Multiple diagnostic tests demonstrate an increased risk of canine heartworm disease in northern Queensland, Australia.

BACKGROUND: Canine heartworm (Dirofilaria immitis) is a life-threatening infection of dogs with a global distribution. Information on the prevalence of D. immitis and associated risk factors for canine heartworm antigen positivity-and thus disease-in Australia is scarce or outdated. The current reference method for D. immitis diagnosis in dogs is via the detection of heartworm antigen in blood using commercially available microwell-based enzyme-linked immunosorbent assays (ELISAs). Heat treatment of canine plasma prior to testing has been suggested to increase test sensitivity. The aim of the current study was to estimate the prevalence of D. immitis in dogs confined to shelters in Queensland, Australia. The impact of heat treatment on antigen test results was also assessed. METHODS: Blood samples (n = 166) were collected directly from dogs in seven shelters across Queensland (latitudinal span of approx. 1700 km) into EDTA blood collection tubes. A commercially available ELISA (DiroCHEK®) was used to detect canine heartworm antigen in untreated and heat-treated plasma. Whole blood was concurrently tested for the presence of microfilariae and D. immitis DNA using a modified Knott's test and real-time PCR, respectively. Risk factors (age, gender, source, location) associated with the odds of positivity for canine heartworm were assessed using binary logistic regression models. RESULTS: A total of 16 dogs (9.6%; 95% confidence interval [CI]: 5.9-15.2%) were positive for canine heartworm based on combined test results. Heat treatment did not impact on the positivity of D. immitis antigen within samples (Cohen's kappa = 0.98), but the optical density was significantly increased in paired plasma samples for D. immitis antigen-positive samples (Wilcoxon matched-pairs signed rank test, two-tailed P < 0.01). Location of the dog in a shelter in northern Queensland was the only risk factor significantly associated with the odds of a dog being more likely to be D. immitis antigen positive (odds ratio: 4.39; 95% CI: 1.26-13.51). All samples positive for the modified Knott's test were also positive for D. immitis DNA by PCR. CONCLUSIONS: This study demonstrated the presence of heartworm-positive dogs in shelters in Queensland, with positive animals significantly more likely to occur in northern Queensland than southern Queensland. Sustained testing for the presence of D. immitis microfilariae and antigen remain important diagnostic tools in areas with known and re-emerging canine heartworm activity.

Human case of subcutaneous nodule because of a novel genetic variation of Dirofilaria sp.

A 75-year-old man presented with a 1-cm large elastic soft subcutaneous nodule on the left side of the umbilicus, which when excised showed presence of a helminthic form within the granulomatous lesions. Morphologically, the helminth was considered to be of the genus Dirofilaria, and the patient showed increased serum antibody titer against canine filaria. The partial DNA sequence of the mitochondrial 12S rRNA gene locus of this clinical isolate showed the highest nucleotide identity (89.6%) with Dirofilaria repens; however, the phylogenetic analysis addressed the haplotype and Dirofilaria ursi as outgroups of the clusters of D. repens and Dirofilaria immitis, which are the causal agents of most human dirofilariasis. As like bear filaria D. ursi, a wide variety of other carnivore-parasitizing filaria species have rarely been reported in humans. The newly detected genetic haplotype in this case may correspond to one of these species of Dirofilaria, though the genetic references are not available thus far.

Effect of sildenafil and pimobendan on intracardiac heartworm infections in four dogs.

Four dogs, referred for management of heartworm (HW) disease, were found to have HWs entangled in their tricuspid valve apparatus. None of the dogs were actively hemolyzing or showed signs of acute cardiovascular collapse that would have necessitated emergency transvenous HW extraction, and surgery was not performed at time of presentation. The dogs received pimobendan and sildenafil within 24 h of identifying HW in the tricuspid valve apparatus, and the HW moved to the pulmonary arteries within 2 days in most cases (median 2 days, range 1-14 days). All dogs survived to discharge from the original hospital admission and were subsequently treated with adulticide (melarsomine) without complication. All dogs were HW antigen negative 6 months after their last melarsomine injection. Four dogs appeared to respond positively to medical management aimed at decreasing pulmonary arterial pressure and improving the right ventricular function, but movement of HW out of the heart for other reasons cannot be excluded. This therapeutic option is not advised when dogs with HW disease are presented for acute collapse, ongoing hemolysis, and hypotension as surgical extraction is still considered the best option in these cases. It remains unknown if medical management is a safe option for all dogs with intracardiac HW without clinical signs of caval syndrome. Controlled prospective studies are required to determine the efficacy and safety of this treatment regimen in comparison with surgical extraction.

Seroepidemiological survey of human exposure to Dirofilaria spp. in Romania and Moldova.

The present study aimed to evaluate the extent of Dirofilaria immitis and D. repens exposure in humans from eastern and southern areas of Romania and central Moldova by serological methods. The serological screening was performed on a total of 450 serum samples (187 from Romania and 263 from Moldova). The sera were collected using a convenience sampling with the help of physicians from the hospitals of the study areas. All samples were analysed by a non-commercial ELISA test for the detection of IgG antibodies against adult somatic antigens of D. immitis and D. repens. The results showed a total of 49 (10.9%; 95% CI = 8.3-14.1%) individuals from Romania and Moldova with a positive response to IgG antibodies against both adult somatic antigens of D. immitis and D. repens. Specifically, 48 (10.7%; 95% CI = 8.0-14.0%) patients were positive for IgG-antibodies against adult somatic antigens of D. immitis, one (0.2%; 95% CI = 0.4-1.2%) against D. repens antigens, and four (0.9%; 95% CI = 0.4-3.3%). were positive for antigens of both parasites. At country level, out of 187 samples from Romania, 13 (6.9%; 95% CI = 4.1-11.5%) were positive for anti-D. immitis IgG with high exposure in the southern part of the country (Bucharest). Of the 263 people from Moldova, 36 (13.7%; 95% CI = 10.0-18.4%) were positive for D. immitis antigens from which three (1.1%, 95% CI = 0.4-3.3%) were positive for the antibodies against antigens of both parasites. Only one sample was found positive for anti-D. repens IgG. Positive IgG-ELISA results were confirmed by Western blot analysis. In addition, for further confirmation, a complementary ELISA was performed for anti-WSP IgG antibodies against Wolbachia endosymbionts. Our findings showed a noticeable exposure of humans from Romania and Moldova to Dirofilaria parasites. Serology can be useful for indicating exposure to Dirofilaria spp. in a healthy population in order to obtain useful data on the epidemiological scenario of human dirofilariosis in Eastern Europe.

Leishmania infantum, Dirofilaria spp. and other endoparasite infections in kennel dogs in central Italy.

Prevalence and risk factors of Leishmania infantum, Dirofilaria spp. and other potentially zoonotic or canine-specific endoparasite infections were assessed in 639 kennel dogs from central Italy. To this end, individual blood and fecal samples were examined using parasitological, immunological and molecular techniques. The presence of compatible clinical pictures, as well as age and gender were considered as putative risks factors. To evaluate risk factors, multivariable analysis with logistic regression and univariable analysis with a Chi square test and a Fischer's exact test were performed. Overall, 52.6% of dogs (95% CI 48.6-56.5) were found positive, while 39.6% of dogs (95% CI 35.8-43.5) were infected by potentially zoonotic species. Leishmania infantum and Dirofilaria repens showed prevalences of 2.5% (95% CI 1.5-4.1) and 2.8% (95% CI 1.7-4.5), respectively. The prevalence of cardiorespiratory parasites was 7.8% (95% CI 5.9-10.3) and included the species Angiostrongylus vasorum, Eucoleus aerophilus, Eucoleus boehmi and D. immitis; the latter showed a prevalence of 0.2% (95% CI 0.001-1). Intestinal parasites were significantly prevalent (38.8%, 95% CI 35-42.7) and they consisted mainly of species of major zoonotic concern, including ancylostomatids, Toxocara canis, Giardia duodenalis, Dipylidium caninum, Taeniidae, Strongyloides stercoralis and Cryptosporidium parvum. Endoparasites were significantly prevalent in clinically suspected dogs. Leishmania infantum and cardiorespiratory nematodes were prevalent in older dogs, while intestinal parasites were prevalent in younger dogs. Results show high dog and public health risks in kennels in central Italy, and suggest the need for more effective control measures.

Evidences of increasing risk of dirofilarioses in southern Italy.

Given the spread of Aedes albopictus from northern to southern Italy, and the lack of updated data on Dirofilaria infections, this study was carried out to assess the infection risk for dogs and cats in Apulia region. During a 2-year study, 175 A. albopictus female specimens and samples of blood from 427 dogs (309 privately owned dogs and 118 shelter dogs) and 12 cats were collected. All blood samples were subjected to a modified Knott method, to a test for the detection of circulating Dirofilaria immitis antigen, and to a Dirofilaria species-specific real-time PCR for the simultaneous detection of D. immitis and Dirofilaria repens, targeting on partial cytochrome oxidase subunit 1 and internal transcribed spacer-2, respectively. Two abdomen and one thorax pools from A. albopictus were positive for D. immitis, with minimum infection rates of 1.14 and 0.51, respectively, and a probability of a single positive specimen to be infected of P = 0.6 % (95 % confidence interval (CI) = 0.12-1.73). Out of 439 examined subjects, 22 (5.0 %) tested positive for Dirofilaria spp. in at least one diagnostic test. A specific D. immitis infestation rate of 3.5 % was found among the privately owned dogs, while shelter dogs tested positive only for D. repens with a prevalence of 3.4 %; one cat tested molecularly positive for D. immitis. There was a significantly higher rate of positivity among guard dogs for D. immitis (odds ratio, 6.24, 95 % CI, 1.26-25.28; P < 0.05). The increasing risk of D. immitis infection in southern Italy is supported by the noteworthy positivity of A. albopictus populations and the cat. Our data highlight the usefulness to include filarioid infestation in routine diagnosis.

Strong expression of TGF-beta in human host tissues around subcutaneous Dirofilaria repens.

Dirofilaria repens and other Dirofilaria species are widely distributed parasitic nematodes of carnivores, which occasionally are transmitted to men, causing subcutaneous nodules. In humans, it usually occurs only as single male or female filariae without production of microfilariae. The non-productive living or dead Dirofilaria worms in subcutaneous biopsies from 15 human patients permitted us to study the role of the pleiotropic and immunoregulatory cytokine transforming growth factor beta (TGF-beta) independent from the influence of microfilariae. Antiserum against latent TGF-beta 1 was used for an immunohistological examination. In the infiltrates around female and male filariae, there occurred strongly TGF-beta-positive macrophages, mast cells, endothelial cells, fibrocytes, and giant cells adjacent to dead worms. In one nodule, secondary lymph follicles were observed with clearly TGF-beta-positive B cells in the mantle zone and weakly positive macrophages and B cells in the germinal centre. A network of CD35-positive follicular dendritic cells was observed in the germinal centre. All Dirofilaria contained Wolbachia endobacteria, which probably had attracted the numerous TGF-beta-negative neutrophils near to the worm. Wolbachia were phagocytosed by neutrophils adjacent to dead filariae. Macrophages and lymphocytes expressed the MHC class II molecule HLA-DR in small accumulations of immune cells in the outer zone of the infiltrate and the mantle zone and germinal centre of secondary lymph follicles. It is concluded that single non-productive Dirofilaria worms elicit a strong expression of TGF-beta. This result is in accordance with observations on Onchocerca volvulus from patients with the hyporeactive (generalised) form.

Identification of immunoreactive proteins of Dirofilaria immitis and D. repens recognized by sera from patients with pulmonary and subcutaneous dirofilariosis.

Human pulmonary and subcutaneous dirofilariosis caused by Dirofilaria immitis and Dirofilaria repens are worldwide diagnosed with increasing frequency. These species are responsible for the development of benign pulmonary and subcutaneous nodules, respectively, that can be confused with lung or cutaneous cancer. The aim of the present work was to identify D. immitis and D. repens proteins differentially recognized by serum samples from individuals with human pulmonary and subcutaneous dirofilariosis, using two-dimensional electrophoresis and mass spectrometry. Twenty-three immunoreactive proteins of D. immitis and 15 of D. repens were identified. The results point to the existence of differential antigenic recognition in each species, both in the number and type of proteins recognized. Individuals with pulmonary dirofilariosis recognized, on the proteome of D. immitis, among others, different isoforms of 6 enzymes involved in glycolysis, 3 redox-related proteins with antioxidant capacity and 3 heat shock proteins. Individuals with subcutaneous dirofilariosis recognized on the proteome of D. repens only 3 glycolytic enzymes, one protein involved in redox processes and one heat shock protein. These data suggest that in cases of pulmonary dirofilariosis there exists a wider recognition of immunoreactive D. immitis proteins related to key survival processes, such as energy generation, the struggle against oxidative stress and molecular repair, than in cases of human subcutaneous dirofilariosis against D. repens. This could contribute to explain the differences described in the capacity of D. immitis and D. repens development and in the frequency of occurrence of pulmonary and subcutaneous dirofilariosis in the human host.

[The first autochthonous case of subcutaneous dirofilariosis in Austria]. / Der erste autochthone Fall einer subkutanen Dirofilariose in Osterreich.

Dirofilaria repens, a common parasite of carnivore mammals, is transmitted by mosquitos and may also infect humans accidentally causing a subcutaneous dirofilariosis. Autochthonous infections of dogs, foxes or cats with D. repens has not been found in Austria so far; however, 12 cases of imported subcutaneous dirofilariosis in man were noticed between 1981 and 2005. The recent case report deals with the 13(th) human case, which has to be considered as the first autochthonously acquired in Austria.

Wolbachia in Dirofilaria repens, an agent causing human subcutaneous dirofilariasis.

Human subcutaneous dirofilariasis is an increasingly reported zoonosis caused by several filarial species, in particular by Dirofilaria (Nochtiella) repens. Like many filarial worms, D. repens harbors the bacterial endosymbiont Wolbachia that has been implicated in the inflammatory features of filarial infection. Immunohistochemical staining against the Wolbachia surface protein (WSP) was carried out on 14 skin nodules and showed numerous bacteria within the intact worms and occasional positive staining within the surrounding inflammatory infiltrate. Serum samples from 11 of these patients resulted positive for total immunoglobulin G titers against WSP as examined in enzyme-linked immunosorbent assay. This is the first description of Wolbachia distribution in D. repens and the first report of specific immune response to Wolbachia in patients with subcutaneous dirofilariasis.

[Dirofilariasis--a rare antroponosis. Review of the literature and a report of two clinical cases].

Dirofilariasis is a rare anthroponotic endemic disease caused by nematodes. The dog is the reservoir of this parasite and men are accidentally infected by mosquitoes. A subcutaneous mass or pulmonary nodules are the typical clinical presentation. Here, we present two cases of human dirofilariosis and a make a review of the literature.

Comparison of serological tests for the detection of natural heartworm infection in cats.

Serological tests were performed on 380 cats with necropsy-confirmed heartworm disease to compare the performance of currently available commercial laboratory and point-of-care heart-worm serological tests in a heartworm-endemic area. Overall, antigen tests detected 79.3% to 86.2% of heartworm infections and were highly specific. Most cats with false-negative antigen tests had a single male worm. Antibody tests detected 62.1% to 72.4% of heartworm infections and had a wider range of false-positive results (1.4% to 19.1%) than antigen tests (0.3% to 2.0%). Serological tests for feline heartworm infection varied in diagnostic performance. Combining results from antigen and antibody tests achieved greater sensitivity than using either test alone.

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 Guaraqueçaba and Ilha das Peças, presented the highest number of heartworm-infected dogs.

[Migrating subcutaneous swellings due to dirofilariasis after a visit to the South of France]. / Migrerende subcutane zwellingen door dirofilariasis na een bezoek aan Zuid-Frankrijk.

In two women, aged 59 and 31 years, who suffered from an itching cutaneous nodule, subcutaneous dirofilariasis was diagnosed. The disease is characterised by recurrent migrating subcutaneous nodules and swellings. Both patients recently visited the South of France. Laboratory examination revealed a moderate eosinophilia. In both patients, filaria antibodies were demonstrated during the course of the disease and a nodule was eventually resected. Parasitological examination revealed Dirofilaria repens. After this the swellings did not recur. Dirofilariasis is a zoonosis common in dogs and cats, and is transmitted by mosquitoes. In humans, the most common clinical manifestation is a migrating subcutaneous nodule in the case of D. repens, and a pulmonary infiltrate (coin lesion on radiographs) in the case of Dirofilaria immitis. Filarial serology is often positive. The diagnosis is confirmed by parasitological examination of the excised nodule. Dirofilariasis is usually innocuous; it disappears after the worm has died but can cause patients to worry.

A factor of inducing IgE from a filarial parasite prevents insulin-dependent diabetes mellitus in nonobese diabetic mice.

Parasitic helminth infections are characterized by eosinophilia and markedly elevated levels of circulating antigen-nonspecific immunoglobulin E (IgE), responses from which concern helminth protection. We previously purified a factor from Dirofilaria immitis that induces antigen-nonspecific IgE in mice and rats. Recombinant DiAg (rDiAg) has various biological activities. It is also known that parasitic helminth infection generates tremendous Th2 responses. The nonobese diabetic (NOD) mouse spontaneously develops Th1 cell-dependent autoimmune diabetes. Here we investigated the effects of rDiAg on the initiation and progression of this disease. rDiAg treatment of 6-week-old NOD females (the age at which insulitis typically begins) completely prevented insulitis and diabetes. Thus, rDiAg impaired the islet Ag-specific Th1 cell response in vivo, and the prevention of diabetes by rDiAg was associated with switching of the response from a Th1 to a Th2 profile. Since rDiAg clearly prevented insulitis by inhibiting the development and further accumulation of pathogenic Th1 cells to islets of Langerhans, we conclude that DiAg is a native Th2 inducer in filarial helminth and that Th1 responses are required for early events in the development of spontaneous autoimmune diabetes. In conclusion, the presence of parasitic helminth infections may play an important role as an immunomodulator in some autoimmune diseases or allergies.

Serological assays on eight cases of human dirofilariasis identified by morphology and DNA diagnostics.

Specimens of Dirofilaria sp. removed from eight Italian patients were identified as D. repens by morphology and confirmed as such by a PCR-based method of DNA analysis. Blood samples were also drawn from the patients so that two serological tests (ELISA and western blot), one based on the recognition of molecular markers recently identified in the somatic antigenic complex of D. repens, could be evaluated. The antigenic complex used in the ELISA only gave a weak sensitivity. However, the western-blot assays, based on the polypeptide molecular markers, were found to have greater sensitivity and should be useful in detecting human cases of dirofilariasis.

Subconjunctival infection with Dirofilaria repens: serological confirmation of cure following surgery.

Cases of zoonotic dirofilariasis infection, caused by Dirofilaria repens, occur widely throughout European, African, Middle Eastern, and Asian countries. The reports of this infection in humans in Spain are limited, and we herein report the case of a 43-year-old man from Elche (Alicante), Spain, who was seen with acute hyperemic reactivity of the temporal limbus of the right eye. A large nematode was visualized on examination and the intact worm was surgically removed. The parasite was identified as a mature but infertile female D repens. The level of serum antibodies against D repens was monitored for 6 months after surgery using immunoenzymatic assays. Serological results confirmed, as expected, the presence of a single worm and the parasitological cure after the surgical removal of the parasite. To our knowledge, this is the fourth autochthonous case of D repens infecting humans in Spain and also the first autochthonous case of subconjunctival localization.

Prevalence of heartworm infection in cats with signs of cardiorespiratory abnormalities.

OBJECTIVE: To determine prevalence of heartworm infection in a population of pet cats with cardiorespiratory abnormalities and to determine relative usefulness of clinical signs and tests in diagnosis of heartworm disease. DESIGN: Prospective case series. ANIMALS: 100 client-owned cats with clinical signs of cardiorespiratory abnormalities. PROCEDURE: Cats were evaluated using CBC, modified Knott test, ELISA for serologic detection of heartworm antigen and antibodies to heartworms, thoracic radiography, and echocardiography. Cats were considered infected if they had circulating microfilaria, heartworm antigens in serum, or if heartworms were detected by echocardiography or on necropsy. Cats were considered suspicious for infection if they had 2 of the following: serum antibodies to heartworms, eosinophilia or basophilia, or indicative radiographic findings. RESULTS: 9 cats were infected with heartworms, resulting in a prevalence of 9%; 26 cats had evidence of heartworm exposure (i.e., serum antibodies to heartworms). Twenty cats were considered suspicious for heartworm infection. Some outdoor exposure was reported twice as often in heartworm-infected cats, compared with noninfected and suspicious cats. However, a third of infected cats were reportedly housed totally indoors. Cough and dyspnea were strong indicators of heartworm disease. Eight of 9 infected cats had serum antibodies to heartworms and heartworm antigen in serum. Thoracic radiography and echocardiography indicated heartworm infection in 6 and 7 of the 9 cats, respectively. CLINICAL IMPLICATIONS: Cough or dyspnea may indicate heartworm disease in cats; serologic tests, echocardiography, and radiography are most useful diagnostic procedures. Although living indoors is protective, it may not preclude heartworm infection in cats.

Presence of Dirofilaria repens and an insect immunocyte (plasmatocyte) in a human subcutaneous nodule, induced by a mosquito bite.

It is well known that the nematode Dirofilaria repens is transmitted to humans by vector mosquito bite. Examination of a fine needle aspiration biopsy drawn from a month-old nodule on the chest of a woman, residing in Garlasco, province of Pavia, Northern Italy, revealed the presence of not only one immature female of D. repens, but also some scattered cells that we believe to be mosquito's blood cells, plasmatocytes (immunocytes). We presume that plasmatocytes were carried into the bite wound with the mosquito's hemolymph that had oozed from a rupture in its mouthparts during feeding. Because Aedes albopictus recently colonized certain areas in the above region, we suspect that the nodule resulted from the bite of this mosquito.

Human humoral immune response to Dirofilaria species.

Dirofilaria immitis and D. repens, the agents of human pulmonary and subcutaneous dirofilariosis respectively, may coexist in areas of Southern Europe, and L3 and L4 of both species develop in subcutaneous tissue. Previous studies have shown that humans develop high levels of specific IgM, IgG and IgE anti-D. immitis. An antigen of approximately 22 kDa (Di22) is a marker of pulmonary dirofilariosis. In this work, we demonstrate that D. repens also induces IgG in infected humans. Polypeptides between 40 and 26 kDa from adult somatic antigenic complex of the later species, are specifically recognized by sera from individuals with subcutaneous dirofilariosis due to D. repens. These findings complement the previous studies on specific antigens for the serological diagnosis of pulmonary dirofilariosis.