The Complexity of Zoonotic Filariasis Episystem and Its Consequences: A Multidisciplinary View.

Vector-borne transmitted helminthic zoonosis affects the health and economy of both developing and developed countries. The concept of episystem includes the set of biological, environmental, and epidemiological elements of these diseases in defined geographic and temporal scales. Dirofilariasis caused by different species of the genus Dirofilaria is a disease affecting domestic and wild canines and felines and man, transmitted by different species of culicid mosquitoes. This complexity is increased because Dirofilaria species harbor intracellular symbiont Wolbachia bacteriae, which play a key role in the embryogenesis and development of dirofilariae and in the inflammatory pathology of the disease. In addition, the vector transmission makes the dirofilariasis susceptible to the influence of the climate and its variations. The present review addresses the analysis of dirofilariasis from the point of view of the episystem, analyzing the complex network of interactions established between biological components, climate, and factors related to human activity, as well as the different problems they pose. The progress of knowledge on human and animal dirofilariasis is largely due to the multidisciplinary approach. Nevertheless, different aspects of the disease need to continue being investigated and cooperation between countries and specialists involved should be intensified.

Surface associated antigens of Dirofilaria immitis adult worms activate the host fibrinolytic system.

Cardiopulmonary dirofilariosis (Dirofilaria immitis) is characterized by apparent contradictory events, like the long-term survival of adult worms in the circulatory system of the infected hosts and the development of life-threatening events like thromboembolisms and others. Thus parasite mechanisms, like the activation of fibrinolytic system, are key to the survival of both the worms and the host. The aim of this study was to investigate the interaction between D. immitis adult worms surface-associated antigens (DiSAA) and the fibrinolytic system of the host. We demonstrate that DiSAA extract is able to bind plasminogen and generate plasmin, with the latter occurring in a tissue plasminogen activator (t-PA) dependent manner. Additionally, 11 plasminogen-binding proteins from DiSAA extract were identified by proteomics and mass spectrometry (MS) (actin-5C, actin-1, enolase, fructose-bisphosphate aldolase, GAPDH, MSP domain protein, MSP 2, beta-galactosidase-binding-lectin, galectin, immunoglobulin I-set domain-containing protein and cyclophilin Ovcyp-2). Because in a previous work we have shown the positive interaction between the excretory/secretory antigens of D. immitis (DiES) and the host fibrinolytic system and many of the molecules identified here are shared by both antigens, we hypothesize that DiSAA cooperate in host fibrinolytic system activation promoting the fibrin clot lysis.

Identification and phylogenetic analysis of Dirofilaria ursi (Nematoda: Filarioidea) from Wisconsin black bears (Ursus americanus) and its Wolbachia endosymbiont.

Dirofilaria ursi is a filarial nematode of American black bears (Ursus americanus Pallas, 1780) that is vectored by black flies (Simuliidae) in many parts of the United States. In northwestern Wisconsin, the prevalence of microfilaremic bears during the fall hunting season was 21% (n = 47). Unsheathed blood microfilariae from Wisconsin bears possess characters consistent with the original description of D. ursi, as do adult worms observed histologically and grossly. Immunohistochemistry was used to identify the Wolbachia endosymbiont in the hypodermis and lateral cords of an adult female D. ursi. Amplification of wsp, gatB, coxA, fbpA, and ftsZ bacterial sequences from parasite DNA confirmed the presence of Wolbachia, and molecular phylogenetic analysis of the Wolbachia ftsZ gene groups the endosymbiont with Wolbachia from D. immitis and D. repens. Phylogenetic analysis of D. ursi 5s rDNA sequence confirms the morphological observations grouping this parasite as a member of Dirofilaria, and within the Dirofilaria - Onchocerca clade of filarial nematodes. This is the first report of Wolbachia characterization and molecular phylogeny information for D. ursi.

What is new about animal and human dirofilariosis?

Dirofilaria immitis and D. repens, the causal agents of cardiopulmonary and subcutaneous dirofilariosis, respectively, affect canine, feline and human populations with an increasing incidence in temperate and tropical areas of the world. In the past decade outstanding advances in the knowledge of dirofilariosis have been achieved. Nevertheless, questions such as the impact of climate change in the transmission and distribution of dirofilariosis, as well as a profound evaluation of both the role of Dirofilaria and Wolbachia and the proteins produced by them in the parasite-host relationship have not been fully addressed; therefore there must be milestones in dirofilariosis research in order to design new strategies and tools for the control of this disease.

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.

What is happening outside North America regarding human dirofilariasis?

The etiologic agents of human dirofilariasis in the Old World are Dirofilaria immitis, which cause pulmonary and subcutaneous nodules, and Dirofilaria repens, which cause ocular lesions. Although reports of new cases of dirofilariasis are sporadic in other parts of the world, a considerable amount of information is generated in Europe regarding human dirofilariasis. Most cases have been detected in the Mediterranean countries, Ukraine, and Russia; however, isolated or short series of cases have been reported in the Balkan Republics and central and northern European countries. Seroepidemiologic studies have provided evidence that humans living in endemic areas present rates of infection similar to those of the autochthonous canine populations. Antibodies against endosymbiont Wolbachia bacteria have been demonstrated recently in human Dirofilaria infections. During D. immitis infections, preadult worms and third- and fourth-stage larvae are often destroyed by the host reaction, releasing a considerable amount of Wolbachia, and a Th1-type response against Wolbachia and/or filarial antigens is mounted. On the contrary, infections with D. repens, in which worms frequently remain intact, no Th1-type response has been observed. As humans are resistant hosts, the Th1-response could have a role in the resistance against parasites. The causes for the rise in the incidence of human dirofilariasis as well as the possible application of Wolbachia antigens in the serodiagnosis of human infections are discussed.

Immunological role of the endosymbionts of Dirofilaria immitis: the Wolbachia surface protein activates canine neutrophils with production of IL-8.

Filarial nematodes, including Dirofilaria immitis and D. repens, harbour intracellular bacteria belonging to the genus Wolbachia. These bacteria have been implicated in the pathogenesis of filarial diseases, possibly through their endotoxins. Recent studies have shown that a major surface protein of Wolbachia (WSP) induces a specific IgG response in hosts infected by D. immitis. WSP from the Wolbachia of D. immitis was produced in recombinant form. The purified protein was used in stimulation assays on canine neutrophils. The assays performed using a modified Boyden chamber showed that WSP stimulates neutrophil chemokinesis. In addition, RT-PCR revealed increased production of chemokine IL-8 by cells incubated with this protein. Neutrophils have been shown to play a major role in the pathogenesis of river blindness, and to accumulate in the nodules of onchocerciasis patients. In dogs infected by D. immitis, neutrophils accumulate in kidneys and in the wall of pulmonary arteries. As shown by our studies, Wolbachia could contribute to these inflammatory phenomena through its surface protein WSP, independently from its endotoxin component.

wsp gene sequences from the Wolbachia of filarial nematodes.

Wolbachia endosymbiotic bacteria are widespread in arthropods and are also present in filarial nematodes. Almost all filarial species so far examined have been found to harbor these endosymbionts. The sequences of only three genes have been published for nematode Wolbachia (i.e., the genes coding for the proteins FtsZ and catalase and for 16S rRNA). Here we present the sequences of the genes coding for the Wolbachia surface protein (WSP) from the endosymbionts of eight species of filaria. Complete gene sequences were obtained from the endosymbionts of two different species, Dirofilaria immitis and Brugia malayi. These sequences allowed us to design general primers for amplification of the wsp gene from the Wolbachia of all filarial species examined. For these species, partial WSP sequences (about 600 base pairs) were obtained with these primers. Phylogenetic analysis groups these nematode wsp sequences into a coherent cluster. Within the nematode cluster, wsp-based Wolbachia phylogeny matches a previous phylogeny obtained with ftsZ gene sequences, with a good consistency of the phylogeny of hosts (nematodes) and symbionts (Wolbachia). In addition, different individuals of the same host species (Dirofilaria immitis and Wuchereria bancrofti) show identical wsp gene sequences.

Effects of tetracycline on the filarial worms Brugia pahangi and Dirofilaria immitis and their bacterial endosymbionts Wolbachia.

Wolbachia endosymbiotic bacteria have been shown to be widespread among filarial worms and could thus play some role in the biology of these nematodes. Indeed, tetracycline has been shown to inhibit both the development of adult worms from third-stage larvae and the development of the microfilaraemia in jirds infected with Brugia pahangi. The possibility that these effects are related to the bacteriostatic activity of tetracycline on Wolbachia symbionts should be considered. Here we show that tetracycline treatment is very effective in blocking embryo development in two filarial nematodes, B. pahangi and Dirofilaria immitis. Embryo degeneration was documented by TEM, while the inhibition of the transovarial transmission of Wolbachia was documented by PCR. Phylogenetic analysis on the ssrDNA sequence of the Wolbachia of B. pahangi confirms that the phylogeny of the bacterial endosymbionts is consistent with that of the host worms. The possibility that tetracycline inhibition of embryo development in B. pahangi and D. immitis is determined by cytoplasmic incompatibility is discussed.