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1. Dirofilaria infections in animals and
humans
Guidelines for the diagnosis of Dirofilaria infection
in dogs and cats

Claudio Genchi, Med Vet, PhD, EVPC Dipl
Dept of Veterinary Science and Public Health, Università
degli Studi di Milano
Bucharest, June 15th, 2013

2. 1. Laboratory and in-clinic serological and blood
diagnosis in dogs and cats
2. Radiography and echocardiography
3. Dog: staging, prognosis and choice of treatment
4. Practical guidelines for HW diagnosis in dogs and
cats

3. 1. Circulating microfilariae: the intensity of
microfilaraemia does not correlate with adult worm
burden
2. Adult female circulating antigens [Ag]: partially
correlate with adult worm burden
Circulating antibodies [Ab]. Not useful in dogs
DOG

4. … furthemore thoracic xR and in some case
echocardiography are useful for staging the
disease, prognosis and choice of the therapy

5. Fresh blood smears: not suitable, insufficient sensitivity and
specificity; no species differentiation
Concentration methods:
1. Filtration
2. Knott test: much more sensitive [about 2 ml of blood] and specific;
species identification
Acid-Phosphatase stain:
1.D. immitis: 2 activity spots around the anal and the excretory pores
2.D. repens: 1 spot around the anal pore
PCR
Mf examinations must always be performed [in dogs,
mf/aemia is usually long lasting: sensitivity: 50-70%]

6. Knott test
2 ml of whole blood
lysis with 0.2% formalin water
solution
centrifugation
discard the surnatant, stain with
ethylene blue or a drop of eosin

7. Species
Length
µm
Width
µm
Features
Dirofilaria immitis 290-330 5-7 No sheath, cephalic end pointed, tail straight with the end pointed.
APh-S: two activity spots located around the anal and the
excretory pores
D. Repens 300-370 6-8 No sheath, cephalic end obtuse, tail sharp and filiform often
ending like an umbrella handle. APh-S: one spot around the anal
pore
Acanthocheilonema
reconditum
260-283 4 No sheath, cephalic end obtuse with a prominent cephalic hook,
tail button hooked and curved. APh-S: activity throughout the
body
A. Dracunculoides 190-247 4-6.5 Sheath, cephalic end obtuse, caudal end sharp and extended.
APh-S: three spots which include an additional spot in the
medium body
1Microfilariae measured after concentration by the Knott test; when using the Difil® test, lengths are shorter. APh-S: acid phosphatase stain
Morphological features of blood microfilariae from filarial worms of
dogs and cats
ESCCAP GL5, VBD

8. D. immitis/D. repens overlap in many regions and
emphasizes the need of a correct differential diagnosis.
Length
µm
Cephalic
end
Caudal
end
D. repens
D. immitis
350-385
A-Ph
290-330
A-Ph
tapered
2 spots
rounded
1 spot
hook-shaped
pointed
Diagnosis

9. CAT: microfilaemia
Often unisexual infection or only pre-adult worms
[sensitivity < 2%]
When present, microfilaemia is short lasting
A negative Knott test is not sufficient to rule out the
infection in cats

10. Serological and whole blood test for heartworm diagnosis
Antigens:
Circulating Ag from adult female worms:
In dogs positive after 6-7 months from infection [after 7-8
mths in cats]
Most test kits very specific and sensitive in case of >2
adult female worms
Commercially available both in-clinic and laboratory test
kits

11. To note that the increased sensitivity of some test kits
has probably decreased their specificity.
Recently false positive results have been observed in
dogs experimentally infected with Angiostrongylus
vasorum.
In case, serological results for D. immitis should be
confirmed or excluded by additional diagnostic tests
(Knott’s test for D. immitis microfilariae, serology or
Baerman for L1 of A. vasorum in faeces) or Rx imaging
frequently delivering pathognomonic findings for heart
dirofilariosis or angiostrongylosis.

12. A. vasorum:
dense alveolar
patterns
D. immitis:
increase
vascular
patterns

13. Serological and whole blood test for heartworm diagnosis
Antibodies [cats]:
Circulating Ab from worms [L4-adult parasites-death
parasites]; positive reactions from 2 months after
infection and after several months [years] after the death
of worms
Not to be used in dogs
In cats more suitable for assessing the infection risk
than to confirm a clinical suspicion
Positive results are not sufficient to role out a diagnosis
of feline HW infection; it must be confirmed by an Ag test
examination or other diagnostic approach

14. ELISAs for lab procedure
[OD value] and in-clinic
Immunomigration, in-
clinic: easy and very
rapid [5-10 min]
whole blood, serum, plasma

15. Occult infections
• Male worms or female worms only:
very unusual in dogs/quite frequent in cats
• Iatrogenic origin: macrocyclic lactones at
high dosage
• Immunological origin: old/very old female
worms no more able to produce Mfs

16. Microfilarie Tick smear: drop of venous blood
on a microscopic slide covered
with a coverslip
Modified Knott test [2 ml of blood]
Filter test
Mf immunohistochemical staining
PCR on mf and adult worms
Not sensitive, not specific
Easy, sensitive, quite specific
Easy, sensitive, quite specific
Very specific and sensitive
Very specific and sensitive
Adult worms Morphology Not easy particularly in case
of female worms
Serology Ag/Ab ELISA
WSP Ab ELISA
Commercial test available
Commercial test not available
Histology Hematoxylin-eosin [morphology]
WSP/immunohistochemical
staining
Not easy
Very specific and sensitive
Dirofilaria immitis

17. Microfilarie Tick smear: drop of venous blood
on a microscopic slide covered
with a coverslip
Modified Knott test [2 ml of blood]
Filter test
Mf histochemical staining
PCR on mf and adult worms
Not sensitive, not specific
Easy, sensitive, quite specific
Easy, sensitive, quite specific
Very specific and sensitive
Very specific and sensitive
Adult worms Morphology Not easy particularly in case
of female worms
Serology Ag/Ab ELISA
WSP Ab ELISA
Commercial test not available
Commercial test not available
Histology Hematoxylin-eosin [morphology]
WSP/immunohistochemical
staining
Not easy, very thin cuticula
indentation
Very specific and sensitive
Dirofilaria repens

18. whole blood smear
one drop of venous blood on a microscopic slide
covered with a coverslip
Advantages Disadvantages
Rapid and inexpensive Very low sensitivity, frequent false
negative, no species diagnosis (it is not
possible to differentiate microfilarie)
Not useful in cats
To note that intensity of microfilaremia is not correlated to the
adult worm burden: in general, high microfilaremic dogs harbour
few worms.

19. Modified Knott test
Advantage Disadvantages
Sensitive in dogs and specific:
microfilarie belonging to different
species can be differentiate
Time consuming, need of a
centrifuge and a skill operator
with good knowledge of Mf
morphology
Specific but of low sensitivity in
cats

20. Advantages Disadvantages
Rapid and sensitive in
dogs
No need for a centrifuge
apparatus
Expensive (tests are sold as kit, Difil
Test Evsco); difficulties in distinguish
morphology; the lysate solution shrinks
the Mf and new measurement
standards are required to differentiate
species
Low sensitivity in cats
Filter tests

21. Histology and histochemistry
Advantages Disadvantages
HISTO: very specific
HISTOCHM: very specific and
sensitive, suitable in case of
“bad” specimens
Costly, time consuming, need for a
skilled laboratory technician

22. Histology: adult worm
longitudinal
ridges
Courteously Laura H. Kramer,
University of Parma
D. repens
D. immitis

23. WSP/immunohistochemical staining

24. Advantages Disadvantages
Very specific and sensitive for
HW diagnosis [when positive, the
test is the definitive prove of
heartworm infection in dogs and
cats]
Able to diagnose an actual
infection [7-8 month-post
infection/7-8 months after the
death of worms]
Costly, not available for other
filarial infections
Does not work in case of
infections caused by male worms
only
Adult female HW
circulating antigen

25. Advantages Disadvantages
Very sensitive
Able to detect the cat exposure
to heartworm infection
Suitable to asses the infection
risk in cats and for
epidemiological survey
Costly
Not fully specific
Difficult to be interpreted
Antibody test

26. Advantages Disadvantages
Very sensitive, specific and
accurate
Able to discriminate all the filarial
species
Costly, time consuming
Need for specialized laboratory
and skilled technicians
PCR

27. PCR
Species-specific PCR
amplifications of Dirofilaria
immitis and D. repens 12S
rDNA and of
Acanthocheilonema
reconditum coxI. a) PCR
amplification using D.
immitis 12S rDNA specific
primers on D. immitis DNA
(lanes 2-4), D. repens (lanes
5-6), A. reconditum (lanes 7-
8) and mixed DNAs (lanes
9-10);
MW
1 2 3 4 5 6
D. immitis D. repens A. recon.
7 8 9 10
Mix 3 DNANeg
a)
c)
MW
1 2 3 4 5 6
A. recon. D. immitis D. repens
7 8 9 10
Mix 3 DNANeg
b) MW
1 2 3 4 5 6
D. repens D. immitis A. recon.
7 8 9 10
Mix 3 DNANeg

28. Differentiation of Dirofilaria immitis and Dirofilaria repens in
canine peripheral blood by real time PCR coupled to High
Resolution Melting Analysis

29. Evaluation of thomboembolic risk
• Lung arterial vessel
conditions
• Worm burden
xR

30. PROGNOSIS
Risk of developing post-adulticide treatment thromboembolism
• Low risk of thromboembolic
complications (low worm
burden and no parenchyma and/
or pulmonary vascular lesions)
Dogs included in this group
must satisfy all this conditions
• No symptoms
• Normal thoracic radiographs
• Low level of circulating antigens
or a negative antigen test with
circulating microfilariae
• No worms visualized by
echocardiography
• No concurrent diseases
• Permission of exercise
restriction
• High risk of thromboembolic
complications
In this group should be
included all the dog that do
not satisfy one or more of
these conditions
• Symptoms related to the disease
(coughing, lipotimias, swelling of
the abdomen)
• Abnormal thoracic radiographs
• High level of circulating antigens
• Worms visualized by
echocardiography
• Concurrent diseases
• No permission of exercise
restriction

31. PROGNOSIS
Risk of developing post-adulticide treatment
thromboembolism
Other factors to be considered:
Age: low risk < 4 year-old; risk > 4 year-old
Residency: low vs high prevalence in dog population

32. DOG:thoracicradiography

33. Echocardiography
ECH detects echogenic walls of the immature or
mature heartworm residing in the lumen of the
pulmonary arterial tree, if within the visual window of
the ultrasound.
The adult parasite is echogenic, producing images of
two, short parallel lines. The pulmonary arteries, right
ventricle or rarely the right atria must be examined
carefully because infections with one or only a few
parasites could be overlooked.

34. DOG:echocardiography

35. Echo Doppler
Tricuspid overflowing showing
atrium-ventricular high rate
pressure
Systolic overflowing trough tricuspid
valve and pulmonary diastolic
overflowing

36. Cat: thoracic radiography

38. Feline HW
Aelurostrongylus abstrusus

39. Aelurostrongylus abstrusus: nematode Metastrongiloidea,
parasite of terminal bronchioles and alveolar ducts of cats
Diagnosis: larvae in faeces throughout Berman apparatus

40. Echocardiography in cats is
high specific and sensitive!
Because the length of worms in
cats is the same than in dogs [M:
12-18 cm; F: 25-30 cm] it is quite
unlikely that worms could not be
visualized by echo.
In red the areas possible to be explored
by echo

41. Cat: echocardiography

42. Mf Knott Ag test Interpretation Comment
Positive Positive Definitive diagnosis of
HW
ThRx can help to manage the disease
Clinical signs and the results of
semiquantitative ELISA tests can help in
discriminating between low and high risk of
thromboembolism
Positive Negative Definitive diagnosis of
HW: very low HW burden
if D. immitis Mf are
present
Filarial infection caused
by other species than D.
immitis
Normal ThRx patterns
Low/very low risk of thromboembolic
complications
Histochemical stain or PCR can be used to
differentiate Mf
Negative Positive Definitive diagnosis of
HW occult infection
Dogs were previously treated incorrectly with
preventive drugs or with macrocyclic lactone
injectable formulations
ThRx and ECHO can help to manage the
disease
Clinical signs and the results of
semiquantitative ELISA tests can help in
discriminating between low and high risk of
thromboembolism
DOG

43. Mf Knott Ag test Ab test Interpretation Comment
Positive Positive Positive Definitive diagnosis of HW ThR and ECHO can help
to manage the disease
Positive Negative Positive Definitive diagnosis of patent
HW if D. immitis if Mf are
present
ThR and ECHO can help
to manage the disease
Positive Negative Negative Filarial infection due to other
species than D. immitis
Histochemical stain or
PCR for differentiate Mf
and give specific
diagnosis
Negative Positive Positive Definitive diagnosis of HW ThR and ECHO can help
to manage the disease
Negative Negative Positive Low adult female worm burden
Immature worms
Aborted infection
Immune response to previous
patent infection, but warms are
already died
ThR and ECHO are
useful to confirm the
suspicion of HW infection
Re-testing after 4-8
months can help to
confirm the suspicion
Cat