india s dog population is estimated over 25 million and 80 % of this population are either partially restricted or community ( stray ) , or feral ( unrestricted ) dogs ( 

 menezes 2008 ) . again , 17 % of indian households were reported to own a pet or domesticated dog ( 

 sudarshan et al . 2006 ) . among the disease producing agents in canines ,  helminths  are one of the predominating group of which dirofilaria immitis is established as the most pathogenic filarial nematode in terms of disease pathogenicity . mosquitoes like culex , aedes , anopheles , mansonia etc are incriminated as intermediate host of this parasite ( 

 bemrick and sandholm 1966 , 

 ludlam et al . 1970 , 

 lok 1988 , 

 arellano et al . 2002 ) . in india , prevalence of this parasite in dogs another filarid , dirofilaria repens , a parasite of subcutaneous tissue of dogs and other canids . this parasite has been reported from many parts of the world including india ( 

 megat abd rani et al . 2010 ) . dirofilaria repens accidentally affects humans and several human cases have been reported from india , mostly from south india ( senthilvel and pillai 1999 , sekhar et al . 2001 , 

 sabu et al . 2005 ) and also reported from assam ( 

 nath et al . laboratory diagnosis of dirofilariasis in live animals is always in forefront in terms of simple demonstration and identification of microfilariae , serology and molecular techniques . several commercial elisa based test kits are available to diagnose heartworm in dogs but these kits are not widely used in our country where heartworm test in every dog is not mandatory . dna based techniques provide an alternative approach which is very sensitive and accurate for identification of the filarial parasites ( 

 favia et al . sometimes microfilariae in circulating blood of heartworm infected dogs are not seen and such condition is termed as  occult infection  . the reason for occult infection of d. immitis in dog with no microfilariae in blood is due either to male or female worm infection , single worm infection , presence of immature females during pre - patent stage of development , geriatric infection and ectopic infection . immune mediated allergic pneumonitis sometimes associated with occult infections produces eosinophilia besides pneumonic sign and symptoms . the present communication is based on the reports of epidemiological study of heartworm disease in north eastern states of india . the study was undertaken systematically for a period of one calendar year from august , 2011 to july , 2012 , in dogs from guwahati and aizawl . guwahati , a city of assam is located at the latitude of 26110 n and longitude of 91440 e having annual rainfall of 15002600 mm with an average altitude of 52 mts msl and aizawl , the capital city of mizoram state of north east located at 234327 n and 92432 e having annual rainfall of 24002962 mm with an average altitude of 1132 mts msl . three categories of dogs were selected for the epidemiological study , working dogs of military and paramilitary force , pet dogs and stray dogs . pet dogs of different breeds and paramilitary dogs mostly of labrador and german shepherd breeds brought to the teaching veterinary clinical complexes ( tvcc ) of the college of veterinary science , assam agricultural university , khanapara and the college of veterinary sciences and ah , central agricultural university , selesih , aizawl , mizoram during the study period were taken for the study . the stray dog population consisted of local non - descript street dogs of either sexes captured from different parts of the city for sterilization by local non - governmental organization like peoples for animal ( pfa ) and three categories of dogs like working ( 103 ) , pet ( 266 ) and stray ( 413 ) totaling 782 numbers were examined . dogs under the study were of either sexes or a total of 488 dogs from different localities of guwahati and 294 dogs from aizawl formed the entire base of study during the programme . blood sampling from hospital dogs was done at clinics soon after presenting the animals by the owners for clinical investigation . in case of stray dogs , approximately 5 ml of blood was drawn from the cephalic vein collected in disodium salt of ethylene diamine tetra acetic acid ( na2edta ) vacuum tubes and stored at 4 c until further use . the prevalence study for d. immitis was conducted on the basis of conventional wet blood film method and knott s concentration technique ( kct ) , immunological with a commercially available elisa test kit ( snap 4dx , idexx labs . inc . , westbrook , usa ) and molecular techniques targeting to amplify the its-2 region of filarial worms rdna developed by 

 rishniw et al . isolation of genomic dna from blood was carried out using the dneasy blood and tissue kit ( quiagen kit , catalogue no 51104 ) and the protocols follows as per handbook provided by the manufacturer . the primer utilized was referred to as pan filarial primers , forward : didr f1 5-agt gcg aat tgc aga cgc att gag-3 and reverse : didr r1 5-agc ggg taa tca cga ctg agt tga -3 were utilized to amplify and differentiate d. immitis , d. repens , brugia malayi , b. pahangi , acanthocheilonema ( dipetalonema ) reconditum and a. dracunculoides . to amplify the targeted ribosomal gene of d. immitis the pcr reaction mixture was consisted as 2.5 l taq polymerase buffer ( 10x ) , 01 l dntp ( 10 mm ) , 0.5 l mgcl2 ( 50 mm ) , 0.75 l of each forward and reverse primer ( 60pm ) , 0.5 l taq polymerase , 3.0 l template dna and by making the final volume upto 25.0 l with nfw . the cycling condition used for amplifying the targeted product consisted of a initial denaturing step at 94 c for 2 min and 32 cycles of denaturing ( 30 s at 94 c ) , annealing ( 30 s at 60 c ) and extension ( 30 s at 72 c ) , a final extension ( 7 min at 72 c ) and a soak at 4 c in a technee-5000 thermal cycler ( bibby scientific ) . the confirmation of the amplified products were made by gel electrophoresis of the pcr product in 1.5 % agarose gel stained with ethidium bromide and visualized under gel doc ( dnr bio - imaging system , minilumi ) . since pcr test can be utilized to detect up to one microfilaria per 250 l of tested blood sample , hence , differences between heartworm positive cases in pcr test and antigen detection test ( snap4dx ) was considered as criterion for calculation of number of occult cases . the study was undertaken systematically for a period of one calendar year from august , 2011 to july , 2012 , in dogs from guwahati and aizawl . guwahati , a city of assam is located at the latitude of 26110 n and longitude of 91440 e having annual rainfall of 15002600 mm with an average altitude of 52 mts msl and aizawl , the capital city of mizoram state of north east located at 234327 n and 92432 e having annual rainfall of 24002962 mm with an average altitude of 1132 mts msl . three categories of dogs were selected for the epidemiological study , working dogs of military and paramilitary force , pet dogs and stray dogs . pet dogs of different breeds and paramilitary dogs mostly of labrador and german shepherd breeds brought to the teaching veterinary clinical complexes ( tvcc ) of the college of veterinary science , assam agricultural university , khanapara and the college of veterinary sciences and ah , central agricultural university , selesih , aizawl , mizoram during the study period were taken for the study . the stray dog population consisted of local non - descript street dogs of either sexes captured from different parts of the city for sterilization by local non - governmental organization like peoples for animal ( pfa ) and three categories of dogs like working ( 103 ) , pet ( 266 ) and stray ( 413 ) totaling 782 numbers were examined . dogs under the study were of either sexes or a total of 488 dogs from different localities of guwahati and 294 dogs from aizawl formed the entire base of study during the programme . blood sampling from hospital dogs was done at clinics soon after presenting the animals by the owners for clinical investigation . in case of stray dogs , approximately 5 ml of blood was drawn from the cephalic vein collected in disodium salt of ethylene diamine tetra acetic acid ( na2edta ) vacuum tubes and stored at 4 c until further use . the prevalence study for d. immitis was conducted on the basis of conventional wet blood film method and knott s concentration technique ( kct ) , immunological with a commercially available elisa test kit ( snap 4dx , idexx labs . inc . , westbrook , usa ) and molecular techniques targeting to amplify the its-2 region of filarial worms rdna developed by 

 rishniw et al . isolation of genomic dna from blood was carried out using the dneasy blood and tissue kit ( quiagen kit , catalogue no 51104 ) and the protocols follows as per handbook provided by the manufacturer . the primer utilized was referred to as pan filarial primers , forward : didr f1 5-agt gcg aat tgc aga cgc att gag-3 and reverse : didr r1 5-agc ggg taa tca cga ctg agt tga -3 were utilized to amplify and differentiate d. immitis , d. repens , brugia malayi , b. pahangi , acanthocheilonema ( dipetalonema ) reconditum and a. dracunculoides . to amplify the targeted ribosomal gene of d. immitis the pcr reaction mixture was consisted as 2.5 l taq polymerase buffer ( 10x ) , 01 l dntp ( 10 mm ) , 0.5 l mgcl2 ( 50 mm ) , 0.75 l of each forward and reverse primer ( 60pm ) , 0.5 l taq polymerase , 3.0 l template dna and by making the final volume upto 25.0 l with nfw . the cycling condition used for amplifying the targeted product consisted of a initial denaturing step at 94 c for 2 min and 32 cycles of denaturing ( 30 s at 94 c ) , annealing ( 30 s at 60 c ) and extension ( 30 s at 72 c ) , a final extension ( 7 min at 72 c ) and a soak at 4 c in a technee-5000 thermal cycler ( bibby scientific ) . the confirmation of the amplified products were made by gel electrophoresis of the pcr product in 1.5 % agarose gel stained with ethidium bromide and visualized under gel doc ( dnr bio - imaging system , minilumi ) . since pcr test can be utilized to detect up to one microfilaria per 250 l of tested blood sample , hence , differences between heartworm positive cases in pcr test and antigen detection test ( snap4dx ) was considered as criterion for calculation of number of occult cases . the parasitological investigations were carried out by employing conventional , serological and molecular methods on 3 different categories of dogs like , working dogs maintained by military and paramilitary forces , pet dogs and stray dogs . the main objective of the study was to determine the occult dirofilariasis due to heartworm disease . the working dogs had highest prevalence ( 60% ) followed by pet ( 29.16% ) and stray dogs ( 17.75% ) ( table 1 ) . occult cases of dirofilaria immitis values in column ( 3 ) is obtained from values of column ( 1)(2 ) the present findings of higher occult cases recorded in working and pet dogs in comparison to stray dogs might be due to the fact that , owners of pet and working dogs are very much concerned about the health status of their animals . hence , there is regularity in their health check - up by a professional often necessitated for anthelmitic medication with a preference for an endecticidal drug like ivermectin . on the other hand , stray dogs are seldom taken care of with such type of medications . earlier , 

 borthakur et al . ( 2006 ) recorded 35.80 % dog affected with occult dirofilariasis in mizoram . their study was based on necropsy finding of confirmed heartworm cases and subjecting the blood sample from the same cases for kct . ( 1986 ) could record up to 61.9 percent occult dirofilariasis in infected dogs , who conducted the study with necropsy findings and elisa test in freeport , grand bahamas . similarly , 

 lai et al . ( 2001 ) from central taiwan , 

 alves et al . ( 1999 ) from the city of receife , pernambae , brazil , 

 labarthe et al . ( 1997 ) in the state rio de janerio , brazil , and 

 yildiz et al . ( 2008 ) from kirikkale , turkey could record 25.53 , 57.1 , 34.64 ( n= 127 ) and 27.46 ( n=172 ) percent occult infection in dogs , respectively . further , they opined that abusive use of microfilaricidal agent contributed substantially to high rate of occult dirofilariasis . 2011 ) reported 17.7 % occult infections in southern coasts of the caspian sea based on 200 numbers of dogs surveyed by antigen test and modified knott s test . in a more recent study , conducted in guwahati , 

 bhattacharjee ( 2011 ) reported 737.5 percent occult cases of dirofilariasis . dogs with 50100 mature worms that received little exercise may never show signs of heartworm disease , also , one may not be able to find microfilaria in the blood of such subjects ( nayar 1990 ) . it is generally known that , an infection may be occult due to presence of either one sex of the parasite or due to longer pre - patent period ( 180 days ) leading to amicrofilaremic . the highest percentage of occult heartworm infection was present in working dogs maintained under military or paramilitary forces .
background : the north eastern region in india is endemic for canine heartworm disease but in clinics accurate diagnosis is some times difficult . the aim of the present study was to determine the prevalence of occult infections for heartworm disease in canine in two geographical regions of north eastern india.methods:a total of 782 numbers of three categories of dogs namely , working dogs of military and paramilitary forces , pet dogs and stray dogs were screened for the presence of heartworm infection from august 2011 to july , 2012 in guwahati ( assam ) and aizawl ( mizoram ) . 
 conventional , immunological and molecular techniques were followed for this epidemiological study . 
 the criteria to determine the occult heartworm cases were based on the differences between heartworm positive cases in pcr test and antigen elisa test.results:the findings revealed an overall 22.69 percent occult case . 
 the working dogs had highest prevalence ( 60% ) followed by pet ( 29.16% ) and stray dogs ( 17.75%).conclusion : the highest percentage of occult heartworm infection was present in working dogs maintained under military or paramilitary forces .