Right atriotomy using total venous inflow occlusion for removal of heartworms in a cat.

Heartworm infection with caval syndrome was detected in a thirteen-year-old male cat. However, removal of the heartworms via a jugular venotomy was infeasible because the size of the jugular vein limited our ability to use flexible alligator forceps. Therefore, a right atriotomy using total venous inflow occlusion was performed to remove the heartworms. The procedure was accomplished successfully, and the cat recovered from its symptoms. The present case suggests that right atriotomy using venous inflow occlusion is practical for removal and prevention of rupture of heartworms.

Myocardial fluid balance in dogs with naturally acquired heartworm infection.

OBJECTIVE: To determine the effect of naturally acquired heartworm (Dirofilaria immitis) infection on myocardial fluid balance as indicated by myocardial water content and the dynamics of transepicardial fluid flow. ANIMALS: 7 dogs infected with adult heartworms and 8 dogs free of heartworm infection. PROCEDURES: Infected dogs had heartworms in the right ventricle, pulmonary artery, or both but no evidence of cardiovascular disease on physical examination. A hemispheric capsule was attached to the epicardial surface of all dogs for determination of transepicardial fluid dynamics and permeability of the epicardium to water and protein. Myocardial water content and hydroxyproline content were assessed at necropsy. RESULTS: Myocardial water content was significantly lower in heartworm-infected dogs. No differences in myocardial hydroxyproline content, transepicardial fluid flow, or epicardial water or protein permeability were detected. CONCLUSIONS AND CLINICAL RELEVANCE: Heartworm infection significantly altered myocardial fluid balance in dogs, possibly because of a change in the myocardial interstitial pressure-volume relationship. These changes may be associated with increased vulnerability to cardiovascular stressors in heartworm-infected dogs.

A lambda gt11 cDNA recombinant that encodes Dirofilaria immitis paramyosin.

The cDNA synthesized from mRNA of Dirofilaria immitis female adult worms was cloned into the expression vector lambda gt11. Screening the library with a hyperimmune rabbit antiserum raised against adult worm homogenates yielded several antigen positive clones. One of these clones, lambda cDi2, was recognized by rabbit antisera raised against either D. immitis L-3, adult, Brugia malayi L-3 or Onchocerca volvulus adult worm antigen, as well as by antisera from humans naturally infected with O. volvulus or Wuchereria bancrofti. Affinity-purified anti-lambda cDi2 antibodies reacted with a 97-kDa protein on Western transfers of adult D. immitis antigen extracts that were reduced with beta-mercaptoethanol. The whole rabbit anti-D. immitis adult antiserum depleted of anti-lambda cDi2 antibodies exhibited decreased reactivity to this 97-kDa band. A monoclonal antibody (IA6) that specifically binds Schistosoma mansoni paramyosin also recognised a 97-kDa protein in D. immitis extracts upon Western transfer. The deduced amino acid sequence of partial DNA sequence from lambda cDi2 showed some similarity to nematode myosin, and gave a stretch of 82 amino acids that is 91.5% identical to Caenorhabditis elegans paramyosin: thus, lambda cDi2 encodes D. immitis paramyosin.

Comparative studies on the melanization response of male and female mosquitoes against microfilariae.

The melanization response of adult male and female Aedes trivittatus and the black-eyed Liverpool strain of Aedes aegypti against intrathoracically inoculated Dirofilaria immitis microfilariae (mff) was assessed at 1, 3, and 5 days postinoculation (PI). The melanization reaction of males is significantly less effective than the response elicited by female mosquitoes. No mff in male A. aegypti and only 17% of mff recovered from A. trivittatus were fully melanized by day 5 PI compared with 80% and 100% complete melanization of recovered mff from A. aegypti and A. trivittatus females, respectively. A significantly greater percentage of mff retained their viability in males, and inoculation of heat-killed mff did not significantly increase the melanization response as compared with female mosquitoes. Males have significantly lower total hemocyte populations and hemolymph volumes than females, and the possible relationship of hemocyte numbers and reduced melanization capabilities in males is discussed.

Microfilaremia from a Dirofilaria-like parasite in Greece. Case report.

In Europe, zoonotic filarial infections in humans are caused by two species, Dirofilaria immitis and Dirofilaria repens. These parasites are associated mainly with embolic infarcts of the pulmonary artery and subcutaneous nodules, respectively. An unusual dirofilarial infection in a Greek patient who showed marked eosinophilia and microfilaremia is presented. Although the identification of Dirofilaria species is not conclusive, this report is the first on a case of microfilaremia from a Dirofilaria infection in an immunocompetent patient.

Cryopreservation of the microfilariae of Brugia malayi, Dirofilaria corynodes, and Wuchereria bancrofti.

Methods are presented for the cryopreservation of a sheathed microfilaria, Brugia malayi, and an unsheathed species, Dirofilaria corynodes. The former survived best when frozen at the rate of -0.8 degree or -0.5 degree C per minute using 9% dimethyl sulfoxide (DMSO) as the cryopreservative. Approximately 52-79% of the thawed microfilariae developed to the third stage in Aedes aegypti mosquitoes versus 79% of the unfrozen specimens. For D. corynodes the optimum freezing rate was -2 degrees or -5 degrees C per minute, and 6% DMSO combined with 0.004 M polyvinylpyrrolidone (PVP) afforded the best cryoprotective effect. The development of thawed microfilariae in mosquitoes ranged from 22-32% versus 29% for unfrozen specimens. In general, the viability of both species of microfilaria was retained best when stored in liquid nitrogen (-196 degrees C). The entire life cycle of B. malayi was completed in the laboratory using cryopreserved microfilariae as the initial source. The cryopreservation of Wuchereria bancrofti also is discussed.

In vitro development of third- and fourth-stage larvae of Dirofilaria immitis: comparison of basal culture media, serum levels and possible serum substitutes.

In vitro development and survival of third-stage larvae of Dirofilaria immitis were compared in four different culture media and in the presence of varying concentrations of four different medium supplements. Motility and the incidence of third- to fourth-stage molting were used as criteria for evaluating different culture conditions. No significant differences in either motility or molting response were detected between larvae cultured in NCTC-135, F12(K), CMRL 1066 or Dulbecco's Modified Eagle's Medium. Fetal calf serum enhanced development and survival of the cultured larvae in dose-dependent fashion. Its effects were maximal at a concentration of 20 percent of the total medium volume. Addition of a commercial medium supplement, NuSerum, also gave a dose-related increase in larval development and viability. The activity of NuSerum in this respect was comparable to that of fetal calf serum. The tripeptide glycylhistidyllysine and bovine serum albumin, fraction V both failed to stimulate development of third-stage D. immitis larvae in vitro.

The incrimination of Aedes (stegomyia) aegypti as the vector of Dirofilaria repens in Nigeria.

Six local species of culicides were identified as the common mosquitoes in Zaria, out of 15 species captured using various adult and larval collection methods. These common culicides are Culex pipiens fatigans, Anopheles gambiae grp., Mansonia africana, Culex pipiens pipiens, Aedes (stegomyia) aegypti and Aedes vittatus. They were each fed directly on a local dog naturally infected with Dirofilaria repens to evaluate their refractoriness/susceptibility to dirofilarial infection. In a number of donor-feeding trials, 39. 4% Culex pipiens fatigans; 58.9% An gambiae grp.; 60.5% Mansonia africana; 1.8% of Culex pipiens pipiens; 23.4% Ae aegypti and 3.3% of Ae vittatus successfully fed on the microfilaraemic host. Only Aedes aegypti was susceptible to the infection as all 40 (100%) Ae aegypti reaching 10-14 day post-blood meal had infective (L(3)) larvae of D. repens. The remaining five species were refractory. The microfilariae in the five non-susceptible mosquitoes were always found trapped in the blood meal in the insects midgut (stomach). These trapped microfilaria were dead by the 2nd day in the insect's midgut. However, in the susceptible Ae aegypti, the microfilariae were set free from the blood meal in the midgut and within 24h migrated to the malpighian tubules (MT) of the mosquitoes. All Ae aegypti dissected 5-7 day post-infective blood meal showed the typical quiescent sausage stage (L(2)) larvae in the malpighian tubules. At day-10 post-blood meal, relatively active infective (L(3)) larvae of D. repens were found in the MT; and by day 12-14, highly motile infective larvae had reached the insect's head and proboscis, with infective larvae occasionally oozing out during dissection through the tip of the proboscis. The rate of development of D. repens to infective larvae was faster in mosquitoes infected in July when the environmental temperature was 24.5 degrees C than those infected in November when the temperature was 22.5 degrees C. The latter were delayed for 4 days. The breeding sources of Ae aegypti, the local vector implicated were also identified. As no particular vector of this zoonotic filaria has been identified previously in Nigeria, these findings could make any control programme more focussed and easier.

Diethylcarbamazine-enhanced activation of complement by intact microfilariae of Dirofilaria immitis and their in vitro products.

Microfilariae of Dirofilaria immitis and their products inhibited hemolytic complement in sera from various animal species. Inhibition of hemolytic complement activity, fluorescent antibody detection of specific complement proteins binding to microfilarial surfaces, and immunoelectrophoretic evaluation of complement protein C3 conversion demonstrated that (1) intact microfilariae depleted hemolytic complement activity maximally in the presence of diethylcarbamazine whereas the complement-fixing activity of microfilarial products was little affected by diethylcarbamazine; (2) complement proteins C3, properdin, and C5 bound to the cuticular surface of microfilariae; and (3) C3 was converted to a faster-migrating species during incubation with microfilariae or their products. The complement-depleting activity of in vitro products from viable microfilariae was soluble in 10% trichloroacetic acid, resistant to beta-elimination with 0.5 M NaOH, susceptible to treatment with 0.2 M periodate, and composed of 56% neutral sugar, 18% protein, 12% hexosamine, and 10% sulfate. The polysulfated, acidic mucopolysaccharide nature of the surfaces of microfilariae and the results presented here indicate that polyanionic components on worm surfaces or shed by microfilariae react with host complement proteins. This interaction may be enhanced by diethylcarbamazine and contribute to the pathology associated with microfilaremias.

The species of the genus Dirofilaria, Railliet & Henry, 1911.

Some authors divide the genus Dirofilaria into two subgenera. The subgenus Dirofilaria consists of 5 apparently valid species (D. ailure, D. freitasi, D. immitis, D. lutrae, D. spectans) and 7 species of questionable validity (D. fausti, D. indica, D. louisianensis, D. magalhaesi, D. nasuae, D. pongoi, D. spirocauda). The subgenus Nochtiella consists of 22 apparently valid species (D. acutiuscula, D. bonnei, D. cancrivori, D. corynodes, D. genettae, D. granulosa, D. incrassata, D. linstowi, D. macacae, D. macrodemos, D. magnilarvatum, D. minor, D. pagumae, D. panamensis, D. repens, D. sachsi, D. striata, D. subdermata, D. sudanensis, D. tawila, D. tenuis, D. ursi) and 8 species of questionable validity (D. aethiops, D. andersoni, D. conjunctivae, D. desportesi, D. hystrix, D. schoutedeni, D. spinosa, D. subcutanea). Actually, 10 species of the genus Dirofilaria (D. asymmetrica, D. digitala, D. kuelzii, D. ochmanni, D. pauliani, D. roemeri, D. scapiceps, D. timidi, D. uniformis, D. websteri) are attributed to other genus.

Importance in France of the infestation by Dirofilaria (Nochtiella) repens in dogs.

The presence of Dirofilaria repens has been mentioned in Europe, in Africa and in Asia, in different carnivores (dogs, foxes, cats, lions). In Europe the nematode has been reported in dogs, cats and foxes and the countries concerned by native cases are Italy, Spain, Greece, Yugoslavia and France. In France, until now, D. repens has been described, among animals, only in dogs. The first undoubted report dates back to 1953. Later, the prevalence was studied through widespread surveys (1986-1988-1989), focal surveys within some canine populations (military dogs) and some sporadic and fortuitous descriptions. The prevalence has been mainly assessed by pointing up the microfilariae in the blood and by identifying it after histochemical staining (for demonstration of the somatic distribution of acid phosphatase activity). The infestation rate was higher than 20% in some military dog populations. The cases occurred in nineteen French departments (Alpes-de-Haute-Provence, Aude, Bouches-du-Rhône, Corrèze, Corse, Dordogne, Gard, Gers, Gironde, Haute-Garonne, Loir-et-Cher, Nièvre, Pyrénées-Orientales, Rhône, Tarnet-Garonne, Var, Vaucluse, Vienne and Region Parisienne). The pathogenicity which is normally denied has to be reconsidered. The pathology of adult worms has not been clearly proved in dogs but the embolized microfilariae can bring about skin troubles. The potential pathogenicity, the necessity of the differentiation with D. immitis and above all, the zoonotic importance of D. repens must lead to search for it more often and to consider it with great interest.

Research of canine filariasis in Trapani province (western Sicily). Morphology on SEM of male Dirofilaria repens.

The prevalence of canine filariasis was investigated in 4 municipalities of Trapani province (Campobello di Mazara, Castelvetrano, Marsala and Mazara del Vallo), an area where 8 human cases of dirofilariasis due to Dirofilaria (Nochtiella) repens have occurred during the last decade. Blood samples were collected late in the afternoon, during August and September 1995, and examined by Knott technique for the presence of microfilariae. Sixtythree (29.3%) out of 215 domestic dogs (98 males, 117 females; 40 sub-adults, 175 adults) proved to be positive for microfilariae: 55 (25.6%) for D. repens, 13 (6.0%) for Dipetalonema reconditum, 1 (0.5%) for D. immitis; 6 (2.8%) mixed infections (5 D. repens + D. reconditum and 1 D. repens + D. immitis) were also found. The surface topography via SEM of an adult male specimen of D. repens found in a dog during necroscopy is also described. The relatively large number of human cases of dirofilariasis appears to be related to the high prevalence of D. repens observed in dogs of Trapani province. This matter should be investigated more extensively in this area.

Development of Dirofilaria and Setaria nematodes in Aedes albopictus.

The development of Dirofilaria repens, D immitis and Setaria labiatopapillosa up to L3 stage was documented in a laboratory colony of Aedes albopictus originating from specimens collected in Civitavecchia (Central Italy). The susceptibility of Ae albopictus and its plastic trophic habits, strongly suggest that this mosquito may contribute to the spreading of these nematodes in the country. It is particularly emphasized the danger for human health of an increased probability of transmission of Dirofilaria in urban areas.

Ivermectin against larval stages of Dirofilaria repens in dogs.

The prophylactic efficacy of ivermectin against Dirofilaria repens infections in dogs was investigated. A first trial was carried out on 15 dogs exposed to four inoculations of L3 larvae at 15-day intervals and treated, in groups of five, with 0, 6 or 12 micrograms/kg body weight of ivermectin given per os 30 and 60 days after the first inoculation. Necropsy, performed about 9 months later, revealed that worm burdens were reduced by 86.6 and 92.8% for the 6 and 12 micrograms/kg dose levels, respectively. In a second trial with an otherwise identical protocol, a dose rate of 24 micrograms/kg of ivermectin was tested in 12 dogs. Only one of the six treated dogs was found worm free at necropsy. The worm burden was reduced by 87.9% in treated animals as opposed to controls. A lengthening of the prepatent periods, which might be considered dose related, was apparent in all treated groups. Ivermectin was not completely effective in preventing establishment of experimental infections with D. repens in dogs.

[Human pulmonary dirofilariasis: parasitological aspects of the 2d case reported in Italy]. / Dirofilariasi polmonare umana: aspetti parassitologici del secondo caso segnalato in Italia.

The parasitological aspects of a case of pulmonary human dirofilariasis, recently reported by Fabbretti and collaborators (1990) in a 58 year-old man from Ferrara province, are described. This is the second case of pulmonary human dirofilariasis recorded in Italy. The nematode was rolled up inside a small pulmonary artery and was associated with a nodular infarcted lesion, discovered during an X-ray check as a "coin lesion". A pulmonary lobectomy was performed on the patient with a presumptive diagnosis of a malignant neoplastic nodule. The exact diagnosis was only made with the histological examination. In spite of the bad preservation of the nematode, it was possible to diagnose it as an immature female of Dirofilaria probably belonging to the species repens.

Scanning electron microscopy of vascular corrosion casts and histologic examination of pulmonary microvasculature in dogs with dirofilariosis.

OBJECTIVE: To characterize structural changes in pulmonary vessels of dogs with dirofilariosis. ANIMALS: 8 dogs with dirofilariosis and 2 unaffected control dogs. PROCEDURE: Pulmonary artery pressure was measured in affected dogs, and dogs then were euthanatized. Scanning electron microscopy was used to examine vascular corrosion casts of pulmonary vasculature. Tissue sections of pulmonary vasculature were evaluated by use of histologic examination. RESULTS: Pulmonary artery pressure was higher in dogs with severely affected pulmonary vessels. In tissue sections, dilatation, as well as lesions in the tunica intima and proliferative lesions resulting in constriction or obstruction, were frequently observed in branches of the pulmonary artery. Numerous dilated bronchial arteries were observed around affected pulmonary arteries. Hyperplastic venous sphincters were observed in small pulmonary veins and venules. In corrosion casts, affected pulmonary lobar arteries had dilatation, pruning, abnormal tapering, constriction, and obstruction. In small arteries and arterioles, surface structures representing aneurisms and edema were seen. Bronchial arteries were well developed and extremely dilated, and they formed numerous anastomoses with pulmonary arteries at all levels, from the pulmonary trunk to peripheral vessels. Capillaries in the lungs were dilated with little structural change. Small pulmonary veins and venules had irregular annular constrictions that were caused by hyperplastic smooth muscle cells of venous sphincters. CONCLUSIONS AND CLINICAL RELEVANCE: Scanning electron microscopy of microvascular casts delineated links between the bronchial and pulmonary circulations in dogs with dirofilariosis. Results of scanning electron microscopy provided a structural explanation for the development of pulmonary circulatory disturbances and pulmonary hypertension in dogs affected by dirofilariosis.

Efficacy of ivermectin against Dirofilaria immitis larvae in dogs 30 and 45 days after induced infection.

Forty-two Beagles, 14 to 15 weeks of age, were injected subcutaneously with 50 infective larvae of Dirofilaria immitis and were allotted by weight, within sex, to 6 treatment groups. Group 1 served as nonmedicated vehicle-treated controls; groups 2 through 5 were given an oral tablet form of ivermectin at dosages of 0.3 micrograms/kg, 1.0 micrograms/kg, 2.0 micrograms/kg, and 3.3 micrograms/kg at 30 days after inoculation; group 6 was given the 2.0 micrograms/kg dosage at 45 days after inoculation. Dogs were euthanatized and necropsied 154 days after treatment (day 139 for dogs in group 6) and examined for heartworms. On the numerical bases of helminths recovered in the groups, the efficacies for preventing heartworm maturation were 0% (group 2), 53.2% (group 3), 97.2% (group 4), 98.1% (group 5), and 63.8% (group 6). Drug-related adverse reactions were not detected.

Studies of Coquillettidia (coquillettidia) crassipes (Vanderwulp, 1881) in relation to transmission of Cardiofilaria nilesi.

Field surveillance of Cq. crassipes was conducted in an open housing estate near Kuala Lumpur using IMR traps baited with chicken and gerbils. Chicken bait attracted more Cq. crassipes. There was significant difference between chicken and gerbil as bait. The mosquitoes preferred to feed at canopy level. The parous rate was 20.41% and the infection rate for Cardiofilaria nilesi was 2.04%. None of the 120 chicken and 5 ducks, in a village close to the open housing estate, examined for microfilariae were positive. Ar. durhami and Ar. subalbatus did not support development of C. nilesi. However, Cq. crassipes is an extremely efficient vector of C. nilesi.

Human ocular infection with Dirofilaria repens (Railliet and Henry, 1911) in an area endemic for canine dirofilariasis.

Dirofilaria repens, which is usually found in canine subcutaneous tissues, is the main causative agent of human dirofilariasis in the Old Word. However, a relationship between animal and human cases of dirofilariasis caused by D. repens in a given area has never been demonstrated. The uneven distribution of D. repens in provinces in Sicily, Italy represented the foundation for this study. We report a human case of ocular infection with D. repens from Trapani Province, where canine dirofilariasis is endemic. The nematode was morphologically and molecularly identified and surgical removal of the parasite was documented. The relationship between the prevalence of D. repens in dogs and the occurrence of human cases of ocular dirofilariasis is discussed on the basis of a review of the historical literature.