Levels of infection, pathology and nodule size of Onchocerca flexuosa (Nematoda: Onchocercidae) in red deer (Cervus elaphus) from northern Spain.

Between 2005 and 2007, the presence of Onchocerca flexuosa (Wedl, 1856) was discovered and investigated in 110 red deer (Cervus elaphus) shot in the Riaño Regional Hunting Reserve, in the province of León (north-western Spain). Nodules containing O. flexuosa were located in the dorsal region and flanks of the deer. These were collected and measured, and some adult parasites were extracted from the nodules and identified by morphology and by obtaining mitochondrial 12S rDNA sequences, which were identical to those of previously published sequences for O. flexuosa. Some nodules were prepared for histology, embedded in paraffin, sectioned and stained with haematoxylin-eosin. Histologically, the worms were found in several compartments separated by an infiltrated fibrous tissue. These compartments were inhabited by several females and males, surrounded by a fibrous capsule. A total of 85.45% (95% confidence interval (CI): 78.86-92.04%) of red deer were parasitized, with a mean intensity of 9.53 ± 12.27 nodules/host, ranging between 1 and 74 nodules/deer. Significant differences in prevalence and intensity of infection were found between young and adult red deer, and also between seasons. However, no significant differences between males and females were observed. Five hundred and ninety-seven nodules were measured (15.81 ± 3.94 mm) and classified by sizes into small ( < 10 mm), medium (10-20 mm) and large (>20 mm). No relation was found between the size of the nodules and the time of infection. The high values found in the studied parameters show that northern Spain is an area of high-intensity infection for deer.

Blackfly vectors of zoonotic onchocerciasis in Japan.

Studies of blackfly vectors of Onchocerca dewittei japonica Uni, Bain & Takaoka (Spirurida: Onchocercidae), a parasite of wild boar implicated in the aetiology of zoonotic onchocerciasis in Japan, and six other zoonotic Onchocerca species of this country are reviewed. Molecular identification of infective larvae found in wild-caught female blackflies showed that Simulium bidentatum (Shiraki) (Diptera: Simuliidae) is a natural vector of O. dewittei japonica, and also Onchocerca sp. sensu Fukuda et al., another parasite of wild boar. Inoculation experiments demonstrated that Simulium arakawae Matsumura and four other Simulium species are putative vectors. Similarly, S. arakawae, S. bidentatum and Simulium oitanum (Shiraki) are putative vectors of Onchocerca eberhardi Uni & Bain and Onchocerca skrjabini Rukhlyadev, parasites of sika deer. Morphometric studies of infective larvae indicated that Onchocerca lienalis Stiles, a bovine species, is transmitted by S. arakawae, Simulium daisense (Takahasi) and Simulium kyushuense Takaoka, and that Onchocerca sp. sensu Takaoka & Bain, another bovine species, is transmitted by S. arakawae, S. bidentatum, S. daisense and S. oitanum. Prosimulium sp. (Diptera: Simuliidae) and Simulium japonicum Matsumura are suspected vectors of Onchocerca suzukii Yagi, Bain & Shoho and O. skrjabini [Twinnia japonensis Rubtsov (Diptera: Simuliidae) may also transmit the latter], parasites of Japanese serow, following detection of the parasites' DNA genes in wild-caught blackflies.

Oswaldofiaria chabaudi n. sp. (Nematoda: Onchocercidae) from a South American tropidurid lizard (Squamata: Iguania) with an update on Oswaldofilariinae.

A new species of Oswaldofilaria is described from Tropidurus torquatus (Tropiduridae: Iguania); its prevalence at the rocky study area at Juiz de Fora, Minas Gerais, Brazil, was approximately 30% and its mean intensity 3.13 +/- 2.51. Oswaldofilaria chaboudi n. sp. is distinct from the thirteen Oswaldofilaria species known in Australia, Africa and South-America in having the following characteristics: oesophagus medium-sized, left spicule 1 mm long and high spicular ratio (about 5), tail extremity ornated in both sexes with a bifurcated projection, and tooth-like structures near phasmids in the female. A long left spicule and high spicular ratio are convergent derived characters also found in a parasite of Australian crocodilians, O. kanbaya, and in several species of the closely related genus Befilaria, such as the Central American B. puertoricensis from polychrotids. Oswaldofilaria in South America is represented by eight species. Within these, a primitive group that is parasitic in Iguanidae, Polychrotidae (Iguania) and Crocodylidae and that possesses a long oesophagus is recognised, together with two distinct derived lines: three species with numerous, aligned precloacal papillae, parasitic in Teiidae (Laterato) and Scincidae (Scincomorpha), and O. chabaudi n. sp., in which this character is absent. Tropidurids (Tropiduris and Plica) had previously been reported in the host range of two oswaldofilarine genera, Oswaldofilaria and Piratuba, and their parasites assigned to known species described from other groups of lizards.

Genetic evidence for the presence of two species of Onchocerca from the wild boar in Japan.

In order to clarify the genetic differences between Onchocerca dewittei japonica, the causative agent of zoonotic onchocerciasis in Japan and a related undescribed Onchocerca sp., both parasitizing wild boar (Sus scrofa) of which the infective larval stages are indistinguishable from each other, we compared the sequences of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene region from four infective larvae (recovered from experimentally infected black flies), one microfilaria, and one adult of O. dewittei japonica, and from one infective larva (recovered from an experimentally infected black fly), one microfilaria, and a pool of several microfilariae of O. sp. The length of the CO1 gene region was 649 bp for all samples but there was a difference of 8.8 to 9.4% in the sequences between the two species although there were intraspecific variations of 0 to 0.5%. The CO1 sequences of O. sp. did not correspond to any of those deposited in the databases. Our study provides evidence that O. dewittei japonica and O. sp. are genetically different from each other.

Scanning electron microscopy of Onchocerca fasciata (Filarioidea: Onchocercidae) adults, microfilariae and eggs with notes on histopathological findings in camels.

BACKGROUND: Onchocerca fasciata is a prevalent filarial species in camelids of Asia and Africa forming nodules in the skin of dromedary and Bactrian camels. In spite of recent advances in the biology and epidemiology of this nematode species, a relatively scant number of studies have focussed on the morphology of this parasite. The main objective of this study was to describe morphological characteristics of adults, microfilariae and eggs of O. fasciata by scanning electron microscopy (SEM), staining and histology. METHODS: From April 2016 to March 2017 dromedary camels (n = 456) were inspected for infection with O. fasciata in a slaughterhouse in Kerman (south of Iran). Adult worms in nodules were isolated by digestion of nodules in collagenase and used for SEM. Skin nodules were also fixed, sectioned and stained with hematoxylin and eosin for histopathology. Skin microfilariae that were isolated from tissues surrounding the nodules were confirmed as O. fasciata by sequencing of the cytochrome c oxidase subunit 1 (cox1) and 12S rRNA genes and used for SEM and Giemsa staining. RESULTS: Single or multiple O. fasciata nodules (1.2-2.2 cm in diameter and 507-845 mg in weight) were found in 30.3% of the examined camels. SEM analysis helped identify 18 papillae in the caudal region of the male. Discontinuous longitudinal cuticular crests were observed in the posterior region of the male. In female nematodes, the ridges had a rounded shape with a height/width ratio of 7/16 in longitudinal sections. Unsheathed skin microfilariae with a rounded anterior extremity measured 210.7 × 2.5 µm on average. Developed eggs containing microfilariae measured 35.9 × 31.0 µm and their smooth shell surface had characteristic tongue-like appendages. In addition to inflammatory reactions surrounding the parasites, accumulation of intracellular ceroid pigment, golden-yellow to brown in colour, was observed within macrophages upon histopathological examination. CONCLUSIONS: We found longitudinal crests on the surface of the posterior region of the male nematode. Measurements of the main morphological features of microfilariae and eggs, and the shape index of ridges (height/width) in female nematodes are described for the first time.

Description, molecular characteristics and Wolbachia endosymbionts of Onchocerca borneensis Uni, Mat Udin & Takaoka n. sp. (Nematoda: Filarioidea) from the Bornean bearded pig Sus barbatus Müller (Cetartiodactyla: Suidae) of Sarawak, Malaysia.

BACKGROUND: The genus Onchocerca Diesing, 1841 includes species of medical importance, such as O. volvulus (Leuckart, 1893), which causes river blindness in the tropics. Recently, zoonotic onchocercosis has been reported in humans worldwide. In Japan, O. dewittei japonica Uni, Bain & Takaoka, 2001 from wild boars is a causative agent for this zoonosis. Many filarioid nematodes are infected with Wolbachia endosymbionts which exhibit various evolutionary relationships with their hosts. While investigating the filarial fauna of Borneo, we discovered an undescribed Onchocerca species in the bearded pig Sus barbatus Müller (Cetartiodactyla: Suidae). METHODS: We isolated Onchocerca specimens from bearded pigs and examined their morphology. For comparative material, we collected fresh specimens of O. d. dewittei Bain, Ramachandran, Petter & Mak, 1977 from banded pigs (S. scrofa vittatus Boie) in Peninsular Malaysia. Partial sequences of three different genes (two mitochondrial genes, cox1 and 12S rRNA, and one nuclear ITS region) of these filarioids were analysed. By multi-locus sequence analyses based on six genes (16S rDNA, ftsZ, dnaA, coxA, fbpA and gatB) of Wolbachia, we determined the supergroups in the specimens from bearded pigs and those of O. d. dewittei. RESULTS: Onchocerca borneensis Uni, Mat Udin & Takaoka n. sp. is described on the basis of morphological characteristics and its genetic divergence from congeners. Molecular characteristics of the new species revealed its close evolutionary relationship with O. d. dewittei. Calculated p-distance for the cox1 gene sequences between O. borneensis n. sp. and O. d. dewittei was 5.9%, while that between O. d. dewittei and O. d. japonica was 7.6%. No intraspecific genetic variation was found for the new species. Wolbachia strains identified in the new species and O. d. dewittei belonged to supergroup C and are closely related. CONCLUSIONS: Our molecular analyses of filarioids from Asian suids indicate that the new species is sister to O. d. dewittei. On the basis of its morphological and molecular characteristics, we propose to elevate O. d. japonica to species level as O. japonica Uni, Bain & Takaoka, 2001. Coevolutionary relationships exist between the Wolbachia strains and their filarial hosts in Borneo and Peninsular Malaysia.

A survey of Onchocerca fasciata infection in camels (Camelus bactrianus) with notes on morphology.

The goal of this study was to provide insight into the pathogenicity of Onchocerca fasciata in Camelus bactrianus to help control onchocerciasis. From November 2015 to January 2016, the prevalence and severity of onchocerciasis were recorded in 152 camels. Nodules containing Onchocerca were collected and observed. Adult parasites were extracted from the nodules and identified via light microscopy as well as by partial sequencing of the cytochrome oxidase subunit I (COI) gene. The sequences were examined and compared to similar sequences from other Onchocerca species. In total, 80.3% of camels were parasitized. The severity of infection varied, as camels harboured between one and fifteen nodules. The morphology and the cuticle differed in both sexes and displayed considerable variation in the thickness and structure of different body parts. Identification was further confirmed using molecular biology methods. This study provides a comprehensive morphological description of Onchocerca fasciata isolated from camels. The prevalence and intensity of infection (assessed via nodules) varied in the Bactrian camels. The structure of the cuticle was an important morphological feature for species differentiation in Onchocerca. Based on our data, the morphological assessment of O. fasciata represents a reliable method to characterize other Onchocerca species.

Morphological and molecular characterization of Onchocerca fasciata (Nematoda, Onchocercidae) from dromedary camels (Camelus dromedarius) in Iran.

Skin nodules of Onchocerca fasciata Railliet and Henry, 1910 (Spirurida, Onchocercidae) are a common finding in dromedary camels, though with a minimal clinical impact. There is little information about the morphology, molecular make-up and pathological impact of this parasite. Onchocerca fasciata nodules (1.3-2.1 cm in diameter and 509-841 mg in weight) were detected on the neck region in 31.5% of dromedary camels examined in Kerman province, southeastern Iran. Of 38 isolated nodules, only 23 (60.5%) contained viable worms. Measurement and morphological analyses were performed on isolated female worms by light microscopy. The identification of O. fasciata specimens was confirmed by sequence analysis of two mitochondrial genes (12S rDNA and cox1), which showed 0.4% divergence from available O. fasciata sequences. In addition, a phylogeny of filarial nematodes was constructed, based on these two mitochondrial genes and five nuclear genes (18S rDNA, 28S rDNA, MyoHC, rbp1, hsp70); this indicated that O. fasciata belongs to clade ONC3 of Onchocercidae, with representatives of the genera Onchocerca and Dirofilaria. Within the genus Onchocerca, O. fasciata is grouped with bovine parasitic species and the human parasitic Onchocerca volvulus, which suggests an impact of domestication on the radiation of the genus. Data provided here on the distribution and morphology of O. fasciata contribute to the molecular identification and phylogenetic position of the species.

Onchocerca eberhardi n. sp. (Nematoda: Filarioidea) from sika deer in Japan; relationships between species parasitic in cervids and bovids in the Holarctic region.

Onchocerca eberhardi n. sp. from the sika deer, Cervus nippon, in Japan is described. Adult worms lived in the carpal ligament; infection reached high levels (up to 25 female and 16 male worms in a single carpal limb). Skin dwelling microfilariae were mainly found in the ears. Prevalence of infection was 81% at the type locality, Mt. Sobo, in Kyushu. The new material was compared to the 31 species of Onchocerca presently known. Onchocerca eberhardi n. sp. females were characterized by a long slender anterior end and a thin esophagus < or =1 mm long with no or only a slight glandular region. The vulva was located near the level of the mid-esophagus and the cuticle had transverse external ridges and internal striae (two striae between adjoining ridges). The most similar species were O. stilesi (re-examined), O. lienalis, and to a lesser extent O. gutturosa, all from bovids (cattle). Two main lineages of Onchocerca are recognized in cervids with either primitive or with derived characteristics (as exemplified by the new species). The species in both lineages are not restricted to cervids but are also found in bovids in the Holarctic region, suggesting that the species diversified in the two host groups simultaneously, when these host groups lived in the some geographic area.

Litomosoides silvai (Nematoda: Onchocercidae) parasitizing Akodon montensis (Rodentia: Cricetidae) in the southern region of Brazil.

In the present study, Litomosoides silvai parasitizing Akodon montensis in the southern region of Brazil is reported for the first time. New morphological information is provided for some structures of this nematode species, such as a flattened cephalic extremity, presence of two dorsal cephalic papillae, female tail with a constriction at its tip, "s" shaped vagina, spicules characteristic of the carinii species group and microfilaria tail constricted at the tip. This nematode was found parasitizing the thoracic cavity with a prevalence of 10% (2/20), mean intensity of 4 (6/2), mean abundance of 0.4 (8/20) and range of infection of 2-6 specimens per host, in southern Brazil. This occurrence of L. silvai in A. montensis is a new geographical record for southern Brazil, in the Upper Paraná Atlantic Forest ecoregion of the northwestern region of Rio Grande do Sul, which is part of the Atlantic Forest biome.

Onchocerca takaokai n. sp. (Nematoda: Filarioidea) in Japanese wild boars (Sus scrofa leucomystax): Description and molecular identification of intradermal females.

Human zoonotic onchocercosis is caused by Onchocerca dewittei japonica, parasitic in wild boars (Sus scrofa leucomystax) in Japan. Previously, microfilariae longer than those of Onchocerca dewittei japonica were observed in skin snips from wild boars during the study of O. dewittei japonica. Moreover, the third-stage larvae (L3) of these longer microfilariae were obtained from the blackfly Simulium bidentatum after experimental injections. Based on morphometric and molecular studies, similar L3 were found in blackflies during fieldwork in Oita, Japan. However, except for O. dewittei japonica, adult worms of Onchocerca have not been found in wild boars. In this study, we discovered adult females of a novel Onchocerca species in the skin of a wild boar in Oita, and named it Onchocerca takaokai n. sp. Females of this new species had longer microfilariae and differed from O. dewittei japonica in terms of their morphological characteristics and parasitic location. The molecular characteristics of the cytochrome c oxidase subunit 1 and 12S rRNA genes of the new species were identical to those of the longer microfilariae and L3 previously detected, but they differed from those of O. dewittei japonica at the species level. However, both species indicated a close affinity among their congeners and Onchocerca ramachandrini, parasitic in the warthog in Africa, was basal in the Suidae cluster of the 12S rRNA tree.

Diagnostic features of zoonotic filariae in tissue sections.

The diagnostic features in tissue sections from patients with zoonotic filarial infections are reviewed. In general, two types of infections are recognized: 1) those presenting radiologically in healthy patients as coin lesions, which are usually removed because a clinical diagnosis of carcinoma of the lung is made and infarcts containing Dirofilaria immitis parasites in pulmonary arteries are found; and 2) those presenting as subcutaneous nodules, in which D. tenuis, a parasite from raccoons, D. repens, from dogs and cats, D. ursi , from bears, and Onchocerca spp, from horses or cattle, require delineation. Moreover, nodules that on microscopic examination are seen to be lymph nodes may harbor Brugia spp. The geographic distribution of these infections, with emphasis on the United States, is discussed.

The cryopreservation and in vitro cultivation of larval Onchocerca volvulus.

A system developed in our laboratory for the in vitro cultivation of larval Onchocerca volvulus is being employed in a series of morphogenetic, physiologic, chemotherapeutic and immunologic investigations of this parasite. Because of the need for a large and readily available supply of living worms for this work, cryogenic techniques are being used for the long-term preservation of larval parasites collected in various endemic areas of Guatemala, C.A. To date, microfilariae have survived frozen storage in human cutaneous tissues (excised nodules and skin snips) for as long as 504 days, and viable larvae, in all stages of development have been recovered from the black fly vectors (Simulium ochraceum and S. metallicum) kept frozen for 396 days. That cryopreservation does not appear to affect these parasites adversely is indicated by the fact that microfilariae derived from frozen tissues do not differ from those obtained from fresh tissues on the basis of: 1) numbers and vigor of emergent microfilariae; 2) survival and morphogenesis of microfilariae during cultivation in vitro for 24 days; 3) glucose utilization during 72 hours of incubation; and 4) their incorporation of 3H-amino acids as determined after 18 hours of incubation. Details of methodology for cryopreservation and in vitro cultivation, together with resultant data, are presented herein.

Descriptive statistics for the larval stages of Onchocerca volvulus in host-seeking Simulium ochraceum.

In four localities of Guatemala with dissimilar intensities of onchocerciasis, filarial larvae were recovered by dissection from host-seeking Simulium ochraceum. Measurements made on over 600 Onchocerca volvulus larvae were used to characterize the early first, late first, second, and third developmental stages. The numerical attributes used to characterize the third larval stage and the associated means were body length (657.3 microns); anterior body width (18.9 microns); posterior body width (20.2 microns); distance from anterior end to nerve ring (87.9 microns), to junction of muscular and glandular esophagus (137.6 microns), and to junction of esophagus and intestine (419.6 microns); and distance from anus to tip of tail (36.8 microns). Ratios to total body length were as follows: distance from anterior end to nerve ring, mean = 0.133, to junction of esophagus and intestine, mean = 0.634, and to anus, mean = 0.945. Differences between these phenotypic features and those reported for African O. volvulus appeared to be insufficient to distinguish the two forms. With very few exceptions, the filarial larvae found in host-seeking S. ochraceum were considered to be O. volvulus.

Morphometric differentiation of Onchocerca volvulus and O. ochengi infective larvae.

By microinjection of cryopreserved microfilariae (mf) into nulliparous flies, a comparison of the lengths of the infective larvae (L3) of Onchocerca volvulus and O. ochengi from the head of Simulium damnosum s.l. (presumed S. sirbanum) has been made. The suitability of S. sirbanum as a host was similar for both Onchocerca spp. The mean length +/- standard deviation of O. ochengi infective larvae measured in aqueous medium after storage of infected flies in liquid nitrogen was 762 +/- 63 microns (n = 39), significantly longer (P much less than 0.0001) than those of a savanna isolate of O. volvulus (676 +/- 56 microns, n = 26). Although the frequency distributions of the lengths of larvae of the 2 species overlapped, a critical value for discrimination of 719 microns applied to normally distributed populations with means and standard deviations of these samples would result in correct classification of 78% of true O. volvulus and 75% of true O. ochengi. A discriminant function analysis incorporating width measurements did not usefully improve the level of accuracy of discrimination. Larvae from flies stored in 70% ethanol and stained with acid haemalum were about 10% shorter, but O. ochengi infective larvae were still proportionately longer than those of O. volvulus (693 +/- 40 microns, n = 45 compared to 580 +/- 38 microns, n = 6, respectively). These data show that the infective L3 of O. volvulus and O. ochengi differ morphologically. Although the population length distributions overlap, by classifying larvae greater than 719 microns long as O. ochengi and those less than 719 microns long as O. volvulus a more accurate estimation of true O. volvulus infection rates in S. damnosum s.l. can be derived than is currently possible.

Comparison of the cuticular structure of parasitic nematodes recognized by immunocytochemical and lectin binding studies.

The cuticle structure of some nematode species was studied by immunogold and lectin-gold techniques. The gold labelling made it possible to distinguish the cuticle layers by the distribution and/or the density of the marker. On the other hand, no labelling pattern was found which led to a clear grouping of the layers into larger 'zones', since there were no subunits consisting of more than one layer which reacted in a characteristic way as compared to the rest of the cuticle. The outer surface of the epicuticle of parasitic adult worms turned out to be highly inert; it did not react with any of the antibodies or lectins tested. The cuticle of parasitic nematodes seems to function as a protection against the host's defense mechanisms rather than as an interaction site. An immunogenic component on the surface was only found in infective larvae. All antibodies and lectins showed a preferential binding to the electron dense layers and fibrous structures (HPL/GalNAc, WGA/GlcNAc) or to the amorphous ground-substance (Con A/Glc, RCA I/Gal).

Morphologic and genetic characterization of Onchocerca lupi infecting dogs.

In the past decades, sporadic cases of ocular Onchocerca infection have been reported in canids in US and Europe. The present study was undertaken to provide a detailed description of the morphologic characteristics of adults and microfilariae and to characterize the 5S ribosomal rRNA gene (5S rDNA) spacer sequences of Onchocerca lupi causing canine onchocercosis. The morphology of O. lupi is unique within the genus, and morphology based cluster analysis indicates that O. lupi is not closely related to the members of domestic cattle or horse clades occurring in North America and Europe. Similarly, the signature of the 5S rDNA spacer sequences of O. lupi does not resemble any other Onchocerca 5S rDNA spacer sequences including those of the members of domestic cattle or horse clades. The adult and microfilarial morphology and sequence signature supports the biological arguments that a distinct species, O. lupi and not O. lienalis, is responsible for canine ocular onchocercosis.

Studies on the Onchocerca (nematoda: Filarioidea) found in cattle in the United States. I. Systematics of O. gutturosa and O. lienalis with a description of O. stilesi sp. n.

Onchocerca gutturosa and O. lienalis are redescribed and comparative morphological descriptions provided. In addition, a third species of Onchocerca is described from cattle in the United States. This third species, Onchocerca stilesi sp. n., localizes in the connective tissues around the stifle joint, and can be distinguished morphologically from O. gutterosa and O. lienalis. Onchocerca gutturosa and O. lienalis apparently have widespread distribution in the eastern United States; however O. stilesi has been reported only from Minnesota.

Olecranon lesions caused by Onchocerca skrjabini in wild Japanese serows (Capricornis crispus).

Wild Japanese serows (Capricornis crispus) were found to have parasitic lesions in tendons that attached the musculus tricepus brachii to the olecranon. Histopathological study of the lesions showed chronic tendinitis with multiple granulation nodules around the worms. The lesions were found in 138 of the 353 serows examined and were more frequent in aged animals than young ones. Transverse ridges on the cuticle of the female midbody, the sizes and morphological features of the spicules, and the arrangement of the caudal papillae of the males showed the parasite to be Onchocerca skrjabini. Therefore, O. skrjabini causes olecranon lesions in addition to fibrous bursa formation in carpal and tarsal regions of the Japanese serows.

The morphology of O. volvulus microfilaria in the northwestern onchocerciasis endemic foci, Ethiopia.

Microfilariae obtained by corneoscleral biopsy machines from persons in the Humera area along the Sudan boarder in Northwestern Ethiopia were stained using hematoxylin. Morphological measurements were made using a microscope fitted with micrometer and camera, as well as map curvimeter. The mean size of microfilariae was 189 microns by 5.8 microns. The percentage of nucleifree cephalic space as percent of total body length was 3.4 and is close to reports in the literature. The measurement of the distance of the anterior end nerve ring as a percent of the total body length (22.1%) is also close to measurements obtained elsewhere. Although the microfilaria encountered in this study are of the short variety, morphological measurements and the absence of sheath suggests that the microfilaria observed belong to O. volvulus.