Domestic dogs in indigenous Amazonian communities: Key players in Leptospira cycling and transmission?

BACKGROUND: Leptospirosis is the world's most common zoonotic disease. Mitigation and control rely on pathogen identification and understanding the roles of potential reservoirs in cycling and transmission. Underreporting and misdiagnosis obscure the magnitude of the problem and confound efforts to understand key epidemiological components. Difficulties in culturing hamper the use of serological diagnostics and delay the development of DNA detection methods. As a result, especially in complex ecosystems, we know very little about the importance of different mammalian host species in cycling and transmission to humans. METHODOLOGY/PRINCIPAL FINDINGS: We sampled dogs from five indigenous Kichwa communities living in the Yasuní National Park in the Ecuadorian Amazon basin. Blood and urine samples from domestic dogs were collected to assess the exposure of these animals to Leptospira and to identify the circulating species. Microscopic Agglutination Tests with a panel of 22 different serovars showed anti-leptospira antibodies in 36 sampled dogs (75%), and 7 serogroups were detected. Two DNA-based detection assays revealed pathogenic Leptospira DNA in 18 of 19 dog urine samples (94.7%). Amplicon sequencing and phylogenetic analysis of 16S rRNA and SecY genes from 15 urine samples revealed genetic diversity within two of three different Leptospira species: noguchii (n = 7), santarosai (n = 7), and interrogans (n = 1). CONCLUSIONS/SIGNIFICANCE: The high prevalence of antibodies and Leptospira DNA provides strong evidence for high rates of past and current infections. Such high prevalence has not been previously reported for dogs. These dogs live in the peridomestic environment in close contact with humans, yet they are free-ranging animals that interact with wildlife. This complex web of interactions may explain the diverse types of pathogenic Leptospira observed in this study. Our results suggest that domestic dogs are likely to play an important role in the cycling and transmission of Leptospira. Future studies in areas with complex ecoepidemiology will enable better parsing of the significance of genotypic, environmental, and host characteristics.

SARS-CoV-2 infection in brown-headed spider monkeys (Ateles fusciceps) at a wildlife rescue center on the coast of Ecuador-South America.

Human populations can be affected in unpredictable ways by the emergence and spread of zoonotic diseases. The COVID-19 (coronavirus disease of 2019) pandemic was a reminder of how devastating these events can be if left unchecked. However, once they have spread globally, the impact of these diseases when entering non-exposed wildlife populations is unknown. The current study reports the infection of brown-headed spider monkeys (Ateles fusciceps) at a wildlife rescue center in Ecuador. Four monkeys were hospitalized, and all tested positive for SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) by RT-qPCR (Quantitative Reverse Transcription PCR). Fecal samples (n = 12) from monkeys at the rescue center also tested positive; three zookeepers responsible for feeding and deworming the monkeys also tested positive, suggesting human-animal transmission. Whole genome sequencing identified most samples' omicron clade 22B BA.5 lineage. These findings highlight the threat posed by an emerging zoonotic disease in wildlife species and the importance of preventing spillover and spillback events during epidemic or pandemic events.IMPORTANCEAlthough COVID-19 (coronavirus disease of 2019) has been primarily contained in humans through widespread vaccination, the impact and incidence of SARS-CoV-2 (Severe acute respiratory syndrome coronavirus) and its transmission and epidemiology in wildlife may need to be addressed. In some natural environments, the proximity of animals to humans is difficult to control, creating perfect scenarios where susceptible wildlife can acquire the virus from humans. In these places, it is essential to understand how transmission can occur and to develop protocols to prevent infection. This study reports the infection of brown-headed spider monkeys with SARS-CoV-2, a red-listed monkey species, at a wildlife recovery center in Ecuador. This study reports the infection of brown-headed spider monkeys with SARS-CoV-2, indicating the potential for transmission between humans and wildlife primates and the importance of preventing such events in the future.

Seroconversion in Galapagos Sea Lions (Zalophus wollebaeki) Confirms the Presence of Canine Distemper Virus in Rookeries of San Cristóbal Island.

BACKGROUND: The emblematic Galapagos sea lion (GSL-Zalophus wollebaeki) has faced an important population decline over the last four decades. There are multiple environmental and biological factors that might be implied in this decrease. Recently, evidence of various zoonotic infectious diseases that can be potential threats has been reported. Considering that in some islands of the archipelago the risk of transmission of infectious diseases may be promoted by the increasing population of domestic dogs, epidemiological vigilance and search of new pathogens are essential. The canine distemper virus (CDV), one of the viral pathogens that generate the most concern for the agencies responsible for the management and conservation of the Galapagos pinnipeds, was detected in the GSL in 2010. However, there is scarce information about its impact on GSL health and about its epidemiology. METHODS: In this study, 110 GSL serum samples were collected during the summer of 2016 and 2017. All samples were exposed to VERO dog SLAM cells expressing the canine SLAM receptor. RESULTS: Our results showed a significative increase (p = 0.04) in the frequency of neutralizing antibodies to CDV in the 2017 (53.1%) samples compared to the 2016 samples (19.6%). CONCLUSIONS: Our work confirmed the continuous and increasing circulation of the CDV in the GSL and highlights the importance of monitoring emerging diseases that can be transmitted from domestic to wildlife species. Vigilance of CDV is essential to understand the role of this virus in GSL mortality and to take informed decisions for wildlife conservation.

Leptospirosis in horses: Sentinels for a neglected zoonosis? A systematic review.

Background and Aim: Leptospirosis is considered a neglected tropical zoonosis in low-income countries due to surveillance system limitations and non-specificity of symptoms. Humans become infected through direct contact with carrier animals or indirectly through Leptospira-contaminated environments. Conventionally, equines have been considered an uncommon source of leptospirosis, but recent publications in Latin America suggest that their role in the maintenance and dispersion of the bacteria could be more relevant than expected, as horses are susceptible to a wide variety of zoonotic Leptospira spp. from domestic and wild animals with which they share the environment. A systematic review of the published literature was conducted to compile the available information on Leptospira spp. in Ecuador, with a special focus on equine leptospirosis, to better understand the epidemiology of the bacterium and identify possible knowledge gaps. Materials and Methods: A systematic review of the published literature was conducted in PubMed, SciELO and Web of Science databases to compile the available information on Leptospira spp. in Ecuador, with a special focus on equine leptospirosis, to better understand the epidemiology of the bacterium. We used a combination of the terms (Leptospira OR Leptospirosis) AND Ecuador, without restrictions on language or publication date. Results: Our literature review reveals that published scientific information is very scarce. Eighteen full-text original scientific articles related to Leptospira or leptospirosis cases in Ecuador were included in the systematic review. Most of the studies reported data obtained from one of the four regions (Coast), and specifically from only one of the 24 Provinces of Ecuador (Manabí), which evidence a large information bias at the geographical level. Furthermore, only the studies focused on humans included clinical signs of leptospirosis and there is only one study that analyzes the presence of Leptospira spp. in water or soil as a risk factor for pathogen transmission. Finally, only one study investigated Leptospira in horses. Conclusion: Since sentinel species can provide useful data on infectious diseases when epidemiologic al information is lacking, and horses could be considered excellent sentinel species to reveal circulating serovars, we propose developing a nationwide surveillance system using horses. This cost-effective epidemiological survey method provides a baseline for implementing specific prevention and control programs in Ecuador and neighboring developing countries.

Domestic dogs in indigenous Amazonian communities: key players in Leptospira cycling and transmission?

Background: Leptospirosis is the world's most common zoonotic disease. Mitigation and control rely on pathogen identification and understanding the roles of potential reservoirs in cycling and transmission. Underreporting and misdiagnosis obscure the magnitude of the problem and confound efforts to understand key epidemiological components. Difficulties in culturing hamper the use of serological diagnostics and delay the development of DNA detection methods. As a result, especially in complex ecosystems, we know very little about the importance of different mammalian host species in cycling and transmission to humans. Methodology/Principal Findings: We sampled five indigenous Kichwa communities living in the Yasuní National Park in the Ecuadorian Amazon basin. Blood and urine samples from domestic dogs were collected to assess the exposure of these animals to Leptospira, and to identify the circulating species. Microscopic Agglutination Tests with a panel of 22 different serovars showed anti-leptospira antibodies in 36 sampled dogs (75%), and 10 serotypes were detected. Two DNA-based detection assays revealed pathogenic Leptospira DNA in 18 of 19 dog urine samples (94.7%). Amplicon sequencing and phylogenetic analysis of 16s rDNA and SecY genes from 15 urine samples revealed genetic diversity within two of three different Leptospira species: noguchii (n=7), santarosai (n=7), and interrogans (n=1). Conclusions/Significance: The high prevalence of antibodies and Leptospira DNA provides strong evidence for high rates of past and current infections. Such high prevalence has not been previously reported for dogs. These dogs live in the peridomestic environment in close contact with humans, yet they are free-ranging animals that interact with wildlife. This complex web of interactions may explain the diverse types of pathogenic Leptospira observed in this study. Our results suggest that domestic dogs are likely to play an important role in the cycling and transmission of Leptospira. Future studies in areas with complex ecoepidemiology will enable better parsing of the significance of genotypic, environmental, and host characteristics.

First detection of Leptospira santarosai in the reproductive track of a boar: A potential threat to swine production and public health.

BACKGROUND: Leptospirosis causes significant economic losses and is an occupational risk in the swine industry, especially in developing tropical regions where social and geoclimatic conditions are favorable for the transmission of this disease. Although vaccination can reduce infection risk, efficacy is diminished if local genetic and antigenic variants of the pathogen are not accounted for in the vaccine. Identifying and characterizing strains hosts, and potential mechanisms of transmission is therefore critical for public health mitigation practices. METHODOLOGY/PRINCIPAL FINDINGS: Our study was conducted on a rural breeding farm in Ecuador, where we used a PCR assay that targets lipL32 to detect Leptospira spp. and targeted gene sequencing to identify Leptospira santarosai in the kidneys, testicles, and ejaculate of a vaccinated boar. MAT results showed low titers against serovars found in the vaccine, but the MAT panel did not include serovars of L. santarosai. The boar showed no symptoms of leptospirosis but did show blood in the semen. However, no postmortem histopathological lesions were observed tissue samples. Vaccinated sows that were artificially inseminated with the semen from this boar had reproductive problems, suggesting that transmission had occurred. This is the first documented case of Leptospira santarosai in the reproductive tract of a boar. CONCLUSIONS/SIGNIFICANCE: As L. santarosai is pathogenic in other livestock species and humans, our finding highlights the need to evaluate the prevalence and epidemiological significance of this pathogen in livestock and consider the possibility of venereal transmission. In addition, further studies are needed to identify and characterize local serovars that may impact diagnosis and vaccination programs to better control leptospirosis in livestock and spillover into the human population.

Occurrence of SARS-CoV-2 reinfections at regular intervals in Ecuador.

SARS-CoV-2 reinfection is defined as a new infection with a different virus variant in an individual who has already recovered from a previous episode of COVID-19. The first case of reinfection in the world was described in August 2020, since then, reinfections have increased over time and their incidence has fluctuated with specific SARS-CoV-2 variant waves. Initially, reinfections were estimated to represent less than 1% of total COVID-19 infections. With the advent of the Omicron variant, reinfections became more frequent, representing up to 10% of cases (based on data from developed countries). The frequency of reinfections in Latin America has been scarcely reported. The current study shows that in Ecuador, the frequency of reinfections has increased 10-fold following the introduction of Omicron, after 22 months of surveillance in a single center of COVID-19 diagnostics. Suspected reinfections were identified retrospectively from a database of RT-qPCR-positive patients. Cases were confirmed by sequencing viral genomes from the first and second infections using the ONT MinION platform. Monthly surveillance showed that the main incidence peaks of reinfections were reached within four to five months, coinciding with the increase of COVID-19 cases in the country, suggesting that the emergence of reinfections is related to higher exposure to the virus during outbreaks. This study performed the longest monitoring of SARS-CoV-2 reinfections, showing an occurrence at regular intervals of 4-5 months and confirming a greater propensity of Omicron to cause reinfections.

Characterization of the Immune Resistance of Severe Acute Respiratory Syndrome Coronavirus 2 Mu Variant and the Robust Immunity Induced by Mu Infection.

BACKGROUND: We have recently revealed that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Mu variant shows a pronounced resistance to antibodies elicited by natural SARS-CoV-2 infection and vaccination. METHODS: However, it remains unclear which mutations determine the resistance of SARS-CoV-2 Mu to antiviral sera. In addition, it is unclear how SARS-CoV-2 Mu infection induces antiviral immunity. RESULTS: In this study, we reveal that the 2 mutations in the SARS-CoV-2 Mu spike protein, YY144-145TSN and E484K, are responsible for the resistance to coronavirus disease 2019 convalescent sera during early 2020 and vaccine sera. CONCLUSIONS: It is notable that the convalescent sera of SARS-CoV-2 Mu-infected individuals are broadly antiviral against Mu as well as other SARS-CoV-2 variants of concern and interest.

Genomic epidemiology of SARS-CoV-2 transmission lineages in Ecuador.

Characterisation of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic diversity through space and time can reveal trends in virus importation and domestic circulation and permit the exploration of questions regarding the early transmission dynamics. Here, we present a detailed description of SARS-CoV-2 genomic epidemiology in Ecuador, one of the hardest hit countries during the early stages of the coronavirus-19 pandemic. We generated and analysed 160 whole genome sequences sampled from all provinces of Ecuador in 2020. Molecular clock and phylogeographic analysis of these sequences in the context of global SARS-CoV-2 diversity enable us to identify and characterise individual transmission lineages within Ecuador, explore their spatiotemporal distributions, and consider their introduction and domestic circulation. Our results reveal a pattern of multiple international importations across the country, with apparent differences between key provinces. Transmission lineages were mostly introduced before the implementation of non-pharmaceutical interventions, with differential degrees of persistence and national dissemination.

Vector-borne zoonotic blood parasites in wildlife from Ecuador: A report and systematic review.

BACKGROUND AND AIM: Ecuador is a hugely diverse country, but information on infectious diseases in local wild animals is scarce. The aim of this study was to screen the presence of blood parasites in free-ranging wild animals admitted to the Wildlife Hospital at Universidad San Francisco de Quito, from April 2012 to January 2019. MATERIALS AND METHODS: We identified blood parasites by microscopic observation of blood smears from free-ranging wildlife species that attended the Wildlife Hospital of Universidad San Francisco de Quito (Ecuador) from April 2012 to January 2019. RESULTS: The microscopic evaluations of animals as potential reservoirs for vector-borne zoonotic blood parasites revealed the presence of Anaplasma spp., Babesia spp., Ehrlichia spp., Hepatozoon spp., microfilaria, Mycoplasma spp., and Trypanosoma spp. in previously unreported wildlife species. In addition, we performed a systematic review to understand the current knowledge gaps in the context of these findings. CONCLUSION: Our data contribute to the knowledge of blood parasites in wildlife from Ecuador. Furthermore, the potential transmission of these parasites to humans and domestic animals, current anthropogenic environmental changes in the region, and the lack of information on this suggest the importance of our results and warrant further investigations on infectious diseases in animals and humans and their relationship with environmental health as key domains of the One Health concept.

SARS-CoV-2 detection and sequencing in heart tissue associated with myocarditis and persistent arrhythmia: A case report.

BACKGROUND: SARS-CoV-2 uses the human cell receptor angiotensin-converting enzyme (ACE2). ACE2 is widely present in the cardiovascular system including the myocardium and the conduction system. COVID-19 patients that present severe symptoms have been reported to have complications involving myocardial injuries caused by the virus. Here we report the detection of SARS-CoV-2 by whole genome sequencing in the endocardium of a patient with severe bradycardia. CASE PRESENTATION: We report a case of a 34-year-old male patient with COVID-19 tested by PCR, he started with gastrointestinal symptoms, however, he quickly deteriorated his hemodynamic state by means of myocarditis and bradycardia. After performing an endocardium biopsy, it was possible to identify the presence of SARS-CoV-2 in the heart tissue and to sequence its whole genome using the ARTIC-Network protocol and a modified tissue RNA extraction method. The patient's outcome was improved after a permanent pacemaker was implanted. CONCLUSIONS: It was possible to identify a SARS-CoV-2 clade 20A in the endocardium of the reported patient.

SARS-CoV-2 genome sequencing from COVID-19 in Ecuadorian patients: a whole country analysis.

SARS-CoV-2, the etiological agent of COVID-19, was first described in Wuhan, China in December 2019 and has now spread globally. Ecuador was the second country in South America to confirm cases and Guayaquil was one of the first cities in the world to experience high mortality due to COVID-19. The aim of this study was to describe the lineages circulating throughout the country and to compare the mutations in local variants, to the reference strain. In this work we used the MinION platform (Oxford Nanopore Technologies) to sequence the whole SARS-CoV-2 genomes of 119 patients from all provinces of Ecuador, using the ARTIC network protocols. Our data from lineage assignment of the one hundred and nineteen whole genomes revealed twenty different lineages. All genomes presented differences in the S gene compared to the Wuhan reference strain, being the D614G amino acid replacement the most common change. The B.1.1.119 lineage was the most frequent and was found in several locations in the Coast and Andean region. Three sequences were assigned to the new B.1.1.7 lineage. Our work is an important contribution to the understanding of the epidemiology of SARS-CoV-2 in Ecuador and South America.

Genomic epidemiology of SARS-CoV-2 transmission lineages in Ecuador.

Characterisation of SARS-CoV-2 genetic diversity through space and time can reveal trends in virus importation and domestic circulation, and permit the exploration of questions regarding the early transmission dynamics. Here we present a detailed description of SARS-CoV-2 genomic epidemiology in Ecuador, one of the hardest hit countries during the early stages of the COVID-19 pandemic. We generate and analyse 160 whole genome sequences sampled from all provinces of Ecuador in 2020. Molecular clock and phylgeographic analysis of these sequences in the context of global SARS-CoV-2 diversity enable us to identify and characterise individual transmission lineages within Ecuador, explore their spatiotemporal distributions, and consider their introduction and domestic circulation. Our results reveal a pattern of multiple international importations across the country, with apparent differences between key provinces. Transmission lineages were mostly introduced before the implementation of non-pharmaceutical interventions (NPIs), with differential degrees of persistence and national dissemination.

Leptospira in river and soil in a highly endemic area of Ecuador.

BACKGROUND: Leptospira are shed into the environment via urine of infected animals. Rivers are thought to be an important risk factor for transmission to humans, though much is unknown about the types of environment or characteristics that favor survival. To address this, we screened for Leptospira DNA in two rivers in rural Ecuador where Leptospirosis is endemic. RESULTS: We collected 112 longitudinal samples and recorded pH, temperature, river depth, precipitation, and dissolved oxygen. We also performed a series of three experiments designed to provide insight into Leptospira presence in the soil. In the first soil experiment, we characterized prevalence and co-occurrence of Leptospira with other bacterial taxa in the soil at dispersed sites along the rivers (n = 64). In the second soil experiment, we collected 24 river samples and 48 soil samples at three points along eight transects to compare the likelihood of finding Leptospira in the river and on the shore at different distances from the river. In a third experiment, we tested whether Leptospira presence is associated with soil moisture by collecting 25 soil samples from two different sites. In our river experiment, we found pathogenic Leptospira in only 4 (3.7%) of samples. In contrast, pathogenic Leptospira species were found in 22% of shore soil at dispersed sites, 16.7% of soil samples (compared to 4.2% of river samples) in the transects, and 40% of soil samples to test for associations with soil moisture. CONCLUSIONS: Our data are limited to two sites in a highly endemic area, but the scarcity of Leptospira DNA in the river is not consistent with the widespread contention of the importance of river water for leptospirosis transmission. While Leptospira may be shed directly into the river, onto the shores, or washed into the river from more remote sites, massive dilution and limited persistence in rivers may reduce the environmental load and therefore, the epidemiological significance of such sources. It is also possible that transmission may occur more frequently on shores where people are liable to be barefoot. Molecular studies that further explore the role of rivers and water bodies in the epidemiology of leptospirosis are needed.

Metagenome of a Bronchoalveolar Lavage Fluid Sample from a Confirmed COVID-19 Case in Quito, Ecuador, Obtained Using Oxford Nanopore MinION Technology.

We report the metagenome analysis of a bronchoalveolar lavage (BAL) fluid sample from a confirmed coronavirus disease 2019 (COVID-19) case in Quito, Ecuador. Sequencing was performed using MinION technology.

Vaginal Microbiota Evaluation and Lactobacilli Quantification by qPCR in Pregnant and Non-pregnant Women: A Pilot Study.

Pregnancy outcomes and women's health are directly affected by vaginal microbiota. This microbiota consists of a dynamic ecosystem of various microbes in different ratios, which in healthy conditions protect the vaginal epithelium from infections. However, cases of vaginal infection are regularly diagnosed in women of reproductive age, contributing to more severe outcomes. Therefore, our main goal was to determine the prevalence of bacterial vaginosis (BV), aerobic vaginitis (AV), and vulvovaginal candidiasis (VVC) among Ecuadorian pregnant and non-pregnant women. A cross-sectional study was conducted among 217 women between 13 and 40 years old seeking primary healthcare in Carlos Andrade Marin Hospital (HCAM), Gynecological-Obstetric Hospital Isidro Ayora (HGOIA) and Center for Teaching Health Cipriana Dueñas during October 2018 to February 2019. The classical characterization of the vaginal microbiota was performed through microscopy by the Nugent criteria to evaluate the presence of BV, healthy and intermediate microbiota, by the criteria of Donders to determine the presence of AV and by the Marot-Leblond criteria to diagnose VVC. DNA extraction from vaginal samples and Polymerase Chain Reaction (PCR) analysis was performed to characterize the presence of Gardnerella spp., Mobiluncus mulieris, Escherichia coli, Enterococcus spp., and Lactobacillus spp. Finally, quantification of the lactobacilli was performed by quantitative real-time PCR (qPCR) for samples from women with normal vaginal microbiota and women with AV. Our results showed 52% of women with healthy microbiota, 7% with intermediate microbiota, and 41% with vaginal dysbiosis, comprising 27% with AV, 8% with BV and 4% with VVC and 2% with co-infections or co-dysbiosis. Additionally, a higher amount of lactobacilli were found in pregnant women when compared to non-pregnant women, while AV cases were characterized by a significant drop of Lactobacillus spp., more precisely, between 1E3 and 1E5 colony forming units (CFU)/ml. Finally, women with normal vaginal microbiota showed an average load of lactobacilli between 1E6 and 1E7 CFU/ml. This pilot study showed no statistically significant differences between pregnant and non-pregnant women, pointing to the possibility to use lactobacilli quantification for the prevention of future vaginal infections.

Genome sequencing of the first SARS-CoV-2 reported from patients with COVID-19 in Ecuador.

SARS-CoV-2, the etiological agent of COVID-19 was first described in Wuhan in December 2019 and has now spread globally. Ecuador was the second country in South America to report confirmed cases. The first case reported in Quito, the capital city of Ecuador, was a tourist who came from the Netherlands and presented symptoms on March 10th, 2020 (index case). In this work we used the MinION platform (Oxford Nanopore Technologies) to sequence the metagenome of the bronchoalveolar lavage (BAL) from this case reported, and subsequently we sequenced the whole genome of the index case and other three patients using the ARTIC network protocols. Our data from the metagenomic approach confirmed the presence of SARS-CoV-2 coexisting with pathogenic bacteria suggesting coinfection. Relevant bacteria found in the BAL metagenome were Streptococcus pneumoniae, Mycobacterium tuberculosis, Staphylococcus aureus and Chlamydia spp. Lineage assignment of the four whole genomes revealed three different origins. The variant HEE-01 was imported from the Netherlands and was assigned to B lineage, HGSQ-USFQ-018, belongs to the B.1 lineage showing nine nucleotide differences with the reference strain and grouped with sequences from the United Kingdom, and HGSQ-USFQ-007 and HGSQ-USFQ-010 belong to the B lineage and grouped with sequences from Scotland. All genomes show mutations in their genomes compared to the reference strain, which could be important to understand the virulence, severity and transmissibility of the virus. Our findings also suggest that there were at least three independent introductions of SARS-CoV-2 to Ecuador.

Critical Knowledge Gaps in Our Understanding of Environmental Cycling and Transmission of Leptospira spp.

Exposure to soil or water contaminated with the urine of Leptospira-infected animals is the most common way in which humans contract leptospirosis. Entire populations can be at high risk of leptospirosis while working in inundated fields, when engaging in aquatic sports, or after periods of heavy rainfall. The risk of infection after contact with these environmental sources depends on the ability of Leptospira bacteria to survive, persist, and infect new hosts. Multiple variables such as soil and water pH, temperature, and even environmental microbial communities are likely to shape the environmental conditions needed by the pathogen to persist. Here we review what is known about the environmental phase of the infectious Leptospira transmission cycle and identify knowledge gaps that will serve as a guide for future research.

PUP MORTALITY AND EVIDENCE FOR PATHOGEN EXPOSURE IN GALAPAGOS SEA LIONS (ZALOPHUS WOLLEBAEKI) ON SAN CRISTOBAL ISLAND, GALAPAGOS, ECUADOR.

The Galapagos sea lion ( Zalophus wollebaeki ), an endangered species, experiences high pup mortality (up to 100%) in years when El Niño events reduce food supply in the Galapagos Islands. Mortality of pups in non-El Niño years is estimated to be 5% in undisturbed colonies. From 2009 to 2012 we observed high pup mortality (up to 67%) in colonies close to the Galapagos capital, Puerto Baquerizo Moreno, where contact with humans, domestic animals, and rats is frequent. Gross postmortem findings from 54 pups included hemorrhagic lesions in liver and congestion in lungs; histopathology suggested a possible association with infectious diseases. Evidence of Leptospira infection was found in five out of seven samples collected in 2010. Canine distemper viral (CDV) RNA was detected in tissues from six sea lions (in 2011-12), four of which were confirmed by nucleotide sequencing. The absence of CDV antibodies in 109 juvenile animals tested in 2014 at urban and remote colonies could indicate that the CDV infection observed in 2011 was likely confined to a few animals. Our results indicated that Galapagos sea lions have been exposed at least to two pathogens, Leptospira and CDV; however, the impact of these infections on the sea lions is unclear.