Evaluating the Risk Landscape of Hawaiian Monk Seal Exposure to Toxoplasma gondii.

Toxoplasmosis is a disease of primary concern for Hawaiian monk seals (Neomonachus schauinslandi), due to its apparently acute lethality and especially heavy impacts on breeding female seals. The disease-causing parasite, Toxoplasma gondii, depends on cats to complete its life cycle; thus, in order to understand how this pathogen infects marine mammals, it is essential to understand aspects of the terrestrial ecosystem and land-to-sea transport. In this study, we constructed a three-tiered model to assess risk of Hawaiian monk seal exposure to T. gondii oocysts: (1) oocyst contamination as a function of cat population characteristics; (2) land-to-sea transport of oocysts as a function of island hydrology, and (3) seal exposure as a function of habitat and space use. We were able to generate risk maps highlighting watersheds contributing the most to oocyst contamination of Hawaiian monk seal habitat. Further, the model showed that free-roaming cats most associated with humans (pets or strays often supplementally fed by people) were able to achieve high densities leading to high levels of oocyst contamination and elevated risk of T. gondii exposure.

TERRESTRIAL PATHOGEN POLLUTANT, TOXOPLASMA GONDII, THREATENS HAWAIIAN MONK SEALS (NEOMONACHUS SCHAUINSLANDI) FOLLOWING HEAVY RUNOFF EVENTS.

Toxoplasmosis is a major threat to Hawaiian monk seals (Neomonachus schauinslandi) in the main Hawaiian Islands where seal habitat overlaps with substantial human and domestic cat populations. As the definitive hosts, members of the Felidae are the sole sources contaminating the environment with infectious oocysts; these oocysts can be transported into the marine environment, thereby threatening marine mammals. To understand environmental factors influencing Hawaiian monk seal exposure to Toxoplasma gondii, we examined monk seal strandings from toxoplasmosis in relationship to location and rainfall patterns throughout the main Hawaiian Islands. Using a case-control study design, we compared mortalities due to toxoplasmosis (cases) with those from other causes (controls). We found that cases were up to 25 times more likely than controls to occur after heavy runoff events. The greatest odds ratio was observed when rainfall occurred 3 wk before strandings, potentially indicating important timelines in the disease process. Our results suggest that heavy rainfall frequently delivers sufficient numbers of oocysts to infect Hawaiian monk seals. With infectious doses of as low as a single oocyst, any contaminated runoff constitutes a risk to Hawaii's endangered monk seal.

Sample preparation and liquid chromatography-tandem mass spectrometry for the analysis of selected Pacific ciguatoxins in blood samples.

Consumption of ciguatoxin-contaminated seafood can lead to ciguatera poisoning (CP). The diagnosis of CP in humans is based on the clinical symptoms after eating the fish from tropical or subtropical areas because no confirmatory clinical tests are available. One of the challenges for ciguatoxin analysis is their extremely low but toxicologically relevant concentration in biological samples. We previously reported a method using acetonitrile to precipitate proteins and extract the ciguatoxins simultaneously in whole blood samples from animals for toxin quantification by N2A cell-based assay. However, a test method for unambiguous confirmation of exposure of marine animals or humans to ciguatoxins is still needed. In the present study, we adopted the acetonitrile extraction method and added sample clean-up in the sample preparation for the determination of Pacific ciguatoxins CTX1B (aka P-CTX-1), 52-epi-54-deoxyCTX1B (aka P-CTX-2), and CTX3C (aka P-CTX-3C) in blood plasma by LC-MS/MS. We investigated sample clean-up, LC mobile phases, LC solvent programming, and settings of the two mass spectrometers (4000 Q TRAP and AB SCIEX Triple Quad 5500) in order to improve the ability to detect the Pacific ciguatoxins at ppt level. Rat blood plasma was used for the method development. Average recoveries of the three toxins in the rat plasma samples ranged from 90% to 116% with relative standard deviations of less than 15%. The method detection limits were still not low enough for the determination of the Pacific ciguatoxins in individual blood samples from Hawaiian monk seals with the two LC-MS systems. The methods were applied to a pooled sample of blood plasma collected from Hawaiian monk seals for confirmation of toxin exposure. This study will benefit monitoring of Pacific ciguatoxins in marine mammals and potentially humans by LC-MS/MS.

Correction to: The One Health Approach to Toxoplasmosis: Epidemiology, Control, and Prevention Strategies.

This article was originally published electronically on the publisher's internet portal (currently SpringerLink) on April 3, 2019 without open access.

The One Health Approach to Toxoplasmosis: Epidemiology, Control, and Prevention Strategies.

One Health is a collaborative, interdisciplinary effort that seeks optimal health for people, animals, plants, and the environment. Toxoplasmosis, caused by Toxoplasma gondii, is an intracellular protozoan infection distributed worldwide, with a heteroxenous life cycle that practically affects all homeotherms and in which felines act as definitive reservoirs. Herein, we review the natural history of T. gondii, its transmission and impacts in humans, domestic animals, wildlife both terrestrial and aquatic, and ecosystems. The epidemiology, prevention, and control strategies are reviewed, with the objective of facilitating awareness of this disease and promoting transdisciplinary collaborations, integrative research, and capacity building among universities, government agencies, NGOs, policy makers, practicing physicians, veterinarians, and the general public.

ESTABLISHING HEMATOLOGY AND SERUM CHEMISTRY REFERENCE INTERVALS FOR WILD HAWAIIAN MONK SEALS ( NEOMONACHUS SCHAUINSLANDI).

Hematology and serum chemistry reference intervals have been previously established for the endangered Hawaiian monk seal ( Neomonachus schauinslandi) as an imperative measure for health assessments. Monitoring the health of the wild population depends upon reference intervals that are context specific; hence we developed reference intervals from fresh samples, as opposed to frozen, from wild monk seals. This study builds on the number of parameters from previous efforts by using samples collected between 2004 and 2015 from wild monk seals. Blood samples were analyzed by a single veterinary diagnostic laboratory within 24 hr of collection from apparently healthy, wild seals during research activities. Reference intervals were determined based on the analytical steps outlined by the American Society for Veterinary Clinical Pathology. These comprehensive hematology and serum chemistry reference intervals enable more consistent and systematic interpretation of results, which will guide individual and population-level health assessment and decision-making research and recovery activities.

Survey for Placental Disease and Reproductive Pathogens in the Endangered Hawaiian Monk Seal ( Neomonachus schauinslandi).

There is considerable temporal and spatial variability in the reproductive rates of Hawaiian monk seals (HMS; Neomonachus schauinslandi). Poor reproductive performance limits the recovery of this endangered species; however, causal factors are not fully understood. There is serologic evidence that HMS are exposed to pathogens that can impact reproductive success, but the prevalence of placental infections in HMS has not been evaluated. Placental tissues ( n=50), including tissues from 25% of known HMS births, were opportunistically collected in 2011 from six Northwestern Hawaiian Islands and three main Hawaiian Islands. Reproductive histories of the sampled females were representative of the breeding population, as determined through comparisons in age of primiparity and mature reproductive rate. Placental tissues were examined histologically and screened by PCR for Coxiella burnetii, Brucella spp., Chlamydia spp., Leptospira spp., herpesviruses, and Toxoplasma gondii. There was no histologic evidence of placental pathology, and molecular analyses were negative. These negative results can be used to estimate pathogen prevalence in the nonsampled population. For an approximate population size of 1,300 HMS, we can estimate with 99% confidence that the prevalence of each pathogen tested is 9% or less. This is low relative to other pinnipeds and indicates that factors other than reproductive pathology, such as resource limitation, may drive variability in HMS reproductive rates. Further investigation into the cumulative impacts of resource limitation and other stressors on HMS reproduction is warranted.

Model recommendations meet management reality: implementation and evaluation of a network-informed vaccination effort for endangered Hawaiian monk seals.

Where disease threatens endangered wildlife populations, substantial resources are required for management actions such as vaccination. While network models provide a promising tool for identifying key spreaders and prioritizing efforts to maximize efficiency, population-scale vaccination remains rare, providing few opportunities to evaluate performance of model-informed strategies under realistic scenarios. Because the endangered Hawaiian monk seal could be heavily impacted by disease threats such as morbillivirus, we implemented a prophylactic vaccination programme. We used contact networks to prioritize vaccinating animals with high contact rates. We used dynamic network models to simulate morbillivirus outbreaks under real and idealized vaccination scenarios. We then evaluated the efficacy of model recommendations in this real-world vaccination project. We found that deviating from the model recommendations decreased the efficiency; requiring 44% more vaccinations to achieve a given decrease in outbreak size. However, we gained protection more quickly by vaccinating available animals rather than waiting to encounter priority seals. This work demonstrates the value of network models, but also makes trade-offs clear. If vaccines were limited but time was ample, vaccinating only priority animals would maximize herd protection. However, where time is the limiting factor, vaccinating additional lower-priority animals could more quickly protect the population.

MODELING A MORBILLIVIRUS OUTBREAK IN HAWAIIAN MONK SEALS (NEOMONACHUS SCHAUINSLANDI) TO AID IN THE DESIGN OF MITIGATION PROGRAMS.

We developed a stochastic susceptible-exposed-infectious-removed (SEIR) model to simulate a range of plausible morbillivirus outbreak scenarios in a randomly mixing population of 170 endangered Hawaiian monk seals (Neomonachus schauinslandi). We then modeled realistic vaccination and quarantine measures to determine the potential efficacy of such mitigation efforts. Morbillivirus outbreaks represent substantial risk to monk seals-91% of simulated baseline outbreaks grew (R0>1), and in one-third of the scenarios all, or nearly all, individuals were infected. Simulated vaccination efforts in response to an outbreak were not effective in substantially reducing infections, largely because of the prolonged interval between vaccination and immunity. Prophylactic vaccination, in contrast, could be an effective tool for preventing outbreaks. Herd immunity is practically achievable because of the small sizes of monk seal populations and the animals' accessibility on shore. Adding realistic spatial structure to the model, as informed by movement of seals tracked in the main Hawaiian Islands with the use of telemetry, greatly reduced the simulated impact of outbreaks (≤10 seals were infected in 62% of spatially structured simulations). Although response vaccination remained relatively ineffective, spatial segregation allowed herd immunity to be achieved through prophylactic vaccination with less effort. In a randomly mixing population of 170 seals, 86% would need to be vaccinated to achieve herd immunity in 95% of simulated outbreaks, compared to only approximately 60% in three spatially segregated subgroups with the same combined abundance. Simulations indicate that quarantining a modest number (up to 20) of ill seals has the potential to extinguish even fast-growing outbreaks rapidly. The efficacy of quarantine, however, is highly dependent upon rapid detection and response. We conclude that prophylactic vaccination combined with a quarantine program supported by vigilant surveillance and rapid, reliable diagnosis could greatly mitigate the threat of a morbillivirus outbreak in Hawaiian monk seals.

Protozoal-related mortalities in endangered Hawaiian monk seals Neomonachus schauinslandi.

Protozoal infections have been widely documented in marine mammals and may cause morbidity and mortality at levels that result in population level effects. The presence and potential impact on the recovery of endangered Hawaiian monk seals Neomonachus schauinslandi by protozoal pathogens was first identified in the carcass of a stranded adult male with disseminated toxoplasmosis and a captive monk seal with hepatitis. We report 7 additional cases and 2 suspect cases of protozoal-related mortality in Hawaiian monk seals between 2001 and 2015, including the first record of vertical transmission in this species. This study establishes case definitions for classification of protozoal infections in Hawaiian monk seals. Histopathology and immunohistochemistry were the primary diagnostic modalities used to define cases, given that these analyses establish a direct link between disease and pathogen presence. Findings were supported by serology and molecular data when available. Toxoplasma gondii was the predominant apicomplexan parasite identified and was associated with 100% of mortalities (n = 8) and 50% of suspect cases (n = 2). Incidental identification of sarcocysts in the skeletal muscle without tissue inflammation occurred in 4 seals, including one co-infected with T. gondii. In 2015, 2 cases of toxoplasmosis were identified ante-mortem and shared similar clinical findings, including hematological abnormalities and histopathology. Protozoal-related mortalities, specifically due to toxoplasmosis, are emerging as a threat to the recovery of this endangered pinniped and other native Hawaiian taxa. By establishing case definitions, this study provides a foundation for measuring the impact of these diseases on Hawaiian monk seals.

ESTIMATING CONTACT RATES OF HAWAIIAN MONK SEALS (NEOMONACHUS SCHAUINSLANDI) USING SOCIAL NETWORK ANALYSIS.

Understanding disease transmission dynamics, which are in part mediated by rates and patterns of social contact, is fundamental to predicting the likelihood, rate of spread, impacts, and mitigation of disease outbreaks in wildlife populations. Contact rates, which are important parameters required for epidemiologic models, are difficult to estimate. The endangered Hawaiian monk seal (Neomonachus schauinslandi) may be particularly vulnerable to morbillivirus outbreaks, due to its low abundance, lack of genetic diversity, and history of isolation from mammalian diseases. Morbillivirus epizootics have had devastating effects on other seal populations. We constructed social networks based on visual observations of individually identifiable monk seals associating onshore to estimate contact rates, assuming random mixing, and also to investigate contact patterns of different age and sex classes. Contact rates estimated from two island populations in 4 yr were remarkably similar, indicating any two individuals have about a one in 1,000 chance of making contact on any given day. Further, contact patterns within and among age and sex classes were statistically different from random. The methods we used could be broadly applied to empirically derive contact rates using association data. These rates are critical for epidemiologic modelling to simulate wildlife disease outbreaks and to inform science-based prevention and mitigation programs.

Fetal distress and in utero pneumonia in perinatal dolphins during the Northern Gulf of Mexico unusual mortality event.

An unusual mortality event (UME) involving primarily common bottlenose dolphins Tursiops truncatus of all size classes stranding along coastal Louisiana, Mississippi, and Alabama, USA, started in early 2010 and continued into 2014. During this northern Gulf of Mexico UME, a distinct cluster of perinatal dolphins (total body length <115 cm) stranded in Mississippi and Alabama during 2011. The proportion of annual dolphin strandings that were perinates between 2009 and 2013 were compared to baseline strandings (2000-2005). A case-reference study was conducted to compare demographics, histologic lesions, and Brucella sp. infection prevalence in 69 UME perinatal dolphins to findings from 26 reference perinates stranded in South Carolina and Florida outside of the UME area. Compared to reference perinates, UME perinates were more likely to have died in utero or very soon after birth (presence of atelectasis in 88 vs. 15%, p < 0.0001), have fetal distress (87 vs. 27%, p < 0.0001), and have pneumonia not associated with lungworm infection (65 vs. 19%, p = 0.0001). The percentage of perinates with Brucella sp. infections identified via lung PCR was higher among UME perinates stranding in Mississippi and Alabama compared to reference perinates (61 vs. 24%, p = 0.01), and multiple different Brucella omp genetic sequences were identified in UME perinates. These results support that from 2011 to 2013, during the northern Gulf of Mexico UME, bottlenose dolphins were particularly susceptible to late-term pregnancy failures and development of in utero infections including brucellosis.

Phocine distemper virus: current knowledge and future directions.

Phocine distemper virus (PDV) was first recognized in 1988 following a massive epidemic in harbor and grey seals in north-western Europe. Since then, the epidemiology of infection in North Atlantic and Arctic pinnipeds has been investigated. In the western North Atlantic endemic infection in harp and grey seals predates the European epidemic, with relatively small, localized mortality events occurring primarily in harbor seals. By contrast, PDV seems not to have become established in European harbor seals following the 1988 epidemic and a second event of similar magnitude and extent occurred in 2002. PDV is a distinct species within the Morbillivirus genus with minor sequence variation between outbreaks over time. There is now mounting evidence of PDV-like viruses in the North Pacific/Western Arctic with serological and molecular evidence of infection in pinnipeds and sea otters. However, despite the absence of associated mortality in the region, there is concern that the virus may infect the large Pacific harbor seal and northern elephant seal populations or the endangered Hawaiian monk seals. Here, we review the current state of knowledge on PDV with particular focus on developments in diagnostics, pathogenesis, immune response, vaccine development, phylogenetics and modeling over the past 20 years.

Cetacean morbillivirus: current knowledge and future directions.

We review the molecular and epidemiological characteristics of cetacean morbillivirus (CeMV) and the diagnosis and pathogenesis of associated disease, with six different strains detected in cetaceans worldwide. CeMV has caused epidemics with high mortality in odontocetes in Europe, the USA and Australia. It represents a distinct species within the Morbillivirus genus. Although most CeMV strains are phylogenetically closely related, recent data indicate that morbilliviruses recovered from Indo-Pacific bottlenose dolphins (Tursiops aduncus), from Western Australia, and a Guiana dolphin (Sotalia guianensis), from Brazil, are divergent. The signaling lymphocyte activation molecule (SLAM) cell receptor for CeMV has been characterized in cetaceans. It shares higher amino acid identity with the ruminant SLAM than with the receptors of carnivores or humans, reflecting the evolutionary history of these mammalian taxa. In Delphinidae, three amino acid substitutions may result in a higher affinity for the virus. Infection is diagnosed by histology, immunohistochemistry, virus isolation, RT-PCR, and serology. Classical CeMV-associated lesions include bronchointerstitial pneumonia, encephalitis, syncytia, and lymphoid depletion associated with immunosuppression. Cetaceans that survive the acute disease may develop fatal secondary infections and chronic encephalitis. Endemically infected, gregarious odontocetes probably serve as reservoirs and vectors. Transmission likely occurs through the inhalation of aerosolized virus but mother to fetus transmission was also reported.