Toxoplasma gondii infections in Hawaiian monk seals

Michelle Barbieri, Katie Colegrove, Stacie Robinson, Cara Field, Angela Amlin, Gregg Levine, Charles Littnan

HMS: The Basics

• Endangered

• Only tropical seal

• Population size 1,400

• Poor genetic diversity

• Interisland movement varies by individual

• Diverse benthic foragers

Northwestern Hawaiian Islands (NWHI)1,100 seals

Main Hawaiian Islands (MHI)300 seals

HMS Research

Threats: NWHI

Northwestern Hawaiian Islands (NWHI)1,100 seals

• Low human presence

• Abundant predators/competition

for prey

• Food limitation

• Poor juvenile survival

• Older age of first reproduction

Threats: MHI

Main Hawaiian Islands (MHI)300 seals

• High human presence - fisheries interactions, intentional killing, disease*

• Fishing reduces competition for prey

• Good nutritional condition

• Primiparity as early as age 4

Contaminants: Below concentrations shown to have effects in other species.

Reproductive Pathogens: Prevalence is 9% or less for:

Brucella, Chlamydia, Coxiella, herpesvirus, Leptospira, Toxoplasma

Specimen Archive:Blood

Swabs

Blubber biopsies

Necropsy tissues

Population Health Monitoring

Infectious Diseases:Leptospira rare

Morbillivirus none

Herpesvirus occasional

Adenovirus rare

Brucella rare

Parasites: Gastrointestinal cestodes, nematodesProtozoa

Necropsy:Trauma

Drowning

Reproductive complications

Hook ingestion

The first live-stranded cases in 2015

• Unique - most HMS w/ toxo found dead

• Both patients died w/in 48 hr of admit

• Taught us about “logging” behavior for early detection

• Illustrated the massive inflammatory response to infection

• NOAA elevated its level of concern about the threat of toxo to species

• Pathology crucial to understanding pathogenesis

Rescuing the first live-stranded seal case of toxoplasmosis, adult female RB24, O’ahu 2015

HMS Deaths from Protozoal Infections

2001-present: 13 known deaths

Case definition for protozoal deaths in HMS:

Gross necropsyHistopathologyImmunohistochemistrySupporting molecular data when avail.Barbieri et al. 2016

Excerpt from Table 1, Barbieri et al. 2016

Deaths: n=13

(red/female, blue/male)

Suspect cases:

n=2 (orange)

Predominantly females

affected

More deaths go

undetected

Population Impacts: Causes of death in the main Hawaiian Islands, 1992-2019

Anthropogenic threats…

• physical injury

• drowning/nets

• Toxoplasmosis

…have the largest impact on the population and are hampering its

growth in this region.

Harting et al. 2020 – Marine Mammal Science

(early view) https://doi.org/10.1111/mms.12742

https://doi.org/10.1111/mms.12742

Molecular analyses & Serology

• Serology (apparently healthy animals sampled opportunistically)• Low seroprevalence in the population (4% or less)

• More likely to be seropositive in MHI than NWHI (Aguirre et al. 2007; HMSRP unpub. data)

• 2018 cases: multiple genotypes and mixed infections (predominantly Type I and III alleles) – suggests multiple oocyst sources and different strains causing lethality than in otters (Type X)

• Molecular analysis across archived stranded seal tissues ongoing and not ready for prime time

Acknowledgements/contributors: Katie Haman, Karen Shapiro, Liz VanWormer, Michael Grigg & lab

Gross lesions

Juveniles/Adults:

▪ Adipose tissue - Firm, mottled yellow-red nodules

▪ Pericardial adipose

▪ Mesentery

▪ Lymph nodes - Enlarged, congested, edematous

▪ Fluid in abdominal cavity

Histologic Lesions

• Mesenteric and epicardial steatitis • Lymphadenitis and splenitis• Pneumonia • Meningoencephalitis• Endometritis• Adrenalitis• Pancreatitis• Myocarditis• Myositis

Most severe and more frequent

Least severe and less frequent

MYRIAD TACHYZOITES!!

Adipose tissue:Inflammation, necrosis, myriad tachyzoites

Meningoencephalitis

Adrenalitis

TACHYZOITES

Fetal Pneumonia

Heart and muscle only minimally affected

Zoite cluster

Endometritis and Necrosis –Pregnant and post parturient females

Juvenile Female – No Inflammation

Adult female that recently aborted

Infections confirmed by Toxoplasma gondii IHC and PCR

Severe Toxoplasma Infections in Species Not Coadapted

South America

Necrotizing lymphadenitis

From: Juan-Salles et al. JZWM 1998

Lymphohistiocytic and necrotizing encephalitis

Australia

Marine Mammals – Toxoplasmosis in Sea Otters

- Incidental infections – cysts- Fatal meningoencephalitis – often Type X

Moderate lymphohistiocytic inflammation –variable #s of organisms

Infection can increase susceptibility to predation

Photos courtesy of Melissa Miller

Photos: M. Miller

Marine Mammals – Toxoplasmosis in Pinnipeds

Myocarditis

California sea lions:- Infections identified

infrequently

- 1975-2009 = 9 cases @ one large rehabilitation hospital

- Myocarditis, encephalitis, few fetal infections

Pacific harbor seals:- Pacific Northwest

- Fatal meningoencephalitis observed more frequently in adults

- Typically co-infection with Sarcocystis neurona

Meningoencephalitis

How are monk seals getting exposed?

Case definition in HMS

Impacts on population

Pathogenesis

Let’s talk a little bit about exposure risk…

Toxoplasmosis cases

RH40M 2010

RT10F 2018+ pup

RK29M 2005sRK60F 2018

R017F 2014

RB24F 2015+ pup

R011F 2007s

RK07M 2010

KA060D03M 2006

RN36F 2015

RTX1F 2010

Toxoplasma cases (& suspects) -2019

Stacie Robinson, PhDForaging Ecologist

HMSRP

RT10F 2018+ pup

RK29M 2005sRK60F 2018

R017F 2014

RB24F 2015+ pup

R011F 2007s

RH40M 2010

RK07M 2010

KA060D03M 2006

RN36F 2015

RTX1F 2010

MHI Cases: Space Use

RN36F2015

RT10F 2018

RK29M2005s

RK60F2018

R017F2014

RB24F2015

Oahu Cases: Space Use +Sediment Outflow

Monk seals exhibit far-reaching space use• Potential for wide area of exposure • Mortality site not necessarily indicative of

exposure site

Monk seals exhibit variable space use• From multiple islands to localized beaches• Variable ability to discern location pre-

mortality

Estimate of space use highly dependent on sightings effort

• Effort varies across time and islands• Potential bias against remote beaches

Conclusions on preliminary spatial risk evaluation

CASE STUDYJanuary 22, 2020: RO28 “Pohaku”

First, a note of immense gratitude:

• The entire NOAA HMSRP and PIRO monk seal team

• Volunteers everywhere

• Ke Kai Ola & The Marine Mammal Center

• Hawaii Marine Animal Response

• Veterinary science collective: Gregg Levine, Cara Field, Katie Colegrove, Meghan Barrett, Deb Fauquier, Karen Shapiro, Pat Conrad, Teri Rowles

• United States Coast Guard

RO28 “Pohaku” Days 1-2

Signalment Adult female, presumed pregnant (due July 2020)

Hx Lethargic behavior for 4 days, rescued 1/22/2020

PE QAR, BCS 3/5, 220 kg, no signs trauma

Imaging No foreign body (hook), uterus WNL, mild enteritis

CBC/Chemistry Mild elevations in TP, globulins, ALT, P and Na

Tx Ceftiofur, famotidine, maropitant, praziquantel

Ddx Infectious/inflammatory: toxoplasmosis vs. gastroenteritis vs. reproductive disease

RO28 “Pohaku”

Protozoal titers T. gondii 1:40,960 (Sarco 1:80, Neo: 1:160)

Tx (SID) Ponazuril, Clindamycin, TMS, Dexamethasone

IV/SC LRS, Vitamin B12 & B complex, cerenia,famotidine, OptiMarine supplement, Vit. E, alpha lipoic acid supplement, herring slurry PO (sedated with diazepam)

RO28 “Pohaku” Days 3-5

Protozoal titers T. gondii 1:40,960 (Sarco 1:80, Neo: 1:160)

Tx Ponazuril, Clindamycin, TMS,Dexamethasone, IV/SC LRS, Vitamin B12 & B complex, cerenia,famotidine, OptiMarine supplement, herring-electrolyte slurry PO (sedated with diazepam)

PE Mentation declined to dull and minimally responsive

Other diagnostics Morbillivirus neg, HMS herpesvirus neg

RO28 “Pohaku” Days 3-5

Protozoal titers T. gondii 1:40,960 (Sarco 1:80, Neo: 1:160)

Tx Ponazuril, Clindamycin, TMS,Dexamethasone, IV/SC LRS, Vitamin B12 & B complex, cerenia,famotidine, OptiMarine supplement, herring-electrolyte slurry PO (sedated with diazepam)

PE Mentation declined to dull and minimally responsive

Other diagnostics Morbillivirus neg, HMS herpesvirus neg

Monk seal RKC1 (“Sole”)

• Carcass found January 26, 2020 • Juvenile male • Found dead on Oahu• Necropsy & histopathology – toxoplasmosis • Serology: T. gondii (1:40,960)

RO28 “Pohaku” Days 5-14

PE Mentation slowly improved, but locomotion on land abnormal; unable to haul out of pool

CBC/Chemistry No significant changes from admit

Tx Tapered off dexamethasone, added carprofen, vitamin E, alpha lipoic acid

Protozoal titers (2wk) T. gondii 1:81,920 (Sarco & Neo: 1:80)

RO28 “Pohaku” Weeks 2-6

Too difficult to handle for oral tubings ~week 3

Showed interest in herring offers in pool ~week 3

Developed unilateral keratopathy ~week 4

Locomotory abilities gradually improved; able to exit pool under own power ~week 4

Transferred to partner facility Ke Kai Ola ~week 4.5

Completed full 6 weeks of treatment for toxoplasmosis

Appetite poor but mentation BAR, locomotion WNL

RO28 “Pohaku”

• Succumbed to congestive heart failure (week 10)

• Post-mortem: Chronic brain damage, relatively low #s of zoites, secondary heart effects

• It wasn’t the end we desired but we can (and will) try again

• Resource intense and not a feasible solution to the threat

• Media, web stories, podcast, plus more to come…

Cerebral necrosis

ConclusionsToxoplasmosis appears to be acutely lethal to most HMS

• Greater impacts to female seals = population level impacts• Massive #s of organisms• Suggests high susceptibility & lack of immune response adaptation• Adipose involvement

• Likely inherent characteristic of T. gondii• Related to genotype? • Could this predispose pregnant seals?

• Are all exposed dying, or are we missing subclinical infections?

First wild HMS with acute toxoplasmosis treated in 2020

Two clusters of cases detected in 2018 and 2020

Exposure risks not immediately apparent given small sample size, diverse diet, varied movement patterns related to rainfall/runoff?

Next steps

Risk model in development

Further examination of genotypes in HMS

Enhanced focus on health assessments, especially adult females, paired with instrumentation

"Logging” seals

Apparently healthy seals

Continued engagement with managers, stakeholders, partners

Management challenges

Sheer number of cats• est. 50,000-300,000 on Oahu alone

Few laws, little enforcement

Characteristics of environment and population

Small but vocal group opposing management• Support measures that lack scientific foundation (e.g., TNR)

NOAA Fisheries is an ocean resource agency but protected species are facing terrestrial threats

Optimism

Social scientists at NOAA PIFSC engaged

Interagency working group • NOAA initiated; meets quarterly

• Messaging campaign on keeping cats indoors

• Legislative actions

• Controlling the source (abandonment)

• Build trust with cat advocates to garner support for stronger measures

Long game: a lap for every cat

MAHALO!