Unexpected Death in Captive Maras

1
Unexpected Death in Captive Maras

• Mexico

2
Background

• 3 captive-born juvenile Maras
• Unexpected death 1999-2000
• Africam Safari (Puebla, Mexico)
• Intralesional protozoal cysts with myriads of
  crescent shaped bradyzoites in the cytoplasm of
  host cells
• Host cells were hypertrophied, occasionally
  multinucleate, surrounded by a thick eosinophilic
  capsule

3
Suspected Protozoal Disease

• Besnoitia sp. (Family Sarcocystidae)
• Indirect life cycle
• Definitive carnivore host
• Intermediate host
• Horses, ruminants, opossums, rodents, rabbits,
  lizards

4
Case Confirmation

• Gross Necropsy Findings
• Histological findings
• Other testing
• Post-mortem urinalysis
• Lung culture
• Electron microscopy of lung

5
Gross Necropsy Findings

• Good body condition (3/3)
• Abundant froth in tracheal lumen (3/3)
• Diffusely reddened and edematous lungs that
  failed to collapse (3/3)
• Multiple, tiny, whitish nodules on serosal and
  cut surfaces of lung (2/3), visceral pericardium,
  diaphragmatic pleura, hepatic and renal capsule
  (1/3)
• Mild or severe bilateral serosanguineous pleural
  effusion (2/3)
• Reddish urine (1/3)
• urinalysis

6
Urinalysis

• Post-mortem
• Marked proteinuria
• Marked hematuria

7
Aerobic Culture of Lung (3)

• Yersinia sp.

8
Histology

• Lung
• Kidney
• Heart
• Skeletal muscle
• Lymph nodes

9
Lung Histopathology

• Severe subacute interstitial pneumonia
• Fibrin exudation into alveolar spaces
• Type II pneumocyte hyperplasia
• Interstitial infiltration of inflammatory cells
  (Mostly macrophages)
• Macrophages, lymphocytes, and syncytia within
  alveoli
• Alveolar edema and hemorrhage
• Lesions were less prominent in Mara 2

10
Kidney Histopathology (3)

• Previously described cysts in the kidney
• Cysts associated with occasional large foci of
  granulomatous interstitial nephritis around lytic
  cysts
• Marked tubular resorbtion of proteins

11
Cardiac Histopathology(3)

• Cysts in myocardium often surrounded by rings of
  lymphocytes and macrophages, few neutrophils and
  occasional multinucleate giant cells

12
Skeletal Muscle Histopathology

• Cysts in skeletal muscle often surrounded by
  rings of lymphocytes and macrophages, few
  neutrophils and occasional multinucleate giant
  cells

13
Lymph Nodes

• Lymphoid depletion (2/3)
• Hyperplasia and histiocytosis with occasional
  syncytia (1/3)

14
EM of Lung

• Cysts consisted of myriads of bradyzoites
  surrounded by a parasitophorous vacuole in the
  host cell cytoplasm
• Bradyzoites were crescent-shaped and had
• a conoid, micronemes, a few rophtries that
  extended well posterior to the nucleus
• Uniform electron dense granular material with
  numerous large granules located posterior to the
  nucleus

15
EM of Lung--cont

• Transverse sections through the apical pole of
  the bradyzoites
• 22 subpellicular microtubules beneath their outer
  membrane
• Host cell
• Hypertrophied and multinucleate
• Projections of cell membrane into the outer cyst
  wall, composed of collagen fibers and electron
  dense granules
• No viral particles were seen

16
Diagnosis

• Pulmonary besnoitasis (disseminated in Mara 3)
• By light microscopy and EM, Besnoitia cysts can
  be differentiated from other tissue cyst forming
  coccidia in the family Sarcocystidae
• Specifically, bradyzoites are surrounded by a
  parasitophorous vacuole within the cytoplasm of a
  hypertrophied and multinucleate fibroblast
  surrounded by a thick capsule composed of
  collagen and electrodense capsules

17
Establishing Endemic Levels

• Retrospective review of all Mara necropsy cases
  at Africam Safari
• 54 Animals, 1996-2003
• 10 more cases that had occasional or low numbers
  of similar cysts in the lungs with minimal or
  moderate inflammation surrounding them
• Severity of these lesions correlated with the
  presence of lytic and mineralized cysts
• One Mara also had cysts in the pancreas and
  peripancreatic adipose tissue

18
Is There An outbreak?

• There was no evidence of Besnotiasis prior to
  1999
• All of the 13 total cases with cysts
• Present in the lungs with concurrent interstitial
  or granulomatous pneumonia

19
Descriptive Epidemiology

• Herd history
• Case description
• Environmental factors

20
Herd History

• Mara colony at park for gt20 years
• 1997 5 animals from Argentina added to group
• 1 male, 4 females
• Reproduction good since introduction (66 births)
• During time of the 3 Mara juvenille deaths
• Herd composed of 37 animals
• 28 births
• 14 deaths (7 adults, 4 juvenilles, 3 neonates)

21
Case Description

• Mara 1
• Female, 2.9kg, 2.5m old (July 1999)
• Mara 2
• Female, 1.7kg, 1.3m old (September 1999)
• Mara 3
• Female, 2.0kg, 1.5m old (December 2000)
• Mara 1 2 born to same dam 4 months apart
• The other Maras present at these times showed no
  clinical signs of disease.

22
Environmental Factors

• Diet
• Water ad libitum
• Oranges, carrots, chopped alfalfa and rabbit
  pellets
• Enclosure
• 120m2
• Fenced with 1.20m high wire mesh and bamboo on
  one side
• Sandy substrate
• Often see squirrels (Spermophilus variegatus),
  Mice (Mus musculus) and various birds inside the
  enclosure.
• Often see opossum (Didelphis virginiana),
  ringtail cat (Bassariscus astutus) and stray cats
  (Felis domesticus) inside the park.

23
1996-2003

• 13 positive animals in an 8 year period
• 54 total deaths
• 24
• 3 animals from current colony of 37
• 8

24
Background of Besnotia

• Species of besnotia have heterogeneous life
  cycles
• Uses predator-prey relationships to ensure
  transmission
• Intermediate host becomes infected by ingesting
  sporulated oocysts in feed or water contaminated
  with feces
• Most besnoitia spp. Have a broad range of
  intermediate hosts
• Cats are definitive hosts of B. darlingi and B.
  wallace
• Role of cats and carnivores in the life cycle of
  other Besnoitia species is unclear

25
Hypothesis

• Wild carnivores in the park?
• Definitive hosts by fecal shedding of oocysts
• Hematophagous insects?
• Transmission by these insects may occur in
  ungulates
• Pre-existing chronic granulomatous inflammation?
• Involved in the development of fatal interstitial
  pneumonia
• Concurrent infection with agent causing
  interstitial pneumonia?
• Undetected by available diagnostic techniques

26
Investigate Hypothesis

• Review literature for similar cases
• Environmental Investigation

27
Literature Review

• No other reports of Besnoitiasis in Maras
• No other reports of Besnoitiasis associated with
  fatal interstitial pneumonia in ANY animal
• Pulmonary Besnoitiasis reported in rabbits with
  little or no surrounding inflammation

28
Environmental Investigation

• Feral cats have been trapped
• Negative for fecal shedding of Besnoitia oocysts
• Sandy substrate of mara exhibit is inviting to
  cats and other carnivores as a latrine area
• Feral predator detection and removal via
  live-trapping has been implemented
• Daily cleaning of feed area

29
Referencias

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  75114-117, 1998
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