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Article Summary
Ruthann West
BIOL 1615, Section 003
Professor Jack Later
Title: Bronchoalveolar Lavage and Pulmonary Histopathology in Harp Seals Infected with
Lungworm
Introduction:
Seals are often found infected with species of Metastrongyloidea, or roundworm.
Otostrongylus Circumlitus and Parafilaroides, types of lungworm, are found in pinnipeds and
can cause pneumonia and other symptoms such as cough, dyspnea, anorexia, emaciation,
dehydration, and depression. There is a correlation between infection intensity and blubber
thickness- in this case, the more infection, the less blubber on the pinniped. The larger of these
two lungworm species is the O. Circumlitus. These stay in the bronchi and bronchioles of the
lungs for the most part, but are sometimes found in the pulmonary arteries and right ventricles of
elephant seals. This species of lungworm is found in all regions of the world, in Harbor seals,
ringed seal, grey seal, northern elephant seal, harp seal, and in one case only, a California sea
lion. The worms are ingested by seals through infected prey. The prey harbor third stage larvae.
These larvae find their way to the bronchi through lymphatic circulation or the pulmonary
arteries. When these reach adulthood, the nematodes (or roundworm) release first stage larvae,
which then travel up to the pharynx and are released through swallowing and digestive action
into the feces. This has been useful for antemortem (before death) study and diagnosis of seals;
The Baermann technique can detect these larvae in feces. This study was done to further view
respiratory effects of infection on young seals, as well as the safety and usefulness of a
bronchoalveolar lavage (BAL) in pinnipeds. The experiment focused on YOY (young of the
Ruthann West
Article Summary
year) seals, as infection is much more prevalent in these (82% prevalence in grey seal/ harbor
seal YOY in Canada, and 81% prevalence in YOY seals from Arctic Canada).
Materials and Methods:
The study used 14 weaned, 12 day old harp seal pups. The were separated into a control
group (seals 1-7) and an experiment group (seals 8-14). They were forced to fast for 10 days and
then fed ad libitum (as desired) shrimp and herring with vitamin and mineral supplements. Third
stage larvae were obtained from American plaice (a type of fish) which had been infected with
first stage larvae earlier.
These larvae were harvested from the fish and introduced to each seal via gastric
intubation with saline, after a 12 hour fast. Control seals received 5 mL saline, and the
experiment seals received 300 larvae with saline.
From day 33 postexposure to day 53, feces were collected from the experiment group
using a soft, sterilized, plastic tube inserted into the rectum. These were tested using the
Baermann technique (feces are placed into a mesh bag and suspended in water at the top of a
filled tube. The larvae are able to move out of the bag and drift to the bottom, where they are
collected). The first BAL was performed at 0 day postexposure (dpe) after anesthesia (2 mg/kg of
aminophylline through injection beforehand, and in some animals, 0.1-0.2 mg/kg Midazolam
hydrochloride. Isoflurane was given using a handmade mask and a Bain nonrebreathing system
to get muscle relaxation). Seals were then intubated with a sterilized 6 mm endotracheal tube. A
sterilized soft urinary catheter was then inserted into the endotracheal tube gently until the
researcher felt some resistance or the length of the catheter was reached. Warm saline was
injected (between 9-17 mL) and withdrawn with a syringe catheter tip attached to the catheter.
Ruthann West
Article Summary
After the lavage was finished, the isoflurane was removed and the endotracheal tube was
extracted after the seals were able to breathe on their own. After complete recovery, the seals
were returned to their tanks. Three more BALs were performed at 20, 34, and 53 dpe. After all
of these were completed, the seals were all euthanized while still under anesthesia with sodium
pentobarbital. Necropsy was performed an each seal. Six samples of lung tissue were taken from
each seal for histopathologic (microscopic) examination (three samples from each lung).
Sample Prep: The BAL fluid from each seal was measured on a scale from 0-5 (0 being clear,
colorless and without mucous). It was poured into EDTA tubes (a type of anticoagulant
preservative) with 3 drops of serum from each seal. Within 2 hours of collection, fluid density
was measured using a refractometer. 2 tubes from each sample were centrifuged x 10 minutes
within 12-36 hours. 1 drop of sediment from each tube was used for 4-6 stained line smears and
searched for leukocytes (a type of white blood cell). Slides were also searched for evidence of
inflammation, mucous, bronchitis, glandular hyperplasia (organ enlargement due to over
reproduction of cells) , alveolar atelectasia (collapse of alveoli, or airsacs of the lungs),
alveolitis (inflammation of alveoli) and other symptoms.
Results:
Infection was confirmed in 3 of the 7 exposed seals. First stage larvae was detected in the
feces of these seals at 42, 38, and 45 dpe respectively. Adult nematodes were found during
necropsy in seals 11 and 12, but none in seal 10. The BAL fluids were generally colorless or very
slightly white, clear or slightly cloudy and with small amounts of mucous. There was no
difference in BAL fluid between the control and experimental groups. However, the density of
the BAL fluid in exposed seals was much higher than the control seals. The leukocyte score
increased in control seals at 34 dpe. The noninflammatory cell count was lower in exposed seals
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Article Summary
at 20 dpe. All of the experimental seals, except seal 9, had at least a 5% increase of eosinophils
(another type of white blood cell), compared to a 0%-1% difference in the control group.
In the lung biopsies taken, presence of bronchitis lesions were shown by mucous
infiltrated with granulocytes and other inflammatory cells. Lesions of obstructive bronchitis were
found in seals 11 and 12. Complete blockage of bronchi by a large amount of mucous containing
white blood cells and hyperplasic pulmonary glands, along with collapsed pulmonary
parenchyma (soft or non-connective tissue) were found in these animals. Alveolitis was found in
all six sections of lung tissue, and was more extensive in the exposed seals than control seals.
This was characterized by the presence of white blood cells in the alveoli.
There was no presence of lungworm in the other organs examined during necropsy.
Enlargement of the tracheobronchial lymph nodes was found in all exposed seals except 9 and
14.
Discussion:
This experiment created a verminous pneumonia in 3/7 harp seals. Because the seals were
so young when captured, it is almost certain that infection was introduced by the experiment.
There were 56 BALs performed in total, without complication or death occurring. This shows
that this is probably a relatively safe procedure for healthy or mostly healthy harp seals. BAL and
histopathology work together well in seals to determine infection and infection intensity, though
one is used antemortem (before death) and one is used postmortem (after death).
The lack of difference in neutrophils and macrophages between control and experimental
groups shows that this species of roundworm probably doesnt affect those cells, or that the
difference was statistically insignificant. The higher number of neutrophils found in both groups
could be attributed to indoor environmental conditions, or indicate that these samples were more
Ruthann West
Article Summary
indicative of the tracheobronchial region than the airways further into the lung. This is
corroborated by the fact that a higher percentage of neutrophils should be expected in the
trachea. The indoor environmental impact on both groups of seals is also shown by leukocytes
found in the alveoli during necropsy.
Results were compared using a statistical approach. The exposed seals higher percentage
of eosinophils was found to be statistically significant, meaning that the infection from O.
Circumlitus is most likely at fault. This means that verminous pneumonia should be considered
in a sick seal with an eosinophil percentage greater than 2% in BAL fluid, but given other causes
of this problem, this pneumonia is not the only possibility.
Seals 9 and 14 had regurgitated the larvae/saline mixture shortly after introduction, which
could acc-ount for their less intense infections. Seal 10 had a positive Baermann test, and a
higher percentage of eosinophils, but no adult nematodes found during the postmortem
examination. Three other seals showed an increase in eosinophils, but had negative Baermann
tests and a lack of adult nematodes. This supports the idea that four seals had a short, low
intensity infection and eliminated the infection by the end of the study on their own. This
suggests that seals are able of limiting their infections in the wild or are naturally resistant to
them.