understanding leptospirosis epidemiology in the Galapagos requires integrated research that includes domestic animals and wildlife. Coordinated efforts will be essential not only to protect the GSL population but also to prevent the potential spread of this pathogen to humans.

Conclusions

Our findings confirm the presence of L. interrogans in GSL at The Malecon rookery living in San Cristóbal Island. Additionally, evidence of exposure to this bacterium was confirmed in GSL serum that was collected in 2016 and 2017 at two different rookeries on the same island. These results support the presence of pathogenic Leptospira in GSL, aligning with previous studies and providing further insights into the pathogen’s dynamics in this endemic species. This highlights the importance of investigating its epidemiology through a One Health approach to mitigate potential health risks for humans, livestock, and synanthropic species in the archipelago.

Methods

This study was carried out in two phases. The aim of the first phase was to establish that the population of Z. wollebaeki continue to be exposed to Leptospira, using serological analyses of samples taken from animals in the largest rookery of GSL. The second phase was designed to confirm the presence of pathogenic leptospiral DNA in urine and tissue samples, and to identify the pathogen species. Phase 1 involved the collection of blood samples between 2016 and 2017 during the breeding season, while phase 2 involved the collection of sand with GSL urine and kidney samples from dead pups necropsied during the breeding season of 2021.

Study site and sample collection

The fieldwork was carried out following the protocols of ethics and animal handling approved by the Galapagos National Park Directorate (GNPD) and the Universidad San Francisco de Quito (USFQ) under research permits PC-16-17 and PC-31-21. The sampling was conducted in “El Malecón” (0°54’08’’S, 89°36’47’’W) and “Punta Pitt” (0°42’50’’S, 89°14’34’’W) rookeries in San Cristobal Island (Fig. 2A) which are the largest in the archipelago and comprise 22.7% of the total population of this species [1, 26]. The “El Malecón” rookery is located within the urban limits of Puerto Baquerizo Moreno, the second most populated port in the Galapagos archipelago, which increases the risk of exposure to zoonotic diseases [27]. Meanwhile, the “Punta Pitt” rookery is located in the protected natural area on the opposite side of the island.

Fig. 2.

Geographic location where samples were collected. Legend: (A) San Cristobal Island in the Galapagos Archipelago (inset), the circles indicate the locations and sample number from the “El Malecon” (pink) and “Punta Pitt” (green) rookeries. (B) The “El Malecon” rookery was sampled at 6 sites: (a) Lobería, (b) Playa Base Naval, (c) Playa de los Marinos, (d) Playa de Oro, (e) Playa Mann, and (f) Punta Carola

Phase 1 of the study was conducted during the summers of 2016 and 2017. We collected 72 and 63 blood samples from juvenile sea lions of the “El Malecón” and “Punta Pitt” rookeries, respectively, by venipuncture of the caudal gluteal vein. Subsequently, these samples were placed in sterile vacuum tubes with EDTA, as an anticoagulant agent. For serum separation, the tubes were centrifuged at 3, 000 g for 15 min; the serum was extracted and preserved at -20 °C for transport to the National Reference Laboratory for Animal Diagnostics (AGROCALIDAD) in Quito-Ecuador.

During Phase 2 conducted from September to October of 2021, we collected sand contaminated with urine from adult GSL (n = 24). Due to logistical constraints these samples were collected only from individuals of the “El Malecón” rookery resting on different beaches used by the rookery (Fig. 2B). To best protect samples from heat and sunlight, the samples of sand with urine (n = 24) were collected early in the morning and immediately after the observed micturition of the individuals. To maximize the probability of finding positive samples while minimizing cost, we pooled samples from 5 different sea lions (approximately 5 g of sand with urine per animal) that we observed urinating. Samples in each pool were from the same beach and collected on the same date. Sand with urine was preserved at 4 °C in Shield 1X (Zymo Research, CA, USA) and transported to our laboratory in Quito (Mainland Ecuador). Additionally, kidney samples were also obtained through necropsies of 24 deceased pups found on the different beaches. All individuals were pups, with a weight between 2.6 and 13.98 kg, 14 males (age: 3–11 months), and 10 females (age: 4 − 11 months). These samples were preserved in Shield 1X (Zymo Research, CA, USA) stored at 4 °C and transported to our laboratory in Quito.

Microaglutination test (MAT) in serum samples

Serum samples were analyzed by the National Reference Laboratory for Animal Diagnostics (AGROCALIDAD), using the microscopic agglutination test (MAT) for Leptospira antibodies. The MAT panel used to test the samples consisted of 20 different available serovars: Icterohaemorrhagiae (ICT), Canicola (CAN), Pomona (POM), Hardjo (HAR), Grippotyphosa (GRIP), Bratislava (BRAT), Autumnalis (AUT), Hebdomadis (HEB), Bataviae (BAT), Cynopteri (CYN), Australis (AUS), Saxkoebing (SAX), Patoc (PAT), Celledoni (CELL), Djasiman (DJA), Shermani (SHE), Tarassovi (TAR), Sejroe (SEJ), Pyrogenes (PYR) and Javanica (JAV). The endpoint reading of the microagglutination reaction was reported as the final dilution of serum at which 50% of Leptospira were observed to be agglutinated by dark-field microscopy. All serum samples that agglutinated from a titer of 1:20 were considered seropositive, since the agglutination of the antibodies reveals possible exposure to Leptospira [28]. Low titers (< 1:100) can indicate: the onset of antibody production in the early stage of an acute infection or a low level of antibodies that persist for a long time after infection [29]. To evaluate the differences seropotitivity between years 2016 and 2017, we performed a 2-sample test for equality of proportions with continuity correction in R v4.3.0 [30].

DNA extraction

DNA from kidneys was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. For sand with urine preserved in Shield 1X, we included filtration and lysis steps. Briefly, filtration was performed using MCE membranes with 0.22 μm pores (Merck, Darmstadt, Germany). After filtration, thick sand particles were removed from the membrane and ¼ of each membrane with Shield 1X from the original sample was placed in tubes with Lysing Matrix B 2 (MP Biomedicals, Graffenstaden, France) that contain 0.1 mm silica spheres. Tubes were vortexed at high speed for 3 min and centrifuged at 15, 000 rpm for 1 min. 250 µL of the supernatant was used for DNA extraction using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions.

Molecular detection of pathogenic Leptospira species

Two qPCR approaches that amplify different target DNA sequences were used for Leptospira DNA detection: a lipL32 gene fragment and a 16 S rDNA fragment using the SNP 111 assay, as described by Barragán and Stoddard [31, 32], respectively. qPCR was performed using the TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA) and each sample was run in triplicate in a CFX96 Touch Deep Well Real-Time PCR Detection System (Bio-Rad Laboratories, CA, USA). DNA extracted from L. interrogans serovar Icterohaemorrhagiae was used as positive control, and a no-template reaction was used as a negative control. Samples that amplified with a Ct value < 40 with one of both assays in any of the replicates were considered positive. To further avoid false-negative results due to PCR inhibitory compounds, sand samples were tested using a PCR assay that amplifies bacterial 16 S rDNA (present in environmental samples) [33], and kidney samples were tested using an assay that amplifies a β-actin gene fragment (present in all mammalian host cells) [34].

Sequencing and identification of pathogenic Leptospira species

Positive samples for pathogenic Leptospira by qPCR were further amplified using a nested PCR approach targeting a secY gene fragment, which has been shown to provide sufficient sequence heterogeneity to allow for phylogenetic interpretation of Leptospira species [35, 36]. Amplicons were purified using AMPure kit (Beckeman Coulter, CA, USA) following the manufacturer’s guidelines and normalized to 200 fmol. Library preparation was performed using the Ligation Sequencing LSK-109 kit (Oxford Nanopore Technologies, Oxford, UK) with the Oxford Nanopore Native Barcoding Expansion 96 kit (EXP-NBD196) and 20 fmol was loaded onto a flowcell (R9.4.1, FLO-MIN106D) on the GridION Mk1 (Oxford Nanopore Technologies, Oxford, UK). The sequencing run was monitored using MinKNOW software v. 22.12.5 and terminated after 24 h. The base calls from resulting reads were made using Guppy v. 6.4.6. with the Super Accuracy (SUP) setting and with a minimum Q-score of 9. Samples were demultiplexed in real-time and the adapters were trimmed using the MinKNOW software v. 22.12.5. Subsequently, quality control (QC) of the FASTq files was performed using NanoPlot [37] to verify run statistics and sequences quality. Nanopore sequencing of PCR products was performed at Centro de Bioinformática of Instituto de Microbiología, Universidad San Francisco de Quito. The consensus sequences were obtained using the Amplicon sorter tool [38] with default parameters, a minimum length of 390 bp, and a maximum length of 500 bp. Identification of Leptospira species was confirmed using the Nucleotide BLAST tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Raw data and partial secY sequences obtained in this study were deposited in NCBI under the accession number PRJNA1012001. A phylogenetic tree was built in MEGA-12 [39] using the Neighbor-Joining method [40], with the Maximum Composite Likelihood model [41] and 500 bootstrap replicates. Finally, the phylogenetic tree was visualized using iTOL [42].

Author contributions

S.M., PM, MC, V.B. experimental work and data analysis, D.P., J.M. and E.D. sample collection, E.D., D.P.R. and V.B. design of the study. S.M., A.G., V.B., D.P., E.D. and T.P.: draft writing and data analysis. All authors reviewed the manuscript.

Funding

This project was funded by the Galapagos Science Center award number 17264 (Barragan) and by the NIH/NIAID - R01AI172924 (Pearson).

Data availability

The data supporting the findings of this study are included within the article. Sequences were deposited in NCBI under the accession number PRJNA1012001. For any additional information, inquiries can be directed at the corresponding author.

Declarations

Ethics approval and consent to participate

Animal sampling protocols followed the strict guidelines established by the Galapagos National Park. These protocols were approved by the Galapagos National Park Directorate (GNPD) and Universidad San Francisco de Quito (USFQ) under research permits PC-16-17 and PC-31-21 (granted to Diego Páez-Rosas).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.