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The Rockefeller Foundation in 1969 stated that schistosomiasis is “the greatest unconquered parasitic disease now affecting man and animals” (Weir, 1969). Three major species of schistosomes, Schistosoma mansoni (in Africa, South America, and the Caribbean), S. haematobium (in Africa and the Middle East), and S. japonicum (in Japan, China, Philippines, and the Celebes), infect man. While S. intercalatum is also a human parasite, it has a limited distribution in West Africa. In addition, many different species of animal and bird schistosomes may penetrate the skin of man within which they die and produce a dermatitis known as swimmer’s itch. The schistosomes are multicellular organisms, and several different stages of the life cycle occur within the human definitive host: the cercariae released in bodies of water by infected snails penetrate the skin, changing into schistosomula. These immature schistosomes pass from the skin into the lungs and then down into the blood vessels of the liver where they develop into adult male or female worms, mate, and migrate into the intestinal (S. mansoni and S. japonicum) or vesical (S. haematobium) venules, where they produce 300–3000 eggs daily over periods of many years.

In the highly competitive world of parasites, where survival is indissolubly tied to the presence of suitable hosts in the same ecological niche, Strongyloides stercoralis occupies a unique position. This versatile nematode has developed a successful relationship with two ecosystems, the terrestrial environment and the mammalian body, by becoming facultatively independent of either (Box 1). It requires external maturation for transmission, but it also has the ability to multiply within a single host. It prefers man, but may infect apes, monkeys, dogs and cats. As a human parasite, S. stercoralis establishes self-perpetuating intestinal colonies and negotiates their survival with the host's defense mechanisms. In this article, Robert Genta reviews the immunobiological aspects of this unusual relationship.

Malaria parasites can be grouped evolutionarily by analysis of DNA composition and genome arrangement. Those that vary widely with regard to host range, morphology, and biological characteristics fit into only a small number of distinctive groups. The DNA of the human parasite Plasmodium falciparum fits into a group that includes rodent and avian malarias and is unlike the DNA of other primate malaria parasites. The DNA of Plasmodium vivax, which is also a human parasite, fits into a distinctly different group that includes Plasmodium cynomolgi, a parasite of monkeys. The evolutionary lines suggested here appear to be consistent with similarities seen among malaria parasites with regard to gene sequence.

Schizont-infected erythrocytes and merozoites were isolated from in vitro cultures of the human parasite, Plasmodium falciparum labeled with various radioactive substrates. The isolated merozoites were viable since they were able to reinvade fresh erythrocytes. On the basis of sensitivity to specific enzymes, eleven proteins synthesised by the parasite, were localised on the surface of the schizont-infected erythrocyte. Eight of these were glycoproteins, six of which appeared to represent three doublets. Five merozoite surface proteins were identified on the basis of their sensitivity to trypsin and chymotrypsin, treatments which also rendered the merozoite incapable of erythrocyte invasion. Merozoites appeared not to contain any glycoproteins; all of the glycoproteins synthesised by the parasite were apparently transported to the surface of the schizont-infected erythrocyte.

Publisher Summary This chapter discusses the evasion of immunity by nematode parasites causing chronic infections. Human nematode infections are long-lasting (chronic), and people living in endemic areas may become infected in early childhood and carry worms for the remainder of their lives. This chapter considers those aspects of the immunological relationship between host and parasite, in field and laboratory studies, that are pertinent to an understanding of the mechanisms by which immunity is avoided and thus, chronicity of infection achieved. Human parasite epidemiologists, veterinary parasitologists, parasite biologists, and immunologists all have an interest in the strategies employed by parasites to evade host immune responses and also in control measures aimed at breaking the transmission of these important pathogens. Much remains to be accomplished in this challenging area of parasite immunology, and a closer liaison between all research groups interested in chronic nematode parasites ensures rapid progress in the near future.

Publisher Summary Malaria is one of the world's most devastating diseases. Its intermittent fevers have been recognized since the beginning of recorded history. Because the parasites that cause malaria are very small, it was not possible to detect these pathogens until after the invention of the microscope. About five years after the finding of the human malaria parasites, birds were found also to be infected with malaria. But it was not until 1898 that malaria was seen in monkeys. It seems odd that bird malarias were found before the monkey malarias and that there was a lapse of 18 years from the discovery of the human parasite until they were found in these lower primates, animals that are recognized to have many similarities to man. Some scientists believe that some of the malarias of man and monkey are the same species, viz., P. malariae of man is the same as P. brasilianum of the Western Hemisphere monkeys. There are many important problems that can, should, and probably will be investigated in this new monkey host–human parasite model. However, the major difficulties have to do with the supply of the experimental animals, both quantitatively and qualitatively.

Bartonella bacilliformis anaemia (Oroya fever) is indigenous only to western South America1. The disease is diagnosed by identifying the morphology of the causative organisms (Bartonella bacilliformis) on the erythrocyte. “On the red blood cells it is unmistakable, and no other human parasite resembles it even slightly.”2

A linguatulid, very similar to the nymphal ‘Pentastoma gracile’ from crocodiles described by Sambon, was found in faecal material from a European resident in Khartoum. There is no evidence concerning the method of infection.I am indebted to Dr E. S. Horgan, until recently Director of the Stack Medical Research Laboratories, for his reply to my inquiries concerning linguatulid infections in the Sudan; and to Prof. J. J. C. Butley, of the London School of Hygiene and Tropical Medicine, for his valuable assistance in obtaining relevant literature.

Schistosoma spindale (Montgomery) is a common trematode parasite of cattle, water buffalo, goats and other mammals over a vast area including southern Africa, South and South-East Asia. It is a close relative of the human parasite S. haematobium and while it has only rarely been reported from man it is a significant producer of cercarial dermatitis in rice field workers. As part of an investigation of S. spindale in Malaysia, a number of laboratory animals were infected in various ways with cercariae of this helminth. One young female white mouse was exposed by tail immersion to numerous cercariae of a local strain in Kuala Lumpur and killed 82 days later with 0.3 ml. of heparinized ‘Nembutal’ (pento-barbital sodium) injected intraperitoneally. The liver and spleen were larger than those usually found in mice with S. spindale infections of similar age. The spleen was soft and yellowish-white and particularly distended over about half its length. Small pieces of liver, spleen, lungs and intestine were fixed in 10% formalin, after which the mouse was perfused with citrated saline according to the method of Duvall and DeWitt1. Eighteen pairs of worms and thirty adult males were recovered in the perfusate; all except one pair and two males had nodular lesions (Fig. 1 b–f) visible to the naked eye or with low magnification. I found several additional worms, including some with such lesions, in the tissues (Fig. 1f), but no affected worms in fifty-seven other mice similarly exposed and perfused. The discovery of these diseased worms was unanticipated. Because their significance was not immediately appreciated, they were fixed with 10% formalin and stored in 70% alcohol as usual. On subsequent examination, it became clear that their nodular lesions were filled with great numbers of cocci (Fig 1g and h) which could by then be identified only as being Gram-positive.

Publisher Summary This chapter describes fluorescent antibody (FA) studies in experimental malarias focusing on Simian–Human malaria antibody relationship. The six patients analyzed were infected only with P. falciparum or P. malariae. The FA responses are average values from 3 to 7 serum samples from each patient, adjusted so that the highest titer against the human parasite is equal to 100. The first three patients had P. falciparum only; it was seen that there were significant titers when P. fieldi antigen was used, as well as other minor cross reactivity. The three patients with P. malariae only also showed significant titers to P. fieldi, and even higher titers to P. brasilianum. This latter is of considerable interest, in that both P. malariae and P. brasilianum are quartan parasites and P. brasilianum has been shown to be infective to humans under experimental conditions. This immunologic cross-reactivity appears to be further indication of a close relationship between these two species. Minor cross-reactions with the other simian and human species were also seen.

microRNAs (miRNAs), a class of short, non-coding RNA can be found in a highly stable, cell-free form in mammalian body fluids. Specific miRNAs are secreted by parasitic nematodes in exosomes and have been detected in the serum of murine and dog hosts infected with the filarial nematodes Litomosoides sigmodontis and Dirofilaria immitis, respectively. Here we identify extracellular, parasite-derived small RNAs associated with Onchocerca species infecting cattle and humans. Small RNA libraries were prepared from total RNA extracted from the nodule fluid of cattle infected with Onchocerca ochengi as well as serum and plasma from humans infected with Onchocerca volvulus in Cameroon and Ghana. Parasite-derived miRNAs were identified based on the criteria that sequences unambiguously map to hairpin structures in Onchocerca genomes, do not align to the human genome and are not present in European control serum. A total of 62 mature miRNAs from 52 distinct pre-miRNA candidates were identified in nodule fluid from cattle infected with O. ochengi of which 59 are identical in the genome of the human parasite O. volvulus. Six of the extracellular miRNAs were also identified in sequencing analyses of serum and plasma from humans infected with O. volvulus. Based on sequencing analysis the abundance levels of the parasite miRNAs in serum or plasma range from 5 to 127 reads/per million total host miRNA reads identified, comparable to our previous analyses of Schistosoma mansoni and L. sigmodontis miRNAs in serum. All six of the O. volvulus miRNAs identified have orthologs in other filarial nematodes and four were identified in the serum of mice infected with L. sigmodontis. We have identified parasite-derived miRNAs associated with onchocerciasis in cattle and humans. Our results confirm the conserved nature of RNA secretion by diverse nematodes. Additional species-specific small RNAs from O. volvulus may be present in serum based on the novel miRNA sequences identified in the nodule fluid. In our analyses comparison to European control serum illuminates the scope for false-positives, warranting caution in criteria that should be applied to identification of biomarkers of infection.

Onchocerciasis, a neglected tropical disease prevalent in western and central Africa, is a major health problem and has been targeted for elimination. The causative agent for this disease is the human parasite Onchocerca volvulus. Onchocerca ochengi and Litomosoides sigmodontis, infectious agents of cattle and rodents, respectively, serve as model organisms to study filarial nematode infections. Biomarkers to determine infection without the use of painful skin biopsies and microscopic identification of larval worms are needed and their discovery is facilitated by an improved knowledge of parasite-specific metabolites. In addition to proteins and nucleic acids, lipids may be suitable candidates for filarial biomarkers that are currently underexplored. To fill this gap, we present the phospholipid profile of the filarial nematodes O. ochengi, O. volvulus and L. sigmodontis. Direct infusion quadrupole time-of-flight (Q-TOF) mass spectrometry was employed to analyze the composition of phospholipids and their molecular species in the three nematode species. Analysis of the phospholipid profiles of plasma or serum of uninfected and infected hosts showed that nematode-specific phospholipids were below detection limits. However, several phospholipids, in particular ether lipids of phosphatidylethanolamine (PE), were abundant in O. ochengi worms and in bovine nodule fluid, suggesting that these phospholipids might be released from O. ochengi into the host, and could serve as potential biomarkers.

Primate--and, specifically, monkey--malaria infections are commonly used for understanding the pathology of and immune response to the human disease because they are thought to resemble most closely the host-parasite relationship found in humans. Plasmodium cynomolgi is used extensively as a model for the human parasite, P. vivax, and P. knowlesi is used primarily as a model for the development of erythrocytic-stage vaccines. Both of these simian parasites can naturally infect man, resulting in mildly symptomatic episodes of the disease. The phylogenetic relationship between these two simian parasites and previously characterized Plasmodium species, including P. vivax, was examined by comparison of the asexually expressed small-subunit ribosomal RNA genes. Our analysis confirmed that P. vivax is most closely related to P. cynomolgi and that it remains an appropriate model of the human pathogen. Furthermore, with P. knowlesi and P. fragile, these two species form a group of closely related species, distant from other Plasmodium species. What is considered to be the most ancient of the human malaria pathogens, P. malariae, was also included in the analysis and does not group at all with other simian or human parasites.

Although Plasmodium vivax is responsible for the majority of malaria infections outside Africa, little is known about its evolution and pathway to humans. Its closest genetic relative, P. vivax-like, was discovered in African great apes and is hypothesized to have given rise to P. vivax in humans. To unravel the evolutionary history and adaptation of P. vivax to different host environments, we generated using long- and short-read sequence technologies 2 new P. vivax-like reference genomes and 9 additional P. vivax-like genotypes. Analyses show that the genomes of P. vivax and P. vivax-like are highly similar and colinear within the core regions. Phylogenetic analyses clearly show that P. vivax-like parasites form a genetically distinct clade from P. vivax. Concerning the relative divergence dating, we show that the evolution of P. vivax in humans did not occur at the same time as the other agents of human malaria, thus suggesting that the transfer of Plasmodium parasites to humans happened several times independently over the history of the Homo genus. We further identify several key genes that exhibit signatures of positive selection exclusively in the human P. vivax parasites. Two of these genes have been identified to also be under positive selection in the other main human malaria agent, P. falciparum, thus suggesting their key role in the evolution of the ability of these parasites to infect humans or their anthropophilic vectors. Finally, we demonstrate that some gene families important for red blood cell (RBC) invasion (a key step of the life cycle of these parasites) have undergone lineage-specific evolution in the human parasite (e.g., reticulocyte-binding proteins [RBPs]).

Blastocystis hominis is a common human parasite with infection rates up to 50% in developing countries, and giardiasis is the commonest intestinal one in Mexico. No doubt, various parasites as Giardia lamblia and Entamoeba histolytica can cause rheumatic diseases. This study coproparasitoscopic analysis evaluated the cysts by B. hominis, G. lamblia, E. hartmani, E. coli and E. histolytica in Mexican rheumatic disease patients. Also, ELISA was used to detect E. histolytica, Ascaris lumbricoides, Toxocara canis, and Trichinella spiralis in Mexican patients with rheumatoid arthritis (RA) and ankylosing spondylitis (AS). Thirty-six patients (24 with AS and 12 with RA) and 77 healthy control individuals were enrolled in this study. The frequencies of protozoan cysts were comparable in rheumatic disease patients (AS and RA) and healthy control donors (33 and 25 vs. 26%, respectively; p > 0.05). The frequency of antibodies to T. canis was significantly higher in AS patients than in healthy control donors (16 vs. 2.6%, respectively; p = 0.027), whereas no differences were observed for the prevalence of antibodies for the other parasites (E. histolytica, A. lumbricoides and T. spiralis) (p > 0.05). This information indicates the need to intensify educational efforts for the prevention of parasite infections associated with AS disease that cannot be controlled only by drugs.

Giardia lamblia (syn. duodenalis, intestinalis) is a globally occurring micro-aerophilic human parasite that causes gastrointestinal disease. Standard treatment of G. lamblia infections is based on the 5-nitroimidazole drugs metronidazole and tinidazole. In two other micro-aerophilic parasites, Entamoeba histolytica and Trichomonas vaginalis, 5-nitroimidazole drugs bind to proteins involved in the thioredoxin-mediated redox network and disrupt the redox equilibrium by inhibiting thioredoxin reductase and depleting intracellular thiol pools. The major aim of this study was to assess whether nitroimidazoles exert a similar toxic effect on G. lamblia physiology. The 5-nitroimidazoles metronidazole and tinidazole were found to bind to the same subset of proteins including thioredoxin reductase. However, in contrast to E. histolytica and T. vaginalis, none of the other proteins bound are candidates for being involved in the thioredoxin-mediated redox network. Translation elongation factor EF-1γ, an essential factor in protein synthesis, was widely degraded upon treatment with 5-nitroimidazoles. 2-Nitroimidazole (azomycin) and the 5-nitroimidazole ronidazole did not bind to any G. lamblia proteins, which is in contrast to previous findings in E. histolytica and T. vaginalis. All nitroimidazoles tested reduced intracellular thiol pools in G. lamblia, but metronidazole, also in contrast to the situation in the other two parasites, had the slightest effect. Taken together, our results suggest that nitroimidazole drugs affect G. lamblia in a fundamentally different way than E. histolytica and T. vaginalis.

Background The protozoan Giardia duodenalis is a common but highly diverse human parasite that comprises a complex of seven morphologically identical genetic assemblages, further divided into sub-assemblages. There is very little information available on the diversity of Giardia sub-assemblages and multi-locus genotypes infecting people in the United Kingdom. In this study we studied the molecular epidemiology of Giardia in symptomatic patients from North West England.

Attempts to control human malaria by immunological means could be compromised by antigenic variability within and between different strains of malarial parasites1. A useful alternative approach might be to block parasite antigens which are important in the mechanisms of invasion of red cells. As the major human parasite Plasmodium falciparum is highly specific for human red cells, isolation of the proteins involved in the recognition of red cells by this parasite might be of particular value. Recent studies suggest that the major red cell sialoglycoproteins (SGPs), glycophorins A, B and possibly C, may carry the sites recognized by the parasite2-4. Furthermore, because certain carbohydrates present on SGPs such as N-acetylglucosamine are able to block invasion by the parasite5, they may be involved in the initial interaction between parasite and red cell. We have now identified parasite proteins which bind to SGP or N-acetylglucosamine on Sepharose 4B columns. Three proteins, of molecular weights (MWs) 140,000 (140K), 70K and 35K, seem to be specifically bound by N-acetylglucosamine.

Surface-sterilized larvae were incubated with nutrient broth cultures of P-fimbriate E. coli. Immunofluorescence microscopy techniques employing monoclonal antibodies [mouse IgG anti-(E. coli P-fimbriae)] revealed that E. coli attach to the larval surface. Transmission electron microscopy also implied that E. coli adhere to the larval surface (Fig. 1). Further experimental work implied that surface-sterilized larvae excrete bacteria from their own intestinal tracts. This was suggested by the fact that a mixed growth of bacteria was obtained following inoculation of 'sterile' larvae onto bloodagar plates. The invasive filariform larvae do not feed, but the rhabditiform larvae in an earlier state of the parasite life cycle are ravenous feeders. Thus, if they ingest enteric bacteria while feeding in the human gut, then it could well be the case that enteric bacteria are excreted by the now transformed and disseminated filariform larvae. Septicaemia may also develop due to enteric bacteria escaping from the bowel when the invasive filariform larvae penetrate the gut wall. Such an investigation into this proposed mechanism was not feasible during this study, but there is no reason to discount it as a possibility. There have been several reports in the literature of helminths acting as vectors of microbial diseases. This has been clearly demonstrated in agriculture with plant parasite nematodes and the microflora of the rhizosphere (Poinar & Hansen, 1986). Enteric bacteria have been associated with human parasite nematodes in several instances, but a vector relationship has only been suggested (Woodruff, 1968). The frequency with which bacterial infections complicate disseminated strongyloidiasis suggests that enteric bacteria can be carried in or on the disseminated larvae, but up until now the precise mechanisms have never been elucidated. Our results suggest that the hypothesis that S . stercorulis larvae can act as vectors of enteric bacteria is indeed valid.

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Rabbits were immunized with the histidine-rich protein (HRP) of the avian malaria parasite, Plasmodium lophurae. The anti-HRP immunoprecipitated the knob protein (KP) from extracts of the human parasite, Plasmodium falciparum. The anti-HRP did not react with any antigens of a laboratory-derived strain of P. falciparum (K-) that does not form knobs. Antisera raised against a membrane-enriched fraction of P. falciparum-infected erythrocytes as well as sera from humans exposed to P. falciparum infections immunoprecipitated HRP from P. lophurae extracts.

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Through the kindness of Dean Charles E. Brookover of the University of Arkansas Medical Department, Little Rock, I received recently a specimen of a nematode which has not heretofore been recorded as a human parasite. The worm was removed from the lip of a girl. The conditions surrounding the case are reported as follows by the attending physician, Dr. Robert Lee Covington of Jefferson, Ark., to whom I am indebted not only for the case history cited here, but also for replies to several inquiries on the matter necessary to perfect the record for publication. Dr. Covington's report is as follows

It seems to be generally ignored thatTrichocephalus dispar, one of the intestinal parasites most common in man, is a parasite of pathologic importance. The majority of textbooks give few data concerning the pathologic changes which may be produced by this organism; some authors treat the matter as if it were not of importance, saying that the organism is harmless, without offering any proof for such an affirmation. This lack of attention toTrichocephalus disparis probably due to its frequent association with the hookworm, the study of which has been such an attractive theme in biology that any symptoms due to the former were accepted as a result of the latter. My purpose is to call attention to the clinical importance ofTrichocephalus disparand to review the facts obtained by various workers, in the hope that this may lead to further study of this important human parasite. METHODS

Prior to 1960, no natural vectors were known for any species of simian malaria. In the course of investigations in Southeast Asia during the last 3 years, natural vectors for five of these malarias have been found; four have been transmitted in the laboratory. Natural vectors for the African and New World species are still unknown. In a series of tables, in the appendix, the authors summarize the literature on the potential experimental vectors. The ecology of simian malaria in Malaya is reviewed with particular reference to members of the Anopheles leucosphyrus group of mosquitoes in which three natural vectors of simian malaria have been identified. Because they are also vectors of human malaria, it is with this group of mosquitoes that the greatest possibility of a liaison between human and simian malaria exists in Southeast Asia. Interest in the vectors of simian malaria has been stimulated by the transmission of the Malayan monkey parasite Plasmodium cynomolgi bastianellii from man to monkeys by the bite of infected mosquitoes. The original infections in man were acquired accidentally in three different laboratories in the United States from experimentally infected Anopheles freeborni mosquitoes (Eyles et al., 1960; Schmidt et al., 1961). Subsequently other strains of Plasmodium cynomolgi, which is closely related to the human parasite P. vivax, were transmitted to man (Coatney et al., 1961) and it has been shown that man-to-man transmission of the parasite can occur under laboratory conditions. It is also known that the Central American monkey parasite, P. brasilianum, is transmissible to man (Contacos et al., 1963). These findings have led to efforts to determine the vectors of simian malaria in nature and to see whether there are ecological barriers Received for publication 8 October 1963. * Presented at the XVI International Congress of Zoology, Washington, D. C., on 21 August 1963 and sponsored by the American Society of Parasitologists and the British Society of Parasitology. t Present address: Division of Entomology, Commonwealth Scientific and Industrial Organization, Brisbane, Australia. which would prevent the transmission of monkey malaria to man under natural conditions in the field. These investigations have been centered mainly in Southeast Asia, particularly in Malaya, but it is pertinent at this stage to review briefly what is known about the vectors of simian malaria the world over. VECTORS OF SIMIAN MALARIA IN AFRICA Four members of the genus Plasmodium have been isolated from subhuman primates in Africa (Table I). Three species come from the great apes, Plasmodium reichenowi; a falciparum parasite of gorillas and chimpanzees; Plasmodium schwetzi, a vivax parasite of gorillas and chimpanzees; and Plasmodium malariae, a quartan parasite, similar to the human quartan parasite, of chimpanzees. The natural vectors of simian malaria are unknown in Africa, and information on the development of parasites under experimental conditions is scanty. Bray (1957) reported gut infections with Plasmodium reichenowi in experimentally fed Anopheles gambiae but mosquitoes dissected for gland infections were all negative. A similar observation was made by Rodhain and Lassman (1940) when they noted that A. atroparvus fed on chimpanzees infected with P. schwetzi developed gut infections, but no

The first human case of Ancylostoma ceylanicum is reported from the Philippines. It occurred in a 53-year-old woman from Laoag, Ilocos Norte, P. I. Twenty-three worms (12 females and 11 males) were recovered. No previous record is known of Ancylostoma ceylanicum (Looss, 1911) as a human parasite in the Philippines. However, Tubangui's (1947) checklist of parasitic worms reported in the Philippines includes Ancylostoma braziliense (de Faria, 1910) as a human parasite. A 53-year-old female patient from Laoag, Ilocos Norte, Philippines, was referred to one of us (B.C.C.) for diagnosis and treatment. Two hours after administration of atebrine, 3 ft of strobila with intact scolex of Taenia saginata and 23 hookworms (11 males, 12 females) were recovered. The hookworms were fixed in 10% formalin. Six female worms and four males were sent to the senior author for study. The worms were transferred to 70% glycerine alcohol and examined in phenolalcohol solution. Measurements are averages in microns unless otherwise stated.

Background\ud \ud Wolbachia endosymbionts are a proven target for control of human disease caused by filarial nematodes. However, little is known about the occurrence of Wolbachia in taxa closely related to the superfamily Filarioidea. Our study addressed the status of Wolbachia presence in members of the superfamily Dracunculoidea by screening the human parasite Dracunculus medinensis and related species from wildlife for Wolbachia.\ud \ud Findings\ud \ud D. medinensis, D. lutrae and D. insignis specimens were all negative for Wolbachia colonization by PCR screening for the Wolbachia ftsZ, 16S rRNA and Wolbachia surface protein (wsp) sequences. The quality and purity of the DNA preparations was confirmed by amplification of nematode 18S rRNA and cytochrome c oxidase subunit I sequences. Furthermore, Wolbachia endobacteria were not detected by whole mount fluorescence staining, or by immunohistochemistry using a Wolbachia-specific antiserum. In contrast, positive control Brugia malayi worms were shown to harbour Wolbachia by PCR, fluorescence staining and immunohistochemistry.\ud \ud Conclusions\ud \ud Three examined species of Dracunculus showed no evidence of Wolbachia endobacteria. This supports that members of the superfamily Dracunculoidea are free of Wolbachia. Within the order Spirurida, these endosymbionts appear restricted to the Filarioidea.