Sara V. Brant

Molecular Systematics of the Avian Schistosome Genus Trichobilharzia (Trematoda: Schistosomatidae) in North America

Abstract Trichobilharzia is a genus of threadlike schistosomes with a cosmopolitan distribution in birds. Species of Trichobilharzia achieve notoriety as major etiological agents of cercarial dermatitis, or swimmers itch. There are 40 species described in the literature, for which the majority lacks molecular sequence information. To better understand the phylogenetic relationships, diversity, species boundaries, host use, and geographic distribution of this genus better, we surveyed 378 birds and over 10,000 snails from North America. The phylogenetic analysis was based on nuclear 18S, 28S rDNA, internal transcribed spacer region and mitochondrial cytochrome oxidase I sequence data. Specimens were recovered that could be related to 6 of the 14 described species of Trichobilharzia from North America (Trichobilharzia physellae, Trichobilharzia querquedulae, Trichobilharzia szidati, Trichobilharzia stagnicolae, Trichobilharzia franki, and Trichobilharzia brantae). An additional 5 lineages were found that could not be related directly to previously described species. Trichobilharzia brantae, transmitted by Gyraulus parvus, grouped outside the clade containing the recognized species of Trichobilharzia. A subgroup of the Trichobilharzia clade designated Clade Q was comprised of closely related species whose adults and eggs are similar, yet the European species use lymnaeids whereas the North American species use physids as snail hosts. This molecular phylogeny provides a useful framework (1) to facilitate identification of worms, including those involved in dermatitis outbreaks; (2) to test hypotheses about the evolution, diversification, host–parasite interactions and character evolution of Trichobilharzia; and (3) to guide future taxonomic revision of Trichobilharzia.

AN APPROACH TO REVEALING BLOOD FLUKE LIFE CYCLES, TAXONOMY, AND DIVERSITY: PROVISION OF KEY REFERENCE DATA INCLUDING DNA SEQUENCE FROM SINGLE LIFE CYCLE STAGES

Revealing diversity among extant blood flukes, and the patterns of relationships among them, has been hindered by the difficulty of determining if specimens described from different life cycle stages, hosts, geographic localities, and times represent the same or different species. Persistent collection of all available life cycle stages and provision of exact collection localities, host identification, reference DNA sequences for the parasite, and voucher specimens eventually will provide the framework needed to piece together individual life cycles and facilitate reconciliation with classical taxonomic descriptions, including those based on single life cycle stages. It also provides a means to document unique or rare species that might only ever be recovered from a single life cycle stage. With an emphasis on the value of new information from field collections of any available life cycle stages, here we provide data for several blood fluke cercariae from freshwater snails from Kenya, Uganda, and Australia. Similar data are provided for adult worms of Macrobilharzia macrobilharzia and miracidia of Bivitellobilharzia nairi. Some schistosome and sanguinicolid cercariae that we recovered have peculiar morphological features, and our phylogenetic analyses (18S and 28S rDNA and mtDNA CO1) suggest that 2 of the new schistosome specimens likely represent previously unknown lineages. Our results also provide new insights into 2 of the 4 remaining schistosome genera yet to be extensively characterized with respect to their position in molecular phylogenies, Macrobilharzia and Bivitellobilharzia. The accessibility of each life cycle stage is likely to vary dramatically from one parasite species to the next, and our examples validate the potential usefulness of information gleaned from even one such stage, whatever it might be.

Can specialized pathogens colonize distantly related hosts? Schistosome evolution as a case study.

Parasites that live in intimate contact with the immune system of their hosts require specialized adaptations to survive in such exposed environments. Once adapted to these demanding environments, it seems such parasites could not easily switch to distantly related hosts [1], and, thus, would be good candidates to diversify congruently with their hosts, i.e., cospeciation. One of the best-known parasite groups is the schistosomes, digenetic (having alternating sexually and asexually reproducing generations in their life cycle) trematodes that live in the vascular system of their vertebrate hosts. Schistosomes achieve notoriety because six of the roughly 100 described species [2,3] cause schistosomiasis, a disease that afflicts 200 million people, mostly in tropical Africa. Schistosomiasis is usually chronic and debilitating in its course, with most of the pathogenesis caused not directly by the adult worms but by the eggs they produce. Eggs become lodged in the viscera and incite tissue responses, often causing pronounced enlargements in the liver and spleen, and abnormalities in blood flow through these organs. If worms colonize the urinary system, hematuria (blood in the urine) and kidney and bladder damage often result. Schistosomiasis negatively affects growth and productivity, and has largely underappreciated, insidious effects on the people with this disease. Adult worms can be killed by drugs, but the limited availability and high cost of these drugs and the potential for emergence of drug resistance are important concerns. Immunity is slow to develop, though hope for an effective vaccine remains high. Schistosomes infect birds or mammals, but one species, Griphobilharzia amoena, often considered the missing link in schistosome evolution, is known to infect freshwater crocodiles [4]. Schistosomes share the habit of living in a vascular habitat with other trematodes, including the Spirorchiidae of turtles and the Sanguinicolidae of fishes. Worms in these three families have two-host life cycles—a snail host and a vertebrate host—and also share a distinctive tegument, or body covering, consisting of two lipid bilayers instead of the typical single bilayer. The double bilayer is believed to be an adaptation for survival in the immunologically hostile environment of the blood [5]. Schistosomes differ from the other two families of blood flukes, though, by being dioecious (having separate male and female worms) and dimorphic (with the two sexes different in morphology), and by having specialized habitat requirements. The remarkable biology of schistosomes has precipitated considerable discussion regarding their origins and their evolution of dioecy (the change from the typical state in trematodes of being hermaphrodites to a state with separate males and females) [2,4,6–10]. The discovery of G. amoena [4], the only species of schistosome known in an ectotherm, gave rise to a hypothesis that schistosomes arose in early ectothermic archosaurs, for example, ancestors of modern crocodiles, and radiated into endothermic archosaurs (birds). This view was supported by a phylogenetic analysis of adult morphology, which placed G. amoena as basal, or ancestral, among schistosomes [11], and challenged the role of endothermy as the pivotal factor driving schistosome diversification [10–12]. Molecular phylogenetic studies to date [7,13,14] have been hampered by an incomplete sampling of the 13 widely recognized schistosome genera, including the provocative and putatively basal G. amoena. The molecular phylogeny in Figure 1 includes representatives of all the commonly recognized genera of schistosomes, and spirorchiids from both freshwater and marine turtles [7]. Included in this molecular phylogeny is G. amoena, specimens of which were recovered from the Australian freshwater crocodile, Crocodylus johnstoni, obtained near Darwin, Australia. Figure 1 Maximum Likelihood Estimated Tree from the Combined Data Partitions of 6,335 Bases Derived from 18S and 28S Ribosomal DNA and Partial Cytochrome Oxidase I Mitochondrial DNA Genes Our analysis shows that G. amoena is, in fact, not a basal schistosome, but is more closely related to spirorchiids from freshwater turtles. This expands the host range of spirorchiids to include reptiles other than turtles, and suggests that schistosomes parasitize only endotherms. Our analysis confirms that the sister group to the schistosomes are the spirorchiids from marine turtles [7], and that the basal schistosomes are parasites of marine birds and snails (Figure 1). This pattern supports the idea that a long-range host switch from turtles to avian hosts occurred in marine habitats, and that schistosomes subsequently colonized birds, mammals, and freshwater snails. This argues against a hypothesis of a long-term association between schistosomes and archosaurs (crocodilians), and suggests that exploitation of endotherms has been the key factor leading to the emergence of schistosomes and their dioecious condition [2,8]. We speculate that the transferal of a spirorchiid protoschistosome to an endothermic host was favored by the partial endothermy [15,16] of their ancestral marine turtle hosts. Endotherms have metabolic rates that are roughly one order of magnitude higher than those of ectotherms of comparable size, and they consequently ingest more food [17]. Nutrients are then conveyed to the liver via the hepatic portal system. Most species of schistosomes live in the hepatic portal system. We argue that schistosomes colonized this specific habitat in endotherms to take advantage of its high nutrient content. In contrast, spirorchiids prefer the heart and arterial system [10], but can be found in widely scattered locations in the vascular system [18,19]. One of the unique demands imposed by the hepatic portal system is the need for the adult worms to move against the flow of blood to reach the small blood vessels surrounding the intestinal wall where oviposition would best lead to expulsion of the eggs in the feces. This requires a difficult combination of strength, to allow movement against the flow of blood, and delicacy, to allow extension of the body into the smallest vessels for oviposition. One solution is to forego the normal digenean condition of hermaphroditism to relegate each of these functions to separate sexes [2]. The muscular schistosome males are specialized to move females to preferred oviposition sites, whereas the delicate, thread-like females [10] are adapted to insinuate into tiny vessels for oviposition. Precision oviposition is crucial for schistosomes because eggs swept into the liver are not only a dead end from the parasites point of view, but they also impose significant pathology on their hosts [10]. Schistosomes remain paradoxical because they are specialized in morphology and habitat, and given the intimacy of contact with their host, it seems these worms would become restricted to particular hosts and consequently exhibit cophylogenetic patterns with those hosts. Our results (Figure 1), however, suggest that, historically, schistosomes have undertaken numerous long-range host switches among both vertebrates and snail hosts [7,13,14,20]. Observations that avian schistosomes can persist for a surprisingly long period in mammalian hosts, and likewise for some mammalian schistosomes in avian hosts, provide further evidence for this propensity [21–23]. We propose that schistosomes retain a pleisiomorphic (ancestral trait), nonspecific immune evasion strategy, such that long-distance host switches occasionally occur. The double tegumental membrane and the ability of the tegument to acquire macromolecules from many different host species [5] may contribute to this switching ability. With the possible exception of some species in derived taxa, such as duck schistosomes of the genus Trichobilharzia (see Figure 1) [24], the phylogeny of schistosomes does not mirror that of its hosts. Free-swimming larval schistosomes, the miracidia (for snails) and cercariae (for vertebrates), no doubt incessantly attempt to colonize new potential hosts, creating opportunities for host switches. Our results suggest that there are fundamental immune evasive mechanisms dictating schistosome success in intravascular environments in humans and other vertebrate hosts that we do not yet fully appreciate. They also suggest that morphological and microhabitat specialization need not preclude successful colonization of new hosts, indicating that congruent patterns in host and parasite phylogenies may be rare in these specialized worms [25,26].

Avian Schistosomes and Outbreaks of Cercarial Dermatitis

SUMMARY Cercarial dermatitis (swimmers itch) is a condition caused by infective larvae (cercariae) of a species-rich group of mammalian and avian schistosomes. Over the last decade, it has been reported in areas that previously had few or no cases of dermatitis and is thus considered an emerging disease. It is obvious that avian schistosomes are responsible for the majority of reported dermatitis outbreaks around the world, and thus they are the primary focus of this review. Although they infect humans, they do not mature and usually die in the skin. Experimental infections of avian schistosomes in mice show that in previously exposed hosts, there is a strong skin immune reaction that kills the schistosome. However, penetration of larvae into naive mice can result in temporary migration from the skin. This is of particular interest because the worms are able to migrate to different organs, for example, the lungs in the case of visceral schistosomes and the central nervous system in the case of nasal schistosomes. The risk of such migration and accompanying disorders needs to be clarified for humans and animals of interest (e.g., dogs). Herein we compiled the most comprehensive review of the diversity, immunology, and epidemiology of avian schistosomes causing cercarial dermatitis.

Schistosomes in the southwest United States and their potential for causing cercarial dermatitis or ‘swimmer's itch’

Cercarial dermatitis or swimmers itch results when cercariae of schistosomes penetrate human skin and initiate inflammatory responses. The parasites typically die in the skin but in some cases may persist and infect other organs. Cercarial dermatitis is caused by a complex and poorly known assemblage of schistosome species, and can occur in any location where people come in contact with water bodies harbouring schistosome-infected snails. In North America, most cases are reported from the upper Midwest. In south-western USA, this phenomenon has not been well studied, and it is not known which schistosome species are present, or if cercarial dermatitis occurs with any regularity. As part of our ongoing studies of schistosome diversity, using morphological traits and sequence data to differentiate species, we have thus far identified eight schistosome genetic lineages from snails from New Mexico and Colorado. We have investigated two cercarial dermatitis outbreaks, one occurring in Stubblefield Lake in northern New Mexico, and one in Prospect Lake in the heart of Colorado Springs, Colorado. The New Mexico outbreak involved either one or two different avian schistosome species, both transmitted by physid snails. The Colorado outbreak was due to Trichobilharzia brantae, a species transmitted by geese and the snail Gyraulus parvus. These outbreaks are in contrast to those in northern states where schistosomes infecting snails of the family Lymnaeidae are more often responsible for outbreaks. Our survey suggests that dermatitis-causing schistosomes are not rare in the southwest, and that there are plenty of opportunities for dermatitis outbreaks to occur in this region.

Additional observations on blood parasites of birds in Costa Rica

Birds from the Area de Conservación Guanacaste in northwestern Costa Rica were surveyed for blood parasites in June 2001 and December 2001–January 2002. Of 354 birds examined, representing 141 species of 35 families and 15 orders, 44 (12.4%) were infected with blood parasites. Species of Haemoproteus (4.8% prevalence), Plasmodium (0.6%), Leucocytozoon (0.3%), Trypanosoma (2.0%), and microfilariae (7.6%) were recorded. Twelve species of birds in this survey were examined for blood parasites for the first time. Several new host-parasite associations were identified.

A molecular approach for identification of paramphistomes from Africa and Asia

Paramphistomiasis is a disease of domestic and wild ruminants caused by some digenetic trematodes of the superfamily Paramphistomoidea. Human infections with certain species of paramphistomes have been reported. Given their ubiquity and their abundance within hosts, it seems likely that the importance of these worms is underestimated globally. Based on the reported difficulties of morphological identification of paramphistomes, the development of alternative approaches to better delineate species is needed. Paramphistome samples examined during this study were cercariae, rediae from snail hosts and adult flukes obtained from slaughterhouses from Kenya, Tanzania, Egypt and Nepal. The results confirm that ITS2 is a good molecular marker for paramphistome identification and can be used to determine the affiliation of samples among the different taxa of Paramphistomoidea. Our 22 samples include eight species of paramphistomes. Ten samples of paramphistome larvae (cercariae or rediae) yielded seven species and the 12 samples of adult flukes from bovines yielded four, only one of which was not also represented among the larvae. We found three cases in which the sequences for adult worms and larvae matched, thus revealing the hosts involved locally in those particular life cycles. We interpret our specimens to include five species from Kenya, two from Nepal, two from Tanzania and one from Egypt. Bulinus forskalii was found infected with three species of paramphistomes. The results of the present study will help construct a framework to facilitate identification and eventually control of this underestimated cosmopolitan group of digeneans.

PHYLOGENY OF SPECIES OF THE GENUS LITOMOSOIDES (NEMATATODA: ONCHOCERCIDAE): EVIDENCE OF RAMPANT HOST SWITCHING

Filarioid nematodes of the genus Litomosoides occur in the abdominal and (or) thoracic cavities of marsupials, rodents, and bats of the Nearctic and Neotropical regions. In this study, the phylogenetic relationships among these nematodes were estimated with a parsimony analysis of morphological characters derived from species descriptions. This nonweighted analysis produced 20 shortest trees. The monophyly of the genus was not supported in that Litomosoides thomomydis and Litomosoides westi failed to group with the other members of the genus. When these 2 taxa (parasites of pocket gophers) were excluded, monophyly of Litomosoides was supported by 2 synapomorphies (structure of the walls and general shape of the stoma); however, ancestor–descendant relationships among the species in the genus were not well resolved. A posteriori reweighting of the characters produced a single tree, different from all 20 most parsimonious trees. Alternative host–parasite evolutionary models were tested against these results supporting the process of host switching as being most important in forming the patterns of mammal–nematode associations that have been detected in this group of nematodes.

Discovery-based studies of schistosome diversity stimulate new hypotheses about parasite biology

This review provides an update of ongoing efforts to expand our understanding of the diversity inherent within the Schistosomatidae, the parasites responsible for causing schistosomiasis and cercarial dermatitis. By revealing more of the species present, particularly among understudied avian schistosomes, we gain increased understanding of patterns of schistosome diversification, and their abilities to colonize new hosts and habitats. Schistosomes reveal a surprising ability to switch into new snail and vertebrate host species, into new intrahost habitats, and may adopt novel body forms in the process. Often these changes are not associated with deep splits or long branches in their phylogeny, suggesting some are of relatively recent origin. Several hypotheses prompted by the new observations are discussed, helping to focus thinking on processes influencing not only schistosome diversification but also their pathogenicity and abundance.

Cercarial dermatitis transmitted by exotic marine snail.

TOC summary: Introduction of exotic hosts can support unexpected emergence of unknown parasites.