High prevalence and concomitant infection of Ranavirus and Eustrongylides sp. in the invasive American Bullfrog in Brazil.

American Bullfrogs, Aquarana catesbeiana, are invasive anuran species distributed worldwide. One of the adverse impacts that this species causes in native communities is as a reservoir host for pathogens and parasites. Here, we report the coinfection of two pathogenic organisms in A. catesbeiana: Ranavirus and the nematode Eustrongylides. Bullfrogs were collected in the wild in a pond close to the urban area of São Paulo, Brazil. The prevalence of both pathogens was high: 77% were infected with ranavirus with a mean viral load of 1010.3 viral copies, and 100% of the bullfrogs were infected by Eustrongylides sp. with a mean intensity of infection of 13.4 specimens per host. Four host specimens (31%) presented pathological signs that seemed to be related to the Eustrongylides sp. infection, such as internal organs adhered to each other due to high intensity and large size of the nematodes, ulcers, and raw flesh wounds caused by the nematode. The pathogenic and concomitant infections have potential zoonotic implications and raise concerns about human infection risks for Eustrongylides infection. Moreover, such infections may represent an additional level of threat to native communities through the potential shifts in patterns of parasite and pathogen transmission. Future research involving the native anuran community is essential to ascertain whether invasive bullfrogs are attenuating or exacerbating diseases such as ranavirosis and eustrongylidiosis.

Gastrointestinal parasites in bullfrogs (Lithobates catesbeianus) in aquaculture production units in the Mexican central highlands.

In Mexico, intensive production of bullfrogs is one of the most important aquaculture activities, due to growing demand for their meat. Frogs can be hosts for several parasites that negatively affect their development and health. The objective of this study was to identify the presence of intestinal parasites in bullfrogs in aquaculture production units. Eighteen bullfrogs aquaculture production units were selected, and 20 animals (n=360) from each farm. Fecal samples were obtained by mucosal scraping and processed using the concentration method. The overall prevalence of intestinal parasites was 70.5%, and all farms had frogs infected by some species of parasite. Two species of parasites were identified: Eimeria sp. and Strongyloides sp. Significant differences were found regarding parasite prevalence between males and females (73.8% vs 58.8%) and regarding tibia length (5.5 vs 6.1 cm) and weight (168 vs 187 g) between parasitized and non-parasitized frogs. In conclusion, the present study showed a high prevalence of intestinal parasites, and morphometric alterations (weight, snout-cloaca length, radio-ulna length, tibia length and distance between parotid glands) were identified in the parasitized animals. These results provided useful information that will enable establishment of adequate control measures to help minimize the adverse effects of these parasites.

Evaluating the Biological and Ecological Factors Influencing Transmission of Larval Digenetic Trematodes: A Test of Second Intermediate Host Specificity of Two North American Halipegus Species.

Host specificity of parasites is a basic principle in parasitology; however, it is not easily measured. Previously, host specificity was calculated as the number of species that a parasite infected, but this is not an accurate description of host usage because some species are capable of being infected but do not contribute to the completion of the life cycle. Instead, measures of host specificity should take into consideration interactions between a parasite and a potential host species as well as interactions between current and subsequent hosts in the life cycle. The objectives of this study were to track the development of 2 trematode species, Halipegus eccentricus and Halipegus occidualis , in 3 phylogenetically and ecologically distinct microcrustacean second intermediate hosts, and then evaluate the extent to which each of these hosts contributed to transmission of each Halipegus species to the next odonate host in the life cycle. All 3 microcrustacean species exposed became infected with both species of Halipegus. The patterns of growth of H. eccentricus and H. occidualis were similar, but there were consistent differences in the rates of growth among the microcrustacean species in both Halipegus species. Regardless of host species infected, all individuals of both species were considered to be developmentally infective to the next host in the life cycle by 19 days postexposure (DPE) when they lost their excretory bladder. Worms of varying sizes were capable of surviving without this structure, suggesting that there is not a strong relationship between the rate of growth of the metacercariae and the development of their osmoregulatory system. Although Halipegus species were capable of living without an excretory bladder at 19 DPE, there were differences in their size and rates at which the 3 microcrustaceans contributed to transmission of the parasites to subsequent odonate hosts. Collectively, under controlled laboratory conditions, there was an approximate 2-fold difference in the average percentage of worms that established in odonates from the ostracod, Cypridopsis sp., than from the harpacticoid copepod, Phyllognathopus sp., and the difference was nearly 3-fold between Cypridopsis sp. and the cyclopoid copepod, Thermocyclops sp. Therefore, despite all 3 microcrustacean species becoming infected, not all species were equally suited for transmission and completion of the life cycle. Differences among the 3 microcrustacean species in cercaria ingestion, metacercarial growth and development, and odonate predation rates on infected microcrustacean species were important factors in determining transmission of the 2 Halipegus species to odonate hosts.

Anuran Host Species Influences Site Fidelity of Halipegus occidualis.

Helminths often demonstrate preferential site selection in which a parasite will only occur in 1 microhabitat or a restricted portion of its fundamental niche within its host. However, factors responsible for helminth site specificity are poorly understood, and very little is known about how these factors vary among multiple host species. Some helminths, such as Halipegus occidualis, have been reported from different habitats (stomach or under the tongue) within multiple anuran host species, suggesting that the site selected varies within anuran species. This study examined the site selection by H. occidualis in 7 definitive anuran host species using experimental infections. Then, the site fidelity of H. occidualis was further tested by transplanting worms from under the tongue to the stomach and vice versa in different anuran host combinations, and the movement of worms was recorded. Halipegus occidualis individuals occupied the habitat under the tongue in 6 of 7 anuran species. However, worms always occupied the stomach of American bullfrogs (Lithobates catesbeianus) and were never found under the tongue or in the mouth of these hosts. More importantly, all worms remained in the original habitat when transplanted from the stomach to the stomach or the buccal cavity to the buccal cavity within another individual of the same amphibian species. However, when worms were transplanted from the stomach to the buccal cavity or vice versa in the same host species, the worms always migrated back to the original habitat. The main contribution of this study is that it experimentally documented the variability in the site fidelity of H. occidualis within multiple definitive host species and determined that site fidelity is not as strongly conserved in this genus as suggested previously. Additionally, this work suggests that the variation in site selection in different host species could lead to speciation of the parasites.

The Alteration of Life History Traits and Increased Success of Halipegus eccentricus Through the Use of a Paratenic Host: A Comparative Study.

Complex life cycles are a hallmark characteristic of many parasites; however, little is known about the process by which life cycles become more complex through the addition of hosts. Paratenic hosts are present in the life cycles of several phylogenetically distinct groups of helminths; this suggests that they may play a key role during this process. This study examined the development of metacercariae of Halipegus eccentricus within intermediate microcrustacean and odonate paratenic hosts. Then a comparative approach was used to evaluate how life history traits of H. eccentricus within the anuran definitive hosts differ between metacercariae of the same age that developed within an intermediate ostracod host or a paratenic odonate host. The results of this study indicate that metacercariae of H. eccentricus do not grow at the same rate in different intermediate hosts, and significant differences exist in growth within intermediate and paratenic hosts. Individuals from odonate paratenic hosts always had larger bodies and suckers than those of metacercariae of the same age that develop within microcrustacean intermediate hosts. Furthermore, metacercariae from odonates were more successful in establishing and migrating in definitive anuran hosts. Last, individuals from paratenic hosts began reproducing earlier within anuran definitive hosts than age-matched worms that develop within the intermediate hosts. Collectively these results suggest that the variation in body and sucker sizes within odonate and microcrustacean hosts may carry over to the definitive host and in the case of H. eccentricus using the paratenic host increases transmission and alters other life history traits within definitive hosts. These results indicate that using a paratenic host can affect the success of parasites in subsequent hosts, and therefore these hosts may provide benefits other than just increasing transmission by bridging an ecological gap.

The absence of zoonotic agents in invasive bullfrogs (Lithobates catesbeianus) in Belgium and The Netherlands.

Exotic invasive bullfrogs (Lithobates catesbeianus) are considered to exert a considerable negative impact on native amphibian communities. This can be due to competition and predation, but they are also a notorious source of the infectious diseases chytridiomycosis and ranavirosis, affecting amphibian populations globally. Little is known regarding their carriage of other microbial agents that might be transferred to humans or other animals. In this study we determined the occurrence of the amphibian pathogens Ranavirus and Batrachochytrium dendrobatidis and of the zoonotic agents Coxiella burnetii, Neospora caninum, Leptospira sp., Toxoplasma gondii, Mycoplasma sp., Campylobacter sp., Salmonella sp. and extended-spectrum beta-lactamase producing Escherichia coli in 164 bullfrogs from three populations in Belgium and The Netherlands. Although B. dendrobatidis was present at a high prevalence of 63%, mean infection loads were low with an average of 10.9 genomic equivalents (SD 35.5), confirming the role of bullfrogs as B. dendrobatidis carriers, but questioning their role as primary reservoirs for B. dendrobatidis transmission to native amphibian communities. All tested samples were negative for the other infectious agents examined. These results suggest a limited role of bullfrogs as carrier of these pathogens.

The role of gamont entry into erythrocytes in the specificity of Hepatozoon species (Apicomplexa: Adeleida) for their frog hosts.

Hepatozoon species are apicomplexan parasites that infect blood cells and viscera of terrestrial vertebrates. One species, Hepatozoon clamatae, primarily infects green frogs, Rana clamitans , whereas another, Hepatozoon catesbianae, primarily infects bullfrogs, Rana catesbeiana , although both species of parasite are capable of infecting either species of frog. The aim of this study was to determine whether the basis for this partial host specificity is manifested at the gamont, or intraerythrocytic, stage of the parasite's life cycle. Blood was drawn from infected frogs and treated in vitro with a saline solution to induce intracellular gamonts to emerge from host erythrocytes. This treated blood was added to in vitro samples of uninfected blood of green frogs and bullfrogs. After 1 hr, samples were analyzed to determine the level of re-entry of the parasites into uninfected erythrocytes. Results obtained using multiple combinations of donor and recipient frogs indicate that extracellular gamonts of both parasite species do not exhibit preference for erythrocytes of 1 frog species over those of another. These results suggest that the basis for the observed host specificity is not determined at the gamont stage and is more likely dependent on another stage in the parasite life cycle.

Introduced bullfrogs and their parasites: Haematoloechus longiplexus (Trematoda) exploits diverse damselfly intermediate hosts on Vancouver Island.

The lung fluke, Haematoloechus longiplexus, is the most prevalent and abundant parasite of introduced bullfrogs on Vancouver Island, British Columbia, Canada. The ecological success of this trematode in invasive bullfrogs is related to the fluke's ability to utilize native intermediate hosts for transmission. The purpose of this study was to identify the odonate (dragonfly/damselfly) species involved in the transmission of H. longiplexus to the introduced bullfrog. The prevalences and mean intensities of 21 species of odonates (nymphs and adults) were examined for metacercariae infections. Haematoloechus longiplexus is a second intermediate host specialist, being found only in damselflies. Six damselfly species exhibiting the "climber" ecological habit were identified as second intermediate hosts of H. longiplexus. Enallagma carunculatum (prevalence = 75.0%, mean intensity = 17.2 ± 10.8), Ischnura cervula (65.2%, 8.9 ± 4.3), Ischnura perparva (45.5%, 15.4 ± 10.3), and Enallagma boreale (40.7%, 4.8 ± 7.8) were the most commonly infected damselfly species. Metacercariae were absent in damselflies collected from sites lacking bullfrogs. Haematoloechus longiplexus was likely introduced along with the bullfrog, and subsequently adapted to the physid snail and diverse damselfly intermediate hosts present in ponds on Vancouver Island.

Do invasive bullfrogs in Victoria, British Columbia, Canada, show evidence of parasite release?

Few studies have examined vertebrate models of invasive species to explore parasite release as a proposed mechanism through which host species might become invasive. In this study, we examined evidence for parasite release in invasive American bullfrogs (Rana catesbeiana/Lithobates catesbeianus) from five sites in Victoria, British Columbia, Canada. We examined helminth species richness, as well as the prevalence, intensity and abundance of lung and kidney fluke infections. These flukes are expected to impose costs on host survival, growth and reproductive output. We compared measures of these parasite taxa with bullfrogs from Ontario and New Brunswick where they are endemic. Helminth species richness in bullfrogs from the Victoria sites was lower than in Ontario bullfrogs, but comparable to reported indices for other endemic populations. The prevalence of lung flukes (Haematoloechus spp.) in bullfrogs from Victoria was twice as high as was observed in the Ontario bullfrogs, and higher than has been reported from other endemic locations. In four of the five study sites in Victoria, numbers of Echinostoma spp. kidney cysts were lower than observed in endemic populations; however, the fifth site had uncharacteristically high numbers of cysts. In this study, there did not appear to be clear evidence to support parasite release using either parasite species numbers, or infection by specific parasite taxa. Instead, the invasive bullfrogs demonstrated high parasite species richness and high levels of infection for parasites known to be harmful to their hosts.

Helminth and leech community structure in tadpoles and caudatan larvae of two amphibian species from Western Nebraska.

Currently no comparative studies exist on helminth and leech community structure among sympatric anuran tadpoles and salamander larvae. During June-August 2007-2009, we examined 50 bullfrog tadpoles, Rana catesbeiana , 50 barred tiger salamander larvae, Ambystoma mavortium , and 3 species of snails from Nevens Pond, Keith County, Nebraska for helminth and leech infections. The helminth and leech compound community of this larval amphibian assemblage consisted of at least 7 species, 4 in bullfrog tadpoles and 4 in barred tiger salamander larvae. Bullfrog tadpoles were infected with 2 species of nematodes ( Gyrinicola batrachiensis and Spiroxys sp.) and 2 types of metacercariae ( Telorchis sp. and echinostomatids), whereas barred tiger salamander larva were infected with 1 species of leech ( Placobdella picta ), 2 species of adult trematodes ( Telorchis corti and Halipegus sp.), and 1 species of an unidentified metacercaria. The component community of bullfrog tadpoles was dominated by helminths acquired through active penetration, or incidentally ingested through respiratory currents, or both, whereas the component community of larval salamanders was dominated by helminths acquired through ingestion of intermediate hosts (χ²  =  3,455.00, P < 0.00001). Differences in amphibian larval developmental time (2-3 yr for bullfrog tadpoles versus 2-5 mo for salamander larvae), the ephemeral nature of intermediate hosts in Nevens Pond, and the ability of bullfrog tadpole to eliminate echinostome infections had significant effects on mean helminth species richness among amphibian species and years (t  =  12.31, P < 0.0001; t  =  2.09, P  =  0.04). Differences in herbivorous and carnivorous diet and time to metamorphosis among bullfrog tadpoles and barred tiger salamander larvae were important factors in structuring helminth communities among the larval stages of these 2 sympatric amphibian species, whereas size was important in structuring helminth and leech communities in larval salamanders, but not in bullfrog tadpoles.

The role of damselflies (Odonata: Zygoptera) as paratenic hosts in the transmission of Halipegus eccentricus (Digenea: Hemiuridae) to anurans.

Halipegus eccentricus is a common hemiurid trematode in the eustachian tubes of North America frogs. However, the life cycle of this species has never been completely elucidated. Studies on H. eccentricus suggest that it has a 3-host life cycle. Here, we show through fieldwork and host specificity experimental infections that the life cycle of Halipegus eccentricus utilizes 4 hosts. Metamorphosed anurans become infected with H. eccentricus by feeding on infected damselflies; worms reside in the stomach of anurans, migrate to the eustachian tubes within 32-39 days post-exposure (DPE), and release eggs 50-60 DPE. Cystophorous cercariae develop in Physa gyrina snails within 32-35 DPE, infect ostracod ( Cypridopsis sp.) second intermediate hosts, and develop to metacercariae. Fifteen- to 19-day-old metacercariae from ostracods are infective to both damselfly larvae and metamorphosed anurans. Field surveys of damselflies and tadpoles, along with laboratory exposure of damselfly larvae, metamorphosed anurans, and tadpoles with infected ostracods, indicated that only metamorphosed anurans and damselflies become infected with H. eccentricus , whereas field-collected tadpoles and laboratory-exposed tadpoles were never infected with H. eccentricus . Because little morphological change occurred in the metacercaria stage of H. eccentricus between the ostracod second intermediate host and damselfly host, and metamorphosed anurans became infected with H. eccentricus metacercariae recovered from both host groups, we suggest that odonates serve as paratenic hosts in this life cycle. Additionally, our field work and experimental infections provide data on the use of odonates as the route of infection by another North American Halipegus sp. that matures in the stomach of frogs. Our data indicate that when the life cycles are known, the use of odonates as the route of infection to anurans is common in the life cycles of Halipegus spp., and all species exhibit remarkable infection site fidelity in their amphibian hosts.

Pathologic findings in larval and juvenile anurans inhabiting farm ponds in Tennessee, USA.

Amphibian populations are declining globally, yet general pathologic surveys for free-ranging amphibians are uncommon. Pathologic surveys are necessary to provide insight into the impacts of humans on emergence of pathogens in amphibian populations. During 2005, 104 American bullfrog (Rana catesbeiana) and 80 green frog (Rana clamitans) larvae and 40 green frog juveniles were collected from farm ponds in Tennessee, and complete necropsies were performed. Diagnostic testing included bacterial culture, virus testing, fecal parasite analysis, and histologic examination. Gross and histologic examination revealed that all individuals, except one bullfrog tadpole, could be classified as clinically normal. The clinically abnormal tadpole had swollen erythemic legs, and was positive for Aeromonas hydrophila but negative for Ranavirus. Parasites were common (43%) among specimens, with myxosporidium and trematodes most often noted. Commensal and opportunistic microorganisms were cultured from the tissues. Ranavirus was detected in 29% of individuals but generally not associated with significant histopathologic changes. Myxosporidia and Ranavirus coinfections occurred in 7 and 26% of green and bullfrog tadpoles, respectively, with the highest coinfection rate (83%) in bullfrog tadpoles during winter. Protozoans were most common in fecal examination. These data can serve as a baseline to evaluate the presence of clinical disease in larval and juvenile amphibians.

Combined effects of agricultural activity and parasites on biomarkers in the bullfrog, Rana catasbeiana.

Agricultural contaminants can have devastating impacts on amphibian survival and development, particularly considering their sensitivity to environmental perturbation. However, it is commonly overlooked that amphibians are infected with various parasites that can influence the overall health of the animal when exposed to a stressful environment. We investigated the interaction of agriculture and parasitism on the health of bullfrogs (Rana catesbeiana) in the field. Nine physiological and immunological biomarkers were related to naturally acquired parasite infections, along a gradient of agricultural activity. Most health biomarkers were affected by agriculture, parasitism, or both. Although bullfrogs residing in agricultural areas were infected with fewer parasite species, reflecting environmentally compromised ecosystems, certain persistent parasites interacted with agricultural disturbance to alter the physiology and immune competence of bullfrogs. The consequences of the combination for animal health highlight the importance of parasitism in ecotoxicological studies. Consideration of parasitism is warranted when evaluating the influence of anthropogenic disturbance on amphibian declines and environmental health.

Alternative life cycle strategies and colonization of young anurans by Gorgoderina attenuata in Nebraska.

Studies on life cycles and epizootiology of North American frog bladder flukes indicate that adult frogs become infected predominantly by ingesting tadpoles or other frogs that serve as second intermediate hosts for gorgoderid metacercariae. Other studies have indicated that newly metamorphosed frogs are rarely infected with these parasites because they are gape-limited predators that cannot feed on large intermediate hosts such as tadpoles and other frogs. We examined the role of potential intermediate hosts in the recruitment of the frog bladder fluke, Gorgoderina attenuata, to metamorphosed northern leopard frogs, Woodhouse's toads, and bullfrogs from western Nebraska. We completed the life cycle of G. attenuata in the laboratory in 3 anuran species by experimentally infecting a variety of hosts. In addition, we generated and compared DNA sequence data from life cycle stages collected from a variety of naturally infected hosts. Our field and laboratory data indicate that in Nebraska G. attenuata has a truncated, 2-host life cycle that includes fingernail clams and anurans. Cercariae are ingested directly by tadpoles; unencysted juvenile worms then develop in the kidneys of tadpoles before moving to, and maturing in, the urinary bladder when tadpoles metamorphose. Additionally, G. attenuata can infect metamorphosed leopard frogs, bullfrogs, and toads when metacercariae in damselfly second intermediate hosts are ingested. These worms can also infect adult bullfrogs when they feed on other infected anurans possessing worms in their kidneys. Comparison of our material to published accounts of G. attenuata morphology and life cycles in Massachusetts suggests that previous work may have inadvertently involved 2 different species of gorgoderids. Our comparative approach to life cycle studies in different anuran life stages and multiple species of hosts suggests that tadpoles and metamorphosed anurans have favored alternative life cycle strategies in this trematode.

Infestation of wild-caught American bullfrogs (Rana catesbeiana) by multiple species of metazoan parasites.

The American bullfrog (Rana catesbeiana) is an aquatic, carnivorous member of the family Ranidae that is used extensively in physiology education programs and in various physiology, toxicology, sensorineural, and genetics research. Eleven bullfrogs purchased from a vendor distributing wild-caught frogs for use in a physiology research protocol were emaciated but otherwise showed no apparent clinical signs of illness. Necropsies performed on selected emaciated frogs indicated heavy infestation with multiple species of endoparasites. Identified helminths included Gorgodera amplicava, Haematolechus breviplexus, Clinostomum spp, Contracaecum spp, Cosmocercoides dukae, and Eustrongyloides spp. Grossly, parasitized bullfrogs showed encysted trematode larvae within skeletal muscle, nematode impaction of the intestinal tract, and lack of coelemic fat stores. Histopathologic lesions were restricted primarily to the gastrointestinal tract and consisted of parasitic granulomas associated with Contracaecum spp. The parasitic lesions may have been associated with the poor body condition of the bullfrogs. Food crickets maintained in-house were negative for parasite larvae or ova. Heavy parasitism of wild-caught bullfrogs may confound research protocols and markedly impair animal health. We encourage researchers to purchase laboratory-bred and -reared bullfrogs and to routinely monitor the parasite status of colony frogs.

Differential Salmonella survival against communities of intestinal amoebae.

Predation from intestinal amoebae may provide selective pressure for the maintenance of high genetic diversity at the Salmonella enterica rfb locus, whereby serovars better escape predators in particular environments depending on the O-antigens they express. Here, the hypothesis that amoebae from a particular intestinal environment collectively prefer one serovar over another is tested. Collections of Acanthamoeba, Tetramitus, Naegleria and Hartmannella were isolated from the intestinal tracts of several vertebrate hosts, including bullfrog tadpoles, goldfish, turtles and bearded dragons, and their feeding preferences were determined. Congeneric amoebae from the same environment had significantly similar feeding preferences. Strikingly, even unrelated amoebae - such as Naegleria and Tetramitus from goldfish - also had significantly similar feeding preferences. Yet amoebae isolated from different environments showed no similarity in prey choice. Thus, feeding preferences of amoebae appear to reflect their environment, not their taxonomic relationships. A mechanism mediating this phenotypic convergence is discussed.

Effects of the nematode Gyrinicola batrachiensis on development, gut morphology, and fermentation in bullfrog tadpoles (Rana catesbeiana): a novel mutualism.

We describe a novel mutualism between bullfrog tadpoles (Rana catesbeiana) and a tadpole-specific gastrointestinal nematode (Gyrinicola batrachiensis). Groups of tadpoles were inoculated with viable or nonviable nematode eggs, and development, morphology, and gut fermentation activity were compared between nematode-infected and uninfected tadpoles. Nematode infection accelerated tadpole development; the mean time to metamorphosis was 16 d shorter and the range of times to metamorphosis was narrower in nematode-infected tadpoles than in uninfected tadpoles. At metamorphosis, infected and uninfected bullfrogs did not differ in body size or condition. Colon width, wet mass of colon contents, and concentrations of most fermentation byproducts (short-chain fatty acids: SCFAs) in the hindgut were greater in infected tadpoles. Furthermore, in vitro fermentation yields for all SCFAs combined were over twice as high in infected tadpoles than in uninfected tadpoles. One explanation for accelerated development in infected tadpoles is the altered hindgut fermentation associated with the nematodes. Energetic contributions of fermentation were estimated to be 20% and 9% of the total daily energy requirement for infected and uninfected tadpoles, respectively. Infection by G. batrachiensis nematodes potentially confers major ecological and evolutionary advantages to R. catesbeiana tadpoles. The mutualism between these species broadens our understanding of the taxonomic diversity and physiological contributions of fermentative gut symbionts and suggests that nematodes inhabiting the gut regions of other ectothermic herbivores might have beneficial effects in those hosts.

Phylogenetic relationships of Hepatozoon (Apicomplexa: Adeleorina) based on molecular, morphologic, and life-cycle characters.

To evaluate higher-level affinities of Hepatozoon species within Apicomplexa, we sequenced the 18S rRNA gene from 2 parasites (Hepatozoon americanum and Hepatozoon canis) of dogs and 1 (Hepatozoon catesbianae) of bullfrogs. Sequences from other apicomplexans among the Sarcocystiidae, Eimeriidae, Theileriidae, Plasmodiidae, Cryptosporiidae, and Babesiidae, a Perkinsus species and 2 dinoflagellates were obtained from GenBank. Phylogenetic analysis indicated that Plasmodium, Cryptosporidium, and Hepatozoon form a monophyletic group distinct from representatives of other apicomplexan families. Although equivocal, our analysis indicated that Plasmodium and Cryptosporidium are sister taxa and that Hepatozoon is basal to them. To evaluate phylogenetic affinities among H. americanum, H. canis, and other species of Hepatozoon, we examined 18 morphologic and life-cycle features of 13 species currently assigned to Hepatozoon. This analysis indicates paraphyly of Hepatozoon (as currently arranged) because Hepatozoon lygosomarum was found most closely related to Hemolivia mauritanicum. These results, combined with results of previous studies, support elevating Hepatozoon to familial level (Hepatozoidae) as originally suggested by Wenyon in 1926. Both DNA sequence data and morphologic and life-cycle characters support a sister-group relationship between H. americanum and H. canis.

The life history and host specificity of Hepatozoon clamatae (Apicomplexa: Adeleorina) and ITS-1 nucleotide sequence variation of Hepatozoon species of frogs and mosquitoes from Ontario.

The life cycle of an intraerythrocytic hemogregarine, Hepatozoon clamatae, was studied in green frogs (Rana clamitans melanota), bullfrogs (Rana catesbeiana), northern leopard frogs (Rana pipiens), and in the mosquito, Culex territans. Gametogenesis, fertilization, and sporogony occurred within cells of the Malpighian tubules of laboratory-reared Cx. territans that had fed on naturally infected frogs. Mature oocysts containing hundreds of sporocysts were observed in mosquitoes 30 days postfeeding. Each sporocyst enclosed 4 sporozoites. Merozoites appeared in the peripheral circulation of laboratory-reared bullfrogs, green frogs and leopard frogs that had been fed sporocysts 35-70 days previously. Attempts to infect American toads (Bufo americanus) and blue-spotted salamanders (Ambystoma laterale) were not successful. Gamonts of this parasite induced nuclear fragmentation or segmentation in host erythrocytes. The life cycle, morphological, and morphometric features of H. clamatae are compared with H. catesbianae, a similar species that also infects ranids. Nucleotide sequence analysis of the internal transcribed spacer region (ITS-1) of these sympatric species revealed that only 6 nucleotide sites of the 129 base pairs of this region were variable among 4 isolates of H. clamatae and 2 isolates of H. catesbianae. A redescription of H. clamatae is presented based on data from this study and from the original description by Stebbins in 1905.

Helminths of two native frog species (Rana chiricahuensis, Rana yavapaiensis) and one introduced frog species (Rana catesbeiana) (Ranidae) from Arizona.

The gastrointestinal tracts, lungs, urinary bladders, and body cavities of Rana catesbeiana (n = 25), Rana chiricahuensis (n = 25), and Rana yavapaiensis (N = 37) from Arizona were examined for helminths. Helminths representing 9 species of trematodes: Cephalogonimus brevicirrus, Glypthelmins quieta, Gorgoderina attenuata, Haematoloechus complexus, Haematoloechus langiplexus, Megalodiscus temperatus, Alaria sp., Clinostomum sp., and an unidentified strigeid; and 4 species of nematodes: Falcaustra catesbeianae, Rhabdias ranae, Physaloptera sp., and an unidentified ascarid were found. The helminth fauna of introduced R. catesbeiana differed markedly from that of native ranids. Helminths of R. chiricahuensis and R. yavapaiensis represent new host records. Arizona is a new locality record for C. brevicirrus, G. attenuata, H. complexus, H. longiplexus, M. temperatus, and R. ranae.