Application of Dirofilaria immitis immunoreactive proteins in serodiagnosis.

Dirofilariasis is a zoonotic global vector-borne disease caused by Dirofilaria immitis. The present study focuses on the somatic and excretory/secretory (E/S) proteins released from adult D. immitis. We aimed to fractionate and identify adult D. immitis immunoreactive proteins. Somatic and E/S extracts were immunoblotted to identify the immunoreactive proteins. In the current study, we used matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF/MS) to characterize the immunogenic proteins. Additionally, we used fast protein liquid chromatography (FPLC) to fractionate and evaluate the immunogenicity of the D. immitis secretome. The most immunoreactive proteins were between 10 and 48 kDa. Six proteins including polyprotein antigen, P22u, pepsin inhibitor Dit33, neutrophil chemotactic factor (DiNCF) precursor, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and heat-shock protein 70 (HSP70) were found in both somatic and E/S extracts. Eluting the FPLC column with NaCl resolved two peaks in which the immunoreactivities of the purified proteins were conserved. Characterization of these proteins could provide a novel perspective for understanding the pathogenesis and diagnosing of this disease.

Evaluation of cardiopulmonary and inflammatory markers in dogs with heartworm infection during treatment with the 2014 American Heartworm Society recommended treatment protocol.

BACKGROUND: Heartworm disease in dogs is a life-threatening parasitic disease. Although adulticide treatment with melarsomine has been proven to be the most effective, complications associated with adulticide treatment are major concerns for clinicians. METHODS: This study evaluated the change in levels of D-dimer, interleukin-6, C-reactive protein and cardiac troponin I in 12 dogs with different severities of heartworm infection treated by the American Heartworm Society (AHS) recommended protocol during the treatment period. The serum levels of several markers were measured on the day of diagnosis (T-60), before the initiation of melarsomine therapy (T0), 1 day after the first injection (T1), 1 week after the first injection (T7), 1 month after the first injection (T30), 1 day after the second injection (T31), 1 day after the third injection (T32), 1 week after the third injection (T39), 1 month after the third injection (T62), 2 months after the third injection (T92), 3 months after the third injection (T122), and 6 months after the third injection (T182). RESULTS: The serum levels of these markers were significantly different at the test time point after melarsomine treatment and also differed significantly according to the stage of heartworm disease in the dogs. CONCLUSION: This study found that monitoring of inflammatory and hemostatic markers in dogs with heartworm disease being treated with melarsomine might be beneficial in predicting the clinical outcomes and complications associated with melarsomine treatment.

Seroreactivity to Dirofilaria antigens in people from different areas of Serbia.

BACKGROUND: The Northern part of Serbia is hyperendemic-endemic for canine dirofilarioses. Considering this fact, many human dirofilarial infections could be expected, however only about 30 cases in Serbia have been described until today. Aims of this survey were to assess the people reactivity to the antigens of D. repens and D. immitis and to identify risk factors for the contact exposure. METHODS: Investigation included sera taken from 297 people (179 women and 118 men) living in different areas of Serbia (Pancevo, Novi Sad, Zajecar, Leskovac, Vranje, Nis, Pirot). Sera were analysed by means of two indirect enzyme-linked immunosorbent (ELISA) home-designed that use as antigens adult somatic/metabolic polyproteins of D. repens (DR) and D. immitis (DI), respectively. The results were elaborated using the statistical method of descriptive and quantitative analysis. RESULTS: Significant differences by area in the reactivity of human sera to dirofilarial antigens were not observed (p = 0.056). A high seroreactivity was demonstrated in people from the towns of northern Serbia (Pancevo = 27,1%; Novi Sad = 16,3%), as well as in people from Zajecar (eastern Serbia = 15,8%) and Vranje (southern Serbia = 15,1%). No differences were evidenced between people reactivity to polyproteins of the two dirofilarial species, nor differences related to the gender of examinees. Factor risks evidenced were: i) place of residence; ii) spending work time outdoors during the mosquito season; iii) spending time outdoors and nearby rivers, lakes, swamps or canals; unespectedly, iv) cat owning. CONCLUSION: The findings emerging from this investigation indicate that clinicians and public health authorities should pay greater attention to this zoonosis. Continuing education and training of physicians will greatly contribute to the knowledge of the actual impact of filarial worms on animal and public health, and allow for the planning of suitable measures to prevent the infections.

First report of Dirofilaria immitis in the Republic of Cape Verde.

In Maio Island, Republic of Cape Verde, a seven-year old mongrel female dog exhibiting severe generalized adenomegaly and a poor body condition was examined during an animal welfare campaign. A blood smear was drawn from peripheral blood collection and several organisms consistent with Dirofilaria immitis microfilariae were identified. Both the antigen test conducted from plasma and the RT-PCR test performed from the blood smear sample were positive for D. immitis. This is, to the best of our knowledge, the first report of D. immitis in Cape Verde. The fact that the dog was autochthonous and had never left the island strongly suggests there might be other animals infected with the parasite. Our finding confirms the existence of the parasite in the canine population and necessarily implies the presence of a competent vector. As a serious cardiopulmonary disease and with the risk of the pathogen spreading rapidly, broader epidemiological studies need to be conducted to determine D. immitis prevalence in the canine population of Maio Island.

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.

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.

Allergic inflammatory reaction is involved in necrosis of human pulmonary dirofilariasis.

AIMS: To determine whether, in view of the massive inflammatory cell infiltration and the rounded rather than wedge-shaped character of pulmonary lesions in dirofilariasis, the inflammatory response against the worm contributes to the coagulative necrosis, in addition to an ischaemic process. METHODS AND RESULTS: The histopathological features of 13 resected dirofilariasis cases with well-defined nodules ranged from 10 to 30 mm were analysed. On routine histology and using immunohistochemistry, the peripheral encapsulating wall showed mild to severe infiltration of eosinophils, lymphocytes and plasma cells and a histiocytic reaction in all cases, often with necrotic eosinophils seen within the necrosis (84.6%) and inflammatory changes in the adjacent lung (38.5%). The CD4+ lymphocyte count (80.8 +/- 33.4) was greater than that of CD8+ lymphocytes (24.5 +/- 16.9) in the central necrosis and vice versa in the wall. In the necrotic regions, disruption of the pulmonary artery (61.5%) and extravasation of the torn worm (23.1%) could be seen. CONCLUSIONS: These findings indicate that an allergic inflammatory reaction, mediated by eosinophils and lymphocytes, is involved in the formation of the dirofilarial necrotizing granuloma rather than infarction caused simply by embolism.

Feline dirofilariosis: antibody response to antigenic fractions containing specific 20 to 30 kDa polypeptides from the adult Dirofilaria immitis somatic antigen.

Fractions from the adult somatic antigen (SA) Dirofilaria immitis complex, containing polypeptides from 20 to 30kDa, previously identified as molecular markers of feline dirofilariosis are isolated by sequential application of gel filtration and anion exchange chromatography. Indirect enzyme-linked immunosorbent assays, employing these fractions (20-26kDa/ELISAF1 and 30kDa/ELISAF7) show multivalent diagnostic capacities: they were able to detect pre-patent infections 2 months after infection, infections in clinical phase, and the fall of antibodies after the worms were removed from the heart, or the application of a ivermectin treatment. The results obtained by the two tests correlated well, in spite of the fact that ELISAF1 was most useful to detect antibodies in sera from cats in the clinical phase, while ELISAF7 has more sensitivity for the early detection of the infections. Both ELISAs were useful in the detection of the decrease of antibodies after the worms were removed by surgery or pharmacological treatment.

IgG response against infective larvae of Dirofilaria immitis in experimentally infected cats.

Somatic antigens from third stage larvae of Dirofilaria immitis (SL3) were used to detect IgG response against heartworm infection in 8 experimentally infected cats. A moderate specific anti-SL3 IgG response was found one month post-infection. Afterwards, antibodies decreased reaching a basal level 4 months post-infection and remained at this level until the end of the study. 6 months post-infection. Western blot analysis showed specific recognition of polypeptides of 79, 73, 60, 52, 40 and 39 kDa by sera from infected cats 1 month post-infection, but not by sera taken prior to the infection. The low antigenicity of the SL3 antigen in the cat should allow the parasite to escape the host's immune response.

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.

Utility of antibodies against a 22 kD molecule of Dirofilaria immitis in the diagnosis of human pulmonary dirofilariasis.

To assess the characteristics of an ELISA test for the diagnosis of human pulmonary dirofilariasis, we studied the sera of 24 subjects with other helmintoses and of 37 patients suffering from non-parasitic focal lung diseases, comparing them with negative and positive sera. ELISA and Western blot with complete somatic antigen and ELISA with protein Di22 (specifically recognized in cases of lung dirofilariasis) were performed. With ELISA SA the false positive rate was 25% in cases with other parasitoses and 30% in cases with focal lung diseases. ELISA Di22 decreases this positivity levels. Only 2 cases with visceral larva migrans (8.3%) and a case with lung nodules metastatic from renal adenocarcinoma (2.7%) were positive. ELISA Di22 therefore greatly decreases the false positive rate of ELISA SA.

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.

Prevalence of Dirofilaria immitis infection in cats in Saitama, Japan.

To clarify Dirofilaria immitis infection among cats in Saitama Prefecture, Japan, 1,840 cats were examined postmortem for adult worms and microfilariae in the blood from 1989 to 1995. As a reference control, 500 dogs from the same area were examined in the same way and period. D. immitis worms were found in 15 cats, one of which had microfilariae in the blood. Prevalence rate of D. immitis infection was 0.8% (15/1,840) in cats and 46.8% (234/500) in dogs examined, whereas it was 4.1% and 64.6% in cats and dogs, respectively, aged 2 years and over. Worm burden per positive cat was 1.5 +/- 0.7 (mean +/- SD), the maximum number of worm was 3 in 2 cats, and 10 cats had a single worm each. All the worm-positive cats were tested for antibodies to feline immunodeficiency virus (FIV) and antigens of feline leukemia virus (FeLV) in sera. Positive rates of coinfection with D. immitis were 26.7% and 13.3% for FIV and FeLV, respectively.

Dirofilaria immitis: experimental infection of rabbits with immature fifth-stage worms.

To establish an animal model for human pulmonary dirofilariasis, we experimentally infected nine rabbits with immature fifth-stage worms of Dirofilaria immitis. The rabbits were infected by subcutaneous transplantation with various numbers of immature worms collected from 110- and 120-day-old infections of dogs. Four of seven rabbits infected with up to four larvae possessed encapsulated worms in the lungs at 196 or 308 days post-transplantation. Two rabbits transplanted with eight worms died of pulmonary hemorrhagic infarction 18 and 28 days post-transplantation. Marked histopathologic changes were observed in the lungs at the site of degenerating worms, which were encapsulated by a fibrous wall. Severe to mild infiltrations with eosinophils, heterophils, lymphocytes, plasma cells, and histiocytes were found in granulomas and their surrounding areas. The findings in these rabbits resemble those reported for human cases of pulmonary dirofilariasis.

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.

[Human dirofilariasis: identification of a 44kD antigen recognized by IgM against Dirofilaria immitis]. / Dirofilariosis humana: identificación de un antígeno de 44 kD reconocido por IgM anti-Dirofilaria immitis.

BACKGROUND: Dirofilaria immitis is a nematode parasite of canids that, in endemic areas, can infect humans. The use of adult somatic antigens for the diagnosis of human dirofilariasis has the disadvantage of a low sensibility because of cross-reactivity with other helminths, specially with IgM antibodies. We describe in this work proteins of the adult somatic antigenic complex that are specifically recognized by human IgM. METHODS: Human sera with different characteristics were analyzed by an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-D. immitis IgM seropositive individuals. Moreover, an enzyme-linked immuno-electro-transfer blot (EITB) was performed for the detection of polypeptides that specifically react with IgM anti-D. immitis. RESULTS: EITB analysis shows that 2 proteins of 44 and 18 kD are recognized by IgM in human sera from an endemic area with a high antibody titer against D. immitis measured by ELISA. Sera from a non-endemic area and from patients suffering from other parasitic and non-parasitic diseases did not recognize these antigens. CONCLUSIONS: We believe that the 44 kD protein could be a good marker of recent D. immitis infection.

Immunoblot analysis of Dirofilaria immitis recognized by infected humans.

To help to develop better diagnostic tests for zoonotic pulmonary dirofilariasis, seven patients with histologically confirmed pulmonary dirofilariasis were evaluated using the immunoblot technique. Six patients exhibited a response to the excretory-secretory (AS) antigen proteins with molecular weights of 20-19.5, 17.5-17 and 14 derived from Dirofilaria immitis adult works. However, when adult worm extracts were used as the antigen, there was no qualitative difference in antigen recognition between the Dirofilaria patients, other patients with non-filarial parasitic infections or with lung cancer or tuberculosis, and normal individuals. All the sera, not only those of the Dirofilaria patients but also those of the non-filarial patients and the controls, strongly cross-reacted with the 18-kDa ES antigen. These findings suggest that the ES antigen of D. immitis provides a more sensitive antigen than does the adult somatic antigen; but the ES antigen and the adult somatic antigen have a common antigenic band at 18 kDa.

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.