The occurrence of Dirofilaria immitis in dogs in South Australia

Heart, lung and samples of blood from 230 dogs were examined for infections of filarial parasites. Dirofilaria immitis worms and microfilariae were detected in one dog. Blood samples from a further 1428 dogs were examined for microfilariae and 22 were found to be infected. Eighteen dogs were infected with D immitis microfilariae and four with Dipetolonema reconditum microfilariae. The histories were available for 18 of the dogs infected with heartworm and only seven dogs had not travelled outside South Australia. It was concluded that heartworm infection was endemic in South Australia but the apparent prevalence was only about 1%.     

Macrocyclic lactones and Dirofilaria immitis microfilariae

Although there has been extensive veterinary focus on both the effectiveness of macrocyclic lactones for heartworm prevention in dogs and their adulticidal effects, little attention has been directed to their effects on heartworm microfilariae. With routine use of macrocyclic lactones, in some cases knowingly, in dogs with existing heartworm infections, veterinarians should recognize the benefits, and possible complications, arising from this behavior. Macrocyclic lactones remain our only class of heartworm prevention available, and preserving their effectiveness is critical. Drugs in this class share common traits: there are currently no Food and Drug Administration-approved microfilaricides in the US marketplace, but because all macrocyclic lactones have microfilaricidal properties (to varying degrees), they are widely used by veterinarians for this purpose. Originally formulated to be used in dogs without patent heartworm infections, all have been demonstrated as safe to use at label doses, and higher, in microfilaremic dogs. All of the product labels indicate that dogs should be tested for heartworm infection before starting preventive therapy. Although microfilaricidal, microfilariae reduction may take many months to occur, and some dogs may never clear. The effects of macrocyclic lactones on the numbers of circulating microfilariae may be due to several different underlying causes (i.e., direct effect on the nervous system, affecting stages found in the uterus of the female worms), but the details of all mechanisms by which microfilariae are killed and/or cleared in dogs treated with macrocyclic lactones have not yet been fully elucidated. Some 10% to 20% of heartworm-infected dogs that begin monthly heartworm preventive treatment without adulticide therapy will have the continued and persistent presence of circulating microfilariae, and the concern is that this may be selecting for resistance to these molecules. The veterinary literature now includes evidence of increased genotypic homozygosity in specific dogs in one area of the country for a marker gene associated with macrocyclic lactone resistance in nematodes of ruminants. This article will review the biology of microfilariae, as well as the evolution of diagnostic testing for heartworm infection. The effects of macrocyclic lactones on microfilaria behavior and survival will be discussed, as well as the use and effects of macrocyclic lactones in microfilaremic dogs, with or without adulticide treatment. The effect of doxycycline on heartworm microfilariae, optimal testing methodologies, and verification of effective clearance of microfilariae after adulticide treatment and microfilaricidal therapy so that dogs do not remain a potential source of infection for other dogs are all covered.     

Epidemiological aspects of filariosis in dogs on the coast of Paraná state, Brazil: with emphasis on Dirofilaria immitis

The present study determined the prevalence and geographical distribution of Dirofilaria immitis and other filariae, from dogs in littoral areas of Paraná state, in Brazil. This survey spanned eight months, between 1998 and 1999, and was also designed to compare the efficacy of different tests for diagnosis of heartworm infection in that area. Blood samples were collected from 256 native-owned dogs distributed along the Paraná coastal area. Five diagnostic procedures were used: direct smear examination, the Knott's modified test, filtration assay, and two heartworm antigen detection kits. A follow-up imaging exam was performed to support the heartworm diagnosis. The imaging diagnosis included radiographic and ultrasonographic exams of six dogs that had positive results for the heartworm antigen detection kits, but showed different microfilarial burdens. The presence and severity of radiographic and ultrasonographic signs were compared with the results obtained in microfilariae detection and antigen tests. Diagnostic parasitology results indicated that 31.25% of the dogs were microfilaremic. Three different microfilariae were recovered: D. immitis, Dipetalonema reconditum, and the third (mf3) was not identified. D. reconditum was the species with the highest prevalence: 22.6%. In general, D. immitis prevalence was 5.47% (28.57% occult infections), but it varied along the coast and the range was from 0 to 20%. No correlation could be established between the overall scores for microfilarial counts (small or large numbers) and the severity of radiographic results or the likelihood of detecting filariae in the pulmonary artery using echocardiography. The finding of a different type of microfilaria (mf) suggested the existence of a third species in Paraná state, whose prevalence was 4.68%. These results show that to obtain a reliable diagnosis of heartworm infection, antigen detection kits are indicated. Knott's test or filtration should be performed to confirm microfilaremia and not for diagnosis of heartworm infection. Imaging tests support parasitology exams and add more about severity of infection. The northern areas, specially Guaraque?aba and Ilha das Pe?as, presented the highest number of heartworm-infected dogs.     

Epidemiology of canine heartworm in its southern distribution limit in South America: Risk factors, inter-annual trend and spatial patterns

This study was aimed at understanding some aspects of the canine heartworm epidemiology in the southern distribution limit of the parasite in South America. With this objective, 19,298 blood samples of owned dogs from 65 localities of 13 municipalities of Buenos Aires Province were tested for Dirofilaria immitis circulating microfilariae and/or female antigens. The overall heartworm prevalence was 1.63% by microhematocrit tube technique (n=19,136), 3.65% by modified Knott (n=713), and 14.41% by antigen test kit (n=118). Microfilaremic dogs showed a median of 1933 microfilariae per millilitre (q1=375, q3=5625, n=100). Male dogs belonging to breeds of short hair and large size recorded significantly higher prevalences than the other categories. Also, the prevalence increased significantly with the age and only dogs younger than 12 months were not found infected. A clear decreasing trend of the annual prevalence was observed during the whole study period, from 3.91% in 2001 to 1.17% in 2006. D. immitis-infected dogs were detected in 32 localities of 9 municipalities (prevalence range: 0.2-6.7%). Generalized linear models were used to assess associations between heartworm prevalence and environmental variables. The resulting significant models were univariate and included variables related with soil cover and human population density. The best model predicted maximum heartworm prevalences around middle values of bare soil cover, and lower at high and low covers. According to our analyses, canine heartworm infection in urban temperate Argentina could be described as relatively low, endemic, and spatially heterogeneous. Host and environmental factors affecting heartworm transmission at local level were identified and discussed.     

Efficacy of combined chemotherapy against gastrointestinal nematodes and Fasciola hepatica in cattle

Direct blood smear examination (using 0.05 ml of whole blood) detected 168 (80.9%) of 204 microfilaremic canine blood samples as determined by the modified Knott test for microfilariae (mff) of Dirofilaria immitis (using 1 ml of whole blood). Direct smear examination detected all of 134 microfilaremias greater than 50 mff ml(-1), but only 31 of 70 (44.3%) microfilaremias having less than 50 mff ml(-1). In a separate retrospective query of a database of 963 dogs with necropsy-confirmed heartworm infections, 834 (86.6%) were positive by the DiroCHEK heartworm antigen test, and 504 (52.3%) were microfilaremic by the modified Knott test. Only 2 (0.4%) of the microfilaremic dogs were DiroCHEK negative and another 18 (3.6%) were very weak positives. Although these microfilaremic dogs were not tested by direct smear, only one of the two DiroCHEK-negative and six of 18 weakly DiroCHEK-positive dogs had microfilaremias so low that a direct smear may have given a false negative result. Significant adverse reactions to either diethylcarbamazine or the macrolide endectocides have not been reported for microfilaremias less than 500 mff ml(-1), thus substitution of the direct smear for a concentration test for mff, such as the modified Knott test or membrane filtration, does not appear to increase the risk of an unexpected adverse reaction to heartworm prophylactic drugs. Such a substitution results in only a very slight decrease (on the order of 0.1%) in the overall sensitivity of heartworm screening, provided a test for mff is run concurrently with an antigen test. If a test for mff is the only screening test used, then substitution of a direct smear for a concentration test may decrease the sensitivity of heartworm screening by nearly 20%, depending on the prevalence of low level microfilaremias in the population of dogs tested.     

Discrimination between six species of canine microfilariae by a single polymerase chain reaction

Canine dirofilariasis caused by Dirofilaria immitis is usually diagnosed by specific antigen testing and/or identification of microfilariae. However, D. immitis and at least six other filariae can produce canine microfilaremias with negative heartworm antigen tests. Discriminating these can be of clinical importance. To resolve discordant diagnoses by two diagnostic laboratories in an antigen-negative, microfilaremic dog recently imported into the US from Europe we developed a simple molecular method of identifying different microfilariae, and subsequently validated our method against six different filariae known to infect dogs by amplifying ribosomal DNA spacer sequences by polymerase chain reaction using common and species-specific primers, and sequencing the products to confirm the genotype of the filariae. We identified the filaria in this dog as D. repens. This is the first case of D. repens infection in the United States. Additionally, we examined microfilariae from five additional antigen-negative, microfilaremic dogs and successfully identified the infecting parasite in each case. Our diagnoses differed from the initial morphological diagnosis in three of these cases, demonstrating the inaccuracy of morphological diagnosis. In each case, microfilariae identified morphologically as A. reconditum were identified as D. immitis by molecular methods. Finally, we demonstrated that our PCR method should amplify DNA from at least two additional filariae (Onchocerca and Mansonella), suggesting that this method may be suitable for genotyping all members of the family Onchocercidae.     

Dirofilaria immitis prevalence in a canine population in the Samana Peninsula (Dominican Republic) - June 2001

Dirofilaria immitis, the etiological agent for heartworm disease, is a zoonotic, vector-borne parasite. The dog is the most common natural host. Heartworm disease is present in all US states, as well as in South and Central America, Asia, Europe, Africa and Australia [. Prevalence of feline heartworm disease - a global review. In: Proceedings of the Heartworm Symposium'95, Auburn, Alabama, USA, 31 March-2 April, 1995. American Heartworm Society, Batavia, USA, pp. 79-86]. According to Menda [Menda, J.A., 1989. Transplacental migration of Dirofilaria immitus microfilariae. Companion Anim. Pract. 19 (6-7), 18-20], 18% of the canine population in the city of Santo Domingo tested positive for heartworm using the Filar Assay Kit. In the year 2000, clinical veterinarians from the Samana Peninsula in the Dominican Republic stated that the incidence of heartworm was minimal (Goodman, personal communication). In this small epidemiological study performed during the summer of 2001, 104 dogs from the Samana Peninsula were tested for heartworm antigen with the WITNESS HW Test and a wet mount evaluation for microfilaria under light microscopy. It was found that 18.2% of the canine population tested positive according to either one or both of the diagnostic methods. It would be advisable to apply prevention measures to control Dirofilaria immitis infection in the canine population of the Samana Peninsula in the Dominican Republic.     

Evaluation of a new in-clinic method for the detection of canine heartworm antigen

Canine heartworm is endemic in many parts of the world, and veterinarians rely on rapid in-clinic antigen tests to screen for this infection. Recently, an in-clinic, instrument-based rotor employing a colloidal gold agglutination immunoassay was launched in the marketplace (VetScan VS2(?) Canine Heartworm (HW) Antigen Test Kit; Abaxis, Inc.). Because of the widespread use of heartworm prevention and possible false negative test results in dogs with low heartworm burdens, the performance of the VetScan VS2(?) HW test and a commercially available in-clinic, membrane-based ELISA test (SNAP(?) Heartworm RT Test; IDEXX Laboratories) was compared using samples from dogs with low heartworm burdens and/or low levels of circulating antigen. Ninety serum samples were evaluated using the two methods. Testing was performed according to the manufacturer's product insert by personnel blinded to sample status. The samples were derived from two populations: dogs with necropsy-confirmed heartworm status (40 with 1-4 female worms, 30 with no worms), and field dogs (20) confirmed positive for antigen by microtiter plate ELISA (PetChek(?) Heartworm PF Antigen Test; IDEXX Laboratories). All 40 dogs with heartworms on necropsy were also confirmed to have circulating antigen by the PetChek HW ELISA. In necropsy-negative dogs (n=30), neither the VetScan VS2 HW nor SNAP HW tests detected heartworm antigen. Of the samples testing positive for antigen by PetChek HW (n=60), the VetScan VS2 HW and SNAP HW tests detected antigen in 15 and 56 samples, respectively. Percent agreement (plus 95% confidence interval) for each test relative to the PetChek HW qualitative result was 50% (40-60%) for VetScan VS2 HW and 96% (89-98%) for SNAP HW. Relative to the presence or absence of female worms at necropsy, agreement was 61% (50-72%) for VetScan VS2 HW and 99% (92-99.6%) for SNAP HW tests. It is clinically important that dogs with low heartworm burdens and/or low levels of circulating heartworm antigen be correctly identified by veterinarians in order to ensure prompt treatment, and the VetScan(?) VS2 HW test does not appear to be as accurate as the SNAP HW or PetChek HW tests when performed on this subset of patients.     

Prevalence and epidemiological aspects of Dirofilaria immitis in dogs from Kayseri Province, Turkey

This study was conducted to determine the prevalence of Dirofilaria immitis infection and to investigate the risk factors related to heartworm disease in dogs from Kayseri, Turkey. Blood samples were collected from 280 dogs from May 2005 to March 2006 and were examined by membrane filtration-acid phosphatase histochemical staining and antigen Elisa techniques to detect circulating microfilariae and antigens of D. immitis, respectively. Of the total of 280 dogs, 27 were positive for D. immitis with a prevalence value of 9.6%. In addition 29.6% of positive dogs determined to have occult D. immitis infections. D. immitis was the only canine filarial parasite present in the study area. The mean number of microfilariae in infected dogs was 4730+/-5479 per ml of blood. The highest heartworm prevalence were observed in 7-10 age group (28.6%) followed by 4-6 (17.1%) and 0.5-3 (4.8%) age groups. The differences between 0.5-3 and other age groups were found significant, whereas no statistically significant difference was observed between 4-6 and 7-10 age groups. The infection was more prevalent in males, larger breeds and the dogs not on prophylaxis. No statistically significant difference was observed between stray and owned dogs. Our results suggest that heartworm treatment and prophylaxis should be considered in Kayseri Province.     

Efficacy of selamectin in the prevention of adult heartworm (Dirofilaria immitis) infection in dogs in northern Italy

The efficacy of a novel avermectin, selamectin, was evaluated for the prevention of heartworm disease (adult Dirofilaria immitis infection) in 120 dogs (aged 9 months to 13 years at enrolment) presented as veterinary patients. The study was conducted at five veterinary practices in a heartworm hyperendemic region of northern Italy. Dogs were allocated randomly in a 2:1 ratio to treatment with either selamectin or ivermectin. Treatments were administered at monthly intervals for 6 months during the heartworm transmission season (May-November). Selamectin was applied topically in a single spot to the skin on each animal's back at the base of the neck in front of the scapulae as a unit dose that provided at least the minimum recommended dosage of 6mgkg(-1) (range, 6-12mgkg(-1)). Ivermectin (6microgkg(-1) of body weight) was administered orally at monthly intervals, in accordance with the manufacturer's product label recommendations. Study day 0 was defined individually for each dog as the day of first treatment administration. Efficacy was assessed on the basis of the absence of D. immitis microfilariae and adult heartworm (D. immitis) antigen in tests conducted on days 180 and 300. There were no adverse clinical signs arising due to treatment with selamectin and no drug-related mortalities. The prevention rate for D. immitis microfilariae and adult heartworm antigen was 100% for both selamectin and ivermectin. Thus, selamectin administered as a unit dose, providing at least the recommended minimum dosage of 6mgkg(-1), at monthly intervals during the heartworm transmission season was safe and 100% effective in the prevention of heartworm disease in dogs presented as veterinary patients.     

Are antigen test kits efficient for detecting heartworm-infected dogs at the southern distribution limit of the parasite in South America? Preliminary results

The aim of this study was to evaluate the performance of commercial heartworm antigen tests in dogs harbouring Dirofilaria immitis microfilariae near its distribution limit in South America. A total of 4934 blood samples of adult dogs from Southern Greater Buenos Aires were examined to detect circulating microfilariae in the buffy coat interface between December 2005 and April 2006. Microfilariae were detected in 88 (1.8%) blood samples and all the microfilariae observed were identified as D. immitis by acid phosphatase stain technique. In a first trial, 69 (78.4%) out of the 88 serum were positive by Speed((R)) Diro. Then, a new test was performed over 25 microfilariae-positive serum samples randomly selected among the 88 previously tested samples and using simultaneously Speed((R)) Diro, Witness((R)) Dirofilaria and Snap((R)) 3dx. This second trial showed identical results for the three different tests, in which 19 (76%) samples were positive. Therefore, more than 20% of microfilaremic dogs were antigen negative. The main hypothesis that could explain our finding is a low worm burden in the study area. According to our preliminary results, it is highly recommendable the complementary use of antigen tests and other procedures to obtain an accurate diagnostic near the distribution limit of the parasite.     

Prevalence of antibodies to tickborne encephalitis and West Nile flaviviruses and the clinical signs of tickborne encephalitis in dogs in the Czech Republic

Blood sera from 151 dogs from areas of the Czech Republic endemic for human tickborne encephalitis (TBE) were examined for the presence of antibodies to TBE and West Nile (WN) flaviviruses by the haemagglutination-inhibition test Antibodies to TBE virus at titres equal to or exceeding 40 were found in five dogs. Antibodies to WN virus were detected in only one dog that also had a high antibody titre to TBE, suggesting this was a cross-reaction between the two closely related viruses. Three of the dogs (all rottweilers) with a TBE titre of 320 had clinical signs of meningoencephalitis or encephalitis. They all survived after treatment for the clinical signs. It was proved by seroconversion that the disease was caused by the TBE virus in one of these three dogs, and it seems very likely that the virus was responsible for the disease in the other two.     

Preliminary findings on the efficacy of selamectin in the treatment of dogs naturally infected with Dirofilaria repens

Subcutaneous dirofilariosis caused by Dirofilaria repens is common in dogs and it is an emerging helminthozoonosis in Europe, Asia, Africa and also in Hungary. Macrocyclic lactones are used for preventing the infection; however, their activity against the microfilariae and mature stages of this species is questionable. Selamectin is widely used for the prophylaxis of heartworm (D. immitis) infection. The objective of the present study was to test the microfilaricidal efficacy of the topical formulation of selamectin in dogs naturally infected with D. repens . A total of 78 Beagle dogs were examined for the presence of circulating microfilariae by Knott's test. Twenty-three of the microfilaraemic dogs were divided into four groups and included in the trial. The dogs received monthly or biweekly selamectin treatment and were subjected to monthly blood testing for a period of 252 or 336 days. At the end of the study, 65% of the dogs were not microfilaraemic and the rest had low number of microfilariae in their blood. These results indicate that chronic spot-on selamectin treatment may be a useful tool also in the control of canine subcutaneous dirofilariosis.     

Immune response to and tissue localization of the Wolbachia surface protein (WSP) in dogs with natural heartworm (Dirofilaria immitis) infection

Human and animal parasitic filarial nematodes, including the agent of canine and feline heartworm disease Dirofilaria immitis, harbour intracellular bacteria of the genus Wolbachia (Rickettsiaies). It is thought that these bacteria play an important role in the pathogenesis and immune response to filarial infection. Immunoglobulin G (total IgG, IgG1, IgG2) production against and immunohistochemical staining of tissues for the Wolbachia surface protein (WSP) from dogs with natural heartworm infection were evaluated. All infected dogs had significant total anti-WSP IgG levels compared to healthy controls. Interestingly, WSP was recognized by the IgG2 subclass in both microfilariemic dogs and in dogs with no circulating microfilariae (occult infection). However, microfilariemic dogs also produced gG1 antibodies. Positive staining for WSP was observed in lungs, liver and kidneys, in particular in glomerular capillaries of naturally infected dogs who had died from heartworm disease. Our results show for the first time that Wolbachia is recognized specifically by D. immitis--infected dogs and that the bacteria is released into host tissue. Furthermore, microfilariemic status appears to effect immune responses to this endosymbiont.     

Efficacy of avermectin B1a against microfilariae of Dirofilaria immitis

Avermectin B1a given at a dose level of 0.05 or 0.1 mg/kg of body weight caused rapid removal of Dirofilaria immitis microfilariae from the blood of dogs with heartworm infections. If the adult worms were also killed with an arsenical (melarsoprol), the removal of microfilariae was permanent.     

Heartworm in dogs in Canada in 1986

In late December 1986, 1224 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1986; 46% of them responded. Veterinarians reported that 150,989 dogs were blood-tested for microfilariae and 869 dogs were found with heartworm. Another 65 dogs were amicrofilaremic but diagnosed with heartworm disease and one was found with heartworm at necropsy to give the total number diagnosed in 1986 as 935 (0.62%).

Heartworm was reported from Manitoba, New Brunswick, Ontario and Quebec, but most (810) of the cases were from Ontario. South-western Ontario continued to be the primary focus of the infection in Canada. There were 103 cases reported from Quebec, mostly from and around Montreal, and 21 cases from Manitoba, from Winnipeg and surrounding areas. Heartworm was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 33% of the cases were observed with clinical signs of heartworm disease and 81% had a history of not having left Canada.

     

Keratitis due to microfilariae in dogs: a newly recognized disease

Parasitic agents have been associated with keratitis, but a diagnosis of parasitic keratitis has not been commonly made in domestic animals. The purpose of this study was to describe the clinical and histopathological findings in seven dogs with chronic keratitis caused by microfilariae diagnosed in Brazil. All dogs presented with superficial corneal opacities of varying degrees affecting the perilimbal and central regions of the cornea, with other opaque areas appearing as crystalline deposits and corneal vascularization. The lesions were bilateral and were associated with mild‐to‐moderate conjunctival hyperemia. There was no history of blepharospasm or pruritus, and no subjects presented with epithelial erosions. Corneal biopsy revealed free microfilariae in the corneal stroma, with varying degrees of inflammation and collagen fiber destruction. The microfilariae were also found in skin lesions by skin snip technique. No adult worms were found in these dogs, and no dogs were on heartworm preventative before diagnosis. Monthly doses of oral ivermectin improved ocular and dermal lesions. One dog showed complete remission with the treatment. The species of the microfilariae was not identified.     

Comparison of serologic tests for detection of antigen in canine heartworm infections

In 30 random-source dogs, we determined sensitivity and specificity of 5 serologic tests for detection of canine heartworm antigens. Seventeen of the dogs were infected naturally with adult Dirofilaria immitis, and 4 of the infected dogs were amicrofilaremic. The ability of the serologic tests to predict whether a dog was infected or uninfected (overall test accuracy) ranged from 73 to 97%. Sensitivity was not affected by circulating D immitis microfilariae, but was markedly influenced by the number of adult D immitis present. False-positive reactions were rare and were not associated with intestinal parasites or Dipetalonema reconditum microfilariae. Modifications of some of the test procedures were necessary to maximize test accuracy and reproducibility. These modifications and other technical details might limit the usefulness of some of the tests in a veterinary practice.     

Prevalence of Dirofilaria immitis in a stray dog population in western New York.

In a survey in 1973 undertaken to determine the prevalence of Dirofilaria immitis in 100 dogs from a public pound in western New York, 2 dogs were found to be positive. Microfilariae of Dipetalonema sp were not present. Hearts and pulmonary blood vessels were dissected for adult worms, and blood samples were examined for microfilariae, using a modified Knott technique. Although adult heartworms and microfilariae have been recovered from wild foxes, coyotes, and wolves in New York, this is the first report of heartworm in domesticated dogs in the state.     

Effects of doxycycline on early infections of Dirofilaria immitis in dogs

The antifilarial effects of tetracycline drugs were first demonstrated when they were found to be highly effective against L(3) and L(4) of Brugia pahangi and Litomosoides sigmodontis in rodent models. Tetracyclines are also now known to have activity against microfilariae and adult Dirofilaria immitis, but assessment of their activity against larval and juvenile heartworms has not been reported previously. This study assessed the effects of doxycycline administered orally at 10mg/kg twice daily for 30-day periods at selected times during the early part of the life cycle of D. immitis in dogs with dual infections of D. immitis and B. pahangi. Twenty beagles were randomly allocated by weight to four groups of five dogs each. On Day 0, each dog was given 50 D. immitis L(3) and 200 B. pahangi L(3) by SC injection. Dogs received doxycycline on Days 0-29 (Group 1); Days 40-69 (Group 2); or Days 65-94 (Group 3). Group 4 served as untreated controls. Blood samples were collected for microfilariae counting and antigen testing. Necropsy for collection of adult heartworms and selected tissues were performed Days 218-222. Heartworms recovered were examined by immunohistology, conventional microscopy/transmission electron microscopy, and molecular biology techniques. No live heartworms were recovered from dogs in Group 1; dogs in Group 2 had 0 to 2 live worms (98.4% efficacy), and dogs in Group 3 had 0-36 live worms (69.6% efficacy). All control dogs had live adult heartworms (25-41). The live worms recovered from dogs in Groups 2 and 3 were less developed and smaller that worms from control dogs. Microfilariae were not detected in any dogs in Groups 1 and 2; one dog in Group 3 had 1 microfilariae/ml at necropsy. All control dogs had microfilariae at necropsy. One dog in Group 1 was antigen positive at one sampling (Day 166). One dog in Group 2 was antigen positive Days 196 and 218-222 and three dogs in Group 3 were antigen positive at one or more samplings All five control dogs were antigen positive at all three sampling times. These findings suggest that doxycycline at 10mg/kg orally twice daily for 30 days has efficacy against migrating tissue-phase larvae and juvenile worms and will delay or restrict microfilarial production.