Detection of heartworm antigen without cross-reactivity to helminths and protozoa following heat treatment of canine serum.

BACKGROUND: Detection of Dirofilaria immitis, or heartworm, through antigen in sera is the primary means of diagnosing infections in dogs. In recent years, the practice of heat-treating serum prior to antigen testing has demonstrated improved detection of heartworm infection. While the practice of heat-treating serum has resulted in earlier detection and improved sensitivity for heartworm infections, it has been suggested that heat treatment may cause cross reactivity with A. reconditum and intestinal helminth infections of dogs. No studies have assessed the potential cross-reactivity of these parasites with heartworm tests before and after heat treatment using blood products and an appropriate gold standard reference. METHODS: Canine sera (n=163) was used to evaluate a heartworm antigen-ELISA (DiroCHEK®) and potential cross-reactivity with common parasitic infections. The heartworm status and additional parasite infections were confirmed by necropsy and adult helminth species verified morphologically or by PCR, and feces evaluated by centrifugal fecal flotation. RESULTS: Intestinal parasites were confirmed in 140 of the dogs by necropsy, and 130 by fecal flotation. Acanthocheilonema reconditum microfilariae were confirmed in 22 dogs. Prevalence of heartworm infection confirmed by necropsy was 35.6% (58/163). In the 105 dogs without heartworms, specificity remained unchanged at 100% both before and after heat treatment despite confirmed infections with A. reconditum, Ancylostoma caninum, Ancylostoma brasiliense, Trichuris vulpis, Toxocara canis, Dipylidium caninum, Spirometra mansonoides, Macracanthorynchus ingens, Cystoisospora sp., Giardia sp., and Sarcocystis sp. CONCLUSIONS: These findings suggest that the use of heat treatment improves sensitivity of heartworm tests and is unlikely to cause false positive antigen results due to Acanthocheilonema reconditum, intestinal helminths, and protozoal parasites in dogs.

Dirofilaria immitis proteins recognized by antibodies from individuals living with microfilaremic dogs.

INTRODUCTION: Dirofilaria immitis is a nematode that affects human health in several countries of the world. This study was conducted to examine whether serum samples from the owners of microfilaremic dogs present immunoreactivity to parasite proteins. METHODOLOGY: Eight serum samples from the owners of microfilaremic dogs were examined. Total proteins were extracted from adult worms and 12% SDS-PAGE was performed. The gel was electroblotted to a nitrocellulose membrane, and a Western blot (WB) was performed. Reactive bands of 22, 33, 39, 49, and 63 kDa in WB were excised from the gel and analyzed by mass spectrometry (MS). RESULTS: The MS results showed the presence of 10 different proteins of D. immitis recognized by the human serum samples. CONCLUSIONS: These results indicate that in endemic areas of D. immitis, owners of infected dogs recognize specific proteins of the parasite, suggesting a possible infection.

Angiogenic response in an in vitro model of dog microvascular endothelial cells stimulated with antigenic extracts from Dirofilaria immitis adult worms.

BACKGROUND: Angiogenesis can occur under pathological conditions when stimuli such as inflammation, vascular obstruction or hypoxia exist. These stimuli are present in cardiopulmonary dirofilariosis (Dirofilaria immitis). The aim of this study was to analyze the capacity of D. immitis antigens to modify the expression of angiogenic factors and trigger the formation of pseudocapillaries (tube-like structures) in an in vitro model of endothelial cells. METHODS: The expression of VEGF-A, sFlt, mEndoglin and sEndoglin in cultures of canine microvascular endothelial cells stimulated with extract of adult worms of D. immitis obtained from an untreated dog (DiSA) and from a dog treated for 15 days with doxycycline (tDiSA), was determined by using commercial kits. The capacity of pseudocapillary formation was evaluated analyzing cell connections and cell groups in Matrigel cell cultures stimulated with DiSA and tDiSA. In both cases non-stimulated cultures were used as controls. RESULTS: First, we demonstrated that worms obtained from the dog treated with doxycycline showed a significantly lower amount of Wolbachia (less than 60%) than worms removed from the untreated dog. Only DiSA was able to significantly increase the expression of the proangiogenic factor VEGF-A in the endotelial cells cultures. None of the D. immitis extracts modified the expression of sFlt. tDiSA extract was able to modify the expression of the endoglins, significantly decreasing the expression of the pro-angiogenic mEndoglin and increasing the anti-angiogenic sEndoglin. The formation of pseudocapillaries was negatively influenced by tDiSA, which reduced the organization and number of cellular connections. CONCLUSIONS: The ability of antigens from adult D. immitis worms to modify the expression of pro and anti-angiogenic factors in endotelial cell cultures was demonstrated, as well as the trend to form pseudocapillaries in vitro. The capacity of stimulation may be linked to the amount of Wolbachia present in the antigenic extracts.

Highly modified and immunoactive N-glycans of the canine heartworm.

The canine heartworm (Dirofilaria immitis) is a mosquito-borne parasitic nematode whose range is extending due to climate change. In a four-dimensional analysis involving HPLC, MALDI-TOF-MS and MS/MS in combination with chemical and enzymatic digestions, we here reveal an N-glycome of unprecedented complexity. We detect N-glycans of up to 7000 Da, which contain long fucosylated HexNAc-based repeats, as well as glucuronylated structures. While some modifications including LacdiNAc, chitobiose, α1,3-fucose and phosphorylcholine are familiar, anionic N-glycans have previously not been reported in nematodes. Glycan array data show that the neutral glycans are preferentially recognised by IgM in dog sera or by mannose binding lectin when antennal fucose and phosphorylcholine residues are removed; this pattern of reactivity is reversed for mammalian C-reactive protein, which can in turn be bound by the complement component C1q. Thereby, the N-glycans of D. immitis contain features which may either mediate immunomodulation of the host or confer the ability to avoid immune surveillance.

Detection of Dirofilaria immitis antigen and antibodies against Anaplasma phagocytophilum, Borrelia burgdorferi and Ehrlichia canis in dogs from ten provinces of China.

Despite the fact vector-borne diseases (VBDs) have been increasingly reported in dogs worldwide, there are only limited reports on VBDs in dogs in China with most being based on molecular detection of active infections. To provide further data on the exposure of dogs in China to VBD agents, we used commercial immunochromatographic assays to test plasma from 637 apparently healthy indoor and breeding colony dogs from 21 veterinary clinics in 10 provinces in China and a commercial dog breeding facility for circulating antigen of Dirofilaria immitis, and for circulating antibodies against Ehrlichia spp., Anaplasma spp., and Borrelia burgdorferi. Overall, we found only low levels of exposure to Ehrlichia spp. (4.7%; 30/637), Anaplasma spp. (1.4%; 9/637), B. burgdorferi (0.9%; 6/637) and D. immitis (0.2%; 1/637) with most of the positive animals coming from the commercial breeding colony (26/103; 25.2%) where ectoparasites were most commonly noted. At least one vector-borne agent was found in dogs from 6 of the 10 provinces investigated. Our results confirm exposure of dogs from around China to a variety of VBDs, even indoor pets seldom observed to harbor ectoparasites.

Heartworm extract induces relaxation of isolated rat thoracic aorta.

Isolated rat thoracic aortic strips undergoing noradrenaline-induced contraction were treated with an adult heartworm (HW) crude extract and then examined for isometric changes in tension. HW extract caused relaxation of endothelium-intact strips, but not endothelium-denuded strips. This effect was inhibited by treatment with NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) and could be reversed by additional treatment with L-arginine. However, HW extract at a high concentration caused slight relaxation of endothelium-denuded strips, and relaxation persisted after L-NAME treatment in endothelium intact-strips. These data suggested that the relaxation induced by HW extract was mainly endothelium-dependent, nitric oxide-mediated, but in part, also endothelium-independent. In addition, a bioassay using isolated rat thoracic aortas may be a useful tool for investigating vasoactive substances in the HW extract.

Interaction of macrocyclic lactones with a Dirofilaria immitis P-glycoprotein.

Dirofilaria immitis, a filarial nematode, causes dirofilariasis or heartworm disease in dogs, cats and wild canids. Effective prevention of the disease is mainly by the use of the macrocyclic lactone class of drugs as heartworm preventives, and no other class of drugs is effective for preventing infection. Macrocyclic lactones have been used for prevention of heartworm infection for more than 26years. However, prevention has been compromised by the development of resistance in recent years. The mechanism of macrocyclic lactone resistance in D. immitis has yet to be established. In other parasitic nematodes, P-glycoproteins (PGPs) have been implicated in macrocyclic lactone resistance. The presence of two polymorphic loci on D. immitis P-glycoprotein-11 (Dim-pgp-11) correlated with loss of efficacy of macrocyclic lactone anthelmintics, suggesting that PGPs may be involved in macrocyclic lactone resistance in D. immitis. We have identified the full length of Dim-Pgp-11 cDNA, expressed it in mammalian cells, and studied the functional activity of the expressed protein. We have characterised its interaction with the four macrocyclic lactone preventives, ivermectin, selamectin, moxidectin and milbemycin oxime, using the transport of different fluorescent substrates. The inhibitory effect of these macrocyclic lactones on the transport of two fluorophore probes, Rhodamine 123 and Hoechst 33342, by Dim-PGP-11 has been studied. The avermectins, ivermectin and selamectin, markedly inhibited Rhodamine 123 transport in a concentration-dependent and saturable manner, whereas the milbemycins, moxidectin and milbemycin oxime, were found to have different inhibition profiles with Rhodamine 123 transport. However, both avermectins and milbemycin preventives inhibited the transport of Hoechst 33342 by Dim-PGP-11 in a concentration-dependent and apparently saturable manner, although differences existed in terms of efficiency and potency of inhibition between the two sub-classes of macrocyclic lactones. We postulate that Dim-PGP-11 may have two to three drug binding sites, as with mammalian Pgp, including the 'R' site for Rhodamine 123 and the 'H' site for Hoechst 33342. The avermectins appear to bind the 'R' binding site unlike the milbemycins, whereas both sub-classes of macrocyclic lactones might interact with the 'H' site of D. immitis PGP-11.

Expression of translationally controlled tumor protein (TCTP) gene of Dirofilaria immitis guided by transcriptomic screening.

Dirofilaria immitis (heartworm) infections affect domestic dogs, cats, and various wild mammals with increasing incidence in temperate and tropical areas. More sensitive antibody detection methodologies are required to diagnose asymptomatic dirofilariasis with low worm burdens. Applying current transcriptomic technologies would be useful to discover potential diagnostic markers for D. immitis infection. A filarial homologue of the mammalian translationally controlled tumor protein (TCTP) was initially identified by screening the assembled transcriptome of D. immitis (DiTCTP). A BLAST analysis suggested that the DiTCTP gene shared the highest similarity with TCTP from Loa loa at protein level (97%). A histidine-tagged recombinant DiTCTP protein (rDiTCTP) of 40 kDa expressed in Escherichia coli BL21 (DE3) showed immunoreactivity with serum from a dog experimentally infected with heartworms. Localization studies illustrated the ubiquitous presence of rDiTCTP protein in the lateral hypodermal chords, dorsal hypodermal chord, muscle, intestine, and uterus in female adult worms. Further studies on D. immitis-derived TCTP are warranted to assess whether this filarial protein could be used for a diagnostic purpose.

Identification of Dirofilaria immitis immunoreactive proteins recognized by sera from infected cats using two-dimensional electrophoresis and mass spectrometry.

The aim of the present work was to identify proteins of Dirofilaria immitis recognized by the immune system of naturally and experimentally infected cats, using two-dimensional electrophoresis and mass spectrometry. Thirty-five immunoreactive proteins of D. immitis were identified. These proteins are involved in metabolism, plasminogen binding, anti-oxidant and detoxificant activity, up-regulation of the Th2 anti-inflammatory response and other processes. The timing evolution of this recognition pattern indicated that at 2 months post-infection a wide recognition of many parasite proteins belonging to many functional groups is still observed, increasing progressively during the course of the infection. The real effect on the vital capacity of D. immitis worms and on the development of pathological events of feline dirofilariosis will be investigated in the future.

Identification of immunoreactive proteins from the dog heartworm (Dirofilaria immitis) differentially recognized by the sera from dogs with patent or occult infections.

Heartworm disease caused by Dirofilaria immitis affects canine and feline hosts. Moreover, the parasite can infect humans, causing pulmonary dirofilariosis. Most affected dogs have patent infections with circulating microfilariae in peripheral blood, although infected dogs sometimes develop occult infections characterized by the absence of microfilariae. Microfilaremic infections (mf+) are associated with a predominant Th2-type immune response, whereas a Th1-type response predominates in amicrofilaremic infections (mf-), suggesting a role for this response in the suppression of circulating microfilariae. However, nothing is known about the molecules involved in the immune regulation of these infections. The objective of the present work was to identify the parasite proteins recognized differentially by the immune response of dogs with patent or occult infections, using two-dimensional electrophoresis and mass spectrometry. Nineteen proteins of D. immitis were identified, of which 6 were immunoreactive against serum samples from both mf+ and mf- dogs, while another two groups of 6 and 7 different proteins were differentially recognized by sera from mf+ or mf- dogs, respectively. The results point to the existence of differential antigen recognition in patent and occult infections due to D. immitis. Several proteins that could be involved in the immune regulation of these infections are identified. Additionally, the findings seem to suggest that some antigens of D. immitis, together with Wolbachia antigens, could contribute to the stimulation of the Th1-type response.

Th1 response in BALB/c mice immunized with Dirofilaria immitis soluble antigens: a possible role for Wolbachia?

The immune response to filarial infection has been shown to be of both the Th1 and Th2 types. Studies aimed at developing immunization strategies against Dirofilaria immitis infection in dogs have shown that protection against larval challenge is of the Th2 type and that several proteins are recognized by immunized or infected animals. The bacterial endosymbiont Wolbachia, harbored by many filarial species including D. immitis, has recently been shown to interact with the host immune system. Specific antibodies to the Wolbachia recombinant surface protein (WSPr) have been observed in cats infected with D. immitis. In this work the authors have determined cytokine production and antibody response in BALB/c mice inoculated with soluble antigens from third stage larvae or from adult worms of D. immitis. Inoculated mice first produced IFN-gamma followed by a peak in IL-4. Specific antibodies to the Wolbachia protein WSPr were exclusively IgG2a, while antibodies against peptides derived from antigens of D. immitis were in the IgG1 and IgE subclasses. The cytokine response is thus similar to that reported for other filarial infection, where Th1 response shifts towards Th2. Antibody response indicates that Wolbachia may induce preferentially a Th1 response during filarial infection, while nematode antigens may be involved in Th2 response. There is thus an overall agreement with current opinions on the role of bacterial versus nematode molecules in driving the response towards the different directions.

Immunological responses of dogs experimentally infected with Dirofilaria immitis.

Three dogs were experimentally infected with Dirofilaria immitis. All dogs were euthanised at 30, 36 and 37 weeks after inoculation of D. immitis for the recovery of adult worms. Three cases accounted to 42.91 % recovery of inoculated worms. Serum samples from dogs experimentally inoculated with D. immitis were analyzed by ELISA and immunoblotting methods. Antibody titers of dogs detected by ELISA peaked between 7 and 14 weeks then decreased between weeks 15 to 24 followed by another increase during weeks 25 to 30 and persisted throughout the remainder of the experiment period. Analysis of adult D. immitis protein stained with Coomassie brilliant blue R-250 indicated separately more than 10 bands, and the major bands were 22, 40, 46, 56, 70, 72 and 89 kDa. Antigenic identification of extracts antigens of adults D. immitis by immunoblotting analysis revealed several bands from pooled sera of patent infection (30 weeks after inoculation). The detected bands were 24, 70, 80 and 110 kDa, 22, 72 and 84 kDa, and 58 and 72 kDa in dogs 1, 2 and 3, respectively. Results of antibody titers reached high levels on the 4th molting stage after inoculation of infective larva (L3), and reinforced previous findings that high molecular weight regions are detected in young animals.

Element composition and biochemical components of Dirofilaria immitis.

Element composition of whole canine heartworms (Dirofilaria immitis) was determined using inductively coupled argon plasma emission spectroscopy. The elements evaluated included Na, K, Ca, Mg, P, Al, B, Ba, Cu, Fe, Mn, Sr, Zn, As, and Pb. The only significant sex difference was calcium concentration which was higher in females. Amino acid analyses of body fluid revealed presence of nine essential and eight non-essential amino acids. Concentrations for male and female heartworms were not significantly different. The spectrum of amino acids was similar to that previously reported for canine plasma. Neurotransmitters or their metabolites detected in whole male and female heartworms using HPLC with electrochemical detector were epinephrine, dihydroxyphenylacetic acid, dopamine, and 5-hydroxyindoleacetic acid. Those not detected were norepinephrine, homovanillic acid, and 5-hydroxytryptamine.

Immunofluorescent localization of intermediate filaments (IFs) in helminths using anti-mammalian IFs monoclonal antibody.

Intermediate filaments (IFs) make up the cytoskeleton of most eukaryotic cells. In vertebrates, a number of IF proteins have been identified, showing distributions unique to tissue or cell type. Information on helminth IFs is limited to some nematode species. To observe immunofluorescent localization of IFs in helminth tissues, we selected a murine hybridoma clone producing IgM antibody to multiple types of mammalian IF proteins and examined cross-reactivity to helminth proteins. The selected monoclonal antibody (HUSM-9) cross-reacted well with IFs from nematode species such as Toxocara canis, Dirofilaria immitis, Anisakis simplex, and Trichinella britovi; strong immunofluorescence on cryostat sections was detected in the hypodermis, cords, body muscle, smooth muscle of the uterus, and other epithelial structures. In platyhelminths, i.e., adult Schistosoma mansoni, larval Taenia taeniaeformis, adult Taenia crassiceps, and Echinococcus multilocularis protoscolex, the reactivity was weaker than in nematodes, and localized in the body wall muscle and subtegumental tissue. Western blotting of 8 M urea extracts of parasites with the antibody detected a pair of clear bands in nematodes but not in S. mansoni or the cestodes. These results might be explained by sparse distribution of IFs in platyhelminths, or low affinity of the used antibody to platyhelminth IF proteins, or both.

Determination of metabolite and regulatory enzyme levels in Dirofilaria immitis and Ascaris suum: a comparative study.

Studies on metabolite levels in Dirofilaria immitis revealed similarities in several metabolites with those of Ascaris suum. The glycogen level in the filariid was however 3-4 times lower than that in A. suum. Levels of three regulatory enzymes were also determined in D. immitis and compared with those in A. suum. The activities of Hexokinase and Phosphofructokinase were similar. However, the levels of Glycogen phosphorylase b appeared to be much lower in the filariid than in A. suum. The subtle but important differences observed may reflect modifications of the parasite enzymes suggesting salient differences in the regulation of energy production from carbohydrates in the worms. The differences may also represent specialization required for the unique life style of the worms in their different locations in their hosts.

Pathologic findings in dogs with shock induced by intravenous administration of heartworm extract.

OBJECTIVE: To examine pathologic findings in dogs with shock induced by IV administration of heartworm (HW) extract. ANIMALS: 22 mixed-breed adult dogs. PROCEDURES: Heartworm extract was administered IV, and pathologic changes in dogs that died or were euthanatized at 24 hours were examined. RESULTS: The most severe lesions observed during initial collapse were centralobular congestion in the liver, hemorrhage and edema in the gallbladder wall, and congestion and hemorrhage in mucous membranes of the gastrointestinal tract. These findings disappeared with recovery from collapse. Hyalinization of venous walls of the liver and cardiac muscle were observed in dogs that died during initial collapse. Focal coagulation necrosis in hepatic cells was seen in dogs that were euthanatized at 24 hours. One dog with profuse bloody diarrhea that died during secondary collapse had severe hemorrhage in mucous membranes of the large intestine. CONCLUSION: Heartworm extract appeared to contain some substances constricting hepatosplanchnic vessels and some toxic substances that injured the smooth muscle of venous walls, cardiac muscle, and hepatic cells directly or indirectly. CLINICAL RELEVANCE: The shock-inducing substances in HW extract may have an important role in the pathophysiologic mechanism of HW disease, and investigation of them may contribute to prevention of the shock reaction attributable to microfilaricide and adulticide use.

The peroxidoxin 2 protein of the human parasite Onchocerca volvulus: recombinant expression, immunolocalization, and demonstration of homologous molecules in other species.

The peroxidoxin protein of the filarial parasite Onchocerca volvulus (OvPXN-2) belongs to a group of highly conserved antioxidant molecules. For a more detailed characterization of this protein and for determination of its expression pattern the OvPXN-2 protein was recombinantly expressed as a His-tagged protein. Under reducing conditions the recombinant protein had an apparent molecular mass of 28 kDa. Considering the size of the His-tag and the FLAG epitope introduced to the recombinant protein, this size is in agreement with that of the native protein identified in O. volvulus extract. Antiserum raised against the recombinant protein was used for immunolocalization. In O. volvulus the antigen is predominantly expressed in the hypodermis and particularly the lateral and median chords show high levels of expression. The protein is also expressed strongly in the hypodermis of infective larvae and more weakly in microfilariae. Related cross-reacting proteins were detected in several Onchocerca species and other filariae. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with Western blotting revealed proteins with almost identical mobility in extracts prepared from O. ochengi, O. gibsoni, and Dirofilaria immitis.

Cloning and characterization of cDNAs encoding beta-tubulin from Dirofilaria immitis and Onchocerca volvulus.

Beta-Tubulin is the target for the benzimidazole anthelmintics. Unfortunately, none of these drugs is clinically useful against adult filariae. However, beta-tubulin has been shown to be a target for antibody-based toxicity to Brugia pahangi. We cloned and characterized cDNAs encoding beta-tubulin from 2 filariae, Dirofilaria immitis and Onchocerca volvulus, to explore possible explanations for benzimidazole insensitivity among adult filariae and the likelihood that epitopes of beta-tubulin could be used as antigens for a broad-spectrum filarial vaccine. The proteins predicted by these cDNAs were almost identical to the beta-tubulin previously reported from B. pahangi but were less similar to a beta-tubulin cDNA from Onchocerca gibsoni. We cloned the genomic locus for the O. volvulus beta-tubulin cDNA and compared its organization to the reported genomic loci for beta-tubulin in B. pahangi and O. gibsoni. The comparison reinforces the conclusion that the published O. gibsoni gene is in a different family, possibly the beta2 family previously described in B. pahangi. The substitution of tyr for phe at position 200 of beta-tubulin is associated with benzimidazole resistance. All 4 filarial beta-tubulins are predicted to encode phe at this position, suggesting that filarial beta-tubulin is not inherently insensitive to the benzimidazoles. A monoclonal antibody that recognizes the COOH terminus of B. pahangi beta-tubulin is lethal to this parasite in culture. The COOH terminal region is the most variable among the different isotypes of beta-tubulin and distinguishes mammalian from nematode tubulins. This region is highly conserved in 3 of the filarial beta-tubulins.

Molecular characterization of a calcium-binding protein from the filarial parasite Dirofilaria immitis.

A full length D. immitis cDNA (nDiCal) encoding a protein with significant similarity to the calreticulin protein family was isolated from a 6-day fourth-stage larval cDNA expression library by immunoscreening, using serum from a rabbit immunized by repeated injection of small numbers of third-stage larvae. nDiCal is 1538 bp long and contains the 21 bp nematode splice leader sequence SL1 at the 5' end. nDiCal encodes for a protein (pDiCal) with a predicted molecular mass of 46 kDa. pDiCal sequence analysis revealed similarities with calreticulin, a protein that typically resides in the endoplasmic reticulum. pDiCal possesses three consensus sequences of the calreticulin family of proteins: a neutral N-terminal region with a putative signal sequence; a proline- and tryptophan-rich P region; and a highly acidic C-terminal region. A 45Ca2+-overlay assay showed that recombinant pDiCal (rDiCal) is a Ca2+-binding protein. Antibodies to rDiCal identified a 56 kDa native antigen in all developmental stages including the excretory-secretory products derived from larvae and adult worms. Localization studies demonstrated the ubiquitous presence of pDiCal with intense expression in the hypodermis and syncitial muscle cells in both male and female adult worms. Labeling was also seen in the developing embryos within the uterus of the female worms. Sera from immune as well as chronically-infected microfilaremic dogs contained antibodies that bind rDiCal. In addition, immunoblot analysis showed that serum from a rabbit immunized with L3 cuticles reacted with rDiCal.