INTRODUCTION
Dirofilaria immitis
heartworm of dog
Class: Nematoda Subclass: Secernentea Order: Spirurida Superfamily: Filarioidea Family: Filariidea Scientific name - Dirofilaria immitis Common name Heartworm of dog
Most
Endemic in temperate, subtropics and tropical countries lives mainly in right ventricle and the pulmonary artery
(Quinn, 1997) 1997)
Worm
Other
locationslocations- Posterior vena cava, anterior chamber of the eye, interdigital cysts and other parts of the body
(Levine, 1980)
Mosquito
borne
Intermediate hosts- approx.70 spp. of mosquitoes hosts(Culex, Aedes, Anopheles, Mansonia etc.)
(Arellano et al.,2002). al.,2002).
In
It causes exercise intolerance, pulmonary thromboembolism, hypertension,endarteritis and pneumonitis Zoonotic: causing Human pulmonary dirofilariosis around the globe Transmission takes place when a potential vector, reservoir of infection and favorable conditions coexist
(McCall et al., 2004) al.,
-5-7% from Terai region - 33% from NE region of India - 7-24% from Kerala From wild animals: animals: Captive lions & tigers (Gupta et al 1996) Vulpes bengaliensis in Nandankanan (Rao & Acharya 1993) Leopard in BTR, WB (Samanta et al 2007)
In dogs:
In man: Occular Dirofilariosis Joseph et al. (1976)., George and Kurian(1978)., Sekhar et al. (2000)., Nadgir et al.(2001) and Gautam et al.(2001) al., 2002 al., Subcutaneous infection Badhe and Sane (1989)., Ittyerah and Mallik (2004)., Padmaja et al.(2005) and Sabu et al.(2005) al.(2005) al.(2005)
Reports from globe -15 % from Sydney, Australia -53.4 % Bahamas -55.9 % from Taichung -23.5 % from Argentina -9.3 % from Turkey -5-15 % from Italy -6 % from Serbia -48 % from Croatia ->50 % from Gran Canaria island -4-47 % from USA
DIAGNOSIS
History Clinical signs Demonstration of microfilariae in peripheral blood. Radiography Cardiography Serological diagnosis Molecular diagnosis
Microfilaria Detection
- Direct blood smear (wet,thick,thin) - Concentration technique a) Knotts method b) Filter method Each methodology: - advantages/disadvantages - efficacy is similar
Radiography
Degree of arterial disease Best assessed by dorsoventral projection Tortuisity and pruning of arteries Caudal lobar arteries-not to be exceed 9th rib Cranial lobal arteries- not to be exceed 3rd rib
Pulmonic
Limitations
Not
The
worms often are limited to the peripheral branches of the pulmonary arteries which are beyond the al., echocardiographic field of view. (Mc Call et al., 2004)
Serological tests
Kits available
ASSURE CH Dirocheck Petcheck HTWM PF Snap Canine Heartworm PF UNIUNI-TEC CHW ICT GOLD HW VetRED
Live female worms with >5 months infection can be detected False positive results technical error False negative results low worm burden
(Courtney and Zeng, 2001)
Limitations
False positive results technical error False negative results low worm burden
(Courtney and Zeng, 2001)
MOLECULAR DIAGNOSIS
ITS region generally display sequence variation between species (Guarro et al.,1999; Henry et al., 2000; al.,1999; al.,
and Turenne et al., 1999). al.,
PCR based differential diagnosis of D. immitis and D. reconditum using primer derived from internal transcribed spacer-2 (ITS2) region. spacerSensitivitySensitivity- 1x 10-2 microfilariae of each species of parasite or even with mixed samples could be detected Similarity of DNA sequence between the ITS2 region of two species was as low as 41.1%
(Mar et al.,2002) al.,2002)
PCRPCR-RFLP of ITS1 allowed differentiation down to the species level for D. immitis, D. repens, Wuchereria bancrofti, Brugia malayi and Brugia pahangi
(Nuchprayoon et al.,2005) al.,2005)
1pg of parasite DNA, which allows for the presence of only a single microfilaria in 250l blood sample to be detected
(Nuchprayoon et al.,2005) al.,2005)
Rishnew et al. (2006) developed molecular al. method based on 18S ribosomal DNA PanPan-filarial primers which amplified fragments of different fragment length of both D. immitis and D. reconditum Different microfilariae of six different filarial parasites of dogs could be detected
(Rishnew et al.,2006) al.,2006)
Critical gap
Conventional approaches for Microfilaria Detection Not adequate Potentially misleading (Mar et al.,2002) al.,2002) Requires experienced personnel Morphological alteration of microfilariae (Sawyer and Weinstein, 1963) Over-estimated,if other species are identified wrongly Over(Scoles and Kambhampati, 1995) No use in occult dirofilariosis ( Song et al., 2002) al.,
HeartGard Plus
(Ivermectin/Pyrantal)
Female worms
Hind end of the male worm showing a characteristic spicules and pappillae
Knott method
Knott method
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Sex of dogs
Male
26
7 (26.9%)
26 (50.0%)
16(30.8%)
Female
2 (25.0%)
6 (11.5%)
4 (7.7%)
Total
34
9 (26.5%)
32 (61.5%)
20(38.5%)
For ITS2
94 C for 5 min - initial denaturation 94 C for 1 min - cycle denaturation 60 C for 30 s - annealing temperature 30 cycles 72 C for 45 s - extension 72 C for 10 min - final extension
M 1 2
504 bp 465 bp
Elution of DNA
(PCR purification kit QIAGEN)
Restriction digestion
1 M M 1
520 bp
481 bp
Colony PCR
1 M M 1
504 bp
465 bp
pellet washed with 500 l chilled PBS twice pellet suspended in 400 l lysis buffer 370 C x 1 h proteinase K (@ 5 mg/ml) 560 C x 5 h standard phenol chloroform extraction
(Sambrook et al., 1989) al.,
Conclusion
Overall incidence of 26.5% at necropsy was recorded Worm burden ranged from 1-16 worms per positive dog (Average 5.7) was 1recorded Female : Male worm- 1.6 : 1 wormNo significant variation in the prevalence rate due to sex of dog ITS1 and ITS2 regions of ribosomal gene of D. immitis was characterized ITS1 and ITS2 regions of ribosomal DNA of Mizoram isolate showed length and sequence polymorphisms ITS1 and ITS2 were used for specific diagnosis of D. immitis
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