Experimental infection of cats with Dirofilaria immitis.

Natural and experimental infections of cats with Dirofilaria immitis have been reported. Experimental infections of D. immitis in cats with the subsequent detection of microfilaremia and immediate skin hypersensitivity to antigen to D. immitis were produced. Cutaneous nodules and chylothorax were also detected in some infected cats. Adult worm recoveries were low and dead worms were found in some cats indicating the unsuitability of the cat as a host for D. immitis. However, one successful mosquito passager of D. immitis from a cat to a dog was accomplished.     

Dirofilaria immitis: identification and partial characterization of parasite antigens in the serum of infected dogs

We have recently developed a sensitive and specific immunodiagnostic test for canine Dirofilaria immitis infection based on detection of soluble parasite antigens in dog sera by monoclonal antibody-based enzyme immunoassay. In addition to their importance as markers of infection, these antigens may contribute to the pathogenesis of heartworm disease in dogs. In the present study, a variety of methods were used to identify and characterize circulating D. immitis antigens. Two antigens were identified in infected dog sera that formed lines of identity in rocket-line immunoelectrophoresis with soluble antigens extracted from adult D. immitis. Circulating D. immitis antigens were also demonstrated in infected dog sera by immunoblot analysis with polyclonal and monoclonal antibodies. These antigens had apparent molecular weights that ranged from 50 to 250 kDa. Most of the circulating D. immitis antigens contained the epitope defined by monoclonal antibody 1418BF2.1 which is used in our enzyme immunoassay for circulating D. immitis antigen. Studies of parasite antigens released during in vitro culture indicated that the circulating D. immitis antigens in dog sera that are detected by our enzyme immunoassay are primarily derived from adult female worms.     

Prevalence of antibodies to Toxoplasma gondii in raccoons (Procyon lotor) from an urban area of Northern Virginia

Raccoons (Procyon lotor) are intermediate hosts for Toxoplasma gondii, and clinical toxoplasmosis in raccoons has been reported. A 2-yr serological survey was conducted to determine the prevalence of antibodies to T. gondii in raccoons collected from Fairfax County, Virginia, a suburban/urban area outside Washington, D.C. Serum samples from 256 raccoons were examined for T. gondii antibodies at a 1:50 dilution using the modified direct agglutination test. Results indicated that 216 (84.4%) of the raccoons had been exposed to T. gondii. Our results indicate that raccoons in this area of Virginia are frequently exposed to T. gondii. Domestic cats were common in the study area and may have served as a source of oocysts for raccoons and the food items of raccoons.     

Prevalence of agglutinating antibodies to Sarcocystis neurona in raccoons (Procyon lotor) from an urban area of Virginia

Equine protozoal myeloencephalitis is the most important protozoan disease of horses in North America and is usually caused by Sarcocystis neurona. Natural and experimentally induced cases of encephalitis caused by S. neurona have been reported in raccoons (Procyon lotor) and raccoons are an intermediate host for this parasite. A 3-yr-long serological survey was conducted to determine the prevalence of agglutinating antibodies to S. neurona in raccoons collected from Fairfax County, Virginia, a suburban-urban area outside Washington, D.C. Samples from 469 raccoons were examined, and agglutinating antibodies (> or = 1:50 dilution) were found in 433 (92.3%) of the raccoons. This study indicates that exposure to S. neurona is high in this metropolitan area.     

Natural infection of Aedes trivittatus (COQ) with Dirofilaria immitis in central Iowa.

Juveniles identified as Dirofilaria immitis were recovered from Aedes trivittatus and Anopheles punctipennis collected CO2-baited CDC light traps in Ames, Iowa, during 10 days in July and August 1975. Ae. trivittatus composed 58.7% of the mosquitoes collected, and D. immitis was recovered from 18 of 393 (4.6%) dissected and from 7 of 31 pools (911 mosquitoes; 0.77%). Approximately 1.0% of the Ae. trivittatus collected harbored infective-stage juveniles, and as many as 27 were recovered from a single mosquito. This is the first report of Ae. trivittatus supporting the development of D. immitis. An. punctipennis was the only other species infected, with 2 of 468 harboring first-stage juveniles. The data suggest that Ae. trivittatus probably is the principal vector of D. immitis in central Iowa.     

Antigenic role of the endosymbionts of filarial nematodes: IgG response against the Wolbachia surface protein in cats infected with Dirofilaria immitis

Filarial nematodes harbour intracellular endosymbiotic bacteria, which have been assigned to the genus Wolbachia. These bacteria appear to play an important role in the pathogenesis of filarial diseases through their lipopolysaccharides. In view of the presence of Wolbachia endosymbionts in the body of filarial nematodes, one might also expect that proteins from these bacteria play an antigenic role in humans and animals affected by filariases. To test this hypothesis, we produced in recombinant form the surface protein WSP and a portion of the cell-cycle protein FTSZ from the Wolbachia of Dirofilaria immitis. Western immunoblot assays were then performed using cat sera to test the immunogenicity of these proteins. Sera were collected from owners' cats, which were either sero-negative or sero-positive for D. immitis and from cats before and after experimental infection with D. immitis. FTSZ was recognized in Western blots by sera from both positive and negative cats and from both uninfected and experimentally infected cats. WSP was recognized only by sera from positive cats and from cats experimentally infected with D. immitis; this protein was not recognized by sera from negative cats and from cats before experimental infection with D. immitis. The results of Western blot assays on WSP thus support the hypothesis that infection with filarial nematodes induces the production of antibodies against Wolbachia proteins.     

Survey of canine heartworm in the city of Recife, Pernambuco, Brazil.

Six hundred and eleven random-source dogs (338 male, 273 female) one year of age or older, from six sections of the city of Recife, Pernambuco, were examined antemortem for circulating microfilariae Dirofilaria immitis and Dipetalonema reconditum adult heartworm (D. immitis) antigen, and examined postmortem for adult heartworms. The prevalence of heartworm infection was 2.3% (14/611), as determined by necropsy for adult worms, and 1% (6/611) had circulating microfilariae of D. immitis; thus, 57.1% of the heartworm-infected dogs had occult infections. The results of serological testing indicated that 1.3% (8/611) of the dogs were positive for adult heartworm antigen. A total of 42 (6.9%) of the dogs had microfilariae of D. reconditum; 40 of these had only D. reconditum and two additional dogs had microfilariae of both species, D. immitis and D. reconditum.     

Hemoparasites of raccoons (Procyon lotor) in Florida

Four hemoparasite species (Babesia lotori, Trypanosoma cruzi, Dirofilaria tenuis and Mansonella llewellyni) were found in raccoons (Procyon lotor) collected from 1972 to 1974 in Duval (n = 14) and Collier (n = 170) counties, Florida (USA). Trypanosoma cruzi was found in thin blood smears from one raccoon at each locality. The prevalence of B. lotori was 79% and 80% in samples taken in December 1973 in Collier and Duval counties, respectively. No patent infections by B. lotori were detected in raccoons collected in Collier County in December 1972, but 42% of the raccoons examined in September 1973 were infected. In Collier County there were no significant differences in the prevalence of B. lotori by host sex or age. In Duval County, overall D. tenuis prevalence was 7%, whereas that of M. llewellyni was 14%; the latter species was not found in Collier County. Adult raccoons had a significantly greater prevalence of D. tenuis (32%) than did subadults and juveniles (7%), and male raccoons showed a significantly greater prevalence (51%) than did females (8%).     

Dirofilaria immitis in coyotes and foxes in Missouri

Wild canid carcasses were obtained during the 1986-1987 and 1987-1988 trapping seasons in Missouri. Hearts and lungs from 293 coyotes (Canis latrans), 85 red foxes (Vulpes vulpes) and 70 gray foxes (Urocyon cinereoargenteus) were examined for Dirofilaria immitis. Age of hosts was determined by radiographic and histologic techniques. Nineteen coyotes (7%) had from 1 to 100 D. immitis and five red foxes (6%) had from 1 to 7 D. immitis, whereas gray foxes had none. This study indicates that heartworm prevalence differs by wild canid species within the same area and during the same time period.     

The first record of Dirofilaria immitis infection in a Humboldt penguin, Spheniscus humboldti

Dirofilaria immitis infection is an important parastic disease in many mammals, especially canids, but has not been reported in bird hosts. Filarial worms were isolated from the lumen of the right atrium of the heart and the connective tissue of the lung of a captive female Humboldt penguin, Spheniscus humboldti, that died at a zoo in Japan. One of these worms was observed morphologically and identified as D. immitis by features such as 4 pairs of cephalic papillae, 1 pair of cervical papillae, esophagus divided into 2 regions, 4 pairs of pre-anal papillae, 5 pairs of post-anal papillae, and unequal spicules. In addition, the partial DNA sequence (234 bp) of mitochondrial CO / gene of the filarial worm was identical to that of D. immitis. This is the first report of D. immitis infection in a bird.     

Dirofilaria immitis: experimental infections in the ferret (Mustela putorius furo)

The ferret, Mustela putorius furo, was found to be susceptible to Dirofilaria immitis infection when exposed to low (14) or high (280-420) numbers of infective larvae harvested from Aedes aegypti. Eight ferrets (half of them cortisonized) were inoculated subcutaneously with 14 larvae each. All of them were subsequently found to harbor D. immitis in the heart, and all but one of them had worms of both sexes. Six of these ferrets were examined for microfilaremia at 31 to 35 weeks after inoculation; 3 were positive (one observed only at postmortem examination) and there was evidence that fertilization of female worms had occurred in one other. Females up to 25.5 cm and males up to 16.0 cm were recovered. There was no evidence that the cortisonization of some ferrets had affected the infections. Both male and female ferrets became infected. Four cortisonized ferrets were inoculated with 280 or 420 larvae of D. immitis (divided equally between subcutaneous and intraperitoneal routes). All of them died 16 to 18 weeks after inoculation, yielding 102 to 125 immature D. immitis. In these lethal infections, worms were recovered from the heart and adjoining vessels, and also from vascular and extravascular sites throughout the body.     

Experimental infection of Dirofilaria immitis in raccoon dogs

Canine heartworm (Dirofilaria immitis) is a nematode that naturally parasitizes in the pulmonary arteries and the right ventricle of domestic dogs (Canis familiaris) as final hosts. Japanese raccoon dogs (Nyctereutes procyonoides viverrinus) also are known to be susceptible to infection by the parasite. However, prevalence of this infection among free-ranging raccoon dogs is low and so is the worm burden. To examine the susceptibility of the raccoon dog to D. immitis infection, 3 raccoon dogs and 2 beagles were inoculated 4 times with 25 third-stage larvae (L3s) of D. immitis at 3-wk intervals. Worms were recovered from 2 raccoon dogs and both domestic dogs. The average percentage of recovery (2.3%) of the raccoon dogs was almost 10 times lower (24.5%) than that of the domestic dogs, but there was no significant difference in the body length of worms recovered from 2 types of hosts. To examine microfilaremia, 2 raccoon dogs were infected with 100 L3s. Microfilaremia was observed for 180 days postinoculation (PI) but disappeared at about 300 days PI. The raccoon dog was mildly susceptible to infection with D. immitis, but surviving worms developed and matured normally.     

Nine more cases of human infections by Dirofilaria repens diagnosed in Italy by morphology and recombinant DNA technology

Dirofilariosis is a mosquito-borne disease of carnivors due, in Italy, to Dirofilaria repens and D. immitis species which are sympatric in many areas. Nine more cases of human infection are here reported, detected in Italian patients living in northern regions (where D. immitis and D. repens are largely present), in Tuscany (where D. repens is more frequent than D. immitis), and in Sicily (where D. immitis is present only sporadically). The nematodes surgically removed (7 female and 2 male specimens) have been identified by a PCR-based method and by morphology as D. repens. Morphological, clinical, and epidemiological data of these human infections are discussed.     

Experimental Dirofilaria immitis-associated glomerulonephritis induced in part by in situ formation of immune complexes in the glomerular capillary wall

Eight dogs were immunized with an aqueous-soluble extract of adult Dirofilaria immitis. Subsequent to at least 7-fold increases in antibody titer, the left renal artery of each dog was infused with 6 mg of D. immitis antigen. Fourteen days after infusion, the left kidney was compared to the right kidney and preinfusion biopsies. All dogs developed glomerular lesions in the left kidney characterized by 1 or more of the following: mesangial cell proliferation, neutrophil infiltration, increased periodic acid-Schiff-positive staining of the mesangium and glomerular basement membrane (GBM), fibrin deposition, and thickening of the GBM. Left kidney glomerular immunofluorescence was positive in 7 of the 8 dogs using polyclonal antisera for canine IgG and C3 in a linear or fine granular pattern. Ultrastructural lesions were present in the left kidney of all dogs and consisted of irregular GBM thickening, intramembranous and mesangial electron-dense deposits, and mesangial and endothelial cell proliferation. Antibodies directed against D. immitis antigen were demonstrated in all kidney eluates from the left kidney. The right kidneys of 3 of the dogs developed lesions; however, in comparison to the left kidney, the lesions in the right kidneys were inconsistent, mild, and focal. The histologic findings in the left kidney were similar to those observed in dogs with naturally occurring D. immitis infections. In sham-immunized control dogs, renal arterial infusion of D. immitis antigen did not cause consistent immune complex glomerulonephritis; however, antigen adherence to glomerular capillary walls was observed by immunofluorescent microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Periodicity of Dirofilaria immitis microfilariae in a dog from Muheza district, Tanzania

Dirofilaria immitis (the dog heartworm) microfilarial periodicity was determined hourly for five days in an infected dog from Kambai village in Muheza district Tanzania. Maximal microfilarial counts were found at 1100 h and minimal at 2200 h. This finding represents the first record of D. immitis microfilarial periodicity in Tanzania.     

Immunisation of mice with fractions derived from the intestines of Dirofilaria immitis

Antigens that are not normally seen by the host but that are nevertheless, accessible to host immune effector molecules and cells such as the native endoantigens associated with the intestinal epithelium of haematophagous tissue-dwelling parasites, could be potentially useful vaccine antigens. In this study, intestines were dissected from adult Dirofilaria immitis, homogenised, and a 105,000 x g pellet obtained and extracted with Triton X-100. The soluble 105,000 x g supernatant from this extract induced partial protection (51%) against a challenge infection of third stage larvae (L3) implanted in micropore chambers. Sera from mice immunised with this soluble detergent extract reacted with proteins ranging in size from 38 to 130 kDa. Immunolocalisation studies indicated the mouse sera reacted primarily to the lumenal surface of the intestines of adult D. immitis, though reactivity to the lateral nerve/epithelial chords, hypodermis and reproductive tracts was also noted, indicating the presence of shared antigens. Tissues of L3s were also recognised by the immunised mouse sera. These mouse sera did not react to a dog blood fraction prepared identically to the D. immitis fraction. Only those sera from D. immitis-infected dogs with heavy or long-term infections were reactive to a single 42 kDa protein. After 24 h incubation in fluorescein isothiocyanate-conjugated serum the intestinal tract of Onchocerca volvulus and D. immitis L3 and L4 fluoresced, indicating the serum had been ingested. These data suggest that filarial gut-associated antigens (apart from the single 42 kDa antigen) are not seen by normally infected hosts, that they can be accessible to antibodies and that they can induce an immune response which is partially protective.     

Serological and molecular studies on Dirofilaria immitis in dogs from Turkey

We estimated the prevalence of Dirofilaria immitis infection in domestic dogs in five Turkish provinces - Sakarya, Kocaeli, Ankara, Elazig and Mersin - using a commercial ELISA kit for detecting circulating antigen and a PCR test for detecting circulating microfilarial DNA. A total of 211 whole-blood and serum samples were collected from dogs of various breeds, ages and life status (owned or stray). Sample population characteristics were recorded and examined for differences in prevalence. Additionally, we collected 15 blood samples from cats (14 owned and 1 stray) from Ankara province and used PCR to detect D. immitis infection. Twenty-seven (12.8%) of 211 dog samples were positive for D. immitis antigen by ELISA. No differences in prevalence were observed by sex (female: 14.4%; male: 10.7%; P>0.05). The prevalence of D. immitis infection varied with age: 11.8% in younger dogs (0.5-2 years) and 17.5% in older dogs (3-5 years). Prevalence between stray dogs (15.2%) and owned dogs (9.3%) did not differ (P>0.05). Prevalence rates were highest in Kocaeli province (18.3%), followed by Ankara (14.8%), Sakarya (12.3%) and Mersin (10.5%) provinces. Prevalence in Elazig province was 0%. No dogs or cats had microfilarial DNA detectable by PCR.     

Increased rat alveolar macrophage expression of functional iNOS induced by a Dirofilaria immitis immunoglobulin superfamily protein.

Dirofilaria immitis is a worldwide filarial nematode causing heartworm disease in dogs and cats. Several mosquito species, which are able to feed both on humans and animals, can transmit this parasite. Inflammatory progression of host tissues induced by parasites are mediated by several molecules, including nitric oxide (NO), which usually exerts deleterious effects on parasites and occasionally on the host. We analyze the in vitro effect of total D. immitis adult worm somatic antigens on na?ve rat alveolar macrophage NO production and further separation of parasite proteins to define specific D. immitis somatic molecules influencing host cell NO secretion. Additionally, we address the possible influence of Wolbachia spp. on the in vitro production of NO by macrophages. Our results demonstrate that D. immitis adult worm soluble antigens are able to specifically induce NO production from host macrophages. Furthermore, we demonstrated that this effect is due to nematode antigens rather than to defined components (LPS and metabolic molecules) derived from its endosymbiont, Wolbachia spp. In addition, we were able to isolate and identify one of the parasite specific components from the DiSo extract, denominated DiID35.3 and putatively belonging to the Immunoglobulin Superfamily Protein (ISP) group, triggering NO release from macrophages in a dose-dependent and specific manner.     

First finding of Dirofilaria repens in a natural population of Aedes albopictus

The invasive mosquito Aedes albopictus (Skuse) (Diptera: Culicidae) has become widespread in Italy during the past decade. Also Italy has foci of canine filariasis caused by Dirofilaria (Spirurida: Onchocercidae), due to subcutaneous D. repens Railliet & Henry as well as the dog heartworm D. immitis (Leidy) transmitted by various vector mosquitoes (Diptera: Culicidae). In 2002, at Fiumicino, west of Rome (Lazio Region), 17% of dogs were found to have D. repens microfilariae in peripheral blood. To evaluate the role of Ae. albopictus as a vector of Dirofilaria in this area, female mosquitoes were collected daily, June-October 2002, landing on dog or human bait in a rural house at Focene. Mosquitoes were maintained at 27 degrees C and 70% RH for 6 days, to allow development or purging of filaria larvae, then identified and frozen for subsequent molecular assay with filaria-specific ribosomal S2-S16 primers. To distinguish specimens harbouring infective L3 Dirofilaria larvae, DNA was extracted separately from the mosquito abdomen and head-thorax. Dirofilaria species were identified by sequencing, confirmed by polymerase chain reaction of positive specimens using primers specific for D. immitis and D. repens. Dirofilaria DNA was detected in 3/154 (2%) of Ae. albopictus females examined: D. repens DNA in head-thorax and abdomen of one collected 27th July; D. immitis in the abdomen of one collected 24th September; DNA of both D. immitis and D. repens in the head-thorax of one collected 11th October 2002. Thus Ae. albopictus is a potential vector of both Dirofilarias in Italy, representing risks for veterinary and human health.     

Prevalence of antibodies to Trypanosoma cruzi in raccoons (Procyon lotor) from an urban area of northern Virginia

Raccoons (Procyon lotor) are reservoir hosts for Trypanosoma cruzi. A 3-yr-long serological survey was conducted to determine the prevalence of antibodies to this zoonotic parasite in raccoons collected from Fairfax County, Virginia, a suburban/urban area outside Washington, D.C. Serum samples from 464 raccoons were examined for T. cruzi antibodies at a 1:40 dilution with an indirect fluorescent antibody test (IFAT) and Brazil strain T. cruzi amastigotes and trypomastigotes as antigen. A positive IFAT test was found in 154 (33%) of the 464 samples. The yearly prevalence was 49 of 132 (37%) in 2000; 19 of 120 (16%) in 2001; and 86 of 212 (41%) in 2002. Our study indicates that raccoons in this area of Virginia are frequently exposed to T. cruzi.